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     

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.     

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.     

Ca2+ effects on ethylene, carbon dioxide and 1-aminocyclopropane-1-carboxylic acid synthase activity

The response of pericarp disks from ripening tomato (Lycopersicon esculentum Mill. cv. Traveler‘76) to CaCl₂, additions was studied to determine the effect of Ca²⁺ on ethylene and CO₂ production. Application of 5 mM CaCl₂ resulted in a 2, 20, 33, 39, and 50% increase in ethylene production in disks obtained from preclimacteric minimum, climacteric rise, climacteric peak, one, and two days postclimacteric fruit, respectively. CaCl₂ concentrations of 10 and 50 mM gave no additional stimulation of ethylene production; CO₂ production at 5 mM CaCl₂ was not different from controls, but is increased at 10 and 50mM CaCl₂. CaCl₂ also increased ethylene production in disks treated with 1-aminocyclopropane-1-carboxylic acid (ACC) or aminoethoxy-vinylglycine. Chloride salts of K⁺, Na⁺, Mg²⁺, Sr²⁺ and La³⁺ did not stimulate ethylene production. SrCl₂ stimulated ethylene production to a lesser degree than CaCl₂. Disks from potato (Solanum tuberosum L. cv. Katahdin) tubers produced greater quantities of ethylene and ACC when 5 mM CaCl₂ was included in the incubation medium (K. B. Evensen, 1983. Physiol. Plant. 60:125–128). Ca²⁺-treated disks had more than three times as much ACC synthase activity as control disks after 18 to 24 h incubation, when ethylene and ACC were maximal. The apparent K_m for S-adenosylmethionine was 13 μM at 29°C, pH 8.0 in extracts from both Ca²⁺-treated and control disks. Inclusion of 1 to 50 mM CaCl₂ in the assay medium did not significantly affect enzyme activity. ACC synthase extracted from control and Ca²⁺-treated disks had a pH optimum of 8.5 and an apparent molecular weight of 72 kdalton, estimated by gel filtration. It is likely that the presence of Ca²⁺ in the buffer allows greater synthesis of ACC synthase as part of the wound-healing response in potato, while in tomato the predominant effect is on membrane stabilization.     

Effects of turgor potential on 1-aminocyclopropane-1-carboxylic acid uptake into the vacuolar compartment and ethylene production in tomato pericarp slices

While solute transport and ethylene production by plant tissue are sensitive to the osmotic concentration of the solution bathing the tissue, the influence of tissue water relations and specifically tissue turgor potential on the kinetics of 1-aminocyclopropane-1-carboxylic acid (ACC) uptake into the vacuolar compartment and ethylene production have not been examined. 1-Aminocyclopropane-1-carboxylic acid transport and ethylene production were examined in tomato (Lycopersicon esculentum Mill. cv. Liberty) pericarp slices incubated in solutions having a range of mannitol, polyethylene glycol 3350 and ethylene glycol concentrations known to affect tissue water relations. Tissue osmotic and turgor potentials were derived from osmolality measurements of cell saps recovered by freeze-thawing and corrected for the contribution of the free-space solution. When relatively nonpermeable (mannitol or polyethylene glycol 3350) osmotica were used, both ACC uptake and ethylene production were greatest at a solution osmolality of 230 milliosmolal where tissue turgor potential ranged between 120 and 140 kPa. At higher and lower turgor potentials, the high-affinity saturating component of ACC uptake and ethylene production were inhibited, and ACC efflux from the vacuolar compartment was increased. The inhibition of ACC uptake was evident as a decrease in V_max with no effect on K_m. Turgor potential changes caused by adjusting solution osmolality with mannitol or polyethylene glycol 3350 were accompanied by changes in the osmotic potential and water potential of the tissue. The effects of turgor potential vs the osmotic and water potentials of tomato pericarp slices were differentiated by comparing responses to nonpermeable osmotica and mixtures of nonpermeable and permeable osmotica. Ethylene glycol-mannitol mixtures had effects on the osmotic potential and water potential of the tissue similar to those of nonpermeable osmotica but had less effect on tissue turgor, ACC transport and ethylene production. Incubating tissue in solutions without nonpermeable osmotica osmotically shocked the tissue. Increasing solution osmolality with ethylene glycol in the absence of nonpermeable osmotica increased tissue turgor and ethylene production. The present study indicates that tissue turgor is an important factor affecting the kinetics of ACC uptake into the vacuolar compartment and ethylene production in tomato pericarp slices.     

Plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase activity implicated in different aspects of plant development

Proper plant development is dependent on the coordination and tight control of a wide variety of different signals. In the study of the plant hormone ethylene, control of the immediate biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is of interest as the level of ethylene can either help or hinder plant growth during times of stress. It is known that ACC can be reversibly removed from the biosynthesis pathway through conjugation into other compounds. We recently reported that plants can also irreversibly remove ACC from ethylene production through the activity of a plant encoded ACC deaminase. Heretofore only found in bacteria, we showed that there was ACC deaminase activity in both Arabidopsis and in developing wood of poplar. Here we extend this original work and show that there is also ACC deaminase activity in tomato plants, and that this activity is regulated during tomato fruit development. Further, using an antisense construct of AtACD1 in Arabidopsis, we investigate the role of ACC deamination during salt stress. Together these studies shed light on a new level of control during ethylene production in a wide variety of plant species and during different plant developmental stages.Key words: tomato fruit ripening, wood development, stress response, hormone, antisense, synthesisHormones are a class of signaling molecules produced and sensed at very low levels; therefore control of their biosynthesis is crucial for proper plant development. The plant hormone ethylene has been studied for over a century and can positively impact plant development, such as in the initiation of fruit ripening, but ethylene accumulation can also induce widespread damage during stress responses.¹ Ethylene is produced in two steps from the S-adenosylmethionine (SAM) that is derived from the Yang cycle.² In the first committed step, SAM is converted into 1-aminocyclopropane-1-carboxcylic acid (ACC) via the action of ACC SYNTHASEs (ACSs).³ ACC is then converted into ethylene by ACC OXIDASEs (ACOs), a particular adaptation of flowering plants.⁴ Once ACC is produced, there are few proven pathways that can divert it from conversion into ethylene. ACC can be conjugated into malonyl-1-aminocyclopropane- 1-carboxylic acid (MACC) through the activity of ACC malonyl transferase⁵ or to 1-(γ-L-glutamyl-amino) cyclopropane-1-carboxylic acid (GACC) via γ-glutamyltranspeptidase.⁶ In bacteria, another pathway exists that can break down ACC obtained from plants through an irreversible deamination process.⁷ Through heterologous expression of bacterial ACC DEAMINASEs (ACDs) in plants it has been possible to engineer plants that have reduced production of ethylene by affecting the native pools of ACC.⁸ Until recently no ACC deaminase pathway has ever been proven in plants, although a number of different plant genomes encode genes which bear sequence homology to bacterial ACDs. Should these genes code for active ACDs, this would provide an additional level of control for ethylene production beyond the activity of ACSs and ACOs. Recently we reported that Arabidopsis and Populus have inherent ACC deaminase activity, and we showed that this activity in Arabidopsis is due, in part, to the product of ACC DEAMINASE1 (AtACD1) (At1g48420).⁹ This discovery raises many questions concerning the role of ACC deaminases during ethylene mediated processes in a number of different plant models. We report here some of our preliminary findings in the areas of tomato fruit ripening and salt stress in Arabidopsis.As precise control of ethylene levels is essential during climacteric fruit development, in parallel with our reported studies we also studied ACC deaminase activity in developing tomato fruit. Ethylene production during ripening in tomato is controlled by ethylene receptor turnover¹⁰ and conjugation of ACC by MACC and GACC.^6,11,12 We found that tomatoes also have inherent ACD activity, and that this activity varies over ripening of the fruit (Fig. 1; solid line). During the immature green stage in tomato development ACC deaminase activity was low. This activity increased significantly during the ‘late breaker’ stage, just prior to the orange/red stage of development, and then decreased during later stages of tomato ripening. Also shown in this figure are the predicted levels of ethylene during fruit development. It is interesting to note that the highest amount of ACC deaminase activity coincides with the drop in ethylene levels soon after the breaker stage (Fig. 1; dashed line; based on Brady¹³). Our data would suggest that, in addition to ethylene receptor turnover and GACC and MACC activity, ACC deaminase activity may also help control ethylene levels. It has already been shown that constitutive expression of a bacterial ACC deaminase in tomato can delay the rate of tomato fruit ripening by reduction of ethylene production.⁸ Although ACD activity is evident during ripening in tomato, the gene responsible has not been identified. Recently a tomato gene with sequence similarity to bacterial ACC deaminases was tested for ACD activity. It was found that, despite the close sequence similarity, this gene (accession number {"type":"entrez-nucleotide","attrs":{"text":"EU639448","term_id":"186920268","term_text":"EU639448"}}EU639448) did not have ACD activity.¹⁴ Therefore, additional work must be done to isolate the gene responsible for the ACD activity we demonstrate in tomato fruit.Open in a separate windowFigure 1Tomato fruits exhibit AC deaminase activity during ripening. A plot of ACC Deaminase activity (Solid Line) with known levels of ethylene production during ripening (Dashed Line; Brady¹³) superimposed over pictures of the corresponding stage of tomato development. *Indicates significant increase in activity (†nmol mg⁻¹ hr⁻¹). AC deaminase activity analysis was performed on total tomato fruit protein as per Penrose and Glick (2003).²¹Our discovery of a plant encoded ACC deaminase in Arabidopsis allows us, for the first time, to downregulate ACC deaminase activity and investigate how this affects plant development. Previously, we showed that downregulation of AtACD1 using antisense resulted in up to a 30% reduction in ACD activity and up to a 2.5-fold increase in the evolution of ethylene.⁹ We showed that this difference in ACD activity was sufficient to alter hypocotyl elongation during Arabidopsis germination on different concentrations of ACC. It was unknown, however, if this difference was sufficient to affect other areas of development, such as stress response, in Arabidopsis. The expression of bacterial ACC deaminases in plants are known to increase plant resistance to a number of stressors due to decreased ethylene evolution.^15–18 Based on microarray data, it is known that AtACD1 expression is upregulated 150% during salt stress¹⁹ and functionally it has been demonstrated that ACC production is increased in salt stressed roots²⁰ and overexpression of bacterial ACDs in canola increases salt tolerance.¹⁸ It was unknown, however, if a reduction in native ACD activity would result in reduced vigour of plants grown on increasing concentrations of sodium chloride. We observed that there was no significant difference in rosette size, leaf production or percent dry weight between wildtype and three independent Arabidopsis lines expressing the AtACD1 antisense construct when grown on MS media without salt (Fig. 2A–C). As the concentration of salt increased in the growth media it was found that the antisense lines also did not differ from wildtype in their growth. The lack of a definitive phenotype under salt stress may mean that the level of reduced ACD activity achieved in the AtACD1 antisense lines was not sufficient to quantifiably affect the development of Arabidopsis. Additionally, as ethylene is not the only factor that affects a plant’s survival during times of salt stress, it is also possible that the plants were able to compensate for increased ethylene production in the AtACD1 antisense lines to promote normal plant development. This finding highlights the complex nature of the different signals involved in a plant’s response to salt stress and the need for a better understanding of the role of plant ACDs and how the plant may compensate for altered ACD activity.Open in a separate windowFigure 2Growth and development of Arabidopsis wildtype and three Antisense AtACD1 lines on increasing concentrations of salt. Stratified wildtype Arabidopsis (Col-0) and three independent transgenic lines expressing an antisense construct of AtACD1 (A1, A2, A3) were sown on 0 mM NaCl (Dark Grey Bars), 100 mM NaCl (White Bars), 125 mM NaCl (Black Bars) and 150 mM NaCl (Light Grey Bars) and allowed to germinate and grow for 2 weeks under long-day conditions (16 h light/8 h dark) at a light intensity of 130 to 190 µE m-2s⁻¹ at the rosette level at 21°C in Econair AC -60 growth chambers. Plants were analyzed for rosette diameter (A), leaf production (B) and percent dry weight (C). Error bars are ± SE.In the known framework of ethylene synthesis our work has shown that plants do have the ability to reduce ethylene synthesis by irreversibly deaminating ACC through the action of a native ACC deaminase. Further to our first study, we show here that there is inherent ACC deaminase activity in tomatoes and that this activity varies during tomato ripening in a manner consistent with a factor that is involved in the regulation of ethylene levels. We also show here that transgenic Arabidopsis lines with a mild reduction in ACD1 activity do not have an obvious affect on mediation of salt stress. This finding, however, does not preclude a role for ACD1 in mediating other aspects of plant development or in affecting plant development during other types of plant stress (i.e., drought). Therefore, there still remain many questions to answer concerning the role of plant encoded ACC deaminases and many exciting avenues of ethylene regulation to pursue. The identification and exploitation of tomato, poplar and other plant ACC deaminases could be used to alter fruit ripening, wood production and stress tolerance—all aspects of plant development that are economically and scientifically important.     

Role of ethylene in the regulation of cell death and leaf loss in ozone-exposed European beech

To test the involvement of ethylene in mediating ozone-induced cell death and leaf loss in European beech ( Fagus sylvatica L.), tree seedlings were exposed to proportionally increased or decreased field ozone levels for up to 6 months. Ozone treatment caused cell death and accelerated leaf loss at higher than ambient levels, but had only minor effects at ambient and no effects at subambient ozone levels. The emission of ethylene, the levels of its precursor, 1-aminocyclopropane-1-carboxylate (ACC), and mRNA levels of specific ACC synthase ( FS-ACS2 ) and ACC oxidase ( FS-ACO1 ) isoforms showed a persistent increase and preceded cell death by approximately 2 weeks. Inhibition of ethylene biosynthesis led to reduced lesion formation whereas application of ACC accelerated ozone-induced cell death and leaf loss. Similar results were obtained when adult beech trees were exposed to 2 × ozone by a whole tree free-air canopy exposure system. The results suggest a role of ethylene in amplifying ozone effects under field conditions in this major European broad-leaved tree species.     

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.     

Calcium effects on ethylene and ethane production and 1-aminocyclopropane-1-carboxylic acid content in potato disks

Potato ( Solanum tuberosum L. cv. Katahdin) disks produce ethyline in increasing amounts from 6 to 24 h incubation in buffer at pH 4.0. Ethylene production is increased 2–3 times in the presence of 50 m M CaCl₂. Levels of endogenous 1-amino-cyclopropane-1-carboxylic acid (ACC) increase in parallel with ethylene production, and ACC levels are 3–5 times higher in calcium-treated disks than in controls. Most of the calcium-induced stimulation of ethylene production can be accounted for by its effect on ACC production, indicating that the primary effect of calcium is on a step of ethylene biosynthesis preceeding ACC production. However, calcium may also have an effect on conversion of ACC to ethylene, since a consistent increase in ACC-de-pendent ethylene production was observed in the presence of calcium. Production of ethane, a marker of lipid peroxidation, was reduced by calcium, so it is possible that membrane stabilization by calcium could be involved in its effects on ethylene production.     

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     

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     

Ethylene levels are regulated by a plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase

Control of the levels of the plant hormone ethylene is crucial in the regulation of many developmental processes and stress responses. Ethylene production can be controlled by altering endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene or by altering its conversion to ethylene. ACC is known to be irreversibly broken down by bacterial or fungal ACC deaminases (ACDs). Sequence analysis revealed two putative ACD genes encoded for in the genome of Arabidopsis thaliana ( A. thaliana ) and we detected ACD activity in plant extracts. Expression of one of these A. thaliana genes ( AtACD1 ) in bacteria indicated that it had ACD activity. Moreover, transgenic plants harboring antisense constructs of the gene decreased ACD activity to 70% of wild-type (WT) levels, displayed an increased sensitivity to ACC and produced significantly more ethylene. Taken together, these results show that AtACD1 can act as a regulator of ACC levels in A. thaliana .     

Changes in ethylene production and 1-aminocyclopropane-1-carboxylic acid content of pollinated carnation flowers

Pollination of flowers of standard carnation (Dianthus caryophyllus L. cv. White Sim) with pollen from flowers of miniature carnations (D. caryophyllus L. cv. Exquisite) caused them to wilt irreversibly within 1 to 2 days. Pollination stimulated a sequential increase in ethylene production by stigmas, ovaries, receptacles, and petals of the flowers. The ACC content of the stigmas increased rapidly in the first few hours after pollination. The possibility that subsequent production of ethylene by other parts of the flower is stimulated by translocated ACC is discussed. Ethylene production and ACC content of other parts of the flower reached their maximum 24 h after pollination. The petal tissues contributed the bulk of the ethylene productionper flower thereafter. There appears to be a qualitative difference between the enzyme in the stigmas converting ACC to ethylene and that in other parts of the flower.     

Selected ion monitoring/isotope dilution mass spectrometric determination of 1-aminocyclopropane-1-carboxylic acid levels in ripening tomato fruit

A new method is described for the quantitation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plants. [2,2,3,3,-2H4]ACC has been synthesized and used as an internal standard for selected ion monitoring/isotope dilution quantitation of this compound in ripening tomato fruit. These data are compared with those derived from the widely used indirect oxidative ACC assay (which underestimated the ACC levels by between two- and fourfold). The greater accuracy, sensitivity (100X), and specificity of the mass spectrometric method will be of considerable benefit to those interested in factors which control ACC and ultimately ethylene levels since it is believed that ACC synthesis and its oxidative metabolism to ethylene are the key points at which ethylene biosynthesis is regulated.     

Subcellular localization of the sites of conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene in plant cells

The subcellular localization of the sites of 1-aminocyclopropane-1-carboxylic acid (ACC) conversion into ethylene was studied by comparing the specific radioactivity of ethylene evolved from the whole cells with that of intra- and extracellular pools of labelled ACC. We demonstrate that some cells cultured in vitro (Vitis vinifera L. cv. Muscat) or leaf tissues (Hordeum vulgare L. and Triticum aestivum L.) have two sites of ethylene production: (i) an external site, converting apoplastic ACC, located at the plasma membrane, and very sensitive to high osmotica and, (ii) an intracellular site, converting internal ACC and remaining unaffected even under severe plasmolysis. In other cells cultured in vitro (Vitis vinifera L. cv. Gamay) and pea leaves (Pisum sativum L.), only the intracellular site operates and ethylene production is almost unaffected by plasmolysis. Protoplasts obtained from plasmolysis-sensitive Muscat cells lose 97% of their capacity for ethylene production compared with the parent cell, while those from plasmolysisinsensitive Gamay cells retain up to 50%. Protoplasts from both Gamay and Muscat cells cultured for 8 d in vitro, recover the full capacity of ethylene production of the initial whole cells, whether or not they are allowed to reform their cell wall. Therefore, we exclude a cooperation between the cell wall and the plasma membrane in ethylene production.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme We are grateful to Dr. Philip John (Reading, UK) for useful discus sions made possible by a North Atlantic Treaty Organization Colla borative Grant (No. 0383/88) and Dr. Yves Meyer (Perpignan, France) for his collaboration in culturing protoplasts.     

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.