F. Christopher H. Franklin

Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica

The Arabidopsis thaliana ASY1 gene is essential for homologous chromosome synapsis. Antibodies specific to Asy1 protein and its homologue BoAsy1 from the related crop species Brassica oleracea have been used to investigate the temporal expression and localization of the protein in both species. Asy1 is initially detected in pollen mother cells during meiotic interphase as numerous punctate foci distributed over the chromatin. As leptotene progresses the signal appears to be increasingly continuous and is closely associated with the axial elements but not to the extended chromatin loops associated with them. By the end of zygotene the signal extends almost the entire length of the synapsed homologues, although not to the telomeres. The protein begins to disappear as the homologues desynapse, until by late diplotene it is no longer associated with the chromosomes. Immunogold labelling in conjunction with electron microscopy established that Asy1 localizes to regions of chromatin that associate with the axial/lateral elements of meiotic chromosomes rather than being a component of the synaptonemal complex itself. These data together with the previously observed asynaptic phenotype of the asy1 mutant suggest that Asy1 is required for morphogenesis of the synaptonemal complex, possibly by defining regions of chromatin that associate with the developing synaptonemal complex structure.

A homologue of the yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1

Abstract.Synapsis of homologous chromosomes is a key event in meiosis as it is essential for normal chromosome segregation and is implicated in the regulation of crossover frequency. We have previously reported the identification and cytological characterisation of a T-DNA-tagged asynaptic mutant of Arabidopsis thaliana. We have demonstrated that this mutant, asy1, is defective in meiosis in both males and females. Cloning and nucleotide sequencing of the ASY1 gene has revealed that it encodes a polypeptide of 596 amino acids that exhibits similarity to the HOP1 gene of Saccharomyces cerevisiae, which is known to encode a protein essential for synaptonemal complex assembly and normal synapsis. Expression studies indicate that, in common with a number of other Arabidopsis meiotic genes, ASY1 exhibits low-level expression in a range of plant tissues. Southern analysis coupled with database searching has resulted in the identification of an ASY1 homologue, ASY2. Although asy1 exhibits a strong asynaptic phenotype, a residual low level of synapsis indicates that ASY1 and ASY2 may exhibit a low degree of functional redundancy.

Identification of the pollen self-incompatibility determinant in Papaver rhoeas

Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility is an important mechanism used in many species to prevent inbreeding; it is controlled by a multi-allelic S locus. ‘Self’ (incompatible) pollen is discriminated from ‘non-self’ (compatible) pollen by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in pollen inhibition and programmed cell death. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS (Papaver rhoeas pollen S), from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single-copy gene linked to the pistil S gene (currently called S, but referred to hereafter as PrsS for Papaver rhoeas stigma S determinant). Sequence analysis indicates that PrsS and PrpS are equally ancient and probably co-evolved. PrpS encodes a novel ∼20-kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver self-incompatibility system. As a novel cell–cell recognition determinant it contributes to the available information concerning the origins and evolution of cell–cell recognition systems involved in discrimination between self and non-self, which also include histocompatibility systems in primitive chordates and vertebrates.

The meiotic protein SWI1 is required for axial element formation and recombination initiation in Arabidopsis

We report the detailed characterization of SWITCH1 (SWI1) an Arabidopsis thaliana protein that has been linked with the establishment of sister chromatid cohesion during meiosis. Using a combination of cytological methods including immunolocalization of meiotic chromosome-associated proteins we show that SWI1 is required for formation of axial elements. Our studies reveal that the swi1-2 mutation prevents the formation of RAD51 foci during meiotic prophase and suppresses the chromosome fragmentation phenotype of the recombination-defective dif1-1 mutant. Together, these data suggest that SWI1 may be required for meiotic recombination initiation. Finally we raised an antibody against SWI1 and showed, by immunolocalization coupled with bromodeoxyuridine incorporation experiments, that SWI1 is expressed exclusively in meiotic G1 and S phase. Thus, SWI1 appears to be required for early meiotic events that are at the crossroad of sister chromatid cohesion, recombination and axial element formation. The possible inter-relationship between these processes and the function of SWI1 are discussed.

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible (‘self’) pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.

Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele–specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele–specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen–pistil self-incompatibility reaction.

A Puromycin-Sensitive Aminopeptidase Is Essential for Meiosis in Arabidopsis thaliana

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.

Activation of a putative MAP kinase in pollen is stimulated by the self-incompatibility (SI) response.

Mitogen‐activated protein kinases (MAPKs) operate downstream of receptor–ligand interactions, playing a pivotal role in responses to extracellular signals. The self‐incompatibility (SI) response in Papaver rhoeas L. triggers a Ca2+‐dependent signalling cascade resulting in inhibition of incompatible pollen. We have investigated the possible involvement of MAPKs in SI. We report the enhanced activation of a 56 kDa protein kinase (p56) in SI‐induced pollen and provide evidence that p56 has MAPK activity. This provides an important advance in our understanding of the SI response. We believe this is the first direct biochemical demonstration of activation of a MAPK during SI.

Identification and cloning of related self-incompatibility S-genes in Papaver rhoeas and Papaver nudicaule

Abstract The primary goal of this study was to identify, clone and analyse new S-gene sequences in order to provide a basis for identifying amino acid residues that confer S-allele specificity. Three new putative S-alleles from Papaver rhoeas and Papaver nudicaule were identified using immunological and PCR methods. cDNAs encoding full-length open reading frames of the P. rhoeasS8 and P. nudicauleSn1 genes were isolated. Nucleotide sequencing of these cDNAs, together with the partial S7 sequence obtained by PCR, was used to derive the corresponding amino acid sequences. It is of interest that the P. nudicaule Sn1 sequence, which is the first S-allele isolated from another species of Papaver, shares a closer sequence identity to the P. rhoeas S3 amino acid sequence than S3 does to S1 from P. rhoeas. The identity of the S8 allele was confirmed by expressing the coding region in Escherichia coli and demonstrating that the recombinant protein, designated S8e, specifically inhibited S8 pollen in an in vitro bioassay.Information from sequence analysis of the S8, Sn1 and partial S7 amino acid sequences revealed important information about Papaver S-proteins. It confirmed previous observations based on only two S-alleles, that whilst exhibiting a high degree of amino acid sequence polymorphism ranging from 51.3% to 63.7%, these molecules probably share very similar secondary structures. These studies also revealed that, in contrast to the S-proteins from the Solanaceae and Brassica, amino acid sequence variation is not found in hypervariable blocks, but instead, is found throughout the S-proteins, interspersed with numerous short strictly conserved segments.

Histone hyperacetylation affects meiotic recombination and chromosome segregation in Arabidopsis.

In this study, the meiotic role of MEIOTIC CONTROL OF CROSSOVERS1 (MCC1), a GCN5-related histone N-acetyltransferase, is described in Arabidopsis. Analysis of the over-expression mutant obtained by enhancer activation tagging revealed that acetylation of histone H3 increased in male prophase I. MCC1 appeared to be required in meiosis for normal chiasma number and distribution and for chromosome segregation. Overall, elevated MCC1 did not affect crossover number per cell, but has a differential effect on individual chromosomes elevating COs for chromosome 4, in which there is also a shift in chiasma distribution, and reducing COs for chromosome 1 and 2. For the latter there is a loss of the obligate CO/chiasma in 8% of the male meiocytes. The meiotic defects led to abortion in about half of the male and female gametes in the mutant. In wild type, the treatment with trichostatin A, an inhibitor of histone deacetylases, phenocopies MCC1 over-expression in meiosis. Our results provide evidence that histone hyperacetylation has a significant impact on the plant meiosis.