Tab 0127

Flower Development
Authors: Alvarez-Buylla, Elena R., Benítez, Mariana, Corvera-Poiré,

Adriana, Chaos Cador, Álvaro, de Folter, Stefan, et al.
Source: The Arabidopsis Book, 2010(8)
Published By: The American Society of Plant Biologists
URL: https://doi.org/10.1199/tab.0127
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The Arabidopsis Book © 2010 American Society of Plant Biologists
First published on March 23, 2010: e0127. 10.1199/tab.0127
Flower Development
Elena R. Alvarez-Buylla,a , 1 Mariana Benítez,a Adriana Corvera-Poiré,a Álvaro Chaos Cador,a Stefan de Folter,b
Alicia Gamboa de Buen,c Adriana Garay-Arroyo,a Berenice García-Ponce,a Fabiola Jaimes-Miranda,a Rigoberto V.
Pérez-Ruiz,a Alma Piñeyro-Nelson,a and Yara E. Sánchez-Corralesa
a
Laboratorio de Genética Molecular, Desarrollo y Evolución de Plantas, Departamento de Ecología Funcional, Instituto de
Ecología, Universidad Nacional Autónoma de México. 3er Circuito Exterior S/N Junto a Jardín Botánico Exterior, Cd. Universi-
taria, Coyoacán, México D.F. 04510, Mexico
b
Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Centro de Investigación y de Estudios Avanzados del
Instituto Politécnico Nacional (CINVESTAV-IPN), Km. 9.6 Libramiento Norte, Carretera Irapuato-León, A.P. 629, CP 36821
Irapuato, Gto. Mexico
c
Laboratorio de Ecofisiología Vegetal, Departamento de Ecología Funcional, Instituto de Ecología, Universidad Nacional
Autónoma de México. 3er Circuito Exterior S/N Junto a Jardín Botánico Exterior, Cd. Universitaria, Coyoacán, México D.F.
04510, Mexico
Authors contributed equally and are listed in alphabetical order.
1
Address correspondence to eabuylla@gmail.com
Flowers are the most complex structures of plants. Studies of Arabidopsis thaliana, which has typical eudicot flowers,
have been fundamental in advancing the structural and molecular understanding of flower development. The main pro-
cesses and stages of Arabidopsis flower development are summarized to provide a framework in which to interpret the
detailed molecular genetic studies of genes assigned functions during flower development and is extended to recent
genomics studies uncovering the key regulatory modules involved. Computational models have been used to study
the concerted action and dynamics of the gene regulatory module that underlies patterning of the Arabidopsis inflores-
cence meristem and specification of the primordial cell types during early stages of flower development. This includes
the gene combinations that specify sepal, petal, stamen and carpel identity, and genes that interact with them. As a dy-
namic gene regulatory network this module has been shown to converge to stable multigenic profiles that depend upon
the overall network topology and are thus robust, which can explain the canalization of flower organ determination and
the overall conservation of the basic flower plan among eudicots. Comparative and evolutionary approaches derived
from Arabidopsis studies pave the way to studying the molecular basis of diverse floral morphologies.
1. INTRODUCTION: WHEN DID THE FLOWER EVOLVE? The Spermatophyte group has been further divided into Gymno-
sperms (originating 380-325 MYBP) and Angiosperms. Accord-
The flower is the most complex structure of plants. Flowers distin- ing to the fossil record, flower-like structures originated 160-147
guish the most recently diverged plant lineage, the angiosperms MYBP (Frohlich, 2006). A general trend within land plant evolution
or flowering plants, from the other land plants (Figure 1). Embryo- is the appearance of heterospory: the existence of a megaga-
phytes originated approximately 450 million years before present metophyte, including the female gametes, and a microgameto-
(MYBP) and have as distinctive features a thick cuticle resistant phyte, including the male gametes, a progressive reduction in
to desiccation, sporopollenin, pores or true stomata that aid in gametophyte size (sexual reproductive structures), and within
gas exchange, a glycolate oxidase system that improves carbon the seed plants, the presence of a diploid embryo. While these
fixation at high oxygen tensions, and importantly, distinctive mul- characteristics are shared among both extant and extinct seed
ticellular diploid (sporophytic) and haploid (gametophytic) stages plant lineages, the defining features of the angiosperm flower are:
within their life cycles (Judd et al., 2002). The major extant land (1) a closed carpel bearing the ovules, which are each generally
plant lineages are Bryophytes (Liverworts, Hornworts and Moss- comprised of two integuments and (2) a nucellus that contains the
es), which do not have a vascular system, and Tracheophytes, embryo sac within which, after double fertilization, a diploid em-
vascular plants. Within the large latter group, Lycophytes, ferns, bryo and a triploid endosperm (nutritional tissue for the embryo)
and seed bearing plants (Spermatophytes) can be distinguished. will develop to form a seed (Judd et al., 2002). Another character-

2 of 57 The Arabidopsis Book
Time scale (MYBP)
420
380
140
450
Seed plants
Vascular plants
Land plants
Bryophytes
Chara
Figure 1. Phylogenetic context of Arabidopsis thaliana: Evolutionary history of land plants.
Phylogenetic tree of land plant evolution with some speciation events shown as colored nodes. White node, origin of land plants; light blue node, origin of
vascular plants; blue node, origin of seed plants; dark blue node, origin of flowering plants. Here, Chara spp. from the green algae order Charales is the
outgroup, since it has been used to root several recent molecular land plant phylogenies. The topology of this tree is based on studies by Soltis et al. (1999)
and Nickrent et al. (2000). Time references in million years before present (MYBP) were taken from Judd et al. (2002).
istic of angiosperms is true hermaphroditism (Judd et al., 2002; model for floral organ determination (Bowman et al., 1989; Coen
Frohlich, 2006). and Meyerowitz, 1991). While much work has been and continues
Flower structure has been studied in a variety of ways. Studies to be done in Antirrhinum and other eudicot species, including
of the natural history and evolutionary biology of flowers have em- Petunia hybrida, the genomic and life-cycle characteristics of Ara-
phasized understanding the ultimate (evolutionary) causes of the bidopsis make it the preferred experimental system for in-depth
wide range of variants such as color, symmetry, meristic arrange- studies on the molecular components underlying cell differentia-
ments (e.g. flower organ number), size, pollination syndrome, etc. tion and morphogenesis during flower development.
Other studies have addressed the cellular, tissue type, morpho- The basic floral architecture is mostly conserved among the
logical and physical factors that can account for both the pheno- so-called core eudicots, that make up over 73% of extant flower-
typic plasticity and developmental constraints in flower form (for ing plants (Drinnan et al., 1994) including Arabidopsis. Flowers
a review of the developmental framework of angiosperm mor- within this group generally have four concentric whorls of organs
phology, see Endress, 2006). A different approach flourished in that are specified, from the outside to the center of the flower, in
the late 1980s and early 1990s, the molecular genetics of flower the sequence: sepals, petals, stamens, and carpels. Arabidopsis
development in two model eudicot species: Arabidopsis thaliana has this typical floral architecture. An interesting exception to the
and Antirrhinum majus (see reviews in: Jack, 2004; Kaufmann et conserved floral ground plan of eudicots is found in a Mexican
al., 2005; Krizek and Fletcher, 2005; Theissen and Melzer, 2007). rainforest monocotyledon, Lacandonia schismatica (Triuridaceae),
Genetic studies of floral homeotic mutants in both plant spe- which bears central stamens surrounded by carpels (Martínez and
cies yielded the now classic combinatorial ABC developmental Ramos, 1989; Vergara-Silva et al., 2003; Ambrose et al., 2006).

Flower Development 3 of 57
SAM
A B
IM
CZ
PZ PZ
SAM RZ
L1 L2 L3 FM
Corpus
IM
Br
IM Distal
P2 C
se se
FM se
5
Br
3 Adaxial Abaxial
se
IM 1 IM 2
Proximal
P3
FM
Br
Figure 2. Schematic representation of the shoot apical meristem (SAM): the inflorescence shoot apical meristem and floral meristem.
(A) Diagram outlining the geometry of the inflorescence shoot apical meristem (IM) and flower meristem (FM) during the first stages of development of
the latter. On the flank of the IM a first bulge that corresponds to the rudimentary bract (Br) appears. In its axil, a second bulge forms and this continues to
grow engulfing the first one and forming the FM proper. Theses stages of FM development correspond to P2 and P3 according to Reddy et al. (2004). The
arrow and arrowhead indicate the first and second visible grooves respectively (see section 2.3 for further detail).
(B) Three distinctive zones make up the IM: the central zone (CZ) which contains the stem cells; the peripheral zone (PZ) on the flanks of the CZ that
gives rise to the bract and floral primordia; and the rib zone (RZ) underneath the CZ that yields stem tissue. Three cell layers are distinguished: L1 and L2
layers constitute the tunica and include portions of both the CZ and the PZ. The rest of the cells form the L3 layer or corpus. In L1 and L2, cell divisions
are anticlinal, while in L3 they occur in all directions (arrows, direction of cell division). The structure is maintained in the FM.
(C) Schematic representation of the boundary zones (blue lines) and axes of polarity during floral development with the differentiation of sepals (se) from
the floral primordium illustrated.
Even though the basic floral architecture is overall conserved Recent integrated approaches to study the concerted action of
among core eudicots, variation in the symmetry and size of flow- the molecular components in flower development (Mendoza and
ers, the number of whorls of each organ type, the number of or- Alvarez-Buylla, 1998; Espinosa-Soto et al., 2004), have led to
gans per whorl, and their arrangement, size, shape and color is a hypothesis that helps explain such robustness and conserva-
common (e.g., Judd et al., 2002). tion at the level of the GRN underlying floral organ specification.
The overall conservation of the flower plan suggests that ro- However structural (e.g., mechanical) constraints could also be
bust gene regulatory network (GRN) modules controlling the ba- involved in conserving floral architecture (see section 4). Ap-
sic features of flower development were established early in the proaches that integrate genetic and structural aspects of flowers
evolution of angiosperms and have persisted in the great major- should be pursued further to understand flower development in
ity of lineages throughout 140 million years of flower evolution. Arabidopsis and other angiosperms.

2. STRUCTURAL ASPECTS OF ARABIDOPSIS FLOWER et al. (2004). The difference could be due to the fact that not all
DEVELOPMENT of the cells estimated to be involved in the latter approaches are
In this section, we provide a summary of structural features of incorporated into the flower meristem proper. Some of them may
Arabidopsis flower development. This is essential background to form a part of the subtending rudimentary bract (Figure 2A; see
the molecular genetics reviewed in section 3. next section for further discussion).
The first cells produced by the RZ following the transition to
flowering are rectangular with their long axis perpendicular to the
2.1 Structural Organization of the Inflorescence Meristem major axis of the stem, but the subsequent elongation of these
and Origin of the Flower Meristem cells reverses this situation (Vaughan, 1955). The RZ gives rise
to stem tissue. The CZ encompasses the reservoir of stem cells
During the vegetative phase of the Arabidopsis life cycle, the that divide less frequently than cells at the periphery (Grandjean
shoot apical meristem (SAM) produces leaves on its flanks and et al., 2004; Reddy et al., 2004). The CZ maintains itself and
on transition to flowering, the shoot bolts and the SAM becomes yields daughter cells that form both the PZ and RZ (Bowman and
the inflorescence shoot apical meristem (IM). On bolting, some of Eshed, 2000). Fifteen stages of Arabidopsis flower development
the pre-existing leaf primordia become cauline leaves subtend- have been distinguished (Smyth et al., 1990). The first stages of
ing lateral inflorescence shoots (paraclades) and the shoot apex flower meristem development are: stage 1, when a flower buttress
starts to produce flowers (Hempel and Feldman, 1995). A primary arises, stage 2 when the flower meristem is formed and stage 3
IM produces lateral meristems that may go on to produce flowers when sepal primordia appear. Recently researchers have been
or secondary inflorescences. Arabidopsis inflorescences are sub- able to study early flower meristem development in greater detail
tended by fully developed bracts, but flowers only by rudimentary (Reddy et al., 2004; Kwiatkowska, 2006; reviewed by Kwiatkows-
ones. It is generally said that the IM generates the floral meri- ka, 2008) and have proposed subdividing stage 1 (see section
stems (FM) on its flanks, but to be more precise, Arabidopsis FM 2.3).
are formed in the axils of the rudimentary bracts (Figure 2A; Long
and Barton 2000; Hepworth et al., 2006; Kwiatkowska, 2006; re-
viewed in Kwiatkowska, 2008). 2.2 Floral Organ Primordia
The SAM of the Arabidopsis inflorescence consists of a small
dome of cells organized into different regions (Figure 2B) with Once a flower primordium is initiated, the geometry changes and
different gene expression profiles (see section 4.1), cellular a rapid and coordinated burst of cell expansion and division oc-
behaviors and structures. The tunica at the SAM surface and curs in three dimensions generating a concentric group of cells
corpus are distinguished on the basis of cell division planes. as an almost spherical flower primordium, from which all floral
In Arabidopsis, the tunica consists of two clonally distinct cell tissues are derived (Bossinger and Smyth, 1996; Reddy et al.,
layers called L1 and L2 (Vaughan, 1952; Steeves and Sussex, 2004; Kwiatkowska, 2006). Jenik and Irish (2000) found that the
1989). Cell divisions within these meristem layers are exclusive- regulation of cell divisions during early and late stages of flower
ly anticlinal and the new cell walls are formed perpendicular to development seems to depend upon different mechanisms. Early
the surface of the meristem. The progeny of cells in the L1 will in flower development, when the floral meristem of Arabidopsis
therefore remain in this same layer within the meristem similar is divided into four concentric rings (each with a characteristic
to the underlying L2 progeny. Since outside the meristem the L1 multigenic expression profile; see section 3.3), cell division pat-
derived cells continue to divide only anticlinally the L1 eventually terns depend upon the cell’s radial position in the floral meristem,
gives rise to epidermal cells. The cells originating from L2 also and not on the future identity of the floral organ to be formed in
divide periclinally (outside the SAM) and contribute for example each ring. After stage 6, during organogenesis, the ABC homeo-
to the leaf mesophyll or stem ground tissue formation during or- tic genes (see section 3.3) seem to control the rate and orienta-
ganogenesis. This is also the germ line in the angiosperm SAM tion of cell divisions. As a result, the continuity of the concentric
(Ruth et al., 1985, Klekowski, 1988; Kwiatkowska, 2008). Below rings is broken giving distinct floral organ primordia within each
the tunica, cell divisions are both anticlinal and periclinal. This whorl, then cells subdifferentiate into distinct types within each or-
region of the SAM is the corpus or L3 from which the innermost gan. The initiation and identity of floral organs are also regulated
tissues, like vascular tissues, are formed (Figure 2B; Brand et by different and largely independent molecular modules. This is
al., 2001). suggested, for example, by the fact that conversion of petals into
The SAM is also organized into three different cytohistologi- sepal-like organs in mutant plants does not alter the number of
cal zones each with characteristic cytoplasmic densities and cell cells involved in their initiation (Crone and Lord, 1994; Bossinger
division rates: the central zone (CZ), the peripheral zone (PZ) sur- and Smyth, 1996).
rounding the CZ and the rib zone (RZ) underneath the CZ (Figure Tissues of floral organs are organized according to coordinat-
2B; Bowman, 1994; Bowman and Eshed, 2000). ed patterns and rates of cell division in the different cell layers of
Flower primordia are derived from the PZ of the IM and are ini- the meristem that dynamically acquire distinct fates. Clonal analy-
tiated from a block of four so-called founder cells (Bossinger and sis shows that L1 contributes to the epidermis, the stigma, part
Smyth, 1996; Reddy et al., 2004). This estimate was based on of the transmitting tract and the integument of the ovules, while
sector boundary analysis. However, using a non-invasive replica L2 and L3 contribute to the mesophyll and other internal tissues
method and a 3-D reconstruction algorithm, Kwiatkowska (2006; (Jenik and Irish, 2000).
2008) argues that more cells are assigned to the flower primor- Sector boundary analysis of surface cells has shown that se-
dium, and this is consistent with the observations by Grandjean pals and carpels are initiated from eight cells, stamens from four

Stage 1 2 3 4 5 6 7 8 9 10
FM
Time (h) 24 30 18 18 6 30 24 24 60 12
Stage 11 12 13 14 15 16 17 18 19 20
Time (h) 30 42 6 18 24 12 192 36 Up to 24 -
Figure 3. Summary of the 20 stages of flower development.
Schematic representation of developmental stages of Arabidopsis flowers. Briefly, the flower primordium is formed at stages 1 and 2. At stage 3, sepal
primordia are already visible and continue growing until they enclose the flower meristem (from stage 4 to 6). Meanwhile, at stage 5, petal and stamen
primordia are beginning to be visible, and the gynoecium starts to form (stage 6). Organ development continues and by stage 9, stigmatic papillae arise
at the top of the gynoecium. At stage 12, petals are similar in length to stamens. Anthesis occurs at stage 13, fertilization occurs, and the flower opens at
stage 14. Siliques reach their maximum size and are green by stage 17, then they loose water and turn yellow (stage 18) until valves separate from dry
siliques (stage 19) and seeds fall (stage 20). Floral meristems (FM), pink; sepals, green; petals, bright pink; stamens, blue; gynoecia, yellow; ovules, dark
green; seeds orange and brown. Duration of each stage in hours (h) is given under the figures (from Smyth et al., 1990).
cells, and petals from two cells (Bossinger and Smyth, 1996). meridional (i.e. radial when viewed from the top of the meristem)
Each organ primordium arises as a set of cells separated by direction (Kwiatkowska, 2006) eventually leading to formation of a
boundary regions of slow-dividing cells (Figure 2C and section shallow crease, which corresponds to the first visible groove and
3.4.2; Breuil-Broyer et al., 2004). Flower development ends when to the P2 stage (according to Reddy et al., 2004) of flower devel-
mature organs are formed and all the flower meristem cells are opment (Figure 2A). This shallow crease corresponds to the axil of
used up (Takeda et al., 2004; Krizek and Fletcher, 2005). the putative rudimentary bract (Kwiatkowska, 2006, 2008). Soon
after the bract is formed, another bulge occurs in its axil in an
upward direction. This second bulging corresponds to the forma-
2.3 Stages of Flower Development tion of a flower primordium proper and to stage P3 according to
Reddy et al. (2004). This stage corresponds to stage 2 according
We provide an illustrated description of 20 states of floral develop- to Smyth et al. (1990). Hence, during early stages of flower devel-
ment and fruit formation (Figures 2-7), mostly based on Bowman opment in Arabidopsis, two types of primordia (bract and flower
(1994), Smyth et al. (1990), Ferrándiz et al. (1999) and Roeder primordium proper) and organ boundaries are observed. The first
and Yanofsky (2006), with updates and substages as proposed boundary is the adaxial boundary of the rudimentary bract, while
by Long and Barton (2000), Reddy et al., 2004; Hepworth et al. the second is the boundary between the IM and the flower primor-
(2006), Kwiatkowska (2006) and Kwiatkowska (2008). dium proper (Figure 2A). The expression patterns of several genes
STAGE 1: The first sign of flower primordium formation is the confirm the developmental stages distinguished here (see more
bulging of the peripheral surface of the IM in a lateral direction. data on gene expression in the next section).
This stage was referred to as P1 by Reddy et al. (2004). It is hy- A significant increase in mitotic activity is observed upon for-
pothesized that a lateral protrusion formed during bulging is a rudi- mation of the primordium. The mitotic activity can be estimated as
mentary bract (Figure 2A; Kwiatkowska, 2006). At this early stage, the increase in the number of cells per 24 h or the accompanying
growth is fast and strongly anisotropic, with maximal growth in a area growth rates on the condition that the mean cell size does

10m
10 m 10 m
10 m 10m
1010m
m 1010m
m
10 10m
m
Figure 4. Stages 1 to 6 of Arabidopsis flower development.
(A) and (B) Inflorescence shoot apical meristem (IM) and floral meristem (FM) at stage 1 and 2 as indicated.
(C) and (D) Stage 3 FM showing abaxial (ab) and adaxial (ad) sepals (se).
(E) At stage 4, lateral sepals (l) shown growing perpendicularly to the abaxial and adaxial ones.
(F) and (G) At stage 5, stamen primordia are visible (arrows) and sepals almost cover the rest of the meristem.
(H) Flower bud where sepals are covering the stamens and the gynoecium primordium.
(I) Section through a stage-6 flower primordium where the gynoecium (g), stamens (st), and sepals (se) are apparent.
Pictures are scanning electron micrographs (SEM), except (D), (G) and (I) which are optical images of histological sections. All pictures are of Columbia-0
wild-type plants.
not increase (Grandjean et al., 2004; Kwiatkowska, 2006; Reddy becomes clearly delimited from the IM, and starts to grow larger
et al., 2004). During these early stages of flower development, very quickly in all directions (Figures 3 and 4A-B; Reddy et al.,
periclinal cell divisions occur in the corpus while L1 and L2 cells 2004; Kwiatkowska, 2006).
only divide anticlinally (Vaughan, 1955). Hence, the two-layered STAGE 3: This stage begins when sepal primordia become vis-
tunica organization is maintained in the flower meristem, but all of ible. By now the flower primordium is 30-35 μm in diameter and is
its cells are mitotically active. becoming stalked with an incipient pedicel. It has also started to
STAGE 2: During this stage, the hemispherical primordium grow vertically. The two lateral (l) sepal primordia appear first, but
continues to grow forming almost a right angle with the surface are soon outgrown by the abaxial (ab) then the adaxial (ad) sepal
of the SAM, which itself lengthens and widens rebuilding the por- primordia. Sepal primordia arise initially as ridges that lengthen
tion of the periphery that has been used for primordium forma- and curve inwards until they begin to overtop the remaining dome-
tion (Figures 3 and 4A-B). At this stage the flower primordium shaped portion of the flower primordium (Figures 3 and 4C-D).

STAGE 4: During this stage, the elongation of the pedicel con- the stamens outstrip the gynoecium in length and self pollination
tinues concurrently with an increase in the diameter of the devel- takes place. The gynoecium is now mature and its three distinct
oping flower primordium to 65-70 μm. The medial sepal primordia regions can be distinguished: an apical stigma, a style, and a bas-
have already partly overtopped the remaining floral meristem al ovary. After pollination, pollen tubes grow to fertilize the ovules,
(Figure 4E). the stamens extend above the stigma, and furrows at both valve/
STAGE 5: This stage is when the petal and stamen primordia replum boundaries appear.
become visible. Primordia of the four medial (long) stamens are STAGE 14: This is also defined as the stage zero hours after
first seen as wide outgrowths on the flanks of the central dome flowering (0 HAF), and it marks the beginning of silique (the fertil-
of the FM. The four petal primordia that arise between the se- ized pistil or fruit) and seed development. Cells in the exocarp
pals close to their base are just visible during this stage. The two continue to divide anticlinally and expand longitudinally in the re-
lateral (short) stamens develop from primordia that appear later plum and the valve, where there is also some expansion in other
during this stage (Figures 3 and 4F-G). directions. There is also division and expansion in the mesocarp
STAGE 6: The sepals grow to completely cover the floral bud and many chloroplasts develop (Figures 3 and 6I).
and the primordia of the four long stamens bulge out and become STAGE 15: The stigma extends above the long anthers. In the
distinct from the central dome of cells that comprise the FM. The carpel walls, cell division and expansion continue. The medial vas-
two lateral stamen primordia arise slightly lower on the dome and cular bundles continue to grow and xylem lignifies, while the lateral
develop later. The petal primordia grow somewhat but are still bundles branch out through the mesocarp (Figures 3 and 6J-K).
relatively small. A rim around the central dome of the flower pri- STAGE 16: At this stage the silique is twice as long as a
mordium now begins to grow upward to produce an oval tube that stage-13 pistil. Petals and sepals wither and tissues in the silique
will become the gynoecium (Figures 3 and 4H-I). continue expanding (Figures 3 and 6L).
STAGE 7: This stage begins when the growing primordia of the STAGE 17: This stage is defined by the abscission of the se-
long stamens become stalked at their base. The stalks give rise to nescent floral organs from the silique, ~2 days after fertilization.
the filaments, and the wider upper region to the anthers. By this The green silique grows to reach its final length and matures, a
stage, petal primordia have become hemispherical although they phase lasting about 8 days making this the longest stage. The
are still relatively small (ca. 25 μm in diameter; Figures 3 and 5A-B). dehiscence zone also differentiates (Figures 3, 7A and 7E; Sub-
STAGE 8: The beginning of stage 8 is defined by another land- stages 17A and 17B, see Roeder and Yanofsky, 2006).
mark in stamen development: anther locules are visible as convex STAGE 18: The silique begins to yellow from the tip to the
protrusions on the inner (adaxial) surface of the long stamens. base. One of the endocarp cell layers (the second from the inside)
At this stage stamens are 55-60 μm long most of which is the lignifies further, and the inner endocarp cell layer disintegrates,
developing anther. Locules also appear soon after in the short while the mesocarp begins to dry out. It has been suggested that
stamens. Petal growth now accelerates and petal primordia be- lignification may contribute to the silique shattering process, act-
come apparent (Figures 3 and 5C-E). ing in a springlike manner to create mechanical tensions (Figures
STAGE 9: This stage begins when the petal primordia elongate. 3 and 7B).
There is a rapid lengthening of all organs especially of petals that STAGE 19: The valves begin to separate from the dry silique,
acquire a tongue-like shape and increase in length from about 45 apparently owing to the lack of cell cohesion at the separation
μm to up to 200 μm. Nectary glands appear and the stamens grow layer.(Figures 3 and 7C).
rapidly. By the end of stage 9, the medial stamens are around 300 STAGE 20: At this stage the valves become separated from
μm long. Most of this growth occurs in the anther region, which still the dry silique and the mature seeds are ready to be dispersed
accounts for over 80% of their total length. At this stage the floral (Figures 3, 7D and 7F).
bud remains completely closed (Figures 3 and 5F-G).
STAGE 10: The rapidly growing petals reach the top of the
lateral stamens. The cap of papillae that will constitute the stigma 2.4 Morphology, Histology and Development of
starts to form at the top of the gynoecium (Figures 3 and 5H-I). Floral Organs
STAGE 11: This stage begins when the upper surface of the
gynoecium develops stigmatic papillae (Figures 3 and 6A-C) al- Sepals: In sepals L1-derived cells form the epidermis, the meso-
though their outward growth is limited at first to regions not in phyll originates from the L2, and the L3 contributes to the vascu-
contact with the overlapping sepals. By the end of this stage petal lature in the basal part (Jenik and Irish, 2000). Sepals and petals
primordia reach the top of the medial stamens. together form the perianth. Both organ types have a simple lami-
STAGE 12: Petals continue to lengthen relatively rapidly. Lat- nar structure, consisting of an epidermis, mesophyll and rather
eral sepals continue to grow while the stamens and gynoecium delicate vascular bundles (veins). The four sepal primordia (the
lengthen coordinately. The anthers have almost reached their abaxial, adaxial, and two lateral sepal primordia) are the first floral
mature length of 350-400 μm and the filaments now lengthen rap- organ primordia to appear. They arise at stage 3 of flower devel-
idly. The upper part of the gynoecium differentiates into the style opment in a cruciform pattern (Figures 4C-D; Smyth et al., 1990;
(Figure 6D) and a sharp boundary separates it from the cap of Bowman, 1994). Whether all four sepals occupy one whorl or the
stigmatic papillae. Stage 12 ends when the sepals open (Figures two lateral sepals occupy a separate outer whorl, has been the
3 and 6D-F). subject of discussion (Figure 4E; Smyth et al., 1990; Bowman,
STAGE 13: Petals become visible between the sepals and 1994; Choob and Penin, 2004), but all sepal primordia are formed
continue to elongate rapidly. The stigma is receptive at this stage at around the same time, shortly after they are specified (Figure
(Figures 3 and 6G-H). Stamen filaments extend even faster so 4E; Bowman, 1994).

(A) Stage 7 in which petal (pe) and stamen (arrowhead) primordia are indicated.
(B) Vertical view of the gynoecium (g) in a stage 7 floral primordium.
(C) to (E) Carpels and stamens at stage 8 of floral development are shown. Filament (f) and anther (a) regions of the stamen are differentiated (C) and
a slot is formed at the tip of the style in the gynoecium (D). Section through the floral bud with sepals (se), petals (pe), stamens (st) and gynoecium (g)
indicated (E).
(F) and (G) Floral bud at stage 9 in which petal primordia (pe) are indicated (F). Section through flower primordium (G) in which XAL1:GUS is shown
staining nectaries (n).
(H) and (I) Stage10 flowers. Flower bud showing the enlarged sepals which cover other floral organs, stalked petals and stamens, and developing carpels
in the center (H). Stigma starts to be formed at the top of the gynoecium (I, arrows)
Bars = 10 μm except in (F) and (H). Images (A), (B), (C), (G) and (I) are of Lansberg erecta ecotype, from Smyth et al. (1990) provided by Dr J. Bowman.
Some sepals were removed from flower buds shown in (A), (B), (C), (D), (F), (H) and (I). All images except (E) and (G) are SEM. (D), (E), (F), (H) are of
Columbia-0 ecotype.
The adaxial and abaxial surfaces of the sepal epidermis are unbranched trichomes (Figure 8E; Smyth et al., 1990; Bowman,
different (Figures 8B and 8D-E). On the abaxial surface, cells 1994; Hase et al., 2000; Krizek et al., 2000).
have irregular shapes and sizes with some quite long cells (with Petals: In the petal primordium the meristematic layer L1 con-
nuclei of various sizes) and fringes of smaller cells. Unlike the tributes to the epidermis and L2 to the mesophyll; as yet cells
adaxial surface, the abaxial surface has stomata and may have originating from L3 have not been found to form part of the petal

(Figure 2B; Jenik and Irish, 2000). These primordia become ap- noecium consists of two valves separated by a false septum with
parent almost at the same time as stamen primordia at stage 5 of ovules arising from parental placental tissue on each side of the
flower development. Visible signs of petal differentiation are seen septum (Bowman, 1994). The valves grow upward from the flower
by stage 9 (Figure 5F; Smyth et al., 1990; Bowman, 1994). The meristem to form a closed cylinder. At early stage 8, the walls of
four petals of Arabidopsis are white and flat and approximately the cylinder are composed of an L1-derived epidermis, one L2-
the same size and shape. They are narrower and greenish toward derived subepidermal layer and a two-cell thick, L3-derived core.
the base (Figure 8C; Takeda et al., 2004). At this stage the distal L2 cells start to divide periclinally (with
Cells on the adaxial surface are conical with epicuticular thicken- respect to the top surface of the cylinder), contributing to the lon-
ings running from the cell base to the apex, whereas those on the gitudinal growth of the carpel (Figure 2B; Jenik and Irish, 2000).
abaxial surface are flatter and more cobblestone-like with cuticular Later the inner surfaces of septal outgrowths within this cylinder
thickening (Figures 8F-G). Stomata are absent from both petal sur- will fuse, the tip will close and ovules will develop along the mar-
faces (Bowman, 1994; Krizek et al., 2000). Cells toward the base of gins of the fused walls (placenta) of the bilocular chamber (Bow-
petals resemble those of stamen filaments (Bowman, 1994). man, 1994; Sessions and Zambryski, 1995). The gynoecium is
Stamens: Primordia appear at stage 5 of flower development oriented in the flower so that the septum coincides with the medial
(Figure 4F) due to periclinal divisions in the subprotodermal cell plane (Figures 4D-E and 4G; Sessions and Zambryski, 1995).
layer (L2) and sometimes in L3 (Crone and Lord, 1994; Jenik and At the distal end of the gynoecium, the stigma, an epidermal
Irish, 2000). Stamen primordia are visible at stage 6. By stage 7, structure composed of stigmatic papillae (bulbous elongated
differentiation can be observed and long stamen primordia ap- cells), functions in pollen binding and recognition and participates
pear stalked at their bases (Figures 4I and 5A-B; Bowman, 1994; in the induction of pollen germination (Figures 6A-B). After germi-
Smyth et al., 1990). At this stage stamen primordia are composed nation, the pollen tubes will grow between the papillar cells into
of an L1-derived epidermis, one layer of L2-derived subepider- the transmitting tract at the center of the style and the septum of
mis, and an L3-derived core (Figure 2B; Jenik and Irish, 2000). the ovary (Bowman, 1994; Sessions and Zambryski, 1995).
Locules appear in the anthers by stage 8 (Figure 5C). Growth At about stage 11, the inner and outer integuments of the
of the internal anther tissue at this stage is due to divisions of ovule are formed. By stage 12, the integuments of the developing
L2-derived cells (Jenik and Irish, 2000). At stage 14, anthers ex- ovule grow to cover the nucellus and megagametogenesis occurs
tend above the stigma (Figure 5I; Bowman, 1994). In the mature (Figures 6E-F; Bowman, 1994).
anther, the L3 cells contribute only to the vasculature (Jenik and Nectaries: These organs produce and secrete nectar. Nectar
Irish, 2000). Stamens of the Arabidopsis flower are not formed is a protein- and carbohydrate-rich solution, which varies in com-
simultaneously: four long medial stamens arise a little earlier than position among different plant species (Davis et al., 1998). Nectar
the two short lateral ones (Smyth et al., 1990). may be a reward for pollinators or for insects that protect the plant
Each stamen consists of two distinct parts, the filament and the against herbivores, or even a lure for animal prey in carnivorous
anther. At the tip of the filaments, the anther develops both repro- plants (Davis et al., 1998; Baum et al., 2001; Lee et al., 2005a).
ductive and non-reproductive tissues that produce, harbor, and In Arabidopsis, the nectarium (multiple nectary) found in in-
release pollen grains upon maturity (Goldberg et al., 1993). The dividual flowers (Davis et al., 1998) is composed of two parts:
anther is a bilocular structure with longitudinal dehiscence (Figure nectary glands that form below the stamen filament, and the
6G; Bowman, 1994). Each locule develops from successive divi- connective tissue linking the glands in a continuum around the
sions of subprotodermal archesporial cells formed in the anther androecium (Bowman, 1994; Baum et al., 2001). The nectarium
primordium that gives rise to three morphologically distinct layers: is always situated in the third whorl of the flower and its location
the endothecium, the middle layer, and the tapetum which sur- is independent of the identity of the other organs occupying this
rounds the pollen mother cells (PMCs). The PMCs undergo meio- whorl. These glands are formed from stage 9 to 17 of flower de-
sis and form the haploid microspores. The tapetum is a source velopment (Figure 5G; Bowman, 1994; Bowman and Smyth 1999;
of nutrients and is indispensable for microspore maturation (Xing Baum et al., 2001; Tapia-López et al., 2008).
and Zachgo, 2008). Anther development and microspore forma-
tion in Arabidopsis is a complex process that has been divided
into 14 stages (See also section 3.4.5; Sanders et al., 1999). 3. MOLECULAR GENETICS OF ARABIDOPSIS FLOWER
Once formed, PMCs are surrounded by a layer of callose. Af- DEVELOPMENT
ter meiosis, the anther contains most of its specialized cells and
tissues, and tetrads of microspores are present within the pollen Plant organogenesis, including flower formation, occurs from ac-
sacs; with microspores in each tetrad surrounded by a callose tively proliferating meristems over the entire life cycle. In the next
wall. Callose dissolves and microspores are released. As pollen section we provide a very brief summary of the molecular mecha-
grains develop, the anther enlarges and is pushed upward in the nisms that maintain an active SAM. In section 3.2, we explain how
flower by the elongating filament (Scott et al., 2004). the flower meristem is specified and becomes determinate after
Carpels: The fourth and innermost whorl is occupied by the the flower organs are formed.
gynoecium that is composed of two fused carpels. Carpel primor-
dia start to form at stage 6 of flower development (Figure 4I) due
to periclinal cell divisions in the L3 layer (Jenik and Irish, 2000). 3.1 Shoot Apical Meristem Proliferation and Maintenance
Carpels enclose and protect the developing ovules, mediate pol-
lination, and after fertilization develop into a fruit within which fer- The balance between cell proliferation and cell recruitment to
tilized ovules develop into seeds (Bowman et al., 1999). The gy- differentiated tissues in the SAM is dependent on mechanisms

100m 10m
10m 100m
100m 10m
10m
100m
100m
100m 100m
100m 100m
(A) to (C) Stage 11 of flower development where the gynoecium develops stigmatic papillae (arrows) (A) and (B). Longitudinal section where sepals (se),
stamen (st), and gynoecium (g) are indicated (C).
(D) to (F) Flower primordium at stage 12. Longitudinal (E) and transverse (F) sections showing all the organs as well as ovules and pollen grains.
(G) and (H) Flower anthesis at early stage 13 when the stigma (arrowhead) is already receptive (G); a close-up view of the stigma (H).
(I) to (L) Flower primordium at stages 14 (I) and 15 where the gynoecium has begun to enlarge to form the silique (J). Close-up of a stage-15 stigma (K)
and stage-16 flowers where sepals and petals are beginning to wither (L).
Bars = 100 μm. All images except (C), (E) and (F) are SEM. Images are of Columbia-0 ecotype, except (A) that is of Landsberg erecta (from Smyth et
al.,1990, provided by Dr. J. Bowman).
regulated by WUSCHEL (WUS; Laux et al., 1996; Sablowski, an imbalance between cells retained within meristems versus
2007). The homeodomain-containing WUS transcription factor those recruited to form lateral organs. clv mutations cause an
has the role of the maintaining the identity of stem cells in the expansion of the WUS expression domain resulting in an en-
organizing center of the CZ; wus mutants lack stem cells in the larged stem cell niche. CLV3 expression is, in turn, positively
SAM (Mayer et al., 1998). WUS expression is limited to the cells regulated by WUS, suggesting that meristem size depends
immediately below the stem cells, an expression domain regu- greatly on a WUS-CLV regulatory loop (Clark et al., 1993, 1995;
lated by the receptor-kinase signaling system that includes the Kayes and Clark, 1998; Brand et al., 2000). Overexpression
CLAVATA1, 2 and 3 (CLV1, 2, 3) gene products (Mayer et al., of CLV3 represses WUS expression and decreases meristem
1998; Brand et al., 2000; Schoof et al., 2000). CLV1 is expressed activity, suggesting that CLV3, a secreted CLE-domain pep-
in most L3 stem cells while CLV3 is expressed in all three stem tide, is the signal that regulates WUS expression via the CLV1/
cell layers but mostly in L1 and L2 stem cells (see Figure 2B; CLV2 LRR protein-kinase transduction complex (Fletcher et al.,
Clark et al., 1997; Fletcher et al., 1999). In clv mutants, there is 1999; Jeong et al., 1999; Trotochaud et al., 1999; Clark, 2001a

and 2001b; Ni and Clark, 2006). It has been shown that other
LRR-protein kinases closely related to CLV1 like BARELY ANY
MERISTEM1 and 2 (BAM1, 2) are also involved in meristem
maintenance possibly by sequestering CLV3 on the flanks of
the meristem where they are expressed (DeYoung et al., 2006;
DeYoung and Clark, 2008).
SHOOT MERISTEMLESS (STM) is a KNOTTED1-like ho-
meobox (KNOX) gene that encodes a protein expressed in the
SAM’s CZ, RZ and regions of the PZ that have not been assigned
to a primordium, i.e. it is expressed throughout the meristem ex-
cept for anlagen, the sites of primordium formation (Figure 2B).
STM promotes the profileration of stem cell derivatives until a
critical cellular mass is attained sufficient to form either leaves or
floral primordia. It also inhibits the expression of ASYMMETRIC
LEAVES1 and 2 (AS1, 2) genes in the SAM, preventing these
cells from undergoing premature differentiation (Byrne et al.,
2000; Byrne et al., 2002). Thus, the STM gene is considered to
play a pivotal role in meristem maintenance (Long et al., 1996;
Carles et al., 2004). ULTRAPETALA1 (ULT1) encodes a cyste-
ine-rich protein with a B-box like domain that restricts the size
of shoot and floral meristems. It functions antagonistically to the
proliferative roles of WUS and STM during most of the Arabidop-
sis life cycle but it in an independent genetic pathway (Carles et
al., 2004).
3.2 Floral Meristem Specification and Determination
The changes in cellular characteristics, growth and geometry ob-

served in the transition of the SAM to an IM (Kwiatkowska, 2006)
are correlated with dynamic changes in the spatial and temporal
expression of certain genes. The Arabidopsis IM produces rudi-
mentary bracts in whose axils flower meristems emerge. STM
and AINTEGUMENTA (ANT) expression patterns correlate with
the development of this rudimentary bract primordium (Long and
Barton, 2000). Figure 7. Stages 17 to 20 of Arabidopsis flower development.
The expression of LEAFY (LFY), a transcription factor found (A) to (D) Photographs of developing and mature siliques at stages 17 (A),
only in plants (Schultz and Haughn, 1991; Weigel et al., 1992; 18 (B), 19 (C), and 20 (D) of flower development.
Maizel et al., 2005; Weigel, 2005) and ANT has been used in (E) SEM of seeds from a silique at stage 17.
order to trace the cells that form the flower primordium (Grand- (F) Close-up view of a seed from a stage-20 dehiscent silique.
jean et el., 2004). First, tens of cells are rapidly recruited to those All photographs are of Columbia-0 ecotype.
already committed to become part of the flower meristem. This
stage may correspond to the upward bulging at the shallow crease
formed between the rudimentary bract and the IM described by
Kwiatkowska (2006). These cells which express LFY then con- a flower meristem has more founder cells. An explanation that
tinue to proliferate. Interpretating this, the first cells that express resolves the discrepancy is that the LFY-expressing cells could
LFY would correspond to the rudimentary bract (but not its axil or include those that eventually form the rudimentary bract, as well
shallow crease), and later the domain of LFY expression would as those which form the flower primordium (Kwiatkowska, 2006;
expand to include the cells committed to the flower primordium reviewed in Kwiatkowska, 2008)
proper (Kwiatkowska, 2006). This interpretation can explain the The gene CUP-SHAPED COTYLEDON2 (CUC2) is ex-
discrepancy in the number of founder cells estimated using sec- pressed in the slow-dividing cells that expand in a latitudinal di-
tor boundary analysis (Bossinger and Smyth, 1996) and using in rection (Reddy et al., 2004) to define the second boundary be-
vivo LFY expression patterns (Grandjean et al., 2004). Bossinger tween the floral primordium proper and the IM (Breuil-Broyer et
and Smyth (1996) concluded that a FM derives from four founder al., 2004). Several regulators of CUC including a miRNA have
cells directly on the surface of the IM (or SAM). In support of this, been described as important components of the GRN involved in
evidence from confocal laser scanning microscopy indicates that this developmental process (Laufs et al., 2004; Aida and Tasaka,
flower primordia are formed from two rows of cells in a radial arc 2006a).
(Reddy et al., 2004). In contrast, the number of cells express- Flower versus inflorescence meristem identity is controlled
ing LFY at these early stages (Reddy et al., 2004) suggest that by a complex GRN that integrates environmental and internal

expressed in the IM that is then transformed into a FM (Shannon

and Meeks-Wagner, 1991; 1993; Weigel et al., 1992, Bowman et
pe al., 1993; Gustafson-Brown et al., 1994; Bradley et al., 1997; Chen
et al., 1997; Moon et al., 2003). On the contrary, if AP1, CAL and
LFY are repressed, the FM attains IM identity (Ratcliffe et al., 1998;
Ratcliffe et al., 1999). TFL1 is an important regulator of inflores-
cence development (Alvarez et al., 1992; Ratcliffe et al., 1998; Par-
cy et al., 2002). It encodes a phosphatidyl ethanolamine-binding
se protein (PEBP) that is transcribed in the center of the IM but the
A B C protein moves to other cells where AP1 and LFY are downregulat-
ed (Bradley et al., 1997; Conti and Bradley, 2007). EMF genes are
required for vegetative growth, but they seem to regulate flowering
time and inflorescence development too (Sung et al., 1992; Aubert
et al., 2001; Yoshida et al., 2001). Loss-of-function mutants in these
genes produce flowers immediately after germination skipping the
vegetative phase (Yang et al., 1995; Chen et al., 1997). EMF1 en-
codes a transcription factor that represses AP1 but not LFY, and
D E EMF2 encodes a novel zinc finger protein related to the polycomb
group (Aubert et al., 2001; Yoshida et al., 2001).
LFY is necessary and sufficient to specify FMI (Weigel et
al., 1992; Weigel and Nilsson, 1995). In lfy mutants, leaves and
secondary shoots are produced instead of flowers (Schultz and
Haughn, 1991; Weigel et al., 1992) and LFY overexpression
causes the conversion of leaves and axillary meristems to flow-
F G ers (Weigel and Nilsson, 1995). LFY is expressed in the leaf pri-
mordia during vegetative growth, but when induced by external
Figure 8. Sepal and petal cell types. (vernalization and light) and/or internal (gibberellins) signals, it is
Scanning electron micrographs (SEM) of wild-type flowers and flower or-
strongly expressed and relocates to the SAM flanks where floral
gans. meristems are formed (Figure 9; Blazquez et al., 1997; Hempel
(A) A mature flower with sepals (se) and petals (pe) fully expanded and et al., 1997; for LFY regulation see: Nilsson et al., 1998; Blazquez
the stigma extending above the long stamens. and Weigel, 2000; Liu et al., 2008). LFY expression persists at
(B) Sepal blade showing simple unbranched trichomes (arrowheads) high levels in the FM until stage 3 of development and then di-
characteristic of the abaxial surface. minishes in the center of the flower (Figure 10; Blazquez et al.,
(C) Mature petal blade consisting of a basal claw and a distal blade. 1997; Wagner et al., 2004). LFY protein abundance, however, is
(D) Adaxial sepal surface with irregular sizes and shapes of cells, some homogenous in the FM, probably because it moves between cells
elongated (800x). (Parcy et al., 1998; Sessions et al., 2000; Wu et al., 2003).
(E) Abaxial sepal surface bearing stomata (arrows) and characteristic LFY and AP1 have overlapping functions in establishing the
elongated cells (500x). FM; while the ap1 mutant has shoots with inflorescence charac-
(F) Adaxial surface of a mature petal blade showing conical cells with epi- teristics, the lfy ap1 double mutant has an almost complete con-
cuticular thickenings running from the base to the apex (800x). version of flowers into shoots (Huala and Sussex, 1992; Bowman
(G) Abaxial petal surface showing flatter, cobblestone-shaped cells with et al., 1993). Both genes when overexpressed cause a terminal
cuticular thickenings. Both petal surfaces lack stomata.
flower phenotype suggesting that each one is sufficient to deter-
mine the IM (Mandel and Yanofsky, 1995a; Weigel and Nilsson,
1995). CAL, the closest paralogue of AP1, and FRUITFULL (FUL)
cues (Figure 9). On induction of flowering, the IM genes, such as from the same gene clade within the MADS-box phylogenetic tree
TERMINAL FLOWER 1 (TFL1; Alvarez et al., 1992; Shannon and (Alvarez-Buylla et al., 2000; Martínez-Castilla and Alvarez-Buylla,
Meeks-Wagner, 1991 and 1993, Ohshima et al., 1997) and EM- 2003; Parenicová et al., 2003), may also act redundantly to AP1 in
BRYONIC FLOWER 1 and 2 (EMF1, 2; Chen et al., 1997; Aubert FM specification. Single cal and ful mutants do not show any FMI
et al., 2001), are repressed in the FM, while the floral meristem disorders, but in combination with ap1 in double or triple mutants,
identity (FMI) genes, mainly LFY, APETALA1 (AP1), APETALA2 the ap1 phenotype is greatly intensified (Bowman et al., 1993;
(AP2), and CAULIFLOWER (CAL), are upregulated (Figure 10; Kempin et al., 1995; Ferrándiz et al., 2000a). FUL is expressed
Blazquez et al., 1997; for review Mandel et al., 1992; Weigel et at the same time as LFY during the establishment of the FMI
al., 1992; Bowman et al., 1993; Kempin et al., 1995; Mandel and (Mandel and Yanofsky, 1995b; Hempel et al., 1997), but is mostly
Yanofsky, 1995a; Blazquez et al., 2006). localized in the IM (Figure 10). Later during flower development,
Mutual repression of the IM and FMI genes seem to underlie FUL is expressed again during carpel and silique development
the co-existence, identity and boundaries of both types of meristem where it plays an important role (Gu et al., 1998). Despite its close
in the SAM in the transition to flowering (Chen et al., 1997; Liljegren similarity to AP1, overexpression of CAL is not able to determine
et al., 1999; Ratcliffe et al., 1999). For example, if genes such as the IM as does overexpression of AP1, indicating that CAL does
TFL1 or EMF1 or 2 are mutated, LFY and/or AP1 are ectopically not interact with the same partners as AP1. The unique functions

of AP1 rely on residues within the K and COOH domains that are is a positive regulator of local cytokinin (CK) biosynthesis and
not found in CAL (Alvarez-Buylla et al., 2006). accumulation (Jasinski et al., 2005; Yanai et al., 2005), and a re-
LFY directly regulates AP1 and CAL transcription by binding pressor of gibberellin (GA) production (Jasinski et al., 2005). On
to the consensus sequence CCANTG (CArG-box; Parcy et al., the other hand, WUS enhances CK activity by repressing ARABI-
1998; Wagner et al., 1999; Wagner et al., 2004; William et al., DOPSIS TYPE A RESPONSE REGULATORS (ARRs) (Leibfried
2004). However, expression reminiscent of AP1 is seen in the lfy et al., 2005). The resulting high CK:auxin ratio and low GA levels
mutant, while it is completely abolished in the double mutant lfy promote indeterminate growth (Shani et al., 2006). While a high
ft (flowering locus t; Ruiz-Garcia et al., 1997; Schmid et al., 2003; auxin concentration restricts STM and CUC expression (see sec-
Wigge et al., 2005). Thus FT, a homolog of TFL1 (Koornneef et al., tion 3.4.2), it also downregulates CK biosynthesis and activity,
1991; Kardailsky et al., 1999), together with FD, a bZip transcrip- thus yielding a high auxin:CK ratio and high levels of GA, which
tion factor (Abe et al., 2005), redundantly regulate AP1 with LFY. induce floral meristem formation. Raising GA levels or response,
AP1 and CAL in turn regulate LFY by positive feedback, allowing for example by crossing with the spindly (spy) mutant, is sufficient
it to exert its transcriptional regulation during flower development to suppress FM reversion to IM in lfy, ap1, ap2 and ag mutants.
(Bowman et al., 1993; Liljegren et al., 1999). Recently, additional This demonstrates the importance of GA in the maintenance of
LFY targets have been found (William et al., 2004), among them FM identity (Okamuro et al., 1996; Okamuro et al., 1997).
LATE MERISTEM IDENTITY1 (LMI1), which encodes a home- Light signal transduction pathways are also involved in FM
odomain leucine-zipper transcription factor and functions as a maintenance. Spontaneous floral reversion in wild-type Arabidop-
FMI gene. Interestingly, LMI1 acts together with LFY to activate sis has only been observed at low frequencies in the first flowers
CAL expression (Figure 9; Saddic et al., 2006). of Landsberg erecta grown in short days. However, long hypocotyl
AP2 encodes a putative transcription factor of a plant-specific (hy1-1), a mutant in which phytochrome activity is blocked, sup-
gene family (AP2/EREBP) with diverse functions (Riechmann presses floral reversion of both lfy and ag single mutants in short
and Meyerowitz, 1998). Mutations in AP2 enhance both ap1 and days (Okamuro et al., 1996). Floral reversion seems to be a devel-
lfy mutant phenotypes, indicating that AP2 also plays a role in opmental abnormality with no apparent adaptative significance,
specifying FMI (Huala and Sussex, 1992; Schultz and Haughn, unless plant resources are somehow saved under certain condi-
1993; Shannon and Meeks-Wagner, 1993; Simpson et al., 1999). tions if flowering is reversed. Further ecological and evolutionary
MADS-box genes are key components of the regulatory mod- developmental studies of Arabidopsis ecotypes will continue to
ule that integrates flowering transition signaling pathways (for re- elucidate the genetic, epigenetic, physiological, and environmen-
view see Jack, 2004; Parcy, 2005, Blazquez et al., 2006), IM and tal mechanisms involved in the maintenance of the FMI.
FM identities (Mandel et al., 1992; Bowman et al., 1993; Mandel
and Yanofsky, 1995a, 1995b), and floral organ specification (see
section 3.3; Coen and Meyerowitz, 1991). To specify the FM, LFY 3.3 Specification of Floral Organs: The ABC Genes
and/or AP1 are also required to downregulate flowering induction
genes such as AGAMOUS-LIKE 24 (AGL24), SUPPRESSOR OF Very soon after FM specification (11-13 days after germination
OVEREXPRESSION OF CO 1 (SOC1), SHORT VEGETATIVE in Landsberg erecta ecotype), the flower meristem is subdivided
PHASE (SVP), and FUL (Figures 9 and 10). Overexpression of into four regions. Each one will give rise to the primordia of the
any of these genes causes FM to revert to IM-like structures as different floral whorls, which from the outside to the inside are:
when LFY and/or AP1 are mutated (Mandel and Yanofsky 1995b; sepals, petals, stamens, and carpels. The genes responsible for
Yu et al., 2004a; Liu et al., 2007). floral whorl specification attain their spatio-temporal pattern as a
Floral reversion is often found in plants heterozygous for lfy- result of regulatory interactions among themselves, interactions
6 (LFY/lfy) and homozygous for agamous-1 (ag-1), suggesting with meristem identity genes and with some other genes, such
a key role for LFY and AG in the maintenance of determinate as WUS and UNUSUAL FLORAL ORGANS (UFO; Levin and
floral meristems (Okamuro et al., 1996). The reason for this is Meyerowitz, 1995). The complexity of the interactions involved is
that late in floral organogenesis AG, induced by WUS, LFY and shown in the ‘floral organ specification gene regulatory network’
PERIANTHIA (PAN) among others, positively regulates KNUCK- (FOS-GRN) model, analyzed in Section 4.1. This model includes
LES (KNU) which in turn represses WUS expression to terminate a set of interacting genes sufficient to pattern the IM and FM dur-
the stem cell niche after a limited number of organs have been ing the first stages of flower development.
formed (Parcy et al., 1998; Busch et al., 1999; Lenhard et al., One of the key FM identity genes is LFY. The protein encoded
2001; Lohmann et al., 2001; Das et al., 2009; Maier et al., 2009; by this gene requires co-factors to set the spatial limits of ex-
Sun et al., 2009). In fact, while WUS expression declines after pression of the floral organ identity genes AP3, PI, and AG. For
stage 6 in wild-type flowers, it persists in pan or ag flowers (Len- example, LFY participates with UFO in the regulation of AP1 and
hard et al., 2001; Lohmann et al., 2001; Das et al., 2009; Maier AP3 transcription (Lee et al., 1997; Chae et al., 2008), and with
et al., 2009). ULT also participates in meristem determinancy to- WUS co-regulates the expression of AG (Lenhard et al., 2001;
gether with AG downregulating WUS (Carles et al., 2004). Lohmann et al., 2001). LFY also regulates the expression of the
Although it is very rare to observe spontaneous or induced SEPALLATA (SEP) genes SEP1, SEP2 and SEP3, additional
reversion from FM to IM, a set of genes that actively maintain FM MADS-box genes required for organ identity specification (Krizek
identity could conform to a “flower developmental module” that and Fletcher, 2005).
prevents reversion. The genetic mechanisms involved in maintain- UFO is expressed in the second and third whorls during flo-
ing FMI are closely linked to hormone balance and environmental ral stage 3, probably restricting the B-gene expression domain
factors (Tooke et al., 2005). For example, we now know that STM to these whorls, together with LFY (Lee et al., 1997; Traas and

Figure 9. Inflorescence shoot apical meristem (IM) versus flower meristem (FM).
Simplified model of a gene regulatory network (GRN) that induces and maintains the FM. Flowering induction genes like FT, SOC1 and AGL24 are highly
expressed in the IM in response to external (vernalization and light) and internal (gibberellins; GA) signals. These proteins in turn promote the expression
of flower meristem identity (FMI) genes, LFY and AP1. Paradoxically, during the establishment of the FM, genes like TFL1 and EMF1 that help to maintain
the IM identity are also expressed, keeping the expression of the FMI genes out of the IM. Later in development, LFY and AP1 repress the expression of
TFL1 and flowering genes SOC1 and AGL24, among others, thus maintaining the FMI. Arrows and bars indicate positive and negative regulatory interac-
tions respectively. (See references in main text).
Doonan, 2003). The UFO gene encodes a protein containing an carpels-carpels, and C-class mutant flowers have sepals-petals-
F-box domain, which is a characteristic of E3 ubiquitin ligases petals-sepals (Coen and Meyerowitz, 1991). It was shown that
that are components of SCF (Skp Cullin F-box containing) com- mutations in all three functions lead to the transformation of all
plexes and mark proteins for proteosome-dependent degradation floral organs into leaf-like organs, suggesting that flowers are
(Deshaies, 1999). It was recently shown that LFY interacts with modified leaves (reviewed in Robles and Pelaz, 2005). The Arabi-
UFO in order to directly bind the AP3 promoter. Furthermore, the dopsis ABC mutants are shown in Figure 11.
proteosome activity mediated by UFO is required for the tran- Hence, three different classes of homeotic genes with over-
scriptional activation of AP3 by LFY (Chae et al., 2008). lapping activities were proposed to be necessary for floral organ
Key components of the GRN that underlies the early pattern- specification. The A function specifies sepals, the A and B func-
ing of the flower meristem are the so-called ABC homeotic genes, tions specify petals, the B and C functions specify stamens and
AP1, AP2, AP3, PI, and AG, which are all transcription factors the C function specifies carpels (Figure 12; Bowman et al., 1991).
belonging to the MADS-box gene family, except AP2 (Coen and The A and C functions negatively regulate each other and the B
Meyerowitz, 1991; Wagner et al., 1999; Ng and Yanofsky, 2001; function is restricted to the second and third whorls. The latter
Lamb et al., 2002). was originally thought to be independent of A and C functions
The classic ABC model was inferred using Arabidopsis and (Bowman et al., 1991; Drews et al., 1991), but it was later shown
Antirrhinum homeotic flower mutants (Coen and Meyerowitz, that the A function gene AP1 regulates the B genes. AP1 binds to
1991). In these mutants two floral organ types are replaced by the promoter of AP3 (Hill et al., 1998; Tilly et al., 1998). AP1 can
two other floral organ types as follows: A- class mutant flowers also specify petals by regulating the spatial domain of B genes
have carpels-stamens-stamens-carpels (from the outermost to together with UFO in the first flowers to arise, and independently
the innermost whorl), B-class mutant flowers have sepals-sepals- of UFO in later flowers (Ng and Yanoksky, 2001).

FUL AGL24 SOC1 TFL1 WUS LFY AP1 AP2 CAL

IM FM
se
3
se
ca
6
st pe
Figure 10. Schematic representation of some inflorescence shoot apical (IM) and flower (FM) meristem gene expression patterns at stages 1, 3 and 6.
Flowering (FUL, AGL24 and SOC), indeterminate (WUS and TFL1), and FMI (LFY, AP1, AP2 and CAL) gene expression patterns based on in situ hy-
bridization data during floral primordium developmental stages 1, 3 and 6. At stage 1, expression patterns correspond to their functions in IM and FM
identities. Sepal (se), petal (pe), stamen (st) and carpel (ca) primordia are indicated. At stages 3 to 6, all with the exception of TFL1 are expressed in the
FM, probably because their respective proteins also affect organ development. FUL will participate in fruit development, LFY will induce all the ABC genes
and AP1 and AP2 are fundamental in sepal and petal formation (see references in main text).
Once identified at the molecular level, the mRNA expression (Figure 11, 12 and 13; Coen and Meyerowitz 1991; Goto and Mey-
patterns of the ABC genes were shown to overlap with the flo- erowitz, 1994; Jack et al., 1994; Honma and Goto, 2000). The fact
ral regions where the corresponding mutants had a phenotype that both single mutants yield the same phenotype shows their
(Yanofsky et al., 1990; Mandel et al., 1992; Goto and Meyerow- interdependence. AP3 and PI are regulated in two steps: they are
itz, 1994; Jack et al., 1994). AP1 and AP2 are A-function genes. first induced by LFY/UFO in response to flowering signals and they
AP1 is expressed in the two outer whorls of the floral meristem later maintain their expression in a self-regulatory loop (Honma
(Figures 10, 12, 13A; Mandel et al., 1992) and is important for and Goto, 2000). The proteins encoded by these two genes form
the establishment of sepal and petal identity as well as the FM heterodimers to exert their B function during petal and stamen
(section 3.2). AP1 expression is first up-regulated by LFY and FT/ development (Figure 14; Jack et al., 1992; Goto and Meyerowitz,
FD (section 3.2), but later is maintained by the B class genes in 1994; Zik and Irish, 2003a) and this oligomerization is necessary
a positive feedback loop (Sundström et al., 2006). Strong ap1 al- for them to move into the nucleus (McGonigle et al., 1996).
leles (ap1-1) often lack petals in the second whorl, while weaker Both genes are also regulated positively in a regulatory loop
mutant alleles of this gene do not have a full homeotic conversion by AP1 and negatively by EARLY BOLTING IN SHORT DAYS
of floral organs (see section 3.2; Irish and Sussex, 1990). (EBS), a gene that encodes a nuclear protein that participates in
In contrast to the MADS-box ABCs, the expression pattern of petal and stamen development and regulates flowering time by
AP2 does not correlate with the site where it exerts its function repressing FT (Gómez-Mena et al., 2001; Piñeiro et al., 2003).
in floral organ identity. AP2 mRNA is found throughout the flower ANT, a member of the AP2 gene family, is another regulator of
meristem (Figures 10 and 12; Jofuku et al., 1994). Recent data the B function, positively inducing AP3 (Klucher et al., 1996; Nole-
has shown that AP2 is repressed at the translational level by a Wilson and Krizek, 2006; see section 3.4.2).
microRNA (miR172), which is active only in whorls 3 and 4 (Chen, The only C-type gene discovered up to now is the MADS-box
2004), thus explaining why the function of AP2 is restricted to the gene AG (Bowman et al., 1989). ag mutant flowers lack stamens
first two whorls of flower organs. In a recent experiment using and carpels, and also bear indeterminate flowers with reiterating
double mutants of ag and an ap2 allele, which is insensitive to sepals and petals (Figure 11), suggesting that AG is important for
repression by miR172, it was shown that both AG and miR172 floral meristem determinancy (see section 3.2), besides its role in
independently downregulate AP2, but miR172 is more important stamen and carpel identity (Yanofsky et al., 1990; Mizukami and
than AG (Zhao et al., 2007). ap2 mutants rarely develop petals Ma, 1997). The regulation of AG has been much studied; at least
and their sepals are transformed into carpelloid structures due to ten proteins repress and five activate it to maintain its expression
ectopic AG expression (Figure 11), which is negatively regulated in the appropriate whorl (Figures 12 and 13A).
by AP2 itself (Drews et al., 1991). AP2 is also implicated in the AG is repressed by a transcriptional co-repressor complex
upregulation of the B genes, AP3 and PI (Zhao et al., 2007). formed by LEUNIG (LUG) and SEUSS (SEU) (Figure 15; Franks
The B class genes (AP3 and PI) are expressed in the second et al., 2002). LUG encodes a transcription protein similar to TUP1
and third whorls and mutant flowers of any or both of these two from yeast and interacts with SEU, which encodes a plant spe-
genes lack petals and stamens, as predicted in the ABC model cific protein (see Table S1; Conner and Liu, 2000; Franks et al.,

A B C D
B B B
A C C A C A
WT SEP ap2 SEP pi SEP ag SEP
E F G H
ap2 pi C ap2 ag B ap3 ag A ap2 pi ag

SEP SEP SEP SEP
Figure 11. Arabidopsis ABC homeotic floral mutants.
Photos of single, double and triple ABC gene mutant flowers. Each photo is accompanied by a small diagram where rectangles represent the A (AP1 and
AP2), B (AP3 and PI), and C (AG) combinatorial transcriptional regulatory functions and the SEP (1, 2, 3, 4) genes active in these mutants. Organs are
listed below from the outer to the inner whorl unless stated otherwise.
(A) Wild-type (WT) flower.
(B) Single ap2 mutant flower composed of carpelloid sepals, stamens, stamens and carpels.
(C) The pi mutant has flowers composed of sepals, sepals, carpels and carpels.
(D) The ag flower has the stamens transformed into petals and the carpels are replaced by another flower repeating the same pattern.
(E) The ap2 pi double mutant displays flowers composed only of sepalloid carpels.
(F) The ap2 ag flowers have leaf-like organs in the first and fourth whorls and mosaic petal/stamen organs in the second and third whorls.
(G) ap3 ag double mutants produce flowers composed of repeated whorls of sepals.
(H) The ap2 pi ag mutant has leaf-like organs with some residual carpel properties. (Photographs provided by Dr. J. Bowman).
2002; Sridhar et al., 2004). Neither of these proteins are able to el by several ENHANCER OF AG-4 (HUA) and HUA ENHANCER
bind DNA sequences and AP1 and SEP3 recruit SEU/LUG to (HEN) genes. All of these genes play a major role in pre-mRNA
the second intron of AG to perform their inhibitory function and processing of AG (Cheng et al., 2003).
prevent the ectopic expression of AG (Sridhar et al., 2006). Re- The ABC proteins exert their regulatory function as multimers.
cently, another transcriptional repressor of AG was identified, In Antirrhinum majus, a ternary complex between A and B func-
LEUNIG_HOMOLOG (LUH). This gene is the closest homolog of tion proteins was found to bind CArG DNA boxes more efficiently
LUG and its inhibitory function on AG is completely dependent on than single proteins (Egea-Cortines et al., 1999). More specifical-
SEU (Sitaraman et al., 2008). ly, a higher-order complex consisting of SQUAMOSA (SQUA, the
Another repressor of AG is BELLRINGER (BLR), a homeodo- AP1 ortholog), DEFICIENS, and GLOBOSA (DEF and GLO are
main protein that binds to regions in the second intron of AG and A. majus AP3 and PI orthologues, respectively) bound DNA more
prevents ectopic AG expression in the two outer whorls of the efficiently than DEF/GLO or SQUA alone (Egea-Cortines et al.,
flower (see Table S1; Bao et al., 2004). AG is also negatively regu- 1999). These results suggest that transcriptional complexes that
lated epigenetically by a histone acetyltransferase GCN5 (Ber- combine A and B function proteins are more stable than those
trand et al., 2003). Other genes that participate in floral organo- formed with proteins of any one function alone.
genesis are repressors of AG, namely RABBIT EARS (RBE, see The fact that the ABC genes are necessary but not sufficient
section 3.4.4), ANT and STERILE APETALA (SAP) (see Table to determine floral organ identity was later confirmed in Arabidop-
S1). AG is also positively regulated at the post-transcriptional lev- sis. Honma and Goto (2001) used a yeast three-hybrid method to

show that SEP3 and AP1 are able to interact with the heterodimer
AP3/PI but not with AP3 or PI alone. Moreover, they described
2 this interaction as essential, since the heterodimer AP3/PI lacks
FM the activation domain necessary for a transcription factor to func-
tion, a domain which both SEP3 and AP1 possess (Honma and
IM Goto, 2001). These findings suggest that the inclusion of SEP3
4 or AP1 together with AP3/PI could result in an active tetrameric
se 2 transcriptional complex (Figure 14). It was also demonstrated that
1 the ABC proteins on their own or combined according to the ABC
ad 3 model (A, AB, BC, or C) were not sufficient to determine floral
organs when expressed in leaves under the action of the 35S
ab
constitutive promoter (Pelaz et al., 2001). However, floral organs
5 could indeed be recovered in leaves once appropriate combina-
tions of genes were expressed (Honma and Goto, 2001; Pelaz et
2 al., 2001).
The SEP genes received their names because the floral or-
FM
gans that develop in all four whorls in triple sep mutants resemble
IM sepals (Pelaz et al., 2000). This sep1 sep2 sep3 triple mutant
4 phenotype is markedly similar to that of double mutants that lack
se 2 both B and C class activity, such as pi ag and ap3 ag (Figure 11G;
1 Bowman et al., 1989; Pelaz et al., 2000) in which the floral meri-
ad 3 stem becomes indeterminate as well. Single or double mutants
for these SEP genes yield flowers indistinguishable from wild
ab
type, thus suggesting that the three SEP genes are functionally
5 redundant and are important in determining three of the four floral
organs: petals, stamens, and carpels (Honma and Goto, 2001;
2 Pelaz et al., 2001; Robles and Pelaz, 2005).
Given that the triple sep1 sep2 sep3 mutant does not show
FM
alterations in sepal identity, an additional gene is likely to be in-
IM volved in sepal specification. Indeed, another SEP-like MADS-
4 box gene, SEP4 (previously AGL3), has now been character-
se 2 ized (Ditta et al., 2004), and the quadruple sep1 sep2 sep3 sep4
1 mutants produce flowers with leaf-like organs in all whorls, thus
ad 3 confirming the SEP genes contribute to each floral organ identity
(Figure 14). Coincidently, SEP genes are expressed in the whole
ab
floral meristem during flower development (Figure 13B; Flanagan
5 and Ma, 1994), are important in regulating B and C gene expres-
sion (Liu et al., 2009), and encode proteins that apparently inter-
act with the ABC proteins (Figure 14; Robles and Pelaz, 2005).
2
Based on data from Antirrhinum and yeast two-hybrid and
FM
three-hybrid protein interactions, and on the phenotypes of the
ABC mutants, three models have been proposed to explain how
IM
4 the MADS domain proteins interact to constitute functional tran-
2 scriptional complexes and bind DNA. None of the models com-
se
1 pletely explains the experimental data available, but the quartet
ad 3 model seems the most plausible (Jack, 2001; de Folter et al.,
2005). This model proposes that MADS domain proteins form
ab tetrameric complexes during floral organ determination (Figure
5 14; Theissen, 2001; Theissen and Saedler, 2001; Becker and
Theissen, 2003; Jack, 2004). Within each transcriptional com-
plex, there would be two MADS dimers, each one binding to a
Figure 12. Expression patterns of the ABC genes during early stages of single CArG-binding site causing the DNA of the promoter re-
Arabidopsis flower development. gion to bend, enabling the MADS dimers to act cooperatively in
SEM of meristems have been colored to show expression patterns of A a tetrameric complex to regulate the gene. For example, binding
class (red, outer whorls), B class (yellow, petal and stamen primordia) of one dimer within the tetramer to DNA could increase the af-
and C class (blue, inner whorls) genes. Five flowers at early stages of finity of the second dimer for local DNA binding (Melzer et al.,
development are marked 1 to 5 (5 being the oldest). Inflorescence shoot 2009). Besides, one of the dimers could function as the activa-
apical meristem (IM), floral meristem (FM) and sepals (se): adaxial (ad) tion domain of the tetramer allowing for efficient transcriptional
and abaxial (ab) are indicated. (Photographs provided by Dr. J. Bowman). activation (Honma and Goto, 2001). Interestingly, several dimers

A Stage 1-2 3 4 5 6
FM se se ca
se se st
AP1
pe
AP3 + PI
AG
B Stage 1-2 3 6
FM
SEP1
SEP2
SEP3
SEP4
Figure 13. Diagram illustrating mRNA expression patterns of Arabidopsis ABC and SEP genes during different stages of flower development.
(A) ABC gene expression patterns illustrated from stage 1 to 6. The A function gene AP1 is expressed (red) in the two outer floral primordia whorls that will
later develop into sepals (se) and petals (pe) (Mandel et al., 1992; Gustafson-Brown et al, 1994; Parcy et al., 1998). The A function gene AP2 is expressed
in all four whorls of the flower (see figure 10; Jofuku et al., 1994). The B function genes (dark yellow) AP3 and PI are expressed from stage 3 in the next two
inner whorls of the flower (Weigel and Meyerowitz, 1993; Parcy et al., 1998). Interestingly PI is also expressed at stages 3 and 4 in cells that will generate
the fourth whorl (light yellow). After stage 5, the pattern of PI expression largely coincides with that of AP3, only in petal and stamen (st) primordia (Goto
and Meyerowitz, 1994). The C function gene AG is expressed (blue) in the two inner whorls that will become the stamens and carpels (ca) (Yanofsky et
al., 1990; Gustafson-Brown et al., 1994; Parcy et al., 1998; Ito et al., 2004).
(B) SEP gene expression pattern during several stages (1 or 2, 3 and 6) of flower development. SEP1 and SEP2 are expressed in all whorls of the flower
(Savidge et al., 1995). SEP3 is first detected in late stage 2 flower primordia and afterwards in petal (pe), stamen (st), and carpel (ca) primordia. The ex-
pression pattern at stage 6 was deduced that from at stage 7 (Mandel and Yanofsky, 1998). SEP4 is weakly expressed in sepal primordia at stage 3 and
strongly expressed in carpel primordia from stage 3 to 6. (Ditta et al., 2004). Both figures have been modified and expanded from Krizek and Fletcher (2005).

genes having cell-specific roles are turned on especially during

SEP
stamen and carpel development that is much more complex than
SEP
A perianth development (Sablowski, 2009).
A
The first examples of genes regulated by the ABC genes were
sepal
two MADS-box genes, AGL1 and AGL5 (renamed the SHAT-
TERPROOF genes (SPH1 and 2, respectively). There is virtu-
ally no expression of either gene in ag mutants (Savidge et al.,
SEP 1995). SHP2 is only expressed in carpels and AG can activate
C
SEP an SPH2:GUS reporter construct; furthermore, AG binds its pro-
C moter in vitro (Savidge et al., 1995).
The first non-ABC gene identified as a target of a MADS A,
carpel B or C protein was NAC-LIKE ACTIVATED BY AP3/PI (NAP),
SEP
C a target of the AP3/PI complex. It is important for the transition
B
between cell division and cell expansion during petal and sta-
B
men development (see section 3.4.5 and Table S1; Sablowski and
stamen
Meyerowitz, 1998).
B A recent study identified two genes negatively regulated by
A PI/AP3 AP3/PI, GATA NITRATE INDUCIBLE, CARBON-METABOLISM-IN-
SEP A C
B API/AP2 AG VOLVED (GCN) and GCN-LIKE (GNL), a GCN paralog (Mara and
B
SEP Irish, 2008). Both genes regulate chlorophyll biosynthesis in plant
1, 2, 3, 4
petal cells. Thus, their downregulation could be important in preventing
chlorophyll accumulation in petals and anthers. The same study
Figure 14. Schematic representation of the interaction of ABC and SEP shows that both GNC and GNL, together with the B class genes,
proteins in the quartet model for Arabidopsis floral organ specification. regulate the expression of a number of other GATA-motif-containing
target genes like HEXOKINASE1 (HXK1; Mara and Irish, 2008).
Possible MADS-domain protein complexes (circles) of the ABC model are SUPERMAN (SUP; Bowman et al., 1992) is upregulated by
sufficient for the specification of each of the four floral organs. In the ABC
AP3/PI and AG and by LFY (Riechmann et al., 1996; Sakai et
model, rectangles represent the A (AP1 and AP2), B (AP3 and PI), and C
al., 2000). SUP encodes a transcription factor with a C2H2-zinc
(AG) combinatorial transcriptional regulatory functions necessary for se-
finger motif and is involved in the maintenance of the stamen/car-
pal, petal, stamen and carpel primordia specification. The green rectangle
pel whorl boundary (Sakai et al., 2000; Dathan et al., 2002; see
below represents the SEP (1, 2, 3 and 4) proteins that interact with pro-
section 3.4.2). While the B genes and LFY seem to regulate early
teins encoded by the ABC genes (unknown for AP2 which has not been
tested) to specify each floral organ (modified from Bowman et al., 1993;
SUP expression, AG and the B genes are involved in maintaining
Robles and Pelaz, 2005). its expression in flowers from stage 5 onward (Sakai et al., 2000).
Recent microarray experiments have proved useful in reveal-
ing new targets of the ABC MADS homeotic genes, as well as
many putative components of the complex networks involved in
and potential tetramers have been documented in a complete floral organogenesis. For example, it was shown that the AP3/PI
Arabidopsis MADS-domain family protein-protein interactome dimer regulates, directly or indirectly, 47 target genes. Only two
via yeast two-hybrid interactions (de Folter et al., 2005). This of these are transcription factors, while most participate in basic
data base has been updated with a yeast three-hybrid screen cellular functions required for stamen and petal development (Zik
for MADS-domain proteins (Immink et al., 2009). Future stud- and Irish, 2003a). By contrast, AG controls, directly or indirect-
ies should test which of the complexes inferred from the MADS ly, the expression of 149 genes most of which are transcription
interactomes are functional and what their roles are during Ara- factors, including other members of the MADS-box gene family.
bidopsis development. Ten of these were also shown to be direct targets of AG using
chromatin immunoprecipitation (ChIP), including AG itself, AP3,
CRC and ATH1, a gene that encodes a BELL-type homeodomain
3.3.1 Target genes of the ABCs protein that participates in the development of the basal region of
shoot organs (Gómez-Mena et al., 2005).
Target genes of the ABC genes link the floral organ specification A more exhaustive experiment used four homeotic mutants
- gene regulatory network (FOS-GRN) with processes in organ (ap1/ap2, ap3, pi and ag) in two types of microarray assays: a
primordia establishment and development (for review of MADS whole genome microarray with approximately 26,090 gene-spe-
target genes, see de Folter and Angenent, 2006). Among the cific oligonucleotides and a flower specific-cDNA microarray with
direct targets of the ABC genes, transcriptional regulators and 5,000-6,000 genes. To summarize the assay results, transcription
hormone-related genes are prominent (Sablowski, 2009). But the factors were neither over or underrepresented as being regulated
sets of target genes change as organ development progresses; by the ABC genes; on the contrary, genes involved in general
at later stages of floral organ development, several components cellular maintenance (DNA recombination and protein synthesis)
of what could be common modules have been found that are in- were underrepresented. Genes specifically expressed in each of
volved in generic developmental processes (see below) during the four different whorls were identified: 13 genes for sepals, 18
sepal, petal, stamen and carpel development. Finally, multiple for petals, 1162 for stamens, and 260 for carpels. As expected

from their structural and cellular complexity, the reproductive floral Regulatory modules controlling distinct components of floral
organs had many more specific target genes than the perianth organ development have been elucidated to different extents de-
organs (Wellmer et al., 2004; Sablowski, 2009). pending on available mutant phenotypes. In correlation with ana-
In another genomic study of early floral stages it was found tomical and morphological complexity, the size and complexity of
that many genes were downregulated in incipient floral primordia the regulatory modules underlying stamen and carpel develop-
while many of them were activated during the differentiation of ment are much greater than those that regulate sepal or petal
floral organs (Wellmer et al., 2006). However, some genes were development. Carpel development is covered in the “Fruit Devel-
overrepresented during all stages analyzed (i.e. transcription fac- opment” chapter (Roeder and Yanofsky, 2006) in this book, and is
tors including the family of MADS-box genes, PIN dependent aux- only briefly considered here.
in transport genes, as well as auxin and GA metabolism genes). In the flower meristem, normal organogenesis depends upon
Even though the MADS box genes were overrepresented, the pro- a homeostatic equilibrium between stem cell specification and
moter regions of the genes expressed during these different stag- cellular differentiation (Green et al., 2005). Plant morphogenesis
es are not enriched in CArG-box sequences compared to random is influenced both by the orientation and rate of cell division, as
samples from the whole genome. This result suggests that MADS- well as by cell expansion and differentiation (see section 2 for
domain transcription factors may be able to bind sequences other a description of floral organ initiation and morphogenesis). How
than CArG motifs, or that they have few direct targets during the the molecular aspects of these processes are coordinated has
developmental stages analyzed (Wellmer et al., 2006). been very difficult to elucidate. However, it is generally accepted
In a different approach, an inducible post-translational version that cells in meristematic regions respond to positional informa-
of AG was used in gene expression profiling to detect AG target tion important for inducing and controlling morphogenesis (Sus-
genes. One of the genes identified that is upregulated by AG is sex, 1954; 1955; Meyerowitz 1997; Hauser et al., 1998). One of
SPOROCYTELESS (SPL). AG is able to bind in vitro to the 3’ re- these positional signals is auxin (see Section 3.4.1; Reinhardt
gion (downstream of the stop codon) of the SPL gene (Ito et al., et al., 2000; Benková et al., 2003; Reinhardt et al., 2003; de
2004). SPL has been described as a key regulator of sporogenesis Reuille et al., 2006). Several mutations that affect the number,
later during stamen and carpel development (see sections 3.4.5, size, and/or shape of one or several floral organs have also been
3.4.6 and Table S1; Schiefthaler et al., 1999; Yang et al., 1999). characterized. Some of these phenotypes are pleiotropic conse-
quences of mutations in genes acting from earlier steps of plant
and flower development. Others are the result of alterations in
3.4 Floral Organogenesis organ specific genes (Figures 16-17). An extensive list of genes
involved in flower organ morphogenesis with their inferred func-
The challenge of inferring the topology of the gene regulatory net- tions, mutant phenotypes and mRNA expression patterns is
work (GRN) underlying the establishment of floral organ primor- given in Table S1.
dia, and their development (cell differentiation, morphogenesis
and growth) is still ahead. However, some key components and
GRN functional modules characterized to date are summarized 3.4.1. Floral meristem and organ primordia positioning: the
in Section 4. Such modules involve several functional feedback role of auxin
loops and underlie different generic developmental processes
mainly: primordia type specification; delimitation; floral organ The shoot apical meristem produces leaves and then flowers in
primordia positioning that depends on fundamentally on auxins; a highly predictable and regular phyllotactic pattern (Tanaka et
primordia number; inter-whorl and within-whorl boundaries; and al., 2006). One of the key compounds that regulate this devel-
primordia and organ adaxial-abaxial polarity (Figures 2C and 15; opmental process is the hormone auxin (Reinhardt et al., 2000).
Irish, 2008). At later stages of floral organ development, subcellu- Increased auxin levels mark the initiation sites for organ primordia
lar differentiation and patterning, as well as overall organogenesis (including those of floral organs) and local application of auxin
takes place and more specific regulatory modules are involved. is sufficient to trigger leaf or flower formation in the shoot apex
The genes within such modules are treated separately for each (Reinhardt et al., 2000; Tanaka et al., 2006). Once the primordium
organ type (Figures 16-17). is established, there is a depletion of auxin around it and another
As a precursor to integrating GRN modules in the above cate- peak of auxin is only able to form in cells at a specific distance
gories, we now provide a synthesis of the molecular genetic stud- from pre-existing primordia, generating a phyllotactic pattern (Re-
ies of how such generic developmental processes are regulated. inhardt et al., 2000; Reinhardt et al., 2003; de Reuille et al., 2006;
Several of these have also been identified as important regulators Tanaka et al., 2006; Berleth et al., 2007; Kuhlemeier, 2007). After
of leaf development, substantiating the proposal of Goethe that all initiation, the primordium grows by cell proliferation and cell ex-
plant organs are elaborations or modifications of a core leaf-like pansion, and the organ differentiates along the apical-basal and
developmental program (for review of common pathways see Sa- dorsal-ventral axes (Heisler et al., 2005; Golz, 2006).
blowski, 2009). ABC floral organ identity genes are also important The overall distribution of auxin depends on its biosynthesis,
in fine-tuning or coordinating the role of genes involved in some metabolism, and directional transport. Most auxin is synthesized
of the generic developmental modules during flower development in young tissues of the shoot and distributed throughout the plant
(Figure 15; Sablowski, 2009). Some genes participate in more by two physiologically distinct pathways. One of them is passive
than one process or module and are important for making con- and occurs only by diffusion through the mature phloem. The oth-
nections between different GRN modules. In such cases, they are er one is an active polar auxin transport (called PAT) that medi-
considered in more than one category. ates cell-to-cell movement of auxin through two different types of

Primordia positioning PHV Primordia polarity

REV
PIN1 AUXIN
AUX1 PHB Adaxial
LAX
PGP19 AXR1
ARFs AS1 JAG Margin
KAN1-4 NUB expansion
ARGOS
AGO1
YAB1-6 Abaxial
ANT
GA STM CLV3 LFY

PAN UFO
CUC1-3 CK CLV1
LUG
ARR WUS AG
SEU
AP1 AP3
AP2 PI
SUP
Primordia identity & growth

B
A C
Sepal Petal Stamen Carpel
Figure 15. Functional gene regulatory modules during early flower development.
Common molecular modules act during early meristem morphogenesis from the SAM both before and after reproduction. During floral organogenesis,
these modules interact among themselves and with the FOS-GRN that includes the floral homeotic genes. Anlagen positioning in the SAM flanks depends
on auxin gradients. Transport and signal transduction proteins, as well as other factors regulated by auxins (letters in blue), participate in the establish-
ment of such gradients and finally determining the position of primordia. The auxin pathway also downregulates some members of the NAC family (CUC1
to 3 are important for organ boundary establishment), which also participate in the positive regulation of STM and KNOX genes. Since WUS maintains
the apical meristem stem cells in a proliferating state with CLV proteins that in turn regulate its expression in a non cell-autonomous negative-positive
feedback loop, and STM prevents meristem cell differentiation by inducing the production of cytokinins (CK) and the ARR transduction pathway (see text),
floral primordia may emerge if cells in the anlagen are able to downregulate STM. This can be achieved by the action of AS1 and ANT. Upregulation of
LFY by the flowering genes (Section 3.2; Figure 9) in conjunction with some KAN and YAB proteins, activate the expression of ABC homeotic genes (in
red) for the establishment of the floral organ primordia identity and growth (gene acronyms in black, see text and Table S1 for full names). Lateral organ
primordia acquire apical/basal, lateral/medial and adaxial/abaxial polarities by the action of protein families that include PHABs (PHB, PHV and REV),
KANs (KANADI1-3, ATS/KAN4), YABs (FIL/YAB1, YAB2, YAB3, INO/YAB4, YAB5 and CRC/YAB6), JAG and NUB (letters in green). Some of these are
organ-specific while others are shared by different floral organ primordia (see section 3.4). Not all the genes involved in each module are depicted, just
some of the most representative ones, which help us to understand how they are interconnected. Arrows and bars indicate positive and negative regulatory
interactions, respectively, and dashed lines a postulated interaction not yet proven. The text color used for the gene names in each module is the same as
in Figures 16, 17, and 19 where specific organ developmental processes are summarized and the ABC genes are shown in boxes on the organ specified
as in the model shown below. Hormones are in purple.
This figure was composed partially from information in Clark (2001b), Blazquez et al. (2006), Hord et al. (2006), Shani et al. (2006) and Feng and
Dickinson (2007).

Petal development
carpel
carpel primordia
primordia stamen
sepal primordia
petal
primordia
Flower stage 5 Flower Stage 6 Flower Stage 12
PETAL PRIMORDIUM FORMATION EARLY PETAL DEVELOPMENT LATE PETAL DEVELOPMENT
FFO ?
EEP1 CUC1/2 cell proliferation ? FRL1
PHB PAN TGA factors
SEU ROXY1 BLR
FIL/YAB1 AG
LUG ANT
SAP
RBE
AS1/2 PTL
JAG cell expansion &
LFY NAP differentiation
UFO AP3
At4g30270
AP1 PI
GNC
GNL
HXK1
Figure 16. Main stages of petal development and some genes involved.
Schemes at the top illustrate three different stages of petal development (for details see section 2). Briefly, GRN modules (genes) in petal development
include those involved in the establishment of the second whorl domain, the specification of petal identity and cell differentiation. CUC genes under the
regulation of miR164c are involved in establishing whorl boundaries. Genes involved in polarity determination like JAG, PHB and YAB1 are also necessary
for petal development. A, B and SEP genes, and the absence of C genes, determine petal identity (AP2 and SEP genes are not shown here for clarity;
see Figures 11 and 14). Petal blades are formed by active cell division at early developmental stages and by cell enlargement and differentiation at later
stages. Some of the genes expressed early need to be continuously expressed throughout petal growth, including ROXY1, SEU, and LUG. Downregulation
of the GNC, GNL, and HXK1 genes inhibits chlorophyll accumulation and expression of photosynthetic genes. At4g30270 might be necessary for correct
cell wall dynamics during petal growth (see text section 3.4.5 and Table S1 for details; Franks et al., 2006; Irish, 2008;). Gene color code as in Figure 15;
arrows and bars indicate positive and negative regulatory interactions, respectively.
proteins, efflux and influx carriers. Some of the genes that encode ers. PIN1 expression is induced by auxin and it encodes a protein
these transporters (or carriers) have been cloned: PIN-FORMED with 10-12 putative transmembrane domains and shares some
(PIN) and P-GLYCOPROTEINS (ABCB/PGP) for auxin efflux, similarity with bacterial transporters (Gälweiler et al., 1998). pin1
and AUXIN1 (AUX1) and its paralogs LIKE-AUX1 (LAX1-3) for mutant plants accumulate high amounts of auxin in vegetative
auxin uptake/influx (Figure 15; Bennett et al., 1996; Friml, 2003; meristems and a deficiency in the apical inflorescence meristem,
Yang et al., 2006; Bandyopadhyay et al., 2007). which results in a defective organ initiation of leaves and flowers,
The PIN gene family encodes eight protein members in total; a phenotype that can be imitated in wild type using auxin efflux
three of them (PIN5, 6, and 8) of unknown function. All of the PIN inhibitors (Okada et al., 1991; Reinhardt et al., 2000). Of the other
proteins characterized until now are asymmetrically distributed on PIN proteins, only pin3 and pin7 loss-of-function mutants have
the plasma membrane and some of them can be found in specific flowers, and these bear fused petals, no stamens, and occasion-
cell types with no pronounced polarity (Vieten et al., 2007). The ally no sepals (Benková et al., 2003). PIN3 is essentially involved
direction of auxin flow is believed to be determined by the asym- in mediating differential shoot growth (Friml et al., 2002) and PIN7
metric cellular localization of PIN proteins (Friml, 2003). The first is important during early embryo development (Friml et al., 2003).
of these proteins to be characterized was PIN1, and its mutation Auxin movement mediated by PIN carrier proteins determines
(pin1) results in pin-shaped inflorescence meristems without flow- the growth axis of the developing organ by establishing an auxin

Anther development
carpel vasculature
epidermis Tds
carpel
primordia stamen
sepal
primordia stomium PG
Ar
region
petal anther stage 2 anther stage 7 anther stage 14
primordia
flower stage 5 flower stage 6-7 flower stage 13-14
2oP (outer) Endothecium

1oP Middle layer
2oP (inner)
Ar Tapetum
1oSp MMC Microspores
STAMEN PRIMORDIUM ARCHESPORIAL CELLS MEIOSIS I AND II ANTHER STAGE 14
miRNA
ETT BAM1/2
ANT MYB33/65 DIF1
AG SPL/NZZ EMS1/EXS DYT1 NST
MS1
AP3-PI AMS ARF6
SEP TPD1 GNE1/2 ARF8
SERK12 FAT TAPETUM
ROXY1/2
Archesporial initiation microsporangium formation
Figure 17. Stages of stamen development with emphasis on the genes implicated in anther formation.
Schemes of some stages of flower development showing representative stages of anther cell differentiation (Sanders et al., 1999) are shown at the top.
At stage 1 of anther development and microspore formation, rounded stamen primordia emerge with three cell layers, L1, L2 and L3. During stage 2, the
archesporial cells (Ar) arise in the four “corners” of the L2 layer and the epidermis in the L1. Before meiosis the Ar cells divide and generate the primary
parietal layer (1oP) and the primary sporogenous layer (1oSp). The 1oP then divides into two secondary parietal layers (outer and inner, 2oP). The outer
layer gives rise to the endothecium, the inner cells to the middle layer and the tapetum. 1oSp produces the microspore mother cell (MMC) that undergoes
meiosis and gives rise to the microspores (Alves-Ferreira et al., 2007). At stage 7, meiosis is completed and the four locules carrying tetrads (Tds) of
microspores are seen. At stage 14, cells shrink and the anther dehisces liberating the pollen grains (PG; Sanders et al., 1999). Some of the known genetic
interactions important during anther development are shown in purple. AG (in red) induces the expression of SPL (the first gene known to be committed to
anther development); later during microsporangium formation the action of the EMS, DYT, MS1 and AMS genes is also indispensable (Feng and Dickinson,
2007). See section 3.4.6 for further explanation and Figure 15 for gene color code. Arrows and bars indicate positive and negative regulatory interactions,
respectively, and dashed lines possible indirect interactions.
gradient with its maximum at the tip (Benková et al., 2003; Tanaka are important in maintaining long-distance auxin transport (Ti-
et al., 2006). As the primordium rapidly expands, auxin is depleted tapiwatanakun et al., 2009). One of the PGP proteins (PGP19)
from the tip. Two hypotheses have been proposed to explain this co-localizes and interacts with PIN1 and the ABCB protein is ap-
observation: either auxin is transported through the primordium parently important in stabilizing plasma membrane microdomains
interior into the vascular network (Benková et al., 2003; Tanaka necessary for enhanced PIN1 activity (Bandyopadhyay et al.,
et al., 2006) or it is depleted from primordial regions as a result of 2007; Titapiwatanakun et al., 2009).
specific reversals in PIN1 polarity (Heisler et al., 2005). Auxin enters the cell passively by simple diffusion and also by
The ABCB/PGPs are also transmembrane proteins that be- the import activity of AUX1 and related LAX proteins. The AUX1
long to the ATP-binding cassette (ABC) transporter superfamily. gene encodes a protein with 11 putative transmembrane domains
In Arabidopsis, three of their members, ABCB1, ABCB4, and (Hobbie, 2006) similar to plant amino acid permeases (Bennett
ABCB9, are able to transport auxin away from apical tissues and et al., 1996). The mutant form (aux1) was identified in a screen

for auxin resistant and agravitropic mutants (Bennett et al., 1996; 1999; Brand et al., 2000; Doerner, 2000). Mutations in genes that
Vieten et al., 2007). The AUX1 protein also has polar subcellular control cell proliferation in the SAM, such as the CLV genes, are
localization in some cells and co-localizes with PIN1 in the shoot similar to ULT in that they have larger SAM and primordia (Fletch-
apical meristem. AUX1/LAX function could be essential for stabi- er, 2001; Carles et al., 2004) and WIGGUM (WIG; Running et al.,
lizing the phyllotactic pattern. The proposed model for AUX1/LAX 1998).
function is that these proteins concentrate auxin in the cytoplasm When WUS is repressed and the number of cells for floral pri-
of cells of the L1 layer, preventing auxin diffusion in the apoplast mordia formation is reduced, organ architecture is compromised
(Bainbridge et al., 2008). suggesting that there is a threshold number of cells required to
PINOID (PID) encodes a Ser/Thr protein kinase (Christensen form a normal organ (Weiss et al., 2005). In fact, the loss of or-
et al., 2000) which has been implicated to function in redirecting gans observed in A-function mutants, or any other AG repressor
subcellular PIN polarities, because the loss of its activity causes mutant could be explained as a result of premature repression
a shift in apical-basal PIN polarity (Friml et al., 2004; Berleth et of WUS by AG in these organs (Crone and Lord, 1994; Liu and
al., 2007; Michniewicz et al., 2007). pid mutants have a defect in Meyerowitz, 1995; Laux et al., 1996).
organ formation similar to that of pin1, but they do produce a few Other mutants that have altered floral organ numbers are pan
flowers (Reinhardt et al., 2003) with altered floral organ numbers (Running and Meyerowitz, 1996; Chuang et al., 1999), ett (Ses-
(more petals but fewer stamens) (Bennett et al., 1995). Recently, sions et al., 1997) and sup (Jacobsen and Meyerowitz, 1997).
Michniewicz et al., (2007) reported that in vivo PIN1 phosphoryla- Both pan and ett have more sepals and petals and fewer sta-
tion is directly dependent on the kinase PID and a phosphatase mens, whereas sup produces more stamens at the expense of
PP2A, which may act directly by dephosphorylating PIN1 or in- carpels (Weiss et al., 2005). Double pan sup mutants however
directly through PID. This phosphorylation status determines the have an attenuated sup phenotype in the fourth whorl, probably
intracellular apical-basal localization of PIN1 and therefore auxin because in this mutant AG is downregulated and the domain of
transport-dependent development. PIN1 is targeted to the apex expression of WUS is expanded (Das et al., 2009).
when it is phosphorylated and to the base when it is dephos- The PAN gene mutation specifically alters floral organ num-
phorylated (Michniewicz et al., 2007; Vieten et al., 2007). ber, yielding fertile plants with a pentamerous meristic pattern
Accumulation of auxin activates downstream processes (Running and Meyerowitz, 1996). PAN encodes a member of
through specific receptors and the combinatorial action of mem- the bZIP class of transcriptional regulators (Chuang et al., 1999)
bers of two large families of transcription factors, AUXIN RE- and is thought to act in the process by which cells assess their
SPONSE FACTORS (ARF) and IAA/AUX (Kuhlemeier, 2007). position within the developing floral meristem. This gene may
The Aux/IAA proteins are degraded when the levels of free auxin affect the switch that commits floral organ primordia cells to en-
rise, resulting in derepression of ARFs. ETTIN (ETT)/ARF3 has a ter an organ initiation program (Running and Meyerowitz, 1996).
dynamic role in patterning by acting in specific cells within floral PAN and WUS expression overlaps and in clv mutants both
meristems and reproductive organs. At early stages, ETT func- genes are ectopically expressed (Chuang et al., 1999; Maier et
tions in determining the number of organ primordia, whereas later al., 2009). WUS overexpression causes PAN overexpansion too
it is involved in the outgrowth and patterning of tissues within or- suggesting that this gene is positively regulated by WUS (Maier
gan primordia (Figure 15; Sessions et al., 1997). ett mutant plants et al., 2009).
show altered flower development; some flowers have missing pet- Interestingly, pentameric symmetry is characteristic of flow-
als and rudimentary radialized stamens, and others have normal ers in early-diverging angiosperm lineages, thus suggesting that
fertile stamens, but radialized petals (Pekker et al., 2005). ETT is PAN may have been involved in changes to meristic patterns
also involved in prepatterning apical and basal boundaries in the during angiosperm diversification; particularly the evolution from
gynoecium primordium (see Table S1; Sessions and Zambryski, pentamerous to tetramerous flowers in the Brassicaceae lineage
1995; Sessions et al., 1997). MONOPTEROS (MP)/ARF5 mu- (Chuang et al., 1999).
tants (mp) have inflorescences with smaller or absent flowers, Organ size is also regulated by the same components in all
similar to pin1 mutants (Przemeck et al., 1996). whorls. The ANT gene encodes a transcription factor of the AP2
family, which seems to be a general regulator of organ size during
organogenesis (Elliott et al., 1996; Klucher et al., 1996; Krizek,
3.4.2. Floral organ primordia number, size, and boundaries 1999; Krizek et al., 2000; Mizukami and Fischer, 2000). The over-
expression of ANT causes increased cell division in sepals and
In Arabidopsis, which is a self-fertilizing (autogamous) and par- increased cell expansion in the inner three whorls, probably af-
tially cleistogamous (before flower bud opens) plant, floral organ fecting both the rate and duration of cell divisions which are im-
size might not be under strong evolutionary pressure compared portant determinants of the final size of lateral organs (Krizek,
to allogamous species. However, it has been an important model 1999; Mizukami and Fischer, 2000; Weiss et al., 2005). ARGOS
to study genes that control size and architectural traits of flowers participates in the same transduction pathway as ANT and acts
(Weiss et al., 2005). downstream of AUXIN RESISTANT 1 (AXR1). Interestingly, in-
Several mutations that affect meristem size and maintenance creased organ size observed in ARGOS overexpression lines is
lead to alterations in flower organ number or size. Mutations in due to an extended period of cell division rather than to an in-
the CLV genes (Clark et al., 1993 and 1995; Kayes and Clark, crease in growth rate (Hu et al., 2003; Weiss et al., 2005). So, it is
1998) cause an increase in meristem size, thus yielding addi- plausible to assume that these two genes (and probably others)
tional whorls and a change in floral organ number with altered affect organ size by transducing signals from plant growth regu-
phyllotaxis (Clark et al., 1993; Clark et al., 1997; Fletcher et al., lators, such as auxin, which is a key player in establishing SAM

primordia and a general regulator of cell proliferation and expan- the KANADI genes (Eshed et al., 2001; Kerstetter et al., 2001),
sion (Figure 15). whereas adaxial cell fate is determined by members of the PHAB
ANT also participates in defining abaxial-adaxial organ polar- family: REVOLUTA (REV), PHABULOSA (PHB), and PHAVOLU-
ity in combination with FILAMENTOUS FLOWER/YABBY1 (FIL/ TA (PHV) (McConnell et al., 2001; Emery et al., 2003; reviewed in
YAB1; Nole-Wilson and Krizek, 2006; see next section) and thus Bowman et al., 2002; Zik and Irish, 2003b; Golz, 2006) together
may be one of the links between the modules controlling primor- with JAGGED (JAG) and NUBBIN (NUB) (Figure 15; Dinneny et
dia growth and the polarity establishment (Figure 15). al., 2004; Dinneny et al., 2006).
Ectopic expression of UFO (Levin and Meyerowitz, 1995) YABBY proteins (YAB) are transcription factors with a Zn-fin-
also causes increased floral organ size (Lee et al., 1997), due ger and a helix-loop-helix (YABBY) domain that are promoters
to increased cell division (Mizukami, 2001; Weiss et al., 2005). of abaxial cell fate in all lateral organs, among other functions
This pathway is regulated by UFO independently of its role in B (Bowman 1999; Sawa et al., 1999; Siegfried et al., 1999). Dur-
gene expression, because ectopic expression of the B genes ing flower development they participate in establishing the pri-
does not induce any increase in organ size, so missexpression mordium domain and meristem patterns, and later in maintaining
of other unknown UFO-dependent factors may account for this abaxial polarity (Siegfried et al., 1999; Goldshmidt et al., 2008).
phenotype (Ni et al., 2004). UFO and two gene enhancers of the FIL/YAB1, YAB2, and YAB3 are expressed in a polar manner in all
ufo- phenotype, FUSED FLORAL ORGANS 1 and 3 (FFO1 and lateral organs of the flower meristem, while CRABS CLAW (CRC/
FFO3), could also participate in establishing and maintaining or- YAB6) is only expressed in carpels and nectaries, and INNER
gan boundaries probably by affecting cell proliferation (Levin et NO OUTER (INO/YAB4) is restricted to outer integuments (see
al., 1998). section 3.4.6 and 3.4.7; Alvarez and Smyth, 1999; Bowman and
Morphological boundaries are established in the early stages Smyth, 1999; Villanueva et al., 1999).
of the formation of a primordium separating it from surrounding KANADI (KAN) genes encode transcription factors of the
tissues, and later from adjacent organ primordia (Figure 2C; Aida GARP family. KAN1, KAN2, and KAN3 have been implicated in
and Tasaka, 2006a). Cells in the boundary are distinctly narrow promoting abaxial cell fates (Eshed et al., 1999; Eshed et al.,
and elongated with low proliferation rates (Aida and Tasaka, 2001; Kerstetter et al., 2001). The kan1 mutant was selected as
2006b). Genes expressed in the boundary may affect both meri- a genetic enhancer of crc gynoecium phenotype, producing a
stem and organ development by upregulating cell differentiation mirror-image of adaxial tissues in the kan1 crc double mutant,
genes and downregulating meristematic genes (Borghi et al., indicating that both genes participate in a redundant manner to
2007). CUC1, 2, and 3 encode NAC-domain transcription factors promote abaxial identity (Eshed et al., 1999). In kan1 kan2 double
that promote morphological separation of lateral organs through mutants, all floral organs are also extremely adaxialized (Eshed
growth repression (Aida et al., 1997; Vroemen et al., 2003; Taoka et al., 2001; Kerstetter et al 2001). Although these KAN genes are
et al., 2004). cuc1 cuc2 double mutant seedlings have fused coty- not necessary for the activation of YAB genes, they are important
ledons with no shoots. However, when adventitious stems are in- in controling their proper abaxial localization (Eshed et al., 2001).
duced in this genotype, flowers have fused sepals and stamens, Even though KAN and YAB genes may have common targets,
fewer petals and stamens number, and reduced fertility (Aida et they also have different ones, since the phenotype of the fil yab3
al., 1997). CUC genes are epigenetically regulated (Laufs et al., double mutant is not quite the same as the extreme phenotype of
2004; Kwon et al., 2006). kan1 kan2 (Bowman et al., 2002).
Other genes, such as LATERAL ORGAN BOUNDARY (LOB) It has been hypothesized that the “default” state of cells is the
and JAGGED LATERAL ORGANS (JLO), members of the LAT- abaxial fate (Sussex 1954, 1955). Genes that belong to the PHAB
ERAL ORGAN BOUNDARY DOMAIN (LBD) gene family, encode family (class III homeodomain-leucine zipper, HD-ZIP III; Sessa
putative transcription factors with a leucine-zipper motif that are et al., 1998; McConnell et al., 2001; Golz, 2006) of transcrip-
also expressed in boundary cells (Shuai et al., 2002; Borghi et al., tion factors, like PHB and PHV, might be activated by a proxi-
2007). JLO along with the CUC genes probably coordinate auxin mal signal coming from the apical meristem. These cells that are
accumulation and loss of meristem-specific gene expression in programmed to yield the adaxial portion of the lateral organ, are
organ anlagen (Takada et al., 2001; Borghi et al., 2007). predicted to in turn haveYAB and KAN genes repressed (Bow-
man et al., 2002). In this respect, semidominant gain-of-function
mutants of PHB and PHV genes cause adaxialization of lateral
3.4.3. Floral organ polarity organs (McConnell and Barton, 1998; McConnell et al., 2001).
PHB, PHV, and REV have similar expression patterns. They are
Establishing organ polarity is an important aspect of morphogen- expressed in the SAM initiating lateral organs and later become
esis and it is sometimes clearly associated with specific functions adaxially restricted as the primordium emerges (McConnell et
of plant organs. Both, adaxial-abaxial and proximal-distal polari- al., 2001; Otsuga et al., 2001; Prigge et al., 2005). Finally, phe-
ties are regulated by genetic circuits that are similar for all lateral notypes of the loss-of-function rev mutants could be interpreted
organs (Figure 2C; Feng and Dickinson, 2007), although each or- as having a partial loss of adaxial identity (Talbert et al., 1995;
gan type has distinct cell types and morphogenesis in the abaxial Otsuga et al., 2001).
versus adaxial surfaces, and in the proximal versus distal regions Besides the PHAB function in polarity, it is also interesting to
(Figures 2C and 8). Organ polarity is also linked to the establish- note that a phb phv cna (corona, another member of the HD-
ment of hormone gradients. ZIP III gene family) triple mutant has a very similar phenotype
Briefly, abaxial fate is conferred by members of the YABBY to those of clv mutants with a distinct increase in organ number
family (Sawa et al., 1999; Siegfried et al., 1999) and by some of in each whorl. This would suggest that HD-ZIP III genes and the

CLV pathway regulate meristem function in a similar manner. The flower development. However, we still do not understand fully how
possible interrelation of these modules could contribute to ho- such functional modules interact with each other.
meostasis between stem cell maintenance and organ formation As it was said before sepal and petal boundary and organ
(Prigge et al., 2005). number establishment are controlled by the CUC and FFO2
NUB and JAG are similar genes which encode C2H2-zinc genes (see Figure 16 and section 3.4.2; Aida et al., 1997; Levin
finger transcription factors that are proposed to play redundant et al., 1998). CUC gene expression is regulated by the miR164c
functions in proliferation and differentiation of adaxial cells, par- (encoded by EEP1) in an organ specific manner (Laufs et al.,
ticularly during anther and carpel development (Dinneny et al., 2004; Baker et al., 2005).
2004; Ohno et al., 2004; Dinneny et al., 2006; Xu et al., 2008). Several genes are involved in establishing and maintaining the
They specifically work together in determining the number of cell sepal and/or petal domain and, in a way, determining the bound-
layers formed in floral organs, and like the PHAB family, they are aries between the organs. One of the main activities of these
not cell-fate genes. Hypothetically, JAG suppresses the prema- genes is to exclude AG expression from the first and second
ture differentiation of tissues by slowing down the cessation of whorl. As stated in section 3.3, AG is repressed by RBE, LUG,
cell division in distal regions of organs until it finally arrests after SEU, ROXY1, AP2, BLR, ANT and SAP (for more information
normal morphogenesis has occurred (Dinneny et al., 2004). about each gene, see Table S1; Figures 15 and 16).
AS1 and AS2 have redundant functions in the establishment Briefly, RBE is mainly involved in boundary and organ number
of adaxial identity (Ori et al., 2000; Sun et al., 2000; Semiarti et determination of both sepals (non-autonomously) and petals, and
al., 2001). AS1 encodes a MYB-domain transcription factor, and in AG exclusion from the second whorl at early stages of flower
AS2 is a member of the LBD gene family (Serrano-Cartagena organ development. But it is also important during late petal de-
et al., 1999; Byrne et al., 2000; Semiarti et al., 2001; Sun et al., velopment as mutants may form filamentous organs in the second
2002). AS1 protein is expressed in organ initials and physically whorl. RBE expression is controlled by both PTL and UFO (Takeda
interacts with AS2 to inhibit KNOX gene expression, thus guiding et al., 2004; Krizek et al., 2006). PTL is a trihelix transcription fac-
primordia toward differentiation (Figure 15; Ori et al., 2000; Byrne tor that is expressed at early stages in four zones between the
et al., 2002; Xu et al., 2003; Guo et al., 2008). initiating sepal primordia and in lateral regions of stamen primor-
Other reviews on polarity determination in embryos and in dial. Later on, PTL expression can be detected at the margins of
leaves are found in other chapters in this series: “Embryogenesis: expanding sepals, petals, and stamens (Brewer et al., 2004). Thus
pattern formation from a single cell” (Berleth and Chatfield, 2002) PTL may delimit the AG expression region indirectly by activating
and “Leaf development” (Tsukaya, 2002). RBE expression (Irish, 2008), and it may also be controlling lateral
outgrowth of mature sepals, petals and stamens defining their final
shape and orientation (Griffith et al, 1999; Brewer et al., 2004).
3.4.4 Sepals and petals UFO is also an important regulator of petal development. Its
action toward RBE may be indirect, as it may be degrading (as
Sepals and petals constitute the sterile perianth in the first and part of an SCF E3 ubiquitin ligase complex) an unknown repres-
second flower whorls, respectively. The sepal whorl or calyx pro- sor of RBE (Irish, 2008). But UFO is an important network link
tects the developing floral bud and in some plants, but not in Ara- between the AG inactivation pathway and the B gene identity de-
bidopsis, it may be involved in fruit development (He et al., 2004). termination pathway, because UFO interacts with LFY to activate
The petal whorl or corolla is generally thought to be important for AP3 expression (See section 3.3; Lee et al., 1997; Samach et
attracting pollinators (Krizek and Fletcher, 2005), but in an au- al., 1999; Chae et al., 2008). Importantly, UFO expression is also
togamous plant such as Arabidopsis, the corolla is generally not required for normal petal blade outgrowth after petal identity has
showy. been established (Laufs et al., 2003), as well as for determination
According to the ABC model, sepal identity specification de- of sepal shape and number in the first whorl (Levin and Meyerow-
pends on the activity of both A and SEP genes (see section 3.3; itz, 1995; Samach et al., 1999).
Coen and Meyerowitz, 1991; Pelaz et al., 2000), and petal identity SEU and LUG also repress AG expression in the first and sec-
specification depends on the overlapping activities of A, B and ond whorls by forming a protein complex with AP1 and SEP3 (see
SEP genes (see section 3.3; Coen and Meyerowitz, 1991; Pelaz section 3.3; Sridhar et al., 2004; Sridhar et al., 2006). But these
et al., 2000). Also, it has been shown that sepal and petal identity genes are also part of the adaxial/abaxial polarity establishment
specification depends, at least in part, on the correct downregula- pathway in the petal GRN, as they are required for normal PHB
tion of AG expression in the second whorl (see below). and FIL expression (Figure 15). SEU and LUG participate in petal
Several molecular components known to influence develop- shape regulation by controlling blade cell number and petal vas-
ment of sepals, influence petals too. But knowledge is still lim- culature development in an AG independent manner (Franks et al,
ited especially of sepal developmental gene networks. However, 2006). Finally, SEU is also involved in auxin response pathways
a basic GRN for petal development can be constructed based on by directly interacting with ETT, and influencing the final shape,
available data (Figure 16). As stated earlier, organ identity deter- number and phyllotaxy of petals (Pfluger and Zambryski, 2004).
mination, boundary establishment, and expression of polarity de- As part of the regulatory network that represses AG expres-
terminants are common features needed for the correct develop- sion, AP2 is itself negatively regulated by miR172 when second
ment of all the flower organs (Figure 15). There are several pieces whorl boundaries are determined (Chen, 2004; Zhao et al., 2007).
of evidence that suggest that genes involved in these processes Besides being a negative regulator of AG, ANT also affects organ
might be acting at the same time (for example, expression pro- number and morphology in the first three whorls (Elliott et al.,
files and in situ hybridization assays), at least momentarily during 1996; Klucher et al., 1996). SAP, another regulator of the mor-

phology of all organs, but mostly of petals, is unexpectedly more Petals have been proposed as an excellent model system in
important in later flowers (Byzova et al., 1999). which to study development because they have a simple orga-
Another important indirect repressor of AG is ROXY1. As a nization and are not essential for survival or reproduction (Irish,
glutaredoxin, ROXY1 seems to be a postranslational modifier of 2008). Although much progress has been made, much work is still
AP2, LUG, UFO and RBE giving them the specificity to repress needed for an integrated and dynamical understanding of petal
AG in the second whorl (Xing et al., 2005; Irish, 2008). ROXY1 development.
is also important for repressing PAN expression and for activat-
ing other TGA factors at different stages of petal development
(Li et al., 2009). 3.4.5 Stamens
Genes that usually work in the establishment of lateral organ
polarity (see section 3.4.3) are also important in determining the Six stamens occupy the third whorl in the Arabidopsis flower. Sta-
polarity of sepals and petals, e.g. PHB, JAG, FIL, YAB3, KAN, men specification depends on the overlapping activities of B, C and
AS1 and AS2 (Figure 16). Experimental data suggest that AS1, SEP MADS-box genes (Coen and Meyerowitz, 1991; Pelaz et al.,
AS2 and JAG are negative regulators of CUC1/2 and PLT (Xu et 2000). A complex network of gene regulatory modules is simulta-
al., 2008). This links the expression of these genes with those neously activated in young stamen primordia, and these are also
important for boundary determination in the GRN of both sepals important for organ morphogenesis (Figure 15). These modules in-
and petals. PHB and FIL expression are also part of the network clude those that regulate adaxial-abaxial primordium polarity (also
and are regulated by SEU and LUG (Franks et al., 2006). Lateral- affecting other vegetative and reproductive lateral organs) including
axis dependent development is determined by the PRESSED genes from the PHAB (PHB, PHV, and REV), KANADI (KAN1-4),
FLOWER (PRS) homeobox gene (Matsumoto and Okada, 2001). and YABBY (FIL/YAB1, YAB2, and YAB3) families. At later stages of
As with some other genes involved, its position in the GRN is stamen development, genes involved in sporogenesis such as SPL
unknown, but by analyzing the mutant phenotypes, it becomes and BAM1/2, and in anther development, such as JAG and NUB,
clear that the same regulatory modules that underlie polarity de- are activated (see Figures 15 and 17 for regulatory modules and
termination are involved in organ shape regulation. genes; Scott et al., 2004; Ma, 2005; Feng and Dickinson, 2007).
In Arabidopsis, as in other plants, several mutants featuring a Among the most striking stamen development mutants is fil
foliose-sepal-syndrome (FSS) (leaf-like sepals) have been isolat- (also called antherless and undeveloped anther) which bears
ed. Ectopic expression of the MADS-box genes AGL24, SVP, and normal filaments with neither anthers nor pollen. The FIL gene is
ZMM19 (from Zea mays), belonging to the STMADS11-clade (ac- YABBY-like and the fil phenotype suggests that the developmen-
cording to Theissen et al., 2000), result in FSS (He et al., 2004). tal programs of the filament and anther are controlled by indepen-
The main feature of these leaf-like sepals is that they are large dent regulatory modules (Sanders et al., 1999).
and have leaf-like stellate trichomes on their outer surface. One As mentioned in section 3.3.1, SPL/NZZ is essential for male
of the characteristics of ap1 mutant plants is that they also have and female reproductive development and is probably the first
large or foliose sepals. Thus, it has been proposed that, in addi- reproductive gene to be activated in the anther or, at least, it is
tion to their roles in floral transition and/or organ determination, the only gene that remains active during most of early anther
AP1, SVP, and AGL24 may also have a role regulating sepal size development. This transcription factor gene is expressed during
(He et al., 2004). But how they interact among themselves or with micro- and megasporogenesis. AG directly induces SPL but AG
other sepal specific genes is still unknown. is not necessary for maintaining its expression (Ito et al., 2004).
Final sepal and/or petal morphology is also determined by spl mutants are not able to produce microsporogenous cells or
FRL1 (Hase et al., 2000; Hase et al., 2005), TSO1 (Hauser et tapetal tissue, and show several alterations in anther wall and nu-
al., 1998), the AP3/PI regulated genes GNC, GNL, At4g30270, cellus development (Schiefthaler et al., 1999; Yang et al., 1999).
HXK1 (Mara and Irish, 2008), and NAP (Sablowski and Meyerow- Interestingly, BAM1 and BAM2, which participate in the first cell
itz, 1998). Except for FRL1, which is involved in endoreduplication division of the archesporial cells and the subsequent periclinal
control, and TSO1, which is likely involved in chromatin remodel- divisions to produce the somatic cell layers, are proposed to form
ling, the position of these genes in the petal GRN has already a regulatory loop with SPL (Figure 17; Hord et al., 2006; Feng and
been established (see Figure 16). Dickinson, 2007). Since SPL maintains the sporogenous activity
Using microarray approaches Wellmer et al. (2004), compared in the microsporogenous cells, and BAM1/2 maintain somatic dif-
gene expression levels within different floral homeotic mutants ferentiation, bam1 bam2 anthers have cells interior to the epider-
(see section 3.3.1). Their first study of stage 2 flowers identi- mis with characteristics of pollen mother cells (Hord et al., 2006).
fied only 13 genes as being sepal-specific and only 18 genes Although SPL is one of the genes expressed the earliest in
expressed exclusively (or predominantly) in petals. However, a stamen development, it is not the only one. Ectopic expression of
more recent study of flowers at stage 3, when sepal primordia SPL in all the whorls of an ag mutant, results in the formation of
have just formed, revealed that 199 genes are upregulated and microsporangia only in the lateral parts of the staminoid ‘petals’,
161 genes downregulated (Figures 3-4; Wellmer et al., 2006). suggesting that microsporangial localization is established inde-
One speculation is that sepals are relatively simple organs and pendently of AG, and that there is at least one other SPL inducer
not many specific genes are involved in their development. But that is expressed in the second whorl, and not in other whorls (Ito
more detailed studies are still required. Results also suggest that et al., 2004; Feng and Dickinson, 2007). Two other genes, JAG
genes regulating sepal and petal development may have been and NUB play a crucial role in the formation of the four-locular
recruited from leaf developmental pathways, and, hence, are not anther architecture, independent of SPL induction. jag nub dou-
specific for the development of these organs. ble mutants do not have a proper microsporangium. Instead, they

IAA
JA
GA
Pollen mitosis Anther dehiscence
Stage flower 10 11 12 13
Filament elongation
Figure 18. Hormones in late stages of stamen development.
At stage 10 of flower development, the auxin (IAA) concentration (yellow arrow) peaks (red gradient) in the stamens. During this period filaments start to
elongate and auxin prevents premature dehiscence. Auxin also participates in later anther dehiscence, probably by inducing JA production (green arrow)
that peaks (dark green gradient) at stages 11 and 12 (Nagpal et al., 2005). JA coordinates filament elongation, pollen maturation, anther dehiscence and
flower opening (Ishiguro et al., 2001). Although it has not been quantified, GA (blue arrow) is involved in filament elongation and participates in microspo-
rogenesis. Pollen development in anthers of GA-biosynthetic mutants is arrested before microspore mitosis (for details see section 3.4.6; Cheng et al.,
2004; Iuchi et al., 2007).
form a finger-like structure that expresses SPL in its tips (Dinneny ROXY1 and ROXY2 function downstream of SPL and upstream of
et al., 2006; Feng and Dickinson, 2007). DYSFUNCTIONAL TAPETUM1 (DYT1). As with other glutaredox-
The correct number of microsporangial initials and the subse- ins, they may need an interaction with glutathione to catalyze bio-
quent production of the tapetal cell and middle cell layer identi- synthetic reactions, suggesting that they may have a role in redox
ties are properties specified by a putative LRR receptor kinase, regulation and/or plant stress defense mechanisms involved in the
EXCESS MICROSPOROCYTES1 (EMS1)/EXTRA SPOROG- control of male gametogenesis (Xing and Zachgo, 2008).
ENOUS CELLS (EXS) (Canales et al., 2002). Until recently, the After tapetal cells are specified, a range of genes are essen-
ligand for EMS1 was unknown, though it was hypothesized that it tial for subsequent development. DYT1 encodes a putative bHLH
could be involved in the same signaling pathway as the TAPETAL transcription factor which functions downstream of SPL and EMS1.
DETERMINANT1 (TPD1) gene. Both tpd1 and tpd1 ems1 mu- However DYT1 is not able to complement the spl or ems1 mutant
tants are similar to the single ems1 mutant with arrested meiotic phenotypes when it is overexpressed, indicating that it is required
cytokinesis and degenerated microsporocytes (Yang et al., 2003). but not sufficient for normal tapetum development. dyt1 exhibits ab-
TPD1, is a small putatively secreted protein that interacts with normal anther morphology with largely vacuolated tapetal cells that
EMS1 and induces its phosphorylation suggesting that TPD1 is eventually collapse. Several tapetum-expressed genes, such as
the ligand of the EMS1 receptor that signals cell fate determina- MALE STERILE 1 (MS1) and ABORTED MICROSPORES (AMS)
tion during sexual cell morphogenesis (Jia et al., 2008). are upregulated by DYT1 (Zhang et al., 2006). In ms1 mutants for
ROXY1 and ROXY2 redundantly regulate anther development in example, tapetal cell abnormalities can be seen and pollen devel-
Arabidopsis (Xing and Zachgo, 2008). Lateral and medial stamens opment is arrested just after microspores are released from the
of roxy1 mutants might be fused and the former are sometimes tetrads (Bowman, 1994; Wilson et al., 2001; Yang et al., 2007a).
missing (Xing et al., 2005). In these mutants, the adaxial anther Other genes that participate in tapetum development include RE-
lobes are affected in sporogenous cell formation during early dif- CEPTOR-LIKE PROTEIN KINASE2 (RPK2), FAT TAPETUM and
ferentiation steps, abaxial lobes develop normally but pollen moth- GUS-NEGATIVE1 and 2 (GNE1, GNE2). RPK2 regulates tapetal
er cells degenerate, while the tapetum overgrows and occupies function and middle layer differentiation (Mizuno et al., 2007). FAT
most of the locule space. Eventually, the tapetum degenerates too. TAPETUM, when mutated, has a middle layer that fails to collapse

after meiosis and shows tapetal-like behavior (Sanders et al., 1999; JA has been shown to be involved in at least three androecial
Ma, 2005). In gne1 and gne2 mutants the sporogenous cells en- developmental pathways: filament elongation, anther dehiscence
ter meiosis, but cytokinesis is frequently arrested. The few highly and pollen production (Mandaokar et al., 2006). Different male
aberrant tetrads formed degenerate early and microsporangia of sterile mutants have been found to be JA biosynthetic mutants
mature anthers end up empty (Sorensen et al., 2002). (McConn and Browse, 1996; Sanders et al., 2000) including: the
Several mutants affecting pollen development have been triple fad mutant (fad3–2 fad7–2 fad8), which lacks the fatty acid
described: pollenless3; three division mutant (tdm1); ms5, ms3 precursors of JA; defective in anther dehiscence 1 (dad1), which
and ms15; determinate infertile1 (dif1); switch1 (swi1); defective- encodes a phospholipase A1 that catalyzes the initial step of
pollen 1; and 6492 among others (Bhatt et al., 1999; Sanders et JA biosynthesis; and dd1, a member of the 12-OXOPHYTODI-
al., 1999; Sorensen 2002). Meiotic cells in pollenless3 anthers ENOATE REDUCTASE (OPR3) gene family (Stintzi and Browse,
undergo a third division without DNA replication generating some 2000; Ishiguro et al., 2001). OPR3/DD1 is expressed in the sto-
cells with unbalanced chromosome numbers (Sanders et al., mium and in the septum cells of the anther that are involved in
1999) or “tetrads” with more than four microspores. dif1 and swi1 pollen release. All these mutant phenotypes can be rescued by
mutants have micro- and megaspores of uneven sizes because exogenous application of JA, suggesting that this hormone plays
the encoded proteins are essential for sister chromatid cohesion an important role in controlling the timing of anther dehiscence.
in male and female meiosis and so mutants are totally infertile Interestingly, DAD1 is a direct target of AG (Ito et al., 2007).
(Bhatt et al., 1999; Parisi et al., 1999; Mercier et al., 2001; Ma, Similarly, the coronatine insensitive 1 (coi1; JA receptor)
2005). Finally, other pollen mutants exhibit abnormal callose de- mutant is defective in both pollen development and anther de-
position (ms32, ms31, ms37, 7219, and 7593). hiscence. Stamens of coi1 flowers have shorter filaments than
There are late-developmental anther mutants that affect an- those of wild-type flowers and anthers are indehiscent contain-
ther dehiscence. In non-dehiscence1 mutant plants, anthers con- ing pollen grains with conspicuous vacuoles (Feys et al., 1994;
tain apparently wild-type pollen but do not dehisce. It has been Xie et al., 1998).
hypothesized that a cell death suppression program, which is Three related polygalacturonases, enzymes involved in pectin
normally responsible for dehiscence, might be inactive in this degradation that promotes cell separation, are also involved in
mutant (Sanders et al., 1999). ms35 is also affected in anther JA-regulated anther dehiscence. ARABIDOPSIS DEHISCENCE
dehiscence, because endothecial cells fail to develop the lignified ZONE POLYGALACTURONASE 1 (ADPG1) and 2 (ADPG2),
secondary walls that after desiccation shrink differentially leading and QUARTET2 (QRT 2) gene expression are distinctly regulated
to the retraction of the anther wall and full opening of the stomi- by JA (Ogawa et al., 2009).
um (Dawson et al., 1999; Scott et al., 2004). MS35, now MYB26 To determine the jasmonate-regulated stamen-specific tran-
(Steiner-Lange, 2003), is expressed during early anther develop- scriptome the expression profiles of JA-treated and untreated opr3
ment and may be a regulator of NAC SECONDARY WALL-PRO- mutants were compared (Mandaokar et al., 2006). It was found
MOTING FACTOR 1 and 2 (NST1, NST2), which have also been that 821 genes were induced (70% of them expressed in the spo-
linked to secondary thickening in the anther endothecium (Yang rophytic tissue) and 480 genes were repressed by JA and 13 tran-
et al., 2007b). In delayed-dehiscence mutants (dd1, dd2, dd3, scription factors were identified that could be important for stamen
dd4, dd5) anther dehiscence and pollen release occurs after the maturation pathway(s). Of these, MYB21, MYB24, and MYB28 are
stigma is no longer receptive preventing successful pollination, JA-responsive genes (Mandaokar et al., 2006). myb21 mutants
but stamens look wild-type and pollen is viable (Goldberg et al., have short filaments, are late to dehisce and have reduced fertility.
1993). On the contrary, in defective-pollen1, 2, and 3, anthers are Though myb24 mutants look like wild type, myb21 myb24 double
able to dehisce, but the pollen is aberrant and unviable. mutants have a more severe phenotype than myb21, suggesting
Recent publications have established that gibberellic acid that these two genes might be redundantly involved in important
(GA), jasmonic acid (JA), and auxins are involved during stamen aspects of JA-dependent stamen development. MYB28 is involved
development (Figure 18; Fleet and Sun, 2005; Nagpal et al., 2005; in amino acid metabolism and it is downregulated by both JA and
Wu et al., 2006; Cecchetti et al., 2008). The GA-deficient mutant, RGA. This study also uncovered several other biochemical path-
ga1-3, produces an abortive anther where microsporogenesis is ways that could be important during stamen and pollen matura-
arrested prior to pollen mitosis (Cheng et al., 2004). Mutations tion. Other results indicate that JA coordinates pollen maturation,
in two GA receptors, GA-INSENSITIVE DWARF1a and b (AtGI- anther dehiscence, and flower opening (Ishiguro et al., 2001). Aux-
D1a, b), affect the elongation of stamens, suggesting that these ins have also been proved to participate in these processes arf6
receptors have specific roles during stamen development (Iuchi arf8 double mutants are defective in anther dehiscence probably
et al., 2007). GA induces the degradation of the DELLA protein because they produce too little JA. Accordingly, this phenotype
REPRESSOR OF GA1-3 (RGA) upon ubiquitination. Microarray can be rescued by application of JA (Nagpal et al., 2005). How-
analysis shows that 38% of the RGA downregulated genes are ever, auxins trigger filament elongation and prevent premature an-
expressed in the male gametophyte at various stages of micro- ther dehiscence and pollen maturation at earlier stages of stamen
sporogenesis (Hou et al., 2008). development. While JA production peaks at stages 11-12 of flower
Temporal coordination of the elongation of filaments, pollen development (see Figure 6 and 18; Nagpal et al., 2005) auxin
maturation, and dehiscence of anthers is important for efficient receptors (TIR1 and AFBs) are already expressed at the end of
fertilization. The expression overlap of RGA-regulated genes and meiosis. Mutants in these genes cause the release of mature pol-
jasmonate-responsive genes during stamen development sug- len grains before filaments elongate. At later stages, the amount
gest a crosstalk between GA and JA signaling pathways in these of JA decreases allowing these processes to continue (Figure 18;
processes (Hou et al., 2008). Cecchetti et al., 2008).

Carpel development
carpel
primordia ovule
sm
sm
O O embryo sac
O O
va
Ar
flower stage 6 flower stage 11 flower stage 12-13

2 synergids
Ar Megasporogenesis MMC Megagametogenesis embryo sac 1 egg cell
1 central cell
3 antipodals
CARPEL PRIMORDIUM ARCHESPORIAL CELLS MEIOSIS AND MITOSIS
Mediate auxin
HEC
processes
ETT AG JAG FIL YAB3
TSL ANT
SEU LUG SHP1/2
SPT
FIL/YAB1 FUL RPL (BLR)
STY
CRC/YAB6 IND ALC

PHB VALVE REPLUM
NGA
INO/YAB4 SUP VALVE MARGIN
Figure 19. Main stages of carpel development and some genes involved.
Three different stages of carpel development are represented by the schemes in the upper part of the figure. Briefly, at stage 6, the central zone of the FM
begins to grow upward and eventually will form the gynoecium. From stages 11 to 13, the ovule primordia (O) arise from the placenta flanking the medial
ridges, and the Archesporial cell (Ar) develops from a single hypodermal cell at the ovule. The Ar then forms the megaspore mother cell (MMC) through
megasporogenesis, and the MMC forms the embryo sac through megagametogenesis. The embryo sac consists of 2 synergids, 1 egg cell, 1 central cell
and 3 antipodal cells. The medial ridges meet in the center of the fruit to form the septum (sm) which divides the gynoecium in two internal compartments.
The mature gynoecium is externally formed by the fusion of two valves (va); internally, it also carries totally differentiated ovules each one containing its
own embryo sac.
Carpel-specific gene networks are shown in blue. For genes and references not in the main text, see Table S1. Part of the network shown here was taken
from Roeder and Yanofsky (2006) and Balanzá et al. (2006). Color codes of interactions and gene/floral organs are according to those of functional mod-
ules identified in Figure 15. Arrows and bars indicate positive and negative regulatory interactions, respectively.
Additional stamen or pollen microarray analyses have been 3.4.6 Carpels and ovules
performed recently. For example, a clear difference was found be-
tween the genes that are expressed in the sporophyte and in pol- Carpels are specified by the C gene AG, and the SHP1, SHP2,
len with 39% of the expressed genes being pollen specific (Honys and STK genes (in an AG independent manner) together with
and Twell, 2003; Pina et al., 2005). The global gene expression the SEP genes (Bowman et al., 1989; Coen and Meyerowitz,
profiles of wild-type reproductive axes have been compared to 1991; Favaro et al., 2003; Pelaz et al., 2000; Pinyopich et al.,
those of the floral mutants ap3, spl/nzz, and ms1 in order to study 2003). They arise in the center of the flower meristem and when
gene expression during stamen development and microspore carpels are fully developed the floral meristematic cells are
formation (Alves-Ferreira et al., 2007). The data suggest that dif- completely consumed. Carpels are the most complex structures
ferent interconnected regulatory modules may control specific within flowers and a GRN underlies their development (Figure
stages of anther and microspore development (for further details 19; Table S1). Comprehensive reviews on carpel and fruit de-
see: Amagai et al., 2003; Cnudde et al., 2003; Honys and Twell, velopment can be found in Bowman et al., (1999), Ferrándiz et
2003; Zik and Irish, 2003a; Wellmer et al., 2004; Pina et al., 2005; al., (1999), Balanzá et al., (2006) and in Roeder and Yanofsky
Alves-Ferreira et al., 2007; Verelst et al., 2007). (2006) in this book.

3.4.7 Nectaries in particular, in flower development (for reviews Alvarez-Buylla et

al., 2007; Grieneisen and Scheres, 2009). At the level of GRN,
Little is known about the molecular genetics of nectary develop- mathematical and computational models provide useful tools for
ment. It is clear that nectaries are ABC-independent because ap2-2 integrating and interpreting molecular genetic information, or for
pi-1 ag-1 triple mutant flowers develop nectaries, although in these detecting gaps and contradictions in the evidence for particular
mutants nectaries are clearly smaller. However, ABC genes may functional regulatory modules. At other levels, two or three-di-
play a role in nectary patterning as pi-1 ag-1 and ap3-3 ag-3 double mensional morphogenetic models of coupled GRNs within cells
mutants lack them (Baum et al., 2001). Also, single mutant lfy and or among cells are useful for understanding spatiotemporal cell
ufo plants show reduced nectary formation (Lee et al., 2005a). patterning in individual organs and overall plant architecture; and
Several genes have been found to be expressed in the nec- this enables novel insights into the mechanisms underlying de-
taries (e.g., SHP1, ALC, SPL, MS35 and XAL1), but no detect- velopmental processes to be made. Such morphogenetic models
able defect is observed in their respective mutants (Figure 5G; are also a way of posing informed non-trivial predictions, testing
Schiefthaler et al., 1999; Roeder and Yanofsky, 2006; Yang et al., hypotheses, uncovering potentially generic mechanisms under-
2007b; Tapia-López et al., 2008). The only gene that has been lying conserved features, and performing in silico investigations
clearly related to nectary development is CRC, which is also im- that guide novel experiments in biological development.
portant for gynoecial development (Alvarez and Smyth, 1999; As the amount of experimental evidence increases and novel
Bowman and Smyth, 1999). The regulation of CRC in the nectar- theoretical approaches and techniques develop, there continue
ies seems to be independent of its expression in the gynoecium. to arise experimentally-grounded models of development and
Expression of this gene is already detectable at stage 6 of flow- theoretical tools useful in posing predictions amenable to further
er development in the area where the nectaries will be formed. experimental testing. These advances contribute to discussions
Thus, CRC may be important for the early specification of nectary of central issues in developmental and evolutionary biology (e.g.,
cells (Bowman and Smyth, 1999). CRC may also be necessary Kauffman, 1969; Berg et al., 2004; Milo et al., 2004; Wagner, 2005;
for maturation or maintenance of the nectaries, because it is ex- Alvarez-Buylla et al., 2007; Balleza et al., 2008). In Arabidopsis,
pressed at high levels when they develop (Bowman and Smyth, early flower development has been studied using dynamic gene
1999) and crc mutants have defects in nectary development. But regulatory network (GRN) models. Such models have helped
CRC is not sufficient for nectary development, because its ectopic capture the logic of gene regulation, mostly at the transcriptional
expression does not yield ectopic nectaries (Lee et al., 2005b). level, during cell-type specification in various systems (e.g. von
Bioinformatic and functional molecular genetic approaches have Dassow et al., 2000; Espinosa-Soto et al., 2004; Huang and Ing-
been taken to discover components of the regulatory network in ber, 2006; Li et al., 2006; Benítez et al., 2008). In this section we
which CRC participates during nectary and carpel development. focus on this modeling approach and present some of the main
A combination of floral homeotic gene activities acting redundant- results derived from network modeling in flower development.
ly with each other, involving AP3, PI and, AG and in combina-
tion with SEP proteins, activate CRC in both organs (Lee et al.,
2005a). Interestingly in another study, CRC was also found to be 4.1 Gene Regulatory Network Models
a direct target gene of AG (Gómez-Mena et al., 2005) and to be
indirectly regulated by LFY and UFO (Lee et al., 2005a). A model In this section we review some central notions in GRN theory
has been proposed in which LFY and UFO activate downstream and the main assumptions that are made and present some of
MADS-box genes which could be working in conjunction with the main results derived from network modeling in flower devel-
region-specific factors to activate CRC during nectary and carpel opment. GRN models are composed of nodes, which stand for
development (Lee et al., 2005a). genes or proteins, and edges or connections, which represent
Evolutionary studies have indicated that the CRC gene may the interactions among nodes (arrows for upregulation and bars
have been recruited as a regulator of nectary development in the for downregulation; for an example see Figure 20). Genes in the
core eudicot plant lineage, but its ancestral function could have GRN model may take different activation states, depending on
been related to carpel development (Lee et al., 2005b). the activation states of their inputs. Given the architecture of the
network and the sign of the interactions, it is possible to define
a set of rules or kinetic functions that govern the GRN dynam-
4. THEORETICAL MODELS: INTEGRATIVE AND DYNAMIC ics, that is, the way activation states of the genes change over
TOOLS FOR UNDERSTANDING FLOWER DEVELOPMENT time. These rules or kinetic functions may be defined and stud-
ied in the context of different mathematical frameworks, some of
As shown throughout this chapter, morphogenetic patterns under- which have been thoroughly reviewed elsewhere (Gibson and
lying flower development arise from complex, often non-additive, Mjolsness, 2004; Alvarez-Buylla et al., 2007). In experimentally-
interactions among several molecular and other kinds of compo- based GRNs, the dynamic rules may be obtained from reported
nents (e.g., cells) and factors (e.g., morphogen gradients, physi- molecular genetics data as well as from functional genomics
cal and geometrical constraints) at different levels of organization. datasets.
Dynamical models can be used to study the concerted action of The kinetic functions of gene activation depend on the states
many elements at different spatio-temporal scales and levels of of the input nodes and are multivariate. These may be modeled
organization; an approach which is becoming both necessary with discrete or continuous functions. In the former, Boolean
and possible for understanding how morphogenetic patterns functions that allow only “0” (OFF; not expressed) or “1” (ON;
emerge and are maintained during development in general, and expressed) values for the nodes have been successfully used

then the elements of the GRN change their activation state ac-
cording to the dynamic rules until they reach an attractor. Kauff-
man (1969) proposed that Boolean GRN attractors correspond
to the activation profiles typical of different cell types and there-
fore that exploring the GRN architecture and dynamics is funda-
mental to understanding cell-type determination processes. This
idea has now been verified experimentally and explored further
(e.g. Albert and Othmer, 2003; Huang and Ingber, 2006; Alvarez-
Buylla et al., 2007).
Another helpful notion in GRN dynamical studies is that of
basins of attraction. Given the dynamic rules of the network,
the set of initial conditions that lead to each of the attractors is
known as its basin of attraction. As we discuss below, the con-
cepts related to a GRN - attractor, initial condition and basin of
attraction - may be useful in addressing some pertinent aspects
of flower development.
4.1.2 Functional Modules in Flower Development
The functional data on genes involved in flower development

reviewed in this Chapter suggest that several regulatory mod-
Figure 20. Floral organ specification gene regulatory network (FOS-GRN) ules act at different stages and in different structures (Figures
model. 9, 15-17 and 19). We define a regulatory module as a set of in-
teracting genes that have more interactions among themselves
The diagram shows GRN topology where circles or nodes correspond to
than with other genes. These modules are semi-autonomous,
genes or proteins, and arrows and bars correspond to positive and nega-
tive regulatory interactions, respectively. The SEP node represents the
meaning that their dynamic outcomes are fairly independent of
SEP1, 2, and 3 genes together. The interactions are updated with respect other modules. In Figure 15 we have presented the best-studied
to previous publications (Espinosa-Soto et al., 2004; Chaos et al., 2006). modules associated with flower development. The approach de-
The GRN attractors or steady states match the gene expression profiles scribed here for the functional module that includes the ABC
that characterize inflorescence meristem regions and flower organ primor- genes could in principle be used for all of these modules as
dia. See text and Table 1 for details and experimental data supporting this sufficient nodes have been identified and their regulatory inter-
model (and Table S2 for the dynamics truth tables). Dotted lines represent actions characterized. Eventually, models of coupled GRN that
interactions predicted by the model. consider several such models together, both within and among
cells, will be possible. For now, we have focused in just one such
regulatory module.
to recover the key qualitative aspects of GRNs (e.g., Albert and In previous studies, we have proposed and analyzed the regu-
Othmer, 2003; Espinosa-Soto et al., 2004). In Boolean networks, latory module, which includes the ABC genes as well as other
parameters of specific kinetic functions are not required. It is ap- components, that is sufficient to regulate the partitioning of the
inflorescence and floral meristems into subregions of primordial
propriate to assume that the GRN nodes are Boolean variables
cells. In the case of the flower meristem, each one of the four
given that: (1) transcriptional regulation may be discrete and take
subregions is composed of the primordial cells that eventually
place in the form of pulses, rather being continuous (Ross et al.
give rise to each of the four types of floral organs: sepals, petals,
1994, Fiering et al., 2000, Ozbudak et al., 2002); (2) the experi-
stamens and carpels.
mental data at hand can be readily formalized as logical rules
We use this functional module as a working example of the
(see detailed discussions in Albert and Othmer, 2003; Espinosa-
protocol that has been used in order to assemble an experi-
Soto et al., 2004; Chaos et al., 2006), while there are no or very
mentally grounded gene regulatory network (GRN) model cor-
few available data on parameters required to postulate continu- responding to a functional module. Then we demonstrate how
ous functions; and (3) in complex GRNs with many components once such a GRN model is postulated, it is possible to follow its
interacting in non-linear manners, the overall topology of the GRN dynamics, and explore how the concerted action of multiple inter-
and the form of the logical rules of gene interaction, rather than connected molecular components eventually lead to stable gene
the details of the kinetic functions, are what determine the qualita- expression profiles that may be compared to those characterizing
tive network dynamics. different cell types. Then we delineate some theoretical approach-
Independently of the mathematical formalism used, dynamical es put forward to model coupled GRN dynamics that may underlie
analyses of GRNs mostly focus on finding the steady gene activa- pattern formation and morphogenesis during the early stages of
tion profiles, that is, the configurations of the network that, once flower development, when the floral meristem is partitioned into
reached, remain in that configuration. These configurations are four concentric rings of primordial cells. Finally, we review other
called attractors. The GRN model may be initialized on a particu- modeling approaches that are useful to study signal transduction
lar gene-activation configuration known as an initial condition and pathways.

4.2 Arabidopsis Flower Organ Specification GRN (FOS-GRN) AG, SEP); some genes that link floral organ specification to other
modules regulating primordia formation and homeostasis (AG,
Soon after flowering is induced, the flower meristem is partioned CLF and WUS); and some regulators of organ boundaries (UFO
into four concentric regions of primordial cells from which floral and LUG; Figures 9, 15 and 20).
organs will later form. During the last decade, an experimentally- The main result obtained from analyzing this GRN is that the
grounded GRN model for flower organ specification (FOS-GRN) postulated network converges to only ten attractors—even though
has been built and investigated (Figure 20; Mendoza and Alva- it can be initialized in more than 130,000 different configurations of
rez-Buylla 1998; Espinosa-Soto et al., 2004; Chaos et al., 2006; gene activation. Furthermore, the attractors—the stable configura-
Alvarez-Buylla et al., 2008). This model incorporates the intri- tions recovered—match gene activation profiles typical of the four
cate regulatory interactions among ABC genes themselves and inflorescence meristem regions (i.e., a region lacking WUS and
among ABC and non-ABC genes that are key to this process. UFO, two regions with either one of these two genes turned on,
This functional module includes: some key regulators underlying and a fourth region with both genes turned on; see Espinosa-Soto
the transition from IM to FM (FT, TFL, EMF, LFY, AP1, FUL); the et al., 2004), as well as those of primordial sepal, petal, stamen
ABCs and some of their interacting genes (AP1, AP3, PI, AP2, and carpel cells (Figure 21). This shows that the FOS-GRN is suf-
Figure 21. Arabidopsis inflorescence and flower development and FOS-GRN.
(A) SEM colored where four regions I1, I2, I3 and I4 are distinguished within the IM. FMs are also seen arising from the flanks of the IM,1 the oldest and
5 the youngest.
(B) I1, I2, I3 and I4 regions of the IM correspond to four of the FOS-GRN attractors. Expressed genes for each attractor are represented as green circles,
while non-expressed genes correspond to red circles (nodes are in the same relative position as in Figure 20. * marks the position of the EMF1 node for
further reference). Note that this model recovers the respective regions in the IM with both WUS and UFO, with either one of these two genes, or with
neither expressed.
(C) SEM colored to distinguish four types of primordial cells in young flower meristems. Each will eventually develop into the different flower organs, from
the flower periphery to the center, sepals (se), petals (pe), stamens (st) and carpels (ca).
(D) The six attractors of the FOS-GRN model match gene expression profiles characteristic of sepal, petal (p1 and p2), stamen (st1 and st2) and carpel
primordial cells. The gene activation profiles of the attractors are congruent with the combinatorial activities of A, B, and C genes described in the ABC
model of floral organ determination (adapted from Alvarez-Buylla et al., 2008).

Figure 22. Basins of attraction for the four flower organ FOS-GRN attractors.
Attractors of FOS-GRN match the gene expression profiles of the four types of floral organ primordia of young floral buds (sepal, petal, stamen and carpel).
The fan diagrams depict the GRN configurations (combinations of 0s and 1s corresponding to gene activation profiles) that lead to each of the attractors.
Points in the outermost layers of these fan diagrams correspond to initial configurations of the network and they are linked to the transitory configurations.
Petal2 and Stamen2 stand for one of the two possible attractors for each one of these organs. Relative position of nodes and their colors as in Figure 21.
* marks the position of the EMF1 node for further reference.
ficient to recover the gene activation profiles required to specify and postulates a set of gene interactions with the ABC genes, that
primordial cells during the first stages of flower organ develop- are also sufficient for FOS. The functions and interactions of the
ment. Therefore the GRN itself constitutes a functional module genes included are reviewed earlier in this chapter.
that robustly leads to the gene configurations that characterize dif- The FOS-GRN was validated by using this model to simulate
ferent regions of inflorescence and flower meristems during early the effect of loss-of-function mutations or overexpression, and
flower development; and this independently of the activation states comparing the results recovered from the model with the gene
of additional genes that are connected to this elucidated regulato- activation profiles determined experimentally in mutant or over-
ry module. Furthermore, various robustness analyses have been expressor lines. The mutants were simulated by fixing the state
performed showing that the recovered attractors are also robust in of the gene to 0 for loss of function, and to 1 for gain of function
response to permanent alterations in the logical functions of gene or overexpression (Figure 20; Table 1 and Table S2). In all cases
interactions and the inclusion of gene duplications. Therefore, tested, the simulated and empirically-reported profiles matched
these results (Espinosa-Soto, et al., 2004; Chaos et al., 2006) sug- (Espinosa-Soto et al., 2004).
gest that FOS dynamically and robustly emerges from complex In addition, this GRN model has enabled investigations to be
networks of molecular components, rather than from a series of made into the sufficiency and necessity of particular gene regula-
linear or hierarchical gene interactions or from the action of partic- tory interactions, which have led to novel predictions. For example,
ular genes. The FOS-GRN model not only recovers the ABC gene these analyses predicted that AG upregulated itself (Espinosa-
combinations that are necessary for FOS, but it also provides a Soto el al., 2004), which seemed somewhat counterintuitive at
dynamic explanation for the formation of such gene combinations, the time, but which was then verified by independent experiments

(Gómez-Mena et al., 2005). Also, computer simulations of the pressing lines, and to postulate novel interactions. Eventually, two
FOS-GRN that show that its attractors are robust to different types or more functional modules may be interconnected via common
of perturbation and to duplications (Espinosa-Soto et al., 2004; components to postulate GRN aggregates. Such an approach will
Chaos et al., 2006) can account for the overall conservation of be useful in beginning to uncover the types of microtopological
the flower structure throughout angiosperm (particularly eudicot) trait that characterize the nodes connecting different functional
evolution (Rudall, 2007; Whipple et al., 2004; Adam et al., 2007). modules, for example.
Since the FOS-GRN model was based on thorough molecular We have illustrated the potential of using dynamic GRN mod-
data and is one of the few well-characterized regulatory modules, els to understand cell differentiation using a relatively small and
it has been used as a “model GRN” for further methodological, well-characterized module. Approaches used for small regulatory
theoretical and conceptual developments in GRN and systems bi- modules that are well-characterized in terms of molecular genet-
ology research (Table 2). The main conclusions obtained from the ics, should feedback from functional genomic efforts that span the
first versions of this GRN have been confirmed. New data regard- dynamics of a larger number of genes or proteins under diverse
ing FOS are continuously being generated (novel data are also conditions and developmental stages or tissues.
summarized in Table 1) and the FOS-GRN constitutes a basic the-
oretical framework in which to integrate it alongside previous data.
Here, we have updated the FOS-GRN taking these novel data into 4.2. Temporal and Spatial Patterns of Cell-fate Attainment
account and conclude that the basic module originally put forward During Early Flower Development
(Espinosa-Soto et al., 2004; Chaos et al., 2006) is robust to the
addition of these newly discovered interactions. We consider, for In real biological systems, populations of meristematic cells differ-
instance, that EMF1 downregulates AG (Calonje et al., 2008), and entiate into different cell types in stereotyped temporal sequences
AP3/PI downregulate AP1 (Sundström et al., 2006), so the postu- and spatial patterns. The first primordial cells to be determined
lated module seems to be robust to the addition of intermediary in the flower meristem are those of sepals, then those of petals,
components or previously missing interactions. stamens and carpels going from the periphery to the center of
Simulations of the updated FOS-GRN have been performed the floral meristem. This suggests that in the population of meri-
with the new software, ATALIA (http://www.ecologia.unam. stematic cells the most probable temporal order in which each
mx/~achaos/Atalia/atalia.htm) developed in the Alvarez-Buylla attractor is visited follows the same sequence (Alvarez-Buylla et
laboratory by A. Chaos-Cador. This tool can be used to readily al., 2008). Recent results from another theoretical approach show
update this and other GRN models and explore their dynamics. that the sequence of floral organ determination can be recovered
We illustrate the use of this software with a visualization of the at- by introducing some level of stochasticity (random noise) in the
tractors’ basins (Figure 22) and a simulation of the updated wild- GRN dynamics, namely, a degree of error in the updating dynami-
type and certain mutant FOS-GRN dynamics (Figure 23). cal rules of the GRN (Alvarez-Buylla et al., 2008). These results
In the simulated FOS-GRN, genes can take only two activation are consistent with a handful of other recent studies showing that
states: 0 for no expression and 1 for expression. Hence, by using stochasticity at the molecular scale may contribute to the forma-
combinations of 0s and 1s, we can describe all the possible initial tion of spatiotemporal patterns in development (see review in Raj
conditions of the GRN. Figure 22 presents the so-called fan dia- and van Oudenaarden, 2008). Studies with the stochastic version
grams that show all the GRN configurations leading to each of the of the FOS-GRN also concluded that the relative position of the
attractors. Knowing the relative sizes of the basins of attraction of basins is important in determining the most probable temporal
each steady state is the key to exploring the robustness of each sequence of cell-fate attainment referred to above (Alvarez-Buylla
attractor in the face of perturbations. et al., 2008). This fascinating result certainly suggests that the
ATALIA can also calculate the attractor that every possible ini- stereotypical temporal pattern of cell fate specification at early
tial condition will eventually reach and show this information in a stages of flower development may be an emergent and robust
tapestry of destinies. In such tapestries, each possible configu- consequence of the complex GRN underlying cell-fate determi-
ration of the GRN is represented by a square in a lattice and is nation and that, in principle, it could take place in the absence
colored according to the attractor it reaches. Moreover, ATALIA of inducing signals, emerging only as a result of the stochastic
can draw a tapestry that represents the difference between the fluctuations that occur during transcriptional regulation (Alvarez-
original wild-type tapestry and a mutant one (Figure 23). For ex- Buylla et al., 2008). Ongoing modeling efforts are explicitly focus-
ample, if we want to know whether an ap2 mutation has a more or ing on spatial domains, and exploring the need and sufficiency of
less drastic effect in terms of the GRN dynamics than a pi muta- different cell-cell communication mechanisms or physical fields
tion, we can analyze the tapestries of ap2 and pi shown in Figure (e.g., created by curvature or tension forces) that could provide
23 and conclude that ap2 mutation has stronger dynamic effects positional information for spatio-temporal cell patterning during
than pi given the GRN postulated up to now. Given the complexity early stages of flower development.
of the network involved, such predictions are impossible to make It is important to mention that the FOS-GRN modeled up to
without a tool like ATALIA. As the regulatory interactions in other now is an abstraction of the qualitative regulatory logic underlying
modules that participate in flower development are gradually un- the IM and FM subregionalization during early stages of flower
covered, for each one the experimental data can be exhaustively development when the ABC patterns are established. However,
mined and formalized in the form of a GRN topology and logi- other regulatory modules for meristem positioning, growth and
cal rules governing its components’ interactions. ATALIA can then polarity, among others, still need to be considered in order to fully
be used to explore their dynamics, validate the proposed GRN understand spatiotemporal cell patterning and morphogenesis of
models by simulating experimental reports of mutants or overex- IM and FM. Some genes interacting with FOS-GRN components

Figure 23. Simulation results for wild type (WT) and two mutants.
(A) Simplified representation of the FOS-GRN. The mutated genes are in red (nodes are in the same relative position as in Figure 20). Mutations were
simulated by constitutively turning “off” (loss-of-function) mutated genes regardless of the dynamical rules.
(B) Floral diagrams showing floral organs of the simulated WT and mutant plants. These correspond to the steady-state gene expression arrays (attractors)
attained in the simulation.
(C) Tapestries of gene configuration destinies corresponding to the simulated WT and mutant lines. In the WT simulation each square in the tapestry
represents an initial condition and they are colored according to the attractor they eventually reach. In the mutant simulation for ap2 and pi, the tapestries
illustrate the difference between the WT tapestry of destinies and that obtained for the mutant simulations. Yellow squares, configuration attained is the
same attractor as in the WT; red squares, configurations that reached a new attractor; purple squares, configurations that attained a pre-existing attractor
but not the same one reached in the WT simulations. Images generated with ATALIA (http://www.ecologia.unam.mx/~achaos/Atalia/atalia.htm).
(e.g. AGL24, BEL, RBE and those described in the last section of that help unravel how such single-cell GRNs are coupled in ex-
Table 1) that do not seem to directly affect cell-type determination plicit cellularized spatial domains and physicochemical fields
in the floral meristem, could link the FOS-GRN with: a) signaling (e.g. Jönsson et al., 2005, Savage et al., 2008; Benítez et al.,
pathways (e.g. Díaz and Alvarez-Buylla, 2006); b) genes involved 2008), including metabolism, signaling, and emergent gradients
in cell growth and proliferation both before and after the partition- of morphogens (e.g., auxin), cell growth and proliferation, me-
ing of the floral meristem into the four concentric regions; and c) chanical forces and cell-cell communication mechanisms. All of
other types of downstream genes or modules that are important these are likely to feedback in non-linear ways from and to the
during cell sub-differentiation and organogenesis at later stages GRNs underlying cell differentiation or proliferation (for example
of flower development. see Hamant et al., 2008).
A complete understanding of flower morphogenesis will con- It is important to keep in mind, for example, that plant cell
tinue to require multidisciplinary approaches and modeling tools growth in meristems is symplastic. This implies that the contacts

Table 1. Summary of evidence for the FOS-GRN gene interactions shown in Figures 20-23 (ChIP, chromosome immunoprecipitation; EMSA, electropho-
retic mobility shift assays; arrows indicate gene induction and bars repression; Espinosa-Soto et al., 2004; Chaos et al., 2006).
INTERACTIONS EXPERIMENTAL EVIDENCE REFERENCE
AG (AT4G18960)Æ
AG ChIP shows that AG interacts in vivo with predicted regulatory sequences of AG . Gómez-Mena et al., 2005.
AP1 (AT1G69120) --| AG Sepals are replaced by carpels, and petals by stamens in ap1 mutants. AG mRNA Bowman et al., 1993; Weigel
found in all flower primordia of ap1-1 plants. First whorl organs are sometimes carpel- and Meyerowitz, 1993; Liu and
loid, and second whorl organs are staminoid in ap1 mutants. Meyerowitz, 1995.
CLF (AT2G23380) --| AG In clf mutants, first whorl sepals are frequently carpelloid, second whorl organs are Goodrich et al., 1997; Calonje et
staminoid petals and AG mRNA is detected in sepals. It is likely that CLF is part of a al., 2008.
complex with EMF2, MSI1, and FIE that epigenetically regulate AG.
LFY (AT5G61850) ÆAG Expression of AG is reduced in lfy-6 flowers. Weigel and Meyerowitz, 1993;
The expression of LFY fused to a strong activation domain produces increased and Parcy et al., 1998; Busch et al.,
ectopic AG expression. 1999; Lohmann et al., 2001.
LFY binds to the first intron of AG, and cooperates with the WUS homeodomain to
activate it.
LUG (AT4G32551)--| AG AG is ectopically expressed in lug-1 mutants. Liu and Meyerowitz, 1995;
LUG functions as a repressor of AG via its the second regulatory intron. Sieburth and Meyerowitz, 1997;
Deyholos and Sieburth, 2000;
Gregis et al., 2006.
SEP3 (AT1G24260) ÆAG There is AG expression in rosette leaves of 35S:SEP3 plants. In addition, 35S:AG Castillejo et al., 2005.
35S:SEP3 plants have more pronounced carpelloid features.
TFL1 (AT5G03840) --| AG Stigmas and styles of terminal flowers in lfy ap1 double mutants are normal if the tfl1 Shannon and Meeks-Wagner,
mutation is added. 1993.
WUS (AT2G17950) ÆAG wus mutants lack carpels and most stamens. In AP3:WUS transgenic plants, second Laux et al., 1996; Lenhard et al.,
whorl organs are carpelloid stamens instead of petals, whereas in AP3:WUS ag 2001; Lohmann et al., 2001.
plants, second and third whorl organs do not differentiate into carpelloid stamens.
AG --| AP1 AP1 mRNA accumulates uniformly in ag-1 mutant flowers. Gustafson-Brown et al., 1994.
FT (AT1G65480) ÆAP1 In ft lfy double mutants, there is no AP1 mRNA unlike in the respective single mutants, Ruiz-García et al., 1997.
suggesting that at least one of these two genes needs to be active for AP1 activation
LFY ÆAP1 AP1 expression is delayed in lfy-6 null mutants, ectopic in 35S:LFY plants and in- Parcy et al., 1998; Liljegren et
creased when LFY-VP16 is induced. al., 1999. Weigel and Nilsson,
LFY directly binds the AP1 promoter and activates this gene. 1995; Wagner et al., 1999.
TFL1 --| AP1 In tfl1 mutants, AP1 is ectopically expressed in the basal lateral meristems and in Gustafson-Brown et al., 1994;
terminal flowers. AP1 expression is also retarded in 35S:TFL1 Ratcliffe et al., 1998.
TFL1 --| AP2 (AT4G36920) The absence of petals in tfl1 ap2 flowers and the presence of petals in tfl1 single mu- Shannon and Meeks-Wagner,
tants suggest there is ectopic AP2 activity in the terminal flowers of tfl1 single mutants. 1993.
AG ÆAP3 (AT3G54340) There is weaker GUS expression in the third whorl of ag-1 AP3:GUS flowers than in Hill et al., 1998; Honma and
the transgenic control. Goto, 2001; Gómez-Mena et al.,
AG may maintain AP3 expression because cauline leaves of 35S:PI 35S:AP3 2005; Zhao et al., 2007.
35S:SEP3 35S:AG are converted into stamen-like organs.
ChIP shows that AG interacts in vivo with predicted regulatory sequences of AP3.
Also, AP3 RNA is absent from the center of the ag-1 meristem.
AP1 ÆAP3 AP3 expression is quite normal in ap1 mutants but is almost undetectable in lfy ap1 Weigel and Meyerowitz, 1993;
double mutants, indicating that AP1 can act with LFY to regulate AP3 expression. Hill et al., 1998; Ng and Yanof-
Futhermore, AP1 seems to bind AP3 cis-regulatory elements. sky, 2001; Lamb et al., 2002.
AP3 ÆAP3 Endogenous AP3 is upregulated in 35S:AP3-GR plants induced with dexamethasone, Hill et al., 1998; Honma and
supporting the notion that AP3 self-activates. Goto, 2000.
(Continued)

Table 1. (continued)

LFY+UFO (AT1G30950) Both the amount and the domain of AP3 expression are reduced in lfy-6 mutants. Weigel and Meyerowitz, 1993;
ÆAP3 ufo-2 plants have less AP3 protein and less AP3 mRNA. Meyerowitz, 1995; Parcy et al.,
Both LFY and UFO have to be overexpressed to induce ectopic expression of AP3. 1998; Lamb et al., 2002; Levin
EMSA show that LFY binds directly to sequences in the AP3 promoter. and Chae, 2008.
ChIP shows that UFO associates with the AP3 promoter. This association was
abolished when ChIP was performed using extracts from lfy-26 plants harboring the
35S:UFO-Myc transgene.
SEP (AT5G15800, In AP3:GUS 35S:PI 35S:AP3 35S:AP1 mutants, AP3-GUS is expressed throughout Honma and Goto, 2001; Cas-
AT3G02310, AT1G24260, the plant supporting the idea that full activation of the B-function genes requires tetra- tillejo et al., 2005.
AT2G03710) ÆAP3 mer formation to include SEP.
The ectopic expression of SEP3 resulted in the induction of ectopic AP3 expression.
Stronger 35S:SEP3 lines are also capable of activating AP3:GUS ectopically
LFY --| EMF1 (AT5G11530) Ectopic LFY expression in emfl-1 mutants increases the severity of the emf phenotype. Chen et al., 1999.
EMF1 --| FT FT RNA levels are higher in the emf1-1 mutant and are detected earlier than in the Moon et al., 2003.
wild type.
AP1 --| FUL (AT5G60910) FRUITFULL is ectopically expressed in ap1 mutants. Mandel and Yanofski, 1995b;
Ferrándiz et al., 2000a.
TFL1 --| FUL TFL1 has been postulated to be an inhibitor but it also is possible that other factors Espinosa-Soto et al., 2004.
have this posttranscriptional inhibitory role. This interaction is necessary as when the
negative posttranscriptional regulation of FUL by TFL1 is not considered, the nonfloral
gene steady states disappear. No experimental evidence.
AP1 Æ LFY In ap1 and ap1 cal double mutants, LFY expression is reduced. Additionally, LFY is Bowman et al., 1993; Kempin
activated earlier in 35S:AP1 plants than in the wild type. et al., 1995; Weigel and Nilson,
1995; Piñeiro and Coupland,
1998; Liljegren et al., 1999.
EMF1 --| LFY Double mutants of the weak emf1-1 allele and lfy-1 bear lfy-like flowers suggesting Yang et al., 1995.
that, for this trait, lfy is epistatic. These genes have antagonistic activities.
FUL Æ LFY Even though LFY expression is similar in wild type and LFY:GUS ful-2 plants, there is Ferrándiz et al., 2000a.
less expression in ful ap1 cal triple mutants than in ap1 cal double mutants, suggest-
ing that the role of FUL in LFY upregulation is only important when AP1 is inactive.
TFL1 --| LFY In tfl1 mutant plants LFY is ectopically expressed in the shoot apex. Weigel et al., 1992; Ratcliffe et
al., 1999.
LFYÆ PI (AT5G20240) Amount and domain of PI expression are reduced in lfy-6 mutants. There is no GUS Weigel and Meyerowitz, 1993;
expression in early lfy PI:GUS flowers. Honma and Goto, 2000.
PI Æ PI AP3 and PI co-immunoprecipitate. Jack et al., 1992; Goto and

AP3 and PI mRNA levels are not maintained in ap3-3 pi-1 double mutants. Meyerowitz, 1994; Honma and
In AP3:GUS 35S:PI 35S:AP3 35S:AP1 mutants, AP3:GUS is expressed throughout Goto, 2001.
the plant supporting the idea that full activation of the B-function genes requires PI
LFY Æ SEP Microarray experiments show that the group of LFY dependent genes includes the Schmid et al., 2003.
homeotic cofactors SEP1-3.
AP1 --| TFL1 In 35S:AP1, TFL1 expression is greatly diminished. TFL1 is ectopically expressed in Liljegren et al., 1999.
ap1 cal double mutants.
AP2 --| TFL1 The tfl1-1 mutation partially suppresses the ap2-1 ap1-1 inflorescence phenotype. Schultz and Haughn, 1993;
Shannon and Meeks-Wagner,
1993.
EMF1 Æ TFL1 In emf1-2 tfl1 double mutants, the emf1-2 mutation is epistatic with respect to flower Chen et al., 1997.
initiation. These genes do not have antagonistic activities. This suggests that EMF1
upregulates TFL1.
LFY --| TFL1 The 35S:LFY plants resemble the tfl1 mutant and have no TFL1 expression. Weigel and Nilsson, 1995;
LFY can inhibit TFL1 at the transcriptional level. TFL1 is also ectopically expressed in Liljegren et al., 1999; Ratcliffe et
lfy mutants. al., 1999.
AG --| WUS There is strong WUS expression in the center of ag floral meristem. Lenhard et al., 2001; Lohmann
et al., 2001.

Table 1. (continued)

SEP --| WUS SEP activity is required for WUS downregulation by AG because sep1 sep2 sep3 triple Pelaz et al., 2000.
mutant plants bear indeterminate flowers.
WUS Æ WUS No experimental evidence. Assumption of model. Espinosa-Soto et al., 2004;
Chaos et al., 2006
UPDATES (Chaos et al., 2006 and this chapter)
EMF1 --| AG In ChIP experiments, EMF1 is associated with sites in the promoter and second Calonje et al., 2008.
intron of AG. EMF1 interferes with transcription by RNA polymerase II and T7 RNA
polymerase in vitro.
AP3 --| AP1 AP1 transcript levels are significantly higher in ap3-3 mutant plants than in both WT Sundström et al., 2006.
and 35S:AP3.
PI --| AP1 ChIP shows that PI binds to target sequences in the AP1 promoter Sundström et al., 2006
MiR172 (AT2G28056, Elevated miR172 accumulation results in floral organ identity defects similar to those Chen et al., 2002; Park et al.,
AT5G04275, AT3G11435) + in loss-of-function ap2 mutants. On the other hand, the miR172 abundance depends 2002; Chen et al., 2004; Zhao et
HEN1 (AT4G20910) --| AP2 on the activity of DICER-like protein HUA ENHANCER 1 (HEN1), which is expressed al., 2007.
through the plant. This observation suggests that a cofactor expressed in the inner
floral whorls is required to give specificity to the HEN1-dependent repression of AP2.
The need for AG inactivity for AP2 function is added to the AP2 logical rules
LFY Æ SEP1-3 Microarray experiments show that the group of LFY dependent genes includes the Schmid et al., 2003.
homeotic cofactors SEP1-3.
INTERACTIONS NOT INCLUDED IN THE MODEL
AGL24 (AT4G24540) + In the agl24 svp double mutant, AG mRNAs are detected in the inflorescence and Gregis et al., 2006.
SVP (AT2G22540) --| AG floral meristems as early as stage 1, indicative of early AG expression. In later stages,
AG is still expressed in all floral organs. Probably, this interaction is part of a different
GRN that ocurs before the cell fate determination
BLR (AT5G02030) --| AG AG is expressed ectopically in blr mutants. BLR directly binds to AG cis elements Bao et al., 2004.
(identified by EMSA). This interaction is probably important in organogenesis.
RBE (AT5G06070) --| AG In rbe mutants, there is ectopic expression of AG in second-whorl cells. This interac- Krizek et al., 2006.
tion may be important in organogenesis.
SEU (AT1G43850) --| AG The direct in vivo association of SEUSS (SEU) with the AG cis-regulatory element was Sridhar et al., 2006.
shown by ChIP. SEU interacts with LUG in a repressor complex to regulate AG, and
LUG is already considered in the GRN model.
AGL24+SVP --| AP3 An in situ analysis shows that in the agl24 svp double mutant, AP3 is expressed in all Gregis et al., 2006.
parts of the floral meristem and later in all floral organs. Probably, this interaction is
part of a different GRN occurring before the cell fate determination.
LFY Æ CAL(AT1G26310) Using posttranslational activation of LFY-GR, it is demonstrated that CAL is a direct William et al., 2004.
LFY target. cis-regulatory elements in the putative CAL promoter are bound by LFY.
AP1 forms heterodimers with CAL and AP1 is already included.
AP3 --| FUL The domain of FUL expression is expanded to the third whorl in stage-3 ap3 mutants, Mandel and Yanofsky, 1995b;
but no direct interaction is detected by ChIP analysis. Sundström et al., 2006.
FT --| FUL FUL is expressed at higher levels in 35S:FT-VP16. It is not considered because this Teper-Bamnolker and Samach,
interaction could be mediated by TFL1 and LFY. 2005.
PNY (AT5G02030) Æ LFY The transcripts of LFY are substantially reduced in shoot apices of pny pnf double Anrar et al., 2008.
mutants after floral induction. pny pnf double mutants do not produce flowers but,
PNF (AT2G27990) Æ LFY
35S:LFY pny pnf plants do produce flowers. This interaction is part of a different GRN.
AP2 Æ PI In situ hybridization shows there is less PI RNA occupying a smaller area in ap2-2 Zhao et al., 2007.
flowers than in wild type. Probably an indirect effect.
AG --- SEP3 ChIP shows that AG interacts in vivo with predicted regulatory sequences of SEP3. Gómez-Mena et al., 2005.
Insufficient experimental data.
FT Æ SEP3 Overexpression of FT causes ectopic expression of SEP3 in leaves. No further experi- Teper-Bamnolker and Samach,
mental evidence. 2005.

Table 2. Some of the contributions that have used the flower organ specification GRN model in order to test, advance or discuss novel conceptual or
methodological approaches.
Contribution Reference
Logical analysis of the flower organ specification (FOS) GRN. Mendoza et al., 1999
Introduction of the transsys formalism to represent GRN and implementation of FOS-GRN in this framework. Kim, 2001
Method for gene network inference based on nonlinear differential equations and logical approaches. Perkins et al., 2004
Predictions were tested using FOS-GRN.
New method for automatically inferring gene regulation functions modeled as logical functions. Bozek et al., 2006
The method is applied to FOS-GRN.
Automatic Petri-net-based method, applied to FOS-GRN, for finding stationary states. Gambin et al., 2006
Analysis of the dynamic role of feedback loops in networks including FOS-GRN. Kwon and Cho, 2007
Application of the GenYsis software to model the discrete and multiple valued FOS-GRN. Garg et al., 2007
Analysis of the effect of feedback loops on the robustness of Boolean networks, such as that of flower organ specification. Kwon and Cho, 2008
Dynamic study of FOS-GRN and other GRNs with the finding that these exhibit a property known as criticality. Balleza et al., 2008
Formal analysis of the main sources of perturbation and their effects in biological regulatory networks, with the Demongeot et al., 2008
FOS-GRN as example.
between cells are preserved because there is no displacement 5. CONCLUSIONS AND PERSPECTIVES
or sliding at middle lamellas that join neighboring cells (Priestley,
1930 and Erickson, 1986; cited in Kwiatkowska, 2008). Therefore, Arabidopsis has been indispensable in unraveling the molecu-
overall plant growth could be modeled using the principles of lar genetic bases of the stereotypical and most conserved as-
solid body mechanics (see review in Kwiatkowska, 2008). How- pects of flower development. It has also been used to resolve
ever plant cells also grow anisotropically which implies a variation some basic mechanisms of floral meristem determination, as
in the directional growth rates at a given point (Baskin, 2005). well as floral organ cell differentiation and morphogenesis. The
Hence, meristem growth has rather been modeled using the prin- challenge ahead will be to understand how modules regulating
ciples of continuum mechanics, computing variables that char- each aspect of flower development are interconnected among
acterize plastic strain (Goodall and Green, 1986; for review see themselves and with signal transduction pathways that respond
Green, 1999). to environmental and internal cues to yield coupled GRN spa-
Some quantitative mesoscopic models for flower develop- tiotemporal dynamics during flower development. Such dynam-
ment and growth in Arabidopsis and other angiosperms have ics likely involve feedback from physical or mechanical forces,
been put forward (e.g., Rolland-Lagan et al., 2003; Lee et al., structural and geometric characteristics of domains of activity
2004; Skryabin et al., 2004; Mündermann et al., 2005). A finite and from cell dynamics (cell growth and division) in complex
element model of the SAM has also shown, for example, that lat- ways still requiring multiple theoretical multilevel models and
eral bulging of the meristem surface leading to the formation of a coordinated experimental research. Different functional mod-
primordium results in a gradient of shear stresses with high shear ules are now being characterized (Figure 24 and Table S1) and
stress at the point where the future primordium emerges (Selker shown to regulate some of the main processes involved in flower
et al., 1992; reviewed in Kwiatkowska 2008). More recently, it was development. Some of these modules or their components may
shown that cells in the Arabidopsis SAM orient their cortical mi- participate in one or more flower developmental process and
crotubules along lines of mechanical stress generated during tis- data on the functions and interactions of genes are becoming
sue formation, and this then affects the mechanical properties of available to enable new dynamic computational models of GRN
the cell, thus establishing a feedback loop (Hamant et al., 2008). and signaling pathways during flower development (Figure 24
This seems to be particularly relevant during the formation of the and Table S1).
groove between the apical meristem and the primordium of lat- Computational models for the gene regulatory module that
eral organs, but less so during growth and differentiation, because underlies patterning of the inflorescence meristem and determi-
the lateral organ primordia are not affected when the microtubular nation of the primordial cell types during early stages of flower
network is disintegrated by a drug (Hamant et al., 2008). This im- organ specification, have demonstrated the potential and need of
plies that the mechanical feedback loop described is likely to act theoretical dynamic approaches in understanding complex GRN
in parallel with the previously described auxin-mediated patterning underlying flower development. But information on each regula-
mechanism (Laufs et al., 2009). Similar morphogenetic mecha- tory module and the interconnections between modules and with
nisms are likely to be at work in flower meristem and floral organ signal transduction pathways is still scanty.
development, and both morphogenetic mechanisms connected to It would be fascinating to unravel which molecular compo-
the functional regulatory modules, including FOS-GRN and others nents, circuits, or sub-networks underlie the development and
that have been partly elucidated and reviewed in this Chapter. evolution of the diversity of flower forms and the variations

Figure 24. The main regulatory gene modules and hormone signaling pathways during flower developmental processes.
Four main developmental processes in flowers shown schematically from FM formation to mature flower formation. 1) Specification of the floral meristem
anlagen. To initiate this process, FMI genes like LFY and AP1 are upregulated. However the position and polarity of these meristems are determined by
other gene families and hormones like auxin (IAA) and gibberellins (GA). 2) Specification of whorls of organ primordia. The ABC identity genes and SEP
are necessary and, together with other genes, sufficient to specify floral organ primordial cells (FOS-GRN module). 3) Organ primordia cell proliferation,
boundary establishment and organ polarity are regulated by additional modules that are presumably coordinated during floral organ primordia formation.
4) Cellular differentiation and organ morphogenesis yield the final shape, size and tissue composition of functional sepals, petals, stamens and carpels.


around the overall conserved “theme” of floral structure among Findings from diverse groups of angiosperms, mostly com-
angiosperms. This will be possible with integrated multidisci- parative analyses of ABC gene expression data among diverse
plinary approaches addressing pending questions. For example, angiosperm groups (especially basal angiosperm taxa), with
in order to understand how a flower meristem forms will require emphasis on the A and B class genes, have already been used
knowledge of the regulatory mechanisms underlying mechanore- to account for the underlying genetic differences in the diver-
ception and cell wall, microfibril and microtubule behaviour. How sity of petal and stamen morphology among extant flowering
are such mechanisms interconnected or coordinated with the cell plants (Kim et al., 2005; Rudall, 2007). Figure 25 shows a dia-
differentiation GRNs as well as with the morphogen-mediated grammatic and very simplified angiosperm phylogeny and the
patterning mechanisms? The challenge ahead consists in inte- variations observed in the domains of expression of the ABC
grating mesoscopic mechanical and morphogen-gradient models class genes in selected species, representative of the morpho-
with experimentally grounded models of the GRNs underlying cell logical diversity present in their respective angiosperm lineages.
behaviour, dynamics and differentiation. The aim is to build multi- Overall, these approaches are helping refine our knowledge of
level computational modeling frameworks that can be used to test flower development, and will be instrumental in understanding
the sufficiency and necessity of contrasting mechanisms, which the canonical GRN modules involved in flower formation and
scale from the biochemical and GRN level to the physical factors discovering variations.
constraining plant growth (Hogeweg, 2002). Ideally, joint efforts in
modeling, bioinformatics and experimentation continually feeding
back on each other should give a better understanding of flower, ACKNOWLEDGMENTS
and more generally, plant development and evolution.
Notwithstanding the usefulness of Arabidopsis, such a grand We greatly acknowledge Diana Romo for her help formatting this chap-
challenge will surely benefit or require comparative experimental ter. We thank Silvia Espinosa-Matias from the Laboratorio de Microscopia
and evolutionary studies of other angiosperms with divergent floral Electronica de Barrido Facultad de Ciencias UNAM for most of the SEMs
structures such as the monocots, other eudicots and basal angio- presented. Many thanks to John L. Bowman, who provided SEM pictures
sperms. Such an approach has been successful in understand- in Figures 4A, 4B, 4C, 4I, 5A (with permission of the American Society of
ing and interpreting morphological traits of plants (Kaplan, 2001). Plant Biology), Figure 11 (with permission of the International Journal De-
Recently, studies in non-model monocots such as orchids (Tsai et velopmental Biology) and Figure 12. Work in Álvarez-Buylla/Garay-Arroyo/
al., 2004; Xu et al., 2006) and commelinids, (Ochiai et al., 2004), in García-Ponce, Gamboa de Buen and de Folter laboratories is financed
maize and rice (Whipple et al., 2004; Xu and Kong, 2007), in mem- by: Mexican Science Council (CONACyT 41848; 81542; 81433; 90565;
bers of the Solanaceae, such as tomato (Hileman et al., 2006; de 82826) and UNAM (PAPIIT 221406; 210408; 223607). The de Folter labo-
Martino et al., 2006), and in basal angiosperms (Soltis et al., 2007) ratory is also financed by CONCyTEG. We also thank the students in our
among others, have started to demonstrate the power of coupling laboratory for continued feedback and useful discussions that have en-
functional and evolutionary questions of a comparative approach riched this work. Particular thanks also go to M. Aldana, C. Espinosa-Soto,
with detailed molecular experimentation in several species. D. Hartasánchez, P. Padilla, and G. Santos for ideas in Section 4.
Figure 25. Angiosperm phylogeny and schematic representation of ABC gene expression patterns of selected taxa.
Schematic phylogeny based on APGII (2003) conventions with variations in the ABC model among angiosperm groups shown (see section 3.3). We
present all rosids and asterids, but taxa comprising basal angiosperms, the magnoliid complex, monocots and core eudicots have been compacted and
simplified. Arabidopsis thaliana belongs to the order Brassicales (bold and underlined). In the ABC model, the A function for sepal specification is main-
tained for all groups, although the class A genes involved in Arabidopsis are not functionally conserved for other taxa and may not be separable from floral
meristem determination. The A function for all lineages was kept to enable comparison with Arabidopsis although a question mark was added to underline
its dubious role. For B function, it should be noted that B class genes have undergone extensive duplications within different angiosperm lineages; while
these duplications do not affect overall B function, on occasion they implicate subfunctionalization of the resulting paralogs (Irish and Litt, 2005; Soltis et
al., 2007). For example, in species of Solanaceae such as tomato (de Martino et al., 2006) and petunia (Vandenbussche et al., 2004), and in the majority
of eudicot taxa in which B function expression has been analyzed, two copies of the AP3 gene are found that have undergone subfunctionalization, AP3
and TM6. Specified floral organs are indicated underneath each ABC model (Theissen and Melzer, 2007). Abbreviations: male organs (mo); female organs
(fo); sepal-like tepals (sl); petal-like tepals (pl); staminodes (sd); stamens (st); carpels (ca); petaloid tepals (te); petals (pe); palea/lemma (pa); lodicules
(lo); sepals (se); sepaloid petals (sp). Symbols used to refer to compacted plant lineages are: Basal tricolpates (i), including orders Ranunculales and
Proteales and families Buxaceae, Sabiaceae and Trochodendraceae; Asparagales (k) including Dioscorales, Liliales and Pandanales; (a) the Commelinid
grade that, in addition to Poales and Commelinales, includes Dasipogonaeae, Arecales and Zingiberales; the Magnoliid complex (h) including Canella-
les, Piperales, Laurales and Magnoliales. Images of rice spikelet, Nymphaea alba and the male Gnetum gnemon reproductive structure were taken from
Yale Virtual Centre for Cellular Expression Profiling of Rice http://bioinformatics.med.yale.edu/riceatlas/anatomy.jspx; http://commons.wikimedia.org/wiki/
Image:Nymphaea_alba.jpg and http://commons.wikimedia.org/wiki/Image:Gnetum_gnemon_male.jpg respectively.

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Flower Development

Authors: Alvarez-Buylla, Elena R., Benítez, Mariana, Corvera-Poiré,

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First published on March 23, 2010: e0127. 10.1199/tab.0127

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Time scale (MYBP)

Figure 1. Phylogenetic context of Arabidopsis thaliana: Evolutionary history of land plants.

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Time (h) 30 42 6 18 24 12 192 36 Up to 24 -

Figure 3. Summary of the 20 stages of ﬂower development.

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Figure 4. Stages 1 to 6 of Arabidopsis ﬂower development.

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Figure 5. Stages 7 to 10 of Arabidopsis ﬂower development.

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Figure 6. Stages 11 to 16 of Arabidopsis ﬂower development.

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3.2 Floral Meristem Speciﬁcation and Determination

The changes in cellular characteristics, growth and geometry ob-

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expressed in the IM that is then transformed into a FM (Shannon

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FUL AGL24 SOC1 TFL1 WUS LFY AP1 AP2 CAL

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ap2 pi C ap2 ag B ap3 ag A ap2 pi ag

Figure 11. Arabidopsis ABC homeotic ﬂoral mutants.

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genes having cell-speciﬁc roles are turned on especially during

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Primordia positioning PHV Primordia polarity

GA STM CLV3 LFY

Primordia identity & growth

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2oP (outer) Endothecium

STAMEN PRIMORDIUM ARCHESPORIAL CELLS MEIOSIS I AND II ANTHER STAGE 14

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Pollen mitosis Anther dehiscence

Figure 18. Hormones in late stages of stamen development.

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flower stage 6 flower stage 11 flower stage 12-13

CARPEL PRIMORDIUM ARCHESPORIAL CELLS MEIOSIS AND MITOSIS

ETT AG JAG FIL YAB3

CRC/YAB6 IND ALC

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