The non-canonically organized members of MIR395 gene family in Brassica juncea are associated with developmentally regulated, sulfate-stress responsive bidirectional promoters that exhibit orientation-dependent differential transcriptional activity.

Several MICRORNA genes belonging to same family or different families are often found in homologous or non-homologous clusters. Among the various classes, head-to-head arranged genes form one of the largest categories of non-canonically organized genes. Such head-to-head arranged, non-canonically organized genes possibly share cis-regulatory region with the intergenic sequence having the potential to function as bi-directional promoter (BDP). The transcriptional regulation of head-to-head arranged genes, especially with bidirectional promoters, remains an enigma. In the past, bidirectional promoters have been characterized for a small set of protein-coding gene pairs in plants; however, to the best of our knowledge, no such study has been carried so far for MICRORNA genes. The present study thus functionally characterizes bidirectional promoters associated with members of MIR395 family, which is evolutionary conserved and is most frequently occurring cluster across plant kingdom. In Arabidopsis thaliana, the MIR395 gene family contains six members with two head-to-head arranged gene pairs- MIR395A-B and MIR395E-F. This organization was found to be conserved at seven loci for MIR395A-B, and eleven loci for MIR395E-F in five Brassica sps. Sequence analysis of the putative bidirectional promoters revealed variation in length, GC content and distribution of strict TATA-box. Comparatively higher level of conservation at both the ends of the bidirectional promoters, corresponding to ca. 250 bp upstream of 5'end of the respective MIRNA precursor, was observed. These conserved regions harbour several abiotic stress (nutrient, salt, drought) and hormone (ABA, ethylene) responsive cis-motifs. Functional characterization of putative bidirectional promoters associated with MIR395A-B and MIR395E-F from Arabidopsis and their respective orthologs from Brassica juncea (Bj_A08 MIR395A-B, Bj_B03 MIR395A-B, Bj_A07.1 MIR395E-F and Bj_A07.2 MIR395E-F) was carried out using a dual-reporter vector with β-glucuronidase (GUS) and Green Fluorescent Protein (GFP). Analysis of transcriptional regulation of the two reporter genes - GUS and GFP during developmental stages confirmed their bidirectional nature. Orientation-dependent differential reporter activity indicated asymmetric nature of the promoters. Comparison of the reporter activity amongst orthologs, paralogs and homeologs revealed regulatory diversification, an outcome expected in polyploid genomes. Interestingly, reporter gene activities driven by selected bidirectional promoters were also observed in anther and siliques apart vegetative tissues indicating role of miR395 in anther and fruit development. Finally, we evaluated the activity of reporter genes driven under transcriptional regulation of bidirectional promoters under normal and sulfate-deprived conditions which revealed asymmetric inducibility under sulfate-starvation, in agreement with the known role of miR395 in sulfate homeostasis.