Cotton is a valuable cash crop in many countries. Cotton fiber is a trichome that develops from a single epidermal cell and serves as an excellent model for understanding cell differentiation and other life processes. Alternative splicing (AS) of genes is a common post-transcriptional regulatory process in plants that is essential for plant growth and development. The process of AS during cotton fiber formation, on the other hand, is mainly unknown. A substantial number of multi-exon genes were discovered to be alternatively spliced during cotton fiber formation in this study, accounting for 23.31% of the total number of genes in Gossypium hirsutum. Retention intron (RI) is not necessarily the most common AS type, indicating that AS genes and processes during fiber development are very temporal and tissue-specific. When compared to fiber samples, AS is more prevalent at the fiber initiation stages and in the ovule, indicating that development stages and tissues use different AS strategies. Genes involved in fiber development have gone through stage-specific AS, demonstrating that AS regulates cotton fiber development. Furthermore, AS can be regulated by trans-regulation elements such as splicing factor and cis-regulation elements such as gene length, exon numbers, and GC content, particularly at exon–intron junction sites. Our findings also suggest that increased DNA methylation may aid in the efficiency of AS, and that gene body methylation is key in AS control. Finally, our research will provide useful information about the roles of AS during the cotton fiber development process.

The paper mulberry is a commonly found tree species with a long history of cultivation. It also serves as a crucial case study for understanding how woody plants adapt to low temperatures. Under cold treatment, we observed a substantial number of alternative splicing (AS) genes, showcasing the intricate landscape of AS events. We have detected all seven types of AS events, with the alternative 3′ splice site (A3) having the most. We observed that many genes that underwent differential AS were significantly enriched in starch and sucrose metabolism and circadian rhythm pathways. Moreover, a considerable proportion of differentially spliced genes (DSGs) also showed differential expression, with 20.38% and 25.65% under 12 h and 24 h cold treatments, respectively. This suggests a coordinated regulation between gene AS and expression, playing a pivotal role in the paper mulberry’s adaptation to cold stress. We further investigated the regulatory mechanisms of AS, identifying 41 serine/arginine-rich (SR) splicing factors, among which 11 showed differential expression under cold treatment, while 29 underwent alternative splicing. Additionally, genes undergoing AS displayed significantly higher DNA methylation levels under cold stress, while normal splicing (non-AS) genes exhibited relatively lower methylation levels. These findings suggest that methylation may play an important role in governing gene AS. Finally, our research will provide useful information on the role of AS in the cold acclimation tolerance of the paper mulberry.