Evolutionary logic and biotechnological potential of alternative splicing in higher eukaryotes: Comparative insights from plants and animals.

Alternative splicing (AS) generates transcript and protein isoforms, but its evolutionary significance cannot be inferred from isoform number alone. This review uses a comparative evidence framework to separate splice-variant origin, functional consequence, and evolutionary retention in plants and animals. We synthesize evidence from long-read sequencing, single-cell isoform profiling, pangenomics, proteogenomics, and perturbation studies. Animal studies emphasize exon-level switches in neural, cardiac, immune, and germline programs, whereas plant studies highlight intron retention, exitron splicing, environmental responses, and polyploidy-associated regulation. These are tendencies shaped by biological and sampling differences, not exclusive properties of either kingdom. Three worked examples illustrate how reproducible regulation, conservation or population evidence, molecular consequences, and targeted perturbation support different levels of inference. Functional effects alone do not establish historical adaptation, and conservation alone does not identify the trait under selection. The framework helps prioritize isoforms for crop improvement, biomarkers, and RNA intervention. Clinical splice modulation and field-evaluated natural rice alleles demonstrate translational potential, while highlighting the distinction between validated outcomes and prospective engineering. Future studies should connect isoform-resolved measurements to causal perturbation and fitness or agronomic performance across relevant environments.