The photoreactivation of 6 - 4 photoproducts in chloroplast and nuclear DNA depends on the amount of the Arabidopsis UV repair defective 3 protein.

BACKGROUND: 6 - 4 photoproducts are the second most common UV-induced DNA lesions after cyclobutane pyrimidine dimers. In plants, they are mainly repaired by photolyases in a process called photoreactivation. While pyrimidine dimers can be deleterious, leading to mutagenesis or even cell death, 6 - 4 photoproducts can activate specific signaling pathways. Therefore, their removal is particularly important, especially for plants exposed to high UV intensities due to their sessile nature. Although photoreactivation in nuclear DNA is well-known, its role in plant organelles remains unclear. In this paper we analyzed the activity and localization of GFP-tagged AtUVR3, the 6 - 4 photoproduct specific photolyase. RESULTS: Using transgenic Arabidopsis with different expression levels of AtUVR3, we confirmed a positive trend between these levels and the rate of 6 - 4 photoproduct removal under blue light. Measurements of 6 - 4 photoproduct levels in chloroplast and nuclear DNA of wild type, photolyase mutants, and transgenic plants overexpressing AtUVR3 showed that the photoreactivation is the main repair pathway responsible for the removal of these lesions in both organelles. The GFP-tagged AtUVR3 was predominantly located in nuclei with a small fraction present in chloroplasts and mitochondria of transgenic Arabidopsis thaliana and Nicotiana tabacum lines. In chloroplasts, this photolyase co-localized with the nucleoid marked by plastid envelope DNA binding protein. CONCLUSIONS: Photolyases are mainly localized in plant nuclei, with only a small fraction present in chloroplasts and mitochondria. Despite this unbalanced distribution, photoreactivation is the primary mechanism responsible for the removal of 6 - 4 photoproducts from nuclear and chloroplast DNA in adult leaves. The amount of the AtUVR3 photolyase is the limiting factor influencing the photoreactivation rate of 6 - 4 photoproducts. The efficient photoreactivation of 6 - 4 photoproducts in 35S: AtUVR3-GFP Arabidopsis and Nicotiana tabacum is a promising starting point to evaluate whether transgenic crops overproducing this photolyase are more tolerant to high UV irradiation and how they respond to other abiotic and biotic stresses under field conditions.

DnaK and DnaJ proteins from Hsp70/40 family are involved in Rubisco biosynthesis in Synechocystis sp. PCC6803 and sustain the enzyme assembly in a heterologous system.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of carbon fixation performed by photosynthetic organisms. Form I of this enzyme found in plants and cyanobacteria is composed of eight large (RbcL) and eight small (RbcS) subunits. To form a functional enzyme, Rubisco subunits need to be properly folded, with the assistance of cellular chaperone machinery, and consecutively assembled in a strictly orchestrated manner, with the help of multiple auxiliary factors. In recent years, multiple Rubisco assembly chaperones and their function in enzyme biogenesis have been extensively characterized. Little is known about the potential specialized factors involved in Rubisco subunits folding at the pre-chaperonin stage, yet this knowledge is greatly needed for the fast and efficient testing of new Rubisco variants.Synechococcus sp. PCC 6803 Rubisco shows limited solubility and a lack of assembly in the Escherichia coli expression system. In this study, we aim to identify which additional chaperones are necessary and sufficient in sustaining the heterologous assembly of native Rubisco. Our findings prove that upon the introduction of Synechocystis DnaK2 to the E. coli system, RbcL is produced in soluble form. The addition of specific DnaJ (Sll1384) enhances this effect. We explain these combined effects based on binding constancies, measured for particular partners in vitro, as well as our analysis of the putative tertiary structure of the proteins. Our results have potential implications for Rubisco engineering.

Photosynthetic Reactions: From Molecules to Function, and from Simple Models to Complex Systems.

Photosynthesis is the basic process for life on Earth-and the one that has changed life history most drastically [...].

Quantum dot clusters as self-assembled antennae with phycocyanine and phycobilisomes as energy acceptors.

In this study, we investigated an experimental and Monte-Carlo computational characterization of self-assembled antennae built using CdTe colloidal quantum dots (QDs). These clusters provide efficient excitation of phycocyanine (PC) or phycobilisomes (PBSs). PBSs are light-harvesting complexes (LHCs) of cyanobacteria, made of several PC units, organized in disks and rods. Each PC contains three separate cofactors. Therefore, we analyzed variations in multi-donor and multi-acceptor systems. The self-assembled QD clusters were formed mostly by electrostatic interactions, possibly due to the introduction of a positive charge on an originally negatively charged nanoparticle surface. Our results suggest that PC may accept energy from multiple nanoparticles localized at a distance significantly longer than the Förster radius. The excitation transfers between particular nanoparticles with possible delocalization. The maximal energy transfer efficiency was obtained for the PC/PBS : QD ratio from 1 to 20 depending on the QD size. This cannot be fully explained using computational simulations; hence, we discussed the hypothesis and explained the observations. Our self-assembled systems may be considered for possible applications in artificial light-harvesting systems because absorption spectra of QDs are different from the absorption characteristics of PC/PBS. In addition, huge clusters of QDs may effectively increase the optical cross-section of so-created nanohybrids.

Insights into the Structure of Rubisco from Dinoflagellates-In Silico Studies.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the best studied enzymes. It is crucial for photosynthesis, and thus for all of biosphere's productivity. There are four isoforms of this enzyme, differing by amino acid sequence composition and quaternary structure. However, there is still a group of organisms, dinoflagellates, single-cell eukaryotes, that are confirmed to possess Rubisco, but no successful purification of the enzyme of such origin, and hence a generation of a crystal structure was reported to date. Here, we are using in silico tools to generate the possible structure of Rubisco from a dinoflagellate representative, Symbiodinium sp. We selected two templates: Rubisco from Rhodospirillum rubrum and Rhodopseudomonas palustris. Both enzymes are the so-called form II Rubiscos, but the first is exclusively a homodimer, while the second one forms homo-hexamers. Obtained models show no differences in amino acids crucial for Rubisco activity. The variation was found at two closely located inserts in the C-terminal domain, of which one extends a helix and the other forms a loop. These inserts most probably do not play a direct role in the enzyme's activity, but may be responsible for interaction with an unknown protein partner, possibly a regulator or a chaperone. Analysis of the possible oligomerization interface indicated that Symbiodinium sp. Rubisco most likely forms a trimer of homodimers, not just a homodimer. This hypothesis was empowered by calculation of binding energies. Additionally, we found that the protein of study is significantly richer in cysteine residues, which may be the cause for its activity loss shortly after cell lysis. Furthermore, we evaluated the influence of the loop insert, identified exclusively in the Symbiodinium sp. protein, on the functionality of the recombinantly expressed R. rubrum Rubisco. All these findings shed new light onto dinoflagellate Rubisco and may help in future obtainment of a native, active enzyme.

Bean and Pea Plastoglobules Change in Response to Chilling Stress.

Plastoglobules (PGs) might be characterised as microdomains of the thylakoid membrane that serve as a platform to recruit proteins and metabolites in their spatial proximity in order to facilitate metabolic channelling or signal transduction. This study provides new insight into changes in PGs isolated from two plant species with different responses to chilling stress, namely chilling-tolerant pea (Pisum sativum) and chilling-sensitive bean (Phaseolus coccineus). Using multiple analytical methods, such as high-performance liquid chromatography and visualisation techniques including transmission electron microscopy and atomic force microscopy, we determined changes in PGs' biochemical and biophysical characteristics as a function of chilling stress. Some of the observed alterations occurred in both studied plant species, such as increased particle size and plastoquinone-9 content, while others were more typical of a particular type of response to chilling stress. Additionally, PGs of first green leaves were examined to highlight differences at this stage of development. Observed changes appear to be a dynamic response to the demands of photosynthetic membranes under stress conditions.

Arabidopsis Phototropins Participate in the Regulation of Dark-Induced Leaf Senescence.

Senescence is the final stage of plant development, affecting individual organs or the whole organism, and it can be induced by several environmental factors, including shading or darkness. Although inevitable, senescence is a complex and tightly regulated process, ensuring optimal remobilization of nutrients and cellular components from senescing organs. Photoreceptors such as phytochromes and cryptochromes are known to participate in the process of senescence, but the involvement of phototropins has not been studied to date. We investigated the role of these blue light photoreceptors in the senescence of individually darkened Arabidopsis thaliana leaves. We compared several physiological and molecular senescence markers in darkened leaves of wild-type plants and phototropin mutants (phot1, phot2, and phot1phot2). In general, all the symptoms of senescence (lower photochemical activity of photosystem II, photosynthetic pigment degradation, down-regulation of photosynthetic genes, and up-regulation of senescence-associated genes) were less pronounced in phot1phot2, as compared to the wild type, and some also in one of the single mutants, indicating delayed senescence. This points to different mechanisms of phototropin operation in the regulation of senescence-associated processes, either with both photoreceptors acting redundantly, or only one of them, phot1, playing a dominant role.

Detailed characterization of Synechocystis PCC 6803 ferredoxin:NADP+ oxidoreductase interaction with model membranes.

Direct interaction of ferredoxin:NADP+ oxidoreductase (FNR) with thylakoid membranes was postulated as a part of the cyclic electron flow mechanism. In vitro binding of FNR to digalactosyldiacylglycerol and monogalactosyldiacylglycerol membranes was also shown. In this paper we deal with the latter interaction in more detail describing the effect for two FNR forms of Synechocystis PCC 6803. The so-called short FNR (sFNR) is homologous to FNR from higher plant chloroplasts. The long FNR (lFNR) form contains an additional domain, responsible for the interaction with phycobilisomes. We compare the binding of both sFNR and lFNR forms to native and non-native lipids. We also include factors which could modulate this process: pH change, temperature change, presence of ferredoxin, NADP+ and NADPH and heavy metals. For the lFNR, we also include phycobilisomes as a modulating factor. The membrane binding is generally faster at lower pH. The sFNR was binding faster than lFNR. Ferredoxin isoforms with higher midpoint potential, as well as NADPH and NADP+, weakened the binding. Charged lipids and high phosphate promoted the binding. Heavy metal ions decreased the rate of membrane binding only when FNR was preincubated with them before injection beneath the monolayer. FNR binding was limited to surface lipid groups and did not influence hydrophobic chain packing. Taken together, FNR interaction with lipids appears to be non-specific, with an electrostatic component. This suggests that the direct FNR interaction with lipids is most likely not a factor in directing electron transfer, but should be taken into account during in vitro studies.

The effect of UV-B on Arabidopsis leaves depends on light conditions after treatment.

BACKGROUND: Ultraviolet B (UV-B) irradiation can influence many cellular processes. Irradiation with high UV-B doses causes chlorophyll degradation, a decrease in the expression of genes associated with photosynthesis and its subsequent inhibition. On the other hand, sublethal doses of UV-B are used in post-harvest technology to prevent yellowing in storage. To address this inconsistency the effect of short, high-dose UV-B irradiation on detached Arabidopsis thaliana leaves was examined. RESULTS: Two different experimental models were used. After short treatment with a high dose of UV-B the Arabidopsis leaves were either put into darkness or exposed to constant light for up to 4 days. UV-B inhibited dark-induced chlorophyll degradation in Arabidopsis leaves in a dose-dependent manner. The expression of photosynthesis-related genes, chlorophyll content and photosynthetic efficiency were higher in UV-B -treated leaves left in darkness. UV-B treatment followed by constant light caused leaf yellowing and induced the expression of senescence-related genes. Irrespective of light treatment a high UV-B dose led to clearly visible cell death 3 days after irradiation. CONCLUSIONS: High doses of UV-B have opposing effects on leaves depending on their light status after UV treatment. In darkened leaves short UV-B treatment delays the appearance of senescence symptoms. When followed by light treatment, the same doses of UV-B result in chlorophyll degradation. This restricts the potential usability of UV treatment in postharvest technology to crops which are stored in darkness.

Correction to "Quantum Dots Assembled with Photosynthetic Antennae on a Carbon Nanotube Platform: A Nanohybrid for the Enhancement of Light Energy Harvesting".

[This corrects the article DOI: 10.1021/acsomega.3c07673.].

Empirical and computational design of iron-sulfur cluster proteins.

Here, we compare two approaches of protein design. A computational approach was used in the design of the coiled-coil iron-sulfur protein, CCIS, as a four helix bundle binding an iron-sulfur cluster within its hydrophobic core. An empirical approach was used for designing the redox-chain maquette, RCM as a four-helix bundle assembling iron-sulfur clusters within loops and one heme in the middle of its hydrophobic core. We demonstrate that both ways of design yielded the desired proteins in terms of secondary structure and cofactors assembly. Both approaches, however, still have much to improve in predicting conformational changes in the presence of bound cofactors, controlling oligomerization tendency and stabilizing the bound iron-sulfur clusters in the reduced state. Lessons from both ways of design and future directions of development are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Time-resolved absorption measurements quantify the competition of energy and electron transfer between quantum dots and cytochrome c.

We applied transient absorption spectroscopy to study the early photodynamics in a system composed of CdTe quantum dots (QDs) and cytochrome c (Cyt c) protein. In the QDs and Cyt c mixtures, about 25 % of the excited QD electrons quickly relax (∼23 ps) to the ground state and roughly 75 % decay on slower time scale - mostly due to quenching by Cyt c. On the basis of the assumed model, we estimated the contribution of electron transfer and other mechanisms to this quenching. The primary quenching mechanism is probably energy transfer but electron transfer makes a significant contribution (∼8 %), resulting in photoreduction of Cyt c. The lifetime of one fraction of reduced Cyt c (35-90 %) is âˆ¼ 1 ms and the lifetime of the remaining fraction was longer than the âˆ¼ 50-ms time window of the experiment. We speculate that, in the former fraction, the back electron transfer from the reduced Cyt c to QDs occurs and the latter fraction of Cyt c is stably reduced.

Quantum Dots Assembled with Photosynthetic Antennae on a Carbon Nanotube Platform: A Nanohybrid for the Enhancement of Light Energy Harvesting.

The construction of artificial systems for solar energy harvesting is still a challenge. There needs to be a light-harvesting antenna with a broad absorption spectrum and then the possibility to transfer harvested energy to the reaction center, converting photons into a storable form of energy. Bioinspired and bioderivative elements may help in achieving this aim. Here, we present an option for light harvesting: a nanobiohybrid of colloidal, semiconductor quantum dots (QDs) and natural photosynthetic antennae assembled on the surface of a carbon nanotube. For that, we used QDs of cadmium telluride and cyanobacterial phycobilisome rods (PBSr) or light-harvesting complex II (LHCII) of higher plants. For this nanobiohybrid, we confirmed composition and organization using infrared spectroscopy, X-ray photoelectron spectroscopy, and high-resolution confocal microscopy. Then, we proved that within such an assembly, there is a resonance energy transfer from QD to PBSr or LHCII. When such a nanobiohybrid was further combined with thylakoids, the energy was transferred to photosynthetic reaction centers and efficiently powered the photosystem I reaction center. The presented construct is proof of a general concept, combining interacting elements on a platform of a nanotube, allowing further variation within assembled elements.

Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP(+) oxidoreductase.

This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP(+) oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed.Electronic supplementary material The online version of this article (doi:10.1007/s10867-012-9262-z) contains supplementary material, which is available to authorized users.

QD:Puf Nanohybrids Are Compatible with Studies in Cells.

Colloidal semiconductor quantum dots (QD), as well as other nanoparticles, are useful in cell studies as fluorescent labels. They may also be used as more active components in various cellular assays, serving as sensors or effectors. However, not all QDs are biocompatible. One of the main problems is their outer coat, which needs to be stable and to sustain hydrophilicity. Here we show that purpose-designed CdSe QDs, covered with a Puf protein, can be efficiently accumulated by HeLa cells. The uptake was measurable after a few hours of incubation with nanoparticles and most of the fluorescence was localised in the internal membrane system of the cell, including the endoplasmic reticulum and the Golgi apparatus. The fluorescence properties of QDs were mostly preserved, although the maximum emission wavelength was slightly shifted, and the fluorescence lifetime was shortened, indicating partial sensitivity of the QDs to the cell microenvironment. QD accumulation resulted in a decrease in cell viability, which was attributed to disturbance of endoplasmic reticulum performance.

De novo design of a non-natural fold for an iron-sulfur protein: alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core.

Using a 'metal-first' approach, we computationally designed, prepared, and characterized a four-iron four-sulfur (Fe(4)S(4)) cluster protein with a non-natural alpha-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe(4)S(4) cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe(4)S(4) clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, alpha-helical coiled-coil binding a single Fe(4)S(4) cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of low-potential, [Fe(4)S(4)](+) (S=1/2), with g values of g(zy)=(1.970, 1.975), and g(x)=2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe(4)S(4)](+) clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as "wires" for coupling any given set of redox enzymes.

Zinc-bacteriochlorophyllide dimers in de novo designed four-helix bundle proteins. A model system for natural light energy harvesting and dissipation.

Photosynthetic organisms utilize interacting pairs of chlorophylls and bacteriochlorophylls as excitation energy donors and acceptors in light harvesting complexes, as photosensitizers of charge separation in reaction centers, and maybe as photoprotective quenching centers that dissipate excess excitation energy under high light intensities. To better understand how the pigment's local environment and spatial organization within the protein tune its ground- and excited-state properties to perform different functions, we prepared and characterized the simplest possible system of interacting bacteriochlorophylls within a protein scaffold. Using HP7, a high-affinity heme-binding protein of the HP class of de novo designed four-helix bundles, we incorporated 13(2)-OH-zinc-bacteriochlorophyllide-a (ZnBChlide), a water-soluble bacteriochlorophyll derivative, into specific binding sites within the four-helix bundle protein core. We capitalized on the rich and informative optical spectrum of ZnBChlide to rigorously characterize its complexes with HP7 and two variants, in which a single heme-binding site is eliminated by replacing histidine residues at positions 7 or 42 by phenylalanine. Surprisingly, we found the ZnBChlide binding capacity of HP7 and its variants to be higher than for heme: up to three ZnBChlide pigments bind per HP7, or two per each single histidine variant. The formation of dimers within HP7 results in dramatic quenching of ZnBChlide fluorescence, reducing its quantum yield by about 80%, and the singlet excited-state lifetime by 2 orders of magnitudes compared to the monomer. Thus, HP7 and its variants are the first examples of a simple protein environment that can isolate a self-quenching pair of photosynthetic pigments in pure form. Unlike its complicated natural analogues, this system can be constructed from the ground up, starting with the simplest functional element, increasing the complexity as needed.

Competition between Photoinduced Electron Transfer and Resonance Energy Transfer in an Example of Substituted Cytochrome c-Quantum Dot Systems.

Colloidal quantum dots (QDs) are nanoparticles that are able to photoreduce redox proteins by electron transfer (ET). QDs are also able to transfer energy by resonance energy transfer (RET). Here, we address the question of the competition between these two routes of QDs' excitation quenching, using cadmium telluride QDs and cytochrome c (CytC) or its metal-substituted derivatives. We used both oxidized and reduced versions of native CytC, as well as fluorescent, nonreducible Zn(II)CytC, Sn(II)CytC, and metal-free porphyrin CytC. We found that all of the CytC versions quench QD fluorescence, although the interaction may be described differently in terms of static and dynamic quenching. QDs may be quenchers of fluorescent CytC derivatives, with significant differences in effectiveness depending on QD size. SnCytC and porphyrin CytC increased the rate of Fe(III)CytC photoreduction, and Fe(II)CytC slightly decreased the rate and ZnCytC presence significantly decreased the rate and final level of reduced FeCytC. These might be partially explained by the tendency to form a stable complex between protein and QDs, which promoted RET and collisional quenching. Our findings show that there is a net preference for photoinduced ET over other ways of energy transfer, at least partially, due to a lack of donors, regenerating a hole at QDs and leading to irreversibility of ET events. There may also be a common part of pathways leading to photoinduced ET and RET. The nature of synergistic action observed in some cases allows the hypothesis that RET may be an additional way to power up the ET.

Interaction of ferredoxin:NADP+ oxidoreductase with model membranes.

The ferredoxin:NADP+ oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR-membrane interaction is discussed.

Nanoparticles as energy donors and acceptors in bionanohybrid systems.

The bionanohybrids are the junctions of at least two objects of different origin: abiotic and biotic. The abiotic part is a nanoparticle (often a fluorescent quantum dot), the biotical one may be a protein (especially fluorescent one or redox-active one), nucleic acid, carbohydrate as well as a simple organic molecule. When such a junction undergoes illumination, the energy transfer between the partners is possible. The nanoparticles, depending on their characteristics, may be donors, acceptors or mediators of the energy transfer. In most cases, the mechanism of the transfer is the Förster resonance energy transfer (FRET) or the electron transfer (ET). Here, we reviewed the newest achievements in the field with special attention paid to those bionanohybrids which allow FRET or ET. Such nanohybrids are important not only for exploration of the mechanism of the partner interaction but mainly for working out nanobiodevices for biosensing and nanotools for modern therapies.