Biomedical subjects

Ralph Bock

Publications and source records attributed to Ralph Bock.

29 records · Page 2Linked to original sources

PubMed2004

Rapid evolution of RNA editing sites in a small non-essential plastid gene.

Chloroplast RNA editing proceeds by C-to-U transitions at highly specific sites. Here, we provide a phylogenetic analysis of RNA editing in a small plastid gene, petL, encoding subunit VI of the cytochrome b6f complex. Analyzing representatives from most major groups of seed plants, we find an unexpectedly high frequency and dynamics of RNA editing. High-frequency editing has previously been observed in plastid ndh genes, which are remarkable in that their mutational inactivation does not produce an obvious mutant phenotype. In order to test the idea that reduced functional constraints allow for more flexible evolution of RNA editing sites, we have created petL knockout plants by tobacco chloroplast transformation. We find that, in the higher plant tobacco, targeted inactivation of petL does not impair plant growth under a variety of conditions markedly contrasting the important role of petL in photosynthesis in the green alga Chlamydomonas reinhardtii. Together with a low number of editing sites in plastid genes that are essential to gene expression and photosynthetic activity, these data suggest that RNA editing sites may evolve more readily in those genes whose transitory loss of function can be tolerated. Accumulated evidence for this 'relative neutrality hypothesis for the evolution of plastid editing sites' is discussed.

Base Sequence↗

PubMed2004

Surprising features of plastid ndhD transcripts: addition of non-encoded nucleotides and polysome association of mRNAs with an unedited start codon.

RNA editing in higher plant plastids is a post- transcriptional RNA maturation process changing single cytidine nucleotides into uridine. In the ndhD transcript of tobacco and several other plant species, editing of an ACG codon to a standard AUG initiator codon is believed to be a prerequisite for translation. In order to test this assumption experimentally, we have analyzed the editing status of ndhD mRNA species in the process of translation. We show that unedited ndhD transcripts are also associated with polysomes in vivo, suggesting that they are translated. This surprising finding challenges the view that ACG to AUG editing is strictly required to make the ndhD message translatable and raises the possibility that ACG can be utilized as an initiator codon in chloroplasts. In addition, we have mapped the termini of the ndhD transcript and discovered a novel form of RNA processing. Unexpectedly, we find that highly specific sequences are added to the 3' end of the ndhD mRNA at high frequency. We propose a model in which these sequences are added by the successive action of a CCA-adding enzyme (tRNA nucleotidyltransferase) and an RNA-dependent RNA polymerase (RdRp) activity. The presence of an RdRp activity may have general implications also for other steps in plastid gene expression.

Base Sequence↗

PubMed2004

Taming plastids for a green future.

Plant genetic engineering will probably contribute to the required continued increase in agricultural productivity during the coming decades, and moreover, plants can potentially provide inexpensive production platforms for pharmaceuticals and nutraceuticals. With the advent of technologies for altering the genetic information inside chloroplasts, a new attractive target for genetic engineering has become available to biotechnologists. Potential advantages over conventional nuclear transformation include high transgene expression levels and increased biosafety because of maternal organelle inheritance in most crops. This review summarizes the state of the art in chloroplast genetic engineering and describes how reverse genetics approaches enhance our understanding of photosynthesis and other important chloroplast functions. Furthermore, promising strategies by which chloroplast genetic engineering might contribute to the successful modification of plant metabolism are discussed.

Chloroplasts↗

PubMed2004

Studying RNA editing in transgenic chloroplasts of higher plants.

RNA editing in plant cell organelles is a posttranscriptional process that changes the identity of individual nucleotides by pyrimidine transitions. C-to-U conversions are the predominantly found modifications in both mitochondrial and chloroplast transcripts of vascular plants. Research on functional, mechanistic, and evolutionary aspects of plant RNA editing has benefited immensely from the development of transgenic technologies for higher plant chloroplasts. This chapter describes methods for the generation of plants with stably transformed chloroplast genomes and their use for studying RNA editing processes in vivo.

Biolistics↗

PubMed2003

Plastid protein synthesis is required for plant development in tobacco.

Chloroplasts fulfill important functions in cellular metabolism. The majority of plastid genome-encoded genes is involved in either photosynthesis or chloroplast gene expression. Whether or not plastid genes also can determine extraplastidic functions has remained controversial. We demonstrate here an essential role of plastid protein synthesis in tobacco leaf development. By using chloroplast transformation, we have developed an experimental system that produces recombination-based knockouts of chloroplast translation in a cell-line-specific manner. The resulting plants are chimeric and, in the presence of translational inhibitors, exhibit severe developmental abnormalities. In the absence of active plastid protein synthesis, leaf blade development is abolished because of an apparent arrest of cell division. This effect appears to be cell-autonomous in that adjacent sectors of cells with translating plastids are phenotypically normal but cannot complement for the absence of plastid translation in mutant sectors. Developmental abnormalities also are seen in flower morphology, indicating that the defects are not caused by inhibited expression of plastid photosynthesis genes. Taken together, our data point to an unexpected essential role of plastid genes and gene expression in plant development and cell division.

Blotting, Southern↗

PubMed2003

High-frequency gene transfer from the chloroplast genome to the nucleus.

Eukaryotic cells arose through endosymbiotic uptake of free-living bacteria followed by massive gene transfer from the genome of the endosymbiont to the host nuclear genome. Because this gene transfer took place over a time scale of hundreds of millions of years, direct observation and analysis of primary transfer events has remained difficult. Hence, very little is known about the evolutionary frequency of gene transfer events, the size of transferred genome fragments, the molecular mechanisms of the transfer process, or the environmental conditions favoring its occurrence. We describe here a genetic system based on transgenic chloroplasts carrying a nuclear selectable marker gene that allows the efficient selection of plants with a nuclear genome that carries pieces transferred from the chloroplast genome. We can select such gene transfer events from a surprisingly small population of plant cells, indicating that the escape of genetic material from the chloroplast to the nuclear genome occurs much more frequently than generally believed and thus may contribute significantly to intraspecific and intraorganismic genetic variation.

Base Sequence↗

PubMed2003

Evidence that cytochrome b559 mediates the oxidation of reduced plastoquinone in the dark.

The function of cytochrome b(559) in photosystem II (PSII) was investigated using a mutant created in tobacco in which the conserved phenylalanine at position 26 in the beta-subunit (PsbF) was changed to serine (Bock, R., Kössel, H., and Maliga, P. (1994) EMBO J. 13, 4623-4628). The mutant grew photoautotrophically, but the amount of PSII was reduced and the ultrastructure of the chloroplast was dramatically altered. Very few grana stacks were formed in the mutant. Although isolated PSII-enriched membrane fragments showed low PSII activity, electron paramagnetic resonance indicated the presence of functional PSII. Difference absorption spectra showed that the cytochrome b(559) contained heme. The plastoquinone pool was largely reduced in dark-adapted leaves of the mutant, based on chlorophyll fluorescence and thermoluminescence measurements. We therefore propose that cytochrome b(559) plays an important role in PSII by keeping the plastoquinone pool and thereby the acceptor side of PSII oxidized in the dark. Structural alterations as induced by the single Phe --> Ser point mutation in the transmembrane domain of PsbF evidently inhibit this function.

Chlorophyll↗

PubMed2003

The calcium sensor CBL1 integrates plant responses to abiotic stresses.

Calcium ions represent both an integrative signal and an important convergence point of many disparate signaling pathways. Calcium-binding proteins, like calcineurin B-like (CBL) proteins, have been implicated as important relays in calcium signaling. Here, we report the in vivo study of CBL1 function in Arabidopsis. Analyses of loss-of-function as well as CBL1-overexpressing lines indicate a crucial function of this calcium sensor protein in abiotic stress responses. Mutation of CBL1 impairs plant responses to drought and salt stresses and affects gene expression of cold-regulated genes, but does not affect abscisic acid (ABA) responsiveness. Conversely, overexpression of CBL1 reduces transpirational water loss and induces the expression of early stress-responsive transcription factors and stress adaptation genes in non-stressed plants. Together, our data indicate that the calcium sensor protein CBL1 may constitute an integrative node in plant responses to abiotic stimuli and contributes to the regulation of early stress-related transcription factors of the C-Repeat-Binding Factor/dehydration-responsive element (CBF/DREB) type.

Abscisic Acid↗

PubMed2002

Temperature sensitivity of RNA editing and intron splicing reactions in the plastid ndhB transcript.

Primary transcripts in higher plant cell organelles undergo a series of essential RNA processing steps, including 5'- and 3'-terminal maturation, intron splicing and RNA editing. Most of the protein factors involved in these RNA processing mechanisms are still unknown. Also, little is known about the environmental and developmental factors regulating RNA processing events in plastids. Here we have tested the influence of the environmental factor temperature on RNA processing in the ndhB mRNA from tobacco plastids. We find that, whereas temperature increase to 37 degrees C fully inhibits RNA editing at only one out of nine sites in the tobacco ndhB transcript, further temperature increase to 42 degrees C selectively blocks editing at two additional sites. As these temperature effects are strictly site-specific, our findings provide evidence for the existence of site-specific editing factors which exhibit differential temperature sensitivity. Furthermore, splicing of the group II intron present in the ndhB pre-mRNA is largely blocked at 42 degrees C, suggesting that chloroplast RNA processing steps are more sensitive to high temperature than chloroplast transcription. Our findings may also suggest that impaired plastid RNA processing contributes to the loss of chloroplast function upon plant growth at high temperature.

Hot Temperature↗

PubMed2002

Lack of the small plastid-encoded PsbJ polypeptide results in a defective water-splitting apparatus of photosystem II, reduced photosystem I levels, and hypersensitivity to light.

Photosystem II is a large pigment-protein complex catalyzing water oxidation and initiating electron transfer processes across the thylakoid membrane. In addition to large protein subunits, many of which bind redox cofactors, photosystem II particles contain a number of low molecular weight polypeptides whose function is only poorly defined. Here we have investigated the function of one of the smallest polypeptides in photosystem II, PsbJ. Using a reverse genetics approach, we have inactivated the psbJ gene in the tobacco chloroplast genome. We show that, although the PsbJ polypeptide is not principally required for functional photosynthetic electron transport, plants lacking PsbJ are unable to grow photoautotrophically. We provide evidence that this is due to the accumulation of incompletely assembled water-splitting complexes, which in turn causes drastically reduced photosynthetic performance and extreme hypersensitivity to light. Our results suggest a role of PsbJ for the stable assembly of the water-splitting complex of photosystem II and, in addition, support a control of photosystem I accumulation through photosystem II activity.

Amino Acid Sequence↗

PubMed2001

The amino acid sequence of a plastid protein is developmentally regulated by RNA editing.

RNA editing in plant organelles post-transcriptionally alters single nucleotides by C-to-U or U-to-C conversions at highly specific sites. Plant editing is generally viewed as a repair mechanism acting at the transcript level by restoring conserved amino acid residues. Here we report that an editing reaction within the ndhB transcript (encoding a plastid NAD(P)H dehydrogenase subunit) is strictly dependent on active photosynthesis. Employing non-photosynthetic mutants, we show that in the absence of photosynthesis, the site remains unedited, whereas it is fully edited when the photosynthetic apparatus is intact. Moreover, the site also remains unedited during the etiolated stage of seedling development, suggesting that two different NdhB proteins are synthesized under photosynthetic versus non-photosynthetic conditions. This is the first case where RNA editing in plants appears to regulate gene expression qualitatively, resulting in the production of two different proteins from one and the same gene in a developmental stage-dependent manner.

Base Sequence↗