Review Article
The Diversity of Self-Incompatibility Systems in Flowering Plants
S. J. Hiscock and S. M. McInnis
School of Biological Sciences, University of Bristol, Bristol, UK
Received: September 2, 2002; Accepted: January 15, 2003
Abstract: Flowering plants are the most successful group of
land plants and dominate the earths vegetation with around
300 000 species. This success is, in part, the consequence of a
set of unique reproductive innovations that evolved with the
flower. Most notable of these innovations were the closed car-
pel and double fertilization. Closed carpels permitted the evolu-
tion of effective mechanisms for pollen selection and discrimi-
nation, while double fertilization leading to endosperm forma-
tion allowed for more efficient utilization of resources because
reserves are only allocated to the seed after fertilization. This
review will focus on the most important and best understood
mechanism of pollen discrimination, self-incompatibility (SI), a
genetically determined pollen recognition system that prevents
self-fertilization and fertilization by other individuals with the
same incompatibility phenotype. In recent years much progress
has been made towards elucidating the molecular mechanisms
of SI operating in three distinct SI systems found in the Brassi-
caceae, Solanaceae and Papaveraceae, respectively. More re-
cent molecular data obtained from the Poaceae, Convolvula-
ceae and Asteraceae, however, suggest that other molecular
mechanisms of SI exist. A survey of classical genetic studies of
SI predicts yet further potential molecular mechanisms of SI.
We discuss the evolutionary implications of this apparent diver-
sity in molecular pathways leading to SI and stress the need for
more molecular studies of different SI systems.
Key words: Self-incompatibility, S genes, S-RNase, S receptor
kinase, S-protein.
Introduction
Most flowering plants have co-sexual (bi-sexual) flowers con-
taining male reproductive structures (the stamens, consisting
of anther and filament) and female reproductive structures
(carpels/pistils, consisting of stigma, style and ovary). Co-sex-
ual flowers greatly increase the efficiency of insect pollination
because deposition of cross pollen on stigmas and removal of
self-pollen from anthers are accomplished during one insect
visit. Co-sexuality, however, also increases the risk of self-pol-
Plant biol. 5 (2003) 23 ± 32
 Georg Thieme Verlag Stuttgart ´ New York
ISSN 1435-8603
23
lination leading to self-fertilization and its detrimental conse-
quence, inbreeding depression. Nevertheless, many co-sexual
species are able to prevent self-fertilization because they are
ªself-incompatibleº (SI). SI is a genetically determined pre-zy-
gotic barrier to fertilization by self- or self-related pollen that
eliminates any risk of inbreeding and therefore optimizes the
potential for out-breeding afforded by insect pollination. It
has been suggested that SI was a feature of the earliest flower-
ing plants that may have been a significant factor in their ex-
traordinary evolutionary success (Whitehouse, 1950). Indeed
today, it is estimated that SI is present in around 60 % of flower-
ing plants (see Hiscock and Kües, 1999).
Darwin (1877) was aware of the phenomenon of SI when he
described ªillegitimateº and ªlegitimateº matings between
the two different flower forms (morphs) in Primula. Primula
has two flower forms, ªpinº flowers have long styles such that
the stigma is placed above the anthers, and ªthrumº flowers
with short styles, and anthers placed above the stigma. ªLegit-
imateº matings (resulting in seed set) are only possible be-
tween pin and thrum flowers, never between pin and pin or
between thrum and thrum ± these being ªillegitimateº mat-
ings. This type of SI, where morphological features of the flow-
er differ between incompatibility types is known as hetero-
morphic SI, in contrast to so-called homomorphic SI where
there are no such differences in floral morphology between
incompatibility types. The Primula system with two flower
morphs is known as distyly and is the most widespread form
of heteromorphic SI (reviewed by Barrett and Cruzan, 1994).
Less common is tristyly where three flower morphs co-exist
as in Lythrum (Lythraceae), again, first described by Darwin
(1877). Heteromorphic SI has a scattered distribution among
flowering plants, being recorded in 25 families and 155 genera
(Ganders, 1979) but will not be dealt with in this review (but
see Barrett and Cruzan, 1994, for review). Instead we will focus
on homomorphic SI systems as more is known about their mo-
lecular genetics and cell biology.
The Genetics of SI
SI (whether homomorphic or heteromorphic) is usually con-
trolled by a single S (self-incompatibility) locus. In homo-
morphic SI systems S is multiallelic, whereas in heteromorphic
systems S is always diallelic (reviewed by de Nettancourt,
1977). In most cases, if pollen and pistil share a common S al-
lele, the pollen is rendered incompatible; exceptions only be-
24 Plant biol. 5 (2003)
ing possible where dominance interactions occur between S
alleles (see below). Classical genetic studies identified two dis-
tinct genetic forms of SI: gametophytic (GSI) and sporophytic
(SSI). In GSI the incompatibility phenotype of the pollen is de-
termined by its own haploid genome, whereas in SSI the in-
compatibility phenotype of the pollen is determined by the
diploid genome of the plant (sporophyte) that produced it. This
important genetic difference has significant consequences for
the behaviour of S alleles in the two SI systems. In GSI, S alleles
are expressed co-dominantly in the pistil whereas in SSI sys-
tems, complex dominance interactions are possible between S
alleles. In SSI, dominance interactions between S alleles can act
independently in pollen and pistil and can allow successful
matings to occur between individuals that share one recessive
S allele. This means that it is theoretically possible to have S
homozygotes within populations of SSI species, a phenomenon
that is theoretically impossible under GSI. Distyly and tristyly
are both under sporophytic control with the two S alleles, S
and s showing complete dominance (for a review of SI genetics
see de Nettancourt, 1977). Within a given angiosperm family,
homomorphic SI is always of one type (Hiscock and Kües,
1999). Thus, GSI is found typically in the Solanaceae, Rosaceae,
Scrophulariaceae and Papaveraceae, whereas SSI is found in
the Brassicaceae, Asteraceae and Convolvulaceae. Over the last
20 years, research into the molecular basis of GSI and SSI has
focused principally on three ªmodelº SI systems and within
these systems generally just one or a few species, often chosen
because of their economic importance. The three model sys-
tems are a) SSI in the Brassicaceae (Brassica oleracea, B. rapa
[syn. campestris] and more recently Arabidopsis lyrata); b) S-
RNase-mediated GSI (principally in species from the Solana-
ceae, such as Nicotiana, Petunia, Solanum and Lycopersicon,
but also in species from the Rosaceae, i.e., Pyrus, Prunus and
Malus and Scrophulariaceae, i.e., Antirrhinum); c) GSI in Pa-
paver (Papaveraceae) which involves an S-protein not related
to S-RNases (for comprehensive reviews see Hiscock and Kües,
1999; McCubbin and Kao, 2000). Evidence from research into
these model SI systems indicates that different genes must
regulate the expression of SI in pollen and pistil. Nevertheless,
so far, only in Brassica have both the pollen and pistil S genes
been characterized and shown to encode different proteins
(see Schopfer and Nasrallah, 2000). A minimum of two genes
is thus predicted to reside at the S-locus, so variants of the S-
locus are now better referred to as S-haplotypes rather than
S-alleles ± the term ªalleleº now only being used to describe
variants of the same S gene.
Despite the understandable emphasis of molecular research
on the three model SI systems there have been some molecular
studies of SI in species from other families. These studies pre-
dict further distinct systems of SI. Following a review of SI in
the three model systems we will discuss these new molecular
findings and highlight genetic evidence from the literature
that together predict a greater diversity for molecular mecha-
nisms of SI than was hitherto thought.
Molecular Studies of SI in Model Systems
a) Sporophytic SI in the Brassicaceae
The SSI system in the Brassicaceae is the only SI system for
which both pollen and stigma S-specificity determinants have
been identified. Early molecular studies on Brassica oleracea
S. J. Hiscock and S. M. McInnis
and B. rapa (syn. campestris) identified two stigma-expressed
genes at the S locus, the S Locus Glycoprotein (SLG) and the S
Receptor (serine-threonine) Kinase (SRK) (Nasrallah et al.,
1994). SLG encodes a secreted glycoprotein that accumulates
within the cell wall of stigmatic papillar cells, while SRK en-
codes a membrane-spanning receptor kinase that localizes to
the plasma membrane of stigmatic papillae. SRK and SLG both
typically have conserved regions and regions of extreme ami-
no acid variability (hypervariable regions) that may be impor-
tant for allelic specificity. Interestingly, SLG and the receptor
domain of SRK have very similar amino acid sequences, par-
ticularly when they are from the same haplotype, where they
are characteristically ~ 90 % identical (Kusaba et al., 1997). This
led to the suggestion that SRK and SLG may interact with the
putative pollen ligand in a concerted manner and were there-
fore likely to have co-evolved (Kusaba et al., 1997). However,
subsequent studies showed that this close within-haplotype
sequence similarity between SRK and SLG did not hold for all
S haplotypes. The amino acid sequences of some SLGs and
SRKs were clearly more similar between S haplotypes than
within the same S haplotype, indicating that recombination
can take place between S haplotypes and that strict sequence
conservation of related pairs of SRK and SLG is not essential for
SSI (Suzuki et al., 2000). Subsequently it was demonstrated
that SLG is dispensable for functional SI in Brassica even
though its presence produces a ªstrongerº SSI response, as
measured by the extent of incompatible pollen tube develop-
ment (Takasaki et al., 2000). Even though the evolutionary
and functional relationship between SRK and SLG is obscure,
amino acid sequence similarities allow them to be assigned to
one of two S haplotype classes (class I and class II) first defined
on the basis of their dominance relationships (discussed in Ku-
saba et al., 1997, 2000). Class I haplotypes are all dominant or
co-dominant, whereas class II haplotypes are recessive in pol-
len to the class I haplotypes. Sequences of SRKs and SLGs are
clearly conserved within each class but diverge significantly
between classes ± class I SLGs and SRKs showing only ~ 65 %
amino acid sequence similarity to class II SRKs and SLGs, com-
pared to the ~ 80 % similarity typical of within-class compari-
sons (Kusaba et al., 2000).
Recently the pollen determinant of SSI was independently
identified in Brassica rapa as a small (6 kD) cysteine-rich pep-
tide located in the pollen coating. Schopfer et al. (1999) named
the peptide S Cysteine Rich protein (SCR), and Takayama et al.
(2000) named it SP11 (S Pollen protein 11). Transgenic gain-of-
function experiments demonstrated that SCR/SP11 was neces-
sary and sufficient for pollen S-specificity and in situ hybridi-
zation studies confirmed sporophytic expression of SCR/SP11
in the anther tapetum (Takayama et al., 2000), the predicted
site of expression for sporophytically derived proteins des-
tined for the pollen coating (Stephenson et al., 1997). Gameto-
phytic SCR/SP11 expression was also detectable in developing
pollen grains (Takayama et al., 2000) but this was later found
to be true only for dominant (class I) alleles; for recessive
(class II) alleles only sporophytic expression could be detected
(Shiba et al., 2002). Interestingly, in heterozygotes with domi-
nant and recessive SCR/SP11 alleles, expression of the recessive
allele was undetectable (Shiba et al., 2002; Kusaba et al., 2002),
suggesting that allelic dominance is a consequence of suppres-
sed expression of recessive alleles in the presence of dominant
ones. A number of SCR/SP11 alleles have now been character-
ized, revealing more extreme sequence polymorphism than
Self-Incompatibility in Plants Plant biol. 5 (2003) 25

was previously observed for either SLGs or SRKs. Typical pair-

wise amino acid sequence comparisons of SCR/SP11 alleles
range from ~ 30 ± 55 % amino acid identity (Watanabe et al.,
2000). All SCR/SP11 proteins possess 8 conserved cysteine resi-
dues that presumably form four disulphide bridges in the ma-
ture protein suggesting that, despite their highly divergent
amino acid sequences, the overall tertiary structure of allelic
forms of SCR/SP11 may be similar.

With both male and female determinants of SSI identified in

Brassica, the biochemical mechanism of pollen recognition
and rejection can potentially be investigated. Hypothetical
models, based on analogies with receptor kinase signalling
systems in animals, predicted an S haplotype-specific pro-
tein±protein interaction between SCR/SP11 and the extracellu-
lar receptor domain of SRK leading to dimerization of activated
SRKs and autophosphorylation on serines and threonines
within the kinase domain (Fig. 1, Schopfer and Nasrallah,
2000). Recently, this model was confirmed independently by
Kachroo et al. (2001) and Takayama et al. (2001). Kachroo et
al. (2001) used in vitro ªpull downº assays to show haplotype-
specific binding between tagged versions of SRK6 and SCR6.
Takayama et al. (2001) demonstrated similar S-haplotype-
specific binding of a radio-iodinated synthetic form of S8-SP11
and SRK8. Interestingly, neither Takayama et al. (2001) nor
Kachroo et al. (2001) could demonstrate any significant af-
finity between SP11/SCR and SLG. However, Takayama et al.
(2001) did detect the presence of SLG8 in SP118/SRK8 com-
plexes, suggesting a haplotype-specific interaction between Fig. 1 Model for the mechanism of SSI in Brassica. Pollen from an
SRK and SLG before or during the SRK-SP11 interaction. This S1S2 individual is inhibited on an S1S3 stigma as a consequence of an
may explain why SLG apparently enhances S-haplotype- S-haplotype-specific interaction between male (pollen) and female
specific rejection of pollen but is itself a non-essential com- (stigma) products of the S1 haplotype. The pollen protein SCR1 is re-
cognized by and binds to the extracellular receptor domain of SRK1
ponent of the SI rejection process (Takasaki et al., 2001). Ta-
thereby inducing dimerization of SRK1 and autophosphorylation on
kayama et al. (2001) also showed that formation of the SRK8/ serine and threonine residues in the kinase domain. Activation of
S8-SP11/SLG8 complex resulted in specific autophosphoryla- the SRK initiates an intracellular signalling cascade within the stig-
tion of serine and threonine residues in the kinase domain of matic papilla cell that leads to localized rejection of the pollen. While
SRK8, the presumed cue for further downstream intracellular just SCR and SRK are required for S-haplotype specific pollen rejec-
signalling events leading to pollen arrest. tion, the strength of the SI response (in terms of numbers of incom-
patible pollen grains failing to germinate) is enhanced by the pres-
ence of SLG from the same haplotype. SLG shares approximately
Progress has already been made in identifying components of
90 % amino acid sequence identity with the receptor domain of SRK.
this signalling cascade using the yeast two-hybrid assay system This sequence identity may be important the association of SRK and
with SRK as ªbaitº (Bower et al., 1996; Gu et al., 1998). ARC1 is SLG within the ªreceptor complexº. Signalling downstream of SRK
an arm-repeat protein required for the SI response in Brassica has yet to be characterized, however ARC1, an arm-repeat protein,
that shows a specific phosphorylation-dependent interaction that binds to the kinase domain of SRK in a phosphorylation-depen-
with the kinase domain of SRK (Gu et al., 1998; Stone et al., dent manner is essential for SI, but as yet its function is unknown.
1999). How ARC1 mediates further downstream signalling
events remains to be determined. Two thioredoxin-h proteins,
THL1 and THL2 also interact with the kinase domain of SRK in
a phosphorylation-independent manner (Bower et al., 1996). SI phenotype to this normally self-compatible species, indicat-
THL1 may act as a negative regulator of SRK because THL1 can ing that all the signalling components downstream of SRK that
inhibit the kinase activity of SRK in the absence of ªactivatingº are required for SI are present and functional in A. thaliana.
components of the pollen coating (presumably SCR/SP11 of the This finding offers endless possibilities for complete dissection
same haplotype) (Cabrillac et al., 2001). However, the require- of the SSI signalling pathway in the Brassicaceae using Arabi-
ment of THL1 for regulated autophosphorylation of SRK during dopsis.
its binding to SCR/SP11 is inconclusive (Takayama et al., 2001).
b) RNase-mediated gametophytic SI
The quest to identify further downstream components of the SI
signalling system in the Brassicaceae has been given a boost by In species from the Solanaceae, Rosaceae and Scrophularia-
the recent engineering of SI lines of Arabidopsis thaliana. Or- ceae, the female determinant of GSI is a secreted ribonuclease
thologues of SRK and SCR were isolated from Arabidopsis lyra- (S-RNase) that accumulates to very high concentrations in the
ta, a close SI relative of A. thaliana and transformed into A. transmitting tract of stigma and style (McClure et al., 1989).
thaliana (Nasrallah et al., 2002). Transfer of the A. lyrata SRK S-RNase alleles have been characterized from many species:
and SCR genes into A. thaliana was sufficient to impart a full Nicotiana, Petunia, Solanum, Physalis and Lycopersicon (Solana-
26 Plant biol. 5 (2003)
ceae), Malus, Pyrus and Prunus (Rosaceae) and Antirrhinum
(Scrophulariaceae) and found to be highly polymorphic. Typi-
cal amino acid sequence identities between S-RNases from
species in the Solanaceae are around 50 % (McCubbin and Kao,
2000). Conserved and hypervariable regions can be identified
within the S-RNase protein and site-directed mutagenesis, and
domain swapping experiments indicate that the hypervariable
regions are important for S allele-specific pollen recognition
and rejection (Matton et al., 1997; Zurek et al., 1997). Interest-
ingly, Solanaceous S-RNases are frequently more similar be-
tween species than they are within species indicating that S-
RNases within this family are extremely ancient and that they
diversified before speciation (McCubbin and Kao, 2000). More
recently, a phylogenetic analysis of all available S-RNase se-
quences suggested that S-RNases from the Solanaceae, Scro-
phulariaceae and Rosaceae have a monophyletic origin, even
though these three families diverged from their common an-
cestor at least 110 million years ago (Igic and Kohn, 2001).
Transgenic loss-of-function and gain-of-function experiments
demonstrated that S-RNases are required for allele-specific
pollen rejection in Nicotiana (Murfett et al., 1994) and Petunia
(Lee et al., 1994), and analyses of mutant S-RNases in Petunia
and Lycopersicon indicate that ribonuclease activity is essential
for pollen tube rejection (Royo et al., 1994; Huang et al., 1994).
Nevertheless, other stylar components also appear to be re-
quired for S-RNase-mediated inhibition of pollen tube growth
(reviewed in McClure et al., 2000) particularly a small aspara-
gine-rich protein, HT. The gene encoding HT is not linked to the
S locus but seems to act as an essential co-factor for SI in Ni-
cotiana (McClure et al., 1999) and Lycopersicon (Kondo et al.,
2002). Indeed a picture is emerging for the existence in vivo
of the S-RNase as a large protein complex consisting of the S-
RNase (probably as a dimer or oligomer), HT protein, NaTTS
protein (the Nicotiana alata homologue of a N. tabaccum
Transmitting Tissue-Specific arabinogalactan glycoprotein),
NaMG-15 protein (the N. alata homologue of a N. tabaccum
stylar glycoprotein, PELPIII) and p11 an 11 kD copper-binding
phytocyanin (McClure et al., 2000; Cruz-Garcia et al., 2003).
S. J. Hiscock and S. M. McInnis
Fig. 2 Cytotoxic/inhibitor model for pollen
rejection during the GSI response in species
from the Solanaceae, Rosaceae and Scro-
phulariaceae (based on Luu et al., 2000). In
these species, S-RNases are the female (pis-
til) determinant of GSI. The GSI response oc-
curs later than the SSI response and pollen
tubes grow into the style before being ar-
rested. Secreted S-RNases (S1 and S2) are
taken up non-specifically by the growing
pollen tubes. Once inside the pollen tube,
S-RNases are recognized by a general RNase
inhibitor and inhibited. If, however, the S-
RNase has the same S genotype as the pol-
len tube, an S-specific RNase interactor
binds to the S-RNase and prevents interac-
tion with the general S-RNase inhibitor. This
S-specific interaction retains the enzymic ac-
tivity of the S-RNase which proceeds to de-
grade rRNA within the pollen tube, thereby
preventing protein synthesis and further pol-
len tube growth.
The functional significance of this S-RNase complex is obscure,
but it may be required for uptake of S-RNases by pollen tubes
(Cruz-Garcia et al., 2003).
The male determinant of GSI in pollen remains elusive. It is
clear that S-RNases play no part in the expression of SI in pol-
len (Dodds et al., 1999) so, as was shown for SSI in the Brassi-
caceae, two different genes at the S-locus must encode the
male and female determinants of SI. Current models for GSI in
the Solanaceae, Rosaceae and Scrophulariaceae evoke a cyto-
toxic response in which S-RNases enter incompatible pollen
tubes and degrade ribosomal RNA and mRNA thereby prevent-
ing further pollen tube growth (Fig. 2; Golz et al., 2000;
McCubbin and Kao, 2000). Such a model predicts two possible
identities for the pollen S-protein. Either it could be an S-
specific RNase translocator or it could be an S-specific RNase
inhibitor ± specific in the sense that if the S-RNase and S-inhib-
itor are encoded by the same S-haplotype, the S-RNase inhibi-
tor is ineffectual. The translocator hypothesis now seems the
less likely hypothesis because S-RNases have been shown to
be taken up in vivo with equal ease by both compatible and in-
compatible pollen tubes (Luu et al., 2000). Moreover, direct
evidence for the inhibitor model has come from studies of pol-
len-part mutants (PPMs) ± self-compatible plants defective in
pollen SI function but not affected in pistil SI function (de Net-
tancourt, 1977; Golz et al., 2000). PPMs generated by X-ray and
gamma mutagenesis were shown to possess a duplicated copy
of the S locus translocated to another chromosome or, more
usually, carried on a small additional chromosomal fragment
(ªcentric fragmentº) generated by the mutagenesis (Golz et
al., 1999, 2001). A proportion of pollen grains from the PPMs
will thus carry two different S alleles and hence, two different
S-RNase inhibitors capable of inhibiting both pistil S-RNases,
thereby accounting for the self-compatible phenotype. The
well-known occurrence of GSI breakdown in tetraploids (de
Nettancourt, 1977; Golz et al., 2000) is also consistent with
the inhibitor model because, provided plants are S heterozy-
gotes, their diploid pollen will carry two different S-RNase in-
hibitors.
Self-Incompatibility in Plants
One current variation on the ªinhibitor modelº suggests that
there may be two pollen components involved in GSI ± a gen-
eral RNase inhibitor (RI) that can inactivate any S-RNase and
an S-allele-specific product that maintains the activity of a
specific S-RNase by blocking RI binding (Luu et al., 2000,
Fig. 2). A possible candidate for such a general inhibitor of S-
RNases has been identified recently in Petunia hybrida by its
ability to bind S-RNases in the yeast two-hybrid system (Sims
and Ordanic, 2001). This novel protein, PhSBP1 (Petunia hy-
brida S-RNase Binding Protein 1) cannot be the pollen S deter-
minant because it does not vary between S haplotypes and its
interaction with S-RNases is not haplotype-specific. How-
ever, the strong binding of PhSBP1 to S-RNases suggests that
it plays a general role in the SI response. Interestingly, PhSBP1
contains a C-terminal RING-HC finger domain and RING do-
main proteins are known to act as E3 ubiquitin ligases that tar-
get specific proteins for ubiquitination and subsequent degra-
dation (Joazeiro and Weissmann, 2000). Sims and Ordanic
(2001) therefore speculate that PhSBP1 may have the role of
an E3 ubiquitin ligase in pollen tubes by targeting all S-RNases
for degradation via the ubiquitin pathway. The pollen S deter-
minant would then be a protein that disrupts the interaction
between PhSBP1 and S-RNases in an S-allele-specific manner
as predicted by Luu et al. (2000) (Fig. 2). A strong candidate
for the pollen S determinant has recently been identified by
Lai et al. (2002) working on Antirrhinum (Scrophulariaceae).
This pollen-expressed gene, AhSLF-S2 (Antirrhinum hispani-
cum S-Locus F-box-S2), exhibits characteristics expected for
the pollen S determinant ± tight linkage to the S locus and al-
lelic polymorphism (Yongbiao Xue, pers. commun.). AhSLF-S2
encodes a putative F-box protein that is expressed specifically
in pollen and in the anther tapetum. Interestingly, F-box pro-
teins, like RING domain proteins, are known to recruit specific
substrates into the ubiquitin-mediated protein degradation
pathway (Craig and Tyers, 1999). However, F-box proteins re-
quire their substrates to be phosphorylated and contain a ly-
sine residue for ubiquitin ligation. All S-RNases contain a con-
served lysine residue in their third conserved (C3) domain and
a pollen-derived Ca2+-dependent protein kinase has been
shown to phosphorylate S-RNases in vitro in a non-haplotype-
specific manner (Kunz et al., 1996). These observations suggest
that the conditions necessary for F-box-mediated degradation
of S-RNases could be met in pollen tubes in vivo. Recently three
more AhSLF alleles have been cloned, AhSLF-S1, AhSLF-S4 and
AhSLF-S5, and like AhSLF-S2, each is tightly linked to a cor-
responding S-RNase gene encoding the S1-RNase, S4-RNase
and S5-RNase, respectively. AhSLF alleles share ~ 90 % amino
acid sequence identity, and each allele is situated alongside
1 ± 2 paralogous AhSLF copies (30 ± 40 % amino acid identity)
at the S locus (Yongbiao Xue, pers. comm.). By virtue of this al-
lelic polymorphism, AhSLF could function as both the S-RNase
inhibitor and the determinant of S-allele specificity (Lai et al.,
2002) making it the strongest candidate for the pollen deter-
minant yet identified. Alternatively AhSLF may determine pol-
len S specificity by interacting with the general S-RNase inhib-
itor (PhSBP1?) according to the model of Luu et al., 2000. Not-
withstanding these two possibilities, the fact that two proteins
involved in the ubiquitin-mediated protein degradation path-
way have been implicated in the pollen part of the GSI reaction
bodes well for molecular confirmation of the inhibitor model
of S-RNase-mediated GSI.
Plant biol. 5 (2003) 27
c) GSI in Papaver
Even though the genetics of GSI is identical in Papaver rhoeas
(common poppy) and species from the Solanaceae, Rosaceae
and Scrophulariaceae, S-RNases do not regulate the GSI re-
sponse in Papaver (reviewed by Jordan et al., 2000 a). Instead
a novel ~ 15 kD stigma-specific secreted protein (S-protein)
has been shown to induce S-haplotype-specific arrest of pollen
tube growth in vitro (Franklin-Tong et al., 1988). The S-protein
gene has been cloned and five alleles characterized (Foote et
al., 1994; Walker et al., 1996; Kurup et al., 1998). Like other S-
proteins, Papaver S-proteins are highly polymorphic, sharing
between ~ 51 ± 64 % amino acid identity, and some are glyco-
sylated. However, the proteins are predicted to possess a vir-
tually identical secondary structure consisting of a series of b-
strands linked by hydrophilic (probably surface) loops. Using
this secondary structure prediction in combination with se-
quence comparisons, Ride et al. (1999) identified a large gene
family in Arabidopsis with homology to Papaver S-proteins. So
far over 50 S-protein homologues (SPHs) have been identified
in the Arabidopsis genome but surveys of predicted open-read-
ing frames suggest there may be as many as 100 SPHs in Arabi-
dopsis (Ride et al., 1999; Jordan et al., 2000 a). The function(s)
of SPHs in Arabidopsis has yet to be determined but prelimi-
nary expression studies of two SPH genes revealed fairly uni-
versal expression (albeit variable) in different tissues, suggest-
ing that these SPH genes have a general role in cell develop-
ment and differentiation (Jordan et al., 2000 a).
In Papaver an in vitro bioassay for SI has allowed biochemical
dissection of the signalling events that accompany the arrest
of incompatible pollen tubes and eventual pollen cell death.
Challenge of growing pollen tubes with incompatible S-pro-
tein (purified or recombinant) initiates a rapid transient in-
crease in cytosolic free Ca2+ (Franklin-Tong et al., 1993) ahead
of tube arrest. Increasing the Ca2+ concentration in growing
pollen tubes with Ca2+ ionophores (such as A23187, or masto-
poran) or by the release of caged Ca2+ can mirror this effect, in-
dicating that GSI in Papaver is mediated by a Ca2+-dependent
signal transduction pathway (Franklin-Tong et al., 1993, 1995).
Ratio-imaging of intracellular free Ca2+ in pollen tubes chal-
lenged by incompatible S-protein demonstrated that the tip-
focussed Ca2+ gradient essential for continuous pollen tube
growth becomes dissipated, such that Ca2+ levels fall at the
tube tip but increase rapidly in subapical regions (Franklin-
Tong et al., 1997). These events coincide with dramatic changes
in the F-actin cytoskeleton, such that after 10 ± 15 min no actin
microfilament bundles can be seen within pollen tubes (Snow-
man et al., 2000). More recent studies have shown that this
phenomenon is associated with a large and sustained depoly-
merization of F-actin (Snowman et al., 2002).
The in vitro GSI response is also accompanied by rapid phos-
phorylation and dephosphorylation of specific pollen proteins.
Two proteins showing hyperphosphorylation in pollen tubes
after challenge with incompatible S-protein are p26 and p68
(Rudd et al., 1996). Phosphorylation of p26 is Ca2+- and calmo-
dulin-dependent, whereas phosphorylation of p68 (which be-
comes phosphorylated later than p26) is Ca2+-independent
(Rudd et al., 1997). The gene encoding p26 has been cloned
and shown to encode an active inorganic pyrophosphatase
(Rudd and Franklin-Tong, 2003) but the role of this enzyme in
the GSI response is at present unclear. Recent studies have re-
28 Plant biol. 5 (2003)
Fig. 3 Model for the mechanism of GSI in Papaver rhoeas. Pollen
tube arrest occurs at the stigma surface and is mediated by a Ca2+-
based signalling system induced by exposure of pollen to a stigmatic
S-protein of the same haplotype. The identity of the pollen S-protein
is unknown but it is proposed to be a receptor that binds to stigmat-
ic S-proteins of the same haplotype. This haplotype-specific binding
is thought to be mediated by SBP (S-binding protein), a heavily gly-
cosylated pollen protein that binds non-specifically to all stigmatic S-
proteins. S-haplotype-specific interaction between pollen and stigma
components then initiates an increase in cytosolic free Ca2+ which is
followed by a Ca2+-dependent hyperphosphorylation of p26, an inor-
ganic pyrophosphatase. This is followed by a Ca2+-independent hyper-
phosphorylation of another pollen protein, p68. Dramatic changes in
the F-actin cytoskeleton are also observed, but it is uncertain whether
these changes are linked to the observed protein phosphorylations.
Eventually the incompatible pollen tube dies, apparently through a
pathway of programmed cell death (apoptosis).
vealed that the eventual death of incompatible pollen tubes
following arrest is most probably an active process (apoptosis)
rather than a passive process (necrosis). Assays for fragmenta-
tion of genomic DNA (FragEL and TUNEL) following S-protein-
induced pollen tube arrest proved positive for this characteris-
tic feature of programmed cell death (PCD), while artificially
elevating Ca2+ levels in pollen tubes also resulted in DNA frag-
mentation (Jordan et al., 2000 a, b), suggesting that Ca2+ may
be the signal for induction of PCD.
Despite this wealth of biochemical data on intracellular signal-
ling associated with the GSI response in Papaver, the pollen S
determinant has yet to be identified. A pollen-specific protein
has been identified that binds to stigmatic S-proteins but this
binding was not S-allele-specific (Hearn et al., 1996). The pol-
len S Binding Protein (SBP) is highly glycosylated, has a mo-
S. J. Hiscock and S. M. McInnis
lecular weight of ~ 70 ± 120 kD, and localizes to the plasma
membrane of the pollen. Even though binding of SBP to the
S-protein is not allele-specific, the interaction between the
two proteins appears crucial to the GSI reaction because S-pro-
tein mutants that exhibit reduced pollen inhibition activity in
the bioassay also show defective SBP binding (Jordan et al.,
2000 a). While SBP is unlikely to be the pollen S-protein, it
may function as an accessory protein that facilitates interac-
tion between the stigma S-protein and the pollen S-specific
receptor (Fig. 3).
Molecular Studies of SI in Other Species
GSI in the grasses
In the grasses (Poaceae), GSI is controlled by two independent
loci S and Z (Lundqvist, 1954; Hayman, 1956). S and Z are
both multiallelic, and full incompatibility requires that both
S and Z alleles match in pollen and pistil. S and Z thus function
in a complementary manner making the two-locus system
mechanistically distinct from the single locus GSI system of
the Solanaceae and Papaveraceae (Lewis, 1979 a). GSI in the
grasses does not appear to involve pistil-expressed RNases, or
Papaver-type S-proteins (Baumann et al., 2000). Some years
ago, Li et al. (1994) identified a pollen gene, Bm2, that ap-
peared to reside at the S locus of Phalaris coerulescens. Bm2
was found to encode a protein with an active thioredoxin do-
main at the C-terminus and a polymorphic, potentially allelic
region at the N-terminus. However, extensive segregation ana-
lyses revealed that Bm2 was not the pollen S determinant be-
cause recombination was observed between Bm2 and S; the
map position of Bm2 is estimated to be ~ 1 cM from the S locus
(Baumann et al., 2000). Interestingly, in situ hybridization
studies revealed that Bm2 was highly expressed in incompati-
ble pollen tubes prior to and during their arrest, suggesting
that Bm2 may play a direct role in the process of pollen tube
inhibition by acting downstream of S- or Z-allele-specific rec-
ognition events (Baumann et al., 2000).
SSI in Ipomoea trifida
Ipomoea trifida (Convolvulaceae) is a close diploid relative of
the commercially important hexaploid sweet potato (I. bata-
tas) and so was selected for genetic analysis of sporophytic SI
in the genus Ipomoea (Kowyama et al., 1994). Both I. trifida and
I. batatas possess a very strong system of SSI characterized by
rapid arrest of pollen development before emergence of a pol-
len tube (Kowyama et al., 2000). Because Ipomoea has a sporo-
phytic SI system orthologues of SRK might be predicted to reg-
ulate the female side of the SI response (Kowyama et al., 1995).
RT-PCR with degenerate primers designed to conserved re-
gions of Brassica SRKs amplified four distinct SRK-like cDNAs
from pools of stigma mRNAs. One of these, IRK1, was expressed
exclusively in stigma tissues and showed a pattern of develop-
mental regulation characteristic of a potential female S gene,
with optimal expression at, or just after, anthesis. However,
segregation analyses revealed that neither IRK1 nor the three
other SRK-like sequences were linked to the S locus (Kowyama
et al., 1996). These data strongly suggest that SSI in Ipomoea is
regulated by a different set of genes to species from the Brassi-
caceae. With this in mind, Kowyama and his co-workers (Ko-
wyama et al., 2000) used 2D-PAGE to detect stigma-specific
proteins associated with defined S-homozygotes; an approach
Self-Incompatibility in Plants
similar to those originally used to identify SLG and S-RNases in
Brassica and Nicotiana, respectively. Four polymorphic protein
spots of ~ 70 kD and pI 4 ± 6 each associated with one of four
different S-haplotypes were detected in Ipomoea stigmas. The
genes for these ªS-locus-linked Stigma Proteinsº (SSPs) were
cloned and found to encode allelic forms of a non-metallo
short-chain alcohol dehydrogenase (SCAD). The four SSP al-
leles had an overall amino acid identity of 95 ± 98 %. Expression
of SSP was shown to be confined to stigmas and developmen-
tally regulated, with maximal expression coinciding with an-
thesis ± characteristics expected for a female S gene. However,
segregation analysis failed to show complete linkage of SSP to
the S locus; recombination analysis placed SSP ~ 1.2 cM from
the S locus (Kowyama et al., 2000). Nevertheless, SSP will no
doubt prove useful as a marker for gene-walks to identify
genes located at the Ipomoea S locus.
SSI in Senecio squalidus
Senecio squalidus is being used as a ªmodelº species in which to
study the molecular basis of SSI in the Asteraceae (Hiscock,
2000 a, b). Even though the detailed genetics of sporophytic SI
were first described in members of the Asteraceae (Hughes
and Babcock, 1950; Gerstel 1950) there have been few subse-
quent studies of SI in this family. As with Ipomoea, initial in-
vestigations in Senecio focused on identifying potential ortho-
logues of SRK in pools of stigmatic mRNAs using degenerate
RT-PCR (Hiscock et al., 2000 a). Three potential SRK ortho-
logues were identified with 35 ± 46 % amino acid identity to
Brassica SRKs. None of these cDNAs, however, were expressed
exclusively in stigmas and none was linked to the S locus (His-
cock et al., 2003). These observations suggest that in Senecio,
as in Ipomoea, SSI operates through a molecular mechanism
different to that found in Brassica. Analysis of Senecio stigma
proteins using isoelectric focusing (IEF) identified polymor-
phic proteins associated with defined S alleles. These ªStigma
S-associated Proteinsº (SSPs) were found to be basic glycopro-
teins (pI 7 ± 8.5) with a molecular weight of ~ 35 kD. Two SSP
genes (SSP1 and SSP2) have been cloned so far and shown to
encode allelic forms of the same gene, sharing ~ 96 % identity
at the amino acid level. The SSP gene is expressed exclusively
in pistils and is developmentally regulated with maximal ex-
pression coinciding with anthesis (Hiscock et al., 2003). These
characteristics make the Senecio SSP gene a good candidate for
a female S gene but, as was demonstrated for Bm2 from Pha-
laris and SSP from Ipomoea, such evidence must be treated
with caution until linkage of the Senecio SSP gene to the S locus
has been established.
Genetic Evidence for Further SI Systems ±
Implications for the Evolution of SI
With the recent advances in the understanding of the molecu-
lar mechanisms of SI in the three model SI systems, it is easy to
forget the many other systems of homomorphic SI that have
been characterized genetically, but for which no molecular,
biochemical or cell biological data are available. Identification
and genetic characterization of many of these somewhat ªun-
usualº and generally complex systems of SI was the work of
Arne Lundqvist and co-workers (Lundqvist, 1975). Following
genetic characterization of the first two-locus SI system in
plants ± the S and Z GSI system that is unique to, and universal
in the grasses (Lundqvist, 1954), Lundqvist turned his atten-
Plant biol. 5 (2003) 29
Fig. 4 Simplified phylogenetic tree of the flowering plants based on
Soltis et al. (1999) showing the relationships between groups with
genetically characterized SI systems. Gametophytic SI (GSI) systems
with multiple loci are associated with more basal groups while spor-
ophytic SI (SSI) systems are confined to higher eudicot groups.
tion to analysing SI systems in primitive groups of dicotyle-
dons and other monocotyledons with the intention of identify-
ing phylogenetic trends in the evolution of SI. To this end he
identified three more multi-locus GSI systems, in the Ranun-
culaceae (Ranunculus acris, R. bulbosus and R. polyanthemos)
(reviewed in Lundqvist, 1990), Chenopodiaceae (Beta vulgaris)
(Lundqvist et al., 1973; Lundqvist, 1975) and Liliaceae (Lilium
martagon) (Lundqvist, 1991), respectively. All three were
shown to be regulated by four complementary self-incompat-
ibility loci. From a molecular mechanistic standpoint, such
complementary multi-locus incompatibility systems are like-
ly to be extremely complex, because, by analogy with the bet-
ter characterized two-locus complementary system of the
grasses, full incompatibility will only arise when alleles from
each of the four loci are matched in pollen and pistil. Different
combinations of alleles will therefore produce a range of in-
compatibilities (dependent on the percentages of compatible
pollen). The overall effect of such a self-incompatibility system
is to greatly increase the likelihood of compatibility between
close relatives (siblings), leading to inbreeding depression. Le-
wis (1979 a, b) considered such multi-locus SI systems to be
more primitive than single locus SI systems which exert great-
er control over bi-parental inbreeding. Thus it might be pre-
dicted that single locus SI systems evolved from, or superseded
previously existing multi-locus SI systems (Lewis, 1979 a, b;
Lundqvist, 1975, 1990), as appears to be the case with fungal
mating type systems (Hiscock and Kües, 1999). The phylo-
genetic distribution of multi-locus and single locus SI systems
in flowering plants supports this prediction (Fig. 4). Known
multi-locus SI systems are confined to the monocots and basal
orders of eudicots, while single locus GSI and SSI systems are
found throughout the higher Rosid and Asterid clades of eudi-
cots. However, the SI status of species from the most basal
clades of angiosperms that form the ANITA Grade (Soltis et al.,
1999) is largely unknown, making it difficult to deduce the
ªprimitiveº SI state. A report of GSI in Illicium (Illiciaceae)
(Thien et al., 1983) is therefore intriguing, because it supports
the prediction that GSI is the primitive SI condition. However,
more detailed genetic analyses of GSI in such species from the
ANITA grade are needed to confirm the presence of GSI in this
basal lineage and to determine the number of loci involved.
Further evidence for the primitive nature of GSI came from a
recent phylogenetic analysis of S-RNases in the Solanaceae,
30 Plant biol. 5 (2003)
Scrophulariaceae and Rosaceae which deduced a monophylet-
ic origin for S-RNases in these distantly related families (Igic
and Kohn, 2001). This finding implies that S-RNase-mediated
GSI was the primitive SI state for ~ 75 % of eudicots, i.e., the en-
tire Rosid and Asterid clades. S-RNase-based GSI must there-
fore have been present before the diversification of the major-
ity of the eudicots, suggesting that this form of GSI has been
lost or eroded continuously during eudicot diversification to
be replaced, in many cases, by other systems of SI, most nota-
bly SSI (both homomorphic and heteromorphic). The apparent
basal nature of S-RNase-mediated GSI in angiosperms raises
important questions about its evolutionary relationship with
the Papaver system, most importantly: which GSI system is
the most basal, and is the Papaver system unique to the Ranun-
culales? From a molecular perspective, given the taxonomic
relatedness of the Papaveraceae and the Ranuculaceae (both
are within the order Ranunculales), it would be interesting
to investigate whether orthologues of the Papaver S gene are
expressed in stigmas and styles of Ranunculus or whether
these tissues express orthologues of S-RNases. Such simple
molecular studies may give some important insights into the
possible origins of these two single locus GSI systems and al-
low conclusions to be drawn about the primitive SI state.
Lundqvist also investigated SI systems in the Caryophyllaceae
and identified a system of SSI (single locus) in Cerastium ar-
vense and Stellaria holostea with traces of gametophytic con-
trol in the pollen and no traces of the dominance interactions
so characteristic of SSI in other families (Lundqvist, 1990,
1994). Lundqvist (1990) therefore believed that here he had
discovered an SI system that could provide critical insights
into the evolution of SSI from GSI by a switch in the timing of
pollen S gene expression, concluding in his final paper that ªse-
quence data for S-genes may shed decisive light on this ques-
tionº (Lundqvist, 1994). The recent finding in Brassica of dual
sporophytic (tapetum) and gametophytic (microspores/pol-
len) expression of SCR/SP11, the male determinant of SSI, is
thus all the more intriguing (Takayama et al., 2000). Perhaps
the gametophytic expression of SCR/SP11 is relictual and re-
flects evolution of the present SSI system from a previous sys-
tem of GSI that utilized SCR and SRK? Interestingly, almost
identical patterns of gene expression have been observed for
the Antirrhinum AhSLF-S2 gene, which is a strong candidate
for the male determinant of S-RNase-mediated GSI (Lai et al.,
2002). What is clear, however, if one accepts Igic and Kohn
(2001)s conclusions, is that the ancestor of the Brassicaceae
must have once possessed and then lost an S-RNase-based sys-
tem of GSI, and subsequently evolved a sporophytic system of
SI operating with a completely different set of genes.
Further insights into the evolutionary relationship between
GSI and SSI can be gained from reports of a cryptic system of
GSI operating in certain lines of Brassica rapa (syn. campestris)
and Raphanus sativus (see Lewis, 1994, for review). The G gene
was identified as a consequence of analysing inbred progeny
arrays generated from ªanomalousº compatible crosses that,
according to their S genotype, should be incompatible. Such
compatible anomalies occur with some regularity in sporo-
phytic SI systems (Lewis, 1994). The G gene, which has two
alleles, usually exists in the homozygous state, especially in
cultivated varieties, and so is normally undetectable. Only in
heteroallelic stocks and wild populations could the activity of
G be detected through anomalous compatible crosses. Lewis
S. J. Hiscock and S. M. McInnis
(1994) further speculated that an underlying gametophytic
system was a feature of all sporophytic SI systems and there is
some evidence, particularly from species in the Asteraceae, to
suggest that this prediction may be correct (Lewis, 1994; His-
cock, 2000 b). This suggests that SSI systems can evolve in the
presence of established GSI systems rather than necessarily
evolving from them. Further genetic studies of this gameto-
phytic-sporophytic variant of SSI will no doubt shed light on
this intriguing possibility.
Conclusions and Perspectives
Substantial progress is being made towards establishing the
molecular basis of SI in the three model SI systems ± SSI in
Brassicaceae, S-RNase-mediated GSI and GSI in Papaver. Never-
theless, it is clear from preliminary molecular analyses of SSI in
Ipomoea and Senecio, and from a wealth of classical genetic
data in the literature, that there are far more molecular mecha-
nisms of homomorphic SI than these three. Future studies of
ªnon-modelº SI systems at a molecular level will make it pos-
sible to begin to start piecing together the evolutionary history
of the different forms of GSI and SSI. It will be particularly in-
teresting to use molecular ªtoolsº arising from studies of SSI
and GSI to investigate the distribution and expression of SI
genes in basal angiosperms, because in these primitive line-
ages will be found clues to the origins of self-incompatibility.
Acknowledgements
We thank Yongbiao Xue for sharing unpublished data with us.
We also thank Adrian Brennan and David Tabah for helpful dis-
cussions, two anonymous referees for useful comments on re-
vising the original manuscript and Tim Colborn for preparing
the figures. Our research is funded by the Biotechnology and
Biological Sciences Research Council (BBSRC).
References
Barrett, S. C. H. and Cruzan, M. B. (1994) Incompatibility in hetero-
stylous plants. In Genetic control of incompatibility and reproduc-
tive development in flowering plants (Williams, E. G., Knox, R. B.,
and Clarke, A. E., eds.), Netherlands: Kluwer Academic Publishers,
pp. 189 ± 219.
Baumann, U., Juttner, J., Bian, X., and Langridge, P. (2000) Self-incom-
patibility in the grasses. Ann. Bot. 85 (A), 203 ± 209.
Bower, M. S., Matias, D. D., Fernandes-Carvalho, E., Mazzurco, M., Gu,
T., Rothstein, S., and Goring, D. R. (1996) Two members of the thio-
redoxin-h family interact with the kinase domain of a Brassica S
locus receptor kinase. Plant Cell 8, 1641 ± 1650.
Cabrillac, D., Cock, M. J., Dumas, C., and Gaude, T. (2001) The S-locus
receptor kinase is inhibited by thioredoxins and activated by pol-
len coat proteins. Nature 410, 220 ± 223.
Craig, K. L. and Tyers, M. (1999) The F-box: a new motif for ubiquitin
dependent proteolysis in cell cycle regulation and signal transduc-
tion. Prog. Biophys. Mol. Biol. 72, 299 ± 328.
Cruz-Garcia, F., and Hancock, N. C., and McClure, B. A. (2003) S-RNase
complexes and pollen rejection. J. Exp. Bot. 54, 123 ± 130.
Darwin, C. (1877) The different forms of flowers and plants of the
same species. London: John Murray.
de Nettancourt, D. (1977) Incompatibility in Angiosperms. Berlin:
Springer-Verlag.
Dodds, P. N., Ferguson, C., Clarke, A. E., and Newbigin, E. (1999) Pol-
len-expressed S-RNases are not involved in self-incompatibility in
Lycopersicon peruvianum. Sex. Plant Reprod. 12, 76 ± 87.
Self-Incompatibility in Plants
Foote, H. C. C., Ride, J. P., Franklin-Tong, V. E., Walker, E. A., Lawrence,
M. J., and Franklin, F. C. H. (1994) Cloning and expression of a dis-
tinctive class of self-incompatibility (S) gene from Papaver rhoeas
L. Proc. Natl. Acad. Sci. USA 91, 2265 ± 2269.
Franklin-Tong, V. E., Lawrence, M. J., and Franklin, F. C. H. (1988) An in
vitro bioassay for the stigmatic product of the self-incompatibility
gene in Papaver rhoeas L. New Phytol. 110, 109 ± 118.
Franklin-Tong, V. E., Ride, J. P., Read, N. D., Trewavas, A. J., and Frank-
lin, F. C. H. (1993) The self-incompatibility response in Papaver
rhoeas is mediated by cytosolic free calcium. Plant J. 4, 163 ± 167.
Franklin-Tong, V. E., Ride, J. P., and Franklin, F. C. H. (1995) Recombi-
nant stigmatic self-incompatibility (S-) protein elicits a Ca2+ tran-
sient in pollen of Papaver rhoeas. Plant J. 8, 299 ± 307.
Franklin-Tong, V. E., Hackett, G., and Hepler, P. K. (1997) Ratio imag-
ing of Ca2+i in the self-incompatibility response in pollen tubes of
Papaver rhoeas. Plant J. 12, 1375 ± 1386.
Ganders, F. R. (1979) The biology of heterostyly. N. Z. J. Bot. 17, 607 ±
635.
Gerstel, D. U. (1950) Self-incompatibility studies in Guayule. Genet-
ics 35, 482 ± 506.
Golz, J. F., Su, V., Clarke, A. E., and Newbigin, E. (1999) A molecular de-
scription of mutations affecting the pollen component of the Ni-
cotiana alata S-locus. Genetics 152, 1123 ± 1135.
Golz, J. F., Clarke, A. E., and Newbigin, E. (2000) Mutational approa-
ches to the study of self-incompatibility: revisiting the pollen-part
mutants. Ann. Bot. 85 (A), 95 ± 103.
Golz, J. F., Oh, H.-Y., Su, V., Kusaba, M., and Newbigin, E. (2001) Genet-
ic analysis of Nicotiana pollen-part mutants is consistent with the
presence of an S-ribonuclease inhibitor at the S locus. Proc. Natl.
Acad. Sci. 98, 15372 ± 15376.
Gu, T., Mazzurco, M., Sulaman, W., Matias, D. D., and Goring, D. R.
(1998) Binding of an arm repeat protein to the kinase domain of
the S-locus receptor kinase. Proc. Natl. Acad. Sci. USA 95, 382 ± 387.
Hayman, D. L. (1956) The genetic control of incompatibility in Pha-
laris coerulescens DESF. Australian J. Biol. Sci. 9, 321 ± 331.
Hearne, M. J., Franklin, F. C. H., and Ride, J. P. (1996) Identification of a
membrane glycoprotein in pollen of Papaver rhoeas which binds
self-incompatibility (S-) proteins. Plant J. 9, 467 ± 475.
Hiscock, S. J. (2000 a) Genetic control of self-incompatibility in Sene-
cio squalidus L. (Asteraceae): a successful colonizing species. Her-
edity 85, 10 ± 19.
Hiscock, S. J. (2000 b) Self-incompatibility in Senecio squalidus L. (As-
teraceae). Ann. Bot. 85 (A), 181 ± 190.
Hiscock, S. J., McInnis, S. M., Tabah, D. A., Henderson, C. A., and Bren-
nan, A. C. E. (2003) Self-incompatibility in Senecio squalidus L. (As-
teraceae) ± the search for S. J. Exp. Bot. 54, 169 ± 174.
Hiscock, S. J., and Kües, U. (1999) Cellular and molecular mechanisms
of sexual incompatibility in plants and fungi. Int. Rev. Cytol. 193,
165 ± 295.
Hughes, M. B. and Babcock, E. B. (1950) Self-incompatibility in Crepis
foetida L. subsp. rhoeadifolia Bieb. Schinz et Keller. Genetics 35,
570 ± 588.
Huang, S., Lee, H.-S., Karunanandaa, B., and Kao, T.-H. (1994) Ribonu-
clease activity of Petunia inflata S proteins is essential for rejection
of self-pollen. Plant Cell 6, 1021 ± 1028.
Igic, B. and Kohn, J. R. (2001) Evolutionary relationships among self-
incompatibility RNases. Proc. Natl. Acad. Sci. USA 98, 13167 ±
13171.
Joazeiro, C. A. P. and Weissmann, A. M. (2000) RING finger proteins:
mediators of ubiquitin ligase activity. Cell 102, 549 ± 552.
Jordan, N. D., Ride, J. P., Rudd, J. J., Davies, E. M., Franklin-Tong, V. E.,
and Franklin, F. C. H. (2000 a) Inhibition of self-incompatible pol-
len in Papaver rhoeas involves a complex series of cellular events.
Ann. Bot. 85 (A), 197 ± 202.
Jordan, N. D., Franklin, F. C. H., and Franklin-Tong, V. E. (2000 b) Evi-
dence for DNA fragmentation triggered in the self-incompatibility
response in pollen of Papaver rhoeas. Plant J. 23, 471 ± 479.
Plant biol. 5 (2003) 31
Kachroo, A., Schopfer, C. R., Nasrallah, M. E., and Nasrallah, J. B. (2001)
Allele-specific receptor-ligand interactions in Brassica self-incom-
patibility. Science 293, 1824 ± 1826.
Kondo, K., Yamamoto, M., Itahashi, R., Sato, T., Egashira, H., Hattori, T.,
and Kowyama, Y. (2002) Insights into the evolution of self-com-
patibility in Lycopersicon from the study of stylar factors. Plant J.
30, 143 ± 153.
Kowyama, Y., Takahashi, H., Muraoka, K., Tani, T., Hara, K., and Shio-
tani, I. (1994) Number, frequency and dominance relationships of
S-alleles in diploid Ipomoea trifida. Heredity 73, 275 ± 283.
Kowyama, Y., Kakeda, K., Nakano, R., and Hattori, T. (1995) SLG/SRK-
like genes are expressed in the reproductive tissues of Ipomoea tri-
fida. Sex. Plant Reprod. 8, 333 ± 338.
Kowyama, Y., Kakeda, K., Kondo, K., Imada, T., and Hattori, T. (1996) A
putative receptor protein kinase gene in Ipomoea trifida. Plant Cell
Physiol. 37, 681 ± 685.
Kowyama, Y., Tsuchiya, T., and Kakeda, K. (2000) Sporophyitc self-in-
compatibility in Ipomoea trifida: a close relative of sweet potato.
Ann. Bot. 85 (A), 191 ± 196.
Kurup, S., Ride, J. P., Jordan, N. D., Fletcher, E. G., Franklin-Tong, V. E.,
and Franklin, F. C. H. (1998) Identification and cloning of related
self-incompatibility S-genes in Papaver rhoeas and Papaver nudi-
caule. Sex. Plant Reprod. 11, 192 ± 198.
Kunz, C., Chang, A., Faure, J.-D., Clarke, A. E., Polya, G. M., and Ander-
son, M. A. (1996) Phosphorylation of style S-RNases by Ca2+-de-
pendent protein kinases from pollen tubes. Sex. Plant Reprod. 9,
25 ± 34.
Kusaba, M., Nishio, T., Satta, Y., Hinata, K., and Ockendon, D. (1997)
Striking sequence similarity in inter- and intra-specific compari-
sons of class I SLG alleles from Brassica oleracea and Brassica cam-
pestris: Implications for the evolution and recognition mechanism.
Proc. Natl. Acad. Sci. USA 94, 7673 ± 7678.
Kusaba, M., Matsushita, M., Okazaki, K., Satta, Y., and Nishio, T.
(2000) Sequence and structural diversity of the S-locus genes from
different lines with the same self-recognition specificities in Bras-
sica oleracea. Genetics 154, 413 ± 420.
Kusaba, M., Tung, C.-W., Nasrallah, M. E., and Nasrallah, J. B. (2002)
Monoallelic expression and dominance interactions in anthers of
self-incompatible Arabidopsis lyrata. Plant Physiol. 128, 17 ± 20.
Lai, Z., Ma, W., Han, B., Liang, L., Zhang, Y., Hong, G., and Xue, Y. (2002)
An F-box gene linked to the self-incompatibility (S) locus of Anti-
rrhinum is expressed specifically in pollen and tapetum. Plant Mol.
Biol. 50, 29 ± 42.
Lee, H.-S., Huang, S., and Kao, T.-H. (1994) S proteins control rejection
of incompatible pollen in Petunia inflata. Nature 367, 560 ± 563.
Lewis, D. (1979 a) Sexual Incompatibility in Plants. Edward Arnold.
Lewis, D. (1979 b) Genetic versatility of incompatibility in plants. N.
Z. J. Bot. 17, 637 ± 644.
Lewis, D. (1994) Gametophytic-sporophytic incompatibility. In Ge-
netic Control of Self-Incompatibility and Reproductive Develop-
ment in Flowering Plants (Williams, E. G., Clarke, A. E., and Knox,
R. B., eds.), Dordrecht: Kluwer, pp. 88 ± 101.
Li, X., Nield, J., Hayman, D., and Langridge, P. (1994) Cloning a puta-
tive self-incompatibility gene from the pollen of the grass Phalaris
coerulescens. Plant Cell 6, 1923 ± 1932.
Lundqvist, A. (1954) Studies on self-sterility in rye, Secale cereale L.
Hereditas 40, 278 ± 294.
Lundqvist, A., ésterbye, U., Larsen, K., and Linde-Laursen, I. (1973)
Complex self-incompatibility systems in Ranunculus acris L. and
Beta vulgaris L. Hereditas 74, 161 ± 168.
Lundqvist, A. (1975) Complex self-incompatibility systems in an-
giosperms. Proc. R. Soc. Lond. B. 188, 235 ± 245.
Lundqvist, A. (1990) The complex S-gene system for control of self-
incompatibility in the buttercup genus Ranunculus. Hereditas 113,
29 ± 46.
32 Plant biol. 5 (2003)
Lundqvist, A. (1990) One-locus sporophytic S-gene system with tra-
ces of gametophytic pollen control in Cerastium arvense ssp. stric-
tum (Carophyllaceae). Hereditas 113, 203 ± 215.
Lundqvist, A. (1991) Four-locus S-gene control of self-incompatibil-
ity made probable in Lilium martagon (Liliaceae). Hereditas 114,
57 ± 63.
Lundqvist, A. (1994) ªSlowº and ªquickº S-alleles without dominance
interaction in the sporophytic one-locus self-incompatibility sys-
tem of Stellaria holostea (Caryophyllaceae). Hereditas 120, 191 ±
202.
Luu, D.-T., Qin, X., Morse, D., and Cappadocia, M. (2000) S-RNase up-
take by compatible pollen tubes in gametophytic self-incompat-
ibility. Nature 407, 649 ± 651.
Matton, D. P., Maes, O., Laublin, G., Xike, Q., Bertrand, C., Morse, D.,
and Cappadocia, M. (1997) Hypervariable domains of self-incom-
patibility RNases mediate allele-specific pollen recognition. Plant
Cell 9, 1756 ± 1766.
McClure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J.,
Sakiyama, F., and Clarke, A. E. (1989) Style self-incompatibility
gene products of Nicotiana alata are ribonucleases. Nature 342,
955 ± 957.
McClure, B. A., Mou, B., Canevascini, S., and Bernatsky, R. (1999) A
small asparagine-rich protein required for S-allele-specific pollen
rejection in Nicotiana. Proc. Natl. Acad. Sci. USA 96, 13548 ± 13553.
McClure, B. A., Cruz-Garcia, F., Beecher, B., and Sulaman, W. (2000)
Factors affecting inter- and intra-specific pollen rejection in Nicoti-
ana. Ann. Bot. 85 (A), 113 ± 123.
McCubbin, A. G. and Kao, T.-H. (2000) Molecular recognition and re-
sponse in pollen and pistil interactions. Ann. Rev. Cell Dev. Biol. 16,
333 ± 364.
Murfett, J., Atherton, T. L., Mou, B., Gasser, C. S., and McClure, B. A.
(1994) S-RNase expressed in transgenic Nicotiana causes S-allele-
specific pollen rejection. Nature 367, 563 ± 566.
Nasrallah, J. B., Stein, J. C., Kandasamy, M. K., and Nasrallah, M. E.
(1994) Signalling the arrest of pollen tube development in self-in-
compatible plants. Science 266, 1505 ± 1508.
Nasrallah, M. E., Liu, P., and Nasrallah, J. B. (2002) Generation of self-
incompatible Arabidopsis thaliana by transfer of two S locus genes
from A. lyrata. Science 297, 247 ± 249.
Ride, J. P., Davies, E. M., Franklin, F. C. H., and Marshall, D. F. (1999)
Analysis of Arabidopsis genome reveals a large new gene family in
plants. Plant Mol. Biol. 39, 927 ± 932.
Royo, J., Kunz, C., Kowyama, Y., Anderson, M., Clarke, A. E., Newbigin,
E. (1994) Loss of histidine residue at the active site of S-locus ribo-
nuclease is associated with self-compatibility in Lycopersicon per-
uvianum. Proc. Natl. Acad. Sci. USA 91, 6511 ± 6514.
Rudd, J. J. and Franklin-Tong, V. E. (2003) Signals and targets of the
self-incompatibility response in pollen of Papaver rhoeas. J. Exp.
Bot. 54, 141 ± 148.
Rudd, J. J., Lord, J. M., Franklin, F. C. H., and Franklin-Tong, V. E. (1996)
Increased phosphorylation of a 26 kD pollen protein is induced by
the self-incompatibility response in Papaver rhoeas. Plant Cell 8,
713 ± 724.
Rudd, J. J., Franklin, F. C. H., and Franklin-Tong, V. E. (1997) Ca2+-inde-
pendent phosphorylation of a 68 kD pollen protein is stimulated
by the self-incompatibility response in Papaver rhoeas. Plant J. 12,
507 ± 514.
Schopfer, C. R., Nasrallah, M. E., and Nasrallah, J. B. (1999) The male
determinant of self-incompatibility in Brassica. Science 266,
1697 ± 1700.
Schopfer, C. R. and Nasrallah, J. B. (2000) Self-incompatibility. Pro-
spects for a novel putative peptide-signaling molecule. Plant Phys-
iol. 124, 935 ± 939.
Shiba, H., Iwano, M., Entani, T., Ishimoto, T., Che, F.-S., Satta, Y., Ito, A.,
Takada, Y., Watanabe, M., Isogai, A., and Takayama, S. (2002) The
dominance of alleles controlling self-incompatibility in Brassica
pollen is regulated at the RNA level. Plant Cell 14, 491 ± 504.
S. J. Hiscock and S. M. McInnis
Sims, T. L. and Ordanic, M. (2001) Identification of a S-ribonuclease-
binding protein in Petunia hybrida. Plant Mol. Biol. 47, 771 ± 783.
Snowman, B. N., Geitmann, A., Clarke, S. R., Staiger, C. J., Franklin, C. H.
F., Emons, A. M. C., and Franklin-Tong, V. E. (2000) Signalling and
the cytoskeleton of pollen tubes of Papaver rhoeas. Ann. Bot. 85
(A), 49 ± 57.
Snowman, B. N., Kovar, D. R., Shevchenko, G., Franklin-Tong, V. E., and
Staiger, C. J. (2002) Signal-mediated depolymerization of actin in
pollen during the self-incompatibility response. Plant Cell 14,
2613 ± 2626.
Soltis, P. S., Soltis, D. E., and Chase, M. W. (1999) Angiosperm phylo-
geny inferred from multiple genes as a tool for comparative biolo-
gy. Nature 402, 402 ± 404.
Stephenson, A. J., Doughty, J., Elleman, C. J., Hiscock, S. J., and Dickin-
son, H. G. (1997) The male determinant of self-incompatibility in
Brassica oleracea is located in the pollen-coating. Plant Journal 12,
1351 ± 1359.
Stone, S. L., Arnoldo, M., and Goring, D. R. (1999) A breakdown of
Brassica self-incompatibility in ARC1 antisense transgenic plants.
Science 286, 1729 ± 1731.
Suzuki, G., Kusaba, M., Matsushita, M., Okazaki, K., and Nishio, T.
(2000) Characterization of Brassica S-haplotypes lacking S-locus
glycoprotein. FEBS Lett. 482, 102 ± 108.
Takasaki, T., Hatakeyama, K., Suzuki, G., Watanabe, M., Isogai, A., and
Hinata, K. (2000) The S receptor kinase determines self-incompat-
ibility in Brassica stigma. Nature 403, 913 ± 916.
Takayama, S., Shiba, H., Iwano, M., Shimosato, H., Che, F.-S., Kai, N.,
Watanabe, M., Suzuki, G., Hinata, K., and Isogai, A. (2000) The pol-
len determinant of self-incompatibility in Brassica campestris.
Proc. Natl. Acad. Sci. 97, 1920 ± 1925.
Takayama, S., Shimosato, H., Shiba, H., Funato, M., Che, F.-E., Wata-
nabe, M., Iwano, M., and Isogai, A. (2001) Direct ligand-receptor
complex interaction controls Brassica self-incompatibility. Nature
413, 534 ± 538.
Thien, L. B., White, D. A., and Yatsu, L. Y. (1983) The reproductive biol-
ogy of a relict ± Illicium floridanum. Am. J. Bot. 70, 717 ± 727.
Walker, E. A., Ride, J. P., Kurup, S., Franklin-Tong, V. E., Lawrence, M. J.,
and Franklin, F. C. H. (1996) Molecular analysis of two functional
homologues of the S3 allele of the Papaver rhoeas incompatibility
gene isolated from different populations. Plant Mol. Biol. 30,
983 ± 994.
Watanabe, M., Ito, A., Takada, Y., Ninmiya, C., Kakizaki, T., Takahata, Y.,
Hatakeyama, K., Hinata, K., Suzuki, G., Takasaki, T., Satta, Y., Shiba,
H., Takayama, S., and Isogai, A. (2000) Highly divergent sequences
of the pollen self-incompatibility (S) gene in class-I S haplotypes of
Brassica campestris (syn. rapa) L. FEBS Lett. 473, 139 ± 144.
Whitehouse, H. L. K. (1950) Multiple allelomorph incompatibility of
pollen and style in the evolution of angiosperms. Ann. Bot. N. S. 14,
198 ± 216.
Zurek, D. M., Mou, B., Beecher, B., and McClure, B. (1997) Exchanging
sequence domains between S-RNases from Nicotiana alata dis-
rupts pollen recognition. Plant J. 11, 797 ± 808.
S. J. Hiscock
School of Biological Sciences
University of Bristol
Woodland Road
Bristol BS8 1UG
UK
E-mail: simon.hiscock@bristol.ac.uk
Section Editor: L. A. C. J. Voesenek