SummaryIn self-incompatible plants, the recognition and subsequent rejection of self-related pollen by the stigma during pollination are genetically controlled by haplotypes of the Slocus complex. We identified two self-compatible Brassica strains that carried nonfunctional S haplotypes and that did not express transcripts of SRK, an S-locus gene that encodes a receptor-like protein kinase. In one of these strains, a deletion generated a null allele of the SRKstructural gene. This deleted haplotype exhibited typical interactions when combined with other normal haplotypes in heterotygotes indicating that other functions of the haplotype were retained. The data support a mechanism of self-incompatibility in which activation of the SRK kinase triggers the signalling cascade that culminates in the arrest of incompatible pollen development.

Type 2A serine/threonine protein phosphatases (PP2A) have been implicated as important mediators of a number of plant growth and developmental processes. In an effort to identify plant PP2A substrates and/or regulators, we performed a yeast two-hybrid screen using an Arabidopsis PP2A catalytic subunit cDNA as bait. All true positives identified by this screen were derived from the same gene, which we have named TAP46 (2A phosphatase associated protein of 46 kD). The TAP46 gene appears to be a single-copy gene and is expressed in all Arabidopsis organs. Transcripts derived from this gene are induced by chilling treatment but not by heat or anaerobic stress. Immunoprecipitation assays using antibodies generated to a peptide spanning amino acids 356 to 366 of TAP46 indicate that TAP46 is associated with a type 2A protein phosphatase in vivo. A search of the database identified TAP46 as a homolog of Saccharomyces cerevisiae TAP42 and mammalian ␣4. These two proteins are known to bind to the catalytic subunit of PP2A and to function in the target-of-rapamycin signaling pathway. Our results identify TAP46 as a plant PP2A-associated protein, with a possible function in the chilling response, and suggest that a target-of-rapamycin-like signaling pathway may exist in plants.

Numerous plant processes ranging from signal transduction to metabolism appear to be mediated, in part, by type 2A protein serinekhreonine phosphatases (PP2A). In an effort to identify factors that control the activity of this enzyme in plants, we have isolated and characterized DNA sequences encoding the B' regulatory subunit of PP2A from Arabidopsis thaliana. Specifically, we used PCR to amplify a segment of Arabidopsis cDNA that encodes a conserved section of the B' polypeptide. This PCR fragment was subsequently used as a probe to screen an Arabidopsis cDNA library and cDNA clones derived from three distinct genes were identified. The AtB'a and AtB'P genes encode highly similar 57-kDa B' regulatory subunits while the third gene, AtB'y, encodes a more divergent 59-kDa B' protein. A comparison of the three Arabidopsis B' polypeptides to those of yeast and animals shows the core region of this protein to be the most conserved while the amino and carboxy termini vary both in length and sequence. Genomic Southern blots indicate that at most the Arabidopsis genome contains five genes encoding the B' regulatory subunit. The three genes identified in this study are expressed in all Arabidopsis organs, albeit at varying levels. In addition, mRNAs derived from the three genes accumulate differentially in response to heat shock. Our results indicate that the activity of plant PP2A might be regulated by a B' type regulatory subunit similar to those found in animals and yeast, and suggest possible roles for B'-containing PP2A complexes within plant cells.

Type 2A serine/threonine protein phosphatases (PP2A) are key components in the regulation of signal transduction and control of cell metabolism. The activity of these protein phosphatases is modulated by regulatory subunits. While PP2A activity has been characterized in plants, little is known about its regulation. We used the polymerase chain reaction to amplify a segment of a cDNA encoding the B regulatory subunit of PP2A from Arabidopsis. The amplified DNA fragment of 372 nucleotides was used as a probe to screen an Arabidopsis cDNA library and a full-length clone (AtB alpha) of 2.1 kbp was isolated. The predicted protein encoded by AtB alpha is 43 to 46% identical and 53 to 56% similar to its yeast and mammalian counterparts, and contains three unique regions of amino acid insertions not present in the animal B regulatory subunit. Genomic Southern blots indicate the Arabidopsis genome contains at least two genes encoding the B regulatory subunit. In addition, other plant species also contain DNA sequences homologous to the B regulatory subunit, indicating that regulation of PP2A activity by the 55 kDa B regulatory subunit is probably ubiquitous in plants. Northern blots indicate the AtB alpha mRNA accumulates in all Arabidopsis tissues examined, suggesting the protein product of the AtB alpha gene performs a basic housekeeping function in plant cells.

We have examined the effect of the protein phosphatase inhibitors okadaic acid and microcystin on pollen-pistil interactions in Brassica. Inhibitor-treated flowers or floral buds were pollinated with untreated pollen and examined for pollen tube growth by fluorescence microscopy. Our results show that type 1 or type 2A serinelthreonine phosphatases play a crucial role in the pollination responses of Brassica. We observed two distinct effects of protein phosphatase inhibitors on pollination: (a) the inhibition of pollen tube growth during cross-pollination in flowers, and (b) the breakdown of self-incompatibility or promotion of pollen tube growth during self-pollination in flower buds just prior to anthesis. Thus, treatment of flower pistils with protein phosphatase inhibitors resulted in the inhibition of pollen tube growth at the surface of the papillar cells of the stigma in crosses between different selfincompatible Brassica oleracea strains, in an interspecific cross between B. oleracea and Brassica campesfris, and in self-pollinations of a self-fertile Brassica napus cultivar. With four different self-incompatibility genotypes, treatment of mature flowers with protein phosphatase inhibitors had no effect on self-pollination response. In contrast, treatment of flower buds just prior to the anthesis stage allowed self-pollen tube invasion of papillar cells. However, the magnitude of this effect was genotype dependent, being most pronounced in the Saz genotype. The data support the conclusion that pollinations in Brassica are controlled in part by the presence of phosphorylated proteins in the papillar cells of the stigma, and that the quantity of these proteins or their levels of phosphorylation changes during stigma development.A successful pollination involves the capture of pollen, the hydration and gennination of individual grains, and the growth of a pollen tube. In members of the Brassicaceae (crucifers) the pollen tube invades the papillar cells of the stigma surface and grows within the secondary papillar cell wall. The pollen tube then grows intercellularly in the transmitting tissues of the stigma, style, and ovary, where the tube grows over the surface of the septum, penetrates the funiculus, and enters the unfertilized ovule to effect fertilization (Hill and Lord, 1987).Although it has been suggested that signals of a chemical, electical, or mechanical nature operate in the pistil and induce the directionality of pollen tube growth (Steer and 1165Steer, 1988), very little is known about the molecular requirements for in vivo pollen germination and pollen tube growth. It is clear that specific cell-cell interactions must be important components of pollination and fertilization. In a11 plants, the pistil presents a bamer to pollen from other species. In addition, plants have evolved intraspecific genetic mechanisms that allow the discrimination between "self" and "nonself" pollen, thus promoting outbreeding and genetic variability. In Brassica, this bamer to self-fertilization known as SI occurs a...