Abstract
Alternative polyadenylation (APA) is a widespread co-transcriptional mechanism that regulates gene expression in growth, development and environmental responses. Pollen development is essential for the reproductive success of flowering plants, yet the contribution of APA to this process remains poorly understood. Here, we combine bulk RNA-seq in multiple tissues with single-nucleus transcriptomics across pollen developmental stages to systematically characterize APA dynamics during Arabidopsis thaliana pollen development. We show that mature pollen exhibits the most tissue-specific APA profile among the examined tissues, characterized by widespread 3' untranslated region (3' UTR) shortening. At single-nucleus resolution, APA patterns display pronounced temporal and cell-type specificity, particularly during the transition from bicellular to tricellular pollen and during vegetative nucleus maturation. Sperm nuclei exhibit the most distinct poly(A) site usage patterns. Moreover, genetic analyses of representative genes showed that altered poly(A) site usage is associated with changes in transcript abundance and pollen development phenotypes. Consistent with these observations, in vivo reporter assays showed that 3' UTR configurations are sufficient to modulate gene expression at the transcript level. Together, our study establishes APA as a structured co-transcriptional regulatory layer during pollen development and provides a framework for understanding 3' end-mediated gene regulation in male gametophytes.