Enhanced Salt Tolerance of Rhizobia-inoculated Soybean Correlates with Decreased Phosphorylation of the Transcription Factor GmMYB183 and Altered Flavonoid Biosynthesis.

Soybean (Glycine max (L.) Merrill) is an important component of the human diet and animal feed, but soybean production is limited by abiotic stresses especially salinity. 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 data mining, ChIP and EMSA, we found that GmMYB183 binds to the promoter of the soybean GmCYP81E11 gene encoding for a Cytochrome P450 monooxygenase which contributes to the accumulation of ononin, a monohydroxy B-ring flavonoid that negatively regulates 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 with wild-type soybean plants, these results correlate with the transcriptional induction of GmCYP81E11 by GmMYB183 and the subsequent accumulation of ononin. Our findings provide molecular insights into how rhizobia enhance salt tolerance of soybean plants.

Quantitative Phosphoproteomic and Metabolomic Analyses Reveal GmMYB173 Optimizes Flavonoid Metabolism in Soybean under Salt Stress.

Salinity causes osmotic stress to crops and limits their productivity. To understand the mechanism underlying soybean salt tolerance, proteomics approach was used to identify phosphoproteins altered by NaCl treatment. Results revealed that 412 of the 4698 quantitatively analyzed phosphopeptides were significantly up-regulated on salt treatment, including a phosphopeptide covering the serine 59 in the transcription factor GmMYB173. Our data showed that GmMYB173 is one of the three MYB proteins differentially phosphorylated on salt treatment, and a substrate of the casein kinase-II. MYB recognition sites exist in the promoter of flavonoid synthase gene GmCHS5 and one was found to mediate its recognition by GmMYB173, an event facilitated by phosphorylation. Because GmCHS5 catalyzes the synthesis of chalcone, flavonoids derived from chalcone were monitored using metabolomics approach. Results revealed that 24 flavonoids of 6745 metabolites were significantly up-regulated after salt treatment. We further compared the salt tolerance and flavonoid accumulation in soybean transgenic roots expressing the 35S promoter driven cds and RNAi constructs of GmMYB173 and GmCHS5, as well as phospho-mimic (GmMYB173S59D ) and phospho-ablative (GmMYB173S59A ) mutants of GmMYB173 Overexpression of GmMYB173S59D and GmCHS5 resulted in the highest increase in salt tolerance and accumulation of cyaniding-3-arabinoside chloride, a dihydroxy B-ring flavonoid. The dihydroxy B-ring flavonoids are more effective as anti-oxidative agents when compared with monohydroxy B-ring flavonoids, such as formononetin. Hence the salt-triggered phosphorylation of GmMYB173, subsequent increase in its affinity to GmCHS5 promoter and the elevated transcription of GmCHS5 likely contribute to soybean salt tolerance by enhancing the accumulation of dihydroxy B-ring flavonoids.