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 biosynthesis in tissues of young and mature avocado fruits

Sitrit, Y., Blumenfeld, A. and Riov, J. 1987. Ethylene biosynthesis in tissues of young and mature avocado fruits.
Avocado (Persea americana Mill.) fruit tissues differ greatly in their capability to pro duce wound ethylene. In fruitlets, the endosperm lacks the ability to produce ethylene because no 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized and no activity of the ethylene-forming enzyme (EFE) is present. The cotyledons (embryo) do not produce significant amounts of ethylene at any of the developmental stages of the fruits, although in both young and mature fruits they contain a relatively high level of ACC synthase (EC 4.4.1.-) activity. Because of the very low EFE activity present in the cotyledons, most of the ACC formed in this tissue is conjugated. Of the various fruitlet tissues, the seed coat has the highest potential to produce ethylene. This is due to a high ACC synthase activity and particularly a high EFE activity. Also, the seed coat is very sensitive to the autocatalytic effect of ethylene. Fruitletpericarp possesses a lower potential to produce ethylene than the seed coat. Towardruit maturiy, the endosperm disappears and the seed coat shrivels and dies so that the pericarp and the cotyledons remain as the only active tissues in the mature fruit. At this stage, the pericarp is the only tissue producing ethylene. Mature precli macteric pericarp has a lower potential to produce ethylene than fruitlet pericarpThe role of ethylene in regulating various physiological processes at different stages of fruit maturation is discussed.     

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.     

Regulation of Ethylene Biosynthesis in Avocado Fruit during Ripening

Preclimacteric avocado (Persea americana Mill.) fruits produced very little ethylene and had only a trace amount of l-aminocyclopropane-1-carboxylic acid (ACC) and a very low activity of ACC synthase. In contrast, a significant amount of l-(malonylamino)cyclopropane-1-carboxylic acid (MACC) was detected during the preclimacteric stage. In harvested fruits, both ACC synthase activity and the level of ACC increased markedly during the climacteric rise reaching a peak shortly before the climacteric peak. The level of MACC also increased at the climacteric stage. Cycloheximide and cordycepin inhibited the synthesis of ACC synthase in discs excised from preclimacteric fruits. A low but measurable ethylene forming enzyme (EFE) activity was detected during the preclimacteric stage. During ripening, EFE activity increased only at the beginning of the climacteric rise. ACC synthase and EFE activities and the ACC level declined rapidly after the climacteric peak. Application of ACC to attached or detached fruits resulted in increased ethylene production and ripening of the fruits. Exogenous ethylene stimulated EFE activity in intact fruits prior to the increase in ethylene production. The data suggest that conversion of S-adenosylmethionine to ACC is the major factor limiting ethylene production during the preclimacteric stage. ACC synthase is first synthesized during ripening and this leads to the production of ethylene which in turn induces an additional increase in ACC synthase activity. Only when ethylene reaches a certain level does it induce increased EFE activity.     

Regulation by CO2 of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climacteric fruits

Cheverry, J. L., Sy, M. O., Pouliquen, J. and Marcellin, P. 1988. Regulation by CO₂ of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climateric fruits. - Physiol. Plant. 72: 535–540.
A high CO₂ concentration (20%) at 20°C rapidly and strongly inhibited the development of the climacteric ethylene burst in apple ( Malus domestica Borkh. cv. Granny Smith) and avocado ( Persea americana Mill. cv. Fuerte) fruits and did not change 1-aminocyclopropane-l-carboxylic acid (ACC) content. Treatment with 20% CO₂ markedly decreased ACC-dependent ethylene biosynthesis at 20°C in climacteric pericarp tissues. It is suggested, therefore, that high CO₂ levels inhibit conversion of ACC to ethylene.
Synthesis of the ethylene forming enzyme (EFE) was enhanced when intact preclimacteric apples or early climacteric avocados were pretreated for 40 h with 10 μ11^-1 ethylene. When CO₂ (20%) and ethylene were both applied, a reduced stimulatory effect of ethylene on EFE synthesis was observed. A high CO₂ concentration enhanced EFE acivity in excised tissues of apples and avocados incubated with ACC (2 m M ) and cycloheximide (1 m M ) or 2–5-norbornadiene (5 ml 1^-1). In the autocatalytic process, 20% CO₂ antagonized the stimulation of EFE synthesis by ethylene, but promoted EFE activity.     

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     

Effect of temperature on ethylene biosynthesis in carnation petals

Carnation petals, at a stage in which they are already producing ethylene, show a sigmoidal dependency of ethylene production on temperature within the range of 0 to 30°C. An Arrhenius plot of these data show a break atca. 22°C in the straight lines connecting the points. The activity of the ethylene-forming enzyme (EFE), measured bothin vitro, using isolated membranes, andin vivo, using petals pretreated with 1-aminocyclopropane-1-carboxylic acid (ACC), shows an exponential dependency on temperature within the same range. Arrhenius plots of EFE activity fail to show any discontinuity.In contrast, ACC synthase activity measuredin vitro shows the same sigmoidal dependency on temperature as that of the intact petals. We suggest, therefore, that ACC synthase activity is the rate-limiting step mediating the influence of temperature on ethylene biosynthesis by carnation petals over the range studied.     

Radiolabeling of a wound-inducible pyridoxal phosphate-utilizing enzyme: evidence for its identification as ACC synthase

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase, a pyridoxal phosphate-utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate-limiting step in the biosynthesis of the plant hormone ethylene. We report the partial purification (400-fold) of ACC synthase from wounded pink tomato pericarp. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase-enriched fraction was achieved by reduction using tritiated sodium borohydride. Evidence that this radiolabeled protein is ACC synthase is presented.     

Induction of 1-Aminocyclopropane-1-carboxylic Acid Synthase in Wounded Mesocarp Tissue of Winter Squash Fruit and the Effects of Ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase activityincreased rapidly after wounding of mesocarp tissue of wintersquash fruit (Cucurbita maxima Duch.) and reached a peak at16 h after excision and then declined sharply. The rise in ACCsynthase activity was followed by increases in the endogenousACC content and the rate of ethylene production. The activityof ethylene forming enzyme (EFE) also increased rapidly in theexcised discs of mesocarp of winter squash fruit. ACC synthase activity was strongly inhibited by aminoethoxyvinylglycinewith a Ki value of 2.1 µM. Michaelis-Menten constant ofACC synthase for S-adenosylmethionine was 13.3 µM. Ethylene suppressed the induction of ACC synthase in the woundedmesocarp tissue. The suppression by ethylene increased withthe increasing concentrations of applied ethylene and the maximumeffect was obtained at about 100 µl 1^–1 ethylene,at which point the induction was suppressed by 54%. Ethylenedid not inhibit ACC synthase activity, nor did it suppress theinduction of EFE, but rather it slightly enhanced the latter. (Received August 24, 1984; Accepted October 29, 1984)     

Isolation of a Tomato Protease that May Be Involved in Proteolysis of 1-Aminocyclopropane-l-Carboxylate Synthase

1-aminocyclopropane- 1-carboxylate （ACC） synthase is a principal enzyme that catalyses the committed step in phytohormone ethylene biosynthesis. Previous evidence indicates that the hypervariable C-terminus of ACC synthase is most likely to be processed proteolytically in vivo. However, the protease responsible has not been identified thus far. In the present study, we detected proteolytic activity against ACC synthase （LeACS2） in tomato （Lycopersicon esculentum Mill.） fruit extract based on a newly established in vitro assay system. Purification of the protease through DEAE, gel filtration and MonoQ chromatography resulted in considerable enrichment of a 64-kDa protein species. Subsequent biochemical analysis of the purified tomato protease revealed that the optimal conditions for its proteolytic activity were at pH 8.0 and at 37 ~C. In addition, the protease activity was blocked completely by the metalloprotease inhibitor 1,10-phenanthroline. The present study represents the first report on the isolation of an ACC synthase- processing protease from plant tissues.     

The effect of plant-hormone pretreatments on ethylene production and synthesis of 1-aminocyclopropane-1-carboxylic acid in water-stressed wheat leaves

Excised wheat (Triticum aestivum L.) leaves, when subjected to drought stress, increased ethylene production as a result of an increased synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and an increased activity of the ethyleneforming enzyme (EFE), which catalyzes the conversion of ACC to ethylene. The rise in EFE activity was maximal within 2 h after the stress period, while rehydration to relieve water stress reduced EFE activity within 3 h to levels similar to those in nonstressed tissue. Pretreatment of the leaves with benzyladenine or indole-3-acetic acid prior to water stress caused further increase in ethylene production and in endogenous ACC level. Conversely, pretreatment of wheat leaves with abscisic acid reduced ethylene production to levels produced by nonstressed leaves; this reduction in ethylene production was accompanied by a decrease in ACC content. However, none of these hormone pretreatments significantly affected the EFE level in stressed or nonstressed leaves. These data indicate that the plant hormones participate in regulation of water-stress ethylene production primarily by modulating the level of ACC.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - BA N⁶-benzyladenine - EFE ethylene-forming enzyme - IAA indole-3-acetic acid     

PRO－LONG涂膜处理对后熟香蕉果实乙烯形成以及其它有关生理变化的影响

香蕉（ＭｕｓａａｃｕｍｉｎａｔａＣｏｌｌａｃｖ．ＤｗａｒｆＣａｖｅｎｄｉｓｈ）果实采后以商业上推荐使用的１．５％Ｐｒｏ－ｌｏｎｇ溶液处理,贮藏于２０℃和７５％相对湿度下,分别测定果实的ＡＣＣ含量、ＭＡＣＣ含量、ＥＦＥ酶活性、乙烯释放、叶绿素含量的变化和果实的硬度变化．结果表明,ＰＲＯ－ＬＯＮＧ处理延缓了香蕉果实果皮的叶绿素降解、硬度的下降以及乙烯释放的增加．在后熟过程中,处理果实的ＡＣＣ含量发生积累．ＡＣＣ含量的高峰在乙烯释放高峰和ＥＦＥ酶活性高峰之前出现．与对照比较,处理果实的ＡＣＣ含量和ＥＦＥ酶活性的高峰延迟了５ｄ出现．在后熟过程中,以Ｐｒｏ－ｌｏｎｇ处理果肉四片,其ＥＦＥ酶活性受部分抑制（抑制率为１９．４５％至４０．５１％）．果实ＭＡＣＣ含量在贮藏起初处于一个较显著水平,随着后熟的发展而逐步增加,但与ＡＣＣ含量的明显增加相比变化是微小的．我们的研究进一步阐明了ＰＲＯ－ＬＯＮＧ涂膜对香蕉果实后熟的影响主要是通过减少氧的供给,部分地抑制了ＥＦＥ酶活性,延缓了乙烯的形成和释放,从而延长了后熟过程．     

Properties and Partial Purification of 1-Aminocyclopropane-1-carboxylate Synthase

We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato (Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on ²H₂O as compared to ACC synthase from H₂O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of l-[³⁵S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding.     

Induction by Electric Currents of Ethylene Biosynthesis in Cucumber (Cucumis sativus L.) Fruit

The effects of an electric current on ethylene biosynthesis were investigated in cucumber (Cucumis sativus L.) fruit that were producing almost no ethylene. Direct currents at 0.5 to 3.0 milliamperes induced much ethylene synthesis, with a rapid continuous increase in the rate, which reached a peak within 5 to 6 hours and then decreased. The rate of production was greater with a stronger current. Ethylene production was not observed after the use of a sine-wave alternating current (60 hertz) at 3 milliamperes, the magnitude at which a direct current had the greatest effect. The activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ethylene forming enzyme (EFE) increased before the rise in ethylene production. ACC synthase and EFE were activated sixfold and fourfold, respectively, by 2 hours. The concentration of ACC increased linearly up to 6 hours and then decreased. Ethylene induction by an electric current was suppressed almost completely by the infiltration of the cucumbers with 5 millimolar aminooxyacetic acid, an inhibitor of ACC synthase, and was also suppressed 70% by 5 millimolar salicylic acid, an inhibitor of EFE. The results indicate that the ethylene induced by the direct current was synthesized via the ACC-ethylene pathway as a result of electrical stress, a new kind of stress to be identified.