Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation.

Diatoms are successful phytoplankton clades able to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity via the nonphotochemical quenching (NPQ) process. While the molecular effectors of NPQ as well as the involvement of the proton motive force (PMF) in its regulation are known, the regulators of the PET/PMF relationship remain unidentified in diatoms. We generated mutants of the H+ /K+ antiporter KEA3 in the model diatom Phaeodactylum tricornutum. Loss of KEA3 activity affects the PET/PMF coupling and NPQ responses at the onset of illumination, during transients and in steady-state conditions. Thus, this antiporter is a main regulator of the PET/PMF coupling. Consistent with this conclusion, a parsimonious model including only two free components, KEA3 and the diadinoxanthin de-epoxidase, describes most of the feedback loops between PET and NPQ. This simple regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory calcium ion (Ca2+ )-binding domain in KEA3 modulating its activity. This circuit is likely tuned by the NPQ-effector proteins, LHCXs, providing diatoms with the required flexibility to thrive in different ocean provinces.

UV-B Perception and Acclimation in Chlamydomonas reinhardtii.

Plants perceive UV-B, an intrinsic component of sunlight, via a signaling pathway that is mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8) and induces UV-B acclimation. To test whether similar UV-B perception mechanisms exist in the evolutionarily distant green alga Chlamydomonas reinhardtii, we identified Chlamydomonas orthologs of UVR8 and the key signaling factor CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). Cr-UVR8 shares sequence and structural similarity to Arabidopsis thaliana UVR8, has conserved tryptophan residues for UV-B photoreception, monomerizes upon UV-B exposure, and interacts with Cr-COP1 in a UV-B-dependent manner. Moreover, Cr-UVR8 can interact with At-COP1 and complement the Arabidopsis uvr8 mutant, demonstrating that it is a functional UV-B photoreceptor. Chlamydomonas shows apparent UV-B acclimation in colony survival and photosynthetic efficiency assays. UV-B exposure, at low levels that induce acclimation, led to broad changes in the Chlamydomonas transcriptome, including in genes related to photosynthesis. Impaired UV-B-induced activation in the Cr-COP1 mutant hit1 indicates that UVR8-COP1 signaling induces transcriptome changes in response to UV-B. Also, hit1 mutants are impaired in UV-B acclimation. Chlamydomonas UV-B acclimation preserved the photosystem II core proteins D1 and D2 under UV-B stress, which mitigated UV-B-induced photoinhibition. These findings highlight the early evolution of UVR8 photoreceptor signaling in the green lineage to induce UV-B acclimation and protection.

UV-B photoreceptor-mediated protection of the photosynthetic machinery in Chlamydomonas reinhardtii.

Life on earth is dependent on the photosynthetic conversion of light energy into chemical energy. However, absorption of excess sunlight can damage the photosynthetic machinery and limit photosynthetic activity, thereby affecting growth and productivity. Photosynthetic light harvesting can be down-regulated by nonphotochemical quenching (NPQ). A major component of NPQ is qE (energy-dependent nonphotochemical quenching), which allows dissipation of light energy as heat. Photodamage peaks in the UV-B part of the spectrum, but whether and how UV-B induces qE are unknown. Plants are responsive to UV-B via the UVR8 photoreceptor. Here, we report in the green alga Chlamydomonas reinhardtii that UVR8 induces accumulation of specific members of the light-harvesting complex (LHC) superfamily that contribute to qE, in particular LHC Stress-Related 1 (LHCSR1) and Photosystem II Subunit S (PSBS). The capacity for qE is strongly induced by UV-B, although the patterns of qE-related proteins accumulating in response to UV-B or to high light are clearly different. The competence for qE induced by acclimation to UV-B markedly contributes to photoprotection upon subsequent exposure to high light. Our study reveals an anterograde link between photoreceptor-mediated signaling in the nucleocytosolic compartment and the photoprotective regulation of photosynthetic activity in the chloroplast.

Global spectroscopic analysis to study the regulation of the photosynthetic proton motive force: A critical reappraisal.

In natural variable environments, plants rapidly adjust photosynthesis for optimum balance between photochemistry and photoprotection. These adjustments mainly occur via changes in their proton motive force (pmf). Recent studies based on time resolved analysis of the Electro Chromic Signal (ECS) bandshift of photosynthetic pigments in the model plant Arabidopsis thaliana have suggested an active role of ion fluxes across the thylakoid membranes in the regulation of the pmf. Among the different channels and transporters possibly involved in this phenomenon, we previously identified the TPK3 potassium channel. Plants silenced for TPK3 expression displayed light stress signatures, with reduced Non Photochemical Quenching (NPQ) capacity and sustained anthocyanin accumulation, even at moderate intensities. In this work we re-examined the role of this protein in pmf regulation, starting from the observation that both TPK3 knock-down (TPK3 KD) and WT plants display enhanced anthocyanin accumulation in the light under certain growth conditions, especially in old leaves. We thus compared the pmf features of young "green" (without anthocyanins) and old "red" (with anthocyanins) leaves in both genotypes using a global fit analysis of the ECS. We found that the differences in the ECS profile measured between the two genotypes reflect not only differences in TPK3 expression level, but also a modified photosynthetic activity of stressed red leaves, which are present in a larger amounts in the TPK3 KD plants.

Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1.

Plants possessing dysfunctional plastids due to defects in pigment biosynthesis or translation are known to repress photosynthesis-associated nuclear genes via retrograde signals from the disturbed organelles toward the nucleus. These signals are thought to be essential for proper biogenesis and function of the plastid. Mutants lacking plastid-encoded RNA polymerase-associated proteins (PAPs) display a genetic arrest in eoplast-chloroplast transition leading to an albino phenotype in the light. Retrograde signaling in these mutants, therefore, could be expected to be similar as under conditions inducing plastid dysfunction. To answer this question, we performed plastome- and genomewide array analyses in the pap7-1 mutant of Arabidopsis (Arabidopsis thaliana). In parallel, we determined the potential overlap with light-regulated expression networks. To this end, we performed a comparative expression profiling approach using light- and dark-grown wild-type plants as relative control for the expression profiles obtained from light-grown pap7-1 mutants. Our data indicate a specific impact of retrograde signals on metabolism-related genes in pap7-1 mutants reflecting the starvation situation of the albino seedlings. In contrast, light regulation of PhANGs and other nuclear gene groups appears to be fully functional in this mutant, indicating that a block in chloroplast biogenesis per se does not repress expression of them as suggested by earlier studies. Only genes for light harvesting complex proteins displayed a significant repression indicating an exclusive retrograde impact on this gene family. Our results indicate that chloroplasts and arrested plastids each emit specific signals that control different target gene modules both in positive and negative manner.

An update on the regulation of photosynthesis by thylakoid ion channels and transporters in Arabidopsis.

In natural, variable environments, plants rapidly adjust photosynthesis for optimal balance between light absorption and utilization. There is increasing evidence suggesting that ion fluxes across the chloroplast thylakoid membrane play an important role in this regulation by affecting the proton motive force and consequently photosynthesis and thylakoid membrane ultrastructure. This article presents an update on the thylakoid ion channels and transporters characterized in Arabidopsis thaliana as being involved in these processes, as well as an outlook at the evolutionary conservation of their functions in other photosynthetic organisms. This is a contribution to shed light on the thylakoid network of ion fluxes and how they help plants to adjust photosynthesis in variable light environments.

A dual strategy to cope with high light in Chlamydomonas reinhardtii.

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition-deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.

Adjustments of embryonic photosynthetic activity modulate seed fitness in Arabidopsis thaliana.

In this work, we dissect the physiological role of the transient photosynthetic stage observed in developing seeds of Arabidopsis thaliana. By combining biochemical and biophysical approaches, we demonstrate that despite similar features of the photosynthetic apparatus, light absorption, chloroplast morphology and electron transport are modified in green developing seeds, as a possible response to the peculiar light environment experienced by them as a result of sunlight filtration by the pericarp. In particular, enhanced exposure to far-red light, which mainly excites photosystem I, largely enhances cyclic electron flow around this complex at the expenses of oxygen evolution. Using pharmacological, genetic and metabolic analyses, we show that both linear and cyclic electron flows are important during seed formation for proper germination timing. Linear flow provides specific metabolites related to oxygen and water stress responses. Cyclic electron flow possibly adjusts the ATP to NADPH ratio to cope with the specific energy demand of developing seeds. By providing a comprehensive scenario of the characteristics, function and consequences of embryonic photosynthesis on seed vigour, our data provide a rationale for the transient building up of a photosynthetic machinery in seeds.

Thylakoid potassium channel is required for efficient photosynthesis in cyanobacteria.

A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b(6)f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.

Targeted Gene Editing of Nuclear-Encoded Plastid Proteins in Phaeodactylum tricornutum via CRISPR/Cas9.

Genome modifications in microalgae have emerged as a crucial and indispensable tool for research in fundamental and applied biology. In particular, CRISPR/Cas9 has gained significant recognition as a highly effective method for genome engineering in these photosynthetic organisms, enabling the targeted induction of mutations in specific regions of the genome. Here, we present a comprehensive protocol for generating knock-out mutants in the model diatom Phaeodactylum tricornutum using CRISPR/Cas9 by both biolistic transformation and bacterial conjugation. Our protocol outlines the step-by-step procedures and experimental conditions required to achieve successful genome editing, including the design and construction of guide RNAs, the delivery of CRISPR/Cas9 components into the algae cells, and the selection of the generated knockout mutants. Through the implementation of this protocol, researchers can harness the potential of CRISPR/Cas9 in P. tricornutum to advance the understanding of diatom biology and explore their potential applications in various fields.

Plastid gene expression during chloroplast differentiation and dedifferentiation into non-photosynthetic plastids during seed formation.

Arabidopsis seed formation is coupled with two plastid differentiation processes. Chloroplast formation starts during embryogenesis and ends with the maturation phase. It is followed by chloroplast dedifferentiation/degeneration that starts at the end of the maturation phase and leads to the presence of small non-photosynthetic plastids in dry seeds. We have analysed mRNA and protein levels of nucleus- and plastid-encoded (NEP and PEP) components of the plastid transcriptional machinery, mRNA and protein levels of some plastid RNA polymerase target genes, changes in plastid transcriptome profiles and mRNA and protein levels of some selected nucleus-encoded plastid-related genes in developing seeds during embryogenesis, maturation and desiccation. As expected, most of the mRNAs and proteins increase in abundance during maturation and decrease during desiccation, when plastids dedifferentiate/degenerate. In contrast, mRNAs and proteins of components of the plastid transcriptional apparatus do not decrease or even still increase during the period of plastid dedifferentiation. Results suggest that proteins of the plastid transcriptional machinery are specifically protected from degradation during the desiccation period and conserved in dry seeds to allow immediate regain of plastid transcriptional activity during stratification/germination. In addition, results reveal accumulation and storage of mRNAs coding for RNA polymerase components and sigma factors in dry seeds. They should provide immediately-to-use templates for translation on cytoplasmic ribosomes in order to enhance RNA polymerase protein levels and to provide regulatory proteins for stored PEP to guaranty efficient plastid genome transcription during germination.

Light-independent regulation of algal photoprotection by CO2 availability.

Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2 conditions. When light energy exceeds CO2 fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2 Concentrating Mechanism (CCM). How light and CO2 signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2 concentrations and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2 and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2 levels.

Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging.

Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales.

Consequences of Mixotrophy on Cell Energetic Metabolism in Microchloropsis gaditana Revealed by Genetic Engineering and Metabolic Approaches.

Algae belonging to the Microchloropsis genus are promising organisms for biotech purposes, being able to accumulate large amounts of lipid reserves. These organisms adapt to different trophic conditions, thriving in strict photoautotrophic conditions, as well as in the concomitant presence of light plus reduced external carbon as energy sources (mixotrophy). In this work, we investigated the mixotrophic responses of Microchloropsis gaditana (formerly Nannochloropsis gaditana). Using the Biolog growth test, in which cells are loaded into multiwell plates coated with different organic compounds, we could not find a suitable substrate for Microchloropsis mixotrophy. By contrast, addition of the Lysogeny broth (LB) to the inorganic growth medium had a benefit on growth, enhancing respiratory activity at the expense of photosynthetic performances. To further dissect the role of respiration in Microchloropsis mixotrophy, we focused on the mitochondrial alternative oxidase (AOX), a protein involved in energy management in other algae prospering in mixotrophy. Knocking-out the AOX1 gene by transcription activator-like effector nuclease (TALE-N) led to the loss of capacity to implement growth upon addition of LB supporting the hypothesis that the effect of this medium was related to a provision of reduced carbon. We conclude that mixotrophic growth in Microchloropsis is dominated by respiratory rather than by photosynthetic energetic metabolism and discuss the possible reasons for this behavior in relationship with fatty acid breakdown via ß-oxidation in this oleaginous alga.

Generation of Mutants of Nuclear-Encoded Plastid Proteins Using CRISPR/Cas9 in the Diatom Phaeodactylum tricornutum.

Genome modifications in microalgae are becoming a widespread and mandatory tool for research in both fundamental and applied biology. Among genome editing methods in these photosynthetic organisms, CRISPR/Cas9 offers a specific, powerful and efficient tool for genome engineering by inducing mutations in targeted regions of the genome. Here we described a protocol that allows the generation of knockout mutants by CRISPR/Cas9 in the diatom Phaeodactylum tricornutum using biolistic transformation.

Photoreceptor-dependent regulation of photoprotection.

In photosynthetic organisms, proteins in the light-harvesting complex (LHC) harvest light energy to fuel photosynthesis, whereas photoreceptor proteins are activated by the different wavelengths of the light spectrum to regulate cellular functions. Under conditions of excess light, blue-light photoreceptors activate chloroplast avoidance movements in sessile plants, and blue- and green-light photoreceptors cause motile algae to swim away from intense light. Simultaneously, LHCs switch from light-harvesting mode to energy-dissipation mode, which was thought to be independent of photoreceptor-signaling up until recently. Recent advances, however, indicate that energy dissipation in green algae is controlled by photoreceptors activated by blue and UV-B light, and new molecular links have been established between photoreception and photoprotection.