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.

The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation.

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

Fine-Tuning of Photosynthesis Requires CURVATURE THYLAKOID1-Mediated Thylakoid Plasticity.

The thylakoid membrane system of higher plant chloroplasts consists of interconnected subdomains of appressed and nonappressed membrane bilayers, known as grana and stroma lamellae, respectively. CURVATURE THYLAKOID1 (CURT1) protein complexes mediate the shape of grana stacks in a dosage-dependent manner and facilitate membrane curvature at the grana margins, the interface between grana and stroma lamellae. Although grana stacks are highly conserved among land plants, the functional relevance of grana stacking remains unclear. Here, we show that inhibiting CURT1-mediated alteration of thylakoid ultrastructure in Arabidopsis (Arabidopsis thaliana) reduces photosynthetic efficiency and plant fitness under adverse, controlled, and natural light conditions. Plants that lack CURT1 show less adjustment of grana diameter, which compromises regulatory mechanisms like the photosystem II repair cycle and state transitions. Interestingly, CURT1A suffices to induce thylakoid membrane curvature in planta and thylakoid hyperbending in plants overexpressing CURT1A. We suggest that CURT1 oligomerization is regulated at the posttranslational level in a light-dependent fashion and that CURT1-mediated thylakoid plasticity plays an important role in fine-tuning photosynthesis and plant fitness during challenging growth conditions.

Thylakoid Membrane Architecture in Synechocystis Depends on CurT, a Homolog of the Granal CURVATURE THYLAKOID1 Proteins.

Photosynthesis occurs in thylakoids, a highly specialized membrane system. In the cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis 6803), the thylakoids are arranged parallel to the plasma membrane and occasionally converge toward it to form biogenesis centers. The initial steps in PSII assembly are thought to take place in these regions, which contain a membrane subcompartment harboring the early assembly factor PratA and are referred to as PratA-defined membranes (PDMs). Loss of CurT, the Synechocystis 6803 homolog of Arabidopsis thaliana grana-shaping proteins of the CURVATURE THYLAKOID1 family, results in disrupted thylakoid organization and the absence of biogenesis centers. As a consequence, PSII is less efficiently assembled and accumulates to only 50% of wild-type levels. CurT induces membrane curvature in vitro and is distributed all over the thylakoids, with local concentrations at biogenesis centers. There it forms a sophisticated tubular network at the cell periphery, as revealed by live-cell imaging. CurT is part of several high molecular mass complexes, and Blue Native/SDS-PAGE and isoelectric focusing demonstrated that different isoforms associate with PDMs and thylakoids. Moreover, CurT deficiency enhances sensitivity to osmotic stress, adding a level of complexity to CurT function. We propose that CurT is crucial for the differentiation of membrane architecture, including the formation of PSII-related biogenesis centers, in Synechocystis 6803.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.

The antimycin A-sensitive pathway of cyclic electron flow: from 1963 to 2015.

Cyclic electron flow has puzzled and divided the field of photosynthesis researchers for decades. This mainly concerns the proportion of its overall contribution to photosynthesis, as well as its components and molecular mechanism. Yet, it is irrefutable that the absence of cyclic electron flow has severe effects on plant growth. One of the two pathways mediating cyclic electron flow can be inhibited by antimycin A, a chemical that has also widely been used to characterize the mitochondrial respiratory chain. For the characterization of cyclic electron flow, antimycin A has been used since 1963, when ferredoxin was found to be the electron donor of the pathway. In 2013, antimycin A was used to identify the PGRL1/PGR5 complex as the ferredoxin:plastoquinone reductase completing the last puzzle piece of this pathway. The controversy has not ended, and here, we review the history of research on this process using the perspective of antimycin A as a crucial chemical for its characterization.

Arabidopsis CURVATURE THYLAKOID1 proteins modify thylakoid architecture by inducing membrane curvature.

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.

Structure and dynamics of thylakoids in land plants.

Thylakoids of land plants have a bipartite structure, consisting of cylindrical grana stacks, made of membranous discs piled one on top of the other, and stroma lamellae which are helically wound around the cylinders. Protein complexes predominantly located in the stroma lamellae and grana end membranes are either bulky [photosystem I (PSI) and the chloroplast ATP synthase (cpATPase)] or are involved in cyclic electron flow [the NAD(P)H dehydrogenase (NDH) and PGRL1-PGR5 heterodimers], whereas photosystem II (PSII) and its light-harvesting complex (LHCII) are found in the appressed membranes of the granum. Stacking of grana is thought to be due to adhesion between Lhcb proteins (LHCII or CP26) located in opposed thylakoid membranes. The grana margins contain oligomers of CURT1 proteins, which appear to control the size and number of grana discs in a dosage- and phosphorylation-dependent manner. Depending on light conditions, thylakoid membranes undergo dynamic structural changes that involve alterations in granum diameter and height, vertical unstacking of grana, and swelling of the thylakoid lumen. This plasticity is realized predominantly by reorganization of the supramolecular structure of protein complexes within grana stacks and by changes in multiprotein complex composition between appressed and non-appressed membrane domains. Reversible phosphorylation of LHC proteins (LHCPs) and PSII components appears to initiate most of the underlying regulatory mechanisms. An update on the roles of lipids, proteins, and protein complexes, as well as possible trafficking mechanisms, during thylakoid biogenesis and the de-etiolation process complements this review.

Sorting of plant vacuolar proteins is initiated in the ER.

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor-ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer-mediated recycling of the plant VSR BP80 starts at the trans-Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co-expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII-mediated transport route. Retention of soluble cargo despite ongoing COPII-mediated bulk flow can only be explained by an interaction with membrane-bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER-anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR-ligand interaction. It also implies that the retromer-mediated recycling route for the VSRs leads from the TGN back to the ER.

Modulating the activities of chloroplasts and mitochondria promotes adenosine triphosphate production and plant growth.

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin-Benson-Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

PGR5-PGRL1-Dependent Cyclic Electron Transport Modulates Linear Electron Transport Rate in Arabidopsis thaliana.

Plants need tight regulation of photosynthetic electron transport for survival and growth under environmental and metabolic conditions. For this purpose, the linear electron transport (LET) pathway is supplemented by a number of alternative electron transfer pathways and valves. In Arabidopsis, cyclic electron transport (CET) around photosystem I (PSI), which recycles electrons from ferrodoxin to plastoquinone, is the most investigated alternative route. However, the interdependence of LET and CET and the relative importance of CET remain unclear, largely due to the difficulties in precise assessment of the contribution of CET in the presence of LET, which dominates electron flow under physiological conditions. We therefore generated Arabidopsis mutants with a minimal water-splitting activity, and thus a low rate of LET, by combining knockout mutations in PsbO1, PsbP2, PsbQ1, PsbQ2, and PsbR loci. The resulting Δ5 mutant is viable, although mature leaves contain only ∼ 20% of wild-type naturally less abundant PsbO2 protein. Δ5 plants compensate for the reduction in LET by increasing the rate of CET, and inducing a strong non-photochemical quenching (NPQ) response during dark-to-light transitions. To identify the molecular origin of such a high-capacity CET, we constructed three sextuple mutants lacking the qE component of NPQ (Δ5 npq4-1), NDH-mediated CET (Δ5 crr4-3), or PGR5-PGRL1-mediated CET (Δ5 pgr5). Their analysis revealed that PGR5-PGRL1-mediated CET plays a major role in ΔpH formation and induction of NPQ in C3 plants. Moreover, while pgr5 dies at the seedling stage under fluctuating light conditions, Δ5 pgr5 plants are able to survive, which underlines the importance of PGR5 in modulating the intersystem electron transfer.