Identification and functional characterization of microRNA regulatory elements in Brassicaceae

As a class of short non-coding RNAs, microRNAs can regulate gene expression by a post-transcriptional pathway through repressing or degrading mRNA. In the evolutionary history, many plant microRNAs are highly conserved from green alge to land plants. In recent years, dramatic studies demonstrate that microRNAs play a crucial in plant growth and development, in response to environmental stresses. Some microRNAs can response to plant hormones, while some others are tissue or cell specific. The understanding of how these microRNAs are regulated at the transcriptional level is just initiated. With the aim to understand the regulatory mechanism of plant microRNA in evolutionary tem, and identify the most relevant cisregulatory elements in some microRNAs for improving the agriculture in the future, this study was carried out. microRNA390 is one of the many conserved microRNAs, it can indirectly regulate the ARFs expression level by targeting TAS3, and consequently regulate lateral organ and later root development in plants. In order to understand the regulatory mechanism of microRNAs in an evolutionary term, microRNA390a and microRNA390b in Arabidopsis were chosen and studied. In 16 phylogenetically related species within Brassicaceae, we analyzed the microRNA promoter sequences and identified overall conserved cREs in microRNA390 promoter regions, and accompanied with functional characterization, we obtained a good view of microRNA390 regulatory network. Based on 454 sequencing technique, took the microRNA sequences of sequenced Arabidopsis as reference, by assembling and aligning the microRNA promoter sequences, calculated the PWM and predicted the putative motifs with both MEME program and PlantCARE database, subsequently compared the motif similarities by TOMTOM program, we eventually obtained the putative ones met the required E-value. In the meantime, we reconstructed the phylogenetic trees of both paralogs by MEGA7 program. We identified 6 and 5 overall conserved cREs. Subsequently, we experimentally validated the putative cREs by Arabidopsis transformation and site-specific mutagenesis. The results we have obtained were as follows: (1) There were totally 29 microRNA loci in 9 families identified to be highly conserved, and totally 104 putative motifs were predicted in their promoter regions. (2) The reconstructed phylogenetic trees based on miRNA390a and miRNA390b promoter sequences respectively were compared with the the phylogenetic relationships (species trees) in known Brassicaceae phylogeny. The data derived from both promoter sequences were inconsistent with Brassicaceae phylogeny. This implied that there might be multiple copies of specific cREs in some specific species, hence the promoter sequences evolution of microRNA is not reflective of species phylogeny. (3) Took Arabidopsis thaliana as model plant, we successfully constructed GUS-fused promoters of miRNA390a and miRNA390b. The GUS histochemical assay indicated that the two paralogs expressed in different tissues in transgenic Arabidopsis. miRNA390a expressed in lateral root primordia, true leaves, cotyledons, as well as in the floral organs, yet it was absent from lateral root tip and shoot apical meristem; whereas miRNA390b specifically expressed on lateral root tips, and a more restricted expression pattern was detected on aerial part of true leaves and floral organ. These differences indicated possible sub-functionalization with respect to their ancestral miR390 during the evolutionary process. (4) Based on the six putative cREs identified in miRNA390a and the reliable WT constructs, we also constructed six GUS-fused promoters that undergone site-specific mutagenesis. The GUS assay demonstrated that the activity of putative cis-elements varied with distance to TSS. Mutations of proximal sites (m2 and m3) enhanced expression thereby M2 and M3 were likely to be silencers; while mutations of distal elements (m5 and m6) tended to decrease the promoter expression, hence M5 and M6 probably work as enhancers. These evidences suggest there was a specific modular cooperativity of miR390a cREs in regulating gene expression and mediating plant development. Furthermore, we treated the 7d-old transgenic seedlings with iron-deficiency, both the GUS assay and qRT-PCR data conferred the iron responsiveness of putative iron-deficiency related E-box M3 and the iron-deficiency responsive cis-element 1 M6.