Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves

We have previously shown that stromal and thylakoid-bound ascorbate peroxid ase (APX) isoenzymes of spinach chloroplasts arise from a common pre-mRNA b y alternative splicing in the C-terminus of the isoenzymes [Ishikawa, Yoshi mura, Tamoi, Takeda and Shigeoka (1997) Biochem. J. 328, 795-800]. To explo re the production of mature, functional mRNA encoding chloroplast APPX isoe nzymes, reverse transcriptase-mediated PCR and S1 nuclease protection analy sis were performed with poly(A)(+) RNA or polysomal RNA from spinach leaves . As a result, four mRNA variants, one form of thylakoid-bound APX (tAPX-I) and three forms of stromal APX (sAPX-I, sAPX-II and sAPX-III), were identi fied. The sAPX-I and sAPX-III mRNA species were generated through the excis ion of intron 11; they encoded the previously identified sAPX protein. Inte restingly, the sAPX-II mRNA was generated by the insertion of intron 11 bet ween exons 11 and 12. The use of this insertional sequence was in frame wit h the coding sequence and would lead to the production of a novel isoenzyme containing a C-terminus in which a seven-residue sequence replaced the las t residue of the previously identified sAPX. The recombinant novel enzyme e xpressed in Escherichia coli showed the same enzymic properties (except for molecular mass) as the recombinant sAPX from the previously identified sAP X-I mRNA, suggesting that the protein translated from the sAPX-II mRNA is f unctional as a soluble APX in vivo. The S1 nuclease protection analysis sho wed that the expression levels of mRNA variants for sAPX and tAPX isoenzyme s are in nearly equal quantities throughout the spinach leaves grown under normal conditions. The present results demonstrate that the expression of c hloroplast APX isoenzymes is regulated by a differential splicing efficienc y that is dependent on the 3'-terminal processing of ApxII, the gene encodi ng the chloroplast APX isoenzymes.