Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

2019; Elsevier BV; Volume: 18; Issue: 11 Linguagem: Inglês

10.1074/mcp.ra119.001704

ISSN

1535-9484

Autores

Erxu Pi, Jia Xu, Huihui Li, Wei Fan, Chengmin Zhu, Tongyao Zhang, Jiachen Jiang, Litao He, Hongfei Lu, Huizhong Wang, B. W. Poovaiah, Liqun Du,

Tópico(s)

Plant Stress Responses and Tolerance

Resumo

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. 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. Soybean (Glycine max (L.) Merrill) is becoming an important ingredient of human diet and animal feeds. It is also used as an important industrial material because of its high content of protein and oil. Hence it is not surprising that the demand for soybean is growing at an unprecedented pace (2Lu 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 and 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, 3Pi 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). Salinity is a major abiotic stress that limits the growth and yields of soybean (4Qu 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). The excessive Na+ ions usually stimulate the accumulation of reactive oxygen species (ROS) 1The abbreviations used are:ROSreactive oxygen specieshpthours post treatmentCHSchalcone synthaseCHIchalcone isomeraseCYPcytochrome P450 monooxygenase. 1The abbreviations used are:ROSreactive oxygen specieshpthours post treatmentCHSchalcone synthaseCHIchalcone isomeraseCYPcytochrome P450 monooxygenase. and triggers oxidative stress in plant cells (5Affenzeller 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, 6Fahnenstich 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). Plants develop two main strategies to rebalance the intracellular ROS, including enzymatic degradation (7Wu 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 using the reductive capacities of small molecules such as ascorbic acid, mannitol and flavonoid (2Lu 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 and 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, 8MartensYan 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). reactive oxygen species hours post treatment chalcone synthase chalcone isomerase cytochrome P450 monooxygenase. reactive oxygen species hours post treatment chalcone synthase chalcone isomerase cytochrome P450 monooxygenase. Flavonoids from plants constitute a huge family with over 10,000 species reported so far (9Aoki T. Akashi T. Ayabe S. Flavonoids of leguminous plants: structure, biological activity, and biosynthesis.J. Plant Res. 2000; 113: 475-488Crossref Google Scholar, 10Jiang 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). Plants synthesize various classes of flavonoids, such as chalcones, flavanones (e.g. naringenin), isoflavone (e.g. ononin,) and flavonols (e.g. quercetin) (9Aoki T. Akashi T. Ayabe S. Flavonoids of leguminous plants: structure, biological activity, and biosynthesis.J. Plant Res. 2000; 113: 475-488Crossref Google Scholar). These flavonoids have been reported to play a wide range of physiological functions in plant defense responses to abiotic stresses, for example, protecting plants from harmful UV radiation (11Shen 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, 12Bandurska 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, 13Soledad 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: 1-8Crossref PubMed Scopus (36) Google Scholar) and eliminating destructive hydrogen peroxide (6Fahnenstich 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, 14Graham 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, 15Jia 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). The hydroxyl groups of flavonoids play a pivotal role in fulfilling their bioactive functions when they are oxidized to quinones during these processes (16Sakao 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, 17Yang 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, 18Chobot 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, 19Kim 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). The flavonoids in soybean also play a critical role in plant-microbe symbiosis (20Tirichine L. Imaizumi-Anraku H. Yoshida S. Murakami Y. Madsen L.H. Miwa H. Nakagawa T. Sandal N. Albrektsen A.S. Kawaguchi M. Downie A. Sato S. Tabata S. Kouchi H. Parniske M. Kawasaki S. Stougaard J. Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development.Nature. 2006; 441: 1153-1156Crossref PubMed Scopus (344) Google Scholar). Some species of flavonoids diffuse into rhizosphere signal the presence of hosts to the rhizobia before the initiation of the rhizobia-legume interaction. Once they are perceived by rhizobia, the bacteria will be induced to express the NodD protein which forms a complex with isoflavones. This complex further activates the transcription of the bacterial nodulation genes (20Tirichine L. Imaizumi-Anraku H. Yoshida S. Murakami Y. Madsen L.H. Miwa H. Nakagawa T. Sandal N. Albrektsen A.S. Kawaguchi M. Downie A. Sato S. Tabata S. Kouchi H. Parniske M. Kawasaki S. Stougaard J. Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development.Nature. 2006; 441: 1153-1156Crossref PubMed Scopus (344) Google Scholar), producing Nod factor which triggers a series of biological responses in the soybean roots and finally leads to the nodule formation on the roots of the legume host (21Zhang J. Subramanian S. Stacey G. Yu O. Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.Plant J. 2009; 57: 171-183Crossref PubMed Scopus (181) Google Scholar). Microbes have been known to play significant roles in enhancing legume tolerance to salinity. Ectomycorrhizal fungi were reported to help their host plants to tolerate salt stress by reducing the accumulation of Na+ and increasing the absorption of K+ (22Chen S.L. Hawighorst P. Sun J. Polle A. Salt tolerance in populus: significance of stress signaling networks, mycorrhization, and soil amendments for cellular and whole-plant nutrition.Environ. Exp. Bot. 2014; 107: 113-124Crossref Scopus (65) Google Scholar, 23Miransari M. Smith D.L. Alleviating salt stress on soybean (Glycine max (L.) Merr.) - Bradyrhizobium japonicum symbiosis, using signal molecule genistein.Eur. J. Soil Biol. 2009; 45: 146-152Crossref Scopus (92) Google Scholar, 24Ruiz-Lozano J.M. Porcel R. Azcon C. Aroca R. Regulation by arbuscular mycorrhizae of the integrated physiological response to salinity in plants: new challenges in physiological and molecular studies.J. Exp. Bot. 2012; 63: 4033-4044Crossref PubMed Scopus (350) Google Scholar). Some rhizosphere bacteria could also enhance the host's salt tolerance using bioactive components such as indolacetic acid, phenolic compounds, flavonoids and antioxidant enzymes in plants (25Bianco C. Defez R. Medicago truncatula improves salt tolerance when nodulated by an indole-3-acetic acid-overproducing Sinorhizobium meliloti strain.J. Exp. Bot. 2009; 60: 3097-3107Crossref PubMed Scopus (177) Google Scholar). We recently found that inoculation of rhizobia could enhance soybean's tolerance to salinity (4Qu 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). However, the molecular mechanism by which rhizobia regulate soybean salt responses is still unclear. To date, three categories of enzymes, including the chalcone synthase (CHS), chalcone isomerase (CHI) and cytochrome P450 monooxygenase (CYP), are documented to play significant roles in the complicated flavonoid metabolic processes where 4-coumaroyl-CoA and malonyl-CoA are first converted to naringenin chalcone by CHS. The chalcone is then converted to flavanone and isoflavone by CHI and CYP, respectively. Interestingly, CHS, CHI and CYP have been shown to play key roles in plant responses to salt stress (26Koes 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, 27Ferreyra M.L.F. Rius S.P. Casati P. Flavonoids: biosynthesis, biological functions, and biotechnological applications.Front. Plant Sci. 2012; 3: 222PubMed Google Scholar, 28Ishida 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). For example, the RNA interference of GmFNSII (coding for CYP93B) significantly reduces the soybean root's tolerance to salinity because of the decreased flavone level and increased accumulation of H2O2 (8MartensYan 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 found that salt tolerance of Arabidopsis and soybean were positively regulated by CHS but negatively regulated by CHI and CYP (3Pi 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). Genes encoding CHSs, CHIs, and CYPs that are involved in various flavonoid biosynthetic steps, are regulated by MYB transcription factors (29Liao 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, 30Broun 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, 31Schwinn 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, 32de VettenLotkowska 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, 33Yi J.X. Derynck M.R. Li X.Y. Telmer P. Marsolais F. Dhaubhadel S. A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean.Plant J. 2010; 62: 1019-1034PubMed Google Scholar). AtMYB75, AtMYB90, MYB112, MYB113, and MYB114 have been shown to play important roles in activating genes of the anthocyanin biosynthetic pathway (32de VettenLotkowska 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, 34Borevitz 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, 35Gonzalez 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 addition, the Scutellaria baicalensis MYB8 was reported to improve drought stress tolerance by regulating flavonoid biosynthesis (36Yuan 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). Moreover, GmMYBJ2 was found to markedly improve the seed germination rate of transgenic Arabidopsis plants undergoing water-deficit stress (37Su 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). Omics studies monitoring the proteome and metabolome which are directly related to the development of phenotype, allow us to obtain a comprehensive insight and to correlate specific nodes of the stress-related molecular networks to the establishment of stress tolerance (38Bassel 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). Furthermore, the quantitative analyses of these high-throughput data at the systemic level provide us with unique opportunities to investigate the precise network structure and signaling kinetics underlying physiological processes during the establishment of plant tolerance to the causative stresses (39Albert R. Network inference, analysis, and modeling in systems biology.Plant Cell. 2007; 19: 3327-3338Crossref PubMed Scopus (131) Google Scholar). In this study, we attempted to explore whether and how rhizobia inoculation affects soybean's phosphoproteome and metabonome, and how these changes help protect soybean from salinity. We identified changes in phosphorylation status of transcription factor MYB183 in response to both salt treatment and rhizobia inoculation. In addition, we documented a mechanism regarding how GmMYB183 regulates the expression of CYP and flavonoid biosynthesis using reverse genetic and metabolomic approaches. Seeds of Glycine max cultivar Union85140, kindly provided by Prof. Lijuan Qiu at the Chinese Academy of Agricultural Sciences, were surface sterilized and germinated between wet filter papers as previously described (3Pi 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 inoculated with Sinorhizobium fredii 15006 (Agricultural Culture Collection of China, http://www.accc.org.cn/show.asp) as described (4Qu 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), and then transplanted into a mixture of pearlite and sphagnum peat (v : v = 1 : 3). The light intensity in the growth chamber was set at 200 μmol×m−2×s−1 with a photo period of 18 h of light per day; and the temperature was set as 25/18 °C for day/night cycle. The seedlings were watered with 1/4 strength of Fahräeus medium every 4 days and deionized water was used to irrigate every 2 days after adding Fahräeus medium. At the second trifoliate leaf stage, the seedlings were treated with 200 mM of NaCl for 24 h as described by Qu et al. (4Qu 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). Next, the samples were collected and immediately stored at −80 °C until further use. The TCA/Acetone extraction method was used to isolate the total proteins from soybean roots as previously described (3Pi 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) for overnight protein extraction at −20 °C. The homogenate was then centrifuged at 7000 × g for 20 min and the pellet was washed three times with 40 ml acetone. After that, the residual acetone was removed by vacuum and 50 mg of white powder from each sample was resuspended in 1 ml of SDT lysis buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, 1 mM PMSF, pH 7.6, including 1× PhosSTOP phosphatase inhibitor mixture from Roche, Basel, Switzerland). The solution was boiled for 15 min in a thermal block, sonicated for 100 s, centrifuged at 13,400 × g for 15 min, and the pellet was discarded. The protein concentration in the supernatant was measured using the BCA (bicinchoninic acid) method and 20 μg of extracted proteins was run in an SDS-PAGE gel for quality assurance. The protein digestion was carried out as previously described (3Pi 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, each sample containing 200 μg of protein was processed through filter aided sample preparation (FASP) procedure to remove the residual SDS. Next, the concentrated proteins were digested with 8 μg of trypsin at 37 °C for 16 to18 h. After digestion, the peptide solution was passed through a Microcon filtration device (MWCO 10 kd) and the OD280 was measured to estimate its concentration. All the above procedures were carried out at 4 °C unless otherwise stated. An aliquot containing 100 μg of digested peptides from each sample was subjected to AB Sciex iTRAQ labeling. The eight-plex iTRAQ labeling was performed according to the manufacturer's instructions. TiO2 beads were used to enrich phosphopeptides as previously described (3Pi 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). The labeled peptides were acidified with 50 μl of DHB buffer (3% 2, 5-dihydroxy benzoic acid, 80% acetonitrile and 0.1% TFA), and then incubated with 25 μg of TiO2 beads (10 μm in diameter, Sangon Biotech, Shanghai, China) for 40 min at room temperature. After the incubation, the TiO2 beads were spun down and the pellets were packed into 10 μl pipette tips. The peptide-TiO2 beads were washed three times with 20 μl of wash solution I (20% acetic acid, 300 mM of octanesulfonic acid sodium salt and 20 mg/ml DHB), then three times with 20 μl of wash solution II (70% water; 30% acetonitrile). The phosphopeptides were finally eluted using freshly prepared ABC buffer (50 mM of ammonium phosphate, pH 10.5). The enriched phosphopeptide solution was lyophilized and redissolved in 20 μl 0.1% TFA solution for Nano RPLC-MS/MS Analysis. For NanoRPLC-MS/MS analysis, 5 μg (≤ 10 μl) phosphopeptides solution was loaded into a two dimensional EASY-nLC1000 system coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, Massachusetts). In the nanoLC separation system, the mobile phase A solution contained 0.1% formic acid in water, and mobile the phase B solution contained 84% acetonitrile and 0.1% formic acid. Before peptide loading, the Thermo EASY SC200 trap column (RP-C18, 3 μm, 100 mm × 75 μm) was pre-equilibrated with 95% mobile phase A for 30 min. The phosphopeptides were first transferred to the Thermo scientific EASY column (2 cm × 100 μm 5 μm-C18) and then separated through the trap column using a gradient of 0% to 55% mobile phase B for 220 min. Then the columns were rinsed with 100% mobile phase B for 8 min and re-equilibrated to the initial conditions for 12 min. The flow rate of the above procedures was set at 0.25 μl per minute. The raw data were extracted by Mascot2.2 and analyzed by Proteome Discoverer1.4 (Thermo Scientific). To identify the phosphopeptides, the mascot data was searched against 74305 entries curated in the peptide database uniprot_Glycine_74305_20140429.fasta (http://www.uniprot.org/, on April 29, 2014). To generate reliable phosphopeptides, the missed cleavages and false discovery rates (FDR) values were set at less than 2 and 0.01, respectively; the mass tolerances for precursor and fragment ions were set at less than 20 ppm and 0.1 Da, respectively. In addition, the carbamidomethyl (C), Itraq8plex (N-term) and iTRAQ8plex (K) were chosen as fixed modifications, whereas the oxidation (M), phospho (ST), and phospho (Y) were selected as variable modifications. The metabolomics analysis was conducted mostly as previously described (40Niehaus T.D. Nguyen T.N. Gidda S.K. ElBadawi-Sidhu M. Lambrecht J.A. McCarty D.R. Downs D.M. Cooper A.J. Fiehn O. Mullen R.T. Hanson A.D. Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids.Plant Cell. 2014; 26: 3010-3022Crossref PubMed Scopus (48) Google Scholar) with appropriate modifications. 5 g of root tissue from each sample was homogenized in liquid nitrogen. For metabolite extraction, 50 mg homogenate was dissolved in 1 ml of pre-cooled methanol: acetonitrile: water solution (2:2:1, v/v/v). The mixtures were briefly vortexed, and then sonicated for 30 min, these vortex-sonication steps were repeated twice. After 60 min incubation at −20 °C, the mixture was centrifuged at 14,000 × g for 15 min. Two microliters of supernatant was injected into the Agilent 1290 Infinity LC system coupled to a Triple TOF 6600 Mass Spectrometer (AB SCIEX, Framingham, Massachusetts). In the UHPLC separation system, the mobile phase A solution contained 25 mM ammonium acetate and 25 mM ammonia in water, and the mobile phase B solution contained 100% acetonitrile. The ACQUITY UPLC BEH Amide separation column (Waters, 1.7 μm, 2.1 mm× 100 mm) was pre-equilibrated with 85% mobile phase B at 4 °C before metabolite loading. The metabolites were separated using a gradient of 85–65% mobile phase B for 12 min. Then, the mobile phase B was kept at 40% for 3 minuntes followed by a 5 min rinse with 85% of mobile phase B. The flow rate of the total mobile phase was set at 300 μl per minute. Ten biological replicates were analyzed for each treatment. The QC (Quality Control) sample was used

Referência(s)