A High-Throughput Screening System for Populus Wood-Associated Transcription Factors and Its Application to Lignin Regulation.

Hua Xu,Li Xu,Shumin Wang,Wenjun Lian,Yang Zhang,Dian Wang,Yamei Zhuang,Congpeng Wang,Gongke Zhou,Xianfeng Tang,Guohua Chai,Lin Du,Jie Meng,Sihui Chen

doi:10.3389/fpls.2021.715809

Abstract

Wood formation of trees is a complex and costly developmental process, whose regulatory network is involved in the protein-protein and protein-DNA interactions. To detect such interactions in wood development, we developed a high-throughput screening system with 517 Gal4-AD-wood-associated transcription factors (TFs) library from Populus alba × P. glandulosa cv “84K.” This system can be used for screening the upstream regulators and interacting proteins of targets by mating-based yeast-one hybrid (Y1H) and yeast-two-hybrid (Y2H) method, respectively. Multiple regulatory modules of lignin biosynthesis were identified based on this Populus system. Five TFs interacted with the 500-bp promoter fragment of PHENYLALANINE AMMONIA-LYASE 2 (PAL2), the first rate-limiting enzyme gene in the lignin biosynthesis pathway, and 10 TFs interacted with PaMYB4/LTF1, a key regulator of lignin biosynthesis. Some of these interactions were further validated by EMSA and BiFC assays. The TF-PaPAL2 promoter interaction and TF-PaMYB4 interaction revealed a complex mechanism governing the regulation of lignin synthesis in wood cells. Our high-throughput Y1H/Y2H screening system may be an efficient tool for studying regulatory network of wood formation in tree species.

Highlights

The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet
PaPAL2, the ortholog of Arabidopsis PAL1 in Populus, is a rate-limiting enzyme that controls the deamination of phenylpropanoid to cinnamic acid during monolignol biosynthesis in woody cells (Wang et al, 2014)
Recent studies in the stem of Populus show that wood formation involves regulatory homeostasis determined by combinations of transcription factors (TFs)-DNA and TF-protein regulations (Petzold et al, 2017; Chen et al, 2019)

Summary

Introduction

The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. Functional characterization of a serial of Arabidopsis mutants shows a three-layered regulatory network for secondary cell wall formation (Zhong et al, 2010; Wang and Dixon, 2012; Nakano et al, 2015). In Arabidopsis stems, SND1 is the highest level regulator of HTFN, and it directly activates the second-layered master switches MYB46 and its paralog MYB83, inducing the expression of multiple cell wall biosynthetic genes. Similar to Arabidopsis SND1-mediated HTFN, in Populus stems PtrSND1-B1 induces PtrMYB21, a homolog of Arabidopsis MYB46/83, to regulate the expression of a number of genes associated with cell wall component and wood biosynthesis. The TF protein-complex regulators (dimers and trimers) are shown to cooperatively or combinatorially mediate the biosynthesis of specific types of lignin in Populus stems (Chen et al, 2019)

Methods

Results

Conclusion