Kazuhiro Ohtsu

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

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.

Regulation of small RNA accumulation in the maize shoot apex.

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial–abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.

Loss of RNA–Dependent RNA Polymerase 2 (RDR2) Function Causes Widespread and Unexpected Changes in the Expression of Transposons, Genes, and 24-nt Small RNAs

Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA–dependent RNA polymerase 2 (RDR2) is a component of the RNA–directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA–seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2–sensitive and RDR2–resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant.

Microdissection of Shoot Meristem Functional Domains

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.

Characterization and expression of the genes for cytochrome c oxidase subunit VIb (COX6b) from rice and Arabidopsis thaliana

Many of the subunits of cytochrome c oxidase (COX) in the mitochondria of higher plants are encoded by nuclear genes. These genes are less characterized compared to mitochondrial-encoded genes. We previously isolated a cDNA encoding COX6b (designated OsCOX6b1 in this study) from the rice nuclear genome and analyzed its expression. The deduced protein had an extended N-terminus compared with human and yeast COX6b proteins. In this study, we identified another COX6b gene (OsCOX6b2) in rice and revealed that it was actually expressed. The deduced protein of this gene did not have an extended N-terminus and had about the same size as the human and yeast proteins. Genomic Southern hybridization analysis revealed that there was at least one OsCOX6b-homologus sequences in the rice genome other than OsCOX6b1 and OsCOX6b2. Furthermore, we identified three COX6b genes in a dicotyledonous plant, Arabidopsis thaliana. One of these genes (AtCOX6b1) was relatively long, with a length similar to that of OsCOX6b1, and the other two (AtCOX6b2 and AtCOX6b3) were shorter, with lengths similar to the length of OsCOX6b2. Genomic Southern hybridization analysis indicated there were no additional COX6b genes in the Arabidopsis genome. The coding regions of OsCOX6b1 and AtCOX6b1 were separated by four introns and those of OsCOX6b2, AtCOX6b2 and AtCOX6b3 were separated by three introns. A Northern hybridization analysis showed that OsCOX6b1, AtCOX6b1 and AtCOX6b3 were expressed in all organs examined, although with some differences in the amount of expression among the organs. OsCOX6b2 and AtCOX6b2 were strongly expressed in roots but most of the transcripts of AtCOX6b2 were degraded. The evolution of COX6b genes from rice and Arabidopsis is discussed.

Laser Microdissection–Mediated Isolation and In Vitro Transcriptional Amplification of Plant RNA

Laser microdissection of cells allows for isolation of specific cells of interest for downstream analyses including transcriptional profiling. Plant cells present unique challenges for laser microdissection due to their cellulosic cell walls and large vacuoles. Here we present protocols for plant tissue preparation, laser microdissection of select plant cells, and linear amplification of RNA from dissected cells. Linear amplification of RNA from dissected cells allows sufficient RNA for subsequent quantitative analysis by RT‐PCR, microarray, or RNA sequencing.

DNA extraction from freeze-dried plant tissue with CTAB in a 96-well format.

This protocol is a modified version of DNA isolation using cetyltrimethylammonium bromide (CTAB) and 96-well plates. It is high-throughput, which facilitates the analysis of large mapping populations. The DNA yield is adequate for at least 100-500 polymerase chain reaction (PCR) procedures.

T7-based RNA amplification for genotyping from maize shoot apical meristem.

INTRODUCTIONThe use of RNA for genotyping analysis can be advantageous because transcriptomes are significantly smaller than genomes and typically contain far fewer repetitive sequences. Laser capture microdissection (LCM) has been used successfully to isolate sequences (especially rare transcripts) that accumulate in specific tissues. The amount of RNA collected in a standard microdissection is often insufficient for global gene expression analysis but can be increased via linear amplification. Upon conversion to cDNA, the product serves as template for 454 sequencing to produce expressed sequence tags for subsequent SNP analysis and detection. This protocol describes how to amplify RNA extracted from laser-dissected and captured tissues and cells.