For some of the processes associated with ripening in tomato, for example carotenoid biosynthesis, the major steps in the pathway have been identified and current understanding of their role is based on more than twenty years of transgenic experiments with single ripening-related genes. More recently, advances in quantitative genetics are beginning to complement these earlier experiments and are allowing a more comprehensive approach for linking genotype and phenotype. Flavour development is discussed extensively in a recent review by Klee and Giovannoni (2011), so here we have focused on the two other key quality attributes: colour and texture.
Chloroplast to chromoplast conversion influences not only fruit colour and associated health-promoting properties of fruits, but also their nutrient and flavour composition, as carotenoids, lipids and branched chain amino acids synthesized in the plastid can form precursors of volatile flavour compounds (Klee and Giovannoni, 2011). At the onset of ripening, the thylakoids, which contain the photosynthetic pigments, disassemble and chlorophyll degradation is one of the earliest observable signs of ripening in tomato and many other fruits. The thylakoid lipids then form plastoglobuli, where carotenoid pigments accumulate. Chromoplast metabolic activity is associated with the biogenesis of carotenoids, many other secondary metabolites, amino acids and fatty acids (Klee and Giovannoni, 2011).
Carotenoid formation has been studied extensively in tomato, and this fruit has become the model system to investigate the underlying biochemistry and molecular biology. All the major steps in the pathway have been elucidated and are reviewed elsewhere (Cazzonelli and Pogson, 2010). They can be summarized as follows. The formation of phytoene is the first committed step in carotenoid biosynthesis and is dependent on the catalytic activity of phytoene synthase (PSY). Phytoene then undergoes two desaturation reactions to form ζ-carotene, catalysed by phytoene desaturase (PDS), which in turn is desaturated to neurosporene and finally lycopene. This carotenoid accumulates in tomato fruit, providing the characteristic red colour. Lycopene can then be cyclized at both ends of the molecule consecutively by lycopene β-cyclase (LYCB) to form β-carotene or cyclized at one end by LYCB and at the other by lycopene ε-cyclase (LYCE) to form α-carotene. These cyclic carotenoids can then be converted to xanthophylls. During tomato ripening, the concentration of carotenoids increases between 10- and 14-fold, due mainly to the accumulation of lycopene (Fraser et al., 1994).
Current understanding of the regulation of carotenoid biosynthesis in fruits, and in plants in general, is incomplete. Links to light signalling and chromatin remodelling were mentioned earlier with respect to genes underlying the hp1 and hp2 mutations, but a detailed picture of the connections between the ripening regulatory network and the carotenoid biosynthetic pathway has remained elusive. However, the sequencing of the tomato and other fruit genomes, transcriptome profiling and network inference, combined with the use of chromatin immunoprecipitation experiments, is beginning to reveal aspects of these molecular circuits. For example, in tomato, the RIN gene product has been demonstrated to be associated with various ripening-related regulatory sequences upstream of PSY1 (Fujisawa et al., 2011; Martel et al., 2011). These regulatory sequences may also bind other ripening-related transcription factors, for example ERFs (Osorio et al., 2011). PSY is under strong ethylene control, as is β-cyclase and the cyclase genes (LCY-b and LCY-e) are strongly down-regulated during ripening, thus preventing lycopene cyclization. Thus, both PSY1 and β-cyclase may be under RIN/ERF control. Understanding the control of carotenoid biosynthesis is important not only for enhancing fruit colour and the associated health benefits, but also for flavour traits because carotenoids are also metabolized during ripening to flavour volatiles (Goff and Klee, 2006).
Fruit texture and shelf life
Texture is an important driver of consumer preferences for fruits: a consumer preference study reported that tomato fruit characteristics that were most disliked were a soft or ‘mealy’ texture, along with poor flavour characteristics such as sour, or lacking aroma (Sinesio et al., 2010). Texture not only affects consumer preference, but also has a significant impact on shelf life and transportability. Understanding the key factors that influence texture and ripening-related softening has therefore been a priority from a horticultural and commercial standpoint. However, this has proved to be far more challenging to unravel than initially expected, primarily because texture reflects many factors, including cell wall structure, cuticle properties, cellular turgor and fruit morphology (Vicente et al., 2007). The best studied of these are the multitude of cell wall changes that are apparent to varying degrees during ripening in essentially all fleshy fruit. Tomato has represented the primary experimental model to elucidate cell wall modification during ripening, as illustrated by an analysis that the authors have performed as part of the recently published work on the tomato genome sequence and ripening-related transcriptome (The Tomato Genome Consortium, 2012).
Plant cell walls are highly complex hydrated matrices, largely comprising a variety of polysaccharide networks and several types of structural glycoproteins, in addition to some minor components including ions and numerous enzymes (Cosgrove, 2005; Lee et al., 2004, 2011b). While the basic classes of wall polymers are common to most cell walls, variation is seen between cell types, such as lignin accumulation in secondary walls of vascular tissue, and structural lipid polymers of the cuticle in epidermal cells, as well as taxonomically (Bonawitz and Chapple, 2010; Burton et al., 2010; Popper et al., 2011; Schreiber, 2010). In dicots, such as tomato, current models suggest that a framework of cellulose microfibrils, coated and cross-linked with the hemicellulose xyloglucan, is embedded within a gel of pectins, including the polymers homogalacturonan (HG), rhamnogalacturonan I and rhamnogalacturonan II. The tomato fruit cell wall is probably the best studied with respect to changes during ripening (Brummell and Harpster, 2001; Seymour et al., 1990), but the precise roles of most of the polysaccharide and glycoprotein components are still poorly understood (McCann and Rose, 2010). Initial models suggested that one or two enzymes, such as polygalacturonase (PG), which can hydrolyse the pectin backbone of HG polymers de-esterified by pectin methylesterase (PME), might play the major role in controlling texture changes in tomato (Brummell, 2006; Giovannoni, 2001) because of the substantial changes in the levels of activity of these enzymes during the ripening process. However, experiments where genes encoding these and other wall remodelling proteins have been silenced in transgenic tomato fruits have not supported this hypothesis. These experiments indicated that although small effects on fruit softening can be achieved by individual gene knockdowns (Brummell and Harpster, 2001; Powell et al., 2003), substantial changes in fruit texture are likely to require the simultaneous modulation of multiple such genes.
The tomato genome sequence contains more than 700 gene models annotated as having cell wall–related functions, of which around 90 have been characterized to varying degrees (Tomato Genome Consortium, 2012; Supplementary Section). Our initial analysis showed that more than 50 of these genes show differential expression during fruit ripening and encode proteins involved in the modification of wall architecture (Figure 2). At the onset of ripening, there is a substantial decrease in the expression of at least four cellulose synthase genes and also those encoding a variety of glycosyl hydrolases. This is in contrast to genes annotated as xyloglucan endotransglucosylase hydrolases (XTHs) (Saladié et al., 2006), where ten members of this family show a ripening-related burst of expression. These new data suggest a more important role for members of the XTH family in ripening-associated cell wall changes than previously suspected.
Figure 2. Selection of the most highly expressed genes encoding proteins involved in cell wall remodelling and degradation in tomato. Expression at mature green (green bar), breaker (yellow bar) and ripe (red bar). Each gene is labelled with its published name where known or its Solyc chromosome address (www.solgenomics.net). Cel, cellulase; FLA, Fasciclin-like arabinogalactan protein; PL, pectate lyase; TBG, beta-galactosidase; PME, pectin methylesterase; EXP, expansin; SlXTH, xyloglucan endotransglucosylase/hydrolase; PG, polygalacturonase; CS, cellulose synthase; LeMAN, beta-mannosidase (Data from Tomato Genome Consortium, 2012).
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Analyses of the tomato genome sequence and gene expression patterns in ripening fruit have supported earlier findings related to genes mainly, or exclusively, expressed in fruit tissue (Tomato Genome Consortium, 2012). Several pectin remodelling enzymes, including PMEs, show fruit-related expression, and they are all down-regulated prior to the full onset of ripening. PME enzymes act to remove methyl groups from the pectic polysaccharides in a blockwise or more random fashion, depending on the gene family member and possibly on the cell wall environment (Jolie et al., 2010). PMEs may act synergistically with PGs, with PME action resulting in the generation of HG substrates that are more susceptible to PG-mediated hydrolysis. However, PME can also potentially act to strengthen the cell wall by facilitating calcium cross-linking between adjacent pectin molecules and therefore cell-to-cell adhesion. This idea was supported by studies of the tomato PMEU1 gene, which encodes the PME1 isoform, the transcript of which is expressed highly at the mature green stage of fruit development, while levels decline substantially at the onset of ripening (Phan et al., 2007). Gene silencing experiments revealed that the loss of PMEU1 expression leads to an enhanced rate of softening during ripening, which suggests that PME action contributes to maintaining fruit firmness, although it is important to note that this possibly reflects an indirect effect. The temporal expression of this PMEU1 gene is similar to others in that the highest levels are at MG or at least prior to ripening (Figure 2), suggesting that a family of such proteins may act in concert, although it may be that there are differences in substrate specificity or other enzyme properties.
In addition to pectin-degrading enzymes, many other cell wall–related structural genes show changes in expression during ripening (Figure 2). A ripening-related increase in α-mannosidase expression has been reported previously in tomato, and it is likely to be involved in the processing of N-glycans present in cell wall–related glycoproteins. Recent transgenic experiments (Meli et al., 2010) show that suppression of the gene enhances fruit shelf life, but the precise mechanism of action is unclear. Furthermore, five members of the arabinogalactan cell surface glycoproteins (AGPs) family showed a substantial decrease in transcript abundance just prior to the onset of ripening. The role of these glycoproteins in tomato fruit ripening is unknown, but in other tissues, they have been proposed to function in cell wall cross-linking or as pectin plasticizers (Ellis et al., 2010).
Water relations, cuticles and fruit softening
In addition to the structural matrices of the cell wall, another important contributor to texture and fruit firmness is cellular turgor. This in turn is governed by the water status within fruit and the relative distribution of water within the cell (the symplast) and in the cell wall (the apoplast). The factors that influence fruit turgor during development and ripening are poorly understood, and indeed, its importance as a target for extending shelf life and generally enhancing texture has long been neglected. However, some recent studies are starting to reveal some of the molecular pathways and structures that are likely involved. The most apparent of these is the cuticle, the waxy layer comprising the polyester cutin and a variety of waxes, which covers the aerial surface of land plants (Jeffree, 2006; Nawrath, 2006). A major function of the cuticle is to limit water loss and desiccation, which would also lead to a loss of cellular turgor and consequently a reduction in tissue biomechanical strength. In fruits, this would be apparent as ‘shrivelling’ during postharvest storage, or as a consequence of fruit cracking. An association between fruit firmness, shelf life and cuticle structure and composition was suggested through the characterization of the delayed fruit deterioration (DFD) tomato, whose fruit show remarkably prolonged resistance to postharvest desiccation and remain firm for many months after harvest (Saladié et al., 2007). The polysaccharide cell walls of DFD fruits appear to undergo the same extensive disassembly during ripening as is seen in normally softening fruits and loss of cell–cell adhesion also occurs to the same extent. However, analysis of the DFD cuticles revealed substantial differences in the amount of cutin as well as of some waxes. The relative contributions of cutin and waxes to resisting transpirational water loss are still not well resolved, but tomato fruit makes an excellent model to address this question as, unlike most plant cuticles, it is astomatous and far thicker than typical leaf cuticles (Buda et al., 2009; Isaacson et al., 2009). Analyses of cutin-deficient tomato mutants suggest that cutin, the major component of cuticles, does not make the most important contribution to limiting water movement across the cuticle and that waxes are relatively more important, while cutin is important in resisting microbial infection (Isaacson et al., 2009). However, the specific molecular components of the cuticle that are the primary determinants of water movement have yet to be determined.
Many other questions remain regarding factors that influence water loss, including the potential role of aquaporin proteins during ripening in regulating the passage of water from the symplast to the apoplast, and the importance of apoplastic solutes in determining the osmolarity of the apoplastic fluid, which would presumably also affect water movement out of the cell. Research into fruit water relations and cuticle biology is likely to be important in the development of strategies to extend shelf life and improve fruit texture traits, as will an improved understanding of the interplay between water relations, cell wall biology and the molecular status of the apoplast.