Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves

The intracellular compartmentation of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized.     

A mutation in the cytosolic O-acetylserine (thiol) lyase induces a genome-dependent early leaf death phenotype in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol) lyase (OAS-TL) catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.     

Characterization of the O-acetylserine(thiol)lyase gene family in Solanum lycopersicum L.

Key message

This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L.

Abstract

Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a β-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.

     

Mitochondrial serine acetyltransferase functions as a pacemaker of cysteine synthesis in plant cells

Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide. Thus, Cys synthesis in plants is significantly different from that in nonphotosynthetic eukaryotes at the cellular level.     

Separation and Ultrastructure of Proplastids from Dark-grown Euglena Cells

A procedure for the separation of proplastids free of mitochondria from dark-grown Euglena cells has been developed. A fraction enriched in proplastids was used for freeze-etching study of proplastid structure. The prolamellar body in freeze-etched replicas appeared sponge-like, with thylakoids, often vesicular, emerging from it. The prolamellar body and the thylakoids were covered by particles of about 100Å in diameter. No larger particles, typical of light-grown chloroplasts, were observed.     

The relevance of compartmentation for cysteine synthesis in phototrophic organisms

In the vascular plant Arabidopsis thaliana, synthesis of cysteine and its precursors O-acetylserine and sulfide is distributed between the cytosol, chloroplasts, and mitochondria. This compartmentation contributes to regulation of cysteine synthesis. In contrast to Arabidopsis, cysteine synthesis is exclusively restricted to chloroplasts in the unicellular green alga Chlamydomonas reinhardtii. Thus, the question arises, whether specification of compartmentation was driven by multicellularity and specified organs and tissues. The moss Physcomitrella patens colonizes land but is still characterized by a simple morphology compared to vascular plants. It was therefore used as model organism to study evolution of compartmented cysteine synthesis. The presence of O-acetylserine(thiol)lyase (OAS-TL) proteins, which catalyze the final step of cysteine synthesis, in different compartments was applied as criterion. Purification and characterization of native OAS-TL proteins demonstrated the presence of five OAS-TL protein species encoded by two genes in Physcomitrella. At least one of the gene products is dual targeted to plastids and cytosol, as shown by combination of GFP fusion localization studies, purification of chloroplasts, and identification of N termini from native proteins. The bulk of OAS-TL protein is targeted to plastids, whereas there is no evidence for a mitochondrial OAS-TL isoform and only a minor part of OAS-TL protein is localized in the cytosol. This demonstrates that subcellular diversification of cysteine synthesis is already initialized in Physcomitrella but appears to gain relevance later during evolution of vascular plants.     

Why are plastids maternally inherited in epilobium? Ultrastructural observations during microgametogenesis

We have investigated the sequential stages of microgametogenesis by electron microscopy, to determine the basis of maternal inheritance of plastids in Epilobium. The development of both the vegetative and generative cells has been followed using a semi-artificial growth system for pollen tubes. The generative cells inside the pollen grain contains numerous mitochondria, 5–8 proplastids, and, in contrast to the vegetative cytoplasm, only a few vacuoles. When the generative cell has divided into the two sperm cells inside the pollen tube, small vesicles deriving from dicytosome cisternae become abundant. These vesicles appear to form vacuoles by fusion which then contain remnants of fibrillar, globular or membranaceous material. It is suggested that this material derives from proplastids as the proplastids disappear either before or shortly after the generative cell has divided, concurrently with the appearance of the ‘remnants’ in the vacuoles. The mitochondria of the sperm cells remain intact.     

Effects of short-term ozone exposure on lung mitochondrial oxidative and energy metabolism

Ozone effects on lung mitochondrial oxidative metabolism were examined after short-term exposure of rats and monkeys to O₃. Exposure of animals to 2 ppm O₃ for 8 hr or to 4 ppm O₃ for 4 hr caused a 15–27% (P < 0.05) depression of lung mitochondrial O₂ consumption, using 2-oxoglutarate, succinate, and glycerol-1-phosphate. but not ascorbate plus Wurster's blue as substrates. Under these exposure conditions (4 ppm 4 hr) the ADP:O ratios dropped 25–36% (P < 0.05) and the respiratory control indices decreased 27–33% (P < 0.02) for oxidation of all substrates examined. Lung mitochondria from control animals were relatively impermeable to added NADH, but those from O₃-exposed animals showed an increased permeability as judged from NADH oxidation at a rate 3-fold higher than the control. Likewise, added cytochrome c caused a 22% (P < 0.01) stimulation of succinate oxidation in exposed lung mitochondria as against 5% (nonsignificant) in controls. Ozone exposure also caused a 20% (P < 0.01) oxidation of thiol groups in lung mitochondria, but no lipid peroxidation products were detectable in O₃-exposed lung tissue. The depression of substrate utilization, coupled phosphorylation and respiratory control observed in lung mitochondria of O₃-exposed animals might be related to alteration of membrane permeability, and inhibition of respiratory enzymes (dehydrogenases) due to oxidation of functional thiol groups.     

Multiple forms of isocitrate lyase in the matrix of Turbatrix aceti mitochondria

The localization of five separate forms of isocitrate lyase (EC 4.1.3.1) in the free-living nematode Turbatrix aceti was determined by analyzing their distribution among subcellular fractions with continuous (Tris-acetate, pH 7.5) polyacrylamide gel electrophoresis. Enzyme activity on gels was detected either by substrate-dependent Schiff's-aldehyde staining or absorbance of phenylhydrazone at 324 nm. Following rate sedimentation of worm homogenates, the highest specific activity for isocitrate lyase was recovered in the pellet containing intact mitochondria. Glyoxysomes (microbodies) were not observed by electron microscopy in this or any other fraction. Selective removal of mitochondrial outer membranes (and hence components in the intermembrane space) was accomplished by two different procedures: (1) passage of mitochondria in a hypersomotic medium through a French pressure cell at 1500 psi, or (2) treatment with 0.6 mg digitonin mg protein. Electron microscopy revealed essentially complete removal for the former procedure, but only partial removal following digitonin treatment. Equilibrium centrifugation on sucrose gradients was necessary to strip the residual outer membranes from the digitonin-treated mitochondria. Mitoplasts resulting from the two procedures were subfractionated into matrix and inner membrane components by high-pressure disruption (14,000 psi) and subsequent rate sedimentation (144,000g, 60 min). Identical electrophoretic patterns were found using both slab and disc gels, whether stained with Schiff's reagent or scanned at 324 nm, in samples taken from clarified homogenates, intact mitochondria, mitoplasts, or matrix fractions. The results indicate that all five forms of the enzyme occur together in the mitochondrial matrices. Their individual functions are not yet known, but they may be involved in the regulation of isocitrate metabolism common to the tricarboxylic acid and glyoxylate cycles occurring within the same mitochondria.     

Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures

Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. In mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography.     

Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells

Alkbh1 is a mammalian homolog of the Escherichia coli DNA repair enzyme AlkB, an Fe(II) and 2-oxoglutarate dependent dioxygenase that removes alkyl lesions from DNA bases. The human homolog ALKBH1 has been associated with six different enzymatic activities including DNA, mRNA, or tRNA hydroxylation, cleavage at abasic (AP) sites in DNA, as well as demethylation of histones. The reported cellular roles of this protein reflect the diverse enzymatic activities and include direct DNA repair, tRNA modification, and histone modification. We demonstrate that ALKBH1 produced in mammalian cells (ALKBH1₂₉₃) is similar to the protein produced in bacteria (ALKBH1_Ec) with regard to its m⁶A demethylase and AP lyase activities. In addition, we find that ALKBH1₂₉₃ forms a covalent adduct with the 5′ product of the lyase product in a manner analogous to ALKBH1_Ec. Localization and subcellular fractionation studies with the endogenous protein in two human cell strains confirm that ALKBH1 is primarily in the mitochondria. Two strains of CRISPR/Cas9-created ALKBH1-deficient HEK293 cells showed increases in mtDNA copy number and mitochondrial dysfunction as revealed by growth measurements and citrate synthase activity assays.     

Redox-optimized ROS balance: A unifying hypothesis

While it is generally accepted that mitochondrial reactive oxygen species (ROS) balance depends on the both rate of single electron reduction of O₂ to superoxide (O₂^?) by the electron transport chain and the rate of scavenging by intracellular antioxidant pathways, considerable controversy exists regarding the conditions leading to oxidative stress in intact cells versus isolated mitochondria. Here, we postulate that mitochondria have been evolutionarily optimized to maximize energy output while keeping ROS overflow to a minimum by operating in an intermediate redox state. We show that at the extremes of reduction or oxidation of the redox couples involved in electron transport (NADH/NAD⁺) or ROS scavenging (NADPH/NADP⁺, GSH/GSSG), respectively, ROS balance is lost. This results in a net overflow of ROS that increases as one moves farther away from the optimal redox potential. At more reduced mitochondrial redox potentials, ROS production exceeds scavenging, while under more oxidizing conditions (e.g., at higher workloads) antioxidant defenses can be compromised and eventually overwhelmed. Experimental support for this hypothesis is provided in both cardiomyocytes and in isolated mitochondria from guinea pig hearts. The model reconciles, within a single framework, observations that isolated mitochondria tend to display increased oxidative stress at high reduction potentials (and high mitochondrial membrane potential, ?Ψ_m), whereas intact cardiac cells can display oxidative stress either when mitochondria become more uncoupled (i.e., low ?Ψ_m) or when mitochondria are maximally reduced (as in ischemia or hypoxia). The continuum described by the model has the potential to account for many disparate experimental observations and also provides a rationale for graded physiological ROS signaling at redox potentials near the minimum.     

Entwicklung und Struktur der Proplastiden

In this study the proplastid development in embryonic cells is described for the apical meristem of Elodea canadensis, embryo sacs from Lilies, and Begonia leaf buds. The formation of these cell organelles originates with submicroscopical particles which consist of a homogeneous stroma with a surrounding double membrane. When these proplastids reach an average size of 1 µ, the inner layer of the membrane begins to invaginate into the stroma. This process is comparable to tubuli formation in mitochondria. Under growth conditions with sufficient exposure to light, the development of the grana and stroma lamellae proceeds without interruption. If the plants are kept in the dark, small vesicles are formed which accumulate in the prolamellar body of the proplastids. After illumination these elementary vesicles merge to form membranes which evolve into grana and stroma lamellae. The structural similarity of the early proplastid stages with the mitochondria seems to indicate that there exists some phylogenetic relationship between the two cell organelles.     

Organic Acid Metabolism in Cellular Organelles of Vigna cylindrica (Mitorisasage) Cotyledons

In starchy cotyledons of Vigna cylindrica (L.) Skeels (Mitorisasage)during seed germination, the enzymes of the glyoxylate cyclewere located in the matrix of mitochondria. Glyoxysomes wereabsent. The glyoxylate cycle in the mitochondria supplies organicacids to the tricarboxylic acid cycle. In mitochondria, isocitratelyase activity was much higher than malate synthase activity.Part of the glyoxylate thus produced in mitochondria may benonenzymatically converted to formate by H₂O₂ and the formatethen converted to CO₂ by peroxidase or by formic dehydrogenase.The activity of superoxide dismutase, which supplies H₂O₂, washigher in mitochondria than in peroxisomes. The remaining glyoxylatein mitochondria is possibly converted to glycine by alanine-glyoxylateaminotransferase or transported to peroxisomes which lackedisocitrate lyase activity but had high malate synthase activity.In peroxisomes, glyoxylate may be also produced from urate,as is suggested by the fairly high activities of uricase, allantoinaseand allantoicase. Judging from the enzyme distribution, Vignaperoxisomes should be capable of producing malate, oxalacetate,citrate, isocitrate and a-ketoglutarate. ¹Present address: Department of Horticulture, College of Agricultureand Animal Science, Yeugnam University, Gyeongsan 632, Korea. (Received May 27, 1987; Accepted October 7, 1987)