review article cotyledon organogenesis

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 clas-

sification 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 develop-

ment throughout the plant kingdom and this review

focuses on debates about the phylogenetic relation-

ship 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 anyplant organ are complex and involve many gene hierar-chies, and co-ordinated spatially and temporally regulatedprogrammes of cell divisions, gene expression, andhormone function. The developmental regulation of leaf

and root growth has been extensively described andreviewed (Fleming, 2005; Hochholdinger and Zimmermann,2008), as have the patterning processes during embryo-

genesis responsible for establishing the meristems, mainlyin 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 devel-opment of the cotyledon as an organ has been neglected,although cotyledons are often the first aerial organ todifferentiate and their initiation is inherent in embryonic

patterning processes. With the explosion in the use ofcotyledon mutants as tools for gene cloning (see Table 1for a summary of cotyledon mutants in Arabidopsis and

other species), many genetic pathways have been eluci-dated, which are usually included in reviews on embry-onic patterning. However, one alternative consideration

which provides a framework for thinking about cotyle-don development, is the similarity between leaf andcotyledon development. Although dicot cotyledons

and leaves have different functions in terms of storageand photosynthesis and morphological differences such astrichomes and stipules on leaves, on the basis ofcomparative morphology, they can be considered homol-

ogous organs according to the following criteria: thehomologous apical positioning of both sets of organs onthe shoot, the presence of meristems at their bases

during primordium growth, similar organ expansionprogrammes and mature morphology, and that intermedi-ate structures exist between cotyledons and leaves (quoted

in Kaplan and Cooke, 1997). In addition to morphologicalhomology, cotyledon and leaf initiation share significantsignalling determinants, most notably resulting from the

asymmetric distribution of auxin; in a similar way to thepre-patterning of cotyledon primordia outgrowth, leafprimordia initiate at positions of maximum auxin concen-

tration/perception resulting from polar PIN1 localization(Reinhardt et al., 2003; Reinhardt, 2005; Heisler et al.,2005; Jonsson et al., 2006). One important difference

* To whom correspondence should be addressed. E-mail: [email protected]

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between cotyledon and leaf initiation resides in phyllo-taxis: dicot cotyledons arise opposite each other, butleaves are subsequently initiated in many differentphyllotactic patterns, however, the similarities provide ananalogous framework for comparing the developmentalgenetic mechanisms in each case. With this analogy asa background, this review consolidates what is knownabout the initiation and outgrowth of cotyledons in themodel eudicot Arabidopsis thaliana and surveys mutantsknown to affect cotyledon development in this and otherspecies.The formal division of angiosperm species based on

morphological characteristics into Dicotyledonae andMonocotyledonae classes was first created by John Rayin his Methodus Platarum Nova in 1682. Cotyledonnumber is one of the characteristics involved in thisdivision, with the Monocotyldonae (monocots) havinga single cotyledon and containing approximately 50 000lineages and the Dicotyledonae comprising about 200 000species. Similarly for leaves and flowers, the cotyledon asan organ displays huge variation in morphology anddevelopmental programmes throughout the plant kingdomand the evolutionary origin of the cotyledon will also be

reviewed here, together with considerations of naturalvariation in cotyledon number and development.

Cotyledon diversity

In dicots, the cotyledons are lateral organs and the shootapical meristem (SAM) is at a central position, whereas inmonocots the cotyledon is terminal and the SAM ata lateral position. Cotyledons are formed during embryo-genesis and are defined as either epigeal, if they emergeabove ground, expand following germination and becomephotosynthetic, or hypogeal, if they remain below ground,do not expand, and remain non-photosynthetic. Cotyle-dons can be ephemeral structures, only persisting fora few days following emergence, or can be retained by theplant throughout its vegetative life. They may havepetioles, or be sessile.For most dicot species, the number of cotyledons is

invariate at two (Fig. 1A), however, exceptions includesome species of Acer, Juglans, and Coffea which havethree cotyledons (Eames, 1961; Duke, 1969) as well asRaphanus (Dube et al., 1981) and Sesamum (Pillai andGoyal, 1983), some members of the Ranales which have

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 Supernumery cotyledons Chaudhury et al., 1993ASYMMETRIC LEAVES1 Distorted cotyledons Byrne et al., 2000DORNROSCHEN/DORNROSCHEN-LIKE Fused cotyledons, variable cotyledon number Chandler et al., 2007aEXTRA COTYLEDON1,2 Homeotic transformation of leaves into cotyledons Conway and Poethig, 1997FACKEL Malformed cotyledons Schrick et al., 2000GURKE Cotyledons reduced or absent Torres-Ruiz et al., 1996HYDRA1 Pleiocotyly Topping et al., 1997laterne (pinoid, enhancer of pinoid) Cotyledons lacking Treml et al., 2005LEC/FUSCA Homeotic transformation of cotyledons into

leavesMeinke et al., 1994; Keith et al.,1994

MACCHI-BOU4 ENHANCER OF PINOID Cotyledon fusion variable cotyledonnumber

Furutani et al., 2007

MONOPTEROS Single cotyledons Berleth and Jurgens, 1993PINOID Fused cotyledons, Pleiocotyly Bennet et al., 1995SHOOTMERISTEMLESS Partially fused cotyleons Aida et al., 1999TOPLESS Temperature-dependent cotyledon defects Long et al., 2002TWIN1 Variable cotyledon number Vernon et al., 2001; Vernon and

Meinke, 2004TIR family Single cotyledons Dharmasiri et al., 2005bCUP-SHAPED COTYLEDON (CUC)family

Fused cotyledons Aida et al., 1997, 1999; Vroemanet al., 2003

HD-ZIP Class III family Single cotyledons or absence Prigge et al., 2005KANADI/YABBY families Altered cotyledon differentiation Izhaki and Bowman, 2007; Eshed

et al., 2001; Siegfried et al., 1999PINFORMED1 family Single and fused cotyledons Friml et al., 2003YUCCA family Single cotyledons Cheng et al., 2007a

Antirrhinum CONNATA Cotyledon fusion Stubbe, 1966CUPULIFORMIS Cotyledon fusion Weir et al., 2004PLEIOCOTYLEDONEA Pleiocotyly Stubbe, 1966

Petunia NO APICAL MERISTEM Partial cotyledon fusion Souer et al., 1996Tomato DEFECTIVE EMBRYO AND MERISTEM Pleiocotyly Keddie et al., 1998

LANCEOLATE Cotyledons sometimes lacking Avasarala et al., 1996POLYCOTYLEDON Pleiocotyly Al Hammadi et al., 2003;

Madishetty et al., 2006Pea SINGLE COTYLEDON Cotyledon fusion Liu et al., 1999

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three or four cotyledons (Datta, 1988) and some species ofPersoonia (Protaceae) (Fletcher, 1909). Conversely, thegenus Pinguicula is anomalous amongst eudicots forsometimes having a single cotyledon (Degtjareva et al.,2006) and some parasitic plants such as those from theOrobanchaceae, Santalales, and Rafflesiales have nocotyledons (Burgher, 1998). Monocots have a scutellum(Fig. 1B), considered by many to be a single cotyledon,which does not emerge above ground and can thereforeonly be shown in a seedling cross-section (Fig. 1B). Asthe scutellum remains underground it does not photosyn-thesize and therefore is not green, in contrast to mostepigeal dicot cotyledons. Diversity exists in monocotcotyledon number, with some species such as Commelina,Dioscorea, Tamus, and Tinantia having a second vestigialcotyledon (Datta, 1988). Gymnosperms, distinct from

angiosperm cotyledon classification, have variable cotyle-don numbers between species, ranging from two, to 24 forPinus maximartinezii; the largest number of cotyledonsknown for a plant (Farjon and Styles, 1997). Cotyledonnumber is also flexible within many gymnosperm species,for example, 5–7 for Pinus radiata and 7–13 for Pinusjeffreyi (Mirov, 1967), or 5 for Pinus sylvestris (Fig. 1C).In addition to cotyledon number, cotyledons can be

classified according to size; dicots usually demonstrateisocotyly: the development of two equally sized cotyle-dons. Anisocotyly, however, is the prolonged growth ofone cotyledon over the other, to give a large macro-cotyledon and a small microcotyledon. This is demon-strated by Claytonia virginica (Cook), Capparis species(Franceschini and Tressens, 2004) and members of theGesneriaceae including Streptocarpus rexii (Mantegazza

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; Monophyllaeahorsfieldii showing the gradual development of unequally sized cotyledons: isocotyls 1 week after germination (D), the start of the expansion ofa single cotyledon 2 weeks after germination (E), and obvious anisocotyly after 3 weeks’ growth (F). Arabidopsis cotyledons in wild type (G) and inthe dornroschen (drn) mutant: a pseudo monocot (H), fused cotyledons (I), tricots (J), cup-shaped cotyledons (K), and a complete absence ofcotyledon in the drn drnl double mutant (L). All scale bars represent 1 mm.

Cotyledon development and evolution 2919


et al., 2007) and Monophyllaea horsfieldii (Tsukaya,1997; Fig. 1D–F), where the macrocotyledon developsfrom a basal meristem to resemble a large foliage leaf upto 30 cm long, with no additional leaves being initiatedbefore floral induction. Syncotyly relates to partial orcomplete fusion of cotyledons, as exists in species ofCalophyllum, Swietenia, Guarea, and Carapa, wherecotyledons are distally fused (Datta, 1988). The corollaryof syncotyly is schizocotyly, applied to cotyledon bi-furcation or lobing which can result in supernumerycotyledons. Schizocotyly occurs in seedlings of theChenopodiaceae, where one of two cotyledons may becleft, to give hemitrocotyly, or one cotyledon of a tricoty-ledonous plant bifurcated to give a hemitetracotyl(Karschon, 1973). Various types of cotyledon syncotylyor pleiocotyly are demonstrated by some developmentalmutants in Arabidopsis such as dornroschen anddornroschen-like, different alleles of which show pleioco-tyly and syncotyly at low penetrance (Fig. 1G–L). Thelarge diversity in cotyledon form and morphologythroughout the plant kingdom argues against a singleconserved ontogenetic programme.

The evolution of cotyledony

Cotyledons exist across the plant kingdom in gymno-sperms, monocots, and eudicots. A distinction should bemade here between dicots as a group, containing all plantswith two cotyledons, where the term dicot is used asa form of morphology only, and the eudicots, a mono-phyletic phyllogenetic group containing most dicot spe-cies. The Angiosperm Phylogeny Group website hasconsolidated current views of land plant phylogeny(http://www.mobot.org/MOBOT/research/APweb/) and itis believed that the gymnosperms diverged about 385MYA (Zimmer et al., 2007) from eudicot progenitors andthe monophyletic monocot group about 200 MYA(Zimmer et al., 2007) (Fig. 2). There has been a long-standing debate as to the origin of monocotyledony, butsince the dicot state exists across all eudicot groups,including the Gnetopsids, it is accepted to be ancestral tomonocotyly (Dean, 2002), suggesting that the monocotstate is derived (is an apomorphy). The exact position ofthe Gnetopsids is also a subject of debate, with theanthophyte hypothesis placing them as a sister group tothe angiosperms, but most molecular evidence placesthem as a sister group to the conifers (Chaw et al., 2000;Fig. 2). Dicotyly in diverse groups could also be the resultof independent coevolution and the debate as to whethermono- or dicotyly represents the more primitive develop-mental programme is ongoing, with several alternativetheories: a single monocot cotyledon could have derivedfrom the fusion of two cotyledons, or dicotyly mightrepresent an evolutionary organ duplication or cotyledonsplitting event from the monocot state. Alternatively, the

monocot cotyledon state could have resulted from the lossor suppression of both cotyledons and subsequent novelapomorphic innovation, or by functional sub-specification,whereby one cotyledon became retarded to form the firstplumular leaf and the other retained cotyledon positionand function, i.e. heterocotyly.The taxonomic allocation into monocots or dicots has

never been entirely clear for some species of paleoherbssuch as Nymphaea (Tillich, 1990), which has a singlecotyledon with two lobes, which could also be interpretedas two fused cotyledons. It has been suggested thatNymphaea is a monocot (Titova and Batygina, 1986),although molecular studies confirm a dicot association(Nickrent and Soltis, 1995). Alternatively, the cotyledonsyncotyly of Nymphaea may represent a transitionalcotyledon form. Some members of the Hydatellaceae havea sheathing structure which is similar to both a singlemonocot cotyledon and to the two fused cotyledons ofNymphaeales. The recent reclassification of the Hydatella-ceae from the monocot order Poales to the Nymphaeales(Sokoloff et al., 2007) illustrates how unclear morpholog-ical characteristics defining the monocot–dicot categoriescan be and Hydatellaceae may demonstrate how a mono-cotyledonous condition could have arisen from thedicotyly present in water lilies. The phenomenon ofdicotyledonous embryo formation in the monocot Aga-panthus and the observation of stable transitional formsbetween mono- and dicotyledonous embryos supports thehypothesis of homology between monocot and dicotcotyledons, and Titova (2003) has advanced the hypothe-sis that, at least in this species, monocotyly derived fromthe dicotylous situation via congenital fusion of twocotyledons and subsequent suppression of the growth ofone of them.The question whether dicotyly is plesiomorphic to

monocotyly depends on the fundamental question of

Fig. 2. Schematic phylogeny of the plant kingdom, showing thebranching of the monocots and gymnosperms from eudicot ancestorsabout 200 and 385 million years’ ago (MYA), respectively. Thedicotyledonous synapomorphy and monocot and gymnosperm cotyle-don apomorphies are represented by circles on the cladogram. Thedebated position of the Gnetales is also shown, according Chaw et al.(2000).

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http://www.mobot.org/MOBOT/research/APweb/

homology between both, i.e. a continuous evolutionaryorigin should give rise to homologous structures (Burgher,1998). Burgher claims that monocot and dicot cotyledonsare not homologous for the following reasons: firstly, bothsets of organs do not share equivalent positions, with theterminal cotyledon of monocots not equivalent to thelateral dicot organs. The second criterion is homology ofstructure; dicot cotyledons are morphologically distinctfrom succeeding leaves, whereas monocot cotyledonsshare homology with succeeding leaves. Assuming ho-mology, intermediate or transitional structures should befound between both sets of cotyledons. Despite occasionalinstances of unstereotypic cotyledons amongst bothmonocots and dicots, these are considered by Burgher tobe exceptions and the structures in question do notcompletely resemble the respective mono- or dicotontogeny. Finally, an assumption of homology is thatmono- and dicot cotyledons cannot co-occur in one plantat the same developmental stage. It has been interpretedthat the third leaf in Nymphaea corresponds to themonocot cotyledon (references quoted in Burgher, 1998),and therefore, that dicot and monocot characteristics co-occur in Nymphaea. These lines of evidence againsta shared homology between mono- and dicot cotyledonsstill leaves the question of the origin of both sets of organsunresolved. The situation is confused since dicot plantsare not a monophyletic group (Soltis and Soltis, 2004) andnot 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 orcoleoptile represents the monocot cotyledon. The grassscutellum and dicot cotyledon are functionally equivalentin being reserve lipid and protein storage organs, but towhat extent the grass scutellum represents the wholecotyledon, part of it or an alternative structure is open(Vernoud et al., 2005). The coleoptile protects theplumule during germination and has also been consideredby some to be equivalent to the cotyledon or part of it(Satoh et al., 1999). This debate is also related to whetherthe coleoptile is part of the scutellum, the first leaf, ora novel organ (reviewed in Vernoud et al., 2005) and isinfluenced by the observation that the development of thescutellum and coleoptile are genetically distinct (Satohet al., 1999). The evolution of gymnosperm cotyledons isnot clear, but they probably arose as a derived orapomorphic character via cotyledon duplication or schiz-ocotyly within an ancestral angiosperm.

Homology between cotyledons and leaves

Despite developmental similarities between cotyledonsand leaves, mutants exist which completely fail to developcotyledons, but correctly initiate leaf primordia. These

include several mutant combinations in Arabidopsis ingenes involved in auxin biosynthesis or PIN1 expression,such as the laterne phenotype, caused by mutations inboth PINOID and MACCHI-BOU 4/ENHANCER OFPINOID (Treml et al., 2005; Furutani et al., 2007) orDORNROSCHEN (DRN) and DORNROSCHEN-LIKE(DRNL) (Chandler et al., 2007), NON-PHOTOTROPHICHYPOCOTYL3 and PINOID or a loss of YUCCA1,YUCCA4, and PINOID (Cheng et al., 2007a, b). Inaddition, some mutants fail to establish a SAM, such asstm in Arabidopsis, no apical meristem in Petunia (Soueret al., 1996), and leaf pattern mutants in pea (Marx,1987), but produce normal cotyledons. This geneticuncoupling of cotyledon and leaf development demon-strates that they must each possess at least partlyindependent developmental programmes.

Transformation of cotyledons into leaves and leavesinto cotyledons

Several mutants in Arabidopsis inform a discussion as towhether leaves and cotyledons are homologous structuresor sui generis organs. These mutant phenotypes eitherdemonstrate partial transformation of leaves into cotyle-dons or cotyledons into leaves, respectively. The LEAFYCOTYLEDON (LEC) class of genes, LEC1 and LEC2, andFUSCA3 (FUS3) provide functions required for normalseed development. When mutated, cotyledons displayfeatures usually associated with vegetative leaves, such astrichomes, storage products, desiccation tolerance, andmore complex vasculature (West et al., 1994; Meinkeet al., 1994; Keith et al., 1994; Stone et al., 2001), andover-expression results in seedlings with embryoniccharacteristics (Stone et al., 2001). The lec cotyledonphenotype has been interpreted as representing partialhomeosis (Meinke et al., 1994), or a disturbance inheterochrony (Keith et al., 1994), leading to an alteredtiming of developmental programmes. Similar to lecmutants, mutants in the HYDRA1 or HYDRA2/FACKELgenes in Arabidopsis have cotyledons with trichomes,thereby having a mixed phase phenotype (Topping et al.,1997; Jang et al., 2000; Schrick et al., 2000). Supplemen-tation is defined as when the first true leaf is cotyledonaryin character and three such mutations have been charac-terized in Arabidopsis (Conway and Poethig, 1997); extracotyledon1 (xtc1), xtc2, and altered meristem program1(amp1). These mutants produce leaves which phyllotacti-cally develop perpendicular to the cotyledons, but possesspartial or complete cotyledonary characteristics such asfew or no trichomes, a simple venation pattern, andprotein, lipid, and starch storage bodies. Some ampmutants show true polycotyly, with a true extra cotyledonbeing produced (Chaudhury et al., 1993), but the basis forthe supplementation phenotypes is the precocious initia-tion of leaves during embryogenesis, and the mutations



are therefore heterochronic, rather than homeotic. How-ever, the simultaneous presence of cotyledon- and leaf-specific characteristics in xtc1, xtc2, amp, and the lecmutants demonstrates that both cotyledons and leavesmust at least share some common regulatory programmes.

Phase change

Phase-change from embryonic to vegetative developmentprogrammes has been little studied, but the question hasbeen addressed in Brassica napus, which has non-dormantembryos (Fernandez, 1997). Precociously germinatingembryos either produce extra cotyledons in a spiralphyllotaxy, possessing stipules, both leaf developmentalcharacteristics, or chimeric organs with sectors of leaftissue, characterized by trichomes and cotyledon tissue.Expression of molecular storage protein markers, nor-mally restricted to the embryo, showed that the fate ofa particular primordium was determined by the identity ofthe cells immediately surrounding it, implicating shortrange signalling pathways in primordium fate determina-tion. As organ identity is related to the age of the embryoin Brassica napus, homeosis of cotyledons and leaves isa consequence of heterochrony, i.e. differentiation isdependent on temporal events in cellular environments. Inanisocotylous plants such as Streptocarpus wendlandii,growth of the macrocotyledon is accompanied by tri-chome initiation in the basal region, which is characteris-tic for leaves (Nishii et al., 2004). Cytokinin applicationcan convert the microcotyledon into a macrocotyledon inthis species (Nishii et al., 2004), suggesting that endoge-nous cytokinin may play a role in cotyledon to leaf phasechange.

Do cotyledons arise from the SAM?

If cotyledons and leaves share homology, the questionarises whether they develop from the SAM or indepen-dently from it (Kaplan and Cooke, 1997; Barton andPoethig, 1993; Bowman and Eshed, 2000). If cotyledonsoriginate from stem cell precursors in the SAM, whatcauses the phase-change switch to the initiation of leafprimordia in a different phyllotaxy to cotyledons? and ifcotyledon development is independent of the SAM, whatare the instructive signals determining cotyledon precursorcell fate? The SAM is usually defined in relation to itsposition between the cotyledons and, although in Arabi-dopsis, fully mature SAM histology becomes apparentonly after cotyledon outgrowth, in some species, such asPinus strobus, the SAM is visible before cotyledoninitiation (Spurr, 1949). In addition, changes in geneexpression domains which pre-pattern cotyledon out-growth and SAM initiation in Arabidopsis are alreadyestablished in the globular embryo, and clonal analysescannot alone define the presence and activity of the SAM

(Kaplan and Cooke, 1997). Long and Barton (1998) argueagainst the origin of both leaves and cotyledons from theSAM, on the basis that most cotyledon cells have neverexpressed STM, in contrast to those of leaf primordia, andthat full SAM structure and activity is establishedfollowing cotyledon outgrowth. In anisocotylous speciessuch as Streptocarpus wendlandii (Nishii et al., 2004), themacrocotyledon enlarges by basal meristem activity, butas no leaves are subsequently produced, only an in-florescence, the meristem cannot be considered as a classi-cal SAM. The question of the origin of the cotyledons cannot be solved by histology alone, partly because of thehuge developmental variation between species, and thedanger of using Arabidopsis as a single paradigm, butonly at the resolution of transcriptional cell profiling insingle cells to identify the cell fate determinants of thecotyledon precursor cell(s).

Cotyledon morphogenesis in Arabidopsis

Cotyledon initiation and outgrowth

The initiation and outgrowth of cotyledons in eudicots asexemplified by Arabidopsis is morphologically visible atthe late globular stage and represents a symmetry-break-ing event, taking the embryo from a radially symmetricalembryo to one having two planes of bilateral symmetry(Chandler et al., 2008b). This re-definition of symmetrycreates two domains within the apical embryo regionwhich are each characterized by diagnostic gene expres-sion patterns: an apical central domain, not to be confusedwith the embryo central domain situated between apicaland basal domains, and an apical peripheral domain, witha central-peripheral axis linking them (Fig. 3A). Thedifferential expression of several genes in these domainsleads to the establishment of the SAM in the central apicaldomain. A feature of cotyledon outgrowth is the necessityto maintain separate boundaries between the centralembryo domain and the enlarging cotyledon primordiumand between the two primordia and the role of the cup-shaped cotyledon (CUC) genes is fundamental in thisprocess: Arabidopsis possesses three redundant NACdomain transcription factors, CUC1, CUC2, and CUC3.Double cuc1 cuc2 and triple cuc1 cuc2 cuc3 mutantsshow complete cotyledon fusion to create cup-shapedcotyledons and seedlings lack a SAM, demonstrating thatCUC gene functions are upstream of STM (Aida et al.,1999; Takada et al., 2001; Vroeman et al., 2003). Theexpression of CUC genes in the medial domain, betweenapical central and peripheral regions, in the heart stageembryo represses cell proliferation and differentiation inthis domain, thereby creating a boundary which separatesthe developing cotyledons. In addition, CUC genespositively regulate STM expression in the central apicaldomain (Aida et al., 1999), where STM also represses

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CUC expression (Fig. 3B), leading to mutually exclusiveexpression domains (Aida et al., 1999). KNAT6 is alsoexpressed at the boundary between the SAM andcotyledons and acts redundantly with STM in organseparation via CUC genes (Belles-Boix et al., 2006). Theboundary of CUC gene expression is also regulated bymiR164 (Laufs et al., 2004) and TCP transcription factors(Koyama et al., 2007) and the STM expression domain isalso positioned by DRN/DRNL (Chandler et al., 2008a). Afundamental aspect of the transition phase of embryogen-esis, when apical and basal organ fate is established, is therepression of root-promoting gene cascades in the apicalembryo domain, which is provided by the activity of theTOPLESS (TPL) and TOPLESS-RELATED (TPR) pro-teins (Long et al., 2006), co-repressors which functiontogether with AUX/IAA proteins in auxin-mediatedtranscriptional repression (Szemenyei et al., 2008).

ASYMMETRIC LEAVES1 (AS1), a MYB domain tran-scription factor is required for normal cotyledon de-velopment and is involved in the control of celldifferentiation. AS1 expression marks the sub-epidermaldomain of the cotyledon initials and is later negativelyregulated by STM in the developing SAM, leading to AS1expression in a sub-epidermal domain of the developingcotyledons but is absent in the central domain (Byrneet al., 2000).

Two redundant homeodomain proteins ATML1 andPDF2 which maintain the identity of L1 cells arenecessary for cotyledon initiation (Abe et al., 2003), andthe serine protease ALE1 and receptor like kinases ALE2and ACR4 have overlapping roles in regulating pro-toderm-specific gene expression and cotyledon develop-ment (Tanaka et al., 2007). This provides somecircumstantial evidence that appropriate protoderm differ-entiation in the embryo is a prerequisite for cotyledonformation. This is further supported by rpk1 toad2 doublemutants, deficient in two receptor-like kinases, redun-dantly required for cotyledon development, which havedefects in the central domain protoderm of globularembryos subjacent to cotyledon defects (Nodine et al.,2007; Nodine and Tax, 2008). How embryo centraldomain protoderm function is linked to cotyledon initia-tion is an open question, but it implicates an additionalsignalling mechanism involved in cotyledon initiation tothose involving the apical central embryo domain.

Establishing central and peripheral embryo domainsand cotyledon differentiation

Continuing the theme of the analogous development ofcotyledons and leaves, polarity or sidedness is establishedin both organs with respect to the SAM, with the adaxialor upper side being that closest to the plant axis or SAMand deriving from the embryo apical central domain, andthe abaxial side the opposite or underside generated by theperipheral embryo domain (Fig. 3C). The adaxial andabaxial surfaces of cotyledons are morphologically dis-tinct with respect to cell size and stomatal density: theadaxial epidermis possesses cells of uniform size and hasa flat surface with low stomatal density, whilst the abaxialsurface has interspersed large and small cells, a roughsurface and high stomatal density (Siegfried et al., 1999).This polarization during cotyledon differentiation, simi-larly to that of leaves, is established by three genefamilies: the class III HD-ZIP genes promote adaxial cellfate and KANADI and YABBY families promote abaxialcell fate (Fig. 3B). These roles are supported in each caseby gene expression domains, loss-of-function and gain-of-function phenotypes.Of the five class III HD-ZIP gene family members,

PHAVOLUTA (PHV) and PHABULOSA (PHB) contributeto a functional apical meristem and REVOLUTA (REV)towards vasculature development, but all are also re-sponsible for specifying the adaxial domain of the leavesand cotyledons. PHV, PHB, and REV are expressed fromthe 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 theperipheral domain and PHV and PHB transcripts localizeto the adaxial side of the emerging and developingcotyledons (Emery et al., 2003; Prigge et al., 2005). The

Fig. 3. Schematic overview of embryo domains and the geneticpathways involved in cotyledon initiation and outgrowth in Arabidop-sis: a late globular/transition stage embryo at the point where thecotyledons begin to bulge out from the embryo flanks and the embryochanges from having radial symmetry to two planes of bilateralsymmetry. The central apical domain is distinguished from theperipheral apical domain and the embryo apical, central, and basaldomains are shown (A). Genetic interactions involved in cotyledonoutgrowth (B). A heart stage embryo with cotyledon adaxial and abaxialsides distinguished (C). The genetic interactions involved in cotyledonpolarization (D).



YABBY (YAB) genes FILAMENTOUS FLOWER andYAB3 are expressed from transition and heart stageembryos onwards, respectively, and subsequently at theabaxial side of the developing cotyledons, in cells derivedfrom the peripheral domain (Sawa et al., 1999; Siegfriedet al., 1999). Similarly, KAN1, KAN2, and KAN3,encoding GARP family plant-specific transcription factors(Eshed et al., 2001) are expressed in abaxial regions ofdeveloping embryos and cotyledons (Kerstetter et al.,2001; Eshed et al., 2004) and KAN4 expression is mainlyovule-specific (McAbee et al., 2006). Although the exactinteraction between class III HD ZIP genes and KANADI/YABBY genes is not clear, the mutually exclusiveexpression domains of class III HD-ZIP genes and YABBYand KANADI genes has led to models of mutualantagonism (Emery et al., 2003; Eshed et al., 2004, Fig.3D). In addition, the boundaries of class III HD-ZIP geneexpression are partly delineated by miRNA control(Williams et al., 2005) and via interaction with the classof 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 abaxializedcotyledon lacking bilateral symmetry (Emery et al., 2003),showing that the genes are necessary for adaxial cell fateand involved in hierarchies leading to the establishment ofbilateral symmetry. Constitutive over-expression ofKAN1, KAN2 or KAN3, or the YABBY genes FILAMEN-TOUS FLOWER1 (FIL) or YAB3 leads to constitutivewild-type abaxial characteristics in cotyledons (Eshedet al., 2001; Siegfried et al., 1999), suggesting that thesegenes promote abaxial cell fate. Constitutive expression ofFIL or YAB3 (Siegfried et al., 1999) or the KAN genes(Eshed et al., 2001) also results in SAM arrest andtogether with the fact that phb-1d mutants have anenlarged meristem, shows that SAM formation correlateswith alterations in expression boundaries of genes de-termining adaxial or abaxial cotyledon fate, and thereby,that SAM maintenance and cotyledon differentiationinvolves two-way signalling between them (Siegfriedet al., 1999; Golz, 2006). Not all single mutant YABBYand KANADI genes display a mutant phenotype, butdouble fil yab3 mutants result in a loss of polarity inleaves, with a uniform mesophyll and the epidermis iscomprised of mosaics of abaxial and adaxial tissue(Siegfried et al., 1999). Increasingly, severe polarityphenotypes are observed in higher order kan mutants(Eshed et al., 2004), such that kan1 kan 2 kan4 triplemutants exhibit radialized leaf-like outgrowths on theabaxial side of the developing cotyledons and hextuplekan1 kan2 kan4 fil yab3 yab5 mutants initiate threenarrow cotyledons (Izhaki and Bowman, 2007). Polaritydefects in higher order class III HD-ZIP or KANADI

mutants (phb phv rev and kan1 kan 2 kan4) can beexplained by an altered PIN1 expression pattern, leadingto altered auxin maxima and, consequently, abnormalorgan initiation (Izhaki and Bowman, 2007).

The hormonal control of cotyledon development

Auxin synthesis, transport and perception

Auxin is a key regulator of cotyledon development asshown by mutant phenotypes of many of the loci in Table1 which are involved with either auxin biosynthesis, polartransport or response, via the auxin receptor TIR1, orgenes known to be up- or downstream of auxin responseor sensitivity. In addition, many of these loci are membersof 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 evidencedemonstrate the importance of auxin synthesis or concen-tration in cotyledon organogenesis: the first is the use ofthe synthetic auxin-responsive DR5 promoter which hasroutinely been used to provide a read-out of auxinconcentration (Ulmasov et al., 1997; Benkova et al.,2003; Friml et al., 2003), although an absence of DR5expression does not prove an absence of auxin maxima.DR5 expression concentrates in the tips of the incipientcotyledons in transition stage embryos and the tips ofdeveloping cotyledons (Cheng et al., 2007a; Fig. 4A) andcorrelates with an accumulation of IAA (Benkova et al.,2003). Secondly, expression of the YUCCA1 andYUCCA4 genes, encoding flavin monooxygenase, a keyenzyme in Trp-dependent auxin biosynthesis is strong inthe central apical domain of the globular embryo andsubsequently peaks in the tips of cotyledons (Cheng et al.,2007a). Auxin synthesized in the central apical domain isredirected by PIN1-mediated polar auxin transport (Frimlet al., 2003).It has been known for many years that treatment of

embryos with various polar auxin inhibitors inhibitscotyledon and SAM development in wheat (Fischer-Iglesias et al., 2001), Brassica napus (Ramesar-Fortnerand Yeung, 2001), Brassica juncea (Liu et al., 1993),Arabidopsis (Benkova et al., 2003) and, more recently, inNorway Spruce (Larsson et al., 2008) and moss (Fujitaet al., 2008). Auxin is transported in Arabidopsis embryosvia the family of PIN auxin efflux transporters (Frimlet al., 2003). PIN1 is polarly localized in the L1 layer ofthe developing embryo, such that the polarity correlateswith auxin accumulation in cotyledon tips (Benkova et al.,2003; Fig. 4B, C). Within the developing cotyledon, PIN1becomes localized on the basal side of developingvasculature cells to direct auxin flow into the centre ofthe primordium and basipetally within the embryo(Benkova et al., 2003). Thus polar auxin transport isresponsible for creating auxin maxima necessary for

2924 Chandler


correct cotyledon specification. The activity of the PINproteins is regulated by the phosphorylation state con-trolled by the PINOID serine/threonine protein kinase(Benjamins et al., 2001) and PP2A phosphatase (Michnie-wicz et al., 2007) and PIN1 polar localization is alsoregulated by ENHANCER OF PINOID/MACCHI BOU4(Treml et al., 2005; Furutani et al., 2007). pin1 pid doublemutants completely lack cotyledons and the embryos arecharacterized by an expansion in the expression domainsof CUC1, CUC2, and STM, demonstrating that thenegative regulation of the expression domains of thesegenes by PIN1 and PID is necessary for normal cotyledoninitiation (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 hydrophobicpocket within the TIR1 protein (Tan et al., 2007), therebystabilizing the interaction between TIR1 and Aux/IAAproteins, targeting them for degradation and therebyrelieving auxin response factor (ARF) transcription factorsfrom transcriptional repression (Quint and Gray, 2006).Higher order mutants between TIR1 and AFB genes showcotyledon development defects (Dharmasiri et al., 2005b),demonstrating that TIR and AFB proteins regulate auxinresponses during cotyledon development (Dharmasiriet al., 2005b). Arabidopsis possesses 23 ARFs (Guilfoyleand Hagen, 2007) which bind to auxin responsiveelements (AREs) in target genes and lead to theiractivation or repression. The DRN promoter containscanonical AREs (Chandler et al., 2008a), providingfurther corroboration that auxin-controlled gene expres-sion is relevant for cotyledon development.

Cytokinins

Some evidence implicates cytokinins in cotyledon de-velopment: in hybrid larch, exogeneous application ofbenzyladenine caused a decrease in cotyledon number(von Aderkas, 2002) and a monocotyledonous mutant of

Catharanthus roseus could be restored to normal dicoty-ledony by the application of kinetin, suggesting that themutant phenotype resulted from developmental-stage-specific cytokinin deficiency (Rai and Kumar, 2000). TheArabidopsis amp mutant has a pleiotropic phenotype,including polycotyly and considerably higher cytokininlevels than the wild type (Chaudury et al., 1993), showingthat appropriate cytokinin levels play a role in normalcotyledon initiation. The DORNROSCHEN (DRN)/ENHANCER OF SHOOT REGENERATION1 (ESR1)and DRN-LIKE/SUPPRESSOR OF PHYTOCHROMEB/BOLITA/ESR2 genes have a role in cotyledon develop-ment, with mutants showing syncotyly and pleiocotyly(Ikeda et al., 2006; Chandler et al., 2007) and they havean additional role in cytokinin-independent shoot regener-ation and are themselves regulated by cytokinins (Bannoet al., 2001; Ikeda et al., 2006).

Cotyledon development in other eudicots andmonocots

Although Arabidopsis has become the developmentalparadigm for eudicot patterning processes, it must bereiterated that it is atypical amongst eudicot embryodevelopment in having an inflexible hallmark cell divisionpattern. Embryo and cotyledon development in otherspecies have not been extensively studied at the molecularlevel, only on a morphological descriptive basis (Ward-law, 1955). Embryos of monocots and eudicots arebasically morphologically indistinguishable at the earlyembryo stages and differences only become apparent atthe globular stage when, in eudicots, the outgrowth of thecotyledons results in a heart-stage embryo and, inmonocots, the embryo is more elongated as the cotyledondefines the primary axis (Burgher, 1998). Homologuesand orthologues of several Arabidopsis genes involved incotyledon organogenesis have been isolated from otherspecies and show a conservation of function: CUPULI-FORMIS (CUP) from Antirrhinum is a CUC orthologue

Fig. 4. Cotyledons develop at sites of auxin maxima: An Arabidopsis heart stage embryo with GFP expression under control of the DR5 auxin-responsive 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 PIN1localization to direct auxin flux in the L1 layer, towards the tip of the developing cotyledons.



(Weir et al., 2004), as is NO APICAL MERISTEM (NAM)in Petunia (Souer et al., 1996) and mutations in thesegenes lead to cotyledon phenotypes. In maize, ZmCUC3 isa CUC3 orthologue and ZmNAM1 and ZmNAM2 areCUC1/CUC2 orthologues (Zimmermann and Werr, 2005)and their expression pattern supports a function forZmCUC3 in boundary specification and for ZmNAM1 andZmNAM2 in SAM establishment, similar to CUC genefunction in Arabidopsis. Demonstrating the orthologousfunction of Arabidopsis genes involved in cotyledondevelopment in monocots is difficult, due to morpholog-ical differences. However, orthologues of STM exist inmaize (Knotted1; Long et al., 1996) and rice (Sentokuet al., 1999) and the maize AS1 homologue is roughsheath2 (RS2; Timmermans et al., 1999; Tsiantis et al.,1999). barren inflorescence2 (bif2) in maize is anorthologue of Arabidopsis PINOID (McSteen et al.,2007) and bif2 homologues from other grass species andtheir comparison with PINOID shows that the sequence,expression, and function is conserved between monocotsand eudicots. PIN1 orthologues have also been isolatedfrom maize (Carraro et al., 2006), but as yet, not subjectto mutagenesis. The DRN homologue in maize, ZmDRN isexpressed in the prospective scutellum (Zimmermann andWerr, 2007), thereby showing an analogous expressionpattern to that in Arabidopsis, where DRN expression pre-patterns cotyledon development in the Arabidopsis globu-lar embryo, and suggesting an orthologous function.There is, therefore, clear conservation of many tran-

scription factor hierarchies controlling cotyledon develop-ment in Arabidopsis in divergent plant species, andinvolving auxin (Table 1). The function of these genes inmonocot cotyledon development is often hard to elucidate,and even if they have no role in cotyledon development,their further characterization by heterologous complemen-tation, together with the isolation of more heterologousgenes will reveal the repertoire of ancestral pathwaysaffecting cotyledon development and those which havesub-functionalized or diverged. An evolutionary develop-mental biology perspective should help to address thisquestion.

Cotyledon development in gymnosperms

Studies on cotyledon development in gymnosperms arefew and are mainly related to cotyledon number (Harrisonand Von Aderkas, 2004) and limited to somatic embryosand morphological descriptions. However, a comparisonof expressed sequence tags from loblolly pine embryoswith those of Arabidopsis revealed a large conservationwith angiosperm embryogenesis-related genes (Cairneyet al., 2006). The variation in cotyledon number withina 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 cotyledonnumber in somatic embryos compared with zygoticembryos. The ring-shaped initiation of multiple cotyle-dons in gymnosperms means that multiple axes ofbilateral symmetry are established by cotyledon initiationand not just two axes as in eudicots.

Pleiocotyly and natural variation in cotyledonnumber

An abnormally large number of cotyledons withina species has been referred to in the literature aspleiocotyly or polycotyly. Pleiocotyly in natural popula-tions has not been extensively studied, although wide-spread examples of spontaneous tricotyledony have beenreviewed by Holtorp (1944) and Haskell (1954) inPopulus (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 sun-flower (Hu et al., 2005). The genetic basis for some ofthese examples is not known, but a tricotyledonousmutant of Catharanthus roseus was found to be controlledby a single recessive mutation (Rai and Kumar, 2001). Intomato, two independent loci causing polycotyledonywere characterized: the polycotyledon (poc) mutation wasmapped (Madishetty et al., 2006) and the mutant showedincreased levels of polar auxin transport, suggesting thatthe cotyledon phenotype is a result of altered auxindistribution (Al-Hammadi et al., 2003). The defectiveembryo and meristems (dem) mutant also lacks a func-tional SAM and is caused by the mutation of a geneencoding a novel protein of unknown function (Keddieet al., 1998). In pea, three independent single cotyledon(sic) loci when mutated, give rise to single or fusedcotyledons (Liu et al., 1999).The frequency of pleiocotyly has been studied as

a complex trait in wild radish populations: Raphanusraphanistrum and Raphanus sativus show either more orfewer than two cotyledons at a frequency of between 0%and 5% (Conner and Agrawal, 2005). Paternal half-siblingfamilies were compared to assess whether the lowphenotypic penetrance was due to stabilizing selection ora lack of genetic variance for the trait. These alternativescould not be distinguished due to the small samples andlow number of phenotypic plants. However, artificialselection has shown that genetic variance for pleiocotylyis not lacking: the frequency of tricotyledonous seedlingsresulting from a cross between wild and cultivatedsunflower 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 polycotylyexists. Similarly, artificial selection for pleiocotyly inAntirrhinum majus was able to increase its frequency

2926 Chandler


from 5% to 64% (Haskell, 1954), which suggests that lackof genetic variation is not the reason for the usuallyinvariant number of cotyledons. The variable numbers ofcotyledons in gymnosperms has been addressed in clonalpopulations of somatic embryos of hybrid larch and wasfound to correlate directly with the diameter of the apicalembryo surface (Harrison and Von Aderkas, 2004).

Breeding for cotyledon number

The possibility exists that additive variance in naturalpopulations of some plants could allow the selection ofpleiocotyly as a trait, although the potential adaptive valueof variable cotyledon numbers has not been well studiedand is dependent on understanding the genetic basis ofnatural variation in cotyledon number. The possibleagricultural benefits of pleiocotyly or manipulating cotyle-don size are greater yield and quality of oils in oil seedplants such as rape or sunflower, where oil is stored in thecotyledons rather than the endosperm. Additional cotyle-dons may even confer an enhancement in seedlingestablishment; a tricotyledonous sunflower mutation hadno detrimental effect on growth and development and ledto three leaves per node, increasing photosynthetic surfacearea and therefore potential productivity (Hu et al., 2005).

Conclusions

The Arabidopsis developmental paradigm has elucidatedmany gene hierarchies regulating cotyledon developmentand has demonstrated the key involvement of auxin. Afurther feature of the control of cotyledon development isredundancy amongst gene families such as the PIN (Friml,2003), class III HD-ZIP (Prigge et al., 2005), YUCCA(Cheng et al., 2007a) or TIR/AFB (Dharmasiri et al.,2005b). Redundancy also exists between transcriptionfactor pairs such as DRN/DRNL (Chandler et al., 2007)and TOAD2/RPK1 (Nodine et al., 2007). This geneticrobustness and plethora of parallel pathways presumablyreflects evolutionary adaptation to ensure buffering againstnegative mutations at a critical point in plant survival,thereby ensuring that the embryonic patterning pro-gramme proceeds successfully. It will be interesting todiscover how conserved this redundancy is amongst othereudicots for which Arabidopsis is not representative.Despite the enormous diversity of cotyledon morphologyand development within and between the monocots andeudicots, the existence of many cotyledon developmentgenes and regulatory pathways are conserved, such asthose involving CUC genes, STM, pathways involvingauxin, although in many cases no monocot mutants areavailable to demonstrate how they affect cotyledon de-velopment. The increased ease of isolating gene homo-logues by heterologous cloning and applying the

information to evolutionary developmental biology willincreasingly reveal the extent of this conservation acrossthe plant kingdom and distinguish pathways which havedivergently evolved. It will also inform the debate on theevolution of cotyledon states.The similarities in the genetic regulation and auxin-

dependent initiation and outgrowth of cotyledons andleaves demonstrates the homology between the structures.However, open questions such as what distinguishes leaffounder cells from cotyledon precursor cells and howcotyledon and leaf development differ, including determi-nants of phase change and the relationship of cotyledoninitiation to the SAM, remain tantalizing foci of research.Natural variation in cotyledon number is under-

researched and understanding the genetic basis of pleioco-tyly could enable breeding agronomically important seedoil crops for increased productivity based on this trait.

Acknowledgements

JWC thanks Melanie Cole for PIN1 and DR5 Arabidopsis embryopictures, Judith Nardmann for maize embryo photos, SiegfriedWerth for photography, Klaus Menrath and Jurgen Hintzsche forproviding plant material, and Wolfgang Werr for critical reading ofthis manuscript. JWC also gratefully acknowledges funding for thiswork from the Deutsche Forschungsgemeinschaft via SFB572.

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