High Temperature Sensitivity of Auxin-induced Ethylene Production in Mung Bean Hypocotyl Sections

High temperature sensitivities of IAA-induced and 1-aminocyclopropane-1-carboxylicacid (ACC)-dependent ethylene production in etiolated mung bean(Vigna radiata [L] Wilczek) hypocotyl sections were comparedat 30,40, 42.5°C. When ethylene production at 30°C wastaken as control, IAA-induced production at 40°C was firstenhanced and then suppressed after 3 h, whereas ACC-dependentproduction was enhanced two-fold throughout the 8 h experimentalperiod. However, when hypocotyl sections treated with 1 mM ACCat 30°C for several hours were transferred to 40°C,the ACC-dependent production rate fell below that at 30°C.An initial transient enhancement of IAA-induced ethylene productionat 40°C was supported by increased ACC synthase activityand thus by ACC content. At 42.5°C, both IAA-induced andACC-dependent production were almost completely suppressed.The results indicate that auxin-induced ethylene productionis affected by high temperatures in two different steps: a)at 40°C, the auxin action gradually deteriorates althoughconversion of ACC to ethylene is not affected at all, and at42.5°C, the conversion is nearly completely suppressed. (Received July 8, 1985; Accepted January 24, 1986)     

The Role of 1-Aminocyclopropane-1-carboxylic acid in the Control of Low Temperature Induced Ethylene Production in Leaf Tissue of Phaseolus vulgaris L .

Ethylene production from leaf discs of dwarf bean (Phaseolausvulgaris L.) was less than 02 nl g^–1 h^–1 at 5 Cbut rapidly increased tenfold on transfer to 25 C. The lowethylene production at 5 C and the potential for overshootproduction on transfer to 25C were not associated with accumulationof the ethylene synthesis intermediate 1-aminocyclopropane-1-carboxylicacid (ACC). Addition of exogenous ACC to leaf discs incubatedat 5C increased ethylene production, while similarly incubatedleaf discs did not synthesize increasing amounts of endogenousACC until they were transferred to 25 C. The basis for theovershoot in ethylene production when leafdiscs were transferredfrom 5 to 25 C appears to reside in changes to the pathwayleading to the synthesis of ACC or an earlier intermediate inthe pathway of ethylene biosynthesis. Ethylene, 1-aminocyclopropane-l-carboxylic acid, Phuseolru vulgaris L., dwarf bean, temperature     

Development of Ethylene Biosynthesis in Pear Fruits at --1 {degrees}C

Knee, M. 1987. Development of ethylene biosynthesis in pearfruits at — 1 °C.—J. exp. Bot. 38: 1724–1733. The regulation of ethylene synthesis in pear fruits was investigated.During storage for 60 d at — 1 °C the rate of ethylenesynthesis increased 100-fold but the concentration of 1-aminocyclopropane-l-carboxylicacid (ACC) increased only 2-fold and ACC synthase activity waslow. On transfer to 15 °C after storage at — 1 °Cethylene synthesis increased 10-fold within 10 h but ACC synthaseactivity only increased rapidly after 24 h; the decline in ACClevels during the first 16 h at 15 °C was insufficient tosustain ethylene synthesis. Ethylene synthesis was further investigatedusing discs cut from the mid cortex of pear fruits. Synthesiswas inhibited by aminoethoxyvinylglycine (AVG) and amino-oxyaceticacid at all stages of ripening. The rate of synthesis and ACCsynthase activity increased rapidly after slicing of pears heldat — 1 °C but more slowly in discs cut from pearsimmediately after harvest. Cycloheximide (CHI) inhibited theseincreases and reversed increases resulting from pre-incubationof discs. A combination of CHI and AVG abolished the capacityof discs to synthesize ACC and ethylene production was curtailed.Cordycepin and actinomycin-D were less effective as inhibitorsof the development of ethylene synthesis and ACC synthase activitythan as inhibitors of incorporation of 5-[³H] uridine into totalRNA or poly A rich RNA. The ability of discs to develop ethylenesynthesis and ACC synthase activity in the presence and absenceof cordycepin increased concurrently during storage of wholefruits at — 1 °C. This suggested that mRNA for ACCsynthase was formed at — 1 °C. Key words: 1-Aminocyclopropane-l-carboxylic acid, ethylene, fruit ripening, Pyrus communis L. (fruit ripening)     

Effect of Ethylene and Culture Environment on Rice Callus Proliferation

Modifications to the gaseous envelope by callus during culturein Petri dishes were shown to reduce growth and promote necrosisof several rice (Oryza sativa L.) cultivars. Incubatingcallusunder a continuous flow of gas mixtures of known compositionsuggested that the inhibition of growth was caused by the accumulationof ethylene, the depletion of oxygen and, to a lesser extent,the accumulation of carbon dioxide. In order to evaluate theimportance of ethylene accumulation aminoethoxyvinylglycine(AVG), 1-aminocyclopropane-l-carboxylic acid (ACC and silvernitrate (AgNO₃), were added to the nutrient medium and ethylenemeasurements performed during callus culture. Ethylene restrictedcallus growth particularly under high (35 °C) as comparedto moderate (25 °C) temperatures and under illuminated ascompared to darkened incubation. Under illuminated incubationat 25 °C AVG (5 mmol m^–3) and AgNO°(50 mmol m^–3)significantly improvedcallus growth (100 and 60% respectively)while ACC (200 mmol m^–3) significantly decreased growth(40%). AVG and AgNO₃ were less effective under dark incubationat 25 °C where ethylene production was lower. Furthermore,callus growth was significantly better in large as comparedto small culture vessels since the ethylene concentration wasdiluted and more oxygen was available for respiration. Bettercontrol of ethylene and increased oxygen availability couldbe a way ofproducing healthy callus for the formation of embryogenictissues of otherwise recalcitrant cultivars of rice (e.g. IndicaIR42) and may be a way of improving manipulation of other cerealspecies. Key words: 1-Aminocyclopropane-1-carboxylic acid, aminoethoxyvinylglycine, callus, ethylene, Oryza sativa, silver nitrate     

Role of Aminocyclopropane-1-carboxylic Acid (ACC) in Control of Ethylene Production in Fresh and Cold-stored Rose Flowers

The relationships between ethylene production, aminocyclopropane-1-carboxylicacid (ACC) content and ethylene-forming-enzyme (EFE) activityduring ageing and cold storage of rose flower petals (Rose hybridaL. cv. Gabriella) were investigated. During flower ageing at20 °C there was a climacteric rise in petal ethylene production,a parallel increase in ACC content, but a continuous decreasein EFE activity. Applied ACC increased petal ethylene productionc. 200-fold. During cold storage of flowers at 1 °C therewere parallel increases in petal ethylene production and ACCcontent, to levels greater than those reached in fresh flowersheld at 20 °C. EFE activity decreased during storage. Immediatelyafter cold-stored flowers were transferred to 20 °C ethyleneproduction and ACC levels were c. four times greater than infreshly cut flowers. These levels increased to maximum valuesof two to four times the maximum values reached during ageingof fresh, unstored, flowers. It was concluded that in rose petalsethylene synthesis is probably regulated by ACC levels and thatcold storage stimulates ethylene synthesis because it increasesthe levels of ACC in the petals. Key words: Rose flower, senescence, ethylene     

Petiole Growth in Ranunculus sceleratus L.: Ethylene Synthesis and Submergence

Petioles of the celery-leaved buttercup (Ranunculus sceleratusL.) elongate in response to treatment with ethylene in air whenthe leaf blades are attached. An enhanced rate of elongationgrowth also occurs when the leaves are submerged. Submergencecauses an increase in extractable ethylene gas within the tissues,and these levels appear to approach those required to saturatethe ethylene-promoted elongation growth response. Coincidentwith a rise in ethylene in the tissues is a dramatic increasein the level of I-aminocyclopropane-1-carboxylic acid (ACC),the precursor of ethylene. Both the petiole and leaf blade tissueshave a similar capacity to evolve ethylene in the presence ofadded ACC. However, in air the leaf blade evolves more ethylenefrom endogenous resources than the petiole. The simultaneousincreases in ethylene and ACC levels in submerged tissues areconsidered in terms of the low diffusivity of ethylene in water,the ‘autocatalytic’ effect of ethylene on ethylenebiosynthesis and the rôle of both carbon dioxide and oxygenfluxes in ethylene metabolism of submerged tissues. Ranunculus sceleratus, celery-leaved buttercup, petiole growth, submergence, ethylene metabolism     

The Effect of Low Temperature on Ethylene Production by Leaf Tissue of Phaseolus vulgaris L.

When leaf discs cut from primary leaves of Phaseolus vulgarisL. cv. Masterpiece plants grown at 25°C were incubated attemperatures below 25 °C, basal and wound ethylene productioncontinued at reduced rates. In both cases detectable levelsof ethylene were produced at 25 °C. When the rates of ethyleneproduction were plotted according to the Arrhenius equationa marked discontinuity was found at 11.4 °C which is consistentwith a membrane phase-transition at the critical chilling temperatureof the plant. Activation energies for the rate-limiting enzymereaction in ethylene production above and below the criticaltemperature have been calculated and the data interpreted asindicating the involvement of membrane-bound enzyme systemsin the biosynthesis of basal and wound ethylene. ethylene, temperature, Arrhenius plot, activation energy, Phaseolus vulgaris L., bean     

Wound-Induced Ethylene Production and 1-Aminocyclopropane-1-carboxylic Acid Synthase in Mesocarp Tissue of Winter Squash Fruit

Discs (9 mm in diameter and 2 mm in thickness) sliced from mesocarpof winter squash fruit (Cucurbita maxima Duch.) upon incubationat 24°C produced ethylene at an increasing rate after alag period of 3 h. 1-Aminocydopropane-l-carboxylic acid (ACC)synthase activity also increased at a rapid rate after lag periodof less than 3 h, reaching a peak 14 h after incubation andthen declining sharply. The rise in ACC synthase activity precededa rapid increase in ACC formation and ethylene production. Inductionof ACC synthase by wounding in sliced discs was strongly suppressedby the application of cycloheximide, actinomycin D and cordycepin,suggesting that the rise in ACC synthase activity may resultfrom de novo synthesis of protein. ACC synthase extracted from wounded tissue of winter squashmesocarp required pyridoxal phosphate for its maximum activity.The optimum pH of the reaction was 8.5. Km value for S-adenosylmethioninewas 120 µM. The reaction was markedly inhibited by aminoethoxyvinylglycinewith Ki value being 2.7 µM. (Received March 23, 1983; Accepted May 23, 1983)     

Ethylene Involvement in Dormancy Release of Ricinodendron rautanenii Seeds

Ricinodendron rautanenii (manketti) seeds respond to as littleas 10^–2 µl I^–1 ethylene. Both dry and imbibedseeds respond to ethylene treatments. The length of the imbibitionperiod prior to the ethylene treatment is not important as seedswhich were simultaneously gassed and imbibed germinated successfully.Seeds were sensitive to the gas at temperatures between 25 and35 °C. No temperature effect was observed in seeds whichwere transferred from temperatures below 20 °C, or above35 °C, to 30 °C. There is no period of carbon dioxidesensitivity associated with the ethylene treatment and the effectsof the ethylene treatment persisted in both dry and imbibedseeds when subsequently maintained (desiccated) at room temperaturefor 8 d Ricinodendron rautanenii, manketti nut, ethylene, germination     

Effect of Ethylene and Ethylene Precursors on Protein Phosphorylation and Xylogenesis in Tuber Explants of Helianthus tuberosus (L.)

Koritsas, V. M. 1988. Effect of ethylene and ethylene precursorson protein phosphorylation and xylogenesis in tuber explantsof Helianthus tuberosus (L.).J. exp. Bot. 39: 375–386. The role of ethylene in protein phosphorylation and xylem-celldifferentiation was studied in explant cultures of Jerusalemartichoke, Helianthus tuberosus (L.). Explants on xylem-inducingmedium growing at 25 °C developed more xylem elements andproduced more ethylene gas within 3 d of culture than controlcultures growing and developing at similar rates. L-methionineand 1-aminocdopropane-1-carboxylic acid (ACC) incorporated intoboth xylem-inducing and control media enhanced xylem differentiation,increased protein levels and stimulated protein kinase activityin the explants. Inhibitors of the ethylene biosynthetic pathway,cobalt nitrate and aminoethoxyinyl glycine (AVG), depressedxylem differentiation, protein phosphorylation and polypeptidesynthesis. Ethylene gas or ACC reversed the effects of the inhibitors.Ethylene production, protein phosphoryilation and xylem-celldifferentiation were all linked. Ethylene thus promotes xylem-celldifferentiation in Jerusalem artichoke explants, the processprobably being regulated by protein phosphorylation. Key words: Ethylene, phosphorylation, xylem differentiation, Jerusalem artichoke explants     

A Relationship between Membrane Permeability and Ethylene Production at High Temperature in Leaf Tissue of Phaseolus vulgaris L.

Leaf discs cut from primary leaves of Phaseolus vulgaris L cvMasterpiece were incubated at temperatures higher than the growthtemperature of 25 °C Both basal and wound ethylene productionincreased up to temperatures of 35–37 5 °C, thereafterdeclining rapidly There was no detectable ethylene productionat temperatures above 42 5 °C Exposure of leaf discs tohigh temperature for 60 mm resulted in a large production ofwound ethylene when they were returned to 25 °C The magnitudeof ethylene production was related to the initial incubationtemperature as was the length of the lag period before maximumproduction was achieved The results are discussed in relationto the requirement for continued membrane integrity for ethyleneproduction ethylene, temperature, membrane permeability, Phaseolus vulgaris L, dwarf bean     

Physiological Responses of Cut Rose Flowers to Exposure to Low Temperature: Changes in Membrane Permeability and Ethylene Production

Senescence of cut rose flowers (Rosa hybrida L. cv. Mercedes)at 22 °C occurred earlier in flowers previously held at2 °C for 10 d or 17 d than in freshly cut flowers. Thisadvanced senescence was observed as an earlier increase in bothethylene production rate and membrane permeability. The risein ethylene production preceded the rise in the level of ionleakage from petals, and this in turn preceded visible symptomsof petal death. Applied ethylene stimulated ion leakage andinhibitors of ethylene synthesis and action (amino-oxyaceticacid and silver thiosulphate respectively) inhibited the normalincrease in ion leakage. The maximum rate of ethylene productionof 22 °C increased markedly in petals of flowers previouslyheld at 2 °C, up to nine times the level in fresh flowers.We conclude that during exposure of rose flowers to 2 °C,in addition to senescence, processes were induced which ledto stimulated ethylene production after transferral to 22 °C.Ethylene apparently caused the subsequent advance in membranepermeability and senescence. Key words: Rose flower, Low temperature, Senescence     

Induction of ethylene biosynthesis in tobacco leaf discs by cell wall disesting enzymes

Cellulysin induces ethylene production in tobacco leaf discs by initiating the formation of 1-aminocyclopropane-1-carboxylic acid. Induction occurred within 30 to 60 min of incubation and was inhibited by aminoethoxyvinylglycine, and the antiproteases, PMSF and soybean trypsin inhibitor. Cycloheximide (CHI) at 2.8 μg/ml and chloramphenicol (CAP) at 100 μg/ml did not inhibit this induction although incorporation of the label from (3,4-¹⁴C)methionine into the acid-insoluble fraction was inhibited by 57%. At 14 μg/ml CHI, and CAP, ethylene production was inhibited by 25% while protein synthesis was inhibited by 75%. We suggest that either the low amounts of protein synthesis that appear to be insensitive to CHI is sufficient to induce ethylene biosynthesis or that Cellulysin activates a preexisting but inactive form of ACC synthase to promote ethylene biosynthesis. Also, induction of ethylene production by microbial enzymes that digests plant cell walls may be an initial protective response of plants that serves to combat microbial infection.     

The Effects of Oxygen, Carbon Dioxide and Ethylene on Ethylene Biosynthesis in Relation to Shoot Extension in Seedlings of Rice (Oryza sativa) and Barnyard Grass (Echinochloa oryzoides)

Exposing dark-grown seedlings for 3 d to oxygen deficiency (0or 5 kPa) or to additions of carbon dioxide (10 kPa) or ethylene(0·1 Pa) slowed shoot extension in Echinochloa oryzoides,while in rice it was promoted by these treatments, except that5 kPa oxygen was without effect. In E. oryzoides this was dueto reduced growth of the mesocotyl, and in rice to enhancedgrowth of the coleoptile. These responses to carbon dioxideand oxygen deficiency were not consequences of increased ethyleneproduction, since this remained unchanged by carbon dioxideand depressed by oxygen shortage in both species. Furthermore,exogenous ethylene and the ethylene action inhibitor 2,5-norbornadieneeach failed to influence extension in anoxic seedlings, indicatingno regulatory role for ethylene in the absence of oxygen. However,concentrations of the ethylene precursor 1 -aminocyclopropane-1-carboxylic acid (ACC) were increased by carbon dioxide and0 kPa or 5 kPa oxygen, although after 72 h without oxygen totalACC production (i.e. changes in ethylene + ACC + MACC) was suppressedin both species. There was little effect on bound ACC [putativemalonyl-ACC (MACC)] formation. Transferring anaerobic (0 kPa)seedlings to oxygenated conditions (21 kPa) resulted in abnormallyfast rates of ethylene formation, possibly due to the accumulationof ACC under anoxia. This post-anoxic ethylene may have contributedto the faster extension by rice coleoptiles and slower extensionby mesocotyls of E. oryzoides compared with those of seedlingsmaintained continuously in air. Echinochloa oryzoides [Ard.] Fritsch, barnyard grass, Oryza sativa L, rice, oxygen shortage, carbon dioxide, ethylene biosynthesis, shoot extension, 1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC, GC-MS     

Ethylene Production by the Kudzu Strains of Pseudomonas syringae pv. phaseolicola Causing Halo Blight in Pueraria lobata (Willd) Ohwi

Significant amounts of ethylene was produced by Pseudomonassolanacearum (all strains), P. syringae pv. phaseolicola (Kudzustrains isolated from Pueraria lobata) and Erwinia rhapontici(2 strains out of 22) out of 24 species, 3 subspecies and 38pathovars of plant pathogenic bacteria tested in yeast extract-peptonebroth. The bean strains of P. syringae pv. phaseolicola causinghalo blight in kindney bean plants did not produce ethylene.The Kudzu strains produced ethylene at a rate of 7 to 100?10^–9nl cell^–1 h^–1, which was 500 to 1,000 times higherthan that of P. solanacearum and several times higher than thatof Penicillium digitatum, the most potent ethylene producerknown among microorganisms. The presence of living cells was essential for ethylene productionby the Kudzu strains. The bacterium effectively produced ethylenefrom amino acids such as glutamate, aspartate and their amides.Although glucose and succinate were also good substrates forethylene biosynthesis, the rate of ethylene production was significantlysmaller than that with glutamate. Methionine, which is knownas the precursor of ethylene in plants, had no effect on ethyleneproduction by the bacterium. 1-Aminocyclopropane-1-carboxylicacid (ACC) also had no effect on ethylene production, and therewas not enough ACC in the bacterial cells to account for thehigh rate of ethylene production. Ethylene production from glutamatewas inhibited by n-propylgallate and EDTA, but not by aminoethoxyvinylglycine.These results indicate that ACC is not involved as an intermediatein the process of ethylene biosynthesis by the bacterium, suggestingthe presence of a pathway different from that of plant tissues. (Received September 4, 1984; Accepted October 27, 1984)     

光敏核不育水稻幼穗的乙烯生成与育性转换

研究了在不同光温组合下光敏核不育水稻（ＰＧＭＲ）农垦５８５（ＮＫ５８Ｓ）及普通水稻品种农垦５８（ＮＫ５８Ｂ）的幼穗乙烯生物合成变化及其与育性表达的关系。结果表明ＮＫ５８Ｓ幼穗乙烯生成受光周期和温度的共同调节,而光周期对ＮＫ５８Ｂ幼穗乙烯生成无明显影响。ＮＫ５８Ｓ幼穗乙烯生成的变化与育性转换光温作用模式完全吻合。其幼穗乙烯释放速率（ＥＲＲ）与花粉可育度（ｌｎｙ）呈极显著负相关。用ＡＶＧ抑制乙烯生成可使ＮＫ５８Ｓ（ＬＤ）不育性明显逆转,而用ＡＣＣ促进乙烯生成又可急剧降低ＮＫ５８Ｓ（ＳＤ）的可育水平。表明幼穗乙烯生成与ＰＧＭＲ育性表达密切相关,乙烯参与育性转换的调控并可能起着重要作用。     

Temperature-stress Pretreatment in Barley Anther Culture

Methods of pretreating anthers at different temperatures priorto culture have been tested, with respect to pollen-callus productionand plant regeneration, in Hordeum vulgare cv. Sabarlis. For callus production, pretreatment of excised spikes (in sealedPetri dishes) was more effective than pretreatment of excisedtillers (in water or in polythene) at both 4 and 25 °C.Pretreatment of individual anthers at these temperatures wasdeleterious. Greater callus yields resulted from pretreatmentat 4 than at 25 °C, both for spikes and tillers, 3–5weeks being required for maximal yields at 4 °C and 3–5days at 25 °C. At 4 °C, a shorter pretreatment was requiredfor spikes than for tillers. Pretreatment of spikes was alsomore effective at 4 than at 7, 14 or 20 °C. Pretreatmentof individual spikelets at 4 °C was as effective as thatof whole spikes. For plant regeneration, calluses derived from pretreatment ofspikes were more effective than those derived from pretreatmentof tillers. More plants resulted from pretreatment at 4 thanat 25 °C, both for spikes and tillers. Maximal pretreatmenttimes for plant regeneration generally exceeded those for callusproduction. Following spike pretreatment at 4 °C the maximumfor plant regeneration exceeded that for callus production byabout 2 weeks. With this optimal pretreatment approximately60 per cent of the calluses gave rise to plantlets. Among this60 per cent, for every three calluses giving albinos, two gavegreen plantlets, equivalent to five green plantlets on averagefor every 100 anthers (= two spikes) cultured. The ratio ofgreen to albino plantlets was lower for all other pretreatments. Hordeum vulgare L., barley, anther culture, pollen callus, pollen plant-production, temperature stress     

Regulation of Auxin-induced Ethylene Biosynthesis. Repression of Inductive Formation of 1-Aminocyclopropane-1-carboxylate Synthase by Ethylene

Changes in the 1-aminocyclopropane-1-carboxylate (ACC) synthaseactivity which regulates auxin-induced ethylene production werestudied in etiolated mung bean hypocotyl segments. Increasesboth in ethylene production and ACC synthase activity in tissuetreated with IAA and BA were severely inhibited by cycloheximide(CHI), 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide,actinomycin D and -amanitin. Aminoethoxyvinylglycine (AVG),a potent inhibitor of the ACC synthase reaction, increased theactivity of the enzyme in the tissue 3- to 4-fold. This stimulationalso was severely inhibited by the above inhibitors. Stimulationof the increase in the enzyme content by AVG was partially suppressedby an exogenous supply of ACC or ethylene. Suppression of theincrease in the enzyme took place with 0.3 µl/liter ethylene,and inhibition was increased to 10 µl/liter, which caused65% suppression. Air-flow incubation of the AVG-treated tissue,which greatly decreased the ethylene concentration surroundingthe tissue, further increased the amount of enzyme. Thus, oneeffect of AVG is to decrease the ethylene concentration insidethe tissue. The apparent half life of ACC synthase activity,measured by the administration of CHI, was estimated as about25 min. AVG lengthened the half life of the activity about 2-fold.Feedback repression by ethylene in the biosynthetic pathwayof auxin-induced ethylene is discussed in relation to the effectof AVG. (Received January 22, 1982; Accepted March 26, 1982)     

Chilling-Induced Ethylene Production in Cucumbers (Cucumis sativus L.)

1-Aminocyclopropane-1-carboxylic acid (ACC) level, ACC synthase activity, and ethylene production in cucumbers (Cucumis sativus L.) remain low while the fruit are held at a temperature which causes chilling injury (2.5°C) and increase rapidly only upon transfer to warmer temperatures. The increase in ACC synthase activity during the warming period is inhibited by cycloheximide but not cordycepin or α-amanitin. Our data indicate that the synthesis of ACC synthase, which results in increased ACC levels and accelerated ethylene production, occurs only upon warming, possibly from a message produced or unmasked during the chilling period. Ethylene production by chilled (2.5°C) cucumbers increased very little upon transfer to 25°C if the fruit were chilled for more than 4 days. The fruit held for 4 days or longer showed a large increase in ACC levels but little ethylene production even in the presence of exogenous ACC. This suggests that the system which converts ACC to ethylene is damaged by prolonged exposure to the chilling temperature. Cucumbers stored at a low but nonchilling temperature (13°C) showed very little change in ACC level, ethylene production, or ACC synthase activity even after transfer to 25°C.