Effect of paraquat on ethylene biosynthesis by intact green Phaseolus vulgaris seedlings

Ethylene production by intact green bean ( Phaseolus vulgaris L. cv. Limburgse vroege) seedlings was investigated in white light and in darkness. In white light both endogenous and 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene production were stimulated. A decrease in the 1-(malonylamino)cyclopropane-1-carboxylic acid (M-ACC) level and a slight increase in the free ACC concentration could be observed in light. The total amount of endogenous ACC was not changed by light. We related the effect of light to the effect of paraquat on ethylene biosynthesis. Paraquat caused a strong increase of endogenous ethylene production in light. However, the conversion of exogenously applied ACC in light was not influenced by the paraquat treatment, although the presence of the herbicide in the chloroplasts was evident through the inhibition of net photosynthesis. In light, paraquat increased the total ACC content. This was due to an enlargement of the free ACC pool. The effects of white light and paraquat on ethylene biosynthesis can be differentiated from one another: white light exerts its influence on the conversion of ACC to ethylene; it also seems to inhibit the malonylation and may act on the formation of ACC itself. Paraquat influences only ACC synthesis.     

Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a ²H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - FW fresh weight - GC/MS coupled gas chromatography/mass spectrometry - HPLC high-performance liquid chromatography     

The effect of light and phytochrome on 1-aminocyclopropane-1-carboxylic Acid metabolism in etiolated wheat seedling leaves

While light-grown wheat leaves produced ethylene at a low rate of <0.1 nanomoles per gram per hour and contained 1-aminocyclopropane-1-carboxylic acid (ACC) at low levels of <2.5 nanomoles per gram, etiolated wheat leaves produced ethylene at a rate of 2 nanomoles per gram per hour and accumulated concentrations of ACC at levels of 40 nanomoles per gram. Upon illumination of 8-day-old etiolated wheat seedlings with white light, the ethylene production rate increased initially, due to the activation of ethylene-forming activity, but subsequently declined to a low level (0.1 nanomoles per gram per hour) at the end of the 6-hour illumination. This light-induced decline in ethylene production rate resulted from a decline (more than 35 nanomoles per gram) in ACC level, which was accompanied by a corresponding increase in 1-(malonylamino)cyclopropane-1-carboxylic acid content. These data indicate that illumination promoted ACC malonylation, resulting in reduced ACC level and consequently reduced ethylene production. However, light did not cause any significant increase in the extractable ACC-malonyltransferase activity. The effect of continuous white light on promotion of ACC malonylation was also observed in intermittent white light or red light. A far-red light treatment following red light partially reversed the red light effect, indicating that phytochrome participates in the promotion of ACC malonylation.     

Accumulation of 1-(malonylamino)cyclopropane-1-carboxylic acid in ethylene-synthesizing tobacco leaves

During the hypersensitive reaction of Samsun NN tobacco to tobacco mosaic virus (TMV) the inoculated leaves synthesize large quantities of ethylene. At the same time, 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), a conjugate of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) accumulates. Smaller amounts of MACC are formed concomitant with ethylene synthesis during the normal development of tobacco leaves. The conjugate appears neither to be hydrolysed to liberate ACC, nor to be transported to other plant parts. Its accumulation thus reflects the history of the operation of the pathway of ethylene synthesis in the leaf. In floating leaf discs exogenously applied ACC was converted only slowly to both ethylene and MACC. More ethylene and less MACC were produced in darkness than in light, suggesting that environmental conditions may influence the ratio at which ACC in converted to either ethylene or MACC.     

The quantification of 1-(malonylamino)cyclopropane-1-carboxylic acid in plant tissues by quantitative mass spectrometry

A method for the quantitation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), a conjugated form of 1-aminocyclopropane-1-carboxylic acid (ACC), in plants is described. [2,2,3,3-²H₄]MACC has been used as an internal standard for selected ion monitoring/isotope dilution quantitation of MACC in wheat seedlings and in tomato leaves. This method is compared with a widely-used two step indirect assay for MACC, which is based upon hydrolysis of MACC to ACC and conversion of ACC by hypochlorite reagent to ethylene which is subsequently quantified by gas chromatography.     

The enzymatic malonylation of 1-aminocyclopropane-1-carboxylic acid in homogenates of mung-bean hypocotyls

Homogenates of hypocotyls of light-grown mung-bean (Vigna radiata (L.) Wilczek) seedlings catalyzed the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) from the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-coenzyme A. Apparent K_m values for ACC and malonyl-CoA were found to be 0.17 mM and 0.25 mM, respectively. Free coenzyme A was an uncompetitive inhibitor with respect to malonyl-CoA (apparent K_i=0.3 mM). Only malonyl-CoA served as an effective acyl donor in the reaction. The d-enantiomers of unpolar amino acids inhibited the malonylation of ACC. Inhibition by d-phenylalanine was competitive with respect to ACC (apparent K_i=1.2 mM). d-Phenylalanine and d-alanine were malonylated by the preparation, and their malonylation was inhibited by ACC. When hypocotyl segments were administered ACC in the presence of certain unpolar d-amino acids, the malonylation of ACC was inhibited while the production of ethylene was enhanced. Thus, a close-relationship appears to exist between the malonylation of ACC and d-amino acids. The cis- as well as the trans-diastereoisomers of 2-methyl- or 2-ethyl-substituted ACC were potent inhibitors of the malonyltransferase. Treatment of hypocotyl segments with indole-3-acetic acid or CdCl₂ greatly increased their content of ACC and MACC, as well as their release of ethylene, but had little, or no, effect on their extractable ACC-malonylating activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

The effect of water stress on 1-(malonylamino)cyclopropane-1-carboxylic acid concentration in plant tissues

Since the discovery of1-(malonylamino)cyclopropane-1-carboxylic acid (MACC)as a major metabolite of both endogenous andexogenously applied 1-aminocyclopropane-1-carboxylicacid (ACC), it has become evident that the formationof MACC from ACC can act to regulate ethyleneproduction in certain tissues. Hence it was suggestedthat MACC could serve as an indicator of water-stresshistory in plant tissues. The accurate quantificationof MACC in plant tissues is essential forunderstanding the role of MACC in the regulation ofethylene biosynthesis.Hoffman et al. [15] described a method for themeasurement of MACC in which MACC was hydrolysed byHCl to ACC, which was then assayed by chemicaloxidation to form ethylene. Attempts have been made byothers to raise monoclonal antibodies to MACC so thatan immunoassay could be developed in order to gain adeeper understanding of stress-induced ethyleneproduction but no further publications have beenforthcoming.Here a method employing GC-MS is compared with theindirect assay for MACC, which is based uponhydrolysis of MACC to ACC and conversion of ACC byhypochlorite reagent to ethylene which is subsequentlyquantified by GC.     

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

Auxin-induced ethylene biosynthesis in subapical stem sections of etiolated seedlings of Pisum sativum L.

1-Aminocyclopropane-1-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections of etiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 mM ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-¹⁴C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections incubated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine - IAA indole-3-acetic acid - SAM S-adenosylmethionine - SMM S-methylmethionine     

Ethylene-promoted malonylation of 1-aminocyclopropane-1-carboxylic acid participates in autoinhibition of ethylene synthesis in grapefruit flavedo discs

The increase in ethylene formation and in 1-aminocyclopropane-1-carboxylic acid (ACC) content in flavedo tissue of grapefruit (Citrus paradisi Macfad. cv. Ruby Red) in response to excision was markedly inhibited by exogenous ethylene. Ethylene treatment inhibited the synthesis of ACC, but increased the tissue's capability to malonylate ACC to N-malonyl-ACC, resulting in further reduction in the endogenous ACC content. The development of extractable ACC-malonyl-transferase activity in the tissue was markedly promoted by treatment with exogenous ethylene. These results indicate that the autoinhibition of ethylene production in this tissue results not only from suppression of ACC synthesis, but also from promotion of ACC malonylation; both processes reduce the availability of ACC for ethylene synthesis.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethyoxyvinylglycine (2-amino-4-(2-aminoexthoxy)-trans-3-butenoic acid) - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid     

Cell-free ethylene-forming systems lack stereochemical fidelity

In-vitro systems for the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene have been reported using pea supernatants, carnation petal microsomes, olive leaf protein and, most recently, pea mitochondria. It has also been shown, in intact tissues of apple, mung bean and pea, that the system responsible for conversion of ACC to ethylene can produce 1-butene from isomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC). This conversion shows a high degree of steroselectivity, and isomer discrimination is therefore a valuable criterion by which to judge the validity of subcellular systems. It is shown here that all in-vitro ethylene-forming systems so far described fail by a wide margin to match the AEC-isomer preference of the corresponding intact tissues with respect to 1-butene generation. This work supports and extends recent reports by McKeon and Yang (1984, Planta 160, 84–87) and by Guy and Kende (1984, Planta 160, 281–287) on the characteristics of ethylene formation by pea homogenates. The vacuolar conversion described by the latter authors is the simplest system yet described that retains appropriate sterochemical fidelity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - DEAE diethylaminoethyl - IAA indole-3-acetic acid     

Enhancement of extractable ethylene at light/dark transition in primary leaves of paclobutrazol-treated Phaseolus vulgaris seedlings

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol], a triazole growth retardant, increased the 1-aminocyclopropane-1-carboxylic acid (ACC) level and resulted in reduced ethylene production, estimated as ethylene release in a closed system or by vacuum-extraction, in the primary leaves of Phaseolus vulgaris L. cv. Juliska seedlings exposed to light. At the light/dark transition, a definite enhancement of the endogenous ethylene level was observed by vacuum-extraction of primary leaves of treated plants and the ethylene deficiency of retardant-treated leaves ceased. The concentration of ACC after the light/dark transition followed the pattern for ethylene, and the increase in ACC content was paralleled by a decrease in malonyl-ACC.
It is concluded that the internal level of ethylene is not necessarily lower in the primary leaves of paclobutrazol-treated bean plants, but under special environmental conditions in vivo it may reach that of the control.     

Relationship between the malonylation of 1-aminocyclopropane-1-carboxylic acid and D-amino acids in mung-bean hypocotyls

In sections from hypocotyls of dark-grown mung-bean (Vigna radiata L.) seedlings, D-phenylalanine and D-methionine (D-met) inhibited the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid from exogenously administered 1-aminocyclopropane-1-carboxylic acid (ACC), resulting in an increase in free ACC content and stimulation of ethylene production, whereas their L-enantiomers had little or no such effect. When the hypocotyls were administered D-Met, it was mainly metabolized to N-malonylmethionine and N-malonylmethionine sulfoxide, and this malonylation process was inhibited to a greater extent by ACC and D-amino acids (phenylalanine and serine) than by L-amino acids. These results indicate that malonylation of D-amino acids and of ACC are intimately interrelated.     

The effects of 2,2′-bipyridine and other metal chelators on the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in rice

Effects of metal chelators, 2,2-bipyridine, 8-hydroxyquinoline and 1,10-phenenthroline, on the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in detached leaves of light-grown rice (Oryza sativa) seedlings and detached shoots of etiolated rice seedlings were investigated. Metal chelators strongly inhibited the in vivo ACC oxidase activity in detached leaves and detached etiolated shoots. This inhibition could be partially recovered by Fe²⁺. Our results support the notion that Fe²⁺ is an essential cofactor for the conversion of ACC to ethylene in vivo.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - BP 2,2-bypyridine - HQ 8-hydroxylquinoline - MJ methyl jasmonate - PA 1,10-phenanthroline - Put putrescine     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Metabolism of 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

In preclimacteric apple fruits ( Malus × domestica Borkh. cv. Golden Delicious) ethylene production is controlled by the rates of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis, and by its metabolism to ethylene by the ethylene-forming enzyme and to 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by malonyl CoA-ACC transferase. The onset of the climacteric in ethylene production is associated with an increase in the activity of the ethylene-forming enzyme in the pulp and with a rise in the activity of ACC synthase. Malonyl transferase activity is very high in the skin of immature fruit, decreases sharply before the onset of the climacteric, and remains nearly constant thereafter. More than 40% of the ACC synthesized in the skin and around 5% in the flesh, are diverted to MACC at early climacteric. At the climacteric peak there are substantial gradients in ethylene production between different portions of the tissue, the inner cortical tissues producing up to twice as much as the external tissues. This increased production is associated with, and apparently due to, increased content of ACC synthase. Less than 1% of the synthesized ACC is diverted to MACC in the flesh of climacteric apples. In contrast, the skin contains high activity of malonyl transferase, and correspondingly high levels [1000 nmol (g dry weight)⁻¹] of MACC.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

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     

Salicylic acid inhibits the biosynthesis of ethylene in detached rice leaves

The effects of salicylic acid (SA) on ethylene biosynthesis in detached rice leaves were investigated. SA at pH 3.5 effectively inhibited ethylene production within 2 h of its application. It inhibited the conversion of ACC to ethylene, but did not affect the levels of ACC and conjugated ACC. Thus, the inhibitory effect of SA resulted from the inhibition of both synthesis of ACC and the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - SA salicylic acid     

Metabolic changes in cherry flower buds associated with breaking of dormancy in early and late blooming cultivars

Changes of metabolic activities during dormancy and breaking of dormancy in the cherry flower buds of early blooming (EB) cultivar ( Prunus avium L. cv. Coeur de Pigeon) and late blooming (LB) cultivar ( Prunus serrulata Lindl. cv. Kwanzan) were determined. The LB buds had higher polyamines, protein and 1-(malonylamino) cyclopropane-1-carboxylic acid (MACC) content than the EB buds. During the dormant state, the DNA, RNA, protein and polyamines in the EB buds were low but increased slowly and steadily, whereas those in the LB buds remained at a consistently higher level. The transition from dormancy to the active state in both cultivars was characterized by a sharp increase in DNA, RNA, protein, polyamines, S-adenosyl-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC) and MACC. After initial swelling and development of flowers, the levels of all these components decreased. Polyamine and ethylene biosyntheses did not seem to be competing for their common substrate, SAM, during flower bud development.