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.     

Daminozide inhibits ethylene production in apple fruit by blocking the conversion of methionine to aminocyclopropane-1-carboxylic acid (ACC)

At harvest, fruit from apple trees sprayed with daminozide (+daminozide) had lower levels of aminocyclopropane-1-carboxylic acid (ACC) and produced significantly lower amounts of ethylene than untreated (–daminozide) fruit. Flesh discs from the fruit of +daminozide and –daminozide trees were fed precursors of ethylene to determine how daminozide inhibits ethylene production. ACC was metabolized to ethylene regardless of treatment. Methionine (MET), however, was only converted to ethylene by –daminozide fruit, and only after the fruit had been maintained at 4 °C for 5 months. +Daminozide fruit failed to convert MET to ethylene at harvest, as well as after cold storage. When daminozide was added to the incubation media of flesh discs it did not inhibit ethylene production or the conversion of ACC to ethylene. The addition of daminozide did, however, inhibit the metabolism of exogenous MET to ethylene. Aminooxyacetate acid (AOA) blocked both the endogenous production of ethylene and that from MET feeds. Daminozide inhibits ethylene production by preventing the conversion of MET to ACC, but it does not appear to act as a simple competitive inhibitor of ACC synthase activity.Abbreviations ACC aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - AOA aminooxyacetic acid - CH cycloheximide - MET methionine - PUT putrescine Author for correspondence     

Effects of 1-aminocyclopropane-1-carboxylic acid and oxygen concentrations on in vivo and in vitro activity of ACC oxidase of sunflower hypocotyl segments

In vivo ethylene production by hypocotyl segments of sunflower seedlings and in vitro activity of 1-aminocyclopropane-1-carboxylic acid oxidase (formerly ethylene-forming enzyme) extacted from the same tissues increase with increasing concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and oxygen. ACC oxidase activity follows Michaelis-Menten kinetics. The apparent Km values of the enzyme towards ACC, estimated in vivo and in vitro, are respectively 219 M and 20.6 M. Both Km values towards O₂ are similar, ca 10.6–11.4%. A decrease in concentration in one of the substrates (ACC or O₂) results in an increase in in vivo apparent Km of ACC oxidase for the other substrate. On the contrary, Km values of the enzyme towards ACC or O₂ estimated in vitro are not dependent upon the concentration of the other substrate (ACC or O₂).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - MACC malonylate 1-aminocyclopropane-1-carboxylic acid - SD standard deviation     

Identification and active site analysis of the 1-aminocyclopropane-1-carboxylic acid oxidase catalysing the synthesis of ethylene in Agaricus bisporus

Background

1-Aminocyclopropane-1-carboxylate oxidase (ACO) is a key enzyme that catalyses the final step in the biosynthesis of the plant hormone ethylene. Recently, the first ACO homologue gene was isolated in Agaricus bisporus, whereas information concerning the nature of the ethylene-forming activity of this mushroom ACO is currently lacking.

Methods

Recombinant ACO from A. bisporus (Ab-ACO) was purified and characterised for the first time. Molecular modelling combined with site-directed mutagenesis and kinetic and spectral analysis were used to investigate the property of Ab-ACO.

Results

Ab-ACO has eight amino acid residues that are conserved in the Fe (II) ascorbate family of dioxygenases, including four catalytic residues in the active site, but Ab-ACO lacks a key residue, S289. In comparison to plant ACOs, Ab-ACO requires ACC and Fe (II) but does not require ascorbate. In addition, Ab-ACO had relatively low activity and was completely dependent on bicarbonate, which could be ascribed to the replacement of S289 by G289. Moreover, the ferrous ion could induce a change in the tertiary, but not the secondary, structure of Ab-ACO.

Conclusions

These results provide crucial experimental support for the ability of Ab-ACO to catalyse ethylene formation in a similar manner to that of plant ACOs, but there are differences between the biochemical and catalytic characteristics of Ab-ACO and plant ACOs.

General significance

This work enhances the understanding of the ethylene biosynthesis pathways in fungi and could promote profound physiological research of the role of ethylene in the regulation of mushroom growth and development.     

Effects of ACC (1-aminocyclopropane-1-carboxylic acid) applied through the roots of maize seedlings on vegetative and early reproductive development of the shoots

Maize plants, grown in aerated solution cultures, were exposed, at different growth stages, to ACC (1-aminocyclopropane-1-carboxylic acid) applied through the roots for up to 9 d. Total uptake of ACC increased with seedling size. During ACC treatment, ethylene evolution, by the shoots, proceeded at an almost constant rate per unit fresh weight that was up to 40-fold faster than that of untreated plants. This stimulation extended several days beyond the period of ACC uptake. The effects on growth and development were assessed when plants were 50–52-d old. ACC application shortened certain stem internodes, leaf-sheaths and laminae. The location of these effects depended on the time of application. The greatest shortening was induced by application, at the 4-leaf stage (10 d-old), prior to elongation of the cone of the shoot apex. This is ascribed to effects on meristematic tissue, in addition to those on elongating cells. An unexpected response to ACC treatment, at the 4-leaf stage, was an increase of up to four leaf-bearing stem nodes compared to untreated plants. This resulted in a parallel elevation of the uppermost ear-bearing axillary shoot to higher nodal positions. The length of leaves high in the canopy (nodes 11–16) was promoted by treating seedlings with ACC. The only clear effect of the ACC treatments on emergent axillary shoots per se was a retardation of silk elongation.     

Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4

A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.     

Purification,properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits

The enzyme which converts 1-aminocyclo-propane-1-carboxylic acid (ACC) into ethylene, ACC oxidase, has been isolated from apple fruits (Malus x domestica Borkh. cv. Golden Delicious), and for the first time stabilized in vitro by 1,10-phenanthroline and purified 170-fold to homogeneity in a five-step procedure. The sodium dodecyl sulfate-denatured and native proteins have similar molecular weights (approx. 40 kDa) indicating that the enzyme is active in its monomeric form. Antibodies raised against a recombinant ACC oxidase over-produced in Escherichia coli from a tomato cDNA recognise the apple-fruit enzyme with high specificity in both crude extracts and purified form. Glycosylation appears to be absent because of (i) the lack of reactivity towards a mixture of seven different biotinylated lectins and (ii) the absence of N-linked substitution at a potential glycosylation site, in a sequenced peptide. Phenylhydrazine and 2-methyl-1-2-dipyridyl propane do not inhibit activity, indicating that ACC oxidase is not a prosthetic-heme iron protein. The partial amino-acid sequence of the native protein has strong homology to the predicted protein of a tomato fruit cDNA demonstrated to encode ACC oxidase.     

变叶海棠及其伴生植物峨眉小檗的水分利用策略

变叶海棠(Malus toringoides)是分布在我国四川省甘孜州炉霍县亚高山地区的野生植物种,其嫩叶可制茶,为一种纯正的天然保健珍品。它根系发达、抗逆性强,也是川西亚高山干旱地区退化植被生态恢复的良好树种。运用氢稳定同位素示踪技术,比较分析了变叶海棠和伴生植物峨眉小檗(Berberis aemulans)茎水与其潜在水源(降水、土壤水和河水)的δD值,结果表明:变叶海棠与峨眉小檗植物水主要来源于降水和深层土壤水。生长在河边的变叶海棠并不利用河水。在干季,降水10-20 mm后,变叶海棠对降水的利用率为33.50%-70.06%,而峨眉小檗为26.17%-45.17%;在雨季,降水10-25 mm后,变叶海棠对降水的利用率为40.64%-69.01%,而峨眉小檗为28.44%-71.41%;无论干季还是雨季,两种植物在雨后对降水利用的格局相似,但变叶海棠对降水的利用率皆显著高于峨眉小檗(P<0.01)。两种植物水分利用策略与其根系分布相一致。为川西甘孜州亚高山干旱地区退化植被生态恢复的树种选择以及变叶海棠的扩繁与利用提供科学理论依据。     

Kinetic and mutagenic evidence for the role of histidine residues in the Lycopersicon esculentum 1-aminocyclopropane-1-carboxylic acid oxidase

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M^–1 min^–1. The pH–inactivation rate data imply the involvement of an amino acid residue with a pK value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.     

Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses

Hyaloscyphaceae is the largest family in Helotiales, Leotiomycetes. It is mainly characterized by minute apothecia with well-differentiated hairs, but its taxonomic delimitation and infrafamilial classification remain ambiguous. This study performed molecular phylogenetic analyses using multiple genes including the ITS-5.8S rDNA, the D1–D2 region of large subunit of rDNA, RNA polymerase II subunit 2, and the mitochondrial small subunit. The primary objective was to evaluate the phylogenetic utility of morphological characters traditionally used in the taxonomy of Hyaloscyphaceae through reassessment of the monophyly of this family and its genera. The phylogenetic analyses inferred Hyaloscyphaceae as being a heterogeneous assemblage of a diverse group of fungi and not supported as monophyletic. Among the three tribes of Hyaloscyphaceae only Lachneae formed a monophyletic lineage. The presence of hairs is rejected as a synapomorphy, since morphologically diversified hairs have originated independently during the evolution of Helotiales. The true- and false-subiculum in Arachnopezizeae are hypothesized to have evolved through different evolutionary processes; the true-subiculum is likely the product of a single evolutionary origin, while the false-subiculum is hypothesized to have originated multiple times. Since Hyaloscyphaceae sensu lato was not resolved as monophyletic, Hyaloscyphaceae sensu stricto is redefined and only applied to the genus Hyaloscypha.     

A simplified method for determining 1-aminocyclopropane-1-carboxylic acid (ACC) in plant tissues using a mass selective detector

A sensitive and specific method is described for the routine assay of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in 100–200 mg fresh weight samples of green or etiolated tissue. The method involves high performance liquid chromatography (HPLC) and gas chromatography linked to mass spectrometry (GCMS) and uses ¹⁴C-labelled ACC as an internal standard, N-benzoyl n-propyl ACC as an easily prepared derivative for HPLC and GCMS, and N-benzoyl isobutyl ACC as an internal standard for GCMS. The procedure is faster and safer than an existing GCMS method and more specific and reliable than indirect assays widely in use. The method has been used to measure ACC in maize roots, young leaves of cucumber, and aerobic or anaerobic seedlings of rice.     

ACC deaminase-containing Arthrobacter protophormiae induces NaCl stress tolerance through reduced ACC oxidase activity and ethylene production resulting in improved nodulation and mycorrhization in Pisum sativum

Induction of stress ethylene production in the plant system is one of the consequences of salt stress which apart from being toxic to the plant also inhibits mycorrhizal colonization and rhizobial nodulation by oxidative damage. Tolerance to salinity in pea plants was assessed by reducing stress ethylene levels through ACC deaminase-containing rhizobacteria Arthrobacter protophormiae (SA3) and promoting plant growth through improved colonization of beneficial microbes like Rhizobium leguminosarum (R) and Glomus mosseae (G). The experiment comprised of treatments with combinations of SA3, G, and R under varying levels of salinity. The drop in plant biomass associated with salinity stress was significantly lesser in SA3 treated plants compared to non-treated plants. The triple interaction of SA3 + G + R performed synergistically to induce protective mechanism against salt stress and showed a new perspective of plant-microorganism interaction. This tripartite collaboration increased plant weight by 53%, reduced proline content, lipid peroxidation and increased pigment content under 200 mM salt condition. We detected that decreased ACC oxidase (ACO) activity induced by SA3 and reduced ACC synthase (ACS) activity in AMF (an observation not reported earlier as per our knowledge) inoculated plants simultaneously reduced the ACC content by 60% (responsible for generation of stress ethylene) in SA3 + G + R treated plants as compared to uninoculated control plants under 200 mM salt treatment. The results indicated that ACC deaminase-containing SA3 brought a putative protection mechanism (decrease in ACC content) under salt stress, apart from alleviating ethylene-induced damage, by enhancing nodulation and AMF colonization in the plants resulting in improved nutrient uptake and plant growth.     

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.     

Ethylene biosynthesis: The role of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, and its possible evolutionary origin

Recent developments in our knowledge of the biochemistry of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, the enzyme responsible for the final stage in the biosynthesis of ethylene, are reviewed. Particular reference is made to the role of carbon dioxide as an essential cofactor, the activity of ACC oxidase in the plant cell, the enzyme catalytic centre, and the role of ACC oxidase in the evolutionary development of ethylene biosynthesis in plants. Evidence is marshalled to support a proposal that the membrane requirement for ACC oxidase that is observed in vivo is attributable to a need for a charged plasma membrane to maintain ascorbate in the reduced state for an ACC oxidase located in the apoplast. It is argued on biochemical grounds that the acquisition of the ACC oxidase was the crucial evolutionary step in the development by seed plants of an ethylene biosynthesis pathway that could easily be regulated, and that signalled the plant's response to stress and pathogen attack.     

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai)

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0∼300 μl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (≧10 μ1/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 μl/kg f.w./h–10 μl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear. Received: 1 July 1998 / Accepted: 6 October 1998