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JXB Advance Access published online on January 30, 2009
Journal of Experimental Botany, doi:10.1093/jxb/ern343
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Heterologous expression analyses of rice OsCAS in Arabidopsis and in yeast provide evidence for its roles in cyanide detoxification rather than in cysteine synthesis in vivo

Kwok Wai Lai1, Chi Ping Yau1, Yu Chung Tse2, Liwen Jiang2,3 and Wing Kin Yip1,3,*

1School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China
2Department of Biology and Molecular Biotechnology Program, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
3State Key Laboratory of Agrobiotechnology, China

* To whom correspondence should be addressed. E-mail: wkyip{at}hkucc.hku.hk

While most dicot plants produce little ethylene in their vegetative stage, many monocots such as rice liberate a relatively large amount of ethylene with cyanide as a co-product in their seedling stage when etiolated. One of the known functions of β-cyanoalanine synthase (CAS) is to detoxify the co-product cyanide during ethylene biosynthesis in higher plants. Based on a tryptic peptide sequence obtained from a partially purified CAS activity protein preparation in etiolated rice seedlings, the full-length putative rice CAS-encoding cDNA sequence (OsCAS), which is homologous to those O-acetylserine sulphydrylase (OASS) genes, was cloned. Unlike most of the CAS genes reported from dicots, the transcription of OsCAS is promoted by auxins but suppressed by ethylene. To address the function and the subcellular localization of this gene product in planta, a binary vector construct consisting of this gene appended with a yellow fluorescent protein-encoding sequence was employed to transform Arabidopsis. Specific activities on CAS and OASS of the purified recombinant protein from transgenic Arabidopsis were 181.04 µmol H2S mg–1 protein min–1 and 0.92 µmol Cys mg–1 protein min–1, respectively, indicating that OsCAS favours CAS activity. The subcellular localization of OsCAS was found mostly in the mitochondria by immunogold electron-microscopy. Chemical cross-linking and in-gel assay on a heterodimer composed of functional and non-functional mutants in a yeast expression system on OsCAS suggested that OsCAS functions as a homodimer, similar to that of OASS. Despite the structural similarity of OsCAS with OASS, it has also been confirmed that OsCAS could not interact with serine-acetyltransferase, indicating that OsCAS mainly functions in cyanide detoxification.

Key words: Cyanide, β-cyanoalanine synthase, ethylene, rice, serine acetyltransferase

Received 17 September 2008; Revised 20 November 2008 Accepted 5 December 2008


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