Unknown,Transcriptomics,Genomics,Proteomics

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A global analysis of gene expression in histological layers of the shoot apical meristem of maize

ABSTRACT: All above ground organs of higher plants are ultimately derived from specialized organogenic structures termed shoot apical meristems (SAMs). The SAM exhibits distinctive structural organization, marked by cell layering. Maize SAMs are comprised of two cell layers, L1 (single cell layered tunica) and L2 (corpus). To identify genes required for layer-specific functions intact maize SAMs were fixed, embedded in paraffin and sectioned. L1 and L2 cells were isolated from these sections via laser capture microdissection (LCM). RNA was isolated from six biological replications of L1 and L2, amplified and hybridized to microarrays spotted with ~37,000 maize cDNA clones. This experiment identified ~700 ESTs that are preferentially expressed in the L1 or the L2 (P <0.001). The L1-up-regulated ESTs included ZmOCL1 and ZmOCL4, which are known to exhibit L1-specific expression in the maize SAM. The L2-up-regulated ESTs included KNOTTED1, whose transcripts are known to accumulate in the L2 but not in the L1 of the maize SAM. Differentially expressed ESTs included genes involved in transcription, signal transduction, transport and metabolism, many of which are novel candidates that are required for layer-specific functions in the maize SAM. Several L1-up-regulated ESTs were annotated as yabby family genes or basic helix-loop-helix transcription factor-like genes, which have not previously been reported as having layer-specific expression in the SAM. Novel WW domain-containing genes (WW genes) were identified in this study. The WW domain mediates protein-protein interactions, often with signal transduction components. These WW genes were substantially up-regulated in the L1 relative to the L2. Keywords: Cell Type Comparison An experimental aim is to identify genes that are differentially expressed in distinct histological cell layers of maize SAM by comparing the transcript accumulation between L1 (single cell layered tunica) and L2 (corpus) using cDNA microarrays that have over 37,000 informative spots from maize.

ORGANISM(S): Zea mays

SUBMITTER: Patrick Schnable

PROVIDER: E-GEOD-9610 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Natural Antisense Transcripts (NATs) can regulate gene expression by virtue of their ability to form double-stranded RNA duplexes. To investigate NATs in the maize transcriptome, cDNAs from seedling of two inbred lines (B73 and Mo17) were hybridized to an oligonucleotide microarray designed to validate the expression of in silico detected NATs and to screen for NATs that can anneal to a random set of 3â UTRs and selected repeats found in 3â UTRs regions. Quantitative Real-Time PCR experiments were conducted to determine the minimum detection threshold of microarray experiment and to thereby identify genes for which both sense and antisense transcripts accumulate to detectable levels. Two independent approaches, strand-specific RT-PCR and S1 nuclease assays were conducted to validate the results of the microarray experiment. Based on these conservative assays, NATs accumulate in seedlings that can anneal to over 70% of a random set of maize genes. In addition, both sense and antisense transcripts anneal to more than 80% of a set of maize repeats. Significantly, sense and antisense transcripts exhibit significant different expression patterns between the two genotypes. Based on these findings we hypothesize that interactions between sense and antisense transcripts may contribute to the differential patterns of gene expression in maize hybrids and to heterosis. Keywords: Global antisense transcripts profiling between two maize inbreds To systematically identify NATs in maize, we employed multiple strategies to computationally identify putative antisense transcripts from our partial genome assembly (MAGIs) and a collection of ESTs we sequenced with known sequence orientation. Additionally, we randomly sampled and surveyed maize UTRs which often harbor transposons. Strand-specific oligonucleotides which can hybridize to the antisense strand were designed and spotted with together with the oligonucleotides hybridizing sense strands on a custom, strand-specific oligoarray, providing the first global expression profiling study of NATs in maize UTRs. In total, 10 biological replication were conducted to compare the global gene expression profiles between two maize genotypes (B73 and Mo17).

Project description:Non-additive gene regulation has been recently suggested as an important factor promoting phenotypic variation and plasticity. In order to obtain a description of gene expression status at an early stage of ear development in a maize (Zea mays L.) F1 hybrid as relative to its parental inbreds, we compared gene expression profiles in immature ears of elite inbred lines B73 and H99 to one of their F1 hybrids (B73xH99) using cDNA microarray technology. Results show several genes expressed at a significantly different level between both inbred lines and their hybrid. In addition, gene expression non-additivity in the hybrid was detected on a broad scale, consisting of both dominance and over-dominance components, indicating that complex non-additive interactions at the molecular level exist in the developing ear of the studied maize hybrid. Non-additively regulated genes belong to a wide range of molecular functions, indicating that several regulatory and metabolic patterns are possibly affected during ear development in the investigated hybrid. We discuss the possibility that observed gene expression non-additivity in immature ear might be an early molecular manifestation of hybrid vigor, the most exploited factor for maize agronomic improvement. We directly contrasted both B73 and H99 inbred lines vs. their F1 heterotic hybrid transcriptomes in immature ear. Our experimental design consisted of 10 cDNA microarray hybridizations, 5 for each combination of hybrid vs. inbred genotypes, involving 20 separate labeling reactions. Labeling dyes were swapped in two of the five replicates for each combination. In each hybridization, control channel was assigned to the F1 hybrid, and differences in transcriptional levels between B73 and H99 were inferred using the hybrid as a common reference sample in an indirect experimental design. To take in account variability in transcript population among individuals, total RNA coming from different isolations, each collected on multiple individuals, were mixed before poly(A+) RNA purification. All hybridizations on microarray slides were then performed using cDNA independently labeled from the poly(A+) RNA purification product for each genotype. Base-two logarithms of expression ratios were subjected to one-class response significance analysis in SAM v. 2.20 software [Tusher et al. 2001]. For each EST the estimates of additive parameter "a" and dominance parameter "d" (middle-parent heterosis) were obtained as a = (L2 – L1)/2 d = (L1 + L2)/2 (where L1 and L2 are mean base-two logarithms of expression ratios of F1 vs. B73 and F1 vs. H99, respectively). Positive values of "a" indicate expression values bigger in B73 than in H99. The dominance/additivity ratio (d/|a|) was also calculated [Falconer 1989]. Evaluation of statistical significance of parameters was done by calculating standard errors of the estimates "a" and "d" as standard errors of linear functions of the means. Significance testing was done correspondingly, using an F-test for linear contrasts. P-values for the families of tests corresponding to each parameter were subjected to global error analyses using a method based on fitting mixture distribution [Allison et al. 2002], allowing to estimate the false discovery rates (FDR) and false negative rates (FNR). Confidence intervals for d/|a| ratios were obtained by Fieller’s method [Piepho and Emrich 2005], allowing to classify the genes into different dominance type classes.

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A global analysis of gene expression in histological layers of the shoot apical meristem of maize

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