TOBACCO PLANTS TRANSFORMED WITH THE O-ACETYLSERINE (THIOL) LYASE GENEOF WHEAT ARE RESISTANT TO TOXIC LEVELS OF HYDROGEN-SULFIDE GAS

O-acetylserine (thiol) lyase (EC4.2.99.8) is the key enzyme in the cys teine biosynthetic pathway of plants and prokaryotes. The gene, cys1, encoding this enzyme was isolated from a wheat (Triticum aestivum L.) cDNA library, and its deduced amino acid sequence found to show 53% se quence identity with the 0-acetyl-serine (thiol) lyase of Escherichia coli and Salmonella typhimurium. The deduced peptide consists of 325 a mino acids (34.1 kDa), contains a conserved motif for the binding of p yridoxal phosphate, a co-factor required for enzymatic activity, and a n N-terminal region of 37 amino acid residues resembling chloroplast t ransit peptides. The identity of cys1 was further demonstrated through complementation of an E. coli cysteine auxotroph, which lacks O-acety lserine (thiol) lyase, by expression of the wheat gene. Northern blot analysis showed that cys1 is highly expressed in green vegetative and reproductive tissues and in the roots of wheat, as well as in the leav es of several plant species. Southern blot analysis demonstrated that the gene exists as a single copy in the wheat genome. Tobacco plants t ransformed with cys1 in the sense orientation (sense plants) or antise nse orientation (antisense plants), under the control of the CaMV 35S promoter, showed high levels of transcripts. The O-acetylserine (thiol ) lyase activity in transgenic plants was determined, and found to be three- to fivefold higher in sense plants than in control plants, but unaffected in antisense transformants. Fumigation experiments with tox ic levels of hydrogen sulphide (H2S) gas showed that while sense trans formants were highly resistant, control and antisense plants were seve rely damaged by the treatment.