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Satoru Kondo, Hiroko Yamada, and Sutthiwal Setha

developed. Ethylene production is also influenced by other phytohormones. For example, the application of methyl jasmonate (MeJA) inhibited the ACC content, ACC oxidase activity, and ethylene production in grain amaranth ( Amaranthus caudatus L.) seeds

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P. Perkins-Veazie, J.K. Collins, V. Russo, and B. Cartwright

Individually, green melon aphids (Aphis gossypi) and anthracnose (Colletotricum lagenarium) can cause serious economic damage to watermelons by reducing stands and marketable yields. Greenhouse-grown watermelon seedlings at the third true leaf stage were infected with anthracnose (106 spores/mL) and/or infested with 30 aphids per plant. At the 5th leaf stage (about 7 days after inoculation/infestation), leaf disks were harvested from plants and indicators of stress measured. Peroxidase activity increased from 0.03 to 0.28 absorbance units/mg protein-minute in leaves with anthracnose. When plants were infested with aphids after anthracnose inoculation, peroxidase activity was 0.40 absorbance units/mg protein-minute. Plants having both aphids and anthracnose had more anthracnose lesions when leaves were infested with aphids prior to anthracnose inoculation. The presence of aphids and/or anthracnose stimulated 1-aminocyclopropane-1-caroxylic acid (ACC) oxidase activity from 28 to 44 nL/g-h, indicating enhanced ethylene production. However, aphids had to be present on plants at least 5 days before ACC oxidase activity was stimulated above control levels. Aphids combined with anthracnose failed to elevate ACC oxidase levels higher than either aphids or anthracnose alone. Both peroxidase activity and ACC oxidase activity in watermelon plants increased with anthracnose infection. Thus, watermelon plants stressed by aphids and anthracnose responded differently from plants stressed individually by aphids or anthracnose.

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Jean-Claude Pech

We have generated transgenic Cantaloupe Charentais melons expressing an ACC oxidase antisense gene in which ethylene production was reduced to less than 1% as compared to control untransformed fruits. As a consequence, some aspects of the ripening process were strongly inhibited (aroma volatiles production, chlorophyll and cell wall degradation, pigmentation of the rind, activation of peduncular abscission zone) while others remained unchanged (coloration of the flesh), allowing us to distinguish between ethylene-dependent and ethylene-independent pathways. Some postharvest characteristics of the transgenic fruit are described in terms of expression of ripening-related genes, respiratory behavior, and biochemical composition. Data also are presented showing that exogenous ethylene treatments could reverse the antisense phenotype.

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Yali Liu, Fuling Hao, Rui Meng, and Weirong Xu

The enzyme ACC oxidase (ACO), encoded by a small multigene family in many plants, catalyzes the terminal step in the ethylene biosynthesis pathway. In this research, based on the total RNA isolated from the flowers of Asia hybrids `Pollyanna' and Oriental hybrids `Sorbonne', we obtained two cDNA fragments of ACO genes (Genbank accession DQ062133 and DQ062134) by RT-PCR technique. The two cDNA fragments were reversely inserted into plant expression vector pWR306 respectively, and constructed two antisense ACO gene expression binary vectors harboring hygromycin phosphotransferase (hptII), glucuronidase (uid A), and a green fluorescent protein (GFP) gene in the T-DNA region. We have developed a system to produce transgenic plants in LiLium via Agrobacterium tumefaciens-mediated transformation of calli. Transformants were subjected to GFP expression analysis, PCR assay, and Southern hybridization to confirm gene integration.

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Keith W. Savin, Stanley C. Baudinette, Michael W. Graham, Ellen L-J. White, Michael Z. Michael, Ann Bayly, Chin-Yi Lu, Stephen F. Chandler, and Edwina C. Cornish

Ethylene is essential for the senescence process in many fruit and flowers. In the last two steps in the biosynthesis of ethylene in plants ACC synthase converts S-adenosyl methionine to 1-aminocyclopropane-1-carboxylic acid(ACC). ACC oxidase (ACO) then degrades ACC to ethylene. Inhibitors of ethylene synthesis, such as amino-oxyacetic acid, and of the response to ethylene, such as silver thiosulphate, delay or prevent senescence. By expression of an antisense version of ACO RNA, we have generated two varieties of transgenic carnation which produce flowers with an extended vase life. These were produced using the cultivars Red Sim and White Sim. Flowers from these plants produce very little ethylene and normally fail to display the inrolling phenotype typical of senescence in this species. At the time after harvest when inrolling would normally lake place (5 days), the antisense ACO flowers produce only barely detectable levels of endogenous ACO mRNA or ACS (ACC Synthase) mRNA. Exposure to exogenous ethylene(100ppm) induces inrolling and production of ACS and ACO mRNA species. Such carnations will be valuable both as a commercial product and as a tool for further exploring the role of ethylene in carnation flower senescence and leaf wound response.

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James R. Gorny and Adel A. Kader

Ethylene biosynthesis of Golden Delicious apple fruit at 20°C is rapidly inhibited by a controlled atmosphere of air + 20% CO2. However, in vitro ACC oxidase activity and ACC content were not significantly different between air and air + 20% CO2 treated fruit, To determine the in vivo effects of CO2 treatment, both in vivo and in vitro enzyme activity essays were performed in en atmosphere of air or air + 20% CO2. Western blots were also performed to quantify the amount of ACC oxidase protein present in the air and air + 20% CO2 treated fruit.

We believe that in vivo cytosolic pH changes, induced by CO2, may reduce the in vivo catalytic capacity of ACC oxidase, end hence significantly reduce ethylene biosynthesis in climacteric tissue,

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James R. Gorny and Adel A. Kader

Preclimacteric `Golden Delicious' apples (Malus domestica Borkh.) were stored at 0 °C in: air; air + 5% CO2; 2% O2 + 98% N2; or 2% O2 + 5% CO2 + 93% N2, and sampled monthly for 4 months to investigate the mechanism(s) by which reduced O2 and/or elevated CO2 atmospheres inhibit C2H4 biosynthesis. Ethylene biosynthesis rates and in vitro ACS activity were closely correlated in all treatments, while in vitro ACO activity significantly increased over time regardless of the treatment. Only a small amount of C2H4 biosynthesis inhibition by lowered O2 and/or elevated CO2 atmospheres could be accounted for by suppressed induction of ACO activity. Western blot analysis demonstrated that apples held for 2 months in lowered O2 and/or elevated CO2 atmospheres had significantly reduced abundance of ACO protein, compared to fruit held in air. Northern blot analysis of ACS and ACO transcript abundance revealed that reduced O2 and/or elevated CO2 atmospheres delay induction and reduce the abundance of both transcripts. Reduced O2 and/or elevated CO2 atmospheres reduce C2H4 biosynthesis by delaying and suppressing expression of ACS at the transcriptional level and by reducing the abundance of active ACO protein. Chemical names used: 1-aminocyclopropane-1-carboxylic acid (ACC), ACC synthase (ACS), ACC oxidase (ACO), ethylene (C2H4), S-adenosylmethionine (AdoMet).

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James R. Gorny and Adel A. Kader

The objective of this study was to compare and contrast the mode of action by which elevated carbon dioxide and/or reduced oxygen atmospheres inhibit ethylene biosynthesis. `Golden Delicious' apple fruit were placed at 0C in one of the following four atmospheres: 1) air; 2) air + 5% CO2; 3) 2% O2 + 98% N2; or 4) 2% O2 + 5% CO2 + 93% N2 and then sampled monthly for 4 months. Ethylene biosynthesis rates and in vitro ACC synthase activities were closely correlated in all treatments. In vitro ACC synthase activity and ethylene biosynthesis rates were lowest in fruit treated with 5% CO2 + 2% O2, while air-treated fruit had the highest ethylene biosynthesis rate and in vitro ACC synthase activity. Fruit treated with air + 5% CO2, or 2% O2 + 98% N2, had intermediate ethylene and in vitro ACC synthase activities. In vitro ACC oxidase was significantly different among treatments, but not as closely correlated with the ethylene biosynthesis rate as in vitro ACC synthase activity. Western blot analysis of the ACC oxidase protein was performed to determine if activity differences among treatments were correlated with the amount of enzyme present in vivo. ACC synthase and ACC oxidase mRNA transcript of abundance was determined via Northern blot analysis. Results will be discussed regarding how ethylene biosynthesis is inhibited at the molecular level by elevated CO2 and/or reduced O2.

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Pankaj Kumar Bhowmik and Toshiyuki Matsui

The molecular and enzymatic changes that follow harvesting of asparagus are important aspects for postharvest deterioration. To define the factors contributing to postharvest deterioration, the early changes in ethylene production and the activities and expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, and phenylalanine ammonia-lyase (PAL) were studied in whole spears and in excised top and bottom portions. As a result of wounding, ethylene production was found higher in both top and bottom portions compared with whole spears but followed the same trend reaching the peak at 16 hours after harvest. ACC synthase was rapidly induced in excised top portions but no significant ACC synthase activity was observed in excised bottom portions. In top portions, ACC synthase reached a peak 8 hours after harvest and thereafter started to decline. In contrast to ACC synthase, ACC oxidase was induced markedly in both top and bottom portions and remained high until 16 hours after excision. On the other hand, PAL greatly increased in activity in bottom portions but not in excised tops. Northern blot analysis showed that increased mRNA levels coincided well with the excision-promoted increases in enzyme activity and ethylene production.

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Keith W. Savin, Stanley C. Baudinette, Michael W. Graham, Michael Z. Michael, Greg D. Nugent, Chin-Yi Lu, Stephen F. Chandler, and Edwina C. Cornish