‘Movers and shakers’ in the regulation of fruit ripening: a cross-dissection of climacteric versus non-climacteric fruit

Abstract

Fruit ripening is a complex and highly coordinated developmental process involving the expression of many ripening-related genes under the control of a network of signalling pathways. The hormonal control of climacteric fruit ripening, especially ethylene perception and signalling transduction in tomato has been well characterized. Additionally, great strides have been made in understanding some of the major regulatory switches (transcription factors such as RIPENING-INHIBITOR and other transcriptional regulators such as COLOURLESS NON-RIPENING, TOMATO AGAMOUS-LIKE1 and ETHYLENE RESPONSE FACTORs), that are involved in tomato fruit ripening. In contrast, the regulatory network related to non-climacteric fruit ripening remains poorly understood. However, some of the most recent breakthrough research data have provided several lines of evidences for abscisic acid- and sucrose-mediated ripening of strawberry, a non-climacteric fruit model. In this review, we discuss the most recent research findings concerning the hormonal regulation of fleshy fruit ripening and their cross-talk and the future challenges taking tomato as a climacteric fruit model and strawberry as a non-climacteric fruit model. We also highlight the possible contribution of epigenetic changes including the role of plant microRNAs, which is opening new avenues and great possibilities in the fields of fruit-ripening research and postharvest biology.

Abscisic acid, climacteric fruit, ethylene, epigenetics, fruit ripening, non-climacteric fruit, strawberry, tomato.

Introduction

Fleshy fruits form an integral part of the human diet and serve as an important source of vitamins, minerals, antioxidants, sugars, and fibre content. The nutritional quality attributes of fruits depend largely on the ripening stage, and an optimum ripeness is advocated for consumption (Symons et al., 2012). Fruit ripening is a complex and genetically controlled process involving changes in colour, texture, flavour, and aroma (Klee and Giovannoni, 2011; Seymour et al., 2013a, b; Shen et al., 2014). Although these changes are advantageous in increasing the palatability, over-ripening may also lead to pathogen attack and cause problems in postharvest handling (storage and marketing) of fruit commodities.

Based on the physiological differences in respiratory pattern during ripening, fleshy fruits have been categorized as climacteric and non-climacteric. Climacteric fruits such as tomato, banana, apple, mango, and kiwi display a well-characterized peak in respiration with a concomitant burst of ethylene at the onset of ripening (Abdul Shukor et al., 1990; Giovannoni, 2004; Kondo et al., 2009; Atkinson et al., 2011; Xu et al., 2012). In contrast, non-climacteric fruits, which include strawberry, melon, and grape, do not show a dramatic change in respiration, and ethylene production remains at a basal level (Bapat et al., 2010; Chai et al., 2011; Symons et al., 2012). Notably, a large body of research data has been accumulated on ethylene regulation of ripening process in climacteric fruits (Cara and Giovannoni, 2008; Bapat et al., 2010; Kondo et al., 2009; Atkinson et al., 2011; Xu et al., 2012). When compared with climacteric fruits, much less research data are available on the hormonal regulatory mechanisms that operate in non-climacteric fruits during ripening. Recent studies have suggested that abscisic acid (ABA) plays an important role in the regulation of ripening-related gene expression in strawberry fruits via perception and signal transduction pathways (Chai et al., 2011; Jia et al., 2011; 2013a).

In this review, we discuss the most recent research findings concerning the genetic and molecular aspects of fleshy fruit ripening. We also highlight some of the important findings related to epigenome modifications and their regulatory mechanisms in fleshy fruit development and ripening.

The physiology and biochemistry of fruit ripening: a brief account

Both climacteric and non-climacteric fruits experience a wide range of developmental changes during the course of their development from ovary to mature fruit. Fruit maturity ensures ripening-related changes associated with colour, fruit softening, and textural changes, increase in specific volatiles, and alterations in the sugar/acid balance (Brummell, 2006; Saladie et al., 2007; Figueroa et al., 2008, 2010, 2012; Handa et al., 2011). Starch and sugars (sucrose, glucose, and fructose) form important carbohydrates in fruits, and changes in their ratio during ripening ultimately determine the fruit quality, especially of fruit sweetness. Accumulation of sugars during ripening has been reported in various fruits (Lo Bianco and Rieger, 2002; Fait et al., 2008; Osorio et al., 2012). In tomato, the dominant soluble metabolites are sugars and organic acids (Grierson and Kader, 1986; Roessner-Tunali et al., 2003). Starch is a transitory carbohydrate reserve, accumulating during early fruit development and then declining to undetectable levels in the ripe fruits (Schaffer and Petreikov, 1997).

Many fruits accumulate organic acids such as citric, malic, and ascorbic acids during ripening. Although the ratio of organic acids to sugar is an important quality attribute at harvest time, their study has received much less attention to date (Osorio and Fernie, 2013). Among organic acids, malate and citrate are the main acids in many climacteric and non-climacteric fruits (Mounet et al., 2009; Osorio et al., 2012; Merchante et al., 2013). It is important to note that malate accumulation and degradation do not follow the typical classification into climacteric or non-climacteric fruits or the overall changes in respiration rates. Certain types of climacteric fruits use malate during the respiratory burst, while others continue accumulating malate throughout ripening (Goodenough et al., 1985; Kortstee et al., 2007). It has been proposed that malate metabolism is important for transitory starch metabolism in normal tomato fruit development (Centeno et al., 2011). A positive correlation between malate levels and the genes involved in starch synthesis was observed in pepper, suggesting a possible conservation of the transitory starch metabolism between climacteric and non-climacteric fruits (Osorio et al., 2012).

Apart from sugars and organic acids, a bewildering array of secondary metabolites such as vitamins, volatiles, flavonoids, and pigments are also been implicated during fruit ripening. Although excellent reviews detailing these metabolites and their interrelationships are available (Bouzayen et al., 2010; Handa et al., 2011; Klee and Giovannoni, 2011; Osorio and Fernie, 2013), it is worth mentioning the two key quality attributes of colour and texture briefly in this review.

The thylakoid disassembly of photosynthetic pigments leading to chlorophyll degradation was reported as one of the earliest changes associated with the onset of fruit ripening in many fruits including tomato (Fraser et al., 1994; Bramley, 2002; Grassi et al., 2013). Carotenoids are isoprenoid molecules that are common to all photosynthetic tissues, and their biosynthesis and regulation occur during fruit ripening alongside the conversion of chloroplast to chromoplast (Bramley, 2002; Grassi et al., 2013). Understanding the regulatory control of carotenoid biosynthesis is important in genetic manipulation studies pertaining to enhancement in fruit quality and colour, especially of lycopene content and their health benefits (Seymour et al., 2013a). Carotenoids are also reported to be metabolized during ripening to flavour volatiles (Goff and Klee, 2006). An increment in carotenoid content between 10- and 14-fold during tomato ripening due to the accumulation of lycopene was reported (Fraser et al., 1994). A recent integrative study combining carotenoid profiles and whole-genome transcriptome analysis has provided insights in to the synthesis and accumulation of carotenoids and the genes involved during four successive stages (corresponding to the white, white-pink, pink, and red-ripe colour of the fruit flesh) of fruit development and ripening in watermelon (Grassi et al., 2013). The data from this study suggested that maintenance of many regulators in the watermelon genome is common with tomato, yet suggest a complex and different regulatory system for isoprenoid biosynthesis between these fruits, possibly revealing different ripening physiologies of climacteric and non-climacteric fruits.

Fruit textural change during ripening is an essential attribute that makes fruit edible, more palatable, and attractive for human consumption as well for vectors of fruit dispersal. Ripening-associated textural changes are complex in nature and usually involve modifications to the cell-wall components, especially the polysaccharides and proteins (Brummell, 2006; Vicente et al., 2007; Negi and Handa, 2008; Osorio et al., 2012). Fruit firmness is also determined by a number of other factors such as cuticle properties, turgor, and free radicals (Chaib et al., 2007; Vicente et al., 2007; Handa et al., 2011). More than 700 gene models annotated in the tomato genome sequence showed cell-wall-related functions and more than 50 of these genes were reported to show differential expression during fruit ripening and to encode proteins involved in the modification of cell-wall properties (Seymour et al., 2013a). Cuticle, the waxy coating layer made up of polyester cutin, and a variety of waxes help prevent or limit water loss from the aerial surface of plant organs (Nawrath, 2006). The recent characterization of a ‘Delayed Fruit Deterioration’ (DFD) tomato cultivar by Saladie et al. (2007) revealed the association between cuticle, shelf-life, and fruit firmness, as these fruits showed prolonged resistance to postharvest desiccation unlike normal fruits. Although the contributions of cutin and waxes to limiting water loss are not well understood, analysis of the DFD fruit cuticle revealed substantial differences in the amount of cutin and waxes (Saladie et al., 2007).

Hormonal control of fruit development and ripening in climacteric fruits

Ethylene biosynthesis genes

In plants, 1-aminocyclopropane-1-carboxylic acid (ACC) is the precursor of ethylene and is formed from the amino acid methionine (Met). Met is S-adenosylated to form S-adenosylmethionine (SAM) by the enzyme SAM synthetase (Fluhr and Mattoo, 1996). SAM is subsequently metabolized to ACC and 5ʹ-methylthioadenosine by ACC synthase (ACS). 5ʹ-Methylthioadenosine is then recycled back to Met by the Met or Yang cycle for another round of ethylene biosynthesis (Van de Poel et al., 2012). ACC is further converted to ethylene by ACC oxidase (ACO). The activities of ACS and ACO enzymes are considered as the rate-limiting steps in ripening-related ethylene production (Cara and Giovannoni, 2008), and are encoded by multigene families. It has been proposed that specific members of the ACS and ACO gene families control two systems of ethylene production in plants, system-1 and system-2 (Lelievre et al., 1997). In tomato, system-1 is autoinhibitory, where perception of ethylene by the plant inhibits further ethylene production or its biosynthesis as found in unripe fruits and vegetative tissues (Barry and Giovannoni, 2007). In contrast, system-2 is autocatalytic, where perception of ethylene stimulates ethylene synthesis as displayed during floral senescence and fruit ripening (Nadeau et al., 1993; Klee and Clark, 2004; Barry and Giovannoni, 2007; Yokotani et al., 2009).

Five different isoforms of ACO have been identified in tomato and are differentially expressed during fruit development and ripening. The ACO1 and ACO4 genes show moderate expression in system-1, while ACO3 is transiently expressed during the transition phase. A 7-fold increase in ACO1 and ACO4 expression that peaked during the orange stage under system-2 has been reported (Barry et al., 1996; Nakatsuka et al., 1998). A total of nine ACS genes were identified in tomato (ACS1A, ACS1B, and ACS2–ACS8) and among them, five are expressed during fruit ripening. During system-1, ACS1A and ACS3 are constitutively expressed at a low level, whereas ACS6 shows a higher expression (Nakatsuka et al., 1998; Barry et al., 2000). ACS2 and ACS4 are highly expressed during system-2 (Nakatsuka et al., 1998; Barry et al., 2000). Therefore, in tomato and other climacteric fruits, ACS genes play an important role in the transition from system-1 to system-2. However, in plum, in addition to the differential regulation of four members of the ACC synthase gene family (PsACS), it is likely that ethylene-overproducer 1 (ETO1)-like, PsEOL1, is also involved (El-Sharkawy et al., 2008). System-1 ethylene synthesis continues throughout plum fruit development until the fruit attains a state of physiological maturity and the transition occurs at this point (62–77 d after bloom). The transition from system-1 to system-2 is induced throughout, and the induction and/or repression of unknown ethylene-independent factors could contribute for the ripening competency of the plum fruit. It has been shown that the PsACS1 and PsACS3a transcripts were expressed during this transition period in an ethylene-independent manner but in an auxin- and/or gibberellin-dependent manner (El-Sharkawy et al., 2008). The existence of ethylene-independent pathways (Alba et al., 2000; Flores et al., 2001; El-Sharkawy et al., 2009) coupled with the co-existence of ethylene-dependent pathways might therefore coordinate the ripening process in climacteric fruits (Pech et al., 2008).

Autocatalytic ethylene synthesis triggers a transcriptional cascade that regulates the expression of many genes involved in quality attributes including softening, textural, and colour changes as well as enhanced flavour and aroma (Atkinson et al., 2011). It has been shown that blocking ethylene synthesis by inhibitors of ethylene perception such as aminoethoxyvinylglycine and 1-methylcyclopropene (1-MCP) delays ripening and the associated respiratory rise. This treatment has now become a widely accepted methodology for manipulation and regulation of fruit ripening and senescence, especially of climacteric fruits (Saltveit, 2005; Lu et al., 2013; Nock and Watkins, 2013). The recent targeted systems biology profiling approach in tomato has revealed a novel regulatory mechanism for ethylene biosynthesis and involves coordination among key enzymes such as 5ʹ-methylthioadenosine nucleosidase, 5ʹ-methylthioribose kinase, and acireductone dioxygenase during post-climacteric tomato ripening (Van de Poel et al., 2012). When compared with the regulation in detached fruits, the post-climacteric red tomato on the plant showed only a moderate regulation of ACC synthase and these key enzymes. Furthermore, treatment of red fruits with 1-MCP and ethephon revealed that the shut-down mechanism in ethylene biosynthesis is developmentally programmed and only moderately ethylene sensitive. It is proposed that the termination of autocatalytic ethylene biosynthesis of system-2 in ripe fruits delays senescence and preserves the fruits until seed dispersal (Van de Poel et al., 2012).

Alternative oxidase (AOX) is encoded by a small nuclear gene family and branches from the cytochrome c oxidase pathway at the level of ubiquinone (Xu et al., 2012). To gain insights into the role played by AOX, and its relationship with ethylene in tomato fruit ripening, transgenic tomato plants 35S-AOX1a and 35S-AOX-RNAi were generated (Xu et al., 2012). In this study, although the AOX-silenced fruits did not show ethylene or respiration bursts, they could reach the red stage, indicating that the climacteric is indispensable for ripening to occur in tomato. Interestingly, the 1-MCP-treated AOX-RNAi fruits failed to ripen. Hence, it is likely that both AOX and ethylene contribute to fruit ripening and that inhibition of both AOX and ethylene is required to halt tomato ripening completely (Xu et al., 2012). Furthermore, the expression analysis of ripening genes such as NEVER-RIPE (NR), COLOURLESS NON-RIPENING (CNR), RIPENING-INHIBITOR (RIN), and NONRIPENING (NOR) showed reduced accumulation of NR and CNR mRNA in 1-MCP-treated AOX-RNAi fruits (Xu et al., 2012).

Ethylene perception and signal transduction

Ethylene is perceived by receptors encoded by a multigene family, the ethylene receptors (ETRs) in Arabidopsis. Advancements in isolation and characterization of ETRs for the first time in the model plant Arabidopsis have paved the way towards understanding their role in fruit ripening, especially of tomato. In tomato, the ETR family consists of seven members, namely LeETR1–7, of which LeETR3 is responsible for the NR mutation (Wilkinson et al., 1995; Zhou et al., 1996; Lashbrook et al., 1998; Klee 2002). The LeETR4–6 genes were reported to be expressed abundantly in reproductive tissues (flowers and fruits) and less in vegetative tissue (Tieman and Klee, 1999). Furthermore, downregulation of LeETR4 led to early fruit ripening in tomato but without affecting the fruit size, yield, or flavour-related chemical composition (Kevany et al., 2008). ETRs are membrane proteins associated with the endoplasmic reticulum and act as negative regulators of the ethylene response pathway (Seymour et al., 2013a) (Fig. 1).

Fig. 1.

The hormonal control (ethylene- and ABA-mediated signalling cascades) of fruit ripening in tomato (green arrows) and strawberry (red arrows). In tomato, the current model of ethylene action suggests that, in the absence of hormone, receptors signal to the negative regulator CTR1 and the response pathway is blocked (top left). Binding of ethylene by the receptors releases the negative regulator allowing the ethylene response to occur (top right). The ethylene binding to ETRs blocks its interaction with CTR releasing the inhibition on EIN2. The activated EIN2 moves to the nuclear membrane and transduces EIN3 and EIN3-like (EIL) proteins. This may lead to the binding of EIN3 and EILs to ethylene response elements present in the promoter region of ethylene response factors (ERFs) by an unknown mechanism, and in turn activate the expression of ripening-related (ethylene-response) genes. Hormones such as auxin/indole acetic acid/AOX may also be involved in this process (dashed green arrows). In strawberry, ABA-mediated signalling cascades follow an ABA–ABAR/CHLH or ABA–PYR1–PP2C–SnRK2 pathway. The latter can function in the nucleus, in which the complex may directly regulate ABA-responsive gene expression by phosphorylation of ABF/AREB-type transcription factors. It is possible that the binding of ABA to FaPYR1 or FaCHLH/ABAR receptors could activate SnRK2 and other ripening-related genes in strawberry fruit. The PP2C protein is a negative regulator of fruit ripening (shown as a tilted-T line). In green strawberry fruit, the relatively low levels of ABA and the resultant high levels of PP2C expression could block signal transduction, whereas in red fruit, high ABA levels could result in relatively low levels of PP2C expression and may evoke ABA signal transduction and thus promote ripening-related genes such as SnRK2, PG1, CHS, ANS, UFGT, and others. Sucrose may function as a signal to promote fruit ripening by stimulating ABA biosynthesis through FaNCED1 (dashed red arrows). However, the functionality of ABFs/AREBs not yet been proved in strawberry (dashed oval shape). ER, endoplasmic reticulum; NM, nuclear membrane; CHL, chloroplast; Suc, sucrose; Glc, glucose; Fru, fructose. ER of this figure was produced using Servier Medical Art (www.servier.com).

The onset of fruit ripening increases LeETR4, LeETR6, and NR expression significantly and these three receptor genes are by far the most highly expressed in ripening fruits. The effect of receptors on ethylene sensitivity has been explained by the negative regulation model. This model predicts that presence of more receptor reduces and less receptor increases ethylene sensitivity (Klee and Giovannoni, 2011). Kevany et al. (2007) used specific antibodies against each of the three abundantly expressed receptors and showed that ethylene binding triggers receptor protein degradation. Although transcriptional activation of genes encoding the receptors occurs during ripening, the actual receptor protein levels significantly drop and remain low throughout ripening. Therefore, it is ethylene binding that keep receptors in a functionally ‘off’ state by receptor protein degradation and is probably mediated via the 26S proteasome-dependent pathway.

In the signalling pathway, it has been reported that the negative regulation of ethylene response by the ethylene receptors work through the action of another gene, Constitutive Triple Response1 (CTR1), which acts downstream of ETR (Kieber et al., 1993). CTR1 encodes a Raf-like Ser/Thr kinase that physically interacts with ETR (Clark et al., 1998). In tomato, four CTR homologues (SlCTR1–4) have been isolated and all homologues with the exception of SlCTR2 complemented the ctr1 mutations in Arabidopsis, with SlCTR1 being the most actively expressed during fruit ripening (Adams-Phillips et al., 2004; Lin et al., 2008). ETHYLENE INSENSITIVE2 (EIN2) has been suggested to transduce the ethylene response to downstream transcription factors such as ETHYLENE INSENSITIVE3 (EIN3). EIN3 and EIN3-like proteins (EILs) belong to a family of DNA-binding proteins and bind to ethylene response elements (EREs) and are involved in the regulation of ethylene-sensitive genes (Solano et al., 1998; Tieman et al., 2001; Yokotani et al., 2009). Ethylene response factors (ERFs) such as ERF1 are plant transcriptional regulators and act downstream of EIN3, and its constitutive expression activates a variety of ethylene-response genes in Arabidopsis (Solano et al., 1998; Yu et al., 2004). It could mediate the ethylene response via binding to a conserved motif, AGCCGCC (GCC-box), located in the promoter region of ethylene-regulated genes (Handa et al., 2011). Furthermore, the ethylene response transduced by EIN2 to downstream transcription factors such as EIN3 and other EILs could activate an ethylene-regulated MAP kinase (transcriptional) cascade (Roman et al., 1995).

The molecular events concerning EIN2 activation of the transacting protein EIN3, EILs and ERFs that regulate the downstream ethylene response genes is not yet fully understood (Handa et al., 2011). However, it has been reported that, in the absence of ethylene, ETR binds to CTR1, which then interacts with EIN2 and inactivates the downstream signals of the ethylene response, and, conversely, ethylene binding to ETR eliminates CTR-regulated suppression of the ethylene signalling pathway (Fig. 1). Therefore, the current models for the mechanism of ethylene perception describe the receptors in a functionally ‘on’ state in the absence of ethylene and conversion to an ‘off’ state in the presence of ethylene, permitting ethylene signalling to proceed (Klee and Giovannoni, 2011; Seymour et al., 2013a) (Fig. 1).

The regulatory network in climacteric fruit ripening

Transcription factor genes as master switches in climacteric fruit ripening

In tomato, the phenotypes rin, nor, and Cnr represent three non-ripening pleiotropic mutants. In these mutants, all aspects of the ripening process (e.g. increased climacteric rise in respiration, carotenoid accumulation, fruit softening, and aroma production) typical in climacteric fruit ripening were found to be inhibited. Although the autocatalytic ethylene synthesis is blocked in these mutants (Barry and Giovannoni, 2007), further experimental studies have indicated that they retain the capacity to synthesize wound-related ethylene, and exogenous ethylene could also partially regain ethylene-regulated gene expression (Barry et al., 2000). Therefore, it is suggested that rin, nor, and Cnr mutants represent ‘lesions in master switches’ and act upstream of ethylene in the ripening cascade that determines the competency of fruit to ripen (Barry and Giovannoni, 2007). Positional cloning revealed that the rin and Cnr loci encode different classes of transcription factors (Vrebalov et al., 2002; Manning et al., 2006). Vrebalov et al. (2002) reported that two MADS-box genes, namely, MADS-RIN and MADS-MC, are associated with the rin locus. The MADS-RIN ectopic expression complemented mutation in the rin fruits, while its downregulation using an antisense gene imparted a non-ripening phenotype similar to the rin mutation, whereas MADS-MC affected flowering characteristics such as sepal and inflorescence development (Vrebalov et al., 2002). The gene at the Cnr locus encodes an SBP-box gene for a transcription factor that probably interacts with the promoter of MADS-box genes (Manning et al. 2006).

Molecular characterization of tomato fruit-ripening regulator RIN and the rin mutant protein has shown that RIN protein accumulates in ripening fruits and is localized in the nucleus of the cell (Ito et al., 2008). Further in vitro analysis has revealed that RIN forms a stable homodimer that binds to MADS domain-specific DNA sites,and the binding sites of RIN highly resemble the Arabidopsis MADS-box protein SEPALLATA (SEP) that controls the identity of floral organs (Ito et al., 2008). The RIN mutant protein that accumulates in mutant fruits exhibits DNA-binding activity similar to wild-type protein but has lost its transcription/activation function and this could account for the inhibition of ripening in mutant fruits (Ito et al., 2008).

The recent chromatin immunoprecipitation approach has provided evidence that MADS-RIN interacts not just with cell-wall metabolism and ethylene synthesis-related gene promoters but also with ethylene signalling, the ethylene response, and carotenoid synthesis genes, in addition to promoters of other ripening-related transcription factors (Martel et al., 2011). Thus, RIN acts as a master regulator of ripening and directly influences many ripening-associated processes via a mechanism that is dependent on the presence of a functional CNR gene. Furthermore, enrichment of ACS2 and ACS4 gene promoters following RIN chromatin immunoprecipitation analysis has indicated that RIN associates with their respective promoters. HB-1 is an HD-zip transcription factor that positively controls the expression of the ethylene-producing enzyme ACO1 during fruit development and ripening (Lin et al., 2008). Although ACO1 is not directly targeted by RIN, the promoter of the ACO regulator, HB-1, is a direct target of RIN and therefore could indirectly influence ACO1 expression (Martel et al., 2011) (Fig. 2A). A similar study by Fujisawa et al. (2011) also confirmed that RIN binds to the CArG boxes in the promoters of the genes involved in cell-wall modification (PG, LeEXP1, TBG4, and LeMAN4) and system-2 ethylene biosynthesis (LeACS2 and LeACS4) both in vivo and in vitro (Fig. 2B). Moreover, the finding that RIN binds to its own promoter suggests the presence of autoregulation for RIN expression (Fujisawa et al., 2011). Taken together, it has been suggested that RIN acts as a master switch that controls fruit softening and ethylene production by the direct transcriptional regulation of the cell-wall-modifying genes and ACS genes, respectively, in tomato (Fig. 2A, B).

Fig. 2.

Schematic representation of the ripening-related transcription factors that influence ethylene-mediated fruit ripening in tomato. (A) The regulatory network involves the combined action of gene products from RIN, CNR, and NOR loci. RIN and TAGL1 are represented as master switches (boxed area) that influence ethylene synthesis by directly binding to the ACS2 and ACS4 promoters, while HB-1 is involved in the direct regulation of ACO1 and further signal transduction to downstream effectors of colour, texture, taste, and smell. Transcription factors like AP2 act as a negative regulator of ripening (shown by the tilted-T line. (B) A model showing the RIN transcription factor as a major switch that regulates downstream target genes. Representation of RIN binding to novel CArG- box promoter sequences of target genes such as PG, TBG4, and LeEXP1 among others is shown. (C, D) The control of epigenetic factors such as methylation that influence tomato fruit ripening. (C) In immature fruits without viable seeds, ripening does not occur and promoter regions of key ripening genes are hypermethylated (coloured ovals) so that the RIN and CNR transcription factors cannot bind and therefore there is no transcription (represented by crossed arrow). (D), In maturing wild-type fruits, the promoters are demethylated (uncoloured ovals) by an unidentified mechanism but influenced by RIN and CNR, leading to transcription. It is ambiguous that whether transcription factor binding facilitates promoter hypomethylation or hypomethylation facilitates transcription factors binding, or whether both influence the fruit transition into a ripening-competent state (represented by both the single-ended and double-ended arrow heads and a question mark) (Zhong et al., 2013).

A recent study confirmed the direct binding of RIN to promoters of five target genes, namely E8, TomloxC, PNAE, PGK, and ADH2, in rin mutant tomato fruits (Qin et al., 2012). Furthermore, TomloxC and ADH2 encoding lipoxygenase (LOX) and alcohol dehydrogenase, respectively, were found to be crucial in the production of the characteristic tomato aroma compounds, and the loss of function of RIN caused deregulation of the LOX pathway and thereby a specific defect in the production of aromatic compounds via the LOX pathway. These findings suggest that RIN modulates aroma formation by the direct regulation of gene expression in the LOX pathway (Qin et al., 2012). Similarly, the transcriptome and in silico promoter analysis of a total of 112 genes showed their downregulation in rin mutant fruits, although the expression patterns of these genes were found to be similar to that of LeMADS-RIN in wild-type fruits. In silico analysis of putative promoters of these genes for the presence of the CArG box along with ERE and ethylene inducibility revealed that genes lacking the CArG box in their promoter regions were indirectly regulated by LeMADS-RIN (Kumar et al., 2012). This study hence provides further insights into the LeMADS-RIN-directed ethylene-dependent as well as ethylene-independent regulation of tomato fruit ripening. Overall, the observed ethylene-independent aspect of ripening suggests that the RIN, NOR, and CNR proteins are candidates for conserved molecular mechanisms in both climacteric and non-climacteric fruit ripening (Osorio and Fernie, 2013).

Many other master switches controlling the ripening process have been identified including the MADS-box genes TDR4 and TAGL1(Itkin et al., 2009; Vrebalov et al., 2009; Jaakola et al., 2010; Seymour et al., 2011). The FRUITFULL (FUL) and SHATTERPROOF (SHP) genes belong to the MADS-box family and in the model plant Arabidopsis control the development and dehiscence of siliques (dry fruit) (Dinneny et al., 2005). Many of these MADS-box transcription factors influence ethylene synthesis by directly binding to ACS2 and ACS4 gene promoters as in the case of RIN and TAGL1. The tomato TDR4 and TAGL1 genes are probably orthologues of FUL and SHP (Seymour et al., 2013a). TAGL1 is involved in the normal ripening of tomato and contributes to ‘fleshiness’, while the role of TDR4 is not yet identified (Itkin et al., 2009; Vrebalov et al., 2009). However, silencing the bilberry version of TDR4 dramatically altered the anthocyanin accumulation in bilberry (Vaccinium myrtillus) fruits (Jaakola et al., 2010). The precise molecular mechanism by which the gene products encoded by RIN, NOR, CNR, TAGL1, and HB-1 operate in the regulatory network remains elusive (Seymour et al., 2013a). However, it is likely that MADS-box proteins act as heterodimers or multimers (Giovannoni, 2007). Additionally, other components of the regulatory network, including transcription factors such as AP2 (acts as a negative regulator) and SIAP2a (belonging to the AP2/ERF superfamily of transcription factors and acting downstream of RIN, NOR, and CNR) are reported to be the major regulators of ripening (Chung et al., 2010; Karlova et al., 2011) (Fig. 2A).

Although most of the transcription factors known to regulate ripening have been identified from tomato, MADS-box genes have also been reported to be involved in the development of many other climacteric (peach, banana, apple, and oil palm) and non-climacteric (grape, bilberry, and strawberry) fruit crops (Yao et al., 1999; Boss et al., 2002; Tadiello et al., 2009; Elitzur et al., 2010; Jaakola et al., 2010; Seymour et al., 2011; Tranbarger et al., 2011; Daminato et al., 2013). Traditionally, non-climacteric fruits were considered as a separate group that did not follow a typical climacteric ripening pattern. However, the identification of MADS-box genes in both climacteric- and non-climacteric fruits suggests that at least some aspects of fruit ripening are shared between these two categories of fruit (Daminato et al., 2013). Recent genome sequence data along with comparative genomic analysis carried out in tomato and hot pepper indicate that the expression of transcription genes (RIN, TAGLI, and NOR) and genes involved in ethylene signalling pathways (NR, ETR4, EIN2, and EIL families) are well conserved during fruit ripening in both fruit types (Kim et al., 2014). However, CNR, Uniform (Golden-like), and HB-1 showed distinct expression patterns in hot pepper and tomato (very low levels of CNR expression in hot pepper, while tomato showed very high expression). Interestingly, it was found that the ethylene biosynthesis genes, especially ACS2, ACS4, and ACO1 were expressed at very low levels in hot pepper. This suggests that the conservation and divergence of the transcription of these genes and their interactions may ultimately determine the physiological differences involved in fleshy fruit ripening (Kim et al., 2014). It is worth mentioning that fleshy fruits themselves are botanically diverse and exhibit sharp differences in fruit morphology and structure. These structural differences may also reflect differences in their ripening mechanisms. Some of these aspects will be discussed in the later part of this review.

Role of biogenic amines (polyamines) in relation to climacteric fruit ripening

Polyamines (putrescine, spermidine, and spermine) are a group of ubiquitous nitrogenous cellular constituents that are essential for cell division and viability, and can influence a myriad of growth and development processes, including fruit ripening and abiotic stress responses in plants (Cherian et al., 2006; Handa and Mattoo, 2010; Kausch et al., 2012). In plants, putrescine is the direct substrate for spermidine and spermine and is synthesized from arginine, a reaction catalysed by arginine decarboxylase or from ornithine by ornithine decarboxylase. Spermidine synthase catalyses spermidine synthesis from putrescine and decarboxylated SAM (dcSAM). The formation of dcSAM from SAM by SAM decarboxylase is a committed and rate-limiting step in the polyamine pathway (Handa and Mattoo, 2010) and has the potential to commit the flux of SAM either into polyamine biosynthesis, ethylene biosynthesis, or both (Osorio and Fernie, 2013). Recent studies employing reverse genetics approaches have provided direct evidence for the involvement of polyamines in fruit ripening (Srivastava et al., 2007; Handa and Mattoo, 2010; Kausch et al., 2012). Kausch et al. (2012) generated transgenic tomato with reduced endogenous methyl jasmonate (MeJA) levels by silencing a fruit ripening-associated LOX, SILoxB, using a truncated LOX gene under the control of the cauliflower mosaic virus 35S promoter. These transgenic fruits were evaluated by nuclear magnetic resonance spectroscopy for the effects of reduced MeJA on cellular metabolites and it was found that MeJA reduction significantly affected overall primary metabolism, especially the aminome (amino acids and polyamines) of ripening fruits. Transgenic tomato plants overexpressing the yeast spermidine synthase gene were shown to have longer fruit shelf-life and delayed decay symptoms during storage (Nambeesan et al., 2010).

Fruit development and ripening in strawberry: the non-climacteric fruit model

Role of ethylene

In contrast to climacteric fruits, less progress has been made in understanding the regulatory mechanisms of fruit ripening in non-climacteric fruits. Strawberry has been widely accepted as a model system for the non-climacteric fruit category, as these fruits do not exhibit a peak in respiration and also the external application of ethylene to green strawberry fruits does not affect the rate of ripening (Given et al., 1988). However, recent experimental evidence suggests that ethylene does play some role in the ripening of non-climacteric fruits such as grape, melon, and strawberry (Chervin et al., 2004; Trainotti et al., 2005; McCollum and Maul, 2007; Pech et al., 2008; Villarreal et al., 2010; Merchante et al., 2013) and may therefore share some elements of the downstream components that are normally associated with ethylene signal transduction (Lee et al., 2010).

Despite its presence in comparatively low concentrations, a characteristic pattern of ethylene production has been reported during different developmental stages of strawberry fruits, such that it is high in green fruits and decreases in white fruits, and finally increases again at the red stage of ripening (Perkins-Veazie et al., 1996; Iannetta et al., 2006). Two ACO genes (FaACO1 and FaACO2) and three ETR genes of type I (FaEtr1 and FaErs1) and type II (FaEtr2) have been reported in strawberry (Trainotti et al., 2005). The pattern of expression of strawberry ACO and related genes associated with ethylene production has been studied recently by various authors (Trainotti et al., 2005; Merchante et al., 2013; Sun et al., 2013). It has been shown that both FaACO1 and FaACO2 genes exhibit high expression in flowers and a decreasing rate of expression in developing young fruits. FaACO1 showed an expression increment from the large green to the white stage, followed by a continuous decrease to reach minimum levels in the red fruits. In contrast, FaACO2 expression was minimal during the white stage of the fruit but thereafter displayed a continuous low increment throughout ripening, suggesting that only a little ethylene is required to trigger ripening-related physiological responses in strawberry (Trainotti et al., 2005).

The two key ethylene biosynthesis genes (ACS and ACO) and their expression patterns during strawberry fruit ripening was found to be developmental and organ specific (achene or receptacle), with the highest expression values in green achenes (FaACS3, FaACS4, and FaACO3) (Merchante et al., 2013). The highest expression of FaACS1 occurred during the green/white stage of active cell expansion and just before changes associated with ripening took place. In the climacteric fruit model (tomato), LeACS1a and LeACS6 are associated with early ethylene production, while LeACS2 and LeACS4 are associated with the climacteric production of ethylene (Cara and Giovannoni, 2008). The protein sequence comparing FaACS1 with LeACS2 and LeACS4 suggests a certain parallel between ripening in the strawberry receptacle with that of the tomato fruit (Merchante et al., 2013). Correlated with the increase in ethylene synthesis in the red-ripe strawberry fruit, there is an increase in the expression of the ETR FaEtr2, which is similar to tomato LeETR4 (Trainotti et al., 2005). A combined analysis of the ethylene biosynthesis-related gene FaSAMS1 and the signalling gene FaCTR1 in Fragaria×ananassa (cv. Camarosa) fruit has provided some new genetic evidence for the role of ethylene in strawberry ripening. Downregulation of the FaSAMS1 or FaCTR1 transcript via the tobacco rattle virus-induced gene silencing technique not only inhibited the development of fruit red colouring and firmness but also affected ethylene levels and the accumulation of several ethylene signalling components, thus demonstrating that FaCTR1 positively regulates strawberry fruit ripening. Furthermore, ethephon treatment could significantly promote natural red development in white fruits and partially rescued both FaCTR1-RNAi and FaSAM1-RNAi associated with fruit anthocyanin biosynthesis (Sun et al., 2013). Taken together, these results suggest that ethylene is required for the normal development of the strawberry fruit, in which it acts differentially in achenes and the receptacle, i.e. in an organ-specific manner. Also, the inhibition of some aspects of strawberry fruit ripening by the ethylene perception inhibitor 1-MCP further points to a role for ethylene in non-climacteric fruit ripening (Villarreal et al., 2010). Recent comparative transcriptome and metabolome studies have revealed that both climacteric (tomato) and non-climacteric (pepper) fruit share similar ethylene-mediated signalling components. However, the regulation of these genes is clearly different in pepper and may reflect regulators other than ethylene (Lee et al., 2010; Osorio et al., 2012). The ethylene biosynthesis genes in climacteric tomato (ACC synthase and ACC oxidase) were not induced in pepper; however, as in tomato, genes downstream of ethylene perception, such as cell-wall metabolism genes, ERF3, and carotenoid biosynthesis genes, were upregulated during fruit ripening (Osorio et al., 2012; Kim et al., 2014).

The observation that an increase in expression of ethylene receptors together with the expression of downstream signalling elements in non-climacteric fruits supports the concept that climacteric and non-climacteric fruits possibly share, although minimally, a common regulatory pathway modulated by ethylene. Mesifurane is an important contributor to strawberry fruit aroma and is controlled by the gene O-methyl transferase (FaOMT). FaOMT promoter sequence analysis revealed the presence of ABA response elements (Zorrilla-Fontanesi et al., 2012). The results from an analysis of transgenic etr-1-1 strawberry lines showed the downregulation of FaOMT as well as the ETR genes (FaETR, FaETR2, FaERS1), especially of the receptacle tissue (Merchante et al., 2013), and therefore a possible connection could be established between ABA and ethylene sensitivity in strawberry fruit ripening.

ABA and sucrose as major regulators of strawberry fruit ripening

ABA is a versatile phytohormone that regulates a broad range of plant traits such as adaptation of plants to adverse conditions, seedling growth, seed dormancy, and fruit development (Hirayama and Shinozaki, 2007; Bastias et al., 2011; Li et al., 2011). To date, two core ABA signalling pathways have been proposed in Arabidopsis, particularly the ABA–PYR/PYL/RCAR-type 2C protein phosphatase (PP2C)–SNF1-related protein kinase 2 (SnRK2) (Fujii et al., 2009) and ABA–ABAR–WRKY40–ABI5 (Shang et al., 2010) pathways. In PYR1–PP2C–SnRK2 pathway, ABA promotes the interaction of PYR1 and PP2C, resulting in PP2C inhibition and SnRK2 activation leading to the transduction of ABA signals through the phosphorylation of downstream factors such as AREB/ABF bZIP-type transcription factors (Fujii et al., 2009; Umezawa et al., 2010). In the other model, ABA stimulates the ABAR–WRKY interaction and relieves the ABI5 gene of inhibition by repressing WRKY40 expression (Shang et al., 2010). The gate–latch–lock mechanism of ABA signal perception and transduction involves three major components: PYR/PYL/RCAR perception, PYR1–PP2C interaction, and inhibition of PP2C activity and activation of SnRK2 (Fujii et al., 2009; Umezawa et al., 2010). In the absence of ABA, PP2C inactivates SnRK2 by direct dephosphorylation, and in response to environmental and/or developmental cues, ABA promotes the interaction of PYR/PYL/RCAR and PP2C, resulting in PP2C inhibition and SnRK2 activation (Umezawa et al., 2010).

For more than 30 years, ABA has been implicated as an inducer of strawberry fruit ripening (Kano and Asahira, 1981). ABA treatment accelerates fruit colour and softening, ethylene production, and increased activity of phenylalanine ammonia-lyase (PAL) enzyme (Jiang and Joyce, 2003). It was even hypothesized that the ABA:auxin ratio could be part of the signal that triggers strawberry fruit ripening (Archbold and Dennis, 1984). Recent evidence shows that ABA promotes sugar metabolism and accumulation in non-climacteric grape berry (Pan et al., 2005), and that 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme involved in ABA biosynthesis, is related to the ripening of several fruits, including grape berry (Zhang et al., 2009a) and tomato (Zhang et al., 2009b). A significant reduction in SlNCED1 activity leading to a decline in the transcription of genes encoding major cell-wall catabolic enzymes was reported suggesting that ABA may affect cell-wall catabolism during tomato fruit ripening (Sun et al., 2012).

ABA levels were found to be increased in the receptacle during strawberry fruit development, and the exogenous application of ABA and DMSO (an ABA accelerator) promoted fruit (receptacle) development, whereas fluoridone (an ABA biosynthesis inhibitor) notably inhibited development, suggesting that ABA accelerates fruit ripening (Jia et al., 2011). A gene homologous to the Arabidopsis ABA receptor gene ABAR, namely the H subunit of magnesium chelatase CHLH (Shen et al., 2006), was isolated from strawberry and named FaCHLH/ABAR (Jia et al., 2011). A rapid increase in soluble sugars, especially of sucrose after the onset of fruit ripening was related to fruit degreening and red colouring in strawberry. Furthermore, it has been shown that treatments with glucose and sucrose both increased FaNCED1 mRNA abundance and the levels of ABA, suggesting a cross-talk between sucrose signal and ABA biosynthesis in strawberry fruit ripening (Jia et al., 2011) (Fig. 1). However, downregulation of the FaCHLH/ABAR gene decreased the soluble sugar (sucrose, glucose, and fructose) levels, suggesting that repression of the FaCHLH/ABAR-mediated ABA signalling pathway may affect ABA-induced sucrose accumulation (Jia et al., 2011). The expression analysis of a set of ABA-responsive genes in FaCHLH/ABAR-RNAi fruits revealed upregulation of a negative signalling regulator (ABI1) and downregulation of the positive ABA signalling regulators (ABI3, ABI4, ABI5, and SnRK2) and the sugar metabolism-/pigment biosynthesis-related genes (SigE, AMY, and CHS), further supporting the positive role for FaCHLH/ABAR receptor-mediated ABA signalling in strawberry fruit ripening (Jia et al., 2011) (Fig. 1).These data therefore provide evidence that the two signalling molecules (sugar and ABA) may cooperatively interact to regulate strawberry fruit ripening and that this correlation could be a core mechanism in the regulation of non-climacteric fruit ripening (Gambetta et al., 2010; Li et al., 2011) (Fig. 1).

Structural biology provides a detailed gate–latch–lock mechanism involved in ABA signal perception and transduction, including ABA/PYR1 perception, PYR1–PP2C interaction, inhibition of PP2C activity, and activation of SnRK2 (Fujii et al., 2009). Chai et al. (2011) cloned a strawberry gene homologous to PYR1 (pyrabactin resistance 1 gene), the initial ABA receptor identified in Arabidopsis (Melcher et al., 2009), and provided strong evidence about the role of FaPYR1 in strawberry fruit ripening. The expression of the FaPYR1 gene shows an up–down–up pattern during fruit development, suggesting that FaPYR1 is mainly involved in early strawberry fruit growth and later reddening (Chai et al., 2011). The loss of red colouring in FaPYR1-RNAi fruit is not rescued by the exogenously applied ABA, while ABA could promote the ripening of the wild-type fruits (Chai et al., 2011). ABI1, ABI3, ABI4, ABI5, and SnRK2 genes were downregulated in FaPYR1-RNAi fruit, which may lead to the suppression of ABA responses and, in turn, destroy ABA-responsive physiological events such as fruit reddening. In addition, certain downregulated ABA signalling-derived events may serve as feedback signals to regulate ABA levels, since ABA accumulates at rather high levels in FaPYR1-downregulated RNAi fruits compared with control fruits (Chai et al., 2011).

The gene ABI1 of strawberry, encoding a Ser/Thr protein phosphatase PP2C1, exhibited an expression pattern that rapidly declined during strawberry fruit development, suggesting a negative role of this gene in fruit ripening (Jia et al., 2013a). Plant PP2Cs are reported to be encoded by a large multigene family with 80 members in Arabidopsis (Xue et al., 2008). In the case of tomato fruit, transcriptional analysis suggested that tomato SlPYL1, SlPYL2, SlPP2C1, SlPP2C5, and SnRK2.3 may be involved in the regulation of fruit ripening (Sun et al., 2011). The mRNA expression levels of FaABI1 were remarkably high in small green fruits, and declined rapidly during fruit development in strawberry, and finally remained at notably low levels at the full red stage (Jia et al., 2013a). In addition, silencing and overexpression of FaABI1 led to the promotion of red colour development but inhibited fruit ripening. Several ripening-related genes of F.×ananassa fruits like the anthocyanin biosynthesis genes such as chalcone syntase (FaCHS), chalcone isomerase (FaCHI), flavanone 3-hydroxylase (FaF3H), dihydroflavonol reductase (FaDFR), anthocyanidin synthase (FaANS), and UDP glucose:flavonoid 3-O-glucosyl transferase (FaUFGT), and cell wall-modifying genes such as polygalacturonase (FaPG1) and pectate lyase (FaPL1), were all remarkably downregulated in overexpressing lines and suggested that ABA perception and signal transduction underlying PYR1–PP2C–SnRK2 might be a core mechanism in non-climacteric fruits (Jia et al., 2013a).

In another study, Jia et al. (2013b) demonstrated that sucrose is an important signal in the regulation of strawberry fruit ripening. This study revealed that exogenous sucrose and its non-metabolizable analogue, turanose, induced ABA accumulation and accelerated fruit ripening. Furthermore, RNAi induced silencing of a sucrose transporter gene, FaSUT1, led to a decrease in both sucrose and ABA content. In contrast, the overexpression of FaSUT1 led to an increase in both sucrose and ABA (Jia et al., 2013b). It is possible that, in green strawberry fruits, the low ABA levels could lead to high PP2C expression and may block signal transduction, whereas in red fruits, the high ABA levels could result in relatively low levels of PP2C expression and thus evoke ABA signal transduction and promote ABA-regulated ripening-related genes such as SnRK2, PG1, CHS, ANS, UFGT, and others (Fig. 1).

Role of other hormones and their cross-talk

Auxins, gibberellins and others

A number of other plant hormones including auxins [auxin (Aux), indole-3-acetic acid (IAA)], gibberellins (GAs), brassinosteroids, and MeJA have been implicated in the ripening of climacteric and non-climacteric fruits (Jones et al., 2002; Jiang and Joyce, 2003; Trainotti et al., 2007; Figueroa et al., 2009; Zhang et al., 2009b; Chai et al., 2011; Jia et al., 2011; Symons et al., 2012; Concha et al., 2013). The identification of elements of signal transduction pathways of these hormones have revealed that some of their components may be part of the cross-talk existing between climacteric and non-climacteric fruits. For example, although lycopene synthesis in tomato is ethylene dependent, it can be enhanced by exogenous ABA treatment. ABA may induce ethylene biosynthesis via the regulation of ACS and ACO gene expression (Zhang et al., 2009b). It is likely that ethylene and ABA may cooperate indirectly to trigger ripening-related colour changes in non-climacteric fruits such as grape berry (Sun et al., 2010).

Auxin

It has been shown that auxin stimulates receptacle expansion during strawberry fruit development and later inhibits fruit ripening (Given et al., 1988). A decrease in auxin levels was reported during ripening (Nitsch, 1950). A role as a repressor of fruit ripening has been ascribed to auxin, since the exogenous application of auxin has been found to decrease the expression of several cell-wall-modifying genes such as pectate lyase, expansins, and polygalacturonase (Medina-Escobar et al., 1997; Figueroa et al., 2009; Villarreal et al., 2009).

Aux/IAAs are nuclear proteins that are characterized by the presence of four conserved domains (I, II, III, and IV) (Abel et al., 1994). Aux/IAA (FaAux/IAA1, FaAux/IAA2) genes from strawberry exhibited high levels of gene expression at green and white fruit stages and then decreased at the turning and ripe stages (Liu et al., 2010). Thus, these two genes could be related to early fruit development and might only play a negative role during fruit ripening (Liu et al., 2010). Similarly, in the case of tomato, the Aux/IAA gene SlIAA9 was reported to be involved mainly in fruit development, since its downregulation resulted in early fruit development and parthenocarpy (Wang et al., 2005). Nevertheless, another Aux/IAA family member in tomato, SlIAA3, showed a lower transcript level in immature green fruits and a higher level in mature green, breaker ripe, and red-ripe stages (Zhang et al., 2007), giving evidence that members of Aux/IAA gene families could play a role in tomato fruit ripening as well as fruit development. A number of genes encoding auxin response factors have been isolated from tomato, and auxin/IAA appears to work in concert with ethylene in tomato fruit development (Jones et al., 2002; Wang et al., 2005; de Jong et al., 2009b). In fact, hormones such as auxin/IAA may also use EIN2 to transduce their response through auxin response factors (Jones et al., 2002; Bouzayen et al., 2010; Handa et al., 2011) (Fig. 1).

Gibberellins

Three bioactive GAs have been detected in different stages of strawberry fruit development (Csukasi et al., 2011). GA₁ was detected only at the green stage, while GA₃ was found in lower levels at the white and red stages, respectively. GA₄ displayed a clear peak at the white stage, suggesting a major role for GA₄ in the developmental processes underlying the transition from green to white and subsequently to red in the receptacle (Csukasi et al., 2011). GA3ox and GA2ox genes encode key enzymes of biosynthesis and inactivation of GAs, respectively, in strawberry (Yamaguchi, 2008). The rate of biosynthesis and deactivation was considered the criteria for determining the concentration of bioactive GAs. The gene expression patterns of both of these genes were found to be consistent with the continued accumulation of GAs in the receptacle between the green and white stages of fruit development. However, a considerable decline in the bioactive forms of these hormones was reported following their inactivation in the red receptacle (Csukasi et al., 2011). In addition, the expression of the FaGA3ox gene in the green receptacle was 40 times higher than in the green achene, suggesting that the receptacle is the main source of GA biosynthesis in strawberry fruits. In contrast, Kang et al. (2013) showed that the same gene in Fragaria vesca (FvGA3ox1) is expressed only at a low level in achene and small green fruit, while the other members (FvGA3ox4, -5, and -6) are expressed many times higher than FvGA3ox1 in the endosperm and seed coat (the ghost tissue) of achene, revealing a central role for these tissues in GA biosynthesis. Furthermore, they proposed a model to illustrate how hormonal signals produced in the endosperm and seed coat coordinate seed, ovary wall, and receptacle fruit development. Negative regulators of GA signalling such as DELLA proteins, which act directly downstream of the GA receptors, have also been reported (Thomas and Sun, 2004). Two DELLA-like genes have been identified in strawberry (Hytonen et al., 2009).

To investigate the cross-talk between auxin and GAs during strawberry fruit development, Csukasi et al. (2011) removed the achenes (the source of auxin) from green fruits and observed that the expression of FaGA3ox was reduced 25-fold when compared with entire fruits, whereas the expression of FaGA2ox was unaffected. These data suggest that auxin may have a central role in controlling the concentrations of GAs in strawberry through regulating the expression of FaGA3ox (Csukasi et al., 2011). Similar changes in the expression levels of GA3ox as a result of changes in auxin content were reported in tomato (Serrani et al., 2008). GAs cooperate with auxin in the regulation of tomato fruit set (de Jong et al., 2009a). Csukasi et al. (2011) found that, in the strawberry receptacle, the action of auxin and GAs are not independent of one another but are coordinated, much like in tomato fruits (Serrani et al., 2008).

During the growth of parthenocarpic tomato fruits, auxin application accelerated the rate of cell division and GA treatment enhanced cell expansion (de Jong et al., 2009a). It was proposed that, during the growth of tomato fruit pericarp, cell elongation and cell division activities are coordinated by a delicate balance between GA and auxin. According to Symons et al. (2012), the level of GA₁ followed a trend similar to that observed for auxin (a rise at the small green stage and then a decline during fruit maturation), except that the GA₁ rise and fall lagged behind that for auxin. Studies in Arabidopsis and tomato showed that auxin acts upstream of GA by stimulating GA biosynthesis during fruit set (Serrani et al., 2008; Dorcey et al., 2009; Fuentes et al., 2012). Csukasi et al. (2011) suggested that the receptacle represents a plausible site where auxin and GA₄ could act in a coordinated and sequential manner with auxin, having a positive effect on GA biosynthesis. During the movement from the ghost to the receptacle, GA and auxin may be transported by plant-specific transport proteins such as PIN via both PIN-dependent and PIN-independent mechanisms (Kang et al., 2013). Upon arriving at the receptacle, auxin and GAs act by stimulating downstream signalling events and aid receptacle growth in strawberry (Kang et al., 2013).

Jasmonates

Jasmonates such as jasmonic acid (JA) and MeJA, are important cellular regulators of a wide range of processes, including biotic and abiotic stress, seed germination, and leaf senescence. JAs have also been found to play a role in fruit ripening. In non-climacteric fruits such as raspberry and blackberry, MeJA treatment increased the soluble solid content/titratable acidity (SSC/TA) ratio, sucrose and glucose concentrations, and anthocyanin content (Wang and Zheng, 2005; Wang et al., 2008). In strawberry fruit (F.×ananassa cv. Camarosa), MeJA was found to stimulate an increase in weight and acquisition of colour through faster chlorophyll degradation and a transient increment in anthocyanin accumulation (Perez et al., 1997). Similarly, in blackberry, raspberry, and apple, MeJA applications led to increased anthocyanin accumulation (Kondo et al., 2000; Wang and Zheng, 2005; Wang et al., 2008). Yao and Tian (2005) showed that MeJA applied to non-climacteric sweet cherry fruit increased the activity of both PAL and peroxidase enzymes in the fruit mesocarp, indicating a stimulation of lignin biosynthesis. Ziosi et al. (2008) reported that, in the climacteric peach fruit, MeJA application altered the expression levels of several cell-wall-modifying genes, suggesting a possible role of JAs in their regulation.

Additionally, JAs stimulate ethylene biosynthesis in several climacteric fruits (Fan et al., 1997; Kondo et al., 2007). In unripe pear and tomato, MeJA stimulated the expression of the ACS and ACO genes, which resulted in increased enzymatic activity and ethylene concentrations (Kondo et al., 2007; Yu et al., 2011). In strawberry fruit, MeJA application at the white stage was found to increase ACO activity, thereby promoting ethylene biosynthesis (Mukkun and Singh, 2009). In the Chilean strawberry (Fragaria chiloensis), exogenous application of MeJA altered the expression profiles of several ripening-related genes including those related to ethylene and JA biosynthesis (Concha et al., 2013). MeJA may accelerate fruit softening through the depolymerization of hemicelluloses and enhanced expression of the xyloglucan endotransglycosylase/hydrolase 1 (XTH1) and endo-1,4-β-glucanase 1 (EG1) genes, resulting in anthocyanin accumulation, given that MeJA upregulates genes of the anthocyanin biosynthesis pathway, including CHS, CHI, F3H, DFR, ANS, and UFGT. Concomitant with the higher amounts of anthocyanins, MeJA also induces the JA biosynthesis genes such as LOX, allene oxidase synthase (AOS) and 12-oxophytodienoate reductase 3 (OPR3) in strawberry (F. chiloensis) fruits (Concha et al., 2013).

Brassinosteroids

The phytohormone brassinosteroid (BR) accelerates fruit ripening (pericarp tissue) and development in tomato (Vidya Vardhini and Rao, 2002; Lisso et al., 2006). BRs are also involved in strawberry fruit ripening (Bombarely et al., 2010). Expressed sequence tags homologous to the receptors and two components of the BR signalling pathway, namely BRASSINOSTEROID INSENSITIVE1 (FaBRI1), BRASSINAZOLE-RESISTANT 1 (FaBRZ1), and SHAGGY-LIKE KINASE (FaBIN2) have been identified (Bombarely et al. 2010). It was noted that the expression level of FaBRI1 in F.×ananassa was low in achenes, and was higher in the receptacle, and increased sharply during ripening (Bombarely et al., 2010). BZR1 is a transcription factor (Lin and Den, 2005) phosphorylated by BIN2, a glycogen synthase kinase-3 (GSK3)-like kinase (He et al., 2002), and, when phosphorylated, BZR1 functions in the nucleus and induces the expression of BR-dependent genes. Bombarely et al. (2010) showed that the expression of FaBZR and FaBIN2 occurs in achenes and receptacles throughout development. However, the expression ratio of FaBZR:FaBIN2 was found to be higher in white achenes and lower in white receptacles, respectively. Further studies focusing on protein interactions may clarify the role of these two players during strawberry fruit development.

BRs have also been implicated in the regulation of grape and strawberry fruit ripening (Symons et al., 2006, 2012). It was reported that application of BRs to tomato pericarp discs could elevate lycopene levels and lower chlorophyll content, indicating that BRs accelerate tomato fruit ripening and senescence (Vidya Vardhini and Rao, 2002). In another study, a tomato d^x mutant displaying reduced dry mass, starch, and sugars due to severe symptoms of BR deficiency was rescued by the application of BR, demonstrating that BR is required for tomato fruit development (Lisso et al., 2006). The perception of BR by the BR receptor BRI1 at the cell surface leads to the dissociation of BRI1 kinase inhibitor from the plasma membrane and association of BRI1 with its co-receptor, BAK1 (Clouse, 2011). In strawberry fruit (F.×ananassa cv. Akihime), FaBRI1 receptor expression was increased in the receptacle and was markedly raised during ripening (Chai et al., 2013). These authors showed that the application of epibrassinolide to receptacles of developmental fruits promoted fruit ripening.

Cytokinins

Relatively little information is available regarding the role of cytokinins (CKs) in non-climacteric fruit ripening. Bombarely et al. (2010) reported two expressed sequence tags sequences related to CK signalling genes in strawberry fruit. They identified two genes, histidine phosphotransfer protein (AHP) and nuclear response regulator (ARR), belonging to the CK signal transduction pathway from a total of six cDNA libraries prepared from fruits of several varieties of F.×ananassa. Kang et al. (2013) compared the transcriptomes of two stages of fruit development (post-fertilization versus pre-fertilization) in F. vesca and described 17 genes related to CK biosynthesis, signalling, and degradation in four different tissues (seed, achene wall, and cortex and pith of the receptacle). Most of these genes (11) were found to be upregulated in different tissues (Kang et al., 2013), suggesting that CKs play some role in the early development of strawberry fruits. CKs are also involved in the early fruit development of tomato fruits (Matsuo et al., 2012).

Hormonal regulation in strawberry fruit differs from other non-climacteric fruits

Unlike other fleshy fruits, there are several aspects of strawberry fruit that must be taken into consideration when we discuss the fruit development and ripening in strawberry. First, strawberry fruit is made up of two organs namely, the receptacle, which is derived from the floral receptacle and the achene, which is the true fruit formed from the ovary. These two organs are very different in terms of their origin, physiological role, and the associated metabolic networks. Due to this heterogeneity in sampling, it is difficult to interpret the results of an analysis of the complete berry (Merchante et al., 2013). Secondly, the relative contribution of each of these organs to the whole berry is highly dependent on the developmental stage of the fruit. For instance, the relative contribution of the achenes to the biomass of the whole fruit is high in green fruits, whereas this contribution is considerably less in red fruits. A third important consideration is the inherent difficulty in measuring independent endogenous production of ethylene in achenes and receptacle. To add to this complexity is the very low endogenous production of this hormone in strawberry (Iannetta et al., 2006).

Transgenic technologies were employed to overcome these bottlenecks by creating transgenic lines (that overexpress Arabidopsis etr1-1 mutant ethylene receptor) with reduced sensitivity to endogenous ethylene and by performing separate analyses in achenes and receptacle, respectively (Merchante et al., 2013). It was found that genes involved in ethylene perception as well as related downstream processes such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis were all differentially expressed in two transgenic tissues, the achenes and receptacles (Merchante et al., 2013). This study further revealed that full ripening of achenes requires ethylene action, especially when the embryo is in the cotyledon stage and the dry pericarp of the achenes is still developing. These authors opined that ripening of the receptacle partially resembles the climacteric ripening of fleshy fruits, considering the fact that, although receptacle is not a true fruit, the enlargement of this organ is important for seed dispersal and human consumption. This functional evolutionary convergence with true fleshy fruits reflects the fact that, despite some differences, many other aspects such as changes in the receptacle and changes related to cell-wall disassembly as well as primary and secondary metabolisms are very much similar to those of true fleshy fruits (Fait et al., 2008; Osorio et al., 2012; Merchante et al., 2013).

Similarly, the levels of different phytohormones such as IAA, GA₁, ABA, and bioactive BR, castasterone, and brassinolide during ripening were studied to elucidate whether there was any consistent pattern of fruit development and ripening in both non-climacteric and climacteric fruits (Symons et al., 2012). However, these investigators could not deduce similar patterns of hormonal signals controlling ripening in grape and strawberry. There was, for example, a strong rise in castasterone levels at the onset of fruit ripening in grape that does not appear to occur during ripening in strawberry. Accordingly, Symons et al. (2012) suggested that BRs do not play an important role during strawberry fruit ripening, although a role during the early phases of fruit development is possible. Marked differences were also noticed with regard to other hormones such as GA₁ and ABA, with the GA₁ level being consistently low in grape except at flowering, while strawberry showed a pronounced peak during the early stages of fruit development. ABA levels showed a steady rise throughout strawberry development, while in grape an initial decline followed by a rise at the onset of fruit ripening was noted (Davies et al., 1997).

Unravelling the role of epigenetic variation and short RNAs in fleshy fruit ripening

Gene expression is a highly regulated and multistep process in plants that ensures proper development and function of tissues in response to varying environmental conditions (Moxon et al., 2008). Post-transcriptional regulatory mechanisms are predicted to be involved in several aspects of plant development including the fruit ripening process. MicroRNAs (miRNAs) are one of the best-characterized and ubiquitous plant short RNAs (sRNAs) (Jones-Rhoades et al., 2006) and are crucial in mediating gene silencing at the post-transcriptional level (Zuo et al., 2012). Most of the plant miRNA sequences identified across plant families are of conserved class; however, some are species specific and non-conserved (Allen et al., 2004; Axtell and Bartel 2005; Moxon et al., 2008). Recent deep sequencing in a sRNAome study in developing tomato fruits covering the period between closed flowers and ripened fruits through the profiling of sRNAs showed differential expression of non-miRNAs during fruit development and ripening (Mohorianu et al., 2011).

In plants, there are several classes of 21–24 nt short RNAs that regulate gene expression. Although sRNAs have mostly been studied in Arabidopsis, several reports have emerged recently about sRNAs in tomato (Moxon et al., 2008; Dalmay, 2010; Zuo et al., 2012; Karlova et al., 2013). Zuo et al. (2012) employed a combination of next-generation sequencing and molecular biology approaches to identify miRNAs involved in tomato fruit ripening and senescence and their possible roles in ethylene response. Interestingly, some targets of miRNAs profiles were predicted to be involved in fruit ripening and softening such as pectate lyase and β-galactosidase, while a few others were predicted to be involved in ethylene biosynthesis and signalling pathway, such as ACS, EIN2 and CTR1. Recent evidence suggests that reprogramming of DNA methylation guides epigenetic inheritance via sRNAs (Moxon et al., 2008; Dalmay, 2010; Calarco et al., 2012). Analysis of sRNA changes in tomato has shown that miRNAs are involved in the regulation of CNR and other genes of ethylene signalling and may play an important role in fruit development and ripening (Moxon et al., 2008; Dalmay, 2010).

Epigenetic inheritance refers to transmission of modified genetic material from one generation to the next under the influence of developmental and environmental cues (Calarco et al., 2012). The first reported epigenetic inheritance in plants involved transposable elements and inheritance of epialleles (McClintock, 1965; Martienssen et al., 1990) through DNA methylation (Becker et al., 2011; Schmitz et al., 2011). A recent study reported that reprogramming of DNA methylation in pollen guides epigenetic inheritance via sRNAs (Calarco et al., 2012).

Recent advancements in genetics have shown that crop improvement strategies could benefit not only by taking into account the DNA sequence variation available among different plant lines but also by making use of the information encoded in the epigenome (Seymour et al., 2008). Epigenetic variations are more common in plant genomes and can affect phenotypes. They are caused by DNA methylation and other forms of chromatin modification (Calarco et al., 2012). The possible role of epigenetic processes in fruit ripening was highlighted by characterization of the CNR locus (Thompson et al., 1999; Eriksson et al., 2004; Manning et al., 2006; Ecker 2013). Cnr is a natural epigenetic (epiallele) mutation resulting from hypermethylation of the promoter of the SBP-box transcription factor rather than any genetic alteration in the DNA sequence. The elevated levels of promoter methylation led to reduced Cnr gene expression in the mutant compared with the wild-type fruit. It has been noted that the Cnr phenotype could be induced by either virus-induced gene silencing of the SBP-box gene or transgene-induced methylation of the appropriate site on the CNR promoter (Manning et al., 2006; Kanazawa et al., 2011). Other than rare reversion events, Cnr epimutants do not ripen and their fruits turn yellow and remain firm and unpalatable. Using different experimental approaches including whole-genome bisulfite sequencing, Zhong et al. (2013) investigated whether global epigenome reprogramming occurs during tomato fruit ripening. First, they demonstrated that exposure of tomatoes to the methyltransferase inhibitor 5-azacytidine, a well-known inhibitor of cytosine DNA methylation in mammalian cells, could prematurely induce fruit ripening. Furthermore, bisulfite sequencing confirmed that the region 5ʹ-upstream of the Cnr gene was demethylated in ripening sectors, whereas in green sectors the 5ʹ-upstream region of Cnr remained hypermethylated.

In addition, the mRNAs of hallmark ripening genes, such as pg2a encoding pectinase polygalacturonase and psy1 encoding phytoene synthase 1, the rate-limiting enzyme in fruit carotenoid synthesis were detected in areas of early ripening sectors of tomato fruit. These findings indicate that inhibition of DNA cytosine methylation removes the developmental constraint that prevents ripening before seeds are mature and further confirmed the uncoupling of the seed-maturation and fruit-development processes. Based on these results, a three-component model for the control of fruit ripening was proposed, in which the ripening hormone ethylene and fruit-specific transcription factors together with epigenetic reprogramming triggers the transition of fruit into a ripening-competent state when seeds become viable (Zhong et al., 2013). The model further explains that, in non-fruit tissues and immature fruit, the promoters of key ripening genes are hypermethylated and this prevents ripening, whereas in maturing wild-type fruits, the relevant promoters become demethylated by an unidentified mechanism, probably influenced by RIN and CNR. An important issue that remains to be addressed is whether transcription factor binding facilitates promoter hypomethylation or whether hypomethylation aids DNA binding, or whether both activities influence the ripening transition (Fig. 2C, D). In future, epigenetic diversity through methylation and miRNAs may be beneficially exploited in breeding programmes for fruit crop improvement.

Future perspectives

Great strides have been made in understanding the molecular basis of fruit development and ripening in climacteric fruits through the judicious use of a varied collection of tomato mutants, especially as modulated by system-1 and system-2 ethylene biosynthesis and signal transduction pathways. The role of other hormones and their possible cross-talk with ethylene may continue to remain a challenging research area. The regulatory control of fruit ripening in non-climacteric fruit is not well understood. However, the ABA perception and signal transduction underlying the coupling of PYR1–PP2C–SnRK2 with sugar metabolism seems to be a favoured core mechanism. Accordingly, reconstitution of the ABA–FaPYR1–FaABI1–FaSnRK2 signalling pathway in vitro will be a challenging task (Jia et al., 2013a).

As the genomes of 13 fleshy fruits have already been fully sequenced (including the recently added hot pepper genome), the resources available for mining new information about ripening and its complex networks are increasing (Pech et al., 2013). It is expected that the identification and characterization of more regulatory elements and binding-site motifs associated with ripening promoters could impact on and widen our knowledge of fruit physiology. Apart from the genome sequence data, other high-throughput tools such as transcriptomics, proteomics, and metabolomics, or a combination of these approaches through systems biology, might lead to a more comprehensive knowledge about the network of regulatory events in fruit ripening. For instance, research results have already started to emerge in this direction, as with the proposed model by Kang et al. (2013) illustrating how hormonal signals coordinate and proceed in the endosperm and different layers of fruit such as ovary wall, seed coat, and receptacle. Unravelling epigenetic events represents an additional new and challenging area of research.

In conclusion, the molecular dissection of climacteric and non-climacteric fruit models has shown that various phytohormones (ethylene, ABA, and others) move in concert to affect fruit textural and organoleptic attributes at the onset of ripening. These ‘movers’ function through the intervention of many protein families (such as cell-wall stabilizing and depolymerizing enzymes) and transcription factors that are tangible key players in the fruit-ripening process. Epigenetic elements intervene as possible ‘shakers’ to re-route the pathways through DNA methylation and other forms of chromatin modifications.

Abbreviations:

Abbreviations:
 
• 1-MCP

  1-methylcyclopropene

 
• ABA

  abscisic acid

 
• ACC

  1-aminocyclopropane-1-carboxylic acid

 
• ACO

  ACC oxidase

 
• ACS

  ACC synthase

 
• AOX

  alternative oxidase

 
• BR

  brassinosteroid

 
• CK

  cytokinin

 
• EIL

  EIN3-like protein

 
• ERE

  ethylene response element

 
• ERF

  ethylene response factor

 
• ETR

  ethylene receptor

 
• GA

  gibberellin

 
• IAA

  indole-3-acetic acid

 
• JA

  jasmonic acid

 
• MeJA

  methyl jasmonate

 
• Met

  methionine

 
• miRNA

  microRNA

 
• SAM

  S-adenosylmethionine

 
• sRNA

  short RNA.

Acknowledgements

We thank Professor Graham Seymour (University of Nottingham, UK) for his critical assessment and valuable suggestions on this manuscript. CRF is supported by the National Commission for Scientific and Technological Research (CONICYT, Chilean Government) through Fondecyt 1140663 and PIA ACT-1110 projects.