Characterization of small RNAs and genes in maize anthers (ms23 sterile)
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ABSTRACT: Successful male gametogenesis involves orchestration of sequential gene regulation for somatic differentiation in pre-meiotic anthers. We report here the cloning of Male Sterile23 (Ms23), encoding an anther-specific predicted basic helix-loop-helix (bHLH) transcription factor required for tapetal differentiation; transcripts localize initially to the precursor secondary parietal cells then predominantly to daughter tapetal cells. In knockout ms23-ref mutant anthers, five instead of the normal four wall layers are observed. Microarray transcript profiling demonstrates a more severe developmental disruption in ms23-ref than in ms32 anthers, which possess a different bHLH defect. RNA-seq and proteomics data together with yeast two-hybrid assays suggest that MS23 along with MS32, bHLH122, and bHLH51 act sequentially as either homo- or heterodimers to choreograph tapetal development. Among them, MS23 is the earliest-acting factor, upstream of bHLH51 and bHLH122, controlling tapetal specification and maturation. In contrast, MS32 is constitutive and independently regulated and is required later than MS23 in tapetal differentiation. We characterized a maize (Zea mays) mutant line, ms23-ref, which the tapetal cells lack a dense cytoplasm and are not Binucleate -- the two characteristics of normal TP during meiosis. The meiocytes in ms23-ref fail to progress beyond meiotic prophase I. By profiling the small RNA population in ms23 mutants, we investigated the impact of ms23 mutation on small RNAs abundance in 0.4 mm, 0.7 mm, 1.0 mm, 1.5 mm and 2.0 mm anther. We also investigated the transcript abundance that were impacted in ms23 mutant by transcriptome profiling. The result provides an avenue for future research to understand the genetic networks and protein interactions among ms23, important for tapetal development in maize anther.
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: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
Project description:Phenotypes of maize male sterile 8 plants are small anthers, meiotic failure, shorter epidermal cells, and non-secreting tapetal cells. Excess callose accumulates around meiotic cells, which later abort. Thousands of transcriptome changes occur including ectopic activation of genes not expressed in fertile siblings, failure to express genes, and differential expression of genes shared with fertile siblings. Sixty-three differentially expressed proteins were identified after 2-D DIGE followed by LC/MS/MS, including those involved in metabolism and cell division. The majority were not identified by differential RNA expression. Keywords: anther development, maize, male-sterile, ms8 4 replicates of ms8 mutant and 4 replicates of ms8 fertile siblings at 3 anther stages. Spike-in controls were included.
Project description:A cascade of basic helix-loop-helix transcription factors guide tapetal cell development in maize anthers, using proteins conserved in Arabidopsis and rice but deployed with a distinctive timing. Anthers were dissected and staged to be 1500 μm in length (+/- 100 μm) and samples were compared between mutants and fertile siblings on an Agilent 4x44 custom microarray. Analysis included MS32 which is another basic helix-loop-helix factor that acts later during tapetal differentiation.
Project description:Phenotypes of maize male sterile 8 plants are small anthers, meiotic failure, shorter epidermal cells, and non-secreting tapetal cells. Excess callose accumulates around meiotic cells, which later abort. Thousands of transcriptome changes occur including ectopic activation of genes not expressed in fertile siblings, failure to express genes, and differential expression of genes shared with fertile siblings. Sixty-three differentially expressed proteins were identified after 2-D DIGE followed by LC/MS/MS, including those involved in metabolism and cell division. The majority were not identified by differential RNA expression. Keywords: anther development, maize, male-sterile, ms8
Project description:Evidence from confocal microscopic reconstruction of maize anther development in fertile, mac1 (excess germ cells) and msca1 (no germ cells) flowers indicates that the male germ line is multiclonal and uses the MAC1 protein to organize the somatic niche. Furthermore, we identified redox status as a determinant of germ cell fate, defining a mechanism distinct from the animal germ cell lineage. Decreasing oxygen or H2O2 increases germ cell numbers, stimulates superficial germ cell formation, and rescues germinal differentiation in msca1 flowers. Conversly, oxidizing environments inhibit germ cell specification and cause ectopic differentiation in deeper tissues. We propose that hypoxia, arising naturally within growing anther tissue, acts as a positional cue to set germ cell fate. key words: anther development, maize, male-sterile Two replicates of laser microdissected pre-meiotic cells were compared to two replicates of whole anthers, which contains pre-meiotic cells and somatic cells, from the same developmental stage and individual wild type plants. In another experiment, laser microdissected pre-meiotic cells from wild type (mac1-heterozygous) anthers were compared to laser microdissected pre-meiotic cells from mac1 (mutant) anthers.
Project description:In maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known. Using laser capture microdissection (LCM), we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products. This dataset includes 24-nt phasiRNA part of data. 24-nt phasiRNAs are found to accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum.
Project description:We isolated pre-meiotic and early meiotic cells from 24 maize anthers, covering a week of development from the day after archesporial (AR) cell specification to the early zygotene stage of meiotic prophase I. Starting material was staged by anther length, and anther stages were densely sampled from throughout this period. High quality reads were obtained from 144 cells.
Project description:Agilent oligonucleotide arrays were used to profile gene expression in dissected maize anthers of 3 types of male-sterile plants and their fertile siblings at four stages of development: after anther initiation, at the rapid mitotic proliferation stage, pre-meiosis, and meiotic prophase I. The male-sterile mutants (ms23, msca, and mac1) lack a range of normal cell types resulting from a temporal progression of anther failure. By combining the data sets from the comparisons between individual sterile and fertile anthers, candidate genes predicted to play important roles during maize anther development were assigned to stages and to likely cell types. Keywords: anther development, maize, male sterility