Project description:In this study we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. These are the RNA-seq datasets used in this study. We profiled changes in gene expression during normal maize ear and tassel development and in developing maize ear primordia upon genetic perturbation of the RAMOSA branching pathway. For the wild-type ear and tassel developmental series, greenhouse-grown B73 inbred plants were used. 10mm ears were collected and sectioned as follows from tip to base along the developmental gradient: tip 1mm sampled (tip; Inflorescence Meristem/Spikelet Pair Meristem), next 1mm discarded, next 1mm sampled (mid; Spikelet Meristem), next 2mm discarded, next 2 mm sampled (base; Floral Meristem), and immediately frozen in liquid nitrogen. Sections from ~30 sampled ears were pooled for each of 2 biological replicates to represent tip, mid, and base stages. Tassels were hand-dissected, measured, separated by stage: 1-2mm (stg1), 3-4mm (stg2), and 5-7mm (stg3), and immediately frozen in liquid N. For each stage, ~20-30 tassels were pooled for each of 2 biological replicates. For ramosa mutant series, segregating families (1:1) of ra1-R, ra2-R, and ra3-fea1 mutant alleles, all introgressed at least 6 times into the B73 inbred background, were grown at CSHL Uplands Farm. Field-grown plants were genotyped and collected 6-7 weeks after germination (V7-V8 stage). First and second ear primordia were immediately hand-dissected, measured, and frozen in liquid nitrogen. For ra1, ra2 and ra3 mutants and wild-type controls, ears were pooled into two size classes: 1) 1mm class included a range of 0.7-1.5mm sized ears and nine ears were pooled for each of 2 biological replicates; 2) 2mm class included a range of 1.8-2.5mm sized ears and six ears were pooled for each of three biological replicates. Wild-type samples were proportional mixtures of heterozygote siblings segregating in ra1, ra2, and ra3 populations. Variability factors (e.g. ear size within class, ear rank on the plant, and time of collection) were distributed evenly across pooled samples.

Project description:Maize LEAFBLADELESS1 (LBL1) and Arabidopsis SUPPRESSOR OF GENE SILENCING3 (SGS3) play orthologous roles in the biogenesis of 21 nucleotide trans-acting short-interfering RNAs (tasiRNAs). The phenotypes conditioned by mutation of lbl1 and SGS3 are, however, strikingly different, suggesting that the activities of these small RNA biogenesis components, or the tasiRNAs and their targets might not be entirely conserved. To investigate the basis for this phenotypic variation, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects all major classes of small RNAs, and reveal unexpected crosstalk between tasiRNA biogenesis and other small RNA pathways regulating miRNAs, retrotransposons, and DNA transposons. We further identified genomic regions generating phased siRNAs, including numerous loci generating 22-nt phased small RNAs from long hairpin RNAs or overlapping antisense transcripts not previously described in other plant species. By combining both analyses, we identified nine TAS loci, all belonging to the conserved TAS3 family. Contrary to other plant species, no TAS loci targeted by a single miRNA were identified. Information from target prediction, RNAseq, and PARE analyses identified the tasiARFs as the major functional tasiRNAs in the maize vegetative apex where they regulate expression of ARF3 homologs. As such, divergence in TAS pathways is unlikely to account for the distinct phenotypes of tasiRNA biogenesis mutants in Arabidopsis and maize. Instead, the data suggests variation in the spatiotemporal regulation of ARF3, or divergence in its function, as a plausible basis for the dramatic phenotypic differences observed upon mutation of SGS3/lbl1 in Arabidopsis and maize. An analysis of tasiRNA biogenesis, activity, and contribution to developmental phenotypes in the maize leaf. Data generated includes small RNA sequencing data and mRNA sequencing data. All data was generated in both wild type and lbl1 mutant maize leaf apices. Three replicates were generated for each genotype for the small RNA data. Two of these replicates were also used for the RNA-seq data.

Project description:Trimethylation of histone H3 lysine 27 (H3K27me3) plays a critical role in regulating gene expression during plant and animal development. We characterized the genome-wide distribution of H3K27me3 in five developmentally distinct tissues in maize plants of two genetic backgrounds, B73 and Mo17, representatives of two distinct heterotic groups. There are numerous differences in the distribution of H3K27me3 among tissues. In contrast, we found the distribution of H3K27me3 among the two genetic backgrounds to be quite similar. The tissue-specific patterns of H3K27me3 are often associated with differences in gene expression among the tissues and most of the imprinted genes that are expressed solely from the paternal allele in endosperm are targets of H3K27me3. Many maize genes with important developmental roles, including numerous genes encoding putative transcription factors, are modified with H3K27me3 in at least one of the tissues that were profiled. A comparison of the H3K27me3 targets in rice, maize, and Arabidopsis provided evidence for conservation of the H3K27me3 targets among plant species. However, there was limited evidence for conserved targeting of H3K27me3 in the two maize subgenomes derived from whole genome duplication. Genomic profiling of H3K27me3 in loss-of-function mutant stocks for Mez2 and Mez3, two of the three putative H3K27me3 methyltranferases present in the maize genome, suggests partial redundancy of this gene family for maintaining H3K27me3 patterns. Only a portion of the targets of H3K27me3 requires Mez2 and/or Mez3 and there was limited evidence for functional consequences of H3K27me3 at these targets. This study provides a catalogue of 6,337 genes that are marked by H3K27me3 in five maize tissues. B73 tissues for h3k27 analysis: Samples are replicated in triplicate with antibody pulldown (IP) and control genomic DNA (input) channels. histone modification mutants for h3k27 analysis: Samples are replicated in triplicate with antibody pulldown (IP) and control genomic DNA (input) channels. Mo17 tissues for h3k27 analysis: Samples contain antibody pulldown (IP) and control genomic DNA (input) channels.

Project description:miRNA levels depend on both biogenesis and turnover. The methyltransferase HEN1 stabilizes plant miRNAs, animal piRNAs, and siRNAs in both kingdoms via 3' terminal methylation. Loss of HEN1 in plants results in non-templated oligo-uridylation and accelerated degradation of miRNAs. In hen1 mutants from Arabidopsis and rice, we found that the patterns of miRNA truncation and uridylation differ substantially among miRNA families, but such patterns for the same miRNA are conserved between species. miR166 and miR163 are truncated predominantly to ~17 and ~16 nt, and subsequently recover via uridylation to approximately their original sizes, 21 and 24 nt, suggesting that in these cases miRNA truncation triggers uridylation. miR171 is untruncated but uridylated to 22 nt in hen1 mutants, gaining the ability to trigger production of phased, secondary siRNAs. Truncated and tailed variants were bound by ARGONAUTE1 (AGO1) in hen1, implying that these events occur while miRNAs are still bound by AGO1. Unexpectedly, a portion of miR158 in wildtype remains unmethylated and thus subject to uridylation and destabilization, suggesting that plants naturally utilize miRNA methylation to modulate miRNA accumulation. Our results suggest that the AGO1-containing RISC complex may undergo programming to reflect each bound miRNA, determining a defined, distinct decay destiny. In this analysis, we sequenced sRNAs from two hen1 mutant alleles in Arabidopsis and three hen1 alleles in rice. In Arabidopsis, the strong hen1-1 allele in the Landsberg erecta (Ler) ecotype is the first hen1 mutant and emerged from an enhancer screen in the hua1-1/hua2-1 background, and hen1-8 in the Columbia (Col) background is a weak allele. In rice, WAVY LEAF1 (WAF1) is the ortholog of Arabidopsis HEN1, and two mutant alleles waf1-1 and waf1-2 each bear a single-base substitution leading to a premature stop codon in the second exon and a non-functional splicing site of the fourth intron, respectively. We identified a third mutant allele of the rice HEN1 gene (Oshen1-3 from the Korean (POSTEC) rice T-DNA mutant population).

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Regulatory Modules Controlling Maize Inflorescence Architecture

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