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Female GametophyteDevelopment inFlowering PlantsWei-Cai Yang,1 Dong-Qiao Shi,1

and Yan-Hong Chen2

1Key Laboratory of Molecular and Developmental Biology, Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences, Beijing 100101, China;email: wcyang@genetics.ac.cn, dqshi@genetics.ac.cn2College of Life Sciences, Nantong University, Zhongxiu Campus, Nantong 226007,China; email: chenyh@ntu.edu.cn

Annu. Rev. Plant Biol. 2010. 61:89–108

First published online as a Review in Advance onFebruary 24, 2010

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev-arplant-042809-112203

Copyright c© 2010 by Annual Reviews.All rights reserved

1543-5008/10/0602-0089$20.00

Key Words

embryo sac, synergid cell, egg cell, central cell, cell fate, ovule

AbstractThe multicellular female gametophyte, a unique feature of higherplants, provides us with an excellent experimental system to addressfundamental questions in biology. During the past few years, we havegained significant insight into the mechanisms that control embryo sacpolarity, gametophytic cell specification, and recognition between maleand female gametophytic cells. An auxin gradient has been shown for thefirst time to function in the female gametophyte to regulate gametic cellfate, and key genes that control gametic cell fate have also been iden-tified. This review provides an overview of these exciting discoverieswith a focus on molecular and genetic data.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 90OVULE DEVELOPMENT . . . . . . . . . . 90THE TRANSITION FROM

SOMATIC TO GERMLINEFATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

SPECIFICATION OF THEFUNCTIONAL MEGASPORE . . . 93

PROGRESSION OF THEGAMETOPHYTIC MITOTICCYCLE . . . . . . . . . . . . . . . . . . . . . . . . . . . 94The Initiation of Female

Gametogenesis . . . . . . . . . . . . . . . . . 94Control of the Gametophytic

Cell Cycle . . . . . . . . . . . . . . . . . . . . . . 94CELLULARIZATION OF THE

EMBRYO SAC . . . . . . . . . . . . . . . . . . . . 96EMBRYO SAC POLARITY AND

GAMETOPHYTIC CELLSPECIFICATION. . . . . . . . . . . . . . . . . 96

THE FUNCTIONAL FEMALEGERM UNIT . . . . . . . . . . . . . . . . . . . . . 99The Egg Cell . . . . . . . . . . . . . . . . . . . . . . 99The Synergid Cell . . . . . . . . . . . . . . . . . 99The Central Cell . . . . . . . . . . . . . . . . . . 101

CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 102

INTRODUCTION

A female gametophyte is a multicellularhaploid structure that develops into an embryoand endosperm after fertilization. In thepast decade, gametophyte development inplants has emerged as an excellent systemto address fundamental questions in biology,such as cell specification, cell-cell interaction,and the developmental role of basic cellularmachinery. Significant progress has been madeto define the genetic components that governgametogenesis. This review focuses on recentadvances in defining genetic control of femalegametophyte development.

OVULE DEVELOPMENT

An ovule is a female organ within the carpel ofa flower that harbors the female gametophyte.

Ovule development starts as a protrusion (pri-mordium) on the edges of the septum of the gy-necium. As the ovule primordium elongates, afinger-like structure (nucellus) is formed. Then,a hypodermal cell at the tip of the nucellus startsto differentiate and forms an archesporial cell,which produces the germline. The archesporialcell enters meiotic development to differentiatea megasporocyte, which becomes distinct by itslarge size and nuclear morphology (Figure 1a).The megasporocyte then undergoes meiosisto give rise to four haploid megaspores. Inmost flowering plants, which include the modelspecies Arabidopsis and rice, micropylar mega-spores undergo programmed cell death, and thechalazal-most megaspore becomes functionaland ultimately forms the female gametophyte,the embryo sac (Figure 1b). Concurrently, epi-dermal cells at the proximal third of the nucel-lus divide parallel to the long axis and form twoprimodia, which become the inner and outer in-teguments, respectively (Figure 1a). These en-close the functional megaspore, which becomesthe embryo sac, forming a narrow opening atthe micropyle where the pollen tube enters af-ter pollination.

While nonfunctional megaspores undergocell death, the functional megaspore increasesin size and undergoes a nuclear division with-out cytokinesis to produce a two-nucleate em-bryo sac (Figure 1c). The two daughter nuclei,now separated to the poles by the formationof a central vacuole (Figure 1d), proceed toa second karyokinesis to form a four-nucleateembryo sac with nuclei in a 2n+2n configu-ration (Figure 1e). As the vacuole increases insize, the third karyokinesis takes place, whichresults in the formation of a huge coenocyticcell with eight nuclei that adopt a 4n+4nconfiguration—the eight-nucleate embryo sac(Figure 1f ). Thereafter, two polar nuclei, onefrom each pole, migrate to the micropylar cyto-plasm of the embryo sac and finally fuse to forma diploid central nucleus. As polar nuclei mi-grate, cell walls are formed simultaneously, di-viding the embryo sac into seven cells with fourcell types: three antipodal cells at the chalazalend, a diploid central cell, two synergids, and

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an egg cell at the micropylar end (Figure 1g).Antipodal cells degenerate shortly before fer-tilization in Arabidopsis (Figure 1h) or undergofurther mitosis as seen in maize. Antipodal cellsare likely dispensable for fertilization. There-fore, the central cell, the egg, and two synergidcells form a female germ unit—a functional unitthat is able to attract a pollen tube, interact withthe tube to trigger sperm release, and completedouble fertilization. As mentioned above, ovuledevelopment involves both sporophytic and ga-metophytic processes, and is an excellent systemto study the basic developmental mechanismsthat control germline formation, cell growthand division, and gametic cell fate specification.

THE TRANSITION FROMSOMATIC TO GERMLINE FATE

In angiosperms, the initial cells of the germline,called archesporial cells, are formed de novofrom the hypodermal L2 cell layer of the ovuleprimordium. Generally, in females, a single hy-podermal L2 cell at the tip of the nucellus dif-ferentiates into the germline cell that enters themeiotic pathway, which ultimately gives rise togametic cells: the egg and the central cell.

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 1Development of the female gametophyte inArabidopsis. Confocal optical section showing(a) nucleus with MMC ( green) and primordia ofinner (it) and outer (ot) integuments;(b) one-nucleate embryo sac ( green) anddegenerating megaspores ( yellow) close to themicropyle; (c) an early two-nucleate embryo sac( green); (d ) a late two-nucleate embryo sac ( green),inner (it) and outer (ot) integuments, and funiculus(fc); (e) a four-nucleate embryo sac ( green); ( f ) anearly eight-nucleate embryo sac ( green), withantipodal nuclei (pink), polar nuclei (blue), eggnucleus (red ), and synergid nuclei ( yellow); ( g) a lateeight-nucleate embryo sac ( green), with antipodalnuclei (pink), polar nuclei (blue), egg nucleus (red ),and synergid nuclei ( yellow); (h) a mature four-celledembryo sac ( green), with the secondary nucleus(blue), egg nucleus (red ), and synergid nuclei( yellow). Scalebar: 10 μm. Confocal images weremodified with PhotoShop to highlight themegaspore mother cell, embryo sac, and nuclei.

The archesporial cell first becomes morpho-logically distinguishable from its surroundingnucellar cells by its larger size and pronouncednucleus, and it is called a megasporocyte when

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its nucleus enters the prophase of meiosis.How the transition from a somatic cell fateto a germline fate is controlled remains un-known. However, several putative componentsthat control this transition have been identi-fied through genetic approaches. In Arabidopsis,the SPOROCYTELESS gene (SPL/NOZZLE)has been implicated in controlling germlinecell fate. In spl mutants, archesporial cells areformed in both anther and ovule primodia, butthey fail to develop further, which results in acomplete lack of germline in male and femaleorgans (63, 75). This indicates that SPL plays anessential role in germline formation. A recentstudy revealed that the floral homeotic regu-lator AGAMOUS (AG) can activate the SPLgene by binding to the CArG-box in its 3′ re-gion (24). SPL expression activated by AG in-duces the ectopic formation of microspores onpetaloid floral organs, which indicates ectopicformation of the male germline. This findingnot only showed that SPL is a direct down-stream target of AG but also demonstrated thatSPL is sufficient to trigger male germline spec-ification in Arabidopsis. Whether SPL is alsosufficient for female germline specification re-mains unknown. SPL encodes a novel nuclearprotein with limited homology to MADS-boxtranscription factors (63, 75). Therefore, howSPL regulates its downstream genes in germlinespecification is of great interest. Recently, re-searchers suggested that SPL may be involved inauxin homeostasis by repressing YUCCA genesin lateral organ development (32). It would beinteresting to investigate whether auxin is alsoinvolved in germline formation.

Several genes that control the numberof cells entering germline fate have alsobeen identified. Mutation in MULTIPLEARCHESPORIAL CELLS 1 (MAC1) resultsin an excessive number of archesporial cellsin maize (64). Similarly, in rice, the multiplesporocyte 1 (msp1) mutants have increased num-bers of both male and female sporocytes (45).These excess sporocytes likely result from ex-cessive archesporial cells, which suggests thatMSP1 is required for archesporial cell fate.The MSP1 gene is expressed in nucellar cells

that surround germline cells but not in the de-veloping germline cells. This supports a rolefor MSP1 in determining germline cell fateby preventing the surrounding cells from be-coming germline cells, similar to MAC1 inmaize (64). MSP1 encodes a leucine-rich re-peat containing receptor-like kinase (LRR-RLK). This implies that signaling betweenthe archesporial cell and its neighboring cellsis controlled by a ligand-receptor signalingcascade, and this plays a critical role in fe-male germline development. These findingsimply that a lateral inhibition mechanism mayact in controlling the number of germlinecells.

Because MSP1 is a membrane receptor ki-nase, it likely exerts its effects by binding to aligand. Recently, a putative ligand OsTDL1Awas shown to bind the extracellular domainof MSP1 by yeast two-hybrid and BiFC ex-periments (78). Similar to MSP1, TDL1A isexpressed exclusively in nucellar cells but notin germline cells. Furthermore, knockdown ofOsTDL1A expression phenocopies the msp1phenotype in the nucellus. A similar mecha-nism, mediated by TPD1-EMS1/EXS1 signal-ing, controls male sporocyte fate (25, 36). Thesedata suggest that specification of germline cellfate and number in plants involves a similarligand-receptor signaling system in both themale and female.

In animals, germline fate is controlled bya germline-specific PIWI-associated miRNA(piRNA) system (33). Emerging data suggestthat miRNA also plays an important role ingermline development in plants. MEIOSISARRESTED AT LEPTOTENE1 (MEL1), agermline-specific member of ARGONAUTE(AGO) genes family, is specifically expressedin archesporial cells and sporogenous cells(SCs), and disappears when SCs enter meiosisin anther and ovule. Ovule development inmel1 mutants is arrested at various stages frompre-meiosis to tetrad. Chromosomes remainuncondensed, and aberrant chromatin modifi-cation is detected in mel1 germline cells, whichsuggests that MEL1 acts on chromatin struc-tures, similar to those of the PIWI subfamily of

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AGOs in Drosophila. This implies that MEL1is required for female germline development,most likely by regulating cell division ofpre-meiotic germline cells, modificationof meiotic chromosomes, and progression ofmeiosis; but it does not affect the initiation,establishment, and early mitotic division ofgermline cells (46).

Interestingly, the MEL1 expression domainis larger than the germline cell in anther andovule primordia. Therefore, archespores maybe produced in excess of subsequent archespo-rial cells, and the fate of archesporial deriva-tives may be controlled by signals from SCs.Therefore, MEL1 may suppress somatic geneexpression during germline development (46).How does MEL1 function? MEL1 may modifychromatin structure to repress the somatic geneexpression program in germline cells. Althoughthe role of MEL1 orthologs in germline forma-tion has not been identified in other plants, thisdemonstrates for the first time that, as in ani-mals, the small RNA-mediated gene silencingpathway mediated by MEL1 plays a key role inplant germline formation (18).

SPECIFICATION OF THEFUNCTIONAL MEGASPORE

The female megasporocyte undergoes meioticdivision to produce four haploid megaspores.Of the four megaspores, one, two, or four mayparticipate in the formation of the final femalegametophyte depending on the plant species.In the more advanced species, only one of thefour megaspores is functional, and the remain-ing three undergo programmed cell death. Thechoice of functional megaspore is position de-pendent and also species dependent. In mostflowering plants, including Arabidopsis and rice,the megaspore that is closer to the maternal tis-sue becomes the functional megaspore; distalmicropylar megaspores undergo programmedcell death. Evolution seems to favor the moredefined monosporic type of gametogenesis, andgenetic mechanisms must have evolved to de-fine such developmental control.

Observed in many species, polarity withinthe megasporocyte is manifested by the po-lar distribution of organelles, the dynamic de-position of callose, and the microtubule cy-toskeleton. So far, the role of this polarity onmegaspore development is unknown. The dis-ruption of this polarity in megasporocytes wasobserved in switch1 (swi1)/dyad ovules in Ara-bidopsis (42, 68). SWI1 encodes a novel proteininvolved in chromatid cohesion establishmentand chromosome structure during meiosis (2,38). Interestingly, dyad mutation causes de-fective meiosis that produces two unreducedmegaspores (diploid) (60). However, only thechalazal megaspore, but not the micropylarmegaspore, expresses functional megaspore-specific markers. This indicates that only thechalazal megaspore is functional and suggestsa position-dependent mechanism. It also im-plies that a decision on cell fate has been madeat this stage of ovule development. In maize,a similar mutation in ameiotic (am1) has beenidentified in which meiosis is replaced by a mi-totic division, as in swi1/dyad. Analysis of am1allelic mutations indicates that the division ofthe megasporocyte is completely blocked insome alleles, or meiosis is initiated but notcompleted in other alleles. However, all mei-otic processes are impaired in am1. Together,these data suggest that SWI and AM1 are es-sential for the switch between meiotic and mi-totic division cycles and likely regulate thetransition through a novel leptotene–zygotenecheckpoint. AM1, which shares 30% iden-tity with SWI1, is a plant-specific chromatin-binding protein with as yet unknown function(54).

Surprisingly, the chalazal unreduced megas-pore in dyad mutants occasionally proceeds toform an unreduced embryo sac that can be fer-tilized to produce triploid seeds (60), whichmay have implications for engineering hybridseed production in agriculture. This is likelylinked to the truncated SWI1 protein producedin dyad. The functional dissection of SWI1 andAM1 will shed light on how meiotic to mitotictransition is regulated.

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PROGRESSION OF THEGAMETOPHYTIC MITOTICCYCLE

The functional megaspore undergoes rapidgrowth, taking up the space left by the de-generating megaspores, and goes through threeconsecutive rounds of karyokinesis to form aneight-nucleate syncytial embryo sac. This sacis cellularized simultaneously to form a seven-celled female gametophyte, composed of fourdifferent cell types: egg, synergid, central, andantipodal. In the past decade, genetic studieshave identified several mutations and genes thatcontrol different stages of embryo sac develop-ment (50). Meanwhile, comparative expression-profiling studies and cell-specific EST sequenc-ing have revealed many genes that are expressedin the female gametophyte (31, 70, 74, 76,77). We discuss progress in each developmen-tal stage and focus on recent important findingsbelow.

The Initiation of FemaleGametogenesis

Little is known about the genetic and molec-ular control of the initiation of female game-togenesis in flowering plants, although manygametophytic mutations block embryo sac de-velopment at the one-nucleate stage. Mutationsin AGL23, a type I MADS-box gene, block thefirst nuclear division of the functional mega-spore (12). AGL23 expression is first detectedin the functional megaspore and persists in theembryo sac. Together these data suggest thatAGL23-regulated transcription is required forearly female gametogenesis. However, AGL23may not be required for the initiation or cell di-vision of the functional megaspore, or cell cycleprogression during subsequent embryo sac de-velopment in Arabidopsis.

Using monoclonal antibodies against cellwall components, female reproductive lineagehas been associated with distinct changes inthe distribution and types of arabinogalactanprotein (AGP) epitopes (11). Whether AGPsfunction in cell surface signaling or recognition

during gametogenesis remains to be clarified.In AGP18 knockdown plants, the functionalmegaspore fails to enlarge and divide. AGP18is expressed in the female germline includingthe functional megaspore, and it is weakly ex-pressed in somatic nucellar cells. This sug-gests that a cell surface proteoglycan is re-quired for functional megaspore developmentin Arabidopsis (1).

Control of the GametophyticCell Cycle

Many female gametophyte ( fem) mutations,which were identified through a distortedMendelian segregation screen, display mitoticarrest of the female gametophytic division cycle(50). This indicates that progression of the mi-totic cycle is critical for the formation of a func-tional gametophyte. Several mutants defectivein the progression of the mitotic division cyclehave been identified and molecular cloning ofthese genes is starting to shed light on how cellcycle progression is regulated during female ga-metogenesis in plants.

The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle–regulated,multiple-subunit E3 ubiquitin-protein ligasethat controls important transitions duringmitotic progression and exit by sequentiallytargeting for degradation many cell cycleregulators, such as cyclins (55). The knockoutof APC/C components often impairs femalegametophyte development. Mutations in eitherNOMEGA, which encodes APC6/CDC16,or APC2, which interacts with APC11 andAPC8/CDC23, cause embryo sac arrest at thetwo-nucleate stage (7, 29). This indicates thatthe APC/C ubiquitin-mediated proteolysispathway plays a role in female gametophyticcell cycle control. Similarly, mutations inregulatory particle triple A ATPase (RPT ) of the26S proteasome arrest embryo sac develop-ment at the one- or two-nucleate stage in therpt5a-4 rpt5b-1 double mutant, which indicatesa defect of the first or second mitosis of thefemale gametophyte (17). Similarly, the doublemutation of two RING-finger E3 ligase genes

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RHF1a and RHF2a results in the interphasearrest of the mitotic cell cycle in the femalegametophyte. RHF1a directly targets a cyclin-dependent kinase inhibitor ICK4/KRP6 forproteasome-mediated degradation (34). Thissuggests that the ubiquitin/26S proteasomesystem helps control cell cycle progressionduring female gametophyte development.

PRL encodes the DNA replication licensingfactor subunit MCM7 that is required in allproliferating cells, including female germlinecells (69). Knockout of PRL function arrestsembryo sacs primarily at the one-nucleate stage.Recently, we identified the slow walker1 (swa1)mutation that causes slow progression of thegametophytic division cycle and female sterilityowing to incomplete development of the femalegametophyte at anthesis (65). Delayed pollina-tion tests showed that a small fraction of swa1ovules are able to form functional female game-tophytes; however, they missed the correct timefor fertilization because of the slow down of fe-male gametophyte development when naturallypollinated, which indicates that coordinateddevelopment between the male and female ga-metophyte is critical for fertility in Arabidopsis.SWA1 encodes a nucleolar WD40-containingprotein that is involved in the processing ofpre-18S rRNA in Arabidopsis, which suggeststhat RNA biogenesis plays a role in theprogression of the gametophytic division cycle.

Mutations in the Arabidopsis retinoblastoma-related (RBR) gene, a key negative regulatorthat controls G1/S transition by repressing E2Ftranscription factors, result in arrested mitosisand uncontrolled nuclear proliferation, whichgives rise to embryo sacs with supernumerarynuclei that are irregular in size and partially en-closed by cell-wall-like structures (14). This in-dicates that cellularization and karyokinesis canbe uncoupled, and they are regulated indepen-dently. Consistently, premature cellularizationhas been observed in hadad (hdd) ovules afterthe first or second gametophytic division (41).Although most proliferating rbr female game-tophytes fail to express cell-specific markers, afraction of ovules in selfed rbr1–1/+ siliquesor rbr1–1/+ siliques that are pollinated with

wild-type pollen form abnormal embryos,which implies that RBR is required for a com-plete differentiation of all gametophytic cells(14, 26). This indicates that rbr mutant ovulesare able to cellularize and form functional eggcells, which suggests that egg cell fate requiresthe completion of a third nuclear division, butnot an arrest in the gametophytic cell cycle.Therefore, RBR connects cell cycle control tocellular differentiation processes.

In the indeterminate gametophyte1 (ig1) mu-tant of maize, female gametophytes have aprolonged phase of free nuclear divisions be-fore cellularization, which leads to a variety ofembryo sac abnormalities, including extra eggcells, extra synergids, and extra central cells withextra polar nuclei (16, 20). The rbr1 mutants ofArabidopsis have a similar phenotype, with addi-tional defects in pollen development. This sug-gests that IG1 restricts the proliferative phaseof female gametophyte development.

Together, these data suggest that timely mi-totic progression of the division cycle is vi-tal for gametogenesis. In addition to the con-served cell cycle machinery, other regulatorymechanisms may play a role in gametophyticcell fate in plants. One such mechanism is pro-tein phosphorelay. The loss of CYTOKINININDEPENDENT1 (CKI1) function also re-sults in the early degeneration of gametophyticcells and excess nuclei (56). CKI1 encodes ahistidine kinase, which suggests that His/Aspphosphorelay signaling plays a role in femalegametogenesis. At the onset of megagameto-genesis, RNA interference depletes CHR11, amember of the ATP-dependent SWI2/SNF2family of chromatin-remodeling factors. Thisdepletion arrests uncellularized embryo sac de-velopment at the one-, two-, four-, or eight-nucleate stages in Arabidopsis, which indicatesa role for chromatin-remodeling in karyoki-nesis during female gametogenesis (22). RBRsuppresses E2F-like protein activity by re-cruiting chromatin-remodeling factors of theSWI2/SNF2 family. Together, CHR11 andRBR regulate the E2F family proteins, andthereby promote nuclear proliferation duringfemale gametogenesis. Furthermore, histone

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acetyltransferase HAM1 and HAM2 act redun-dantly during ovule development because ham1ham2 double mutant embryo sacs arrest at theone-nucleate stage with a huge nucleus (30),which indicates that gene suppression and chro-matin compaction requiring hypoacetylation ofhistones in the functional megaspore block thefirst karyokinesis during megagametogenesis.

CELLULARIZATION OF THEEMBRYO SAC

Cellularization of the syncytial embryo sacis controlled temporally and spatially duringovule development, and it is critical for gameto-phytic cell fate specification. Several genes havebeen implicated in controlling this process. Ingemini pollen2 ( gem2), the cellularization of theembryo sac is impaired: resulting in embryosacs that contain five nuclei at the micropylarpole and three at the chalazal pole. Moreover,no cell boundaries or only partial cellulariza-tions are observed between nuclei. Most ma-ture mutant embryo sacs contain one or twoextremely large nuclei, which might arise fromthe fusion of free nuclei at the micropylar pole(52). However, gem2 plants also display a vari-ety of division defects in pollen development,which include division asymmetry and incom-plete cytokinesis. Together, these data suggestthat GEM2 plays a critical role in coordinatingkaryokinesis and cytokinesis during gametoge-nesis. Interestingly, the partial cellularization ofgem2 ovules can develop further and form anembryo sac with a large cell that possesses vac-uolar characteristics of an egg cell, which sug-gests that egg cell fate can occur in a partiallycellularized embryo sac, and local cellulariza-tion may reinforce a gradient of cell fate deter-minants that are established in the coenocyticembryo sac (52).

In addition, gem1/mor1 and two in one (tio)mutants display similar or even more severeovule phenotypes as gem2. GEM1/MOR1 en-codes a microtubule-associated protein (73),and TIO encodes an essential phragmoplast-associated protein that is homologous to theFUSED (Fu) Ser/Thr protein kinase of the

hedgehog signaling complex in animals. BothGEM1/MOR1 and TIO have a general effecton cell plate formation in somatic and repro-ductive cells. The incomplete cell plate in tiopollen is positioned correctly, which suggeststhat TIO is not required for the positioning orestablishment of the cell plate but has a specificrole in cell plate expansion (47). In addition,knockout of TUBG1 and 2 genes, which encodeγ-tubulin, results in uncellularized embryo sacswith aberrant morphology, positioning, andnumber of nuclei (53). In plant cells, the lateralexpansion of phragmoplast and cell plate is reg-ulated by a kinesin-MAPKKK pathway duringcytokinesis; mutations in two kinesin-like pro-teins, AtNACK1 and AtNACK2, that bind andactivate the MAPKK kinase NPK1 result in themispositioning of nuclei and the formation ofnonfunctional gametophytes (72). Therefore,they all play a role in positioning phragmoplastand cell plate expansion, thereby controlling thecellularization of the embryo sac. However, itis not clear how the cell plate expansion pheno-type is associated with the karyokinesis defect.

EMBRYO SAC POLARITY ANDGAMETOPHYTIC CELLSPECIFICATION

The embryo sac is a polarized structure withantipodal cells at the chalazal end and the eggapparatus at the micropylar end. Gametophyticcells within the embryo sac are also highly po-larized. The nucleus of the egg cell is locatedtoward the chalazal end of the embryo sac,whereas the nuclei of the synergid and cen-tral cells are located toward the micropylar end.Therefore, the egg cell, and the central cell andsynergids have opposite polarity. The oppositepolarity of the central cell and egg brings theirnuclei into close proximity, which may facili-tate their fusion with the sperm nuclei duringdouble fertilization. The final polarity of theembryo sac is a result of coordinated nucleardivision and positioning, expansion of the cen-tral vacuole, and cellularization. This polaritycan be traced back to the four-nucleate stage,when two pairs of nuclei, separated by a large

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central vacuole, migrate to opposite ends of thesyncytial embryo sac. At the chalazal pole, thenuclei are positioned one above the other withrespect to the micropylar-chalazal axis. Mean-while, the nuclei generally locate side by sideat the micropylar end. Comparatively large nu-cleoli distinguish the two polar nuclei in theeight-nucleate stage. The migration and pre-cise positioning of the nuclei and morphologi-cal differentiation of the polar nuclei suggest anearly distinction between the nuclei before cel-lularization. Polar expression of genes has alsobeen observed. For example, the DEMETER(DME) gene, a key regulator of gene imprint-ing during endosperm development, is polarlyexpressed in the micropylar domain of the em-bryo sac: first in the polar nuclei and synergidnuclei before cellularization and then restrictedto the central cell after cellularization. FIS2, adownstream gene of DME, is expressed only inthe polar nuclei but not in the future synergidor egg, or antipodal nuclei (9). In rbr mutantembryo sacs, FIS2 expression is lost or occa-sionally deregulated, which indicates the RBR-controlled FIS2 polarity is nessessary for thedifferentiation of the central cell in the embryosac (26). This polarity further suggests a rolefor positional cues in gametic cell specification.Compared to embryo sac polarity, the polar-ity within gametophytic cells is obviously estab-lished after cellularization of the eight-nucleateembryo sac. The differentiation of egg and syn-ergid cell fate also suggests that a lateral inhi-bition mechanism may exist for gametophyticcell specification after cellularization (19).

Although the cellular and molecular basisof gametophytic cell specification in the em-bryo sac is largely unknown, emerging evidencesupports the involvement of positional and lat-eral inhibition mechanisms in determining ga-metophytic cell fate. Several studies in maizeand Arabidopsis support the idea of a positionalmechanism. In the maize ig1 mutant, embryosacs undergo extra rounds of free nuclear divi-sions, which result in extra egg cells, extra cen-tral cells, and extra polar nuclei (17, 23). The fi-nal fate of the extra nuclei as either egg nuclei orpolar nuclei depends on their relative position

in the embryo sac. IG1 encodes a LATERALORGAN BOUNDARIES (LOB) domain pro-tein with high similarity to ASYMMETRICLEAVES2 (17), which controls leaf symmetryin Arabidopsis. During female gametogenesis,IG1 is expressed in functional, but not nonfunc-tional, megaspores; it is strong in antipodal cellsand the egg; and no signal is detected in thesynergids (16). Thus IG1 is asymmetrically ex-pressed in the embryo sac, which supports theexistence of embryo sac polarity.

Recently, Sundaresan and colleagues dis-covered an asymmetric auxin gradient in thesyncytial embryo sac that plays a key role ingametic cell specification (49). Using the syn-thetic DR5:GFP or DR5:GUS reporter that isresponsive to auxin, the auxin response can betraced by monitoring reporter expression. Dur-ing megasporogenesis, GFP or GUS expressionis detected at the distal tip of the nucellus andincreases in nucellar cells that surround the de-veloping embryo sac at the early one-nucleatestage. As the ovule develops, auxin level in-creases within the micropylar domain of theembryo sac at the two- to eight-nucleate stages.Interestingly, the auxin response distribution isless polarized in the cellularized embryo sac,because the reporter is expressed in all gameto-phytic cells. These data suggest a micropylar-chalazal gradient of auxin in embryo sacs. Con-sistently, the disruption of auxin responses bydownregulating AUXIN RESPONSE FACTOR(AFR) gene expression, or auxin synthesis byectopic expression of the auxin biosyntheticYUCCA1 gene, impairs gametophytic cell iden-tities at the micropylar domain. Specifically,synergids adopt the fate of the egg cell in ARFknockdown embryo sacs, and ectopic YUCCA1expression results in the misspecification of allgametophytic cells. However, no abnormalitiesin embryo sac polarity or changes in central celland antipodal cell fates are observed. This sug-gests that such an auxin gradient is essential forgametophytic cell specification but not for em-bryo sac polarity and central cell and antipo-dal cell fates. Furthermore, auxin polar trans-porters of the PIN family are not expressedin the embryo sac, suggesting that the auxin

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gradient is correlated with location-specificauxin biosynthesis and diffusion (49).

A fundamental question of female gameto-genesis is, How is gametic cell fate determined?As discussed above, embryo sac polarity and anauxin gradient play a key role in gametophyticcell fate. The manipulation of auxin responsesor synthesis results in the switching of gameticand nongametic cell fates (49). To identify genesthat control egg cell fate, Gross-Hardt and col-leagues (19) mutagenized an egg cell-specificmarker line and then screened for expressionchanges of the egg cell-specific marker. Theyidentified three mutants, lachesis (lis), clotho (clo)and atropos (ato), that showed deregulation ofthe marker (19, 40). In lis embryo sacs, the ex-pression of the egg–specific marker is expandedto the synergids and central cell. Consistentlylis synergids display egg cell morphology de-fects and downregulate synergid-specific geneexpression. Pollen tube attraction is compro-mised, and reduced as well. These results in-dicate that the synergids have adopted an eggcell fate in lis embryo sacs. Interestingly, theET884 synergid-specific marker is ectopicallyexpressed throughout lis embryo sacs. Similarly,the central cell in lis embryo sacs also adoptsan egg cell fate. Polar nuclei rarely fuse andcellularize separately to give rise to small un-inucleate cells that are morphologically indis-tinguishable from the egg cell. Compromisedcentral cell fate is further suggested by thedownregulation of expression from the cen-tral cell-specific MEA promoter. Surprisingly,the antipodal cells of lis embryo sacs adopt acentral cell fate as evidenced by their fusion,and the downregulation of an antipodal-specificmarker, and the activation of a central cell-specific marker (19). A similar phenotype is alsofound in the clo/gfa1 mutant (40). Unlike thatof the ig1 mutant in maize, the accessory cell(synergid and antipodal cell) fates have beenmis-specified because no supernumerary nucleior cells are observed in lis or clo/gfa1 embryosacs. One idea that accounts for these pheno-types is that accessory cells are gradually re-cruited as gametic cells. This demonstrates acentral role for LIS and CLO/GFA1 in gametic

cell fate specification during female gameto-phyte development. Furthermore, LIS nuclearlocalization requires the CLO/GFA1 protein.The findings above have several profound im-plications, as pointed out by Gross-Hardt andcolleagues (19). First, all gametophytic cells arecompetent to adopt gametic cell fate, and LISis involved in a mechanism that represses ga-metic cell fate in accessory cells. Second, theremight be an intracellular signaling mechanismthat senses the number of nuclei in a given cell.Third, there are two levels of cell fate regula-tion: one between the gametic cell and acces-sory cells, and the other between the egg celland central cell. Together, these genes might beinvolved in an as yet unknown signaling path-way that operates in gametic cells and preventsaccessory cells from adopting a gametic cell fate.

Interestingly, LIS, CLO/GFA1, and ATO allencode components of RNA splicing machin-ery. LIS is homologous to the yeast splicingfactor PRP4; CLO/GFA1 is a plant homologof yeast Snu114p, likely a component of theU5 snRNP of the spliceosome, and is requiredfor the cell-specific expression of LIS gene (35,40). This implies that LIS is downstream ofCLO/GFA1 in the pathway controlling ga-metic cell specification. In addition, LIS andCLO/GFA1 are colocalized to nuclear speck-les; this suggests they may form a complex aswell. ATO is homologus to SF3a60, a proteinthat is implicated in prespliceosome formation.These findings suggest that the RNA splicingmachinery plays an important role in gameticcell specification in the female gametophyte, al-though the underlying mechanisms remain tobe elucidated.

The LIS gene is expressed strongly in repro-ductive tissues and remains high in gametic cellsbut downregulated in accessory cells shortly af-ter cellularization. Combined with its mutantphenotype, this has led to a lateral inhibitionmodel in which, upon differentiation, gameticcells generate an inhibitory signal that is trans-mitted to adjacent cells to prevent excess ga-metic cell formation (19). This model can ex-plain the maintenance of only one egg cell inthe embryo sac after the initial specification of

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cell fate. However, once the egg cell is differ-entiated, a lateral inhibition mechanism may benecessary to maintain cell fates because acces-sory cells can differentiate into gametic cellslater if this inhibitory mechanism is not present(19).

EOSTRE, which encodes a BELL-likehomeodomain protein (BLH1), plays a role inrestricting synergid cell fate. In the eostre mu-tant, the female gametophyte is arrested at mul-tiple stages that range from one-nucleate tomature embryo sacs, and some mutant embryosacs collapse completely. The mutant embryosacs often display mispositioned nuclei duringthe syncytial stage. Interestingly, a portion ofeostre embryo sacs displays abnormal cell spec-ification, in which one of the two synergids ex-hibits polarity characteristic of the egg cell andexpresses an egg cell-specific marker. This in-dicates that one synergid has adopted an eggcell fate, and therefore that there are two eggcells in a single embryo sac, whereas the initialcentral cell and the egg cell fate are not af-fected (51). Furthermore, the extra egg cellcan be fertilized after pollination, which indi-cates that it is fully functional. Molecular anal-ysis showed that the eostre phenotype is causedby the misexpression of the EOSTRE gene,which is not expressed in the ovule in wildtype. BELL functions by forming a BELL-KNAT heterodimer whose activity is regulatedby ovate family proteins (OFPs). Consistently,the eostre phenotype can be reversed by a muta-tion in the class II knox gene KNAT3 and phe-nocopied by disruption of AtOFP5, a regulatorof BLH1-KNAT3 heterodimers (51). Together,these data suggest a role for BLH-KNAT3complex and AtOFP in gametic cell specifi-cation. Other BELL-like genes may substitutethe EOSTRE function to promote egg cell fatein female gametophytes because the loss ofEOSTRE function has no effect on female ga-metophyte development. In addition, it wouldbe interesting to know whether the aberrant nu-clear configuration observed in eostre syncytialembryo sacs or the polarity change in one ofthe synergid cells has any role in gametic cellspecification.

THE FUNCTIONAL FEMALEGERM UNIT

The Egg Cell

Egg cell specification is a result of the in-terplay between the auxin gradient and theLIS-mediated mechanism as discussed above.Although many egg-specific transcripts havebeen identified through single cell library andEST analysis, key genes that control egg cellfunction have not been identified. GAMETEEXPRESSED3 (GEX3) is expressed specificallyin the egg cell of the female gametophyte andin pollen, and encodes a plasma membrane-localized protein with unknown function thathas homologs in other plants. Both knockdownand overexpression of GEX3 impair the femalecontrol of micropylar pollen tube guidance (3).Although the underlying mechanism is still un-known, this demonstrates a role for the egg cellin the female control of pollen tube guidance,in addition to fertilization.

The Synergid Cell

Synergid cells are key components of the fe-male germ unit (FGU) and are located, side byside with the egg cell, in the micropylar por-tion of the embryo sac. Typically, the synergidcells display an opposite polarity compared tothe egg cell, they have no or discontinuous cellwall at the chalazal end, and they are sealed by aspecialized cell wall-like structure—the filiformapparatus at the micropylar opening. Often,one of the two synergids undergoes cell deathupon arrival of the pollen tube. The synergidslikely play important roles in the attraction andrecognition of the pollen tube, sperm release,and transportation. Therefore, genes that areinvolved in those processes may be expressed insynergids. However, the mechanisms by whichsynergid cells develop their unique features arepoorly understood.

Mutations that affect synergid developmentand function and genes that are specifically ex-pressed in the synergids have been identified(70). MYB98, which encodes a R2R3-type MYB

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transcription factor, is specifically expressed inthe synergids. Mutation in the MYB98 genespecifically abolishes the formation of the fil-iform apparatus and has no effect on other as-pects of ovule development (28). Thus, MYB98is required specifically for the formation ofthe filiform apparatus during synergid cell dif-ferentiation. Consistently, recent studies showthat MYB98 binds to a specific DNA sequence(TAAC) and regulates a subset of genes thatencode secreted proteins targeted to the fili-form apparatus (59). Most synergid-expressedgenes that are downregulated in myb98 en-code small defensin-like cycteine-rich proteins(CRPs) that are secreted into the filiform ap-paratus, which suggests that they play a rolein either the formation or the function of thefiliform apparatus (59). Many of these genesare also weakly expressed in egg and/or cen-tral cells. Interestingly, myb98 female gameto-phytes that lack the filiform apparatus also losetheir ability to guide the pollen tube to the mi-cropyle; therefore, MYB98 may also play a rolein the production of the guidance cue. Together,these data strongly suggest that MYB98 actsas a synergid-specific transcriptional regulatorto activate downstream genes that are requiredfor pollen tube guidance and filiform apparatusformation.

To attract and recognize the pollen tube,synergids need the ability to secrete pollen at-tracting signals and receive the pollen tube. Re-cent studies have provided strong evidence ofthis. Using laser ablation, synergids, but notany other gametophytic cells, were shown tobe required for pollen tube guidance in Toreniafournieri (21). Also in T. fournieri, CRPs namedLUREs have been identified and are secretedby synergids to attract pollen tubes in a semi–in vivo assay (48). In Arabidopsis, CRPs are en-coded by a large gene family and expressed inthe synergids and secreted into the filiform ap-paratus as discussed above. However, geneticdata that support their role as the guidance cueare still lacking.

Synergid cells also play an essential rolefor pollen tube reception. FERONIA (FER)

(allelic to SIRENE, SRN) is a key gene thatcontrols the recognition between the synergidsand the pollen tube. In fer/srn ovules, the pollentube enters a synergid and overgrows withinthe embryo sac (23, 61), which suggests thatFER/SRN plays a role in pollen tube receptionby the synergid. FER encodes a LRR-RLK thataccumulates specifically on the plasma mem-brane of the synergid cells (15). Therefore,FER may be a synergid-specific membrane re-ceptor which, upon binding to a signal from thepollen tube, triggers a signaling cascade withinthe synergids to prepare for fertilization andalso sends a signal to stop pollen tube growth(15, 37). In support of this idea, additionalmutations are also reported in Arabidopsis. Inthe lorelei (lre) mutant, pollen tubes that reachembryo sacs often do not rupture but continueto grow in the embryo sac, reminiscent ofthe fer/srn phenotype. Moreover, lre embryosacs often attract additional pollen tubes.LRE encodes a small plant-specific putativeglucosylphosphatidylinositol-anchored proteinand is expressed in synergid cells prior to fer-tilization (76). Pollen tubes of anxur1/anxur2double mutants rupture before arriving atthe synergid cells (39). Both ANXUR1 and2 genes encode receptor-like kinases thatare specifically expressed in pollen tubes. Inaddition, mutations in ABERRANT PEROX-ISOME MORPHOLOGY2/ABSTINENCEBY MUTUAL CONSENT (APM2/AMC),which is required for peroxisome transport,impair pollen tube reception (5). This suggestsanother signaling cascade, independent of theFER/SRN-ANXUR kinase pathway that re-quires intact peroxisome function in both maleand female gametophytes, although the under-lying mechanism remains to be elucidated.

Synergid cell death associated with fertil-ization is a common phenomenon, which hasbeen described in many plant species and in-variably involves the collapse of vacuoles, a dra-matic decrease in cell volume, and completedisintegration of the plasma membrane andmost organelles. The underlying mechanismshave not been revealed so far. In Arabidopsis,

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synergid cell death is initiated upon pollen tubearrival but before pollen tube discharge, whichsuggests that pollen tube-synergid contact trig-gers a signaling cascade that induces synergidcell death (62). In gfa2 mutant ovules, the po-lar nuclei fail to fuse and the synergid per-sists after pollination. These ovules can attractpollen tubes but are not fertilized. This indi-cates that synergid cell death associated withfertilization did not occur, which suggests arole for GFA2 in promoting synergid cell death(10). The GFA2 gene encodes a mitochondrion-located DnaJ domain–containing protein sim-ilar to yeast Mdj1p that functions as a chaper-one in the mitochondrial matrix. Consistently,GFA2 partially complements a yeast mdj1 mu-tant, which suggests a role for mitochondriain synergid cell death in plants. How GFA2acts in the mitochondria in promoting syn-ergid cell death remains unknown. Interest-ingly, cell deaths of nonfunctional megasporesand antipodal cells are not affected in gfa2 mu-tants; this suggests that cell death pathwaysare different between synergids and antipodalcells.

The Central Cell

Molecular mechanisms that control the spec-ification and differentiation of the central cellare poorly understood. Genetic evidence sug-gests that Type I MADS-box genes play an im-portant role in central cell development. In thediana (dia, agl61) mutant, polar nuclei of thecentral cell are not fused, and central cell mor-phology is aberrant. The mutant embryo sac isable to attract a pollen tube but fertilization ofthe central cell does not occur (4, 71). Egg- andsynergid-specific markers, but not central cell-specific markers, are expressed in dia ovules,which indicates that egg and synergid fates arespecified, and that central cell fate is impaired inthe mutant. DIA is expressed exclusively in thelate central cell, and the DIA protein is localizedin the polar nuclei and the central cell nucleus.All these data suggest that DIA is required forcentral cell differentiation and function. DIA

forms a heterodimer with AGL80, and its nu-clear localization requires AGL80 because DIAnuclear localization is lost in the agl80 mu-tant. AGL80 is also expressed in the polar nu-clei and the secondary nucleus of the centralcell, and a mutation in the AGL80/FEM111gene in Arabidopsis specifically affects centralcell maturation. Polar nucleoli and vacuole mat-uration fail and lead to endosperm developmentarrests after fertilization. The egg, synergid,and antipodal cells are correctly specified (57).Therefore, both DIA and AGL80, most likelyforming a heterodimer, are required for po-lar nuclear fusion and central cell differentia-tion. Furthermore, AGL80, by interacting withanother Type I MADS-box protein AGL62,also plays a critical role in endosperm develop-ment. AGL62 is expressed in the syncytial en-dosperm and is suppressed by the FIS Polycombcomplex just before endosperm cellularization.Mutation in AGL62 causes precocious cellular-ization of the endosperm (13, 27). Together,these Type I MADS-box transcription factorsare critical for central cell and endosperm de-velopment in Arabidopsis. It would be interest-ing to know the downstream genes controlledby the DIA-AGL80 complex. Likely candidateswould be DD46 and DME, which are not ex-pressed in agl80 mutant ovules (57). However,there is no MADS-box binding site, the CArGbox [CC(A/T)6GG] found in either gene, whichsuggests that they may activate these down-stream genes indirectly. Therefore, the DIA-AGL80 complex may ultimately activate thetranscription of Polycomb group genes in thecentral cell to repress endosperm developmentprior to fertilization.

In magatama3 (maa3) mutant ovules, the po-lar nuclei fail to fuse at pollination and contain asmaller nucleolus that lacks the vacuolar-like in-ternal structure as compared to that of the wild-type. Consequently, central cell development isarrested in the mutant, which ultimately resultsin defective micropylar pollen tube guidance inArabidopsis (57). The MAA3 gene encodes a ho-molog of yeast SPLICING ENDONUCLE-ASE1 (SEN1) helicase involved in processing

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of a variety of RNA species in yeasts. There-fore, MAA3 may regulate the RNA metabolismthat is responsible for nucleolar organizationof the central cell and micropylar pollen tubeguidance.

The fusion of the polar nuclei is an im-portant step in central cell development. Elec-tron microscopy revealed that the fusion ofthe polar nuclei begins with contact with en-doplasmic reticulum (ER) membranes that arecontinuous with the outer nuclear membranesof the polar nuclei. First, the fusion withthe ER membranes gives rise to a continu-ous outer nuclear membrane that brings theinner nuclear membranes into close contact,which leads to its final fusion. So far, little isknown about the molecular basis of polar nu-clear membrane fusion, although many muta-tions and genes have been identified (10, 50,58). Interestingly, most of the genes whosemutation blocks polar nuclear fusion encodemitochondrial proteins. Similarly, mutationin GLUCOSE 6-PHOSPHATE/PHOSPHATETRANSLOCATOR1 (GPT1) also blocks polarnuclear fusion in the central cell (44). One pos-sibility is that high respiratory activity is re-quired for central cell development because thecentral cell has such a large cytoplasm. Alter-natively, an intracellular feedback mechanismamong the organellar and nuclear genomes maycoordinate central cell development. GFA2 hasbeen implicated in the membrane fusion. Ingfa2 mutants, the polar nuclei fail to fuse; theirouter nuclear membranes come into contactbut do not fuse (10). This suggests that GFA2,either directly or indirectly, is required forthe membrane fusion of the polar nuclei inArabidopsis.

As the polar nuclei fuse, central cell spec-ification and functional differentiation occur.In glauce ( glc) mutant embryo sacs, gameto-phytic cells develop and are specified nor-mally as manifested by the correct expressionof gametophytic cell-specific markers; how-ever, the central cell cannot be fertilized (43).

Genetic analysis suggests that GLC is epistaticto MEA and plays an essential role in the ma-ternal control of embryo and endosperm de-velopment, which suggests that it might play arole in central cell competence for fertilization.The molecular nature of GLC remains to berevealed.

In addition to its role in fertilization, thecentral cell also plays a role in pollen tube guid-ance. Pollen tube guidance is abolished in sev-eral mutants that disrupt central cell develop-ment. This includes maa1 and maa3 in whichthe polar nuclei fail to fuse (66, 67), which indi-cates a defect in central cell development or/andthe maturation of the female gametophyte. Incontrast, in the central cell guidance (ccg) mu-tant, central cell development is not affectedbecause it does not display any morphologicalabnormality and expresses a central cell-specificmarker (8). CCG is expressed in the central cellof the mature embryo sac specifically and en-codes a nuclear protein that might play a rolein regulating the expression of a subset of genesthat are necessary for pollen tube guidance inthe central cell.

CONCLUSIONS

Molecular mechanisms that control thegermline, gametic cell specification, and cell-cell interactions in plant gametogenesis arebeginning to be revealed. Genes that controlthese processes have been identified (Table 1).The haploid female gametophyte providesus with an exciting system to investigatedevelopmental roles of the RNA splicingmachinery; signaling pathways mediated bymembrane receptor kinases like MSP1, FER,and ANUXR; and genetic control of cell fates.Emerging combinatory approches that employgenetics, single-cell based genomics, andbiochemistry will undoubtedly facilitate deci-phering the genetic complexity and molecularmechanisms that control female gametogenesisin angiosperms.

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Table 1 List of genes discussed in the text

Gene name Protein function Biological function ReferenceSPOROCYTELESS(SPL/NOZZLE)

Transcription regulator Germline cell fate 24, 63, 75

AGAMOUS (AG) MADS-box protein binds to CArG-boxDNA sequence

Activation of the SPL gene,meristem determinacy andfloral organ identity

24

MULTIPLE ARCHESPORIALCELLS 1 (MAC1)

Not available Germline cell number 64

MULTIPLE SPOROCYTE 1(MSP1)

Leucine rich repeat–containing receptorprotein kinase

Numbers of male and femalesporocytes

45

OsTDL1A Putative ligand of MSP1 Numbers of male and femalesporocytes

78

MEIOSIS ARRESTED ATLEPTOTENE1 (MEL1)

A germline-specific member ofARGONAUTE genes family

Germline development;regulator of early meiosis

46

SWITCH1 (SWI1)/DYAD Novel protein Chromatid cohesionestablishment and chromosomestructure in meiosis

2, 38

AMEIOTIC (AM1) A plant-specific chromatin-bindingprotein, with 30% identity with SWI1

Regulator of leptotene tozygotene transition in meiosis

54

AGL23 Type I MADS-box family transcriptionfactor

Early female gametogenesis 12

ARABINOGALACTANPROTEIN 18 (AGP18)

A cell surface arabinogalactan proteoglycan Functional megasporedevelopment

1

ANAPHASE-PROMOTINGCOMPLEX/CYCLOSOME(APC/C)

Multiple-subunit E3 ubiquitin-proteinligase

Mitotic progression 7, 17, 29, 34

NOMEGA/APC6/CDC16 A component of the Anaphase PromotingComplex

Cell cycle control 7, 29

REGULATORY PARTICLETRIPLE A ATPASE (RPT)

A regulatory subunit of the 20Sproteosome; a member of the AAAsuperfamily

Cell cycle control 17

RING-FINGER E3 LIGASE 1aand 1b (RHF1a and RHF2a)

Ring-finger E3 ligase that targets acyclin-dependent kinase inhibitorICK4/KRP6 for proteosome-mediateddegradation

Cell cycle control 34

SLOW WALKER 1 (SWA1) A nucleolar WD40-containing proteincontrolling pre-18S rRNA processing

Cell cycle progression 65

RETINOBLASTOMA-RELATED(RBR)

Negative regulator of G1/S transition Cell cycle control 14, 26

HADAD (HDD) Not available Cellularization of the embryo sac 41INDETERMINATEGAMETOPHYTE1 (IG1)

LATERAL ORGAN BOUNDARIES(LOB) domain protein

Cell cycle control 16, 20

CYTOKININ INDEPENDENT1(CKI1)

Histidine kinase Cytokinin signaling 56

CHROMATIN-REMODELINGFACTOR 11 (CHR11)

A member of the ATP-dependentSWI2/SNF2 family of chromatin-remodeling factors

Cell division control 22

GEMINI POLLEN2 (GEM2) Not available Cell division control 52(Continued )

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Table 1 (Continued )

Gene name Protein function Biological function ReferenceGEM1/MOR1 A microtubule-associated protein Cell plate formation 73TWO IN ONE (TIO) A phragmoplast-associated protein Cell plate formation 47AtNACK1 and AtNACK2 M-phase-specific kinesin-like protein Phragmoplast and cell plate

expansion72

DEMETER (DME) DNA glycosylase Gene imprinting duringendosperm development

9

FERTILIZATION-INDEPENDENT SEED2(FIS2)

C2H2 zinc finger–containing polycombgroup protein

Suppressor of endospermdevelopment

26

LACHESIS (LIS) RNA splicing factor Gametic cell specification 19CLOTHO (CLO)/GFA1 RNA processing Gametic cell specification 35, 40ATROPOS (ATO) A component of prespliceosome and RNA

splicing machineryGametic cell specification

EOSTRE A BELL-like homeodomain protein Synergid cell fate 51GAMETE EXPRESSED3 (GEX3) A plasma membrane protein Pollen tube guidance 3MYB98 MYB family transcription factor Filiform apparatus formation

and pollen tube guidance59

FERONIA (FER) or SIRENE(SRN)

Leucine rich repeat–containing receptorprotein kinase

Synergid-pollen tube interaction 15, 23, 37, 61

LORELEI (LRE) Putativeglucosylphosphatidylinositol-anchoredprotein

Pollen tube attraction 76

ANXUR1 and 2 Pollen-specific receptor-like protein kinase Pollen tube release 39ABERRANT PEROXISOMEMORPHOLOGY2/ABSTINENCE BY MUTUALCONSENT (APM2/AMC)

Src homology (SH3)-domain containingperoxisomal membrane protein

Peroxisome transport and pollentube reception

5

GFA2 Mitochondrion-located DnaJdomain–containing protein

Synergid cell death 10

DIANA (DIA, AGL61) Type I MADS-box transcription factor,forming a heterodimer with AGL80

Central cell differentiation andfunction

4, 71

AGL80/ FEM111 Type I MADS-box transcription factor,forming a heterodimer with DIA

Central cell differentiation andfunction

57

AGL62 Type I MADS-box transcription factor Central cell and endospermdevelopment

13, 27

MAGATAMA3 (MAA3) RNA helicase involved in RNA splicing Central cell maturation andpollen tube guidance

57

GLUCOSE6-PHOSPHATE/PHOSPHATETRANSLOCATOR1 (GPT1)

Transmembrane glucose 6–phosphatetranslocator

Central cell development 44

GLAUCE (GLC) Not available Central cell function 43CENTRAL CELL GUIDANCE(CCG)

Putative transcription regulator Pollen tube guidance 8

MAGATAMA 1 (MAA1) Not available Central cell development andpollen tube guidance

66

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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

The authors acknowledge financial support from the National Science Foundation of China toY. W. C. (30830063) and D. Q. S. (3060032), and also from the Chinese Academy of Sciences toW. C. Y. (KSCX2-YW-N-048).

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Annual Review ofPlant Biology

Volume 61, 2010Contents

A Wandering Pathway in Plant Biology: From Wildflowers toPhototropins to Bacterial VirulenceWinslow R. Briggs � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Structure and Function of Plant PhotoreceptorsAndreas Moglich, Xiaojing Yang, Rebecca A. Ayers, and Keith Moffat � � � � � � � � � � � � � � � � � � � � �21

Auxin Biosynthesis and Its Role in Plant DevelopmentYunde Zhao � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �49

Computational Morphodynamics: A Modeling Framework toUnderstand Plant GrowthVijay Chickarmane, Adrienne H.K. Roeder, Paul T. Tarr, Alexandre Cunha,

Cory Tobin, and Elliot M. Meyerowitz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �65

Female Gametophyte Development in Flowering PlantsWei-Cai Yang, Dong-Qiao Shi, and Yan-Hong Chen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

Doomed Lovers: Mechanisms of Isolation and Incompatibility in PlantsKirsten Bomblies � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 109

Chloroplast RNA MetabolismDavid B. Stern, Michel Goldschmidt-Clermont, and Maureen R. Hanson � � � � � � � � � � � � � � 125

Protein Transport into ChloroplastsHsou-min Li and Chi-Chou Chiu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

The Regulation of Gene Expression Required for C4 PhotosynthesisJulian M. Hibberd and Sarah Covshoff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 181

Starch: Its Metabolism, Evolution, and Biotechnological Modificationin PlantsSamuel C. Zeeman, Jens Kossmann, and Alison M. Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 209

Improving Photosynthetic Efficiency for Greater YieldXin-Guang Zhu, Stephen P. Long, and Donald R. Ort � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 235

HemicellulosesHenrik Vibe Scheller and Peter Ulvskov � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 263

Diversification of P450 Genes During Land Plant EvolutionMasaharu Mizutani and Daisaku Ohta � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 291

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Evolution in Action: Plants Resistant to HerbicidesStephen B. Powles and Qin Yu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 317

Insights from the Comparison of Plant Genome SequencesAndrew H. Paterson, Michael Freeling, Haibao Tang, and Xiyin Wang � � � � � � � � � � � � � � � � 349

High-Throughput Characterization of Plant Gene Functions by UsingGain-of-Function TechnologyYouichi Kondou, Mieko Higuchi, and Minami Matsui � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 373

Histone Methylation in Higher PlantsChunyan Liu, Falong Lu, Xia Cui, and Xiaofeng Cao � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 395

Genetic and Molecular Basis of Rice YieldYongzhong Xing and Qifa Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 421

Genetic Engineering for Modern Agriculture: Challenges andPerspectivesRon Mittler and Eduardo Blumwald � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 443

Metabolomics for Functional Genomics, Systems Biology, andBiotechnologyKazuki Saito and Fumio Matsuda � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 463

Quantitation in Mass-Spectrometry-Based ProteomicsWaltraud X. Schulze and Bjorn Usadel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 491

Metal Hyperaccumulation in PlantsUte Kramer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 517

Arsenic as a Food Chain Contaminant: Mechanisms of Plant Uptakeand Metabolism and Mitigation StrategiesFang-Jie Zhao, Steve P. McGrath, and Andrew A. Meharg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 535

Guard Cell Signal Transduction Network: Advances in UnderstandingAbscisic Acid, CO2, and Ca2+ SignalingTae-Houn Kim, Maik Bohmer, Honghong Hu, Noriyuki Nishimura,

and Julian I. Schroeder � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 561

The Language of Calcium SignalingAntony N. Dodd, Jorg Kudla, and Dale Sanders � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 593

Mitogen-Activated Protein Kinase Signaling in PlantsMaria Cristina Suarez Rodriguez, Morten Petersen, and John Mundy � � � � � � � � � � � � � � � � � 621

Abscisic Acid: Emergence of a Core Signaling NetworkSean R. Cutler, Pedro L. Rodriguez, Ruth R. Finkelstein, and Suzanne R. Abrams � � � � 651

Brassinosteroid Signal Transduction from Receptor Kinases toTranscription FactorsTae-Wuk Kim and Zhi-Yong Wang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 681

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Directional Gravity Sensing in GravitropismMiyo Terao Morita � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 705

Indexes

Cumulative Index of Contributing Authors, Volumes 51–61 � � � � � � � � � � � � � � � � � � � � � � � � � � � 721

Cumulative Index of Chapter Titles, Volumes 51–61 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 726

Errata

An online log of corrections to Annual Review of Plant Biology articles may be found athttp://plant.annualreviews.org

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