The mechanisms of photoinhibition and repair in plants under high light conditions and interplay with abiotic stressors.

This review comprehensively examines the phenomenon of photoinhibition in plants, focusing mainly on the intricate relationship between photodamage and photosystem II (PSII) repair and the role of PSII extrinsic proteins and protein phosphorylation in these processes. In natural environments, photoinhibition occurs together with a suite of concurrent stress factors, including extreme temperatures, drought and salinization. Photoinhibition, primarily caused by high irradiance, results in a critical imbalance between the rate of PSII photodamage and its repair. Central to this process is the generation of reactive oxygen species (ROS), which not only impair the photosynthetic apparatus first PSII but also play a signalling role in chloroplasts and other cellulular structures. ROS generated under stress conditions inhibit the repair of photodamaged PSII by suppressing D1 protein synthesis and affecting PSII protein phosphorylation. Furthermore, this review considers how environmental stressors exacerbate PSII damage by interfering with PSII repair primarily by reducing de novo protein synthesis. In addition to causing direct damage, these stressors also contribute to ROS production by restricting CO2 fixation, which also reduces the intensity of protein synthesis. This knowledge has significant implications for agricultural practices and crop improvement under stressful conditions.

Photo-oxidative damage of photosystem I by repetitive flashes and chilling stress in cucumber leaves.

Photosystem I (PSI) is an essential protein complex for oxygenic photosynthesis and is also known to be an important source of reactive oxygen species (ROS) in the light. When ROS are generated within PSI, the photosystem can be damaged. The so-called PSI photoinhibition is a lethal event for oxygenic phototrophs, and it is prevented by keeping the reaction center chlorophyll (P700) oxidized in excess light conditions. Whereas regulatory mechanisms for controlling P700 oxidation have been discovered already, the molecular mechanism of PSI photoinhibition is still unclear. Here, we characterized the damage mechanism of PSI photoinhibition by in vitro transient absorption and electron paramagnetic resonance (EPR) spectroscopy in isolated PSI from cucumber leaves that had been subjected to photoinhibition treatment. Photodamage to PSI was induced by two different light treatments: 1. continuous illumination with high light at low (chilling) temperature (C/LT) and 2. repetitive flashes at room temperature (F/RT). These samples were compared to samples that had been illuminated with high light at room temperature (C/RT). The [FeS] clusters FX and (FA FB) were destructed in C/LT but not in F/RT. Transient absorption spectroscopy indicated that half of the charge separation was impaired in F/RT, however, low-temperature EPR revealed the light-induced FX signal at a similar size as in the case of C/RT. This indicates that the two branches of electron transfer in PSI were affected differently. Electron transfer at the A-branch was inhibited in F/RT and also partially in C/LT, while the B-branch remained active.

Structural insight into the functional regulation of Elongation factor Tu by reactive oxygen species in Synechococcus elongatus PCC 7942.

In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Šand 2.0 Šin Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis.

Outdoor cultivation of Picochlorum sp. in a novel V-shaped photobioreactor on the Caribbean island Bonaire.

Microalgae are a promising renewable feedstock that can be produced on non-arable land using seawater. Their biomass contains proteins, lipids, carbohydrates, and pigments, and can be used for various biobased products, such as food, feed, biochemicals, and biofuels. For such applications, the production costs need to be reduced, for example, by improving biomass productivity in photobioreactors. In this study, Picochlorum sp. (BPE23) was cultivated in a prototype of a novel outdoor V-shaped photobioreactor on Bonaire (12°N, 68°W). The novel photobioreactor design was previously proposed for the capture and dilution of sunlight at low-latitude locations. During several months, the biomass productivity of the local thermotolerant microalgae was determined at different dilution rates in continuous dilution and batch dilution experiments, without any form of temperature control. Reactor temperatures increased to 35°C-45°C at midday. In the continuous dilution experiments, high average biomass productivities of 28-31 g m-2 d-1 and photosynthetic efficiencies of 3.5%-4.3% were achieved. In the batch dilution experiments, biomass productivities were lower (17-23 g m-2 d-1), as microalgal cells likely experienced sudden light and temperature stress after daily reactor dilution. Nonetheless, dense cultures were characterized by high maximum photosynthetic rates, illustrating the potential of Picochlorum sp. for fast growth under outdoor conditions.

Psb28 protein is indispensable for stable accumulation of PSII core complexes in Arabidopsis.

Enhancing the efficiency of photosynthesis represents a promising strategy to improve crop yields, with keeping the steady state of PSII being key to determining the photosynthetic performance. However, the mechanisms whereby the stability of PSII is maintained in oxygenic organisms remain to be explored. Here, we report that the Psb28 protein functions in regulating the homeostasis of PSII under different light conditions in Arabidopsis thaliana. The psb28 mutant is much smaller than the wild-type plants under normal growth light, which is due to its significantly reduced PSII activity. Similar defects were seen under low light and became more pronounced under photoinhibitory light. Notably, the amounts of PSII core complexes and core subunits are specifically decreased in psb28, whereas the abundance of other representative components of photosynthetic complexes remains largely unaltered. Although the PSII activity of psb28 was severely reduced when subjected to high light, its recovery from photoinactivation was not affected. By contrast, the degradation of PSII core protein subunits is dramatically accelerated in the presence of lincomycin. These results indicate that psb28 is defective in the photoprotection of PSII, which is consistent with the observation that the overall NPQ is much lower in psb28 compared to the wild type. Moreover, the Psb28 protein is associated with PSII core complexes and interacts mainly with the CP47 subunit of PSII core. Taken together, these findings reveal an important role for Psb28 in the protection and stabilization of PSII core in response to changes in light environments.

Rapid Continuous 3D Printing via Orthogonal Dual-Color Photoinitiation and Photoinhibition.

Stereolithographic additive manufacturing technology has developed from point-by-point scanning exposure to layer-by-layer masking curing and even volumetric printing. Rapid prototyping is one of the important goals pursued by researchers. A continuous three-dimensional (3D) printing system based on the dual-color photoinitiation and photoinhibition is proposed with the aim of further improving printing speed. The process of continuous 3D printing is realized through the anti-polymerization layer between the cured part and the window generated by the ultraviolet (UV) light sheet (355 nm), and dynamic masking with the blue light (470 nm). The volume of the anti-polymerization layer can be adjusted by the intensity ratio of the incident lights (IUV, 0/Iblue,0) and the size of UV laser spot to enhance the reflow filling rate of the liquid resin. For the orthogonal Gaussian anti-polymerization layer, an intensity ratio of 28.6 allows for an inhibition volume of 97.1% of the desired rectangular anti-polymerization zone with a height of 1 mm. The simulation analysis of continuous 3D printing process by flow-structure interaction reveals that the increase of the thickness of the anti-polymerization layer effectively improves the filling rate of the resin and the cross-sectional area of printing, and reduces the stress of the cured part. The experiments with two different 3D structures printing demonstrate that the filling rate and the stress have virtually no effect on the printing process at a large-scale thickness of the anti-polymerization layer, and the printing speed is capable of reaching 200 µm/s. Certainly, the printing volume and complexity can be further improved with the improvement of the system and the optimization of the resin.

Chlorophyll Fluorescence on the Fast Timescale.

Chlorophyll fluorescence is a rapid and noninvasive tool used for probing the activity of photosynthesis that can be used in vivo and in the field. It is highly relevant to the demands of high-throughput crop phenotyping and can be automated or manually applied. In this chapter, we describe protocols and advice for making fast timescale fluorescence measurements using handheld equipment in the laboratory or in the field in the context of phenotyping. While interpretation of some measured parameters requires caution for the purpose of identifying underlying mechanisms, we demonstrate this technique is appropriate for some applications where convenience, rapidity, and sensitivity are required.

S2P2-the chloroplast-located intramembrane protease and its impact on the stoichiometry and functioning of the photosynthetic apparatus of A. thaliana.

S2P2 is a nuclear-encoded protease, potentially located in chloroplasts, which belongs to the zinc-containing, intramembrane, site-2 protease (S2P) family. In A. thaliana cells, most of the S2P proteases are located within the chloroplasts, where they play an important role in the development of chloroplasts, maintaining proper stoichiometric relations between polypeptides building photosynthetic complexes and influencing the sensitivity of plants to photoinhibitory conditions. Among the known chloroplast S2P proteases, S2P2 protease is one of the least known. Its exact location within the chloroplast is not known, nor is anything known about its possible physiological functions. Therefore, we decided to investigate an intra-chloroplast localization and the possible physiological role of S2P2. To study the intra-chloroplast localization of S2P2, we used specific anti-S2P2 antibodies and highly purified chloroplast fractions containing envelope, stroma, and thylakoid proteins. To study the physiological role of the protease, we used two lines of insertion mutants lacking the S2P2 protease protein. Here, we present results demonstrating the thylakoid localization of S2P2. Moreover, we present experimental evidence indicating that the lack of S2P2 in A. thaliana chloroplasts leads to a significant decrease in the level of photosystem I and photosystem II core proteins: PsaB, PsbA, PsbD, and PsbC, as well as polypeptides building both the main light-harvesting antenna (LHC II), Lhcb1 and Lhcb2, as well as Lhcb4 and Lhcb5 polypeptides, constituting elements of the minor, peripheral antenna system. These changes are associated with a decrease in the number of PS II-LHC II supercomplexes. The consequence of these disorders is a greater sensitivity of s2p2 mutants to photoinhibition. The obtained results clearly indicate that the S2P2 protease is another thylakoid protein that plays an important role in the proper functioning of A. thaliana chloroplasts, especially in high-light-intensity conditions.

Accounting for photosystem I photoinhibition sheds new light on seasonal acclimation strategies of boreal conifers.

The photosynthetic acclimation of boreal evergreen conifers is controlled by regulatory and photoprotective mechanisms that allow conifers to cope with extreme environmental changes. However, the underlying dynamics of photosystem II (PSII) and photosystem I (PSI) remain unresolved. Here, we investigated the dynamics of PSII and PSI during the spring recovery of photosynthesis in Pinus sylvestris and Picea abies using a combination of chlorophyll a fluorescence, P700 difference absorbance measurements, and quantification of key thylakoid protein abundances. In particular, we derived a new set of PSI quantum yield equations, correcting for the effects of PSI photoinhibition. Using the corrected equations, we found that the seasonal dynamics of PSII and PSI photochemical yields remained largely in balance, despite substantial seasonal changes in the stoichiometry of PSII and PSI core complexes driven by PSI photoinhibition. Similarly, the previously reported seasonal up-regulation of cyclic electron flow was no longer evident, after accounting for PSI photoinhibition. Overall, our results emphasize the importance of considering the dynamics of PSII and PSI to elucidate the seasonal acclimation of photosynthesis in overwintering evergreens. Beyond the scope of conifers, our corrected PSI quantum yields expand the toolkit for future studies aimed at elucidating the dynamic regulation of PSI.

Mechanisms of photoprotection in overwintering evergreen conifers: Sustained quenching of chlorophyll fluorescence.

Evergreen conifers growing in high-latitude regions must endure prolonged winters that are characterized by sub-zero temperatures combined with light, conditions that can cause significant photooxidative stress. Understanding overwintering mechanisms is crucial for addressing winter adversity in temperate forest ecosystems and enhancing the ability of conifers to adapt to climate change. This review synthesizes the current understanding of the photoprotective mechanisms that conifers employ to mitigate photooxidative stress, particularly non-photochemical "sustained quenching", the mechanism of which is hypothesized to be a recombination or deformation of the original mechanism employed by conifers in response to short-term low temperature and intense light stress in the past. Based on this hypothesis, scattered studies in this field are assembled and integrated into a complete mechanism of sustained quenching embedded in the adaptation process of plant physiology. It also reveals which parts of the whole system have been verified in conifers and which have only been verified in non-conifers, and proposes specific directions for future research. The functional implications of studies of non-coniferous plant species for the study of coniferous trees are also considered, as a wide range of plant responses lead to sustained quenching, even among different conifer species. In addition, the review highlights the challenges of measuring sustained quenching and discusses the application of ultrafast-time-resolved fluorescence and decay-associated spectra for the elucidation of photosynthetic principles.

Addressing the long-standing limitations of double exponential and non-rectangular hyperbolic models in quantifying light-response of electron transport rates in different photosynthetic organisms under various conditions.

The models used to describe the light response of electron transport rate in photosynthesis play a crucial role in determining two key parameters i.e., the maximum electron transport rate (J max) and the saturation light intensity (I sat). However, not all models accurately fit J-I curves, and determine the values of J max and I sat. Here, three models, namely the double exponential (DE) model, the non-rectangular hyperbolic (NRH) model, and a mechanistic model developed by one of the coauthors (Z-P Ye) and his coworkers (referred to as the mechanistic model), were compared in terms of their ability to fit J-I curves and estimate J max and I sat. Here, we apply these three models to a series of previously collected Chl a fluorescence data from seven photosynthetic organisms, grown under different conditions. Our results show that the mechanistic model performed well in describing the J-I curves, regardless of whether photoinhibition/dynamic down-regulation of photosystem II (PSII) occurs. Moreover, both J max and I sat estimated by this model are in very good agreement with the measured data. On the contrary, although the DE model simulates quite well the J-I curve for the species studied, it significantly overestimates both the J max of Amaranthus hypochondriacus and the I sat of Microcystis aeruginosa grown under NH4 +-N supply. More importantly, the light intensity required to achieve the potential maximum of J (J s) estimated by this model exceeds the unexpected high value of 105 µmol photons m-2 s-1 for Triticum aestivum and A. hypochondriacus. The NRH model fails to characterize the J-I curves with dynamic down-regulation/photoinhibition for Abies alba, Oryza sativa and M. aeruginosa. In addition, this model also significantly overestimates the values of J max for T. aestivum at 21% O2 and A. hypochondriacus grown under normal condition, and significantly underestimates the values of J max for M. aeruginosa grown under NO3 -N supply. Our study provides evidence that the 'mechanistic model' is much more suitable than both the DE and NRH models in fitting the J-I curves and in estimating the photosynthetic parameters. This is a powerful tool for studying light harvesting properties and the dynamic down-regulation of PSII/photoinhibition.

Dysfunction of Chloroplast Protease Activity Mitigates pgr5 Phenotype in the Green Algae Chlamydomonas reinhardtii.

Researchers have described protection mechanisms against the photoinhibition of photosystems under strong-light stress. Cyclic Electron Flow (CEF) mitigates electron acceptor-side limitation, and thus contributes to Photosystem I (PSI) protection. Chloroplast protease removes damaged protein to assist with protein turn over, which contributes to the quality control of Photosystem II (PSII). The PGR5 protein is involved in PGR5-dependent CEF. The FTSH protein is a chloroplast protease which effectively degrades the damaged PSII reaction center subunit, D1 protein. To investigate how the PSI photoinhibition phenotype in pgr5 would be affected by adding the ftsh mutation, we generated double-mutant pgr5ftsh via crossing, and its phenotype was characterized in the green algae Chlamydomonas reinhardtii. The cells underwent high-light incubation as well as low-light incubation after high-light incubation. The time course of Fv/Fm values in pgr5ftsh showed the same phenotype with ftsh1-1. The amplitude of light-induced P700 photo-oxidation absorbance change was measured. The amplitude was maintained at a low value in the control and pgr5ftsh during high-light incubation, but was continuously decreased in pgr5. During the low-light incubation after high-light incubation, amplitude was more rapidly recovered in pgr5ftsh than pgr5. We concluded that the PSI photoinhibition by the pgr5 mutation is mitigated by an additional ftsh1-1 mutation, in which plastoquinone pool would be less reduced due to damaged PSII accumulation.

High Myristic Acid in Glycerolipids Enhances the Repair of Photodamaged Photosystem II under Strong Light.

Cyanobacteria inhabit areas with a broad range of light, temperature and nutrient conditions. The robustness of cyanobacterial cells, which can survive under different conditions, may depend on the resilience of photosynthetic activity. Cyanothece sp. PCC 8801 (Cyanothece), a freshwater cyanobacterium isolated from a Taiwanese rice field, had a higher repair activity of photodamaged photosystem II (PSII) under intense light than Synechocystis sp. PCC 6803 (Synechocystis), another freshwater cyanobacterium. Cyanothece contains myristic acid (14:0) as the major fatty acid at the sn-2 position of the glycerolipids. To investigate the role of 14:0 in the repair of photodamaged PSII, we used a Synechocystis transformant expressing a T-1274 encoding a lysophosphatidic acid acyltransferase (LPAAT) from Cyanothece. The wild-type and transformant cells contained 0.2 and 20.1 mol% of 14:0 in glycerolipids, respectively. The higher content of 14:0 in the transformants increased the fluidity of the thylakoid membrane. In the transformants, PSII repair was accelerated due to an enhancement in the de novo synthesis of D1 protein, and the production of singlet oxygen (1O2), which inhibited protein synthesis, was suppressed. The high content of 14:0 increased transfer of light energy received by phycobilisomes to PSI and CP47 in PSII and the content of carotenoids. These results indicated that an increase in 14:0 reduced 1O2 formation and enhanced PSII repair. The higher content of 14:0 in the glycerolipids may be required as a survival strategy for Cyanothece inhabiting a rice field under direct sunlight.

Anthocyanins act as a sugar-buffer and an alternative electron sink in response to starch depletion during leaf senescence: a case study on a typical anthocyanic tree species, Acer japonicum.

We hypothesized that anthocyanins act as a sugar-buffer and an alternative electron sink during leaf senescence to prevent sugar-mediated early senescence and photoinhibition. To elucidate the role of anthocyanin, we monitored seasonal changes in photosynthetic traits, sugar, starch and N contents, pigment composition, and gene expression profiles in leaves exposed to substantially different light conditions within a canopy of an adult fullmoon maple (Acer japonicum) tree. Enhancement of starch amylolysis accompanied by cessation of starch synthesis occurred in the same manner independent of light conditions. Leaf sugar contents increased, but reached upper limits in the late stage of leaf senescence, even though leaf anthocyanins further increased after complete depletion of starch. Sun-exposed leaves maintained higher energy consumption via electron flow than shade-grown leaves during leaf N resorption. Thus, anthocyanins accumulated in sun-exposed leaves might have a regulative role as a sugar-buffer, retarding leaf senescence, and an indirect photoprotective role as an alternative sink for electron consumption to compensate declines in other metabolic processes such as starch and protein synthesis. In this context, anthocyanins may be key substrates protecting both outer-canopy leaves (against photoinhibition) and inner-canopy leaves (via shading by outer-canopy leaves) from high light stress during N resorption.

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been studied, and its involvement in state transitions and PSII repair is known. Yet, little is known about the phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, we applied proteomics analysis to generate a map of the phosphorylation sites of the PSI-LHCI proteins in Chlorella ohadii cells that were grown under low or extreme high-light intensities (LL and HL). Furthermore, we analyzed the content of oxidized tryptophans and PSI-LHCI protein degradation products in these cells, to estimate the light-induced damage to PSI-LHCI. Our work revealed the phosphorylation of 17 of 22 PSI-LHCI subunits. The analyses detected the extensive phosphorylation of the LHCI subunits Lhca6 and Lhca7, which is modulated by growth light intensity. Other PSI-LHCI subunits were phosphorylated to a lesser extent, including PsaE, where molecular dynamic simulation proposed that a phosphoserine stabilizes ferredoxin binding. Additionally, we show that HL-grown cells accumulate less oxidative damage and degradation products of PSI-LHCI proteins, compared with LL-grown cells. The significant phosphorylation of Lhca6 and Lhca7 at the interface with other LHCI subunits suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other subunits.

Interplay among photoreceptors determines the strategy of coping with excess light in tomato.

This study investigates photoreceptor's role in the adaption of photosynthetic apparatus to high light (HL) intensity by examining the response of tomato wild type (WT) (Solanum lycopersicum L. cv. Moneymaker) and tomato mutants (phyA, phyB1, phyB2, cry1) plants to HL. Our results showed a photoreceptor-dependent effect of HL on the maximum quantum yield of photosystem II (Fv/Fm) with phyB1 exhibiting a decrease, while phyB2 exhibiting an increase in Fv/Fm. HL resulted in an increase in the efficient quantum yield of photosystem II (ΦPSII) and a decrease in the non-photochemical quantum yields (ΦNPQ and ΦN0) solely in phyA. Under HL, phyA showed a significant decrease in the energy-dependent quenching component of NPQ (qE), while phyB2 mutants showed an increase in the state transition (qT) component. Furthermore, ΔΔFv/Fm revealed that PHYB1 compensates for the deficit of PHYA in phyA mutants. PHYA signaling likely emerges as the dominant effector of PHYB1 and PHYB2 signaling within the HL-induced signaling network. In addition, PHYB1 compensates for the role of CRY1 in regulating Fv/Fm in cry1 mutants. Overall, the results of this research provide valuable insights into the unique role of each photoreceptor and their interplay in balancing photon energy and photoprotection under HL condition.

Plastid terminal oxidase (PTOX) protects photosystem I and not photosystem II against photoinhibition in Arabidopsis thaliana and Marchantia polymorpha.

The plastid terminal oxidase PTOX controls the oxidation level of the plastoquinone pool in the thylakoid membrane and acts as a safety valve upon abiotic stress, but detailed characterization of its role in protecting the photosynthetic apparatus is limited. Here we used PTOX mutants in two model plants Arabidopsis thaliana and Marchantia polymorpha. In Arabidopsis, lack of PTOX leads to a severe defect in pigmentation, a so-called variegated phenotype, when plants are grown at standard light intensities. We created a green Arabidopsis PTOX mutant expressing the bacterial carotenoid desaturase CRTI and a double mutant in Marchantia lacking both PTOX isoforms, the plant-type and the alga-type PTOX. In both species, lack of PTOX affected the redox state of the plastoquinone pool. Exposure of plants to high light intensity showed in the absence of PTOX higher susceptibility of photosystem I to light-induced damage while photosystem II was more stable compared with the wild type demonstrating that PTOX plays both, a pro-oxidant and an anti-oxidant role in vivo. Our results shed new light on the function of PTOX in the protection of photosystem I and II.

Improved capacity for the repair of photosystem II via reinforcement of the translational and antioxidation systems in Synechocystis sp. PCC 6803.

In the cyanobacterium Synechocystis sp. PCC 6803, translation factor EF-Tu is inactivated by reactive oxygen species (ROS) via oxidation of Cys82 and the oxidation of EF-Tu enhances the inhibition of the repair of photosystem II (PSII) by suppressing protein synthesis. In our present study, we generated transformants of Synechocystis that overexpressed a mutated form of EF-Tu, designated EF-Tu (C82S), in which Cys82 had been replaced by a Ser residue, and ROS-scavenging enzymes individually or together. Expression of EF-Tu (C82S) alone in Synechocystis enhanced the repair of PSII under strong light, with the resultant mitigation of PSII photoinhibition, but it stimulated the production of ROS. However, overexpression of superoxide dismutase and catalase, together with the expression of EF-Tu (C82S), lowered intracellular levels of ROS and enhanced the repair of PSII more significantly under strong light, via facilitation of the synthesis de novo of the D1 protein. By contrast, the activity of photosystem I was hardly affected in wild-type cells and in all the lines of transformed cells under the same strong-light conditions. Furthermore, transformed cells that overexpressed EF-Tu (C82S), superoxide dismutase, and catalase were able to survive longer under stronger light than wild-type cells. Thus, the reinforced capacity for both protein synthesis and ROS scavenging allowed both photosynthesis and cell proliferation to tolerate strong light.