Sequence and functional analysis of MIR319 promoter homologs from Brassica juncea reveals regulatory diversification and altered expression under stress.

KEY MESSAGE: Extensive regulatory divergence during development, abiotic stress and ABA regime observed amongst promoter homologs and homeologs of MIR319 from Brassica juncea. Gene duplication followed by sub-functionalization, neo-functionalization, and pseudogenization are routes to functional and adaptive diversification. The influence of polyploidy on protein-coding genes is well investigated but little is known about their impact on transcriptional regulation of MIRNA gene family. The present study was therefore performed with an aim to uncover regulatory diversification of MIR319 homologs and homeologs in Brassica juncea. We employed comparative genomics to identify and isolate six promoter homologs of MIR319 from B. juncea. Regulatory diversification was studied using analysis of reporter activity driven by BjMIR319 promoters in a heterologous system employing promoter-reporter fusion constructs. MIR319 is known to play important roles in leaf and flower development, and multiple stress responses. Reporter activity was therefore monitored during development, hormonal and stress regimes. In-silico analyses revealed differential distribution of cis-regulatory motifs and functional analysis revealed distinct spatiotemporal expression patterns. The significance of presence of selected cis-regulatory motifs corresponding to heat, cold, salt and ABA stress were further functionally validated. It was observed that promoter of Bj -MIR319a-A01 was upregulated in response to cold and salt stress, while promoter of Bj -MIR319c-A04 (D1) and Bj -MIR319c-A05 (FL) were downregulated in response to high temperature. In summary, comparative analysis of homologous promoters from Brassica juncea, an allopolyploid revealed extensive sequence and functional diversity. Spatiotemporal activity of reporter gene driven by BjMIR319 promoter was distinct, and partially overlapping with from those reported previously for A. thaliana. The present study clearly demonstrates regulatory divergence amongst promoter homologs of MIR319 in Brassica juncea during development and stress response, and underlines the urgent need for dissection of promoter function and detailed characterization including identification of interacting trans-factors. Genbank accession numbers: MT379853-MT379858.