Exploring LHCSR3 expression and its role in Chlamydomonas reinhardtii under osmotic stress: Implications for non-photochemical quenching mechanism.

Plants have a protective mechanism called non-photochemical quenching to prevent damage caused by excessive sunlight. A critical component of this mechanism is energy-dependent quenching (qE). In Chlamydomonas reinhardtii, the protein expression called light-harvesting complex stress-related protein 3 (LHCSR3) is crucial for the qE mechanism. LHCSR3 expression is observed in various conditions that result in photooxidation, such as exposure to high light or nutrient deprivation, where the amount of captured light surpasses the maximum photosynthetic capacity. Although the role of LHCSR3 has been extensively studied under high light (HL) conditions, its function during nutrient starvation remains unclear. In this study, we demonstrate that LHCSR3 expression can occur under light intensities below saturation without triggering qE, particularly when nutrients are limited. To investigate this, we cultivated C. reinhardtii cells under osmotic stress, which replicates conditions of nutrient scarcity. Furthermore, we examined the photosynthetic membrane complexes of wild-type (WT) and npq4 mutant strains grown under osmotic stress. Our analysis revealed that LHCSR3 expression might modify the interaction between the photosystem II core and its peripheral light-harvesting complex II antennae. This alteration could potentially impede the transfer of excitation energy from the antenna to the reaction center.

Metabolomic response to high light from pgrl1 and pgr5 mutants of Chlamydomonas reinhardtii.

Chlamydomonas (C.) reinhardtii metabolomic changes in cyclic electron flow-dependent mutants are still unknown. Here, we used mass spectrometric analysis to monitor the changes in metabolite levels in wild-type, cyclic electron-deficient mutants pgrl1 and pgr5 grown under high-light stress. A total of 55 metabolites were detected using GC-MS analysis. High-light stress-induced selective anaplerotic amino acids in pgr5. In addition, pgr5 showed enhancement in carbohydrate, polyamine, and polyol metabolism by 2.5-fold under high light. In response to high light, pgr5 triggers an increase in several metabolites involved in regulating osmotic pressure. Among these metabolites are glycerol pathway compounds such as glycerol-3-phosphate and glyceryl-glycoside, which increase significantly by 1.55 and 3.07 times, respectively. In addition, pgr5 also enhanced proline and putrescine levels by 2.6- and 1.36-fold under high light. On the other hand, pgrl1-induced metabolites, such as alanine and serine, are crucial for photorespiration when subjected to high-light stress. We also observed a significant increase in levels of polyols and glycerol by 1.37- and 2.97-fold in pgrl1 under high-light stress. Both correlation network studies and KEGG pathway enrichment analysis revealed that metabolites related to several biological pathways, such as amino acid, carbohydrate, TCA cycle, and fatty acid metabolism, were positively correlated in pgrl1 and pgr5 under high-light stress conditions. The relative mRNA expression levels of genes related to the TCA cycle, including PDC3, ACH1, OGD2, OGD3, IDH3, and MDH4, were significantly upregulated in pgrl1 and pgr5 under HL. In pgr5, the MDH1 level was significantly increased, while ACS1, ACS3, IDH2, and IDH3 levels were reduced considerably in pgrl1 under high-light stress. The current study demonstrates both pgr5 and prgl1 showed a differential defense response to high-light stress at the primary metabolites and mRNA expression level, which can be added to the existing knowledge to explore molecular regulatory responses of prg5 and pgrl1 to high-light stress.

High light-induced changes in whole-cell proteomic profile and its correlation with the organization of thylakoid super-complex in cyclic electron transport mutants of Chlamydomonas reinhardtii.

Light and nutrients are essential components of photosynthesis. Activating the signaling cascades is critical in starting adaptive processes in response to high light. In this study, we have used wild-type (WT), cyclic electron transport (CET) mutants like Proton Gradient Regulation (PGR) (PGRL1), and PGR5 to elucidate the actual role in regulation and assembly of photosynthetic pigment-protein complexes under high light. Here, we have correlated the biophysical, biochemical, and proteomic approaches to understand the targeted proteins and the organization of thylakoid pigment-protein complexes in the photoacclimation. The proteomic analysis showed that 320 proteins were significantly affected under high light compared to the control and are mainly involved in the photosynthetic electron transport chain, protein synthesis, metabolic process, glycolysis, and proteins involved in cytoskeleton assembly. Additionally, we observed that the cytochrome (Cyt) b6 expression is increased in the pgr5 mutant to regulate proton motive force and ATPase across the thylakoid membrane. The increased Cyt b6 function in pgr5 could be due to the compromised function of chloroplast (cp) ATP synthase subunits for energy generation and photoprotection under high light. Moreover, our proteome data show that the photosystem subunit II (PSBS) protein isoforms (PSBS1 and PSBS2) expressed more than the Light-Harvesting Complex Stress-Related (LHCSR) protein in pgr5 compared to WT and pgrl1 under high light. The immunoblot data shows the photosystem II proteins D1 and D2 accumulated more in pgrl1 and pgr5 than WT under high light. In high light, CP43 and CP47 showed a reduced amount in pgr5 under high light due to changes in chlorophyll and carotenoid content around the PSII protein, which coordinates as a cofactor for efficient energy transfer from the light-harvesting antenna to the photosystem core. BN-PAGE and circular dichroism studies indicate changes in macromolecular assembly and thylakoid super-complexes destacking in pgrl1 and pgr5 due to changes in the pigment-protein complexes under high light. Based on this study, we emphasize that this is an excellent aid in understanding the role of CET mutants in thylakoid protein abundances and super-complex organization under high light.

Photosynthetic efficiency and transcriptome analysis of Dunaliella salina under hypersaline: a retrograde signaling mechanism in the chloroplast.

Understanding the molecular mechanisms of environmental salinity stress tolerance and acclimation strategies by photosynthetic organisms facilitates accelerating the genetic improvement of tolerant economically important crops. In this study, we have chosen the marine algae Dunaliella (D.) salina, a high-potential and unique organism that shows superior tolerance against abiotic stresses, especially hypersaline conditions. We have grown the cells in three different salt concentrations 1.5M NaCl (control), 2M NaCl, and 3M NaCl (hypersaline). Fast chlorophyll fluorescence analysis showed increased initial fluorescence (Fo) and decreased photosynthetic efficiency, indicating hampered photosystem II utilization capacity under hypersaline conditions. Also, the reactive oxygen species (ROS) localization studies and quantification revealed elevated accumulation of ROS was observed in the chloroplast in the 3M condition. Pigment analysis shows a deficit in chlorophyll content and increased carotenoid accumulation, especially lutein and zeaxanthin content. This study majorly explored the chloroplast transcripts of the D. salina cell as it is the major environmental sensor. Even though most of the photosystem transcripts showed moderate upregulation in hypersaline conditions in the transcriptome study, the western blot analysis showed degradation of the core as well as antenna proteins of both the photosystems. Among the upregulated chloroplast transcripts, chloroplast Tidi, flavodoxin IsiB, and carotenoid biosynthesis-related protein transcripts strongly proposed photosynthetic apparatus remodeling. Also, the transcriptomic study revealed the upregulation of the tetrapyrrole biosynthesis pathway (TPB) and identified the presence of a negative regulator of this pathway, called the s-FLP splicing variant. These observations point towards the accumulation of TPB pathway intermediates PROTO-IX, Mg-PROTO-IX, and P-Chlide, those earlier reported as retrograde signaling molecules. Our comparative transcriptomic approach along with biophysical and biochemical studies in D. salina grown under control (1.5 M NaCl) and hypersaline (3M NaCl) conditions, unveil an efficient retrograde signaling mechanism mediated remodeling of photosynthetic apparatus.

Long- and short-term acclimation of the photosynthetic apparatus to salinity in Chlamydomonas reinhardtii. The role of Stt7 protein kinase.

Salt stress triggers an Stt7-mediated LHCII-phosphorylation signaling mechanism similar to light-induced state transitions. However, phosphorylated LHCII, after detaching from PSII, does not attach to PSI but self-aggregates instead. Salt is a major stress factor in the growth of algae and plants. Here, our study mainly focuses on the organization of the photosynthetic apparatus to the long-term responses of Chlamydomonas reinhardtii to elevated NaCl concentrations. We analyzed the physiological effects of salt treatment at a cellular, membrane, and protein level by microscopy, protein profile analyses, transcripts, circular dichroism spectroscopy, chlorophyll fluorescence transients, and steady-state and time-resolved fluorescence spectroscopy. We have ascertained that cells that were grown in high-salinity medium form palmelloids sphere-shaped colonies, where daughter cells with curtailed flagella are enclosed within the mother cell walls. Palmelloid formation depends on the presence of a cell wall, as it was not observed in a cell-wall-less mutant CC-503. Using the stt7 mutant cells, we show Stt7 kinase-dependent phosphorylation of light-harvesting complex II (LHCII) in both short- and long-term treatments of various NaCl concentrations-demonstrating NaCl-induced state transitions that are similar to light-induced state transitions. The grana thylakoids were less appressed (with higher repeat distances), and cells grown in 150 mM NaCl showed disordered structures that formed diffuse boundaries with the flanking stroma lamellae. PSII core proteins were more prone to damage than PSI. At high salt concentrations (100-150 mM), LHCII aggregates accumulated in the thylakoid membranes. Low-temperature and time-resolved fluorescence spectroscopy indicated that the stt7 mutant was more sensitive to salt stress, suggesting that LHCII phosphorylation has a role in the acclimation and protection of the photosynthetic apparatus.

Synthesis and anti-Alzheimer potential of novel α-amino phosphonate derivatives and probing their molecular interaction mechanism with acetylcholinesterase.

Pleiotropic interference may be a prerequisite for the efficient limitation of the progression of multi-factorial diseases such as Alzheimer's disease (AD). Concept of designing the single chemical entity acting on two or more targets of interest has potential advantage in AD therapy. In line with this, rational design and synthesis of frame work of hybrids bearing 2,3-disubstituted quinazolinone, vanillin and α-amino phosphonate scaffolds (5a─v) were carried out. A congeneric set of twenty-two synthetic derivatives (5a─v) were evaluated for their cholinesterase inhibitory, antioxidant, DNA nicking, DNA protection, neuroprotective and Aß aggregation modulatory activities. Amongst tested activities, the most significant and worth mentioning is that the analogues 5m, 5p and 5u were found to be the most potent, selective, and mixed type inhibitors of EeAChE with IC50 values of 0.296 ± 0.030, 0.289 ± 0.027, and 0.306 ± 0.028 µM, respectively. Further, the biophysical approaches indicated that the compounds 5m, 5p, and 5u have a strong binding affinity towards AChE. Kinetic and Molecular docking studies have revealed that the most active congeners were well oriented in the AChE active site by interacting with both catalytic active site (CAS) and peripheral anionic site (PAS). A few parameters derived from molecular dynamics (MD) simulation trajectories emphasized the stability of AChE-5p and 5m complexes throughout the 100 ns simulations, and the local conformational changes of the residues of AChE validate the stability of AChE-5p and 5m complexes. Further, these derivatives significantly impacted ABTS radical scavenging capacities and maximal DNA protection activity. Importantly, Thioflavin T (ThT) assay and FE-SEM study demonstrated compounds 5m, 5p and 5u as effective Aß1-42 fibril modulators at molecular level by the formation of micro size co-assembled mature structures, thus efficiently abolishing the cytotoxicity of Aß1-42. Finally, these active compounds are determined to be non-toxic and highly neuroprotective against H2O2-induced cell death in SK-N-SH cell lines. Furthermore, in silico ADMET prediction studies have revealed that the targeted analogues satisfied most of the characteristics of CNS acting drugs. These multi-functional efficacies indicated worthiness of these α-amino phosphonate derivatives being chosen for further pharmacokinetics, toxicity, and behavioral research to test their potential for AD treatment.

High light-induced changes in thylakoid supercomplexes organization from cyclic electron transport mutants of Chlamydomonas reinhardtii.

The localization of carotenoids and macromolecular organization of thylakoid supercomplexes have not been reported yet in Chlamydomonas reinhardtii WT and cyclic electron transport mutants (pgrl1 and pgr5) under high light. Here, the various pigments, protein composition, and pigment-protein interactions were analyzed from the cells, thylakoids, and sucrose density gradient (SDG) fractions. Also, the supercomplexes of thylakoids were separated from BN-PAGE and SDG. The abundance of light-harvesting complex (LHC) II trimer complexes and pigment-pigment interaction were changed slightly under high light, shown by circular dichroism. However, a drastic change was seen in photosystem (PS)I-LHCI complexes than PSII complexes, especially in pgrl1 and pgr5. The lutein and ß-carotene increased under high light in LHCII trimers compared to other supercomplexes, indicating that these pigments protected the LHCII trimers against high light. However, the presence of xanthophylls, lutein, and ß-carotene was less in PSI-LHCI, indicating that pigment-protein complexes altered in high light. Even the real-time PCR data shows that the pgr5 mutant does not accumulate zeaxanthin dependent genes under high light, which shows that violaxanthin is not converting into zeaxanthin under high light. Also, the protein data confirms that the LHCSR3 expression is absent in pgr5, however it is presented in LHCII trimer in WT and pgrl1. Interestingly, some of the core proteins were aggregated in pgr5, which led to change in photosynthesis efficiency in high light.

Temperature-induced reversible changes in photosynthesis efficiency and organization of thylakoid membranes from pea (Pisum sativum).

High temperature can induce a substantial adverse effect on plant photosynthesis. This study addressed the impact of moderately high temperature (35 °C) on photosynthetic efficiency and thylakoid membrane organization in Pisum sativum. The Chl a fluorescence curves showed a significant change, indicating a reduction in photosynthetic efficiency when pea plants were exposed to moderate high-temperature stress. The pulse-amplitude modulation measurements showed decreased non-photochemical quenching while the non-regulated energy dissipation increased in treated compared to control and recovery plants. Both parameters indicated that the photosystem (PS)II was prone to temperature stress. The PSI donor side limitation increased in treated and recovery plants compared to control, suggesting the donor side of PSI is hampered in moderate-high temperature. Further, the PSI acceptor side increased in recovery plants compared to control, suggesting that the cyclic electron transport is repressed after temperature treatment but revert back to normal in recovery conditions. Also, the content of photoprotective carotenoid pigments like lutein and xanthophylls increased in temperature-treated leaves. These results indicate the alteration of macro-organization of thylakoid membranes under moderately elevated temperature, whereas supercomplexes restored to the control levels under recovery conditions. Further, the light harvesting complex (LHC)II trimers, and monomers were significantly decreased in temperature-treated plants. Furthermore, the amount of PSII reaction center proteins D1, D2, PsbO, and Cyt b6 was reduced under moderate temperature, whereas the content of LHC proteins of PSI was stable. These observations suggest that moderately high temperature can alter supercomplexes, which leads to change in the pigment-protein organization.

Change in the photochemical and structural organization of thylakoids from pea (Pisum sativum) under salt stress.

Salt can induce adverse effects, primarily on the photosynthetic process, ultimately influencing plant productivity. Still, the impact of salt on the photosynthesis process in terms of supercomplexes organization of thylakoid structure and function is not understood in Pea (Pisum sativum). To understand the structure and function in the leaves and thylakoids under salt (NaCl) treatment, we used various biophysical and biochemical techniques like infrared gas analyzer, chlorophyll a fluorescence, circular dichroism, electron microscopy, blue native gels, and western blots. The net photosynthetic rate, transpiration rate, and stomatal conductance were reduced significantly, whereas the water use efficiency was enhanced remarkably under high salt conditions (200 mM NaCl). The photochemical efficiency of both photosystem (PS) I and II was reduced in high salt by inhibiting their donor and acceptor sides. Interestingly the non-photochemical quenching (NPQ) is reduced in high salt; however, the non-regulated energy dissipation (NO) of PSII increased, leading to inactivation of PSII. The obtained results exhibit inhibition of NAD(P)H dehydrogenase (NDH) mediated pathway-dependent cyclic electron transport under salinity caused a decrease in proton motive force of ΔpH and Δψ. Further, the electron micrographs show the disorganization of grana thylakoids under salt stress. Furthermore, the macro-organization and supercomplexes of thylakoids were significantly affected by high salt. Specifically, the mega complexes, PSII-LHCII, PSI-LHCI, and NDH complexes were notably reduced, ultimately altering the electron transport. The reaction center proteins of oxygen-evolving complexes, D1 and D2 proteins were affected to high salt indicating changes in photochemical activities.

Autophagy Induced Accumulation of Lipids in pgrl1 and pgr5 of Chlamydomonas reinhardtii Under High Light.

Chlamydomonas (C.) reinhardtii is a potential microalga for lipid production. Autophagy-triggered lipid metabolism in microalgae has not being studied so far from a mutant of proton gradient regulation 1 like (PGRL1) and proton gradient regulation 5 (PGR5). In this study, C. reinhardtii cells (wild-type CC124 and cyclic electron transport dependant mutants pgrl1 and pgr5) were grown photoheterotrophically in high light 500 µmol photons m-2 s-1, where pgr5 growth was retarded due to an increase in reactive oxygen species (ROS). The lipid contents were increased; however, carbohydrate content was decreased in pgr5. Further, the Nile Red (NR) fluorescence shows many lipid bodies in pgr5 cells under high light. Similarly, the electron micrographs show that large vacuoles were formed in high light stress despite the grana stacks structure. We also observed increased production of reactive oxygen species, which could be one reason the cells underwent autophagy. Further, a significant increase of autophagy ATG8 and detections of ATG8-PE protein was noticed in pgr5, a hallmark characteristic for autophagy formation. Consequently, the triacylglycerol (TAG) content was increased due to diacylglycerol acyltransferases (DGAT) and phospholipid diacylglycerol acyl-transference (PDAT) enzymes' expression, especially in pgr5. Here the TAG synthesis would have been obtained from degraded membrane lipids in pgr5. Additionally, mono, polyunsaturated, and saturated fatty acids were identified more in the high light condition. Our study shows that the increased light induces the reactive oxygen species, which leads to autophagy and TAG accumulation. Therefore, the enhanced accumulation of TAGs can be used as feedstock for biodiesel production and aqua feed.

Role of cyclic electron transport mutations pgrl1 and pgr5 in acclimation process to high light in Chlamydomonas reinhardtii.

Light is crucial for photosynthesis, but the amount of light that exceeds an organism's assimilation efficacy can lead to photo-oxidative damage and even cell death. In Chlamydomonas (C). reinhardtii cyclic electron flow (CEF) is very important for the elicitation of non-photochemical quenching (NPQ) by controlling the acidification of thylakoid lumen. This process requires the cooperation of proton gradient regulation (PGR) proteins, PGRL1 and PGR5. Here, we compared the growth pattern and photosynthetic activity between wild type (137c, t222+) and mutants impaired in CEF (pgrl1 and pgr5) under photoautotrophic and photoheterotrophic conditions. We have observed the discriminative expression of NPQ in the mutants impaired in CEF of pgrl1 and pgr5. The results obtained from the mutants showed reduced cell growth and density, Chl a/b ratio, fluorescence, electron transport rate, and yield of photosystem (PS)II. These mutants have reduced capability to develop a strong NPQ indicating that the role of CEF is very crucial for photoprotection. Moreover, the CEF mutant exhibits increased photosensitivity compared with the wild type. Therefore, we suggest that besides NPQ, the fraction of non-regulated non-photochemical energy loss (NO) also plays a crucial role during high light acclimation despite a low growth rate. This low NPQ rate may be due to less influx of protons coming from the CEF in cases of pgrl1 and pgr5 mutants. These results are discussed in terms of the relative photoprotective benefit, related to the thermal dissipation of excess light in photoautotrophic and photoheterotrophic conditions.

Non-photochemical quenching-dependent acclimation and thylakoid organization of Chlamydomonas reinhardtii to high light stress.

Light is essential for all photosynthetic organisms while an excess of it can lead to damage mainly the photosystems of the thylakoid membrane. In this study, we have grown Chlamydomonas reinhardtii cells in different intensities of high light to understand the photosynthetic process with reference to thylakoid membrane organization during its acclimation process. We observed, the cells acclimatized to long-term response to high light intensities of 500 and 1000 µmol m-2 s-1 with faster growth and more biomass production when compared to cells at 50 µmol m-2 s-1 light intensity. The ratio of Chl a/b was marginally decreased from the mid-log phase of growth at the high light intensity. Increased level of zeaxanthin and LHCSR3 expression was also found which is known to play a key role in non-photochemical quenching (NPQ) mechanism for photoprotection. Changes in photosynthetic parameters were observed such as increased levels of NPQ, marginal change in electron transport rate, and many other changes which demonstrate that cells were acclimatized to high light which is an adaptive mechanism. Surprisingly, PSII core protein contents have marginally reduced when compared to peripherally arranged LHCII in high light-grown cells. Further, we also observed alterations in stromal subunits of PSI and low levels of PsaG, probably due to disruption of PSI assembly and also its association with LHCI. During the process of acclimation, changes in thylakoid organization occurred in high light intensities with reduction of PSII supercomplex formation. This change may be attributed to alteration of protein-pigment complexes which are in agreement with circular dichoism spectra of high light-acclimatized cells, where decrease in the magnitude of psi-type bands indicates changes in ordered arrays of PSII-LHCII supercomplexes. These results specify that acclimation to high light stress through NPQ mechanism by expression of LHCSR3 and also observed changes in thylakoid protein profile/supercomplex formation lead to low photochemical yield and more biomass production in high light condition.