Generation and Functional Characteristics of CRISPR/Cas9-Edited Triple-Gene Mutants in Poplar.

Phototropins (PHOTs), as blue light receptors, play a pivotal role in plant light signal perception and adaptive regulation, yet their functional characteristics in trees remain poorly understood. In this study, the PHOT gene family was identified in , and it included three members, , , and , all of which were highly expressed in mature leaves. Using CRISPR/Cas9 gene editing technology, triple-gene mutations in the () were generated, providing initial insights into the functions of PHOTs in trees. Compared to the wild type (WT), triple-gene mutants displayed curved and wrinkled leaves, reduced leaf area, and delayed phototropic responses, indicating the central role of PHOTs in blue light signal perception. The stomatal aperture recovery rate in mutants was only 40% of that observed in WT, accompanied by significant downregulation of the gene transcription levels, confirming the conservation of the PHOT-BLUS1-H⁺-ATPase signaling axis in stomatal regulation. Transcriptome of triple-gene mutants revealed 1413 differentially expressed genes, of which were enriched in auxin response (upregulation of family genes), jasmonic acid (downregulation of genes), and light signaling pathways, suggesting that PHOTs could regulate plant adaptability by integrating light signals and hormone homeostasis. Overall, this study achieved the knockouts of three family genes, and characteristics of triple-gene mutants elucidated the multifunctional roles of PHOTs in leaf development, phototropism, and stomatal movement in poplar. Our work provides a foundation for deciphering light signaling networks and molecular breeding in woody plants.