Alexandra-Viola Bohne

RAP, the Sole Octotricopeptide Repeat Protein in Arabidopsis , Is Required for Chloroplast 16S rRNA Maturation

Chloroplast gene expression is mainly regulated by nucleus-encoded helical repeat proteins, like PPR, TPR, and OPR proteins. This study identifies a molecular role for RAP, the only OPR protein in Arabidopsis, in chloroplast 16S rRNA maturation. Furthermore, it provides evidence that the nucleoid is the relevant suborganellar location for rRNA maturation in the chloroplast. The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5′ region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

Reciprocal regulation of protein synthesis and carbon metabolism for thylakoid membrane biogenesis.

A subunit of the chloroplast pyruvate dehydrogenase complex, which serves as a metabolic enzyme, also has a dual function as an RNA-binding protein and influences mRNA translation.

An intermolecular disulfide‐based light switch for chloroplast psbD gene expression in Chlamydomonas reinhardtii

Expression of the chloroplast psbD gene encoding the D2 protein of the photosystem II reaction center is regulated by light. In the green alga Chlamydomonas reinhardtii, D2 synthesis requires a high-molecular-weight complex containing the RNA stabilization factor Nac2 and the translational activator RBP40. Based on size exclusion chromatography analyses, we provide evidence that light control of D2 synthesis depends on dynamic formation of the Nac2/RBP40 complex. Furthermore, 2D redox SDS-PAGE assays suggest an intermolecular disulfide bridge between Nac2 and Cys11 of RBP40 as the putative molecular basis for attachment of RBP40 to the complex in light-grown cells. This covalent link is reduced in the dark, most likely via NADPH-dependent thioredoxin reductase C, supporting the idea of a direct relationship between chloroplast gene expression and chloroplast carbon metabolism during dark adaption of algal cells.

Development of photosynthetic biomaterials for in vitro tissue engineering

Tissue engineering has opened a new therapeutic avenue that promises a revolution in regenerative medicine. To date, however, the translation of engineered tissues into clinical settings has been highly limited and the clinical results are often disappointing. Despite decades of research, the appropriate delivery of oxygen into three-dimensional cultures still remains one of the biggest unresolved problems for in vitro tissue engineering. In this work, we propose an alternative source of oxygen delivery by introducing photosynthetic scaffolds. Here we demonstrate that the unicellular and photosynthetic microalga Chlamydomonas reinhardtii can be cultured in scaffolds for tissue repair; this microalga shows high biocompatibility and photosynthetic activity. Moreover, Chlamydomonas can be co-cultured with fibroblasts, decreasing the hypoxic response under low oxygen culture conditions. Finally, results showed that photosynthetic scaffolds are capable of producing enough oxygen to be independent of external supply in vitro. The results of this study represent the first step towards engineering photosynthetic autotrophic tissues.

Quantitative LC–MS Provides No Evidence for m6dA or m4dC in the Genome of Mouse Embryonic Stem Cells and Tissues

Until recently, it was believed that the genomes of higher organisms contain, in addition to the four canonical DNA bases, only 5-methyl-dC (m5 dC) as a modified base to control epigenetic processes. In recent years, this view has changed dramatically with the discovery of 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNA from stem cells and brain tissue. N6 -methyldeoxyadenosine (m6 dA) is the most recent base reported to be present in the genome of various eukaryotic organisms. This base, together with N4 -methyldeoxycytidine (m4 dC), was first reported to be a component of bacterial genomes. In this work, we investigated the levels and distribution of these potentially epigenetically relevant DNA bases by using a novel ultrasensitive UHPLC-MS method. We further report quantitative data for m5 dC, hmdC, fdC, and cadC, but we were unable to detect either m4 dC or m6 dA in DNA isolated from mouse embryonic stem cells or brain and liver tissue, which calls into question their epigenetic relevance.

The nucleoid as a site of rRNA processing and ribosome assembly

Protein biosynthesis is one of the key elements of gene expression in living cells. All proteins are synthesized on ribonucleoprotein complexes, the ribosomes. In bacteria and their derivatives—eukaryotic mitochondria and plastids—ribosomes consist of a small and a large subunit which together comprise more than 50 ribosomal proteins and usually two to four ribosomal RNAs (rRNAs). Synthesis of the required rRNAs and proteins, and their correct folding, maturation/modification and assembly into functional particles are highly coordinated. However, whereas ribosomal composition and many mechanistic aspects of their biogenesis are well understood, little is known about the spatial organization of the procedure in bacteria and organelles. In eukaryotes, the individual processes involved occur in defined regions of the cell: ribosomal proteins are synthesized in the cytosol, but most rRNAs are transcribed, processed and modified in the nucleolus, a distinct subnuclear compartment (Lafontaine and Tollervey, 2001; Boisvert et al., 2007). Only the transcription of the small 5S rRNA occurs in the nucleoplasm. After their synthesis, ribosomal proteins and assembly factors are imported into the nucleus, where they are combined with the appropriate rRNAs. Subsequently, small and large ribosomal subunits are exported into the cytosol, where they pair up to form functional ribosomes.

Chloroplast Gene Expression—Translation

Translation has often been shown to represent the rate-limiting step of chloroplast gene expression. Genetic and biochemical analyses indicate that numerous nucleus-encoded protein factors in concert with their cognate target sites on chloroplast mRNAs are involved in determining protein-specific synthesis rates. In this chapter, we summarize the constituents of the chloroplast translational apparatus as well as the molecular principles underlying its spatiotemporal regulation.

Metabolic Control of Chloroplast Gene Expression: An Emerging Theme

In all organisms, regulation of gene expression plays an essential part in acclimation to changes in the environment and the metabolic status of cells. However, little is known about the mechanisms by which nutritional and metabolic signals mediate this control. Nonetheless, it is clear that bacterial and eukaryotic gene expression is modulated by carbon metabolism (Wellen and Thompson, 2012; Baranska et al., 2013), and that DNA replication and/or transcription are adjusted in accordance with the demands of the organism as reflected in the levels of specific metabolites.

Plastid Transformation in Algae

Pioneering work from the late 1980s using the green alga Chlamydomonas reinhardtii has paved the way for biolistic chloroplast transformation in general. Since then, the continuous development of a molecular toolkit has made this chlorophyte alga the prime organism for algal transplastomic biotechnological applications. However, comparatively little progress has been made with the stable genetic manipulation of members of other algal groups with the red alga Porphyridium UTEX 637 representing a rare exception. In this chapter, we summarize the basic molecular principles of chloroplast transformation in algae as well as current approaches to optimize foreign gene expression in Chlamydomonas.