The never ripe mutation blocks ethylene perception in tomato.

Seedlings of tomato fruit ripening mutants were screened for their ability to respond to ethylene. Ethylene induced the triple response in etiolated hypocotyls of all tomato ripening mutants tested except for one, Never ripe (Nr). Our results indicated that the lack of ripening in this mutant is caused by ethylene insensitivity. Segregation analysis indicated that Nr-associated ethylene insensitivity is a single codominant trait and is pleiotropic, blocking senescence and abscission of flowers and the epinastic response of petioles. In normal tomato flowers, petal abscission and senescence occur 4 to 5 days after the flower opens and precede fruit expansion. If fertilization does not occur, pedicel abscission occurs 5 to 8 days after petal senescence. If unfertilized, Nr flowers remained attached to the plant indefinitely, and petals remained viable and turgid more than four times longer than their normal counterparts. Fruit development in Nr plants was not preceded by petal senescence; petals and anthers remained attached until they were physically displaced by the expanding ovary. Analysis of engineered 1-aminocyclopropane-1-carboxylate (ACC) synthase-overexpressing plants indicated that they are phenotypic opposites of Nr plants. Constitutive expression of ACC synthase in tomato plants resulted in high rates of ethylene production by many tissues of the plant and induced petiole epinasty and premature senescence and abscission of flowers, usually before anthesis. There were no obvious effects on senescence in leaves of ACC synthase overexpressers, suggesting that although ethylene may be important, it is not sufficient to cause tomato leaf senescence; other signals are clearly involved.     

Members of the tomato LeEIL (EIN3-like) gene family are functionally redundant and regulate ethylene responses throughout plant development

The plant hormone ethylene regulates many aspects of growth, development and responses to the environment. The Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) protein is a nuclear-localized component of the ethylene signal-transduction pathway with DNA-binding activity. Loss-of-function mutations in this protein result in ethylene insensitivity in Arabidopsis. To gain a better understanding of the ethylene signal-transduction pathway in tomato, we have identified three homologs of the Arabidopsis EIN3 gene (LeEILs). Each of these genes complemented the ein3-1 mutation in transgenic Arabidopsis, indicating that all are involved in ethylene signal transduction. Transgenic tomato plants with reduced expression of a single LeEIL gene did not exhibit significant changes in ethylene response; reduced expression of multiple tomato LeEIL genes was necessary to reduce ethylene sensitivity significantly. Reduced LeEIL expression affected all ethylene responses examined, including leaf epinasty, flower abscission, flower senescence and fruit ripening. Our results indicate that the LeEILs are functionally redundant and positive regulators of multiple ethylene responses throughout plant development.     

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.     

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.     

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.     

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     

Expression of multiple forms of polygalacturonase gene during ripening in banana fruit.

The activity of polygalacturonase (PG, E.C 3.2.1.15) during ripening in climacteric fruits has been positively correlated with softening of the fruit tissue and differential expression of its gene is suspected to be regulated by the plant hormone ethylene. We have cloned four partial cDNAs, MAPG1 (acc. no. AF311881), MAPG2 (acc. no. AF311882), MAPG3 (acc. no. AF542382) and MAPG4 (acc. no. AY603341) for PG genes and studied their differential expression during ripening in banana. MAPG3 and MAPG4 are believed to be ripening related and regulated by ethylene whereas MAPG2 is associated more with senescence. MAPG1 shows constitutive expression and is not significantly expressed in fruit tissue. The genomic clone MAGPG (acc. No. AY603340) includes the complete MAPG3 gene, which consists of four exons and three introns. The structure of the gene has more similarity to tomato abscission PG rather than tomato fruit PG. It is concluded that softening during ripening in banana fruit results from the concerted action of at least four PG genes, which are differentially expressed during ripening.     

Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.