ACC Oxidase Gene Research Articles

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.

VARIETAL VARIATION IN MICROSATELLITE AND CAPS FOR ETHYLENE-RELATED GENES AND ITS POSSIBLE ASSOCIATION WITH AGRONOMIC CHARACTERS IN MELON (CUCUMIS MELO)

Shelf-life of fruit is one of the most important traits in melon breeding. Shelf-life of climacteric fruits is closely related with ethylene whose biosynthesis is controlled by ACC synthase and ACC oxidase genes. As the first step to develop selection markers for shelf-life in melon (Cucumis melo), microsatellite and cleaved amplified polymorphic sequence (CAPS) polymorphisms were analyzed in two ACC synthase genes (CMe-ACS1, CMe-ACS2) and two ACC oxidase genes (CMe-ACO1, CMe-ACO3), resulting in the establishment of six microsatellite and two CAPS markers. Five markers were developed in two genes, CMe-ACS1 and CMe-ACO1, which were expressed during fruit ripening. A highly polymorphic marker was found in CMe-ACS2, where the number of simple sequence repeat of (TA) was highly variable among cultivars, ranging from seven to thirty-seven. Insertion/deletion of (T)n and (A)n was also detected in CMe-ACS2. CAPS and microsatellite markers specific to vars. makuwa and conomon, local varieties in East Asia, were detected in four markers, Acs1-c1, Acs2-ms1, Aco1-ms2 and Aco3-ms1, and could be useful markers for phylogenetic analysis in melon.

Ethylene responsiveness in carnation styles is associated with stigma receptivity

Sensitivity to ethylene increases as flowers mature, and this is associated with autocatalytic ethylene production that initiates petal senescence. The coordination of the senescence process within the flower requires interorgan communication. To determine if differential ethylene responsiveness among floral organs is involved in regulating senescence signaling, ethylene biosynthesis and the expression of ethylene biosynthetic genes were investigated in styles, ovaries, and petals from 6 stages of flowers following ethylene treatment. As flowers matured, all floral organs investigated became more responsive to ethylene. Ovaries were the first flower organ that had detectable increases in ethylene production following ethylene treatment. In stage 4 and 5 flowers, the highest levels of ethylene production were detected in petals, while styles had the highest ethylene production at stage 6. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) genes were induced by ethylene treatment, and transcript abundance of DCACS1, DCACS2 and DCACO1 increased in all floral organs as flowers matured. DCACS3, a pollination-responsive ACS gene, did not show a significant increase in transcript levels following ethylene treatment except in styles. Patterns of gene expression in ethylene-treated styles and petals corresponded with increases in ethylene biosynthesis, with increases in ACS mRNA abundance detected at stage 1 or 2 in petals and at stages 4, 5, or 6 in styles. The largest increases in ethylene production and gene expression occurred in styles at stages 5 and 6 and corresponded to the stages at which the styles were first receptive to pollination. Reasons for differential ethylene sensitivity among the flower organs during development are discussed.

A Harvest-induced ACC Oxidase Gene from Tips of Harvested Asparagus Spears and its Expression During Storage

A Harvest-induced ACC Oxidase Gene from Tips of Harvested Asparagus Spears and its Expression During Storage

Genetics of ethylene biosynthesis and restriction fragment length polymorphisms (RFLPs) of ACC oxidase and synthase genes in melon ( Cucumis melo L.).

We investigated the genetics of ethylene biosynthesis and its linkage to the RFLPs of the ACC oxidase and synthase genes in melon ( Cucumis melo L.). The results suggested that the A(0) and B(0) fragments of RFLP-MEL1 of the ACC oxidase gene were two alleles from a single locus, as were the B and C fragments of RFLP-MEACS1 of the ACC synthase gene. The B(0) allele seemed to be partially dominant over the A(0) allele, whereas B and C alleles appeared to map to quantitative trait loci (QTLs), which most likely contributed to ethylene production. Both RFLPs were linked to ethylene production rates, but they were not linked to each other. The interaction effects of the ACC oxidase and synthase genes on ethylene production were revealed by segregation of RFLP-MEL1 and RFLP-MEACS1. The results of single-copy-reconstruction assays suggested that the ACC oxidase gene is a single copy, whereas the ACC synthase gene is a component of a multigene family in the melon genome. The abscission phenotype appeared to be controlled by an independent locus, with the abscission (full-slip) allele dominant over the non-abscission (not full-slip) allele. These results may facilitate efforts toward mapping the quantitative trait loci (QTLs) of ethylene production. The RFLPs may be used in marker-assisted selection in developing melons with a more-desirable low ethylene production rate for enhancing postharvest storage life.

Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) Heynh.

An unforeseen side-effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol-1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. 'Columbia' was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol-1) with 0.35 mmol mol-1 CO2 in N2. Ethylene in full-sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol-1 O2. At the same time, silique ACC oxidase mRNA increased three-fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis.

Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.

Expression of ACC Oxidase Gene in Ethanol-Treated Banana Fruit

Expression of ACC Oxidase Gene in Ethanol-Treated Banana Fruit

Agrobacterium tumefaciens-mediated transformation to alter ethylene and cytokinin biosynthesis in broccoli

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. Postharvest treatment of broccoli with 6-benzylaminopurine delays senescence, whilst exogenous ethylene has been shown to accelerate this process following harvest. To alter ethylene biosynthesis, broccoli was transformed, using Agrobacterium tumefaciensmediated transformation, with an antisense ACC oxidase gene from broccoli driven by the asparagine synthetase promoter from asparagus. In addition, broccoli was transformed with the chimeric gene construct SAG12-IPT to alter cytokinin biosynthesis during harvest-induced senescence. Transformation was achieved using both hypocotyl and cotyledonary petiole explants. The presence of an antisense ACC oxidase gene enhanced transformation efficiency, but Ag + incorporated into the medium did not. The transgenic nature of these plants was confirmed by PCR and Southern analyses.

IAA and N(6)-benzyladenine inhibit ethylene-regulated expression of ACC oxidase and ACC synthase genes in mungbean hypocotyls.

Regulation of expression of 1-aminocyclopropane-1-carboxylate (ACC) oxidase (VR-ACO1) and ACC synthase (VR-ACS1) genes by ethylene, indole-3-acetic acid (IAA), and N(6)-benzyladenine (BA), was investigated in mungbean hypocotyl tissues. Exogenous ethylene markedly increased transcript level of VR-ACO1 and reduced that of VR-ACS1, whereas aminooxyacetic acid (AOA), an inhibitor of ethylene biosynthesis, decreased the level of VR-ACO1 mRNA and increased that of VR-ACS1, indicating that expression of VR-ACO1 and VR-ACS1 genes are under positive and negative feedback control by ethylene, respectively. However, IAA treatment reduced the level of VR-ACO1 transcripts and increased that of VR-ACS1, although the hormone greatly induced ethylene production. We have demonstrated that, in a system that separates the effect of IAA proper from the effect of IAA-induced ethylene, the amount of IAA-induced ethylene was enough to cause accumulation of VR-ACO1 mRNA and decrease of VR-ACS1 mRNA. We have also shown that the responsiveness of VR-ACO1 and VR-ACS1 to exogenous ethylene was greatly reduced in the presence of IAA. In addition, BA abolished ethylene responsiveness with respect to expression of VR-ACO1 and VR-ACS1. Based on these results, we suggest that IAA and BA inhibit ethylene action, resulting in suppression of VR-ACO1 expression and induction of VR-ACS1 expression.

Differential expression of ACC synthase and ACC oxidase genes in mung bean leaves under saline and oxidative stresses

We investigated the differential expression of seven ACC synthase and two ACC oxidase genes in mung bean leaves. Among these, onlyACS2,ACO1, andACO 2 were expressed in etiolated leaves. When seedlings were de-etiolated, the expression level ofACO1 decreased by 65%, expression ofACO2 disappeared, while that ofACS2 was unchanged. For de-etiolated leaves treated with NaCl in the dark, the photochemical efficiency of PSII was not altered, and no genes were newly induced within the first 12 h. However, in the presence of light, transcripts ofACS1, ACS3, ACS4, andACS6 were newly accumulated, and the expression levels ofACS2 andACO1 were increased. The kinetics of transcript accumulation in response to methyl viologen (MV) treatment in the light were similar to those observed in the NaCl-treated leaves in the presence of light, suggesting that changes in the latter were caused by oxidative, rather than saline, stress. However, transcripts from most of the genes began accumulating more slowly in MV-treated leaves, with those ofACS1 andACS6 accumulating in much lower amounts than in NaCl-treated leaves. The reverse was true forACS4 transcripts.

DIFFERENTIAL SENSITIVITY TO ETHYLENE OF THE VARIOUS RIPENING PATHWAYS OF ETHYLENE-SUPPRESSED CANTALOUPE MELONS

The availability of ethylene-suppressed transgenic melon fruit harboring an ACC oxidase gene in antisense orientation (Ayub et al., 1996) allowed to discriminate between ethylene-dependent and ethylene-independent ripening pathways. It has been observed that pulp coloration, accumulation of sugars, and loss of acidity are ethylene independent processes, whereas rind yellowing, flesh softening, development of the peduncular abscission (Guis et al., 1997), aroma production (Bauchot et al., 1998) and respiratory burst (not published) are ethylene dependent. The present study is aimed at characterizing the sensitivity to ethylene of softening of the flesh and yellowing of the rind by treating ethylene-suppressed melons with ethylene. The kinetics of changes have been followed under various ethylene concentrations and upon transfer to air. This has allowed an estimation of the threshold levels of ethylene required for initiating the processes and the saturating levels for full velocity. The effects of the cessation of the ethylene treatment have also been determined.

Characterization and expression of two members of the peach 1-aminocyclopropane-1-carboxylate oxidase gene family.

The characterization and expression of PP-ACO1 and PP-ACO2, two members of the peach 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) gene family, are reported. PP-ACO1 is organized in 4 exons interrupted by 3 introns, whereas PP-ACO2 has only 2 of the 3 introns present in PP-ACO1. Comparison of the deduced amino acid sequences of PP-ACO1 and PP-ACO2 reveals a 77.7% identity. PP-ACO1 and PP-ACO2 show highest degree of similarity with petunia (PH-ACO3; 84.1%) and apple (85.4%) ACO genes, respectively. PP-ACO1 is expressed in flowers, fruitlet abscission zones, mesocarp and in young fully expanded leaves. PP-ACO1 transcript accumulation strongly increases during fruitlet abscission, in ripe mesocarp and senescing leaves, and is enhanced by propylene. PP-ACO2 mRNA accumulation is detected in fruits only during early development and is unaffected by propylene treatment. Both ACO genes are expressed in epicotyl and roots of growing seedlings, although a stronger accumulation of PP-ACO2 mRNA is observed.

Structure and ethylene-induced expression of the 1-aminocyclopro-pane-1-carboxylate oxidase gene in mung bean (Vigna radiate L.)

Previously we isolated two closely related 1-aminocyclopropane-i-carboxylate (ACC) oxidase cDNAs, pVR-ACOl, and pVR-ACO2, from mung bean seedlings. Ethylene caused a marked increase in the accumulation ofVR-ACO1 transcript, whereas the ethylene-induction ofVR-ACO 2 was much lower in the hypocotyl and root tissues. In the current study, genomic Southern blot analysis confirmed the existence of two ACC oxidase genes in the mung bean genome. To gain information concerning the structure of the ethylene-responsiveVR-ACO1 gene as well as to investigate its promoter sequence, we also obtained and sequenced the genomic clone encoding VR-ACO1. TheVR-ACO1 gene consists of four exons interrupted by three introns whose junctions are in agreement with the consensus intron/exon borders of plant genes. Individual sizes of the three introns are 100, 413, and 205 bp. The coding region ofVR-ACO1 comprises 951 bp encoding a protein of 317 amino acid residues, with a predicted molecular mass of 36 kDa. The major start site of transcription is located 66 nucleotides upstream from the 5’-end of the coding region of theVR-ACO1 gene; the putative TATA box is found at the 27th nucleotide upstream from this start site. The nuclear run-on transcription assay indicated that ethylene induces the expression ofVR-ACO1 at the transcriptional level in mung bean hypocotyls. Comparing the sequence of the 5’-flanking region ofVR-ACO1 with known ethylene responsive ele-ments (EREs) revealed two copies of sequence (TTCAA) that displays a high degree of identity with the primary ethylene responsive element (PERE) found in theCST1, E4, andERF1 genes. In addition, the promoter contains three octanucleotide sequence motifs reminiscent of the EIN3 binding site that exists inArabidopsis ERF1 and tobaccoERF2. We also discuss the possible molecular mechanism of regulation of ACC oxidase gene expression by ethylene in mung bean seedlings.

Regulation of genes encoding ethylene biosynthetic enzymes in peach (Prunus persica L.) fruit by carbon dioxide and 1-methylcyclopropene

Abstract We have cloned one member (PP-ACS1) of the 1-aminocyclopropane-1-carboxylate (ACC) synthase and two members (PP-ACO1 and PP-ACO2) of the ACC oxidase gene families in peach (Prunus persica L.) fruit and studied their expression characteristics during fruit ripening and treatment with CO2 and 1-methylcyclopropene (MCP), inhibitors of ethylene action. Northern analysis showed that the abundance of PP-ACS1, PP-ACO1 and PP-ACO2 mRNAs increased with fruit ripening in parallel with increases in ethylene production and activities of ACC synthase and ACC oxidase. The abundance of PP-ACO2 mRNA was much lower than that of PP-ACO1. CO2 and MCP treatment inhibited ethylene production, ACC synthase activity, and accumulation of PP-ACS1 mRNA. Although CO2 had little effect on ACC oxidase activity, it inhibited the accumulation of PP-ACO1 and PP-ACO2 mRNAs to the same levels as MCP. Wound-induced ethylene production, ACC synthase activity, and the abundance of PP-ACS1 mRNA were blocked and stimulated by CO2 and MCP, respectively. CO2 and MCP had no effect on wound-induced ACC oxidase activity but inhibited the accumulation of its mRNA. Wound-induced activities of ACC synthase and ACC oxidase, and abundance of their mRNAs were inhibited and stimulated, respectively, by exogenous ethylene. The translational inhibitor, cycloheximide inhibited wound-induced ethylene biosynthesis but super-induced the accumulation of PP-ACS1 and PP-ACO1 mRNAs, suggesting that their induction is a primary response to the inducer. These results suggest that the expression of PP-ACS1 and PP-ACO1 genes play a key role in the regulation of ethylene biosynthesis in peach fruit during ripening and in response to wounding. The results also indicate that wound-induced PP-ACS1 and PP-ACO1 genes are under negative and positive control, respectively. Further, using MCP we provide evidence indicating that CO2 does not regulate ACC synthase activity and expression of the PP-ACS1 gene in peach fruit during ripening and in response to wounding by antagonizing ethylene action.

A Wound-induced ACC Oxidase Gene of Moso Bamboo Shoot

A Wound-induced ACC Oxidase Gene of Moso Bamboo Shoot

Microsatellite and CAPS Markers for Ethylene-related Genes, 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Synthase and ACC Oxidase Genes, and their Variation in Melon (Cucumis melo L.).

To establish DNA markers for ethylene-related genes in melon (Cucumis melo L.), we searched for microsatellite and cleaved amplified polymorphic sequence (CAPS) polymorphisms in two ACC synthase genes (CMe-ACS1, CMe-ACS2) and two ACC oxidase genes (CMe-ACO1, CMe-ACO3), and found two CAPSs and six microsatellite markers. Five markers were detected in two genes, CMe-ACS1 and CMe-ACO1, which were expressed during fruit ripening. A highly polymorphic marker was found in CMe-ACS2, where the number of simple sequence repeats (TA) was highly variable among cultivars, ranging from seven to thirty-seven. Insertion/deletion of (T)n and (A)n were also detected in CMe-ACS2. Specific CAPS and microsatellites for vars. makuwa and conomon, local varieties in East Asia, were detected in four markers, Acs1-c1, Acs2-ms1, Aco1-ms2 and Aco3-ms1, and could be useful markers for phylogenetic analysis in melon.

MELON REGENERATION AND TRANSFORMATION USING AN APPLE ACC OXIDASE ANTISENSE GENE

The efficiency of melon regeneration protocols using cotyledon and leaf melon explants was evaluated in terms of number of regenerated plants, ploidy and transformation level. Young leaf explants were found to regenerate at a higher rate of diploidy, and they were selected as a recipient tissue for transformation. Agrobacterium tumefaciens gene transfer methods showed a good efficiency of transformation around 3%. A construction containing an antisense ACC oxidase gene, named pAP4, the last enzyme of the ethylene biosynthetic pathway, was used for transforming melon leaves. In the transgenic plants, the pAP4 gene was detected and the fruits showed a low ethylene production.

Expression of genes responsible for ethylene production and wilting are differently regulated in carnation ( Dianthus caryophyllus L.) petals

Carnation petals exhibit autocatalytic ethylene production and wilting during senescence. The autocatalytic ethylene production is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes, whereas the wilting of petals is related to the expression of the cysteine proteinase (CPase) gene. So far, it has been believed that the ethylene production and wilting are regulated in concert in senescing carnation petals, since the two events occurred closely in parallel with time. In the present study, we investigated the expression of these genes in petals of a transgenic carnation harboring a sense ACC oxidase transgene and in petals of carnation flowers treated with 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS). In petals of the transgenic carnation flowers, treatment with exogenous ethylene caused accumulation of the transcript for CPase and in-rolling (wilting), whereas it caused no or little accumulation of the transcripts for ACC oxidase and ACC synthase and negligible ethylene production. In petals of the flowers treated with DPSS, the transcripts for ACC synthase and ACC oxidase were accumulated, but no significant change in the level of the transcript for CPase was observed. These results suggest that the expression of ACC synthase and ACC oxidase genes, which leads to ethylene production, is differentially regulated from the expression of CPase, which leads to wilting, in carnation petals.

Purification and characterisation of two ACC oxidases expressed differentially during leaf ontogeny in white clover

Two isoforms of ACC oxidase (ACO) (EC 1.4.3), expressed differentially during leaf ontogeny in white clover (Trifolium repens L.), have been identified and purified to homogeneity. One isoform, designated MGI, was purified from mature green leaf tissue while the second isoform, designated SEII, was purified from senescent leaf tissue. The isolation and purification of these isoforms were achieved using a combination of hydrophobic interaction chromatography, anion exchange chromatography, chromatofocusing and gel filtration column chromatography. The Mr of both MGI and SEII was determined to be 37.5 kDa by gel filtration, and 37 kDa (MGI), 35 kDa (SEII) by SDS‐PAGE, indicating that both isoforms are active as monomers. During purification, both isoforms were recognised by a polyclonal antibody directed against a recombinant polypeptide derived from a white clover ACO gene expressed in mature green leaf tissue, TR‐ACO2. In addition to molecular mass, differences between the two isoforms were observed in terms of pH optima, isoelectric point (pI), Km for ACC, optimal requirements for the co‐substrate ascorbate, and NaHCO3 and Fe2+ as co‐factors. The identification of distinct ACC oxidases from the same tissue at different developmental stages shows that the now widely observed transcriptional regulation of the ACO gene family in higher plants is also expressed in terms of differential regulation of enzyme isoforms.