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. 2019 Nov;18(11):2225-2243.
doi: 10.1074/mcp.RA119.001704. Epub 2019 Aug 28.

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

Erxu Pi  1 , Jia Xu  2 , Huihui Li  2 , Wei Fan  3 , Chengmin Zhu  2 , Tongyao Zhang  2 , Jiachen Jiang  2 , Litao He  2 , Hongfei Lu  4 , Huizhong Wang  2 , B W Poovaiah  5 , Liqun Du  6
Affiliations

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

Erxu Pi et al. Mol Cell Proteomics. 2019 Nov.

Abstract

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.

Keywords: GmMYB183; Label-free quantification; flavonoid; iTRAQ; metabolomics; phosphoproteome; phosphorylation; salt tolerance; soybean; transcription.

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Figures

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Graphical abstract
Fig. 1.
Fig. 1.
Phosphorylation motifs enriched from differentially phosphorylated peptides after salt treatment. A, and B, are phosphorylation motifs extracted from the phosphopeptides in the Up group and Down group, respectively, when the soybean roots were inoculated with rhizobia. C, and D, show phosphorylation motifs extracted from the phosphopeptides in the Up and Down groups when the rhizobia-inoculated root was treated with NaCl. E, GmMYB183 could be a substrate of GmCK2α. Phosphorylation assay was carried out as described in the experimental procedures. WT and S61D: recombinant proteins of the N-terminal 168 aa of GmMYB183-S61D and GmMYB183-WT fused to a Trx- and a 6 x His-tags at its N terminus, and a 6 x His-tag at its C terminus. Data indicated that the Ser61 is the unique site in GmMYB183.
Fig. 2.
Fig. 2.
GmMYB183-RFP localizes in the nucleus of soybean root cells. The original roots of soybean seedling were removed and infected with A. rhizogenes carrying a GmMYB183-RFP expressing construct, transgenic roots with red fluorescence were selected using a Nikon SMZ1500 fluorescence stereoscope (A, B). The subcellular localization of the GmMYB183-RFP fusion protein was observed using a Zeiss LSM710 confocal microscope (C).
Fig. 3.
Fig. 3.
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 indicates 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 ± S.E. (n = 3). H, Transcription of GmMYB183 in soybean roots treated with R(-)Na(-), R(+)Na(-), R(-)Na(+) and R(+)Na(+). Data represent mean values ± S.E., 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(-)].
Fig. 4.
Fig. 4.
HPLC-based analysis of ononin contents in transgenic roots of soybean. A, The chromatographic spectrum observed from ononin standard separation. B, to (N) The chromatographic spectrum observed from HPLC separation of transgenic GmCYP81E11-OE R(-)Na(-) (B), GmCYP81E11-OE R(+)Na(-) (C), GmCYP81E11-OE R(+)Na(+) (D), GmCYP81E11-KD R(-)Na(-) (E), GmCYP81E11-KD R(+)Na(-) (F), GmCYP81E11-KD R(+)Na(+) (G), GmMYB183 OE-WT R(-)Na(-) (H), GmMYB183 OE-S61D R(-)Na(-) (I), GmMYB183 OE-S61D R(+)Na(-) (J), GmMYB183 OE-S61A R(-)Na(-) (K), GmMYB183-KD R(-)Na(-) (L), GmMYB183-KD R(+)Na(-) (M), GmMYB183-KD R(+)Na(+) (N) roots.
Fig. 5.
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 GmMYB183 (OE-WT), GmMYB183S61D (OE-S61D), GmMYB183S61A (OE-S61A) and RNAi-mediated knockdown construct of GmMYB183 (KD). Transgenic roots were confirmed by DsRed fluorescence as described in the 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 root samples from three plants and the experiments are repeated three times with similar results; an asterisk indicates a significant difference from the control (EV) based on p ≤ 0.05 of student's t test;double asterisks indicates a significant difference from the control (EV) based on p ≤ 0.01 of student's t test.
Fig. 6.
Fig. 6.
The phosphorylated peptide GYAsxDDA in GmMYB183 protein is highly conserved in several plants. The peptide GYAsxDDA was found in homologous proteins of GmMYB183 in several plants including Arachis ipaensis, Medicago truncatula, Glycine max, Cajanus cajan, Phaseolus vulgaris, Vigna angularis, Prunus persica, Malus domestica, Morus notabilis and Citrus clementine.
Fig. 7.
Fig. 7.
A hypothetical model for transcription factors GmMYB183 in regulating soybean's responses to salinity. After the perception of salinity or rhizobia inoculation signal, the phosphorylation of GmMYB183 was inhibited. Furthermore, the expression of its downstream gene GmCYP81E11 was downregulated and the GmCYP81E11-induced monohydroxy B-ring flavonoid (ononin) declined. Finally, the adjusted flavonoids appropriately reduced the ROS for enhancing the soybean's tolerance to salinity.
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References

    1. Pi E., Zhu C., Fan W., Huang Y., Qu L., Li Y., Zhao Q., Qiu L., Wang H., Poovaiah B. W., and Du L. (2018) Quantitative phosphoproteomic and metabonomic analyses reveal GmMYB173 optimizes flavonoid metabolism in soybean to survive salt stress. Mol. Cell. Proteomics 17, 1209–1224 - PMC - PubMed
    1. Lu Y. H., Lam H. M., Pi E. X., Zhan Q. L., Tsai S. N., Wang C. M., Kwan Y. W., and Ngai S. M. (2013) Comparative metabolomics in Glycine max and Glycine soja under salt stress to reveal the phenotypes of their offspring. J. Agr. Food Chem. 61, 8711–8721 - PubMed
    1. Pi E. X., Qu L. Q., Hu J. W., Huang Y. Y., Qiu L. J., Lu H., Jiang B., Liu C., Peng T. T., Zhao Y., Wang H. Z., Tsai S. N., Ngai S. M., and Du L. Q. (2016) Mechanisms of soybean roots' tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars. Mol. Cell. Proteomics 15, 266–288 - PMC - PubMed
    1. Qu L. Q., Huang Y. Y., Zhu C. M., Zeng H. Q., Shen C. J., Liu C., Zhao Y., and Pi E. X. (2016) Rhizobia-inoculation enhances the soybean's tolerance to salt stress. Plant Soil 400, 209–222
    1. Affenzeller M. J., Darehshouri A., Andosch A., Lutz C., and Lutz-Meindl U. (2009) Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata. J. Exp. Bot. 60, 939–954 - PMC - PubMed

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