Genetic Regulation of Fruit Development and Ripening

Fruit development and ripening are unique to plants and represent an important component of human and animal diets. Recent discoveries have shed light on the molecular basis of developmental ripening control, suggested common regulators of climacteric and nonclimacteric ripening physiology, and defined a new role for MADS box genes in this late stage of floral development. Analyses of fruit-ripening mutants and ripening-related gene expression suggest higher levels of a developmental regulatory cascade that remain to be defined. Examination of the molecular basis of ethylene signaling in tomato has demonstrated conservation of the basic model defined in Arabidopsis, yet with modifications in gene family composition and expression that may represent adaptations to promote successful fruit development and seed dispersal. The role of light signaling in fruit carotenoid accumulation is being examined and may represent a target for practical manipulation of fruit pigmentation and nutrient content. The continuing development of genomics tools, including ESTs and cDNA microarrays, for important fruit crops should foster accelerated discovery in fruit development and ripening research. By anatomical definition, the fruit is a mature ovary and therefore typically includes carpel tissues in part or in whole. Many fleshy fruit species important to humans additionally develop mature fruit tissues, including extracarpellary floral components. Examples include strawberry, pineapple, mulberry, and pome fruit (apple, pear), in which the receptacle, bracts, calyx, and floral tube (the fused base of floral organs), respectively, constitute the majority of mature fruit tissue. Even species with fruit derived from carpel tissue exclusively can display a range of developmental programs, spanning the relatively uniform single expanded carpel or drupe of stone fruit to the differentiated carpel tissues giving rise to the peel (flavedo) and multicarpel flesh of citrus and banana. Evolutionary pressures have resulted in a variety of developmental manifestations of fruit tissues, resulting in structures that range in design and function from hardened fruit capsules or pods that forcefully expel seeds at maturation, to forms optimized for seed movement by wind, water, animal fur, or gravity, to those implementing developmental programs that yield succulent and flavorful tissues for organisms that consume and disperse the associated seed. Tanksley (this issue) discusses the impact of domestication on selection for fruit genes that influence size and shape early in fruit development in addition to discoveries regarding their underlying molecular functions. The focus here will be on recent advances in our understanding of developmental and signaling pathways that affect later fruit maturation and ripening. Although dehiscent and dry fruit types (e.g., cereals) represent the majority of plant species, fruit developmental studies to date have focused primarily on fleshy species because of their importance in the human diet. Particular emphasis has been placed on tomato as an especially tractable system for molecular genetic analysis of fleshy fruit development and ripening (Giovannoni, 2001). Arabidopsis also has proven exceptionally informative as a model system for floral development in general (Lohmann and Weigel, 2002) and gene identification and the subsequent functional analysis of carpel identity–, development-, and maturation-associated genes (Pinyopich et al., 2003, and references therein). In addition, the Arabidopsis silique is a dehiscent fruit characteristic of the legumes and thus represents another exceptionally important fruit type in terms of human and animal food. Other systems will be mentioned where appropriate, but the majority of this review will focus on Arabidopsis and tomato as the major systems underlying many recent discoveries in fruit development and ripening. Since the initial description of a requirement for the AGAMOUS (AG) protein for carpel and stamen determination (Bowman et al., 1989), a large family of Arabidopsis MADS box genes has been reported and in many cases functionally defined (Alvarez-Buylla et al., 2000, and references therein). For example, the redundant SEPALLATA genes (SEP1, SEP2, and SEP3) can be eliminated via mutation individually with minimal impact on floral development, yet the triple mutant results in the conversion of all floral organs to sepals, indicating roles in normal petal, stamen, and carpel development (Pelaz et al., 2000). Spatial constraint of AG expression via negative regulation by the APETALA2 (AP2) EREBP-like protein provided early evidence that additional transcription factors also play important roles in floral and carpel development, in part via the regulation of MADS box genes (Drews et al., 1991). Additional insight into the molecular basis of carpel determination in the developing flower came through the recent discovery that two previously described MADS box SHATTERPROOF genes (SHP1 and SHP2; Liljegren et al., 2000), originally associated with carpel dehiscence and residing in the same phylogenetic clade as AG, provide functional redundancy to AG in carpel determination (Pinyopich et al., 2003). AG, SHP1, and SHP2 also are functionally redundant with the SEEDSTICK MADS box gene as determinants of ovule identity. Normal ovule development in turn influences later carpel expansion as a response to successful fertilization (Ferrandiz et al., 2000). SHP1 and SHP2 in particular are capable of functioning in carpel and ovule determination, carpel expansion, and dehiscence of the mature fruit. This level of functional redundancy suggests that the main determinant of the primary in vivo developmental roles of members of this group of related MADS box genes is through differential gene expression (Pinyopich et al., 2003). Given the number and similarity of plant MADS box genes, this paradigm may be repeated, possibly for functions that are unique to fruit development of specific or related sets of plant species, suggesting that it may be useful to identify and characterize MADS box genes expressed in developing fruit. Given the diversity of fruit development programs across the plant kingdom and molecular insights developed in Arabidopsis (especially with respect to MADS box genes), it will be important to determine how this family has evolved in number, function, and target genes to facilitate fruit form and development in diverse plant species. Antisense repression of the tomato AG homolog TAG1 caused homeotic conversion of inner floral whorls similar to that observed in Arabidopsis ag mutants, whereas ectopic expression resulted in the development of red fleshy sepals, suggestive of ripe fruit tissues and consistent with a role in carpel determination (Pnueli et al., 1994b). Independent antisense repression of two tomato SEP homologs, TM5 (Pnueli et al., 1994a) and TM29 (Apomah-Dwamena et al., 2002), resulted in a range of anticipated and unanticipated phenotypes (based on the Arabidopsis model) suggestive of less functional redundancy than was seen in Arabidopsis. Specifically, TM5 repression resulted in partial conversions of carpels, stamens, and petals to less specialized structures and resulted in additional organ whorls, whereas TM29 repression yielded green stamens and petals in addition to parthenocarpic fruit from which additional shoots emerged. Although mutations in SEP genes indicate redundant functions in the determination and development of the three inner floral whorls of Arabidopsis, the use of full-length cDNAs for antisense of both TM5 and TM29 (and the high degree of sequence similarity in the MADS box domain) limits our ability to define specific roles for these genes in tomato in the absence of additional characterization of transgene effects at the molecular level. Nevertheless, mutations in crop MADS box genes have been useful in defining a MADS box role in tomato pedicel abscission zone formation (Mao et al., 2000) and the functional basis of parthenocarpic (seedless) fruit development in apple (Yao et al., 2001). An additional tomato MADS box gene regulating fruit ripening (Vrebalov et al., 2002) is described below. The ripening of fruit organs represents the terminal stage of development in which the matured seeds are released. In the dehiscent fruit of the Arabidopsis silique, this process is facilitated by senescence of the mature carpel tissue followed by separation of the valves at an abscission cell layer (termed the dehiscence zone) that is formed between the valve-replum boundary. The MADS box SHP1 and SHP2 genes were shown originally to regulate the formation of the dehiscence zone (Liljegren et al., 2000) under the negative regulation of the FRUITFUL (FUL) and REPLUMLESS gene products, which together limit SHP expression to the dehiscence zone (Ferrandiz et al., 2000; Roeder et al., 2003). The SEEDSTICK MADS box gene was demonstrated recently to be required for the formation of the funiculus/seed abscission zone that allows separation of the seed from the carpel to facilitate seed dispersal at dehiscence (Pinyopich et al., 2003). Major Developmental Changes during Tomato Fruit Development and Ripening. Relative changes in cell division, cell expansion, respiration, ethylene synthesis, fruit softening, and carotenoid accumulation are shown over the course of fruit development. The time from anthesis (a) to mature green (MG; fully expanded unripe fruit with mature seed), breaker (BR; first visible carotenoid accumulation), and red ripe (RR) can vary substantially among cultivars. The time line shown would be for a medium-/large-fruit cultivar such as the breeding line MH1 (5 to 7 cm diameter mature fruit). dpa, days after anthesis. Examples of common climacteric fruits that require ethylene for ripening include tomato, apple, banana, and most stone fruits, whereas nonclimacteric fruits, including grape, citrus, and strawberry, are capable of ripening in the absence of increased ethylene synthesis. Interestingly, climacteric fruit span a wide range of angiosperm evolution, including both dicots (e.g., tomato) and monocots (e.g., banana). Nevertheless, members of the same (e.g., melon) or closely related (e.g., melon and watermelon) species are reported to include both climacteric and nonclimacteric varieties. The molecular distinctions underlying climacteric versus nonclimacteric ripening are poorly understood. Nevertheless, it seems likely that at least in instances of the same or closely related species with examples of both climacteric and nonclimacteric types, that nonclimacteric phenotypes may represent mutations in ethylene synthesis or signaling as opposed to more complex distinctions. Indeed, nonclimacteric melons are notoriously difficult to harvest compared with their climacteric counterparts because of reduced abscission, suggesting a defect in ethylene synthesis or response and a mature phenotype consistent with incomplete ripening (Perin et al., 2002). In this regard, it is especially important when selecting a system for the analysis of nonclimacteric ripening to be certain that the ripening physiology of the candidate species is well characterized and consistent with nonclimacteric ripening as opposed to inhibited ripening resulting from reduced ethylene synthesis or Although the specific role of climacteric in fruit ripening the of ethylene as a of ripening in climacteric species likely to facilitate and ripening. is regarding specific ripening in a number of climacteric and nonclimacteric species, yet is the regulation of ripening in nonclimacteric fruit or the regulation of ethylene in their climacteric Recent evidence of the MADS box regulation of ripening in both tomato and suggests common regulatory early in both climacteric and nonclimacteric species (Vrebalov et al., 2002). The of the molecular basis of such early and common represents an in fruit ripening research. Tomato has as the primary model for climacteric fruit ripening for a of and The importance of tomato as an has resulted in of and breeding that have yielded and including many that affect fruit development and ripening can be and at the Tomato and size and and of genetic and including et al., et al., 2002) and Fruit ESTs from for and Fruit ESTs from for is the most system for nonclimacteric resulting in the identification and characterization of ripening-related genes that affect cell and et al., and 2002). The of has genetic analysis in this species, is et al., and are Recent and of and to a degree species suggests the of their roles as for nonclimacteric the of these crops will limit their as basic in plant tissues results from The in fruit ethylene synthesis include the conversion of to via and the subsequent of to ethylene by In tomato and most characterized both are by least genes are expressed in tomato fruit et al., et al., 2000). and are under developmental and are for the of ripening are at the of and this is by mutation at the et al., 2000). Fruit for the mutation to the in ethylene and fruit are capable of to as shown by the of gene and The a MADS box transcription and the of mutant phenotypes described has been to a function in ripening over climacteric ethylene synthesis via the of and in addition to a regulatory process the of ethylene influence (Vrebalov et al., 2002). is under ethylene and thus ethylene of climacteric in response to ethylene resulting from and The tomato fruit is for ethylene synthesis and is in response to ripening ethylene et al., 2000). Although most plant tissues an of two tomato fruit genes also are during ripening in response to ethylene and thus to ethylene synthesis et al., for the of Tomato Fruit Ripening. Fruit mutations for the genes or are The and mutants are all with The and mutants are from reported and with triple mutant in and with associated of carotenoid accumulation also is of Arabidopsis ethylene response mutants, for and of their genes have resulted in the development of an of ethylene by and 2000; and 2000). have and characterized genes from tomato in an to the degree of conservation of the basic signaling defined in Arabidopsis and to (especially related to fruit development, and in crop species. The of tomato and plants via the of Arabidopsis ethylene demonstrated the functional conservation for this component of ethylene signaling et al., The first ethylene in tomato was through the of the fruit-ripening et al., of and ethylene in the mutant et al., to a candidate in which tomato of Arabidopsis ethylene genes were and for to et al., to be a tomato gene similar to the Arabidopsis et al., a mutation that ethylene et al., expression analysis of and additional tomato that and are most in ripening fruit tissues et al., et al., et al., of gene antisense suggested functional redundancy similar to that reported for the Arabidopsis gene expression was resulting in unique manifestations et al., 2000). Specifically, in expression normal phenotypes as a of the of repression was for by the expression of was tomato resulting in ethylene response and accelerated via the ectopic expression of The impact of repression was in that it suggested that in tomato a single a role in ethylene in to Arabidopsis, in which genes be to ethylene phenotypes and The increased and expression during ripening also suggests a response to a for to facilitate the of ethylene in ripening fruit Although ethylene genes have been in Arabidopsis, have been to date in tomato and in Arabidopsis have been shown to with the protein et al., et al., and mutation in results in of all ethylene indicating a negative regulatory role in ethylene signaling et al., tomato homolog was from ripening fruit and shown to be capable of functioning in Arabidopsis ethylene via of the mutation et al., 2002). also was shown to be during ripening and in response to indicating of the of ethylene signaling in addition to during fruit ripening. of the tomato yielded an additional and a was from a cDNA as a gene has been reported to date in Arabidopsis. of the additional tomato genes are more similar at the and sequence levels to than to gene in the Arabidopsis suggesting the of additional genes function in tomato and The Arabidopsis and related genes transcription factors that represent of ethylene signaling et al., tomato genes capable of the Arabidopsis ethylene phenotype were during ripening or by ethylene et al., 2001). of gene via antisense resulted in whereas repression of all three genes resulted in ethylene for all Although this of genes is suggestive of in tomato family size or it that additional genes be of ethylene signaling in tomato suggests that at the early of the during ripening. ethylene exceptionally et al., and may in the formation of a complex with et al., et al., species tissues are to high of ethylene as part of normal developmental or response may have gene family size expression to ethylene and during of increased ethylene synthesis. model in which the to the of ethylene is consistent with a negative regulatory that may to ethylene signaling during climacteric ripening The molecular basis of ethylene synthesis and regulation has been a of ripening analysis in the with more recent emphasis to characterization of ethylene Antisense repression of tomato et al., and et al., genes and similar in melon et al., the role of these genes in regulating climacteric ethylene synthesis. is well that ethylene is for ripening and that a developmental to to ethylene be et al., et al., 2001). fruit typically in response to of ripening including the of in demonstrated that of climacteric fruit ripening are by developmental factors that be with ethylene synthesis et al., Developmental mutations that affect all of the tomato fruit-ripening process have been for and include the mutation is similar to in that fruit to climacteric ethylene or yet to ethylene at the molecular level to in response to ethylene and the and were on the tomato genetic as first in et al., The MADS The of tomato in the mutant is shown with the The in the of and the of this gene in addition to the and transcription of the that constitute the of the mutant is shown in the with of the normal and shown (Vrebalov et al., 2002). of the tomato provided the first molecular insight into the developmental regulation of climacteric ethylene synthesis and fruit ripening. The identification of a MADS box cDNA with suggests the that MADS box may represent a function in the regulation of ripening in both climacteric and nonclimacteric species (Vrebalov et al., 2002). analysis of this gene through antisense repression in is in and because MADS box genes have been shown to as or higher et al., it is that additional tomato MADS box genes may in ripening. than members of the tomato MADS box family are as ESTs tomato to and and cDNA that for at least of these genes are expressed in early ripening or fully ripe fruit tomato gene expression on to which was shown previously to be expressed in and ripe fruit (Vrebalov et al., 2002), in addition to these genes and are thus for genes MADS box that may with TAG1 and have been in (Pnueli et al., et al., 2002), and it is additional MADS box genes may have been in these in both ripening of mature carpel tissues of MADS of and domain) of MADS box gene were in analysis the and are tomato genes, and SEP2, and are Arabidopsis The gene is at the of ripening influence by indicating higher regulatory (Vrebalov et al., 2002). gene expression analysis in and fruit suggests an of genes, including that to developmental and ethylene in but fruit et al., expression has been characterized and is to of ripening levels in mature green fruit at a time consistent with the of ripening and normal expression in response to ethylene et al., et al., The regulation of expression in and the absence of expression in with the to ripening in mutant via a minimal regulatory that during fruit ripening. In this ethylene a of ripening genes or in with developmental by the gene represents a regulatory in which but developmental In this gene expression via the of ethylene synthesis. Although tomato ripening genes have been for expression changes in relatively have been characterized in characterization of ripening gene expression in will facilitate the of developmental regulation during which is by both mutations in that The has been and a transcription with to MADS box gene and The of this sequence also should promote the of the developmental regulatory fruit ripening Additional tomato ripening mutants are and the of their should in the expansion of our understanding of ripening (Giovannoni, 2001). For example, the mutation also results in ripening et al., can be by ethylene in and the mutation affect the expression of et al., 2002), suggesting a function of or in a regulatory cell and associated changes have been a major focus of ripening the of the tomato fruit has been reported to represent of ripening fruit and results in cell in with the of ripening and et al., and references therein). expression is inhibited substantially by both the and with additional influence by ethylene et al., antisense repression et al., and ectopic expression in unripe fruit et al., that is for Nevertheless, a in ripe fruit to in antisense fruit to the of antisense The of the that the primary determinant of tomato fruit caused to turn to the and functional analysis of cell by and et al., 2001). of from fruit cell ripening by of to is expressed ripening and is by ethylene as ripening Although repression of tomato fruit via antisense resulted in increased to the of was et al., In addition to have been characterized in ripening fruit. of the ripening-related as or and pedicel and fruit abscission, respectively, but influence fruit et al., et al., a ripening-related and tomato have a impact on fruit when via antisense et al., 2002), as repression of the ripening et al., et al., The of cell is by an complex of cell and many of which are by that likely to the of the molecular basis of fruit cell Although has been in the of specific cell during fruit the molecular basis of fruit is poorly and an of and represent the primary of ripe fruit pigmentation in that carotenoid synthesis have been from tomato and to a number of previously defined pigmentation mutants et al., and references therein). Examples include the resulting in of the gene and of or reduced expression of the carotenoid resulting in the fruit of the mutants et al., 2002), and and mutations of the resulting in and et al., 2000). Although a has been the of the carotenoid synthesis in recent regulation of through the is a is that is by ethylene during indicating a major for fruit carotenoid accumulation et al., 2000). In addition, analysis of associated with tomato fruit carotenoid that in addition to to carotenoid et al., 2003). has been shown to affect carotenoid accumulation in a number of species, including et that light was required for normal ripe fruit pigmentation but affect ripening Tomato and mutants, characterized by increased green fruit and in addition to increased ripe fruit have been shown to be to light et al., expression of an in tomato resulted in phenotypes similar to those by and the role of light in fruit carotenoid accumulation and The has been and shown to the tomato homolog of the Arabidopsis negative of light additional molecular evidence for the regulation of carotenoid synthesis via light et al., Indeed, this with the of on fruit suggests that manipulation of genes may be a useful for fruit pigmentation and associated The of genomics the of more and in ripening in a cDNA et an that to development in of the first of expression analysis in fruit. Although the of a major fruit crop to be review of the is an of where are likely to in the than tomato ESTs have been developed and in this the of fruit crop species are cDNA defining at least tissue types and including ESTs from fruit at of development et al., 2002). similar number of ESTs are from In is the species for which the number of fruit ESTs are more fruit ESTs than for Interestingly, are to for many of the most important fruit species in terms of ESTs derived from and can be informative in their in that the and of ESTs a can as a of gene expression et al., et al., 2003). the majority of tomato and ESTs are derived from such expression analysis should be and has been developed for tomato such for additional fruit species, it will to the expression of genes across species on a genomics for example, of between climacteric and nonclimacteric species to identify both and unique ripening functions. Although expression analysis can be useful for expression in tissues from which ESTs have been this is a for gene expression tomato cDNA has been developed with that gene has been developed to and of resulting from the use of the tomato to et al., 2001). and are to use these for analysis of fruit development and ripening The recent development of genomics for tomato will promote and expanded discovery in this model of fruit development and ripening. to for with of the and for to and for with and to for in is by from the and the of for and the and Development and the of