Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

A comparison of 1-aminocyclopropane-1-carboxylate synthase in vitro translation product and in-vivo-labeled protein in ripening tomatoes

We determined the time course of increases in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in ripening tomato (Lycopersicon esculentum (L.) Mill.) pericarp discs following wounding and treatment with 75 mM LiCl. Over the course of 24 h, we detected oscillations in the amount of enzyme activity from an initial peak at 6 h to a subsequent, even higher level at 18 h. In-vitro translation products derived from poly(A)⁺ RNAs isolated at various times of treatment and in-vivo-labeled proteins were immunoprecipitated using antibodies specific for ACC synthase. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that wounding and treatment with LiCl induced an accumulation of translatable ACC-synthase-specific mRNAs. In addition, single, prominent bands were apparent for both in-vivo and in-vitro samples but their molecular masses differed. It appears that the in-vitro translation product is a polypeptide of 56 kDa while the in-vivo-labeled enzyme has a molecular mass of 47 kDa. The authors greatly appreciate the skilled technical assistance of Renate deZacks and Gail Robinson. This research was supported by the National Science Foundation through Grant No. DCB-8718873 and by the Department of Energy through Contract No. DE-AC02-76ER-01338.     

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.     

S-methylmethionine is both a substrate and an inactivator of 1-aminocyclopropane-1-carboxylate synthase

S-methyl-l-methionine (SMM) is ubiquitous in the tissues of flowering plants, but its precise function remains unknown. It is both a substrate and an inhibitor of the pyridoxal 5^′-phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase, due to its structural similarity to the natural substrate of this enzyme, S-adenosyl-l-methionine. In the reaction with ACC synthase, SMM can either be transaminated to yield 4-dimethylsulfonium-2-oxobutyrate; converted to α-ketobutyrate, ammonia, and dimethylsulfide; or inactivate the enzyme covalently after elimination of dimethylsulfide. These results suggest a previously unrecognized role for SMM in the regulation of ACC synthase activity in plants.     

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.     

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)⁺ RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3-end was intact, it lacked a portion of sequence at the 5-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - HPLC high-pressure liquid chromatography - kDa kilodalton - kb kilobase - mAb monoclonal antibody - Met methionine - PCR polymerase chain reaction - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.     

Identification and characterization of three putative genes for 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments

The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean.     

Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp

This is the first report documenting the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium. This enzyme, previously found in free-living bacteria, yeast and fungi, degrades ACC, the immediate precursor of ethylene in higher plants. Thirteen different rhizobial strains were examined by Southern hybridization, Western blots and ACC deaminase enzyme assay. Five of them tested positive for ACC deaminase. Induction of the expression of ACC deaminase was examined in one of the positively tested strains, Rhizobium leguminosarum bv. viciae 128C53K. This rhizobial ACC deaminase had a trace basal level of expression without ACC, but could be induced by a concentration of ACC as low as 1 μM. The more ACC added to this Rhizobium the higher the expression level of the ACC deaminase. This revised version was published online in June 2006 with corrections to the Cover Date.     

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     

Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid

A simple and sensitive chemical assay was developed for 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. The assay is based on the liberation of ethylene from ACC at pH 11.5 in the presence of pyridoxal phosphate, MnCl₂ and H₂O₂. This assay was used to detect ACC in extracts of tomato fruits (Lycopersicon esculentum Mill.) and to measure the activity of a soluble enzyme from tomato fruit that converted S-adenosylmethionine (SAM) to ACC. The enzyme had a K_m of 13 M for SAM, and conversion of SAM to ACC was competitively and reversibly inhibited by aminoethoxyvinylglycine (AVG), an analog of rhizobitoxine. The K_i value for AVG was 0.2 M. The level of the ACC-forming enzyme activity was positively correlated with the content of ACC and the rate of ethylene formation in wild-type tomatoes of different developmental stages. Mature fruits of the rin (non-ripening) mutant of tomato, which only produce low levels of ethylene, contained much lower levels of ACC and of the ACC-forming enzyme activity than wild-type tomato fruits of comparable age.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid - SAM S-adenosyl-L-methionine Michigan Agricultural Experiment Station No. 8876     

An auxin-responsive 1-aminocyclopropane-1-carboxylate synthase is responsible for differential ethylene production in gravistimulated<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L. flower stems

The regulation of gravistimulation-induced ethylene production and its role in gravitropic bending was studied in Antirrhinum majus L. cut flower stems. Gravistimulation increased ethylene production in both lower and upper halves of the stems with much higher levels observed in the lower half. Expression patterns of three different 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) genes, an ACC oxidase (ACO) and an ethylene receptor (ETR/ERS homolog) gene were studied in the bending zone of gravistimulated stems and in excised stem sections following treatment with different chemicals. One of the ACS genes (Am-ACS3) was abundantly expressed in the bending zone cortex at the lower side of the stems within 2 h of gravistimulation. Am-ACS3 was not expressed in vertical stems or in other parts of (gravistimulated) stems, leaves or flowers. Am-ACS3 was strongly induced by indole-3-acetic acid (IAA) but not responsive to ethylene. The Am-ACS3 expression pattern strongly suggests that Am-ACS3 is responsible for the observed differential ethylene production in gravistimulated stems; its responsiveness to IAA suggests that Am-ACS3 expression reflects changes in auxin signalling. Am-ACS1 also showed increased expression in gravistimulated and IAA-treated stems although to a much lesser extent than Am-ACS3. In contrast to Am-ACS3, Am-ACS1 was also expressed in non-bending regions of vertical and gravistimulated stems and in leaves, and Am-ACS1 expression was not confined to the lower side cortex but evenly distributed over the diameter of the stem. Am-ACO and Am-ETR/ERS expression was increased in both the lower and upper halves of gravistimulated stems. Expression of both Am-ACO and Am-ETR/ERS was responsive to ethylene, suggesting regulation by IAA-dependent differential ethylene production. Am-ACO expression and in vivo ACO activity, in addition, were induced by IAA, independent of the IAA-induced ethylene. IAA-induced growth of vertical stem sections and bending of gravistimulated flowering stems were little affected by ethylene or 1-methylcyclopropene treatments, indicating that the differential ethylene production plays no pivotal role in the kinetics of gravitropic bending.     

Iron: an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

The activity of the ethylene-forming enzyme (EFE) in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells was almost completely abolished within 10 min by 0.4 mM of the metal-chelating agent 1,10-phenanthroline. Subsequent addition of 0.4 mM FeSO₄ immediately reversed this inhibition. A partial reversion was also obtained with 0.6 mM CuSO₄ and ZnSO₄, probably as a consequence of the release of iron ions from the 1,10-phenanthroline complex. The inhibition was not reversed by Mn²⁺ or Mg²⁺. Tomato cells starved of iron exhibited a very low EFE activity. Addition of Fe²⁺ to these cells caused a rapid recovery of EFE while Cu²⁺, Zn²⁺ and other bivalent cations were ineffective. The recovery of EFE activity in iron-starved cells was insensitive to cycloheximide and therefore does not appear to require synthesis of new protein. The EFE activity in tomato cells was induced by an elicitor derived from yeast extract. Throughout the course of induction, EFE activity was blocked within 10–20 min by 1,10-phenanthroline, and the induced level was equally rapidly restored after addition of iron. We conclude that iron is an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in vivo.