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Fruit Growth-Related Genes in Tomato
Fruit Growth-Related Genes in Tomato
Review Paper
Abstract
Tomato (Solanum lycopersicum Mill.) represents a model species for all fleshy fruits due to its biological cycle and the
availability of numerous genetic and molecular resources. Its importance in human nutrition has made it one of the
most valuable worldwide commodities. Tomato fruit size results from the combination of cell number and cell size,
which are determined by both cell division and expansion. As fruit growth is mainly driven by cell expansion, cells
from the (fleshy) pericarp tissue become highly polyploid according to the endoreduplication process, reaching a DNA
content rarely encountered in other plant species (between 2C and 512C). Both cell division and cell expansion are
under the control of complex interactions between hormone signalling and carbon partitioning, which establish cru-
cial determinants of the quality of ripe fruit, such as the final size, weight, and shape, and organoleptic and nutritional
traits. This review describes the genes known to contribute to fruit growth in tomato.
Key words: Cell cycle, cell division, cell expansion, development, endoreduplication, fruit, genetic control, growth, metabolic
control, hormones, tomato.
Introduction
The fruit is a plant organ specific to angiosperms that protects In general, the term fruit refers to the fleshy, edible portion
the ovule and seed during embryo development and ensures that is the major component of fruits, such as grape, banana,
seed dispersal after maturation. At the botanical level, most apple, citrus, peach, strawberry, melon, and tomato. All of
fruits develop from mature ovaries and, therefore, include these species are produced worldwide, and continue to rely
carpellar tissues. The physiological function of seed disper- on breeding schemes for agronomic traits. Particular traits of
sal accounts for a significant part of the adaptive success of interest are enhanced yield and quality, increased time of fruit
angiosperms on Earth. Indeed, a wide diversity of fruit size, storage and longer shelf-life, optimized cultural practices, and
form, and composition, as well as a variety of fruit dispersion pest and pathogen resistance. The common feature of fleshy
mechanisms, have emerged as the result of strong environ- fruits is the fine balance between sugars and organic acids,
mental selective pressures. For example, the Solanaceae fam- and the accumulation of water, minerals, pigments, aromatic
ily, which encompasses nearly 10 000 species, has very diverse compounds, and vitamins that confer upon the fruit its juici-
types of fruits, with capsules, drupes, pyrenes, berries, and ness and attractiveness. Consequently, fleshy fruits represent
several types of dehiscent non-capsular fruits occurring in a major source of vitamins, fibre, carbohydrates, and phyto-
>90 genera (Knapp, 2002). nutrient compounds essential for human nutrition.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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Page 2 of 12 | Azzi et al.
Tomato (Solanum lycopersicum Mill.) fruit, a multicarpel- ~17–20 rounds of cell divisions that occur during a pre-anthe-
lar berry, has arisen as the model species for fleshy fruits, due sis period located within the L3 layer of the floral meristem,
to certain advantages for use in both agronomical and funda- but which involve virtually no cell expansion (Coombe, 1976;
mental research (Gillaspy et al., 1993; Tanksley, 2004; Klee Ho, 1992). Obviously, the number of cells formed in the ovary
and Giovannoni, 2011). These advantages include a short before anthesis is critical for the final size of fruit, and such
life cycle, high multiplication rate, self-pollination, and ease a positive correlation is frequently observed (Tanksley, 2004).
of mechanical crossing. This highly favourable biology has From flower initiation to the double fertilization occurring in
been widely exploited to generate segregating populations ovules (Gillaspy et al., 1993; Brukhin et al., 2003), the mor-
such as recombinant inbred lines (RILs) or near isogenic lines phogenesis and growth of carpels and ovules require the spa-
(NILs). Tomato has proven amenable for marker-assisted tial and temporal synthesis and action of auxin, cytokinin,
mapping using crosses between small and round, wild toma- and gibberellins (GAs). In order to keep the ovary in a tem-
toes and domesticated varieties of various sizes and shapes porally protected and dormant state, abscisic acid (ABA) and
that has enriched our knowledge of the genetic control of ethylene work to stop growth within the ovary shortly before
fruit development (Grandillo et al., 1999; Causse et al., anthesis when the ovary has reached its mature size (Vriezen
2007; Muños et al., 2011; Rodriguez et al., 2011). A large et al., 2008). Growth resumes only after successful pollination
spectrum of mutants is now available (Menda et al., 2004; and fertilization of ovules triggers fruit set—the first phase of
Watanabe et al., 2007; Just et al., 2013) for screening and gene fruit development—through the action of ovary-synthesized
Fruit Development
Cell expansion
Anthesis 3 5 8 12 15 20 25 35 40 dpa
35 dpa
8 dpa
0 dpa
Fig. 1. Fruit development in tomato. The fruit originates from the development of the ovary. Following fertilization (concomitant with anthesis) and fruit
set, fruit growth starts with a period of very active cell division inside the ovary that lasts for 8–10 days post-anthesis (dpa). After anthesis and in <8–10 d,
the number of cell layers across the pericarp has doubled compared with that at anthesis and becomes fixed until the end of development. Fruit growth
then proceeds into the cell expansion phase and continues until ripening (~35 dpa). Cell enlargement starts concomitantly with cell division as early as 1
or 2 dpa and contributes to the increase in fruit size.
Endopolyploidy is the occurrence of different ploidy levels 1996; Joubès et al., 1999; Cheniclet et al., 2005; Bertin et al.,
within an organism. In plants, it results mostly from endore- 2007). Ploidy levels as high as 512C (where C is the haploid
duplication that is estimated to occur in >90% of angiosperms DNA content) have been observed to occur in a large array
(Nagl, 1976; D’Amato, 1984). Endoreduplication occurs in of tomato genotypes. This extraordinary extent of endore-
the absence of any obvious DNA condensation and decon- duplication occurring in tomato fruit is unmatched by any
densation steps that lead to the production of chromosomes other plant species and makes tomato an excellent model
with 2n chromatids or without any change in chromosome to study the role of endoreduplication as a determinant of
number (Joubès and Chevalier, 2000; Edgar and Orr-Weaver, fruit size. Cheniclet et al. (2005) demonstrated that the mean
2001). As a consequence, hypertrophic nuclei arise from suc- ploidy level achieved in pericarp cells correlates not only with
cessive cycles of DNA replication without segregation of the large variation in the fruit weight that can be encoun-
sister chromatids, thereby resulting in the production of poly- tered in the various genotypes, but also with the mean cell
tene chromosomes (Bourdon et al., 2012). The physiological size inside the pericarp. The endoreduplication-associated
and developmental importance of endoreduplication is still a cell expansion in fruit is characterized by profound cellular
matter of debate. However, the frequent observation that cell modifications. It is evident that the formation of polytene
size and ploidy levels are highly and positively correlated in chromosomes impacts on nuclear, nucleolar, and chroma-
many different plant species, organs, and cell types (Chevalier tin organization within hypertrophied nuclei delimited by a
et al., 2011) suggests a role for endoreduplication in the con- highly invaginated nuclear envelope (Bourdon et al., 2012).
trol of cell size, according to the ‘karyoplasmic ratio theory’. These profound nuclear grooves are filled with numerous
This theory, which was formulated as early as the beginning mitochondria, whose number increases according to nuclear
of the 20th century, states that there is a causal relationship DNA content. Apparently, endoreduplication triggers effi-
between nuclear and cytoplasmic growth to maintain a con- cient communication between the nucleus and the cytoplasm,
stant ratio of nucleus to cell volume (Sugimoto-Shirasu and despite the increase in nuclear volume. The cytoplasmic vol-
Roberts, 2003; Chevalier et al., 2014). ume of cells also correlates with ploidy levels, which indicates
In the course of tomato fruit development, endoreduplica- that the maintenance of the nuclear to cytoplasmic ratio to
tion produces high levels of nuclear DNA ploidy within the drive cell growth is consistent with the ‘karyoplasmic ratio’
mesocarp and the locular jelly-like tissue (Bergervoet et al., theory. In addition, Bourdon et al. (2012) provided evidence
Page 4 of 12 | Azzi et al.
for the existence of a strong relationship between endoredu- complex activity as it confers its stability, its localization, and
plication and rRNA and mRNA transcription. its substrate specificity (Inzé and De Veylder, 2006). Specific
As a morphogenetic factor, endoreduplication thus con- CDK–CYC complexes operate at the boundaries between the
tributes to maintain homeostasis of cytoplasmic components various phases of the cell cycle in order to phosphorylate tar-
through the establishment of a highly structured cellular sys- get proteins. These post-translational modifications which can
tem, where multiple physiological functions are integrated to be either inhibitory or activating, are essential for the progres-
support cell growth during fruit development. sion through cell cycle boundaries (De Veylder et al., 2007). To
allow a subtle regulation of cell cycle progression, the kinase
activities of the CDK–CYC complexes are regulated in sev-
Modifying fruit growth with cell cycle and eral ways: (i) CDK activity is finely tuned by phosphorylation
and dephosphorylation of the CDK on conserved residues by
endocycle regulatory genes
specific kinases and phosphatases; (ii) the proteolytic destruc-
Cell division and cell expansion associated with endoredu- tion of the cyclin subunit by the ubiquitin–proteasome system
plication provide, respectively, the building blocks for the (UPS) is sufficient to abolish activity of the complex, as CDK
fruit organ and the growth-driving force that contribute to alone does not display kinase activity without its cyclin part-
establish the final fruit weight/size. Therefore, modifying fruit ner; and (iii) the CDK–CYC complexes are inactivated by the
growth by targeting genes involved in mitosis and endoredu- specific binding of CDK inhibitors (Churchmann et al., 2006;
Anthesis
A B C 3 5 8
Cell division
Ovary Dev.
Fruit set
Auxin signaling:
SlPIN4
D 3 5 8 12 15 20 25 35
Cell expansion
Fig. 2. Genes involved in tomato fruit growth. Mapping of QTLs, cloning of the associated genes, in planta functional analyses, and characterization of
mutants have identified genes that impact fruit growth both positively and negatively. Theses genes are reported according to the respective stages of
fruit development in which they are involved: (A) ovary development; (B) fruit set; (C) phase of cell division; and (D) phase of cell expansion.
Tomato fruit growth | Page 5 of 12
In tomato, several functional analyses have attempted to complex activity, which is necessary to permit the youngest
modulate the CDK–CYC complex activity during fruit devel- cells of the outermost layers of pericarp (namely the exo-
opment, either through direct targeting of CDK gene expres- carp) to enter into the phase of endoreduplication-driven cell
sion or through post-translational regulation of the complex expansion. In a similar manner, overexpression of CDKA;1
(Fig. 2C). Czerednik et al. (2012) generated transgenic plants induced mitosis across the pericarp and altered the endore-
aimed at down-regulating the canonical CDKA;1, the key duplication index (Czerednik et al., 2014), thus supporting
player in cell cycle progression, in a fruit-specific manner the idea that CDKA plays an important part in the onset of
using an artificial microRNA (amiCDKA) and the TPRP endoreduplication and subsequent cell and fruit growth.
promoter (Fernandez et al., 2009). The amiCDKA plants pro- The inhibitory effect of phosphorylating CDKA on the
duced smaller fruits than those of the wild type, and which Tyr15 residue through the action of the WEE1 kinase rep-
had a thinner pericarp due to an overall decreased number resents a good example of a post-translational regulatory
of cell layers within the exocarp (displaying the smallest mechanism impairing the cell cycle, and potentially trigger-
cells). In contrast, the mesocarp displayed normal, enlarged ing the endocycle (Fig. 2D). Gonzalez et al. (2007) generated
cells without any significant difference in ploidy levels. transgenic tomato plants in which WEE1 was ectopically
A similar phenotype was obtained with fruit-specific over- repressed. Smaller fruits were produced displaying a thin-
expression of either mitosis-associated CDKB1 or CDKB2 ner pericarp composed of smaller cells. There was a strong
(Czerednik et al., 2012). Interestingly, the expression of reduction in endoreduplication as a result of impaired WEE1
as already described for a strong overexpression of KRP and its orthologues in relation to ion transport remains to be
genes in other plant tissues or organs (De Veylder et al., 2001; established, and how this could interfere with the control of
Jasinski et al., 2002; Zhou et al., 2003). However, the same cell proliferation is an intriguing question.
construct was ineffective during the phase of cell expansion fw3.2 is only the second major QTL for fruit size/weight
and did not alter the final size of fruits, in contrast to pre- to be fine mapped and cloned in tomato (Chakrabarti et al.,
vious reports describing plant dwarfism. The mean cell size 2013) (Fig. 2C). The gene underlying this QTL encodes a
within the pericarp was unaffected, demonstrating that cell P450 enzyme of the CYP78A subfamily, previously identified
size was, at least partially, uncoupled from DNA ploidy levels. as KLUH (Anastasiou et al., 2007). The effect of SlKLUH
Nevetheless, endoreduplication occurred in these transgenic is to enlarge fruit volume through an increase in cell number
fruit, but to a far lesser extent than in the wild type. This sug- within the pericarp and septum tissues. Repressing SlKLUH
gests that DNA ploidy levels were sufficient to support cell using an RNA interference (RNAi) strategy led to decreased
growth, which is in agreement with the ‘karyoplasmic ratio fruit and seed size and, consequently, plant architecture was
theory’ (Schnittger et al., 2003; Chevalier et al., 2014). modified with a higher number and length of side shoots
(Chakrabarti et al., 2013). The cloning of the gene and its
function characterization will be an important step towards
Modifying fruit growth by genes controlling elucidating the biological function of FW3.2.
fw11.3 is another important locus governing fruit weight in
fruit size and shape
thus resulting in reduced cell elongation (Hackbusch et al., seeds occur concomitantly according to a precise, genetically
2005; Wang et al., 2007, 2011). The indirect influence of controlled process mediated by phytohormones (Gillaspy
OVATE on fruit shape through regulation of GA biosynthe- et al., 1993).
sis in tomato remains to be demonstrated. Recent reviews have highlighted the role of plant hormones
The sun mutation causes an elongated-fruit phenotype. and their interplay in the control of fruit development (Ruan
A gene duplication event mediated by a retrotransposon et al., 2012; Kumar et al., 2014). Here, we focus on the most
placed the SUN gene under the control of the defensin relevant genes, whose functional characterization in tomato
gene DEFL1 promoter, thus leading to high expression in revealed an involvement in the growth period of fruit devel-
fruit (Xiao et al., 2008; Jiang et al., 2009). The SUN protein opment. Auxin and GA appear to be the predominant hor-
belongs to the IQ67 domain-containing plant protein family mones required for fruit initiation in response to fertilization,
(Xiao et al., 2008) of unknown function. SUN overexpression since exogenous applications of both hormones lead to fruit
results in increased cell number in the longitudinal direction initiation and parthenocarpic development (de Jong et al.,
and reduced cell number in the transverse direction of the 2009). A role for cytokinin, ethylene, and ABA in fruit for-
fruit, thus leading to fruit elongation (Wu et al., 2011). mation has been demonstrated, but is less well documented
The number of fruit locules is determined by the number of thus far (Kumar et al., 2014).
carpels within the flower. Wild species of tomato produce fruits Early fruit development is governed by the allocation of
with 2–4 locules, whereas cultivated varieties can develop >15 auxin to tissues and cells in order to initiate signal transduc-
during fruit initiation and growth resulted in plants severely plants, whereas they were enlarged in RNAi-silenced plants.
affected in vegetative development and reduced pollen viabil- The cell number within the carpel was reduced, leading to a
ity (Olimpieri et al., 2011). Ovaries from these SlGA20ox1- smaller carpel, thus suggesting that IMA encodes an inhibi-
silenced plants remained fertile and developed normally tor of cell division. Consequently, plants overexpressing IMA
after cross-pollination with wild-type pollen, but remained produced smaller flowers and fruits, whereas RNAi-silenced
parthenocarpic. The individual silencing of SlGA20ox1, plants produced fruits composed of supernumerary ovaries.
SlGA20ox2, and SlGA20ox3 genes confirmed the effects on In addition, IMA repressed the expression of WUS which
vegetative development but, again, no effects on fruit set were controls the meristem organizing centre and the determinacy
observed (Xiao et al., 2006). Altogether, these data confirmed of the nucellus during ovule development (Sicard et al., 2008)
the pleiotropic developmental role of GAs, but suggested that (Fig. 2A).
the expression of more than one GA20ox gene is required to
control fruit set in tomato. Interestingly, the heterologous
Metabolic control of fruit development
overexpression in tomato of CgGA20ox1 from citrus clearly
demonstrated the expected influence of GA and GA20ox The early stages of fruit development represent a critical
activity on fruit set and development (Garcia-Hurtado et al., period whereby traits, such as organoleptic composition,
2012). The overexpression of CgGA20ox1 resulted in elevated are established and ultimately dictate the final quality of the
GA4 content and boosted vegetative development, as shown fruit. Water, organic acids (primarily citrate and malate), and
at the expense of starch storage. Fruit displayed reduced res- central enzyme in ascorbate biosynthesis, exhibited defects
piratory rates, which were accompanied by lower ATP levels in cell expansion and the biosynthesis of non-cellulosic cell
and ATP/ADP ratios in fruit extracts, indicating profound wall polysaccharides (Gilbert et al., 2009) (Fig. 2D). Taken
metabolic perturbations. together, these findings indicate an ascorbate-mediated link
The role of sucrose synthase (SuSy) in tomato fruit devel- among the energy-generating processes of respiration and
opment has been studied by silencing a fruit-specific isoform photosynthesis, primary metabolism, and developmental
(D’Aoust et al., 1999) (Fig. 2D). The inhibition of SuSy activ- processes, all of which are crucial for fruit growth in tomato.
ity affected fruit set and very early fruit development related Another example of a gene potentially modifying pri-
to the reduced unloading capacity for sucrose. This led the mary carbon metabolism and photosynthesis in fruit is the
authors to propose that SuSy participates in the control of chloroplastic isoform of fructose 1,6-bisphosphatase (cp-
sucrose import capacity of young tomato fruit and, conse- FBPase), an important enzyme in control of the Calvin cycle.
quently, influences fruit set and development. Unfortunately, Obiadalla-Ali et al. (2004) generated tomato plants where
independent trials using equivalent transgenic plants failed the activity of this isoform was repressed specifically in fruit
to reproduce these results, and the lack of reports associating (Fig. 2D). Although overall carbohydrate metabolism was
SuSy isoforms and QTLs for fruit weight or sugar content only slightly altered, fruit growth and final fruit size were sig-
raised doubts about the validity of these conclusions (Carrari nificantly reduced, suggesting that cp-FBPase contributes to
and Fernie, 2006). fruit photosynthesis in providing carbon for fruit growth.