Two chloroplast thioredoxin systems differentially modulate photosynthesis in Arabidopsis depending on light intensity and leaf age.

Various regulatory mechanisms have evolved in plants to optimize photosynthetic activity under fluctuating light. Thioredoxins (TRX) are members of the regulatory network balancing activities of light and carbon fixation reactions in chloroplasts. We have studied the impact of two chloroplast TRX systems, the ferredoxin-dependent TRX reductase (FTR) and the NADPH-dependent TRX reductase C (NTRC) on regulation of photosynthesis by mutants lacking or overexpressing a component of either system. Plants were subjected to image-based phenotyping and chlorophyll fluorescence measurements that allow long-term monitoring of the development and photosynthetic activity of the rosettes, respectively. Our experiments demonstrate that NTRC and FTR systems respond differently to variation of light intensity. NTRC was an indispensable regulator of photosynthesis in young leaves, at light-intensity transitions and under low light intensities limiting photosynthesis, whereas steady-state exposure of plants to growth or higher light intensities diminished the need of NTRC in regulation of photosynthesis. In fluctuating light, overexpression of NTRC increased the quantum yield of Photosystem II (YII) at low light and stimulated the relaxation of non-photochemical quenching (NPQ) after high light exposure, indicating that overexpression of NTRC improves leaf capacity to convert light energy to chemical energy under these conditions. Overexpression of chimeric protein (NTR-TRXf) containing both the thioredoxin reductase and TRXf activity on an ntrc mutant background, did not completely recover either growth or steady-state photosynthetic activity, whereas OE-NTR-TRXf plants exposed to fluctuating light regained the wild-type level of Y(II) and NPQ.

Multilevel regulation of non-photochemical quenching and state transitions by chloroplast NADPH-dependent thioredoxin reductase.

In natural growth habitats, plants face constant, unpredictable changes in light conditions. To avoid damage to the photosynthetic apparatus on thylakoid membranes in chloroplasts, and to avoid wasteful reactions, it is crucial to maintain a redox balance both within the components of photosynthetic electron transfer chain and between the light reactions and stromal carbon metabolism under fluctuating light conditions. This requires coordinated function of the photoprotective and regulatory mechanisms, such as non-photochemical quenching (NPQ) and reversible redistribution of excitation energy between photosystem II (PSII) and photosystem I (PSI). In this paper, we show that the NADPH-dependent chloroplast thioredoxin system (NTRC) is involved in the control of the activation of these mechanisms. In plants with altered NTRC content, the strict correlation between lumenal pH and NPQ is partially lost. We propose that NTRC contributes to downregulation of a slow-relaxing constituent of NPQ, whose induction is independent of lumenal acidification. Additionally, overexpression of NTRC enhances the ability to adjust the excitation balance between PSII and PSI, and improves the ability to oxidize the electron transfer chain during changes in light conditions. Thiol regulation allows coupling of the electron transfer chain to the stromal redox state during these changes.

Regulation of cyclic electron flow by chloroplast NADPH-dependent thioredoxin system.

Linear electron transport in the thylakoid membrane drives photosynthetic NADPH and ATP production, while cyclic electron flow (CEF) around photosystem I only promotes the translocation of protons from stroma to thylakoid lumen. The chloroplast NADH dehydrogenase-like complex (NDH) participates in one CEF route transferring electrons from ferredoxin back to the plastoquinone pool with concomitant proton pumping to the lumen. CEF has been proposed to balance the ratio of ATP/NADPH production and to control the redox poise particularly in fluctuating light conditions, but the mechanisms regulating the NDH complex remain unknown. We have investigated potential regulation of the CEF pathways by the chloroplast NADPH-thioredoxin reductase (NTRC) in vivo by using an Arabidopsis knockout line of NTRC as well as lines overexpressing NTRC. Here, we present biochemical and biophysical evidence showing that NTRC stimulates the activity of NDH-dependent CEF and is involved in the regulation of generation of proton motive force, thylakoid conductivity to protons, and redox balance between the thylakoid electron transfer chain and the stroma during changes in light conditions. Furthermore, protein-protein interaction assays suggest a putative thioredoxin-target site in close proximity to the ferredoxin-binding domain of NDH, thus providing a plausible mechanism for redox regulation of the NDH ferredoxin:plastoquinone oxidoreductase activity.

Chloroplast thioredoxin systems: prospects for improving photosynthesis.

Thioredoxins (TRXs) are protein oxidoreductases that control the structure and function of cellular proteins by cleavage of a disulphide bond between the side chains of two cysteine residues. Oxidized thioredoxins are reactivated by thioredoxin reductases (TR) and a TR-dependent reduction of TRXs is called a thioredoxin system. Thiol-based redox regulation is an especially important mechanism to control chloroplast proteins involved in biogenesis, in regulation of light harvesting and distribution of light energy between photosystems, in photosynthetic carbon fixation and other biosynthetic pathways, and in stress responses of plants. Of the two plant plastid thioredoxin systems, the ferredoxin-dependent system relays reducing equivalents from photosystem I via ferredoxin and ferredoxin-thioredoxin reductase (FTR) to chloroplast proteins, while NADPH-dependent thioredoxin reductase (NTRC) forms a complete thioredoxin system including both reductase and thioredoxin domains in a single polypeptide. Chloroplast thioredoxins transmit environmental light signals to biochemical reactions, which allows fine tuning of photosynthetic processes in response to changing environmental conditions. In this paper we focus on the recent reports on specificity and networking of chloroplast thioredoxin systems and evaluate the prospect of improving photosynthetic performance by modifying the activity of thiol regulators in plants.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.

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 CF1 γ 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.

Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in vivo function of reductase and thioredoxin domains.

Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC) contains both reductase (NTRd) and thioredoxin (TRXd) domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive TRXd, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modeling of the three-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protective pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for sustainable bioenergy production.

Deletion of chloroplast NADPH-dependent thioredoxin reductase results in inability to regulate starch synthesis and causes stunted growth under short-day photoperiods.

Plastid-localized NADPH-dependent thioredoxin reductase C (NTRC) is a unique NTR enzyme containing both reductase and thioredoxin domains in a single polypeptide. Arabidopsis thaliana NTRC knockout lines (ntrc) show retarded growth, especially under short-day (SD) photoperiods. This study identified chloroplast processes that accounted for growth reduction in SD-acclimated ntrc. The strongest reduction in ntrc growth occurred under photoperiods with nights longer than 14 h, whereas knockout of the NTRC gene did not alter the circadian-clock-controlled growth of Arabidopsis. Lack of NTRC modulated chloroplast reactive oxygen species (ROS) metabolism, but oxidative stress was not the primary cause of retarded growth of SD-acclimated ntrc. Scarcity of starch accumulation made ntrc leaves particularly vulnerable to photoperiods with long nights. Direct interaction of NTRC and ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was confirmed by yeast two-hybrid analysis. The ntrc line was not able to maximize starch synthesis during the light period, which was particularly detrimental under SD conditions. Acclimation of Arabidopsis to SD conditions also involved an inductive rise of ROS production in illuminated chloroplasts that was not counterbalanced by the activation of plastidial anti-oxidative systems. It is proposed that knockout of NTRC challenges redox regulation of starch synthesis, resulting in stunted growth of the mutant lines acclimated to the SD photoperiod.

Posttranslational influence of NADPH-dependent thioredoxin reductase C on enzymes in tetrapyrrole synthesis.

The NADPH-dependent thioredoxin reductase C (NTRC) is involved in redox-related regulatory processes in chloroplasts and nonphotosynthetic active plastids. Together with 2-cysteine peroxiredoxin, it forms a two-component peroxide-detoxifying system that acts as a reductant under stress conditions. NTRC stimulates in vitro activity of magnesium protoporphyrin IX monomethylester (MgPMME) cyclase, most likely by scavenging peroxides. Reexamination of tetrapyrrole intermediate levels of the Arabidopsis (Arabidopsis thaliana) knockout ntrc reveals lower magnesium protoporphyrin IX (MgP) and MgPMME steady-state levels, the substrate and the product of MgP methyltransferase (CHLM) preceding MgPMME cyclase, while MgP strongly accumulates in mutant leaves after 5-aminolevulinic acid feeding. The ntrc mutant has a reduced capacity to synthesize 5-aminolevulinic acid and reduced CHLM activity compared with the wild type. Although transcript levels of genes involved in chlorophyll biosynthesis are not significantly altered in 2-week-old ntrc seedlings, the contents of glutamyl-transfer RNA reductase1 (GluTR1) and CHLM are reduced. Bimolecular fluorescence complementation assay confirms a physical interaction of NTRC with GluTR1 and CHLM. While ntrc contains partly oxidized CHLM, the wild type has only reduced CHLM. As NTRC also stimulates CHLM activity in vitro, it is proposed that NTRC has a regulatory impact on the redox status of conserved cysteine residues of CHLM. It is hypothesized that a deficiency of NTRC leads to a lower capacity to reduce cysteine residues of GluTR1 and CHLM, affecting the stability and, thereby, altering the activity in the entire tetrapyrrole synthesis pathway.

Retrograde signaling from functionally heterogeneous plastids.

Structural and functional components of chloroplast are encoded by genes localized both to nuclear and plastid genomes of plant cell. Development from etioplasts to chloroplasts is triggered by light receptors that activate the expression of photosynthesis-associated nuclear genes (PhaNGs). In addition to photoreceptor-mediated pathways, retrograde signals from the chloroplast to the nucleus activate or repress the expression of nuclear genes involved in acclimatory or stress responses in plant leaves. A plant mesophyll cell contains up to 100 chloroplasts that function autonomously, raising intriguing questions about homogeneity and coordination of retrograde signals transmitted from chloroplast to nucleus. We have previously demonstrated that the knockout of the chloroplast regulatory protein, chloroplast NADPH-dependent thioredoxin reductase (NTRC) leads to a heterogeneous population of chloroplasts with a range of different functional states. The heterogeneous chloroplast population activates both redox-dependent and undifferentiated plastid-generated retrograde signaling pathways in the mutant leaves. Transcriptome data from the ntrc knockout lines suggest that the induction of the redox-dependent signaling pathway depends on light conditions and leads to activation of stress-responsive gene expression. Analysis of mutants in different developmental stages allows to dissect signals from normal and anomalous chloroplasts. Thus, the signals derived from anomalous chloroplasts repress expression of PhaNGs as well as genes associated with light receptor signaling and differentiation of stomata, implying interaction between retrograde pathways and plant development. Analysis of the nuclear gene expression in mutants of retrograde signaling pathways in ntrc background would reveal the components that mediate signals generated from heterogeneous plastids to nucleus.

Purification and analysis of polyhistidine-tagged human parvovirus B19 VP1 and VP2 expressed in insect cells.

Human parvovirus B19 is an autonomously replicating human pathogen with a specific tropism for human erythroid progenitor cells. There is an interest in producing empty nucleocapsids of B19 as they can be used as tools in molecular biology and diagnostics. Native B19 virus particles are formed from two structural viral proteins, VP1 and VP2. The VP2 protein alone is able to self assemble and consequently form virus-like particles (VLPs) in heterologous expression systems. Purification of recombinant VLPs has been conducted using various traditional methods. These include laborious and time-consuming, e.g. cesium chloride or sucrose gradient ultracentrifugation steps, allowing limited working volumes to be processed. Therefore, an alternative purification method enabling process scale-up was developed and evaluated. Polyhistidine-tagged versions of B19 VP1 and VP2 capsid proteins were engineered and produced using the baculovirus expression system. The recombinant protein products were purified by immobilized metal-ion affinity chromatography (IMAC) and analyzed by SDS-PAGE, immunoblotting, electron microscopy, and enzyme-linked immunosorbent assays. Further, the immunological properties of the recombinant proteins were evaluated. The results showed that the VP2 fusion protein assembled into capsid-like structures and that both VP1 and VP2 following purification by IMAC have potential as antigens for diagnosis of a B19 infection.

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles.

Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy.

Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment. The results showed that approximately 10 and 9 fluorescent units were associated with the corresponding CPV and B19 VLPs. In summary, we were able to estimate the number of fluorescent subunits in a baculovirus containing a GFP-fusion with its gp64 envelope protein and in two different parvo-VLPs containing EGFP-fused with their VP2 capsid proteins.

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles.

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.

Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and past-immunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2 s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5 x 10(-8) cm2 s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a past-immunity serum sample gave an average diffusion coefficient of 3.5 x 10(-8) cm2 s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7 x 10(-7) to 1.6 x 10(-7) cm2 s(-1), which is much smaller than that observed with acute phase or past-immunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and past-immunity sera. FCS measurement of VLP-immune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.

Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.

Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.