Abstract
Citrus (Citrus unshiu Marcov.) dehydrin in response to chilling stress was overexpressed in tobacco (Nicotiana tabacum L.), and the cold stress tolerance of transgenics at low temperature was analyzed. The freezing at −4 °C for 3 h of 24 independent lines indicated that a phenotype expressing citrus dehydrin showed less electrolyte leakage than the control. Dehydrin protein content was correlated with freezing tolerance in transgenics. Dehydrin-expressing tobacco exhibited earlier germination and better seedling growth than the control at 15 °C. Cell fractionation experiments suggested that the protein was predominantly expressed in mitochondria and the soluble fraction. Malondialdehyde production enhanced by chilling stress was lower in tobacco plants expressing citrus dehydrin than in control phenotypes. Dehydrin protein, purified from Escherichia coli expressing citrus dehydrin cDNA, prevented peroxidation of soybean (Glycine max L.) liposomes in vitro. The inhibitory activity of dehydrin against liposome oxidation was stronger than that of albumin, glutathione, proline, glycine betaine, and sucrose. These results suggest that dehydrin facilitates plant cold acclimation by acting as a radical-scavenging protein to protect membrane systems under cold stress.
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Abbreviations
- AAPH:
-
2, 2′-azobis (2-amidinopropane)
- BSA:
-
bovine serum albumin
- CuCOR19:
-
Citrus unshiu cold-regulated gene encoding a 19-kDa protein
- GUS:
-
β-glucuronidase
- MDA:
-
malondialdehyde
- pBIT:
-
GUS control tobacco
- pC19T:
-
CuCOR19-expressing tobacco
- WT:
-
wild-type tobacco
References
Adonaylo VN, Oteiza PI (1999) Pb2+ promotes lipid oxidation and alterations in membrane physical properties. Toxicology 132:19–32
Alonso A, Queiroz CS, Magalhaes AC (1997) Chilling stress leads to increased cell membrane rigidity in roots of coffee (Coffea arabica L.) seedlings. Biochim Biophys Acta 1323:75–84
Barclay KD, McKersie BD (1994) Peroxidation reactions in plant membranes: effects of free fatty acids. Lipids 29:877–882
Boldyrev AA, Dupin AM, Batrukova MA, Bavykina NI, Korshunova GA, Shvachkin YP (1989) A comparative study of synthetic carnosine analogs as antioxidants. Comp Biochem Physiol B 94: 237–240
Borovskii GB, Stupnikova IV, Antipina AI, Downs CA, Voinikov VK (2000) Accumulation of dehydrin-like-proteins in the mitochondria of cold-treated plants. J Plant Physiol 156:797–800
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 72:248–254
Chaudiere J, Ferrari-Iliou R (1999) Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem Toxicol 37:949–962
Chen H-M, Muramoto K, Yamauchi F, Nokihara K (1996) Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. J Agric Food Chem 44:2619–2623
Close TJ (1997) Dehydrins: a commonalty in the response of plants to dehydration and low temperature. Physiol Plant 100:291–296
Danyluk J, Perron A, Houde M, Limin A, Fowler B, Benhamou N, Sarhan F (1998) Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. Plant Cell 10:623–638
Fryer MJ, Andrews JR, Oxborough K, Blowers DA, Baker NR (1998) Relationship between CO2 assimilation, photosynthetic electron transport, and active O2 metabolism in leaves of maize in the field during periods of low temperature. Plant Physiol 116:571–580
Griffiths G, Leverentz M, Silkowski H, Gill N, Sanchez-Serrano JJ (2000) Lipid hydroperoxide levels in plant tissues. J Exp Bot 349:1363–1370
Guy CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu Rev Plant Physiol Plant Mol Biol 41:187–223
Hakansson G, van der Mark F, Bonnett HT, Glimelius K (1988) Variant mitochondrial protein and DNA patterns associated with cytoplasmic male-sterile lines of Nicotiana. Theor Appl Genet 76:431–437
Hamill JD, Rounsley S, Spencer A, Todd G, Rhodes MJC (1991) The use of the polymerase chain reaction in plant transformation studies. Plant Cell Rep 10: 221–224
Hara M, Wakasugi Y, Ikoma Y, Yano M, Ogawa K, Kuboi T (1999) cDNA sequence and expression of a cold-responsive gene in Citrus unshiu. Biosci Biotechnol Biochem 63: 433–437
Hara M, Terashima S, Kuboi T (2001) Characterization and cryoprotective activity of cold-responsive dehydrin from Citrus unshiu. J Plant Physiol 158:1333–1339
Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends Plant Sci 6:431–438
Hong Z, Lakkineni K, Zhang Z, 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
Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403
Ismail AM, Hall AE, Close TJ (1999a) Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence. Proc Natl Acad Sci USA 96:13566–13570
Ismail AM, Hall AE, Close TJ (1999b) Purification and partial characterization of a dehydrin involved in chilling tolerance during seedling emergence of cowpea. Plant Physiol 120:237–244
Kaye C, Neven L, Hofig A, Li QB, Haskell D, Guy C (1998) Characterization of a gene for spinach CAP160 and expression of two spinach cold-acclimation proteins in tobacco. Plant Physiol 116:1367–1377
Kazuoka T, Oeda K (1994) Purification and characterization of COR85-oligomeric complex from cold-acclimated spinach. Plant Cell Physiol 35:601–611
Kodama H, Hamada T, Horiguchi G, Nishimura M, Iba K (1994) Genetic enhancement of cold tolerance by expression of a gene for chloroplast ω-3 fatty acid desaturase in transgenic tobacco. Plant Physiol 105:601–605
Koop HU, Steinmueller K, Wagner H, Roessler C, Eibl C, Sacher L (1996) Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199:193–201
Moller IM (2001) Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annu Rev Plant Physiol Plant Mol Biol 52:561–591
Morgenthaler JJ, Marsden MP, Price CA (1975) Factors affecting the separation of photosynthetically competent chloroplasts in gradients of silica sols. Arch Biochem Biophys 168:289–301
Murata N, Ishizaki-Nishizawa O, Higashi S, Hayashi H, Tasaka Y, Nishida I (1992) Genetically engineered alteration in the chilling sensitivity of plants. Nature 356:710–713
Peskan T, Oelmueller R (2000) Heterotrimeric G-protein β-subunit is localized in the plasma membrane and nuclei of tobacco leaves. Plant Mol Biol 42:915–922
Rinne PLH, Kaikuranta PLM, van der Plas LHW, van der Schoot C (1999) Dehydrins in cold-acclimated apices of birch (Betula pubescens Ehrh.): production, localization and potential role in rescuing enzyme function during dehydration. Planta 209:377–388
Roxas VP, Lodhi SA, Carrett DK, Mahan JR, Allen RD (2000) Stress tolerance in transgenic tobacco seedlings that overexpress glutathione S-transferase/glutathione peroxidase. Plant Cell Physiol 41:1229–1234
Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T, Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y, Shinozaki K (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J 31:279–292
Soriani M, Pietraforte D, Minetti M (1994) Antioxidant potential of anaerobic human plasma: role of serum albumin and thiols as scavengers of carbon radicals. Arch Biochem Biophys 312:180–188
Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571–599
Thompson JE, Legge RL, Barber RF (1987) The role of free radicals in senescence and wounding. New Phytol 105:317–344
Uemura M, Yoshida S (1983) Isolation and identification of plasma membrane from light-grown winter rye seedlings (Secale cereale L. cv Puma). Plant Physiol 73:586–597
Wisniewski M, Webb R, Balsamo R, Close TJ, Yu XM, Griffith, M (1999) Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiol Plant 105:600–608
Acknowledgement
This study was supported in part by a Grant-in-Aid (No. 12460078) for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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Hara, M., Terashima, S., Fukaya, T. et al. Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco. Planta 217, 290–298 (2003). https://doi.org/10.1007/s00425-003-0986-7
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DOI: https://doi.org/10.1007/s00425-003-0986-7