Wolfram Thiele

Rpl33, a Nonessential Plastid-Encoded Ribosomal Protein in Tobacco, Is Required under Cold Stress Conditions

Plastid genomes contain a conserved set of genes encoding components of the translational apparatus. While knockout of plastid translation is lethal in tobacco (Nicotiana tabacum), it is not known whether each individual component of the plastid ribosome is essential. Here, we used reverse genetics to test whether several plastid genome–encoded ribosomal proteins are essential. We found that, while ribosomal proteins Rps2, Rps4, and Rpl20 are essential for cell survival, knockout of the gene encoding ribosomal protein Rpl33 did not affect plant viability and growth under standard conditions. However, when plants were exposed to low temperature stress, recovery of Rpl33 knockout plants was severely compromised, indicating that Rpl33 is required for sustaining sufficient plastid translation capacity in the cold. These findings uncover an important role for plastid translation in plant tolerance to chilling stress.

ATP Synthase Repression in Tobacco Restricts Photosynthetic Electron Transport, CO2 Assimilation, and Plant Growth by Overacidification of the Thylakoid Lumen

The authors use a transgenic approach to show that a close adjustment of ATP synthase activity to linear electron flux is essential for fine-tuning the proton motive force. If ATP synthase activity is too low, lumen overacidification restricts linear electron flux and initiates photoprotective mechanisms (nonphotochemical quenching) in low light, diminishing the quantum efficiency of CO2 fixation. Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b6f complex to the metabolic demand for ATP and NADPH. While the cytochrome b6f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclear-encoded γ-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastid-encoded atpB gene (encoding the essential β-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b6f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO2 fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed.

The plastid‐specific ribosomal proteins of Arabidopsis thaliana can be divided into non‐essential proteins and genuine ribosomal proteins

Plastid translation occurs on bacterial-type 70S ribosomes consisting of a large (50S) subunit and a small (30S) subunit. The vast majority of plastid ribosomal proteins have orthologs in bacteria. In addition, plastids also possess a small set of unique ribosomal proteins, so-called plastid-specific ribosomal proteins (PSRPs). The functions of these PSRPs are unknown, but, based on structural studies, it has been proposed that they may represent accessory proteins involved in translational regulation. Here we have investigated the functions of five PSRPs using reverse genetics in the model plant Arabidopsis thaliana. By analyzing T-DNA insertion mutants and RNAi lines, we show that three PSRPs display characteristics of genuine ribosomal proteins, in that down-regulation of their expression led to decreased accumulation of the 30S or 50S subunit of the plastid ribosomes, resulting in plastid translational deficiency. In contrast, two other PSRPs can be knocked out without visible or measurable phenotypic consequences. Our data suggest that PSRPs fall into two types: (i) PSRPs that have a structural role in the ribosome and are bona fide ribosomal proteins, and (ii) non-essential PSRPs that are not required for stable ribosome accumulation and translation under standard greenhouse conditions.

The Plastid Genome-Encoded Ycf4 Protein Functions as a Nonessential Assembly Factor for Photosystem I in Higher Plants

Photosystem biogenesis in the thylakoid membrane is a highly complicated process that requires the coordinated assembly of nucleus-encoded and chloroplast-encoded protein subunits as well as the insertion of hundreds of cofactors, such as chromophores (chlorophylls, carotenoids) and iron-sulfur clusters. The molecular details of the assembly process and the identity and functions of the auxiliary factors involved in it are only poorly understood. In this work, we have characterized the chloroplast genome-encoded ycf4 (for hypothetical chloroplast reading frame no. 4) gene, previously shown to encode a protein involved in photosystem I (PSI) biogenesis in the unicellular green alga Chlamydomonas reinhardtii. Using stable transformation of the chloroplast genome, we have generated ycf4 knockout plants in the higher plant tobacco (Nicotiana tabacum). Although these mutants are severely affected in their photosynthetic performance, they are capable of photoautotrophic growth, demonstrating that, different from Chlamydomonas, the ycf4 gene product is not essential for photosynthesis. We further show that ycf4 knockout plants are specifically deficient in PSI accumulation. Unaltered expression of plastid-encoded PSI genes and biochemical analyses suggest a posttranslational action of the Ycf4 protein in the PSI assembly process. With increasing leaf age, the contents of Ycf4 and Y3IP1, another auxiliary factor involved in PSI assembly, decrease strongly, whereas PSI contents remain constant, suggesting that PSI is highly stable and that its biogenesis is restricted to young leaves.

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-b6 f 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 b6 f 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 b6 f complex and have an identical assimilation capacity as wild type leaves, both cytochrome b6 f complex contents and assimilation capacities of mature and old leaves are strongly reduced in the mutant, indicating that the cytochrome b6 f 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 b6 f complex by adaptive up-regulation of complex synthesis. We propose that efficient cytochrome b6 f 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 b6 f complex biogenesis occurs at the post-transcriptional level.

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.

LCAA, a Novel Factor Required for Magnesium Protoporphyrin Monomethylester Cyclase Accumulation and Feedback Control of Aminolevulinic Acid Biosynthesis in Tobacco

Low Chlorophyll Accumulation A (LCAA) antisense plants were obtained from a screen for genes whose partial down-regulation results in a strong chlorophyll deficiency in tobacco (Nicotiana tabacum). The LCAA mutants are affected in a plastid-localized protein of unknown function, which is conserved in cyanobacteria and all photosynthetic eukaryotes. They suffer from drastically reduced light-harvesting complex (LHC) contents, while the accumulation of all other photosynthetic complexes per leaf area is less affected. As the disturbed accumulation of LHC proteins could be either attributable to a defect in LHC biogenesis itself or to a bottleneck in chlorophyll biosynthesis, chlorophyll synthesis rates and chlorophyll synthesis intermediates were measured. LCAA antisense plants accumulate magnesium (Mg) protoporphyrin monomethylester and contain reduced protochlorophyllide levels and a reduced content of CHL27, a subunit of the Mg protoporphyrin monomethylester cyclase. Bimolecular fluorescence complementation assays confirm a direct interaction between LCAA and CHL27. 5-Aminolevulinic acid synthesis rates are increased and correlate with an increased content of glutamyl-transfer RNA reductase. We suggest that LCAA encodes an additional subunit of the Mg protoporphyrin monomethylester cyclase, is required for the stability of CHL27, and contributes to feedback-control of 5-aminolevulinic acid biosynthesis, the rate-limiting step of chlorophyll biosynthesis.

Elimination of a group II intron from a plastid gene causes a mutant phenotype

Group II introns are found in bacteria and cell organelles (plastids, mitochondria) and are thought to represent the evolutionary ancestors of spliceosomal introns. It is generally believed that group II introns are selfish genetic elements that do not have any function. Here, we have scrutinized this assumption by analyzing two group II introns that interrupt a plastid gene (ycf3) involved in photosystem assembly. Using stable transformation of the plastid genome, we have generated mutant plants that lack either intron 1 or intron 2 or both. Interestingly, the deletion of intron 1 caused a strong mutant phenotype. We show that the mutants are deficient in photosystem I and that this deficiency is directly related to impaired ycf3 function. We further show that, upon deletion of intron 1, the splicing of intron 2 is strongly inhibited. Our data demonstrate that (i) the loss of a group II intron is not necessarily phenotypically neutral and (ii) the splicing of one intron can depend on the presence of another.

Inducible repression of nuclear-encoded subunits of the cytochrome b6f complex in tobacco reveals an extraordinarily long lifetime of the complex

De novo biogenesis of the cytochrome b6f complex is restricted to young leaves of tobacco. The biogenesis of the cytochrome b6f complex in tobacco (Nicotiana tabacum) seems to be restricted to young leaves, suggesting a high lifetime of the complex. To directly determine its lifetime, we employed an ethanol-inducible RNA interference (RNAi) approach targeted against the essential nuclear-encoded Rieske protein (PetC) and the small M subunit (PetM), whose function in higher plants is unknown. Young expanding leaves of both PetM and PetC RNAi transformants bleached rapidly and developed necroses, while mature leaves, whose photosynthetic apparatus was fully assembled before RNAi induction, stayed green. In line with these phenotypes, cytochrome b6f complex accumulation and linear electron transport capacity were strongly repressed in young leaves of both RNAi transformants, showing that the M subunit is as essential for cytochrome b6f complex accumulation as the Rieske protein. In mature leaves, all photosynthetic parameters were indistinguishable from the wild type even after 14 d of induction. As RNAi repression of PetM and PetC was highly efficient in both young and mature leaves, these data indicate a lifetime of the cytochrome b6f complex of at least 1 week. The switch-off of cytochrome b6f complex biogenesis in mature leaves may represent part of the first dedicated step of the leaf senescence program.

The Thylakoid Membrane Protein CGL160 Supports CF1CF0 ATP Synthase Accumulation in Arabidopsis thaliana

The biogenesis of the major thylakoid protein complexes of the photosynthetic apparatus requires auxiliary proteins supporting individual assembly steps. Here, we identify a plant lineage specific gene, CGL160, whose homolog, atp1, co-occurs with ATP synthase subunits in an operon-like arrangement in many cyanobacteria. Arabidopsis thaliana T-DNA insertion mutants, which no longer accumulate the nucleus-encoded CGL160 protein, accumulate less than 25% of wild-type levels of the chloroplast ATP synthase. Severe cosmetic or growth phenotypes result under either short day or fluctuating light growth conditions, respectively, but this is ameliorated under long day constant light growth conditions where the growth, ATP synthase activity and photosynthetic electron transport of the mutants are less affected. Accumulation of other photosynthetic complexes is largely unaffected in cgl160 mutants, suggesting that CGL160 is a specific assembly or stability factor for the CF1CF0 complex. CGL160 is not found in the mature assembled complex but it does interact specifically with subunits of ATP synthase, predominantly those in the extrinsic CF1 sub-complex. We suggest therefore that it may facilitate the assembly of CF1 into the holocomplex.