Architecture of chloroplast TOC-TIC translocon supercomplex.

Chloroplasts rely on the translocon complexes in the outer and inner envelope membranes (the TOC and TIC complexes, respectively) to import thousands of different nuclear-encoded proteins from the cytosol1-4. Although previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes5-7, the overall architectures of the TOC-TIC supercomplexes and the mechanism of preprotein translocation are unclear. Here we report the cryo-electron microscopy structure of the TOC-TIC supercomplex from Chlamydomonas reinhardtii. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly identified small inner-membrane proteins (Simp1-3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate molecule is located at the Tic214-Toc90 interface and stabilizes their assembly. Four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Multiple potential pathways found in the TOC-TIC supercomplex may support translocation of different substrate preproteins into chloroplasts.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology.

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

BnaPPT1 is essential for chloroplast development and seed oil accumulation in Brassica napus.

INTRODUCTION: Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis. OBJECTIVES: This study investigated PPTs' functions in plant growth and seed oil biosynthesis in oilseed crop Brassica napus. METHODS: We created over-expression and mutant material of BnaPPT1. The plant development, oil content, lipids, metabolites and ultrastructure of seeds were compared to evaluate the gene function. RESULTS: The plastid membrane localized BnaPPT1 was found to be required for normal growth of B. napus. The plants grew slower with yellowish leaves in BnaA08.PPT1 and BnaC08.PPT1 double mutant plants. The results of chloroplast ultrastructural observation and lipid analysis show that BnaPPT1 plays an essential role in membrane lipid synthesis and chloroplast development in leaves, thereby affecting photosynthesis. Moreover, the analysis of primary metabolites and lipids in developing seeds showed that BnaPPT1 could impact seed glycolytic metabolism and lipid level. Knockout of BnaA08.PPT1 and BnaC08.PPT1 resulted in decreasing of the seed oil content by 2.2 to 9.1%, while overexpression of BnaC08.PPT1 significantly promoted the seed oil content by 2.1 to 3.3%. CONCLUSION: Our results suggest that BnaPPT1 is necessary for plant chloroplast development, and it plays an important role in maintaining plant growth and promoting seed oil accumulation in B. napus.

Bioaccessibility of essential lipophilic nutrients in a chloroplast-rich fraction (CRF) from agricultural green waste during simulated human gastrointestinal tract digestion.

An in vitro gastrointestinal human digestion model, with and without additional rapeseed oil, was used to measure the bioaccessibility of the major lipophilic nutrients enriched in chloroplasts: ß-carotene; lutein; α-tocopherol; and α-linolenic acid. Chloroplast-rich fraction (CRF) material for this work was prepared from post-harvest pea vine field residue (pea vine haulm, or PVH), an abundant source of freely available, underutilised green biomass. PVH was either steam sterilised (100 °C for 4 min) and then juiced (heat-treated PVH, or HPVH), or was juiced fresh and the juice heated (90 °C for 3 min) (heat-treated juice, or HJ); the CRF from all biomass treatments was recovered from the juice by centrifugation. The impact of postharvest heat treatment of the biomass (HPVH), or of heat treatment of the juice (HJ) derived from the biomass, on the retention and bioaccessibility of the target nutrients was determined. The results showed that both heat treatments increased the apparent retention of ß-carotene, lutein, α-tocopherol, and α-linolenic acid in the CRF material during digestion. The presence of edible oil during digestion did not dramatically affect the retention of these nutrients, but it did increase the bioaccessibility of ß-carotene, lutein, and α-tocopherol from CRF material derived from heated biomass or juice. The presence of oil also increased the bioaccessibility of ß-carotene, but not of lutein, α-tocopherol, or α-linolenic acid, from fresh CRF material.

Transforming Escherichia coli Proteomembranes into Artificial Chloroplasts Using Molecular Photocatalysis.

During the light-dependent reaction of photosynthesis, green plants couple photoinduced cascades of redox reactions with transmembrane proton translocations to generate reducing equivalents and chemical energy in the form of NADPH (nicotinamide adenine dinucleotide phosphate) and ATP (adenosine triphosphate), respectively. We mimic these basic processes by combining molecular ruthenium polypyridine-based photocatalysts and inverted vesicles derived from Escherichia coli. Upon irradiation with visible light, the interplay of photocatalytic nicotinamide reduction and enzymatic membrane-located respiration leads to the simultaneous formation of two biologically active cofactors, NADH (nicotinamide adenine dinucleotide) and ATP, respectively. This inorganic-biologic hybrid system thus emulates the cofactor delivering function of an active chloroplast.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

Isolation and functional characterization of a glucose-6-phosphate/phosphate translocator (IbG6PPT1) from sweet potato (Ipomoea batatas (L.) Lam.).

Sweet potato (Ipomoea batatas (L.) Lam.) is a good source of carbohydrates, an excellent raw material for starch-based industries, and a strong candidate for biofuel production due to its high starch content. However, the molecular basis of starch biosynthesis and accumulation in sweet potato is still insufficiently understood. Glucose-6-phosphate/phosphate translocators (GPTs) mediate the import of glucose-6-phosphate (Glc6P) into plastids for starch synthesis. Here, we report the isolation of a GPT-encoding gene, IbG6PPT1, from sweet potato and the identification of two additional IbG6PPT1 gene copies in the sweet potato genome. IbG6PPT1 encodes a chloroplast membrane-localized GPT belonging to the GPT1 group and highly expressed in storage root of sweet potato. Heterologous expression of IbG6PPT1 resulted in increased starch content in the leaves, root tips, and seeds and soluble sugar in seeds of Arabidopsis thaliana, but a reduction in soluble sugar in the leaves. These findings suggested that IbG6PPT1 might play a critical role in the distribution of carbon sources in source and sink and the accumulation of carbohydrates in storage tissues and would be a good candidate gene for controlling critical starch properties in sweet potato.

Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. I. 31P-NMR Spectroscopy.

Build-up of the energized state of thylakoid membranes and the synthesis of ATP are warranted by organizing their bulk lipids into a bilayer. However, the major lipid species of these membranes, monogalactosyldiacylglycerol, is a non-bilayer lipid. It has also been documented that fully functional thylakoid membranes, in addition to the bilayer, contain an inverted hexagonal (HII) phase and two isotropic phases. To shed light on the origin of these non-lamellar phases, we performed 31P-NMR spectroscopy experiments on sub-chloroplast particles of spinach: stacked, granum and unstacked, stroma thylakoid membranes. These membranes exhibited similar lipid polymorphism as the whole thylakoids. Saturation transfer experiments, applying saturating pulses at characteristic frequencies at 5 °C, provided evidence for distinct lipid phases-with component spectra very similar to those derived from mathematical deconvolution of the 31P-NMR spectra. Wheat-germ lipase treatment of samples selectively eliminated the phases exhibiting sharp isotropic peaks, suggesting easier accessibility of these lipids compared to the bilayer and the HII phases. Gradually increasing lipid exchanges were observed between the bilayer and the two isotropic phases upon gradually elevating the temperature from 5 to 35 °C, suggesting close connections between these lipid phases. Data concerning the identity and structural and functional roles of different lipid phases will be presented in the accompanying paper.

Protocol for evaluating protein relocalization from the plasma membrane to chloroplasts.

We present a protocol for analyzing and evaluating the relocalization of proteins from the plasma membrane to chloroplasts. Some plant membrane-bound proteins carry dual targeting signals, e.g., a membrane-anchoring N-myristoylation motif and a chloroplast transit peptide. These proteins are predominantly targeted to membranes; upon certain cues, however, they can undergo detachment from membranes and relocalization to chloroplasts. This protocol combines imaging and biochemical analyses to track in a reliable and quantitative manner the relocalization of proteins between subcellular organelles. For complete details on the use and execution of this protocol, please refer to Medina-Puche et al. (2020).

Isolation of Plastoglobules for Lipid Analyses.

Plastoglobules are plastid compartments designed for the storage of neutral lipids. They share physical and structural characteristics with cytosolic lipid droplets. Hence, special care must be taken to avoid contamination by cytosolic lipid droplets during plastoglobule purification. We describe the isolation of pure plastoglobules from Arabidopsis thaliana leaves, and the methods we use to determine their lipid composition. After preparation of a crude chloroplast fraction, plastoglobules are isolated from plastid membranes by two steps of ultracentrifugation on discontinuous sucrose gradients. For lipid analyses, total lipids are then extracted by a standard chloroform-methanol protocol, and polar lipids are separated from neutral lipids by liquid-liquid extraction. While polar lipid classes are subsequently separated by thin-layer chromatography (TLC) with the classical Vitiello solvent mix, a double TLC development has to be performed for neutral lipids, to separate phytyl and steryl esters. Lipids are quantified by gas chromatography after conversion of the fatty acids into methyl esters.

Identification of Chlorophyll Metabolism- and Photosynthesis-Related Genes Regulating Green Flower Color in Chrysanthemum by Integrative Transcriptome and Weighted Correlation Network Analyses.

Green chrysanthemums are difficult to breed but have high commercial value. The molecular basis for the green petal color in chrysanthemum is not fully understood. This was investigated in the present study by RNA sequencing analysis of white and green ray florets collected at three stages of flower development from the F1 progeny of the cross between Chrysanthemum × morifolium "Lüdingdang" with green-petaled flowers and Chrysanthemum vistitum with white-petaled flowers. The chlorophyll content was higher and chloroplast degradation was slower in green pools than in white pools at each developmental stage. Transcriptome analysis revealed that genes that were differentially expressed between the two pools were enriched in pathways related to chlorophyll metabolism and photosynthesis. We identified the transcription factor genes CmCOLa, CmCOLb, CmERF, and CmbHLH as regulators of the green flower color in chrysanthemum by differential expression analysis and weighted gene co-expression network analysis. These findings can guide future efforts to improve the color palette of chrysanthemum flowers through genetic engineering.

Antioxidant and Signaling Role of Plastid-Derived Isoprenoid Quinones and Chromanols.

Plant prenyllipids, especially isoprenoid chromanols and quinols, are very efficient low-molecular-weight lipophilic antioxidants, protecting membranes and storage lipids from reactive oxygen species (ROS). ROS are byproducts of aerobic metabolism that can damage cell components, they are also known to play a role in signaling. Plants are particularly prone to oxidative damage because oxygenic photosynthesis results in O2 formation in their green tissues. In addition, the photosynthetic electron transfer chain is an important source of ROS. Therefore, chloroplasts are the main site of ROS generation in plant cells during the light reactions of photosynthesis, and plastidic antioxidants are crucial to prevent oxidative stress, which occurs when plants are exposed to various types of stress factors, both biotic and abiotic. The increase in antioxidant content during stress acclimation is a common phenomenon. In the present review, we describe the mechanisms of ROS (singlet oxygen, superoxide, hydrogen peroxide and hydroxyl radical) production in chloroplasts in general and during exposure to abiotic stress factors, such as high light, low temperature, drought and salinity. We highlight the dual role of their presence: negative (i.e., lipid peroxidation, pigment and protein oxidation) and positive (i.e., contribution in redox-based physiological processes). Then we provide a summary of current knowledge concerning plastidic prenyllipid antioxidants belonging to isoprenoid chromanols and quinols, as well as their structure, occurrence, biosynthesis and function both in ROS detoxification and signaling.

Crystal structure of higher plant heme oxygenase-1 and its mechanism of interaction with ferredoxin.

Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.

Response of chloroplast pigments, sugars and phenolics of sweet cherry leaves to chilling.

The aim of the present study was to evaluate the effect of post-flowering chilling of sweet cherry (Prunus avium L.) on the content of biochemical parameters in the leaf (chloroplast pigments, sugars and phenolics). The effect of chilling was investigated in two experiments. Potted 2-year-old trees of cv. 'Grace Star' and 'Schneiders' were exposed to one, two or three consecutive overnight chillings at an average air temperature of 4.7 °C (Experiment I), but in the following year only trees of 'Grace Star' were chilled at 2.2 °C (Experiment II), 3 to 7 weeks after flowering. The analysis of the biochemical parameters was performed by high performance liquid chromatography combined with electrospray ionization mass spectrometry. Chilling at 4.7 °C caused little or no stress, while 2.2 °C induced more intense stress with increased zeaxanthin, sugar and phenolic content in leaves, while exposure of trees to higher temperatures and closer to flowering showed no changes. Two or three consecutive overnight chilling periods increased the phenolic content and enhanced the accumulation of zeaxanthin in the leaves. Sucrose, sorbitol, fructose, total sugar, and total flavonoid content in leaves increased within 48 h after chilling. Zeaxanthin epoxidized within 24 h after one and 48 h after one and two consecutive overnight chillings.

Head-Group Acylation of Chloroplast Membrane Lipids.

Head group-acylated chloroplast lipids were discovered in the 1960s, but interest was renewed about 15 years ago with the discovery of Arabidopsides E and G, acylated monogalactosyldiacylglycerols with oxidized fatty acyl chains originally identified in Arabidopsis thaliana. Since then, plant biologists have applied the power of mass spectrometry to identify additional oxidized and non-oxidized chloroplast lipids and quantify their levels in response to biotic and abiotic stresses. The enzyme responsible for the head-group acylation of chloroplast lipids was identified as a cytosolic protein closely associated with the chloroplast outer membrane and christened acylated galactolipid-associated phospholipase 1 (AGAP1). Despite many advances, critical questions remain about the biological functions of AGAP1 and its head group-acylated products.

Photosynthesis-assisted remodeling of three-dimensional printed structures.

The mechanical properties of engineering structures continuously weaken during service life because of material fatigue or degradation. By contrast, living organisms are able to strengthen their mechanical properties by regenerating parts of their structures. For example, plants strengthen their cell structures by transforming photosynthesis-produced glucose into stiff polysaccharides. In this work, we realize hybrid materials that use photosynthesis of embedded chloroplasts to remodel their microstructures. These materials can be used to three-dimensionally (3D)-print functional structures, which are endowed with matrix-strengthening and crack healing when exposed to white light. The mechanism relies on a 3D-printable polymer that allows for an additional cross-linking reaction with photosynthesis-produced glucose in the material bulk or on the interface. The remodeling behavior can be suspended by freezing chloroplasts, regulated by mechanical preloads, and reversed by environmental cues. This work opens the door for the design of hybrid synthetic-living materials, for applications such as smart composites, lightweight structures, and soft robotics.

Expressing Phosphate Transporter PvPht2;1 Enhances P Transport to the Chloroplasts and Increases Arsenic Tolerance in Arabidopsis thaliana.

Arsenic (As) contamination in soils is of great concerns due to its toxicity to plants. As an analogue, phosphorus plays an important role in protecting plants from As toxicity. In this study, we identified a new phosphate transporter 2 (PHT2), PvPht2;1, from As-hyperaccumulator Pteris vittata and analyzed its functions in As and P transport in a yeast mutant, and model plant Arabidopsis thalian. PvPht2;1 contained 12 transmembrane domains, sharing high identity with PHT2 genes in diverse plants. Further, independent of external P or As levels, PvPht2;1 was mainly expressed in P. vittata fronds with the expression being 3-4 folds higher than that in the roots and rhizomes. Localized to the chloroplasts based on GFP-fused PvPht2;1 in model plant tobacco, PvPht2;1 functioned as a low-affinity P transporter. Under As exposure, PvPht2;1 yeast transformants showed comparable growth with the control while high-affinity P transporter PvPht1;3 transformants showed better growth, suggesting that PvPht2;1 transported P but slower than PvPht1;3 transporter. Expressing PvPht2;1 in A. thaliana increased its shoot P concentration without influencing its As accumulation. Further, the chloroplasts' P content in transgenic A. thaliana increased by 37-59% than wild-type (WT) plants. Under As exposure, the photosynthesis of PvPht2;1-expressing A. thaliana remained stable but that of WT plants decreased. The data indicate that, under As stress, expressing PvPht2;1 in A. thaliana enhanced its P transport to the chloroplasts and protected its photosynthesis. In short, highly expressed in the fronds and not impacted by As exposure, chloroplast-located PvPht2;1 may have protected As-hyperaccumulator P. vittata from As toxicity by efficiently transporting only P to its chloroplasts.

Effect of steam sterilisation on lipophilic nutrient stability in a chloroplast-rich fraction (CRF) recovered from postharvest, pea vine field residue (haulm).

Postharvest, pea vine field residue (haulm) was steam-sterilised and then juiced; a chloroplast-rich fraction (CRF) was recovered from the juice by centrifugation. The stability of selected nutrients (ß-carotene, lutein, and α-tocopherol) in the freeze-dried CRF material was measured over 84 days; the impact of temperature (-20 °C, 4 °C, 25 °C and 40 °C), light and air on nutrient stability was established. All three nutrients were stable at -20 °C and 4 °C in the presence or absence of air; this stability was lost at higher temperatures in the presence of air. The extent and rate of nutrient breakdown significantly increased when the CRF samples were exposed to light. ß-Carotene appeared to be more susceptible to degradation than lutein and α-tocopherol at 40 °C in the presence of air, but when CRF was exposed to light all three nutrients measured were significantly broken down during storage at 25 °C or 40 °C, whether exposed to air or not.

Dual role of the active site 'lid' regions of protochlorophyllide oxidoreductase in photocatalysis and plant development.

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR-Pchlide-NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo-electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation - a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.

Ectopic expression of human apolipoprotein D in Arabidopsis plants lacking chloroplastic lipocalin partially rescues sensitivity to drought and oxidative stress.

The chloroplastic lipocalin (LCNP) is induced in response to various abiotic stresses including high light, dehydration and low temperature. It contributes to protection against oxidative damage promoted by adverse conditions by preventing accumulation of fatty acid hydroperoxides and lipid peroxidation. In contrast to animal lipocalins, LCNP is poorly characterized and the molecular mechanism by which it exerts protective effects during oxidative stress is largely unknown. LCNP is considered the ortholog of human apolipoprotein D (APOD), a protein whose lipid antioxidant function has been characterized. Here, we investigated whether APOD could functionally replace LCNP in Arabidopsis thaliana. We introduced APOD cDNA fused to a chloroplast transit peptide encoding sequence in an Arabidopsis LCNP KO mutant line and challenged the transgenic plants with different abiotic stresses. We demonstrated that expression of human APOD in Arabidopsis can partially compensate for the lack of the plastid lipocalin. The results are consistent with a conserved function of APOD and LCNP under stressful conditions. However, if the results obtained with the drought and oxidative stresses point to the protective effect of constitutive expression of APOD in plants lacking LCNP, this effect is not as effective as that conferred by LCNP overexpression. Moreover, when investigating APOD function in thylakoids after high light stress at low temperature, it appeared that APOD could not contribute to qH, a slowly reversible form of non-photochemical chlorophyll fluorescence quenching, as described for LCNP. This work provides a base of understanding the molecular mechanism underlying LCNP protective function.