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

## TL;DR

This study uses CRISPR to create triple mutants in poplar PHOT genes, revealing their roles in leaf development, light response, and stomatal regulation.

## Contribution

The first CRISPR-edited triple-gene mutants of PHOTs in poplar, uncovering their multifunctional roles in light signaling and plant adaptability.

## Key findings

- Triple-gene ptrphots mutants showed curved leaves, reduced area, and delayed phototropic responses.
- Stomatal aperture recovery in mutants was 40% of wild type, with downregulated BLUS1 gene expression.
- Transcriptome analysis revealed 1413 differentially expressed genes linked to auxin, jasmonic acid, and light signaling.

## Abstract

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 Populus trichocarpa, and it included three members, PtrPHOT1, PtrPHOT2.1, and PtrPHOT2.2, all of which were highly expressed in mature leaves. Using CRISPR/Cas9 gene editing technology, triple-gene mutations in the PtrPHOT1/2.1/2.2 (PtrPHOTs) were generated, providing initial insights into the functions of PHOTs in trees. Compared to the wild type (WT), triple-gene ptrphots 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 BLUS1 gene transcription levels, confirming the conservation of the PHOT-BLUS1-H⁺-ATPase signaling axis in stomatal regulation. Transcriptome of triple-gene ptrphots mutants revealed 1413 differentially expressed genes, of which were enriched in auxin response (upregulation of SAUR family genes), jasmonic acid (downregulation of JAZ 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 PtrPHOTs family genes, and characteristics of triple-gene ptrphots 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.