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.     

Is lipoxygenase involved in the formation of ethylene from ACC?

Freezing or desiccation of winter rape leaves ( Brassica napus L. var. oleifera (cv. Górczanski) stimulated both lipoxygenase (EC 1.13.11.12) activity and ethylene formation during the post-stress period. The effect depended on the degree of membrane injury. In tissues showing injury less than 50% (as checked with the electrical conductivity method) both activities increased according to the degree of stress-induced damage. In leaves injured to a higher degree both activities decreased. Light and low temperature (5°C) inhibited the development of both lipoxygenase activity and ethylene formation in leaf disks stored for 20 h. Ethylene formation was also observed in a model system where soybean lipoxygenase was added to a mixture containing 1-aminocyclopropane-1-carboxylic acid and linoleic or linolenic acid as substrate for lipoperoxide formation. Changes in pH and temperature conditions of the incubation mixture caused similar differences in the lipoxygenase activity and ethylene formation. We propose that the stimulation of lipoxygenase-catalysed oxidation of polyunsaturated fatty acids (increasing free radical formation) leads to an increased ethylene production from ACC.     

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.     

Uniconazole inhibits stress-induced ethylene in wheat and soybean seedlings

Previous studies have shown that uniconazole inhibits ethylene synthesis and protects plants from various stresses. The present research was conducted to delineate the mechanism of ethylene inhibition by uniconazole [(E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol]. Following heat stress of 48°C for 3 h, the shoots of the control wheat seedlings became desiccated, and the seedlings lost 23% of their fresh mass 8 h after stress. The control soybean seedlings had epinastic unifoliate leaves 5 h after foliar application (4.4 g.a.i./ha) of the herbicide triclopyr [(3,5,6-trichloro-2-pyridinyl)oxyacetic acid]. Soil drench applications of uniconazole, a potent member of the triazole family, reduced these symptoms associated with heat and herbicide stress in wheat (5.0 mg/L) and soybean (0.4 mg/L) seedlings, respectively.Basal ethylene production was inhibited 32 and 48% by uniconazole in the wheat and acotyledonous soybean seedlings, respectively. Following a 48°C heat stress, 1-aminocyclopropane-1-carboxylic acid (ACC) levels increased 40% in both the control and uniconazole-treated wheat seedlings. After triclopyr application, ACC levels increased 400% in both the control and uniconazoletreated soybean seedlings. The increased ACC levels, following stress, were accompanied by increased ethylene production from the control, but not from the uniconazole-treated wheat and acotyledonous soybean seedlings. Uniconazole treatment did not significantly change the basal or stress-induced N-malonyl-1-aminocyclopropane-1-carboxylic acid (MACC) levels compared to controls. These results suggest that uniconazole inhibits ethylene synthesis by interfering with the conversion of ACC to ethylene in wheat and acotyledonous soybean seedlings. Ethylene production and ACC conversion were not inhibited by uniconazole in excised soybean cotyledons. These results indicate that different ethylene-forming enzyme (EFE) systems operate in the soybean acotyledonous seedling and cotyledon, and the system in the former is inhibited by uniconazole.     

Uniconazole inhibits stress-induced ethylene in wheat and soybean seedlings

Previous studies have shown that uniconazole inhibits ethylene synthesis and protects plants from various stresses. The present research was conducted to delineate the mechanism of ethylene inhibition by uniconazole [(E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol]. Following heat stress of 48°C for 3 h, the shoots of the control wheat seedlings became desiccated, and the seedlings lost 23% of their fresh mass 8 h after stress. The control soybean seedlings had epinastic unifoliate leaves 5 h after foliar application (4.4 g.a.i./ha) of the herbicide triclopyr [(3,5,6-trichloro-2-pyridinyl)oxyacetic acid]. Soil drench applications of uniconazole, a potent member of the triazole family, reduced these symptoms associated with heat and herbicide stress in wheat (5.0 mg/L) and soybean (0.4 mg/L) seedlings, respectively. Basal ethylene production was inhibited 32 and 48% by uniconazole in the wheat and acotyledonous soybean seedlings, respectively. Following a 48°C heat stress, 1-aminocyclopropane-1-carboxylic acid (ACC) levels increased 40% in both the control and uniconazole-treated wheat seedlings. After triclopyr application, ACC levels increased 400% in both the control and uniconazoletreated soybean seedlings. The increased ACC levels, following stress, were accompanied by increased ethylene production from the control, but not from the uniconazole-treated wheat and acotyledonous soybean seedlings. Uniconazole treatment did not significantly change the basal or stress-induced N-malonyl-1-aminocyclopropane-1-carboxylic acid (MACC) levels compared to controls. These results suggest that uniconazole inhibits ethylene synthesis by interfering with the conversion of ACC to ethylene in wheat and acotyledonous soybean seedlings. Ethylene production and ACC conversion were not inhibited by uniconazole in excised soybean cotyledons. These results indicate that different ethylene-forming enzyme (EFE) systems operate in the soybean acotyledonous seedling and cotyledon, and the system in the former is inhibited by uniconazole.     

Effects of Free Radical Scavengers on Stress Ethylene in Soybean Leaves Hypersensitively Reacting to Tobacco Necrosis Virus

The hypersensitive reaction of soybean cuttings to tobacco necrosisvirus is characterized by a large stimulation of stress ethyleneinvolving a marked accumulation of free 1-aminocyclopropane-1-carboxylicacid (ACC) and a moderate increase in ethylene-forming enzyme(EFE) activity. The scavengers of hydroxyl radicals (OH^{dot})sodium benzoate, sodium formate, mannitol and dimethylsulphoxide,did not affect stress ethylene biosynthesis. Propyl gallate,an inhibitor of lipoxygenase enzymes, substantially reducedthe release of stress ethylene from hypersensitive leaves. Thisreduction was not attributable to an inhibitory effect on EFEactivity, but to a strong reduction of free ACC accumulationin leaf tissues. The results suggest that OH^{dot} and the lipoxygenasesystem are not involved in stress ethylene produced during thehypersensitive reaction of soybean to this virus. Glycine max Merr, soybean, ethylene, free radicals, hypersensitivity, tobacco necrosis virus     

春小麦水分胁迫响应中的ACC、MACC合成及乙烯的释放

水分胁迫使两个抗旱性不同的春小麦 (TriticumaestivumL .)品种“8139”(抗旱性较弱 )和“5 0 4”(抗旱性较强 )叶片ACC和MACC含量于胁迫初期下降后期升高 ,ACC合酶活性持续升高 ,乙烯释放量在 8139中下降而在5 0 4中先大幅升高而后下降。两种作用效果相反的抑制剂MGBG (抑制SAMDC活性 )和AOA (抑制ACC合酶活性 )均明显影响了两品种春小麦叶片以上各指标的变化。结果表明 ,水分胁迫下作物乙烯的释放量并不与其合成直接前体ACC的量成正相关 ;胁迫乙烯在抗性品种中于胁迫初期的升高可能是植物胁迫信号传导的响应之一 ,是一种干旱适应现象 ,可能与作物的干旱忍耐形成有关 ,而MACC具有调节胁迫乙烯释放的特殊生理作用。     

Changes m Ethylene Production in Relation to l-Aminocyclopropane-l-Carboxylic Acid and Its Malonyl Conjugate in Waterlogged Wheat Plants

When wheat seedlings were subjected to waterlogging, 1-aminocyelopropane-l-carboxylic acid (ACC), an ethylene precursor, accumulated in large quantity in roots. In shoots, ACC and ethylene production also increased, but declined with the prolonged periods of waterlogging. However, ACC content in roots maintained in high level during the whole period of waterlogging. Drainage caused a drastic drop in both ACC content and ethylene production in waterlogged plants to control level. 1-(malonylamino) cyclopropane-l-carboxylic acid (MACC) level in roots subjected to waterlogging showed little changes. However, MACC content in shoots kept increasing during the 9-days period of waterlogging. At later period of waterlogging (longer than 5 days) when ACC and ethylene production bad dropped, the. level of MACC continued to increase. Draining stopped this increasing, but did not reduced its level. When exogenous ACC was introduced into the leaves via transpiration stream, the ability of leaves of waterlogged plant to convert ACC to MACC was much higher than control. The data presented showed that at the later stage of waterlogging, the conversien of a great quantity of ACC to MACC in waterlogged wheat plants is the cause of the reduction of ethylene production and ACC content. It was suggested that the formation of MACC is another way of regulation in ethylene biosynthesis. Among leaves of different ages, the enhancement of ethylene, ACC and MACC content was more pronounced in older leaves than in younger laves during the waterlogging period. The physiological significance of adaptation to waterlogging stress was discussed.     

Changes in 1-(malonylamino)cyclopropane-1-carboxylic acid content in wilted wheat leaves in relation to their ethylene production rates and 1-aminocyclopropane-1-carboxylic acid content

In excised wheat (Triticum aestivum L.) leaves, water-deficit stress resulted in a rapid increase, followed by a decrease, in ethylene production rates and in the levels of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. However, the level of N-malonyl-ACC (MACC), the major metabolite of ACC, increased gradually, then leveled off. This increase in MACC was much greater than the decrease in ACC level. The MACC levels were positively correlated with severity of water stress. Once established, the MACC levels did not decrease even after the stressed tissues were rehydrated. Administration of labeled ACC and MACC showed that the conjugation of ACC to MACC was essentially irreversible. Repeated wilting treatments following the first wilting and rehydration cycle resulted in no further increase in ethylene production and in the levels of ACC and MACC. However, when benzyladenine was supplied during the preceding rehydration process, subsequent wilting treatment resulted in a rise in MACC level and a rapid rise followed by a decline in ethylene production rates and in the level of ACC. The magnitude of these increases was, however, smaller in these rewilted tissues than that observed in the first wilting treatment. Since MACC accumulates with water stress and is not appreciably metabolized, the MACC level is a good indicator of the stress history in the detached leaves used.     

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.     

Regulation of ethylene biosynthesis after short-term heat treatment in etiolated pea stems

Incubation of plant tissues at a constant elevated temperature greatly inhibits both basal and wound ethylene production. However, recovery from heat treatment is relatively rapid and is followed by stimulated ethylene production. The present investigation examines the kinetics of ethylene production after short-term heal treatment and the regulation of heat-altered ethylene production. Subapical stem segments of 7-day-old etiolated pea L. cv. Alaska) seedlings were analyzed for ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) oxidation, and ACC and l-(malonylamino)cyclopropane-l-carboxylic acid (MACC) content after a 2-min 40°C heat pulse. The short-term heat pulse transiently inhibited ethylene production and ACC oxidation accompanied by a slight ACC accumulation within a 30-min time period. Conjugation to MACC did not appear to play an integral role in heat-regulated ethylene production. It was concluded that the major factor affecting ethylene production after heat treatment is the temporary inactivation of ACC oxidation. The possible roles of ACC synthase, ACC oxidase and lipoxygenase in regulating ethylene production after heat treatment are discussed.     

Effect of paclobutrazol on water stress-induced ethylene biosynthesis and polyamine accumulation in apple seedling leaves

Ethylene biosynthesis and polyamine content were determined in [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol] (paclobutrazol) pre-treated and non-treated water-stressed apple seedling leaves. Paclobutrazol reduced water loss, and decreased endogenous putrescine spermidine content. Gibberellic acid (GA) counteracted the inhibitory effect of paclobutrazol on polyamine content. Paclobutrazol also prevented accumulation of water stress-induced 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), ethylene production and polyamines in apple leaves. α-Difluoromethylarginine (DFMA), but not α-difluoromethylornithine (DFMO), inhibited the rise of putrescine and spermidine in stressed leaves. S-Adenosylmethionine (SAM) was maintained at a steady state level even when ethylene and the polyamines were actively synthesized in stressed apple seedling leaves. The conversion of ACC to ethylene did not appear to be affected by paclobutrazol treatment.     

Comparative Study of the Metabolism of 1-Aminocyclopropane-1-carboxylic Acid and Senescence of Water-Stressed and ABA-Treated Excised Rice Leaves

Changes in the metabolism of 1-aminocyclopropane-l-carboxylicacid (ACC) during senescence in the light in turgid, water-stressed,and ABA-treated, excised rice leaves were examined. The decreasesin levels of Chl and protein were more rapid in the water-stressedand in the ABA-treated leaves than in the turgid leaves. Inturgid leaves, levels of proline remained very low, but theyincreased considerably as a result of water stress or treatmentwith ABA. The production of ethylene was strongly inhibitedby water stress and by ABA through the inhibition of the synthesisof ACC and/or the conversion of ACC to ethylene. In turgid leaves,the level of 1-(malonylamino)cyclopropane-l-carboxylic acid(MACC) increased with time during incubation in the light. Waterstress resulted in a pattern of accumulation of MACC similarto that in the turgid control. However, ABA blocked the malonylationof ACC. (Received July 27, 1989; Accepted March 12, 1990)     

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     

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.     

Effect of MGBG on Growth of the Callus Tissue,Induction of Somatic Embryogenesis of Alfalfa and Their Ethylene Biosynthesis

MGBG [methylglyoxal bis(guanylhydrazone)], when added to the initial of callus subculture, promoted ethylene production in callus grawn in Bsh medium, and enhanced the ACC (1-aminocyclopropane-l-carboxylic acid) level and the ACC synthase activity both in the callus grown in Bsh medimn, and in the suspension cultures in Bsg liquid medium for induction of somatic embryogenesis. However, MGBG reduced the malonyl-ACC (MACC) level of the suspension tissues. The treatments of MGBG caused growth of the cultured tissues and induction efficiency of somatic embryogenesis to reduce. Moreover, the effects of MGBG was enhanced when it was added at induction medium of somatic embryogenesis. It could be concluded that this inhibitory effect of MGBG could be due to its promotion of ethylene, ACC levels and ACC synthase activity which has shown to inhibit somatic embryogenesis. The reduction of MACC level could be also involved in the MGBG effect on induction of the embryogenesis.     

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.     

瓠瓜劝苗的乙烯释放与ACC,乙烯利的吸收和运转

以~14G—ACC饲喂瓠瓜幼苗基部叶片,其放射性在植株各部位呈不均匀分布,大部分集中于生长旺盛的生长点与正在形成的花芽。饲喂后4h内,ACC主要以游离形式存在,24h后,出现ACC的结合物MACC.     

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.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine - EDTA ethylenediaminetetraacetic acid     

The physiological role of lipoxygenase in ethylene formation from 1-aminocyclopropane-1-carboxylic acid in oat leaves

In order to understand the physiological significance of the in-vitro lipoxygenase (EC 1.13.11.12)-mediated ethylene-forming system (J.F. Bousquet and K.V. Thimann 1984, Proc. Natl. Acad. Sci. USA 81, 1724–1727), its characteristics were compared to those of an in-vivo ethylene-forming system. While oat (Avena sativa L.) leaves, as other plant tissues, preferentially converted only one of the 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) isomers to 1-butene, the lipoxygenase system converted all four AEC isomers to 1-butene with nearly equal efficiencies. While the in-vivo ethylene-forming system of oat leaves was saturable with ACC with a K_m of 16 M, the lipoxygenase system was not saturated with ACC even at 10 mM. In contrast to the in-vivo results, only 10% of the ACC consumed in the lipoxygenase system was converted to ethylene, indicating that the reaction is not specific for ethylene formation. Increased ACC-dependent ethylene production in oat leaves following pretreatment with linoleic acid has been inferred as evidence of the involvement of lipoxygenase in ethylene production. We found that pretreating oat leaves with linoleic acid resulted in increased ACC uptake and thereby increased ethylene production. A similar effect was observed with oleic acid, which is not a substrate of lipoxygenase. Since linoleic acid hydroperoxide can substitute for lipoxygenase and linoleic acid in this system, it is assumed that the alkoxy radicals generated during the decomposion of linoleic acid hydroperoxide are responsible for the degradation of ACC to ethylene. Our results collectively indicate that the reported lipoxygenase system is not the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - Epps N-(2-hydroxyethyl)-piperazine-N-3-propanesulfonic acid - LH linoleic acid - LOOH linoleic acid hydroperoxide - pyridoxal-P pyridoxal-phosphate This work was presented at the 12th International Conference on Plant Growth Substances, Heidelberg, FRG, August 1985 (Abstract No. PO 5-52)