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Expression of an NADP-malic enzyme gene in rice (Oryza sativa. L) is induced by environmental stresses; over-expression of the gene in Arabidopsis confers salt and osmotic stress tolerance

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Abstract

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plants, and has recently been implicated in plant defense such as in responses to wounding and UV-B radiation. In this study, we isolated a complementary DNA (cDNA) clone by using the differential display method and screening of a root cDNA library of rice (Oryza sativa. L) under carbonate (NaHCO3) stress, and identified it as one of the rice NADP-ME genes (we named it NADP-ME 2 , GenBank accession no. AB053295). The 5′ end of NADP-ME 2 was obtained by the 5′-RACE method, and the full-length cDNA had a length of 2217 bp encoding 593 amino acids. Expression of NADP-ME 2 mRNA in roots was induced by stress from carbonates (NaHCO3 and Na2CO3), NaCl, and environmental pH changes. NADP-ME 2 transcripts increased during 72-h exposures to NaHCO3, NaCl, and PEG stresses. Furthermore, NADP-ME activities in leaves and roots of rice seedlings increased by more than 50% in the presence of carbonates (NaHCO3 and Na2CO3), NaCl, and PEG. These results indicate that rice NADP-ME 2 responds to salts and osmotic stresses. Transgenic Arabidopsis plants over-expressing NADP-ME 2 were obtained through transformation, screening, Northern analysis and in situ NADP-ME activity assay. Transgenic Arabidopsis plants over-expressing NADP-ME 2 grew well in 1/2 × MS medium with 100 mM NaCl or 4% mannitol, whereas growth of wild-type (WT) Arabidopsis seedlings was strongly inhibited. In addition, under 125 mM NaCl stress, the root lengths of transgenic lines were about twice as long as those of the WT. These results suggest that NADP-ME 2 has a role in enhancing tolerance of plants to salt and osmotic stress.

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Abbreviations

CAM:

Crassulacean acid metabolism

CaMV35S:

Cauliflower mosaic virus 35S

cDNA:

Complementary DNA

DIG:

Digoxigenin

E. coli :

Escherichia coli

ME:

Malic enzyme

NADP-ME:

NADP-malic enzyme

NBT/BCIP:

5-bromo-4-chloro-3-indoxyl phosphate/nitroblue tetrazolium chloride

ORF:

Open reading frame

PEG6000:

Polyethylene glycol 6000

PEPCase:

Phosphoenolpyruvate carboxylase

PMSF:

Phenylmethylsulfonyl fluoride

ROS:

Reactive oxygen species

RT-PCR:

Reverse transcription polymerase chain reaction

WT:

Wild-type

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Acknowledgements

This work was supported by a Grant-in-aid for Scientific Research (18658001) and by a grant from Heiwa Nakajima Foundation to T.T. This work was also supported by the National High Technology Research and Development Program (863 Program, 2002AA241111) from the People’s Republic of China to S.K.L and Hei Long Jiang Province Natural Science Foundation Project (C200518) to Y.X.C.

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Authors and Affiliations

  1. Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin, 150040, P. R. China

    Shenkui Liu, Yuxiang Cheng, Xinxin Zhang & Qingjie Guan

  2. Asian Natural Environmental Science Center (ANESC), The University of Tokyo, 1-1-1 Midori-cho, Nishitokyo, Tokyo, 188-0002, Japan

    Shenkui Liu, Xinxin Zhang, Shunsaku Nishiuchi, Kenichi Hase & Tetsuo Takano

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  1. Shenkui Liu
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  2. Yuxiang Cheng
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Correspondence to Tetsuo Takano.

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Liu, S., Cheng, Y., Zhang, X. et al. Expression of an NADP-malic enzyme gene in rice (Oryza sativa. L) is induced by environmental stresses; over-expression of the gene in Arabidopsis confers salt and osmotic stress tolerance. Plant Mol Biol 64, 49–58 (2007). https://doi.org/10.1007/s11103-007-9133-3

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