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

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 (GmMYB173_S59D ) and phospho-ablative (GmMYB173_S59A ) mutants of GmMYB173 Overexpression of GmMYB173_S59D 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.

Keywords: Flavonoid Metabolism; GmMYB173; Metabolomics; Metabonomics; Oxidative stress; Phosphoproteome; Phosphoproteomics; Protein Modification*; Soybean; Stress response; iTRAQ.

Fig. 1.

Phosphorylation motifs deduced from peptides with different modification levels following salt stress and verification of GmMYB173 as a substrate of GmCK2α. A, Phosphorylation motifs expressed as positional weight matrices extracted from the phosphopeptides in the Up group using motif-X software. B, Phosphorylation motifs extracted from the phosphopeptides in the Down group. C, GmMYB173 could be a substrate of GmCK2α. Phosphorylation assay was carried out as described in experimental procedure section. S59A and WT: recombinant proteins of the N-terminal 167 aa of GmMYB173-S59A and GmMYB173-WT fused to a Trx- and a 6 × His-tags at its N-terminal end, and a 6 × His-tag at its C-terminal end. Data indicated that the Ser59 is the unique site in GmMYB173.

Fig. 2.

GmMYB173-RFP is localized in the nucleus of soybean root cells. The transgenic roots with red fluorescence were selected by Nikon SMZ1500 fluorescent stereo microscope (A, B), and then used for subcellular localization analysis with Zeiss LSM710 confocal microscope (C).

Fig. 3.

GmMYB173 binds to the promoter of GmCHS5 via an MYB-binding element and regulates its expression. A, Position of MYB TF binding motifs, (G/A/T)(G/A/T)T(C/A)(A/G)(A/G)(G/T)(T/A), boxes in the promoter of GmCHS5. Fragments selected for amplification in ChIP-qPCR assay are presented proportionally underneath the promoter; sequences for probes in EMSA (solid boxes) are listed. B, C, Chromatin immuno-precipitation assay verified the GmMYB173 binding to promoter of GmCHS5 in vivo. ChIP assay reveals that GmMYB173 binds the GmCHS5 promoter (B); ChIP-qPCR-based in vivo binding assay with promoter fragments spanning the GmCHS5-P3 fragment (C, NC: Negative control, root sample transformed with the empty vector, FC: fold change). EMSA assay showed that GmMYB173 specially binds to the GmCHS5-P3 fragment from the GmCHS5 promoter (D), and Phosphorylation at S59 of GmMYB173 enhances this interaction (E). (GmCHS5-P3, ttttGTTGATGCATGGGGTAGATAGAGATGCATA) or two mutated version with its GGTAGATA core sequence changed to GCCAGATA (M1) or GGCCGATA (M2) was labeled as probe. EMSAs were carried as described in the section of Experimental Procedures. pET32a-Trx tag: the 184 aa Trx protein with two his-tags at its C terminus expressed from the empty pET32b vector. Recombinant proteins of the N-terminal 167 aa of GmMYB173, GmMYB173-S59A, and GmMYB173-S59D are fused to a Trx- and a 6 × His-tags at its N-terminal end, and a 6 × His-tag at its C-terminal end. Competitor is 5-hundred folds of unlabeled double strand GmCHS5-P3 fragment. F, G, Transcription of GmMYB173 (F) and GmCHS5 (G) in untransformed control, GmMYB173-OE (overexpression) and GmMYB173-KD (knock-down) roots; expression of GmCHS5 was found to be correlated with expression of GmMYB173. H, Transcription of GmMYB173 in soybean roots treated with 200 mm NaCl. Data represent mean values ± S.E., each sample was analyzed with three biological replicates. An asterisk indicates a significant difference based on P (≤ 0.05) of the Student's t test.

Fig. 4.

HPLC-based analysis of cyanidin 3-arabinoside chloride (C3A) contents in transgenic roots of soybean. A, The chromatographic spectrum observed from C3A standard separation. B, The chromatographic spectrum observed from HPLC separation of transgenic GmMYB173-OE roots without salt treatment. C–H The chromatographic spectrum observed from HPLC separation of transgenic GmCHS5-OE control (C), GmCHS5-OE salt stress (D), GmMYB173-OE control (E), GmMYB173-OE salt stress (F), GmMYB173_S59D control (G), GmMYB173_S59D salt stress (H) roots.

Fig. 5.

Effects of salt treatment on transgenic roots of composite soybean plants. Composite soybean plants were generated by infection with A. rhizogenes strain K599 carrying empty vector (EV), overexpression constructs of GmMYB173 (OE-WT), GmMYB173_S59D (OE-S59D), GmMYB173_S59A (OE-S59A) and RNAi-mediated knock down construct of GmMYB173 (KD). Transgenic roots were confirmed by DsRed fluorescence as described in materials and methods section. One-week old composite plants were treated in 1/4 fold Fahräeus medium without (control) or with 200 mm NaCl [NaCl (+)] for 4 weeks. A, Plants are typical representatives of three repeats of salt treatments. B, Fresh weights of transgenic roots: data expressed are means ± s.d. of three repeats; an asterisk indicates a significant difference from the control (EV) based on p ≤ 0.05 of student's t test.

Fig. 6.

The phosphorylated peptide GYAsADDA in GmMYB173 protein is highly conserved in several plants. The peptide GYAsADDA was found in homologs of GmMYB173 in several plants, including Glycine max, Phaseolus vulgaris, Cajanus cajan, Vigna angularis, Arachis hypogaes, Medicago truncatula and Citrus arietinum.

Fig. 7.

A hypothetical model illustrating how the transcription factor GmMYB173 regulates the expression of target gene GmCHS5 during soybean response to salinity. After the perception of salinity signal, the GmMYB173 is phosphorylated by casein kinase II and then activates the transcription of downstream target genes including GmCHS5. These enzymes catalyze the production of dihydroxy B-ring flavonoids (such as cyanidin 3-arabinoside chloride), and the accumulation of these flavonoids help reduce the ROS and the establishment of soybean tolerance to salinity.