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     

Co-suppression of the <Emphasis Type="Italic">EIN2</Emphasis>-homology gene <Emphasis Type="Italic">LeEIN2</Emphasis> inhibits fruit ripening and reduces ethylene sensitivity in tomato

EIN2 (ethylene insensitive 2) is a very important component in the ethylene signal transduction pathway. Recently, the genomic DNA and full-length cDNA of LeEIN2 (tomato EIN2) have been isolated in our laboratory. To reveal the function of LeEIN2, transgenic tomato plants with reduced expression levels of LeEIN2 were produced. The fruit ripening and expressions of ripening-related genes encoding polygalacturonase and TomLoxB were inhibited in the LeEIN2-silenced transgenic plants compared to the wild-type Ailsa Craig. In the seedling ethylene response assay, the transgenic tomato plants with reduced LeEIN2 expression exhibited ethylene insensitivity. These results indicate that LeEIN2 plays a critical role in regulating tomato fruit ripening and is a positive regulator in ethylene signal transduction pathway.     

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.     

SPINDLY interacts with EIN2 to facilitate ethylene signalling-mediated fruit ripening in tomato

The post-translational modification of proteins enables cells to respond promptly to dynamic stimuli by controlling protein functions. In higher plants, SPINDLY (SPY) and SECRET AGENT (SEC) are two prominent O-glycosylation enzymes that have both unique and overlapping roles; however, the effects of their O-glycosylation on fruit ripening and the underlying mechanisms remain largely unknown. Here we report that SlSPY affects tomato fruit ripening. Using slspy mutants and two SlSPY-OE lines, we provide biological evidence for the positive role of SlSPY in fruit ripening. We demonstrate that SlSPY regulates fruit ripening by changing the ethylene response in tomato. To further investigate the underlying mechanism, we identify a central regulator of ethylene signalling ETHYLENE INSENSITIVE 2 (EIN2) as a SlSPY interacting protein. SlSPY promotes the stability and nuclear accumulation of SlEIN2. Mass spectrometry analysis further identified that SlEIN2 has two potential sites Ser771 and Thr821 of O-glycans modifications. Further study shows that SlEIN2 is essential for SlSPY in regulating fruit ripening in tomatoes. Collectively, our findings reveal a novel regulatory function of SlSPY in fruit and provide novel insights into the role of the SlSPY-SlEIN2 module in tomato fruit ripening.     

The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening

In the years since we last reviewed the use of mutants to study tomato fruit ripening ( Grierson et al. 1987 ), considerable information has been gained by the cloning, sequencing and identification of many mRNAs implicated in this developmental process. Genes involved in cell wall degradation, colour change and ethylene synthesis have been cloned, and antisense techniques have been developed and used to produce genetically engineered mutant fruit deficient in these aspects of ripening (see Gray et al. 1992 ). Recently, a previously cloned ripening gene has been used to complement a naturally occurring fruit colour mutant, yellow flesh ( Fray & Grierson 1993a ), and a ripening impaired mutant, ripening inhibitor, has been used to identify several new ripening-related mRNAs ( Picton et al. 1993b ). The chromosomal region bearing the ripening inhibitor mutation has been subjected to high-resolution mapping ( Churchill, Giovannoni & Tanksley 1993 ) and chromosome walking experiments are in progress to identify this gene.     

Ethylene and fruit ripening

The latest advances in our understanding of the relationship between ethylene and fruit ripening are reviewed. Considerable progress has been made in the characterisation of genes encoding the key ethylene biosynthetic enzymes, ACC synthase (ACS) and ACC oxidase (ACO) and in the isolation of genes involved in the ethylene signal transduction pathway, particularly those encoding ethylene receptors ( ETR ). These have allowed the generation of transgenic fruit with reduced ethylene production and the identification of the Nr tomato ripening mutant as an ethylene receptor mutant. Through these tools, a clearer picture of the role of ethylene in fruit ripening is now emerging. In climacteric fruit, the transition to autocatalytic ethylene production appears to result from a series of events where developmentally regulated ACO and ACS gene expression initiates a rise in ethylene production, setting in motion the activation of autocatalytic ethylene production. Differential expression of ACS and ACO gene family members is probably involved in such a transition. Finally, we discuss evidence suggesting that the NR ethylene perception and transduction pathway is specific to a defined set of genes expressed in ripening climacteric fruit and that a distinct ETR pathway regulates other ethylene-regulated genes in both immature and ripening climacteric fruit as well as in non-climacteric fruit. The emerging picture is one where both ethylene-dependent and -independent pathways coexist in both climacteric and non-climacteric fruits. Further work is needed in order to dissect the molecular events involved in individual ripening processes and to understand the regulation of the expression of both ethylene-dependent and -independent genes.     

The Tomato E8 Gene Influences Ethylene Biosynthesis in Fruit but Not in Flowers

We investigated the function of the tomato (Lycopersicon esculentum) E8 gene. Previous experiments in which antisense suppression of E8 was used suggested that the E8 protein has a negative effect on ethylene evolution in fruit. E8 is expressed in flowers as well as in fruit, and its expression is high in anthers. We introduced a cauliflower mosaic virus 35S-E8 gene into tomato plants and obtained plants with overexpression of E8 and plants in which E8 expression was suppressed due to co-suppression. Overexpression of E8 in unripe fruit did not affect the level of ethylene evolution during fruit ripening; however, reduction of E8 protein by cosuppression did lead to elevated levels during ripening. Levels for ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ACC oxidase mRNA were increased approximately 7-fold in fruit of plants with reduced E8 protein. Levels of ACC synthase 2 mRNA were increased 2.5-fold, and ACC synthase 4 mRNA was not affected. Reduction of E8 protein in anthers did not affect the accumulation of ACC or of mRNAs encoding enzymes involved in ethylene biosynthesis. Our results suggest that the product of the E8 reaction participates in feedback regulation of ethylene biosynthesis during fruit ripening.     

Ripening of fleshy fruit: Molecular insight and the role of ethylene

Development and ripening in fruit is a unique phase in the life cycle of higher plants which encompasses several stages progressively such as fruit development, its maturation, ripening and finally senescence. During ripening phase, several physiological and biochemical changes take place through differential expression of various genes that are developmentally regulated. Expression and/or suppression of these genes contribute to various changes in the fruit that make it visually attractive and edible. However, in fleshy fruit massive losses accrue during post harvest handling of the fruit which may run into billions of dollars worldwide. This encouraged scientists to look for various ways to save these losses. Genetic engineering appears to be the most promising and cost effective means to prevent these losses. Most fleshy fruit ripen in the presence of ethylene and once ripening has been initiated proceeds uncontrollably. Ethylene evokes several responses during ripening through a signaling cascade and thousands of genes participate which not only sets in ripening but also responsible for its spoilage. Slowing down post ripening process in fleshy fruit has been the major focus of ripening-related research. In this review article, various developments that have taken place in the last decade with respect to identifying and altering the function of ripening-related genes have been described. Role of ethylene and ethylene-responsive genes in ripening of fleshy fruit is also included. Taking clues from the studies in tomato as a model fruit, few case studies are reviewed.     

Ethylene regulation of fruit ripening: Molecular aspects

Progress in ethylene regulating fruit ripening concerning itsperception and signal transduction and expression of ACC synthaseand ACC oxidase genes is reviewed. ACC synthase and ACC oxidasehave been characterized and their genes cloned from various fruittissues. Both ACC synthase and ACC oxidase are encoded bymultigene families, and their activities are associated withfruit ripening. In climacteric fruit, the transition toautocatalytic ethylene production appears to be due to a seriesof events in which ACC sythase and ACC oxidase genes have beenexpressed developmentally. Differential expression of ACCsynthase and ACC oxidase gene family members is probably involvedin such a transition that ultimately controls the onset of fruitripening.In comparison to ACC synthase and ACC oxidase, less is knownabout ethylene perception and signal transduction because of thedifficulties in isolating and purifying ethylene receptors orethylene-binding proteins using biochemical methods. However, theidentification of the Nr tomato ripening mutant as anethylene receptor, the applications of new potent anti-ethylenecompounds and the generation of transgenic fruits with reducedethylene production have provided evidence that ethylenereceptors regulate a defined set of genes which are expressedduring fruit ripening. The properties and functions of ethylenereceptors, such as ETR1, are being elucidated.Application of molecular genetics, in combination withbiochemical approaches, will enable us to better understand theindividual steps leading from ethylene perception and signaltransduction and expression of ACC synthase and ACC oxidase genefamily member to the physiological responses.     

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     

A novel tomato F‐box protein,SlEBF3, is involved in tuning ethylene signaling during plant development and climacteric fruit ripening

Ethylene is instrumental to climacteric fruit ripening and EIN3 BINDING F‐BOX (EBF) proteins have been assigned a central role in mediating ethylene responses by regulating EIN3/EIL degradation in Arabidopsis. However, the role and mode of action of tomato EBFs in ethylene‐dependent processes like fruit ripening remains unclear. Two novel EBF genes, SlEBF3 and SlEBF4, were identified in the tomato genome, and SlEBF3 displayed a ripening‐associated expression pattern suggesting its potential involvement in controlling ethylene response during fruit ripening. SlEBF3 downregulated tomato lines failed to show obvious ripening‐related phenotypes likely due to functional redundancy among SlEBF family members. By contrast, SlEBF3 overexpression lines exhibited pleiotropic ethylene‐related alterations, including inhibition of fruit ripening, attenuated triple‐response and delayed petal abscission. Yeast‐two‐hybrid system and bimolecular fluorescence complementation approaches indicated that SlEBF3 interacts with all known tomato SlEIL proteins and, consistently, total SlEIL protein levels were decreased in SlEBF3 overexpression fruits, supporting the idea that the reduced ethylene sensitivity and defects in fruit ripening are due to the SlEBF3‐mediated degradation of EIL proteins. Moreover, SlEBF3 expression is regulated by EIL1 via a feedback loop, which supposes its role in tuning ethylene signaling and responses. Overall, the study reveals the role of a novel EBF tomato gene in climacteric ripening, thus providing a new target for modulating fleshy fruit ripening.