Metabolism of plastid terpenoids. II. Regulation of phytoene synthesis in plastid stroma isolated from higher plants

In plastid stroma the synthesis of phytoene was regulated by several factors, (i) MgCl₂ or MnCl₂ alone and the combination of both oriented selectively the incorporation of isopentenyl pyrophosphate (IPP) between phytoene and other isoprenoids including geranylgeraniol, geranyllinalool, and solanesol (ii) neutral detergents (Tween-80, Triton X-100), polyethyleneglycol and digalactosyldiglyceride liposomes stimulated the incorporation of IPP into phytoene (iii) ATP or different glycolytic intermediates enhanced the synthesis of phytoene from IPP.     

Metabolism of plastid terpenoids: in vitro inhibition of phytoene synthesis by phenethyl pyrophosphate derivatives

The inhibition of partially purified phytoene synthetase activity from Capsicum annuum chromoplasts was investigated using aminophenethyl pyrophosphate and azidophenethyl pyrophosphate. These compounds were effective inhibitors of phytoene synthesis and kinetic analysis showed that they were competitive with respect to the substrate isopentenyl pyrophosphate. These data were strengthened by the ability of azidophenethyl pyrophosphate to photoinactivate irreversibly the activity of the enzyme complex. These results suggest that the primary targets of these analogs are at the level of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthetase.     

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.     

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.

     

Light-Stimulated Carotenoid Biosynthesis during Transformation of Maize Etioplasts Is Regulated by Increased Activity of Isopentenyl Pyrophosphate Isomerase

Light-stimulated carotenoid biosynthesis associated with the transformation of etioplasts to chloroplasts was investigated after dark-grown maize (Zea mays) seedlings were transferred into light. These studies focused on the enzymes of the pathway to detect those enzyme activities that were stimulated in the light and thus that were responsible for increased biosynthesis of carotenoids. In preliminary experiments, norflurazon, an inhibitor of phytoene desaturase, was used to prevent phytoene being further metabolized to carotenoids. Light-dependent stimulation of phytoene accumulation indicated that the light-regulated steps are located in the pathway leading to phytoene synthesis. The use of the 14C- labeled precursors mevalonic acid, isopentenyl pyrophosphate, and farnesyl pyrophosphate pointed to increased activity of an enzyme involved in the biosynthetic steps between isopentenyl pyrophosphate and farnesyl pyrophosphate. Determination of the activities of all five enzymes of the pathway involved in the sequence from mevalonic acid to phytoene revealed that the only enzyme activity stimulated by light was isopentenyl pyrophosphate isomerase. Over a 3-h period of illumination, this enzyme activity, like carotenoid biosynthesis, was stimulated 2.8-fold.     

Developmental and stress regulation of gene expression for plastid and cytosolic isoprenoid pathways in pepper fruits.

Plant cells synthesize a myriad of isoprenoid compounds in different subcellular compartments, which include the plastid, the mitochondria, and the endoplasmic reticulum cytosol. To start the study of the regulation of these parallel pathways, we used pepper (Capsicum annuum) fruit as a model. Using different isoprenoid biosynthetic gene probes from cloned cDNAs, we showed that only genes encoding the plastid enzymes (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and capasanthin-capsorubin synthase) are specifically triggered during the normal period of development, at the ripening stage. This pattern of expression can be mimicked and precociously induced by a simple wounding stress. Concerning the cytosol-located enzymes, we observed that the expression of the gene encoding farnesyl pyrophosphate synthase is constitutive, whereas that of farnesyl pyrophosphate cyclase (5-epi-aristolochene synthase) is undetectable during the normal development of the fruit. The expression of these later genes are, however, only selectively triggered after elicitor treatment. The results provide evidence for developmental control of isoprenoid biosynthesis occurring in plastids and that cytoplasmic isoprenoid biosynthesis is regulated, in part, by environmental signals.     

Biosynthesis of phytoene from isopentenyl pyrophosphate by a Neurospora enzyme system.

An enzyme system catalyzing the conversion of isopentenyl pyrophosphate to phytoene has been isolated from Neurospora crassa mycelia. This enzyme system shows an absolute requirement for Mg^?, but no other cofactors. Cultures of N. crassa exhibit a low level of phytoene synthesizing activity when grown in the dark. A 2-min in vivo blue light irradiation results in a ninefold increase in activity after 24 h. This increase is dependent on the duration of the light treatment and is inhibited by cycloheximide. A similar blue light-induced elevation of phytoene synthesizing activity was demonstrated in an albino-1 mutant. This enzyme activity was not found in either dark-grown or irradiated cultures of an albino-2 or an albino-3 mutant.     

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.     

Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa

The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-¹⁴C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [¹⁴C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [¹⁴C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [¹⁴C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [¹⁴C]geranylgeranyl pyrophosphate from [1-¹⁴C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino-1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway.     

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     

Carotenoid synthesis and phytoene synthase activity during mating of Blakeslea trispora

Carotenoid formation was investigated in wild type and carotenogenic mutants of Blakeslea trispora after mating (−) and (+) strains. The highest yields of carotenoids, especially β-carotene was observed following mating. In vitro incorporation of geranylgeranyl pyrophosphate into phytoene and β-carotene corresponded to increased carotenogenesis in the mated strains. Immuno determination of phytoene synthase protein levels revealed that the amounts of this enzyme is concurrent with the increases in carotenoid content. In fungi, phytoene synthase together with lycopene cyclase are encoded by a fusion gene crtYB or carRA with two individual domains. These domains were both heterologously expressed in an independent manner and antisera raised against both. These antisera were used, to assess protein levels in mated and non-mated B. trispora. The phytoene synthase domain was detected as an individual soluble protein with a molecular weight of 40 kDa and the lycopene cyclase an individual protein of mass about 30 kDa present in the membrane fraction following sub-cellular fractionation. This result demonstrates a post-translational cleavage of the protein transcribed from a single mRNA into independent functional phytoene synthase and lycopene cyclase.     

Cloning,sequencing and expressing the carotenoid biosynthesis genes,lycopene cyclase and phytoene desaturase,from the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 in Escherichia coli

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and β-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% (Erwinia herbicola), 37.4% (Erwinia uredovora) and 22.9% (Synechococcus sp.), and those between phytoene desaturase genes are 50.3% (E. herbicola), 54.7% (E. uredovora) and 39.6% (Rhodobacter capsulatus).     

Phytoene production utilizing the isoprenoid biosynthesis capacity of Thermococcus kodakarensis

Phytoene (C₄₀H₆₄) is an isoprenoid and a precursor of various carotenoids which are of industrial value. Archaea can be considered to exhibit a relatively large capacity to produce isoprenoids, as they are components of their membrane lipids. Here, we aimed to produce isoprenoids such as phytoene in the hyperthermophilic archaeon Thermococcus kodakarensis. T. kodakarensis harbors a prenyltransferase gene involved in the biosynthesis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are precursors of squalene and phytoene, respectively. However, homologs of squalene synthase and phytoene synthase, which catalyze their condensation reactions, are not found on the genome. Therefore, a squalene/phytoene synthase homolog from an acidothermophilic archaeon Sulfolobus acidocaldarius, Saci_1734, was introduced into the T. kodakarensis chromosome under the control of a strong promoter. Production of the Saci_1734 protein was confirmed in this strain, and the generation of phytoene was detected (0.08–0.75 mg L⁻¹ medium). We then carried out genetic engineering in order to increase the phytoene production yield. Disruption of an acetyl-CoA synthetase I gene involved in hydrolyzing acetyl-CoA, the precursor of phytoene, together with the introduction of a second copy of Saci_1734 led to a 3.4-fold enhancement in phytoene production.

     

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.     

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, β-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for β-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.     

Enzymatic synthesis of carotenes by cell-free preparations of fruit of several genetic selections of tomatoes

Several mutants of tomatoes are known in which the carotene content of the fruit is markedly altered qualitatively and quantitatively from that found in the standard red tomato variety. These selections are: rr (yellow flesh, low carotene); tt (tangerine, orange, proneurosporene and prolycopene); at at (apricot, low in acyclic carotenes); og^c og^c (crimson, high in lycopene); Verkerk 377-2αα (probably identical to vircscent orange vo vo, high in ζ-carotene); B B (H_i-β, high in β-carotene), and Del Del (Hi-δ, high in δ-carotenc). Studies of carotene synthesis from [1-¹⁴C]isopentenyl pyrophosphate, [¹⁴C]phytoene, and [¹⁴C]lycopcne by soluble enzyme systems obtained from fruits of these selections have shown unexpected enzyme activities. All selections evidence activity for the synthesis of phytoene. All mutants have also been found to contain an enzyme system for the synthesis of β-carotenefrom lycopene. Three of the selections analyzed (rr, at at, and og^c og^c) also contain an enzyme system for the conversion of lycopene to α-carotene and the variants rr and tt contain an enzyme for the synthesis of poly-cis-carotencs from isopentenyl pyrophosphate and phytocne.The reasons for the discrepancies that are observed between carotene composition of fruit of field-grown tomato selections and enzyme activities for carotene synthesis by cell-free preparations obtained from these fruits are not presently known. It is obvious, however, that either inhibitors are present, cofactors are missing, or there are permeability barriers to substrate or cofactor transport into plastids of selections in which enzyme activities are not expressed in field-grown fruit. Further investigations will be required for clarification of this problem.     

New functional assignment of the carotenogenic genes crtB and crtE with constructs of these genes from Erwinia species

The role of carotenoid genes crtB and crtE has been functionally assigned. These genes were cloned from Erwinia into Escherichia coli or Agrobacterium tumefaciens. Their functions were elucidated by assaying early isoprenoid enzymes involved in phytoene formation. In vitro reactions from extracts of E. coli carrying the crtE gene or a complete carotenogenic gene cluster in which crtB was deleted showed an elevated conversion of farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP). These results strongly indicate that the crtE gene encodes GGPP synthase. Introduction of the crtB gene into A. tumefaciens led to the conversion of GGPP into phytoene. This activity was absent in similar transformants with the crtE gene. Thus, the crtB gene probably encodes phytoene synthase, which was further supported by demonstration that phytoene accumulated in E. coli harboring both the crtB and crtE genes.