Confocal laser scanning microscopy of ROS in Arabidopsis thaliana (L.) Heynh. TROL-FNR mutants

Confocal laser scanning microscopy of ROS in Arabidopsis thaliana (L.) Heynh. TROL-FNR mutants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Confocal laser scanning microscopy of ROS in Arabidopsis thaliana (L.) Heynh. TROL-FNR mutants Ena Dumančić, Lea Vojta, Hrvoje Fulgosi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6220323/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Thylakoid rhodanase-like protein (TROL) is located in thylakoid membranes like a hinge between the protein complexes of photosynthetic electron transport chain (PETC) and the nicotinamide adenine dinucleotide phosphate (NADPH) synthesis. TROL is the docking site for the flavoenzyme ferredoxin-NADP + oxidoreductase (FNR). As proposed in our previous researches, TROL-FNR complex plays an important role in maintaining redox equilibrium in chloroplasts, and even in entire plant cells. To further test this hypothesis and confirm our previous results, we monitored ROS propagation in the leaves of Arabidopsis wild type (WT), TROL knock-out (KO), and TROL ΔRHO mutant pants in situ , by using confocal laser scanning microscopy with the specific fluorescent probes for the three different ROS: O 2 ˙ˉ, H 2 O 2 , 1 O 2 . Plants were grown under the conditions of normal substrate moisture and under the drought stress conditions. Results Under the drought stress conditions, TROL KO line showed successful detoxification of O 2 ˙ˉ, while ΔRHO line showed successful detoxification of H 2 O 2 . Conclusion This research once again proves the involvement of the dynamical TROL-FNR complex formation in redox equilibrium maintenance and the distribution of energy. Abiotic stress Arabidopsis ferredoxin:NADP+ oxidoreductase linear electron transport redox homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 BACKGROUND Photosynthesis is a very important biochemical process that is carried out by photoautotrophic organisms. They use the energy of photons, as well as water molecules as the electron donor and the source of protons, in order to fix carbon dioxide (CO 2 ) from the atmosphere to finally produce simple sugars [ 19 ]. Very important part of the photosynthetic electron transfer chain (PETC), is the last, reversible, linear step of electron transfer from the small soluble protein ferredoxin (Fd) to the nicotinamide adenine dinucleotide phosphate (NADP + ) [ 9 ]. This step is catalyzed by a dimerized flavoenzyme ferredoxin:NADP + oxidoreductase (FNR) that can be attached to photosynthetic membranes via different proteins [ 3 , 4 , 11 ], but herein we focus on the interaction with the thylakoid rhodanese like protein (TROL) [ 2 , 13 ]. FNR bound to TROL efficiently catalyzes NADPH biosynthesis, while soluble form prevents reactive oxygen species (ROS) formation and damage of molecular structures, suggesting protective role of this complex [ 11 , 22 ]. Besides its fundamental role in sustaining life on Earth, photosynthesis is also the primary source of ROS production in plants. Also, as in other organisms, ROS are generated in various cellular compartments other than chloroplasts and have the ability to transition between them (Noctor, 2016). Because of that, in this study, we focused on ROS propagation and scavenging at the whole-cell level, including ROS not directly linked to PETC. Although TROL is part of the PETC within chloroplasts, we proposed its influence on electron management and redox homeostasis system of the whole plant [ 24 ]. This role arises from the necessity of preventing ROS damage across all cellular compartments, particularly under stress conditions [ 36 ]. TROL consists of the two transmembrane domains which span trough the thylakoid membrane, an inactive RHO domain in the thylakoid lumen, the C-terminal stromal domain that binds FNR, the ITEP domain characteristic for vascular plants, and the N-terminal stromal domain with chloroplast targeting pre-sequence [ 22 , 23 ]. Upstream of the ITEP domain PEPE domain is located. This domain contains characteristic proline-valine-proline repeat rich region that enables flexibility [ 11 ]. TROL is located in the thylakoid membranes like a hinge between the end of the protein complexes of the photosynthetic electron transport chain (PETC) and the precursor of the Calvin Cycle, NADP + [ 23 ]. Inactive RHO domain differs from the active one by the aspartate residue replacing cysteine 207 in the active site. Inactive RHO domain of TROL protein might be responsible for the transmembrane signal transduction from lumen to stroma that influences TROL-FNR dynamical binding, and consequently partitioning of photosynthetic electrons. It possibly causes conformational changes in the ITEP domain, which leads to release of FNR when overreduction of PETC occurs [ 16 ]. In PETC, photosystem I (PSI) and photosystem II (PSII) are the main sources of ROS propagation (singlet oxygen ( 1 O 2 ) and superoxide radical (O 2 ˙ˉ)) (Fig. 7 ), especially under stress conditions [ 8 ]. Besides chloroplasts, ROS are generated in peroxisomes, mitochondria, cytosol, apoplast, endoplasmic reticulum and cell wall [ 25 ]. It is well known that ROS in certain amounts serve as secondary messengers, but increased amounts can, due to their high reactivity, cause damage to DNA, RNA, lipids, proteins, and the whole cells. To prevent this critical damage, a balance between ROS propagation and scavenging needs to be maintained. This is one of the reasons why plants have developed various antioxidant defense mechanisms, since they are sessile organisms [ 17 ]. Excess amounts of electrons in PETC cause energy imbalance, which can be manifested either as overoxidation, causing saturation of linear electron flow (LEF), or overreduction, causing excessive photooxidation, which leads to ROS over propagation and damage of cellular ultrastructures. This imbalance of energy does not remain limited just within chloroplasts, it disturbs homeostasis of the whole plant [ 37 , 38 ]. At this point, dynamical binding of FNR to the TROL and the consequent ability of this complex to regulate redox status comes to focus [ 2 , 12 , 19 ]. Although ROS are short-lived and primarily generated in chloroplasts, they can travel between cellular compartments through membrane diffusion, aquaporins, and vesicle transport. Additionally, rather than physically moving between compartments, ROS can transmit redox signals and influence the antioxidant system by modifying oxidized glutathione, thiols, transcription factors, PRX, TRX, and GRX. In addition to that, ROS propagation multiplies over time. Furthermore, mitochondria-chloroplast contact sites and peroxisome-mitochondria interactions create specialized microdomains that facilitate efficient ROS exchange, allowing them to affect different organelles without requiring long-distance diffusion [ 26 , 27 ]. It is also worth mentioning that FNR reductive activity, which ensures transfer of electrons from NADPH back to Fd, is important for handling part of the redox imbalance [ 5 ]. It has been proposed that when the NADP + /NADPH ratio changes and PETC is over reduced, FNR detaches from TROL due to conformational changes facilitated by the transmembrane signal, which is possibly mediated by the lumen-located RHO domain. Consequently, FNR is starting to reduce Fd, and other ROS scavengers and alternative electron sinks are activated (Fig. 7 ) [ 22 ]. Besides electron transfer to NADP + , Fd, with the help of the Fd-dependent thioredoxin reductase (FTR), transfers electrons to redox regulating enzymes thioredoxins (TRX). In general, the equilibrium between ROS formation and scavenging is under control of nonenzymatic (carotenoids, glutathione (GSH), ascorbate (ASC)) and enzymatic antioxidants. Cu/Zn and Fe superoxide dismutases (SOD) are the first line defense enzymatic antioxidants that act against superoxide anion by catalyzing its conversion to hydrogen peroxide (H 2 O 2 ). H 2 O 2 can cause formation of another ROS, namely hydroxyl radical (OH∙) (Fig. 7 ). The amounts of H 2 O 2 are under control of ascorbate peroxidases (APX) and thiol-dependent peroxidases (TPX). APX use ascorbate as electron donor for H 2 O 2 reduction and consequent formation of monodehydroascorbate (MDA) that is then oxidized to the dehydroascorbate (DHA). MDA and DHA reductases use GSH as an electron donor to regenerate ASC, while regeneration of GSH is under control of the NADPH-dependent GSH reductase (GR). In the TPX group peroxiredoxins (PRX) and GSH peroxidases (GPX) are included. Maintenance of chloroplast redox equilibrium is linked to the H 2 O 2 scavenging system via the disulfide reductase activity of the NADPH-dependent redox system (NTRC), as well as TRX and glutaredoxins (GRX) that enable TPX activity. NADPH serves as an electron donor for H 2 O 2 antioxidative enzymes, while on the other side, H 2 O 2 serves as an electron sink, indicating the role of these molecules in the regulation of the redox status trough antioxidant systems [ 6 ]. We live in times of huge global climate changes including lack of rainfall and extremely high temperatures, resulting in severe drought periods. Besides this, the global population is in constant increase, dragging along elevated request for food, therefore plant cultivation and crop production must fulfil high demands. All those and many more other reasons elevate the importance of research with the goal of plant yield improvement and increased plant tolerance and resistance to stress [ 7 ]. Photosynthesis is one of the primary targets in research within this scope, since it is directly affected by changes in climate, and is also proportional to crop productivity. Under conditions of stress, like drought, above mentioned balance in transport of electrons is disturbed and ROS are propagated and distributed across cells with redox maintaining violation even beyond photosynthesis processes and chloroplasts. The goal of this research was to further test the hypothesis that the dynamic interaction of FNR with TROL is responsible for the activation of alternative electron sinks in vascular plant photosynthesis and consequent redox homeostasis maintenance in the whole plant. We also wanted to confirm our previous results obtained by the electron paramagnetic resonance spectroscopy (EPR) measurements [ 22 ], this time by using in situ ROS detection approach. Because ROS are short-lived, every detection method has certain limitations. Moreover, detecting ROS at specific sites and under precise conditions while determining their origin remains a significant challenge. To address this, we incubated plants in darkness before measurement. Since electron transport processes are nearly inactive in the dark, this approach allowed us to examine ROS propagation, distribution, and redox state at the cellular level while minimizing dominant ROS production in the PETC. Additionally, dark incubation with ROS-detecting fluorescent probes helped eliminate potential ROS formation caused by the fluorescent probes themselves. Also, laser excitation wavelength was in the green spectral region which is the least efficient for driving photosynthesis and auto producing ROS from fluorescent probes. Taken together, this experiment demonstrated how TROL and its various mutations influence the redox state at the whole-plant level. Till this day various methods have been established for detection of ROS in cells: EPR, optical spectroscopy, chromatography, and confocal laser scanning microscopy. The latter was used in this research. METHODS Plant material and growth conditions Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col-0) plants (originally obtained from the European Arabidopsis stock center, NASC, Loughborough, UK) were used as model organisms. Wild type (WT) and 2 mutant lines: TROL KO mutant line with the mutation on the chromosome 4 in the gene At4g01050 (T-DNA element SAIL_27_B04 insertion into the last intron at the position 2278 of At4g01050 ) which does not express TROL protein [ 11 ] and ΔRHO (13 amino acid (203–215) deletion in the RHO domain) were used [ 10 , 20 ]. Seeds were propagated and plants were grown in our laboratory under controlled conditions. 15 plants of each mutant line and the WT were grown in the growing system (Arasystem 3600 KIT, Betatech, Belgium), trays were divided into WT and two mutant lines. The substrate (A400, Stender, Germany) was distributed in araflats (specially designed arrays consisting of 51 of individual pot cavities, perfectly suited for growing Arabidopsis plants at optimal densities) and soaked in trays overnight prior to sowing. Plant seeds were vernalized 2 days prior to sowing. Sowed seeds were covered with transparent foil until germination. Growth conditions were 21 ℃, 440 ± 20 ppm CO 2 , 12 h day/night photoperiod (equinox May 21st ), under the LED illumination of CI-800 Programmable LED Experimentation System (CID Bio-Science, Inc., Camas, WA, USA) which mimics the sun light on the geographic position of Birmingham city, West midlands, UK (latitude 54.00, longitude − 2.00). Plants were grown for 1 month under the normal humidity conditions, and then for 2 more weeks under the arid conditions. The difference between humid and arid conditions was maintained by the gravimetric method, between 2.5–3.0 kg for humid and 2.0–2.4 kg for arid conditions. Plants were supplemented with ¼ Hoagland’s solution (1.25 ml/L Ca(NO 3 ) 2 x 4H 2 O, 1.25 ml/L KNO 3 , 0.25 ml/L KH 2 PO 4 , 0.5 ml/L MgSO 4 x 7 H 2 O, 0.25 ml/L micronutrients (2.86 g/L H 3 BO 3 , 1.81 g/L MnCl 2 x 4H 2 O, 0.22 g/L ZnSO 4 x 7H 2 O, 0.08 g/L CuSO 4 x 5H 2 O, 0.02 g/L NaMoO 4 x H 2 O g/L), 0.25 ml/L Fe-EDTA (10.4 g/L EDTA, 7.8 g/L FeSO 4 x 7 H 2 O, 56.1 g/L KOH g/L) [ 14 ] every two weeks. Substrate chemical composition was determined and checked by accredited analytical laboratory of the Department for Plant Nutrition, Division of Agroecology, Faculty of Agriculture, University of Zagreb, Svetošimunska cesta 25, 10000 Zagreb, Croatia. Chemical composition analyses revealed subtle changes in the macronutrient composition between the different substrate lots. Those differences affected reproducible plant growth and development. To offset those differences, we supplemented both substrate types with chemically well-defined Hoaglands nutrient solution. Together with that, reproducible growth conditions (light, temperature, CO 2 level), enabled detection of the subtle differences in biochemical processes, morphology, and photosynthesis of different TROL mutant lines, grown under different substrate moisture conditions, which was the goal of this research. Chemicals Fluorescent probes dyhidroethidium (DHE) and Singlet oxygen sensor green (SOSG) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and Spy-LHP from Dojindo Molecular Technologies Inc. (Rockville, MD, USA). Sample preparation Plants of each line were put in the dark 2 h before experiment. The whole leaf was cut out from the plant and rinsed in 50 mM HEPES buffer (pH 7.5) and after that incubated 30 minutes in desired fluorescent probe (250 µM DHE, 50 µM Spy-LHP, or 50 µM SOSG), while control samples were incubated in 50 mM HEPES buffer. After incubation, the leaf was again rinsed in 50 mM HEPES buffer and transferred on a glass slide in the drop of 50 mM HEPES buffer and covered with cover glass. Protocol was modified according to Prasad et al . 2020. Confocal microscopy The amount and arrangement of ROS within leaves were visualized using Confocal microscope Leica TCS SP8 Laser Scanning Confocal Microscope (Leica Microsystems GmbH, Germany) (Fig. 1 , 3 , 5 ). The excitation for SOSG was 504 nm, and emission 520–560 nm, for the Spy-LHP excitation was 524 nm, emission 535–580 nm, and for the DHE excitation was 480 nm, emission at 560–610 nm. Chloroplast autofluorescence was determined at 650–750 nm for all fluorescent probes. In the beginning of each experiment, proper laser intensity was set by using control samples in which leaves were incubated in 50 mM HEPES buffer without fluorescent probes. All confocal microscopy experiments include images of chloroplasts autofluorescence (red field signal), bright field images (gray field signal), and detected ROS with fluorescent probes (green field signal). For each fluorescent probe and control, 3 visual fields in different plane levels were recorded for each of three leaf replicas. The experiment was carried out in situ . Image analysis Images obtained with confocal laser scanning microscopy (Fig. 1 , 3 , 5 ) were analyzed by using ImageJ ( Fiji ) software. Green field fluorescent signal strength of fluorescent probes reactions with ROS was measured in total counts. The average value of control images was subtracted from the average value of belonging reaction groups to evaluate signal intensity. Ten measurements of each fluorescent probe and control sample for every growth condition and plant lines were used on the three leaf replicas. Statistical analysis All the data were expressed as mean ± standard deviation. Statistical analysis was carried out with GraphPad Prism (v.9.0.0.121). Comparison of groups were performed by using one-way ANOVA with post-hoc analysis Dunnett´s test. The statistical significance is indicated as * at P < 0,05 confidence level. RESULTS In this work we examined the propagation and the distribution of the three common ROS, superoxide anion, hydrogen peroxide, and singlet oxygen in the leaves of the model plant Arabidopsis thaliana (L.) Heyn. ecotype Columbia (Col-0) (WT), and the two mutant lines TROL KO and TROL ΔRHO. We examined the influence of different soil moisture conditions on ROS accumulation at the level of whole leaves. We further tested our hypothesis that arises from the previous work in our laboratory carried out by Vojta et al. 2015 and Vojta et al. 2023., which postulates that alternative pathways of electron partitioning (Fig. 7 ) under different environmental conditions depend on the TROL-FNR complex dynamical formation. We investigated ROS distribution under the influence of TROL not just on the chloroplast level but on the whole leaves tissue. In this work we used confocal laser scanning microcopy in combination with specific fluorescent probes for visualization of three different ROS species [ 17 ]. ROS are usual byproducts generated in the various metabolic processes. Formation of ROS is caused by partial reduction or energy transfer to O 2 . The major site of O 2 ˙ˉ propagation by the excess electron spillage to O 2 , is the PSI in whose vicinity on the thylakoid membrane TROL is positioned (Fig. 7 ). Such proximity of O 2 ˙ˉ propagation site and the TROL-FNR complex further points toward the protective role hypothesis. In addition to chloroplasts, ROS are generated in various cellular sites. These include mitochondria, particularly in the respiratory ETC under stress or when ATP synthesis is impaired, peroxisomes, especially under high light (HL) intensity when photorespiration is elevated, NADPH oxidases in the plasma membrane, peroxidases in the cell wall, xanthine oxidase reaction sites, and autoxidation of redox compounds under stress conditions [ 28 , 29 , 30 ]. In our experimental setup for detection of O 2 ˙ˉ we used redox sensitive fluorescent probe DHE. Under the conditions of drought stress there was a significant reduction in the amount of O 2 ˙ˉ in the TROL KO mutant line in comparison with the WT. Although the amount of O 2 ˙ˉ is also reduced for the ΔRHO mutant line under drought stress conditions in comparison with the WT, the difference was not significant and not pronounced as is in the case of TROL KO mutant line (Fig. 2 ). These results confirm protective role and detoxification activity dependent on the TROL-FNR complex dynamical binding that is activated under the stress conditions when alternative electron sink is preferred over the LEF and it implies positive influence on whole leaves level (Fig. 7 ). Under normal moisture conditions both mutant lines show unsignificant increase of O 2 ˙ˉ propagation (Fig. 2 ). These results indicate that ΔRHO mutation has no effect on O 2 ˙ˉ scavenging, at least not on such high level as in TROL KO line. Results also indicate that under unstressed conditions there is no increased scavenging activity (Fig. 2 ). In plants some ROS, especially H 2 O 2 , serve as secondary messengers in various signaling and gene expression pathways. In chloroplasts, H 2 O 2 is mostly generated on the PSII at low rate, in the Mehler reaction, photorespiration, and as a product of O 2 ˙ˉ reduction, with the latter also occurring in various cellular compartments. Other sources of H₂O₂ production include mitochondria during aerobic respiration, peroxisomes during photorespiration and β-oxidation, oxidase enzymes in the cytoplasm, peroxidases in the cell wall, and auxin oxidation [ 31 , 32 , 33 ]. SOD is one of the first and most abundant defense antioxidant mechanisms against O 2 ˙ˉ. Since SOD produces great amount of H 2 O 2 from superoxide (can be seen from the scale difference on Y axis of Figs. 2 and 4 ), the protection against peroxide in cells is very high and efficient. According to that, Fig. 4 shows efficient scavenging of peroxide under drought conditions. In this work, propagation and distribution of H 2 O 2 was monitored by using highly selective fluorescent probe Spy-LHP. Under the drought stress conditions, ΔRHO showed significantly less amount of H 2 O 2 in comparison with the WT. On the other hand, the TROL KO mutant line did not show significantly elevated amounts of H 2 O 2 (Fig. 4 ). These results indicate the possible involvement of the RHO domain in transmembrane redox signaling from lumen to stroma and consequent regulation of TROL-FNR complex dynamical binding and TROL complex formation with tAPX, antioxidant enzyme that scavenges H 2 O 2 , all with a goal to form alternative electron sinks that could protect cells under the stress conditions. We also observed that the TROL KO line is not as efficient in H 2 O 2 detoxification as it is in O 2 ˙ˉ scavenging. Under the conditions of normal moisture, compared with the WT, in ΔRHO mutant line H 2 O 2 was equally accumulated, while in TROL KO line was elevated. However, the increase in H 2 O 2 was not statistically significant in this case (Fig. 4 ). This again indicates that alternative electron sinks are activated under the conditions of stress when the protection against ROS is needed. The formation of 1 O 2 , which represents the first excited electronic state of O 2 , is mostly located in the vicinity of the PSII where interaction of O 2 with triplet chlorophyll occurs. Additionally, by triggering lipid peroxidation, ¹O₂ can amplify its own production. Certain peroxidases and lipoxygenases can also generate ¹O₂ from fatty acids. And these reactions are not related only to chloroplasts [ 34 , 35 ]. 1 O 2 is specific ROS because it is not generated by electron transfer to O 2 . The two main mechanisms for 1 O 2 detoxification are physical and chemical quenching. Highly selective fluorescent probe SOSG was used for 1 O 2 signal strength detection. Under the conditions of normal growth substrate moisture, the amount of 1 O 2 was significantly reduced in ΔRHO mutant line in comparison with the WT. Unsignificant changes in the amount of 1 O 2 could be observed in the TROL KO line. Under the drought stress conditions, both mutant lines showed equally elevated, yet unsignificant 1 O 2 content (Fig. 6 ). Knowledge gathered so far together with the results of this work indicates that formation of TROL-FNR complex is triggered in situations when LEF and NADP + synthesis is preferred, while when stress conditions are present, releasing of FNR from TROL enables various alternative electron sinks and detoxification mechanisms in order to maintain whole plant redox homeostasis. DISCUSION Plants are confronting many severe environmental conditions, but they are successful in overcoming them as they have evolved numerous short- and long-term adaptation and acclimation mechanisms. Dynamical binding and positioning of the photosynthetic complexes can change depending on growth conditions, especially stress conditions like drought. Drought limits electron donation by reducing the photolysis of water, which impairs electron flow through the ETC. This leads to ETC over-reduction, increasing excitation pressure and electron leakage. To dissipate excess electrons, photorespiration and the water-water cycle become crucial, CEF and antioxidant enzymes are upregulated to maintain redox balance [ 39 , 40 ]. One of the mechanisms presented by our group is that soluble FNR handles oxidative stress better than when docked to TROL, consequently enabling TROL KO plants to propagate lower amounts of ROS. Different TROL mutations affect TROL-FNR binding dynamic, and some of the mutations cause more efficient ROS scavenging. TROL-FNR pair regulates electron transport and partitioning dynamic in accord with the energy requirements, particularly NADPH synthesis, and perhaps ATP. Since LET products are used in various metabolic reactions, TROL-FNR regulation of electron transport consequently influences whole organism. It is not the TROL that is directly involved in ROS scavenging and energy production, but the flavoenzyme FNR. TROL mutant lines are involved in the FNR binding and release from the vicinity of the photosystem I. It is also to be expected that the RHO domain of TROL has influence on electron partitioning by possible transmembrane redox signal transduction from lumen to stroma with consequent influence on the dynamical binding of the TROL-FNR complex. It appears that scavenging reactions and alternative electron partitioning, driven by changes in the dynamic binding of the TROL-FNR pair, influence ROS metabolism and the maintenance of redox homeostasis at the whole plant level. Firstly, we established that O 2 ˙ˉ formation in TROL KO mutant line was reduced, while generation of the other ROS was mostly enhanced (Fig. 2 , 4 , 6 ). There is a significant reduction in the amount of O 2 ˙ˉ in TROL KO mutant line in comparison with the WT under the drought stress. The same results were obtained in the ΔRHO mutant line, but on the less pronounced scale. Under the normal humidity, in both mutant lines, the amount of O 2 ˙ˉ was elevated in comparison to the WT (Fig. 2 ). When growth conditions were compared, less O 2 ˙ˉ could be detected in TROL KO and ΔRHO under drought stress, in comparison with the WT (Fig. 2 ), indicating the protective role of FNR when TROL is not present. Alternative electron sinks are activated and by successful scavenging of propagated O 2 ˙ˉ (Fig. 7 ) there is less spillage of electrons to O 2 , as earlier proposed [ 1 , 22 , 24 ]. It is important to mention that under growth-light conditions (GL), the electron transport rate is not lower in KO. In fact, under GL, TROL KO plants are growing more successfully than the WT, which is consistent with earlier PAM measurements that showed that electron transport rate is not lower in KO [ 11 ]. TROL-FNR binding becomes rate limited in linear electron transport rate at quite high photosynthetically active radiation (PAR). Enzyme FNR is involved in other electron transport reactions besides linear photosynthetic electron transport (Fig. 7 ). In addition, it takes 2 reduced ferredoxin molecules to generate 1 NADPH. TROL docks FNR in the vicinity of photosystem I, so reduced ferredoxin can efficiently transfer electrons to NADP + . When TROL is not present, FNR is not efficiently docked to the thylakoid membranes and electrons are distributed in alternative pathways, which take electrons more rapidly (Fig. 7 .) [ 22 ]. This however happens at very high light intensities, or when plants are exposed to more than one stressor (combined stress). Additionally, the reduction of the negative stress effects enabled by the TROL-FNR pair in the PETC seems to be so significant that it positively influences the maintenance of a balanced redox state across the entire cell. With these results in mind, we wanted to investigate whether other ROS are also susceptible to scavenging influenced by the lack of TROL. Under drought stress conditions TROL KO shows elevated amounts of H 2 O 2 in comparison with the WT. Contrary to that, ΔRHO shows significantly less amount (Fig. 4 ). This result indicates reduced ROS scavenging efficiency, other than O 2 ˙ˉ in TROL KO plants, while other defensive mechanisms against H 2 O 2 could be present and more expressed in ΔRHO mutant line. RHO domain was proposed to have a role in transmembrane redox signal transduction that affects dynamical binding of TROL and FNR and consequent flow of electrons [ 11 ]. In addition, as proposed in Vojta et al. 2023, TROL most likely forms a complex with the tAPX that is an important antioxidant enzyme in the process of H 2 O 2 detoxification. The existence of tAPX isoform is proven in the vicinity of PSI which is the place of H 2 O 2 and O 2 ˙ˉ generation. Spectrofotometrical measurement also showed increased APX activity in the TROL KO and ΔRHO mutant lines under drought conditions in comparison with WT (Dumančić and Fulgosi, unpublished data). Under normal growth substrate moisture, ΔRHO mutant line showed equal amount of H 2 O 2 as the WT, while TROL KO mutant line showed unsignificant elevation (Fig. 4 ). Contrary to the TROL KO, Arabidopsis plants that overexpress TROL (TROL OX) failed to successfully cope with the ROS propagation, owing to the fact that more FNR was bound to the membrane in this plants [ 24 ]. Results presented in this work are in accordance with mentioned results and also with the previously demonstrated successful O 2 ˙ˉ scavenging in TROL KO plants even when they were treated with methyl-viologen (herbicide that strongly induces formation of O 2 ˙ˉ), as shown by the EPR [ 18 , 22 , 24 ]. Considering all mentioned findings, the proposal that TROL-FNR dynamic interaction has a significant role in maintaining redox homeostasis is even more strengthened (Fig. 7 ). The positioning of TROL on the thylakoid membranes plays crucial role in controlling redox balance in chloroplasts, which is expected, as chloroplasts are the primary sites of ROS generation in plants. However, its significance extends beyond this. By regulating ROS equilibrium and electron partitioning, TROL prevents redox imbalance affecting areas beyond the chloroplasts. This is achieved by controlling electron flow and partitioning, thereby reducing ROS formation and preventing subsequent chain reactions that lead to ROS multiplication and intercellular ROS transfer. Oxidative stress could regulate release of FNR form thylakoids [ 12 , 15 ] and this regulation could be achieved through the RHO domain in combination with reduced PQ by redox sensing from lumen trough thylakoid membrane and influencing TROL-FNR dynamic binding on the stromal side trough conformational changes induced in ITEP domain [ 16 , 20 , 21 ]. Shown in this research, ΔRHO mutant line exhibits a potential to efficiently remove H 2 O 2 . ΔRHO mutation might also influence TROL-FNR complex interaction with the tAPX. APX is antioxidant enzyme involved in H 2 O 2 scavenging and its thylakoidal (tAPX) form is positioned in the vicinity of PSI, indicating possible interaction and cooperation with TROL [ 24 ]. Exact mechanisms of ROS scavenging influenced by the TROL-FNR complex dynamics remain to be determined. According to the results of this research, neither of tested mutant lines possesses the capability of detoxification of 1 O 2 under drought stress conditions. Further research should be carried out to answer these questions. CONCLUSION When considering improvement of plant/crop stress tolerance and protection, TROL-FNR complex is inevitable to take into consideration because of various dynamical interactions through which it can regulate electron transport in different pathways of PETC and ROS scavenging in chloroplast and beyond. The exact mechanisms of ROS detoxification pathways that involve TROL-FNR complex remain to be elucidated. To conclude, this research brings additional evidence for the involvement of TROL-FNR interaction in the ROS-protection under stress conditions. Once more, we showed that dynamical binding of FNR to TROL significantly participates in the maintenance of redox equilibrium by activating various detoxication mechanisms against ROS (precisely O 2 ˙ˉ and H 2 O 2 ) formed under the, in this case, drought stress conditions. Declarations Supplementary Information Not applicable. Acknowledgements Not applicable. Author contributions E.D. grew plant material, prepared specimens for confocal microscopy, collected and analyzed data, wrote the initial drafts. L.V. advised on experimental setup, wrote and revised the manuscript. H.F. supervised the research, participated in planning the experiments, writing process, and revised the manuscript. H.F. led the project and raised the project funds. Funding This work was funded by the Croatian Science Foundation (HRZZ) Grants: IP-2020-02-8590 and DOK-2021-02-2018 to HF. Availability of data and materials The datasets generated and analysed during the current study are available in the Figshare repository https://doi.org/10.6084/m9.figshare.25736829.v3. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Allen JF. Cyclic, pseudocyclic and noncyclic photophosphorylation: new links in the chain. 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Front Plant Sci. 2020;10:467919. Rac A. Molecular analysis of the FNR-TROL interaction elements. PhD thesis. University of Zagreb, Faculty of Science, Department of Biology; 2021. Tanaka A, Makino A. Photosynthetic research in plant science. Plant Cell Physiol. 2009;50:681–3. Vojta L, Fulgosi H. Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana . Physiol Plant. 2019;166:300–8. Vojta L, Fulgosi H. Energy conductance from thylakoid complexes to stromal reducing equivalents. Adv Photosynthesis-Fundamental Aspects. 2012;175–90. Vojta L, Carić D, Cesar V, Antunović Dunić J, Lepeduš H, Kveder M, et al. TROL-FNR interaction reveals alternative pathways of electron partitioning in photosynthesis. Sci Rep. 2015;5:10085. Vojta L, Horvat L, Fulgosi H. Balancing chloroplast redox status-regulation of FNR binding and release. Period Biol. 2012;114:25–31. Vojta L, Rac-Justament A, Zechmann B, Fulgosi H. Thylakoid rhodanese-like protein–ferredoxin: NADP + oxidoreductase interaction is integrated into plant redox homeostasis system. Antioxidants. 2023;12(10):1838. Sharma P, Jha AB, Dubey RS, Pessarakli M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot. 2012;1:217037. Sandalio LM, Peláez-Vico MA, Molina-Moya E, Romero-Puertas MC. Peroxisomes as redox-signaling nodes in intracellular communication and stress responses. Plant Physiol. 2021;186(1):22–35. Noctor G, Foyer CH. Intracellular redox compartmentation and ROS-related communication in regulation and signaling. Plant Physiol. 2016;171(3):1581–92. Huang H, Ullah F, Zhou DX, Yi M, Zhao Y. Mechanisms of ROS regulation of plant development and stress responses. Front Plant Sci. 2019;10:800. Jiménez-Quesada MJ, Traverso JÁ, Alché JDD. NADPH oxidase-dependent superoxide production in plant reproductive tissues. Front Plant Sci. 2016;7:359. Bowler C, Van Camp W, Van Montagu M, Inzé D, Asada K. Superoxide dismutase in plants. CRC Crit Rev Plant Sci. 1994;13(3):99–218. Niu L, Liao W. Hydrogen peroxide signaling in plant development and abiotic responses: crosstalk with nitric oxide and calcium. Front Plant Sci. 2016;7:230. Smirnoff N, Arnaud D. Hydrogen peroxide metabolism and functions in plants. New phytol. 2019;221(3):1197–214. Černý M, Habanova H, Berka M, Luklova M, Brzobohatý B. Hydrogen peroxide: its role in plant biology and crosstalk with signalling networks. Int J Mol Sci. 2018;19(9):2812. Laloi C, Havaux M. Key players of singlet oxygen-induced cell death in plants. Front. Plant Sci. 2015;6:39. Dogra V, Kim C. Singlet oxygen metabolism: from genesis to signaling. Front Plant Sci. 2020;10:1640. Tripathy BC, Oelmüller R. Reactive oxygen species generation and signaling in plants. Plant Signal Behav. 2012;7(12):1621–33. Hüner NP, Bode R, Dahal K, Hollis L, Rosso D, Krol M, et al. Chloroplast redox imbalance governs phenotypic plasticity: the grand design of photosynthesis revisited. Front. Plant Sci. 2012;3:255. Xu Z, Zhang R, Yang M, Law YS, Sun F, Hon NL, et al. A balance between the activities of chloroplasts and mitochondria is crucial for optimal plant growth. Antioxidants. 2021;10(6):935. Golding AJ, Johnson GN. Down-regulation of linear and activation of cyclic electron transport during drought. Planta. 2023;218(1):107–14. Qiao M, Hong C, Jiao Y, Hou S, Gao H. Impacts of drought on photosynthesis in major food crops and the related mechanisms of plant responses to drought. Plants. 2024;13(13):1808. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6220323","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432170851,"identity":"2d86199a-38ad-42ca-8514-1284f1e711d7","order_by":0,"name":"Ena Dumančić","email":"","orcid":"","institution":"Ruđer Bošković Institute","correspondingAuthor":false,"prefix":"","firstName":"Ena","middleName":"","lastName":"Dumančić","suffix":""},{"id":432170852,"identity":"b79d50f4-eb7a-4638-a849-c62b6fcc7d2a","order_by":1,"name":"Lea Vojta","email":"","orcid":"","institution":"Ruđer Bošković Institute","correspondingAuthor":false,"prefix":"","firstName":"Lea","middleName":"","lastName":"Vojta","suffix":""},{"id":432170853,"identity":"4cbe6482-8c34-4e07-951c-8fc1d2d8aa5a","order_by":2,"name":"Hrvoje Fulgosi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApUlEQVRIiWNgGAWjYBAC9gbmBiAlIcfGwMPATJQWngOMYC3GJGthSGwgXotEYvOLD38s0vukzx5gLtxDnJY2y5ltErltfHkJzDOeEaHFHqjFmLcBqIWHx4CZ5wCRthjz/JFIZyNFS/NjHjaJBBK08DxsYwT6xbCNhy/hMHFa2JMPf/jwp05evof34GOitAABmwSMRaQGBgbmD8SqHAWjYBSMghEKAORQLaIHZJk2AAAAAElFTkSuQmCC","orcid":"","institution":"Ruđer Bošković Institute","correspondingAuthor":true,"prefix":"","firstName":"Hrvoje","middleName":"","lastName":"Fulgosi","suffix":""}],"badges":[],"createdAt":"2025-03-13 13:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6220323/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6220323/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79079087,"identity":"44e43a31-9d84-4f65-b971-ac4094203e79","added_by":"auto","created_at":"2025-03-24 07:56:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2621502,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning microscopy images of superoxide radical (DHE). (\u003cstrong\u003ea\u003c/strong\u003e) under normal humidity growth conditions (\u003cstrong\u003eb\u003c/strong\u003e) under drought growth conditions. Samples are visualized under the same settings to enable comparison: HC PL APO CS2 63x/1.40 oil and identical camera settings. Representative images are shown. The contrast of green field images was enhanced in favor of better visibility and results interpretation equally across the whole image by GIMP 2.10.38. Measurements were made on original, unprocessed images. Scale bars: 25 μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6220323/v1/6f1387aa42eb0627155551e0.png"},{"id":79078190,"identity":"d3ddf221-ec7b-4d86-935d-4580e75508fe","added_by":"auto","created_at":"2025-03-24 07:48:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21494,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning microscopy signal strength detection results for superoxide anion. Statistically significant decrease of the O\u003csub\u003e2\u003c/sub\u003e˙ˉ amount in the TROL KO mutant line under the drought stress condition indicates protective role of the TROL-FNR complex dynamical binding and alternative electrons sink formation. Error bars represent mean ± standard deviation with statistical significance indicated as * at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0,05 confidence level.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6220323/v1/fa8f923a7fd292f655091d55.png"},{"id":79079096,"identity":"3e9a1988-a2e8-4a6e-b499-73b8a5f22335","added_by":"auto","created_at":"2025-03-24 07:56:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2557705,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning microscopy images of hydrogen peroxide (Spy-LHP). (\u003cstrong\u003ea\u003c/strong\u003e) under normal humidity growth conditions (\u003cstrong\u003eb\u003c/strong\u003e) under drought growth conditions. Samples are visualized under the same settings to enable comparison: HC PL APO CS2 63x/1.40 oil and identical camera settings. Representative images are shown. The contrast of green field images was enhanced in favor of better visibility and results interpretation equally across the whole image by GIMP 2.10.38. Measurements were made on original, unprocessed images. Scale bars: 25 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6220323/v1/c4b9edd5151928616acdf788.png"},{"id":79080274,"identity":"beaf4d78-ff45-42cf-b8ae-df8463ed8a4c","added_by":"auto","created_at":"2025-03-24 08:12:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21733,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning microscopy signal strength detection results for hydrogen peroxide. Statistically significant decrease of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e amount in the ΔRHO mutant line under the drought stress condition indicates defense role of the TROL RHO domain against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003eby transmembrane signal transduction that influence TROL-FNR complex dynamical binding and TROL complex formation with tAPX. Error bars represent mean ± standard deviation with statistical significance indicated as * at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0,05 confidence level.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6220323/v1/75eadaa32a00009f1fd3f1b0.png"},{"id":79079092,"identity":"061e9815-8a0b-463e-a81c-c08eee5cee9d","added_by":"auto","created_at":"2025-03-24 07:56:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2847388,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning microscopy images of singlet oxygen (SOSG). (\u003cstrong\u003ea\u003c/strong\u003e) under normal humidity growth conditions (\u003cstrong\u003eb\u003c/strong\u003e) under drought growth conditions. Samples are visualized under the same settings to enable comparison: HC PL APO CS2 63x/1.40 oil and identical camera settings. Representative images are shown. The contrast of green field images was enhanced in favor of better visibility and results interpretation equally across the whole image by GIMP 2.10.38. Measurements were made on original, unprocessed images. Scale bars: 25 μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6220323/v1/c802269202f33441579743c7.png"},{"id":79080534,"identity":"d7760b77-a493-49a9-910f-193c9058d9b8","added_by":"auto","created_at":"2025-03-24 08:20:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21478,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning microscopy signal strength detection results for singlet oxygen. Statistically significant decrease of the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e amount in the ΔRHO mutant line under normal humidity conditions is observed. Error bars represent mean ± standard deviation with statistical significance indicated as * at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0,05 confidence level.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6220323/v1/755242a86fc84bd13a77b9e8.png"},{"id":79079089,"identity":"59e06e39-2409-4809-9460-7af84f9725e4","added_by":"auto","created_at":"2025-03-24 07:56:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":76618,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosynthetic ROS propagation, possible electron transfer pathways, and the role of the TROL-FNR pair. (\u003cstrong\u003ea\u003c/strong\u003e) ROS propagation sites in the photosynthetic electron transport chain by excitation and reduction of O\u003csub\u003e2\u003c/sub\u003e; (\u003cstrong\u003eb\u003c/strong\u003e) existing electron transfer pathways from Fd\u003csub\u003ered \u003c/sub\u003ein the vicinity of PSI. When FNR is docked to thylakoid membranes by the TROL, linear electron transport pathway is preferred. In the absence of FNR binding to TROL, e.g. in the TROL KO plants, electrons from Fd\u003csub\u003ered\u003c/sub\u003e can be distributed to some of the alternative pathways. This transfer is more rapid than the linear electron transport pathway, efficiently preventing electron transfer to O\u003csub\u003e2\u003c/sub\u003e, and consequently lower O\u003csub\u003e2\u003c/sub\u003e˙ˉ propagation. Dynamical binding of the TROL-FNR protein pair influences the distribution of electrons in different pathways depending on energy status and needs of the cell.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6220323/v1/35b061f137ade55de60f856d.png"},{"id":83228956,"identity":"2e158b31-7ce6-428d-a6ad-492a42e95261","added_by":"auto","created_at":"2025-05-21 12:32:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9939971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6220323/v1/44b96234-9b6e-4ab2-9c05-90639406a9cc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Confocal laser scanning microscopy of ROS in Arabidopsis thaliana (L.) Heynh. TROL-FNR mutants","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003ePhotosynthesis is a very important biochemical process that is carried out by photoautotrophic organisms. They use the energy of photons, as well as water molecules as the electron donor and the source of protons, in order to fix carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) from the atmosphere to finally produce simple sugars [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Very important part of the photosynthetic electron transfer chain (PETC), is the last, reversible, linear step of electron transfer from the small soluble protein ferredoxin (Fd) to the nicotinamide adenine dinucleotide phosphate (NADP\u003csup\u003e+\u003c/sup\u003e) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This step is catalyzed by a dimerized flavoenzyme ferredoxin:NADP\u003csup\u003e+\u003c/sup\u003e oxidoreductase (FNR) that can be attached to photosynthetic membranes via different proteins [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], but herein we focus on the interaction with the thylakoid rhodanese like protein (TROL) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. FNR bound to TROL efficiently catalyzes NADPH biosynthesis, while soluble form prevents reactive oxygen species (ROS) formation and damage of molecular structures, suggesting protective role of this complex [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Besides its fundamental role in sustaining life on Earth, photosynthesis is also the primary source of ROS production in plants. Also, as in other organisms, ROS are generated in various cellular compartments other than chloroplasts and have the ability to transition between them (Noctor, 2016). Because of that, in this study, we focused on ROS propagation and scavenging at the whole-cell level, including ROS not directly linked to PETC. Although TROL is part of the PETC within chloroplasts, we proposed its influence on electron management and redox homeostasis system of the whole plant [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This role arises from the necessity of preventing ROS damage across all cellular compartments, particularly under stress conditions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTROL consists of the two transmembrane domains which span trough the thylakoid membrane, an inactive RHO domain in the thylakoid lumen, the C-terminal stromal domain that binds FNR, the ITEP domain characteristic for vascular plants, and the N-terminal stromal domain with chloroplast targeting pre-sequence [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Upstream of the ITEP domain PEPE domain is located. This domain contains characteristic proline-valine-proline repeat rich region that enables flexibility [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. TROL is located in the thylakoid membranes like a hinge between the end of the protein complexes of the photosynthetic electron transport chain (PETC) and the precursor of the Calvin Cycle, NADP\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Inactive RHO domain differs from the active one by the aspartate residue replacing cysteine 207 in the active site. Inactive RHO domain of TROL protein might be responsible for the transmembrane signal transduction from lumen to stroma that influences TROL-FNR dynamical binding, and consequently partitioning of photosynthetic electrons. It possibly causes conformational changes in the ITEP domain, which leads to release of FNR when overreduction of PETC occurs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn PETC, photosystem I (PSI) and photosystem II (PSII) are the main sources of ROS propagation (singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) and superoxide radical (O\u003csub\u003e2\u003c/sub\u003e˙ˉ)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), especially under stress conditions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Besides chloroplasts, ROS are generated in peroxisomes, mitochondria, cytosol, apoplast, endoplasmic reticulum and cell wall [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It is well known that ROS in certain amounts serve as secondary messengers, but increased amounts can, due to their high reactivity, cause damage to DNA, RNA, lipids, proteins, and the whole cells. To prevent this critical damage, a balance between ROS propagation and scavenging needs to be maintained. This is one of the reasons why plants have developed various antioxidant defense mechanisms, since they are sessile organisms [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Excess amounts of electrons in PETC cause energy imbalance, which can be manifested either as overoxidation, causing saturation of linear electron flow (LEF), or overreduction, causing excessive photooxidation, which leads to ROS over propagation and damage of cellular ultrastructures. This imbalance of energy does not remain limited just within chloroplasts, it disturbs homeostasis of the whole plant [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. At this point, dynamical binding of FNR to the TROL and the consequent ability of this complex to regulate redox status comes to focus [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although ROS are short-lived and primarily generated in chloroplasts, they can travel between cellular compartments through membrane diffusion, aquaporins, and vesicle transport. Additionally, rather than physically moving between compartments, ROS can transmit redox signals and influence the antioxidant system by modifying oxidized glutathione, thiols, transcription factors, PRX, TRX, and GRX. In addition to that, ROS propagation multiplies over time. Furthermore, mitochondria-chloroplast contact sites and peroxisome-mitochondria interactions create specialized microdomains that facilitate efficient ROS exchange, allowing them to affect different organelles without requiring long-distance diffusion [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It is also worth mentioning that FNR reductive activity, which ensures transfer of electrons from NADPH back to Fd, is important for handling part of the redox imbalance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It has been proposed that when the NADP\u003csup\u003e+\u003c/sup\u003e/NADPH ratio changes and PETC is over reduced, FNR detaches from TROL due to conformational changes facilitated by the transmembrane signal, which is possibly mediated by the lumen-located RHO domain. Consequently, FNR is starting to reduce Fd, and other ROS scavengers and alternative electron sinks are activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBesides electron transfer to NADP\u003csup\u003e+\u003c/sup\u003e, Fd, with the help of the Fd-dependent thioredoxin reductase (FTR), transfers electrons to redox regulating enzymes thioredoxins (TRX). In general, the equilibrium between ROS formation and scavenging is under control of nonenzymatic (carotenoids, glutathione (GSH), ascorbate (ASC)) and enzymatic antioxidants. Cu/Zn and Fe superoxide dismutases (SOD) are the first line defense enzymatic antioxidants that act against superoxide anion by catalyzing its conversion to hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can cause formation of another ROS, namely hydroxyl radical (OH∙) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The amounts of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are under control of ascorbate peroxidases (APX) and thiol-dependent peroxidases (TPX). APX use ascorbate as electron donor for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reduction and consequent formation of monodehydroascorbate (MDA) that is then oxidized to the dehydroascorbate (DHA). MDA and DHA reductases use GSH as an electron donor to regenerate ASC, while regeneration of GSH is under control of the NADPH-dependent GSH reductase (GR). In the TPX group peroxiredoxins (PRX) and GSH peroxidases (GPX) are included. Maintenance of chloroplast redox equilibrium is linked to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging system via the disulfide reductase activity of the NADPH-dependent redox system (NTRC), as well as TRX and glutaredoxins (GRX) that enable TPX activity. NADPH serves as an electron donor for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e antioxidative enzymes, while on the other side, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e serves as an electron sink, indicating the role of these molecules in the regulation of the redox status trough antioxidant systems [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe live in times of huge global climate changes including lack of rainfall and extremely high temperatures, resulting in severe drought periods. Besides this, the global population is in constant increase, dragging along elevated request for food, therefore plant cultivation and crop production must fulfil high demands. All those and many more other reasons elevate the importance of research with the goal of plant yield improvement and increased plant tolerance and resistance to stress [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Photosynthesis is one of the primary targets in research within this scope, since it is directly affected by changes in climate, and is also proportional to crop productivity. Under conditions of stress, like drought, above mentioned balance in transport of electrons is disturbed and ROS are propagated and distributed across cells with redox maintaining violation even beyond photosynthesis processes and chloroplasts.\u003c/p\u003e \u003cp\u003eThe goal of this research was to further test the hypothesis that the dynamic interaction of FNR with TROL is responsible for the activation of alternative electron sinks in vascular plant photosynthesis and consequent redox homeostasis maintenance in the whole plant. We also wanted to confirm our previous results obtained by the electron paramagnetic resonance spectroscopy (EPR) measurements [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], this time by using \u003cem\u003ein situ\u003c/em\u003e ROS detection approach. Because ROS are short-lived, every detection method has certain limitations. Moreover, detecting ROS at specific sites and under precise conditions while determining their origin remains a significant challenge. To address this, we incubated plants in darkness before measurement. Since electron transport processes are nearly inactive in the dark, this approach allowed us to examine ROS propagation, distribution, and redox state at the cellular level while minimizing dominant ROS production in the PETC. Additionally, dark incubation with ROS-detecting fluorescent probes helped eliminate potential ROS formation caused by the fluorescent probes themselves. Also, laser excitation wavelength was in the green spectral region which is the least efficient for driving photosynthesis and auto producing ROS from fluorescent probes. Taken together, this experiment demonstrated how TROL and its various mutations influence the redox state at the whole-plant level. Till this day various methods have been established for detection of ROS in cells: EPR, optical spectroscopy, chromatography, and confocal laser scanning microscopy. The latter was used in this research.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003ePlant material and growth conditions\u003c/p\u003e \u003cp\u003e \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (L.) Heynh. ecotype Columbia (Col-0) plants (originally obtained from the European Arabidopsis stock center, NASC, Loughborough, UK) were used as model organisms. Wild type (WT) and 2 mutant lines: TROL KO mutant line with the mutation on the chromosome 4 in the gene \u003cem\u003eAt4g01050\u003c/em\u003e (T-DNA element SAIL_27_B04 insertion into the last intron at the position 2278 of \u003cem\u003eAt4g01050\u003c/em\u003e) which does not express TROL protein [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and ΔRHO (13 amino acid (203\u0026ndash;215) deletion in the RHO domain) were used [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Seeds were propagated and plants were grown in our laboratory under controlled conditions. 15 plants of each mutant line and the WT were grown in the growing system (Arasystem 3600 KIT, Betatech, Belgium), trays were divided into WT and two mutant lines. The substrate (A400, Stender, Germany) was distributed in araflats (specially designed arrays consisting of 51 of individual pot cavities, perfectly suited for growing Arabidopsis plants at optimal densities) and soaked in trays overnight prior to sowing. Plant seeds were vernalized 2 days prior to sowing. Sowed seeds were covered with transparent foil until germination. Growth conditions were 21 ℃, 440\u0026thinsp;\u0026plusmn;\u0026thinsp;20 ppm CO\u003csub\u003e2\u003c/sub\u003e, 12 h day/night photoperiod (equinox May 21st ), under the LED illumination of CI-800 Programmable LED Experimentation System (CID Bio-Science, Inc., Camas, WA, USA) which mimics the sun light on the geographic position of Birmingham city, West midlands, UK (latitude 54.00, longitude \u0026minus;\u0026thinsp;2.00). Plants were grown for 1 month under the normal humidity conditions, and then for 2 more weeks under the arid conditions. The difference between humid and arid conditions was maintained by the gravimetric method, between 2.5\u0026ndash;3.0 kg for humid and 2.0\u0026ndash;2.4 kg for arid conditions. Plants were supplemented with \u0026frac14; Hoagland\u0026rsquo;s solution (1.25 ml/L Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e x 4H\u003csub\u003e2\u003c/sub\u003eO, 1.25 ml/L KNO\u003csub\u003e3\u003c/sub\u003e, 0.25 ml/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.5 ml/L MgSO\u003csub\u003e4\u003c/sub\u003e x 7 H\u003csub\u003e2\u003c/sub\u003eO, 0.25 ml/L micronutrients (2.86 g/L H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 1.81 g/L MnCl\u003csub\u003e2\u003c/sub\u003e x 4H\u003csub\u003e2\u003c/sub\u003eO, 0.22 g/L ZnSO\u003csub\u003e4\u003c/sub\u003e x 7H\u003csub\u003e2\u003c/sub\u003eO, 0.08 g/L CuSO\u003csub\u003e4\u003c/sub\u003e x 5H\u003csub\u003e2\u003c/sub\u003eO, 0.02 g/L NaMoO\u003csub\u003e4\u003c/sub\u003e x H\u003csub\u003e2\u003c/sub\u003eO g/L), 0.25 ml/L Fe-EDTA (10.4 g/L EDTA, 7.8 g/L FeSO\u003csub\u003e4\u003c/sub\u003e x 7 H\u003csub\u003e2\u003c/sub\u003eO, 56.1 g/L KOH g/L) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] every two weeks. Substrate chemical composition was determined and checked by accredited analytical laboratory of the Department for Plant Nutrition, Division of Agroecology, Faculty of Agriculture, University of Zagreb, Svetošimunska cesta 25, 10000 Zagreb, Croatia. Chemical composition analyses revealed subtle changes in the macronutrient composition between the different substrate lots. Those differences affected reproducible plant growth and development. To offset those differences, we supplemented both substrate types with chemically well-defined Hoaglands nutrient solution. Together with that, reproducible growth conditions (light, temperature, CO\u003csub\u003e2\u003c/sub\u003e level), enabled detection of the subtle differences in biochemical processes, morphology, and photosynthesis of different TROL mutant lines, grown under different substrate moisture conditions, which was the goal of this research.\u003c/p\u003e \u003cp\u003eChemicals\u003c/p\u003e \u003cp\u003eFluorescent probes dyhidroethidium (DHE) and Singlet oxygen sensor green (SOSG) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and Spy-LHP from Dojindo Molecular Technologies Inc. (Rockville, MD, USA).\u003c/p\u003e \u003cp\u003eSample preparation\u003c/p\u003e \u003cp\u003ePlants of each line were put in the dark 2 h before experiment. The whole leaf was cut out from the plant and rinsed in 50 mM HEPES buffer (pH 7.5) and after that incubated 30 minutes in desired fluorescent probe (250 \u0026micro;M DHE, 50 \u0026micro;M Spy-LHP, or 50 \u0026micro;M SOSG), while control samples were incubated in 50 mM HEPES buffer. After incubation, the leaf was again rinsed in 50 mM HEPES buffer and transferred on a glass slide in the drop of 50 mM HEPES buffer and covered with cover glass. Protocol was modified according to Prasad \u003cem\u003eet al\u003c/em\u003e. 2020.\u003c/p\u003e \u003cp\u003eConfocal microscopy\u003c/p\u003e \u003cp\u003eThe amount and arrangement of ROS within leaves were visualized using Confocal microscope Leica TCS SP8 Laser Scanning Confocal Microscope (Leica Microsystems GmbH, Germany) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The excitation for SOSG was 504 nm, and emission 520\u0026ndash;560 nm, for the Spy-LHP excitation was 524 nm, emission 535\u0026ndash;580 nm, and for the DHE excitation was 480 nm, emission at 560\u0026ndash;610 nm. Chloroplast autofluorescence was determined at 650\u0026ndash;750 nm for all fluorescent probes. In the beginning of each experiment, proper laser intensity was set by using control samples in which leaves were incubated in 50 mM HEPES buffer without fluorescent probes. All confocal microscopy experiments include images of chloroplasts autofluorescence (red field signal), bright field images (gray field signal), and detected ROS with fluorescent probes (green field signal). For each fluorescent probe and control, 3 visual fields in different plane levels were recorded for each of three leaf replicas. The experiment was carried out \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eImage analysis\u003c/p\u003e \u003cp\u003eImages obtained with confocal laser scanning microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) were analyzed by using ImageJ (\u003cem\u003eFiji\u003c/em\u003e) software. Green field fluorescent signal strength of fluorescent probes reactions with ROS was measured in total counts. The average value of control images was subtracted from the average value of belonging reaction groups to evaluate signal intensity. Ten measurements of each fluorescent probe and control sample for every growth condition and plant lines were used on the three leaf replicas.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis was carried out with GraphPad Prism (v.9.0.0.121). Comparison of groups were performed by using one-way ANOVA with post-hoc analysis Dunnett\u0026acute;s test. The statistical significance is indicated as * at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0,05 confidence level.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eIn this work we examined the propagation and the distribution of the three common ROS, superoxide anion, hydrogen peroxide, and singlet oxygen in the leaves of the model plant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (L.) Heyn. ecotype Columbia (Col-0) (WT), and the two mutant lines TROL KO and TROL ΔRHO. We examined the influence of different soil moisture conditions on ROS accumulation at the level of whole leaves. We further tested our hypothesis that arises from the previous work in our laboratory carried out by Vojta \u003cem\u003eet al.\u003c/em\u003e 2015 and Vojta \u003cem\u003eet al.\u003c/em\u003e 2023., which postulates that alternative pathways of electron partitioning (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) under different environmental conditions depend on the TROL-FNR complex dynamical formation. We investigated ROS distribution under the influence of TROL not just on the chloroplast level but on the whole leaves tissue. In this work we used confocal laser scanning microcopy in combination with specific fluorescent probes for visualization of three different ROS species [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eROS are usual byproducts generated in the various metabolic processes. Formation of ROS is caused by partial reduction or energy transfer to O\u003csub\u003e2\u003c/sub\u003e. The major site of O\u003csub\u003e2\u003c/sub\u003e˙ˉ propagation by the excess electron spillage to O\u003csub\u003e2\u003c/sub\u003e, is the PSI in whose vicinity on the thylakoid membrane TROL is positioned (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Such proximity of O\u003csub\u003e2\u003c/sub\u003e˙ˉ propagation site and the TROL-FNR complex further points toward the protective role hypothesis. In addition to chloroplasts, ROS are generated in various cellular sites. These include mitochondria, particularly in the respiratory ETC under stress or when ATP synthesis is impaired, peroxisomes, especially under high light (HL) intensity when photorespiration is elevated, NADPH oxidases in the plasma membrane, peroxidases in the cell wall, xanthine oxidase reaction sites, and autoxidation of redox compounds under stress conditions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our experimental setup for detection of O\u003csub\u003e2\u003c/sub\u003e˙ˉ we used redox sensitive fluorescent probe DHE. Under the conditions of drought stress there was a significant reduction in the amount of O\u003csub\u003e2\u003c/sub\u003e˙ˉ in the TROL KO mutant line in comparison with the WT. Although the amount of O\u003csub\u003e2\u003c/sub\u003e˙ˉ is also reduced for the ΔRHO mutant line under drought stress conditions in comparison with the WT, the difference was not significant and not pronounced as is in the case of TROL KO mutant line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results confirm protective role and detoxification activity dependent on the TROL-FNR complex dynamical binding that is activated under the stress conditions when alternative electron sink is preferred over the LEF and it implies positive influence on whole leaves level (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Under normal moisture conditions both mutant lines show unsignificant increase of O\u003csub\u003e2\u003c/sub\u003e˙ˉ propagation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results indicate that ΔRHO mutation has no effect on O\u003csub\u003e2\u003c/sub\u003e˙ˉ scavenging, at least not on such high level as in TROL KO line. Results also indicate that under unstressed conditions there is no increased scavenging activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn plants some ROS, especially H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, serve as secondary messengers in various signaling and gene expression pathways. In chloroplasts, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is mostly generated on the PSII at low rate, in the Mehler reaction, photorespiration, and as a product of O\u003csub\u003e2\u003c/sub\u003e˙ˉ reduction, with the latter also occurring in various cellular compartments. Other sources of H₂O₂ production include mitochondria during aerobic respiration, peroxisomes during photorespiration and β-oxidation, oxidase enzymes in the cytoplasm, peroxidases in the cell wall, and auxin oxidation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. SOD is one of the first and most abundant defense antioxidant mechanisms against O\u003csub\u003e2\u003c/sub\u003e˙ˉ. Since SOD produces great amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e from superoxide (can be seen from the scale difference on Y axis of Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), the protection against peroxide in cells is very high and efficient. According to that, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows efficient scavenging of peroxide under drought conditions. In this work, propagation and distribution of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was monitored by using highly selective fluorescent probe Spy-LHP. Under the drought stress conditions, ΔRHO showed significantly less amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in comparison with the WT. On the other hand, the TROL KO mutant line did not show significantly elevated amounts of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results indicate the possible involvement of the RHO domain in transmembrane redox signaling from lumen to stroma and consequent regulation of TROL-FNR complex dynamical binding and TROL complex formation with tAPX, antioxidant enzyme that scavenges H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, all with a goal to form alternative electron sinks that could protect cells under the stress conditions. We also observed that the TROL KO line is not as efficient in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detoxification as it is in O\u003csub\u003e2\u003c/sub\u003e˙ˉ scavenging. Under the conditions of normal moisture, compared with the WT, in ΔRHO mutant line H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was equally accumulated, while in TROL KO line was elevated. However, the increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was not statistically significant in this case (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This again indicates that alternative electron sinks are activated under the conditions of stress when the protection against ROS is needed.\u003c/p\u003e \u003cp\u003eThe formation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, which represents the first excited electronic state of O\u003csub\u003e2\u003c/sub\u003e, is mostly located in the vicinity of the PSII where interaction of O\u003csub\u003e2\u003c/sub\u003e with triplet chlorophyll occurs. Additionally, by triggering lipid peroxidation, \u0026sup1;O₂ can amplify its own production. Certain peroxidases and lipoxygenases can also generate \u0026sup1;O₂ from fatty acids. And these reactions are not related only to chloroplasts [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e is specific ROS because it is not generated by electron transfer to O\u003csub\u003e2\u003c/sub\u003e. The two main mechanisms for \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e detoxification are physical and chemical quenching. Highly selective fluorescent probe SOSG was used for \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e signal strength detection. Under the conditions of normal growth substrate moisture, the amount of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e was significantly reduced in ΔRHO mutant line in comparison with the WT. Unsignificant changes in the amount of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e could be observed in the TROL KO line. Under the drought stress conditions, both mutant lines showed equally elevated, yet unsignificant \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Knowledge gathered so far together with the results of this work indicates that formation of TROL-FNR complex is triggered in situations when LEF and NADP\u003csup\u003e+\u003c/sup\u003e synthesis is preferred, while when stress conditions are present, releasing of FNR from TROL enables various alternative electron sinks and detoxification mechanisms in order to maintain whole plant redox homeostasis.\u003c/p\u003e"},{"header":"DISCUSION","content":"\u003cp\u003ePlants are confronting many severe environmental conditions, but they are successful in overcoming them as they have evolved numerous short- and long-term adaptation and acclimation mechanisms. Dynamical binding and positioning of the photosynthetic complexes can change depending on growth conditions, especially stress conditions like drought. Drought limits electron donation by reducing the photolysis of water, which impairs electron flow through the ETC. This leads to ETC over-reduction, increasing excitation pressure and electron leakage. To dissipate excess electrons, photorespiration and the water-water cycle become crucial, CEF and antioxidant enzymes are upregulated to maintain redox balance [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. One of the mechanisms presented by our group is that soluble FNR handles oxidative stress better than when docked to TROL, consequently enabling TROL KO plants to propagate lower amounts of ROS. Different TROL mutations affect TROL-FNR binding dynamic, and some of the mutations cause more efficient ROS scavenging. TROL-FNR pair regulates electron transport and partitioning dynamic in accord with the energy requirements, particularly NADPH synthesis, and perhaps ATP. Since LET products are used in various metabolic reactions, TROL-FNR regulation of electron transport consequently influences whole organism. It is not the TROL that is directly involved in ROS scavenging and energy production, but the flavoenzyme FNR. TROL mutant lines are involved in the FNR binding and release from the vicinity of the photosystem I. It is also to be expected that the RHO domain of TROL has influence on electron partitioning by possible transmembrane redox signal transduction from lumen to stroma with consequent influence on the dynamical binding of the TROL-FNR complex. It appears that scavenging reactions and alternative electron partitioning, driven by changes in the dynamic binding of the TROL-FNR pair, influence ROS metabolism and the maintenance of redox homeostasis at the whole plant level.\u003c/p\u003e \u003cp\u003eFirstly, we established that O\u003csub\u003e2\u003c/sub\u003e˙ˉ formation in TROL KO mutant line was reduced, while generation of the other ROS was mostly enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). There is a significant reduction in the amount of O\u003csub\u003e2\u003c/sub\u003e˙ˉ in TROL KO mutant line in comparison with the WT under the drought stress. The same results were obtained in the ΔRHO mutant line, but on the less pronounced scale. Under the normal humidity, in both mutant lines, the amount of O\u003csub\u003e2\u003c/sub\u003e˙ˉ was elevated in comparison to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). When growth conditions were compared, less O\u003csub\u003e2\u003c/sub\u003e˙ˉ could be detected in TROL KO and ΔRHO under drought stress, in comparison with the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating the protective role of FNR when TROL is not present. Alternative electron sinks are activated and by successful scavenging of propagated O\u003csub\u003e2\u003c/sub\u003e˙ˉ (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) there is less spillage of electrons to O\u003csub\u003e2\u003c/sub\u003e, as earlier proposed [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It is important to mention that under growth-light conditions (GL), the electron transport rate is not lower in KO. In fact, under GL, TROL KO plants are growing more successfully than the WT, which is consistent with earlier PAM measurements that showed that electron transport rate is not lower in KO [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. TROL-FNR binding becomes rate limited in linear electron transport rate at quite high photosynthetically active radiation (PAR). Enzyme FNR is involved in other electron transport reactions besides linear photosynthetic electron transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In addition, it takes 2 reduced ferredoxin molecules to generate 1 NADPH. TROL docks FNR in the vicinity of photosystem I, so reduced ferredoxin can efficiently transfer electrons to NADP\u003csup\u003e+\u003c/sup\u003e. When TROL is not present, FNR is not efficiently docked to the thylakoid membranes and electrons are distributed in alternative pathways, which take electrons more rapidly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This however happens at very high light intensities, or when plants are exposed to more than one stressor (combined stress). Additionally, the reduction of the negative stress effects enabled by the TROL-FNR pair in the PETC seems to be so significant that it positively influences the maintenance of a balanced redox state across the entire cell. With these results in mind, we wanted to investigate whether other ROS are also susceptible to scavenging influenced by the lack of TROL. Under drought stress conditions TROL KO shows elevated amounts of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in comparison with the WT. Contrary to that, ΔRHO shows significantly less amount (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This result indicates reduced ROS scavenging efficiency, other than O\u003csub\u003e2\u003c/sub\u003e˙ˉ in TROL KO plants, while other defensive mechanisms against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e could be present and more expressed in ΔRHO mutant line. RHO domain was proposed to have a role in transmembrane redox signal transduction that affects dynamical binding of TROL and FNR and consequent flow of electrons [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, as proposed in Vojta \u003cem\u003eet al.\u003c/em\u003e 2023, TROL most likely forms a complex with the tAPX that is an important antioxidant enzyme in the process of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detoxification. The existence of tAPX isoform is proven in the vicinity of PSI which is the place of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e˙ˉ generation. Spectrofotometrical measurement also showed increased APX activity in the TROL KO and ΔRHO mutant lines under drought conditions in comparison with WT (Dumančić and Fulgosi, unpublished data). Under normal growth substrate moisture, ΔRHO mutant line showed equal amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as the WT, while TROL KO mutant line showed unsignificant elevation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eContrary to the TROL KO, Arabidopsis plants that overexpress TROL (TROL OX) failed to successfully cope with the ROS propagation, owing to the fact that more FNR was bound to the membrane in this plants [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Results presented in this work are in accordance with mentioned results and also with the previously demonstrated successful O\u003csub\u003e2\u003c/sub\u003e˙ˉ scavenging in TROL KO plants even when they were treated with methyl-viologen (herbicide that strongly induces formation of O\u003csub\u003e2\u003c/sub\u003e˙ˉ), as shown by the EPR [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Considering all mentioned findings, the proposal that TROL-FNR dynamic interaction has a significant role in maintaining redox homeostasis is even more strengthened (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The positioning of TROL on the thylakoid membranes plays crucial role in controlling redox balance in chloroplasts, which is expected, as chloroplasts are the primary sites of ROS generation in plants. However, its significance extends beyond this. By regulating ROS equilibrium and electron partitioning, TROL prevents redox imbalance affecting areas beyond the chloroplasts. This is achieved by controlling electron flow and partitioning, thereby reducing ROS formation and preventing subsequent chain reactions that lead to ROS multiplication and intercellular ROS transfer.\u003c/p\u003e \u003cp\u003eOxidative stress could regulate release of FNR form thylakoids [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and this regulation could be achieved through the RHO domain in combination with reduced PQ by redox sensing from lumen trough thylakoid membrane and influencing TROL-FNR dynamic binding on the stromal side trough conformational changes induced in ITEP domain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Shown in this research, ΔRHO mutant line exhibits a potential to efficiently remove H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. ΔRHO mutation might also influence TROL-FNR complex interaction with the tAPX. APX is antioxidant enzyme involved in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging and its thylakoidal (tAPX) form is positioned in the vicinity of PSI, indicating possible interaction and cooperation with TROL [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Exact mechanisms of ROS scavenging influenced by the TROL-FNR complex dynamics remain to be determined. According to the results of this research, neither of tested mutant lines possesses the capability of detoxification of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e under drought stress conditions. Further research should be carried out to answer these questions.\u003c/p\u003e "},{"header":"CONCLUSION","content":"\u003cp\u003eWhen considering improvement of plant/crop stress tolerance and protection, TROL-FNR complex is inevitable to take into consideration because of various dynamical interactions through which it can regulate electron transport in different pathways of PETC and ROS scavenging in chloroplast and beyond. The exact mechanisms of ROS detoxification pathways that involve TROL-FNR complex remain to be elucidated. To conclude, this research brings additional evidence for the involvement of TROL-FNR interaction in the ROS-protection under stress conditions. Once more, we showed that dynamical binding of FNR to TROL significantly participates in the maintenance of redox equilibrium by activating various detoxication mechanisms against ROS (precisely O\u003csub\u003e2\u003c/sub\u003e˙ˉ and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) formed under the, in this case, drought stress conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.D. grew plant material, prepared specimens for confocal microscopy, collected and analyzed data, wrote the initial drafts. L.V. advised on experimental setup, wrote and revised the manuscript. H.F. supervised the research, participated in planning the experiments, writing process, and revised the manuscript. H.F. led the project and raised the project funds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Croatian Science Foundation (HRZZ) Grants: IP-2020-02-8590 and DOK-2021-02-2018 to HF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available in the \u003cem\u003eFigshare\u003c/em\u003e repository \u0026nbsp;https://doi.org/10.6084/m9.figshare.25736829.v3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAllen JF. 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Antioxidants. 2021;10(6):935.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGolding AJ, Johnson GN. Down-regulation of linear and activation of cyclic electron transport during drought. Planta. 2023;218(1):107\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiao M, Hong C, Jiao Y, Hou S, Gao H. Impacts of drought on photosynthesis in major food crops and the related mechanisms of plant responses to drought. Plants. 2024;13(13):1808.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Abiotic stress, Arabidopsis, ferredoxin:NADP+ oxidoreductase, linear electron transport, redox homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-6220323/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6220323/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThylakoid rhodanase-like protein (TROL) is located in thylakoid membranes like a hinge between the protein complexes of photosynthetic electron transport chain (PETC) and the nicotinamide adenine dinucleotide phosphate (NADPH) synthesis. TROL is the docking site for the flavoenzyme ferredoxin-NADP\u003csup\u003e+\u003c/sup\u003e oxidoreductase (FNR). As proposed in our previous researches, TROL-FNR complex plays an important role in maintaining redox equilibrium in chloroplasts, and even in entire plant cells. To further test this hypothesis and confirm our previous results, we monitored ROS propagation in the leaves of \u003cem\u003eArabidopsis\u003c/em\u003e wild type (WT), TROL knock-out (KO), and TROL ΔRHO mutant pants \u003cem\u003ein situ\u003c/em\u003e, by using confocal laser scanning microscopy with the specific fluorescent probes for the three different ROS: O\u003csub\u003e2\u003c/sub\u003e˙ˉ, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. Plants were grown under the conditions of normal substrate moisture and under the drought stress conditions.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUnder the drought stress conditions, TROL KO line showed successful detoxification of O\u003csub\u003e2\u003c/sub\u003e˙ˉ, while ΔRHO line showed successful detoxification of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis research once again proves the involvement of the dynamical TROL-FNR complex formation in redox equilibrium maintenance and the distribution of energy.\u003c/p\u003e","manuscriptTitle":"Confocal laser scanning microscopy of ROS in Arabidopsis thaliana (L.) Heynh. TROL-FNR mutants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 07:48:09","doi":"10.21203/rs.3.rs-6220323/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53fe2e07-bc47-4fb7-b58b-8a5067411453","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-21T12:23:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-24 07:48:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6220323","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6220323","identity":"rs-6220323","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}