Integrating 3D imaging, GWAS and single-cell transcriptome approaches to elucidate root system architecture in Populus.

Roots are essential for nutrient uptake and structural stability in trees. Despite their critical role, the genetic determinants underlying root system architecture (RSA) remain poorly understood. In this study, we employed an integrated approach combining automated three-dimensional (3D) spatial imaging, multi-omics analyses, genetic transformation, and molecular experiments to investigate the genetic architecture and regulatory networks governing RSA in Simon poplar (Populus simonii). Here, using a panel of 303 P. simonii accessions collected from different geographical regions in China, we performed a genome-wide association study (GWAS) on 96 RSA traits and identified S-phase kinase-associated protein 2B (PsiSKP2B) as a candidate gene co-localized by six traits. By integrating the findings from GWAS, transcriptome, and single-cell RNA-seq (scRNA-seq) analyses, we identified PsiSKP2B as a key regulator of meristematic tissue cells involved in lateral root (LR) development. Overexpression of PsiSKP2B in 84k (Populus alba × Populus glandulosa) had a substantial effect on RSA traits, increasing the number and density of LRs by 65.9% and 98.6%, respectively, compared with wild-type plants. Our in vitro and in vivo assays revealed that PsiSKP2B modulates LR development by interacting with WUSCHEL RELATED HOMEOBOX 4 (PsiWOX4) or ZINC FINGER HOMEODOMAIN 9 (PsiZHD9), both of which are specifically expressed in atrichoblast cells, thereby activating a regulatory feedback loop. These findings highlight an atrichoblast-dependent regulatory mechanism through which PsiSKP2B governs LR development. Our study not only introduces an advanced image recognition methodology for quantifying RSA traits in P. simonii but also provides a comprehensive multi-omics framework for elucidating the genetic and molecular basis of RSA.