ALTERNATIVE MESSENGER-RNA SPLICING OF 3'-TERMINAL EXONS GENERATES ASCORBATE PEROXIDASE ISOENZYMES IN SPINACH (SPINACIA-OLERACEA) CHLOROPLASTS

We have isolated two cDNA clones encoding spinach (Spinacia oleracea) stromal and thylakoid-bound ascorbate peroxidase isoenzymes [Ishikawa, Sakai, Yoshimura, Takeda and Shigeoka (1996) FEES Lett. 384, 289-293] . The gene (ApxII) encoding both chloroplastic ascorbate peroxidase is oenzymes was isolated and the organization of the gene was determined. Alignment between the cDNAs and the gene for chloroplastic ascorbate peroxidase isoenzymes indicates that both enzymes arise from a common pre-mRNA by alternative splicing of two 3'-terminal exons. Genomic Sou thern-blot analysis supported this finding. The gene spanned nearly 8. 5 kbp and contained 13 exons split by 12 introns. The penultimate exon 12 (residues 7376-7530) for the stromal ascorbate peroxidase mRNA con sisted of one codon for Asp(365) before the TAA termination codon, and the entire 3'-untranslated region, including a potential polyadenylat ion signal (AATAAA). The final exon 13 (residues 7545-7756) for the th ylakoid-bound ascorbate peroxidase mRNA consisted of the corresponding coding sequence of the hydrophobic C-terminal region, the TGA termina tion codon and the entire 3'-untranslated region, including a potentia l polyadenylation signal (AATATA). Both exons were interrupted by a 14 bp non-coding sequence. Northern-blot and reverse transcription-PCR a nalysis showed that the transcripts for stromal and thylakoid-bound as corbate peroxidase are present in spinach leaves.