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Satoru Kondo, Hiroko Yamada, and Sutthiwal Setha

aminoethoxyvinylglycine (AVG), which is a 1-aminocyclopropane-1-carboxylate (ACC) synthase inhibitor ( Lieberman, 1979 ), along with 1-methylcyclopropene (1-MCP), which blocks ethylene receptors and retards ethylene action ( Blankenship and Dole, 2003 ), have been

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Carl D. Schlagnhaufer, Richard N. Arteca, and Eva J. Pell

Ethylene production is involved in many plant physiological processes including stress responses and is frequently associated with foliar senescence. Ethylene emission is a common plant response to many biotic and abiotic stresses. We have cloned two ACC synthase cDNAs (OIP-1, PAC-1) from the leaves of ozone treated Solanum tuberosum L. plants. Plants treated with ozone produced ethylene within 1 hour following treatment initiation. Levels continued to increase reaching a peak after 2 h. PAC-1 was expressed after 1 hour reaching a maximum by 2 hours and showed a marked decline after 4 h. OIP-1 was first expressed after 2 hours and high levels of expression continued up to 4 hours following treatment initiation. Leaves treated with CuCl2 produced high levels of ethylene within 0.5 hour after treatment initiation. Ethylene levels continued to increase reaching a peak after 2 hours with no change after 4 h. PAC-1 was expressed after 0.5 hour reaching a peak at 1 hour and showed a progressive decline from 2 to 4 h. However, OIP-1 expression was first detected 2 hours following treatment initiation and high levels of expression continued up 4 h. Leaves exposed to Alternaria solani produced increased levels of ethylene 1 day following inoculation reaching a peak after 3 days. PAC-1 was expressed at a low level 1 day after inoculation and expression remained constant for the duration of the experiment, whereas, OIP-1 was not expressed until day 4.

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Chih-Hsien Lei, Jon T. Lindstrom, and William R. Woodson

At anthesis, petunia pollen contains large amounts of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This ACC is thought to contribute to the rapid burst of ethylene produced by the pistil following pollination. An analysis of ACC content in developing anthers revealed that ACC began to accumulate the day before anthesis, indicating its synthesis was a late event in pollen development. We employed degenerate DNA primers to conserved amino acid sequences of ACC synthesis to amplify a cDNA from anther mRNA by RT-PCR. The resulting cDNA (pACS2) was sequenced and found to represent ACC synthase. Use of pACS2 as a hybridization probe revealed an increase in ACC synthase mRNA concomitant with the increase in ACC content. Further analysis indicated the ACC synthase mRNA was localized specifically to the haploid pollen grain. In an attempt to determine the function of ACC in pollen maturation or pollen–pistil interactions, we have generated a series of transgenic petunias designed to inhibit the accumulation of ACC in pollen. For these experiments, we have employed a pollen-specific promoter (LAT52) from tomato to drive the expression of antisense pACS2 or the coding region of ACC deaminase. The results of the experiments will be discussed.

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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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Gabriel O. Sozzi, Adela A. Fraschina, Agustín A. Navarro, Osvaldo Cascone, L. Carl Greve, and John M. Labavitch

α-l-Arabinofuranosidases (α-Af) are plant enzymes that have the capacity to release terminal arabinofuranosyl residues from a wide variety of pectic and hemicellulosic polymers, as well as different glycoconjugates. Our interest in α-Af is related to its potential role in ripening-related loss of arabinose from tomato fruit cell walls. Using both control (cv. VF 36) and ACC synthase antisense (A11.1) tomatoes (Lycopersicon esculentum Mill.), we demonstrate that tomato α-Af activity is present during the entire ontogeny of the fruit. Immature 10-day-old fruit displayed 6-fold more α-Af activity on a per gram fresh weight basis, than mature green fruit. In VF 36 fruit, α-Af activity increased 45% from mature green 4 (48 days post anthesis) to light red stages (55 days) when fruit ripened on the vine. In contrast, no similar increase was detected in ACC synthase antisense fruit that do not ripen in the same time frame. However, when A11.1 fruit were detached at 48 days after anthesis and treated continuously with 100 mL·L-1 ethylene the fruit ripened and α-Af increased, as in ripening normal fruit. The α-Af activity pattern is similar to that reported for tomato β-galactosidases. The increasing α-Af activity during ripening and the decreased activity in antisense ACC synthase fruit after reaching the mature green stage suggest a role for ethylene in the ripening-related synthesis or activation of this enzyme.

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Jon T. Lindstrom, Chih-Hsien Lei, Michelle L. Jones, and William R. Woodson

Mature pollen from Petunia hybrida contains significant levels of 1-aminocyclopropane-1-carboxylic acid (ACC), and this ACC is thought to play a role in pollination-induced ethylene by the pistil. We investigated the developmental accumulation of ACC in anthers and pollen. The level of ACC in anthers was very low until the day before anthesis, at which time it increased 100-fold. A 1.1-kb partial ACC synthase cDNA clone (pPHACS2) was amplified from total RNA isolated from mature anthers by reverse transcriptase, followed by polymerase chain reaction using oligonucleotide primers synthesized to conserved amino acid sequences in ACC synthases. The expression of pPHACS2 mRNA during anther development was correlated with the accumulation of ACC and was localized to the pollen grain. The pPHACS2 cDNA was used to identify the PH-ACS2 gene from a library of genomic DNA fragments from Petunia hybrida. PH-ACS2 encoded an ACC synthase transcript of four exons interrupted by three introns. The ACC synthase protein encoded by the PH-ACS2 gene shared >80% homology with ACC synthases from tomato (LE-ACS3) and potato (ST-ACS1a). A chimeric PH-ACS2 promoter-β-glucuronidase (GUS) gene was used to transform petunia and transgenic plants were analyzed for GUS activity. GUS staining was localized to mature pollen grains and was not detected in other tissues. Despite similarities to LE-ACS3, we did not detect GUS activity under conditions of anaerobic stress or in response to auxin. A series of 5-prime-flanking DNA deletions revealed that sequences within the PH-ACS2 promoter were responsible for pollen-specific expression.

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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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James R. Gorny and Adel A. Kader

1 To whom reprint requests should be addressed. We thank Shang Fa Yang for the generous gift of cDNA clones for ACC synthase and ACC oxidase (ACC-O), as well as polyclonal antibodies specific to the ACC-O protein. The cost of publishing this paper

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Akihiro Itai, Takaaki Igori, Naoko Fujita, Mayumi Egusa, Motoichiro Kodama, and Hideki Murayama

synthase gene ( PpACS4 ). To get ACC synthase genes related to pathogen infection, total RNA was extracted from leaves 0 h and 12 h after inoculation according to the hot borate method ( Wan and Wilkins, 1994 ). First-strand cDNA was synthesized from 1 μg