Photoautotrophic cultivation of a Chlamydomonas reinhardtii mutant with zeaxanthin as the sole xanthophyll.

BACKGROUND: Photosynthetic microalgae are known for their sustainable and eco-friendly potential to convert carbon dioxide into valuable products. Nevertheless, the challenge of self-shading due to high cell density has been identified as a drawback, hampering productivity in sustainable photoautotrophic mass cultivation. To address this issue, mutants with altered pigment composition have been proposed to allow a more efficient light diffusion but further study on the role of the different pigments is still needed to correctly engineer this process. RESULTS: We here investigated the Chlamydomonas reinhardtii Δzl mutant with zeaxanthin as the sole xanthophyll. The Δzl mutant displayed altered pigment composition, characterized by lower chlorophyll content, higher chlorophyll a/b ratio, and lower chlorophyll/carotenoid ratio compared to the wild type (Wt). The Δzl mutant also exhibited a significant decrease in the light-harvesting complex II/Photosystem II ratio (LHCII/PSII) and the absence of trimeric LHCIIs. This significantly affects the organization and stability of PSII supercomplexes. Consequently, the estimated functional antenna size of PSII in the Δzl mutant was approximately 60% smaller compared to that of Wt, and reduced PSII activity was evident in this mutant. Notably, the Δzl mutant showed impaired non-photochemical quenching. However, the Δzl mutant compensated by exhibiting enhanced cyclic electron flow compared to Wt, seemingly offsetting the impaired PSII functionality. Consequently, the Δzl mutant achieved significantly higher cell densities than Wt under high-light conditions. CONCLUSIONS: Our findings highlight significant changes in pigment content and pigment-protein complexes in the Δzl mutant compared to Wt, resulting in an advantage for high-density photoautotrophic cultivation. This advantage is attributed to the decreased chlorophyll content of the Δzl mutant, allowing better light penetration. In addition, the accumulated zeaxanthin in the mutant could serve as an antioxidant, offering protection against reactive oxygen species generated by chlorophylls.

Photosystem II monomeric antenna CP26 plays a key role in nonphotochemical quenching in Chlamydomonas.

Thermal dissipation of excess excitation energy, called nonphotochemical quenching (NPQ), is 1 of the main photoprotective mechanisms in oxygenic photosynthetic organisms. Here, we investigated the function of the monomeric photosystem II (PSII) antenna protein CP26 in photoprotection and light harvesting in Chlamydomonas reinhardtii, a model organism for green algae. We used CRISPR/Cas9 genome editing and complementation to generate cp26 knockout mutants (named k6#) that did not negatively affect CP29 accumulation, which differed from previous cp26 mutants, allowing us to compare mutants specifically deprived of CP26, CP29, or both. The absence of CP26 partially affected PSII activity, causing reduced growth at low or medium light but not at high irradiances. However, the main phenotype observed in k6# mutants was a more than 70% reduction of NPQ compared to the wild type (Wt). This phenotype was fully rescued by genetic complementation and complemented strains accumulating different levels of CP26, demonstrating that ∼50% of CP26 content, compared to the Wt, was sufficient to restore the NPQ capacity. Our findings demonstrate a pivotal role for CP26 in NPQ induction, while CP29 is crucial for PSII activity. The genetic engineering of these 2 proteins could be a promising strategy to regulate the photosynthetic efficiency of microalgae under different light regimes.

Acclimation strategies of the green alga Chlorella vulgaris to different light regimes revealed by physiological and comparative proteomic analyses.

Acclimation to different light regimes is at the basis of survival for photosynthetic organisms, regardless of their evolutionary origin. Previous research efforts largely focused on acclimation events occurring at the level of the photosynthetic apparatus and often highlighted species-specific mechanisms. Here, we investigated the consequences of acclimation to different irradiances in Chlorella vulgaris, a green alga that is one of the most promising species for industrial application, focusing on both photosynthetic and mitochondrial activities. Moreover, proteomic analysis of cells acclimated to high light (HL) or low light (LL) allowed identification of the main targets of acclimation in terms of differentially expressed proteins. The results obtained demonstrate photosynthetic adaptation to HL versus LL that was only partially consistent with previous findings in Chlamydomonas reinhardtii, a model organism for green algae, but in many cases similar to vascular plant acclimation events. Increased mitochondrial respiration measured in HL-acclimated cells mainly relied on alternative oxidative pathway dissipating the excessive reducing power produced due to enhanced carbon flow. Finally, proteins involved in cell metabolism, intracellular transport, gene expression, and signaling-including a heliorhodopsin homolog-were identified as strongly differentially expressed in HL versus LL, suggesting their key roles in acclimation to different light regimes.

Photosynthesis 2.0.

Photosynthesis is a process that provides the continuous income of energy needed to sustain life on our planet [...].

Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii.

Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned ß-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii, and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii. Increased ß-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L-1 (4.5 ± 0.1 mg g-1 CDW) in mixotrophic and 23.5 mg L-1 (1.09 mg L-1 h-1) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii. This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.

The transcription factor VviNAC60 regulates senescence- and ripening-related processes in grapevine.

Grapevine (Vitis vinifera L.) is one of the most widely cultivated fruit crops because the winemaking industry has huge economic relevance worldwide. Uncovering the molecular mechanisms controlling the developmental progression of plant organs will prove essential for maintaining high-quality grapes, expressly in the context of climate change, which impairs the ripening process. Through a deep inspection of transcriptomic data, we identified VviNAC60, a member of the NAC transcription factor family, as a putative regulator of grapevine organ maturation. We explored VviNAC60 binding landscapes through DNA affinity purification followed by sequencing and compared bound genes with transcriptomics datasets from grapevine plants stably and transiently overexpressing VviNAC60 to define a set of high-confidence targets. Among these, we identified key molecular markers associated with organ senescence and fruit ripening. Physiological, metabolic, and promoter activation analyses showed that VviNAC60 induces chlorophyll degradation and anthocyanin accumulation through the upregulation of STAY-GREEN PROTEIN 1 (VviSGR1) and VviMYBA1, respectively, with the latter being upregulated through a VviNAC60-VviNAC03 regulatory complex. Despite sharing a closer phylogenetic relationship with senescence-related homologs to the NAC transcription factor AtNAP, VviNAC60 complemented the nonripening(nor) mutant phenotype in tomato (Solanum lycopersicum), suggesting a dual role as an orchestrator of both ripening- and senescence-related processes. Our data support VviNAC60 as a regulator of processes initiated in the grapevine vegetative- to mature-phase organ transition and therefore as a potential target for enhancing the environmental resilience of grapevine by fine-tuning the duration of the vegetative phase.

Engineering astaxanthin accumulation reduces photoinhibition and increases biomass productivity under high light in Chlamydomonas reinhardtii.

BACKGROUND: Astaxanthin is a highly valuable ketocarotenoid with strong antioxidative activity and is natively accumulated upon environmental stress exposure in selected microorganisms. Green microalgae are photosynthetic, unicellular organisms cultivated in artificial systems to produce biomass and industrially relevant bioproducts. While light is required for photosynthesis, fueling carbon fixation processes, application of high irradiance causes photoinhibition and limits biomass productivity. RESULTS: Here, we demonstrate that engineered astaxanthin accumulation in the green alga Chlamydomonas reinhardtii conferred high light tolerance, reduced photoinhibition and improved biomass productivity at high irradiances, likely due to strong antioxidant properties of constitutively accumulating astaxanthin. In competitive co-cultivation experiments, astaxanthin-rich Chlamydomonas reinhardtii outcompeted its corresponding parental background strain and even the fast-growing green alga Chlorella vulgaris. CONCLUSIONS: Metabolic engineering inducing astaxanthin and ketocarotenoids accumulation caused improved high light tolerance and increased biomass productivity in the model species for microalgae Chlamydomonas reinhardtii. Thus, engineering microalgal pigment composition represents a powerful strategy to improve biomass productivities in customized photobioreactors setups. Moreover, engineered astaxanthin accumulation in selected strains could be proposed as a novel strategy to outperform growth of other competing microalgal strains.

Astaxanthin and eicosapentaenoic acid production by S4, a new mutant strain of Nannochloropsis gaditana.

BACKGROUND: Astaxanthin is a ketocarotenoid with high antioxidant power used in different fields as healthcare, food/feed supplementation and as pigmenting agent in aquaculture. Primary producers of astaxanthin are some species of microalgae, unicellular photosynthetic organisms, as Haematococcus lacustris. Astaxanthin production by cultivation of Haematococcus lacustris is costly due to low biomass productivity, high risk of contamination and the requirement of downstream extraction processes, causing an extremely high price on the market. Some microalgae species are also primary producers of omega-3 fatty acids, essential nutrients for humans, being related to cardiovascular wellness, and required for visual and cognitive development. One of the main well-known producers of omega-3 fatty eicosapentaenoic acid (EPA) is the marine microalga Nannochloropsis gaditana (named also Microchloropsis gaditana): this species has been already approved by the Food and Drug Administration (FDA) for human consumption and it is characterized by a fast grow phenotype. RESULTS: Here we obtained by chemical mutagenesis a Nannochloropsis gaditana mutant strain, called S4, characterized by increased carotenoid to chlorophyll ratio. S4 strain showed improved photosynthetic activity, increased lipid productivity and increased ketocarotenoids accumulation, producing not only canthaxanthin but also astaxanthin, usually found only in traces in the WT strain. Ketocarotenoids produced in S4 strain were extractible in different organic solvents, with the highest efficiency observed upon microwaves pre-treatment followed by methanol extraction. By cultivation of S4 strain at different irradiances it was possible to produce up to 1.3 and 5.2 mgL-1 day-1 of ketocarotenoids and EPA respectively, in a single cultivation phase, even in absence of stressing conditions. Genome sequencing of S4 strain allowed to identify 199 single nucleotide polymorphisms (SNP): among the mutated genes, mutations in a carotenoid oxygenase gene and in a glutamate synthase gene could explain the different carotenoids content and the lower chlorophylls content, respectively. CONCLUSIONS: By chemical mutagenesis and selection of strain with increased carotenoids to chlorophyll ratio it was possible to isolate a new Nannochloropsis gaditana strain, called S4 strain, characterized by increased lipids and ketocarotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for different industrial applications.

A synthetic switch based on orange carotenoid protein to control blue-green light responses in chloroplasts.

Synthetic biology approaches to engineer light-responsive systems are widely used, but their applications in plants are still limited due to the interference with endogenous photoreceptors and the intrinsic requirement of light for photosynthesis. Cyanobacteria possess a family of soluble carotenoid-associated proteins named orange carotenoid proteins (OCPs) that, when activated by blue-green light, undergo a reversible conformational change that enables the photoprotection mechanism that occurs on the phycobilisome. Exploiting this system, we developed a chloroplast-localized synthetic photoswitch based on a protein complementation assay where two nanoluciferase fragments were fused to separate polypeptides corresponding to the OCP2 domains. Since Arabidopsis (Arabidopsis thaliana) does not possess the prosthetic group needed for the assembly of the OCP2 complex, we first implemented the carotenoid biosynthetic pathway with a bacterial ß-carotene ketolase enzyme (crtW) to generate keto-carotenoid-producing plants. The photoswitch was tested and characterized in Arabidopsis protoplasts and stably transformed plants with experiments aimed to uncover its regulation by a range of light intensities, wavelengths, and its conversion dynamics. Finally, we applied the OCP-based photoswitch to control transcriptional responses in chloroplasts in response to green light illumination by fusing the two OCP fragments with the plastidial SIGMA FACTOR 2 and bacteriophage T4 anti-sigma factor AsiA. This pioneering study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses upon exposure to specific light spectra.

LPA2 protein is involved in photosystem II assembly in Chlamydomonas reinhardtii.

Photosynthetic eukaryotes require the proper assembly of photosystem II (PSII) in order to strip electrons from water and fuel carbon fixation reactions. In Arabidopsis thaliana, one of the PSII subunits (CP43/PsbC) was suggested to be assembled into the PSII complex via its interaction with an auxiliary protein called Low PSII Accumulation 2 (LPA2). However, the original articles describing the role of LPA2 in PSII assembly have been retracted. To investigate the function of LPA2 in the model organism for green algae, Chlamydomonas reinhardtii, we generated knockout lpa2 mutants by using the CRISPR-Cas9 target-specific genome editing system. Biochemical analyses revealed the thylakoidal localization of LPA2 protein in the wild type (WT), whereas lpa2 mutants were characterized by a drastic reduction in the levels of D1, D2, CP47 and CP43 proteins. Consequently, reduced PSII supercomplex accumulation, chlorophyll content per cell, PSII quantum yield and photosynthetic oxygen evolution were measured in the lpa2 mutants, leading to the almost complete impairment of photoautotrophic growth. Pulse-chase experiments demonstrated that the absence of LPA2 protein caused reduced PSII assembly and reduced PSII turnover. Taken together, our data indicate that, in C. reinhardtii, LPA2 is required for PSII assembly and proper function.

High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance.

Microalgae represent a carbon-neutral source of bulk biomass, for extraction of high-value compounds and production of renewable fuels. Due to their high metabolic activity and reproduction rates, species of the genus Chlorella are highly productive when cultivated in photobioreactors. However, wild-type strains show biological limitations making algal bioproducts expensive compared to those extracted from other feedstocks. Such constraints include inhomogeneous light distribution due to high optical density of the culture, and photoinhibition of the surface-exposed cells. Thus, the domestication of algal strains for industry makes it increasingly important to select traits aimed at enhancing light-use efficiency while withstanding excess light stress. Carotenoids have a crucial role in protecting against photooxidative damage and, thus, represent a promising target for algal domestication. We applied chemical mutagenesis to Chlorella vulgaris and selected for enhanced tolerance to the carotenoid biosynthesis inhibitor norflurazon. The NFR (norflurazon-resistant) strains showed an increased carotenoid pool size and enhanced tolerance towards photooxidative stress. Growth under excess light revealed an improved carbon assimilation rate of NFR strains with respect to WT. We conclude that domestication of Chlorella vulgaris, by optimizing both carotenoid/chlorophyll ratio and resistance to photooxidative stress, boosted light-to-biomass conversion efficiency under high light conditions typical of photobioreactors. Comparison with strains previously reported for enhanced tolerance to singlet oxygen, reveals that ROS resistance in Chlorella is promoted by at least two independent mechanisms, only one of which is carotenoid-dependent.

CO2 supply modulates lipid remodelling, photosynthetic and respiratory activities in Chlorella species.

Microalgae represent a potential solution to reduce CO2 emission exploiting their photosynthetic activity. Here, the physiologic and metabolic responses at the base of CO2 assimilation were investigated in conditions of high or low CO2 availability in two of the most promising algae species for industrial cultivation, Chlorella sorokiniana and Chlorella vulgaris. In both species, high CO2 availability increased biomass accumulation with specific increase of triacylglycerols in C. vulgaris and polar lipids and proteins in C. sorokiniana. Moreover, high CO2 availability caused only in C. vulgaris a reduced NAD(P)H/NADP+ ratio and reduced mitochondrial respiration, suggesting a CO2 dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria. Several rearrangements of the photosynthetic machinery were observed in both species, differing from those described for the model organism Chlamydomonas reinhardtii, where adaptation to carbon availability is mainly controlled by the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. Acclimation strategies to cope with a fluctuating inorganic carbon supply are thus diverse among green microalgae, and these results suggest new biotechnological strategies to boost CO2 fixation.

Incorporating a molecular antenna in diatom microalgae cells enhances photosynthesis.

Diatom microalgae have great industrial potential as next-generation sources of biomaterials and biofuels. Effective scale-up of their production can be pursued by enhancing the efficiency of their photosynthetic process in a way that increases the solar-to-biomass conversion yield. A proof-of-concept demonstration is given of the possibility of enhancing the light absorption of algae and of increasing their efficiency in photosynthesis by in vivo incorporation of an organic dye which acts as an antenna and enhances cells' growth and biomass production without resorting to genetic modification. A molecular dye (Cy5) is incorporated in Thalassiosira weissflogii diatom cells by simply adding it to the culture medium and thus filling the orange gap that limits their absorption of sunlight. Cy5 enhances diatoms' photosynthetic oxygen production and cell density by 49% and 40%, respectively. Cy5 incorporation also increases by 12% the algal lipid free fatty acid (FFA) production versus the pristine cell culture, thus representing a suitable way to enhance biofuel generation from algal species. Time-resolved spectroscopy reveals Förster Resonance Energy Transfer (FRET) from Cy5 to algal chlorophyll. The present approach lays the basis for non-genetic tailoring of diatoms' spectral response to light harvesting, opening up new ways for their industrial valorization.

Heterologous expression of cyanobacterial Orange Carotenoid Protein (OCP2) as a soluble carrier of ketocarotenoids in Chlamydomonas reinhardtii.

Photosynthetic organisms evolved different mechanisms to protect themselves from high irradiances and photodamage. In cyanobacteria, the photoactive Orange Carotenoid-binding Protein (OCP) acts both as a light sensor and quencher of excitation energy. It binds keto-carotenoids and, when photoactivated, interacts with phyco-bilisomes, thermally dissipating the excitation energy absorbed by the latter, and acting as efficient singlet oxygen quencher. Here, we report the heterologous expression of an OCP2 protein from the thermophilic cyanobacterium Fischerella thermalis (FtOCP2) in the model organism for green algae, Chlamydomonas reinhardtii. Robust expression of FtOCP2 was obtained through a synthetic redesigning strategy for optimized expression of the transgene. FtOCP2 expression was achieved both in UV-mediated mutant 4 strain, previously selected for efficient transgene expression, and in a background strain previously engineered for constitutive expression of an endogenous ß-carotene ketolase, normally poorly expressed in this species, resulting into astaxanthin and other ketocarotenoids accumulation. Recombinant FtOCP2 was successfully localized into the chloroplast. Upon purification it was possible to demonstrate the formation of holoproteins with different xanthophylls and keto-carotenoids bound, including astaxanthin. Moreover, isolated ketocarotenoid-binding FtOCP2 holoproteins conserved their photoconversion properties. Carotenoids bound to FtOCP2 were thus maintained in solution even in absence of organic solvent. The synthetic biology approach herein reported could thus be considered as a novel tool for improving the solubility of ketocarotenoids produced in green algae, by binding to water-soluble carotenoids binding proteins.

VviNAC33 promotes organ de-greening and represses vegetative growth during the vegetative-to-mature phase transition in grapevine.

Plants undergo several developmental transitions during their life cycle. In grapevine, a perennial woody fruit crop, the transition from vegetative/green-to-mature/woody growth involves transcriptomic reprogramming orchestrated by a small group of genes encoding regulators, but the underlying molecular mechanisms are not fully understood. We investigated the function of the transcriptional regulator VviNAC33 by generating and characterizing transgenic overexpressing grapevine lines and a chimeric repressor, and by exploring its putative targets through a DNA affinity purification sequencing (DAP-seq) approach combined with transcriptomic data. We demonstrated that VviNAC33 induces leaf de-greening, inhibits organ growth and directly activates the expression of STAY-GREEN PROTEIN 1 (SGR1), which is involved in Chl and photosystem degradation, and AUTOPHAGY 8f (ATG8f), which is involved in the maturation of autophagosomes. Furthermore, we show that VviNAC33 directly inhibits AUXIN EFFLUX FACILITATOR PIN1, RopGEF1 and ATP SYNTHASE GAMMA CHAIN 1T (ATPC1), which are involved in photosystem II integrity and activity. Our results show that VviNAC33 plays a major role in terminating photosynthetic activity and organ growth as part of a regulatory network governing the vegetative-to-mature phase transition.

Identification of distinct pH- and zeaxanthin-dependent quenching in LHCSR3 from Chlamydomonas reinhardtii.

Under high light, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating absorbed energy, which is called nonphotochemical quenching. In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via a pH drop and serves as a quenching site. Using a combined in vivo and in vitro approach, we investigated quenching within LHCSR3 from Chlamydomonas reinhardtii. In vitro two distinct quenching processes, individually controlled by pH and zeaxanthin, were identified within LHCSR3. The pH-dependent quenching was removed within a mutant LHCSR3 that lacks the residues that are protonated to sense the pH drop. Observation of quenching in zeaxanthin-enriched LHCSR3 even at neutral pH demonstrated zeaxanthin-dependent quenching, which also occurs in other light-harvesting complexes. Either pH- or zeaxanthin-dependent quenching prevented the formation of damaging reactive oxygen species, and thus the two quenching processes may together provide different induction and recovery kinetics for photoprotection in a changing environment.

Green plants and algae rely on sunlight to transform light energy into chemical energy in a process known as photosynthesis. However, too much light can damage plants. Green plants prevent this by converting the extra absorbed light into heat. Both the absorption and the dissipation of sunlight into heat occur within so called light harvesting complexes. These are protein structures that contain pigments such as chlorophyll and carotenoids. The process of photoprotection starts when the excess of absorbed light generates protons (elementary particles with a positive charge) faster than they can be used. This causes a change in the pH (a measure of the concentration of protons in a solution), which in turn, modifies the shape of proteins and the chemical identity of the carotenoids. However, it is still unclear what the exact mechanisms are. To clarify this, Troiano, Perozeni et al. engineered the light harvesting complex LHCSR3 of the green algae Chlamydomonas reinhardtii to create mutants that either could not sense changes in the pH or contained the carotenoid zeaxanthin. Zeaxanthin is one of the main carotenoids accumulated by plants and algae upon high light stress. Measurements showed that both pH detection and zeaxanthin were able to provide photoprotection independently. Troiano, Perozeni et al. further found that pH and carotenoids controlled changes to the organisation of the pigment at two separate locations within the LHCSR3, which influenced whether the protein was able to prevent photodamage. When algae were unable to change pH or carotenoids, dissipation was less effective. Instead, specific molecules were produced that damage the cellular machinery. The results shed light onto how green algae protect themselves from too much light exposure. These findings could pave the way for optimising dissipation, which could increase yields of green algae by up to 30%. This could lead to green algae becoming a viable alternative for food, biofuels and feedstock.

Chlamydomonas reinhardtii LHCSR1 and LHCSR3 proteins involved in photoprotective non-photochemical quenching have different quenching efficiency and different carotenoid affinity.

Microalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, their limited biomass productivity represents a bottleneck that needs to be overcome to meet the applicative potential of these organisms. One of the domestication targets for improving their productivity is the proper balance between photoprotection and light conversion for carbon fixation. In the model organism for green algae, Chlamydomonas reinhardtii, a photoprotective mechanism inducing thermal dissipation of absorbed light energy, called Non-photochemical quenching (NPQ), is activated even at relatively low irradiances, resulting in reduced photosynthetic efficiency. Two pigment binding proteins, LHCSR1 and LHCSR3, were previously reported as the main actors during NPQ induction in C. reinhardtii. While previous work characterized in detail the functional properties of LHCSR3, few information is available for the LHCSR1 subunit. Here, we investigated in vitro the functional properties of LHCSR1 and LHCSR3 subunits: despite high sequence identity, the latter resulted as a stronger quencher compared to the former, explaining its predominant role observed in vivo. Pigment analysis, deconvolution of absorption spectra and structural models of LHCSR1 and LHCR3 suggest that different quenching efficiency is related to a different occupancy of L2 carotenoid binding site.

Evolutionary divergence of photoprotection in the green algal lineage: a plant-like violaxanthin de-epoxidase enzyme activates the xanthophyll cycle in the green alga Chlorella vulgaris modulating photoprotection.

The xanthophyll cycle is the metabolic process by which the carotenoid violaxanthin is de-epoxidated to zeaxanthin, a xanthophyll with a crucial photoprotective role in higher plants and mosses. The role of zeaxanthin is still unclear in green algae, and a peculiar violaxanthin de-epoxidating enzyme was found in the model organism Chlamydomonas reinhardtii. Here, we investigated the molecular details and functions of the xanthophyll cycle in the case of Chlorella vulgaris, one of the green algae most considered for industrial cultivation, where resistance to high light stress is a prerequisite for sustainable biomass production. Identification of the violaxanthin de-epoxidase enzyme in C. vulgaris was performed by genome mining and in vitro analysis of the catalytic activity of the gene product identified. The photoprotective role of zeaxanthin was then investigated in vivo and in isolated pigment-binding complexes. The results obtained demonstrate the functioning, even though with a different pH sensitivity, of a plant-like violaxanthin de-epoxidase enzyme in C. vulgaris. Differently from C. reinhardtii, zeaxanthin accumulation in C. vulgaris was found to be crucial for photoprotective quenching of excitation energy harvested by both photosystem I and II. These findings demonstrate an evolutionary divergence of photoprotective mechanisms among Chlorophyta.

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin; however, a ß-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic redesign of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.3 mg/L/day. Astaxanthin productivity in engineered C. reinhardtii shown here might be competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments were much higher in cell wall-deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host.

Monomeric light harvesting complexes enhance excitation energy transfer from LHCII to PSII and control their lateral spacing in thylakoids.

Proper assembly of plant photosystem II, in the appressed region of thylakoids, allows for both efficient light harvesting and the dissipation of excitation energy absorbed in excess. The core moiety of wild type supercomplex is associated with monomeric antennae that, in turn, bind peripheral trimeric LHCII complexes. Acclimation to light environment dynamics involves structural plasticity within PSII-LHCs supercomplexes, including depletion in LHCII and CP24. Here, we report on the acclimation of NoM, an Arabidopsis mutant lacking monomeric LHCs but retaining LHCII trimer. Lack of monomeric LHCs impaired the operation of both photosynthetic electron transport and state transitions, despite the fact that NoM underwent a compensatory over-accumulation of the LHCII complement compared to the wild type. Mutant plants displayed stunted growth compared to the wild type when probed over a range of light conditions. When exposed to short-term excess light, NoM showed higher photosensitivity and enhanced singlet oxygen release than the wild type, whereas long-term acclimation under stress conditions was unaffected. Analysis of pigment-binding supercomplexes showed that the absence of monomeric LHCs did affect the macro-organisation of photosystems: large PSI-LHCII megacomplexes were more abundant in NoM, whereas the assembly of PSII-LHCs supercomplexes was impaired. Observation by electron microscopy (EM) and image analysis of thylakoids highlighted impaired granal stacking and membrane organisation, with a heterogeneous distribution of PSII and LHCII compared to the wild type. It is concluded that monomeric LHCs are critical for the structural and functional optimisation of the photosynthetic apparatus.