Characterization of BRS1 Functions in Plant Stress Responses


 Background: Brassinosteroid-insensitive 1 suppressor 1 (BRS1), is a serine carboxypeptidase that mediates brassinosteroid signaling and participates in multiple developmental processes in Arabidopsis. However, little is known about the precise role of BRS1 in this context. Results: In this study, we analyzed transcriptional and proteomic profiles of Arabidopsis seedlings overexpressing BRS1 and found that this gene is involved in both biotic and abiotic stress responses and redox regulation. Further proteomic evidence shows that BRS1 regulates cell redox by indirectly interacting with cytosolic NADP+-dependent isocitrate dehydrogenase (cICDH). We identified two novel splice products of BRS1, which might play important roles in development and stress responses in plants. Conclusions: This study highlights the role of BRS1 in plant redox regulation and stress responses, which extends our understanding of extracellular serine carboxypeptidases.


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around 20 times in the brs1-1D mutant (Fig. 2a), which is consistent with the presence 95 of four copies of CaMV 35S enhancers inserted in the promoter region of BRS1 (Fig.  96 2b) [4]. 97 Interestingly, the transcriptional level of the first exon in brs1-1 maintained a similar 98 level to that of the wild type (Fig. 2c), indicating there is a not feedback loop to enhance 99 BRS1 transcription. We also saw no increased expression of the homologs of BRS1 100 when BRS1 expression was disrupted (Supplementary Figure S1). 101 Notably, we detected novel splice variants in brs1-1D, which retained multiple 102 introns compared to the wild type (Fig. 2c). The two new splice products (4 and 5) made 103 up a lower proportion of the total BRS1 transcripts in brs1-1D, then the level still equal 104 to the total transcripts of BRS1 in wild type (Fig. 2c), indicating these novel splice 105 products may have important functions. 106

Overexpression of BRS1 alters multiple stress responses 107
Gene Ontology (GO) enrichment analysis of DEGs from brs1-1D found a significant 108 enrichment in genes associated with responding to salicylic acid (SA) and jasmonic 109 acid (JA) (Fig. 3). We also found enrichment in genes associated with both biotic (innate 110 immune responses, bacterium, fungus, and chitin) and abiotic (water deprivation, cold 111 and hyperosmotic salinity) stresses (Fig. 3). In agreement with these findings, genes 112 associated with redox regulation and cell death were also enriched. These results 113 strongly suggest that BRS1 participates in the responses to both abiotic and biotic 114 stressors. 115

BRS1 regulates redox-related proteins 116
We next performed proteomic analysis on seedlings using two-dimensional difference 117 gel electrophoresis (2D-DIGE) (Fig. 4a). 19 proteins were assigned as differentially 118 expressed proteins, and 14 proteins were identified, of which 5 proteins were involved 119 in redox regulation (Table 1), supporting that BRS1 is involved in redox regulation. 120 The 2D-DIGE revealed two spots (6 and 7), which were most changed compared to 121 wild type, showing a decrease and increase in brs1-1D, respectively (Fig. 4). These 122 spots corresponded to the same protein, the cytosolic NADP+-dependent isocitrate 123 dehydrogenase (cICDH, at1g65930) (Table 1), a critical redox regulator [19]. Together, 124 these data suggest that BRS1 is involved in redox regulation 125

BRS1 participates in redox regulation by interacting with cICDH 126
To confirm that BRS1 regulates cICDH, the enzyme activity of cICDH was measured 127 in BRS1 mutants. We found that cICDH activity was significantly increased in brs1-1D 128 compared to wild type plants (Fig. 5a), whilst no change was seen in brs1-1 plants. 129 These findings are consistent with our earlier results that showed only overexpression 130 of BRS1 altered transcription. Altogether this suggests that BRS1 regulates the activity 131 of cICDH. 132 To understand how the secretory protein BRS1 could regulate cICDH, which is 133 localized to the cytosol, the cellular localization of BRS1 was evaluated. We observed 134 expression of BRS1 at the membrane and cell wall of mature epidermal cells (Fig. 5b), 135 consistent with localization to the extracellular space [4,5]. Unexpectedly, we also 136 found cICDH-GFP to be localized to the membrane of Nicotiana benthamiana leaves 137 ( Fig. 5c), indicating a potential interaction might occur in this area. However, yeast 138 two-hybrid and pull-down assays found no direct interaction between cICDH and BRS1 139 (Supplementary Table S2 Our results suggest that the BRS1-related stress response is involved in SA and JA 147 signaling (Fig. 3). These are key hormones required for the induction of plant defenses 148 in response to pathogens and insects [21,22]. Similarly, an apoplastic SCPL in rice, 149 OsBISCPL1, also induces a stress response via SA and JA signaling [9], indicating 150 SCPLs may use a common mechanism to regulate stress responses. 151 The trigger effector production by apoplastic proteases used be the key mechanism 152 to induce plant stress response [13]. Our results showed that systemic stress response 153 was induced upon BRS1 over-expression (Fig. 3) in brs1-1D (Fig. 2c), implying a splicing-based functional variation of BRS1, which 173 may play a special role in those highly redundant gene family to overcome their 174 redundancy nature in changing environments. 175

Conclusions 176
In this study, transcriptomic and proteomic analyses revealed that BRS1 plays a role in 177 regulating plant responses to biotic and abiotic stress. We find that BRS1 likely 178 participates in redox regulation in cells through indirect interaction with cICDH.

Identification of Differentially Expressed Genes (DEGs) and GO enrichment 213
analysis 214 Identification all of differential genes in brs1-1 and brs1-1D compared to control WS2, 215 respectively. The corrected read count data of genes were imported into the R package 216 DESeq2 (v1.26.0) to identify DEGs with the standard of a fold change ≥ 2.0, a false 217 discovery rate (FDR)-adjusted P-value < 0.05, and expression (FPKM ≥ 1) in at least 218 one sample for each comparison [30]. 219 The GO descriptions were obtained by AnnotationHub ("AH75734"), and used the 220 R package clusterProfiler (v3.14.0) with the "enrichGO" function for GO enrichment 221 analysis. The statistical significance of the enrichment of GO was examined using the 222 hypergeometric distribution test, followed by multiple-test correction using the 223 Benjamini-Hochberg method. GO terms with q -value < 0.01 for further enrichment 224 analysis. 225 in each image, and then spots that showed significant differential expression were 258 determined by ANOVA and Student's t-test (p <0.05). 19 spots with significant 259 differential expression were selected for mass spectrometric identification. 260

Protein Identification 261
Coomassie brilliant blue staining was performed on the scanned 2-D-DIGE gel, and 262 then differential protein spots were found by position comparison, but it was difficult 263 to detect proteins with low background expression. Therefore, a 2-DE gel prepared with 264 1 mg of internal standard protein was used for staining to show spots that could not be 265 determined from the 2D-DIGE gel. 266 After 19 differential protein spots were excised from 2-D-DIGE gel, each spot was 267 destained in destaining buffer (25 mM ammonium bicarbonate, 50% v/v acetonitrile). 268

Availability of data and materials 295
All data generated or analyzed in this study are included in this article and the 296 supplemental files. The raw data of RNA sequencing were submitted to the NCBI 297 database with the bioproject ID: PRJNA657702. 298

Competing interests 299
The authors declare that they have no competing interests. 300