Molecular biology of fruit ripening and its manipulation with antisense genes

Considerable progress in tomato molecular biology has been made over the past five years. At least 19 different mRNAs which increase in amount during tomato fruit ripening have been cloned and genes for enzymes involved in cell wall degradation (polygalacturonase and pectinesterase) and ethylene synthesis (ACC synthase) have been identified by conventional procedures. Transgenic plants have been used to identify regions of DNA flanking fruit-specific, ripening-related and ethylene-regulated genes and trans-acting factors which bind to these promoters have also been identified.Antisense genes expressed in transgenic plants have proved to be highly effective for inhibiting the specific expression of ripening-related genes. These experiments have changed our understanding of how softening occurs in tomato fruit. Antisense techniques have also been used to identify genes encoding enzymes for carotenoid biosynthesis (phytoene synthase) and ethylene biosynthesis (the ethylene-forming enzyme). The altered characteristics of fruit transformed with specific antisense genes, such as retarded ripening and resistance to splitting, may prove to be of value to fruit growers, processors and ultimately the consumer.     

Genetic mapping of ripening and ethylene-related loci in tomato

 The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response. Received: 26 October 1998 / Accepted: 13 November 1998     

The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening

Tomato mRNA was extracted from individual fruits at different stages of development and ripening, translated in a rabbit reticulocyte lysate and the protein products analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicate that there are at least two classes of mRNA under separate developmental control. One group of approximately six mRNAs is present during fruit growth and then declines at the mature-green stage. Another group of between four and eight mRNAs increases substantially in amount at the onset of ripening, after the start of enhanced ethylene synthesis by the fruit, and continues to accumulate as ripening progresses. Studies of protein synthesis in vivo show that several new proteins are synthesised by ripening fruits including the fruit-softening enzyme polygalacturonase. One of the ripening-related mRNAs is shown to code for polygalacturonase, by immunoprecipitation with serum from rabbits immunised against the purified tomato enzyme. Polygalacturonase mRNA is not detectable in green fruit but accumulates during ripening. It is proposed that the ripening-related mRNAs are the products of a group of genes that code for enzymes important in the ripening process.Abbreviation SDS sodium dodecyl sulfate     

Inhibition of expression of tomato-ripening genes at high temperature

Abstract. Ripening tomato fruits incubated at 35°C fail to achieve normal pigmentation, soften little and show a marked decline in ethylene evolution. Labelling studies in vivo indicate that protein synthesis continues throughout incubation at 35°C although the spectrum of labelled proteins is different to that observed at 25°C. Translation of mRNAs in vitro shows traces of several 'heat-shock' mRNAs at 35°C and the loss of several others normally found in fruit ripened at 25°C. Using ripening-related cDNA clones as hybridization probes the expression of 12 ripening-related genes was followed during incubation at 25°C and 35°C. In general, there was a marked decline in the amounts of these mRNAs following incubation of ripening fruit at 35°C. In particular, mRNA homologous to pTOM 6, a cDNA clone coding for polygalacturonase, a major cell wall degrading enzyme, showed a rapid decline following incubation at 35°C and after 72-h at elevated temperature was undetectable. There was no recovery of expression during 120 h at 35°C and the application of exogenous ethylene did not overcome the inhibition of ripening or lead to the renewed accumulation of polygalacturonase mRNA. It is proposed that the failure to soften normally at elevated temperature is due, in part, to the suppression of polygalacturonase mRNA and that the inhibition of other facets of ripening at 35°C is due to the inhibition or reduced expression of other, as yet unidentified, ripening-related genes.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Identification of <Emphasis Type="Italic">Solanum habrochaites</Emphasis> loci that quantitatively influence tomato fruit ripening-associated ethylene emissions

The phytohormone ethylene is essential for ripening of climacteric fruits such as tomato. While many of the genes responsible for ethylene synthesis and perception have been identified, the regulatory network controlling autocatalytic climacteric ethylene synthesis is not well understood. In order to better understand the regulation of ripening-associated ethylene, we have exploited the genetic variation within Solanum Sect. Lycopersicon. In particular, we have used a near-isogenic population of S. habrochaites introgression lines to identify chromosome segments affecting ethylene emissions during ripening. S. habrochaites fruits produce much larger quantities of ethylene during ripening than do cultivated S. lycopersicum tomatoes. A total of 17 segments were identified; 3 had emissions more than twice the level of the tomato parent, 11 had less than a twofold increase and 3 had significantly reduced emissions at one or more ripening stages. While several of these segments co-segregate with known ethylene-related genes, many do not correspond to known genes. Thus, they may identify novel modes of regulation. These results illustrate the utility of wild relatives and their introgression lines to understand regulation of fruit ripening-related processes.     

Silver ions inhibit the ethylene-stimulated production of ripening-related mRNAs in tomato

Abstract. Silver ions effectively inhibited both the initiation and the continuation of tomato ( Lyeopersicon esculentum Mill) ripening. Studies of protein synthesis in vivo showed that application of 2 mol m⁻³ silver thiosulphate to mature green fruit prevented the appearance of several novel proteins associated with ripening, including the softening enzyme polygalacturonase. However, total protein synthesis, as judged by the incorporation of [³⁵S] methionine into proteins, continued unabated after silver treatment. Ripening was also arrested when silver was supplied after ripening had begun. The accumulation of several ripening-related mRNAs, including that for polygalacturonase, was studied by translation in vitro and using cDNA clones as hybridization probes. Silver was shown to prevent the appearance of polygalaturonase mRNA when supplied to mature green fruit and to cause a rapid reduction in the concentration of mRNA for polygalacturonase and other ripening-related proteins when supplied after ripening had begun. It is proposed that silver exerts its effects due to interaction with the ethylene perception mechanism. The results suggest that perception of ethylene is vital not only for the initiation of ripening but also for the continued expression of genes required for ripening.     

Suppression of Ripening-Associated Gene Expression in Tomato Fruits Subjected to a High CO2 Concentration

High concentrations of CO2 block or delay the ripening of fruits. In this study we investigated the effects of high CO2 on ripening and on the expression of stress- and ripening-inducible genes in cherry tomato (Lycopersicon esculentum Mill.) fruit. Mature-green tomato fruits were submitted to a high CO2 concentration (20%) for 3 d and then transferred to air. These conditions effectively inhibited ripening-associated color changes and ethylene production, and reduced the protein content. No clear-cut effect was observed on the expression of two proteolysis-related genes, encoding polyubiquitin and ubiquitin-conjugating enzyme E2, respectively. Exposure of fruit to high CO2 also resulted in the strong induction of two genes encoding stress-related proteins: a ripening-regulated heat-shock protein and glutamate decarboxylase. Induction of these two genes indicated that high CO2 had a stress effect, most likely through cytosolic acidification. In addition, high CO2 blocked the accumulation of mRNAs for genes involved in the main ripening-related changes: ethylene synthesis (1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase), color (phytoene synthase), firmness (polygalacturonase), and sugar accumulation (acid invertase). The expression of ripening-specific genes was affected by CO2 regardless of whether their induction was ethylene- or development-dependent. It is proposed that the inhibition of tomato fruit ripening by high CO2 is due, in part, to the suppression of the expression of ripening-associated genes, which is probably related to the stress effect exerted by high CO2.     

Genetic identification and genomic organization of factors affecting fruit texture

Fleshy fruits are an essential part of the human diet providing vital vitamins, minerals and other health-promoting compounds. The texture of the ripe fruit has a significant effect on quality and influences consumer acceptance, shelf-life, resistance, and transportability. The development of rational approaches to improve texture and shelf-life depend on understanding the biological basis of fruit ripening. Until recently, work has focused on the isolation of ripening-related genes from a variety of fleshy fruits. However, little is known about the genes that regulate this complex developmental process or whether similar regulatory genes are active in all fruiting species. A major breakthrough would be the identification of generic genes associated with texture and other aspects of ripening in fleshy fruits. In tomato, a small number of single gene mutations exist, such as ripening-inhibitor (rin), non-ripening (nor), Never-ripe (Nr), and Colourless non-ripening (Cnr) which have pleiotropic effects resulting in the reduction or almost complete abolition of ripening. These mutations probably represent lesions in regulatory genes. The cloning of the wild-type alleles of RIN and NOR is reported by Moore et al. in this issue. This review focuses on the texture characteristics of the Cnr mutant. A possible framework for the molecular regulation of fruit texture is discussed and quantitative genetic approaches to determining the generic attributes of fruit texture are explored.