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.     

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     

The tomato Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 gene.

Fruit ripening represents a complex system of genetic and hormonal regulation of eukaryotic development unique to plants. We are using tomato ripening mutants as tools to elucidate genetic components of ripening regulation and have recently demonstrated that the Never-ripe (Nr) mutant is insensitive to the plant growth regulator ethylene (M.B. Lanahan, H.-C. Yen, J.J. Giovannoni, H.J. Klee [1994] Plant Cell 6:521-530). We report here ethylene sensitivity over a range of concentrations in normal and Nr tomato seedlings and show that the Nr mutant retains residual sensitivity to as little as 1 part per million of ethylene. Analysis of ripening-related gene expression in normal and mutant ethylene-treated fruit demonstrates that Nr exerts its influence on development at least in part at the level of ethylene-inducible gene expression. We have additionally used cloned tomato and Arabidopsis sequences known to influence ethylene perception as restriction fragment length polymorphism probes, and have identified a tomato locus linked to Nr that hybridizes to the Arabidopsis ETR1 gene at low stringency, suggesting the possibility that Nr may be homologous to ETR1.     

Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model

The hormone ethylene regulates many aspects of plant growth and development, including fruit ripening. In transgenic tomato (Lycopersicon esculentum) plants, antisense inhibition of ethylene biosynthetic genes results in inhibited or delayed ripening. The dominant tomato mutant, Never-ripe (Nr), is insensitive to ethylene and fruit fail to ripen. The Nr phenotype results from mutation of the ethylene receptor encoded by the NR gene, such that it can no longer bind the hormone. NR has homology to the Arabidopsis ethylene receptors. Studies on ethylene perception in Arabidopsis have demonstrated that receptors operate by a "receptor inhibition" mode of action, in which they actively repress ethylene responses in the absence of the hormone, and are inactive when bound to ethylene. In ripening tomato fruit, expression of NR is highly regulated, increasing in expression at the onset of ripening, coincident with increased ethylene production. This expression suggests a requirement for the NR gene product during the ripening process, and implies that ethylene signaling via the tomato NR receptor might not operate by receptor inhibition. We used antisense inhibition to investigate the role of NR in ripening tomato fruit and determine its mode of action. We demonstrate restoration of normal ripening in Nr fruit by inhibition of the mutant Nr gene, indicating that this receptor is not required for normal ripening, and confirming receptor inhibition as the mode of action of the NR protein.     

Isolation of a set of ripening-related genes from strawberry: their identification and possible relationship to fruit quality traits

The ripening of strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, is a complex developmental process that involves many changes in gene expression. To understand how these changes relate to the biochemistry and composition of the fruit the specific genes involved have been examined. A high-quality cDNA library prepared from ripe strawberry fruit was differentially screened for ripening-related clones using cDNA from ripe and white fruits. From 112 up-regulated clones obtained in the primary screen, 66 differentially expressed clones were isolated from the secondary screen. The partial sequences of these cDNAs were compared with database sequences and 26 families of non-redundant clones were identified. Northern analysis confirmed that all of these cDNAs were ripening-enhanced. The expression of many of their corresponding genes was negatively regulated in auxin-treated fruit. These sequences, several of which are novel to fruits, encode proteins involved in key metabolic events including anthocyanin biosynthesis, cell wall degradation, sucrose and lipid metabolism, protein synthesis and degradation, and respiration. These findings are discussed in relation to the role of these genes in determining fruit quality characteristics. Received: 19 January 1998 / Accepted: 5 February 1998     

Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits

Xyloglucan endotransglucosylase/hydrolase (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan modifying enzyme, has been proposed to have a role during tomato and apple fruit ripening by loosening the cell wall. Since the ripening of climacteric fruits is controlled by endogenous ethylene biosynthesis, we wanted to study whether XET activity was ethylene-regulated, and if so, which specific genes encoding ripening-regulated XTH genes were indeed ethylene-regulated. XET specific activity in tomato and apple fruits was significantly increased by the ethylene treatment, as compared with the control fruits, suggesting an increase in the XTH gene expression induced by ethylene. The 25 SlXTH protein sequences of tomato and the 11 sequences MdXTH of apple were phylogenetically analyzed and grouped into three major clades. The SlXTHs genes with highest expression during ripening were SlXTH5 and SlXTH8 from Group III-B, and in apple MdXTH2, from Group II, and MdXTH10, and MdXTH11 from Group III-B. Ethylene was involved in the regulation of the expression of different SlXTH and MdXTH genes during ripening. In tomato fruit fifteen different SlXTH genes showed an increase in expression after ethylene treatment, and the SlXTHs that were ripening associated were also ethylene dependent, and belong to Group III-B (SlXTH5 and SlXTH8). In apple fruit, three MdXTH showed an increase in expression after the ethylene treatment and the only MdXTH that was ripening associated and ethylene dependent was MdXTH10 from Group III-B. The results indicate that XTH may play an important role in fruit ripening and a possible relationship between XTHs from Group III-B and fruit ripening, and ethylene regulation is suggested.     

Hormonal regulation during plant fruit development

The modern concept of the hormonal regulation of fruit set, growth, maturation, and ripening is considered. Pollination and fertilization induce ovule activation by surmounting the blocking action of ethylene and ABA to be manifested in auxin accumulation. Active fruit growth by pericarp cell division and elongation is due to the syntheses of auxin in the developing seed and of gibberellins in the pericarp. In climacteric fleshy fruits, the maturation is controlled by ethylene via so-called System 1 combining the possibilities of autoinhibition and autocatalysis by ethylene of its own biosynthesis. Transition of tomato fruits from maturation to ripening is characterized by highly active synthesis of ethylene and its receptors due to the functioning of regulatory System 2 resulting in the up-regulation of much greater number of ethylene-inducible genes. In peach fruits, the hormonal regulation of ripening includes also an active auxin involvement in the ethylene biosynthesis, which is combined with the ethylene-induced expression of genes encoding both auxin biosynthesis and the response to auxin. Ethylene induces the expression of genes responsible for the fruit softening, its taste, color, and flavor. Nonclimacteric fleshy fruits produce very small amounts of ethylene; its evolution increases only by the very end of ripening and can be described by a reduced System 1. The ripening of nonclimacteric fruits only weakly depends on ethylene but is stimulated by abscisic acid.