Plants with altered microRNA metabolism have pleiotropic developmental defects, but direct evidence for microRNAs regulating specific aspects of plant morphogenesis has been lacking. In a genetic screen, we identified the JAW locus, which produces a microRNA that can guide messenger RNA cleavage of several TCP genes controlling leaf development. MicroRNA-guided cleavage of TCP4 mRNA is necessary to prevent aberrant activity of the TCP4 gene expressed from its native promoter. In addition, overexpression of wild-type and microRNA-resistant TCP variants demonstrates that mRNA cleavage is largely sufficient to restrict TCP function to its normal domain of activity. TCP genes with microRNA target sequences are found in a wide range of species, indicating that microRNA-mediated control of leaf morphogenesis is conserved in plants with very different leaf forms.Although much is known about how organs acquire their particular fate, we are only starting to learn how organs are sculpted, even if they are just flat sheets such as wings or leaves. An elegant study recently demonstrated that making a flat organ is not a trivial problem: snapdragon leaves are normally flat, but they become crinkly in plants lacking the CINCINNATA (CIN) gene 1 . In cin mutants, differential regulation of cell division across the leaf is disturbed, causing negative leaf curvature. CIN RNA itself is expressed in a dynamic pattern, in front of and perhaps overlapping the mitotic arrest zone, suggesting a direct role of CIN in regulating leaf morphogenesis.Although it is unknown how expression of CIN, which encodes a TCP transcription factor 2 , is regulated, a specific RNA pattern can result from differential transcription or changes in transcript stability. A post-transcriptional mechanism that has only recently been recognized is that of plant mRNA cleavage initiated by partially or fully complementary microRNAs (miRNAs) 3,4 . The mechanism of cleavage is similar, or identical, to cleavage guided by short interfering RNAs (siRNAs) 5 .The double-stranded ribonucleases Dicer in animals and DICER-LIKE1 (DCL1) in plants process miRNAs-which are usually 21-22 nucleotides long-from longer precursor RNAs with fold-back structure 4,6,7 . Additional factors required for accumulation of miRNAs include members of the Argonaute family and HEN1 protein 8,9 . The importance of miRNAs for plant development is supported by the abnormalities seen in several mutants or transgenic plants with general defects in miRNA accumulation or activity [9][10][11][12] . However, although biochemical studies have demon- Fig. 2b). b, Seedlings, individual leaves and leaf rosettes of Columbia wild-type and jaw-1D plants. Leaves were mounted between glass plates and illuminated from below. Dark green areas indicate overlapping leaf parts after flattening. c, Expression changes of TCP genes in jaw-1D estimated from Affymetrix arrays (grey bars) or from RT-qPCR (black bars). See Supplementary Information for absolute values. NP, termed 'not present' by MAS software. Note th...

Most plant microRNAs (miRNAs) have perfect or near-perfect complementarity with their targets. This is consistent with their primary mode of action being cleavage of target mRNAs, similar to that induced by perfectly complementary small interfering RNAs (siRNAs). However, there are natural targets with up to five mismatches. Furthermore, artificial siRNAs can have substantial effects on so-called off-targets, to which they have only limited complementarity. By analyzing the transcriptome of plants overexpressing different miRNAs, we have deduced a set of empirical parameters for target recognition. Compared to artificial siRNAs, authentic plant miRNAs appear to have much higher specificity, which may reflect their coevolution with the remainder of the transcriptome. We also demonstrate that miR172, previously thought to act primarily by translational repression, can efficiently guide mRNA cleavage, although the effects on steady-state levels of target transcripts are obscured by strong feedback regulation. This finding unifies the view of plant miRNA action.

Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).

In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentified, with the PINOID kinase being the only known molecular component. Here, we identify PP2A phosphatase as an important regulator of PIN apical-basal targeting and auxin distribution. Genetic analysis, localization, and phosphorylation studies demonstrate that PP2A and PINOID both partially colocalize with PINs and act antagonistically on the phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. Thus, in plants, polar sorting by the reversible phosphorylation of cargos allows for their conditional delivery to specific intracellular destinations. In the case of PIN proteins, this mechanism enables switches in the direction of intercellular auxin fluxes, which mediate differential growth, tissue patterning, and organogenesis.

SummaryComprehensive analysis of gene function requires the detailed examination of mutant alleles. In Arabidopsis thaliana, large collections of sequence-indexed insertion and chemical mutants provide potential loss-offunction alleles for most annotated genes. However, limitations for phenotypic analysis include gametophytic or early sporophytic lethality, and the ability to recombine mutant alleles in closely linked genes, especially those present as tandem duplications. Transgene-mediated gene silencing can overcome some of these shortcomings through tissue-specific, inducible and partial gene inactivation, or simultaneous targeting of several, sequence-related genes. In addition, gene silencing is a convenient approach in species or varieties for which exhaustive mutant collections are not yet available. Typically, gene function is reduced posttranscriptionally, effected by small RNAs that act in a sequence-specific manner by base pairing to complementary mRNA molecules. A recently introduced approach is the use of artificial microRNAs (amiRNAs). Here, we review various strategies for small RNA-based gene silencing, and describe in detail the design and application of amiRNAs in many plant species.