Laser-Capture Microdissection, a Tool for the Global Analysis of Gene Expression in Specific Plant Cell Types: Identification of Genes Expressed Differentially in Epidermal Cells or Vascular Tissues of Maize[W]

Abstract

Laser-capture microdissection (LCM) allows for the one-step procurement of large homogeneous populations of cells from tissue sections. In mammals, LCM has been used to conduct cDNA microarray and proteomics studies on specific cell types. However, LCM has not been applied to plant cells, most likely because plant cell walls make it difficult to separate target cells from surrounding cells and because ice crystals can form in the air spaces between cells when preparing frozen sections. By fixing tissues, using a cryoprotectant before freezing, and using an adhesive-coated slide system, it was possible to capture large numbers ( Ͼ 10,000) of epidermal cells and vascular tissues (vascular bundles and bundle sheath cells) from ethanol:acetic acid-fixed coleoptiles of maize. RNA extracted from these cells was amplified with T7 RNA polymerase and used to hybridize a microarray containing ‫ف‬ 8800 maize cDNAs. Approximately 250 of these were expressed preferentially in epidermal cells or vascular tissues. These results demonstrate that the combination of LCM and microarrays makes it feasible to conduct high-resolution global gene expression analyses of plants. This approach has the potential to enhance our understanding of diverse plant cell type-specific biological processes.

Isolation, Characterization, and Pericycle-Specific Transcriptome Analyses of the Novel Maize Lateral and Seminal Root Initiation Mutant rum1

“…None of the previously published cell-type-specific transcriptome analyses in plants (Asano et al, 2002;Birnbaum et al, 2003;Nakazono et al, 2003) dissected the root pericycle. Our analyses identified 90 genes that were preferentially expressed in the wild-type pericycle and 73 genes that were preferentially expressed in the rum1 pericycle.…”

“…The apical 0.3 cm of the root apex, including the meristematic zone and the distal elongation zone, were discarded. The remaining differentiation and elongation zones of the roots were collected in 0.5-cm samples, fixed, and embedded in TissueTek OCT medium according to the protocol described by Nakazono et al (2003). The corresponding parts of three roots were embedded in one cryomold representing one biological replicate.…”

“…Microarray probe synthesis and hybridization of four spotted 12k maize cDNA microarray slides (Generation II, Version A) from the Iowa State MicroArray Facility (http://www.plantgenomics.iastate.edu/maizechip/) were conducted as described by Nakazono et al (2003). Three independent biological replications from each genotype were profiled on the four arrays.…”

“…Extracted RNA was amplified according to the protocol of Nakazono et al (2003), first synthesizing double-stranded cDNA of the mRNA followed by RNA synthesis via in vitro transcription with T7-polymerase. The amplification procedure was repeated twice to increase the amplified RNA yield.…”

“…Such microarray experiments provide average gene expression profiles of all cells in an organ and may potentially mask differential gene expression in a particular cell type. Laser capture microdissection (LCM) technology allows for the analysis of cell-type-specific gene expression profiles (Asano et al, 2002;Kerk et al, 2003;Nakazono et al, 2003). In this approach, frozen or fixed cells of interest are physically linked to a thermoplastic film with a low-power laser beam.…”