Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis

Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (E_GSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal E_GSH dynamics, we show that stromal E_GSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.     

Post‐harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers

Plastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non‐photosynthetic plastids are generally discouraging. Here, we report the expression of two thioredoxin genes (trx f and trx m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately two to three orders of magnitude lower than in the leaf. However, we demonstrate that a simple post‐harvest light treatment of microtubers developed in vitro or soil‐grown tubers induces up to 55 times higher accumulation of the recombinant protein in just seven to ten days. After the applied treatment, the Trx f levels in microtubers and soil‐grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for eight months maintained the capacity of increasing the foreign protein levels after the light treatment. Post‐harvest cold induction (up to five times) at 4°C was also detected in microtubers. We conclude that plastid transformation and post‐harvest light treatment could be an interesting approach for the production of foreign proteins in potato.     

Copper stress‐induced changes in leaf soluble proteome of Cu‐sensitive and tolerant Agrostis capillaris L. populations

Changes in leaf soluble proteome were explored in 3‐month‐old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1–50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO₄ spiked‐nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2‐DE (linear 4–7 pH gradient). Analysis of CCB‐stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC‐MS/MS. In both populations, Cu excess impacted both light‐dependent (OEE, cytochrome b6‐f complex, and chlorophyll a‐b binding protein), and ‐independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin‐NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S‐containing amino‐acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 ( http//proteomecentral.proteomexchange.org/dataset/PXD001930 ).     

Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN‐PAGE separation of membrane protein complexes and selective MS that the accumulation of one‐helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light‐grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.     

Extreme variation in rates of evolution in the plastid Clp protease complex

Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multi‐subunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid‐encoded gene ( clpP1 ) and multiple nuclear genes. The clpP1 gene is highly conserved across most green plants, but it is by far the fastest evolving plastid‐encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history of clpP1 molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find that clpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g. a loss of introns and RNA ‐editing sites) within seed plants. Although clpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely true. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genus Silene , which has highly elevated and heterogeneous rates of clpP1 evolution. We confirmed that clpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear‐encoded Clp subunits, even in one of the most divergent Silene species. Additionally, there is a tight correlation between amino acid substitution rates in clpP1 and the nuclear‐encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex.     

Physiological relevance of plant 2‐Cys peroxiredoxin overoxidation level and oligomerization status

Peroxiredoxins are ubiquitous thioredoxin‐dependent peroxidases presumed to display, upon environmental constraints, a chaperone function resulting from a redox‐dependent conformational switch. In this work, using biochemical and genetic approaches, we aimed to unravel the factors regulating the redox status and the conformation of the plastidial 2‐Cys peroxiredoxin (2‐Cys PRX) in plants. In Arabidopsis, we show that in optimal growth conditions, the overoxidation level mainly depends on the availability of thioredoxin‐related electron donors, but not on sulfiredoxin, the enzyme reducing the 2‐Cys PRX overoxidized form. We also observed that upon various physiological temperature, osmotic and light stress conditions, the overoxidation level and oligomerization status of 2‐Cys PRX can moderately vary depending on the constraint type. Further, no major change was noticed regarding protein conformation in water‐stressed Arabidopsis, barley and potato plants, whereas species‐dependent up‐ and down‐variations in overoxidation were observed. In contrast, both 2‐Cys PRX overoxidation and oligomerization were strongly induced during a severe oxidative stress generated by methyl viologen. From these data, revealing that the oligomerization status of plant 2‐Cys PRX does not exhibit important variation and is not tightly linked to the protein redox status upon physiologically relevant environmental constraints, the possible in planta functions of 2‐Cys PRX are discussed.     

Plastid‐associated polyamines: their role in differentiation,structure, functioning,stress response and senescence

Polyamines are low‐molecular weight biogenic amines. They are a specific group of cell growth and development regulators. In the past decade biochemical, molecular and genetic studies have contributed much to a better understanding of the biological role of polyamines in the plant cell. Substantial evidence has also been added to our understanding of the role of polyamines in plastid development. In developing chloroplasts, polyamines serve as a nitrogen source for protein and chlorophyll synthesis. In chloroplast structure, thylakoid proteins linked to polyamines belong mainly to antenna proteins of light‐harvesting chlorophyll a/b–protein complexes. The fact that LHCII oligomeric forms are much more intensely labelled by polyamines, in comparison to monomeric forms, suggests that polyamines participate in oligomer stabilisation. In plastid metabolism, polyamines modulate effectiveness of photosynthesis. The role of polyamines in mature chloroplasts is also related to the photo‐adaptation of the photosynthetic apparatus to low and high light intensity and its response to environmental stress. The occurrence of polyamines and enzymes participating in their metabolism at every stage of plastid development indicates that polyamines play a role in plastid differentiation, structure, functioning and senescence.     

Stabilization of oligomeric structure of α‐crystallin by Zn+2 through intersubunit bridging

α‐Crystallin, the major protein of mammalian eye lens, is a member of the small heat shock protein family and is a molecular chaperone. We previously reported that its molecular chaperone function as well as stability increased in presence of Zn⁺². Despite the effect of Zn⁺² on the structure and function of α‐crystallin, evidence for direct interaction between them remained elusive. We now present the MALDI mass spectrometric data that shows direct evidence of Zn⁺² binding to recombinant αA‐ and αB‐crystallin. The binding stoichiometry was over three Zn⁺² per subunit of α‐crystallin at zinc/protein molar ratio of 20. Observation of multiple Zn⁺² binding is consistent with the large increase in thermodynamic stability. Sequence‐based analysis of αA‐ and αB‐crystallin predicted both proteins to be nonzinc binding proteins. Our dynamic light scattering data shows that Zn⁺² stabilizes the oligomeric structure of α‐crystallin by bridging neighboring subunits in multiple centers. Despite the low affinity binding, the intersubunit bridging by multiple Zn⁺² makes the oligomer so stable that oligomer breakdown does not occur even at 6M urea. The subunit bridging has been supported by our FRET data that showed absence of subunit exchange in presence of zinc. MALDI data also showed that the interaction of α‐crystallin with Zn⁺² is quite different from other bivalent metal ions. Bound Zn⁺² could be easily removed by dialysis of the complex. The relevance of such weak interaction on the stability of the oligomeric structure of α‐crystallin and its function in the eye lens has been discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 105–116, 2011.     

Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.     

Nucleus‐encoded mRNAs for Chloroplast Proteins GapA,PetA, and PsbO are Trans‐spliced in the Flagellate Euglena gracilis Irrespective of Light and Plastid Function

Euglena gracilis is a fresh‐water flagellate possessing secondary chloroplasts of green algal origin. In contrast with organisms possessing primary plastids, mRNA levels of nucleus‐encoded genes for chloroplast proteins in E. gracilis depend on neither light nor plastid function. However, it remains unknown, if all these mRNAs are trans‐spliced and possess spliced leader sequence at the 5′‐end and if trans‐splicing depends on light or functional plastids. This study revealed that polyadenylated mRNAs encoding the chloroplast proteins glyceraldehyde‐3‐phosphate dehydrogenase (GapA), cytochrome f (PetA), and subunit O of photosystem II (PsbO) are trans‐spliced irrespective of light or plastid function.     

Crosstalk between chloroplast thioredoxin systems in regulation of photosynthesis

Thioredoxins (TRXs) mediate light‐dependent activation of primary photosynthetic reactions in plant chloroplasts by reducing disulphide bridges in redox‐regulated enzymes. Of the two plastid TRX systems, the ferredoxin‐TRX system consists of ferredoxin‐thioredoxin reductase (FTR) and multiple TRXs, while the NADPH‐dependent thioredoxin reductase (NTRC) contains a complete TRX system in a single polypeptide. Using Arabidopsis plants overexpressing or lacking a functional NTRC, we have investigated the redundancy and interaction between the NTRC and Fd‐TRX systems in regulation of photosynthesis in vivo. Overexpression of NTRC raised the CO₂ fixation rate and lowered non‐photochemical quenching and acceptor side limitation of PSI in low light conditions by enhancing the activation of chloroplast ATP synthase and TRX‐regulated enzymes in Calvin–Benson cycle (CBC). Overexpression of NTRC with an inactivated NTR or TRX domain partly recovered the phenotype of knockout plants, suggesting crosstalk between the plastid TRX systems. NTRC interacted in planta with fructose‐1,6‐bisphosphatase, phosphoribulokinase and CF₁γ subunit of the ATP synthase and with several chloroplast TRXs. These findings indicate that NTRC‐mediated regulation of the CBC and ATP synthesis occurs both directly and through interaction with the ferredoxin‐TRX system and is crucial when availability of light is limiting photosynthesis.     

Multi‐level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ‐subunit through the ferredoxin–thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild‐type levels as irradiance was increased. This effect was caused by an altered redox state of the γ‐subunit under low, but not high, light. The low light‐specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non‐photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin–thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.     

Identification of inositol 1,4,5‐trisphosphate‐binding proteins by heparin‐agarose affinity purification and LTQ ORBITRAP MS in Oryza sativa

Inositol 1,4,5‐trisphosohate (IP₃) and its receptors play a pivotal role in calcium signal transduction in mammals. However, no homologs of mammalian IP₃ receptors have been found in plants. In this study, we isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP₃‐binding proteins by heparin‐agarose affinity purification. The IP₃‐binding activities of these protein fractions were determined by [³H] IP₃‐binding assay. SDS‐PAGE and MS analysis were then performed to characterize these proteins. We have identified 297 proteins from the eluates of heparin‐agarose column chromatography, which will provide insight into the IP₃ signaling pathways in plants. All MS data have been deposited in the ProteomeXchange with identifier PXD000763 ( http://proteomecentral.proteomexchange.org/dataset/PXD000763 ).     

Nanoscale elongating control of the self‐assembled protein filament with the cysteine‐introduced building blocks

Self‐assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self‐assembling proteins, named “Nanolego.” Nanolego consists of “structural elements” of a structurally stable symmetrical homo‐oligomeric protein and “binding elements,” which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self‐assembly process. The stabilization method is mediated by disulfide bonds between Cysteine‐residues incorporated into the binding elements, and the termination method uses “capping Nanolegos,” in which some of the binding elements in the Nanolego are absent for the self‐assembled ends. With these technologies, we successfully constructed timing‐controlled and size‐regulated filament‐shape complexes via Nanolego self‐assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.