Cloning and expression of 1-aminocyclopropane-1-carboxylate synthase cDNA from Rosa (Rosa X hybrida)

Abstract

Phytohormone ethylene regulates a variety of physiological processes in plant growth and development including fruit ripening, seed germination, leaf and flower senescence. Three enzymes, namely S-adenosyl-methionine synthase, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase are involved in the catalysis of methionine via S-adenosyl-methionine, ACC into ethylene. Previous studies suggest that ACC synthase plays a key regulatory role in ethylene biosynthesis. In the present study, the role of ACC synthase in flower petal senescence was investigated. A cDNA library from senescing petals of Rosa hybrid cv. Kardinal prepared in λcDNA ZAP Express Vector was probed with a rose-specific 400bp probe developed using degenerate primers by RT-PCR, and eight putative positive clones were isolated. Inserts in these clones varied from 1200 to 1800bp. Except for difference in length, the sequences of these clones were identical. Full-length clone, RKacc7 of 1750bp contains an open reading frame of 480 amino acids (58 KDa) which is proceeded by an untranslated leader of 269bp and ends with a 38bp sequence at the 3'-end. The deduced amino acid sequence of the polypeptide contains the eleven conserved amino acid residues, the substrate and pyridoxal phosphate binding sites that are characteristic of all ACC synthases. The in vitro transcripts from the full-length clones when translated in rabbit reticulocyte lysates exhibited a 55 KDa polypeptide which comigrated with a polypeptide synthesized from mRNA fraction isolated from senescing petals and were both immunoselected by anti-ACC synthase antibodies. RKacc7 was cloned into pET plasmid for over expression in E.coli. Fusion polypeptides with a His-Tag at the amino terminus and a S-Tag at the carboxyl terminus were purified by affinity chromatography. In vivo synthesized polypeptides were detected either by S-Tag based detection system or with anti-ACC antibodies in western blot. RT-PCR based studies showed that in planta RKacc7 is specifically expressed in rose petals, ovary and sepals. Genomic southern blots probed with RKacc7 showed multiple bands beyond those expected from restriction sites in cDNA or even after taking into account intron lengths. The number and sizes of these DNA fragments are consistent with a pattern expected from a multigene family.