Overexpression of GmProT1 and GmProT2 increases tolerance to drought and salt stresses in transgenic Arabidopsis

doi:10.1016/S2095-3119(15)61288-6

摘要/Abstract

Abstract: The proline transporter protein (ProT) plays an important role in protective stress responses in various plants. However, its function in abiotic stress responses in soybean (Glycine max) remains obscure. In the present study, two soybean ProT genes, namely GmProT1 and GmProT2, were isolated by homologous cloning. GmProT1 and GmProT2 encode polypeptides of 435 and 433 amino acids, respectively. The GmProT1 and GmProT2 proteins showed high similarity to other ProT proteins. GmProT1 and GmProT2 transcripts were detected in different soybean tissues including roots, stems, leaves, flowers, and developmental seeds, and during diverse developmental stages. GmProT1 was strongly expressed in seeds 35 days after flowering. Quantitative real-time PCR analysis showed that the two genes were highly expressed in leaves and could be strongly induced in response to salt and drought conditions and ABA treatment. Transgenic Arabidopsis thaliana plants overexpressing the two genes were generated, which showed that GmProT genes attenuate damage from salt and drought stress. In addition, transgenic Arabidopsis plants accumulated proline in response to salt and osmotic stress. Transcription levels of salinity-responsive gene (RD29B and S0S3) and drought-induced gene (CDPK1) were higher in the transgenic lines than that of wild type plants. Our work provides evidence that GmProT genes function in the response to abiotic stresses and may affect the synthesis and response system of proline.

Key words: Glycine max , salt and drought stresses , GmProT1 , GmProT2

GUO Na, XUE Dong, ZHANG Wei, ZHAO Jin-ming, XUE Chen-chen, YAN Qiang, XUE Jin-yan, WANG Hai-tang, ZHANG Yu-mei, XING Han. [J]. Journal of Integrative Agriculture, 2016, 15(8): 1727-1743.

GUO Na, XUE Dong, ZHANG Wei, ZHAO Jin-ming, XUE Chen-chen, YAN Qiang, XUE Jin-yan, WANG Hai-tang, ZHANG Yu-mei, XING Han. Overexpression of GmProT1 and GmProT2 increases tolerance to drought and salt stresses in transgenic Arabidopsis[J]. Journal of Integrative Agriculture, 2016, 15(8): 1727-1743.

参考文献

Bates L, Waldren R, Teare I. 1973. Rapid determination of free proline for water-stress studies. Plant and Soil, 39, 205–207.

Clough S J, Bent A F. 1998. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16, 735–743.

Grallath S, Weimar T, Meyer A, Gumy C, Suter-Grotemeyer M, Neuhaus J M, Rentsch D. 2005. The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns. Plant Physiology, 137, 117–126.

Hamilton E W, Heckathorn S A. 2001. Mitochondrial adaptations to NaCl. Complex I is protected by anti-oxidants and small heat shock proteins, whereas complex II is protected by proline and betaine. Plant Physiology, 126, 1266–1274.

Hasanuzzaman M, Alam M M, Rahman A, Hasanuzzaman M, Nahar K, Fujita M. 2014. Exogenous proline and glycine betaine mediated upregulation of antioxidant defense and glyoxalase systems provides better protection against salt-induced oxidative stress in two rice (Oryza sativa L.) varieties. BioMed Research International, 2014, 757219.

Hasanuzzaman M, Fujita M. 2013. Exogenous sodium nitroprusside alleviates arsenic-induced oxidative stress in wheat (Triticum aestivum L.) seedlings by enhancing antioxidant defense and glyoxalase system. Ecotoxicology, 22, 584–596.

Hoque M A, Banu M N, Nakamura Y, Shimoishi Y, Murata Y. 2008. Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells. Journal of Plant Physiology, 165, 813–824.

Hoque M A, Okuma E, Banu M N, Nakamura Y, Shimoishi Y, Murata Y. 2007. Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities. Journal of Plant Physiology, 164, 553–561.

Hua X J, van de Cotte B, Van Montagu M, Verbruggen N. 1997. Developmental regulation of pyrroline-5-carboxylate reductase gene expression in Arabidopsis. Plant Physiology, 114, 1215–1224.

Igarashi Y, Yoshiba Y, Takeshita T, Nomura S, Otomo J, Yamaguchi-Shinozaki K, Shinozaki K. 2000. Molecular cloning and characterization of a cDNA encoding proline transporter in rice. Plant Cell Physiology, 41, 750–756.

Igarashi Y, Yoshiba Y, Takeshita T, Nomura S, Otomo J, Yamaguchi-Shinozaki K, Shinozaki K. 2000. Molecular cloning and characterization of a cDNA encoding proline transporter in rice. Plant Cell Physiology, 41, 750–756.

Jian B, Liu B, Bi Y, Hou W, Wu C, Han T. 2008. Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Molecular Biology, 9, 59.

Khedr A H, Abbas M A, Wahid A A, Quick W P, Abogadallah G M. 2003. Proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of Pancratium maritimum L. to salt-stress. Journal of Experimental Botany, 54, 2553–2562.

Kidokoro S, Watanabe K, Ohori T, Moriwaki T, Maruyama K, Mizoi J, Myint Phyu Sin Htwe N, Fujita Y, Sekita S, Shinozaki K, Yamaguchi-Shinozaki K. 2015. Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression. The Plant Journal (for Cell and Molecular Biology), 81, 505–518.

Kornbrot D. 2000. Statistical software for microcomputers: SigmaPlot 2000 and SigmaStat2. The British Journal of Mathematical and Statistical Psychology, 53, 335–337.

Krasensky J, Jonak C. 2012. Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. Journal of Experimental Botany, 63, 1593–1608.

Kumar A A, Mishra P, Kumari K, Panigrahi K C. 2012. Environmental stress influencing plant development and flowering. Frontiers in Bioscience (Scholar Edition), 4, 1315–1324.

Lehmann S, Funck D, Szabados L, Rentsch D. 2010. Proline metabolism and transport in plant development. Amino Acids, 39, 949–962.

Liu J, Ishitani M, Halfter U, Kim C S, Zhu J K. 2000. The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proceedings of the National Academy of Sciences of the United States of America, 97, 3730–3734

Liu X, Bush D R. 2006. Expression and transcriptional regulation of amino acid transporters in plants. Amino Acids, 30, 113–120.

Maiti R K, Satya P. 2014. Research advances in major cereal crops for adaptation to abiotic stresses. GM Crops & Food, 5, 259–279.

McNeil S D, Nuccio M L, Hanson A D. 1999. Betaines and related osmoprotectants. Targets for metabolic engineering of stress resistance. Plant Physiology, 120, 945–950.

Mizoi J, Ohori T, Moriwaki T, Kidokoro S, Todaka D, Maruyama K, Kusakabe K, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki K. 2013. GmDREB2A;2, a canonical DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2-type transcription factor in soybean, is posttranslationally regulated and mediates dehydration-responsive element-dependent gene expression. Plant Physiology, 161, 346–361.

Nanjo T, Fujita M, Seki M, Kato T, Tabata S, Shinozaki K. 2003. Toxicity of free proline revealed in an arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase. Plant Cell Physiology, 44, 541–548.

Nounjan N, Nghia P T, Theerakulpisut P. 2012. Exogenous proline and trehalose promote recovery of rice seedlings from salt-stress and differentially modulate antioxidant enzymes and expression of related genes. Journal of Plant Physiology, 169, 596–604.

Ortiz-Lopez A, Chang H, Bush D R. 2000. Amino acid transporters in plants. Biochimica et Biophysica Acta, 1465, 275–280.

Popova O V, Dietz K J, Golldack D. 2003. Salt-dependent expression of a nitrate transporter and two amino acid transporter genes in Mesembryanthemum crystallinum. Plant Molecular Biology, 52, 569–578.

Rentsch D, Hirner B, Schmelzer E, Frommer W B. 1996. Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant. The Plant Cell, 8, 1437–1446.

Schwacke R, Grallath S, Breitkreuz K E, Stransky E, Stransky H, Frommer W B, Rentsch D. 1999. LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen. The Plant Cell, 11, 377–392.

Sharma S, Villamor J G, Verslues P E. 2011. Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential. Plant Physiology, 157, 292–304.

Silva-Ortega C O, Ochoa-Alfaro A E, Reyes-Aguero J A, Aguado-Santacruz G A, Jimenez-Bremont J F. 2008. Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear. Plant Physiology and Biochemistry (Societe Francaise de Physiologie Vegetale), 46, 82–92.

Silveira J A, Viegas Rde A, da Rocha I M, Moreira A C, Moreira Rde A, Oliveira J T. 2003. Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves. Journal of Plant Physiology, 160, 115–123.

Sobahan M A, Arias C R, Okuma E, Shimoishi Y, Nakamura Y, Hirai Y, Mori I C, Murata Y. 2009. Exogenous proline and glycinebetaine suppress apoplastic flow to reduce Na(+) uptake in rice seedlings. Bioscience, Biotechnology, and Biochemistry, 73, 2037–2042.

Strizhov N, Abraham E, Okresz L, Blickling S, Zilberstein A, Schell J, Koncz C, Szabados L. 1997. Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. The Plant Journal (for Cell and Molecular Biology), 12, 557–569.

Szekely G, Abraham E, Cseplo A, Rigo G, Zsigmond L, Csiszar J, Ayaydin F, Strizhov N, Jasik J, Schmelzer E, Koncz C, Szabados L. 2008. Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. The Plant Journal (for Cell and Molecular Biology), 53, 11–28.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.

Tarczynski M C, Jensen R G, Bohnert H J. 1993. Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science, 259, 508–510.

Teh C Y, Mahmood M, Shaharuddin N A, Ho C L. 2014. In vitro rice shoot apices as simple model to study the effect of NaCl and the potential of exogenous proline and glutathione in mitigating salinity stress. Plant Growth Regulation, 75, 771–781.

Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, Higgins D G. 1997. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882.

Tran L S, Quach T N, Guttikonda S K, Aldrich D L, Kumar R, Neelakandan A, Valliyodan B, Nguyen H T. 2009. Molecular characterization of stress-inducible GmNAC genes in soybean. Molecular Genetics and Genomics, 281, 647–664.

Ueda A, Shi W, Sanmiya K, Shono M, Takabe T. 2001. Functional analysis of salt-inducible proline transporter of barley roots. Plant Cell Physiolog, 42, 1282–1289.

Verbruggen N, Villarroel R, Van Montagu M. 1993. Osmoregulation of a pyrroline-5-carboxylate reductase gene in Arabidopsis thaliana. Plant Physiology, 103, 771–781.

Haake V, Cook D, Riechmann J L, Pineda O, Thomashow M F, Zhang J Z. 2002. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiology, 130, 639–648.

Waditee R, Hibino T, Tanaka Y, Nakamura T, Incharoensakdi A, Hayakawa S, Suzuki S, Futsuhara Y, Kawamitsu Y, Takabe T, Takabe T. 2002. Functional characterization of betaine/proline transporters in betaine-accumulating mangrove. The Journal of Biological Chemistry, 277, 18373–18382.

Wang H, Zhou L, Fu Y, Cheung M Y, Wong F L, Phang T H, Sun Z, Lam H M. 2012. Expression of an apoplast-localized BURP-domain protein from soybean (GmRD22) enhances tolerance towards abiotic stress. Plant, Cell & Environment, 35, 1932–1947.

Wang W, Vinocur B, Altman A. 2003. Plant responses to drought, salinity and extreme temperatures: Towards genetic engineering for stress tolerance. Planta, 218, 1–14.

Wilkins M R, Gasteiger E, Bairoch A, Sanchez J C, Williams K L, Appel R D, Hochstrasser D F. 1999. Protein identification and analysis tools in the ExPASy server. Methods in Molecular Biology, 112, 531–552.

Xu J, Xue C, Xue D, Zhao J, Gai J, Guo N, Xing H. 2013. Overexpression of GmHsp90s, a heat shock protein 90 (Hsp90) gene family cloning from soybean, decrease damage of abiotic stresses in Arabidopsis thaliana. PLOS ONE, 8, e69810.

Yamaguchi-Shinozaki K, Shinozaki K. 1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. The Plant Cell, 6, 251–264.

Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K. 1997. Regulation of levels of proline as an osmolyte in plants under water stress. Plant Cell Physiology, 38, 1095–1102.

Zhang F, Li S, Yang S, Wang L, Guo W. 2015. Overexpression of a cotton annexin gene, GhAnn1, enhances drought and salt stress tolerance in transgenic cotton. Plant Molecular Biology, 87, 47–67.

Zhou G A, Chang R Z, Qiu L J. 2010. Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis. Plant Molecular Biology, 72, 357–367.