Search Results (18)

Mitochondrial biogenesis requires coordinated expression from both nuclear and mitochondrial genomes. To understand the consequences of mitochondrial genome loss, we generated a mitochondrial DNA-depleted line (crm⁻) in Chlamydomonas reinhardtii via long-term ethidium bromide treatment. We then examined how mtDNA disruption affects mitochondrial ultrastructure, chloroplast function, and the mitochondrial transcription termination factor (mTERF) gene family. Our results reveal that mitochondrial dysfunction is associated with severe organelle remodeling, including mitochondrial elongation, matrix condensation, and cristae collapse. Consequently, mitochondria reduce the electron sink capacity which appears to over-reduce the chloroplast electron transport chain, correlating with causing damage to photosystem II (PSII), as indicated by higher plastoquinone PQ redox state and PSII excitation pressure and lower non-photochemical quantum yield [Y(NPQ)]. Furthermore, we identified and characterized eight nuclear-encoded mTERF genes in C. reinhardtii (CrmTERFs). Phylogenetic analysis grouped them into three clades with potential functional conservation. Collinearity analysis suggested potential evolutionary relationships between mTERF genes in Chlamydomonas and Marchantia polymorpha. Gene ontology annotation linked CrmTERFs to transcription termination and RNA biosynthesis regulation. Additionally, in silico prediction identified twelve putative miRNAs targeting seven of the eight CrmTERFs, with CrmTERF3 as the only exception, providing candidates for future experimental validation. This study provides the first comprehensive analysis of the nuclear encoded mTERF gene family in Chlamydomonas and demonstrates that mtDNA loss is correlated with mTERF genes expression, as well as mitochondrial structure and chloroplast photoprotective impairments. These findings suggest a potential role for CrmTERFs in mitochondrial retrograde signaling and organellar crosstalk, though functional validation is required to establish causality. Full article

Xanthophylls are carotenoids with diverse roles in stress protection across all taxa of life. This review highlights chloroplast-localized xanthophylls (with a focus on zeaxanthin) of plants by presenting an overview of the protective effects of xanthophylls as well as the role of carotenoids as precursors of multiple plant stress hormones. It also examines the roles of xanthophylls and stress hormones in signaling cascades between the chloroplast and nuclear genes that control plant growth, development, and stress defenses. This overview addresses the biosynthetic pathways of xanthophylls and carotenoid-derived plant stress hormones, functions of xanthophylls in photoprotection of photosynthesis, carotenoids as essential human micronutrients, and roles of xanthophylls in membrane integrity. Attention is given to the involvement of zeaxanthin in both abiotic and biotic defense as well as its impact on components of the biotic defense system with contrasting targets. Examples for the multiple principal loops of signaling cascades between the chloroplast and nucleus, which are based on chloroplast redox state and modulated by xanthophylls, are summarized. This review integrates the role of chloroplast carotenoids in controlling light-use efficiency and providing photoprotection with their system-wide regulatory effects as precursors of carotenoid-derived plant stress hormones and modulators of chloroplast redox state. A better understanding of these connections is needed to guide development of plant lines with improved resilience and productivity in complex, changing, and challenging environments. Full article

Spirodela polyrhiza is a suitable model organism for investigating plant developmental influences due to its intracolonial variations in response to various environmental fluctuations, like nutrient deficiency. In this study, transmission electron microscopy was used to examine age-dependent variation in chloroplast ultrastructure, while pigment levels (chlorophyll and anthocyanins), starch accumulation, and metabolic activity (photosynthetic and respiratory rates) were measured to determine metabolic responses to sulfur deficiency. For a comprehensive insight into electron transport efficiency and the redox states of the photosynthetic apparatus, rapid light curves, chlorophyll fluorescence (JIP test parameters), and modulated reflection at 820 nm were analyzed. Under S deficit, mother fronds relied on stored reserves to maintain functional PSII but accumulated reduced PQ pools, slowing electron flow beyond PSII. The first-generation daughter fronds, despite having higher baseline photosynthetic capacity, exhibited the largest decline in photosynthetic indicators (e.g., rETR fell about 50%), limitations in the water-splitting complex, and reduced PSI end-acceptor capacity that resulted in donor- and acceptor-side bottlenecks of electron transport. The youngest granddaughter fronds avoided these bottlenecks by absorbing less light per PSII, channeling electrons through the alternative pathway to balance PQ pools and redox-stable PSI while diverting more carbon into starch and anthocyanin production up to 5-fold for both. These coordinated and age-specific adjustments that provide response flexibility may help maintain photosynthetic function of the colony and facilitate rapid recovery when sulfur becomes available again. Full article

Aspirin (Asp) is extensively used in human health as an anti-inflammatory, antipyretic, and anti-thrombotic drug. In this study, we investigated if the foliar application of Asp on tomato plants has comparable beneficial effects on photosynthetic function to that of salicylic acid (SA), with which it shares similar physiological characteristics. We assessed the consequences of foliar Asp-spray on the photosystem II (PSII) efficiency of tomato plants, and we estimated the reactive oxygen species (ROS) generation and the chloroplast ultrastructural changes. Asp acted as an osmoregulator by increasing tomato leaf water content and offering antioxidant protection. This protection kept the redox state of plastoquinone (PQ) pull (qp) more oxidized, increasing the fraction of open PSII reaction centers and enhancing PSII photochemistry (Φ_PSII). In addition, Asp foliar spray decreased reactive oxygen species (ROS) formation, decreasing the excess excitation energy on PSII. This resulted in a lower singlet oxygen (¹O₂) generation and a lower quantum yield for heat dissipation (Φ_NPQ), indicating the photoprotective effect provided by Asp, especially under excess light illumination. Simultaneously, we observed a decrease in stomatal opening by Asp, which reduced the transpiration. Chloroplast ultrastructural data revealed that Asp, by offering a photoprotective effect, decreased the need for the photorespiration process, which reduces photosynthetic performance. It is concluded that Asp shares similar physiological characteristics with SA, having an equivalent beneficial impact to SA by acting as a biostimulant of the photosynthetic function for an enhanced crop yield. Full article

The redox state of the plastoquinone (PQ) pool in thylakoids plays an important role in the regulation of chloroplast metabolism. In the light, the PQ pool is mostly reduced, followed by oxidation after light cessation. It has been believed for a long time that dark oxidation depends on oxygen, although the precise mechanisms of the process are still unknown and debated. In this work, we analyzed PQ pool oxidation kinetics in isolated pea (Pisum sativum) thylakoids by tracking the changes in the area above the OJIP fluorescence curve (A_fl) over time intervals from 0.1 s to 10 min in the dark following illumination. A_fl served as an indirect measure of the redox state of the PQ pool that enabled quantification of the rate of PQ pool oxidation. The results showed a two-phase increase in A_fl. The “fast” phase appeared to be linked to electron flow from the PQ pool to downstream acceptors of the photosynthetic electron transport chain. The “slow” phase involved oxidation of PQH₂ through oxygen-dependent mechanisms. Adding octyl gallate, an inhibitor of plastid terminal oxidase (PTOX), to isolated thylakoid suspensions decreased the rate of the “slow” phase of PQ pool oxidation in the dark after illumination. The addition of either H₂O₂ or catalase, an enzyme that decomposes H₂O₂, revealed that H₂O₂ accelerates oxidation of the PQ pool. This indicates that under conditions that favor H₂O₂ accumulation, H₂O₂ can contribute substantially to PQ pool oxidation in the dark after illumination. The contribution of PTOX and H₂O₂ to the modulation of the PQ pool redox state in plants in the dark after illumination is discussed. Full article

Chloroplasts play crucial roles in biotic and abiotic stress responses, regulated by nuclear gene expression through changes in the cellular redox state. Despite lacking the N-terminal chloroplast transit peptide (cTP), nonexpressor of pathogenesis-related genes 1 (NPR1), a redox-sensitive transcriptional coactivator was consistently found in the tobacco chloroplasts. Under salt stress and after exogenous application of H₂O₂ or aminocyclopropane-1-carboxylic acid, an ethylene precursor, transgenic tobacco plants expressing green fluorescent protein (GFP)-tagged NPR1 (NPR1-GFP) showed significant accumulation of monomeric nuclear NPR1, irrespective of the presence of cTP. Immunoblotting and fluorescence image analyses indicated that NPR1-GFP, with and without cTP, had similar molecular weights, suggesting that the chloroplast-targeted NPR1-GFP is likely translocated from the chloroplasts to the nucleus after processing in the stroma. Translation in the chloroplast is essential for nuclear NPR1 accumulation and stress-related expression of nuclear genes. An overexpression of chloroplast-targeted NPR1 enhanced stress tolerance and photosynthetic capacity. In addition, compared to the wild-type lines, several genes encoding retrograde signaling-related proteins were severely impaired in the Arabidopsis npr1-1 mutant, but were enhanced in NPR1 overexpression (NPR1-Ox) transgenic tobacco line. Taken together, chloroplast NPR1 acts as a retrograding signal that enhances the adaptability of plants to adverse environments. Full article

Chloroplast ascorbate peroxidases exert an important role in the maintenance of hydrogen peroxide levels in chloroplasts by using ascorbate as the specific electron donor. In this work, we performed a functional study of the stromal APX in rice (OsAPX7) and demonstrated that silencing of OsAPX7 did not impact plant growth, redox state, or photosynthesis parameters. Nevertheless, when subjected to drought stress, silenced plants (APX7i) show a higher capacity to maintain stomata aperture and photosynthesis performance, resulting in a higher tolerance when compared to non-transformed plants. RNA-seq analyses indicate that the silencing of OsAPX7 did not lead to changes in the global expression of genes related to reactive oxygen species metabolism. In addition, the drought-mediated induction of several genes related to the proteasome pathway and the down-regulation of genes related to nitrogen and carotenoid metabolism was impaired in APX7i plants. During drought stress, APX7i showed an up-regulation of genes encoding flavonoid and tyrosine metabolism enzymes and a down-regulation of genes related to phytohormones signal transduction and nicotinate and nicotinamide metabolism. Our results demonstrate that OsAPX7 might be involved in signaling transduction pathways related to drought stress response, contributing to the understanding of the physiological role of chloroplast APX isoforms in rice. Full article

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO₂ assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have. Full article

One of the earliest hallmarks of plant immune response is production of reactive oxygen species (ROS) in different subcellular compartments, which regulate plant immunity. A suitable equilibrium, which is crucial to prevent ROS overaccumulation leading to oxidative stress, is maintained by salicylic acid (SA), a chief regulator of ROS. However, ROS not only act downstream of SA signaling, but are also proposed to be a central component of a self-amplifying loop that regulates SA signaling as well as the interaction balance between different phytohormones. The exact role of this crosstalk, the position where SA interferes with ROS signaling and ROS interferes with SA signaling and the outcome of this regulation, depend on the origin of ROS but also on the pathosystem. The precise spatiotemporal regulation of organelle-specific ROS and SA levels determine the effectiveness of pathogen arrest and is therefore crucial for a successful immune response. However, the regulatory interplay behind still remains poorly understood, as up until now, the role of organelle-specific ROS and SA in hypersensitive response (HR)-conferred resistance has mostly been studied by altering the level of a single component. In order to address these aspects, a sophisticated combination of research methods for monitoring the spatiotemporal dynamics of key players and transcriptional activity in plants is needed and will most probably consist of biosensors and precision transcriptomics. Full article

Salvia sclarea L. is a Cd²⁺ tolerant medicinal herb with antifungal and antimicrobial properties cultivated for its pharmacological properties. However, accumulation of high Cd²⁺ content in its tissues increases the adverse health effects of Cd²⁺ in humans. Therefore, there is a serious demand to lower human Cd²⁺ intake. The purpose of our study was to evaluate the mitigative role of excess Zn²⁺ supply to Cd²⁺ uptake/translocation and toxicity in clary sage. Salvia plants were treated with excess Cd²⁺ (100 μM CdSO₄) alone, and in combination with Zn²⁺ (900 μM ZnSO₄), in modified Hoagland nutrient solution. The results demonstrate that S. sclarea plants exposed to Cd²⁺ toxicity accumulated a significant amount of Cd²⁺ in their tissues, with higher concentrations in roots than in leaves. Cadmium exposure enhanced total Zn²⁺ uptake but also decreased its translocation to leaves. The accumulated Cd²⁺ led to a substantial decrease in photosystem II (PSII) photochemistry and disrupted the chloroplast ultrastructure, which coincided with an increased lipid peroxidation. Zinc application decreased Cd²⁺ uptake and translocation to leaves, while it mitigated oxidative stress, restoring chloroplast ultrastructure. Excess Zn²⁺ ameliorated the adverse effects of Cd²⁺ on PSII photochemistry, increasing the fraction of energy used for photochemistry (Φ_PSII) and restoring PSII redox state and maximum PSII efficiency (Fv/Fm), while decreasing excess excitation energy at PSII (EXC). We conclude that excess Zn²⁺ application eliminated the adverse effects of Cd²⁺ toxicity, reducing Cd²⁺ uptake and translocation and restoring chloroplast ultrastructure and PSII photochemical efficiency. Thus, excess Zn²⁺ application can be used as an important method for low Cd²⁺-accumulating crops, limiting Cd²⁺ entry into the food chain. Full article

Photorespiration, an essential component of plant metabolism, is concerted across four subcellular compartments, namely, chloroplast, peroxisome, mitochondrion, and the cytoplasm. It is unclear how the pathway located in different subcellular compartments respond to stress occurring exclusively in one of those. We attempted to assess the inter-organelle interaction during the photorespiratory pathway. For that purpose, we induced oxidative stress by menadione (MD) in mitochondria and photo-oxidative stress (high light) in chloroplasts. Subsequently, we examined the changes in selected photorespiratory enzymes, known to be located in other subcellular compartments. The presence of MD upregulated the transcript and protein levels of five chosen photorespiratory enzymes in both normal and high light. Peroxisomal glycolate oxidase and catalase activities increased by 50% and 25%, respectively, while chloroplastic glycerate kinase and phosphoglycolate phosphatase increased by ~30%. The effect of MD was maximum in high light, indicating photo-oxidative stress was an influential factor to regulate photorespiration. Oxidative stress created in mitochondria caused a coordinative upregulation of photorespiration in other organelles. We provided evidence that reactive oxygen species are important signals for inter-organelle communication during photorespiration. Thus, MD can be a valuable tool to modulate the redox state in plant cells to study the metabolic consequences across membranes. Full article

In the chloroplast, Calvin–Benson–Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges is simultaneous. In oxidized CP12, the C₂₃–C₃₁ pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C₆₆–C₇₅ pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C₆₆–C₇₅ pair than for the C₂₃–C₃₁ pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12. Full article

Thioredoxins (Trxs) are small, ubiquitous enzymes that catalyze disulphide–dithiol interchange in target enzymes. The large set of chloroplast Trxs, including f, m, x and y subtypes, use reducing equivalents fueled by photoreduced ferredoxin (Fdx) for fine-tuning photosynthetic performance and metabolism through the control of the activity of redox-sensitive proteins. Although biochemical analyses suggested functional diversity of chloroplast Trxs, genetic studies have established that deficiency in a particular Trx subtype has subtle phenotypic effects, leading to the proposal that the Trx isoforms are functionally redundant. In addition, chloroplasts contain an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. Interestingly, Arabidopsis mutants combining the deficiencies of x- or f-type Trxs and NTRC display very severe growth inhibition phenotypes, which are partially rescued by decreased levels of 2-Cys peroxiredoxins (Prxs). These findings indicate that the reducing capacity of Trxs f and x is modulated by the redox balance of 2-Cys Prxs, which is controlled by NTRC. In this study, we explored whether NTRC acts as a master regulator of the pool of chloroplast Trxs by analyzing its functional relationship with Trxs y. While Trx y interacts with 2-Cys Prxs in vitro and in planta, the analysis of Arabidopsis mutants devoid of NTRC and Trxs y suggests that Trxs y have only a minor effect, if any, on the redox state of 2-Cys Prxs. Full article

Glutathione and reactive oxygen species (ROS) play important roles, within different cell compartments, in activating plant defense and the development of resistance. In mitochondria, the accumulation of ROS and the change of glutathione towards its oxidized state leads to mitochondrial dysfunction, activates cell death, and triggers resistance. The accumulation of glutathione in chloroplasts and peroxisomes at the early stages of plant pathogen interactions is related to increased tolerance and resistance. The collapse of the antioxidative system in these two cell compartments at the later stages leads to cell death through retrograde signaling. The cytosol can be considered to be the switchboard during biotic stress where glutathione is synthesized, equally distributed to, and collected from different cell compartments. Changes in the redox state of glutathione and the accumulation of ROS in the cytosol during biotic stress can initiate the activation of defense genes in nuclei through pathways that involve salicylic acid, jasmonic acid, auxins, and abscisic acid. This review dissects the roles of glutathione in individual organelles during compatible and incompatible bacterial, fungal, and viral diseases in plants and explores the subcelluar roles of ROS, glutathione, ascorbate, and related enzymes in the development of resistance. Full article

2-Cysteine peroxiredoxins (2-CysPRX) are highly abundant thiol peroxidases in chloroplasts and play key roles in reactive oxygen species (ROS) defense and redox signaling. Peroxide-dependent oxidation of cysteines induces conformational changes that alter the ability for protein–protein interactions. For regeneration, 2-CysPRXs withdraw electrons from thioredoxins (TRXs) and participate in redox-dependent regulation by affecting the redox state of TRX-dependent targets, for example, in chloroplast metabolism. This work explores the redox conformation-specific 2-CysPRX interactome using an affinity-based pull down with recombinant variants arrested in specific quaternary conformations. This allowed us to address a critical and poorly explored aspect of the redox-regulatory network and showed that the interaction of TRXs, their interaction partners, and 2-CysPRX occur under contrasting redox conditions. A set of 178 chloroplast proteins were identified from leaf proteins and included proteins with functions in photosynthesis, carbohydrate, fatty acid and amino acid metabolism, and defense. These processes are known to be deregulated in plants devoid of 2-CysPRX. Selected enzymes like LIPOXYGENASE 2, CHLOROPLAST PROTEIN 12-1, CHORISMATE SYNTHASE, ß-CARBONIC ANHYDRASE, and FERREDOXIN-dependent GLUTAMATE SYNTHASE 1 were subjected to far Western, isothermal titration calorimetry, and enzyme assays for validation. The pull down fractions frequently contained TRXs as well as their target proteins, for example, FRUCTOSE-1,6-BISPHOSPHATASE and MALATE DEHYDROGENASE. The difference between TRX-dependent indirect interactions of TRX targets and 2-CysPRX and direct 2-CysPRX binding is hypothesized to be related to quaternary structure formation, where 2-CysPRX oligomers function as scaffold for complex formation, whereas TRX oxidase activity of 2-CysPRX controls the redox state of TRX-related enzyme activity. Full article