Mechanism of the drought tolerance of a transgenic soybean overexpressing the molecular chaperone BiP

  • Flaviane Silva Coutinho
  • Danilo Silva dos Santos
  • Lucas Leal Lima
  • Camilo Elber Vital
  • Lázaro Aleixo Santos
  • Maiana Reis Pimenta
  • João Carlos da Silva
  • Juliana Rocha Lopes Soares Ramos
  • Angela Mehta
  • Elizabeth Pacheco Batista Fontes
  • Humberto Josué de Oliveira RamosEmail author
Research Article


Drought is one of major constraints that limits agricultural productivity. Some factors, including climate changes and acreage expansion, indicates towards the need for developing drought tolerant genotypes. In addition to its protective role against endoplasmic reticulum (ER) stress, we have previously shown that the molecular chaperone binding protein (BiP) is involved in the response to osmotic stress and promotes drought tolerance. Here, we analyzed the proteomic and metabolic profiles of BiP-overexpressing transgenic soybean plants and the corresponding untransformed line under drought conditions by 2DE-MS and GC/MS. The transgenic plant showed lower levels of the abscisic acid and jasmonic acid as compared to untransformed plants both in irrigated and non-irrigated conditions. In contrast, the level of salicylic acid was higher in transgenic lines than in untransformed line, which was consistent with the antagonistic responses mediated by these phytohormones. The transgenic plants displayed a higher abundance of photosynthesis-related proteins, which gave credence to the hypothesis that these transgenic plants could survive under drought conditions due to their genetic modification and altered physiology. The proteins involved in pathways related to respiration, glycolysis and oxidative stress were not signifcantly changed in transgenic plants as compared to untransformed genotype, which indicate a lower metabolic perturbation under drought of the engineered genotype. The transgenic plants may have adopted a mechanism of drought tolerance by accumulating osmotically active solutes in the cell. As evidenced by the metabolic profiles, the accumulation of nine primary amino acids by protein degradation maintained the cellular turgor in the transgenic genotype under drought conditions. Thus, this mechanism of protection may cause the physiological activities including photosynthesis to be active under drought conditions.


Stress abiotic Amino acid metabolism Proteomic Metabolomic 



The authors would like to thank to NuBioMol (Center of Analyses of Biomolecules-UFV, Brazil) for the infrastructure and technical assistance. This study was supported by the National Institute of Science and Technology in Plant-Pest Interaction (INCT-IPP), The Brazilian Soybean Genome Consortium (GENOSOJA), the Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Supplementary material

12298_2019_643_MOESM1_ESM.docx (850 kb)
Supplementary material 1 (DOCX 849 kb)


  1. Aayudh D, Rushton PJ, Rohila JS (2017) Metabolomic profiling of soybeans (Glycine max L.) reveals the importance of sugar and nitrogen metabolism under drought and heat stress. Plants 6(2):21Google Scholar
  2. Ahmad P, Rasool S, Gul A, Akram NA, Ashraf M, Gucel S (2016) Jasmonates: multifunctional roles in stress tolerance. Front Plant Sci 7:813PubMedGoogle Scholar
  3. Alia MP, Matysik J (2001) Effect of proline on the production of singlet oxygen. Amino Acids 21:195–200CrossRefPubMedGoogle Scholar
  4. Alvim FC, Carolino SMB, Cascardo JCM, Nunes CC, Martinez CA, Otoni WC, Fontes EPB (2001) Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress. Plant Physiol 126:1042–1054CrossRefPubMedGoogle Scholar
  5. Ashraf M, Foolad MR (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot 59:206–216CrossRefGoogle Scholar
  6. Azooz MM, Youssef MM (2010) Evaluation of heat shock and salycilic acid treatments as inducers of drought stress tolerance in hassawi wheat. Am J Plant Physiol 5:56–70CrossRefGoogle Scholar
  7. Bao Y, Howell SH (2017) The unfolded protein response supports plant development and defense as well as responses to abiotic stress. Front Plant Sci. Google Scholar
  8. Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24:23–58CrossRefGoogle Scholar
  9. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  10. Carvalho HH, Brustolini OJB, Pimenta RP, Mendes GC, Gouveia BC, Silva PA, Silva JCF, Mota CS, Ramos JRLS, Fontes EPB (2014) The molecular chaperone binding protein BiP prevents leaf dehydration-induced cellular homeostasis disruption. PLoS ONE 9:86661CrossRefGoogle Scholar
  11. Cascardo JCM, Buzeli RAA, Almeida RS, Otoni WC, Fontes EPB (2001) Differential expression of the soybean BiP gene family. Plant Sci 160:273–281CrossRefPubMedGoogle Scholar
  12. CONAB-National Supply Company (2014) Follow-up of the harvest 2013/2014. Accessed 10 Apr 2014
  13. Costa MDL, Reis PAB, Valente MAS, Irsigler AST, Carvalho CM, Loureiro ME, Aragão FJL, Boston RS, Fietto LG, Fontes EPB (2008) A new branch of endoplasmic reticulum stress signaling and the osmotic signal converge on plant–specific asparagine–rich proteins to promote cell death. J Biol Chem 283:20209–20219CrossRefPubMedGoogle Scholar
  14. Cuadros-Inostroza Á, Caldana C, Redestig H, Kusano M, Lisec J, Peña-Cortés H, Willmitzer L, Hannah MA (2009) TargetSearch - a Bioconductor package for the efficient preprocessing of GC-MS metabolite profiling data. BMC Bioinformatics 10(1):428CrossRefPubMedGoogle Scholar
  15. Dai A (2013) Increasing drought under global warming in observations and models. Nat Clim Change 3:52–58CrossRefGoogle Scholar
  16. Das A, Eldakak M, Paudel B, Kim WD, Hemmati H, Basu C, Rohila JS (2016) Leaf proteome analysis reveals prospective drought and heat stress response mechanisms in soybean. Biomed Res 2016:6021047Google Scholar
  17. De Ronde JA, Van Der Mescht A, Steyn HSF (2000) Proline accumulation in response to drought and heat stress in cotton. Afr Crop Sci J 8:85–92CrossRefGoogle Scholar
  18. Dias MC, Brüggemann W (2010) Limitations of photosynthesis in Phaseolus vulgaris under drought stress: gas exchange, chlorophyll fluorescence and Calvin cycle enzymes. Photosynthetica 48(1):96–102CrossRefGoogle Scholar
  19. Djilianov D, Georgieva T, Moyankova D, Atanassov A, Shinozaki K, Smeeken SCM, Verma PDS, Murata N (2005) Improved abiotic stress tolerance in plants by accumulation of osmoprotectants. Gene Transf Approach Biotechnol Biotechnol Equip 19:63–71CrossRefGoogle Scholar
  20. Fernie AR, Carrari F, Sweetlove LJ (2004) Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr Opin Plant Biol 7:254–261CrossRefPubMedGoogle Scholar
  21. Flexas J, Galmes J, Ribas-Carbo M, Medrano H (2005) The effects of water stress on plant respiration. Plant respiration: from cell to ecosystem. Springer, Dordrecht, pp 85–94Google Scholar
  22. Foyer CH, Lam HM, Nguyen HT, Siddique KHM, Varshney R, Colmer TD, Cowling W, Bramley H, Mori TA, Hodgson JM, Cooper JW, Miller AJ, Kunert K, Vorster J, Cullis C, Ozga JA, Wahlqvist ML, Liang Y, Shou H, Shi K, Yu J, Fodor N, Kaiser BN, Wong FL, Valliyodan B, Considine MJ (2016) Neglecting legumes has compromised global food and nutritional security. Nat Plants 2:1–10Google Scholar
  23. Gupta AS, Heinen JL, Holaday AS, Burke JJ, Allen RD (1993) Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide-dismutase. Proc Natl Acad Sci USA 90:1629–1633CrossRefPubMedGoogle Scholar
  24. Haigh NG, Johnson AE (2002) A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane. J Cell Biol 156:261–270CrossRefPubMedGoogle Scholar
  25. Hamman BD, Hendershot LM, Johnson AE (1998) BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation. Cell 92:747–758CrossRefPubMedGoogle Scholar
  26. Häusler RE, Ludewig F, Krueger S (2014) Amino acids—a life between metabolism and signaling. Plant Sci 229:225–237CrossRefPubMedGoogle Scholar
  27. Hildebrandt TM, Nesi AN, Araújo WL, Braun HP (2015) Amino acid catabolism in plants. Mol Plant 8(11):1563–1579CrossRefPubMedGoogle Scholar
  28. Hirakuri MH, Lazzarotto JJ (2014) Soybean agribusiness in the world and Brazilian contexts. Embrapa Soy, Londrina. Accessed 24 Mar 2018
  29. Hong Z, Jin H, Tzfira T, Li J (2008) Multiple mechanism–mediated retention of a defective brassinosteroid receptor in the endoplasmic reticulum of Arabidopsis. P. Cell. 20:3418–3429CrossRefGoogle Scholar
  30. Huang B, Gao H (2000) Growth and carbohydrate metabolism of creeping bentgrass cultivars in response to increasing temperatures. Crop Sci 40:1115–1120CrossRefGoogle Scholar
  31. Huang T, Jander G (2017) Abscisic acid-regulated protein degradation causes osmotic stress induced accumulation of branched-chain amino acids in Arabidopsis thaliana. Planta 246:737–747CrossRefPubMedGoogle Scholar
  32. Khan MS, Ahmad D, AdilKhan M (2015) Utilization of genes encoding osmoprotectants in transgenic plants for enhanced abiotic stress tolerance. Electron J Biotech 8:257–266CrossRefGoogle Scholar
  33. Ku YS, Au-Yeung WK, Yung YL, Li MW, Wen CQ, Liu X, Lam HM (2013) Drought stress and tolerance in soybean. In: Board JE (ed) A comprehensive survey of international soybean research—genetics, physiology, agronomy and nitrogen relationships. InTech, New York, pp 209–237Google Scholar
  34. Kurepa J, Wang S, Li Y, Smalle J (2009) Proteasome regulation, plant growth and stress tolerance. Plant Signal Behav 4(10):924–927CrossRefPubMedGoogle Scholar
  35. Leborgne-Castel N, Jelitto-Van Dooren EPWM, Crofts AJ, Denecke J (1999) Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress. Plant Cell 11:459–470CrossRefPubMedGoogle Scholar
  36. Less H, Galili G (2008) Principal transcriptional programs regulating plant amino acid metabolism in response to abiotic stresses. Plant Physiol 147:316–330CrossRefPubMedGoogle Scholar
  37. Lisec J, Schauer N, Kopka J, Willmitzer L, Fernie AR (2006) Gas chromatography mass spectrometry-based metabolite profiling in plants. Nat Protoc 1:387–396CrossRefPubMedGoogle Scholar
  38. Lisec J, Peña-Cortés H, Willmitzer L, Hannah MA (2009) TargetSearch—a bioconductor package for the efficient preprocessing of GC-MS metabolite profiling data. BMC Bioinform 10:428CrossRefGoogle Scholar
  39. Meyer RF, Boyer JS (1981) Osmoregulation, solute distribution, and growth in soybean seedlings having low water potentials. Planta 151:482–489CrossRefPubMedGoogle Scholar
  40. Müller M, Munné-Bosch S (2011) Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Plant Methods 7:37CrossRefPubMedGoogle Scholar
  41. Nambara E, Kawaide H, Kamiya Y, Naito S (1998) Characterization of an Arabidopsis thaliana mutant that has a defect in ABA accumulation: ABA dependent and ABA-independent accumulation of free amino acids during dehydration. Plant Cell Physiol 39:853–858CrossRefPubMedGoogle Scholar
  42. Oliveira GLT (2016) The geopolitics of Brazilian soybeans. J Peasant Stud 43(2):348–372CrossRefGoogle Scholar
  43. Oliver MJ, Guo L, Alexander DC, Ryals JA, Wone BWM, Cushman JC (2011) A sister group contrast using untargeted global metabolomic analysis delineates the biochemical regulation underlying desiccation tolerance in sporobolusstapfianus. Plant Cell 23:1231–1248CrossRefPubMedGoogle Scholar
  44. Parry MAJ, Andralojc PJ, Khan S, Lea PJ, Keys AJ (2002) Rubisco activity: effects of drought stress. Ann Bot 89:833–839CrossRefPubMedGoogle Scholar
  45. Pincus D, Chevalier MW, Aragon T, Van Anken E, Vidal SE, El-Samad H (2010) BiP Binding to the ER-stress sensor ire1 tunes the homeostatic behavior of the unfolded protein response. PLoS Biol 1:1. Google Scholar
  46. Reddya AR, Chaitanyaa KV, Vivekanandanb M (2004) Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. J Plant Physiol 161:1189–1202CrossRefGoogle Scholar
  47. Reis PAB, Fontes EPB (2012) N-rica proteína (NRP) mediada sinalização morte celular: um novo ramo da resposta ao estresse ER com implicações para a biotecnologia vegetal. Plant Signal Behav 7:628–632CrossRefPubMedGoogle Scholar
  48. Reis PAB, Rosado GL, Silva LA, Oliveira LC, Oliveira LB, Costa MD, Alvim FC, Fontes EPB (2011) The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway. Plant Physiol 157:1853–1865CrossRefPubMedGoogle Scholar
  49. Reis PAB, Carpinetti PA, Freitas PPJ, Santos EGD, Camargos LF, OliveiraI HT, Silva JCF, Carvalho HH, Dal-Bianco M, Ramos JLRS, Fontes EPB (2016) Functional and regulatory conservation of the soybean ER stress-induced DCD/NRP-mediated cell death signaling in plants. BMC PlantBiol 16:156CrossRefGoogle Scholar
  50. Ribas-Carbo M, Taylor LN, Giles L, Busquets S, Finnegan PM, Day DA, Lambers H, Medrano H, Berry JA, Flexas J (2005) Effects of water stress on respiration in soybean leaves. Plant Physiol 139:466–473CrossRefPubMedGoogle Scholar
  51. Rodrigues SM, Andrade MO, Gomes APS, DaMatta FM, Baracat-Pereira MC, Fontes EPB (2006) Arabidopsis and tobacco plants ectopically expressing the soybean antiquitin-like ALDH7 gene display enhanced tolerance to drought, salinity, and oxidative stress. J Biol Chem 57:1909–1918Google Scholar
  52. Ruberti C, Kim SJ, Stefano G, Brandizzi F (2015) Unfolded protein response in plants: one master, many questions. Curr Opin Plant Biol 27:59–66CrossRefPubMedGoogle Scholar
  53. Scholander PE, Hammel HT, Bradstreet ED, Hemmingsen EA (1965) Sap pressure in vascular plants. Science 148:339–346CrossRefPubMedGoogle Scholar
  54. Shevchenko A, Tomas H, HavlĭsOlsen JV, Mann M (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860CrossRefGoogle Scholar
  55. Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417CrossRefPubMedGoogle Scholar
  56. Shulaev V, Cortes D, Miller G, Mittler R (2008) Metabolomics for plant stress response. Physiol Plant 132:199–208CrossRefPubMedGoogle Scholar
  57. Silva MA, Jifon JL, Silva JAG, Sharma V (2007) Use of physiological parameters as fast tools to screen for drought tolerance in sugarcane. Braz J Plant Physiol 19:193–201CrossRefGoogle Scholar
  58. Silva PA, Silva JCF, Caetano HDN, Machado JPB, Mendes GC, Reis PAB, Brustolini OJB, Dal-Bianco M, Fontes EPB (2015) Comprehensive analysis of the endoplasmic reticulum stress response in the soybean genome: conserved and plant-specific features. BMC Genom 16:783CrossRefGoogle Scholar
  59. Silvente S, Sobolev AP, Lara M (2012) Metabolite adjustments in drought tolerant and sensitive soybean genotypes in response to water stress. PLoS ONE 7:e38554CrossRefPubMedGoogle Scholar
  60. Stewart GR, Larher F (1980) Accumulation of amino acids and related compounds in relation to environmental stress. Biochem Plants 5:609–635Google Scholar
  61. Tripathi P, Rabara RC, Reese RN, Miller MA, Rohila JS, Subramanian S, Shen QJ, Morandi D, Bücking H, Shulaev V, Rushton PJ (2016) A toolbox of genes, proteins, metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes. BMC Genom 17:1–22CrossRefGoogle Scholar
  62. Umeda M, Hara C, Matsubayashi Y, Li HH, Liu Q, Tadokoro F, Aotsuka S, Uchimiya H (1994) Expressed sequence tags from cultured cells of rice 106 (Oryza sativa L.) under stressed conditions: analysis of genes engaged in ATP generating pathways. Plant Mol Biol 25:469–478CrossRefPubMedGoogle Scholar
  63. Valente MAS, Faria JQA, Ramos JRLS, Reis PAB, Pinheiro GL, Piovesan ND, Morais AT, Menezes CC, Cano MAO, Fietto LG, Loureiro ME, Aragao FJL, Fontes EBP (2009) The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco. J Exp Bot 60:533–546CrossRefPubMedGoogle Scholar
  64. Wang D, Weaver ND, Kesarwani M, Dong X (2005) Induction of protein secretory pathway is required for systemic acquired resistance. Science 308:1036–1040CrossRefPubMedGoogle Scholar
  65. Wehmeyer N, Vierling E (2000) The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerance. Plant Physiol 122:1099–1108CrossRefPubMedGoogle Scholar
  66. You J, Chan Z (2015) ROS regulation during abiotic stress responses in crop plants. Front Plant Sci. Google Scholar
  67. Zhu JK (2016) Abiotic stress signaling and responses in plants. Cell 167(2):313–324CrossRefPubMedGoogle Scholar
  68. Zhu T, Budworth P, Han B, Brown D, Chang HS, Zou G, Wang X (2001) Toward elucidating the global expression patterns of developing Arabidopsis: parallel analysis of 8300 genes by a high-density oligonucleotide probe array. Plant Physiol Biochem 39:221–242CrossRefGoogle Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2019

Authors and Affiliations

  • Flaviane Silva Coutinho
    • 1
    • 2
  • Danilo Silva dos Santos
    • 1
  • Lucas Leal Lima
    • 1
    • 2
  • Camilo Elber Vital
    • 2
  • Lázaro Aleixo Santos
    • 2
  • Maiana Reis Pimenta
    • 1
  • João Carlos da Silva
    • 1
  • Juliana Rocha Lopes Soares Ramos
    • 1
  • Angela Mehta
    • 3
  • Elizabeth Pacheco Batista Fontes
    • 1
  • Humberto Josué de Oliveira Ramos
    • 1
    • 2
    Email author
  1. 1.Laboratory of Plant Molecular Biology, Department of Biochemistry and Molecular BiologyUniversidade Federal de Viçosa, BIOAGRO/INCT-IPPViçosaBrazil
  2. 2.Center of Analyses of Biomolecules, NuBioMolUniversidade Federal de ViçosaViçosaBrazil
  3. 3.Embrapa Recursos Genéticos e BiotecnologiaBrasíliaBrazil

Personalised recommendations