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

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

doi:10.3389/fpls.2021.715809

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

Front Plant Sci. 2022 Jan 14:12:715809. doi: 10.3389/fpls.2021.715809. eCollection 2021.

Authors

Yamei Zhuang^{1

2}, Sihui Chen², Wenjun Lian¹, Li Xu¹, Dian Wang^{2

3}, Congpeng Wang^{1

2}, Jie Meng^{1

2}, Xianfeng Tang², Hua Xu², Shumin Wang², Lin Du², Yang Zhang², Gongke Zhou^{1

2

4}, Guohua Chai^{1

2

4}

Affiliations

¹ College of Resources and Environment, Qingdao Agricultural University, Qingdao, China.
² Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
³ College of Agronomy, Qingdao Agricultural University, Qingdao, China.
⁴ Academy of Dongying Efficient Agricultural Technology and Industry on Saline and Alkaline Land in Collaboration With Qingdao Agricultural University, Dongying, China.

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.

Keywords: Populus; Y1H; Y2H; lignin; transcription factors; wood.