An oligonucleotide/oligosaccharide-binding-fold protein enhances the alternative splicing event producing thylakoid membrane-bound ascorbate peroxidase in Nicotiana tabacum

The stromal and thylakoid membrane-bound ascorbate peroxidase isoforms are produced by the alternative splicing event of the 3′-terminal region of the APXII gene in spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum), but not in Arabidopsis (Arabidopsis thaliana). However, all alternative splicing variants were detected in APXII gene-transformed Arabidopsis, indicating the occurrence of its regulatory mechanisms in Arabidopsis. The efficiency of this alternative splicing event in producing thylakoid membrane-bound ascorbate peroxidase mRNA is regulated by a splicing regulatory cis element, but trans splicing regulatory factor(s) for alternative splicing remain unclear. To identify this factor, we conducted a forward genetic screen using Arabidopsis in combination with a luciferase reporter system to evaluate the alternative splicing efficiency of thylakoid membrane-bound ascorbate peroxidase mRNA production. We isolated 9 mutant lines that showed low efficiency of the AS in producing thylakoid membrane-bound ascorbate peroxidase mRNA compared with that in the control plants. From one mutant [APXII alternative splicing inhibition (apsi1)], the causal gene responsible for the phenotype, AT5G38890 (oligonucleotide/oligosaccharide-binding-fold protein, APSI1), was identified. The levels of thylakoid membrane-bound ascorbate peroxidase mRNA from the transformed APXII gene decreased and increased in APSI1 knockout and APSI1-overexpressing plants, respectively. APSI1 was localized to the nucleus and specifically bound to the splicing regulatory cis element sequence. Tobacco plants that disrupted the closest homologs of APSI1 showed low levels of endogenous thylakoid membrane-bound ascorbate peroxidase mRNA. These results indicate that APSI1 is an enhancing component of the alternative splicing event of APXII.