Plants to be Studied:
Principal - Zea mays
Project Objectives:
- - Develop and optimize laser capture microdissection microarray procedures for the analyses of maize vegetative shoot apical meristems (SAMs).
- - Determine if the publicly available maize microarry platforms contain adequate representation of genes whose expression is enriched in the vegetative SAM, and if not, a maize SAM-enriched EST discovery project will be initiated.
- - Identify genes differentially expressed in meristematic and leaf primordial developmental domains.
- - Determine the tissue- and cell-specific expression profiles of genes that are differentially expressed in maize meristems and leaf primordia.
Experimental Approaches:
The bulk of the proposed activities in the first 6 to 12 months of this project are dedicated to optimization of the LCM-microarray technology to the specific SAM tissues targeted in the experimental plan. Three specific tasks will be undertaken:
- - Comparison and optimization of the LCM procedure for paraffin-embedded versus cryosectioned SAM tissues
- - Determination of whether LCM is a useful procedure for harvesting of SAM-specific transcripts
- - Beta testing of the utility and performance of the publicly available maize oligo-arrays and cDNA microarrays
Microarray hybridizations will compare LCM-captured SAM transcripts to transcripts collected from whole maize seedlings, and to mixed sources of RNA (excluding tissues containing inflorescence or floral meristems. These controls are designed towards the identification of maize transcripts that are specifically abundant in the vegetative SAM, and will determine whether or not the available microarray platforms will require supplementation with SAM-specific clones in order to be useful for the purposes of this project. If the available platforms are found to be bereft of SAM-specific sequences, a maize SAM EST discovery project will be initiated during the first year of this project.
Laser capture microdissection microarray analyses will compare global patterns of gene expression in maize shoot meristems and developing leaf primordia. RNA will be extracted from meristematic cells that are harvested from sectioned vegetative shoot meristems or young leaf primordia via laser capture microscopy. Following amplification of the extracted RNA, labeled cDNA will be used in microarray analyses. Genes that exhibit differential expression in a variety of developmental domains (including meristematic L1 vs L2 layers; apical meristematic zones; KNOX accumulating meristem cells vs. KNOX-off; adaxial vs. abaxial domains of leaf primordia), leaf mutations (narrow sheath; ragged seedling2; leafbladeless1 and rough sheath2), and growth conditions (NPA leaf -arrested meristems vs unarrested) will be identified. Approximately 200 of these differentially-expressed genes will be selected for further investigation and verification of gene expression and function.
Approximately 200 differentially-expressed genes will be selected for verification of gene expression patterns using real time RT-PCR and Northern gel blot analyses. Subsequently, maize in situ hybridization analyses will be performed on approximately 25 high-priority genes, in order to identify their organ, tissue, and domain-specific expression patterns.
Information/Materials to be Generated:
- Maize EST clones
- Microarray profiles of global gene expression
- Detailed expression patterns of genes specific to, or enriched in, plant meristems.
Practical Applications of Research:
These experiments will microdissect gene expression patterns in meristems and leaf primordia, and promise to provide novel insight into mechanisms of plant development.
Detailed project description (PDF) - 2 MB : Part of the project proposal
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