Regulation of tissue-specific alternative splicing of chloroplastic ascorbate peroxidase isoenzymes in spinach

Abstract

In higher plants, four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, -III) encoding stromal APX (sAPX), are produced by the alternative splicing of the 3¿-terminal region [Yoshimura et al. (1999) Biochem. J., 338, 41-48]. The same observation is also true in the cases of chloroplastic ascorbate peroxidases (chlAPX) isoenzymes in pumpkin, M. crystallinum, and tobacco, indicating that the alternative splicing of chlAPX isoenzymes is a universal regulation mechanism in higher plants. To explore the regulation mechanism of alternative splicing, the expression of chlAPX isoenzymes in the leaf, stem, and root of spinach was analyzed. The expression ratio of four types of chlAPX mRNA variants was different in a tissue-specific manner, whereas the transcript levels of total chlAPX were almost equal in each tissue. The expression ratio of sAPX mRNAs (sAPX-I, -II, -III) to tAPX mRNA was almost equal in leaf; in root, however, the ratio of sAPX mRNAs to tAPX mRNA was greatly enhanced due to the decrease in tAPX-I mRNA and the increase in sAPX-III mRNA. A putative splicing regulatory cis-element (SRE), which was highly conserved in the sequences of chlAPX genes from higher plants, was identified. In fact, gel-shift analysis showed a tissue-specific interaction between SRE and a nuclear protein prepared from each tissue. These results indicate that the tissue-specific alternative splicing of chlAPX isoenzymes is regulated by SRE as a splicing enhancer. We are progressing in the identification of a trans-acting factor to regulate the alternative splicing mechanism.