The microEGo Project: Unraveling Post-Transcriptional Regulation Mechanisms on Eucalyptus Wood Formation

Wood is an abundant, carbon-neutral renewable resource with superlative economic, social and ecological value for many national economies. This composite, highly variable tissue presents anatomical, chemical and physical features determined upon complex ontogenetic processes, requiring the spatiotemporal transcriptional and post-transcriptional regulation of a large number of genes. A remarkable example is the Angiosperms’ response to gravitropic stimulus, leading to formation of tension wood, an excellent model to study the biosynthesis of xylem cell walls (xylogenesis).

In plants, the post-transcriptional regulation by microRNAs has been recognized to play crucial roles in diverse biological processes such as development, cellular differentiation, and responses to abiotic and biotic stress, including cambium differentiation. These endogenously expressed small RNAs down-regulate the expression of their target genes at the post-transcriptional level by cleaving mRNAs predominantly.

Using two Eucalyptus xylogenesis models of study (seasonal variation and tension wood), the microEGo project  (https://sites.google.com/site/microegopublic/; PTDC/AGR-GLP/098179/2008) pursues the identification of miRNA by combining the sequencing of 15 E.globulus smallRNA libraries with in silico miRNA prediction of candidates identified over the E. grandis genome sequence. CRAVELA framework (www.cravela.org) was used to perform an exhaustive genome-wide analysis. Cross validation of these parallel strategies allowed the prediction of several hundreds of robust candidates, filtered and ranked according their intrinsic, structural and transcriptional potential features. Northern blot was used to validate ten novel top-scored miRNA on E. globulus developing xylem. Target gene prediction and experimental validation (using modified 5'-RACE of cleaved miRNA targets) suggested microRNA post-transcriptional control over genes associated with transcription factor regulatory activity.