Glucose Inhibits ACC Oxidase Activity and Ethylene Biosynthesis in Ripening Tomato Fruit

Experiments were carried out to evaluate the effect of glucose on ripening and ethylene biosynthesis in tomato fruit (Lycopersicon esculentum Mill.). Fruit at the light-red stage were vacuum infiltrated with glucose solutions post-harvest and changes in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, ACC oxidase, and ethylene production monitored over time. ACC oxidase activity was also measured in pericarp discs from the same fruits that were treated either with glucose, fructose, mannose, or galactose. While control fruit displayed a typical peak of ethylene production, fruit treated with glucose did not. Glucose appeared to exert its effect on ethylene biosynthesis by suppressing ACC oxidase activity. Fructose, mannose, and galactose did not inhibit ACC oxidase activity in tomato pericarp discs. Glucose treatment inhibited ripening-associated colour development in whole fruit. The extent of inhibition of colour development was dependent upon the concentration of glucose. These results indicate that glucose may play an important role in ethylene-associated regulation of fruit ripening.     

Ethylene biosynthesis and respiration in strawberry fruit treated with diazocyclopentadiene and IAA

Diazocyclopentadiene (DACP), an inhibitor of ethylene action, was used to investigate the role of ethylene receptor(s) in regulating ethylene biosynthesis and respiration in strawberry fruit. DACP stimulated ethylene production of fruit at all stages of maturity. This stimulation was mainly due to an increase in ACC content in the tissue without significantly changing ACC oxidase activity. DACP did not induce any change in respiration in fruit at various stages of maturity except the early green stage (green I). We also investigated the possible interaction of DACP and IAA in ethylene production. Results indicated that all treatments increased ethylene production compared to the control although the absolute ethylene production differed in the order IAA plus DACP > only DACP > only IAA > control. IAA stimulated ethylene production without change of ACC oxidase activity at 1 day after treatment in strawberry fruit at pink stage. Results suggest that ethylene biosynthesis in nonclimacteric strawberry fruit at various stages of maturity may be regulated by ethylene receptor(s) with inhibition of ethylene production. DACP may release this inhibitory effect, and resulting in increasing ethylene production. Results also indicated that respiration may not be regulated by an ethylene receptor in strawberry fruit at most stages of maturity. DACP and IAA showed interaction in regulation of ethylene production which was caused by an increase in ACC content, not ACC oxidase activity.     

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     

Biosynthesis of ethylene in higher plants: the metabolic site of inhibition by phosphate*

Abstract. Phosphate inhibited endogenous as well as 1-aminocyclopropane-1-carboxylic acid (ACC)-stimulated ethylene synthesis in slices of tomato fruit, segments of carrot root and pea hypocotyls. ACC concentrations of up to 10 mol m^?3 did not overcome this inhibition. Phosphate inhibited the conversion of ¹⁴C ACC to ethylene in tomato fruit and vegetative tissue. Enzymatic conversion of ACC to ethylene by pea seedling homogenate was also inhibited by phosphate with a linear concentration dependency. The formation of ACC from S-adenosylmethionine (SAM) by extracts of pink tomatd fruit was slightly, but not significantly, affected by phosphate. However, the SAM to ACC conversion was greater when extracts from tomato fruit were made in phosphate rather than in HEPES-KOH buffer. Non-enzymatic ethylene synthesis from ACC in a model system was stimulated by phosphate. We suggest that phosphate is an inhibitor of ethylene biosynthesis in higher plants and that one site of its control is the conversion of ACC to ethylene.     

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

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

Recovery of ethylene biosynthesis in diazocyclopentadiene (DACP)-treated tomato fruit

Diazocyclopentadiene (DACP), a competitive ethylene action inhibitor binds irreversibly to the ethylene receptor to reduce tissue responses to ethylene. Tomato fruit (Lycopersicon esculentum Mill cv lsquo;Rondellorsquo;) were treated with DACP at the mature green stage. Ethylene biosynthesis and respiration rate were depressed. Color changes from green to red were delayed. Compared to the control, ACC content increased and ACC oxidase activity in vivo decreased in DACP-treated fruit. Thus, decrease of ethylene production caused by DACP treatment was due to the reduction of ACC oxidase activity. The decline in ripening subsequently recovered after DACP treatment. Results from the Northern analysis for gene expression of ACC synthase and ACC oxidase, showed that expression of both genes declined in DACP-treated fruit, and then recovered. Therefore the recovery of ethylene production was due to the recovery in gene expression and activity of ACC oxidase. We conclude that the effects of DACP on ethylene biosynthesis are on expression of ACC synthase and ACC oxidase genes, and/or regulation of ACC oxidase activity.     

Cobalt inhibition of thigmomorphogenesis in Bryonia dioica: possible role and mechanism of ethylene production

Rubbing-induced inhibition of elongation in Bryonia dioica was completely prevented by 10⁻⁷ M cobalt chloride. Cellular redistribution of peroxidases, mainly characterized by transiently enhanced membrane-binding of soluble peroxidases, occurred as an immediate consequence of rubbing and was not inhibited by Co²⁺. Ethylene synthesis and 1-aminocyclopropane-1-carboxylic acid (ACC) conversion readily increased upon rubbing and fell soon afterwards, but ACC conversion then increased again progressively. Co²⁺ did not drastically counteract these changes, except for the second rise in ACC conversion which was completely eliminated. The rubbing-induced rise in ethyiene production and ACC conversion was closely correlated to microsomal ACC conversion and peroxidase activity, but only during the first hours after rubbing. The presented approach enables us to correlate stress-induced ethylene production to membrane-binding of peroxidases. It is suggested that ACC conversion in Bryonia dioica is triggered by two different, sequentially ordered mechanisms. The difference in the effects of Co²⁺ on elongation and ethylene production is discussed with respect to the role of ethylene in thigmomorphogensis.     

钙对香蕉采后乙烯释放的抑制

本实验用CaCl_2溶液对香蕉(Musa acuminata cf. 'Dwarf Davendish')组织进行真空浸透处理,研究Ca~(2 )对香蕉采后乙烯释放、EFE活性、ACC水平以及ACC/MACC比值的影响。结果表明,Ca~(2 )处理可抑制香蕉果皮和果肉组织乙烯生成,对抑制果皮的乙烯生成尤为明显。Ca~(2 )处理还可降低内源ACC水平,抑制EFE活性。结果还显示,Ca~(2 )处理对组织中ACC/MACC比值有一定影响。     

Coleoptile removal-induced ethylene production in winter rye seedlings

Coleoptile removal-induced ethylene production was investigated in light-grown winter rye seedlings. Removal of the coleoptile induced 1-aminocyclopropane-l-carboxylic acid (ACC) synthesis and ethylene production by primary leaves and caused an inhibition of elongation growth of the leaves. The activity of ethylene-forming enzyme (EFE) was associated with the increase in ethylene evolution. Both rise in ethylene and ACC production, as well as EFE activity were inhibited by cycloheximide. Wounding the tissue 40 min after the initial treatment resulted in the second increase in ethylene evolution. Derooting of the seedlings without coleoptile removal did not induce ethylene production. It is suggested that the coleoptile represents a barrier for wound-induced ethylene production from actively growing leaf tissue.     

Methyl jasmonate effects on ethylene synthesis and organ-specific senescence in Helianthus annuus seedlings

Both methyl jasmonate (MJ) and ethylene have been implicated in promoting senescence, but the specific roles of each and the mechanisms by which they act are not well known. We tested the possibility that MJ and ethylene interact to promote senescence. In sunflower seedlings, the ability of MJ to affect ethylene metabolism was investigated in hypocotyls, cotyledons, and leaves. 1-aminocylcopropane-1-carboxylic acid (ACC)-dependent ethylene production was promoted to different extents depending on the organ and the age of the tissue. Newly emerged hypocotyls were sensitive to MJ, but became desensitized as the cotyledons emerged. The cotyledons increased and peaked in MJ sensitivity from emergence to the production of the primary leaves. Leaves were found to be somewhat insensitive to MJ treatment compared to cotyledons at all ages tested. In cotyledons, MJ also promoted ACC and ethylene production. However the changes in ACC, and ACC-dependent ethylene production were not directly correlated with those in ethylene production with respect to MJ concentration or tissue age. Moreover, changes in ACC-dependent ethylene production did not correlate with in vitro ACC oxidase activity. We hypothesized that MJ affects ethylene production by increasing the spatial access of ACC to ACC oxidase perhaps through increased membrane permeability. Ethylene was not involved in the MJ-induced loss of chlorophyll. But the breakdown of cell integrity and cell membranes (estimated by monitoring conductivity of the solution that bathed the cotyledons) was greatly and synergistically promoted by the combination of MJ and ethylene. Promotion of membrane breakdown by MJ and ethylene could be inhibited by treatments with ethylene inhibitors (STS or CoCl₂), and neither MJ nor ACC treatment alone could induce as much membrane breakdown as both together. We suggest that MJ and ethylene interact to accelerate some aspects of senescence in specific organs for nutrient remobilization for the benefit of the whole plant.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MJ methyl jasmonate - STS silver thiosulphate     

ACC conversion to ethylene by sunflower seeds in relation to maturation,germination and thermodormancy

Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene was studied in sunflower (Helianthus annuus L., cv. Mirasol) seeds in relation to germinability. Ethylene production from ACC decreased during seed maturation, and non-dormant mature seeds were practically unable to synthesize ethylene until germination and growth occurred, indicating that ethylene forming enzyme (EFE) activity developed during tissue imbibition and growth. ACC conversion to ethylene was reduced by the presence of pericarp, and in young seedlings it was less in cotyledons than in growing axes.ACC conversion to ethylene by cotyledons from young seedlings was optimal at c. 30°C, and was strongly inhibited at 45°C. Pretreatment of imbibed seeds at high temperature (45°C) induced a thermodormancy and a progressive decrease in EFE activity.Abscisic acid and methyl-jasmonate, two growth regulators which inhibit seed germination and seedling growth, and cycloheximide were also shown to inhibit ACC conversion to ethylene by cotyledons of 3-day-old seedlings and by inbibed seeds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - CH cycloheximide - EFE ethylene forming enzyme - IAA indole-3-acetic acid - Me-Ja methyl-jasmonate     

Characterization of ethylene production in developing strawberry fruit

Ethylene production, ACC content, and ACC oxidase activity were determined in strawberry fruit harvested at different stages of development and in fruit harvested green and developed in vitro in solutions containing sucrose. In fruit harvested at progressive stages of development from green through full ripe, ethylene production and ACC oxidase activity decreased whereas ACC content increased between the white and pink stages. Fruit detached at the green stage and developed to full ripe by immersion of the cut pedicel in sucrose solutions exhibited an increase in ACC content, decreased ethylene production, and no change in ACC oxidase activity. Detached green fruit provided with sucrose containing 0.5 mM silver (STS) had elevated ethylene production and more ACC oxidase activity than did fruit incubated without the silver salt. Green fruit provided with sucrose containing 1 mM ACC showed markedly increased ACC content, ACC oxidase activity, and ethylene production. These increases were noted following 4 days incubation in ACC, and were more pronounced after 11 days, at which time fruit of all treatments had attained a full-ripe stage of development. Calyx tissue exhibited more ACC oxidase activity, less ACC content, and similar ethylene production compared with receptacle tissue. ACC synthase could not be detected in fruit harvested at different developmental stages or in fruit detached and developed in vitro.abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - HQS 8-hydroxyquinoline hemisulfate - SAM S-adenosyl methionine - STS silver thiosulfate     

Inhibition of activity of the ethylene-forming enzyme by α(p-chlorophenoxy)isobutyric acid

(p-Chlorophenoxy)isobutyric acid (PCIB) inhibited indole-3-acetic acid (IAA)-induced ethylene production in etiolated mung bean hypocotyl sections. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC) was not significantly affected by PCIB, indicating that PCIB exerted its effect primarily by inhibiting the activity of the ethylene-forming enzyme (EFE). This conclusion was supported by the observations that PCIB inhibited the conversion of exogenously applied ACC to ethylene. The inhibitory effect of PCIB was already evident with 0.05 mM PCIB, and it increased with time after application of the inhibitor. PCIB also significantly inhibited ethylene production in apple fruit tissues, but it only slightly reduced the level of endogenous ACC. Similar to mung bean, EFE activity in apple tissue was significantly inhibited by PCIB. The possibility that PCIB also inhibits auxin-induced ACC synthase activity is discussed.     

Blue light-induced acidification of the medium of a nitrate-starved Chlorella mutant

Sunflower ( Helianthus annuus L.) seedlings were grown in aeroponic chambers which allowed for easy access to and easy harvesting of undamaged roots. In different portions of these roots we followed the rate of ethylene production, levels of 1-aminocyclopropane-1-carboxylic acid (ACC), N-malonyl-ACC and ACC oxidase mRNA and activity of ACC oxidase. ACC oxidase was measured with an in vitro assay, ACC and N-malonyl-ACC by selected ion monitoring gas chromatography-mass spectrometry. Ethylene production was highest in the tip of the root and tower in the middle and basal (part nearest the hypocotyl) portions of the root. The levels of ACC and ACC oxidase mRNA mirrored the levels of ethylene production. The lowest quantities of N-malonyl-ACC were found in the root tips. Upon gentle transfer of seedlings from an aeroponic system to treatment tubes, ACC content transiently increased; the greatest increase occurred in the tips. This brief rise in ACC content was not correlated with an increase in ethylene production. ACC oxidase activity was lowest in the tip and higher in the middle and base; the opposite of the pattern of ethylene production. Treating the seedlings with ACC produced a rapid rise in ACC content and ethylene production and inhibited root elongation. ACC oxidase activity was not induced by ACC treatment.     

Rapidly induced ethylene formation after wounding is controlled by the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis

Bean leaves from Phaseolus vulgaris L. var. Pinto 111 react to mechanical wounding with the formation of ethylene. The substrate for wound ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC). It is not set free by decompartmentation but is newly synthesized. ACC synthesis starts 8 to 10 min after wounding at 28°C, and 15 to 20 min after wounding at 20°C. Aminoethoxyvinylglycine (AVG), a potent inhibitor of ethylene formation from methionine via ACC, inhibits wound ethylene synthesis by about 95% when applied directly after wounding (incubations at 20°C). AVG also inhibits the accumulation of ACC in wounded tissue. AVG does not inhibit conversion of ACC to ethylene. Wound ethylene production is also inhibited by cycloheximide, n-propyl gallate, and ethylenediaminetetraacetic acid.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine - EDTA ethylenediaminetetraacetic acid     

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.