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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.
Plant Mol Biol 1997 Mar
PMID:Effects of chilling on the expression of ethylene biosynthetic genes in Passe-Crassane pear (Pyrus communis L.) fruits. 910 8

A cDNA encoding the banana 1-aminocyclopropane-1-carboxylate (ACC) oxidase has previously been isolated from a cDNA library that was constructed by extracting poly(A)+ RNA from peels of ripening banana. This cDNA, designated as pMAO2, has 1,199 bp and contains an open reading frame of 318 amino acids. In order to identify ripening-related promoters of the banana ACC oxidase gene, pMAO2 was used as a probe to screen a banana genomic library constructed in the lambda EMBL3 vector. The banana ACC oxidase MAO2 gene has four exons and three introns, with all of the boundaries between these introns and exons sharing a consensus dinucleotide sequence of GT-AG. The expression of MAO2 gene in banana begins after the onset of ripening (stage 2) and continuous into later stages of the ripening process. The accumulation of MAO2 mRNA can be induced by 1 microliter/l exogenous ethylene, and it reached steady state level when 100 microliters/l exogenous ethylene was present.
Biochem Mol Biol Int 1997 Apr
PMID:Characterization and expression analysis of a banana gene encoding 1-aminocyclopropane-1-carboxylate oxidase. 913 25

1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyses the terminal step in ethylene biosynthesis, is encoded by a small multigene family in tomato that is differentially expressed in response to developmental and environmental cues. In this study we report the isolation and sequencing of approximately 2 kb of 5'-flanking sequence of three tomato ACC oxidase genes (LEACO1, LEACO2, LEACO3) and the occurrence of class I and class II mobile element-like insertions in promoter and intron regions of two of them. The LEA CO1 upstream region contains a 420-bp direct repeat which is present in multiple copies in the tomato genome and is very similar to sequences in the promoters of the tomato E4 and 2A11 genes. The region covering the repeats resembles the remnant of a retrotransposon. Two copies of a small transposable element, belonging to the Stowaway inverted repeat element family, have been found in the 5'-flanking sequence and the third intron of LEACO3.
Mol Gen Genet 1997 Apr 16
PMID:Identification of transposon-like elements in non-coding regions of tomato ACC oxidase genes. 915 Feb 64

The fruit of Actinidia chinensis, a diploid relative of kiwifruit, showed an increased rate of ripening in response to the application of exogenous ethylene. Moreover, late in ripening the fruit produced a burst of ethylene biosynthesis. Thus ripening is climacteric, and there is a clear temporal separation of ethylene sensitivity and ethylene production. RNase protection assays were used to monitor transcript levels of ethylene biosynthetic genes during fruit development and ethylene-induced ripening. The application of exogenous ethylene correlated with increased transcript levels for three different S-adenosyl-L-methionine (SAM) synthetase genes and for the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family. Transcription of an ACC synthase gene was not affected by exogenous ethylene. However, ACC synthase transcript levels increased during subsequent ethylene production by the fruit, consistent with this being the control step for the onset of climacteric ethylene production. ACC oxidase transcripts increased significantly both prior to and during climacteric ethylene production, while only one of the three SAM synthetase transcripts was induced during the late ethylene burst. We propose that the regulation of SAM synthetase transcripts by ethylene may occur as part of the methionine salvage pathway.
Plant Mol Biol 1997 May
PMID:Expression of ethylene biosynthetic genes in Actinidia chinensis fruit. 917 11

Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity. Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.
Plant Mol Biol 1997 May
PMID:Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence. 917 15

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.
Plant Mol Biol 1997 Aug
PMID: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). 929 Jun 38

In an effort towards understanding the biochemical properties and physiological functions of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologues, we have isolated three ACC oxidase clones from sunflower (Helianthus annuus) seedlings. ACCO1 is a cDNA clone while ACCO2 and ACCO3 and reverse transcriptase-polymerase chain reaction clones. Southern analysis indicated the existence of at least three members in the sunflower ACC oxidase gene family. Expression studies showed that ACCO3 was equally expressed in leaves, hypocotyl, and roots of sunflower seedlings, but it constituted only a small amount of the total ACC oxidase transcripts. In contrast, ACCO1 and ACCO2 were differentially expressed in these organs. ACCO1 mRNA was most abundant in roots, whereas ACCO2 was the major homologue in leaves and in hypocotyl. The levels of total ACC oxidase transcripts in these organs were also determined. High ACC oxidase transcript levels were associated with tissue containing rapidly dividing cells. Wounding and silver ion treatments of hypocotyls increased ACC oxidase mRNA levels and ACC oxidase activity; these events being consistent with the increases in ethylene production. In contrast, ACC oxidase protein levels were not affected by these treatments, suggesting that either a translational regulation and/or a rapid turn-over of the protein is involved in both wound- and silver ion-induced gene expression. Contrary to data in the literature, we found that auxins, ethephon and ACC did not affect ACC oxidase mRNA levels in sunflower hypocotyls. The complexity of ACC oxidase regulation and the significance of organ differential expression of ACC oxidase genes are discussed.
Plant Mol Biol 1997 Aug
PMID:Differential and wound-inducible expression of 1-aminocylopropane-1-carboxylate oxidase genes in sunflower seedlings. 929 Jun 44

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6 beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone, gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those for e8 protein, hyoscyamine-6 beta-hydroxylase and ethylene-forming enzyme. These results provide further support that a common ancestral gene is responsible for the appearance of this family of dioxygenases. Hydroxylase assays and RNA blot hybridization studies showed that enzyme activity followed closely the levels of d4h transcripts, occurring predominantly in young leaves and in much lower levels in stems and fruits. In contrast, etiolated seedlings which contained considerable levels of d4h transcripts had almost undetectable hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid increase of enzyme activity without a significant further increase in d4h transcripts over those detected in dark-grown seedlings. These results suggest that the activating effect of light may occur at a point downstream of transcription which remains to be elucidated.
Plant Mol Biol 1997 Aug
PMID:Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don. 929 Jun 45

ACC (1-aminocyclopropane-1-carboxylate) oxidase genes are differentially expressed in melon during development and in response to various stresses. We investigated the molecular basis of their transcription by analyzing the 5' untranslated regions of the ACC oxidase genes CM-ACO1 and CM-ACO3. In order to determine how their temporal and spatial expression patterns were established, we fused the promoter regions of CM-ACO1 (726 bp) and CM-ACO3 (2260 bp) to the beta-glucuronidase (GUS) reporter gene and examined their regulation in transgenic tobacco plants. The CM-ACO1 promoter was able to drive GUS expression in response to wounding, and to treatment with ethylene or copper sulfate. It was also rapidly induced (8-12 h postinoculation) in tobacco leaves inoculated with the hypersensitive response (HR)-inducing bacterium Ralstonia solanacearum. Expression was also observed during compatible interactions but was delayed. In contrast, the CM-ACO3 promoter was not expressed in response to infection, but was up-regulated during flower development. Both promoters were regulated during leaf senescence but in different patterns. The CM-ACO1-driven GUS activity increased sharply concomitantly with the onset of chlorophyll breakdown, while the CM-ACO3 promoter drove strong GUS expression in green, fully expanded leaves and this declined at the onset of senescence. This result is consistent with the expression patterns of these two genes in senescent melon leaves. These data suggest that the regulation of expression of CM-ACO1 is related preferentially to stress responses, whereas CM-ACO3 seems to be associated with developmental processes. The possible role of ethylene is discussed, particularly in the regulation of the CM-ACO1 gene in response to stress and during senescence.
Mol Gen Genet 1997 Oct
PMID:Differential activation of two ACC oxidase gene promoters from melon during plant development and in response to pathogen attack. 939 45

The enzyme ACC oxidase catalyses the last step of ethylene biosynthesis in plants. Expression of the melon ACC oxidase gene, CM-ACO1, is rapidly induced (within 10 min) by ethylene treatment or upon wounding in leaves. The inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), inhibited the accumulation of ethylene-induced CM-ACO1 mRNA transcripts, while wound-induced expression of the gene was not affected. The 5'-untranslated region of the CM-ACO1 gene was fused to the beta-glucuronidase (GUS) reporter gene and the corresponding transgenic tobacco plants were analysed. Two separate regions of the CM-ACO1 promoter activated GUS expression in response to ethylene treatment and wounding. These results suggest that induction of CM-ACO1 gene expression occurs via two separate signal transduction pathways in response to wounding and ethylene treatment.
Plant Mol Biol 1997 Dec
PMID:Wound and ethylene induction of the ACC oxidase melon gene CM-ACO1 occurs via two direct and independent transduction pathways. 942 25


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