Transcriptome analysis of a spontaneous male-sterile mutant in Betula platyphylla and functional characterization of the key gene BpSPL2 in regulating inflorescence development.

key messageA key gene, BpSPL2, identified from male-sterile mutants of Betula platyphylla, was demonstrated to play a crucial role in regulating male inflorescence development. Pollen represents a significant allergen in birch (Betula platyphylla Suk.). Its male inflorescence develops over nearly one year and overwinters as uninucleate microspores. Therefore, elucidating the molecular basis of male sterility in this species is essential for breeding pollen-free varieties. In this study, we used wild-type (WT) plants and two types of male inflorescences (WLM and MM) derived from the same spontaneous mutant to perform phenotypic observations and transcriptome analyses at three key developmental stages: microspore mother cell, tetrad/microspore, and mature pollen before pollination. Both WLM and MM displayed impaired pollen grain maturation and defective anther dehiscence at stage 6. As floral development progressed, the numbers of differentially expressed genes (DEGs) between the mutant inflorescences and WT increased markedly. KEGG enrichment analysis of these DEGs revealed that pathways, such as phenylpropanoid biosynthesis and ABC transporters, may play critical roles in birch male flower development. GO enrichment identified 31 candidate genes linked to pollen exine formation, sporopollenin biosynthetic process, and anther development (e.g., BpSPL8: SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8, BpAPY7: Apyrase 7, BpCYP703A2: CYTOCHROME P450 703A2). Phylogenetic and co-expression analyses identified BpSPL2 as a core regulator. Overexpression of BpSPL2 arrested microsporogenesis at the microsporocyte stage, preventing tetrad formation. The arrested cells subsequently degraded during overwintering, ultimately yielding no microspores or pollen. Conversely, BpSPL2 suppression promoted microsporogenesis, and caused inflorescence and anther enlargement. These results establish BpSPL2 as a key temporal regulator of microsporogenesis in B. platyphylla. This study systematically investigated the potential molecular mechanisms underlying two distinct abnormal male flower phenotypes on a spontaneous mutant individual and elucidated the central regulatory function of BpSPL2 in male flower development. These findings provide a theoretical foundation and genetic resources for breeding pollen-free varieties of B. platyphylla.