M Bouzayen
email: bouzayen@ensat.fr

A Latche

P Nath

J C Pech

J C Pech Ge

NR E R Etr

P M Cytosol

Cw

Mechanism of Fruit Ripening

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 Fig. 16.1 Schematic representation depicting the molecular mechanisms controlling the ripening of climacteric fruit. The fruit ripening process is a genetically regulated developmental process involving the activation of a high number of primary and secondary metabolic pathways that all contribute to the overall sensory and nutritional quality of the fruit. This process involves the expression of ripening-related genes that encode enzymes (proteins) involved in the various ripening pathways (e.g., softening, color development). The whole process is under the control of hormonal and environmental signals, amongst which ethylene plays a major role

Climacteric and Non-Climacteric Fruit Ripening

Fruit can be divided into two groups according to the regulatory mechanisms underlying the ripening process. Climacteric fruit, such as tomato, apple, pear, and melon (Table 16.1), are characterized by a ripening-associated increase in respiration and in ethylene production. By contrast, non-climacteric fruits, such as orange, grape, and pineapple (Table 16.1), are characterized by the lack of ethylene-associated respiratory peak. At the onset of ripening, climacteric fruit present a peak in respiration, and a concomitant burst of ethylene production. The relationship existing between the climacteric respiration and fruit ripening has been questioned following the discovery that ripening on the vine of a number of fruit may occur in the absence of any increase in respiration [START_REF] Salveit | Internal carbon dioxide and ethylene levels in ripening tomato fruit attached to or detached from the plant[END_REF][START_REF] Shellie | The lack of a respiratory rise in muskmelon fruit ripening on the plant challenges the definition of climacteric behaviour[END_REF]. More recently, it has been reported that the presence or absence of a respiratory climacteric on the vine depends upon prevailing environmental conditions [START_REF] Bower | Culture conditions and detachment of the fruit influence the effect of ethylene on the climacteric respiration of melon[END_REF]. These observations indicate that the respiratory climacteric is probably not an absolute trigger of the ripening process, but secondary and consequential to the process of ripening. An ethylene burst that precedes respiratory climacteric has been shown during the ripening of banana [START_REF] Pathak | Expression and activities of ethylene biosynthesis enzymes during ripening in banana fruits and effect of 1 MCP treatment[END_REF]. Two distinct ethylene biosynthesis systems have been described. System 1 corresponds to low ethylene production in the pre-climacteric period of climacteric fruit, and is present throughout the development of non-climacteric fruit. System 2 refers to an auto-stimulated massive ethylene production called "autocatalytic synthesis", and is specific to climacteric fruit. Therefore, the major ethylene-related differences between climacteric and non-climacteric fruit is the presence or absence of autocatalytic ethylene production [START_REF] Mcmurchie | Treatment of fruit with propylene gives information about the biogenesis of ethylene[END_REF][START_REF] Alexander | Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening[END_REF]. The ethylene biosynthetic pathway is now well established (Fig. 16.1; Yang and Hoffmann 1984). This ripening hormone is synthesized from methionine via S-adenosyl-L-methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC). Two major enzymes are involved in the biosynthetic pathway, namely, ACC synthase (ACS), which converts SAM into ACC, and ACC oxidase (ACO), which converts ACC into ethylene. The corresponding genes have been identified and characterized [START_REF] Sato | Cloning of the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants[END_REF][START_REF] Hamilton | Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants[END_REF][START_REF] Hamilton | Identification of a tomato gene for the ethyleneforming enzyme by expression in yeast[END_REF]. Both ACO and ACS are encoded by a multigene family of five and nine members, respectively in tomato, with expressions differentially regulated during fruit development and ripening [START_REF] Barry | Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato[END_REF][START_REF] Barry | The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF]. While LeACO1 and LeACO4 genes are up-regulated at the onset of ripening, and continue being active throughout ripening, LeACO3 displays only transient activation at the breaker stage of fruit ripening (Fig. 16.2). It was shown that Le ACS6 and LeACS1A are expressed at the pre-climacteric stage (system 1), while at the transition to ripening, LeACS4 and LeACS1A are the most active genes (Fig. 16.2). Subsequently, LeACS4 continues to express highly during climacteric phase, whereas the expression of LeACS1A declines. The rise in ripening-associated ethylene production results in the induction of LeACS2, and the inhibition of Le ACS6 and LeACS1A expression. This fine tuning of the ACS genes is thought to be critical for the switch from pre-climacteric system 1 to climacteric system 2. Noteworthy is that system 1 is characterized by inhibitory feedback of ethylene in its own biosynthetic pathway, whereas the transition to system 2 is characterized by autocatalytic production. The requirement for ethylene to trigger the ripening of climacteric fruit has been clearly demonstrated by down-regulating ACO and ACS genes in transgenic plants using an antisense strategy. The ethylene-suppressed lines showed strongly delayed ripening in tomato [START_REF] Oeller | Reversible inhibition of tomato fruit senescence by antisense RNA[END_REF][START_REF] Picton | Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene[END_REF], and in other fruits, e.g., melon [START_REF] Ayub | Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits[END_REF] and apple [START_REF] Dandekar | Effect of down-regulation of ethylene biosynthesis on fruit flavor complex in apple fruit[END_REF]). However, ethylene-independent ripening pathways exist in climacteric fruit, as illustrated in melon fruit, where part of softening, sugar accumulation, and coloration of the flesh occur in ethylenesuppressed fruit [START_REF] Flores | The use of ethylene-suppressed lines to assess differential sensitivity to ethylene of the various ripening pathways in Cantaloupe melons[END_REF]. These results have led to the conclusion that climacteric (ethylene-dependent) and non-climacteric (ethylene-independent) regulation coexists in climacteric fruit (Pech et al. 2008a).

Although the ripening of non-climacteric fruit is not associated with any significant change in ethylene production, some ethylene-dependent processes do exist in this type of fruit. In grape berries, the ethylene synthesis pathway is activated at the inception of the ripening, the so-called veraison stage. Treatments with exogenous ethylene stimulate the long-term expression of genes related to anthocyanin synthesis, and ethylene signals appear to be involved in the regulation of vascular flux, acid content, and in some steps of aroma volatile production [START_REF] Mailhac | Ethylene and grape berry ripening[END_REF]. In citrus, another class of typically non-climacteric fruit, the existence of an autocatalytic system of ethylene production similar to that of climacteric fruit has been suggested [START_REF] Katz | Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in nonclimacteric Citrus fruit[END_REF]), and it is well known that these types of fruit have the ability to respond to exogenous ethylene in terms of chlorophyll degradation. Moreover, in all non-climacteric fruits, exogenous ethylene accelerates senescence LeACS, Lycopersicon esculentum ACC synthase; LeACO, Lycopersicon esculentum ACC oxidase; LeETR and NR, ethylene receptors. Eth+ and Eth-refer to the stimulation and repression, respectively of gene or protein expression (adapted from [START_REF] Barry | Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato[END_REF][START_REF] Barry | The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF][START_REF] Kevany | Ethylene receptor degradation controls the timing of ripening in tomato fruit[END_REF] via the deterioration of cell membranes. While the role of ethylene in climacteric fruit ripening is beginning to be well understood, the main signaling pathways involved in non-climacteric ripening remain very poorly understood.

Ethylene Perception and Signal Transduction

Breakthrough advances in the field of ethylene perception have been made possible by the use of the model plant Arabidopsis, and the implementation of molecular genetics strategies. Following the identification of the ethylene-insensitive mutants, named ETR1 [START_REF] Bleecker | Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana[END_REF], the gene encoding the ethylene receptor was isolated by positional cloning [START_REF] Chang | Arabidopsis ethylene-response gene ETR: similarity of product to two component regulators[END_REF]). The ethylene receptor was the first plant hormone receptor to be isolated and characterized, and this paved the way toward the isolation of the other components of the ethylene transduction pathway [START_REF] Klee | Ethylene signal transduction in fruits and flowers[END_REF]. Based on these discoveries, the use of Arabidopsis has been critical in helping to isolate the ethylene receptor from other plant species, and to understand the role of the receptors in the ripening process. The ethylene receptors are encoded by a small multigene family for structurally distinct but functionally redundant proteins working either as hetero-or homo-multimers. In tomato, six ethylene receptor genes have been isolated and found to be expressed in all plant tissues, three of these showing a net increase during ripening, while two express constitutively (Fig. 16.2). Interestingly, it was demonstrated that the tomato Never ripe (Nr) mutation, which results in impaired ripening, occurs in one of the ethylene receptor genes. Recent studies demonstrated that the ethylene receptors are rapidly degraded during fruit ripening, while the transcription rate remains high, and that the receptor level determines the timing of ripening [START_REF] Kevany | Ethylene receptor degradation controls the timing of ripening in tomato fruit[END_REF]). Moreover, the suppression of the ethylene receptor LeETR4 led to an early ripening of tomato fruits [START_REF] Kevany | Fruit-specific suppression of the ethylene receptor LeETR4 results in early-ripening tomato fruit[END_REF].

In more applied terms, the search for ethylene antagonists led to the discovery of 1-methylcyclopropene (MCP), a powerful antagonist of ethylene action [START_REF] Sisler | Inhibition of ethylene responses by 1-methylcyclopropene and 3-methylcyclopropene[END_REF]). This compound is now widely used both by academic researchers as a tool for understanding ethylene-regulated developmental processes [START_REF] Blankenship | 1-methylcyclopropene: a review[END_REF], and by the producers and shippers of fresh fruit and flowers on a commercial scale for extending the shelf life of these products. MCP probably represents the most remarkable innovation in the past two decades in the field of post-harvest horticulture (http://www.hort.cornell.edu/department/faculty/watkins/ ethylene/).

The CTR1 gene (Constitutive Triple Response), first isolated from Arabidopsis, encodes another major component of ethylene signaling lying downstream of the receptor acting as a negative regulator of the ethylene transduction pathway [START_REF] Kieber | CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases[END_REF]. The tomato CTR1 gene (Sl-CTR1) was first isolated from fruit tissue [START_REF] Leclercq | Lectr1, a tomato ctr1-like gene, demonstrates ethylene signaling ability in Arabidopsis and novel expression patterns in tomato[END_REF], and in spite of being a negative regulator of ethylene responses, its transcripts are up-regulated during fruit ripening, commensurate with the rise in ethylene production. Subsequently, it was shown that the CTR family was composed of four genes in tomato, each displaying a specific pattern of expression during ripening and in response to ethylene, with Sl-CTR1 being the most actively expressed during fruit ripening [START_REF] Adams-Phillips | Evidence that CTR1-mediated ethylene signal transduction in tomato is encoded by a multigene family whose members display distinct regulatory features[END_REF]). Strikingly, reverse genetic strategies have to date failed to show any impact of altered CTR1 expression on the fruit ripening process, indicating a potential functional redundancy among the CTR genes.

Control of Ethylene Response in Fruit

Because of the tremendous change in the expression level of a large number of genes during fruit ripening, and in order to gain better insight into the control mechanisms underlying this process, differential screening approaches were attempted to isolate and characterize ethylene-regulated genes [START_REF] Lincoln | Regulation of gene expression by ethylene during Lycopersicon esculentum (tomato) fruit development[END_REF]. Genes encoding cell wall-degrading, ethylene production, and pigment biosynthesis enzymes were among the first ethylene-responsive genes to be isolated from tomato fruit. Later, a set of early ethylene-regulated genes were isolated from mature green tomatoes that are responsive to exogenous ethylene, but not yet producing elevated levels of ripening-associated ethylene [START_REF] Zegzouti | Ethylene-regulated gene expression in tomato fruit: characterization of novel ethylene-responsive and ripening-related genes isolated by differential display[END_REF]. Expression studies revealed that the ethylene-responsive genes can be up-regulated, down-regulated, or transiently induced following short periods of hormone treatment, supporting the idea that ethylene can act as negative or positive regulator of gene expression [START_REF] Gupta | Differential expression of genes during banana fruit development, ripening and 1-MCP treatment: presence of distinct fruit specific, ethylene induced and ethylene repressed expression[END_REF][START_REF] Kesari | Ethylene-induced ripening in banana evokes expression of defense and stress related genes in fruit tissue[END_REF]. Noteworthy is that many of the early ethylene-responsive genes encode putative regulatory proteins involved in transduction pathways and transcriptional or post-transcriptional regulation, indicating that the ethylene control of the ripening process operates in a complex multilevel way. More recently, the work by Giovannoni's group [START_REF] Alba | Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development[END_REF]) demonstrated the importance of ethylene control during tomato fruit development. In the tomato Nr mutant, impaired in ethylene sensing and fruit ripening, up to one third of ripening-associated genes showed altered expression compared to wild type [START_REF] Alba | Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development[END_REF]. Moreover, in a non-climacteric fruit like strawberry, microarray analyses comparing akene and receptacle tissues show high levels of ethylene response factor (ERF) and ethylene regulated (ER) gene expression in akene tissue, suggesting a role for ethylene in the maturation of the akene [START_REF] Aharoni | Microarray gene expression analysis during strawberry achenes and receptacle maturation[END_REF]. Together, these data demonstrate the important role of ethylene in fruit ripening in both climacteric and non climacteric fruit. However, the mechanistic insight into how ethylene acts to bring about the activation of all the ripening-associated metabolic pathways remains unclear. Ethylene is known to have numerous effects on a wide range of developmental processes, including germination, flower and leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell death, and responsiveness to abiotic stress and pathogen attack [START_REF] Johnson | The ethylene gas signaling pathway in plants: a molecular perspective[END_REF][START_REF] Bleecker | Ethylene: a gaseous signal molecule in plants[END_REF][START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF]. This diversity of plant responses to ethylene raises the question on how this phytohormone selects the desired target genes with respect to their tissue and developmental specificity. This question becomes even more relevant when considering that the ethylene transduction pathway is linear in its upstream part from the receptor to ein3, the first transcription regulator. It is therefore tempting to speculate that most of the diversity of ethylene responses may arise largely from fine tuning of the expression and/or activity of ERFs, transcriptional regulator proteins lying downstream of EIN3 (Fig. 16.3). Indeed, ERFs belong to one of the largest families of transcription factors in plants [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF], thus offering different branching possibilities to channel the hormone signaling to a variety of responses. The diversity and complexity of ethylene responses can also arise from the cross-talk between ethylene and other hormones [START_REF] Rosado | The Arabidopsis tetratricopeptide repeat-containing protein TTL1 is required for osmotic stress responses and abscisic acid sensitivity[END_REF][START_REF] Stepanova | Multilevel interactions between ethylene and auxin in Arabidopsis roots[END_REF].

ERF genes encode a type of trans-acting factors unique to plants that specifically bind the GCC box, a conserved motif of the cis-acting element found in the promoter of ethylene-responsive genes (Ohme-Takagi and Shinshi 1995; [START_REF] Solano | Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1[END_REF] Fig. 16.3 The hormone-dependent transcriptional regulation associated with fruit ripening. A main focus is made on ethylene and auxin, aiming at exemplifying the importance of cross-talk between hormone signaling. Ethylene transduction cascade leads to the activation of EIN3-Like (EIL) genes, which activates primary target genes (ethylene-response factors, ERFs). ERFs in turn activate the expression of secondary ripening-related genes. Other signals, such as auxin, are also involved in this process. Some auxin response factors (ARFs) and Aux/IAA transcription factors are also ethylene-responsive, and therefore are likely to participate in the expression of ripeningrelated genes [START_REF] Jones | Downregulation of an ARF-like gene in the tomato results in a pleiotropic phenotype including darkgreen and blotchy ripening fruit[END_REF] and the ERF family is part of the AP2/ERF superfamily of transcription factors, which also contains the AP2 and RAV families [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF]. Since ERFs belong to a large multigene family, it is expected that members of this family have varied functionality, and diverse binding activities. Using combined reverse genetics and transcriptomics approaches, intensive studies are in progress to uncover the specific role of each ERF in the ripening process, and to establish the set of target genes regulated by each member of this transcription factor family. In the long term, the objective of these studies is to set up tools enabling targeted control of the ripening process, thus allowing engineering fruit ripening in specific ways, such as slowing down the loss of firmness, while enhancing desired metabolic pathways.

Hormone Cross-Talk and Fruit Ripening

As mentioned above, fruit ontogeny and ripening are genetically regulated processes involving a complex multi-hormonal control (Fig. 16.3). While the roles of ethylene in triggering and regulating the ripening of climacteric fruit have been clearly demonstrated, little is known on the roles of other hormones. Phytohormones exert their effect on plant development via a chain of transduction pathways that ultimately activates specific transcription factors, which in turn regulate the expression of a set of target genes. In order to uncover the role of hormones that act in concert with ethylene to regulate tomato fruit development, a screen for transcription factors showing differential expression from fruit set through ripening led to the isolation of a number of genes encoding auxin transcriptional regulators of the ARF and Aux/IAA type [START_REF] Jones | Downregulation of an ARF-like gene in the tomato results in a pleiotropic phenotype including darkgreen and blotchy ripening fruit[END_REF]. Among the isolated auxin-response factors, some showed fruit-specific and ethylene-regulated expression that clearly correlated with their pattern of ethylene responsiveness, suggesting a cross-talk between ethylene and auxin throughout fruit development [START_REF] Jones | Downregulation of an ARF-like gene in the tomato results in a pleiotropic phenotype including darkgreen and blotchy ripening fruit[END_REF][START_REF] Wang | LeIAA9, a tomato Aux/IAA gene involved in fruit development and leaf morphogenesis[END_REF]. Combined reverse genetic and transcriptomic approaches have been carried out to uncover the functional significance of these genes. Molecular and physiological characterization of transgenic tomato plants under-and over-expressing these transcription factors confirmed their crucial role in both early and late stages of fruit development. Important quality traits, such as sugar content, firmness, and parthenocarpy, are strongly affected in the transgenic lines [START_REF] Jones | Downregulation of an ARF-like gene in the tomato results in a pleiotropic phenotype including darkgreen and blotchy ripening fruit[END_REF][START_REF] Wang | LeIAA9, a tomato Aux/IAA gene involved in fruit development and leaf morphogenesis[END_REF]. These genes offer new potential targets for improving fruit quality, either by marker-assisted selection or by biotechnological means.

Biochemical Changes and Sensory Traits Associated with Fruit Ripening

One of the major factors associated with the post-harvest deterioration of fruit is the rate of softening. Excessive softening results in shorter shelf life during storage, transportation and distribution, and increased wastage. A number of genes potentially involved in cell wall degradation, rearrangement and structure have been isolated, and most of these have been studied in the tomato model. However, unexpectedly, it has been shown that the suppression of candidate genes, such as those encoding polygalacturonase, pectin-methyl-esterase, and b-glucanase, did not have a major impact on the evolution of fruit firmness [START_REF] Giovannoni | Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening[END_REF][START_REF] Tieman | An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit[END_REF]Brummell et al. 1999a). Up to 40% reduction of tomato fruit softening has been achieved by down-regulating the TBG4 b-galactosidase gene [START_REF] Smith | Down-regulation of tomato b-galactosidase 4 results in decreased fruit softening[END_REF], but in antisense TBG4 fruit, TBG3 gene expression was also reduced, indicating a possible cooperation of the two genes. Expansins are cell wall proteins that loosen cell walls by reversibly disrupting hydrogen bonds between cellulose microfibrils and matrix polysaccharides. The LeExp1 (tomato expansin 1) gene encodes a protein that is specifically expressed in ripening fruit. Down-regulation resulted in strong reduction of softening throughout ripening, probably by alteration of the microfibril-/matrix glycan interface that facilitates access of cell wall hydrolases to the matrix glycan substrates (Brummell et al. 1999b). Another class of cell wall-degrading enzymes, pectate lyases, appears to have a more important role in ripening than previously expected. In strawberry, a non-climacteric fruit, suppression of the pectate lyase mRNA resulted in significantly firmer fruits (Jime ´nez-Bermu ´dez et al. 2002), with the highest reduction in softening being shown to occur during the transition from the white to the red stage. Within the gene families of cell wall-degrading genes of climacteric fruit, some members are regulated by ethylene, while others are not, confirming the coexistence of ethylene-dependent and -independent processes [START_REF] Flores | The use of ethylene-suppressed lines to assess differential sensitivity to ethylene of the various ripening pathways in Cantaloupe melons[END_REF][START_REF] Nishiyama | Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon[END_REF]. In general, it appears that fruit softening involves many genes that encode a variety of cell wall-degrading enzymes and non-enzymatic proteins. Each protein, and each protein isoform, may play a specific role in softening and textural changes. Pigments are essential for the attractiveness of fruits, accumulating most often in the skin during the ripening process, although many climacteric fruits accumulate pigments also in their pulp tissue. The most important pigments of fruit are carotenoids and anthocyanins. Beside their role in pigmentation, they are important for human health as a source of vitamin A and antioxidant compounds. Carotenoids comprise carotenes, such as lycopene and b-carotene, and xanthophylls, such as lutein. They are derived from terpenoids, and are synthesized in fruit at a high rate during the transition from chloroplast to chromoplast. Many genes involved in the biosynthesis of carotenoids have been cloned [START_REF] Cunningham | Genes and enzymes of carotenoids biosynthesis in plants[END_REF][START_REF] Hirschberg | Carotenoid biosynthesis in flowering plants[END_REF], and extensive information is available on the regulation of carotenoid formation during fruit ripening [START_REF] Bramley | Regulation of carotenoid formation during tomato fruit ripening and development[END_REF]. Anthocyanins belong to the flavonoid subclass of phenolic compounds. The flavonoid biosynthetic pathway has been elucidated in plants, and many enzymes and corresponding genes have been isolated and characterized [START_REF] Winkel-Shirley | Flavonoids biosynthesis. A colourful model for genetics, biochemistry, cell biology, and biotechnology[END_REF]. In grape, where anthocyanins are crucial for the quality of wine, it has been demonstrated that ethylene (or the ethylene generator ethephon) stimulates berry coloration, demonstrating that this hormone is involved in the regulation of anthocyanin biosynthesis genes [START_REF] El-Kereamy | Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries[END_REF]. A number of factors and signals influence the accumulation of anthocyanins and the expression of related genes, including photochrome and light, hormones (gibberellins, methyl jasmonate), and various stresses such as wounding and low temperature [START_REF] Mol | Signal perception, transduction, and gene expression involved in anthocyanin biosynthesis[END_REF]. Environmental conditions and orchard management, including irrigation, pruning, and fertilization, are also known to strongly impact on fruit coloration.

Aroma volatiles contribute strongly to the overall sensory quality of fruit and vegetables. Extensive studies have been focused on the identification of volatile compounds, and to the elucidation of some of the biosynthetic routes either by bioconversion or by tracing of precursors [START_REF] Sanz | Aroma biochemistry of fruits and vegetables[END_REF][START_REF] D'auria | Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri[END_REF][START_REF] Dudareva | Biochemistry of plant volatiles[END_REF]). In recent years, research efforts have been directed toward the isolation of the corresponding genes in fruits and vegetables [START_REF] Aharoni | Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays[END_REF][START_REF] Yahyaoui | Molecular and biochemical characteristics of a gene encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening[END_REF][START_REF] Beekwilder | Functional characterization of enzymes forming volatile esters from strawberry and banana[END_REF][START_REF] El-Sharkawy | Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity[END_REF]Pech et al. 2008b). Aroma is generally a complex mixture of a wide range of compounds. Each product has a distinctive aroma, which is function of the proportion of the key volatiles, and the presence or absence of unique components. The most important classes of aromas are monoterpenes, sesquiterpenes, and compounds derived from lipids, sugars, and amino acids. Ethylene is known to control the rate of ripening, the duration of storage life, and most of the ripening events in climacteric fruit. Therefore, breeders have "incidentally" reduced ethylene synthesis or action by generating genotypes with extended shelf life. Because many genes of aroma biosynthesis are ethylene-regulated [START_REF] El-Sharkawy | Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity[END_REF]Manriquez et al. 2006), this has often resulted in a severe loss of flavor in long-keeping genotypes that have commonly been generated by breeding with non-ripening mutants [START_REF] Mcglasson | Influence of the non-ripening mutants rin and nor on the aroma of tomato fruit[END_REF][START_REF] Aubert | Investigation of volatiles in Charentais cantaloupe melons (Cucumis melo var. cantalupensis). Characterization of aroma constituents in some cultivars[END_REF]. One major challenge for the future is to uncouple the down-regulation of ethylene from inhibition of aroma volatile production.

Molecular Markers and QTL Mapping of Fruit Ripening Traits

The advent of genetic approaches based on quantitative trait loci (QTLs) opens new prospects toward genetic improvement of fruit. Indeed, most fruit quality traits are under multigenic control, and the QTL approach allows the localization on genetic maps of loci responsible for at least part of the phenotypic variation, and enables the quantification of their individual effects. Because of the low molecular polymorphism observed in cultivated tomato, which is usually used as model species in fruit research, the majority of these studies [START_REF] Tanksley | Seed banks and molecular maps: unlocking genetic potential from the wild[END_REF][START_REF] Causse | QTL analysis of fruit quality in fresh market tomato: a few chromosome regions control the variation of sensory and instrumental traits[END_REF] rely on interspecific progeny. Surprisingly, in spite of their characteristics inferior to those of cultivated species, wild species can possess alleles useful for improving fruit traits. A good example is given by a QTL improving fruit color, detected in a Solanum habrochaites (Lycopersicon hirsutum), a green-fruited species. The molecular markers localized in the vicinity of this QTL are now being used in marker-assisted selection to create parent lines with increased potential, or in contrast, to avoid certain unfavorable traits [START_REF] Fulton | Quantitative trait loci (QTL) affecting sugars, organic acids and other biochemical properties possibly contributing to flavor, identified in four advanced backcross populations of tomato[END_REF]. A fruit weight QTL, common to several studies, has been precisely localized and then cloned by chromosome walking [START_REF] Frary | fw2.2: a quantitative trait locus key to the evolution of tomato fruit size[END_REF]. Another QTL controlling sugar concentration in fruit has also been cloned [START_REF] Fridman | A recombination hotspot delimits a wild species QTL for tomato sugar content to 484-bp within an invertase gene[END_REF], and the gene responsible for this QTL has been shown to encode a cell wall invertase [START_REF] Fridman | Zooming in on a quantitative trait for tomato yield using interspecific introgressions[END_REF]).

As emphasized above, the climacteric character represents an important determinant of the ripening rate and storability. Because genetically compatible climacteric and non-climacteric types of melon are available, it has been possible to study the inheritance of the climacteric character. A segregating population resulting from a cross between a typical climacteric-type Charentais melon (Cucumis melo var. cantalupensis cv. Ve´drantais) and a non-climacteric melon, Songwhan Charmi PI 161375 (Cucumis melo var. chinensis), has been generated and used to study the segregation of the formation of the abscission layer (Al) of the peduncle and ethylene production [START_REF] Pe ´rin | Molecular and genetic characterisation of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit[END_REF]. It was found that the climacteric character was controlled by two duplicated independent loci (Al-3 and Al-4), and the intensity of ethylene production was controlled by at least four QTLs localized in other genomic regions. None of the QTLs matched with known genes of the ethylene biosynthetic or transduction pathways. Recently, it was reported that some introgression lines generated from two non-climacteric melons, Piel de Sapo (var. inodorus) and Songwhan Charmi PI 161375 (var. chinensis) possessed a climacteric character [START_REF] Obando | Creating climacteric melon fruit from non-climacteric parentals: postharvest implications[END_REF]). The QTLs associated with ethylene production and respiration rate in this work have not been mapped at the same position as the Al loci described by Pe ´rin et al. (2002). Collectively, these data suggest that different and complex genetic regulation exists for the climacteric character.

Improvement of fruit quality arose in some cases randomly, like in the apple, where a chance seedling, Golden Delicious, was discovered with good agronomic characters. It has been crossed with old apple varieties having good sensory attributes to generate new apple cultivars that combine good agronomic and good sensory characters [START_REF] Vaysse | Recognizing apple and pear varieties. Centre Technique Interprofessionnel des Fruits Le ´gumes[END_REF]. Similarly, the poor-keeping qualities of Delicious have been improved by crossing with long-keeping apples (Rall's Janet), giving rise to the Fuji group of apples [START_REF] Vaysse | Recognizing apple and pear varieties. Centre Technique Interprofessionnel des Fruits Le ´gumes[END_REF]. Likewise, in Charentaistype melons, long or mid-shelf life commercial genotypes are available. Some of these have been generated using a non-ripening melon named "Vauclusien". However, the long shelf life character is often associated with poor sensory qualities [START_REF] Aubert | Investigation of volatiles in Charentais cantaloupe melons (Cucumis melo var. cantalupensis). Characterization of aroma constituents in some cultivars[END_REF]. Low ethylene production is generally correlated with long storage life. The delayed ripening of these genotypes was found to result in alteration of ethylene biosynthetic or response genes. The amount of ethylene in ripening Fuji apples parallels the transcript levels of the ripening-specific ACS gene, MdACS1 [START_REF] Harada | An allele of the 1aminocyclopropane-1-carboxylate synthase gene accounts for the low level of ethylene production in climacteric fruits of some apple cultivars[END_REF]. An allele of this gene (MdACS1-2) contains an insertion of a retro-transposon-like sequence in the 5 0 -flanking region, and is transcribed at a lower level than the wild-type allele MdACS1-1. Cultivars that are homozygous for the MdACS1-2 allele have low ethylene production and long storage life [START_REF] Sunako | An allele of the ripening-specific 1-aminocyclopropane-1-carboxylate synthase gene (ACS1) in apple fruit with a long storage life[END_REF]. Two ERF genes (MdERF1 and MdERF2) have been isolated from ripening apple fruit. The MdERF1 gene has been shown to express predominantly in ripening fruit, and MdERF2 exclusively in ripening fruit [START_REF] Wang | MdERFs, two ethylene-response factors involved in apple fruit ripening[END_REF]. Expression of both genes was repressed by treatment with 1-MCP. Apple cultivars with low ethylene production had a tendency to show lower expression of these two MdERF genes than those with high ethylene production. By screening different cantaloupe melons, [START_REF] Zheng | Ethylene production, shelf-life and evidence of RFLP polymorphisms linked to ethylene genes in melon (Cucumis melo L.)[END_REF] reported a correlation between ethylene production and post-harvest decay. In addition, using ACO cDNA probes, they were able to demonstrate that low ethylene production was associated with the presence of an RFLP ACO allele Ao, whereas high ethylene production was associated with the Bo allele in homozygous conditions [START_REF] Zheng | Genetics of ethylene biosynthesis and restriction fragment length polymorphism (RFLPs) of ACC oxidase and synthase genes in melon (Cucumis melo L.)[END_REF].

Amongst climacteric fruits, there are genetic differences in the capacity to induce the ripening process. The most striking case is given by fruit cultivars that require exposure to post-harvest low temperatures for ripening. Some winter pear varieties, such as D'Anjou, Beurre Bosc, and Passe Crassane, require chilling temperatures for the induction of autocatalytic ethylene production [START_REF] Blankenship | Development of ethylene biosynthesis and ethyleneinduced ripening in D'Anjou pears during the cold requirement for ripening[END_REF][START_REF] Morin | Conse ´quences d'un se ´jour au froid sur le me ´tabolisme de l'e ´thyle `ne au cours de la maturation de la poire Passe Crassane apre `s re ´colte[END_REF][START_REF] Knee | Development of ethylene biosynthesis in pear fruit at -1 C[END_REF]). Furthermore, it has been reported that the cold-requirement character can be transmitted by breeding, as exemplified by crossing of Passe-Crassane pears and a cold-independent variety, Old Home, to give a mixed population of cold-dependent and cold-independent hybrids [START_REF] El-Sharkawy | Differential regulation of ACC synthase genes in cold-dependent and -independent ripening in pear fruit[END_REF]. Cold requirement appears to be linked to the possibility of inducing ethylene biosynthesis genes. In Passe Crassane pears, a 3-month chilling treatment at 0 C strongly stimulated ACC oxidase activity, and to a lesser extent, ACC synthase activity [START_REF] Lelie `vre | Ethylene and fruit ripening[END_REF]. It has been shown that the presence of some ACS alleles was correlated with the chilling requirements for ripening, and with the induction of autocatalytic ethylene production [START_REF] El-Sharkawy | Differential regulation of ACC synthase genes in cold-dependent and -independent ripening in pear fruit[END_REF]).

Natural Mutants Affected in the Ripening Phenotype

Among the reasons why tomato has emerged as a model species for studying fleshy fruit development is the presence of well-characterized, spontaneous mutants or wild-allele variants that have been recovered from production fields or breeding programs. A number of genes corresponding to various mutations have been isolated by positional cloning [START_REF] Giovannoni | Fruit ripening mutants yield insights into ripening control[END_REF]. The first ripening-impaired mutant to be characterized at the molecular level is Never-ripe (Nr), which bears a dominant mutation that affects the ethylene response, and results in fruit producing reduced amounts of ethylene and retaining very low ethylene responsiveness [START_REF] Lanahan | The Never ripe mutation blocks ethylene perception in tomato[END_REF]. It was shown that the NR gene encodes an ethylene receptor from the ERS family devoid of receiver domain [START_REF] Wilkinson | An ethylene inducible component of signal transduction encoded by Never-ripe[END_REF]. The Green-ripe (Gr) mutant corresponds also to a dominant ripening mutation lying in a gene encoding a new component of ethylene signaling [START_REF] Barry | Ripening inhibition in the tomato Green-ripe mutant results from ectopic expression of a novel protein which disrupts ethylene signal transduction[END_REF], corresponding to the Reversion To Ethylene Sensitivity1 (RTE1) shown to interact and regulate the ETR1 ethylene receptor in Arabidopsis [START_REF] Resnick | REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis[END_REF][START_REF] Zhou | RTE1 is a Golgi-associated and ETR1-dependent negative regulator of ethylene responses[END_REF].

One of the tomato mutations most commonly used by the breeders affects the transcriptional control of fruit ripening. The ripening-inhibitor (rin) mutation is a recessive mutation that blocks the ripening process, and prevents ethylene production and responsiveness. In the last decade, the rin locus has been widely used for generating long shelf life commercial varieties. The rin mutation encodes a MADS box-type transcription factor that is present in both climacteric and nonclimacteric fruit [START_REF] Vaysse | Recognizing apple and pear varieties. Centre Technique Interprofessionnel des Fruits Le ´gumes[END_REF], suggesting that it probably acts upstream of the climacteric switch. The Colorless non-ripening (Cnr) mutant is a dominant mutant corresponding to an epigenetic mutation that alters the methylation of the promoter of a SPB box transcription factor [START_REF] Manning | A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[END_REF]. Although it has been proposed that both rin and cnr act upstream of ethylene production [START_REF] Giovannoni | Fruit ripening mutants yield insights into ripening control[END_REF], the location of these two transcription factors in the ripening regulatory network is not clear.

A number of other mutants affect the fruit composition in terms of secondary metabolites. Because of the ease of visual screening, most of the mutants affected in fruit composition are altered in pigment accumulation. The color change from green to red associated with ripening in tomato results from both chlorophyll degradation and carotenoid pigment accumulation. The numerous tomato mutants affected in pigmentation represent a valuable genetic resource, which has been exploited to facilitate the identification of the genes involved in carotenoid biosynthetic pathways, and understanding the complex mechanisms regulating pigment accumulation [START_REF] Bramley | Regulation of carotenoid formation during tomato fruit ripening and development[END_REF]. The role of light has been reported in the regulation of fruit pigmentation [START_REF] Giovannoni | Molecular biology of fruit maturation and ripening[END_REF]. The yellow-flesh (r) mutation that results in the absence of carotenoid accumulation corresponds to a deletion within the ethyleneregulated phytoene synthase-1 gene [START_REF] Fray | Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression[END_REF]. The delta mutant displays an orange color resulting from the accumulation of d-carotene at the expenses of lycopene [START_REF] Tomes | Delta-carotene in the tomato[END_REF], due to a dominant mutation within the CrtL-e gene encoding a lycopene e-cyclase [START_REF] Ronen | Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is downregulated during ripening and is elevated in the mutant Delta[END_REF]. The Beta (B) partially dominant mutation also results in orange color, due to the accumulation of b-carotene instead of lycopene. The gene responsible for the B mutation encodes a fruit-and flowerspecific lycopene, b-cyclase, capable of converting lycopene into b-carotene. Its expression is strongly increased in the B mutant [START_REF] Ronen | An alternative pathway to b-carotene formation in plant chloroplasts discovered by map-based cloning of beta and old-gold color mutations of tomato[END_REF]. Deep-red fruit of old-gold and old-gold-crimson mutants are null mutations of an allele of the B gene [START_REF] Ronen | An alternative pathway to b-carotene formation in plant chloroplasts discovered by map-based cloning of beta and old-gold color mutations of tomato[END_REF]. Tangerine is a recessive mutation conferring orange color by accumulation of pro-lycopene instead of normal lycopene. It corresponds to an impairment of the expression of a carotenoid isomerase gene that is suspected to enable carotenoid biosynthesis in the dark, and in non-photosynthetic tissues [START_REF] Isaacson | Cloning of tangerine from tomato reveals a carotenoid isomerise essential for the production of b-carotene and xanthophylls in plants[END_REF]. The hp1 and hp2 mutants exhibiting elevated content of flavonoid and carotenoid are mutated in Damaged DNA Binding Protein1 [START_REF] Liu | Manipulation of light signal transduction as means of modifying fruit nutritional quality in tomato[END_REF], and Detiolated1 [START_REF] Mustilli | Phenotype of the tomato high pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1[END_REF]) genes, respectively. The corresponding genes in Arabidopsis encode nuclear-localized light signal transduction proteins.

Conclusions and Future Directions

While tremendous progress has been made in understanding the mechanisms of fruit ripening, a number of questions remain unanswered. In climacteric fruit, amongst the major issues that remain to be addressed are the role of hormones other than ethylene, and the way in which they interact with ethylene signaling to control different aspects of fruit ripening. The mechanism by which ethylene selects specific ripening-regulated genes is another important topic that needs to be investigated. In non-climacteric fruit, the detailed mechanisms that regulate the ripening process remain largely unknown, although molecular data are accumulating. So far, the regulation of gene expression during fruit ripening has been viewed mostly at the transcriptional level. Recent studies on the ethylene receptor [START_REF] Kevany | Ethylene receptor degradation controls the timing of ripening in tomato fruit[END_REF]) illustrate that post-transcriptional regulation plays an essential role, and deserves more attention. Regulation of gene expression by epigenetic variations is now recognized as an important determinant of plant development. Epigenetic variations do not affect the primary DNA sequence, but consist of DNA methylation or histone modifications that affect gene expression generally at the level of chromatin organization. In fruit, the Cnr mutation is the only well-characterized, natural and stably inherited epigenetic mutation [START_REF] Seymour | Genetics and epigenetics of fruit development and ripening[END_REF]). Research efforts are now being directed toward understanding the epigenetic regulation of the fruit ripening process. It is predictable that the answers to these questions will require cooperation between fruit physiologists, molecular biologists, and geneticists.

The recent development of high-throughput technology for analyzing genome structure and functions is starting to have an impact on fruit research. A number of national and multinational programs are attempting to combine genomics, proteomics, metabolomics, and reverse genetic approaches to unravel the molecular mechanisms of fruit development [START_REF] Wang | Regulatory features underlying pollination-dependent and -independent tomato fruit set revealed by transcript and primary metabolite profiling[END_REF]. The implementation of these genome-wide [START_REF] Alba | ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development[END_REF]) and metabolomic technologies [START_REF] Overy | Application of metabolite profiling to the identification of traits in a population of tomato introgression lines[END_REF], together with bioinformatics tools, is expected to provide new understanding of the fruit developmental program, and reveal the networks of interactions between different pathways leading to the accumulation of fruit quality traits. The most important programs are being implemented on the tomato model species. A multinational consortium has been established recently, which has made available centralized facilities for tomato ESTs and derived DNA chips [START_REF] Mueller | The SOL Genomics Network: a comparative resource for Solanaceae biology and beyond[END_REF]. This is enabling the elucidation of global changes in gene expression during fruit development and ripening, and researchers to mine and analyze the expression profiling data in order to cluster the complete set of genes involved in specific metabolic and regulatory mechanisms. By comparing differences between natural variants, ripening mutants, or introgression lines, genes will be identified that are essential for specific aspects of fruit ripening, with their corresponding impact on fruit metabolism [START_REF] Fei | Comprehensive EST analysis of tomato and comparative genomics of fruit ripening[END_REF][START_REF] Overy | Application of metabolite profiling to the identification of traits in a population of tomato introgression lines[END_REF]. In addition, reverse genetics approaches for highthroughput functional identification of target genes are being developed, amongst which the emerging TILLING (targeting induced local lesions in genomes) technology is most promising. The completion of the tomato genome sequencing project, and the availability of the tomato genome sequence in the near future will represent a major breakthrough likely to change our understanding in the area of the fundamentals of fruit growth and development, and open new avenues to address the varied topics in fruit research.

  Fig. 16.2 Schematic representation describing the expression of ethylene biosynthesis and ethylene perception genes during the transition to climacteric in tomato. System 1 refers to preclimacteric ethylene production, and System 2 to climacteric autocatalytic ethylene production. LeACS, Lycopersicon esculentum ACC synthase; LeACO, Lycopersicon esculentum ACC oxidase; LeETR and NR, ethylene receptors. Eth+ and Eth-refer to the stimulation and repression, respectively of gene or protein expression (adapted from[START_REF] Barry | Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato[END_REF][START_REF] Barry | The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF][START_REF] Kevany | Ethylene receptor degradation controls the timing of ripening in tomato fruit[END_REF]

Table 16 .

 16 1 A list of representative climacteric and non-climacteric fruit. A more extensive list is provided by[START_REF] Watkins | Ethylene synthesis: mode of action, consequences, and control[END_REF]

	Climacteric fruits	Non-climacteric fruits
	Apple (Malus domestica Borkh.)	Asian pear (Pyrus serotina Rehder)
	Apricot (Prunus armeniaca L.)	Cactus pear (Opuntia amyclaea Tenore)
	Avocado (Persea americana Mill.)	Carambola (Averrhoa carambola L.)
	Banana (Musa sapientum L.)	Cashew (Anacardium occidentale L.)
	Cherimoya (Annona cherimola Mill.)	Cherry (Prunus avium L.)
	Corossol (Annona muricata L.)	Cucumber (Cucumis sativus L.)
	Durian (Durio zibethinus Murr.)	Grape (Vitis vinifera L.)
	Feijoa (Feijoa sellowiana Berg.)	Grapefruit (Citrus grandis Osbech)
	Fig (Ficus carica L.)	Lime (Citrus aurantifolia Swingle)
	Guava (Psidium guajava L.)	Limon (Citrus limonia Burm.)
	Kiwifruit (Actinidia sinensis Planch.)	Litchee (Litchi sinensis Sonn.)
	Mango (Mangifera indica L.)	Mandarin (Citrus reticulata Blanco)
	Melon Cantaloup and Honeydew (Cucumis melo L.) Mangoustan (Garcinia mangostana L.)
	Papaya (Carica papaya L.)	Olive (Olea europaea L.)
	Passion fruit (Passiflora edulis Sims.)	Orange (Citrus sinensis Osbeck)
	Peach (Prunus persica Batsch)	Pepper (Capsicum annuum L.)
	Pear (Pyrus communis L.)	Pineapple (Ananas comosus Merr.)
	Persimmon (Diospyros kaki Thunb.)	Pomegranate (Punica granatum L.)
	Physalis (Physalis peruviana L.)	Rambutan (Nephelium lappaceum L.)
	Plum (Prunus domestica L.)	Raspberry (Rubus idaeus L.)
	Sapota (Manilkara achras Fosb.)	Strawberry (Fragaria sp.)
	Tomato (Solanum lycopersicum L.)	Tamarillo (Cyphomandra betacea Sendtu)
		Watermelon (Citrullus lanatus Mansf.)

Auxin, Light…) Nucleus Ripening-related genes EIL Ethylene synthesis Taste (Sugars, acid, tannin…) Aroma Color Respiration Cell wall degradation Membrane degradation Autocatalysis Primary targets Signal amplification and diversification ERE SIGNAL TRANSDUCTION ARF Aux/IAA ERF3 GCC ERFn ERF1 ERF2 TRANSCRIPTIONAL REGULATION TGTCTC

  . ERFs are known to be the last actors of the ethylene signaling pathway,

	Ethylene	Other Hormone and non-hormone signals
		(e.g.

Acknowledgments The authors greatly acknowledge the financial support of the IFCPAR-CEFIPRA Indo-French programme (Grant 3303-02).