Identification and expression of the chloroplast clpP gene in the conifer Pinus contorta

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Abstract
The clpP gene from the conifer Pinus contorta was identified and isolated from a chloroplast genomic library by heterologous hybridisation to the second exon of the chloroplast clpP gene in tobacco. DNA sequencing of two overlapping clones revealed an uninterrupted 615 bp open-reading frame with 41 to 65% similarity to the clpP genes in five other chloroplast genomes and Escherichia coli. The 615 bp sequence in P. contorta contained perfectly matched motifs for the serine and histidine active sites of the ClpP protease in E. coli. The location of the clpP gene was determined using a physical map of the P. contorta chloroplast genome, and was found to lie within a 10 kb region between the psbE/F and rpoB genes. Sequencing of the regions adjacent to the clpP gene revealed the first exon of the rps12 gene located 135 bp downstream. The genomic position of the first exon of the rps12 gene in relation to the clpP gene is conserved for all other chloroplast clpP genes identified so far. Northern blot analysis showed that the clpP gene in both P. contorta and P. sylvestris was present in several transcript of different length, ranging from 0.8 to 2.4 kb. The two longer transcripts in P. contorta also included the first exon of the rps12 gene. Mapping of the 5′ end of the clpP transcripts by primer extension, however, revealed a single transcription initiation site 53 bp upstream of the first ATG codon. Analysis of total RNA isolated from the two pine species grown in darkness or moderate light conditions (250 μmol photons m-2 s-1) showed no significant difference in the level of expression of the clpP gene. The results suggest that the clpP gene in conifers is part of an operon which includes the first exon of the rps12 and the entire rpl20 gene, and is expressed in a light-independent manner as a polycistronic precursor which later undergoes post-transcriptional processing to give the mature monocistronic clpP mRNA.