Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from different maize tissues (including leaves, ears and tassels) collected from wild-type plants of the B73 variety. The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study.
Project description:RNA-directed DNA methylation (RdDM) in plants is a well-characterized example of RNA interference-related transcriptional gene silencing. To determine the relationships between RdDM and heterochromatin in the repeat-rich maize (Zea mays) genome, we performed whole-genome analyses of several heterochromatic features: dimethylation of lysine 9 and lysine 27 (H3K9me2 and H3K27me2), chromatin accessibility, DNA methylation, and small RNAs; we also analyzed two mutants that affect these processes, mediator of paramutation1 and zea methyltransferase2.
Project description:Anthers are the male reproductive floral organs, but loss of the 24-nt class of phased small-interfering RNAs (phasiRNAs) may confer temperature-sensitive male sterility in maize. 24-nt phasiRNAs from 176 loci are abundant coordinately in fertile maize anthers after the last periclinal divisions are complete, all four wall layers are formed, and meiosis has initiated in the pollen mother cells. In this study, we found male sterile maize ms23, ms32, and bhlh122 mutants with various degrees of tapetal defects lack 24-PHAS mRNA precursors and 24-nt phasiRNAs, which were not affected in another male sterile mutant, bhlh51. Transcription from most of 24-PHAS loci depends on bHLH122 while bHLH122 and Dcl5 transcription depends on both Ms23 and Ms32. Multiple lines of evidence suggest that 24-nt phasiRNAs biogenesis is primarily controlled at multiple layers by MS23 and MS32, with distinctive action on 24-PHAS transcription by bHLH122, creating a transcription factor cascade in maize tapetal 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
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from different maize tissues (including leaves, ears and tassels) collected from wild-type plants of the B73 variety. The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from leaves, ears and tassels of maize variety B73 (wild-type). Plants were grown in a flood irrigated plot at the University of Arizona (Tucson, AZ, USA) in 2007 and organs were pooled from several plants for each library. Young leaves were collected from 6-weeks-old seedlings. Post-meiotic immature ears were harvested from 10- and 11-week old plants while pre-meiotic tassels were collected from 8-week old plants. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen) and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Lyudmila Sidorenko and Vicki Chandler for providing the plant material and Kan Nobuta for assistance with the computational methods.
Project description:These data include RNA-seq, circRNA-seq, and small RNA-seq of transcriptome, Ribo-seq of translatome and protein protein binary interactions by recombination-based library vs. library yeast-2-hybrid throughout the lifecycle of the maize inbred line B73.
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:Through hierarchical clustering of transcript abundance data across a diverse set of tissues and developmental stages in maize, we have identified a number of coexpression modules which describe the transcriptional circuits of maize development.