Assessment of photosynthetic activity in dense microalgae cultures using oxygen production.

Microalgae are photosynthetic microorganisms playing a pivotal role in primary production in aquatic ecosystems, sustaining the entry of carbon in the biosphere. Microalgae have also been recognized as sustainable source of biomass to complement crops. For this objective they are cultivated in photobioreactors or ponds at high cell density to maximize biomass productivity and lower the cost of downstream processes. Photosynthesis depends on light availability, that is often not constant over time. In nature, sunlight fluctuates over diurnal cycles and weather conditions. In high-density microalgae cultures of photobioreactors outdoors, on top of natural variations, microalgae are subjected to further complexity in light exposure. Because of the high-density cells experience self-shading effects that heavily limit light availability in most of the mass culture volume. This limitation strongly affects biomass productivity of industrial microalgae cultivation plants with important implications on economic feasibility. Understanding how photosynthesis responds to cell density is informative to assess functionality in the inhomogeneous light environment of industrial photobioreactors. In this work we exploited a high-sensitivity Clark electrode to measure microalgae photosynthesis and compare cultures with different densities, using Nannochloropsis as model organism. We observed that cell density has a substantial impact on photosynthetic activity, and demonstrated the reduction of the cell's light-absorption capacity by genetic modification is a valuable strategy to increase photosynthetic functionality on a chlorophyll-basis of dense microalgae cultures.

Biological carbon capture from biogas streams: Insights into Cupriavidus necator autotrophic growth and transcriptional profile.

Recycling carbon-rich wastes into high-value platform chemicals through biological processes provides a sustainable alternative to petrochemicals. Cupriavidus necator, known for converting carbon dioxide (CO2) into polyhydroxyalkanoates (PHA) was studied for the first time using biogas streams as the sole carbon source. The bacterium efficiently consumed biogenic CO2 from raw biogas with methane at high concentrations (50%) proving non-toxic. Continuous addition of H2 and O2 enabled growth trends comparable to glucose-based heterotrophic growth. Transcriptomic analysis revealed CO2-adaptated cultures exhibited upregulation of hydrogenases and Calvin cycle enzymes, as well as genes related to electron transport, nutrient uptake, and glyoxylate cycle. Non-adapted samples displayed activation of stress response mechanisms, suggesting potential lags in large-scale processes. These findings showcase the setting of growth parameters for a pioneering biological biogas upgrading strategy, emphasizing the importance of inoculum adaptation for autotrophic growth and providing potential targets for genetic engineering to push PHA yields in future applications.

Cupriavidus necator as a platform for polyhydroxyalkanoate production: An overview of strains, metabolism, and modeling approaches.

Cupriavidus necator is a bacterium with a high phenotypic diversity and versatile metabolic capabilities. It has been extensively studied as a model hydrogen oxidizer, as well as a producer of polyhydroxyalkanoates (PHA), plastic-like biopolymers with a high potential to substitute petroleum-based materials. Thanks to its adaptability to diverse metabolic lifestyles and to the ability to accumulate large amounts of PHA, C. necator is employed in many biotechnological processes, with particular focus on PHA production from waste carbon sources. The large availability of genomic information has enabled a characterization of C. necator's metabolism, leading to the establishment of metabolic models which are used to devise and optimize culture conditions and genetic engineering approaches. In this work, the characteristics of available C. necator strains and genomes are reviewed, underlining how a thorough comprehension of the genetic variability of C. necator is lacking and it could be instrumental for wider application of this microorganism. The metabolic paradigms of C. necator and how they are connected to PHA production and accumulation are described, also recapitulating the variety of carbon substrates used for PHA accumulation, highlighting the most promising strategies to increase the yield. Finally, the review describes and critically analyzes currently available genome-scale metabolic models and reduced metabolic network applications commonly employed in the optimization of PHA production. Overall, it appears that the capacity of C. necator of performing CO2 bioconversion to PHA is still underexplored, both in biotechnological applications and in metabolic modeling. However, the accurate characterization of this organism and the efforts in using it for gas fermentation can help tackle this challenging perspective in the future.

Improving photosynthetic efficiency toward food security: Strategies, advances, and perspectives.

Photosynthesis in crops and natural vegetation allows light energy to be converted into chemical energy and thus forms the foundation for almost all terrestrial trophic networks on Earth. The efficiency of photosynthetic energy conversion plays a crucial role in determining the portion of incident solar radiation that can be used to generate plant biomass throughout a growth season. Consequently, alongside the factors such as resource availability, crop management, crop selection, maintenance costs, and intrinsic yield potential, photosynthetic energy use efficiency significantly influences crop yield. Photosynthetic efficiency is relevant to sustainability and food security because it affects water use efficiency, nutrient use efficiency, and land use efficiency. This review focuses specifically on the potential for improvements in photosynthetic efficiency to drive a sustainable increase in crop yields. We discuss bypassing photorespiration, enhancing light use efficiency, harnessing natural variation in photosynthetic parameters for breeding purposes, and adopting new-to-nature approaches that show promise for achieving unprecedented gains in photosynthetic efficiency.

Modulation of xanthophyll cycle impacts biomass productivity in the marine microalga Nannochloropsis.

Life on earth depends on photosynthetic primary producers that exploit sunlight to fix CO2 into biomass. Approximately half of global primary production is associated with microalgae living in aquatic environments. Microalgae also represent a promising source of biomass to complement crop cultivation, and they could contribute to the development of a more sustainable bioeconomy. Photosynthetic organisms evolved multiple mechanisms involved in the regulation of photosynthesis to respond to highly variable environmental conditions. While essential to avoid photodamage, regulation of photosynthesis results in dissipation of absorbed light energy, generating a complex trade-off between protection from stress and light-use efficiency. This work investigates the impact of the xanthophyll cycle, the light-induced reversible conversion of violaxanthin into zeaxanthin, on the protection from excess light and on biomass productivity in the marine microalgae of the genus Nannochloropsis. Zeaxanthin is shown to have an essential role in protection from excess light, contributing to the induction of nonphotochemical quenching and scavenging of reactive oxygen species. On the contrary, the overexpression of zeaxanthin epoxidase enables a faster reconversion of zeaxanthin to violaxanthin that is shown to be advantageous for biomass productivity in dense cultures in photobioreactors. These results demonstrate that zeaxanthin accumulation is critical to respond to strong illumination, but it may lead to unnecessary energy losses in light-limiting conditions and accelerating its reconversion to violaxanthin provides an advantage for biomass productivity in microalgae.

Structure and function of bark and wood chloroplasts in a drought-tolerant tree (Fraxinus ornus L.).

Leaves are the most important photosynthetic organs in most woody plants, but chloroplasts are also found in organs optimized for other functions. However, the actual photosynthetic efficiency of these chloroplasts is still unclear. We analyzed bark and wood chloroplasts of Fraxinus ornus L. saplings. Optical and spectroscopic methods were applied to stem samples and compared with leaves. A sharp light gradient was detected along the stem radial direction, with blue light mainly absorbed by the outer bark, and far-red-enriched light reaching the underlying xylem and pith. Chlorophylls were evident in the xylem rays and the pith and showed an increasing concentration gradient toward the bark. The stem photosynthetic apparatus showed features typical of acclimation to a low-light environment, such as larger grana stacks, lower chlorophyll a/b and photosystem I/II ratios compared with leaves. Despite likely receiving very few photons, wood chloroplasts were photosynthetically active and fully capable of generating a light-dependent electron transport. Our data provide a comprehensive scenario of the functional features of bark and wood chloroplasts in a woody species and suggest that stem photosynthesis is coherently optimized to the prevailing micro-environmental conditions at the bark and wood level.

Oxygenic photosynthetic responses of cyanobacteria exposed under an M-dwarf starlight simulator: Implications for exoplanet's habitability.

Introduction: The search for life on distant exoplanets is expected to rely on atmospheric biosignatures detection, such as oxygen of biological origin. However, it is not demonstrated how much oxygenic photosynthesis, which on Earth depends on visible light, could work under spectral conditions simulating exoplanets orbiting the Habitable Zone of M-dwarf stars, which have low light emission in the visible and high light emission in the far-red/near-infrared. By utilizing cyanobacteria, the first organisms to evolve oxygenic photosynthesis on our planet, and a starlight simulator capable of accurately reproducing the emission spectrum of an M-dwarf in the range 350-900 nm, we could answer this question. Methods: We performed experiments with the cyanobacterium Chlorogloeopsis fritschii PCC6912, capable of Far-Red Light Photoacclimation (FaRLiP), which allows the strain to harvest far-red in addition to visible light for photosynthesis, and Synechocystis sp. PCC6803, a species unable to perform this photoacclimation, comparing their responses when exposed to three simulated light spectra: M-dwarf, solar and far-red. We analysed growth and photosynthetic acclimation features in terms of pigment composition and photosystems organization. Finally, we determined the oxygen production of the strains directly exposed to the different spectra. Results: Both cyanobacteria were shown to grow and photosynthesize similarly under M-dwarf and solar light conditions: Synechocystis sp. by utilizing the few photons in the visible, C. fritschii by harvesting both visible and far-red light, activating the FaRLiP response. Discussion: Our results experimentally show that an M-dwarf light spectrum could support a biological oxygen production similar to that in solar light at the tested light intensities, suggesting the possibility to discover such atmospheric biosignatures on those exoplanets if other boundary conditions are met.

Functional analysis of PsbS transmembrane domains through base editing in Physcomitrium patens.

Plants exposed to light fluctuations are protected from photodamage by non-photochemical quenching (NPQ), a reversible mechanism that enables dissipation of excess absorbed energy as heat, which is essential for plant fitness and crop productivity. In plants NPQ requires the presence of the membrane protein PsbS, which upon activation interacts with antenna proteins, inducing their dissipative conformation. Here, we exploited base editing (BE) in the moss Physcomitrium patens to introduce specific amino acid changes in vivo and assess their impact on PsbS activity, targeting transmembrane regions to investigate their role in essential protein-protein interactions. This approach enabled the recognition of residues essential for protein stability and the identification of a hydrophobic cluster of amino acids impacting PsbS activity. This work provides new information on the molecular mechanism of PsbS while also demonstrating the potential of BE approaches for in planta gene function analysis.

An increase in the membrane lipids recycling by PDAT overexpression stimulates the accumulation of triacylglycerol in Nannochloropsis gaditana.

Oleaginous microalgae represent potential feedstocks for the sustainable production of lipids thanks to their ability to accumulate triacylglycerols (TAGs). TAG accumulation in several algal species is strongly induced under specific conditions such as nutrient deprivation and high light which, however, also negatively impact growth. Genetic modification of lipogenic pathways can potentially enhance TAG accumulation without negatively affecting growth, avoiding the trade-off between biomass and lipid productivity. In this study, the phospholipid: diacylglycerol acyltransferase (PDAT), an enzyme involved in membrane lipid recycling, was overexpressed in the seawater alga Nannochloropsis gaditana. PDAT overexpression induced increased TAG content in actively growing algae cultures while no effects were observed in conditions naturally stimulating strong lipid accumulation such as high light and nitrogen starvation. The increase of TAG content was confirmed also in a strain cultivated in industrially relevant conditions even though PDAT overexpression, if too strong, the gene overexpression becomes detrimental for growth in the longer term. Results overall suggest that genetic modulation of the PDAT gene represents a promising strategy to increase microalgae lipid content by minimizing negative effects on biomass productivity.

Role of serine/threonine protein kinase STN7 in the formation of two distinct photosystem I supercomplexes in Physcomitrium patens.

Reversible thylakoid protein phosphorylation provides most flowering plants with dynamic acclimation to short-term changes in environmental light conditions. Here, through generating Serine/Threonine protein kinase 7 (STN7)-depleted mutants in the moss Physcomitrella (Physcomitrium patens), we identified phosphorylation targets of STN7 kinase and their roles in short- and long-term acclimation of the moss to changing light conditions. Biochemical and mass spectrometry analyses revealed STN7-dependent phosphorylation of N-terminal Thr in specific Light-Harvesting Complex II (LHCII) trimer subunits (LHCBM2 and LHCBM4/8) and provided evidence that phospho-LHCBM accumulation is responsible for the assembly of two distinct Photosystem I (PSI) supercomplexes (SCs), both of which are largely absent in STN7-depleted mutants. Besides the canonical state transition complex (PSI-LHCI-LHCII), we isolated the larger moss-specific PSI-Large (PSI-LHCI-LHCB9-LHCII) from stroma-exposed thylakoids. Unlike PSI-LHCI-LHCII, PSI-Large did not demonstrate short-term dynamics for balancing the distribution of excitation energy between PSII and PSI. Instead, PSI-Large contributed to a more stable increase in PSI antenna size in Physcomitrella, except under prolonged high irradiance. Additionally, the STN7-depleted mutants revealed altered light-dependent phosphorylation of a monomeric antenna protein, LHCB6, whose phosphorylation displayed a complex regulation by multiple kinases. Collectively, the unique phosphorylation plasticity and dynamics of Physcomitrella monomeric LHCB6 and trimeric LHCBM isoforms, together with the presence of PSI SCs with different antenna sizes and responsiveness to light changes, reflect the evolutionary position of mosses between green algae and vascular plants, yet with clear moss-specific features emphasizing their adaptation to terrestrial low-light environments.

Knowledge of Regulation of Photosynthesis in Outdoor Microalgae Cultures Is Essential for the Optimization of Biomass Productivity.

Microalgae represent a sustainable source of biomass that can be exploited for pharmaceutical, nutraceutical, cosmetic applications, as well as for food, feed, chemicals, and energy. To make microalgae applications economically competitive and maximize their positive environmental impact, it is however necessary to optimize productivity when cultivated at a large scale. Independently from the final product, this objective requires the optimization of biomass productivity and thus of microalgae ability to exploit light for CO2 fixation. Light is a highly variable environmental parameter, continuously changing depending on seasons, time of the day, and weather conditions. In microalgae large scale cultures, cell self-shading causes inhomogeneity in light distribution and, because of mixing, cells move between different parts of the culture, experiencing abrupt changes in light exposure. Microalgae evolved multiple regulatory mechanisms to deal with dynamic light conditions that, however, are not adapted to respond to the complex mixture of natural and artificial fluctuations found in large-scale cultures, which can thus drive to oversaturation of the photosynthetic machinery, leading to consequent oxidative stress. In this work, the present knowledge on the regulation of photosynthesis and its implications for the maximization of microalgae biomass productivity are discussed. Fast mechanisms of regulations, such as Non-Photochemical-Quenching and cyclic electron flow, are seminal to respond to sudden fluctuations of light intensity. However, they are less effective especially in the 1-100 s time range, where light fluctuations were shown to have the strongest negative impact on biomass productivity. On the longer term, microalgae modulate the composition and activity of the photosynthetic apparatus to environmental conditions, an acclimation response activated also in cultures outdoors. While regulation of photosynthesis has been investigated mainly in controlled lab-scale conditions so far, these mechanisms are highly impactful also in cultures outdoors, suggesting that the integration of detailed knowledge from microalgae large-scale cultivation is essential to drive more effective efforts to optimize biomass productivity.

Protein dynamics and lipid affinity of monomeric, zeaxanthin-binding LHCII in thylakoid membranes.

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from 13C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.

Inactivation of mitochondrial complex I stimulates chloroplast ATPase in Physcomitrium patens.

Light is the ultimate source of energy for photosynthetic organisms, but respiration is fundamental for supporting metabolism during the night or in heterotrophic tissues. In this work, we isolated Physcomitrella (Physcomitrium patens) plants with altered respiration by inactivating Complex I (CI) of the mitochondrial electron transport chain by independently targeting on two essential subunits. Inactivation of CI caused a strong growth impairment even in fully autotrophic conditions in tissues where all cells are photosynthetically active, demonstrating that respiration is essential for photosynthesis. CI mutants showed alterations in the stoichiometry of respiratory complexes while the composition of photosynthetic apparatus was substantially unaffected. CI mutants showed altered photosynthesis with high activity of both Photosystems I and II, likely the result of high chloroplast ATPase activity that led to smaller ΔpH formation across thylakoid membranes, decreasing photosynthetic control on cytochrome b6f in CI mutants. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.

Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. II. Structure and Functions.

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

Acclimation of photosynthetic apparatus in the mesophilic red alga Dixoniella giordanoi.

Eukaryotic algae are photosynthetic organisms capable of exploiting sunlight to fix carbon dioxide into biomass with highly variable genetic and metabolic features. Information on algae metabolism from different species is inhomogeneous and, while green algae are, in general, more characterized, information on red algae is relatively scarce despite their relevant position in eukaryotic algae diversity. Within red algae, the best-known species are extremophiles or multicellular, while information on mesophilic unicellular organisms is still lacunose. Here, we investigate the photosynthetic properties of a recently isolated seawater unicellular mesophilic red alga, Dixoniella giordanoi. Upon exposure to different illuminations, D. giordanoi shows the ability to acclimate, modulate chlorophyll content, and re-organize thylakoid membranes. Phycobilisome content is also largely regulated, leading to almost complete disassembly of this antenna system in cells grown under intense illumination. Despite the absence of a light-induced xanthophyll cycle, cells accumulate zeaxanthin upon prolonged exposure to strong light, likely contributing to photoprotection. D. giordanoi cells show the ability to perform cyclic electron transport that is enhanced under strong illumination, likely contributing to the protection of Photosystem I from over-reduction and enabling cells to survive PSII photoinhibition without negative impact on growth.

Role of an ancient light-harvesting protein of PSI in light absorption and photoprotection.

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

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.

Conformational Dynamics of Light-Harvesting Complex II in a Native Membrane Environment.

Photosynthetic light-harvesting complexes (LHCs) of higher plants, moss, and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr light-harvesting complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states.

Light excess stimulates Poly-beta-hydroxybutyrate yield in a mangrove-isolated strain of Synechocystis sp.

Poly-ß-hydroxybutyrate (PHB) is a biodegradable biopolymer that may replace fossil-based plastics reducing its negative environmental impact. One highly sustainable strategy to produce these biopolymers is the exploitation of photosynthetic microorganisms that use sunlight and CO2 to produce biomass and subsequently, PHB. Exploring environmental biological diversity is a powerful tool to find resilient microorganisms potentially exploitable to produce bioproducts. In this work, a cyanobacterium (Synechocystis sp.) isolated from a contaminated area close to an important industrial complex was shown to produce PHB under different culture conditions. Carbon, nutrients supply and light intensity impact on biomass and PHB productivity were assessed, showing that the highest yield of PHB achieved was 241 mg L-1 (31%dcw) under high light intensity. Remarkably this condition not only stimulated PHB accumulation by 70% compared to other conditions tested but also high cellular duplication rate, maximizing the potential of this strain for PHB production.

The chloroplast NADH dehydrogenase-like complex influences the photosynthetic activity of the moss Physcomitrella patens.

Alternative electron pathways contribute to regulation of photosynthetic light reactions to adjust to metabolic demands in dynamic environments. The chloroplast NADH dehydrogenase-like (NDH) complex mediates the cyclic electron transport pathway around PSI in different cyanobacteria, algae, and plant species, but it is not fully conserved in all photosynthetic organisms. In order to assess how the physiological role of this complex changed during plant evolution, we isolated Physcomitrella patens lines knocked out for the NDHM gene that encodes a subunit fundamental for the activity of the complex. ndhm knockout mosses indicated high PSI acceptor side limitation upon abrupt changes in illumination. In P. patens, pseudo-cyclic electron transport mediated by flavodiiron proteins (FLVs) was also shown to prevent PSI over-reduction in plants exposed to light fluctuations. flva ndhm double knockout mosses had altered photosynthetic performance and growth defects under fluctuating light compared with the wild type and single knockout mutants. The results showed that while the contribution of NDH to electron transport is minor compared with FLV, NDH still participates in modulating photosynthetic activity, and it is critical to avoid PSI photoinhibition, especially when FLVs are inactive. The functional overlap between NDH- and FLV-dependent electron transport supports PSI activity and prevents its photoinhibition under light variations.