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Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance

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Abstract

Two cysteine proteinase inhibitors (cystatins) from Arabidopsis thaliana, designated AtCYSa and AtCYSb, were characterized. Recombinant GST-AtCYSa and GST-AtCYSb were expressed in Escherichia coli and purified. They inhibit the catalytic activity of papain, which is generally taken as evidence for cysteine proteinase inhibitor function. Northern blot analyses showed that the expressions of AtCYSa and AtCYSb gene in Arabidopsis cells and seedlings were strongly induced by multiple abiotic stresses from high salt, drought, oxidant, and cold. Interestingly, the promoter region of AtCYSa gene contains a dehydration-responsive element (DRE) and an abscisic acid (ABA)-responsive element (ABRE), which identifies it as a DREB1A and AREB target gene. Under normal conditions, AtCYSa was expressed in 35S: DREB1A and 35S: AREB1 plants at a higher level than in WT plants, while AtCYSa gene was expressed in 35S: DREB2A plants at the same level as in WT plants. Under stress conditions (salt, drought and cold), AtCYSa was expressed more in all three transgenic plants than in WT plants. Over-expression of AtCYSa and AtCYSb in transgenic yeast and Arabidopsis plants increased the resistance to high salt, drought, oxidative, and cold stresses. Taken together, these data raise the possibility of using AtCYSa and AtCYSb to genetically improve environmental stresses tolerance in plants.

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Abbreviations

ABA:

Abscisic acid

ABRE:

ABA-responsive element

AREB:

ABRE binding protein

Cystatins:

Cysteine proteinase inhibitors

DRE:

Dehydration-responsive element

DREB:

DRE binding protein

GST:

Glutathione S-transferase

PCD:

Programmed cell death

Pis:

Proteinase inhibitors

PMSF:

Phenylmethylsulfonyl fluoride

TCA:

Trichloroacetic acid

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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. Additional support was provided by the Heilongjiang Provincial Program for Distinguished Young Scholars (JC200609) to S.L. We thank Dr. K. Yamaguchi-Shinozaki for valuable suggestions.

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Authors and Affiliations

  1. Asian Natural Environment Science Center (ANESC), The University of Tokyo, Midori Cho 1-1-1, Nishitokyo City, Tokyo, 188-0002, Japan

    Xinxin Zhang & Tetsuo Takano

  2. Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin, 150040, Peoples’ Republic of China

    Xinxin Zhang & Shenkui Liu

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  1. Xinxin Zhang
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  2. Shenkui Liu
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Correspondence to Tetsuo Takano.

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Zhang, X., Liu, S. & Takano, T. Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance. Plant Mol Biol 68, 131–143 (2008). https://doi.org/10.1007/s11103-008-9357-x

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