# Genome-Wide Identification, Drought-Responsive Expression, and EAR-Mediated Regulatory Network Construction of TOPLESS Genes in Populus ussuriensis Kom

## TL;DR

This study identifies and characterizes drought-related TOPLESS genes in Populus ussuriensis, showing their role in stress adaptation and regulatory networks.

## Contribution

First genome-wide analysis of TPL/TPR genes in P. ussuriensis with functional validation and regulatory network construction.

## Key findings

- 21 TPL/TPR genes were identified and classified into five subfamilies in P. ussuriensis.
- Most PuTPLs showed significant upregulation under drought stress in a tissue-specific manner.
- Transgenic lines overexpressing PuTPLs exhibited enhanced drought tolerance with reduced damage markers.

## Abstract

Drought stress significantly impairs plant growth and productivity, which triggers complex adaptive responses mediated by diverse gene families. Among these, the TOPLESS (TPL)/TPL-related (TPR) family of transcriptional corepressors plays a crucial role by recruiting epigenetic modifiers through interactions with EAR motif-containing proteins. However, genome-wide studies of this corepressor family and its associated regulatory networks with EAR motif-containing repressors remain limited. This study aimed to characterize the TPL/TPR transcriptional corepressor family in Populus ussuriensis Kom., elucidate their regulatory networks with EAR motif-containing repressors, and validate their functional roles in drought stress adaptation. To this end, we identified 21 TPL/TPR genes in P. ussuriensis (PuTPLs), classified them into five subfamilies, and found they are evolutionarily conserved with Arabidopsis thaliana and Populus trichocarpa, harboring characteristic CTLH and WD40 domains. Given that TPL/TPR proteins are recruited by transcription factors containing repression motifs, we constructed a putative TPL/TPR-EAR motif interaction network representing a core paradigm of negative regulation. Expression profiling under drought stress showed significant upregulation of most PuTPLs in a tissue-specific and temporal manner. Functional validation using transgenic P. ussuriensis lines overexpressing five PuTPLs demonstrated enhanced drought tolerance, evidenced by reduced electrolyte leakage and malondialdehyde content and increased proline accumulation. Our study provides the first comprehensive genome-wide analysis of the TPL/TPR family in P. ussuriensis, establishes a core EAR-mediated negative regulatory network, and validates the critical role of these genes in drought stress adaptation, providing valuable resources for future mechanistic research and breeding of stress-resistant trees.