Methods are described for the removal of the sporophytic pollen grain coating of Brassica olracea and for the isolation of coat polypeptides. The coat contains a small number of proteins ranging from 6 to 45 kDa. Many of the larger proteins are glycosylated, while all carry hig positive charges resulting in pI values from 8.5 to 11. Polypeptides with pI values of 9.5, 9.0, and 8.5 possess strong esterase activity. No major differences could be detected in either pI values or molecular masses of pollen-coating polypeptides from grains carrying different sporophytically expressed S (self-incompatibility) alleles. Mixing pollen coat proteins with smatic extracts results in a conspicuous binding interaction involving female S-locus-specific and perhaps S-locus-related glycoproteins. This interaction, which is reversed by heating in the presence of SDS, results in an apparent charge shift of the female glycoprotein(s) of up to 2 pI units. The male participant in this interaction has been isolated by using a combination of fast protein liquid chromatography and reverse-phase HPLC and was shown to be a 7-kDa nonglycosylated peptide. Experiments with whole pollen cultured in vitro show challenge with stigmatic extracts to stimulate the release of gametophytic and sporophytic polypeptides and to result in the formation of a conspicuous interaction product, demonstrating the 7-kDa peptide to be freely available within the coating of pollen in vivo.The self-incompatibility (SI) systems of higher plants are emerging as a family of unique signaling systems evolved from mechanisms already operative in the pollen/pistil interaction (1). Thus, in Nicotiana and other members of the Solanaceae, self-pollen is rejected by a mechanism involving a stylar RNase (2, 3), an enzyme common to the styles of many plants and hypothesized to play a role in defense against pathogens. In most species, SI is regulated by a simple genetic system based on few loci and large numbers of alleles (4, 5); generally, if pollen and stigma carry alleles in common, the pollen/stigma interaction is disrupted. The genetics of SI in Brassica and other members of the Cruciferae and Compositae is not so straightforward; the pollen S phenotype is determined by the S-allelic constitution of the parent plant, rather than that of the haploid grain (6).Through a series of elegant molecular studies, Nasrallah et al. (7) have demonstrated two types of sequence to be linked to the S locus of Brassica. One (S locus glycoprotein; SLG) encodes a 55-kDa glycoprotein (S-locus-specific glycoprotein; SLSG) expressed in the stigmatic papillae, and the other encodes a transmembrane kinase (S receptor kinase; SRK) expressed in the organs ofboth sexes. Sequence comparisons suggest that the SRK features a domain, which may be extracellular, with maintained homology to the SLG. Further, other gene families with considerable homology to the SLG are also present in the genome (8-10) but unlinked to the S locus (S locus related; SLR). Interestingly, reporter constructs driven...

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P 2 ] is a phospholipid that has a role in controlling membrane trafficking events in yeast and animal cells. The function of this lipid in plants is unknown, although its synthesis has been shown to be up-regulated upon osmotic stress in plant cells. PtdIns(3,5)P 2 is synthesized by the PIKfyve/Fab1 family of proteins, with two orthologs, FAB1A and FAB1B, being present in Arabidopsis (Arabidopsis thaliana). In this study, we attempt to address the role of this lipid by analyzing the phenotypes of plants mutated in FAB1A and FAB1B. It was not possible to generate plants homozygous for mutations in both genes, although single mutants were isolated. Both homozygous single mutant plant lines exhibited a leaf curl phenotype that was more marked in FAB1B mutants. Genetic transmission analysis revealed that failure to generate double mutant lines was entirely due to inviability of pollen carrying mutant alleles of both FAB1A and FAB1B. This pollen displayed severe defects in vacuolar reorganization following the first mitotic division of development. The presence of abnormally large vacuoles in pollen at the tricellular stage resulted in the collapse of the majority of grains carrying both mutant alleles. This demonstrates a crucial role for PtdIns(3,5)P 2 in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P 2 production being central to cellular responses to changes in osmotic conditions.

Self-incompatibility (SI) inBrassica species is controlled by a single polymorphic locus ( S ) with multiple specificities. Two stigmatically expressed genes that have been cloned from this region encode the S locus glycoprotein (SLG) and S receptor kinase (SRK). Both appear to be essential for the operation of SI. It is believed that rejection of incompatible pollen grains is effected by recognition events between an as yet unidentified S locus-encoded pollen coating-borne protein and the SLG/SRK. We previously identified a small pollen coat protein PCP7 (renamed here PCP-A1, for pollen coat protein, class A, 1) that binds with high affinity to SLGs irrespective of S genotype. Here, we report the cloning of PCP-A1 from Brassica oleracea and demonstrate that it is unlinked to the S locus. In situ localization of PCP-A1 transcripts revealed that they accumulate specifically in pollen at the late binucleate/trinucleate stage of development rather than in the tapetum, which previously was taken to be the principal source of the pollen coat. PCP-A1 is characterized by the presence of a structurally important motif consisting of eight cysteine residues shared by the plant defensins. Based on the presence of this motif and other data, homology modeling has been used to produce a putative structure for PCP-A1. Protein-protein interaction analyses demonstrate that SLG exists in monomeric and dimeric forms, both of which bind PCP-A1. Evidence is also presented for the existence of putative membrane-associated PCP-A1 binding proteins in stigmatic tissue. INTRODUCTIONFlowering plants have evolved a range of both morphological and molecular mechanisms that prevent self-pollination. It has been estimated that up to 50% of angiosperms possess such self-incompatibility (SI) systems (Darlington and Mather, 1949; Brewbaker, 1959), highlighting their evolutionary importance (Stebbins, 1950(Stebbins, , 1957 Whitehouse, 1950;de Nettancourt, 1977). The most advanced SI systems are thought to be those that are controlled by a single multiallelic locus ( S locus), typified by Brassica , Nicotiana , and Papaver spp. In Nicotiana and Papaver spp, in which pollen compatibility is controlled by the gametophyte, the grain is rejected if the S allele it carries is shared by the stigma. Importantly, pollen compatibility in Brassica spp is regulated by the sporophyte, and thus the S alleles of the pollen parent determine whether the grain is accepted or rejected. In recent years, advances have been made toward elucidating the molecular basis of SI in these and other species (reviewed in Franklin et al., 1995;Hiscock et al., 1996;Kao and McCubbin, 1996).The Brassica S locus has been estimated to span several hundred kilobases of DNA (Boyes and Nasrallah, 1993). To date, two polymorphic stigmatically expressed S locus genes that appear to be required for the operation of SI have been cloned; one encodes a transmembrane receptor-like protein kinase (Stein et al., 1991) termed SRK (for S receptor kinase), and the other encodes a glycoprotein (Na...

A thin film of Nafion, of approximately 5 μm thickness, asymmetrically deposited onto a 6 μm thick film of poly(ethylene terephthalate) (PET) fabricated with a 5, 10, 20, or 40 μm microhole, is shown to exhibit prominent ionic diode behavior involving cation charge carrier ("cationic diode"). The phenomenon is characterized via voltammetric, chronoamperometric, and impedance methods. Phenomenologically, current rectification effects are comparable to those observed in nanocone devices where space-charge layer effects dominate. However, for microhole diodes a resistive, a limiting, and an overlimiting potential domain can be identified and concentration polarization in solution is shown to dominate in the closed state.

Summary The establishment of pollen–pistil compatibility is strictly regulated by factors derived from both male and female reproductive structures. Highly diverse small cysteine‐rich proteins (CRPs) have been found to play multiple roles in plant reproduction, including the earliest stages of the pollen–stigma interaction. Secreted CRPs found in the pollen coat of members of the Brassicaceae, the pollen coat proteins (PCPs), are emerging as important signalling molecules that regulate the pollen–stigma interaction.Using a combination of protein characterization, expression and phylogenetic analyses we identified a novel class of Arabidopsis thaliana pollen‐borne CRPs, the PCP‐Bs (for pollen coat protein B‐class) that are related to embryo surrounding factor (ESF1) developmental regulators. Single and multiple PCP‐B mutant lines were utilized in bioassays to assess effects on pollen hydration, adhesion and pollen tube growth.Our results revealed that pollen hydration is severely impaired when multiple PCP‐Bs are lost from the pollen coat. The hydration defect also resulted in reduced pollen adhesion and delayed pollen tube growth in all mutants studied.These results demonstrate that At PCP‐Bs are key regulators of the hydration ‘checkpoint’ in establishment of pollen–stigma compatibility. In addition, we propose that interspecies diversity of PCP‐Bs may contribute to reproductive barriers in the Brassicaceae.