Objective GRF transcription factors play conserved roles in plant development and stress responses, yet their molecular contributions to drought tolerance in poplar remain understood. Here, we functionally characterize PtoGRF9 in Populus tomentosa, elucidate its role in the growth-defence balance and provide a target for molecular breeding of drought resistant poplar.

Method We first cloned PtoGRF9 from Populus tomentosa, performed phylogenetic, structural, promoter and expression analyses; then, Agrobacterium-mediated leaf-disc transformation was employed to generate both over-expression (PtoGRF9-OE) and RNAi-silenced (ptogrf9-RNAi) 84K poplar lines, which were then subjected to drought stress to evaluate photosynthetic performance and antioxidant enzyme activities. Finally, DNA affinity purification sequencing (DAP-seq) data were integrated to identify and preliminarily validate the downstream target genes of PtoGRF9.

Result (1) PtoGRF9 encodes a protein of 457 amino acids that contains one QLQ domain and two WRC domains. (2) The PtoGRF9 promoter contains multiple cis-acting elements involved in plant growth and development, stress signaling, and biotic/abiotic stress responses. Reverse transcription-quantitative PCR (RT-qPCR) assays showed that PtoGRF9 has a relatively high expression level in developing leaves and is significantly induced by drought stress. (3) Under short-term drought, PtoGRF9-OE lines exhibited significantly higher photosynthetic performance and peroxidase activities than WT, whereas ptogrf9-RNAi lines displayed the opposite trend. (4) DNA affinity purification sequencing (DAP-seq) and RT-qPCR analyses identified the wax-biosynthesis and stomatal-development genes SHN1, ERD7, ERECTA and PIP1 as potential PtoGRF9 targets.

Conclusion PtoGRF9 is a transcription factor that positively regulates drought tolerance in poplar and plays a key role in maintaining the balance between leaf development and stress resistance. Our findings not only provide new insights into the molecular mechanisms underlying poplar growth, development, and stress tolerance but also identify a key candidate gene for drought-tolerant molecular breeding.