ACC Oxidase Activity Research Articles

Purification and characterization of ACC oxidase from Artocarpus altilis

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase was purified to homogeneity from breadfruit (Artocarpus altilis) by a six-step chromatography procedure to yield an 87-fold increase in purification with a recovery of 9.5%. SDS-PAGE revealed that the purified enzyme had a molecular mass of 42.3 kDa and a Km of 28.2 μM. The enzyme showed marked inhibition by cobalt sulphate, sodium metabisulphite, sodium dithionite, n-propyl gallate, zinc sulphate and hydrogen peroxide. Dithiothreitol (DTT) enhanced enzyme activity when it was included in the assay medium. Optimum activity of ACC oxidase was obtained at a pH of 7.2 at 28 °C.

Ethylene metabolism in Scots pine (Pinus sylvestris) shoots during the year.

Ethylene evolution, concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC conjugates, activities of ACC synthase and ACC oxidase, and cambial growth as measured by tracheid production were monitored from November to July in 1-year-old shoots, and between July and September in current-year shoots, of Scots pine (Pinus sylvestris L.). Needles, buds and four stem parts (cortex, phloem, cambial region and mature xylem) were surveyed. Ethylene evolution was quantified by gas chromatography. Free ACC and bound ACC (after acidic hydrolysis of ACC conjugates) were quantified by combined gas chromatography-mass spectrometry, with [2H]ACC as an internal standard. Activities of ACC synthase and ACC oxidase were measured in crude protein extracts. We detected high activities of ACC synthase in needles and buds, but not in stem fractions except the phloem. In July, during the period of intensive shoot growth, we found ACC only in buds and needles. In contrast, ACC oxidase activity was high in stem tissues, particularly in the cambial region during the period of rapid tracheid production, but no ACC oxidase activity was detected in needles and buds. Nevertheless, needles evolved large amounts of ethylene. Ethylene was produced by all stem fractions, and the peak rate of ethylene evolution in the cambial region coincided with the period of maximal tracheid production. Conjugated ACC was present in every fraction except mature xylem. The concentration of conjugated ACC decreased when rates of tracheid production and ethylene evolution were high, suggesting that conjugated ACC may serve as a source for ACC in the cambial region. The regulation of ethylene biosynthesis in Scots pine shoots is discussed.

Characterisation of the ACC oxidase activity in transgenic auxin overproducing tomato during ripening

As part of our studies on the role of auxin in regulating the ethylenebiosynthesis during fruit ripening, in this paper we describe the functionalproperties of the ACC oxidase activity extracted from transgenic tomato(Lycopersicum esculentum Mill. cv. Ailsa craig)overexpressing the tryptophan monooxygenase or iaaM protein fromAgrobacterium tumefaciens that increases the auxin levels.Maximal activity was recovered by extracting the enzyme at pH 8.0 from fruitspicked three days after the onset of the colour change. The enzyme exhibits ahalf-life of 85 min, two relative maxima at 30 and 38°C, an optimum pH of 7.9 and an apparent Km forACC of 118 μM. Our results also show the first evidence of anallosteric type kinetic of the ACC oxidase activity with respect to itscosubstrate ascorbate, with an apparent Km of 12.5mM,estimated as the concentration which gave 50% Vmax.

Wound-induced ethylene synthesis in stem tissue of harvested broccoli and its effect on senescence and ethylene synthesis in broccoli florets

Abstract Broccoli ( Brassica oleracea , var. italica ) florets (flower buds) senesced rapidly after harvest at ambient temperatures. ACC (1-aminocyclopropane-1-carboxylic acid) synthase (ACS) was induced rapidly in the cut surface of stem tissue in the first 2 mm thick and the basal portions of curds soon after harvest, leading to an accumulation of ACC. The abundance of one ACS mRNA (BO-ACS1) increased in the first layer, the inner layer of stem (2–4 mm, second layer), and the basal portion of curds, while the transcripts for another (BO-ACS2) increased only in the first layer. Conjugated ACC (malonyl ACC, MACC) levels increased in all portions as senescence progressed. In florets, ACC synthase activity and BO-ACS1 transcripts were detected with no significant changes observed during senescence. ACC levels in florets stayed low throughout the experimental period, whereas MACC levels were much higher than those of ACC. The marked rise in ACC oxidase (ACO) activity in florets was detected almost in parallel with a significant increase in ethylene production. The abundance of ACO transcripts (BO-ACO1 and BO-ACO2) increased concurrently with the rise in ACC oxidase activity. These findings suggest that ACC and ethylene synthesized in the stem in response to wounding may have involved the enhanced activity of ACC oxidase and increased abundance of its transcripts in florets.

Chilling induced ethylene biosynthesis in ‘Hayward’ kiwifruit following storage

‘Hayward’ kiwifruit were stored at 0, 5, 10, 15 and 20°C for 5, 12 and 17 days before rewarming to 20°C for 10 more days. Ethylene and CO 2 production, ACC, ACC synthase (ACS) and ACC oxidase (ACO) activities, flesh and core firmness, soluble solids content (SSC) and flesh colour were measured. Kiwifruit stored at 0, 5, 10 and 15°C did not ripen, produce ethylene or show increases in ACS or ACO activity. Fruit stored for 5 days at the above temperatures, then rewarmed to 20°C, did not show any change during the following 10 days. Rewarmed fruit, pre-stored at 0–10°C for 12 days, started autocatalytic ethylene production within 24 h, followed by fruit ripening. Fruit stored at 15°C for 12 days needed 72 h to start ethylene autocatalyse and did not fully ripen during 10 days at 20°C. After 17 days storage at 0–15°C kiwifruit started autocatalytic ethylene production with no delay upon exposure to 20°C. Autocatalytic ethylene production correlated with increased ACC content, and increased activities of ACS and ACO. Fruit held continuously at 20°C started autocatalytic ethylene production after 19 days, with concomitant increases in ACC content, ACS and ACO activities and ripening. Respiration increased after rewarming, concomitantly with the increase in ethylene production. We concluded that exposing kiwifruit to chilling temperatures (0–10°C) for 12 days advanced ethylene biosynthesis and ripening when compared with fruit held continuously at 20°C. The advanced ethylene biosynthesis was due to increase ACS and ACO activities immediately upon rewarming of the fruit.

Stimulation of ‘Fuji’ apple skin color by ethephon and phosphorus–calcium mixed compounds in relation to flavonoid synthesis

The effect of ethephon (2-chloroethyl phosphonic acid) and a seniphos-like substance (SLS) (a mixture of 310 g/l P 2O 5, 56 g/l CaO and 30 g/l N: 1% NO 3 and 2% NH 3) on the coloring of skin of ‘Fuji’ apple were studied. Both treatments applied 4 weeks before commercial harvest, caused an enhancement of red peel color and an increase in concentration of flavonoid compounds. Anthocyanin, proanthocyanidins and flavonols showed different accumulation pattern with different treatments during fruit maturation, but eventually became similar concentrations at harvest. ACC (1-aminocyclopropane-1-carboxylic acid) oxidase activity and internal ethylene concentration were greatly increased by the ethephon treatment but slightly by SLS treatment. The ethephon treatment increased greatly the chalcone isomerase (CHI) activity, and moderately phenylalanine-ammonia lyase (PAL) activity, but showed no effect on UDP-3- O-galactosyltransferase (UFGalT), all of which are involved in flavonoid biosynthesis. On the other hand, the SLS increased PAL and UFGalT activities greatly and CHI activity moderately.

EFFECT OF GAMMA IRRADIATION ON SOFTENING CHANGES AND ENZYME ACTIVITIES DURING RIPENING OF PAPAYA FRUIT

The aim of this work was to study the role of cell wall and ethylene related enzymes to papaya fruit firmness. Irradiation treatment was used as an imposed stress to cause changes in firmness. Physiologically mature papaya fruits were irradiated (500 Gy) and allowed to ripen at 22C and 90% RH. Polygalacturonase (PG), pectinmethylesterase (PME), β-galactosidase, cellulase and 1-aminocyclopropane-1-carboxylate oxidase (ACC-oxidase) activities were followed during ripening and correlated to changes in firmness, skin and pulp color, respiration and ethylene production. The firmness of irradiated fruits was retained at least 2 days longer than in normal fruits and also had a slower rate of softening. Total soluble solids (°Brix), cellulase activity and ethylene production were not altered by irradiation treatment. The activity patterns of PG, PME and β-galactosidase were related to pulp softening and affected by irradiation. ACC-oxidase activity was influenced by irradiation treatment, but its changes were not temporally related to those in firmness. It was concluded that irradiation had no direct effect on firmness but it acted by altering the ripening induced synthesis of cell wall enzymes, mainly PME. Cette etude examine l'effet d'une irradiation gamma sur la fermete des papayes reliee a l'action de plusieurs enzymes connues importantes dans le metabolisme de la membrane cellulaire. Des papayes sont irradiees et entreposees pour une maturation a 22°C et 90% d'humidite relative. Les activites enzymatiques (polygalacturonase, pectinesterase, β-galactosidase, cellulase, oxidase) sont suivies pendant la maturation et correlees aux modifications dans la fermete, la couleur de la peau et la pulpe, la respiration et la production d'ethylene. L'irradiation n'a pas d'effet direct sur la fermete du fruit mais agit sur les enzymes des membranes cellulaires.

Changes of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Activity in Stressed Pinus Sylvestris Needles

Stimulation of ethylene biosynthesis in pine needles by hydrogen peroxide and sodium bisulfite coincided with the activation of ACC oxidase at the level of protein synthesis. Decrease in ethylene production at high concentrations of sodium bisulfite (above 7 mM) was apparently due to inhibition of ACC oxidase activity. Treatment of pine needles with aminotriazole caused an inhibition of both ethylene production and ACC oxidase activity. Both methylviologen and methyl jasmonate stimulated ACC oxidase activity in a concentration-dependent manner with no parallel changes in ethylene production. The presented results suggest that ACC oxidase plays an important role in regulation of ethylene formation in pine needles in response to different stimuli.

Effect of Nitrate and Aminoethoxyvinylglycine on Cicer Arietinum L. Nodules

The chickpea (Cicer arietinum L.) cv. HC-1 was raised in earthen pots filled with dune sand in screenhouse. At vegetative stage, i.e. 40 – 45 d after sowing, 10, 20 and 40 mM NO3− was applied through rooting medium. After 24 h of NO3− treatments an ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG) in concentration 5 μM was given. A conspicuous increase in (5 – 9 fold) ethylene evolution in nodules was noticed after NO3− treatments. This rise was parallel to the increase in 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC oxidase activity. On the contrary, a sharp decline in ACC content, ACC oxidase activity and ethylene evolution was observed when AVG was given. A decrease of in acetylene reduction assay (ARA) with NO3− treatments was associated with decline in cytosolic pH (from 6.12 to 5.45), leghemoglobin (Lb) content, accumulation of H2O2 and with the loss of membrane integrity. The lipid peroxidation, followed as MDA production and electrolyte leakage increased with NO3− treatments, however, the level of MDA was brought down in AVG-treated nodules. Results confirm that ethylene might be involved in mechanism by which the functioning of nodules is adversely affected by NO3−.

Regulation of genes encoding ethylene biosynthetic enzymes in peach (Prunus persica L.) fruit by carbon dioxide and 1-methylcyclopropene

Abstract We have cloned one member (PP-ACS1) of the 1-aminocyclopropane-1-carboxylate (ACC) synthase and two members (PP-ACO1 and PP-ACO2) of the ACC oxidase gene families in peach (Prunus persica L.) fruit and studied their expression characteristics during fruit ripening and treatment with CO2 and 1-methylcyclopropene (MCP), inhibitors of ethylene action. Northern analysis showed that the abundance of PP-ACS1, PP-ACO1 and PP-ACO2 mRNAs increased with fruit ripening in parallel with increases in ethylene production and activities of ACC synthase and ACC oxidase. The abundance of PP-ACO2 mRNA was much lower than that of PP-ACO1. CO2 and MCP treatment inhibited ethylene production, ACC synthase activity, and accumulation of PP-ACS1 mRNA. Although CO2 had little effect on ACC oxidase activity, it inhibited the accumulation of PP-ACO1 and PP-ACO2 mRNAs to the same levels as MCP. Wound-induced ethylene production, ACC synthase activity, and the abundance of PP-ACS1 mRNA were blocked and stimulated by CO2 and MCP, respectively. CO2 and MCP had no effect on wound-induced ACC oxidase activity but inhibited the accumulation of its mRNA. Wound-induced activities of ACC synthase and ACC oxidase, and abundance of their mRNAs were inhibited and stimulated, respectively, by exogenous ethylene. The translational inhibitor, cycloheximide inhibited wound-induced ethylene biosynthesis but super-induced the accumulation of PP-ACS1 and PP-ACO1 mRNAs, suggesting that their induction is a primary response to the inducer. These results suggest that the expression of PP-ACS1 and PP-ACO1 genes play a key role in the regulation of ethylene biosynthesis in peach fruit during ripening and in response to wounding. The results also indicate that wound-induced PP-ACS1 and PP-ACO1 genes are under negative and positive control, respectively. Further, using MCP we provide evidence indicating that CO2 does not regulate ACC synthase activity and expression of the PP-ACS1 gene in peach fruit during ripening and in response to wounding by antagonizing ethylene action.

Physiology of pre-cut mango. I. ACC and ACC oxidase activity of slices subjected to osmotic dehydration

Physiologically mature mangoes were ripened for 6 days at 24°C and 98% relative humidity. Slices from these fruits were osmotically dehydrated by immersion in sucrose 65°Brix at 30°C and 211 mbar vacuum during 30 min. Slices not subjected to osmotic dehydration (NOD) and slices with osmotic dehydration (OD) were stored at 24, 13 or 5°C. The respiration rate of both slice types was affected by the storage temperature. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthesis indicated activity of ACC synthase in both slices as well as in the whole fruit. ACC oxidase activity was greater in OD slices as compared to NOD and that was associated to better membrane stability. OD favored compaction of external layer in slices. No ethylene was detected in slices; however, the tissues did not lose their ability to synthesize ethylene. Results suggest that OD under vacuum may be beneficial as a pre-treatment of mango slices for longer shelf life under refrigeration.

ACC oxidase is found in seedlings of two (Coniferales, Gnetales) of the four gymnosperm orders

Leaf material and seedlings of species representing all gymnosperm orders were tested for 1‐aminocyclopropane‐1‐carboxylate (ACC) oxidase activity. Seedlings of Pinus nigra, Pinus radiata, Pseudotsuga menziesii, Cupressocyparis leylandii, Ephedra major and E. nevadensis showed high level in vitro ACC oxidase activity. The enzyme from seedlings of Pinus nigra var. nigra (Arnold) was shown to resemble the angiosperm enzyme in a requirement for ascorbate, carbon dioxide and Fe(II). In contrast, seedlings of Ginkgo biloba, Dioon edule, Zamia furfuraceae and Cycas revoluta showed no detectable ACC oxidase activity. Leaf material from species representing all orders of gymnosperms was also tested for ACC oxidase activity in vitro, but none could be demonstrated. The results presented here support an origin of ACC oxidase in a common ancestor of the angiosperms, Gnetales and Coniferales.

Ethylene evolution and antioxidant defence mechanism in Cicer arietinum roots in the presence of nitrate and aminoethoxyvinylglycine

The plants of chickpea ( Cicer arietinum L.) cv. HC-1 were raised in earthen pots filled with 5 kg washed river sand under natural conditions of the screen house. Forty to forty-five days after sowing, the roots were exposed to 10, 20 and 40 mM NO 3 – through rooting medium with the aim for studying NO 3 – induced changes in ethylene production and root functioning. Aminoethoxyvinylglycine (AVG, 5 μM), an ethylene biosynthesis inhibitor, was added after 24 h of NO 3 – treatments. Three days after the treatments, NO 3 – applied at 10 mM induced a significant increase in 1-amino cyclopropane-1-carboxylic acid (ACC) content (49 %) and in vivo ACC oxidase activity (100 %) which were associated with an increase in ethylene production (25 %) and with an important increase in both H 2O 2 (60 %) and malondialdehyde (MDA, 1 270 %). In contrast, supply of NO 3 – at 40 mM resulted in an important increase in ethylene production (400 %) by inducing significant enhancement of ACC content (123 %), ACC oxidase activity (128 %) and with simultaneous increase of H 2O 2 (160 %) and MDA (1 900 %). The reversible membrane alteration showed a linear correlation with ethylene production. This was confirmed by a sharp decline in NO 3 – induced ethylene evolution by AVG. An oxidative damage by NO 3 – to the defence mechanism was evident from the increase in H 2O 2 content which is apparent from the decrease in activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.11.1.9) to 23–67, 39–43, 33–55 and 26–45 %, respectively. The AVG treatment showed partial recovery in the activities of these enzymes and decrease in H 2O 2 content (17–32 %). It is inferred that ethylene evolution due to NO 3 – is the key marker interfering with the functioning of roots along with the weakening of antioxidant defence mechanism.

Ethylene feedback mechanisms in tomato and strawberry fruit tissues in relation to fruit ripening and climacteric patterns

Exposing pericarp tissue excised from immature tomato fruit to 4.5 mmol l 1 C2H4 revealed a negative C2H4 feedback mechanism in relation to its biosynthesis since ACC concentration and C2H4 production by the tissue were reduced. An opposite trend (positive C2H4 feedback mechanism) was observed in pericarp tissue excised from fruit at the pink stage. At the mature-green stage however, tissue showed a transition from negative to positive C2H4 feedback mechanism with the onset of tissue ripening. In strawberry tissues excised from green, white and half-coloured fruits however, C2H4 application caused a short-term increase in C2H4 production followed by a sharp reduction to the control level along with a marked reduction in ACC levels. In both tomato and strawberry fruit tissues, C2H4 application significantly induced ACC oxidase (ACO) activity at all ripening stages, as measured by in vivo ACC conversion to C2H4. This strongly suggests that ACC synthesis is the limiting step in C2H4 autocatalysis and the only limiting step in C2H4 autoinhibition. In tomato pericarp tissues, C2H4 autoinhibition and autocatalysis caused by C2H4 application in immature and pink fruits, respectively, were eliminated when tissues were transferred to air and re-occurred when tissues were returned back to C2H4. These responses did not occur in all strawberry tissues due to the sharp reduction in C2H4 production with the time course of C2H4 application. Inhibiting C2H4 action with STS pretreatment inhibited both negative and positive C2H4 feedback mechanisms in both tomato and strawberry tissues indicating that C2H4 feedback mechanism is one sort of C2H4 action. In addition, only tomato fruit tissue showed significant increases in CO2 production with C2H4 application. In contrast to the nonclimacteric behaviour of strawberry fruit which exhibits only a negative C2H4 feedback mechanism, these data strongly suggest that the transition of the C2H4 feedback mechanism from negative to positive, which occurs in tomato fruit only with ripening initiation and progress, may be the reason behind the climacteric behaviour of tomato fruit. © 2000 Elsevier Science B.V. All rights reserved.

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase.

Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination.

Conditioning period, CO2 and GR24 influence ethylene biosynthesis and germination of Striga hermonthica

Germination of witchweed (Striga hermonthica [Del.] Benth), an important root parasite on poaceous crops, requires pretreatment ‘conditioning’ in a warm moist environment and a subsequent exposure to a stimulant. The roles of conditioning period, CO2 and a strigol analogue (GR24) in ethylene biosynthesis and germination of the parasite were investigated. Conditioning increased the seeds’ capacity to oxidize exogenous 1‐aminocyclopropane‐1‐carboxylic acid (ACC). Exogenous CO2 increased the seeds capacity to oxidize ACC by 3‐ to 9‐fold. A combination of GR24 and ACC increased ethylene production by more than 3‐fold in comparison with the rates obtained using these compounds separately. Aminoethoxyvinylglycine (AVG) completely inhibited ethylene induction by GR24, but not by ACC. A GR24 treatment, made subsequent to conditioning in GR24, did not induce ethylene. However, seeds conditioned in GR24 and then given 1 mM ACC produced 293 nl l−1 ethylene. ACC oxidase (ACCO) activity in crude extracts was increased by conditioning and CO2. The enzyme displayed an absolute requirement for ascorbate. Absence of exogenous Fe2+ reduced enzyme activity only by 14%. GR24 applied during conditioning reduced germination in response to a subsequent GR24 treatment. ACC was, invariably, less effective in inducing S. hermonthica germination than GR24 even at concentrations which induce more ethylene than concurrent GR24 treatments. The results are consistent with a model in which conditioning removes a restriction on the ethylene biosynthetic pathway in S. hermonthica seeds. GR24 modulates the key enzymes in ethylene biosynthesis. The stimulant suppresses ethylene biosynthesis in unconditioned seeds and promotes it in conditioned ones. Germination of S. hermonthica results from the joint action of GR24 and the ethylene it induces.

Ripening Physiology in `Navaho' Thornless Blackberries: Color, Respiration, Ethylene Production, Softening, and Compositional Changes

Fruit were harvested from an erect, thornless blackberry (Rubus L. subgenus Rubus Watson, `Navaho' to study ripening changes. Soluble solids content increased between the red (unripe) and dull-black (overripe) stages of ripening while titratable acidity decreased sharply between the mottled and shiny-black ripeness stages. Anthocyanin content increased sharply between the mottled and shiny-black stages. Firmness of drupelet and receptacle tissues decreased between the mottled and shiny-black stages of ripeness. In whole blackberries, total uronic acids decreased, and water soluble uronic acids increased between the green-red and shiny-black ripeness stages. Volatile production paralleled ripening changes, and was highest in dull-black fruit, with alcohols and aldehydes predominating. Respiration of intact fruit maintained in water decreased between the green and red ripeness stages and increased at the mottled (part-black) and black ripeness stages. Ethylene production remained below 10 nmol·kg-1·h-1 until the dull-black (overripe) stage of maturity. Free 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC oxidase did not increase in berries until the shiny-black stage, corresponding with the onset of detectable ethylene production. ACC oxidase activity decreased in the drupelet tissue (0.5 to 0.01 μmol·kg-1·h-1) and increased in the receptacle tissue (2 to 3.8 μmol·kg-1·h-1) as fruit changed from red to dull black. These results indicate that ripening in blackberries may be initiated in the receptacle tissue. Ripening in blackberries is likely independent of ethylene, but ethylene may regulate berry detachment from pedicels, thus controlling timing of fruit harvests.

Control of ethylene biosynthesis and softening in ‘Cox’s Orange Pippin’ apples during low-ethylene, low-oxygen storage

Abstract The response of apples (Malus domestica Borkh.) cv. Cox’s Orange Pippin (‘Cox’) to low ethylene, controlled atmosphere (CA) storage was examined in two seasons. The content of 1-aminocyclopropane-1-carboxylic acid (ACC) and the activity of ACC oxidase (measured as the capacity for formation of ethylene from exogenous ACC) in cortical tissue of apple fruits from low ethylene containers, remained constant for about the first 20 weeks of storage, before increasing. Removal of ethylene from the storage atmosphere was more effective at reducing softening in

A reliable system for the transformation of cantaloupe charentais melon ( Cucumis melo L. var. cantalupensis) leading to a majority of diploid regenerants

An efficient system of transformation leading to a majority of transformed diploid plants from leaf explants of Cucumis melo L. var. Cantalupensis (cv. Védrantais) was developed. Several regeneration protocols using cotyledon or leaf explants were analysed with particular emphasis on the regeneration efficiency and the ploidy level of the regenerated melon plants. The use of leaf explants excised from 10 day-old seedlings, cultured in Murashige and Skoog’s medium supplemented with 1 μM 6-benzylaminopurine (BAP) and 1 μM 6-(γ,γ-dimethylallylamino)-purine (2iP), resulted in a high regeneration frequency (73%). In these conditions, more than 84% of the regenerated plants were found to be diploid. Addition of an Agrobacterium-mediated transformation step did not significantly change the percentage (81.8%) of diploid plants regenerated. This protocol was successfully used to produce diploid transgenic melon plants expressing the antisense ACC oxidase gene, encoding ACC oxidase which catalyses the last step of ethylene biosynthesis. Ethylene production and ACC oxidase activity of the leaf explants from transgenic plants was reduced by more than 80% as compared to the control untransformed tissues. This transformation/regeneration method could be routinely used for the introduction of other genes of interest in melon.

Effect of Methyl Jasmonate on Senescence of Broccoli Florets.

Broccoli (Brassica oleracea L. cv Italica) florets senesced rapidly after harvest at ambient temperatures. During senescence at 20°C, the rate of ethylene production of florets significantly increased, concurrent with a rapid yellowing of sepals ascribed to chlorophyll degradation. 1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase activity in florets also rapidly increased to a peak ; the enzyme activity then declined sharply, paralleling the increased pattern of ethylene production. Treatment of florets with 1 mM methyl jasmonate (MJ) significantly promoted ethylene production and ACC oxidase activity during senescence, attaining a higher peak faster than the control. Chlorophyll loss and a concurrent enhancement of ACC synthase activity by exogenous 1 mM MJ were accelerated to a greater extent than those of the control. The rise in ethylene production was reduced and delayed by treatment with 10 mM diethyldithiocarbamate (DIECA) that is thought to be an inhibitor of jasmonate biosynthesis. These results suggest that jasmonates may be associated with promoting senescence by enhancing ethylene production in broccoli florets.