Fluorescence quenching in thylakoid membranes induced by single-walled carbon nanotubes.

The distinct photochemical and electrochemical properties of single-walled carbon nanotubes (SWCNTs) boosted the research interest in nanomaterial utilization in different in vivo and in vitro photosynthetic biohybrid setups. Aiming to unravel the yet not fully understood energetic interactions between the nanotubes and photosynthetic pigment-protein assemblies in an aqueous milieu, we studied SWCNT effects on the photochemical reactions of isolated thylakoid membranes (TMs), Photosystem II (PSII)-enriched membrane fragments and light-harvesting complexes (LHCII). The SWCNTs induced quenching of the steady-state chlorophyll fluorescence in the TM-biohybrid systems with a corresponding shortening of the average fluorescence lifetimes. The effect was not related to changes in the integrity and macroorganization of the photosynthetic membranes. Moreover, we found no evidence for direct excitation energy exchange between the SWCNTs and pigment-protein complexes, since neither the steady-state nor time-resolved fluorescence of LHCII-biohybrid systems differed from the corresponding controls. The attenuation of the fluorescence signal in the TM-biohybrid systems indicates possible leakage of photosynthetic electrons toward the nanotubes that most probably occurs at the level of the PSII acceptor site. Although it is too early to speculate on the nature of the involved electron donors and intermediate states, the observed energetic interaction could be exploited to increase the photoelectron capture efficiency of natural biohybrid systems for solar energy conversion.

Nanodiamond Particles Reduce Oxidative Stress Induced by Methyl Viologen and High Light in the Green Alga Chlamydomonas reinhardtii.

Widely used in biomedical and bioanalytical applications, the detonation nanodiamonds (NDs) are generally considered to be biocompatible and non-toxic to a wide range of eukaryotic cells. Due to their high susceptibility to chemical modifications, surface functionalisation is often used to tune the biocompatibility and antioxidant activity of the NDs. The response of photosynthetic microorganisms to redox-active NDs is still poorly understood and is the focus of the present study. The green microalga Chlamydomonas reinhardtii was used to assess the potential phytotoxicity and antioxidant activity of NDs hosting hydroxyl functional groups at concentrations of 5-80 µg NDs/mL. The photosynthetic capacity of microalgae was assessed by measuring the maximum quantum yield of PSII photochemistry and the light-saturated oxygen evolution rate, while oxidative stress was assessed by lipid peroxidation and ferric-reducing antioxidant capacity. We demonstrated that hydroxylated NDs might reduce cellular levels of oxidative stress, protect PSII photochemistry and facilitate the PSII repair under methyl viologen and high light associated stress conditions. Factors involved in this protection may include the low phytotoxicity of hydroxylated NDs in microalgae and their ability to accumulate in cells and scavenge reactive oxygen species. Our findings could pave the way for using hydroxylated NDs as antioxidants to improve cellular stability in algae-based biotechnological applications or semi-artificial photosynthetic systems.

Single-walled carbon nanotubes protect photosynthetic reactions in Chlamydomonas reinhardtii against photoinhibition.

Single-walled carbon nanotubes (SWCNTs) are among the most exploited carbon allotropes in nanosensing, bioengineering, and photobiological applications, however, the interactions of nanotubes with the photosynthetic process and structures are still poorly understood. We found that SWCNTs are not toxic to the photosynthetic apparatus of the model unicellular alga Chlamydomonas reinhardtii and demonstrate that this carbon nanomaterial can protect algal photosynthesis against photoinhibition. The results show that the inherent phytotoxicity of the nanotubes may be overcome by an intentional selection of nanomaterial characteristics. A low concentration (2 µg mL-1) of well-dispersed, purified and small SWCNTs did not alter the growth and pigment accumulation of the cultures. Indeed, under the photoinhibitory conditions of our experiments, SWCNT-enriched samples were characterized by a lower rate of PSII inactivation, reduced excitation pressure in PSII, a higher rate of photosynthetic electron transport, and an increased non-photochemical quenching in comparison with the controls. In addition, SWCNTs change the distribution of energy between the photosystems in favour of PSII (state 1). The underlying mechanism of this action is not yet understood but possibly, electrons or energy can be exchanged between the redox active nanotubes and photosynthetic components, and probably other redox active intra-chloroplast constituents. Alternatively, nanotubes may promote the formation of an NPQ conformation of PSII. Our results provided evidence for such electron/energy transfer from photosynthetic structures toward the nanotubes. The discovered photoprotective effects can potentially be used in photobiotechnology to maintain the photosynthetic activity of microorganisms under unfavourable conditions.

Mapping Single Walled Carbon Nanotubes in Photosynthetic Algae by Single-Cell Confocal Raman Microscopy.

Carbon nanotubes (CNTs) are among the most exploited carbon allotropes in the emerging technologies of molecular sensing and bioengineering. However, the advancement of algal nanobiotechnology and nanobionics is hindered by the lack of methods for the straightforward visualization of the CNTs inside the cell. Herein, we present a handy and label-free experimental strategy based on visible Raman microscopy to assess the internalization of single-walled carbon nanotubes (SWCNTs) using the model photosynthetic alga Chlamydomonas reinhardtii as a recipient. The relationship between the properties of SWCNTs and their biological behavior was demonstrated, along with the occurrence of excitation energy transfer from the excited chlorophyll molecules to the SWCNTs. The non-radiative deactivation of the chlorophyll excitation promoted by the SWCNTs enables the recording of Raman signals originating from cellular compounds located near the nanotubes, such as carotenoids, polyphosphates, and starch. Furthermore, the outcome of this study unveils the possibility to exploit SWCNTs as spectroscopic probes in photosynthetic and non-photosynthetic systems where the fluorescence background hinders the acquisition of Raman scattering signals.

Photosystem-II D1 protein mutants of Chlamydomonas reinhardtii in relation to metabolic rewiring and remodelling of H-bond network at QB site.

Photosystem II (PSII) reaction centre D1 protein of oxygenic phototrophs is pivotal for sustaining photosynthesis. Also, it is targeted by herbicides and herbicide-resistant weeds harbour single amino acid substitutions in D1. Conservation of D1 primary structure is seminal in the photosynthetic performance in many diverse species. In this study, we analysed built-in and environmentally-induced (high temperature and high photon fluency - HT/HL) phenotypes of two D1 mutants of Chlamydomonas reinhardtii with Ala250Arg (A250R) and Ser264Lys (S264K) substitutions. Both mutations differentially affected efficiency of electron transport and oxygen production. In addition, targeted metabolomics revealed that the mutants undergo specific differences in primary and secondary metabolism, namely, amino acids, organic acids, pigments, NAD, xanthophylls and carotenes. Levels of lutein, ß-carotene and zeaxanthin were in sync with their corresponding gene transcripts in response to HT/HL stress treatment in the parental (IL) and A250R strains. D1 structure analysis indicated that, among other effects, remodelling of H-bond network at the QB site might underpin the observed phenotypes. Thus, the D1 protein, in addition to being pivotal for efficient photosynthesis, may have a moonlighting role in rewiring of specific metabolic pathways, possibly involving retrograde signalling.

Features of cues and processes during chloroplast-mediated retrograde signaling in the alga Chlamydomonas.

Retrograde signaling is an intracellular communication process defined by cues generated in chloroplast and mitochondria which traverse membranes to their destination in the nucleus in order to regulate nuclear gene expression and protein synthesis. The coding and decoding of such organellar message(s) involve gene medleys and metabolic components about which more is known in higher plants than the unicellular organisms such as algae. Chlamydomonas reinhardtii is an oxygenic microalgal model for genetic and physiological studies. It harbors a single chloroplast and is amenable for generating mutants. The focus of this review is on studies that delineate retrograde signaling in Chlamydomonas vis a vis higher plants. Thus, communication networks between chloroplast and nucleus involving photosynthesis- and ROS-generated signals, functional tetrapyrrole biosynthesis intermediates, and Ca2+-signaling that modulate nuclear gene expression in this alga are discussed. Conceptually, different signaling components converge to regulate either the same or functionally-overlapping gene products.

Comparative analyses of H2 photoproduction in magnesium- and sulfur-starved Chlamydomonas reinhardtii cultures.

Magnesium (Mg)-deprived Chlamydomonas reinhardtii cells are capable to sustain hydrogen (H2 ) photoproduction at relatively high photosystem II (PSII) activity levels for an extended time period as compared with sulfur (S)-deprived cells. Herein, we present a comparative study of H2 photoproduction induced by Mg and S shortage to unravel the specific rearrangements of the photosynthetic machinery and cell metabolism occurring under the two deprivation protocols. The exhaustive analysis of photosynthetic activity and regulatory pathways, respiration and starch metabolism revealed the specific rearrangements of the photosynthetic machinery and cellular metabolism, which occur under the two deprivation conditions. The obtained results allowed us to conclude that the expanded time period of H2 production upon Mg-deprivation is due to the less harmful effects that Mg-depletion has on viability and metabolic performance of the cells. Unlike S-deprivation, the photosynthetic light and dark reactions in Mg-deprived cells remained active over the whole H2 production period. However, the elevated PSII activity in Mg-deprived cells was counteracted by the operation of pathways for O2 consumption that maintain anaerobic conditions in the presence of active water splitting.

The plastoquinol-plastoquinone exchange mechanism in photosystem II: insight from molecular dynamics simulations.

In the photosystem II (PSII) of oxygenic photosynthetic organisms, the reaction center (RC) core mediates the light-induced electron transfer leading to water splitting and production of reduced plastoquinone molecules. The reduction of plastoquinone to plastoquinol lowers PSII affinity for the latter and leads to its release. However, little is known about the role of protein dynamics in this process. Here, molecular dynamics simulations of the complete PSII complex embedded in a lipid bilayer have been used to investigate the plastoquinol release mechanism. A distinct dynamic behavior of PSII in the presence of plastoquinol is observed which, coupled to changes in charge distribution and electrostatic interactions, causes disruption of the interactions seen in the PSII-plastoquinone complex and leads to the "squeezing out" of plastoquinol from the binding pocket. Displacement of plastoquinol closes the second water channel, recently described in a 2.9 Å resolution PSII structure (Guskov et al. in Nat Struct Mol Biol 16:334-342, 2009), allowing to rule out the proposed "alternating" mechanism of plastoquinol-plastoquinone exchange, while giving support to the "single-channel" one. The performed simulations indicated a pivotal role of D1-Ser264 in modulating the dynamics of the plastoquinone binding pocket and plastoquinol-plastoquinone exchange via its interaction with D1-His252 residue. The effects of the disruption of this hydrogen bond network on the PSII redox reactions were experimentally assessed in the D1 site-directed mutant Ser264Lys.

Water Collective Dynamics in Whole Photosynthetic Green Algae as Affected by Protein Single Mutation.

In the context of the importance of water molecules for protein function/dynamics relationship, the role of water collective dynamics in Chlamydomonas green algae carrying both native and mutated photosynthetic proteins has been investigated by neutron Brillouin scattering spectroscopy. Results show that single point genetic mutation may notably affect collective density fluctuations in hydrating water providing important insight on the transmission of information possibly correlated to biological functionality. In particular, we highlight that the damping factor of the excitations is larger in the native compared to the mutant algae as a signature of a different plasticity and structure of the hydrogen bond network.

Synthetic biology and biomimetic chemistry as converging technologies fostering a new generation of smart biosensors.

Biosensors are powerful tunable systems able to switch between an ON/OFF status in response to an external stimulus. This extraordinary property could be engineered by adopting synthetic biology or biomimetic chemistry to obtain tailor-made biosensors having the desired requirements of robustness, sensitivity and detection range. Recent advances in both disciplines, in fact, allow to re-design the configuration of the sensing elements - either by modifying toggle switches and gene networks, or by producing synthetic entities mimicking key properties of natural molecules. The present review considered the role of synthetic biology in sustaining biosensor technology, reporting examples from the literature and reflecting on the features that make it a useful tool for designing and constructing engineered biological systems for sensing application. Besides, a section dedicated to bioinspired synthetic molecules as powerful tools to enhance biosensor potential is reported, and treated as an extension of the concept of biomimetic chemistry, where organic synthesis is used to generate artificial molecules that mimic natural molecules. Thus, the design of synthetic molecules, such as aptamers, biomimetics, molecular imprinting polymers, peptide nucleic acids, and ribozymes were encompassed as "products" of biomimetic chemistry.

Photosynthesis at the forefront of a sustainable life.

The development of a sustainable bio-based economy has drawn much attention in recent years, and research to find smart solutions to the many inherent challenges has intensified. In nature, perhaps the best example of an authentic sustainable system is oxygenic photosynthesis. The biochemistry of this intricate process is empowered by solar radiation influx and performed by hierarchically organized complexes composed by photoreceptors, inorganic catalysts, and enzymes which define specific niches for optimizing light-to-energy conversion. The success of this process relies on its capability to exploit the almost inexhaustible reservoirs of sunlight, water, and carbon dioxide to transform photonic energy into chemical energy such as stored in adenosine triphosphate. Oxygenic photosynthesis is responsible for most of the oxygen, fossil fuels, and biomass on our planet. So, even after a few billion years of evolution, this process unceasingly supports life on earth, and probably soon also in outer-space, and inspires the development of enabling technologies for a sustainable global economy and ecosystem. The following review covers some of the major milestones reached in photosynthesis research, each reflecting lasting routes of innovation in agriculture, environmental protection, and clean energy production.

Structure/function/dynamics of photosystem II plastoquinone binding sites.

Photosystem II (PSII) continuously attracts the attention of researchers aiming to unravel the riddle of its functioning and efficiency fundamental for all life on Earth. Besides, an increasing number of biotechnological applications have been envisaged exploiting and mimicking the unique properties of this macromolecular pigment-protein complex. The PSII organization and working principles have inspired the design of electrochemical water splitting schemes and charge separating triads in energy storage systems as well as biochips and sensors for environmental, agricultural and industrial screening of toxic compounds. An intriguing opportunity is the development of sensor devices, exploiting native or manipulated PSII complexes or ad hoc synthesized polypeptides mimicking the PSII reaction centre proteins as biosensing elements. This review offers a concise overview of the recent improvements in the understanding of structure and function of PSII donor side, with focus on the interactions of the plastoquinone cofactors with the surrounding environment and operational features. Furthermore, studies focused on photosynthetic proteins structure/function/dynamics and computational analyses aimed at rational design of high-quality bio-recognition elements in biosensor devices are discussed.

Mutations of photosystem II D1 protein that empower efficient phenotypes of Chlamydomonas reinhardtii under extreme environment in space.

Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga, Chlamydomonas reinhardtii, which has been widely used as a model of photosynthetic organisms. The algal mutants contained specific amino acid substitutions in the functionally important regions of the pivotal Photosystem II (PSII) reaction centre D1 protein near the QB binding pocket and in the environment surrounding Tyr-161 (YZ) electron acceptor of the oxygen-evolving complex. Using real-time measurements of PSII photochemistry, here we show that during the space flight while the control strain and two D1 mutants (A250L and V160A) were inefficient in carrying out PSII activity, two other D1 mutants, I163N and A251C, performed efficient photosynthesis, and actively re-grew upon return to Earth. Mimicking the neutron irradiation component of cosmic rays on Earth yielded similar results. Experiments with I163N and A251C D1 mutants performed on ground showed that they are better able to modulate PSII excitation pressure and have higher capacity to reoxidize the QA (-) state of the primary electron acceptor. These results highlight the contribution of D1 conformation in relation to photosynthesis and oxygen production in space.

A powerful molecular engineering tool provided efficient Chlamydomonas mutants as bio-sensing elements for herbicides detection.

This study was prompted by increasing concerns about ecological damage and human health threats derived by persistent contamination of water and soil with herbicides, and emerging of bio-sensing technology as powerful, fast and efficient tool for the identification of such hazards. This work is aimed at overcoming principal limitations negatively affecting the whole-cell-based biosensors performance due to inadequate stability and sensitivity of the bio-recognition element. The novel bio-sensing elements for the detection of herbicides were generated exploiting the power of molecular engineering in order to improve the performance of photosynthetic complexes. The new phenotypes were produced by an in vitro directed evolution strategy targeted at the photosystem II (PSII) D1 protein of Chlamydomonas reinhardtii, using exposures to radical-generating ionizing radiation as selection pressure. These tools proved successful to identify D1 mutations conferring enhanced stability, tolerance to free-radical-associated stress and competence for herbicide perception. Long-term stability tests of PSII performance revealed the mutants capability to deal with oxidative stress-related conditions. Furthermore, dose-response experiments indicated the strains having increased sensitivity or resistance to triazine and urea type herbicides with I(50) values ranging from 6 × 10(-8) M to 2 × 10(-6) M. Besides stressing the relevance of several amino acids for PSII photochemistry and herbicide sensing, the possibility to improve the specificity of whole-cell-based biosensors, via coupling herbicide-sensitive with herbicide-resistant strains, was verified.

Trapping of the quenched conformation associated with non-photochemical quenching of chlorophyll fluorescence at low temperature.

The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures between 5 and 25 degrees C and during rapid jumps of the leaf temperature. At 5 degrees C, NPQ relaxed very slowly in the dark and was sustained for up to 30 min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to 25 degrees C, the quenched state relaxed within 1 min, characteristic for qE, the energy-dependent component of NPQ. Measurements of the membrane permeability (delta A515) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending the model of Horton and co-workers (FEBS Lett 579:4201-4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE.

Electrokinetic and light scattering properties of pea and Chlamydomonas reinhardtii thylakoid membranes: effect of phytohemagglutinin.

The effects of phytohemagglutinin (PHA) and illumination on the surface charge densities and 90 degrees light scattering properties of pea and Chlamydomonas reinhardtii thylakoids were investigated. The electrophoretic mobility (EPM) of pea thylakoids decreased after treatment by various concentrations of PHA at ionic strengths of I = 0.01 and I = 0.02, while that of C. reinhardtii thylakoids remained stable except for a drop after treatment by PHA at a concentration of 6 ng/mL in a medium with an ionic strength of I = 0.01. Illumination did not influence the EPM of untreated thylakoids. However, if the EPM of thylakoids had been retarded by pretreatment with PHA, light exposure stimulated a recovery of the reduced negative surface charge density up to at least the initial values. In addition to reducing EPM, PHA also induced a decrease of the basal light scattering property of pea thylakoids, which is an indicator of thylakoid aggregation. The physiological role of the membrane surface charges of thylakoid particles in lectin regulated processes of thylakoid stacking and activity is discussed.