Microdissection of Shoot Meristem Functional Domains

Abstract

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize.

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

“…Six biological replicates were harvested, each comprising three to five whole SAMs from either mutant or wild-type plants; RNA extracted from the captured SAM cells was linearly amplified prior to its use in microarray hybridizations (Supplemental Table S1; "Materials and Methods"). Two maize gene arrays (SAM 1.1 and SAM 3.0 comprising 28,671 total elements and approximately 23,000 unique maize genes [described in Brooks et al, 2009]) were used in SAM-specific transcript profiling analyses. For each array platform, six biological replicates were hybridized with dye swapping to minimize dye-labeling bias.…”

“…For each array platform, six biological replicates were hybridized with dye swapping to minimize dye-labeling bias. Data normalization and evidence test of differential expression were performed as described (Brooks et al, 2009).…”

“…A PCRbased reverse genetic strategy exploiting the maize Mutator (Mu) transposon system (described in Brooks et al, 2009) utilized a combination of PVE1-specific and Mu transposon primers to identify two independent Mu transposon-insertion alleles of PVE1. Sequence analyses of plants homozygous for the pve1-R allele were found to contain a Mu1-like element inserted at base pair 614 of the coding region of PVE1 (Fig.…”

“…Maize seedlings harvested at 14 d after germination were fixed in formalinacetic-alcohol, paraffin embedded, sectioned at 10 mm, and stained in either toluidine blue O or Safranin Fast Green using Johanssen's method as described (Brooks et al, 2009). …”

“…Maize SAM 1.1 (GPL3333) and SAM 3.0 (GPL3538) gene chips containing a combined total of 28,671 cDNAs were used in this experiment (Brooks et al, 2009). Detailed information about these chips is available at http://www.…”