Search Results (99)

Thylakoid rhodanese-like protein (TROL) serves as a thylakoid membrane hinge linking photosynthetic electron transport chain (PETC) complexes to nicotinamide adenine dinucleotide phosphate (NADPH) synthesis. TROL is the docking site for the flavoenzyme ferredoxin-NADP⁺ oxidoreductase (FNR). Our prior work indicates that the TROL-FNR complex maintains redox equilibrium in chloroplasts and systemically in plant cells. Improvement in the knowledge of redox regulation mechanisms is critical for engineering stress-tolerant plants in times of elevated global drought intensity. To further test this hypothesis and confirm our previous results, we monitored light-independent ROS propagation in the leaves of Arabidopsis wild type (WT), TROL knock-out (KO), and TROL ΔRHO (RHO-domain deletion mutant) mutant plants in situ by using confocal laser scanning microscopy with specific fluorescent probes for the three different ROS: O₂^·−, H₂O₂, and ¹O₂. Plants were grown under the conditions of normal substrate moisture and under drought stress conditions. Under the drought stress conditions, the TROL KO line showed ≈32% less O₂^·− while the TROL ΔRHO line showed ≈49% less H₂O₂ in comparison with the WT. This research confirms the role of dynamical TROL-FNR complex formation in redox equilibrium maintenance by redirecting electrons in alternative sinks under stress and also points it out as promising target for stress-tolerant plant engineering. Full article

Copper (Cu) is an essential micronutrient for cyanobacteria, where it functions as a cofactor in key proteins involved in photosynthesis and antioxidant defense. However, at elevated concentrations, Cu becomes toxic, exhibiting algicidal effects by disrupting metal homeostasis and competing for metal-binding sites on critical cellular proteins. Due to the considerable morphological and physiological diversity within the phylum Cyanobacteria, the thresholds for Cu deficiency or toxicity vary considerably among strains. Maintaining Cu homeostasis in cyanobacterial cells is a complex process involving multiple layers of regulation. It begins at the extracellular polysaccharide layer, involves specialized membrane-bound proteins (in the outer, plasma, and thylakoid membranes), and results in transcriptional regulation in response to intracellular Cu status. This review summarizes the current understanding of Cu uptake and efflux pathways in cyanobacteria and explores how these mechanisms contribute to maintaining cellular Cu balance. The knowledge gained may contribute to the application of cyanobacteria in bioremediation strategies and/or the targeted use of Cu in the control of harmful cyanobacterial blooms. Full article

The chloroplast signal recognition particle (cpSRP) components cpSRP43 and cpSRP54 not only form a complex with light-harvesting chlorophyll (Chl)-binding proteins to direct them to the thylakoid membrane, but also serve other functions. cpSRP43 independently acts as a chaperone for some tetrapyrrole biosynthesis (TBS) enzymes, while cpSRP54 participates in the co-translational targeting of plastid-encoded proteins. However, it remains unclear to what extent the two cpSRP components are coregulated—despite their distinct functions—and whether both participate in genomes-uncoupled (GUN)-mediated retrograde signaling. Here, we demonstrate that cpSRP43 and cpSRP54 accumulation is strongly interdependently controlled: overexpression of one protein increases the level of the other, while a deficiency in one of the two proteins leads to a simultaneous decrease in the other component. Disruption of this balance, e.g., by combining the overexpression of one component with a knockout of the other, results in severe chlorosis, stunted growth, and reduced levels of Chl and tetrapyrrole intermediates. Moreover, cpSRP43 deficiency exacerbates the pale-green phenotype of gun4 and gun5 mutants, highlighting a synergistic impact on TBS; however, cpSRP43 overexpression fails to rescue these defects. Remarkably, loss of cpSRP43 does not affect the expression of nuclear-encoded photosynthetic genes under intrinsic plastid stress, clearly demonstrating that cpSRP43 is not involved in plastid-to-nucleus retrograde signaling. Overall, our findings underscore that the fine-tuned expression of cpSRP43 and cpSRP54 is crucial for proper chloroplast function and pigment biosynthesis, while cpSRP43 alone does not participate in the retrograde signaling pathway. Full article

The absence of α-carbonic anhydrase 2 (α-CA2) in Arabidopsis thaliana leads to higher contents of chlorophylls a and b, and to a reduced chlorophyll a/b ratio, suggesting an increased PSII antenna compared to the wild type (WT). The evaluation of the OJIP kinetics of chlorophyll fluorescence in leaves of WT and α-carbonic anhydrase 2 knockout (α-CA2-KO) plants revealed higher apparent photosystem II (PSII) light-harvesting antenna size in the mutants. The higher levels of both Lhcb1 and Lhcb2 proteins in α-CA2-KO plants compared to WT plants were demonstrated using immunoblotting. Gene expression analysis showed increased lhcb1 expression levels in mutants, whereas the lhcb2 and lhcb3 genes were downregulated. The content of hydrogen peroxide (H₂O₂) in leaves, as well as the production of H₂O₂ within the thylakoid membranes (“membrane” H₂O₂) was lower in α-CA2-KO plants as compared with WT plants. The expression levels of the genes encoding regulating proteins, which are involved in retrograde chloroplast–nucleus signaling, were lower in the α-CA2-KO than in the WT. The changes in the PSII light-harvesting complex size in the absence of α-CA2 correlates with the decreased accumulation of H₂O₂ in the leaves of mutants. It is suggested that this led to lower expression levels of the genes related to retrograde signal transduction from the chloroplast to the nucleus. The results of this study support previous conclusions regarding the involvement of α-CA2 in photosynthetic processes and its location within the chloroplasts of Arabidopsis. Full article

The rapid regulatory mechanism of light-induced state transitions (STs) in oxygenic photosynthesis is particularly appealing for membrane-based applications. This interest stems from the unique ability of the thylakoid membrane protein cytochrome b₆f (cytb₆f) to increase or decrease its hydrophobic thickness (d_P) in parallel with the reduction or oxidation of the PQ pool induced by changes in light quality. This property appears to be the long-sought biophysical driver behind the reorganizations of membrane proteins during STs. This study decisively advances the hydrophobic mismatch (HMM) model for cytb₆f-driven STs by thoroughly analyzing thirteen X-ray crystal and eight cryo-electron microscopy cytb₆f structures. It uncovers the lipid nanoenvironments that cytb₆f, with different hydrophobic thicknesses, selectively attracts. Under optimal, stationary conditions for photosynthesis in low light, when there is hydrophobic matching between the hydrophobic thicknesses of cytb₆f d_P and that of the bulk thylakoid lipid phase d_L, d_P = d_L, cytb₆f predominantly binds to anionic lipids—several phosphatidylglycerol (PG) molecules and one sulfoquinovosyldiacylglycerol (SQDG) molecule. Upon the induction of the transition to State 2, when d_P increases and induces a positive HMM (d_P > d_L), the neutral, non-bilayer-forming lipid monogalactosyldiacylglycerol (MGDG) replaces some of the bound PGs. Upon the induction of the transition to State 1, when d_P decreases and induces a negative HMM (d_P < d_L), PGs and SQDG detach from their binding sites, and two neutral, bilayer-forming lipids such as digalactosyldiacylglycerol (DGDG) occupy two sites. Additionally, this research uncovers two lipid-mediated signaling pathways from Chla to the center of flexibility, the Phe/Tyr124^fg-loop-suIV residue—one of which involves β-carotene. This study identifies two novel types of lipid raft-like nanodomains that are devoid of typical components, such as sphingomyelin and cholesterol. These findings firmly validate the HMM model and underscore the STs as the first recognized functional process that fully utilizes the unique and evolutionarily conserved composition of just four thylakoid lipid classes. This research contributes to our understanding of membrane dynamics in general and STs in particular. It introduces a novel and simple approach for reversible protein reorganization driven purely by biophysical mechanisms, with promising implications for various membrane-based applications. Full article

Bioenergetic membranes of mitochondria, thylakoids, and chromatophores are primary sites of ATP production in living cells. These membranes contain an electron transport chain (ETC) in which electrons are shuttled between a series of redox proteins during the generation of ATP via oxidative phosphorylation. The phospholipid composition of these membranes, which often include negative lipids, plays a role in determining the electrostatics of their surface owing to the spatial distribution of their charged head groups. Cardiolipin (CDL) is a phospholipid commonly associated with bioenergetic membranes and is also a significant contributor to the negative surface charge. Interactions between cytochromes and phospholipid head groups in the membrane can in principle affect the rate of its travel between ETC components, hence influencing the rate of ATP turnover. Here, we use molecular dynamic (MD) simulations that feature an accelerated membrane model, termed highly mobile membrane mimetic (HMMM), to study protein–lipid interactions during the diffusion of cytochrome c₂ between redox partners in a bioenergetic membrane. We observe a “skipping” mode of diffusion for cytochromes along with a bias for binding to anionic lipids, particularly with a strong preference for CDL. During diffusion, cytochrome c₂ maintains a relatively fixed tilt with respect to the membrane normal with wider fluctuations in its angle with respect to the plane of the membrane. The obtained results describing the behavior of cytochrome c₂ on a representative bioenergetic membrane have direct ramifications in shuttling motions of other similar electron-carrying elements in other bioenergetic membranes, which are composed of a significant amount of anionic lipids. The mode of surface-restricted diffusion reported here would modulate rapid electron transfer between the ETC complexes anchored in bioenergetic membranes by reducing the search space between them. Full article

The green unicellular algae Chlamydomonas reinhardtii contains 12–13 carbonic anhydrases (CAs). For a long time, the two closely related α-CAs of the periplasmic membrane CAH1 and CAH2 were considered to be the CAs with the highest CO₂ hydration activity. The recombinant protein α-CA CAH3 (rCAH3) from the thylakoid lumen obtained in the present study showed more than three times higher activity compared to CAH1 and more than 11 times higher compared to previous studies with rCAH3. Long-term sustainability of the enzyme was observed at alkaline pH (>8), with maintenance of half of its activity at 4 °C for up to 50 days. Thermostability of rCAH3 indicated the retention of the activity at 20 °C for one hour at pH 9–10 with its ~50% decrease at pH 6–7. However, the residual activity of rCAH3 after incubation at an extremely high temperature (75 °C) for 15 min led to the formation of the double-hump graph with maxima at pH 6 and 9. The enzyme demonstrated high sensitivity to ethoxyzolamide and acetazolamide at nM concentrations, to Zn²⁺ and Cu²⁺ cations at 1 mM concentrations, and L-cysteine was able to completely inhibit CA activity of rCAH3 through reduction of sulfhydryl groups. Esterase activity of rCAH3 was well detected with values comparable to those of bovine CAII, but with a maximum at pH 8 instead of pH 9, which is usual for bovine CAII. The results indicated that CAH3 may be the most active CA of C. reinhardtii and that its role in the photosynthetic apparatus function could have been underestimated in previous works. Full article

Rice lesion mimic mutants are important materials for studying the mechanisms of cell death. In-depth research on these mutants can provide insights into the molecular mechanisms underlying rice growth and development, offering a theoretical basis for crop improvement. In this study, rice variety Wuyunjing 21 (WYJ21) was mutagenized with ethyl methanesulfonate to obtain a lesion mimic mutant, lmm28. Unlike wild-type (WT) plants, the lmm28 mutant exhibits brown lesions on the leaves starting from the early tillering stage. The size of the lesions increases as the plant grows. Additionally, the lmm28 mutant shows significantly reduced plant height, tiller number, number of effective panicles, seed setting rate, and 1000-grain weight compared to the WT. Leaf staining of the mutant revealed an accumulation of reactive oxygen species and cell death in the lesion leaves. Transmission electron microscopy images showed that, in the lmm28 mutant, the nuclear boundaries in leaf cells became indistinct and damage to the chloroplast membrane structures was observed, with thylakoid disorganization occurring in some chloroplasts. Genetic analysis and map-based cloning localized the candidate gene of the mutant to a 167.79 kb region on chromosome 5. After analyzing the annotated genes within this region, the candidate gene was preliminarily identified as OsBON3. Sequencing analysis revealed that, in lmm28, a base change from GT to GC occurred at the 5′ splice junction of the 15th intron of OsBON3. Further analysis, using cDNA amplification of exons 14–16 followed by sequencing, showed that the mutation at the splice recognition site caused the incorrect splicing of OsBON3 pre-mRNA, leading to an increased number of transcripts in lmm28. The transcript containing an inserted intron is present at much higher levels than the normal transcript, which may lead to a reduction in the protein levels containing the functional vWA domain. Therefore, the vWA domain of OsBON3 is likely crucial for maintaining ROS homeostasis in rice and plays a key role in regulating its growth and development. Full article

Wheat is one of the most important crops to ensure food production globally. Understanding the mechanism of leaf senescence in wheat plays a crucial role in improving its productivity and resilience under various stress scenarios. In this study, we investigated biochemical, functional, and ultrastructural changes during leaf senescence in wheat genotypes with contrasting drought tolerance. For this, key parameters such as chlorophyll and total protein content, membrane stability, malondialdehyde level, and the activity of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, benzidine peroxidase, and catalase) were comparatively analyzed during both natural and drought-induced senescence. Additionally, the expression of superoxide dismutase isoform genes functioning in different cellular compartments was studied, alongside ultrastructural changes in flag leaves. The experiments involved genotypes of bread wheat (Triticum aestivum L.) and durum (Triticum durum Desf.) wheat. The plants were grown in controlled environment chambers under control and drought conditions using a completely randomized design. After the booting stage, irrigation was discontinued for drought-treated plants. Flag leaves were sampled at 7, 14, 21, 28, and 35 days after anthesis. Drought-tolerant genotypes exhibited slower chlorophyll degradation, lower lipid peroxidation, enhanced membrane stability, and stronger antioxidant responses, allowing them to maintain cellular function longer, whereas sensitive genotypes showed accelerated leaf senescence. Transcript levels of FeSOD increased significantly post-flowering but declined as senescence progressed, while MnSOD expression exhibited a rise towards the later stages of ontogenesis across all studied genotypes. Ultrastructural analysis revealed progressive damage to chloroplast membranes, thylakoid structures, and mesophyll cell walls under stress conditions, particularly in sensitive genotypes. These findings contribute to a deeper understanding of the physiological and molecular responses of wheat to drought stress, offering potential targets for improving crop performance in water-limited environments. Full article

Photosynthesis, as the primary source of energy for all life forms, plays a crucial role in maintaining the global balance of energy, entropy, and enthalpy in living organisms. Among its various building blocks, photosystem I (PSI) is responsible for light-driven electron transfer, crucial for generating cellular reducing power. PSI acts as a light-driven plastocyanin-ferredoxin oxidoreductase and is situated in the thylakoid membranes of cyanobacteria and the chloroplasts of eukaryotic photosynthetic organisms. Comprehending the structure and function of the photosynthetic machinery is essential for understanding its mode of action. New insights are offered into the structure and function of PSI and its associated light-harvesting proteins, with a specific focus on the remarkable structural conservation of the core complex and high plasticity of the peripheral light-harvesting complexes. Full article

The plastid stroma-localized chaperone HSP90C plays a crucial role in maintaining optimal proteostasis within chloroplasts and participates in protein translocation processes. While existing studies have revealed HSP90C’s direct interaction with the Sec translocase-dependent client pre-protein PsbO1 and the SecY1 subunit of the thylakoid membrane-bound Sec1 translocase channel system, its direct involvement with the extrinsic homodimeric Sec translocase subunit, SecA1, remains elusive. Employing bimolecular fluorescence complementation (BiFC) assay and other in vitro analyses, we unraveled potential interactions between HSP90C and SecA1. Our investigation revealed dynamic interactions between HSP90C and SecA1 at the thylakoid membrane and stroma. The thylakoid membrane localization of this interaction was contingent upon active HSP90C ATPase activity, whereas their stromal interaction was associated with active SecA1 ATPase activity. Furthermore, we observed a direct interaction between these two proteins by analyzing their ATP hydrolysis activities, and their interaction likely impacts their respective functional cycles. Additionally, using PsbO1, a model Sec translocase client pre-protein, we studied the intricacies of HSP90C’s possible involvement in pre-protein translocation via the Sec1 system in chloroplasts. The results suggest a complex nature of the HSP90C-SecA1 interaction, possibly mediated by the Sec client protein. Our studies shed light on the nuanced aspects of HSP90C’s engagement in orchestrating pre-protein translocation, and we propose a potential collaborative role of HSP90C with SecA1 in actively facilitating pre-protein transport across the thylakoid membrane. Full article

Salt stress significantly impacts the functions of the photosynthetic apparatus, with varying degrees of damage to its components. Photosystem II (PSII) is more sensitive to environmental stresses, including salinity, than photosystem I (PSI). This study investigated the effects of different salinity levels (0 to 200 mM NaCl) on the PSII complex in isolated thylakoid membranes from hydroponically grown pea (Pisum sativum L.) and maize (Zea mays L.) plants treated with NaCl for 5 days. The data revealed that salt stress inhibits the photochemical activity of PSII (H₂O → BQ), affecting the energy transfer between the pigment–protein complexes of PSII (as indicated by the fluorescence emission ratio F₆₉₅/F₆₈₅), Q_A reoxidation, and the function of the oxygen-evolving complex (OEC). These processes were more significantly affected in pea than in maize under salinity. Analysis of the oxygen evolution curves after flashes and continuous illumination showed a stronger influence on the PSIIα than PSIIβ centers. The inhibition of oxygen evolution was associated with an increase in misses (α), double hits (β), and blocked centers (S_B) and a decrease in the rate constant of turnover of PSII reaction centers (K_D). Salinity had different effects on the two pathways of Q_A reoxidation in maize and pea. In maize, the electron flow from Q_A- to plastoquinone was dominant after treatment with higher NaCl concentrations (150 mM and 200 mM), while in pea, the electron recombination on Q_AQ_B- with oxidized S₂ (or S₃) of the OEC was more pronounced. Analysis of the 77 K fluorescence emission spectra revealed changes in the ratio of the light-harvesting complex of PSII (LHCII) monomers and trimers to LHCII aggregates after salt treatment. There was also a decrease in pigment composition and an increase in oxidative stress markers, membrane injury index, antioxidant activity (FRAP assay), and antiradical activity (DPPH assay). These effects were more pronounced in pea than in maize after treatment with higher NaCl concentrations (150 mM–200 mM). This study provides insights into how salinity influences the processes in the donor and acceptor sides of PSII in plants with different salt sensitivity. Full article

Extreme weather events, including drought, have occurred worldwide with increasing frequency and severity in recent years. Drought stress is the main limiting factor for agricultural development in many regions, including tobacco—an important economic crop and a model plant for scientific research. As it is adapted to the tropics, tobacco is highly susceptible to drought stress, with resultant decreases in yield and quality. Glycine betaine (GB) is an osmoregulatory substance that can enhance plant resistance to various abiotic stresses. Here, we investigate the protective mechanism of genetically engineered glycine betaine (GB) on tobacco photosynthesis under drought stress induced by 30% PEG-6000. This study used transgenic tobacco (T) accumulating GB and wild-type tobacco (WT) to investigate the protective effects conferred by the genetic engineering of GB synthesis on tobacco photosynthesis under drought stress (induced by 30% PEG-6000). The results showed that the net photosynthetic rate of the tobacco plants significantly decreased under drought stress, and the degree of decrease was significantly lower in the T line than in the WT line. GB accumulation improved the resistance of photosynthesis to drought stress. Furthermore, under drought stress, the photosynthesis improvement in the T line was related to the accumulation of GB, leading to maintenance of the water status, the promotion of osmotic regulation, and an enhancement in antioxidant enzyme activities, which reduced membrane peroxidation and thereby increased the thylakoid membrane’s protein content and function, especially the photosystem II (PSII) function. The results provide a theoretical basis for further research on genetic engineering related to GB synthesis and the field application of exogenous GB. Full article

This study aimed to investigate the regulation of fucoxanthin (FX) biosynthesis under various nitrogen conditions to optimize FX productivity in Phaeodactylum tricornutum. Apart from light, nitrogen availability significantly affects the FX production of microalgae; however, the underlying mechanism remains unclear. In batch culture, P. tricornutum was cultivated with normal (NN, 0.882 mM sodium nitrate), limited (LN, 0.22 mM), and high (HN, 8.82 mM) initial nitrogen concentrations in f/2 medium. Microalgal growth and photosynthetic pigment production were examined, and day 5 samples were subjected to fucoxanthin–chlorophyll a/c-binding protein (FCP) proteomic and transcriptomic analyses. The result demonstrated that HN promoted FX productivity by extending the exponential growth phase for higher biomass and FX accumulation stage (P1), showing a continuous increase in FX accumulation on day 6. Augmented FX biosynthesis via the upregulation of carotenogenesis could be primarily attributed to enhanced FCP formation in the thylakoid membrane. Key proteins, such as LHC3/4, LHCF8, LHCF5, and LHCF10, and key genes, such as PtPSY, PtPDS, and PtVDE, were upregulated under nitrogen repletion. Finally, the combination of low light and HN prolonged the P1 stage to day 10, resulting in maximal FX productivity to 9.82 ± 0.56 mg/L/day, demonstrating an effective strategy for enhancing FX production in microalgae cultivation. Full article

Potato scab, a global soil-borne disease caused by Streptomyces, is pivotal in developing resistant cultivars due to its complex resistance mechanisms. This study investigates the transcriptomic responses in potato to common scab using resistant variety CS10 and susceptible CS11 post S. scabie inoculation (0 d and 10 d, 12 cDNA libraries). Differential expression analysis identified 147 key DEGs (Differentially Expressed Genes) essential in disease recognition, signal transduction, and defense. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses revealed several significant metabolic pathways, such as ADP binding, heme binding, chloroplast thylakoid membrane, photosynthesis, glutathione metabolism, and homologous recombination, among others. Notably, the correlation between chloroplast pathways (GO:0019745) and photosynthesis (map00195) highlights photosynthesis’s role in potato scab response, while the oxygen transport (GO:0031408)-related glutathione metabolism pathway (map00480) emphasizes antioxidant defenses. Furthermore, three potential resistance genes were validated: Ethylene Response Factor ERF010 (LOC102589042), Disease Resistance Protein RPP13 (LOC102605863), and Cytochrome P450 83B1 (LOC102604056), demonstrating the linkage between metabolic pathways and pathogen response. These findings offer insights into potato’s molecular resistance mechanisms against potato scab, supporting the breeding of resistant varieties and comprehensive disease management, thus advancing sustainable agriculture. Full article