Intercellular communication mediated by CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides and their receptors is crucial for plant development and environmental adaptation. In this study, 45 and 89 CLEs were identified in Populus tomentosa and Populus alba × Populus glandulosa, respectively, and, together with the 52 CLEs in Populus trichocarpa, the chromosome localization, gene and protein characteristics, collinearity and gene duplication events, cis-acting regulatory elements in promoters and evolutionary relationships of CLEs in these three poplar species were analyzed. The CLEs of three poplar species were divided into four subfamilies. Among them, the CLEs in subfamilies I, II and IV were A-type CLEs, while those in subfamily III were B-type CLEs. During the evolutionary process of poplar, the selection pressure faced by whole-genome duplication or segmental duplication was purifying selection, and the duplication events led to the expansion of the CLE family in poplar. The exogenous addition of a certain concentration of poplar CLE13 peptides inhibits the root growth of Arabidopsis thaliana and poplar and simultaneously reduces the expression levels of ARFs and LBDs in the roots. In addition, drought stress induces the expression of PtrCLE13A. The overexpression of preCLE13A significantly enhances the osmotic and drought tolerance in Populus tomentosa. These results have provided valuable information for further research on the molecular mechanisms of CLE peptide signaling pathways in the woody model plant poplar regarding plant growth and stress resistance.

Signaling mediated by CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides and their receptors is essential for plants to adapt to abiotic stress. To address the global issue of drought-induced growth inhibition and mortality in poplar (Populus spp.), this study investigated the function of the PtrCLE1A gene from Populus trichocarpa Torr. et Gray in drought tolerance regulation. We employed gene cloning, expression vector construction, and genetic transformation of poplar, combined with bioinformatics analysis, subcellular localization, phenotypic observation, physiological index measurement, and gene expression analysis. The results demonstrated that both PtrCLE1A and PtrCLE1B encode pre-propeptides containing a signal peptide, with an identical mature peptide sequence (RLSPGGPDPRHH), and their putative receptors are PtrCLV1/2. Furthermore, the PtrCLE1A pre-propeptide was localized around the plasma membrane in tobacco (Nicotiana benthamiana Domin) mesophyll cells, consistent with its predicted function. PtrCLE1A and PtrCLE1B are primarily expressed in the roots and xylem of P. trichocarpa. Additionally, only the PtrCLE1A promoter contained drought-responsive cis-elements, and its expression was induced by drought stress in root, xylem, and leaf tissues of P. trichocarpa. Overexpression of the PtrCLE1A gene in Populus tomentosa Carrière (triploid) significantly increased adventitious root length under osmotic stress. Overexpression lines exhibited 22.00% to 22.92% longer adventitious roots than EV lines at 50/100 mM mannitol, and 65.12% to 73.17% longer at 150 mM mannitol. The OE lines also exhibited higher photosynthetic capacity and instantaneous water use efficiency (iWUE), along with reduced membrane damage under drought conditions, indicating enhanced drought resistance. This study provides new genetic resources and a theoretical foundation for molecular breeding of drought-tolerant poplar.