Enhanced function of non-photoinhibited photosystem II complexes upon PSII photoinhibition.

Light induced photosystem (PS)II photoinhibition inactivates and irreversibly damages the reaction center protein(s) but the light harvesting complexes continue the collection of light energy. Here we addressed the consequences of such a situation on thylakoid light harvesting and electron transfer reactions. For this purpose, Arabidopsis thaliana leaves were subjected to investigation of the function and regulation of the photosynthetic machinery after a distinct portion of PSII centers had experienced photoinhibition in the presence and absence of Lincomycin (Lin), a commonly used agent to block the repair of damaged PSII centers. In the absence of Lin, photoinhibition increased the relative excitation of PSII and decreased NPQ, together enhancing the electron transfer from still functional PSII centers to PSI. In contrast, in the presence of Lin, PSII photoinhibition increased the relative excitation of PSI and led to strong oxidation of the electron transfer chain. We hypothesize that plants are able to minimize the detrimental effects of high-light illumination on PSII by modulating the energy and electron transfer, but lose such a capability if the repair cycle is arrested. It is further hypothesized that dynamic regulation of the LHCII system has a pivotal role in the control of excitation energy transfer upon PSII damage and repair cycle to maintain the photosynthesis safe and efficient.

Plants acclimate to Photosystem I photoinhibition by readjusting the photosynthetic machinery.

Photosynthetic light reactions require strict regulation under dynamic environmental conditions. Still, depending on environmental constraints, photoinhibition of Photosystem (PSII) or PSI occurs frequently. Repair of photodamaged PSI, in sharp contrast to that of PSII, is extremely slow and leads to a functional imbalance between the photosystems. Slow PSI recovery prompted us to take advantage of the PSI-specific photoinhibition treatment and investigate whether the imbalance between functional PSII and PSI leads to acclimation of photosynthesis to PSI-limited conditions, either by short-term or long-term acclimation mechanisms as tested immediately after the photoinhibition treatment or after 24 h recovery in growth conditions, respectively. Short-term acclimation mechanisms were induced directly upon inhibition, including thylakoid protein phosphorylation that redirects excitation energy to PSI as well as changes in the feedback regulation of photosynthesis, which relaxed photosynthetic control and excitation energy quenching. Longer-term acclimation comprised reprogramming of the stromal redox system and an increase in ATP synthase and Cytochrome b6 f abundance. Acclimation to PSI-limited conditions restored the CO2 assimilation capacity of plants without major PSI repair. Response to PSI inhibition demonstrates that plants efficiently acclimate to changes occurring in the photosynthetic apparatus, which is likely a crucial component in plant acclimation to adverse environmental conditions.

Simultaneous Ozone and High Light Treatments Reveal an Important Role for the Chloroplast in Co-ordination of Defense Signaling.

Plants live in a world of changing environments, where they are continuously challenged by alternating biotic and abiotic stresses. To transfer information from the environment to appropriate protective responses, plants use many different signaling molecules and pathways. Reactive oxygen species (ROS) are critical signaling molecules in the regulation of plant stress responses, both inside and between cells. In natural environments, plants can experience multiple stresses simultaneously. Laboratory studies on stress interaction and crosstalk at regulation of gene expression, imply that plant responses to multiple stresses are distinctly different from single treatments. We analyzed the expression of selected marker genes and reassessed publicly available datasets to find signaling pathways regulated by ozone, which produces apoplastic ROS, and high light treatment, which produces chloroplastic ROS. Genes related to cell death regulation were differentially regulated by ozone versus high light. In a combined ozone + high light treatment, the light treatment enhanced ozone-induced cell death in leaves. The distinct responses from ozone versus high light treatments show that plants can activate stress signaling pathways in a highly precise manner.

Chlorophyll a fluorescence illuminates a path connecting plant molecular biology to Earth-system science.

For decades, the dynamic nature of chlorophyll a fluorescence (ChlaF) has provided insight into the biophysics and ecophysiology of the light reactions of photosynthesis from the subcellular to leaf scales. Recent advances in remote sensing methods enable detection of ChlaF induced by sunlight across a range of larger scales, from using instruments mounted on towers above plant canopies to Earth-orbiting satellites. This signal is referred to as solar-induced fluorescence (SIF) and its application promises to overcome spatial constraints on studies of photosynthesis, opening new research directions and opportunities in ecology, ecophysiology, biogeochemistry, agriculture and forestry. However, to unleash the full potential of SIF, intensive cross-disciplinary work is required to harmonize these new advances with the rich history of biophysical and ecophysiological studies of ChlaF, fostering the development of next-generation plant physiological and Earth-system models. Here, we introduce the scale-dependent link between SIF and photosynthesis, with an emphasis on seven remaining scientific challenges, and present a roadmap to facilitate future collaborative research towards new applications of SIF.

Modulating the activities of chloroplasts and mitochondria promotes adenosine triphosphate production and plant growth.

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin-Benson-Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

Specific thylakoid protein phosphorylations are prerequisites for overwintering of Norway spruce (Picea abies) photosynthesis.

Coping of evergreen conifers in boreal forests with freezing temperatures on bright winter days puts the photosynthetic machinery in great risk of oxidative damage. To survive harsh winter conditions, conifers have evolved a unique but poorly characterized photoprotection mechanism, a sustained form of nonphotochemical quenching (sustained NPQ). Here we focused on functional properties and underlying molecular mechanisms related to the development of sustained NPQ in Norway spruce (Picea abies). Data were collected during 4 consecutive years (2016 to 2019) from trees growing in sun and shade habitats. When day temperatures dropped below -4 °C, the specific N-terminally triply phosphorylated LHCB1 isoform (3p-LHCII) and phosphorylated PSBS (p-PSBS) could be detected in the thylakoid membrane. Development of sustained NPQ coincided with the highest level of 3p-LHCII and p-PSBS, occurring after prolonged coincidence of bright winter days and temperatures close to -10 °C. Artificial induction of both the sustained NPQ and recovery from naturally induced sustained NPQ provided information on differential dynamics and light-dependence of 3p-LHCII and p-PSBS accumulation as prerequisites for sustained NPQ. Data obtained collectively suggest three components related to sustained NPQ in spruce: 1) Freezing temperatures induce 3p-LHCII accumulation independently of light, which is suggested to initiate destacking of appressed thylakoid membranes due to increased electrostatic repulsion of adjacent membranes; 2) p-PSBS accumulation is both light- and temperature-dependent and closely linked to the initiation of sustained NPQ, which 3) in concert with PSII photoinhibition, is suggested to trigger sustained NPQ in spruce.

PGR5 and NDH-1 systems do not function as protective electron acceptors but mitigate the consequences of PSI inhibition.

Avoidance of photoinhibition at photosystem (PS)I is based on synchronized function of PSII, PSI, Cytochrome b6f and stromal electron acceptors. Here, we used a special light regime, PSI photoinhibition treatment (PIT), in order to specifically inhibit PSI by accumulating excess electrons at the photosystem (Tikkanen and Grebe, 2018). In the analysis, Arabidopsis thaliana WT was compared to the pgr5 and ndho mutants, deficient in one of the two main cyclic electron transfer pathways described to function as protective alternative electron acceptors of PSI. The aim was to investigate whether the PGR5 (pgr5) and the type I NADH dehydrogenase (NDH-1) (ndho) systems protect PSI from excess electron stress and whether they help plants to cope with the consequences of PSI photoinhibition. First, our data reveals that neither PGR5 nor NDH-1 system protects PSI from a sudden burst of electrons. This strongly suggests that these systems in Arabidopsis thaliana do not function as direct acceptors of electrons delivered from PSII to PSI - contrasting with the flavodiiron proteins that were found to make Physcomitrella patens PSI resistant to the PIT. Second, it is demonstrated that under light-limiting conditions, the electron transfer rate at PSII is linearly dependent on the amount of functional PSI in all genotypes, while under excess light, the PGR5-dependent control of electron flow at the Cytochrome b6f complex overrides the effect of PSI inhibition. Finally, the PIT is shown to increase the amount of PGR5 and NDH-1 as well as of PTOX, suggesting that they mitigate further damage to PSI after photoinhibition rather than protect against it.

Photoinhibition of Photosystem I Provides Oxidative Protection During Imbalanced Photosynthetic Electron Transport in Arabidopsis thaliana.

Photosynthesis involves the conversion of sunlight energy into stored chemical energy, which is achieved through electron transport along a series of redox reactions. Excess photosynthetic electron transport might be dangerous due to the risk of molecular oxygen reduction, generating reactive oxygen species (ROS) over-accumulation. Avoiding excess ROS production requires the rate of electron transport to be coordinated with the capacity of electron acceptors in the chloroplast stroma. Imbalance between the donor and acceptor sides of photosystem I (PSI) can lead to inactivation, which is called PSI photoinhibition. We used a light-inducible PSI photoinhibition system in Arabidopsis thaliana to resolve the time dynamics of inhibition and to investigate its impact on ROS production and turnover. The oxidation state of the PSI reaction center and rates of CO2 fixation both indicated strong and rapid PSI photoinhibition upon donor side/acceptor side imbalance, while the rate of inhibition eased during prolonged imbalance. PSI photoinhibition was not associated with any major changes in ROS accumulation or antioxidant activity; however, a lower level of lipid oxidation correlated with lower abundance of chloroplast lipoxygenase in PSI-inhibited leaves. The results of this study suggest that rapid activation of PSI photoinhibition under severe photosynthetic imbalance protects the chloroplast from over-reduction and excess ROS formation.

The unique photosynthetic apparatus of Pinaceae: analysis of photosynthetic complexes in Picea abies.

Pinaceae are the predominant photosynthetic species in boreal forests, but so far no detailed description of the protein components of the photosynthetic apparatus of these gymnosperms has been available. In this study we report a detailed characterization of the thylakoid photosynthetic machinery of Norway spruce (Picea abies (L.) Karst). We first customized a spruce thylakoid protein database from translated transcript sequences combined with existing protein sequences derived from gene models, which enabled reliable tandem mass spectrometry identification of P. abies thylakoid proteins from two-dimensional large pore blue-native/SDS-PAGE. This allowed a direct comparison of the two-dimensional protein map of thylakoid protein complexes from P. abies with the model angiosperm Arabidopsis thaliana. Although the subunit composition of P. abies core PSI and PSII complexes is largely similar to that of Arabidopsis, there was a high abundance of a smaller PSI subcomplex, closely resembling the assembly intermediate PSI* complex. In addition, the evolutionary distribution of light-harvesting complex (LHC) family members of Pinaceae was compared in silico with other land plants, revealing that P. abies and other Pinaceae (also Gnetaceae and Welwitschiaceae) have lost LHCB4, but retained LHCB8 (formerly called LHCB4.3). The findings reported here show the composition of the photosynthetic apparatus of P. abies and other Pinaceae members to be unique among land plants.

Consequences of photosystem-I damage and repair on photosynthesis and carbon use in Arabidopsis thaliana.

Natural growth environments commonly include fluctuating conditions that can disrupt the photosynthetic energy balance and induce photoinhibition through inactivation of the photosynthetic apparatus. Photosystem II (PSII) photoinhibition is efficiently reversed by the PSII repair cycle, whereas photoinhibited photosystem I (PSI) recovers much more slowly. In the current study, treatment of the Arabidopsis thaliana mutant proton gradient regulation 5 (pgr5) with excess light was used to compromise PSI functionality in order to investigate the impact of photoinhibition and subsequent recovery on photosynthesis and carbon metabolism. The negative impact of PSI photoinhibition on CO2 fixation was especially deleterious under low irradiance. Impaired starch accumulation after PSI photoinhibition was reflected in reduced respiration in the dark, but this was not attributed to impaired sugar synthesis. Normal chloroplast and mitochondrial metabolisms were shown to recover despite the persistence of substantial PSI photoinhibition for several days. The results of this study indicate that the recovery of PSI function involves the reorganization of the light-harvesting antennae, and suggest a pool of surplus PSI that can be recruited to support photosynthesis under demanding conditions.

Chloroplastic ATP synthase optimizes the trade-off between photosynthetic CO2 assimilation and photoprotection during leaf maturation.

In the present study, we studied the role of chloroplastic ATP synthase in photosynthetic regulation during leaf maturation. We measured gas exchange, chlorophyll fluorescence, P700 redox state, and the electrochromic shift signal in mature and immature leaves. Under high light, the immature leaves displayed high levels of non-photochemical quenching (NPQ) and P700 oxidation ratio, and higher values for proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes but lower values for the activity of chloroplastic ATP synthase (gH+) than the mature leaves. Furthermore, gH+ was significantly and positively correlated with CO2 assimilation rate and linear electron flow (LEF), but negatively correlated with pmf and ΔpH. ΔpH was significantly correlated with LEF and the P700 oxidation ratio. These results indicated that gH+ was regulated to match photosynthetic capacity during leaf maturation, and the formation of pmf and ΔpH was predominantly regulated by the alterations in gH+. In the immature leaves, the high steady-state ΔpH increased lumen acidification, which, in turn, stimulated photoprotection for the photosynthetic apparatus via NPQ induction and photosynthetic control. Our results highlighted the importance of chloroplastic ATP synthase in optimizing the trade-off between CO2 assimilation and photoprotection during leaf maturation.

FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors.

Plant-type ferredoxins in Arabidopsis transfer electrons from the photosystem I to multiple redox-driven enzymes involved in the assimilation of carbon, nitrogen, and sulfur. Leaf-type ferredoxins also modulate the switch between the linear and cyclic electron routes of the photosystems. Recently, two novel ferredoxin homologs with extra C-termini were identified in the Arabidopsis genome (AtFdC1, AT4G14890; AtFdC2, AT1G32550). FdC1 was considered as an alternative electron acceptor of PSI under extreme ferredoxin-deficient conditions. Here, we showed that FdC1 could interact with some, but not all, electron acceptors of leaf-type Fds, including the ferredoxin-thioredoxin reductase (FTR), sulfite reductase (SiR), and nitrite reductase (NiR). Photoreduction assay on cytochrome c and enzyme assays confirmed its capability to receive electrons from PSI and donate electrons to the Fd-dependent SiR and NiR but not to the ferredoxin-NADP+ oxidoreductase (FNR). Hence, FdC1 and leaf-type Fds may play differential roles by channeling electrons from photosystem I to different downstream electron acceptors in photosynthetic tissues. In addition, the median redox potential of FdC1 may allow it to receive electrons from FNR in non-photosynthetic plastids.

Photoinhibition of photosystem I in Nephrolepis falciformis depends on reactive oxygen species generated in the chloroplast stroma.

We studied how high light causes photoinhibition of photosystem I (PSI) in the shade-demanding fern Nephrolepis falciformis, in an attempt to understand the mechanism of PSI photoinhibition under natural field conditions. Intact leaves were treated with constant high light and fluctuating light. Detached leaves were treated with constant high light in the presence and absence of methyl viologen (MV). Chlorophyll fluorescence and P700 signal were determined to estimate photoinhibition. PSI was highly oxidized under high light before treatments. N. falciformis showed significantly stronger photoinhibition of PSI and PSII under constant high light than fluctuating light. These results suggest that high levels of P700 oxidation ratio cannot prevent PSI photoinhibition under high light in N. falciformis. Furthermore, photoinhibition of PSI in N. falciformis was largely accelerated in the presence of MV that promotes the production of superoxide anion radicals in the chloroplast stroma by accepting electrons from PSI. From these results, we propose that photoinhibition of PSI in N. falciformis is mainly caused by superoxide radicals generated in the chloroplast stroma, which is different from the mechanism of PSI photoinhibition in Arabidopsis thaliana and spinach. Here, we provide some new insights into the PSI photoinhibition under natural field conditions.

Regulation of cyclic electron flow by chloroplast NADPH-dependent thioredoxin system.

Linear electron transport in the thylakoid membrane drives photosynthetic NADPH and ATP production, while cyclic electron flow (CEF) around photosystem I only promotes the translocation of protons from stroma to thylakoid lumen. The chloroplast NADH dehydrogenase-like complex (NDH) participates in one CEF route transferring electrons from ferredoxin back to the plastoquinone pool with concomitant proton pumping to the lumen. CEF has been proposed to balance the ratio of ATP/NADPH production and to control the redox poise particularly in fluctuating light conditions, but the mechanisms regulating the NDH complex remain unknown. We have investigated potential regulation of the CEF pathways by the chloroplast NADPH-thioredoxin reductase (NTRC) in vivo by using an Arabidopsis knockout line of NTRC as well as lines overexpressing NTRC. Here, we present biochemical and biophysical evidence showing that NTRC stimulates the activity of NDH-dependent CEF and is involved in the regulation of generation of proton motive force, thylakoid conductivity to protons, and redox balance between the thylakoid electron transfer chain and the stroma during changes in light conditions. Furthermore, protein-protein interaction assays suggest a putative thioredoxin-target site in close proximity to the ferredoxin-binding domain of NDH, thus providing a plausible mechanism for redox regulation of the NDH ferredoxin:plastoquinone oxidoreductase activity.

Phosphorylation-induced lateral rearrangements of thylakoid protein complexes upon light acclimation.

Understanding the mechanistic basis of balanced excitation energy distribution between photosystem II and photosystem I (PSII and PSI) requires detailed investigation of the thylakoid light-harvesting system composed of energetically connected LHCII trimers. The exact mechanisms controlling the excitation energy distribution remain elusive, but reversible phosphorylation is known to be one important component. Here, we addressed the role of grana margins in regulation of excitation energy distribution, as these thylakoid domains host all the complexes of photosynthetic light reactions with dynamic response to environmental cues. First, the effect of detergents for the thylakoid membrane connectivity is explained. We show that a specific interaction between the separate LHCII trimers as well as between the LHCII trimers and the PSII and PSI-LHCI complexes is a prerequisite for energetically connected and functional thylakoid membrane. Second, we demonstrate that the optimization of light reactions under changing light conditions takes place in energetically connected LHCII lake and is attained by lateral rearrangements of the PSII-LHCII and PSI-LHCI-LHCII complexes depending especially on the phosphorylation status of the LHCII protein isoform LHCB2.

Switching off photoprotection of photosystem I - a novel tool for gradual PSI photoinhibition.

Photosystem I (PSI) has evolved in anaerobic atmospheric conditions and until today remains susceptible to oxygen. To minimize the probability of damaging side reactions, plants have evolved sophisticated mechanisms to control electron transfer, and PSI becomes inhibited only when malfunctions of these regulatory mechanisms occur. Because of the complicated induction of PSI photoinhibition, a detailed investigation into the process and following reactions are still largely missing. Here, we introduce the theoretical framework and a novel method for an easy and controlled induction of PSI photoinhibition in vivo. The method mimics the PSI damage mechanisms of fluctuating light-sensitive mutant plants (stn7, pgr5) which cannot control electron donation to PSI. Because PSII and PSI have different light absorption properties, electrons accumulate in the intersystem electron transfer chain (ETC), if PSII is preferentially excited. A saturating light pulse given upon an over-reduced ETC leads to the saturation of PSI electron acceptors, ultimately leading to the production of reactive oxygen species and photoinhibition of PSI. By adjusting the time of the light treatment, PSI can be gradually photoinhibited, providing a novel tool to holistically investigate the PSI photoinhibition phenomenon.

Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane.

Conversion of solar energy into chemical energy in plant chloroplasts concomitantly modifies the thylakoid architecture and hierarchical interactions between pigment-protein complexes. Here, the thylakoids were isolated from light-acclimated Arabidopsis leaves and investigated with respect to the composition of the thylakoid protein complexes and their association into higher molecular mass complexes, the largest one comprising both photosystems (PSII and PSI) and light-harvesting chlorophyll a/b-binding complexes (LHCII). Because the majority of plant light-harvesting capacity is accommodated in LHCII complexes, their structural interaction with photosystem core complexes is extremely important for efficient light harvesting. Specific differences in the strength of LHCII binding to PSII core complexes and the formation of PSII supercomplexes are well characterized. Yet, the role of loosely bound L-LHCII that disconnects to a large extent during the isolation of thylakoid protein complexes remains elusive. Because L-LHCII apparently has a flexible role in light harvesting and energy dissipation, depending on environmental conditions, its close interaction with photosystems is a prerequisite for successful light harvesting in vivo. Here, to reveal the labile and fragile light-dependent protein interactions in the thylakoid network, isolated membranes were subjected to sequential solubilization using detergents with differential solubilization capacity and applying strict quality control. Optimized 3D-lpBN-lpBN-sodium dodecyl sulfate-polyacrylamide gel electrophoresis system demonstrated that PSII-LHCII supercomplexes, together with PSI complexes, hierarchically form larger megacomplexes via interactions with L-LHCII trimers. The polypeptide composition of LHCII trimers and the phosphorylation of Lhcb1 and Lhcb2 were examined to determine the light-dependent supramolecular organization of the photosystems into megacomplexes.

Interaction between photosynthetic electron transport and chloroplast sinks triggers protection and signalling important for plant productivity.

The photosynthetic light reactions provide energy that is consumed and stored in electron sinks, the products of photosynthesis. A balance between light reactions and electron consumption in the chloroplast is vital for plants, and is protected by several photosynthetic regulation mechanisms. Photosystem I (PSI) is particularly susceptible to photoinhibition when these factors become unbalanced, which can occur in low temperatures or in high light. In this study we used the pgr5 Arabidopsis mutant that lacks ΔpH-dependent regulation of photosynthetic electron transport as a model to study the consequences of PSI photoinhibition under high light. We found that PSI damage severely inhibits carbon fixation and starch accumulation, and attenuates enzymatic oxylipin synthesis and chloroplast regulation of nuclear gene expression after high light stress. This work shows that modifications to regulation of photosynthetic light reactions, which may be designed to improve yield in crop plants, can negatively impact metabolism and signalling, and thereby threaten plant growth and stress tolerance.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.

Comparative analysis of mutant plants impaired in the main regulatory mechanisms of photosynthetic light reactions - From biophysical measurements to molecular mechanisms.

Chlorophyll (chl) fluorescence emission by photosystem II (PSII) and light absorption by P700 reaction center chl a of photosystem I (PSI) provide easy means to probe the function of the photosynthetic machinery. The exact relationship between the measured optical variables and the molecular processes have, however, remained elusive. Today, the availability of mutants with distinct molecular characterization of photosynthesis regulatory processes should make it possible to gain further insights into this relationship, yet a systematic comparative analysis of such regulatory mutants has been missing. Here we have systematically compared the behavior of Dual-PAM fluorescence and P700 variables from well-characterized photosynthesis regulation mutants. The analysis revealed a very convincing relationship between the given molecular deficiency in the photosynthetic apparatus and the original fluorescence and P700 signals obtained by using varying intensities of actinic light and by applying a saturating pulse. Importantly, the specific information on the underlying molecular mechanism, present in these authentic signals of a given photosynthesis mutant, was largely nullified when using the commonly accepted parameters that are based on further treatment of the original signals. Understanding the unique relationship between the investigated molecular process of photosynthesis and the measured variable is an absolute prerequisite for comprehensive interpretation of fluorescence and P700 measurements. The data presented here elucidates the relationships between the main regulatory mechanisms controlling the photosynthetic light reactions and the variables obtained by fluorescence and P700 measurements. It is discussed how the full potential of optical photosynthesis measurements can be utilized in investigation of a given molecular mechanism.