Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+release.

Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K+-permeable channel was coined fast-activating chloroplast cation channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K+ conductance domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K+ uptake. Nuclear POLLUX ion channels cofunction with Ca2+ channels to generate Ca2+ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca2+ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca2+ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant's stress-related Ca2+ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.

Stromal NADH supplied by PHOSPHOGLYCERATE DEHYDROGENASE3 is crucial for photosynthetic performance.

During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin-Benson-Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.

Chloroplast Outer Membrane ß-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 ß-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 ß-barrel protein, Toc75, has been shown to use this pathway. The route other ß-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 ß-barrels that do not use transit peptides also enter the chloroplast using components of the general import pathway.

Evolution of protein transport to the chloroplast envelope membranes.

Chloroplasts are descendants of an ancient endosymbiotic cyanobacterium that lived inside a eukaryotic cell. They inherited the prokaryotic double membrane envelope from cyanobacteria. This envelope contains prokaryotic protein sorting machineries including a Sec translocase and relatives of the central component of the bacterial outer membrane ß-barrel assembly module. As the endosymbiont was integrated with the rest of the cell, the synthesis of most of its proteins shifted from the stroma to the host cytosol. This included nearly all the envelope proteins identified so far. Consequently, the overall biogenesis of the chloroplast envelope must be distinct from cyanobacteria. Envelope proteins initially approach their functional locations from the exterior rather than the interior. In many cases, they have been shown to use components of the general import pathway that also serves the stroma and thylakoids. If the ancient prokaryotic protein sorting machineries are still used for chloroplast envelope proteins, their activities must have been modified or combined with the general import pathway. In this review, we analyze the current knowledge pertaining to chloroplast envelope biogenesis and compare this to bacteria.

The Arabidopsis T-DNA mutant SALK_008491 carries a 14-kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress.

In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light-dependent reaction and CO2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer-DNA (T-DNA) insertion mutant line SALK_008491, previously known as nhd1-1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non-photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2 pool, we identified an ~14-kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1. Besides NHD1, which encodes for a putative plastid Na+/H+ antiporter, the stromal NAD-dependent D-3-phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow-up studies employing respective single mutants and complementation with overlapping transformation-competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion-carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T-DNA lines. Although second-site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T-DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development.

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein-protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochondria results in 7%-10% of plants altered in leaf morphology, heat tolerance, and mitochondrial genome stability. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH1. MSH1 depletion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane.

Translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75) is the core component of the chloroplast protein import machinery. It belongs to the Omp85 family whose members exist in various Gram-negative bacteria, mitochondria, and chloroplasts of eukaryotes. Chloroplasts of Viridiplantae contain another Omp85 homolog called outer envelope protein 80 (OEP80), whose exact function is unknown. In addition, the Arabidopsis thaliana genome encodes truncated forms of Toc75 and OEP80. Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent. The bipartite targeting sequence-dependent sorting of Toc75 has been demonstrated but the targeting mechanisms of other chloroplast Omp85 homologs remain largely unexplored. This study was aimed to address these unresolved issues in order to further our understanding of chloroplast evolution. Sequence alignments and recently determined structures of bacterial Omp85 homologs were used to predict structures of chloroplast Omp85 homologs. The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80. Phylogenetic analyses using Omp85 homologs from various cyanobacteria and chloroplasts provided strong support for the grouping of Toc75 and OEP80 sister to cyanoOmp85. However, this support was diminished when the analysis included Omp85 homologs from other bacteria and mitochondria. Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.