Characterization of the three Arabidopsis thaliana RAD21 cohesins reveals differential responses to ionizing radiation.

The RAD21/REC8 gene family has been implicated in sister chromatid cohesion and DNA repair in several organisms. Unlike most eukaryotes, Arabidopsis thaliana has three RAD21 gene homologues, and their cloning and characterization are reported here. All three genes, AtRAD21.1, AtRAD21.2, and AtRAD21.3, are expressed in tissues rich in cells undergoing cell division, and AtRAD21.3 shows the highest relative level of expression. An increase in steady-state levels of AtRAD21.1 transcript was also observed, specifically after the induction of DNA damage. Phenotypic analysis of the atrad21.1 and atrad21.3 mutants revealed that neither of the single mutants was lethal, probably due to the redundancy in function of the AtRAD21 genes. However, AtRAD21.1 plays a critical role in recovery from DNA damage during seed imbibition, prior to germination, as atrad21.1 mutant seeds are hypersensitive to radiation damage.

TETRASPORE encodes a kinesin required for male meiotic cytokinesis in Arabidopsis.

A key step in pollen formation is the segregation of the products of male meiosis into a tetrad of microspores, each of which develops into a pollen grain. Separation of microspores does not occur in tetraspore (tes) mutants of Arabidopsis thaliana, owing to the failure of male meiotic cytokinesis. tes mutants thus generate large 'tetraspores' containing all the products of a single meiosis. Here, we report the positional cloning of the TES locus and details of the role played by the TES product in male cytokinesis. The predicted TES protein includes an N-terminal domain homologous to kinesin motors and a C-terminus with little similarity to other proteins except for a small number of plant kinesins. These include the Arabidopsis HINKEL protein and NACK1 and two from tobacco (Nishihama et al., 2002), which are involved in microtubule organization during mitotic cytokinesis. Immunocytochemistry shows that the characteristic radial arrays of microtubules associated with male meiotic cytokinesis fail to form in tes mutants. The TES protein therefore is likely to function as a microtubule-associated motor, playing a part either in the formation of the radial arrays that establish spore domains following meiosis, or in maintaining their stability.

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.

Parent-of-origin effects on seed development in Arabidopsis thaliana.

Many flowering plants are polyploid, but crosses between individuals of different ploidies produce seeds that develop abnormally and usually abort. Often, seeds from interploidy crosses develop differently depending on whether the mother or father contributes more chromosome sets, suggesting that maternal and paternal genomes are not functionally equivalent. Here we present the first cytological investigation of seed development following interploidy crosses in Arabidopsis thaliana. We find that crosses between diploid and tetraploid plants in either direction, resulting in double the normal dose of maternal or paternal genomes in the seed, produce viable seeds containing triploid embryos. However, development of the seed and in particular the endosperm is abnormal, with maternal and paternal genomic excess producing complementary phenotypes. A double dose of maternal genomes with respect to paternal contribution inhibits endosperm development and ultimately produces a smaller embryo. In contrast, a double dose of paternal genomes promotes growth of the endosperm and embryo. Reciprocal crosses between diploids and hexaploids, resulting in a triple dose of maternal or paternal genomes, produce seeds that begin development with similar but more extreme phenotypes than those with a double dose, but these invariably abort. One explanation of our observations is that seeds with maternal or paternal excess contain different doses of maternally or paternally expressed imprinted loci affecting endosperm development.

Origin of allelic diversity in antirrhinum S locus RNases.

In many plant species, self-incompatibility (SI) is genetically controlled by a single multiallelic S locus. Previous analysis of S alleles in the Solanaceae, in which S locus ribonucleases (S RNases) are responsible for stylar expression of SI, has demonstrated that allelic diversity predated speciation within this family. To understand how allelic diversity has evolved, we investigated the molecular basis of gametophytic SI in Antirrhinum, a member of the Scrophulariaceae, which is closely related to the Solanaceae. We have characterized three Antirrhinum cDNAs encoding polypeptides homologous to S RNases and shown that they are encoded by genes at the S locus. RNA in situ hybridization revealed that the Antirrhinum S RNase are primarily expressed in the stylar transmitting tissue. This expression is consistent with their proposed role in arresting the growth of self-pollen tubes. S alleles from the Scrophulariaceae form a separate group from those of the Solanaceae, indicating that new S alleles have been generated since these families separated (approximately 40 million years). We propose that the recruitment of an ancestral RNase gene into SI occurred during an early stage of angiosperm evolution and that, since that time, new alleles subsequently have arisen at a low rate.

The characterisation of tapetum-specific cDNAs isolated from a Lilium henryi L. meiocyte subtractive cDNA library.

Differential screening of a meiocyte subtractive cDNA library from Lilium henryi L. has identified a group of 16 anther-specific partial cDNAs. Three of these sequences, LHM2, LHM6 and LHM7 have been further characterised. Hybridisation in situ with antisense riboprobes of LHM2, LHM6, and LHM7 gives a strong, clear signal which, contrary to expectations, is localised to the tapetal layer surrounding the meiocytes and not the meiocytes themselves. Developmental slot blots demonstrate that mRNAs corresponding to the three LHM cDNAs are transcribed from prophase of meiosis I to the uninucleate microspore stage, while Northern analysis reveals these tapetally expressed cDNAs to correspond with transcripts of some 500 bp. Although LHM2 is less abundant than LHM6 and LHM7, the pattern of developmental expression, and the size range of the transcripts suggests that all three cDNAs may be related. The deduced polypeptide products of LHM6 and LHM7 share 71% identity over a conserved region of 38 residues. Inverse polymerase chain reaction was used to obtain the full sequence for LHM7. Its deduced protein sequence has a signal peptide indicating it may be secreted; the cleaved protein has a molecular weight of 8.9 kDa. Furthermore, the LHM7 protein has significant levels of homology with tapetally expressed proteins from Arabidopsis thaliana, Antirrhinum majus and Lycopersicon esculentum. All contain a highly conserved pattern of cysteine residues present in seed and non-specific lipid transfer proteins. The function of this gene product is discussed in the perspective of current models of another development.

Ultrastructural and physiological differences between buds and mature flowers of Petunia hybrida prior to and following pollination.

The structural events accompanying the maturation of the pistil of Petunia hybrida have been studied in detail, together with the changes in the protein spectrum of the transmitting tissue that occur over this period. These events have been considered in terms of the acquisition of the self-incompatibility response, which occurs while the pistil is enclosed in the bud. Apart from several minor differences, the young pistils differ only from the mature in that their transmitting tissue cells fail to respond to pollination by undergoing characteristic ultrastructural changes. Data from electrofocusing indicates that several proteins, mobilised in the mature transmitting tissue some three hours after pollination, are absent from bud pistils. It is proposed that the pollination-stimulated release of certain polypeptides, believed to be involved in the self-incompatibility response, does not take place in young pistils. These observations are considered with reference to currently-accepted models of the operation of the self-incompatibility mechanism in Petunia.