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     

Biosynthesis of ethylene: the effect of phosphate*

Abstract Biosynthesis of ethylene in tomato and avocado fruit slices, carrot root, pea seedling and tomato shoot segments, Penicillium expansum and Escherichia coli was found to be inhibited by inorganic phosphate. Compared with microbial systems, relatively high concentrations of phosphate in the incubating medium were necessary to bring about a significant inhibition of ethylene production in higher plants. The degree of inhibition in higher plants correlated with the increased internal cellular concentration of phosphate and not with that of the incubating medium. Phosphate concentrations inhibitory for ethylene biosynthesis did not affect the respiration of tomato fruit slices. The phosphate effect was reversible, confined to only the biological systems and was not due to a change in the ionic strength. The differential inhibitory effects of aminoethoxyvinylglycine on ethylene biosynthesis in tomato fruit slices of various stages of ripening, were markedly influenced by high phosphate concentrations. The data indicate a biological significance to the phosphate control of ethylene biosynthesis.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

No difference in the regulation pattern of calcium on ethylene biosynthesis between wild-type and never-ripe tomato fruit at mature green stage

This work investigated how calcium regulates the ethylene biosynthesis in the fruits of wild-type tomato (Lycopersicon esculentum L.) and their ethylene receptor never-ripe (Nr) mutants. In Nr tomato, the ethylene perception was blocked. When both materials were treated with calcium, the content of 1-aminocyclopropane-1-carboxylic acid (ACC)/malonyl-ACC and the activity of ACC oxidase (ACO) in tomato fruit discs increased, whereas the production of ethylene, content of malondialdehyde, and membrane permeability decreased. Calcium treatment did not affect the activity of ACC synthase, which is the first committed step in the ethylene biosynthesis pathway. The expression of LeACO1 in mature green fruit was inhibited significantly by calcium treatment in wild-type and Nr tomatoes, but the expression of LeACS2, the key ACC synthase gene in ethylene synthesis during tomato fruit maturing, was not affected. These results revealed that the effect of calcium on ethylene biosynthesis in tomato mature green fruit was independent of ethylene perception. The results also revealed that the targeting step of calcium preventing ethylene production was located at the ACC conversion to ethylene, by means of inhibiting ACC availability for ACO through enhancing cell membrane integrity and by means of preventing LeACO1 gene expression. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 60–67. The text was submitted by the authors in English.     

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

Auxin-induced Ethylene Production and Its Inhibition by Aminoethyoxyvinylglycine and Cobalt Ion

Auxin is known to stimulate greatly both C₂H₄ production and the conversion of methionine to ethylene in vegetative tissues, while amino-ethoxyvinylglycine (AVG) or Co²⁺ ion effectively block these processes. To identify the step in the ethylene biosynthetic pathway at which indoleacetic acid (IAA) and AVG exert their effects, [3-¹⁴C]methionine was administered to IAA or IAA-plus-AVG-treated mung bean hypocotyls, and the conversion of methionine to S-adenosylmethionine (SAM), 1-amino-cyclopropane-1-carboxylic acid (ACC), and C₂H₄ was studied. The conversion of methionine to SAM was unaffected by treatment with IAA or IAA plus AVG, but active conversion of methionine to ACC was found only in tissues which were treated with IAA and which were actively producing ethylene. AVG treatment abolished both the conversion of methionine to ACC and ethylene production. These results suggest that in the ethylene biosynthetic pathway (methionine → SAM → ACC → C₂H₄) IAA stimulates C₂H₄ production by inducing the synthesis or activation of ACC synthase, which catalyzes the conversion of SAM to ACC. Indeed, ACC synthase activity was detected only in IAA-treated tissues and its activity was completely inhibited by AVG. This conclusion was supported by the observation that endogenous ACC accumulated after IAA treatment, and that this accumulation was completely eliminated by AVG treatment. The characteristics of Co²⁺ inhibition of IAA-dependent and ACC-dependent ethylene production were similar. The data indicate that Co²⁺ exerts its effect by inhibiting the conversion of ACC to ethylene. This conclusion was further supported by the observation that when Co²⁺ was administered to IAA-treated tissues, endogenous ACC accumulated while ethylene production declined.     

{alpha}-Aminoisobutyric acid : A probable competitive inhibitor of conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

Of 16 compounds related to 1-aminocyclopropane-1-carboxylicacid (ACC), aminoisobutyric acid (AIB) inhibited the productionof endogenous ethylene in the cotyledonary segments of cocklebur(Xanthium pennsylvanicum Wallr.) seeds most strongly. AIB at4 mM inhibited the formation of ethylene by about 50%, althoughthe O₂ uptake of the segments was not affected even at 20 mM.AIB also inhibited ethylene formation in the stem segments ofetiolated pea (Pisum sativum L. cv. Alaska) seedlings. Kineticanalysis with cell free extracts from etiolated pea shoots revealedthat AIB competitively inhibits the conversion of ACC into ethylene. (Received May 26, 1980; )     

Inhibition of ethylene synthesis in tomato plants subjected to anaerobic root stress

Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon esculentum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO²⁺ prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co²⁺ had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. All three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co²⁺, conversion of both root- and petiole-synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low O₂ roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants.     

Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled ¹⁴C-Arg, ¹⁴C-Orn, L-[U-¹⁴C]Met, ¹⁴C-SAM and ¹⁴C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-¹⁴C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of ¹⁴C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of ¹⁴C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene biosynthesis, and (3) cellular flux of SAM in plants is homeostatically regulated based on its demand for competing pathways.     

Biosynthesis of auxin-induced ethylene in mung bean hypocotyls

The pathway of ethylene biosynthesis in auxin-treated mung beanhypocotyls was investigated by comparing the specific radioactivitiesof ethylene produced and S-adenosylmethionine (SAM) in the tissuefollowing the administration of 3,4-¹⁴C-methionine, and by analyzingthe methionine metabolites. When the rate of auxin-induced ethyleneproduction was low due to a low concentration of auxin, thespecific radioactivity of ethylene released was always higherthan that of SAM in the tissue. When the tissue was treatedwith auxin, the tissue produced and accumulated a methioninemetabolite which was converted into ethylene more efficientlythan methionine. The metabolite was identified as 1-aminocyclopropane-l-carboxylicacid (ACC) by means of paper and thin-layer chromatography,high voltage paper electrophoresis and co-crystallization. ACCformation was neither inhibited by low oxygen nor by the inhibitoryprotein of ethylene synthesis, but inhibited by aminoethoxyvinylglycine(AVG). ACC application to the tissue greatly reduced incorporationof 3,4-¹⁴C-methionine into ethylene. The control tissue thatwas not treated with auxin also converted ACC into ethyleneindicating that the enzyme which converts ACC into ethyleneis already present in the tissue and that auxin induced productionof the enzymatic system responsible for the conversion of methionineinto ACC. Ethylene synthesis from ACC was not inhibited by AVG,abscisic acid, cycloheximide or actinomycin D, but inhibitedby low oxygen and the inhibitory protein. (Received November 21, 1979; )     

1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis

1-Aminocyclopropanecarboxylate (ACC) synthase, which catalyzes the conversion of S-adenosylmethionine (SAM) to ACC and methylthioadenosine, was demonstrated in tomato extract. Methylthioadenosine was then rapidly hydrolyzed to methylthioribose by a nucleosidase present in the extract. ACC synthase had an optimum pH of 8.5, and a K_m of 20 μm with respect to SAM. S-Adenosylethionine also served as a substrate for ACC synthase, but at a lower efficiency than that of SAM. Since S-adenosylethionine had a higher affinity for the enzyme than SAM, it inhibited the reaction of SAM when both were present. S-Adenosylhomocysteine was, however, an inactive substrate. The enzyme was activated by pyridoxal phosphate at a concentration of 0.1 μm or higher, and competitively inhibited by aminoethoxyvinylglycine and aminooxyacetic acid, which are known to inhibit pyridoxal phosphate-mediated enzymic reactions. These results support the view that ACC synthase is a pyridoxal enzyme. The biochemical role of pyridoxal phosphate is catalyzing the formation of ACC by α,γ-elimination of SAM is discussed.     

Effect of silver ions on ethylene biosynthesis by tomato fruit tissue

Mature-green tomato fruit (Lycopersicon esculentum Mill.) were treated asymmetrically with 2 millimolar silver thiosulfate (STS) through a cut portion of the peduncle while still attached to the plant. One-half of the fruit received silver and remained green while the other half ripened normally and was silver-free (less than 0.01 parts per billion). Harvested mature-green fruit were also treated with STS through the cut pedicel. Green tissue from silver-treated fruit had levels of 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate ethylene precursor) slightly less or similar to that of turning or red-ripe tissue from the same fruit, and similar to that of mature-green tissue from control fruit. Ethylene production was higher in green tissue from silver-treated fruit than from either red tissue from the same fruit, or mature-green tissue from control fruit. By inhibiting ACC synthesis with aminoethoxyvinyl glycine, and by applying ACC ± silver to excised disks of pericarp tissue from control or silver-treated tomatoes, we showed that short-term silver treatment did not affect the biological conversion of ACC to ethylene, while long-term treatment stimulated both the conversion of ACC to ethylene and the synthesis of ACC.     

The modulation of the conversion of l-aminocyclopropane-l-carboxylic acid to ethylene by light

Endogenous ethylene production of tobacco leaves was similar in light and in darkness. However, the rate of conversion of exogenously applied l-aminocyclopropane-l-carboxylic acid (ACC) to ethylene was reversibly inhibited by light. Virus-stimulated ethylene production, during the hypersensitive reaction of tobacco leaves to tobacco mosaic virus, was likewise inhibited by light. Under such circumstances ethylene production is limited at the level of the conversion of ACC to ethylene. Inhibition of the increase in ACC-stimulated ethylene production by cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide after shifting leaf discs from light to darkness indicated that de novo protein synthsis was involved. Regulation of ACC-dependent ethylene production by reversible oxidation/reduction of essential SH groups, as suggested by Gepstein and Thimann (1980, Planta 149, 196–199) could be excluded. Instead, regulation of the ACC-converting enzyme at the level of both synthesis/degradation and activation/inactivation is suggested. Phytochrome was not involved in light inhibition, but low intensities of either red or blue light decreased the rate of ACC conversion. Dichlorophenyldimethylurea counteracted the inhibitory effect of light, indicating that (part of) the photosynthetic system is involved in the light inhibition. The ethylene production of Pharbitis cotyledons grown in darkness or light, either in the presence of absence of the inhibitor of carotenoid synthesis, SAN 9789 (norflurazon), supported this view.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DCMU dichlorophenyldimethylurea - MDMP 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide - SAM S-adenosylmethionine - SH groups sulfhydryl groups - TCA trichloroacetic acid - TMV tobacco mosaic virus     

Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4-[C]methionine into spermidine in aged orange peel discs

The flux of radioactivity from 3,4-[(14)C]methionine into S-adenosyl-l-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylene by 100 millimolar phosphate and 2 millimolar Co(2+) was studied in aged peel discs of orange (Citrus sinensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightly reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-[(14)C]methionine into spermidine.     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Suppression of wound-induced ethylene production in muskmelon fruit tissue after vacuum infiltration with aqueous buffer

Buffered solutions are used commonly to introduce chemical inhibitors and promoters of ethylene synthesis into plant tissues. Vacuum infiltration of preclimacteric muskmelon (Cucumis melo L.) fruit tissue with a buffer (50 mM MES, pH 6.1) immediately after excision inhibited the wound-induced increase in ethylene production, but it did not suppress the accumulation of 1-aminocyclopropane-l-carboxylic acid (ACC) during the 48 h following injury. The inhibition of ethylene production by infiltration was not reversed by treatment with ACC. If the injured tissue was allowed to age for 3 h before treatment, wound-induced ethylene production in tissue samples was not inhibited by vacuum infiltration with aqueous buffer. The results indicate that infiltration of melon fruit tissue with a liquid medium does not block the development of wound-induced ethylene production by either limiting ACC or inhibiting the ongoing conversion of ACC to ethylene. Liquid infiltration of the tissue appears to interfere with the initiation of physiological events during the first 3 h after wounding that are critical for the subsequent conversion of ACC to ethylene.     

Auxin-induced ethylene biosynthesis in subapical stem sections of etiolated seedlings of Pisum sativum L.

1-Aminocyclopropane-1-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections of etiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 mM ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-¹⁴C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections incubated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine - IAA indole-3-acetic acid - SAM S-adenosylmethionine - SMM S-methylmethionine     

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     

Ethylene synthesis and growth of tomato hypocotyls: Induction by auxin and fusicoccin and inhibition by vanadate

The effects of fusicoccin (FC) on growth and ethylene synthesis of tomato (Lycopersicon esculentum Mill.) hypocotyls were compared to those of indole-3-acetic acid (IAA). Fusicoccin promoted both growth and ethylene production maximally at <2M. Growth was stimulated to a slightly greater extent by FC as compared to IAA, while ethylene synthesis rates in response to FC were about 50% less than those induced by IAA. Cycloheximide (0.5 M) inhibited auxin-induced growth by 80% but had no effect on FC-induced growth; ethylene production was inhibited to the same extent (58%) when induced by either IAA or FC. Both IAA and FC caused tissue contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-ACC to increase, indicating that like IAA, FC induces ethylene synthesis by stimulating the formation of ACC. Orthovanadate, a potent inhibitor of proton-translocating plasma membrane ATPases, reduced both IAA- and FC-induced growth and ethylene synthesis at concentrations less than 1 mM, with ethylene synthesis being approximately 10 times more sensitive to inhibition than growth. Vanadate did not affect tissue ACC levels, slightly reduced total ACC production, and inhibited conversion of ACC to ethylene. However, significant inhibition of in vivo ethylene-forming enzyme activity required high concentrations of vanadate (1 mM) and was less effective than inhibition by cobaltous ion. The site of action of vanadate in inhibiting ethylene synthesis remains unclear, but the ion did not prevent the elevation of tissue ACC levels in response to IAA or FC. It is unlikely, therefore, that stimulation of plasma membrane H⁺-ATPase activity is required for the induction of ACC synthase by IAA and FC.