Identification and analysis of drought-responsive F-box genes in upland rice and involvement of OsFBX148 in ABA response and ROS accumulation

Identification and analysis of drought-responsive F-box genes in upland rice and involvement of OsFBX148 in ABA response and ROS accumulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification and analysis of drought-responsive F-box genes in upland rice and involvement of OsFBX148 in ABA response and ROS accumulation Yifan Wang, Fang Chen, Yuyang Chen, Kaiwen Ren, Dan Zhao, Kun Li, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4598345/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2024 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Background: Upland rice varieties exhibit significant genetic diversity and broad environmental adaptability, making them ideal candidates for identifying consistently expressed stress-responsive genes. F-box proteins typically function as part of the SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes to precisely regulate gene expression and protein level, playing essential roles in the modulation of abiotic stress responses. Therefore, utilizing upland rice varieties for screening stress-responsive F-box genes is a highly advantageous approach. Results: Through mRNA-seq analysis in the Brazilian upland rice (cv. IAPAR9), the research identified 29 drought-responsive F-box genes. Gene distribution and duplication analysis revealed these genes are distributed on 11 of the 12 chromosomes and 10 collinear gene pairs were identified on different chromosomes. 13 cis-elements or binding sites were identified in the promoters of the 29 drought-responsive F-box genes. These F-box proteins possess F-box domain and several other domains, and they are mostly unstable proteins with subcellular localization in cytoplasm, nucleus, chloroplasts, mitochondria and endoplasmic reticulum. Most of drought-responsive F-box genes exhibited expression in various tissues such as root, stem, leaf, leaf sheath and panicle except for OsFBO10 and OsFBX283 . These genes exhibited various responses to abiotic stresses such as osmotic, cold, heat, and salt stresses, along with ABA treatment. Importantly, a frame-shift mutation in OsFBX148 was created in the ZH11 variety, leading to altered ABA signal transduction and ROS accumulation. The study further elucidated the interaction of OsFBX148 with SKP1 family proteins OSK4/7/17 to form the SCF complex, dependent on the F-box domain. Conclusions: The research identified and analyzed 29 drought-responsive F-box genes in upland rice and provides valuable insights into the role of OsFBX148 in ABA and ROS responses. It establishes a basis for future exploration of F-box genes in improving resistance to abiotic stresses, especially drought. Upland rice abiotic stress transcriptome-wide identification OsFBX148 ABA response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background As sessile organisms, plants are constantly exposed to a variety of abiotic stresses, among which drought is one of the most severe factors adversely affecting plant growth, development, and productivity worldwide [1]. With climate change intensifying drought events in recent years [2], understanding the complex molecular mechanisms underlying plant drought responses has become a critical area of research. Plants have evolved complex regulatory networks to coordinate dynamic changes and cope with environmental challenges [3]. A key mechanism by which plants fine-tune their stress responses is through the selective proteolysis of regulatory proteins by the ubiquitin-proteasome system (UPS) [4]. In this system, ubiquitin is first activated by E1 activation enzymes, then transferred to E2 conjugation enzymes. E3 ligases use their specific substrate-binding domains to recognize and target proteins, catalyzing the covalent attachment of ubiquitin to lysine residues on these target proteins. Though this rapid and reversible process, plants can fine-tune the abundance of specific regulatory proteins to cope with drought stress [5-7]. F-box proteins are integral components of Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complexes in plants, which are responsible for the ubiquitination and subsequent proteasomal degradation of target substrate proteins. These complexes play crucial roles in diverse biological processes [8]. Within the SCF complex, F-box proteins interact with Skp1 proteins through their F-box domain [9]. The F-box domain acts as a recognition module, allowing the SCF complex to specifically bind to and ubiquitinate particular substrates [10]. By regulating gene expression or protein specific degradation within the SCF complex, F-box protein can precisely regulate plant response to abiotic stress. However, Skp1 possesses numerous members as a protein family, different F-box proteins may interact with different Skp1 members, thus, whether a particular F-box protein functions in SCF complex or which Skp1 proteins it interacts with still need to be deeply explored. F-box proteins also play a critical role in regulating abscisic acid (ABA) signal transduction. The phytohormone ABA coordinates a broad range of physiological and developmental processes and mediates responses to drought stress [11]. A key mechanism by which ABA exerts its regulatory effects is through the ubiquitin-proteasome system, specifically via the F-box protein family [12, 13]. In the context of ABA signaling, numerous F-box proteins have been identified as important regulators. For instance, the F-box protein TaFBA1 in wheat acts as a negative regulator of ABA signaling by physically interacting with the ABA receptor RCAR1 and the ABA-responsive transcription factor ABI5. Through these protein-protein interactions, TaFBA1 modulates the expression of ABA-inducible genes, thereby reducing the plant's sensitivity to ABA and its capacity to respond to drought stress [14]. Similarity, the Arabidopsis protein RIFP1, a member of the F-box E3 ligase family, interacts with the ABA receptor RCAR3 in the nucleus. This interaction increases the degradation rate of RCAR3, thereby disrupting the normal transduction of the ABA signal. Furthermore, mutation of the RIFP1 gene also leads to corresponding changes in the expression of ABA responsive genes. Therefore, RIFP1 is considered to be a negative regulator of ABA signal transduction [15]. Ongoing research on the complex interplay between F-box proteins, ABA, and the UPS continues to shed light on the dynamic regulation of stress tolerance in plants. Elucidating these regulatory mechanisms holds great promise for engineering crop plants with improved resilience to environmental challenges. However, as a superfamily, the F-box genes’ functions are not comprehensively revealed. A lot of work still need to do. Through genome-wide identification and analysis, F-box gene families have been characterized in a variety of plant species. In Arabidopsis thaliana, 694 F-box genes have been identified, with functional studies implicating these proteins in diverse processes such as plant development, hormone signaling, and biotic/abiotic stress responses [16]. Similarly extensive F-box gene families have been identified in other plant species. In rice ( Oryza sativa ), 779 putative F-box genes have been annotated [17], while in maize [18], soybean [19], apple ( Malus domestica ) [20], cotton ( Gossypium hirsutum. L) [21], Medicago truncatula [22], and tomato ( Solanum lycopersicum ) [23], 359, 517, 509, 592, 972 and 139 F-box genes were identified, respectively. The identification and comparative analyses of F-box gene families across diverse taxa have provided valuable insights into the evolution and functional diversification of this important protein family. As more genome sequences become available, future studies will undoubtedly uncover additional F-box genes and elucidate their roles in various cellular processes and organismal adaptations. However, few studies have been reported about the F-box genes in upland rice. Upland rice, also known as "dryland rice", is adapted to grow in rainfed, non-flooded conditions, requiring significantly less water than its lowland counterparts. Except for the differences in morphological and biochemical traits, upland rice and lowland rice varieties exhibit distinct patterns of gene expression when exposed to drought stress [24-26]. Therefore, utilizing upland rice materials to screen and identify genes related to drought regulation is highly feasibility. In this study, we used the upland rice variety IAPAR9 for drought treatment followed by transcriptome sequencing, identifying 29 drought-responsive F-box genes. We then performed comprehensive analysis on these 29 genes, including phylogenetic tree analysis, gene structure prediction, characterization of chromosome distributions, cis-element analysis of gene promoters, tissue-specific expression profiling, and expression profiling under abiotic stresses. Additionally, we conducted preliminary functional studies on the selected gene OsFBX148 . This work lays a strong foundation for further research on functions of F-box genes in rice, particularly in the context of drought stress response. The insights gained from this study will facilitate the development of rice varieties with improved drought resistance. Results Identification, phylogenetic analysis and chromosomal location of the drought-responsive F-box genes mRNA-seq was conducted using upland rice IAPAR9 following drought treatment in our previous study (accession number SRP273943), followed by functional annotation of differentially expressed genes. To identify the drought-responsive F-box genes in rice, we performed further analysis of the transcriptome data. A total of 36 differentially expressed F-box genes were initially screened. However, protein domain analysis revealed that seven of these genes lacked the F-box domain. Therefore, these seven genes were excluded. Consequently, we identified 29 drought-responsive F-box genes in IAPAR9 (Table S1) . To investigate the phylogenetic relationships of these 29 F-box proteins between rice and other plants, the amino acid sequences of these proteins were used as queries for a BLAST search against GenBank to retrieve homologous sequences from all species. An unrooted phylogenetic tree was then constructed based on the alignments (Fig. 1) . The BLAST analysis showed that most of the F-box proteins have highly homologous genes within the rice genome, and these homologous genes are mostly F-box proteins as well, indicating a high level of homology among the F-box proteins. This may be due to the large number and relatively high conservation of F-box proteins in rice [17]. Furthermore, the homologs of these 29 F-box proteins were found exclusively in plants, not in animals or microorganisms, suggesting that these F-box proteins are plant-specific. Among these 29 F-box genes, the homologous genes of OsFBX342 , OsFBX150 , OsFBK8 , OsFBX261 , OsFBX281 , OsFBD11 , OsFBX283 , OsFBX258 , OsFBX438 , OsFBX345 , OsFBX44 , OsFBX315 , OsFBX220 , OsFBDUF13 , OsFBX31 , OsFBX40 , OsFBXPP2-B1 , OsFBX357 and OsFBX109 exist only in Oryza plants, indicating their high conservation within this genus. Additionally, OsFBO10 has 73 homologs across diverse plant species, indicating a widespread presence in the plant kingdom. OsFBL28 has 13 homologs, OsFBX281 has 14 homologs, OsFBX283 has 14 homologs, OsFBL27 has 11 homologs, and OsFBX148 has 23 homologs. The gene with the fewest homologs has only 2 (Table S2) . These results together suggest that F-box proteins are highly conserved and plant-specific, indicating their potential roles in specific plant functions. The 29 drought responsive F-box genes are distributed on 11 of the 12 chromosomes in rice (Fig. 2a) . The number of F-box genes varies drastically across the chromosome, ranging from 0 to 5. Chromosomes 2 and 4 have largest number of F-box members, with five genes (17.24%) each. Chromosomes 6, 8 and 11 contain three genes (10.34%). There are two genes (6.90%) located on chromosomes 3, 7, 9 and 10, respectively. Only one gene (3.45%) is located on chromosome 1 and chromosome 12. Notably, no drought responsive F-box genes are found on chromosome 5. These results suggest that the drought responsive F-box genes are unevenly distributed across the rice chromosomes. Gene duplication is considered a major driver of genomic innovation and the emergence of novel biological features and capabilities in organisms [27, 28]. The additional genetic material provided by duplicated genes allows for the exploration of new functional possibilities through mutation and selection, ultimately expanding the coding and regulatory capacity of the genome. To investigate the presence of gene duplication events among the 29 F-box genes, we performed segmental duplication analysis. A total of 10 collinear gene pairs were identified on different chromosomes, indicating segmental duplication events (Fig. 2b) . Chromosome 4 had the highest number of duplicated F-box gene pairs, with seven pairs. This is followed by three pairs on chromosome 2, two pairs each on chromosomes 3, 11 and 12, and one pair each on chromosomes 7, 8, 9, and 10 (Table S3) . These findings show that segmental duplication has played an important role in the expansion of the F-box family genes in rice. Analysis of basic physicochemical properties and domain of drought-responsive F-box genes The physical and chemical properties of drough-responsive F-box proteins were analyzed using ExPasy (https://web.expasy.org/protparam/). This analysis included parameters such as amino acid length, isoelectric point (PI), protein molecular weight (MW), protein instability index, and grand average of hydropathicity (GRAVY) (Table 1) . The results showed significant variability in the protein sequences of different F-box proteins. Most F-box proteins had an amino acid length of less than 1000 residues, except for OsFBX150 and OsFBX345. Among these, OsFBX150 exhibited the longest amino acid length at 1526 residues with a molecular weight of 159241.75 Da, while OsFBX77 had the shortest length with only 178 residues and a weight of 18611.57 Da. The isoelectric point ranged from 5.27 (OsFBX44) to 10.74 (OsFBX357), with 15 F-box proteins having acidic points below 7 and the remaining 14 being relatively alkaline. The protein instability index, an important indicator of protein stability, indicated that most drought-responsive F-box proteins were unstable, except for OsFBDUF13 and OsFBX148, with instability indices of 38.71 and 31.67, respectively. This instability may be linked to their role in dynamically regulating target proteins under drought conditions. The hydrophilicity analysis revealed that 23 F-box proteins had a GRAVY value below 0, indicating a hydrophilic nature, while the remaining 6 F-box proteins were hydrophobic with a GRAVY value above 0. Furthermore, WoLF PSORT was used to predict the subcellular localization of these drought-responsive F-box proteins. Apart from OsFBL27, which localizes to the endoplasmic reticulum, and OsFBX109, which localizes to the mitochondria, the other F-box proteins were found to be localized in the nucleus, cytoplasm, and chloroplasts. Phylogenetic relationships, conserved motifs identification, conserved domains analysis, and gene structure analysis were conducted on the complete sequences of 29 F-box proteins (Fig. 3) . A total of 9 conserved motifs were identified in these sequences. Notably, Motif 2 was present in all F-box proteins (Fig. 3b) , indicating its role as the F-box domain and potential importance in their function. Motif 1 was found in all F-box proteins except for OsFBXPP2-B1 and OsFBX563, suggesting a relatively conserved nature compared to motif 2. Additionally, motif 5 was unique to OsFBX261, OsFBXPP2-B1, OsFBX438, and OsFBX148, while motif 4 was specific to OsFBK8 and OsFBK25, which clustered together phylogenetically, possibly indicating specialized physiological functions. Although motif 3 was shared among evolutionarily related members OsFBX261, OsFBXPP2-B1, OsFBX438, and OsFBX148, it was also present in OsFBX345. Similarly, motif 9 showed a similar pattern, predominantly found in OsFBX261, OsFBXPP2-B1, and OsFBX438, but also in OsFBX281, suggesting both universal and specific functions for these motifs. Furthermore, motifs 6, 7, and 8 exhibited irregular distribution among different genes, indicating potential diversification of function among the F-box proteins. The F-box domain located at the N-terminal of the SCF complex is evolutionarily conserved [17]. The analysis revealed that all 29 F-box proteins possessed this conserved domain (Fig. 3c) . Additionally, 11 other domains were identified, including the FBD, Ketch and Tub domains (Fig. 3c) . FBD domain-containing F-box proteins in Arabidopsis were found to play roles in ethylene/abscisic acid signaling, plant–pathogen interactions, and root development [29]. The Ketch domain, which is the most abundant type of F-box proteins, has various functions in plants, such as regulating seed germination, development, the phenylpropanoid pathway, ultraviolet radiation tolerance, drought and heat stress resistance, and root architecture [9, 30-32]. The Tub domain, named after the TUBBY protein in mice, binds specifically to negatively charged phosphoinositide species and is involved in ABA, abiotic stress, and immunity regulation in plants [33-37]. Other domains like DUF, KIX, and PP2 were also found in F-box proteins. The identification of multiple domains suggests that F-box proteins in rice may have diverse mechanisms and functions. The structures of the exons, introns and untranslated regions (UTRs) of the 29 F-box genes were visualized by use of TBtools software (Fig. 3d) . The results showed that the closer the genetic relationship and phylogenesis, the more similar the gene structure. Cis-elements in the promoters of F-box genes in response to drought stress The transcriptional expression of genes is to some extent regulated by cis-elements located in the promoter regions upstream of the encoding genes [38]. Thus, the 2-kb putative promoter regions of the 29 F-box genes were analyzed using the online database PlantCARE. A total of 13 cis-elements or binding sites were identified, including important elements involved in abiotic stress responses such as dehydration, low temperature, and salt stresses. Additionally, elements involved in abscisic acid responsiveness and MYB binding site associated with drought inducibility were identified (Fig. 4) . Apart from core promoter elements and common cis-acting element detected in most F-box gene promoters, other cis-elements related to plant growth and development were also discovered. These included regulatory elements associated with meristem specific activation, differentiation of palisade mesophyll cells, and meristem expression. These results assuredly suggest that the 29 drought-responsive F-box genes are not only involved in drought stress regulation, but also in other abiotic stress and various aspects of plant growth and development. Expression patterns analysis of F-box genes in response to drought stress in different tissues Studying the tissue-specific expression of genes helps predict their functions in unique tissue. This analysis can provide insight into the regulatory mechanisms of genes, contributing to a better understanding of gene regulation throughout the plant. In a study analyzing the expression patterns of 29 drought-responsive F-box genes, qRT-PCR was conducted using total RNA extracted from various tissues of rice, including root, stem, leaf, leaf sheath and panicle. The results revealed significant differences in expression levels among the F-box genes across different tissues (Fig. 5) . Notably, the expression of OsFBO10 and OsFBX283 could not be detected by qRT-PCR, suggesting their low expression level and potentially minor functional roles. The remaining 27 F-box genes were all successfully detected. Among these, OsFBX148 , OsFBX31 , OsFBDUF13 , OsFBX77 , OsFBX438 , OsFBX315 and OsFBX150 exhibited predominant expression in the root compared to other tissues. OsFBX40 , OsFBX281 , OsFBX563 , OsFBD8 and OsFBX220 showed relatively higher expression levels in the stem compared to other tissues. In rice leaf, eight genes, OsFBX357 , OsFBXPP2-B1 , OsFBL28 , OsFBT6 , OsFBL27 , OsFBK8 , OsFBX261 and OsFBX258 , were found to be expressed more than in other tissues. Additionally, OsFBX132 and OsFBX109 showed higher expression levels in the leaf sheath, while OsFBX44 and OsFBD11 were more expressed in the panicle. Several F-box genes exhibited relatively high expression levels in multiple tissues. For instance, OsFBX281 showed high expression in both the stem and panicle, while OsFBX563 had high expression in the stem, leaf, leaf sheath, and panicle. Furthermore, OsFBX132 was expressed in both the stem and leaf sheath, and OsFBX44 had high expression levels in both leaf and panicle. These results suggest that although these F-box genes are responsive to drought stress, they may have distinct regulatory roles in various plant tissues. Expression profiles of F-box genes in response to drought stress to abiotic stress as well as ABA treatment To validate the accuracy of transcriptional expression changes for the drought-responsive genes, identified in mRNA-seq data, 6-week-old rice plants were grown under normal conditions and then subjected to drought treatment. Leaf samples were collected under moderate and severe drought conditions for further analysis. Subsequent qRT-PCR experiments revealed that the expression patterns of most genes were consistent with the transcriptome sequencing data (Fig. 6a) . However, genes such as OsFBX40 , OsFBX345 , OsFBX261 , and OsFBK25 exhibited expression changes that contradicted the mRNA-seq results. This discrepancy could potentially be attributed to variations in rice growth stages and cultivation environments. The study once again confirmed that these 29 drought-responsive genes are indeed influenced by drought stress. Previous research has indicated that F-box proteins are involved in various abiotic stresses, such as hyperosmotic, salt, high and low temperature stress in plants [39]. To investigate whether the transcriptional expression of the 29 drought-responsive F-box genes can be influenced by other abiotic stress factors, ZH11 seedlings were individually subjected to PEG, cold, heat and NaCl treatments, followed by qRT-PCR experiments. The expression of OsFBO10 and OsFBX283 genes could not be detected even with qRT-PCR methods, which aligns with the results of tissue-specific expression analysis where the expression of these two genes was also undetectable. Under hyperosmotic stress induced by PEG-6000 treatment, the transcript levels of 26 F-box genes were altered, except for OsFBD11 (Fig. 6b) . Among these, 2 genes were downregulated and 15 genes were upregulated at three different time points. 19 genes were upregulated at both the 3-hour and 6-hour time points post-treatment, with the majority reaching peak expression levels after 6 hours, indicating their potential roles in the early stages of osmotic stress response. These findings suggest that F-box genes may indeed be involved in responses to hyperosmotic stress. Cold stress served as an abiotic factor that led to an increase in the expression of 18 F-box genes after 3- and 6-hour treatment periods (Fig. 6c) . Out of these genes, 13 showed sustained upregulation, while 4 displayed continuous downregulation. The expression patterns of the remaining 5 genes fluctuated without any clear trends. These findings indicate that the F-box genes may be involved in the regulation of cold stress. 27 F-box genes were impacted by heat stress induced by a 42℃ treatment (Fig. 6d) . Out of these, the expression of 19 genes was consistently upregulated throughout all three time points of treatment, while the expression of 5 genes was consistently downregulated across the same time points. Specifically, OsFBX77 and OsFBX281 showed initial upregulation at 3- and 6-hour treatment conditions, followed by a return to levels similar to the control. On the other hand, OsFBD11 exhibited a slight decrease after the 3-hour treatment, followed by a subsequent increase of 1.5-fold. These findings suggest that these genes may play a crucial role in the ongoing regulation of heat stress response. Under salt stress treatment, all 27 F-box genes showed changes in expression levels (Fig. 6e) . Fourteen genes displayed sustained upregulation at all three time points, while three genes exhibited sustained downregulation. The majority of upregulated genes reached their peak expression levels at the 6-hour time point. Eight genes initially showed downregulation after 3 hours of NaCl treatment, but their expression was upregulated after 6 hours, with fold changes exceeding 2-fold. These findings highlight the intricate regulation of gene expression under salt stress, with potential involvement of F-box genes. ABA, an essential plant hormone, plays a pivotal role in seed germination, growth, development, and response to abiotic stresses [11]. Analysis of cis-elements in F-box gene promoters revealed the presence of ABA-responsive elements. Subsequent qRT-PCR experiments confirmed that the transcriptional expression of most F-box genes was upregulated in response to ABA, with the exception of OsFBX345 (Fig. 6f) . While OsFBXPP2-B1 and OsFBX281 initially decreased and then increased, OsFBK8 and OsFBX258 exhibited an initial increase followed by a decrease. Notably, OsFBX150 showed no consistent trend in expression levels. These findings suggest that these drought responsive F-box genes, at least part of them, may regulate stress responses in an ABA-dependent manner. Under various abiotic stresses, the expression of OsFBX132 , OsFBX148 , OsFBX31 , OsFBDUF13 , OsFBD8 , OsFBX44 , OsFBL27 , OsFBX261 , and OsFBK25 genes was upregulated, while OsFBK8 and OsFBX258 genes were downregulated. This indicates that these genes may have similar and significant regulatory roles under different abiotic stress conditions. ABA, as a broad-spectrum plant antistress hormone, is crucial in regulating plant stress responses. The study revealed that 21 genes were induced and up-regulated by ABA, with 12 of them showing up-regulation by nearly 10-fold or more. This implies that these genes are likely regulated or at least partially in an ABA-dependent manner to control stress responses. Most of the drought-responsive F-box genes identified are believed to have important regulatory roles in managing plant responses to abiotic stresses, highlighting the essential and critical function of F-box genes in regulating plant responses to such stresses. OsFBX148 is a member of SCF complex and involved in ABA response and ROS accumulation of rice The above studies have demonstrated that OsFBX148 is significantly up-regulated in response to drought and ABA treatment, as evidenced by transcription data and qRT-PCR analysis. In order to further explore its biological role, a mutant allele of Osfbx148 was generated by inserting a nucleotide T into the first exon of the gene (Fig. S1) . Uniformly germinated ZH11 and Osfbx148 seeds were cultivated on half-strength MS medium supplemented with 2.5 and 5 μM ABA, respectively, for a period of 4-5 days. Under normal conditions, there was no observable difference between ZH11 and Osfbx148 . However, upon ABA treatment, it was noted that the root and shoot length of Osfbx148 were notably shorter compared to ZH11 (Fig. 7) . These findings suggest that OsFBX148 plays a role in ABA response and acts as a negative regulator of ABA signal transduction. Exposure of plants to abiotic stress leads to a significant increase in reactive oxygen species (ROS) levels within the cell. To investigate the function of OsFBX148 in controlling ROS accumulation, experiments were carried out using detached leaves from 40-day-old ZH11 and Osfbx148 plants. The leaves were subjected to treatments with ABA, NaCl, and H 2 O 2 , followed by staining with DAB and NBT (Fig. 8) . The results revealed that leaves carrying the Osfbx148 mutant allele exhibited a deeper coloration when exposed to ABA and NaCl. Interestingly, when treated with H 2 O 2 , only DAB staining resulted in color change, while NBT staining did not, suggesting an accumulation of more hydrogen peroxide than singlet oxygen under H 2 O 2 treatment. These findings suggest that Osfbx148 plants had higher ROS levels compared to ZH11 plants, indicating the role of OsFBX148 in ROS regulation and increased ROS accumulation in Osfbx148 mutants under stress conditions. The study investigated the role of OsFBX148, an F-box protein, in the formation of the SCF complex responsible for specific substrate binding through interaction with SKP1. A yeast two-hybrid experiment was conducted to assess the interaction between OsFBX148 and the SKP1 protein, using 6 randomly selected OSKs proteins (OSK1/4/7/15/16/17). Results indicated that OsFBX148 could interact with OSK4/7/17 (Fig. 9a) , but this interaction was lost upon truncation of the F-box domain of OsFBX148 (Fig. 9b) . These findings suggest that OsFBX148 may function by contributing to the formation of the SCF complex. Overall, OsFBX148 plays a role in ABA response and ROS accumulation in rice through its involvement in the SCF complex. Discussion As the substrate-recognition components of SCF E3 ubiquitin ligase complexes, F-box proteins are responsible for targeting specific substrates for ubiquitination and subsequent degradation by the 26S proteasome. They play crucial roles in various cellular processes, including hormone signaling, development, and stress responses [8]. Therefore, the identification and characterization of F-box genes have been a focus of extensive research in the plant kingdom. The availability of numerous plant genome sequences has enabled the systematic identification and comparative analysis of F-box genes across diverse plant species. One of the earliest and most comprehensive studies on plant F-box genes was conducted in the model plant Arabidopsis thaliana, which identified a total of 694 F-box genes, making it one of the largest F-box gene families among eukaryotes [16]. Similar genome-wide surveys have been carried out in other major plant species. These studies have consistently found that the F-box gene family is significantly expanded in plants compared to other eukaryotes, with the number of F-box genes ranging from several hundred to over a thousand, depending on the plant species [21]. Transcriptome analysis, enabled by RNA sequencing (RNA-seq), provides a comprehensive view of the genes expressed in a plant under specific environmental conditions. By comparing the transcriptional profiles of plants exposed to stress treatments versus control conditions, researchers can identify differentially expressed genes (DEGs) that are induced or repressed in response to the stress [40]. These DEGs often include members of gene families known to play important roles in stress tolerance, such as transcription factors, kinases, transporters, and antioxidant enzymes. In this study，we screened 29 differentially expressed F-box genes by RNA-seq using upland rice IAPAR9, indicating that several F-box genes were responsive to drought stress. This result is consistent with many previous reports that F-box genes are involved in the regulation of plant drought stress response [8]. Analyzing the physicochemical properties and structural features of proteins can help predict their potential biological functions. Our analysis of the physicochemical properties of 29 F-box proteins showed that the pI values of these proteins range from 5.27 to 10.74, and their molecular weights, except for OsFBX77 and OsFBX148, are mostly greater than 40 kD, which is generally consistent with previous reports [41]. However, through further analysis of the instability indices of these 29 proteins, we found that the instability indices, except for OsFBDUF13 and OsFBX148, are all greater than 40, indicating that these proteins have relatively low stability. This may be related to their rapid response to drought stress and their functions in signal transduction regulation [42]. Unstable proteins are more easily degraded, which allows the cell to quickly modulate the abundance of these critical proteins to respond to changes in external drought stress [43]. This dynamic regulation facilitates the plant's rapid perception and reaction to environmental stresses. The subcellular localization analysis showed that the 29 F-box proteins are localized in the cytoplasm, nucleus, chloroplasts, mitochondria, and endoplasmic reticulum, which is consistent with previous reports [14, 15, 44, 45]. The F-box domain is a conserved structural domain of F-box proteins, usually located at the N-terminus. The C-terminal domain is related to the specific binding of its substrates, reflecting the functional specificity of different F-box proteins. The analysis results show that all 29 F-box proteins contain the conserved F-box domain, but not every protein's C-terminus contains other structural domains. This may be because the F-box domain itself can be sufficient to mediate the recognition and binding of specific substrates [19]. It's also possible that some F-box proteins lack additional structural domains because they rely on interactions with other proteins to determine substrate specificity, rather than requiring extra structural domains. Cis-acting elements in the promoters of the 29 drought-responsive F-box genes were also analyzed in rice. We identified elements related to the regulation of plant growth, development, and abiotic stress responses, such as drought, low temperature, high salinity, and ABA response elements. Based on this, the present study used various abiotic stress treatments including drought, osmotic, high salinity, high temperature, low temperature, and ABA to investigate whether these 29 genes responded to the above treatments. The results showed that except for 2 genes, OsFBX10 and OsFBX283, which could not be detected, the remaining 27 F-box genes were responded to the treatments. This suggests that these F-box genes may indeed play a role in regulating abiotic stress responses, which is generally consistent with previous research [11]. The gene expression profile reveals the potential functions of the gene in different plant tissues. Therefore, we investigated the tissue expression of the 29 F-box genes, and the results showed that 27 of the genes were expressed in the roots, stems, leaves, sheaths, and panicles of rice. This is consistent with the previously reported expression patterns of F-box genes in different rice tissues. As a component of the SCF complex, F-box proteins are able to interact with SKP1 and determine the specific substrate binding and degradation of this complex, thereby regulating plant growth, development, stress response and signal transduction [46, 47]. This study found that OsFBX148 interacts with the SKP1 proteins OSK4/7/17 to form an SCF complex, which is consistent with previous reports [9]. In this study, we found that the promoter of the OsFBX148 gene contains two ABA-responsive cis-acting elements, suggesting that this gene may play a role in regulating ABA responses. The qRT-PCR detection results also confirmed this conclusion. Previous studies have reported that F-box proteins can interact with key proteins in the ABA signaling pathway, such as PYL and ABI5, to regulate the ABA signaling pathway and thereby control plant responses to ABA. The results of the phenotypic study in this research show that the response of OsFBX148 gene mutant seedlings to ABA is enhanced, indicating that OsFBX148 negatively regulates ABA signal transduction. Thus, we identified a novel F-box protein factor involved in the regulation of ABA signaling and provided new insights into the molecular mechanisms of plant ABA signal regulation. Conclusions The study identified 29 drought-responsive F-box genes from the upland rice variety IAPAR9, and analyzed the evolutionary relationships, chromosomal distributions, gene duplication events, promoter cis-acting elements, as well as the physicochemical properties and structural domains of the encoded proteins. Homologous analysis revealed that most homologous proteins of 29 drought-responsive F-box proteins were also F-box-containing proteins and they were exclusively exsist in plants. Ten collinear gene pairs were identified, indicating the numerous members in F-box superfamily may be related to segmental duplication. Thirteen cis-acting elements or binding sites were identified, suggesting their roles in ABA and abiotic stress responses. Function analysis of FBD, Ketch, Tub and any other domains found in the 29 drought-responsive F-box proteins revealed their roles in hormone signal transduction and stress responses. The physicochemical properties analysis also revealed the instability of drought-responsive F-box proteins and this may be linked to their role in dynamically regulating target proteins under drought conditions. Gene expression analysis showed that 27of 29 drought-responsive F-box genes are expressed in root, stem, leaf, sheath and panicle and they can response to drought, cold, heat, salt and ABA. Furthermore, a preliminary functional analysis of OsFBX148 was conducted, revealing that OsFBX148 interacts with OSK4/7/17 to form an SCF complex, and plays a role in regulating ABA signal transduction and ROS accumulation in plants. These findings lay the foundation for further exploring the functions of F-box proteins in stress responses. Materials And Methods Plant Materials The Brazilian upland rice (cv. IAPAR9) was used for drought treatment and RNA sequencing. The ZhongHua11 (ZH11) rice variety was used for total RNA extraction and gene expression analysis, as well as tissue-specific expression analysis, after various abiotic stress treatments. ZH11 and Osfbx148 were used for function characterization of OsFBX148. Identification of drought-responsive F-box genes in upland rice To identify drought-responsive genes, the upland rice variety IAPAR9 was subjected to drought stress. Leaf tissue was collected for total RNA extraction followed by transcriptome sequencing. Differentially expressed genes affected by drought stress were analyzed, and the function annotating was also performed. Among these differentially expressed genes, 36 putative F-box genes were found by searching “F-box” as key words. Structural domain analysis was then conducted on these 36 genes through CD-search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to identify their conserved domains and exclude those without the F-box domain. Cis-elements analysis in the promoters of drought responsive F-box genes The 2000 base pairs promoter sequences upstream of ATG start codon of 29 F-box genes were extracted from the rice genome sequence and submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) for cis-elements analysis. The files obtained from the PlantCARE website were subjected to an advanced filtering process, with Arabidopsis thaliana selected as the species and existing cis-acting elements chosen. The data was then visualized using the TBtools software for further analysis. Phylogenetic tree analysis In order to gain more insight into the evolutionary relationships of the 29 F-box proteins, the amino acid sequences of these proteins were submitted to Protein BLAST in NCBI BLAST (https://www.ncbi.nlm.nih.gov/) to retrieve homologous protein sequences from other species. Eventually, only the sequences sharing more than 85% similarity were used for subsequent analysis. The resulting 250 protein sequences from different varieties were compared with the amino acid sequences of the 29 F-box proteins in rice using MEGA11 software (https://www.megasoftware.net/) and a phylogenetic tree was constructed by NJ (Neighbor-Joining) method. Later, FigTree v1.4.3 software (https://www.softsea.com/review/FigTree.html) was used to optimize the phylogenetic tree, such as font format and branch color differentiation, so as to obtain a more intuitive phylogenetic tree and make an in-depth analysis of the evolutionary relationship of these F-box proteins. Chromosomal localization The positional information of these 29 F-box genes on chromosomes was obtained by analyzing the rice gene annotation file. Subsequently, the advanced gene localization visualization feature of TBtools software was utilized to plot the position map of these genes on chromosomes. Furthermore, the gene structure was displayed using the advanced gene structure view feature of TBtools, based on the genome annotation files and obtained NCBI CD-Search conserved domains. Phylogeny and domain analysis The amino acid sequences of the 29 F-box proteins were aligned by MEGA 11 software, and simple phylogenetic trees were constructed using maximum likelihood. The conserved motifs of these 29 F-box proteins were retrieved by online tool MEME (http://meme-suite.org/tools/meme) with the selected MEME motif number set to 10. The obtained results were downloaded for subsequent analysis. The phylogenetic trees, gene annotation files, protein sequence files and MEME motifs files of these 29 F-box proteins were visualized through the Gene Structure View tool in TBtools software. The final graphs were modified in detail using Adobe Illustrator 2021 software to more intuitively present the evolutionary relationships, gene structure, protein motifs and conserved domains of these proteins. Physicochemical properties and subcellular localization prediction The molecular weight (MW), isoelectric point (pI), grand average of hydropathicity (GRAVY), and instability index of the 29 F-box proteins were predicted and analyzed using the ExPASy website (https://web.expasy.org/protparam/). And the subcellular localization of F-box proteins was predicted by WoLF PSORT (https://wolfpsort.hgc.jp/). Seedlings treatment and gene expression analysis To study the effects of different abiotic stress factors on the expression of F-box genes (the primer sequences are listed in Table S4 ), 2-week-old ZH11 seedlings grown hydroponically in growth chamber were separately treated with 20% PEG6000, 4°C, 42°C, 150 mM NaCl and 30 μM ABA. Shoot samples were collected at 0, 3 h, 6 h, and 12 h for total RNA isolation and the following gene expression analysis. Total RNA was isolated using the traditional Trizol reagent method with minor modifications [48] . Briefly, samples were ground in liquid nitrogen-cooled mortar, stirred well with 1 mL Trizol reagent in RNase-free 1.5 mL centrifuge tubes, incubated at room temperature for 5 min, and then centrifuged at 12,000 rpm for 10 min. The supernatant was mixed with 200 μL trichlormethane, and centrifuged at 12,000 rpm for 15 min. 500 μL of the upper aqueous phase was mixed with an equal volume of isopropanol, incubated at -20°C for 30 min, and then centrifuged at 12,000 rpm for 10 min. The pellet was washed with 1 mL 75% ethanol and resuspended in 30 μL DEPC-treated water. RNA quality and concentration were detected using NanoDrop 2000 (Thermo Fisher Scientific, USA). 2 μg of total RNA was used for reverse transcription using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Shiga, Japan). Quantitative PCR was then performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), with the reaction mixture containing 5 μL 2× ChamQ Universal SYBR qPCR Master Mix, 0.4 μL each of forward and reverse primers, 1 μL cDNA template, and 3.2 μL water in a total volume of 10 μL. The qRT-PCR was performed on an ABI StepOne Plus (Thermo Fisher Scientific, USA) with the following program: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s, with a melting curve analysis using the instrument's default program. Relative gene expression level was calculated using the 2-ΔΔCT method [49]. Stress treatment and ROS staining For detached leaves treatment, leaves from 40-day-old rice plants grown in soil under normal conditions were excised into 1-2 cm pieces and placed in MS liquid medium containing 5 μM ABA, 150 mM NaCl, or 1% H 2 O 2 , and incubated for 3 days under normal growth conditions. For germinated seeds treatment by ABA, simultaneously harvested and dried ZH11 and Osfbx148 seeds were placed in Petri dishes with moistened filter paper and germinated in the dark at 28°C for 2 days. Seedlings with uniform radicle length were selected and transferred to MS liquid medium containing 2.5 μM or 5 μM ABA, and grown under normal conditions (28°C, 16 h light/8 h dark) for 4 days. Photographs were taken, and the shoot and root lengths were measured. For DAB and NBT staining, DAB and NBT were prepared in distilled water to a working concentration of 1 mg/mL. The leaves of ZH11 and Osfbx148 before and after treatment were submerged in the DAB or NBT solution and vacuum-infiltrated overnight. The solutions were then discarded, and the leaves were decolorized in a graded ethanol series (100%, 90%, 75%) for 10 minutes each until the chlorophyll was completely removed. Photographs were taken after decolorization. Yeast two hybrid The CDS sequences of OsFBX148 and OSKs were respectively cloned into the pPGBKT7 and pGADT7 vectors. The two constructs were then co-transformed with salmon sperm DNA into AH109 yeast competent cells. The transformed cells were plated on SD2 yeast medium and incubated at 28°C for 2 days. Single colonies were then picked, serially diluted, and spotted on SD4 medium. Yeast growth was observed and photographed. To study the effect of the F-box domain on the interaction between OsFBX148 and OSKs, the OsFBX148 sequence with the F-box domain deleted was cloned into pPGBKT7 and co-transformed with OSKs into AH109 yeast competent cells. The remaining steps were the same as above. Abbreviations SCF: SKP1-CUL1-F-box; ZH11: ZhongHua11; qRT-PCR: quantitative real-time PCR; ROS: reactive oxygen species; UPS: ubiquitin-proteasome system; ABA: abscisic acid; PI: isoelectric point; MW: molecular weight; GRAVY: grand average of hydropathicity; UTR: untranslated region; DAB: diaminobenzidine; NBT: nitrotetrazolium blue chloride; Y2H: yeast two-hybrid; DEG: differentially expressed gene; NJ: neighbor-joining; NCBI: national center for biotechnology information Declarations Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Conflicts of Interest : The authors declare no conflict of interest. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements: Not applicable. Funding: This work was supported by the National Natural Science Foundation of China (32360075), the Scientific Research Project of Department of Science and Technology of Hubei Province (2022CFB694), "Yazhou Bay" Elite Talent Science and Technology Program of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2023-35), the Incubation Project of Hubei Minzu University (PY22005). The research was finished in Hubei Key Laboratory of Biological Resources Protection and Utilization and Yazhou Bay Seed Laboratory of Hainan Province. Author Contributions: Y.L designed the research. Y.W, F.C, X.W, and Y.C completed the bioinformatics analysis in this research. Y.L and Y.W conducted the qRT-PCR experiment. F.C, K.R and D.Z conducted ABA response, ROS accumulation and Y2H experiments. Y.L, Z.X, Z.H and Q.T dealed with the figures. Y.L wrote the main manuscript text. M.P and Q.F polished the manuscript. Y.Z revised the work. K.L and H.L provided reasonable advices. 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Tables Table 1 The basic physicochemical properties of drought-responsive F-box proteins (*Grand Average of Hydropathicity) Protein Name Length(aa) pI MW(Da) Instability index Subcellular localization GRAVY * OsFBX342 415 6.75 45858.32 41.77 Cytosol -0.089 OsFBX150 1526 7.56 159241.75 52.23 Cytosol -0.114 OsFBX77 178 9.39 18611.57 47.80 chloroplast 0.185 OsFBK8 391 5.39 40366.16 48.52 nucleus -0.162 OsFBL28 276 10.48 31021.08 60.61 chloroplast -0.245 OsFBX261 299 5.3 31611.02 51.7 Cytosol -0.064 OsFBO10 509 6.60 56258.32 54.11 nucleus -0.209 OsFBT6 462 9.35 51529.37 61.74 nucleus -0.495 OsFBX281 463 5.75 51844.52 59.04 nucleus -0.163 OsFBX563 677 8.46 73677.52 44.98 nucleus 0.188 OsFBD11 526 8.91 57806.71 42.88 Cytosol 0.121 OsFBX283 541 8.37 61342.95 58.67 nucleus -0.236 OsFBX258 406 5.41 42650.39 47.20 Cytosol -0.011 OsFBX438 302 5.68 34389.78 43.93 nucleus -0.394 OsFBD8 475 9.28 51705.91 57.37 nucleus 0.034 OsFBX345 1078 5.51 123349.83 43.55 chloroplast -0.136 OsFBX44 422 5.27 47173.74 64.42 Cytosol -0.255 OsFBK25 383 6.11 40796.99 47.69 Cytosol -0.192 OsFBX315 447 9.69 49837.08 55.97 chloroplast -0.286 OsFBX220 438 5.81 48728.64 40.16 nucleus -0.108 OsFBL27 720 5.33 79222.78 55.31 Endoplasmic reticulum -0.010 OsFBDUF13 377 7.13 41363.62 38.71 Cytosol -0.347 OsFBX148 202 6.89 21999.98 31.67 nucleus -0.274 OsFBX31 362 6.50 39846.30 52.41 Cytosol -0.171 OsFBX40 439 7.18 48691.19 46.82 Cytosol 0.045 OsFBXPP2-B1 323 5.73 35345.82 45.40 chloroplast -0.322 OsFBX357 330 10.74 36817.10 73.13 chloroplast -0.377 OsFBX109 443 9.54 47664.37 49.32 mitochondrion 0.042 OsFBX132 311 7.58 33156.89 53.81 Cytosol -0.053 Additional Declarations No competing interests reported. Supplementary Files Additionalfile1FigureS1GeneandproteinstructuresofOsFBX148.docx Additional file 1: Fig. S1Gene and protein structures of OsFBX148. aGene structure of OsFBX148 . The red box represents the exons of Os FBX148 and the black box represents the introns of Os FBX148 . b A close-up view protein structures of OsFBX148 and Osfbx148 . The single-base insertion at the position 188 downstream of the ATG start codon on the OsFBX148 genomic DNA was shown in the left panel. The newly created premature stop codon TGA on the OsFBX148 ORF sequence was underlined. The full-length and truncated proteins corresponding to the entire ORF sequence and the mutated ORF sequence of OsFBX148 were shown in the right panel. Black frame represents a portion of protein with a changed sequence due to the frame shift resulting from the single-base insertion. The numbers above the diagrams indicated the coordinates relative to the start codon ATG (left panel) or start amino acid 1 (right panel). Additionalfile2TableS1DifferentiallyexpressedFboxgenesundercontrolanddroughtconditionsdetectedbymRNAseqtechnology.docx Additional file 2: Table S1 Differentially expressed F-box genes under control and drought conditions detected by mRNA-seq technology Additionalfile3TableS2DroughtresponsiveFboxproteinsandtheirhomogousproteinsindifferentspecies.docx Additional file 3: Table S2 Drought-responsive F-box proteins and their homogous proteins in different species (Red font represents the homogous protein contains F-box domain) Additionalfile4TableS3DetailsoftheFboxgenepairsingeneduplicationevents.docx Additional file 4: Table S3 Details of the F-box gene pairs in gene duplication events Additionalfile5TableS4Theprimersequencesofthe29droughtresponsiveFboxgenesforqRTPCR.docx Additional file 5: Table S4 The primer sequences of the 29 drought-responsive F-box genes for qRT-PCR Cite Share Download PDF Status: Published Journal Publication published 25 Nov, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 26 Sep, 2024 Reviews received at journal 25 Sep, 2024 Reviewers agreed at journal 07 Sep, 2024 Reviews received at journal 13 Aug, 2024 Reviewers agreed at journal 03 Aug, 2024 Reviewers agreed at journal 02 Aug, 2024 Reviewers agreed at journal 11 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers invited by journal 22 Jun, 2024 Editor assigned by journal 21 Jun, 2024 Submission checks completed at journal 21 Jun, 2024 First submitted to journal 18 Jun, 2024 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. 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University","correspondingAuthor":false,"prefix":"","firstName":"Yifeng","middleName":"","lastName":"Zhou","suffix":""},{"id":325591868,"identity":"c17db39a-5bf7-4c00-b50c-e08959e18664","order_by":14,"name":"Yanke Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACPjB5gEGOjZn54IOEChvCWtigWoz52duSDR6cSSNeS+LMnjNmkg/bDhGhRSLH8HPBGRvGDTdyzCoS2A4w8Ld3JxDSYiw940Yas8GNtLIbCTx3GCTOnN1ASIuBNM+Hw2wGN5K33UiQeMZgIJFLUIvxb54P/3kMbiSYFSQYHCZKi5k0z40DEpI9R8wYEhKI0cLzrMya50yyASiQJRIOpPEQ9As/e/Lm2zzH7OrbgFH58ec/Gzn+9l78WhgYOAxQuDwElIMA+wMiFI2CUTAKRsGIBgBD70o4iLauKwAAAABJRU5ErkJggg==","orcid":"","institution":"Hubei Minzu University","correspondingAuthor":true,"prefix":"","firstName":"Yanke","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2024-06-18 08:06:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4598345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4598345/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05820-z","type":"published","date":"2024-11-25T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60130683,"identity":"8f539e0a-d2c2-4b75-add0-43759911cb62","added_by":"auto","created_at":"2024-07-12 07:04:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":516028,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of drought-responsive F-box proteins and their homologous proteins from various species. The F-box proteins were grouped into 13 distinct subgroups. Each group in the phylogenetic tree is represented by a distinctive color. The font colors of 29 drought-responsive F-box proteins were marked with red.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/4ff625e0f827fce81f4633d6.png"},{"id":60132195,"identity":"c0ec5049-c443-430a-9abe-7f8636928f2c","added_by":"auto","created_at":"2024-07-12 07:20:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":348394,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal localizations and collinearity analysis of drought-responsive F-box genes on twelve chromosomes of rice. \u003cstrong\u003ea\u003c/strong\u003e 29 drought-responsive F-box genes are unequally localized on 11 Chromosomes of rice. \u003cstrong\u003eb\u003c/strong\u003e Circos figure for the chromosome locations with the 29 drought-responsive F-box genes segmental duplication links.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/f1bcb81d3d4e9574375a8d0e.png"},{"id":60130688,"identity":"c1ba7554-dad9-4483-897d-a2610dfe7645","added_by":"auto","created_at":"2024-07-12 07:04:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187426,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree, conserved motifs, structural domains, and gene structures of drought-responsive F-box genes. \u003cstrong\u003ea\u003c/strong\u003e The phylogenetic tree of 29 drought-responsive F-box proteins. \u003cstrong\u003eb, c\u003c/strong\u003e The conserved motifs and structural domains of 29 F-box proteins; different conserved motifs and structural domains are marked by different colors. \u003cstrong\u003ed\u003c/strong\u003e Intron and exon structures of 29 F-box genes; exons and untranslated regions (UTRs) are represented by yellow and green boxes, introns are represented by gray lines.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/d78b2dbad7eb4def59bc1dc6.png"},{"id":60130686,"identity":"a44dc9d6-190a-4aaf-a966-a3e49df3b7eb","added_by":"auto","created_at":"2024-07-12 07:04:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":328100,"visible":true,"origin":"","legend":"\u003cp\u003eCis-elements identified in promoters of the 29 drought-responsive F-box genes. Different cis-elements are represented by different colored boxes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/65e680be1bd1bff57a8cea15.png"},{"id":60131540,"identity":"4a37d52b-2e94-497e-a477-8b8183c749a8","added_by":"auto","created_at":"2024-07-12 07:12:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":220499,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap showing the expression levels of the 29 F-box genes in root, stem, leaf, leaf sheath and \u0026nbsp;panicle. The gray colors indicate that the expression of the gene cannot be detected. The color scale is indicated in the right part of the heat map.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/ae4d4e6801779c58ddf703b8.png"},{"id":60130691,"identity":"6e35d7fe-b279-4cb0-abca-3e1695b6a4ce","added_by":"auto","created_at":"2024-07-12 07:04:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":473831,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional expression validation of the 29 drought-responsive F-box genes and their expression profiles in response to other abiotic stress as well as ABA. Six-week-old ZH11 were subjected to moderate drought (MD) and severe drought (SD), leaves were collected for qRT-PCR analysis (\u003cstrong\u003ea\u003c/strong\u003e). CK, no drought stress. For analysis of expression profiles of the 29 F-box genes in response to other abiotic stress as well as ABA , two-week-old ZH11 seedlings grown hydroponically in growth chamber were separately exposed to 20% PEG6000 (\u003cstrong\u003eb\u003c/strong\u003e), 4°C (\u003cstrong\u003ec\u003c/strong\u003e), 42°C (\u003cstrong\u003ed\u003c/strong\u003e), 150 mM NaCl (\u003cstrong\u003ee\u003c/strong\u003e), and 30 μM ABA (\u003cstrong\u003ef\u003c/strong\u003e). Shoot samples were collected at 0, 3 h, 6 h, and 12 h, and then total RNA was isolated and subjected to qRT-PCR assays.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/85ba08f8dc1395ebc6d7ccb6.png"},{"id":60130694,"identity":"5ad839ef-d30f-4008-afcd-d64dbb163c13","added_by":"auto","created_at":"2024-07-12 07:04:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":570639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOsFBX148\u003c/em\u003emutation plants increased ABA sensitivity. \u003cstrong\u003ea\u003c/strong\u003eGrowth performance of \u003cem\u003eOsfbx148 \u003c/em\u003eand ZH11 seedlings grown on 1/2 MS medium with or without ABA. Relative shoot length (\u003cstrong\u003eb\u003c/strong\u003e) and relative root length (\u003cstrong\u003ec\u003c/strong\u003e) of \u003cem\u003eOsfbx148\u003c/em\u003e and ZH11 seedlings grown on 1/2 MS with or without ABA at 4 days after germination. Experiments in (a) were repeated three times with similar results. Data presented in (b) and (c) are the means ± SD from three independent experiments and difference between \u003cem\u003eOsfbx148\u003c/em\u003e and ZH11 plants are indicated by asterisks (*p\u0026lt;0.05) according to Student t test.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/3a11795b7aba5cbe5cd87707.png"},{"id":60130693,"identity":"6ec35f85-3d11-4935-8c0a-37414cc20df7","added_by":"auto","created_at":"2024-07-12 07:04:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1131595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOsFBX148\u003c/em\u003e mutation plants increased ABA, NaCl and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensitivity. \u003cstrong\u003ea\u003c/strong\u003e Sensitivity of \u003cem\u003eOsfbx148\u003c/em\u003e plants to exogenous ABA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Leaf fragments were collected from 8-week-old \u003cem\u003eOsfbx148\u003c/em\u003e and ZH11 plants grown under normal condition and were exposed to 1/2MS medium with or without 150 mM NaCl, 5 μM ABA and 1% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 72 h. \u003cstrong\u003eb\u003c/strong\u003e In situ detection of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and superoxide anion in leaves treated with ABA or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by DAB and NBT staining, respectively.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/6b53c030d492b1ce293bacef.png"},{"id":60132900,"identity":"59fc78fd-113c-4d0a-9412-34f42bd73f47","added_by":"auto","created_at":"2024-07-12 07:28:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":447940,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction between OsFBX148 and OSK4/7/17 in yeast. \u003cstrong\u003ea \u003c/strong\u003eFull length protein of OsFBX148 can interact with OSK4/7/17 in yeast. \u003cstrong\u003eb\u003c/strong\u003e OsFBX148 protein without F-box domain cannot interact with OSK4/7/17 in yeast.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/62672c51d1a797c3378935e5.png"},{"id":70381199,"identity":"7024c7b8-c8bc-42d1-97a7-a870d05f1bfd","added_by":"auto","created_at":"2024-12-02 16:04:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6381073,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/be40687b-ebc6-47d9-a5aa-e1ef2e797dd6.pdf"},{"id":60130681,"identity":"9703fef2-bc4e-4042-a21b-37942256bac6","added_by":"auto","created_at":"2024-07-12 07:04:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Fig. S1\u003c/strong\u003eGene and protein structures of OsFBX148. \u003cstrong\u003ea\u003c/strong\u003eGene structure of \u003cem\u003eOsFBX148\u003c/em\u003e. The red box represents the exons of Os\u003cem\u003eFBX148 \u003c/em\u003eand the black box represents the introns of Os\u003cem\u003eFBX148\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e A close-up view protein structures of OsFBX148 and \u003cem\u003eOsfbx148\u003c/em\u003e. The single-base insertion at the position 188 downstream of the ATG start codon on the \u003cem\u003eOsFBX148\u003c/em\u003e genomic DNA was shown in the left panel. The newly created premature stop codon TGA on the \u003cem\u003eOsFBX148\u003c/em\u003e ORF sequence was underlined. The full-length and truncated proteins corresponding to the entire ORF sequence and the mutated ORF sequence of OsFBX148 were shown in the right panel. Black frame represents a portion of protein with a changed sequence due to the frame shift resulting from the single-base insertion. The numbers above the diagrams indicated the coordinates relative to the start codon ATG (left panel) or start amino acid 1 (right panel).\u003c/p\u003e","description":"","filename":"Additionalfile1FigureS1GeneandproteinstructuresofOsFBX148.docx","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/e92c8f7b798ec67e2dc34225.docx"},{"id":60131539,"identity":"116d3bbe-7689-4cf3-b7ad-3b2fd284b406","added_by":"auto","created_at":"2024-07-12 07:12:56","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Table S1 \u003c/strong\u003eDifferentially expressed F-box genes under control and drought conditions detected by mRNA-seq technology\u003c/p\u003e","description":"","filename":"Additionalfile2TableS1DifferentiallyexpressedFboxgenesundercontrolanddroughtconditionsdetectedbymRNAseqtechnology.docx","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/fe43e05e2cc4f8f20336b308.docx"},{"id":60131537,"identity":"a2395197-7bcd-4896-b2c3-754a5a57bdd2","added_by":"auto","created_at":"2024-07-12 07:12:56","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":39045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: Table S2 \u003c/strong\u003eDrought-responsive F-box proteins and their homogous proteins in different species (Red font represents the homogous protein contains F-box domain)\u003c/p\u003e","description":"","filename":"Additionalfile3TableS2DroughtresponsiveFboxproteinsandtheirhomogousproteinsindifferentspecies.docx","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/e35514533c26143629013379.docx"},{"id":60130685,"identity":"6bb58883-99e0-44b7-bc44-6850da9713cc","added_by":"auto","created_at":"2024-07-12 07:04:56","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4: Table S3 \u003c/strong\u003eDetails of the F-box gene pairs in gene duplication events\u003c/p\u003e","description":"","filename":"Additionalfile4TableS3DetailsoftheFboxgenepairsingeneduplicationevents.docx","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/5a70d13ea1f0f1bc0891b625.docx"},{"id":60131542,"identity":"00c4247d-7f40-45cc-a2ad-3fc10d95e06e","added_by":"auto","created_at":"2024-07-12 07:12:56","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":18855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 5: Table S4 \u003c/strong\u003eThe primer sequences of the 29 drought-responsive F-box genes for qRT-PCR\u003c/p\u003e","description":"","filename":"Additionalfile5TableS4Theprimersequencesofthe29droughtresponsiveFboxgenesforqRTPCR.docx","url":"https://assets-eu.researchsquare.com/files/rs-4598345/v1/4d3ce30e7ccff9cbbd154b27.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification and analysis of drought-responsive F-box genes in upland rice and involvement of OsFBX148 in ABA response and ROS accumulation","fulltext":[{"header":"Background","content":"\u003cp\u003eAs sessile organisms, plants are constantly exposed to a variety of abiotic stresses, among which drought is one of the most severe factors adversely affecting plant growth, development, and productivity worldwide [1]. With climate change intensifying drought events in recent years [2], understanding the complex molecular mechanisms underlying plant drought responses has become a critical area of research. Plants have evolved complex regulatory networks to coordinate dynamic changes and cope with environmental challenges [3]. A key mechanism by which plants fine-tune their stress responses is through the selective proteolysis of regulatory proteins by the ubiquitin-proteasome system (UPS) [4]. In this system, ubiquitin is first activated by E1 activation enzymes, then transferred to E2 conjugation enzymes. E3 ligases use their specific substrate-binding domains to recognize and target proteins, catalyzing the covalent attachment of ubiquitin to lysine residues on these target proteins. Though this rapid and reversible process, plants can fine-tune the abundance of specific regulatory proteins to cope with drought stress [5-7].\u003c/p\u003e\n\u003cp\u003eF-box proteins are integral components of Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complexes in plants, which are responsible for the ubiquitination and subsequent proteasomal degradation of target substrate proteins. These complexes play crucial roles in diverse biological processes [8]. Within the SCF complex, F-box proteins interact with Skp1 proteins through their F-box domain [9]. The F-box domain acts as a recognition module, allowing the SCF complex to specifically bind to and ubiquitinate particular substrates [10]. By regulating gene expression or protein specific degradation within the SCF complex, F-box protein can precisely regulate plant response to abiotic stress. However, Skp1 possesses numerous members as a protein family, different F-box proteins may interact with different Skp1 members, thus, whether a particular F-box protein functions in SCF complex or which Skp1 proteins it interacts with still need to be deeply explored.\u003c/p\u003e\n\u003cp\u003eF-box proteins also play a critical role in regulating abscisic acid (ABA) signal transduction. The phytohormone ABA coordinates a broad range of physiological and developmental processes and mediates responses to drought stress [11]. A key mechanism by which ABA exerts its regulatory effects is through the ubiquitin-proteasome system, specifically via the F-box protein family [12, 13]. In the context of ABA signaling, numerous F-box proteins have been identified as important regulators. For instance, the F-box protein TaFBA1 in wheat acts as a negative regulator of ABA signaling by physically interacting with the ABA receptor RCAR1 and the ABA-responsive transcription factor ABI5. Through these protein-protein interactions, TaFBA1 modulates the expression of ABA-inducible genes, thereby reducing the plant\u0026apos;s sensitivity to ABA and its capacity to respond to drought stress [14]. Similarity, the Arabidopsis protein RIFP1, a member of the F-box E3 ligase family, interacts with the ABA receptor RCAR3 in the nucleus. This interaction increases the degradation rate of RCAR3, thereby disrupting the normal transduction of the ABA signal. Furthermore, mutation of the\u0026nbsp;\u003cem\u003eRIFP1\u003c/em\u003e gene also leads to corresponding changes in the expression of ABA responsive genes. Therefore, RIFP1 is considered to be a negative regulator of ABA signal transduction [15]. Ongoing research on the complex interplay between F-box proteins, ABA, and the UPS continues to shed light on the dynamic regulation of stress tolerance in plants. Elucidating these regulatory mechanisms holds great promise for engineering crop plants with improved resilience to environmental challenges. However, as a superfamily, the F-box genes\u0026rsquo; functions are not comprehensively revealed. A lot of work still need to do.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThrough genome-wide identification and analysis, F-box gene families have been characterized in a variety of plant species. In Arabidopsis thaliana, 694 F-box genes have been identified, with functional studies implicating these proteins in diverse processes such as plant development, hormone signaling, and biotic/abiotic stress responses [16]. Similarly extensive F-box gene families have been identified in other plant species. In rice (\u003cem\u003eOryza sativa\u003c/em\u003e), 779 putative F-box genes have been annotated [17], while in maize [18], soybean [19], apple (\u003cem\u003eMalus domestica\u003c/em\u003e) [20], cotton (\u003cem\u003eGossypium hirsutum.\u0026nbsp;\u003c/em\u003eL) [21], Medicago truncatula [22], and tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) [23], 359, 517, 509, 592, 972 and 139 F-box genes were identified, respectively. The identification and comparative analyses of F-box gene families across diverse taxa have provided valuable insights into the evolution and functional diversification of this important protein family. As more genome sequences become available, future studies will undoubtedly uncover additional F-box genes and elucidate their roles in various cellular processes and organismal adaptations. However, few studies have been reported about the F-box genes in upland rice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUpland rice, also known as \u0026quot;dryland rice\u0026quot;, is adapted to grow in rainfed, non-flooded conditions, requiring significantly less water than its lowland counterparts. Except for the differences in morphological and biochemical traits, upland rice and lowland rice varieties exhibit distinct patterns of gene expression when exposed to drought stress [24-26]. Therefore, utilizing upland rice materials to screen and identify genes related to drought regulation is highly feasibility. In this study, we used the upland rice variety IAPAR9 for drought treatment followed by transcriptome sequencing, identifying 29 drought-responsive F-box genes. We then performed comprehensive analysis on these 29 genes, including phylogenetic tree analysis, gene structure prediction, characterization of chromosome distributions, cis-element analysis of gene promoters, tissue-specific expression profiling, and expression profiling under abiotic stresses. Additionally, we conducted preliminary functional studies on the selected gene \u003cem\u003eOsFBX148\u003c/em\u003e. This work lays a strong foundation for further research on functions of F-box genes in rice, particularly in the context of drought stress response. The insights gained from this study will facilitate the development of rice varieties with improved drought resistance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification, phylogenetic analysis and chromosomal location of the drought-responsive F-box genes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emRNA-seq was conducted using upland rice IAPAR9 following drought treatment in our previous study (accession number SRP273943), followed by functional annotation of differentially expressed genes. To identify the drought-responsive F-box genes in rice, we performed further analysis of the transcriptome data. A total of 36 differentially expressed F-box genes were initially screened. However, protein domain analysis revealed that seven of these genes lacked the F-box domain. Therefore, these seven genes were excluded. Consequently, we identified 29 drought-responsive F-box genes in IAPAR9 \u003cstrong\u003e(Table S1)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo investigate the phylogenetic relationships of these 29 F-box proteins between rice and other plants, the amino acid sequences of these proteins were used as queries for a BLAST search against GenBank to retrieve homologous sequences from all species. An unrooted phylogenetic tree was then constructed based on the alignments \u003cstrong\u003e(Fig. 1)\u003c/strong\u003e. The BLAST analysis showed that most of the F-box proteins have highly homologous genes within the rice genome, and these homologous genes are mostly F-box proteins as well, indicating a high level of homology among the F-box proteins. This may be due to the large number and relatively high conservation of F-box proteins in rice [17]. Furthermore, the homologs of these 29 F-box proteins were found exclusively in plants, not in animals or microorganisms, suggesting that these F-box proteins are plant-specific. Among these 29 F-box genes, the homologous genes of \u003cem\u003eOsFBX342\u003c/em\u003e, \u003cem\u003eOsFBX150\u003c/em\u003e, \u003cem\u003eOsFBK8\u003c/em\u003e, \u003cem\u003eOsFBX261\u003c/em\u003e, \u003cem\u003eOsFBX281\u003c/em\u003e, \u003cem\u003eOsFBD11\u003c/em\u003e, \u003cem\u003eOsFBX283\u003c/em\u003e, \u003cem\u003eOsFBX258\u003c/em\u003e, \u003cem\u003eOsFBX438\u003c/em\u003e, \u003cem\u003eOsFBX345\u003c/em\u003e, \u003cem\u003eOsFBX44\u003c/em\u003e, \u003cem\u003eOsFBX315\u003c/em\u003e, \u003cem\u003eOsFBX220\u003c/em\u003e, \u003cem\u003eOsFBDUF13\u003c/em\u003e, \u003cem\u003eOsFBX31\u003c/em\u003e, \u003cem\u003eOsFBX40\u003c/em\u003e, \u003cem\u003eOsFBXPP2-B1\u003c/em\u003e, \u003cem\u003eOsFBX357\u003c/em\u003e and \u003cem\u003eOsFBX109\u003c/em\u003e exist only in \u003cem\u003eOryza\u003c/em\u003e plants, indicating their high conservation within this genus. Additionally, \u003cem\u003eOsFBO10\u003c/em\u003e has 73 homologs across diverse plant species, indicating a widespread presence in the plant kingdom. \u003cem\u003eOsFBL28\u003c/em\u003e has 13 homologs, OsFBX281 has 14 homologs, \u003cem\u003eOsFBX283\u003c/em\u003e has 14 homologs, \u003cem\u003eOsFBL27\u003c/em\u003e has 11 homologs, and \u003cem\u003eOsFBX148\u003c/em\u003e has 23 homologs. The gene with the fewest homologs has only 2 \u003cstrong\u003e(Table S2)\u003c/strong\u003e. These results together suggest that F-box proteins are highly conserved and plant-specific, indicating their potential roles in specific plant functions.\u003c/p\u003e\n\u003cp\u003eThe 29 drought responsive F-box genes are distributed on 11 of the 12 chromosomes in rice \u003cstrong\u003e(Fig. 2a)\u003c/strong\u003e.\u0026nbsp;The number of F-box genes varies drastically across the chromosome, ranging from 0 to 5. Chromosomes 2 and 4 have largest number of F-box members, with five genes (17.24%) each. Chromosomes 6, 8 and 11 contain three genes (10.34%). There are two genes (6.90%) located on chromosomes 3, 7, 9 and 10, respectively. Only one gene (3.45%) is located on chromosome 1 and chromosome 12. Notably, no drought responsive F-box genes are found on chromosome 5. These results suggest that the drought responsive F-box genes are unevenly distributed across the rice chromosomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGene duplication is considered a major driver of genomic innovation and the emergence of novel biological features and capabilities in organisms\u0026nbsp;[27, 28]. The additional genetic material provided by duplicated genes allows for the exploration of new functional possibilities through mutation and selection, ultimately expanding the coding and regulatory capacity of the genome. To investigate the presence of gene duplication events among the 29 F-box genes, we performed segmental duplication analysis. A total of 10\u0026nbsp;collinear gene pairs were identified on different chromosomes, indicating segmental duplication events \u003cstrong\u003e(Fig. 2b)\u003c/strong\u003e. Chromosome 4 had the highest number of duplicated F-box gene pairs, with seven pairs. This is followed by three pairs on chromosome 2, two pairs each on chromosomes 3, 11 and 12, and one pair each on chromosomes 7, 8, 9, and 10\u003cstrong\u003e\u0026nbsp;(Table S3)\u003c/strong\u003e.\u0026nbsp;These findings show that segmental duplication has played an important role in the expansion of the F-box family genes in rice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of basic physicochemical properties and domain of drought-responsive F-box genes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physical and chemical properties of drough-responsive F-box proteins were analyzed using ExPasy (https://web.expasy.org/protparam/). This analysis included parameters such as amino acid length, isoelectric point (PI), protein molecular weight (MW), protein instability index, and grand average of hydropathicity (GRAVY)\u003cstrong\u003e\u0026nbsp;(Table 1)\u003c/strong\u003e. The results showed significant variability in the protein sequences of different F-box proteins. Most F-box proteins had an amino acid length of less than 1000 residues, except for OsFBX150 and OsFBX345. Among these, OsFBX150 exhibited the longest amino acid length at 1526 residues with a molecular weight of 159241.75 Da, while OsFBX77 had the shortest length with only 178 residues and a weight of 18611.57 Da. The isoelectric point ranged from 5.27 (OsFBX44) to 10.74 (OsFBX357), with 15 F-box proteins having acidic points below 7 and the remaining 14 being relatively alkaline. The protein instability index, an important indicator of protein stability, indicated that most drought-responsive F-box proteins were unstable, except for OsFBDUF13 and OsFBX148, with instability indices of 38.71 and 31.67, respectively. This instability may be linked to their role in dynamically regulating target proteins under drought conditions.\u0026nbsp;The hydrophilicity analysis revealed that 23 F-box proteins had a GRAVY value below 0, indicating a hydrophilic nature, while the remaining 6 F-box proteins were hydrophobic with a GRAVY value above 0. Furthermore, WoLF PSORT was used to predict the subcellular localization of these drought-responsive F-box proteins. Apart from OsFBL27, which localizes to the endoplasmic reticulum, and OsFBX109, which localizes to the mitochondria, the other F-box proteins were found to be localized in the nucleus, cytoplasm, and chloroplasts.\u003c/p\u003e\n\u003cp\u003ePhylogenetic relationships, conserved motifs identification, conserved domains analysis, and gene structure analysis were conducted on the complete sequences of 29 F-box proteins \u003cstrong\u003e(Fig. 3)\u003c/strong\u003e. A total of 9 conserved motifs were identified in these sequences. Notably, Motif 2 was present in all F-box proteins \u003cstrong\u003e(Fig. 3b)\u003c/strong\u003e, indicating its role as the F-box domain and potential importance in their function. Motif 1 was found in all F-box proteins except for OsFBXPP2-B1 and OsFBX563, suggesting a relatively conserved nature compared to motif 2. Additionally, motif 5 was unique to OsFBX261, OsFBXPP2-B1, OsFBX438, and OsFBX148, while motif 4 was specific to OsFBK8 and OsFBK25, which clustered together phylogenetically, possibly indicating specialized physiological functions. Although motif 3 was shared among evolutionarily related members OsFBX261, OsFBXPP2-B1, OsFBX438, and OsFBX148, it was also present in OsFBX345. Similarly, motif 9 showed a similar pattern, predominantly found in OsFBX261, OsFBXPP2-B1, and OsFBX438, but also in OsFBX281, suggesting both universal and specific functions for these motifs. Furthermore, motifs 6, 7, and 8 exhibited irregular distribution among different genes, indicating potential diversification of function among the F-box proteins.\u003c/p\u003e\n\u003cp\u003eThe F-box domain located at the N-terminal of the SCF complex is evolutionarily conserved [17]. The analysis revealed that all 29 F-box proteins possessed this conserved domain \u003cstrong\u003e(Fig. 3c)\u003c/strong\u003e. Additionally, 11 other domains were identified, including the FBD, Ketch and Tub domains \u003cstrong\u003e(Fig. 3c)\u003c/strong\u003e. FBD domain-containing F-box proteins in Arabidopsis were found to play roles in ethylene/abscisic acid signaling, plant\u0026ndash;pathogen interactions, and root development [29]. The Ketch domain, which is the most abundant type of F-box proteins, has various functions in plants, such as regulating seed germination, development, the phenylpropanoid pathway, ultraviolet radiation tolerance, drought and heat stress resistance, and root architecture [9, 30-32]. The Tub domain, named after the TUBBY protein in mice, binds specifically to negatively charged phosphoinositide species and is involved in ABA, abiotic stress, and immunity regulation in plants [33-37]. Other domains like DUF, KIX, and PP2 were also found in F-box proteins. The identification of multiple domains suggests that F-box proteins in rice may have diverse mechanisms and functions.\u003c/p\u003e\n\u003cp\u003eThe structures of the exons, introns and untranslated regions (UTRs) of the 29 F-box genes were visualized by use of TBtools software\u0026nbsp;\u003cstrong\u003e(Fig. 3d)\u003c/strong\u003e. The results showed that the closer the genetic relationship and phylogenesis, the more similar the gene structure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCis-elements in the promoters of F-box genes in response to drought stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptional expression of genes is to some extent regulated by cis-elements located in the promoter regions upstream of the encoding genes [38]. Thus, the 2-kb putative promoter regions of the 29 F-box genes were analyzed using the online database PlantCARE. A total of 13 cis-elements or binding sites were identified, including important elements involved in abiotic stress responses such as dehydration, low temperature, and salt stresses. Additionally, elements involved in abscisic acid responsiveness and MYB binding site associated with drought inducibility were identified \u003cstrong\u003e(Fig. 4)\u003c/strong\u003e. Apart from core promoter elements and common cis-acting element detected in most F-box gene promoters, other cis-elements related to plant growth and development were also discovered. These included regulatory elements associated with meristem specific activation, differentiation of palisade mesophyll cells, and meristem expression. These results assuredly suggest that the 29 drought-responsive F-box genes are not only involved in drought stress regulation, but also in other abiotic stress and various aspects of plant growth and development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression patterns analysis of F-box genes in response to drought stress in different tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudying the tissue-specific expression of genes helps predict their functions in unique tissue. This analysis can provide insight into the regulatory mechanisms of genes, contributing to a better understanding of gene regulation throughout the plant. In a study analyzing the expression patterns of 29 drought-responsive F-box genes, qRT-PCR was conducted using total RNA extracted from various tissues of rice, including\u0026nbsp;root, stem, leaf, leaf sheath and panicle. The results revealed significant differences in expression levels among the F-box genes across different tissues \u003cstrong\u003e(Fig. 5)\u003c/strong\u003e. Notably, the expression of \u003cem\u003eOsFBO10\u003c/em\u003e and \u003cem\u003eOsFBX283\u0026nbsp;\u003c/em\u003ecould not be detected by qRT-PCR, suggesting their low expression level and potentially minor functional roles. The remaining 27 F-box genes were all successfully detected. Among these, \u003cem\u003eOsFBX148\u003c/em\u003e, \u003cem\u003eOsFBX31\u003c/em\u003e, \u003cem\u003eOsFBDUF13\u003c/em\u003e, \u003cem\u003eOsFBX77\u003c/em\u003e, \u003cem\u003eOsFBX438\u003c/em\u003e, \u003cem\u003eOsFBX315\u003c/em\u003e and \u003cem\u003eOsFBX150\u003c/em\u003e exhibited predominant expression in the root compared to other tissues. \u003cem\u003eOsFBX40\u003c/em\u003e, \u003cem\u003eOsFBX281\u003c/em\u003e, \u003cem\u003eOsFBX563\u003c/em\u003e, \u003cem\u003eOsFBD8\u003c/em\u003e and \u003cem\u003eOsFBX220\u003c/em\u003e showed relatively higher expression levels in the stem compared to other tissues. In rice leaf, eight genes, \u003cem\u003eOsFBX357\u003c/em\u003e, \u003cem\u003eOsFBXPP2-B1\u003c/em\u003e, \u003cem\u003eOsFBL28\u003c/em\u003e, \u003cem\u003eOsFBT6\u003c/em\u003e, \u003cem\u003eOsFBL27\u003c/em\u003e, \u003cem\u003eOsFBK8\u003c/em\u003e, \u003cem\u003eOsFBX261\u003c/em\u003e and \u003cem\u003eOsFBX258\u003c/em\u003e, were found to be expressed more than in other tissues. Additionally, \u003cem\u003eOsFBX132\u003c/em\u003e and \u003cem\u003eOsFBX109\u0026nbsp;\u003c/em\u003eshowed higher expression levels in the leaf sheath, while \u003cem\u003eOsFBX44\u003c/em\u003e and \u003cem\u003eOsFBD11\u003c/em\u003e were more expressed in the panicle. Several F-box genes exhibited relatively high expression levels in multiple tissues. For instance, \u003cem\u003eOsFBX281\u003c/em\u003e showed high expression in both the stem and panicle, while \u003cem\u003eOsFBX563\u003c/em\u003e had high expression in the stem, leaf, leaf sheath, and panicle. Furthermore, \u003cem\u003eOsFBX132\u003c/em\u003e was expressed in both the stem and leaf sheath, and \u003cem\u003eOsFBX44\u003c/em\u003e had high expression levels in both leaf and panicle. These results suggest that although these F-box genes are responsive to drought stress, they may have distinct regulatory roles in various plant tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression profiles of F-box genes in response to drought stress to abiotic stress as well as ABA treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the accuracy of transcriptional expression changes for the drought-responsive genes, identified in mRNA-seq data, 6-week-old rice plants were grown under normal conditions and then subjected to drought treatment. Leaf samples were collected under moderate and severe drought conditions for further analysis. Subsequent qRT-PCR experiments revealed that the expression patterns\u003c/p\u003e\n\u003cp\u003eof most genes were consistent with the transcriptome sequencing data \u003cstrong\u003e(Fig. 6a)\u003c/strong\u003e. However, genes such as \u003cem\u003eOsFBX40\u003c/em\u003e, \u003cem\u003eOsFBX345\u003c/em\u003e, \u003cem\u003eOsFBX261\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;OsFBK25\u0026nbsp;\u003c/em\u003eexhibited expression changes that contradicted the mRNA-seq results. This discrepancy could potentially be attributed to variations in rice growth stages and cultivation environments.\u0026nbsp;The study once again confirmed that these 29 drought-responsive genes are indeed influenced by drought stress.\u003c/p\u003e\n\u003cp\u003ePrevious research has indicated that F-box proteins are involved in various abiotic stresses, such as hyperosmotic, salt, high and low temperature stress in plants\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e[39]. To investigate whether the transcriptional expression of the 29 drought-responsive F-box genes can be influenced by other abiotic stress factors, ZH11 seedlings were individually subjected to PEG, cold, heat and NaCl treatments, followed by qRT-PCR experiments. The expression of \u003cem\u003eOsFBO10\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOsFBX283\u003c/em\u003e genes could not be detected even with qRT-PCR methods, which aligns with the results of tissue-specific expression analysis where the expression of these two genes was also undetectable. Under hyperosmotic stress induced by PEG-6000 treatment, the transcript levels of 26 F-box genes were altered, except for \u003cem\u003eOsFBD11\u003c/em\u003e \u003cstrong\u003e(Fig. 6b)\u003c/strong\u003e. Among these, 2 genes were downregulated and 15 genes were upregulated at three different time points. 19 genes were upregulated at both the 3-hour and 6-hour time points post-treatment, with the majority reaching peak expression levels after 6 hours, indicating their potential roles in the early stages of osmotic stress response. These findings suggest that F-box genes may indeed be involved in responses to hyperosmotic stress.\u003c/p\u003e\n\u003cp\u003eCold stress served as an abiotic factor that led to an increase in the expression of 18 F-box genes after 3- and 6-hour treatment periods \u003cstrong\u003e(Fig. 6c)\u003c/strong\u003e. Out of these genes, 13 showed sustained upregulation, while 4 displayed continuous downregulation. The expression patterns of the remaining 5 genes fluctuated without any clear trends. These findings indicate that the F-box genes may be involved in the regulation of cold stress.\u003c/p\u003e\n\u003cp\u003e27 F-box genes were impacted by heat stress induced by a 42℃ treatment \u003cstrong\u003e(Fig. 6d)\u003c/strong\u003e. Out of these, the expression of 19 genes was consistently upregulated throughout all three time points of treatment, while the expression of 5 genes was consistently downregulated across the same time points. Specifically, \u003cem\u003eOsFBX77\u003c/em\u003e and \u003cem\u003eOsFBX281\u0026nbsp;\u003c/em\u003eshowed initial upregulation at 3- and 6-hour treatment conditions, followed by a return to levels similar to the control. On the other hand, \u003cem\u003eOsFBD11\u003c/em\u003e exhibited a slight decrease after the 3-hour treatment, followed by a subsequent increase of 1.5-fold. These findings suggest that these genes may play a crucial role in the ongoing regulation of heat stress response.\u003c/p\u003e\n\u003cp\u003eUnder salt stress treatment, all 27 F-box genes showed changes in expression levels \u003cstrong\u003e(Fig. 6e)\u003c/strong\u003e. Fourteen genes displayed sustained upregulation at all three time points, while three genes exhibited sustained downregulation. The majority of upregulated genes reached their peak expression levels at the 6-hour time point. Eight genes initially showed downregulation after 3 hours of NaCl treatment, but their expression was upregulated after 6 hours, with fold changes exceeding 2-fold. These findings highlight the intricate regulation of gene expression under salt stress, with potential involvement of F-box genes.\u003c/p\u003e\n\u003cp\u003eABA, an essential plant hormone, plays a pivotal role in seed germination, growth, development, and response to abiotic stresses [11]. Analysis of cis-elements in F-box gene promoters revealed the presence of ABA-responsive elements. Subsequent qRT-PCR experiments confirmed that the transcriptional expression of most F-box genes was upregulated in response to ABA, with the exception of \u003cem\u003eOsFBX345\u003c/em\u003e \u003cstrong\u003e(Fig. 6f)\u003c/strong\u003e. While \u003cem\u003eOsFBXPP2-B1\u003c/em\u003e and \u003cem\u003eOsFBX281\u003c/em\u003e initially decreased and then increased, \u003cem\u003eOsFBK8\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOsFBX258\u003c/em\u003e exhibited an initial increase followed by a decrease. Notably, \u003cem\u003eOsFBX150\u003c/em\u003e showed no consistent trend in expression levels. These findings suggest that these drought responsive F-box genes, at least part of them, may regulate stress responses in an ABA-dependent manner.\u003c/p\u003e\n\u003cp\u003eUnder various abiotic stresses, the expression of \u003cem\u003eOsFBX132\u003c/em\u003e, \u003cem\u003eOsFBX148\u003c/em\u003e, \u003cem\u003eOsFBX31\u003c/em\u003e, \u003cem\u003eOsFBDUF13\u003c/em\u003e, \u003cem\u003eOsFBD8\u003c/em\u003e, \u003cem\u003eOsFBX44\u003c/em\u003e, \u003cem\u003eOsFBL27\u003c/em\u003e, \u003cem\u003eOsFBX261\u003c/em\u003e, and \u003cem\u003eOsFBK25\u003c/em\u003e genes was upregulated, while \u003cem\u003eOsFBK8\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOsFBX258\u0026nbsp;\u003c/em\u003egenes were downregulated. This indicates that these genes may have similar and significant regulatory roles under different abiotic stress conditions. ABA, as a broad-spectrum plant antistress hormone, is crucial in regulating plant stress responses. The study revealed that 21 genes were induced and up-regulated by ABA, with 12 of them showing up-regulation by nearly 10-fold or more. This implies that these genes are likely regulated or at least partially in an ABA-dependent manner to control stress responses. Most of the drought-responsive F-box genes identified are believed to have important regulatory roles in managing plant responses to abiotic stresses, highlighting the essential and critical function of F-box genes in regulating plant responses to such stresses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsFBX148 is a member of SCF complex and involved in ABA response and ROS accumulation of rice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe above studies have demonstrated that \u003cem\u003eOsFBX148\u003c/em\u003e is significantly up-regulated in response to drought and ABA treatment, as evidenced by transcription data and qRT-PCR analysis. In order to further explore its biological role, a mutant allele of \u003cem\u003eOsfbx148\u003c/em\u003e was generated by inserting a nucleotide T into the first exon of the gene \u003cstrong\u003e(Fig. S1)\u003c/strong\u003e. Uniformly germinated ZH11 and \u003cem\u003eOsfbx148\u003c/em\u003e seeds were cultivated on half-strength MS medium supplemented with 2.5 and 5 \u0026mu;M ABA, respectively, for a period of 4-5 days. Under normal conditions, there was no observable difference between ZH11 and \u003cem\u003eOsfbx148\u003c/em\u003e. However, upon ABA treatment, it was noted that the root and shoot length of \u003cem\u003eOsfbx148\u0026nbsp;\u003c/em\u003ewere notably shorter compared to ZH11 \u003cstrong\u003e(Fig. 7)\u003c/strong\u003e. These findings suggest that OsFBX148 plays a role in ABA response and acts as a negative regulator of ABA signal transduction.\u003c/p\u003e\n\u003cp\u003eExposure of plants to abiotic stress leads to a significant increase in reactive oxygen species (ROS) levels within the cell. To investigate the function of OsFBX148 in controlling ROS accumulation, experiments were carried out using detached leaves from 40-day-old ZH11 and \u003cem\u003eOsfbx148\u003c/em\u003e plants. The leaves were subjected to treatments with ABA, NaCl, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, followed by staining with DAB and NBT \u003cstrong\u003e(Fig. 8)\u003c/strong\u003e. The results revealed that leaves carrying the \u003cem\u003eOsfbx148\u003c/em\u003e mutant allele exhibited a deeper coloration when exposed to ABA and NaCl. Interestingly, when treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, only DAB staining resulted in color change, while NBT staining did not, suggesting an accumulation of more hydrogen peroxide than singlet oxygen under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment. These findings suggest that \u003cem\u003eOsfbx148\u003c/em\u003e plants had higher ROS levels compared to ZH11 plants, indicating the role of OsFBX148 in ROS regulation and increased ROS accumulation in \u003cem\u003eOsfbx148\u003c/em\u003e mutants under stress conditions.\u003c/p\u003e\n\u003cp\u003eThe study investigated the role of OsFBX148, an F-box protein, in the formation of the SCF complex responsible for specific substrate binding through interaction with SKP1. A yeast two-hybrid experiment was conducted to assess the interaction between OsFBX148 and the SKP1 protein, using 6 randomly selected OSKs proteins (OSK1/4/7/15/16/17). Results indicated that OsFBX148 could interact with OSK4/7/17 \u003cstrong\u003e(Fig. 9a)\u003c/strong\u003e, but this interaction was lost upon truncation of the F-box domain of OsFBX148 \u003cstrong\u003e(Fig. 9b)\u003c/strong\u003e. These findings suggest that OsFBX148 may function by contributing to the formation of the SCF complex.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, OsFBX148 plays a role in ABA response and ROS accumulation in rice through its involvement in the SCF complex.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs the substrate-recognition components of SCF E3 ubiquitin ligase complexes, F-box proteins are responsible for targeting specific substrates for ubiquitination and subsequent degradation by the 26S proteasome. They play crucial roles in various cellular processes, including hormone signaling, development, and stress responses\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e[8]. Therefore, the identification and characterization of F-box genes have been a focus of extensive research in the plant kingdom. The availability of numerous plant genome sequences has enabled the systematic identification and comparative analysis of F-box genes across diverse plant species. One of the earliest and most comprehensive studies on plant F-box genes was conducted in the model plant Arabidopsis thaliana, which identified a total of 694 F-box genes, making it one of the largest F-box gene families among eukaryotes [16]. Similar genome-wide surveys have been carried out in other major plant species. These studies have consistently found that the F-box gene family is significantly expanded in plants compared to other eukaryotes, with the number of F-box genes ranging from several hundred to over a thousand, depending on the plant species\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e[21]. Transcriptome analysis, enabled by RNA sequencing (RNA-seq), provides a comprehensive view of the genes expressed in a plant under specific environmental conditions. By comparing the transcriptional profiles of plants exposed to stress treatments versus control conditions, researchers can identify differentially expressed genes (DEGs) that are induced or repressed in response to the stress [40]. These DEGs often include members of gene families known to play important roles in stress tolerance, such as transcription factors, kinases, transporters, and antioxidant enzymes. In this study，we screened 29 differentially expressed F-box genes by RNA-seq using upland rice IAPAR9, indicating that several F-box genes were responsive to drought stress. This result is consistent with many previous reports that F-box genes are involved in the regulation of plant drought stress response [8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalyzing the physicochemical properties and structural features of proteins can help predict their potential biological functions. Our analysis of the physicochemical properties of 29 F-box proteins showed that the pI values of these proteins range from 5.27 to 10.74, and their molecular weights, except for OsFBX77 and OsFBX148, are mostly greater than 40 kD, which is generally consistent with previous reports [41]. However, through further analysis of the instability indices of these 29 proteins, we found that the instability indices, except for OsFBDUF13 and OsFBX148, are all greater than 40, indicating that these proteins have relatively low stability. This may be related to their rapid response to drought stress and their functions in signal transduction regulation [42].\u0026nbsp;Unstable proteins are more easily degraded, which allows the cell to quickly modulate the abundance of these critical proteins to respond to changes in external drought stress [43]. This dynamic regulation facilitates the plant\u0026apos;s rapid perception and reaction to environmental stresses. The subcellular localization analysis showed that the 29 F-box proteins are localized in the cytoplasm, nucleus, chloroplasts, mitochondria, and endoplasmic reticulum, which is consistent with previous reports [14, 15, 44, 45]. The F-box domain is a conserved structural domain of F-box proteins, usually located at the N-terminus. The C-terminal domain is related to the specific binding of its substrates, reflecting the functional specificity of different F-box proteins. The analysis results show that all 29 F-box proteins contain the conserved F-box domain, but not every protein\u0026apos;s C-terminus contains other structural domains. This may be because the F-box domain itself can be sufficient to mediate the recognition and binding of specific substrates [19]. It\u0026apos;s also possible that some F-box proteins lack additional structural domains because they rely on interactions with other proteins to determine substrate specificity, rather than requiring extra structural domains. Cis-acting elements in the promoters of the 29 drought-responsive F-box genes were also analyzed in rice. We identified elements related to the regulation of plant growth, development, and abiotic stress responses, such as drought, low temperature, high salinity, and ABA response elements. Based on this, the present study used various abiotic stress treatments including drought, osmotic, high salinity, high temperature, low temperature, and ABA to investigate whether these 29 genes responded to the above treatments. The results showed that except for 2 genes, OsFBX10 and OsFBX283, which could not be detected, the remaining 27 F-box genes were responded to the treatments. This suggests that these F-box genes may indeed play a role in regulating abiotic stress responses, which is generally consistent with previous research [11]. The gene expression profile reveals the potential functions of the gene in different plant tissues. Therefore, we investigated the tissue expression of the 29 F-box genes, and the results showed that 27 of the genes were expressed in the roots, stems, leaves, sheaths, and panicles of rice. This is consistent with the previously reported expression patterns of F-box genes in different rice tissues.\u003c/p\u003e\n\u003cp\u003eAs a component of the SCF complex, F-box proteins are able to interact with SKP1 and determine the specific substrate binding and degradation of this complex, thereby regulating plant growth, development, stress response and signal transduction [46, 47]. This study found that OsFBX148 interacts with the SKP1 proteins OSK4/7/17 to form an SCF complex, which is consistent with previous reports [9]. In this study, we found that the promoter of the\u0026nbsp;\u003cem\u003eOsFBX148\u003c/em\u003e gene contains two ABA-responsive cis-acting elements, suggesting that this gene may play a role in regulating ABA responses. The qRT-PCR detection results also confirmed this conclusion. Previous studies have reported that F-box proteins can interact with key proteins in the ABA signaling pathway, such as PYL and ABI5, to regulate the ABA signaling pathway and thereby control plant responses to ABA.\u0026nbsp;The results of the phenotypic study in this research show that the response of \u003cem\u003eOsFBX148\u003c/em\u003e gene mutant seedlings to ABA is enhanced, indicating that OsFBX148 negatively regulates ABA signal transduction. Thus, we identified a novel F-box protein factor involved in the regulation of ABA signaling and provided new insights into the molecular mechanisms of plant ABA signal regulation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe study identified 29 drought-responsive F-box genes from the upland rice variety IAPAR9, and analyzed the evolutionary relationships, chromosomal distributions, gene duplication events, promoter cis-acting elements, as well as the physicochemical properties and structural domains of the encoded proteins. Homologous analysis revealed that most homologous proteins of 29 drought-responsive F-box proteins were also F-box-containing proteins and they were exclusively exsist in plants. Ten collinear gene pairs were identified, indicating the numerous members in F-box superfamily may be related to segmental duplication. Thirteen cis-acting elements or binding sites were identified, suggesting their roles in ABA and abiotic stress responses. Function analysis of FBD, Ketch, Tub and any other domains found in the 29 drought-responsive F-box proteins revealed their roles in hormone signal transduction and stress responses. The physicochemical properties analysis also revealed the instability of drought-responsive F-box proteins and this may be linked to their role in dynamically regulating target proteins under drought conditions. Gene expression analysis showed that 27of 29 drought-responsive F-box genes are expressed in root, stem, leaf, sheath and panicle and they can response to drought, cold, heat, salt and ABA. Furthermore, a preliminary functional analysis of OsFBX148 was conducted, revealing that OsFBX148 interacts with OSK4/7/17 to form an SCF complex, and plays a role in regulating ABA signal transduction and ROS accumulation in plants. These findings lay the foundation for further exploring the functions of F-box proteins in stress responses.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Brazilian upland rice (cv. IAPAR9) was used for drought treatment and RNA sequencing. The ZhongHua11 (ZH11) rice variety was used for total RNA extraction and gene expression analysis, as well as tissue-specific expression analysis, after various abiotic stress treatments. ZH11 and \u003cem\u003eOsfbx148\u003c/em\u003e were used for function characterization of OsFBX148. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of drought-responsive F-box genes in upland rice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify drought-responsive genes, the upland rice variety IAPAR9 was subjected to drought stress. Leaf tissue was collected for total RNA extraction followed by transcriptome sequencing. Differentially expressed genes affected by drought stress were analyzed, and the function annotating was also performed. Among these differentially expressed genes, 36 putative F-box genes were found by searching \u0026ldquo;F-box\u0026rdquo; as key words. Structural domain analysis was then conducted on these 36 genes through CD-search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to identify their conserved domains and exclude those without the F-box domain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCis-elements analysis in the promoters of drought responsive F-box genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 2000 base pairs promoter sequences upstream of ATG start codon of 29 F-box genes were extracted from the rice genome sequence and submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) for cis-elements analysis. The files obtained from the PlantCARE website were subjected to an advanced filtering process, with Arabidopsis thaliana selected as the species and existing cis-acting elements chosen. The data was then visualized using the TBtools software for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic tree analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to gain more insight into the evolutionary relationships of the 29 F-box proteins, the amino acid sequences of these proteins were submitted to Protein BLAST in NCBI BLAST (https://www.ncbi.nlm.nih.gov/) to retrieve homologous protein sequences from other species. Eventually, only the sequences sharing more than 85% similarity were used for subsequent analysis. The resulting 250 protein sequences from different varieties were compared with the amino acid sequences of the 29 F-box proteins in rice using MEGA11 software (https://www.megasoftware.net/) and a phylogenetic tree was constructed by NJ (Neighbor-Joining) method. Later, FigTree v1.4.3 software (https://www.softsea.com/review/FigTree.html) was used to optimize the phylogenetic tree, such as font format and branch color differentiation, so as to obtain a more intuitive phylogenetic tree and make an in-depth analysis of the evolutionary relationship of these F-box proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromosomal localization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe positional information of these 29 F-box genes on chromosomes was obtained by analyzing the rice gene annotation file. Subsequently, the advanced gene localization visualization feature of TBtools software was utilized to plot the position map of these genes on chromosomes. Furthermore, the gene structure was displayed using the advanced gene structure view feature of TBtools, based on the genome annotation files and obtained NCBI CD-Search conserved domains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogeny and domain analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amino acid sequences of the 29 F-box proteins were aligned by MEGA 11 software, and simple phylogenetic trees were constructed using maximum likelihood. The conserved motifs of these 29 F-box proteins were retrieved by online tool MEME (http://meme-suite.org/tools/meme) with the selected MEME motif number set to 10. The obtained results were downloaded for subsequent analysis. The phylogenetic trees, gene annotation files, protein sequence files and MEME motifs files of these 29 F-box proteins were visualized through the Gene Structure View tool in TBtools software. The final graphs were modified in detail using Adobe Illustrator 2021 software to more intuitively present the evolutionary relationships, gene structure, protein motifs and conserved domains of these proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical properties and subcellular localization prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular weight (MW), isoelectric point (pI), grand average of hydropathicity (GRAVY), and instability index of the 29 F-box proteins were predicted and analyzed using the ExPASy website (https://web.expasy.org/protparam/). And the subcellular localization of F-box proteins was predicted by WoLF PSORT (https://wolfpsort.hgc.jp/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeedlings treatment and gene expression analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the effects of different abiotic stress factors on the expression of F-box genes (the primer sequences are listed in \u003cstrong\u003eTable S4\u003c/strong\u003e), 2-week-old ZH11 seedlings grown hydroponically in growth chamber were separately treated with 20% PEG6000, 4\u0026deg;C, 42\u0026deg;C, 150 mM NaCl and 30 \u0026mu;M ABA. Shoot samples were collected at 0, 3 h, 6 h, and 12 h for total RNA isolation and the following gene expression analysis.\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using the traditional Trizol reagent method with minor modifications [48]\u003cstrong\u003e.\u003c/strong\u003e Briefly, samples were ground in liquid nitrogen-cooled mortar, stirred well with 1 mL Trizol reagent in RNase-free 1.5 mL centrifuge tubes, incubated at room temperature for 5 min, and then centrifuged at 12,000 rpm for 10 min. The supernatant was mixed with 200 \u0026mu;L trichlormethane, and centrifuged at 12,000 rpm for 15 min. 500 \u0026mu;L of the upper aqueous phase was mixed with an equal volume of isopropanol, incubated at -20\u0026deg;C for 30 min, and then centrifuged at 12,000 rpm for 10 min. The pellet was washed with 1 mL 75% ethanol and resuspended in 30 \u0026mu;L DEPC-treated water. RNA quality and concentration were detected using NanoDrop 2000 (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003cp\u003e2 \u0026mu;g of total RNA was used for reverse transcription using PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Shiga, Japan). Quantitative PCR was then performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), with the reaction mixture containing 5 \u0026mu;L 2\u0026times; ChamQ Universal SYBR qPCR Master Mix, 0.4 \u0026mu;L each of forward and reverse primers, 1 \u0026mu;L cDNA template, and 3.2 \u0026mu;L water in a total volume of 10 \u0026mu;L. The qRT-PCR was performed on an ABI StepOne Plus (Thermo Fisher Scientific, USA) with the following program: 95\u0026deg;C for 30 s, followed by 40 cycles of 95\u0026deg;C for 10 s and 60\u0026deg;C for 30 s, with a melting curve analysis using the instrument\u0026apos;s default program. Relative gene expression level was calculated using the 2-\u0026Delta;\u0026Delta;CT method\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e[49].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStress treatment and ROS staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor detached leaves treatment, leaves from 40-day-old rice plants grown in soil under normal conditions were excised into 1-2 cm pieces and placed in MS liquid medium containing 5 \u0026mu;M ABA, 150 mM NaCl, or 1% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and incubated for 3 days under normal growth conditions. For germinated seeds treatment by ABA, simultaneously harvested and dried ZH11 and \u003cem\u003eOsfbx148\u003c/em\u003e seeds were placed in Petri dishes with moistened filter paper and germinated in the dark at 28\u0026deg;C for 2 days. Seedlings with uniform radicle length were selected and transferred to MS liquid medium containing 2.5 \u0026mu;M or 5 \u0026mu;M ABA, and grown under normal conditions (28\u0026deg;C, 16 h light/8 h dark) for 4 days. Photographs were taken, and the shoot and root lengths were measured.\u003c/p\u003e\n\u003cp\u003eFor DAB and NBT staining, DAB and NBT were prepared in distilled water to a working concentration of 1 mg/mL. The leaves of ZH11 and \u003cem\u003eOsfbx148\u0026nbsp;\u003c/em\u003ebefore and after treatment were submerged in the DAB or NBT solution and vacuum-infiltrated overnight. The solutions were then discarded, and the leaves were decolorized in a graded ethanol series (100%, 90%, 75%) for 10 minutes each until the chlorophyll was completely removed. Photographs were taken after decolorization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast two hybrid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CDS sequences of OsFBX148 and OSKs were respectively cloned into the pPGBKT7 and pGADT7 vectors. The two constructs were then co-transformed with salmon sperm DNA into AH109 yeast competent cells. The transformed cells were plated on SD2 yeast medium and incubated at 28\u0026deg;C for 2 days. Single colonies were then picked, serially diluted, and spotted on SD4 medium. Yeast growth was observed and photographed. To study the effect of the F-box domain on the interaction between OsFBX148 and OSKs, the OsFBX148 sequence with the F-box domain deleted was cloned into pPGBKT7 and co-transformed with OSKs into AH109 yeast competent cells. The remaining steps were the same as above.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSCF: SKP1-CUL1-F-box; ZH11: ZhongHua11; qRT-PCR: quantitative real-time PCR; ROS: reactive oxygen species; UPS: ubiquitin-proteasome system; ABA: abscisic acid; PI: isoelectric point; MW: molecular weight; GRAVY: grand average of hydropathicity; UTR: untranslated region; DAB: diaminobenzidine; NBT: nitrotetrazolium blue chloride; Y2H: yeast two-hybrid; DEG: differentially expressed gene; NJ: neighbor-joining; NCBI: national center for biotechnology information\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (32360075), the Scientific Research Project of Department of Science and Technology of Hubei Province (2022CFB694), \u0026quot;Yazhou Bay\u0026quot; Elite Talent Science and Technology Program of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2023-35), the Incubation Project of Hubei Minzu University (PY22005). The research was finished in Hubei Key Laboratory of Biological Resources Protection and Utilization and Yazhou Bay Seed Laboratory of Hainan Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eY.L designed the research. Y.W, F.C, X.W, and Y.C completed the bioinformatics analysis in this research. Y.L and Y.W conducted the qRT-PCR experiment. F.C, K.R and D.Z conducted ABA response, ROS accumulation and Y2H experiments. Y.L, Z.X, Z.H and Q.T dealed with the figures. Y.L wrote the main manuscript text. M.P and Q.F polished the manuscript. Y.Z revised the work. K.L and H.L provided reasonable advices. 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genome\u003c/strong\u003e. \u003cem\u003eMolecular Genetics and Genomics \u003c/em\u003e2015, \u003cstrong\u003e290\u003c/strong\u003e:1435-1446.\u003c/li\u003e\n\u003cli\u003eZhang S, Tian Z, Li H, Guo Y, Zhang Y, Roberts JA, Zhang X, Miao Y: \u003cstrong\u003eGenome-wide analysis and characterization of F-box gene family in Gossypium hirsutum L\u003c/strong\u003e. \u003cem\u003eBMC genomics \u003c/em\u003e2019, \u003cstrong\u003e20\u003c/strong\u003e:1-16.\u003c/li\u003e\n\u003cli\u003eSong JB, Wang YX, Li HB, Li BW, Zhou ZS, Gao S, Yang ZM: \u003cstrong\u003eThe F-box family genes as key elements in response to salt, heavy mental, and drought stresses in Medicago truncatula\u003c/strong\u003e. \u003cem\u003eFunctional \u0026amp; integrative genomics \u003c/em\u003e2015, \u003cstrong\u003e15\u003c/strong\u003e:495-507.\u003c/li\u003e\n\u003cli\u003eMo F, Zhang N, Qiu Y, Meng L, Cheng M, Liu J, Yao L, Lv R, Liu Y, Zhang Y: \u003cstrong\u003eMolecular characterization, gene evolution and expression analysis of the F-box gene family in tomato (Solanum lycopersicum)\u003c/strong\u003e. \u003cem\u003eGenes \u003c/em\u003e2021, \u003cstrong\u003e12\u003c/strong\u003e(3):417.\u003c/li\u003e\n\u003cli\u003eLuo Z, Xiong J, Xia H, Ma X, Gao M, Wang L, Liu G, Yu X, Luo L: \u003cstrong\u003eTranscriptomic divergence between upland and lowland ecotypes contributes to rice adaptation to a drought\u003c/strong\u003e\u003cstrong\u003e‐prone agroecosystem\u003c/strong\u003e. \u003cem\u003eEvolutionary Applications \u003c/em\u003e2020, \u003cstrong\u003e13\u003c/strong\u003e(9):2484-2496.\u003c/li\u003e\n\u003cli\u003eLuo L: \u003cstrong\u003eBreeding for water-saving and drought-resistance rice (WDR) in China\u003c/strong\u003e. \u003cem\u003eJournal of experimental botany \u003c/em\u003e2010, \u003cstrong\u003e61\u003c/strong\u003e(13):3509-3517.\u003c/li\u003e\n\u003cli\u003eXia H, Zheng X, Chen L, Gao H, Yang H, Long P, Rong J, Lu B, Li J, Luo L: \u003cstrong\u003eGenetic 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One \u003c/em\u003e2013, \u003cstrong\u003e8\u003c/strong\u003e(7):e68672.\u003c/li\u003e\n\u003cli\u003eJia Y, Gu H, Wang X, Chen Q, Shi S, Zhang J, Ma L, Zhang H, Ma H: \u003cstrong\u003eMolecular cloning and characterization of an F-box family gene CarF-box1 from chickpea (Cicer arietinum L.)\u003c/strong\u003e. \u003cem\u003eMolecular biology reports \u003c/em\u003e2012, \u003cstrong\u003e39\u003c/strong\u003e:2337-2345.\u003c/li\u003e\n\u003cli\u003eFeder A, Burger J, Gao S, Lewinsohn E, Katzir N, Schaffer AA, Meir A, Davidovich-Rikanati R, Portnoy V, Gal-On A: \u003cstrong\u003eA Kelch domain-containing F-Box coding gene negatively regulates flavonoid accumulation in muskmelon\u003c/strong\u003e. \u003cem\u003ePlant Physiology \u003c/em\u003e2015, \u003cstrong\u003e169\u003c/strong\u003e(3):1714-1726.\u003c/li\u003e\n\u003cli\u003eZhang X, Gou M, Guo C, Yang H, Liu C-J: \u003cstrong\u003eDown-regulation of Kelch domain-containing F-box protein in Arabidopsis enhances the production of (poly) phenols and tolerance to ultraviolet radiation\u003c/strong\u003e. \u003cem\u003ePlant Physiology \u003c/em\u003e2015, \u003cstrong\u003e167\u003c/strong\u003e(2):337-350.\u003c/li\u003e\n\u003cli\u003eWang M, Xu Z, Kong Y: \u003cstrong\u003eThe tubby-like proteins kingdom in animals and plants\u003c/strong\u003e. \u003cem\u003eGene \u003c/em\u003e2018, \u003cstrong\u003e642\u003c/strong\u003e:16-25.\u003c/li\u003e\n\u003cli\u003eBao Y, Song W-M, Jin Y-L, Jiang C-M, Yang Y, Li B, Huang W-J, Liu H, Zhang H-X: \u003cstrong\u003eCharacterization of Arabidopsis Tubby-like proteins and redundant function of AtTLP3 and AtTLP9 in plant response to ABA and osmotic stress\u003c/strong\u003e. \u003cem\u003ePlant molecular biology \u003c/em\u003e2014, \u003cstrong\u003e86\u003c/strong\u003e:471-483.\u003c/li\u003e\n\u003cli\u003eLi Z, Wang X, Cao X, Chen B, Ma C, Lv J, Sun Z, Qiao K, Zhu L, Zhang C: \u003cstrong\u003eGhTULP34, a member of tubby-like proteins, interacts with GhSKP1A to negatively regulate plant osmotic stress\u003c/strong\u003e. \u003cem\u003eGenomics \u003c/em\u003e2021, \u003cstrong\u003e113\u003c/strong\u003e(1):462-474.\u003c/li\u003e\n\u003cli\u003eThulasi Devendrakumar K, Copeland C, Adamchek C, Zhong X, Huang X, Gendron JM, Li X: \u003cstrong\u003eArabidopsis Tubby domain\u003c/strong\u003e\u003cstrong\u003e‐containing F\u003c/strong\u003e\u003cstrong\u003e‐box proteins positively regulate immunity by modulating PI4K\u003c/strong\u003e\u003cstrong\u003e\u0026beta; protein levels\u003c/strong\u003e. \u003cem\u003eNew Phytologist \u003c/em\u003e2023, \u003cstrong\u003e240\u003c/strong\u003e(1):354-371.\u003c/li\u003e\n\u003cli\u003eKou Y, Qiu D, Wang L, Li X, Wang S: \u003cstrong\u003eMolecular analyses of the rice tubby-like protein gene family and their response to bacterial infection\u003c/strong\u003e. \u003cem\u003ePlant cell reports \u003c/em\u003e2009, \u003cstrong\u003e28\u003c/strong\u003e:113-121.\u003c/li\u003e\n\u003cli\u003eTroukhan M, Tatarinova T, Bouck J, Flavell RB, Alexandrov NN: \u003cstrong\u003eGenome-wide discovery of cis-elements in promoter sequences using gene expression\u003c/strong\u003e. \u003cem\u003eOMICS A Journal of Integrative Biology \u003c/em\u003e2009, \u003cstrong\u003e13\u003c/strong\u003e(2):139-151.\u003c/li\u003e\n\u003cli\u003eAbd-Hamid N-A, Ahmad-Fauzi M-I, Zainal Z, Ismail I: \u003cstrong\u003eDiverse and dynamic roles of F-box proteins in plant biology\u003c/strong\u003e. \u003cem\u003ePlanta \u003c/em\u003e2020, \u003cstrong\u003e251\u003c/strong\u003e:1-31.\u003c/li\u003e\n\u003cli\u003eHan R, Rai A, Nakamura M, Suzuki H, Takahashi H, Yamazaki M, Saito K: \u003cstrong\u003eDe novo deep transcriptome analysis of medicinal plants for gene discovery in biosynthesis of plant natural products\u003c/strong\u003e. \u003cem\u003eMethods in enzymology \u003c/em\u003e2016, \u003cstrong\u003e576\u003c/strong\u003e:19-45.\u003c/li\u003e\n\u003cli\u003eSadat MA, Ullah MW, Bashar KK, Hossen QMM, Tareq MZ, Islam MS: \u003cstrong\u003eGenome-wide identification of F-box proteins in Macrophomina phaseolina and comparison with other fungus\u003c/strong\u003e. \u003cem\u003eJournal of Genetic Engineering and Biotechnology \u003c/em\u003e2021, \u003cstrong\u003e19\u003c/strong\u003e(1):46.\u003c/li\u003e\n\u003cli\u003eVierstra RD: \u003cstrong\u003eThe ubiquitin\u0026ndash;26S proteasome system at the nexus of plant biology\u003c/strong\u003e. \u003cem\u003eNature reviews Molecular cell biology \u003c/em\u003e2009, \u003cstrong\u003e10\u003c/strong\u003e(6):385-397.\u003c/li\u003e\n\u003cli\u003eDreher K, Callis J: \u003cstrong\u003eUbiquitin, hormones and biotic stress in plants\u003c/strong\u003e. \u003cem\u003eAnnals of botany \u003c/em\u003e2007, \u003cstrong\u003e99\u003c/strong\u003e(5):787-822.\u003c/li\u003e\n\u003cli\u003eCheng C, Wang Z, Ren Z, Zhi L, Yao B, Su C, Liu L, Li X: \u003cstrong\u003eSCFAtPP2-B11 modulates ABA signaling by facilitating SnRK2. 3 degradation in Arabidopsis thaliana\u003c/strong\u003e. \u003cem\u003ePLoS genetics \u003c/em\u003e2017, \u003cstrong\u003e13\u003c/strong\u003e(8):e1006947.\u003c/li\u003e\n\u003cli\u003eKim H, Yu S-i, Jung SH, Lee B-h, Suh MC: \u003cstrong\u003eThe F-box protein SAGL1 and ECERIFERUM3 regulate cuticular wax biosynthesis in response to changes in humidity in Arabidopsis\u003c/strong\u003e. \u003cem\u003eThe Plant Cell \u003c/em\u003e2019, \u003cstrong\u003e31\u003c/strong\u003e(9):2223-2240.\u003c/li\u003e\n\u003cli\u003edel Pozo JC, Estelle M: \u003cstrong\u003eF-box proteins and protein degradation: an emerging theme in cellular regulation\u003c/strong\u003e. \u003cem\u003ePlant Molecular Biology \u003c/em\u003e2000, \u003cstrong\u003e44\u003c/strong\u003e:123-128.\u003c/li\u003e\n\u003cli\u003eWu R, Song K, Jing R, Du L: \u003cstrong\u003eThe de-ubiquitinase UBQUITIN SPECIFIC PROTEASE 15 (UBP15) interacts with the SCF E3 complex adaptor ARABIDOPSIS SKP1 HOMOLOGUE 1 (ASK1) to regulate petal size and fertility in Arabidopsis thaliana\u003c/strong\u003e. \u003cem\u003ePlant Science \u003c/em\u003e2024:112112.\u003c/li\u003e\n\u003cli\u003eWilkins TA, Smart LB: \u003cstrong\u003eIsolation of RNA from plant tissue\u003c/strong\u003e. \u003cem\u003eA laboratory guide to RNA: isolation, analysis, and synthesis \u003c/em\u003e1996:21-42.\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD: \u003cstrong\u003eAnalysis of relative gene expression data using real-time quantitative PCR and the 2\u0026minus; \u0026Delta;\u0026Delta;CT method\u003c/strong\u003e. \u003cem\u003eMethods (San Diego, Calif) \u003c/em\u003e2001, \u003cstrong\u003e25\u003c/strong\u003e(4):402-408.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eThe basic physicochemical properties of drought-responsive F-box proteins (*Grand Average of Hydropathicity)\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eProtein Name\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eLength(aa)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003epI\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eMW(Da)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003eInstability index\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eSubcellular localization\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003eGRAVY\u003csup\u003e*\u003c/sup\u003e\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX342\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e415\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e6.75\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e45858.32\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e41.77\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eCytosol\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.089\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX150\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1526\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e7.56\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e159241.75\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e52.23\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eCytosol\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.114\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX77\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e178\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e9.39\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e18611.57\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e47.80\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003echloroplast\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.185\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBK8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e391\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.39\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e40366.16\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e48.52\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003enucleus\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.162\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBL28\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e276\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e10.48\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e31021.08\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e60.61\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003echloroplast\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.245\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX261\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e299\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e31611.02\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e51.7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eCytosol\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.064\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBO10\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e509\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e6.60\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e56258.32\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e54.11\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003enucleus\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.209\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBT6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e462\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e9.35\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e51529.37\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e61.74\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003enucleus\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.495\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX281\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e463\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.75\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e51844.52\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e59.04\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003enucleus\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.163\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX563\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e677\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e8.46\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e73677.52\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e44.98\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003enucleus\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.188\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBD11\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e526\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e8.91\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e57806.71\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e42.88\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eCytosol\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.121\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX283\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e541\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e8.37\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e61342.95\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e58.67\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003enucleus\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.236\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX258\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e406\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.41\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e42650.39\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e47.20\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eCytosol\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.011\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX438\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e302\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.68\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e34389.78\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e43.93\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003enucleus\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.394\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBD8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e475\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e9.28\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e51705.91\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e57.37\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003enucleus\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.034\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX345\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1078\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.51\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e123349.83\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e43.55\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003echloroplast\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.136\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX44\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e422\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.27\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e47173.74\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e64.42\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eCytosol\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.255\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBK25\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e383\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e6.11\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e40796.99\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e47.69\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eCytosol\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.192\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX315\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e447\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e9.69\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e49837.08\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e55.97\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003echloroplast\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.286\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBX220\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv 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align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.33\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e79222.78\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e55.31\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eEndoplasmic reticulum\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.010\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOsFBDUF13\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e377\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv 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Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Upland rice, abiotic stress, transcriptome-wide identification, OsFBX148, ABA response","lastPublishedDoi":"10.21203/rs.3.rs-4598345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4598345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Upland rice varieties exhibit significant genetic diversity and broad environmental adaptability, making them ideal candidates for identifying consistently expressed stress-responsive genes. F-box proteins typically function as part of the SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes to precisely regulate gene expression and protein level, playing essential roles in the modulation of abiotic stress responses. Therefore, utilizing upland rice varieties for screening stress-responsive F-box genes is a highly advantageous approach.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThrough mRNA-seq analysis in the Brazilian upland rice (cv. IAPAR9), the research identified 29 drought-responsive F-box genes. Gene distribution and duplication analysis revealed these genes are distributed on 11 of the 12 chromosomes and 10 collinear gene pairs were identified on different chromosomes. 13 cis-elements or binding sites were identified in the promoters of the 29 drought-responsive F-box genes. These F-box proteins possess F-box domain and several other domains, and they are mostly unstable proteins with subcellular localization in cytoplasm, nucleus, chloroplasts, mitochondria and endoplasmic reticulum. Most of drought-responsive F-box genes exhibited expression in various tissues such as root, stem, leaf, leaf sheath and panicle except for \u003cem\u003eOsFBO10\u003c/em\u003e and \u003cem\u003eOsFBX283\u003c/em\u003e. These genes exhibited various responses to abiotic stresses such as osmotic, cold, heat, and salt stresses, along with ABA treatment. Importantly, a frame-shift mutation in \u003cem\u003eOsFBX148\u003c/em\u003e was created in the ZH11 variety, leading to altered ABA signal transduction and ROS accumulation. The study further elucidated the interaction of OsFBX148 with SKP1 family proteins OSK4/7/17 to form the SCF complex, dependent on the F-box domain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003eThe research identified and analyzed 29 drought-responsive F-box genes in upland rice and provides valuable insights into the role of OsFBX148 in ABA and ROS responses. It establishes a basis for future exploration of F-box genes in improving resistance to abiotic stresses, especially drought.\u003c/p\u003e","manuscriptTitle":"Identification and analysis of drought-responsive F-box genes in upland rice and involvement of OsFBX148 in ABA response and ROS accumulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 07:04:51","doi":"10.21203/rs.3.rs-4598345/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-26T09:08:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-25T15:54:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53006652227930242376720238649080204958","date":"2024-09-08T03:27:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-14T00:30:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133396866820929219580995713821796534495","date":"2024-08-03T22:03:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134037140427571681739213623078964642160","date":"2024-08-02T20:52:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207585049672105186424274014752867192498","date":"2024-07-11T09:37:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274615579079452016783805883286427405972","date":"2024-07-08T06:59:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-22T20:47:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-21T11:48:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-21T11:46:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-06-18T08:05:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6cfdef21-41ca-4a8c-b2b7-03445bf8e25f","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T15:58:54+00:00","versionOfRecord":{"articleIdentity":"rs-4598345","link":"https://doi.org/10.1186/s12870-024-05820-z","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-11-25 15:56:51","publishedOnDateReadable":"November 25th, 2024"},"versionCreatedAt":"2024-07-12 07:04:51","video":"","vorDoi":"10.1186/s12870-024-05820-z","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05820-z","workflowStages":[]},"version":"v1","identity":"rs-4598345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4598345","identity":"rs-4598345","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}