A molecular biology study of 1-aminocyclopropane-1-carboxylate synthase in tomatoes

by Shiu, Oi-yin

(Uncorrected OCR) Abstract of thesis entitled

A MOLECULAR BIOLOGY STUDY OF 1.AMINOCYCLOPROPANE??ARBOXYLATE SYNTHASE IN TOMATOES

submitted by

Shiu Oi Yin

for the degree of Doctor of Philosophy at The University of Hong Kong

in April 1996

The gaseous hormone ethylene plays an important role in plant development.

Ethylene biosynthesis is induced in seed germination, fruit ripening, flower fading,

and leaf senescence; it is also produced in response to wounding, flooding and

infection by pathogens. The key regulatory enzyme of ethylene biosynthesis is 1-

aminocyclopropane-l-carboxylic acid (ACC) synthase. ACC synthase is encoded

by a multigene family whose members are differentially regulated under various

conditions. In tomato, the DNA sequences of three complete genomic clones are

known, but more than five ACC synthases have been identified in tomato. Amongst

all the described ACC synthase genes in tomato, LE-ACS2 and LE-ACS4 are

expressed during fruit ripening and in wound-induced tomato pericarp tissue. The

LE-ACS3 gene is induced in the roots of the tomato plant during flooding and in

tomato cell suspension cultures when treated with auxins. The LE-ACS5 gene is

induced by auxin and the expression of the twins, LE-ACSIA and LE-ACSIB,

remains unclear.

In this study, a novel ACC synthase gene in tomato was isolated, sequenced

and analyzed at the nucleic acid level. Its nucleotide sequence contains 4459 base

pairs with 2463 bp and 393 bp at the 5' ~d 3' untranslated regions, respectively. The coding sequence translated for 467 residues and was interrupted by two introns. The deduced protein is 53.1 kDa in molecular weight with a pI value of 8.35. Seven conserved regions and an active center in ACC synthases were present. Besides, the eleven invariant amino acids conserved in various aminotransferases and synthases were found in the deduced polypeptide.

Expression of the gene was studied by ribonuclease protection assay. The gene was expressed mainly in vegetative tissues and therefore it is different from all the genes mentioned in former publications. The gene showed positive regulation by indole-3-acetic acid in seedling hypocotyls. The wounding effect in the intact leaves induced the ethylene production and the transcription of the gene. These changes were observed at two hours after wounding. The levels of ethylene production and accumulation of mRNA were increased up to four hours after induction and then declined almost to normal at eight hours.

Phylogenetic analysis showed that this gene was highly homologous to two ACC synthase members in potato, ST-ACSIA and ST-ACSIB. These two genes were reported to be induced in the indole-3-acetic acid treated seedling hypocotyls, but depressed during wounding in the potato leaves. This gene was also closely related to LE-ACS3 in tomato with 83% homology. Since the potato and tomato belongs to the Solanaceae family, it is believed that they inherited a similar set of ACC synthase genes during evolution.