Inactivation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase,which catalyses the final step in the biosynthesis of ethylene,showed a non-linear time-course in vitro and activity decayedwith a half-life of around 14 min. This loss of activity wasstudied using tomato ACC oxidase purified from Escherichia coiltransformed with the cDNA clone pTOM13. Inactivation was notdue to end-product inhibition by dehydroascorbic acid or cyanide.Preincubatlon of enzyme in the combined presence of Fe²⁺ ascorbateand ACC, which together allowed catalytic turnover, resultedin almost total loss of ACC oxidase activity. Enzyme Inactivatedby catalysis could not be reactivated by passage through SephadexG-25 or by treating with combina tions of DTT and CO₂ A non-lineartime-course and inactivation in the presence of all substratesand cofactors was also shown for the enzyme assayed in vivowith melon fruit discs. Using the purified tomato enzyme a distinctascorbate-dependent inactivation was also observed, which occurredIn the absence of catalysis and was prevented, although notreversed, by catalase. This ascorbate-dependent inactivationmay thus be due to H₂O₂ attack on ACC oxidase. Key words: 1-aminocyclopropane-1-carboxylate (ACC) oxidase, catalase, catalytic inactivation, ethylene     

1-aminocyclopropane-1-carboxylate oxidase of apple fruit is periplasmic

Immunocytological studies have previously shown that 1-aminocyclopropane-1-carboxylate oxidase (ACO), the enzyme which catalyses the last step of ethylene biosynthesis, is located in the cell wall of apple and tomato fruit cells. In the present study, a combination of cell fractionation and immunocytological methods have been used in order to determine a precise location within this space. Western blotting assays indicated that more than 70% of ACO antigens of the whole cell are recovered in freshly prepared protoplasts and that these ACO antigens are completely removed upon treatment of protoplasts with proteinase K. Immunocytolabelling showed a periplasmic ACO-antigen signal in protoplasts which is completely absent in proteinase K-treated protoplasts. Taken together, these data demonstrate that, in apple fruit, ACO is located at the external face of the plasma membrane. Possible interactions between the plasma membrane and ACO activity are discussed.Key words: ACC oxidase, Malus domestica, apple fruit protoplasts, plasma membrane, immunocytolocalization.      

Subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in tomato cells

The localization of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase was examined in suspension-cultured cells of tomato (Lycopersicon esculentum Mill.), using cell-fractionation techniques, followed by immunoblot analysis with monospecific antibodies raised against a tomato ACC oxidase expressed in Escherichia coli. When assayed in vivo, ACC oxidase had a low activity in untreated tomato cells but was strongly induced when the cells were supplied with its substrate, ACC. Immunoblots showed that this induction was accompanied by the accumulation of a single protein corresponding to ACC oxidase, with an apparent molecular mass (M_r) of 36 kDa. The level of this protein in induced cells, estimated by immunoblotting, was compared with that in protoplasts and vacuoles, and with that in various particulate and soluble fractions obtained by differential centrifugation of cell homogenates. It was found that the ACC oxidase antigen was absent from the vacuole, and that most of it was localized in the cytoplasm of the protoplasts without being associated with membranes. Measurements of ACC oxidase activity in preparations of protoplasts and vacuoles supported these results.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid We thank Martin Regenass (Friedrich Miescher-Institut, Basel, Switzerland) for maintaining the cell cultures and Georg Felix (Friedrich Miescher-Institut, Basel, Switzerland) for helpful discussions. This work was supported, in part, by the Swiss National Science Foundation, Grant 31-26492.89.     

Expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase gene stimulates shoot regeneration in Cucumis melo

The role of ethylene in shoot regeneration was investigated using transgenic Cucumis melo plants expressing an antisense 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene. ACC oxidase catalyses the last step of ethylene biosynthesis. Leaf and cotyledon explants from the transgenic plants exhibited low ACC oxidase activity and ethylene production, whereas the regeneration capacity of the tissues was greatly enhanced (3.5- and 2.8-fold, respectively) compared to untransformed control tissues. Addition of ethylene released by 50 or 100 μm 2-chloroethylphosphonic acid dramatically reduced the shoot regeneration rate of the transgenic tissues. The results clearly demonstrate that ethylene plays an important role in C. melo morphogenesis in vitro. Received: 23 April 1997 / Revision received: 9 June 1997 / Accepted: 2 July 1997     

Cloning and expression of 1-aminocyclopropane-1-carboxylate oxidase cDNA induced by thidiazuron during somatic embryogenesis of alfalfa (Medicago sativa)

Embryogenic callus (EC) induced from petioles of alfalfa (Medicago sativa L. cv. Jinnan) on B5h medium turned green, compact and non-embryogenic when the kinetin (KN) in the medium was replaced partially or completely by thidiazuron (TDZ). The application of CoCl₂, which is an inhibitor of 1-aminocyclopropane-1-carboxylate oxidase (ACO), counteracted the effect of TDZ. Ethylene has been shown to be involved in the modulation of TDZ-induced morphogenesis responses. However, very little is known about the genes involved in ethylene formation during somatic embryogenesis (SE). To investigate whether ethylene mediated by ACO is involved in the effect of TDZ on inhibition of embryogenic competence of the alfalfa callus. In this study we cloned full-length ACO cDNA from the alfalfa callus, named MsACO, and observed changes in this gene expression during callus formation and induction of SE under treatment with TDZ or TDZ plus CoCl₂. RNA blot analysis showed that during the EC subcultural period, the expression level of MsACO in EC was significantly increased on the 2nd day, rose to the highest level on the 8th day and remained at this high level until the 21st day. However, the ACO expression in the TDZ (0.93 μM)-treated callus was higher than in the EC especially on the 8th day. Moreover the ACO expression level increased with increasing TDZ concentration during the subcultural/maintenance period of the callus. It is worth noting that comparing the treatment with TDZ alone, the treatment with 0.93 μM TDZ plus 50 μM CoCl₂ reduced both of the ACO gene expressions and ACO activity in the treated callus. These results indicate that the effect of TDZ could be counteracted by CoCl₂ either on the ACO gene expression level or ACO activity. Thus, a TDZ inhibitory effect on embryogenic competence of alfalfa callus could be mediated by ACO gene expression.     

Li+-regulated 1-aminocyclopropane-1-carboxylate synthase gene expression in Arabidopsis thaliana

In Arabidopsis thaliana, 1-aminocyclopropane-1-carboxylate synthase (ACS) is encoded by a multigene family consisting of at least five members whose expression is induced by hormones, developmental signals, and protein synthesis inhibition. Li⁺, known to interfere with the phosphoinositide (PI) second messenger system by inhibiting the activity of inositol-phosphate phosphatases, is one of the strongest inducers of ACC synthase activity in plants. Treatment of etiolated Arabidopsis seedlings with LiCl results in a rapid induction of the ACS5 gene. Also, LiCl represses the cycloheximide (CHX)-induced accumulation of the ACS2 mRNA. The effects of Li⁺ on the expression of ACS5 and ACS2 are specific, dose-dependent, and can be reversed by Ca²⁺ and mimicked by the protein kinase inhibitor K-252a. The results suggest that the regulation of some ACS genes by various inducers may involve protein kinase activity, which in turn may be controlled through an inositol 1,4,5-triphosphate (IP₃)-mediated Ca²⁺ mobilization. Since plants contain no Li⁺, the cation appears to unmask pre-existing biochemical capacity that may be utilized by various unknown transducers during plant growth and development.