Objective To reveal the genome-wide expression dynamics of male floral buds in Populus tomentosa from floral induction to pollen maturation, and to identify key regulatory genes controlling floral development, so as to provide a theoretical basis for understanding the molecular mechanisms of flower development in perennial woody plants and to offer candidate gene resources for molecular breeding aimed at regulating flowering time and reducing pollen allergens in Populus.

Method Male floral bud samples at eight consecutive developmental stages (MS1–MS8) of P. tomentosa were subjected to transcriptome sequencing. Principal component analysis was used to assess sample reproducibility, differentially expressed genes (DEGs) were screened, and transcription factor families were statistically analyzed. Mfuzz time-series clustering was performed to classify expression trend clusters, followed by GO enrichment analysis. Promoter cis-element prediction was carried out for transcription factors selected from key clusters. WGCNA was applied to construct co-expression networks and to screen for developmental stage-related modules, which were then analyzed by GO enrichment. Select key genes for RT-qPCR validation to assess the accuracy of RNA-seq data.

Result (1) The number of DEGs varied markedly across developmental transitions: the comparison between MS8 and MS7 (from overwintering dormancy to pollen maturation initiation) showed the largest number of DEGs (10 863 up-regulated, 10 890 down-regulated), suggesting that massive transcriptional reprogramming after dormancy release might be the molecular basis for rapid progression of reproductive development; whereas the comparison between MS4 and MS3 (from bud swelling to dormancy transition) showed the fewest DEGs (1 480 up-regulated, 3 178 down-regulated), indicating that this stage might be relatively quiescent. (2) Mfuzz time-series clustering divided all expressed genes into 10 trend clusters. Among them, Cluster 1 was highly expressed during floral induction and then continuously down-regulated, enriched in cell wall synthesis and cytoskeleton organization; cluster 4 peaked at stamen primordium formation, involved in nucleic acid metabolism and chromatin remodeling; cluster 6 showed specific high expression at the sporogenous cell formation stage, mainly enriched in stress response and hormone response-related terms; Cluster 8 was highly expressed at the pollen maturation stage, significantly enriched in reproductive processes such as pollen tube growth and pollen germination. This reveals the temporal functional transition of gene expression at different developmental stages. (3) WGCNA identified three stage-specific co-expression modules (MEpink, MEmagenta, MEbrown), which were closely associated with early cell wall formation and phenylpropanoid metabolism, vesicle transport and energy supply during dormancy, and cytoskeleton organization and extracellular matrix formation during pollen maturation, respectively, confirming at the network level the synergy between module functions and developmental progression. (4) Promoter analysis showed that the promoter regions of 16 key differentially expressed transcription factors from clusters 1 and 8 were extensively enriched in light-responsive and multiple hormone-responsive cis-elements, implying that light and hormone signals may coordinately regulate male floral bud development. (5) RT-qPCR validated the expression trends of six flower-development-related genes (PtoMYC2, PtoSEP2, PtoSEP4, PtoSOC1, PtoSVP, PtoTCP9), which were highly consistent with RNA-seq data (r > 0.9). Different MADS-box family members (PtoSEP2、PtoSVP、PtoSEP4、PtoSOC1) exhibited distinct expression patterns in male floral bud development.

Conclusion During male floral bud development of P. tomentosa, gene expression changes most dramatically at the dormancy-release stage and is relatively stable during the dormancy-transition stage, indicating that dormancy release is a critical node of transcriptional reprogramming. This study provides a comprehensive dynamic transcriptome atlas of male floral bud development across eight stages in P. tomentosa, revealing stage-specific regulatory characteristics of gene expression. These findings provide a basis for understanding the stage-specific regulation of flower development in perennial woody plants and offer candidate genes for molecular breeding to control flowering time and reduce pollen allergy in Populus.