Habitat-specific alternative splicing shifts and the 5mC methylation landscape of differentially spliced genes in Riccia fluitans.

Phenotypic plasticity in amphibious plants was considered a key evolutionary strategy for survival in fluctuating environments; however, the epigenetic mechanisms and post-transcriptional regulations governing the transition between aquatic and terrestrial life forms were previously poorly understood. In the present study, the interplay between 5mC DNA methylation and alternative splicing (AS) was investigated in Riccia fluitans, a species exhibiting distinct morphological and transcriptomic adaptations to different habitats. To achieve this, Illumina short-read sequencing was integrated with Oxford Nanopore Technologies direct RNA and direct DNA long-read sequencing. A total of 19,045 AS events were detected, comprising 4,927 A3SS, 3,580 A5SS, 169 MXE, 5,814 RI, and 4,555 SE. Among these, 1,251 events were found to be specific to the water form, 845 events were identified as specific to the land form, and 448 events were determined to differ significantly between the two forms. Furthermore, it was demonstrated by 5mC DNA methylation profiling that methylation conserved in both forms was concentrated in CG motifs, whereas habitat-specific methylation was found to occur primarily in the dynamic CHH context, with higher levels being reached in the water form. A decrease in methylation levels in the flanking regions of genes undergoing shifts in AS was observed, which was interpreted as an indication that environment-dependent reprogramming of 5mC methylation co-occurred with the regulation of AS.