. We recently found that rhizobia inoculation enhances soybean tolerance to salt stress, but the underlying mechanisms are unaddressed. Here, we used quantitative phosphoproteomic and metabonomic approaches to identify changes in phosphoproteins and metabolites in soybean roots treated with rhizobia inoculation and salt. Results revealed differential regulation of 800 phosphopeptides, at least 32 of these phosphoproteins or their homologous were reported be involved in flavonoid synthesis or trafficking, and 27 out of 32 are transcription factors. We surveyed the functional impacts of all these 27 transcription factors by expressing their phospho-mimetic/ablative mutants in the roots of composite soybean plants and found that phosphorylation of GmMYB183 could affect the salt tolerance of the transgenic roots. Using ChIP and EMSA, we found GmMYB183 binds to the promoter of the soybean GmCYP81E11 gene encoding for a Cytochrome P450 monooxygenase contributes to the accumulation of ononin, a monohydroxy B-ring flavonoid negatively regulate soybean tolerance to salinity. Phosphorylation of GmMYB183 was inhibited by rhizobia inoculation, overexpression of GmMYB183 enhanced the expression of GmCYP81E11 and rendered salt sensitivity to the transgenic roots. Plants deficient in GmMYB183 function are more tolerant to salt stress as compared to wild-type soybean plants which correlated with the transcriptional induction of GmCYP81E11 and the subsequent accumulation of ononin. Our findings provide molecular insights into how rhizobia enhance salt tolerance of soybean plants.
Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
Figure 1: GmMYB183 Binds to a MYB-Specific Cis-Element Present in the Promoter of GmCYP81E11. (A) Distribution of MYB binding motifs (G/A/T)(G/A/T)T(C/A)(A/G)(A/G)(G/T)(T/A) in the promoter of GmCYP81E11. Position of the probe (underlined sequence) used for ChIP-based binding assay is shown below the gene. Position Weight Matrix of MYB binding motifs was curated in the JASPAR database (http://jaspar.genereg.net/cgi-bin/jaspar_db.pl?rm=browse&db=core&tax_group=plants). (B) and (C), ChIP-qPCR assays indicating that GmMYB183 binds to the promoter sections containing the MYB-binding motifs in vivo. The empty vector was used as a negative control (NC). An asterisk indicates a significant difference (P ≤ 0.05) to the NC according to Student‘s t test. FC means fold change. (D) and (E), EMSA assays showed that GmMYB183 specially binds to the GmCYP81E11-P1 fragment from the GmCYP81E11 promoter (D), and phosphorylation at S61 of GmMYB183 enhances this interaction (E). GmCYP81E11-P1 (ttttATGTATTAGTGATTAAGTTTAATAACGTGA) or a mutated version with its ATTAAGTT core sequence changed to ATAAAGTT (GmCYP81E11-P1M) was labeled as a probe, and 200 or 500 folds of unlabeled double strand GmCYP81E115- P1 fragment was set as the competitor. (F) and (G) The transcription levels of GmMYB183 (F) and GmCYP81E11 (G) in soybean transgenic roots expressing empty vector (EV), GmMYB183-overexpression (GmMYB183-OE) and RNAi (GmMYB183-KD) constructs, respectively. Data presented are mean ± SE (n=3). (H) Transcription of GmMYB183 in soybean roots treated with R(-)Na(-), R(+)Na(-), R(- )Na(+) and R(+)Na(+). Data represent mean values ± SE, each sample was analyzed with three biological replicates. An asterisk indicates a significant difference (P ≤ 0.05, Student‘s t-test) between treatment [R(+)Na(-), R(-)Na(+) and R(+)Na(+)] and the control [R(-)Na(-)].