Laser Microdissection of Narrow Sheath Mutant Maize Uncovers Novel Gene Expression in the Shoot Apical Meristem

Abstract

Microarrays enable comparative analyses of gene expression on a genomic scale, however these experiments frequently identify an abundance of differentially expressed genes such that it may be difficult to identify discrete functional networks that are hidden within large microarray datasets. Microarray analyses in which mutant organisms are compared to nonmutant siblings can be especially problematic when the gene of interest is expressed in relatively few cells. Here, we describe the use of laser microdissection microarray to perform transcriptional profiling of the maize shoot apical meristem (SAM), a ~100-μm pillar of organogenic cells that is required for leaf initiation. Microarray analyses compared differential gene expression within the SAM and incipient leaf primordium of nonmutant and narrow sheath mutant plants, which harbored mutations in the duplicate genes narrow sheath1 (ns1) and narrow sheath2 (ns2). Expressed in eight to ten cells within the SAM, ns1 and ns2 encode paralogous WUSCHEL1-like homeobox (WOX) transcription factors required for recruitment of leaf initials that give rise to a large lateral domain within maize leaves. The data illustrate the utility of laser microdissection-microarray analyses to identify a relatively small number of genes that are differentially expressed within the SAM. Moreover, these analyses reveal potentially conserved WOX gene functions and implicate specific hormonal and signaling pathways during early events in maize leaf development.

Transcription Repressor HANABA TARANU Controls Flower Development by Integrating the Actions of Multiple Hormones, Floral Organ Specification Genes, and GATA3 Family Genes inArabidopsis

“…Tissue fixation and in situ hybridizations were performed as described (Zhang et al, 2007) with minor modifications. Western Blue plus 1 mM tetramisole was used instead of 5-Bromo-4-Chloro-3-Indolyl Phosphate/ Nitroblue tetrazolium as the substrate solution to reduce the hybridization background.…”

Ontogeny of the Maize Shoot Apical Meristem

“…Among these are four transcripts encoding known stem cell regulators, including three CLASS I KNOX genes and a GATA domain protein homologous to HANABA TARANU in Arabidopsis (Figures 4C and 4D; see Supplemental Data Set 8 online) Zhao et al, 2004;Mukherjee et al, 2009;reviewed in Hay and Tsiantis, 2010). Upregulated transcripts encoding proteins that function in organogenesis and cell proliferation of lateral organs include WUSHEL-RELATED HOMEOBOX3A (WOX3A), the YABBY gene DROOPING LEAF2 (DL2), the lateral organ boundary gene INDETERMINATE GA-METOPHYTE1 (IG1), and two GRF-LIKE transcripts (Kim et al, 2003;Nardmann et al, 2004;Yamaguchi et al, 2004;Evans, 2007;Nardmann et al, 2007;Zhang et al, 2007;Zhang et al, 2008). In addition to three AUXIN RESPONSE FACTOR (ARF) class transcription factors, several transcripts that encode hormone biosynthetic proteins are also upregulated in stage 14 SAMs, including seven gibberellin metabolic transcripts implicated in lateral organ growth and development.…”

PUNCTATE VASCULAR EXPRESSION1Is a Novel Maize Gene Required for Leaf Pattern Formation That Functions Downstream of the Trans-Acting Small Interfering RNA Pathway

“…plantgenomics.iastate.edu/maizechip/ or MicroArray Data Interface (http:// schnablelab.plantgenomics.iastate.edu:8080/madi/). The MIAME (for minimum information about a microarray experiment) guidelines utilized, hybridization protocols, and array scanning procedure were as described (Zhang et al, 2007;Brooks et al, 2009). …”

“…Median centering was used to normalize data across slides from each channel (Yang et al, 2002). A mixed linear model was applied to the normalized data to identify the differentially expressed genes among rgd2-R mutant and wild-type samples as described (Dudoit et al, 2002;Zhang et al, 2007). The resulting P values from the tests for SAM-type effects were converted to q values using the method of Storey and Tibshirani (2003) to estimate the false discovery rate associated with any P value threshold for significance.…”

“…SAM tissues and leaf primordia (P1-P4) tissue were harvested by LM from rgd2 mutant and wild-type siblings; cDNA was synthesized from amplified RNA as described (51). Gene-specific primers were designed (Supplemental Table S3) for use with SYBR-Green (Quanta) in qRT-PCR as described (Zhang et al, 2007). Three biological replicates were examined, and samples were normalized to UBIQUITIN or to 18S rRNA transcript accumulation as described using Bio-Rad iQ5 version 1.0 software (Livak and Schmittgen, 2001).…”