The enzymatic malonylation of 1-aminocyclopropane-1-carboxylic acid in homogenates of mung-bean hypocotyls

Homogenates of hypocotyls of light-grown mung-bean (Vigna radiata (L.) Wilczek) seedlings catalyzed the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) from the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-coenzyme A. Apparent K_m values for ACC and malonyl-CoA were found to be 0.17 mM and 0.25 mM, respectively. Free coenzyme A was an uncompetitive inhibitor with respect to malonyl-CoA (apparent K_i=0.3 mM). Only malonyl-CoA served as an effective acyl donor in the reaction. The d-enantiomers of unpolar amino acids inhibited the malonylation of ACC. Inhibition by d-phenylalanine was competitive with respect to ACC (apparent K_i=1.2 mM). d-Phenylalanine and d-alanine were malonylated by the preparation, and their malonylation was inhibited by ACC. When hypocotyl segments were administered ACC in the presence of certain unpolar d-amino acids, the malonylation of ACC was inhibited while the production of ethylene was enhanced. Thus, a close-relationship appears to exist between the malonylation of ACC and d-amino acids. The cis- as well as the trans-diastereoisomers of 2-methyl- or 2-ethyl-substituted ACC were potent inhibitors of the malonyltransferase. Treatment of hypocotyl segments with indole-3-acetic acid or CdCl₂ greatly increased their content of ACC and MACC, as well as their release of ethylene, but had little, or no, effect on their extractable ACC-malonylating activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Ethylene-promoted malonylation of 1-aminocyclopropane-1-carboxylic acid participates in autoinhibition of ethylene synthesis in grapefruit flavedo discs

The increase in ethylene formation and in 1-aminocyclopropane-1-carboxylic acid (ACC) content in flavedo tissue of grapefruit (Citrus paradisi Macfad. cv. Ruby Red) in response to excision was markedly inhibited by exogenous ethylene. Ethylene treatment inhibited the synthesis of ACC, but increased the tissue's capability to malonylate ACC to N-malonyl-ACC, resulting in further reduction in the endogenous ACC content. The development of extractable ACC-malonyl-transferase activity in the tissue was markedly promoted by treatment with exogenous ethylene. These results indicate that the autoinhibition of ethylene production in this tissue results not only from suppression of ACC synthesis, but also from promotion of ACC malonylation; both processes reduce the availability of ACC for ethylene synthesis.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethyoxyvinylglycine (2-amino-4-(2-aminoexthoxy)-trans-3-butenoic acid) - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid     

Evidence that brassinosteroid stimulates auxin-induced ethylene synthesis in mung bean hypocotyls between S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid

Brassinosteroid (BR) stimulation of auxin-induced ethylene production and the particular step at which BR acts to promote such synthesis were studied in mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Increasing concentrations of methionine alone and in combination with 3 μ M BR and 10 μ M IAA had a minimal effect on ethylene production. With increasing concentrations of 1-aminocyclopro-pane-1-carboxylic acid (ACC), however, ethylene production increased. BR or IAA further enhanced ethylene production with maximum rates occurring when these compounds were added together with ACC. The addition of 10 μ M CoCl₂ in conjunction with BR and/or IAA resulted in 85–97% inhibition of ethylene production. When 20 μ M cycloheximide was used in conjunction with BR and/or IAA there was a complete inhibition of ethylene production. Total inhibition also resulted when 1.0 μ M aminoethoxy-vinylglycine (AVG) was used in combination with BR and/or IAA. AVG alone had no effect on ACC conversion to ethylene.     

Immunopurification and characterization of a 40-kD 1-aminocyclopropane-1-carboxylic acid N-malonyltransferase from mung bean seedling hypocotyls.

1-Aminocyclopropane-1-carboxylic acid (ACC) N-malonyltransferase catalyzes the transfer of the malonyl group from malonyl coenzyme A to ACC to form malonyl ACC. Using partially purified ACC N-malonyltransferase from the hypocotyls of mung bean (Vigna radiata) seedlings, we produced two mouse monoclonal antibodies (1H5 and 2G3) to this enzyme. These antibodies bind to sites other than the active site of the enzyme because monoclonal antibody-bound ACC N-malonyltransferase still exhibits full catalytic activity. A monoclonal antibody column was constructed using 1H5 and protein G Sepharose. The ACC N-malonyltransferase purified from this monoclonal antibody column has a molecular mass of 40 kD, which is different from that reported previously. The enzyme has a higher electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of the reducing agent dithiothreitol. The optimum temperature of this 40-kD ACC N-malonyltransferase is 45 degrees C and the apparent Kms for ACC and malonyl coenzyme A are 66.7 and 40 microns, respectively.     

Relationship between stimulatory effect of methyl jasmonate on ethylene production and 1-aminocyclopropane-1-carboxylic acid content in tomatoes

The effect of methyl jasmonate (JA-Me) applied in concentration 1.0 % in lanolin paste to detached tomato fruits at the mature green, advanced mature green and light red stages on the ethylene production and l-aminocyclopropane-l-carboxylic acid (ACC) content was investigated at different times after treatment. JA-Me stimulated ethylene production in all stages of ripening, but the level of ACC increased or decreased in comparison with control depending on the stage of ripening. Higher level of ACC in JA-Me treated tissue was found in mature green stage and fully ripened tomatoes-treated at advanced green stage; lower one in light red stage — treated at advanced green stage and fully ripened stage - treated at light red stage.     

The catalytic mechanism of 1-aminocyclopropane-1-carboxylic acid oxidase

It has been proposed that 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase catalyzes the oxidation of ACC to ethylene via N-hydroxyl-ACC as an intermediate. However, due to its chemical instability the putative intermediate has never been isolated. Here, we have shown that a purified recombinant ACC oxidase can utilize alpha-aminoisobutyric acid (AIB), an analog of ACC, as an alternative substrate, converting AIB into CO2, acetone, and ammonia. We chemically synthesized the putative intermediate compound, N-hydroxyl-AIB (HAIB), and tested whether it serves as an intermediate in the oxidation of AIB. When [1-(14)C]AIB was incubated with ACC oxidase in the presence of excess unlabeled HAIB as a trap, no labeled HAIB was detected. By comparing the acetone production rates employing HAIB and AIB as substrates, the conversion of HAIB to acetone was found to be much slower than that of using AIB as substrate. Based on these observations, we conclude that ACC oxidase does not catalyze via the N-hydroxylation of its amino acid substrate. ACC oxidase also catalyzes the oxidation of other amino acids, with preference for the D-enantiomers, indicating a stereoselectivity of the enzyme.     

Metabolism of 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

In preclimacteric apple fruits ( Malus × domestica Borkh. cv. Golden Delicious) ethylene production is controlled by the rates of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis, and by its metabolism to ethylene by the ethylene-forming enzyme and to 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by malonyl CoA-ACC transferase. The onset of the climacteric in ethylene production is associated with an increase in the activity of the ethylene-forming enzyme in the pulp and with a rise in the activity of ACC synthase. Malonyl transferase activity is very high in the skin of immature fruit, decreases sharply before the onset of the climacteric, and remains nearly constant thereafter. More than 40% of the ACC synthesized in the skin and around 5% in the flesh, are diverted to MACC at early climacteric. At the climacteric peak there are substantial gradients in ethylene production between different portions of the tissue, the inner cortical tissues producing up to twice as much as the external tissues. This increased production is associated with, and apparently due to, increased content of ACC synthase. Less than 1% of the synthesized ACC is diverted to MACC in the flesh of climacteric apples. In contrast, the skin contains high activity of malonyl transferase, and correspondingly high levels [1000 nmol (g dry weight)⁻¹] of MACC.     

Formation and transport of 1-aminocyclopropane-1-carboxylic acid in pea plants

The formation and transport of free 1-aminocyclopropane-1-carboxylic acid (ACC) and conjugated ACC [1-(malonylamino)cyclopropane-1-carboxylic acid; M-ACC] was studied in pea plants. Excision and dark incubation induced ACC and M-ACC synthesis in stem segments, including the second node. At similar rates as in segments, ACC and M-ACC were formed near the cut surface in stems after decapitation, leading to a transient increase in both compounds in the node adjacent to the cut. The maximum level of M-ACC at 6 hr exceeded that of ACC at 3 hr. Seven days after decapitation, total M-ACC in the shoot returned to the level in the control plants. Over the same period of time, M-ACC accumulated in the roots in amounts comparable to those previously observed in the shoot. It is concluded that M-ACC formed near the cut is transported basipetally, and that the roots act as a sink. Both the increase in ACC and M-ACC in the node after decapitation and the degree to which growth of lateral shoots was inhibited by ACC applied to the cut end increased with advancing age of the plant.     

A simple and sensitive assay for 1-aminocyclopropane-1-carboxylic acid

A simple, rapid, and sensitive method for the quantitative determination of 1-amino-cyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plant tissues, is described. The assay is based on the liberation of ethylene from ACC with NaOCl in the presence of Hg²⁺; ethylene is assayed by gas chromatography. The yield is normally 80% and can be determined by internal standards. The method is quite specific and can detect as little as 5 pmol of ACC.     

Subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase in apple fruit

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase catalyzes the oxidation of ACC to the gaseous plant hormone, ethylene. Although the enzyme does not contain a typical N-terminal consensus sequence for the transportation across the endoplasmic reticulum (ER), it has recently been shown to locate extracellularly by immunolocalization study. It was of interest to examine whether the enzyme contains a signal peptide that is overlooked by structure prediction. We observed that the in vitro translated apple ACC oxidase was not co-processed or imported by the canine pancreatic rough microsomes, a system widely used to identify signal peptide for protein translocation across ER, suggesting that apple ACC oxidase does not contain a signal peptide for ER transport. A highly specific polyclonal antibody raised against the recombinant apple ACC oxidase was used to examine the subcellular localization of the enzyme in apple fruit (Malus domestica, var. Golden Delicious). The location of ACC oxidase appeared to be mainly in the cytosol of the apple fruit pericarp tissue as was demonstrated by electron microscopy using immunogold-labeled antibodies. The pre-immune serum or pre-climacteric fruit control gave essentially no positive signal. Based on these observations, we conclude that ACC oxidase is a cytosolic protein.     

Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M(r) 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M(r) 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s-1. The presence of 7 mM Cu2+ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.     

Role of IAA-oxidase in the formation of ethylene from 1-aminocyclopropane-1-carboxylic acid

The IAA-oxidase system of olive tree (Olea europea) in the presence of its substrate, IAA, and cofactors, DCP and Mn², forms ethylene from 1-aminocyclopropane-l-carboxylic acid (ACC) bound as a Schiffs base to pyridoxal phosphate. Similarly, olive leaf discs upon incubation with ACC liberate considerable amounts of ethylene. The results suggest that this IAA-oxidase system may be the one active in the last step in the biosynthesis of ethylene from methionine.     

Antifungal antibiotics and Siba inhibit 1-aminocyclopropane-1-carboxylic acid synthase activity

The antifungal antibiotics Sinefungin and A9145C isolated from Streptomyces griseolus and the synthetic nucleoside Siba, which are analogs of S-adenosylmethionine, inhibit the activity of 1-aminocyclopropane 1-carboxylic acid synthase from tomato fruits. Sinefungin and Siba were shown to be more potent inhibitors than A9145C. In extracts of green fruits, the enzyme activity was inhibited by Sinefungin with an I50 of 1 microM, which was similar to that caused by aminoethoxyvinylglycine, and by Siba with an I50 of 100 microM; in extracts from red tomatoes, the I50's were 25 microM and 100 microM, respectively. The inhibition of ACC synthase by these analogs could be reversed by gel filtration chromatography.     

Effect of 1-aminocyclopropane-1-carboxylic acid on maize kernel development in vitro

Pollination stimulates ethylene production in maize ears, and the application of ethephon during the pollination period can cause kernel abortion. The objective of this study was to determine if kernel abortion could be induced in vitro by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Adding ACC to the culture medium resulted in the evolution of ethylene which caused abortion and reduced mature kernel mass. The effect of ethylene on kernel abortion and dry matter accumulation was partially negated by the addition of the ethylene-binding site inhibitor, 2,5-norbornadiene (NBD). The effect of ethylene on kernel abortion was greatest during the early stage of kernel development and was intensified by an increase in media sucrose concentration. These data suggest that ethylene could regulate kernel abortion in maize.Contribution of the Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108. Paper No. 17,088, Scientific Journal Series, Minnesota Agriculture Experimental Station.     

Inhibition of apple 1-aminocyclopropane-1-carboxylic acid oxidase, by cyclopropane-1,1-dicarboxylic acid and trans-2-phenylcyclopropane-1-carboxylic acid

Cyclopropane-1,1-dicarboxylic acid (CDA) and trans-2-phenylcyclopropane-1-carboxylic acid (PCCA) are the main representatives of a group of compounds that are structural analogues of 1-aminocyclopropane-1-carboxylic acid (ACC) and have been proved to have an inhibitory effect on the wound ethylene produced by Lycopersicum esculentum fruit discs. During the experiments, that were carried out in this work the inhibition pattern of PCCA and CDA were studied when tested on partially purified apple ACO and their Ki values were determined. A mechanistic proposal was given, in order to explain the kinetic behaviour of the inhibitors. The common feature of these molecules is their cyclopropane ring, with different substitutes mainly at the positions C1 and C2. Two other compounds with similar structure where also tested as inhibitors, in order to clarify the relationship between structure and activity. These compounds are: 2-methyl cyclopropanecarboxylic acid (MCA), and cyclopropanecarboxylic acid (CCA).     

Tissue localization of a submergence-induced 1-aminocyclopropane-1-carboxylic acid synthase in rice

At least two 1-aminocyclopropane-1-carboxylic acid synthase genes (ACS) are implicated in the submergence response of rice (Oryza sativa). Previously, the OS-ACS5 gene has been shown to be induced during short- as well as long-term complete submergence of seedlings and to be controlled by a balance of gibberellin and abscisic acid in both lowland and deepwater rice. This study demonstrates that OS-ACS5 mRNA is localized in specific tissues and cells both during normal development and in response to complete submergence. The temporal and spatial regulation of OS-ACS5 expression is presented by in situ hybridization and histochemical analysis of beta-glucuronidase (GUS) activity in transgenic rice carrying an OS-ACS5-gus fusion. Whole-mount in situ hybridization revealed that in air-grown rice seedlings, OS-ACS5 was expressed at a low level in the shoot apex, meristems, leaf, and adventitious root primordia, and in vascular tissues of nonelongated stems and leaf sheaths. In response to complete submergence, the expression in vascular bundles of young stems and leaf sheaths was strongly induced. The results of histochemical GUS assays were consistent with those found by whole-mount in situ hybridization. Our findings suggest that OS-ACS5 plays a role in vegetative growth of rice under normal conditions and is also recruited for enhanced growth upon complete submergence. The possible implication of OS-ACS5 in root-shoot communication during submergence stress and its putative role in aerenchyma formation upon low-oxygen stress are discussed.     

The site of 1-aminocyclopropane-1-carboxylic acid synthesis in senescing carnation petals

To study the cause of the uneven production of ethylene by upper and basal portions of detached petals of carnation ( Dianthus caryophyllus L. cv. White Sim), the petals were divided and exposed to ethylene (30 μl 1^-1 for 16 h). The treatment induced rapid wilting and autocatalytic ethylene production in the basal portion similar to that induced in entire petals. In contrast to the response in entire petals and the basal portions, the upper portions responded to ethylene by delayed wilting and much lower ethylene production. Aminocyclopropane carboxylic acid (ACC)-synthase activity in the basal portion of the petals was 38 to 400 times that in the upper portion. In untreated detached petal pieces from senescing carnation flowers, ethylene production by the upper portion declined after 6 h while the basal portion was still producing ethylene at a steady rate 18 h later. Application of ACC to the upper portion of senescing petals increased their ethylene production. α-Aminooxyacetic acid (0.5 m M ), reduced the ethylene production of the senescing basal portion more than that of the upper portion. Endogenous ACC content in basal portions of senescing carnation petals was 3 to 4 times higher than in the upper parts. When detached senescing petals were divided immediately after detaching, the endogenous ACC levels in upper portions remained steady or declined during 24 h after division, while in the basal portions the ACC level rose steadily as in the intact petals. There was no change in the conjugated ACC in either portion after 24 h. Benzyladenine (BA) applied as a pretreatment to entire preclimacteric petals greatly reduced the development of ACC-synthase activity of the basal portion, but had little effect on the activity in the upper portion of the petal. In both portions, however, BA effectively reduced the conversion of ACC to ethylene.     

Effect of 1-aminocyclopropane-1-carboxylic acid on maize kernel development in vitro

Pollination stimulates ethylene production in maize ears, and the application of ethephon during the pollination period can cause kernel abortion. The objective of this study was to determine if kernel abortion could be induced in vitro by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Adding ACC to the culture medium resulted in the evolution of ethylene which caused abortion and reduced mature kernel mass. The effect of ethylene on kernel abortion and dry matter accumulation was partially negated by the addition of the ethylene-binding site inhibitor, 2,5-norbornadiene (NBD). The effect of ethylene on kernel abortion was greatest during the early stage of kernel development and was intensified by an increase in media sucrose concentration. These data suggest that ethylene could regulate kernel abortion in maize.     

Transport of 1-aminocyclopropane-1-carboxylic acid into isolated maize mesophyll vacuoles

Intracellular transport of the ethylene precursor, I-aminocyclopropane-1-carboxylic acid (ACC) can change the ACC concentration in cell compartments and impact ethylene biosynthesis. Transport of ACC into isolated maize ( Zea mays L.) mesophyll vacuoles was studied by silicon layer flotation filtering. The transport of ACC across the tonoplast was stimulated 2. 4- to 8. 1-fold by 5 m M MgATP, showed saturation kinetics with an apparent K_m for ACC of 20 μ M , and was optimal at 25°C. Transport of ACC was sensitive to the pH of the medium, falling as external pH rose. Effectors known to inhibit proton-translocating ATPases (N, N-dicyclohexylcarbodiimide) and to collapse the electrical (thiocyanate, valinomycin) and chemical (carbonylcyanide m -chlorophenylhydrazone, gramicidin) potential gradients for protons across the tonoplast all reduced ACC transport. The nonhydrolyzable MgATP analog. Mg adenylyl-imidodiphosphate, stimulated ACC transport as effectively as MgATP. Other nucleotides (MgADP, MgCTP, MgUTP, MgGTP) and MgPP_i had little or no effect. These results suggest that ACC uptake into isolated maize mesophyll vacuoles is carrier mediated, is dependent upon an electrochemical potential gradient for protons and is specifically regulated, but not necessarily energized, by MgATP