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A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (1O2*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of 1O2* in photodamage and cell signaling, few studies directly correlate 1O2* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of 1O2* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of 1O2* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of 1O2* in chlorophyll-containing photosynthetic membranes. We find that the apparent 1O2* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of 1O2* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of 1O2* during high light. Upon saturation of NPQ, the concentration of 1O2* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).

Energy status and nutrients regulate photosynthetic protein expression. The unicellular green alga Chromochloris zofingiensis switches off photosynthesis in the presence of exogenous glucose (+Glc) in a process that depends on hexokinase (HXK1). Here, we show that this response requires that cells lack sufficient iron (-Fe). Cells grown in -Fe+Glc accumulate triacylglycerol (TAG) while losing photosynthesis and thylakoid membranes. However, cells with an iron supplement (+Fe+Glc) maintain photosynthesis and thylakoids while still accumulating TAG. Proteomic analysis shows that known photosynthetic proteins are most depleted in heterotrophy, alongside hundreds of uncharacterized, conserved proteins. Photosynthesis repression is associated with enzyme and transporter regulation that redirects iron resources to (a) respiratory instead of photosynthetic complexes and (b) a ferredoxin-dependent desaturase pathway supporting TAG accumulation rather than thylakoid lipid synthesis. Combining insights from diverse organisms from green algae to vascular plants, we show how iron and trophic constraints on metabolism aid gene discovery for photosynthesis and biofuel production.

Cyanobacteria and chloroplasts of algae and plants harbor specialized thylakoid membranes (TMs) that convert sunlight into chemical energy. These membranes house PSII and I, the vital protein-pigment complexes that drive oxygenic photosynthesis. In the course of their evolution, TMs have diversified in structure. However, the core machinery for photosynthetic electron transport remained largely unchanged, with adaptations occurring primarily in the light-harvesting antenna systems. Whereas TMs in cyanobacteria are relatively simple, they become more complex in algae and plants. The chloroplasts of vascular plants contain intricate networks of stacked grana and unstacked stroma thylakoids. This review provides an in-depth view of TM architectures in phototrophs and the determinants that shape their forms, as well as presenting recent insights into the spatial organization of their biogenesis and maintenance. Its overall goal is to define the underlying principles that have guided the evolution of these bioenergetic membranes. [ABSTRACT FROM AUTHOR]

Oxygenic photosynthesis evolved billions of years ago, becoming Earth's main source of biologically available carbon and atmospheric oxygen. Since then, phototrophic organisms have diversified from prokaryotic cyanobacteria into several distinct clades of eukaryotic algae and plants through endosymbiosis events. This diversity can be seen in the thylakoid membranes, complex networks of lipids, proteins, and pigments that perform the light-dependent reactions of photosynthesis. In this review, we highlight the structural diversity of thylakoids, following the evolutionary history of phototrophic species. We begin with a molecular inventory of different thylakoid components and then illustrate how these building blocks are integrated to form membrane networks with diverse architectures. We conclude with an outlook on understanding how thylakoids remodel their architecture and molecular organization during dynamic processes such as biogenesis, repair, and environmental adaptation. [ABSTRACT FROM AUTHOR]

An unknown filamentous cyanobacterium was isolated from the Yuqiao Reservoir, a eutrophic drinking water reservoir in Tianjin city, northern China. The cyanobacterium shares some morphological similarities with most Leptolyngbya-like species, having thin trichomes that can facultatively produce sheaths, with one trichome per sheath, and contain peripheral thylakoids. Based on a combination of morphological, molecular, ultrastructural and ecological evidence, we propose the description of a new cyanobacterial taxon, Cyanoleptonema yuqiaoensis gen. & sp. nov. which expands our understanding of cyanobacterial diversity. Phylogenetic analyses based on 16S rRNA gene sequences placed this freshwater strain into a well-supported clade closely related to the marine or saline genera Sodalinema and Baaleninema. Genetic identity between Cyanoleptonema and other cyanobacteria, including Sodalinema and Baaleninema, was found to be less than 95% in 16S rRNA. The genus was further distinguished by morphological and ecological characteristics, including cell size and the ratio between cell length and width. [ABSTRACT FROM AUTHOR]

Diclofenac, often detected in environmental samples, poses a potential hazard to the aquatic environment. The present study aimed to understand the effect of this drug on photosynthetic apparatus, which is a little-known aspect of its phytotoxicity. Chloroplasts and thylakoids isolated from spinach (Spinacia oleracea) were used for this study and treated with various concentrations of diclofenac (from 125 to 4000 μM). The parameters of chlorophyll a fluorescence (the OJIP test) as measurements for both the intact chloroplasts and the thylakoid membranes revealed that isolated thylakoids showed greater sensitivity to the drug than chloroplasts. The relatively high concentration of diclofenac that is required to inhibit chloroplast and thylakoid functions suggests a narcotic effect of that drug on photosynthetic membranes, rather than a specific interaction with a particular element of the electron transport chain. Using confocal microscopy, we confirmed the degradation of the chloroplast structure after DCF treatment, which has not been previously reported in the literature. In conclusion, it can be assumed that diclofenac's action originated from a non-specific interaction with photosynthetic membranes, leading to the disruption in the function of the electron transport chain. This, in turn, decreases the efficiency of photosynthesis, transforming part of the PSII reaction centers into heat sinks and enhancing non-photochemical energy dissipation. [ABSTRACT FROM AUTHOR]

Chlorophyll (Chl) plays a crucial role in photosynthesis, functioning as a photosensitizer. As an integral component of this process, energy absorbed by this pigment is partly emitted as red fluorescence. This signal can be readily imaged by fluorescence microscopy and provides a visualization of photosynthetic activity. However, due to limited resolution, signals cannot be assigned to specific subcellular/organellar membrane structures. By correlating fluorescence micrographs with transmission electron microscopy, researchers can identify sub‐cellular compartments and membranes, enabling the monitoring of Chl distribution within thylakoid membrane substructures in cyanobacteria, algae, and higher plant single cells. Here, we describe a simple and effective protocol for correlative light‐electron microscopy (CLEM) based on the autofluorescence of Chl and demonstrate its application to selected photosynthetic model organisms. Our findings illustrate the potential of this technique to identify areas of high Chl concentration and photochemical activity, such as grana regions in vascular plants, by mapping stacked thylakoids. [ABSTRACT FROM AUTHOR]

"Drawing inspiration from nature" offers a wealth of creative possibilities for designing cutting‐edge materials with improved properties and performance. Nature‐inspired thylakoid‐based nanoarchitectures, seamlessly integrate the inherent structures and functions of natural components with the diverse and controllable characteristics of nanotechnology. These innovative biomaterials have garnered significant attention for their potential in various biomedical applications. Thylakoids possess fundamental traits such as light harvesting, oxygen evolution, and photosynthesis. Through the integration of artificially fabricated nanostructures with distinct physical and chemical properties, novel photosynthetic nanoarchitectures can be catalytically generated, offering versatile functionalities for diverse biomedical applications. In this article, an overview of the properties and extraction methods of thylakoids are provided. Additionally, the recent advancements in the design, preparation, functions, and biomedical applications of a range of thylakoid‐based photosynthetic nanoarchitectures are reviewed. Finally, the foreseeable challenges and future prospects in this field is discussed. [ABSTRACT FROM AUTHOR]

SUMMARY: The determination of physiological tolerance ranges of photosynthetic species and of the biochemical mechanisms underneath are fundamental to identify target processes and metabolites that will inspire enhanced plant management and production for the future. In this context, the terrestrial green algae within the genus Prasiola represent ideal models due to their success in harsh environments (polar tundras) and their extraordinary ecological plasticity. Here we focus on the outstanding Prasiola antarctica and compare two natural populations living in very contrasting microenvironments in Antarctica: the dry sandy substrate of a beach and the rocky bed of an ephemeral freshwater stream. Specifically, we assessed their photosynthetic performance at different temperatures, reporting for the first time gnsd values in algae and changes in thylakoid metabolites in response to extreme desiccation. Stream population showed lower α‐tocopherol content and thicker cell walls and thus, lower gnsd and photosynthesis. Both populations had high temperatures for optimal photosynthesis (around +20°C) and strong constitutive tolerance to freezing and desiccation. This tolerance seems to be related to the high constitutive levels of xanthophylls and of the cylindrical lipids di‐ and tri‐galactosyldiacylglycerol in thylakoids, very likely related to the effective protection and stability of membranes. Overall, P. antarctica shows a complex battery of constitutive and plastic protective mechanisms that enable it to thrive under harsh conditions and to acclimate to very contrasting microenvironments, respectively. Some of these anatomical and biochemical adaptations may partially limit photosynthesis, but this has a great potential to rise in a context of increasing temperature. Significance Statement: By using Prasiola antarctica as a model system, this work highlights the relevance of constitutive protection mechanisms involved on chloroplast functioning in harsh environments, especially the high proportion of cylindrical galactolipids and xanthophylls, and also reports the first measured gnsd ('gm') values in algae. This species shows a remarkable plasticity to ensure its success in contrasting terrestrial and fresh‐water microenvironments due to anatomical and biochemical changes, such as engrossed cell walls and accumulation of tocopherol. [ABSTRACT FROM AUTHOR]

To increase the understanding of simple thin filamentous cyanobacteria in harsh environmental areas, we previously isolated and identified four strains (XN101, XN102, GS121, NX122) from desert soils and hot spring in China. As a result, two new Oculatellacean genera of these four strains, Gansulinema gen. nov. and Komarkovaeasiopsis gen. nov., are described based on a polyphasic approach. The ultrastructure of these strains showed a similar arrangement of peripheral thylakoids with three to four parallel layers, indicating that they belonged to the orders Nodosilineales, Oculatellales, or Leptolyngbyales. In the 16S rRNA gene phylogeny, two sequences of the Gansulinema strains and the two sequences of the Komarkovaeasiopsis strains formed two independent and robust clusters, within the order Oculatellales. The 16S rRNA gene sequences of strains of Komarkovaeasiopsis and Gansulinema showed low identity to each other (≤93.2%) and to other sequences of the Oculatellacean genera (≤94.5% and ≤93.3%, respectively). Furthermore, the 16S–23S internal transcribed spacer rRNA region secondary structures of strains of Komarkovaeasiopsis and Gansulinema were not consistent with all existing descriptions of Oculatellacean taxa. These data suggest that cyanobacterial communities are rich sources of new taxa in under‐exploited areas, such as desert soils and hot spring in China. [ABSTRACT FROM AUTHOR]

Nanohybrid photosystems have advantages in converting solar energy into electricity, while natural photosystems based solar‐powered energy‐storage device is still under developed. Here, we fabricate a new kind of photo‐rechargeable zinc‐ion hybrid capacitor (ZHC) benefiting from light‐harvesting carbon dots (CDs) and natural thylakoids for realizing solar energy harvesting and storage simultaneously. Under solar light irradiation, the embedded CDs in thylakoids (CDs/Thy) can convert the less absorbed green light into highly absorbed red light for thylakoids, besides, Förster resonance energy transfer (FRET) between CDs and Thy also occurs, which facilitates the photoelectrons generation during thylakoids photosynthesis, thereby resulting in 6‐fold photocurrent output in CDs/Thy hybrid photosystem, compared to pristine thylakoids. Using CDs/Thy as the photocathode in ZHCs, the photonic hybrid capacitor shows photoelectric conversion and storage features. CDs can improve the photo‐charging voltage response of ZHCs to ≈1.2 V with a remarkable capacitance enhancement of 144 % under solar light. This study provides a promising strategy for designing plant‐based photonic and electric device for solar energy harvesting and storage. [ABSTRACT FROM AUTHOR]

The spatial separation of photosystems I and II (PSI and PSII) is thought to be essential for efficient photosynthesis by maintaining a balanced flow of excitation energy between them. Unlike the thylakoid membranes of plant chloroplasts, cyanobacterial thylakoids do not form tightly appressed grana stacks that enforce strict lateral separation. The coexistence of the two photosystems provides a ground for spillover—excitation energy transfer from PSII to PSI. Spillover has been considered as a pathway of energy transfer from the phycobilisomes to PSI and may also play a role in state transitions as means to avoid overexcitation of PSII. Here, we demonstrate a significant degree of energy spillover from PSII to PSI in reconstituted membranes and isolated thylakoid membranes of Thermosynechococcus (Thermostichus) vulcanus and Synechocystis sp. PCC 6803 by steady-state and time-resolved fluorescence spectroscopy. The quantum yield of spillover in these systems was determined to be up to 40%. Spillover was also found in intact cells but to a considerably lower degree (20%) than in isolated thylakoid membranes. The findings support a model of coexistence of laterally separated microdomains of PSI and PSII in the cyanobacterial cells as well as domains where the two photosystems are energetically connected. The methodology presented here can be applied to probe spillover in other photosynthetic organisms. [ABSTRACT FROM AUTHOR]

Diclofenac, often detected in environmental samples, poses a potential hazard to the aquatic environment. The present study aimed to understand the effect of this drug on photosynthetic apparatus, which is a little-known aspect of its phytotoxicity. Chloroplasts and thylakoids isolated from spinach (Spinacia oleracea) were used for this study and treated with various concentrations of diclofenac (from 125 to 4000 μM). The parameters of chlorophyll a fluorescence (the OJIP test) as measurements for both the intact chloroplasts and the thylakoid membranes revealed that isolated thylakoids showed greater sensitivity to the drug than chloroplasts. The relatively high concentration of diclofenac that is required to inhibit chloroplast and thylakoid functions suggests a narcotic effect of that drug on photosynthetic membranes, rather than a specific interaction with a particular element of the electron transport chain. Using confocal microscopy, we confirmed the degradation of the chloroplast structure after DCF treatment, which has not been previously reported in the literature. In conclusion, it can be assumed that diclofenac’s action originated from a non-specific interaction with photosynthetic membranes, leading to the disruption in the function of the electron transport chain. This, in turn, decreases the efficiency of photosynthesis, transforming part of the PSII reaction centers into heat sinks and enhancing non-photochemical energy dissipation.

We fabricated a photo-driven bioanode on laser-induced graphene (LIG) using a facile one-step laser-scribed technique. Nb 4 C 3 T x MXene was decorated on the LIG surface to induce a high surface area for immobilizing the photocatalyst. The thylakoid membrane extracted from Spinacia oleracea utilizes its intrinsic photosynthetic and catalytic capability for efficient energy conversion. A platinum-based gas diffusion electrode is used as the cathode material, allowing for charge transfer and oxygen reduction to produce water. The bioelectrochemical activities of the as-fabricated thylakoid-based biofuel cells were investigated. Upon illumination, the assembled biofuel cell exhibited an open circuit voltage of 0.45 V, a maximum photocurrent density of 68.69 µA/cm2, and a power density of 7.24 µW/cm2. The unique features of thylakoid membranes, Nb 4 C 3 T x MXene, and the LIG substrate play a critical role in producing high-performance photo-biofuel cells. Our approach presents a promising strategy for harnessing the biochemical energy of plants to generate bioelectricity. [ABSTRACT FROM AUTHOR]

A recent study by Catherine Demoulin, Yannick Lara, Alexandre Lambion, and Emmanuelle Javaux has provided evidence for the presence of thylakoids, membrane-bound compartments where photosynthesis takes place, in microfossils that may be cyanobacteria. The researchers used transmission electron microscopy to examine fossils from Australia, Canada, and the Democratic Republic of the Congo. The discovery of preserved thylakoids within these microfossils establishes a minimum age of about 1.75 billion years ago for the divergence of cyanobacteria with and without thylakoids. This finding contributes to our understanding of the history of photosynthesis and the organisms that played a role in the oxygenation of the atmosphere and oceans. [Extracted from the article]

Background: The phosphorylation of the Light-Harvesting Complex of photosystem II (LHCII) driven by STATE TRANSITION 7 (STN7) kinase is a part of one of the crucial regulatory mechanisms of photosynthetic light reactions operating in fluctuating environmental conditions, light in particular. There are evidenced that STN7 can also be activated without light as well as in dark-chilling conditions. However, the biochemical mechanism standing behind this complex metabolic pathway has not been deciphered yet., Results: In this work, we showed that dark-chilling induces light-independent LHCII phosphorylation in runner bean (Phaseolus coccineus L.). In dark-chilling conditions, we registered an increased reduction of the PQ pool which led to activation of STN7 kinase, subsequent LHCII phosphorylation, and possible LHCII relocation inside the thylakoid membrane. We also presented the formation of a complex composed of phosphorylated LHCII and photosystem I typically formed upon light-induced phosphorylation. Moreover, we indicated that the observed steps were preceded by the activation of the oxidative pentose phosphate pathway (OPPP) enzymes and starch accumulation., Conclusions: Our results suggest a direct connection between photosynthetic complexes reorganization and dark-chilling-induced activation of the thioredoxin system. The proposed possible pathway starts from the activation of OPPP enzymes and further NADPH-dependent thioredoxin reductase C (NTRC) activation. In the next steps, NTRC simultaneously activates ADP-glucose pyrophosphorylase and thylakoid membrane-located NAD(P)H dehydrogenase-like complex. These results in starch synthesis and electron transfer to the plastoquinone (PQ) pool, respectively. Reduced PQ pool activates STN7 kinase which phosphorylates LHCII. In this work, we present a new perspective on the mechanisms involving photosynthetic complexes while efficiently operating in the darkness. Although we describe the studied pathway in detail, taking into account also the time course of the following steps, the biological significance of this phenomenon remains puzzling., (© 2024. The Author(s).)

The prediction of the cheese yield (%CY) traits for curd, solids, and retained water and the amount of fat, protein, solids, and energy recovered from the milk into the curd (%REC) by Bayesian models, using Fourier-transform infrared spectroscopy (FTIR), can be of significant economic interest to the dairy industry and can contribute to the improvement of the cheese process efficiency. The yields give a quantitative measure of the ratio between weights of the input and output of the process, whereas the nutrient recovery allows to assess the quantitative transfer of a component from milk to cheese (expressed in % of the initial weight). The aims of this study were: (1) to investigate the feasibility of using bulk milk spectra to predict %CY and %REC traits, and (2) to quantify the effect of the dairy industry and the contribution of single-spectrum wavelengths on the prediction accuracy of these traits using vat milk samples destined to the production of Grana Padano Protected Designation of Origin cheese. Information from 72 cheesemaking days (in total, 216 vats) from 3 dairy industries were collected. For each vat, the milk was weighed and analyzed for composition (total solids [TS], lactose, protein, and fat). After 48 h from cheesemaking, each cheese was weighed, and the resulting whey was sampled for composition as well (TS, lactose, protein, and fat). Two spectra from each milk sample were collected in the range between 5,011 and 925 cm -1 and averaged before the data analysis. The calibration models were developed via a Bayesian approach by using the BGLR (Bayesian Generalized Linear Regression) package of R software. The performance of the models was assessed by the coefficient of determination (R 2 VAL ) and the root mean squared error (RMSE VAL ) of validation. Random cross-validation (CVL) was applied [80% calibration and 20% validation set] with 10 replicates. Then, a stratified cross-validation (SCV) was performed to assess the effect of the dairy industry on prediction accuracy. The study was repeated using a selection of informative wavelengths to assess the necessity of using whole spectra to optimize prediction accuracy. Results showed the feasibility of using FTIR spectra and Bayesian models to predict cheesemaking traits. The R 2 VAL values obtained with the CVL procedure were promising in particular for the %CY and %REC for protein, ranging from 0.44 to 0.66 with very low RMSE VAL (from 0.16 to 0.53). Prediction accuracy obtained with the SCV was strongly influenced by the dairy factory industry. The general low values gained with the SCV do not permit a practical application of this approach, but they highlight the importance of building calibration models with a dataset covering the largest possible sample variability. This study also demonstrated that the use of the full FTIR spectra may be redundant for the prediction of the cheesemaking traits and that a specific selection of the most informative wavelengths led to improved prediction accuracy. This could lead to the development of dedicated spectrometers using selected wavelengths with built-in calibrations for the online prediction of these innovative traits., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Main Conclusion: A structural re-modeling of the thylakoid system, including granum size and regularity, occurs in chlorophyll-deficient wheat mutants affected by photosynthetic membrane over-reduction. In the chloroplast of land plants, the thylakoid system is defined by appressed grana stacks and unstacked stroma lamellae. This study focuses on the variations of the grana organization occurring in outdoor-grown wheat mutants characterized by low chlorophyll content and a tendency for photosynthetic membrane over-reduction. Triticum aestivum ANK-32A and Triticum durum ANDW-7B were compared to their corresponding WT lines, NS67 and LD222, respectively. Electron micrographs of chloroplasts were used to calculate grana ultrastructural parameters. Photosynthetic parameters were obtained by modulated chlorophyll fluorescence and applying Light Curves (LC) and Rapid Light Curves (RLC) protocols. For each photosynthetic parameter, the difference Δ(RLC-LC) was calculated to evaluate the flexible response to light in the examined lines. In the mutants, fewer and smaller disks formed grana stacks characterized by a marked increase in lateral and cross-sectional irregularity, both negatively correlated with the number of layers per granum. A relationship was found between membrane over-reduction and granum structural irregularity. The possible acclimative significance of a greater proportion of stroma-exposed grana domains in relieving the excess electron pressure on PSI is discussed., (© 2024. The Author(s).)

Background: Chlorophyll (Chl) is an agronomic trait associated with photosynthesis and yield. Gibberellin 2-oxidases (GA2oxs) have previously been shown to be involved in Chl accumulation. However, whether and how the PbrGA2ox proteins (PbrGA2oxs) mediate Chl accumulation in pear (Pyrus spp.) is scarce., Results: Here, we aimed to elucidate the role of the pear GA2ox gene family in Chl accumulation and the related underlying mechanisms. We isolated 13 PbrGA2ox genes (PbrGA2oxs) from the pear database and identified PbrGA2ox1 as a potential regulator of Chl accumulation. We found that transiently overexpressing PbrGA2ox1 in chlorotic pear leaves led to Chl accumulation, and PbrGA2ox1 silencing in normal pear leaves led to Chl degradation, as evident by the regreening and chlorosis phenomenon, respectively. Meanwhile, PbrGA2ox1-overexpressing (OE) tobacco plants discernably exhibited Chl built-up, as evidenced by significantly higher Pn and Fv/Fm. In addition, RNA sequencing (RNA-seq), physiological and biochemical investigations revealed an increase in abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA) concentrations and signaling pathways; a marked elevation in reducing and soluble sugar contents; and a marginal decline in the starch and sucrose levels in OE plants. Interestingly, PbrGA2ox1 overexpression did not prominently affect Chl synthesis. However, it indeed facilitated chloroplast development by increasing chloroplast number per cell and compacting the thylakoid granum stacks. These findings might jointly contribute to Chl accumulation in OE plants., Conclusion: Overall, our results suggested that GA2oxs accelerate Chl accumulation by stimulating chloroplast development and proved the potential of PbrGA2ox1 as a candidate gene for genetically breeding biofortified pear plants with a higher yield., (© 2024. The Author(s).)

Starch is one of the major carbohydrate storage compounds in plants. The biogenesis of starch granules starts with the formation of initials, which subsequently expand into granules. Several coiled-coil domain-containing proteins have been previously implicated with the initiation process, but the mechanisms by which they act remain largely elusive. Here, we demonstrate that one of these proteins, the thylakoid-associated MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1), specifically determines the subchloroplast location of initial formation. The expression of MFP1 variants "mis"-targeted to specific locations within chloroplasts in Arabidopsis results in distinctive shifts in not only how many but also where starch granules are formed. Importantly, "re" localizing MFP1 to the stromal face of the chloroplast's inner envelope is sufficient to generate starch granules in this aberrant position. These findings provide compelling evidence that a single protein MFP1 possesses the capacity to direct the initiation and biosynthesis machinery of starch granules., Competing Interests: Competing interests statement:The authors declare no competing interest.

Reactive oxygen species (ROS) are produced by energy transfer and electron transport in plant chloroplast thylakoids at non-toxic levels under normal growth conditions, but at threatening levels under adverse or fluctuating environmental conditions. Among chloroplast ROS, singlet oxygen and superoxide anion radical, respectively, produced by photosystem II (PSII) and PSI, are known to be the major ROS under several stress conditions. Both are very unlikely to diffuse out of chloroplasts, but they are instead capable of triggering ROS-mediated chloroplast operational retrograde signalling to activate defence gene expression in concert with hormones and other molecular compounds. Therefore, their detection, identification and localization in vivo or in biological preparations is a priority for a deeper understanding of their role in (concurrent) regulation of plant growth and defence responses. Here, we present two EPR spin traps, abbreviated as TEMPD-HCl and DEPMPO, to detect and identify ROS in complex systems, such as isolated thylakoids, together with some hints and cautions to perform reliable spin trapping experiments., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Confocal laser scanning microscopy (CLSM) is an advanced microscopy technique based on fluorescence technology which produces sharp images of a specimen in a single focal plane. The optical sectioning by CLSM allows to have z-stacks which can be further processed into 3D reconstructions. These then provide the option of variable perspectives and additional precise data evaluation on structural and anatomical alterations. Here, we used CLSM to image the thylakoids of cyanobacteria and the endoplasmic reticulum (ER) in moss protonemata as an example. Then, out of the confocal z-stacks, we create 3D constructions of the membranes and their alterations to present a holistic, structural view from different angles., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)