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Signaling in pollen–pistil interactions
seminars in C E L L & D E V E L OP M E N T A L B I OL OG Y , Vol 10, 1999: pp. 139]147 Article No. scdb.1999.0289, available online at http:rrwww.idealibrary.com on
Signaling in pollen–pistil interactions Thierry Gaude† and Sheila McCormickU
A complex set of cell]cell interactions is required to achieve fertilization. The pollen grain must be recognized by the pistil, take up water, and grow a pollen tube directionally through the style in order to deliver the sperm to the ovule. In many families of flowering plants, self-fertilization can be prevented by recognition mechanisms that allow self-pollen rejection by the pistil. The self-incompatibility response is under the genetic control of a single multi-allelic locus, the S (Self-incompatibility) locus. There are two major classes of self-incompatibility systems. Gametophytic self-incompatibility has been well characterized in the Solanaceae and in the Papaveraceae, while sporophytic self-incompatibility has been well characterized in the Brassicaceae. In this review article, we present recent advances in understanding the signals mediating pollen recognition and pollen tube growth, in both compatible and incompatible interactions.
pistil and then rehydrate by acquiring water from the style exudate or from the stigma papillar cells, in order to resume active metabolism. Hydrated pollen grains germinate by extruding a pollen tube. The pollen tube grows through the transmitting tissue of the style to the ovary. In the ovary, the pollen tube delivers the two male gametes into the embryo sac where the double fertilization takes place. The numerous cellular interactions that take place between the pollen Žor pollen tube. and the different tissues of the pistil all along the fertilization process, are likely to involve complex cross-talk between diverse molecules. These interactions can be as complex as protein]protein interactions, exemplified by ligand]protein kinase signaling, or can involve molecules as simple as water, calcium, lipids and sugars. Several recent reviews of pollenrpistil interactions and self-incompatibility are available.1 ] 7 In this article we summarize recent studies on selected aspects of pollenrpistil recognition and signal transduction.
Key words: pollen]pistil interactions r receptor-like kinases r self-incompatibility r S-locus r SRNases Q1999 Academic Press
Compatible pollinations Introduction Pollen hydration involves the uptake of water. Because pollen from Arabidopsis cer mutants is defective in hydration, it was suggested that lipids act as signals for water uptake.3,6 More recently, studies with stigmaless Nicotiana alata plants were used to show that certain classes of lipids Ž cis-unsaturated triacylglycerides. could replace the requirement of stigma exudate for pollen hydration.8 Membrane-associated water channels, or aquaporins, may also play a role in water uptake.9 In self-incompatible Brassica plants with a recessive mutation Ž mod . in an aquaporin, pollen that would not normally hydrate and germinate could do so when placed on mod stigmas.10 Lastly, N. alata pollen that is germinated in medium with a defined oilrwater interface will grow towards the water, suggesting that water concentration itself is
IN FLOWERING PLANTS, the success of sexual reproduction relies on a series of interactions between the pollen grain Žthe male partner. and the different tissues of the pistil Žthe female partner.. At the time of dispersal, mature pollen grains of most angiosperms are highly dehydrated structures. During compatible pollination, pollen grains adhere to the U
From the Plant Gene Expression Center, USDA r ARS-UCBerkeley, 800 Buchanan St., Albany, CA 94710, USA and †Research unit for Reproduction et Developpement des Plantes, UMR ´ 9938 CNRS-INRA-ENSL, Ecole Normale Superieure de Lyon, 46 ´ allee ´´ d’Italie, 69364 Lyon Cedex 07, France. Q1999 Academic Press 1084-9521r 99r 020139q 09 $30.00r 0
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a cue for pollen growth. Pollen tubes showed no directional growth if the oilrwater interface was emulsified.11 It is not yet clear which, if any, components of the extracellular matrix of the style play an important role in pollen tube growth. For example, contradictory results were obtained from the study of an abundant glycoprotein, termed TTS Žfor transmitting tissue-specific .. Although it was reported that the N. tabacum TTS was a growth stimulant and pollen tube attractant,12 studies performed with the probable homolog Ž97% homology. of TTS from N. alata13 showed only an inhibitory role for TTS. The importance of calcium in pollen tube growth has been well-characterized.14 Although it is still largely unknown if or how the asymmetric gradient is initiated by signals from the style, recent experiments are starting to dissect the downstream events. For example, the sub-cellular location of a Ca2q-dependent protein kinase activity is correlated with the direction of pollen tube growth.15 Furthermore, release of caged inositol 1,4,5-triphosphate in sub-apical regions of the growing pollen tube resulted in pollen tube reorientation and an increase in Ca2q levels in that region.16 Because calmodulin is rather uniformly distributed in the growing point of pollen tubes, it is presumed to be involved in pollen tube growth rather than in orienting the direction of pollen tube growth.17 Lastly, there is some evidence that other classes of pollen receptor-like kinase genes can transduce signals from the pistil tissue. The tomato receptor-like kinases, LePRK1 and LePRK2, have extracellular domains composed of 5]6 leucine-rich repeats ŽLRRs.. LRRs are believed to mediate protein]protein interactions. LePRK1 and LePRK2 are localized to the pollen tube plasma membrane and at least LePRK2 is partially dephosphorylated when pollen membranes are incubated in the presence of style extracts.18 The ligandŽs. for these kinases is unknown, as are details of the downstream signal transduction pathway.
are anchored in the soil by their roots and therefore cannot actively search out their sexual partners. One might think that the high level of inbreeding would be associated with a loss of genetic variability and that self-pollination might therefore have been deleterious for the evolution of angiosperms. However, angiosperms are the most successful groups of terrestrial flora both in terms of the number of species Žapprox. 300 000 described so far. and by their diversity of forms and ecological niches. This success is thought to depend partly on the acquisition by the angiosperms of mechanisms which strongly limit or even prevent self-fertilization. The most sophisticated and widespread of these mechanisms is self-incompatibility ŽSI., which leads to the rejection of self-pollen by the pistil. Several SI systems have been selected during the course of evolution and SI is thought to occur in slightly more than 50% of angiosperm species.19 In most cases, self-incompatibility is controlled by a single multi-allelic locus, the S-locus Ž S for Self-incompatibility.. Classical genetics has established that there are two types of SI systems, classified according to pollen behavior with respect to the pistil tissues. When the incompatibility phenotype of the pollen is determined by its own haploid S genotype, the system is defined as a gametophytic SI system ŽGSI.. When the incompatibility phenotype of the pollen is determined by the diploid S genotype of the mother plant Žthe sporophyte., the system is defined as a sporophytic SI system ŽFigure 1.. In all cases, pollen rejection occurs when the same S allele specificity is expressed both by the pollen and the pistil tissue Žstigma, style or ovary.. In both systems, pollen]pistil recognition has been generally considered to involve signal and receptor interactions and recent molecular data supports this assumption. Strikingly, self-compatible species can also exist within a family in which self-incompatibility is usually the rule. For example, in the Brassicaceae, Arabidopsis thaliana is fully self-compatible. Because of the complexity of self-incompatibility, it was hypothesized that self-compatible species evolved from self-incompatible species, presumably due to mutations or deletions in the S-locus or in genes controlling the expression of S-locus genes.19 This hypothesis has recently found support from a comparative mapping study of the S-locus region of the self-incompatible species Brassica campestris with its homolog in A. thaliana.20 Sequences similar to the Brassica S locus genes were not detected within the corresponding chromosomal region in A. thaliana, or in other re-
Pollenr r pistil signaling and selfincompatibility Angiosperms display several traits which should have promoted or imposed self-pollination, and thus selffertilization. For example, they are largely hermaphroditic Žapprox. 96% of species carry male and female organs on the same individual, and among these species, 75% have hermaphroditic flowers. and 140
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cluding the Solanaceae, the Liliaceae, the Rosaceae, the Papaveraceae and the Poaceae. Rejection of a self-pollen grain generally leads to the arrest of pollen tube growth in the transmitting tissue of the style. However, in poppy and grasses, pollen tube growth is stopped earlier at the stigmatic surface. Much of the molecular work on GSI has been carried out in two Solanaceae species Ž N. alata and Petunia inflata. and in poppy Ž Papaver rhoeas.. The Solanaceae Although the existence of molecules associated with the incompatibility response was demonstrated as early as in 1960 in Petunia hybrida,21 it was only in the mid eighties that the first S gene of GSI systems was cloned, from N. alata.22 This S gene is specifically expressed in the pistil and encodes a glycoprotein which is abundantly produced Žseveral micrograms per pistil. and is secreted in the extracellular space, principally in the stigma and transmitting tissue of the style. Analysis of the amino acid sequences of different S-allele glycoproteins in N. alata revealed the existence of significant homology between the S glycoproteins and the extracellular ribonucleases ŽRNases. T2 of Aspergillus oryzae and Rh of Rhizopus niveus.23 The S glycoproteins indeed have RNase activity and thus are now referred to as S RNases. Interestingly, stylar S RNases are not restricted to the SI system of the Solanaceae. S RNases and S locus ribonuclease genes have been characterized in the Rosaceae 24 ] 26 Že.g. Prunus, Pyrus, and Malus . and in the Scrophulariaceae27 Že.g. Antirrhinum.. A common feature of S RNases is that their primary structure is composed of alternating variable and conserved regions, with the presence of one Žin the Rosaceae. 26 or two Žin the Scrophulariaceae and Solanaceae. 27,28 hypervariable ŽHV. regions. S RNases of different species or of the same species but from different S alleles are highly polymorphic. For example, in the Solanaceae, amino acid identity between different S glycoproteins ranges from 38 to 98%. This level of polymorphism is unusual for most genes; such sequence diversity is expected for products of genes that are involved in self-recognition mechanisms to promote outcrossing.29 A new S allele arising in a population is favored, because pollen expressing this new S allele will have high probability to encounter a stigma bearing a different S allele. Both the cosegregation of stylar S glycoproteins with S alleles and their polymorphic sequences suggested involvement of S RNases in SI; only transgenic
Figure 1. Self-incompatibility systems. Ža. Gametophytic self-incompatibility ŽGSI.: pollen behavior is determined by the haploid genome of the pollen grain. Pollen grains carrying the same S allele specificity Ž S1 in our example. as one expressed by the diploid pistil Ž S1S2 . are rejected. Incompatible pollen tubes are generally arrested in the style. Žb. Sporophytic self-incompatibility ŽSSI.: pollen behavior is determined by the diploid genome of the pollenproducing plant Ž S1S2 .. In our example, S3 pollen grains from the S1 S3 plant are rejected because the S1 allele is expressed both in the pollen-producing plant and the S1S2 pistil. The SI response occurs only if S1 is dominant to, or co-dominant with, S3 in the pollen while S1 is dominant to, or co-dominant with, S2 in the style. Incompatible pollen grains either fail to hydrate and germinate or if they do germinate, pollen tube growth is arrested at the stigma surface.
gions of the Arabidopsis genome, despite the otherwise high degree of synteny at the submegabase scale between these two regions.
Gametophytic SI (GSI) Gametophytic systems are the most common and have been described in more than 60 families, in141
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experiments provided direct evidence, in both N. alata21,30 and P. inflata.28,31 In Petunia, a loss-of-func tion approach Žbased on an anti-sense strategy. and a gain-of-function approach Žusing a sense strategy. was used to prove that S RNases are sufficient for the recognition and rejection of self-pollen by the pistil.31 Moreover, RNase activity of S glycoproteins is necessary for self-pollen rejection, because transgenic plants expressing a mutated S3 protein lacking RNase activity are not able to reject S3 pollen.32 The origin of S allele specificity was presumed to reside either in the carbohydrate side chains of S RNases or in the hypervariable regions of the polypeptide sequences. The role of the carbohydrate moiety was investigated by expressing a mutated non-glycosylated S3 protein in transgenic plants. These transgenic plants produced a normal level of the non-glycosylated S3 RNase but were still capable of rejecting S3 pollen grains. Thus, the glycan chains do not confer S allele specificity and are not necessary for S RNase function in recognition and rejection of the self-pollen grain.33 To examine the role of HV regions in S allele specificity, chimeric S RNases composed of the major part of the S3 protein but containing HV regions from the S1 protein were constructed. The resulting transgenic plants lost the ability to reject S3 pollen but did not acquire the ability to reject S1 pollen.28 This result suggests that HV regions of S proteins are necessary but not sufficient for the establishment of S allele specificity. However, based on a similar approach, contradictory data were obtained 34 in another Solanaceous species, Solanum chacoense. In this species, S11 and S13 RNases only differ by four amino acid residues, which are located in the HV region. Plants transformed with chimeric gene constructs in which the S11 RNase had those four amino acids changed to those of the S13 RNase exhibited an S13 phenotype.34 Thus in S. chacoense it appears that the HV region alone can determine allelic specificity, although whether these results are generalizable has been questioned.35 To test how general the S. chacoense findings are, similar experiments could be attempted with other nearly identical alleles. For example, the Japanese pear S3 and S5 RNases are 95% identical and differ only in the HV region amino acids.26 Furthermore, structural analyses36 of additional S RNases should prove useful in targeting regions of different S RNases before attempting further allelic specificity changes in transgenic plants. A remarkable feature of all these transgenic experiments is that only the SI phenotype of the pistil was affected, while the pollen phenotype remained un-
changed. This indicates that the male component of the SI response is not the S RNase and strongly supports the assumption that pollen and pistil S molecules are encoded by two different genes located at the S locus. In spite of numerous attempts to identify the male S locus component, so far, it is unknown. In the Solanaceae, two hypotheses have been proposed to explain the molecular basis of self-pollen rejection, both of which rely on a cytotoxic effect.28 S RNases are thought to enter the pollen tube in order to hydrolyse ribosomal and messenger RNAs, thus blocking protein synthesis and therefore blocking pollen tube growth.28 The first hypothesis presumes that the pollen S product is a receptor present at the surface of the pollen tube Žin the wall or plasma membrane.. This receptor would allow the allelespecific recognition and translocation of stylar S RNases into the pollen tube. In this ‘receptor’ model, only the stylar S RNase of the same allele as that of the pollen enters the pollen tube and inhibits its growth. The second hypothesis presumes that the pollen S product is a cytosolic RNase inhibitor which inactivates all S RNases except the one sharing the same S allele specificity as that of the pollen. In this ‘inhibitor’ model, S RNases present in the style enter the pollen tube independently of their S allele specificity, but only the S RNase recognized as ‘self’ inside the pollen tube will not be inhibited and thus can degrade pollen RNA. No direct evidence that may support either of the two models has been reported. In fact, two recent papers challenge the cytotoxic model. Despite the common perception that incompatible pollen tubes irretrievably arrest in the upper one-third of the style, incompatible pollen tubes of N. alata can continue to grow, and can resume a rapid rate of growth if the incompatible style they are growing through is grafted to a compatible style.37 Furthermore, both ribosomes and polysomes continue to be visible after self- incom patible pollinations,38 suggesting that the target of the S RNase must be highly specific and that pollen tube arrest is not due to a general degradation of pollen RNA Žribosomal or mRNA.. The Papaveraceae In Papaver rhoeas, stigma S proteins are also secreted glycoproteins, but they do not have RNase activity or any other known catalytic activity.39 However, using an in vitro pollen germination assay, stigma S proteins have been shown to inhibit pollen tube growth 142
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in an S allele-specific manner.39 Moreover, S proteins are also capable of interacting with a pollen plasma membrane protein called SBP Ž S Binding Protein.,40 but with no S-allele specificity. In order to incorporate this interaction into the SI response, a model proposes that pollen SBP and stigma S proteins must interact in an S allele-specific manner with an as yet unknown pollen component encoded by the S locus. In an attempt to define the regions of the S protein that mediate interaction with pollen components, mutant derivatives were constructed and the expressed proteins assayed for pollen inhibition using the in vitro pollen germination assay. Mutation of the only hypervariable amino acid in a hydrophilic region Žloop 6. of the stigmatic S1 protein resulted in a complete loss of S1 pollen inhibition.41 This result suggests that loop 6 participates in pollen recognition. However, changing the hypervariable amino acid in S1 to that present in either of two different S alleles Ž S3 or S8 . was not able to confer a new allelic specificity on the mutant protein. Thus these results in poppy are analogous to those in Petunia and N. alata28,30 and in contrast to those in S. chacoense.34 In the poppy, there is considerable information42 available on the signal transduction pathway that occurs in pollen after the initial recognition response. When growing pollen tubes are challenged with incompatible S proteins, a transient calcium concentration increase occurs within seconds, followed by a calcium-dependent phosphorylation of a 26-kDa pollen protein, and subsequently by a calcium-independent phosphorylation of a 68-kDa pollen protein. Although it is not yet understood how these phosphorylations result in pollen tube arrest, it is interesting to note that incubation of poppy pollen with profilin can also influence the phosphorylation state of the 26-kDa pollen protein.43 Profilin is known to modulate the actin cytoskeleton but may also play a role in modulating pollen tube growth via interactions with protein kinases or their substrates.
rapid arrest of its growth at the stigma surface. Molecular analysis of several S genotypes has revealed that the S- locus has a com plex organization.44 ] 47 The S-locus is composed of several physically linked genes and in this respect resembles the major histocompatibility complex in mammals. This analogy suggested the term ‘haplotypes’48 to designate different alleles of the S-locus. Two S-locus genes have been studied in detail. The first gene linked to the S-locus, S-Locus Glycoprotein, SLG, encodes a glycoprotein secreted into the cell wall of stigmatic papillae.49 The second gene, S-locus Receptor Kinase, SRK, encodes a receptor-like kinase.50 On the basis of cDNA sequence analysis, SRK is predicted to be a plasma membrane protein consisting of three domains: an extracellular domain that shares homology with SLG, a single transmembrane region and a cytoplasmic domain with consensus sequences of serinerthreonine kinases. The cytoplasmic domain of SRK has been overexpressed in Escherichia coli and shown to have serinerthreonine kinase activity.51 It is presumed that the extracellular domain acts as a receptor, in analogy with membrane receptor kinases characterized in animal systems that recognize and transduce peptide signals controlling processes, such as development, cell growth, differentiation and death.52 What about the pollen component of SI? While SLG and SRK are essentially expressed in stigmas and, at a low level, in anthers, a third gene, S-Locus Anther, SLA, mapped to the S locus and is specifically expressed in anthers.53 It was first thought that SLA could encode the long-sought pollen determinant of SI. However, fully self-incompatible B. oleracea lines were shown to carry a non-functional SLA gene.54 Two other anther-expressed candidates that mapped to the S locus of a different haplotype45 were also eliminated as potential pollen determinants of SI. One candidate was similarly expressed in both self-incompatible and self-compatible lines, while the other showed no sequence variation between different haplotypes. In a complementary approach to identify the pollen ligand, Brassica pollen coating was fractionated and interspersed between pollen and pistil, of the same and different haplotypes. A fraction that was enriched for PCPs Žsmall, cysteine-rich proteins similar to plant defensins. had the ability to alter pollen hydration and germination in an allele-specific manner.55 However, in other work, the gene encoding a particular pollen coat protein of this class, PCP-Al, was unlinked to the S locus and furthermore was shown to be gametophytically expressed.56 Fur-
Sporophytic SI (SSI) Although sporophytic self-incompatibility is less widespread than gametophytic self-incompatibility, it is present in plant families which are more highly evolved, such as the Brassicaceae, the Asteraceae, and the Caryophyllaceae. Most studies concerning molecular aspects of sporophytic SI have concentrated on species in the Brassicaceae. Self-pollen rejection results in a blockage of pollen tube emergence or a 143
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thermore, although purified PCP-Al interacts with SLG, the interaction shows no allelic specificity. The pollen component of SI is still unknown and will remain so, until a pollen protein interacting with stigma S molecules and exhibiting S allelic specificity is shown to be encoded by a gene linked to the S locus. What is the recognition determinant in the pistil? For a given haplotype, the SLG and extracellular domain of SRK, eSRK, share between 83 and 90% identity at the amino acid level.44,57 This sequence conservation strongly contrasts with the high degree of divergence Žup to 30%. between SLGs Žor SRKs. from different haplotypes. Perhaps selection pressure acts to maintain a high homology between SLG and SRK alleles within a given haplotype because both SLG and SRK are required for recognition of the pollen determinant. To complicate matters, in the case of at least one SLG gene, SLG2 , alternative transcripts can encode two SLG proteins, a secreted and a membrane-anchored form.58 The S2 haplotype is a class II, or so-called pollen recessive haplotype. However, three other pollen recessive haplotypes from B. rapa do not show this alternative transcript pattern.59 Because of the abundance of SLGs in stigma papillae, molecular control of the SI response was initially thought to depend mainly on SLG.49 Indeed, the level of SLG mRNA reaches its maximum one day prior to anthesis, precisely at the stage when the flowers become self-incompatible. The role played by SLG in the SI response has been recently questioned, because there are self-incompatible plants Žof the S2 haplotype. that express a very low level of SLG. Furthermore, naturally self-compatible variants of the S15 and Sc haplotypes express a high level of SLG.60,61 A recent analysis of the S15 haplotype in B. oleracea has revealed the existence of a new SLG gene in this haplotype ŽCabrillac et al., submitted paper.. We have found that the S locus in the S15 haplotype contains at least three members of the S gene family, all of which are expressed in the stigma: SRK and two different SLG genes, designated SLGA and SLGB. Both SLGA and SLGB are interrupted by a single intron but while SLGA encodes both soluble and membrane-anchored forms of SLG, SLGB only encodes soluble SLG proteins. Interestingly, we found that the two other pollen-recessive S haplotypes, S2 and S5 , carry only one or the other of the SLG genes, indicating either that these genes are redundant in the S15 haplotype or that they are not required for the self-incompatibility response. These observations
suggest that SRK, rather than SLG, plays a key role in the SI reaction. This hypothesis gained more support from the analysis of self-compatible variants of rapeseed and cabbage.62,63 These plants express normal levels of SLGs, but they carry mutations that lead either to the absence of SRK transcripts or to the production of truncated transcripts. These data indicate that the expression of a functional SRK is necessary for the SI response. In the S3 haplotype, SRK is a 120-kDa glycoprotein that is specifically expressed in the stigma and anchored in the plasma membrane.57,65 SRK 3 has also been shown to encode a truncated, soluble form of SRK 3 corresponding to its extracellular domain: the eSRK 3 protein.65 Alternative splicing of SRK transcripts may generate eSRK 3 . This result is particularly interesting because of the identification of soluble truncated forms of receptor kinases in some animal signal transduction pathways.66,67 In the case of the animal receptor kinases, the soluble forms inhibit signal transduction by limiting binding of the ligand to the transmembrane receptor. A similar role may be played by eSRK in the regulation of the SI response in Brassica. The expression of recombinant SRK proteins in a membranous environment, using the insect cellrbaculovirus system, has allowed the analysis of how SRK functions at a molecular level ŽGiranton et al., in preparation.. This system has already been used for the successful expression of animal kinase receptors.68 ] 71 The experimental determination of the topology of recombinant SRK in microsomal membranes supports a model in which the S domain of SRK is directed towards the periplasm at the surface of stigma cells, and thus is probably involved in the recognition of a ligand.50,57,64 Kinase assays performed on microsomes showed that recombinant SRK autophosphorylated on serine and threonine residues but not on tyrosine residues. Autophosphorylation was constitutive since it did not require addition of pollen or stigma extracts to the phosphorylation buffer. The co-expression of two different SRK constructs, which exhibit either a functional or a kinase defective activity and could be recovered independently, revealed that SRK phosphorylation occurred in trans, suggesting the existence of constitutive homo-oligomers of membrane-anchored recombinant SRK. Furthermore, cross-linking experiments indicated that SRK, but neither eSRK nor SLG, exists constitutively as an oligomeric complex in planta. Our analyses of the enzymatic properties of recombinant SRK expressed in insect cells, and the investigation of SRK’s oligomeric status in planta therefore 144
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6. Perspective Although analyses of pollen]pistil interactions and SI mechanisms have progressed, there is still a great deal to be learned. Unfortunately, we still do not know the identity of both signaling partners Žmale and female. for any of the interactions discussed in this review. In the gametophytic self-incompatibility arena, the exciting results suggesting that allelic specificity can be changed Žin S. chacoense. needs to be duplicated in other plants and with other S alleles. Because transgenic plant analyses have been unsuccessful in definitively proving which components are involved in sporophytic self-incompatibility, it will be crucial to try other, perhaps more biochemical, approaches. It is clear that detailed studies of several haplotypes will help elucidate those features that are common and therefore more likely to be critical. Figure 2. Model of the pollen]stigma interaction, which leads to the SI response in Brassicaceae. A pollen ligand encoded by the S-locus Žas yet unknown. is thought to interact with the membrane receptor SRK. SRK, probably existing as a dimer, may be associated with other molecules either encoded by the S-locus ŽSLG, eSRK. or not Žfor example a putative co-receptor, designated CoR.. By analogy with animal kinase receptors, binding of the pollen ligand may modify the phosphorylation level, the conformation, andror the oligomerization status of SRK. This step, for which the molecular details are still unknown, may allow the recruitment of cytoplasmic targets leading to SI response.
References 1. Gasser CS, Robinson-Beers K Ž1993. Pistil development. Plant Cell 5:1231]1239 2. Bedinger PA, Hardeman KJ, Loukides CA Ž1994. Traveling in style: the cell biology of pollen. Trends Cell Biol 4:132]138 3. Preuss D Ž1995. Being fruitful: genetics of reproduction in Arabidopsis. Trends Genet 11:147]153 4. Cheung AY Ž1996. Pollen]pistil interactions during pollentube growth. Trends Plant Sci 1:45]51 5. McCormick S Ž1998. Self- incompatibility and other pollen]pistil interactions. Curr Opin Plant Biol 1:18]25 6. Pruitt RE Ž1997. Molecular mechanics of smart stigmas. Trends Plant Sci 2:328]329 7. Mahlo R Ž1998. Pollen tube guidance}the long and winding road. Sex. Plant Reprod 11:242]244 8. Wolter-Arts M, Lush WM, Mariani C Ž1998. Lipids are required for directional pollen- tube growth. Nature 392:818]821 9. Schaffner AR Ž1998. Aquaporin function, structure and expression: are there more surprises to surface in water relations? Planta 204:131]139 10. Ikeda S, Nasrallah JB, Dixit R, Preiss S, Nasrallah ME Ž1997. An aquaporin-like gene required for the Brassica self-incompatibility response. Science 276:1564]1566 11. Lush WM, Grieser F, Wolters-Arts M Ž1998. Directional guidance of Nicotiana alata pollen tubes in vitro and on the stigma. Plant Physiol 118:733]741 12. Cheung AY, Wang H, Wu HM Ž1995. A floral transmitting tissue-specific glycoprotein attracts pollen tubes and stimulates their growth. Cell 82:383]393 13. Sommer-Knudsen J, Lush WM, Bacic A, Clarke AE Ž1998. Re-evaluation of the role of a transmitting tract-specific glycoprotein on pollen tube growth. Plant J 13:529]535 14. Trewavas AJ, Mahlo R Ž1998. Ca2q signalling in plant cells: the big network. Curr Opin Plant Biol 1:428]433 15. Moutinho A, Trewavas AJ, Malho R Ž1998. Relocation of Ca2q-dependent protein kinase activity during pollen tube reorientation. Plant Cell 10:1499]1509 16. Mahlo R Ž1998. Role of 1,4,5-inositol triphosphate-induced Ca2q release in pollen tube orientation. Sex Plant Reprod 11:231]235
suggest that signal transduction during SI response might be mediated by modification of a preexisting SRK oligomeric complex rather than by ligand-dependent dimerization of SRK molecules. Similar conclusions have been drawn in animals from the study of TGF-b signal transduction via its serinerthreonine kinase receptors. These receptors are able to homooligomerize even in the absence of ligand and the type II receptors exhibit constitutive serinerthreonine kinase activity.72,73 These data highlight the analogies existing between animal and plant signal transduction pathways. Moreover, they allow us to reconsider the classical model in which the SI response is initiated by ligand-dependent dimerization of SRK. An alternative model ŽFigure 2. proposes that SRK can exist as a dimer even in non-pollinated stigma. The interaction of SRK with its ligand could either allow the recruitment of cytoplasmic targets following a conformational change of the SRK kinase domain, or favor the recruitment of a yet unknown co-receptor also involved in signal transduction ŽFigure 2.. 145
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17. Mountinho A, Love J, Trewavas AJ, Malho R Ž1998. Distribution of calmodulin protein and mRNA in growing pollen tubes. Sex Plant Reprod 11:131]139 18. Muschietti JP, Eyal Y, McCormick S Ž1998. Pollen tube localization implies a role in pollen]pistil interactions for the tomato receptor-like kinases LePRK1 and LePRK2. Plant Cell 10:319]330 19. Mayo O, Leach CR Ž1987. Stability of self-incompatibility systems. Theor Appl Genet 74:789]792 20. Conner JA, Conner P, Nasrallah ME, Nasrallah JB Ž1998. Comparative mapping of the Brassica S locus region and its homeolog in Arabidopsis. Implications for the evolution of mating systems in the Brassicaceae. Plant Cell 10:801]812 21. Newbigin E, Anderson MA, Clarke A Ž1993. Gametophytic self-incompatibility systems. Plant Cell 5:1315]1324 22. Anderson MA, Cornish EC, Mau SL, Williams EG, Hoggart R, Atkinson A, Bonig I, Grego B, Simpson R, Roche PJ, Haley JD, Penschow JD, Niall HD, Tregear GW, Coghlan JP, Crawford RJ, Clarke AE Ž1986. Cloning of cDNA for a stylar glycoprotein associated with expression of self-incompatibility in Nicotiana alata. Nature 321:38]44 23. McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ, Sakiyama F, Clarke AE Ž1989. Style self-incompatibility gene products of Nicotiana alata are ribonucleases. Nature 342:955]957 24. Tao R, Yamane H, Sassa H, Mori H, Gradziel TM, Dandekar AM, Sugiura A Ž1997. Identification of stylar RNases associated with gametophytic self-incompatibility in almond Ž Prunus dulcis.. Plant Cell Physiol 38:304]311 25. Sassa H, Nishio T, Kowyama Y, Hirano H, Koba T, Ikehashi H Ž1996. Self-incompatibility Ž S . alleles of the Rosaceae encode members of a distinct class of the T2rS ribonuclease superfamily. Mol Gen Genet 250:547]557 26. Ishimizu T, Shinkawa T, Sakiyama F, Norioka S Ž1998. Primary structural features of rosaceous S-RNases associated with gametophytic self-incompatibility. Plant Mol Biol 37:931]941 27. Xue Y, Carpenter R, Dickinson HG, Coen ES Ž1996. Origin of allelic diversity in Antirrhinum S locus RNases. Plant Cell 8:805]814 28. Kao T-H, McCubbin AG Ž1996. How flowering plants discriminate between self and non-self pollen to prevent inbreeding. Proc Natl Acad Sci USA 93:12059]12065 29. Clark AG, Kao TH Ž1991. Excess non-synonymous substitution at shared polymorphic sites among self-incompatibility alleles of Solanaceae. Proc Nad Acad Sci USA 88:9823]9827 30. Murfett J, Atherton T, Mou B, Gasser C, McClure B Ž1994. S-RNase expressed in transgenic Nicotiana causes S-allelespecific pollen rejection. Nature 367:563]566 31. Lee H-S, Huang S, Kao T-H Ž1994. S proteins control rejection of incompatible pollen in Petunia inflata. Nature 367:560]563 32. Huang S, Lee H-S, Karunanandaa B, Kao T-H Ž1994. Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self pollen. Plant Cell 6:1021]1028 33. Karunanandaa B, Huang S, Kao T-H Ž1994. Carbohydrate moiety of the Petunia inflata S3 protein is not required for self-incompatibility interactions between pollen and pistil. Plant Cell 6:1933]1940 34. Matton DP, Maes O, Laublin G, Xike Q, Bertrand C, Morse D, Cappadocia M Ž1997. Hypervariable domains of self-incompatibility RNases mediate allele-specific pollen recognition. Plant Cell 9:1757]1766 35. Verica JA, McCubbin AG, Kao, TH Ž1998. Are the hypervariable regions of S RNases sufficient for allele-specific recognition of pollen? Plant Cell 10:314]316 36. Parry S, Newbigin E, Craik D, Nakamura KT, Bacic A, Oxley D Ž1998. Structural analysis and molecular model of a self-in-
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compatibility RNase from wild tomato. Plant Physiol 116:463]469 Lush M, Clarke AE Ž1997. Observations of pollen tube growth in Nicotiana alata and their implications for the mechanism of self-incompatibility. Sex Plant Reprod 10:27]35 Walles B, Han, SP Ž1998. Ribosomes in incompatible pollen tubes in the Solanaceae. Physiol Plant 103:461]465 Foote HCC, Ride JP, Franklin-Tong VE, Walker EA, Lawrence MJ, Franklin FCH Ž1994. Cloning and expression of a novel self-incompatibility Ž S- . gene from Papaver rhoeas L. Proc Natl Acad Sci USA 91:2265]2269 Hearn MJ, Franklin CH, Ride JP Ž1996. Identification of a membrane glycoprotein in pollen of Papaver rhoeas which binds stigmatic self-incompatibility Ž S- . proteins. Plant J 9:467]475 Kakeda K, Jordan ND, Conner A, Ride JP, Franklin-Tong VE, Franklin FCH Ž1998. Identification of residues in a hydrophylic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen. Plant Cell 10:1723]1731 Rudd JJ, Franklin FCH, Franklin-Tong VE Ž1997. Ca2q independent phosphorylation of a 68-kDa pollen protein is stimulated by the self-incompatibility response in Papaver rhoeas. Plant J 12:507]514 Clarke SR, Staiger CJ, Gibbon BC, Franklin-Tong VE Ž1998. A potential signaling role for profilin in pollen of Papaver rhoeas. Plant Cell 10:967]979 Nasrallah JB, Nasrallah ME Ž1993. Pollen]stigma signaling in the sporophytic self-incompatibility response. Plant Cell 5:1325]1335 Yu K, Schafer U, Glavin TL, Goring DR, Rothstein SJ Ž1996. Molecular characterization of the S locus in two self-incompatible Brassica napus lines. Plant Cell 8:2369]2380 Boyes DC, Nasrallah ME, Vrebalov J, Nasrallah JB Ž1997. The self-incompatibility Ž S . haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin. Plant Cell 9:237]247 Suzuki G, Watanabe M, Kai N, Matsuda N, Toryama K, Takayama S, Isogai A, Hinata K Ž1997. Three members of the S multigene family are linked to the S locus of Brassica. Mol Gen Genet 256:257]264 Boyes DC, Nasrallah JB Ž1993. Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea. Mol Gen Genet 236:369]373 Nasrallah JB, Kao T-H, Goldberg ML, Nasrallah ME Ž1985. A cDNA clone encoding an S-locus specific glycoprotein from Brassica oleracea. Nature 318:617]618 Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB Ž1991. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc Natl Acad Sci USA 88:8816]8820 Goring DR, Rothstein SJ Ž1992. The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serinerthreonine kinase. Plant Cell 4:1273]1281 Heldin C-H Ž1995. Dimerization of cell surface receptors in signal transduction. Cell 80:213]223 Boyes DC, Nasrallah JB Ž1995. An anther specific gene encoded by an S-locus haplotype of Brassica produces complementary and differentially regulated transcripts. Plant Cell 7:1283]1294 Pastuglia M, Rufflo-Chable V, Delorme V, Gaude T, Dumas C, Cock JM Ž1997. A functional S locus anther gene is not required for the self-incompatibility response in Brassica oleracea. Plant Cell 9:2065]2076 Stephenson AG, Doughty J, Dixon S, Elleman CJ, Hiscock SJ, Dickinson HG Ž1997. The male determinant of self-incompatibility in Brassica oleracea is located in the pollen coating. Plant J 12:1351]1359 Doughty J, Dixon S, Hiscok SJ, Willis AC, Parkin IAP, Dickin-
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son HG Ž1998. PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression. Plant Cell 10:1333]1347 Delorme V, Giranton J-L, Hatzfeld Y, Friry A, Heizmann P, Ariza MJ, Dumas C, Gaude T, Cock JM Ž1995. Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: Identification of a membrane-localized protein encoded by the S locus receptor kinase gene. Plant J 7:429]440 Tantikanjana T, Nasrallah ME, Stein JC, Chen C-H, Nasrallah JB Ž1993. An alternative transcript of the S locus glycoprotein gene in a class II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein. Plant Cell 5:657]666 Hatakeyama K, Takasaki T, Watanabe M, Hinata K Ž1998. Molecular characterization of S locus genes, SLG and SRK, in a pollen-recessive self-incompatibility haplotype of Brassica rapa L. Genetics 149:1587]1597 Gaude T, Friry A, Heizmann P, Mariac C, Rougier M, Fobis I, Dumas C Ž1993. Expression of a self-incompatibility gene in a self-compatible line of Brassica oleracea. Plant Cell 5:75]86 Gaude T, Rougier M, Heizmann P, Ockendon DJ, Dumas C Ž1995. Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in the class II S haplotypes of Brassica oleracea. Plant Mol Biol 27:1003]1014 Goring DR, Glavin TL, Schafer U, Rothstein SJ Ž1993. An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion. Plant Cell 5:531]539 Nasrallah JB, Rundle SJ, Nasrallah ME Ž1994. Genetic evidence for the requirement of the Brassica S-locus receptor kinase gene in the self-incompatibility response. Plant J 5:373]384 Stein JC, Dixit R, Nasrallah ME, Nasrallah JB Ž1996. SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco. Plant Cell 8:429]445
65. Giranton J-L, Ariza M-J, Dumas C, Cock JM, Gaude T Ž1995. The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SRK extracellular domain in Brassica oleracea. Plant J 8:101]108 66. Duan D-SR, Pazin MJ, Fretto U, Williams LT Ž1991. A functional soluble extracellular region of the platelet-derived growth factor ŽPDGF. receptor antagonizes PDGF-stimulated responses. J Biol Chem 266:413]418 67. Flickinger TW, Mailhe NJ, King HJ Ž1992. An alternatively processed mRNA from the avian c-erB gene encodes a soluble, truncated form of the receptor that can block liganddependent transformation. Mol Cell Biol 12:883]893 68. Greenfield C, Patel G, Clark S, Jones N, Waterfield MD Ž1988. Expression of the human EGF receptor with ligandstimulatable kinase activity in insect cells using a baculovirus vector. EMBO J 7:139]146 69. Paul JL, Tavare ´ J, Denton RM, Steiner DF Ž1990. Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain. J Biol Chem 265:13074]13083 70. Ventura F, Doody J, Liu F, Wrana JL, Massague ´ J Ž1994. Reconstitution and transphosphorylation of TGF-b receptor complexes. EMBO J 13:5581]5589 71. Gaymard F, Cerutti M, Horeau C, Lemaillet G, Urbach S, Ravallec M, Devauchelle G, Sentenac H, Thibaud JB Ž1996. The baculovirusrinsect cell system as an alternative to Xenopus oocytes. First characterization of the AKT1 Kq channel from Arabidopsis thaliana. J Biol Chem 271:22863]22870 72. Wrana JL, Attisano L, Wieser R, Ventura F, Massague ´ J Ž1994. Mechanism of activation of TGF- b receptor. Nature 370:341]347 73. Gilboa L, Wells RG, Lodish HF, Henis YI Ž1998. Oligomeric structure of type I and II transforming growth factor b receptors: homodimers form in the ER and persist at the plasma membrane. J Cell Biol 140:767]777
147
Signaling in pollen–pistil interactions Thierry Gaude† and Sheila McCormickU
A complex set of cell]cell interactions is required to achieve fertilization. The pollen grain must be recognized by the pistil, take up water, and grow a pollen tube directionally through the style in order to deliver the sperm to the ovule. In many families of flowering plants, self-fertilization can be prevented by recognition mechanisms that allow self-pollen rejection by the pistil. The self-incompatibility response is under the genetic control of a single multi-allelic locus, the S (Self-incompatibility) locus. There are two major classes of self-incompatibility systems. Gametophytic self-incompatibility has been well characterized in the Solanaceae and in the Papaveraceae, while sporophytic self-incompatibility has been well characterized in the Brassicaceae. In this review article, we present recent advances in understanding the signals mediating pollen recognition and pollen tube growth, in both compatible and incompatible interactions.
pistil and then rehydrate by acquiring water from the style exudate or from the stigma papillar cells, in order to resume active metabolism. Hydrated pollen grains germinate by extruding a pollen tube. The pollen tube grows through the transmitting tissue of the style to the ovary. In the ovary, the pollen tube delivers the two male gametes into the embryo sac where the double fertilization takes place. The numerous cellular interactions that take place between the pollen Žor pollen tube. and the different tissues of the pistil all along the fertilization process, are likely to involve complex cross-talk between diverse molecules. These interactions can be as complex as protein]protein interactions, exemplified by ligand]protein kinase signaling, or can involve molecules as simple as water, calcium, lipids and sugars. Several recent reviews of pollenrpistil interactions and self-incompatibility are available.1 ] 7 In this article we summarize recent studies on selected aspects of pollenrpistil recognition and signal transduction.
Key words: pollen]pistil interactions r receptor-like kinases r self-incompatibility r S-locus r SRNases Q1999 Academic Press
Compatible pollinations Introduction Pollen hydration involves the uptake of water. Because pollen from Arabidopsis cer mutants is defective in hydration, it was suggested that lipids act as signals for water uptake.3,6 More recently, studies with stigmaless Nicotiana alata plants were used to show that certain classes of lipids Ž cis-unsaturated triacylglycerides. could replace the requirement of stigma exudate for pollen hydration.8 Membrane-associated water channels, or aquaporins, may also play a role in water uptake.9 In self-incompatible Brassica plants with a recessive mutation Ž mod . in an aquaporin, pollen that would not normally hydrate and germinate could do so when placed on mod stigmas.10 Lastly, N. alata pollen that is germinated in medium with a defined oilrwater interface will grow towards the water, suggesting that water concentration itself is
IN FLOWERING PLANTS, the success of sexual reproduction relies on a series of interactions between the pollen grain Žthe male partner. and the different tissues of the pistil Žthe female partner.. At the time of dispersal, mature pollen grains of most angiosperms are highly dehydrated structures. During compatible pollination, pollen grains adhere to the U
From the Plant Gene Expression Center, USDA r ARS-UCBerkeley, 800 Buchanan St., Albany, CA 94710, USA and †Research unit for Reproduction et Developpement des Plantes, UMR ´ 9938 CNRS-INRA-ENSL, Ecole Normale Superieure de Lyon, 46 ´ allee ´´ d’Italie, 69364 Lyon Cedex 07, France. Q1999 Academic Press 1084-9521r 99r 020139q 09 $30.00r 0
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a cue for pollen growth. Pollen tubes showed no directional growth if the oilrwater interface was emulsified.11 It is not yet clear which, if any, components of the extracellular matrix of the style play an important role in pollen tube growth. For example, contradictory results were obtained from the study of an abundant glycoprotein, termed TTS Žfor transmitting tissue-specific .. Although it was reported that the N. tabacum TTS was a growth stimulant and pollen tube attractant,12 studies performed with the probable homolog Ž97% homology. of TTS from N. alata13 showed only an inhibitory role for TTS. The importance of calcium in pollen tube growth has been well-characterized.14 Although it is still largely unknown if or how the asymmetric gradient is initiated by signals from the style, recent experiments are starting to dissect the downstream events. For example, the sub-cellular location of a Ca2q-dependent protein kinase activity is correlated with the direction of pollen tube growth.15 Furthermore, release of caged inositol 1,4,5-triphosphate in sub-apical regions of the growing pollen tube resulted in pollen tube reorientation and an increase in Ca2q levels in that region.16 Because calmodulin is rather uniformly distributed in the growing point of pollen tubes, it is presumed to be involved in pollen tube growth rather than in orienting the direction of pollen tube growth.17 Lastly, there is some evidence that other classes of pollen receptor-like kinase genes can transduce signals from the pistil tissue. The tomato receptor-like kinases, LePRK1 and LePRK2, have extracellular domains composed of 5]6 leucine-rich repeats ŽLRRs.. LRRs are believed to mediate protein]protein interactions. LePRK1 and LePRK2 are localized to the pollen tube plasma membrane and at least LePRK2 is partially dephosphorylated when pollen membranes are incubated in the presence of style extracts.18 The ligandŽs. for these kinases is unknown, as are details of the downstream signal transduction pathway.
are anchored in the soil by their roots and therefore cannot actively search out their sexual partners. One might think that the high level of inbreeding would be associated with a loss of genetic variability and that self-pollination might therefore have been deleterious for the evolution of angiosperms. However, angiosperms are the most successful groups of terrestrial flora both in terms of the number of species Žapprox. 300 000 described so far. and by their diversity of forms and ecological niches. This success is thought to depend partly on the acquisition by the angiosperms of mechanisms which strongly limit or even prevent self-fertilization. The most sophisticated and widespread of these mechanisms is self-incompatibility ŽSI., which leads to the rejection of self-pollen by the pistil. Several SI systems have been selected during the course of evolution and SI is thought to occur in slightly more than 50% of angiosperm species.19 In most cases, self-incompatibility is controlled by a single multi-allelic locus, the S-locus Ž S for Self-incompatibility.. Classical genetics has established that there are two types of SI systems, classified according to pollen behavior with respect to the pistil tissues. When the incompatibility phenotype of the pollen is determined by its own haploid S genotype, the system is defined as a gametophytic SI system ŽGSI.. When the incompatibility phenotype of the pollen is determined by the diploid S genotype of the mother plant Žthe sporophyte., the system is defined as a sporophytic SI system ŽFigure 1.. In all cases, pollen rejection occurs when the same S allele specificity is expressed both by the pollen and the pistil tissue Žstigma, style or ovary.. In both systems, pollen]pistil recognition has been generally considered to involve signal and receptor interactions and recent molecular data supports this assumption. Strikingly, self-compatible species can also exist within a family in which self-incompatibility is usually the rule. For example, in the Brassicaceae, Arabidopsis thaliana is fully self-compatible. Because of the complexity of self-incompatibility, it was hypothesized that self-compatible species evolved from self-incompatible species, presumably due to mutations or deletions in the S-locus or in genes controlling the expression of S-locus genes.19 This hypothesis has recently found support from a comparative mapping study of the S-locus region of the self-incompatible species Brassica campestris with its homolog in A. thaliana.20 Sequences similar to the Brassica S locus genes were not detected within the corresponding chromosomal region in A. thaliana, or in other re-
Pollenr r pistil signaling and selfincompatibility Angiosperms display several traits which should have promoted or imposed self-pollination, and thus selffertilization. For example, they are largely hermaphroditic Žapprox. 96% of species carry male and female organs on the same individual, and among these species, 75% have hermaphroditic flowers. and 140
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cluding the Solanaceae, the Liliaceae, the Rosaceae, the Papaveraceae and the Poaceae. Rejection of a self-pollen grain generally leads to the arrest of pollen tube growth in the transmitting tissue of the style. However, in poppy and grasses, pollen tube growth is stopped earlier at the stigmatic surface. Much of the molecular work on GSI has been carried out in two Solanaceae species Ž N. alata and Petunia inflata. and in poppy Ž Papaver rhoeas.. The Solanaceae Although the existence of molecules associated with the incompatibility response was demonstrated as early as in 1960 in Petunia hybrida,21 it was only in the mid eighties that the first S gene of GSI systems was cloned, from N. alata.22 This S gene is specifically expressed in the pistil and encodes a glycoprotein which is abundantly produced Žseveral micrograms per pistil. and is secreted in the extracellular space, principally in the stigma and transmitting tissue of the style. Analysis of the amino acid sequences of different S-allele glycoproteins in N. alata revealed the existence of significant homology between the S glycoproteins and the extracellular ribonucleases ŽRNases. T2 of Aspergillus oryzae and Rh of Rhizopus niveus.23 The S glycoproteins indeed have RNase activity and thus are now referred to as S RNases. Interestingly, stylar S RNases are not restricted to the SI system of the Solanaceae. S RNases and S locus ribonuclease genes have been characterized in the Rosaceae 24 ] 26 Že.g. Prunus, Pyrus, and Malus . and in the Scrophulariaceae27 Že.g. Antirrhinum.. A common feature of S RNases is that their primary structure is composed of alternating variable and conserved regions, with the presence of one Žin the Rosaceae. 26 or two Žin the Scrophulariaceae and Solanaceae. 27,28 hypervariable ŽHV. regions. S RNases of different species or of the same species but from different S alleles are highly polymorphic. For example, in the Solanaceae, amino acid identity between different S glycoproteins ranges from 38 to 98%. This level of polymorphism is unusual for most genes; such sequence diversity is expected for products of genes that are involved in self-recognition mechanisms to promote outcrossing.29 A new S allele arising in a population is favored, because pollen expressing this new S allele will have high probability to encounter a stigma bearing a different S allele. Both the cosegregation of stylar S glycoproteins with S alleles and their polymorphic sequences suggested involvement of S RNases in SI; only transgenic
Figure 1. Self-incompatibility systems. Ža. Gametophytic self-incompatibility ŽGSI.: pollen behavior is determined by the haploid genome of the pollen grain. Pollen grains carrying the same S allele specificity Ž S1 in our example. as one expressed by the diploid pistil Ž S1S2 . are rejected. Incompatible pollen tubes are generally arrested in the style. Žb. Sporophytic self-incompatibility ŽSSI.: pollen behavior is determined by the diploid genome of the pollenproducing plant Ž S1S2 .. In our example, S3 pollen grains from the S1 S3 plant are rejected because the S1 allele is expressed both in the pollen-producing plant and the S1S2 pistil. The SI response occurs only if S1 is dominant to, or co-dominant with, S3 in the pollen while S1 is dominant to, or co-dominant with, S2 in the style. Incompatible pollen grains either fail to hydrate and germinate or if they do germinate, pollen tube growth is arrested at the stigma surface.
gions of the Arabidopsis genome, despite the otherwise high degree of synteny at the submegabase scale between these two regions.
Gametophytic SI (GSI) Gametophytic systems are the most common and have been described in more than 60 families, in141
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experiments provided direct evidence, in both N. alata21,30 and P. inflata.28,31 In Petunia, a loss-of-func tion approach Žbased on an anti-sense strategy. and a gain-of-function approach Žusing a sense strategy. was used to prove that S RNases are sufficient for the recognition and rejection of self-pollen by the pistil.31 Moreover, RNase activity of S glycoproteins is necessary for self-pollen rejection, because transgenic plants expressing a mutated S3 protein lacking RNase activity are not able to reject S3 pollen.32 The origin of S allele specificity was presumed to reside either in the carbohydrate side chains of S RNases or in the hypervariable regions of the polypeptide sequences. The role of the carbohydrate moiety was investigated by expressing a mutated non-glycosylated S3 protein in transgenic plants. These transgenic plants produced a normal level of the non-glycosylated S3 RNase but were still capable of rejecting S3 pollen grains. Thus, the glycan chains do not confer S allele specificity and are not necessary for S RNase function in recognition and rejection of the self-pollen grain.33 To examine the role of HV regions in S allele specificity, chimeric S RNases composed of the major part of the S3 protein but containing HV regions from the S1 protein were constructed. The resulting transgenic plants lost the ability to reject S3 pollen but did not acquire the ability to reject S1 pollen.28 This result suggests that HV regions of S proteins are necessary but not sufficient for the establishment of S allele specificity. However, based on a similar approach, contradictory data were obtained 34 in another Solanaceous species, Solanum chacoense. In this species, S11 and S13 RNases only differ by four amino acid residues, which are located in the HV region. Plants transformed with chimeric gene constructs in which the S11 RNase had those four amino acids changed to those of the S13 RNase exhibited an S13 phenotype.34 Thus in S. chacoense it appears that the HV region alone can determine allelic specificity, although whether these results are generalizable has been questioned.35 To test how general the S. chacoense findings are, similar experiments could be attempted with other nearly identical alleles. For example, the Japanese pear S3 and S5 RNases are 95% identical and differ only in the HV region amino acids.26 Furthermore, structural analyses36 of additional S RNases should prove useful in targeting regions of different S RNases before attempting further allelic specificity changes in transgenic plants. A remarkable feature of all these transgenic experiments is that only the SI phenotype of the pistil was affected, while the pollen phenotype remained un-
changed. This indicates that the male component of the SI response is not the S RNase and strongly supports the assumption that pollen and pistil S molecules are encoded by two different genes located at the S locus. In spite of numerous attempts to identify the male S locus component, so far, it is unknown. In the Solanaceae, two hypotheses have been proposed to explain the molecular basis of self-pollen rejection, both of which rely on a cytotoxic effect.28 S RNases are thought to enter the pollen tube in order to hydrolyse ribosomal and messenger RNAs, thus blocking protein synthesis and therefore blocking pollen tube growth.28 The first hypothesis presumes that the pollen S product is a receptor present at the surface of the pollen tube Žin the wall or plasma membrane.. This receptor would allow the allelespecific recognition and translocation of stylar S RNases into the pollen tube. In this ‘receptor’ model, only the stylar S RNase of the same allele as that of the pollen enters the pollen tube and inhibits its growth. The second hypothesis presumes that the pollen S product is a cytosolic RNase inhibitor which inactivates all S RNases except the one sharing the same S allele specificity as that of the pollen. In this ‘inhibitor’ model, S RNases present in the style enter the pollen tube independently of their S allele specificity, but only the S RNase recognized as ‘self’ inside the pollen tube will not be inhibited and thus can degrade pollen RNA. No direct evidence that may support either of the two models has been reported. In fact, two recent papers challenge the cytotoxic model. Despite the common perception that incompatible pollen tubes irretrievably arrest in the upper one-third of the style, incompatible pollen tubes of N. alata can continue to grow, and can resume a rapid rate of growth if the incompatible style they are growing through is grafted to a compatible style.37 Furthermore, both ribosomes and polysomes continue to be visible after self- incom patible pollinations,38 suggesting that the target of the S RNase must be highly specific and that pollen tube arrest is not due to a general degradation of pollen RNA Žribosomal or mRNA.. The Papaveraceae In Papaver rhoeas, stigma S proteins are also secreted glycoproteins, but they do not have RNase activity or any other known catalytic activity.39 However, using an in vitro pollen germination assay, stigma S proteins have been shown to inhibit pollen tube growth 142
Signaling in pollen]pistil interactions
in an S allele-specific manner.39 Moreover, S proteins are also capable of interacting with a pollen plasma membrane protein called SBP Ž S Binding Protein.,40 but with no S-allele specificity. In order to incorporate this interaction into the SI response, a model proposes that pollen SBP and stigma S proteins must interact in an S allele-specific manner with an as yet unknown pollen component encoded by the S locus. In an attempt to define the regions of the S protein that mediate interaction with pollen components, mutant derivatives were constructed and the expressed proteins assayed for pollen inhibition using the in vitro pollen germination assay. Mutation of the only hypervariable amino acid in a hydrophilic region Žloop 6. of the stigmatic S1 protein resulted in a complete loss of S1 pollen inhibition.41 This result suggests that loop 6 participates in pollen recognition. However, changing the hypervariable amino acid in S1 to that present in either of two different S alleles Ž S3 or S8 . was not able to confer a new allelic specificity on the mutant protein. Thus these results in poppy are analogous to those in Petunia and N. alata28,30 and in contrast to those in S. chacoense.34 In the poppy, there is considerable information42 available on the signal transduction pathway that occurs in pollen after the initial recognition response. When growing pollen tubes are challenged with incompatible S proteins, a transient calcium concentration increase occurs within seconds, followed by a calcium-dependent phosphorylation of a 26-kDa pollen protein, and subsequently by a calcium-independent phosphorylation of a 68-kDa pollen protein. Although it is not yet understood how these phosphorylations result in pollen tube arrest, it is interesting to note that incubation of poppy pollen with profilin can also influence the phosphorylation state of the 26-kDa pollen protein.43 Profilin is known to modulate the actin cytoskeleton but may also play a role in modulating pollen tube growth via interactions with protein kinases or their substrates.
rapid arrest of its growth at the stigma surface. Molecular analysis of several S genotypes has revealed that the S- locus has a com plex organization.44 ] 47 The S-locus is composed of several physically linked genes and in this respect resembles the major histocompatibility complex in mammals. This analogy suggested the term ‘haplotypes’48 to designate different alleles of the S-locus. Two S-locus genes have been studied in detail. The first gene linked to the S-locus, S-Locus Glycoprotein, SLG, encodes a glycoprotein secreted into the cell wall of stigmatic papillae.49 The second gene, S-locus Receptor Kinase, SRK, encodes a receptor-like kinase.50 On the basis of cDNA sequence analysis, SRK is predicted to be a plasma membrane protein consisting of three domains: an extracellular domain that shares homology with SLG, a single transmembrane region and a cytoplasmic domain with consensus sequences of serinerthreonine kinases. The cytoplasmic domain of SRK has been overexpressed in Escherichia coli and shown to have serinerthreonine kinase activity.51 It is presumed that the extracellular domain acts as a receptor, in analogy with membrane receptor kinases characterized in animal systems that recognize and transduce peptide signals controlling processes, such as development, cell growth, differentiation and death.52 What about the pollen component of SI? While SLG and SRK are essentially expressed in stigmas and, at a low level, in anthers, a third gene, S-Locus Anther, SLA, mapped to the S locus and is specifically expressed in anthers.53 It was first thought that SLA could encode the long-sought pollen determinant of SI. However, fully self-incompatible B. oleracea lines were shown to carry a non-functional SLA gene.54 Two other anther-expressed candidates that mapped to the S locus of a different haplotype45 were also eliminated as potential pollen determinants of SI. One candidate was similarly expressed in both self-incompatible and self-compatible lines, while the other showed no sequence variation between different haplotypes. In a complementary approach to identify the pollen ligand, Brassica pollen coating was fractionated and interspersed between pollen and pistil, of the same and different haplotypes. A fraction that was enriched for PCPs Žsmall, cysteine-rich proteins similar to plant defensins. had the ability to alter pollen hydration and germination in an allele-specific manner.55 However, in other work, the gene encoding a particular pollen coat protein of this class, PCP-Al, was unlinked to the S locus and furthermore was shown to be gametophytically expressed.56 Fur-
Sporophytic SI (SSI) Although sporophytic self-incompatibility is less widespread than gametophytic self-incompatibility, it is present in plant families which are more highly evolved, such as the Brassicaceae, the Asteraceae, and the Caryophyllaceae. Most studies concerning molecular aspects of sporophytic SI have concentrated on species in the Brassicaceae. Self-pollen rejection results in a blockage of pollen tube emergence or a 143
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thermore, although purified PCP-Al interacts with SLG, the interaction shows no allelic specificity. The pollen component of SI is still unknown and will remain so, until a pollen protein interacting with stigma S molecules and exhibiting S allelic specificity is shown to be encoded by a gene linked to the S locus. What is the recognition determinant in the pistil? For a given haplotype, the SLG and extracellular domain of SRK, eSRK, share between 83 and 90% identity at the amino acid level.44,57 This sequence conservation strongly contrasts with the high degree of divergence Žup to 30%. between SLGs Žor SRKs. from different haplotypes. Perhaps selection pressure acts to maintain a high homology between SLG and SRK alleles within a given haplotype because both SLG and SRK are required for recognition of the pollen determinant. To complicate matters, in the case of at least one SLG gene, SLG2 , alternative transcripts can encode two SLG proteins, a secreted and a membrane-anchored form.58 The S2 haplotype is a class II, or so-called pollen recessive haplotype. However, three other pollen recessive haplotypes from B. rapa do not show this alternative transcript pattern.59 Because of the abundance of SLGs in stigma papillae, molecular control of the SI response was initially thought to depend mainly on SLG.49 Indeed, the level of SLG mRNA reaches its maximum one day prior to anthesis, precisely at the stage when the flowers become self-incompatible. The role played by SLG in the SI response has been recently questioned, because there are self-incompatible plants Žof the S2 haplotype. that express a very low level of SLG. Furthermore, naturally self-compatible variants of the S15 and Sc haplotypes express a high level of SLG.60,61 A recent analysis of the S15 haplotype in B. oleracea has revealed the existence of a new SLG gene in this haplotype ŽCabrillac et al., submitted paper.. We have found that the S locus in the S15 haplotype contains at least three members of the S gene family, all of which are expressed in the stigma: SRK and two different SLG genes, designated SLGA and SLGB. Both SLGA and SLGB are interrupted by a single intron but while SLGA encodes both soluble and membrane-anchored forms of SLG, SLGB only encodes soluble SLG proteins. Interestingly, we found that the two other pollen-recessive S haplotypes, S2 and S5 , carry only one or the other of the SLG genes, indicating either that these genes are redundant in the S15 haplotype or that they are not required for the self-incompatibility response. These observations
suggest that SRK, rather than SLG, plays a key role in the SI reaction. This hypothesis gained more support from the analysis of self-compatible variants of rapeseed and cabbage.62,63 These plants express normal levels of SLGs, but they carry mutations that lead either to the absence of SRK transcripts or to the production of truncated transcripts. These data indicate that the expression of a functional SRK is necessary for the SI response. In the S3 haplotype, SRK is a 120-kDa glycoprotein that is specifically expressed in the stigma and anchored in the plasma membrane.57,65 SRK 3 has also been shown to encode a truncated, soluble form of SRK 3 corresponding to its extracellular domain: the eSRK 3 protein.65 Alternative splicing of SRK transcripts may generate eSRK 3 . This result is particularly interesting because of the identification of soluble truncated forms of receptor kinases in some animal signal transduction pathways.66,67 In the case of the animal receptor kinases, the soluble forms inhibit signal transduction by limiting binding of the ligand to the transmembrane receptor. A similar role may be played by eSRK in the regulation of the SI response in Brassica. The expression of recombinant SRK proteins in a membranous environment, using the insect cellrbaculovirus system, has allowed the analysis of how SRK functions at a molecular level ŽGiranton et al., in preparation.. This system has already been used for the successful expression of animal kinase receptors.68 ] 71 The experimental determination of the topology of recombinant SRK in microsomal membranes supports a model in which the S domain of SRK is directed towards the periplasm at the surface of stigma cells, and thus is probably involved in the recognition of a ligand.50,57,64 Kinase assays performed on microsomes showed that recombinant SRK autophosphorylated on serine and threonine residues but not on tyrosine residues. Autophosphorylation was constitutive since it did not require addition of pollen or stigma extracts to the phosphorylation buffer. The co-expression of two different SRK constructs, which exhibit either a functional or a kinase defective activity and could be recovered independently, revealed that SRK phosphorylation occurred in trans, suggesting the existence of constitutive homo-oligomers of membrane-anchored recombinant SRK. Furthermore, cross-linking experiments indicated that SRK, but neither eSRK nor SLG, exists constitutively as an oligomeric complex in planta. Our analyses of the enzymatic properties of recombinant SRK expressed in insect cells, and the investigation of SRK’s oligomeric status in planta therefore 144
Signaling in pollen]pistil interactions
6. Perspective Although analyses of pollen]pistil interactions and SI mechanisms have progressed, there is still a great deal to be learned. Unfortunately, we still do not know the identity of both signaling partners Žmale and female. for any of the interactions discussed in this review. In the gametophytic self-incompatibility arena, the exciting results suggesting that allelic specificity can be changed Žin S. chacoense. needs to be duplicated in other plants and with other S alleles. Because transgenic plant analyses have been unsuccessful in definitively proving which components are involved in sporophytic self-incompatibility, it will be crucial to try other, perhaps more biochemical, approaches. It is clear that detailed studies of several haplotypes will help elucidate those features that are common and therefore more likely to be critical. Figure 2. Model of the pollen]stigma interaction, which leads to the SI response in Brassicaceae. A pollen ligand encoded by the S-locus Žas yet unknown. is thought to interact with the membrane receptor SRK. SRK, probably existing as a dimer, may be associated with other molecules either encoded by the S-locus ŽSLG, eSRK. or not Žfor example a putative co-receptor, designated CoR.. By analogy with animal kinase receptors, binding of the pollen ligand may modify the phosphorylation level, the conformation, andror the oligomerization status of SRK. This step, for which the molecular details are still unknown, may allow the recruitment of cytoplasmic targets leading to SI response.
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suggest that signal transduction during SI response might be mediated by modification of a preexisting SRK oligomeric complex rather than by ligand-dependent dimerization of SRK molecules. Similar conclusions have been drawn in animals from the study of TGF-b signal transduction via its serinerthreonine kinase receptors. These receptors are able to homooligomerize even in the absence of ligand and the type II receptors exhibit constitutive serinerthreonine kinase activity.72,73 These data highlight the analogies existing between animal and plant signal transduction pathways. Moreover, they allow us to reconsider the classical model in which the SI response is initiated by ligand-dependent dimerization of SRK. An alternative model ŽFigure 2. proposes that SRK can exist as a dimer even in non-pollinated stigma. The interaction of SRK with its ligand could either allow the recruitment of cytoplasmic targets following a conformational change of the SRK kinase domain, or favor the recruitment of a yet unknown co-receptor also involved in signal transduction ŽFigure 2.. 145
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