An Overview of Pentatricopeptide Repeat (PPR) Proteins in the Moss Physcomitrium patens and Their Role in Organellar Gene Expression.

Pentatricopeptide repeat (PPR) proteins are one type of helical repeat protein that are widespread in eukaryotes. In particular, there are several hundred PPR members in flowering plants. The majority of PPR proteins are localized in the plastids and mitochondria, where they play a crucial role in various aspects of RNA metabolism at the post-transcriptional and translational steps during gene expression. Among the early land plants, the moss Physcomitrium (formerly Physcomitrella) patens has at least 107 PPR protein-encoding genes, but most of their functions remain unclear. To elucidate the functions of PPR proteins, a reverse-genetics approach has been applied to P. patens. To date, the molecular functions of 22 PPR proteins were identified as essential factors required for either mRNA processing and stabilization, RNA splicing, or RNA editing. This review examines the P. patens PPR gene family and their current functional characterization. Similarities and a diversity of functions of PPR proteins between P. patens and flowering plants and their roles in the post-transcriptional regulation of organellar gene expression are discussed.

Diurnal control of intracellular distributions of PAS-Histidine kinase 1 and its interactions with partner proteins in the moss Physcomitrium patens.

In multi-step phosphorelay (MSP) signaling, upon reception of various environmental signals, histidine kinases (HKs) induce autophosphorylation and subsequent phosphotransfer to partner histidine-containing phosphotransfer proteins (HPts). Recently, we reported that (i) two Per-Arnt-Sim (PAS) domain-containing HKs (PHK1 and PHK2) of the moss Physcomitrium (Physcomitrella) patens suppressed red light-induced branching of protonema tissue, and (ii) they interacted with partner HPts (HPt1 and HPt2) in the nucleus in the dark while cytoplasmic interactions also occurred under red light. Here we demonstrate that PHK1 is diurnally regulated, i.e., it is localized and interacts with HPt1 and HPt2 in the nucleus at night whereas these activities reversibly expand and become nucleocytoplasmic in the day. In the dark, PHK1 interacts with HPts only in the nucleus, even in subjective daytime, indicating that endogenous regulation by the circadian clock is not involved. These results suggest that PHK1 is a regulator of moss' adaptation to a light environment on a daily timescale. We discuss a possible regulatory mechanism for the branching of protonema.

Physcomitrium patens pentatricopeptide repeat protein PpPPR_32 is involved in the accumulation of psaC mRNA encoding the iron sulfur protein of photosystem I.

Pentatricopeptide repeat (PPR) proteins are involved in RNA metabolism and also play a role in posttranscriptional regulation during plant organellar gene expression. Although a hundred of PPR proteins exist in the moss Physcomitrium patens, their functions are not fully understood. Here, we report the function of P-class PPR protein PpPPR_32 in P. patens. A transient expression assay using green fluorescent protein demonstrated that the N-terminal region of PpPPR_32 functions as a chloroplast-targeting transit peptide, indicating that PpPPR_32 is localized in chloroplasts. PpPPR_32 knockout mutants grew autotrophically but with reduced protonema growth and the poor formation of photosystem I (PSI) complexes. Quantitative real-time reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in the transcript level of the psaC gene encoding the iron sulfur protein of PSI but no alteration to the transcript levels of other PSI genes. This suggests that PpPPR_32 is specifically involved in the expression level of the psaC gene. Our results indicate that PpPPR_32 is essential for the accumulation of psaC transcript and PSI complexes.

Red light-regulated interaction of Per-Arnt-Sim histidine kinases with partner histidine-containing phosphotransfer proteins in Physcomitrium patens.

Multi-step phosphorelay (MSP) is a broadly distributed signaling system in organisms. In MSP, histidine kinases (HKs) receive various environmental signals and transmit them by autophosphorylation followed by phosphotransfer to partner histidine-containing phosphotransfer proteins (HPts). Previously, we reported that Per-Arnt-Sim (PAS) domain-containing HK1 (PHK1) and PHK2 of the moss Physcomitrium (Physcomitrella) patens repressed red light-induced protonema branching, a critical step in the moss life cycle. In plants, PHK homolog-encoding genes are conserved only in early-diverging lineages such as bryophytes and lycophytes. PHKs-mediated signaling machineries attract attention especially from an evolutionary viewpoint, but they remain uninvestigated. Here, we studied the P. patens PHKs focusing on their subcellular patterns of localization and interaction with HPts. Yeast two-hybrid analysis, a localization assay with a green fluorescent protein, and a bimolecular fluorescence complementation analysis together showed that PHKs are localized and interact with partner HPts mostly in the nucleus, as unprecedented features for plant HKs. Additionally, red light triggered the interactions between PHKs and HPts in the cytoplasm, and light co-repressed the expression of PHK1 and PHK2 as well as genes encoding their partner HPts. Our results emphasize the uniqueness of PHKs-mediated signaling machineries, and functional implications of this uniqueness are discussed.

Plastid Transformation in Physcomitrium (Physcomitrella) patens: An Update.

The moss Physcomitrium (Physcomitrella) patens performs efficient homologous recombination in both the nucleus and plastid enabling the study of individual gene function by generating precise inactivation or modification of genes. Polyethylene glycol (PEG)-mediated transformation of protoplasts is routinely used to study the nuclear gene function of P. patens. PEG-mediated protoplast transformation is also applied for plastid transformation of this moss. The efficiency of plastid transformation is quite reliable and one or two homoplasmic transplastomic lines are obtained in a plastid transformation experiment (5 × 105 protoplasts) by selection for spectinomycin resistance.

Moss PPR-SMR protein PpPPR_64 influences the expression of a psaA-psaB-rps14 gene cluster and processing of the 23S-4.5S rRNA precursor in chloroplasts.

KEY MESSAGE: Moss PPR-SMR protein PpPPR_64 is a pTAC2 homolog but is functionally distinct from pTAC2. PpPPR_64 is required for psaA gene expression and its function may have evolved in mosses. The pentatricopeptide repeat (PPR) proteins are key regulatory factors responsible for the control of plant organellar gene expression. A small subset of PPR proteins possess a C-terminal small MutS-related (SMR) domain and have diverse roles in plant organellar biogenesis. However, the function of PPR-SMR proteins is not fully understood. Here, we report the function of PPR-SMR protein PpPPR_64 in the moss Physcomitrium patens. Phylogenetic analysis indicated that PpPPR_64 belongs to the same clade as the Arabidopsis PPR-SMR protein pTAC2. PpPPR_64 knockout (KO) mutants grew autotrophically but with reduced protonemata growth and the poor formation of photosystems' antenna complexes. Quantitative reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in transcript levels of the psaA-psaB-rps14 gene cluster but no alteration to transcript levels of most photosynthesis- and non-photosynthesis-related genes. In addition, RNA processing of 23S-4.5S rRNA precursor was impaired in the PpPPR_64 KO mutants. This suggests that PpPPR_64 is specifically involved in the expression level of the psaA-psaB-rps14 gene and in processing of the 23S-4.5S rRNA precursor. Our results indicate that PpPPR_64 is functionally distinct from pTAC2 and is a novel PPR-SMR protein required for proper chloroplast biogenesis in P. patens.

Substitutional RNA Editing in Plant Organelles.

RNA editing by cytidine (C) to uridine (U) conversions frequently occurs in land plant mitochondria and plastids. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins in a sequence-specific manner. In the moss Physcomitrella patens, all PPR editing factors possess the DYW-deaminase domain at the C-terminus. Here, we describe methods for the direct sequencing of cDNA to detect RNA editing events and the RNA electrophoresis mobility shift assay (REMSA) to analyze the specific binding of PPR editing factors to their target RNA.

Two Novel PLS-Class Pentatricopeptide Repeat Proteins Are Involved in the Group II Intron Splicing of Mitochondrial Transcripts in the Moss Physcomitrella patens.

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that function in posttranscriptional regulation as gene-specific regulators of RNA metabolism in plant organelles. Plant PPR proteins are divided into four classes: P, PLS, E and DYW. The E- and DYW-class proteins are mainly implicated in RNA editing, whereas most of the P-class proteins predominantly participate in RNA cleavage, splicing and stabilization. In contrast, the functions of PLS-class proteins still remain obscure. Here, we report the function of PLS-class PpPPR_31 and PpPPR_9 in Physcomitrella patens. The knockout (KO) mutants of PpPPR_31 and PpPPR_9 exhibited slower protonema growth compared to the wild type. The PpPPR_31 KO mutants showed a considerable reduction in the splicing of nad5 intron 3 and atp9 intron 1. The PpPPR_9 KO mutants displayed severely reduced splicing of cox1 intron 3. An RNA electrophoresis mobility shift assay showed that the recombinant PpPPR_31 protein bound to the 5' region of nad5 exon 4 and the bulged A region in domain VI of atp9 group II intron 1 while the recombinant PpPPR_9 bound to the translated region of ORF622 in cox1 intron 3. These results suggest that a certain set of PLS-class PPR proteins may influence the splicing efficiency of mitochondrial group II introns.

The L motifs of two moss pentatricopeptide repeat proteins are involved in RNA editing but predominantly not in RNA recognition.

Pentatricopeptide repeat (PPR) proteins, composed of PPR motifs repeated in tandem, are sequence-specific RNA binding proteins. Recent bioinformatic studies have shown that the combination of polar amino acids at positions 5 and last in each PPR motif recognizes RNA bases, and an RNA recognition code for PPR proteins has been proposed. Subsequent studies confirmed that the P (canonical length) and S (short) motifs bind to specific nucleotides according to this code. However, the contribution of L (long) motifs to RNA recognition is mostly controversial, owing to the presence of a nonpolar amino acid at position 5. The PLS-class PPR protein PpPPR_56 is a mitochondrial RNA editing factor in the moss Physcomitrella patens. Here, we performed in vitro RNA binding and in vivo complementation assays with PpPPR_56 and its variants containing mutated L motifs to investigate their contributions to RNA recognition. In vitro RNA binding assay showed that the original combination of amino acids at positions 5 and last in the L motifs of PpPPR_56 is not required for RNA recognition. In addition, an in vivo complementation assay with RNA editing factors PpPPR_56 and PpPPR_78 revealed the importance of nonpolar amino acids at position 5 of C-terminal L motifs for efficient RNA editing. Our findings suggest that L motifs function as non-binding spacers, not as RNA-binding motifs, to facilitate the formation of a complex between PLS-class PPR protein and RNA. As a result, the DYW domain, a putative catalytic deaminase responsible for C-to-U RNA editing, is correctly placed in proximity to C, which is to be edited.

The P-class pentatricopeptide repeat protein PpPPR_21 is needed for accumulation of the psbI-ycf12 dicistronic mRNA in Physcomitrella chloroplasts.

Chloroplast gene expression is controlled by numerous nuclear-encoded RNA-binding proteins. Among these, pentatricopeptide repeat (PPR) proteins are known to be key players in post-transcriptional regulation in chloroplasts. However, the functions of many PPR proteins remain unknown. In this study, we characterized the function of a chloroplast-localized P-class PPR protein PpPPR_21 in Physcomitrella patens. Knockout (KO) mutants of PpPPR_21 exhibited reduced protonemata growth and lower photosynthetic activity. Immunoblot analysis and blue-native gel analysis showed a remarkable reduction of the photosystem II (PSII) reaction center protein and poor formation of the PSII supercomplexes in the KO mutants. To assess whether PpPPR_21 is involved in chloroplast gene expression, chloroplast genome-wide microarray analysis and Northern blot hybridization were performed. These analyses indicated that the psbI-ycf12 transcript encoding the low molecular weight subunits of PSII did not accumulate in the KO mutants while other psb transcripts accumulated at similar levels in wild-type and KO mutants. A complemented PpPPR_21KO moss transformed with the cognate full-length PpPPR_21cDNA rescued the level of accumulation of psbI-ycf12 transcript. RNA-binding experiments showed that the recombinant PpPPR_21 bound efficiently to the 5' untranslated and translated regions of psbImRNA. The present study suggests that PpPPR_21 may be essential for the accumulation of a stable psbI-ycf12mRNA.

Light-regulated PAS-containing histidine kinases delay gametophore formation in the moss Physcomitrella patens.

Two-component systems (TCSs) are signal transduction mechanisms for responding to various environmental stimuli. In angiosperms, TCSs involved in phytohormone signaling have been intensively studied, whereas there are only a few reports on TCSs in basal land plants. The moss Physcomitrella patens possesses several histidine kinases (HKs) that are lacking in seed plant genomes. Here, we studied two of these unique HKs, PAS-histidine kinase 1 (PHK1) and its paralog PHK2, both of which have PAS (Per-Arnt-Sim) domains, which are known to show versatile functions such as sensing light or molecular oxygen. We found homologs of PHK1 and PHK2 only in early diverged clades such as bryophytes and lycophytes, but not in seed plants. The PAS sequences of PHK1 and PHK2 are more similar to a subset of bacterial PAS sequences than to any angiosperm PAS sequences. Gene disruption lines that lack either PHK1 or PHK2 or both formed gametophores earlier than the wild-type, and consistently, more caulonema side branches were induced in response to light in the disruption lines. Therefore, PHK1 and PHK2 delay the timing of gametophore development, probably by suppressing light-induced caulonema branching. This study provides new insights into the evolution of TCSs in plants.

The DYW Domains of Pentatricopeptide Repeat RNA Editing Factors Contribute to Discriminate Target and Non-Target Editing Sites.

In land plant organelles, many transcripts are modified by cytidine to uridine RNA editing. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins via their sequence-specific RNA-binding motifs. In the moss Physcomitrella patens, all PPR editing factors have C-terminal E and DYW domains. To examine the contribution of E and DYW domains in RNA editing, we performed a complementation assay using mutated PpPPR_56 and PpPPR_71, which are responsible for mitochondrial editing sites. This assay showed that both E and DYW domains are required for RNA editing at the target sites, and that the conserved zinc-binding signature and the terminal triplet of the DYW domain are essential for editing. In addition, DYW domain-swapping experiments demonstrated that DYW domains are functionally different between PpPPR_56 and other mitochondrial PPR editing factors, and that residues 37-42 of the DYW domain are involved in site-specific editing. Our results suggest that PPR-DYW proteins specifically recognize their target editing sites via PPR motifs and the DYW domain.

An evolutionarily conserved P-subfamily pentatricopeptide repeat protein is required to splice the plastid ndhA transcript in the moss Physcomitrella patens and Arabidopsis thaliana.

Pentatricopeptide repeat (PPR) proteins are known to play important roles in post-transcriptional regulation in plant organelles. However, the function of the majority of PPR proteins remains unknown. To examine their functions, Physcomitrella patens PpPPR_66 knockout (KO) mutants were generated and characterized. The KO mosses exhibited a wild-type-like growth phenotype but showed aberrant chlorophyll fluorescence due to defects in chloroplast NADH dehydrogenase-like (NDH) activity. Immunoblot analysis suggested that disruption of PpPPR_66 led to a complete loss of the chloroplast NDH complex. To examine whether the loss of PpPPR_66 affects the expression of plastid ndh genes, the transcript levels of 11 plastid ndh genes were analyzed by reverse transcription PCR. This analysis indicated that splicing of the ndhA transcript was specifically impaired while mRNA accumulation levels as well as the processing patterns of other plastid ndh genes were not affected in the KO mutants. Complemented PpPPR_66 KO lines transformed with the PpPPR_66 full-length cDNA rescued splicing of the ndhA transcript. Arabidopsis thaliana T-DNA tagged lines of a PPR_66 homolog (At2 g35130) showed deficient splicing of the ndhA transcript. This indicates that the two proteins are functionally conserved between bryophytes and vascular plants. An in vitro RNA-binding assay demonstrated that the recombinant PpPPR_66 bound preferentially to the region encompassing a part of exon 1 to a 5' part of the ndhA group II intron. Taken together, these results indicate that PpPPR_66 acts as a specific factor to splice ndhA pre-mRNA.

The 5' untranslated region of the rbp1 mRNA is required for translation of its mRNA under low temperatures in the cyanobacterium Synechococcus elongatus.

The unicellular cyanobacterium Synechococcus elongatus has three RNA-binding protein (Rbp) genes, rbp1, rbp2 and rbp3. The rbp1 gene was upregulated by cold treatment while rbp2 and rbp3 expression decreased remarkably after exposure to cold temperatures. To investigate the mechanism underlying cold-induced rbp1 expression, a series of rbp1-luxAB transcriptional fusion constructs were expressed in S. elongatus PCC 7942 under cold conditions. The results showed that the region from -33 to -3 of the transcription initiation site contains an essential sequence for basal transcription of the rbp1 gene and that the 120-bp region (-34 to -153) does not contain critical cis-elements required for cold-shock induction. In contrast, mutational analysis carrying the 5'-untranslated region (UTR) of rbp1-luxAB translational fusions indicated that the 5'-UTR of rbp1 plays an important role in cold induction of the rbp1 gene product. Taken together, we conclude that the cold induction of rbp1 may be regulated at a posttranscriptional level rather than at the transcriptional level.

RNA Editing and Its Molecular Mechanism in Plant Organelles.

RNA editing by cytidine (C) to uridine (U) conversions is widespread in plant mitochondria and chloroplasts. In some plant taxa, "reverse" U-to-C editing also occurs. However, to date, no instance of RNA editing has yet been reported in green algae and the complex thalloid liverworts. RNA editing may have evolved in early land plants 450 million years ago. However, in some plant species, including the liverwort, Marchantia polymorpha, editing may have been lost during evolution. Most RNA editing events can restore the evolutionarily conserved amino acid residues in mRNAs or create translation start and stop codons. Therefore, RNA editing is an essential process to maintain genetic information at the RNA level. Individual RNA editing sites are recognized by plant-specific pentatricopeptide repeat (PPR) proteins that are encoded in the nuclear genome. These PPR proteins are characterized by repeat elements that bind specifically to RNA sequences upstream of target editing sites. In flowering plants, non-PPR proteins also participate in multiple RNA editing events as auxiliary factors. C-to-U editing can be explained by cytidine deamination. The proteins discovered to date are important factors for RNA editing but a bona fide RNA editing enzyme has yet to be identified.

P-class pentatricopeptide repeat protein PTSF1 is required for splicing of the plastid pre-tRNA(I) (le) in Physcomitrella patens.

Pentatricopeptide repeat (PPR) proteins are widely distributed in eukaryotes and are mostly localized in mitochondria or plastids. PPR proteins play essential roles in various RNA processing steps in organelles; however, the function of the majority of PPR proteins remains unknown. To examine the function of plastid PPR proteins, PpPPR_4 gene knock-out mutants were characterized in Physcomitrella patens. The knock-out mosses displayed severe growth retardation and reduced effective quantum yield of photosystem II. Immunoblot analysis showed that knock-out of PpPPR_4 resulted in a strongly reduced level of plastid-encoded proteins, such as photosystem II reaction center protein D1, the ß subunit of ATP synthase, and the stromal enzyme, Rubisco. To further investigate whether knock-out of the PpPPR_4 gene affects plastid gene expression, we analyzed steady-state transcript levels of protein- and rRNA-coding genes by quantitative RT-PCR. This analysis showed that the level of many protein-coding transcripts increased in the mutants. In contrast, splicing of a spacer tRNA(I) (le) precursor encoded by the rrn operon was specifically impaired in the mutants, whereas the accumulation of other plastid tRNAs and rRNAs was not largely affected. Thus, the defect in tRNA(I) (le) splicing leads to a considerable reduction of mature tRNA(I) (le) , which may be accountable for the reduced protein level. An RNA mobility shift assay showed that the recombinant PpPPR_4 bound preferentially to domain III of the tRNA(I) (le) group-II intron. These results provide evidence that PpPPR_4 functions in RNA splicing of the tRNA(I) (le) intron, and hence PpPPR_4 was named plastid tRNA splicing factor 1 (PTSF1).

Plastid genome sequences of Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia reveal remarkable structural conservation among chlorarachniophyte species.

Chlorarachniophyte algae have complex plastids acquired by the uptake of a green algal endosymbiont, and this event is called secondary endosymbiosis. Interestingly, the plastids possess a relict endosymbiont nucleus, referred to as the nucleomorph, in the intermembrane space, and the nucleomorphs contain an extremely reduced and compacted genome in comparison with green algal nuclear genomes. Therefore, chlorarachniophyte plastids consist of two endosymbiotically derived genomes, i.e., the plastid and nucleomorph genomes. To date, complete nucleomorph genomes have been sequenced in four different species, whereas plastid genomes have been reported in only two species in chlorarachniophytes. To gain further insight into the evolution of endosymbiotic genomes in chlorarachniophytes, we newly sequenced the plastid genomes of three species, Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia. Our findings reveal that chlorarachniophyte plastid genomes are highly conserved in size, gene content, and gene order among species, but their nucleomorph genomes are divergent in such features. Accordingly, the current architecture of the plastid genomes of chlorarachniophytes evolved in a common ancestor, and changed very little during their subsequent diversification. Furthermore, our phylogenetic analyses using multiple plastid genes suggest that chlorarachniophyte plastids are derived from a green algal lineage that is closely related to Bryopsidales in the Ulvophyceae group.

Diversity of plant circadian clocks: Insights from studies of Chlamydomonas reinhardtii and Physcomitrella patens.

Arabidopsis thaliana has long been the model plant of choice for elucidating the mechanisms of the circadian clock. Recently, relevant results have accumulated in other species of green plant lineages, including green algae. This mini-review describes a comparison of the mechanism of the A. thaliana clock to those of the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens, focusing on commonalities and divergences of subsystems of the clock. The potential of such an approach from an evolutionary viewpoint is discussed.

Molecular characterization of three PRORP proteins in the moss Physcomitrella patens: nuclear PRORP protein is not essential for moss viability.

RNase P is a ubiquitous endonuclease that removes the 5' leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5'-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5'-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNAAsp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNA(Arg) (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNA(Arg) (CCG) and pre-tRNA(Arg) (UCU) but they cleaved pre-tRNA(Arg) (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical.