Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

2018; Elsevier BV; Volume: 17; Issue: 6 Linguagem: Inglês

10.1074/mcp.ra117.000417

ISSN

1535-9484

Autores

Erxu Pi, Chengmin Zhu, Wei Fan, Yingying Huang, Liqun Qu, Yangyang Li, Qinyi Zhao, Feng Ding, Lijuan Qiu, Huizhong Wang, B. W. Poovaiah, Liqun Du,

Tópico(s)

Plant Stress Responses and Tolerance

Resumo

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. 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. Salinity is one of the main abiotic stresses affecting the growth and productivity of crops (1.Sobhanian H. Aghaei K. Komatsu S. Changes in the plant proteome resulting from salt stress: Toward the creation of salt-tolerant crops?.J. Proteomics. 2011; 74: 1323-1337Crossref PubMed Scopus (115) Google Scholar). In saline soils, the sodium ion causes most of the damages to plant cells at the molecular level through ionic toxicity and homeostasis disruption (2.Zhu J.K. Plant salt tolerance.Trends Plant Sci. 2011; 6: 66-71Abstract Full Text Full Text PDF Scopus (2790) Google Scholar, 3.Katiyar-Agarwal S. Zhu J. Kim K. Agarwal M. Fu X. Huang A. Zhu J.K. The plasma membrane Na+/H+ antiporter SOS1 interacts with RCD1 and functions in oxidative stress tolerance in Arabidopsis.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 18816-18821Crossref PubMed Scopus (194) Google Scholar, 4.Qu L.Q. Huang Y.Y. Zhu C.M. Zeng H.Q. Shen C.J. Liu C. Zhao Y. Pi E.X. Rhizobia-inoculation enhances the soybean's tolerance to salt stress.Plant Soil. 2016; 400: 209-222Crossref Scopus (28) Google Scholar). Furthermore, the salt stress interferes with the photosynthesis in chloroplasts and the electron transport in mitochondria and accelerates the production of reactive oxygen species (ROS) and simultaneously causes oxidative stress (5.Fahnenstich H. Scarpeci T.E. Valle E.M. Flugge U.I. Maurino V.G. Generation of hydrogen peroxide in chloroplasts of Arabidopsis overexpressing glycolate oxidase as an inducible system to study oxidative stress.Plant Physiol. 2008; 148: 719-729Crossref PubMed Scopus (107) Google Scholar, 6.Affenzeller M.J. Darehshouri A. Andosch A. Lutz C. Lutz-Meindl U. Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata.J. Exp. Bot. 2009; 60: 939-954Crossref PubMed Scopus (208) Google Scholar). Plants have evolved two main strategies to eliminate these excessive ROS, the enzymatic degradation, for example elimination of ROS by superoxide dismutase or ascorbate peroxidase (7.Wu T. Pi E.X. Tsai S.N. Lam H.M. Sun S.M. Kwan Y.W. Ngai S.M. GmPHD5 acts as an important regulator for crosstalk between histone H3K4 di-methylation and H3K14 acetylation in response to salinity stress in soybean.BMC Plant Biol. 2011; 11: 178Crossref PubMed Scopus (28) Google Scholar), and deoxidization by small molecules such as ascorbic acid, mannitol, and flavonoids (8.Lu Y.H. Lam H.M. Pi E.X. Zhan Q.L. Tsai S.N. Wang C.M. Kwan Y.W. Ngai S.M. Comparative metabolomics in Glycine max Glycine soja under salt stress to reveal the phenotypes of their offspring.J. Agr. Food Chem. 2013; 61: 8711-8721Crossref PubMed Scopus (69) Google Scholar, 9.MartensYan J.H. Wang B.A. Jiang Y.N. Cheng L.J. Wu T.L. GmFNSII-controlled soybean flavone metabolism responds to abiotic stresses and regulates plant salt tolerance.Plant Cell Physiol. 2014; 55: 74-86Crossref PubMed Scopus (53) Google Scholar). Compared with that of ascorbic acid and mannitol, the role of the flavonoid family in plant tolerance to salinity is just beginning to unfold (9.MartensYan J.H. Wang B.A. Jiang Y.N. Cheng L.J. Wu T.L. GmFNSII-controlled soybean flavone metabolism responds to abiotic stresses and regulates plant salt tolerance.Plant Cell Physiol. 2014; 55: 74-86Crossref PubMed Scopus (53) Google Scholar, 10.Yamasaki H. Sakihama K. Ikehara N. Flavonoid-peroxidase reaction as a detoxification mechanism of plant cells against H2O2.Plant Physiol. Bioch. 1997; 115: 1405-1412Crossref Scopus (375) Google Scholar, 11.Chen Y. Lin F.Z. Yang H. Yue L. Hu F. Wang J.L. Luo Y.Y. Cao F.L. Effect of varying NaCl doses on flavonoid production in suspension cells of Ginkgo biloba: relationship to chlorophyll fluorescence, ion homeostasis, antioxidant system and ultrastructure.Acta Physiol. Plant. 2014; 36: 3173-3187Crossref Scopus (31) Google Scholar), although more than 10,000 species of flavonoids have been isolated from plants thus far (12.Aoki T. Akashi T. Ayabe S-i. Flavonoids of leguminous plants: structure, biological activity, and biosynthesis.J. Plant Res. 2000; 113: 475-488Crossref Google Scholar, 13.Jiang Y.N. Wang B.A. Li H. Yao L.M. Wu T.L. Flavonoid production is effectively regulated by RNA1 interference of two flavone synthase genes from Glycine max.J. Plant Biol. 2010; 53: 425-432Crossref Scopus (21) Google Scholar). It is well known that all flavonoids are derivatives of chalcone, an intermediate that is directly synthesized from p-coumaroyl-CoA and alonyl-CoA to 15-carbon skeletons at a 3:1 ratio (12.Aoki T. Akashi T. Ayabe S-i. Flavonoids of leguminous plants: structure, biological activity, and biosynthesis.J. Plant Res. 2000; 113: 475-488Crossref Google Scholar). Derived from chalcone, various categories of flavonoids can be synthesized, including flavanones (e.g. naringenin), isoflavone (e.g. formononetin), flavonols (e.g. quercetin) and others, based on their structural variation in the C-ring and the functional groups at C-3 and C-4 (supplemental Fig. S1) (12.Aoki T. Akashi T. Ayabe S-i. Flavonoids of leguminous plants: structure, biological activity, and biosynthesis.J. Plant Res. 2000; 113: 475-488Crossref Google Scholar). Most of the natural flavonoids are conjugated with glycosides (11.Chen Y. Lin F.Z. Yang H. Yue L. Hu F. Wang J.L. Luo Y.Y. Cao F.L. Effect of varying NaCl doses on flavonoid production in suspension cells of Ginkgo biloba: relationship to chlorophyll fluorescence, ion homeostasis, antioxidant system and ultrastructure.Acta Physiol. Plant. 2014; 36: 3173-3187Crossref Scopus (31) Google Scholar), such as naringenine-7-rhamnosidoglucoside (naringin) and daidzein-7-O-β-d-glucopyranoside (daidzin). Flavonoids have been found to have a wide array of physiological functions in plant tolerant responses to abiotic stresses, including protection against harmful UV light (14.Shen X. Zhou Y. Duan L. Li Z. Eneji A.E. Li J. Silicon effects on photosynthesis and antioxidant parameters of soybean seedlings under drought and ultraviolet-B radiation.J. Plant Physiol. 2010; 167: 1248-1252Crossref PubMed Scopus (237) Google Scholar, 15.Bandurska H. Niedziela J. Chadzinikolau T. Separate and combined responses to water deficit and UV-B radiation.Plant Sci. 2013; 213: 98-105Crossref PubMed Scopus (100) Google Scholar, 16.Soledad O.N. Florencia M.M. Laura F.M. Raul D.G. Balbina A.A. Pia O.F. Potassium phosphite increases tolerance to UV-B in potato. Plant.Physiol. Bioch. 2015; 88: 1-8Google Scholar) and hydrogen peroxide scavenging (5.Fahnenstich H. Scarpeci T.E. Valle E.M. Flugge U.I. Maurino V.G. Generation of hydrogen peroxide in chloroplasts of Arabidopsis overexpressing glycolate oxidase as an inducible system to study oxidative stress.Plant Physiol. 2008; 148: 719-729Crossref PubMed Scopus (107) Google Scholar, 17.Graham T.L. Graham M.Y. Subramanian S. Yu O. RNAi silencing of genes for elicitation or biosynthesis of 5-deoxyisoflavonoids suppresses race-specific resistance and hypersensitive cell death in Phytophthora sojae infected tissues.Plant Physiol. 2007; 144: 728-740Crossref PubMed Scopus (94) Google Scholar, 18.Jia L.G. Sheng Z.W. Xu W.F. Li Y.X. Liu Y.G. Xia Y.J. Zhang J.H. Modulation of anti-oxidation ability by proanthocyanidins during germination of Arabidopsis thaliana seeds.Mol. Plant. 2012; 5: 472-481Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 19.Nakabayashi R. Yonekura-Sakakibara K. Urano K. Suzuki M. Yamada Y. Nishizawa T. Matsuda F. Kojima M. Sakakibara H. Shinozaki K. Michael A.J. Tohge T. Yamazaki M. Saito K. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids.Plant J. 2014; 77: 367-379Crossref PubMed Scopus (674) Google Scholar). The hydroxyl groups of flavonoids play a critical role during the ROS scavenging processes (20.Chobot V. Huber C. Trettenhahn G. Hadacek F. (±)-catechin: chemical weapon, antioxidant, or stress regulator?.J. Chem. Ecol. 2009; 35: 980-996Crossref PubMed Scopus (58) Google Scholar, 21.Sakao K. Fujii M. Hou D.X. Acetyl derivate of quercetin increases the sensitivity of human leukemia cells toward apoptosis.BioFactors. 2009; 35: 399-405Crossref PubMed Scopus (24) Google Scholar, 22.Kim H.J. Suh H.J. Kim J.H. Park S. Joo Y.C. Kim J.S. Antioxidant activity of glyceollins derived from soybean elicited with Aspergillus sojae.J. Agric. Food Chem. 2010; 58: 11633-11638Crossref PubMed Scopus (59) Google Scholar, 23.Yang X. Kang S.M. Jeon B.T. Kim Y.D. Ha J.H. Kim Y.T. Jeon Y.J. Isolation and identification of an antioxidant flavonoid compound from citrus-processing by-product.J. Sci. Food Agric. 2011; 91: 1925-1927Crossref PubMed Scopus (39) Google Scholar). Three categories of enzymes, including the chalcone synthase (CHS) 1The abbreviations used are: CHS, chalcone synthase; CHI, chalcone isomerase; CPM, cytochrome P450 monooxygenase; TF, transcription factor. 1The abbreviations used are: CHS, chalcone synthase; CHI, chalcone isomerase; CPM, cytochrome P450 monooxygenase; TF, transcription factor., chalcone isomerase (CHI), and cytochrome P450 monooxygenase (CPM), play critical roles in flavonoid synthesis. CHS can catalyze the conversion of 4-coumaroyl-CoA and malonyl-CoA to chalcone. The chalcone could be directly converted to flavanone by CHI. Then, CPM catalyzes the conversion of flavanones (e.g. naringenin) to isoflavones (e.g. formononetin). Interestingly, CHS, CHI, and CPM usually play key roles in plant response to salt stress (24.Koes R. Verweij W. Quattrocchio F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways.Trends Plant Sci. 2005; 10: 236-242Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar, 25.Ferreyra M.L.F. Rius S.P. Casati P. Flavonoids: biosynthesis, biological functions, and biotechnological applications.Front Plant Sci. 2012; 3: 222PubMed Google Scholar, 26.Ishida J.K. Wakatake T. Yoshida S. Takebayashi Y. Kasahara H. Wafula E. dePamphilis C.W. Namba S. Shirasu K. Local auxin biosynthesis mediated by a YUCCA flavin monooxygenase regulates Haustorium development in the Parasitic plant Phtheirospermum japonicum.Plant Cell. 2016; 28: 1795-1814Crossref PubMed Scopus (67) Google Scholar). Piao et al. demonstrated that AtCHS expression was significantly increased concomitantly with the accumulation of anthocyanins in Arabidopsis plants subjected to salt stress (27.Piao H.L. Lim J.H. Kim S.J. Cheong G.W. Hwang I. Constitutive over-expression of AtGSK1 induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis.Plant J. 2001; 27: 305-314Crossref PubMed Google Scholar). Martens Yan et al. found that the RNA interference of GmFNSII (coding for CYP93B, an enzyme in cytochrome P450 monooxygenase subfamily) significantly reduced the tolerance of soybean roots to salinity because of the decreased flavone level and higher H2O2 accumulation (9.MartensYan J.H. Wang B.A. Jiang Y.N. Cheng L.J. Wu T.L. GmFNSII-controlled soybean flavone metabolism responds to abiotic stresses and regulates plant salt tolerance.Plant Cell Physiol. 2014; 55: 74-86Crossref PubMed Scopus (53) Google Scholar). Our recent study documented that salt tolerance of Arabidopsis and soybean were positively regulated by CHS and negatively regulated by CHI and CPM (28.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. Du L.Q. Mechanisms of soybean roots' tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars.Mol. Cell. Proteomics. 2016; 15: 266-288Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). It is noteworthy that CHS is a single copy gene in the Arabidopsis genome (29.Schmutz J. Cannon S.B. Schlueter J. Ma J.X. Mitros T. Nelson W. Hyten D.L. Song Q.J. Thelen J.J. Cheng J.L. Xu D. Hellsten U. May G.D. Yu Y. Sakurai T. Umezawa T. Bhattacharyya M.K. Sandhu D. Valliyodan B. Lindquist E. Peto M. Grant D. Shu S.Q. Goodstein D. Barry K. Futrell-Griggs M. Abernathy B. Du J.C. Tian Z.X. Zhu L.C. Gill N. Joshi T. Libault M. Sethuraman A. Zhang X.C. Shinozaki K. Nguyen H.T. Wing R.A. Cregan P. Specht J. Grimwood J. Rokhsar D. Stacey G. Shoemaker R.C. Jackson S.A. Genome sequence of the palaeopolyploid soybean.Nature. 2010; 463: 178-183Crossref PubMed Scopus (3042) Google Scholar), and our results confirmed that the AtCHS knockout mutant is extremely sensitive to salt stress, demonstrating that chalcone and its downstream derivatives play a key role in protecting plants from the damage caused by saline stresses (28.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. Du L.Q. Mechanisms of soybean roots' tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars.Mol. Cell. Proteomics. 2016; 15: 266-288Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). CHS genes in leguminous plants is comprised of a multiple gene family, for instance there are at least nine CHS genes in soybean (29.Schmutz J. Cannon S.B. Schlueter J. Ma J.X. Mitros T. Nelson W. Hyten D.L. Song Q.J. Thelen J.J. Cheng J.L. Xu D. Hellsten U. May G.D. Yu Y. Sakurai T. Umezawa T. Bhattacharyya M.K. Sandhu D. Valliyodan B. Lindquist E. Peto M. Grant D. Shu S.Q. Goodstein D. Barry K. Futrell-Griggs M. Abernathy B. Du J.C. Tian Z.X. Zhu L.C. Gill N. Joshi T. Libault M. Sethuraman A. Zhang X.C. Shinozaki K. Nguyen H.T. Wing R.A. Cregan P. Specht J. Grimwood J. Rokhsar D. Stacey G. Shoemaker R.C. Jackson S.A. Genome sequence of the palaeopolyploid soybean.Nature. 2010; 463: 178-183Crossref PubMed Scopus (3042) Google Scholar). Expressions of different CHS family members exhibit distinct temporal and spatial patterns in response to developmental and environmental cues (30.Wingender R. Rohrig H. Horicke C. Wing D. Schell J. Differential regulation of soybean chalcone synthase genes in plant defense, symbiosis and upon environmental stimuli.Mol. Gen. Genet. 1989; 218: 315-322Crossref PubMed Scopus (95) Google Scholar), implying CHS members in soybean might be involved in different physiological processes. Identifying the specific roles of a CHS gene in soybean is a challenge to traditional genetic and physiological studies. Genes encoding CHS, CHI, and CPM that are involved in different flavonoid biosynthetic steps, are usually regulated by MYB transcription factors (TFs) (31.Broun P. Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis.Curr. Opin. Plant Biol. 2005; 8: 272-279Crossref PubMed Scopus (330) Google Scholar, 32.Liao Y. Zou H.F. Wang H.W. Zhang W.K. Ma B. Zhang J.S. Chen S.Y. Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants.Cell Res. 2008; 18: 1047-1060Crossref PubMed Scopus (188) Google Scholar, 33.Schwinn K.E. Boase M.R. Bradley J.M. Lewis D.H. Deroles S.C. Martin C.R. Davies K.M. MYB and bHLH transcription factor transgenes increase anthocyanin pigmentation in petunia and lisianthus plants, and the petunia phenotypes are strongly enhanced under field conditions.Front. Plant Sci. 2014; 5: 603Crossref PubMed Scopus (47) Google Scholar, 34.Lotkowska M.E. Tohge T. Fernie A.R. Xue G.P. Balazadeh S. Mueller-Roeber B. The Arabidopsis transcription factor MYB112 promotes anthocyanin formation during salinity and under high light stress.Plant Physiol. 2015; 169: 1862-1880PubMed Google Scholar). The MYBs can be grouped into four types, MYB1R, R2R3-MYB, R1R2R3-MYB, and 4RMYB, based on the variation in the repeat numbers of the MYB DNA-binding domains (32.Liao Y. Zou H.F. Wang H.W. Zhang W.K. Ma B. Zhang J.S. Chen S.Y. Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants.Cell Res. 2008; 18: 1047-1060Crossref PubMed Scopus (188) Google Scholar). Among them, the R2R3-MYB subfamily is well known to regulate flavonoid metabolism (35.Yuan Y. Qi L.J. Yang J. Wu C. Liu Y.J. Huang L.Q. A Scutellaria baicalensis R2R3-MYB gene, SbMYB8, regulates flavonoid biosynthesis and improves drought stress tolerance in transgenic tobacco.Plant Cell Tiss. Org. 2015; 120: 961-972Crossref Scopus (46) Google Scholar). In Arabidopsis, AtMYB75, AtMYB90, MYB112, MYB113, and MYB114 have been shown to play important roles in activating the transcription of genes in the anthocyanin biosynthetic pathway (34.Lotkowska M.E. Tohge T. Fernie A.R. Xue G.P. Balazadeh S. Mueller-Roeber B. The Arabidopsis transcription factor MYB112 promotes anthocyanin formation during salinity and under high light stress.Plant Physiol. 2015; 169: 1862-1880PubMed Google Scholar, 36.Borevitz J.O. Xia Y. Blount J. Dixon R.A. Lamb C. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis.Plant Cell. 2000; 12: 2383-2394Crossref PubMed Scopus (1128) Google Scholar, 37.Gonzalez A. Zhao M. Leavitt J.M. Lloyd A.M. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.Plant J. 2008; 53: 814-827Crossref PubMed Scopus (1088) Google Scholar). In Scutellaria baicalensis, SbMYB8 was reported to improve drought tolerance by regulating flavonoid biosynthesis (35.Yuan Y. Qi L.J. Yang J. Wu C. Liu Y.J. Huang L.Q. A Scutellaria baicalensis R2R3-MYB gene, SbMYB8, regulates flavonoid biosynthesis and improves drought stress tolerance in transgenic tobacco.Plant Cell Tiss. Org. 2015; 120: 961-972Crossref Scopus (46) Google Scholar). Soybean GmMYBJ2 was found to notably improve the seed germination rate of transgenic Arabidopsis undergoing water-deficit stress (38.Su L.T. Wang Y. Liu D.Q. Li X.W. Zhai Y. Sun X. Li X.Y. Liu Y.J. Li J.W. Wang Q.Y. The soybean gene, GmMYBJ2, encodes a R2R3-type transcription factor involved in drought stress tolerance in Arabidopsis thaliana.Acta Physiol. Plant. 2015; 37: 138Crossref Scopus (11) Google Scholar). Usually, the MYB-type TFs interact with other transcription factors such as bHLH (37.Gonzalez A. Zhao M. Leavitt J.M. Lloyd A.M. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.Plant J. 2008; 53: 814-827Crossref PubMed Scopus (1088) Google Scholar) and WD40 proteins to regulate the production of anthocyanins (25.Ferreyra M.L.F. Rius S.P. Casati P. Flavonoids: biosynthesis, biological functions, and biotechnological applications.Front Plant Sci. 2012; 3: 222PubMed Google Scholar, 39.Walker A.R. Davison P.A. Bolognesi-Winfield A.C. James C.M. Srinivasan N. Blundell T.L. Esch J.J. Marks M.D. Gray J.C. The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein.Plant Cell. 1999; 11: 1337-1350Crossref PubMed Scopus (722) Google Scholar). The physiological responses to salinity rely on diverse interplays, such as those between kinases and protein phosphorylation (28.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. Du L.Q. Mechanisms of soybean roots' tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars.Mol. Cell. Proteomics. 2016; 15: 266-288Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), between MYB-type TFs and salt-responsive enzymes (e.g. CHS, CHI, and CPM) (32.Liao Y. Zou H.F. Wang H.W. Zhang W.K. Ma B. Zhang J.S. Chen S.Y. Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants.Cell Res. 2008; 18: 1047-1060Crossref PubMed Scopus (188) Google Scholar, 40.Zhang Z.Y. Liu X. Wang X.D. Zhou M.P. Zhou X.Y. Ye X.G. Wei X.N. An R2R3 MYB transcription factor in wheat, TaPIMP1, mediates host resistance to Bipolaris sorokiniana and drought stresses through regulation of defense- and stress-related genes.New Phytol. 2012; 196: 1155-1170Crossref PubMed Scopus (136) Google Scholar), and those between enzymes and metabolites (24.Koes R. Verweij W. Quattrocchio F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways.Trends Plant Sci. 2005; 10: 236-242Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar). All these form complicated networks to help plants adapt to the ever-changing environments (41.Bassel G.W. Gaudinier A. Brady S.M. Hennig L. Rhee S.Y. De Smet I. Systems analysis of plant functional, transcriptional, physical interaction, and metabolic networks.Plant Cell. 2012; 24: 3859-3875Crossref PubMed Scopus (74) Google Scholar). Hence, it is essential to gain an in-depth and integrated knowledge for understanding the nature of these complicated responses. Omics studies, including the proteomics and metabolomics, allow researchers to obtain comprehensive insight and to connect the nodes of the molecular networks underlying plants' abiotic tolerances (41.Bassel G.W. Gaudinier A. Brady S.M. Hennig L. Rhee S.Y. De Smet I. Systems analysis of plant functional, transcriptional, physical interaction, and metabolic networks.Plant Cell. 2012; 24: 3859-3875Crossref PubMed Scopus (74) Google Scholar). Interestingly, the quantitative analyses of omics provide us precise network structure and kinetic data to simulate the dynamics of a biological process (42.Albert R. Network inference, analysis, and modeling in systems biology.Plant Cell. 2007; 19: 3327-3338Crossref PubMed Scopus (131) Google Scholar, 43.Taus T. Kocher T. Pichler P. Paschke C. Schmidt A. Henrich C. Mechtler K. Universal and confident phosphorylation site localization using phosphoRS.J. Proteome Res. 2011; 10: 5354-5362Crossref PubMed Scopus (568) Google Scholar). Using phosphoproteomic and proteomic approaches, our previous studies revealed that several key proteins (such as MYB TFs, CHS, CHI and CPM) are involved in soybean tolerance to salinity (28.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. Du L.Q. Mechanisms of soybean roots' tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars.Mol. Cell. Proteomics. 2016; 15: 266-288Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We also proposed a salt tolerance pathway involving flavonoid metabolism, mostly mediated by phosphorylated MYB TFs. In this study, we are introducing a mechanism by which the phosphorylation of GmMYB173 regulates flavonoid syntheses via expressional control of GmCHS5 and enhances the tolerance of soybean to salt stress. Seeds of Glycine max cultivar Union85140 were surface sterilized and germinated in wet filter papers as previously described (28.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. Du L.Q. Mechanisms of soybean roots' tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars.Mol. Cell. Proteomics. 2016; 15: 266-288Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Two days after germination, the seedlings were transplanted in pearlite and sphagnum peat soil (v:v = 1:3). After transferring to the growth chamber, the light intensity was set at 200 μmol × m−2 × s−1 with a photo period of 18 h per day. The temperature was adjusted to 25/18 °C for day/night cycle. The seedlings were watered with 1/4 fold Fahräeus medium every 4 days and deionized water was also used to irrigate every 2 days after application of abovementioned medium. At the three-trifoliate stage, the seedlings were treated with 200 mm NaCl for 24 h. Then the samples were collected and immediately stored at −80 °C until further use. The TCA/Acetone extraction method was used to isolate total proteins from soybean roots as previously described (28.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. Du L.Q. Mechanisms of soybean roots' tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars.Mol. Cell. Proteomics. 2016; 15: 266-288Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Five grams of root tissue for each sample was ground into fine powder in liquid nitrogen. The powder was thoroughly suspended in 45 ml of pre-cooled TCA/Acetone (v:v = 1:9) and kept at −20 °C overnight for protein extraction. The homogenate was centrifuged at 7000 × g for 20 min and the pellet was washed three times with 40 ml acetone. Then the residual acetone was removed by applying vacuum and 50 mg white powder of each sample was resuspended in 1 ml SDT lysis buffer (4% SDS, 100 mm Tris-HCl, 1 mm DTT, 1 mm PMSF, pH7.6, including 1 × PhosSTOP phosphatase inhibitor mixture from Roche, Mannheim, Germany). The solution was boiled for 15 min in a water bath, sonicated for 100 s, centrifuged at 13,400 × g for 15 min. The protein concentration in supernatant was quantified via BCA (bicinchoninic acid) method and 20 μg was used to run a SDS-PAGE gel for QC test. The protein digestion procedure was performed as previously described (28.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. Du L.Q. Mechanisms of soybean roots' tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars.Mol. Cell. Proteomics. 2016; 15: 266-288Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Before digestion, 200 μg of protein for each sample were processed to remove residual SDS in the samples by filter aided sample preparation (FASP) method. Next, the concentrated proteins were digested with 8 μg of trypsin at 37 °C for 16–18 h. After digestion, the peptide solution was passed through a Microcon filtration device (MWCO 10 kd) and the peptide concentration was quantified by measuring OD280. All the procedures were carried out at 4 °C unless exceptions were stated. One hundred micrograms of digested peptide for each sample was subjected to AB Sciex iTRAQ labeling. The eight-plex iTRAQ labeling was performed according to the manufacturer's instructions (28.Pi E.X.

Referência(s)