Journal of Experimental Botany, Vol. 66, No. 4 pp. 1075–1086, 2015
doi:10.1093/jxb/eru527 Advance Access publication 7 January 2015
Review Paper
Fruit growth-related genes in tomato
Lamia Azzi1,*, Cynthia Deluche1,*, Frédéric Gévaudant1, Nathalie Frangne1, Frédéric Delmas1, Michel Hernould1
and Christian Chevalier2,†
1 University of Bordeaux, UMR1332 Biologie du Fruit et Pathologie, INRA Bordeaux Aquitaine, CS20032, F-33882 Villenave d’Ornon
cedex, France
2 INRA, UMR1332 Biologie du Fruit et Pathologie, INRA Bordeaux Aquitaine, CS20032, F-33882, Villenave d’Ornon cedex, France
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: [email protected]
† Received 1 October 2014; Revised 5 December 2014; Accepted 10 December 2014
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 crucial 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
the ovule and seed during embryo development and ensures
seed dispersal after maturation. At the botanical level, most
fruits develop from mature ovaries and, therefore, include
carpellar tissues. The physiological function of seed dispersal accounts for a significant part of the adaptive success of
angiosperms on Earth. Indeed, a wide diversity of fruit size,
form, and composition, as well as a variety of fruit dispersion
mechanisms, have emerged as the result of strong environmental selective pressures. For example, the Solanaceae family, which encompasses nearly 10 000 species, has very diverse
types of fruits, with capsules, drupes, pyrenes, berries, and
several types of dehiscent non-capsular fruits occurring in
>90 genera (Knapp, 2002).
In general, the term fruit refers to the fleshy, edible portion
that is the major component of fruits, such as grape, banana,
apple, citrus, peach, strawberry, melon, and tomato. All of
these species are produced worldwide, and continue to rely
on breeding schemes for agronomic traits. Particular traits of
interest are enhanced yield and quality, increased time of fruit
storage and longer shelf-life, optimized cultural practices, and
pest and pathogen resistance. The common feature of fleshy
fruits is the fine balance between sugars and organic acids,
and the accumulation of water, minerals, pigments, aromatic
compounds, and vitamins that confer upon the fruit its juiciness and attractiveness. Consequently, fleshy fruits represent
a major source of vitamins, fibre, carbohydrates, and phytonutrient 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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1076 | Azzi et al.
Tomato (Solanum lycopersicum Mill.) fruit, a multicarpellar berry, has arisen as the model species for fleshy fruits, due
to certain advantages for use in both agronomical and fundamental research (Gillaspy et al., 1993; Tanksley, 2004; Klee
and Giovannoni, 2011). These advantages include a short
life cycle, high multiplication rate, self-pollination, and ease
of mechanical crossing. This highly favourable biology has
been widely exploited to generate segregating populations
such as recombinant inbred lines (RILs) or near isogenic lines
(NILs). Tomato has proven amenable for marker-assisted
mapping using crosses between small and round, wild tomatoes and domesticated varieties of various sizes and shapes
that has enriched our knowledge of the genetic control of
fruit development (Grandillo et al., 1999; Causse et al.,
2007; Muños et al., 2011; Rodriguez et al., 2011). A large
spectrum of mutants is now available (Menda et al., 2004;
Watanabe et al., 2007; Just et al., 2013) for screening and gene
identification by reverse genetic tools, such as the powerful
technology of Tilling (Targeting Induced Local Lesions In
Genomes; Okabe et al., 2011). Functional analyses of candidate genes are routinely conducted, since tomato is highly
suitable to stable transformation via Agrobacterium tumefaciens, and susceptible to transient gene expression via agroinjection (Orzaez et al., 2006). Additionally, the repression of
target genes can be obtained by virus-induced gene silencing
(VIGS) (Orzaez et al., 2009) or by a genome-editing strategy using the RNA-guided DNA endonuclease system called
the clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated9 (Cas9) system (Brooks
et al., 2014). Finally, the release in 2012 of the full tomato
genome sequence represented an extraordinary breakthrough
for developmental studies in fleshy fruits (Tomato Genome
Consortium, 2012).
The development of plant organs requires mechanisms of
differentiation by which tissue identity is imposed on cells as
well as mechanisms regulating growth that dictate final organ
size. The determination of organ size thus relies on the fine
regulation of the number and size of cells that is determined
by the cell division and cell expansion processes, respectively.
These processes respond to spatial and temporal controls that
come under the influence of internal (genotypic) and external
(environmental) cues. As a general feature of angiosperms,
cell expansion is frequently ascribed to nuclear endopolyploidization in several plant organs, according to the process of endoreduplication (Chevalier et al., 2011, 2014; De
Veylder et al., 2011).
This review addresses the current knowledge on fruit development in tomato, in particular describing those genes that
are involved in the developmental processes governing fruit
growth.
Tomato fruit development and the
contribution of endopolyploidization to
fruit growth
Fruits typically develop from pre-existing organs, such as
carpels inside flowers. In tomato, carpels are formed by
~17–20 rounds of cell divisions that occur during a pre-anthesis period located within the L3 layer of the floral meristem,
but which involve virtually no cell expansion (Coombe, 1976;
Ho, 1992). Obviously, the number of cells formed in the ovary
before anthesis is critical for the final size of fruit, and such
a positive correlation is frequently observed (Tanksley, 2004).
From flower initiation to the double fertilization occurring in
ovules (Gillaspy et al., 1993; Brukhin et al., 2003), the morphogenesis and growth of carpels and ovules require the spatial and temporal synthesis and action of auxin, cytokinin,
and gibberellins (GAs). In order to keep the ovary in a temporally protected and dormant state, abscisic acid (ABA) and
ethylene work to stop growth within the ovary shortly before
anthesis when the ovary has reached its mature size (Vriezen
et al., 2008). Growth resumes only after successful pollination
and fertilization of ovules triggers fruit set—the first phase of
fruit development—through the action of ovary-synthesized
auxins and GAs (Ruan et al., 2012, and references therein).
Fruit growth is the longest phase of fruit development,
as it ranges from 5 to 8 weeks depending on the genotype.
Growth proceeds first by a period of intense mitotic activity
according to a spatially and temporally organized pattern of
cell division. Active cell division within the pericarp is usually restricted to an initial period of 1–2 weeks after fruit set
(Fig. 1). Remarkably, cell division occurs within discrete cell
layers with well-defined planes of division fulfilling specific
purposes. For instance, the two subepidermal layers of the
pericarp undergo several rounds of periclinal divisions, thus
leading to an increase in the number of pericarp cell layers,
whereas the two epidermal cell layers undergo anticlinal divisions in response to the resulting increase in fruit volume
(Cheniclet et al., 2005). These various types of cell divisions
are differently regulated, because cell divisions promoting
cell layer formation occur only within 5–8 days post-anthesis (dpa), whereas randomly oriented cell divisions occur for
longer periods up to 10–18 dpa (Cheniclet et al., 2005). Cell
divisions in growing fruit account for as much as 80–97% of
newly formed cells present after anthesis. How this spatiotemporal pattern of development is related to gene expression, metabolic profiles, and cellular characteristics remains
poorly understood (Lemaire-Chamley et al., 2005; Bourdon
et al., 2011, 2012), primarily due to the complex nature of
fruit in relation to differing cell type.
It has been reported that a second phase of growth related
to cell expansion occurs separately after the cell division
phase (Gillaspy et al., 1993). In fact, cell expansion starts
within very few days after fruit set, concomitantly with cell
division (Cheniclet et al., 2005), and lasts for the entire period
of fruit growth (Fig. 1). At the end of the cell expansion
phase, individual cells in the fleshy part (mesocarp tissue)
of the fruit have reached spectacular volumes, exhibiting a
>30 000-fold increase from initial cell volume, and can correspond to a >0.5 mm increase in diameter (Cheniclet et al.,
2005). This cell enlargement mostly occurs through dramatic
increases in the vacuolar compartment and cell vacuolation index. Remarkably, this spectacular cell hypertrophy is
closely correlated to an increase in nuclear DNA levels due to
endopolyploidization.
Tomato fruit growth | 1077
Fruit Development
Cell expansion
Fruit set
Anthesis
Cell division
3
5
Ripening
8
12
15
20
25
35
40
dpa
35 dpa
8 dpa
0 dpa
7-8
Cell layers
14
Cell layers
14
Cell layers
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
within an organism. In plants, it results mostly from endoreduplication that is estimated to occur in >90% of angiosperms
(Nagl, 1976; D’Amato, 1984). Endoreduplication occurs in
the absence of any obvious DNA condensation and decondensation steps that lead to the production of chromosomes
with 2n chromatids or without any change in chromosome
number (Joubès and Chevalier, 2000; Edgar and Orr-Weaver,
2001). As a consequence, hypertrophic nuclei arise from successive cycles of DNA replication without segregation of
sister chromatids, thereby resulting in the production of polytene chromosomes (Bourdon et al., 2012). The physiological
and developmental importance of endoreduplication is still a
matter of debate. However, the frequent observation that cell
size and ploidy levels are highly and positively correlated in
many different plant species, organs, and cell types (Chevalier
et al., 2011) suggests a role for endoreduplication in the control of cell size, according to the ‘karyoplasmic ratio theory’.
This theory, which was formulated as early as the beginning
of the 20th century, states that there is a causal relationship
between nuclear and cytoplasmic growth to maintain a constant ratio of nucleus to cell volume (Sugimoto-Shirasu and
Roberts, 2003; Chevalier et al., 2014).
In the course of tomato fruit development, endoreduplication produces high levels of nuclear DNA ploidy within the
mesocarp and the locular jelly-like tissue (Bergervoet et al.,
1996; Joubès et al., 1999; Cheniclet et al., 2005; Bertin et al.,
2007). Ploidy levels as high as 512C (where C is the haploid
DNA content) have been observed to occur in a large array
of tomato genotypes. This extraordinary extent of endoreduplication occurring in tomato fruit is unmatched by any
other plant species and makes tomato an excellent model
to study the role of endoreduplication as a determinant of
fruit size. Cheniclet et al. (2005) demonstrated that the mean
ploidy level achieved in pericarp cells correlates not only with
the large variation in the fruit weight that can be encountered in the various genotypes, but also with the mean cell
size inside the pericarp. The endoreduplication-associated
cell expansion in fruit is characterized by profound cellular
modifications. It is evident that the formation of polytene
chromosomes impacts on nuclear, nucleolar, and chromatin organization within hypertrophied nuclei delimited by a
highly invaginated nuclear envelope (Bourdon et al., 2012).
These profound nuclear grooves are filled with numerous
mitochondria, whose number increases according to nuclear
DNA content. Apparently, endoreduplication triggers efficient communication between the nucleus and the cytoplasm,
despite the increase in nuclear volume. The cytoplasmic volume of cells also correlates with ploidy levels, which indicates
that the maintenance of the nuclear to cytoplasmic ratio to
drive cell growth is consistent with the ‘karyoplasmic ratio’
theory. In addition, Bourdon et al. (2012) provided evidence
1078 | Azzi et al.
Modifying fruit growth with cell cycle and
endocycle regulatory genes
Cell division and cell expansion associated with endoreduplication provide, respectively, the building blocks for the
fruit organ and the growth-driving force that contribute to
establish the final fruit weight/size. Therefore, modifying fruit
growth by targeting genes involved in mitosis and endoreduplication has been considered for tomato (Fig. 2).
In Eukaryotes, the cell cycle is composed of four distinct
phases: an undifferentiated DNA pre-synthesis phase with a
2C nuclear DNA content, termed the G1 phase; the S phase
during which DNA is synthesized, with a nuclear DNA content intermediate between 2C and 4C; a second undifferentiated phase (a DNA post-synthetic phase) with a 4C nuclear
DNA content, termed the G2 phase; and the M phase, or
mitotic phase. Progression of the cell cycle is ensured by the
activity of core regulators consisting of conserved heterodimeric protein complexes. These complexes are formed by
a catalytic subunit referred to as cyclin-dependent kinase
(CDK) and a regulatory cyclin (CYC) subunit. The presence of the regulatory CYC is essential for the CDK–CYC
Anthesis
B
C
Fruit set
Ovary Dev.
A
complex activity as it confers its stability, its localization, and
its substrate specificity (Inzé and De Veylder, 2006). Specific
CDK–CYC complexes operate at the boundaries between the
various phases of the cell cycle in order to phosphorylate target proteins. These post-translational modifications which can
be either inhibitory or activating, are essential for the progression 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 several ways: (i) CDK activity is finely tuned by phosphorylation
and dephosphorylation of the CDK on conserved residues by
specific kinases and phosphatases; (ii) the proteolytic destruction of the cyclin subunit by the ubiquitin–proteasome system
(UPS) is sufficient to abolish activity of the complex, as CDK
alone does not display kinase activity without its cyclin partner; and (iii) the CDK–CYC complexes are inactivated by the
specific binding of CDK inhibitors (Churchmann et al., 2006;
Torres Acosta et al., 2011).
The endoreduplication cycle (endocyle) consists of successive rounds of DNA synthesis (S phases) in the absence of
mitosis, and thus represents a partial and modified cell cycle.
Consequently, cell cycle and endocycle progression involves
control of CDK–CYC activity levels by common regulatory
mechanisms on the molecular level, such as those mentioned
above (De Veylder et al., 2011). Central to this regulation is
the maintenance of a certain threshold of CDK–CYC activity to allow the commitment to mitosis. When mitotic CDK
complexes do not form or their activity is suppressed, this
threshold is not exceeded and the level of CDK–CYC activity is insufficient to drive cells into mitosis; endoreduplication
can then take place.
Cell division
for the existence of a strong relationship between endoreduplication and rRNA and mRNA transcription.
As a morphogenetic factor, endoreduplication thus contributes to maintain homeostasis of cytoplasmic components
through the establishment of a highly structured cellular system, where multiple physiological functions are integrated to
support cell growth during fruit development.
Auxin signaling:
SlPIN4, SlTIR1, SlARF7,
SlARF8, SlIAA9
Carpel identity and
Cell division control:
TAGL1,
FAS, LC,
SlWUS,
SlIMA
3
5
8
Cell cycle control:
SlCDKA;1, SlCDKB1, SlCDKB2
Cell division control:
FW2.2, SlKLUH/FW3.2, FW11.3,
OVATE, SUN, SlIMA
GA signaling:
SlGA20ox1, SlDELLA1
D
Cell expansion
Auxin signaling:
SlPIN4
3
5
8
Endocycle control:
SlCCS52A, SlWEE1, SlKRP1
Auxin signaling:
SlIAA17
ABA biosynthesis:
NOTABILIS/NCED1, FLACCA
12
15
20
25
35
Primary metabolism:
HXK1, SuSY, LIN5, TIV1
mMDH, cpFBP
Regulation of primary metabolism:
SPA
Ascorbate biosythesis:
Gal-LDH, GME
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 | 1079
In tomato, several functional analyses have attempted to
modulate the CDK–CYC complex activity during fruit development, either through direct targeting of CDK gene expression or through post-translational regulation of the complex
(Fig. 2C). Czerednik et al. (2012) generated transgenic plants
aimed at down-regulating the canonical CDKA;1, the key
player in cell cycle progression, in a fruit-specific manner
using an artificial microRNA (amiCDKA) and the TPRP
promoter (Fernandez et al., 2009). The amiCDKA plants produced smaller fruits than those of the wild type, and which
had a thinner pericarp due to an overall decreased number
of cell layers within the exocarp (displaying the smallest
cells). In contrast, the mesocarp displayed normal, enlarged
cells without any significant difference in ploidy levels.
A similar phenotype was obtained with fruit-specific overexpression of either mitosis-associated CDKB1 or CDKB2
(Czerednik et al., 2012). Interestingly, the expression of
CDKA;1 was greatly repressed in overexpressing CDKB1
and CDKB2 lines in accordance with the phenotype of the
amiCDKA line. More recently, tomato fruits overexpressing CDKA;1 have been reported (Czerednik et al., 2014).
CDKA1-overexpressing fruits were visually indistinguishable
from those of the wild type, with similar fruit weights and
diameters. However, they exhibited a larger placental area,
a thicker pericarp, and a reduced number of seeds. Across
the pericarp, the number of constituting cell layers was significantly increased, as well as the number of cells per mm2,
which was also observed in the placenta. Overall, the observations showed that mean cell size was smaller, and was accompanied by a decrease in endoreduplication levels in cells from
the mesocarp, placenta, and jelly-like tissues. These data provided another example of the relationship between endoreduplication and final cell size, suggesting a pivotal role for
CDKA1 in the regulation of mitotic activities during fruit
development. Additionally, CDKA1 overexpression affected
the production of seeds in developing fruits that indirectly
affected pericarp cell expansion, which is normally driven by
seed-produced phytohormones.
Altering CDKA or CDKB1/B2 gene expression in tomato
fruits produced phenotypes that highlighted differential
effects on cell division, cell expansion, and endoreduplication (Czerednik et al., 2012, 2014). In general, the observed
data are difficult to interpret, most probably because they are
associated with the specific tissular and developmental pattern of expression of the TPRP promoter, which was used
to drive gene misexpression. The TPRP promoter is a fruitspecific promoter whose expression starts early in the ovary
and reaches a maximum during the cell expansion phase of
fruit development (Fernandez et al., 2009). As a consequence,
CDKB1 and CDKB2 were overexpressed outside their natural timing of expression (Joubès et al., 1999, 2001), which
may have greatly affected the availability of regulatory cyclins for proper composition of CDK–CYC complexes, and/
or created an artificial competition for regulatory cyclins
among the misexpressed CDKs. In the absence of measured
CDK activity, we speculate that the phenotypes observed by
down-regulating CDKA genes, or up-regulating CDKB genes
are caused by the lack of endocycle-specific CDKA–CYC
complex activity, which is necessary to permit the youngest
cells of the outermost layers of pericarp (namely the exocarp) to enter into the phase of endoreduplication-driven cell
expansion. In a similar manner, overexpression of CDKA;1
induced mitosis across the pericarp and altered the endoreduplication index (Czerednik et al., 2014), thus supporting
the idea that CDKA plays an important part in the onset of
endoreduplication and subsequent cell and fruit growth.
The inhibitory effect of phosphorylating CDKA on the
Tyr15 residue through the action of the WEE1 kinase represents a good example of a post-translational regulatory
mechanism impairing the cell cycle, and potentially triggering the endocycle (Fig. 2D). Gonzalez et al. (2007) generated
transgenic tomato plants in which WEE1 was ectopically
repressed. Smaller fruits were produced displaying a thinner pericarp composed of smaller cells. There was a strong
reduction in endoreduplication as a result of impaired WEE1
kinase activity leading to an enhanced CDK–CYC activity. The WEE1 phosphorylation activity on its CDK targets
appears to be an important mode of regulation for the promotion of endoreduplication during fruit development, and
contributes to cell size determination, which ultimately influences final fruit size (Chevalier et al., 2011, 2014).
The completion of mitosis and progression from mitosis
back into interphase requires the loss of CDK–CYC complex activity, which occurs through proteolytic destruction of
the cyclin moiety by the UPS. This process involves a specific E3-type ubiquitin ligase named the anaphase-promoting
complex/cyclosome (APC/C), which is activated through its
association with CDH1/FZR-type proteins (Heyman and De
Veylder, 2012). In plants, the commitment towards endoreduplication involves the selective destruction of mitotic cyclins,
thereby preventing the formation of a proper mitotic CDK–
CYC complex, and thus impairing the associated kinase
activity. This process is achieved by the CCS52A-mediated
activation of the APC/C (Cebolla et al., 1999). The ectopic
loss of function of CCS52A in transgenic tomato plants led
to the production of smaller fruits, when compared with the
wild type (Mathieu-Rivet et al., 2010) (Fig. 2D). The DNA
ploidy levels in these fruits were shifted towards lower levels,
which corresponded to a decrease in mean cell size and an
increase in cell number. Conversely, the ectopic overexpression of SlCCS52A in tomato plants resulted in fruits slightly
smaller than those of the wild type. The cytological analyses
of these fruits revealed that the overexpression of SlCCS52A
first slowed down early fruit growth, which then resumed
as highly endoreduplicated nuclei, and consequently larger
cells, were generated. The resulting accelerated fruit growth
(Mathieu-Rivet et al., 2010) was in agreement with the
expected involvement of SlCCS52A in endoreduplicationdriven cell expansion.
The last example in modulating the CDK–CYC complex
activity during tomato fruit development refers to the work
of Nafati et al. (2011). SlKRP1 (Fig. 2D), encoding a tomato
CDK-specific inhibitor, was overexpressed in a fruit- and
cell expansion-specific manner using the PEPC2 promoter
(Fernandez et al. 2009). Increasing SlKRP1 significantly
reduced the extent of endoreduplication within the mesocarp,
1080 | Azzi et al.
as already described for a strong overexpression of KRP
genes in other plant tissues or organs (De Veylder et al., 2001;
Jasinski et al., 2002; Zhou et al., 2003). However, the same
construct was ineffective during the phase of cell expansion
and did not alter the final size of fruits, in contrast to previous reports describing plant dwarfism. The mean cell size
within the pericarp was unaffected, demonstrating that cell
size was, at least partially, uncoupled from DNA ploidy levels.
Nevetheless, endoreduplication occurred in these transgenic
fruit, but to a far lesser extent than in the wild type. This suggests that DNA ploidy levels were sufficient to support cell
growth, which is in agreement with the ‘karyoplasmic ratio
theory’ (Schnittger et al., 2003; Chevalier et al., 2014).
Modifying fruit growth by genes controlling
fruit size and shape
Since its introduction in Europe in the 16th century, the
domestication and extensive breeding of tomato led to the
creation of cultivated varieties bearing enlarged fruits compared with the wild ancestors (Paran and van der Knaap,
2007). The diversity of fruit size in tomato has been attributed to nearly 30 quantitative trait loci (QTLs; Grandillo
et al., 1999).
The Fruit Weight QTL of chromosome 2, number 2 (fw2.2)
was the first QTL-associated gene that was identified and
cloned (Alpert et al., 1995; Alpert and Tanksley, 1996; Frary
et al., 2000). As a major QTL, fw2.2 accounts for as much as
a 30% difference in fruit fresh weight between the domesticated tomato and its wild relatives. FW2.2 acts as a negative
regulator of cell proliferation (Fig. 2C), thus explaining its
major effect on fruit size in tomato. In addition, the role of
fw2.2 in the determination of organ size is conserved within
the plant kingdom, in both monocotyledonous and dicotyledonous species, such as avocado, maize, soybean, and cherry
(Dahan et al., 2010; Guo et al., 2010; Libault et al., 2010; De
Franceschi et al., 2013). Unfortunately, no clear biochemical
function has been attributed so far to FW2.2 and its orthologues, especially in relation to the regulation of the cell cycle/
cell division.
FW2.2 belongs to a multigene family encompassing 17
homologues in tomato (hereafter referred to as FW2.2-Like
or FWL genes). FW2.2 and FWL proteins all contain the
uncharacterized PLAC8 motif, which was originally identified in proteins from mammalian placenta (Guo et al., 2010).
The PLAC8 motif contains two conserved cysteine-rich
domains separated by a variable region that are predicted to
be transmembrane segments. The original tomato FW2.2 protein possesses these two transmembrane-spanning domains
that fix the protein to the plasmalemma (Cong and Tanksley,
2006). From previous reports, it appears that the cysteine-rich
domains may be involvled in the transport of heavy metals
such as cadmium and zinc, as identified in the plant cadmium
resistance proteins from Arabidopsis (Song et al., 2010). This
type of protein may multimerize into a homopentamer to
form a transmembrane pore, thereby facilitating metal cation
transport (Guo et al., 2010). The association between FW2.2
and its orthologues in relation to ion transport remains to be
established, and how this could interfere with the control of
cell proliferation is an intriguing question.
fw3.2 is only the second major QTL for fruit size/weight
to be fine mapped and cloned in tomato (Chakrabarti et al.,
2013) (Fig. 2C). The gene underlying this QTL encodes a
P450 enzyme of the CYP78A subfamily, previously identified
as KLUH (Anastasiou et al., 2007). The effect of SlKLUH
is to enlarge fruit volume through an increase in cell number
within the pericarp and septum tissues. Repressing SlKLUH
using an RNA interference (RNAi) strategy led to decreased
fruit and seed size and, consequently, plant architecture was
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
elucidating the biological function of FW3.2.
fw11.3 is another important locus governing fruit weight in
tomato (Van der Knaap and Tanksley, 2003) (Fig. 2C). The
fine mapping of fw11.3 demonstrated that it overlaps with
fasciated (fas) which is a locus governing fruit shape on chromosome 11, but fw11.3 and fas are not allelic (Huang and van
der Knaap, 2011). Unlike fw2.2 and fas, the large-fruit allele
of fw11.3 is partially dominant. The anticipated cloning of
FW11.3 will yield a third example of a cloned QTL regulating
fruit weight.
Besides increasing fruit size, the domestication of tomato
resulted in a high diversity in fruit shape. From the invariable, round-shaped fruits of wild species, cultivated tomato
has diversified into round, flat, rectangular, ellipsoid, obovoid
(pear-shaped), heart, oxheart, and long (bell pepper-shaped)
fruits (Monforte et al., 2014). This diversity in tomato fruit
shape originates from only four mutated genes, OVATE,
SUN, FASCIATED (FAS), and LOCULE NUMBER (LC)
(Rodríguez et al., 2011). Both OVATE and SUN control fruit
elongation, OVATE is a negative regulator of growth leading to shorter fruit, whereas SUN is a positive regulator of
growth resulting in elongated fruit (Fig. 2C). FAS and LC
control the number of fruit locules, which ultimately influences both fruit shape and fruit size (Fig. 2A).
OVATE was the first gene associated with fruit shape to be
identified by positional cloning (Liu et al., 2002). It belongs
to the ovate family protein (OFP) family of as yet unclear
function (Liu et al., 2002; Wang et al., 2011). The effects of
the ovate mutation on fruit shape vary from elongated fruits
to pear- or round-shaped fruits depending on the genetic
background carrying the ovate mutation (Gonzalo and van
der Knaap, 2008). This suggests that OVATE is not responsible for the observed phenotype and probably interacts with
other genes in an epistatic manner. The ovate pear-shaped
fruit phenotype was complemented both by a genomic DNA
fragment covering the OVATE gene and by its ectopic overexpression to revert to round-shaped fruits (Liu et al., 2002).
As a result, the ovate mutation is likely to be a loss-of-function mutation of a negative regulator of plant growth whose
function remains to be elucidated. Interestingly, it has been
shown in Arabidopsis that OVATE-like proteins act as transcriptional repressors that affect, in particular, the expression
of AtGA20ox1, a key player in the GA biosynthetic pathway,
Tomato fruit growth | 1081
thus resulting in reduced cell elongation (Hackbusch et al.,
2005; Wang et al., 2007, 2011). The indirect influence of
OVATE on fruit shape through regulation of GA biosynthesis in tomato remains to be demonstrated.
The sun mutation causes an elongated-fruit phenotype.
A gene duplication event mediated by a retrotransposon
placed the SUN gene under the control of the defensin
gene DEFL1 promoter, thus leading to high expression in
fruit (Xiao et al., 2008; Jiang et al., 2009). The SUN protein
belongs to the IQ67 domain-containing plant protein family
(Xiao et al., 2008) of unknown function. SUN overexpression
results in increased cell number in the longitudinal direction
and reduced cell number in the transverse direction of the
fruit, thus leading to fruit elongation (Wu et al., 2011).
The number of fruit locules is determined by the number of
carpels within the flower. Wild species of tomato produce fruits
with 2–4 locules, whereas cultivated varieties can develop >15
locules. As a result, not only can the shape of such fruits be
greatly impacted, but this can also account for a tremendous
increase in fruit size by as much as 50% (Tanksley, 2004). The
QTL fas was identified as a trait governing extreme fruit size
that increases the number of locules from two to more than
seven, whereas the QTL lc has a weaker effect (Lippman and
Tanksley, 2001; Barrero et al., 2006). FAS codes for a YABBYlike transcription factor (Cong et al., 2008), whereas LC has
been located in a non-coding region between two putative candidate genes, WUSCHEL, which is a member of the plant-specific WUS homeobox (WOX) transcription factor family, and
a gene encoding a WD40 repeat protein (Muños et al., 2011).
The function of most of the WOX genes studied so far can
be ascribed to promotion of cell division and/or prevention of
premature differentiation (van der Graaff et al., 2009). More
specifically, WUS is involved in maintaining stem cell fate and
meristem size, and, therefore, it is possible that the function of
WUSCHEL is affected in lc. FAS and LC can interact epistatically to produce fruits with an extremely high locule number
(Barrero and Tanksley, 2004). Both FAS and LC control floral
meristem size and ultimately the development of supernumerary carpels (locules) leading to larger fruits (Cong et al., 2008;
Muños et al., 2011). In this context, the link between fruit size,
as influenced by FAS and LC, and the activities regulating meristem size and cell division have been clearly established.
Functional analyses using TOMATO AGAMOUSLIKE1 (TAGL1), which is the orthologue of the duplicated
SHATTERPROOF (SHP) MADS box gene of Arabidopsis
thaliana, demonstrated the involvement of this transcription factor in the regulation of fruit development (Vrebalov
et al., 2009) (Fig. 2A). Tomato plants repressing TAGL1 by
RNAi produced smaller fruits with a thinner pericarp which
displayed fewer cell layers, and altered ripening-related fruit
pigmentation, indicating that TAGL1 is important for regulating both fruit growth and the ripening process.
Hormonal regulation of fruit growth
After successful flower pollination and ovule fertilization, the
fruit and seed initiation, a stage that is commonly referred to
as fruit set, and subsequent development of both fruit and
seeds occur concomitantly according to a precise, genetically
controlled process mediated by phytohormones (Gillaspy
et al., 1993).
Recent reviews have highlighted the role of plant hormones
and their interplay in the control of fruit development (Ruan
et al., 2012; Kumar et al., 2014). Here, we focus on the most
relevant genes, whose functional characterization in tomato
revealed an involvement in the growth period of fruit development. Auxin and GA appear to be the predominant hormones required for fruit initiation in response to fertilization,
since exogenous applications of both hormones lead to fruit
initiation and parthenocarpic development (de Jong et al.,
2009). A role for cytokinin, ethylene, and ABA in fruit formation has been demonstrated, but is less well documented
thus far (Kumar et al., 2014).
Early fruit development is governed by the allocation of
auxin to tissues and cells in order to initiate signal transduction pathways (Fig. 2B). Recent evidence has indicated that
the PIN-FORMED (PIN) auxin efflux transport proteins are
involved in the processes of fruit set and early fruit development in tomato (Pattison and Catala, 2012). Silencing of the
tomato SlPIN4 gene, which is predominantly expressed in
flower buds and young developing fruit, produced seedless
(parthenocarpic) fruits of reduced size exhibiting precocious
development (Mounet et al., 2012). The auxin signalling
pathway involves an auxin receptor called the TRANSPORT
INHIBITOR RESPONSE1 (TIR1) protein. In the presence
of auxin, TIR1 recruits the AUXIN/INDOLE-3-ACETIC
ACID (Aux/IAA) transcriptional repressors and triggers
their degradation by the 26S proteasome. The degradation
of Aux/IAA repressor proteins releases the Aux/IAA-bound
auxin response factors (ARFs), thereby initiating the auxin
response through auxin responsive element-mediated gene
transcription. The misexpression in tomato of the auxin
receptor TIR1 gene, as well as specific members of the Aux/
IAA and ARF gene family, alters the normal flower-to-fruit
transition and results in uncoupling fruit set from pollination and fertilization and gives rise to parthenocarpic fruits
(Wang et al., 2005; de Jong et al., 2009; Ren et al., 2011).
Recently, Su et al. (2014) reported that tomato plants silenced
for the Aux/IAA transcriptional repressor SlIAA17 displayed
larger fruits with thicker pericarp tissue (Fig. 2D). This phenotype stemmed from enhanced cell expansion due to high
ploidy levels. This suggests an enhancement of the endoreduplication process in the absence of the optimal quantity of
SlIAA17 during fruit development.
In tomato, fruit set is partly mediated by GAs according to a complex hormonal cross-talk with auxin (Serrani
et al., 2008) (Fig. 2B). Auxin synthesized both in the ovary
and in the apical shoot prevents unpollinated ovaries from
developing by reducing transcript levels of genes encoding
GA biosynthetic enzymes, in particular the GA 20-oxidases
(Serrani et al., 2007). Pollination then triggers the up-regulation of transcripts for GA 20-oxidases that successively
synthesize active GA1 and GA4. The GA 20-oxidase activity thus appears as the limiting factor for active GA biosynthesis and, consequently, for fruit set. Functional analyses in
tomato aimed at the constitutive repression of SlGA20ox1
1082 | Azzi et al.
during fruit initiation and growth resulted in plants severely
affected in vegetative development and reduced pollen viability (Olimpieri et al., 2011). Ovaries from these SlGA20ox1silenced plants remained fertile and developed normally
after cross-pollination with wild-type pollen, but remained
parthenocarpic. The individual silencing of SlGA20ox1,
SlGA20ox2, and SlGA20ox3 genes confirmed the effects on
vegetative development but, again, no effects on fruit set were
observed (Xiao et al., 2006). Altogether, these data confirmed
the pleiotropic developmental role of GAs, but suggested that
the expression of more than one GA20ox gene is required to
control fruit set in tomato. Interestingly, the heterologous
overexpression in tomato of CgGA20ox1 from citrus clearly
demonstrated the expected influence of GA and GA20ox
activity on fruit set and development (Garcia-Hurtado et al.,
2012). The overexpression of CgGA20ox1 resulted in elevated
GA4 content and boosted vegetative development, as shown
by longer hypocotyls and roots and increased plant height.
In addition, flowers from the the transgenic plants exhibited
a protruding stigma due to a longer style and fruit displayed
parthenocarpic development.
The GA signal transduction pathway requires the recognition of GA by its receptor called GA INSENSITIVE
DWARF1 (GID1). The GID1–GA complex interacts with the
nuclear repressor DELLA to target it for ubiquitin-dependent proteolytic degradation by the 26S proteasome. The
repression of GA-responsive genes is then released to initiate GA signal transduction. Silencing of the SlDELLA1 gene
in tomato produced very similar vegetative and reproductive
phenotypes to those described for GA20ox1-overexpressing
plants (Marti et al., 2007), as fruits were facultative parthenocarpic, smaller in size, and elongated in shape (Fig. 2B).
A number of ABA-deficient mutants have proven valuable
toward elucidating the role of ABA in fruit growth. Three
ABA biosynthetic mutants have been described: sitiens (sit),
flacca (flc), and notabilis (not) that lack, respectively, an
ABA-aldehyde oxidase (Harrison et al., 2011), a molybdenum-cofactor sulphurase (Sagi et al., 2002), and 9-cis epoxycarotenoid dioxygenase1 (NCED1) (Burbidge et al., 1999)
(Fig. 2D). Although the mutants have been characterized on
the molecular level, unfortunately little was reported on the
effects of these mutations on fruit growth. Nitsch et al. (2012)
reported the phenotypic characterization of not/flc double
mutant lines. The fruits of these double mutants have considerably reduced ABA levels, and displayed smaller fruit size
and cell size, especially within the pericarp. The consequence
of increasing ethylene levels, while lowering ABA, suggested
that ABA stimulates fruit growth by restricting ethylene levels
in normal fruits.
The INHIBITOR OF MERISTEM ACTIVITY (IMA)
protein is a mini zinc finger (MIF) protein harbouring an unusual zinc-finger domain that was identified as an important
effector of a signalling pathway involving multiple hormones
that links cell division, cell differentiation, and hormonal control of development in tomato (Sicard et al., 2008) (Fig. 2A,
C). IMA regulates the meristem activity and the processes of
flower and ovule development. Carpel primordia within the
floral meristem were much smaller in IMA-overexpressing
plants, whereas they were enlarged in RNAi-silenced plants.
The cell number within the carpel was reduced, leading to a
smaller carpel, thus suggesting that IMA encodes an inhibitor of cell division. Consequently, plants overexpressing IMA
produced smaller flowers and fruits, whereas RNAi-silenced
plants produced fruits composed of supernumerary ovaries.
In addition, IMA repressed the expression of WUS which
controls the meristem organizing centre and the determinacy
of the nucellus during ovule development (Sicard et al., 2008)
(Fig. 2A).
Metabolic control of fruit development
The early stages of fruit development represent a critical
period whereby traits, such as organoleptic composition,
are established and ultimately dictate the final quality of the
fruit. Water, organic acids (primarily citrate and malate), and
minerals accumulate inside the vacuole of expanding cells
(Coombe, 1976) while starch accumulates transiently and
is converted subsequently to reducing sugars (Wang et al.,
1993). Fruit softening, colouring, and sweetening then occur
during the ripening phase (Giovannoni, 2004; Gapper et al.,
2013). Fruit development and fruit weight are intimately connected to its composition of primary and secondary metabolites (Carrari and Fernie, 2006; Tohge et al., 2014). Therefore,
modifying the expression of metabolism-associated genes
has been investigated as a means to induce variations in fruit
composition and size.
The development of fruit as a sink organ is more dependent
upon the allocation of photo-assimilates than on the fruit’s
own photosynthetic capacity. Modification of photo-assimilate supply substantially affects fruit development and size
through the modulation of cell number and cell size (Bohner
and Bangerth, 1988; Bertin et al., 2002). When tomato plants
were submitted to extended darkness, fruit growth was
severely impaired as the result of a strong repression of cell
cycle genes inside fruit tissues (Baldet et al., 2002). Conversely,
increasing photo-assimilate availability within the fruit by
reducing the number of fruit per truss led to an enhancement
in both flower and fruit growth rates. This corresponded to a
greater cell number inside the carpel, due to an enhancement
of mitotic activities (Baldet et al., 2006). The percentage variation observed in fruit fresh weight resulting from modulating fruit load is even as high as that which can be achieved by
genetic modification (Prudent et al., 2009). Hence, the modification of carbon metabolism and photo-assimilate partitioning by the manipulation of key enzymatic activities, such
as those involved in primary carbon metabolism and photosynthesis, was expected to have an impact on fruit growth.
In order to investigate this, the Arabidopsis Hexokinase 1
(AtHXK1) gene was constitutively overexpressed in tomato
plants (Menu et al., 2004) (Fig. 2D). Overexpressing lines
exhibited marked phenotypic and biochemical changes in
developing fruits, such as reduced fruit size and a decrease in
cell expansion. The carbon supply required to support these
processes was lower throughout development, most probably
due to decreased photosynthesis. Consequently, any sucrose
provided to these fruits would be used to fuel cell metabolism
Tomato fruit growth | 1083
at the expense of starch storage. Fruit displayed reduced respiratory rates, which were accompanied by lower ATP levels
and ATP/ADP ratios in fruit extracts, indicating profound
metabolic perturbations.
The role of sucrose synthase (SuSy) in tomato fruit development has been studied by silencing a fruit-specific isoform
(D’Aoust et al., 1999) (Fig. 2D). The inhibition of SuSy activity affected fruit set and very early fruit development related
to the reduced unloading capacity for sucrose. This led the
authors to propose that SuSy participates in the control of
sucrose import capacity of young tomato fruit and, consequently, influences fruit set and development. Unfortunately,
independent trials using equivalent transgenic plants failed
to reproduce these results, and the lack of reports associating
SuSy isoforms and QTLs for fruit weight or sugar content
raised doubts about the validity of these conclusions (Carrari
and Fernie, 2006).
The QTL Lin5 has been identified as a major QTL controlling fruit weight and sugar content (Fridman et al., 2000),
and the associated gene was found to code for a cell wallbound invertase (Fridman et al., 2004). When Lin5 was RNAi
silenced, fruit yield was greatly reduced, as well as fruit size,
seed size, and seed number (Zanor et al., 2009) (Fig. 2D). In
these transgenic plants, metabolic changes were largely confined to sugar metabolism, since sucrose content increased
while glucose and fructose contents decreased, as observed
at the red ripe stage. Silencing of the vacuolar invertase TIV1
gene by an antisense strategy in tomato gave overall similar
results. Production of smaller fruits corresponded to high
rates of sucrose accumulation and decreased hexose sugar
concentrations during the last stage of development (Klann
et al., 1996) (Fig. 2D). Interestingly, changes in the concentration of osmotically active soluble sugars occurred during the
phase of cell expansion of fruit growth and affected fruit size.
This suggests that the concentration of osmotically active
sugars is tied to water influx, which is an important determinant of fruit enlargement.
In order to establish a link between glycolysis, synthesis of
hexose phosphates, and their conversion into organic acids,
transgenic tomato plants were silenced for the mitochondrial
tricarboxylic acid (TCA) cycle-associated malate dehydrogenase (mMDH) gene (Nunes-Nesi et al., 2005) (Fig. 2D).
RNAi-mMDH plants showed not only enhanced chloroplastic electron transport rates and photosynthetic activity,
but also increased fruit dry matter, indicating that the repression of mMDH improves carbon assimilation. Interestingly,
transgenic fruit accumulated the redox-related compound
ascorbate and displayed an increased capacity to use l-galactono-lactone which is the immediate precursor of ascorbate
biosynthesis, as a respiratory substrate. Correspondingly,
silencing of the tomato l-galactono-1,4-lactone dehydrogenase (Gal-LDH), which converts l-galactono-lactone into
ascorbate, substantially modified mitochondrial function,
including alteration of the ascorbate redox state and the TCA
cycle (Alhagdow et al., 2007) (Fig. 2D). Consequently, plant
and fruit growth were greatly reduced, since cell expansion
was affected. Additionally, fruit from tomato plants silenced
for the GDP-d-mannose 3,5-epimerase (GME), which is the
central enzyme in ascorbate biosynthesis, exhibited defects
in cell expansion and the biosynthesis of non-cellulosic cell
wall polysaccharides (Gilbert et al., 2009) (Fig. 2D). Taken
together, these findings indicate an ascorbate-mediated link
among the energy-generating processes of respiration and
photosynthesis, primary metabolism, and developmental
processes, all of which are crucial for fruit growth in tomato.
Another example of a gene potentially modifying primary carbon metabolism and photosynthesis in fruit is the
chloroplastic isoform of fructose 1,6-bisphosphatase (cpFBPase), an important enzyme in control of the Calvin cycle.
Obiadalla-Ali et al. (2004) generated tomato plants where
the activity of this isoform was repressed specifically in fruit
(Fig. 2D). Although overall carbohydrate metabolism was
only slightly altered, fruit growth and final fruit size were significantly reduced, suggesting that cp-FBPase contributes to
fruit photosynthesis in providing carbon for fruit growth.
More recently, the search for genomic regions spanning
QTLs connected to yield-associated traits identified nine candidate genes located to tomato chromosome 4 (Bermúdez
et al., 2008). Among these genes, a DnaJ chaperone-likeencoding gene was isolated and appeared to be associated
with changes in primary metabolites across tomato fruit development. The in planta functional analysis aimed at silencing
this gene, subsequently named SPA for Sugar PartitioningAffecting (Fig. 2D), showed that the ripe fruit weight, the
number of fruits per plant, and the harvest index were significantly higher in transgenic than in wild-type plants (Bermúdez
et al., 2014). SPA as a putative chaperone protein was shown
to act through a mechanism involving the regulation of phosphoglucomutase, sugar kinase, and invertase enzyme activities
during tomato fruit growth, thus regulating the harvest index
by affecting the source–sink carbon distribution.
Conclusion
In this review, we focused mainly on developmental and cellular processes that relate to the determination of growthrelated traits in tomato fruit, and we described various genetic
and functional analyses that provide insight into factors modifying fruit size. Although studies aimed at deciphering the
genetic control of fruit size and shape have provided intriguing results, many questions remain unanswered from the
available data. Association genetics and functional genomics approaches have allowed the localization of major QTLs,
and the genes controlling these traits have subsequently been
identified. Since we are still far from understanding the influence of genes, such as FW2.2, FW3.2, FW11.3, or OVATE,
SUN, and LC, on the process of fruit development, such as
cell proliferation, in-depth functional analyses are required
to unravel the molecular bases of their respective regulatory
properties.
Acknowledgements
The authors wish to express their deepest thanks to Professor Mark Hooks
(University of Bordeaux) for reading and editing the manuscript.
1084 | Azzi et al.
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