Illumina miRNA profiles with anatomical study revealed involvement of miR397a in Citrus adaptation to long-term B-toxicity via modulating secondary cell wall biosynthesis
Our previous studies indicated that long-term B-toxicity was mainly limited to leaves of Citrus species; alternations of cell wall structure in vascular bundles were involved in tolerance to B-toxicity. Here, miRNAs and their expression pattern were first identified from B-treated Citrus sinensis (tolerant) and C. grandis (intolerant) leaves with high-throughput sequencing in order to identify miRNAs that might be involved in tolerance to B-toxicity. Results: 51 (23 conserved and 28 novel) miRNAs in C. grandis and 20 (6 conserved and 14 novel) in C. sinensis were differentially expressed after B-toxic treatment, respectively. Illumina sequencing results were validated by stem-loop qRT-PCR, and 82.5% qRT-PCR data coincided with those from direct sequencing. Among the differentially expressed miRNAs, miR395a and miR397a were the most significantly up-regulated in B-toxic C. grandis leaves, yet both were down-regulated in B-toxic C. sinensis ones. With modified 5’-RACE, four auxin response factor genes were confirmed as the real targets of miR160a, and two laccase (LAC) genes as those of miR397a, respectively. Localization of cell wall polysaccharides indicated that up-regulation of LAC4 resulted in secondary deposition of cell wall polysaccharides in regions near the pits of vessel elements in C. sinensis, and that down-regulation of both LAC17 and LAC4, via up-regulating their negative regulator miR397a, led to poorly developed vessel elements in C. grandis.Our findings demonstrated that miR397a played a pivotal role in woody Citrus tolerance to B-toxicity by targeting LAC17 and LAC4, of which both were responsible for secondary cell wall synthesis. Overall design: Four libaries were constructed from C. grandis and C. sinensis leaves from seedlings treated with 10 (B-sufficient, control) and 400 (B-toxicity) µM boron acid, and sequenced by illumina sequencing.
External Link: /pubmed:26962011