Agriculture Reference
In-Depth Information
lipidic pollen coat free from contamination by gametophytic proteins from within
pollen grains. Experimental evidence that the pollen
S
-specificity component
is indeed in the pollen coat was gained using a pollination bioassay (Stephenson
et al.
, 1997). In this assay, stigmatic papillae were treated with either cross- or self-
pollen coat, pollinated with cross-pollen, and the ability of the pollen to hydrate was
assessed. It was found that pre-treatment with self-pollen coat, but not cross pollen
coat, significantly reduced hydration of cross pollen on the stigma (Stephenson
et al.
, 1997). These results strongly suggest that the molecule that encodes pollen
S
-haplotype specificity does indeed reside in the pollen coat. Subsequent fraction-
ation of pollen coat proteins, followed by assessing their activity in this bioassay,
determined that the pollen
S
-determinant is a member of a group of basic, cysteine-
rich proteins of the PCP family with a molecular mass less than 10 kDa (Stephenson
et al.
, 1997).
The pollen
S
-determinant is predicted to have a number of other characteristics.
It must exhibit sequence polymorphism between
S
-haplotypes and it has to be ge-
netically and physically linked to the
S
-locus. Using these criteria, two separate
groups undertook a comprehensive characterisation of the
Brassica S
-locus region
surrounding
SLG
and
SRK
to identify potential candidates. In the first published re-
port, a 76-kb region of the
S
-locus of the
S
9
-haplotype of
B. campestris
was cloned
and completely sequenced. In addition to the
SLG
and
SRK
genes, it was found to
contain 12 genes that are expressed in anthers and/or pistils (Suzuki
et al.
, 1999).
One of these genes (
SP11
) attracted particular interest as a potential candidate for
encoding the pollen
S
-determinant, as it encodes a protein with similar character-
istics to those predicted using the above bioassy, being a small, basic cysteine-rich
protein. Further, this gene is located in the immediate 3
flanking region of
SRK
9
and it is expressed predominantly in anthers (Suzuki
et al.
, 1999). Independently, a
second allele of
SP11
was identified in a 13-kb region between
SRK
and
SLG
in the
S
8
-haplotype of
B. campestris
, though this gene was termed
SCR
(
S
-locus cysteine-
rich) (Schopfer
et al.
, 1999). Additional alleles of
SP11/SCR
have been isolated and
sequenced (Schopfer
et al.
, 1999; Takayama
et al.
, 2000) and found to be extremely
polymorphic, with overall amino acid sequence identities of between 26 and 46%.
These proteins are 74-77 amino acids in length and are hydrophilic, except for an
N-terminal stretch of 19 amino acids. The hydrophobic N-terminus is highly con-
served and is predicted to encode a signal peptide that leads to secretion of these
proteins. The mature, secreted protein is predicted to be 8.4-8.6 kDa and a pI of
8.1-8.4, with only 12 amino acids conserved between haplotypes and 8 of these are
cysteines hypothesised to form disulphide bonds involved in intramolecular struc-
ture. Moreover,
in situ
hybridisation of anther sections showed that SCR/SP11 is
expressed in the tapetum of the anther (Takayama
et al.
, 2000). This sporophytic ex-
pression pattern explains why in
Brassica
the SI phenotype of pollen is determined
by the genotype of the pollen parent.
Proof that
SP11
/
SCR
is indeed the pollen
S
-determinant has been attained using
both the bioassay approach and, conclusively, transgenic experiments (Schopfer
et al.
, 1999; Takayama
et al.
, 2000). In a classic gain-of-function experiment, the
