Abstract
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.
Highlights
Salinity is one of the main abiotic stresses affecting the growth and productivity of crops [1]
Five motifs ([sDxE], [Ds], [sxs], [sxE] and [sxP]) were only found in the Up group and five motifs ([sDD], [DsxE], [sDx], [ss] and [sE]) were only detected in the Down group. These differentially phosphorylated motifs were searched against relevant databases to identify kinases which could target them as substrates, and our results indicate that [sP], [sDxE], [sxs], [sxE] are potential substrates of plant ERK1 and ERK2 kinases, [sxD] is recognized by casein kinase-II (CK2), sPxR is a substrate of CDK1/2/4/6 kinase, growth associated histone HI kinase, and Cdc2 kinase [48]
Phosphorylation of GmMYB173S59 Improve Salt Tolerance of Transgenic Soybean Roots—We compared the salt tolerance of transgenic roots, our results showed that roots expressing GmMYB173S59D (OE-S59D) showed higher tolerance than those transformed with GmMYB173S59A (OE-S59A) and GmMYB173 (OE-WT) constructs (Fig. 5)
Summary
Salinity is one of the main abiotic stresses affecting the growth and productivity of crops [1]. A 234-fold enrichment was observed in qRT-PCR amplification of ChIPed DNA from soybean roots expressing 35S::GmMYB173–2xHA/Ubq10::RFP using primer pairs spanning the MYB-binding site CHS5-P3 in the GmCHS5 promoter as compared with ChIPed DNA from soybean roots carrying the empty vector (Fig. 3B, 3C).
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