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Enhanced tolerance to drought and salt stresses in transgenic faba bean (Vicia faba L.) plants by heterologous expression of the PR10a gene from potato

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Abstract

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We report for the first time that expression of potato PR10a gene in faba bean causes enhanced tolerance to drought and salinity.

Abstract

Grain legumes such as soybean (Glycine max L. Merrill), pea (Pisum sativum L.) and faba bean (Vicia faba L.) are staple sources of protein for human and animal nutrition. Among grain legumes, faba bean is particularly sensitive to abiotic stress (in particular osmotic stress due to lack of water or enhanced soil salinity) and often suffers from severe yield losses. Many stress responsive genes have been reported with an effect on improving stress tolerance in model plants. Pathogenesis-related proteins are expressed by all plants in response to pathogen infection and, in many cases, in response to abiotic stresses as well. The PR10a gene isolated from the potato cultivar Desiree was selected for this study due to its role in enhancing salt and/or drought tolerance in potato, and transferred into faba bean cultivar Tattoo by Agrobacterium tumefaciens-mediated transformation system based upon direct shoot regeneration after transformation of meristematic cells derived from embryo axes. The transgene was under the control of the constitutive mannopine synthase promoter (p-MAS) in a dicistronic binary vector, which also contained luciferase (Luc) gene as scorable marker linked by internal ribosome entry site elements. Fertile transgenic faba bean plants were recovered. Inheritance and expression of the foreign genes were demonstrated by PCR, RT-PCR, Southern blot and monitoring of Luciferase activity. Under drought condition, after withholding water for 3 weeks, the leaves of transgenic plants were still green, while non-transgenic plants (WT) wilted and turned brown. Twenty-four hours after re-watering, the leaves of transgenic plants remained green, while WT plants did not recover. Moreover, the transgenic lines displayed higher tolerance to NaCl stress. Our results suggested that introducing a novel PR10a gene into faba bean could be a promising approach to improve its drought and salt tolerance ability, and that MAS promoter is not only constitutive, but also wound-, auxin/cytokinin- as well as stress-inducible.

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Abbreviations

bar :

Bialaphos resistance gene

PPT:

Phosphinothricin

BAP:

6-Benzylaminopurine

IBA:

Indole-3-butyric acid

CTAB:

Cetyltrimethylammoniumbromide

PR:

Pathogenesis-related

References

  • Agarwal S, Pandey V (2004) Antioxidant enzyme responses to NaCl stress in Cassia angustifolia. Biol Plant 48:555–560

    Article  CAS  Google Scholar 

  • Agrawal GK, Rakwal R, Tamogami S, Yonekura M, Kubo A, Saji H (2002) Chitosan activates defense/stress response(s) in the leaves of Oryza sativa seedlings. Plant Physiol Biochem 40:1061–1069

    Article  CAS  Google Scholar 

  • Armengaud P, Thiery L, Buhot N, Grenier-De MG, Savoure A (2004) Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features. Physiol Plant 120:442–450

    Article  PubMed  CAS  Google Scholar 

  • Awade A, Metz-Boutigue MH, Le Ret M, Genot G, Amiri I, Burkard G (1991) The complete amino acid sequence of the pathogenesis-related (PR2) protein induced in chemically stressed bean leaves. Biochim Biophys Acta 1077:241–244

    Article  PubMed  CAS  Google Scholar 

  • Banu MNA, Hoque MA, Watanabe-Sugimoto M, Matsuoka K, Nakamura Y, Shimoishi Y, Murata Y (2009) Proline and glycinebetaine induce antioxidant defense gene expression and suppress cell death in cultured tobacco cells under salt stress. J Plant Physiol 166:146–156

    Article  PubMed  CAS  Google Scholar 

  • Bates LS, Waldren RP, Teare LD (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207

    Article  CAS  Google Scholar 

  • Bhatnagar-Mathur P, Vadez V, Sharma KK (2008) Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects. Plant Cell Rep 27:411–424

    Article  PubMed  CAS  Google Scholar 

  • Bond DA (1987) Recent developments in breeding of field beans (Vicia faba L.). Plant Breeding 99:1–26

    Article  Google Scholar 

  • Böttinger P, Steinmetz A, Schieder O, Pickardt T (2001) Agrobacterium-mediated transformation of Vicia faba. Mol Breeding 8(3):243–254

    Article  Google Scholar 

  • Crowell DN, John ME, Russel D, Amasona R (1992) Characterization of a stress induced, developmentally regulated gene family from soybean. Plant Mol Biol 18:459–466

    Article  PubMed  CAS  Google Scholar 

  • Dash M, Panda SK (2001) Salt stress induced changes in growth and enzyme activities in germinating Phaseolus mungo seeds. Biol Plant 44:587–589

    Article  CAS  Google Scholar 

  • Dorokhov YL, Skulachev MV, Ivanov PA, Zvereva SD, Tjulkina LG, Merits A, Gleba YY, Hohn T, Atabekov JG (2002) Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc Natl Acad Sci USA 99:5301–5306

    Article  PubMed  CAS  Google Scholar 

  • Eapen S (2008) Advances in development of transgenic pulse crops. Biotechnol Adv 26:162–168

    Article  PubMed  CAS  Google Scholar 

  • El-Banna A, Hajirezaei MR, Wissing J, Ali Z, Vaas L, Heine-Dobbernack E, Jacobsen HJ, Schumacher H, Kiesecker H (2010) Over-expression of PR10a leads to increased salt and osmotic tolerance in potato cell cultures. J Biotechnol 150:277–287

    Article  PubMed  CAS  Google Scholar 

  • Guevara-García A, Mosqueda-Cano G, Argüello-Astorga G, Simpson J, Herrera-Estrella L (1993) Tissue-specific and wound-inducible pattern of expression of the mannopine synthase promoter is determined by the interaction between positive and negative cis-regulatory elements. Plant J 4(3):495–505

    Article  PubMed  Google Scholar 

  • Gutierrez N, Giménez MJ, Palomino C, Avila CM (2011) Assessment of candidate reference genes for expression studies in Vicia faba L. by real-time quantitative PCR. Mol Breeding 28:13–24

    Article  Google Scholar 

  • Hanafy MS, Pickardt T, Kiesecker H, Jacobsen HJ (2005) Agrobacterium-mediated transformation of faba bean (Vicia faba L.) using embryo axes. Euphytica 142:227–236

    Article  CAS  Google Scholar 

  • Hanafy MS, Jacobsen HJ, Böttinger P, Pickardt T (2008) Agrobacterium-mediated genetic transformation of faba bean, Vicia faba L. In: Kirti PB (ed) Handbook of new technologies for genetic improvement of legumes. The Haworth Press, Taylor & Francis Group, New York, pp 287–300

  • Hashimoto M, Kisseleva L, Sawa S, Furukawa T, Komatsu S, Koshiba T (2004) A novel rice PR10 Protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway. Plant Cell Physiol 45:550–559

    Article  PubMed  CAS  Google Scholar 

  • Hellens RP, Edwards A, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832

    Article  PubMed  CAS  Google Scholar 

  • Hong ZL, Lakkineni K, Zhang ZM, Verma DPS (2000) Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol 122:1129–1136

    Article  PubMed  CAS  Google Scholar 

  • Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218

    Article  CAS  Google Scholar 

  • Ishimoto M, Rahman S, Hanafy MS, Khalafalla MM, El-Shemy HA, Nakamoto Y, Kita Y, Takanashi K, Matsuda F, Murano Y, Funabashi T, Miyagawa H, Wakasa K (2010) Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation. Mol Breeding 25:313–326

    Article  CAS  Google Scholar 

  • Jain S, Srivastava S, Sarin NB, Kav NNV (2006) Proteomics reveals elevated levels of PR 10 proteins in saline-tolerant peanut (Arachis hypogaea) calli. Plant Physiol Biochem 44:253–259

    Article  PubMed  CAS  Google Scholar 

  • Jain S, Kumar D, Jain M, Chaudhary P, Deswal R, Sarin NB (2012) Ectopic overexpression of a salt stress-induced pathogenesis-related class 10 protein (PR10) gene from peanut (Arachis hypogaea L.) affords broad spectrum abiotic stress tolerance in transgenic tobacco. Plant Cell Tiss Organ Cult 109:19–31

    Article  CAS  Google Scholar 

  • Jellouli N, Ben Jouira H, Daldoul S, Chenennaoui S, Ghorbel A, Ben Salem A, Gargouri A (2010) Proteomic and transcriptomic analysis of grapevine PR10 expression during salt stress and functional characterization in yeast. Plant Mol Biol Rep 28:1–8

    Article  CAS  Google Scholar 

  • Kav NNV, Srivastava S, Goonewardene L, Blade SF (2004) Proteome-level changes in the roots of Pisum sativum L. in response to salinity. Ann Appl Biol 145:217–230

    Article  CAS  Google Scholar 

  • Kieft JS, Zhou K, Jubin R, Doudna JA (2001) Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 7:194–206

    Article  PubMed  CAS  Google Scholar 

  • Langridge WHR, Fitzgerald KJ, Koncz C, Schell J, Szalay AA (1989) Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones. Proc Natl Acad Sci USA 86(9):3219–3223 PMCID: PMC287101

    Article  PubMed  CAS  Google Scholar 

  • Li H, Flachowsky H, Fischer TC, Hanke V, Forkmann G, Treutter D, Schwab W, HoVmann T, Szankowski I (2007) Maize Lc transcription factor causes induction of anthocyanins, distinct proanthocyanidins and phenylpropanoids in apple Malus domestica Borkh.). Planta 226:1243–1254

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Zhu J-K (1997) Proline accumulation and salt-stress-induced gene expression in a salt-hyper sensitive mutant of Arabidopsis. Plant Physiol 114:591–596

    Article  PubMed  CAS  Google Scholar 

  • Liu JJ, Ekramoddoullah AKM, Yu X (2003) Differential expression of multiple PR10 proteins in western white pine following wounding, fungal infection and cold-hardening. Physiol Plant 119:544–553

    Article  CAS  Google Scholar 

  • Ma SS, Gong QQ, Bohnert HJ (2006) Dissecting salt stress pathways. J Exp Bot 57:1097–1107

    Article  PubMed  CAS  Google Scholar 

  • Marineau C, Matton DP, Brisson N (1987) Differential accumulation of potato tuber mRNAs during the hypersensitive response induced by arachidonic acid elicitor. Plant Mol Biol 9:335–342

    Article  CAS  Google Scholar 

  • McGee JD, Hamer JE, Hodges TK (2001) Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea. Mol Plant Microbe Interact 14:877–886

    Article  PubMed  CAS  Google Scholar 

  • OuYang B, Yang T, Li H, Zhang L, Zhang Y, Zhang J, Fei Z, Ye Z (2007) Identification of early salt stress response genes in tomato root by suppression subtractive hybridization and microarray analysis. J Exp Bot 58:507–520

    Article  PubMed  CAS  Google Scholar 

  • Pellegrineschi A, Pulleman M, Sullivan S, Trethowan R, Reynolds M (2005) Using transgenic plants as a source of genetic diversity for breeding greater drought tolerance into wheat. ISB news report, July

  • Pickardt T, Saalbach I, Waddell D, Meixner M, Müntz K, Schieder O (1995) Seed specific expression of the 2S albumin gene from Brazil nut (Bertholletia excelsa) in transgenic Vicia narbonensis. Mol Breeding 1:295–301

    Article  CAS  Google Scholar 

  • Popelka JC, Terryn N, Higgins TJV (2004) Gene technology for grain legumes: can it contribute to the food challenge in developing countries? Plant Sci 167:195–206

    Article  CAS  Google Scholar 

  • Przymusinski R, Rucinska R, Gwozdz EA (2004) Increased accumulation of pathogenesis-related proteins in response of lupine roots to various abiotic stresses. Environ Exp Bot 52:53–61

    Article  CAS  Google Scholar 

  • Rakwal R, Agrawal GK, Yonekura M (1999) Separation of proteins from stressed rice Oryza sativa L. leaf tissues by two dimensional polyacrylamide gel electrophoresis, induction of pathogenesis-related and cellular protectant proteins by jasmonic acid, UV irradiation and copper chloride. Electrophoresis 20:3472–3478

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol 3(3):217–223

    PubMed  CAS  Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417

    Article  PubMed  CAS  Google Scholar 

  • Solleti SK, Bakshi S, Purkayastha J, Panda SK, Sahoo L (2008) Transgenic cowpea (Vigna unguiculata) seeds expressing a bean alpha-amylase inhibitor 1 confer resistance to storage pests, bruchid beetles. Plant Cell Rep 27:1841–1850

    Article  PubMed  CAS  Google Scholar 

  • Somssich IE, Schmelzer E, Bollmann J, Hahlbrock K (1986) Rapid activation by fungal elicitor of genes encoding “pathogenesis-related” proteins in cultured parsley cells (two-dimensional gels/doned cDNAs/RNA blot hybridization/nuclear run-off transcription). Proc Nat Acad Sci USA 83:2427–2430

    Article  PubMed  CAS  Google Scholar 

  • Srivastava S, Fristensky B, Kav NNK (2004) Constitutive expression of a PR-10 protein enhances the germination of Brassica napus under saline conditions. Plant Cell Physiol 45:1320–1324

    Article  PubMed  CAS  Google Scholar 

  • Sul IW, Korban SS (1996) A highly efficient method for isolating genomic DNA from plant tissues. Plant Tissue Culture Biotechnol 2:113–116

    Google Scholar 

  • Tanaka N, Che FS, Watanabe N, Fujiwara S, Takayama S, Isogai A (2003) Flagellin from an incompatible strain of Acidovorax avenae mediates H2O2 generation accompanying hypersensitive cell death and expression of PAL, Cht-1, and PBZ1, but not of LOX in rice. Mol Plant Microbe Interact 16(5):422–428

    Article  PubMed  CAS  Google Scholar 

  • Terenin IM, Dmitriev SE, Andreev DE, Royall E, Belsham GJ, Roberts LO, Shatsky IN (2005) A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry. Mol Cell Biol 25:7879–7888

    Article  PubMed  CAS  Google Scholar 

  • Tran LSP, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16:2481–2498

    Article  PubMed  CAS  Google Scholar 

  • Vaas L, Marheine M, Seufert S, Schumacher HM, Kiesecker H, Heine-Dobbernack E (2012) Impact of pr-10a overexpression on the cryopreservation success of Solanum tuberosum suspension cultures. Plant Cell Rep 31(6):1061–1071. doi:10.1007/s00299-011-1225-5

    Article  PubMed  CAS  Google Scholar 

  • Van Loon LC, Van Strien EA (1999) The families of pathogenesis-related proteins, their activities and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol 55:85–97

    Article  Google Scholar 

  • Van Loon LC, Pierpoint WC, Boller Th, Conejero V (1994) Recommendations for naming plant pathogenesis-related proteins. Plant Mol Biol Rep 12:245–264

    Article  Google Scholar 

  • Xianjun P, Xingyong M, Weihong F, Man S, Liqin C, Alam I, Lee B, Dongmei Q, Shihua S, Gongshe L (2011) Improved drought and salt tolerance of Arabidopsis thaliana by transgenic expression of a novel DREB gene from Leymus chinensis. Plant Cell Rep 30:1493–1502

    Article  PubMed  Google Scholar 

  • Xiong L, Zhu J-K (2002) Molecular and genetic aspects of plant responses to osmotic stress. Plant, Cell Environ 25:131–139

    Article  CAS  Google Scholar 

  • Zhou W, Li Y, Zhao BC, Ge RC, Shen YZ, Wang G, Huang ZJ (2009) Overexpression of TaSTRG gene improves salt and drought tolerance in rice. J Plant Physiol 166:1660–1671

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This study was supported by Alexander von Humboldt foundation (AvH), Germany. M.S. Hanafy was holder of a fellowship for experienced researcher from AvH (Georg Forster program). The authors thank Carolyn Lee-Parsons (Northeastern University) for language editing.

Conflict of interest

The authors have declared no conflict of interest.

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Author notes

  1. Moemen S. Hanafy

    Present address: Faculty of Science and Humanities, Biology Department, Salman bin Abdulaziz University (SAU), 11942, Al-Kharj, Saudi Arabia

Authors and Affiliations

  1. Plant Biotechnology Department, Leibniz University Hannover, Institute of Plant Genetics, Herrenhäuser Str. 2, 30419, Hannover, Germany

    Moemen S. Hanafy, Hans-Jörg Jacobsen & Fathi S. Hassan

  2. Plant Biotechnology Department, National Research Centre (NRC), Tahrir Str. Dokki, 12311, Cairo, Egypt

    Moemen S. Hanafy

  3. Faculty of Agriculture, Department of Genetics, Kafr El-Sheikh University, 33516, Kafr El-Sheikh, Egypt

    Antar El-Banna

  4. German Collection of Microorganisms and Cell Cultures DSMZ GmbH, Inhoffenstr. 7b, 38124, Braunschweig, Germany

    Heinz Martin Schumacher

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  1. Moemen S. Hanafy
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  2. Antar El-Banna
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Correspondence to Fathi S. Hassan.

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Communicated by P. Kumar.

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Hanafy, M.S., El-Banna, A., Schumacher, H.M. et al. Enhanced tolerance to drought and salt stresses in transgenic faba bean (Vicia faba L.) plants by heterologous expression of the PR10a gene from potato. Plant Cell Rep 32, 663–674 (2013). https://doi.org/10.1007/s00299-013-1401-x

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