Propagation through cuttings is a well-established and effective technique for plant multiplication. This study explores the regeneration of poplar roots using spatial transcriptomics to map a detailed developmental trajectory. Mapping of the time-series transcriptome data revealed notable alterations in gene expression during root development, particularly in the activation of cytokinin-responsive genes. Our analysis identified six distinct clusters during the second and third stages, each corresponding to specific anatomical regions with unique gene expression profiles. Auxin response cis-elements (AuxREs) were prevalent in the promoters of these cytokinin-responsive genes, indicating a regulatory interplay between auxin and cytokinin. Pseudo-temporal trajectory analysis mapped the differentiation from cambium cells to root primordium cells, revealing a complex pattern of cell differentiation. SAC56 and LOS1 emerged as potential novel biomarkers for enhancing root regeneration, with distinct spatial expression patterns confirmed by in situ hybridization. This comprehensive spatial analysis enhances our understanding of the molecular interactions driving root regeneration and provides insights for improving plant propagation techniques.

In this study, we report the cloning and functional characterization of an early responsive gene, BplERD15, from Betula platyphylla Suk to dehydration. BplERD15 is located in the same branch as Morus indica Linnaeus ERD15 and Arabidopsis Heynh ERD15 in the phylogenetic tree built with ERD family protein sequences. The tissue-specific expression patterns of BplERD15 were characterized using qRT-PCR and the results showed that the transcript levels of BplERD15 in six tissues were ranked from the highest to the lowest levels as the following: mature leaves (ML) > young leaves (YL) > roots (R) > buds (B) > young stems (YS) > mature stems (MS). Multiple drought experiments were simulated by adding various osmotica including polyethylene glycol, mannitol, and NaCl to the growth media to decrease their water potentials, and the results showed that the expression of BplERD15 could be induced to 12, 9, and 10 folds, respectively, within a 48 h period. However, the expression level of BplERD15 was inhibited by the plant hormone abscisic acid in the early response and then restored to the level of control. The BplERD15 overexpression (OE) transgenic birch lines were developed and they did not exhibit any phenotypic anomalies and growth deficiency under normal condition. Under drought condition, BplERD15-OE1, 3, and 4 all displayed some drought tolerant characteristics and survived from the drought while the wild type (WT) plants withered and then died. Analysis showed that all BplERD15-OE lines had significant lower electrolyte leakage levels as compared to WT. Our study suggests that BplERD15 is a drought-responsive gene that can reduce mortality under stress condition.

Cold is an abiotic stress that can greatly affect the growth and survival of plants. Here, we reported that an AP2/ERF family gene, BplERF1, isolated from Betula platyphylla played a contributing role in cold stress tolerance. Overexpression of BplERF1 in B. platyphylla transgenic lines enhanced cold stress tolerance by increasing the scavenging capability and reducing H₂O₂ and malondialdehyde (MDA) content in transgenic plants. Construction of BplERF-mediated multilayered hierarchical gene regulatory network (ML-hGRN), using Top-down GGM algorithm and the transcriptomic data of BplERF1 overexpression lines, led to the identification of five candidate target genes of BplERF1 which include MPK20, ERF9, WRKY53, WRKY70, and GIA1. All of them were then verified to be the true target genes of BplERF1 by chromatin-immunoprecipitation PCR (ChIP-PCR) assay. Our results indicate that BplERF1 is a positive regulator of cold tolerance and is capable of exerting regulation on the expression of cold signaling and regulatory genes, causing mitigation of reactive oxygen species.