ACC Synthesis as the Activated Step Responsible for the Rise of Ethylene Production Accompanying Citrus Exocortis Viroid Infection in Tomato Plants

Ethylene production was stimulated during the period when systemic symptoms appeared in tomato plants infected with citrus exocortis viroid (CEV). Neither methionine nor S-adenosylmethionine increased ethylene production in leaf discs. In contrast, 1-aminocyclopropane-l-carboxylic acid (ACC) stimulated ethylene production notably. Whether viroid infection acted upon ACC production, its conversion to ethylene, or both, was studied by determining the time course of the concentration of ACC and its in vivo production and conversion rates. During early symptoms, ACC synthesis increased and then remained steady during the development of symptoms, but no difference in the capacity of conversion of ACC to ethylene between healthy and CEV-infected tissues was observed. This indicates that ethylene production in tomato leaves showing systemic symptoms to CEV is activated at the level of ACC production.     

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。     

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     

Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase

Ethylene evolved during compatible or susceptible disease interactions may hasten and/or worsen disease symptom development; if so, the prevention of disease-response ethylene should reduce disease symptoms. We have examined the effects of reduced ethylene synthesis on Verticillium wilt (causal organism, Verticillium dahliae) of tomato by transforming tomato with ACC deaminase, which cleaves ACC, the immediate biosynthetic precursor of ethylene in plants. Three promoters were used to express ACC deaminase in the plant: (i) CaMV 35S (constitutive expression); (ii) rolD (limits expression specifically to the site of Verticillium infection, i.e. the roots); and (iii) prb-1b (limits expression to certain environmental cues, e.g. disease infection). Significant reductions in the symptoms of Verticillium wilt were obtained for rolD- and prb-1b-, but not for 35S-transformants. The pathogen was detected in stem sections of plants with reduced symptoms, suggesting that reduced ethylene synthesis results in increased disease tolerance. The effective control of formerly recalcitrant diseases such as Verticillium wilt may thus be obtained by preventing disease-related ethylene production via the tissue-specific expression of ACC deaminase.     

The Production of Marker-Free Genetically Engineered Broccoli with Sense and Antisense ACC synthase 1 and ACC oxidases 1 and 2 to Extend Shelf-Life

The production of transgenic broccoli (Brassica oleracea) with increased shelf-life using an Agrobacterium rhizogenes-mediated co-transformation protocol is reported. An Agrobacterium rhizogenes Ri vector, pRi1855:GFP was constructed to allow expression of the green fluorescent protein to identify insertion of Ri T_L-DNA into plant cells. The Brassica oleracea ACC synthase 1 and ACC oxidase 1 and 2 cDNAs in sense and antisense orientations were co-transformed into GDDH33, a doubled haploid calabrese-broccoli cultivar. Transformation efficiency was 3.26%, producing 150 transgenic root lines, of which 18 were regenerated into mature plants. The floral buds from T₀ broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. Buds from T₀ lines transformed with ACC oxidase 1 and 2 constructs produced significantly less post-harvest ethylene at 20 °C than the untransformed plants and chlorophyll loss was significantly reduced over a 96 h post-harvest period. The T₀ plants transformed with sense and antisense ACC synthase 1 had a significantly reduced 24 h post-harvest ethylene peak and delayed chlorophyll loss. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by a regression. This demonstrates that the shelf-life of a very perishable vegetable may be increased up to 2 days at 20 °C by reducing post-harvest ethylene production.     

1-Methylcyclopropene and ethylene as regulators of in vitro organogenesis in kiwi explants

In this paper, we study the influence of ACC, AVG and 1-MCP on in vitro organogenesis of kiwi (Actinidia deliciosa) explants and on ethylene metabolism. Results indicated that increasing ethylene production and accumulation in the head space of the culture vessel by adding ACC to the culture medium inhibited organogenesis, except for rooting, which increased and stimulated ACC oxidase activity threefold, whereas AVG increased the length and number of shoots and leaves and inhibited about 80% ACC synthase activity compared with the reference explants. 1-MCP exerts a stimulatory effect analogous to AVG, increasing ACC synthase activity and organogenesis of kiwi explants. This effect is not reverted by the addition of ACC to the culture medium. Therefore, ethylene production and its accumulation in the headspace of the culture vessels seems to be responsible for the inhibition of shoot development as well as increasing rooting in in vitro cultured kiwi explants.     

Ethylene and ACC levels in developing grains are related to the poor appearance and milling quality of rice

The possible relationship between the levels of ethylene and 1-aminocylopropane-1-carboxylic acid (ACC) in the grains and the quality of rice (Oryza sativa L.) were investigated by using 12 rice cultivars. The results showed that both the ethylene evolution rate and ACC content in grains during the grain filling period correlated negatively with head rice production and positively with chalky kernels, chalky size, and chalkiness. The levels of ethylene and ACC were not significantly correlated with alkali spreading value and amylose content. Application of ethephon, an ethylene-releasing agent, or ACC to panicles at the early grain filling stage significantly reduced the rates of brown rice, milled rice, and head rice, and significantly increased the percentage of chalky kernels, chalky size, and chalkiness. Application of aminoethoxyvinylglycine, an inhibitor of ACC synthase, had the opposite effect. Chalkiness appears to be a senescence related phenomenon which is stimulated by ethylene. The results suggest that ethylene and ACC in grains play an important role in regulating rice quality, and that grain appearance and milling quality would be improved though the reduction of ethylene and ACC in grains during grain filling.     

Formation of stress ethylene depends both on ACC synthesis and on the activity of free radical-generating system

Leaves of soybean ( Glyxine max. L., var. Progress) were subjected to desiccation, which brought about varying degree of membrane damage as checked with the conductivity method. Progress of injury up to 30% was associated with promotion of ethylene synthesis and with accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-l-carboxylic acid (MACC) in the cells, as well as with activation of lipoxygenase, the enzyme which is involved in lipid peroxidation and which is capable of forming activated oxygen. The stress-induced promotion of ethylene synthesis was inhibited by the ACC synthase inhibitor aminooxyacetate (AOA). as well as by n-propyl gallate (PG), a free radical scavenger and inhibitor of lipoxygenase. Pretreatment of non-stressed soybean leaves with different concentrations of PG also resulted in the corresponding inhibition of lipoxygenase activity and ethylene formation, the former effect being less pronounced than the latter one. In the tissues pretreated with propyl gallate, the ACC level was not affected, whereas the MACC substantially increased. In leaves showing 40% membrane damage neither lipoxygenase activity nor ethylene synthesis increased any further, despite a further increase in the ACC and MACC levels. Therefore, we propose that there are two prerequisites for effective in vivo synthesis of stress ethylene: promotion of ACC synthesis and activation of a free radical-generating system, which is responsible for the non-enzymatic conversion of ACC to ethylene. The latter effect seems to be due to the activation of the membrane-associated lipoxygenase, which depends on stress-induced alterations in membrane properties.     

Daminozide inhibits ethylene production in apple fruit by blocking the conversion of methionine to aminocyclopropane-1-carboxylic acid (ACC)

At harvest, fruit from apple trees sprayed with daminozide (+daminozide) had lower levels of aminocyclopropane-1-carboxylic acid (ACC) and produced significantly lower amounts of ethylene than untreated (–daminozide) fruit. Flesh discs from the fruit of +daminozide and –daminozide trees were fed precursors of ethylene to determine how daminozide inhibits ethylene production. ACC was metabolized to ethylene regardless of treatment. Methionine (MET), however, was only converted to ethylene by –daminozide fruit, and only after the fruit had been maintained at 4 °C for 5 months. +Daminozide fruit failed to convert MET to ethylene at harvest, as well as after cold storage. When daminozide was added to the incubation media of flesh discs it did not inhibit ethylene production or the conversion of ACC to ethylene. The addition of daminozide did, however, inhibit the metabolism of exogenous MET to ethylene. Aminooxyacetate acid (AOA) blocked both the endogenous production of ethylene and that from MET feeds. Daminozide inhibits ethylene production by preventing the conversion of MET to ACC, but it does not appear to act as a simple competitive inhibitor of ACC synthase activity.Abbreviations ACC aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - AOA aminooxyacetic acid - CH cycloheximide - MET methionine - PUT putrescine Author for correspondence     

Expression of ACC synthase is enhanced earlier than that of ACC oxidase during fruit ripening of mume (Prunus mume)

Mume (Japanese apricot: Prunus mume Sieb. et Zucc.) is a climacteric fruit that produces large amounts of ethylene as it ripens. Ripening is accompanied by marked increases in the activities of two ethylene-biosynthetic enzymes, namely, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. To study the molecular aspects of ripening of mume, we isolated cDNA clones for proteins that we considered likely to be involved in the biosynthesis and perception of ethylene during ripening, namely, ACC synthase, ACC oxidase and the ethylene receptor. Northern blotting analysis revealed the markedly increased expression of ACC synthase prior to that of ACC oxidase and the increase in ethylene production during ripening. Overall, the levels of the mRNAs for the genes corresponded closely to the levels of activity of the ethylene-biosynthetic enzymes. Exposure of mature green mume fruit to ethylene for 12 h induced strong expression of ACC synthase, as well as of ACC oxidase. Wounding of the pericarp of mume fruit induced the expression of ACC synthase but not of ACC oxidase. The rate of ethylene production increased only slightly after wounding. These results suggest that expression of the genes for ACC synthase and ACC oxidase must be activated sequentially for maximum production of ethylene during ripening of mume fruit and that several mechanisms regulate the expression of ethylene-biosynthetic genes during ripening.     

Vitrification of carnation in vitro: Changes in ethylene production,ACC level and capacity to convert ACC to ethylene

Carnation tissue was allowed to vitrify in liquid culture and ethylene production, ACC content and capacity to convert ACC to ethylene were measured in comparison to tissue developing normally on solid medium. Flask atmospheres of liquid cultures accumulated ethylene at a higher rate during the first four days. Daily ethylene production by vitrifying material decreased later. Ethylene emission by vitrifying tissues always remained above controls when subcultured daily to fresh medium. Explants and microsomal preparations from vitrifying carnations converted ACC to ethylene at a higher degree from the first day in liquid medium. ACC level markedly increased in vitrifying tissues during the first two days of liquid culture. Raising the level of ethylene in the atmosphere of solid cultures did not induce vitrification symptoms nor did use of inhibitors of ethylene biosynthesis in liquid cultures prevent the process. The role of ethylene in vitrification is reappraised.     

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)