Hormonal Induction of ACC Synthase

Ethylene is a plant hormone involved in many aspects of plant growth, development and response to stress conditions. The synthesis of ethylene requires the conversion of S-adenosylmethionine (AdoMet) to 1-aminocyclopropane-l-carboxylic acid (ACC) which gives rise to ethylene. The enzyme catalyzing this reaction is ACC synthase which is believed to be the rate limiting step in the ethylene biosynthetic pathway. Indole-3-acetic acid (IAA) and brassinosteroid (BR) have been shown to stimulate ACC synthase levels in etiolated mung bean hypocotyl tissues. The study of ACC synthase and how plant hormones modify its levels in tissues have been hampered by the inability to obtain purified enzyme. Three recent reports have described the purification of ACC synthase from wounded tomato pericarp tissue, winter squash and zucchini fruit tissue. Although these procedures appear to have obtained pure enzyme the quantities obtained were inadequate for extensive characterization due to the lability of the preparation. We have recently reported a procedure for the purification of ACC synthase from etiolated mung bean hypocotyl segments. This procedure was made possible by the ability to stimulate enzyme levels dramatically with hormone treatments and to stabilize the enzyme with high phosphate concentrations. The four step procedure resulted in 1,050-fold purification with 25% yield and consisted of stepwise elution from hydroxylapatite, gradient elution from Phenyl Sepharose CL-4B, gradient elution from hydroxylapatite and Fast Performance Liquid Chromatography (FPLC) on a MonoQ anion exchange column. The purified enzyme migrated as a single band of molecular weight 65,000 on denaturing polyacrylamide gel electrophoresis. The molecular weight of the native enzyme by Bio-Gel A-0.5 M chromatography was 125,000, indicating that the enzyme probably exists as a dimer of identical 65,000 MW subunits. ACC synthase exhibited a pH optimum of 8.0 for activity and a Km for AdoMet of 55 uM at 30 ° C with no substrate inhibition up to 2 mM. Aminooxyacetic acid was found to be a competitive inhibitor of ACC synthase with a Ki of 2 uM. ACC synthase exhibited an Arrhenius activation energy of 12 kcal /mol degree and was inactivated at temperatures in excess of 40 °C. The specific activity of pure ACC synthase was 21 umol of ACC formed/ mg protein/ hour when determined under optimal conditions with 400 uM AdoMet. We have recently obtained the N-terminal sequence for ACC synthase and have made an oligonucleotide probe. Sixty-three different stable cell lines which secrete monoclonal antibodies specific for ACC synthase as determined by enzyme linked immunosorbent assay (ELISA) have been made. Thus far we have selected six of the best clones, purified antibodies and characterized them. These clones have the ability to immunoprecipitate ACC synthase, give strong positive reactions in ELISA and recognize one band (65 KD) on western blots. A discussion on the use of these probes to determine if IAA and BR are promoting de novo production of ACC synthase and whether this appearance is regulated at the level of gene transcription or a subsequent step of protein synthesis will be discussed.