Molecular Mechanism of microRNA396 Mediating Pistil Development in Arabidopsis^1,^[W]

Abstract

The precise spatial and temporal expression of regulatory genes that control tissue patterning and cell fate is important for plant development. Misexpression of certain key regulatory genes causes developmental abnormalities in plants. There is increasing evidence that small RNA molecules are important participants in the control of gene expression, providing sequence specificity for targeted regulation of key developmental factors at the posttranscriptional level. MicroRNAs (miRNAs) are 21- to 24-nucleotide noncoding RNAs that negatively regulate gene expression by pairing with their target mRNAs. They are produced from primary miRNAs that are transcribed from MIRNA genes. After the miRNA duplexes are released from the nucleus, mature miRNAs are recruited into an RNA-induced silencing complex associated with ARGONAUTE (AGO) proteins, where they suppress target mRNAs by complementary matching for cleavage and/or translational repression (Reinhart et al., 2002; Carrington and Ambros, 2003; Bartel, 2004; Brodersen et al., 2008; Lanet et al., 2009).

Several plant miRNAs have been shown to function in plant development. The lack of miRNA processing protein(s) can cause severe developmental phenotypes. For example, the weaker dicer-like1 (dcl1) alleles produce various aberrant morphological phenotypes, including extra whorls of stamens, an indefinite number of carpels, female sterility, altered ovule development, and reduced plant height, indicating that miRNA metabolism is essential for normal plant development (Schauer et al., 2002). ago1 null mutants exhibit morphological defects similar to those of dcl1, hua enhancer1, and hyponastic leaves1 (hyl1) mutants (Vaucheret et al., 2004). The specific functions of miRNAs in floral development have been characterized. For example, Arabidopsis (Arabidopsis thaliana) microRNA160a (miR160a) mutant produced floral organs in carpels (Liu et al., 2010). Overexpression of miR164 led to flowers with fused sepals, which resembled the flowers of its target mutants, cuc1cuc2 (for cup-shaped cotyledon1cup-shapedcotyledon2; Mallory et al., 2004). Enhanced expression of the miR164-resistant version of mCUC1 resulted in flowers with more petals than those of the wild type (Baker et al., 2005). Plants ectopically expressing miR166 showed extreme fasciation of the inflorescence meristem and a reduced or filamentous gynoecium (Kim et al., 2005; Williams et al., 2005). Constitutive expression of miR159 or miR167, which led to reduced expressions of their target genes (MYB33 and MYB65; Auxin Responsive Factor6 [ARF6] and ARF8), caused male sterility in Arabidopsis (Achard et al., 2004; Millar and Gubler, 2005; Ru et al., 2006; Wu et al., 2006). Elevated miR172 accumulation resulted in floral organ identity defects similar to those in its target gene mutant (apetala2; Aukerman and Sakai, 2003; Chen, 2004).

GROWTH-REGULATING FACTOR (GRF) genes are a class of plant-specific transcription regulators. In Arabidopsis, there are nine GRF genes that can be divided into five subfamilies: group I (GRF1 and GRF2), group II (GRF3 and GRF4), group III (GRF5 and GRF6), group IV (GRF7 and GRF8), and group V (GRF9; Kim et al., 2003). Among them, GRF1, GRF2, GRF3, GRF4, GRF7, GRF8, and GRF9 are the direct targets of miR396 (Jones-Rhoades and Bartel, 2004). It has been revealed that miR396 is involved in leaf development by controlling the levels of its GRF targets (Liu et al., 2009; Yang et al., 2009; Rodriguez et al., 2010; Wang et al., 2011; Debernardi et al., 2012). A GRF protein consists of two regions, the QLQ and WRC domains. The QLQ domain is responsible for protein interaction, while the WRC domain comprises a functional nuclear localization signal and a zinc-finger motif that functions in DNA binding (Kim et al., 2003). GRF genes are involved in regulating leaf growth and morphology (Kim et al., 2003; Horiguchi et al., 2005). The GRF-INTERACTING FACTOR1 (GIF1) protein was identified to interact with GRF1 as a transcription coactivator to regulate leaf development (Kim and Kende, 2004). Horiguchi et al. (2005) revealed that both GRF5 and GRF9 interact with GIF1 to regulate leaf development. GIF1 contains two domains: the SNH and QG domains. The SNH domain is responsible for the interaction with the QLQ domain of GRF. The GIF gene family has three members, GIF1, GIF2, and GIF3, which have overlapping functions in determining organ (leaf and petal) size (Kim and Kende, 2004; Lee et al., 2009).

Another group (Hewezi et al., 2012) revealed the functions of miR396 in reprogramming root cells during infection by a parasitic cyst nematode. Here, we demonstrate that the products of all seven GRF targets can interact with GIFs that may function as cotranscription factors. Overexpression of miR396 caused reduced expressions of GRF genes, which disrupted the formation of the GRF/GIF complex, leading to pistil anomalies. These results indicate that miR396-directed regulation is critical for pistil development.

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