ETHYLENE AND FRUIT RIPENING

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1 Annual Plant Reviews (2012) 44, doi: / ch11 Chapter 11 ETHYLENE AND FRUIT RIPENING Jean-Claude Pech 1,2, Eduardo Purgatto 3, Mondher Bouzayen 1,2 and Alain Latché 1,2 1 Université de Toulouse, INP-ENSA Toulouse, Génomique et Biotechnologie des Fruits, Avenue de l Agrobiopole, BP 32607, Castanet-Tolosan 31326, France 2 INRA, Génomique et Biotechnologie des Fruits, Chemin de Borde Rouge, Castanet-Tolosan 31326, France 3 Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Departamento de Alimentos e Nutrição Experimental, Av. Professor Lineu Prestes 580, bl 14, , São Paulo, Brazil Abstract: The ripening of fleshy fruit is a developmentally regulated process unique to plants during which the majority of the sensory quality attributes are elaborated including aroma, flavour, texture and nutritional compounds. In climacteric fruit, the plant hormone ethylene is the key regulator of the ripening process as exemplified by the dramatic inhibition of fruit ripening that results from the down-expression of ACC (1-amino-cyclopropane-1-carboxylic acid) synthase and ACC oxidase genes involved in ethylene biosynthesis. By contrast, the ripening of non-climacteric fruit is not dependent on ethylene but rather on cues of unknown nature though ethylene may contribute at least partly to the control of some aspects of the ripening process. The expression of the ripening-associated genes is regulated by a network of signalling pathways among which ethylene perception and transduction play a primary role. Building on the knowledge gained on the Arabidopsis thaliana model system, the importance of ethylene signalling in fruit ripening has been extensively studied. This chapter summarizes the present knowledge on the role of ethylene in fruit ripening and addresses the molecular mechanisms involved in ethylene perception and responses. It also highlights recent advances and prospects on the means by which the ethylene transduction pathway leads to diversified physiological responses and how ethylene signalling interacts with other hormones to activate the expression of ripening-related genes. While this review mostly refers to the tomato as major model for fruit research, it also gives insight on the ripening process in other fruit species, including nonclimacteric types. Annual Plant Reviews Volume 44: The Plant Hormone Ethylene, First Edition. Edited by Michael T. McManus. C 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd. 275

2 276 The Plant Hormone Ethylene Keywords: fruit ripening; climacteric; non-climacteric; ACC (1-aminocyclopropane-1-carboxylic acid) synthase; ACC oxidase; genetic determinism; ethylene receptors; ethylene signalling; ethylene-response factors; hormone crosstalk 11.1 Introduction Fruit ripening corresponds to a developmentally regulated process, which is accompanied by a number of biochemical events, such as changes in colour, sugar, acidity, texture, and aroma volatiles that are crucial for the sensory quality. All biochemical and physiological changes that take place during fruit ripening are driven by a cascade of molecular events starting with the activation of signalling pathways. These lead to the stimulation of specific transcriptional regulators responsible for the coordinated expression of fruit ripening-related genes directly involved in the biochemical processes (Giovannoni, 2001, 2004; Lin et al., 2009). In climacteric fruit, the plant hormone ethylene is considered to be the major signalling molecule that controls most aspects of fruit ripening. By contrast, in non-climacteric fruit, ethylene is not the trigger of the ripening process, which appears to depend on signals not yet elucidated (e.g. other hormones, developmental factors, etc.). It is clear, however, that ethylene-independent events also exist in climacteric fruit and that, conversely, the ripening of non-climacteric fruit comprises ethylene-dependent events (Lelièvre et al., 1997a; Alexander & Grierson, 2002; Mailhac & Chervin, 2006; Barry & Giovannoni, 2007). The cascade of events leading to the activation of ripening-related genes and highlights the prominent role played by ethylene in concert with other signals as summarized in Figure Regulation of ethylene production during ripening of climacteric fruit The requirement for ethylene in the ripening of climacteric fruit has long been recognized (Abeles et al., 1992) and discrimination between climacteric and non-climacteric fruits has been made on the basis of the presence or absence of the climacteric rise in respiration and of autocatalytic ethylene production (Biale & Young, 1981). The main specificity of climacteric fruits is their capacity to produce ethylene through a so-called autocatalytic process in which ethylene stimulates its own biosynthesis (McMurchie et al., 1972). Hence, the concept of two systems of ethylene production has emerged. System 1 is characterized by low levels of ethylene production and is present throughout the ripening of non-climacteric fruit. It operates in climacteric fruit during the period preceding the climacteric rise in respiration. System 2 operates at the onset of the climacteric and is responsible for auto-catalytic ethylene production.

3 Ethylene and Fruit Ripening 277 Perception Ethylene Other signals (Auxin, ABA, light, etc.) C 2 H 4 ACO ACC ACS SAM ER ETR, NR CTR1 Signal Transduction Receptors YANG's CYCLE EIN2 Autocatalysis TRANSCRIPTIONAL REGULATION Primary targets Signal amplification and diversification Ethylene-dependent and -independent ripening-related genes Figure 11.1 Schematic representation of the molecular events leading to the expression of ripening-related genes. In climacteric fruit, ethylene through its perception and signal transduction pathway plays a major role in the activation of the transcriptional machinery resulting in the expression of a number of ethylene-dependent genes. The process is fed by autocatalytic production of ethylene, and other hormonal and non-hormonal signals play a secondary role. In non-climacteric fruit, signals other than ethylene are essential and the majority of ripening-related genes are ethylene independent. ER, endoplasmic reticulum Regulation of ethylene biosynthesis genes during the System 1 to System 2 transition The main feature of System 1 is its negative feedback regulation by ethylene itself (auto-inhibition). In contrast, System 2, which operates in climacteric fruit, is responsible for the high levels of ethylene production prevailing at the climacteric phase and is characterized by a positive feedback

4 278 The Plant Hormone Ethylene regulation by ethylene referred to as autocatalytic or climacteric ethylene production. The molecular mechanisms of autocatalytic ethylene production were first identified by studying the expression of genes involved in ethylene biosynthesis, ACC oxidase (ACO) and ACC synthase (ACS), during the transition from System 1 to System 2 in ripening tomatoes. It was shown that System 1 relied on the expression of LeACS6 and LeACS1A with the two genes being negatively regulated by ethylene during the transition to System 2. Subsequently, the up-regulation of LeACS2 and LeACS4 through positive feedback by ethylene is responsible for the activation of System 2 (Nakatsuka et al., 1998; Barry et al., 2000). However, System 2 is not controlled solely via autocatalytic regulation (Yokotani et al., 2009) since the expression of LeACS2 and LeACS4 was shown to be also regulated by an ethylene-independent developmental factor. Interestingly, it has been demonstrated recently that LeACS2 is regulated by RIN (ripening inhibitor), a MADS box transcription factor, capable of binding to the LeACS2 promoter (Ito et al., 2008). Among the five ACO genes encountered in tomato, three of them are expressed in ripening fruit. LeACO1 and LeACO4 display ethylene-dependent regulation (Barry et al., 1996) and are expressed in immature green fruit and exhibit strong up-regulation at the climacteric peak of ethylene production. LeACO3 is induced transiently at the breaker stage (Barry et al., 1996). The main changes in ACS and ACO gene expression during tomato fruit ripening are summarized in Figure This regulation seems to operate in climacteric fruit from other species including apple where, among three MdACO genes, MdACO1 expression is fruit specific and preferentially expressed during ripening (Binnie & McManus, 2009). The nature of the developmental factors responsible for the transition from System 1 to System 2 is not known. However, in some winter varieties of pears such as D Anjou, Beurré Bosc and Passe Crassane, the transition occurs only after exposure, at post-harvest, to low temperatures since in the absence of cold treatment, the fruit remains locked in System 1 (Lelièvre et al., 1997b; Blankenship & Richardson, 1985; Morin et al., 1985). Detailed analysis of the expression of ACS genes in Passe Crassane pears indicates that in immature fruit, System 1 ethylene relies on the expression of PcACS3 while the cold treatment induces the expression of another ACS gene, PcACS1a, in an ethylene-dependent manner (El-Sharkawy et al., 2004). This enhanced expression results in increased ethylene production, which in turn activates the expression of three ethylene-inducible ACS genes, PcACS4, PcACS5 and PcACS2a. The elevated levels of ethylene produced by System 2 causes a negative feedback of System 1 with down-regulation of PcACS3 expression. In this type of pear fruit, PcACS1a might be a control point for the onset of System 2 and subsequent ripening because of its characteristic pattern of expression throughout the cold-induced transition period (El-Sharkawy et al., 2004). In tomato, where cold treatment is not required for ripening, LeACS4 seems to play the major role in the transition phase (Barry et al., 2000). A cold treatment, although not absolutely required, is also capable of hastening

5 Ethylene and Fruit Ripening 279 System 1 System 2 Non-climacteric Pre-climacteric Climacteric Ethylene (-) Imature green Mature green Breaker - Red ripe LeACS6 LeACS6 Developmental factors LeACS1A (+) (+) LeACO1,4 LeACS1A LeACS2 RIN (+) LeACS4 LeACO1,4 LeACS1A LeACS2 LeACS4 LeACO1,4 (+) (+) (+) Ethylene Autocatalysis LeACO3 Figure 11.2 Transition from System 1 to System 2 of ethylene production during ripening of tomato fruit. The transition is the result of a cascade of changes in the expression level of several members of the ACS and ACO gene families. At the immature green stage, System 1 ethylene production is prevailing and LeACS6, the main gene responsible for System 1 ethylene production, is highly expressed. Later, LeACS6 gene expression is inhibited through a negative feedback mechanism ( ). At the mature green stage, developmental factors up-regulate (+) the expression of LeACS2 and LeACS4, the main genes responsible for System 2 ethylene production. The expression of LeACS2 is also up-regulated by the RIN transcription factor (+). Thereafter, at breaker and red-ripe stages, ethylene, through a positive feedback mechanism (+), stimulates the expression of LeACS2 and LeACS4 as well as that of two ACO genes, LeACO1 and LeACO4. (Data used for drawing the figures have been taken from Barry et al. (1996 and 2000) and Ito et al. (2008).) and synchronizing the onset of the climacteric rise of ethylene production and ripening of Bartlett pears, both on the tree (Wang et al., 1971) and detached (Looney, 1972). Similar effects have also been reported for Conference pears (Knee, 1987) and apple varieties including Granny Smith (Jobling et al., 1991) and Golden Delicious (Knee et al., 1983). In Granny Smith apples, cold

6 280 The Plant Hormone Ethylene treatment also induces ACO activity (Jobling et al., 1991; Larrigaudière & Vendrell, 1993) and ACO protein accumulation (Lelièvre et al., 1995). Some fruits such as avocadoes fail to produce climacteric ethylene and cannot ripen until they are detached from the tree. Others show acceleration of ethylene production upon detachment, such as plums, apples and passion fruit, though some varieties do not display such acceleration (Lin & Walsh, 2008). Increased sensitivity of respiratory activity to ethylene has also been observed in melon fruit upon detachment (Bower et al., 2002). The effect of detachment has been ascribed to a tree factor whose nature still remains unknown although several hypotheses have been formulated. In avocado, auxin transported through the peduncle has been considered to act as a ripening inhibitor (Tingwa & Young, 1975). In persimmon, detachment of the fruit has been considered to cause a drought-induced synthesis of ethylene in the calyx associated with an ethylene-independent induction of an ACS gene, DkACS2. Ethylene produced in the calyx then diffuses to other parts of the fruit tissue and results in fruit ripening (Nakano et al., 2003). High humidity delayed fruit ripening and induction of DkACS2, thus demonstrating the role of water loss in the regulation of ethylene synthesis in detached persimmon fruit. Water loss has also been correlated with the onset of climacteric ethylene in detached avocadoes (Adato & Gazit, 1974), indicating that water stress could be a general stimulator of autocatalytic ethylene production in ripening fruit, at least after detachment. Together, these data, while stressing the prominent role of ethylene signalling, clearly point out to the involvement of multiple cues of various nature acting in concert with ethylene to trigger and coordinate the climacteric ripening ACS gene alleles are major determinants of ethylene biosynthesis and shelf-life of climacteric fruit The correlation between storage life and ethylene biosynthesis has been most extensively studied in apple and pear fruits and shows that low ethylene production is generally correlated with long storage life. A complex pattern of regulation of ACS genes seems to play an active role in determining the ripening and the post-harvest changes in these species. In apple fruit, ripening-associated ethylene production is related to the level of expression of the MdACS1 fruit-specific gene, and it was established that the post-harvest changes are determined by the presence of one of the two allelic forms, MdACS1-1 and MdACS1-2 (Sunako etal., 1999; Harada etal., 2000). MdACS1-2 contains an insertion of a retro-transposon-like sequence that confers lower transcription, and cultivars homozygous for the MdACS1-2 allele have low ethylene production and longer shelf-life (Harada et al., 2000; Sato et al., 2004). However, the differences in ethylene production observed within the same allelic forms suggest the involvement of additional ripening-associated

7 Ethylene and Fruit Ripening 281 ACS genes. Indeed, MdACS3, whose expression is under a negative feedback regulation by ethylene, is another key player. The expression of MdACS3 was found to precede that of MdACS1 and to gradually decrease as MdACS1 expression increases (Wang et al., 2009). Three ACS3 genes are present in apple (MdACS3a, MdACS3b and MdACS3c) among which only MdACS3a is expressed in fruit, while the expression of MdACS3b and MdACS3c during ripening is likely to be impeded by the transposon-like insertion in the 5 flanking region. A single nucleotide mutation was found in the coding region of MdACS3a, leading to an amino acid substitution (Gly289 Val) in the active site that inactivates the enzyme and so cultivars homozygous for this SNP mutation in MdACS3a have a longer shelf-life with very low expression of ripening-related genes. The picture is made more complex with the identification in some long shelf-life apple cultivars, of a null variant, designated Mdacs3a, that is not transcribed. Therefore, the MdACS3a gene is another major determinant of ethylene production and shelf-life in apple fruit and plays a crucial role in regulating fruit ripening (Wang et al., 2009). In Japanese pear, two ACS genes, pppacs1 and pppacs2, were identified and RFLP (restriction fragment length polymorphism) markers were tightly linked to the locus that determines the rate of ethylene production (Itai et al., 1999). Cultivars that produce high levels of ethylene possess at least two copies of pppacs1 and those producing moderate levels of ethylene have at least one additional copy of pppacs2 (Itai et al., 1999) Genetic determinism of the climacteric character A promising way to assess the genetic determinism of the climacteric character is to cross genotypes within the same species that have either a climacteric or non-climacteric behaviour, with melon one of the very few species that comprise such compatible genotypes. Crosses between a typical climacterictype Charentais melon (Cucumis melo var. cantalupensis cv. Védrantais) and a non-climacteric melon, Songwhan Charmi PI (Cucumis melo var. chinensis), have led to the generation of a population segregating for the formation of the abscission layer (Al) in the peduncle, which is associated with climacteric ethylene production (Perin et al., 2002). It was found that the climacteric character was controlled by two duplicated independent loci (Al-3 and Al-4). The intensity of ethylene production was controlled by at least four quantitative trait loci (QTLs) localized in other genomic regions, but none of these matched the chromosomal position of ethylene biosynthetic or transduction pathways. Of particular interest is the observation that some near isogenic lines (NILs) of melon generated by crossing two non-climacteric melons, Piel de Sapo (var. inodorus) and Songwhan Charmi PI (var. chinensis), did exhibit a climacteric character associated with a higher sensitivity to chilling injury (Obando et al., 2007). The QTLs associated with ethylene production and respiration rate in this cross were located at linkage group

8 282 The Plant Hormone Ethylene III (LGIII) at a position that differed from the Al loci described by Perin et al. (2002). These data indicate that the climacteric character is controlled at the genetic level by multiple loci and that it corresponds to a complex regulatory process Transcriptional control of ethylene biosynthesis genes Several genes have been identified that control ethylene production at the transcriptional level. The RIN gene encodes a putative MADS box transcription factor that controls tomato fruit ripening, with its mutated version (rin) conferring a non-ripening character (Vrebalov et al., 2002). Assays in vivo revealed that RIN binds to the promoter region of LeACS2 (Ito et al., 2008), but since LeACS2 gene expression was also shown to be ethylene-dependent (Barry et al., 2000), transcription is, therefore, induced by both RIN-dependent and ethylene-dependent mechanisms. A tomato AGAMOUS-LIKE1 (TAGL1), another MADS box gene whose down-regulation results in yellow fruit with reduced carotenoids and thin pericarp, has recently been shown to control fruit expansion and ripening (Itkin et al., 2009; Vrebalov et al., 2009). Interestingly, TAGL1-repressed fruit also produce lower amounts of ethylene with a reduced expression of LeACS2 suggesting that TAGL1 may be another important regulator of ripening-associated ethylene production. Ethylene-response factors (ERFs) are transcriptional regulators acting downstream of the ethylene transduction pathway (see the upcoming sections) and are encoded by one of the largest families of transcription factors (see Chapter 7 for a general description). Because of their position downstream of the ethylene transduction pathway, they are suitable candidates for mediating the expression of ACS and ACO genes, which are known to be ethylene regulated during fruit ripening. It was reported recently that downregulation of LeERF2 in tomato resulted in the suppression of the positive feedback of ethylene production (Zhang et al., 2009a). Biochemical analysis showed that LeERF2 interacts with the GCC box-conserved motif found in the promoter of tobacco NtACS3 and with the dehydration-responsive element present in the promoter of tomato LeACO3. ERF2 can, therefore, be considered as a major player in the auto-stimulated ethylene production. Another transcription factor encoded by the LeHB-1 gene belonging to class-i HD-Zip proteins is able to bind the promoter of LeACO1 (Lin et al., 2008) and its silencing via virus-induced gene silencing (VIGS) technology results in down-regulation of LeACO1 expression associated with delayed fruit ripening. Putative binding sites for LeHB-1 are also present in the promoter region of ripening-related genes such as LeACO2, polygalacturonase (PG), LeMADS- RIN and NAC-NOR. Altered transcriptional control that impacts fruit ripening can also result from epigenetic changes. A clear example is given by the Cnr (colourless non ripening) mutation corresponding to an epigenetic

9 Ethylene and Fruit Ripening 283 change that alters the promoter methylation of a SQUAMOSA promoterbinding (SPB) protein and results in colourless fruits with a substantial loss of cell-to-cell adhesion (Manning et al., 2006). In the CNR mutant, the expression of ethylene-related genes such as ACO1, E8 and Never-Ripe (NR), and several other ripening-related genes is inhibited (Thompson et al., 1999), indicating that CNR is acting upstream of ethylene synthesis and action. The characterization of all these induced or natural mutations occurring in genes encoding transcriptional regulators clearly indicates that transcription factors are key players in relaying ripening-inducing signals and controlling the expression of ethylene biosynthesis genes. Therefore, they represent an important control step of the ripening process Role of ethylene in ripening of non-climacteric fruit Non-climacteric fruit do not produce autocatalytic ethylene during ripening and no factors have yet been identified that play a similar role to ethylene for triggering and coordinating the ripening process. However, some studies do report an increase in ethylene production in non-climacteric fruit that has been suggested to play a role in ripening. In grapes, a small increase in ethylene production occurs at the veraison stage when berries reach the onset of colour changes (Chervin et al., 2004, 2008). Treatment of grape berries with 1-MCP, an inhibitor of ethylene perception, did affect anthocyanin accumulation and berry swelling and caused a decrease in acidity, suggesting that ethylene might be required for the full accomplishment of the ripening process. Accordingly, exogenous ethylene treatment of grape berries has been shown to stimulate the long-term expression of anthocyanin biosynthesis genes (El-Kereamy et al., 2003) and the transcription of an alcohol dehydrogenase gene (Tesnière et al., 2004). The role of ethylene in inducing colour changes in the flavedo tissue of citrus fruit, a non-climacteric fruit, has long been recognized (Goldschmidt et al., 1993, Goldschmidt, 1997). Moreover, autocatalytic ethylene production has been observed in the early stages of fruit development (Katz et al., 2004) and the expression of some genes, including chlorophyllase, was found to be ethylene regulated (Jacob-Wilk et al., 1999). In strawberry, which is generally considered as non-climacteric fruit, an increase in ethylene production associated with a raise in respiration has been observed when the fruit reaches the red-ripe stage (Iannetta et al., 2006). In addition, ethylene produced by red-ripe fruit is regulated by positive feedback, indicative of an autocatalytic ethylene production. However, ACO and ethylene receptor genes, including a homologue of LeETR4 from tomato, do not show up-regulation at the red-ripe stage, but at a much earlier stage (Trainotti et al., 2005). The late expression of a fruit-specific peptide methionine sulphoxide reductase gene, which is a homologue of the ethylene-responsive

10 284 The Plant Hormone Ethylene ripening-related gene of tomato, ER4, is better correlated to the later phases of strawberry ripening (Lopez et al., 2006). Altogether, these data suggest a putative involvement of ethylene in at least some aspects of the ripening process but the timing of the increase in ethylene production observed in non-climacteric fruit does not tightly correlate with the ripening phase as it does in climacteric fruit. It seems unlikely that the control of the whole ripening process in non-climacteric fruit depends on a single factor or one major hormone, but it rather requires the interaction among multiple cues many of which are yet to be identified. The presence of regulatory mechanisms common to both climacteric and non-climacteric fruits is supported by the ripening-associated pattern of expression in tomato of the promoter of capsanthin/capsorubin synthase and fibrillin genes, used as ripening markers in the non-climacteric pepper (Kuntz et al., 1998). In the transgenic tomato, the promoter-gus expression was strongly up-regulated during fruit ripening in a similar pattern to the induction of these genes in pepper fruits suggesting that climacteric and nonclimacteric fruits may share common regulatory mechanisms. Further attempts to elucidate the molecular factors that differentiate climacteric and non-climacteric fruits have been performed using high throughput transcriptomic data via comparative in silico profiling of gene expression in climacteric tomato and grapes (Fei et al., 2004). Also, comparative transcriptome analysis among fruits of the Solanaceae family, with one climacteric example, the tomato, and one non-climacteric example, the pepper, has been carried out using heterologous microarray hybridization (Lee et al., 2010). However, the differences observed could also arise from the different genetic backgrounds among species or genera such that the outcomes of these studies are still not conclusive. The determinism of the climacteric or non-climacteric character has also been studied by genetic approaches as exemplified in Section Manipulation of Ethylene Biosynthesis and Ripening Manipulation of ethylene biosynthesis and ripening The chemical control of ethylene production emerged after the sequential elucidation of the steps of ethylene biosynthesis (Lieberman, 1979; Yu et al., 1979). A number of inhibitors of ethylene synthesis were discovered and used extensively as experimental tools to dissect ethylene physiology. Only one of them, aminoethoxyvinylglycine (AVG), an inhibitor of ACS, is used commercially (under the brand name Retain R ) to reduce pre-harvest drop and delay fruit ripening. Ethylene is involved in the promotion of senescence, abscission and ripening of horticultural products, and so by reducing ethylene biosynthesis and action, it is possible to slow down the post-harvest deterioration of fruit and vegetables, thus extending shelf-life and limiting post-harvest losses.

11 Ethylene and Fruit Ripening 285 Very high inhibition of ethylene production (around 99% inhibition at the peak of ethylene production) has been achieved by sense or antisense suppression of ACS expression in tomato (Oeller et al., 1991). As a result, the development of red colour was inhibited as well as softening and aroma production, and treating with ethylene completely restored the ripening process. Strong reduction has also been achieved using antisense constructs of the ACO gene (Picton et al., 1993). However, this reduced ethylene synthesis only had a partial effect on fruit ripening, and the major alterations in ripening were only significant after detachment (Murray et al., 1993). Any residual ethylene was probably responsible for the development of some of the ripening processes such as softening, which is consistent with the observation that minimal ethylene is able to stimulate PG gene expression in antisense ACS tomatoes (Sitrit & Bennett, 1998). Other strategies have comprised decreasing the pool of the ethylene precursor S-adenosylmethione (SAM) by the overexpression of S-adenosyl-methionine hydrolase (SAMase) gene or in converting ACC into ammonia and -ketobutyric acid by the overexpression of a ACC deaminase gene (Klee, 1993; Good et al., 1994; Kramer et al., 1997). However, the inhibition of ethylene production was not sufficient to significantly affect the ripening process. Cantaloupe melon of the Charentais type is the second fruit (after tomato) where very high (>99.5%) inhibition of ethylene production has been achieved using an antisense ACO gene (Ayub et al., 1996). Silva et al. (2004) have inserted an antisense construct of an apple ACO gene in the same variety of cantaloupe Charentais melon, Védrantais, and described similar inhibitory effects on the ripening process. Further, low concentrations of ethylene (2.5 5 L/L) applied to antisense ACO melons were able to restore the original ripening phenotype (Flores et al., 2001). Manipulating ethylene metabolism through such transgenic approaches has been less attractive for perennial species as it takes many years to obtain fruit. However, apple can be transformed (James et al., 1989, 1996) and thus has become a target for the genetic analysis of ethylene-related genes. Apple fruits have been obtained from plants silenced for either ACS or ACO (Defilippi et al., 2004), and such ethylene-suppressed fruits have been shown to be significantly firmer and displayed an increased shelf-life as compared to controls. Headspace analysis of aroma production, an ethylene-associated event, showed a reduction in ester and alcohol production in the ethylenesuppressed lines. Ethylene suppression also affected malic acid degradation, sucrose and fructose concentrations as well as phenolics (Dandekar et al., 2004). One important conclusion emerging from the experimental approaches reported in the previous paragraphs is that only drastic inhibition of ethylene synthesis (>99%) can lead to significant alteration of the ripening process. These strongly ethylene-inhibited fruit could open the way for new postharvest handling procedures in which fruit can be harvested at full development with minimal risk of over-ripening, stored for the desired period of time and allowed to ripen on command using exogenous ethylene. When

12 286 The Plant Hormone Ethylene inhibition of ethylene production is not severe but still significant (around 85% to 95%), ripening is not inhibited. In such cases, fruit can be harvested at the mature green or breaker stage and ripening may proceed at a slow rate with reduced risks of over-ripening Ethylene-dependent and -independent aspects of climacteric ripening The generation of transgenic fruit with reduced ethylene production has also provided a powerful tool to uncover the ripening aspects that are ethylene dependent or independent (Pech et al., 2008) (Figure 11.3). Some ripening pathways, such as colouration of the flesh and accumulation of sugars and organic acids, were not affected by ethylene suppression. The sugar content could even be higher since antisense ACO fruit could be kept on the vine for longer without abscission or risk of over-ripening. The softening of the flesh was not completely abolished by ethylene suppression indicating that it is partly ethylene independent (Guis et al., 1997). Moreover, the ethylenedependent events of the ripening process exhibit differential sensitivity to ethylene (Flores et al., 2001). The threshold level for de-greening of the rind is 1 ppm, while 2.5 ppm is required to trigger the ethylene-dependent component of the softening process. The saturating level of ethylene for all these events is less than 5 ppm, which is far lower than the internal ethylene concentrations found in the fruit at the climacteric peak (around 100 ppm). Ethylene-suppressed melons fail to produce aroma volatiles due to the inhibition of most of the steps of the biosynthesis pathway leading to ester volatiles (Flores et al., 2002). Detachment of the fruit influences the development of the respiratory climacteric (Bower et al., 2002). Fruit remaining attached to the vine, although producing higher levels of ethylene, exhibit a reduced climacteric rise in respiration when compared with detached fruit. The response of antisense ACO fruit to exogenous ethylene, in terms of respiration, is higher in detached than in attached fruit. It has been shown that antisense ACO melons display enhanced tolerance to low-temperature disorders during and after storage at 2 C when compared with the wild-type fruit (Ben Amor et al., 1999). The existence of both ethylene-dependent and -independent pathways was further confirmed at the molecular level by comparative analysis of gene expression between wild-type and AS melons (Hadfield et al., 2000). This study identified a set of genes whose expression was unchanged and two other categories corresponding to genes whose expression was down- and up-regulated by ethylene suppression, clearly indicating that both ethylene-dependent and -independent pathways of gene regulation do co-exist in climacteric fruit.

13 Ethylene and Fruit Ripening 287 Transcriptional cascade signal amplification and diversification Ethylene biosynthesis Auxin C 2 H 4 Developmental factors EIN3 RIN? LeHB-1 Aux/IAA PERE ERFs ERFs ERFs TAGI NOR Associated transcription factors ACS, ACO Nucleus ACO ACS Ripening-related genes Other signs (ABA, light, etc.) Ripening-related changes Aroma Colour Respiration Texture Taste Figure 11.3 Model for nuclear events leading to ethylene-dependent and -independent gene expression during fruit ripening. Several signalling molecules (hormones, developmental factors, etc.), among which ethylene is the most important, act through perception and transduction pathways to stimulate the expression of ripening-related genes. Ethylene regulates the expression of EIN3/EIN3-like (EILs) transcription factors that bind to the primary ethylene-response elements (PEREs) of the promoters of ethylene-response factors (ERFs). ERFs bind to GCC-boxes in the promoter region of several ripening-related genes resulting in signal amplification. Aux/IAAs transcription factors regulate the activity of auxin-response factors (ARFs) to promote the expression of auxin-responsive genes. Auxins can also regulate the expression of some ERFs, indicating a crosstalk between the two hormones. Developmentally regulated transcription factors, such as RIN, TAG1 and LeHB-1, regulate fruit ripening upstream of ethylene by controlling the expression not only of some ACS and ACO, but also of some ripening-related genes. Other signals, not well characterized and often acting in interaction, mediate the expression of another pool of genes.

14 288 The Plant Hormone Ethylene 11.7 Ethylene perception and transduction effects in fruit ripening Ethylene perception Soon after the identification of the ethylene receptors in Arabidopsis (see Chapter 5), it was discovered that the Never-ripe (Nr) mutant of tomato (which had been known for many years) corresponded to a mutation in the ethylene receptor, thus conferring ethylene insensitivity (Wilkinson et al., 1995). In tomato, ethylene receptors correspond to a family of genes comprising six members that have been classified into two subfamilies based on their structural features. In the tomato, LeETR1, LeETR2 and NR belong to subfamily 1, while LeETR4, LeETR5 and LeETR6 belong to subfamily 2 (Klee, 2004; Cara & Giovannoni, 2008). Ethylene receptors of the two families have been identified in a number of climacteric fruits including pear (El-Sharkawy et al., 2003), peach (Rasori et al., 2002), kiwi fruit (Yin et al., 2008) and the non-climacteric fruits grape (Deluc et al., 2007) and strawberry (Trainotti et al., 2005). The ethylene receptor genes are differentially expressed in organs and tissues, but none of them seem to have strict organ-specificity. However, some receptors are more highly expressed in certain tissues. For instance, NR, LeETR4 and LeETR5 exhibit a significant increase in ripening tomato fruit (Klee, 2002) and expression of NR is stimulated by ethylene (Wilkinson et al., 1995). Increased expression during fruit ripening and after ethylene treatment has been demonstrated for three receptors in pears, PcETR1a, PcERS1a, PcETR5 (El-Sharkawy et al., 2003), and one in peach fruit, PpERS1 (Rasori et al., 2002). In ripening kiwifruit, there is a diverse response of the five members of the ethylene receptor family to internal and external ethylene such that AdERS1a, AdETR2 and AdETR3 expression increases at the climacteric stage and transcripts are induced by external ethylene treatment, while AdERS1b showed no response to ethylene. AdETR1 was negatively regulated by internal and external ethylene (Yin et al., 2008). Interestingly, non-climacteric fruits exhibit increased expression of ethylene receptor genes concomitant with the increase in ethylene synthesis (Trainotti et al., 2005; Deluc et al., 2007). Receptors and other elements of the ethylene transduction pathway are also differentially regulated in early or late ripening plums showing typical climacteric or suppressed climacteric ripening patterns, respectively (El-Sharkawy et al., 2007). The physiological significance of the increase in expression of receptor genes during fruit ripening is not clear given the negative regulation concept governing how the receptors work. Accordingly, fruit ripening would require lower amounts of ethylene receptors to increase its sensitivity and response to ethylene. However, it has been shown that the levels of transcript and protein accumulation are disconnected such that the protein levels reach a maximum in immature fruit and decrease at the early stages of ripening, when the level of the corresponding mrna increases (Kevany et al., 2007). In addition,

15 Ethylene and Fruit Ripening 289 ethylene treatment of immature fruit results in degradation of the LeETR4 and LeETR6 receptor proteins via a proteasome-dependent pathway. Thus, a model has been proposed, which suggests that the timing of fruit ripening is related to the capacity to sense cumulative effects of ethylene through the gradual degradation of the receptor proteins (Kevany et al., 2007). The Nr mutant has been well characterized and it corresponds to a dominant mutation that affects the ethylene response and results in fruit producing reduced amounts of ethylene and retaining very low ethylene responsiveness (Lanahan et al., 1994). The NR gene encodes an ethylene receptor from the ERS (ethylene response sensor) family devoid of the receiver domain (Wilkinson et al., 1995). Another mutant, Green-ripe (Gr) also corresponds to a dominant ripening mutation that occurs in a gene encoding another component of ethylene signalling (Barry & Giovannoni, 2006) that corresponds to the REVERSION TO ETHYLENE SENSITIVITY1 (RTE1) shown to interact with and regulate the ETR1 ethylene receptor in Arabidopsis (Resnick et al., 2006; Zhou et al., 2007; see Chapter 5). Down-regulation of NR and LeETR1 receptors has been performed in tomato fruit with little effect on fruit ripening (Tieman et al., 2000; Whitelaw et al., 2002) probably due to functional compensation. For instance, in NR antisense lines, high expression of LeETR4 was detected (Tieman etal., 2000). However, suppression of LeETR4 and LeETR6 expression did result in accelerated fruit ripening but severely affected plant growth (Kevany et al., 2007), while fruit-specific suppression of LeETR4 resulted in early-ripening fruit without affecting plant growth (Kevany et al., 2008). Interestingly, antisense inhibition of the NR gene was capable of restoring normal ripening to the tomato NR mutant, supporting the evidence that the ethylene receptors act as negative regulators of ethylene action (Hackett et al., 2000). Beside post-translational regulation, ethylene perception is also controlled by a Green-Ripe (GR) protein, a tomato homologue of RTE1 from Arabidopsis, which acts as negative regulator of the ethylene response. The Gr mutant fails to ripen as a consequence of inhibition of ethylene responsiveness (Barry et al., 2005) and the GR protein is proposed to interact with and regulate the ethylene receptor(s) possibly via receptor-copper interaction (Zhou et al., 2007). The various modes of ethylene receptor regulation has been reviewed by Kendrick and Chang (2008) Chemical control of the post-harvest ethylene response in fruit ripening The discovery that some olefin compounds counteract the role of ethylene has been published in the early 1970s (Sisler & Pian, 1973). In fact, many compounds have been found that counteract the action of ethylene, but the most efficient has proved to be 1-methylcyclopropene (1-MCP) (Sisler, 2006). The 1-MCP molecule induces a covalent modification of the ethylene receptor and the affinity of 1-MCP for the receptor is related to specific chemical

16 290 The Plant Hormone Ethylene properties such as the strain of alkene molecule, pyramidalization and enantiomeric selectivity (Pirrung et al., 2008). This potent inhibitor of ethylene responses has been approved for commercial uses under the trade name of EthylBloc TM for use on ornamentals and SmartFresh TM for use on edible horticultural products, mainly fruit. As a gas, 1-MCP can only be used after harvest and many papers have been published on the effect of 1-MCP on the post-harvest behaviour of many horticultural products. A compendium of physiological processes or disorders in fruits, vegetables and ornamental products that are delayed or decreased, increased or unaffected by application of 1-MCP is available at website: faculty/watkins/ethylene/, and the role of 1-MCP is described in more detail in Chapter Ethylene signal transduction In Arabidopsis, a single gene, CTR1 (constitutive triple response 1), encoding a Raf-like protein kinase, acts downstream of the ethylene receptors as a negative regulator of ethylene signalling by repressing a positive regulator, EIN2 (see Chapter 6). In tomato, CTR is encoded by a family of three genes, one of them, LeCTR1, is highly expressed during ripening and upon ethylene treatment (Leclercq et al., 2002; Adams-Phillips et al., 2004). A PpCTR1 gene has also been isolated in peach fruit that shows up-regulation at early stages of ripening (Begheldo et al., 2008). The ethylene receptor NR has been shown to interact, at the endoplasmic reticulum, with all three tomato CTR proteins, but it is unknown whether the different LeCTRs are functionally redundant or have unique roles (Zhong et al., 2008). So far, no ripening phenotype has been observed by down- or up-regulating CTR genes in tomato. The only observation is that VIGS of CTR resulted in an epinastic phenotype and up-regulation of chitinase gene, an ethylene-inducible gene in vegetative tissues (Liu et al., 2002). Targeted inhibition of individual members of the family would help understand the functional role of CTR in fruit ripening. Downstream of CTR is the ETHYLENE INSENSITIVE 2 gene (EIN2) that shows similarity to natural resistance-associated macrophage proteins (NRAMP) metal ion carriers and is proposed to also play a role in the signal transduction of other hormones (Alonso et al., 1999; see Chapter 5). The expression of LeEIN2 is constant at different stages of fruit development in tomato and is not regulated by ethylene. Nevertheless, in tomato fruit downregulation of LeEIN2 gene using VIGS-mediated silencing results in a delay in fruit development and ripening in common with a down-regulation of ethylene- and ripening-related genes. In addition, the content of auxin and the expression of auxin-regulated genes were lower in EIN2-silenced fruit when compared with wild type, indicating that EIN2 might act as point of crosstalk between ethylene and auxin signalling (Zhu et al., 2006). However,

17 Ethylene and Fruit Ripening 291 in peach a transient increase in PpEIN2like gene expression has been detected at the early stages of ripening (Begheldo et al., 2008) The transcriptional cascade leading to the regulation of ethylene-responsive and ripening-related genes The ethylene transduction pathway leads to the regulation of ripening via a transcriptional cascade comprising primary (ETHYLENE-INSENSITIVE3 (EIN3) and EIN3-like (EIL)) and secondary response factors (ETHYLENE- RESPONSE FACTORS (ERF)). Four EIL genes have been identified in tomato with only LeEIL4 being up-regulated during tomato fruit ripening (Tieman et al., 2001; Yokotani et al., 2003). Further, transgenic tomato plants with reduced expression of a single LeEIL gene did not exhibit significant changes in ethylene responses suggesting a functional redundancy among family members and the down-regulation of multiple tomato LeEIL genes was necessary to significantly reduce ethylene sensitivity and to affect fruit ripening (Tieman et al., 2001). Overexpression of LeEIL1 in the Nr mutant, where its expression is low, restored fruit ripening and stimulated the expression of some, but not all, ethylene-responsive genes (Chen et al., 2004). EIN3 and EIL proteins were shown to bind to the primary ethylene-response element present in the promoter of target ERF genes (Solano et al., 1998). In addition to tomato, EIN3-like genes have been isolated from banana where five genes have been identified with only one, MA-EIL2, being ripening and ethylene regulated (Mbéguié-A-Mbéguié et al., 2008). Another major control point of the ethylene-signalling pathway is the post-translational modification of the EIN3-like proteins (Chao et al., 1997; Kendrick & Chang, 2008). In Arabidopsis, EBF (EIN3-binding F-box) proteins were shown to negatively regulate ethylene signalling via mediating the degradation of EIN3/EIL proteins (Potuschak et al., 2003; see Chapter 5). The identification of two tomato F-box genes, SlEBF1 and SlEBF2, from the EBF subfamily was reported recently and the expression of the two genes was shown to be regulated by both ethylene and auxin (Yang et al., 2010). Silencing of SlEBF1 and SlEBF2 expression caused a constitutive ethylene response phenotype and accelerated fruit ripening (Yang et al., 2010). The data indicate that the coordinated regulation of SlEBF1 and SlEBF2 is fundamental to tomato growth and that the dynamic regulation of these genes is essential for fruit ripening. The data also support the hypothesis that protein degradation via the ubiquitin/26s proteasome pathway is also an important control point of fruit ripening. However, it remains to be determined whether the EBF regulation of fruit ripening observed in tomato also operates in other types of fleshy fruits. The ERFs correspond to a large family of transcription factors that regulate the expression of target genes through the binding to cis-regulatory elements containing a conserved GCC motif (Ohme-Takagi & Shinshi, 1995; see Chapter 7). So far, only a limited number of ERF genes have been characterized

18 292 The Plant Hormone Ethylene in species bearing fleshy fruit. However, searches in silico has recently identified up to 85 ERF genes from tomato and the differential expression displayed by these ERF genes during fruit development and in response to abiotic stresses suggests diverse roles (Sharma et al., 2010). However, due to the high likelihood of functional redundancy among these family members, data demonstrating their role in mediating ethylene responses during fruit ripening are scarce. However, a ripening-associated pattern of expression has been shown for LeERF2 (Tournier et al., 2003) and LeERF3b (Chen et al., 2008) in tomato fruit. Further, tomato-expressing and -antisense LeERF2 displayed a significant decrease in the expression of LeACS and LeACO genes when compared with wild-type and tomato-overexpressing LeERF2 (Zhang et al., 2009a), indicating that LeERF2 is a positive regulator in the feedback loop of ethylene production. However, the effect of down-regulation of LeERF2 on fruit ripening was not reported. Seven PsERF genes have been isolated and are expressed at all stages of fruit development in two Japanese plum varieties (El-Sharkawy et al., 2009) with a sharp but transient up-regulation during fruit ripening. Although there were some differences between varieties, the expression of most ERFs was inhibited by the ethylene antagonist 1-MCP, indicating that ethylene was a major regulator of PsERFs expression. In common with tomato, expression is also influenced by other hormones including auxins, cytokinins and gibberellins (GAs). Two MdERFs have been isolated from apple fruit, with MdERF1 being expressed predominantly in ripening fruit with low expression in non-fruit tissues, while MdERF2 is expressed exclusively in ripening fruit (Wang et al., 2007). The increased expression in ripening fruit was repressed by 1-MCP treatment, again indicating that transcription is positively regulated by the ethylene signalling. Moreover, cultivars with low ethylene production tend to show lower MdERFs expression compared to those displaying high ethylene production. The role of ERFs in controlling fruit ripening was also shown in tomato, where LeERF1 was reported to control fruit ripening and softening (Li et al., 2007). Nevertheless, the binding of ERF to the promoter of a ripeningrelated gene has not been demonstrated so far, and therefore the specific role of each ERF in the ripening process is far from being understood Hormonal crosstalk in fruit ripening The exogenous application of hormones, including auxins, GAs and abscisic acid (ABA), has long suggested the existence, in concert with ethylene signalling, of additional regulatory mechanisms that control fruit ripening. The introduction of auxins into unripe strawberry fruit through the peduncle delayed subsequent fruit ripening as assessed by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness (Given et al., 1998). As well, the inhibition of fruit softening following exogenous application of