Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-mRNA in higher plants

Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (ch1APX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element, (SRE), which is highly conserved in the sequences of ch1APX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of ch1APX pre-mRNA is regulated by the splicing enhancer SRE.