Abstract

Pentatricopeptide repeat (PPR) proteins are encoded by the nuclear genome as a large gene family in land plants. PPR proteins play essential roles in organelle-related functions, mostly in RNA-processing steps in plastids and mitochondria. In the moss Physcomitrella patens, there is also a large gene family, but the moss PPR proteins are likely to be divergent from those of higher plants. To investigate the function of plastid PPR proteins, we have generated and characterized a PPR protein gene disruptant of P. patens. The PPR531-11-disrupted mosses displayed abnormal phenotypic characteristics, such as a significantly smaller protonemal colony, different chloroplast morphology, and incomplete thylakoid membrane formation. In addition, the quantum yield of photosystem II was reduced in the disrupted mosses. To further investigate whether disruption of the PPR531-11 gene affects chloroplast gene expression, we performed Northern blot and reverse transcription polymerase chain reaction analyses. These analyses revealed that PPR531-11 has a role in intergenic RNA cleavage between clpP and 5'-rps12 and in the splicing of clpP pre-mRNA. Western blot analysis showed that disruption of PPR531-11 resulted in a reduced level of ClpP, photosystem II reaction center protein D1, and the stromal enzyme, ribulose-bisphosphate carboxylase/oxygenase. These reductions might result in the severely retarded growth of the protonemal colony. Taken together, we propose a model where PPR531-11 function affects the steady-state level of ClpP, which regulates the formation and maintenance of thylakoid membranes in chloroplasts. This is the first evidence of a PPR protein controlling the protein expression level of ClpP.

Highlights

  • Tetratricopeptide repeat motif, a well characterized protein interaction motif that is composed of 34 amino acids [2]

  • Many chloroplast genes of land plants are cotranscribed as polycistronic pre-RNAs, which are extensively processed into shorter mature RNA species [13]

  • Structure of the PPR531-11 Gene— In a previous study [3], we showed that two PPR proteins, PPR513-10 and PPR566-6, are plastid-localized in P. patens

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Summary

A Novel PPR Protein in Moss

PPR531-11 (an isoform of PPR513-10) protein gene disruptant. A chloroplast PPR gene disrupted moss displayed abnormal phenotypic characteristics, such as significantly retarded growth of the protonemal colonies and smaller chloroplasts with an abnormal thylakoid membrane structure. Disruption of PPR531-11 resulted in aberrant RNA processing of clpP pre-mRNA. This is the first identification of an RNA species targeted by the moss chloroplast PPR protein

EXPERIMENTAL PROCEDURES
RESULTS
Results in Significantly Reduced
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