Marja C. P. Timmermans

microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity

In both animals and plants, many developmentally important regulatory genes have complementary microRNAs (miRNAs), which suggests that these miRNAs constitute a class of developmental signalling molecules. Leaves of higher plants exhibit a varying degree of asymmetry along the adaxial/abaxial (upper/lower) axis. This asymmetry is specified through the polarized expression of class III homeodomain/leucine zipper (HD-ZIPIII) genes. In Arabidopsis, three such genes, PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV), are expressed throughout the incipient leaf, but become adaxially localized after primordium emergence. Downregulation of the HD-ZIPIII genes allows expression of the KANADI and YABBY genes, which specify abaxial fate. PHB, PHV and REV transcripts contain a complementary site for miRNA165 and miRNA166, which can direct their cleavage in vitro. Here we show that miRNA166 constitutes a highly conserved polarizing signal whose expression pattern spatially defines the expression domain of the maize hd-zipIII family member rolled leaf1 (rld1). Moreover, the progressively expanding expression pattern of miRNA166 during leaf development and its accumulation in phloem suggests that miRNA166 may form a movable signal that emanates from a signalling centre below the incipient leaf.

Pattern formation via small RNA mobility

MicroRNAs and trans-acting siRNAs (ta-siRNAs) have important regulatory roles in development. Unlike other developmentally important regulatory molecules, small RNAs are not known to act as mobile signals during development. Here, we show that low-abundant, conserved ta-siRNAs, termed tasiR-ARFs, move intercellularly from their defined source of biogenesis on the upper (adaxial) side of leaves to the lower (abaxial) side to create a gradient of small RNAs that patterns the abaxial determinant AUXIN RESPONSE FACTOR3. Our observations have important ramifications for the function of small RNAs and suggest they can serve as mobile, instructive signals during development.

Direct Repression of KNOX Loci by the ASYMMETRIC LEAVES1 Complex of Arabidopsis

KNOTTED1-like homeobox (KNOX) genes promote stem cell activity and must be repressed to form determinate lateral organs. Stable KNOX gene silencing during organogenesis is known to involve the predicted DNA binding proteins ASYMMETRIC LEAVES1 (AS1) and AS2 as well as the chromatin-remodeling factor HIRA. However, the mechanism of silencing is unknown. Here, we show that AS1 and AS2 form a repressor complex that binds directly to the regulatory motifs CWGTTD and KMKTTGAHW present at two sites in the promoters of the KNOX genes BREVIPEDICELLUS (BP) and KNAT2. The two binding sites act nonredundantly, and interaction between AS1-AS2 complexes at these sites is required to repress BP. Promoter deletion analysis further indicates that enhancer elements required for BP expression in the leaf are located between the AS1-AS2 complex binding sites. We propose that AS1-AS2 complexes interact to create a loop in the KNOX promoter and, likely through recruitment of HIRA, form a repressive chromatin state that blocks enhancer activity during organogenesis. Our model for AS1-AS2–mediated KNOX gene silencing is conceptually similar to the action of an insulator. This regulatory mechanism may be conserved in simple leafed species of monocot and dicot lineages and constitutes a potential key determinant in the evolution of compound leaves.

Small RNAs are on the move

A key feature of RNA interference is its ability to spread from cell to cell. Such non-cell-autonomous gene silencing has been characterized extensively in both plants and animals, but the identity of the mobile silencing signal has remained elusive. Several recent studies now shed light on the identity of this signal in plants, and indicate that small RNA molecules—from short-interfering RNAs to microRNAs—are capable of moving between cells and through the vasculature. The movement of small, 21–24-nucleotide RNA species has implications for biological processes ranging from developmental patterning and stress responses to epigenetic inheritance.

Maize rough sheath2 and Its Arabidopsis Orthologue ASYMMETRIC LEAVES1 Interact with HIRA, a Predicted Histone Chaperone, to Maintain knox Gene Silencing and Determinacy during Organogenesis

Plant shoots are characterized by indeterminate growth resulting from the action of a population of stem cells in the shoot apical meristem (SAM). Indeterminacy within the SAM is specified in part by the class I knox homeobox genes. The myb domain proteins rough sheath2 (RS2) and ASYMMETRIC LEAVES1 (AS1) from maize (Zea mays) and Arabidopsis thaliana, respectively, are required to establish determinacy during leaf development. These proteins are part of a cellular memory system that in response to a stem cell–derived signal keeps knox genes in an off state during organogenesis. Here, we show that RS2/AS1 can form conserved protein complexes through interaction with the DNA binding factor ASYMMETRIC LEAVES2, a predicted RNA binding protein (RIK, for RS2-Interacting KH protein), and a homologue of the chromatin-remodeling protein HIRA. Partial loss of HIRA function in Arabidopsis results in developmental defects comparable to those of as1 and causes reactivation of knox genes in developing leaves, demonstrating a direct role for HIRA in knox gene repression and the establishment of determinacy during leaf formation. Our data suggest that RS2/AS1 and HIRA mediate the epigenetic silencing of knox genes, possibly by modulating chromatin structure. Components of this process are conserved in animals, suggesting the possibility that a similar epigenetic mechanism maintains determinacy during both plant and animal development.

Specification of adaxial cell fate during maize leaf development

Dorsoventral (adaxial/abaxial) polarity of the maize leaf is established in the meristem and is maintained throughout organ development to coordinate proper outgrowth and patterning of the leaf. rolled leaf1 (rld1) and leafbladeless1 (lbl1) are required for the specification of the adaxial/upper leaf surface. rld1 encodes a class III homeodomain-leucine zipper (HD-ZIPIII) protein whose adaxial expression is spatially defined by miRNA166-directed transcript cleavage on the abaxial side. The semi-dominant Rld1-Original (Rld1-O) mutation, which results from a single nucleotide substitution in the miRNA166 complementary site, leads to persistent expression of mutant transcripts on the abaxial site. This causes the adaxialization or partial reversal of leaf polarity. By contrast, recessive mutations in lbl1 cause the formation of abaxialized leaves. The lbl1 and Rld1-O mutations mutually suppress each other, indicating that these two genes act in the same genetic pathway. Adaxial and meristematic expression of rld1 is reduced in lbl1 mutants, indicating that lbl1 acts upstream of rld1 to specify adaxial fate during primordium development. However, rld1 expression in the vasculature of lbl1 is normal, suggesting that the specification of adaxial/abaxial polarity during vascular and primordia development is governed by separate but overlapping pathways. We also show that members of the maize yabby gene family are expressed on the adaxial side of incipient and developing leaf primordia. This expression pattern is unlike that observed in Arabidopsis, where YABBY expression is correlated with abaxial cell fate. The yabby expression patterns in lbl1 and Rld1-O mutants suggest that the yabby genes act downstream in the same pathway as lbl1 and rld1. Moreover, our observations suggest that maize yabby genes may direct lateral organ outgrowth rather than determine cell fate. We propose that a single genetic pathway involving lbl1, rld1 and the yabby genes integrates positional information within the SAM, and leads to adaxial/abaxial patterning and mediolateral outgrowth of the leaf.

Signals and prepatterns: new insights into organ polarity in plants

The flattening of leaves results from the interaction between upper (adaxial) and lower (abaxial) domains in the developing primordium. These domains are specified by conserved, overlapping genetic pathways involving several distinct transcription factor families and small regulatory RNAs. Polarity determinants employ a series of antagonistic interactions to produce mutually exclusive cell fates whose positioning is likely refined by signaling across the adaxial-abaxial boundary. Signaling candidates include a mobile small RNA-the first positional signal described in adaxial-abaxial polarity. Possible mechanisms to polarize the incipient primordium are discussed, including meristem-derived signaling and a model in which a polarized organogenic zone prepatterns the adaxial-abaxial axis.

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.

The ASYMMETRIC LEAVES complex maintains repression of KNOX homeobox genes via direct recruitment of Polycomb-repressive complex2

Polycomb-repressive complexes (PRCs) ensure the correct spatiotemporal expression of numerous key developmental regulators. Despite their pivotal role, how PRCs are recruited to specific targets remains largely unsolved, particularly in plants. Here we show that the Arabidopsis ASYMMETRIC LEAVES complex physically interacts with PRC2 and recruits this complex to the homeobox genes BREVIPEDICELLUS and KNAT2 to stably silence these stem cell regulators in differentiating leaves. The recruitment mechanism resembles the Polycomb response element-based recruitment of PRC2 originally defined in flies and provides the first such example in plants. Combined with recent studies in mammals, our findings reveal a conserved paradigm to epigenetically regulate homeobox gene expression during development.