Chloroplasts Of Plant Cells Research Articles

Purification of Chloroplast Envelope, Thylakoids, and Stroma from Angiosperm Leaves.

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.

Mitochondrial DNA: From Forensic Genetics to the Origins of Humanity

Mitochondria are cellular organelles that provide cellular energy. Along with chloroplasts of plant cells, mitochondria are the only organelles that have genetic material. These peculiarities make mitochondrial DNA (DNA) one of the main research topics in the field of Cytology and Genetics. Interest shared by Criminalistics, which uses mDNA analysis for forensic identification of DNA traces in which nuclear DNA analysis is not possible. In addition to being used in forensic investigation, mtDNA analysis has also been widely used in paleoanthropological investigation through the elaboration of human phylogenies, which retroact to only one female ancestor, known as mitochondrial Eve. In this context, this manuscript aims to address the relevance of this organelle in the field of Criminalistics and the Sciences of Origins, presenting the scientific, philosophical and theological implications arising from the in-depth analysis of this cellular structure.

Chloroplasts in plant cells show active glassy behavior under low-light conditions

Plants have developed intricate mechanisms to adapt to changing light conditions. Besides phototropism and heliotropism (differential growth toward light and diurnal motion with respect to sunlight, respectively), chloroplast motion acts as a fast mechanism to change the intracellular structure of leaf cells. While chloroplasts move toward the sides of the plant cell to avoid strong light, they accumulate and spread out into a layer on the bottom of the cell at low light to increase the light absorption efficiency. Although the motion of chloroplasts has been studied for over a century, the collective organelle motion leading to light-adapting self-organized structures remains elusive. Here, we study the active motion of chloroplasts under dim-light conditions, leading to an accumulation in a densely packed quasi-2D layer. We observe burst-like rearrangements and show that these dynamics resemble systems close to the glass transition by tracking individual chloroplasts. Furthermore, we provide a minimal mathematical model to uncover relevant system parameters controlling the stability of the dense configuration of chloroplasts. Our study suggests that the meta-stable caging close to the glass transition in the chloroplast monolayer serves a physiological relevance: Chloroplasts remain in a spread-out configuration to increase the light uptake but can easily fluidize when the activity is increased to efficiently rearrange the structure toward an avoidance state. Our research opens questions about the role that dynamical phase transitions could play in self-organized intracellular responses of plant cells toward environmental cues.

Structural features of chloroplast trigger factor determined at 2.6 Å resolution.

The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis-trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Å resolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts.

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants.

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme’s sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle’s genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an ‘Fe-only’ nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.

Hydrogen sulfide: An emerging signaling molecule regulating drought stress response in plants.

Hydrogen sulfide (H2 S) is a small, reactive signaling molecule that is produced within chloroplasts of plant cells as an intermediate in the assimilatory sulfate reduction pathway by the enzyme sulfite reductase. In addition, H2 S is also produced in cytosol and mitochondria by desulfhydration of l-cysteine catalyzed by l-cysteine desulfhydrase (DES1) in the cytosol and from β-cyanoalanine in mitochondria, in a reaction catalyzed by β-cyano-Ala synthase C1 (CAS-C1). H2 S exerts its numerous biological functions by post-translational modification involving oxidation of cysteine residues (RSH) to persulfides (RSSH). At lower concentrations (10-1000 μmol L-1 ), H2 S shows huge agricultural potential as it increases the germination rate, the size, fresh weight, and ultimately the crop yield. It is also involved in abiotic stress response against drought, salinity, high temperature, and heavy metals. H2 S donor, for example, sodium hydrosulfide (NaHS), has been exogenously applied on plants by various researchers to provide drought stress tolerance. Exogenous application results in the accumulation of polyamines, sugars, glycine betaine, and enhancement of the antioxidant enzyme activities in response to drought-induced osmotic and oxidative stress, thus, providing stress adaptation to plants. At the biochemical level, administration of H2 S donors reduces malondialdehyde content and lipoxygenase activity to maintain the cell integrity, causes abscisic acid-mediated stomatal closure to prevent water loss through transpiration, and accelerates the photosystem II repair cycle. Here, we review the crosstalk of H2 S with secondary messengers and phytohormones towards the regulation of drought stress response and emphasize various approaches that can be addressed to strengthen research in this area.

Endosymbiotic Origin of Chloroplasts in Plant Cells’ Evolution

The theory of endosymbiotic origin of chloroplasts has become basal in present-day biology. In this regard, the emergence of eukaryotic photosynthesis has been established as a result of phagocytal capture of cyanobacteria by some ancestral eukaryotic cell. The phenomenon of endosymbiosis is an indispensable element of cell evolution theory. The process of endosymbiogenesis belongs to the crucial factors of biological progress that gave rise to astonishing diversity and complexity of the eukaryotic organisms. The modern theory of evolution is built on the joint base of the phenomena of endosymbiosis, parallel gene transfer, the theory of natural selection, and the data of phylogenomics. Ultimately, eukaryogenesis has integrated different supramolecular complexes in the cells, created the diversity of organelles, and has led to biosphere oxygenation and modern diversity of living forms.

A Candidate Secreted Effector Protein of Rubber Tree Powdery Mildew Fungus Contributes to Infection by Regulating Plant ABA Biosynthesis.

Powdery mildew infects a wide range of crops and economic plants, causing substantial losses. Rubber trees (Hevea brasiliensis) are the primary source of natural rubber, and powdery mildew infection causes significant losses to natural rubber yields. How the causal agent, Erysiphe quercicola, establishes successful infection in rubber trees is largely unknown. Previously, 133 candidate secreted effector proteins (CSEPs) were identified in powdery mildew fungus. In this study, we characterize a CSEP named EqCSEP01276 for its function in suppressing host plant defense responses. We show that EqCSEP01276 is a secreted protein and is able to disturb the localization of 9-cis-epoxycarotenoid dioxygenase 5 (HbNCED5), a key enzyme in abscisic acid (ABA) biosynthesis in plant cell chloroplasts of H. brasiliensis. We also show that this effector inhibits ABA biosynthesis, and that in H. brasiliensis ABA is a positive regulator of the plant immune response against powdery mildew. Our study reveals a strategy by which powdery mildew fungus manipulates plant ABA-mediated defense for a successful infection.

Photosynthetic Electrochemical Cell Based on RuO2 Nanosheets Modified Bio-Anode

Thylakoids are the photosynthetic complexes that absorb light energy to produce photosynthetic electrons (PEs) in the chloroplast of plant cells. PE production starts from water splitting at photosystem II (PSII) in the thylakoid membrane and they are excited by absorbing photon energy. Over the last decade, many researchers have attempted to extract PEs from thylakoid by enhancing attachment of thylakoid membranes to an electrode or using artificial mediators. However, these have some limitations including increased ohmic resistance from the length of tethering molecules and energy loss due to artificial mediators. In this study, we propose a photosynthetic electrochemical cell based on RuO2 nanosheet modified electrode for the photosynthetic energy harvesting from thylakoids. RuO2 is known as one of the most conductive metal oxides. Metal oxides have partially positive charges that can improve attachment of negatively charged thylakoids to the electrode. Also, partially positively and negatively charged characteristics of RuO2 surfaces induce ensemble docking between electron carriers and electrode surface, which can attract both negatively charged electron carriers and positively charged electron carriers. Furthermore, RuO2 nanosheets show a unique characteristic of proton adsorption, which can sustain positive electrical potential during PEs extraction. For photosynthetic energy harvesting, the photosynthetic electrochemical cell was constructed with anode based on thylakoid and air-cathode based on Pt-C, which separated by a proton exchange membrane. The light was controlled by a 100 W halogen lamp to operate the photosystem so that excited PEs can be extracted to the electrode. The surface potential of RuO2 nanosheets modified electrodes was 250 mV vs. Ag/AgCl, which shifted positively more than 100 mV than that of the bare Au electrode. From the inspection of RuO2 lattice by x-ray absorption spectroscopy, positively shifted surface potential is the result of proton adsorption. A positively biased electrode surface can induce a large amount of PEs extraction from the electron carriers than the Au electrode. When the light is illuminated on the thylakoids deposited electrodes, 8.9 ± 6.3 mV and 88.7 ± 13.8 mV of the surface potentials were measured from the Au and the RuO2 modified electrode, respectively. These lowered electrode potentials are due to the redox potential of the electron carriers on the electron transport chain of the thylakoid membrane. The open-circuit voltages (OCV, the subtracted potential between the cathode and the anode) were measured 575.1 ± 6.4 mV and 424.0 ± 17.2 mV from the Au and the RuO2 electrode, respectively. Although OCV of the RuO2 electrode is lower than the Au electrode, the power density of the RuO2 electrode shows 4 times higher than the Au electrode, and this is due to the enhanced PEs extraction by both the surface charge and the proton adsorption characteristics of RuO2 nanosheets.

Targeted delivery of nanomaterials with chemical cargoes in plants enabled by a biorecognition motif

Current approaches for nanomaterial delivery in plants are unable to target specific subcellular compartments with high precision, limiting our ability to engineer plant function. We demonstrate a nanoscale platform that targets and delivers nanomaterials with biochemicals to plant photosynthetic organelles (chloroplasts) using a guiding peptide recognition motif. Quantum dot (QD) fluorescence emission in a low background window allows confocal microscopy imaging and quantitative detection by elemental analysis in plant cells and organelles. QD functionalization with β-cyclodextrin molecular baskets enables loading and delivery of diverse chemicals, and nanoparticle coating with a rationally designed and conserved guiding peptide targets their delivery to chloroplasts. Peptide biorecognition provides high delivery efficiency and specificity of QD with chemical cargoes to chloroplasts in plant cells in vivo (74.6 ± 10.8%) and more specific tunable changes of chloroplast redox function than chemicals alone. Targeted delivery of nanomaterials with chemical cargoes guided by biorecognition motifs has a broad range of nanotechnology applications in plant biology and bioengineering, nanoparticle-plant interactions, and nano-enabled agriculture.

The critical function of the plastid rRNA methyltransferase, CMAL, in ribosome biogenesis and plant development

Methylation of nucleotides in ribosomal RNAs (rRNAs) is a ubiquitous feature that occurs in all living organisms. The formation of methylated nucleotides is performed by a variety of RNA-methyltransferases. Chloroplasts of plant cells result from an endosymbiotic event and possess their own genome and ribosomes. However, enzymes responsible for rRNA methylation and the function of modified nucleotides in chloroplasts remain to be determined. Here, we identified an rRNA methyltransferase, CMAL (Chloroplast MraW-Like), in the Arabidopsis chloroplast and investigated its function. CMAL is the Arabidopsis ortholog of bacterial MraW/ RsmH proteins and accounts to the N4-methylation of C1352 in chloroplast 16S rRNA, indicating that CMAL orthologs and this methyl-modification nucleotide is conserved between bacteria and the endosymbiont-derived eukaryotic organelle. The knockout of CMAL in Arabidopsis impairs the chloroplast ribosome accumulation and accordingly reduced the efficiency of mRNA translation. Interestingly, the loss of CMAL leads not only to defects in chloroplast function, but also to abnormal leaf and root development and overall plant morphology. Further investigation showed that CMAL is involved in the plant development probably by modulating auxin derived signaling pathways. This study uncovered the important role of 16S rRNA methylation mediated by CMAL in chloroplast ribosome biogenesis and plant development.

Symptomatic plant viroid infections in phytopathogenic fungi

Viroids are pathogenic agents that have a small, circular noncoding RNA genome. They have been found only in plant species; therefore, their infectivity and pathogenicity in other organisms remain largely unexplored. In this study, we investigate whether plant viroids can replicate and induce symptoms in filamentous fungi. Seven plant viroids representing viroid groups that replicate in either the nucleus or chloroplast of plant cells were inoculated to three plant pathogenic fungi, Cryphonectria parasitica, Valsa mali, and Fusarium graminearum By transfection of fungal spheroplasts with viroid RNA transcripts, each of the three, hop stunt viroid (HSVd), iresine 1 viroid, and avocado sunblotch viroid, can stably replicate in at least one of those fungi. The viroids are horizontally transmitted through hyphal anastomosis and vertically through conidia. HSVd infection severely debilitates the growth of V. mali but not that of the other two fungi, while in F. graminearum and C. parasitica, with deletion of dicer-like genes, the primary components of the RNA-silencing pathway, HSVd accumulation increases. We further demonstrate that HSVd can be bidirectionally transferred between F. graminearum and plants during infection. The viroids also efficiently infect fungi and induce disease symptoms when the viroid RNAs are exogenously applied to the fungal mycelia. These findings enhance our understanding of viroid replication, host range, and pathogenicity, and of their potential spread to other organisms in nature.

Molecular and Functional Diversity of RNA Editing in Plant Mitochondria.

RNA editing is a fundamental biochemical process relating to the modification of nucleotides in messenger RNAs of functional genes in cells. RNA editing leads to re-establishment of conserved amino acid residues for functional proteins in nuclei, chloroplasts, and mitochondria. Identification of RNA editing factors that contributes to target site recognition increases our understanding of RNA editing mechanisms. Significant progress has been made in recent years in RNA editing studies for both animal and plant cells. RNA editing in nuclei and mitochondria of animal cells and in chloroplast of plant cells has been extensively documented and reviewed. RNA editing has been also extensively documented on plant mitochondria. However, functional diversity of RNA editing factors in plant mitochondria is not overviewed. Here, we review the biological significance of RNA editing, recent progress on the molecular mechanisms of RNA editing process, and function diversity of editing factors in plant mitochondrial research. We will focus on: (1) pentatricopeptide repeat proteins in Arabidopsis and in crop plants; (2) the progress of RNA editing process in plant mitochondria; (3) RNA editing-related RNA splicing; (4) RNA editing associated flower development; (5) RNA editing modulated male sterile; (6) RNA editing-regulated cell signaling; and (7) RNA editing involving abiotic stress. Advances described in this review will be valuable in expanding our understanding in RNA editing. The diverse functions of RNA editing in plant mitochondria will shed light on the investigation of molecular mechanisms that underlies plant development and abiotic stress tolerance.

WHITE PANICLE3, a Novel Nucleus-Encoded Mitochondrial Protein, Is Essential for Proper Development and Maintenance of Chloroplasts and Mitochondria in Rice.

Mitochondria and chloroplasts are interacting organelles that play important roles in plant development. In addition to a small number proteins encoded by their own genomes, the majority of mitochondrial and chloroplast proteins are encoded in the cell nucleus and imported into the organelle. As a consequence, coordination between mitochondria, chloroplasts, and the nucleus is of crucial importance to plant cells. Variegated mutants are chloroplast-defective mutants and are considered to be ideal models for studying the intercommunication between these organelles. Here, we report the isolation of WHITE PANICLE3 (WP3), a nuclear gene involved in variegation, from a naturally occurring white panicle rice mutant. Disrupted expression of WP3 in the mutant leads to severe developmental defects in both chloroplasts and mitochondria, and consequently causes the appearance of white-striped leaves and white panicles in the mutant plants. Further investigation showed that WP3 encodes a protein most likely targeted to mitochondria and is specifically expressed in rice panicles. Interestingly, we demonstrate that the recessive white-panicle phenotype in the wp3 mutant is inherited in a typical Mendelian manner, while the white-striped leaf phenotype in wp3 is maternally inherited. Our data collectively suggest that the nucleus-encoded mitochondrial protein, WP3, plays an essential role in the regulation of chloroplast development in rice panicles by maintaining functional mitochondria. Therefore, the wp3 mutant is an excellent model in which to explore the communication between the nucleus, mitochondria, and chloroplasts in plant cells.

Intrinsically Disordered Proteome of Human Membrane-Less Organelles.

It is recognized now that various proteinaceous membrane-less organelles (PMLOs) are commonly found in cytoplasm, nucleus, and mitochondria of various eukaryotic cells (as well as in the chloroplasts of plant cells). Being different from the "traditional" membrane-encapsulated organelles, such as chloroplasts, endoplasmic reticulum, Golgi apparatus, lysosomes, mitochondria, nucleus, and vacuoles, PMLOs solve the cellular need to facilitate and regulate molecular interactions via reversible and controllable isolation of target molecules in specialized compartments. PMLOs possess liquid-like behavior and are believed to be formed as a result of biological liquid-liquid phase transitions (LLPTs, also known as liquid-liquid phase separation), where an intricate interplay between RNA and intrinsically disordered proteins (IDPs) or hybrid proteins containing ordered domains and intrinsically disordered protein regions (IDPRs) may play an important role. This review analyzes the prevalence of intrinsic disorder in proteins associated with various PMLOs found in human cells and considers some of the functional roles of IDPs/IDPRs in biogenesis of these organelles.

Temporal Proteomics of Inducible RNAi Lines of Clp Protease Subunits Identifies Putative Protease Substrates.

The Clp protease in the chloroplasts of plant cells is a large complex composed of at least 13 nucleus-encoded subunits and one plastid-encoded subunit, which are arranged in several ring-like structures. The proteolytic P-ring and the structurally similar R-ring form the core complex that contains the proteolytic chamber. Chaperones of the HSP100 family help with substrate unfolding, and additional accessory proteins are believed to assist with Clp complex assembly and/or to promote complex stability. Although the structure and function of the Clp protease have been studied in great detail in both bacteria and chloroplasts, the identification of bona fide protease substrates has been very challenging. Knockout mutants of genes for protease subunits are of limited value, due to their often pleiotropic phenotypes and the difficulties with distinguishing primary effects (i.e. overaccumulation of proteins that represent genuine protease substrates) from secondary effects (proteins overaccumulating for other reasons). Here, we have developed a new strategy for the identification of candidate substrates of plant proteases. By combining ethanol-inducible knockdown of protease subunits with time-resolved analysis of changes in the proteome, proteins that respond immediately to reduced protease activity can be identified. In this way, secondary effects are minimized and putative protease substrates can be identified. We have applied this strategy to the Clp protease complex of tobacco (Nicotiana tabacum) and identified a set of chloroplast proteins that are likely degraded by Clp. These include several metabolic enzymes but also a small number of proteins involved in photosynthesis.

The Mitochondrial DNA-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination.

In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.

A Noncrystallization Approach toward Uniform Thylakoids-like 2D "Nano-coins" and Their Grana-like 3D Suprastructures.

Two-dimensional (2D) circular shape nanostructures (e.g., "nano-coins") are ubiquitously present in thylakoids and grana within chloroplasts of plant cells in nature. The design and fabrication of 2D nano-coins with controlled sizes and thicknesses yet remain challenging tasks. Herein, we present a noncrystallization approach to achieve 2D nano-coins from assemblies of a set of zwitterionic giant surfactants. Distinguished from traditional crystallization approaches where the 2D nanostructures with specific crystallographic symmetries are fabricated, the noncrystallization assembly of giant surfactants results in 2D nano-coins that are derived from the separation of assembled 3D multiple lamellar cylindrical colloids with uniform diameters. The diameters and thicknesses of these nano-coins can be readily tailored by varying the molecular length of giant surfactants' tails. The formation of 2D nano-coins or 3D cylindrical colloid suprastructures is controlled by tuning the pH value of added selective solvents. This new strategy opens a door for controlling the shape, size, and size distribution of assembled nanostructures with different hierarchies.

Crystal Structure of the Chloroplastic Oxoene Reductase ceQORH from Arabidopsis thaliana.

Enzymatic and non-enzymatic peroxidation of polyunsaturated fatty acids give rise to accumulation of aldehydes, ketones, and α,β-unsaturated carbonyls of various lengths, known as oxylipins. Oxylipins with α,β-unsaturated carbonyls are reactive electrophile species and are toxic. Cells have evolved several mechanisms to scavenge reactive electrophile oxylipins and decrease their reactivity such as by coupling with glutathione, or by reduction using NAD(P)H-dependent reductases and dehydrogenases of various substrate specificities. Plant cell chloroplasts produce reactive electrophile oxylipins named γ-ketols downstream of enzymatic lipid peroxidation. The chloroplast envelope quinone oxidoreductase homolog (ceQORH) from Arabidopsis thaliana was previously shown to reduce the reactive double bond of γ-ketols. In marked difference with its cytosolic homolog alkenal reductase (AtAER) that displays a high activity toward the ketodiene 13-oxo-9(Z),11(E)-octadecadienoic acid (13-KODE) and the ketotriene 13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-KOTE), ceQORH binds, but does not reduce, 13-KODE and 13-KOTE. Crystal structures of apo-ceQORH and ceQORH bound to 13-KOTE or to NADP+ and 13-KOTE have been solved showing a large ligand binding site, also observed in the structure of the cytosolic alkenal/one reductase. Positioning of the α,β-unsaturated carbonyl of 13-KOTE in ceQORH-NADP+-13-KOTE, far away from the NADP+ nicotinamide ring, provides a rational for the absence of activity with the ketodienes and ketotrienes. ceQORH is a monomeric enzyme in solution whereas other enzymes from the quinone oxidoreductase family are stable dimers and a structural explanation of this difference is proposed. A possible in vivo role of ketodienes and ketotrienes binding to ceQORH is also discussed.

Factor IX Expressed in Lettuce Chloroplasts Induces Oral Tolerance in Hemophilia B Mice

Inhibitor formation represents a major complication of factor replacement therapy for hemophilia. There are currently no prophylactic protocols against this antibody response. To address this problem, we sought to develop an antigen-specific immune tolerance protocol that does not rely on immune suppressive drugs and that would be feasible in pediatric patients. Oral tolerance fits these requirements but is hampered by vulnerability of the protein antigen to degradation in the stomach, cost of production, and inadequate delivery across the gut epithelium to the immune system (the gut-associated lymphoid tissue or GALT). We had hypothesized that transgenic expression of coagulation factors fused to a transmucosal carrier in the chloroplasts of plant cells would provide high level and cost-effective antigen production, bioencapsulation, and transmucosal delivery. Although we demonstrated this concept by of expression cassettes for factor VIII (FVIII) and factor IX (FIX) antigens fused to cholera toxin B (CTB) subunit in tobacco, further advancement of this system to the clinic is not feasible. Translation of this approach would require generation of transgenic edible plant cells expressing these fusion proteins. Therefore, in this study, we expressed CTB-FIX using species-specific chloroplast vectors regulated by endogenous psbA sequences, a requirement for optimal transgene expression (Plant Physiology 152: 2088-2104). Transplastomic plants were homoplasmic (inserted into all copies of chloroplast genomes in each plant cell) as evidenced by Southern blots. CTB-FIX antigen levels reached ~1mg antigen/g of lyophilized leaf cells and was stable with proper folding, disulfide bond formation, and CTB pentamer assembly when stored at ambient temperature for up to 2 years. Oral gavage of these cells (twice per week for 2 months) to hemophilia B mice (FIX gene deletion, C3H/HeJ background, n=7-11/experimental group) delivered CTB-FIX efficiently to the gut epithelium and immune system (as shown by immunohistochemistry), and induced LAP+ regulatory T cells in response to intravenous challenge with recombinant FIX protein. Moreover, FIX-CTB antigen doses of 1.5 mg, 5 mg, or 15 mg all effectively prevented inhibitor/IgG formation against intravenous FIX and averted anaphylaxis and associated fatal reactions by suppression of FIX-specific IgE production. Average inhibitor titers were 15-fold lower for CTB-FIX fed compared to control mice. Of orally treated mice, >80% had inhibitor titers that were undetectable to <2 BU, while controls formed high-titer inhibitors that were >5BU, with 9/11 showing >10 BU. Induction of tolerance in such a broad dose range should greatly facilitate translation of the approach. Our results support initiation of clinical studies on oral tolerance for hemophilia B using transplastomic lettuce. DisclosuresHerzog:Spark Therapeutics: Patents & Royalties: Patent licenses; Novo Nordisk: Research Funding. Daniell:Novo Nordisk: Research Funding.