The enigma of introns: Intronic miRNA-directed mechanisms and alternative splicing diversify alternative oxidase potential in Vitis vinifera

Alternative oxidase (AOX) transcript levels were associated with efficiently balanced respiration and validated to assist selection on multiple-resilience. Consistently, AOX has also been identified as a target for weakening the survival capabilities of parasites and microorganisms responsible for severe human diseases. Despite the unique features of AOX in Vitis vinifera, particularly intense constitutive expression of AOX2 in the presence of unusually large introns that challenge the dogma of gene expression in eukaryotes, V. vinifera has been overlooked in AOX research. This study uncovered two distinct alternative splicing variants of the AOX: AOX1a-Alternative variant attributed to unusual retention of the intron-4 in the 3´UTR, and AOX2-alternative variant, which is intron-1-dependent, involving the skipping of exon-1. The AOX2-alternative variant differed in that cystine-I changed to serine, which is linked to different metabolite stimulation. However, molecular docking suggested that AOX2 and the variant proteins exhibit the same catalytic activities and binding affinities for ubiquinol. The unique large introns in AOX2 exhibited 16 miRNAs, including the master regulator of development and stress responses, mir-398. Among these, nine were conserved and validated in other plant species, whereas seven were considered potential novel miRNA candidates. Transcriptome analyses revealed down- and up-regulation of AOX1a-Alternative during shrivelling and water deficiency, and up-regulation of AOX2-Alternative with increasing temperatures. Consistent with previous studies, AOX1a and AOX1d were linked to biotic and abiotic stress, whereas AOX2 showed constitutive or developmental regulation. This study encourages hypothesis-driven advanced research on early mechanisms and functionality of newly discovered alternative splicing events and intronic miRNAs. Given functional marker-assisted breeding, it strengthened the requirement to consider overall AOX transcript levels as markers for predicting multiple-resilient phenotypes.