Principal Investigator: María Rosa Ponce.
Investigators: Verónica Aguilera Díaz.
Instituto de Bioingeniería. Universidad Miguel Hernández.

Identification of novel microRNA pathway elements acting as developmental controls in Arabidopsis thaliana

Postranscriptional repression mediated by microRNAs (miRNAs) is a recent addition to the inventory of mechanisms of gene expression regulation acting as developmental controls in multicellular eukaryotes. In the model plant Arabidopsis thaliana, one of the miRNA machinery elements is encoded by the ARGONAUTE1 (AGO1) gene, whose ago1 mutant alleles perturb many developmental processes and often cause lethality or sterility. We isolated two viable and moderately fertile mutants, ago1-51 and ago1-52, and obtained their double mutant combinations with alleles of other miRNA machinery genes and miRNA target genes. All the double mutants displayed synergistic phenotypes, as expected from the functional relationship among the mutations involved. This prompted us to search for synergistic interactions in double mutants involving ago1-51 and already obtained but scarcely studied mutants. We also mutagenized ago1-52 seeds using ethyl methanesulfonate in order to isolate mutations modifying the phenotype caused by ago1-52. In addition, we performed microarray analyses on several mutants isolated in our laboratory, which are affected in miRNA machinery genes. This allowed us to identify several hundred genes similarly misregulated in the mutants, some of which are either experimentally proven or in silico predicted miRNA targets, and others had not been predicted or demonstrated to be miRNA targets.

We propose a genetic and molecular approach to identify novel miRNA pathway elements acting as developmental controls in Arabidopsis thaliana. Our goals include (a) to use the mutants identified in our screening in order to positionally clone and functionally characterize at least one of the genes that modulate the phenotype of the ago1-52 mutant, and (b) to experimentally demonstrate that several of the genes found upregulated in our microarray analyses are novel miRNA targets, as well as to identify their regulator miRNAs.