Chloroplast Thylakoid Membranes Research Articles

Get the Ball Rolling: Update and Perspective on the Role of Chloroplast Plastoglobule-associated Protein under Abiotic Stress.

Plastid-localized plastoglobules (PGs) are monolayer lipid droplets typically associated with the outer envelope of thylakoid membranes in chloroplasts. The size and number of PGs can vary significantly in response to different environmental stimuli. Since the early 21st century, a variety of proteins attached to the surface of PGs have been identified and experimentally characterized using advanced biotechnological techniques, revealing their biological functions. This article aims to review the latest discoveries regarding PG-associated proteins and explore their dynamics under both single and combined abiotic stress conditions, providing insights into the critical role of plastid lipid droplets in plant adaptation to global climate-related challenges.

Localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus to thylakoid subdomains in Arabidopsis.

Thylakoid membranes in chloroplasts and cyanobacteria harbor the multisubunit protein complexes that catalyze the light reactions of photosynthesis. In plant chloroplasts, the thylakoid membrane system comprises a highly organized network with several subcompartments that differ in composition and morphology: grana stacks, unstacked stromal lamellae, and grana margins at the interface between stacked and unstacked regions. The localization of components of the photosynthetic apparatus among these subcompartments has been well characterized. However, less is known about the localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus, the partitioning of proteins between two recently resolved components of the traditional margin fraction (refined margins and curvature), and the effects of light on these features. In this study, we analyzed the partitioning of numerous thylakoid biogenesis and repair factors among grana, curvature, refined margin, and stromal lamellae fractions of Arabidopsis thylakoid membranes, comparing the results from illuminated and dark-adapted plants. Several proteins previously shown to localize to a margin fraction partitioned in varying ways among the resolved curvature and refined margin fractions. For example, the ALB3 insertase and FtsH protease involved in photosystem II (PSII) repair were concentrated in the refined margin fraction, whereas TAT translocon subunits and proteins involved in early steps in photosystem assembly were concentrated in the curvature fraction. By contrast, two photosystem assembly factors that facilitate late assembly steps were depleted from the curvature fraction. The enrichment of the PSII subunit OE23/PsbP in the curvature fraction set it apart from other PSII subunits, supporting the previous conjecture that OE23/PsbP assists in PSII biogenesis and/or repair. The PSII assembly factor PAM68 partitioned differently among thylakoid fractions from dark-adapted plants and illuminated plants and was the only analyzed protein to convincingly do so. These results demonstrate an unanticipated spatial heterogeneity of photosystem biogenesis and repair functions in thylakoid membranes and reveal the curvature fraction to be a focal point of early photosystem biogenesis.

Real-time monitoring of stromal NADPH levels in Arabidopsis using a metagenome-derived NADPH-binding fluorescent protein

The light irradiation to the plant chloroplasts drives NADPH and ATP synthesis in the stroma via the electron transport chains within the thylakoid membranes. Conventional methods for assessing photosynthetic light reactions are often invasive or require specific conditions. While detection markers do not significantly affect plant growth itself, developing a method for the real-time and non-invasive detection of NADPH is a highly impactful and important research area in plant physiology and biochemistry. This study introduces a genetically encoded NADPH-binding blue fluorescent protein (mBFP) targeted to the chloroplast stroma or thylakoid membrane in Arabidopsis thaliana and Nicotiana benthamiana. Using two-photon microscopy, we monitored real-time stromal NADPH levels in transgenic leaves of Arabidopsis in response to light exposure. A mutant mBFP construct targeted to the thylakoid membrane allowed us to detect the stromal NADPH levels in real time under different light conditions. This in planta biosensor provides a non-invasive tool for studying photosynthetic responses to light more quantitatively and holds potential for optimizing light conditions in controlled-environment agriculture, such as indoor vertical farms, to improve crop productivity.

Functional organization of 3D plant thylakoid membranes as seen by high resolution microscopy

In the field of photosynthesis, only a limited number of approaches of super-resolution fluorescence microscopy can be used, as the functional architecture of the thylakoid membrane in chloroplasts is probed through the natural fluorescence of chlorophyll molecules. In this work, we have used a custom-built fluorescence microscopy method called Single Pixel Reconstruction Imaging (SPiRI) that yields a 1.4 gain in lateral and axial resolution relative to confocal fluorescence microscopy, to obtain 2D images and 3D-reconstucted volumes of isolated chloroplasts, obtained from pea (Pisum sativum), spinach (Spinacia oleracea) and Arabidopsis thaliana. In agreement with previous studies, SPiRI images exhibit larger thylakoid grana diameters when extracted from plants under low-light regimes. The three-dimensional thylakoid architecture, revealing the complete network of the thylakoid membrane in intact, non-chemically-fixed chloroplasts can be visualized from the volume reconstructions obtained at high resolution. From such reconstructions, the stromal connections between each granum can be determined and the fluorescence intensity in the stromal lamellae compared to those of neighboring grana.

Temporal dynamics of chloroplast biogenesis revealed initiation of photosynthesis-related gene expression and protein complexes during alfalfa seed germination

The chloroplast biogenesis occurs in cotyledon during alfalfa seed germination before true leaf formation, and is extremely important for the followed plant development and growth. In this study, we conducted a simulation of alfalfa seed germination in the soil by using tin foil and focused on 10 pivotal time points of chloroplast biogenesis in cotyledons before and after light exposure, which showed significant differences in multispectral images, and covered the whole process of chloroplast biogenesis from proplastid, etioplast to mature chloroplast. We revealed three phases that referred to the programmed involvements of photosynthesis promotion, ultrastructure maturity, transcriptomic expression, and protein complex construction, and observed distinct transcriptional expressions of genes from nuclear and chloroplast genomes. In phase I at dark germination before light exposure, chloroplast-encoded genes showed up-regulated expressions together with the importation of chloroplast proteins. In phase II for the first day after light exposure, nuclear-encoded genes’ expressions were initiated at 2 h after light exposure (E2h), followed by swift assembly of chloroplast thylakoid membrane protein complexes, and roaring Fv/Fm and contents of chlorophyll a, chlorophyll b and carotenoid. The initiation at E2h was pronounced by the observation of gradual accumulation of single lamella, and facilitated the formation of granum stacks (thylakoid) at E8h in phase II. In phase III from the second day after light exposure, chloroplast became gradually complete with the fully established photosynthetic capacity. Altogether, our results layed a theoretical foundation for enhancing potential photosynthetic efficiency in alfalfa and related species.

Orchestration of Photosynthesis-Associated Gene Expression and Galactolipid Biosynthesis during Chloroplast Differentiation in Plants.

The chloroplast thylakoid membrane is composed of membrane lipids and photosynthetic protein complexes, and the orchestration of thylakoid lipid biosynthesis and photosynthesis-associated protein accumulation is considered important for thylakoid development. Galactolipids consist of ∼80% of the thylakoid lipids, and their biosynthesis is fundamental for chloroplast development. We previously reported that the suppression of galactolipid biosynthesis decreased the expression of photosynthesis-associated nuclear-encoded genes (PhAPGs) and photosynthesis-associated plastid-encoded genes (PhAPGs). However, the mechanism for coordinative regulation between galactolipid biosynthesis in plastids and the expression of PhANGs and PhAPGs remains largely unknown. To elucidate this mechanism, we investigated the gene expression patterns in galactolipid-deficient Arabidopsis seedlings during the de-etiolation process. We found that galactolipids are crucial for inducing both the transcript accumulation of PhANGs and PhAPGs and the accumulation of plastid-encoded photosynthesis-associated proteins in developing chloroplasts. Genetic analysis indicates the contribution of the GENOMES UNCOUPLED1 (GUN1)-mediated plastid-to-nucleus signaling pathway to PhANG regulation in response to galactolipid levels. Previous studies suggested that the accumulation of GUN1 reflects the state of protein homeostasis in plastids and alters the PhANG expression level. Thus, we propose a model that galactolipid biosynthesis determines the protein homeostasis in plastids in the initial phase of de-etiolation and optimizes GUN1-dependent signaling to regulate the PhANG expression. This mechanism might contribute to orchestrating the biosynthesis of lipids and proteins for the biogenesis of functional chloroplasts in plants.

INIBIDORES DO FOTOSSISTEMA II: UMA PERSPECTIVA ALELOQUÍMICA

PHOTOSYSTHEM II INHIBITORS: AN ALLELOCHEMICAL PERSPECTIVE. The process of photosynthesis involves the absorption of light energy by photosynthetic pigments found in the thylakoid membrane of chloroplasts in order to produce chemical energy. In the presence of photosynthetic inhibitors, plant development is affected mainly by the reduction of the electron chain, which leads to growth inhibition. In this context, chlorophyll a (Chl a) fluorescence is an important technique used to identify the effects of inhibitors on the photosynthetic apparatus employing the JIP-test, which correlates the Chl a fluorescence transient to biophysical parameters, providing valuable information about the efficiency of photosystems I and II. Natural products have been highlighted as photosynthesis inhibitors due to the continued use of synthetic herbicides, which leads to the development of invasive plants resistant to these pesticides, in addition to the toxicity caused to humans and the environment. Thus, this review describes the main applications of extracts and isolated secondary metabolites obtained from plants and microorganisms in the investigation of electron transport inhibition on photosystem II.

Thylakoid Lumen; from “proton bag” to photosynthetic functionally important compartment

This mini review provides an update of the thylakoid lumen, shedding light on its intricate structure, unique proteome, and potential physiological significance. This compartment within the thylakoid membranes of chloroplasts was originally perceived as “empty”, only providing a site for proton accumulation to support ATP formation. Instead, recent investigations have revealed that the lumen houses a specific set of proteins each with potentially critical roles. The structure of this compartment has been shown to be dynamic, with changes in size and organization influenced by light exposure, impacting protein mobility and function. Noteworthy, some of the lumen proteins are permanently or transiently in contact with protein complexes located in the thylakoid membrane, such as PSII (PsbP-like and PsbQ-like proteins) cytochrome b6f, and PSI. Meanwhile, other lumen proteins seems to be more “independent” such as proteases, immunophilins, stress-related proteins, pentapeptide repeat proteins, and many others with unknown functions. All these proteins play crucial roles in maintaining photosynthetic machinery, adapting to environmental stress, and regulating cellular processes. Understanding the lumen’s function is vital as it holds promise for uncovering novel regulatory interactions and signaling pathways within the chloroplast.

Quantifying the Energy Spillover between Photosystems II and I in Cyanobacterial Thylakoid Membranes and Cells.

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.

Alterations in Carbohydrate Metabolism and Modulation of Thermo-Tolerance in Tomato under Heat Stress

Heat stress affects the source and sink activities, growth and development, economic yield and harvest index of plants negatively. Heat stress has got severe effects on the biochemical reactions of photosynthesis which included irreversible damages of RuBisCO, oxygen evolving complexes, PSII reaction centres and disruptions of chloroplast ultrastructure and thylakoid membrane. Sugars are products of photosynthesis in plants which serve as substrates for energy metabolism and are required for synthesis of cellulose and starch polysaccharides. Glucose, fructose, sucrose, mannose, trehalose, maltose etc. are the sugars present in plants. Abiotic stresses like temperature, drought, flood and salinity causes crucial differences in the carbohydrate metabolism. Sugars acts as primary signaling molecules and regulate signals that control the expression of many genes and enzymes involved in carbohydrate metabolism. Decrease in crop productivity under heat stress is chiefly associated with alterations in carbohydrate accumulation and resulting defects in assimilation partitioning from source to sink. It is essential to understand the responses and acclimatization processes to extreme temperatures in plants, such that the mechanisms underlying can be used for developing heat-tolerant varieties. Sugars such as glucose, sucrose, fructan, raffinose and trehalose not only acts structural component and metabolic resources also regulate genes associated with heat stress tolerance and reduce the chances of crop loss. The external application of compounds like osmoregulators, phytohormones, signaling molecules, etc., have exhibited positive responses in stress tolerance due to its growth promoting and antioxidant activities.

Chloroplast Damage and Photosynthetic System Disorder Induced Chlorosis in the Leaves of Rice Seedlings under Excessive Biuret

Excessive biuret in fertilizer causes leaf albinism in direct-seeded rice fields. This study aimed to provide a comprehensive understanding of the underlying physiology and molecular mechanisms of leaf chlorosis via biuret using morphophysiological and transcriptome analyses. The induction of biuret in albino rice leaves was examined in a net-growing cultivation bed. Some key morphophysiological indices were measured including biuret content, blade ultrastructure, chlorophyll content, and chlorophyll fluorescence parameters. Candidate genes in the chlorotic leaves under biuret stress were also excavated using transcriptome analysis. Furthermore, physiological and biochemical analyses of the changes in enzyme activities and intermediate metabolite contents in relation to the phenotypic changes in the leaves were carried out. The chlorotic leaves of rice seedlings showed higher biuret accumulation, and the leaves suffered severe damage with higher malondialdehyde contents and low chlorophyll contents. Abnormal chloroplast ultrastructures and thylakoid membrane structure loss were observed in chlorotic leaves under biuret exposure. The related genes involved in the chloroplast development, photosynthesis (including antenna proteins), and carbon fixation pathways were significantly downregulated, which suggests that photosynthesis was destroyed in the chlorotic leaves of rice seedlings. Biuret disturbed the photosynthetic system in chloroplast thylakoid membranes by inhibiting chloroplast development, thereby promoting the formation of the chlorotic leaf phenotype in rice seedlings. Our results promote the understanding of the molecular mechanism of rice in response to biuret toxicity.

LHCII - a protein like a 'Swiss Army knife' with many mechanisms and functions.

The review highlights the relationship between the molecular organization of the light-harvesting complex of photosystem II (LHCII) and sunlight utilization by higher plants. The molecular form of LHCII switches rapidly and reversibly during diurnal changes of light intensity, from low (ca. 10) to high [ca. 1,000 μmol(photon) m-2 s-1], so the sensitivity of LHCII to light may control the balance between light harvesting and photoprotection state. Our understanding and concept of this mechanism are based on the knowledge of the structure and photophysics of different LHCII molecular forms: monomer, dimer, trimer, and aggregate. It is proposed that LHCII monomers, dimers, and lateral aggregates are fundamental blocks of excess light-dissipation machinery. Trimer is exceptionally well suited to play a physiological role of an antenna complex. A correlation between the LHCII molecular form and the presence of xanthophyll cycle pigment violaxanthin and zeaxanthin in the complex structure is also shown. Moreover, the role of LHCII protein phosphorylation in thylakoid membrane architecture is also discussed. The dual function of LHCII has been studied in the natural thylakoid membranes of chloroplasts, in the artificial lipid-LHCII model membranes, and by suspension of LHCII in a detergent solution.

Chimeric deletion mutation of rpoC2 underlies the leaf-patterning of Clivia miniata var. variegata.

The deletion mutated rpoC2 leads to yellow stripes of Clivia miniata var. variegata by down regulating the transcription of 28 chloroplast genes and disturbing chloroplast biogenesis and thylakoid membrane development. Clivia miniata var. variegata (Cmvv) is a common mutant of Clivia miniata but its genetic basis is unclear. Here, we found that a 425bp deletion mutation of chloroplast rpoC2 underlies the yellow stripes (YSs) of Cmvv. Both RNA polymerase PEP and NEP coexist in seed-plant chloroplasts and the β″ subunit of PEP is encoded by rpoC2. The rpoC2 mutation changed the discontinuous cleft domain required to form the PEP central cleft for DNA binding from 1103 to 59aa. RNA-Seq revealed that 28 chloroplast genes (cpDEGs) were all down-regulated in YSs, of which, four involved in chloroplast protein translation and 21 of photosynthesis system (PS)I, PSII, cytochrome b6/f complex and ATP synthase are crucial for chloroplast biogenesis/development. The accuracy and reliability of RNA-Seq was verified by qRT-PCR. Moreover, the chlorophyll (Chl) a/b content, ratio of Chla/Chlb and photosynthetic rate (Pn) of YS decreased significantly. Meanwhile, chloroplasts of the YS mesophyll cells were smaller, irregular in shape, contain almost no thylakoid membrane, and even proplastid was found in YS. These findings indicate that the rpoC2 mutation down-regulated expression of the 28 cpDEGs, which disturb chloroplast biogenesis and its thylakoid membrane development. Thus, there are not enough PSI and II components to bind Chl, so that the corresponding areas of the leaf are yellow and show a low Pn. In this study, the molecular mechanism of three phenotypes of F1 (Cmvv ♀ × C. miniata ♂) was revealed, which lays a foundation for the breeding of variegated plants.

A paler shade of green: engineering cellular chlorophyll content to enhance photosynthesis in crowded environments.

In natural ecosystems, plants compete for space, nutrients and light. The optically dense canopies limit the penetration of photosynthetically active radiation and light often becomes a growth-limiting factor for the understory. The reduced availability of photons in the lower leaf layers is also a major constraint for yield potential in canopies of crop monocultures. Traditionally, crop breeding has selected traits related to plant architecture and nutrient assimilation rather than light use efficiency. Leaf optical density is primarily determined by tissue morphology and by the foliar concentration of photosynthetic pigments (chlorophylls and carotenoids). Most pigment molecules are bound to light-harvesting antenna proteins in the chloroplast thylakoid membranes, where they serve photon capture and excitation energy transfer toward reaction centers of photosystems. Engineering the abundance and composition of antenna proteins has been suggested as a strategy to improve light distribution within canopies and reduce the gap between theoretical and field productivity. Since the assembly of the photosynthetic antennas relies on several coordinated biological processes, many genetic targets are available for modulating cellular chlorophyll levels. In this review, we outline the rationale behind the advantages of developing pale green phenotypes and describe possible approaches toward engineering light-harvesting systems.

Cryoresistance Differences Between Species of Autotrops From Polar Regions Sensed by Chlorophyll Fluorescence

Lichens from the cold regions of Earth exhibit high, but different degree of tolerance to drought and freeze stresses. Therefore, three lichen species Xanthoria elegans, Umbilicaria cylindrica, Usnea sphacelata, and a representative of extremophilic cyanobacteria Nostoc commune were selected to measure temperature response curves of chlorophyll fluorescence parameters in cooling experiment using a constant rate of cooling from 20 to –45°C. Two chlorophyll fluorescence parameters (FV/FM – potential yield of photosynthetic processes in photosystem II and ΦPSII – effective quantum yield) were measured with a modulated fluorometer. For all experimental species, the temperature-response curves of II FV/FM and ΦPSII showed typical triphasic shape: plateau (temperature decrease from 20 to –5°C), decline, and a shoulder reaching the critical point. The phase typically formed a S-curve of different shape as dependent on species and chlorophyll fluorescence parameter. U. cylindrica was the most cryoresistant in terms of photosynthetic processes ongoing in thylakoid membrane of chloroplast at below zero temperature.

Inhibitors of ATP Synthase as New Antibacterial Candidates.

ATP, the power of all cellular functions, is constantly used and produced by cells. The enzyme called ATP synthase is the energy factory in all cells, which produces ATP by adding inorganic phosphate (Pi) to ADP. It is found in the inner, thylakoid and plasma membranes of mitochondria, chloroplasts and bacteria, respectively. Bacterial ATP synthases have been the subject of multiple studies for decades, since they can be genetically manipulated. With the emergence of antibiotic resistance, many combinations of antibiotics with other compounds that enhance the effect of these antibiotics have been proposed as approaches to limit the spread of antibiotic-resistant bacteria. ATP synthase inhibitors, such as resveratrol, venturicidin A, bedaquiline, tomatidine, piceatannol, oligomycin A and N,N-dicyclohexylcarbodiimide were the starting point of these combinations. However, each of these inhibitors target ATP synthase differently, and their co-administration with antibiotics increases the susceptibility of pathogenic bacteria. After a brief description of the structure and function of ATP synthase, we aim in this review to highlight therapeutic applications of the major bacterial ATP synthase inhibitors, including animal's venoms, and to emphasize their importance in decreasing the activity of this enzyme and subsequently eradicating resistant bacteria as ATP synthase is their source of energy.

Ante- and post-mortem cellular injury dynamics in hybrid poplar foliage as a function of phytotoxic O3 dose.

After reaching phytotoxic levels during the last century, tropospheric ozone (O3) pollution is likely to remain a major concern in the coming decades. Despite similar injury processes, there is astounding interspecific-and sometimes intraspecific-foliar symptom variability, which may be related to spatial and temporal variation in injury dynamics. After characterizing the dynamics of physiological responses and O3 injury in the foliage of hybrid poplar in an earlier study, here we investigated the dynamics of changes in the cell structure occurring in the mesophyll as a function of O3 treatment, time, phytotoxic O3 dose (POD0), leaf developmental stage, and mesophyll layer. While the number of Hypersensitive Response-like (HR-like) lesions increased with higher O3 concentrations and POD0, especially in older leaves, most structural HR-like markers developed after cell death, independent of the experimental factors. The pace of degenerative Accelerated Cell Senescence (ACS) responses depended closely on the O3 concentration and POD0, in interaction with leaf age. Changes in total chlorophyll content, plastoglobuli and chloroplast shape pointed to thylakoid membranes in chloroplasts as being especially sensitive to O3 stress. Hence, our study demonstrates that early HR-like markers can provide reasonably specific, sensitive and reliable quantitative structural estimates of O3 stress for e.g. risk assessment studies, especially if they are associated with degenerative and thylakoid-related injury in chloroplasts from mesophyll.

Characterization of a TatA/TatB binding site on the TatC component of the Escherichia coli twin arginine translocase.

The twin arginine transport (Tat) pathway exports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of chloroplasts. In Escherichia coli and other Gram-negative bacteria, the Tat machinery comprises TatA, TatB and TatC components. A Tat receptor complex, formed from all three proteins, binds Tat substrates, which triggers receptor organization and recruitment of further TatA molecules to form the active Tat translocon. The polytopic membrane protein TatC forms the core of the Tat receptor and harbours two binding sites for the sequence-related TatA and TatB proteins. A 'polar' cluster binding site, formed by TatC transmembrane helices (TMH) 5 and 6 is occupied by TatB in the resting receptor and exchanges for TatA during receptor activation. The second binding site, lying further along TMH6, is occupied by TatA in the resting state, but its functional relevance is unclear. Here we have probed the role of this second binding site through a programme of random and targeted mutagenesis. Characterization of three stably produced TatC variants, P221R, M222R and L225P, each of which is inactive for protein transport, demonstrated that the substitutions did not affect assembly of the Tat receptor. Moreover, the substitutions that we analysed did not abolish TatA or TatB binding to either binding site. Using targeted mutagenesis we introduced bulky substitutions into the TatA binding site. Molecular dynamics simulations and crosslinking analysis indicated that TatA binding at this site was substantially reduced by these amino acid changes, but TatC retained function. While it is not clear whether TatA binding at the TMH6 site is essential for Tat activity, the isolation of inactivating substitutions indicates that this region of the protein has a critical function.

Understanding the assembly mechanism of the twin arginine transport system.

The twin-arginine protein translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Substrate binding promotes a conformation rearrangement of the Tat complex. TatA is then recruited to the substrate activated TatBC complex, where it polymerizes to form the substrate translocation pathway. The central challenge in understanding Tat transport is to determine how the Tat components are assembled to form the active translocon allowing folded substrates to be transferred across the membrane bilayer. In this work, we used molecular dynamics simulations to describe the TatA oligomerization mechanism and how the translocation pathway is formed. Our results show that the Tat complex becomes more dynamic after substrate binding. The presence of the substrate induces a “breathing” movement, increasing the separation between two adjacent TatC subunits. Moreover, our results also suggest that substrate binding induces an asymmetry in the Tat Complex, favouring TatA aggregation around the interface opposite to the TatC subunit where the signal peptide is bound. The TatA oligomer is anchored to the Tat complex through contacts with the TM1 and the TM6 of the adjacent TatC subunits, which promotes a local weakening of the membrane bilayer. Our extensive coarse-grained MD simulations provide a molecular description of the Tat translocon assembly and new insights into the Tat transport cycle.

Arabidopsis Sec14 proteins (SFH5 and SFH7) mediate interorganelle transport of phosphatidic acid and regulate chloroplast development

Lipids establish the specialized thylakoid membrane of chloroplast in eukaryotic photosynthetic organisms, while the molecular basis of lipid transfer from other organelles to chloroplast remains further elucidation. Here we revealed the structural basis of Arabidopsis Sec14 homology proteins AtSFH5 and AtSFH7 in transferring phosphatidic acid (PA) from endoplasmic reticulum (ER) to chloroplast, and whose function in regulating the lipid composition of chloroplast and thylakoid development. AtSFH5 and AtSFH7 localize at both ER and chloroplast, whose deficiency resulted in an abnormal chloroplast structure and a decreased thickness of stacked thylakoid membranes. We demonstrated that AtSFH5, but not yeast and human Sec14 proteins, could specifically recognize and transfer PA invitro. Crystal structures of the AtSFH5-Sec14 domain in complex with L-α-phosphatidic acid (L-α-PA) and 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) revealed that two PA ligands nestled in the central cavity with different configurations, elucidating the specific binding mode of PA to AtSFH5, different from the reported phosphatidylethanolamine (PE)/phosphatidylcholine (PC)/phosphatidylinositol (PI) binding modes. Quantitative lipidomic analysis of chloroplast lipids showed that PA and monogalactosyldiacylglycerol (MGDG), particularly the C18 fatty acids at sn-2 position in MGDG were significantly decreased, indicating a disrupted ER-to-plastid (chloroplast) lipid transfer, under deficiency of AtSFH5 and AtSFH7. Our studies identified the role and elucidated the structural basis of plant SFH proteins in transferring PA between organelles, and suggested a model for ER-chloroplast interorganelle phospholipid transport from inherent ER to chloroplast derived from endosymbiosis of a cyanobacteriumproviding a mechanism involved in the adaptive evolution of cellular plastids.