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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  Pa MYB4/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- Pa MYB4 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.

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