Genome-Wide Identification and Expression Analysis of LOX Gene Family in Rice (Oryza sativa L.) Under Abiotic Stress Conditions

Genome-Wide Identification and Expression Analysis of LOX Gene Family in Rice (Oryza sativa L.) 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Under Abiotic Stress Conditions Ahana Deb Tusti, Md. Nazmul Hasan, Jeba Faizah Rahman, Md. Hammadul Hoque, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8728944/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background Lipoxygenases ( LOX s) are enzymes that facilitate the oxygenation of polyunsaturated fatty acids (PUFAs) to create oxylipins through hydroperoxides that are involved in methyl jasmonate (MeJA) signaling pathway and are essential for seed germination, growth and development, defense mechanisms, and responses to stress. This study was designed for systematic analysis and exploration of expression patterns of the LOX gene family of rice. Results Twelve LOX genes were identified, and phylogenetic and structural analyses grouped them into 9-LOXs, Type I 13-LOXs, and Type II 13-LOXs subfamilies with conserved lipoxygenase domains, motif and gene structure patterns. These genes were dispersed unevenly across seven chromosomes. Analysis of gene duplication found four tandem and segmental duplication events predominantly driving their moderate expansion, with Ka/Ks ratios suggesting purifying selection. Syntenic analysis across three genomes indicated relatively conserved roles of orthologous gene pairs of this family. The cis-acting regulatory elements revealed 46 CRE motifs responsive to light, cellular development, hormones and stress. Notably, 151 putative miRNAs were identified as potential post-transcriptional regulators. PPI analysis highlighted 10 key nodes with high interaction degrees. GO enrichment classified the functions of OsLOX s into biological, metabolic and cellular processes. RNA-seq expression profiling demonstrated OsLOX s expression across diverse tissues, developmental stages and stress. RT-qPCR data analysis further validated that OsLOX genes exhibited temporally variable differential expression under multiple abiotic stress conditions including drought, salinity, saline-alkalinity, heat and cold, and in response to IAA hormonal treatment. Conclusion In conclusions, drought and cold stress significantly upregulated certain OsLOX s, enabling rice plants to confront these adverse conditions. This study will underscore the path for forthcoming in-vivo analyses to uncover the molecular processes of LOX genes involved in tolerance to stress in rice. LOX Phylogeny Cis-elements Expression profiling GO enrichment PPI Abiotic stress RT-qPCR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Background Rice ( Oryza sativa ) is considered as the preeminent cereal grain globally, feeding above 50% of the earth’s population [ 1 ]. Multitude of abiotic challenges resulting from environmental and climate changes such as salinity, drought, submergence, extreme temperatures, nutrient deficiencies and heavy metal accumulation [ 2 ], severely disrupt physiological [ 3 ], biochemical [ 4 ], molecular [ 5 ], and metabolic processes in rice plants [ 6 ], thereby adversely impacting their growth and development, productivity, and overall, an annual yield reduction of 32% (approximately 3 million tons) worldwide [ 1 ]. Abiotic stress response in rice plants occurs via induction of complex gene network modules [ 7 ], and the adaptation in changing environmental situations is well-regulated by the perception and stress signals transduction [ 8 ]. Rice is the first species to have its entire genome sequenced among cereal grains. Initial draft genomes for the japonica and indica subspecies (regarded as 93–11) were released in 2002. Although, the Japonica cultivar Nipponbare was the inaugural high-quality reference genome for rice, and indeed for any cereal species, which was released in 2005 [ 9 ]. Several studies have identified functionally annotated and validated expression profiles of stress responsive genes in rice through the analysis of genomes and transcriptomics data [ 10 – 12 ]. Therefore, characterizing the genetic background of rice thorough a genome wide investigation of stress-responsive families is essential for elucidating molecular mechanisms of stress tolerance and for breeding varieties with high adaptability under environmentally constrained conditions [ 11 , 13 , 14 ]. Lipoxygenases (LOXs) are an enzyme that is present in both plants and mammals. They are members of the iron-containing fatty acid dioxygenase family and include linoleate: oxygen oxidoreductase (EC 1.13.11.12) [ 15 ]. It enables the conversion of polyunsaturated fatty acids (PUFAs) from plants into linoleic acid (LA, C18:2ω6), linolenic acid (ALA, C18:3ω3), and arachidonic acid (AA, C20:4ω6) through the process of oxygenation and dehydrogenation. This process results in the formation of unsaturated hydroperoxides [ 16 ]. These intermediate products undergo several metabolic reactions to generate oxygenated derivatives including jasmonic acid (JA), reactive oxylipins containing epoxides, aromatic compounds conjugated carbonyls or aldehydes, and leaf aldehydes and diethylene ethers which have antibacterial and antifungal activities [ 17 ]. Additionally, certain PUFAs are metabolized by α-dioxygenase (α-DOX) enzymes to produce α-hydroxy or α-peroxy PUFAs [ 18 ]. Two spatially separated LOX pathways: 9-LOX and 13-LOX have been observed in flora. 13-LOX pathway was classified further as Type I 13-LOX and Type II 13-LOX, present on cytoplasm and chloroplast respectively [ 16 , 19 ]. The N-terminal of LOX gene contains a conserved PLAT/LH2 (polycystin-1, lipoxygenase, alpha-toxin/lipoxygenase homology) domain, while the C-terminal of true LOX genes contains a characteristics lipoxygenase domain composed of histidine amino acid rich region [His-(X)4-His-(X)4-His-(X)17-His-(X)8-His] [ 20 ]. Numerous signal molecules are generated via LOX-mediated pathways; among these, the major signalling compounds are C6-volatile compounds and jasmonates which contribute to plant physiological responses encompass germination [ 21 ], growth and development [ 22 ], fruit ripening [ 23 ], reactions to both biotic and abiotic stressors [ 24 – 28 ], wounding [ 29 ], senescence and cell death [ 30 ], and synthesis of stress responsive hormones such as jasmonic acid (JA) and abscisic acid (ABA) [ 31 , 32 ]. Plants express numerous LOX genes during stress, which induces physiological and anatomical modifications to accommodate adverse conditions [ 33 ]. LOX is a polygenic family studied and characterized in several plants. Understanding the specific physiological function of LOX genes has been challenging given the prevalence of several isozymes [ 34 ]. The occurrence of these distinct LOX isoforms is contingent upon the tissue, sub-cellular spaces, and stages of embryogenesis, and their catalytic features, tissue-specific gene expression, and amino acid sequences vary [ 35 ]. Prior research has determined a cumulative sum of 6 LOX family members in Arabidopsis [ 36 ], 36 members in soybean [ 37 ], 11 members in radish and tea tree [ 38 , 39 ], 12 members in Foxtail millet [ 40 ], 18 members in melon [ 41 ], 14 members in strawberries [ 42 ], 20 members in poplar and Artemisia annua and so on. Among many C3 photosynthetic cereal crops, durum wheat has been shown to upregulate TdLpx-A2 during hyperosmotic stress and thereby counteract excess ROS generation by TdLOX2 to mitigate plant oxidative damage [ 43 ]. CRISPR/Cas9-mediated overexpression line of soybean demonstrated GmLOX6 increases salinity tolerance through JA-mediated biosynthesis pathway [ 44 ]. C4 model plant foxtail millet exhibited a substantial upregulation of SiLOX7 during the exposure of saline condition in two stress-tolerant varieties [ 40 ]. In oriental melon, suppression of CmLOX10 has been demonstrated to enhance drought susceptibility via jasmonic acid-mediated stomatal closure and feedback involving CmMYC2 [ 27 ]. Cloning of the computationally identified promoter region of CmLOX08 elucidated the mechanisms underlying resistance to abiotic stresses through signaling molecules and stress-inducible core promoters [ 45 ]. Further study on Medicago truncatula confirmed that exogenous MtLOX24 overexpression in Arabidopsis mitigates the MeJA-induced oxidative damage, suggesting its role to insect related abiotic stress responses (L. Xu et al., 2024). Although numerous research has examined rice transcriptome data and co-expression patterns in response to various abiotic and biotic challenges, a systematic examination of the LOX gene family and its specific response to abiotic stress remains lacking. A comprehensive characterization and expression profiling of LOX gene family of rice may offer significant understanding of its function under abiotic stressors, attributable to the presence of lipoxygenase enzymes. Thus, our approach can effectively identify and characterize LOX gene family, by uncovering their evolutionary relationships, expression profiles, and potential functionalities. Additional, functional characterization of selective LOX s in response to abiotic and hormonal stress will yield insights for further examination of rice LOX gene family. Materials and methods Sequence curation of Lipoxygenase genes in Oryza sativa genome and physicochemical properties analysis A multi-step method employing diverse bioinformatics resources and databases was applied to discover LOX family members in rice. TAIR database ( https://www.arabidopsis.org/ )[ 47 ] was utilized to obtain the protein sequences for six Arabidopsis thaliana genes ( At1g55020, At3g45140, At1g17420, At1g72520, At3g22400, At1g67560 ) that are members of lipoxygenase gene family [ 48 ]. The rice LOX proteins were identified by undertaking a BLASTP search against the Oryza sativa v7.0 genome dataset in the Phytozome database ( https://phytozome-next.jgi.doe.gov/info/Osativa_v7_0 ) [ 49 ]. For the sake of comprehensive identification, these AtLOX protein sequences were additionally tested against the Rice Genome Annotation Project (RGAP) database ( https://rice.uga.edu/ )[ 50 ] and NCBI protein database ( https://www.ncbi.nlm.nih.gov/protein )[ 51 ] individually to ensure no potential member was overlooked. To identify all the possible LOX genes, Hidden Markov Model (HMM) profile for Lipoxygenase domain (PF00305) and PLAT/LH2 domain (PF01477) were acquired from the Pfam database[ 52 ] with HMMER program (v3.4) [ 53 ]. The e-value threshold was established at <1e-5. To further validate the presence and integrity of conserved domains, the presumed LOX sequences were analyzed using SMART ( http://smart.embl-heidelberg.de/ ) [ 54 ], NCBI CDD ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) [ 55 ], and InterPro ( http://www.ebi.ac.uk/interpro/ ) [ 56 ]. The genes were renamed as ‘ OsLOX ’ with the ‘ Os ’ prefix indicating Oryza sativa and sequentially numbered according to chromosomal positions (from top to bottom), following an established naming procedure [ 57 ]. Gene lengths, CDS lengths, locus IDs, strand locations, and coding sequence (CDS) coordinates (5′ to 3′) were all taken from the Phytozome database [ 49 ]. Further, to ascertain similarities in the protein sequences of OsLOX s, multiple sequence alignments were conducted using the ClustalW alignment function of MEGA 11[ 58 ] and results were visualized using the software GeneDoc v.2.7 [ 59 ]. The physicochemical properties of the genes, including the theoretical isoelectric point (pI), index of instability, grand average of hydropathy (GRAVY), and molecular weight of the proteins were assessed using OsLOX protein sequences in ProtParam hosted by ExPASy ( https://web.expasy.org/protparam/ ) [ 60 ]. In addition, for determining the proteins subcellular distribution, the protein sequences FASTA file were uploaded to WoLF PSORT ( https://wolfpsort.hgc.jp/ ) [ 61 ] and CELLO v.2.5 ( http://cello.life.nctu.edu.tw/ ) [ 62 ]. TBtools version v2.225 [ 63 ] was employed to visualize the anticipated protein signals of each gene. Phylogenetic relationship analysis To generate the tree of evolutionary relationship of LOX proteins from several plant species, the completely discovered sequences of LOX proteins of Arabidopsis thaliana [ 36 ], Zea mays [ 64 ], Brassica rapa [ 65 ], Sorgum bicolor [ 66 ], Setaria italica [ 40 ] and Glycine max [ 37 ] were used to keep the rate uniform across sites. Peptide sequences of LOX genes of Zea mays (B73 reference genome), Sorgum bicolor, Setaria italica and Glycine max were acquired from Phytozome [ 49 ] and Brassicaceae database ( http://brassicadb.cn ) [ 67 ]. TAIR database [ 47 ] was employed for retrieval of peptide sequences of LOX genes from Brassica rapa and Arabidopsis thaliana genome correspondingly. By employing the MUSCLE alignment and the Maximum Likelihood Method with a 1000 bootstrap value to substantiate branch values, an unrooted phylogenetic tree was constructed in MEGA 11 [ 58 ]. The tool iTOL ( https://itol.embl.de/ ) [ 68 ] was employed for tree annotation, manipulation and visualization. Gene structure and conserved motif analysis The organization of exon-intron of OsLOX s were efficiently identified and graphically depicted utilizing Gene Structure Display Server (GSDS2.0) ( http://gsds/gao-lab.org ) [ 69 ] by comparing the coding sequences (CDS) and genomic sequences lacking the UTR (untranslated region) as input resources on the server. MEME Version 5.5.8 (Multiple Em for Motif Elicitation; https://meme-suite.org/meme/tools/meme ) [ 70 ] was utilized to identify conserved motifs on proteins, employing default parameters with the exception of selecting a maximum of 15 motifs. MEME online interface used the motif scanning method (MSA) to visualize the motifs. The role of each of the 15 detected motifs was determined by evaluating them using Pfam [ 52 ]. Chromosomal distribution analysis MapChart software[ 71 ] was employed to create a rudimentary chromosomal distribution diagram of OsLOX genes based on their location across the 12 rice chromosomes. The details on gene loci, length for the physical map and CDS coordinates obtained from the Phytozome [ 49 ] were employed for pinpointing the location and position on the map. Prediction of gene duplication and collinearity analysis For estimating the divergence time of OsLOX genes and investigating evolutionary patterns, the non-synonymous (Ka) and synonymous (Ks) substitution rates for duplicated OsLOX gene pairs were calculated using TBtools version v2.225 [ 63 ]. Segmental duplications were defined as instances in which gene pairs exhibited more than 90% sequence similarity, while tandem duplications were defined as two or more homologous genes within a 100 kb region on the same chromosome [ 72 ]. Selection was inferred from Ka/Ks ratios, and divergence time was determined using the formula T = Ks/2λ × 10 − 6 (million years). λ is defined as a constant rate of 1.5 ×10 − 8 substitutions per site per annum for plants with dicotyledons [ 73 ]. Multiple Collinearity Scan Toolkit (MCScanX) [ 74 ] was used to detect gene duplication events. The Oryza sativa genomic sequence and GFF annotation file were utilized for self-alignment in the investigation of intraspecific collinearity. Interspecific collinearity analysis involved comparing the genomic sequences and GFF annotation files of Arabidopsis thaliana and Zea mays with the genome sequences of Oryza sativa . The syntenic relationship was then determined using the syntenic analysis maps generated by Dual Systeny Plotter technique ( https://github.com/CJ-Chen/TBtools ). Finally, the result was visualized using TBtools v2.225. Cis -regulatory elements analysis of OsLOX s The 2000 bp upstream sequences of each OsLOX promoter region were obtained from the Phytozome v13 database [ 49 ] to investigate the cis-acting regulatory elements (CREs) and their functions. To identify the prospective cis-regulatory elements (CREs) within the promoter region of OsLOX genes, the PlantCARE database ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) [ 75 ] was employed and the potential CREs were visualized using TBtools v2.225 [ 63 ]. Specification putative miRNA targeting OsLOX genes To identify potential miRNAs that target the LOX genes in rice, psRNATarget ( https://www.zhaolab.org/psRNATarget ) [ 76 ] was utilized by uploading the CDS sequences of OsLOX s. The analysis focused on the mature micro-RNAs of rice listed in miRbase ( https://www.mirbase.org/ ) [ 77 ]. The associations between these miRNAs and their target OsLOX genes were illustrated through Cytoscape software [ 78 ]. Gene Ontology Analysis To ascertain the functional relationship of the discovered OsLOX genes, gene ontology (GO) enrichment analysis was performed in collaboration the Plant Transcription Factor Database ( https://plantregmap.gao-lab.org/go.php ) [ 79 ]. ChiPlot ( https://www.chiplot.online ) [ 80 ] was employed for data visualization. Prediction of phosphorylation sites For the prediction of phosphorylation sites, the protein sequences were used as input using the NetPhos-3.1 website ( https://services.healthtech.dtu.dk/services/NetPhos-3.1/ ) [ 81 ]. Results with a combined score greater than 0.5 were reliable. Protein-protein interaction (PPI) network prediction STRING version-12.0 ( https://string-db.org/ ) [ 82 ] was employed to predict the PPI network of OsLOX proteins based on the homologous proteins from Arabidopsis thaliana . The following parameters of STRING tool were specified for the analysis: (i) full STRING network, as network type, (ii) the meaning of network edges as evidence, (iii) minimum interaction score 0.4 (medium confidence parameter), and (iv) maximum number of interactions in first shell < 10. Cytoscape software [ 78 ] was employed to visualize the predicted interaction network. Further, putative functions of the interacting protein families and result of KEGG enrichment analysis of PPI node proteins were retrieved. Secondary and tertiary structure analysis of OsLOX proteins The STRIDE program ( https://webclu.bio.wzw.tum.de/stride/ ) [ 83 ] with default parameters was employed to predict OsLOX protein’s secondary structure. PROTEUS Structure Prediction Server 2.0 ( http://www.proteus2.ca/proteus2/ ) [ 84 ] was employed to forecast the turns, coils, 310 helices, extended beta sheets, and alpha helices in the context of structural analysis. Additionally, investigation of membrane-spanning motif was done by using DeepTMHMM 2.0 ( https://services.healthtech.dtu.dk/services/TMHMM-2.0/ ) [ 85 ]. Next, the trRosetta webserver ( https://yanglab.qd.sdu.edu.cn/trRosetta/ ) [ 86 ] were employed for generating the tertiary structure of OsLOX proteins. The predicted tertiary structures were refined using the GalaxyRefine2 module of the GalaxyWEB server ( http://galaxy.seoklab.org/ ) [ 87 ]. Energy minimization was done employing Swiss-PdbViewer v4.1 ( http://www.expasy.org/spdbv/ ) [ 88 ]. To verify the reliability of the protein structures, PROCHECK tool ( https://servicesn.mbi.ucla.edu/PROCHECK ) [ 89 ] and ERRAT servers ( https://servicesn.mbi.ucla.edu/ERRAT/ ) [ 90 ] were utilized for assessing model quality. In addition, Z-scores and energy plots were assessed using ProSA-web ( https://prosa.services.came.sbg.ac.at/prosa.php ) [ 91 ]. Finally, the 3D structures were visualized using the Pymol 3.1 software [ 92 ]. Spatiotemporal expression profiling of the genes in rice using RNA-seq data The RGAP database [ 50 ] and Rice Expression Database ( https://ngdc.cncb.ac.cn/red/index/ ) [ 93 ] were employed for collecting RNA-seq expression data of twelve OsLOX transcripts in various tissues throughout several development phases. Plant Public RNA-seq Database ( https://plantrnadb.com/ricerna/ ) [ 94 ] was employed with RGAP Locus IDs of OsLOXs to collect transcriptomic data of rice during different time points for biotic and abiotic stress conditions. To quantify gene expression, sample transcript value was normalized using Log2 transformation method (FPKM + 1) [ 95 ] for each gene. GraphPad Prism 10.5.0 ( https://www.graphpad.com/ ) [ 96 ] software was used to visualize a heat map derived from the expression profile data. Plant germination, abiotic stress treatments, and total RNA isolation Seeds of the indica rice variety BRRI Dhan 105 were procured from the Bangladesh Rice Research Institute (BRRI), with only the healthy, mature, and high-quality seeds chosen for this experiment. Thoroughly cleaned seeds were positioned in a petri dish with wet tissue paper to initiate germination. After three to four days, germinated seedlings were transferred into a hydroponic cultivation system. In the growth chamber, environmental conditions were regulated at a temperature of 25 ± 2°C, with a photoperiod of 16 hours of light and 8 hours of darkness, and a light intensity of 1500–2000 lux. 21 days old seedlings were subjected to several abiotic stress treatments which included salt (150 mM NaCl), drought (3 mM PEG 6000), saline-alkalinity (60 mM NaHCO 3 ), heat (42°C), cold (4°C) and IAA (98mg/L dissolved in NaOH followed by distilled water) for 24h; and samples were taken at two time intervals: 12 hours and 24 hours, to ensure optimal responsiveness.The untreated seedlings were employed as a control. Fresh young leaves collected 12h and 24h post-treatment were thoroughly cleaned with 70% ethanol and distilled water for removing any pollutants prior to RNA extraction. Total RNA of treated and controlled frozen leaf tissue samples was extracted following the FavorPrep™ Tri-RNA Reagent user guide's procedure. The removal of genomic DNA contamination was conducted with DNase I from Thermo Fisher Scientific Corporation’s Invitrogen™ DNA-free™ DNA Removal Kit. The first-strand cDNA of the mRNA was produced with the ABscript II cDNA First Strand Synthesis Kit (ABclonal, Inc. USA), in accordance with the manufacturer's instructions. Expression profiling under abiotic stress conditions using quantitative real-time PCR data The primer design for the RT-qPCR was conducted using the NCBI Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ )[ 97 ] and the OligoAnalyzer Tool ( https://sg.idtdna.com/pages/tools/oligoanalyzer ) of Integrated DNA Technologies, Inc. The product length was maintained between 160 and 220 bp (Table S1 ). To examine the expression of each of the twelve OsLOX genes expression regarding treatments, the RT-qPCR was conducted employing a 96-well plate format on the BioRad CFX96™ Real-Time PCR Detection System. Eukaryotic elongation factor 1 alpha (eEF-1α)[ 98 ] was chosen for internal reference. The study utilized the GoTaq® qPCR Master Mix (2X) from Promega Corporation, USA. For each 15 µL reaction mixture, we incorporated 7.5 µL of GoTaq® qPCR Master Mix (2X), 2 µL of 10-fold diluted cDNA, 1 µL of gene-specific primers, and 3.5 µL of nuclease-free water. Each reaction was conducted under the following conditions: initial denaturation for 2 minutes at 95°C, followed by 40 cycles of denaturation for 15 seconds at 95°C, annealing for 30 seconds, and extension at 72°C for 40 seconds. The annealing temperature was 63.4°C for OsLOX1 , 61.6°C for OsLOX2 and OsLOX10 ; 63.5°C for OsLOX3, OsLOX8 and OsLOX5 ; 62.6°C for OsLOX4, OsLOX9 and OsLOX11 ; 61.1°C for OsLOX6 , 61.3°C for OsLOX7 and OsLOX12 ; 59.6°C for eEF-1α . All reactions were performed in triplicate for each experimental condition and melting curve analysis was conducted after PCR amplification. The double delta Ct value method was used to determine the relative gene expression levels [ 99 ]. To ascertain the mean values of various treatments, technical replication was implemented and further, data processing and statistical analysis was performed using Microsoft Office 365 and GraphPad Prism 10.5.0 [ 96 ]. In each treatment group, the experimental data were represented as the mean ± standard deviation (SD) of the measured values. GraphPad Prism v10.5.0 was employed to generate bar graphs. A significance test was conducted utilizing two-way ANOVA, succeeded by Dunnett’s test. The asterisks (*) above the bars denote statistically significant differences (p < 0.05, Dunnett’s test) among various time points. Results Sequence curation of Lipoxygenase genes in Oryza sativa genome and physicochemical properties analysis A cumulative of twelve LOX gene family members were identified in the genome of Oryza sativa . Six AtLOX ’s protein sequences were used as a reference to perform BLASTP searches against the Oryza sativa v7_JGI dataset on Phytozome [ 49 ]. A total of 24 matches were identified, and of the 24 transcripts analyzed, 17 were confirmed to include both the Lipoxygenase domain and the PLAT/LH2 domain through sequence analysis using a Hidden Markov Model (HMM) profile, as these are the defining characteristics of LOX genes. These 17 transcripts correspond to the twelve LOX genes that display alternative splice variants. A BLASTP search was conducted in the RGAP [ 50 ] and NCBI protein database [ 51 ] to ensure that no potential LOX protein sequence was overlooked. The representative transcripts for the genes in this investigation were selected from the Phytozome annotated primary transcripts. Based on chromosomal number and location, the genes were named sequentially ( OsLOX1 – OsLOX12 ), where ‘ Os ’ prefix was used for Oryza sativa , followed by LOX for Lipoxygenase [ 64 ]. Information regarding the conserved domains positions within the twelve OsLOX proteins is stated in Table S2 . To get a comprehensive idea regarding physicochemical properties of OsLOXs proteins, sequence characterization was done. The proteins length ranged from 347 to 942 aa, along with CDS length ranging from 1041 bp (OsLOX12) to 2826 bp (OsLOX9). The molecular weights of the deduced OsLOX proteins varied from 39,243.14 Da for OsLOX12 to 104,687.15 Da for OsLOX11. The proteins exhibited an average molecular weight of 92,146.14 Da. Analysis of the instability index revealed that four out of seventeen transcripts had an instability score below 40, indicating their stability. It was determined that 24% of the genes were stable, while 76% were unstable. Isoelectric point (pI) study revealed that eight OsLOX proteins had pI value 7.0, signifying their basic characteristics. Furthermore, the negative GRAVY value of all the proteins indicates that the OsLOX proteins were likely hydrophilic. Subcellular localization prediction was conducted to further the understanding of the biological processes and functions of OsLOX proteins within various organelles. This confirmed the predominant presence of the highest quantity of OsLOX proteins in the chloroplast, cytoplasm and mitochondria. Both the vacuole and the plasma membrane contain OsLOX proteins; however, only OsLOX12 was found in the vacuole while OsLOX6 and OsLOX12 were found in the plasma membrane (Figure S1 ). All the attributes of OsLOX gene family members along with their physicochemical properties are mentioned in Table 1 . MSA revealed that all OsLOX genes exhibit significant sequence similarity, possessing two highly conserved domains at their N- and C-termini: the PLAT/LH2 Domain and the Lipoxygenase Domain. Position of the characteristic conserved domains of OsLOX proteins is highlighted in Fig. 1 . Table 1 Characteristics attributes of the members of Lipoxygenase gene family in Oryza sativa L. SL No Gene Name Locus ID Transcript Strand Chr No CDS Coordinates (5′ to 3′) Length (bp) Protein (aa) MW(Da) pI Instability Index GRAVY Value Localization Gene cDNA CDS 1 OsLOX1 LOC_Os02g10120 LOC_Os02g10120.1 R 2 5282626 − 5276620 6007 3224 2781 927 103585.62 5.73 46.57 (unstable) -0.317 Cyt a , Mit a , Chl b 2 OsLOX2 LOC_Os03g08220 LOC_Os03g08220.1 F 3 4187107–4193539 6433 3456 2757 919 101959.30 6.95 45.90 (unstable) -0.318 Mit a , Cyt ab 3 OsLOX3 LOC_Os03g49260 LOC_Os03g49260.1* F 3 28049441–28053725 4285 3144 2604 868 97984.80 6.41 39.96 (stable) -0.359 Cyt a , Mit a , Chl ab LOC_Os03g49260.2 F 3 28049440–28053725 4285 3132 2592 864 97499.11 6.34 39.21 (stable) -0.379 Nuc ab , Mit ab , Chl b 4 OsLOX4 LOC_Os03g49380 LOC_Os03g49380.1* F 3 28106903–28113300 6398 3179 2634 878 98697.82 6.82 37.42 (stable) -0.323 Cyt ab LOC_Os03g49380.2 F 3 28107728–28113286 6398 2210 1482 494 55535.22 9.17 42.81 (unstable) -0.449 Cyt ab , Mit b , Chl ab 5 OsLOX5 LOC_Os03g52860 LOC_Os03g52860.1 F 3 30315455–30318972 3518 2941 2613 871 97183.72 6.16 32.87 (stable) -0.285 Cyt ab 6 OsLOX6 LOC_Os04g37430 LOC_Os04g37430.1 F 4 22305064–22309320 4257 3032 2394 798 89304.87 10.02 56.45 (unstable) -0.525 Nuc a , Mit ab , PM a , Chl b 7 OsLOX7 LOC_Os05g23880 LOC_Os05g23880.1 F 5 13734067–13740568 6502 3825 2544 848 95343.12 7.20 44.94 (unstable) -0.434 Cyt a , Chl b 8 OsLOX8 LOC_Os08g39840 LOC_Os08g39840.1 R 8 25224080 − 25216363 7718 3251 2775 925 102819.00 5.91 49.81 (unstable) -0.389 Chl ab 9 OsLOX9 LOC_Os08g39850 LOC_Os08g39850.1* F 8 25240906–25250046 9374 3322 2826 942 104494.47 6.54 47.55 (unstable) -0.349 Chl ab , Mit a LOC_Os08g39850.2 F 8 25244414–25250279 9374 2778 2472 824 92381.69 5.70 41.44 (unstable) -0.365 Cyt ab , Chl a , Nuc b LOC_Os08g39850.4 F 8 25244414–25250046 9374 2701 2472 824 92381.69 5.70 41.44 (unstable) -0.365 Cyt ab , Chl ab , Nuc b 10 OsLOX10 LOC_Os11g36719 LOC_Os11g36719.1 F 11 21675027–21684774 9748 3013 2607 869 98325.68 5.37 40.98 (unstable) -0.417 Cyt a , Nuc b , Chl b 11 OsLOX11 LOC_Os12g37260 LOC_Os12g37260.1 R 12 22860200 − 22854611 5590 3218 2769 923 104687.15 5.87 46.98 (unstable) -0.494 Cyt a , Mit a , Chl ab , Nuc ab 12 OsLOX12 LOC_Os12g37320 LOC_Os12g37320.1* F 12 22920107–22923778 3672 1390 1077 359 40772.79 8.35 48.15 (unstable) -0.720 Chl ab , Nuc ab , Mit ab , Vac b LOC_Os12g37320.2 F 12 22920107–22923778 3672 1520 1041 347 39243.14 9.11 52.31 (unstable) -0.694 Chl ab , Nuc ab , Mit a , PM a , Vac b *Primary transcripts CDS: Coding Sequence; MW: Molecular weight, pI: Isoelectric point; GRAVY: Grand average of hydropathy; bp: base pair; aa: amino acid; R: reverse strand; F: forward strand; Cyt: Cytoplasm; Mit: Mitochondria; PM: Plasma membrane; Chl: Chloroplast, Vac: Vacuole. a Subcellular localization prediction by CELLO v.2.5 ( http://cello.life.nctu.edu.tw/ ). b Subcellular localization prediction by WoLF PSORT ( https://wolfpsort.hgc.jp/ ). Phylogenetic relationship analysis In order to investigate the evolutionary relationships among the LOX genes from different species, a phylogenetic tree was generated by aligning the full-length amino acid sequences of the highly homologous LOX protein sequences from: Oryza sativa, Arabidopsis thaliana [ 36 ], Zea mays [ 64 ], Brassica rapa [ 65 ], Sorgum bicolor [ 66 ], Setaria italica [ 40 ] and Glycine max [ 37 ]. An unrooted tree was generated via the Maximum Likelihood approach in MEGA11 [ 58 ]. Poisson correlation and pairwise deletion were selected as the mode and gap, respectively, with 1000 bootstrap repetitions employed as the validation parameter. The phylogenetic tree (Fig. 2 ) illustrated that the twelve OsLOX proteins were categorized into two subfamilies: 9-LOXs and 13-LOXs, with the latter further subdivided into Type I and Type II. In detail, the subfamily 9-LOXs contains 4 OsLOX proteins (OsLOX3, OsLOX4, OsLOX5 and OsLOX10) along with 2 AtLOX, 3 BrrLOX, 7 SiLOX, 5 SbLOX, 6 ZmLOX, and 22 GmLOX proteins. Maximum of 7 OsLOX (OsLOX1, OsLOX2, OsLOX6, OsLOX8, OsLOX9, OsLOX11 and OsLOX12) proteins belonged to 13-LOX Type I; this subfamily also contained 4 AtLOX, 12 BrrLOX, 5 SiLOX, 3 SbLOX, 6 ZmLOX, and 12 GmLOX proteins. Only the OsLOX7 protein from the rice genome is present in 13-LOX Type II, which also comprises 2 GmLOX, 1 ZmLOX, 1 SbLOX, and 1 SiLOX proteins. Subfamily 13-LOX Type II solely included monocots, except for one GmLOX which further suggests that these genes were exclusive to monocotyledons[ 64 ]. Gene structure and conserved motif analysis Exon-intron structure is a significant evolutionary characteristic of genes that offers essential insights into functional diversity. The analysis of exon-intron structures (Fig. 3 a) indicated that the overall number of coding sequences (CDS) or exons varied from 3 to 10, while the number of introns ranged from 2 to 9. OsLOX10 possessed the lowest number of exons and introns, comprising 3 exons and 2 introns, whereas OsLOX3 exhibited the greatest amount, consisting of 10 exons and 9 introns. There was no intron-less gene present. The diversity in the lipoxygenase gene family of rice indicates varied functional roles and evolutionary divergence within this gene family. For identifying conserved motif distribution and diversity analysis of OsLOX genes, web-based tool MEME [ 70 ] were employed. 15 conserved motifs were found and designated as motifs 1 through 15 (Fig. 3 .b). Majority of the motifs were shared by the members due to the strong sequence similarity between OsLOX peptide sequences. Among the 15 motifs, motifs 4, 5, 8, 11 and 14 were shared by all the OsLOX proteins. Apart from OsLOX7, motif 15 was found in the remaining 11 LOX proteins. OsLOX12 is devoid of all other motifs, indicating its uniqueness within subfamily 13-LOX Type I. Except OsLOX6, remaining motifs were shared by the members of all subfamilies. Functional analysis of the motifs revealed that all the motifs belong to Lipoxygenase iron binding catalytic domain profile (Table S3 ). Chromosomal distribution analysis The investigation of chromosomal mapping analysis of OsLOX genes indicated that all OsLOX s are distributed randomly and unevenly throughout 7 of the 12 chromosomes in rice (Fig. 4 ). No LOX members were mapped onto chromosomes 1, 6, 7, 9, and 10. Chromosome 3 contained 4 LOX genes apiece, while chromosomes 8 and 12 each possessed 2 LOX genes. A single LOX gene was present on each of the following chromosomes: 2, 4, 5, 11. OsLOX1 and OsLOX2 are located on the p arm of their respective chromosomes, while the remaining genes are located on the q arm. OsLOX7 were positioned near the centromere. Table S4 lists the chromosomal locations, positions, and orientations of all the OsLOX s. Gene duplication prediction and collinearity analysis Gene duplication study aids in understanding the evolution of plants by emergence of novel gene functions and expansion of gene families. Findings revealed the evolutionary links of OsLOX genes and the existence of selective pressure, including neutral, positive, and negative/purifying selections (Table 2 ). Four pairs of duplicated genes were identified from McScanX result. The result revealed that two pairs of genes ( OsLOX3 / OsLOX5 & OsLOX2 / OsLOX6 ) were segmentally duplicated, while two pairs found in the 100 kb region of same chromosome ( OsLOX8 / OsLOX9 & OsLOX11 / OsLOX12 ) were tandem duplicated. To estimate the selection pressure: neutral selection was denoted by Ka/Ks = 1, purifying selection by Ka/Ks 1 [ 72 ]. Duplicated pairs Ka/Ks fraction were all constantly remaining below 1, indicating a notable presence of strong purifying selection. Findings also showed that duplication events presumably took place between 46.94596 and 4.72241 million years ago (MYA), demonstrating the more ancient duplication events have occurred in OsLOX3 / OsLOX5 ; in contrast, OsLOX8/OsLOX9 was the most recent duplicated pair, indicating recent divergence events of tandem duplicated pairs. Table 2 Calculations of divergence time and Ka/Ks ratios in the duplicated pairs of OsLOX s. Duplicated gene pair Ka Ks Ka/Ks Time (MYA) Purifying assortment Duplicate type OsLOX3 / OsLOX5 0.177696 1.408379 0.126171 46.94596 Yes Segmental OsLOX2 / OsLOX6 0.325064 1.242597 0.2616 41.41991 Yes Segmental OsLOX8 / OsLOX9 0.060522 0.141672 0.427195 4.72241 Yes Tandem OsLOX11 / OsLOX12 0.12902 0.185634 0.695022 6.18780 Yes Tandem Intraspecific collinearity analysis was conducted to elucidate the potential regulatory functions associated with the highly conserved gene order of the OsLOX gene family. Circos tool was used to display the linked gene pairs. This study identified 4 collinear gene pairs within rice genome. Chr3 and Chr4 contained two segmentally duplicated pairs while Chr8 and Chr12 contained two tandemly duplicated pairs (Fig. 5 a). Comparative interspecific collinearity analysis maps were constructed among rice LOX s and two other plant species ( Zea mays as a monocot and Arabidopsis thaliana as a dicot species) for studying the potential evolutionary relationship between identified members of these species. Seven orthologous pairs were identified (Fig. 5 b, Table 3 ). The Ka/Ks ratio ranged from 0.363 to 1.345, with a mean of 0.323. However, an exception was found between rice and Arabidopsis due to high sequence divergence value (pS > = 0.75) which led to Ks saturation. Highest quantity of orthologous gene pairs identified between Zea mays and rice demonstrated the substantial impact of duplication events in monocot species, leading to the expansion and functional diversity of the LOX gene family during evolution. Table 3 Duplication events involving rice LOX genes and other species. Duplicated Gene 1 Duplicated Gene 1 Ka Ks Ka/Ks Time (MYA) Purifying assortment Duplicate type OsLOX2 AT1G72520.1 (A.thaliana) 0.2799 pS > = 0.75 N/A N/A Possible Segmental OsLOX1 Zm00001eb206040_T001 (Zea mays) 0.1899 0.3632 0.523 12.106 Yes Segmental OsLOX2 Zm00001eb005920_T003 0.0694 0.4996 0.139 16.654 Yes Tandem OsLOX3 Zm00001eb216870_T004 0.2613 1.3459 0.1942 44.862 Yes Segmental OsLOX6 Zm00001eb423430_T001 0.1535 0.439 0.3497 14.634 Yes Segmental OsLOX6 Zm00001eb081610_T002 0.146 0.4135 0.3532 13.782 Yes Segmental OsLOX8 Zm00001eb035010_T001 0.184 0.414 0.4444 13.801 Yes Tandem Cis -regulatory elements (CREs) analysis of OsLOX s To investigate the potential regulatory mechanisms of OsLOX s in the presence of a variety of stimuli and to comprehend the pivotal role they play in transcriptional regulation and gene expression, the CREs of the promoter region of each OsLOX s (up to 2.0 kb upstream of the translation start site) were searched against PlantCARE database [ 100 ]. There were 46 cis-regulatory elements found within the 5′ UTR promoter region of OsLOX s and grouped into seven different functional categories, namely elements responsible for i) Light (14.5% of all identified CREs), ii) Abiotic challenges (4.6%), iii) Hormonal regulation (12%), iv) Cellular development (2%), v) Promoter associated (63.7%), vi) Biotic challenges (2.2%), and vii) Miscellaneous functions (1%). The aspects of hormonal regulation were categorized into a) Methyl jasmonate (MeJA), b) Salicylic acid, c) Auxin, d) Gibberellin, and e) Abscisic acid responsive elements. The entire list of identified CREs along with their respective functions was included in Table S5 . The prevalence of presumed CREs within the promoter region of OsLOX s were depicted in Fig. 6 . Various abiotic stress related elements are involved in anaerobic, anoxic, cold, drought and defense responses. The abiotic stress responsive cis -elements identified in the promoter region of OsLOX s include ARE, TC-rich repeats, MBS, LTR and GC-motifs. Furthermore, biotic stress responsive cis-elements including the W box, WUN-motif, and WRE3 were implicated in responses to fungal elicitors and wounding. Additionally, all OsLOX genes were characterized by the presence of core promoter elements TATA box and CAAT box. They predominantly operate within the promoter and enhancer regions. Hormonal responsive CREs include ABRE, TGA-element, ARE-motif, P-box, AuxRR-core, TATC-box, TGACG-motif, CGTCA-motif and TCA-element, which were essential for five distinct hormones. The seed specific regulation, zein metabolism, circadian control, meristem and endosperm expression were all dependent on cellular development responsive CREs including O2-site, CAT-box, GCN4-motif, RY-element, and Circadian. Light responsive elements include Box 4, Gap-box, G-box, AE-box, I-box, TCCC-motif, AT1-motif, GT1-motif, ATCT-motif, Sp1, LS7, GA-motif, TCT-motif, GATA-motif, ACE, LAMP-element, GTGGC-motif, and MRE. In addition to these, CCAAT-box, A-box, HD-Zip 3, and AT-rich element exemplify cis-elements with diverse roles. The notable abundance of CREs within the promoter region of OsLOX s indicates their involvement in the transcriptional regulation. Furthermore, a variety of hormones and stress-related CREs point to a direct relationship between the gene’s activity in rice under various stress conditions. To completely understand the intricate interactions between CREs of the OsLOX gene family and the related signaling pathways in controlling rice stress responses, further research is required. Specification putative miRNA targeting OsLOX s MicroRNAs (miRNAs) are essential in both plants and animals as post-transcriptional regulators of gene expression. miRNAs downregulation signifies an increased expression of the targeted mRNA. The psRNATarget database[ 76 ] was employed for identifying miRNAs targeting the LOX genes in rice. The study identified 151 potential candidate miRNAs with a length of 20–24 nucleotides that target OsLOX s. The lowest quantity of miRNAs (1) targeted OsLOX12 , while the maximum quantity of miRNAs (25) targeted OsLOX6 . The total count of miRNAs that targeted OsLOX s was as follows: 19 for OsLOX 1, 4 for OsLOX2 , 14 for OsLOX3 , 17 for OsLOX4 , 10 for OsLOX5 , 25 for OsLOX6 , 20 for OsLOX7 , 12 for OsLOX8 , 15 for OsLOX9 , 6 for OsLOX10 , 8 for OsLOX11 ¸and 1 for OsLOX12 . The regulatory interactions between putative miRNAs and their targeted LOX s are illustrated in Fig. 7 . Among all the miRNAs, 25 targeted several OsLOX s, while the rest miRNAs were exclusive to each gene (Table S6 ). The majority of miRNA-mediated OsLOX repression was accomplished by mRNA cleavage, with only a small percentage of targets experiencing inhibition during the translation phase. Consequently, this demonstrates that the discovered miRNAs might have the potential to post-transcriptionally influence the production of OsLOX s via cleaving mRNA and suppressing translation. Gene Ontology Analysis Gene ontology enrichment analysis was conducted to determine a variety of regulatory activities of OsLOX genes. It establishes a framework classifying genes into three distinct groups based on their biological processes, cellular components and molecular functions (Fig. 8 ). It has been estimated that there are 22 GO terms across all OsLOX s. Among the three groups, the biological process was the most frequent as it contains 16 GO IDs. However, there were three GO IDs in each of the classes of molecular functions and cellular components. Moreover, the functional analysis involving the corresponding GO terms for biological process regions identified that GO terms responsible for the following processes: the biosynthesis and metabolism of oxylipin; the oxidation and modification of lipids; the biosynthesis and metabolism of fatty acids; the biosynthesis and metabolism of carboxylic acids; the biosynthesis and metabolism of organic acids; and the metabolism of oxoacids. GO terms associated with molecular processes found out they are responsible for oxidoreductase and dioxygenase activity, which are characteristic features of LOX genes across diverse species. Cellular components associated with these GO terms are located within the cytoplasm, plastid, and chloroplast. GO analysis regarding the predicted OsLOX genes with their respective p-values are listed in Table S7 . Phosphorylation sites prediction The NetPhos 3.1 service predicted the phosphorylation sites for serine (Ser), threonine (Thr), and tyrosine (Tyr) of eukaryotic proteins through neural network integration. The OsLOX protein family possesses multiple unique phosphorylation sites throughout its amino acid sequences, amounting to a total of 1473 phosphorylation sites. In particular, 778 of the total phosphorylation sites were made up of serine residues, with threonine and tyrosine accounting for 552 and 143 residues, respectively. The most serine phosphorylation site was OsLOX8 with 114, while the least number was OsLOX12 with 35. The most likely phosphorylation site had a value of 0.998 (much larger than the threshold value of 0.500) [ 101 ],which indicates predominant kinase specificity; the most threonine phosphorylation sites was OsLOX6 with 73, and the least abundant was OsLOX12 with 24, of which the most likely phosphorylation site had a value of 0.994; the most abundant tyrosine phosphorylation site was OsLOX7 with 20, while the least abundant was OsLOX9 with only 4, of which the most likely phosphorylation site had a value of 0.988. It is suggested that the OsLOX s functional variety is determined by proteins with distinct architectures and kinase-specific phosphorylation sites (Fig. 9 ). Protein-protein interaction (PPI) network prediction Analysis of PPI network demonstrated the possible regulatory roles of OsLOX proteins based on the strong homology of orthologous Arabidopsis STRING proteins. Apart from OsLOX11, the family was found to be homologous with 10 Arabidopsis proteins (CYP74A1, CYP74A2, CYP74A3, CYP74A4, SLM1, AOC, Q2RAM0_ORYSJ, Q2R2W3_ORYSJ, PLA2-II, and PLA2-III). Additionally, KEGG enrichment analysis indicated that proteins that interact with OsLOX s may have the potential to participate in hormone-mediated pathways and external stress responses (Fig. 10 , Table S8 ). Secondary and tertiary structure analysis of OsLOX proteins Secondary structural analysis of OsLOXs revealed the percentages and locations of different constituents; as well as the location of transmembrane helix (Table S9 ). With exception of OsLOX1, the predominant secondary structure was the alpha-helix, closely succeeded by the turn, coil, beta-sheet, and 310-helix. OsLOX1 has the highest turn in its structure which indicates a compact and flexible protein structure, it might be involved in binding or signaling processes. Moreover, the stability of OsLOXs is suggested by the higher proportion of helical structures [ 102 ], and structures such as random coils are essential for signaling cascades [ 103 ]. Only OsLOX6 had single membrane spanning motif (MSM) at 664 to 673bp position (Fig. 11 ). The trRosetta webserver [ 86 ] is a deep learning algorithm that emplys a neural network for predicting inter-residue geometry (orientations and distances) and builds tertiary models using RosettaTTAfold. GalaxyRefine [ 87 ] further refined the generated models, and then Swiss PdbViewer [ 88 ] was utilized for energy minimization. Figure 12 depicts the predicted 3D structures of OsLOX proteins, generated using Pymol 3.1 Software [ 92 ]. Multiple validation tools were implemented to assess the structural reliability of predicted protein models (S10 table). Ramachandran plot analysis through PROCHECK [ 89 ] revealed that more than 90% of the residues were situated within the most favorable and additionally allowed regions, while fewer than 1.5% were positioned in disallowed regions, indicating a high-quality stereochemical conformation (Figure S2 ). ERRAT analysis [ 90 ] further supported this with models exhibiting an overall quality factor exceeding 87, suggesting minimal structural errors (Figure S3 ). ProSA-web analysis [ 91 ] yielded Z-scores that are within the typically observed range for experimentally determined native proteins and serve as evidence for the reliability of the predicted structures (Figure S4 ). The corresponding energy plots confirmed that all residues were associated with favorable (low) energy values, indicative of structurally stable regions (Figure S5 ). The validation results collectively affirm the high quality and dependability of the produced tertiary protein models. Spatiotemporal expression profiling of the genes in rice using RNA-seq data Gene expression analysis measures the functional activity and expression level of active gene products (functional RNA or protein) in different cells and tissues. This is a highly specific molecular indicator of biological activity and metabolite levels. Changes associated with gene expression patterns are linked to corresponding alteration in development, disease progression, and adaptation to external stresses. In this study, RNA sequencing data of 12 OsLOX s in different tissues were retrieved from RGAP database [ 50 ] and Rice Expression Database [ 93 ] to investigate spatiotemporal effects during developmental stages for corresponding genes. The generated heatmap visualized the RNA transcript profiles with normalized expression values of RNA-seq FPKM (Fig. 13 ). The analysis identified low to moderate expression levels in most tissues, with several members exhibiting strong tissue-specific expression. For instance, OsLOX1 displayed high level of expression in leaf (7 DBH to 7 days DAF) and shoot (7-day seedling and 14 DAG), this indicates its potential role in leaf development. OsLOX8 and OsLOX9 were upregulated in anthers during flowering stage indicating its role in pollen maturation. In the same manner, OsLOX4 and OsLOX6 were moderately upregulated in roots which implies potential participatory role in root growth or defense signaling. OsLOX2 was upregulated in anther and glumes, while OsLOX5 exhibited a mild upregulation in root (14 DS and 21 DAS). OsLOX4 showed notable upregulation during early germination (8 h after imbibition), whereas other members were downregulated in germinating seed and mature seed tissues. OsLOX6 showed overall moderate upregulatory expression for most tissues. OsLOX3, 10, 11 , and 12 expression level were downregulated for all tissues. To get a better understanding of how OsLOX s respond to various stresses, the expression patterns of OsLOX s were examined in response to various biotic and abiotic stressors. The Plant Public RNA-seq Database [ 94 ] was employed to acquire RNA-seq data for the investigation. GraphPad Prism 10.5.0 [ 96 ] was used to generate the corresponding heatmaps. Log2 values of relative expression are represented by the color gradient on the right side of the heatmap (Fig. 14 ). In response to infection by bacterial leaf streak pathogen ( Xanthomonas oryzae pv. Oryzae) , OsLOX1 , 2, 4, 5, 6, 7, 8 , and 9 were notably upregulated, while OsLOX3. 10, 11 and 12 displayed downregulation. In the event of infection by the rice blast fungus (Magnaporthe oryzae), OsLOX1 , 2, 4, 6, 7, 8, 9 , and 11 exhibited consistent upregulation from 8 h to 72 h post-inoculation, suggesting their active participation in defense signaling against fungal invasion. Remaining genes were downregulated. Additionally, infection by rice stripe virus (RSV) led the upregulation of OsLOX1 , 2, 4, 6, 7 , and 11 at 3 to 15 days post-inoculation (DPI). OsLOX8 was upregulated in 3 DPI, but prolonged infection led to supression of the gene. Infection by necrotrophic fungus Rhizoctonia solani (Sheath blight) resulted in upregulation of OsLOX2, 6, 7 , and 11 at 1 to 3 DPI, OsLOX8 and 9 showed delayed upregulation (3 DPI); in contrast OsLOX3, 5, 10 and 12 were markedly downregulated. Rice black streaked dwarf virus (RBSDV) infection triggered a high upregulation of OsLOX1 , 2, 4, 7, and 11 , indicating a possible role in antiviral defense. Pyricularia oryzae is another fungal pathogen responsible for rice blast disease, exhibited upregulation of all genes of OsLOX family apart from OsLOX3, 5, 10 , and 12 . Similarly, fungal blast causing Magnaporthe grisea increased the expression of OsLOX2, 3, 4, 5, 6 , and 7 . Moreover, Meloidogyne graminicola (root knot nematode) infection induced the expression of OsLOX1, 2, 4, 7, 8, 9 , and 11 at both 3 and 7 days after infection (DAI). Striga infection also led to upregulation of OsLOX4, 5 , and 6 at infection stage of 3 and 7 days, while OsLOX1, 10, 11 , and 12 remained significantly downregulated. Xylaria striata is an endophyte that exhibits improved disease resistance by upregulating OsLOX4, 5 and 9. Plant infected with rice root nematode (RRN) Hirschmanniella showed upregulation of OsLOX3, 6 , and 9 . In conclusion, these results address potential role of OsLOX s in rice defense mechanisms against pathogens and biotic stress conditions. Treatment with abiotic stress caused a noticeable change in OsLOX s gene expression patterns over a range of time periods. In response to submergence stress, OsLOX2, 4 , 8, 9 and 11 were upregulated consistently at all time points, suggesting a potential role in submergence adaptation (Fig. 15 ). The remaining genes were consistently downregulated. During drought stress, OsLOX4 and 11 were consistently upregulated under 1 to 24 hours of exposure. OsLOX1 and 2 showed upregulation during first 3 hours of treatment, OsLOX8 , 9, and 12 showed upregulation during 3 to 12 hours of treatment. OsLOX3, 5, 6, 7 , and 10 were predominantly downregulated, indicating their suppression under dehydration conditions. Similar distinct patterns were observed under saline conditions. OsLOX1 and 2 showed highly upregulated expression across 1 to 24 hours of ionic imbalance. Short-term salt exposure (1–5 h) induced moderate upregulation of OsLOX4 , 7 , and 11 , OsLOX5 were upregulated during the 2nd hour of treatment, but continued exposure to salt led to downregulation. OsLOX9 were moderately expressed during 5 to 10h of salinity, whereas OsLOX3, 6, 10 and 12 were consistently downregulated. In the case of saline alkalinity, OsLOX2, 4, 6 and OsLOX9 remained highly upregulated, suggesting sensitivity to alkaline ion toxicity. Cold treatment at 4°C resulted in a strong and constant upregulation of OsLOX4, 8, 9, and 11 from 1 to 168 hours, indicating a possible role in cold acclimation. OsLOX1 and 7 started with high induction (upregulatory expression) from 0 to 24 hours, but further cold treatment led to downregulation of the transcripts. With the remaining genes showing downregulation across all time points suggest cold mediated transcriptional repression. Heat stress caused constant upregulation of OsLOX1 and 2 from 30 to 240 mins, whereas OsLOX 4 and 7 were moderately upregulated, indicating heat-responsive activation. Rest of the genes were downregulated during thermal treatment. These differential expression patterns indicate OsLOX family members participate actively in regulating rice adaptation to environmental stressors. In addition to numerous abiotic treatments, several hormones including auxin, gibbereline, absicic acid, jasmonic acid, salicylic acid, kinetins, and others aid plants during growth and development. During the biosynthetic process, LOX genes produce JA. Therefore, understanding transcript expression patterns under hormone treatment will aid in our comprehension of the optimal conditions for plant growth. In response to JA treatment, OsLOX2 , OsLOX8 , and OsLOX11 were consistently upregulated at 6, 12, and 24 hours in shoots, highlighting their involvement in JA-mediated signaling. OsLOX4 and 9 showed moderate expression patterns whereas remaining genes were mainly downregulated across JA time points. During ABA treatment, OsLOX4, 8 and 9 were upregulated, with the rest being in downregulation implies that ABA does not activate these isoforms. In response to auxin treatment, OsLOX3, 4, 5, 6 and 8 demonstrated high upregulation in root tissue for weekly treatment. Overall, the OsLOX gene family displayed distinct and stress-specific transcriptional responses which implies functional divergence for adverse environmental conditions. Expression profiling under abiotic stress conditions using quantitative real-time PCR data The relative expression patterns of OsLOX s were assessed in rice seedlings leaves throughout two different time periods to explore the effects of drought, salinity, saline-alkalinity, heat, cold and IAA (indole-3-acetic acid) stress conditions. Figure 16 illustrates a bar chart representing their real-time expression data under various stress situations after 12 and 24 hours. As shown in Fig. 16 , OsLOX1 transcript was significantly upregulated under drought, heat and salt stress and downregulated expression was observed for saline-alkalinity and IAA treatment at 12h. Response to drought and salt stress showed a contrasting downregulatory expression at 24, whereas response persists for saline-alkalinity and IAA treatment. Heat stress response alleviated after prolonged exposure. Cold treatment resulted in no change in transcript expression pattern compared to control. OsLOX2 , drought and cold response were significantly upregulated at both time points, with cold response demonstrating seven-fold upregulation at 24h. Salinity induced significant upregulation after 24h, while saline-alkalinity showed decrease to increase expression pattern. Heat and IAA treatment resulted in higher upregulation at 12h but OsLOX2 transcript displayed no significant result at 24h. OsLOX3 exhibited substantial upregulation of transcript during drought and cold stress. Drought stress caused approximately four-fold significant higher expression at both times, but cold stress issued approximately seven-fold increase of relative expression after 24h. Salt, saline-alkalinity, and heat resulted in significant downregulation under these stressful situations and the downregulation sustained. IAA treatment induced downregulation at 12h but further exposure led to no relative change of gene activity. OsLOX4, 5, 6 , and 8 showed similar expression patterns like OsLOX3 . For OsLOX5 , drought stress instigated significant upregulation of gene activity from two-fold to approximately four-fold; and cold stress resulting in less than one-fold to more than six-fold transcriptional upregulatory activity. The remaining stress significantly downregulated the transcript, and the downregulation was continuous. OsLOX5 transcript under drought conditions affected a significant upregulation from more than two-fold to approx. five-fold. Although, there was upregulation of cold-treated transcript at 12h, it was not significant until 24 where it showed approx. three-fold upregulation of relative expression. Like previously stated transcript, salt, saline-alkalinity, and heat treatment showed significant downregulation at both time points. IAA treatment resulted in significant downregulation at only 12h. OsLOX6 exhibited significantly higher level of expression change for drought conditions, the relative expression value fluctuated from more than three-fold to slightly higher than one-fold. Cold stress elevated the expression at 24h. Remaining stress resulted in significant downregulation, and it persisted till 24h. OsLOX8 expression was significantly enhanced under drought condition for both sampling time; under cold condition expression increased at 12h but it became significant only at 24h. Other stress followed the significant downregulatory pattern. A change for salt-induced stress was discovered in OsLOX7 , it showed significantly higher levels of expression at 12h; but contrasting effect was observed during 24h with significant downregulation of the transcript. Drought caused significant upregulation of gene activity at 12h (with approx. three-fold change), which slightly decreased at 24h but remained significant. Cold condition caused significant upregulation at 12, IAA depicted significant upregulation at 24h with delayed response. Significant downregulation was observed for heat treatment at 12h and for saline-alkaline treatment at 24h. Regarding the transcript profile of OsLOX9 , it showed significant upregulation under drought and cold conditions. Heat treatment demonstrated significant downregulation at both time periods; significant downregulation was noticed in salt and saline-alkaline treated samples of 24h. Exception of IAA treatment with notable upregulation was observed at 24h. Moreover, a decrease to increase expression pattern was observed in OsLOX10 with a significantly higher expression level of over fourfold under cold stress. Rest of the stress including drought situation was significantly downregulated for this transcript. OsLOX11 had considerably significant upregulation levels of approx. eight-fold during heat (only 24h) and cold (both period) stress conditions. Salt and saline-alkalinity showed significant increase to decrease pattern of expression. Drought and IAA induced significant upregulation at only 12h. No significant changes were observed in OsLOX12 transcript for drought, cold and IAA stress situations. Salt, saline-alkalinity resulted in significant downregulation after prolonged period. Heat stress exhibited consistently significant downregulation. However, under salt and cold conditions the RNA-seq data presented in Fig. 15 did not exhibit consistent findings with RT-qPCR data for all gene transcripts. Drought, saline-alkalinity and heat stress induced expression of RT-qPCR relative expression was more or less consistent with RNA-seq data. Thus, these findings show the greatest time dependent differential expression pattern of OsLOX s, suggesting its significant role in the adaptation to abiotic stress in rice. Discussion Lipoxygenase is a significant dioxygenase encoding protein family that catalyzes the regio and stereoselective dioxygenation of polyunsaturated fatty acids (PUFAs) and serves as precursors for oxylipins. It actively participates in various growth and development phases and enhances resistance to harsh external environmental conditions across diverse plant genotypes [ 104 ]. This present study identified 12 distinct OsLOX genes in rice whereas fourteen genes had been reported previously [ 105 ]. Identified genes possess both characteristic domains, the PLAT/LH2 and Lipoxygenase domains at their respective N and C termini which is consistent with previously identified true LOX family members [ 37 , 106 ]. Compared to C3 and C4 plants, Oryza sativa has more numbers of gene than in Arabidopsis (six) (Umate, 2011), Sesame (7) [ 107 ], Radish (11) [ 39 ], and fewer than Maize (13) [ 108 ], Wheat (44) [ 57 ], and Cotton (64) [ 109 ]. These differences in the quantity of LOX s are not proportionate with genome size, suggesting that the LOX gene has not been conserved throughout the evolutionary process. Physicochemical analysis revealed these genes differ in sequence length, molecular weight, isoelectric point, and GRAVY score, highlighting the heterogeneity of the gene family. The negative GRAVY score of the proteins verifies their hydrophilic nature [ 110 ]. The subcellular localization of the proteins indicated their presence in the chloroplast, cytoplasm, mitochondria, nucleus, and plasma membrane. The localization discrepancies of the proteins predicted by the two independent tools. According to the findings of CELLO v.2.5 [ 62 ], most proteins are found in the cytoplasm, whereas WoLF PSORT[ 61 ] indicated their localization in chloroplast, mitochondria and vacuolar membrane. Additional verification of protein subcellular localization necessitates validation by a GFP-fused transient expression system employing an Agro-infiltration method [ 111 ]. Hence, it could be said that different LOX gene members serve catalytic functions in different organelles. In a multispecies phylogenetic tree constructed with both monocot and dicot species, genes in a subgroup often have similar functions. OsLOX proteins were categorized into two distinct subfamilies: 9-LOXs and 13-LOXs, with the latter further subdivided into Type I and Type II, consistent with Wheat [ 57 ] and Artemisia annua [ 112 ]. However, LOX genes in sorghum [ 66 ] and pepper [ 23 ] were categorized into 9-LOX, 13-LOX and an unknown subfamily [ 113 ]. Seven OsLOX genes were present in Type I 13-LOXs, but one OsLOX gene was present in Type II 13-LOXs with no AtLOX sequence belonging to this group; it indicates certain divergences between LOX gene families of monocots and dicots. The structural divergence of introns and exons in plant species is crucial for evolution [ 114 ]. All OsLOX gene family members were identified to have exons ranging from 3–10, and the intron number ranged from 2–9. Identification is consistent with the pattern of Arabidopsis thaliana [ 36 ] and Brassica rapa [ 28 ]. There was no intron-less gene which indicates that they are highly expressed and have not undergone recent evolution. Analysis of conserved motifs revealed that the substantial sequence similarity among OsLOX proteins resulted in shared motifs across all three phylogenetic catagories. Except for OsLOX6 and OsLOX12, all members of Type I 13-LOX exhibited 15 motifs; the absence of a total of 5 and 9 motifs in these two genes, respectively, indicates their uniqueness within Type I 13-LOXs. The analysis of chromosomal distribution found that all OsLOX genes are dispersed randomly and unevenly throughout 7 of the 12 chromosomes. No OsLOX member was mapped onto chromosomes 1, 6, 7, 9, and 10. Previous study identified five tandemly duplicated pairs in tomato [ 115 ]. Three tandem and three segmental duplicated gene pairs were observed in LOX gene family of poplar [ 106 ]. Our research revealed that of the four duplicated gene pairs, two were tandem duplicates and two were segmental duplicates. It is possible to assert that the expansion of the LOX gene family in the rice genome is being driven by both segmental and tandem duplicated gene clusters. Seven orthologous gene pairings were found among rice, Arabidopsis thaliana and Zea mays . The observed cases of gene duplication across various chromosomes indicate segmental duplication, which serves as the principal driver for diversification. The evolution of the duplicated LOX pairs was influenced by purifying selection, as evidenced by Ka/Ks values < 1 [ 72 ]. A total of 46 categories of cis-regulatory elements were identified and categorized into seven functional groups through further investigation of the promoter region of the OsLOX gene family. These elements include those for cellular development (ABRE, AuxRR-core, ARE-motif, P-box, TATC-box, etc.), light response (CAT-box, Box 4, Sp1, Gap-box, MRE etc.), phytohormones (ABRE, TGA-element, AuxRR-core, P-box, TATC-box, etc.), biotic stress (W box, WUN-motif and WRE3), abiotic stress (ARE, TC-rich repeats, GC-motif, MBS and LTR, etc.) and Promoter associated (TATA box and CAAT box). The most prevalent CREs were associated with promoters, where multiple transcription factors bind and it indicates the complex regulatory mechanisms OsLOX s. Our results are consistent with previous research that has emphasized the multifaceted role of LOX genes in the stress response [ 25 , 38 , 66 , 109 ]. miRNAs are critical regulators of gene expression that are essential for the regulation of plant responses to abiotic and biotic stresses in cross species [ 116 ]. A total of 151 unique candidate miRNAs targeting members of the rice OsLOX gene family were identified. 25 miRNAs targeted several OsLOX s, while the other miRNAs were restricted to individual genes. The majority of miRNA-mediated OsLOX silencing resulted in mRNA cleavage, whereas a minor proportion of targets experienced translational inhibition and subsequent transcript degradation, thereby suggesting post-transcriptional regulation and translation inhibition. According to results of gene ontology prediction, the OsLOX proteins of rice function in response to oxylipin, lipid, carboxylic acid, fatty acid and diverse metabolic processes, in addition to participating in organic acid and biosynthetic processes alongside several partners. Furthermore, these findings indicate that oxidoreductase and dioxygenase activities are significant functions of OsLOX genes, as revealed by GO analysis regarding molecular function, largely occurring within plant cells, specifically in the plastid, chloroplast, and cytoplasm. Protein-protein interactions are essential for numerous intracellular and extracellular processes [ 117 ]. Multiple Arabidopsis proteins interact with the OsLOX proteins. Among these, CYP74A1 to CYP74A4 functions as Allene oxide synthase 4 which is a growth regulator and signaling molecules in plant defense. PLA2-II, PLA2-III, Q2R2W3_ORYSJ acts as phospholipase or its homologs. AOC interacts with auxin-mediated signaling pathway & implicated in NaCl stress response. OsLOX genes are abundantly phosphorylated at serine, threonine and tyrosine residues. Identifying the putative phosphorylation sites of proteins helps in understanding of the signal transduction, which is essential for plant development and adaptability to environmental stress [ 118 ]. Analysis of secondary structures showed that except OsLOX1, alpha helix is the most prevalent secondary structure which surpasses others in dominance. It ensures the stable conformation of proteins, like our findings [ 102 ]. Only OsLOX6 was identified to possess a singular membrane-spanning motif (MSM). The tertiary structures of the OsLOX proteins were predicted using several techniques, which validated the high quality of all structure predictions and might be used in further research. The information obtained from the RNA-seq database across various rice tissues was utilized to elucidate the functional roles of OsLOX s during growth and developmental phases. OsLOX s were discovered as differently expressed using tissue-specific expression pattern analysis. The analysis showed that among the tissues studied, OsLOX1 exhibited higher expression in leaf (7 DBH to 7 DAH), shoot (7-day seedling) and shoot (14-day seedling). Anther and Anther (flowering) stage showed upregulation of OsLOX2, 8 and 9 which might indicate the specific role on reproduction. OsLOX5 demonstrated mild regulatory pattern in root (14 DS and 21 DAS) whereas, OsLOX4 were significantly upregulated during early germination stages (8 h after imbibition). Expression levels were moderately upregulated for OsLOX6 for most tissues. All other genes ( OsLOX3, 10, 11 , and 12 ) exhibited lowest expression for due tissues. These findings are consistent with predominant expression of BnaLOX2 in stamens, indicating reproductive function [ 119 ]. Proteome profiling of LOX gene showed expression in mature seeds, seedling stage, early germination in Arabidopsis, tomato, soybean and cucumber [ 120 – 124 ]. Additionally, RNA-seq data was processed to examine the role of OsLOXs under various biotic stress conditions. Our analysis demonstrated how OsLOX s response to diverse biotic factors including viral, bacterial, and fungal infections, as well as nematode inoculation. When induced with Xanthomonas oryzae pv. oryzae infection, strong upregulation of OsLOX1 , 2, 4, 5, 6, 7, 8 , and 9 were shown and this activates JA-related or oxylipin pathways typical of antibacterial responses. Fungal infections caused by Magnaporthe oryzae , which is rice blast responsible organims, also triggered significant upregulation of several OsLOXs (OsLOX1 , 2, 4, 6, 7, 8, 9 , and 11) . Necrotrophic fungal infection caused by Rhizoctonia solani induces sheath blight in rice and elevated the expression of OsLOX2, 6, 7 , and 11 . Fungus Pyricularia oryzae is also responsible for rice blast disease, and it showed higher activity of all genes of OsLOX family apart from OsLOX3, 5, 10 , and 12 . Viral infections (RSV and RBSDV) led to upregulation of OsLOX1 , 2, 4, 6, 7 , and 11 , highlighting a possible conserved antiviral role. Root-associated pathogens further emphasized context specific responses: nematode Meloidogyne graminicola upregulated the expression of OsLOX1, 2, 4, 7, 8, 9 , and 11 , while the beneficial endophyte Xylaria striata enhanced expression of OsLOX4, 5 and 9 . Taken together, these patterns address dynamic and distinct role of OsLOX s depending on pathogen type, supporting their central role in coordinating defense mechanisms under biotic stressors. This present finding is consistent with previous studies where Arabidopsis thaliana AtLOX proteins showed defence mechanism against bacteria and pathogens via oxylipins through lateral root development [ 18 ]. AtLOX2 and AtLOX3 mutants were shown to control early-stage plant nematode infections [ 125 ]. ZmLOX3 of maize acts as a root-specific suppressor of primary defence signaling pathways and provides resistance against nematodes [ 126 ]. In papaya, the hydroperoxides generated by 13-LOX exhibited significant antifungal activity against blight disease [ 127 ]. Furthermore, the expression profile of OsLOX s was examined in the context of drought, salinity, saline-alkalinity, heat, cold and IAA (indole-3-acetic acid) in two different time points and the data of RT-qPCR analysis was compared with RNA-seq to ascertain the role under different abiotic stress conditions. The expression profiles of the majority of genes were broadly similar between the RNA-Seq and RT-qPCR results, despite the minor deviations observed during salt and cold stress. In RT-qPCR analysis, certain reference or housekeeping genes may occasionally exhibit significant responses to changes in experimental conditions or tissue types [ 128 , 129 ]. In our study, we used eEF-1α to validate the qPCR result for gene expression [ 98 ]. Because of drought and cold stress, OsLOX genes showed tolerance at different time points with upregulated transcripts. OsLOX genes were identified as pivotal in modulating responses to drought and cold conditions. Under drought stress, OsLOX1-9 and OsLOX11 genes showed significant upregulation, which indicates their possible involvement in drought tolerance mechanisms. In contrast, OsLOX10 and 12 were downregulated or showed no significant change under drought conditions, indicating gene-specific divergence within the LOX family. Under cold stress, several transcripts exhibited significant and sustained upregulation. OsLOX2-11 had significantly higher expression levels at almost both sampling periods, which aid plants in surviving cold environment. In contrast, OsLOX1 and 12 did not exhibit significant changes under cold treatment. Expression was predominantly suppressive in nature under saline condition. OsLOX3-6, 8–10 and 12 showed significant downregulation, whereas OsLOX1, 2, 7 and 11 displayed time varying upregulation pattern. Under saline-alkalinity, with exception of OsLOX2 and 11 , all genes exhibited significant downregulation. Except for OsLOX1, 2 and 11 , heat stress reported downregulation for OsLOX gene family, which suggest suppression of non-essential processes and dominating stress responsive pathways. For IAA treatment, OsLOX1, 3–8 , and 10–11 significantly downregulated. OsLOX2 and 9 showed upregulation and OsLOX12 exhibited no significant changes across treatments. Similar studies have shown upregulatory expression pattern due to drought stress in Amorpha fruticosa L. (AfLOX4) and downregulation due to saline alkaline conditions, which is persistent with our study [ 130 ]. Several genes of maize exhibited transient expression patterns during cold stress, presumably signifying a delayed involvement in cold-responsive networks. ZmLOX2 was approximately 70-fold upregulated in response to drought stress [ 108 ]. Expression pattern in foxtail millet contrasted with rice for salt and drought condition [ 40 ]. Based on expression intensity, upregulation was observed for salt stress and under drought stress, downregulation was observed for SiLOX2, SiLOX6, SiLOX8 and SiLOX9 . In cotton [ 109 ], most LOX genes are associated with heat and salt stress; however, GhLOX18 was exclusively induced under cold stress, whereas some GhLOX genes exhibited altered expression in response to heat. Additionally, CmLOX10 of oriental melon significantly increases drought tolerance by mediating a JA biosynthesis pathway while only CmLOX13 was suppressed by high temperature [ 27 , 41 ]. CaLOX1 of pepper showed strong tolerance under salinity and drought [ 23 ]. Under combined drought-salt stress, upregulation of sesame SiLOX5 was evident [ 107 ]. Overall, drought and cold emerged as the most potent inducers of OsLOX s gene expression, while salt, saline-alkalinity, heat and IAA decreased expression. These findings suggest that OsLOX s are differentially regulated by abiotic stresses, with certain genes playing key roles in drought and cold stress adaptation, whereas others show stress-specific and time-dependent regulation. Conclusion This study presents a thorough characterization of Lipoxygenase gene family in rice and their expression profiles under a variety of abiotic stress conditions. A total of twelve OsLOX members in rice were identified and categorized into three subfamilies according to structural similarities. These genes are located on seven different chromosomes and distinct conserved motifs are present. Through synteny analysis it was observed that genes underwent significant purifying selection and orthologous gene pairs evolved through segmental duplication. Notably, cis-regulatory elements analysis linked these elements to light, biotic and abiotic stress tolerance, hormonal regulation, cellular developments and core promoter associated elements. miRNA analysis helps understand the function of miRNA-modulated OsLOX activity. Gene ontology analysis showed the functional activity of OsLOX s in different physiological processes, whereas PPI analysis identified homologous proteins with Arabidopsis signaling mileu. Expression profiling through RNA-seq data demonstrated OsLOX s tissue-specific expression and revealed the function of OsLOX s in response to various biotic and abiotic stresses. Further, the OsLOX s expression profiling under abiotic stress treatments was evaluated by RT-qPCR data and it revealed time-dependent differential regulation of OsLOX genes, with drought and cold stress consistently inducing upregulation. In contrast, salt, saline-alkalinity, heat, and IAA treatments predominantly triggered sustained downregulation, with only a few transcripts showing transient or delayed induction. These findings expand the comprehension of the LOX gene family, establish a basis for future functional investigations of the OsLOX gene family and thereby offer molecular insights for enhancing rice resilience through genetic improvement and stress-tolerant variety development. Abbreviations LOX s Lipoxygenase PUFAs poly unsaturated fatty acids MeJA methyl jasmonate CREs cis-regulatory elements RNA-Seq data RNA (cDNA) Sequencing data FPKM Fragment Per Kilobase of exon per Million mapped read PEG polyethylene glycol RT-qPCR quantitative real time polymerase chain reaction. Declarations Ethics approval and consent to participate Plant materials ( Oryza sativa L.) used in this article were obtained from the Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh. All plant materials were provided free of charge and maintained in accordance with the international guidelines. This article does not contain any studies on human participants or animals and does not involve any endangered or protected species. Consent for publication All authors agreed to publish. Availability of data and materials Data will be made available upon request. Competing interests The authors declare that they have no competing interests. Funding The authors declare that no funds, grants, or additional support were received during the preparation of this manuscript. Authors' contributions ADT and MNH contributed toward conceptualization and experimental design of this research. ADT performed data curation, software handling, data analysis and interpretation, laboratory study, writing and preparing the original draft. MNH and JFR handled software, investigation, and manuscript revision. MHH and AC conducted investigation and validation. SHP supervised the project, provided scientific direction, edited and revised the final manuscript. All authors reviewed and approved the final manuscript prior to its submission. Acknowledgment The authors express their sincere gratitude for the support from the Plant Genetic Engineering (PGE) Laboratory, Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh, which was instrumental conducting this research. 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Kim BR, Nam HY, Kim SU, Kim S, Il, Chang YJ. Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett. 2003;25. https://doi.org/10.1023/A:1026298032009 . Zhang Y, Wang K, Wang Z, Li XF, Li M, Zhu F, et al. The lipoxygenase gene AfLOX4 of Amorpha fruticosa L. is a potential regulator of drought stress tolerance pathways under saline and alkaline conditions. Acta Physiol Plant. 2023;45. https://doi.org/10.1007/s11738-023-03542-7 . Additional Declarations No competing interests reported. Supplementary Files FigureS1.docx Figure S1. The heatmap illustrates the predicted sub-cellular localization of OsLOX proteins. The names of the corresponding cellular organelles are displayed at the bottom of the heatmap, whereas the names of each OsLOX protein are displayed on the left side. The presence of protein signals corresponding to the genes is shown by the color intensity on the right side of the heatmap. FigureS2.docx Figure S2. Ramachandran plot of the 3D structures of OsLOXs. PROCHECK was utilized to create the Ramachandran plot. The hues red, yellow, light yellow, and white represent residues in the most favored, additional allowed, generously allowed, and disallowed regions respectively. FigureS3.docx Figure S3. ERRAT plot of the 3D structures of OsLOXs. The plot was generated via the ERRAT server. Proteins that are capable of being excluded with a 95% confidence level are indicated by yellow bars, while those with a 99% confidence level are indicated by red bars. The segment with the least error rate is indicated by white bars. FigureS4.docx Figure S4. Z-score representation of the 3D structures of OsLOXs. The Z-scores of proteins derived from X-ray crystallography are displayed in light blue, whereas those obtained from nuclear magnetic resonance (NMR) spectroscopy are represented in dark blue. Each protein's Z-score is displayed as a black dot. FigureS5.docx Figure S5. Energy plot of the 3D structures of OsLOXs. The plot was created employing ProSA-Web. The dark green line indicates the average energy across each fragment of 40 residues, while the light green line represents the average energy across each fragment of 10 residues. S1Table.docx Table S1. Primer list for real-time quantitative PCR analysis S2Table.docx Table S2. Characteristic conserved domains position of OsLOX proteins. S3Table.docx Table S3. Identified conserved motifs with their width, symbol and description S4Table.docx Table S4. The chromosomal locations, positions, and orientations of all OsLOXs. S5Table.docx Table S5. List of all identified cis-regulatory elements discovered in the 5’ UTR region of OsLOX s along with their corresponding functions. S6Table.docx Table S6. List of miRNAs that target various LOX genes along with mode of inhibition. S7Table.xlsx Table S7. Details of the GO analysis of the predicted OsLOX genes. S8Table.docx Table S8. Predicted protein family’s interaction with OsLOX genes. S9Table.docx Table S9. Secondary structure analysis of the OsLOX proteins in rice. S10Table.docx Table S10. Tertiary structure validation score of OsLOX proteins via different tools. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 20 Feb, 2026 Reviewers invited by journal 19 Feb, 2026 Editor invited by journal 02 Feb, 2026 Editor assigned by journal 01 Feb, 2026 Submission checks completed at journal 01 Feb, 2026 First submitted to journal 29 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8728944","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594717305,"identity":"543366ae-1450-4883-9213-3f7dbdf2dd7c","order_by":0,"name":"Ahana Deb Tusti","email":"","orcid":"","institution":"Shahjalal University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ahana","middleName":"Deb","lastName":"Tusti","suffix":""},{"id":594717306,"identity":"9365e60e-8231-4251-ab1a-79ccee2a96dd","order_by":1,"name":"Md. Nazmul Hasan","email":"","orcid":"","institution":"Daffodil International University","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Nazmul","lastName":"Hasan","suffix":""},{"id":594717307,"identity":"367505f6-a425-4e35-83dc-1e17ba3a3b69","order_by":2,"name":"Jeba Faizah Rahman","email":"","orcid":"","institution":"Chittagong Veterinary and Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Jeba","middleName":"Faizah","lastName":"Rahman","suffix":""},{"id":594717308,"identity":"981dc24e-b398-4716-9db0-62606c89735c","order_by":3,"name":"Md. Hammadul Hoque","email":"","orcid":"","institution":"Shahjalal University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Hammadul","lastName":"Hoque","suffix":""},{"id":594717309,"identity":"9a9e21c8-a56b-4ad1-b7a9-d78f7b1dcf7a","order_by":4,"name":"Anindita Chakraborty","email":"","orcid":"","institution":"Shahjalal University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Anindita","middleName":"","lastName":"Chakraborty","suffix":""},{"id":594717310,"identity":"b5fad78c-8cd7-4ac6-b9cc-da74083c3307","order_by":5,"name":"Shamsul Haque Prodhan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYDACCTBpkcDAwAOkK6CiPIS1SIC0MDYwnAGy2UjSwthGhBb52c1PN/5sk8gzZ+A9/uDnvLrE+fMbGB+8bcOtxeDOMbPbvG0SxZYNfImNvdsOJ244xsBsOBefFokEs9uMbRKJG+6/MWxm3HYgcQMbA5s0Lx4t8jPSv938CdJygAeoZQ7QYW0M7L/xaWG4kWN2gxeupYE5seEYAxszPi0GN3LKbvOck0jcCfTLzJ5jh403HEtslpxzDq/Dtt38UWaTuJ2B98CHHzV1svObDx/88KYMj8Pg1iGYwCglChgQVjIKRsEoGAUjFQAAXglVdwdSnPgAAAAASUVORK5CYII=","orcid":"","institution":"Shahjalal University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Shamsul","middleName":"Haque","lastName":"Prodhan","suffix":""}],"badges":[],"createdAt":"2026-01-29 08:24:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8728944/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8728944/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103298623,"identity":"12247821-cb3f-4c0d-b7e3-40da865391b1","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":700103,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of OsLOX proteins. The PLAT/LH2 and Lipoxygenase domain are distinguished by bold lines at the N and C termini, respectively. Amino acids (identical or similar) are also shaded.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/9c0a6c11e6f026768aefd10f.png"},{"id":103298625,"identity":"81e66d6c-d0ed-423b-bf08-598dc4fd6c99","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2411822,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationship of LOX proteins in \u003cem\u003eOryza sativa,\u003c/em\u003e \u003cem\u003eArabidopsis thaliana, Zea mays, Brassica rapa, Sorgum bicolor, Setaria italica\u003c/em\u003e and \u003cem\u003eGlycine max\u003c/em\u003e. Maximum likelihood approach with 1000 bootstrap values were employed to generate the tree in MEGA11 software. The subfamilies 9-LOX, 13-LOX Type I, and 13-LOX Type II are represented by the violet, yellow, and blue clades, respectively. Red Color highlighted the OsLOX proteins.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/a15906fc136876b5a6ffcfd6.png"},{"id":103298640,"identity":"181baf73-bff4-4f8d-a138-09e8ab92017d","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":370657,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic representation of conserved motifs and gene structure in twelve rice \u003cem\u003eOsLOX\u003c/em\u003es. a) Black lines denote introns, while orange boxes indicate exons. The lengths of exons and introns can be inferred using the scale provided below. b) 15 distinct colored boxes illustrate the conserved motifs of OsLOXs. The legends below denote the protein sequence consensus for the respective motifs.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/5c372acea13aeefaeab57183.png"},{"id":103506327,"identity":"ab8559d4-5781-4d2f-b26c-d03e416d9215","added_by":"auto","created_at":"2026-02-26 13:35:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91335,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution of \u003cem\u003eLOX\u003c/em\u003e genes throughout rice genome. MapChart software was used to generate the map. At the top, chromosome number was indicated with sequential numbering, and the centromere location was indicated by \"C.\" Left side of the figure shows referencing scale for comparing the location of each gene.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/ad41a6698880b8d2d941dd62.png"},{"id":103298624,"identity":"bb84b6c1-2c19-49a3-9db5-e1a4aa506d5e","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2977995,"visible":true,"origin":"","legend":"\u003cp\u003eDuplication events of \u003cem\u003eOsLOX\u003c/em\u003es within the genome of \u003cem\u003eOryza sativa\u003c/em\u003e and synteny analysis of \u003cem\u003eOsLOX\u003c/em\u003es between two different genomes. a) The intraspecific collinearity analysis of the replicative \u003cem\u003eOsLOX\u003c/em\u003es in rice. The red lines denote segmental duplications, whereas the blue lines signify tandem duplicates. b) Interspecific synteny analysis of \u003cem\u003eLOX\u003c/em\u003e genes of \u003cem\u003eOryza sativa, Arabidopsis thaliana, \u003c/em\u003eand\u003cem\u003e Zea mays. \u003c/em\u003eThe red lines denote collinear pairings in various species. The chromosomes of different species were indicated by different color bars. The grey lines in the background denote the collinear blocks of the entire genome across various species.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/89b0ca497a3978e339d2fafd.png"},{"id":103298626,"identity":"2087f5a7-cbb4-49e1-93cc-4b3c3bfd9df5","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1917369,"visible":true,"origin":"","legend":"\u003cp\u003eIdentified cis-regulatory elements within \u003cem\u003eOsLOX\u003c/em\u003es\u003cem\u003e \u003c/em\u003epromoter region. The scale at the end indicates the upstream nucleotides of the translation initiation site. The colorful boxes on the right represent functions linked to distinct cis-acting elements of the corresponding genes.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/e94d18c7076026ca99029fb3.jpg"},{"id":103298628,"identity":"924a2c50-1557-4943-b6cc-88e9c8872321","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":892789,"visible":true,"origin":"","legend":"\u003cp\u003eRecognition of potential miRNAs that target \u003cem\u003eOsLOX\u003c/em\u003es. Cytoscape was employed to create a graphic depiction of the interaction between miRNA and \u003cem\u003eOsLOX\u003c/em\u003es. The miRNAs are depicted in green boxes, the Os\u003cem\u003eLOX\u003c/em\u003es are represented by elliptical shapes of various hues, and the regulatory link is indicated by arrows.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/a83f414df48c23f6b43099e7.png"},{"id":103298639,"identity":"4ed22c0a-46d3-49da-8c49-88ddf9b61127","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1111811,"visible":true,"origin":"","legend":"\u003cp\u003eGene Ontology enrichment analysis of differentially expressed genes (DEGs) associated with \u003cem\u003eOsLOX\u003c/em\u003es. The classification of \u003cem\u003eOsLOX\u003c/em\u003e gene functions is displayed on the right side of the circos plot. The quantity of genes associated with a specific GO ID, the total number of genes, differential genes, expected values, and rich factors are presented in a unique color scheme. The scaling of the -log10 (p-value) is represented in three separate colors: red, yellow and green.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/39876c457933228359a59138.png"},{"id":103298648,"identity":"7e18cb18-cbd4-402f-9909-06202ce99780","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1835676,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of predicted phosphorylation sites of OsLOX proteins. Vertical red, green, and blue lines in the middle graph reflect projected phosphorylation site scores for serine, threonine, and tyrosine, respectively. The average of the anticipated score threshold of 0.5 is shown by the horizontal pink line.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/66c1aec23bd960cfbb84cccc.png"},{"id":103298651,"identity":"5cede616-3e55-439b-9064-64be07228c96","added_by":"auto","created_at":"2026-02-24 07:43:37","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":5125118,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the protein-protein interaction network and functional pathways associated with the \u003cem\u003eOsLOX\u003c/em\u003e gene family. a) PPI network of the \u003cem\u003eOsLOX\u003c/em\u003e family; b) KEGG enrichment study of proteins within PPI nodes.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/b7c616e87aa39c8a0fd4b72e.png"},{"id":103298650,"identity":"f943e9cb-f9cf-415b-a9c5-c549d50c2cfc","added_by":"auto","created_at":"2026-02-24 07:43:37","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":6064767,"visible":true,"origin":"","legend":"\u003cp\u003eSecondary structure analysis of the OsLOXs. The structure was generated employing the STRIDE tool, and the membrane-spanning motif was found by the DeepTMHMM v2.0 server. The transmembrane helix is represented by the black box. The icons for secondary structure legends are displayed in the lower-right corner.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/cd9aca77d58b201b1cc8ac4e.png"},{"id":103298649,"identity":"5c0852a4-4e5f-47b4-bec9-4f8f6809332e","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":22532202,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted 3D structures of OsLOX proteins represented in a gradient of rainbow colors from N (blue) to C terminus (red) utilizing PyMOL v3.1 software.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/ba75fb48f49bfba8fa096099.png"},{"id":103298636,"identity":"368c238a-485c-4641-9b13-a0d35b3b884f","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":1372534,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap depicting the expression profiling of \u003cem\u003eOsLOX\u003c/em\u003es in rice based on tissue types. The color bar on the right side of the heatmap indicates relative expression values (FPKM), with red signifying high expression, black indicating moderate expression, and green representing low expression. Abbreviations: 7 DBH - seven days before heading; 7 DAF - seven days after flowering; 21 DAS - 21 days after sowing; 14 DAG - 14 days after germination; 5 DAP - five days after pollination.\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/b5aab619a2b05c99d53dae74.png"},{"id":103506114,"identity":"dedb528f-5cce-4c55-95f9-0bb40cc3e817","added_by":"auto","created_at":"2026-02-26 13:34:07","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":1718573,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap depicting the expression profiles of \u003cem\u003eOsLOX\u003c/em\u003es under diverse biotic stressors. RNA-Seq data obtained from the Plant Public RNA-seq Database (PPRD) was utilized to create the heatmap using GraphPad Prism 10.5.0 software. The right-side scale of the heatmap denotes relative expression values derived from the log2 transformed FPKM values. The colors red, green, and black represent high, low, and moderate expression levels, respectively.Heatmap depicting the expression profiles of \u003cem\u003eOsLOX\u003c/em\u003es under diverse biotic stressors. RNA-Seq data obtained from the Plant Public RNA-seq Database (PPRD) was utilized to create the heatmap using GraphPad Prism 10.5.0 software. The right-side scale of the heatmap denotes relative expression values derived from the log2 transformed FPKM values. The colors red, green, and black represent high, low, and moderate expression levels, respectively.\u003c/p\u003e","description":"","filename":"Figure14.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/87c22a6adef5d3722fe304bb.png"},{"id":103298652,"identity":"4d18ab5f-29a5-43e4-b272-afe35fe30a72","added_by":"auto","created_at":"2026-02-24 07:43:38","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":2429197,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap illustrating the expression profiling of \u003cem\u003eOsLOX\u003c/em\u003es in reaction to various abiotic stress conditions. RNA-Seq data obtained from the Plant Public RNA-seq Database (PPRD) was utilized to generate the heatmap in GraphPad Prism version 10.5.0. The right-side scale of the heatmap illustrates relative expression values based on the log2 FPKM values. The colors red, green, and black represent high, low, and moderate expression levels, respectively.\u003c/p\u003e","description":"","filename":"Figure15.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/397ecf14b0f1f7cc998ebab8.png"},{"id":103505669,"identity":"5d1bb9f6-4c62-4a74-b435-1ce2ebb4fd83","added_by":"auto","created_at":"2026-02-26 13:32:33","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":3483909,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression analysis of \u003cem\u003eOsLOX\u003c/em\u003es in response to a variety of abiotic stress conditions. The Y-axis represents the relative expression levels of each gene assessed via RT-qPCR in the leaves of the rice plant. The X-axis represents the various abiotic stress conditions under which the relative expression analysis was performed. Data was analyzed using GraphPad Prism 10.5.0 and Microsoft Office 365. Statistically significant differences were identified between pairs of measurements using two-way ANOVA, followed by Dunnett's test. To indicate significant differences, different means were annotated with a corresponding number of asterisks (*). *, **, and **** stand for the different significance levels (p ≤ 0.05, p ≤ 0.01, and p \u0026lt; 0.001, respectively).\u003c/p\u003e","description":"","filename":"Figure16.png","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/b11a25d21a9ae9218620a5ea.png"},{"id":103298634,"identity":"2c7c00aa-c50c-4000-a236-530fd2d4ad2b","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":194430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1.\u003c/strong\u003e The heatmap illustrates the predicted sub-cellular localization of OsLOX proteins. The names of the corresponding cellular organelles are displayed at the bottom of the heatmap, whereas the names of each OsLOX protein are displayed on the left side. The presence of protein signals corresponding to the genes is shown by the color intensity on the right side of the heatmap.\u003c/p\u003e","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/7700dda7523c338506a6e836.docx"},{"id":103506827,"identity":"6fc650b0-7557-4190-979d-e049fb8f1c2e","added_by":"auto","created_at":"2026-02-26 13:39:37","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":637877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2. \u003c/strong\u003eRamachandran plot of the 3D structures of OsLOXs. PROCHECK was utilized to create the Ramachandran plot. The hues red, yellow, light yellow, and white represent residues in the most favored, additional allowed, generously allowed, and disallowed regions respectively.\u003c/p\u003e","description":"","filename":"FigureS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/b4c4ecdcd74908c2637795ab.docx"},{"id":103505913,"identity":"aa9c2729-6a12-4b08-a5ce-a29cdf9c94a8","added_by":"auto","created_at":"2026-02-26 13:33:28","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7516387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3. \u003c/strong\u003eERRAT plot of the 3D structures of OsLOXs. The plot was generated via the ERRAT server. Proteins that are capable of being excluded with a 95% confidence level are indicated by yellow bars, while those with a 99% confidence level are indicated by red bars. The segment with the least error rate is indicated by white bars.\u003c/p\u003e","description":"","filename":"FigureS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/71cb5259d8bdab6b10d5a591.docx"},{"id":103298645,"identity":"fa2c82a3-8968-4f2d-9ed1-d62ab38a4355","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":519378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S4.\u003c/strong\u003e Z-score representation of the 3D structures of OsLOXs. The Z-scores of proteins derived from X-ray crystallography are displayed in light blue, whereas those obtained from nuclear magnetic resonance (NMR) spectroscopy are represented in dark blue. Each protein's Z-score is displayed as a black dot.\u003c/p\u003e","description":"","filename":"FigureS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/56aca7885bd70438f0505853.docx"},{"id":103298638,"identity":"dfb25332-b29d-423b-a591-8a69f44fab98","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2441135,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S5.\u003c/strong\u003e Energy plot of the 3D structures of OsLOXs. The plot was created employing ProSA-Web. The dark green line indicates the average energy across each fragment of 40 residues, while the light green line represents the average energy across each fragment of 10 residues.\u003c/p\u003e","description":"","filename":"FigureS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/294ec2a1b0b4f5cd85cc6785.docx"},{"id":103298644,"identity":"88b28b33-da64-4188-a670-f5417800c578","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003e Primer list for real-time quantitative PCR analysis\u003c/p\u003e","description":"","filename":"S1Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/315e45b9d66662a9b3b00765.docx"},{"id":103298641,"identity":"e53c94c6-8efe-466d-9872-62fc660896cc","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2. \u003c/strong\u003eCharacteristic conserved domains position of OsLOX proteins.\u003c/p\u003e","description":"","filename":"S2Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/82e10d2df8806ec89d25693b.docx"},{"id":103298633,"identity":"e7bab040-9a43-4234-84eb-4bea82105ee0","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":296145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3. \u003c/strong\u003eIdentified conserved motifs with their width, symbol and description\u003c/p\u003e","description":"","filename":"S3Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/d6ac549a1cc32780723757da.docx"},{"id":103298647,"identity":"4a9ef827-7839-4349-a616-a9cbf349e710","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":15539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4.\u003c/strong\u003e The chromosomal locations, positions, and orientations of all OsLOXs.\u003c/p\u003e","description":"","filename":"S4Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/f82986ebe6756d6969810aab.docx"},{"id":103298630,"identity":"85edde6a-4895-4fc7-b53a-22e60fda1455","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":19829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S5.\u003c/strong\u003e List of all identified cis-regulatory elements discovered in the 5’ UTR region of \u003cem\u003eOsLOX\u003c/em\u003es along with their corresponding functions.\u003c/p\u003e","description":"","filename":"S5Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/9c41b2684d73560946919ceb.docx"},{"id":103298642,"identity":"cc947d69-91c7-4c99-b1e9-72c1fa95db62","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":29528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S6. \u003c/strong\u003eList of miRNAs that target various LOX genes along with mode of inhibition.\u003c/p\u003e","description":"","filename":"S6Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/081167563815db7089a8dc53.docx"},{"id":103505895,"identity":"0cd58965-25c0-4ba0-9b90-065822698690","added_by":"auto","created_at":"2026-02-26 13:33:24","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":13235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S7.\u003c/strong\u003e Details of the GO analysis of the predicted \u003cem\u003eOsLOX\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"S7Table.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/87fb972c272ce217e7179656.xlsx"},{"id":103298635,"identity":"a4c1206d-bca8-4251-82de-da1340e92578","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":15316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S8. \u003c/strong\u003ePredicted protein family’s interaction with \u003cem\u003eOsLOX\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"S8Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/dec2cb4479ff38526f202cd1.docx"},{"id":103298632,"identity":"403f9dc0-9f4f-46f3-a3a6-1a043ed3f680","added_by":"auto","created_at":"2026-02-24 07:43:35","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":16416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S9.\u003c/strong\u003e Secondary structure analysis of the OsLOX proteins in rice.\u003c/p\u003e","description":"","filename":"S9Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/7300c8216739ab308e7863f6.docx"},{"id":103298643,"identity":"fc783e2b-2cdd-4137-b3d2-b4ac6d87b72c","added_by":"auto","created_at":"2026-02-24 07:43:36","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":16559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S10. \u003c/strong\u003eTertiary structure validation score of OsLOX proteins via different tools.\u003c/p\u003e","description":"","filename":"S10Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-8728944/v1/d299345370b75359027df389.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Identification and Expression Analysis of LOX Gene Family in Rice (Oryza sativa L.) Under Abiotic Stress Conditions","fulltext":[{"header":"Background","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e) is considered as the preeminent cereal grain globally, feeding above 50% of the earth\u0026rsquo;s population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Multitude of abiotic challenges resulting from environmental and climate changes such as salinity, drought, submergence, extreme temperatures, nutrient deficiencies and heavy metal accumulation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], severely disrupt physiological [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], biochemical [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], molecular [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and metabolic processes in rice plants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], thereby adversely impacting their growth and development, productivity, and overall, an annual yield reduction of 32% (approximately 3\u0026nbsp;million tons) worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Abiotic stress response in rice plants occurs via induction of complex gene network modules [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and the adaptation in changing environmental situations is well-regulated by the perception and stress signals transduction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Rice is the first species to have its entire genome sequenced among cereal grains. Initial draft genomes for the \u003cem\u003ejaponica\u003c/em\u003e and \u003cem\u003eindica\u003c/em\u003e subspecies (regarded as 93\u0026ndash;11) were released in 2002. Although, the \u003cem\u003eJaponica\u003c/em\u003e cultivar \u003cem\u003eNipponbare\u003c/em\u003e was the inaugural high-quality reference genome for rice, and indeed for any cereal species, which was released in 2005 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Several studies have identified functionally annotated and validated expression profiles of stress responsive genes in rice through the analysis of genomes and transcriptomics data [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, characterizing the genetic background of rice thorough a genome wide investigation of stress-responsive families is essential for elucidating molecular mechanisms of stress tolerance and for breeding varieties with high adaptability under environmentally constrained conditions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLipoxygenases (LOXs) are an enzyme that is present in both plants and mammals. They are members of the iron-containing fatty acid dioxygenase family and include linoleate: oxygen oxidoreductase (EC 1.13.11.12) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It enables the conversion of polyunsaturated fatty acids (PUFAs) from plants into linoleic acid (LA, C18:2ω6), linolenic acid (ALA, C18:3ω3), and arachidonic acid (AA, C20:4ω6) through the process of oxygenation and dehydrogenation. This process results in the formation of unsaturated hydroperoxides [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These intermediate products undergo several metabolic reactions to generate oxygenated derivatives including jasmonic acid (JA), reactive oxylipins containing epoxides, aromatic compounds conjugated carbonyls or aldehydes, and leaf aldehydes and diethylene ethers which have antibacterial and antifungal activities [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, certain PUFAs are metabolized by α-dioxygenase (α-DOX) enzymes to produce α-hydroxy or α-peroxy PUFAs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTwo spatially separated LOX pathways: 9-LOX and 13-LOX have been observed in flora. 13-LOX pathway was classified further as Type I 13-LOX and Type II 13-LOX, present on cytoplasm and chloroplast respectively [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The N-terminal of \u003cem\u003eLOX\u003c/em\u003e gene contains a conserved PLAT/LH2 (polycystin-1, lipoxygenase, alpha-toxin/lipoxygenase homology) domain, while the C-terminal of true \u003cem\u003eLOX\u003c/em\u003e genes contains a characteristics lipoxygenase domain composed of histidine amino acid rich region [His-(X)4-His-(X)4-His-(X)17-His-(X)8-His] [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Numerous signal molecules are generated via LOX-mediated pathways; among these, the major signalling compounds are C6-volatile compounds and jasmonates which contribute to plant physiological responses encompass germination [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], growth and development [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], fruit ripening [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], reactions to both biotic and abiotic stressors [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], wounding [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], senescence and cell death [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and synthesis of stress responsive hormones such as jasmonic acid (JA) and abscisic acid (ABA) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Plants express numerous LOX genes during stress, which induces physiological and anatomical modifications to accommodate adverse conditions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eLOX\u003c/em\u003e is a polygenic family studied and characterized in several plants. Understanding the specific physiological function of \u003cem\u003eLOX\u003c/em\u003e genes has been challenging given the prevalence of several isozymes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The occurrence of these distinct \u003cem\u003eLOX\u003c/em\u003e isoforms is contingent upon the tissue, sub-cellular spaces, and stages of embryogenesis, and their catalytic features, tissue-specific gene expression, and amino acid sequences vary [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Prior research has determined a cumulative sum of 6 \u003cem\u003eLOX\u003c/em\u003e family members in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], 36 members in soybean [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], 11 members in radish and tea tree [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], 12 members in \u003cem\u003eFoxtail millet\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], 18 members in melon [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], 14 members in strawberries [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], 20 members in \u003cem\u003epoplar and Artemisia annua\u003c/em\u003e and so on. Among many C3 photosynthetic cereal crops, durum wheat has been shown to upregulate \u003cem\u003eTdLpx-A2\u003c/em\u003e during hyperosmotic stress and thereby counteract excess ROS generation by \u003cem\u003eTdLOX2\u003c/em\u003e to mitigate plant oxidative damage [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. CRISPR/Cas9-mediated overexpression line of soybean demonstrated \u003cem\u003eGmLOX6\u003c/em\u003e increases salinity tolerance through JA-mediated biosynthesis pathway [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. C4 model plant foxtail millet exhibited a substantial upregulation of \u003cem\u003eSiLOX7\u003c/em\u003e during the exposure of saline condition in two stress-tolerant varieties [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In oriental melon, suppression of \u003cem\u003eCmLOX10\u003c/em\u003e has been demonstrated to enhance drought susceptibility via jasmonic acid-mediated stomatal closure and feedback involving \u003cem\u003eCmMYC2\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Cloning of the computationally identified promoter region of \u003cem\u003eCmLOX08\u003c/em\u003e elucidated the mechanisms underlying resistance to abiotic stresses through signaling molecules and stress-inducible core promoters [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Further study on \u003cem\u003eMedicago truncatula\u003c/em\u003e confirmed that exogenous \u003cem\u003eMtLOX24\u003c/em\u003e overexpression in \u003cem\u003eArabidopsis\u003c/em\u003e mitigates the MeJA-induced oxidative damage, suggesting its role to insect related abiotic stress responses (L. Xu et al., 2024).\u003c/p\u003e \u003cp\u003eAlthough numerous research has examined rice transcriptome data and co-expression patterns in response to various abiotic and biotic challenges, a systematic examination of the \u003cem\u003eLOX\u003c/em\u003e gene family and its specific response to abiotic stress remains lacking. A comprehensive characterization and expression profiling of \u003cem\u003eLOX\u003c/em\u003e gene family of rice may offer significant understanding of its function under abiotic stressors, attributable to the presence of lipoxygenase enzymes. Thus, our approach can effectively identify and characterize \u003cem\u003eLOX\u003c/em\u003e gene family, by uncovering their evolutionary relationships, expression profiles, and potential functionalities. Additional, functional characterization of selective \u003cem\u003eLOX\u003c/em\u003es in response to abiotic and hormonal stress will yield insights for further examination of rice \u003cem\u003eLOX\u003c/em\u003e gene family.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eSequence curation of Lipoxygenase genes in\u003c/b\u003e \u003cb\u003eOryza sativa\u003c/b\u003e \u003cb\u003egenome and physicochemical properties analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA multi-step method employing diverse bioinformatics resources and databases was applied to discover \u003cem\u003eLOX\u003c/em\u003e family members in rice. TAIR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] was utilized to obtain the protein sequences for six \u003cem\u003eArabidopsis thaliana\u003c/em\u003e genes (\u003cem\u003eAt1g55020, At3g45140, At1g17420, At1g72520, At3g22400, At1g67560\u003c/em\u003e) that are members of \u003cem\u003elipoxygenase\u003c/em\u003e gene family [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The rice LOX proteins were identified by undertaking a BLASTP search against the \u003cem\u003eOryza sativa\u003c/em\u003e v7.0 genome dataset in the Phytozome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/info/Osativa_v7_0\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/info/Osativa_v7_0\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. For the sake of comprehensive identification, these AtLOX protein sequences were additionally tested against the Rice Genome Annotation Project (RGAP) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rice.uga.edu/\u003c/span\u003e\u003cspan address=\"https://rice.uga.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and NCBI protein database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/protein\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/protein\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] individually to ensure no potential member was overlooked. To identify all the possible \u003cem\u003eLOX\u003c/em\u003e genes, Hidden Markov Model (HMM) profile for Lipoxygenase domain (PF00305) and PLAT/LH2 domain (PF01477) were acquired from the Pfam database[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] with HMMER program (v3.4) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The e-value threshold was established at \u0026lt;1e-5. To further validate the presence and integrity of conserved domains, the presumed \u003cem\u003eLOX\u003c/em\u003e sequences were analyzed using SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], NCBI CDD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], and InterPro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The genes were renamed as \u0026lsquo;\u003cem\u003eOsLOX\u003c/em\u003e\u0026rsquo; with the \u0026lsquo;\u003cem\u003eOs\u003c/em\u003e\u0026rsquo; prefix indicating \u003cem\u003eOryza sativa\u003c/em\u003e and sequentially numbered according to chromosomal positions (from top to bottom), following an established naming procedure [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Gene lengths, CDS lengths, locus IDs, strand locations, and coding sequence (CDS) coordinates (5\u0026prime; to 3\u0026prime;) were all taken from the Phytozome database [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Further, to ascertain similarities in the protein sequences of \u003cem\u003eOsLOX\u003c/em\u003es, multiple sequence alignments were conducted using the ClustalW alignment function of MEGA 11[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] and results were visualized using the software GeneDoc v.2.7 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe physicochemical properties of the genes, including the theoretical isoelectric point (pI), index of instability, grand average of hydropathy (GRAVY), and molecular weight of the proteins were assessed using OsLOX protein sequences in ProtParam hosted by ExPASy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In addition, for determining the proteins subcellular distribution, the protein sequences FASTA file were uploaded to WoLF PSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] and CELLO v.2.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw/\u003c/span\u003e\u003cspan address=\"http://cello.life.nctu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. TBtools version v2.225 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] was employed to visualize the anticipated protein signals of each gene.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic relationship analysis\u003c/h2\u003e \u003cp\u003eTo generate the tree of evolutionary relationship of LOX proteins from several plant species, the completely discovered sequences of LOX proteins of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], \u003cem\u003eZea mays\u003c/em\u003e [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], \u003cem\u003eBrassica rapa\u003c/em\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], \u003cem\u003eSorgum bicolor\u003c/em\u003e [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], \u003cem\u003eSetaria italica\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] were used to keep the rate uniform across sites. Peptide sequences of \u003cem\u003eLOX\u003c/em\u003e genes of \u003cem\u003eZea mays\u003c/em\u003e (B73 reference genome), \u003cem\u003eSorgum bicolor, Setaria italica\u003c/em\u003e and \u003cem\u003eGlycine max\u003c/em\u003e were acquired from Phytozome [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and Brassicaceae database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://brassicadb.cn\u003c/span\u003e\u003cspan address=\"http://brassicadb.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. TAIR database [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] was employed for retrieval of peptide sequences of \u003cem\u003eLOX\u003c/em\u003e genes from \u003cem\u003eBrassica rapa\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e genome correspondingly. By employing the MUSCLE alignment and the Maximum Likelihood Method with a 1000 bootstrap value to substantiate branch values, an unrooted phylogenetic tree was constructed in MEGA 11 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The tool iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] was employed for tree annotation, manipulation and visualization.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGene structure and conserved motif analysis\u003c/h3\u003e\n\u003cp\u003eThe organization of exon-intron of \u003cem\u003eOsLOX\u003c/em\u003es were efficiently identified and graphically depicted utilizing Gene Structure Display Server (GSDS2.0) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds/gao-lab.org\u003c/span\u003e\u003cspan address=\"http://gsds/gao-lab.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] by comparing the coding sequences (CDS) and genomic sequences lacking the UTR (untranslated region) as input resources on the server. MEME Version 5.5.8 (Multiple Em for Motif Elicitation; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] was utilized to identify conserved motifs on proteins, employing default parameters with the exception of selecting a maximum of 15 motifs. MEME online interface used the motif scanning method (MSA) to visualize the motifs. The role of each of the 15 detected motifs was determined by evaluating them using Pfam [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eChromosomal distribution analysis\u003c/h3\u003e\n\u003cp\u003eMapChart software[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] was employed to create a rudimentary chromosomal distribution diagram of \u003cem\u003eOsLOX\u003c/em\u003e genes based on their location across the 12 rice chromosomes. The details on gene loci, length for the physical map and CDS coordinates obtained from the Phytozome [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] were employed for pinpointing the location and position on the map.\u003c/p\u003e\n\u003ch3\u003ePrediction of gene duplication and collinearity analysis\u003c/h3\u003e\n\u003cp\u003eFor estimating the divergence time of \u003cem\u003eOsLOX\u003c/em\u003e genes and investigating evolutionary patterns, the non-synonymous (Ka) and synonymous (Ks) substitution rates for duplicated \u003cem\u003eOsLOX\u003c/em\u003e gene pairs were calculated using TBtools version v2.225 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Segmental duplications were defined as instances in which gene pairs exhibited more than 90% sequence similarity, while tandem duplications were defined as two or more homologous genes within a 100 kb region on the same chromosome [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Selection was inferred from Ka/Ks ratios, and divergence time was determined using the formula T\u0026thinsp;=\u0026thinsp;Ks/2λ\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e (million years). λ is defined as a constant rate of 1.5 \u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e substitutions per site per annum for plants with dicotyledons [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Multiple Collinearity Scan Toolkit (MCScanX) [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] was used to detect gene duplication events. The \u003cem\u003eOryza sativa\u003c/em\u003e genomic sequence and GFF annotation file were utilized for self-alignment in the investigation of intraspecific collinearity. Interspecific collinearity analysis involved comparing the genomic sequences and GFF annotation files of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e with the genome sequences of \u003cem\u003eOryza sativa\u003c/em\u003e. The syntenic relationship was then determined using the syntenic analysis maps generated by Dual Systeny Plotter technique (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/CJ-Chen/TBtools\u003c/span\u003e\u003cspan address=\"https://github.com/CJ-Chen/TBtools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Finally, the result was visualized using TBtools v2.225.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCis\u003c/b\u003e \u003cb\u003e-regulatory elements analysis of\u003c/b\u003e \u003cb\u003eOsLOX\u003c/b\u003e\u003cb\u003es\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 2000 bp upstream sequences of each OsLOX promoter region were obtained from the Phytozome v13 database [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] to investigate the cis-acting regulatory elements (CREs) and their functions. To identify the prospective cis-regulatory elements (CREs) within the promoter region of \u003cem\u003eOsLOX\u003c/em\u003e genes, the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] was employed and the potential CREs were visualized using TBtools v2.225 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpecification putative miRNA targeting\u003c/b\u003e \u003cb\u003eOsLOX\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify potential miRNAs that target the \u003cem\u003eLOX\u003c/em\u003e genes in rice, psRNATarget (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.zhaolab.org/psRNATarget\u003c/span\u003e\u003cspan address=\"https://www.zhaolab.org/psRNATarget\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] was utilized by uploading the CDS sequences of \u003cem\u003eOsLOX\u003c/em\u003es. The analysis focused on the mature micro-RNAs of rice listed in miRbase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mirbase.org/\u003c/span\u003e\u003cspan address=\"https://www.mirbase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The associations between these miRNAs and their target \u003cem\u003eOsLOX\u003c/em\u003e genes were illustrated through Cytoscape software [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eGene Ontology Analysis\u003c/h3\u003e\n\u003cp\u003eTo ascertain the functional relationship of the discovered \u003cem\u003eOsLOX\u003c/em\u003e genes, gene ontology (GO) enrichment analysis was performed in collaboration the Plant Transcription Factor Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plantregmap.gao-lab.org/go.php\u003c/span\u003e\u003cspan address=\"https://plantregmap.gao-lab.org/go.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. ChiPlot (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e] was employed for data visualization.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePrediction of phosphorylation sites\u003c/h2\u003e \u003cp\u003eFor the prediction of phosphorylation sites, the protein sequences were used as input using the NetPhos-3.1 website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/NetPhos-3.1/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/NetPhos-3.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Results with a combined score greater than 0.5 were reliable.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtein-protein interaction (PPI) network prediction\u003c/h3\u003e\n\u003cp\u003eSTRING version-12.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e] was employed to predict the PPI network of OsLOX proteins based on the homologous proteins from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The following parameters of STRING tool were specified for the analysis: (i) full STRING network, as network type, (ii) the meaning of network edges as evidence, (iii) minimum interaction score 0.4 (medium confidence parameter), and (iv) maximum number of interactions in first shell\u0026thinsp;\u0026lt;\u0026thinsp;10. Cytoscape software [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] was employed to visualize the predicted interaction network. Further, putative functions of the interacting protein families and result of KEGG enrichment analysis of PPI node proteins were retrieved.\u003c/p\u003e\n\u003ch3\u003eSecondary and tertiary structure analysis of OsLOX proteins\u003c/h3\u003e\n\u003cp\u003eThe STRIDE program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://webclu.bio.wzw.tum.de/stride/\u003c/span\u003e\u003cspan address=\"https://webclu.bio.wzw.tum.de/stride/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] with default parameters was employed to predict OsLOX protein\u0026rsquo;s secondary structure. PROTEUS Structure Prediction Server 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.proteus2.ca/proteus2/\u003c/span\u003e\u003cspan address=\"http://www.proteus2.ca/proteus2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e] was employed to forecast the turns, coils, 310 helices, extended beta sheets, and alpha helices in the context of structural analysis. Additionally, investigation of membrane-spanning motif was done by using DeepTMHMM 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/TMHMM-2.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/TMHMM-2.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNext, the trRosetta webserver (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://yanglab.qd.sdu.edu.cn/trRosetta/\u003c/span\u003e\u003cspan address=\"https://yanglab.qd.sdu.edu.cn/trRosetta/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e] were employed for generating the tertiary structure of OsLOX proteins. The predicted tertiary structures were refined using the GalaxyRefine2 module of the GalaxyWEB server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://galaxy.seoklab.org/\u003c/span\u003e\u003cspan address=\"http://galaxy.seoklab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Energy minimization was done employing Swiss-PdbViewer v4.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.expasy.org/spdbv/\u003c/span\u003e\u003cspan address=\"http://www.expasy.org/spdbv/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. To verify the reliability of the protein structures, PROCHECK tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://servicesn.mbi.ucla.edu/PROCHECK\u003c/span\u003e\u003cspan address=\"https://servicesn.mbi.ucla.edu/PROCHECK\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e] and ERRAT servers (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://servicesn.mbi.ucla.edu/ERRAT/\u003c/span\u003e\u003cspan address=\"https://servicesn.mbi.ucla.edu/ERRAT/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e] were utilized for assessing model quality. In addition, Z-scores and energy plots were assessed using ProSA-web (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://prosa.services.came.sbg.ac.at/prosa.php\u003c/span\u003e\u003cspan address=\"https://prosa.services.came.sbg.ac.at/prosa.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Finally, the 3D structures were visualized using the Pymol 3.1 software [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSpatiotemporal expression profiling of the genes in rice using RNA-seq data\u003c/h2\u003e \u003cp\u003eThe RGAP database [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and Rice Expression Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/red/index/\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/red/index/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e] were employed for collecting RNA-seq expression data of twelve \u003cem\u003eOsLOX\u003c/em\u003e transcripts in various tissues throughout several development phases. Plant Public RNA-seq Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plantrnadb.com/ricerna/\u003c/span\u003e\u003cspan address=\"https://plantrnadb.com/ricerna/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e] was employed with RGAP Locus IDs of OsLOXs to collect transcriptomic data of rice during different time points for biotic and abiotic stress conditions. To quantify gene expression, sample transcript value was normalized using Log2 transformation method (FPKM\u0026thinsp;+\u0026thinsp;1) [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e] for each gene. GraphPad Prism 10.5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.graphpad.com/\u003c/span\u003e\u003cspan address=\"https://www.graphpad.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e] software was used to visualize a heat map derived from the expression profile data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePlant germination, abiotic stress treatments, and total RNA isolation\u003c/h2\u003e \u003cp\u003eSeeds of the \u003cem\u003eindica\u003c/em\u003e rice variety BRRI Dhan 105 were procured from the Bangladesh Rice Research Institute (BRRI), with only the healthy, mature, and high-quality seeds chosen for this experiment.\u003c/p\u003e \u003cp\u003eThoroughly cleaned seeds were positioned in a petri dish with wet tissue paper to initiate germination. After three to four days, germinated seedlings were transferred into a hydroponic cultivation system. In the growth chamber, environmental conditions were regulated at a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, with a photoperiod of 16 hours of light and 8 hours of darkness, and a light intensity of 1500\u0026ndash;2000 lux. 21 days old seedlings were subjected to several abiotic stress treatments which included salt (150 mM NaCl), drought (3 mM PEG 6000), saline-alkalinity (60 mM NaHCO\u003csub\u003e3\u003c/sub\u003e), heat (42\u0026deg;C), cold (4\u0026deg;C) and IAA (98mg/L dissolved in NaOH followed by distilled water) for 24h; and samples were taken at two time intervals: 12 hours and 24 hours, to ensure optimal responsiveness.The untreated seedlings were employed as a control. Fresh young leaves collected 12h and 24h post-treatment were thoroughly cleaned with 70% ethanol and distilled water for removing any pollutants prior to RNA extraction. Total RNA of treated and controlled frozen leaf tissue samples was extracted following the FavorPrep\u0026trade; Tri-RNA Reagent user guide's procedure. The removal of genomic DNA contamination was conducted with DNase I from Thermo Fisher Scientific Corporation\u0026rsquo;s Invitrogen\u0026trade; DNA-free\u0026trade; DNA Removal Kit. The first-strand cDNA of the mRNA was produced with the ABscript II cDNA First Strand Synthesis Kit (ABclonal, Inc. USA), in accordance with the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExpression profiling under abiotic stress conditions using quantitative real-time PCR data\u003c/h2\u003e \u003cp\u003eThe primer design for the RT-qPCR was conducted using the NCBI Primer-BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e] and the OligoAnalyzer Tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sg.idtdna.com/pages/tools/oligoanalyzer\u003c/span\u003e\u003cspan address=\"https://sg.idtdna.com/pages/tools/oligoanalyzer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) of Integrated DNA Technologies, Inc. The product length was maintained between 160 and 220 bp (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To examine the expression of each of the twelve \u003cem\u003eOsLOX\u003c/em\u003e genes expression regarding treatments, the RT-qPCR was conducted employing a 96-well plate format on the BioRad CFX96\u0026trade; Real-Time PCR Detection System. Eukaryotic elongation factor 1 alpha (eEF-1α)[\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e] was chosen for internal reference. The study utilized the GoTaq\u0026reg; qPCR Master Mix (2X) from Promega Corporation, USA. For each 15 \u0026micro;L reaction mixture, we incorporated 7.5 \u0026micro;L of GoTaq\u0026reg; qPCR Master Mix (2X), 2 \u0026micro;L of 10-fold diluted cDNA, 1 \u0026micro;L of gene-specific primers, and 3.5 \u0026micro;L of nuclease-free water. Each reaction was conducted under the following conditions: initial denaturation for 2 minutes at 95\u0026deg;C, followed by 40 cycles of denaturation for 15 seconds at 95\u0026deg;C, annealing for 30 seconds, and extension at 72\u0026deg;C for 40 seconds. The annealing temperature was 63.4\u0026deg;C for \u003cem\u003eOsLOX1\u003c/em\u003e, 61.6\u0026deg;C for \u003cem\u003eOsLOX2\u003c/em\u003e and \u003cem\u003eOsLOX10\u003c/em\u003e; 63.5\u0026deg;C for \u003cem\u003eOsLOX3, OsLOX8\u003c/em\u003e and \u003cem\u003eOsLOX5\u003c/em\u003e; 62.6\u0026deg;C for \u003cem\u003eOsLOX4, OsLOX9\u003c/em\u003e and \u003cem\u003eOsLOX11\u003c/em\u003e; 61.1\u0026deg;C for \u003cem\u003eOsLOX6\u003c/em\u003e, 61.3\u0026deg;C for \u003cem\u003eOsLOX7\u003c/em\u003e and \u003cem\u003eOsLOX12\u003c/em\u003e; 59.6\u0026deg;C for \u003cem\u003eeEF-1α\u003c/em\u003e. All reactions were performed in triplicate for each experimental condition and melting curve analysis was conducted after PCR amplification. The double delta Ct value method was used to determine the relative gene expression levels [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. To ascertain the mean values of various treatments, technical replication was implemented and further, data processing and statistical analysis was performed using Microsoft Office 365 and GraphPad Prism 10.5.0 [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. In each treatment group, the experimental data were represented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of the measured values. GraphPad Prism v10.5.0 was employed to generate bar graphs. A significance test was conducted utilizing two-way ANOVA, succeeded by Dunnett\u0026rsquo;s test. The asterisks (*) above the bars denote statistically significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Dunnett\u0026rsquo;s test) among various time points.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSequence curation of Lipoxygenase genes in\u003c/strong\u003e \u003cstrong\u003eOryza sativa\u003c/strong\u003e \u003cstrong\u003egenome and physicochemical properties analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA cumulative of twelve \u003cem\u003eLOX\u003c/em\u003e gene family members were identified in the genome of \u003cem\u003eOryza sativa\u003c/em\u003e. Six \u003cem\u003eAtLOX\u003c/em\u003e\u0026rsquo;s protein sequences were used as a reference to perform BLASTP searches against the \u003cem\u003eOryza sativa\u003c/em\u003e v7_JGI dataset on Phytozome [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. A total of 24 matches were identified, and of the 24 transcripts analyzed, 17 were confirmed to include both the Lipoxygenase domain and the PLAT/LH2 domain through sequence analysis using a Hidden Markov Model (HMM) profile, as these are the defining characteristics of LOX genes. These 17 transcripts correspond to the twelve LOX genes that display alternative splice variants. A BLASTP search was conducted in the RGAP [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e] and NCBI protein database [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e] to ensure that no potential LOX protein sequence was overlooked. The representative transcripts for the genes in this investigation were selected from the Phytozome annotated primary transcripts. Based on chromosomal number and location, the genes were named sequentially (\u003cem\u003eOsLOX1\u003c/em\u003e \u0026ndash; \u003cem\u003eOsLOX12\u003c/em\u003e), where \u0026lsquo;\u003cem\u003eOs\u003c/em\u003e\u0026rsquo; prefix was used for \u003cem\u003eOryza sativa\u003c/em\u003e, followed by \u003cem\u003eLOX\u003c/em\u003e for \u003cem\u003eLipoxygenase\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]. Information regarding the conserved domains positions within the twelve OsLOX proteins is stated in Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eTo get a comprehensive idea regarding physicochemical properties of OsLOXs proteins, sequence characterization was done. The proteins length ranged from 347 to 942 aa, along with CDS length ranging from 1041 bp (OsLOX12) to 2826 bp (OsLOX9). The molecular weights of the deduced OsLOX proteins varied from 39,243.14 Da for OsLOX12 to 104,687.15 Da for OsLOX11. The proteins exhibited an average molecular weight of 92,146.14 Da. Analysis of the instability index revealed that four out of seventeen transcripts had an instability score below 40, indicating their stability. It was determined that 24% of the genes were stable, while 76% were unstable. Isoelectric point (pI) study revealed that eight OsLOX proteins had pI value\u0026thinsp;\u0026lt;\u0026thinsp;7.0, categorizing them as acidic, while four OsLOX-encoded proteins displayed pI values\u0026thinsp;\u0026gt;\u0026thinsp;7.0, signifying their basic characteristics. Furthermore, the negative GRAVY value of all the proteins indicates that the OsLOX proteins were likely hydrophilic. Subcellular localization prediction was conducted to further the understanding of the biological processes and functions of OsLOX proteins within various organelles. This confirmed the predominant presence of the highest quantity of OsLOX proteins in the chloroplast, cytoplasm and mitochondria. Both the vacuole and the plasma membrane contain OsLOX proteins; however, only OsLOX12 was found in the vacuole while OsLOX6 and OsLOX12 were found in the plasma membrane (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). All the attributes of OsLOX gene family members along with their physicochemical properties are mentioned in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. MSA revealed that all OsLOX genes exhibit significant sequence similarity, possessing two highly conserved domains at their N- and C-termini: the PLAT/LH2 Domain and the Lipoxygenase Domain. Position of the characteristic conserved domains of OsLOX proteins is highlighted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eCharacteristics attributes of the members of Lipoxygenase gene family in \u003cem\u003eOryza sativa\u003c/em\u003e L.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSL No\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eGene Name\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLocus ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eTranscript\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eStrand\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eChr No\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCDS Coordinates (5\u0026prime; to 3\u0026prime;)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eLength (bp)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eProtein (aa)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMW(Da)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003epI\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eInstability Index\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eGRAVY Value\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLocalization\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ecDNA\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCDS\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os02g10120\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os02g10120.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5282626\u0026thinsp;\u0026minus;\u0026thinsp;5276620\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6007\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3224\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n2781\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e927\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e103585.62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46.57 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.317\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003ea\u003c/sup\u003e, Mit\u003csup\u003ea\u003c/sup\u003e, Chl\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g08220\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g08220.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4187107\u0026ndash;4193539\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6433\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3456\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n2757\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n919\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e101959.30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45.90 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.318\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMit\u003csup\u003ea\u003c/sup\u003e, Cyt\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX3\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g49260\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g49260.1*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28049441\u0026ndash;28053725\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4285\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3144\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2604\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e868\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97984.80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39.96 (stable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.359\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003ea\u003c/sup\u003e, Mit\u003csup\u003ea\u003c/sup\u003e, Chl\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g49260.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28049440\u0026ndash;28053725\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4285\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3132\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2592\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e864\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97499.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39.21 (stable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.379\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNuc\u003csup\u003eab\u003c/sup\u003e, Mit\u003csup\u003eab\u003c/sup\u003e, Chl\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX4\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g49380\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g49380.1*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28106903\u0026ndash;28113300\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6398\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3179\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2634\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n878\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e98697.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37.42 (stable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.323\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g49380.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28107728\u0026ndash;28113286\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6398\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2210\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n1482\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e494\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e55535.22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42.81 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.449\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003eab\u003c/sup\u003e, Mit\u003csup\u003eb\u003c/sup\u003e, Chl\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX5\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g52860\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os03g52860.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30315455\u0026ndash;30318972\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3518\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2941\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n2613\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n871\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97183.72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32.87 (stable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.285\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX6\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os04g37430\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os04g37430.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22305064\u0026ndash;22309320\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4257\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3032\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n2394\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e798\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e89304.87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56.45 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.525\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNuc\u003csup\u003ea\u003c/sup\u003e, Mit\u003csup\u003eab\u003c/sup\u003e, PM\u003csup\u003ea\u003c/sup\u003e, Chl\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX7\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os05g23880\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os05g23880.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13734067\u0026ndash;13740568\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6502\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3825\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n2544\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e848\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95343.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e44.94 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.434\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003ea\u003c/sup\u003e, Chl\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX8\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os08g39840\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os08g39840.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25224080\u0026thinsp;\u0026minus;\u0026thinsp;25216363\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7718\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3251\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2775\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e925\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e102819.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e49.81 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.389\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChl\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX9\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eLOC_Os08g39850\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os08g39850.1*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25240906\u0026ndash;25250046\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9374\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3322\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e2826\u003c/div\u003e\n\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;942\u003c/div\u003e\n\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e104494.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e47.55 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.349\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChl\u003csup\u003eab\u003c/sup\u003e, Mit\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os08g39850.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25244414\u0026ndash;25250279\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9374\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2778\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2472\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n824\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92381.69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e41.44 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.365\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003eab\u003c/sup\u003e, Chl\u003csup\u003ea\u003c/sup\u003e, Nuc\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os08g39850.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n25244414\u0026ndash;25250046\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9374\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2701\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;2472\u003c/div\u003e\n\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n824\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92381.69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e41.44 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.365\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003eab\u003c/sup\u003e, Chl\u003csup\u003eab\u003c/sup\u003e, Nuc\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX10\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os11g36719\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os11g36719.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21675027\u0026ndash;21684774\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9748\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;2607\u003c/div\u003e\n\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e869\u003c/div\u003e\n\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e98325.68\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40.98 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.417\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003ea\u003c/sup\u003e, Nuc\u003csup\u003eb\u003c/sup\u003e, Chl\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX11\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os12g37260\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os12g37260.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22860200\u0026thinsp;\u0026minus;\u0026thinsp;22854611\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5590\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3218\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2769\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;923\u003c/div\u003e\n\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e104687.15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46.98 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.494\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCyt\u003csup\u003ea\u003c/sup\u003e, Mit\u003csup\u003ea\u003c/sup\u003e, Chl\u003csup\u003eab\u003c/sup\u003e, Nuc\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX12\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLOC_Os12g37320\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os12g37320.1*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22920107\u0026ndash;22923778\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3672\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1390\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1077\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e359\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40772.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48.15 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.720\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChl\u003csup\u003eab\u003c/sup\u003e, Nuc\u003csup\u003eab\u003c/sup\u003e, Mit\u003csup\u003eab\u003c/sup\u003e, Vac\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC_Os12g37320.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e22920107\u0026ndash;22923778\u003c/div\u003e\n\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3672\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1520\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1041\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e347\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39243.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e52.31 (unstable)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-0.694\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChl\u003csup\u003eab\u003c/sup\u003e, Nuc\u003csup\u003eab\u003c/sup\u003e, Mit\u003csup\u003ea\u003c/sup\u003e, PM\u003csup\u003ea\u003c/sup\u003e, Vac\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"16\"\u003e*Primary transcripts\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"16\"\u003eCDS: Coding Sequence; MW: Molecular weight, pI: Isoelectric point; GRAVY: Grand average of hydropathy; bp: base pair; aa: amino acid; R: reverse strand; F: forward strand; Cyt: Cytoplasm; Mit: Mitochondria; PM: Plasma membrane; Chl: Chloroplast, Vac: Vacuole.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"16\"\u003e\u003csup\u003ea\u003c/sup\u003e Subcellular localization prediction by CELLO v.2.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw/\u003c/span\u003e\u003c/span\u003e).\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"16\"\u003e\u003csup\u003eb\u003c/sup\u003e Subcellular localization prediction by WoLF PSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003c/span\u003e).\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003ePhylogenetic relationship analysis\u003c/h2\u003e\n\u003cp\u003eIn order to investigate the evolutionary relationships among the LOX genes from different species, a phylogenetic tree was generated by aligning the full-length amino acid sequences of the highly homologous LOX protein sequences from: \u003cem\u003eOryza sativa, Arabidopsis thaliana\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e], \u003cem\u003eZea mays\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e], \u003cem\u003eBrassica rapa\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e], \u003cem\u003eSorgum bicolor\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e], \u003cem\u003eSetaria italica\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] and \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. An unrooted tree was generated via the Maximum Likelihood approach in MEGA11 [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. Poisson correlation and pairwise deletion were selected as the mode and gap, respectively, with 1000 bootstrap repetitions employed as the validation parameter. The phylogenetic tree (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) illustrated that the twelve OsLOX proteins were categorized into two subfamilies: 9-LOXs and 13-LOXs, with the latter further subdivided into Type I and Type II. In detail, the subfamily 9-LOXs contains 4 OsLOX proteins (OsLOX3, OsLOX4, OsLOX5 and OsLOX10) along with 2 AtLOX, 3 BrrLOX, 7 SiLOX, 5 SbLOX, 6 ZmLOX, and 22 GmLOX proteins. Maximum of 7 OsLOX (OsLOX1, OsLOX2, OsLOX6, OsLOX8, OsLOX9, OsLOX11 and OsLOX12) proteins belonged to 13-LOX Type I; this subfamily also contained 4 AtLOX, 12 BrrLOX, 5 SiLOX, 3 SbLOX, 6 ZmLOX, and 12 GmLOX proteins. Only the OsLOX7 protein from the rice genome is present in 13-LOX Type II, which also comprises 2 GmLOX, 1 ZmLOX, 1 SbLOX, and 1 SiLOX proteins. Subfamily 13-LOX Type II solely included monocots, except for one GmLOX which further suggests that these genes were exclusive to monocotyledons[\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eGene structure and conserved motif analysis\u003c/h2\u003e\n\u003cp\u003eExon-intron structure is a significant evolutionary characteristic of genes that offers essential insights into functional diversity. The analysis of exon-intron structures (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) indicated that the overall number of coding sequences (CDS) or exons varied from 3 to 10, while the number of introns ranged from 2 to 9. \u003cem\u003eOsLOX10\u003c/em\u003e possessed the lowest number of exons and introns, comprising 3 exons and 2 introns, whereas \u003cem\u003eOsLOX3\u003c/em\u003e exhibited the greatest amount, consisting of 10 exons and 9 introns. There was no intron-less gene present. The diversity in the lipoxygenase gene family of rice indicates varied functional roles and evolutionary divergence within this gene family.\u003c/p\u003e\n\u003cp\u003eFor identifying conserved motif distribution and diversity analysis of \u003cem\u003eOsLOX\u003c/em\u003e genes, web-based tool MEME [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e] were employed. 15 conserved motifs were found and designated as motifs 1 through 15 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.b). Majority of the motifs were shared by the members due to the strong sequence similarity between OsLOX peptide sequences. Among the 15 motifs, motifs 4, 5, 8, 11 and 14 were shared by all the OsLOX proteins. Apart from OsLOX7, motif 15 was found in the remaining 11 LOX proteins. OsLOX12 is devoid of all other motifs, indicating its uniqueness within subfamily 13-LOX Type I. Except OsLOX6, remaining motifs were shared by the members of all subfamilies. Functional analysis of the motifs revealed that all the motifs belong to Lipoxygenase iron binding catalytic domain profile (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eChromosomal distribution analysis\u003c/h2\u003e\n\u003cp\u003eThe investigation of chromosomal mapping analysis of \u003cem\u003eOsLOX\u003c/em\u003e genes indicated that all \u003cem\u003eOsLOX\u003c/em\u003es are distributed randomly and unevenly throughout 7 of the 12 chromosomes in rice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). No \u003cem\u003eLOX\u003c/em\u003e members were mapped onto chromosomes 1, 6, 7, 9, and 10. Chromosome 3 contained 4 \u003cem\u003eLOX\u003c/em\u003e genes apiece, while chromosomes 8 and 12 each possessed 2 \u003cem\u003eLOX\u003c/em\u003e genes. A single LOX gene was present on each of the following chromosomes: 2, 4, 5, 11. \u003cem\u003eOsLOX1\u003c/em\u003e and \u003cem\u003eOsLOX2\u003c/em\u003e are located on the p arm of their respective chromosomes, while the remaining genes are located on the q arm. \u003cem\u003eOsLOX7\u003c/em\u003e were positioned near the centromere. Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e lists the chromosomal locations, positions, and orientations of all the \u003cem\u003eOsLOX\u003c/em\u003es.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eGene duplication prediction and collinearity analysis\u003c/h2\u003e\n\u003cp\u003eGene duplication study aids in understanding the evolution of plants by emergence of novel gene functions and expansion of gene families. Findings revealed the evolutionary links of \u003cem\u003eOsLOX\u003c/em\u003e genes and the existence of selective pressure, including neutral, positive, and negative/purifying selections (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Four pairs of duplicated genes were identified from McScanX result. The result revealed that two pairs of genes (\u003cem\u003eOsLOX3\u003c/em\u003e/\u003cem\u003eOsLOX5\u003c/em\u003e \u0026amp; \u003cem\u003eOsLOX2\u003c/em\u003e/\u003cem\u003eOsLOX6\u003c/em\u003e) were segmentally duplicated, while two pairs found in the 100 kb region of same chromosome (\u003cem\u003eOsLOX8\u003c/em\u003e/\u003cem\u003eOsLOX9\u003c/em\u003e \u0026amp; \u003cem\u003eOsLOX11\u003c/em\u003e/\u003cem\u003eOsLOX12\u003c/em\u003e) were tandem duplicated. To estimate the selection pressure: neutral selection was denoted by Ka/Ks\u0026thinsp;=\u0026thinsp;1, purifying selection by Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1, and positive selection by Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;1 [\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e]. Duplicated pairs Ka/Ks fraction were all constantly remaining below 1, indicating a notable presence of strong purifying selection. Findings also showed that duplication events presumably took place between 46.94596 and 4.72241\u0026nbsp;million years ago (MYA), demonstrating the more ancient duplication events have occurred in \u003cem\u003eOsLOX3\u003c/em\u003e/\u003cem\u003eOsLOX5\u003c/em\u003e; in contrast, \u003cem\u003eOsLOX8/OsLOX9\u003c/em\u003e was the most recent duplicated pair, indicating recent divergence events of tandem duplicated pairs.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eCalculations of divergence time and Ka/Ks ratios in the duplicated pairs of \u003cem\u003eOsLOX\u003c/em\u003es.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDuplicated gene pair\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKa\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKs\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKa/Ks\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTime (MYA)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePurifying assortment\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDuplicate type\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX3\u003c/em\u003e/\u003cem\u003eOsLOX5\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.177696\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.408379\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.126171\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46.94596\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSegmental\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX2\u003c/em\u003e/\u003cem\u003eOsLOX6\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.325064\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.242597\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.2616\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e41.41991\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSegmental\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX8\u003c/em\u003e/\u003cem\u003eOsLOX9\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.060522\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.141672\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.427195\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.72241\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTandem\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX11\u003c/em\u003e/\u003cem\u003eOsLOX12\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.12902\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.185634\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.695022\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.18780\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTandem\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIntraspecific collinearity analysis was conducted to elucidate the potential regulatory functions associated with the highly conserved gene order of the \u003cem\u003eOsLOX\u003c/em\u003e gene family. Circos tool was used to display the linked gene pairs. This study identified 4 collinear gene pairs within rice genome. Chr3 and Chr4 contained two segmentally duplicated pairs while Chr8 and Chr12 contained two tandemly duplicated pairs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Comparative interspecific collinearity analysis maps were constructed among rice \u003cem\u003eLOX\u003c/em\u003es and two other plant species (\u003cem\u003eZea mays\u003c/em\u003e as a monocot and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e as a dicot species) for studying the potential evolutionary relationship between identified members of these species. Seven orthologous pairs were identified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The Ka/Ks ratio ranged from 0.363 to 1.345, with a mean of 0.323. However, an exception was found between rice and Arabidopsis due to high sequence divergence value (pS\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;0.75) which led to Ks saturation. Highest quantity of orthologous gene pairs identified between \u003cem\u003eZea mays\u003c/em\u003e and rice demonstrated the substantial impact of duplication events in monocot species, leading to the expansion and functional diversity of the \u003cem\u003eLOX\u003c/em\u003e gene family during evolution.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eDuplication events involving rice LOX genes and other species.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDuplicated Gene 1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDuplicated Gene 1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKa\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKs\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKa/Ks\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTime (MYA)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePurifying assortment\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDuplicate type\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eAT1G72520.1\u003c/em\u003e \u003cstrong\u003e(A.thaliana)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.2799\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epS\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;0.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePossible\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSegmental\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eZm00001eb206040_T001\u003c/em\u003e \u003cstrong\u003e(Zea mays)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.1899\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.3632\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.523\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.106\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSegmental\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eZm00001eb005920_T003\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0694\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.4996\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.139\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16.654\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTandem\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX3\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eZm00001eb216870_T004\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.2613\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.3459\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.1942\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e44.862\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSegmental\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX6\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eZm00001eb423430_T001\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.1535\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.439\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.3497\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.634\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSegmental\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX6\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eZm00001eb081610_T002\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.146\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.4135\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.3532\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.782\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSegmental\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eOsLOX8\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eZm00001eb035010_T001\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.184\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.414\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.4444\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.801\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTandem\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"8\"\u003e\u003cstrong\u003eCis\u003c/strong\u003e\u003cstrong\u003e-regulatory elements (CREs) analysis of\u003c/strong\u003e \u003cstrong\u003eOsLOX\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTo investigate the potential regulatory mechanisms of \u003cem\u003eOsLOX\u003c/em\u003es in the presence of a variety of stimuli and to comprehend the pivotal role they play in transcriptional regulation and gene expression, the CREs of the promoter region of each \u003cem\u003eOsLOX\u003c/em\u003es (up to 2.0 kb upstream of the translation start site) were searched against PlantCARE database [\u003cspan class=\"CitationRef\"\u003e100\u003c/span\u003e]. There were 46 cis-regulatory elements found within the 5\u0026prime; UTR promoter region of \u003cem\u003eOsLOX\u003c/em\u003es and grouped into seven different functional categories, namely elements responsible for i) Light (14.5% of all identified CREs), ii) Abiotic challenges (4.6%), iii) Hormonal regulation (12%), iv) Cellular development (2%), v) Promoter associated (63.7%), vi) Biotic challenges (2.2%), and vii) Miscellaneous functions (1%). The aspects of hormonal regulation were categorized into a) Methyl jasmonate (MeJA), b) Salicylic acid, c) Auxin, d) Gibberellin, and e) Abscisic acid responsive elements. The entire list of identified CREs along with their respective functions was included in Table \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e. The prevalence of presumed CREs within the promoter region of \u003cem\u003eOsLOX\u003c/em\u003es were depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eVarious abiotic stress related elements are involved in anaerobic, anoxic, cold, drought and defense responses. The abiotic stress responsive \u003cem\u003ecis\u003c/em\u003e-elements identified in the promoter region of \u003cem\u003eOsLOX\u003c/em\u003es include ARE, TC-rich repeats, MBS, LTR and GC-motifs. Furthermore, biotic stress responsive cis-elements including the W box, WUN-motif, and WRE3 were implicated in responses to fungal elicitors and wounding. Additionally, all \u003cem\u003eOsLOX\u003c/em\u003e genes were characterized by the presence of core promoter elements TATA box and CAAT box. They predominantly operate within the promoter and enhancer regions. Hormonal responsive CREs include ABRE, TGA-element, ARE-motif, P-box, AuxRR-core, TATC-box, TGACG-motif, CGTCA-motif and TCA-element, which were essential for five distinct hormones. The seed specific regulation, zein metabolism, circadian control, meristem and endosperm expression were all dependent on cellular development responsive CREs including O2-site, CAT-box, GCN4-motif, RY-element, and Circadian. Light responsive elements include Box 4, Gap-box, G-box, AE-box, I-box, TCCC-motif, AT1-motif, GT1-motif, ATCT-motif, Sp1, LS7, GA-motif, TCT-motif, GATA-motif, ACE, LAMP-element, GTGGC-motif, and MRE. In addition to these, CCAAT-box, A-box, HD-Zip 3, and AT-rich element exemplify cis-elements with diverse roles. The notable abundance of CREs within the promoter region of \u003cem\u003eOsLOX\u003c/em\u003es indicates their involvement in the transcriptional regulation. Furthermore, a variety of hormones and stress-related CREs point to a direct relationship between the gene\u0026rsquo;s activity in rice under various stress conditions. To completely understand the intricate interactions between CREs of the \u003cem\u003eOsLOX\u003c/em\u003e gene family and the related signaling pathways in controlling rice stress responses, further research is required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecification putative miRNA targeting\u003c/strong\u003e \u003cstrong\u003eOsLOX\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroRNAs (miRNAs) are essential in both plants and animals as post-transcriptional regulators of gene expression. miRNAs downregulation signifies an increased expression of the targeted mRNA. The psRNATarget database[\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e] was employed for identifying miRNAs targeting the \u003cem\u003eLOX\u003c/em\u003e genes in rice. The study identified 151 potential candidate miRNAs with a length of 20\u0026ndash;24 nucleotides that target \u003cem\u003eOsLOX\u003c/em\u003es. The lowest quantity of miRNAs (1) targeted \u003cem\u003eOsLOX12\u003c/em\u003e, while the maximum quantity of miRNAs (25) targeted \u003cem\u003eOsLOX6\u003c/em\u003e. The total count of miRNAs that targeted \u003cem\u003eOsLOX\u003c/em\u003es was as follows: 19 for \u003cem\u003eOsLOX\u003c/em\u003e1, 4 for \u003cem\u003eOsLOX2\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e for \u003cem\u003eOsLOX3\u003c/em\u003e, 17 for \u003cem\u003eOsLOX4\u003c/em\u003e, 10 for \u003cem\u003eOsLOX5\u003c/em\u003e, 25 for \u003cem\u003eOsLOX6\u003c/em\u003e, 20 for \u003cem\u003eOsLOX7\u003c/em\u003e, 12 for \u003cem\u003eOsLOX8\u003c/em\u003e, 15 for \u003cem\u003eOsLOX9\u003c/em\u003e, 6 for \u003cem\u003eOsLOX10\u003c/em\u003e, 8 for \u003cem\u003eOsLOX11\u003c/em\u003e\u0026cedil;and 1 for \u003cem\u003eOsLOX12\u003c/em\u003e. The regulatory interactions between putative miRNAs and their targeted \u003cem\u003eLOX\u003c/em\u003es are illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e. Among all the miRNAs, 25 targeted several \u003cem\u003eOsLOX\u003c/em\u003es, while the rest miRNAs were exclusive to each gene (Table \u003cspan class=\"InternalRef\"\u003eS6\u003c/span\u003e). The majority of miRNA-mediated \u003cem\u003eOsLOX\u003c/em\u003e repression was accomplished by mRNA cleavage, with only a small percentage of targets experiencing inhibition during the translation phase. Consequently, this demonstrates that the discovered miRNAs might have the potential to post-transcriptionally influence the production of \u003cem\u003eOsLOX\u003c/em\u003es via cleaving mRNA and suppressing translation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eGene Ontology Analysis\u003c/h2\u003e\n\u003cp\u003eGene ontology enrichment analysis was conducted to determine a variety of regulatory activities of \u003cem\u003eOsLOX\u003c/em\u003e genes. It establishes a framework classifying genes into three distinct groups based on their biological processes, cellular components and molecular functions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). It has been estimated that there are 22 GO terms across all \u003cem\u003eOsLOX\u003c/em\u003es. Among the three groups, the biological process was the most frequent as it contains 16 GO IDs. However, there were three GO IDs in each of the classes of molecular functions and cellular components. Moreover, the functional analysis involving the corresponding GO terms for biological process regions identified that GO terms responsible for the following processes: the biosynthesis and metabolism of oxylipin; the oxidation and modification of lipids; the biosynthesis and metabolism of fatty acids; the biosynthesis and metabolism of carboxylic acids; the biosynthesis and metabolism of organic acids; and the metabolism of oxoacids. GO terms associated with molecular processes found out they are responsible for oxidoreductase and dioxygenase activity, which are characteristic features of \u003cem\u003eLOX\u003c/em\u003e genes across diverse species. Cellular components associated with these GO terms are located within the cytoplasm, plastid, and chloroplast. GO analysis regarding the predicted \u003cem\u003eOsLOX\u003c/em\u003e genes with their respective p-values are listed in Table \u003cspan class=\"InternalRef\"\u003eS7\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003ePhosphorylation sites prediction\u003c/h2\u003e\n\u003cp\u003eThe NetPhos 3.1 service predicted the phosphorylation sites for serine (Ser), threonine (Thr), and tyrosine (Tyr) of eukaryotic proteins through neural network integration. The OsLOX protein family possesses multiple unique phosphorylation sites throughout its amino acid sequences, amounting to a total of 1473 phosphorylation sites. In particular, 778 of the total phosphorylation sites were made up of serine residues, with threonine and tyrosine accounting for 552 and 143 residues, respectively. The most serine phosphorylation site was OsLOX8 with 114, while the least number was OsLOX12 with 35. The most likely phosphorylation site had a value of 0.998 (much larger than the threshold value of 0.500) [\u003cspan class=\"CitationRef\"\u003e101\u003c/span\u003e],which indicates predominant kinase specificity; the most threonine phosphorylation sites was OsLOX6 with 73, and the least abundant was OsLOX12 with 24, of which the most likely phosphorylation site had a value of 0.994; the most abundant tyrosine phosphorylation site was OsLOX7 with 20, while the least abundant was OsLOX9 with only 4, of which the most likely phosphorylation site had a value of 0.988. It is suggested that the \u003cem\u003eOsLOX\u003c/em\u003es functional variety is determined by proteins with distinct architectures and kinase-specific phosphorylation sites (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eProtein-protein interaction (PPI) network prediction\u003c/h2\u003e\n\u003cp\u003eAnalysis of PPI network demonstrated the possible regulatory roles of OsLOX proteins based on the strong homology of orthologous \u003cem\u003eArabidopsis\u003c/em\u003e STRING proteins. Apart from OsLOX11, the family was found to be homologous with 10 \u003cem\u003eArabidopsis\u003c/em\u003e proteins (CYP74A1, CYP74A2, CYP74A3, CYP74A4, SLM1, AOC, Q2RAM0_ORYSJ, Q2R2W3_ORYSJ, PLA2-II, and PLA2-III). Additionally, KEGG enrichment analysis indicated that proteins that interact with \u003cem\u003eOsLOX\u003c/em\u003es may have the potential to participate in hormone-mediated pathways and external stress responses (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e, Table \u003cspan class=\"InternalRef\"\u003eS8\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eSecondary and tertiary structure analysis of OsLOX proteins\u003c/h2\u003e\n\u003cp\u003eSecondary structural analysis of OsLOXs revealed the percentages and locations of different constituents; as well as the location of transmembrane helix (Table \u003cspan class=\"InternalRef\"\u003eS9\u003c/span\u003e). With exception of OsLOX1, the predominant secondary structure was the alpha-helix, closely succeeded by the turn, coil, beta-sheet, and 310-helix. OsLOX1 has the highest turn in its structure which indicates a compact and flexible protein structure, it might be involved in binding or signaling processes. Moreover, the stability of OsLOXs is suggested by the higher proportion of helical structures [\u003cspan class=\"CitationRef\"\u003e102\u003c/span\u003e], and structures such as random coils are essential for signaling cascades [\u003cspan class=\"CitationRef\"\u003e103\u003c/span\u003e]. Only OsLOX6 had single membrane spanning motif (MSM) at 664 to 673bp position (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe trRosetta webserver [\u003cspan class=\"CitationRef\"\u003e86\u003c/span\u003e] is a deep learning algorithm that emplys a neural network for predicting inter-residue geometry (orientations and distances) and builds tertiary models using RosettaTTAfold. GalaxyRefine [\u003cspan class=\"CitationRef\"\u003e87\u003c/span\u003e] further refined the generated models, and then Swiss PdbViewer [\u003cspan class=\"CitationRef\"\u003e88\u003c/span\u003e] was utilized for energy minimization. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e depicts the predicted 3D structures of OsLOX proteins, generated using Pymol 3.1 Software [\u003cspan class=\"CitationRef\"\u003e92\u003c/span\u003e]. Multiple validation tools were implemented to assess the structural reliability of predicted protein models (S10 table). Ramachandran plot analysis through PROCHECK [\u003cspan class=\"CitationRef\"\u003e89\u003c/span\u003e] revealed that more than 90% of the residues were situated within the most favorable and additionally allowed regions, while fewer than 1.5% were positioned in disallowed regions, indicating a high-quality stereochemical conformation (Figure \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). ERRAT analysis [\u003cspan class=\"CitationRef\"\u003e90\u003c/span\u003e] further supported this with models exhibiting an overall quality factor exceeding 87, suggesting minimal structural errors (Figure \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). ProSA-web analysis [\u003cspan class=\"CitationRef\"\u003e91\u003c/span\u003e] yielded Z-scores that are within the typically observed range for experimentally determined native proteins and serve as evidence for the reliability of the predicted structures (Figure \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e). The corresponding energy plots confirmed that all residues were associated with favorable (low) energy values, indicative of structurally stable regions (Figure \u003cspan class=\"InternalRef\"\u003eS5\u003c/span\u003e). The validation results collectively affirm the high quality and dependability of the produced tertiary protein models.\u003c/p\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003eSpatiotemporal expression profiling of the genes in rice using RNA-seq data\u003c/h2\u003e\n\u003cp\u003eGene expression analysis measures the functional activity and expression level of active gene products (functional RNA or protein) in different cells and tissues. This is a highly specific molecular indicator of biological activity and metabolite levels. Changes associated with gene expression patterns are linked to corresponding alteration in development, disease progression, and adaptation to external stresses. In this study, RNA sequencing data of 12 \u003cem\u003eOsLOX\u003c/em\u003es in different tissues were retrieved from RGAP database [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e] and Rice Expression Database [\u003cspan class=\"CitationRef\"\u003e93\u003c/span\u003e] to investigate spatiotemporal effects during developmental stages for corresponding genes. The generated heatmap visualized the RNA transcript profiles with normalized expression values of RNA-seq FPKM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe analysis identified low to moderate expression levels in most tissues, with several members exhibiting strong tissue-specific expression. For instance, \u003cem\u003eOsLOX1\u003c/em\u003e displayed high level of expression in leaf (7 DBH to 7 days DAF) and shoot (7-day seedling and 14 DAG), this indicates its potential role in leaf development. \u003cem\u003eOsLOX8\u003c/em\u003e and \u003cem\u003eOsLOX9\u003c/em\u003e were upregulated in anthers during flowering stage indicating its role in pollen maturation. In the same manner, \u003cem\u003eOsLOX4\u003c/em\u003e and \u003cem\u003eOsLOX6\u003c/em\u003e were moderately upregulated in roots which implies potential participatory role in root growth or defense signaling. \u003cem\u003eOsLOX2\u003c/em\u003e was upregulated in anther and glumes, while \u003cem\u003eOsLOX5\u003c/em\u003e exhibited a mild upregulation in root (14 DS and 21 DAS). \u003cem\u003eOsLOX4\u003c/em\u003e showed notable upregulation during early germination (8 h after imbibition), whereas other members were downregulated in germinating seed and mature seed tissues. \u003cem\u003eOsLOX6\u003c/em\u003e showed overall moderate upregulatory expression for most tissues. \u003cem\u003eOsLOX3, 10, 11\u003c/em\u003e, and \u003cem\u003e12\u003c/em\u003e expression level were downregulated for all tissues.\u003c/p\u003e\n\u003cp\u003eTo get a better understanding of how \u003cem\u003eOsLOX\u003c/em\u003es respond to various stresses, the expression patterns of \u003cem\u003eOsLOX\u003c/em\u003es were examined in response to various biotic and abiotic stressors. The Plant Public RNA-seq Database [\u003cspan class=\"CitationRef\"\u003e94\u003c/span\u003e] was employed to acquire RNA-seq data for the investigation. GraphPad Prism 10.5.0 [\u003cspan class=\"CitationRef\"\u003e96\u003c/span\u003e] was used to generate the corresponding heatmaps. Log2 values of relative expression are represented by the color gradient on the right side of the heatmap (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e). In response to infection by bacterial leaf streak pathogen (\u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. \u003cem\u003eOryzae)\u003c/em\u003e, \u003cem\u003eOsLOX1\u003c/em\u003e, \u003cem\u003e2, 4, 5, 6, 7, 8\u003c/em\u003e, and \u003cem\u003e9\u003c/em\u003e were notably upregulated, while \u003cem\u003eOsLOX3. 10, 11\u003c/em\u003e and \u003cem\u003e12\u003c/em\u003e displayed downregulation. In the event of infection by the rice blast fungus \u003cem\u003e(Magnaporthe oryzae), OsLOX1\u003c/em\u003e, \u003cem\u003e2, 4, 6, 7, 8, 9\u003c/em\u003e, and \u003cem\u003e11\u003c/em\u003e exhibited consistent upregulation from 8 h to 72 h post-inoculation, suggesting their active participation in defense signaling against fungal invasion. Remaining genes were downregulated. Additionally, infection by rice stripe virus (RSV) led the upregulation of \u003cem\u003eOsLOX1\u003c/em\u003e, \u003cem\u003e2, 4, 6, 7\u003c/em\u003e, and \u003cem\u003e11\u003c/em\u003e at 3 to 15 days post-inoculation (DPI). \u003cem\u003eOsLOX8\u003c/em\u003e was upregulated in 3 DPI, but prolonged infection led to supression of the gene. Infection by necrotrophic fungus \u003cem\u003eRhizoctonia solani\u003c/em\u003e (Sheath blight) resulted in upregulation of \u003cem\u003eOsLOX2, 6, 7\u003c/em\u003e, and \u003cem\u003e11\u003c/em\u003e at 1 to 3 DPI, \u003cem\u003eOsLOX8\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e showed delayed upregulation (3 DPI); in contrast \u003cem\u003eOsLOX3, 5, 10\u003c/em\u003e and \u003cem\u003e12\u003c/em\u003e were markedly downregulated. Rice black streaked dwarf virus (RBSDV) infection triggered a high upregulation of \u003cem\u003eOsLOX1\u003c/em\u003e, \u003cem\u003e2, 4, 7, and 11\u003c/em\u003e, indicating a possible role in antiviral defense. \u003cem\u003ePyricularia oryzae\u003c/em\u003e is another fungal pathogen responsible for rice blast disease, exhibited upregulation of all genes of OsLOX family apart from \u003cem\u003eOsLOX3, 5, 10\u003c/em\u003e, and \u003cem\u003e12\u003c/em\u003e. Similarly, fungal blast causing \u003cem\u003eMagnaporthe grisea\u003c/em\u003e increased the expression of \u003cem\u003eOsLOX2, 3, 4, 5, 6\u003c/em\u003e, and \u003cem\u003e7\u003c/em\u003e. Moreover, \u003cem\u003eMeloidogyne graminicola\u003c/em\u003e (root knot nematode) infection induced the expression of \u003cem\u003eOsLOX1, 2, 4, 7, 8, 9\u003c/em\u003e, and \u003cem\u003e11\u003c/em\u003e at both 3 and 7 days after infection (DAI). Striga infection also led to upregulation of \u003cem\u003eOsLOX4, 5\u003c/em\u003e, and \u003cem\u003e6\u003c/em\u003e at infection stage of 3 and 7 days, while \u003cem\u003eOsLOX1, 10, 11\u003c/em\u003e, and \u003cem\u003e12\u003c/em\u003e remained significantly downregulated. \u003cem\u003eXylaria striata\u003c/em\u003e is an endophyte that exhibits improved disease resistance by upregulating \u003cem\u003eOsLOX4, 5\u003c/em\u003e and \u003cem\u003e9.\u003c/em\u003e Plant infected with rice root nematode (RRN) \u003cem\u003eHirschmanniella\u003c/em\u003e showed upregulation of \u003cem\u003eOsLOX3, 6\u003c/em\u003e, and \u003cem\u003e9\u003c/em\u003e. In conclusion, these results address potential role of \u003cem\u003eOsLOX\u003c/em\u003es in rice defense mechanisms against pathogens and biotic stress conditions.\u003c/p\u003e\n\u003cp\u003eTreatment with abiotic stress caused a noticeable change in \u003cem\u003eOsLOX\u003c/em\u003es gene expression patterns over a range of time periods. In response to submergence stress, \u003cem\u003eOsLOX2, 4\u003c/em\u003e, 8, 9 and \u003cem\u003e11\u003c/em\u003e were upregulated consistently at all time points, suggesting a potential role in submergence adaptation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e). The remaining genes were consistently downregulated. During drought stress, \u003cem\u003eOsLOX4\u003c/em\u003e and \u003cem\u003e11\u003c/em\u003e were consistently upregulated under 1 to 24 hours of exposure. \u003cem\u003eOsLOX1\u003c/em\u003e and \u003cem\u003e2\u003c/em\u003e showed upregulation during first 3 hours of treatment, \u003cem\u003eOsLOX8\u003c/em\u003e, 9, and \u003cem\u003e12\u003c/em\u003e showed upregulation during 3 to 12 hours of treatment. \u003cem\u003eOsLOX3, 5, 6, 7\u003c/em\u003e, and \u003cem\u003e10\u003c/em\u003e were predominantly downregulated, indicating their suppression under dehydration conditions. Similar distinct patterns were observed under saline conditions. \u003cem\u003eOsLOX1\u003c/em\u003e and \u003cem\u003e2\u003c/em\u003e showed highly upregulated expression across 1 to 24 hours of ionic imbalance. Short-term salt exposure (1\u0026ndash;5 h) induced moderate upregulation of \u003cem\u003eOsLOX4\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, and \u003cem\u003e11\u003c/em\u003e, \u003cem\u003eOsLOX5\u003c/em\u003e were upregulated during the 2nd hour of treatment, but continued exposure to salt led to downregulation. \u003cem\u003eOsLOX9\u003c/em\u003e were moderately expressed during 5 to 10h of salinity, whereas \u003cem\u003eOsLOX3, 6, 10\u003c/em\u003e and \u003cem\u003e12\u003c/em\u003e were consistently downregulated. In the case of saline alkalinity, \u003cem\u003eOsLOX2, 4, 6\u003c/em\u003e and \u003cem\u003eOsLOX9\u003c/em\u003e remained highly upregulated, suggesting sensitivity to alkaline ion toxicity. Cold treatment at 4\u0026deg;C resulted in a strong and constant upregulation of \u003cem\u003eOsLOX4, 8, 9, and 11\u003c/em\u003e from 1 to 168 hours, indicating a possible role in cold acclimation. \u003cem\u003eOsLOX1\u003c/em\u003e and 7 started with high induction (upregulatory expression) from 0 to 24 hours, but further cold treatment led to downregulation of the transcripts. With the remaining genes showing downregulation across all time points suggest cold mediated transcriptional repression. Heat stress caused constant upregulation of \u003cem\u003eOsLOX1\u003c/em\u003e and \u003cem\u003e2\u003c/em\u003e from 30 to 240 mins, whereas \u003cem\u003eOsLOX 4\u003c/em\u003e and \u003cem\u003e7\u003c/em\u003e were moderately upregulated, indicating heat-responsive activation. Rest of the genes were downregulated during thermal treatment. These differential expression patterns indicate \u003cem\u003eOsLOX\u003c/em\u003e family members participate actively in regulating rice adaptation to environmental stressors.\u003c/p\u003e\n\u003cp\u003eIn addition to numerous abiotic treatments, several hormones including auxin, gibbereline, absicic acid, jasmonic acid, salicylic acid, kinetins, and others aid plants during growth and development. During the biosynthetic process, LOX genes produce JA. Therefore, understanding transcript expression patterns under hormone treatment will aid in our comprehension of the optimal conditions for plant growth. In response to JA treatment, \u003cem\u003eOsLOX2\u003c/em\u003e, \u003cem\u003eOsLOX8\u003c/em\u003e, and \u003cem\u003eOsLOX11\u003c/em\u003e were consistently upregulated at 6, 12, and 24 hours in shoots, highlighting their involvement in JA-mediated signaling. \u003cem\u003eOsLOX4\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e showed moderate expression patterns whereas remaining genes were mainly downregulated across JA time points. During ABA treatment, \u003cem\u003eOsLOX4, 8 and 9\u003c/em\u003e were upregulated, with the rest being in downregulation implies that ABA does not activate these isoforms. In response to auxin treatment, \u003cem\u003eOsLOX3, 4, 5, 6\u003c/em\u003e and \u003cem\u003e8\u003c/em\u003e demonstrated high upregulation in root tissue for weekly treatment. Overall, the \u003cem\u003eOsLOX\u003c/em\u003e gene family displayed distinct and stress-specific transcriptional responses which implies functional divergence for adverse environmental conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003eExpression profiling under abiotic stress conditions using quantitative real-time PCR data\u003c/h2\u003e\n\u003cp\u003eThe relative expression patterns of \u003cem\u003eOsLOX\u003c/em\u003es were assessed in rice seedlings leaves throughout two different time periods to explore the effects of drought, salinity, saline-alkalinity, heat, cold and IAA (indole-3-acetic acid) stress conditions. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e illustrates a bar chart representing their real-time expression data under various stress situations after 12 and 24 hours.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e, \u003cem\u003eOsLOX1\u003c/em\u003e transcript was significantly upregulated under drought, heat and salt stress and downregulated expression was observed for saline-alkalinity and IAA treatment at 12h. Response to drought and salt stress showed a contrasting downregulatory expression at 24, whereas response persists for saline-alkalinity and IAA treatment. Heat stress response alleviated after prolonged exposure. Cold treatment resulted in no change in transcript expression pattern compared to control. \u003cem\u003eOsLOX2\u003c/em\u003e, drought and cold response were significantly upregulated at both time points, with cold response demonstrating seven-fold upregulation at 24h. Salinity induced significant upregulation after 24h, while saline-alkalinity showed decrease to increase expression pattern. Heat and IAA treatment resulted in higher upregulation at 12h but \u003cem\u003eOsLOX2\u003c/em\u003e transcript displayed no significant result at 24h. \u003cem\u003eOsLOX3\u003c/em\u003e exhibited substantial upregulation of transcript during drought and cold stress. Drought stress caused approximately four-fold significant higher expression at both times, but cold stress issued approximately seven-fold increase of relative expression after 24h. Salt, saline-alkalinity, and heat resulted in significant downregulation under these stressful situations and the downregulation sustained. IAA treatment induced downregulation at 12h but further exposure led to no relative change of gene activity. \u003cem\u003eOsLOX4, 5, 6\u003c/em\u003e, and \u003cem\u003e8\u003c/em\u003e showed similar expression patterns like \u003cem\u003eOsLOX3\u003c/em\u003e. For \u003cem\u003eOsLOX5\u003c/em\u003e, drought stress instigated significant upregulation of gene activity from two-fold to approximately four-fold; and cold stress resulting in less than one-fold to more than six-fold transcriptional upregulatory activity. The remaining stress significantly downregulated the transcript, and the downregulation was continuous. \u003cem\u003eOsLOX5\u003c/em\u003e transcript under drought conditions affected a significant upregulation from more than two-fold to approx. five-fold. Although, there was upregulation of cold-treated transcript at 12h, it was not significant until 24 where it showed approx. three-fold upregulation of relative expression. Like previously stated transcript, salt, saline-alkalinity, and heat treatment showed significant downregulation at both time points. IAA treatment resulted in significant downregulation at only 12h. \u003cem\u003eOsLOX6\u003c/em\u003e exhibited significantly higher level of expression change for drought conditions, the relative expression value fluctuated from more than three-fold to slightly higher than one-fold. Cold stress elevated the expression at 24h. Remaining stress resulted in significant downregulation, and it persisted till 24h. \u003cem\u003eOsLOX8\u003c/em\u003e expression was significantly enhanced under drought condition for both sampling time; under cold condition expression increased at 12h but it became significant only at 24h. Other stress followed the significant downregulatory pattern.\u003c/p\u003e\n\u003cp\u003eA change for salt-induced stress was discovered in \u003cem\u003eOsLOX7\u003c/em\u003e, it showed significantly higher levels of expression at 12h; but contrasting effect was observed during 24h with significant downregulation of the transcript. Drought caused significant upregulation of gene activity at 12h (with approx. three-fold change), which slightly decreased at 24h but remained significant. Cold condition caused significant upregulation at 12, IAA depicted significant upregulation at 24h with delayed response. Significant downregulation was observed for heat treatment at 12h and for saline-alkaline treatment at 24h. Regarding the transcript profile of \u003cem\u003eOsLOX9\u003c/em\u003e, it showed significant upregulation under drought and cold conditions. Heat treatment demonstrated significant downregulation at both time periods; significant downregulation was noticed in salt and saline-alkaline treated samples of 24h. Exception of IAA treatment with notable upregulation was observed at 24h. Moreover, a decrease to increase expression pattern was observed in \u003cem\u003eOsLOX10\u003c/em\u003e with a significantly higher expression level of over fourfold under cold stress. Rest of the stress including drought situation was significantly downregulated for this transcript. \u003cem\u003eOsLOX11\u003c/em\u003e had considerably significant upregulation levels of approx. eight-fold during heat (only 24h) and cold (both period) stress conditions. Salt and saline-alkalinity showed significant increase to decrease pattern of expression. Drought and IAA induced significant upregulation at only 12h. No significant changes were observed in \u003cem\u003eOsLOX12\u003c/em\u003e transcript for drought, cold and IAA stress situations. Salt, saline-alkalinity resulted in significant downregulation after prolonged period. Heat stress exhibited consistently significant downregulation.\u003c/p\u003e\n\u003cp\u003eHowever, under salt and cold conditions the RNA-seq data presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e did not exhibit consistent findings with RT-qPCR data for all gene transcripts. Drought, saline-alkalinity and heat stress induced expression of RT-qPCR relative expression was more or less consistent with RNA-seq data. Thus, these findings show the greatest time dependent differential expression pattern of \u003cem\u003eOsLOX\u003c/em\u003es, suggesting its significant role in the adaptation to abiotic stress in rice.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLipoxygenase is a significant dioxygenase encoding protein family that catalyzes the regio and stereoselective dioxygenation of polyunsaturated fatty acids (PUFAs) and serves as precursors for oxylipins. It actively participates in various growth and development phases and enhances resistance to harsh external environmental conditions across diverse plant genotypes [\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e]. This present study identified 12 distinct \u003cem\u003eOsLOX\u003c/em\u003e genes in rice whereas fourteen genes had been reported previously [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Identified genes possess both characteristic domains, the PLAT/LH2 and Lipoxygenase domains at their respective N and C termini which is consistent with previously identified true \u003cem\u003eLOX\u003c/em\u003e family members [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. Compared to C3 and C4 plants, \u003cem\u003eOryza sativa\u003c/em\u003e has more numbers of gene than in Arabidopsis (six) (Umate, 2011), Sesame (7) [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e], Radish (11) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and fewer than Maize (13) [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e], Wheat (44) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and Cotton (64) [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. These differences in the quantity of \u003cem\u003eLOX\u003c/em\u003es are not proportionate with genome size, suggesting that the \u003cem\u003eLOX\u003c/em\u003e gene has not been conserved throughout the evolutionary process. Physicochemical analysis revealed these genes differ in sequence length, molecular weight, isoelectric point, and GRAVY score, highlighting the heterogeneity of the gene family. The negative GRAVY score of the proteins verifies their hydrophilic nature [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. The subcellular localization of the proteins indicated their presence in the chloroplast, cytoplasm, mitochondria, nucleus, and plasma membrane. The localization discrepancies of the proteins predicted by the two independent tools. According to the findings of CELLO v.2.5 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], most proteins are found in the cytoplasm, whereas WoLF PSORT[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] indicated their localization in chloroplast, mitochondria and vacuolar membrane. Additional verification of protein subcellular localization necessitates validation by a GFP-fused transient expression system employing an Agro-infiltration method [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e]. Hence, it could be said that different \u003cem\u003eLOX\u003c/em\u003e gene members serve catalytic functions in different organelles.\u003c/p\u003e \u003cp\u003eIn a multispecies phylogenetic tree constructed with both monocot and dicot species, genes in a subgroup often have similar functions. OsLOX proteins were categorized into two distinct subfamilies: 9-LOXs and 13-LOXs, with the latter further subdivided into Type I and Type II, consistent with Wheat [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and \u003cem\u003eArtemisia annua\u003c/em\u003e [\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e]. However, \u003cem\u003eLOX\u003c/em\u003e genes in sorghum [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] and pepper [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] were categorized into 9-LOX, 13-LOX and an unknown subfamily [\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e]. Seven \u003cem\u003eOsLOX\u003c/em\u003e genes were present in Type I 13-LOXs, but one \u003cem\u003eOsLOX\u003c/em\u003e gene was present in Type II 13-LOXs with no \u003cem\u003eAtLOX\u003c/em\u003e sequence belonging to this group; it indicates certain divergences between \u003cem\u003eLOX\u003c/em\u003e gene families of monocots and dicots. The structural divergence of introns and exons in plant species is crucial for evolution [\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e]. All \u003cem\u003eOsLOX\u003c/em\u003e gene family members were identified to have exons ranging from 3\u0026ndash;10, and the intron number ranged from 2\u0026ndash;9. Identification is consistent with the pattern of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and \u003cem\u003eBrassica rapa\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. There was no intron-less gene which indicates that they are highly expressed and have not undergone recent evolution. Analysis of conserved motifs revealed that the substantial sequence similarity among OsLOX proteins resulted in shared motifs across all three phylogenetic catagories. Except for OsLOX6 and OsLOX12, all members of Type I 13-LOX exhibited 15 motifs; the absence of a total of 5 and 9 motifs in these two genes, respectively, indicates their uniqueness within Type I 13-LOXs.\u003c/p\u003e \u003cp\u003eThe analysis of chromosomal distribution found that all \u003cem\u003eOsLOX\u003c/em\u003e genes are dispersed randomly and unevenly throughout 7 of the 12 chromosomes. No \u003cem\u003eOsLOX\u003c/em\u003e member was mapped onto chromosomes 1, 6, 7, 9, and 10. Previous study identified five tandemly duplicated pairs in tomato [\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e]. Three tandem and three segmental duplicated gene pairs were observed in \u003cem\u003eLOX\u003c/em\u003e gene family of poplar [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. Our research revealed that of the four duplicated gene pairs, two were tandem duplicates and two were segmental duplicates. It is possible to assert that the expansion of the \u003cem\u003eLOX\u003c/em\u003e gene family in the rice genome is being driven by both segmental and tandem duplicated gene clusters. Seven orthologous gene pairings were found among rice, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e. The observed cases of gene duplication across various chromosomes indicate segmental duplication, which serves as the principal driver for diversification. The evolution of the duplicated \u003cem\u003eLOX\u003c/em\u003e pairs was influenced by purifying selection, as evidenced by Ka/Ks values\u0026thinsp;\u0026lt;\u0026thinsp;1 [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA total of 46 categories of cis-regulatory elements were identified and categorized into seven functional groups through further investigation of the promoter region of the \u003cem\u003eOsLOX\u003c/em\u003e gene family. These elements include those for cellular development (ABRE, AuxRR-core, ARE-motif, P-box, TATC-box, etc.), light response (CAT-box, Box 4, Sp1, Gap-box, MRE etc.), phytohormones (ABRE, TGA-element, AuxRR-core, P-box, TATC-box, etc.), biotic stress (W box, WUN-motif and WRE3), abiotic stress (ARE, TC-rich repeats, GC-motif, MBS and LTR, etc.) and Promoter associated (TATA box and CAAT box). The most prevalent CREs were associated with promoters, where multiple transcription factors bind and it indicates the complex regulatory mechanisms \u003cem\u003eOsLOX\u003c/em\u003es. Our results are consistent with previous research that has emphasized the multifaceted role of \u003cem\u003eLOX\u003c/em\u003e genes in the stress response [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. miRNAs are critical regulators of gene expression that are essential for the regulation of plant responses to abiotic and biotic stresses in cross species [\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e]. A total of 151 unique candidate miRNAs targeting members of the rice \u003cem\u003eOsLOX\u003c/em\u003e gene family were identified. 25 miRNAs targeted several \u003cem\u003eOsLOX\u003c/em\u003es, while the other miRNAs were restricted to individual genes. The majority of miRNA-mediated \u003cem\u003eOsLOX\u003c/em\u003e silencing resulted in mRNA cleavage, whereas a minor proportion of targets experienced translational inhibition and subsequent transcript degradation, thereby suggesting post-transcriptional regulation and translation inhibition.\u003c/p\u003e \u003cp\u003eAccording to results of gene ontology prediction, the OsLOX proteins of rice function in response to oxylipin, lipid, carboxylic acid, fatty acid and diverse metabolic processes, in addition to participating in organic acid and biosynthetic processes alongside several partners. Furthermore, these findings indicate that oxidoreductase and dioxygenase activities are significant functions of \u003cem\u003eOsLOX\u003c/em\u003e genes, as revealed by GO analysis regarding molecular function, largely occurring within plant cells, specifically in the plastid, chloroplast, and cytoplasm. Protein-protein interactions are essential for numerous intracellular and extracellular processes [\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e]. Multiple \u003cem\u003eArabidopsis\u003c/em\u003e proteins interact with the OsLOX proteins. Among these, \u003cem\u003eCYP74A1\u003c/em\u003e to \u003cem\u003eCYP74A4\u003c/em\u003e functions as Allene oxide synthase 4 which is a growth regulator and signaling molecules in plant defense. \u003cem\u003ePLA2-II, PLA2-III, Q2R2W3_ORYSJ\u003c/em\u003e acts as phospholipase or its homologs. \u003cem\u003eAOC\u003c/em\u003e interacts with auxin-mediated signaling pathway \u0026amp; implicated in NaCl stress response. \u003cem\u003eOsLOX\u003c/em\u003e genes are abundantly phosphorylated at serine, threonine and tyrosine residues. Identifying the putative phosphorylation sites of proteins helps in understanding of the signal transduction, which is essential for plant development and adaptability to environmental stress [\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis of secondary structures showed that except OsLOX1, alpha helix is the most prevalent secondary structure which surpasses others in dominance. It ensures the stable conformation of proteins, like our findings [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. Only OsLOX6 was identified to possess a singular membrane-spanning motif (MSM). The tertiary structures of the OsLOX proteins were predicted using several techniques, which validated the high quality of all structure predictions and might be used in further research.\u003c/p\u003e \u003cp\u003eThe information obtained from the RNA-seq database across various rice tissues was utilized to elucidate the functional roles of \u003cem\u003eOsLOX\u003c/em\u003es during growth and developmental phases. \u003cem\u003eOsLOX\u003c/em\u003es were discovered as differently expressed using tissue-specific expression pattern analysis. The analysis showed that among the tissues studied, \u003cem\u003eOsLOX1\u003c/em\u003e exhibited higher expression in leaf (7 DBH to 7 DAH), shoot (7-day seedling) and shoot (14-day seedling). Anther and Anther (flowering) stage showed upregulation of \u003cem\u003eOsLOX2, 8\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e which might indicate the specific role on reproduction. \u003cem\u003eOsLOX5\u003c/em\u003e demonstrated mild regulatory pattern in root (14 DS and 21 DAS) whereas, \u003cem\u003eOsLOX4\u003c/em\u003e were significantly upregulated during early germination stages (8 h after imbibition). Expression levels were moderately upregulated for \u003cem\u003eOsLOX6\u003c/em\u003e for most tissues. All other genes (\u003cem\u003eOsLOX3, 10, 11\u003c/em\u003e, and \u003cem\u003e12\u003c/em\u003e) exhibited lowest expression for due tissues. These findings are consistent with predominant expression of \u003cem\u003eBnaLOX2\u003c/em\u003e in stamens, indicating reproductive function [\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e]. Proteome profiling of \u003cem\u003eLOX\u003c/em\u003e gene showed expression in mature seeds, seedling stage, early germination in Arabidopsis, tomato, soybean and cucumber [\u003cspan additionalcitationids=\"CR121 CR122 CR123\" citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, RNA-seq data was processed to examine the role of OsLOXs under various biotic stress conditions. Our analysis demonstrated how \u003cem\u003eOsLOX\u003c/em\u003es response to diverse biotic factors including viral, bacterial, and fungal infections, as well as nematode inoculation. When induced with \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. \u003cem\u003eoryzae\u003c/em\u003e infection, strong upregulation of \u003cem\u003eOsLOX1\u003c/em\u003e, \u003cem\u003e2, 4, 5, 6, 7, 8\u003c/em\u003e, and \u003cem\u003e9\u003c/em\u003e were shown and this activates JA-related or oxylipin pathways typical of antibacterial responses. Fungal infections caused by \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e, which is rice blast responsible organims, also triggered significant upregulation of several \u003cem\u003eOsLOXs (OsLOX1\u003c/em\u003e, \u003cem\u003e2, 4, 6, 7, 8, 9\u003c/em\u003e, and \u003cem\u003e11)\u003c/em\u003e. Necrotrophic fungal infection caused by \u003cem\u003eRhizoctonia solani\u003c/em\u003e induces sheath blight in rice and elevated the expression of \u003cem\u003eOsLOX2, 6, 7\u003c/em\u003e, and \u003cem\u003e11\u003c/em\u003e. Fungus \u003cem\u003ePyricularia oryzae\u003c/em\u003e is also responsible for rice blast disease, and it showed higher activity of all genes of \u003cem\u003eOsLOX\u003c/em\u003e family apart from \u003cem\u003eOsLOX3, 5, 10\u003c/em\u003e, and \u003cem\u003e12\u003c/em\u003e. Viral infections (RSV and RBSDV) led to upregulation of \u003cem\u003eOsLOX1\u003c/em\u003e, \u003cem\u003e2, 4, 6, 7\u003c/em\u003e, and \u003cem\u003e11\u003c/em\u003e, highlighting a possible conserved antiviral role. Root-associated pathogens further emphasized context specific responses: nematode \u003cem\u003eMeloidogyne graminicola\u003c/em\u003e upregulated the expression of \u003cem\u003eOsLOX1, 2, 4, 7, 8, 9\u003c/em\u003e, and \u003cem\u003e11\u003c/em\u003e, while the beneficial endophyte \u003cem\u003eXylaria striata\u003c/em\u003e enhanced expression of \u003cem\u003eOsLOX4, 5\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e. Taken together, these patterns address dynamic and distinct role of \u003cem\u003eOsLOX\u003c/em\u003es depending on pathogen type, supporting their central role in coordinating defense mechanisms under biotic stressors. This present finding is consistent with previous studies where \u003cem\u003eArabidopsis thaliana AtLOX\u003c/em\u003e proteins showed defence mechanism against bacteria and pathogens via oxylipins through lateral root development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eAtLOX2\u003c/em\u003e and \u003cem\u003eAtLOX3\u003c/em\u003e mutants were shown to control early-stage plant nematode infections [\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e]. \u003cem\u003eZmLOX3\u003c/em\u003e of maize acts as a root-specific suppressor of primary defence signaling pathways and provides resistance against nematodes [\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e]. In papaya, the hydroperoxides generated by 13-LOX exhibited significant antifungal activity against blight disease [\u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the expression profile of \u003cem\u003eOsLOX\u003c/em\u003es was examined in the context of drought, salinity, saline-alkalinity, heat, cold and IAA (indole-3-acetic acid) in two different time points and the data of RT-qPCR analysis was compared with RNA-seq to ascertain the role under different abiotic stress conditions. The expression profiles of the majority of genes were broadly similar between the RNA-Seq and RT-qPCR results, despite the minor deviations observed during salt and cold stress. In RT-qPCR analysis, certain reference or housekeeping genes may occasionally exhibit significant responses to changes in experimental conditions or tissue types [\u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e, \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e]. In our study, we used \u003cem\u003eeEF-1α\u003c/em\u003e to validate the qPCR result for gene expression [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBecause of drought and cold stress, \u003cem\u003eOsLOX\u003c/em\u003e genes showed tolerance at different time points with upregulated transcripts. \u003cem\u003eOsLOX\u003c/em\u003e genes were identified as pivotal in modulating responses to drought and cold conditions. Under drought stress, \u003cem\u003eOsLOX1-9\u003c/em\u003e and \u003cem\u003eOsLOX11\u003c/em\u003e genes showed significant upregulation, which indicates their possible involvement in drought tolerance mechanisms. In contrast, \u003cem\u003eOsLOX10\u003c/em\u003e and \u003cem\u003e12\u003c/em\u003e were downregulated or showed no significant change under drought conditions, indicating gene-specific divergence within the \u003cem\u003eLOX\u003c/em\u003e family. Under cold stress, several transcripts exhibited significant and sustained upregulation. \u003cem\u003eOsLOX2-11\u003c/em\u003e had significantly higher expression levels at almost both sampling periods, which aid plants in surviving cold environment. In contrast, \u003cem\u003eOsLOX1\u003c/em\u003e and \u003cem\u003e12\u003c/em\u003e did not exhibit significant changes under cold treatment. Expression was predominantly suppressive in nature under saline condition. \u003cem\u003eOsLOX3-6, 8\u0026ndash;10\u003c/em\u003e and \u003cem\u003e12\u003c/em\u003e showed significant downregulation, whereas OsLOX1, 2, 7 and 11 displayed time varying upregulation pattern. Under saline-alkalinity, with exception of \u003cem\u003eOsLOX2\u003c/em\u003e and \u003cem\u003e11\u003c/em\u003e, all genes exhibited significant downregulation. Except for \u003cem\u003eOsLOX1, 2\u003c/em\u003e and \u003cem\u003e11\u003c/em\u003e, heat stress reported downregulation for \u003cem\u003eOsLOX\u003c/em\u003e gene family, which suggest suppression of non-essential processes and dominating stress responsive pathways. For IAA treatment, \u003cem\u003eOsLOX1, 3\u0026ndash;8\u003c/em\u003e, and \u003cem\u003e10\u0026ndash;11\u003c/em\u003e significantly downregulated. \u003cem\u003eOsLOX2\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e showed upregulation and \u003cem\u003eOsLOX12\u003c/em\u003e exhibited no significant changes across treatments. Similar studies have shown upregulatory expression pattern due to drought stress in \u003cem\u003eAmorpha fruticosa\u003c/em\u003e L. \u003cem\u003e(AfLOX4)\u003c/em\u003e and downregulation due to saline alkaline conditions, which is persistent with our study [\u003cspan citationid=\"CR130\" class=\"CitationRef\"\u003e130\u003c/span\u003e]. Several genes of maize exhibited transient expression patterns during cold stress, presumably signifying a delayed involvement in cold-responsive networks. \u003cem\u003eZmLOX2\u003c/em\u003e was approximately 70-fold upregulated in response to drought stress [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. Expression pattern in foxtail millet contrasted with rice for salt and drought condition [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Based on expression intensity, upregulation was observed for salt stress and under drought stress, downregulation was observed for \u003cem\u003eSiLOX2, SiLOX6, SiLOX8\u003c/em\u003e and \u003cem\u003eSiLOX9\u003c/em\u003e. In cotton [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e], most \u003cem\u003eLOX\u003c/em\u003e genes are associated with heat and salt stress; however, \u003cem\u003eGhLOX18\u003c/em\u003e was exclusively induced under cold stress, whereas some GhLOX genes exhibited altered expression in response to heat. Additionally, \u003cem\u003eCmLOX10\u003c/em\u003e of oriental melon significantly increases drought tolerance by mediating a JA biosynthesis pathway while only \u003cem\u003eCmLOX13\u003c/em\u003e was suppressed by high temperature [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003eCaLOX1\u003c/em\u003e of pepper showed strong tolerance under salinity and drought [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Under combined drought-salt stress, upregulation of sesame \u003cem\u003eSiLOX5\u003c/em\u003e was evident [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. Overall, drought and cold emerged as the most potent inducers of \u003cem\u003eOsLOX\u003c/em\u003es gene expression, while salt, saline-alkalinity, heat and IAA decreased expression. These findings suggest that \u003cem\u003eOsLOX\u003c/em\u003es are differentially regulated by abiotic stresses, with certain genes playing key roles in drought and cold stress adaptation, whereas others show stress-specific and time-dependent regulation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a thorough characterization of Lipoxygenase gene family in rice and their expression profiles under a variety of abiotic stress conditions. A total of twelve OsLOX members in rice were identified and categorized into three subfamilies according to structural similarities. These genes are located on seven different chromosomes and distinct conserved motifs are present. Through synteny analysis it was observed that genes underwent significant purifying selection and orthologous gene pairs evolved through segmental duplication. Notably, cis-regulatory elements analysis linked these elements to light, biotic and abiotic stress tolerance, hormonal regulation, cellular developments and core promoter associated elements. miRNA analysis helps understand the function of miRNA-modulated \u003cem\u003eOsLOX\u003c/em\u003e activity. Gene ontology analysis showed the functional activity of \u003cem\u003eOsLOX\u003c/em\u003es in different physiological processes, whereas PPI analysis identified homologous proteins with Arabidopsis signaling mileu. Expression profiling through RNA-seq data demonstrated \u003cem\u003eOsLOX\u003c/em\u003es tissue-specific expression and revealed the function of \u003cem\u003eOsLOX\u003c/em\u003es in response to various biotic and abiotic stresses. Further, the \u003cem\u003eOsLOX\u003c/em\u003es expression profiling under abiotic stress treatments was evaluated by RT-qPCR data and it revealed time-dependent differential regulation of \u003cem\u003eOsLOX\u003c/em\u003e genes, with drought and cold stress consistently inducing upregulation. In contrast, salt, saline-alkalinity, heat, and IAA treatments predominantly triggered sustained downregulation, with only a few transcripts showing transient or delayed induction. These findings expand the comprehension of the \u003cem\u003eLOX\u003c/em\u003e gene family, establish a basis for future functional investigations of the \u003cem\u003eOsLOX\u003c/em\u003e gene family and thereby offer molecular insights for enhancing rice resilience through genetic improvement and stress-tolerant variety development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eLOX\u003c/em\u003es\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipoxygenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePUFAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epoly unsaturated fatty acids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMeJA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethyl jasmonate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCREs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecis-regulatory elements\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNA-Seq data\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRNA (cDNA) Sequencing data\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFPKM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFragment Per Kilobase of exon per Million mapped read\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolyethylene glycol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative real time polymerase chain reaction.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant materials (\u003cem\u003eOryza sativa\u003c/em\u003e L.) used in this article were obtained from the Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh. All plant materials were provided free of charge and maintained in accordance with the international guidelines. This article does not contain any studies on human participants or animals and does not involve any endangered or protected species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or additional support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADT\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;MNH\u003c/strong\u003e contributed toward conceptualization and experimental design of this research. \u003cstrong\u003eADT\u003c/strong\u003e performed data curation, software handling, data analysis and interpretation, laboratory study, writing and preparing the original draft.\u003cstrong\u003e\u0026nbsp;MNH\u003c/strong\u003e and \u003cstrong\u003eJFR\u003c/strong\u003e handled software, investigation, and manuscript revision. \u003cstrong\u003eMHH and AC\u0026nbsp;\u003c/strong\u003econducted investigation and validation. \u003cstrong\u003eSHP\u0026nbsp;\u003c/strong\u003esupervised the project, provided scientific direction, edited and revised the final manuscript. All authors reviewed and approved the final manuscript prior to its submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude for the support from the Plant Genetic Engineering (PGE) Laboratory, Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh, which was instrumental conducting this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAzad M, Tohidfar M, Ghanbari Moheb Seraj R, Mehralian M, Esmaeilzadeh-Salestani K. Identification of responsive genes to multiple abiotic stresses in rice (Oryza sativa): a meta-analysis of transcriptomics data. 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The lipoxygenase gene AfLOX4 of Amorpha fruticosa L. is a potential regulator of drought stress tolerance pathways under saline and alkaline conditions. Acta Physiol Plant. 2023;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11738-023-03542-7\u003c/span\u003e\u003cspan address=\"10.1007/s11738-023-03542-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"LOX, Phylogeny, Cis-elements, Expression profiling, GO enrichment, PPI, Abiotic stress, RT-qPCR","lastPublishedDoi":"10.21203/rs.3.rs-8728944/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8728944/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLipoxygenases (\u003cem\u003eLOX\u003c/em\u003es) are enzymes that facilitate the oxygenation of polyunsaturated fatty acids (PUFAs) to create oxylipins through hydroperoxides that are involved in methyl jasmonate (MeJA) signaling pathway and are essential for seed germination, growth and development, defense mechanisms, and responses to stress. This study was designed for systematic analysis and exploration of expression patterns of the \u003cem\u003eLOX\u003c/em\u003e gene family of rice.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTwelve \u003cem\u003eLOX\u003c/em\u003e genes were identified, and phylogenetic and structural analyses grouped them into 9-LOXs, Type I 13-LOXs, and Type II 13-LOXs subfamilies with conserved lipoxygenase domains, motif and gene structure patterns. These genes were dispersed unevenly across seven chromosomes. Analysis of gene duplication found four tandem and segmental duplication events predominantly driving their moderate expansion, with Ka/Ks ratios suggesting purifying selection. Syntenic analysis across three genomes indicated relatively conserved roles of orthologous gene pairs of this family. The cis-acting regulatory elements revealed 46 CRE motifs responsive to light, cellular development, hormones and stress. Notably, 151 putative miRNAs were identified as potential post-transcriptional regulators. PPI analysis highlighted 10 key nodes with high interaction degrees. GO enrichment classified the functions of \u003cem\u003eOsLOX\u003c/em\u003es into biological, metabolic and cellular processes. RNA-seq expression profiling demonstrated \u003cem\u003eOsLOX\u003c/em\u003es expression across diverse tissues, developmental stages and stress. RT-qPCR data analysis further validated that \u003cem\u003eOsLOX\u003c/em\u003e genes exhibited temporally variable differential expression under multiple abiotic stress conditions including drought, salinity, saline-alkalinity, heat and cold, and in response to IAA hormonal treatment.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn conclusions, drought and cold stress significantly upregulated certain \u003cem\u003eOsLOX\u003c/em\u003es, enabling rice plants to confront these adverse conditions. This study will underscore the path for forthcoming in-vivo analyses to uncover the molecular processes of \u003cem\u003eLOX\u003c/em\u003e genes involved in tolerance to stress in rice.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Identification and Expression Analysis of LOX Gene Family in Rice (Oryza sativa L.) Under Abiotic Stress Conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 07:43:09","doi":"10.21203/rs.3.rs-8728944/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"288265629357924026932319635371323170478","date":"2026-02-21T02:45:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-20T03:15:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-02T07:38:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-01T22:37:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-01T22:35:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2026-01-29T07:52:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e9505a54-57e4-4437-b208-7eadf2b1606c","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T01:53:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 07:43:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8728944","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8728944","identity":"rs-8728944","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}