The Arabidopsis mTERF-repeat MDA1 protein plays a dual function in transcription and stabilization of specific chloroplast transcripts within the psbE and ndhH operons.

The mTERF gene family encodes for nucleic acid binding proteins that are predicted to regulate organellar gene expression in eukaryotes. Despite the implication of this gene family in plant development and response to abiotic stresses, a precise molecular function was assigned to only a handful number of its c. 30 members in plants. Using a reverse genetics approach in Arabidopsis thaliana and combining molecular and biochemical techniques, we revealed new functions for the chloroplast mTERF protein, MDA1. We demonstrated that MDA1 associates in vivo with components of the plastid-encoded RNA polymerase and transcriptional active chromosome complexes. MDA1 protein binds in vivo and in vitro with specificity to 27-bp DNA sequences near the 5'-end of psbE and ndhA chloroplast genes to stimulate their transcription, and additionally promotes the stabilization of the 5'-ends of processed psbE and ndhA messenger (m)RNAs. Finally, we provided evidence that MDA1 function in gene transcription likely coordinates RNA folding and the action of chloroplast RNA-binding proteins on mRNA stabilization. Our results provide examples for the unexpected implication of DNA binding proteins and gene transcription in the regulation of mRNA stability in chloroplasts, blurring the boundaries between DNA and RNA metabolism in this organelle.

The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants.

To understand the biogenesis of the thylakoid membrane in higher plants and to identify auxiliary proteins required to build up this highly complex membrane system, we have characterized the allelic nuclear mutants high chlorophyll fluorescence222-1 (hcf222-1) and hcf222-2 and isolated the causal gene by map-based cloning. In the ethyl methanesulfonate-induced mutant hcf222-1, the accumulation of the cytochrome b6f (Cytb6f) complex was reduced to 30% compared with the wild type. Other thylakoid membrane complexes accumulated to normal levels. The T-DNA knockout mutant hcf222-2 showed a more severe defect with respect to thylakoid membrane proteins and accumulated only 10% of the Cytb6f complex, accompanied by a reduction in photosystem II, the photosystem II light-harvesting complex, and photosystem I. HCF222 encodes a protein of 99 amino acids in Arabidopsis (Arabidopsis thaliana) that has similarities to the cysteine-rich zinc-binding domain of DnaJ chaperones. The insulin precipitation assay demonstrated that HCF222 has disulfide reductase activity in vitro. The protein is conserved in higher plants and bryophytes but absent in algae and cyanobacteria. Confocal fluorescence microscopy showed that a fraction of HCF222-green fluorescent protein was detectable in the endoplasmic reticulum but that it also could be recognized in chloroplasts. A fusion construct of HCF222 containing a plastid transit peptide targets the protein into chloroplasts and was able to complement the mutational defect. These findings indicate that the chloroplast-targeted HCF222 is indispensable for the maturation and/or assembly of the Cytb6f complex and is very likely involved in thiol-disulfide biochemistry at the thylakoid membrane.

Expression of a nuclear-encoded psbH gene complements the plastidic RNA processing defect in the PSII mutant hcf107 in Arabidopsis thaliana.

The helical-repeat RNA-binding protein HCF107 is required for processing, stabilization and translation of plastid-encoded psbH mRNA. The psbH gene encodes a small, hydrophilic subunit of the PSII complex and is part of the plastidic psbB-psbT-psbH-petB-petD transcription unit. In Arabidopsis hcf107 mutants, only trace amounts of PSII proteins can be detected. Beside drastically reduced synthesis of PsbH, the synthesis of CP47 was also reduced in these mutants, although the corresponding psbB transcripts accumulate to wild type levels. This situation raises the question, whether the reduction of CP47 is a direct consequence of the mutation, based on targeting of HCF107 to the psbB mRNA, or a secondary affect due to the absent PsbH. To clarify this issue we introduced a chimeric psbH construct comprising a sequence encoding a chloroplast transit peptide into the hcf107-2 background. We found that the nucleus-localized psbH was able to complement the mutant defect resulting in photoautotrophic plants. The PSII proteins CP47 and D1 accumulated to barely half of the wild type level. Further experiments showed that cytosolically synthesized PsbH was imported into chloroplasts and assembled into PSII complexes. Using this approach, we showed that the tetratricopeptide repeat protein HCF107 of Arabidopsis is only responsible for expression of PsbH and not for synthesis of CP47. In addition the data suggest the necessity of the small, one-helix membrane spanning protein PsbH for the accumulation of CP47 in higher plants.

pAUL: a gateway-based vector system for adaptive expression and flexible tagging of proteins in Arabidopsis.

Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags.

How to build functional thylakoid membranes: from plastid transcription to protein complex assembly.

Chloroplasts are the endosymbiotic descendants of cyanobacterium-like prokaryotes. Present genomes of plant and green algae chloroplasts (plastomes) contain ~100 genes mainly encoding for their transcription-/translation-machinery, subunits of the thylakoid membrane complexes (photosystems II and I, cytochrome b (6) f, ATP synthase), and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Nevertheless, proteomic studies have identified several thousand proteins in chloroplasts indicating that the majority of the plastid proteome is not encoded by the plastome. Indeed, plastid and host cell genomes have been massively rearranged in the course of their co-evolution, mainly through gene loss, horizontal gene transfer from the cyanobacterium/chloroplast to the nucleus of the host cell, and the emergence of new nuclear genes. Besides structural components of thylakoid membrane complexes and other (enzymatic) complexes, the nucleus provides essential factors that are involved in a variety of processes inside the chloroplast, like gene expression (transcription, RNA-maturation and translation), complex assembly, and protein import. Here, we provide an overview on regulatory factors that have been described and characterized in the past years, putting emphasis on mechanisms regulating the expression and assembly of the photosynthetic thylakoid membrane complexes.

The atypical short-chain dehydrogenases HCF173 and HCF244 are jointly involved in translational initiation of the psbA mRNA of Arabidopsis.

The related proteins D1 and D2 together build up the photosystem II reaction center. Synthesis of D1 (PsbA) is highly regulated in all photosynthetic organisms. The mechanisms and specific protein factors involved in controlled expression of the psbA gene in higher plants are highly elusive. Here, we report on the identification of a chloroplast-located protein, HCF244 (for high chlorophyll fluorescence244), which is essentially required for translational initiation of the psbA messenger RNA in Arabidopsis (Arabidopsis thaliana). The factor is highly conserved between land plants, algae, and cyanobacteria. HCF244 was identified by coexpression analysis of HCF173, which encodes a protein that is also necessary for psbA translational initiation and in addition for stabilization of this messenger RNA. Phenotypic characterization of the mutants hcf244 and hcf173 suggests that the corresponding proteins operate cooperatively during psbA translation. Immunolocalization studies detected the majority of the two proteins at the thylakoid membrane. Both HCF244 and HCF173 are members of the atypical short-chain dehydrogenase/reductase superfamily, a modified group, which has lost enzyme activity but acquires new functions in the metabolism of the cell.

Recruitment of a ribosomal release factor for light- and stress-dependent regulation of petB transcript stability in Arabidopsis chloroplasts.

Land plant genomes encode four functional ribosomal peptide chain release factors (Prf) of eubacterial origin, two (PrfA and PrfB homologs) for each endosymbiotic organelle. Formerly, we have shown that the Arabidopsis thaliana chloroplast-localized PrfB homolog, PrfB1, is required not only for termination of translation but also for stabilization of UGA stop codon-containing chloroplast transcripts. A previously undiscovered PrfB-like protein, PrfB3, is localized to the chloroplast stroma in a petB RNA-containing complex and found only in vascular plants. Highly conserved positions of introns unequivocally indicate that PrfB3 arose from a duplication of PrfB1. Notably, PrfB3 is lacking the two most important tripeptide motifs characteristic for all eubacterial and organellar PrfB homologs described so far: the stop codon recognition motif SPF and the catalytic center GGQ for peptidyl-tRNA hydrolysis. Complementation studies, as well as functional and molecular analyses of two allelic mutations in Arabidopsis, both of which lead to a specific deficiency of the cytochrome b6f complex, revealed that PrfB3 is essentially required for photoautotrophic growth. Plastid transcript, polysome, and translation analyses indicate that PrfB3 has been recruited in vascular plants for light- and stress-dependent regulation of stability of 3' processed petB transcripts to adjust cytochrome b6 levels.

HCF208, a homolog of Chlamydomonas CCB2, is required for accumulation of native cytochrome b6 in Arabidopsis thaliana.

The cytochrome b(6) subunit of the cytochrome b(6)f complex is a multiheme protein. Two b-type hemes are bound non-covalently to the protein, whereas the third heme (heme c(n)) is covalently attached via an atypical thioether bond. To understand the maturation of cytochrome b(6) and to identify the assisting factors, we characterized the ethyl methanesulfonate-induced nuclear mutant hcf208. This Arabidopsis mutant shows a high chlorophyll fluorescence phenotype and does not accumulate the major cytochrome b(6)f complex subunits. Transcript levels and patterns of the four major polypeptides of the complex are equal to those of the wild type. The mutant cytochrome b(6) polypeptide shows a faster migration behavior in SDS-PAGE compared with the wild type and it has no peroxidase activity. The HCF208 locus was mapped and the gene was cloned. Sequence analysis revealed that HCF208 is a homolog of the Chlamydomonas reinhardtii CCB2 protein, which is a factor mediating attachment of heme c(n) to the cytochrome b(6) polypeptide as part of a novel heme biogenesis pathway, called system IV. Blue Native PAGE revealed residual amounts of the cytochrome b(6)f complex dimer in hcf208; however, this form is unable to participate in electron transport reactions.

The nuclear-encoded factor HCF173 is involved in the initiation of translation of the psbA mRNA in Arabidopsis thaliana.

To gain insight into the biogenesis of photosystem II (PSII) and to identify auxiliary factors required for this process, we characterized the mutant hcf173 of Arabidopsis thaliana. The mutant shows a high chlorophyll fluorescence phenotype (hcf) and is severely affected in the accumulation of PSII subunits. In vivo labeling experiments revealed a drastically decreased synthesis of the reaction center protein D1. Polysome association experiments suggest that this is primarily caused by reduced translation initiation of the corresponding psbA mRNA. Comparison of mRNA steady state levels indicated that the psbA mRNA is significantly reduced in hcf173. Furthermore, the determination of the psbA mRNA half-life revealed an impaired RNA stability. The HCF173 gene was identified by map-based cloning, and its identity was confirmed by complementation of the hcf phenotype. HCF173 encodes a protein with weak similarities to the superfamily of the short-chain dehydrogenases/reductases. The protein HCF173 is localized in the chloroplast, where it is mainly associated with the membrane system and is part of a higher molecular weight complex. Affinity chromatography of an HCF173 fusion protein uncovered the psbA mRNA as a component of this complex.

HCF153, a novel nuclear-encoded factor necessary during a post-translational step in biogenesis of the cytochrome bf complex.

We have isolated the nuclear photosynthetic mutant hcf153 which shows reduced accumulation of the cytochrome b(6)f complex. The levels and processing patterns of the RNAs encoding the cytochrome b(6)f subunits are unaltered in the mutant. In vivo protein labeling experiments and analysis of polysome association revealed normal synthesis of the large chloroplast-encoded cytochrome b(6)f subunits. The mutation resulted from a T-DNA insertion and the affected nuclear gene was cloned. HCF153 encodes a 15 kDa protein containing a chloroplast transit peptide. Sequence similarity searches revealed that the protein is restricted to higher plants. A HCF153-Protein A fusion construct introduced into hcf153 mutant plants was able to substitute the function of the wild-type protein. Fractionation of intact chloroplasts from these transgenic plants suggests that most or all of the fusion protein is tightly associated with the thylakoid membrane. Our data show that the identified factor is a novel protein that could be involved in a post-translational step during biogenesis of the cytochrome b(6)f complex. It is also possible that HCF153 is necessary for translation of one of the very small subunits of the cytochrome b(6)f complex.

RNA-binding properties of HCF152, an Arabidopsis PPR protein involved in the processing of chloroplast RNA.

The nonphotosynthetic mutant of Arabidopsis hcf152 is impaired in the processing of the chloroplast polycistronic transcript, psbB-psbT-psbH-petB-petD, resulting in nonproduction of the essential photosynthetic cytochrome b6f complex. The nucleus-encoded HCF152gene was identified to encode a pentatricopeptide repeat (PPR) protein composed primarily of 12 PPR motifs, similar to other proteins of this family that were identified in mutants defected in chloroplast gene expression. To understand the molecular mechanism of how HCF152 modulates chloroplast gene expression, the molecular and biochemical properties should be revealed. To this end, HCF152 and several truncated versions were produced in bacteria and analyzed for RNA-binding and protein-protein interaction. It was found that two HCF152 polypeptides bind to form a homodimer, and that this binding is impaired by a single amino acid substitute near the carboxyl terminus, replacing leucine with proline. Recombinant HCF152 bound with higher affinity RNA molecules, resembling the petB exon-intron junctions, as well as several other molecules. The highest affinity was found to RNA composed of the poly(A) sequence. When truncated proteins composed of different numbers of PPR motifs were analyzed for RNA-binding, it was found that two PPR motifs were required for RNA-binding, but had very low affinity. The affinity to RNA increased significantly when proteins composed of more PPR motifs were analyzed, displaying the highest affinity with the full-length protein composed of 12 PPR motifs. Together, our data characterized the nuclear-encoded HCF152 to be a chloroplast RNA-binding protein that may be involved in the processing or stabilization of the petB transcript by binding to the exon-intron junctions.

HCF152, an Arabidopsis RNA binding pentatricopeptide repeat protein involved in the processing of chloroplast psbB-psbT-psbH-petB-petD RNAs.

The psbB-psbT-psbH-petB-petD operon of higher plant chloroplasts is a heterogeneously composed transcriptional unit that undergoes complex RNA processing events until the mature oligocistronic RNAs are formed. To identify the nucleus-encoded factors required for the processing and expression of psbB-psbT-psbH-petB-petD transcripts, we performed mutational analysis using Arabidopsis. The allelic nuclear mutants hcf152-1 and hcf152-2 were identified that are affected specifically in the accumulation of the plastidial cytochrome b(6)f complex. In both mutants, reduced amounts of spliced petB RNAs (encoding the cytochrome b(6) subunit) were detected, thus explaining the observed protein deficiencies. Additionally, mutant hcf152-1 is affected in the accumulation of transcripts cleaved between the genes psbH and petB. As a result of a close T-DNA insertion, the HCF152 gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF152 encodes a pentatricopeptide repeat (PPR) protein with 12 putative PPR motifs that is located inside the chloroplast. The protein shows a significant structural, but not primary, sequence similarity to the maize protein CRP1, which is involved in the processing and translation of the chloroplast petD and petA RNAs. In addition, we found that HCF152 is an RNA binding protein that binds certain areas of the petB transcript. The protein possibly exists in the chloroplast as a homodimer and is not associated with other proteins to form a high molecular mass complex.