SELF-INCOMPATIBILITY IN PLANTS - Semantic Scholar€¦ · Sexual reproduction in many ﬂowering...

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Self-Incompatibility in PlantsSeiji Takayama and Akira IsogaiLaboratory of Intercellular Communications, Graduate School of Biological Sciences,Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101,Japan; email: [email protected], [email protected]

Annu. Rev. Plant Biol.2005. 56:467–89

doi: 10.1146/annurev.arplant.56.032604.144249

Copyright c© 2005 byAnnual Reviews. All rightsreserved

First published online as aReview in Advance onJanuary 18, 2005

1543-5008/05/0602-0467$20.00

Key Words

self-/nonself-recognition, signal transduction, receptor kinase, F-boxprotein, Ca2+ signaling

AbstractSexual reproduction in many flowering plants involves self-incompatibility (SI), which is one of the most important systems toprevent inbreeding. In many species, the self-/nonself-recognition ofSI is controlled by a single polymorphic locus, the S-locus. Moleculardissection of the S-locus revealed that SI represents not one system, buta collection of divergent mechanisms. Here, we discuss recent advancesin the understanding of three distinct SI mechanisms, each controlledby two separate determinant genes at the S-locus. In the Brassicaceae,the determinant genes encode a pollen ligand and its stigmatic recep-tor kinase; their interaction induces incompatible signaling(s) withinthe stigma papilla cells. In the Solanaceae-type SI, the determinantsare a ribonuclease and an F-box protein, suggesting the involvement ofRNA and protein degradation in the system. In the Papaveraceae, theonly identified female determinant induces a Ca2+-dependent signalingnetwork that ultimately results in the death of incompatible pollen.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 468BRASSICACEAE-TYPE

SELF-INCOMPATIBILITY. . . . . . . 468Female Determinant . . . . . . . . . . . . . . . 470Male Determinant . . . . . . . . . . . . . . . . . 470Interaction between Male and

Female Determinants . . . . . . . . . . . 472Signal Transduction in the

Incompatible Stigmatic Cell . . . . . 472SOLANACEAE-TYPE

SELF-INCOMPATIBILITY. . . . . . . 474Female Determinant . . . . . . . . . . . . . . . 474Male Determinant . . . . . . . . . . . . . . . . . 476Mechanisms of S-haplotype-

Specific Pollen Inhibition . . . . . . . 478PAPAVERACEAE-TYPE SELF-

INCOMPATIBILITY . . . . . . . . . . . . . 479Female Determinant . . . . . . . . . . . . . . . 479Male Determinant . . . . . . . . . . . . . . . . . 479Signaling Cascade in the

Incompatible Pollen. . . . . . . . . . . . .479CONCLUSIONS AND FUTURE

PERSPECTIVES . . . . . . . . . . . . . . . . . 482

INTRODUCTION

Self-incompatibility (SI) is one of the most im-SI:self-incompatibility

GSI: gametophyticself-incompatibility

SSI: sporophyticself-incompatibility

portant systems used by many flowering plantsto prevent self-fertilization and thereby gen-erate and maintain genetic diversity within aspecies (6). The SI response is comprised of aself- and nonself-recognition process betweenpollen and pistil that is followed by selective in-hibition of the self-pollen (tube) development.Classic genetic studies have established thatthe self-/nonself-recognition in most speciesis controlled by a single multiallelic locus, theS-locus, and that pollen inhibition occurs whenthe same “S-allele” specificity is expressed byboth pollen and pistil.

After 20 years of intense molecular stud-ies focused on the entities of the S-locus,the molecules involved in the SI recognitionwere finally identified in certain plant species.

These identified determinant genes have di-verse structures, suggesting that the SI does notrepresent one mechanism but encompasses acollection of divergent systems. The only unify-ing scheme that has emerged from these stud-ies is that the S-locus consists of at least twolinked transcriptional units arranged in pairs,with one functioning as the female determi-nant and the other as the male (Figure 1). Thismultigene complex at the S-locus is inherited asone segregating unit, and therefore the variantsof the gene complex are called “S-haplotypes.”Self-/nonself-recognition operates at the levelof protein-protein interaction of the two de-terminants and the SI response occurs whenboth determinants are issued from the sameS-haplotypes.

Both the female and male determinants werefirst identified in the Brassicaceae (Figure 1).Recent studies also identified the male de-terminant in the Solanaceae, Rosaceae, andScrophulariaceae, all of which share the samefemale determinant molecule. Additionally, thefemale determinant was identified from stud-ies on the Papaveraceae. In spite of thisunifying scheme of a multiallelic two-generecognition system, the identified determinantsbear no similarity to one another, suggestingthat SI evolved independently and probablymultiple times in different lineages of the an-giosperms. There are numerous excellent re-views of SI (6, 10, 12, 20, 27, 32, 47, 68, 80, 93).The present review highlights recent works toupdate the reader on our current understand-ing of the molecular mechanisms of SI in theseplant species.

BRASSICACEAE-TYPESELF-INCOMPATIBILITY

Classic genetic studies in the early 1950s un-raveled two distinct forms of SI, the gameto-phytic (GSI) and the sporophytic (SSI), whichwere distinguished by the genetic behavior ofthe pollen’s SI phenotype (6, 47, 68). The pollenSI phenotype in GSI is determined by its ownhaploid genome, whereas in SSI the pollen SIphenotype is determined by the diploid genome

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Figure 1A schematic drawing of the S-locus and a list of the identified female and male determinant genes. TheS-locus contains at least two genes, one encoding the male determinant that is carried by the pollen grain,and the other encoding the female determinant that is expressed in the pistil. Both the male and femaledeterminants are polymorphic and inherited as one segregating unit. The variants of this gene complex arecalled S-haplotypes. The recognition of self/nonself operates at the level of the protein-protein interactionsbetween the two determinants and an incompatible response occurs when both determinants are issued fromthe same S-haplotype. Thus far, both determinants have been identified in the Brassicaceae and Solanaceae.Only the female determinant has been identified in the Papaveraceae.

of its parent (sporophyte). According to thisclassification, the SI in the Brassicaceae belongsto SSI and, so far, is the only SSI system inwhich the mechanism has been characterizedat the molecular level (20, 27, 80, 93). Morethan 30 and 50 S-haplotypes have been identi-fied in B. rapa (syn. campestris) and in B. oleracea,

respectively (54, 56). In the self-incompatibleplants of this family, pollen tubes do not developproperly on the stigma that express the sameS-haplotypes as the pollen’s parent. Self-pollenrejection results in abrogated pollen hydration,or a rapid arrest of the pollen tube growth atthe stigma surface.

www.annualreviews.org · Self-Incompatibility 469

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Female Determinant

Searches for the female determinant be-gan with the immunological identificationof an S-haplotype-specific antigen in thestigma, followed by the biochemical identifi-cation of stigma glycoproteins called S-locusglycoproteins (SLGs) that cosegregate withS-haplotypes. SLGs are 50–60-kDa secretedglycoproteins with several N-linked oligosac-charides and twelve conserved cysteine residues(52, 81). The identification of SLGs led to theisolation of the second S-locus gene, the S-locus receptor kinase (SRK) gene (72). SRKconsists of an SLG-like extracellular domain(S-domain), a transmembrane domain, and anintracellular serine/threonine kinase domain.

SLG and SRK exhibit a number of char-acteristics that would be expected for thefemale determinant of SI. First, they are pre-dominantly produced in the stigma papilla cells,which come into direct contact with pollen.Second, their expression occurs just prior toflower opening and coincides with the timingof SI acquisition by the stigma. Third, they ex-hibit allelic sequence diversity among all of theS-haplotypes examined.

A gain-of-function experiment clarified theinvolvement of SLGs and SRKs in the SIresponse (79). Transgenic B. rapa expressingSRK28 (SRK of the S28-haplotype) acquired S28-haplotype specificity in the stigma and rejectedthe S28 pollen. In contrast, transgenic plants ex-pressing SLG28 did not display S28-haplotypespecificity. When both SLG28 and SRK28 wereintroduced, however, the transformants ex-hibited stronger incompatibility against S28

pollen and produced fewer seeds. These re-sults demonstrate that SRK alone determinesthe S-haplotype specificity of the stigma, andthat SLG enhances the activity of SRK. In an-other gain-of-function experiment performedin B. napus, the role of SRK as the female de-terminant was also confirmed (69). However,no enhancing role for SLG was detected in theexperiment. Thus, the requirement for SLG inthe SI response may be variable among the dif-ferent S-haplotypes. In support of this view, sev-

eral S-homozygous lines of Brassica, and othergenera of the Brassicaceae, Arabidopsis lyrata,lack SLG expression, even though they still ex-hibit a strong SI phenotype (35, 77, 78).

Male Determinant

The first important clue for identifying the maledeterminant was obtained from a pollinationbioassay, which demonstrated that the biologi-cal activity responsible for SI resides in a smallprotein fraction (<10 kDa) of the pollen coat(73). Although isolating the active componentwas unsuccessful, two genetic approaches, thecloning and sequencing of the S-locus regionand the polymorphic gene search using fluo-rescent differential display, succeeded in iden-tifying the male determinant genes, which werenamed SP11 (S-locus protein 11) or SCR (S-locuscysteine-rich) (62, 76, 82).

SP11/SCR encodes the secreted forms ofsmall, basic, cysteine-rich proteins. SP11/SCRexhibits an extensive S-haplotype-associatedpolymorphism in which the alleles share a rel-atively conserved signal sequence but have ma-ture proteins that are highly variable (19.5% to94% amino acid identity), suggesting a strongpositive selection for diversification. Only afew residues are highly, but not absolutely,conserved among most S-haplotypes. Namely,these include eight cysteine residues (hereafterdesignated as C1 through C8), a glycine residuebetween C1 and C2, and an aromatic residuebetween C3 and C4 (63, 82, 92).

The identity of SP11/SCR as the maleS-determinant was definitively established bygain-of-function experiments and by direct ac-tivity testing using a pollination bioassay (62,65, 82, 83). In all of the gain-of-function exper-iments, pollen from the transformants with theSP11/SCR transgene acquired the S-haplotypespecificity of the transgene. In the pollinationbioassay, the pretreatment of the stigma withbacterially expressed or chemically synthesized“self” SP11/SCR inhibited the hydration andpenetration of “cross” pollen. These resultsclearly suggest that SP11/SCR is the sole male


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Figure 2Molecular model of the self-incompatibility (SI) response in the Brassicaceae. The S-locus consists of threegenes, SRK, SP11, and SLG. The SRK receptor kinase is the female determinant and spans the plasmamembrane of the stigma papilla cell. SP11 is the male determinant and is predominantly expressed in theanther tapetum and accumulates in the pollen coat during pollen maturation. Upon pollination, SP11penetrates the papilla cell wall and binds SRK in an S-haplotype-specific manner. This binding induces theautophosphorylation of SRK, triggering a signaling cascade that results in the rejection of self-pollen. SLG isnot essential for the self-/nonself-recognition but localizes in the papilla cell wall and enhances the SIreaction in some S-haplotypes. The signaling cascade downstream of SRK has not yet been characterized,but the essential positive effectors include MLPK and ARC1. MLPK localizes papilla cell membrane andmay form a signaling complex with SRK. ARC1, an E3 ubiquitin ligase, binds to the kinase domain of SRKin a phosphorylation-dependent manner and may target unknown substrates for ubiquitination. Theproteasomal degradation of these substrates could result in pollen rejection.

determinant that directly induces incompatiblereactions in the stigma papilla cells.

In situ hybridization analyses demonstratethat SP11/SCR is expressed sporophytically inthe anther tapetum cells and gametophyticallyin microspores (82) (Figure 2). In some S-haplotypes, the SP11/SCR expression is de-tected only in the anther tapetal cell layer (63).Because the tapetal cell layer is a diploid tis-sue that nourishes developing pollen grainsand provides the components of pollen coat-ing, the expression pattern of SP11/SCR eas-

ily explains the sporophytic nature of BrassicaSI. In fact, immunohistochemical studies sug-gest that the SP11/SCR protein is secreted ina cluster from the tapetal cells into the antherlocule and translocated to the pollen surface(25).

Recently, NMR analysis revealed the ter-tiary solution structure of the SP11 protein ofthe S8-haplotype (S8-SP11) (48), which specif-ically induces the SI response on S8 stigma ata dose of as little as 50 fmol per stigma (83).S8-SP11 folds into an α/β sandwich structure


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made up of a twisted three-stranded β-sheetlayer backed by another layer formed by an α-helix with flanking loops. Four disulfide bonds(C1–C8, C2–C5, C3–C6, and C4–C7) stabi-lize the structure. Although S8-SP11 adopts afold similar to plant defensins, the edges of theloop region are extensively stabilized by disul-fide bonds and hydrophobic packing. This fea-ture seems to be unique to SP11/SCR. Theconserved aromatic (tyrosine) residue makeshydrophobic contact with the sulfur atom ofC7, stabilizing the L1 loop structure betweenthe α helix and the β2 strand. Another con-served glycine residue helps form a stable typeII β-turn at the flanking segment, connectingthe β1 strand to the α helix. Structure-based se-quence alignment and homology modeling ofallelic SP11/SCR suggest that the L1 loop re-gion forms the hypervariable (HV) domain thatbulges out from the protein body and serves asan S-haplotype-specific site. However, the L1loop region contributes only the protein sur-face area of 470 A2, which is too small to con-fer the high affinity observed with the stigmareceptor (see below), suggesting that an addi-tional interface(s) is required (48). In support ofthis was an experiment that used site-directed(alanine-scanning) mutagenesis, and the resultssuggest that both the C3-C4 region (corre-sponding to L1 loop region) and the C5-C6region (corresponding to L2 loop region) con-tribute to the SRK binding (4). Similarly, mu-tations of each of the conserved cysteines ora tyrosine abolished the activity of SP11/SCR,whereas the replacement of a conserved glycineresidue with valine was tolerated. A domain-swapping experiment was also conducted, butthe results were more perplexing. For exam-ple, the SCR6 (SCR from the S6-haplotype)protein in which the C5-C6 region is derivedfrom SCR13 acquired the S13-haplotype speci-ficity, whereas the SCR13 protein in which theC5-C6 region is derived from SCR6 retainedthe S13-haplotype specificity. Although furtherstudies are required, these results support thefact that the arrangement of specificity deter-minants might vary significantly between SCRvariants.

Interaction between Male and FemaleDeterminants

Two different biochemical approaches demon-strated S-haplotype-specific interactions be-tween the male (SP11/SCR) and the female(SRK) determinants. In one experiment, the in-teractions between tagged versions of recombi-nant SRK and SP11/SCR were analyzed. Theextracellular domain of SRK6-FLAG (eSRK6-FLAG) was expressed in tobacco leaves, and theSCR (SCR-Myc-His6) was expressed in bac-teria. The eSRK6-FLAG was shown by bothpull-down and enzyme-linked immunosorventassay (ELISA) to interact more strongly withSCR6-Myc-His6 than with SCR13-Myc-His6

(28). In another experiment, 125I-labeled S8-SP11 (125I-S8-SP11) was used to monitor the in-teraction with the stigmatic receptor (83). The125I-S8-SP11 specifically bound the stigmaticmicrosomal membranes of the S8 homozygote.Scatchard analysis indicated the presence ofboth a high-affinity (Kd = 0.7 nM, Bmax =180 fmol/mg protein) and a low-affinity (Kd =250 nM, Bmax = 3 pmol/mg protein) bindingsite. Cross-linking experiments revealed thatthe high-affinity binding site consisted of pro-teins of 110 and 60 kDa. Immunoprecipitationexperiments suggested that the 110-kDa pro-tein is SRK8. The 60-kDa protein is assumedto be SLG8 or a truncated form of SRK (desig-nated eSRKs), but currently its identity is notclear. Furthermore, in an in vitro phosphory-lation assay, autophosphorylation of SRK8 onthe stigma plasma membrane was induced byS8-SP11, but not by S9-SP11, at a kineticallyrelevant concentration. This result clearly in-dicates that SP11/SCR alone can activate SRKin an S-haplotype-specific manner (83).

Signal Transduction in theIncompatible Stigmatic Cell

Now that the molecular basis of self-pollenrecognition, which is the first step of theself-incompatibility response, has been estab-lished, the present focus of research has turnedto characterizing the downstream signaling


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pathway(s) (Figure 2). The question that needsto be answered is how is the SRK activationtransduced into the inhibition of self-relatedpollen? Presently, two molecules have beenidentified as positive signaling mediators of thispathway.

One is the arm repeat–containing pro-tein, ARC1 (Armadillo-repeat-containing 1), astigma protein first identified in a yeast two-hybrid screen as a protein interacting with thecytoplasmic domain of SRK (17, 51). This in-teraction, which requires the C-terminal armrepeats region of ARC1 and an active SRKkinase domain, results in the phosphorylationof ARC1 in vitro. Suppression of ARC1 ex-pression by antisense cDNA causes a partialloss of the SI response, suggesting that ARC1functions as a positive effector of SI signal-ing (75). However, the incomplete loss of theSI response implies that another branch ofthe signaling pathway exists, although it couldalso be attributed to residual ARC1 expres-sion in the transgenic stigmas. Scrutinizing theARC1 sequence has suggested that it containsa U-box motif, a modified RING-finger; fur-thermore, recent analyses demonstrated thatARC1 has U-box-dependent E3 ubiquitin lig-ase activity (74). When expressed in culturedtobacco cells, ARC1 was distributed through-out the cytosol, but localized to the protea-some/COP9 signalosome in the presence ofan active SRK kinase domain. In the pis-til, levels of ubiquitinated protein increasedafter incompatible pollinations, but the in-crease was not apparent in ARC1 antisense-suppressed pistils. Furthermore, proteasomeinhibition disrupts the SI response. There-fore, it was proposed that ARC1 is activatedby SRK to promote the ubiquitination andproteasomal degradation of stigmatic proteinsthat support pollen germination and/or pollentube growth. Other scenarios are possible,however, because ubiquitination has functionsunrelated to protein degradation, such as sub-cellular targeting of proteins. Identifying ARC1substrates will therefore be an essential nextstep in further dissecting the process of pollenrejection.

Another molecule is the M locus proteinkinase (MLPK), which was recently identi-fied after re-examining the modifier (m) gene,a recessive mutant gene responsible for theself-compatibility of B. rapa var Yellow Sarson(49), that was once thought to encode anaquaporin-like protein, MIP-MOD (24), al-though this turned out not to be the case (14).MLPK is a protein kinase belonging to thesubfamily of the receptor-like cytoplasmic ki-nase (RLCK), which has a common mono-phyletic origin with receptor-like kinases buthas no apparent signal sequence or transmem-brane domain (66). MLPK from Yellow Sarsonhas a missense mutation in the conserved kinasesubdomain VIa, resulting in the loss of kinaseactivity. The mm plants exhibit a completelyself-compatible phenotype, and the transientexpression of MLPK can restore the abil-ity of mm papilla cells to reject self-pollen.These results suggest that MLPK is a posi-tive mediator of SI signaling, and that MLPKlocalizes upstream in the signaling pathway,assuming that the pathway from SRK di-vides into multiple routes. In addition, MLPKhas a typical plant N-myristoylation motif[MGXXXS/T(R)] at the N terminus, and ispresent in the plasma membrane of the stigma,suggesting that MLPK acts in the vicinity ofSRK. If ARC1 is also the primary componentof SRK signaling, then the SRK signal trans-ferred to MLPK must return to SRK becauseARC1 is a direct downstream effector of SRK.Taking the various factors together, MLPK mayform a signaling complex with SRK that medi-ates the rejection response (49). Very little isknown about the function of RLCKs, althoughthe genome of Arabidopsis thaliana contains asmany as 150 RLCKs (66). MLPK is the firstexample showing that a RLCK member medi-ates the signaling of receptor-like kinases. Fur-ther studies will address the precise relationshipbetween SRK and MLPK in SI signaling.

In addition to these positive mediators ofSI signaling, several identified components areexpected to negatively regulate the pathway.Two thioredoxin-h proteins, THL1 and THL2,are interacting stigma proteins that were


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identified in a yeast two-hybrid screen. THL1and THL2 interact with a conserved cysteineat the transmembrane domain of SRK in aphosphorylation-independent manner (1, 51).In vitro phosphorylation experiments demon-strate that THL1 inhibits the autophospho-rylation activity of SRK in the absence of an“activating” component of the pollen coat (pre-sumably SP11/SCR) (3). These results sug-gest that THL1 and THL2 may function asnegative regulators, preventing the constitutiveactivation of the SI pathway. However, the rela-tionship between THL1/2 and SRK activationrequires further study because a different ex-perimental system has not revealed any involve-ment of thioredoxins in the SP11/SCR-inducedSRK activation process (83). Another negative-regulator candidate is a protein phosphatase,KAPP (kinase-associated protein phosphatase),which interacts with the kinase domains ofmany receptor-like kinases. In Arabidopsis, a se-ries of transformation experiments suggest thatKAPP plays a general role in the downreg-ulation of various receptor kinases. Recently,a KAPP homolog was isolated from a stigmacDNA library of B. oleracea (90). This KAPP ho-molog interacts with, and is phosphorylated by,the kinase domain of SRK in vitro. In addition,KAPP dephosphorylated SRK as a substrate,suggesting its negative role in SRK downreg-ulation. Recently, a yeast two-hybrid screenidentified two kinds of additional interactingproteins, calmodulins 1 and 2, and a sortingnexin, SNX1. The calmodulins interact with anamphiphilic helix in the SRK subdomain VIa. Inanimal cells, both calmodulin and sorting nexinare implicated in the downregulation of recep-tor kinase activity. As with KAPP, the calmod-ulins and SNX1 interact in vitro with diversemembers of plant receptor kinases, suggest-ing their general role in the receptor-kinase-mediated signaling pathways.

Another important finding was obtainedfrom the comparative analysis of the self-compatible A. thaliana and its close relativethe self-incompatible A. lyrata (35). Despitethe fact that the S-locus region of A. lyratacontained functional SRK and SP11/SCR

genes, the relevant genomic region ofA. thaliana contained truncated and nonfunc-tional SRK and SP11/SCR, suggesting thatthe self-compatibility of A. thaliana might bedue to the inactivation of the S-determinantgenes. To support this, the transfer of SRK andSCR genes of A. lyrata are sufficient to imparta self-incompatible phenotype in A. thaliana(53). This transformation clearly demonstratesthat the entire signaling cascade leading toinhibition of self-related pollen is retained inA. thaliana. Thus, the self-incompatible line ofA. thaliana is an ideal tool for future geneticand molecular dissection of the SRK-mediatedsignal transduction cascade.

SOLANACEAE-TYPESELF-INCOMPATIBILITY

The Solanaceae, Rosaceae, and Scrophulari-aceae families all share a female S-determinant,an S-RNase (12, 32). The S-RNase was firstidentified in the Solanaceae so we refer to thisS-RNase-mediated type of SI as Solanaceae-type SI. The Solanaceae-type SI is under ga-metophytic control (GSI) and the rejection ofself-pollen occurs during pollen tube growthin the style. Recently, the genomic sequencesaround the S-RNase genes were thoroughly an-alyzed in these taxa, with the net result of finallyidentifying the elusive male S-determinant.The molecular nature of the identified male S-determinant suggests a new model of how thesedeterminants are involved in the specific rejec-tion of self-pollen.

Female Determinant

The female determinants were first identified inthe self-incompatible Nicotiana alata as the styleglycoproteins of ∼30 kDa that cosegregate withthe S-haplotype in genetic crosses. This en-abled the identification and cloning of many re-lated proteins from members of the Solanaceaeand other families. Sequence data revealed thatthe style proteins contain a region homologousto the catalytic domain of the fungal T2-typeribonucleases. Further studies confirmed that


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these proteins possess ribonuclease activity andthus are referred to as S-RNases (45). S-RNasesare expressed exclusively in the pistil, with theprotein localized mostly in the upper segmentof the style where inhibition of the self-pollentubes occurs. The function of the S-RNases inSI was directly confirmed by gain- and loss-of-function experiments (38, 50). These experi-ments demonstrated that the S-RNase is thesole female determinant responsible for the S-haplotype specificity of the pistil. S-RNases areglycoproteins with one or more N-linked gly-can chains. An engineered S3-RNase of Petu-nia inflata that was engineered so that only theN-glycosylation site was knocked out, retainedits activity to reject S3 pollen, suggesting thatthe S-haplotype specificity determinant of S-RNases resides in the protein backbone and notin the glycan chains.

The S-RNases are highly divergent, withamino acid sequence identity ranging from 38%to 98% in the Solanaceae species (47). Despitethis high sequence diversity, the S-RNases con-tain a number of conserved regions. S-RNasesfrom the Solanaceae contain five highly con-served regions, designated C1 through C5, andthose from the Rosaceae and the Scrophular-iaceae have similar structural features exceptthat the C4 region is absent. C2 and C3 arethe regions similar to fungal RNase T2, andeach contains a conserved catalytic histidineresidue. There are two hypervariable regionsin the S-RNases from the Solanaceae, termedHVa and HVb, whereas only one hypervariableregion, corresponding to HVa of the solana-ceous Sf11-RNases, has been detected in therosaceous S-RNases. The crystal structures ofa solanaceous Sf11-RNase from Nicotiana alataand a rosaceous S3-RNase from Pyrus pyrifoliahave been determined (23, 41). Both S-RNaseshave very similar structures consisting of eighthelices and seven β-strands, and this topol-ogy is typical of the RNase T2 family of en-zymes. The amino acid residues constitutingthe substrate-binding sites of these S-RNasescan be geometrically superimposed over thoseof the RNase T2 enzymes. The most remark-able difference between the two S-RNases is in

their HV regions. The HVa and HVb regionsof solanaceous Sf11-RNase are composed of along, positively charged loop followed by a partof an α-helix and a short, negatively charged α-helix, respectively. Because these HV regionsare geometrically close to one another and ex-posed to the molecular surface, they both areexpected to form a domain interacting with themale S-determinant. Domain-swapping exper-iments of S-RNases support this model (42, 43).S11-RNase and S13-RNase of S. chacoense differonly in 10 amino acids, 3 of which are locatedin the HVa and 1 in the HVb region. Whenthe amino acids of HVa and HVb of S11-RNasewere changed to those of S13-RNase, transgenicplants expressing this hybrid S-RNase rejectedS13 pollen but not S11 pollen. These resultsclearly suggest that the HVa and HVb regionsplay a key role in determining the S-haplotypespecificity, despite the fact that the involvementof other regions cannot be ruled out. In contrastto the solanaceous S-RNases, the HV region ofthe rosaceous S3-RNase comprises a positivelycharged long loop followed by a short α-helix,but the α-helix corresponding to HVb does notexist (41). In the rosaceous S-RNase, the HVaregion alone may form the interacting domaincorresponding to the HVa and HVb regions ofthe solanaceous S-RNases.

S-haplotype-specific pollen rejection re-quires high levels of S-RNase expression. Theconcentration of S-RNase in the extracellularmatrix is estimated at 10–50 mg/ml, and onlythe transformants with an equivalent amountof S-RNase expression are able to acquire newS-haplotype specificities. The ribonuclease ac-tivity of S-RNases is essential for pollen re-jection (18). Furthermore, a radioactive tracerexperiment showed that pollen RNA is de-graded specifically after incompatible pollina-tion (44). Thus, S-RNases function as highlyspecific cytotoxins that inhibit the growth ofincompatible pollen (Figure 3).

Although S-RNase is the sole female fac-tor determining the S-haplotype specificity ofthe pistil, a requirement of other stylar fac-tors for the full function of S-RNase has beensuggested (5). One such factor is HT-B, a


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Figure 3Molecular model of the self-incompatibility response in the Solanaceae, Rosaceae, and Scrophulariaceae.The S-locus consists of two genes, S-RNase and SLF/SFB. S-RNase is the female determinant and issecreted in large amounts into the extracellular matrix of the style. In a pollinated style, S-RNase isincorporated into the pollen tubes and functions as a cytotoxin that degrades pollen RNA. Although theS-RNase enters the pollen tubes regardless of their S-haplotypes, RNA degradation occurs only inself-pollen tubes. SLF/SFB is the male determinant and is a member of the F-box family of proteins, whichgenerally function as a component of an E3-ubiquitin ligase complex. Thus, SLF/SFB is expected to beinvolved in ubiquitin-mediated protein degradation of nonself-S-RNases.

small asparagine-rich protein that was origi-nally identified during a differential screen per-formed to identify stylar genes expressed in theself-incompatible Nicotiana alata but not in theself-compatible Nicotiana plumbaginifolia (46).Homologs of HT-B were also identified in twoother genera of the Solanaceae, Lycopersicon andSolanum (33, 34, 55). In a comparative anal-ysis of self-incompatible and self-compatibletaxa of Lycopersicon, the expression of HT-Bgene was not detected in all self-compatibletaxa (33, 34). A requirement for HT-B pro-tein was demonstrated by an RNAi suppres-sion experiment in self-incompatible Solanumchacoense. Two HT-B-suppressed transformantsexpressed S-RNase normally but did not show

S-haplotype-specific pollen rejection. Theseresults suggest that the HT-B protein is im-plicated in the SI response, although its exactfunction remains unclear (55).

Male Determinant

The molecular nature of the male determi-nant and the molecular mechanisms of howS-RNases degrade pollen RNA in an S-haplotype-specific manner were long-standingmysteries of S-RNase-mediated SI. One plau-sible model was the “inhibitor model,” in whichthe pollen S-determinant was postulated to bean inhibitor that could inhibit all S-RNaseswith the exception of the cognate S-RNase (31,


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45). The immunocytochemical observationsthat all S-RNases could enter the pollen tuberegardless of S-haplotype supported this model(40). Some refined “inhibitor models” were alsoproposed (39, 40), but such an S-RNase in-hibitor has yet to be identified.

The male determinant was recently identi-fied through genomic analyses of the S-locusregion. Genomic analyses were first conductedon solanaceous species such as Petunia inflata,Petunia hybrida, Lycopersicon peruvianum, andNicotiana alata. However, the S-locus of thesespecies is located in the subcentromeric re-gion and surrounded by abundant repetitivesequences that have hampered chromosomalwalking (8, 91). The first clue for the maledeterminant was obtained from sequence anal-ysis of the S-locus region of Antirrhinum his-panicum, a member of the Scrophulariaceae.The region of the S2-haplotype contained anovel F-box protein gene, AhSLF-S2 (A. his-panicum S-locus F-box of S2-haplotype), whichis specifically expressed in anther and pollengrains of S2-haplotype (36). However, no geneallelic to AhSLF-S2 has been identified in otherS-haplotypes, and a gene with an extremelyhigh sequence similarity (97.9% amino acid se-quence identity) has been found in other lineswith different S-haplotypes. It was thus un-clear whether AhSLF-S2 encoded the pollen S-determinant. Genomic analysis of the S-locusof Prunus mume, a member of the Rosaceae, re-veals that the ∼60-kb genomic region aroundthe S-RNase gene contains as many as fourF-box genes (7). Among them, only one F-box gene, termed PmSLF, fulfills the condi-tions of a pollen S-determinant gene: (a) it islocated within the highly divergent genomicregion of the S-locus, (b) it exhibits S-haplotype-specific diversity (78% to 81%amino acid identity), and (c) it is specifically ex-pressed in pollen (7). Around the same time,polymorphic F-box genes were also found inthe S-locus region of Prunus dulcis, Prunusavium, and Prunus cerasus, and were indepen-dently named SFB (S-haplotype-specific F-box)(86, 95). SLF/SFB from Prunus species ful-

filled all conditions required of the pollen S-determinant. Aligning deduced amino acid se-quences of SLF/SFBs of these Prunus speciesrevealed the presence of two hypervariable re-gions, HVa and HVb, at the C terminus (32,86). Two self-compatible haplotypes of P. aviumand P. mume encoded partial loss-of-functionmutations in SLF/SFB, which lack both HVaand HVb regions (87). This fact provides addi-tional evidence that the SLF/SFB is the pollenS-determinant.

The conclusive evidence that SLF/SFB en-codes the pollen S-determinant was finallyobtained from transformation experiments inPetunia inflata (67). A thorough search of thepollen S-determinant in a huge S-locus re-gion (328-kb BAC contig of S2-haplotype)identified a polymorphic F-box gene, namedPiSLF, ∼161-kb downstream of the S-RNasegene. Although the genomic region outsidethis contig contained two more polymorphicF-box genes that were genetically linked tothe S-locus, the PiSLF exhibited the high-est sequence diversity. To ascertain whetherPiSLF encodes the pollen S-determinant, awell-documented phenomenon termed “com-petitive interaction” was utilized. Competitiveinteraction is often observed in tetraploidplants. Among the diploid pollen grains pro-duced, those carrying two different S-haplo-types (heteroallelic pollen), but not two of thesame S-haplotypes (homoallelic pollen), fail tofunction in SI, although the molecular mecha-nism of the breakdown is unknown (6, 9). Con-sistent with this phenomenon, the transforma-tion of S1S1, S1S2, and S2S3 plants with theS2-allele of PiSLF (PiSLF2) caused breakdownof their pollen function in SI. Furthermore,genotypic analyses of the progeny from self-pollinations of S1S2/PiSLF2 and S2S3/PiSLF2

revealed that only S1 and S3 pollen carrying thePiSLF2 transgene (corresponding to heteroal-lelic pollen), but not S2 pollen carrying PiSLF2

(corresponding to homoallelic pollen), becameself-compatible. These results conclusivelydemonstrate that SLF/SFB is the long-soughtpollen S-determinant.


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Mechanisms of S-haplotype-SpecificPollen Inhibition

In spite of the fact that both female and male de-terminants have been identified, the molecularmechanisms regulating how these molecules in-teract and specifically inhibit self-pollen growthremain unclear. The fact that RNase activityis required for the function of S-RNases, andthat S-RNases are taken up by both self- andnonself-pollen tubes, suggests that S-RNasesfunction inside pollen tubes as specific cy-totoxins degrading the RNA of self-pollen(Figure 3). On the other hand, SLF/SFB con-tains a motif, called the F-box, which is bestknown for mediating interactions with otherproteins that make up an enzyme complexreferred to as the E3 ubiquitin ligase com-plex (15). E3 ubiquitin ligases act in con-junction with the E2 enzymes to ubiquitinatetarget proteins, which in many cases are de-graded by the 26S proteasome. Recent bio-chemical studies suggest the involvement ofAhSLF-S2 in this protein degradation path-way, although it remains to be clarified whetherAhSLF-S2 from Antirrhinum is an orthologof PiSLF (57, 67). AhSLF-S2 interacts withASK1- and CULLIN1-like proteins, which arethe expected components of the SCF complex.AhSLF-S2 interacts with both self- and nonself-S-RNases, but appears to mediate degradationof only nonself-S-RNases. Although such in-teraction and degradation have not been re-ported for Prunus and Petunia SLF/SFBs, ifthis is the case then SLF/SFBs should inter-act with all S-RNases but ubiquitinate onlynonself-S-RNases.

To explain the molecular mechanisms forthis specificity, some hypothetical models thatare compatible with the “inhibitor models” havebeen presented (7, 57, 67, 87). One modelpostulates that SLF/SFBs contain two sepa-rate interaction domains, like the classical “in-hibitor model.” One domain would bind tothe hypervariable domain of its cognate S-RNase in an S-haplotype-specific way, andthe other domain would bind to a domaincommon to all S-RNases. The S-haplotype-

specific interaction is expected to somehow sta-bilize, or at least not alter, the S-RNase ac-tivity, and the general interaction would leadto the polyubiquitination and degradation ofS-RNases. Another mechanism postulates theinvolvement of another molecule, such as a gen-eral inhibitor in a modified “inhibitor model.”To support this, a pollen-expressed RING-finger protein, PhSBP1 (P. hybrida S-RNase-binding protein), interacts specifically withS-RNases in an S-haplotype-nonspecific man-ner (70). Because many RING-finger domainproteins also function as E3 ubiquitin ligases,PhSBP1 is postulated to be involved in the gen-eral degradation of S-RNases. In such cases,SLF/SFB is expected to bind to its cognate S-RNase as a pseudosubstrate and protect it fromubiquitination and subsequent degradation(12).

However, none of these models can ex-plain the phenomenon of “competitive in-teraction.” These models assume that theS-haplotype-specific binding between S-RNaseand its cognate SLF/SFB is thermodynami-cally favored over general binding between S-RNase and nonself-SLF/SFB (or PhSBP1), andthat the S-haplotype-specific binding somehowprecludes S-RNases from ubiquitination, per-mitting RNase activity. In “competitive interac-tion,” two SLF/SFBs in the heteroallelic pollenshould each preferentially bind to their re-spective S-RNases in an S-haplotype-specificmanner, leaving the RNases active. Therefore,in contrast to experimental observations, thesemodels predict incompatibility for heteroal-lelic pollen. To explain the “competitive in-teraction,” a refined version of the “inhibitormodel” has been proposed in which the maleS-determinants are predicted to form a multi-mer prior to interacting with the S-haplotype-specific binding site of S-RNases (39).However, SLF/SFBs are not likely to form amultimer during the interaction process. Thus,although both the female and male determi-nants have been identified, exactly how S-haplotype-specific pollen inhibition is achievedremains a mystery.


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PAPAVERACEAE-TYPESELF-INCOMPATIBILITY

SI in the field poppy, Papaver rhoeas, is also un-der gametophytic control (GSI) in that the S-phenotype of pollen is determined by its haploidS-genotype. However, the identified S-protein(female determinant) and the mechanisms in-volved in pollen inhibition differ dramaticallyfrom those in the Solanaceae. The overall num-ber of S-haplotypes in P. rhoeas is estimated ataround 66 (37). Although the exact nature ofthe male determinant is not known, a reliablein vitro bioassay system was developed in whichpollen germination and pollen tube growth canbe inhibited by the recombinant S-protein inan S-haplotype-specific manner, thus allowingthe biochemical events that take place in thepollen following self-recognition to be studiedin detail.

Female Determinant

The bioassay system that reproduces the SIreaction of P. rhoeas in vitro allowed theidentification of biologically active stigmaticS-determinants (11). The S-proteins are small,secreted proteins (∼15 kDa), some of which aremodified by N-glycosylation (12, 47). Thus far,five allelic stigmatic Papaver S-protein geneshave been cloned. The S-proteins are highlypolymorphic and share between 51.3% and63.7% amino acid sequence identity. Neverthe-less, they have four conserved cysteine residuesand a predicted conserved secondary structurethat is comprised of six β-strands and two α-helices connected by seven hydrophilic surfaceloops. In contrast to the S–determinants in theBrassicaceae and Solanaceae, amino acid se-quence variation is not found in hypervariableblocks, but rather throughout the S-proteins.

Because some recombinant S-proteins pro-duced in Escherichia coli inhibit pollen germi-nation in an S-haplotype-specific manner, theS-proteins must be the sole female determinantin Papaver SI. This also suggests that the glycanchains are not required for the S-determinantfunction. Further studies, using site-directed

mutagenesis, revealed that some residues lo-cated in predicted surface loop 6 are crucialfor pollen recognition. Mutations of the onlyamino acid residue (Asp-79 in loop 6 of S1) thatis variable across five available S alleles, and ofadjacent highly conserved amino acids (Asp-77and Asp-78), resulted in complete loss of abilityof S1-protein to inhibit S1 pollen (30).

Male Determinant

When S-proteins were challenged on pollentubes, the fastest change occurred in the shankregion approximately 50 µm behind the pollentube tip, where the concentration of intracellu-lar free calcium ([Ca2+]i) increased within onlya few seconds (Figure 4). Because this rapidalteration in [Ca2+]i is induced by Ca2+ influx(13), the pollen S-determinant is expected to bea membrane-located receptor that is somehowassociated with channels that conduct Ca2+.Biochemical studies suggest the presence ofa candidate receptor, which was named SBP(S-protein binding protein). SBP is a pollen-specific integral membrane proteoglycan of 70–120 kDa that binds specifically to stigmaticS-proteins but apparently does so in a non-S-haplotype-specific manner. Therefore, it hasbeen proposed that SBP acts as an accessory re-ceptor rather than as the pollen S-determinant(19). Biochemical analysis using site-directedmutagenesis reveals that all S-protein mutantsthat exhibit a reduced ability to inhibit incom-patible pollen also exhibit reduced SBP bindingactivity, suggesting a direct involvement of SBPin the SI reaction (26).

Signaling Cascade in theIncompatible Pollen

The S-haplotype-specific interaction betweenstigmatic S-protein and its putative pollen re-ceptor somehow induces a rapid increase of[Ca2+]i in the pollen grains (tubes). The [Ca2+]i

increase occurs in the shank region of thepollen tube within a few seconds after the chal-lenge of an incompatible S-protein and contin-ues for several minutes (13) (Figure 4). The


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Figure 4Molecular model of the self-incompatibility response in the Papaveraceae. Only the female determinantgene has been identified, which encodes a secreted stigma protein named S-protein. S-protein interacts withthe assumed S-haplotype-specific pollen receptor (the putative male determinant) and induces Ca2+ influxin the shank of the pollen tube. SBP is an integral proteoglycan of the pollen plasma membranes and isexpected to function as an accessory receptor. Ca2+-influx stimulates increases in [Ca2+]i, with somecontribution from the intracellular stores as well as from extracellular sources. These increases in [Ca2+]itrigger the downstream signaling cascades that result in rapid growth inhibition and ultimately the death ofincompatible pollen tubes.

increase of [Ca2+]i in the subapical region ac-companies the rapid loss of the oscillating high[Ca2+]i gradient in the apical region of thepollen tubes. Most biochemical and physiolog-ical studies suggest that [Ca2+]i acts as a secondmessenger that triggers multiple intracellularsignaling cascades, resulting in rapid inhibitionof pollen tube growth and ultimately the deathof the incompatible pollen. Thus, recent studieshave focused on identifying the signaling com-ponents downstream of the initial Ca2+ signalsand their targets in pollen. These studies areexpected to give insight into how the growth of

the pollen tube is regulated, not only in the SIresponse, but also in a more general context.

The oscillating apical high [Ca2+]i is typi-cal of all tip-growing cells such as pollen tubesand neurons, although their biological signifi-cance is unclear. Thus, the loss of the oscillating[Ca2+]i gradient will likely play a part in the ini-tial inhibition of pollen tube growth.

One of the most rapid and dramatic phys-iological changes observed during the SI re-sponse is the dynamic rearrangement of theactin cytoskeleton in the pollen tube. Within1–2 min postchallenge by an incompatible


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S-protein, many F-actin bundles are lost andlater punctate foci of actin accumulate in thecortex (16). Quantitative analysis of F-actindemonstrates that the SI-induced actin al-terations are due to the depolymerization ofF-actin, which starts within 1 min after SI chal-lenge, with the reduction of F-actin reaching∼74% after 60 min (71). Similar depolymer-ization of actin was achieved in pollen bytreatments that increase [Ca2+]i artificially, sug-gesting that the actin depolymerization is notjust a consequence of pollen tube growth ar-rest, but is part of the Ca2+-mediated SI signal-ing cascade. Furthermore, recent biochemicalstudies have suggested that two kinds of actin-binding proteins, profilin and gelsolin, areinvolved in this Ca2+-induced actin depolymer-ization process (22).

Another early target of the SI response isp26, a cytosolic 26-kDa pollen protein, thephosphorylation of which is induced within90 sec of an incompatible S-protein challenge,with a further increase occurring during thenext 400 sec. Because this timing coincides withthe increase of [Ca2+]i in SI-induced pollenand the protein kinase activity responsible forp26 phosphorylation is dependent on Ca2+

and calmodulin, the phosphorylation of p26 isalso expected to be apart of the Ca2+-mediatedSI signaling cascade (12). Sequence analysesreveal that p26 shares approximately 80%amino acid sequence identity of plant-solubleinorganic pyrophosphatases, and its activitywas confirmed by biochemical assays on therecombinant p26 protein. Furthermore, underconditions of raised [Ca2+]i, when p26 is phos-phorylated, its pyrophosphatase activity is re-duced (12, 59), indicating that p26 activity willlikely be affected by the SI response. Solubleinorganic pyrophosphatases drive cellularbiosynthetic reactions generating ATP andbiopolymers, such as long-chain carbohydratesand proteins. Thus, it has been proposed thatthe inactivation of p26 during the SI responseleads to inhibition of the pollen tube growthby depleting biopolymers required for tipgrowth. This proposal, however, needs to beexperimentally tested.

Another target of SI signaling that dis-

PCD: programmedcell death

plays a slightly delayed response is the puta-tive mitogen-activated protein kinase (MAPK),p56 (60). In-gel kinase assays identified p56as a 56-kDa protein kinase in pollen that isspecifically activated after an SI challenge. Theactivation of p56 was detected 5 min after in-compatible S-protein treatment, with peak ac-tivity at ∼10 min. Pretreatment of growingpollen with the calcium channel blocker, lan-thanum, inhibited the activation of p56, sug-gesting that p56 activation is also downstreamof the Ca2+-mediated SI signaling cascade. Al-though p56 has not yet been cloned, severalpieces of biochemical evidence suggest thatp56 is a MAPK. For example, p56 reacts withthe TEY antibody that specifically binds acti-vated MAPKs, and the kinase activity of p56is abolished by apigenin, a specific inhibitorof MAPKs. Because the arrest of pollen tubegrowth precedes p56 activation, p56 is unlikelyto play a role in the early inhibition events. Onespeculative hypothesis suggests that p56 mightactivate a PCD signaling cascade (see below)because some data have emerged suggesting arole for MAPK activation in the induction ofPCD in plants.

Recently, compelling evidence has sug-gested that SI challenge ultimately triggersPCD in incompatible pollen tubes (84). Nu-clear DNA fragmentation, which is a hallmarkof PCD, was observed in incompatible pollentubes. Fragmentation was first detected 4 h af-ter an incompatible S-protein challenge andincreased to approximately 80% of affectedpollen tubes within 14 h postchallenge. Re-cently, this DNA fragmentation was shown tobe inhibited by pretreatment with the tetrapep-tide DEVD, an inhibitor of caspase-3. Althoughno caspase homologue has been found in plantgenomes, the result suggests the involvement ofcaspase-like activity in this signaling cascade.Furthermore, DEVD treatment revealed thebiphasic nature of SI signaling. The SI-inducedarrest of pollen tube growth is very rapid andoccurs within 5 min of SI induction. The ar-rest was also observed in DEVD-pretreatedpollen tubes. However, growth restarted in the


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tubes 15–45 min after the arrest, suggestingthat by inhibiting the caspase-like activity, theinitial growth arrest can be recommenced.Thus, there seems to be a biphasic SI response:rapid inhibition of pollen tube growth, followedby caspase-like protease activation, which pre-sumably makes inhibition irreversible. Cy-tochrome c leakage from the mitochondria, aclassic marker for PCD in many organisms,was also stimulated by SI induction in incom-patible pollen tubes. Cytochrome c leakagewas detected as early as 10 min after SI in-duction and increased until 120 min. To ob-tain further evidence of the involvement ofcaspase-like activity in this SI signaling, the en-zymatic activity that cleaves poly(ADP-ribose)polymerase (PARP) was analyzed. The PARP-cleavage activity was detected in incompatiblepollen tubes 2 h after SI induction and in-creased over time. Finally, each of these PCDhallmarks, i.e., nuclear DNA fragmentation,cytochrome c leakage, and PARP-cleavage ac-tivity, can be sequentially induced by artificiallyincreasing [Ca2+]i in the pollen tubes, suggest-ing that these are all a part of the series ofreactions that make up the Ca2+-mediated SIsignaling cascade.

CONCLUSIONS AND FUTUREPERSPECTIVES

It is clear that the Brassicaceae, Solanaceae,and Papaveraceae have developed completelydifferent self-/nonself-recognition systems.Nonetheless, the S-loci controlling theserecognition reactions have structural com-monalities, i.e., they contain at least twopolymorphic determinant genes surrounded byhighly divergent intergenic sequences (7, 10,64, 94). Significant sequence heterogeneity ofthe S-locus explains how intergenic recombi-nation is suppressed between two determinantgenes, which results in the breakdown ofSI. A more difficult issue to resolve is hownew S-haplotypes evolve. In these two-genesystems, the female and male determinantsmust coevolve to maintain their interaction.Phylogenetic analyses of SRK and SP11/SCR

in Brassica produced almost identical treetopologies for these two genes (61, 63, 92).These analyses provide evidence for the co-evolution of the female and male determinantgenes and suggest that new S-haplotypes arisefrom pre-existing S-haplotypes. Several inter-esting schemes outlining how such a processmight occur have been proposed (4, 10, 42,47, 88), although all schemes are hypothetical.Detailed comparative analyses characteriz-ing more S-haplotypes, and additional struc-ture-function relationship analyses of mutateddeterminants, should provide clues to thisdifficult issue.

Similar multiallelic two-gene recognitionsystems are also evident in the mating-type lociof fungi, which prevent self-matings (2, 29).The mating-type loci can be diallelic or mul-tiallelic, similar to that seen for the S-loci. Forexample, in the phytopathogenic basidiomyceteUstilago maydis, the fusion of haploid cellsand the maintenance of dikaryotic filamentousgrowth are controlled by two unlinked mating-type loci, termed a and b. Multiallelic genes atthese loci (a locus exists in diallelic forms and blocus exists in at least 18 allelic forms) specifya large number of different mating types, andmating can only be completed between indi-viduals that differ at both loci. The a locus con-tains two linked genes for lipopeptide ligandsand their receptors, and is therefore analogousto the S-locus of the Brassicaceae. The b locuscontains two genes for homeodomain proteins(bE and bW), and bE and bW from differentalleles can form an active bE/bW heterodimerthat is able to maintain filamentous growth. Inmultiallelic two-gene recognition systems, twocontrary means can exist to lead to the sameend of self-rejection, namely, “self-recogni-tion” versus “nonself-recognition,” or in otherwords, “opposition” versus “complementation”(Figure 1). In the mating-type recognition offungi, the latter means are used, i.e., when twogenes from different alleles are present in themating cells, their interaction leads to compati-ble reactions. However, in the plant SI systems,at least in the Brassicaceae and the Papaveraceaetypes, the former means are used, i.e., when


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two genes from the same haplotypes meet, theirinteraction leads to incompatible reactions toreject self-pollen.

Classic genetic analyses reveal the pres-ence of two SI types, GSI and SSI. Molecularanalyses show that GSI contains at least twomechanisms, the Solanaceae type and the Pa-paveraceae type. For SSI, although the molec-ular mechanism has only been elucidated in theBrassicaceae, the presence of different mech-anisms has been suggested. In the Convolvu-laceae, the entire S-locus region has beencloned and sequenced in Ipomoea trifida, whichhas an SSI system (85). However, no homol-ogous gene for the Brassica SRK or SP11/SCRhas been found in the S-locus region. In theAsteraceae, a candidate gene for the femaledeterminant, which was named SSP (stigmaS-associated protein), has been identified, al-though it bears no resemblance to either SRKor SLG (21). Current evidence supports theview that SI evolved independently and proba-bly multiple times in different lineages, with its

recognition genes recruited in the family lin-eage by duplication and modification of pre-existing genes that perform other functions(probably other cell-cell recognition or com-munication functions) in the plant. This viewis also supported by the observation that thedeterminant genes identified thus far typicallybelong to large gene families that include mem-bers expressed in nonreproductive tissues (15,58, 66, 89).

One great advantage of the studies on theself-/nonself-recognition mechanisms in SI isthat both the female and male determinantsinvolved in recognition are encoded in pairsin a single locus. Although not easy tasks dueto the complex structures of the S-loci, sev-eral studies are attempting to identify novelS-determinant genes. These studies will notonly lead to a greater understanding of howflowering plants discriminate self/nonself dur-ing fertilization, but should also shed lighton the processes used by plants for cell-cellcommunication.

SUMMARY POINTS

1. In many species, the specificity of the SI response is determined by the haplotypes of theS-locus, which contains at least two separate multiallelic genes, the female and the maledeterminant genes.

2. SI does not represent one system, but rather a collection of divergent mechanisms,suggesting that SI evolved independently in several lineages.

3. In the Brassicaceae, the determinant genes encode a pollen ligand and its stigmatic re-ceptor kinase, and their interaction induces incompatible signaling(s) within the stigmapapilla cells.

4. In the Solanaceae, Rosaceae, and Scrophulariaceae, the determinants are a ribonucleaseand an F-box protein, suggesting the involvement of RNA degradation and proteindegradation within the system.

5. In the Papaveraceae, the only identified female determinant induces a Ca2+-dependentsignaling network that ultimately results in the death of incompatible pollen.

ACKNOWLEDGMENTS

We first and foremost would like to thank all of the researchers whose dedicated studies havegreatly added to our current knowledge in this field and apologize for being unable to individuallycite their work due to limited space. We would also like to thank former and current membersof the Laboratory of Intercellular Communications at Nara Institute of Science and Technology


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for their various contributions, which enabled us to write this review. We are grateful to EmeritusProfessor Kokichi Hinata at Tohoku University and Dr. Masao Watanabe at Iwate University fortheir helpful discussion. Research in the author’s lab is supported by grants from the Ministry ofEducation, Culture, Sports, Science, and Technology of Japan, and from the Japan Society for thePromotion of Science.

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AR242-PP56-18 ARI 28 March 2005 18:33

93. Watanabe M, Takayama S, Isogai A, Hinata K. 2003. Recent progresses on self-incompatibilityresearch in Brassica species. Breed. Sci. 53:199–208

94. Wheeler MJ, Armstrong SA, Franklin-Tong VE, Franklin FCH. 2003. Genomic organizationof the Papaver rhoeas self-incompatibility S1 locus. J. Exp. Bot. 54:131–39

95. Yamane H, Ikeda K, Ushijima K, Sassa H, Tao R. 2003. A pollen-expressed gene for a novelprotein with an F-box motif that is very tightly linked to a gene for S-RNase in two speciesof cherry, Prunus cerasus and P. avium. Plant Cell Physiol. 44:764–69


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Contents ARI 29 March 2005 21:29

Annual Review ofPlant Biology

Volume 56, 2005

Contents

Fifty Good YearsPeter Starlinger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

PhytoremediationElizabeth Pilon-Smits � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �15

Calcium Oxalate in Plants: Formation and FunctionVincent R. Franceschi and Paul A. Nakata � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �41

Starch DegradationAlison M. Smith, Samuel C. Zeeman, and Steven M. Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �73

CO2 Concentrating Mechanisms in Algae: Mechanisms,Environmental Modulation, and EvolutionMario Giordano, John Beardall, and John A. Raven � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �99

Solute Transporters of the Plastid Envelope MembraneAndreas P.M. Weber, Rainer Schwacke, and Ulf-Ingo Flugge � � � � � � � � � � � � � � � � � � � � � � � � � � � � 133

Abscisic Acid Biosynthesis and CatabolismEiji Nambara and Annie Marion-Poll � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 165

Redox Regulation: A Broadening HorizonBob B. Buchanan and Yves Balmer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 187

Endocytotic Cycling of PM ProteinsAngus S. Murphy, Anindita Bandyopadhyay, Susanne E. Holstein,

and Wendy A. Peer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 221

Molecular Physiology of Legume Seed DevelopmentHans Weber, Ljudmilla Borisjuk, and Ulrich Wobus � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

Cytokinesis in Higher PlantsGerd Jürgens � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 281

Evolution of Flavors and ScentsDavid R. Gang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

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Biology of Chromatin DynamicsTzung-Fu Hsieh and Robert L. Fischer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 327

Shoot BranchingPaula McSteen and Ottoline Leyser � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 353

Protein Splicing Elements and Plants: From Transgene Containmentto Protein PurificationThomas C. Evans, Jr., Ming-Qun Xu, and Sriharsa Pradhan � � � � � � � � � � � � � � � � � � � � � � � � � � � 375

Molecular Genetic Analyses of Microsporogenesis andMicrogametogenesis in Flowering PlantsHong Ma � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 393

Plant-Specific Calmodulin-Binding ProteinsNicolas Bouche, Ayelet Yellin, Wayne A. Snedden, and Hillel Fromm � � � � � � � � � � � � � � � � � � � � 435

Self-Incompatibility in PlantsSeiji Takayama and Akira Isogai � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 467

Remembering Winter: Toward a Molecular Understanding ofVernalizationSibum Sung and Richard M. Amasino � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 491

New Insights to the Function of Phytopathogenic Baterial Type IIIEffectors in PlantsMary Beth Mudgett � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 509

INDEXES

Subject Index � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 533

Cumulative Index of Contributing Authors, Volumes 46–56 � � � � � � � � � � � � � � � � � � � � � � � � � � � 557

Cumulative Index of Chapter Titles, Volumes 46–56 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 562

ERRATA

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

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SELF-INCOMPATIBILITY IN PLANTS - Semantic Scholar€¦ · Sexual reproduction in many ﬂowering...

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