Expression and functional analysis of genes involved in early anther development

Anthers contain specialized reproductive and somatic cells required for pollen development. Molecular genetic studies have uncovered genes that are important for anther development. However, little is known about most of the genes that are active during anther development, including possible relationships between these genes and known key regulators. In Arabidopsis, two previously isolated male sterile mutants are dramatically altered in anther development. The sporocyteless ( spl)/nozzle (nzz) mutant is defective in the differentiation of primary sporogenous cells into microsporocytes and does not properly form the somatic cell layers. The excess microsporocytes1 (ems1)/extrasporogenous cell ( exs) mutants produce excess microsporocytes at the expense of the tapetum, suggesting that EMS1 promotes tapetum differentiation. To gain further insights into possible gene functions and interactions during anther cell differentiation, mRNA expression profile of the wild-type anther (stage 4-6) was compared with that of the spl and ems1 anthers. The significance analysis of microarray (SAM) method was used to identify 1954 genes that were differentially expressed in the ems1/exs and/or spl/nzz anthers compared with the wild-type anthers. These genes were grouped into 14 different co-expression clusters using the K-mean clustering method. Biological relevance of these clusters were assessed using genes with known and putative functions. To obtain clues about possible co-regulation within co-expression clusters, we searched for enriched cis-regulatory motifs in putative promoters. These results, together with the published literature, were then used to develop a model of gene regulatory networks for anther development. The hypotheses in this model can be tested experimentally in order to gain further understanding of molecular mechanisms controlling anther cell differentiation. We have characterized the meiotic function of one of the 1954 genes identified our microarray study.;During meiosis, pairing, synapsis and recombination play an important role for precise chromosome segregation into four gametes. In yeast, there are two genetically separable pathways for the formation of crossovers (COs), the MSH4-MSH5-dependent and MUS81-MMS4/EME1-dependent pathways. The last key intermediate of the MSH4-MSH5-dependent pathway is known as the double Holliday junction. Despite identification of several key genes that regulate the formation of crossover pathway intermediates, only a few genes are known for the resolution of double Holliday junctions. In recent years, Arabidopsis has emerged as an excellent model organism for studying the meiotic process. Here we describe the identification of the PARTING DANCERS (PTD) gene, as a gene with an elevated expression level in meiocytes. Analysis of two independently generated T-DNA insertional lines in PTD showed that the mutants had reduced fertility. Further cytological analysis of male meiosis in the ptd mutants revealed defects in meiosis, including reduced formation of chiasmata, the cytological appearance of COs. The residual chiasmata in the mutants were distributed randomly, indicating that the ptd mutants are defective for CO formation in the interference-sensitive pathway. Presence of normal level of synaptonemal complexes and late recombination nodules at pachytene stage suggests that the mutant was defective in bivalent formation in a post-synaptic manner. Furthermore, PTD appears to be the founding member of a gene family conserved in plants. According to our results, in connection with the current model of homologous recombination in meiosis, PTD promotes the formation of COs in the MSH4-MSH5-dependent pathway, possibly by regulating the resolution of double Holliday junction intermediates in favor of CO formation.;Since PTD is a novel gene and its function has not been described previously it is imperative to understand its function in relation to other genes, which are already known to be involved in CO formation. Meiotic CO formation starts with Double Strand Break (DSB) formation, which is induced by a protein called SPO11. These DSBs are later repaired by RAD51. Our genetic analyses indicated that in Arabidopsis, PTD acts downstream of SPO11 and RAD51. In addition, our data indicated that PTD might function in the same pathway as the AtMSH4 and AtMER3/RCK genes, which are important for interference sensitive CO formation in Arabidopsis. Functions of SPO11, RAD51, MSH4 and MER3 have been described in other organisms and their functions appear to be conserved in these orgasinsms. Therefore, our findings suggest that PTD belongs to a cache of conserved meiotic mechinary.