J. Mark Cock

The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins

The self-incompatibility response in Brassica allows recognition and rejection of self-pollen by the stigmatic papillae. The transmembrane S-locus receptor kinase (SRK), a member of the receptor-like kinase superfamily in plants, mediates recognition of self-pollen on the female side, whereas the S-locus cysteine-rich protein (SCR) is the male component of the self-incompatibility response. SCR is presumably located in the pollen coat, and is thought to be the SRK ligand. Although many receptor-like kinases have been isolated in plants, the mechanisms of signal transduction mediated by these molecules remain largely unknown. Here we show that SRK is phosphorylated in vivo within one hour of self-pollination. We also show that, in vitro, autophosphorylation of SRK is prevented by the stigma thioredoxin THL1 in the absence of a ligand. This inhibition is released in a haplotype-specific manner by the addition of pollen coat proteins. Our data indicate that SRK is inhibited by thioredoxins and activated by pollen coat proteins.

Analysis of gene expression in rose petals using expressed sequence tags

Single‐pass sequences were obtained from the 5′‐ends of a total of 1794 rose petal cDNA clones. Cluster analysis identified 242 groups of sequences and 635 singletons indicating that the database represents a total of 877 genes. Putative functions could be assigned to 1151 of the transcripts. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens. The cDNA library and expressed sequence tag database described here represent a valuable resource for future research aimed at improving economically important rose characteristics such as flower form, longevity and scent.

Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response.

The male component of the self-incompatibility response in Brassica has recently been shown to be encoded by the S locus cysteine-rich gene (SCR). SCR is related, at the sequence level, to the pollen coat protein (PCP) gene family whose members encode small, cysteine-rich proteins located in the proteo-lipidic surface layer (tryphine) of Brassica pollen grains. Here we show that the Arabidopsis genome includes two large gene families with homology to SCR and to the PCP gene family, respectively. These genes are poorly predicted by gene-identification algorithms and, with few exceptions, have been missed in previous annotations. Based on sequence comparison and an analysis of the expression patterns of several members of each family, we discuss the possible functions of these genes. In particular, we consider the possibility that SCR-related genes in Arabidopsis may encode ligands for the S gene family of receptor-like kinases in this species.

Receptor kinase signalling in plants and animals: distinct molecular systems with mechanistic similarities

Plant genomes encode large numbers of receptor kinases that are structurally related to the tyrosine and serine/threonine families of receptor kinase found in animals. Here, we describe recent advances in the characterisation of several of these plant receptor kinases at the molecular level, including the identification of receptor complexes, small polypeptide ligands and cytosolic proteins involved in signal transduction and receptor downregulation. Phylogenetic analysis indicates that plant receptor kinases have evolved independently of the receptor kinase families found in animals. This hypothesis is supported by functional studies that have revealed differences between receptor kinase signalling in plants and animals, particularly concerning their interactions with cytosolic proteins. Despite these dissimilarities, however, plant and animal receptor kinases share many common features, such as their single membrane-pass structure, their inclusion in membrane-associated complexes, the involvement of dimerisation and trans autophosphorylation in receptor activation, and the existence of inhibitors and phosphatases that downregulate receptor activity. These points of convergence may represent features that are essential for a functional receptor-kinase signalling system.

Biosynthesis of the major scent components 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene by novel rose O-methyltransferases

In Chinese rose species and in many modern varieties, two methylated phenolic derivatives, 3,5‐dimethoxytoluene and 1,3,5‐trimethoxybenzene, are major scent components. We show that cell‐free extracts of rose petals catalyse the synthesis of 3,5‐dimethoxytoluene and 1,3,5‐trimethoxybenzene by methylation of precursor molecules. An expressed sequence tag approach was used to identify four highly similar O‐methyltransferase sequences expressed specifically in petals and anthers. Thin layer chromatography analysis showed that the activities of these enzymes with different substrates and the proportions of reaction products produced closely mimicked those observed using cell‐free petal extracts, indicating that orcinol O‐methyltransferases are responsible for the biosynthesis of 3,5‐dimethoxytoluene and 1,3,5‐trimethoxybenzene from un‐methylated precursors in this organ.

The S15 Self-Incompatibility Haplotype in Brassica oleracea Includes Three S Gene Family Members Expressed in Stigmas

Self-incompatibility in Brassica is controlled by a single, highly polymorphic locus that extends over several hundred kilobases and includes several expressed genes. Two stigma proteins, the S locus receptor kinase (SRK) and the S locus glycoprotein (SLG), are encoded by genes located at the S locus and are thought to be involved in the recognition of self-pollen by the stigma. We report here that two different SLG genes, SLGA and SLGB, are located at the S locus in the class II, pollen-recessive S15 haplotype. Both genes are interrupted by a single intron; however, SLGA encodes both soluble and membrane-anchored forms of SLG, whereas SLGB encodes only soluble SLG proteins. Thus, including SRK, the S locus in the S15 haplotype contains at least three members of the S gene family. The protein products of these three genes have been characterized, and each SLG glycoform was assigned to an SLG gene. Evidence is presented that the S2 and S5 haplotypes carry only one or the other of the SLG genes, indicating either that they are redundant or that they are not required for the self-incompatibility response.

Comparison of the expression patterns of two small gene families of S gene family receptor kinase genes during the defence response in Brassica oleracea and Arabidopsis thaliana.

SFR2, a member of the S gene family of receptor kinases, has been shown to be rapidly induced by wounding and bacterial infection suggesting that this gene may play a role in the defence response in Brassica. In this study we have compared the response of SFR2 to that of two other members of the SFR gene family in Brassica (SFR1 and SFR3) and to the closely-related ARK genes of Arabidopsis. Different patterns of mRNA accumulation were observed for different members of these families. SFR1 transcripts only accumulated in response to bacterial infection and their abundance was not significantly affected by wounding. Neither treatment induced accumulation of SFR3 transcripts. ARK1 and ARK3 resembled SFR2 in that their mRNAs accumulated in response to both wounding and bacterial infection. Both SFR1 and SFR2 mRNAs accumulated in response to exogenously applied salicylic acid (SA) and SA was shown to be required for induction of expression from the SFR2 promoter in Arabidopsis. However, the timing of the increase in endogenous SA levels following bacterial infiltration in Brassica indicates that the accumulation of SFR mRNA in the first few hours after infiltration does not occur in response to an increase in SA levels. We discuss the possibility that induction of SFR gene expression by SA may contribute to potentialization of the defence response. Taken together with previous studies that indicate a possible role during development, the data presented here suggest that the SFR and ARK gene families may have overlapping roles in both defence and during development.

SLR3: A modified receptor kinase gene that has been adapted to encode a putative secreted glycoprotein similar to theS locus glycoprotein

A new member of the S gene family,SLR3 (S-Locus Related 3), was identified inBrassica oleracea. This gene had a novel pattern of expression compared with previously described members of the family, being expressed in petals, sepals and vegetative apices, in addition to stigmas and anthers. Moreover, use ofSLR3-derived probes in RNA blot and RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) experiments has identified transcripts of genes closely related toSLR3 in leaves, cotyledons and, at high levels in developing anthers.SLR3 is not linked to theS locus but is linked to two or three closely related genes. Sequence analysis of theSLR3 gene indicates that it is derived from an ancestral receptor kinase gene that has been modified by a series of deletion events. As a result of these modifications,SLR3 is predicted to encode a secreted glycoprotein lacking both transmembrane and kinase domains. The putativeSLR3 protein differs from the products of most other S gene family members in that several of the highly conserved cysteines have been lost. Within theS gene family, modification of receptor kinase genes by deletion may represent a general mechanism for the generation of genes encoding secreted glycoproteins.

S-locus glycoproteins are expressed along the path of pollen tubes in Brassica pistils

In Brassica, self-incompatibility has been correlated with S-locus glycoproteins (SLG) localized at the site of pollen rejection, the stigmatic papillae. In this paper we present immunocytochemical evidence that during development of the pistil SLG proteins accumulate not only in the stigmatic papillae, but also in the transmitting tissue of stigma, style and ovary, along the pathway followed by the pollen tube. This observation was confirmed by the biochemical characterization of stylar and ovarian SLG proteins and by the detection of SLG transcripts in style/ovary tissues by polymerase chain reaction analysis. Thus we demonstrate similarities between the pattern of SLG gene expression in Brassica (sporophytic self-incompatibility system) and that of S products in Nicotiana (gametophytic self-incompatibility system) which may reflect relationships between the self-incompatibility systems. An active role of SLG gene products during the pollen-tube growth in the pistil is discussed.

Identification of a gene linked to the Brassica S (self-incompatibility) locus by differential display.

Self-incompatibility in Brassica is controlled by a complex locus, the S locus, that includes several expressed genes. Two S locus genes, SLG and SRK, are expressed in the stigma and have been implicated in self-pollen recognition. The male component of this recognition system is also predicted to be encoded by a gene at the S locus but this gene has not been identified to date. In this study, we have used differential display to screen for polymorphic, S-locus-linked genes that are expressed in anthers. This approach has allowed the identification of a gene, named S5J, which was shown to segregate completely with the S locus. We discuss the possible role of this gene in the self-incompatibility response and evaluate the utility of differential display for the identification of genes at specific genetic loci.