Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato
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GDP-Mannose 3′,5′-epimerase (GME; EC 188.8.131.52) catalyses the conversion of GDP-d-mannose to GDP-l-galactose, an important step in the ascorbic acid (AsA) biosynthesis pathway in higher plants. In this study, two members of the GME gene family were isolated from tomato (Solanum lycopersicum). Both SlGME genes encode 376 amino acids and share a 92% similarity with each other. Semi-quantitative RT-PCR indicated that SlGME1 was constantly expressed in various tissues, whereas SlGME2 was differentially expressed in different tissues. Transient expression of fused SlGME1-GFP (green fluorescent protein) and SlGME2-GFP in onion cells revealed the cytoplasmic localisation of the two proteins. Transgenic plants over-expressing SlGME1 and SlGME2 exhibited a significant increase in total ascorbic acid in leaves and red fruits compared with wild-type plants. They also showed enhanced stress tolerance based on less chlorophyll content loss and membrane-lipid peroxidation under methyl viologen (paraquat) stress, higher survival rate under cold stress, and significantly higher seed germination rate, fresh weight, and root length under salt stress. The present study demonstrates that the overexpression of two members of the GME gene family resulted in increased ascorbate accumulation in tomato and improved tolerance to abiotic stresses.
KeywordsAscorbic acid GDP-Mannose 3′,5′-epimerase Abiotic stress Solanum lycopersicum
Green fluorescent protein
Gene splicing with overlap extension PCR
The authors would like to thank Prof. Wenwu Guo for his critical reviews of the manuscript. This study was supported by grants from the State Major Basic Research Development Program (No. 2011CB100600), High-Tech Research and Development Program (No. 2007AA10Z131) and National Natural Science Foundation of China (Nos. 30800756 and 30921002).
- Alhagdow M, Mounet F, Gilbert L, Nunes-Nesi A, Garcia V, Just D, Petit J, Beauvoit B, Fernie A, Rothan C (2007) Silencing of the mitochondrial ascorbate synthesizing enzyme l-galactono-1, 4-lactone dehydrogenase affects plant and fruit development in tomato. Plant Physiol 145:1408–1422CrossRefPubMedGoogle Scholar
- Du Y, Yao Y, Liu J (2006) Molecular characterization of two rice cDNAs encoding GDP-mannose-3′,5′-epimerase and their expression patterns. Prog Biochem Biophys 33:368–376Google Scholar
- Gilbert L, Alhagdow M, Nunes-Nesi A, Quemener B, Guillon F, Bouchet B, Faurobert M, Gouble B, Page D, Garcia V (2009) The GDP-D-mannose 3,5-epimerase (GME) plays a key role at the intersection of ascorbate and non cellulosic cell wall biosynthesis in tomato. Plant J 60:499–508CrossRefPubMedGoogle Scholar
- Nishikirni M, Fukuyama R, Minoshima S, Shimizu N, Yagi K (1994) Cloning and chromosomal mapping of the human non-functional gene for l-gulono-g-lactone oxidase, the enzyme for l-ascorbic acid biosynthesis missing in man. J Biol Chem 269:13685–13688Google Scholar
- Wolucka B, Persiau G, Van Doorsselaere J, Davey M, Demol H, Vandekerckhove J, Van Montagu M, Zabeau M, Boerjan W (2001) Partial purification and identification of GDP-mannose 3′,5′-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc Natl Acad Sci USA 98:14843–14848CrossRefPubMedGoogle Scholar