Ethylene synthesis regulated by biphasic induction of 1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase genes is required for hydrogen peroxide accumulation and cell death in ozone-exposed tomato

We show that above a certain threshold concentration, ozone leads to leaf injury in tomato (Lycopersicon esculentum). Ozone-induced leaf damage was preceded by a rapid increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, ACC content, and ethylene emission. Changes in mRNA levels of specific ACC synthase, ACC oxidase, and ethylene receptor genes occurred within 1 to 5 h. Expression of the genes encoding components of ethylene biosynthesis and perception, and biochemistry of ethylene synthesis suggested that ozone-induced ethylene synthesis in tomato is under biphasic control. In transgenic plants containing an LE-ACO1 promoter-beta-glucuronidase fusion construct, beta-glucuronidase activity increased rapidly at the beginning of the O(3) exposure and had a spatial distribution resembling the pattern of extracellular H(2)O(2) production at 7 h, which coincided with the cell death pattern after 24 h. Ethylene synthesis and perception were required for active H(2)O(2) production and cell death resulting in visible tissue damage. The results demonstrate a selective ozone response of ethylene biosynthetic genes and suggest a role for ethylene, in combination with the burst of H(2)O(2) production, in regulating the spread of cell death.     

Effects of methyl jasmonate (MeJA) on the dark-induced senescence in oat (Avena sativa L.) leaf segments

To investigate the relationship between methyl jasmonate (MeJA) and ethylene in leaf senescence, we studied the effects of MeJA on ethylene production and ethylene biosynthetic enzyme activities in oat(Avena sativa L.) leaf segments incubated in darkness. MeJA promoted dark-induced senescence judged from the contents of chlorophyll and protein, and increased ethylene production 6 times of the control. MeJA also increased the activities of ethylene biosynthetic enzymes, 1-aminocyclopropane carboxylic acid (ACC) synthase and ACC oxidase as compared to control. In MeJA-treated leaf segments, ACC synthase activity reached its maximum level at 24 h of incubation and ACC oxidase activity peaked at 6 h of incubation. Aminoethoxyvinylglycine (AVG) and Co²⁺, inhibitors of ACC synthase and ACC oxidase respectively, reduced MeJA-induced ethylene production. They also delayed leaf senescence that was promoted by the treatment of MeJA. From these results, we can suggest that MeJA increased the activities of ACC synthase and ACC oxidase, these increased activities lead to increase in ethylene production and this increased ethylene production might promote dark-induced leaf senescence.     

Manipulation of ethylene biosynthesis

The plant hormone ethylene is involved in many plant processes ranging from seed germination to leaf and flower senescence and fruit ripening. Ethylene is synthesized from methionine, via S-adenosyl-L-methionine (SAM) and 1-amino-cyclopropane-1-carboxylic acid (ACC). The key ethylene biosynthetic enzymes are ACC synthase (ACS) and ACC oxidase (ACO). Manipulation of ethylene biosynthesis by chemicals and gene technology is discussed. Biotechnological modification of ethylene synthesis is a promising method to prevent spoilage of agricultural and horticultural products.     

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.     

The Role in Ozone Phytotoxicity of the Evolution of Ethylene upon Induction of 1-Aminocydopropane-1-carboxylic Acid Synthase by Ozone Fumigation in Tomato Plants

The rate of evolution of ethylene by tomato plants was rapidlyincreased by O₃ fumigation. The time course of the increasein 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activitywas the same as that in the rate of evolution of ethylene, suggestingthat ACC synthase activity might be a rate-limiting step inthe evolution of ethylene that is caused by O₃ fumigation. Therate of the O₃-induced evolution of ethylene was increased bythe application of ACC to tomato plants, suggesting the involvementof ACC oxidase in the O₃-induced evolution of ethylene. Treatmentof plants with tiron inhibited the evolution of ethane, butnot of ethylene. These results indicated that evolution of ethylenein O₃-treated tomato plants might result from enzymatic reactionscatalyzed by both ACC synthase and ACC oxidase, but not fromstimulation by O₃ of the peroxidation of lipids mediated byfree radicals. Pretreatment of leaves with aminoethoxyvinylglycine (AVG), aninhibitor of ACC synthase, significantly inhibited the evolutionof ethylene that was induced by O₃ and concomitantly reducedthe extent of O₃-induced visible damage to leaves. Treatmentwith 2,5-norbonadiene, an inhibitor of the action of ethylene,strongly reduced the extent of visible damage caused by O₃,even though it did not suppress the evloution of ethylene. Theseresults indicate that ethylene acts on certain metabolic processesto cause visible damage. (Received September 7, 1995; Accepted December 18, 1995)     

Ethylene regulation of fruit ripening: Molecular aspects

Progress in ethylene regulating fruit ripening concerning itsperception and signal transduction and expression of ACC synthaseand ACC oxidase genes is reviewed. ACC synthase and ACC oxidasehave been characterized and their genes cloned from various fruittissues. Both ACC synthase and ACC oxidase are encoded bymultigene families, and their activities are associated withfruit ripening. In climacteric fruit, the transition toautocatalytic ethylene production appears to be due to a seriesof events in which ACC sythase and ACC oxidase genes have beenexpressed developmentally. Differential expression of ACCsynthase and ACC oxidase gene family members is probably involvedin such a transition that ultimately controls the onset of fruitripening.In comparison to ACC synthase and ACC oxidase, less is knownabout ethylene perception and signal transduction because of thedifficulties in isolating and purifying ethylene receptors orethylene-binding proteins using biochemical methods. However, theidentification of the Nr tomato ripening mutant as anethylene receptor, the applications of new potent anti-ethylenecompounds and the generation of transgenic fruits with reducedethylene production have provided evidence that ethylenereceptors regulate a defined set of genes which are expressedduring fruit ripening. The properties and functions of ethylenereceptors, such as ETR1, are being elucidated.Application of molecular genetics, in combination withbiochemical approaches, will enable us to better understand theindividual steps leading from ethylene perception and signaltransduction and expression of ACC synthase and ACC oxidase genefamily member to the physiological responses.     

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.     

Ethylene biosynthesis in a chilling-sensitive<Emphasis Type="Italic">Arabidopsis</Emphasis> mutant,<Emphasis Type="Italic">chs4-2</Emphasis>

We investigated chilling-induced changes in ethylene levels in Arabidopsis to find plants with distinct patterns of ethylene production in the cold-related biosynthetic pathway. The sensitive mutants identified here includedchs1-2,chs4-2, andchs6-2. Among these, plants of thechs4-2 mutant produced more ethylene than did the wild type after both were transferred from 4°C or 10°C to 22°C. This mutant also showed less freezing tolerance and more electrolyte leakage than the wild-type plants. Our results suggest a relationship between ethylene biosynthesis and chilling sensitivity in the mutant To determine which of the enzymes involved in ethylene biosynthesis were induced by chilling, we tested the activities of ACC synthase and ACC oxidase in both mutant and wild-type plants, and found greater activity by ACC synthase as well as a higher ACC content in the mutants after all the plants were transferred from 10°C to 22°C. However, ACC oxidase activity did not differ between mutant and wild-type plants in response to chilling treatment Therefore, we conclude thatchs4-2 mutants produce more ethylene than do other mutants or the wild type during their recovery from chilling conditions. Furthermore, we believe that ACC synthase is the key enzyme involved in this response.     

The mechanism involved in ethylene-enhanced ethylene synthesis in carnations

The plant hormone ethylene triggers and enhanced ethylene synthesis in certain ripening fruits and senescing flowers. Unlike most carnation (Dianthus caryophyllus L.) cultivars exhibiting climacteric rise in ethylene production at the onset of senescence, cv. Sandrosa does not show this phenomenon naturally. In order to understand the mechanism of autocatalytic ethylene production, we exposed carnation flowers cv. Sandrosa to ethylene which resulted in an enhanced capacity for ethylene synthesis in the petals. A short time response of one hour was measured for an increase in ACC oxidase activity, about five hours in advance of an increase in ACC synthase activity and ethylene production. The observed enhancement was dependent on the presence of exogeneous ethylene, and could be partially inhibited by prior treatment of the petals with -amanitin or cycloheximide. The results of the present study suggest that in response to ethylene, activation of an existing enzyme is taking place first. This is followed by an increase in expression of ACC oxidase and ACC synthase mRNAs.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DTT dithiothreitol - PMSF phenyl-methylsulfonyl fluoride - SAM S-adenosyl-L-methionine     

Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in non-climacteric<Emphasis Type="Italic"> Citrus</Emphasis> fruit

Mature citrus fruits, which are classified as non-climacteric, evolve very low amounts of ethylene during ripening but respond to exogenous ethylene by ripening-related pigment changes and accelerated respiration. In the present study we show that young citrus fruitlets attached to the tree produce high levels of ethylene, which decrease dramatically towards maturation. Upon harvest, fruitlets exhibited a climacteric-like rise in ethylene production, preceded by induction of the genes for 1-aminocyclopropane-1-carboxylate (ACC) synthase 1 (CsACS1), ACC oxidase 1 (CsACO1) and the ethylene receptor CsERS1. This induction was advanced and augmented by exogenous ethylene or propylene, indicating an autocatalytic system II-like ethylene biosynthesis. In mature, detached fruit, very low rates of ethylene production were associated with constitutive expression of the ACC synthase 2 (CsACS2) and ethylene receptor CsETR1 genes (system I). CsACS1 gene expression was undetectable at this stage, even following ethylene or propylene treatment, and CsERS1 gene expression remained constant, indicating that no autocatalytic response had occurred. The transition from system II-like behavior of young fruitlets to system I behavior appears to be under developmental control.Abbreviations ACC 1-Aminocyclopropane-1-carboxylate - CsACS1, CsACS2 ACC synthase - CsACO1 ACC oxidase - CsERS1, CsETR1 Ethylene receptors - DAFB Days after full bloom - 1-MCP 1-Methylcyclopropene     

Assessment of the Germination Potential of Brassica oleracea Seeds Treated with Karrikin 1 and Cyanide,Which Modify the Ethylene Biosynthetic Pathway

The relationship between ethylene and cyanide (HCN) and karrikin 1 (KAR1) in dormancy release was studied in secondary dormant Brassica oleracea L. (Chinese cabbage) seeds. Freshly harvested seeds of Brassica oleracea usually have poor germination potential. Karrikin1 (KAR1) and cyanide (HCN) are able to stimulate seed germination. However, the stimulatory effects of these two chemicals depend on the activation of the ethylene biosynthesis pathway and on ethylene perception. In this study, KAR1 and HCN application increased the activity of ethylene and of two ethylene biosynthesis enzymes, ACC synthase (ACS) and ACC oxidase (ACO). KAR1 and HCN collectively promoted the accumulation of 1 aminocyclopropane-1-carboxylic acid (ACC). In the presence of NO (nitric oxide) and KAR1, ACS and ACO activities reached their maximum levels after 36 and 42 h, respectively. Ethylene inhibitors suppressed seed germination by approximately 55%, whereas the respiratory inhibitors SHAM and NaN3 inhibited seed germination by 5–10% in the presence of HCN and KAR1. KAR1 and HCN collectively reduced the abscisic acid (ABA) content in seeds, increased the gibberellic acid (GA) content and released seed dormancy. The expression of ethylene biosynthesis genes and ethylene receptor genes (BOACO1, BOACS1, BOACS3, BOACS4, BOACS5, BOACS7, BOACS9, BOACS11, BOETR1 and BOETR2) provided further evidence of the involvement of ethylene in KAR1 and HCN-induced germination. BOACO1, BOACS1, BOACS5, BOACS7, BOACS9, BOACS11, BOETR1 and BOETR2 genes were up regulated in the presence of KAR1 and HCN, while the remaining genes were down regulated. The expression of various ethylene biosynthesis and ethylene receptor genes suggested functional diversification and variations in seed sensitivity in the presence of KAR1 and HCN. Therefore, in the current study, KAR1 and HCN application effectively induced the germination of B. oleracea seeds (approximately 97% germination rate) after 6 days by modifying the ethylene biosynthetic pathway.

     

Characterization of ethylene production in developing strawberry fruit

Ethylene production, ACC content, and ACC oxidase activity were determined in strawberry fruit harvested at different stages of development and in fruit harvested green and developed in vitro in solutions containing sucrose. In fruit harvested at progressive stages of development from green through full ripe, ethylene production and ACC oxidase activity decreased whereas ACC content increased between the white and pink stages. Fruit detached at the green stage and developed to full ripe by immersion of the cut pedicel in sucrose solutions exhibited an increase in ACC content, decreased ethylene production, and no change in ACC oxidase activity. Detached green fruit provided with sucrose containing 0.5 mM silver (STS) had elevated ethylene production and more ACC oxidase activity than did fruit incubated without the silver salt. Green fruit provided with sucrose containing 1 mM ACC showed markedly increased ACC content, ACC oxidase activity, and ethylene production. These increases were noted following 4 days incubation in ACC, and were more pronounced after 11 days, at which time fruit of all treatments had attained a full-ripe stage of development. Calyx tissue exhibited more ACC oxidase activity, less ACC content, and similar ethylene production compared with receptacle tissue. ACC synthase could not be detected in fruit harvested at different developmental stages or in fruit detached and developed in vitro.abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - HQS 8-hydroxyquinoline hemisulfate - SAM S-adenosyl methionine - STS silver thiosulfate     

Biosynthesis of stress ethylene in soybean seedlings: Similarities to endogenous ethylene biosynthesis

The similarity of stress ethylene biosynthesis in whole plants to endogenous ethylene biosynthesis was investigated using two inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and cobalt chloride (Co²⁺); and the intermediates, methionine, S -adenosylmethionine (SAM), and 1-aminocyclopropane-1-carboxylic acid (ACC), of basal ethylene biosynthesis. Stress ethylene production induced by ozone, cadmium, or 2,4-dichlorophenoxyacetic acid was inhibited in hydroponically-grown soybean seedlings in a concentration-dependent manner by both AVG and CO²⁺. The ethylene intermediates evoked responses in intact seedlings similar to that described for endogenous ethylene production in isolated vegetative tissue. The addition of SAM to the hydroponic system relieved AVG inhibition of stress ethylene production. Feeding ACC to the seedlings resulted in increased ethylene production independent of stress application or prior AVG inhibition. Cobalt inhibition of stress ethylene production was relieved by increasing concentrations of ACC. A short lag period of 12–18 min was observed in stress ethylene production following a 30-min ozone exposure. Addition of cycloheximide partially inhibited ozone-induced ethylene production.
These results suggest a common pathway in whole plants for stress ethylene production and endogenous ethylene biosynthesis.