Huang Jing-Hao, Qi Yi-Ping, Wen Shou-Xing, Guo Peng, Chen Xiao-Min, Chen Li-Song
Institute of Plant Nutritional Physiology and Molecular Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Pomological Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China.
Sci Rep. 2016 Mar 10;6:22900. doi: 10.1038/srep22900.
The mechanisms underlying tolerance to B-toxicity in plants are still controversial. Our previous studies indicated that B-toxicity is mainly limited to leaves in Citrus and that alternations of cell-wall structure in vascular bundles are involved in tolerance to B-toxicity. Here, miRNAs and their expression patterns were first identified in B-treated Citrus sinensis (tolerant) and C. grandis (intolerant) leaves via high-throughput sequencing. Candidate miRNAs were then verified with molecular and anatomical approaches. The results showed that 51 miRNAs in C. grandis and 20 miRNAs in C. sinensis were differentially expressed after B-toxic treatment. MiR395a and miR397a were the most significantly up-regulated miRNAs in B-toxic C. grandis leaves, but both were down-regulated in B-toxic C. sinensis leaves. Four auxin response factor genes and two laccase (LAC) genes were confirmed through 5'-RACE to be real targets of miR160a and miR397a, respectively. Up-regulation of LAC4 resulted in secondary deposition of cell-wall polysaccharides in vessel elements of C. sinensis, whereas down-regulation of both LAC17 and LAC4, led to poorly developed vessel elements in C. grandis. Our findings demonstrated that miR397a plays a pivotal role in woody Citrus tolerance to B-toxicity by targeting LAC17 and LAC4, both of which are responsible for secondary cell-wall synthesis.
植物对硼毒性的耐受机制仍存在争议。我们之前的研究表明，硼毒性在柑橘中主要局限于叶片，并且维管束中细胞壁结构的变化与对硼毒性的耐受性有关。在此，通过高通量测序首次在经硼处理的甜橙（耐硼）和柚（不耐硼）叶片中鉴定了miRNA及其表达模式。然后用分子和解剖学方法对候选miRNA进行了验证。结果表明，硼毒性处理后，柚中有51个miRNA和甜橙中有20个miRNA差异表达。MiR395a和miR397a是硼毒性处理的柚叶片中上调最显著的miRNA，但在硼毒性处理的甜橙叶片中均下调。通过5'-RACE证实，四个生长素响应因子基因和两个漆酶（LAC）基因分别是miR160a和miR397a的真实靶标。LAC4的上调导致甜橙导管分子中细胞壁多糖的次生沉积，而LAC17和LAC4的下调导致柚中导管分子发育不良。我们的研究结果表明，miR397a通过靶向LAC17和LAC4在木本柑橘对硼毒性的耐受性中起关键作用，这两个基因均负责次生细胞壁的合成。
