Download MOLECULAR RECOGNITION AND RESPONSE IN POLLEN AND

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Annu. Rev. Cell Dev. Biol. 2000. 16:333–64
c 2000 by Annual Reviews. All rights reserved
Copyright MOLECULAR RECOGNITION AND RESPONSE
IN POLLEN AND PISTIL INTERACTIONS
Andrew G. McCubbin and Teh-hui Kao
Department of Biochemistry and Molecular Biology, 403 Althouse Laboratory,
The Pennsylvania State University, University Park, Pennsylvania 16802-4500;
e-mail: [email protected]; [email protected]
Key Words self/non-self discrimination, signal transduction, S-locus,
receptor kinase, RNases
■ Abstract Many bisexual flowering plants possess a reproductive strategy called
self-incompatibility (SI) that enables the female tissue (the pistil) to reject self but
accept non-self pollen for fertilization. Three different SI mechanisms are discussed,
each controlled by two separate, highly polymorphic genes at the S-locus. For the
Solanaceae and Papaveraceae types, the genes controlling female function in SI,
the S-RNase gene and the S-gene, respectively, have been identified. For the Brassicaceae type, the gene controlling male function, SCR /SP11, and the gene controlling
female function, SRK, have been identified. The S-RNase based mechanism involves
degradation of RNA of self-pollen tubes; the S-protein based mechanism involves a signal transduction cascade in pollen, including a transient rise in [Ca2+]i and subsequent
protein phosphorylation/dephosphorylation; and the SRK (a receptor kinase) based
mechanism involves interaction of a pollen ligand, SCR /SP11, with SRK, followed
by a signal transduction cascade in the stigmatic surface cell.
CONTENTS
INTRODUCTION: Self-Incompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SOLANACEAE TYPE SELF-INCOMPATIBILITY . . . . . . . . . . . . . . . . . . . . . . . .
The S-RNase Gene Encodes Pistil S-Haplotype Specificity . . . . . . . . . . . . . . . . .
Structure-Function Relationships of S-RNases . . . . . . . . . . . . . . . . . . . . . . . . . .
Approaches to Identifying the Gene that Controls Pollen S-Haplotype Specificity .
The Generation of New S-Haplotype Specificities . . . . . . . . . . . . . . . . . . . . . . . .
Models for S-RNase Mediated Self-Incompatibility Response . . . . . . . . . . . . . . .
Modifier Loci that Modulate the SI Response . . . . . . . . . . . . . . . . . . . . . . . . . . .
PAPAVERACEAE TYPE SELF-INCOMPATIBILITY . . . . . . . . . . . . . . . . . . . . . .
The S-Gene Controls Stigma Function in Self-Incompatibility . . . . . . . . . . . . . . .
Structural Features of S-Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Identification of Amino Acid Residues of S-Proteins Involved in Recognition . . . .
Biochemical Responses in Pollen Following Self-Recognition . . . . . . . . . . . . . . .
Protein Kinase Activity Implicated in the SI Response . . . . . . . . . . . . . . . . . . . .
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S-Protein-Binding Proteins in Pollen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Model for Self-Incompatibility Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
BRASSICACEAE TYPE SELF-INCOMPATIBILITY . . . . . . . . . . . . . . . . . . . . . .
The SLG Gene–a Polymorphic Stigma-Expressed Gene at the S-Locus . . . . . . . . .
The SRK Gene Encodes Stigma S-Haplotype Specificity . . . . . . . . . . . . . . . . . . .
The SCR/SP11 Gene Encodes Pollen S-Haplotype Specificity . . . . . . . . . . . . . . .
Structural Organization of the S-Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Model for Self-Incompatibility Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONCLUSIONS AND FUTURE PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . .
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INTRODUCTION: Self-Incompatibility
Many flowering plant species that produce bisexual flowers have evolved mechanisms to circumvent the tendency toward self-fertilization, which is created by the
close proximity of the male (anther) and female (pistil) reproductive organs. These
mechanisms are collectively termed self-incompatibility (SI). SI enables the pistil
to distinguish between self (genetically related) and non-self (genetically unrelated) pollen of the same species. Depending on the type of mechanism, rejection
of self pollen (tubes) by the pistil occurs either at the stigmatic surface or in the
style. Non-self pollen is accepted by the pistil and its tube grows down through
the style to reach the ovary where fertilization takes place. Thus SI prevents selffertilization and consequent inbreeding and allows outcrosses to generate genetic
diversity within a species.
Classic genetic studies carried out in the early 20th century revealed two major
types of homomorphic SI systems, gametophytic and sporophytic (de Nettancourt
1977). The term homomorphic indicates that all individuals of a self-incompatible
species produce flowers of the same morphological character. The self/non-self
discrimination between pollen and pistil is determined by one or more highly polymorphic loci. This article focuses on two of the families that possess gametophytic
self-incompatibility (GSI), Solanaceae and Papaveraceae, and one of the families
that possess sporophytic self-incompatibility (SSI), Brassicaceae. Although SI in
each of these families is controlled by a single genetic locus, the S-locus, the mechanisms employed are completely different, at least at the level of recognition of self
and non-self pollen. It should be noted, however, that two other families that possess GSI, the Rosaceae and Scrophulariaceae, most likely employ the same mechanism as the Solanaceae, based on the sequence similarity of the proteins they use to
control pistil function in SI (Sassa et al 1996, Xue et al 1996, Ishimizu et al 1998).
The S-locus of both the Brassicaceae and Solanaceae is now known to be a
multigene complex, and the S-locus of the Papaveraceae is likely to be such a
complex. Thus the term haplotype is used to denote variants of the locus, and the
term allele is used to denote variants of a given polymorphic gene at the S-locus.
For self-incompatible species in the Solanaceae and Papaveraceae, the pistil distinguishes between self and non-self pollen based on whether the S-haplotype of
the haploid pollen matches either of the two S-haplotypes of the diploid pistil.
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That is, the SI phenotype of pollen (gametophyte) is determined by its own
S-genotype. When haplotypes match, pollen is recognized by the pistil as self and
rejected, whereas if haplotypes differ, pollen is accepted for fertilization. Thus
crosses between two plants are compatible as long as their S-genotypes differ in
one of the two S-haplotypes (Figure 1). In self-incompatible species of the Brassicaceae, the SI phenotype of pollen is determined by the S-genotype of its diploid
parent (sporophyte). In the simplest case, the pollen is recognized by the pistil
as self if either of the two S-haplotypes carried by its parent matches one of the
two S-haplotypes carried by the pistil. Thus crosses between two plants are possible only if their S-genotypes do not share any haplotype in common (Figure 1).
However, often complex relationships exist between the two S-haplotypes carried
by the pollen and pistil, such that one could be dominant over or recessive to the
other, or they could interact to result in mutual weakening (Thompson & Taylor
1966).
The following discussion centers on recent efforts to understand the molecular
basis of the self/non-self recognition between pollen and pistil, and the ensuing
Figure 1 Illustration of the genetic basis of gametophytic and sporophytic selfincompatibility. For the gametophytic type, the SI phenotype of pollen is determined
by the S-haplotype of its haploid genome, thus each pollen grain carries the product of
one S-haplotype. For the sporophytic type, the SI phenotype of pollen is determined by
the S-genotype of its diploid parent, thus each pollen grain carries the products of two
S-haplotypes. In both types, matching of one S-haplotype between pollen and pistil results
in rejection of pollen. The scenario depicted for sporophytic SI assumes that S1-haplotype
is co-dominant with, or dominant over, S3-haplotype.
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biochemical events that result in the inhibition of germination or tube growth of
self pollen.
SOLANACEAE TYPE SELF-INCOMPATIBILITY
The genera in the Solanaceae (nightshade) family include Nicotiana, Petunia,
Solanum, and Lycopersicon. The great majority of the wild species in this family
are self-incompatible. For example, of the 19 known taxa of the Petunia genus,
15 are self-incompatible and 4 are self-compatible (Tsukamoto et al 1998). However, most commercial cultivars are self-compatible because the SI trait was
selected out at early stages of breeding in order to produce inbred lines homozygous for desirable traits. For example, tomato (Lycopersicon esculentum),
tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and garden petunia
(Petunia hybrida) are all self-compatible. Much of the molecular information
about this type of SI has been obtained from L. peruvianum, N. alata, P. inflata, and
S. chacoense.
The S-RNase Gene Encodes Pistil S-Haplotype Specificity
The approach used to identify the gene that controls S-haplotype specificity of the
pistil was based on the prediction that proteins involved in self/non-self recognition
must exhibit a high degree of allelic sequence diversity. This prediction was borne
out when pistil proteins that co-segregate with S-haplotypes were identified based
on their differences in molecular mass and/or isoelectric point. For example, pistils
of S1S2 genotype of P. inflata produce a 24-kDa and a 25-kDa protein, the former
co-segregating with the S1-haplotype and the latter with the S2-haplotype (Ai et al
1990). These proteins were initially called S-proteins but have been renamed
S-RNases since the discovery that they have RNase activity (see below).
The S-RNase gene, in addition to being linked to the S-locus, also exhibits a
number of characteristics expected of the gene that determines S-haplotype specificity of the pistil. First, it is exclusively expressed in the pistil, with the protein
localized mostly in the upper segment of the style where inhibition of self pollen
tubes occurs. Second, it is expressed at a very low level in the pistils of immature
buds, which are unable to reject self pollen, and the increase in expression during
subsequent flower development coincides with the acquisition of SI by the pistil. (This expression pattern makes it possible in some self-incompatible species
to obtain plants homozygous for a particular S-haplotype by selfing at immature
bud stages.) Third, the sequences of its allelic products, S-RNases, are highly
divergent, with amino acid sequence identity ranging from 38 to 98%.
The function of the S-RNase gene in SI has been directly confirmed by gainof-function and loss-of-function experiments. These experiments showed that the
S-haplotype specificity of the pistils of transgenic plants could be altered by the
expression of a sense or antisense S-RNase transgene (Lee et al 1994, Murfett
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et al 1994). For example, in P. inflata, when the S3-RNase gene was introduced
into plants of S1S2 genotype, those transgenic plants that produced S3-RNase in
the pistil gained the ability to reject S3 pollen. Conversely, when an antisense
S3-RNase gene was introduced into plants of S2S3 genotype, transgenic plants in
which the production of S3-RNase in the pistil was suppressed lost the ability to
reject S3 pollen. These experiments thus demonstrated that the S-RNase gene is
solely responsible for the S-haplotype specificity of the pistil.
Structure-Function Relationships of S-RNases
Despite the sequence diversity among S-RNases, five regions of sequence conservation, C1 to C5, have been identified (Ioerger et al 1991, Tsai et al 1992). C2
and C3 share a high degree of sequence similarity with the corresponding regions
of two fungal RNases, RNase T2 and RNase Rh. It is this similarity that led to
the discovery that S-proteins are RNases (McClure et al 1989). The finding that
RNases are employed by the pistil to reject self pollen raised the possibility that
the RNase activity is responsible for growth inhibition of self pollen tubes. To
examine this possibility in P. inflata, a mutant S3-RNase gene, with the codon
for one of the two catalytic histidines replaced with an asparagine codon, was
introduced into S1S2 plants, and the transgenic plants were analyzed for their
ability to reject S3 pollen (Huang et al 1994). The results showed that production of this mutant S3-RNase did not confer the ability to reject S3 pollen on the
transgenic plants. Thus the RNase activity is an integral part of the function of
S-RNases.
S-RNases are glycoproteins with one or more N-linked glycan chains. The
structure of the glycan chains of some S-RNases has been determined (Woodward
et al 1992, Oxley et al 1998, Parry et al 1998), and the similarities in the structure
for different S-RNases suggest that the glycan chains are unlikely to encode the
S-haplotype specificity. Indeed, when an engineered S3-RNase gene of P. inflata,
with the asparagine codon for the only N-glycosylation site of the protein replaced
with an aspartic codon, was introduced into P. inflata plants of S1S2 genotype, this
non-glycosylated S3-RNase functioned as well as wild-type S3-RNase in rejecting
S3 pollen (Karunanandaa et al 1994). Thus the S-haplotype specificity determinant
of S-RNases resides in the protein backbone and not in the glycan side chains.
Sequence comparison of S-RNases has revealed two hypervariable regions,
termed HVa and HVb, which are also the most hydrophilic regions of S-RNases
(Ioerger et al 1991, Tsai et al 1992). These two characteristics led to the hypothesis
that HVa and HVb are the prime candidates for the determinant of S-haplotype
specificity. Consistent with this hypothesis, HVa and HVb form a continuous surface on one side of S-RNases in a three-dimensional structure model ( Parry et al
1998) based on the coordinates of RNase Rh (Kurihara et al 1992), which share
similar predicted secondary structures with S-RNases. To examine the role of HVa,
HVb, and other regions of S-RNases in S-haplotype specificity, chimeric S-RNase
genes were constructed and introduced into transgenic plants for analysis of the
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S-haplotype specificity displayed by each of these hybrid S-RNases. In constructing these chimeric genes, one allele of the S-RNase gene was used as the backbone,
and the sequence for a region of another allele was swapped into the corresponding region of the backbone allele. When pairs of S-RNases with a high degree
of sequence diversity were used for domain swapping, every region swapped
(including HVa and HVb) led to the loss of the S-haplotype specificity of the
allele used as the backbone. Moreover, no gain of the new S-haplotype specificity
of the allele source for the newly introduced region was found, despite the fact
that all these hybrid S-RNases exhibited normal levels of RNase activity (Kao &
McCubbin 1996, Zurek et al 1997). These results also suggest that the RNase
activity of S-RNases is necessary but not sufficient for their function in SI.
Two S-RNases of S. chacoense, S11-RNase and S13-RNase, which are among the
most similar pairs of S-RNases characterized so far (Saba-El-Leil et al 1994), were
also used for construction of chimeric S-RNase genes (Matton et al 1997). These
two S-RNases differ only in 10 amino acids, 3 of which are located in HVa and 1
in HVb. When the amino acids of HVa and HVb of S11-RNase were changed to
those of S13-RNase, transgenic plants that produced this hybrid S-RNase rejected
S13 pollen, but not S11 pollen. These results appear to suggest that HVa and HVb
together are sufficient for S-haplotype specificity. However, since any domain
swapping experiment can only address the role of those amino acids exchanged
between the two proteins under study, it remains possible that amino acids outside
HVa and HVb and conserved between S11-RNase and S13-RNase are also involved
in S-haplotype specificity (Verica et al 1998). Nonetheless, it is clear from all
domain swapping experiments that the HVa and HVb regions play a key role in
S-haplotype specificity.
Approaches to Identifying the Gene that Controls Pollen
S-Haplotype Specificity
Several lines of evidence have clearly suggested that a gene other than the
S-RNase gene controls the pollen function in SI. First, some self-compatible mutations mapped to the S-locus affect only pollen function (pollen-part mutations),
whereas others affect only pistil function (pistil-part mutations) (de Nettancourt
1977). Second, when a new allele of the S-RNase gene was expressed in pollen
of transgenic plants, no change in the SI phenotype of the pollen was observed
(Dodds et al 1999). Third, when the antisense and sense S3-RNase genes driven
by the promoter of the S3-RNase gene were introduced into transgenic plants
(as described above), the SI phenotype of the pistil but not pollen was affected
(Lee et al 1994). Fourth, some pollen part mutants result from their pollen carrying two different S-haplotypes, and analysis of such mutants has shown that some
carry a centric fragment containing part of the S-locus without the S-RNase gene
(Golz et al 1999). Fifth, a chromosomal region (larger than 30 kb) containing
the S4-RNase gene was deleted from a self-compatible cultivar of Pyrus serotina
(Japanese pear), a species of the Rosaceae that also employs S-RNases in SI, and
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this deletion affected the pistil function but not pollen function (Sassa et al 1997;
H Sassa, personal communication).
The pollen S-gene is expected to exhibit the following characteristics. First, it
is genetically very tightly linked to the S-RNase gene, as recombination between
the genes controlling pollen and pistil functions has never been observed and
would inevitably result in the breakdown of SI in the progeny (de Nettancourt
1977). Second, it shows a high degree of allelic sequence diversity, at least in the
region(s) determining S-haplotype specificity. Third, its allelic products interact
with S-RNases in an S-haplotype-specific manner. This has been demonstrated in
a dominant-negative experiment (McCubbin et al 1997). When a mutant S3-RNase
gene of P. inflata, with the codon for one of the catalytic histidines replaced with
an arginine codon, was introduced into S2S3 plants, this mutant S3-RNase (without
RNase activity) was found to render wild-type S3-RNase unable to completely
reject S3 pollen, but not to affect the ability of wild-type S2-RNase to reject S2
pollen.
RNA differential display and subtractive hybridization have been used to identify a number of pollen-expressed genes of P. inflata that show S-haplotypespecific sequence polymorphism and are tightly linked to the S-RNase gene (Dowd
et al 2000, McCubbin et al 2000). Whether any of these genes is the pollen
S-gene remains to be determined. Regardless, they can serve as molecular markers for the physical mapping of the S-locus and cloning of the DNA in this
chromosomal region–an approach that has proven to be successful in the identification of the gene encoding pollen S-haplotype specificity in Brassica (see
below).
The Generation of New S-Haplotype Specificities
Several lines of evidence suggest that SI in the Solanaceae is controlled by two
separate but tightly linked genes at the S-locus: the as yet unidentified pollen
S-gene controlling pollen function and the S-RNase gene controlling pistil function. Moreover, it has been clearly shown that SI in Brassica is controlled by two
separate genes. This situation poses a conundrum in formulating models to explain
how new S-haplotypes are generated during the course of evolution. Accumulation
of amino acid changes has been implicated in this process (Clark & Kao 1991,
Saba-El-Leil et al 1994, Matton et al 1997); however, mutation in either the pollen
or pistil gene thereby changing it to a new S-haplotype specificity would cause
breakdown of SI.
A tantalizing possible solution to this problem recently emerged from domainswapping experiments. Using the same pair of S-RNases from S. chacoense (S11and S13-RNases) described above, Matton et al (1999) showed that when three of
the four amino acids in the HVa and HVb regions that differ between these two
RNases were swapped from S13-RNase into S11-RNase, the resulting S-RNase
possessed dual S-haplotype specificity. That is, this hybrid S-RNase rejected
both S11 and S13 pollen. Based on this finding, Matton et al (1999) propose a
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Figure 2 Two models for explaining the generation of a new S-haplotype specificity
without loss of self-incompatibility: (A) a three-mutational-step model; (B) a twomutational-step model. F, pistil; M, pollen. See text for details.
three-mutational-step model for the generation of new S-haplotype specificities
(Figure 2A ). Assume that a point mutation in an S-RNase gene of Sa-haplotype
results in an S-RNase with both Sa- and Sb-haplotypes. A subsequent point mutation
in the pollen S-gene of the plant with dual pistil S-haplotypes would enable the
pollen product to recognize the new (Sb) but not the original (Sa) haplotype specificity. Finally, further mutation of the S-RNase gene with dual specificity results
in the loss of the original specificity. This process would generate a new S-haplotype specificity without the loss of SI at any intermediate stage. Conceivably,
the initial mutation could occur in the pollen S-gene to result in a protein with
dual specificity. Thus a reciprocal version of the above process would be equally
possible.
The domain swapping results described above can also be explained by an alternative model. As the sites of S-RNases involved in S11-haplotype specificity do
not appear to completely overlap with those involved in S13-haplotype specificity
(Matton et al 1999), a given S-RNase or pollen S-protein may possess one or more
latent specificity(ies) aside from the one actively involved in the SI interaction. For
example, an S-RNase of Sa-haplotype may have a latent Sb-haplotype specificity,
which would be uncovered if the pollen S-gene of Sa-haplotype mutates such that
it recognizes amino acids specifying Sb-haplotype, but not those involved in the
original haplotype specificity (Figure 2B ). Once this change in the S-haplotype
specificity of pollen has occurred, selection pressure to conserve amino acids of
Sab-RNase involved in the original (Sa) haplotype specificity would be lost, so
further mutation could occur, thereby causing the loss of the original specificity.
According to this model, a new S-haplotype specificity could be generated in
only two mutational steps, and SI would remain intact throughout the mutational
process.
It should be noted that plants whose pistils or pollen possess dual specificity in
nature have never been reported. At present, we can do little more than hypothesize
about the mechanism by which new S-haplotypes are generated. Identification of
the pollen S-gene and subsequent sequence comparisons between the S-RNase
gene and the pollen S-gene from different S-haplotypes are likely to provide key
information about this process.
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Models for S-RNase Mediated Self-Incompatibility Response
The intrinsic RNase activity of S-RNases is essential for their function in SI;
however, the precise manner in which S-RNases inhibit the growth of self pollen
tubes is not clear. One question is on what type of RNA molecules do S-RNases act?
S-RNases do not appear to have any substrate specificity in vitro (Singh et al 1991),
but it is not known whether this is the case in vivo. It is thought that rRNA genes
are not expressed in angiosperm pollen tubes (McClure et al 1990, Mascarenhas
1993) and that protein synthesis during the growth of pollen tubes is carried out by
ribosome components that are fully formed at pollen maturity. Thus degradation of
rRNA by S-RNases would provide a plausible mechanism for inhibition of pollen
tube growth. Indeed, degradation of rRNA in incompatible pollen tubes has been
observed (McClure et al 1990); however, whether degradation is a cause or an effect
of the growth arrest of self pollen tubes cannot be clearly discerned. Experiments
that involve grafting of incompatibly pollinated styles onto compatible ones have
shown that a certain percentage of arrested incompatible pollen tubes can resume
growth in compatible styles (Lush & Clarke 1997). This finding suggests that if
rRNA is the target of S-RNases, the rRNA genes must be transcribed in pollen
tubes. Alternatively, the target of S-RNases may be a different class of RNA
molecules, perhaps mRNA.
The identity of the pollen S-gene is a key missing piece of the puzzle in
understanding the mechanism of the S-RNase mediated SI response. There are
conceivably two ways by which a pollen S-allele product can work together with
S-RNases to elicit S-haplotype specific inhibition of pollen tube growth: it may
serve as a gatekeeper (Figure 3A ) or as an RNase inhibitor (Figure 3B ). An in vitro
pollen germination assay has been used to test the former possibility by determining whether entry of S-RNases into pollen tubes is S-haplotype specific (Gray et al
1991). The results showed that S-RNases enter the cytoplasm of both self and
non-self pollen tubes; however, because the growth of both kinds of pollen tubes
is inhibited in this assay, these in vitro results may not reflect the in vivo situation.
One theoretical problem in considering the pollen S-protein as an RNase inhibitor is
that, if this were the case, a pollen S-protein would have to recognize all S-RNases
except self S-RNase and inhibit the RNase activity of all non-self S-RNases. Given
the high degrees of sequence diversity between S-RNases, this seems unlikely. A
possible solution to this problem would be to envisage that a pollen S-protein contains an RNase inhibitor domain and an S-haplotype specificity domain and that it
interacts with self and non-self S-RNases differently (Figure 3B ). The specificity
domain would interact with the matching specificity domain of self S-RNase and
in so doing would physically occlude the RNase inhibitory interaction. In the case
of non-self S-RNases, the RNase inhibitor domain of a pollen S-protein would
interact with the active site of these S-RNases because there is no matching in
their specificity domains, and in so doing would inhibit the RNase activity.
Mutational studies have produced a number of self-compatible mutants that
are defective in pollen but not in pistil function in SI. Interestingly, the majority
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of the pollen-part mutants appear to have a centric fragment or a chromosomal
duplication that contains part or all of the S-locus region. Moreover, only those
carrying an extra S-locus region of an S-haplotype differing from that carried by the
endogenous chromosomes lose pollen function in SI (de Nettancourt 1977, Golz
et al 1999). This finding is consistent with the RNase inhibitor model because two
different pollen S-proteins produced in the same pollen tube would together inhibit
the RNase activity of all S-RNases. Ultimate understanding of the mechanism will
have to await the identification and characterization of the pollen S-gene.
Modifier Loci that Modulate the SI Response
The S-locus encodes all the determinants of S-haplotype specificity. However,
there is evidence for the existence of unlinked loci, termed modifier loci, that
modulate the SI response. Early studies of the Solanaceae demonstrated that multiple loci are required for a full manifestation of SI and that some of these can act
differently on different S-haplotypes (East 1932). An S-RNase gene that fails to
function in a self-compatible line of P. hybrida has been shown to be functional
when introgressed into a self-incompatible P. inflata background, suggesting that
the self-compatible P. hybrida background either lacks some factors that are required for the function of the S-RNase gene or contains some factors that suppress
the function of the S-RNase gene (Ai et al 1991). Studies involving the introgression of S-locus bearing chromosomal fragments from self-incompatible L.
hirsutum into self-compatible L. esculentum lines have also demonstrated a requirement for additional non-S-linked factors in the SI response (Bernatzky et al
1995).
Recent reports have begun to provide information on the mechanisms by which
some of these modifier loci act. A modifier locus of P. axillaris has been implicated
in the breakdown of pistil function in SI, and it specifically affects the expression
of one of the three alleles of the S-RNase gene examined (Tsukamoto et al 1999).
The identity of the modifier gene(s) involved has not been determined. To identify
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Figure 3 Models of the S-RNase mediated self-incompatibility response. (A) Gatekeeper
model. (B) RNase inhibitor model. A pistil cell synthesizes and secretes both S1- and
S2-RNases into the transmitting tract of the pistil where S1 and S3 pollen tubes are growing.
In (A), pollen S-allele products are predicted to be membrane- or wall-bound receptors for
S-RNases. The pollen S1-protein allows only the S1-RNase to enter the cytoplasm of the
S1 pollen tube, which results in degradation of its RNA and cessation of tube growth; the
pollen S3-protein allows neither S1- nor S2-RNase to enter the cytoplasm of the S3 pollen
tube. In (B), pollen S-allele products are predicted to be cytosolic RNase inhibitors. Both S1and S2-RNases enter the S1 and S3 pollen tubes, but the pollen S1- and S3-proteins interact
differently with self and non-self S-RNases, resulting in inhibition of RNase activity of all
non-self S-RNases (i.e. S1- and S2-RNases in the S3 pollen tube; S2-RNase in the S1 pollen
tube), but not that of self S-RNase (i.e. S1-RNase in the S1 pollen tube). Consequently, the
growth of the S1 but not S3 pollen tube is inhibited.
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non-S-RNase factors that are required for SI in Nicotiana, McClure et al (1999)
used two closely related species, a self-compatible line of N. alata (which contains
the non-S-RNase factors) and self-compatible N. plumbaginifolia (which does not
contain these factors) to perform a differential screen of pistil expressed genes.
Among the cDNAs selected, one, designated HT, has been shown by antisense
experiments to be essential for SI; suppression of its expression did not affect the
transcript or protein level of the SC10-RNase gene but led to the inability of the
pistil to reject SC10 pollen. The HT cDNA is predicted to encode a protein of 101
amino acids, containing a stretch of 20 asparagine and aspartate residues near the
C terminus. Unfortunately, database searches do not provide any insight into the
cellular function of this protein, and no direct interaction of HT with S-RNases has
been detected. Nonetheless, HT represents the first modifier gene of gametophytic
SI identified, and the elucidation of its role in SI and those of other modifier genes is
of great importance for a full understanding of the mechanism of S-RNase based SI.
PAPAVERACEAE TYPE SELF-INCOMPATIBILITY
This type of SI mechanism has been studied almost exclusively in Papaver rhoeas
(field poppy). This species is recalcitrant to transformation, making it difficult
to use transgenic approaches to examine the function of genes implicated in SI.
However, a reliable and efficient in vitro bioassay for the SI response has been
developed in which pollen germination or tube growth can be shown to be inhibited
by stigmatic extracts in an S-haplotype-specific manner (Franklin-Tong et al 1988).
This assay has been used to identify S-proteins (stigma proteins that are involved
in pollen rejection), study the structure-function relationships of S-proteins, and
characterize self-pollination specific biochemical events that occur in pollen.
The S-Gene Controls Stigma Function in Self-Incompatibility
Unlike S-RNases of the Solanaceae, S-proteins of P. rhoeas are present at very
low levels in the pistil (nanograms per pistil as opposed to micrograms per pistil
for S-RNases). Nonetheless, the allelic sequence diversity of S-proteins is also
very high, allowing identification of stigma proteins that are unique to particular
S-haplotypes by isoelectric focusing. Proteins associated with S1- and S3-haplotypes have been identified and their N-terminal sequences have been determined.
The sequence information has been used to design oligonucleotide probes for
isolating their corresponding cDNAs.
Structural Features of S-Proteins
Five alleles of the Papaver S-gene have now been cloned and sequenced ( Foote et al
1994, Walker et al 1996, Kurup et al 1998). The S-proteins of P. rhoeas do not
share any sequence similarity with S-RNases, despite the fact that both Solanaceae
and Papaveraceae possess GSI. Papaver S-proteins are small (∼15 kDa) secreted
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proteins, some of which are N-glycosylated. Because recombinant proteins produced in Escherichia coli were found to inhibit pollen tube growth in an S-haplotype-specific manner in the in vitro bioassay (Franklin-Tong et al 1995), the glycan
chains are unlikely to be required for the function of S-proteins. Like S-RNases,
S-proteins are highly polymorphic, sharing between 51.3 and 63.7% amino acid
sequence identity. However, they are predicted to possess a virtually identical
secondary structure, which is made up of a series of six β strands followed by
two α-helical regions located at the C terminus, all linked together by seven
hydrophilic loops ( Walker et al 1996, Kurup et al 1998). Significant sequence
similarity has been found between S-proteins and a large number of open reading
frames of the Arabidopsis genome (Ride et al 1999). Indeed it has been estimated
that there are likely to be approximately one hundred S-protein homologues (SPHs)
in Arabidopsis, but none are present in the current EST databases, suggesting that
they may be expressed at very low levels, only at certain developmental stages,
and /or only in response to certain environmental or biotic stimuli. The function
of the SPH genes in Arabidopsis, which is a self-compatible species, is unclear at
the present time.
Identification of Amino Acid Residues of S-Proteins
Involved in Recognition
Because recombinant S-proteins produced in E. coli are functional in the in vitro
bioassay, it has been possible to engineer mutant S-proteins for the identification
of amino acid residues that are required for the function of S-proteins in the SI
response (Kakeda et al 1998). The majority of the mutant S1-proteins generated
contain amino acid changes in the predicted surface loops. Most of these changes
have little or no effect on the ability of S1-protein to inhibit germination or tube
growth of S1 pollen. However, some residues in loop 6 were found to be essential.
For example, changes of the only hypervariable amino acid residue in this loop
and of several highly conserved amino acids adjacent to this residue resulted
in complete loss of the ability of S1-protein to inhibit S1 pollen. These results
suggest that loop 6 may be directly involved in recognition events essential for the
SI response.
Biochemical Responses in Pollen Following Self-Recognition
The in vitro bioassay system has also enabled studies of the biochemical events
that occur in pollen during the SI response. These include changes in pollen gene
expression (Franklin-Tong et al 1990), protein phosphorylation (Franklin-Tong
et al 1992), and cytosolic calcium levels (Franklin-Tong et al 1993) following
challenge of pollen with self S-protein.
Actinomycin D, a transcription inhibitor, has no effect on pollen germination,
presumably because mature pollen contains all the RNAs required for pollen tube
growth. Application of actinomycin D does, however, partially alleviate pollen
inhibition by self S-protein (Franklin-Tong et al 1990), suggesting that de novo
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pollen gene expression is required for a full SI response. In vitro translation of
mRNAs isolated from pollen treated with self or non-self S-protein has revealed
several proteins specific to an incompatible reaction (Franklin-Tong et al 1990).
Several pollen cDNA clones have been identified whose expression appears to be
correlated with the SI response in pollen (Franklin-Tong et al 1992). However,
it is not known if these genes are directly involved in the SI response or whether
they are expressed as a result of the SI response.
The activation of pollen response genes in the SI response suggests that the
interaction between an S-protein and its receptor molecule in the pollen (presumably the product of the pollen S-gene with the same S-haplotype) activates a signal
transduction mechanism that is responsible for the SI response. The involvement
of cytosolic free calcium ([Ca2+]i) as a second messenger in many signal transduction processes in plants is well established (Hepler & Wayne 1985, Trewavas
& Gilroy 1991), and a number of studies have implicated Ca2+ signaling in pollen
germination and pollen tube growth (Franklin-Tong 1999). For example, growing
pollen tubes have a [Ca2+]i gradient at their tips (Obermeyer & Weisenseel 1991,
Miller et al 1992), whereas non-growing tubes lack this gradient. The role of
Ca2+ signaling in the SI response of P. rhoeas has been investigated using Ca2+
-selective dyes (Franklin-Tong et al 1993, 1995). Inhibition of pollen tube growth
resulting from the addition of self S-protein is preceded by a transient increase in
[Ca2+]i in pollen tubes. This elevation originates from the nuclear complex and the
endoplasmic reticulum associated with this region, suggesting that [Ca2+]i may be
involved in the regulation of gene expression (Franklin-Tong et al 1993).
Protein Kinase Activity Implicated in the SI Response
The discovery that [Ca2+]i is associated with the SI response has prompted examination of differential protein phosphorylation in pollen tubes challenged with self
or non-self S-proteins. A number of proteins have been found to be either phosphorylated or dephosphorylated when comparing pollen tubes that are challenged
with self S-protein with those that are challenged with non-self S-protein(s) (Rudd
et al 1996). Two proteins whose phosphorylation is specifically increased as a
consequence of the SI response have been characterized in some detail. One, p26,
is a 26-kDa protein with a pI of 6.2 and exhibits Ca2+-dependent phosphorylation.
This phosphorylation occurs within 90 s of S-protein application to the in vitro
bioassay, with further increase occurring at 400 s. This timing coincides with the
transient increase in [Ca2+]i on treatment with self S-protein (10–∼400 s), suggesting that phosphorylation of p26 may be stimulated directly by the elevation of
[Ca2+]i, probably through activation of a CaM- or Ca2+-dependent protein kinase
(Rudd et al 1996). The other protein, p68, is 68 kDa in size with pI in the range
of 6.10 to 6.45. This protein is also phosphorylated in response to self S-protein,
but in a Ca2+-independent manner. The timing of this phosphorylation is different from that of p26, i.e. barely detectable at 240 s but much increased at 400 s
(Rudd et al 1997). The difference in the timing of phosphorylation of p26 and
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p68 suggests that p68 is likely to be downstream of p26 in the signal cascade.
Given that growth inhibition of the pollen tube occurs very rapidly, within 1–2
min (coinciding with the phosphorylation of p26), this might suggest that p68 is
not involved in growth inhibition, but perhaps in later events involving cell death.
S-Protein-Binding Proteins in Pollen
The technique of Western ligand blotting has identified a pollen protein, SBP
(70–120 kDa in size), that binds to pistil S-proteins of all S-haplotypes examined
(Hearn et al 1996). Phase partitioning has demonstrated that SBP is an integral
plasma membrane protein, as one might expect of a cell surface receptor. The
interaction between SBP and S-proteins appears to require glycan moieties as the
interaction was abolished by periodate treatment of the pollen protein blots prior
to incubation with S-proteins (Hearn et al 1996). To examine the role of SBP in
the SI response, mutant S1-proteins were used in both in vitro bioassay for the
SI response and the Western ligand blotting assay for SBP-binding activity. In
most cases, there was a good correlation between SBP binding and the SI response
(Jordan et al 1999). For example, amino acid changes in the predicted loop 6 of
S1-protein were found simultaneously to reduce greatly the ability of S1-protein
to bind SBP and to affect the in vitro SI response. Similar results were obtained
with amino acid changes in loop 2, although the reduction was to a lesser degree.
However, deletion of the first 16 amino acids of S1-protein did not affect the binding
activity to SBP and yet completely abolished the ability of the protein to inhibit
self pollen. The authors suggest that the lack of activity in the SI response is due
to the large deletion adversely affecting the protein structure–although binding to
SBP was not affected. Of the first 16 amino acids only the first 4 are variable
between different S-proteins, and when these alone were deleted, the resultant
mutant protein retained wild-type activity in both SBP binding and SI assays
(cited in Jordan et al 1999). Because the remaining 12 amino acids are conserved
between S-proteins, perhaps an alternative explanation for these results is that a
second non-S-specific interaction involving these amino acids and an unidentified
protein are essential for the SI response. Nonetheless, it would be interesting to
determine whether S1-protein lacking the first 16 amino acids has a dominantnegative effect over the wild-type S1-protein, which, if so, might be interpreted to
be a result of competition for SBP.
SBP has been proposed to function as an accessory receptor, whose role is to
modulate the interaction of S-proteins with the pollen S receptor (Jordan
et al 1999). The authors suggest that SBP and S-proteins together would form a
recognition complex analogous to those found in some mammalian systems, such
as the fibroblast growth factor (FGF ) signaling system, where a family of membrane proteins, the heparin sulfate proteoglycans (HSPG), function as accessory
receptors (Spivak-Kroizman et al 1994). In this case, low-affinity high-capacity
binding of FGF to glycan side chains of HSPG facilitates oligomerization of the
ligand, which then interacts with the high-affinity FGF receptor triggering a signal.
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Model for Self-Incompatibility Interaction
The results reported to date suggest that SI in P. rhoeas, unlike SI in the Solanaceae,
is controlled by a signal transduction mechanism (Figure 4 ). It can be envisaged
that the stigmatic S-protein acts as a signaling molecule, interacting with its receptor on the plasma membrane of the pollen grain or tube. The identity of the
pollen receptor (presumably encoded by the pollen S-gene) controlling the specificity of this interaction is unknown, but there is evidence that one or more non
Figure 4 A model of S-protein mediated self-incompatibility response. S1- and S2proteins are produced and secreted by the stigmatic surface cell. In the wall of S1 and
S3 pollen, both S-proteins interact with an accessory receptor protein, SBP; however, the
interaction with the pollen receptor (a pollen S-protein) can occur only between the S1protein/SBP complex and the pollen S1-protein. This interaction leads to a cascade of
signal transduction events in the S1 pollen, resulting in inhibition of germination or tube
growth.
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S-specific accessory proteins are involved, one of which has been suggested to
be SBP discussed above. In an incompatible pollination, the S-protein (perhaps
complexed with SBP) would bind to its cognate receptor, triggering a cascade of
signal transduction events. The first step in this cascade has been suggested to be
a transient increase in [Ca2+]i in the pollen. This presumably activates CaM- or
Ca2+-dependent protein kinases and consequent phosphorylation of protein substrates of these enzymes. At the end of the cascade, changes in gene expression
are thought to occur. It is interesting to consider that there might be an amalgamation of two separate processes at work in this system. One is growth inhibition
which results from a recognition event between the stigma and pollen proteins and
induction of a signal cascade, and the other is a cell death pathway not specific
to SI, which is activated indirectly by the cessation of growth. Tentative evidence
for this comes in the form of phosphoprotein p68, the phosphorylation of which
occurs later than the cessation of pollen tube growth and after the point at which
the growth inhibition is reversible.
BRASSICACEAE TYPE SELF-INCOMPATIBILITY
For the families that possess SSI, so far the Brassicaceae is the only one studied
at the molecular level. The species that have been used in most of the studies are
B. campestris (Chinese cabbage), B. oleracea (cabbage, cauliflower, kale, Brussels
sprout, broccoli, etc), and B. napus (oil seed rape). More than 100 haplotypes have
been identified in these species.
The SLG Gene–a Polymorphic Stigma-Expressed
Gene at the S-Locus
As recognition and rejection of self pollen occurs on the stigmatic surface, searches
for the female component of SI focused on stigmatic proteins that co-segregate with
S-haplotypes. The first such proteins identified were SLGs (S-locus glycoproteins).
These proteins are abundant in the stigma and exhibit a number of characteristics
expected of proteins that would determine the S-haplotype specificity of the stigma.
First, they are predominantly produced in the stigma and located in the wall of
the epidermal cells (papillae) of the stigma, which comes into direct contact with
pollen. Second, they are present in very small amounts in the stigmas of immature
buds which are self-compatible, and the timing of the sharp increase in amount
just prior to flower opening coincides with the timing of the acquisition of SI by
the stigma. Third, their sequences are highly divergent, with pair-wise sequence
identity ranging from 65 to 97.5% (Kusaba et al 1997). Thus for a number of
years since its identification the SLG gene has been thought to be required for the
S-haplotype specificity of the stigma.
However, early attempts to demonstrate the function of SLG by gain- and lossof-function approaches did not yield conclusive results. For the loss-of-function
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experiments, introduction of an antisense SLG gene into transgenic plants led
to the breakdown of SI, but the transcript levels of both SLG and SRK (S-locus
receptor kinase) were reduced (Shiba et al 1995). Analysis of a naturally occurring
self-compatible mutant (scf1) whose stigma, but not pollen, function was defective showed that the transcript and protein levels of both SLG and SLRs (S-locus
related) were reduced (Nasrallah et al 1992). The simultaneous reduction in the
expression of SLG and other related genes made it difficult to draw any conclusion
about the function of SLG from these experiments. In the gain-of-function experiments, introduction of an SLG gene of a new S-haplotype did not confer the new
S-haplotype specificity on the stigma of the transgenic plants, and instead, caused
the breakdown of SI. This phenotype was attributed to a phenomenon termed
homology-dependent gene silencing, as the expression of the transgene and the
endogenous SLG, SLRs, and SRK was suppressed (Conner et al 1997). In a few
cases where SLG of a new S-haplotype was produced in transgenic plants because the transgene and its recipient plants were from different Brassica species,
the analysis of the SI behavior was complicated by interspecific incompatibility
(Toriyama et al 1991, Nishio et al 1992).
Several lines of evidence obtained recently cast doubt on the requirement of
SLG for S-haplotype specificity of the stigma. First, mature flowers of a line of
self-incompatible B. oleracea were found to produce lower levels of SLG than
those produced by immature buds (which are unable to reject self pollen) of other
self-incompatible lines (Gaude et al 1995). Second, from sequence analysis of a
large number of SLGs, it was found that several pairs of SLGs from genetically
distinct S-haplotypes are virtually identical in their sequences (Kusaba et al 1997,
Kusaba & Nishio 1999). For example, SLG23 and SLG29 of B. oleracea differ
only in three amino acids and none are in the three hypervariable regions thought
to be involved in S-haplotype specificity. Third, SLG was found to be deleted
in a self-incompatible line of B. oleracea (Okazaki et al 1999). Fourth, SLG43 of
B. campestris was introduced into S52S60 plants of B. campestris, and the transgenic
plants producing SLG43 at a normal level failed to acquire the ability to reject
S43 pollen (Takasaki et al 1999). However, the results from this transformation
experiment were interpreted to mean that SLG alone is not sufficient, rather than
not required, for S-haplotype specificity.
Recent transformation experiments by Takasaki et al (2000) have clearly shown
that SLG is not required for the S-haplotype specificity of the stigma, but may
nonetheless play a role in the enhancement of the SI response. These experiments
also involve SRK and are discussed below.
The SRK Gene Encodes Stigma S-Haplotype Specificity
SRK is the second highly polymorphic gene identified at the Brassica S-locus;
this was accomplished by virtue of the sequence similarity between SRK and
SLG (Stein et al 1991). SRK has the hallmarks of a receptor kinase: it consists
of an extracellular domain, a single transmembrane domain, and a cytoplasmic
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domain that has serine/threonine kinase activity (Goring & Rothstein 1992). The
extracellular domain shares extensive sequence similarity with SLG, and is called
the S-domain. Like SLG, SRK also exhibits properties expected of the gene that
determines the S-haplotype specificity of the stigma. First, SRK is predominantly
expressed in the stigmatic papillae (albeit at a level at least 100 times lower than
the expression level of SLG). Second, SRK spans the plasma membrane of the
papillar cell (Delorme et al 1995) and might serve as a receptor for transducing a
pollen signal to the cytoplasm of the papillar cell. Third, SRK shows a high degree
of allelic sequence polymorphism, as does SLG.
Results from analyses of self-compatible lines of Brassica are consistent with
the notion that SRK is essential for the stigma function in SI. First, SRK of a
self-compatible line of B. napus has a 1-bp deletion, resulting in a truncated SRK
(Goring et al 1993). Second, a chromosomal region of the S-locus containing SRK
is deleted from a self-compatible mutant of B. oleracea whose stigma function is
defective (Nasrallah et al 1994). Third, an SRK gene engineered to encode a kinasedeficient mutant has an S-haplotype-specific dominant-negative effect on wild-type
SRK, which results in partial breakdown of SI in transgenic plants that produced
the wild-type SRK and mutant SRK of the same S-haplotype (Stahl et al 1998).
The most direct means to ascertain the function of SRK in SI is by gain-offunction experiments. However, initial attempts to determine whether SRK of a
new S-haplotype, when expressed in transgenic plants, could confer on them the
new S-haplotype specificity also had the same co-suppression problem encountered in the gain-of-function experiments with SLG. Only very recently has the
function of SRK in SI been definitively established. Takasaki et al (2000) were able
to overcome the problem of homology-dependent gene silencing by introducing
SLG28 and SRK28 of B. campestris into S52S60 and S60S60 plants, respectively, because the SLG and SRK of S52- and S60-haplotypes have a low degree of sequence
similarity with SLG28 and SRK28. They found that production of SRK28 alone, but
not of SLG28 alone, in the stigmas of the transgenic plants conferred the ability
to reject S28 pollen. These results provide the first direct evidence that SRK is
the sole determinant of the S-haplotype specificity of the stigma and rule out the
involvement of SLG in this function.
So, what might the function(s) of SLG be? In the transgenic experiments
carried out by Takasaki et al (2000), the strength of self pollen rejection by SRK28
(as measured by the average seed set per flower) in plants that carried the SLG
gene of different S-haplotypes was also examined. A good correlation was found
between the strength of self pollen rejection and the degree of amino acid sequence
identity between the S-domain of SRK28 and the SLG produced in the same stigma.
The higher the identity, the stronger the rejection. The authors propose that SLG,
although not required for the S-haplotype specificity, interacts with the S-domain
of SRK to facilitate the process of the recognition reaction between SRK and the
pollen determinant of the S-haplotype specificity. The strength of the association
between SLG and SRK might then decrease as the sequence identity between them
decreases. However, since the level of SRK28 produced in the transgenic plants
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was only approximately one third of the normal level in plants carrying one copy of
the SRK28 gene, it remains possible that SLG is not required for a full manifestation
of the SI response when SRK is produced at normal levels.
SLG has also been shown to be required for adhesion of pollen to the surface
of the papillar cell, the first step in the process of compatible pollination (Luu et al
1999). The force of pollen-stigma adhesion was found to be reduced when stigmas
of S2- and S5-haplotypes were pretreated with an anti-SLG2 antibody (which crossreacted with SLG5), and this reduction was attributed to the masking of the binding
sites of SLG for pollen surface molecules during adhesion. A good candidate for
such a molecule is a pollen coat protein, PCP-A1 (pollen coat protein-A1), which
has been shown to interact with SLG in vitro in an S-haplotype-independent manner
(Doughty et al 1993).
The SCR/SP11 Gene Encodes Pollen S-Haplotype Specificity
As in the Solanaceae SI system, several lines of evidence suggest that stigma and
pollen functions in Brassica SI are controlled by different genes at the S-locus.
First, some self-compatible mutants are defective only in pollen function or only
in stigma function (Hinata & Okasaki 1986, Nasrallah et al 1992). Second, downregulation of the expression of SRK, or production of a dominant-negative form
of SRK, in transgenic plants affects their stigma but not pollen function (Conner
et al 1997, Stahl et al 1998). Third, expression of SRK of a new S-haplotype in
transgenic plants confers the new S-haplotype specificity to the stigma but not to
pollen (Takasaki et al 2000).
The pollen coat has long been suspected to be the site where the determinant
of pollen S-haplotype specificity is located (for a review of pollen coating, see
Doughty et al 1992) for these reasons: (a) The SI interaction occurs at the stigmatic
surface, which makes direct contact with the pollen coat; (b) the pollen coat
contains sporophytically derived materials (released from the tapetum), consistent
with the sporophytic control of the pollen SI behavior. A pollination bioassay was
used to ascertain whether the pollen coating indeed contained the S-determinant
(Stephenson et al 1997). In this assay, self or cross pollen coating was first applied
to the papillar cell of a stigma, and the ability of cross pollen to hydrate and
germinate on the stigma was subsequently examined. The results showed that
pretreatment of self pollen coating, but not cross pollen coating, of a stigma led
to a significant reduction of hydration/germination of cross pollen on its surface.
Thus a stigma either accepts or rejects a pollen grain based on the S-haplotype of
the pollen coating applied to the papillar surface. The implication is that the pollen
coating contains molecule(s) that can induce the SI response in the stigma. HPLC
fractionation of the pollen coating coupled with the bioassay led to the suggestion
that the pollen S-determinant is a basic, cysteine-rich protein of the PCP family
with a molecular mass less than 10 kDa (Stephenson et al 1997).
One approach toward identifying the gene encoding the pollen S-determinant
is to examine the region of the S-locus that contains SRK and SLG in order
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to identify genes that exhibit the properties expected of such a gene. The first
potential candidate, SLA (S-locus anther), was identified from the S2-haplotype of
B. oleracea (Boyes & Nasrallah 1995). SLA is located just downstream of SLG2
and is expressed exclusively in the anther and microspores of the S2-haplotype,
but not in a self-compatible line of B. napus of the S2-haplotype, which contains a
retrotransposon-like insertion in SLA. However, subsequent examination of some
self-incompatible lines of B. oleracea revealed that they also carried an allele
of the SLA gene with a similar retrotransposon insertion (Pastuglia et al 1997).
Therefore, SLA is unlikely to be the gene encoding the pollen S-determinant. Two
additional S-linked genes, SLL1 and SSL2 (S-locus linked gene 1 and 2), have
also been ruled out as potential candidates because they exhibit little or no allelic
sequence polymorphism (Yu et al 1996).
A 76-kb region of the S-locus containing SRK and SLG of the S9-haplotype of B. campestris was completely sequenced, and found to contain 11 genes
(in addition to SRK, SLG, and SLL2) that are expressed in anther and/or pistil
(Suzuki et al 1999). The authors postulate that one of these genes, SP11, is a potential candidate for encoding the pollen S-determinant because (a) it encodes a protein
characteristic of PCP family of proteins (small, basic cysteine-rich proteins with
eight cysteines), (b) it is located in the immediate 30 flanking region of SRK9, and
(c) it is expressed predominantly in the anther (Suzuki et al 1999). Another allele of
SP11 was identified independently from the sequencing of a 13-kb region between
SRK and SLG of the S8-haplotype of B. campestris (Schopfer et al 1999); however,
this gene was given a different name, SCR (S-locus cysteine-rich). cDNA clones for
S6-, S12-, S13-, and S52-alleles of SCR /SP11 have also been isolated and sequenced
(Schopfer et al 1999, Takayama et al 2000). SCRs/SP11s are small proteins 74–77
amino acids in length and are hydrophilic except for an N-terminal stretch of 19
amino acids. The mature protein is predicted to be a secreted protein with 8.4–8.6
kDa and a pI of 8.1–8.4. Sequence comparison of these SCRs/SP11s has revealed
that while the putative signal peptide is highly conserved, the mature protein is
highly divergent, with conservation limited to just 12 amino acids, 8 of which are
cysteines. Overall amino acid sequence identities are between 26 and 46%.
That SCR/SP11 determines the S-haplotype specificity of pollen has been definitively established by a gain-of-function experiment (Schopfer et al 1999) and a
pollination bioassay (Takayama et al 2000). The SCR8 promoter was used to drive
the expression of SCR6 cDNA in transgenic B. oleracea plants of S2S2 genotype,
and transgenic plants expressing the transgene were used to pollinate wild-type
S6S6 and S22S22 plants. In all cases, the pollen of the transgenic plants expressing
the SCR6 cDNA was rejected by S6S6 pistils but accepted by S22S22 pistils (Schopfer
et al 1999). In the pollination bioassay much like that used by Stephenson et al
(1997) described above, recombinant SCR/SP11 of the S9-haplotype was applied
to papillar cells of stigmas of the S9-haplotype and the S8-haplotype, and the protein was found to elicit the SI response only in the former (self ) stigmas, which
resulted in inhibition of hydration of cross pollen (Takayama et al 2000). Moreover, in situ hybridization of anther sections showed that SCR/SP11 is expressed
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in the tapetum of the anther (Takayama et al 2000). This sporophytic expression
pattern explains why in Brassica the SI phenotype of pollen is determined by the
genotype of the pollen parent.
Structural Organization of the S-Locus
The Brassica S-locus, by definition, is composed of all the components that specify
an S-haplotype. For SI to be maintained, these components must remain as a tightly
linked genetic unit. In recent years, a number of studies have shed light on how
this might be accomplished. Physical maps have been constructed of the S-locus
region of several S-haplotypes from B. oleracea (Boyes et al 1997), B. campestris
(Boyes et al 1997, Suzuki et al 1999), and B. napus (Cui et al 1999). Considerable
structural divergence has been found in this region of different S-haplotypes. For
example, the distance between SLG and SRK varies from as short as 6 kb (Cui et al
1999) to as long as over 200 kb (Boyes & Nasrallah 1993). Using direct sequencing,
cDNA selection techniques, and RNA differential display, a number of genes have
been identified in the S-locus region (Cui et al 1999, Suzuki et al 1999, Casselman
et al 2000). Retroelements (deletion derivatives of transposable elements) have
also been found in this region (Cui et al 1999). The structural heteromorphism
between S-haplotypes appears to be a result of chromosomal rearrangements
(duplications and inversions), compounded by the accumulation of transposons
and retroelements, and haplotype-specific genes. Recombination is believed to be
suppressed in the S-locus region owing to this structural heteromorphism, which
allows the male and female genes that determine the S-haplotype specificity to
remain linked as a genetic unit.
The genes downstream of SLG have been found to be co-linear between different S-haplotypes, and this finding has led to the speculation that one border of
the S-locus may lie in this region (Cui et al 1999). This has been confirmed by
comprehensive recombinational and physical mapping analyses of the S-locus
of the S8-haplotype of B. campestris (Casselman et al 2000). The entire region examined constitutes about 740–1000 kb and 1.46 cM. The two recombination breakpoints flanking a sub-region containing SLG, SCR/SP11, and SRK
are separated by approximately 50 kb, with one of the breakpoints located 2 to
4 kb downstream of SLG. Surprisingly, kilobase to centimorgan ratios across the
S-locus region analyzed are comparable to those found for the entire genome of
Brassica (500–700/cM). These results might suggest that there is no suppression
of recombination in the S-locus region. Casselman et al (2000) point out, however, that recombination has never been observed between SRK and SLG even in
the S-haplotypes where these two genes are separated by >200 kb. They suggest
that recombination suppression in a physically compressed S-haplotype such as
S8, where SLG, SCR/SP11, and SRK all lie within 13 kb, operates only within this
region. In other haplotypes where the essential components of the SI system are
spread over a larger region, suppression of recombination would necessarily cover
a much larger region.
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For the four S-haplotypes reported so far, SCR/SP11 is located between SLG and
SRK, although its distance to SLG or SRK is not conserved (Schopfer et al 1999,
Suzuki et al 1999, Takayama et al 2000). The necessity of the presence of SLG in
this S-locus region has been brought into question by the recent finding of Takasaki
et al (2000) that SRK alone is sufficient to determine S-haplotype specificity of the
stigma. However, their results also show that SLG may be necessary for a full SI
response. Moreover, in most cases, for a given S-haplotype, SLG shows the highest
degree of sequence similarity with the S-domain of SRK of the same S-haplotype,
suggesting that SLG and SRK may have co-evolved. Lastly, no recombination has
ever been detected between SLG and SRK. Thus SLG should be considered an
integral part of the S-locus.
A comparative mapping study has also been conducted between the
B. campestris S8-haplotype and the Arabidopsis genome (Conner et al 1998).
Both species are members of the same family (Brassicaceae), although Arabidopsis is self-compatible. A region of Arabidopsis chromosome 1 (in the immediate
vicinity of the ethylene response gene ETR1) was found to be homologous to the
Brassica S-locus. A high degree of synteny was found at the sub-megabase scale
between the two homologous regions, but all the Brassica S-locus genes are absent
from Arabidopsis, both in the homologous region and in the rest of the genome.
The authors propose that this is likely to be the result of deletion of the S-locus
genes from Arabidopsis.
Model for Self-Incompatibility Interaction
A working model for SI in Brassica, like Papaver, envisages a recognition event
followed by a signal transduction cascade (Figure 5). Unlike Papaver, however, the recognition event occurs at the stigma surface, and the cascade takes
place in the stigmatic papillar cell rather than in the pollen grain. The Brassica
model dictates that the pollen S-haplotype determinant, SCR/SP11, interacts in an
S-haplotype-specific manner with the extracellular (S-) domain of SRK, which
spans the plasma membrane of the papillar cell. Although this interaction is as yet
to be biochemically demonstrated, it seems likely. What is uncertain is whether
SLG interacts with the S-domain of SRK and/or with SCR/SP11. By analogy with
animal receptor kinases, SRK is generally thought to function as a dimer, although
this has not been shown experimentally. Some animal receptor kinases are active
as monomers, which remains a possibility with SRK. One possible explanation
for the enhancement of the SRK-mediated SI response by SLG as observed by
Takasaki et al (2000) could be that SRK is active as a homodimer but is also
active as a heterodimer in combination with an SLG molecule. Given the high
degree of sequence similarity between SLG and the S-domain of SRK of the same
S-haplotype, it is possible that SLG also interacts with SCR/SP11. However, if
so, one would expect this interaction to compete with the binding of SCR/SP11
to SRK, potentially having a dominant-negative effect, as SLG is >100-fold more
abundant than SRK.
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Figure 5 A model of SRK-mediated self-incompatibility response. A pollen grain with
the SI phenotype of S1S3 and a pollen grain with the SI phenotype of S4S5 land on the
papillar cell of a stigma with S1S2-genotype. All four allelic forms of SCR/SP11 produced
by the pollen grains are taken up by the papillar cell, but the only interaction with the
S-domain of an SRK is between SCR1 and SRK1. This interaction sets off a cascade of
signal transduction events, including phosphorylation of ARC1, in the papillar cell, which
results in inhibition of germination of the pollen grain carrying S1- and S3-haplotypes.
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Activation of SRK by self SCR/SP11 presumably causes autophosphorylation
of SRK and subsequent phosphorylation of intracellular substrates of SRK. One of
these substrates has been positively identified as ARC1, a stigma protein that shows
a phosphorylation-dependent interaction with the cytosolic domain of SRK in vitro
(Gu et al 1998). Suppression of ARC1 production in the stigma of transgenic plants
causes breakdown of SI (Stone et al 1999). Unfortunately, the sequence of ARC1
does not give any clues as to what its cellular function may be. The identity of
other SRK substrates, if any, and downstream components of the signaling cascade
is unknown.
Another key question is how does this sequence of signaling events ultimately
lead to the inhibition of pollen germination or tube growth? To date, there has been
only one report concerning a potential mechanism for pollen inhibition. Ikeda et al
(1997) studied a self-compatible line of B. campestris, which has mutations in both
the S-locus (Nasrallah et al 1994) and an unlinked locus, called MOD. Comparing
this line with its self-incompatible counterpart, the authors determined that the
mutation (recessive) in the MOD locus results from a chromosomal deletion and
identified a putative aquaporin (water channel) gene within this deleted region. The
authors suggest that this aquaporin-like protein is encoded by the MOD gene and
propose that it regulates the availability of water at the stigma surface. Specifically,
in the SI response, SRK activation would lead to activation of this aquaporin-like
protein and an increase in the water flow away from the pollen, thus preventing
pollen hydration. As attractive as this model may appear, further experiments
are necessary to substantiate its validity. The reasons are as follows. First, since
the full extent of the chromosomal deletion in the mod background has not been
reported, it is not known whether the aquaporin-like gene is the only gene deleted
in this region, or whether there are other unidentified genes that are also deleted.
Second, the aquaporin-like protein has not been reported to indeed possess water
(or any other molecule) channel activity, as its sequence seems to suggest. Third,
the aquaporin-like gene has not been shown to be able to restore SI to mod/mod
plants. Nonetheless, even if the aquaporin-like gene turns out not to be the MOD
gene, its linkage to the MOD locus will allow its use as a molecular marker for the
eventual identification of the MOD gene.
CONCLUSIONS AND FUTURE PERSPECTIVES
Considerable knowledge about the gene controlling pistil function in SI has been
obtained for all three types of SI mechanisms discussed here. The identification
of these genes was greatly facilitated by the vast amount of information obtained
from genetic studies carried out in the first half of the last century. The recent
use of transgenic approaches or in vitro bioassay not only has provided conclusive
evidence that these genes encode the pistil S-haplotype determinant in their respective SI systems, but also allows dissection of structure/function relationships
of their gene products.
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However, despite the impressive progress that has been made, our understanding
of the mechanism of any of these SI systems is still far from complete. For
the Solanaceae and Papavaraceae types, a key missing piece of the puzzle is the
pollen S-gene. For the Brassicaceae type, as well as the Papaveraceae type, the
intricacies of the signal cascades triggered in the SI response are largely unknown
as is how these cascades lead to inhibition of germination and/or tube growth
of self pollen. However, recent identification of SCR/SP11, the determinant of
pollen S-haplotype specificity, and ARC1, one of the most upstream components
of the signal cascade, in Brassicaceae represents a significant advancement toward
addressing these questions.
It is clear that even though the S-haplotype specificity of these three types of SI
is determined by the S-locus alone, protein factors encoded by unlinked loci are
required for a full manifestation of the SI response. If these proteins are involved
only in SI, and not essential for other developmental processes, mutations in their
genes would not be lethal but would cause breakdown of SI. These mutant alleles
may constitute the modifier genes that have been identified from studies of selfcompatible lines of self-incompatible species (e.g., the MOD gene of Brassica and
the HT gene of Nicotiana). Many cultivated species, although self-compatible,
are derived from their SI ancestors, with the SI trait having been selected out in
the domestication process. In some cases, the cause of the breakdown of SI is
likely to be a result of mutations in one or more of the modifier genes. Thus
comparative study of self-compatible species and their wild self-incompatible
relatives may prove to be an effective means for the identification of non-S-linked
genes that encode components of an SI system. One of the goals of SI research
is to introduce SI into self-compatible crop plant species to facilitate hybrid seed
production. Identification of modifier genes will make this goal more attainable
because transfer of the S-locus genes alone into these species is unlikely to be
sufficient to confer SI.
To date, the RNase-based SI mechanism employed by the Solanaceae is the
only SI mechanism discussed here that has been found in more than one family,
i.e. in the Rosaceae and Scrophulariaceae. An interesting but unresolved question
is whether S-RNase-based SI evolved before the divergence of these three families
or evolved independently in these families. An important gap in our knowledge of
SI systems comes in the form of the two-locus gametophytic SI system found in
the Poaceae (grass) family. This family contains economically important species
(maize, rice, rye, etc), but efforts to identify the genes involved in SI have not been
successful. (Initial reports of the cloning of the pollen S-gene in a grass species
have now been retracted; see Langridge et al 1999). SI systems, while performing
the same reproductive function, are proving to be diverse in their mechanisms. It
is interesting to note that DNA sequences with similarity to genes that determine
S-haplotype specificity in the three types of SI mechanisms discussed here have
been identified through the Arabidopsis genome sequencing projects, and it seems
likely that the study of SI mechanisms will provide information relevant to other
developmental processes such as cell-cell recognition and cell death. The extent
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of crossover between SI systems and other recognition processes involved in plant
growth, development, or defense is also likely to be revealed during the next few
years.
ACKNOWLEDGMENTS
The research in self-incompatibility carried out in our laboratory was supported
by grants from the National Science Foundation.
Visit the Annual Reviews home page at www.AnnualReviews.org
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