Download Cotyledon organogenesis - Journal of Experimental Botany

Journal of Experimental Botany, Vol. 59, No. 11, pp. 2917–2931, 2008
doi:10.1093/jxb/ern167 Advance Access publication 8 July, 2008
REVIEW ARTICLE
Cotyledon organogenesis
John W. Chandler*
Department of Developmental Biology, University of Cologne, Gyrhofstrasse 17, D-50923 Cologne, Germany
Received 15 April 2008; Revised 15 May 2008; Accepted 19 May 2008
Abstract
The cotyledon represents one of the bases of classification within the plant kingdom, providing the
name-giving difference between dicotyledonous and
monocotyledonous plants. It is also a fundamental
organ and there have been many reports of cotyledon
mutants in many species. The use of these mutants
where they have arisen in Arabidopsis has allowed us
to unravel some of the complexities of embryonic
patterning and cotyledon development with a high
degree of resolution. The cloning of genes involved in
cotyledon development from other species, together
with physiological work, has supported the hypothesis
that there exists a small number of orthologous gene
hierarchies, particularly those involving auxin. The
time is therefore appropriate for a summary of the
regulation of cotyledon development gleaned from
cotyledon mutants and regulatory pathways in the
model species Arabidopsis and what can be inferred
from cotyledon mutants in other species. There is an
enormous variation in cotyledon form and development throughout the plant kingdom and this review
focuses on debates about the phylogenetic relationship between mono- and dicotyledony, discusses
gymnosperm cotyledon development and pleiocotyly
in natural populations, and explores the limits of
homology between cotyledons and leaves.
Key words: Arabidopsis, auxin, cotyledon, dicots, evolution,
monocots, natural variation, phylogeny.
Introduction
The patterning mechanisms responsible for generating any
plant organ are complex and involve many gene hierarchies, and co-ordinated spatially and temporally regulated
programmes of cell divisions, gene expression, and
hormone function. The developmental regulation of leaf
and root growth has been extensively described and
reviewed (Fleming, 2005; Hochholdinger and Zimmermann,
2008), as have the patterning processes during embryogenesis responsible for establishing the meristems, mainly
in Arabidopsis (Laux et al., 2004; Jenik and Barton,
2005; Jenik et al., 2007; Nawy et al., 2008), and
maize (Vernoud et al., 2005). However, the development of the cotyledon as an organ has been neglected,
although cotyledons are often the first aerial organ to
differentiate and their initiation is inherent in embryonic
patterning processes. With the explosion in the use of
cotyledon mutants as tools for gene cloning (see Table 1
for a summary of cotyledon mutants in Arabidopsis and
other species), many genetic pathways have been elucidated, which are usually included in reviews on embryonic patterning. However, one alternative consideration
which provides a framework for thinking about cotyledon development, is the similarity between leaf and
cotyledon development. Although dicot cotyledons
and leaves have different functions in terms of storage
and photosynthesis and morphological differences such as
trichomes and stipules on leaves, on the basis of
comparative morphology, they can be considered homologous organs according to the following criteria: the
homologous apical positioning of both sets of organs on
the shoot, the presence of meristems at their bases
during primordium growth, similar organ expansion
programmes and mature morphology, and that intermediate structures exist between cotyledons and leaves (quoted
in Kaplan and Cooke, 1997). In addition to morphological
homology, cotyledon and leaf initiation share significant
signalling determinants, most notably resulting from the
asymmetric distribution of auxin; in a similar way to the
pre-patterning of cotyledon primordia outgrowth, leaf
primordia initiate at positions of maximum auxin concentration/perception resulting from polar PIN1 localization
(Reinhardt et al., 2003; Reinhardt, 2005; Heisler et al.,
2005; Jönsson et al., 2006). One important difference
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2918 Chandler
Table 1. Loci, mutations and redundant gene families affecting cotyledon organogenesis or pleiocotyly in various species
Species
Locus or mutant
Cotyledon phenotype
Reference
Arabidopsis
ALTERED MERISTEM PROGRAM1
ASYMMETRIC LEAVES1
DORNRÖSCHEN/DORNRÖSCHEN-LIKE
EXTRA COTYLEDON1,2
FACKEL
GURKE
HYDRA1
laterne (pinoid, enhancer of pinoid)
LEC/FUSCA
Supernumery cotyledons
Distorted cotyledons
Fused cotyledons, variable cotyledon number
Homeotic transformation of leaves into cotyledons
Malformed cotyledons
Cotyledons reduced or absent
Pleiocotyly
Cotyledons lacking
Homeotic transformation of cotyledons into
leaves
Cotyledon fusion variable cotyledon
number
Single cotyledons
Fused cotyledons, Pleiocotyly
Partially fused cotyleons
Temperature-dependent cotyledon defects
Variable cotyledon number
Chaudhury et al., 1993
Byrne et al., 2000
Chandler et al., 2007a
Conway and Poethig, 1997
Schrick et al., 2000
Torres-Ruiz et al., 1996
Topping et al., 1997
Treml et al., 2005
Meinke et al., 1994; Keith et al.,
1994
Furutani et al., 2007
MACCHI-BOU4 ENHANCER OF PINOID
MONOPTEROS
PINOID
SHOOTMERISTEMLESS
TOPLESS
TWIN1
Antirrhinum
Petunia
Tomato
Pea
TIR family
CUP-SHAPED COTYLEDON (CUC)
family
HD-ZIP Class III family
KANADI/YABBY families
Single cotyledons or absence
Altered cotyledon differentiation
PINFORMED1 family
YUCCA family
CONNATA
CUPULIFORMIS
PLEIOCOTYLEDONEA
NO APICAL MERISTEM
DEFECTIVE EMBRYO AND MERISTEM
LANCEOLATE
POLYCOTYLEDON
Single and fused cotyledons
Single cotyledons
Cotyledon fusion
Cotyledon fusion
Pleiocotyly
Partial cotyledon fusion
Pleiocotyly
Cotyledons sometimes lacking
Pleiocotyly
SINGLE COTYLEDON
Cotyledon fusion
Single cotyledons
Fused cotyledons
between cotyledon and leaf initiation resides in phyllotaxis: dicot cotyledons arise opposite each other, but
leaves are subsequently initiated in many different
phyllotactic patterns, however, the similarities provide an
analogous framework for comparing the developmental
genetic mechanisms in each case. With this analogy as
a background, this review consolidates what is known
about the initiation and outgrowth of cotyledons in the
model eudicot Arabidopsis thaliana and surveys mutants
known to affect cotyledon development in this and other
species.
The formal division of angiosperm species based on
morphological characteristics into Dicotyledonae and
Monocotyledonae classes was first created by John Ray
in his Methodus Platarum Nova in 1682. Cotyledon
number is one of the characteristics involved in this
division, with the Monocotyldonae (monocots) having
a single cotyledon and containing approximately 50 000
lineages and the Dicotyledonae comprising about 200 000
species. Similarly for leaves and flowers, the cotyledon as
an organ displays huge variation in morphology and
developmental programmes throughout the plant kingdom
and the evolutionary origin of the cotyledon will also be
Berleth and Jürgens, 1993
Bennet et al., 1995
Aida et al., 1999
Long et al., 2002
Vernon et al., 2001; Vernon and
Meinke, 2004
Dharmasiri et al., 2005b
Aida et al., 1997, 1999; Vroeman
et al., 2003
Prigge et al., 2005
Izhaki and Bowman, 2007; Eshed
et al., 2001; Siegfried et al., 1999
Friml et al., 2003
Cheng et al., 2007a
Stubbe, 1966
Weir et al., 2004
Stubbe, 1966
Souer et al., 1996
Keddie et al., 1998
Avasarala et al., 1996
Al Hammadi et al., 2003;
Madishetty et al., 2006
Liu et al., 1999
reviewed here, together with considerations of natural
variation in cotyledon number and development.
Cotyledon diversity
In dicots, the cotyledons are lateral organs and the shoot
apical meristem (SAM) is at a central position, whereas in
monocots the cotyledon is terminal and the SAM at
a lateral position. Cotyledons are formed during embryogenesis and are defined as either epigeal, if they emerge
above ground, expand following germination and become
photosynthetic, or hypogeal, if they remain below ground,
do not expand, and remain non-photosynthetic. Cotyledons can be ephemeral structures, only persisting for
a few days following emergence, or can be retained by the
plant throughout its vegetative life. They may have
petioles, or be sessile.
For most dicot species, the number of cotyledons is
invariate at two (Fig. 1A), however, exceptions include
some species of Acer, Juglans, and Coffea which have
three cotyledons (Eames, 1961; Duke, 1969) as well as
Raphanus (Dube et al., 1981) and Sesamum (Pillai and
Goyal, 1983), some members of the Ranales which have
Cotyledon development and evolution 2919
Fig. 1. Variation in cotyledon morphology amongst the plant kingdom. A typical eudicot seedling; Raphanus sativus (A). A monocot seedling;
a transverse section through a Zea mays (maize) kernel, showing the coleoptile (col), the scutellum, considered to be the single monocot cotyledon
(sc) and the endosperm (end) (B). A gymnosperm seedling: Pinus sylvestris with five cotyledons (C). An anisocotyledonous plant; Monophyllaea
horsfieldii showing the gradual development of unequally sized cotyledons: isocotyls 1 week after germination (D), the start of the expansion of
a single cotyledon 2 weeks after germination (E), and obvious anisocotyly after 3 weeks’ growth (F). Arabidopsis cotyledons in wild type (G) and in
the dornröschen (drn) mutant: a pseudo monocot (H), fused cotyledons (I), tricots (J), cup-shaped cotyledons (K), and a complete absence of
cotyledon in the drn drnl double mutant (L). All scale bars represent 1 mm.
three or four cotyledons (Datta, 1988) and some species of
Persoonia (Protaceae) (Fletcher, 1909). Conversely, the
genus Pinguicula is anomalous amongst eudicots for
sometimes having a single cotyledon (Degtjareva et al.,
2006) and some parasitic plants such as those from the
Orobanchaceae, Santalales, and Rafflesiales have no
cotyledons (Burgher, 1998). Monocots have a scutellum
(Fig. 1B), considered by many to be a single cotyledon,
which does not emerge above ground and can therefore
only be shown in a seedling cross-section (Fig. 1B). As
the scutellum remains underground it does not photosynthesize and therefore is not green, in contrast to most
epigeal dicot cotyledons. Diversity exists in monocot
cotyledon number, with some species such as Commelina,
Dioscorea, Tamus, and Tinantia having a second vestigial
cotyledon (Datta, 1988). Gymnosperms, distinct from
angiosperm cotyledon classification, have variable cotyledon numbers between species, ranging from two, to 24 for
Pinus maximartinezii; the largest number of cotyledons
known for a plant (Farjon and Styles, 1997). Cotyledon
number is also flexible within many gymnosperm species,
for example, 5–7 for Pinus radiata and 7–13 for Pinus
jeffreyi (Mirov, 1967), or 5 for Pinus sylvestris (Fig. 1C).
In addition to cotyledon number, cotyledons can be
classified according to size; dicots usually demonstrate
isocotyly: the development of two equally sized cotyledons. Anisocotyly, however, is the prolonged growth of
one cotyledon over the other, to give a large macrocotyledon and a small microcotyledon. This is demonstrated by Claytonia virginica (Cook), Capparis species
(Franceschini and Tressens, 2004) and members of the
Gesneriaceae including Streptocarpus rexii (Mantegazza
2920 Chandler
et al., 2007) and Monophyllaea horsfieldii (Tsukaya,
1997; Fig. 1D–F), where the macrocotyledon develops
from a basal meristem to resemble a large foliage leaf up
to 30 cm long, with no additional leaves being initiated
before floral induction. Syncotyly relates to partial or
complete fusion of cotyledons, as exists in species of
Calophyllum, Swietenia, Guarea, and Carapa, where
cotyledons are distally fused (Datta, 1988). The corollary
of syncotyly is schizocotyly, applied to cotyledon bifurcation or lobing which can result in supernumery
cotyledons. Schizocotyly occurs in seedlings of the
Chenopodiaceae, where one of two cotyledons may be
cleft, to give hemitrocotyly, or one cotyledon of a tricotyledonous plant bifurcated to give a hemitetracotyl
(Karschon, 1973). Various types of cotyledon syncotyly
or pleiocotyly are demonstrated by some developmental
mutants in Arabidopsis such as dornröschen and
dornröschen-like, different alleles of which show pleiocotyly and syncotyly at low penetrance (Fig. 1G–L). The
large diversity in cotyledon form and morphology
throughout the plant kingdom argues against a single
conserved ontogenetic programme.
The evolution of cotyledony
Cotyledons exist across the plant kingdom in gymnosperms, monocots, and eudicots. A distinction should be
made here between dicots as a group, containing all plants
with two cotyledons, where the term dicot is used as
a form of morphology only, and the eudicots, a monophyletic phyllogenetic group containing most dicot species. The Angiosperm Phylogeny Group website has
consolidated current views of land plant phylogeny
(http://www.mobot.org/MOBOT/research/APweb/) and it
is believed that the gymnosperms diverged about 385
MYA (Zimmer et al., 2007) from eudicot progenitors and
the monophyletic monocot group about 200 MYA
(Zimmer et al., 2007) (Fig. 2). There has been a longstanding debate as to the origin of monocotyledony, but
since the dicot state exists across all eudicot groups,
including the Gnetopsids, it is accepted to be ancestral to
monocotyly (Dean, 2002), suggesting that the monocot
state is derived (is an apomorphy). The exact position of
the Gnetopsids is also a subject of debate, with the
anthophyte hypothesis placing them as a sister group to
the angiosperms, but most molecular evidence places
them as a sister group to the conifers (Chaw et al., 2000;
Fig. 2). Dicotyly in diverse groups could also be the result
of independent coevolution and the debate as to whether
mono- or dicotyly represents the more primitive developmental programme is ongoing, with several alternative
theories: a single monocot cotyledon could have derived
from the fusion of two cotyledons, or dicotyly might
represent an evolutionary organ duplication or cotyledon
splitting event from the monocot state. Alternatively, the
Fig. 2. Schematic phylogeny of the plant kingdom, showing the
branching of the monocots and gymnosperms from eudicot ancestors
about 200 and 385 million years’ ago (MYA), respectively. The
dicotyledonous synapomorphy and monocot and gymnosperm cotyledon apomorphies are represented by circles on the cladogram. The
debated position of the Gnetales is also shown, according Chaw et al.
(2000).
monocot cotyledon state could have resulted from the loss
or suppression of both cotyledons and subsequent novel
apomorphic innovation, or by functional sub-specification,
whereby one cotyledon became retarded to form the first
plumular leaf and the other retained cotyledon position
and function, i.e. heterocotyly.
The taxonomic allocation into monocots or dicots has
never been entirely clear for some species of paleoherbs
such as Nymphaea (Tillich, 1990), which has a single
cotyledon with two lobes, which could also be interpreted
as two fused cotyledons. It has been suggested that
Nymphaea is a monocot (Titova and Batygina, 1986),
although molecular studies confirm a dicot association
(Nickrent and Soltis, 1995). Alternatively, the cotyledon
syncotyly of Nymphaea may represent a transitional
cotyledon form. Some members of the Hydatellaceae have
a sheathing structure which is similar to both a single
monocot cotyledon and to the two fused cotyledons of
Nymphaeales. The recent reclassification of the Hydatellaceae from the monocot order Poales to the Nymphaeales
(Sokoloff et al., 2007) illustrates how unclear morphological characteristics defining the monocot–dicot categories
can be and Hydatellaceae may demonstrate how a monocotyledonous condition could have arisen from the
dicotyly present in water lilies. The phenomenon of
dicotyledonous embryo formation in the monocot Agapanthus and the observation of stable transitional forms
between mono- and dicotyledonous embryos supports the
hypothesis of homology between monocot and dicot
cotyledons, and Titova (2003) has advanced the hypothesis that, at least in this species, monocotyly derived from
the dicotylous situation via congenital fusion of two
cotyledons and subsequent suppression of the growth of
one of them.
The question whether dicotyly is plesiomorphic to
monocotyly depends on the fundamental question of
Cotyledon development and evolution 2921
homology between both, i.e. a continuous evolutionary
origin should give rise to homologous structures (Burgher,
1998). Burgher claims that monocot and dicot cotyledons
are not homologous for the following reasons: firstly, both
sets of organs do not share equivalent positions, with the
terminal cotyledon of monocots not equivalent to the
lateral dicot organs. The second criterion is homology of
structure; dicot cotyledons are morphologically distinct
from succeeding leaves, whereas monocot cotyledons
share homology with succeeding leaves. Assuming homology, intermediate or transitional structures should be
found between both sets of cotyledons. Despite occasional
instances of unstereotypic cotyledons amongst both
monocots and dicots, these are considered by Burgher to
be exceptions and the structures in question do not
completely resemble the respective mono- or dicot
ontogeny. Finally, an assumption of homology is that
mono- and dicot cotyledons cannot co-occur in one plant
at the same developmental stage. It has been interpreted
that the third leaf in Nymphaea corresponds to the
monocot cotyledon (references quoted in Burgher, 1998),
and therefore, that dicot and monocot characteristics cooccur in Nymphaea. These lines of evidence against
a shared homology between mono- and dicot cotyledons
still leaves the question of the origin of both sets of organs
unresolved. The situation is confused since dicot plants
are not a monophyletic group (Soltis and Soltis, 2004) and
not all monocots possess a single cotyledon (Tillich,
1995).
With respect to homology between monocot and dicot
cotyledons, there is debate as to whether the scutellum or
coleoptile represents the monocot cotyledon. The grass
scutellum and dicot cotyledon are functionally equivalent
in being reserve lipid and protein storage organs, but to
what extent the grass scutellum represents the whole
cotyledon, part of it or an alternative structure is open
(Vernoud et al., 2005). The coleoptile protects the
plumule during germination and has also been considered
by some to be equivalent to the cotyledon or part of it
(Satoh et al., 1999). This debate is also related to whether
the coleoptile is part of the scutellum, the first leaf, or
a novel organ (reviewed in Vernoud et al., 2005) and is
influenced by the observation that the development of the
scutellum and coleoptile are genetically distinct (Satoh
et al., 1999). The evolution of gymnosperm cotyledons is
not clear, but they probably arose as a derived or
apomorphic character via cotyledon duplication or schizocotyly within an ancestral angiosperm.
Homology between cotyledons and leaves
Despite developmental similarities between cotyledons
and leaves, mutants exist which completely fail to develop
cotyledons, but correctly initiate leaf primordia. These
include several mutant combinations in Arabidopsis in
genes involved in auxin biosynthesis or PIN1 expression,
such as the laterne phenotype, caused by mutations in
both PINOID and MACCHI-BOU 4/ENHANCER OF
PINOID (Treml et al., 2005; Furutani et al., 2007) or
DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE
(DRNL) (Chandler et al., 2007), NON-PHOTOTROPHIC
HYPOCOTYL3 and PINOID or a loss of YUCCA1,
YUCCA4, and PINOID (Cheng et al., 2007a, b). In
addition, some mutants fail to establish a SAM, such as
stm in Arabidopsis, no apical meristem in Petunia (Souer
et al., 1996), and leaf pattern mutants in pea (Marx,
1987), but produce normal cotyledons. This genetic
uncoupling of cotyledon and leaf development demonstrates that they must each possess at least partly
independent developmental programmes.
Transformation of cotyledons into leaves and leaves
into cotyledons
Several mutants in Arabidopsis inform a discussion as to
whether leaves and cotyledons are homologous structures
or sui generis organs. These mutant phenotypes either
demonstrate partial transformation of leaves into cotyledons or cotyledons into leaves, respectively. The LEAFY
COTYLEDON (LEC) class of genes, LEC1 and LEC2, and
FUSCA3 (FUS3) provide functions required for normal
seed development. When mutated, cotyledons display
features usually associated with vegetative leaves, such as
trichomes, storage products, desiccation tolerance, and
more complex vasculature (West et al., 1994; Meinke
et al., 1994; Keith et al., 1994; Stone et al., 2001), and
over-expression results in seedlings with embryonic
characteristics (Stone et al., 2001). The lec cotyledon
phenotype has been interpreted as representing partial
homeosis (Meinke et al., 1994), or a disturbance in
heterochrony (Keith et al., 1994), leading to an altered
timing of developmental programmes. Similar to lec
mutants, mutants in the HYDRA1 or HYDRA2/FACKEL
genes in Arabidopsis have cotyledons with trichomes,
thereby having a mixed phase phenotype (Topping et al.,
1997; Jang et al., 2000; Schrick et al., 2000). Supplementation is defined as when the first true leaf is cotyledonary
in character and three such mutations have been characterized in Arabidopsis (Conway and Poethig, 1997); extra
cotyledon1 (xtc1), xtc2, and altered meristem program1
(amp1). These mutants produce leaves which phyllotactically develop perpendicular to the cotyledons, but possess
partial or complete cotyledonary characteristics such as
few or no trichomes, a simple venation pattern, and
protein, lipid, and starch storage bodies. Some amp
mutants show true polycotyly, with a true extra cotyledon
being produced (Chaudhury et al., 1993), but the basis for
the supplementation phenotypes is the precocious initiation of leaves during embryogenesis, and the mutations
2922 Chandler
are therefore heterochronic, rather than homeotic. However, the simultaneous presence of cotyledon- and leafspecific characteristics in xtc1, xtc2, amp, and the lec
mutants demonstrates that both cotyledons and leaves
must at least share some common regulatory programmes.
Phase change
Phase-change from embryonic to vegetative development
programmes has been little studied, but the question has
been addressed in Brassica napus, which has non-dormant
embryos (Fernandez, 1997). Precociously germinating
embryos either produce extra cotyledons in a spiral
phyllotaxy, possessing stipules, both leaf developmental
characteristics, or chimeric organs with sectors of leaf
tissue, characterized by trichomes and cotyledon tissue.
Expression of molecular storage protein markers, normally restricted to the embryo, showed that the fate of
a particular primordium was determined by the identity of
the cells immediately surrounding it, implicating short
range signalling pathways in primordium fate determination. As organ identity is related to the age of the embryo
in Brassica napus, homeosis of cotyledons and leaves is
a consequence of heterochrony, i.e. differentiation is
dependent on temporal events in cellular environments. In
anisocotylous plants such as Streptocarpus wendlandii,
growth of the macrocotyledon is accompanied by trichome initiation in the basal region, which is characteristic for leaves (Nishii et al., 2004). Cytokinin application
can convert the microcotyledon into a macrocotyledon in
this species (Nishii et al., 2004), suggesting that endogenous cytokinin may play a role in cotyledon to leaf phase
change.
Do cotyledons arise from the SAM?
If cotyledons and leaves share homology, the question
arises whether they develop from the SAM or independently from it (Kaplan and Cooke, 1997; Barton and
Poethig, 1993; Bowman and Eshed, 2000). If cotyledons
originate from stem cell precursors in the SAM, what
causes the phase-change switch to the initiation of leaf
primordia in a different phyllotaxy to cotyledons? and if
cotyledon development is independent of the SAM, what
are the instructive signals determining cotyledon precursor
cell fate? The SAM is usually defined in relation to its
position between the cotyledons and, although in Arabidopsis, fully mature SAM histology becomes apparent
only after cotyledon outgrowth, in some species, such as
Pinus strobus, the SAM is visible before cotyledon
initiation (Spurr, 1949). In addition, changes in gene
expression domains which pre-pattern cotyledon outgrowth and SAM initiation in Arabidopsis are already
established in the globular embryo, and clonal analyses
cannot alone define the presence and activity of the SAM
(Kaplan and Cooke, 1997). Long and Barton (1998) argue
against the origin of both leaves and cotyledons from the
SAM, on the basis that most cotyledon cells have never
expressed STM, in contrast to those of leaf primordia, and
that full SAM structure and activity is established
following cotyledon outgrowth. In anisocotylous species
such as Streptocarpus wendlandii (Nishii et al., 2004), the
macrocotyledon enlarges by basal meristem activity, but
as no leaves are subsequently produced, only an inflorescence, the meristem cannot be considered as a classical SAM. The question of the origin of the cotyledons can
not be solved by histology alone, partly because of the
huge developmental variation between species, and the
danger of using Arabidopsis as a single paradigm, but
only at the resolution of transcriptional cell profiling in
single cells to identify the cell fate determinants of the
cotyledon precursor cell(s).
Cotyledon morphogenesis in Arabidopsis
Cotyledon initiation and outgrowth
The initiation and outgrowth of cotyledons in eudicots as
exemplified by Arabidopsis is morphologically visible at
the late globular stage and represents a symmetry-breaking event, taking the embryo from a radially symmetrical
embryo to one having two planes of bilateral symmetry
(Chandler et al., 2008b). This re-definition of symmetry
creates two domains within the apical embryo region
which are each characterized by diagnostic gene expression patterns: an apical central domain, not to be confused
with the embryo central domain situated between apical
and basal domains, and an apical peripheral domain, with
a central-peripheral axis linking them (Fig. 3A). The
differential expression of several genes in these domains
leads to the establishment of the SAM in the central apical
domain. A feature of cotyledon outgrowth is the necessity
to maintain separate boundaries between the central
embryo domain and the enlarging cotyledon primordium
and between the two primordia and the role of the cupshaped cotyledon (CUC) genes is fundamental in this
process: Arabidopsis possesses three redundant NAC
domain transcription factors, CUC1, CUC2, and CUC3.
Double cuc1 cuc2 and triple cuc1 cuc2 cuc3 mutants
show complete cotyledon fusion to create cup-shaped
cotyledons and seedlings lack a SAM, demonstrating that
CUC gene functions are upstream of STM (Aida et al.,
1999; Takada et al., 2001; Vroeman et al., 2003). The
expression of CUC genes in the medial domain, between
apical central and peripheral regions, in the heart stage
embryo represses cell proliferation and differentiation in
this domain, thereby creating a boundary which separates
the developing cotyledons. In addition, CUC genes
positively regulate STM expression in the central apical
domain (Aida et al., 1999), where STM also represses
Cotyledon development and evolution 2923
Fig. 3. Schematic overview of embryo domains and the genetic
pathways involved in cotyledon initiation and outgrowth in Arabidopsis: a late globular/transition stage embryo at the point where the
cotyledons begin to bulge out from the embryo flanks and the embryo
changes from having radial symmetry to two planes of bilateral
symmetry. The central apical domain is distinguished from the
peripheral apical domain and the embryo apical, central, and basal
domains are shown (A). Genetic interactions involved in cotyledon
outgrowth (B). A heart stage embryo with cotyledon adaxial and abaxial
sides distinguished (C). The genetic interactions involved in cotyledon
polarization (D).
CUC expression (Fig. 3B), leading to mutually exclusive
expression domains (Aida et al., 1999). KNAT6 is also
expressed at the boundary between the SAM and
cotyledons and acts redundantly with STM in organ
separation via CUC genes (Belles-Boix et al., 2006). The
boundary of CUC gene expression is also regulated by
miR164 (Laufs et al., 2004) and TCP transcription factors
(Koyama et al., 2007) and the STM expression domain is
also positioned by DRN/DRNL (Chandler et al., 2008a). A
fundamental aspect of the transition phase of embryogenesis, when apical and basal organ fate is established, is the
repression of root-promoting gene cascades in the apical
embryo domain, which is provided by the activity of the
TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins (Long et al., 2006), co-repressors which function
together with AUX/IAA proteins in auxin-mediated
transcriptional repression (Szemenyei et al., 2008).
ASYMMETRIC LEAVES1 (AS1), a MYB domain transcription factor is required for normal cotyledon development and is involved in the control of cell
differentiation. AS1 expression marks the sub-epidermal
domain of the cotyledon initials and is later negatively
regulated by STM in the developing SAM, leading to AS1
expression in a sub-epidermal domain of the developing
cotyledons but is absent in the central domain (Byrne
et al., 2000).
Two redundant homeodomain proteins ATML1 and
PDF2 which maintain the identity of L1 cells are
necessary for cotyledon initiation (Abe et al., 2003), and
the serine protease ALE1 and receptor like kinases ALE2
and ACR4 have overlapping roles in regulating protoderm-specific gene expression and cotyledon development (Tanaka et al., 2007). This provides some
circumstantial evidence that appropriate protoderm differentiation in the embryo is a prerequisite for cotyledon
formation. This is further supported by rpk1 toad2 double
mutants, deficient in two receptor-like kinases, redundantly required for cotyledon development, which have
defects in the central domain protoderm of globular
embryos subjacent to cotyledon defects (Nodine et al.,
2007; Nodine and Tax, 2008). How embryo central
domain protoderm function is linked to cotyledon initiation is an open question, but it implicates an additional
signalling mechanism involved in cotyledon initiation to
those involving the apical central embryo domain.
Establishing central and peripheral embryo domains
and cotyledon differentiation
Continuing the theme of the analogous development of
cotyledons and leaves, polarity or sidedness is established
in both organs with respect to the SAM, with the adaxial
or upper side being that closest to the plant axis or SAM
and deriving from the embryo apical central domain, and
the abaxial side the opposite or underside generated by the
peripheral embryo domain (Fig. 3C). The adaxial and
abaxial surfaces of cotyledons are morphologically distinct with respect to cell size and stomatal density: the
adaxial epidermis possesses cells of uniform size and has
a flat surface with low stomatal density, whilst the abaxial
surface has interspersed large and small cells, a rough
surface and high stomatal density (Siegfried et al., 1999).
This polarization during cotyledon differentiation, similarly to that of leaves, is established by three gene
families: the class III HD-ZIP genes promote adaxial cell
fate and KANADI and YABBY families promote abaxial
cell fate (Fig. 3B). These roles are supported in each case
by gene expression domains, loss-of-function and gain-offunction phenotypes.
Of the five class III HD-ZIP gene family members,
PHAVOLUTA (PHV) and PHABULOSA (PHB) contribute
to a functional apical meristem and REVOLUTA (REV)
towards vasculature development, but all are also responsible for specifying the adaxial domain of the leaves
and cotyledons. PHV, PHB, and REV are expressed from
the early globular stages, but as the cotyledon develops,
its adaxial side derives from cells from the central domain,
whilst the abaxial side results from cells from the
peripheral domain and PHV and PHB transcripts localize
to the adaxial side of the emerging and developing
cotyledons (Emery et al., 2003; Prigge et al., 2005). The
2924 Chandler
YABBY (YAB) genes FILAMENTOUS FLOWER and
YAB3 are expressed from transition and heart stage
embryos onwards, respectively, and subsequently at the
abaxial side of the developing cotyledons, in cells derived
from the peripheral domain (Sawa et al., 1999; Siegfried
et al., 1999). Similarly, KAN1, KAN2, and KAN3,
encoding GARP family plant-specific transcription factors
(Eshed et al., 2001) are expressed in abaxial regions of
developing embryos and cotyledons (Kerstetter et al.,
2001; Eshed et al., 2004) and KAN4 expression is mainly
ovule-specific (McAbee et al., 2006). Although the exact
interaction between class III HD ZIP genes and KANADI/
YABBY genes is not clear, the mutually exclusive
expression domains of class III HD-ZIP genes and YABBY
and KANADI genes has led to models of mutual
antagonism (Emery et al., 2003; Eshed et al., 2004, Fig.
3D). In addition, the boundaries of class III HD-ZIP gene
expression are partly delineated by miRNA control
(Williams et al., 2005) and via interaction with the class
of LITTLE ZIPPER (ZPR) genes (Wenkel et al., 2007;
Fig. 3D).
Dominant gain-of-function alleles of PHV, PHB, and
REV alleles cause the adaxialization of lateral organs
(McConnell et al., 2001; Emery et al., 2003). Conversely,
a triple phb phv rev mutant develops a single abaxialized
cotyledon lacking bilateral symmetry (Emery et al., 2003),
showing that the genes are necessary for adaxial cell fate
and involved in hierarchies leading to the establishment of
bilateral symmetry. Constitutive over-expression of
KAN1, KAN2 or KAN3, or the YABBY genes FILAMENTOUS FLOWER1 (FIL) or YAB3 leads to constitutive
wild-type abaxial characteristics in cotyledons (Eshed
et al., 2001; Siegfried et al., 1999), suggesting that these
genes promote abaxial cell fate. Constitutive expression of
FIL or YAB3 (Siegfried et al., 1999) or the KAN genes
(Eshed et al., 2001) also results in SAM arrest and
together with the fact that phb-1d mutants have an
enlarged meristem, shows that SAM formation correlates
with alterations in expression boundaries of genes determining adaxial or abaxial cotyledon fate, and thereby,
that SAM maintenance and cotyledon differentiation
involves two-way signalling between them (Siegfried
et al., 1999; Golz, 2006). Not all single mutant YABBY
and KANADI genes display a mutant phenotype, but
double fil yab3 mutants result in a loss of polarity in
leaves, with a uniform mesophyll and the epidermis is
comprised of mosaics of abaxial and adaxial tissue
(Siegfried et al., 1999). Increasingly, severe polarity
phenotypes are observed in higher order kan mutants
(Eshed et al., 2004), such that kan1 kan 2 kan4 triple
mutants exhibit radialized leaf-like outgrowths on the
abaxial side of the developing cotyledons and hextuple
kan1 kan2 kan4 fil yab3 yab5 mutants initiate three
narrow cotyledons (Izhaki and Bowman, 2007). Polarity
defects in higher order class III HD-ZIP or KANADI
mutants (phb phv rev and kan1 kan 2 kan4) can be
explained by an altered PIN1 expression pattern, leading
to altered auxin maxima and, consequently, abnormal
organ initiation (Izhaki and Bowman, 2007).
The hormonal control of cotyledon development
Auxin synthesis, transport and perception
Auxin is a key regulator of cotyledon development as
shown by mutant phenotypes of many of the loci in Table
1 which are involved with either auxin biosynthesis, polar
transport or response, via the auxin receptor TIR1, or
genes known to be up- or downstream of auxin response
or sensitivity. In addition, many of these loci are members
of redundant gene families, such as PINFORMED (PIN;
Friml et al., 2003), YUCCA (Cheng et al., 2007a) and TIR
(Dharmasiri et al., 2005b). Two lines of evidence
demonstrate the importance of auxin synthesis or concentration in cotyledon organogenesis: the first is the use of
the synthetic auxin-responsive DR5 promoter which has
routinely been used to provide a read-out of auxin
concentration (Ulmasov et al., 1997; Benkovà et al.,
2003; Friml et al., 2003), although an absence of DR5
expression does not prove an absence of auxin maxima.
DR5 expression concentrates in the tips of the incipient
cotyledons in transition stage embryos and the tips of
developing cotyledons (Cheng et al., 2007a; Fig. 4A) and
correlates with an accumulation of IAA (Benkovà et al.,
2003). Secondly, expression of the YUCCA1 and
YUCCA4 genes, encoding flavin monooxygenase, a key
enzyme in Trp-dependent auxin biosynthesis is strong in
the central apical domain of the globular embryo and
subsequently peaks in the tips of cotyledons (Cheng et al.,
2007a). Auxin synthesized in the central apical domain is
redirected by PIN1-mediated polar auxin transport (Friml
et al., 2003).
It has been known for many years that treatment of
embryos with various polar auxin inhibitors inhibits
cotyledon and SAM development in wheat (FischerIglesias et al., 2001), Brassica napus (Ramesar-Fortner
and Yeung, 2001), Brassica juncea (Liu et al., 1993),
Arabidopsis (Benkovà et al., 2003) and, more recently, in
Norway Spruce (Larsson et al., 2008) and moss (Fujita
et al., 2008). Auxin is transported in Arabidopsis embryos
via the family of PIN auxin efflux transporters (Friml
et al., 2003). PIN1 is polarly localized in the L1 layer of
the developing embryo, such that the polarity correlates
with auxin accumulation in cotyledon tips (Benkovà et al.,
2003; Fig. 4B, C). Within the developing cotyledon, PIN1
becomes localized on the basal side of developing
vasculature cells to direct auxin flow into the centre of
the primordium and basipetally within the embryo
(Benkovà et al., 2003). Thus polar auxin transport is
responsible for creating auxin maxima necessary for
Cotyledon development and evolution 2925
Fig. 4. Cotyledons develop at sites of auxin maxima: An Arabidopsis heart stage embryo with GFP expression under control of the DR5 auxinresponsive promoter, representing a maximum for auxin concentration, perception or response marking the tips of the developing cotyledons (A).
PIN1–GFP expression under control of the PIN1 promoter in a transition stage (B) or heart stage Arabidopsis embryo (C), showing polar PIN1
localization to direct auxin flux in the L1 layer, towards the tip of the developing cotyledons.
correct cotyledon specification. The activity of the PIN
proteins is regulated by the phosphorylation state controlled by the PINOID serine/threonine protein kinase
(Benjamins et al., 2001) and PP2A phosphatase (Michniewicz et al., 2007) and PIN1 polar localization is also
regulated by ENHANCER OF PINOID/MACCHI BOU4
(Treml et al., 2005; Furutani et al., 2007). pin1 pid double
mutants completely lack cotyledons and the embryos are
characterized by an expansion in the expression domains
of CUC1, CUC2, and STM, demonstrating that the
negative regulation of the expression domains of these
genes by PIN1 and PID is necessary for normal cotyledon
initiation (Furutani et al., 2004).
The auxin receptor has been identified as TIR1
(Kepinski and Leyser, 2005; Dharmasiri et al., 2005a),
a member of a family of related AFB F-box proteins
(Dharmasiri et al., 2005b). Auxin binds to a hydrophobic
pocket within the TIR1 protein (Tan et al., 2007), thereby
stabilizing the interaction between TIR1 and Aux/IAA
proteins, targeting them for degradation and thereby
relieving auxin response factor (ARF) transcription factors
from transcriptional repression (Quint and Gray, 2006).
Higher order mutants between TIR1 and AFB genes show
cotyledon development defects (Dharmasiri et al., 2005b),
demonstrating that TIR and AFB proteins regulate auxin
responses during cotyledon development (Dharmasiri
et al., 2005b). Arabidopsis possesses 23 ARFs (Guilfoyle
and Hagen, 2007) which bind to auxin responsive
elements (AREs) in target genes and lead to their
activation or repression. The DRN promoter contains
canonical AREs (Chandler et al., 2008a), providing
further corroboration that auxin-controlled gene expression is relevant for cotyledon development.
Cytokinins
Some evidence implicates cytokinins in cotyledon development: in hybrid larch, exogeneous application of
benzyladenine caused a decrease in cotyledon number
(von Aderkas, 2002) and a monocotyledonous mutant of
Catharanthus roseus could be restored to normal dicotyledony by the application of kinetin, suggesting that the
mutant phenotype resulted from developmental-stagespecific cytokinin deficiency (Rai and Kumar, 2000). The
Arabidopsis amp mutant has a pleiotropic phenotype,
including polycotyly and considerably higher cytokinin
levels than the wild type (Chaudury et al., 1993), showing
that appropriate cytokinin levels play a role in normal
cotyledon initiation. The DORNRÖSCHEN (DRN)/
ENHANCER OF SHOOT REGENERATION1 (ESR1)
and DRN-LIKE/SUPPRESSOR OF PHYTOCHROMEB/
BOLITA/ESR2 genes have a role in cotyledon development, with mutants showing syncotyly and pleiocotyly
(Ikeda et al., 2006; Chandler et al., 2007) and they have
an additional role in cytokinin-independent shoot regeneration and are themselves regulated by cytokinins (Banno
et al., 2001; Ikeda et al., 2006).
Cotyledon development in other eudicots and
monocots
Although Arabidopsis has become the developmental
paradigm for eudicot patterning processes, it must be
reiterated that it is atypical amongst eudicot embryo
development in having an inflexible hallmark cell division
pattern. Embryo and cotyledon development in other
species have not been extensively studied at the molecular
level, only on a morphological descriptive basis (Wardlaw, 1955). Embryos of monocots and eudicots are
basically morphologically indistinguishable at the early
embryo stages and differences only become apparent at
the globular stage when, in eudicots, the outgrowth of the
cotyledons results in a heart-stage embryo and, in
monocots, the embryo is more elongated as the cotyledon
defines the primary axis (Burgher, 1998). Homologues
and orthologues of several Arabidopsis genes involved in
cotyledon organogenesis have been isolated from other
species and show a conservation of function: CUPULIFORMIS (CUP) from Antirrhinum is a CUC orthologue
2926 Chandler
(Weir et al., 2004), as is NO APICAL MERISTEM (NAM)
in Petunia (Souer et al., 1996) and mutations in these
genes lead to cotyledon phenotypes. In maize, ZmCUC3 is
a CUC3 orthologue and ZmNAM1 and ZmNAM2 are
CUC1/CUC2 orthologues (Zimmermann and Werr, 2005)
and their expression pattern supports a function for
ZmCUC3 in boundary specification and for ZmNAM1 and
ZmNAM2 in SAM establishment, similar to CUC gene
function in Arabidopsis. Demonstrating the orthologous
function of Arabidopsis genes involved in cotyledon
development in monocots is difficult, due to morphological differences. However, orthologues of STM exist in
maize (Knotted1; Long et al., 1996) and rice (Sentoku
et al., 1999) and the maize AS1 homologue is rough
sheath2 (RS2; Timmermans et al., 1999; Tsiantis et al.,
1999). barren inflorescence2 (bif2) in maize is an
orthologue of Arabidopsis PINOID (McSteen et al.,
2007) and bif2 homologues from other grass species and
their comparison with PINOID shows that the sequence,
expression, and function is conserved between monocots
and eudicots. PIN1 orthologues have also been isolated
from maize (Carraro et al., 2006), but as yet, not subject
to mutagenesis. The DRN homologue in maize, ZmDRN is
expressed in the prospective scutellum (Zimmermann and
Werr, 2007), thereby showing an analogous expression
pattern to that in Arabidopsis, where DRN expression prepatterns cotyledon development in the Arabidopsis globular embryo, and suggesting an orthologous function.
There is, therefore, clear conservation of many transcription factor hierarchies controlling cotyledon development in Arabidopsis in divergent plant species, and
involving auxin (Table 1). The function of these genes in
monocot cotyledon development is often hard to elucidate,
and even if they have no role in cotyledon development,
their further characterization by heterologous complementation, together with the isolation of more heterologous
genes will reveal the repertoire of ancestral pathways
affecting cotyledon development and those which have
sub-functionalized or diverged. An evolutionary developmental biology perspective should help to address this
question.
Cotyledon development in gymnosperms
Studies on cotyledon development in gymnosperms are
few and are mainly related to cotyledon number (Harrison
and Von Aderkas, 2004) and limited to somatic embryos
and morphological descriptions. However, a comparison
of expressed sequence tags from loblolly pine embryos
with those of Arabidopsis revealed a large conservation
with angiosperm embryogenesis-related genes (Cairney
et al., 2006). The variation in cotyledon number within
a given gymnosperm species correlates with embryo size,
which alters from year to year (Butts and Buchholz,
1940). There is a greater degree of variation in cotyledon
number in somatic embryos compared with zygotic
embryos. The ring-shaped initiation of multiple cotyledons in gymnosperms means that multiple axes of
bilateral symmetry are established by cotyledon initiation
and not just two axes as in eudicots.
Pleiocotyly and natural variation in cotyledon
number
An abnormally large number of cotyledons within
a species has been referred to in the literature as
pleiocotyly or polycotyly. Pleiocotyly in natural populations has not been extensively studied, although widespread examples of spontaneous tricotyledony have been
reviewed by Holtorp (1944) and Haskell (1954) in
Populus (Rajora and Zsuffa, 1986), Prosopis cieraria
(Nagesh et al., 2001a), Acacia mellifera (Nagesh et al.,
2001b), Tectona grandis (Nagesh and Kardam, 2004),
Sapindus emarginatus (Nagesh et al., 2004), and sunflower (Hu et al., 2005). The genetic basis for some of
these examples is not known, but a tricotyledonous
mutant of Catharanthus roseus was found to be controlled
by a single recessive mutation (Rai and Kumar, 2001). In
tomato, two independent loci causing polycotyledony
were characterized: the polycotyledon (poc) mutation was
mapped (Madishetty et al., 2006) and the mutant showed
increased levels of polar auxin transport, suggesting that
the cotyledon phenotype is a result of altered auxin
distribution (Al-Hammadi et al., 2003). The defective
embryo and meristems (dem) mutant also lacks a functional SAM and is caused by the mutation of a gene
encoding a novel protein of unknown function (Keddie
et al., 1998). In pea, three independent single cotyledon
(sic) loci when mutated, give rise to single or fused
cotyledons (Liu et al., 1999).
The frequency of pleiocotyly has been studied as
a complex trait in wild radish populations: Raphanus
raphanistrum and Raphanus sativus show either more or
fewer than two cotyledons at a frequency of between 0%
and 5% (Conner and Agrawal, 2005). Paternal half-sibling
families were compared to assess whether the low
phenotypic penetrance was due to stabilizing selection or
a lack of genetic variance for the trait. These alternatives
could not be distinguished due to the small samples and
low number of phenotypic plants. However, artificial
selection has shown that genetic variance for pleiocotyly
is not lacking: the frequency of tricotyledonous seedlings
resulting from a cross between wild and cultivated
sunflower lines increased from 2% in the F2 generation,
to about 50% in the F5 generation (Hu et al., 2005),
showing that additive genetic variation for polycotyly
exists. Similarly, artificial selection for pleiocotyly in
Antirrhinum majus was able to increase its frequency
Cotyledon development and evolution 2927
from 5% to 64% (Haskell, 1954), which suggests that lack
of genetic variation is not the reason for the usually
invariant number of cotyledons. The variable numbers of
cotyledons in gymnosperms has been addressed in clonal
populations of somatic embryos of hybrid larch and was
found to correlate directly with the diameter of the apical
embryo surface (Harrison and Von Aderkas, 2004).
Breeding for cotyledon number
information to evolutionary developmental biology will
increasingly reveal the extent of this conservation across
the plant kingdom and distinguish pathways which have
divergently evolved. It will also inform the debate on the
evolution of cotyledon states.
The similarities in the genetic regulation and auxindependent initiation and outgrowth of cotyledons and
leaves demonstrates the homology between the structures.
However, open questions such as what distinguishes leaf
founder cells from cotyledon precursor cells and how
cotyledon and leaf development differ, including determinants of phase change and the relationship of cotyledon
initiation to the SAM, remain tantalizing foci of research.
Natural variation in cotyledon number is underresearched and understanding the genetic basis of pleiocotyly could enable breeding agronomically important seed
oil crops for increased productivity based on this trait.
The possibility exists that additive variance in natural
populations of some plants could allow the selection of
pleiocotyly as a trait, although the potential adaptive value
of variable cotyledon numbers has not been well studied
and is dependent on understanding the genetic basis of
natural variation in cotyledon number. The possible
agricultural benefits of pleiocotyly or manipulating cotyledon size are greater yield and quality of oils in oil seed
plants such as rape or sunflower, where oil is stored in the
cotyledons rather than the endosperm. Additional cotyledons may even confer an enhancement in seedling
establishment; a tricotyledonous sunflower mutation had
no detrimental effect on growth and development and led
to three leaves per node, increasing photosynthetic surface
area and therefore potential productivity (Hu et al., 2005).
JWC thanks Melanie Cole for PIN1 and DR5 Arabidopsis embryo
pictures, Judith Nardmann for maize embryo photos, Siegfried
Werth for photography, Klaus Menrath and Jürgen Hintzsche for
providing plant material, and Wolfgang Werr for critical reading of
this manuscript. JWC also gratefully acknowledges funding for this
work from the Deutsche Forschungsgemeinschaft via SFB572.
Conclusions
References
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