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Ralph Bock

Publications and source records attributed to Ralph Bock.

At least 19 recordsLinked to original sources
PubMed2026

Synergistic HMGN1 and VP64 Fusions Potentiate High-Precision and PAM-Flexible Base Editing.

RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.

Gene Editing↗
PubMed2025

Reverse genetics in the Arabidopsis chloroplast genome identifies rps16 as a transcribed pseudogene.

The plastid (chloroplast) genomes of seed plants contain a conserved set of ribosomal protein genes. The rps16 gene represents an exception: It has been lost from the plastid genomes of gymnosperms and several lineages of angiosperms, and may have undergone pseudogenization in a few other lineages, including members of the Brassicaceae family. Here we report a reverse genetic approach to test the annotated rps16 gene in the Arabidopsis plastid genome for functionality. Employing the recently developed plastid transformation technology for the model plant Arabidopsis, we have deleted the putative rps16 gene from the Arabidopsis plastid genome. We report that the resulting transplastomic plants display wild-type-like growth and photosynthetic performance under a wide range of conditions. Moreover, genome-wide analyses of chloroplast transcript levels and ribosome footprints revealed unaltered plastid translational activity in Δrps16 mutants compared with wild-type plants. We conclude that the annotated rps16 gene in the plastid genome of Arabidopsis is a transcribed pseudogene that has been replaced in evolution by a nuclear gene copy that supplies functional S16 protein to chloroplasts.

Arabidopsis↗
PubMed2025

Development of chloroplast transformation for five species in the genus Nicotiana.

Technologies for the stable genetic transformation of the plastid (chloroplast) genome are currently restricted to a small number of species. The development of highly efficient tissue culture, regeneration, and selection procedures represents the major hurdle that needs to be overcome to extend the species range of the transplastomic technology. Here, we report the development of efficient plastid transformation protocols for five species in the genus Nicotiana: the model species N. benthamiana, the tree tobacco N. glauca, the ornamental plants N. langsdorffii and N. longiflora, and the wild species N. otophora. We have optimized medium composition for efficient regeneration from leaf explants in all five species and determined suitable selection conditions for plastid transformation. We successfully isolated multiple transplastomic lines for each species and also generated lines that express the fluorescent reporter protein DsRed. Molecular and genetic analyses confirmed the homoplasmic state of the transplastomic lines and demonstrated maternal inheritance of the transgenes. Our work makes plastid genome engineering available for a set of new species and enables new applications in horticultural research and ecology. It also informs the future development of plastid transformation technology for other species.

Nicotiana↗
PubMed2007

The plastome-encoded PsaJ subunit is required for efficient Photosystem I excitation, but not for plastocyanin oxidation in tobacco.

The functions of several small subunits of the large photosynthetic multiprotein complex PSI (Photosystem I) are not yet understood. To elucidate the function of the small plastome-encoded PsaJ subunit, we have produced knockout mutants by chloroplast transformation in tobacco (Nicotiana tabacum). PsaJ binds two chlorophyll-a molecules and is localized at the periphery of PSI, close to both the Lhca2- and Lhca3-docking sites and the plastocyanin-binding site. Tobacco psaJ-knockout lines do not display a visible phenotype. Despite a 25% reduction in the content of redox-active PSI, neither growth rate nor assimilation capacity are altered in the mutants. In vivo, redox equilibration of plastocyanin and PSI is as efficient as in the wild-type, indicating that PsaJ is not required for fast plastocyanin oxidation. However, PsaJ is involved in PSI excitation: altered 77 K chlorophyll-a fluorescence emission spectra and reduced accumulation of Lhca3 indicate that antenna binding and exciton transfer to the PSI reaction centre are impaired in DeltapsaJ mutants. Under limiting light intensities, growth of DeltapsaJ plants is retarded and the electron-transport chain is far more reduced than in the wild-type, indicating that PSI excitation might limit electron flux at sub-saturating light intensities. In addition to defining in vivo functions of PsaJ, our data may also have implications for the interpretation of the crystal structure of PSI.

Kinetics↗
PubMed2006

Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming.

Transgene expression from the chloroplast (plastid) genome offers several attractions to plant biotechnologists, including high-level accumulation of foreign proteins, transgene stacking in operons and a lack of epigenetic interference with the stability of transgene expression. In addition, the technology provides an environmentally benign method of plant genetic engineering, because plastids and their genetic information are maternally inherited in most crops and thus are largely excluded from pollen transmission. During the past few years, researchers in both the public and private sectors have begun to explore possible areas of application of plastid transformation in plant biotechnology as a viable alternative to conventional nuclear transgenic technologies. Recent proof-of-concept studies highlight the potential of plastid genome engineering for the expression of resistance traits, the production of biopharmaceuticals and metabolic pathway engineering in plants.

Agriculture↗
PubMed2006

Knock-out of the plastid-encoded PetL subunit results in reduced stability and accelerated leaf age-dependent loss of the cytochrome b6f complex.

The cytochrome-b6f complex, a key component of the photosynthetic electron transport chain, contains a number of very small protein subunits whose functions are not well defined. Here we have investigated the function of the 31-amino acid PetL subunit encoded in the chloroplast genome in all higher plants. Chloroplast-transformed petL knock-out tobacco plants display no obvious phenotype, suggesting that PetL is not essential for cytochrome b6f complex biogenesis and function (Fiebig, A., Stegemann, S., and Bock, R. (2004) Nucleic Acids Res. 32, 3615-3622). We show here that, whereas young mutant leaves accumulate comparable amounts of cytochrome b6f complex and have an identical assimilation capacity as wild type leaves, both cytochrome b6f complex contents and assimilation capacities of mature and old leaves are strongly reduced in the mutant, indicating that the cytochrome b6f complex is less stable than in the wild type. Reduced complex stability was also confirmed by in vitro treatments of isolated thylakoids with chaotropic reagents. Adaptive responses observed in the knockout mutants, such as delayed down-regulation of plastocyanin contents, indicate that plants can sense the restricted electron flux to photosystem I yet cannot compensate the reduced stability of the cytochrome b6f complex by adaptive up-regulation of complex synthesis. We propose that efficient cytochrome b6f complex biogenesis occurs only in young leaves and that the capacity for de novo synthesis of the complex is very low in mature and aging leaves. Gene expression analysis indicates that the ontogenetic down-regulation of cytochrome b6f complex biogenesis occurs at the post-transcriptional level.

Adaptation, Physiological↗
PubMed2006

A leaf-based regeneration and transformation system for maize (Zea mays L.).

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world's major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.

DNA, Plant↗
PubMed2006

Alternative complex formation of the Ca-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis.

Intracellular release of calcium ions belongs to the earliest events in cellular stress perception. The molecular mechanisms integrating signals from different environmental cues and translating them into an optimized response are largely unknown. We report here the functional characterization of CIPK1, a protein kinase interacting strongly with the calcium sensors CBL1 and CBL9. Comparison of the expression patterns indicates that the three proteins execute their functions in the same tissues. Physical interaction of CIPK1 with CBL1 and CBL9 targets the kinase to the plasma membrane. We show that, similarly to loss of CBL9 function, mutation of either CBL1 or CIPK1 renders plants hypersensitive to osmotic stress. Remarkably, in contrast to the cbl1 mutant and similarly to the cbl9 mutant, loss of CIPK1 function impairs abscisic acid (ABA) responsiveness. We therefore suggest that, by alternative complex formation with either CBL1 or CBL9, the kinase CIPK1 represents a convergence point for ABA-dependent and ABA-independent stress responses. Based on our genetic, physiological and protein-protein interaction data, we propose a general model for information processing in calcium-regulated signalling networks.

Abscisic Acid↗
PubMed2006

Experimental reconstruction of functional gene transfer from the tobacco plastid genome to the nucleus.

Eukaryotic cells arose through the uptake of free-living bacteria by endosymbiosis and their gradual conversion into organelles (plastids and mitochondria). Capture of the endosymbionts was followed by massive translocation of their genes to the genome of the host cell. How genes were transferred from the (prokaryotic) organellar genome to the (eukaryotic) nuclear genome and how the genes became functional in their new eukaryotic genetic environment is largely unknown. Here, we report the successful experimental reconstruction of functional gene transfer between an organelle and the nucleus, a process that normally occurs only on large evolutionary timescales. In consecutive genetic screens, we first transferred a chloroplast genome segment to the nucleus and then selected for gene activation in the nuclear genome. We show that DNA-mediated gene transfer can give rise to functional nuclear genes if followed by suitable rearrangements in the nuclear genome. Acquisition of gene function involves (1) transcriptional activation by capture of the promoter of an upstream nuclear gene and (2) utilization of AT-rich noncoding sequences downstream of the plastid gene as RNA cleavage and polyadenylation sites. Our results reveal the molecular mechanisms of how organellar DNA transferred to the nucleus gives rise to functional genes and reproduce in the laboratory a key process in the evolution of eukaryotic cells.

Base Sequence↗
PubMed2006

Deficiency in phylloquinone (vitamin K1) methylation affects prenyl quinone distribution, photosystem I abundance, and anthocyanin accumulation in the Arabidopsis AtmenG mutant.

Phylloquinone (vitamin K(1)) is synthesized in cyanobacteria and in chloroplasts of plants, where it serves as electron carrier of photosystem I. The last step of phylloquinone synthesis in cyanobacteria is the methylation of 2-phytyl-1,4-naphthoquinone by the menG gene product. Here, we report that the uncharacterized Arabidopsis gene At1g23360, which shows sequence similarity to menG, functionally complements the Synechocystis menG mutant. An Arabidopsis mutant, AtmenG, carrying a T-DNA insertion in the gene At1g23360 is devoid of phylloquinone, but contains an increased amount of 2-phytyl-1,4-naphthoquinone. Phylloquinone and 2-phytyl-1,4-naphthoquinone in thylakoid membranes of wild type and AtmenG, respectively, predominantly localize to photosystem I, whereas excess amounts of prenyl quinones are stored in plastoglobules. Photosystem I reaction centers are decreased in AtmenG plants under high light, as revealed by immunoblot and spectroscopic measurements. Anthocyanin accumulation and chalcone synthase (CHS1) transcription are affected during high light exposure, indicating that alterations in photosynthesis in AtmenG affect gene expression in the nucleus. Photosystem II quantum yield is decreased under high light. Therefore, the loss of phylloquinone methylation affects photosystem I stability or turnover, and the limitation in functional photosystem I complexes results in overreduction of photosystem II under high light.

Anthocyanins↗
PubMed2006

Tobacco plastid ribosomal protein S18 is essential for cell survival.

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Deltarps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.

Cell Survival↗
PubMed2006

Identification of small non-coding RNAs from mitochondria and chloroplasts.

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.

Animals↗
PubMed2006

Sequence of the tomato chloroplast DNA and evolutionary comparison of solanaceous plastid genomes.

Tomato, Solanum lycopersicum (formerly Lycopersicon esculentum), has long been one of the classical model species of plant genetics. More recently, solanaceous species have become a model of evolutionary genomics, with several EST projects and a tomato genome project having been initiated. As a first contribution toward deciphering the genetic information of tomato, we present here the complete sequence of the tomato chloroplast genome (plastome). The size of this circular genome is 155,461 base pairs (bp), with an average AT content of 62.14%. It contains 114 genes and conserved open reading frames (ycfs). Comparison with the previously sequenced plastid DNAs of Nicotiana tabacum and Atropa belladonna reveals patterns of plastid genome evolution in the Solanaceae family and identifies varying degrees of conservation of individual plastid genes. In addition, we discovered several new sites of RNA editing by cytidine-to-uridine conversion. A detailed comparison of editing patterns in the three solanaceous species highlights the dynamics of RNA editing site evolution in chloroplasts. To assess the level of intraspecific plastome variation in tomato, the plastome of a second tomato cultivar was sequenced. Comparison of the two genotypes (IPA-6, bred in South America, and Ailsa Craig, bred in Europe) revealed no nucleotide differences, suggesting that the plastomes of modern tomato cultivars display very little, if any, sequence variation.

Amino Acid Sequence↗
PubMed2006

Evolutionary origin of a plant mitochondrial group II intron from a reverse transcriptase/maturase-encoding ancestor.

Group II introns are widespread in plant cell organelles. In vivo, most if not all plant group II introns do not self-splice but require the assistance of proteinaceous splicing factors. In some cases, a splicing factor (also referred to as maturase) is encoded within the intronic sequence and produced by translation of the (excised) intron RNA. However, most present-day group II introns in plant organellar genomes do not contain open reading frames (ORFs) for splicing factors, and their excision may depend on proteins encoded by other organellar introns or splicing factors encoded in the nuclear genome. Whether or not the ancestors of all of these noncoding organellar introns originally contained ORFs for maturases is currently unknown. Here we show that a noncoding intron in the mitochondrial cox2 gene of seed plants is likely to be derived from an ancestral reverse transcriptase/maturase-encoding form. We detected remnants of maturase and reverse transcriptase sequences in the 2.7 kb cox2 intron of Ginkgo biloba, the only living species of an ancient gymnosperm lineage, suggesting that the intron originally harbored a splicing factor. This finding supports the earlier proposed hypothesis that the ancient group II introns that invaded organellar genomes were autonomous genetic entities in that they encoded the factor(s) required for their own excision and mobility.

Amino Acid Sequence↗
PubMed2006

Elimination of deleterious mutations in plastid genomes by gene conversion.

Asexual reproduction is believed to be detrimental, mainly because of the accumulation of deleterious mutations over time, a hypothesis known as Muller's ratchet. In seed plants, most asexually reproducing genetic systems are polyploid, with apomictic species (plants forming seeds without fertilization) as well as plastids and mitochondria providing prominent examples. Whether or not polyploidy helps asexual genetic systems to escape Muller's ratchet is unknown. Gene conversion, particularly when slightly biased, represents a potential mechanism that could allow asexual genetic systems to reduce their mutation load in a genome copy number-dependent manner. However, direct experimental evidence for the operation of gene conversion between genome molecules to correct mutations is largely lacking. Here we describe an experimental system based on transgenic tobacco chloroplasts that allows us to analyze gene conversion events in higher plant plastid genomes. We provide evidence for gene conversion acting as a highly efficient mechanism by which the polyploid plastid genetic system can correct deleterious mutations and make one good genome out of two bad ones. Our finding that gene conversion can be biased may provide a molecular link between asexual reproduction, high genome copy numbers and low mutation rates.

Base Sequence↗
PubMed2005

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants.

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.

Arabidopsis↗
PubMed2005

Cytoplasm and chloroplasts are not suitable subcellular locations for beta-zein accumulation in transgenic plants.

Zeins, the main storage proteins of maize that accumulate in the endoplasmic reticulum of the endosperm cells, are particularly interesting because they are rich in the essential sulphur amino acids. Overexpression of certain zein genes in plants such as alfalfa would be expected to improve the nutritional characteristics of this crop. Recently, significant accumulation values have been reached, but still far from those considered useful for nutritional purposes. This study investigates whether targeting to compartments other than the endoplasmic reticulum (cytosol and chloroplasts) could result in increasing beta-zein accumulation in transgenic plants. To address beta-zein to the cytosol, the fragment which codes for the signal peptide has been removed. beta-zein has also been targeted to alfalfa and tobacco chloroplasts by a transit peptide signal. Both tobacco, as a model plant species, and alfalfa have been transformed with the assembled constructs. An alternative route to accumulate beta-zein in the chloroplasts is to synthesize beta-zein directly in the plastid lumen. Thus, the beta-zein gene has also been inserted into tobacco plastid DNA. The beta-zein gene in each different type of transformed plant was properly transcribed, as determined by northern blot analysis, but no accumulation of beta-zein was detected, either in the cytoplasm or in the chloroplasts of alfalfa and tobacco transformed plants. Therefore, it is concluded that chloroplasts and the cytosol are not favourable subcellular locations for zein protein accumulation.

Animals↗