Overexpression of the clpP 5′-untranslated region in a chimeric context causes a mutant phenotype, suggesting competition for a clpP-specific RNA maturation factor in tobacco chloroplasts

The plastid ribosomal RNA (rrn) operon promoter was fused with DNA segments encoding the leader sequence (5'-untranslated region [UTR]) of plastid mRNAs to compare their efficiency in mediating translation of a bacterial protein neomycin phosphotransferase (NPTII) in tobacco (Nicotiana tabacum) chloroplasts. In young leaves, NPTII accumulated at 0.26% and 0.8% of the total soluble leaf protein from genes with the clpP and atpB 5'-UTR, respectively. Interestingly, expression of NPTII from the promoter with the clpP 5'-UTR (0.26% NPTII) caused a mutant (chlorotic) phenotype, whereas plants accumulating approximately 0.8% NPTII from the atpB 5'-UTR were normal green, indicating that the mutant phenotype was independent of NPTII accumulation. Low levels of monocistronic clpP mRNA and accumulation of intron-containing clpP transcripts in the chlorotic leaves suggest competition between the clpP 5'-UTR in the chimeric transcript and the native clpP pre-mRNA (ratio 16:1) for an mRNA maturation factor. Because maturation of 11 other intron-containing mRNAs was unaffected in the chlorotic leaves, it appears that the factor is ClpP specific. The mutant phenotype is correlated with reduced levels (approximately 2 times) of the ClpP1 protease subunit, supporting an important role for ClpP1 in chloroplast development.