The Plant Cell, Vol. 13, 1477, July 2001, www.plantcell.org © 2001 American Society of Plant Biologists

IN THIS ISSUE

Multidrug transporters form a largeclass of membrane proteins present inthe cells of most organisms. These pro-teins bind to a variety of potentially cy-totoxic compounds and remove them

from the cell in an ATP- or proton-dependent process (Zhelenova et al.,2000). Traditionally, multidrug trans-porters have been divided into four su-perfamilies: the ATP binding cassette(ABC) superfamily, the major facilitatorsuperfamily, the small multidrug resis-tance family, and the resistance-nodu-lation-cell division family. Brown et al.(1999) defined a fifth family, called themultidrug and toxic compound extru-sion (MATE) family of transporters. TheMATE family is characterized by thepresence of 12 putative transmembranesegments and by the absence of “sig-nature sequences” specific to the othermultidrug transporter superfamilies.MATE proteins are believed to functionas proton-dependent efflux transport-ers, based on the genetic characteriza-tion of two family members, NorM from

Vibrio parahaemolyticus

and its ho-molog YdeH from

Escherichia coli

. Ex-pression of these proteins in

E. coli

confers resistance to various antibioticsand antimicrobial agents that is depen-dent on the maintenance of a protongradient across the plasma membrane.MATE genes are abundant in bacteriaand plants—the Arabidopsis genomecontains at least 54 MATE family mem-bers—but have not been found in mam-mals. Aside from NorM and YdeH, verylittle functional information is availableon these proteins.

In this issue of

The Plant Cell

,

Dieneret al. (pages 1625–1637)

describe thefunctional analysis of the MATE gene

ALF5

in Arabidopsis. The

alf5

mutant ex-hibits greatly inhibited formation of lateralroots when grown in commercial Bactoagar (Figure 1). Interestingly, it was found

Move It on Out with MATEs

that the mutant produced roots that wereindistinguishable from wild-type rootswhen grown in soil, in a different brand ofagar, or in extensively washed Bactoagar, suggesting that the

alf5

mutationcaused sensitivity to a soluble contami-nant present in the Bacto agar. The

alf5

locus was cloned and found to contain a29-bp deletion in a gene,

ALF5

, that en-codes a MATE family integral membraneprotein. Gene expression analysis using

AFL5

fused to the

�

-glucuronidase re-porter gene in transgenic plants indicatedthat the

ALF5

gene is highly expressed inthe root epidermis and cortex. In addi-tion, expression of

ALF5

in yeast con-ferred resistance to the toxic cationtetramethylammonium, supporting theconclusion that ALF5 is a functionalMATE efflux transporter.

PLANTS AS “GREEN LIVERS”

Plant cells, like the cells of most organ-isms, are capable of removing a largenumber of potentially toxic compoundsfrom the cytoplasm. In plants, thesecompounds are either sequestered invacuoles or transported to the cell wall.Toxic compounds may be of xenobioticorigin or produced endogenously (e.g.,phenolics, flavonoids, and phytoalex-ins). The bronze-colored phenotype ofthe

Bronze2

mutation in maize, for ex-ample, is caused by the inability of themutant to transport anthocyanin from

the cytosol to the vacuoles.

Bronze2

en-codes a glutathione transferase (Marrs etal., 1995), and the mutant is unable tocarry out conjugation of anthocyaninwith glutathione, a necessary step be-fore transport of conjugated glutathioneto the vacuole.

Sandermann (1992) likened plant me-tabolism of toxic compounds to that of

the mammalian liver because of thepresence and activity of cytochromeP450 monooxygenases and glutathionetransferases, which resemble the twomajor enzyme systems of the liver. Plantcytochrome P450s and glutathionetransferases are involved in the first twophases of detoxification of a number ofpolychlorinated and polycyclic hydrocar-bons and related xenobiotic compoundsas well as endogenous toxins. In phase I,cytochrome P450s prepare a substratefor phase II via hydroxylation, and phaseII glutathione transferases carry out theconjugation of the hydroxylated com-pound to reduced glutathione. Subse-quently, phase III involves the transportof the glutathione conjugate out of thecytoplasm to the vacuole or cell wall.There is good evidence that multidrugtransporters of the ABC superfamily areinvolved in the transport of glutathioneconjugates in plant cells (Rea et al.,1998; Theodoulou, 2000). Sandermann(1992) described plants as “green livers”that might act as a global sink for envi-ronmental pollutants of this nature. Thepresence of MATE efflux proteins inplants, which are presumed to carry outtransport of lipophilic cations and relatedcompounds that are not glutathione con-jugates, broadens the scope of this con-cept and opens up more possibilities forplant biotechnology.

EVOLUTIONARY PLANT TRICKS

Despite the wide range of chemicallyand structurally distinct substrates formultidrug transporters, transporters ofall five superfamilies show a preference

for hydrophobic (lipophilic) cations, suchas quaternary ammonium antiseptics(Stermitz et al., 2000). Lipophilic cat-ions, such as berberine alkaloids, are

1478 The Plant Cell

commonly produced by plants. Lewis(1999) proposed that berberine alka-loids represent a larger group of cat-ionic toxins that fueled the evolution ofmicrobial multidrug transporters. Inter-estingly, Stermitz et al. (2000) found

that several plant species in the genusBerberis, which produce berberine, alsoproduce an inhibitor of multidrug trans-porters, identified as 5

�

-methoxyhyd-nocarpin. Berberine exhibited relativelyweak antimicrobial action, presumably

because of its efflux from bacterial cellsby multidrug transporters. 5

�

-Methoxy-hydnocarpin had no antimicrobial activityalone, but it strongly potentiated the ac-tion of berberine and other toxins againstthe growth of

Staphylococcus aureus

.This finding suggests that the mainfunction of some microbial multidrugtransporters is resistance against plant-produced antimicrobial compounds.

Aside from plant–pathogen interac-tions, one habitat in which bacteria arelikely to encounter toxic plant com-pounds is in root nodules. In

Rhizobiumetli

, multidrug transporters have beenfound to play an important role in nodu-lation of bean (

Phaseolus vulgaris

).

RmrA

encodes an

R. etli

multidrug ef-flux pump gene that is induced by fla-vonoids released from the roots of

P.vulgaris

, and mutations in this genewere found to reduce nodulation in thebean by an average of 40% (Gonzalez-Pasayo and Martinez-Romero, 2000).

Another habitat in which bacteriamight commonly encounter plant toxinsis in the stomachs of herbivores. In

E

.

coli

, the transcription repressor MarRbinds various phenolic compoundssuch as salicylate and regulates the ex-pression of two multidrug transportersto produce a more effective effluxpump system. Sulavik et al. (1995) sug-gested that drug resistance in

E. coli

isthus enhanced when the bacteria re-side in an omnivore gut rich in plant an-timicrobial compounds.

DIVERSITY OF FUNCTIONS FOR MULTIDRUG TRANSPORTERS

It is apparent that multidrug transport-ers constitute large superfamilies inplants, as in other organisms. The Ara-bidopsis genome contains at least 60open reading frames for ABC trans-porters (Davies and Coleman, 2000). Aswith the less well known MATE family,the function of the majority of thesegenes is unknown, but characteriza-

IN THIS ISSUE

Figure 1. Roots of Wild-Type (Top) and alf5 Mutant Plants Grown in a Commercial Agar.

As a result of the disruption of the ALF5 gene, which encodes a MATE family efflux transporter,the roots of the mutant are sensitive to a contaminant in the agar. The figure was provided byGerald Fink.

July 2001 1479

pression of the

EmrAB

transportergene is relieved by binding of the EmrRrepressor to various neutral compounds.It is interesting to speculate that multi-drug sensors also will be found to con-trol the expression of plant transportergenes such as

ALF5

, whose principalfunction appears to be the efflux oftoxic compounds.

Nancy A. EckardtNews and Reviews Editor

neckardt@aspp.org

REFERENCES

Brown, M.H., Paulsen, I.T., and Skurray,R.A.

(1999). The multidrug efflux proteinNorM is a prototype of a new family oftransporters. Mol. Microbiol.

31,

393–395.

Davies, T.G.E., and Coleman, J.O.D.

(2000).The

Arabidopsis thaliana

ATP-binding cas-sette proteins: An emerging superfamily.Plant Cell Environ.

23,

431–443.

Debeaujon, I., Peeters, A.J.M., Léon-Kloosterziel, K.M., and Koornneef, M.

(2001). The

TRANSPARENT TESTA12

gene of Arabidopsis encodes a multidrugsecondary transporter-like protein requiredfor flavonoid sequestration in vacuoles ofthe seed coat endothelium. Plant Cell

13,

853–871.

Diener, A.C., Gaxiola, R.A., and Fink, G.R.

(2001). Arabidopsis

ALF5

, a multidrugefflux transporter gene family member,confers resistance to toxins. Plant Cell

13,

1625–1637

.

Gaedeke, N., Klein, M., Kolukisaoglu, U.,Forestier, C., Muller, A., Ansorge, M.,Becker, D., Mamnun, Y., Kuchler, K.,Schulz, B., Mueller-Roeber, B., andMartinoia, E.

(2001). The

Arabidopsisthaliana

ABC transporter AtMRP5 controlsroot development and stomata movement.EMBO J.

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1875–1887.

Gonzalez-Pasayo, R., and Martinez-Romero, E.

(2000). Multiresistance genesof

Rhizobium etli

CFN42. Mol. Plant-Microbe Interact.

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572–577.

Leonhardt, N., Vavasseur, A., and Forestier,C.

(1999). ATP binding cassette modula-tors control abscisic acid–regulated slow

IN THIS ISSUE

tion of a few family members suggestsa multiplicity of functions in plantgrowth and development within the su-perfamily, in addition to their role in thetransport of xenobiotic compounds.

Sidler et al. (1998) showed that an Ar-abidopsis ABC transporter, AtPGP1, isinvolved in the regulation of hypocotylelongation during photomorphogene-sis. Under certain light conditions, plantsoverexpressing

PGP1

developed longerhypocotyls, whereas plants with inhib-ited expression of

PGP1

producedshorter hypocotyls compared with thewild type. Hypocotyl elongation in thedark was unaffected by alterations in

PGP1

expression. In wild-type plants, the

AtPGP1

gene was found to be expressedin the plasma membrane of root andshoot apices, and the authors proposedthat AtPGP1 is involved in the transportof a signal molecule, such as a peptidehormone, from the shoot apical region.

Some mammalian ABC transporters,such as the cystic fibrosis transmem-brane conductance regulator (CFTR)and the sulfonylurea receptor (SUR),have been shown to act as ion chan-nels and/or channel regulators. CFTRfunctions as an outwardly rectifying Cl

�

channel that also regulates other ionchannels, and the SUR acts as an ATP-dependent K

�

channel (Theodoulou,2000). Some researchers have begunto look for such activity among ABCtransporters in plants and have foundevidence that ABC proteins may func-tion as ion channel regulators inguard cells. Gaedeke et al. (2001) andLeonhardt et al. (1999) have investi-gated a slow ion channel in Arabidopsisguard cells that shows CFTR-like char-acteristics and that may coordinate theefflux of K

�

and other ions during sto-matal closure.

The MATE transporter superfamilyalso may cover a diverse range of func-tions in plant growth and development.Debeaujon et al. (2001) recently reportedthat the

TRANSPARENT TESTA12

(

TT12

) gene encodes another MATEfamily member in Arabidopsis. The

function of TT12 appears to be incontrolling the vacuolar sequestrationof flavonoids in the seed coat (testa)endothelium. Because of their highchemical reactivity, flavonoids are toxicendogenous compounds that must beremoved from the cytoplasm after theirsynthesis and sequestered in the vacu-ole or cell wall. There is evidence thatthey function as protectants against UVlight damage, oxidative stress, andpathogen attack. The mutant seeds,lacking the function of the TT12 MATEprotein, appear to be unable to trans-port and accumulate flavonoids in thevacuoles of the seed coat endothelium.The seeds are pale in color and alsoshow reduced seed dormancy, sup-porting the idea that flavonoids play animportant role in seed biology (Winkel-Shirley, 1998). Thus, it appears that wecan expect a multiplicity of functions ingrowth and development for the manyother plant MATE family members. Sur-prisingly, Diener et al. found a secondopen reading frame at the

alf5

locus,

LAL5

, which lies immediately down-stream of

ALF5

and encodes a polypep-tide with 83% identity to ALF5. It is notknown if

LAL5

is expressed, and thisneeds to be determined, but the au-thors reported that the gene appearedto be intact in the

alf5

mutant. If thegene were expressed, it would appearto be functionally distinct from

ALF5

.The discovery of multidrug sensors

that regulate the expression of somemicrobial multidrug transporters sug-gests that the main function of thesetransporters is the efflux of xenobiotictoxins (Lewis, 1999). At least three mul-tidrug sensors have been identified.

BmrR

is a transcription factor in

Bacil-lus subtilis

that activates the expressionof the multidrug transporter gene

Bmr

in response to binding a number of hy-drophobic cations, many of which arealso substrates of the Bmr protein. In

S.aureus

, the QacA multidrug transportergene is repressed by QacR, and bind-ing of QacR to various cations induces

QacA

expression. And in

E. coli

, re-

1480 The Plant Cell

anion channels in guard cells. Plant Cell

11,

1141–1151.

Lewis, K.

(1999). In search of natural sub-strates and inhibitors of MDR pumps. J.Mol. Microbiol. Biotechnol.

3,

247–254.

Marrs, K.A., Alfenito, M.R., Lloyd, A.M.,and Walbot, V.

(1995). A glutathione S-con-jugate transferase involved in vacuolartransfer encoded by the maize

Bronze-

2.Nature

375,

397–400.

Rea, P.A., Li, Z.-S., Lu, Y.-P., andDrozdowicz, Y.M.

(1998). From vacuolarGS-X pumps to multispecific ABC trans-porters. Annu. Rev. Plant Physiol. PlantMol. Biol.

49,

727–760.

Sandermann, H.

(1992). Plant metabolism of

xenobiotics. Trends Biochem. Sci.

17,

82–84.

Sidler, M., Hassa, P., Hasan, S., Ringli, C.,and Dudler, R.

(1998). Involvement of anABC transporter in a developmental path-way regulating hypocotyl cell elongation inthe light. Plant Cell

10,

1623–1636.

Stermitz, F.R., Lorenz, P., Tawara, J.N.,Zenewicz, L.A., and Lewis, K.

(2000).Synergy in a medicinal plant: Antimicrobialaction of berberine potentiated by 5

�

-methoxyhydnocarpin, a multidrug pumpinhibitor. Proc. Natl. Acad. Sci. USA

97,

1433–1437.

Sulavik, M.C., Gambino, L.F., and Miller,P.F.

(1995). The MarR repressor of the

multiple antibiotic resistance (

mar

) operonin

Escherichia coli

: A prototypic memberof a family of bacterial regulatory proteinsinvolved in sensing phenolic compounds.Mol. Med.

1,

436–446.

Theodoulou, F.L.

(2000). Plant ABC transport-ers. Biochim. Biophys. Acta

1465,

79–103.

Winkel-Shirley, B.

(1998). Flavonoids inseeds and grains: Physiological function,agronomic importance and the genetics ofbiosynthesis. Seed Sci. Res.

8,

415–422.

Zhelenova, E.E., Markham, P., Edgar, R.,Bibi, E., Neyfakh, A.A., and Brennan,R.G.

(2000). A structure-based mecha-nism for drug binding by multidrug trans-porters. Trends Biochem. Sci.

25,

39–43.

APO2001: A Sexy Apomixer in Como

Long before promiscuity was discov-ered to bear significant risk, many flow-ering plants had partially abandonedthe pleasures of their sexual life toevolve one of the most intriguing repro-ductive alternatives found in nature.Apomixis is an asexual method of re-production through seed that circum-vents meiosis and fertilization toculminate in the autonomous develop-ment of an embryo. Thus, unlike sexualreproduction, which yields geneticallydiverse progeny, apomixis producesclonal offspring. The production ofclonal, genetically identical, seed bearsgreat potential for applications in plantbreeding and seed production (Hannaand Bashaw, 1987; Savidan, 1992;Koltunow et al., 1995). Apomixis hasevolved several times independentlyfrom sexual ancestors and can beviewed as a modification of the sexualreproductive program. In angiosperms,sexual reproduction entails complex in-teractions between a variety of tissues.Female reproductive development oc-

curs in a specialized organ, the ovule,where usually a single cell becomescommitted to the reproductive path-way (the megaspore mother cell, orMMC). After meiosis, a single reducedproduct, the functional megaspore, di-vides mitotically to form the mature fe-male gametophyte or embryo sac. Theusually seven-celled embryo sac con-tains the egg cell and the binucleatecentral cell, both of which get fertilized.In the male reproductive organs, meio-sis produces a tetrad of reducedspores, all of which divide mitotically toform the male gametophytes (pollen).The male gametophyte consists of twosperm cells, which are contained in alarge vegetative cell that delivers thesperm cells to the female gametophyte.During double fertilization, one spermfuses with the egg to form the zygoteand the second sperm fuses with thecentral cell to form the endosperm.

In apomictic plants, this sexual de-velopmental program is bypassed orderegulated at various steps (Koltunow,

1993; Grossniklaus, 2001): (1) meiosisis altered or absent to produce an unre-duced female gametophyte with the fullcomplement of maternal chromosomes(apomeiosis); (2) fertilization is avoided,producing an autonomous embryo (par-thenogenesis); and (3) endosperm de-velopment is initiated autonomously orsexually; in the latter case, embryo sacdevelopment or fertilization is often mod-ified to adjust to a different genomiccontext (Savidan, 2000; Grossniklauset al., 2001). In contrast to the modifiedfemale reproductive program, pollenformation usually is unaffected inapomicts.

During the last two decades, the in-troduction of apomixis into sexual cropshas been perceived as one of the mostpromising challenges faced by agricul-tural biotechnology. Apomixis could al-low the fixation of any genotype, howevercomplex, including that of high yieldingF1 hybrids. The enormous potential ofthis trait was realized as early as the1930s by Navashin and Karpenko

MEETING REPORT

July 2001 1481

(cited by Asker, 1971). Simplified apo-mixis breeding programs would allowan immediate fixation of any genotypeand the production of self-perpetuatingimproved hybrids and could promisesocial and economic benefits thatwould challenge those of the GreenRevolution (Vielle-Calzada et al., 1996a;Grossniklaus et al., 1998a).

The 2nd International Conference onApomixis took place in Como, Italy,from April 24 to 28, 2001. The scientificprogram was organized by Lucia Co-lombo, Thomas Dresselhaus, YvesSavidan, and Rod Scott and was spon-sored by the European Union, the Foodand Agriculture Organization of theUnited Nations, the Institut de Recherchepour de Développement (IRD), the Ital-ian National Research Center, andmany private sponsors. Following onthe success of the 1st InternationalApomixis Meeting held at College Sta-tion, Texas, in 1995, the Como confer-ence attracted 170 participants from 27countries. All meeting abstracts of in-vited speakers and poster sessions areavailable at http://www.apomixis.de.

Leo Beukeboom (University of Lei-den, The Netherlands) gave the open-ing lecture on “Origin and Genetics ofParthenogenesis in Animals.” Asexualreproduction is a widespread phenom-enon across the animal and plant king-doms. There are differences in both theterminology and the mechanisms ofasexual reproduction in plants and ani-mals. Parthenogenesis (virgin birth),first observed by Bonnett in 1745, isthe development of an egg without fer-tilization. Asexual reproduction in ani-mals can occur either by mitoticparthenogenesis (apomixis) or by mei-otic parthenogenesis (automixis). Incontrast to the phenomenon in plants,apomixis in animals is rarely facultative,and most forms of automixis occur ex-clusively in animals. A wide range ofcytological mechanisms underlie mi-totic or meiotic parthenogenesis in ani-mals. Beukeboom presented examplesof asexual reproduction in freshwater

flatworms (

Polycelis nigra

), in which Bchromosomes may be associated withparthenogenetic lineages. Althoughmuch is known of the molecular mech-anisms of fertilization in animals, re-markably little is known about themechanisms of parthenogenesis.

MECHANISMS AND EVOLUTIONOF APOMIXIS

Early studies demonstrated that apo-mixis is controlled genetically. Typi-cally, a single dominant mendelian traitis associated with apomixis, althoughmore complex modes of inheritancehave been reported (see accompanyingInsight article). Cytological descriptionsemphasized distinctions between dif-ferent apomictic mechanisms and haveled to a simplified classification that isbased on the origin and the location ofcells initiating apomictic development(reviewed in Koltunow, 1993). Indiplospory, the MMC undergoes an ab-errant meiosis or divides mitotically,producing unreduced spores that even-tually form the embryo sac containingan egg cell with the full genetic comple-ment of the mother. In apospory, a cellin the ovule other than the MMC pro-duces the unreduced embryo sac.Anna Koltunow (Commonwealth Scien-tific and Industrial Research Organiza-tion, Adelaide, Australia) described theenormous variability existing in apomic-tic processes. In Hieracium, apomicti-cally derived embryo sacs usually areproduced through apospory. However,several loci modify the timing of apo-mictic initiation, the frequency at whichapomictic embryo sacs are formed,and the mode of progression ofapomictic development (Koltunow etal., 1998, 2000; Bicknell et al., 2000).These findings indicate that the majorlocus associated with apomixis mightcreate a competence for a varietyof reproductive developmental pro-cesses in the ovule. Koltunow hypoth-

esized that sporophytic cells in theovule play an active role in modulatingand controlling reproductive develop-ment. Transformation-induced alter-ations in ovule development that resultin significant changes of the mode ofapomixis in Hieracium add support tothis hypothesis.

For Yves Savidan (IRD, Montpellier,France), close examination of develop-mental processes in the ovule indicatesthat apomictic embryo sacs developmore precociously than their sexuallyderived counterparts. However, em-bryo sac development is always nor-mal, no matter how strongly meioticprocesses are disturbed. This suggestsa crucial role for the timing of develop-mental events during reproduction andimplies that all types of gametophyticapomixis may relate to the ectopic ex-pression of the same master regulatorygene(s). In this view, distinct mecha-nisms would result solely from differ-ences in the timing of apomicticinduction within the ovule. DanielGrimanelli (IRD and Centro Internacionalde Mejoramiento de Maíz y Trigó[CIMMYT], México) has cytologicallyanalyzed the apomictic mechanism of

Tripsacum dactyloides

, focusing onchromosome and chromatin dynamicsand the characteristics of the cyto-skeleton during apomeiosis. A widephenotypic variability occurs duringapomictic initiation, even within a singlegenotype. Many of the developmentalabnormalities characteristic of apomei-otic processes in Tripsacum have strik-ing similarities to defects found inmeiotic mutants of maize. Althoughthese meiotic mutants have a strongimpact on fertility, similar abnormalitiesdo not compromise apomictic seed for-mation. This suggests that develop-mental events occurring after apomicticinitiation can compensate for, and res-cue, meiotic defects.

Whereas in Tripsacum the MMC pro-ceeds directly to divide mitotically, indandelions (

Taraxacum

sp) the MMCenters prophase of meiosis I without

MEETING REPORT

1482 The Plant Cell

subsequent chromosome pairing. Thisattempted first division results in a res-titution nucleus enclosing a completeset of chromosomes that undergoes thesecond meiotic division (diplospory).Hans de Jong (Wageningen Univer-sity, The Netherlands) presented a de-tailed genetic analysis of apomixisbased on interploidy crosses betweensexual diploids and apomictic triploidsof Taraxacum. The occurrence of re-combinants in which the initiation ofapomixis and the autonomous devel-opment of embryo and endosperm wereuncoupled indicates that apomixis iscontrolled by at least three loci. A dom-inant trait linked to a microsatellitemarker and located on one of the nu-cleolar organizer chromosomes appearsto control diplospory. Interestingly,this marker is absent in sexual diploidprogeny, suggesting that haploid pollengrains cannot transmit this trait. EmidioAlbertini (University of Perugia, Italy)confirmed that apospory in

Poa praten-sis

can be uncoupled from the mecha-nisms controlling the parthenogeneticdevelopment of embryos. Although par-thenogenesis is seemingly contingenton apomeiosis, the reverse is not thecase. The variability of the degree ofparthenogenesis suggests that it islikely not controlled by a discrete locusand may be under complex control.

Apomixis is reported in more than400 species belonging to 40 families.However, it is particularly prevalentwithin the Asteraceae, Poaceae, andRosaceae. Focusing on the distributionand evolutionary history of apomixis,John Carman (Apomyx, Inc., Logan,UT) emphasized that only 127 of morethan 14,000 genera of flowering plantscontain apomictic species, most ofwhich have appeared during or afterthe Pleistocene. Carman suggests thatapomicts may have arisen by wide hy-bridization of ancestral sexual parentshaving distinct phenotypic traits relatedto reproduction (Carman, 2001). To testthis hybridization-derived floral asyn-chrony hypothesis, Carman’s group

has documented variation of reproduc-tive traits among sexual relatives ofwell-known apomicts (Tripsacum andAntennaria), finding that they are het-erozygous and polygenic. Many formsof reproductive variation, including apo-mixis, may have arisen after hybridiza-tion of sexual ancestors with divergentreproductive traits. In many agamiccomplexes which are composed of in-dividuals of varying ploidy levels, dip-loid genotypes usually are sexual andpolyploid genotypes usually are apo-mictic. Tim Sharbel (Max Planck Institutefor Chemical Ecology, Jena, Germany)studied the evolution of apomixis andpolyploidy in the

Arabis holboellii

ag-amic complex. Using chloroplast hap-lotypes identified in diploid, aneuploid,and triploid individuals, he concludedthat polyploidy arose repeatedly andindependently within this complex(Sharbel and Mitchell-Olds, 2001). Thevariation in reproductive mode andpopulation structure suggests that apo-mixis might have a single evolutionaryorigin, followed by multiple instances ofphenotypic expression of this trait.

BREEDING APOMIXIS INTOSEXUAL CROPS

Among the grasses, apomixis occurs inseveral economically important foragegenera (e.g., Pennisetum, Brachiaria,Paspalum, and Poa). At least threegroups have attempted the introgres-sion of apomixis into sexual crops viawide hybridization using backcross-ing (BC) strategies combined withembryological or cytogenetic studies.For close to 20 years, Wayne Hanna(United States Department of Agricul-ture–Agricultural Research Service,Tifton, GA) and his colleagues haveattempted the transfer of apomixisfrom

Pennisetum squamulatum

topearl millet (

P. glaucum

) (Hanna et al.,1998). By screening large popula-tions to identify partially male fertile

apomictic plants, backcrossing hasprogressed to the BC7 generation.Apomictic tetraploid BC7 plants have28 or 29 chromosomes and closelyresemble pearl millet. However, theyform very little viable seed, a problempossibly related to the dosage sensi-tivity of endosperm development(Morgan et al., 1998). Seed sterilitymay be overcome by exploiting thegenetic diversity found within the tet-raploid germplasm pool.

Following the pioneering work of D.F.Petrov, who realized the potential of in-trogressing apomixis into maize bywide hybridization more than 50 yearsago, Victor Sokolov’s group (Institute ofCytology and Genetics, Novosibirsk,Russia) attempted to transfer apomixisfrom T. dactyloides to maize using tet-raploid female parents. F1 hybrids hav-ing 20 chromosomes from maize and36 from Tripsacum were apomictic butmale sterile. Recurrent backcrossingto male diploid or tetraploid maizeresulted in apomictic genotypes invari-ably containing the same nine Trip-sacum chromosomes. The absenceof any additional Tripsacum chromo-somes resulted in the loss of apomixis,suggesting polygenic control. Moreover,apomeiosis and parthenogenesis segre-gate and are controlled by different loci(Sokolov and Khatypova, 2001). In-spired by the work conducted in Rus-sia, Yves Savidan launched an initiativeto transfer apomixis from Tripsacum tomaize in the early 1990s. Large popula-tion screening, flow cytometry, and ge-nomic in situ hybridization were used toobtain BC3 plants that have 20 chromo-somes from maize and 18 from Trip-sacum. Because little sexuality waspresent in BC3 plants, the acquisition ofsubsequent BC populations was diffi-cult (Savidan, 2000). A plant with a chro-mosome responsible for apomixis hasyet to be found among the BC4 gener-ation. Olivier Leblanc (IRD-CIMMYT,Mexico) indicated that the maize ge-nome severely alters the expression ofapomixis in members of these BC pop-

MEETING REPORT

July 2001 1483

ulations. Specific attributes necessaryfor the proper expression of apomixis inmaize could be related to gene dosageeffects of specific modifiers or to par-ent-of-origin–dependent expression (i.e.,genomic imprinting) of key regulatorygenes that control embryo sac develop-ment and/or early seed formation.

ISOLATION OF GENESCONTROLLING APOMIXIS

Basic knowledge of the genetic andmolecular regulation of female repro-ductive development in apomictic spe-cies remains poor. Although the in-heritance of the trait has been studiedin several species, efforts to isolate thegenes that control apomixis are at anearly stage. The development of thegenetic and molecular tools necessaryto establish an apomictic model systemis well under way (Bicknell, 1994,2001), and the first mutants altered inapomictic developmental pathways havebeen isolated. Ross Bicknell and histeam (Institute for Food and Crop Re-search, Lincoln, New Zealand) haveimplemented �-ray and insertional mu-tagenesis strategies in Hieracium spp.At least two mutants have been iso-lated that have lost the ability to formapomictic seed but still reproduce sex-ually, indicating that apomixis and sex-uality can be uncoupled in Hieracium.In both mutants, the differentiation ofaposporous initials early during ovuledevelopment is affected. Further char-acterization promises insights into themolecular nature of the genes involvedin apomictic initiation.

Peggy Ozias-Akins and co-workers(University of Georgia, Tifton) havemapped apomixis to a single locus inboth Pennisetum ciliare and Pennise-tum squamulatum (Ozias-Akins et al.,1998; Roche et al., 1999), two species re-lated to pearl millet. Detailed analysis ofP. squamulatum revealed no recombi-nation between 12 markers and the

apomixis locus. Interestingly, no se-quences that cross-hybridize to four ofthese markers are present in sexual in-dividuals of a segregating population,suggesting that the region is hemizy-gous or highly divergent in theapomicts. On the basis of marker con-servation between the two species, bac-terial artificial chromosome cloneslinked to apomixis were identified, re-vealing a series of duplications thatmight explain the absence of meiotic re-combination in this chromosomal re-gion. Informative bacterial artificialchromosome clones have been physi-cally mapped using fluorescence in situhybridization. Fulvio Pupilli’s group(Consiglio Nazionale delle Richerche,Perugia, Italy) has mapped the apomixislocus in Paspalum simplex using selectedrice probes as restriction fragment lengthpolymorphism anchor markers and am-plified fragment length polymorphism(AFLP) markers. They confirmed thatapomixis segregates as a single domi-nant trait and identified five rice markerstightly linked to apomixis (Pupilli et al.,2001). As in Pennisetum, the genomic re-gion linked to apomixis shows no signsof meiotic recombination. The authorsconcluded that the apomixis locus iscontained in a chromosomal area that issyntenic to a 15-centimorgan region onrice chromosome 12.

Genes specific to apomictic develop-ment may be identified by comparinggene expression in sexual and apomic-tic ovules among closely related geno-types of the same species (Vielle-Calzada et al., 1996b). Sexual andapomictic genotypes have been char-acterized in the genus Brachiaria. JulioRodrigues from Vera Carneiros’s group(Embrapa, Brasilia, Brazil) reported on adifferential display strategy to isolatedifferentially expressed transcripts dur-ing specific stages of ovary develop-ment in Brachiaria brizantha. Severaldozen fragments were specific to eithersexuals or apomicts and are being ana-lyzed further. Similarly, Gianni Barcaccia(University of Padova, Italy) is investi-

gating gene expression during flower-ing of mutants of alfalfa that fre-quently form unreduced 2n egg cells. Acollection of 40 polymorphic cDNA-AFLP clones was isolated by differen-tial display.

In a special lecture on “Approachesto Capturing Wild Apomictic GenesCombining Genetics and Genomics,”Michael Freeling (University of Califor-nia, Berkeley) presented a strategy toidentify regulatory regions. Nick Kaplinski,a graduate student with Freeling,developed a sliding window–type al-gorithm that identifies regions of con-served noncoding sequences bothwithin and between species. Such aninverse genetics approach can be usedto identify regions that are under func-tional selective pressure.

OVULE AND FEMALEGAMETOPHYTE DEVELOPMENT

The initiation of apomictic developmentoccurs at early stages of ovule devel-opment, during the differentiation ofmeiotic or apomeiotic products. Whatleads to the meiotic or apomeioticcommitment of cells within the ovule?Do megaspores sense their positionand communicate with other sporo-phytic cells? It is becoming apparentthat meiosis and female gametophytedevelopment are controlled by regula-tory genes that may be deregulated inspace and time in apomicts (Koltunow,1993; Grossniklaus, 2001). In fact,many events relevant to apomixis, suchas the initiation of embryo sac develop-ment, autonomous activation of theegg cell, and modified fertilizationmechanisms, are functions of the de-veloping female gametophyte. Yet, weknow little about the genetic control ofthese events, even in sexual modelspecies. A better understanding of themolecular mechanisms underlying em-bryo sac development and its interac-tions with sporophytic cells in the ovule

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could enhance our understanding ofapomixis dramatically.

Ueli Grossniklaus and his team (Uni-versity of Zürich, Switzerland) use Ara-bidopsis as a model system to identifygenes involved in megasporogenesisand embryo sac development. Using anenhancer detection strategy (Sundaresanet al., 1995), they identified mutants af-fected in female gametophyte develop-ment and double fertilization. Some ofthem may be relevant to the develop-mental alterations that ensure the normalendosperm formation observed in manyapomicts. For example, a new signalingpathway between male and female ga-metophytes was identified by the fero-nia mutant: a wild-type pollen tube wasunable to release its sperm cells into amutant feronia embryo sac, suggestingan active role of the female gameto-phyte in this process. Grossniklaus andcolleagues also identified genes ex-pressed in specific cell types of the ovuleand embryo sac, many of which en-code regulatory proteins. Promoters con-trolling expression in the ovule at thesite of apomictic initiation, the MMC, orthe egg could be used to induce ele-ments of apomixis by expressing can-didate genes in specific cell types.

In Arabidopsis, female meiosis resultsin three cells that die, whereas a singlemegaspore produces the embryo sac.Wei-Cai Yang (Institute of MolecularAgrobiology, Singapore) used transpo-son mutagenesis (Sundaresan et al.,1995) to investigate cell fate determina-tion among the megaspores. He identi-fied a tagged mutant with embryo sacsconsisting of large multinucleated cells.These cells may be derived from sev-eral surviving megaspores, indicating adefect in cell specification, or may re-sult from aberrant cellularization eventsin the embryo sac. The isolation of reg-ulatory genes implicated in cell fate de-termination may be related directly to thedevelopmental alternatives associatedwith apomictic initiation. In that regard,the role of transcription factors implicatedin the regulation of ovule development

in sexual species is particularly rele-vant. The work of Rebecca Favaro andcolleagues in Lucia Colombos’s group(University of Milan, Italy) demonstratedthat the expression of a MADS boxgene (AGL11) during ovule and seedformation in Arabidopsis is controlledby an intron that is crucial for determin-ing the temporal and spatial context inwhich AGL11 acts.

CELL CYCLE AND MEIOSIS

Critical aspects of cell cycle regulationand meiosis differ between apomictsand sexuals. Unlike sexually reproduc-ing plants, apomicts do not undergo re-combination during meiosis I, and theyproduce unreduced female gametes.Greater understanding of the (mis)regu-lation of the cell cycle and meiosis willfacilitate the development of systems toinduce parthogenesis and apomeiosis.

To maintain genetic stability and fertil-ity, polyploid crops (e.g., wheat) mustboth pair and segregate the closelyrelated chromosomes correctly duringmeiosis. In polyploids, homologous chro-mosomes must accurately distinguisheach other from homeologous chromo-somes. Peter Shaw (John Innes Centre,Norwich, UK) described how the Ph1 lo-cus controls the specificity of somaticand meiotic chromosome association inwheat. The Ph1 locus restricts chromo-some pairing and recombination to truehomologs. Thus, in hexaploid wheat withPh1 deletions, pairing and recombinationcan occur between homeologs and ho-mologs. In the absence of Ph1, nonho-mologously associated centromeres failto separate at the beginning of meiosis.Shaw discussed how the Ph1 locus pro-motes the specificity rather than theinduction of centromere association(Martinez-Perez et al., 2001).

Screens for male sterility in Arabi-dopsis by Hong Ma’s group (Pennsyl-vania State University, University Park)identified genes regulating chromo-

some segregation during male meio-sis. Two male sterile mutants (ask1 andsds) were shown to have abnormalchromosome segregation during meio-sis I. Premature homolog dissociationoccurs in the sds mutant near the endof prophase I, whereas the homologsseem to remain attached at anaphase Iin the ask1 mutant. The ASK1 gene ismost similar to yeast SKP1, which is asubunit of the SKp1-Cullin-1-F-box(SCF) ubiquitin ligase complex. Al-though the yeast and human SKP1genes regulate the mitotic cell cycle, itwas not known until recently that theseproteins also could be required for mei-osis (Yang et al., 1999). Moleculardetermination of the developmentalmechanisms underlying male and fe-male meiosis is under way by TomGerats’ team (University of Gent, Bel-gium), which is undertaking cDNA-AFLP transcript profiling in Petunia.Transcript profiling techniques wereapplied to male meiosis, focusing onthe identification of genes involved insynapsis and recombination. Repro-ducible cDNA-AFLP patterns were ob-tained using RNA from only threedeveloping anthers. In a pilot study,�100 (5%) differentially expressedtranscripts were identified, includingpreviously characterized meiotic genes(e.g., DMC1-like protein).

Fifty years ago, Böcher reported un-reduced pollen development in the apo-mict A. holboellii (Böcher, 1951). AlexeiKravtchenko (Russian Academy of Sci-ences, Novosibirsk, Russia) conducteda cytological reexamination of unre-duced pollen formation in diploid andtriploid A. holboellii accessions. Thefirst meiotic division was equational,and omission of the second divisionproduced unreduced microspores. Forboth triploid and diploid plants, pollenviability was highly variable. Yet, sometriploids exhibited greater than 90%pollen fertility, an advantage for apo-mixis research on A. holboellii.

Although many meiotic mutants havebeen described in plants, only six genes

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involved in meiosis have been cloned.Christine Horlow (Institut National de laRecherche Agronomique, Versailles,France) showed that the ArabidopsisSWITCH1 (SWI1) protein is required forboth sister chromatid cohesion andbivalent formation (Mercier et al., 2001).Horlow has identified a second swi1 al-lele (swi1-2) that affects both male andfemale meiosis, whereas swi1-1 was re-ported to be female specific (Montamayoret al., 2000). The cytological behaviorof swi1-2 during metaphase in malemeiocytes is intriguing: instead of thenormal 5 bivalents, 20 chromatids areobserved that segregate aberrantly.

Cell cycle control is a complex pro-cess that is mediated largely by proteinkinases, which activate proteins withspecific functions during the cell cycle.Danny Geelen (University of Gent, Bel-gium) reviewed current research on cellcycle progression that is under way inDirk Inzé’s laboratory (Joubes et al.,2000; Stals et al., 2000; de Veylder etal., 2001). Transgenic studies in whichthe function of key cell cycle regulatorsis disrupted demonstrated that the cellcycle is integrated with plant develop-ment. The Inzé group has conductedan AFLP-based screening for cell cyclegenes in tobacco BY-2 cells using anaphidicoline blocker. Approximately 500cell cycle–modulated expressed se-quence tags (ESTs) have been identi-fied, and 50% of the clones isolatedexhibited no significant homology withknown proteins. Jim Murray (Universityof Cambridge, UK) focused on the rolesof D-type cyclins (cycD) in modulatingcell division rates that affect growthand development (Meijer and Murray,2000). The CycD genes play an impor-tant role in deciding whether a cell entersa division cycle. Cytokinin activates celldivision through the induction of CycD3at the G1-S cell cycle transition (Riou-Khamlichi et al., 1999). Overexpression ofCycD2 reduces the length of the G1phase, causing faster cell cycling and ac-celerated plant development (Cockcroftet al., 2000). The constitutive overexpres-

sion of the CycD2 cyclin in the shootmer-istemless Arabidopsis mutant led to arestoration of vegetative growth andlong-lived plants.

FERTILIZATIONAND PARTHENOGENESIS

Parthenogenesis is a critical element ofapomixis whereby an (unreduced) eggcell initiates embryogenesis without fer-tilization. The molecular mechanisms thattrigger parthenogenesis in apomicticplants remain largely unknown. HelmutBäumlein (Institut für Pflanzengenetikund Kulturpflanzenforschung, Gatersle-ben, Germany) discussed embryologicaland molecular studies on apomeiosis inPoa pratensis and parthenogenesis inwheat that his group conducted inclose cooperation with Fritz Matzk. Ge-netic and ploidy analysis (Matzk et al.,2000) of crosses between obligate sex-ual and apomictic Poa lines demon-strated that apomixis was dominantand that apomeiosis and partheno-genesis are not linked. A molecularapproach based on SMART subtrac-tive suppression hybridization identifiedcDNAs from (apo)meiotic stages spe-cific to obligate sexual and apomicticlines. The “Salmon system” of wheatproduces a high fraction of parthenoge-netic offspring, and isogenic sexual linesare available (Matzk, 1996). Bäumleinand co-workers isolated candidate eggcell–specific genes from an egg cellcDNA library of these sexual and par-thenogenetic lines.

In apomicts, embryogenesis occurscompletely without the contributionof the paternal genome. ThomasDresselhaus (University of Hamburg,Germany) presented data suggestingthat maize differs from Arabidopsis withregard to the time of activation of thepaternal genome during early embryo-genesis (Vielle-Calzada et al., 2000). Inmaize, the switch from maternal tozygotic control of gene expression

appears to occur 18 to 24 hr after fer-tilization. Using reproductive cells iso-lated in vitro, Dresselhaus and co-workers developed a procedure that al-lows for the isolation of mRNAs presentin individual cell types of the female ga-metophyte or the zygote. This ap-proach led to the identification ofdifferentially expressed genes (Cordtset al., 2001) and is being implementedin Tripsacum, allowing comparativestudies of gene expression betweensexual and apomictic cells.

Jean-Emmanuel Faure (Ecole NormaleSuperieure, Lyon, France) presented acytological study of Arabidopsis fertiliza-tion using confocal laser scanning mi-croscopy (Christensen et al., 1997).Characterization of the time course offertilization indicates that (1) synergidsdegenerate at �7 hr after pollination(HAP), (2) there is a rapid change in eggcell polarity, (3) karyogamy occurs 8 to 9HAP, and (4) the first division of the pri-mary endosperm nucleus occurs 9 to 12HAP. Faure’s group identified mutantsthat affect fertilization among 4000�-ray–mutagenized M1 plants. Theyconfirmed phenotypes for a collection of200 families containing putative early fer-tilization mutants. Maura Cardarelli (Uni-versity La Sapienza, Rome, Italy)discussed the effects of the Agrobacte-rium rhizogenes rolB gene on gametedevelopment and embryogenesis. rolBexpression driven by AtDMC1 causedsterility that was likely attributable to adelay in anther dehiscence, whereasrolB expression under the control ofthe FBP7 promoter caused floral de-fects and a slight delay in anther de-hiscence.

EMBRYOGENESIS

Despite the existence of large collec-tions of mutants that affect plant em-bryogenesis, the molecular basisunderlying the developmental stepsleading to egg cell activation and early

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1486 The Plant Cell

embryo development remains poorlyunderstood. Bob Goldberg (The SeedInstitute and University of California,Los Angeles) discussed a genomics ap-proach toward the understanding ofplant embryogenesis. The giant em-bryos of scarlet runner bean allowedthe microdissection and isolation ofcDNAs from either the embryo properor the suspensor. EST sequencing wasused to identify differentially expressedgenes in the embryo proper (2863ESTs, 50% unique) and the suspensor(3138 ESTs, 59% unique). Approxi-mately 10 to 12% of the ESTs exhibitedno homology with genes currently in da-tabases. On the basis of these expres-sion data, the suspensor appears to bethe major source of gibberellic acid in thedeveloping embryo. The Seed Institutealso uses Affymetrix chips for transcriptprofiling in Arabidopsis wild-type andmutant seed. The profiles show highlycomplex changes during the early stepsof seed development.

Sacco de Vries (Wageningen Univer-sity, The Netherlands) discussed the roleof the Arabidopsis somatic embryogen-esis receptor–like kinase (AtSERK1) inovule and embryo development. SERKencodes a leucine-rich repeat trans-membrane receptor kinase (Schmidt etal., 1997) that is similar to animal recep-tors. The use of fluorescence spectralimaging microscopy to study physicalinteractions in plant cells showed thatAtSERK1 can oligomerize in vivo (Shahet al., 2001) and interact directly with thekinase-associated protein phosphatase(KAPP) phosphatase. Plants overex-pressing AtSERK1 have an increasedpotential for somatic embryogenesis inculture. To investigate the potential toinduce embryogenesis in planta, thede Vries team developed a multiplexmarker system for testing AtSERK1–overexpressing F2 plants. Fixed het-erozygosity is indicative of apomixis. In-deed, lines expressing AtSERK1 in theovule produced progeny without a re-combination event among 14 markerstested, whereas control crosses did not

yield such progeny among more than1000 F2 plants. This could be attribut-able to apomictic embryo initiation orsuppressed recombination in theseplants. Ed Schmidt (Genetwister Tech-nologies, Wageningen, The Nether-lands) focused on the family oftransmembrane Receptor Kinases–likeSERK (RKS) genes. There are 16 RKSmembers in Arabidopsis, which can begrouped into three classes. Gene-twister Technologies characterizes thedevelopmental function of the RKSfamily using transgenic plants, whicheither overexpress or cosuppress thedifferent RKS genes.

Kim Boutilier’s presentation (PlantResearch International, Wageningen,The Netherlands) focused on the engi-neering of adventitious embryony. Us-ing subtractive hybridization, Boutilierisolated an embryo-expressed genecalled BABY BOOM (BBM) from mi-crospore embryo cultures of Brassicanapus. BBM encodes an AP2 domaintranscription factor. When expressedconstitutively, it induces the spontane-ous formation of somatic embryos inyoung seedlings. Because the BBMoverexpression phenotype is restrictedto seedlings, Boutilier uses tissue-spe-cific promoters with the aim of induc-ing somatic embryos in ovules.Although L1 expression of BBM ex-tends the tissue range and penetranceof somatic embryo production, it is stillrestricted to seedlings. BBM-express-ing lines show a cytokinin overproduc-tion phenotype, and mutants such asamp1 (Chin-Atkins et al., 1996) andother backgrounds affecting cytokininlevels enhance the BBM somatic em-bryo phenotype.

Gabriella Consonni (Universita degliStudi di Milano, Italy) presented a char-acterization of the maize mutant fusedleaves (fdl). The fdl mutant causes or-gan fusion and affects both embryoorganization and seedling growth: epi-dermal cells of the coleoptile and firstleaf, and the first and second leaves arejoined by a single cell wall.

ENDOSPERM DEVELOPMENT

For agricultural applications, it is es-sential that endosperm development inengineered apomicts is normal. Mostapomictic seed formation requires fer-tilization of the central cell (pseudog-amy). This poses a problem for thetransfer of apomixis to sexual crops be-cause maize and likely most cereals re-quire a ratio of maternal to paternalgenomes of 2m:1p for normal endo-sperm development (Lin, 1984). Fertili-zation of an unreduced central cell witha reduced sperm will yield a 4m:1p ratio,which is expected to cause seed abor-tion. In contrast, natural apomicts areeither insensitive to unbalanced endo-sperm or circumvent the problem by al-tering embryo sac development or themechanism of fertilization (Grossniklauset al., 2001).

Abed Chaudhury (CommonwealthScientific and Industrial Research Or-ganization, Canberra, Australia) pro-vided an overview of ongoing researchon the fis (fertilization-independent seed)class of Arabidopsis mutants. The FISclass includes MEDEA (MEA), FIS2,and FERTILIZATION-INDEPENDENT EN-DOSPERM (FIE) (Grossniklaus et al.,1998b; Luo et al., 1999; Ohad et al.,1999). Mutations in these genes lead tomaternal effect seed abortion, and mu-tant central cells are capable of en-dosperm development in the absenceof fertilization, a component of apo-mixis (reviewed in Grossniklaus et al.,2001). Chaudhury’s group made pro-moter::GUS fusions for all three FISclass genes, showing that the expres-sion of MEA and FIS2 is similar but dif-fers significantly from the FIE pattern(Luo et al., 2000). Only maternally inher-ited copies are expressed early in seeddevelopment, suggesting regulation bygenomic imprinting. Seed abortion canbe prevented if a demethylated pater-nal genome is introduced. This effect isindependent of FIS gene activity (Luo etal., 2000). Interestingly, a demethylated

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paternal genome appears to have aneffect on gene expression from the ma-ternal genome.

Robert Fischer (The Seed Institute andUniversity of California, Berkeley) fo-cused on the suppression of endospermdevelopment by the FIS genes. FIE andMEA encode WD40 and suvar3-9, en-hancer-of-zeste, Trithorax (SET) domainproteins of the Polycomb group. FIEand MEA proteins interact directly, liketheir mammalian and Drosophila ho-mologs, which form a multiproteincomplex (Luo et al., 2000; Spillane etal., 2000; Yadegari et al., 2000). Fischerreviewed data showing silencing of thepaternal mea locus in the endosperm(Kinoshita et al., 1999; Vielle-Calzada etal., 1999). He discussed how thesefindings fit with the parental conflicttheory for the evolution of genomicimprinting (Haig and Westoby, 1991)and with the observation that manyapomicts require fertilization of thecentral cell for endosperm formation.

Problems associated with the engi-neering of autonomous endosperm inapomicts also were reviewed by RodScott (University of Bath, UK). En-dosperm abortion may be caused bythe nonequivalence of paternal andmaternal genomes at imprinted loci.Autonomous endosperm developmentobserved in the Arabidopsis fie mutantcan be enhanced by combining it withhypomethylation (Vinkenoog et al.,2000). On the basis of the parental con-flict theory (Haig and Westoby, 1991),Scott proposes that endosperm size inplants is an assay for gametic “gender”as exemplified by interploidy crosses(Scott et al., 1998). In Arabidopsis,modifications of gametic gender maybe achieved through changes in themethylation profile, which either “ma-ternalize” or “paternalize” the gameticgenome, or as a result of mutants suchas fie, which paternalize maternal ga-metes (Adams et al., 2000; Vinkenooget al., 2000).

Fred Berger (Institut National de laRecherche Agronomique, Lyon, France)

discussed the recent work of Boisnard-Lorig et al. (2001), in which a HIS-TONE::YFP fusion protein was used todemonstrate that syncytial endospermis divided into three distinct mitotic do-mains. Enhancer detection screens(Haseloff, 1999) were used to isolateGFP-based endosperm markers, suchas KS117, which is expressed initiallyin the entire endosperm but then is re-stricted to the chalazal region. Using thismarker, it was found that in fis classmutants endosperm polarization is dis-turbed (Sorensen et al., 2001). TheKS117 line has been used in a �-raymutagenesis screen for mutants that al-ter the expression of KS117. Among 4000M1 plants, 60 lines with disturbedKS117 GFP expression were identified.

José Gutierrez (University of Oxford,UK) presented the isolation of can-didate imprinted genes that areexpressed differentially in maize en-dosperm depending on parental origin.Allelic display polymerase chain reac-tion was used to screen for imprintedmaize genes in reciprocal crosses be-tween different inbred lines of maize.Loci that exhibit either maternal-spe-cific or paternal-specific expressionwere identified at early endospermstages (10 days after pollination; 15candidates) and at later endospermstages (30 days after pollination; 31candidates). The group of Angelo Viotti(Consiglio Nazionale delle Richerche,Milan, Italy) investigates epigeneticphenomena in maize endosperm. Somealleles of the �-zein and �-tubulingenes derived from specific inbredlines are subject to genomic imprinting(Lund et al., 1995a, 1995b). Two of thesix �-tubulin genes displayed a correla-tion between DNA demethylation at thelocus and increased RNA accumulationin the endosperm. �-zein and �-tubulingenes are hypomethylated only whentransmitted through the female. Viotti’sresults confirm that the imprinting sta-tus of certain zein and tubulin alleles inmaize endosperm can be influenced byinteractions of parental factors and are

cross and genotype dependent (Ciceriet al., 2000).

Hilde-Gunn Opsahl-Ferstad (Agricul-tural University of Norway, Aas) fo-cused on the defective kernel1 (dek1)and crinkly4 (cr4) genes that regulatecell identity in the cereal endosperm(Becraft et al., 2001; Olsen, 2001). TheOlsen group is studying several of thePioneer Hi-Bred “Trait Utility System forMaize” Mutator-induced mutants to iso-late genes involved in the control ofaleurone cell identity. Promoter studiesof the LIPID TRANSFER PROTEIN1(LTP1) and LTP2 upstream sequenceshave shown LTP2 to direct expression incereals similar to the patterns describedin dicots, whereas the LTP1 directs adifferent expression pattern.

ECONOMIC AND ECOLOGICAL IMPLICATIONS OF APOMIXIS

The long gestation period for the de-velopment of apomixis technology(Savidan, 2000) has allowed reflectionon both economic (Jefferson, 1994;Bicknell and Bicknell, 1999) and eco-logical issues (van Dijk and vanDamme, 2000) regarding the deploy-ment of apomictic crops in agriculture.Apomictic varieties are being devel-oped for a few (mainly forage grass)species. Jorge Gonzalez (UniversidadAutónoma Agraria Antonio Narro, Sal-tillo, México) presented such a breed-ing program for new disease-tolerantbuffelgrass (Penn. ciliare) cultivars.Contiguous planting of a single cultivarof this obligate apomict over large geo-graphic areas in the United States andMexico since 1949 led to genetic vulner-ability to blight disease. To develop newdisease-resistant cultivars, Gonzalez andcolleagues crossed sexual lines withthe apomictic clone Zaragoza-115.Progeny testing and multilocation eval-uation trials over several years led tothe release of the hybrid AN-17-PS forcommercial seed production last year.

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Given current controversies regard-ing transgenic crop plants in agricul-ture, Peter van Dijk (NetherlandsInstitute of Ecology, Heteren) sug-gested that biosafety issues, whichwill undoubtedly arise (Ellstrand, 2001),should be considered well in advanceof future apomixis technology deploy-ment. He looked at the deployment ofapomixis technology from an evolution-ary and population genetics viewpoint(van Dijk and van Damme, 2000). Threehypothetical problems were identifiedwith apomictic crops: (1) invasiveweeds, (2) novel weeds, and (3) infec-tious apomixis, which might reduce ge-netic diversity by freezing the gene pool.Overall, van Dijk concluded that if pollenproduction is necessary in engineeredapomictic crops, pollen flow of a domi-nant apomixis transgene should beprevented. The use of inducible orconditional systems to control apo-mixis could be a means to preventsuch gene flow.

In recent years, there has been an in-creased industrial interest in the devel-opment of apomixis technology. MarcAlbertsen (Pioneer Hi-Bred, IA) pro-vided a well-balanced perspective onthe factors that will determine how apo-mixis is deployed in commercial or sub-sistence farming. Albertsen raised thequestion of who will have access toproprietary apomixis technology (andthe necessary supporting technologies)and under what terms. Both intellectualproperty management and freedom-to-operate issues will have a majorbearing on which beneficiaries will haveaccess to apomixis technology. From astrictly commercial perspective, ap-proaches are needed that allow finan-cial returns on technology investmentand to ensure that “donated” technol-ogy is not used competitively againstthe original developers and their cus-tomers (G. Graaf, A. Bennett, B. Wright,and D. Zilberman, unpublished data;see http://www.cnr.berkeley.edu/csrd/technology/ipcmech/). From a lesscommercial perspective, there are hu-

manitarian arguments for the provisionof proprietary technologies to improvethe well-being of poorer clients such assubsistence farmers in developingcountries. Albertsen concluded that nosingle approach may encompass bothrequirements and that new strategiesto deal with intellectual property rightsand licensing practices will have to bedeveloped.

Richard Jefferson (Center for the Ap-plication of Molecular Biology to Interna-tional Agriculture, Canberra, Australia)continued the theme of how apomixistechnology will be applied in agricultureby focusing on the challenge to deliverapomixis technology as a public good.This issue arose at a meeting funded bythe Rockefeller Foundation in 1998 whenthe Bellagio Declaration was issued bymany of the leading apomixis research-ers (http://billie.btny.purdue.edu/apomixis).Jefferson focused on how current in-tellectual property management limitsthe application of enabling technologiessuch as apomixis to solely commercialobjectives. He expressed concern that,unless the research community paidgreater attention to the terms of re-search agreements and promoted non-exclusive licensing, a small number ofmultinational companies could exercisemonopolistic control over apomixistech-nology worldwide. The current ex-clusionary intellectual property man-agement of both the private and publicsector are nonsustainable businessstrategies. Many publicly funded bod-ies are adopting exclusive licensingmodels that are more suited to com-mercial than to public good objectives.Access to proprietary enabling technol-ogies could be achieved by a consor-tium approach (enabling technologycooperative) that would develop propri-etary technologies but adopt a broadnonexclusive licensing policy, allowingit to act as a clearinghouse for innova-tive proprietary technologies (G. Graaf,A. Bennett, B. Wright, and D. Zilberman,unpublished data; see http://www.cnr.berkeley.edu/csrd/technology/ipcmech/).

CONCLUSION

Research presented at the 2nd Interna-tional Apomixis Conference coalescedaround several themes, some of whichare new and some of which have a longbut unfinished history in apomixis re-search. A wide range of topics—theimportance of existing apomicticmechanisms and their evolutionary im-plications, the isolation of genes con-trolling apomixis, the molecular andgenetic basis of ovule and female ga-metophyte development, and the eco-nomic and ecological implications ofapomixis—were discussed during ple-nary talks. It is increasingly apparentthat sexuality and apomixis are interre-lated and that they need to be inves-tigated simultaneously to obtain acomplete understanding of plant repro-duction at the developmental, ecologi-cal, and evolutionary levels. Indeed, anumber of changes were evident in thecontent and focus of apomixis researchsince the 1st International ApomixisMeeting. There is now a greater accep-tance that genetic and molecular ap-proaches to study sexuality can yieldinsights into the regulation and compo-nents of apomixis. This was particularlyevident in the number of new groupsentering this field. In addition to the im-pact of sexual model species such asArabidopsis and maize, it is evidentthat a number of apomicts (Hieracium,Taraxacum, Tripsacum, Paspalum, andA. holboellii) are emerging as modelsthat are amenable to molecular and ge-netic analyses. A number of new tech-niques (e.g., flow cytometry, transcriptprofiling, differential screening meth-ods, and enhancer detection) will in-crease our knowledge of apomixis inthe coming years and bring us a stepcloser to the engineering of apomixis insexual crops.

Charles SpillaneUniversity of Zürich,

CH-8008 Zürich, Switzerland

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Jean-Philippe Vielle-CalzadaCINVESTAV-Plant Biotechnology Unit

36500 Irapuato, Mexico

Ueli GrossniklausUniversity of Zürich,

CH-8008 Zürich, Switzerlandgrossnik@botinst.unizh.ch

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How to Avoid Sex: The Genetic Control ofGametophytic Apomixis

Apomixis is the natural ability of morethan 400 plant species to reproduceasexually through seed (Nogler, 1984a).Sexual embryos result from the unionof male and female gametes, which pro-duces genetically varied offspring. Incontrast, apomictic embryos are formedwithout paternal contribution. Therefore,apomictic offspring carry the full geneticconstitution of the mother and form astable clone, a feature of great value forplant breeding and seed production.

For many years, apomixis was stud-ied only by a small group of interestedbotanists (Nogler, 1984a; Asker andJerling, 1992) and visionary plantbreeders (Petrov et al., 1979; Hannaand Bashaw, 1987; Savidan, 1992).However, because of its tremendouspotential for agriculture, apomixis re-search has attracted much more atten-

tion during the last few years (Koltunowet al., 1995; Vielle-Calzada et al., 1996;Grossniklaus et al., 1998). If apomixiscould be introduced into sexual crops,it would greatly simplify breedingschemes and allow the fixation of anygenotype (however complex), includ-ing that of F1 hybrids. Apomixis tech-nology could play a major role infeeding the growing population of ourplanet (Jefferson, 1994; Thoenissen,2001) provided that it will be freelyaccessible to all users, especially re-source-poor farmers in developing coun-tries, requiring innovative approaches fortechnology generation, patenting, andlicensing (http://billie.btny.purdue.edu/apomixis).

Current apomixis research focuseson elucidating the genetic basis andmolecular mechanisms that control

apomictic reproduction (see accom-panying Meeting Report). Two majorcomplementary approaches are beingpursued: (1) to identify genes control-ling individual elements of apomixis inwell-defined sexual model species (re-viewed in Grossniklaus, 2001), and (2)to unravel the genetic control of apo-mixis in natural apomicts (reviewed inSavidan, 2000). For nearly two de-cades, the genetic control of apomixishad been elucidated in very few spe-cies. Recently, however, inheritancestudies for several natural apomictshave been published that shed newlight on the genetic control of this im-portant developmental process (vanDijk et al., 1999; Bicknell et al., 2000;Matzk et al., 2000; Noyes and Rieseberg,2000; Pupilli et al., 2001; Quarin et al.,2001).

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DEVELOPMENTAL ASPECTSOF APOMIXIS

Apomixis occurs in many species frommore than 40 plant families and isthought to have evolved multiple timesfrom sexual ancestors. Therefore, it islikely that the controls of sexual andapomictic reproduction are closely in-terrelated. Sexual reproduction andapomixis are not mutually exclusive,and both processes can occur in paral-lel, as it is typical of facultativeapomicts, which produce a mixture ofapomictic and sexual progeny. Al-though the mechanisms leading toapomictic reproduction are diverse(Koltunow, 1993; Crane, 2001), theyshare the common feature that ances-tral sexual processes are deregulatedin space and time (Grossniklaus, 2001).Two distinct types of apomixis havebeen described (Gustafsson, 1947a,1947b): (1) sporophytic apomixis (ad-ventitious embryony), in which an em-bryo forms directly from an unreducedsporophytic cell, and (2) gametophyticapomixis, which involves the formationof an unreduced embryo sac (femalegametophyte). Although no less inter-esting, the genetics of sporophyticapomixis has not been investigated ingreat detail, which is why we focus ongametophytic apomixis in this article.

Sexual reproduction involves thegeneration and fusion of reduced ga-metes (Figure 1). Female gametogene-sis and double fertilization occur withinthe ovule, a specialized reproductiveorgan (reviewed in Drews et al., 1998;Grossniklaus and Schneitz, 1998; Yangand Sundaresan, 2000). Usually a sin-gle cell within the ovule, the megasporemother cell (MMC) becomes committedto the sexual pathway, undergoes mei-osis, and forms a tetrad of four reducedspores. Only one of these will divideand ultimately form the mature embryosac containing the female gametes.Double fertilization involves two pairs ofgametic cells: the egg cell fuses with

one sperm to form the embryo and giverise to the next generation, and thecentral cell fuses with a second spermto form the endosperm, a nutritive tis-sue important for seed developmentand/or germination.

During gametophytic apomixis, severalof these developmental steps are by-passed or altered (Figure 1; Koltunow,1993; Vielle-Calzada et al., 1996). (1)Chromosome reduction is circum-vented (apomeiosis) such that unre-duced cells initiate embryo sacdevelopment. These unreduced cellscan originate from an aberrant or miss-ing meiosis of the MMC (diplospory).Alternately, the unreduced embryo sacforms directly from a cell within theovule other than the MMC (apospory)and the sexual products degenerate.(2) The unreduced egg cell initiates em-bryogenesis in the absence of fertiliza-tion (parthenogenesis). (3) The centralcell either develops autonomously or, inthe majority of apomicts, requires fertil-ization to initiate development (pseu-dogamy). The development of normalendosperm in apomicts is important forseed viability and often requires specialadaptations of embryo sac developmentor fertilization (reviewed in Grossniklauset al., 2001). Recent studies suggestthat some of these developmentalsteps are under independent geneticcontrol, at least in some apomicts (vanDijk et al., 1999; Matzk et al., 2000;Noyes and Rieseberg, 2000).

THE INHERITANCE OF APOMIXIS

Genetic studies largely depend oncrosses and recombination events nei-ther of which is easily obtained inapomicts. However, because mostapomicts produce normal, reducedpollen, the inheritance of apomixis canbe investigated by analyzing the segre-gation ratios in crosses with relatedsexuals. Such analyses are difficult be-cause gametophytic apomicts are al-

most without exception polyploids,causing complex modes of inheritance.An assessment of the breeding systemin the hybrids requires cytological ob-servations or, at least, time-consumingprogeny tests. Variation in the expres-sivity of apomixis may create an addi-tional complication. Moreover, much ofthe earlier work was done on the Ro-saceae, which are extremely difficult toanalyze because the multiple MMCsformed in sexuals make the distinctionbetween reduced and unreduced em-bryo sacs difficult. As a consequence,for many years the genetics of apo-mixis seemed unclear, complex, and id-iosyncratic. However, since the end ofthe 1970s, a clear general pattern in theinheritance of various types of gameto-phytic apomixis has emerged, first in theRanunculaceae and Poaceae and laterin the Compositae.

Using more suitable apomictic spe-cies and focusing on one element ofapomixis, apomeiosis, pioneer studiesby Nogler in the buttercup species Ra-nunculus auricomus and by Savidan inthe grass Panicum maximum indicatedthat apospory in these two speciessegregated as a single dominant men-delian factor (Savidan, 1982; Nogler,1984b, and references therein). Subse-quent investigations showed that bothapospory and diplospory in other spe-cies also fitted this segregation model(Table 1). Although a dominant mende-lian factor can represent any geneticconstitution from a single gene to anentire chromosome (e.g., mammaliansex determination), these observationsoften were taken as evidence for mono-genic inheritance. According to thismodel, apomictic plants possess thesimplex genotype Aaaa, carrying in ad-dition to the dominant apomeiosis al-lele A several recessive alleles forsexual reproduction. Apomictic plantsthus carry the potential for sexual re-production, but in a more or less re-pressed state, because of the presenceof the dominant apomixis factor. Lim-ited penetrance of the apomixis factor

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explains the occurrence of facultativeapomixis. The presence of recessivesexual alleles explains how a cross be-tween two facultative apomicts cangenerate abundant purely sexual off-spring. Although the occurrence ofapomixis fits this model, the degree ofapomixis often is dependent on envi-ronmental conditions (Nogler, 1984a)and/or on modifier genes (Bicknell et al.,2000). These as yet unspecified factorsneed further investigation in the future.Moreover, it remains to be determined

whether this general model also appliesto the many apomicts in the Rosaceae.

THE APOMEIOSIS LOCUS IS LOCATED IN A RECOMBINATIONALLYSUPPRESSED REGION

The segregation model described abovehas been supported and refined by theisolation of molecular markers that arelinked to the presumed apomixis loci in

several species (Table 1). In all cases inwhich it has been critically tested to date,a strong suppression of recombinationaround the apomeiosis locus has beenfound. For instance, strict cosegregationwith apomeiosis of many more molecularmarkers than expected was found inaposporous Pennisetum squamulatum(Ozias-Akins et al., 1998) and diplo-sporous Erigeron annuus (Noyes andRieseberg, 2000). In Brachiaria decum-bens (Pessino et al., 1998), Tripsacumdactyloides (Grimanelli et al., 1998a), and

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Figure 1. Scheme of Sexual and Apomictic Reproduction.

Reduced stages of the life cycle are shown in shaded ovals, and unreduced stages are shown in rectangular boxes. The key developmentalevents that are affected or altered in apomictic species are highlighted in hexagons.

1494 The Plant Cell

Paspalum simplex (Pupilli et al., 2001),comparative mapping with maize or ricemarkers showed a lack of recombinationin the region associated with theapospory locus. Markers that werespread over a region ranging from 15 to40 centimorgans in the sexual relativesstrictly cosegregated in these apomicts.Repression of recombination could frus-trate map-based cloning efforts becauseclosely linked markers may be at greatphysical distances from the apomixis loci.

Because suppressed recombinationoccurs in both dicot and monocot spe-cies, it may be a general characteristicof apomeiosis loci. This could be re-lated to their function as observed inother complex loci containing severalgenes involved in a common process(coadapted gene complexes), such asthe heterostyly supergene in Primula(Ernst, 1936), the self-incompatibility(S) loci in Brassica (Lewis, 1962;Awadalla and Charlesworth, 1999),the mating-type locus in Chlamydo-monas (Ferris and Goodenough,1994), and the major histocompatibility

locus in humans (O’hUigin et al., 2000).Alternately, it could be an evolutionaryby-product of long term asexual repro-duction (Judson and Normark, 1996;Welch and Meselson, 2000). In Penni-setum species, markers that are linkedto apospory in the apomicts could notbe detected by hybridization in sexualrelatives (Ozias-Akins et al., 1998;Roche et al., 1999), indicating that theapomicts were either hemizygous forthe apomixis locus (A---) or that the al-leles were highly divergent (A a� a� a�),as was observed for the Brassica S locus(Boyes et al., 1997; Suzuki et al., 1999).

ONE MASTER APOMIXIS GENE OR SEVERAL INDEPENDENTAPOMIXIS GENES?

Apomictic development deviates fromthe sexual pathway in apomeiosis, par-thenogenesis, and often endospermdevelopment (autonomy, altered em-bryo sac development, or altered fertili-

zation). Are these elements of apomixisall controlled by a single gene or byseveral genes? In the pioneering stud-ies on R. auricomus and P. maximum,parthenogenesis was strictly associ-ated with apospory. Hence, apomixisas a whole was inherited as a singlemendelian trait (Savidan, 1982; Nogler,1984b). Similarly, in Hieracium pilosel-loides, all three elements are inheritedas a single genetic trait (Bicknell et al.,2000). In these species, apomixis couldbe regulated by a single master regula-tory gene controlling all elements or bya gene complex of several tightly linkedgenes that are recombinationally locked.In other species, however, crosses be-tween sexuals and apomicts haveyielded progeny combining elements ofboth the sexual and the apomictic de-velopmental pathways. In Taraxacumofficinale, hybrids were recovered thatdisplayed diplospory and autonomousendosperm development but that lackedparthenogenesis (van Dijk et al., 1999).Such “apomixis recombinants” alsohave been reported in Poa pratensis

INSIGHT

Table 1. Inheritance of Elements of Gametophytic Apomixis (Apomeiosis and Parthenogenesis) in Members of the Ranunculaceae, Poaceae, and Compositae

SpeciesApomeiosisType Family

InferredGenotype

Most CloselyLinked MolecularMarker

Evidence forSuppressionRecombination Reference

ApomeiosisRanunculus auricomus Apospory Ranunculaceae Aaaa – – Nogler, 1984bPanicum maximum Apospory Poaceae Aaaa – – Savidan, 1982Pennisetum squamulatum Apospory Poaceae Aaaa 0 cM Yes Ozias-Akins et al.,1998Brachiaria decumbens Apospory Poaceae Aaaa 1.2 cM ? Pessino et al., 1998Paspalum simplex Apospory Poaceae Aaaa 0 cM Yes Pupilli et al., 2001Hieracium piloselloides Apospory Compositae Aaa – – Bicknell et al., 2000Hieracium aurantiacum Apospory Compositae Aaa – – Bicknell et al., 2000Tripsacum dactyloides Diplospory Poaceae Aaaa 0 cM Yes Grimanelli et al., 1998a, 1998bErigeron annuus Diplospory Compositae Aaa 0 cM Yes Noyes and Rieseberg, 2000Taraxacum officinale Diplospory Compositae Aaa 4.4 cM ? van Dijk et al., unpublished

ParthenogenesisPoa pratensis Apospory Poaceae Pppp 6.6 cM ? Barcaccia et al., 1998Erigeron annuus Diplospory Compositae Ppp 7.3 cM No Noyes and Rieseberg, 2000

The genetic models are based on segregation analyses of sexual � apomictic crosses and in most cases supported by cosegregation of closelylinked molecular markers. No distinction is made between disomic (Aa/aa) and tetrasomic (Aaaa) inheritance. The distance between the apomixislocus and the most closely linked marker is indicated in centimorgans (cM). (–), not investigated; (?), no conclusive data.

July 2001 1495

(Matzk et al., 2000), which suggeststhat the elements of apomixis in thesespecies are regulated by differentgenes. In Erigeron annuus, separategenes for diplospory (A) and partheno-genesis (P) have been mapped geneti-cally (Noyes and Rieseberg, 2000).Apomicts in this species carry the sim-plex genotype for both genes (Aaa andPpp). In contrast to the diplospory A lo-cus, no suppression of recombinationwas observed around the parthenogen-esis P locus (Table 1). In light of thesenew findings, it is likely that coadaptedgene complexes are present in thosespecies in which all elements of apo-mixis cosegregate.

SEGREGATION DISTORTION OF APOMIXIS LOCI

As mentioned above, gametophyticapomicts are usually polyploid, whereasrelated sexuals are diploid. Is gameto-phytic apomixis incompatible with dip-loidy? Again, the pioneering work on R.auricomus by Nogler appears to havegeneral relevance. Nogler showed thatdiploid offspring that developed par-thenogenetically from reduced diploidegg cells of tetraploid apomicts (dihap-loids) or diploids produced through an-ther culture were able to reproduceapomictically (Nogler, 1982). This showsthat apomixis and diploidy are not in-compatible, a finding that has beenconfirmed in several other species(Bicknell, 1997; Kojima and Nagato,1997). However, what matters is the or-igin of the diploid offspring, becausezygotic diploids derived from the fusionof haploid egg cells and haploid spermnever reproduced apomictically in Ra-nunculus. Nogler hypothesized that theapospory (A) locus was recessive lethalin the gametes. Consequently, the A lo-cus could be transmitted via diploid ga-metes to generate polyploid apomictsbut not via haploid gametes to gener-ate diploid apomicts. It is also possible

that mutations closely linked to the Alocus cause haploid gamete nonfunc-tionality. The net result is that haploidgametes carrying the A locus do notcontribute to offspring production, re-sulting in segregation distortion of theA locus.

More recently, additional evidencehas been obtained for segregation dis-tortion of apomixis loci in other plantspecies, such as Tripsacum dacty-loides, Pennisetum squamulatum, andE. annuus (Grimanelli et al., 1998b;Ozias-Akins et al., 1998; Noyes andRieseberg, 2000). Transmission studiesof markers linked to apomixis loci in E.annuus indicate different causes ofnontransmission: the parthenogenesislocus P in E. annuus was not transmit-ted because of selection against hap-loid gametes, as was observed for theA locus in R. auricomus. The diplosporylocus A, in contrast, was not transmit-ted because of meiotic drive. In thesetriploid apomicts, the nondiplosporychromosomes seem to pair preferen-tially, leaving the diplospory chromo-some as a univalent that always endsup in a diploid pollen grain (Noyes andRieseberg, 2000). In Hieracium pilosel-loides, different crossing schemes indi-cate that apomixis can be transmittedvia both haploid and diploid gametes,but post-zygotic lethality rather thansegregation distortion causes the ab-sence of apomixis in diploids (Bicknellet al., 2000).

CONCLUSIONS

Apomixis is a complex trait involvingthe modification of several steps of nor-mal sexual development. Recent re-ports on the inheritance of apomixishave revealed several common fea-tures. (1) The genetic control of apo-mixis is dominant; this is true for allelements of apomixis studied so far.This observation is often taken as evi-dence for apomixis being caused by a

mutated gene, but it is also compatiblewith the misexpression of wild-typegenes playing key regulatory roles insexual development (Koltunow, 1993;Grossniklaus, 2001). The latter view issupported by the fact that both sexualand apomictic reproduction coexist infacultative apomicts and that apomicticplants can be obtained from sexualprogenitors through hybridization (re-viewed in Carman, 2001) or simplechromosome doubling (Quarin et al.,2001). (2) The apomeiosis locus residesin a recombinationally suppressed re-gion, suggesting that even in species inwhich the elements of apomixis are un-der common control, a complex of sev-eral coadapted genes may be present.(3) The apomixis locus is usually asso-ciated with gametic or zygotic lethalityor its transmission is reduced by someother mechanism, with the result thathaploid gametes do not produce prog-eny, thereby maintaining or driving thepolyploidization associated with apo-mictic species.

As many reproductive mutants insexual species indicate, disturbancesof sexual development toward ele-ments of apomixis can easily causeabortion and sterility. Selection hasperfected natural apomicts, refining thedevelopmental modifications and inte-grating them into a functional develop-mental system with high fertility that islikely controlled by coadapted genecomplexes. Although many of the fea-tures discussed in this article pose ob-stacles to understanding the molecularbasis of natural apomixis, a comple-mentary approach using both sexualand apomictic model systems bearsgreat promise that apomixis can even-tually be harnessed to contribute tosustained agricultural production.

ACKNOWLEDGMENTS

We thank Margaret Collinge, ChristophRingli, and three anonymous reviewers for

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1496 The Plant Cell

comments on the manuscript, and Peter vanBaarlen and Daniel Grimanelli for helpfuldiscussions.

Ueli GrossniklausGian A. Nogler

Institute of Plant BiologyUniversity of ZürichZollikerstrasse 107

CH-8008 Zürich, Switzerlandgrossnik@botinst.unizh.ch

Peter J. van DijkNetherlands Institute of Ecology

P.O. Box 406666 ZG Heteren, The Netherlands

pjvandijk@cto.nioo.knaw.nl

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DOI 10.1105/tpc.13.7.1491 2001;13;1491-1498Plant Cell

Ueli Grossniklaus, Gian A. Nogler and Peter J. van DijkHow to Avoid Sex: The Genetic Control of Gametophytic Apomixis

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