ABSTRACT: Global gene expression profiles of seven stages representing 29 days of anther development are analyzed using a 44K oligonucleotide array querying ~80% of maize protein-coding genes. Each anther stage expresses ~10,000 constitutive and ~10,000 or more transcripts restricted to one or a few stages. Keywords: anther development, maize Overall design: 4 replicates of 7 samples (6 anther stages plus mature pollen) were hybridized to a 44K Agilent array according to the A-optimal design recommended by Kerr and Churchill for 7 samples.
INSTRUMENT(S): Zea mays 1x44K Agilent array - designed by Walbot Lab
Project description:Global gene expression profiles of seven stages representing 29 days of anther development are analyzed using a 44K oligonucleotide array querying ~80% of maize protein-coding genes. Each anther stage expresses ~10,000 constitutive and ~10,000 or more transcripts restricted to one or a few stages. Keywords: anther development, maize 4 replicates of 7 samples (6 anther stages plus mature pollen) were hybridized to a 44K Agilent array according to the A-optimal design recommended by Kerr and Churchill for 7 samples.
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 Overall design: 3 biological replicates of 3 different male-sterile mutants and their fertile siblings at 4 stages of anther development (36 arrays). Comparisons were done between male-sterile and fertile sibs at the same development stage.
Project description:Four tissues (leaves, 1 mm anthers, 1.5 mm anthers, pollen) from one hybrid and one inbred maize line were hybridized with the W23 inbred on Agilent oligonucleotide microarrays with 21K elements. Tissue-specific gene expression patterns were documented with leaves having the most tissue-specific transcripts. Haploid pollen expresses about half as many genes as the other samples. High overlap of gene expression was found between leaves and anthers. Anther and pollen transcript expression showed high conservation among the three lines while leaves had more divergence. Antisense transcripts represented about 6 to 14 percent of total transcriptome by tissue type but were similar across lines. Gene Ontology (GO) annotations were assigned and tabulated. Enrichment in GO terms related to cell cycle functions was found for the identified antisense transcripts. Microarray results were validated via quantitative real time PCR and by hybridization to a second oligonucleotide microarray platform. Keywords: genetic background comparison Overall design: We analyzed 4 tissues from 3 maize lines, each with 3 biological replicates, with W23 as a reference, and hybridized to 24 arrays.
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 Overall design: 4 replicates of ms8 mutant and 4 replicates of ms8 fertile siblings at 3 anther stages. Spike-in controls were included.
Project description:MuDR/Mu are a highly active transposon family moving by either cut only (or cut-and-paste) in strictly somatic tissues or net replicative transposition (absence of excision alleles) in reproductive tissues. Aside from the MuDR-encoded MURA and MURB proteins, other factors required for Mu transposition, particularly those contributing to the developmentally specific behavior, have yet to be identified. To address this question and assess the impact of a highly active transposon on the transcriptome, RNA was extracted from anthers at three developmental stages in Mu-active and -inactive stocks and compared on a 44,000 element oligonucleotide array. We found that approximately 30,000 unique genes are expressed at each stage. Of the ~10% (ca. 3000) differentially regulated transcripts per stage, there was approximately equal representation by the active or inactive individuals. Keywords: anther development, maize, Mu, transposon Overall design: Dye swap design with 4 replicates comparing Mu-active to -inactive lines at 3 anther stages. Spike-in controls were included.
Project description:The maize spikelet contains two florets, each of which contains three anthers. Morphologically, the anthers in the upper and lower florets proceed through the same developmental program. To test for global differences in gene expression between these anthers and to identify genes that are coordinately regulated during development, RNA isolated from upper and lower floret anthers at six developmental stages was used to hybridize cDNA microarrays. Approximately 9% of the tested genes exhibited statistically significant differences in gene expression between anthers in the upper and lower florets. This suggests that several basic biological processes are differentially regulated between upper and lower floret anthers, including photosynthesis, translation, and metabolism. To identify genes that are coordinately regulated during anther development, cluster analyses were performed. A variety of patterns of gene expression were observed. Five genes involved in programmed cell death were up-regulated at the time the tapetum begins to degenerate (i.e., the early microspore stage). This finding strongly supports the hypothesis that tapetal degeneration occurs via programmed cell death. In addition, many genes involved in protein synthesis exhibited differential expression patterns during anther development. Each microarray slide was scanned multiple times. This approach led to the identification an additional 30 to 40% of statistically significant differences in gene expression as compared to a single-scan strategy. Keywords: time course Overall design: Experimental aims: 1) To develop a cDNA microarray scanning approach that utilizes multiple scans of different intensities, thus maximizing the power to detecting statistically significant differences in gene expression; 2) To apply the multiple scan approach to identify differentially expressed genes in: a) the upper floret of (N)Ky21 at the selected stages of anther development; b) the lower floret of (N)Ky21 at the selected stages of anther development; and c) the upper vs. lower floret at each selected stage of anther development.
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. We examined transcript abundance data at 50 developmental stages/tissues of maize, from embryogenesis to senescence, using a custom Affymetrix Unigene array. The VE stage is germination and emergence Vn leaf stage refers to when the collar of the nth leaf is visible.
Project description:Saha2011- Genome-scale metabolic network of
Zea mays (iRS1563)
This model is described in the article:
Zea mays iRS1563: a
comprehensive genome-scale metabolic reconstruction of maize
Saha R, Suthers PF, Maranas
PLoS ONE 2011; 6(7): e21784
The scope and breadth of genome-scale metabolic
reconstructions have continued to expand over the last decade.
Herein, we introduce a genome-scale model for a plant with
direct applications to food and bioenergy production (i.e.,
maize). Maize annotation is still underway, which introduces
significant challenges in the association of metabolic
functions to genes. The developed model is designed to meet
rigorous standards on gene-protein-reaction (GPR) associations,
elementally and charged balanced reactions and a biomass
reaction abstracting the relative contribution of all biomass
constituents. The metabolic network contains 1,563 genes and
1,825 metabolites involved in 1,985 reactions from primary and
secondary maize metabolism. For approximately 42% of the
reactions direct literature evidence for the participation of
the reaction in maize was found. As many as 445 reactions and
369 metabolites are unique to the maize model compared to the
AraGEM model for A. thaliana. 674 metabolites and 893 reactions
are present in Zea mays iRS1563 that are not accounted for in
maize C4GEM. All reactions are elementally and charged balanced
and localized into six different compartments (i.e., cytoplasm,
mitochondrion, plastid, peroxisome, vacuole and extracellular).
GPR associations are also established based on the functional
annotation information and homology prediction accounting for
monofunctional, multifunctional and multimeric proteins,
isozymes and protein complexes. We describe results from
performing flux balance analysis under different physiological
conditions, (i.e., photosynthesis, photorespiration and
respiration) of a C4 plant and also explore model predictions
against experimental observations for two naturally occurring
mutants (i.e., bm1 and bm3). The developed model corresponds to
the largest and more complete to-date effort at cataloguing
metabolism for a plant species.
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Project description: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:Mutant maize lines expressing RNAi gene silencing constructs directed to two distinct classes of chromatin associated proteins show heightened sensitivity to UV-B including sunburned necrosis and down regulation of chromatin genes (Casati and Walbot, 2006). We have available transgenic maize expressing an RNAi for these chromatin factors: a subunit of a chromatin remodeling complex R, which is a SWI2/SNF2-like protein containing a SNF2 N and a helicase C domain (CHC101, AI746001); and a methyl-CpG-binding protein (MDB101, AI737448). To better understand the processes that involve chromatin remodeling proteins in UV-B responses, we conducted transcriptome analysis of chromatin remodeling knockdown plants in under two radiation regimes: plants were illuminated with UV-B lamps using fixtures mounted 30 cm above the plants (Phillips, F40UVB 40 W and TL 20 W/12) for 8 h; leaf samples were collected immediately after the radiation treatment. The bulbs were covered with CA filters to exclude wavelengths lower than 280 nm (UV-B). As a control, the bulbs were covered with PE filters to exclude wavelengths lower than 320 nm (minus UV-B). We analyzed genes that are changed upon UV-B irradiation in the transgenic plants and compared to the responses in non transgenic plants. To differentiate between differences due to background specific genes expression, we used non-transgenic sibling as controls. For these experiments, we used Long oligonucleotides arrays from Agilent Technologies. These studies will help to dilucidate how alteration in chromatin remodeling protein proteins affect gene expression, and the transcriptome analysis will help to understand why transgenic plants are hypersensity to UV-B. Keywords: genetic modification-stress response Overall design: Array hybridizations were carried out according to the manufacturer's instructions. For the experiments, Agilent Technologies microarrays were used, they contained 43,451 maize 60-mer probes and each slide had the same pattern of probes in quadruplicate (4x44 version), that were used to hybridize independent biological samples. Specifically, each array was hybridized with two samples, each of 0.825 µg labeled target cRNA, for 17 hours at 60°C. The samples were hybridized in a double loop design arrangement, as described by Kerr and Churchill et (2001). Data were acquired with an Agilent G2565BA scanner. Hybridizations for all 12 comparisons were highly correlated as assessed from correlations between median signal intensities (r2 = 0.97 for both dyes; data not shown) and between log2 ratios of the signals (r2 = 0.94; data not shown). Each sample was analyzed using 4 different biological replicates, and were hybridize to different arrays.