Role ofMicroRNAmiR319 in Plant Development

Originally identified in a genetic screen, microRNA miR319 regulates transcription factors of the TCP family. The balance between miR319 and its targets controls leaf morphogenesis and several other plant developmental processes. High levels of miR319 or low TCP activity causes an excess of cell proliferation that generates a crinkled simple leaf in Arabidopsis and snapdragon or supercompound organ in tomato. In contrast, reduced miR319 levels or high TCP activity reduces leaf and petal size, results in a simple tomato leaf, and is lethal in extreme cases. Insights into the gene networks that are controlled by the miR319regulated TCPs demonstrate their participation in multiple biological pathways, from hormone biosynthesis and signaling to cell proliferation and differentiation. 1 Discovery of miR319 Through a Genetic Screen MicroRNAs (miRNAs) are a conspicuous group of small RNAs present in animals and plants, which are defined by their unique biogenesis as they are processed from an imperfect fold-back precursor (Meyers et al. 2008). In both plants and animals, miRNAs have been discovered by three methods: direct cloning and sequencing, genetic screenings, and bioinformatic predictions. The first plant miRNAs were identified by isolating, cloning, and sequencing small RNA populations (Reinhart et al. 2002; Llave et al. 2002; Park et al. 2002). This approach bursts recently with the development of deep-sequencing strategies [e.g., (Fahlgren et al. 2007; Rajagopalan et al. 2006; Lu et al. 2005, 2006)]. Cloning C. Schommer (*) • E.G. Bresso • S.V. Spinelli • J.F. Palatnik (*) Instituto de Biologı́a Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina e-mail: schommer@ibr.gov. ar; palatnik@ibr.gov. ar R. Sunkar (ed.), MicroRNAs in Plant Development and Stress Responses, Signaling and Communication in Plants 15, DOI 10.1007/978-3-642-27384-1_2, # Springer-Verlag Berlin Heidelberg 2012 29 and sequencing have led to the discovery of many of the currently known plant miRNAs (miRBASE 17.0, www.mirbase.org). Several plant miRNAs have also been identified by bioinformatics approaches based on the conservation of the small RNA sequence in the context of a fold-back precursor in different species [e.g., (Jones-Rhoades and Bartel 2004)]. In contrast, very few have been recognized from genetic screenings, probably due to the small size of the small RNA or redundancy with other miRNA-coding genes that have similar or identical sequences. One exception is miR319a, which was isolated during an activation tagging experiment in which transgenic Arabidopsis plants were screened for morphological changes (Weigel et al. 2000). In this type of screen, plants are transformed with a modified T-DNA vector containing a viral enhancer cassette derived from the 35S promoter of the cauliflower mosaic virus. The T-DNA harboring the viral enhancers is integrated randomly into the Arabidopsis genome causing the transcriptional activation of nearby genes and generating dominant gain-of-function mutations (Weigel et al. 2000). One of the recovered mutants that stood out for its unusual morphology was jawD (Fig. 1). The dominant jaw-D phenotype is governed by its pronounced jagged and wavy leaves. Four independent alleles (jaw-D1 to jaw-D4) were isolated from this and other activation tagging screens (Weigel et al. 2000; Palatnik et al. 2003). For all alleles, the insertion site was determined to be on chromosome IV between the protein-coding genes At4g23710 (coding for vacuolar synthase subunit G2) and At4g23720 (coding for a protein of unknown function). Surprisingly, the region responsible for the jaw-D phenotype was mapped to 1.6 kb in the intergenic region located between these two genes, where no open reading frame could be found, suggesting that JAW might be a nonprotein-coding gene (Weigel et al. 2000). Microarray experiments comparing wild type with jaw-D transcriptomes showed that a group of five TCP transcription factors was significantly downregulated in the jaw-D mutant. Interestingly, a TCP mutant in snapdragon called cincinnata had also defects in leaf development (Nath et al. 2003) similar to those described in jaw-D, indicating that the decrease in TCP levels was underlying the jaw-D phenotype. An alignment of these TCPs showed that apart from the typical TCP domain, they shared a highly conserved short stretch toward the Cterminal part of the protein (Palatnik et al. 2003). Conspicuously, the conservation of this region was found to be exceptionally high at RNA level and the site consisting of 21 nucleotides was found in TCPs of about 20 analyzed species, including CINCINNATA from snapdragon (Fig. 1). A short complementary sequence to this conserved motif was identified in the genomic region in the jaw-D mutant in proximity to the insertion site of the viral enhancers. The sequence was located at the base of a predicted RNA fold-back structure. Small RNA blots and sequencing of small RNA further demonstrated that a small RNA with the predicted sequence was actually overexpressed in jaw-D plants, now known as miR319 (Palatnik et al. 2003). The whole strategy also allowed the identification of miRNA targets by microarrays, an approach that was subsequently used in animals [e.g., (Lim et al. 2005)]. 30 C. Schommer et al.