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REVIEW
Gynoecium and fruit development in Arabidopsis
Humberto Herrera-Ubaldo and Stefan de Folter*
ABSTRACT
Flowering plants produce flowers and one of the most complex floral
structures is the pistil or the gynoecium. All the floral organs
differentiate from the floral meristem. Various reviews exist on
molecular mechanisms controlling reproductive development, but
most focus on a short time window and there has been no recent
review on the complete developmental time frame of gynoecium and
fruit formation. Here, we highlight recent discoveries, including the
players, interactions and mechanisms that govern gynoecium and
fruit development in Arabidopsis. We also present the currently
known gene regulatory networks from gynoecium initiation until fruit
maturation.
KEY WORDS: Gynoecium, Fruit, Gene regulatory networks,
Transcription factors, Hormones
Introduction
Multicellular life on Earth is inconceivable without plants. Our
planet harbors close to 400,000 vascular plant species, of which
approximately 94% are seed plants (Govaerts, 2001; Willis, 2017),
which produce and reproduce themselves via seed. The vast
majority of seed plants are angiosperms, which produce flowers,
such as Arabidopsis thaliana of the Brassicaceae family (Fig. 1A).
Flower and seed formation are adaptive advantages of plant sexual
reproduction, which requires male and female gametes. In general,
flowers have four different types of floral organs placed in four
whorls, from outside to inside: sepals, petals, stamens and carpels
(see Glossary, Box 1). The stamen, also called the androecium, is
the ‘male’part of the flower that produces the male gametophyte
from which gametes differentiate. The ‘female’part of the flower is
formed by one or more carpels. In Arabidopsis, two congenitally
fused carpels form the pistil, which is also called gynoecium (see
Glossary, Box 1). Ovules are formed inside the gynoecium and the
female gametophyte, which produces the female gametes, develops
within each ovule (see Glossary, Box 1). Upon fertilization, seed
development begins and, in most plant species, the gynoecium turns
into a fruit (reviewed by Alvarez-Buylla et al., 2010; Dresselhaus
et al., 2016).
The identification of AGAMOUS (AG) as a transcription factor
controlling male and female reproductive organ development in
plants (androecium and gynoecium, respectively), marked the
beginning of a journey towards the understanding of the molecular
aspects of flower formation (Bowman et al., 1989; Irish, 2017;
Yanofsky et al., 1990). During three decades of research, many
master regulators, such as SEPALLATAs (SEPs), CRABS
CLAW (CRC), SPATULA (SPT), SHATTERPROOFs (SHPs),
FRUITFULL (FUL) and SEEDSTICK (STK), among many others,
have emerged as crucial regulators of reproductive development,
especially of gynoecium development (reviewed by Alvarez-Buylla
et al., 2010; Bowman et al., 1999; Ferrándiz et al., 2010; Reyes-
Olalde et al., 2013; Roeder and Yanofsky, 2006; Simonini and
Østergaard, 2019; Zúñiga-Mayo et al., 2019). Besides the
identification of more transcription factors involved in gynoecium
development, information on genes acting downstream of them
have also been discovered (reviewed by Pajoro et al., 2014).
Together, these discoveries have opened the road for charting gene
regulatory networks (GRNs). In addition, several datasets from
chromatin immunoprecipitation followed by sequencing (ChIP-seq)
experiments, have allowed genome-wide analysis of binding events
for many master regulators that participate in the transition to
reproduction and flower development (Chen et al., 2018). Now,
the fine-tuning aspects of flower development are starting to be
revealed.
Over the years, many review articles on flower and fruit
development have been published (Alvarez-Buylla et al., 2010;
Ballester and Ferrándiz, 2017; Bowman et al., 1999; Ferrándiz et al.,
2010; Marsch-Martínez and de Folter, 2016; Reyes-Olalde and de
Folter, 2019; Reyes-Olalde et al., 2013; Roeder and Yanofsky,
2006; Sehra and Franks, 2015; Simonini and Østergaard, 2019;
Smyth et al., 1990; Zúñiga-Mayo et al., 2019). However, there is
currently no recent review that includes all the current GRNs known
to date for gynoecium and fruit development. In 2015, we proposed
approaches and tools to construct a comprehensive GRN for
gynoecium development (Chávez Montes et al., 2015). Therefore,
this Review focuses on recent discoveries and the integration of the
GRNs that guide the development of Arabidopsis, starting from
gynoecium initiation until fruit maturation.
From a floral meristem to gynoecium initiation (stages 1-6)
Floral meristem
Plants possess the ability to form new organs continuously
(reviewed by Gaillochet and Lohmann, 2015; Sablowski, 2007).
When environmental and endogenous genetic cues are met, the
shoot apical meristem (SAM; see Glossary, Box 1) transitions to an
inflorescence meristem (IM; see Glossary, Box 1) (Fig. 1B,E),
marking the beginning of the reproductive phase of the plant
(reviewed by Andrés and Coupland, 2012). At the flanks of the IM,
auxin induces differentiation to give rise to flower primordia, each
with a floral meristem (FM; see Glossary, Box 1). In Arabidopsis,
the most recently formed flower primordium is referred to as
‘stage 1’(Smyth et al., 1990). When the next flower primordium is
formed, the previous one is called stage 2, and so on. At floral stage
3, the flower primordium increases in size and sepal primordia
become visible (Fig. 1B,C,F). The FM gives rise to the different
floral organs present in a mature flower (Fig. 1D,F) (reviewed by
Denay et al., 2017). During the next stages, sepals continue to grow;
during stage 5, petal and stamen primordia appear, whereas stage 6
is characterized by the complete coverage by the sepals of the
FM and internal flower primordia, including the gynoecium
Unidad de Genomica Avanzada (UGA-Langebio), Centro de Investigacion y de
Estudios Avanzados del Instituto Politecnico Nacional (CINVESTAV-IPN), Km. 9.6
Libramiento Norte, Carretera Irapuato-Leon, Irapuato 36824, Guanajuato, Mexico.
*Author for correspondence (stefan.defolter@cinvestav.mx)
H.H., 0000-0002-5408-4022; S.d., 0000-0003-4363-7274
1
© 2022. Published by The Company of Biologists Ltd
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Development (2022) 149, dev200120. doi:10.1242/dev.200120
DEVELOPMENT
primordium (see Glossary, Box 1), which becomes visible at stage 6
(Fig. 1F,G) (Alvarez-Buylla et al., 2010; Denay et al., 2017; Smyth
et al., 1990).
Floral meristem maintenance and termination
In general, the genes that regulate meristem maintenance in the
SAM and the FM are shared (reviewed by Chang et al., 2020;
Gaillochet and Lohmann, 2015). The role of the WUSCHEL-
CLAVATA3 (WUS-CLV3) circuit in plant stem cell niche
maintenance is key (Brand et al., 2000; Schoof et al., 2000). The
activity of WUS, together with LEAFY (LFY) and others,
converges on the regulation of AG, which starts to be expressed at
stage 3 in the FM (Fig. 2A). In turn, AG starts to repress WUS and
activates several genes that negatively regulate WUS expression
(Fig. 2).
The meristematic activity of the FM ends when the gynoecium
primordium is formed at stage 6; however, the programs controlling
FM termination begin at stage 3. The molecular mechanisms
underlying FM termination have recently been reviewed (Chang
et al., 2020; Lee et al., 2019; Shang et al., 2019; Xu et al., 2019;
Zúñiga-Mayo et al., 2019). The GRNs that control FM termination
(Fig. 2) and gynoecium initiation (Fig. 2B) contain at least 15
transcription factors and various other proteins. AG plays a leading
role in both these processes, as illustrated in the ABC and quartet
model for floral organ formation (reviewed by Coen and
Meyerowitz, 1991; Theißen and Saedler, 2001). AG is therefore
observed in the GRNs as the main hub (Fig. 2A). AG activates
pathways related to auxin and cytokinin signaling (Ó’Maoiléidigh
et al., 2018; Yamaguchi et al., 2017). Cytokinin signaling, in turn,
also controls AG (Gómez-Felipe et al., 2021; Rong et al., 2018).
The effects of AG in the control of FM termination and gynoecium
development must be tightly controlled to perform those functions
properly. Indeed, the mechanisms that control AG expression
are diverse, involving transcription factors, microRNAs, histone
deacetylase complexes and RNA-binding complexes (reviewed by
Pelayo et al., 2021). Interactions with other proteins and the
formation of protein complexes provide additional layers of control
of AG activity (Box 2). For example, the interaction with SEP3, via
the formation of dimers or tetramers, controls different biological
processes (Hugouvieux et al., 2018; Lai et al., 2020).
New nodes in the GRNs
Over the past few years, some new nodes have been included in the
networks that underlie gynoecium initiation. AINTEGUMENTA
(ANT), which has reported roles in the establishment of
flower primordia and the development of floral organs, has
now been identified to function at very early stages of flower
formation (stages 3-6), and the roles of ANT are shared with
AINTEGUMENTA-LIKE 6 (AIL6) (Krizek et al., 2020, 2021). At
stage 3, both proteins directly regulate AG, as well as other genes,
such as MONOPTEROS (MP) and REVOLUTA (REV), which
are related to meristem activity regulation and new primordia
formation (Krizek et al., 2021) (Fig. 2A). At stage 6, ANT positively
regulates SPT, an important gene for tissue development in
the medial domain, as described below (Fig. 2B) (Krizek et al.,
2020).
Epigenetic silencing of AG is illustrated by the participation of
POLYCOMB-GROUP (PcG) complexes with CURLY LEAF
(CLF), EMBRYONIC FLOWERING 1 (EMF1) and EMF2. In a
similar way, APETALA 2 (AP2) recruits TOPLESS (TPL) and
HISTONE DEACETYLASE 19 (HDA19) to repress AG (Fig. 2A)
(Pelayo et al., 2021). Now, some evidence points to the involvement
of PcG in silencing of WUS (Sun et al., 2019) and the modification
of chromatin in the control of auxin biosynthesis by AG and CRC
(Fig. 2) (Yamaguchi et al., 2018).
KNUCKLES (KNU), a C2H2 zinc-finger transcription factor,
also functions during FM termination in addition to its role in
repressing WUS in the FM (Fig. 2B). A recent analysis of its
expression pattern has revealed the presence of KNU in the CLV3
expression domain. The extended functions of KNU include
suppression of the expression of several floral meristem
regulators, such as CLV1 and CLV3, at stage 6 (Kwas
niewska
et al., 2021; Shang et al., 2021).
The transcription factor ETTIN (ETT), also known as AUXIN
RESPONSE FACTOR 3 (ARF3), is involved in gynoecium
patterning (Heisler et al., 2001). An additional function of
ETT is to inhibit cytokinin biosynthesis by repressing
ISOPENTENYLTRANSFERASE (IPT) and LONELY GUY (LOG)
genes, and the gene encoding the cytokinin receptor
ARABIDOPSIS HISTIDINE KINASE 4 (AHK4) during stages 5
Box 1. Glossary
Carpel. The female reproductive structure of a flower, consisting of an
ovary, a stigma and a style.
Carpel margin meristem (CMM). A meristematic region in the medial
domain, a zone located where the carpel margins fused. The CMM is the
origin of the medial tissues: placenta, ovules, septum, style and stigma.
Clearly visible at stages 7 and 8.
Chalaza. The basal part of an ovule opposite to the micropyle; where the
integuments, the nucellus and the funiculus are joined.
Floral meristem (FM). The progenitor of all the flower organs.
Fruit dehiscence. The opening of a mature fruit to release the seeds.
Funiculus. The stalk that attaches an ovule or seed to the placenta.
Gynoecium. The female part of a flower. In Arabidopsis, the gynoecium
consists of two congenitally fused carpels.
Gynoecium primordium. A dome-shaped group of cells, first visible at
stage 6.
Inflorescence meristem (IM). The region at the tip of the growing shoot
containing meristematic cells that generates the flowers.
Lateral domain. The two regions that are on either side of the medial
domain, corresponding to the carpel walls or valves. Visible from stage 7
onwards.
Medial domain. In Arabidopsis, the two carpels are fused vertically at
their margins, and these fused margins correspond to the medial domain
of the gynoecium. Visible from stage 7 onwards (oval-shaped hollow
tube).
Nucellus. The central part of the ovule that contains the embryo sac.
Ovule. A structure that contains the female reproductive cells, after
fertilization ovules become seeds. Ovule primordia arise at stage 9.
Placenta. The region within the ovary to which the ovules and seeds are
attached.
Replum. Located at the outer parts of the septum in the medial domain.
The valves are attached to them.
Septum. In Arabidopsis, there is a false septum that divides the two
carpels. Contains the transmitting tract.
Shoot apical meristem (SAM). The region at the tip of the growing shoot
containing meristematic cells that generates the aerial organs (leaves
and branches).
Silique. A type of dry fruit that has two fused carpels in Arabidopsis,
characteristic of the Brassicaceae family.
Stigma. Specialized epidermal cells located at the top of the gynoecium,
which capture the pollen grains, allowing germination and the first steps
of pollen tube growth.
Style. The tissue that connects the stigma with the ovary.
Transmitting tract. Specialized tissue derived from PCD in the style and
some septum cells in the ovary. Possesses ECM that provides nutrients,
guidance and support to the growing pollen tubes.
Valves. The outer tissue of the ovary; the carpel walls.
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REVIEW Development (2022) 149, dev200120. doi:10.1242/dev.200120
DEVELOPMENT
and 6. Through these mechanisms, ETT contributes to FM
termination (Fig. 2B) (Zhang et al., 2018).
Conversely, cytokinin also activates transcription factors important
for gynoecium initiation and patterning, which starts around stage 6
(Fig. 2B). For example, the master regulator AG is regulated by
cytokinin via the Arabidopsis response regulators type-B (ARRs)
(Gómez-Felipe et al., 2021; Rong et al., 2018). Furthermore, other
transcription factors that act in parallel downstream of AG are CRC,
SHP2 and SPT, which are important for gynoecium initiation and
patterning, as well as being positively regulated by ARR type-B
transcription factors (Fig. 2B) (Gómez-Felipe et al., 2021).
Gene expression: additional nodes in the GRNs?
Many transcription factors involved in gynoecium development
(Reyes-Olalde et al., 2013) are also expressed during gynoecium
initiation. In total, over 60 genes are expressed at stage 4 and over 65
genes at stage 6 (Table S1) (Herrera-Ubaldo et al., 2018 preprint;
Jiao and Meyerowitz, 2010). Current studies are revealing novel
roles for well-known regulators; additionally, previously unreported
regulators and interactions are continuously being discovered.
Therefore, we expect that these GRNs will expand in the future.
This expansion of GRNs also applies to the GRNs described for the
subsequent stages of gynoecium and fruit development.
Gynoecium domains, medial and lateral (stages 7 and 8)
The transition from a gynoecium primordium into two, well-defined
domains (lateral and medial; see Glossary, Box 1) marks the
beginning of patterning and the formation of internal tissues
(Fig. 1G; Fig. 3). During stages 7 and 8, the activation and
maintenance of the carpel margin meristem (CMM; see Glossary,
Box 1) is crucial for the subsequent development of specialized
structures: in the lateral domains, valves (see Glossary, Box 1)
develop to protect the ovules, and seeds are subsequently formed in
the medial domain. Additional tissues, such as the septum and the
transmitting tract (see Glossary, Box 1), also form in the medial
domain to facilitate fertilization (Fig. 1G).
The process of gynoecium differentiation into medial and lateral
domains requires several transcription factors and hormonal signals
(Reyes-Olalde and de Folter, 2019; Reyes-Olalde et al., 2013). One
important regulatory module involves the integration of auxin and
cytokinin signaling with the activity of the transcription factors
HECs and SPT in the medial domain around floral stages 7 and 8
(Müller et al., 2017; Reyes-Olalde et al., 2017; Schuster et al., 2015)
(Fig. 3C). To summarize, auxin signaling is present in the lateral
domains and cytokinin signaling in the medial domain in the
CMM. In the medial domain, cytokinin signaling induces auxin
biosynthesis and auxin transporters to create an auxin flux to
transport auxin to the lateral domain. Auxin-induced proteins in the
lateral domain repress cytokinin signaling genes to limit them to the
medial domain, such as the cytokinin signaling inhibitor
ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN
6 (AHP6) and the auxin response factor ETT (Heisler et al., 2001;
Reyes-Olalde et al., 2017). The created auxin flux is important for
the apical-basal growth of the gynoecium. Cytokinin biosynthesis in
Stage 6
Stage 7 Stage 8 Stage 9 Stage 10 Stage 11 Stage 12
Gynoecium
primordium Lateral domain
Medial domain
Carpel margin
meristem (CMM)
Val v es
You n g
replum
Ovule
primordium
Medial
region Ovules
Septum
Repl um
Transmitting tract
Val v es
EF
35
12
IM
G Gynoecium development
C Floral budB Inflorescence
Stage 3
Stage 4
Stage 6
Stage 2
Stage 5
Stage 4
Sepal
Petal
Stage 6 Stage 13Stage 4Stage 3
Sepals
Peta ls
Stamen
Gynoecium
Key
tage 4
D Mature flower
Stamen
Gynoecium
A A. thaliana
Fig. 1. Overview of gynoecium development in Arabidopsis.(A-F) Once the reproductive phase has initiated, the plant Arabidopsis thaliana (Col-0)
(A) produces an inflorescence with an inflorescence meristem (IM) at the apical tip (top view; B), and the IM produces on its flanks floral buds with floral meristems
(FMs) (B,C,E). The FM gives rise to all the floral organs present in a mature flower: sepals, petals and stamens, with the gynoecium at the center (D,F).
(E) Schematic of an IM and the initial stages of flower development (longitudinal view). Numbers indicate different stages of FMs. At floral stage 3, sepal primordia
become visible and at stage 6 the primordia of petal, stamens and gynoecium are visible. (G) Transverse gynoecia sections showing key tissues and regions
during gynoecium development at stages 6-12. Scale bars: 1 cm (A); 25 µm (B); 40 µm (C); 200 µm (D). Images in B and C are taken from Zuniga-Mayo et al.
(2019). Image in D is from Zuniga-Mayo et al. (2012).
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REVIEW Development (2022) 149, dev200120. doi:10.1242/dev.200120
DEVELOPMENT
the CMM is assumed to be regulated by SHOOT MERISTEMLESS
(STM), as has been shown for other meristems (Jasinski et al.,
2005; Yanai et al., 2005). STM is a KNOTTED (KNAT)1-LIKE
homeodomain transcription factor important for CMM formation
and activity (Scofield et al., 2007). The STM gene is activated by the
CUP-SHAPED COTYLEDON proteins (CUCs) (Kamiuchi et al.,
2014). A positive regulatory loop between STM and CUC genes has
been reported in the SAM and could be present in the CMM as well
(Spinelli et al., 2011). The CUC transcription factors seem to also be
important for SPT expression in the basal part of the gynoecium
(Nahar et al., 2012).
The current GRN that controls activities in the CMM has been
derived from functional studies of genes and reporter lines
(reviewed by Reyes-Olalde and de Folter, 2019; Reyes-Olalde
et al., 2013). The study and integration of additional regulators is
currently in progress; there are more than 50 transcription factors
related to gynoecium development with reported expression in this
region (Table S1) (Herrera-Ubaldo et al., 2018 preprint). The
coordination of biochemical and genetic processes underlying
meristematic activity in the CMM, and its further differentiation,
probably involves the participation of many proteins and pathways
that are yet to be characterized (Kivivirta et al., 2021; Villarino et al.,
2016; Wynn et al., 2011).
Ovule initiation and patterning (stages 9 and 10)
One of the most important functions of the gynoecium is to provide
protection to the developing ovules. The determination of ovule
identity occurs at the flanks of the CMM. Ovule primordia are
formed at stage 9 by periclinal cell divisions within the epidermal
tissue of the placenta (see Glossary, Box 1) (Fig. 1G; Fig. 4). Like
previous examples, the GRNs involve the coordinated action of
several transcription factors and hormonal signaling pathways that
control the main aspects of ovule development: the establishment of
boundaries and primordia (Fig. 4A), the control of ovule initiation
and number, and ovule patterning (Fig. 4B,C) (Barro-Trastoy et al.,
2020b; Cucinotta et al., 2014, 2020).
Regulation of ovule initiation
The intricate mechanisms that guide ovule initiation are far from
fully understood, but advances have been made (Barro-Trastoy
et al., 2020b; Cucinotta et al., 2020). Indeed, the identification of
enzymes and proteins that act at different levels outside the GRN has
shed light on fine aspects of the molecular mechanisms, and opens
new questions and directions regarding our understanding of ovule
development. For example, factors have been recently identified that
control the spacing of ovules: two secreted peptides and their
ERECTA (ER) family receptor kinases coordinate regular ovule
primordia initiation coupled to fruit growth from the placenta and
carpel walls (Kawamoto et al., 2020).
Processes that occur at the cell-wall level have also been
discovered. A recent study has reported on the involvement of
placenta-expressed genes encoding CELL WALL INVERTASE
(CWIN) 2 and 4 as positive regulators of ovule initiation. CWINs
hydrolyze sucrose into glucose and fructose, and have additional
roles in sugar signaling. Specific suppression of the activity of these
enzymes leads to a significant reduction in ovule number, as well as
ovule and seed abortion. Based on transcriptome analysis in
CWIN2- and CWIN4-silenced lines, it has been shown that the
ovule-identity gene STK, as well as auxin signaling-related genes
and genes encoding hexose transporters are downregulated (Liao
et al., 2020).
Also at the membrane level, the localization of PIN auxin
transporters in the placenta epidermis is crucial for ovule initiation
(Fig. 2A). The distribution of PIN1 and auxin fluxes is very
dynamic in the placenta and affects not only one ovule primordium,
but also the neighboring ovule primordium. Analysis of marker
lines and auxin response has revealed that ovules initiate
asynchronously and follow this trend through later stages (Yu
et al., 2020).
A Stage 3: FM maintenance and termination
B Stage 6: Gynoecium initiation
HEN1 DCL1
LFY+WUS
TPL+HDA19+AP2
ATX 1
ANT/AIL6
PcG RBL SQN ULT
HUA1/2
HEN2/4
PAN
AG
Proliferation
Auxin
distribution
WUS CLV3
YUC1/4 SUP
AG
KNU
CLV3
ANT
SP
T
ETT
WUS AHK4 LOG
3/4/7 STM IPT3/5/7
type-A
ARRs ARR1/10/12
SHP2
CRC
Other
type-B
ARRs
DRNL
AHP6
PIN7
PIN3 TRN2
Cytokinin
signaling
Auxin
maxima
Gynoecium
development
YUC1/4
miR172
Fig. 2. Floral meristem termination and gynoecium establishment (stages
3-6). (A) A gene regulatory network for floral meristem (FM) maintenance and
termination at stage 3. (B) AGAMOUS (AG) orchestrates gynoecium identity
establishment by integrating hormonal signals with the activation/repression of
transcription factors (Xu et al., 2019; Zuniga-Mayo et al., 2019). Dashed lines
indicate indirect regulation. Colored words indicate different hormone-related
processes: auxin signaling (blue); cytokinin signaling (green). AHK4,
ARABIDOPSIS HISTIDINE PROTEIN KINASE 4; AHP6, HISTIDINE
PHOSPHOTRANSFER PROTEIN 6; AIL6, AINTEGUMENTA-LIKE6; ANT,
AINTEGUMENTA; AP2, APETALA2; ARRs, ARABIDOPSIS RESPONSE
REGULATORs; ARR1/10/12, RESPONSE REGULATOR 1, 10, 12; ATX1,
ARABIDOPSIS HOMOLOG OF TRITHORAX 1; CLV3, CLAVATA3; CRC,
CRABS CLAW; DCL1, DICER LIKE1; DRNL, DORNROSCHEN-LIKE; ETT,
ETTIN; HEN1/2/4, HUA ENHANCER 1, 2, 4; HDA19, HISTONE
DEACETYLASE 19; HUA1/2, HUA1, 2 (At5g23150); IPT3/5/7,
ISOPENTENYLTRANSFERASE 3, 5, 7; KNU, KNUCKLES; LFY, LEAFY;
LOG3/4/7, LONELY GUY 3, 4, 7; miR172, microRNA172; PAN, PERIANTHIA;
PcG, Polycomb-group; PIN3/7, PIN-FORMED 3, 7; RBL, REBELOTE; SHP2,
SHATTERPROOF 2; SPT, SPATULA; SQN, SQUINT; STM, SHOOT
MERISTEMLESS; SUP, SUPERMAN; TPL, TOPLESS; TRN2, TORNADO 2;
ULT, ULTRAPETALA; WUS, WUSCHEL; YUC1/4, YUCCA 1, 4.
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DEVELOPMENT
Ovule number
On average around 60 ovules are formed inside the gynoecium
(reviewed by Barro-Trastoy et al., 2020b; Cucinotta et al., 2014,
2020; Yuan and Kessler, 2019). However, ovule numbers can range
between 40 and 80, depending on the accession of Arabidopsis
(Yuan and Kessler, 2019). Some modules of the GRN that control
ovule number can also control gynoecium size (reviewed by
Cucinotta et al., 2020). A recent study has uncovered a previously
unidentified positive regulator of ovule number: NEW
ENHANCER OF ROOT DWARFISM1 (NERD1) (Yuan and
Kessler, 2019).
Transcription factors, such as MP, ANT and CUCs, are
interconnected with hormone homeostasis mechanisms, mainly
with auxins, cytokinins, brassinosteroids and gibberellins, to
regulate ovule number (Fig. 4A) (reviewed by Barro-Trastoy
et al., 2020b; Cucinotta et al., 2020; Qadir et al., 2021). As
mentioned above, auxin positively regulates ovule primordia
initiation and cytokinin signaling positively regulates ovule
number (Bartrina et al., 2011; Cerbantez-Bueno et al., 2020;
Cucinotta et al., 2018; Galbiati et al., 2013; Reyes-Olalde et al.,
2017; Zúñiga-Mayo et al., 2018). It has now been shown that
cytokinin metabolism in the epidermis of the placenta is also
important for ovule number (Werner et al., 2021). Furthermore,
brassinosteroids and gibberellins positively and negatively regulate
ovule number, respectively (Barro-Trastoy et al., 2020a;
Huang et al., 2013; Nole-Wilson et al., 2010; Gomez et al., 2018,
2019).
Ovule patterning
After the establishment of ovule identity and ovule number
definition (Fig. 4A), ovule patterning takes place, which involves
differentiation of the funiculus, chalaza and nucellus (see Glossary,
Box 1) at stage 10 (Fig. 4B), and continues with the formation of the
inner and outer integuments at stage 11 (Fig. 4C). Various recent
reviews on these stages are available (Barro-Trastoy et al., 2020b;
Pinto et al., 2019), and an in-depth discussion of this topic goes
beyond the scope of this article. However, one important
transcription factor, STK, is the master regulator of ovule identity
(Favaro et al., 2003; Pinyopich et al., 2003), although this gene has
many additional functions, as described below. Various other genes
involved in ovule patterning have additional functions during
gynoecium development.
Gynoecium patterning (stages 11 and 12)
During stages 11 and 12, gynoecium patterning continues to form
all the tissues, and the gynoecium attains the final shape suitable for
pollination (Fig. 1D). During this period, there is active patterning
in the main axes: the abaxial-adaxial with funiculus, chalaza
and nucellus (summarized in Fig. 5); the medial-lateral with the
valves, valve margins, replum (see Glossary, Box 1), septum and
transmitting tract (Fig. 5A); and the apical-basal with stigma (see
Glossary, Box 1), style (see Glossary, Box 1), ovary and gynophore
(Fig. 5B) (Chávez Montes et al., 2015; Deb et al., 2018; Marsch-
Martínez and de Folter, 2016; Simonini and Østergaard, 2019;
Zúñiga-Mayo et al., 2019). Most of the best-characterized regulators
in these GRNs are transcription factors that affect tissue
differentiation.
Medial-lateral patterning
In a mature gynoecium, the medial-lateral axis is composed of the
valves (the carpel walls), which protect the developing ovules. After
pollination, the valves are attached to the replum (Fig. 1F,G;
Fig. 5A). The formation of this axis (valves-replum-valves) requires
the concerted action of medial factors, such as BP, RPL,
WUSCHEL-RELATED HOMEOBOX 13 (WOX13) and/or NO
TRASMITTING TRACT (NTT), which possess meristematic-
related functions and repress the action of lateral factors, such as
FILAMENTOUS FLOWER/YABBY3 (FIL/YAB3) and the
ASYMMETRIC LEAVES 1/2 (AS1/2) (Alonso-Cantabrana et al.,
2007; Dinneny et al., 2005; Marsch-Martínez et al., 2014; Romera-
Branchat et al., 2013). Additionally, the so-called ‘boundary
factors’(CUCs and KNAT2/6) participate in between, marking
the division of the meristematic and lateral organ functions
(Fig. 5A) (reviewed by Ballester and Ferrándiz, 2017; Simonini
and Østergaard, 2019).
Inside the ovary, transmitting tract formation is controlled by
NTT (Crawford et al., 2007), the basic helix-loop-helix (bHLH)
proteins CESTA/HALF FILLED (CES/HAF), BRASSINOSTEROID
ENHANCED EXPRESSION 1 and 3 (BEE1, BEE3) (Crawford
and Yanofsky, 2011), HECs (Gremski et al., 2007), SPT
Box 2. Protein interactions: a combinatorial model for
gynoecium patterning?
The concerted action of transcription factors (MADS-box and
APETALA 2), to guide the establishment of floral organ identity is
elegantly represented by the ‘ABCDE’model (Theißen and Saedler,
2001). For gynoecium patterning, the involvement of numerous
transcription factor families surpasses those for floral organ
specification. The crucial action and effect of transcription factors may
suggest a combinatorial model for gynoecium patterning; some crucial
transcription factors [i.e. SEUSS (SEU) and LEUNIG (LUG)] may
dimerize to act as a chassis for higher-order complexes in the early
stages (Azhakanandam et al., 2008). In terms of protein interactions, the
best-studied tissues are the stigma and style, where transcription factors,
such as INDEHISCENT (IND), SPATULA (SPT) and NGATHA (NGA)
participate (Ballester et al., 2021; Simonini et al., 2018).
We have made efforts to find such protein complexes. The
identification of physical interactions between several transcription
factors in combination with gene expression and functional information
is useful to represent the networks and interaction dynamics during the
development of the gynoecium (Herrera-Ubaldo et al., 2018 preprint).
Figure shows protein-protein interaction networks at two early stages of
gynoecium development.
The involvement of thesetranscription factors is just the beginning of the
story;previous workshave extendedregulatorynetworks beyondthe range
of transcription factors alone.For example, AGAMOUS (AG) interacts with
histone deacetylases and other proteins (Smaczniak et al., 2012).
Stage 7: Lateral domain
Stage 7: Medial domain
Stage 6:
Gynoecium primordium
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DEVELOPMENT
(Heisler et al., 2001) and STK (Di Marzo et al., 2020b; Herrera-
Ubaldo et al., 2019) (Fig. 5A). In addition, several of these factors
(NTT, SPT and STK) control the development of other tissues in the
gynoecium that are related to transmitting tract formation; they
control programmed cell death (PCD) and the deposition of
extracellular matrix (ECM) (reviewed by Crawford and Yanofsky,
2008; Pereira et al., 2021).
Some processes occurring between stages 11 and 12 highlight the
importance of cell wall modifications in medial-lateral gynoecium
patterning. The leading role of ETT in gynoecium morphology is
clear; ETT is known to participate, in coordination with auxin input,
in the guidance of early patterning events (Fig. 2B; Fig. 3) (Sessions
et al., 1997; Simonini et al., 2016). Now, an additional pathway of
ETT participation has been identified, the regulation of pectin
methylesterase (PME) activity in the valves (Andres-Robin et al.,
2018). PME activity in valve cell walls increases the levels of
demethylesterified pectins, allowing the reduction of cell wall
stiffness and the elongation of the valves.
Inside the gynoecium, other modifications need to occur in the
medial domain to allow the formation of the transmitting tract.
Recently, NTT and STK have been shown to participate in the
modulation of the cell wall composition by regulating a gene
encoding a putative mannanase enzyme, as well as other genes
encoding proteins involved in lipid biosynthesis and transport
(Herrera-Ubaldo et al., 2019).
Apical-basal patterning
Major changes must be coordinated to achieve the distinct apical-
basal features (stigma, style and ovary) observed in a mature
gynoecium. The ovary is a bilateral structure –in contrast to the
style, which exhibits radial symmetry (Fig. 1F,G). During floral
stage 12, the style differentiates and the bilateral-to-radial transition
is controlled by the modulation of auxin flux, as well as cytokinin
sensitivity (Fig. 5B) (Carabelli et al., 2021; Moubayidin and
Ostergaard, 2014). First, the transcription factors SPT and
INDEHISCENT (IND) promote apolar localization of PIN
proteins (auxin efflux transporters normally localized to cell poles
to create polar auxin transport; Moubayidin and Ostergaard, 2014).
SPT and HECs then control the expression of the adaxial-identity
genes HOMEOBOX ARABIDOPSIS THALIANA 3 (HAT3) and
ARABIDOPSIS THALIANA HOMEOBOX 4 (ATHB4), which
orchestrate the final steps of the radicalization process (Fig. 5B)
(Carabelli et al., 2021). In another study focusing on style
development using genetics and protein-protein interaction
experiments, it has been suggested that ETT, IND,
BREVIPEDICELLUS (BP), REPLUMLESS (RPL) and SEUSS
(SEU) work synergistically to regulate style morphology (Fig. 5B)
(Simonini et al., 2018).
Sequential activation and function of transcription factors is
required for stigma development: the NGATHA (NGA) and
HECATE (HEC) protein families cooperatively regulate stigma
A Stage 7 B Stage 8 C
Medial
domain
Lateral
domain
Lateral
domain
STM IPTs
AHP6
DRNL
MP
ANT
HEC1
ETT
ARR-A
Auxin
TAA 1
Auxin
Auxin
flux
PIN1
CUCs
PINs
Cytokinin
SPT ARR-B
Lateral domain
Medial domain
Carpel margin meristem
Key
Fig. 3. Carpel margin meristem activation and maintenance (stages 7 and 8). (A) Schematic of a young gynoecium at stage 7, when two different domains –
lateral (green) and medial (yellow) –are visible. (B) At stage 8, the carpel margin meristem (red) becomes specified. (C) The activity of the CMM is maintained by
the activity of the bHLH transcription factors SPATULA (SPT) and HECATE 1 (HEC1), via the activation of auxin synthesis and transport. Genes related to
cytokinin signaling are also involved (Reyes-Olalde and de Folter,2019). Upper panels represent longitudinal sections of the dashed lines in the bottom panels of
the gynoecium images. Dashed lines in the regulatory network indicate indirect regulation, or interactions found in other tissues that are likely occurring in the
gynoecium. Colored words indicate different hormone-related processes: auxin signaling (blue); cytokinin signaling (green). Purple lines in B and C indicate the
regions in which the processes occur. AHP6, HISTIDINE PHOSPHOTRANSFER PROTEIN 6; ANT, AINTEGUMENTA; ARRs, ARABIDOPSIS RESPONSE
REGULATORs; CUCs, CUP-SHAPED COTYLEDONs; DRNL, DORNROSCHEN-LIKE; ETT, ETTIN; HEC1, HECATE 1; IPTs,
ISOPENTENYLTRANSFERASEs; MP, MONOPTEROS; PIN1, PIN-FORMED 1; SPT, SPATULA; STM, SHOOT MERISTEMLESS; TAA1, TRYPTOPHAN
AMINOTRANSFERASE OF ARABIDOPSIS 1.
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DEVELOPMENT
development through the activation of IND, which later interacts
with NGA (and probably HECs) to activate SPT. SPT is then
probably integrated into the NGA-IND-HEC complex to activate
target genes (Ballester et al., 2021). Recently, three angiosperm-
specific regulators of the style and stigma have been identified
called STIGMA AND STYLE STYLIST 1-3 (SSS1-3), which
belong to a previously unreported family. They are expressed in the
apical tissues of the gynoecium and act downstream of the NGA
transcription factors to fine-tune the development of the stigma and
style (Fig. 5B) (Li et al., 2020).
Pollination and fertilization (stage 13)
Pollination and fertilization involve a continuous and active
communication between female and male tissues over several
steps: pollen hydration, pollen germination, pollen tube growth,
pollen tube attraction to the ovule, pollen tube reception, sperm cell
delivery and gamete activation (reviewed by Cascallares et al.,
2020).
As discussed earlier, the formation of the transmitting tract
includes regulation of PCD and ECM deposition (reviewed by
Pereira et al., 2021), as well as other changes in cell wall
composition in the medial region (Herrera-Ubaldo and de Folter,
2018). The ECM provides nutrition, adhesion and guidance during
pollen tube growth. During fertilization, the transmitting tract
secrets chemical signals, such as arabinogalactan-proteins (AGPs),
whereas other signals, such as LURE proteins, derive from the
ovules to attract pollen tubes (reviewed by Johnson et al., 2019;
Pereira et al., 2021).
Checkpoints during the pollination process control the
acceptance or rejection of the pollen prior to fertilization
(reviewed by Dresselhaus et al., 2016) and various small peptides
and receptors are involved in these processes (reviewed by Zhang
et al., 2021). Recently, some new discoveries have been made
towards the understanding of communication and signaling
cascades during pollination. One such discovery is the existence
of an autocrine signaling pathway acting at the surface of stigmatic
papillae, inducing reactive oxygen species (ROS) production; ROS
levels are reduced upon pollination owing to an antagonistic peptide
from the pollen coat, allowing pollen hydration and germination
(Liu et al., 2021; Zhou et al., 2021). Another discovery also involves
Nucellus
Chalaza
Funiculus
C Stage 11B Stage 10A Stage 9
Ovule
primordium
Boundary
SPL
REV
INO
SUP
BEL1
PHB
PHV
CNA
ETT+ATS
Auxin
UCN
Brassinosteroids
Gibberellins
GID1
PHB
miR166
INO
BRI1
BZR1
WUS
ATS+GAI
RGA
STK
PIN1
Cytokinin
WUS
AHK2/3/4
WUS
SPL
ANT
BEL1
PIN1
Auxin
maximum
CKX3/5
GibberellinsCytokinin
SAUR8/10/12
CYP85A2
DET2
BRI1
Brassinosteroids
BIN2
BZR1
ARGOS
ANT
CUC1/2
PIN1
miR164
MP
YUC1
Auxin
YUC4
UGTs
AHK2/3/4
CRF2
GAI
RGA
RGL2
GID1
Ovule number
Ovule primordium, ovuleTransmitting tract
Val v e
SeptumRepl um
Key
Fig. 4. Ovule identity specification and patterning (stages 9-11). (A-C) The beginning of ovule development (determination of number and position) is
controlled by a complex network that involves the action of at least four hormones and several transcription factors. The regulatory networks controlling ovule
primordia initiation (A), patterning (B) and morphogenesis (C) (Barro-Trastoy et al., 2020b; Cucinotta et al., 2020) are summarized. Colored words indicate
different hormone-related processes: auxin signaling (blue); cytokinin signaling (green); gibberellins (purple); brassinosteroids (orange). AHK2/3/4,
ARABIDOPSIS HISTIDINE PROTEIN KINASE 2, 3, 4; ANT, AINTEGUMENTA; ARGOS, AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE; ATS,
ABERRANT TESTA SHAPE; BEL1, BELL 1; BIN2, BRASSINOSTEROID-INSENSITIVE 2; BRI1, BRASSINOSTEROID INSENSITIVE 1; BZR1,
BRASSINAZOLE RESISTANT 1; CKX3/5, CYTOKININ OXIDASE/DEHYDROGENASE 3, 5; CNA, CORONA; CRF2, CYTOKININ RESPONSE FACTOR 2;
CUC1/2, CUP-SHAPED COTYLEDON 1, 2; CYP85A2, cytochrome p450 enzyme; DET2, ATDET2; ETT, ETTIN; GAI, GIBBERELLIC ACID INSENSITIVE;
GID1, GA INSENSITIVE DWARF; INO, INNER NO OUTER; miR164, microRNA164; miR166, microRNA166; MP, MONOPTEROS; PHB, PHABULOSA; PHV,
PHAVOLUTA; PIN1, PIN-FORMED 1; REV, REVOLUTA; RGA, REPRESSOR OF GA; RGL2, RGA-LIKE 2; SAUR8/10/12, SAUR-like auxin-responsive protein
family; SPL, SPOROCYTELESS; STK, SEEDSTICK; SUP, SUPERMAN; UCN, UNICORN; UGTs, UDP-glucosyl transferases; WUS, WUSCHEL; YUC1/4,
YUCCA 1, 4.
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REVIEW Development (2022) 149, dev200120. doi:10.1242/dev.200120
DEVELOPMENT
the receptor kinase FERONIA (FER) in the control of male-
female interactions. FER, located at the entrance to the female
gametophyte, mediates de-esterified pectins and the production of
nitric oxide (NO), which is necessary for when the first pollen tube
arrives at the ovule; NO accumulation stops the attraction of further
pollen tubes (Duan et al., 2020). The process of pollination, and the
subsequent events, can only occur in a specific time window,
delimited by stigmatic receptivity. Two transcription factors, KIRA1
(KIR1) and ORESARA1 (ORE1), belonging to the NAC family
promote PCD in the stigma cells, thereby controlling stigma
longevity (Gao et al., 2018).
Seed and fruit development (stages 14-20)
After fertilization, the gynoecium becomes a fruit, and its
development in Arabidopsis continues over 12 days to finally
release fully formed, fertile seeds. During the final stages of fruit
development, the fruit grows, the seeds mature and developmental
programs prepare for fruit dehiscence (see Glossary, Box 1) and
seed dispersal (Alvarez-Buylla et al., 2010; Ballester and Ferrándiz,
2017; Roeder and Yanofsky, 2006). After fertilization (stage 13),
the fruit grows mainly longitudinally to reach its maximum size by
stage 17, followed by the start of senescence of the fruit (stage 18),
and finally fruit dehiscence and seed release (stage 20) (Fig. 6A-C).
Longitudinal growth
The involvement of cytokinin in fruit elongation has recently been
reported: phenotypic alterations observed in fruit length of the stk
mutant, which produces short fruits, resemble those of fruits of the
ckx7 mutant (encoding for the enzyme CYTOKININ OXIDASE/
DEHYDROGENASE 7), in which cytokinin degradation is affected
(Di Marzo et al., 2020a). Therefore, cytokinin has a negative effect
on fruit elongation. This additional function of STK also integrates
pathways that control fruit size because STK positively regulates
CKX7 expression in the fruit and indirectly controls FUL, which is
A Stage 12: Medial-lateral patterning B Stage 12: Apical-basal patterning
ARF6/8SPT+HECs STY NTT
BEEs+HAF
Tra n sm it ti n g tr a ct f or m at io n
Repl um Septum Va lve OvuleTransmitting tract Stigma StyleGynophore
Key
AS1/2 KNAT2/6
NTT
ARF4 ETT
KAN AP2
BP
JAG FIL YAB3
FUL CUC1/2RPL
STM
SHP1/2
Replu m
Val ve
margin
Val ve s LL SL
SPT
IND ALC
ETT
HEC1+SPT
Cytokinin
TCP15 AHP6
Auxin
Medial region
development
Stigma
and
style
formation
ARF6/8
CRC
SSSs
HAT3,
ATH B 4 SPT+HECs
Cytokinin
SHP1/2
ANT+FIL
SEU+LUG
Auxin
maxima PINs
PID
(P)
Auxin YUCs SHI/STYs
NGAs
ETT,
SPT+IND
HECs SPLs TCP15
Fig. 5. Medial-lateral and apical-basal gynoecium patterning fertilization (stage 12). (A) In the outer part of the gynoecium, lateral and medial factors
cooperate or compete to define the valves, valve margins and replum. The valve margin is divided in the lignification (LL) and separation layer (SL). The formation
of the transmitting tract is coordinated by several transcription factors. Dashed line represents the transverse section depicted in B. (B) In the apical-basal axis,
stigma and style formation is guided by regulation that converges in the production of auxins. Cooperation between transcription factors and hormones
participates in maintaining the ovary (Alonso-Cantabrana et al., 2007; Deb et al., 2018; Marsch-Martı
nez and de Folter, 2016; Simonini and Østergaard, 2019).
Colored words indicate different hormone-related-processes: auxin signaling (blue); cytokinin signaling (green). ‘(P)’indicates that PID phosphorylates PIN
proteins. AHP6, HISTIDINE PHOSPHOTRANSFER PROTEIN 6; ALC, ALCATRAZ; ANT, AINTEGUMENTA; AP2, APETALA 2; ARF4/6/8, AUXIN RESPONSE
FACTOR 4, 6, 8; AS1/2, ASYMMETRIC LEAVES 1, 2; ATHB4, ARABIDOPSIS THALIANA HOMEOBOX 4; BEEs, BR-ENHANCED EXPRESSIONS; BP,
BREVIPEDICELLUS; CRC, CRABS CLAW; CUC1/2, CUP-SHAPED COTYLEDON 1, 2; ETT, ETTIN; FIL, FILAMENTOUS FLOWER; FUL, FRUITFULL; HAF,
HALF FILLED; HAT3, HOMEOBOX ARABIDOPSIS THALIANA 3; HECs, HECATEs; IND, INDEHISCENT; JAG, JAGGED; KAN, KANADI; KNAT2/6,
KNOTTED-LIKE FROM ARABIDOPSIS THALIANA 2, 6; LUG, LEUNIG; NGAs, NGATHAs; NTT, NO TRANSMITTING TRACT; PID, PINOID; PINs, PIN-
FORMED; RPL, REPLUMLESS; SEU, SEUSS; SHI, SHORT INTERNODES; SHP1/2, SHATTERPROOF 1, 2; SPLs, SQUAMOSA PROMOTER BINDING
PROTEIN-LIKEs; SPT, SPATULA; SSSs, STIGMA AND STYLE STYLISTs; STM, SHOOT MERISTEMLESS; STYs, STYLISHs; TCP15, TEOSINTE
BRANCHED, CYCLOIDEA/PCF 15; YAB3, YABBY 3; YUCs, YUCCAs.
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REVIEW Development (2022) 149, dev200120. doi:10.1242/dev.200120
DEVELOPMENT
involved in a pathway with miR172 and AP2 (Fig. 6E) (Di Marzo
et al., 2020a; Ripoll et al., 2015). Additionally, STK regulates fruit
growth by controlling the expression of α-XYLOSIDASE 1 (XYL1),
which is involved in the modification of xyloglucans (XyGs),
which form a component of cell walls. Indeed, XyG modifications
contribute to the mechanical properties of the cell wall, affecting its
extensibility and thus cell growth. The activity of XYL1 is also
required in the growing seeds in order for them to achieve their final
size (Di Marzo et al., 2021).
The involvement of hormones in fruit development and
maturation goes beyond just auxin and cytokinin. Gibberellins
induce fruit growth in Arabidopsis (Dorcey et al., 2009) and
regulate the formation of the separation layer in the valve margins
(Fig. 6D,F) (Arnaud et al., 2010). The role of hormones, such as
abscisic acid, ethylene and brassinosteroids, are better described for
fleshy fruit development (reviewed by Li et al., 2021; McAtee et al.,
2013; Sotelo-Silveira et al., 2014).
Related to longitudinal growth of the silique (see Glossary,
Box 1) or fruit, a recent study has reported a spatiotemporal map of
fruit growth at the cellular level that captures quantitative data to
measure cell division and cell expansion. Analysis of the ntt mutant
affected in seed-set revealed that final fruit length correlates with the
number of seeds (Ripoll et al., 2019).
Preparation for dehiscence
Early studies on fruit development have described the initial models
for fruit patterning related to fruit opening (dehiscence or pod
shattering), highlighting the key roles of the transcription factors
FUL, SHPs, RPL, ALC and IND (Ballester and Ferrándiz, 2017;
Liljegren et al., 2004). The dehiscence zone, located on the valve
margins, are specialized cell layers that allow valve detachment.
SHP proteins control the expression of IND, which guides the
formation of the lignification layer (Fig. 6D,F). Additionally, SHPs
control ALCATRAZ (ALC), which guides the formation of the
separation layer (reviewed by Ballester and Ferrándiz, 2017).
Moreover, FUL controls the formation of a lignified layer in the
valves (Liljegren et al., 2004).
Another role of STK is the control of lignification in the
funiculus, which is necessary for seed abscission (Balanzà et al.,
2016). In the funiculus, STK, together with the co-repressor SEU,
represses the expression of HEC3,SPT and ALC (Fig. 6F). A rather
similar process occurs in valve margin lignification, whereby SHPs
regulate IND,SPT and ALC, but the regulation is inverted (i.e. SHPs
activate gene expression) (Fig. 5A; Fig. 6F).
Important roles of hormones have been uncovered as well. IND
regulates the expression of PINOID (PID) and WAG2 to control
auxin transport outside the separation layer in the valve margins,
creating a so-called ‘auxin minimum’that is important for dehiscence
zone formation (Sorefan et al., 2009). However, further studies
regarding the participation of auxin in the control of dehiscence,
using reporter lines to track auxin distribution have revealed that, at
early fruit-development stages (stage 14-16), auxin is present at the
valve margin for specifying the dehiscence zone (van Gelderen et al.,
2016). Perhaps the ‘auxin minimum’at later fruit stages (e.g. stage
17B) is still functionally relevant for cell separation (reviewed by
Ballester and Ferrándiz, 2017; de Folter, 2016). The involvement of
cytokinin signaling during fruit development has also been studied.
The valve margins display cytokinin signaling, which is lost in
mutants that are indehiscent (ind and shp1 shp2). When cytokinin is
applied to these mutants, fruit dehiscence is restored (Marsch-
Martínez et al., 2012). Furthermore, replum development is also
dependent on the presence of cytokinin; with less cytokinin or
Repl um
A Fruit development
B Stage 13 C Stage 17B
Ovule
Septum
Val v e
Seed
Septum
Repl um
Val v e
Val v e
margin
Stage
13 14 15 16 17A 17B 18 19 20
Lignified valve layer Lignified margin layer Separation layer
SeptumVal v e Replum
Key
EFG Dehiscence
ARF6/8 FUL STK
miR172C IND CKX7
Cytokinin
degradation
AP2
cis-Zeatin
trans-Zeatin
Cell
elongation
Fruit
elongation
STK+SEU
HEC3
SPT
ALC
Seed abscission
zone
IND
SHPs
SPT
ALC
Val ve
margins
SHPs
IND
GA3ox1
Gibberellins Cytokinin
Auxin
depletion
PIN3
WAG 2
PID
Val ve
margins
D
Fig. 6. Fruit growth and dehiscence (stages 14-20). (A) After fertilization, the
gynoecium becomes a fruit. During stages 14-19, the fruit grows and prepares to
release the seeds. (B-D) Internally, the gynoecium-to-fruit transition involves
changes in tissue conformation. One of the most relevant isthe formation of the
seeds, as well as the differentiation of the lignification (LL) and separation (SL)
layers in the valve margins prior to dehiscence. D shows a detailed view of the
boxed area in C. (E-G) At the molecular level, fruit elongation relies on cell
elongation (E) and dehiscence (F), which are controlled by transcription factors
with hormonal input. (G) The specification of the seed abscission zone and the
valve margins share someregulatory elements, such as SEEDSTICK (STK) and
SHATTERPROOFs (SHPs). Dashed lines indicate indirect regulation. Colored
words indicate different hormone-related processes: auxin signaling (blue);
cytokinin signaling (green); gibberellins (purple) (Balanzaet al., 2016; Di Marzo
et al., 2020a; Marsch-Martı
nez and de Folter, 2016; van Gelderen et al., 2016).
ALC, ALCATRAZ; AP2, APETALA2; ARF6/8, AUXIN RESPONSE FACTOR 6,
8; CKX7, CYTOKININ OXIDASE/DEHYDROGENASE 7; FUL, FRUITFULL;
GA3ox1, GIBBERELLIN 3-beta-dioxygenase 1; HEC3, HECATE 3;
IND, INDEHISCENT; miR172C, microRNA172C; PID, PINOID; PIN3,
PIN-FORMED3; SPT, SPATULA; SEU, SEUSS; WAG2, WAG2.
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REVIEW Development (2022) 149, dev200120. doi:10.1242/dev.200120
DEVELOPMENT
cytokinin signaling, the width of the replum is reduced and vice versa
(Marsch-Martínez et al., 2012; Reyes-Olalde et al., 2017). In a double
Arabidopsis ntt rpl mutant that lacks the replum, replum formation is
restored upon cytokinin application, suggesting that cytokinin
signaling acts downstream of NTT and RPL (Zúñiga-Mayo et al.,
2018). In addition, increased replum width has also been observed
when NTT is overexpressed (Marsch-Martínez et al., 2014).
In addition to the hormonal and genetic control of dehiscence, a
recent study adds the importance of environmental input.
Temperature affects the expression of IND; higher temperature
causes chromatin modifications at the IND locus, which results in
increased IND expression that accelerates valve margin
development (Fig. 6G) (Li et al., 2018).
Gene expression: additional nodes in the GRNs?
The concerted action of GRNs during these stages will ultimately
guide fruit growth and shape. Although a lot of information is
available, further players are likely still to be discovered. A recent
study has conducted transcriptome analysis to reveal gene
expression dynamics during fruit growth and maturation,
including silique samples, from 3, 6, 9 and 12 days after
pollination (Mizzotti et al., 2018). This dataset is supported by
another study, which has analyzed the pre-fertilization stages: carpel
initiation (stage 5), elongation of carpel walls (stage 9), gynoecia
during female meiosis (stage 11), and gynoecia before anthesis
(stage 12) (Kivivirta et al., 2021). These two studies (and others,
e.g. Klepikova et al., 2016; Villarino et al., 2016; Wynn et al., 2011)
now provide a broad gene expression atlas for the complete
developmental window from gynoecium-fruit development. In
both works, samples included complete gynoecia and fruits, so
additional work is required to resolve gene expression further or
dissect transcriptomic data at the tissue- and single-cell level.
Conclusions
The current knowledge on gynoecium and fruit development is
expanding. We have given an overview of the GRNs known to date.
In the future, it is likely that these GRNs will expand because we
know that many more transcription factors are expressed at the
specific stages discussed (Table S1). Furthermore, many recent
works have provided new datasets and information related to
gynoecium formation, such as transcriptomics during development
(Kivivirta et al., 2021; Mizzotti et al., 2018) or at specific times or
scenarios (Krizek et al., 2021; Liao et al., 2020; Martínez-Fernández
et al., 2020; Yu et al., 2020); variation by genome-wide association
studies (Yuan and Kessler, 2019) or quantitative trait locus analysis
(Kawamoto et al., 2020); and protein-protein interactions (Herrera-
Ubaldo et al., 2018 preprint). The presented GRNs are based on data
of Arabidopsis it is likely that these GRNs have variations and/or
rewiring that mayexplain existing diversity in gynoecia and fruits of
other species (Box 3).
Current technologies allow the study of morphogenesis in
Arabidopsis in unprecedented detail. Live imaging, lineage
tracking and geometric analysis, in combination with single-cell
transcriptome profiling, are changing how we study plants and
paving the way to study development in four dimensions. Some
important advances have been made during early flower
development, such as the construction of a 4D atlas that integrates
cell growth quantification and gene expression data (Refahi et al.,
2021); a 3D gene expression atlas of the FM based on the spatial
reconstruction of single-nucleus RNA-sequencing data (Neumann
et al., 2021 preprint); and a study of sepal (Zhu et al., 2020) and
stamen growth (Silveira et al., 2021). In the case of ovule formation,
the generation of a 3D reference atlas (Vijayan et al., 2021) or the
analysis of the relationship between organ geometry and cell fate
(Hernandez-Lagana et al., 2021) provide comprehensive views of
ovule development.
These works are related to reproductive structures, but the field
can also take advantage of broader studies performed at the whole-
plant level. For example, the protein interactome for hormone-
related proteins (Altmann et al., 2020) or the Arabidopsis proteome
with samples from whole flowers and fruits (as well as carpels,
seeds, valves and septum samples; Mergner et al., 2020), provide
useful resources and valuable information to be integrated.
Additionally, a recent dataset has facilitated the study of protein
complexes at the pan-plant level (McWhite et al., 2020), and
another allows the comparison of transcriptomic programs
across land plants (Julca et al., 2021). Finally, the Plant Cell Atlas
initiative will provide insight into the content and processes taking
place in each cell type in the plant (Plant Cell Atlas Consortium
et al., 2021; Rhee et al., 2019). The integration of all these data inthe
Box 3. Evolution of gene regulatory networks (GRNs)
directing gynoecium development
The study of the origin of the carpel at the morphological level is an active
topic of research (Endress, 2019; Endress and Doyle, 2015; Endress
and Igersheim, 2000; Scutt et al., 2006). Some evidence points to the
tube-like (ascidiate) or leaf-like (plicate) origin of the carpel. The
information from Arabidopsis is the reference for comparative studies.
The identification of key regulators (as individuals or families) and their
conservation across the tree of life and its evolution (Pfannebecker et al.,
2017a,b) has allowed the tracing of the history of the main regulators
(Becker, 2020; Ferrándiz and Fourquin, 2014).
Current work is focused on deciphering the origin of regulators and
their connections in GRNs that guide the variation in the shape and
function of fruits. Two basic aspects of GRN evolution are: (1) the rise of
nodes and connections (see figure, green dots and lines); and
(2) rewiring of the edges (blue lines). Furthermore, variation in cell
division rates or cell expansion may explain shape diversity in related
species (Dong et al., 2020; Łangowski et al., 2016).
The construction of genetic and protein interaction networks that
retrieves proteins of different clades gives an idea of how regulatory
networks gain complexity. Furthermore, information from other fruit
species, such as Solanaceae (Ortiz-Ramírez et al., 2018) and others
(Gomariz-Fernández et al., 2017; Pabón-Mora et al., 2014; Simonini
et al., 2018), could give insights into gynoecium evolution.
Initial network
New nodes and
new interactions
Gene duplication and divergence
10
REVIEW Development (2022) 149, dev200120. doi:10.1242/dev.200120
DEVELOPMENT
coming years will reveal many hidden aspects of morphogenesis
during gynoecium and fruit formation and will achieve a detailed
picture of the mechanisms involved, at a similar level to other
systems, such as plant embryo patterning (Harnvanichvech et al.,
2021).
Acknowledgements
We apologize to researchers in the field whose work has not been cited owing to
space constraints. We thank Andrea Gomez-Felipe and Victor Zuniga-Mayo for
providing images for Fig. 1B,C and Fig. 1D, respectively. We also thank the two
anonymous reviewers for their input.
Competing interests
The authors declare no competing or financial interests.
Funding
Work in the S.d.F. laboratory is financed by the Mexican National Council of Science
and Technology (Consejo Nacional de Ciencia y Tecnologı
a; CONACYT) grants
(CB-2012-177739, INFR-2015-253504, FC-2015-2/1061 and CB-2017-2018-A1-S-
1012). H.H.-U. acknowledges a postdoc fellowship from CONACYT. S.d.F.
acknowledges the Fundacion Marcos Moshinsky, and also acknowledges
participation in the European Union Horizon 2020 H2020-MSCA-RISE-2020
EVOfruland project (101007738) and H2020-MSCA-RISE-2019 MAD project
(872417).
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REVIEW Development (2022) 149, dev200120. doi:10.1242/dev.200120
DEVELOPMENT
