Small RNAs are important regulators of gene expression. In maize, adaxial/abaxial (dorsoventral) leaf polarity is established by an abaxial gradient of microRNA166 (miR166), which spatially restricts the expression domain of class III homeodomain leucine zipper (HD-ZIPIII) transcription factors that specify adaxial/upper fate. Here, we show that leafbladeless1 encodes a key component in the trans-acting small interfering RNA (ta-siRNA) biogenesis pathway that acts on the adaxial side of developing leaves and demarcates the domains of hd-zipIII and miR166 accumulation. Our findings indicate that tasiR-ARF, a ta-siRNA, and miR166 establish opposing domains along the adaxial-abaxial axis, thus revealing a novel mechanism of pattern formation.Supplemental material is available at www.genesdev.org.Received January 8, 2007; revised version accepted February 20, 2007. In both animals and plants, many developmentally important regulatory genes are predicted targets of microRNAs (miRNAs), which suggests that such small RNAs constitute a class of developmental determinants (Alvarez-Garcia and Miska 2005;Jones-Rhoades et al. 2006). Patterning and outgrowth of lateral organs in plants depend on the specification of adaxial/abaxial (dorsoventral) polarity in the incipient primordium. This asymmetry is established through the polarized expression of class III homeodomain leucine zipper (HD-ZIPIII) transcription factors that specify adaxial/upper cell fate (McConnell et al. 2001;Emery et al. 2003;Juarez et al. 2004a). The adaxial-specific expression of hd-zipIII family members is delineated by the expression pattern of a 21-nucleotide (nt) miRNA, miR166, which directs the cleavage of hd-zipIII transcripts (Juarez et al. 2004a;Kidner and Martienssen 2004). In maize, miR166 accumulates most abundantly immediately below the incipient leaf, but a gradient of miR166 extends into the abaxial side of the initiating organ that establishes organ polarity (Juarez et al. 2004a).Specification of adaxial/abaxial organ polarity in maize also requires the activity of leafbladeless1 (lbl1). Recessive mutations in lbl1 lead to a variable abaxialization of leaves (Timmermans et al. 1998). The weak lbl1-ref allele causes a partial loss of adaxial identity revealed as patches of abaxial cells on the upper leaf surface, whereas leaves of the severe ragged seedling1 allele (lbl1-rgd1) are often radially symmetric and completely abaxialized (Fig. 1A). Expression of the hd-zipIII family member rld1 is reduced in lbl1 mutants. Conversely, increased levels of hd-zipIII expression in Rld1-O mutants, which carry a miR166-insensitive allele of rld1, can fully suppress the vegetative defects of lbl1 (Juarez et al. 2004b). lbl1 thus contributes to organ polarity by regulating the accumulation of rld1 transcripts on the adaxial side of the developing leaf.Here, we show that lbl1 encodes a homolog of SUPPRESSOR-OF-GENE-SILENCING3 (SGS3), which is specifically required for the biogenesis of trans-acting small interfering RNAs (ta-siRNAs) (Peragine et al. 2...
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.
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.
Leaves of higher plants exhibit a varying degree of asymmetry along their dorsoventral axis. This asymmetry is specified via the polarized expression of class III homeodomain‐leucine zipper (hd‐zipIII) genes, which specify dorsal cell fate. Through detailed in situ hybridization analyses we were able to show that this polarized hd‐zipIII expression is set up by the graded expression pattern of a 21‐nucleotide microRNA, miR166. More recently, we have shown that this miR166 gradient is generated through the opposing activity of a different type of small regulatory RNA, the transacting siRNA tasiR‐ARF. A further twist to the story is added by the observation that tasiR‐ARF in turn is regulated by a third small RNA, miR390. Our observations identify a novel mechanism of pattern formation in which cell fates along a developmental axis are specified through the opposing activity of distinct small RNAs. tasiR‐ARF defines the dorsal side of the leaf by restricting the expression domain of miR166, which in turn delineates the ventral side by restricting expression of the hd‐zipIII transcription factors. Importantly, comparison of the expression patterns of small RNA precursor transcripts to those of the mature small RNA provides evidence that small regulatory RNAs can be mobile, and this offers the intriguing possibility that small RNAs may function as morphogen‐type signals in development.
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