Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.)

Abstract

All above-ground plant organs are derived from shoot apical meristems (SAMs). Global analyses of gene expression were conducted on maize (Zea mays L.) SAMs to identify genes preferentially expressed in the SAM. The SAMs were collected from 14-day-old B73 seedlings via laser capture microdissection (LCM). The RNA samples extracted from LCM-collected SAMs and from seedlings were hybridized to microarrays spotted with 37 660 maize cDNAs. Approximately 30% (10 816) of these cDNAs were prepared as part of this study from manually dissected B73 maize apices. Over 5000 expressed sequence tags (ESTs) (about 13% of the total) were differentially expressed (P < 0.0001) between SAMs and seedlings. Of these, 2783 and 2248 ESTs were up- and down-regulated in the SAM, respectively. The expression in the SAM of several of the differentially expressed ESTs was validated via quantitative RT-PCR and/or in situ hybridization. The up-regulated ESTs included many regulatory genes including transcription factors, chromatin remodeling factors and components of the gene-silencing machinery, as well as about 900 genes with unknown functions. Surprisingly, transcripts that hybridized to 62 retrotransposon-related cDNAs were also substantially up-regulated in the SAM. Complementary DNAs derived from the LCM-collected SAMs were sequenced to identify additional genes that are expressed in the SAM. This generated around 550 000 ESTs (454-SAM ESTs) from two genotypes. Consistent with the microarray results, approximately 14% of the 454-SAM ESTs from B73 were retrotransposon-related. Possible roles of genes that are preferentially expressed in the SAM are discussed.

Figure 1

Laser capture microdissection (LCM) of shoot apical meristem (SAMs) from 14-day-old maize seedlings. A SAM before (a) and after (b) LCM. Leaf primordia are numbered according to their relative developmental ages, wherein P0 (0) corresponds to the incipient leaf forming at the flank of the SAM. Bar: 100 μm.

Figure 2

A fold change distribution of the significant expressed sequence tags (ESTs). Percentages of the up- (black) and down-regulated (gray) ESTs relative to the total number of significant ESTs (2783 up- and 2248 down-regulated; Table 1) in each fold change (FC) category are indicated. Numbers of ESTs in each category are also presented at the top of each bar.

Figure 3

Quantitative RT-PCR (qRT-PCR) analyses on seven significantly up-regulated expressed sequence tags (ESTs) derived from the microarray analysis. Fold changes [shoot apical meristem (SAM)/seedling] from qRT-PCR analyses (black bars) and the microarray analysis (gray bars) are indicated on a logarithmic scale with seven significantly up-regulated ESTs derived from the microarray analysis: RDR2, AGO4a (accession no. DV493575), AGO4b (accession no. DV943274), AGO4c (accession no. DV492375), DDM1 (Table 3), MADS, and B3. AGO4a, AGO4b, and AGO4c have similar but distinct sequences (data not shown), indicating that these ESTs were derived from paralogous loci (Table 3). Means of the two biological replications (RDR2) and means + SD of the three biological replications (AGO4a, AGO4b, AGO4c, DDM, MADS, and B3) are shown. For Cinful and Tekay, one of the seedling samples (replication 4, Appendix S1) did not yield fluorescence above the threshold level whereas the corresponding SAM sample in the same replication did yield fluorescence above threshold level. Fold changes of the single replications for Cinful and Tekay were 7904 and 446, respectively.

Figure 4

Transcript accumulations of maize homologs of RDR2 and VAP in the shoot apical meristem (SAM), inflorescence meristems, and leaf primordia. Fourteen-day-old maize shoot apices were analyzed by in situ hybridization using antisense probes prepared from maize homologs of RDR2 (a–d) and VAP (g, h) cDNA clones. A 21-day-old ear inflorescence apex (e) and a 28-day-old bolting tassel inflorescence (f) were also analyzed for the RDR2 homolog. (a), (b) and (e)–(h) are images of longitudinal sections; (c) and (d) are transverse sections. (b), (d), and (h) are enlarged images of (a), (c), and (g), respectively. Leaf primordia are numbered as described in Figure 1. Red arrowheads demonstrate RDR2 homolog transcript accumulation in leaf primordial margins; red asterisks demonstrate RDR2 homolog mRNA accumulation in the abaxial domains of P4 (4) and P5 (5) leaf primordia. Bar: 100 μm.