Abstract
Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) 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 chlAPX 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 chlAPX pre-mRNA is regulated by the splicing enhancer SRE.
Highlights
Chloroplasts are potentially a powerful source of oxidants, and sites within the cells are at greatest risk of photooxidative damage [1, 2]
Stem, and Root of Spinach—To determine whether the transcription level of ApxII varied in several tissues, we analyzed the steady-state transcript levels of total chloroplastic APX (chlAPX) mRNA, which were expressed as the sum of the four types of mRNA variants encoding stromal APX (sAPX) and thylakoidbound APX (tAPX), in the leaves, stems, and roots of mature spinach by Northern blotting using tAPX cDNA as a probe
Pumpkin, and M. crystallinum, four types of mRNA variants for chlAPX are produced from only one gene by alternative splicing of the 3Ј-terminal region (Fig. 1) [5, 6]
Summary
Chloroplasts are potentially a powerful source of oxidants, and sites within the cells are at greatest risk of photooxidative damage [1, 2]. Alternative splicing events in the 3-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoidbound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner.
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