Outer Envelope Membrane Of Chloroplasts Research Articles

ER-associated VAP27-1 and VAP27-3 proteins functionally link the lipid-binding ORP2A at the ER-chloroplast contact sites

The plant endoplasmic reticulum (ER) contacts heterotypic membranes at membrane contact sites (MCSs) through largely undefined mechanisms. For instance, despite the well-established and essential role of the plant ER-chloroplast interactions for lipid biosynthesis, and the reported existence of physical contacts between these organelles, almost nothing is known about the ER-chloroplast MCS identity. Here we show that the Arabidopsis ER membrane-associated VAP27 proteins and the lipid-binding protein ORP2A define a functional complex at the ER-chloroplast MCSs. Specifically, through in vivo and in vitro association assays, we found that VAP27 proteins interact with the outer envelope membrane (OEM) of chloroplasts, where they bind to ORP2A. Through lipidomic analyses, we established that VAP27 proteins and ORP2A directly interact with the chloroplast OEM monogalactosyldiacylglycerol (MGDG), and we demonstrated that the loss of the VAP27-ORP2A complex is accompanied by subtle changes in the acyl composition of MGDG and PG. We also found that ORP2A interacts with phytosterols and established that the loss of the VAP27-ORP2A complex alters sterol levels in chloroplasts. We propose that, by interacting directly with OEM lipids, the VAP27-ORP2A complex defines plant-unique MCSs that bridge ER and chloroplasts and are involved in chloroplast lipid homeostasis.

Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants

Gravity controls directional growth of plants, and the classical starch-statolith hypothesis proposed more than a century ago postulates that amyloplast sedimentation in specialized cells initiates gravity sensing, but the molecular mechanism remains uncharacterized. The LAZY proteins are known as key regulators of gravitropism, and lazy mutants show striking gravitropic defects. Here, we report that gravistimulation by reorientation triggers mitogen-activated protein kinase (MAPK) signaling-mediated phosphorylation of Arabidopsis LAZY proteins basally polarized in root columella cells. Phosphorylation of LAZY increases its interaction with several translocons at the outer envelope membrane of chloroplasts (TOC) proteins on the surface of amyloplasts, facilitating enrichment of LAZY proteins on amyloplasts. Amyloplast sedimentation subsequently guides LAZY to relocate to the new lower side of the plasma membrane in columella cells, where LAZY induces asymmetrical auxin distribution and root differential growth. Together, this study provides a molecular interpretation for the starch-statolith hypothesis: the organelle-movement-triggered molecular polarity formation.

Selective autophagy regulates chloroplast protein import and promotes plant stress tolerance.

Chloroplasts are plant organelles responsible for photosynthesis and environmental sensing. Most chloroplast proteins are imported from the cytosol through the translocon at the outer envelope membrane of chloroplasts (TOC). Previous work has shown that TOC components are regulated by the ubiquitin-proteasome system (UPS) to control the chloroplast proteome, which is crucial for the organelle's function and plant development. Here, we demonstrate that the TOC apparatus is also subject to K63-linked polyubiquitination and regulation by selective autophagy, potentially promoting plant stress tolerance. We identify NBR1 as a selective autophagy adaptor targeting TOC components, and mediating their relocation into vacuoles for autophagic degradation. Such selective autophagy is shown to control TOC protein levels and chloroplast protein import and to influence photosynthetic activity as well as tolerance to UV-B irradiation and heat stress in Arabidopsis plants. These findings uncover the vital role of selective autophagy in the proteolytic regulation of specific chloroplast proteins, and how dynamic control of chloroplast protein import is critically important for plants to cope with challenging environments.

Pepper mild mottle virus coat protein interacts with pepper chloroplast outer envelope membrane protein OMP24 to inhibit antiviral immunity in plants.

Pepper mild mottle virus (PMMoV) is a devastating viral pathogen of pepper (Capsicum annuum) but it is unclear whether and how peppers protect against PMMoV infection. The expression of the chloroplast outer membrane protein 24 (OMP24) of C. annuum was upregulated under PMMoV infection and it interacted with PMMoV coat protein (CP). Silencing of OMP24 in either C. annuum or Nicotiana benthamiana facilitated PMMoV infection, whereas overexpression of N. benthamiana OMP24 in transgenic plants inhibited PMMoV infection. Both C. annuum OMP24 (CaOMP24) and N. benthamiana OMP24 (NbOMP24) localized to the chloroplast and have a moderately hydrophobic transmembrane domain that is necessary for their localization. Overexpression of CaOMP24 induced stromules, perinuclear chloroplast clustering, and accumulation of reactive oxygen species (ROS), the typical defense responses of chloroplasts transferring the retrograde signaling to the nucleus to regulate resistance genes. The expression of PR1 and PR2 was also upregulated significantly in plants overexpressing OMP24. Self-interaction of OMP24 was demonstrated and was required for OMP24-mediated plant defense. Interaction with PMMoV CP interfered with the self-interaction of OMP24 and impaired OMP24-induced stromules, perinuclear chloroplast clustering and ROS accumulation. The results demonstrate the defense function of OMP24 in pepper during viral infection and suggest a possible mechanism by which PMMoV CP modulates the plant defense to facilitate viral infection.

Live-cell imaging of the chloroplast outer envelope membrane using fluorescent dyes.

We established a live-cell imaging method to visualize the chloroplast outer envelope membrane by staining living cells with fluorescent dyes. This method does not require genetic transformation and allows the observation of the chloroplast outer envelope membrane in various plant species.

Organellar Glue: A Molecular Tool to Artificially Control Chloroplast-Chloroplast Interactions.

Organelles can physically interact to facilitate various cellular processes such as metabolite exchange. Artificially regulating these interactions represents a promising approach for synthetic biology. Here, we artificially controlled chloroplast–chloroplast interactions in living plant cells with our organelle glue (ORGL) technique, which is based on reconstitution of a split fluorescent protein. We simultaneously targeted N-terminal and C-terminal fragments of a fluorescent protein to the chloroplast outer envelope membrane or cytosol, respectively, which induced chloroplast–chloroplast interactions. The cytosolic C-terminal fragment likely functions as a bridge between two N-terminal fragments, thereby bringing the chloroplasts in close proximity to interact. We modulated the frequency of chloroplast–chloroplast interactions by altering the ratio of N- and C-terminal fragments. We conclude that the ORGL technique can successfully control chloroplast–chloroplast interactions in plants, providing a proof of concept for the artificial regulation of organelle interactions in living cells.

Tic12, a 12-kDa essential component of the translocon at the inner envelope membrane of chloroplasts in Arabidopsis.

The complexes translocon at the outer envelope membrane of chloroplasts and translocon at the inner envelope membrane of chloroplasts (TIC) mediate preprotein translocation across the chloroplast outer and inner envelope membranes, respectively. Tic20, Tic56, Tic100, and Tic214 form a stable one-megadalton TIC whose function is essential for Arabidopsis thaliana and Chlamydomonas reinhardtii. Tic20 plays a central role in preprotein translocation by forming a protein-conducting channel. Tic56, Tic100, and Tic214 are also indispensable for TIC function, but whether other components are required for this process remain unclear. Here, we demonstrate that a 12-kDa protein named Tic12 is part of the TIC in A. thaliana and participates in preprotein translocation across the inner envelope membrane. Tic12 was tightly associated with the TIC but disassociated under high-salt conditions in combination with Triton X-100. Site-specific UV crosslinking experiments revealed that Tic12 and Tic20 directly interact with the transit peptide of a translocating preprotein. The tic12 null mutants are albino and seedling lethal, similar to the other tic null mutants. Tic12 and Tic20 were also involved in preprotein translocation in (Pisum sativum) pea chloroplasts. Thus, Tic12 is an essential constituent that forms the functional core together with Tic20 in the one-megadalton TIC.

Chloroplast protein import machinery and quality control.

Most chloroplast proteins are nucleus-encoded, translated on cytoplasmic ribosomes as precursor proteins, and imported into chloroplasts through TOC and TIC, the translocons of the outer and inner chloroplast envelope membranes. While the composition of the TOC complex is well established, there is still some controversy about the importance of a recently identified TIC complex consisting of Tic20, Tic214, Tic100, and Tic56. TOC and TIC form a supercomplex with a protein channel at the junction of the outer and inner envelope membranes through which preproteins are pulled into the stroma by the ATP-powered Ycf2 complex consisting of several FtsH-like ATPases and/or by chloroplast Hsp proteins. Several components of the TOC/TIC system are moonlighting proteins with additional roles in chloroplast gene expression and metabolism. Chaperones and co-chaperones, associated with TOC and TIC on the cytoplasmic and stromal side of the chloroplast envelope, participate in the unfolding and folding of the precursor proteins and act together with the ubiquitin-proteasome system in protein quality control. Chloroplast protein import is also intimately linked with retrograde signaling, revealing altogether an unsuspected complexity in the regulation of this process.

Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves

Each plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplast genome using peptide–DNA complexes composed of plasmid DNA and a fusion peptide consisting of a cell-penetrating peptide (CPP) and a chloroplast transit peptide (cpPD complex). However, how cpPD complexes are transported into the chloroplast from outside the cell remains unclear. Here, to characterize the route by which these cpPD complexes move into chloroplasts, we tracked their movement from the extracellular space to the chloroplast stroma using a fluorescent label and confocal laser scanning microscopy (CLSM). Upon infiltration of cpPD complexes into the extracellular space of Arabidopsis thaliana leaves, the complexes reached the chloroplast surface within 6h. The cpPD complexes reached were engulfed by the chloroplast outer envelope membrane and gradually integrated into the chloroplast. We detected several cpPD complexes localized around chloroplast nucleoids and observed the release of DNA from the cpPD. Our results thus define the route taken by the cpPD complexes for gene delivery from the extracellular space to the chloroplast stroma.

Ultrastructure of the green alga Dunaliella salina strain CCAP19/18 (Chlorophyta) as investigated by quick-freeze deep-etch electron microscopy

The single-celled green alga Dunaliella salina is a model system for studies on stress biology, in particular regarding secondary carotenoid accumulation. Under non-stress conditions the cells are green, but under abiotic stress the cells turn orange, because they switch their metabolism and accumulate β-Carotene in globules in the chloroplast. For the first time, Quick-freeze deep-etch electron microscopy was used to visualize cellular structures in green and orange cells of D. salina strain CCAP19/18. This allowed us to present an in-depth analysis of the cellular ultrastructure describing and comparing the features of the two cell types. Our images illustrate the presence of a pericellular matrix for this strain of D. salina. The pericellular matrix was spongy and strands of unknown material anchored it into the plasma membrane. The cytoplasm contained a variety of vesicles, vacuoles, and acidocalcisomes. We could show for strain CCAP19/18 that cytoplasmic lipid bodies were often in close proximity to and sometimes in contact with the outer chloroplast envelope membrane and with the endoplasmic reticulum. Major visible differences between green and orange cells were in the chloroplast: the orange cells have greatly reduced amounts of thylakoid membranes and greatly increased numbers of β-Carotene globules. We showed that the β-Carotene globules often made point contacts with thylakoid membranes, and frequently laid side-by-side along the thylakoid membrane surface, providing support to studies that indicated exchange of molecules between β-Carotene globules and thylakoid membranes. A novel finding was the β-Carotene globule duplets, suggesting intermediate stages in β-Carotene globule morphogenesis. Overall, the β-Carotene globules appear to be similar to plant plastoglobuli. We provide this description of the cellular ultrastructure features as a resource in the context of the recent publication of the genome of D. salina strain CCAP19/18 to expand on the knowledge regarding this novel reference strain.

Two young genes reshape a novel interaction network in Brassica napus.

New genes often drive the evolution of gene interaction networks. In Brassica napus, the widely used genic male sterile breeding system 7365ABC is controlled by two young genes, Bnams4b and BnaMs3. However, the interaction mechanism of these two young genes remains unclear. Here, we confirmed that Bnams4b interacts with the nuclear localised E3 ligase BRUTUS (BTS). Ectopic expression of AtBRUTUS (AtBTS) and comparison between Bnams4b -transgenic Arabidopsis and bts mutants suggested that Bnams4b may drive translocation of BTS to cause various toxic defects. BnaMs3 gained an exclusive interaction with the plastid outer-membrane translocon Toc33 compared with Bnams3 and AtTic40, and specifically compensated for the toxic effects of Bnams4b . Heat shock treatment also rescued the sterile phenotype, and high temperature suppressed the interaction between Bnams4b and BTS in yeast. Furthermore, the ubiquitin system and TOC (translocon at the outer envelope membrane of chloroplasts) component accumulation were affected in Bnams4b -transgenic Arabidopsis plants. Taken together, these results indicate that new chimeric Bnams4b carries BTS from nucleus to chloroplast, which may disrupt the normal ubiquitin-proteasome system to cause toxic effects, and these defects can be compensated by BnaMs3-Toc33 interaction or environmental heat shock. It reveals a scenario in which two population-specific coevolved young genes reshape a novel interaction network in plants.

Chloroplast Outer Membrane β-Barrel Proteins Use Components of the General Import Apparatus.

Chloroplasts evolved from a cyanobacterial endosymbiont that resided within a eukaryotic cell. Due to their prokaryotic heritage, chloroplast outer membranes contain transmembrane β-barrel proteins. While most chloroplast proteins use N-terminal transit peptides to enter the chloroplasts through the translocons at the outer and inner chloroplast envelope membranes (TOC/TIC), only one β-barrel protein, Toc75, has been shown to use this pathway. The route other β-barrel proteins use has remained unresolved. Here we use in vitro pea (Pisum sativum) chloroplast import assays and transient expression in Nicotiana benthamiana to address this. We show that a paralog of Toc75, outer envelope protein 80 kD (OEP80), also uses a transit peptide but has a distinct envelope sorting signal. Our results additionally indicate that β-barrels that do not use transit peptides also enter the chloroplast using components of the general import pathway.

Ubiquitin-dependent chloroplast-associated protein degradation in plants.

Chloroplasts contain thousands of nucleus-encoded proteins that are imported from the cytosol by translocases in the chloroplast envelope membranes. Proteolytic regulation of the translocases is critically important, but little is known about the underlying mechanisms. We applied forward genetics and proteomics in Arabidopsis to identify factors required for chloroplast outer envelope membrane (OEM) protein degradation. We identified SP2, an Omp85-type β-barrel channel of the OEM, and CDC48, a cytosolic AAA+ (ATPase associated with diverse cellular activities) chaperone. Both proteins acted in the same pathway as the ubiquitin E3 ligase SP1, which regulates OEM translocase components. SP2 and CDC48 cooperated to bring about retrotranslocation of ubiquitinated substrates from the OEM (fulfilling conductance and motor functions, respectively), enabling degradation of the substrates by the 26S proteasome in the cytosol. Such chloroplast-associated protein degradation (CHLORAD) is vital for organellar functions and plant development.

Biogenesis of chloroplast outer envelope membrane proteins.

Most organisms on Earth use glucose, a photosynthetic product, as energy source. The chloroplast, the home of photosynthesis, is the most representative and characteristic organelle in plants and is enclosed by the outer envelope and inner envelope membranes. The chloroplast biogenesis and unique functions are very closely associated with proteins in the two envelope membranes of the chloroplast. Especially, the chloroplast outer envelope membrane proteins have important roles in signal transduction, protein import, lipid biosynthesis and remodeling, exchange of ions and numerous metabolites, plastid division, movement, and host defense. Therefore, biogenesis of these membrane proteins of chloroplast outer envelope membrane is very important for biogenesis of the entire chloroplast proteome as well as plant development. Most proteins among the outer envelope membrane proteins are encoded by the nuclear genome and are post-translationally targeted to the chloroplast outer envelope membrane. In this process, cytoplasmic receptor and import machineries are required for efficient and correct targeting of these membrane proteins. In this review, we have summarized recent advances on the sorting, targeting, and insertion mechanisms of the outer envelope membrane proteins of chloroplasts and also provide future direction of the study on these topics.

The passion fruit liana (Passiflora edulis Sims, Passifloraceae) is tolerant to ozone

Passiflora edulis Sims is a liana species of high economic interest and is an interesting model plant for understanding ozone action on disturbed vegetation. In this work we hypothesized that P. edulis has adaptive responses to oxidative stress that enable it to tolerate ozone damage based on its capacity to grow under a diversity of environmental conditions and to dominate disturbed areas. We exposed seedlings to three levels of ozone in a Free-Air Controlled Exposure (FACE) system (22, 41 and 58 ppb h AOT40 and 13.52, 17.24 and 20.62 mmol m−2 POD0, over 97 days) for identifying its tolerance mechanisms. Anatomical (leaf blade structure and fluorescence emission of chloroplast metabolites), physiological (leaf gas exchange, growth rate and biomass production) and biochemical (pigments, total sugars, starch, enzymatic and non-enzymatic antioxidant metabolites, reactive oxygen species and lipid peroxidation derivatives) responses were assessed. Ozone caused decreased total number of leaves, hyperplasia and hypertrophy of the mesophyll cells, and accelerated leaf senescence. However, O3 did not affect carbohydrates content, net photosynthetic rate, or total biomass production, indicating that the carboxylation efficiency and associated physiological processes were not affected. In addition, P. edulis showed higher leaf contents of ascorbic acid, glutathione (as well high ratio between their reduced and total forms), carotenoids, and flavonoids located in the chloroplast outer envelope membrane. Our results indicate that P. edulis is an O3-tolerant species due to morphological acclimation responses and an effective antioxidant defense system represented by non-enzymatic antioxidants, which maintained the cellular redox balance under ozone.

Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons: Import of Folded Proteins.

The degree of residual structure retained by proteins while passing through biological membranes is a fundamental mechanistic question of protein translocation. Proteins are generally thought to be unfolded while transported through canonical proteinaceous translocons, including the translocons of the outer and inner chloroplast envelope membranes (TOC and TIC). Here, we readdressed the issue and found that the TOC/TIC translocons accommodated the tightly folded dihydrofolate reductase (DHFR) protein in complex with its stabilizing ligand, methotrexate (MTX). We employed a fluorescein-conjugated methotrexate (FMTX), which has slow membrane transport rates relative to unconjugated MTX, to show that the rate of ligand accumulation inside chloroplasts is faster when bound to DHFR that is actively being imported. Stromal accumulation of FMTX is ATP dependent when DHFR is actively being imported but is otherwise ATP independent, again indicating DHFR/FMTX complex import. Furthermore, the TOC/TIC pore size was probed with fixed-diameter particles and found to be greater than 25.6 Å, large enough to support folded DHFR import and also larger than mitochondrial and bacterial protein translocons that have a requirement for protein unfolding. This unique pore size and the ability to import folded proteins have critical implications regarding the structure and mechanism of the TOC/TIC translocons.

Molecular Topology of the Transit Peptide during Chloroplast Protein Import.

Chloroplast protein import is directed by the interaction of the targeting signal (transit peptide) of nucleus-encoded preproteins with translocons at the outer (TOC) and inner (TIC) chloroplast envelope membranes. Studies of the energetics and determinants of transit peptide binding have led to the hypothesis that import occurs through sequential recognition of transit peptides by components of TOC and TIC during protein import. To test this hypothesis, we employed a site-specific cross-linking approach to map transit peptide topology in relation to TOC-TIC components at specific stages of import in Arabidopsis thaliana and pea (Pisum sativum). We demonstrate that the transit peptide is in contact with Tic20 at the inner envelope in addition to TOC complex components at the earliest stages of chloroplast binding. Low levels of ATP hydrolysis catalyze the commitment of the preprotein to import by promoting further penetration across the envelope membranes and stabilizing the association of the preprotein with TOC-TIC. GTP hydrolysis at the TOC receptors serves as a checkpoint to regulate the ATP-dependent commitment of the preprotein to import and is not essential to drive preprotein import. Our results demonstrate the close cooperativity of the TOC and TIC machinery at each stage of transit peptide recognition and membrane translocation during protein import.

New Perspectives on Chloroplast Protein Import.

Virtually all chloroplasts in extant photosynthetic eukaryotes derive from a single endosymbiotic event that probably occurred more than a billion years ago between a host eukaryotic cell and a cyanobacterium-like ancestor. Many endosymbiont genes were subsequently transferred to the host nuclear genome, concomitant with the establishment of a system for protein transport through the chloroplast double-membrane envelope. Presently, 2,000-3,000 different nucleus-encoded chloroplast proteins must be imported into the chloroplast following their synthesis in the cytosol. The TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope membrane of chloroplasts) complexes are protein translocation machineries at the outer and inner envelope membranes, respectively, that facilitate this chloroplast protein import with the aid of a TIC-associated ATP-driven import motor. All the essential components of this protein import system seemed to have been identified through biochemical analyses and subsequent genetic studies that initiated in the late 1990s. However, in 2013, the Nakai group reported a novel inner envelope membrane TIC complex, for which a novel ATP-driven import motor associated with this TIC complex is likely to exist. In this mini review, I will summarize these recent discoveries together with new, or reanalyzed, data presented by other groups in recent years. Whereas the precise concurrent view of chloroplast protein import is still a matter of some debate, it is anticipated that the entire TOC/TIC/ATP motor system, including any novel components, will be conclusively established in the next decade. Such findings may lead to an extensively revised view of the evolution and molecular mechanisms of chloroplast protein import.

Structural components involved in plastid protein import.

Import of preproteins into chloroplasts is an essential process, requiring two major multisubunit protein complexes that are embedded in the outer and inner chloroplast envelope membrane. Both the translocon of the outer chloroplast membrane (Toc), as well as the translocon of the inner chloroplast membrane (Tic) have been studied intensively with respect to their individual subunit compositions, functions and regulations. Recent advances in crystallography have increased our understanding of the operation of these proteins in terms of their interactions and regulation by conformational switching. Several subdomains of components of the Toc translocon have been studied at the structural level, among them the polypeptide transport-associated (POTRA) domain of the channel protein Toc75 and the GTPase domain of Toc34. In this review, we summarize and discuss the insight that has been gained from these structural analyses. In addition, we present the crystal structure of the Toc64 tetratrico-peptide repeat (TPR) domain in complex with the C-terminal domains of the heat-shock proteins (Hsp) Hsp90 and Hsp70.

Chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha.

Under low-light conditions, chloroplasts localize along periclinal cell walls at temperatures near 20 °C, but they localize along anticlinal cell walls near 5 °C. This phenomenon is known as the cold-positioning response. We previously showed that chloroplasts move as aggregates rather than individually during the cold-positioning response in the fern Adiantum capillus-veneris. This observation suggested that chloroplasts physically interact with each other during the cold-positioning response. However, the physiological processes underlying chloroplast aggregation are unclear. In this report, we characterized chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha. Confocal laser microscopy observations of transgenic liverwort plants expressing a fluorescent fusion protein that localizes to the chloroplast outer envelope membrane (OEP7-Citrine) showed that neighboring chloroplast membranes did not fuse during the cold-positioning response. Transmission electron microscopy analysis revealed that a distance of at least 10nm was maintained between neighboring chloroplasts during aggregation. These results indicate that aggregated chloroplasts do not fuse, but maintain a distance of at least 10nm from each other during the cold-positioning response.