Wound Ethylene Research Articles

Initiation of Tomato Fruit Ripening with Copper

Abstract A vacuum infiltration technique that allowed precise control of both infiltration rate and amount of solution administered to whole tomato (Lycopersicon esculentum) fruit was developed. Controlled volumes of 5 mm solutions of CuSO4, Cu(NO3)2, HgCl2, CaSO4, KNO3, (NH4)2C2O4, Na2HPO4, citrate and 1 mm EDTA or EGTA were infiltrated into intact, mature-green tomato fruit and evaluated with regard to their effect on the pattern of tomato ripening. Copper significantly accelerated lycopene accumulation and influenced both the timing and magnitude of climacteric ethylene production. Infiltration with HgCl2 elicited similar effects as copper, but severe phytotoxicity was observed. In contrast, CaSO4, KNO3, and chelators had no significant effect on the pattern of ripening. Copper initiated wound ethylene production in the ripening mutant rin that reached up to 50% of the wound levels observed in normal fruit, but rin was not induced to ripen.

Effect of Red Light on the Synthesis of Wound Ethylene in Etiolated Pea Shoots

Apical segments from 7-day-old etiolated pea seedlings (Pisum sativum L. cv. Vorbote) produced 3 to 4 nl C(2)H(4) h(-1)g(-1) f.w. during the first 5 hours after excision. This formation of wound ethylene was due to a sharp increase of ACC concentration in the segments after exision. A 15 min red light pulse (approx. 0.22 W m(-2)) applied 3 hours before excision prevented the increase of ACC and inhibited wound ethylene formation. The lower ACC level was not caused by a higher rate of metabolism of ACC to MACC and also the capacity for the conversion of ACC to ethylene was not affected by the red light pulse. We therefore relate the lower ethylene formation after red light to an inhibition of wound ACC formation. The red light effect on wound ethylene production was reversible by far red light, so it can be explained as a phytochrome effect. Exogenous ethylene had a similar effect on the synthesis of wound ethylene as red light. Possible relations between these two effects are discussed.

Inhibition of Ethylene Synthesis in Tomato Plants Subjected to Anaerobic Root Stress

Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon esculentum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO(2+) prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co(2+) had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. All three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co(2+), conversion of both root- and petiole-synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low O(2) roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants.

Ethylene as an Effector of Wound-Induced Resistance to Cellulase in Oat Leaves

Peeling the abaxial epidermis from oat leaves (Avena sativa var. Victory) induces the formation of wound ethylene and the development of resistance to cellulolytic digestion of mesophyll cell walls. Ethylene release begins between 1 and 2 hours after peeling in the light or dark. Aminoethoxyvinylglycine (AVG, 0.1 millimolar), CoCl(2) (1.0 millimolar), propyl gallate (PG, 1.0 millimolar) or aminooxyacetic acid (AOA, 1.0 millimolar) inhibits, whereas AgNO(3) stimulates wound ethylene formation. Incubation on inhibitors of ethylene biosynthesis (AVG, CoCl(2), PG, AOA) or action (AgNO(3), hypobaric pressure or the trapping of ethylene with HgClO(4)) also prevents the development of wound-induced resistance to enzymic cell wall digestion. 1-Aminocyclopropane-1-carboxylic acid (ACC, 1.0 millimolar) reverses AVG (0.1 millimolar) inhibition of the development of resistance. Exogenous ethylene partially induces the development of resistance in unwounded oat leaves.These results suggest that peeling of oat leaves induces ethylene biosynthesis, which in turn effects changes in the mesophyll cells resulting in the development of resistance to cellulolytic digestion.

Stimulation of Ethylene Production in Citrus Leaf Discs by Mannitol

Wound ethylene formation induced in leaf tissue of citrus (Citrus sinensis [L.] Osbeck cv. "Washington Navel") by excision was significantly stimulated by mannitol after a lag period of about 6 hours. The extent of stimulation was dependent upon the concentration of mannitol (10 to 100 millimolar). This increased ethylene production was not simply due to osmotic effect or water stress as other osmoticums tested failed to exert such an effect. The stimulatory effect of mannitol resulted from both the enhancement of 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. The effect on the latter step was particularly pronounced in aged discs. The use of labeled mannitol showed that it was taken up by the leaf discs, utilized for respiration, and metabolized to sucrose, but no radioactivity was detected in the ethylene.

Autoinhibition of Ethylene Production in Citrus Peel Discs

Wound ethylene formation induced in flavede tissue of citrus fruit (Citrus paradisi MacFad. cv. Ruby Red) by slicing was almost completely inhibited by exogenous ethylene. The inhibition lasted for at least 6 hours after removal of exogenous ethylene and was then gradually relieved. The extent of inhibition was dependent upon the concentration of ethylene (1 to 10 microliters/liter) and the duration of treatment. The increase in wound ethylene production in control discs was paralleled by an increase in 1-aminocyclopropane-1-carboxylic acid (AAC) content, whereas in ethylene-treated discs there was little increase in ACC content. Application of ACC completely restored ethylene production in ethylene-pretreated discs, indicating that the conversion of ACC to ethylene is not impaired by the presence of ethylene. Thus, autoinhibition of ethylene synthesis was exerted by reducing the availability of ACC. Ethylene treatment resulted in a decrease in extractable ACC synthase activity, but this decrease was too small to account for the marked inhibition of ACC formation. The data indicate that autoinhibition of ethylene production in citrus flavede discs results from suppression of ACC formation through repression of the synthesis of ACC synthase and inhibition of its activity.

Enhancement of Wound-Induced Ethylene Synthesis by Ethylene in Preclimacteric Cantaloupe

Although intact fruits of unripe cantaloupe (Cucumis melo L.) produce very little ethylene, a massive increase in ethylene production occurred in response to excision. The evidence indicates that this wound ethylene is produced from methionine via 1-aminocyclopropanecarboxylic acid (ACC) as in ripening fruits. Excision induced an increase in both ACC synthase and the enzyme converting ACC to ethylene. Ethylene further increased the activity of the enzyme system converting ACC to ethylene. The induction by ethylene required a minimum exposure of 1 hour; longer exposure had increasingly larger effect. The response was saturated at approximately 3 microliters per liter ethylene and was inhibited by Ag(+). Neither ethylene nor ACC had a promotive or inhibitory effect on ACC synthase beyond the effect attributable to wounding.

Fruit Age and Changes in Abscisic Acid Content, Ethylene Production, and Abscission Rate of Cotton Fruits

The relationships of fruit age, abscisic acid (ABA) concentration, ethylene evolution, and abscission rates were studied in an effort to determine why cotton (Gossypium hirsutum L., cv. Deltapine 16) fruits rarely abscise more than 15 days after anthesis. Because abscission of cotton fruits is increased by conditions that limit photosynthesis, greenhouse-grown plants with fruits of various ages were placed in dim light for 3 days to induce high rates of fruit abscission. Abscission rates, ABA concentrations, and ethylene evolution rates were determined for fruits of various ages. Almost all of the young fruits abscised, but abscission rate declined with age until almost no abscission was observed in fruits that were 15 or more days past anthesis.Dim light increased the ABA concentrations of fruits that were 6 to 11 days old but did not increase ABA concentrations in fruits that were younger or older. The concentration of ABA declined with fruit age from peak values at 4 and 6 days after anthesis. Dim light also increased ethylene evolution from fruits up to 10 days old but had little effect on ethylene production or abscission of fruits more than 11 days old. Ethylene evolution declined with fruit age from peak values at 4 and 6 days after anthesis. Fruits of various ages (from plants not exposed to dim light) were sliced to induce high rates of wound ethylene production. The results indicated that the capacity for ethylene production declined with fruit age, parallel with a decline in abscission rate. Decreases in ABA concentration and ethylene evolution with fruit age indicate that change in the capacity to synthesize these hormones, especially in response to stress, is one cause of the decline in abscission rates as cotton fruits become older.

Effect of Wounding on ‘Fuerte’ Avocado Ripening1

Abstract Wounded mature ‘Fuerte’ fruit (Persea americana Mill.) ripened faster than non-wounded fruit when stored at 14°C. Significant differences were not observed in respiration or ethylene production between wounded and non-wounded fruit when stored at 20° but the former softened faster and showed greater polygalacturonase activity. Wounded and non-wounded fruit ripened at similar rates when stored after wounding for 10 days at 5° and thereafter transferred to either 14° or 20°. No “wound ethylene” could be detected immediately after wounding at any temperature and is not the earliest event occurring during ripening. Effects of wounding in metabolic processes of ripening are observed better at a moderate continuous storage temperature of 14° than at 20°.

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.

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.

Mechanism of effect of aging on membrane transport in leaf strips of Centranthus ruber: Possible ethylene involvement in cutting shock

Cerulenin has been used to investigate the mechanism by which leaf strips of Centranthus ruber (L.) Lam et D.C. increase their capacity for solute uptake during a period of incubation in CaSO4 ("aging"). α-Aminoisobutyric acid was used to assess uptake capability. The leaf strips developed their uptake capacity for at least 8-10 h after excision. Cerulenin, if added to the aging medium immediately after cutting or at any time during the aging process, almost completely halted this development, but did not bring about loss of the uptake capacity already achieved. That cerulenin was specifically interfering with fatty-acid biosynthesis was indicated by the fact that it drastically depressed incorporation of labelled acetate into the lipid fraction but did not affect the incorporation of labelled alanine. Cycloheximide strongly inhibited the development of uptake capacity, but sensitivity to actinomycin D was not evident for at least 2 h. The results are consistent with the concept that slicing leads to immediate damage to tissue membranes and that "aging" is a process of membrane repair, involving renewed synthesis of membrane lipids and membrane proteins.The damage to membranes associated with cutting may involve wound ethylene. This is indicated by the following findings: Treatment of aged leaf strips with Ethrel (2-chloroethylphosphonic acid) resulted in drastic loss of their acquired uptake capacity. The strips recovered from such Ethrel-induced loss of uptake capacity while aging in CaSO4 as they can after cutting. Cerulenin halted recovery of uptake capacity after ethrel treatment just as it did after cutting. Treatment of leaves before cutting with amino-ethoxyvinylglycine somewhat improved the uptake performance of leaf strips immediately after excision.

Biosynthesis of Wound Ethylene

Untreated mung bean hypocotyls produced very little C(2)H(4) but, upon treatment with 10 millimolar Cu(2+) or 10 millimolar Cu(2+) + 10 millimolar Ca(2+), C(2)H(4) production increased 20- and 40-fold, respectively, within 6 hours. This increase in C(2)H(4) production was preceded and paralleled by an increase in 1-aminocyclopropanecarboxylic acid (ACC) content, but the level of S-adenosylmethionine (SAM) was unaffected, suggesting that the conversion of SAM to ACC is a key reaction in the production of wound-induced C(2)H(4). This view was further supported by the observation that application of aminoethoxyvinylglycine, a known inhibitor of the conversion of SAM to ACC, eliminated the increases in ACC formation and in C(2)H(4) production. A significant increase in C(2)H(4) production was observed in the albedo tissue of orange in response to excision, and it was paralleled by an increase in ACC content. In columella tissue of unripe green tomato fruit, massive increases in the C(2)H(4) production rate (from 0 to 12 nanoliters per gram per hour), in ACC content (from 0.05 to 12 nmoles per gram), and in ACC synthase activity (from 0 to 6.4 units per milligram protein) occurred during the 9-hour incubation period following excision. Infiltration with 0.1 millimolar cycloheximide, an inhibitor of protein synthesis, completely blocked wound-induced C(2)H(4) production, ACC formation, and development of ACC synthase activity. These data indicate that wounding induces the synthesis of ACC synthase, which is the rate-controlling enzyme in the pathway of C(2)H(4) biosynthesis and, thereby, causes accumulation of ACC and increase in C(2)H(4) production.

Regulation of wound ethylene synthesis in plants

Ethylene is produced by plants at specific stages of their life cycle and is involved in the regulation of many developmental processes, such as fruit ripening, flower fading, leaf abscission, growth in aquatic plants and initiation of flowering in bromeliads1,2. Ethylene is also formed when plants are subjected to stress by, for example, wounding, noxious chemicals, drought or waterlogging1–3. In both cases the biosynthetic pathway begins with methionine 1–5. It proceeds to ethylene by way of S-adenosylmethionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC), a recently discovered intermediate6,7, in ripening fruit7,8 and auxin-treated pea stem sections9,10. We now report that wound-induced ethylene synthesis also proceeds by way of ACC and that it is regulated at the level of the ACC-forming enzyme (ACC synthase). Wounding of pericarp tissue of tomato fruit (Lycopersicon esculentum Mill.) results in rapid enhancement of the activity of ACC synthase and, consequently, in a greatly increased rate of ACC synthesis.

Studies of Rapidly Induced Wound Ethylene Synthesis by Excised Sections of Etiolated Pisum sativum L., cv. Alaska

The rate of wound ethylene synthesis was reduced by more than 85% when 9-millimeter subapical sections of etiolated 7-day-old Pisum sativum L., cv. Alaska seedlings were incubated in water during the 26-minute induction period prior to wound ethylene synthesis, but the rate of synthesis was unaffected if sections were incubated in water during the actual synthesis of wound ethylene. The characteristic timing of the wound response was unaffected by either treatment. The ability of various chemical solutions and aqueous plant extracts to alter the rate of wound ethylene synthesis was studied by first incubating subapical pea stem sections in solutions under anaerobic conditions (anaerobiosis delays the induction and synthesis of wound ethylene; Plant Physiol 61: 675-679), and then measuring wound ethylene synthesis after the tissue was transferred to air. Solutions of several reported precursors of ethylene synthesis, such as methionine, homoserine, or propanal, did not reverse the water-caused reduction of wound ethylene synthesis. A water-soluble, heat-stable factor in extracts from pea seedlings, and solutions of 23 nanomolar triacontanol, 10 micromolar kinetin, or 10 micromolar benzyladenine prevented the reduction of wound ethylene synthesis, but were ineffective if administered after an initial 15-minute anaerobic water incubation. This suggested that the active solutions may have only prevented the loss of some ephemeral, though necessary factor, rather than actually containing the substrate or inducer of wound ethylene synthesis. Attempts to isolate and characterize the active fraction from aqueous tissue extracts were unsuccessful. Free radical quenchers, inhibitors of protein synthesis, and rhizobitoxine, an inhibitor of ethylene synthesis from methionine, all reduced wound ethylene synthesis when administered in solutions which previously had maintained wound ethylene synthesis.

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska

Increased ethylene synthesis was rapidly induced throughout the apical meristematic region of etiolated seedlings of Pisum sativum L., cv. Alaska by cuts made 1 centimeter from the apical hook. The wound signal was transmitted at about 2 millimeters per minute. Accumulation of substance(s) at the cut surfaces of excised sections, as the result of interrupted translocation, did not initiate or significantly contribute to wound-induced ethylene synthesis, nor was the cut surface the site of enhanced ethylene synthesis. Cutting subapical sections into shorter pieces showed that cells less than 2 millimeters from a cut surface produced about 30% less ethylene than cells greater than 2 millimeters from a cut surface.

Role of Wound Ethylene in Fruit Set of Hand-pollinated Muskmelons1

Abstract Application of a lanolin paste containing the aminoethoxy analog of rhizobitoxine, aminoethoxyvinylglycine (AVG), to the base of perfect flowers improved fruit set in emasculated, hand-pollinated flowers of muskmelon (Cucumis melo L.). Applications of indoleacetic acid (IAA) improved fruit set slightly, but reduced seed yields. Combinations of IAA and AVG were more effective than IAA alone, but were less effective than AVG alone at the optimum dosage. AVG prevented would ethylene production by excised, hand-pollinated flowers. The results support the contention that would ethylene production by emasculated, hand-pollinated flowers is an important factor in lowering fruit set.

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska

Wound-induced ethylene synthesis by subapical stem sections of etiolated Pisum sativum L., cv. Alaska seedlings, as described by Saltveit and Dilley (Plant Physiol 1978 61: 447-450), was half-saturated at 3.6% (v/v) O(2) and saturated at about 10% O(2). Corresponding values for CO(2) production during the same period were 1.1% and 10% O(2), respectively. Anaerobiosis stopped all ethylene evolution and delayed the characteristic pattern of wound ethylene synthesis. Exposing tissue to 3.5% CO(2) in air in a flow-through system reduced wound ethylene synthesis by 30%. Enhancing gas diffusivity by reducing the total pressure to 130 mm Hg almost doubled the rate of wound ethylene synthesis and this effect was negated by exposure to 250 mul liter(-1) propylene. Applied ethylene or propylene stopped wound ethylene synthesis during the period of application, but unlike N(2), no lag period was observed upon flushing with air. It is concluded that the characteristic pattern of wound-induced ethylene synthesis resulted from negative feedback control by endogenous ethylene.No wound ethylene was produced for 2 hours after excision at 10 or 38 C. Low temperatures prolonged the lag period, but did not prevent induction of the wound response, since tissue held for 2 hours at 10 C produced wound ethylene immediately when warmed to 30 C. In contrast, temperatures above 36 C prevented induction of wound ethylene synthesis, since tissue cooled to 30 C after 1 hour at 40 C required 2 hours before ethylene production returned to normal levels. The activation energy between 15 and 36 C was 12.1 mole kilocalories degree(-1).

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska

A rapidly induced, transitory increase in the rate of ethylene synthesis occurred in wounded tissue excised from actively growing regions of etiolated barley, cucumber, maize, oat, pea, tomato, and wheat seedlings. Cutting intact stems or excising 9-mm segments of tissue from near the apex of 7-day-old etiolated Pisum sativum L., cv. Alaska seedlings induced a remarkably consistent pattern of ethylene production. At 25 C, wound-induced ethylene production by segments excised 9 mm below the apical hook increased linearly after a lag of 26 minutes from 2.7 nanoliters per g per hour to the first maxium of 11.3 nanoliters per g per hour at 56 minutes. The rate of production then decreased to a minimum at 90 minutes, increased to a lower second maximum at 131 minutes, and subsequently declined over a period of about 100 minutes to about 4 nanoliters per g per hour. Removal of endogenous ethylene, before the wound response commenced, had no effect on the kinetics of ethylene production. Tissue containing large amounts of dissolved ethylene released it as an exponential decay with no lag period. Rapidly induced wound ethylene is synthesized by the tissue and is not merely the result of facilitated diffusion of ethylene already present in the tissue through the newly exposed cut surfaces. Previously wounded apical sections did not exhibit a second response when rewounded. No significant correlation was found between wound-induced ethylene synthesis and either CO(2) or ethane production.

Patterns of Ethylene and Carbon Dioxide Evolution during Cotton Explant Abscission

The relationship between abscission and the evolution of ethylene and CO(2) was examined in explants and explant segments of cotton seedlings (Gossypium hirsutum L. cv. Acala SJ-1) under both static and flow system conditions, and in the presence and absence of mercuric perchlorate. Explant excision was immediately followed by increased ethylene evolution (wound ethylene); senescence was also accompanied by increased ethylene evolution (senescence ethylene). One or two ethylene peaks were found to interrupt the low background rate of ethylene evolution during the period between excision and senescence. The first intermediate ethylene peak coincided with a rise in CO(2) evolution; however, precedence could not be established. No statistical correlations were discovered between either intermediate ethylene peak and abscission. The best statistical correlation was found between wound ethylene and abscission at 12 hr after excision. No positive correlations were found between senescence ethylene and abscission. Implications of these results for the understanding of the role of ethylene in explant abscission are discussed.Relationships between a number of different explant treatments and ethylene evolution were also examined. Ethylene production in response to indoleacetic acid applications, abscisic acid applications, and different types of wounding is summarized. It was concluded that the results of the standard abscission bioassay (conducted in Petri dishes) have not been influenced by unnatural ethylene accumulations.