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.
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 beta-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 micromol H(2)S mg(-1) protein min(-1) and 0.92 micromol 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.
Lai
Kwok Wai
KW
School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Yau
Chi Ping
CP
Tse
Yu Chung
YC
Jiang
Liwen
L
Yip
Wing Kin
WK
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism*; D003545:Cysteine / Q000096:biosynthesis*; D005030:Ethylenes / Q000096:biosynthesis; D018506:Gene Expression Regulation, Plant; D047468:Immunoprecipitation; D008658:Inactivation, Metabolic; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D018517:Plant Roots / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D036226:Seedlings / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment
D001426:Bacterial Proteins; D003486:Cyanides; D005030:Ethylenes; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; C036216:ethylene; D008190:Lyases; C019955:beta-cyanoalanine synthase; D003545:Cysteine