Photosynthesis regulation in response to fluctuating light in the secondary endosymbiont alga Nannochloropsis gaditana

In nature, photosynthetic organisms are exposed to highly dynamic environmental conditions where the excitation energy and electron flow in the photosynthetic apparatus need to be continuously modulated. Fluctuations in incident light are particularly challenging since they drive oversaturation of photosynthesis, with consequent oxidative stress and photoinhibition. Plants and algae have evolved several mechanisms to modulate their photosynthetic machinery to cope with light dynamics, such as thermal dissipation of excited chlorophyll states (Non-Photochemical Quenching, NPQ) and regulation of electron transport. The regulatory mechanisms involved in the response to light dynamics have adapted during evolution and exploring biodiversity is a valuable strategy for expanding our understanding of their biological roles. In this work, we investigated the response to fluctuating light in Nannochloropsis gaditana, a eukaryotic microalga of the phylum Heterokonta originating from a secondary endosymbiotic event. N. gaditana is negatively affected by light fluctuations, leading to large reductions in growth and photosynthetic electron transport. Exposure to light fluctuations specifically damages photosystem I, likely because of ineffective regulation of electron transport in this species. The role of Non-Photochemical Quenching, also assessed using a mutant strain specifically depleted of this response, was instead found to be minor, especially in responding to the fastest light fluctuations

Antenna complexes protect Photosystem I from Photoinhibition

Background Photosystems are composed of two moieties, a reaction center and a peripheral antenna system. In photosynthetic eukaryotes the latter system is composed of proteins belonging to Lhc family. An increasing set of evidences demonstrated how these polypeptides play a relevant physiological function in both light harvesting and photoprotection. Despite the sequence similarity between antenna proteins associated with the two Photosystems, present knowledge on their physiological role is mostly limited to complexes associated to Photosystem II. Results In this work we analyzed the physiological role of Photosystem I antenna system in Arabidopsis thaliana both in vivo and in vitro. Plants depleted in individual antenna polypeptides showed a reduced capacity for photoprotection and an increased production of reactive oxygen species upon high light exposure. In vitro experiments on isolated complexes confirmed that depletion of antenna proteins reduced the resistance of isolated Photosystem I particles to high light and that the antenna is effective in photoprotection only upon the interaction with the core complex. Conclusions We show that antenna proteins play a dual role in Arabidopsis thaliana Photosystem I photoprotection: first, a Photosystem I with an intact antenna system is more resistant to high light because of a reduced production of reactive oxygen species and, second, antenna chlorophyll-proteins are the first target of high light damages. When photoprotection mechanisms become insufficient, the antenna chlorophyll proteins act as fuses: LHCI chlorophylls are degraded while the reaction center photochemical activity is maintained. Differences with respect to photoprotection strategy in Photosystem II, where the reaction center is the first target of photoinhibition, are discussed

Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein

<p>Abstract</p> <p>Background</p> <p>Reactive oxygen species (ROS) are unavoidable by-products of oxygenic photosynthesis, causing progressive oxidative damage and ultimately cell death. Despite their destructive activity they are also signalling molecules, priming the acclimatory response to stress stimuli.</p> <p>Results</p> <p>To investigate this role further, we exposed wild type <it>Arabidopsis thaliana </it>plants and the double mutant <it>npq1lut2 </it>to excess light. The mutant does not produce the xanthophylls lutein and zeaxanthin, whose key roles include ROS scavenging and prevention of ROS synthesis. Biochemical analysis revealed that singlet oxygen (¹O₂) accumulated to higher levels in the mutant while other ROS were unaffected, allowing to define the transcriptomic signature of the acclimatory response mediated by ¹O₂which is enhanced by the lack of these xanthophylls species. The group of genes differentially regulated in <it>npq1lut2 </it>is enriched in sequences encoding chloroplast proteins involved in cell protection against the damaging effect of ROS. Among the early fine-tuned components, are proteins involved in tetrapyrrole biosynthesis, chlorophyll catabolism, protein import, folding and turnover, synthesis and membrane insertion of photosynthetic subunits. Up to now, the <it>flu </it>mutant was the only biological system adopted to define the regulation of gene expression by ¹O₂. In this work, we propose the use of mutants accumulating ¹O₂by mechanisms different from those activated in <it>flu </it>to better identify ROS signalling.</p> <p>Conclusions</p> <p>We propose that the lack of zeaxanthin and lutein leads to ¹O₂accumulation and this represents a signalling pathway in the early stages of stress acclimation, beside the response to ADP/ATP ratio and to the redox state of both plastoquinone pool. Chloroplasts respond to ¹O₂accumulation by undergoing a significant change in composition and function towards a fast acclimatory response. The physiological implications of this signalling specificity are discussed.</p

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR

Cultivation of Gongolaria barbata (Fucales, Phaeophyceae) with a seaweed‐derived biostimulant in order to improve photophysiological fitness and promote fertility to advance the restoration of marine macroalgal forests

As a result of several anthropogenic factors, Cystoseira sensu lato forests have declined or become regionally extinct in many coastal regions of the Mediterranean. Given the low natural recovery of lost populations, research efforts have been encouraged to develop sustainable and efficient restoration of macroalgal forests on a large scale. By promoting growth and fertility of collected thallus branches under controlled laboratory conditions, the availability of seedlings for restoration could be ensured without jeopardizing natural populations. Here we investigated the effect of a commercial algal biostimulant (AlgatronCifo®) on the photophysiology, growth and fertility of Gongolaria barbata (Stackhouse) Kuntze (Fucales, Phaeophyceae). In a factorial laboratory experiment, two different temperatures (10 oC and 14 °C) and two culture media [i.e. seawater (SW) and Algatron (AT)] were tested. The photosynthetic performance of G. barbata doubled after three weeks of culture with AT, while it decreased by 25% when cultivated in SW. The highest photosynthetic performance and growth were achieved at 14oC with AT, where fertile receptacles also developed, followed by seedling settlements. The thalli cultured in AT had similar or better photosynthetic performance than the initial control thalli. AT-cultured thalli had a greater ability to quench energy via photochemical pathways (qP) than those from the SW, which on the contrary, had higher levels of non-photochemical responses (qN, NPQmax). This limited photosynthetic performance was probably linked to the higher P-limitation experienced under that treatment. The algal biostimulant enhanced the physiological performance and induced fertility of G. barbata, demonstrating its valorization potential and setting a new path for improved restoration applications

Higher order photoprotection mutants reveal the importance of \u394pH-dependent photosynthesis-control in preventing light induced damage to both photosystem II and photosystem I

Although light is essential for photosynthesis, when in excess, it may damage the photosynthetic apparatus, leading to a phenomenon known as photoinhibition. Photoinhibition was thought as a light-induced damage to photosystem II; however, it is now clear that even photosystem I may become very vulnerable to light. One main characteristic of light induced damage to photosystem II (PSII) is the increased turnover of the reaction center protein, D1: when rate of degradation exceeds the rate of synthesis, loss of PSII activity is observed. With respect to photosystem I (PSI), an excess of electrons, instead of an excess of light, may be very dangerous. Plants possess a number of mechanisms able to prevent, or limit, such damages by safe thermal dissipation of light energy (non-photochemical quenching, NPQ), slowing-down of electron transfer through the intersystem transport chain (photosynthesis-control, PSC) in co-operation with the Proton Gradient Regulation (PGR) proteins, PGR5 and PGRL1, collectively called as short-term photoprotection mechanisms, and the redistribution of light between photosystems, called state transitions (responsible of fluorescence quenching at PSII, qT), is superimposed to these short term photoprotective mechanisms. In this manuscript we have generated a number of higher order mutants by crossing genotypes carrying defects in each of the short-term photoprotection mechanisms, with the final aim to obtain a direct comparison of their role and efficiency in photoprotection. We found that mutants carrying a defect in the \u394pH-dependent photosynthesis-control are characterized by photoinhibition of both photosystems, irrespectively of whether PSBS-dependent NPQ or state transitions defects were present or not in the same individual, demonstrating the primary role of PSC in photoprotection. Moreover, mutants with a limited capability to develop a strong PSBS-dependent NPQ, were characterized by a high turnover of the D1 protein and high values of Y(NO), which might reflect energy quenching processes occurring within the PSII reaction center

A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing

Conservation of core complex subunits shaped the structure and function of photosystem I in the secondary endosymbiont alga Nannochloropsis gaditana

Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained