Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

Ferredoxin C2 is required for chlorophyll biosynthesis and accumulation of photosynthetic antennae in Arabidopsis.

Ferredoxins (Fd) are small iron-sulphur proteins, with sub-types that have evolved for specific redox functions. Ferredoxin C2 (FdC2) proteins are essential Fd homologues conserved in all photosynthetic organisms and a number of different FdC2 functions have been proposed in angiosperms. Here we use RNAi silencing in Arabidopsis thaliana to generate a viable fdC2 mutant line with near-depleted FdC2 protein levels. Mutant leaves have ~50% less chlorophyll a and b, and chloroplasts have poorly developed thylakoid membrane structure. Transcriptomics indicates upregulation of genes involved in stress responses. Although fdC2 antisense plants show increased damage at photosystem II (PSII) when exposed to high light, PSII recovers at the same rate as wild type in the dark. This contradicts literature proposing that FdC2 regulates translation of the D1 subunit of PSII, by binding to psbA transcript. Measurement of chlorophyll biosynthesis intermediates revealed a build-up of Mg-protoporphyrin IX, the substrate of the aerobic cyclase. We localise FdC2 to the inner chloroplast envelope and show that the FdC2 RNAi line has a disproportionately lower protein abundance of antennae proteins, which are nuclear-encoded and must be refolded at the envelope after import.

Unraveling Hidden Components of the Chloroplast Envelope Proteome: Opportunities and Limits of Better MS Sensitivity.

The chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling the exchange of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides etc.) of the plant cell. However, unraveling the contents of the chloroplast envelope proteome remains a difficult challenge; many proteins constituting this functional double membrane system remain to be identified. Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.4% of the whole cell proteome). In other words, most envelope proteins are so rare at the cell, chloroplast, or even envelope level, that they remained undetectable using targeted MS studies. Cross-contamination of chloroplast subcompartments by each other and by other cell compartments during cell fractionation, impedes accurate localization of many envelope proteins. The aim of the present study was to take advantage of technologically improved MS sensitivity to better define the proteome of the chloroplast envelope (differentiate genuine envelope proteins from contaminants). This MS-based analysis relied on an enrichment factor that was calculated for each protein identified in purified envelope fractions as compared with the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1269 proteins were detected in purified envelope fractions, of which, 462 could be assigned an envelope localization by combining MS-based spectral count analyses with manual annotation using data from the literature and prediction tools. Many of such proteins being previously unknown envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.

Calmodulin is involved in the dual subcellular location of two chloroplast proteins.

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

The N-terminal and third transmembrane domain of PsCor413im1 are essential for targeting to chloroplast envelope membrane.

Cold-regulated (COR) genes, located downstream of the C-repeat binding factors (CBFs) in cold signaling pathways, play a central role in plant response to cold stress. In our previous studies, a Cor413 chloroplast envelope membrane protein, PsCor413im1, was identified from the cold-tolerant plant Phlox subulata. Its overexpression enhanced cold tolerance and altered AtCor15 expression in Arabidopsis. In the present study, the function of PsCor413im1 was further investigated. Transmission electron microscope observation showed that the chloroplast envelope membrane of cold-treated transgenic Arabidopsis seedlings was more stable than that of cold-treated wild-type seedlings. Subcellular localization of green fluorescent protein as a marker revealed that the N-terminal and putative third transmembrane domain (TMD) of PsCor413im1 were essential for its targeting of the chloroplast envelope membrane. Furthermore, overexpression of PsCor413im1 fragments containing N-terminal and third TMD also altered the expression of AtCor15 genes in Arabidopsis. Overall, our results suggest that PsCor413im1 may stabilize the chloroplast envelope membrane under cold stress, and its N-terminal and third TMD are important for its targeting capability and function.

Sequential regiospecific gem-diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

Polyprenylated acylphloroglucinol derivatives, such as xanthones, are natural plant products with interesting pharmacological properties. They are difficult to synthesize chemically. Biotechnological production is desirable but it requires an understanding of the biosynthetic pathways. cDNAs encoding membrane-bound aromatic prenyltransferase (aPT) enzymes from Hypericum sampsonii seedlings (HsPT8px and HsPTpat) and Hypericum calycinum cell cultures (HcPT8px and HcPTpat) were cloned and expressed in Saccharomyces cerevisiae and Nicotiana benthamiana, respectively. Microsomes and chloroplasts were used for functional analysis. The enzymes catalyzed the prenylation of 1,3,6,7-tetrahydroxyxanthone (1367THX) and/or 1,3,6,7-tetrahydroxy-8-prenylxanthone (8PX) and discriminated nine additionally tested acylphloroglucinol derivatives. The transient expression of the two aPT genes preceded the accumulation of the products in elicitor-treated H. calycinum cell cultures. C-terminal yellow fluorescent protein fusions of the two enzymes were localized to the envelope of chloroplasts in N. benthamiana leaves. Based on the kinetic properties of HsPT8px and HsPTpat, the enzymes catalyze sequential rather than parallel addition of two prenyl groups to the carbon atom 8 of 1367THX, yielding gem-diprenylated patulone under loss of aromaticity of the gem-dialkylated ring. Coexpression in yeast significantly increased product formation. The patulone biosynthetic pathway involves multiple subcellular compartments. The aPTs studied here and related enzymes may be promising tools for plant/microbe metabolic pathway engineering.

Tribute Roland Douce, 1939-2018.

On November 4, 2018, Roland Douce, Professor Emeritus at the University of Grenoble, France, died at the age of 79. In Grenoble, where he spent most of his scientific career, Roland Douce created a world-renowned school of plant science, studying the structure, functions, and interactions of plant organelles involved in photosynthesis, respiration, and photorespiration. His main achievements concern the chemical and functional characterization of chloroplast envelope membranes, the demonstration of the uniqueness of plant mitochondria, and the integration of metabolism within the plant cell, among manifold activities. Roland Douce devoted his whole life to science and research with passion and enthusiasm: he was a true charismatic leader.

Substrate channeling in oxylipin biosynthesis through a protein complex in the plastid envelope of Arabidopsis thaliana.

Oxygenated membrane fatty acid derivatives termed oxylipins play important roles in plant defense against biotic and abiotic cues. Plants challenged by insect pests, for example, synthesize a blend of different defense compounds that include volatile aldehydes and jasmonic acid (JA), among others. Because all oxylipins are derived from the same pathway, we investigated how their synthesis might be regulated, focusing on two closely related atypical cytochrome P450 enzymes designated CYP74A and CYP74B, respectively, allene oxide synthase (AOS) and hydroperoxide lyase (HPL). These enzymes compete for the same substrate but give rise to different products: the final product of the AOS branch of the oxylipin pathway is JA, while those of the HPL branch comprise volatile aldehydes and alcohols. AOS and HPL are plastid envelope enzymes in Arabidopsis thaliana but accumulate at different locations. Biochemical experiments identified AOS as a constituent of complexes also containing lipoxygenase 2 (LOX2) and allene oxide cyclase (AOC), which catalyze consecutive steps in JA precursor biosynthesis, while excluding the concurrent HPL reaction. Based on published X-ray data, the structure of this complex was modelled and amino acids involved in catalysis and subunit interactions predicted. Genetic studies identified the microRNA 319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes and CORONATINE INSENSITIVE 1 (COI1) as controlling JA production through the LOX2-AOS-AOC2 complex. Together, our results define a molecular branch point in oxylipin biosynthesis that allows fine-tuning of the plant's defense machinery in response to biotic and abiotic stimuli.

Programmed chloroplast destruction during leaf senescence involves 13-lipoxygenase (13-LOX).

Leaf senescence is the terminal stage in the development of perennial plants. Massive physiological changes occur that lead to the shut down of photosynthesis and a cessation of growth. Leaf senescence involves the selective destruction of the chloroplast as the site of photosynthesis. Here, we show that 13-lipoxygenase (13-LOX) accomplishes a key role in the destruction of chloroplasts in senescing plants and propose a critical role of its NH2-terminal chloroplast transit peptide. The 13-LOX enzyme identified here accumulated in the plastid envelope and catalyzed the dioxygenation of unsaturated membrane fatty acids, leading to a selective destruction of the chloroplast and the release of stromal constituents. Because 13-LOX pathway products comprise compounds involved in insect deterrence and pathogen defense (volatile aldehydes and oxylipins), a mechanism of unmolested nitrogen and carbon relocation is suggested that occurs from leaves to seeds and roots during fall.

ALLENE OXIDE SYNTHASE and HYDROPEROXIDE LYASE, Two Non-Canonical Cytochrome P450s in Arabidopsis thaliana and Their Different Roles in Plant Defense.

The channeling of metabolites is an essential step of metabolic regulation in all living organisms. Multifunctional enzymes with defined domains for metabolite compartmentalization are rare, but in many cases, larger assemblies forming multimeric protein complexes operate in defined metabolic shunts. In Arabidopsis thaliana, a multimeric complex was discovered that contains a 13-lipoxygenase and allene oxide synthase (AOS) as well as allene oxide cyclase. All three plant enzymes are localized in chloroplasts, contributing to the biosynthesis of jasmonic acid (JA). JA and its derivatives act as ubiquitous plant defense regulators in responses to both biotic and abiotic stresses. AOS belongs to the superfamily of cytochrome P450 enzymes and is named CYP74A. Another CYP450 in chloroplasts, hydroperoxide lyase (HPL, CYP74B), competes with AOS for the common substrate. The products of the HPL reaction are green leaf volatiles that are involved in the deterrence of insect pests. Both enzymes represent non-canonical CYP450 family members, as they do not depend on O2 and NADPH-dependent CYP450 reductase activities. AOS and HPL activities are crucial for plants to respond to different biotic foes. In this mini-review, we aim to summarize how plants make use of the LOX2-AOS-AOC2 complex in chloroplasts to boost JA biosynthesis over volatile production and how this situation may change in plant communities during mass ingestion by insect pests.

Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii.

The supply of inorganic carbon (Ci; CO2 and HCO3 (-)) is an environmental rate-limiting factor in aquatic photosynthetic organisms. To overcome the difficulty in acquiring Ci in limiting-CO2 conditions, an active Ci uptake system called the CO2-concentrating mechanism (CCM) is induced to increase CO2 concentrations in the chloroplast stroma. An ATP-binding cassette transporter, HLA3, and a formate/nitrite transporter homolog, LCIA, are reported to be associated with HCO3 (-) uptake [Wang and Spalding (2014) Plant Physiol 166(4):2040-2050]. However, direct evidence of the route of HCO3 (-) uptake from the outside of cells to the chloroplast stroma remains elusive owing to a lack of information on HLA3 localization and comparative analyses of the contribution of HLA3 and LCIA to the CCM. In this study, we revealed that HLA3 and LCIA are localized to the plasma membrane and chloroplast envelope, respectively. Insertion mutants of HLA3 and/or LCIA showed decreased Ci affinities/accumulation, especially in alkaline conditions where HCO3 (-) is the predominant form of Ci. HLA3 and LCIA formed protein complexes independently, and the absence of LCIA decreased HLA3 mRNA accumulation, suggesting the presence of unidentified retrograde signals from the chloroplast to the nucleus to maintain HLA3 mRNA expression. Furthermore, although single overexpression of HLA3 or LCIA in high CO2 conditions did not affect Ci affinity, simultaneous overexpression of HLA3 with LCIA significantly increased Ci affinity/accumulation. These results highlight the HLA3/LCIA-driven cooperative uptake of HCO3 (-) and a key role of LCIA in the maintenance of HLA3 stability as well as Ci affinity/accumulation in the CCM.

Single-Pixel Densitometry Revealed the Presence of Peptidoglycan in the Intermembrane Space of the Moss Chloroplast Envelope in Conventional Electron Micrographs.

Chloroplasts are believed to be descendants of ancestral cyanobacteria that have a peptidoglycan layer between the outer and the inner membranes. In particular, cyanelles having peptidoglycan in Cyanophora paradoxa are considered as evidence for the endosymbiotic origin of chloroplasts. The moss Physcomitrella patens has a complete set of genes involved in the synthesis of peptidoglycan, but a peptidoglycan layer has not been observed by conventional electron microscopy to date. Recently, a new metabolic labeling technique using a fluorescent probe was applied to visualize putative peptidoglycan surrounding the chloroplasts. The exact localization of the peptidoglycan, however, has not been clearly identified. Here we examined conventional electron micrographs of two types of moss materials (mutants or ampicillin-treated plants), one presumably having peptidoglycan and the other presumably lacking peptidoglycan, and analyzed in detail, by single-pixel densitometry, the electron density of the chloroplast envelope membranes and the intermembrane space. Statistical analysis showed that the relative electron density within the intermembrane space with respect to that of the envelope membranes was significantly higher in the materials presumably having peptidoglycan than in the materials presumably devoid of peptidoglycan. We consider this difference as bona fide evidence for the presence of peptidoglycan between the outer and the inner envelope membranes in the wild-type chloroplasts of the moss, although its density is lower than that in bacteria and cyanelles. We will also discuss this low-density peptidoglycan in the light of the phylogenetic origin of peptidoglycan biosynthesis enzymes.

The Sec2 translocase of the chloroplast inner envelope contains a unique and dedicated SECE2 component.

Biogenesis of chloroplasts involves a series of protein trafficking events. Nuclear-encoded proteins are imported into the organelle, and then trafficked to various chloroplast locations by systems that are directly homologous to bacterial systems. Although the thylakoid-based systems have been studied extensively, much less is known about the systems that reside and function in the inner envelope membrane. One such system, the Sec2 system, is homologous to both the thylakoid-based Sec1 system and bacterial Sec systems, and may mediate both integration and translocation across the inner envelope. At a minimum, this system is expected to include three components, but only two, SCY2 and SECA2, have been identified in Arabidopsis. Bioinformatics and protein modeling were used to identify the protein encoded by At4g38490 as a candidate for the missing component (SECE2). Cellular localization, biochemistry, protein interaction assays in yeast, and co-immunoprecipitation experiments were used to establish that this protein is an integral membrane protein of the inner envelope, and specifically interacts with the SCY2 component in vivo. Sequence analyses indicated that SECE2 proteins are found in a variety of plants, and differ from the thylakoid SECE1 proteins in a stroma-exposed helical domain, which may contribute to their specificity. Finally, a genetic analysis indicated that SECE2 plays an essential role in plant growth and development.

Functional characterization of the chloroplast ferric chelate oxidoreductase enzyme.

Iron (Fe) has an essential role in the biosynthesis of chlorophylls and redox cofactors, and thus chloroplast iron uptake is a process of special importance. The chloroplast ferric chelate oxidoreductase (cFRO) has a crucial role in this process but it is poorly characterized. To study the localization and mechanism of action of cFRO, sugar beet (Beta vulgaris cv Orbis) chloroplast envelope fractions were isolated by gradient ultracentrifugation, and their purity was tested by western blotting against different marker proteins. The ferric chelate reductase (FCR) activity of envelope fractions was studied in the presence of NAD(P)H (reductants) and FAD coenzymes. Reduction of Fe(III)-ethylenediaminetetraacetic acid was monitored spectrophotometrically by the Fe(II)-bathophenanthroline disulfonate complex formation. FCR activity, that is production of free Fe(II) for Fe uptake, showed biphasic saturation kinetics, and was clearly associated only to chloroplast inner envelope (cIE) vesicles. The reaction rate was > 2.5 times higher with NADPH than with NADH, which indicates the natural coenzyme preference of cFRO activity and its dependence on photosynthesis. FCR activity of cIE vesicles isolated from Fe-deficient plants also showed clear biphasic kinetics, where the KM of the low affinity component was elevated, and thus this component was down-regulated.

Transplastomic integration of a cyanobacterial bicarbonate transporter into tobacco chloroplasts.

Improving global yields of agricultural crops is a complex challenge with evidence indicating benefits in productivity are achieved by enhancing photosynthetic carbon assimilation. Towards improving rates of CO2 capture within leaf chloroplasts, this study shows the versatility of plastome transformation for expressing the Synechococcus PCC7002 BicA bicarbonate transporter within tobacco plastids. Fractionation of chloroplast membranes from transplastomic tob(BicA) lines showed that ~75% of the BicA localized to the thylakoid membranes and ~25% to the chloroplast envelope. BicA levels were highest in young emerging tob(BicA) leaves (0.12 µmol m(-2), ≈7mg m(-2)) accounting for ~0.1% (w/w) of the leaf protein. In these leaves, the molar amount of BicA was 16-fold lower than the abundant thylakoid photosystem II D1 protein (~1.9 µmol m(-2)) which was comparable to the 9:1 molar ratio of D1:BicA measured in air-grown Synechococcus PCC7002 cells. The BicA produced had no discernible effect on chloroplast ultrastructure, photosynthetic CO2-assimilation rates, carbon isotope discrimination, or growth of the tob(BicA) plants, implying that the bicarbonate transporter had little or no activity. These findings demonstrate the utility of plastome transformation for targeting bicarbonate transporter proteins into the chloroplast membranes without impeding growth or plastid ultrastructure. This study establishes the span of experimental measurements required to verify heterologous bicarbonate transporter function and location in chloroplasts and underscores the need for more detailed understanding of BicA structure and function to identify solutions for enabling its activation and operation in leaf chloroplasts.

Isolation of the Inner and Outer Membranes of the Chloroplast Envelope from Angiosperms.

The outer and the inner membranes of the chloroplast envelope, also called OEM and IEM, have distinct lipid and protein compositions. They host molecular systems involved in the biogenesis of the organelle, its cellular function, and its communication with other compartments. Here we describe a method for the isolation of these two membranes starting from intact chloroplast preparations, with two alternative procedures based on the starting material. One was developed from spinach leaves, the other from pea leaves. The two procedures differ in the method used to isolate and rupture chloroplasts and separate each membrane.

Functional Analysis of Chloroplast Iron Uptake and Homeostasis.

Iron has a crucial role in plastid biology. Iron is a required cofactor for the operation of the photosynthetic functions and other metabolic pathways. Despite the importance of the iron homeostasis in chloroplasts, the functional analysis of the plastidial iron uptake and homeostasis still lack a consensus methodology. Here, we describe a sequence of subsequent techniques that can be applied in functional characterization of proteins involved in iron uptake and incorporation into chloroplasts as well as of the non-transport protein members of the chloroplast iron homeostasis. Since the ferrous iron ligation of bathophenantroline disulfonate is specific and not disrupted by the presence of other transition metals, it offers a simple way for iron quantification both in solubilized chloroplast samples as well as in ferric chelate reductase activity measurements.

Simulated Analysis of Influence of Changes in H+-ATPase Activity and Membrane CO2 Conductance on Parameters of Photosynthetic Assimilation in Leaves.

Photosynthesis is an important process in plants which influences their development and productivity. Many factors can control the efficiency of photosynthesis, including CO2 conductance of leaf mesophyll, which affects the CO2 availability for Rubisco. It is known that electrical stress signals can decrease this conductance, and the response is probably caused by inactivation of H+-ATPase in the plasma membrane. In the current work, we analyzed the influence of both CO2 conductance in the plasma membrane, and chloroplast envelopes and H+-ATPase activity on photosynthetic CO2 assimilation, using a two-dimensional mathematical model of photosynthesis in leaves. The model included a description of assimilation on the basis of the Farquhar-von Caemmerer-Berry model, ion transport through the plasma membrane, diffusion of CO2 in the apoplast, and transport of CO2 through the plasma membrane and chloroplast envelope. The model showed that the photosynthetic CO2 assimilation rate was mainly dependent on the plasma membrane and chloroplast envelope conductance; direct influence of the H+-ATPase activity (through changes in pH and CO2/HCO3- concentration ratio) on this rate was weak. In contrast, both changes in CO2 conductance of the plasma membrane and chloroplast envelopes and changes in the H+-ATPase activity influenced spatial heterogeneity of the CO2 assimilation on the leaf surface in the simulated two-dimensional system. These effects were also observed under simultaneous changes in the CO2 conductance of the plasma membrane and H+-ATPase activity. Qualitatively similar influence of changes in the CO2 conductance of the plasma membrane and chloroplast envelopes, and changes in the H+-ATPase activity on photosynthesis were shown for two different densities of stomata in the simulated leaf; however, lowering the density of stomata decreased the assimilation rate and increased the heterogeneity of assimilation. The results of the model analysis clarify the potential influence of H+-ATPase inactivation on photosynthesis, and can be the basis for development of new methods for remote sensing of the influence of electrical signals.

OPDAT1, a plastid envelope protein involved in 12-oxo-phytodienoic acid export for jasmonic acid biosynthesis in Populus.

Twelve-oxo-phytodienoic acid (OPDA), the cyclopentenone precursor of jasmonic acid (JA), is required for the wounding response of plants. OPDA is derived from plastid-localized α-linolenic acid (α-LeA; 18:3) via the octadecanoid pathway, and is further exported from plastids to the cytosol for JA biosynthesis. However, the mechanism of OPDA transport from plastids has yet to be elucidated. In the current study, a plastid inner envelope-localized protein, designated 12-oxo-Phtyodienoic Acid Transporter 1 (OPDAT1), was identified and shown to potentially be involved in OPDA export from plastids, in Populus trichocarpa. Torr. OPDAT1 is expressed predominantly in young leaves of P. trichocarpa. Functional expression of OPDAT1 in yeast cells revealed that OPDAT1 is involved in OPDA transport. Loss-of-function of OPDAT1 in poplar resulted in increased accumulation of OPDA in the extracted plastids and a reduction in JA concentration, whereas an OPDAT1-overexpressing line showed a reverse tendency in OPDA accumulation and JA biosynthesis. OPDAT1 transcripts were rapidly induced by mechanical wounding of leaves, and an opdat1 mutant transgenic plant displayed increased susceptibility to spider mite (Tetranychus urticae) infestation. Collectively, these data suggest that OPDAT1 is an inner envelope transporter for OPDA, and this has potential implications for JA biosynthesis in poplar under environmental stresses.

Chloroplast import of an intermembrane space protein is facilitated by translocon components Toc75 and Tic236.

Chloroplasts are divided into six subcompartments: the outer membrane, intermembrane space, and inner membrane of the envelope, the stroma, the thylakoid membrane, and the thylakoid lumen. Compared with our knowledge of protein import into other subcompartments, extremely little is known about how proteins are imported into the intermembrane space of the envelope. Tic22 was one of the first proteins identified as localizing to the intermembrane space and the only one for which import has been analyzed in some detail. However, conflicting results have been obtained concerning whether the general translocon is used to import Tic22 into the intermembrane space. Taking advantage of available translocon component mutants, we reanalyzed import of Tic22. We reveal reduced in vitro import of Tic22 preprotein (prTic22) into chloroplasts isolated from the Arabidopsis mar1 and tic236 mutants, which are functional knockdown mutants of the outer-membrane channel Toc75 and the intermembrane space linker Tic236, respectively. Import competition experiments also showed that prTic22 import was reduced by excess amounts of a stroma-targeted preprotein. Our results indicate that prTic22 uses at least part of the general translocon for import into the intermembrane space.