Metabolism of plastid terpenoids. I. Biosynthesis of phytoene in plastid stroma isolated from higher plants

Using prephytoene pyrophosphate and isopentenyl pyrophosphate as substrates, the synthesis of phytoene was demonstrated for the first time in chlorolasts, etioplasts and amyloplasts. The stromal fraction was the sole site of phytoene synthesis. These data were complemented by the fact that the enzymatic activity in each plastid stroma was progressively lost after immunoprecipitation with an antiserum directed against Capsicum chromoplast phytoene synthetase enzyme complex.     

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate) or [¹⁴C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.Abbreviastions Chlorophyll a_GC Chlorophyll a, esterified with geranylgeraniol - GGPP geranylgeranyl pyrophosphate - HPLC high pressure liquid chromatography - IPP isopentenyl pyrophosphate     

The site of carotenogenic enzymes in chromoplasts from Narcissus pseudonarcissus L.

The membranes from the chromoplasts of Narcissus pseudonarcissus L. which are derived from the inner envelope membrane are the site of -carotene synthesis from [1-¹⁴C]isopentenyl diphosphate. The enzymes involved are partly peripheral membrane proteins (prenyltransferase, phytoene synthase) and partly integral membrane proteins (cis-trans isomerase, dehydrogenase(s), cyclase(s)). Metabolic channeling is suggested.Abbreviations IPP isopentenyl diphosphate - GGPP geranylgeranyl diphosphate     

Nuclear—organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis

The immutans (im) variegation mutant of Arabidopsis thaliana contains green- and white-sectored leaves due to the action of a nuclear recessive gene. The mutation is somatically unstable, and the degree of sectoring is influenced by light and temperature. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white sectors are heteroplastidic and contain non-pigmented plastids that lack organized lamellar structures, as well as small pigmented plastids and/or rare normal chloroplasts. This indicates that the plastids in im white cells are not affected equally by the nuclear mutation and that the expression of immutans is ‘plastid autonomous’. In contrast to other variegation mutants with heteroplastidic cells, the defect in im is not maternally inherited. immutans thus represents a novel type of nuclear gene-induced variegation mutant. It has also been found that the white tissues of immutans accumulate phytoene, a non-colored C₄₀ carotenoid intermediate. This suggests that immutans controls, either directly or indirectly, the activity of phytoene desaturase (PDS), the enzyme that converts phytoene to zeta-carotene in higher plants. However, im is not the structural gene for PDS. A secondary effect of carotenoid deficiency, both in immutans and in wild-type plants treated with a herbicide that blocks carotenoid synthesis, is an increase in acid ribonuclease activity in white tissue. It is concluded that the novel variegation generated by the immutans mutation should offer great insight into the complex circuitry that regulates nuclear—organelle interactions.     

The partial purification and properties of a phytoene synthesizing enzyme system.

An enzyme system catalyzing the synthesis of phytoene from isopentenyl pyrophosphate has been isolated from tomato fruit plastids and purified approximately 350-fold in specific activity. This enzyme system has a molecular weight of approximately 200,000. The rate of phytoene formation is maximal at pH 7.0 and 23 °C and the apparent K_m for isopentenyl pyrophosphate is 10 μm The rates of phytoene synthesis when geranylgeranyl pyrophosphate and isopentenyl pyrophosphate were used as substrates were 0.08 and 0.17 nmol of phytoene/mg of protein/h, respectively. The enzyme complex showed an absolute requirement for Mn²⁺, but not for NADP⁺. At a concentration of 2 mm, NADP⁺ produced only a 1.5- to 3-fold stimulation, and this effect varied from preparation to preparation. The addition of NADPH to the incubation mixture produced inhibition of phytoene synthesis and there was no evidence for the concomitant accumulation of lycopersene. The acid labiles produced on acid treatment of the incubation mixture indicated that geranylgeranyl pyrophosphate was formed by the enzyme complex. The enzyme system is stabilized in the presence of 30% glycerol and 10 mm dithiothreitol and it can be stored at ?20 °C for over 1 month without significant loss of activity. However, the enzyme activity for phytoene formation is heat labile, and it is not stable when attempts are made to purify it further by ion-exchange chromatography.     

Carotenoid synthesis and pleiotropic effects in carotenoid-deficient seedlings of maize

Plastid-envelope membranes from seedlings ofZea mays L. made carotenoid-deficient by either norflurazon treatment or mutation lack an activity permitting conversion of phytoene to -carotene. This activity in membrane fractions was measured by coincubation in vitro with a soluble system from spinach chloroplasts capable of converting [¹⁴C]isopentenyl pyrophosphate into phytoene. When grown in light, the carotenoid-deficient seedlings lack many soluble chloroplast proteins, including NADP-dependent malic enzyme (EC 1.1.1.40), pyruvate phosphate dikinase (EC 2.7.9.1), and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), but apparently still contain the soluble activities permitting synthesis of phytoene.Abbreviations IPP isopentenyl pyrophosphate - LHCP light-harvesting chlorophylla/b-binding protein - norflurazon 4-chloro-5(methylamine)-2-(,,-trifluoro-m-tolyl)-3-(2H)-pyrazinone - TLC thin-layer chromatography - Tris 3-amino-2-(dihydroxymethyl)-1,3-propanediol     

Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B₁), and pyridoxine (vitamin B₆) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.     

Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells

Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30–40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W₃BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants are identical to those obtained from dark-grown wild-type cells. Light-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated with one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methyl-amino)-2-(α,α,α,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lower steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amino acids; of these six are undectable in W₃BUL and are, therefore, candidates for coding by plastid DNA.     

A plastid terminal oxidase associated with carotenoid desaturation during chromoplast differentiation

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development.     

Isoprenoid biosynthesis in Synechocystis sp. strain PCC6803 is stimulated by compounds of the pentose phosphate cycle but not by pyruvate or deoxyxylulose-5-phosphate

The photosynthetic cyanobacterium Synechocystis sp. strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-C-methyl-D-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in Escherichia coli, or by deoxyxylulose-5-phosphate, the first pathway intermediate in E. coli. However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds. Fosmidomycin (at 1 micro M and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect [(14)C]IPP incorporation stimulated by DHAP plus GA3P. To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium. The combined results suggest that the MEP pathway, as described for E. coli, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in Synechocystis sp. strain PCC6803. Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis.     

RNA synthesis in synchronously growing populations of HeLa S3 cells. I. Rate of total RNA synthesis and its relationship to DNA synthesis

The rate of RNA synthesis in synchronously growing HeLa S3 cells was determined as a function of position in the cell generation cycle. Measurements throughout the cycle of both the rate of incorporation of radioactively-labeled uridine and of the total amount of RNA indicate that (1) the rate of RNA synthesis is constant (or increases only slightly) during G₁, approximately doubles during the first half of S, and then remains constant during the remainder of S and G₂, and (2) cells attain the average G₁ rate of RNA synthesis very early in G¹, and maintain the average G₂ rate until mitosis. If the initiation of DNA synthesis is blocked, the acceleration of RNA synthesis is markedly reduced or eliminated. Further experiments in which DNA synthesis was inhibited at different times in S, or to varying degrees from the beginning of S, suggest that the extent to which RNA synthesis is accelerated depends on the amount of DNA duplicated. These data also indicate that duplication of the first half, and in particular the first few per cent, of the DNA complement results in a disproportionate acceleration of RNA synthesis. The possibility that fluctuations in the sizes of precursor pools may lead to misinterpretation of labeled-uridine incorporation data was examined. Experiments indicate that in this system pool fluctuations do not cause invalid measures of RNA synthesis. It is concluded that RNA synthesis occurs throughout interphase, but undergoes a two-fold increase in rate which is dependent on the duplication of DNA.     

Novel Bioassay for the Discovery of Inhibitors of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) and Terpenoid Pathways Leading to Carotenoid Biosynthesis

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.     

Effects of acclimation to altitude on oxygen affinity and organic phosphate concentrations in pigeon blood.

Hematocrit ratio, hemoglobin concentration and blood oxygen affinity, Bohr effect factor and Hill coefficient, adenosine triphosphate and inositol pentaphosphate (IPP) concentrations were studied in blood of adult pigeons exposed first at 140 m, and then for 3 weeks at 4000 m in an altitude chamber. At altitude, the hematocrit ratio and hemoglobin concentration significantly increased, IPP concentration decreased, and P₅₀ did not change. A lower mean red cell age and a higher hemoglobin concentration may account for the unchanged P₅₀. Adaptation to hypoxia of the tissue oxygen supply was shown by a greater blood O₂ capacitance (ΔC_HbO₂/Δ_o2) in the physiological range of oxygen partial pressures.     

Formation of terpenoid products in Ginkgo biloba L. cultivated cells

Ginkgolides are diterpenes arising from the terpenoid precursor: geranylgeranyl pyrophosphate (GGPP). Incorporation of [1-¹⁴C] isopentenylpyrophosphate ([1-¹⁴C]IPP) into GGPP was monitored throughout the cultivation cycle of G. biloba L. cultivated cells. Because incorporation of [1-¹⁴C]IPP into GGPP had never been monitored in G. biloba, in either the whole plant or cultivated cell system, modifications to existing protocols were necessary. Modifications consisted of extracting the cells with an extraction buffer supplemented with Triton-X-100. Farnesylpyrophosphate (FPP) was the major product formed. The amount of GGPP detected was about one tenth that of FPP.Abbreviations CHAPS 3-[(3-cholamidopropyl) dimethyl ammonio]-1-propane-sulphonate - DTT [1-4 dithiothreitol] - FPP farnesylpyrophosphate - GGPP geranylgeranylpyrophosphate - IPP [1-¹⁴C] isopentenylpyrophosphate - PVPP polyvinylpolypyrrolidone - Tris Tris(hydroxymethyl)aminomethane     

Toxicity of A Novel Neonicotinoid Insecticide Paichongding to Earthworm Eisenia fetida

Traditional neonicotinoid insecticides are used worldwide. Paichongding (IPP), as a novel neonicotinoid pesticide, has been widely used in China. However, the ecotoxicity of IPP to non-target invertebrates in soil ecosystem has not been reported yet. In this study, acute toxicity of IPP to earthworm Eisenia fetida, as well as the antioxidant response after IPP exposure, was evaluated. In the filter paper contact test, the LC₅₀ at 24 hr and 48 hr for IPP were 14.98 μg/cm² and 7.59 μg/cm², respectively. In artificial soil test, the LC₅₀ (lethal concentration) at 14 days and 28 days for IPP were 541.07 mg/kg and 238.51 mg/kg, respectively. The LC₅₀ of IPP is much higher than that of traditional neonicotinoid insecticides. However, earthworm body weight assessment demonstrated that the growth of earthworm was inhibited by extended exposure to IPP at sublethal doses. The activities of antioxidative enzymes superoxide dismutase and catalase in earthworms were significantly induced after IPP exposure. Malondialdehyde, a biomarker of lipid peroxidation, was also increased after IPP exposure. Although the results indicated that IPP had potentially adverse effect on earthworms, its toxicity was much lower than traditional neonicotinoids.     

Bleaching herbicide flurtamone interferes with phytoene desaturase

The mode of action of the furanone herbicide flurtamone and derivatives was investigated with cress seedlings and with the unicellular cyanobacterium Anacystis. Either in the light or in the dark these compounds inhibited the formation of α- and β-carotene and all of the xanthophylls in the seedlings. Instead, phytoene, a precursor of colored carotenoids, was accumulated. In illuminated seedlings photooxidative destruction of chlorophyll was observed. The I₅₀ value of flurtamone inhibition of carotenoid biosynthesis in intact Anacystis cells and the K₁ value for interaction of flurtamone with phytoene desaturase with Anacystis thylakoids were 30 and 18 nanomoles, respectively. Concentrations of flurtamone which strongly inhibited carotenoid synthesis had no direct peroxidative activities and did not inhibit photosynthetic electron transport.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

Terpenoid metabolism in plastids : sites of phytoene synthetase activity and synthesis in plant cells

The biosynthesis of phytoene from prephytoene pyrophosphate has been localized exclusively in the plastid compartment of ruptured protoplasts derived from Triticum leaves and Capsicum fruits.

The phytoene synthetase activity in Triticum leaves deficient in plastid ribosomes was comparable to those obtained in normal leaves. In addition, the stimulation of phytoene synthetase activity observed in green Capsicum fruit after 2-(4-chlorophenylthio)triethylamine hydrochloride treatment was not abolished by chlororamphenicol and lincomycin, in contrast to the inhibition observed after cycloheximide treatment.

These data conclusively show that phytoene synthetase is localized exclusively in the plastid compartment in higher plants and that its synthesis is not performed on 70S ribosomes.