Isolation and characterization of four ethylene perception elements and their expression during ripening in pears (Pyrus communis L) with/without cold requirement.

Pear (Pyrus communis L.) are climacteric fruit: their ripening is associated with a burst of autocatalytic ethylene production. Some late pear cultivars, such as Passe-Crassane (PC) require a long (80 d) chilling treatment before the fruit will produce autocatalytic ethylene and ripen. As the cold requirement is linked to the capacity to respond to ethylene (or its analogue, propylene), three pear cDNAs homologous to the Arabidopsis ethylene receptor genes At-ETR1, At-ERS1, and At-ETR2, designated Pc-ETR1a (AF386509), Pc-ERS1a (AF386515), and Pc-ETR5 (AF386511), respectively, have been isolated. A pear homologue of the Arabidopsis ethylene signal transduction pathway gene At-CTR1, called Pc-CTR1 (AF386508) has also been isolated. The search of the genomic sequences for Pc-ETR1a and Pc-ERS1a resulted in the isolation of four related genomic clones Pc-DETR1a (AF386525), Pc-DETR1b (AF386520), Pc-DERS1a (AF386517), and Pc-DERS1b (AF386522). Analysis of transcript levels for the four cDNAs in PC and pear fruit genotypes with little or no cold requirement revealed that Pc-ETR1a expression increased during chilling treatment, and Pc-ETR1a, Pc-ERS1a, Pc-ETR5, and Pc-CTR1 expression increased during fruit ripening and after ethylene treatment. Whether the differences in the ethylene response elements studied here are the cause or an effect of the cold requirement in PC fruit is discussed.

Isolation and characterization of mRNAs differentially expressed during ripening of wild strawberry (Fragaria vesca L.) fruits.

Wild strawberry (Fragaria vesca L.) is an attractive model system for studying ripening in non-climacteric fruit, because of its small diploid genome, its short reproductive cycle, and its capacity for transformation. We have isolated eight ripening-induced cDNAs from this species after differential screening of a cDNA library. The predicted polypeptides of seven of the clones exhibit similarity to database protein sequences, including acyl carrier protein, caffeoyl-CoA 3-O-methyltransferase, sesquiterpene cyclase, major latex protein, cystathionine gamma-synthase, dehydrin and an auxin-induced gene. A ninth cDNA clone that was constitutively expressed is predicted to encode a metallothionein-like protein. None of these proteins appear to be directly related to events generally associated with ripening such as cell wall metabolism or the accumulation of sugars and pigments, rather, their putative functions are indicative of the wide range of processes upregulated during fruit ripening.

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.

ER5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding.

We report the isolation by differential display of a novel tomato ethylene-responsive cDNA, designated ER5. RT-PCR analysis of ER5 expression revealed an early (15 min) and transient induction by ethylene in tomato fruit, leaves and roots. ER5 mRNA accumulated during 2 h of ethylene treatment and thereafter underwent a dramatic decline leading to undetectable expression after 5 h of treatment. The full-length cDNA clone of 748 bp was obtained and DNA sequence analysis showed strong homologies to members of the atypical hydrophobic group of the LEA protein family. The predicted amino acid sequence shows 67%, 64%, 64%, and 61% sequence identity with the tomato Lemmi9, soybean D95-4, cotton Lea14-A, and resurrection plant pcC27-45 gene products, respectively. As with the other members of this group, ER5 encodes a predominantly hydrophobic protein. Prolonged drought stress stimulates ER5 expression in leaves and roots, while ABA induction of this ethylene-responsive clone is confined to the leaves. The use of 1-MCP, an inhibitor of ethylene action, indicates that the drought induction of ER5 is ethylene-mediated in tomato roots. Finally, wounding stimulates ER5 mRNA accumulation in leaves and roots. Among the Lea gene family this novel clone is the first to display an ethylene-regulated expression.

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit.

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus x domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.

Purification, properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits.

The enzyme which converts 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene, ACC oxidase, has been isolated from apple fruits (Malus x domestica Borkh. cv. Golden Delicious), and for the first time stabilized in vitro by 1,10-phenanthroline and purified 170-fold to homogeneity in a five-step procedure. The sodium dodecyl sulfate-denatured and native proteins have similar molecular weights (approx. 40 kDa) indicating that the enzyme is active in its monomeric form. Antibodies raised against a recombinant ACC oxidase over-produced in Escherichia coli from a tomato cDNA recognise the apple-fruit enzyme with high specificity in both crude extracts and purified form. Glycosylation appears to be absent because of (i) the lack of reactivity towards a mixture of seven different biotinylated lectins and (ii) the absence of N-linked substitution at a potential glycosylation site, in a sequenced peptide. Phenylhydrazine and 2-methyl-1-2-dipyridyl propane do not inhibit activity, indicating that ACC oxidase is not a prosthetic-heme iron protein. The partial amino-acid sequence of the native protein has strong homology to the predicted protein of a tomato fruit cDNA demonstrated to encode ACC oxidase.

Synthesis and assembly of soybean beta-conglycinin in vitro.

The construction of SP6-derived expression plasmids that encode normal and modified beta-conglycinin subunits is described. With the exception of an additional methionine at their NH2-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids corresponded to mature alpha and beta subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH2-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal alpha subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified beta-conglycinin subunits. The results also show that structural changes at the NH2-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.

5'CATGCAT-3' Elements Modulate the Expression of Glycinin Genes.

Promoters of most seed proteins in legumes contain one or more 5'-CATGCAT-3' elements. To test if these elements have a function in the expression of these genes, the 2.3 kilobase pairs Gy2 glycinin promoter was ligated to a beta-glucuronidase reporter sequence and transformed into tobacco. Elimination of a 5'-CATGCAT-3' element 101 base pairs upstream from the transcription start site in the construction caused about a 10-fold reduction in the amount of beta-glucuronidase activity compared with when the element was present in the gene. Elimination of 1.9 kilobase pairs from the 5'-end of the promoter caused a two-to threefold reduction in activity. The results show that the 5'-CATGCAT-3' element plays a role in regulating the amount of expression from the gene, but that there are also other factors farther upstream from the gene that affect the level of expression.

Protein synthesis associated with quiescence and senescence in auxin-starved pear cells.

Pear fruit cells (Pyrus communis L. cv Passe Crassane) stopped dividing when subcultured in a bioreactor under auxin starvation in the presence of 0.37 molar mannitol. The cessation of cell division was preceded by the accumulation of a specific basic polypeptide of 24 kilodalton. Readdition of 2.3 micromolar 2,4-dichlorophenoxyacetic acid (2,4-D) neither caused a resumption of cell division nor depressed the accumulation of this polypeptide. Under complete auxin starvation, cells began to die at day 18. In vivo radioactive labeling of proteins followed by two-dimensional electrophoresis showed that during auxin starvation the synthesis of some polypeptides including the 24 kilodalton one (referred to as homeostasis-related proteins, HRPs) was decreased while the synthesis of some others (referred as senescence-related proteins, SRPs) was increased. Readdition of 2.3 micromolar 2,4-D postponed the onset of cell death by 10 to 15 days while supplementation with 7.6 micromolar abscisic acid advanced cell death by 8 days. Two-dimensional analysis of protein synthesis indicated that both hormones interact on the synthesis of these two groups of polypeptides. The levels of most HRPs were maintained or increased in the presence of auxin, while the levels of the SRPs were decreased by auxin and increased by abscisic acid. Short and long-term effects of 2,4-D and abscisic acid on the synthesis of specific polypeptides were observed, allowing a discrimination between the direct and indirect effect of both hormones on the development of cell senescence.