Lessons on signalling in plant self-incompatibility systems - Intercellular Communication in Plants - page 263

SCR 6 cDNA was introduced under control of the SCR 2 promoter into B. oleraceae
S 2 S 2 plants. Pollen expressing the SCR 6 transgene was found to be fully incompatible
when used to pollinate wild-type S 6 S 6 and fully compatible on wild-type S 22 S 22
stigmas. In a pollination bioassay, recombinant S 9 SCR / SP11 was applied to S 9 S 9
and S 8 S 8 stigmas and the protein was found to elicit the SI response only in the former
(self) stigmas, causing the inhibition of hydration of cross-pollen (Takayama et al. ,
2000).

10.4.4 Regulation of SRK

Receptor kinase activity is generally tightly regulated to avoid their spontaneous
activation. In the case of SRK, this is an important consideration as non-specific
activation could easily lead to sterility (Kemp & Doughty, 2003). Multiple mecha-
nisms have been identified and characterised in animal systems that act often in a
concerted manner to down-regulate kinase activity (Cock et al. , 2002). In vitro , both
the truncated kinase domain and the full-length SRK protein autophosphorylate
in the absence of the cognate ligand (Goring & Rothstein, 1992; Giranton et al. ,
2000). In vivo ,however, SRK requires the addition of pollen coat for autophos-
phorylation (Cabrillac et al. , 2001), suggesting that some mechanism of repressing
SRK is present in the stigma. Several SRK interacting proteins have been identi-
fied which may potentially regulate SRK activity. Two thioredoxin-h-like proteins
(THL1 and THL2) were identified in yeast two-hybrid screens using the kinase
domain of SRK as bait (Bower et al. , 1996). THL1 inhibits constitutive autophos-
phorylation of SRKin the absence of the SCR ligand (Cabrillac et al. , 2001) and this
interaction requires the presence of a conserved cysteine residue on the cytoplasmic
side of the SRK transmembrane domain (Mazzurco et al. , 2001). This inhibition can
be overcome in a haplotype-specific manner by addition of SCR and suggests that
THL1 (and possibly THL2) acts as basal state inhibitors of SRK. Ligand-mediated
activation through binding of SCR to the extracellular domain of SRK presumably
leads to a conformational change, causing derepression of kinase activity. In a su-
perficially contradictory study, synthetic SCR 8 (but not SCR 9 )was found to increase
phosphorylation of SRK 8 extracted from microsomal membranes in the absence of
thioredoxin or other regulators of SRK activity (Takayama et al. , 2001). One likely
explanation for this is that the enzymatic properties of the SRK kinase domain may
be substantially altered as a result of the conformational change induced by binding
to SCR. The ligand-induced 'active state' might be expected to represent a more
effective conformation of this protein with regards to kinase activity.
It is likely that multiple mechanisms regulate SRK activity. SRK also interacts in
vitro with Arabidopsis kinase-associated protein phosphatase (KAPP) (Braun et al. ,
1997) and recently a stigma-expressed Brassica homologue of KAPP has been
shown to be phosphorylated by, and to dephosphorylate, the SRK kinase domain
(Vanoosthuyse et al. , 2003). These results, together with the observation that in vivo
SRK is phosphorylated approximately 1 h after incompatible pollination (Cabrillac
et al. , 2001), suggest that KAPP may play a role in SRK down-regulation in a