Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China.
Proc Natl Acad Sci U S A. 2013 Jun 25;110(26):10848-53. doi: 10.1073/pnas.1308936110. Epub 2013 Jun 10.
Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome-based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.
漆酶早在 1959 年就被提议催化单酚氧化聚合。由于漆酶基因的功能冗余，支持这一假说的遗传证据一直难以捉摸。拟南芥双突变体证明了漆酶参与木质素生物合成。我们之前在毛白杨中鉴定了一组漆酶基因是 microRNA (miRNA) ptr-miR397a 的靶基因。为了阐明 ptr-miR397a 及其靶基因的作用，我们对漆酶基因家族进行了特征分析，鉴定了 49 个漆酶基因模型，其中 29 个被预测为 ptr-miR397a 的靶基因。我们在转基因毛白杨中过表达了 Ptr-MIR397a。在所有测试的九条转基因株系中，9 条转基因株系的 17 个 PtrLACs 的表达都被 RNA-seq 分析下调。这些漆酶基因表达严重减少的转基因株系导致总漆酶活性下降约 40%。这些转基因株系中 Ptr-MIR397a 的过表达也降低了木质素含量，而所有单酚生物合成基因转录本的水平保持不变。通过自下而上的图形高斯模型算法构建的分层遗传调控网络（GRN）为 ptr-miR397a 作为木质素生物合成的负调控因子提供了额外的支持。基于全转录组的差异基因表达分析表明，在过表达的转基因中，以前未被识别的转录因子及其靶基因在一个扩展的分层 GRN 中发挥作用，包括 ptr-miR397a 和漆酶，它们共同调节木质素生物合成在木材形成中。Ptr-miR397a、漆酶和该网络的其他调控成分可能为遗传操纵木质素含量提供了额外的策略。
