Abstract

The fast-growing arbor poplar is widely distributed across the world and is susceptible to nitrogen availability. The WRKY transcription factor is an important regulatory node of stress tolerance as well as nutrient utilization. However, the potential response mechanism ofWRKYgenes toward nitrogen is poorly understood. Therefore, the identification ofWRKYgenes on thePopulus trichocarpagenome was performed, and 98PtWRKYs(i.e.,PtWRKY1toPtWRKY98) were identified. Phylogenetic analysis and the promoter cis-acting element detection revealed that PtWRKYs have multiple functions, including phosphorus and nitrogen homeostasis. By constructing multilayer-hierarchical gene regulatory networks (ML-hGRNs), it was predicted that many WRKY transcription factors were involved in the nitrogen response, such as PtWRKY33 and PtWRKY95. They mainly regulated the expression of primary nitrogen-responsive genes (NRGs), such asPtNRT2.5A,PtNR2andPtGLT2. The integrative analysis of transcriptome and RT-qPCR results show that the expression levels of 6 and 15PtWRKYswere regulated by nitrogen availability in roots and leaves, respectively, and those were also found in ML-hGRN. Our study demonstrates that PtWRKYs respond to nitrogen by regulating NRGs, which enriches the nitrate-responsive transcription factor network and helps to uncover the hub of nitrate and its related signaling regulation.Keywords:WRKY transcription factors;Populus;nitrogen response;expression pattern