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Transcriptome variation plays an important role in affecting the phenotype of an organism. However, an understanding of the underlying mechanisms regulating transcriptome variation in segregating populations is still largely unknown. We sought to assess and map variation in transcript abundance in maize shoot apices in the intermated B73×Mo17 recombinant inbred line population. RNA–based sequencing (RNA–seq) allowed for the detection and quantification of the transcript abundance derived from 28,603 genes. For a majority of these genes, the population mean, coefficient of variation, and segregation patterns could be predicted by the parental expression levels. Expression quantitative trait loci (eQTL) mapping identified 30,774 eQTL including 96 trans-eQTL “hotspots,” each of which regulates the expression of a large number of genes. Interestingly, genes regulated by a trans-eQTL hotspot tend to be enriched for a specific function or act in the same genetic pathway. Also, genomic structural variation appeared to contribute to cis-regulation of gene expression. Besides genes showing Mendelian inheritance in the RIL population, we also found genes whose expression level and variation in the progeny could not be predicted based on parental difference, indicating that non-Mendelian factors also contribute to expression variation. Specifically, we found 145 genes that show patterns of expression reminiscent of paramutation such that all the progeny had expression levels similar to one of the two parents. Furthermore, we identified another 210 genes that exhibited unexpected patterns of transcript presence/absence. Many of these genes are likely to be gene fragments resulting from transposition, and the presence/absence of their transcripts could influence expression levels of their ancestral syntenic genes. Overall, our results contribute to the identification of novel expression patterns and broaden the understanding of transcriptional variation in plants., Author Summary Phenotypes are determined by the expression of genes, the environment, and the interaction of gene expression and the environment. However, a complete understanding of the inheritance of and genome-wide regulation of gene expression is lacking. One approach, called expression quantitative trait locus (eQTL) mapping provides the opportunity to examine the genome-wide inheritance and regulation of gene expression. In this paper, we conducted high-throughput sequencing of gene transcripts to examine gene expression in the shoot apex of a maize biparental mapping population. We quantified expression levels from 28,603 genes in the population and showed that the vast majority of genes exhibited the expected pattern of Mendelian inheritance. We genetically mapped the expression patterns and identified genomic regions associated with gene expression. Notably, we detected gene expression patterns that exhibited non-Mendelian inheritance. These included 145 genes that exhibited expression patterns in the progeny that were similar to only one of the parents and 210 genes with unexpected presence/absence expression patterns. The findings of non-Mendelian inheritance underscore the complexity of gene expression and provide a framework for understanding these complexities.

Transcriptome variation plays an important role in affecting the phenotype of an organism. However, an understanding of the underlying mechanisms regulating transcriptome variation in segregating populations is still largely unknown. We sought to assess and map variation in transcript abundance in maize shoot apices in the intermated B73 × Mo17 recombinant inbred line population. RNA-based sequencing (RNA-seq) allowed for the detection and quantification of the transcript abundance derived from 28,603 genes. For a majority of these genes, the population mean, coefficient of variation, and segregation patterns could be predicted by the parental expression levels. Expression quantitative trait loci (eQTL) mapping identified 30,774 eQTL including 96 trans-eQTL "hotspots," each of which regulates the expression of a large number of genes. Interestingly, genes regulated by a trans-eQTL hotspot tend to be enriched for a specific function or act in the same genetic pathway. Also, genomic structural variation appeared to contribute to cis-regulation of gene expression. Besides genes showing Mendelian inheritance in the RIL population, we also found genes whose expression level and variation in the progeny could not be predicted based on parental difference, indicating that non-Mendelian factors also contribute to expression variation. Specifically, we found 145 genes that show patterns of expression reminiscent of paramutation such that all the progeny had expression levels similar to one of the two parents. Furthermore, we identified another 210 genes that exhibited unexpected patterns of transcript presence/absence. Many of these genes are likely to be gene fragments resulting from transposition, and the presence/absence of their transcripts could influence expression levels of their ancestral syntenic genes. Overall, our results contribute to the identification of novel expression patterns and broaden the understanding of transcriptional variation in plants.

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., Author Summary All the organs of plant shoots are derived from the shoot apical meristem (SAM), a pool of plant stem cells that are both organogenic and self-sustaining. These dual SAM functions take place in distinct yet adjacent meristematic domains; leaves are derived from the peripheral zone (PZ) of the SAM whereas cells lost during organogenesis are replenished from the central zone (CZ). Deciphering the global patterns of differential gene expression within these discrete SAM functional domains is integral toward understanding the molecular-signaling networks that regulate plant development. We utilized laser-microdissection technology to isolate tissues from the SAM crown and center (SAM-proper) and from initiating leaves (P0/P1) at the SAM periphery for use in microarray comparisons of gene expression within these SAM functional domains. Nine hundred and sixty-two maize genes were differentially expressed, confirming that the distinct functions of these meristematic domains involve widespread differences in gene expression. Genes involved in cell division, cell wall biosynthesis, chromatin structure, and RNA binding are especially upregulated in initiating leaves, whereas genes regulating protein stability and DNA repair are upregulated in the SAM proper. Mutations in a D-cyclin gene that was upregulated in the P0/P1 render narrow-leafed mutant plants with defective stomatal patterning, providing functional genetic data for a previously uncharacterized maize gene.

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., Author Summary Unlike animals, plants exhibit a prolonged period of organogenesis, generating new leaves throughout their life cycle. This ability to maintain an embryo-like state is dependent upon the activity of shoot meristems, whose dual functions are to supply an inner core of pluripotent cells that sustain the shoot meristem while simultaneously generating new leaves derived from cells at the meristem periphery. Deciphering the complex combinations of molecular signals that transform meristematic cells into leaf primordia is a central question in plant developmental biology. In this study, we used the power of focused laser light to microdissect shoot meristems from neighboring leaf and stem tissue in the maize plant. Once isolated, we compared patterns of gene expression in normal shoot meristems to those of genetically mutant shoot meristems that form abnormal, narrow leaves. Out of more than 21,000 maize genes analyzed, 66 genes were identified as misexpressed in the mutant shoot meristems. All but one of the differentially expressed genes are previously unstudied in maize, and the majority are predicted to function during cell division, growth, or developmental signaling. Many of these novel genes are expressed in specific domains of the shoot meristem, consistent with their predicted function during maize leaf initiation.

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., Author Summary Shoot apical meristems (SAMs) are ultimately responsible for generating all above-ground plant tissues. Recent studies highlighted the effects of chromatin remodeling on the expression of various genes important to SAM development. The transposons that comprise a substantial portion of many eukaryotic genomes are typically transcriptionally silenced, presumably to promote genome stability. We demonstrate that a loss of a key component of the RNA–dependent DNA Methylation (RdDM) silencing pathway affects the expression of not only transposons but also thousands of genes, including nearly 80% of the chromatin-associated genes. Surprisingly, the expression of many transposons and genes is down-regulated via the loss of this component of the silencing pathway. In this study, we have shown that a maize mutation of RDR2 causes significant changes in SAM morphology. In combination, these observations indicate the complexity of transcriptome regulation and the crucial roles of RDR2 on transcriptome regulation, chromatin modification, and SAM development.

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., Author Summary Small RNAs regulate many key developmental processes. Consistent with a prominent role in development, miRNAs exhibit complex and distinctive expression patterns. In this study, we identify regulatory mechanisms that allow for the precise spatial accumulation of developmentally important small RNAs in plants. Plants generate new leaves throughout their lifetime. These arise on the flank of a specialized stem cell niche, termed meristem, at the plant's growing tip. Each newly formed leaf becomes polarized and develops distinct adaxial (top) and abaxial (bottom) sides. The establishment of adaxial–abaxial polarity requires a complex genetic network, including miRNAs and trans-acting siRNAs. We used a focused laser to microdissect regions of the shoot apical meristem and developing leaves of maize to analyze the expression profiles of the small RNA precursor molecules. By comparing these expression profiles to the accumulation patterns of the mature small RNAs, we show that precursor genes are subject to tissue-specific regulation and exhibit diverse expression patterns during leaf development. Our findings suggest that mechanisms exist to regulate the biogenesis, stability, and possibly even the intercellular movement of small RNAs. Such regulation should be considered when designing artificial miRNAs and has implications for the roles miRNAs play during plant and animal development.