Structure of the multi-subunit chloroplast RNA polymerase.

Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.

Photosynthesis in the Biomass Model Species Lemna minor Displays Plant-Conserved and Species-Specific Features.

Lemnaceae are small freshwater plants with extraordinary high growth rates. We aimed to test whether this correlates with a more efficient photosynthesis, the primary energy source for growth. To this end, we compared photosynthesis properties of the duckweed Lemna minor and the terrestrial model plant Arabidopsis thaliana. Chlorophyll fluorescence analyses revealed high similarity in principle photosynthesis characteristics; however, Lemna exhibited a more effective light energy transfer into photochemistry and more stable photosynthesis parameters especially under high light intensities. Western immunoblot analyses of representative photosynthesis proteins suggested potential post-translational modifications in Lemna proteins that are possibly connected to this. Phospho-threonine phosphorylation patterns of thylakoid membrane proteins displayed a few differences between the two species. However, phosphorylation-dependent processes in Lemna such as photosystem II antenna association and the recovery from high-light-induced photoinhibition were not different from responses known from terrestrial plants. We thus hypothesize that molecular differences in Lemna photosynthesis proteins are associated with yet unidentified mechanisms that improve photosynthesis and growth efficiencies. We also developed a high-magnification video imaging approach for Lemna multiplication which is useful to assess the impact of external factors on Lemna photosynthesis and growth.

Biogenic signals from plastids and their role in chloroplast development.

Plant seeds do not contain differentiated chloroplasts. Upon germination, the seedlings thus need to gain photoautotrophy before storage energies are depleted. This requires the coordinated expression of photosynthesis genes encoded in nuclear and plastid genomes. Chloroplast biogenesis needs to be additionally coordinated with the light regulation network that controls seedling development. This coordination is achieved by nucleus to plastid signals called anterograde and plastid to nucleus signals termed retrograde. Retrograde signals sent from plastids during initial chloroplast biogenesis are also called biogenic signals. They have been recognized as highly important for proper chloroplast biogenesis and for seedling development. The molecular nature, transport, targets, and signalling function of biogenic signals are, however, under debate. Several studies disproved the involvement of a number of key components that were at the base of initial models of retrograde signalling. New models now propose major roles for a functional feedback between plastid and cytosolic protein homeostasis in signalling plastid dysfunction as well as the action of dually localized nucleo-plastidic proteins that coordinate chloroplast biogenesis with light-dependent control of seedling development. This review provides a survey of the developments in this research field, summarizes the unsolved questions, highlights several recent advances, and discusses potential new working modes.

Effectiveness of Light-Quality and Dark-White Growth Light Shifts in Short-Term Light Acclimation of Photosynthesis in Arabidopsis.

Photosynthesis needs to run efficiently under permanently changing illumination. To achieve this, highly dynamic acclimation processes optimize photosynthetic performance under a variety of rapidly changing light conditions. Such acclimation responses are acting by a complex interplay of reversible molecular changes in the photosynthetic antenna or photosystem assemblies which dissipate excess energy and balance uneven excitation between the two photosystems. This includes a number of non-photochemical quenching processes including state transitions and photosystem II remodeling. In the laboratory such processes are typically studied by selective illumination set-ups. Two set-ups known to be effective in a highly similar manner are (i) light quality shifts (inducing a preferential excitation of one photosystem over the other) or (ii) dark-light shifts (inducing a general off-on switch of the light harvesting machinery). Both set-ups result in similar effects on the plastoquinone redox state, but their equivalence in induction of photosynthetic acclimation responses remained still open. Here, we present a comparative study in which dark-light and light-quality shifts were applied to samples of the same growth batches of plants. Both illumination set-ups caused comparable effects on the phosphorylation of LHCII complexes and, hence, on the performance of state transitions, but generated different effects on the degree of state transitions and the formation of PSII super-complexes. The two light set-ups, thus, are not fully equivalent in their physiological effectiveness potentially leading to different conclusions in mechanistic models of photosynthetic acclimation. Studies on the regulation of photosynthetic light acclimation, therefore, requires to regard the respective illumination test set-up as a critical parameter that needs to be considered in the discussion of mechanistic and regulatory aspects in this subject.

Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis.

The initial greening of angiosperms involves light activation of photoreceptors that trigger photomorphogenesis, followed by the development of chloroplasts. In these semi-autonomous organelles, construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here, we show that the expression of PAP8, an essential subunit of the plastid-encoded RNA polymerase (PEP) in Arabidopsis thaliana, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 protein is localized and active in both plastids and the nucleus, and particularly required for the formation of late photobodies. In the pap8 albino mutant, phytochrome-mediated signalling is altered, degradation of the chloroplast development repressors PIF1/PIF3 is disrupted, HY5 is not stabilized, and the expression of the photomorphogenesis regulator GLK1 is impaired. PAP8 translocates into plastids via its targeting pre-sequence, interacts with the PEP and eventually reaches the nucleus, where it can interact with another PEP subunit pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome B-mediated signalling cascade and the reshaping of the PEP activity, it may coordinate nuclear gene expression with PEP-driven chloroplastic gene expression during chloroplast biogenesis.

Determination of the DNA/RNA-Associated Subproteome from Chloroplasts and Other Plastid Types.

Plastids of plant and algae cells are of endosymbiotic origin. They possess their own genome and a sophisticated protein machinery to express it. Studies over the recent years uncovered that the regulation of plastid gene expression is highly complex involving a multiplicity of regulatory protein factors that are mostly imported from the cytosol. Proper expression of the chloroplast genome in coordination with nuclear genome was found to be absolutely essential for efficient growth and development of plants especially during early steps of photomorphogenesis, but also at later stages of the plant life cycle. Protein factors being responsible for such essential steps, therefore, are highly interesting for fundamental science as well as for industrial applications targeting crop improvement and yield increase. Nevertheless, many proteins involved in regulation of plastid gene expression are still unidentified and/or uncharacterized. This asks for appropriate methods to analyze this special subproteome. Here, we describe suitable methods that proved to be successful in the analysis of the plastid subproteome of DNA/RNA-binding proteins.

PAP genes are tissue- and cell-specific markers of chloroplast development.

MAIN CONCLUSION: Expression of PAP genes is strongly coordinated and represents a highly selective cell-specific marker associated with the development of chloroplasts in photosynthetically active organs of Arabidopsis seedlings and adult plants. Transcription in plastids of plants depends on the activity of phage-type single-subunit nuclear-encoded RNA polymerases (NEP) and a prokaryotic multi-subunit plastid-encoded RNA polymerase (PEP). PEP is comprised of the core subunits α, ß, ß' and ß″ encoded by rpoA, rpoB/C1/C2 genes located on the plastome. This core enzyme needs to interact with nuclear-encoded sigma factors for proper promoter recognition. In chloroplasts, the core enzyme is surrounded by additional 12 nuclear-encoded subunits, all of eukaryotic origin. These PEP-associated proteins (PAPs) were found to be essential for chloroplast biogenesis as Arabidopsis inactivation mutants for each of them revealed albino or pale-green phenotypes. In silico analysis of transcriptomic data suggests that PAP genes represent a tightly controlled regulon, whereas wetlab data are sparse and correspond to the expression of individual genes mostly studied at the seedling stage. Using RT-PCR, transient, and stable expression assays of PAP promoter-GUS-constructs, we do provide, in this study, a comprehensive expression catalogue for PAP genes throughout the life cycle of Arabidopsis. We demonstrate a selective impact of light on PAP gene expression and uncover a high tissue specificity that is coupled to developmental progression especially during the transition from skotomorphogenesis to photomorphogenesis. Our data imply that PAP gene expression precedes the formation of chloroplasts rendering PAP genes a tissue- and cell-specific marker of chloroplast biogenesis.

Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1.

Plants possessing dysfunctional plastids due to defects in pigment biosynthesis or translation are known to repress photosynthesis-associated nuclear genes via retrograde signals from the disturbed organelles toward the nucleus. These signals are thought to be essential for proper biogenesis and function of the plastid. Mutants lacking plastid-encoded RNA polymerase-associated proteins (PAPs) display a genetic arrest in eoplast-chloroplast transition leading to an albino phenotype in the light. Retrograde signaling in these mutants, therefore, could be expected to be similar as under conditions inducing plastid dysfunction. To answer this question, we performed plastome- and genomewide array analyses in the pap7-1 mutant of Arabidopsis (Arabidopsis thaliana). In parallel, we determined the potential overlap with light-regulated expression networks. To this end, we performed a comparative expression profiling approach using light- and dark-grown wild-type plants as relative control for the expression profiles obtained from light-grown pap7-1 mutants. Our data indicate a specific impact of retrograde signals on metabolism-related genes in pap7-1 mutants reflecting the starvation situation of the albino seedlings. In contrast, light regulation of PhANGs and other nuclear gene groups appears to be fully functional in this mutant, indicating that a block in chloroplast biogenesis per se does not repress expression of them as suggested by earlier studies. Only genes for light harvesting complex proteins displayed a significant repression indicating an exclusive retrograde impact on this gene family. Our results indicate that chloroplasts and arrested plastids each emit specific signals that control different target gene modules both in positive and negative manner.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development.

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle.

Chloroplasts are the sunlight-collecting organelles of photosynthetic eukaryotes that energetically drive the biosphere of our planet. They are the base for all major food webs by providing essential photosynthates to all heterotrophic organisms including humans. Recent research has focused largely on an understanding of the function of these organelles, but knowledge about the biogenesis of chloroplasts is rather limited. It is known that chloroplasts develop from undifferentiated precursor plastids, the proplastids, in meristematic cells. This review focuses on the activation and action of plastid RNA polymerases, which play a key role in the development of new chloroplasts from proplastids. Evolutionarily, plastids emerged from the endosymbiosis of a cyanobacterium-like ancestor into a heterotrophic eukaryote. As an evolutionary remnant of this process, they possess their own genome, which is expressed by two types of plastid RNA polymerase, phage-type and prokaryotic-type RNA polymerase. The protein subunits of these polymerases are encoded in both the nuclear and plastid genomes. Their activation and action therefore require a highly sophisticated regulation that controls and coordinates the expression of the components encoded in the plastid and nucleus. Stoichiometric expression and correct assembly of RNA polymerase complexes is achieved by a combination of developmental and environmentally induced programmes. This review highlights the current knowledge about the functional coordination between the different types of plastid RNA polymerases and provides working models of their sequential expression and function for future investigations.

Identification of Early Nuclear Target Genes of Plastidial Redox Signals that Trigger the Long-Term Response of Arabidopsis to Light Quality Shifts.

Natural illumination conditions are highly variable and because of their sessile life style, plants are forced to acclimate to them at the cellular and molecular level. Changes in light intensity or quality induce changes in the reduction/oxidation (redox) state of the photosynthetic electron chain that acts as a trigger for compensatory acclimation responses comprising functional and structural adjustments of photosynthesis and metabolism. Such responses include redox-controlled changes in plant gene expression in the nucleus and organelles. Here we describe a strategy for the identification of early redox-regulated genes (ERGs) in the nucleus of the model organism Arabidopsis thaliana that respond significantly 30 or 60 min after the generation of a reduction signal in the photosynthetic electron transport chain. By comparing the response of wild-type plants with that of the acclimation mutant stn7, we could specifically identify ERGs. The results reveal a significant impact of chloroplast redox signals on distinct nuclear gene groups including genes for the mitochondrial electron transport chain, tetrapyrrole biosynthesis, carbohydrate metabolism, and signaling lipid synthesis. These expression profiles are clearly different from those observed in response to the reduction of photosynthetic electron transport by high light treatments. Thus, the ERGs identified are unique to redox imbalances in photosynthetic electron transport and were then used for analyzing potential redox-responsive cis-elements, trans-factors, and chromosomal regulatory hot spots. The data identify a novel redox-responsive element and indicate extensive redox control at transcriptional and chromosomal levels that point to an unprecedented impact of redox signals on epigenetic processes.

A novel proteinase, SNOWY COTYLEDON4, is required for photosynthetic acclimation to higher light intensities in Arabidopsis.

Excess light can have a negative impact on photosynthesis; thus, plants have evolved many different ways to adapt to different light conditions to both optimize energy use and avoid damage caused by excess light. Analysis of the Arabidopsis (Arabidopsis thaliana) mutant snowy cotyledon4 (sco4) revealed a mutation in a chloroplast-targeted protein that shares limited homology with CaaX-type endopeptidases. The SCO4 protein possesses an important function in photosynthesis and development, with point mutations rendering the seedlings and adult plants susceptible to photooxidative stress. The sco4 mutation impairs the acclimation of chloroplasts and their photosystems to excess light, evidenced in a reduction in photosystem I function, decreased linear electron transfer, yet increased nonphotochemical quenching. SCO4 is localized to the chloroplasts, which suggests the existence of an unreported type of protein modification within this organelle. Phylogenetic and yeast complementation analyses of SCO4-like proteins reveal that SCO4 is a member of an unknown group of higher plant-specific proteinases quite distinct from the well-described CaaX-type endopeptidases RAS Converting Enzyme1 (RCE1) and zinc metallopeptidase STE24 and lacks canonical CaaX activity. Therefore, we hypothesize that SCO4 is a novel endopeptidase required for critical protein modifications within chloroplasts, influencing the function of proteins involved in photosynthesis required for tolerance to excess light.

Environmental control of plant nuclear gene expression by chloroplast redox signals.

Plant photosynthesis takes place in specialized cell organelles, the chloroplasts, which perform all essential steps of this process. The proteins involved in photosynthesis are encoded by genes located on the plastid and nuclear genomes. Proper function and regulation of light harvesting and energy fixation thus requires a tight coordination of the gene expression machineries in the two genetic compartments. This is achieved by a bi-directional exchange of information between nucleus and plastids. Signals emerging from plastids report the functional and developmental state of the organelle to the nucleus and initiate distinct nuclear gene expression profiles, which trigger responses that support or improve plastid functions. Recent research indicated that this signaling is absolutely essential for plant growth and development. Reduction/oxidation (redox) signals from photosynthesis are key players in this information network since they do report functional disturbances in photosynthesis, the primary energy source of plants. Such disturbances are caused by environmental fluctuations for instance in illumination, temperature, or water availability. These environmental changes affect the linear electron flow of photosynthesis and result in changes of the redox state of the components involved [e.g., the plastoquinone (PQ) pool] or coupled to it (e.g., the thioredoxin pool). Thus, the changes in redox state directly reflect the environmental impact and serve as immediate plastidial signals to the nucleus. The triggered responses range from counterbalancing reactions within the physiological range up to severe stress responses including cell death. This review focuses on physiological redox signals from photosynthetic electron transport (PET), their relation to the environment, potential transduction pathways to the nucleus and their impact on nuclear gene expression.