Research Update
SRY represses a negative regulator of male
development. Proc. Natl. Acad. Sci. U. S. A.
90, 3368–3372
6 Vidal, V.P. et al. (2001) Sox9 induces testis
development in XX transgenic mice. Nat. Genet.
28, 216–217
7 Zhang, J. et al. (1999) In vitro binding and
expression studies demonstrate a role for the
mouse Sry Q-rich domain in sex determination.
Int. J. Dev. Biol. 43, 219–227
8 Capel, B. et al. (1993) Circular transcripts of the
testis-determining gene Sry in adult mouse testis.
Cell 73, 1019–1030
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9 Collignon, J. et al. (1996) A comparison of the
properties of Sox-3 with Sry and two related
genes, Sox-1 and Sox-2. Development
122, 509–520
10 Whitfield, L.S. et al. (1993) Rapid sequence
evolution of the mammalian sex-determining
gene SRY. Nature 364, 713–715
11 Tucker, P.K. and Lundrigan, B.L. (1993) Rapid
evolution of the sex determining locus in Old
World mice and rats. Nature 364, 715–717
12 Hacker, A. et al. (1995) Expression of Sry, the
mouse sex determining gene. Development
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13 Bergstrom, D.E. et al. (2000) Related function of
mouse SOX3, SOX9, and SRY HMG domains
assayed by male sex determination. Genesis
28, 111–124
Claire A. Canning
Robin Lovell-Badge*
Division of Developmental Genetics,
MRC National Institute for Medical Research,
The Ridgeway, Mill Hill, London, UK NW7 1AA.
*e-mail: rlovell@nimr.mrc.ac.uk
Receptor–ligand interaction demonstrated in Brassica
self-incompatibility
Noni (V.E.) Franklin-Tong
There are very few examples in plants
where both the receptor and the ligand that
interacts with it have been identified. The
self-incompatibility (SI) system, which
involves the recognition and rejection of
‘self’ pollen, is controlled by the S locus.
Molecular analysis of SI in Brassica
identified two stigmatic components,
SLG and SRK, and a pollen component,
SCR/SP11, at the S locus. Two recent papers
demonstrated that SCR and SRK interact,
providing not only a major breakthrough
in our understanding of the SI response,
but also in our knowledge about
receptor–ligand interactions in plant cells.
Sex is always considered to be an exciting
and risqué topic. It is also crucially
important from the purely biological point
of view of producing the next generation.
Even though many plants can reproduce
by asexual means, sexual reproduction
remains vital to the general fitness of a
population. Indeed, it is thought that
at least part of the great success of
angiosperms (flowering plants) as a group
is owing to their evolution of complex
strategies to prevent self-fertilization and
to ensure out-breeding. This is especially
important if one considers the physical
proximity between the anthers producing
male pollen and the stigma (the female
receptive organ) in most plants (Fig. 1).
Self-incompatibility (SI) is one of the
most widespread of mechanisms used by
flowering plants. SI is usually determined
genetically by a single S locus, with
multiple S alleles. This locus is
remarkably polymorphic; as many as
41 S alleles have been identified in a
http://tig.trends.com
single population [1]. When pollen lands
on a stigma, if it carries the same S allele
as the stigma, it is recognized as ‘self’,
and rejected. If it is ‘non-self’, it grows
normally and will probably achieve
fertilization (Fig. 1). This interaction was
alluded to as a cell–cell recognition system
long before the molecular basis for SI had
been determined.
In the past few years in particular,
major advances have been made in the
identification of components involved in
the rejection of self pollen in a variety of
SI systems, including Brassica (which
include many crop species such as
cabbages, oil-seed rape, mustard and
turnips), the Solanaceae (which include
tobacco, potato, tomato and the
ornamental Petunia) and Papaver (the
field poppy, commonly found in cornfields).
Interestingly, all three of these SI systems
operate using very different mechanisms
[2]. However, it is only relatively recently,
as a result of considerable effort, that the
S-locus components on both the pollen and
stigma side in the Brassica SI system have
been identified. This is, so far, the only
SI system where both the male and female
recognition components are known. Their
identification has enabled the first steps to
be made to investigate the nature of this
very precise interaction.
Brassica S-locus components
Molecular analysis of SI in Brassica
identified two stigmatic components
encoded at the S locus: a secreted S-locus
glycoprotein (SLG) and an S-locus receptor
kinase (SRK). SRK, which functions as
the female determinant of SI, has an
(a)
(b)
Anther
'Cross'
pollen
(e.g. S3 S4)
Style
'Self'
pollen
(S1 S2)
Ovule
Pistil
S genotype
S1 S2
TRENDS in Genetics
Fig. 1. Cartoon of how self-incompatibility (SI) operates.
This plant carries two S alleles: S1 and S2, so it has the
genotype S1S2. The haploid pollen will carry alleles S1 and
S2. If SI is sporophytically determined (as it is in Brassica),
the pollen from this plant will have the phenotype S1S2. If
SI is gametophytically determined (as it is in Papaver and
Nicotiana) the pollen will have the phenotype S1 or S2.
(a) An incompatible scenario (red). Pollen from an
S1S2 plant, if it lands on a stigma of a flower from the
same plant, or on that of another plant carrying matching
S alleles (i.e. S1S2), will be ‘self’ or ‘incompatible’.
Incompatible pollen is inhibited at a specific stage during
pollination. In Brassica and Papaver, this is very early and
occurs on the stigma surface; in the Solanaceae it is late,
and occurs in the style. As a consequence, no seed is set.
(b) A compatible situation (blue). Pollen from plants that
carry different S alleles (e.g. S3S4) that land on a stigma of
a S1S2 plant are not ‘recognized’ since their S alleles do
not match. This pollen can therefore hydrate, germinate
and grow through the stigma and style, and fertilize the
ovules to make seed.
0168-9525/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02613-0
Research Update
114
(a)
Extent of the S locus ~20–400 kb
~15 kb
SRK
SLG
SCR/SP11
SLG
domain
Kinase
domain
Transmembrane
domain
(b)
PCP
SCR/SP11
SCR/SP11
interacts
with SRK
Pollen
grain
SLG
P
SRK
Aquaporin
P
?
ARC1
Stigmatic papilla
TRENDS in Genetics
extracellular domain containing a region
of high homology to SLG [2–4]. The male
determinant at the S locus is a small,
secreted, cysteine-rich pollen coat protein,
which, confusingly, has been given two
independent names: SCR and SP11 [5,6]. A
cartoon, showing the S-locus components,
is shown in Fig. 2a. Other components that
are thought to be involved in SI, but that
are not at the S locus, have also been
identified [2] (Fig. 2b).
It is thought that interaction of
SCR/SP11 and SRK triggers a signal
transduction cascade in the stigma that
results in rapid inhibition of pollen
growth, usually before or soon after pollen
germination [2] (Fig. 2b). Consistent with
this idea, there is evidence that pollen
coat proteins can trigger phosphorylation
of SRK [7]. The role of SLG in the
SI response is rather more equivocal.
In a recent breakthrough, two groups
headed by June Nasrallah (Cornell
University, Ithaca, NY, USA) and
http://tig.trends.com
TRENDS in Genetics Vol.18 No.3 March 2002
Fig. 2. The Brassica self-incompatibility system.
(a) Cartoon of the Brassica S locus. The S locus is
estimated to be between 20 and 400 kb, depending
on the S genotype. The female secreted S-locus
glycoprotein (SLG; dark green), and the female S-locus
receptor kinase (SRK) define the extent of the S locus.
SRK has a domain that has considerable homology
to SLG, a membrane-spanning domain (orange)
and a kinase domain (dark blue). The male S-locus
component, SCR/SP11 (red) is located between SLG
and SRK. The distance between SCR/SP11 and SRK is
approximately 15 kb, but will vary, depending on the
S genotype concerned. (b) How the Brassica SI
system is thought to operate. In Brassica, the stigmatic
S component, the SRK is located in the stigmatic
papillae. SRK (blue) has an extracellular domain, a
single-pass transmembrane region, and an intracellular
kinase domain (dark blue star). The pollen S component,
SCR/SP11 (red circles), is located in the pollen coat of
the pollen grain. PCPs (pink trapezoids) are also located
here. The secreted SLG (dark green) has high homology
to the extracellular domain of SRK, but is not essential
for the SI reaction. When a pollen grain alights on the
papilla surface, the pollen coat (yellow) flows to form a
layer between the pollen and stigma. When S alleles
carried by SCR/SP11 match an S allele carried by the
stigma, an incompatible SI reaction is triggered. The
papers reviewed here provide evidence that SCR/SP11
interact with SRK. This interaction stimulates
phosphorylation of the kinase domain of SRK. Following
activation, SRK phosphorylates ARC1. Further details of
the intracellular signalling cascade within the papilla cell
are not known. However, evidence suggests that it could
regulate the activity of aquaporins, which could limit the
uptake of water necessary for pollen hydration and
germination. Adapted from Ref. [15], with permission
from American Society of Plant Physiologists.
Akira Isogai (Nara Institute of Science and
Technology, Nara, Japan) independently
demonstrated the interaction of the pollen
ligand with the stigma receptor [8,9]. A
number of important findings were
reported in these two papers. Both labs
demonstrated that SCR/SP11, when used
to coat the stigma, was sufficient to inhibit
pollen that was normally compatible, but
only if it matched the S allele now coating
the stigma; that is, when ‘like’ pollen
interacts with ‘like’ stigma, the SI response
is activated. Isogai’s lab analysed SP11
in detail, using MALDI-TOF mass
spectroscopy, and used this information to
synthesize SP11 chemically. They elegantly
demonstrated that – as predicted – the
eight cysteines present in this molecule
formed four disulfide bridges. Moreover,
they showed that the artificially produced
SP11 is biologically active. The availability
of a pure SP11 proved extremely useful.
SRK and SCR/SP11 interact
Both labs also demonstrated, using
different approaches, that SRK interacts
with SCR/SP11 [8,9]. Nasrallah’s lab used
an immunoprecipitation approach to
show S-specific interactions between the
‘ectodomain’ of SRK and SCR [8]. Isogai’s
lab used a slightly different approach,
labelling SP11 by iodination and then
testing its binding to stigmatic microsomal
membranes. Both labs demonstrated an
S-allele-specific interaction, with a large
difference in the strength of binding when
the S alleles matched, compared with a
situation using different S alleles [8,9].
There is a discrepancy between the
data, but this is most probably due to a
technical problem. On balance, it appears
that there is no requirement for the
kinase domain of SRK. This is interesting,
as it contrasts with other studies of
receptor–ligand interactions in plants,
where an active kinase domain is
required for the interaction of both
CLAVATA1 with CLAVATA3, and FLS2
with flagellin [10,11].
Receptor–ligand interactions identified in
plant cells
There is very little information regarding
receptor–ligand interactions in plant
systems. Although a large number of
putative transmembrane receptor-like
kinases (RLKs) have now been identified
in plants [12], the great majority are socalled ‘orphans’ without an identified
ligand. Similarly, although a number of
ligands have been identified, their cognate
receptors have yet to be found. There are
just a handful of examples where RLK
receptor–ligand interactions have been
demonstrated clearly in plant systems.
These have emerged over the past year or
so, and include interactions between the
BRI1 receptor kinase with brassinolide,
a brassinosteroid [13]; the CLAVATA1
receptor kinase (involved in meristematic
determination) and CLAVATA3 [10];
the FLAGELLIN-SENSING 2 (FLS2)
receptor kinase with flagellin [11].
The SCR/SP11–SRK data therefore are
quite significant, especially because the
only interaction where binding affinities
had been measured previously was the
interaction of the BRI1 receptor with
brassinolide [13], which had a dissociation
constant (Kd) of 7.4 nM. The stoichiometric
measurements of the interaction of SP11
with its cognate receptor suggest that
there are two binding sites: a high-affinity
binding site (Kd = 1.2 nM) and a low affinity
site (Kd = 32 nM) [9]. The data for SRK6
interactions with SCR6 [8] give a Kd of
0.04 nM, which is considerably higher.
However, there are several notable
differences in the way that these studies
were carried out, any of which could
Research Update
potentially explain the discrepancy
observed. One obvious difference is the
fact that different Brassica species and
different S-alleles were studied. It is,
therefore, difficult to make any definitive
statements or draw any real comparisons
at present, except to say that these
interactions appear to be in roughly
the same range as some animal
receptor–ligand binding affinities.
SCR/SP11 also interacts with SLG
Both labs tested the ability of SCR/SP11
to interact with the other stigmatic
component, SLG, as the role of this
component in the SI response is rather
unclear. Both papers present data that,
although not definitive, clearly indicate
that SCR/SP11 also interacts with SLG,
but not as strongly as to SRK [8,9]. Because
both labs, using different approaches,
came to the same conclusion, this seems
a probable scenario. Furthermore,
Takayama’s data [9], using cross-linked
SP11 to immunoprecipitate SLG, suggest
a very close association between SRK and
SLG. They propose a model whereby SRK
and SLG form a high-affinity receptor
complex that interacts with SCR/SP11.
The fact that SLG and SCR/SP11 interact
is not altogether surprising, because the
‘ectodomain’ of SRK shares significant
homology with SLG; indeed, this was how
SRK was identified. However, the biological
significance of the interaction between SLG
and SCR/SP11 is unclear, especially as SLG
is not necessary for the SI response, at least
in certain haplotypes [14].
SCR/SP11 can stimulate SRK
autophosphorylation
Finally, it has been demonstrated [9] that
SP11 can induce autophosphorylation of
TRENDS in Genetics Vol.18 No.3 March 2002
SRK in an S-allele-specific manner.
Although it was demonstrated previously
that pollen coat proteins can elicit this
response [7], this is the first time that it
has been established that the pollen
S receptor itself (and alone) can stimulate
phosphorylation of this stigmatic receptor
kinase. This is, therefore, an important
observation, because it provides insight
into the nature of the interaction
between SP11 and SRK. It is assumed
that the interaction triggers a signalling
cascade as a consequence of the Brassica
SI response.
Conclusion
The demonstration that SCR and SRK
interact not only provides a major
breakthrough in our understanding of the
SI response, but also in our knowledge
about receptor–ligand interactions in
plant cells. This lays the foundation
for a more detailed understanding of
receptor–ligand interactions in general. It
also will allow a detailed analysis of the
signal transduction cascade assumed to be
triggered by the SRK–SCR interaction.
115
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8
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10
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12
13
Acknowledgements
Work in the author’s laboratory is
funded by the UK Biotechnological
and Biological Science Research
Council (B.B.S.R.C).
References
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Noni (V.E.) Franklin-Tong
Wolfson Laboratory for Plant Molecular
Biology, School of Biosciences,
University of Birmingham, Edgbaston,
Birmingham, UK B15 2TT.
e-mail: V.E.Franklin-Tong@bham.ac.uk
QTL for timing: a natural diversity of clock genes
Neeraj Salathia, Kieron Edwards and Andrew J. Millar
Conventional, forward genetics has
identified several molecular components of
circadian clocks. Many additional loci and
genetic interactions have recently been
implicated in rhythmic control by a major
effort in mapping quantitative trait loci
(QTL) in the mouse. Reconciling the QTL
with previous results both from QTL and
mutagenesis will be a challenge for
rhythm researchers.
http://tig.trends.com
Most eukaryotes and some prokaryotes
have evolved biological clocks that
regulate behaviour and physiology
rhythmically, in a daily sequence that can
anticipate the environmental cycle. The
clock is termed ‘circadian’, meaning ‘about
daily’, because it does not keep exactly
24-hour days. In Nature, daily light and
temperature cycles reset the clock and
synchronize it with the 24-hour rotation
of the Earth. Exactly when the daily
round starts, relative to dawn (‘phase
angle’), and which rhythms occur in
what sequence, are probably affected
by the array of selective pressures in
particular habitats.
Many induced mutations have now
been studied to bring some understanding
of ‘clock genes’ (genes that are required to
construct the circadian clock) in the five
0168-9525/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02633-6