Carbohydrates Stimulate Ethylene Production in Tobacco Leaf Discs : II. Sites of Stimulation in the Ethylene Biosynthesis Pathway

Galactose, sucrose, and glucose (50 millimolar) applied to tobacco leaf discs (Nicotiana tabacum L. cv `Xanthi') during a prolonged incubation (5-6 d) markedly stimulated ethylene production which, in turn, could be inhibited by aminoethoxyvinylglycine (2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid) (AVG) or Co²⁺ ions. These three tested sugars also stimulated the conversion of l-[3,4-¹⁴C]methionine to [¹⁴C]1-amino-cyclopropane-1-carboxylic acid (ACC) and to [¹⁴C]ethylene, thus indicating that the carbohydrates-stimulated ethylene production proceeds from methionine via the ACC pathway. Sucrose concentrations above 25 mm considerably enhanced ACC-dependent ethylene production, and this enhancement was related to the increased respiratory carbon dioxide. However, sucrose by itself could directly promote the step of ACC conversion to ethylene, since low sucrose concentrations (1-25 mm) enhanced ACC-dependent ethylene production also in the presence of 15% CO₂.     

Regulation of Auxin-induced Ethylene Production in Mung Bean Hypocotyls: Role of 1-Aminocyclopropane-1-Carboxylic Acid

Ethylene production in mung bean hypocotyls was greatly increased by treatment with 1-aminocyclopropane-1-carboxylic acid (ACC), which was utilized as the ethylene precursor. Unlike auxin-stimulated ethylene production, ACC-dependent ethylene production was not inhibited by aminoethoxyvinylglycine, which is known to inhibit the conversion of S-adenosylmethionine to ACC. While the conversion of methionine to ethylene requires induction by auxin, the conversion of methionine to S-adenosylmethionine and the conversion of ACC to ethylene do not. It is proposed that the conversion of S-adenosylmethionine to ACC is the rate-limiting step in the biosynthesis of ethylene, and that auxin stimulates ethylene production by inducing the synthesis of the enzyme involved in this reaction.     

Lipoxygenase-generated hydroperoxides account for the nonphysiological features of ethylene formation from 1-aminocyclopropane-1-carboxylic acid by microsomal membranes of carnations

Several lines of evidence indicate that the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by microsomal membranes from carnation flowers is attributable to hydroperoxides generated by membrane-associated lipoxygenase (EC 1.13.11.12). As the flowers senesce, the capability of isolated microsomal membranes to convert ACC to ethylene changes. This pattern of change, which is distinguishable from that for senescing intact flowers, shows a close temporal correlation with levels of lipid hydroperoxides formed by lipoxygenase in the same membranes. Specific inhibitors of lipoxygenase curtail the formation of lipid hydroperoxides and the production of ethylene from ACC to much the same extent, whereas treatment of microsomes with phospholipase A₂, which generates fatty-acid substrates for lipoxygenase, enhances the production of hydroperoxides as well as the conversion of ACC to ethylene. Lipoxygenase-generated lipid hydroperoxides mediate the conversion of ACC to ethylene in a strictly chemical system and also enhance ethylene production by microsomal membranes. The data collectively indicate that the in-vitro conversion ACC to ethylene by microsomal membranes of carnation flowers is not reflective of the reaction mediated by the native in-situ ethylene-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EDTA ethylenediaminetetraacetic acid     

Galactose inhibits the conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in aged tobacco leaf discs

d-Galactose has been shown to have toxic and growth inhibitory effects in plants. When applied at levels of 50 millimolar to tobacco (Nicotiana tabacum L. cv Xanthi) leaf discs galactose caused a rapid increase in ethylene production during the first 2 days of incubation, followed by a rapid return to the basal level on the third day. This pattern of galactose-stimulated ethylene production was accompanied by increased formation of 1-aminocyclopropane-1-carboxylic acid (ACC), which accumulated without being metabolized to ethylene or to the ACC-conjugate. The inhibitory effect of galactose (50 millimolar) on the conversion of ACC of ethylene was relieved partially by d-glucose or sucrose (50 millimolar), and completely by CO₂ (10%), which were shown to enhance this conversion by themselves. Consequently, application of galactose plus any one of these compounds increased ethylene production and decreased free ACC levels. The data suggest that galactose toxicity may result in both an increased ethylene production as well as in accumulation of free ACC in aged discs. The increased ethylene production rates and ACC levels may, in turn, play a role in the development of symptoms associated with galactose toxicity.     

Inhibition of ethylene biosynthesis in carnation petals by cytokinin

Pretreatment of detached carnation petals (Dianthus caryophyllus cv White Sim) for 24 hours with 0.1 millimolar of the cytokinins n⁶-benzyl-adenine (BA), kinetin, and zeatin blocked the conversion of externally supplied 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene and delayed petal senescence by 8 days. The normal enhanced wilting and increase in endogenous levels of ACC and ethylene production following exposure of petals to ethylene (16 μl/l for 10 hours), were not observed in BA-pretreated petals. In carnation foliage leaves pretreated with 0.1 mm BA, a reduction rather than inhibition of the conversion of exogenous ACC to ethylene was observed. This indicates that foliage leaves respond to cytokinins in a different way than petals. A constant 24-hour treatment with BA (0.1 mm) was not able to reduce ethylene production of senescing carnation petals, while 2 mm aminoxyacetic acid, a known inhibitor of ACC synthesis, or 10 mm propyl gallate, a free radical scavenger, decreased ethylene production significantly.     

Auxin-induced Ethylene Production and Its Inhibition by Aminoethyoxyvinylglycine and Cobalt Ion

Auxin is known to stimulate greatly both C₂H₄ production and the conversion of methionine to ethylene in vegetative tissues, while amino-ethoxyvinylglycine (AVG) or Co²⁺ ion effectively block these processes. To identify the step in the ethylene biosynthetic pathway at which indoleacetic acid (IAA) and AVG exert their effects, [3-¹⁴C]methionine was administered to IAA or IAA-plus-AVG-treated mung bean hypocotyls, and the conversion of methionine to S-adenosylmethionine (SAM), 1-amino-cyclopropane-1-carboxylic acid (ACC), and C₂H₄ was studied. The conversion of methionine to SAM was unaffected by treatment with IAA or IAA plus AVG, but active conversion of methionine to ACC was found only in tissues which were treated with IAA and which were actively producing ethylene. AVG treatment abolished both the conversion of methionine to ACC and ethylene production. These results suggest that in the ethylene biosynthetic pathway (methionine → SAM → ACC → C₂H₄) IAA stimulates C₂H₄ production by inducing the synthesis or activation of ACC synthase, which catalyzes the conversion of SAM to ACC. Indeed, ACC synthase activity was detected only in IAA-treated tissues and its activity was completely inhibited by AVG. This conclusion was supported by the observation that endogenous ACC accumulated after IAA treatment, and that this accumulation was completely eliminated by AVG treatment. The characteristics of Co²⁺ inhibition of IAA-dependent and ACC-dependent ethylene production were similar. The data indicate that Co²⁺ exerts its effect by inhibiting the conversion of ACC to ethylene. This conclusion was further supported by the observation that when Co²⁺ was administered to IAA-treated tissues, endogenous ACC accumulated while ethylene production declined.     

The Conversion of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid to 1-Aminocyclopropane-1-Carboxylic Acid in Plant Tissues

Since 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the major conjugate of 1-aminocyclopropane-1-carboxylic acid (ACC) in plant tissues, is a poor ethylene producer, it is generally thought that MACC is a biologically inactive end product of ACC. In the present study we have shown that the capability of watercress (Nasturtium officinale R. Br) stem sections and tobacco (Nicotiana tabacum L.) leaf discs to convert exogenously applied MACC to ACC increased with increasing MACC concentrations (0.2-5 millimolar) and duration (4-48 hours) of the treatment. The MACC-induced ethylene production was inhibited by CoCl₂ but not by aminoethoxyvinylglycin, suggesting that the ACC formed is derived from the MACC applied, and not from the methionine pathway. This was further confirmed by the observation that radioactive MACC released radioactive ACC and ethylene. A cell-free extract, which catalyzes the conversion of MACC to ACC, was prepared from watercress stems which were preincubated with 1 millimolar MACC for 24 hours. Neither fresh tissues nor aged tissues incubated without external MACC exhibited enzymic activity, confirming the view that the enzyme is induced by MACC. The enzyme had a K_m of 0.45 millimolar for MACC and showed maximal activity at pH 8.0 in the presence of 1 millimolar MnSO₄. The present study indicates that high MACC levels in the plant tissue can induce to some extent the capability to convert MACC to ACC.     

Modulation of ethylene synthesis in acotyledonous soybean and wheat seedlings

The characteristics of ethylene production and ACC conversion in 8-day-old soybean seedlings were examined and a relationship between cytochrome P-450 activity and ethylene-forming enzyme (EFE) activity was found. An atmosphere containing 10% carbon monoxide (CO) significantly inhibited ethylene production and ACC conversion in control soybean seedlings, but had only a slight effect on soybean seedlings treated with uniconazole. Foliar application of triclopyr, a pyridine analogue of the phenoxy herbicides, significantly increased ethylene production and ACC conversion in control, but not in uniconazoletreated seedlings. Triclopyr treatment also resulted in a three-fold increase in extractable cytochrome P-450 of 5-day-old etiolated soybeans. At equimolar concentrations tetcyclacis was more effective than uniconazole in reducing shoot elongation and endogenous ethylene production. Although uniconazole and tetcyclacis did not inhibit ACC conversion in nonherbicide-treated soybean seedlings, they did prevent the observed increase in ACC-dependent EFE activity following triclopyr application. However, the rate of ACC conversion in etiolated soybean segments was sensitive to uniconazole, and tetcyclacis inhibited the rate of ACC conversion by 2.6-fold in etiolated soybean segments within 4 h after treatment. Microsomal membranes were isolated from 5-day-old naphthalic anhydride-treated etiolated wheat shoots as this tissue contains much higher cytochrome P-450 levels than soybean shoots. Optical difference spectroscopy demonstrated that ACC generated binding spectrum characteristic of a reverse-type-I cytochrome P-450 substrate when combined with reduced microsomes. In vitro conversion of ACC to ethylene by microsomal membranes was NADPH-dependent, inhibited by CO, and had an apparent K_m and V_max of 45 M and 0.345 nl/mg protein/h, respectively. These results suggest that cytochrome P-450-mediated monooxygenase reactions may be intimately involved in the conversion of ACC to ethylene in young soybean and wheat seedlings.     

Inhibition by 1-Aminocyclobutane-l-Carboxylate of the Activity of 1-Aminocyclopropane-l-Carboxylate Oxidase Obtained from Senescing Petals of Carnation {Dianthus caryophyllus L.) Flowers

We partially purified 1-aminocyclopropane-l-carboxy-late (ACC)oxidase from senescing petals of carnation {Dianthus caryophyllusL. cv. Nora) flowers and investigated its general characteristics,and, in particular, the inhibition of its activity by ACC analogs.The enzyme had an optimum pH at 7-7.5 and required Fe²⁺, ascorbateand NaHCO₃ for its maximal activity. The K_m for ACC was calculatedas 111-125 µM in the presence of NaHCO₃. Its M_r was estimatedto be 35 and 36 kDa by gel-filtration chromatography on HPLCand SDS-PAGE, respectively, indicating that the enzyme existsin a monomeric form. These properties were in agreement withthose reported previously with ACC oxidases from different planttissues including senescing carnation petals. Among six ACCanalogs tested, l-aminocyclobutane-l-carboxylate (ACBC) inhibitedmost severely the activity of ACC oxidase from carnation petals.ACBC acted as a competitive inhibitor with the K_i of 20-31 µM.The comparison between the K_m for ACC and the K_i for ACBC indicatedthat ACBC had an affinity which was ca. 5-fold higher than thatof ACC. Whereas ACC inactivated carnation ACC oxidase in a time-dependentmanner during incubation, ACBC did not cause the inactiva-tionof the enzyme. Preliminary experiments showed that ACBC andits N-substituted derivatives delayed the onset of senescencein cut carnation flowers. (Received August 19, 1996; Accepted November 26, 1996)     

Free Radical-mediated Formation of Ethylene from 1-Aminocyclopropane-1-carboxylic Acid: A Spin-frap Study

The ability of free radicals to convert l-aminocyclopropane-l-carboxylicacid (ACC) to ethylene under strictly chemical conditions hasbeen investigated using the aerobic xanthine/xanthine oxidasereaction and the Fenton reaction. Ethylene is formed when 1mM ACC is added to either of these reactions. Ethylene productionby the xanthine/xanthine oxidase system can be stimulated byH₂O₂ and inhibited by both catalase and superoxide dismutase,suggesting that the hydroxyl radical (OH?) formed by the Haber-Weissreaction is reacting with ACC to form ethylene. Ethylene productionfrom ACC by the Fenton reagent, which also produces OH?, showsa strong dependence upon H₂O₂. Involvement of the OH? radicalwas confirmed by spin-trap studies using 5,5-dimethyl-l-pyrroline-l-oxide(DMPO). Only the hydroxyl adduct of DMPO was detectable in boththe xanthine/xanthine oxidase reaction and the Fenton reaction.When ACC was added to the Fenton reaction, an additional adductof DMPO was detectable, which, on the basis of its hyperfinesplitting constants, can be tentatively identified as the DMPOadduct of a carbon-centered free radical. The data are consistentwith the view that formation of ethylene from ACC entails attackby OH? and the resultant formation of a carbon-centered radical,possibly of ACC. The chemical conversion of ACC to ethyleneis less efficient than that characteristic of senescing tissues,in which the reaction is enzymatically mediated. (Received October 1, 1981; Accepted November 17, 1981)     

Synergistic enhancement of ethylene production and germination with kinetin and 1-aminocyclopropane-1-carboxylic Acid in lettuce seeds exposed to salinity stress

Relief of salt (0.1 molar NaCl) stress on germination of lettuce (Lactuca sativa L., cv Mesa 659) seeds occurred with applications of 0.05 millimolar kinetin (KIN) and 1 to 10 millimolar 1-aminocyclopropane 1-carboxylic acid (ACC). Treatment with KIN enhanced the pregermination ethylene production under saline condition. A synergistic or an additive enhancement of pregermination ethylene production and germination occurred under saline condition in the presence of KIN and a saturating dose (10 millimolar) of ACC. No KIN-ACC synergism was noted in ethylene production or germination under nonsaline condition. Addition of 1 millimolar aminoethoxyvinylglycine (AVG) inhibited the KIN-enhanced pregermination ethylene production (85 to 89%) and germination (58%) under saline condition but not the synergistic effect of KIN + ACC on ethylene production. Under nonsaline condition, AVG had no effect on germination even though ethylene production was strongly inhibited. Alleviation of salt stress by KIN was inhibited in a competitive manner by 2,5-norbornadiene (NBD) (0.02-0.2 milliliter per liter), and the addition of ACC and/or ethylene reduced this inhibition. An increase in the pregermination ethylene production and germination occurred also by cotylenin E (CN) under saline condition. However, neither AVG (1 millimolar) nor NBD (0.02 to 0.2 milliliter per liter) prevented the relief of salt stress by CN. Thus, KIN may alleviate salt stress on germination by promoting both ACC production and its conversion to ethylene. Rapid utilization of ACC may be the basis for the synergistic or the additive effect of KIN plus ACC. The need for ethylene production and action for the relief of salt stress is circumvented by a treatment with CN.     

The modulation of the conversion of l-aminocyclopropane-l-carboxylic acid to ethylene by light

Endogenous ethylene production of tobacco leaves was similar in light and in darkness. However, the rate of conversion of exogenously applied l-aminocyclopropane-l-carboxylic acid (ACC) to ethylene was reversibly inhibited by light. Virus-stimulated ethylene production, during the hypersensitive reaction of tobacco leaves to tobacco mosaic virus, was likewise inhibited by light. Under such circumstances ethylene production is limited at the level of the conversion of ACC to ethylene. Inhibition of the increase in ACC-stimulated ethylene production by cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide after shifting leaf discs from light to darkness indicated that de novo protein synthsis was involved. Regulation of ACC-dependent ethylene production by reversible oxidation/reduction of essential SH groups, as suggested by Gepstein and Thimann (1980, Planta 149, 196–199) could be excluded. Instead, regulation of the ACC-converting enzyme at the level of both synthesis/degradation and activation/inactivation is suggested. Phytochrome was not involved in light inhibition, but low intensities of either red or blue light decreased the rate of ACC conversion. Dichlorophenyldimethylurea counteracted the inhibitory effect of light, indicating that (part of) the photosynthetic system is involved in the light inhibition. The ethylene production of Pharbitis cotyledons grown in darkness or light, either in the presence of absence of the inhibitor of carotenoid synthesis, SAN 9789 (norflurazon), supported this view.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DCMU dichlorophenyldimethylurea - MDMP 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide - SAM S-adenosylmethionine - SH groups sulfhydryl groups - TCA trichloroacetic acid - TMV tobacco mosaic virus     

Ethylene production by shoot-forming and unorganized crown-gall tumor tissues of Nicotiana and Lycopersicon cultured in vitro

We have studied ethylene biosynthesis in cloned crown-gall cell lines of Nicotiana tabacum L., N. glutinosa L., and Lycopersicon esculentum (L.) Mill. transformed by the A6 strain of Agrobacterium tumefaciens (Smith and Townsend) Conn. or a tms (shooty) mutant strain, A66. Both the synthesis of the ethylene precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) and the conversion of ACC to ethylene were affected by crown-gall transformation. All A6-transformed cell lines contained about 50 times more ACC than the A66-transformed cell lines, indicating that the tms genes stimulate ACC synthesis. On the other hand, A6-transformed N. tabacum and L. esculentum cell lines showed a very low capacity to convert ACC to ethylene when compared with A66-transformed cells of the same species. These differences in ACC-dependent ethylene formation were stable and could not be modified by supplying auxin to the culture medium. In contrast, both the A6- and A66-transformed N. glutinosa cell lines showed a low capacity for ACC-dependent ethylene production. Thus, the low-ethylene-forming phenotype did not seem to be under direct control of the tms genes and appeared to be part of the host response to crown-gall transformation. All cell lines exhibiting the low-ethylene-forming phenotype grew as unorganized tissues in culture, whereas cell lines showing a high capacity to convert ACC to ethylene formed shoots. Thus, ACC-dependent ethylene formation may be useful for studying host factors important in determining tumor phenotype.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA -naphthalencacetic acid     

Inhibition of the Conversion of 1-Aminocyclopropane-1-carboxylic Acid to Ethylene by Structural Analogs, Inhibitors of Electron Transfer, Uncouplers of Oxidative Phosphorylation, and Free Radical Scavengers

Cyclopropane carboxylic acid (CCA) at 1 to 5 millimolar, unlike related cyclopropane ring analogs of 1-aminocyclopropane-1-carboxylic acid (ACC) which were virtually ineffective, inhibited C₂H₄ production, and this inhibition was nullified by ACC. Inhibition by CCA is not competitive with ACC since there is a decline, rather than an increase, in native endogenous ACC in the presence of CCA. Similarly, short-chain organic acids from acetic to butyric acid and α-aminoisobutyric acid inhibited C₂H₄ production at 1 to 5 millimolar and lowered endogenous ACC levels. These inhibitions, like that of CCA, were overcome with ACC. Inhibitors of electron transfer and oxidative phosphorylation effectively inhibited ACC conversion to C₂H₄ in pea and apple tissues. The most potent inhibitors were 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) which virtually eliminated ACC-stimulated C₂H₄ production in both tissues. Still other inhibitors of the conversion of ACC to C₂H₄ were putative free radical scavengers which reduced chemiluminescence in the free radical-activated luminol reaction. These inhibitor studies suggest the involvement of a free radical in the reaction sequence which converts ACC to C₂H₄. Additionally, the potent inhibition of this reaction by uncouplers of oxidative phosphorylation (DNP and CCCP) suggest the involvement of ATP or the necessity for an intact membrane for C₂H₄ production from ACC. In the latter case, CCCP may be acting as a proton ionophore to destroy the membrane integrity necessary for C₂H₄ production.     

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     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid by microsomal membranes from senescing carnation flowers

Isolated membranes from the petals of senescing carnation flowers (Dianthus caryophyllus L. cv. White-Sim) catalyze the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. A microsomal membrane fraction obtained by centrifugation at 131,000 g for 1 h proved to be more active than the membrane pellet isolated by centrifugation at 10,000 g for 20 min. The ethylene-producing activity of the microsomal membranes is oxygen-dependent, heat-denaturable, sensitive to n-propyl gallate, and saturable with ACC. Corresponding cytosol fractions from the petals are incapable of converting ACC to ethylene. Moreover, the addition of soluble fraction back to the membrane fraction strongly inhibits the ACC to ethylene conversion activity of the membranes. The efficiency with which isolated membranes convert ACC to ethylene is lower than that exhibited by intact flowers based on the relative yield of membranes per flower. This may be due to the presence of the endogenous soluble inhibitor of the reaction, for residual soluble fraction inevitably remains trapped in membrane vesicles isolated from a homogenate.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA aminoxyacetic acid - AVG aminoethoxyvinylglycine - EPPS N-2-hydroxyethylpiperazine propane sulfonic acid     

The site of 1-aminocyclopropane-1-carboxylic acid synthesis in senescing carnation petals

To study the cause of the uneven production of ethylene by upper and basal portions of detached petals of carnation ( Dianthus caryophyllus L. cv. White Sim), the petals were divided and exposed to ethylene (30 μl 1^-1 for 16 h). The treatment induced rapid wilting and autocatalytic ethylene production in the basal portion similar to that induced in entire petals. In contrast to the response in entire petals and the basal portions, the upper portions responded to ethylene by delayed wilting and much lower ethylene production. Aminocyclopropane carboxylic acid (ACC)-synthase activity in the basal portion of the petals was 38 to 400 times that in the upper portion. In untreated detached petal pieces from senescing carnation flowers, ethylene production by the upper portion declined after 6 h while the basal portion was still producing ethylene at a steady rate 18 h later. Application of ACC to the upper portion of senescing petals increased their ethylene production. α-Aminooxyacetic acid (0.5 m M ), reduced the ethylene production of the senescing basal portion more than that of the upper portion. Endogenous ACC content in basal portions of senescing carnation petals was 3 to 4 times higher than in the upper parts. When detached senescing petals were divided immediately after detaching, the endogenous ACC levels in upper portions remained steady or declined during 24 h after division, while in the basal portions the ACC level rose steadily as in the intact petals. There was no change in the conjugated ACC in either portion after 24 h. Benzyladenine (BA) applied as a pretreatment to entire preclimacteric petals greatly reduced the development of ACC-synthase activity of the basal portion, but had little effect on the activity in the upper portion of the petal. In both portions, however, BA effectively reduced the conversion of ACC to ethylene.     

Ethylene Biosynthesis and Cadmium Toxicity in Leaf Tissue of Beans (Phaseolus vulgaris L.)

Stress ethylene production in bean (Phaseolus vulgaris L., cv. Taylor's Horticultural) leaf tissue was stimulated by Cd²⁺ at concentrations above 1 micromolar. Cd²⁺-induced ethylene biosynthesis was dependent upon synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Activity of ACC synthase and ethylene production rate peaked at 8 h of treatment. The subsequent decline in enzyme activity was most likely due to inactivation of the enzyme by Cd²⁺, which inhibited ACC synthase activity in vitro at concentrations as low as 0.1 micromolar. Decrease in ethylene production rate was accompanied by leakage of solutes and increasing inhibition of ACC-dependent ethylene production. Ca²⁺, present during a 2-hour preincubation, reduced the effect of Cd²⁺ on leakage and ACC conversion. This suggests that Cd²⁺ exerts its toxicity through membrane damage and inactivation of enzymes. The possibility of an indirect stimulation of ethylene biosynthesis through a wound signal from injured cells is discussed.     

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: II. GRADIENT-PURIFIED MITOCHONDRIA LACK CYANIDE-INSENSITIVE RESPIRATION

Wheat leaves normally produced very little ethylene, but following a water deficit stress which caused a loss of 9% initial fresh weight, ethylene production increased more than 30-fold within 4 hours and declined rapidly thereafter. The changes in ethylene production were paralleled by an increase and subsequent decrease in 1-aminocyclopropanecarboxylic acid (ACC) content. The level of S-adenosylmethionine was unaffected, suggesting that the conversion of S-adenosylmethionine to ACC is a key reaction in the production of water stress-induced ethylene. This view was further supported by the observation that application of ACC to nonstressed leaf tissue caused a 70-fold increase in ethylene production, while aminoethoxyvinylglycine, a known inhibitor of the conversion of S-adenosylmethionine to ACC, inhibited ACC accumulation as well as the surge in ethylene production if the inhibitor was applied prior to the stress treatment. Cycloheximide, an inhibitor of protein synthesis, effectively blocked both ethylene production and ACC formation, suggesting that water stress induces de novo synthesis of ACC synthase, which is the rate-controlling enzyme in the pathway of ethylene biosynthesis.     

Activity of Ageing Carnation Flower Parts and the Effects of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid-Induced Ethylene

Peak levels of 1-aminocyclopropane-l-carboxylic acid (ACC) in flower parts of ageing carnations (Dianthus caryophyllus L. cv Scanea 3C) were detected 6 to 9 days after flower opening. The ethylene climacteric and the first visible sign of wilting was observed 7 days after opening. The concentration of conjugated ACC in these same tissues peaked at day three with reduction of 70% by day 4. From day 5 to day 9 all parts followed a diurnal pattern of increasing in conjugate levels 1 day and decreasing the next. Concentrations of conjugated ACC were significantly higher than those of ACC in all ageing parts. Preclimacteric petals treated with ACC or 1-(malonylamino)-cycloprane-1-carboxylic acid (MACC), started to senesce 30 to 36 hours after treatment. When petals were treated with MACC plus by 0.1 millimolar aminoethyoxyvinylglycine, premature senescence was induced, while ethylene production was suppressed relative to MACC-treated petals. Petals treated with MACC and silver complex produced ethylene, but did not senesce. The MACC-induced ethylene was inhibited by the addition of 1.0 millimolar CoC1₂. These results demonstrate MACC-induced senescence in preclimacteric petals. The patterns of ACC and MACC detected in the flower parts support the view that an individual part probably does not export an ethylene precursor to the remainder of the flower inducing senescence.