Planta DOI 10.1007/s00425-014-2057-7

Review

Unraveling the signal scenario of fruit set Mariana Sotelo‑Silveira · Nayelli Marsch‑Martínez · Stefan de Folter 

Received: 12 November 2013 / Accepted: 5 March 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Long-term goals to impact or modify fruit quality and yield have been the target of researchers for many years. Different approaches such as traditional breeding, mutation breeding, and transgenic approaches have revealed a regulatory network where several hormones concur in a complex way to regulate fruit set and development, and these networks are shared in some way among species with different kinds of fruits. Understanding the molecular and biochemical networks of fruit set and development could be very useful for breeders to meet the current and future challenges of agricultural problems.

Introduction Fruits are essential for human diet and present a wide variety of forms, with both dry and fleshy types (Fig. 1). In the M. Sotelo‑Silveira · S. de Folter (*)  Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Km. 9.6 Libramiento Norte, Carretera Irapuato‑León, CP 36821 Irapuato, Guanajuato, Mexico e-mail: [email protected] M. Sotelo‑Silveira e-mail: [email protected] M. Sotelo‑Silveira  Laboratorio de Bioquímica, Departamento de Biología Vegetal, Facultad de Agronomía, Universidad de la República (UdelaR), Garzón 780, CP 12900 Montevideo, Uruguay N. Marsch‑Martínez  Departamento de Biotecnología y Bioquímica, CINVESTAVIPN, Km. 9.6 Libramiento Norte, Carretera Irapuato‑León, CP 36821 Irapuato, Guanajuato, Mexico e-mail: [email protected]

last years, the process of fruit development has been the subject of many studies aimed at investigating the biochemical and genetic factors that control fruit growth and maturation (Alabadi et al. 2009; Giovannoni 2004; Ozga and Reinecke 2003; Seymour et al. 2013). Traditional breeding, mutation breeding, and transgenic approaches have been used to improve quality and overcome common agricultural problems, but unraveling the mysteries of metabolic interactions and their relationships to crop phenotypes will help to focus breeding strategies and improve efficiency and outcome (Giovannoni 2006). Several studies at the transcriptomic level have explained the role of phytohormones in fruit set regulation demonstrating that many phytohormones regulate this process in a complex way (Pandolfini 2009). Studies on dry fruits (siliques) of Arabidopsis have identified a suite of transcription factors involved in their development and dehiscence (Roeder and Yanofsky 2006; Sundberg and Ferrándiz 2009). In most cases, fruit develop from the gynoecium, the female reproductive organ. The gynoecium is a complex structure that requires the action of many genes that control the correct development of distinct tissues and cell types, giving rise to, e.g., the stigma, style, ovary, and the internal structures (Reyes-Olalde et al. 2013). These tissues must develop correctly to allow fertilization, growth and protection of the developing seeds, and finally, when the fruit and seeds are mature, to aid seed dispersal. During gynoecium and fruit development, many transcription factors are expressed in a dynamic fashion, as, for instance, shown in an expression profile study during the dry Arabidopsis fruit development (de Folter et al. 2004). Studies in tomato have also revealed a regulatory network involved in the developmental regulation of fruit set and ripening in these fleshy fruits (Ampomah-Dwamena et al. 2002; Bemer et al. 2012; Fujisawa and Ito 2013; Fujisawa et al. 2012, 2013; Giovannoni 2004; Itkin et al.

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Fig. 1  Examples of fruit diversity in shape, size, color, and texture

2009; Karlova et al. 2011; Mazzucato et al. 2008; Qin et al. 2012; Vrebalov et al. 2009). Intriguingly, the Arabidopsis orthologs or homologs of the tomato transcription factors, various belonging to the MADS domain family, confer floral organ and fruit tissue identity (Alvarez-Buylla et al. 2010; Roeder and Yanofsky 2006). New strategies for crop improvement could arise using these new findings. This review will focus mainly on the advances made in understanding the signals controlling development after fertilization, particularly in fruit set and mainly in Arabidopsis and tomato as the major models with recent discoveries in fruit development, although other systems will be mentioned where appropriate.

Parthenocarpy, a paradox to normal fruit set and development Successful sexual plant reproduction depends on fruit set, an essential process that can be defined as the activation of a developmental program, which will convert the pistil or gynoecium into a developing fruit with seeds. This process includes two different and coordinated steps: the fertilization of the ovule and the growth of structures that will protect the developing seeds. In most species, the coordination between these two events relies on the signal(s) that promotes fruit growth, which exclusively originates from the developing seeds. So, it is generally accepted that in the absence of pollination and fertilization, the ovary will cease cell division and abscise (Carbonell-Bejerano et al. 2010; Gillaspy et al. 1993; Ozga and Reinecke 2003; Talon et al. 1992). However, some species develop seedless fruits without fertilization. This kind of development, called

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parthenocarpy, is a paradox to normal fruit set and development, and represents a wonderful model of study that is useful to elucidate the molecular and genetic bases underlying fruit set and early fruit development (Dorcey et al. 2009; Table 1). Natural parthenocarpy has been proposed to be the result of conditions that induce a threshold concentration of growth substances in the ovary, which is sufficient to promote growth in the absence of pollination and fertilization (Fos et al. 2000). This kind of parthenocarpy has a genetic basis and it happens in numerous species of commercial interest. In tomato, several parthenocarpic mutants (pat, pat-2, pat-3, pat-4) were described to be due to mutations in different genes (Gorguet et al. 2005). The pat mutation induces homeotic conversions of the stamens, although is not allelic with PISTILLATA (PI) or APETALA3 (AP3), which in Arabidopsis guide the development of these tissues (Mazzucato et al. 2008). In pat-2, parthenocarpy is probably due to a higher synthesis of active GA in the developing unpollinated ovaries as a result of the accumulation of GA20 (Fos et al. 2000). Several apple (Malus domestica) mutants are known to produce only apetalous flowers that readily go on to develop into parthenocarpic fruits, which is caused by a loss-of-function mutation in the PI MADS-box transcription factor (Yao et al. 2001). In tomato, downregulation of the SEPALLATA (SEP) ortholog TM29 MADS-box gene produces parthenocarpic fruits, suggesting that it may limit parthenocarpic fruit development (Ampomah-Dwamena et al. 2002). Interestingly, the Arabidopsis SEP genes do not present this function. These distinct effects in the different species might be attributed to differences in fruit structure and fruit development processes between tomato and Arabidopsis (AmpomahDwamena et al. 2002). A similar case was described for the floral organ identity gene PI, which is associated with parthenocarpic fruit development in apple, as mentioned above, but not in Arabidopsis (Yao et al. 2001). Other examples are seedless crops obtained by traditional breeding methods such as seedless grapes, citrus, cucumber, and watermelon, although some of them are not parthenocarpic, as is the case of grape in which fertilization takes place but the seeds abort afterwards (Gustafson 1942; Pandolfini et al. 2009). Some examples of parthenocarpy point out to the central role of the ovules during fruit set and growth. Ovules are complex structures that are found in all seed-bearing plants, which have one or two protective integuments and the megagametophyte (i.e., embryo sac) (Battaglia et al. 2009). Double fertilization, i.e., fertilization of the egg cell and the central cell, occurs in the mature embryo sac, and it is considered as the primary site from where fruit set is triggered (Berger et al. 2008; Fuentes and Vivian-Smith 2009). The integuments have two roles after fertilization:

Antisense downregulation Emasculation of quadruple mutant Overexpression of CcGA20ox1 in tomato

Gibberellin signaling

Gibberellin signaling

Gibberellin biosynthesis

SIDELLA

AtDELLA

CcGA20ox1

Increased GA20ox leading to higher content of GA and parthenocarpy in tomato 13-hydroxylation pathway of GA biosynthesis is enhanced before anthesis and parthenocarpy in tomato Parthenocarpy in tomato Parthenocarpy in apple Increased levels of Kaempferol and parthenocarpy in Arabidopsis

Stimulates one or more steps of GA biosynthesis Single recessive mutations/not identified gene

Stimulates one or more steps of GA biosynthesis Single recessive mutation/not identified gene Downregulation of TM29 Loss-of-function (pi) Overexpression of AtCYP78A9

Prevention of pistil growth before fertilization

Prevention of pistil growth before fertilization

Communication between seed and valves during fruit development. Expression in inner integument of developing ovules, embryo development, epidermis of embryo, upregulated after fertilization

pat-3/pat-4

TM29

PI

AtCYP78A9

Higher content of GA4 than wild type and parthenocarpy in Arabidopsis Reduced GA1 and GA4 content and seed abortion and shorter siliques than wild type Arabidopsis

pat-2

AtGA2-oxidases Gibberellin inactivation/suppress elongation of Knock out all AtGA2-oxidases the pistil prior to fertilization GA biosynthesis Highly expressed in developing Double knock out AtGA20ox1 seeds AtGA20ox2

Higher content of GA4 than wild type and parthenocarpy after emasculation in tomato

(Ampomah-Dwamena et al. 2002) (Yao et al. 2001) (Ito and Meyerowitz 2000; Marsch-Martinez et al. 2002; Sotelo-Silveira et al. 2013a, b)

(Fos et al. 2001)

(Fos et al. 2000)

(Rieu et al. 2008b)

(Rieu et al. 2008a)

(García-Hurtado et al. 2012)

(Martí et al. 2007)

(Ren et al. 2011)

(Mounet et al. 2012)

(Molesini et al. 2009)

AUCSIA –silenced plants have an increased level of auxin at preanthesis and produced parthenocarpy in tomato Parthenocarpic fruits present slight modifications on auxin homeostasis and minor alterations in ARF and Aux/IAA gene expression levels Altered expression levels of auxin-responsive genes and parthenocarpy in tomato GA constitutive response and parthenocarpy in tomato Parthenocarpy in tomato

Silencing PIN4 tomato gene

(Wang et al. 2005)

Parthenocarpy, early fruit growth in tomato

Downregulation using antisense and constitutive promoter RNAi silencing with phloem-specific promoter

Overexpression of SlTIR1

SlTIR

SlPIN4

SlAUCSIA

SlIAA9

(Goetz et al. 2006, 2007a)

References

(de Jong et al. 2009b)

Parthenocarpy in Arabidopsis and tomato

Effect observed (fruit phenotype/altered hormonal content)

RNAi silencing. Constitutive promoter Parthenocarpy in tomato

arf8-4 (At) Expression of AtARF8-4 mutated gene

Auxin signaling/prevention of pistil growth before fertilization. Expressed in ovules that were not fertilized, downregulated after fertilization Auxin signaling/prevention of pistil growth before fertilization Auxin signaling/prevention of pistil growth before fertilization Auxin signaling/prevention of pistil growth before fertilization. Expressed in the ovary, downregulated after pollination Auxin efflux transport protein. Expressed in the tomato floral bud and in the young developing ovary Auxin receptor

AtARF8

SlARF7

Genetic modification

Function

Gene

Table 1  Summary of genes reported to contribute to the development of seedless fruits without fertilization and their relation to hormones or other metabolites

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(Ingrosso et al. 2011) At = Arabidopsis thaliana; Sl = Solanum lycopersicum; Cc = Citrus cinensis; As = Anona squamosal

(Schijlen et al. 2007)

Stilbene synthesis GSTS

Parthenocarpy in tomato Overexpression of grape STS

Flavonoid biosynthesis SICHS

Parthenocarpy in tomato

(Carmi et al. 2003) Parthenocarpy in tomato

Transgenic expression with ovary/fruit specific promoters RNAi silencing

IaaM

Auxin response in Agrobacterium rhizogenes

(Pandolfini et al. 2002; Rotino et al. 1997) Parthenocarpy in tomato, eggplant, cucumber, raspberry

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rolB

(Lora et al. 2011) (Payne et al. 2004) Parthenocarpy in Annona squamosa Parthenocarpy and ectopic organs growing from the gynoecia of Arabidopsis Outer integument development

AsINO AtKNUCKLES

Deletion of INO locus Transcriptional repressor of cellular proliferation AtKnuckles and floral determinacy control Expressed in carpel primordial, ovules and anthers before anthesis Auxin biosynthesis in Pseudomonas syringae pv. Transgenic expression under ovule specific promoter savastanoi

Function Gene

Table 1  continued

Genetic modification

Effect observed (fruit phenotype/altered hormonal content)

References

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one is to accommodate the embryo sac by its expansion, and the other is to coordinate the growth of both fruit and seeds (Fuentes and Vivian-Smith 2009). These roles were attributed from the fact that Arabidopsis mutants with arrest of integument initiation and outgrowth such as aintegumenta (ant; lack inner and outer integuments), aberrant testa shape (ats; contains single integument), inner no outer (ino; outer integument elongates opposite to the ovule primordium), short integuments1 (sin1; both integuments are short), bell1 (bel1), and apetala2 (ap2) are associated with aborted embryo sac development and reduced fertility (Baker et al. 1997; Elliott et al. 1996; Lang et al. 1994; León-Kloosterziel et al. 1994; Modrusan et al. 1994; Ray et al. 1994; Villanueva et al. 1999). In the case of bel1 and ap2, ovule integuments are converted into carpelloid structures (Modrusan et al. 1994; Pinyopich et al. 2003; Ray et al. 1994). Two of these mutants have been reported to affect parthenocarpic fruit development of the fruit without fertilization (fwf; arf8-4) mutant (Goetz et al. 2006). First, the ats loss-of-function mutation enhances the fwf parthenocarpic phenotype, suggesting that modification of ovule integument structure influences parthenocarpic fruit growth (Vivian-Smith et al. 2001). Second, parthenocarpic fruit development was also enhanced in the bel1 fwf double mutant, and a higher frequency of carpelloid structures was observed compared to the bel1 single mutant (Tiwari et al. 2011). Parthenocarpy together with carpelloid structure phenotypes has also been observed in the Arabidopsis knuckles (knu) mutant (Payne et al. 2004), in the tomato tm29 line (Ampomah-Dwamena et al. 2002) and in some Capsicum annuum genotypes (Ampomah-Dwamena et al. 2002; Payne et al. 2004; Tiwari et al. 2011). This may point to a consistent regulatory link between both traits and suggests that on the one hand, carpelloid structures enhance parthenocarpic fruit development and on the other hand, carpelloid structure development is enhanced in the absence of seed-set (Ampomah-Dwamena et al. 2002; Payne et al. 2004; Tiwari et al. 2011). A phenocopy of the Arabidopsis ino mutant was detected in a spontaneous seedless mutant of Annona squamosa (Thai seedless; Ts), in which ovules lack the outer integument (Lora et al. 2011). Interestingly, in this Ts A. squamosa mutant, the INO gene could not be detected, indicating apparent deletion of the locus in this mutant (Lora et al. 2011), further supporting a link between integuments and parthenocarpy. Interestingly, other organs apart from the ovaries have been reported to have an effect in parthenocarpic development in tomato. Medina et al. (2013) showed that ablation of the anthers early in development produced parthenocarpy (Medina et al. 2013), suggesting that the signaling that controls fruit set goes beyond the female reproductive tissues at least in this species. Metabolites such as phenylpropanoids have been implied in parthenocarpic development or reduction of

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seed-set in species like Arabidopsis, tomato, tobacco, and pear (Fischer et al. 1997; Ingrosso et al. 2011; Mahajan et al. 2011; Mizzotti et al. 2011; Nishitani et al. 2012; Schijlen et al. 2007). Moreover, parthenocarpy is also triggered by the overexpression of CYP78A9 in Arabidopsis, a P450 monooxygenase, which belongs to the CYP78A subfamily (Ito and Meyerowitz 2000; Marsch-Martinez et al. 2002; Sotelo-Silveira et al. 2013a). Interestingly, alterations in the flavonoid pathway were detected in the plants overexpressing CYP78A9, though these alterations were not identified as the cause of the parthenocarpy (Sotelo-Silveira et al. 2013a). The CYP78A9 expression pattern and mutant phenotypes (i.e., outer integument arrest in double knock out mutants and overgrowth in the overexpression mutant) denoted the involvement of the gene during integument development (Sotelo-Silveira et al. 2013a). Furthermore, a suggestive connection was detected between the function of this gene and the signaling process that takes place in the outer integument during fruit set (Sotelo-Silveira et al. 2013b).

Hormonal regulation of fruit set Plant hormones play a significant role in the processes that lead to mature fruit and viable seed production. Auxins, gibberellins (GAs), cytokinins (CKs), abscisic acid (ABA), and ethylene have been implicated at various stages of fruit growth (Alabadi et al. 2009; Balanza et al. 2006; Bencivenga et al. 2012; de Jong et al. 2009a; Marsch-Martinez et al. 2012; McAtee et al. 2013; Ozga and Reinecke 2003; Sun 2008; Sundberg and Ferrándiz 2009). The precise spatial and temporal control of biosynthesis, signaling and action of these hormones, and their interactions that lead to normal fruit development, are only beginning to be understood. In this sense, the expression pattern of some hormone biosynthetic enzymes helped to elucidate their role during fruit development, e.g., the case for GA biosynthesis that was found to take place not only in valves where they have a growth effect but also in the ovules after fertilization (Dorcey et al. 2009; Garcia-Martinez et al. 1997). The main advances in understanding how hormones control fruit set are related to: how fruit growth is prevented before fertilization, what happens with the pistil if fertilization does not occur, and which are the key hormones that trigger fruit growth after fertilization (Carbonell-Bejerano et al. 2010, 2011; Dorcey et al. 2009; McAtee et al. 2013). Work in Arabidopsis and tomato showed the importance of some ARF (Auxin Response Factors ARF8 and ARF7) and DELLA (negative regulators of GA responses) proteins restraining the growth of the pistil before fertilization (Dorcey et al. 2009; Fuentes et al. 2012; Goetz et al. 2006,

2007b). However, this restriction is sustained only for a certain period of time after which a default developmental senescence program is triggered in pistils if pollination and fertilization do not occur (Carbonell-Bejerano et al. 2011). Vivian-Smith and Koltunow (1999) have demonstrated that Arabidopsis pistils have a receptive period after anthesis in which they can respond either to pollination or hormone applications and develop into fruits. Carbonell-Bejerano et al. (2011) showed that this response window was related to ethylene synthesis at the onset of ovule senescence. In contrast, in tomato, ethylene biosynthesis after fruit initiation was related to the release of the dormant state of the ovary (Pascual et al. 2009; Vriezen et al. 2008). Progress has also been made in establishing the sequence of events that occur after fertilization. Dorcey et al. (2009) proposed a model in which auxins and gibberellins (GAs) are the main signals in ovules that trigger fruit development and that they act in a hierarchical manner, i.e., auxin followed by GA (Fig. 2). The main role proposed for auxins in this model is the derepression of ovary growth after fertilization (reviewed in Alabadi et al. 2009; Fig. 2). The evidence supporting this model will be further discussed below (Table 2). Vivian-Smith and Koltunow (1999) showed the fruit growth induction potential of auxins, CKs, and GAs by experiments in which they treated unfertilized Arabidopsis pistils with these hormones. The hormones were able to trigger parthenocarpic fruit development, but not one of the treatments resembled the exact shape, silique length or growth rate of a natural pollinated pistil. Dorcey et al. (2009) showed through analysis of DR5::GFP (a marker that transcriptionally responds to auxin) transgenic plants that auxin response is activated in ovules soon after fertilization. Direct measurements of IAA content on hand pollinated Arabidopsis pistils showed that they have fivefold more IAA per mg of tissue than only emasculated wild type Arabidopsis pistils (Fuentes et al. 2012), further supporting the DR5::GFP response after fertilization. This increase of auxin level after fertilization was also detected in other species pointing out to a conserved role of auxins inducing fruit set (de Jong et al. 2009a). Moreover, transcriptional profiling during tomato fruit initiation showed that auxin signaling genes like ARFs and Aux/IAAs were induced after pollination but not after GA3 treatment (Vriezen et al. 2008). This evidence reinforces the idea that auxin signaling precedes gibberellin responses after fertilization, and this is conserved at least among Arabidopsis and tomato. Another observation that supports the importance of auxins during the process of fruit set is that it is possible to uncouple fruit and seed development by manipulation of auxin action at different levels. At the biosynthesis level, the auxin synthesizing gene iaaM of Pseudomonas

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Fig. 2  Model for fruit set upon fertilization. In the ovule, at preanthesis, specific auxin/indole-3-acetic acid (AUX/IAA) regulatory proteins form heterodimers with auxin response factors (ARFs) repressing auxin signaling and ovary growth. Successful fertilization leads to a localized burst of auxin. The binding of auxin to F-box receptor proteins then leads to the degradation of Aux/IAA proteins via the ubiquitin–proteasome pathway releasing ARFs. Free ARFs subsequently regulate auxin-responsive genes, directly or indirectly inducing GA biosynthesis. GA leads to the degradation of DELLA proteins via the ubiquitin–proteasome pathway, removing the repressing effect of DELLAs in GA signaling inside and outside the ovule. This results in GA-triggered fruit set. Based on (Alabadi et al. 2009; Dorcey et al. 2009)

syringae pv. savastanoi under the control of an ovule specific promoter conferred parthenocarpy in several horticultural crops including tomato, eggplant, cucumber, and raspberry (Donzella et al. 2000; Mezzetti et al. 2004; Pandolfini et al. 2002; Rotino et al. 1997, 2005; Wang et al. 2011; Yin et al. 2006). There are also various examples in which the manipulation of the auxin signaling pathway leads to parthenocarpic fruit development, for instance, the downregulation of IAA9 (Wang et al. 2005), the arf8 mutation in Arabidopsis (Goetz et al. 2006), the arf8-4 mutant allele introduced in tomato (Goetz et al. 2007b), and ARF7 silencing in tomato (de Jong et al. 2009a, b). ARF7 was described as a key regulator of the auxin/GA crosstalk during tomato fruit development (de Jong et al. 2009a, 2011). In tomato, silencing of AUCSIA genes added more evidence about fruit set control via auxins. These genes are expressed in the ovary and are drastically downregulated after pollination.

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AUCSIA-silenced plants generally displayed facultative parthenocarpy and an increase in auxin (IAA) level in preanthesis floral buds (Molesini et al. 2009). Experiments that altered auxin transport in tomato also showed the importance of this hormone in the program of fruit development. Treatments with the auxin transport inhibitor 1-N-Naphthylphthalamic acid (NPA) induced the growth of the fruit in the absence of fertilization (Serrani et al. 2010). Parthenocarpic fruits were also obtained by altering auxin sensitivity by specifically expressing the rolB gene of Agrobacterium rhizogenes in the tomato ovary (Carmi et al. 2003). Furthermore, silencing of the auxin efflux transporter SlPIN4 gene (PIN-FORMED4) also leads to parthenocarpic fruits due to premature fruit growth before fertilization (Mounet et al. 2012). Alterations in fruit set have also been obtained when auxin perception is affected. Different auxin receptors have been described, such as, TRANSPORT INHIBITOR 1 (TIR1) and AUXIN BINDING PROTEIN 1 (ABP1) (Sauer and Kleine-Vehn 2011; Scherer 2011), and it has been demonstrated that the tomato SITIR1 plays an important role in tomato fruit set. Its overexpression altered the content of numerous auxin-responsive genes and produced parthenocarpy (Ren et al. 2011). The DIAGEOTROPICA (DGT) gene, a cyclophilin, was described as a mediator in some auxin responses (Oh et al. 2006). The autopollinated dgt tomato mutant has reduced fruit set in comparison to normal fruit-set when it is hand pollinated, probably due to a defect in pollen release (Balbi and Lomax 2003). In addition, when external auxin was applied to flowers, the dgt mutant showed reduced auxin sensitivity observed as reduced fruit growth compared with the parthenocarpic development presented in the wild type. This suggests that fruit growth in response to either exogenous or internal auxin progresses through different pathways (Mignolli et al. 2012). Remarkable advances have been made in the past few years related to how GA biosynthesis, response and signaling pathway influences fruit initiation (Carrera et al. 2012; Dorcey et al. 2009; Fuentes et al. 2012; García-Hurtado et al. 2012; Hu et al. 2008; Mariotti et al. 2011). Endogenous bioactive GAs have been shown to play a fundamental role in fruit development. Fruit set in the absence of pollination was induced by external application of active gibberellins (GA1 or GA3) in several horticultural species and Arabidopsis. Furthermore, restricted fruit growth was seen when GA inhibitors were applied (Serrani et al. 2007). The increased hormonal content in parthenocarpic plants suggests that endogenous GA concentration in developing ovaries is the limiting factor controlling parthenocarpic development (Fos et al. 2000; Talon et al. 1992). Blocking GA inactivation, by knocking-out the five GA inactivating, GA 2-oxidase, enzymes, leads to the formation of parthenocarpic fruits (Rieu et al.

GA3 in unpollinated pistil

NPA in unpollinated pistil

2,4-D in unpollinated pistil

No DR5revGFP signal

(Aloni et al. 2006; Benkova et al. 2003; Dorcey et al. 2009; Fuentes et al. 2012)

(Dorcey et al. 2009)

(Dorcey et al. 2009)

(Dorcey et al. 2009)

GA2-ox inactivation enzymes were upregulated 24 h after fertilization

Upregulation of AtGA20ox1 after 6 h of the treatment Upregulation of AtGA20ox1 after 6 h of the treatment Inhibition of the GA biosynthesis genes

Upregulation of AtGA20ox2, and ProRGA:GFP-RGA AtGA3ox1 after 6 h of the treatment signal disappear after 48 h of treatment ProRGA:GFP-RGA signal disappear after 24 h of the treatment

No DR5revGFP signal

Inhibition of the GA biosynthesis genes

Upregulation of AtGA20ox1, AtGA20ox2, and AtGA3ox1 after 2 h of the treatment

ProRGA:GFP-RGA signal disappear after 24 h of the treatment

No DR5revGFP signal

No DR5revGFP signal

DR5revGFP signal extended to the valves

Increased DR5revGFP signal 24 h after fertilization in chalaza, micropila and funiculus Increased IAA content in hand pollinated pistils Conjugated auxin detected in embryos Increased DR5revGFP signal 24hs after fertilization in chalaza, micropila and funiculus Increased DR5revGFP signal 24hs after fertilization in chalaza, micropila and funiculus

After fertilization

References

(Aloni et al. 2006; Dorcey et al. 2009)

Expression of gibberellin biosynthesis genes in valves GA20ox1 and GA3ox1 inhibited

GA20ox1 and GA3ox1 inhibited

ProRGA:GFP-RGA signal (high intensity at the base of the ovule and funiculus) ProRGA:GFP-RGA Upregulation of AtGA20ox1 and signal disappear after AtGA3ox1 after 24 h fertilization AtGA20ox3, AtGA20ox5,AtGA3ox3, 48 h of treatment and AtGA3ox4 were upregulated 48 to 72 h after fertilization GA2-ox inactivation enzymes were upregulated 24 h after fertilization

No DR5revGFP signal

Before fertilization

Expression of gibberellin biosynthesis Gibberellin response in genes in ovules/young seeds ovules/young seeds

No DR5revGFP signal

Auxin response in ovules/ Auxin response in young seeds valves

Treatment

Table 2  Auxin content and response and expression of GA biosynthesis and signaling genes in ovules and valves of wild type Arabidopsis pistils under different treatments uncover the hierarchy of auxin inducing fruit set

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2008a). Moreover, the GA biosynthetic GA 20-oxidase and GA 3-oxidase enzymes are required for silique elongation (Hu et al. 2008; Rieu et al. 2008a). Moreover, alterations in the type of active GA form can lead to fruit growth without pollination as occurs in transgenic Micro-Tom plants overexpressing CcGA20ox1 from citrus that have higher GA4 contents, which is normally a minor active GA in tomato (García-Hurtado et al. 2012). Dorcey et al. (2009) showed the importance of the GA signaling pathway by showing that emasculation of a quadruple-DELLA Arabidopsis mutant results in fertilization-independent fruit growth. Furthermore, Fuentes et al. (2012) added new insights into the relative importance of individual DELLA proteins in the control of fruit initiation, further supporting the hypothesis of Dorcey et al. (2009). The SIDELLA loss-of-function tomato mutant procera (pro) showed a GA constitutive response and presented parthenocarpic development. The data obtained by studying this mutant suggests that control of fruit set and development by GA-auxin crosstalk is mediated by SlDELLA and the auxin signaling factor SlARF7 (Carrera et al. 2012). As discussed before, Dorcey et al. (2009) showed evidence supporting a model for Arabidopsis in which fertilization would trigger auxin-mediated promotion of GA synthesis specifically in the ovule. Silique growth starts when the GAs synthesized in the ovules are transported to the valves to promote GA signaling in this tissue (Fig. 2). Fuentes et al. (2012) observed that Arabidopsis pistils from mutants impaired in gibberellin perception were not able to develop fruits in response to auxin treatment. Furthermore, by application of GA to mutants with impaired ovule development, (Carbonell-Bejerano et al. 2010) reinforced the idea of the need of viable ovules to conserve fruit set responsiveness of the pistil to pollen and to GAs in unfertilized pistils. Tiwari et al. (2012) demonstrated that in Capsicum annuum, both auxin and GAs are important to trigger fruit set and have the same hierarchical behavior as in the model described for Arabidopsis and tomato (Dorcey et al. 2009; Tiwari et al. 2012). On the other hand, CK has been related to meristematic activity in very young Arabidopsis gynoecia (Bartrina et al. 2011; Marsch-Martinez et al. 2012), and parthenocarpic development (Vivian-Smith and Koltunow 1999). The existence of two CK peaks in tomato, the first one, at anthesis unlinked to pollination, and the second 5 days after anthesis, suggested that CK levels might be necessary for: (a) the growth and/or maintenance of unpollinated ovaries until successful pollination occurs, and (b) the induction of cell division during tomato fruit development (Matsuo et al. 2012). Ding et al. (2013) showed that CK application to unpollinated tomato ovaries induced parthenocarpic fruit development, but the inducing effect was blocked when CK was applied in combination with a GA

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biosynthesis inhibitor. Both pollination and CK-induced fruits presented increased amounts of GA1 and GA3 GA1+3 and indole-3-acetic (IAA), and this was accompanied with elevated expression of GA and IAA biosynthesis genes and downregulation of GA inactivation genes (Ding et al. 2013). Taken together the evidence of Matsuo et al. (2012) and Ding et al. (2013) suggests that in tomato, CKs may play not only a specific role controlling cell division but also modulating IAA and GA metabolism. On the other hand, Mariotti et al. (2011) showed that in developing tomato fruits, the levels of CKs increased soon after pollination or auxin treatment. More integrative work can help to decipher the crosstalk between these and other hormones during fruit set.

Conclusions In the last years, molecular analyses of pollinationdependent and pollination-independent fruit set have revealed that fruit growth is under control of a complex hormonal regulatory network. Moreover, ovules play a key role in controlling fruit growth, triggering the signaling cascade that initiates fruit growth after fertilization, but also the senescence program that determines the length of the receptive period of the pistil (Carbonell-Bejerano et al. 2010, 2011; Dorcey et al. 2009). Moreover, evidence in Arabidopsis and Annona has highlighted the importance of the ovule integuments, because parthenocarpic fruit development occurs when they are absent or abnormally developed. Therefore, a new role for these tissues as the place in which restricting signals are being distributed and/or generated has been proposed (Lora et al. 2011; Tiwari et al. 2011; Vivian-Smith et al. 2001). However, some open questions remain, such as the nature of the signals and how they interact with the known players of the model. In this respect, metabolomic and transcriptomic analyses performed in fertilized, dissected tissues, can help to uncover different molecules and genes during the process and hopefully discover the signals and genetic players involved. We can speculate that despite the molecular complexity of the process, a hierarchical series of events or many parallel processes are being controlled by a robust signaling pathway where auxin plays the prominent role, but other hormones are integrated in a synergistic or antagonistic way. The general model proposed in Arabidopsis by Dorcey et al. (2009) could explain also the more general aspects of fruit set in tomato (Alabadi et al. 2009; Dorcey et al. 2009; McAtee et al. 2013). However, evidence suggesting that other type of metabolites could be involved in fruit set regulation came from the manipulation of the phenylpropanoid pathway in tomato and tobacco (Ingrosso et al. 2011; Mahajan et al. 2011; Schijlen et al. 2007), and from data

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obtained with the overexpression of CYP78A9 in Arabidopsis (Ito and Meyerowitz 2000; Sotelo-Silveira et al. 2013a, b). Do these metabolites participate in some way in the crosstalk between auxins and GAs? Moreover, other questions regarding CK action regarding fruit set remain open: Do they only specifically control cell division during early embryo development? Or do they modulate auxin signaling, transport, or homeostasis? More insights in the role of this hormone will arise from studies during fruit set on mutants altered in CKs synthesis and perception, as well as the CKs behavior in the already described mutants known to act in the crosstalk. Acknowledgments We would like to thank the anonymous reviewers for their very helpful comments on this review. We thank the Mexican National Council of Science and Technology (CONACyT) for a PhD fellowship to MSS (229496). This work in the de Folter laboratory is financed by the CONACyT Grant 177739.

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