PPR proteins of green algae

Abstract

Using the repeat finding algorithm FT-Rep, we have identified 154 pentatricopeptide repeat (PPR) proteins in nine fully sequenced genomes from green algae (with a total of 1201 repeats) and grouped them in 47 orthologous groups. All data are available in a database, PPRdb, accessible online at http://giavap-genomes.ibpc.fr/ppr. Based on phylogenetic trees generated from the repeats, we propose evolutionary scenarios for PPR proteins. Two PPRs are clearly conserved in the entire green lineage: MRL1 is a stabilization factor for the rbcL mRNA, while HCF152 binds in plants to the psbH-petB intergenic region. MCA1 (the stabilization factor for petA) and PPR7 (a short PPR also acting on chloroplast mRNAs) are conserved across the entire Chlorophyta. The other PPRs are clade-specific, with evidence for gene losses, duplications, and horizontal transfer. In some PPR proteins, an additional domain found at the C terminus provides clues as to possible functions. PPR19 and PPR26 possess a methyltransferase_4 domain suggesting involvement in RNA guanosine methylation. PPR18 contains a C-terminal CBS domain, similar to the CBSPPR1 protein found in nucleoids. PPR16, PPR29, PPR37, and PPR38 harbor a SmR (MutS-related) domain similar to that found in land plants pTAC2, GUN1, and SVR7. The PPR-cyclins PPR3, PPR4, and PPR6, in addition, contain a cyclin domain C-terminal to their SmR domain. PPR31 is an unusual PPR-cyclin containing at its N terminus an OctotricoPeptide Repeat (OPR) and a RAP domain. We consider the possibility that PPR proteins with a SmR domain can introduce single-stranded nicks in the plastid chromosome.

Keywords: chloroplast; cyclin; evolution; green algae; mitochondrion; pentatricopeptide repeat; small MutS-related; tRNA methyltransferase.

Figure 1. Sequence logos of the PPR repeats in Chlorophyta (top panel, based on 1201 sequences) and Arabidopsis (lower panel; 5000 sequences).

Figure 2. Sub-tree extracted from the phylogenetic tree of PPR proteins computed from average inter-repeat distances, showing the relationship between the MT4-domain containing PPR19 and PPR26, and the relationship to PPR20 and PPR1/HCF152. PPR repeats are indicated by red boxes, Methyltransferase_4 domains by blue boxes, Zn-finger CCCH domains in PPR19 by white dots.

Figure 3. Sub-trees from the average-distance tree of PPR proteins showing the relationship between PPR17 and PPR18 (A) and between PPR24 and PPR25 (B). (B) also shows PPR14_MCA1. For clarity, Ostreococcus proteins have been indicated in green. Note the C-terminal domains in PPR18 (CBS) and PPR17/24 (SmR).

Figure 4. Multiple sequence alignment of the putative targets of MCA1, as listed in Table 2. Nucleotides matching the sequence GAGAAGAAAA are written in red and putative -10 box promoter consensus are written in blue. The petA initiation codon, when close enough, is boxed.

Figure 5. (A) Schematic comparison of the distribution of the PPR repeats in the MCA1 protein of Chlorophyceae vs. Trebouxiophyceae. The phylogenetically related repeats are connected by arrows. Key residues critical for the nucleotide recognition are indicated, as well as the putative target sequence (in red) as deduced from the ?PPR code.? (B) Alignment of the petA 5?UTRs from O. tauri and M. pusilla RCC299. The arrow points to the 3? end of the trnQ, located immediately upstream of petA and the petA initiation codon is boxed. A region conserved between the two organisms is written in bold and underlined. The MCA1 binding site in Chlamydomonas is shown below for comparison.

Figure 6. Multiple sequence alignment of the HCF152-C domain of PPR1/HCF152 proteins. The number of residues trimmed from the alignment at the C terminus is indicated in parentheses. For sequences marked by a *, we used manually curated gene models because those in the official annotation were C-terminally truncated. The Pro residue mutated in hcf152-2 is marked by an arrow.

Figure 7. Phylogenetic tree of the SmR and SmR-like domains of PPR-SmR and PPR-cyclins (names in green and yellow, respectively), along with representative SmR domains from subfamilies 1 (white), 2 (blue), and 3 (purple). E. coli YdaL was used as an outgroup.

Figure 8. (A) A section of the average-distance PPR protein tree showing the grouping of PPR-cyclins. (B) Phylogenetic tree generated using only the cyclin domains of PPR-cyclins. Note that they form a clade distinct from those of cytoplasmic cyclins.

Figure 9. Phylogenetic trees (A) of the MT4 domains of PPR-MT4 along with representative TrmB and trm8 proteins; (B) of the PPR domains in PPR-MT4 proteins. Note that in both trees PPR19 tends to group with the PPR-MT4 of secondary endosymbionts, and PPR26 with the PPR-MT4 of Amoebas.

Figure 10. Excerpts from the PPR repeat tree, showing examples of probable intra-protein repeat duplication, in PPR22 (A) and in PPR20 (B).