Transcriptome profiling of nla-mutant Arabidopsis reveals possible post-translational regulation of key factors cross-linking circadian-rhythm and anthocyanin pathways under boron toxicity

Transcriptome profiling of nla-mutant Arabidopsis reveals possible post-translational regulation of key factors cross-linking circadian-rhythm and anthocyanin pathways under boron toxicity | 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 Article Transcriptome profiling of nla-mutant Arabidopsis reveals possible post-translational regulation of key factors cross-linking circadian-rhythm and anthocyanin pathways under boron toxicity Doga Selin Kayihan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7334872/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Understanding molecular responses against boron (B) stress is one of the goals for improving excess B management of cereals. Since earlier findings demonstrated the differential regulation of protein degradation genes under B toxicity, this study focused on the interaction between toxic B responsive regulations and Nitrogen Limitation Adaptation ( NLA ) gene encoding an E3 ubiquitin ligase. Therefore, WT and nla mutant Arabidopsis thaliana plants were grown under mild and moderate levels of B toxicity and RNA sequencing was performed in these plants. Accordingly, ribosome was overrepresented for upregulated genes in nla mutants under all conditions when compared to WT whereas alpha-linolenic acid metabolism, plant-pathogen interaction and MAPK signaling pathways were downregulated. Inhibition of glucosinolate biosynthesis and induction of phenylpropanoid pathway as well as enrichment of circadian rhythm were among the most prominent results for B-stressed nla mutants compared to nla mutant Arabidopsis under control condition (C). Interestingly, unlike WT, B-induced accumulation of anthocyanin was not observed in nla mutants. This was attributed to switching from flavonoid to lignin biosynthesis in the phenylpropanoid pathway. Also, the impairment of crosstalk between circadian rhythm and anthocyanin pathways might explain this phenomenon because two pathways are crosslinked by ORE1, one of the targets of NLA. Biological sciences/Molecular biology Biological sciences/Plant sciences anthocyanin Arabidopsis thaliana Boron toxicity NITROGEN LIMITATION ADAPTATION nla mutant RNA sequencing transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Boron (B) micronutrient, which is essential for the healthy development of plants [1], readily becomes toxic depending on its concentration in the soil [2]. Thus, many crops suffer from B toxicity which, in turn, leads to yield-loss worldwide due to consequences like delay in emergence and foliation, reduced stem height, dry matter weight, 1000-kernel weight, and number of spikes per plant. Indeed, excessive B levels were reported in the soils of many countries including United States, Australia, Turkey, Russia, India, Israel, Mexico, Egypt, Iraq, Morocco, Syria, Libya, Jordan, Malaysia, Chile, Pakistan, Peru, Hungary, Italy and Serbia [3]. The macroscopic side effects of B excess have been attributed to its ability to form ester bonds with several metabolites having multiple hydroxyl groups in the cis-configuration including ribose sugar [4,5]. Namely, B impairs cell division and development by binding to ribose, both as the free sugar and as a constituent of RNA and interferes with primary metabolism by binding to ribose in ATP or NAD(P)H. In addition, it reduces cytosolic pH, thus affects protein conformation and biosynthesis [5]. Plant cells combat these toxic effects of B through a variety of mechanisms including B efflux by B-transporters which export excess B from tissues [6]. Another way plants have been suggested to use is the reduction of free B in cytosol by complexing with anthocyanins which is followed by the subsequent compartmentalization of B-anthocyanin complex in vacuoles and/or efflux of this complex from the cell by ABC transporters [7]. Supportively, induced accumulation of anthocyanin by high B levels was postulated in several studies [8, 9, 10, 11]. One main approach to improve the management of B by cereals is the development of agricultural plants adjusted to its broad range concentrations [12], which may be realized by understanding molecular responses against B toxicity [13]. A previous study demonstrated the differential regulation of protein degradation pathway in wheat cultivars having different tolerance capacities against B toxicity [14]. This allowed us to focus our study on proteasome members in Arabidopsis thaliana . NITROGEN LIMITATION ADAPTATION (NLA) is one of the post-translational regulators of different macronutrient-transporters. In fact, it is an E3 ubiquitin ligase [15] directing ubiquitination of a nitrate transporter (NRT1.7) [16] which is responsible for loading of N in source leaves for the remobilization of N to N-demanding tissues [17]. On the other hand, NLA is crucial for Pi homeostasis due to its contribution to the regulation of phosphate transporters (e.g PHT1) that uptake Pi from the rhizosphere [18]. Phosphate starvation leads to post-transcriptional degradation of NLA mRNAs by means of the microRNA miR827 and, in turn, to the increase of phosphate by the accumulation of phosphate transporters PHT1.4 [19]. In this polyubiquitination process, NLA specifically requires PHO2, an E2 conjugase. Furthermore, NLA has an SPX-domain which interacts with nuclear transcription factors that control the phosphate starvation response pathway [20] suggesting the NLA is also localized in nucleus [21, 22, 18]. All domains of NLA are essential for proper functioning as well as its localization to the plasma membrane and/or nucleus [21]. In addition to these certain roles of NLA in regulation of the membrane transporters, some other functions also were proposed. For instance, it was reported that NLA was involved in molecular responses against biotic stress sources like Pseudomonas syringae [23] and Heterodera schachtii by interacting with protein substrates like pathogenesis-related proteins, MAP kinases, and transcription factors [24]. Finally, NLA was found to target ORE1 which is a key NAC transcription factor regulating age-dependent leaf senescence in A. thaliana [25]. The interaction of NLA with ORE1 in the nucleus regulates its stability and ORE1 level arranged through NLA mediated polyubiquitination using PHO2 determines leaf senescence during nitrogen deficiency [25]. In other words, NLA seems to target a variety of proteins in subcellular compartments. “NITROGEN LIMITATION ADAPTATION” name comes from the inability of nla mutant plants to develop adaptive responses such as anthocyanin accumulation under nitrogen (N)-limited conditions which results in early senescence [22]. Interestingly, this repressed-anthocyanin-accumulation phenomenon of nla mutant was not observed in phosphorous deficient plants [26]. Thus, while NLA contributes to the regulation of different transporters, the respective pathways are likely to be regulated differently where involving mechanisms remain to be discovered. In this study, as a preliminary work, we found the remarkable induction of NLA gene expression in Arabidopsis thaliana through increasing levels of B toxicity (Table S1a). For this reason, in order to gain a more precise insight about the possible relationship between NLA gene and B responsive regulations at molecular level, transcriptome profiling of nla mutant Arabidopsis thaliana exposed to toxic B was determined. Our data might benefit the determination of candidate genes for molecular breeding, and genetic manipulation of plants for B toxicity. Materials and Methods Plant material and Growth condition The seeds of Arabidopsis thaliana ecotype Columbia-0 (Col-0) were provided by Assistant Prof. Dr. Emre Aksoy at Middle East Technical University in Turkey. The seeds of homozygous nla mutant Arabidopsis (N864802) were procured from Nottingham Arabidopsis Stock Centre. Both wild type (Col-0) and nla mutant seeds were surface sterilized with 70% EtOH solution for 2 minutes, then with 15% NaOCl solution for 10 minutes, and then rinsed three times with sterile distilled water. Then, seeds were sown onto half MS media [27] containing 100 µM H 3 BO 3 (control), 1 mM H 3 BO 3 (mild B stress, 1B) and 2 mM H 3 BO 3 (moderate B stress, 2B). After 3 days of stratification at 4°C in dark, they were transferred to plant growth room providing 21 ± 2°C, 16 h light/8 h dark photoperiod with 150 µmol m -2 sec -1 illumination, 60±5% relative humidity and grown for 2 weeks. Photosynthetic pigment and anthocyanin measurement The growth rate of fourteen days old nla seedlings were phenotypically compared with Col-0 under control and toxic B conditions. Following measurement of the fresh weights of leaf tissues, contents of photosynthetic pigments were determined using the method of [28]. Accordingly, 2 ml of pure acetone was added onto seedlings in a tube followed by incubation at 4°C overnight. The contents of pigments were calculated by the following equations after measuring the samples spectrophotometrically at 661.6, 644.8 and 470 nm. The contents of anthocyanin, as a flavonoid pigment, were determined in seedlings under B stress according to [29]. Accordingly, the seedlings were homogenized with 1 mL of extraction buffer (400 mL of 37% H 2 O 2 , 2.6 ml of dH 2 O, and 12 ml of 100% methanol). They were transferred and incubated into tubes at 22 °C for 10 min. They were centrifuged at 15,000 × g for 5 min and supernatant was read at 530 and 657 nm. The following equation was used for anthocyanin content: [A530-(A657/3)]/FW. RNA-seq library construction and sequencing Total RNAs were isolated from 300 mg leaf tissues of Arabidopsis seedlings [30]. The cDNA libraries were generated from the rRNA-depleted RNA samples using an MGIEasy RNA Directional Library Prep Set by Intergen. Annotation was conducted using TAIR10 FASTA sequence (https://www.arabidopsis.org). RNA quantity and purity were evaluated by the BioSpec-nano UV–VIS spectrophotometer (Shimadzu Europa GmbH). RNA integrity was assessed using the Agilent 2100 Bioanalyser (Agilent Technologies, USA) prior to the RNA-seq. Two hundred nanograms of total RNA was used for sequencing on the MGI DNBSEQ-G400RS instrument (MGI, Shenzhen, China). The dsDNA library quantity was evaluated using a Qubit® dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, US) and a Qubit® Fluorometer (Thermo Fisher Scientific, US). Genes presenting a log2 (fold change>1) with adjusted p‐value (≤ 0.05) for each comparison were considered differentially expressed. Principal component analysis (PCA) was performed to assess the correlation between replicates and treatments using ClustVis. The set of differentially expressed genes (DEGs) was identified from pairwise comparisons. Venn Diagrams comparing the number of DEG across samples were created using webtools (https://bioinformatics.psb.ugent.be/webtools/Venn/). Gene ontology (GO) analysis of specific groups of DEGs was performed using ShinyGO (0.82) [31]. All DEGs were mapped to terms in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) because pathway-based analysis improved our understanding of the molecular functions of genes [32]. Read counts (gene expression quantification) per gene were obtained using featureCounts from Rsubread, and analyzed using DESeq2 (Love et al., 2014) (Bioconductor packages in R) after adjustment for false-discovery rate (FDR q values < 0.05) and Relative Log Expression (RLE) normalization. To gain a further overview, the network analyses of the selected DEGs were performed using the STRING database [33]. qRT-PCR analysis To validate the reliability of the transcriptome analysis, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed for randomly selected genes (Figure S1a). The same total RNAs used in RNA-seq were used as templates. 500 ng RNA was used to obtain first strand complementary DNA (cDNA) by using iScript cDNA Synthesis Kit (BioRad). Every sample contained 1 µl of cDNA, 10 µl of iTaq universal SYBR Green super mix (2X) (Bio-Rad), 1 µl of forward primer (0.5 µM final concentration) and 1 µl of reverse primer (0.5 µM final concentration) and nuclease free water for a final volume of 20 µl. This mixture was used for qRT-PCR experiments (Tianlong, Gentier 96E). Primers were designed from exon-exon boundary of the sequences of each gene by using NCBI database and they are shown in Table S1. Conditions of qRT-PCR were initial denaturation at 95 °C for 30 s, followed by 95 °C for 10 s, 60 °C for 1 min for 40 cycles. Conditions of melting curve were at 95 °C for 5s, at 65 °C for 1 min, and cooling for 40 °C for 30s. Expression levels of genes were normalized by using actin ( ACT2 ) gene. The relative fold changes of expressions genes were calculated by 2 -deltadeltaCt . Data statistics RNA-seq experiment was conducted in replicates of two. Experiments of photosynthetic pigment and anthocyanin and qRT-PCR were performed with at least three biological replicates. One-way ANOVA by SPSS statistical program was used to analyze the data obtained from photosynthetic pigment and anthocyanin measurement. The data of qRT-PCR were statistically analyzed by using non-parametric versions of the t-test. They were presented as mean ± standard error of mean (SEM). Results Lower anthocyanin levels of nla mutant A. thaliana than WT under all B conditions Excess B reduced overall seedling growth and caused varying degrees of leaf curling and chlorosis in all Arabidopsis seedlings (Figure S1b). Photosynthetic pigments were affected by B toxicity in both genotypes (Table 1). In WT, chlorophyll a and carotenoid concentrations gradually declined with increasing B applications, whereas chlorophyll b remained relatively stable. In contrast, the concentrations of all photosynthetic pigments (chlorophyll a, b, and carotenoids) significantly decreased in nla mutants under both 1B and 2B conditions. Consequently, no significant differences in these pigment concentrations were detected between the two toxic B treatments in nla mutant A. thaliana . Anthocyanin accumulation exhibited opposing trends in the two genotypes (Table 1). In WT, anthocyanin content slightly decreased under 1B but increased markedly under 2B compared to the control. In nla mutants, anthocyanin levels increased steadily with B concentration; however, their overall anthocyanin content remained considerably lower than those in WT under all conditions, including control. Under 2B, WT had over 4.5 times more anthocyanin than nla mutant A. thaliana . Table 1. Effects of B toxicity on physio-biochemical parameters in A. thaliana seedlings Treatment C 1B 2B Col-0 nla Col-0 nla Col-0 nla Chl a (µg ml -1 g -1 ) 1081±112 a 929±122 ab 879±61 b 538±83 d 766±75 bc 658±206 cd Chl b (µg ml -1 g -1 ) 337±42 a 288±67 ab 306±57 a 198±22 c 312±42 a 225±58 bc Carotenoid (µg ml -1 g -1 ) 328±33 a 287±21 ab 269±17 bc 207±42 d 247±18b cd 219±56 cd Anthocyanin (Abs g-1) 0.030±0,006 b 0.006±0.001 d 0.022±0.002 a 0.010±0.003 de 0.070±0.006 c 0.015±0.001 e Values followed by different letters are significantly different at P < 0.05 level. Transcriptome profiling identified 4698 and 530 DEGs in B-stressed nla mutant Arabidopsis compared to WT-C and nla-C Next, the molecular response of nla mutant and WT under B toxicity conditions were determined by carrying out global gene expression using RNA-sequencing. Accordingly, Principial Component Analysis (PCA) of the normalized gene expressions showed an obvious separation of WT group from nla mutant groups (Figure 1). The principal components explained 70.4% of the total variance (52.1% by PC1 and 18.3% by PC2). In general, biological replicates were closely grouped. One replicate of the 1B samples was slightly associated with mutant control. Differentially expressed genes (DEGs) in response to different B conditions were screened with the threshold of adjusted P value < 0.05. When compared to WT under control (C) condition (hereafter WT-C), 2568, 2320 and 4323 DEGs were determined in nla mutant plants under C, 1B and 2B conditions, respectively. When compared to nla mutant plants under C conditions (hereafter nla-C), 181 and 403 DEGs were detected in nla plants under 1B and 2B conditions, respectively. When compared to WT-C, 826, 893, 1751 genes were differentially upregulated (Figure 2a) and 1742, 1427, 2572 genes were downregulated (Figure 2b) in nla mutants under C, 1B and 2B conditions, respectively. A total of 4698 (1971 up and 2727 down) DEGs were identified in B stressed nla mutants when compared to WT-C. From these DEGs, 673 were commonly upregulated, and 1272 were commonly downregulated in nla mutants exposed to toxic B conditions (Figure 2a, 2b). 177, 148 and 927 genes were specifically upregulated whereas 204, 97 and 862 genes were specifically downregulated under C, 1B and 2B conditions, respectively (Figure 2a, 2b). A total of 530 (243 up and 287 down) DEGs were identified in B stressed nla mutants when compared to nla-C (Figure 2c, 2d). From these DEGs, 16 were commonly upregulated, and 38 were commonly downregulated in nla mutants exposed to toxic-B (Figure 2c, 2d). 116 and 111 genes were specifically upregulated whereas 11 and 238 genes were specifically downregulated under 1B and 2B conditions, respectively (Figure 2c, 2d). GO and KEGG Enrichment Analyses of DEGs in nla under C and toxic B conditions relative to WT-C According to Gene Ontology (GO) analysis, “ribosome” was overrepresented for upregulated genes in nla mutant Arabidopsis under C condition (Figure 3a). In terms of molecular function, the processes represented by the GO terms “Pectate lyase activity”, “Histone methyltransferase activity”, “Histone-lysine N-methyltransferase activity”, “Lysine N-methyltransferase activity”, “Protein-lysine N-methyltransferase activity”, “Protein methyltransferase activity”, “N-methyltransferase activity”, “RRNA binding”, “Structural constituent of ribosome”, “MRNA binding”, “RNA binding” were significantly enriched (Figure S2). In the biological process ontology, the major terms were “Phylloquinone biosynthetic proc”, “Vitamin K biosynthetic proc.”, “Vitamin K metabolic proc”, “Translation”, “Peptide biosynthetic proc.”, “Peptide metabolic proc.”, “Cellular amide metabolic proc.” and “Organonitrogen compound biosynthetic proc.” (Figure S3). According to GO analysis, “Linoleic acid metabolism”, “Alpha-Linolenic acid metabolism”, “Tryptophan metabolism”, “Plant pathogen interaction” “MAPK signaling pathway-plant”, “Plant hormone signal transduction”, “Endocytosis” and “Biosynthesis of secondary metabolites” were overrepresented for downregulated genes in nla mutants under control condition (Figure 3b). According to KEGG analyses, lipoxygenases (e.g., EC 1.13.11.58 in LA and EC 1.13.11.12 in LA and ALA) were highlighted in “Linoleic acid metabolism” and “Alpha-Linolenic acid metabolism” pathways (Figue S4 and S5). In the biological process ontology, the major terms were “Cellular response to hypoxia”, “Cellular response to decreased oxygen levels”, “Response to hypoxia”, “Response to oxygen levels” and “Response to wounding” (Figure S4). Regarding molecular function, the major terms were “Xyloglucan:xyloglucosyl transferase activity” and “Glucosyltransferase activity” (Figure S5). According to GO analysis, “Phenylpropanoid biosynthesis”, “ribosome” and “Biosynthesis of secondary metabolites” were overrepresented for upregulated genes in nla mutant Arabidopsis under 1B condition compared to WT-C (Figure 3c). In terms of molecular function, the processes represented by the GO terms “NAD+ nucleosidase activity”, “NAD(P)+ nucleosidase activity”, “NAD+ nucleotidase cyclic ADP-ribose generating”, “RRNA binding”, “Structural constituent of ribosome” were significantly enriched (Figure S6). In the biological process ontology, the major terms were “Coumarin metabolic proc.” and “Translational elongation” (Figure S7). “Glucosinolate biosynthesis”, “Alpha-Linolenic acid metabolism”, “Plant-pathogen interaction”, “MAPK signaling pathway-plant”, “Plant hormone signal transduction” and “Biosynthesis of secondary metabolites” were overrepresented for downregulated genes in nla mutants under 1B when compared to WT control (Figure 3d). In the biological process ontology, the major terms were “Cellular response to hypoxia”, “Cellular response to decreased oxygen levels”, “Response to hypoxia”, “Response to oxygen levels” (Figure S8). In terms of molecular function, the processes represented by the GO terms “Linoleate 13S-lipoxygenase activity”, “Allene-oxide cyclase activity”, “Diacylglycerol kinase activity” and “Xyloglucan:xyloglucosyl transferase activity” were significantly enriched (Figure S9). According to GO analysis, “ribosome”, “Ribosome biogenesis in eukaryotes”, “Pyrimidine metabolism” and “RNA degradation” were overrepresented for upregulated genes in nla mutant Arabidopsis under 2B condition when compared to WT control (Figure 3e). In terms of molecular function, the processes represented by the GO terms “Large ribosomal subunit rRNA binding”, “MRNA 5-prime-UTR binding”, “Small ribosomal subunit rRNA binding”, “RRNA binding”, “SnoRNA binding” and “Structural constituent of ribosome” were significantly enriched (Figure S10). In the biological process ontology, the major terms were “Ribosomal large subunit biogenesis”, “Ribosome biogenesis”, “Translation”, “Peptide biosynthetic proc.”, “RRNA processing”, “RRNA metabolic proc.”, “Amide biosynthetic proc.”, “Peptide metabolic proc.”, “Ribonucleoprotein complex biogenesis” and “Cellular amide metabolic proc.” (Figure S11). “Glucosinolate biosynthesis”, “Taurine and hypotaurine metabolism”, “Linoleic acid metabolism”, “Alpha-Linolenic acid metabolism” and “Plant-pathogen interaction” were overrepresented for downregulated genes in nla mutants under 2B when compared to WT control (Figure 3f). KEGG analysis was performed for Taurine and hypotaurine metabolism pathway and given as supplementary file (Figure S12). In the biological process ontology, the major terms were “Cellular response to hypoxia”, “Cellular response to decreased oxygen levels”, “Response to hypoxia”, “Response to oxygen levels” (Figure S13). In terms of molecular function, the processes represented by the GO terms “Calmodulin binding”, “Protein serine kinase activity”, “Protein serine/threonine/tyrosine kinase activity”, “Protein serine/threonine kinase activity”, “Protein kinase activity”, “Phosphotransferase activity alcohol group as acceptor” and “Kinase activity” were significantly enriched (Figure S14). GO and KEGG Enrichment Analyses of DEGs in nla under toxic B conditions relative to nla-C According to GO analysis, “Alpha-Linolenic acid metabolism”, “Phenylpropanoid biosynthesis”, and “Biosynthesis of secondary metabolites” were overrepresented for upregulated genes in nla mutant Arabidopsis under 1B condition compared to nla control (Figure 4a). KEGG analysis was performed for Alpha-Linolenic acid metabolism and Phenylpropanoid biosynthesis pathways and given as supplementary file (Figure S15 and S16). In terms of molecular function, the processes represented by the GO terms “Linoleate 13S-lipoxygenase activity”, “Xyloglucan:xyloglucosyl transferase activity”, “Hydroquinone:oxygen oxidoreductase activity”, “Ferric-chelate reductase activity”, “Oxidoreductase activity acting on single donors with incorporation of molecular oxygen” and “Polysaccharide binding” were significantly enriched (Figure S17). In the biological process ontology, the major terms were “Cellular response to hypoxia”, “Cellular response to decreased oxygen levels”, “Response to hypoxia”, “Response to oxygen levels” and “Phenylpropanoid metabolic proc.” (Figure S18). On the other hand, “Photosynthesis-antenna proteins”, “Glucosinolate biosynthesis” and “2-Oxocarboxylic acid metabolism” were overrepresented for downregulated genes in nla mutants under 1B compared to nla control (Figure 4b). In the biological process ontology, the major terms were “Reg. of salicylic acid mediated signaling pathway”, “Systemic acquired resistance”, “Cellular response to salicylic acid stimulus”, “Salicylic acid mediated signaling pathway”, “Response to virus”, “Response to salicylic acid” and “Cellular response to organic cyclic compound” (Figure S19). In terms of molecular function, the processes represented by the GO terms “Chlorophyll binding”, “Beta-glucosidase activity” and “Glucosidase activity” were significantly enriched (Figure S20). According to GO analysis, “Circadian rhythm-plant” was overrepresented for upregulated genes in nla mutant Arabidopsis under 2B condition compared to nla control (Figure 4c). KEGG analysis was performed for Circadian rhythm-plant pathway and given as supplementary file (Figure S21). In terms of molecular function, the processes represented by the GO terms “Large ribosomal subunit rRNA binding” and “RRNA binding” were significantly enriched (Figure S22). In the biological process ontology, the major terms were “Negative reg. of circadian rhythm”, “Reg. of circadian rhythm”, “Photomorphogenesis”, “Cellular response to oxidative stress”, “Cellular response to chemical stress” and “Cellular response to light stimulus” (Figure S23). On the other hand, “Glucosinolate biosynthesis”, “Monobactam biosynthesis”, “Photosynthesis-antenna proteins”, “2-Oxocarboxylic acid metabolism”, “Sulfur metabolism”, “Valine leucine and isoleucine biosynthesis” and “Alpha-Linolenic acid metabolism” were overrepresented for downregulated genes in nla mutants under 2B compared to nla control (Figure 4d). In the biological process ontology, the major terms were “S-glycoside biosynthetic proc.”, “Glycosinolate biosynthetic proc.”, “Glucosinolate biosynthetic proc.”, “S-glycoside metabolic proc”, “Glycosinolate metabolic proc.”, “Glucosinolate metabolic proc.” and “Sulfur compound biosynthetic proc.” (Figure S24). In terms of molecular function, the processes represented by the GO terms “Adenylylsulfate kinase activity”, “Oxidoreductase activity acting on other nitrogenous compounds as donors” and “Glutathione binding” were significantly enriched (Figure S25). GO Enrichment Analysis of DEGs in nla under 2B condition relative to 1B condition According to GO analysis, “Base excision repair” was overrepresented for upregulated genes in nla mutant Arabidopsis under 2B condition relative to 1B (Figure 4e). In terms of molecular function, the processes represented by the GO terms “NAD+ ADP-ribosyltransferase activity” was significantly enriched (Figure S26). In the biological process ontology, no significant enrichment was found. On the other hand, “Alpha-Linolenic acid metabolism”, “Sulfur metabolism”, “Plant-pathogen interaction”, “Cysteine and methionine metabolism” and “Phenylpropanoid biosynthesis” were overrepresented for downregulated genes in nla mutant Arabidopsis under 2B condition relative to 1B (Figure 4f). In terms of molecular function, the processes represented by the GO terms “Allene-oxide cyclase activity”, “Polysaccharide binding”, “Symporter activity”, “Secondary active transmembrane transporter activity”, “Heme binding”, “Calmodulin binding” and “Carbohydrate binding” were significantly enriched (Figure S27). In the biological process ontology, the major terms were “Cellular response to hypoxia”, “Cellular response to decreased oxygen levels”, “Response to hypoxia”, “Response to oxygen levels”, “Response to wounding” and “Response to bacterium” (Figure S28). GO and KEGG Enrichment Analyses of commonly and specifically up- and down-regulated DEGs 426 genes were commonly upregulated in mutant plants under control, 1B and 2B conditions when compared to WT-C (Figure 2a). These DEGs were overrepresented by “Ribosome”. Their GO Biological Process analysis showed that “Phylloquinone biosynthetic proc”, “Phylloquinone metabolic proc”, “Vitamin K biosynthetic proc.” and “Vitamin K metabolic proc” were significantly enriched (Figure 5a). Also, 247 genes were commonly upregulated under 1B and 2B conditions (Figure 2a). These DEGs were overrepresented by “Ribosome”. According to GO Biological Process, “Cytoplasmic translational elongation”, “Maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA 5.8S rRNA LSU-rRNA)”, “Maturation of SSU-rRNA”, “Ribosomal small subunit biogenesis” “Cytoplasmic translation”, “Ribosome assembly” and “Translational elongation” terms were significantly enriched (Figure 5b). On the other hand, 177 genes were specifically upregulated in nla mutant Arabidopsis under C condition (Figure 2a). But no significant enrichment was found for these DEGs. According to GO Biological Process “Photosynthetic electron transport chain” was significantly enriched (Figure 5c). Also, 148 genes were specifically upregulated under 1B condition (Figure 2). These DEGs were overrepresented by “Phenylpropanoid biosynthesis” and “Biosynthesis of various plant secondary metabolites”. According to GO Biological Process, “Coumarin metabolic proc.”, “Suberin biosynthetic proc.”, “Response to iron ion” and “Hydrogen peroxide catabolic proc.” were significantly enriched (Figure 5d). 927 genes were specifically upregulated under 2B condition. These DEGs were overrepresented by “Lysine biosynthesis” and “Ribosome”. KEGG analysis was performed for Lysine biosynthesis pathway and given as supplementary file (Figure S29). According to GO Biological Process, “Ribosomal large subunit biogenesis” and “RRNA processing” were significantly enriched (Figure 5e). On the other hand, 1042 genes were commonly downregulated in mutant plants under control, 1B and 2B conditions when compared to WT-C (Figure 2b). These DEGs were overrepresented by “Alpha-Linolenic acid metabolism”, “Plant-pathogen interaction”, “MAPK signaling pathway-plant” and “Plant hormone signal transduction”. According to GO Biological Process, “Cellular response to hypoxia”, “Cellular response to decreased oxygen levels”, “Response to hypoxia”, “Response to oxygen levels” were significantly enriched (Figure 5f). Also, 230 genes were commonly downregulated by 1B and 2B conditions but not C (Figure 2b). These DEGs were overrepresented by “Glucosinolate biosynthesis” and “2-Oxocarboxylic acid metabolism”. Their GO Biological Process analysis showed that “Defense response to insect”, “S-glycoside biosynthetic proc.” and “Glycosinolate biosynthetic proc.” were significantly enriched (Figure 5g). 204 genes were downregulated only in nla mutants under control condition. But no significant enrichment was found for these DEGs. According to GO Biological Process, “Response to red or far red light” and “Hormone-mediated signaling pathway” were significantly enriched (Figure 5h). However, 97 genes were specifically downregulated under 1B condition. But no significant enrichment was found for these DEGs as well as for their GO Biological Process. Also, 862 genes were specifically downregulated under 2B condition. These DEGs were overrepresented by “Taurine and hypotaurine metabolism”, “Butanoate metabolism”, “Sulfur metabolism”, “Nitrogen metabolism” and “Alanine aspartate and glutamate metabolism”. According to GO Biological Process, “Toxin metabolic proc.”, “Sulfur compound biosynthetic proc.”, “Sulfur compound metabolic proc.” and “Response to bacterium” were significantly enriched (Figure 5i). 16 genes were commonly upregulated in mutant plants under 1B and 2B conditions when compared to nla-C (Figure 2c). These DEGs were overrepresented by “Alpha-Linolenic acid metabolism”, “Porphyrin metabolism” and “Phenylpropanoid biosynthesis”. According to GO Biological Process, “Lignin metabolic proc.” and “Phenylpropanoid metabolic proc.” were significantly enriched (Figure 6a). Also, 116 genes were specifically upregulated in mutant plants under 1B condition compared to nla control. In the biological process ontology, the major terms were “Cellular response to hypoxia”, “Cellular response to decreased oxygen levels”, “Response to hypoxia”, “Response to oxygen levels” and “Response to wounding” (Figure 6b). 111 genes were specifically upregulated in mutant plants under 2B condition compared to nla control. These DEGs was overrepresented by “Circadian rhythm-plant”. In the biological process ontology, the major term was “Negative reg. of circadian rhythm” (Figure 6c). 38 genes were commonly downregulated in mutant plants under 1B and 2B conditions when compared to nla-C. These DEGs were overrepresented by “Glucosinolate biosynthesis” and “Photosynthesis-antenna proteins” where Lhca2 and Lhcb1were significantly downregulated according to KEGG analysis (Figure S30). In the biological process ontology, the major terms were “Positive reg. of defense response to oomycetes” and “Systemic acquired resistance” (Figure 6d). In terms of molecular function, the process represented by the GO terms was “Chlorophyll binding” (Figure 6e). Also, 11 genes were specifically downregulated in mutant plants under 1B condition compared to nla control. GO analyses, except GO Molecular Function, couldn’t carried out due to “too few genes”. In terms of molecular function, the processes represented by the GO terms were “Glucosinolate glucohydrolase activity”, “Thioglucosidase activity”, “NAD+ diphosphatase activity”, “ADP-ribose diphosphatase activity” and “NADH pyrophosphatase activity” (Figure 6f). On the other hand, 238 genes were specifically downregulated in mutant plants under 2B condition compared to nla control. These DEGs were overrepresented by “Glucosinolate biosynthesis”, “Monobactam biosynthesis” and “Sulfur metabolism”. In the biological process ontology, the major terms were “S-glycoside biosynthetic proc.” and “Glycosinolate biosynthetic proc.” (Figure 6g). GO Enrichment Analysis of commonly and specifically regulated DEGs between nla under B toxicity vs WT-C and nla under B toxicity vs nla-C The DEGs of nla mutants under B toxicity relative to WT control were compared with the DEGs of nla mutants under B toxicity relative to nla control and venn diagrams were generated for upregulated and downregulated DEGs (Figure 2e, Figure 2f). Accordingly, 8 genes (AT1G07610, AT1G72030, AT2G05510, AT4G19430, AT3G19450, AT4G37070, AT3G01420, AT5G66690; overrepresented by “Alpha-Linolenic acid metabolism” and “Phenylpropanoid biosynthesis”) were commonly upregulated in these two major groups (Figure S31). In 1B condition, 50 genes (overrepresented by “Cutin suberine and wax biosynthesis” and “Phenylpropanoid biosynthesis”) were commonly upregulated (Figure S32) whereas 345 (enriched by “Phenylpropanoid biosynthesis” and “Ribosome”) and 82 genes (enriched by “Cellular response to hypoxia” according to GO Biological Process) were specifically upregulated respectively (Figure S33 and S34). In 2B condition, 89 genes (enriched by “Large ribosomal subunit rRNA binding” according to GO Molecular Function) were commonly upregulated (Figure S35) whereas 1084 (overrepresented by “Ribosome” and “Lysine biosynthesis”) and 38 genes (overrepresented by “Circadian rhythm-plant”) were specifically upregulated in “nla vs WT-C” and “nla vs nla-C” respectively (Figure S36 and S37). On the other hand, 22 genes (overrepresented by “Glucosinolate biosynthesis”) were commonly downregulated in these two major groups (Figure S38). In terms of 1B condition, 28 genes (overrepresented by “Glucosinolate biosynthesis”) were commonly downregulated (Figure S39) whereas 299 (overrepresented by “Glucosinolate biosynthesis”) and 21 genes (enriched by “Reg. of growth rate”, “Extracellular transport” and “Positive reg. of DNA-binding transcription factor activity” according to GO Biological Process) were specifically downregulated respectively (Figure S40 and S41). In terms of 2B condition, 147 genes (overrepresented by “Glucosinolate biosynthesis”) were commonly downregulated (Figure S42) whereas 945 (overrepresented by “Taurine and hypotaurine metabolism”) and 129 genes (overrepresented by “Glucosinolate biosynthesis” and “Alpha-Linolenic acid metabolism”) were specifically downregulated in respectively (Figure S43 and S44). Most significantly regulated DEGs in nla under B toxicity When compared to WT under control condition, the most significantly upregulated genes were AT2G42140 (VQ17; VQ motif-containing protein; log2fc 6.8-fold), AT1G05807 (not annotated; log2fc 5.5-fold) and AT3G43505 (LCR30; Low-molecular-weight cysteine-rich 30; log2fc 12.5-fold) under control, 1B and 2B conditions, respectively. When compared to WT under control condition, the most significantly downregulated genes were AT4G28405 (not annotated; log2fc 7.5-fold), AT2G07752 (pre-tRNA tRNA-Glu (anticodon: TTC); log2fc 7.9-fold) and AT5G12030 (AT-HSP17.6A; HEAT SHOCK PROTEIN 17.6; log2fc 8.2-fold) under control, 1B and 2B conditions, respectively. When compared to nla mutant under control condition, the most significantly upregulated genes were AT2G17660 (NOI3; Member of the RIN4-like/NOI family; log2fc 4.7-fold) and AT2G20463 (defensin-like (DEFL) family protein; log2fc 10-fold) under 1B and 2B conditions, respectively. When compared to nla under control condition, the most significantly downregulated genes were AT2G42140 (VQ17; VQ motif-containing protein; log2fc 7.6-fold) and AT3G22231 (PATHOGEN AND CIRCADIAN CONTROLLED 1; PCC1; log2fc 7.7-fold) under 1B and 2B conditions, respectively. The differential regulations of the transcription factors (TFs) in nla under B toxicity 45, 38 and 92 TFs were upregulated in nla mutant plants under C, 1B and 2B conditions when compared to WT-C, respectively. Neither significant enrichment nor cluster was found for these TFs. On the other hand, 180, 146 and 207 TFs were downregulated in nla mutant plants under C, 1B and 2B conditions, respectively, when compared to WT-C. No cluster was found for these TFs however 180 downregulated TFs under C condition were enriched by “MAPK signaling pathway-plant” and “Plant-pathogen interaction” (Figure S45). Also, 12 TFs were upregulated and generated one cluster including WRKY40, ZAT10, ERF11, ABR1, WRKY28, NAC019 and DREB1C in nla mutant plants under 1B condition when compared to nla-C (Figure S46). All of these TFs were specifically regulated under 1B. Only 2 TFs (WRKY54 and CRF2/Ethylene-responsive transcription factor) were downregulated in nla mutant Arabidopsis under 1B condition when compared to nla mutant Arabidopsis under control condition. 17 TFs were upregulated in nla mutant under 2B condition when compared to nla mutant under control condition. Neither significant enrichment nor cluster was found for these TFs. However, 16 TFs enriched by “MAPK signaling pathway-plant” and “Plant-pathogen interaction” were downregulated and generated one cluster including WRKY33, WRKY53, WRKY22, WRKY54, WRKY60, ERF6, ERF2, ERF094, MYB52 and NAC102 in nla mutant under 2B condition when compared to nla-C (Figure S47). Except WRKY54, all these TFs were specifically regulated on 2B. KEGG analysis was performed for MAPK signaling pathway-plant and given as supplementary file (Figure S48). The differential regulations of Boron Transporters in nla under B toxicity The relative expression levels of B efflux transporters, AtBOR1 (AT2G47160) and ATBOR4 (AT1G15460), as well as boric acid channel for boron uptake AtNIP5;1 (AT4G10380) remained stable in nla plants under B toxicity compared to both nla mutant and WT Arabidopsis under control conditions. However, B exporter BOR2 (AT3G62270) was significantly induced (more than two-fold) under both 1B and 2B conditions compared to WT control and under 1B compared to nla mutant control. On the other hand, the expression level of tonoplast aquaporin TIP5;1 (AT3G47440) gene was downregulated (more than 4-fold) in nla mutant Arabidopsis under 1B condition when compared with WT control. A network was detected by using the STRING (12.0) [33] between NLA and AtTIP5 (Figure S49). Discussion Excess B in soil occurred by natural or anthropogenic influences leads to toxicity in many plants and gives rise to yield loss of important cereal crops especially in arid and semi-arid regions of the world [34, 35]. Rather than the impractical remediation of soil, the development of tolerant varieties has been accepted as a long-term sustainable solution. Thus, the main mechanisms adopted by the tolerant, hyperaccumulator and other plant species to counteract B toxicity are matters of interest for plant breeders and biotechnologists [35, 36]. A prior study exhibited the differential regulation of protein degradation pathway in B-toxicity-sensitive and -tolerant wheat cultivars under B toxicity [14]. In this study, as a preliminary work, we found the remarkable induction of NLA gene expression in Arabidopsis thaliana through increasing levels of B toxicity (Table S1a). Therefore, in the present study, global expression profile was investigated in nla mutant Arabidopsis thaliana exposed to toxic concentrations of B to elucidate the intricate mechanisms governing NLA-related B toxicity response at the molecular level. Accordingly, PCA was performed to elucidate the dynamics of transcriptomic variations across the experimental conditions. The results derived from PCA exhibited an obvious separation of wild type group from nla mutant groups suggesting that mutation of nla gene significantly disturbed the transcriptome of Arabidopsis leaves. A clear cluster was observed under 2B condition that agrees with the respective phenotype on this condition. High number of DEGs in nla plants under control condition without B treatments underscored the effect of the nla mutation in A. thaliana . NLA encodes a ubiquitin E3 ligase [15] that mediates degradation of PHT1s at plasma membranes. SPX-domain of NLA interacts with nuclear transcription factors that control the phosphate starvation response pathway [20]. On the other hand, nla mutant plants are unable to develop adaptive responses under nitrogen (N)-limited conditions, hence the name “nitrogen limitation adaptation”, which results in early senescence [22]. In fact, it is directing ubiquitination of a nitrate transporter (NRT1.7) [16] which is responsible for loading of N in source leaves for the remobilization of N to N-demanding tissues [17]. In addition to its certain roles in regulation of the membrane transporters, NLA is involved in mediating immune responses by interacting with protein substrates like pathogenesis-related proteins, mitogen activated protein kinases (MAPKs), and transcription factors [24]. In other words, NLA may have many protein targets yet to be discovered. In the current study, it was not surprising to obtain that “ribosome” pathway was overrepresented for upregulated genes in nla mutant Arabidopsis under control condition compared to WT-C since NLA is directly involved in proteasome system and lack of its expression can lead to reorganization of protein translation process. In agreement with this, one of the most represented categories of molecular function was histone methyltransferase activity. The enrichment of histone methyltransferase activity may reflect rearrangement of transcriptional regulation of mutants because histone methylation plays a central role in regulating chromatin state and gene expression in A. thaliana , as in other organisms, and is involved in a variety of physiological and developmental processes [37]. In the biological process, upregulation of phylloquinone (vitamin K1) biosynthesis may refer to alterations in photosynthetic mechanism of mutants. Because phylloquinone is an essential electron carrier in photosystem I and its concentration is highly correlated with chlorophyll [38]. However, according to the physiological findings, photosynthetic pigments in nla mutant Arabidopsis under control condition were very similar to WT under C condition as supported by the results of Peng and coauthors [39]. Another highly enriched pathway was for pectate lyase, an enzyme involved in degradation of pectin, a major constituent of the dicot primary cell wall. It depolymerizes pectin by cleavage between-(1,4) glycosidic bonds and participates in the remodeling of pectin during organogenesis, especially during fruit ripening [40]. It has primarily been investigated as virulence factor and in tissue maceration by necrotrophic pathogens. Plant pectate lyase like proteins (PLLs) have specific roles in accommodating infection threads during symbiosis or organ growth and senescence. In leaves, PLLs were mostly expressed in the vasculature system including leaf veins [41]. Moreover, a recent report demonstrated that a pectate lyase plays an important role during vascular development in Arabidopsis [40]. Another PLL has been suggested to remove homogalacturonan, thus potentially enabling further callose synthase to access and modify the cell wall during sieve pore maturation [42]. In the present study, the enrichment of pectate lyase activity may be related to the possible cell wall modification in nla mutants due to NLA mutation-altered immunity. Supportively, compared with wild type plants, higher deposition of callose in nla mutants conferred resistance to pathogen by reinforcing plant cell walls to arrest pathogen infection [43]. On the other hand, mostly downregulated pathways were linoleic acid (LA) and alpha-Linolenic acid (ALA) metabolisms in nla control plants compared to WT-C. LA and ALA are the two most common polyunsaturated fatty acids in plants [44]. According to KEGG analyses, lipoxygenases (LOX) were highlighted in both pathways (Figue S4 and S5). Indeed, lipoxygenases are key regulators for lipid peroxidation, which is crucial for plant senescence and defense pathways. Oxidation products mediated by LOX, such as jasmonate (JA), play a major role in the responses to biotic and abiotic stress. Interestingly, it was found that nla mutants have high SA and JA even under non-infectious conditions [43]. This may be an explanation for the repression of the JA pathway in nla mutants under C condition in this study (Figure S5). Compatible with that, interrelated pathways like tryptophan metabolism, plant pathogen interaction, MAPK signaling pathway-plant and plant hormone signal transduction were also overrepresented for downregulated genes in nla mutants under control condition. There are multiple MAPK pathways that are involved in hormone signaling and trigger various stress responses where stress signal is transduced based on the DEGs is largely due to MAPK [45]. Since pathogenesis-related proteins and MAPKs are among the substrates of NLA [24], failure of their degradation in mutant plants may have caused their regulation to occur at transcriptional level. Overall repression of these pathways may indicate the unstressed mode of nla plants on C condition. 426 genes commonly upregulated in mutant plants under all conditions (C, 1B and 2B) when compared to WT-C were overrepresented by ribosome and phylloquinone biosynthetic process was significantly enriched in these plants. Moreover, translation related pathways were commonly enriched in both B conditions. B interferes with transcription and/or translation by binding to cis hydroxyls on ribose molecules in RNA species [36]. That could explain the powerful enrichment of rRNA-mRNA binding and translational elongation process. Moreover, it has been suggested that B also interferes with aminoacylation steps of the tRNAs and causes uncharged tRNA stress, thus, the general amino acid control pathway contributes to B toxicity [46]. Supportively, the most significantly repressed gene by 1B compared to WT-C was “pre-tRNA tRNA-Glu” which is the precursor of Glutamyl-tRNA, member of aminoacyl-tRNA biosynthesis pathway. Besides, 148 DEGs specifically regulated under 1B condition were overrepresented by “Phenylpropanoid biosynthesis” and “Biosynthesis of various plant secondary metabolites”. Consistently, similar pathways were highly induced in response to B-induced stress in maize [47], in wheat [14] and in mulberry [48]. Plants produce secondary metabolites against different stress conditions. Thus, the activation of these pathways appears to be implicated in maintenance of the homeostasis. On the other hand, lysine biosynthesis pathway was overrepresented for specifically up-regulated 927 DEGs in 2B (Figure S29). The essential amino acid lysine is synthesized in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine [49]. Lysine is also a precursor for glutamate, an important signaling amino acid that regulates plant growth and responses to the environment [50]. Also, 1042 genes commonly downregulated in mutant plants under all conditions (control, 1B and 2B) when compared to WT-C were overrepresented by ALA metabolism, plant-pathogen interaction, MAPK signaling pathway-plant and plant hormone signal transduction. It is worth mentioning that 2B strongly repressed protein serine/threonine kinase activity when compared to WT-C. MAPKs are a large family of enzymes that phosphorylate their protein targets on serine or threonine residues. It is known that borate might form ester‑like complexes with these residues [51] which may mechanically affect MAPKs’ binding to their target transcription factors (TFs) and subsequently the interaction between TFs and target genes. The TF interaction with the target gene might be modulated also by the direct linkage of B to the TF. So, depending on the target gene and the type of TF (activator or repressor) the linkage with B could regulate gene expression [52]. On the other hand, glucosinolate biosynthesis pathway was commonly enriched in both B conditions. Glucosinolates are categorized into tryptophan-derived indole, tyrosine or phenylalanine derived aromatic, or aliphatic glucosinolates. Aliphatic glucosinolates are synthesized from methionine whereas aliphatic glucosinolates are synthesized from alanine, valine, and leucine. Also, glutathione is involved in the biosynthesis of glucosinolates as a sulfur donor [45]. In B-stressed plants most of the down-regulated DEGs were obtained by 2B where 862 specifically regulated DEGs were enriched by taurine and hypotaurine metabolism, butanoate metabolism and sulfur metabolism which were associated to toxin metabolic process and sulfur compound biosynthetic process by GO Biological Process. Taurine metabolism also is interrelated with glutathione metabolism (Figure S12). These findings corroborated the downregulation of biosynthesis of secondary metabolites, plant hormone signal transduction, degradation of valine, leucine and isoleucine obtained in A. thaliana under toxic B condition [53]. When compared to nla mutants under control condition (nla-C), only 16 genes were commonly upregulated in mutant plants under 1B and 2B conditions and overrepresented by ALA metabolism. In contrast to many genes related to production of JA in this pathway including LOX (EC.1.13.11.12) tended to downregulate in nla-C compared to WT-C (Figure S5), lipoxygenases (EC.1.13.11.12 and EC.1.13.11.92) were upregulated in mutants under 1B (Figure S15) compared to nla-C. Moreover, the latter belonging to a branch of ALA pathway other than JA was commonly upregulated by 1B and 2B. Senescence-related hormones like JA and ethylene were promoted by B excess also in barley and wheat [54, 14]. On the other hand, B stress significantly enriched lignin and phenylpropanoid metabolic process in nla plants (Figure 6a). Increased level of phenylpropanoid metabolites like flavonoids including anthocyanin, is one of the adaptive measures that plants employ to defend against stress. B toxicity-promoted accumulation of these metabolites was obtained in earlier studies [8, 9, 10, 11, 55]. Moreover, anthocyanin, in addition to capability for compartmentalizing excess B into vacuoles [7], exhibits a photoprotective role in mesophyll cells when chloroplast functionality has been compromised by B toxicity [11]. However, nla mutant Arabidopsis plants did not exhibit nitrogen deficiency-induced anthocyanin accumulation; instead showed higher lignin production whereas they still show phosphorous deficiency-induced anthocyanin accumulation [26], suggesting that different signaling cascades are involved in anthocyanin responses against different nutrients. Also, we found that the overall anthocyanin content remained considerably lower than those in Col-0 under the normal and high B conditions (Table 1). In line with this, B stress induced lignin path rather than anthocyanin in the phenylpropanoid pathway in nla mutants by means of the upregulation of cinnamyl-alcohol dehydrogenase (EC 1.1.1.195), peroxidase (EC 1.11.1.7) and coniferyl alcohol glucosyl transferase (EC 2.4.1.111) (Figure S16). Thus, B-stressed nla plants seem to trigger a similar signaling network with the nla plants switching from anthocyanin to lignin biosynthesis path by nitrogen limitation. 111 specifically upregulated genes in mutant plants under 2B condition compared to nla control were overrepresented by negative regulation of the circadian rhythm (Figure 6c). Circadian clocks in plants, as in all eukaryotes, rely on photoreceptors to sense light and gate the timing of central metabolic pathways relative to the outside environment. According to KEGG analysis (Figure S21), upregulation of CCA1 (CIRCADIAN CLOCK ASSOCIATED1) and LHY (LATE ELONGATED HYPOCOTYL) in redlight mediated regulation of antenna proteins and CRY, SPA and HY5 in blue light mediated regulation of photomorphogenesis may at least reveal the B stress-altered photosynthetic machinery as a strategy for nla plants survival since, as in many plant species, the correct synchronization of the clock with the environment contributes to ensuring survival under fluctuating environmental conditions [56]. More importantly, LHY and CCA are among the key transcription factors in the circadian feedback loop that has a potential role in the regulation of PAP1-mediated UV-B protection as seen in KEGG circadian rhythm-plant pathway (Figure S21). ATPAP1 or ARABIDOPSIS THALIANA PRODUCTION OF ANTHOCYANIN PIGMENT 1 is a MYB75 transcription factor involving in anthocyanin metabolism. This interaction cross-links the circadian rhythm and anthocyanin pathway as evidenced by Hu and coauthors who noted the connection between flavonoids and the plant clock [57]. Moreover, circadian control of ORE1 by PRR9, another component of circadian feedback loop, positively regulates leaf senescence by enhancing expression of senescence related genes like SAG29 as well as chlorophyll catabolic enzymes in A. thaliana [58]. ORE1 homeostasis appears to be impaired due to lack of its post-translational regulation by NLA which, in turn, may have disturbed PRR9 tuning and, eventually, the crosstalk between circadian rhythm and anthocyanin pathways in nla plants. This could be an explanation for their inhibited accumulation of anthocyanin and switching to lignin biosynthesis in response to B toxicity. GO enrichment analysis revealed that base excision repair was overrepresented for upregulated genes in nla mutants under 2B condition when compared to 1B where NAD+ ADP-ribosyltransferase activity was significantly enriched. Oxidative stress has been widely determined in plants subjected to B excess [59, 60, 8, 61, 62]. Higher induction of NAD+ ADP-ribosyltransferase activity at 2B condition may imply that oxidative DNA damage is more pronounced in higher B condition, and that DNA repair system is activated since ADP-ribosylation of nuclear proteins, under conditions of low to moderate DNA damage, facilitates DNA repair and promotes cell survival [63]. 38 commonly downregulated genes in mutant plants under 1B and 2B conditions compared to nla -C were overrepresented by photosynthesis-antenna proteins and glucosinolate biosynthesis. Significant enrichment of chlorophyll binding as molecular function (Figure 6e), and downregulation of light harvesting complexes (Lhca2 and Lhcb1) (Figure S30) were in accordance with affected circadian rhythm. Similarly, LHC participants in photosynthesis–antenna proteins pathway were downregulated under high boron which were attributed to a decrease in light absorption, consequently reducing the photosynthesis rate [64]. Photosynthesis is one of the main metabolic processes impaired by B excess due to biochemical limitations including the decline of electron transport rate, reduced CO 2 use efficiency and impairment of photosystem II (PSII) efficiency (reviewed by [35] beside alterations of photosynthetic pigment contents [65, 8, 66]. On the other hand, chlorophyll degradation is typically accompanied by anthocyanin accumulation during leaf senescence. Indeed, anthocyanin biosynthesis and chlorophyll degradation are linked through the action of NAC transcription factors such as ORE1 [67]. Normally, ORE1 activates leaf senescence related genes including SAG29 and genes encoding chlorophyll catabolic enzymes. In this study, nla plants showed inhibited photosynthesis as confirmed by chlorosis to a degree and decreased content of photosynthetic pigments under B toxicity (Table 1, Figure S1). Yet, the inability of their photoprotective adaptation due to reduced anthocyanin accumulation may have directed the mutant plants to minimize their photosynthetic apparatus on toxic B condition. Similar to DEGs compared to WT control condition, glucosinolate biosynthesis was the most strongly enriched pathway in toxic B treated nla plants compared to nla controls. It is notable that repression of glucosinolate pathway was obtained despite the induction of the phenylpropanoid pathway since both represent the secondary metabolism involved in plant defense. The response-shift between the phenylpropanoid and glucosinolate biosynthesis pathways has been suggested to occur depending on the intensity of stress factors in plants [45]. Production of phenylpropanoids has been proposed as an earlier line of defense and, moreover, shown to be limited by glucosinolate pathway [68] which collaborates with the inverse regulation of these pathways in the present study. Likewise, the B toxicity and sulfur deficiency caused differential expression of the same set of genes involved in glucosinolate biosynthetic processes, sulfur metabolism, and osmotic stress [69]. Transcription factors (TFs) emerge as genuine conductors of gene expression symphonies under the influence of many environmental stresses [70]. In this study, one cluster was obtained under 1B condition when compared to nla plants on the control condition. These TFs including WRKY40, ZAT10, ERF11, ABR1, WRKY28, NAC019 and DREB1C were specifically upregulated on 1B. On the other hand, 2B led to repression of one cluster including WRKY33, WRKY53, WRKY22, WRKY54, WRKY60, ERF6, ERF2, ERF094, MYB52 and NAC102 when compared to nla plants on the control condition. These TFs were enriched by “MAPK signaling pathway-plant” and “Plant-pathogen interaction”. These outcomes corroborated the findings of several studies related to transcriptomic regulation in response to B toxicity including increase in expression level of DREB, ERF, NAC and repressions of MYB in poplar [71], significant enrichment of MYB in wheat [64], differential regulation of WRKY and MYB in wheat [14], downregulation of WRKY in barley [54]. The present study indicates that most of the differentially regulated ERF TFs tend to decrease in accordance with the enrichment of signaling pathways for repressed TFs since ERF has been associated with signaling of phytohormones [48]. Notably, WRKY TFs appear to have major roles in both induced and repressed network in nla mutants under B stress due to their central positions according to STRING analysis (Figure S46 and S47). Especially significant downregulation of WRKYs in MAPK signaling pathway (Figure S48) supports the view that B interferes with transcriptional regulation by binding to the serine/threonine residues of the kinase-substrates. The differential regulation of WRKY33 may have also contributed to repressed anthocyanin accumulation of nla mutants under B toxicity. Because a recent study has revealed that WRKY33 negatively regulates anthocyanin biosynthesis [72]. In this case, under Pi limited condition PHR1 (the key transcription factor controlling Pi-starvation response) interacts with WRKY33, and thereby the protein level of WRKY33 decreases; the repression of DFR (dihydroflavonol 4-reductase, a rate-limiting enzyme in anthocyanin production) expression by WRKY33 is thus attenuated, leading to anthocyanin accumulation in A. thaliana . On the other hand, NLA as a negative post-translational regulator of PHR1 involves in phosphate starvation response in A. thaliana [73]. Seedlings of nla mutants display accumulation of anthocyanin under Pi-deficiency. Take in together, in nla mutants, Pi starvation enhances PHR1 protein level which leads to decrease of WRKY33 protein level and subsequent induction of DFR and eventually anthocyanin accumulation. Thus, the findings of the current study may be used to speculate that the low level of WRKY33 protein confers anthocyanin accumulation under B toxicity in WT plants whereas nla mutants fail to induce anthocyanin accumulation because of the impaired PAP1-promoted DFR stimuli. This impairment may arise from an abnormal induction of a factor other than WRKY33 interacting with PAP1 due to disrupted post-translational regulations of NLA. In other words, an NLA-target (i.e., ORE1) may be responsible for this interrupted chain of reactions. Stable level of PAP1 and DFR transcripts despite of the significant downregulation of WRKY33 in nla mutants under B toxicity (Figure S48) strengthens this hypothesis. Interestingly, the most significantly upregulated gene in nla-C compared to WT-C was the most significantly downregulated gene in nla mutant under 1B condition compared to nla-C. This is VQ17 which is a VQ motif-containing protein. The plant specific VQ motif-containing proteins have been recently discovered as a class of plant regulatory proteins interacting with WRKY transcription factors capable of modulating their activity as transcriptional regulators and playing important roles in plant growth, development and stress response [74, 75]. A weak interaction of VQ17 with WRKY33 was postulated in A. thaliana [76]. Moreover, overexpression of VQ17 highly stunted growth of A. thaliana [76]. Overall, these multiple cross-talking signaling pathways in B-stressed nla mutants modulated by transcription factors as well as transcriptional regulators and post-translational modifications need further exploration at both RNA and protein level. The stable expression levels of B efflux transporters (AtBOR1 and ATBOR4) and boric acid channel (AtNIP5;1) were obtained in nla plants under B toxicity compared to both mutant and WT plants under control conditions. However, B exporter BOR2 was induced more than two-fold by both 1B and 2B compared to WT-C and by 1B compared to nla-C. Interestingly, AtTIP5;1 was downregulated more than 4-fold in nla plants under 1B condition when compared with WT-C. AtTIP5 is an aquaporin family member localized to the cell tonoplast membrane and confers high B tolerance lowering the cytoplasmic B concentration by means of vacuolar compartmentation of B [77]. The links between nutrient transporters and aquaporins have been demonstrated to be important to maintain cell homeostasis. For instance, deficiencies of micronutrients induce their tonoplast transporters for remobilization from the vacuole and also reduce plasma membrane aquaporins presence [78]. B toxicity is expected to lead to upregulation of B exporter and vacuolar influx transporters. In the present case, AtTIP5 expression was lower in only nla plants under 1B condition compared to WT-C. This may be related to a collaborative relationship between the transporters and the channels. Supportively, a network analysis generated using the STRING to investigate the functional interaction between NLA and AtTIP5 displayed an interrelationship between NIP-TIP cluster, PHT and NLA (Figure S49). However, much more research is needed to confirm this co-coordinative regulation of aquaporins and B transporters under B stress. In conclusion, this study investigated the global transcription profile of nla mutants under B stress and conferred statements about NLA related B toxicity responses for the first time. Since NLA is a contributor of protein degradation pathway, overall enrichment of ribosome pathway was expected in all mutant plants. Besides, common enrichment of translation related pathways in both B stress conditions was associated with B interference with RNA binding processes as well as aminoacyl-tRNA biosynthesis pathway. Likewise, B may be responsible for the reduced activity of MAPKs due to forming complexes with serine or threonine residues of their target TFs, thereby preventing their action on these TFs such as WRKYs. Physiological findings revealed that anthocyanin accumulation was inhibited in nla mutants, unlike WT plants under B stress. This was attributed to the powerful induction of lignin path rather than anthocyanin in the phenylpropanoid pathway in nla mutants. One reason for switching from anthocyanin to lignin biosynthesis may be the disruption of the crosstalk between circadian rhythm and anthocyanin pathways in nla plants whose ORE1 homeostasis appears to be impaired due to lack of its post-translational regulation by NLA. Stable transcription level of anthocyanin biosynthesis genes, PAP1 and DFR, despite of the significant downregulation of WRKY33 under B toxicity suggested the fact that lack of post-translational regulation of a factor other than WRKY33 by NLA led to inhibition of DFR stimuli by interacting with PAP1 in nla mutants. This alternative interaction of PAP1 with this NLA target (known like ORE1 or unknown) remains to be discovered. Eventually, our data might benefit the determination of candidate genes for molecular breeding, and genetic manipulation of plants for B toxicity. Declarations Author Contribution Author performed the experiments and data analysis and wrote the manuscript. Data availability The RNA-seq data have been uploaded to the NCBI SRA (Sequence Read Archive) database with reference accession PRJNA1130029 (Temporary Submission ID: SUB15556101). The corresponding author may be contacted if someone wants to request the data from this study. Funding Declaration This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. References Warington, K. The effect of boric acid and borax on the broad bean and certain other plants. Ann. Bot. 37 , 629–672 (1923). Eaton., F. M. Deficency, toxicity and accumulation of borron in plants, J. Agric. Res. 69 , 237-277 (1944). Brdar-Jokanovic, M. Boron toxicity and deficiency in agricultural plants. Int. J. Mol. Sci. 21 ,1424. (2020). Bolanos, L., Lukaszewski, K., Bonilla, I. & Blevins, D. Why boron? Plant Physiology and Biochemistry 42 , 907–912 (2004). Reid, R. J., Hayes, J. E., Post, A., Stangoulis, J. C. R. & Graham, R. D. A critical analysis of the causes of boron toxicity in plants. Plant Cell and Environment 27 , 1405–1414 (2004). Miwa, K & Fujiwara, T. Role of overexpressed BOR4, a boron exporter, in tolerance to high level of boron in shoots. Soil Sci. Plant Nutr. 57 , 558–565 (2011). Landi, M., Tattini, M. & Gould, K.S. Multiple functional roles of anthocyanins in plant-environment interactions. Environ. Exp. Bot. 119 , 4-17 (2015). Kayıhan DS, Kayıhan C, Çiftçi YÖ (2016). Excess boron responsive regulations of antioxidative mechanism at physio-biochemical and molecular levels in Arabidopsis thaliana. Plant Physiology and Biochemistry 109: 337-345. Kayıhan, C. The involvement of the induction of anthocyanin biosynthesis and transport in toxic boron responsive regulation in Arabidopsis thaliana. Turk. J. Bot. 45 (3), 1 (2021). Cervilla, L. M. et al. Parameters symptomatic for boron toxicity in leaves of tomato plants. J. Bot. 1-17 (2012). Landi, M., Guidi, L., Pardossi, A., Tattini, M. & Gould, K. S. Photoprotection by foliar anthocyanins mitigates effects of boron toxicity in sweet basil (Ocimum basilicum). Planta 240 (5), 941–953 (2014). Jefferies, S. P. et al. Mapping of chromosome regions conferring boron toxicity tolerance in barley (Hordeum vulgare L.). Theoretical and Applied Genetics 98 , 1293–1303 (1999). Camacho-Cristóbal, J. J. et al. The expression of several cell wall-related genes in Arabidopsis roots is down-regulated under boron deficiency. Environ. Exp. Bot. 63 , 351–358 (2008). Kayıhan, C., Öz, M. T., Eyidoğan, F., Yücel, M. & Öktem, H. A. Physiological, biochemical, and transcriptomic responses to boron toxicity in leaf and root tissues of contrasting wheat cultivars. Plant Mol. Biol. Rep. 35 (1), 97–109 (2017). Kant, S., Peng, M. & Rothstein, S. J. Genetic regulation by NLA and microRNA 827 for maintaining nitrate-dependent phosphate homeostasis in Arabidopsis. PLoS Genetics 7 , e1002021 (2011). Liu, W., Sun Q., Wang, K., Du, Q. & Li, W. X. Nitrogen Limitation Adaptation (NLA) is involved in source-tosink remobilization of nitrate by mediating the degradation of NRT1.7 in Arabidopsis. New Phytologist 214 , 734–744 (2017). Fan, S. C., Lin, C. S., Hsu, P. K., Lin, S. H. & Tsay, Y. F. The Arabidopsis nitrate transporter NRT1.7 expressed in phloem, is responsible for source-to-sink remobilization of nitrate. Plant Cell 21, 2750–2761. (2009). Lin, W. Y., Huang, T. K. & Chiou, T. J. NITROGENLIMITATION ADAPTATION, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis. Plant Cell 25 , 4061–4074 (2013). Park B. S., Seo, J. S. & Chua, N. H. NITROGEN LIMITATION ADAPTATION recruits PHOSPHATE 2 to target the phosphate transporter PT2 for degradation during the regulation of Arabidopsis phosphate homeostasis. Plant Cell 26 , 454–464 (2014). Shi, J., Hu, H., Zhang, K., Zhang, W., Yu, Y., Wu, Z., & Wu, P. The paralogous SPX3 and SPX5 genes redundantly modulate Pi homeostasis in rice. J. Exp. Bot. 65 , 859–870 (2014). Hannam, C. et al. Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway. Botany 96 (2), 79-96 (2018). Peng, M., Hannam, C., Gu H., Bi, Y. M. & Rothstein S. J. A mutation in NLA, which encodes a RING-type ubiquitin ligase, disrupts the adaptability of Arabidopsis to nitrogen limitation. Plant J. 50 , 320-337 (2007a). Yaeno, T., & Iba, K. BAH1/NLA, a RING-type ubiquitin E3 ligase, regulates the accumulation of salicylic acid and immune responses to Pseudomonas syringae DC3000. Plant Physiol. 148 , 1032–1041 (2008). Hewezi, T., Piya, S., Qi, M., Balasubramaniam, M., Rice, J. H., Baum, T. J. Arabidopsis miR827 mediates post-transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility. Plant Journal 88 , 179–192 (2016). Park, B. S. et al. Arabidopsis NITROGEN LIMITATION ADAPTATION regulates ORE1 homeostasis during senescence induced by nitrogen deficiency. Nature Plants 4 , 898–903 (2018). Peng, M., Hudson, D., Schofield, A., Tsao, R., Yang, R., Gu, H., Bi, Y-M., & Rothstein, S.J. Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene. J. Exp. Bot. 59 , 2933-2944 (2008). Murashige, T. & Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant . 15 , 473-497 (1962). Lichtenthaler, H. K. Chlorophylls and carotenoids: pigments of photosynthetic membranes. Methods Enzymol. 148 , 350-382 (1987). Mancinelli, A. L., Yang, C. P. H., Lindquist, P., Anderson, O. R. & Rabino, I. Photocontrol of anthocyanin synthesis III. The action of streptomycin on the synthesis of chlorophyll and antyocyanin. Plant Physiol. 55 , 251-257 (1975). Chomczynski, P. & Sacchi, N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem 162 , 156-159 (1987). Ge, S. X., Jung, D. & Yao, R. Bioinformatics 36 , 2628–2629 (2020). Kanehisa, M., Furumichi, M., Sato, Y., Matsuura, Y. and Ishiguro-Watanabe, M.; KEGG: biological systems database as a model of the real world. Nucleic Acids Res. 53, D672-D677 (2025) Szklarczyk, D. et al. The STRING database in 2023: protein–protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res . 51 (D1):D, 638-646 (2023). Javed, M.B.; Malik, Z.; Kamran, M.; Abbasi, G.H.; Majeed, A.; Riaz, M.; Bukhari, M.A.; Mustafa, A.; Ahmar, S.; Mora-Poblete, F.; et al. Assessing Yield Response and Relationship of Soil Boron Fractions with Its Accumulation in Sorghum and Cowpea under Boron Fertilization in Different Soil Series. Sustainability 2021 , 13 , 4192. Landi, M. et al. Boron toxicity in higher plants: an update. Planta 250 , 1011–1032 (2019). Reid, R. Can we really increase yields by making crop plants tolerant to boron toxicity? Plant Science 178 , 9-11 (2010). Hu, H., & Du, J. Structure and mechanism of histone methylation dynamics in Arabidopsis. Curr. Opin. Plant Biol. 67 , 102211 (2022). Reumann S. Biosynthesis of vitamin K1 (phylloquinone) by plant peroxisomes and its integration into signaling molecule synthesis pathways. Subcell. Biochem . 69 , 213-29 (2013). Peng, M. et al. Genome-wide analysis of Arabidopsis responsive transcriptome to nitrogen limitation and its regulation by the ubiquitin ligase gene NLA. Plant Mol. Biol. 65 , 775–797 (2007b). Bai, Y. et al. A pectate lyase gene plays a critical role in xylem vascular development in Arabidopsis. Int. J. Mol. Sci. 24 , 10883 (2023). Palusa, S. G. & Golovkin, M. Organ-specific, developmental, hormonal and stress regulation of expression of putative pectate lyase genes in Arabidopsis. New Phytol. 174 , 537–550 (2007). Kalmbach, L. et al. Putative pectate lyase PLL12 and callose deposition through polar CALS7 are necessary for long-distance phloem transport in Arabidopsis. Current Biology 33 , 926 - 939 (2023). Val-Torregrosa, B. et al. Loss-of-function of NITROGEN LIMITATION ADAPTATION confers disease resistance in Arabidopsis by modulating hormone signaling and camalexin content. Plant Science 323 , 111374 (2022). Harwood, J. L. Plant acyl lipids: structure, distribution and analysis. In: (ed. Stumpf PK) The Biochemistry of Plants. Vol 4. New York, USA: Academic Press Inc. Publishing House, 24–30 (1980). Waskow, A., Guihur, A., Howling, A. & Furno, I. RNA Sequencing of Arabidopsis thaliana seedlings after non-thermal plasma-seed treatment reveals upregulation in plant stress and defense pathways. Int. J. Mol. Sci. 23 , 3070 (2022). Uluisik, I. et al. Boron stress activates the general amino acid control mechanism and inhibits protein synthesis. PLoS ONE 6 (11), e27772 (2011). Chen, F., Gao, J., Li, W. & Fang, P. Transcriptome profiles reveal the protective role of seed coating with zinc against boron toxicity in maize (Zea mays L.). Journal of Hazardous Materials 423 127105 (2022). Zou, J. et al. Genome-wide transcriptome profiling of mulberry (Morus alba) response to boron deficiency and toxicity reveal candidate genes associated with boron tolerance in leaves. Plant Physiology and Biochemistry 207 ,108316 (2024). Azevedo, R. & Lea, P. Lysine metabolism in higher plants. Amino Acids 20 , 261–279 (2001). G. New insights into the regulation and functional significance of lysine metabolism in plants. Annu. Rev. Plant Biol. 53 , 27-43 (2002). Goldbach, H. E. & Wimmer, M. A. Boron in plants and animals: Is there a role beyond cell‑wall structure? J. Plant Nutr. Soil Sci. 170 , 39‑48 (2007). Gonzalez-Fontes, A. et al. Is boron involved solely in structural roles in vascular plants? Plant Signal Behav . 3 , 24–26 (2008). Kayıhan, C. et al. Transcriptional profiling and proteomic validation revealed higher boron tolerance in Arabidopsis thaliana exposed to salt pre-treatment. South African Journal of Botany 180 , 588-605, (2025). Öz, M. T., Yilmaz, R., Eyidogan, F., Graaff, L. & Yucel, M. & Oktem, H. A. Microarray analysis of late response to boron toxicity in barley (Hordeum vulgare L.) leaves. Turk J. Agric. For. 33 , 191–202 (2009). Zhang, Q. et al. The impact of boron nutrient supply in mulberry (Morus alba) response to metabolomics, enzyme activities, and physiological parameters. Plant Physiol. Biochem. 200 , 107649 (2023). Jang, J., Lee, S., Kim, J. I., Lee, S. & Kim, J. A. The roles of circadian clock genes in plant temperature stress responses. Int. J. Mol. Sci. 25 , 918 (2024). Hu, T., Gao, Z. Q., Hou, J. M., Tian, S. K., Zhang, Z. X., Yang, L. & Liu, Y. Identification of biosynthetic pathways involved in flavonoid production in licorice by RNA-seq based transcriptome analysis. Plant Growth Regulation 92 , 15–28 (2020). Kim, H. et al. Circadian control of ORE1 by PRR9 positively regulates leaf senescence in Arabidopsis. Proceedings of the National Academy of Sciences, USA 115 , 8448–8453 (2018). Molassiotis, A., Sotiropoulos, T., Tanou, G., Diamantidis, G. & Therios, I. Boron-induced oxidative damage and antioxidant and nucleolytic responses in shoot tips culture of the apple rootstock EM 9 (Malus domestica Borkh). Environ. Exp. Bot. 56 (1), 54–62 (2006). Ardic, M., Sekmen, A., Tokur, S., Ozdemir, F. & Turkan, I. Antioxidant responses of chickpea plants subjected to boron toxicity. Plant Biol. 11 (3), 328–338 (2009). Çatav, Ş. S., Genç, T. O., Oktay, M. K. & Küçükakyüz, K. Effect of boron toxicity on oxidative stress and genotoxicity in wheat (Triticum aestivum L.). Bull Environ. Contam. Toxicol. 100 (4), 502–508 (2018). Simón-Grao, S. et al. Arbuscular mycorrhizal symbiosis improves tolerance of Carrizo citrange to excess boron supply by reducing leaf B concentration and toxicity in the leaves and roots. Ecotoxicol. Environ. Saf. 173 , 322–330 (2019). Gibson, B. A. & Kraus, W. L. New insights into the molecular and cellular functions of poly (ADPribose) and PARPs. Nat. Rev. Mol. Cell Biol. 13 , 411 (2012). Khan, M. K. et al. Insight into the Boron Toxicity Stress-Responsive Genes in Boron-Tolerant Triticum dicoccum Shoots Using RNA Sequencing. Agronomy 13 , 631 (2023). Huang, J. H. et al. Effects of boron toxicity on root and leaf anatomy in two Citrus species differing in boron tolerance. Trees 28 (6), 1653–1666 (2014). Sarafi, E., Siomos, A., Tsouvaltzis, P., Therios, I. & Chatzissavvidis, C. Boron toxicity effects on the concentration of pigments, carbohydrates and nutrient elements in six non-grafted pepper cultivars (Capsicum annuum L.). Indian J. Plant Physiol. 23 (3), 474–485 (2018). Pei, Z., Huang, Y., Ni, J., Liu, Y. & Yang, Q. For a colorful life: recent advances in anthocyanin biosynthesis during leaf senescence. Biology 13 , 329 (2024). Kim, J. I., Zhang, X., Pascuzzi, P. E., Liu, C. J. & Chapple, C. Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome-dependent degradation of PAL. New Phytol. 225 , 154–168 (2020). Kayihan, C., Aksoy, E., And, M. & Su, N. Boron toxicity induces sulfate transporters at transcriptional level in Arabidopsis thaliana. Turkish Journal of Botany 47 , No. 1 (2023). Latchman, D. S. Transcription factors: An overview. Int. J. Biochem. Cell Biol. 29 , 1305–1312 (1997). Yıldırım, K. & Uylaş, S., Genome-wide transcriptome profiling of black poplar (Populus nigra L.) under boron toxicity revealed candidate genes responsible in boron uptake, transport and detoxification. Plant Physiol. Biochem. 109 , 146–155 (2016). Tao, H. et al. WRKY33 negatively regulates anthocyanin biosynthesis and cooperates with PHR1 to mediate acclimation to phosphate starvation. Plant Commun. 13 , 5(5):100821 (2024). Park, S. H., Jeong, J. S., Huang, C. H., Park, B. S. & Chua, N. H. Inositol polyphosphates-regulated polyubiquitination of PHR1 by NLA E3 ligase during phosphate starvation response in Arabidopsis. New Phytol. 237 (4):1215-1228 (2023). Guo, J., Chen, J., Yang, J., Yu, Y., Yang, Y. & Wang, W. Identification, characterization and expression analysis of the VQ motif-containing gene family in tea plant (Camellia sinensis). BMC Genomics 19 (1), 710 (2018). Garrido-Gala, J., Higuera, J. J., Muñoz-Blanco, J. et al. The VQ motif-containing proteins in the diploid and octoploid strawberry. Sci. Rep. 9 , 4942 (2019). Cheng, Y. et al. Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors. Plant Physiol. 159 (2), 810-25 (2012). Pang, Y. et al. Overexpression of the tonoplast aquaporin AtTIP5;1 conferred tolerance to boron toxicity in Arabidopsis. J. Genet. Genomics 37 , 389–397 (2010). Barzana, G. et al. Interrelations of nutrient and water transporters in plants under abiotic stress. Physiologia Plantarum 171 , 595-619 (2021). Additional Declarations No competing interests reported. Supplementary Files NLARNASEQsupplementary08.08dk.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7334872","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":514942725,"identity":"8a78b597-1477-4735-af22-a7ee2f297741","order_by":0,"name":"Doga Selin Kayihan","email":"data:image/png;base64,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","orcid":"","institution":"Başkent University","correspondingAuthor":true,"prefix":"","firstName":"Doga","middleName":"Selin","lastName":"Kayihan","suffix":""}],"badges":[],"createdAt":"2025-08-09 15:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7334872/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7334872/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91563360,"identity":"62b95db4-ca1d-45bd-88b9-7c03042aca8c","added_by":"auto","created_at":"2025-09-17 19:06:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70677,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of normalized gene expression values of WT on C condition and \u003cem\u003enla\u003c/em\u003e on C, 1B and 2B conditions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7334872/v1/1bad1a1194d55fd133eff187.png"},{"id":91563362,"identity":"522f1196-3ff1-4744-9734-3091a2963510","added_by":"auto","created_at":"2025-09-17 19:06:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":414005,"visible":true,"origin":"","legend":"\u003cp\u003ea. Venn diagram showing the numbers of upregulated DEGs in nla under C, 1B and 2B conditions compared to WT-C.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eb. Venn diagram showing the numbers of downregulated DEGs in nla under C, 1B and 2B conditions compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ec. Venn diagram showing the numbers of upregulated DEGs in nla under 1B and 2B conditions compared to nla-C\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ed. Venn diagram showing the numbers of downregulated DEGs in nla under 1B and 2B conditions compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ee. Venn diagram showing the numbers of the upregulated DEGs of nla under B toxicity compared to WT-C (nla vs WT-C) and the upregulated DEGs of nla under B toxicity compared to nla-C (nla vs nla-C). Note that the DEGs of nla-C vs WT-C were excluded.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ef. Venn diagram showing the numbers of the downregulated DEGs of nla under B toxicity compared to WT-C (nla vs WT-C) and the downregulated DEGs of nla under B toxicity compared to nla-C (nla vs nla-C). Note that the DEGs of nla-C vs WT-C were excluded.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7334872/v1/2a337019f8e66c1499ea054c.png"},{"id":91564707,"identity":"aa995178-e28d-4a28-b67b-599499eda474","added_by":"auto","created_at":"2025-09-17 19:14:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171656,"visible":true,"origin":"","legend":"\u003cp\u003ea. KEGG enrichment analysis of upregulated genes in nla under C compared to WT-C\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eb. KEGG enrichment analysis of downregulated genes in nla under C compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ec. KEGG enrichment analysis of upregulated genes in nla under 1B compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ed. KEGG enrichment analysis of downregulated genes in nla under 1B compared to WT-C\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ee. KEGG enrichment analysis of upregulated genes in nla under 2B compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ef. KEGG enrichment analysis of downregulated genes in nla under 2B compared to WT-C\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7334872/v1/6cb4464ddd4a2c7f0106508e.png"},{"id":91563364,"identity":"48aa55b3-328a-4a0a-9462-03eb7fc7aa90","added_by":"auto","created_at":"2025-09-17 19:06:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":175416,"visible":true,"origin":"","legend":"\u003cp\u003ea. KEGG enrichment analysis of upregulated genes in nla under 1B compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eb. KEGG enrichment analysis of downregulated genes in nla under 1B compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ec. KEGG enrichment analysis of upregulated genes in nla under 2B compared to nla-C\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ed. KEGG enrichment analysis of downregulated genes in nla under 2B compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ee. KEGG enrichment analysis of upregulated genes in nla under 2B compared to nla under 1B\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ef. KEGG enrichment analysis of downregulated genes in nla under 2B compared to nla under 1B\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7334872/v1/0ec8fa4d774df563334cd996.png"},{"id":91563366,"identity":"2b2fa2ac-6ff1-4594-8dae-01d340977423","added_by":"auto","created_at":"2025-09-17 19:06:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":610259,"visible":true,"origin":"","legend":"\u003cp\u003ea. GO Biological Process analysis of 426 commonly upregulated DEGs in nla under C, 1B and 2B conditions compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eb. GO Biological Process analysis of 247 commonly upregulated DEGs in nla under 1B and 2B conditions but not C compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ec. GO Biological Process analysis of 177 specifically upregulated DEGs in nla under C condition compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ed. GO Biological Process analysis of 148 specifically upregulated DEGs in nla under 1B condition compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ee. GO Biological Process analysis of 927 specifically upregulated DEGs in nla under 2B condition compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ef. GO Biological Process analysis of 1042 commonly down-regulated DEGs in nla under C, 1B and 2B conditions compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eg. GO Biological Process analysis of 230 commonly down-regulated DEGs in nla under 1B and 2B conditions but not C compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eh. GO Biological Process analysis of specifically downregulated 204 DEGs in nla under C condition compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ei. GO Biological Process analysis of specifically downregulated 862 DEGs in nla under 2B condition compared to WT-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7334872/v1/5ab1996066415514cc1ac018.png"},{"id":91565301,"identity":"3b77651b-3e32-45b3-ba3c-741089d30c8c","added_by":"auto","created_at":"2025-09-17 19:22:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":355613,"visible":true,"origin":"","legend":"\u003cp\u003ea. GO Biological Process analysis of 16 commonly upregulated DEGs in nla under 1B and 2B conditions compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eb. GO Biological Process analysis of 116 specifically upregulated DEGs in nla under 1B conditions compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ec. GO Biological Process analysis of 111 specifically upregulated DEGs in nla under 2B conditions compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ed. GO Biological Process analysis of 38 commonly downregulated DEGs in nla under 1B and 2B conditions compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ee. GO Molecular Function analysis of 38 commonly downregulated DEGs in nla under 1B and 2B conditions compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ef. GO Molecular Function analysis of 11 specifically downregulated DEGs in nla under 1B conditions compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eg. GO Biological Process analysis of 238 specifically downregulated DEGs in nla under 2B conditions compared to nla-C\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7334872/v1/c7ed47b7521cc83e9e5c282e.png"},{"id":93577329,"identity":"a5a9be5e-73a8-413f-8792-aa23fa6a2dfa","added_by":"auto","created_at":"2025-10-15 09:39:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3158371,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7334872/v1/78afb534-15f0-4647-962c-0617838f4348.pdf"},{"id":91564710,"identity":"85560c1d-9f74-4384-82fd-ec11f6e122fa","added_by":"auto","created_at":"2025-09-17 19:14:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3555035,"visible":true,"origin":"","legend":"","description":"","filename":"NLARNASEQsupplementary08.08dk.docx","url":"https://assets-eu.researchsquare.com/files/rs-7334872/v1/bb617ef9762c856b6d4523bf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptome profiling of nla-mutant Arabidopsis reveals possible post-translational regulation of key factors cross-linking circadian-rhythm and anthocyanin pathways under boron toxicity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBoron (B) micronutrient, which is essential for the healthy development of plants [1], readily becomes toxic depending on its concentration in the soil [2]. Thus, many crops suffer from B toxicity which, in turn, leads to yield-loss worldwide due to consequences like delay in emergence and foliation, reduced stem height, dry matter weight, 1000-kernel weight, and number of spikes per plant. Indeed, excessive B levels were reported in the soils of many countries including United States, Australia, Turkey, Russia, India, Israel, Mexico, Egypt, Iraq, Morocco, Syria, Libya, Jordan, Malaysia, Chile, Pakistan, Peru, Hungary, Italy and Serbia [3]. The macroscopic side effects of B excess have been attributed to its ability to form ester bonds with several metabolites having multiple hydroxyl groups in the cis-configuration including ribose sugar [4,5]. Namely, B impairs cell division and development by binding to ribose, both as the free sugar and as a constituent of RNA and interferes with primary metabolism by binding to ribose in ATP or NAD(P)H. In addition, it reduces cytosolic pH, thus affects protein conformation and biosynthesis [5]. Plant cells combat these toxic effects of B through a variety of mechanisms including\u0026nbsp;B efflux by B-transporters which export excess B from tissues [6]. Another way plants have been suggested to use is the reduction of free B in cytosol by complexing with anthocyanins which is followed by the subsequent compartmentalization of B-anthocyanin complex in vacuoles and/or efflux of this complex from the cell by ABC transporters [7]. Supportively, induced accumulation of anthocyanin by high B levels was postulated in several studies [8, 9, 10, 11].\u003c/p\u003e\n\u003cp\u003eOne main approach to improve the management of B by cereals is the development of agricultural plants adjusted to its broad range concentrations [12], which may be realized by understanding molecular responses against B toxicity [13]. A previous study demonstrated the differential regulation of protein degradation pathway in wheat cultivars having different tolerance capacities against B toxicity [14]. This allowed us to focus our study on proteasome members in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. NITROGEN LIMITATION ADAPTATION (NLA) is one of the post-translational regulators of different macronutrient-transporters. In fact, it is an E3 ubiquitin ligase [15] directing ubiquitination of a nitrate transporter (NRT1.7) [16] which is responsible for loading of N in source leaves for the remobilization of N to N-demanding tissues [17]. On the other hand, NLA is crucial for Pi homeostasis due to its contribution to the regulation of phosphate transporters (e.g PHT1) that uptake Pi from the rhizosphere [18]. Phosphate starvation leads to post-transcriptional degradation of NLA mRNAs by means of the microRNA miR827 and, in turn, to the increase of phosphate by the accumulation of phosphate transporters PHT1.4 [19]. In this polyubiquitination process, NLA specifically requires PHO2, an E2 conjugase. Furthermore, NLA has an SPX-domain which interacts with nuclear transcription factors that control the phosphate starvation response pathway [20] suggesting the NLA is also localized in nucleus [21, 22, 18]. All domains of NLA are essential for proper functioning as well as its localization to the plasma membrane and/or nucleus [21].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to these certain roles of NLA in regulation of the membrane transporters, some other functions also were proposed. For instance, it was reported that NLA was involved in molecular responses against biotic stress sources like \u003cem\u003ePseudomonas syringae\u003c/em\u003e [23] and \u003cem\u003eHeterodera schachtii\u003c/em\u003e by interacting with protein substrates like pathogenesis-related proteins, MAP kinases, and transcription factors [24]. Finally, NLA was found to target ORE1\u0026nbsp;which is a key NAC transcription factor regulating age-dependent leaf senescence in \u003cem\u003eA. thaliana\u003c/em\u003e [25]. The interaction of NLA with ORE1 in the nucleus regulates its stability and ORE1 level arranged through NLA mediated polyubiquitination using PHO2 determines leaf senescence during nitrogen deficiency [25]. In other words, NLA seems to target a variety of proteins in subcellular compartments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;NITROGEN LIMITATION ADAPTATION\u0026rdquo; name comes from the inability of \u003cem\u003enla\u003c/em\u003e mutant plants to develop adaptive responses such as anthocyanin accumulation under nitrogen (N)-limited conditions which results in early senescence [22]. Interestingly, this repressed-anthocyanin-accumulation phenomenon of \u003cem\u003enla\u003c/em\u003e mutant was not observed in phosphorous deficient plants [26]. Thus, while NLA contributes to the regulation of different transporters, the respective pathways are likely to be regulated differently where involving mechanisms remain to be discovered. In this study, as a preliminary work, we found the remarkable induction of \u003cem\u003eNLA\u003c/em\u003e gene expression in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e through increasing levels of B toxicity (Table S1a). For this reason, in order to gain a more precise insight about the possible relationship between \u003cem\u003eNLA\u003c/em\u003e gene and B responsive regulations at molecular level, transcriptome profiling of \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e exposed to toxic B was determined. Our data might benefit the determination of candidate genes for molecular breeding, and genetic manipulation of plants for B toxicity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant material and Growth condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe seeds of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e ecotype Columbia-0 (Col-0) were provided by Assistant Prof. Dr. Emre Aksoy at Middle East Technical University in Turkey. The seeds of homozygous \u003cem\u003enla\u003c/em\u003e mutant Arabidopsis (N864802) were procured from Nottingham Arabidopsis Stock Centre. Both wild type (Col-0) and \u003cem\u003enla\u003c/em\u003e mutant seeds were surface sterilized with 70% EtOH solution for 2 minutes, then with 15% NaOCl solution for 10 minutes, and then rinsed three times with sterile distilled water. Then, seeds were sown onto half MS media [27] containing 100 \u0026micro;M H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e (control), 1 mM H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e (mild B stress, 1B) and 2 mM H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(moderate B stress, 2B). After 3 days of stratification at 4\u0026deg;C in dark, they were transferred to plant growth room providing 21 \u0026plusmn; 2\u0026deg;C, 16 h light/8 h dark photoperiod with 150 \u0026micro;mol m\u003csup\u003e-2\u003c/sup\u003e sec\u003csup\u003e-1\u003c/sup\u003e illumination, 60\u0026plusmn;5% relative humidity and grown for 2 weeks. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotosynthetic pigment and anthocyanin measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth rate of fourteen days old \u003cem\u003enla\u003c/em\u003e seedlings were phenotypically compared with Col-0 under control and toxic B conditions. Following measurement of the fresh weights of leaf tissues, contents of photosynthetic pigments were determined using the method of [28]. Accordingly, 2 ml of pure acetone was added onto seedlings in a tube followed by incubation at 4\u0026deg;C overnight. The contents of pigments were calculated by the following equations after measuring the samples spectrophotometrically at 661.6, 644.8 and 470 nm. The contents of anthocyanin, as a flavonoid pigment, were determined in seedlings under B stress according to [29]. Accordingly, the seedlings were homogenized with 1 mL of extraction buffer (400 mL of 37% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2.6 ml of dH\u003csub\u003e2\u003c/sub\u003eO, and 12 ml of 100% methanol). They were transferred and incubated into tubes at 22 \u0026deg;C for 10 min. They were centrifuged at 15,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min and supernatant was read at 530 and 657 nm. The following equation was used for anthocyanin content: [A530-(A657/3)]/FW.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq library construction and sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNAs were isolated from 300 mg leaf tissues of \u003cem\u003eArabidopsis\u003c/em\u003e seedlings [30]. The cDNA libraries were generated from the rRNA-depleted RNA samples using an MGIEasy RNA Directional Library Prep Set by Intergen. Annotation was conducted using TAIR10 FASTA sequence (https://www.arabidopsis.org). RNA quantity and purity were evaluated by the BioSpec-nano UV\u0026ndash;VIS spectrophotometer (Shimadzu Europa GmbH). RNA integrity was assessed using the Agilent 2100 Bioanalyser (Agilent Technologies, USA) prior to the RNA-seq. Two hundred nanograms of total RNA was used for sequencing on the MGI DNBSEQ-G400RS instrument (MGI, Shenzhen, China).\u003c/p\u003e\n\u003cp\u003eThe dsDNA library quantity was evaluated using a Qubit\u0026reg; dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, US) and a Qubit\u0026reg; Fluorometer (Thermo Fisher Scientific, US). Genes presenting a log2 (fold change\u0026gt;1) with adjusted p‐value (\u0026le; 0.05) for each comparison were considered differentially expressed. Principal component analysis (PCA) was performed to assess the correlation between replicates and treatments using ClustVis. The set of differentially expressed genes (DEGs) was identified from pairwise comparisons. Venn Diagrams comparing the number of DEG across samples were created using webtools (https://bioinformatics.psb.ugent.be/webtools/Venn/).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGene ontology (GO) analysis of specific groups of DEGs was performed using ShinyGO (0.82) [31]. All DEGs were mapped to terms in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) because pathway-based analysis improved our understanding of the molecular functions of genes [32]. Read counts (gene expression quantification) per gene were obtained using featureCounts from Rsubread, and analyzed using DESeq2 (Love et al., 2014) (Bioconductor packages in R) after adjustment for false-discovery rate (FDR q values \u0026amp;lt; 0.05) and Relative Log Expression (RLE) normalization. To gain a further overview, the network analyses of the selected DEGs were performed using the STRING database [33].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the reliability of the transcriptome analysis, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed for randomly selected genes (Figure S1a). The same total RNAs used in RNA-seq were used as templates.\u0026nbsp;500 ng RNA was used to obtain first strand complementary DNA (cDNA) by using iScript cDNA Synthesis Kit (BioRad). Every sample contained 1 \u0026micro;l of cDNA, 10 \u0026micro;l of iTaq universal SYBR Green super mix (2X) (Bio-Rad), 1 \u0026micro;l of forward primer (0.5 \u0026micro;M final concentration) and 1 \u0026micro;l of reverse primer (0.5 \u0026micro;M final concentration) and nuclease free water for a final volume of 20 \u0026micro;l. This mixture was used for qRT-PCR experiments (Tianlong, Gentier 96E). Primers were designed from exon-exon boundary of the sequences of each gene by using NCBI database and they are shown in Table S1. Conditions of qRT-PCR were initial denaturation at 95 \u0026deg;C for 30 s, followed by 95 \u0026deg;C for 10 s, 60 \u0026deg;C for 1 min for 40 cycles. Conditions of melting curve were at 95 \u0026deg;C for 5s, at 65 \u0026deg;C for 1 min, and cooling for 40 \u0026deg;C for 30s. Expression levels of genes were normalized by using actin (\u003cem\u003eACT2\u003c/em\u003e) gene. The relative fold changes of expressions genes were calculated by 2\u003csup\u003e-deltadeltaCt\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData statistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq experiment was conducted in replicates of two. Experiments of photosynthetic pigment and anthocyanin and qRT-PCR were performed with at least three biological replicates. One-way ANOVA by SPSS statistical program was used to analyze the data obtained from photosynthetic pigment and anthocyanin measurement. The data of qRT-PCR were statistically analyzed by using non-parametric versions of the t-test. They were presented as mean \u0026plusmn; standard error of mean (SEM).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLower anthocyanin levels of \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eA. thaliana\u003c/em\u003e than WT under all B conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcess B reduced overall seedling growth and caused varying degrees of leaf curling and chlorosis in all \u003cem\u003eArabidopsis\u003c/em\u003e seedlings (Figure S1b). Photosynthetic pigments were affected by B toxicity in both genotypes (Table 1). In WT, chlorophyll a and carotenoid concentrations gradually declined with increasing B applications, whereas chlorophyll b remained relatively stable. In contrast, the concentrations of all photosynthetic pigments (chlorophyll a, b, and carotenoids) significantly decreased in \u003cem\u003enla\u003c/em\u003e mutants under both 1B and 2B conditions. Consequently, no significant differences in these pigment concentrations were detected between the two toxic B treatments in \u003cem\u003enla\u0026nbsp;\u003c/em\u003emutant \u003cem\u003eA. thaliana\u003c/em\u003e. Anthocyanin accumulation exhibited opposing trends in the two genotypes (Table 1). In WT, anthocyanin content slightly decreased under 1B but increased markedly under 2B compared to the control. In \u003cem\u003enla\u003c/em\u003e mutants, anthocyanin levels increased steadily with B concentration; however, their overall anthocyanin content remained considerably lower than those in WT under all conditions, including control. Under 2B, WT had over 4.5 times more anthocyanin than \u003cem\u003enla\u0026nbsp;\u003c/em\u003emutant\u003cem\u003e\u0026nbsp;A. thaliana\u003c/em\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Effects of B toxicity on physio-biochemical parameters in \u003cem\u003eA. thaliana\u003c/em\u003e seedlings\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"696\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e1B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e2B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCol-0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003enla\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCol-0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003enla\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCol-0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003enla\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eChl a (\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1081\u0026plusmn;112\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e929\u0026plusmn;122\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e879\u0026plusmn;61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e538\u0026plusmn;83\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e766\u0026plusmn;75\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e658\u0026plusmn;206\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eChl b (\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e337\u0026plusmn;42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e288\u0026plusmn;67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e306\u0026plusmn;57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e198\u0026plusmn;22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e312\u0026plusmn;42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e225\u0026plusmn;58\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCarotenoid (\u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e328\u0026plusmn;33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e287\u0026plusmn;21\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e269\u0026plusmn;17\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e207\u0026plusmn;42\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e247\u0026plusmn;18b\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e219\u0026plusmn;56\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAnthocyanin (Abs g-1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.030\u0026plusmn;0,006\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.006\u0026plusmn;0.001\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.022\u0026plusmn;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.010\u0026plusmn;0.003\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.070\u0026plusmn;0.006\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.015\u0026plusmn;0.001\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues followed by different letters are significantly different at P \u0026lt; 0.05 level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome profiling identified 4698 and 530 DEGs in B-stressed \u003cem\u003enla\u003c/em\u003e mutant Arabidopsis compared to WT-C and nla-C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, the molecular response of \u003cem\u003enla\u003c/em\u003e mutant and WT under B toxicity conditions were determined by carrying out global gene expression using RNA-sequencing. Accordingly, Principial Component Analysis (PCA) of the normalized gene expressions showed an obvious separation of WT group from \u003cem\u003enla\u003c/em\u003e mutant groups (Figure 1).\u0026nbsp;The principal components explained 70.4% of the total variance (52.1% by PC1 and 18.3% by PC2). In general, biological replicates were closely grouped. One replicate of the 1B samples was slightly associated with mutant control. Differentially expressed genes (DEGs) in response to different B conditions were screened with the threshold of adjusted P value \u0026lt; 0.05. When compared to WT under control (C) condition (hereafter WT-C), 2568, 2320 and 4323 DEGs were determined in \u003cem\u003enla\u003c/em\u003e mutant plants under C, 1B and 2B conditions, respectively. When compared to \u003cem\u003enla\u003c/em\u003e mutant plants under C conditions (hereafter nla-C), 181 and 403 DEGs were detected in \u003cem\u003enla\u003c/em\u003e plants under 1B and 2B conditions, respectively.\u003c/p\u003e\n\u003cp\u003eWhen compared to WT-C, 826, 893, 1751 genes were differentially upregulated (Figure 2a) and 1742, 1427, 2572 genes were downregulated (Figure 2b) in \u003cem\u003enla\u003c/em\u003e mutants under C, 1B and 2B conditions, respectively. A total of 4698 (1971 up and 2727 down) DEGs were identified in B stressed \u003cem\u003enla\u003c/em\u003e mutants when compared to WT-C. From these DEGs, 673 were commonly upregulated, and 1272 were commonly downregulated in \u003cem\u003enla\u003c/em\u003e mutants exposed to toxic B conditions (Figure 2a, 2b). 177, 148 and 927 genes were specifically upregulated whereas 204, 97 and 862 genes were specifically downregulated under C, 1B and 2B conditions, respectively (Figure 2a, 2b).\u003c/p\u003e\n\u003cp\u003eA total of 530 (243 up and 287 down) DEGs were identified in B stressed \u003cem\u003enla\u003c/em\u003e mutants when compared to nla-C (Figure 2c, 2d). From these DEGs, 16 were commonly upregulated, and 38 were commonly downregulated in \u003cem\u003enla\u003c/em\u003e mutants exposed to toxic-B (Figure 2c, 2d). 116 and 111 genes were specifically upregulated whereas 11 and 238 genes were specifically downregulated under 1B and 2B conditions, respectively (Figure 2c, 2d).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGO and KEGG Enrichment Analyses of DEGs in \u003cem\u003enla\u003c/em\u003e under C and toxic B conditions relative to WT-C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Gene Ontology (GO) analysis, \u0026ldquo;ribosome\u0026rdquo; was overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e under C condition (Figure 3a).\u0026nbsp;In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Pectate lyase activity\u0026rdquo;, \u0026ldquo;Histone methyltransferase activity\u0026rdquo;, \u0026ldquo;Histone-lysine N-methyltransferase activity\u0026rdquo;, \u0026ldquo;Lysine N-methyltransferase activity\u0026rdquo;, \u0026ldquo;Protein-lysine N-methyltransferase activity\u0026rdquo;, \u0026ldquo;Protein methyltransferase activity\u0026rdquo;, \u0026ldquo;N-methyltransferase activity\u0026rdquo;, \u0026ldquo;RRNA binding\u0026rdquo;, \u0026ldquo;Structural constituent of ribosome\u0026rdquo;, \u0026ldquo;MRNA binding\u0026rdquo;, \u0026ldquo;RNA binding\u0026rdquo; were significantly enriched (Figure S2). In the biological process ontology, the major terms were \u0026ldquo;Phylloquinone biosynthetic proc\u0026rdquo;,\u0026nbsp;\u0026ldquo;Vitamin K biosynthetic proc.\u0026rdquo;, \u0026ldquo;Vitamin K metabolic proc\u0026rdquo;, \u0026ldquo;Translation\u0026rdquo;, \u0026ldquo;Peptide biosynthetic proc.\u0026rdquo;, \u0026ldquo;Peptide metabolic proc.\u0026rdquo;, \u0026ldquo;Cellular amide metabolic proc.\u0026rdquo; and \u0026ldquo;Organonitrogen compound biosynthetic proc.\u0026rdquo; (Figure S3). According to GO analysis, \u0026ldquo;Linoleic acid metabolism\u0026rdquo;, \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo;, \u0026ldquo;Tryptophan metabolism\u0026rdquo;, \u0026ldquo;Plant pathogen interaction\u0026rdquo; \u0026ldquo;MAPK signaling pathway-plant\u0026rdquo;, \u0026ldquo;Plant hormone signal transduction\u0026rdquo;, \u0026ldquo;Endocytosis\u0026rdquo; and \u0026ldquo;Biosynthesis of secondary metabolites\u0026rdquo; were overrepresented for downregulated genes in \u003cem\u003enla\u003c/em\u003e mutants under control condition (Figure 3b). According to KEGG analyses, lipoxygenases (e.g., EC 1.13.11.58 in LA and EC 1.13.11.12 in LA and ALA) were highlighted in \u0026ldquo;Linoleic acid metabolism\u0026rdquo; and \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo; pathways (Figue S4 and S5). In the biological process ontology, the major terms were \u0026ldquo;Cellular response to hypoxia\u0026rdquo;, \u0026ldquo;Cellular response to decreased oxygen levels\u0026rdquo;, \u0026ldquo;Response to hypoxia\u0026rdquo;, \u0026ldquo;Response to oxygen levels\u0026rdquo; and \u0026ldquo;Response to wounding\u0026rdquo; (Figure S4). Regarding molecular function, the major terms were \u0026ldquo;Xyloglucan:xyloglucosyl transferase activity\u0026rdquo; and \u0026ldquo;Glucosyltransferase activity\u0026rdquo; (Figure S5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to GO analysis, \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo;, \u0026ldquo;ribosome\u0026rdquo; and \u0026ldquo;Biosynthesis of secondary metabolites\u0026rdquo; were overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutant Arabidopsis under 1B condition compared to WT-C (Figure 3c). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;NAD+ nucleosidase activity\u0026rdquo;, \u0026ldquo;NAD(P)+ nucleosidase activity\u0026rdquo;, \u0026ldquo;NAD+ nucleotidase cyclic ADP-ribose generating\u0026rdquo;, \u0026ldquo;RRNA binding\u0026rdquo;, \u0026ldquo;Structural constituent of ribosome\u0026rdquo; were significantly enriched (Figure S6).\u0026nbsp;In the biological process ontology, the major terms were \u0026ldquo;Coumarin metabolic proc.\u0026rdquo; and \u0026ldquo;Translational elongation\u0026rdquo; (Figure S7). \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo;, \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo;, \u0026ldquo;Plant-pathogen interaction\u0026rdquo;, \u0026ldquo;MAPK signaling pathway-plant\u0026rdquo;, \u0026ldquo;Plant hormone signal transduction\u0026rdquo; and \u0026ldquo;Biosynthesis of secondary metabolites\u0026rdquo; were overrepresented for downregulated genes in \u003cem\u003enla\u003c/em\u003e mutants under 1B when compared to WT control\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Figure 3d). In the biological process ontology, the major terms were \u0026ldquo;Cellular response to hypoxia\u0026rdquo;, \u0026ldquo;Cellular response to decreased oxygen levels\u0026rdquo;, \u0026ldquo;Response to hypoxia\u0026rdquo;, \u0026ldquo;Response to oxygen levels\u0026rdquo; (Figure S8). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Linoleate 13S-lipoxygenase activity\u0026rdquo;, \u0026ldquo;Allene-oxide cyclase activity\u0026rdquo;, \u0026ldquo;Diacylglycerol kinase activity\u0026rdquo; and \u0026ldquo;Xyloglucan:xyloglucosyl transferase activity\u0026rdquo; were significantly enriched (Figure S9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to GO analysis, \u0026ldquo;ribosome\u0026rdquo;, \u0026ldquo;Ribosome biogenesis in eukaryotes\u0026rdquo;, \u0026ldquo;Pyrimidine metabolism\u0026rdquo; and \u0026ldquo;RNA degradation\u0026rdquo; were overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutant Arabidopsis under 2B condition when compared to WT control\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Figure 3e). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Large ribosomal subunit rRNA binding\u0026rdquo;, \u0026ldquo;MRNA 5-prime-UTR binding\u0026rdquo;, \u0026ldquo;Small ribosomal subunit rRNA binding\u0026rdquo;, \u0026ldquo;RRNA binding\u0026rdquo;, \u0026ldquo;SnoRNA binding\u0026rdquo; and \u0026ldquo;Structural constituent of ribosome\u0026rdquo; were significantly enriched (Figure S10).\u0026nbsp;In the biological process ontology, the major terms were \u0026ldquo;Ribosomal large subunit biogenesis\u0026rdquo;, \u0026ldquo;Ribosome biogenesis\u0026rdquo;, \u0026ldquo;Translation\u0026rdquo;, \u0026ldquo;Peptide biosynthetic proc.\u0026rdquo;, \u0026ldquo;RRNA processing\u0026rdquo;, \u0026ldquo;RRNA metabolic proc.\u0026rdquo;, \u0026ldquo;Amide biosynthetic proc.\u0026rdquo;, \u0026ldquo;Peptide metabolic proc.\u0026rdquo;, \u0026ldquo;Ribonucleoprotein complex biogenesis\u0026rdquo; and \u0026ldquo;Cellular amide metabolic proc.\u0026rdquo; (Figure S11). \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo;, \u0026ldquo;Taurine and hypotaurine metabolism\u0026rdquo;, \u0026ldquo;Linoleic acid metabolism\u0026rdquo;, \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo; and \u0026ldquo;Plant-pathogen interaction\u0026rdquo; were overrepresented for downregulated genes in \u003cem\u003enla\u003c/em\u003e mutants under 2B when compared to WT control\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Figure 3f). KEGG analysis was performed for Taurine and hypotaurine metabolism pathway and given as supplementary file (Figure S12). In the biological process ontology, the major terms were \u0026ldquo;Cellular response to hypoxia\u0026rdquo;, \u0026ldquo;Cellular response to decreased oxygen levels\u0026rdquo;, \u0026ldquo;Response to hypoxia\u0026rdquo;, \u0026ldquo;Response to oxygen levels\u0026rdquo; (Figure S13). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Calmodulin binding\u0026rdquo;, \u0026ldquo;Protein serine kinase activity\u0026rdquo;, \u0026ldquo;Protein serine/threonine/tyrosine kinase activity\u0026rdquo;, \u0026ldquo;Protein serine/threonine kinase activity\u0026rdquo;, \u0026ldquo;Protein kinase activity\u0026rdquo;, \u0026ldquo;Phosphotransferase activity alcohol group as acceptor\u0026rdquo; and \u0026ldquo;Kinase activity\u0026rdquo; were significantly enriched (Figure S14).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGO and KEGG Enrichment Analyses of DEGs in \u003cem\u003enla\u003c/em\u003e under toxic B conditions relative to nla-C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to GO analysis, \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo;, \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo;, and \u0026ldquo;Biosynthesis of secondary metabolites\u0026rdquo; were overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e under 1B condition compared to \u003cem\u003enla\u003c/em\u003e control (Figure 4a). KEGG analysis was performed for Alpha-Linolenic acid metabolism and Phenylpropanoid biosynthesis pathways and given as supplementary file (Figure S15 and S16). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Linoleate 13S-lipoxygenase activity\u0026rdquo;, \u0026ldquo;Xyloglucan:xyloglucosyl transferase activity\u0026rdquo;, \u0026ldquo;Hydroquinone:oxygen oxidoreductase activity\u0026rdquo;, \u0026ldquo;Ferric-chelate reductase activity\u0026rdquo;, \u0026ldquo;Oxidoreductase activity acting on single donors with incorporation of molecular oxygen\u0026rdquo; and \u0026ldquo;Polysaccharide binding\u0026rdquo; were significantly enriched (Figure S17). In the biological process ontology, the major terms were \u0026ldquo;Cellular response to hypoxia\u0026rdquo;, \u0026ldquo;Cellular response to decreased oxygen levels\u0026rdquo;, \u0026ldquo;Response to hypoxia\u0026rdquo;, \u0026ldquo;Response to oxygen levels\u0026rdquo; and \u0026ldquo;Phenylpropanoid metabolic proc.\u0026rdquo; (Figure S18). On the other hand, \u0026ldquo;Photosynthesis-antenna proteins\u0026rdquo;, \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo; and \u0026ldquo;2-Oxocarboxylic acid metabolism\u0026rdquo; were overrepresented for downregulated genes in \u003cem\u003enla\u003c/em\u003e mutants under 1B compared to \u003cem\u003enla\u003c/em\u003e control (Figure 4b). In the biological process ontology, the major terms were \u0026ldquo;Reg. of salicylic acid mediated signaling pathway\u0026rdquo;, \u0026ldquo;Systemic acquired resistance\u0026rdquo;, \u0026ldquo;Cellular response to salicylic acid stimulus\u0026rdquo;, \u0026ldquo;Salicylic acid mediated signaling pathway\u0026rdquo;, \u0026ldquo;Response to virus\u0026rdquo;, \u0026ldquo;Response to salicylic acid\u0026rdquo; and \u0026ldquo;Cellular response to organic cyclic compound\u0026rdquo; (Figure S19). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Chlorophyll binding\u0026rdquo;, \u0026ldquo;Beta-glucosidase activity\u0026rdquo; and \u0026ldquo;Glucosidase activity\u0026rdquo; were significantly enriched (Figure S20). According to GO analysis, \u0026ldquo;Circadian rhythm-plant\u0026rdquo; was overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e under 2B condition compared to \u003cem\u003enla\u003c/em\u003e control\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Figure 4c). KEGG analysis was performed for Circadian rhythm-plant pathway and given as supplementary file (Figure S21). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Large ribosomal subunit rRNA binding\u0026rdquo; and \u0026ldquo;RRNA binding\u0026rdquo; were significantly enriched (Figure S22). In the biological process ontology, the major terms were \u0026ldquo;Negative reg. of circadian rhythm\u0026rdquo;, \u0026ldquo;Reg. of circadian rhythm\u0026rdquo;, \u0026ldquo;Photomorphogenesis\u0026rdquo;, \u0026ldquo;Cellular response to oxidative stress\u0026rdquo;, \u0026ldquo;Cellular response to chemical stress\u0026rdquo; and \u0026ldquo;Cellular response to light stimulus\u0026rdquo; (Figure S23). On the other hand, \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo;, \u0026ldquo;Monobactam biosynthesis\u0026rdquo;, \u0026ldquo;Photosynthesis-antenna proteins\u0026rdquo;, \u0026ldquo;2-Oxocarboxylic acid metabolism\u0026rdquo;, \u0026ldquo;Sulfur metabolism\u0026rdquo;, \u0026ldquo;Valine leucine and isoleucine biosynthesis\u0026rdquo; and \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo; were overrepresented for downregulated genes in \u003cem\u003enla\u003c/em\u003e mutants under 2B compared to \u003cem\u003enla\u003c/em\u003e control\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Figure 4d). In the biological process ontology, the major terms were \u0026ldquo;S-glycoside biosynthetic proc.\u0026rdquo;, \u0026ldquo;Glycosinolate biosynthetic proc.\u0026rdquo;, \u0026ldquo;Glucosinolate biosynthetic proc.\u0026rdquo;, \u0026ldquo;S-glycoside metabolic proc\u0026rdquo;, \u0026ldquo;Glycosinolate metabolic proc.\u0026rdquo;, \u0026ldquo;Glucosinolate metabolic proc.\u0026rdquo; and \u0026ldquo;Sulfur compound biosynthetic proc.\u0026rdquo; (Figure S24). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Adenylylsulfate kinase activity\u0026rdquo;, \u0026ldquo;Oxidoreductase activity acting on other nitrogenous compounds as donors\u0026rdquo; and \u0026ldquo;Glutathione binding\u0026rdquo; were significantly enriched (Figure S25).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGO Enrichment Analysis of DEGs in \u003cem\u003enla\u003c/em\u003e under 2B condition relative to 1B condition\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to GO analysis, \u0026ldquo;Base excision repair\u0026rdquo; was overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e under 2B condition relative to 1B (Figure 4e). In terms of molecular function, the processes represented by the GO terms \u0026ldquo;NAD+ ADP-ribosyltransferase activity\u0026rdquo; was significantly enriched (Figure S26). In the biological process ontology, no significant enrichment was found. On the other hand, \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo;, \u0026ldquo;Sulfur metabolism\u0026rdquo;, \u0026ldquo;Plant-pathogen interaction\u0026rdquo;, \u0026ldquo;Cysteine and methionine metabolism\u0026rdquo; and \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo; were overrepresented for downregulated genes in \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e under 2B condition relative to 1B (Figure 4f). \u0026nbsp;In terms of molecular function, the processes represented by the GO terms \u0026ldquo;Allene-oxide cyclase activity\u0026rdquo;, \u0026ldquo;Polysaccharide binding\u0026rdquo;, \u0026ldquo;Symporter activity\u0026rdquo;, \u0026ldquo;Secondary active transmembrane transporter activity\u0026rdquo;, \u0026ldquo;Heme binding\u0026rdquo;, \u0026ldquo;Calmodulin binding\u0026rdquo; and \u0026ldquo;Carbohydrate binding\u0026rdquo; were significantly enriched (Figure S27). In the biological process ontology, the major terms were \u0026ldquo;Cellular response to hypoxia\u0026rdquo;, \u0026ldquo;Cellular response to decreased oxygen levels\u0026rdquo;, \u0026ldquo;Response to hypoxia\u0026rdquo;, \u0026ldquo;Response to oxygen levels\u0026rdquo;, \u0026ldquo;Response to wounding\u0026rdquo; and \u0026ldquo;Response to bacterium\u0026rdquo; (Figure S28).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGO and KEGG Enrichment Analyses of commonly and specifically up- and down-regulated DEGs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e426 genes were commonly upregulated in mutant plants under control, 1B and 2B conditions when compared to WT-C (Figure 2a). These DEGs were overrepresented by \u0026ldquo;Ribosome\u0026rdquo;. Their GO Biological Process analysis showed that \u0026ldquo;Phylloquinone biosynthetic proc\u0026rdquo;, \u0026ldquo;Phylloquinone metabolic proc\u0026rdquo;, \u0026ldquo;Vitamin K biosynthetic proc.\u0026rdquo; and \u0026ldquo;Vitamin K metabolic proc\u0026rdquo; were significantly enriched (Figure 5a). Also, 247 genes were commonly upregulated under 1B and 2B conditions (Figure 2a). These DEGs were overrepresented by \u0026ldquo;Ribosome\u0026rdquo;. According to GO Biological Process, \u0026ldquo;Cytoplasmic translational elongation\u0026rdquo;, \u0026ldquo;Maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA 5.8S rRNA LSU-rRNA)\u0026rdquo;, \u0026ldquo;Maturation of SSU-rRNA\u0026rdquo;, \u0026ldquo;Ribosomal small subunit biogenesis\u0026rdquo; \u0026ldquo;Cytoplasmic translation\u0026rdquo;, \u0026ldquo;Ribosome assembly\u0026rdquo; and \u0026ldquo;Translational elongation\u0026rdquo; terms were significantly enriched (Figure 5b). On the other hand, 177 genes were specifically upregulated in \u003cem\u003enla\u003c/em\u003e mutant Arabidopsis under C condition (Figure 2a). But no significant enrichment was found for these DEGs.\u0026nbsp;According to GO Biological Process \u0026ldquo;Photosynthetic electron transport chain\u0026rdquo; was significantly enriched (Figure 5c). Also, 148 genes were specifically upregulated under 1B condition (Figure 2). These DEGs were overrepresented by \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo; and \u0026ldquo;Biosynthesis of various plant secondary metabolites\u0026rdquo;. According to GO Biological Process, \u0026ldquo;Coumarin metabolic proc.\u0026rdquo;, \u0026ldquo;Suberin biosynthetic proc.\u0026rdquo;, \u0026ldquo;Response to iron ion\u0026rdquo; and \u0026ldquo;Hydrogen peroxide catabolic proc.\u0026rdquo; were significantly enriched (Figure 5d). 927 genes were specifically upregulated under 2B condition. These DEGs were overrepresented by \u0026ldquo;Lysine biosynthesis\u0026rdquo; and \u0026ldquo;Ribosome\u0026rdquo;. KEGG analysis was performed for Lysine biosynthesis pathway and given as supplementary file (Figure S29). According to GO Biological Process, \u0026ldquo;Ribosomal large subunit biogenesis\u0026rdquo; and \u0026ldquo;RRNA processing\u0026rdquo; were significantly enriched (Figure 5e).\u003c/p\u003e\n\u003cp\u003eOn\u0026nbsp;the other hand, 1042 genes were commonly downregulated in mutant plants under control, 1B and 2B conditions when compared to WT-C (Figure 2b). These DEGs were overrepresented by \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo;, \u0026ldquo;Plant-pathogen interaction\u0026rdquo;, \u0026ldquo;MAPK signaling pathway-plant\u0026rdquo; and \u0026ldquo;Plant hormone signal transduction\u0026rdquo;. According to GO Biological Process, \u0026ldquo;Cellular response to hypoxia\u0026rdquo;, \u0026ldquo;Cellular response to decreased oxygen levels\u0026rdquo;, \u0026ldquo;Response to hypoxia\u0026rdquo;, \u0026ldquo;Response to oxygen levels\u0026rdquo; were significantly enriched (Figure 5f). Also, 230 genes were commonly downregulated by 1B and 2B conditions but not C (Figure 2b). These DEGs were overrepresented by \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo; and \u0026ldquo;2-Oxocarboxylic acid metabolism\u0026rdquo;. Their GO Biological Process analysis showed that \u0026ldquo;Defense response to insect\u0026rdquo;, \u0026ldquo;S-glycoside biosynthetic proc.\u0026rdquo; and \u0026ldquo;Glycosinolate biosynthetic proc.\u0026rdquo; were significantly enriched (Figure 5g). 204 genes were downregulated only in nla mutants under control condition. But no significant enrichment was found for these DEGs.\u0026nbsp;According to GO Biological Process, \u0026ldquo;Response to red or far red light\u0026rdquo; and \u0026ldquo;Hormone-mediated signaling pathway\u0026rdquo; were significantly enriched (Figure 5h). However, 97 genes were specifically downregulated under 1B condition. But no significant enrichment was found for these DEGs as well as for their GO Biological Process. Also, 862 genes were specifically downregulated under 2B condition. These DEGs were overrepresented by \u0026ldquo;Taurine and hypotaurine metabolism\u0026rdquo;, \u0026ldquo;Butanoate metabolism\u0026rdquo;, \u0026ldquo;Sulfur metabolism\u0026rdquo;, \u0026ldquo;Nitrogen metabolism\u0026rdquo; and \u0026ldquo;Alanine aspartate and glutamate metabolism\u0026rdquo;.\u0026nbsp;According to GO Biological Process, \u0026ldquo;Toxin metabolic proc.\u0026rdquo;, \u0026ldquo;Sulfur compound biosynthetic proc.\u0026rdquo;, \u0026ldquo;Sulfur compound metabolic proc.\u0026rdquo; and \u0026ldquo;Response to bacterium\u0026rdquo; were significantly enriched (Figure 5i).\u003c/p\u003e\n\u003cp\u003e16 genes were commonly upregulated in mutant plants under 1B and 2B conditions when compared to nla-C (Figure 2c). These DEGs were overrepresented by \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo;, \u0026ldquo;Porphyrin metabolism\u0026rdquo; and \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo;. According to GO Biological Process, \u0026ldquo;Lignin metabolic proc.\u0026rdquo; and \u0026ldquo;Phenylpropanoid metabolic proc.\u0026rdquo; were significantly enriched (Figure 6a). Also, 116 genes were specifically upregulated in mutant plants under 1B condition compared to \u003cem\u003enla\u003c/em\u003e control. In the biological process ontology, the major terms were \u0026ldquo;Cellular response to hypoxia\u0026rdquo;, \u0026ldquo;Cellular response to decreased oxygen levels\u0026rdquo;, \u0026ldquo;Response to hypoxia\u0026rdquo;, \u0026ldquo;Response to oxygen levels\u0026rdquo; and \u0026ldquo;Response to wounding\u0026rdquo; (Figure 6b). 111 genes were specifically upregulated in mutant plants under 2B condition compared to \u003cem\u003enla\u003c/em\u003e control. These DEGs was overrepresented by \u0026ldquo;Circadian rhythm-plant\u0026rdquo;. In the biological process ontology, the major term was \u0026ldquo;Negative reg. of circadian rhythm\u0026rdquo; (Figure 6c).\u003c/p\u003e\n\u003cp\u003e38 genes were commonly downregulated in mutant plants under 1B and 2B conditions when compared to nla-C. These DEGs were overrepresented by \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo; and \u0026ldquo;Photosynthesis-antenna proteins\u0026rdquo; where Lhca2 and Lhcb1were significantly downregulated according to KEGG analysis (Figure S30). In the biological process ontology, the major terms were \u0026ldquo;Positive reg. of defense response to oomycetes\u0026rdquo; and \u0026ldquo;Systemic acquired resistance\u0026rdquo; (Figure 6d). In terms of molecular function, the process represented by the GO terms was \u0026ldquo;Chlorophyll binding\u0026rdquo; (Figure 6e). Also, 11 genes were specifically downregulated in mutant plants under 1B condition compared to \u003cem\u003enla\u003c/em\u003e control. GO analyses, except GO Molecular Function, couldn\u0026rsquo;t carried out due to \u0026ldquo;too few genes\u0026rdquo;. In terms of molecular function, the processes represented by the GO terms were \u0026ldquo;Glucosinolate glucohydrolase activity\u0026rdquo;, \u0026ldquo;Thioglucosidase activity\u0026rdquo;, \u0026ldquo;NAD+ diphosphatase activity\u0026rdquo;, \u0026ldquo;ADP-ribose diphosphatase activity\u0026rdquo; and \u0026ldquo;NADH pyrophosphatase activity\u0026rdquo; (Figure 6f). On the other hand, 238 genes were specifically downregulated in mutant plants under 2B condition compared to \u003cem\u003enla\u003c/em\u003e control. These DEGs were overrepresented by \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo;, \u0026ldquo;Monobactam biosynthesis\u0026rdquo; and \u0026ldquo;Sulfur metabolism\u0026rdquo;. In the biological process ontology, the major terms were \u0026ldquo;S-glycoside biosynthetic proc.\u0026rdquo; and \u0026ldquo;Glycosinolate biosynthetic proc.\u0026rdquo; (Figure 6g). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGO Enrichment Analysis of commonly and specifically regulated DEGs between \u003cem\u003enla\u003c/em\u003e under B toxicity vs WT-C and \u003cem\u003enla\u003c/em\u003e under B toxicity vs nla-C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DEGs of \u003cem\u003enla\u003c/em\u003e mutants under B toxicity relative to WT control were compared with the DEGs of \u003cem\u003enla\u003c/em\u003e mutants under B toxicity relative to \u003cem\u003enla\u003c/em\u003e control and venn diagrams were generated for upregulated and downregulated DEGs (Figure 2e, Figure 2f). Accordingly, 8 genes (AT1G07610, AT1G72030, AT2G05510, AT4G19430, AT3G19450, AT4G37070, AT3G01420, AT5G66690; overrepresented by \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo; and \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo;) were commonly upregulated in these two major groups (Figure S31). In 1B condition, 50 genes (overrepresented by \u0026ldquo;Cutin suberine and wax biosynthesis\u0026rdquo; and \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo;) were commonly upregulated (Figure S32) whereas 345 (enriched by \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo; and \u0026ldquo;Ribosome\u0026rdquo;) and 82 genes (enriched by \u0026ldquo;Cellular response to hypoxia\u0026rdquo; according to GO Biological Process) were specifically upregulated respectively (Figure S33 and S34). In 2B condition, 89 genes (enriched by \u0026ldquo;Large ribosomal subunit rRNA binding\u0026rdquo; according to GO Molecular Function) were commonly upregulated (Figure S35) whereas 1084 (overrepresented by \u0026ldquo;Ribosome\u0026rdquo; and \u0026ldquo;Lysine biosynthesis\u0026rdquo;) and 38 genes (overrepresented by \u0026ldquo;Circadian rhythm-plant\u0026rdquo;) were specifically upregulated in \u0026ldquo;nla vs WT-C\u0026rdquo; and \u0026ldquo;nla vs nla-C\u0026rdquo; respectively (Figure S36 and S37).\u003c/p\u003e\n\u003cp\u003eOn the other hand, 22 genes (overrepresented by \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo;) were commonly downregulated in these two major groups (Figure S38). In terms of 1B condition, 28 genes (overrepresented by \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo;) were commonly downregulated (Figure S39) whereas 299 (overrepresented by \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo;) and 21 genes (enriched by \u0026ldquo;Reg. of growth rate\u0026rdquo;, \u0026ldquo;Extracellular transport\u0026rdquo; and \u0026ldquo;Positive reg. of DNA-binding transcription factor activity\u0026rdquo; according to GO Biological Process) were specifically downregulated respectively (Figure S40 and S41). In terms of 2B condition, 147 genes (overrepresented by \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo;) were commonly downregulated (Figure S42) whereas 945 (overrepresented by \u0026ldquo;Taurine and hypotaurine metabolism\u0026rdquo;) and 129 genes (overrepresented by \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo; and \u0026ldquo;Alpha-Linolenic acid metabolism\u0026rdquo;) were specifically downregulated in respectively (Figure S43 and S44).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMost significantly regulated DEGs in nla\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eunder B toxicity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen compared to WT under control condition, the most significantly upregulated genes were AT2G42140 (VQ17; VQ motif-containing protein; log2fc 6.8-fold), AT1G05807 (not annotated; log2fc 5.5-fold) and\u0026nbsp;AT3G43505 (LCR30; Low-molecular-weight cysteine-rich 30; log2fc 12.5-fold) under control, 1B and 2B conditions, respectively.\u0026nbsp;When compared to WT under control condition, the most significantly downregulated genes were AT4G28405 (not annotated; log2fc 7.5-fold), AT2G07752 (pre-tRNA tRNA-Glu (anticodon: TTC); log2fc 7.9-fold) and AT5G12030 (AT-HSP17.6A; HEAT SHOCK PROTEIN 17.6; log2fc 8.2-fold) under control, 1B and 2B conditions, respectively. When compared to \u003cem\u003enla\u003c/em\u003e mutant under control condition, the most significantly upregulated genes were AT2G17660 (NOI3; Member of the RIN4-like/NOI family; log2fc 4.7-fold) and\u0026nbsp;AT2G20463 (defensin-like (DEFL) family protein; log2fc 10-fold) under 1B and 2B conditions, respectively. When compared to nla under control condition, the most significantly downregulated genes were AT2G42140 (VQ17; VQ motif-containing protein; log2fc 7.6-fold) and\u0026nbsp;AT3G22231\u0026nbsp;(PATHOGEN AND CIRCADIAN CONTROLLED 1; PCC1; log2fc 7.7-fold) under 1B and 2B conditions, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe differential regulations of the transcription factors (TFs) in nla\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eunder B toxicity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e45, 38 and 92 TFs were upregulated in \u003cem\u003enla\u003c/em\u003e mutant plants under C, 1B and 2B conditions when compared to WT-C, respectively. Neither significant enrichment nor cluster was found for these TFs. On the other hand, 180, 146 and 207 TFs were downregulated in \u003cem\u003enla\u003c/em\u003e mutant plants under C, 1B and 2B conditions, respectively, when compared to WT-C. No cluster was found for these TFs however 180 downregulated TFs under C condition were enriched by \u0026ldquo;MAPK signaling pathway-plant\u0026rdquo; and \u0026ldquo;Plant-pathogen interaction\u0026rdquo; (Figure S45). Also, 12 TFs were upregulated and generated one cluster including WRKY40, ZAT10, ERF11, ABR1, WRKY28, NAC019 and DREB1C in \u003cem\u003enla\u003c/em\u003e mutant plants under 1B condition when compared to nla-C (Figure S46). All of these TFs were specifically regulated under 1B. Only 2 TFs (WRKY54 and CRF2/Ethylene-responsive transcription factor) were downregulated in \u003cem\u003enla\u003c/em\u003e mutant Arabidopsis under 1B condition when compared to \u003cem\u003enla\u003c/em\u003e mutant Arabidopsis under control condition. 17 TFs were upregulated in \u003cem\u003enla\u003c/em\u003e mutant under 2B condition when compared to \u003cem\u003enla\u003c/em\u003e mutant under control condition. Neither significant enrichment nor cluster was found for these TFs. However, 16 TFs enriched by \u0026ldquo;MAPK signaling pathway-plant\u0026rdquo; and \u0026ldquo;Plant-pathogen interaction\u0026rdquo; were downregulated and generated one cluster including WRKY33, WRKY53, WRKY22, WRKY54, WRKY60, ERF6, ERF2, ERF094, MYB52 and NAC102 in \u003cem\u003enla\u003c/em\u003e mutant under 2B condition when compared to nla-C (Figure S47). Except WRKY54, all these TFs were specifically regulated on 2B. KEGG analysis was performed for MAPK signaling pathway-plant and given as supplementary file (Figure S48).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe differential regulations of Boron Transporters in nla\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eunder B toxicity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe relative expression levels of B efflux transporters, \u003cem\u003eAtBOR1\u003c/em\u003e (AT2G47160) and \u003cem\u003eATBOR4\u003c/em\u003e (AT1G15460), as well as boric acid channel for boron uptake \u003cem\u003eAtNIP5;1\u003c/em\u003e (AT4G10380) remained stable in \u003cem\u003enla\u003c/em\u003e plants under B toxicity compared to both \u003cem\u003enla\u003c/em\u003e mutant and WT Arabidopsis under control conditions. However, B exporter \u003cem\u003eBOR2\u003c/em\u003e (AT3G62270) was significantly induced (more than two-fold) under both 1B and 2B conditions compared to WT control and under 1B compared to \u003cem\u003enla\u003c/em\u003e mutant control. On the other hand, the expression level of tonoplast aquaporin \u003cem\u003eTIP5;1\u003c/em\u003e (AT3G47440) gene was downregulated (more than 4-fold) in \u003cem\u003enla\u003c/em\u003e mutant Arabidopsis under 1B condition when compared with WT control. A network was detected by using the STRING (12.0) [33] between NLA and AtTIP5 (Figure S49).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eExcess B in soil occurred by natural or anthropogenic influences leads to toxicity in many plants and gives rise to yield loss of important cereal crops especially in arid and semi-arid regions of the world [34, 35]. Rather than the impractical remediation of soil, the development of tolerant varieties has been accepted as a long-term sustainable solution. Thus, the main mechanisms adopted by the tolerant, hyperaccumulator and other plant species to counteract B toxicity are matters of interest for plant breeders and biotechnologists [35, 36]. A prior study exhibited the differential regulation of protein degradation pathway in B-toxicity-sensitive and -tolerant wheat cultivars under B toxicity [14]. In this study, as a preliminary work, we found the remarkable induction of \u003cem\u003eNLA\u003c/em\u003e gene expression in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e through increasing levels of B toxicity (Table S1a). Therefore, in the present study, global expression profile was investigated in \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e exposed to toxic concentrations of B to elucidate the intricate mechanisms governing NLA-related B toxicity response at the molecular level. Accordingly, PCA was performed to elucidate the dynamics of transcriptomic variations across the experimental conditions. The results derived from PCA exhibited an obvious separation of wild type group from \u003cem\u003enla\u003c/em\u003e mutant groups suggesting that mutation of \u003cem\u003enla\u003c/em\u003e gene significantly disturbed the transcriptome of \u003cem\u003eArabidopsis\u003c/em\u003e leaves. A clear cluster was observed under 2B condition that agrees with the respective phenotype on this condition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHigh number of DEGs in \u003cem\u003enla\u003c/em\u003e plants under control condition without B treatments underscored the effect of the \u003cem\u003enla\u003c/em\u003e mutation in \u003cem\u003eA. thaliana\u003c/em\u003e. NLA encodes a ubiquitin E3 ligase [15] that mediates degradation of PHT1s at plasma membranes. SPX-domain of NLA interacts with nuclear transcription factors that control the phosphate starvation response pathway [20]. On the other hand, \u003cem\u003enla\u003c/em\u003e mutant plants are unable to develop adaptive responses under nitrogen (N)-limited conditions, hence the name \u0026ldquo;nitrogen limitation adaptation\u0026rdquo;, which results in early senescence [22]. In fact, it is directing ubiquitination of a nitrate transporter (NRT1.7) [16] which is responsible for loading of N in source leaves for the remobilization of N to N-demanding tissues [17]. In addition to its certain roles in regulation of the membrane transporters, NLA is involved in mediating immune responses by interacting with protein substrates like pathogenesis-related proteins, mitogen activated protein kinases (MAPKs), and transcription factors [24]. In other words, NLA may have many protein targets yet to be discovered. In the current study, it was not surprising to obtain that \u0026ldquo;ribosome\u0026rdquo; pathway was overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e under control condition compared to WT-C since NLA is directly involved in proteasome system and lack of its expression can lead to reorganization of protein translation process.\u0026nbsp;In agreement with this, one of the most represented categories of molecular function was histone methyltransferase activity. The enrichment of histone methyltransferase activity may reflect rearrangement of transcriptional regulation of mutants because histone methylation plays a central role in regulating chromatin state and gene expression in \u003cem\u003eA. thaliana\u003c/em\u003e, as in other organisms, and is involved in a variety of physiological and developmental processes [37]. In the biological process, upregulation of phylloquinone (vitamin K1) biosynthesis may refer to alterations in photosynthetic mechanism of mutants. Because phylloquinone is an essential electron carrier in photosystem I and its concentration is highly correlated with chlorophyll [38]. However, according to the physiological findings, photosynthetic pigments in \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e under control condition were very similar to WT under C condition as supported by the results of Peng and coauthors [39].\u003c/p\u003e\n\u003cp\u003eAnother highly enriched pathway was for pectate lyase,\u0026nbsp;an enzyme involved in degradation of pectin, a major constituent of the dicot primary cell wall. It depolymerizes pectin by cleavage between-(1,4) glycosidic bonds and participates in the remodeling of pectin during organogenesis, especially during fruit ripening [40]. It has primarily been investigated as virulence factor and in tissue maceration by necrotrophic pathogens. Plant pectate lyase like proteins (PLLs) have specific roles in accommodating infection threads during symbiosis or organ growth and senescence. In leaves, PLLs were mostly expressed in the vasculature system including leaf veins [41]. Moreover, a recent report demonstrated that a pectate lyase plays an important role during vascular development in \u003cem\u003eArabidopsis\u003c/em\u003e [40]. Another PLL has been suggested to remove homogalacturonan, thus potentially enabling further callose synthase to access and modify the cell wall during sieve pore maturation\u0026nbsp;[42]. In the present study, the enrichment of pectate lyase activity may be related to the possible cell wall modification in \u003cem\u003enla\u003c/em\u003e mutants due to \u003cem\u003eNLA\u003c/em\u003e mutation-altered immunity. Supportively, compared with wild type plants, higher deposition of callose in \u003cem\u003enla\u003c/em\u003e mutants conferred resistance to pathogen by\u0026nbsp;reinforcing plant cell walls to arrest pathogen infection [43]. On the other hand, mostly downregulated pathways were linoleic acid (LA) and alpha-Linolenic acid (ALA) metabolisms in \u003cem\u003enla\u003c/em\u003e control plants compared to WT-C. LA and ALA are the two most common polyunsaturated fatty acids in plants [44]. According to KEGG analyses, lipoxygenases (LOX) were highlighted in both pathways (Figue S4 and S5). Indeed, lipoxygenases are key regulators for lipid peroxidation, which is crucial for plant senescence and defense pathways. Oxidation products mediated by LOX, such as jasmonate (JA), play a major role in the responses to biotic and abiotic stress. Interestingly, it was found that \u003cem\u003enla\u003c/em\u003e mutants have high SA and JA even under non-infectious conditions [43]. This may be an explanation for the repression of the JA pathway in \u003cem\u003enla\u003c/em\u003e mutants under C condition in this study (Figure S5). Compatible with that, interrelated pathways like tryptophan metabolism, plant pathogen interaction, MAPK signaling pathway-plant and plant hormone signal transduction were also overrepresented for downregulated genes in \u003cem\u003enla\u003c/em\u003e mutants under control condition. There are multiple MAPK pathways that are involved in hormone signaling and trigger various stress responses where stress signal is transduced based on the DEGs is largely due to MAPK [45]. Since pathogenesis-related proteins and MAPKs are among the substrates of NLA [24], failure of their degradation in mutant plants may have caused their regulation to occur at transcriptional level. \u0026nbsp;Overall repression of these pathways may indicate the unstressed mode of \u003cem\u003enla\u003c/em\u003e plants on C condition.\u003c/p\u003e\n\u003cp\u003e426 genes commonly upregulated in mutant plants under all conditions (C, 1B and 2B) when compared to WT-C were overrepresented by ribosome and phylloquinone biosynthetic process was significantly enriched in these plants. Moreover, translation related pathways were commonly enriched in both B conditions. B interferes with transcription and/or translation by binding to cis hydroxyls on ribose molecules in RNA species [36]. That could explain the powerful enrichment of rRNA-mRNA binding and translational elongation process. Moreover, it has been suggested that B also interferes with aminoacylation steps of the tRNAs and causes uncharged tRNA stress, thus, the general amino acid control pathway contributes to B toxicity [46]. Supportively, the most significantly repressed gene by 1B compared to WT-C was \u0026ldquo;pre-tRNA tRNA-Glu\u0026rdquo; which is the precursor of Glutamyl-tRNA, member of aminoacyl-tRNA biosynthesis pathway.\u003c/p\u003e\n\u003cp\u003eBesides, 148 DEGs specifically regulated under 1B condition were overrepresented by \u0026ldquo;Phenylpropanoid biosynthesis\u0026rdquo; and \u0026ldquo;Biosynthesis of various plant secondary metabolites\u0026rdquo;. Consistently, similar pathways were highly induced in response to B-induced stress in maize [47], in wheat [14] and in mulberry [48]. Plants produce secondary metabolites against different stress conditions. Thus, the activation of these pathways appears to be implicated in maintenance of the homeostasis. On the other hand, lysine biosynthesis pathway was overrepresented for specifically up-regulated 927 DEGs in 2B (Figure S29). The essential amino acid lysine is synthesized in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine [49]. Lysine is also a precursor for glutamate, an important signaling amino acid that regulates plant growth and responses to the environment [50]. Also, 1042 genes commonly downregulated in mutant plants under all conditions (control, 1B and 2B) when compared to WT-C were overrepresented by ALA metabolism, plant-pathogen interaction, MAPK signaling pathway-plant and plant hormone signal transduction. It is worth mentioning that 2B strongly repressed protein serine/threonine kinase activity when compared to WT-C. MAPKs are a large family of enzymes that phosphorylate their protein targets on serine or threonine residues. It is known that borate might form ester‑like complexes with these residues\u0026nbsp;[51]\u0026nbsp;which may mechanically affect MAPKs\u0026rsquo; binding to their target transcription factors (TFs) and subsequently the interaction between TFs and target genes. The TF interaction with the target gene might be modulated also by the direct linkage of B to the TF. So, depending on the target gene and the type of TF (activator or repressor) the linkage with B could regulate gene expression [52]. On the other hand, glucosinolate biosynthesis pathway was commonly enriched in both B conditions.\u0026nbsp;Glucosinolates are categorized into tryptophan-derived indole, tyrosine or phenylalanine derived aromatic, or aliphatic glucosinolates. Aliphatic glucosinolates are synthesized from methionine whereas aliphatic glucosinolates are synthesized from alanine, valine, and leucine. Also, glutathione is involved in the biosynthesis of glucosinolates as a sulfur donor [45]. In B-stressed plants most of the down-regulated DEGs were obtained by 2B where 862 specifically regulated DEGs were enriched by taurine and hypotaurine metabolism, butanoate metabolism and sulfur metabolism which were associated to toxin metabolic process and sulfur compound biosynthetic process by GO Biological Process. Taurine metabolism also is interrelated with glutathione metabolism (Figure S12). These findings corroborated the downregulation of biosynthesis of secondary metabolites, plant hormone signal transduction, degradation of valine, leucine and isoleucine obtained in \u003cem\u003eA. thaliana\u003c/em\u003e under toxic B condition [53].\u003c/p\u003e\n\u003cp\u003eWhen compared to \u003cem\u003enla\u003c/em\u003e mutants under control condition (nla-C), only 16 genes were commonly upregulated in mutant plants under 1B and 2B conditions and overrepresented by ALA metabolism. In contrast to many genes related to production of JA in this pathway including LOX (EC.1.13.11.12) tended to downregulate in nla-C compared to WT-C (Figure S5), lipoxygenases (EC.1.13.11.12 and EC.1.13.11.92) were upregulated in mutants under 1B (Figure S15) compared to nla-C. Moreover, the latter belonging to a branch of ALA pathway other than JA was commonly upregulated by 1B and 2B. Senescence-related hormones like JA and ethylene were promoted by B excess also in barley and wheat [54, 14]. On the other hand, B stress significantly enriched lignin and phenylpropanoid metabolic process in \u003cem\u003enla\u003c/em\u003e plants (Figure 6a). Increased level of phenylpropanoid metabolites like flavonoids including anthocyanin, is one of the adaptive measures that plants employ to defend against stress. B toxicity-promoted accumulation of these metabolites was obtained in earlier studies [8, 9, 10, 11, 55]. Moreover, anthocyanin, in addition to capability for compartmentalizing excess B into vacuoles\u0026nbsp;[7], exhibits a photoprotective role in mesophyll cells when chloroplast functionality has been compromised by B toxicity [11]. However, \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e plants did not exhibit nitrogen deficiency-induced anthocyanin accumulation; instead showed higher lignin production whereas they still show phosphorous deficiency-induced anthocyanin accumulation [26], suggesting that different signaling cascades are involved in anthocyanin responses against different nutrients. Also, we found that the overall anthocyanin content remained considerably lower than those in Col-0 under the normal and high B conditions (Table 1). In line with this, B stress induced lignin path rather than anthocyanin in the phenylpropanoid pathway in \u003cem\u003enla\u003c/em\u003e mutants by means of the upregulation of cinnamyl-alcohol dehydrogenase (EC 1.1.1.195), peroxidase (EC 1.11.1.7) and coniferyl alcohol glucosyl transferase (EC 2.4.1.111) (Figure S16). Thus, B-stressed nla plants seem to trigger a similar signaling network with the \u003cem\u003enla\u003c/em\u003e plants switching from anthocyanin to lignin biosynthesis path by nitrogen limitation.\u003c/p\u003e\n\u003cp\u003e111 specifically upregulated genes in mutant plants under 2B condition compared to \u003cem\u003enla\u003c/em\u003e control were overrepresented by negative regulation of the circadian rhythm (Figure 6c). Circadian clocks in plants, as in all eukaryotes, rely on photoreceptors to sense light and gate the timing of central metabolic pathways relative to the outside environment. According to KEGG analysis (Figure S21), upregulation of CCA1 (CIRCADIAN CLOCK ASSOCIATED1) and LHY (LATE ELONGATED HYPOCOTYL) in redlight mediated regulation of antenna proteins and CRY, SPA and HY5 in blue light mediated regulation of photomorphogenesis may at least reveal the B stress-altered photosynthetic machinery\u0026nbsp;as a strategy for \u003cem\u003enla\u003c/em\u003e plants survival since, as in many plant species, the correct synchronization of the clock with the environment contributes to ensuring survival under fluctuating environmental conditions [56]. More importantly, LHY and CCA are among the key transcription factors in the circadian feedback loop that has a potential role in the regulation of PAP1-mediated UV-B protection as seen in KEGG circadian rhythm-plant pathway (Figure S21).\u0026nbsp;ATPAP1 or ARABIDOPSIS THALIANA PRODUCTION OF ANTHOCYANIN PIGMENT 1 is a MYB75 transcription factor involving in anthocyanin metabolism. This interaction cross-links the circadian rhythm and anthocyanin pathway as evidenced by Hu and coauthors who noted the connection between flavonoids and the plant clock [57]. Moreover, circadian control of ORE1 by PRR9, another component of circadian feedback loop, positively regulates leaf senescence by enhancing expression of senescence related genes like SAG29 as well as chlorophyll catabolic enzymes in \u003cem\u003eA. thaliana\u003c/em\u003e [58]. ORE1 homeostasis appears to be impaired due to lack of its post-translational regulation by NLA which, in turn, may have disturbed PRR9 tuning and, eventually, the crosstalk between circadian rhythm and anthocyanin pathways in \u003cem\u003enla\u003c/em\u003e plants. This could be an explanation for their inhibited accumulation of anthocyanin and switching to lignin biosynthesis in response to B toxicity.\u003c/p\u003e\n\u003cp\u003eGO enrichment analysis revealed that base excision repair was overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutants under 2B condition when compared to 1B where NAD+ ADP-ribosyltransferase activity was significantly enriched. Oxidative stress has been widely determined in plants subjected to B excess [59, 60, 8, 61, 62]. Higher induction of NAD+ ADP-ribosyltransferase activity at 2B condition may imply that oxidative DNA damage is more pronounced in higher B condition, and that DNA repair system is activated since\u0026nbsp;ADP-ribosylation of nuclear proteins, under conditions of low to moderate DNA damage, facilitates DNA repair and promotes cell survival [63].\u003c/p\u003e\n\u003cp\u003e38 commonly downregulated genes in mutant plants under 1B and 2B conditions compared to \u003cem\u003enla\u003c/em\u003e-C were overrepresented by photosynthesis-antenna proteins and glucosinolate biosynthesis. Significant enrichment of chlorophyll binding as molecular function (Figure 6e), and downregulation of light harvesting complexes (Lhca2 and Lhcb1) (Figure S30) were in accordance with affected circadian rhythm. Similarly, LHC participants in\u0026nbsp;photosynthesis\u0026ndash;antenna proteins pathway were downregulated under high boron which were attributed to a decrease in light absorption, consequently reducing the photosynthesis rate [64]. Photosynthesis is one of the main metabolic processes impaired by B excess due to biochemical limitations including the decline of electron transport rate, reduced CO\u003csub\u003e2\u003c/sub\u003e use efficiency and impairment of photosystem II (PSII) efficiency (reviewed by [35] beside alterations of photosynthetic pigment contents [65, 8, 66]. On the other hand, chlorophyll degradation\u003cem\u003e\u0026nbsp;\u003c/em\u003eis typically accompanied by anthocyanin accumulation during leaf senescence. Indeed,\u0026nbsp;anthocyanin biosynthesis and chlorophyll degradation are linked through the action of NAC transcription factors such as ORE1 [67]. Normally, ORE1 activates leaf senescence related genes including SAG29 and genes encoding chlorophyll catabolic enzymes. In this study, \u003cem\u003enla\u003c/em\u003e plants showed inhibited photosynthesis as confirmed by chlorosis to a degree and decreased content of photosynthetic pigments under B toxicity (Table 1, Figure S1). Yet, the inability of their photoprotective adaptation due to reduced anthocyanin accumulation may have directed the mutant plants to minimize their photosynthetic apparatus on toxic B condition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar to DEGs compared to WT control condition, glucosinolate biosynthesis was the most strongly enriched pathway in toxic B treated \u003cem\u003enla\u003c/em\u003e plants compared to \u003cem\u003enla\u003c/em\u003e controls. It is notable that repression of glucosinolate pathway was obtained despite the induction of the phenylpropanoid pathway since both represent the secondary metabolism involved in plant defense. The response-shift between the phenylpropanoid and glucosinolate biosynthesis pathways has been suggested to occur depending on the intensity of stress factors in plants [45]. Production of phenylpropanoids has been proposed as an earlier line of defense and, moreover, shown to be limited by glucosinolate pathway [68] which collaborates with the inverse regulation of these pathways in the present study. Likewise, the B toxicity and sulfur deficiency caused differential expression of the same set of genes involved in glucosinolate biosynthetic processes, sulfur metabolism, and osmotic stress [69].\u003c/p\u003e\n\u003cp\u003eTranscription factors (TFs) emerge as genuine conductors of gene expression symphonies under the influence of many environmental stresses [70]. In this study, one cluster was obtained under 1B condition when compared to \u003cem\u003enla\u003c/em\u003e plants on the control condition. These TFs including WRKY40, ZAT10, ERF11, ABR1, WRKY28, NAC019 and DREB1C were specifically upregulated on 1B. On the other hand, 2B led to repression of one cluster including WRKY33, WRKY53, WRKY22, WRKY54, WRKY60, ERF6, ERF2, ERF094, MYB52 and NAC102 when compared to \u003cem\u003enla\u003c/em\u003e plants on the control condition. These TFs were enriched by \u0026ldquo;MAPK signaling pathway-plant\u0026rdquo; and \u0026ldquo;Plant-pathogen interaction\u0026rdquo;. These outcomes corroborated the findings of several studies related to transcriptomic regulation in response to B toxicity including increase in expression level of DREB, ERF, NAC and repressions of MYB in poplar [71], significant enrichment of MYB in wheat [64], differential regulation of WRKY and MYB in wheat [14], downregulation of WRKY in barley [54]. The present study indicates that most of the differentially regulated ERF TFs tend to decrease in accordance with the enrichment of signaling pathways for repressed TFs since ERF has been associated with signaling of phytohormones [48]. Notably, WRKY TFs appear to have major roles in both induced and repressed network in \u003cem\u003enla\u003c/em\u003e mutants under B stress due to their central positions according to STRING analysis (Figure S46 and S47). Especially significant downregulation of WRKYs in MAPK signaling pathway (Figure S48) supports the view that B interferes with transcriptional regulation by binding to the serine/threonine residues of the kinase-substrates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe differential regulation of WRKY33 may have also contributed to repressed anthocyanin accumulation of \u003cem\u003enla\u003c/em\u003e mutants under B toxicity. Because a recent study has revealed that WRKY33 negatively regulates anthocyanin biosynthesis [72]. In this case, under Pi limited condition PHR1\u0026nbsp;(the key transcription factor controlling Pi-starvation response) interacts with WRKY33, and thereby the protein level of WRKY33 decreases; the repression of DFR (dihydroflavonol 4-reductase, a rate-limiting enzyme in anthocyanin production) expression by WRKY33 is thus attenuated, leading to anthocyanin accumulation in \u003cem\u003eA. thaliana\u003c/em\u003e. On the other hand, NLA as a negative post-translational regulator of PHR1 involves in phosphate starvation response in \u003cem\u003eA. thaliana\u003c/em\u003e [73]. Seedlings of \u003cem\u003enla\u003c/em\u003e mutants display accumulation of anthocyanin under Pi-deficiency. Take in together, in \u003cem\u003enla\u003c/em\u003e mutants, Pi starvation enhances PHR1 protein level which leads to decrease of WRKY33 protein level and subsequent induction of DFR and eventually anthocyanin accumulation. Thus, the findings of the current study may be used to speculate that the low level of WRKY33 protein confers anthocyanin accumulation under B toxicity in WT plants whereas \u003cem\u003enla\u003c/em\u003e mutants fail to induce anthocyanin accumulation because of the impaired PAP1-promoted DFR stimuli. This impairment may arise from an abnormal induction of a factor other than WRKY33 interacting with PAP1 due to disrupted post-translational regulations of NLA. In other words, an NLA-target (i.e., ORE1) may be responsible for this interrupted chain of reactions. Stable level of PAP1 and DFR transcripts despite of the significant downregulation of WRKY33 in \u003cem\u003enla\u003c/em\u003e mutants under B toxicity (Figure S48) strengthens this hypothesis. Interestingly, the most significantly upregulated gene in nla-C compared to WT-C was the most significantly downregulated gene in \u003cem\u003enla\u003c/em\u003e mutant under 1B condition compared to nla-C. This is VQ17 which is a VQ motif-containing protein. The plant specific VQ motif-containing proteins have been recently discovered as a class of plant regulatory proteins interacting with WRKY transcription factors capable of modulating their activity as transcriptional regulators and playing important roles in plant growth, development and stress response [74, 75]. A weak interaction of VQ17 with WRKY33 was postulated in \u003cem\u003eA. thaliana\u003c/em\u003e [76]. Moreover, overexpression of VQ17 highly stunted growth of \u003cem\u003eA. thaliana\u0026nbsp;\u003c/em\u003e[76]. Overall, these multiple cross-talking signaling pathways in B-stressed \u003cem\u003enla\u003c/em\u003e mutants modulated by transcription factors as well as transcriptional regulators and post-translational modifications need further exploration at both RNA and protein level.\u003c/p\u003e\n\u003cp\u003eThe stable expression levels of B efflux transporters (AtBOR1 and ATBOR4) and boric acid channel (AtNIP5;1) were obtained in \u003cem\u003enla\u003c/em\u003e plants under B toxicity compared to both mutant and WT plants under control conditions. However, B exporter BOR2 was induced more than two-fold by both 1B and 2B compared to WT-C and by 1B compared to nla-C. Interestingly, AtTIP5;1 was downregulated more than 4-fold in \u003cem\u003enla\u003c/em\u003e plants under 1B condition when compared with WT-C. AtTIP5 is an aquaporin family member localized to the cell tonoplast membrane and confers high B tolerance lowering the cytoplasmic B concentration by means of vacuolar compartmentation of B [77]. The links between nutrient transporters and aquaporins have been demonstrated to be important to maintain cell homeostasis. For instance, deficiencies of micronutrients induce their tonoplast transporters for remobilization from the vacuole and also reduce plasma membrane aquaporins presence [78]. B toxicity is expected to lead to upregulation of B exporter and vacuolar influx transporters. In the present case, AtTIP5 expression was lower in only \u003cem\u003enla\u003c/em\u003e plants under 1B condition compared to WT-C. This may be related to a collaborative relationship between the transporters and the channels. Supportively, a network analysis generated using the STRING to investigate the functional interaction between NLA and AtTIP5 displayed an interrelationship between NIP-TIP cluster, PHT and NLA (Figure S49). However, much more research is needed to confirm this co-coordinative regulation of aquaporins and B transporters under B stress.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study investigated the global transcription profile of \u003cem\u003enla\u003c/em\u003e mutants under B stress and conferred statements about NLA related B toxicity responses for the first time. Since NLA is a contributor of protein degradation pathway, overall enrichment of ribosome pathway was expected in all mutant plants. Besides, common enrichment of translation related pathways in both B stress conditions was associated with B interference with RNA binding processes as well as aminoacyl-tRNA biosynthesis pathway. Likewise, B may be responsible for the reduced activity of MAPKs due to forming complexes with serine or threonine residues of their target TFs, thereby preventing their action on these TFs such as WRKYs. Physiological findings revealed that anthocyanin accumulation was inhibited in \u003cem\u003enla\u003c/em\u003e mutants, unlike WT plants under B stress. This was attributed to the powerful induction of lignin path rather than anthocyanin in the phenylpropanoid pathway in \u003cem\u003enla\u003c/em\u003e mutants. One reason for switching from anthocyanin to lignin biosynthesis may be the disruption of the crosstalk between circadian rhythm and anthocyanin pathways in \u003cem\u003enla\u003c/em\u003e plants whose ORE1 homeostasis appears to be impaired due to lack of its post-translational regulation by NLA. Stable transcription level of anthocyanin biosynthesis genes, PAP1 and DFR, despite of the significant downregulation of WRKY33 under B toxicity suggested the fact that lack of post-translational regulation of a factor other than WRKY33 by NLA led to inhibition of DFR stimuli by interacting with PAP1 in \u003cem\u003enla\u003c/em\u003e mutants. This alternative interaction of PAP1 with this NLA target (known like ORE1 or unknown) remains to be discovered. Eventually, our data might benefit the determination of candidate genes for molecular breeding, and genetic manipulation of plants for B toxicity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor performed the experiments and data analysis and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe RNA-seq data have been uploaded to the NCBI SRA (Sequence Read Archive) database with reference accession PRJNA1130029 (Temporary Submission ID: SUB15556101). The corresponding author may be contacted if someone wants to request the data from this study.\u003c/p\u003e\n\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWarington, K. The effect of boric acid and borax on the broad bean and certain other plants. \u003cem\u003eAnn. Bot.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 629\u0026ndash;672 (1923).\u003c/li\u003e\n\u003cli\u003eEaton., F. M. Deficency, toxicity and accumulation of borron in plants, \u003cem\u003eJ. Agric. Res.\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 237-277 (1944).\u003c/li\u003e\n\u003cli\u003eBrdar-Jokanovic, M. Boron toxicity and deficiency in agricultural plants. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e,1424. (2020).\u003c/li\u003e\n\u003cli\u003eBolanos, L., Lukaszewski, K., Bonilla, I. \u0026amp; Blevins, D. Why boron? \u003cem\u003ePlant Physiology and Biochemistry\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 907\u0026ndash;912 (2004).\u003c/li\u003e\n\u003cli\u003eReid, R. J., Hayes, J. E., Post, A., Stangoulis, J. C. R. \u0026amp; Graham, R. D. A critical analysis of the causes of boron toxicity in plants. \u003cem\u003ePlant Cell and Environment\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1405\u0026ndash;1414 (2004).\u003c/li\u003e\n\u003cli\u003eMiwa, K \u0026amp; Fujiwara, T. Role of overexpressed BOR4, a boron exporter, in tolerance to high level of boron in shoots. \u003cem\u003eSoil Sci. Plant Nutr. \u003c/em\u003e\u003cstrong\u003e57\u003c/strong\u003e, 558\u0026ndash;565 (2011).\u003c/li\u003e\n\u003cli\u003eLandi, M., Tattini, M. \u0026amp; Gould, K.S. Multiple functional roles of anthocyanins in plant-environment interactions. \u003cem\u003eEnviron. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 4-17 (2015).\u003c/li\u003e\n\u003cli\u003eKayıhan DS, Kayıhan C, \u0026Ccedil;ift\u0026ccedil;i Y\u0026Ouml; (2016). Excess boron responsive regulations of antioxidative mechanism at physio-biochemical and molecular levels in Arabidopsis thaliana. Plant Physiology and Biochemistry 109: 337-345.\u003c/li\u003e\n\u003cli\u003eKayıhan, C. The involvement of the induction of anthocyanin biosynthesis and transport in toxic boron responsive regulation in Arabidopsis thaliana. Turk. J. Bot. 45 (3), 1 (2021).\u003c/li\u003e\n\u003cli\u003eCervilla, L. M. et al. Parameters symptomatic for boron toxicity in leaves of tomato plants. \u003cem\u003eJ. Bot. \u003c/em\u003e1-17 (2012).\u003c/li\u003e\n\u003cli\u003eLandi, M., Guidi, L., Pardossi, A., Tattini, M. \u0026amp; Gould, K. S. Photoprotection by foliar anthocyanins mitigates effects of boron toxicity in sweet basil (Ocimum basilicum). \u003cem\u003ePlanta\u003c/em\u003e \u003cstrong\u003e240 \u003c/strong\u003e(5), 941\u0026ndash;953 (2014).\u003c/li\u003e\n\u003cli\u003eJefferies, S. P. et al. Mapping of chromosome regions conferring boron toxicity tolerance in barley (Hordeum vulgare L.). \u003cem\u003eTheoretical and Applied Genetics\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, 1293\u0026ndash;1303 (1999).\u003c/li\u003e\n\u003cli\u003eCamacho-Crist\u0026oacute;bal, J. J. et al. The expression of several cell wall-related genes in Arabidopsis roots is down-regulated under boron deficiency. \u003cem\u003eEnviron. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 351\u0026ndash;358 (2008).\u003c/li\u003e\n\u003cli\u003eKayıhan, C., \u0026Ouml;z, M. T., Eyidoğan, F., Y\u0026uuml;cel, M. \u0026amp; \u0026Ouml;ktem, H. A. Physiological, biochemical, and transcriptomic responses to boron toxicity in leaf and root tissues of contrasting wheat cultivars. \u003cem\u003ePlant Mol. Biol. Rep.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e(1), 97\u0026ndash;109 (2017).\u003c/li\u003e\n\u003cli\u003eKant, S., Peng, M. \u0026amp; Rothstein, S. J. Genetic regulation by NLA and microRNA 827 for maintaining nitrate-dependent phosphate homeostasis in Arabidopsis. \u003cem\u003ePLoS Genetics\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e1002021 (2011).\u003c/li\u003e\n\u003cli\u003eLiu, W., Sun Q., Wang, K., Du, Q. \u0026amp; Li, W. X. Nitrogen Limitation Adaptation (NLA) is involved in source-tosink remobilization of nitrate by mediating the degradation of NRT1.7 in Arabidopsis. \u003cem\u003eNew Phytologist \u003c/em\u003e\u003cstrong\u003e214\u003c/strong\u003e, 734\u0026ndash;744 (2017).\u003c/li\u003e\n\u003cli\u003eFan, S. C., Lin, C. S., Hsu, P. K., Lin, S. H. \u0026amp; Tsay, Y. F. The Arabidopsis nitrate transporter NRT1.7 expressed in phloem, is responsible for source-to-sink remobilization of nitrate. Plant Cell 21, 2750\u0026ndash;2761. (2009).\u003c/li\u003e\n\u003cli\u003eLin, W. Y., Huang, T. K. \u0026amp; Chiou, T. J. NITROGENLIMITATION ADAPTATION, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis. \u003cem\u003ePlant Cell\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 4061\u0026ndash;4074 (2013).\u003c/li\u003e\n\u003cli\u003ePark B. S., Seo, J. S. \u0026amp; Chua, N. H. NITROGEN LIMITATION ADAPTATION recruits PHOSPHATE 2 to target the phosphate transporter PT2 for degradation during the regulation of Arabidopsis phosphate homeostasis. \u003cem\u003ePlant Cell\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 454\u0026ndash;464 (2014).\u003c/li\u003e\n\u003cli\u003eShi, J., Hu, H., Zhang, K., Zhang, W., Yu, Y., Wu, Z., \u0026amp; Wu, P. The paralogous SPX3 and SPX5 genes redundantly modulate Pi homeostasis in rice. \u003cem\u003eJ. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 859\u0026ndash;870 (2014).\u003c/li\u003e\n\u003cli\u003eHannam, C. et al. Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway. \u003cem\u003eBotany\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e(2), 79-96 (2018).\u003c/li\u003e\n\u003cli\u003ePeng, M., Hannam, C., Gu H., Bi, Y. M. \u0026amp; Rothstein S. J. A mutation in NLA, which encodes a RING-type ubiquitin ligase, disrupts the adaptability of Arabidopsis to nitrogen limitation. \u003cem\u003ePlant J.\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 320-337 (2007a).\u003c/li\u003e\n\u003cli\u003eYaeno, T., \u0026amp; Iba, K. BAH1/NLA, a RING-type ubiquitin E3 ligase, regulates the accumulation of salicylic acid and immune responses to Pseudomonas syringae DC3000. \u003cem\u003ePlant Physiol.\u003c/em\u003e \u003cstrong\u003e148\u003c/strong\u003e, 1032\u0026ndash;1041 (2008).\u003c/li\u003e\n\u003cli\u003eHewezi, T., Piya, S., Qi, M., Balasubramaniam, M., Rice, J. H., Baum, T. J. Arabidopsis miR827 mediates post-transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility. \u003cem\u003ePlant Journal\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 179\u0026ndash;192 (2016).\u003c/li\u003e\n\u003cli\u003ePark, B. S. et al. Arabidopsis NITROGEN LIMITATION ADAPTATION regulates ORE1 homeostasis during senescence induced by nitrogen deficiency. \u003cem\u003eNature Plants\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 898\u0026ndash;903 (2018). \u003c/li\u003e\n\u003cli\u003ePeng, M., Hudson, D., Schofield, A., Tsao, R., Yang, R., Gu, H., Bi, Y-M., \u0026amp; Rothstein, S.J. Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene. \u003cem\u003eJ. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 2933-2944 (2008).\u003c/li\u003e\n\u003cli\u003eMurashige, T. \u0026amp; Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. \u003cem\u003ePhysiol. Plant\u003c/em\u003e. \u003cstrong\u003e15\u003c/strong\u003e, 473-497 (1962).\u003c/li\u003e\n\u003cli\u003eLichtenthaler, H. K. Chlorophylls and carotenoids: pigments of photosynthetic membranes. \u003cem\u003eMethods Enzymol.\u003c/em\u003e \u003cstrong\u003e148\u003c/strong\u003e, 350-382 (1987).\u003c/li\u003e\n\u003cli\u003eMancinelli, A. L., Yang, C. P. H., Lindquist, P., Anderson, O. R. \u0026amp; Rabino, I. Photocontrol of anthocyanin synthesis III. The action of streptomycin on the synthesis of chlorophyll and antyocyanin. \u003cem\u003ePlant Physiol.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 251-257 (1975).\u003c/li\u003e\n\u003cli\u003eChomczynski, P. \u0026amp; Sacchi, N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. \u003cem\u003eAnal. Biochem\u003c/em\u003e \u003cstrong\u003e162\u003c/strong\u003e, 156-159 (1987).\u003c/li\u003e\n\u003cli\u003eGe, S. X., Jung, D. \u0026amp; Yao, R. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 2628\u0026ndash;2629 (2020).\u003c/li\u003e\n\u003cli\u003eKanehisa, M., Furumichi, M., Sato, Y., Matsuura, Y. and Ishiguro-Watanabe, M.; KEGG: biological systems database as a model of the real world. Nucleic Acids Res. 53, D672-D677 (2025)\u003c/li\u003e\n\u003cli\u003eSzklarczyk, D. et al. The STRING database in 2023: protein\u0026ndash;protein association networks and functional enrichment analyses for any sequenced genome of interest. \u003cem\u003eNucleic Acids Res\u003c/em\u003e. \u003cstrong\u003e51 \u003c/strong\u003e(D1):D, 638-646 (2023).\u003c/li\u003e\n\u003cli\u003eJaved, M.B.; Malik, Z.; Kamran, M.; Abbasi, G.H.; Majeed, A.; Riaz, M.; Bukhari, M.A.; Mustafa, A.; Ahmar, S.; Mora-Poblete, F.; et al. Assessing Yield Response and Relationship of Soil Boron Fractions with Its Accumulation in Sorghum and Cowpea under Boron Fertilization in Different Soil Series. \u003cem\u003eSustainability\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e13\u003c/em\u003e, 4192. \u003c/li\u003e\n\u003cli\u003eLandi, M. et al. Boron toxicity in higher plants: an update. \u003cem\u003ePlanta\u003c/em\u003e \u003cstrong\u003e250\u003c/strong\u003e, 1011\u0026ndash;1032 (2019). \u003c/li\u003e\n\u003cli\u003eReid, R. Can we really increase yields by making crop plants tolerant to boron toxicity? \u003cem\u003ePlant Science\u003c/em\u003e \u003cstrong\u003e178\u003c/strong\u003e, 9-11 (2010).\u003c/li\u003e\n\u003cli\u003eHu, H., \u0026amp; Du, J. Structure and mechanism of histone methylation dynamics in Arabidopsis. \u003cem\u003eCurr. Opin. Plant Biol.\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 102211 (2022).\u003c/li\u003e\n\u003cli\u003eReumann S. Biosynthesis of vitamin K1 (phylloquinone) by plant peroxisomes and its integration into signaling molecule synthesis pathways. \u003cem\u003eSubcell. Biochem\u003c/em\u003e. \u003cstrong\u003e69\u003c/strong\u003e, 213-29 (2013).\u003c/li\u003e\n\u003cli\u003ePeng, M. et al. Genome-wide analysis of Arabidopsis responsive transcriptome to nitrogen limitation and its regulation by the ubiquitin ligase gene NLA. \u003cem\u003ePlant Mol. Biol.\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 775\u0026ndash;797 (2007b). \u003c/li\u003e\n\u003cli\u003eBai, Y. et al. A pectate lyase gene plays a critical role in xylem vascular development in Arabidopsis. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 10883 (2023). \u003c/li\u003e\n\u003cli\u003ePalusa, S. G. \u0026amp; Golovkin, M. Organ-specific, developmental, hormonal and stress regulation of expression of putative pectate lyase genes in Arabidopsis. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e174\u003c/strong\u003e, 537\u0026ndash;550 (2007).\u003c/li\u003e\n\u003cli\u003eKalmbach, L. et al. Putative pectate lyase PLL12 and callose deposition through polar CALS7 are necessary for long-distance phloem transport in Arabidopsis. \u003cem\u003eCurrent Biology \u003c/em\u003e\u003cstrong\u003e33\u003c/strong\u003e, 926 - 939 (2023).\u003c/li\u003e\n\u003cli\u003eVal-Torregrosa, B. et al. Loss-of-function of NITROGEN LIMITATION ADAPTATION confers disease resistance in Arabidopsis by modulating hormone signaling and camalexin content. \u003cem\u003ePlant Science \u003c/em\u003e\u003cstrong\u003e323\u003c/strong\u003e, 111374 (2022).\u003c/li\u003e\n\u003cli\u003eHarwood, J. L. Plant acyl lipids: structure, distribution and analysis. In: (ed. Stumpf PK) The Biochemistry of Plants. Vol 4. New York, USA: Academic Press Inc. Publishing House, 24\u0026ndash;30 (1980).\u003c/li\u003e\n\u003cli\u003eWaskow, A., Guihur, A., Howling, A. \u0026amp; Furno, I. RNA Sequencing of Arabidopsis thaliana seedlings after non-thermal plasma-seed treatment reveals upregulation in plant stress and defense pathways. \u003cem\u003eInt. J. Mol. Sci. \u003c/em\u003e\u003cstrong\u003e23\u003c/strong\u003e, 3070 (2022).\u003c/li\u003e\n\u003cli\u003eUluisik, I. et al. Boron stress activates the general amino acid control mechanism and inhibits protein synthesis. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e(11), e27772 (2011).\u003c/li\u003e\n\u003cli\u003eChen, F., Gao, J., Li, W. \u0026amp; Fang, P. Transcriptome profiles reveal the protective role of seed coating with zinc against boron toxicity in maize (Zea mays L.). \u003cem\u003eJournal of Hazardous Materials\u003c/em\u003e \u003cstrong\u003e423\u003c/strong\u003e 127105 (2022).\u003c/li\u003e\n\u003cli\u003eZou, J. et al. Genome-wide transcriptome profiling of mulberry (Morus alba) response to boron deficiency and toxicity reveal candidate genes associated with boron tolerance in leaves. \u003cem\u003ePlant Physiology and Biochemistry\u003c/em\u003e \u003cstrong\u003e207\u003c/strong\u003e,108316 (2024).\u003c/li\u003e\n\u003cli\u003eAzevedo, R. \u0026amp; Lea, P. Lysine metabolism in higher plants. \u003cem\u003eAmino Acids\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 261\u0026ndash;279 (2001). \u003c/li\u003e\n\u003cli\u003eG. New insights into the regulation and functional significance of lysine metabolism in plants. \u003cem\u003eAnnu. Rev. Plant Biol.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 27-43 (2002).\u003c/li\u003e\n\u003cli\u003eGoldbach, H. E. \u0026amp; Wimmer, M. A. Boron in plants and animals: Is there a role beyond cell‑wall structure? \u003cem\u003eJ. Plant Nutr. Soil Sci.\u003c/em\u003e \u003cstrong\u003e170\u003c/strong\u003e, 39‑48 (2007).\u003c/li\u003e\n\u003cli\u003eGonzalez-Fontes, A. et al. Is boron involved solely in structural roles in vascular plants? \u003cem\u003ePlant Signal Behav\u003c/em\u003e. \u003cstrong\u003e3\u003c/strong\u003e, 24\u0026ndash;26 (2008).\u003c/li\u003e\n\u003cli\u003eKayıhan, C. et al. Transcriptional profiling and proteomic validation revealed higher boron tolerance in Arabidopsis thaliana exposed to salt pre-treatment. \u003cem\u003eSouth African Journal of Botany\u003c/em\u003e \u003cstrong\u003e180\u003c/strong\u003e, 588-605, (2025).\u003c/li\u003e\n\u003cli\u003e\u0026Ouml;z, M. T., Yilmaz, R., Eyidogan, F., Graaff, L. \u0026amp; Yucel, M. \u0026amp; Oktem, H. A. Microarray analysis of late response to boron toxicity in barley (Hordeum vulgare L.) leaves. \u003cem\u003eTurk J. Agric. For.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 191\u0026ndash;202 (2009).\u003c/li\u003e\n\u003cli\u003eZhang, Q. et al. The impact of boron nutrient supply in mulberry (Morus alba) response to metabolomics, enzyme activities, and physiological parameters. \u003cem\u003ePlant Physiol. Biochem.\u003c/em\u003e \u003cstrong\u003e200\u003c/strong\u003e, 107649 (2023).\u003c/li\u003e\n\u003cli\u003eJang, J., Lee, S., Kim, J. I., Lee, S. \u0026amp; Kim, J. A. The roles of circadian clock genes in plant temperature stress responses. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 918 (2024). \u003c/li\u003e\n\u003cli\u003eHu, T., Gao, Z. Q., Hou, J. M., Tian, S. K., Zhang, Z. X., Yang, L. \u0026amp; Liu, Y. Identification of biosynthetic pathways involved in flavonoid production in licorice by RNA-seq based transcriptome analysis. \u003cem\u003ePlant Growth Regulation\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 15\u0026ndash;28 (2020).\u003c/li\u003e\n\u003cli\u003eKim, H. et al. Circadian control of ORE1 by PRR9 positively regulates leaf senescence in Arabidopsis. \u003cem\u003eProceedings of the National Academy of Sciences, USA\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 8448\u0026ndash;8453 (2018).\u003c/li\u003e\n\u003cli\u003eMolassiotis, A., Sotiropoulos, T., Tanou, G., Diamantidis, G. \u0026amp; Therios, I. Boron-induced oxidative damage and antioxidant and nucleolytic responses in shoot tips culture of the apple rootstock EM 9 (Malus domestica Borkh). \u003cem\u003eEnviron. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e(1), 54\u0026ndash;62 (2006).\u003c/li\u003e\n\u003cli\u003eArdic, M., Sekmen, A., Tokur, S., Ozdemir, F. \u0026amp; Turkan, I. Antioxidant responses of chickpea plants subjected to boron toxicity. \u003cem\u003ePlant Biol.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e(3), 328\u0026ndash;338 (2009).\u003c/li\u003e\n\u003cli\u003e\u0026Ccedil;atav, Ş. S., Gen\u0026ccedil;, T. O., Oktay, M. K. \u0026amp; K\u0026uuml;\u0026ccedil;\u0026uuml;kaky\u0026uuml;z, K. Effect of boron toxicity on oxidative stress and genotoxicity in wheat (Triticum aestivum L.). \u003cem\u003eBull Environ. Contam. Toxicol.\u003c/em\u003e \u003cstrong\u003e100 \u003c/strong\u003e(4), 502\u0026ndash;508 (2018).\u003c/li\u003e\n\u003cli\u003eSim\u0026oacute;n-Grao, S. et al. Arbuscular mycorrhizal symbiosis improves tolerance of Carrizo citrange to excess boron supply by reducing leaf B concentration and toxicity in the leaves and roots. \u003cem\u003eEcotoxicol. Environ. Saf.\u003c/em\u003e \u003cstrong\u003e173\u003c/strong\u003e, 322\u0026ndash;330 (2019).\u003c/li\u003e\n\u003cli\u003eGibson, B. A. \u0026amp; Kraus, W. L. New insights into the molecular and cellular functions of poly (ADPribose) and PARPs. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 411 (2012).\u003c/li\u003e\n\u003cli\u003eKhan, M. K. et al. Insight into the Boron Toxicity Stress-Responsive Genes in Boron-Tolerant Triticum dicoccum Shoots Using RNA Sequencing. \u003cem\u003eAgronomy\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 631 (2023).\u003c/li\u003e\n\u003cli\u003eHuang, J. H. et al. Effects of boron toxicity on root and leaf anatomy in two Citrus species differing in boron tolerance. \u003cem\u003eTrees\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e(6), 1653\u0026ndash;1666 (2014).\u003c/li\u003e\n\u003cli\u003eSarafi, E., Siomos, A., Tsouvaltzis, P., Therios, I. \u0026amp; Chatzissavvidis, C. Boron toxicity effects on the concentration of pigments, carbohydrates and nutrient elements in six non-grafted pepper cultivars (Capsicum annuum L.). \u003cem\u003eIndian J. Plant Physiol.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e(3), 474\u0026ndash;485 (2018).\u003c/li\u003e\n\u003cli\u003ePei, Z., Huang, Y., Ni, J., Liu, Y. \u0026amp; Yang, Q. For a colorful life: recent advances in anthocyanin biosynthesis during leaf senescence. \u003cem\u003eBiology\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 329 (2024).\u003c/li\u003e\n\u003cli\u003eKim, J. I., Zhang, X., Pascuzzi, P. E., Liu, C. J. \u0026amp; Chapple, C. Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome-dependent degradation of PAL. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e225\u003c/strong\u003e, 154\u0026ndash;168 (2020).\u003c/li\u003e\n\u003cli\u003eKayihan, C., Aksoy, E., And, M. \u0026amp; Su, N. Boron toxicity induces sulfate transporters at transcriptional level in Arabidopsis thaliana. \u003cem\u003eTurkish Journal of Botany\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, No. 1 (2023).\u003c/li\u003e\n\u003cli\u003eLatchman, D. S. Transcription factors: An overview. \u003cem\u003eInt. J. Biochem. Cell Biol.\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 1305\u0026ndash;1312 (1997).\u003c/li\u003e\n\u003cli\u003eYıldırım, K. \u0026amp; Uylaş, S., Genome-wide transcriptome profiling of black poplar (Populus nigra L.) under boron toxicity revealed candidate genes responsible in boron uptake, transport and detoxification. \u003cem\u003ePlant Physiol. Biochem. \u003c/em\u003e\u003cstrong\u003e109\u003c/strong\u003e, 146\u0026ndash;155 (2016).\u003c/li\u003e\n\u003cli\u003eTao, H. et al. WRKY33 negatively regulates anthocyanin biosynthesis and cooperates with PHR1 to mediate acclimation to phosphate starvation. \u003cem\u003ePlant Commun.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 5(5):100821 (2024).\u003c/li\u003e\n\u003cli\u003ePark, S. H., Jeong, J. S., Huang, C. H., Park, B. S. \u0026amp; Chua, N. H. Inositol polyphosphates-regulated polyubiquitination of PHR1 by NLA E3 ligase during phosphate starvation response in Arabidopsis. \u003cem\u003eNew Phytol.\u003c/em\u003e \u003cstrong\u003e237\u003c/strong\u003e(4):1215-1228 (2023).\u003c/li\u003e\n\u003cli\u003eGuo, J., Chen, J., Yang, J., Yu, Y., Yang, Y. \u0026amp; Wang, W. Identification, characterization and expression analysis of the VQ motif-containing gene family in tea plant (Camellia sinensis). \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e(1), 710 (2018).\u003c/li\u003e\n\u003cli\u003eGarrido-Gala, J., Higuera, J. J., Mu\u0026ntilde;oz-Blanco, J. et al. The VQ motif-containing proteins in the diploid and octoploid strawberry. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 4942 (2019).\u003c/li\u003e\n\u003cli\u003eCheng, Y. et al. Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors. \u003cem\u003ePlant Physiol.\u003c/em\u003e\u003cstrong\u003e159\u003c/strong\u003e (2), 810-25 (2012).\u003c/li\u003e\n\u003cli\u003ePang, Y. et al. Overexpression of the tonoplast aquaporin AtTIP5;1 conferred tolerance to boron toxicity in Arabidopsis. \u003cem\u003eJ. Genet. Genomics\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 389\u0026ndash;397 (2010).\u003c/li\u003e\n\u003cli\u003eBarzana, G. et al. Interrelations of nutrient and water transporters in plants under abiotic stress. \u003cem\u003ePhysiologia Plantarum\u003c/em\u003e \u003cstrong\u003e171\u003c/strong\u003e, 595-619 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"anthocyanin, Arabidopsis thaliana, Boron toxicity, NITROGEN LIMITATION ADAPTATION, nla mutant, RNA sequencing, transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-7334872/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7334872/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding molecular responses against boron (B) stress is one of the goals for improving excess B management of cereals. Since earlier findings demonstrated the differential regulation of protein degradation genes under B toxicity, this study focused on the interaction between toxic B responsive regulations and Nitrogen Limitation Adaptation (\u003cem\u003eNLA\u003c/em\u003e) gene encoding an E3 ubiquitin ligase. Therefore, WT and \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e plants were grown under mild and moderate levels of B toxicity and RNA sequencing was performed in these plants. Accordingly, ribosome was overrepresented for upregulated genes in \u003cem\u003enla\u003c/em\u003e mutants under all conditions when compared to WT whereas alpha-linolenic acid metabolism, plant-pathogen interaction and MAPK signaling pathways were downregulated. Inhibition of glucosinolate biosynthesis and induction of phenylpropanoid pathway as well as enrichment of circadian rhythm were among the most prominent results for B-stressed \u003cem\u003enla\u003c/em\u003e mutants compared to \u003cem\u003enla\u003c/em\u003e mutant \u003cem\u003eArabidopsis\u003c/em\u003e under control condition (C). Interestingly, unlike WT, B-induced accumulation of anthocyanin was not observed in \u003cem\u003enla\u003c/em\u003e mutants. This was attributed to switching from flavonoid to lignin biosynthesis in the phenylpropanoid pathway. Also, the impairment of crosstalk between circadian rhythm and anthocyanin pathways might explain this phenomenon because two pathways are crosslinked by ORE1, one of the targets of NLA.\u003c/p\u003e","manuscriptTitle":"Transcriptome profiling of nla-mutant Arabidopsis reveals possible post-translational regulation of key factors cross-linking circadian-rhythm and anthocyanin pathways under boron toxicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 19:06:29","doi":"10.21203/rs.3.rs-7334872/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae864617-7bf8-4591-a6e7-11a98e0721aa","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54694115,"name":"Biological sciences/Molecular biology"},{"id":54694116,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-10-15T09:38:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 19:06:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7334872","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7334872","identity":"rs-7334872","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}