UNDERSTANDING THE EXPANSION OF microRNA NETWORKS IN PLANTS

HEDIN, NICOLAS; PALATNIK, JAVIER F; RODRIGUEZ, RAMIRO E; LARRAN, A; DEBERNARDI, JUAN MANUEL

Congreso; 55th Annual Meeting Argentine Society for Biochemistry and Molecular Biology.; 2019

MicroRNAs (miRNAs) are a type of small RNAs that play a key role in the developmental programs of higher organisms. Several studies suggest that, among all the miRNAs conserved in plants, miR396 is the one that more target genes has gained throughout the evolution. While the miR396-GRF node is present in all vascular plants, different lineages have independently incorporated other target genes into the miR396 regulatory network, which is revealed by the emergence of binding sites with substantial differences in their sequences. In this context, some interesting questions arise. For example, how the cis-context of miR396 binding site affects its efficiency, and also which is the biological relevance of these new nodes that evolution has incorporated. In the first instance, we built sensors carrying different miR396 binding sites. We generated sensors with target sites either within the GFP coding region (CDS) or within the 3' UTR. Transient expression analysis in Nicotiana benthamiana showed that fluorescence levels were considerably different for the vectors used, and quantitative real-time PCR assays confirmed these observations. These results indicate that there may be a difference in the performance of miR396 in a context-dependent manner. In addition, it is well known that overexpression of miR396 causes a significant reduction of leaf size in Arabidopsis thaliana and other plant species, mainly due to the inactivation of GRF transcription factors. However, in silico studies predict that miR396 could regulate genes with a sensitive response to photoperiod. Therefore, we analyzed miR396a and miR396b overexpressing plants grown at differential light conditions, in contrast to grf mutants. Interestingly, we observed that the phenotype of plants overexpressing these miRNAs was significantly affected by the different environmental conditions tested. Consequently, our studies open new avenues for the empirical exploration of new target genes of the evolutionary-conserved miR396.