RNA-seq of ptrlbd39/22 double mutants

We analyzed laser capture microdissection-based transcriptomes of Populus trichocarpa TW-forming tissues representing various stages of TW development. We revealed that PtrLBD39 is the most significantly tension stress-induced transcription factor gene, which has a phylogenetically paired LBD homologous gene, PtrLBD22. These two genes were sharply induced by tension stress and were highly expressed in the TW-forming tissues. CRISPR-based knockout of PtrLBD39/22 led to a severely inhibited TW formation with low cellulose and high lignin, whereas overexpression of PtrLBD39 resulted in an opposite effect. Transcriptomic analyses of CRISPR-based double mutants of PtrLBD39/22 showed that these two genes regulate a set of TW-related genes. Moreover, chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PtrLBD39 direct targets. We integrated transcriptomic analyses and ChIP-seq assays to construct a transcriptional regulatory network (TRN) mediated by PtrLBD39. The network consists of three layers including transcription factors, secondary cell wall component genes and other potential regulators. Our work suggested that PtrLBD39 and PtrLBD22 specifically regulate TW formation by mediating a TW-specific TRN in Populus.

本研究针对毛果杨（Populus trichocarpa）不同发育阶段的张力木（Tension Wood, TW）形成组织，开展了基于激光捕获显微切割（Laser Capture Microdissection, LCM）的转录组分析。研究发现，PtrLBD39是受张力胁迫诱导最为显著的转录因子基因，其系统发育上存在配对的LBD同源基因PtrLBD22。这两个基因均受张力胁迫强烈诱导，并在张力木形成组织中高表达。通过CRISPR技术对PtrLBD39/22进行双基因敲除后，植株的张力木形成受到严重抑制，同时伴随纤维素含量降低、木质素含量升高；而过表达PtrLBD39则呈现出相反的表型。对PtrLBD39/22的CRISPR双突变体开展转录组分析后发现，这两个基因可调控一系列与张力木相关的基因。此外，本研究通过染色质免疫共沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-seq）鉴定了PtrLBD39的直接靶基因。本研究整合转录组分析与ChIP-seq实验结果，构建了由PtrLBD39介导的转录调控网络（Transcriptional Regulatory Network, TRN），该网络包含转录因子、次生细胞壁组分基因及其他潜在调控因子三个层级。本研究结果表明，PtrLBD39与PtrLBD22可通过介导毛果杨中张力木特异性的转录调控网络，特异性调控张力木的形成。