Separated Transcriptomes of Male Gametophyte and Tapetum in Rice
ABSTRACT: In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on the spatiotemporal expression patterns and gene ontology (GO) categories of anther-expressed genes, some noteworthy expression patterns are discussed in connection with various important biological events of anther development. The separated transcriptomes of rice microspore/pollen and tapetum were measured at the premeiosis, meiosis, tetrad, uninuclear, bicellular, and tricelluar stages by using laser microdissection (LM)-mediated microarray.
Project description:Tissue-specific transcriptional profiling of the abscission layer (AL) at the base of young flower in rice using laser micro-dissection: NIL(qSH1) vs. Nipponbare. We used two rice varieties, NIL(qSH1) and Nipponbare. NIL(qSH1) is a nearly isogenic line containing the seed shattering gene qSH1. Seed shattering is easy in NIL(qSH1), but it is not in Nipponbare. So, we used some stages of young flower in NIL(qSH1) and some in Nipponbare. Four regions: 1. abscission layer region of NIL(qSH1), 2. upper abscission region of NIL(qSH1), 3. lower abscission layer region of NIL(qSH1), and 4. abscission layer region of Nipponbare. Sample experiments: NIL(qSH1) AL vs. Nipponbare AL, NIL(qSH1) AL vs. NIL(qSH1) upper region of AL, and NIL(qSH1) AL vs. NIL(qSH1) lower region of AL.
Project description:Using microarray, the anther transcript profiles of the three indica rice CMS lines revealed 622 differentially expression genes (DEGs) in each of the three CMS lines. GO and Mapman analysis indicated that these DEGs were mainly involved in lipid metabolic and cell wall organization. Comprised with the gene expression of sporophytic and gametophytic CMS lines, 303 DEGs were differentially expressed and 56 of them were down-regulated in all the CMS lines. Co-expression network analysis suggested that many genes were significantly differentially expressed in the CMS lines. These down-regulated DEGs in the CMS lines were found to be involved in tapetum or cell wall formation and their suppressed expression might be related to male sterility. The present study will give some information for the nuclear gene regulation by different cytoplasmic genotypes and provide some candidate genes for pollen development in rice. Overall design: Examine gene expression profiles of young panicles of three male sterile lines and their maitainer line.
Project description:Transcriptomic analysis of single, double and triple mutant anthers of bhlh010, bhlh089 and bhlh091. We examine here three recently duplicated Arabidopsis bHLH genes, bHLH010, bHLH089 and bHLH091, using evolutionary, genetic, morphological and transcriptomic approaches, and uncover their redundant functions in anther development. These three genes are relatively highly expressed in the tapetum of the Arabidopsis anther; single mutants at each of the bHLH010, bHLH089 and bHLH091 loci are developmentally normal, but the various double and triple combinations progressively exhibit increasingly defective anther phenotypes (abnormal tapetum morphology, delayed callose degeneration, and aborted pollen development), indicating their redundant functions in male fertility. Note: Samples in SRA were assigned the same sample accession. This is incorrect as there are different samples, hence “Source Name” was replaced with new values. Comment[ENA_SAMPLE] contains the original SRA sample accessions.
Project description:The CLAVATA3/ESR-RELATED (CLE) peptide hormones are required for numerous plant growth and developmental processes. However, little is known regarding the function and working mechanism of the CLEs in the anther. Here, using RNA in situ hybridization analyses, we identified 7 CLE genes that are specifically expressed in the tapetum and microsporocytes in the anther, and the dominant-negative mutant plants of each of these genes exhibited significantly reduced anther size, pollen number, and abnormal pollen wall formation. Further transcriptomic and proteomic studies on cle19, DN-CLE19, and CLE19-OX mutant lines revealed that CLE19 affected the expression of more than 1,000 genes at the RNA level and 595 at the protein level, including genes involved in pollen coat and pollen exine formation, lipid metabolism, pollen germination, and hormone metabolism processes. Phenotypic analyses of mutants of the CLE19 downstream genes GRP20, ACOS5 and MEE48 revealed that the formation of pollen exine was affected in these mutants, confirming that these genes function downstream of CLE19 in the regulation of pollen wall formation. These findings demonstrate the function and downstream genes of CLE19 and redundant genes, providing insights into working pathways of the peptide hormones in pollen development. Overall design: mRNA-seq data of wild type (WT and C24), CLE19::DNCLE19 and 35S::CLE19 transgenic plants stage 4-10 anther were generated by deep sequencing by the Illumina™ Hi-seq 2000 system
Project description:Small RNA diversity and function has been widely characterized in various tissues of the sporophytic generation of the angiosperm model Arabidopsis thaliana. In contrast, there is limited knowledge about small RNA diversity and their roles in developing male gametophytes. We thus carried out small RNA sequencing on RNA isolated from four stages of developing Arabidopsis thaliana pollen. Spores from 4 stages of pollen development (UNM: Uninucleate microspore – BCP: Bicellular pollen – TCP: Tricellular pollen – MP: Mature pollen) were isolated using a percoll gradient-based method (Honys and Twell, 2004) and the small RNA fraction for each sample was isolated and sequenced by Illumina technology. Reference: Honys, D. and Twell, D. (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol. 5/11/R85.
Project description:Despite their importance, there remains a paucity of large scale expression-based studies of reproductive development in the species belonging to the Triticeae. As a first step to address this deficiency, a gene expression atlas of triticale reproductive development was generated using the 55K Affymetrix GeneChip® Wheat Genome Array. The global transcriptional profiles of the anther/pollen, ovary and stigma were analyzed at concurrent developmental stages and co-regulated as well as preferentially expressed genes were identified. Data analysis revealed both novel and conserved regulatory factors underlying Triticeae floral development and function. Triticale reproductive tissues (anther, ovary, stigma) were collected at 4 successive stages using pollen development as a developmental reference: tetrad (TET), uninucleate microspore (UNM), bi-cellular pollen (BCP), and tri-cellular pollen (TCP). Mature pollen grains (MPG) were also collected. Three biological replicates were analyzed for each tissue using the 55K Affymetrix GeneChip® Wheat Genome Array.
Project description:Transcriptomes from multiple pre-meiotic stages of wild type, mac1, and msca1 maize anthers were characterized by microarray hybridization. The goal was to characterize the developmental progression as the anther specifies five cell types and grows rapidly precedeing meiotic entry. The stages characterized were immature anther primordia (0.15 mm long in maize) containing just stem cells, through somatic and germinal cell fate specification (0.20 and 0.25 mm), mitotic proliferation (0.4 mm), and finally the birth of the middle layer and tapetum (0.7 mm). To obtain cell-type specific markers, at 0.7 mm we also compared whole anthers to collections of laser-microdissected anther cell types including the archesporial cells (pre-meiotic germinal cells), nutritive layers (middle layer and tapetum) and structural layers (endothecium and epidemis) of the anther lobe. keyword: anther development, maize, male-sterile Three loop designs covered the early stages (up to 0.7 mm) with two replicates for each comparison. The first loop had 0.2 mm long anthers and compared wild type versus mac1 mutant versus msca1 mutant in a three vertex loop design. The second loop had four vertices and compared 0.15 mm WT anther primordia, 0.25 mm WT anthers, 0.4 mm WT anthers and finally 0.4 mm mac1 mutant anthers. The third had 0.7 mm anthers in a three vertex loop with the nutritive layers (middle layer and tapetum) at one vertex, the germinal pre-meiotic cells at another vertex, and whole anthers at a third vertex. The whole anther samples were also, separately and outside of the loop, compared in four replicates to the structural layers (endothecium and epidermis).
Project description:This study was initiated with the objective of identifying the anther/tapetum specific promoters from cotton floral buds. Cotton is an important commercial crop. Hybrid cotton varieties are developed to obtain improved yield and fiber quality. Most of the hybrid seed production in cotton is carried out by hand emasculation, which requires large amount of manpower, resulting in high cost of hybrid seed. We are developing barnase-barstar based male sterility system, which would be a better alternative for hybrid development. The tapetum specific promoters are main requirement for such a system. The study was thus carried out to identify genes expressed in the anthers. Cotton bud sizes were correlated with tapetum development. RNA was isolated from following tissues: • Anther tissues from buds at pre-meiotic stage of development (Tapetum absent) • Buds without anther tissues at pre-meiotic stage of development • Anther tissues from buds during meiosis (Tapetum present) • Buds without anther tissues during meiosis • Anther tissues from buds at post-meiotic stage of development (Tapetum degenerated) • Buds without anther tissues at post-meiotic stage of development • Leaf tissues • Seedling 5 days after germination Biotin labeled cRNA was hybridized on Affymertix cotton Genechip Genome array following Affymetrix protocols. Three biological replicates were maintained.
Project description:Arabidopsis thaliana MYB80 (formerly MYB103) is expressed in the tapetum and microspores between anther developmental stages 6 and 10. MYB80 encodes a MYB transcription factor that is essential for tapetal and pollen development. In order to identify the genes regulated by MYB80, microarray technology was employed to analyze the expression levels of genes that were differentially regulated in the myb80 mutant and wild- type anthers. Plant Cell 23:2209–2224 (2011) three mutant chips vs three wild-type chips
Project description:A series of microarray experiments by using RNA sources from UDT1-1 mutant type- and wild type-anthers at stages from meiosis to young microspore. In conjuction, microarray experiments by using RNA sources from wild type_Palea/lemmas in the flowering stage and wild type anther at pollen mitosis were performed in a time course design and compared to the microarrays of UDT1-1 mutant type- and wild type-anthers. Keywords: time-course