ACC Oxidase Transcripts Research Articles

Effect of pollination on accumulation of ACC synthase and ACC oxidase transcripts, ethylene production and flower petal abscission in geranium (Pelargonium x hortorum L.H. Bailey).

Self-pollination of diploid zonal geranium (Pelargonium x hortorum L.H. Bailey) florets leads to a dramatic rise in ethylene production, followed by abscission within 4 h. Neither wounding of the stigma, pollination with tetraploid pollen, nor heat-killed self pollen could elicit as much ethylene production and petal abscission as self-pollination. A cDNA sharing sequence identity with ACC synthase (GACS2) and three different cDNAs sharing sequence identity with ACC oxidase (GACO1, GACO2, GACO3) were isolated from geranium pistils. Transcripts hybridizing with these probes increased slightly in response to self-pollination, but the degree of accumulation in response to various treatments did not correlate with ethylene production. When calculated on a per-plant-part basis, transcripts hybridizing with GACS2 were equally distributed among the stigma+style, sterile ovary, and ovary tissues, but transcripts hybridizing with the three ACC oxidase clones were differentially distributed. All transcripts were differentially expressed among the other tissues of the plant, with GACO1 being the most widely distributed. Ethylene production in geranium pistils was not autocatalytic. Propylene failed to induce ethylene production and ethylene did not induce the accumulation of ACC synthase or ACC oxidase transcripts. ACC accumulated in the stigma and style, and to a smaller extent in the sterile ovary, after pollination. These data support a model of pollination-induced ethylene production by post-transcriptional regulation of ethylene biosynthetic gene expression.

Expression and internal feedback regulation of ACC synthase and ACC oxidase genes in ripening tomato fruit.

We have examined whether or not a positive feedback regulation of gene expression for 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase also operates in ripening tomato (Lycopersicon esculentum) fruit during the burst of ethylene production. Two cDNA fragments for ACC synthase and one for ACC oxidase were cloned with high homology to already known genes involved in ethylene biosynthesis in ripening tomato fruit. Accumulation of mRNAs which hybridize to these cDNA probes were induced in mature green fruit within two days by treatment with propylene. In the fruit ripened from the turning stage, red color development, ethylene production, ACC content, and activities of ACC synthase and ACC oxidase increased as maturity progressed. The abundance of two ACC synthase and one ACC oxidase mRNAs in the fruit increased from the turning to pink stage and were followed by a slight decline towards the red stage. These increases in mRNAs abundance with ripening were prevented to a large extent by treatment with the ethylene action inhibitor, 1-methylcyclopropene (MCP). This was most pronounced in the fruit treated with MCP at the turning stage, in which the accumulation of ACC synthase and ACC oxidase transcripts was almost completely eliminated in the first two d, precisely the same stage at which the control fruit had the greatest level of each mRNA accumulation. The inhibition of transcript accumulation recovered to the control level within two to four d. MCP also decreased ethylene biosynthetic activity, although this decrease did not reflect the reduction in the mRNAs accumulation. These results suggest that a strong positive feedback regulation is involved in ethylene biosynthesis at the gene transcriptional level in tomato fruit, even at the stage with a burst of ethylene production.

Effects of chilling on the expression of ethylene biosynthetic genes in Passe-Crassane pear (Pyrus communis L.) fruits.

Passe-Crassane pears require a 3-month chilling treatment at 0 degrees C to be able to produce ethylene and ripen autonomously after subsequent rewarming. The chilling treatment strongly stimulated ACC oxidase activity, and to a lesser extent ACC synthase activity. At the same time, the levels of mRNAs hybridizing to ACC synthase and ACC oxidase probes increased dramatically. Fruit stored at 18 degrees C immediately after harvest did not exhibit any of these changes, while fruit that had been previously chilled exhibited a burst of ethylene production associated with high activity of ACC oxidase and ACC synthase upon rewarming. ACC oxidase mRNA strongly accumulated in rewarmed fruits, while ACC synthase mRNA level decreased. The chilling-induced accumulation of ACC synthase and ACC oxidase transcripts was strongly reduced when ethylene action was blocked during chilling with 1-methylcyclopropene (1-MCP). Upon rewarming ACC synthase and ACC oxidase transcripts rapidly disappeared in 1-MCP-treated fruits. A five-week treatment of non-chilled fruits with the ethylene analog propylene led to increased expression of ACC oxidase and to ripening. However, ethylene synthesis, ACC synthase activity and ACC synthase mRNAs remained at very low level. Our data indicate that ACC synthase gene expression is regulated by ethylene only during, or after chilling treatment, while ACC oxidase gene expression can be induced separately by either chilling or ethylene.

The requirements for Ca2+, protein phosphorylation, and dephosphorylation for ethylene signal transduction in Pisum sativum L.

The role of Ca2+ and protein phosphorylation in the transduction of the ethylene signal resulting in induction of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase has been studied in peas (Pisum sativum L.) by a pharmacological approach. 2,5-Norbornadiene (NBD) and aminoethoxyvinylglycine (AVG) reduced the basal level of ACC oxidase transcript and its enzyme activity. Only NBD was shown to inhibit the ethylene response, the accumulation of ACC oxidase transcript and the stimulation of its enzyme activity. Ethylene influenced 45Ca2+ influx into the segment tissues from pea epicotyls, and ethylene glycol-bis(beta-aminoethyl ether)N,N,N'N'-tetraacetic acid (EGTA) a Ca2+ chelator, inhibited the ethylene response. Ca2+ depletion by pretreatment with EGTA also blocked the ethylene response, which almost completely recovered when Ca2+ was added exogenously after Ca2+ depletion. Ca2+ channel blockers, verapamil, and LaCl3, used to certify the role of extracellular Ca2+, all inhibited the ethylene response. A protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), and protein phosphatase inhibitors, vanadate and okadaic acid, also inhibited the ethylene response. The results of the present study suggest that Ca2+ influx from the extracellular space, protein phosphorylation, and dephosphorylation are required for the induction of ACC oxidase by ethylene.

Controlled-atmosphere Suppression of ACC Synthase and ACC Oxidase in `Golden Delicious' Apples during Long-term Cold Storage

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).

Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato.

The tomato ACC oxidase gene family is comprised of three members designated AC01, AC02 and AC03. These are highly homologous throughout the protein coding regions but do show a degree of sequence divergence within the 3' untranslated regions. These regions have been cloned and used as gene-specific probes to analyse the differential expression of the tomato ACC oxidase gene family in various tissues at different stages of development. Results indicate that all three genes are transcriptionally active and display a high degree of inducibility in a number of organs at various stages of the life cycle. Both AC01 and Ac03 transcripts accumulate during the senescence of leaves, fruit and flowers. In addition, it appears that AC01 is wound-inducible in leaves. All three ACC oxidase genes are expressed during flower development, with each showing a temporally distinct pattern of accumulation. In addition, the ACC oxidase transcripts are also spatially regulated throughout flower development; AC01 is predominantly expressed in the petals and the stigma and style, AC02 expression is mainly restricted to tissues associated with the anther cone whereas AC03 transcripts accumulate in all of the floral organs examined apart from the sepals. ACC oxidase enzyme assays and Western blot analysis indicate that both enzyme activity and ACC oxidase protein increase with transcript abundance in several tissues. The physiological role of the differential expression of the ACC oxidase gene family, in relation to the regulation of ethylene synthesis, during these various developmental processes is discussed.

Structure and expression of cDNAs encoding 1-aminocyclopropane-1-carboxylate oxidase homologs isolated from excised mung bean hypocotyls

By screening a mung bean (Vigna radiata L.) hypocotyl cDNA library using a combination of apple (pAE12) and tomato (pTOM13) 1-aminocyclopropane 1-carboxylate (ACC)-oxidase cDNAs as probes, putative ACC-oxidase clones were isolated. Based on restriction-enzyme map and DNA-sequencing analyses, they can be divided into two homology classes, represented by pVR-ACO1 and pVR-ACO2. While pVR-ACO1 and pVR-ACO2 exhibit close homology in their coding regions, their 3'-noncoding regions are divergent. pVR-ACO1 is a 1312-bp full-length clone and contains a single open reading frame encoding 317 amino acids (MW = 35.8 kDa), while pVR-ACO2 is 1172 bp long and is a partial cDNA clone encoding 308 amino acids. These two deduced amino-acid sequences share 83% identity, and display considerable sequence conservation (73-86%) to other ACC oxidases from various plant species. Northern blot analyses of RNAs isolated from hypocotyl, leaf, and stem tissues using gene-specific probes indicate that the pVR-ACO1 transcript is present in all parts of the seedling and that the expression in hypocotyls is further increased following excision. The maximum induction of ACC-oxidase transcripts occurred at about 6 h after excision, while the maximum enzyme activity was observed at 24 h. When excised hypocotyls were treated with ethylene a further enhanced level of transcripts was observed. Aminooxyacetic acid, an inhibitor of ACC-synthase activity, and 2,5-norbornadiene, an inhibitor of ethylene action, suppressed the wound-induced accumulation of ACC-oxidase mRNA, while an addition of ethylene in these tissues restored the accumulation of ACC-oxidase mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)