James Doughty

PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression.

Self-incompatibility (SI) in Brassica 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.

A 7-kDa pollen coating-borne peptide from Brassica napus interacts with S-locus glycoprotein and S-locus-related glycoprotein

Two S(self-incompatibility)-family glycoproteins have been identified in stigmas of self-compatible (SC) Brassica napus L. by their ability to interact, in vitro, with a peptide fraction from the pollen coating containing a PCP7-like peptide, PCP7 (pollen coat peptide 7[kDa]) being a pollen coat peptide from self-incompatible (SI) Brassica oleracea L. which has been shown to interact with S-locus glycoproteins (SLGs). Electrophoretic purification and N-terminal amino-acid sequencing of these stigmatic glycoproteins confirmed one to be an SLG and the other to be a class 1 S-locus-related glycoprotein (SLR1). This is the first reported isolation of SLG and SLR1 proteins from SC B. napus and the first time that a PCP7-like peptide has been shown to interact with an S-class glycoprotein other than SLG. On the basis of these findings we suggest that an ability to interact with PCP7 or a PCP7-like peptide is a property of SLGs, SLR1s and possibly other S glycoproteins and may thus provide a novel route to the identification of these glycoproteins in stigmatic extracts. The levels of the SLG in stigmas of SC B. napus were relatively much lower than the levels of the SLR1-the opposite to the situation in SI B. oleracea. This finding is discussed in terms of translational control of SLGs and SLR1, and the possible implications for self-incompatibility in B. oleracea and self-compatibility in B. napus.

Arabidopsis FAB1/PIKfyve Proteins Are Essential for Development of Viable Pollen

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] 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)P2 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)P2 in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P2 production being central to cellular responses to changes in osmotic conditions.

Double fertilization in Arabidopsis thaliana involves a polyspermy block on the egg but not the central cell

In animal reproduction, thousands of sperm may compete to fertilize a single egg, but polyspermy blocks prevent multiple fertilization that would otherwise lead to death of the embryo. In flowering plants, successful seed development requires that only two sperm are delivered to the embryo sac, where each must fertilize a female gamete (egg or central cell) to produce the embryo and endosperm. Therefore, polyspermy must be avoided, not only to prevent abnormalities in offspring, but to ensure double fertilization. It is not understood how each sperm fertilizes only one female gamete, nor has the existence of polyspermy barriers been directly tested in vivo. Here, we sought evidence for polyspermy blocks in angiosperms using the polyspermic tetraspore (tes) mutant of Arabidopsis, which allows in-vivo challenge of egg and central cell with multiple male gametes. We show that tes mutant pollen tubes can transmit more than one sperm pair to an embryo sac, and that sperm from more than one pair can participate in fertilization. We detected endosperms but not embryos with ploidies that could only result from multiple fertilization. Our results therefore demonstrate an in-vivo polyspermy block on the egg, but not the central cell of a flowering plant.

Pollen coatings – chimaeric genetics and new functions

Abstract Pollen coatings have long been assumed to play a pivotal role in pollen-stigma interactions, but until now little clear evidence supporting such a function has been available. Recently, however, the use of isolated coatings of Brassica sp. in experiments in vivo has unequivocally demonstrated that the pollen coat layer is responsible for activation of the stigmatic surface, and that it contains the male determinant of the self-incompatibility system. Surprisingly, molecular analysis of the Brassica pollen coat reveals this layer to include both sporophytic and gametophytic components, the latter including a family of small highly-charged proteins which interact with stigmatic molecules known to be encoded by the S(incompatibility)-locus. Most recently, work on Brassica and Arabidopsis suggests that the adhesive function of the coating is more complex than suspected and involves both stigmatic factors and the exine surface itself. Despite this new insight into the genetics and function of pollen coatings, the mechanisms by which components of these layers are formed in the tapetum and translocated to the pollen grain surface remain far from clear.

Flavonoids and the regulation of seed size in Arabidopsis

Understanding how seed size is regulated in angiosperms is a key goal for plant science as seed size is an important component of overall seed yield. Angiosperm seeds comprise three clearly defined components, i.e. the embryo, endosperm and seed coat, with each having a distinct genetic composition which exerts different influences on seed development. Complex cross-talk and integration of signals from these different regions of the seed together determine its final size. The present review considers some of the major regulators of seed size, with a particular emphasis on the role of the seed coat in modulating endosperm proliferation and cellularization. The innermost layer of the seed coat, the endothelium, synthesizes flavonoids which are held to provide a defensive function against microbes, act as feeding deterrents, provide UV protection and to have a role in seed dormancy. A growing body of data suggests that flavonoids may also play a fundamental role in regulating communication between the seed coat and the endosperm. In the present review, we discuss how this may be achieved in the light of the fact that several flavonoids are known to be potent auxin transport regulators.

A Golgi and tonoplast localized S-acyl transferase is involved in cell expansion, cell division, vascular patterning and fertility in Arabidopsis.

S-acylation of eukaryotic proteins is the reversible attachment of palmitic or stearic acid to cysteine residues, catalysed by protein S-acyl transferases that share an Asp-His-His-Cys (DHHC) motif. Previous evidence suggests that in Arabidopsis S-acylation is involved in the control of cell size, polarity and the growth of pollen tubes and root hairs. Using a combination of yeast genetics, biochemistry, cell biology and loss of function genetics the roles of a member of the protein S-acyl transferase PAT family, AtPAT10 (At3g51390), have been explored. In keeping with its role as a PAT, AtPAT10 auto-S-acylates, and partially complements the yeast akr1 PAT mutant, and this requires Cys192 of the DHHC motif. In Arabidopsis AtPAT10 is localized in the Golgi stack, trans-Golgi network/early endosome and tonoplast. Loss-of-function mutants have a pleiotropic phenotype involving cell expansion and division, vascular patterning, and fertility that is rescued by wild-type AtPAT10 but not by catalytically inactive AtPAT10C192A. This supports the hypothesis that AtPAT10 is functionally independent of the other Arabidopsis PATs. Our findings demonstrate a growing importance of protein S-acylation in plants, and reveal a Golgi and tonoplast located S-acylation mechanism that affects a range of events during growth and development in Arabidopsis.

Proteins of the pollen coat of Brassica oleracea

Summary In higher plants, proteins in the coat of the pollen grain are assumed to play an important role in the interaction between pollen and stigma upon pollination. A polyclonal antiserum was raised against a mixture of these proteins. The antiserum strongly reacted with proteins extracted from the pollen coat and from whole stamens, whereas there was only a faint cross-reactivity to proteins from other tissues. Western blot analysis and immunolocalisation of pollen before and after rinsing with cyclohexane, a treatment that selectively removes the coat layer, showed that the proteins were exclusively located in the pollen coat. The same proved to hold for pollen coat proteins from other Brassica species. The availability of an antiserum creates the opportunity to identify coat protein encoding sequences in a cDNA library of anthers.

Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.

Histone H2B Monoubiquitination Mediated by HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 Is Involved in Anther Development by Regulating Tapetum Degradation-Related Genes in Rice

Histone H2B monoubiquitination is essential for anther development. Histone H2B monoubiquitination (H2Bub1) is an important regulatory mechanism in eukaryotic gene transcription and is essential for normal plant development. However, the function of H2Bub1 in reproductive development remains elusive. Here, we report rice (Oryza sativa) HISTONE MONOUBIQUITINATION1 (OsHUB1) and OsHUB2, the homologs of Arabidopsis (Arabidopsis thaliana) HUB1 and HUB2 proteins, which function as E3 ligases in H2Bub1, are involved in late anther development in rice. oshub mutants exhibit abnormal tapetum development and aborted pollen in postmeiotic anthers. Knockout of OsHUB1 or OsHUB2 results in the loss of H2Bub1 and a reduction in the levels of dimethylated lysine-4 on histone 3 (H3K4me2). Anther transcriptome analysis revealed that several key tapetum degradation-related genes including OsC4, rice Cysteine Protease1 (OsCP1), and Undeveloped Tapetum1 (UDT1) were down-regulated in the mutants. Further, chromatin immunoprecipitation assays demonstrate that H2Bub1 directly targets OsC4, OsCP1, and UDT1 genes, and enrichment of H2Bub1 and H3K4me2 in the targets is consistent to some degree. Our studies suggest that histone H2B monoubiquitination, mediated by OsHUB1 and OsHUB2, is an important epigenetic modification that in concert with H3K4me2, modulates transcriptional regulation of anther development in rice.