Poplar is one of the fastest-growing temperate trees in the world and is widely used in ornamental horticulture for shade. The root is essential for tree growth and development and its utilization potential is huge. Calcium (Ca), as a signaling molecule, is involved in the regulation of plant root development. However, the detailed underlying regulatory mechanism is elusive. In this study, we analyzed the morphological and transcriptomic variations of 84K poplar (Populus alba × P. glandulosa) in response to different calcium concentrations and found that low Ca²⁺ (1 mmol · L^-1) promoted lateral root development, while deficiency (0.1 mmol · L^-1 Ca²⁺) inhibited lateral root development. Co-expression analysis showed that Ca²⁺ channel glutamate receptors (GLRs) were present in various modules with significance for root development. Two GLR paralogous genes, PagGLR3.3a and PagGLR3.3b, were mainly expressed in roots and up-regulated under Ca²⁺ deficiency. The CRISPR/Cas9-mediated signal gene (crispr-PagGLR3.3a, PagGLR3.3b) and double gene (crispr-PagGLR3.3ab) mutants presented more and longer lateral roots. Anatomical analysis showed that crispr-PagGLR3.3ab plants had more xylem cells and promoted the development of secondary vascular tissues. Further transcriptomic analysis suggested that knockout of PagGLR3.3a and PagGLR3.3b led to the up-regulation of several genes related to protein phosphorylation, auxin efflux, lignin and hemicellulose biosynthesis as well as transcriptional regulation, which might contribute to lateral root growth. This study not only provides novel insight into how the Ca²⁺ channels mediated root growth and development in trees, but also provides a directive breeding of new poplar species for biofuel and bioenergy production.

杨树是世界上生长最快的温带树木之一，广泛用于观赏园艺的遮荫。根是树木生长发育所必需的，其利用潜力巨大。钙（Ca）作为信号分子，参与植物根系发育的调节。然而，具体的潜在监管机制尚不清楚。本研究分析了84K杨树（Populus alba × P. mudulosa）对不同钙浓度的响应，发现低Ca ²⁺ (1 mmol·L ^-1 ) 促进侧根发育，而钙缺乏则促进侧根发育。 (0.1 mmol·L ^-1 Ca ²⁺ )抑制侧根发育。共表达分析表明Ca ²⁺通道谷氨酸受体(GLR) 存在于对根发育具有重要意义的各种模块中。两个GLR旁系同源基因PagGLR3.3a和PagGLR3.3b主要在根中表达，并在Ca ²⁺缺乏下上调。CRISPR/Cas9介导的信号基因（crispr-PagGLR3.3a、PagGLR3.3b）和双基因（crispr-PagGLR3.3ab）突变体呈现出更多、更长的侧根。解剖分析表明crispr-PagGLR3.3ab植株具有更多的木质部细胞，促进次生维管组织的发育。进一步的转录组分析表明，敲除PagGLR3.3a和PagGLR3.3b会导致与蛋白质磷酸化、生长素外流、木质素和半纤维素生物合成以及转录调控相关的几个基因上调，这可能有助于侧根生长。这项研究不仅提供了关于Ca ²⁺通道如何介导树木根部生长和发育的新见解，而且还为用于生物燃料和生物能源生产的新杨树品种育种提供了指导。