The Arabidopsis DRYK, AtYAK1 regulates the development of the male gametophyte
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ABSTRACT: Pollen is the male gametophyte of land plants. Proper development and maturation of pollen is necessary for the successful reproduction of seed plants. This process involves sophisticated coordination between sporophytic and gametophytic tissues in anthers. To advance the mechanistic studies of anther development, additional players need to be discovered for a comprehensive understanding of the underlying regulatory network. Here we show that the Arabidopsis dual specificity tyrosine phophorylated and regulated kinase (DRYK), AtYAK1, is essential for development of rosette leaves and the male but not female gametophyte in Arabidopsis. Arabidopsis mutant plants carrying a mutation in AtYAK1 produce developmentally stalled microspores, likely because of the defects in the two consecutive mitosis steps in the post-meiotic maturation process of pollen. The mutation of AtYAK1 has a significant effect on gene expression programs in developing pollen. Transcritpome analysis of atyak1 revealed downstream genes in families of protein kinases, transporters and transcription factors, which potentially contribute to pollen development. This study represents the first molecular characterization of DYRK in the plant kingdom. Our results also imply that the regulation of cytokinesis by DYRKs is evolutionally conserved in fungus, fruit fly, animals and plants. 2 biological replicates were performed for wt and 2 different alleles of ayak1 mutants (atyak1-1 and atyak1-2)
Project description:Pollen is the male gametophyte of land plants. Proper development and maturation of pollen is necessary for the successful reproduction of seed plants. This process involves sophisticated coordination between sporophytic and gametophytic tissues in anthers. To advance the mechanistic studies of anther development, additional players need to be discovered for a comprehensive understanding of the underlying regulatory network. Here we show that the Arabidopsis dual specificity tyrosine phophorylated and regulated kinase (DRYK), AtYAK1, is essential for development of rosette leaves and the male but not female gametophyte in Arabidopsis. Arabidopsis mutant plants carrying a mutation in AtYAK1 produce developmentally stalled microspores, likely because of the defects in the two consecutive mitosis steps in the post-meiotic maturation process of pollen. The mutation of AtYAK1 has a significant effect on gene expression programs in developing pollen. Transcritpome analysis of atyak1 revealed downstream genes in families of protein kinases, transporters and transcription factors, which potentially contribute to pollen development. This study represents the first molecular characterization of DYRK in the plant kingdom. Our results also imply that the regulation of cytokinesis by DYRKs is evolutionally conserved in fungus, fruit fly, animals and plants.
Project description:Aims We aim to use transcriptome analysis to establish on a genome-wide scale the identity and regulatory clusters of genes that specify microgametogenesis from the haploid microspore to mature functional pollen in Arabidopsis. Background Pollen as the haploid male gametophyte plays a vital role in plant fertility and crop production through the ability to deliver the male gametes in fertilisation. Despite the obvious importance for plant fertility and crop production we have a very limited understanding of the regulatory mechanisms that have evolved to specify male gametophyte development and functions and less than 150 genes have been identified that are gametophytically expressed in the anther.The availability of functional genomic resources now provides the opportunity to undertake a comprehensive approach to describing cellular development in terms of the transcriptome. This approach is particularly powerful where the complete transcriptome of a single developing cell can be analysed. The male gametophyte is a uniquely accessible cell type for such studies, enabling RNA analysis from distinct purified cell populations during development.The proposed experiments are designed to support a current application (P19208, Twell) to investigate the gametophytic transcriptome and transcription factor networks. The results obtained will extend our knowledge of the contribution of haploid gene expression to anther development and will be used directly to extend BBSRC funded work (P15086, Wilson) to investigate the role and targets the MALE STERILE 1 gene (MS1). In particular the data will be used in collaboration to extract haploid gene expression from datasets of transcriptome analysis of staged flower buds of wild type (Ler) and ms1. This work will also complement BBSRC funded work on sporogenesis (G13338, Dickinson and Scott) and meiosis (G15941, Franklin and Jones) that are focussed on earlier steps in anther development. Biological material and methods. Isolated microspores and pollen at 4 different developmental stages will be analysed. We will isolate spores from developmentally staged buds of Ler grown under defined growth conditions. Buds from several batches of 100 plants will be rapidly sorted into 4 groups according to developmental age, uninucleate microspores (UM), bicellular pollen (BP) tricellular pollen (TP) and mature pollen. Spores will be released by gentle mechanical tissue disruption and purified by filtration and purification of spores. We are confident that our spore isolation procedures are rigorous since we could not detect even trace expression of highly abundant sporophytic transcripts such RbcS and Cab transcripts in microarray data from pollen RNA. Experiment Overall Design: Number of plants pooled:over 100
Project description:Aims We aim to use transcriptome analysis to establish on a genome-wide scale the identity and regulatory clusters of genes that specify microgametogenesis from the haploid microspore to mature functional pollen in Arabidopsis. Background Pollen as the haploid male gametophyte plays a vital role in plant fertility and crop production through the ability to deliver the male gametes in fertilisation. Despite the obvious importance for plant fertility and crop production we have a very limited understanding of the regulatory mechanisms that have evolved to specify male gametophyte development and functions and less than 150 genes have been identified that are gametophytically expressed in the anther.The availability of functional genomic resources now provides the opportunity to undertake a comprehensive approach to describing cellular development in terms of the transcriptome. This approach is particularly powerful where the complete transcriptome of a single developing cell can be analysed. The male gametophyte is a uniquely accessible cell type for such studies, enabling RNA analysis from distinct purified cell populations during development.The proposed experiments are designed to support a current application (P19208, Twell) to investigate the gametophytic transcriptome and transcription factor networks. The results obtained will extend our knowledge of the contribution of haploid gene expression to anther development and will be used directly to extend BBSRC funded work (P15086, Wilson) to investigate the role and targets the MALE STERILE 1 gene (MS1). In particular the data will be used in collaboration to extract haploid gene expression from datasets of transcriptome analysis of staged flower buds of wild type (Ler) and ms1. This work will also complement BBSRC funded work on sporogenesis (G13338, Dickinson and Scott) and meiosis (G15941, Franklin and Jones) that are focussed on earlier steps in anther development. Biological material and methods. Isolated microspores and pollen at 4 different developmental stages will be analysed. We will isolate spores from developmentally staged buds of Ler grown under defined growth conditions. Buds from several batches of 100 plants will be rapidly sorted into 4 groups according to developmental age, uninucleate microspores (UM), bicellular pollen (BP) tricellular pollen (TP) and mature pollen. Spores will be released by gentle mechanical tissue disruption and purified by filtration and purification of spores. We are confident that our spore isolation procedures are rigorous since we could not detect even trace expression of highly abundant sporophytic transcripts such RbcS and Cab transcripts in microarray data from pollen RNA. Keywords: development_or_differentiation_design
Project description:Transcriptome profiling of three developmental stages of immature male gametophyte intobacco (Nicotiana tabacum) Total RNA isolated from tobacco microspores and early and late bicellular pollen was hybridised on Agilent Tobacco Gene Expression Microarray 4x44K in two biological replicates per sample
Project description:The development of viable gametes is especially susceptible to heat in all higher plants, including cotton (Gossypium hirsutum cv. Sicot 71), resulting in substantial reduction in lint quantity and quality at temperatures above 32 °C. Male reproductive cells are especially vulnerable to heat. This study demonstrates a relatively small impact of heat on leaves compared with a profound impact during early and late male gametophyte development. To investigate the mechanisms leading to heat sensitivity, the proteome of pollen was analyzed after two distinct phases of development (tetrads or binucleate microspores) had been exposed for 5 d to 36/25 °C (day/night) or 40/30 °C. The resulting mature pollen grains were collected for quantitative label-free shotgun proteomic analysis. A total of 868 proteins was quantified. Interestingly, Hsp70s were highly induced in response to extreme heat indicating key roles of this family to cope with heat stress.
Project description:Following our initial transcriptomic analyses of the male gametophyte development (Honys and Twell, Genome Biol 5:R85, 2004), we identified several candidate genes for the function of transcriptional regulators of the male gametophyte development. Two transcription factors with verified function (bZIP and C2H2 ZF) were selected for the identification of their regulons. Mature pollen was isolated as published previously (Honys and Twell Plant Physiol 132:640-652, 2003) from Col-0 wild type plants and T-DNA insertion mutants (SAIL) homozygous for tested genes. 6 samples were used in this experiment.
Project description:In Arabidopsis thaliana, DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA (sRNA) transcripts. These transcripts are processed by DICER-LIKE3 into 24-nt small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nt epigenetically activated siRNAs (easiRNAs), which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernable pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV in the Brassicaceae species Capsella rubella, caused post-meiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were 2 depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nt and 24-nt siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella rubella compared to Arabidopsis thaliana, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models.
Project description:To investigate the potential function of miRNAs during male gametogenesis in rice, we first obtained gene expression profiles by using Affymetrix microarray technologies. In genome-scale, we found a common characteristic shared by rice and Arabidopsis, which vast of genes are down regulated from Pb to Pc stage. next, a total of 13363 genes were detected during the gametophyte development in rice, which contains 2925 pollen-enriched/-specific genes including 107 transcription factors. We also analyzed the microarray-based expression patterns of genes in miRNA pathways, and found 3 pollen-specific AGO genes (AGO12, AGO13 and AGO17). This subseries contains three datasets of gene expression in pollen development stages: uninucleate microspore (UNM/Pa), bicellular pollen (BCP/Pb) and tricellular pollen (TCP/Pc)
Project description:Pollen development from the microspore involves a series of coordinated cellular events, and the resultant mature pollen is specialized in function that it can quickly germinate and produces a polar-growth pollen tube derived from the vegetative cell to deliver two sperms for fertilization. Understanding the molecular program underlying pollen development and germination still remains a major challenge for plant biology. We used Affymetrix GeneChip Rice Genome Array to comprehensively analyzed the dynamic changes in the transcriptomes of rice pollen at five sequential developmental stages from microspores to germinated pollen. Among the 51,279 transcripts on the array, we found 25,062 pollen-preferential transcripts, among which 2,203 were development stage-enriched. The diversity of transcripts decreased greatly from microspores to mature and germinated pollen, whereas the number of stage-enriched transcripts displayed a U-type change, with the lowest at the bicellular pollen stage; and a transition of overrepresented stage-enriched transcript groups associated with different functional categories, which indicates a shift in gene expression program at the bicellular pollen stage. About 54% of the now-annotated rice F-box protein genes were expressed preferentially in pollen. The transcriptome profile of germinated pollen was significantly and positively correlated with that of mature pollen. Analysis of expression profiles and coexpressed features of the pollen-preferential transcripts related to cell cycle, transcription, the ubiquitin/26S proteasome system, phytohormone signalling, the kinase system and defense/stress response revealed five expression patterns, which are compatible with changes in major cellular events during pollen development and germination. A comparison of pollen transcriptomes between rice and Arabidopsis revealed that 56.6% of the rice pollen preferential genes had homologs in Arabidopsis genome, but 63.4% of these homologs were expressed, with a small proportion being expressed preferentially, in Arabidopsis pollen. Rice and Arabidopsis pollen had non-conservative transcription factors each. These results supply novel insights into the molecular program and key components of the regulatory network regulating pollen development and germination. KEYWORDS: rice (Oryza sativa L.), pollination and fertilization, stigma, molecular functions, signaling, microarray, stress response
Project description:During the reproductive stage, tomatoes require nighttime temperatures below 21°C and daytime temperatures below 32°C for healthy flower development and fruit set. Prolonged exposure to temperatures above the optimal range can cause abnormalities in pollen development, leading to reduced fertility or sterility. This project aims to identify alterations in proteome expression and elucidate heat tolerance mechanisms affecting pollen development under elevated temperature conditions. Two tomato varieties—the heat-tolerant ‘Black Vernissage’ and the heat-sensitive ‘Micro-Tom’—were exposed to three temperature treatments: 26 °C (control), 32 °C, and 37 °C (heat-treated). Flower buds containing meiotic pollen mother cells prior to the tetrad stage and free microspores were harvested, and single cell type samples were collected using laser microdissection. Tandem mass tag (TMT) proteomics was employed to identify differential proteomic responses in pollen mother cells and free microspores between stress-treated and non-treated plants.