Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen-methyl

The effect of acifluorfen-methyl on tetrapyrrole synthesis in greening chloroplasts of Cucumis sativus was examined. Formation of Mg-proto-porphyrin IX from δ-aminolevulinate was reduced 98% by 10 micromolar acifluorfen-methyl. Conversion of protoporphyrin IX to Mg-protoporphyrin IX was unaffected, but protoporphyrin IX synthesis from δ-aminolevulinate was blocked, indicating a site of inhibition prior to the Mg-chelatase. The enzymic oxidation of protoporphyrinogen IX to protoporphyrin IX was highly sensitive to acifluorfen-methyl, indicating that the site of action of the herbicide is the protoporphyrinogen oxidase. (© 1989 FMC Corporation. All rights reserved.)     

Induction of delta-Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena gracilis by N-Methyl Mesoporphyrin IX

N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable δ-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 × 10⁻⁶ molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and ⁵⁹Fe incorporation into heme was inhibited by N-methyl mesoporphyrin IX, indicating that, as in animal cells, N-methyl mesoporphyrin IX acts specifically to block iron insertion into protoporphyrin IX. Chlorophyll synthesis in wild-type cells was not affected within the first 6 hours after administration of N-methyl mesoporphyrin IX.     

delta-Aminolevulinic Acid Synthase of Euglena gracilis: Regulation of Activity

δ-Aminolevulinic acid (ALA), a key precursor of the tetrapyrroles heme and chlorophyll, is capable of being synthesized by two different routes in cells of the unicellular green alga Euglena gracilis: from the intact carbon skeleton of glutamate, and via the condensation of glycine and succinyl CoA, mediated by the enzyme ALA synthase. The regulatory properties of ALA synthase were examined in order to establish its role in Euglena.

Partially purified Euglena ALA synthase, unlike the case with the bacterial or animal-derived enzyme, does not exhibit allosteric inhibition by the tetrapyrrole pathway products heme, protoporphyrin IX, and porphobilinogen, at concentrations up to 100 micromolar.

In aplastidic mutant cells, extractable ALA synthase activity is constant during exponential growth, and decreases to low levels as the cells reach the stationary state. Rapid exponential decline of ALA synthase (t_1/2 = 55 min) occurs after administration of 43 micromolar cycloheximide, but not 6.2 millimolar chloramphenicol. These results suggest that, as in other eukaryotic cells, ALA synthase is synthesized on cytoplasmic ribosomes and is subject to rapid turnover in vivo.

Extractable ALA synthase activity increases 2.5-fold within 6 hours after administration of 100 millimolar ethanol, a stimulator of mitochondrial development, and 4.5-fold within 12 hours after administration of 1 millimolar 4,6-dioxoheptanoic acid, which blocks ALA utilization, suggesting that activity is controlled in vivo by a feedback induction-repression mechanism, coupled with rapid enzyme turnover.

In heterotrophically grown wild-type cells, low levels of ALA synthase rapidly increase 4.5-fold within 12 hours after cells are transferred from the light to the dark, and decrease exponentially (t_1/2 = 75 min) when cells are transferred from the dark to light. The dark levels are equal to those in light- or dark-grown aplastidic mutant cells. The low level occurring in light-grown wild-type cells is not altered by the presence of 10 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which blocks photosynthetic O₂ production. The decrease that occurs on dark-to-light transfer can be diminished by 12- or 24-hour prior incubation with 6.2 millimolar chloramphenicol, which also retards chlorophyll synthesis after the transfer to light.

The positive relationship of ALA synthase activity to degree of mitochondrial expression, and the inverse relationship to plastid development and chlorophyll synthesis, suggests that ALA synthase functions to provide precursors to nonplastid tetrapyrroles in Euglena. In light-grown, wild-type cells, the diminished levels of ALA synthase may be due to the ability of developing plastids to export heme or a heme precursor to other cellular regions, which thereby supplants the necessity for ALA formation via the ALA synthase route.

     

Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings

Cytokinin promotes morphological and physiological processes including the tetrapyrrole biosynthetic pathway during plant development. Only a few steps of chlorophyll (Chl) biosynthesis, exerting the phytohormonal influence, have been individually examined. We performed a comprehensive survey of cytokinin action on the regulation of tetrapyrrole biosynthesis with etiolated and greening barley seedlings. Protein contents, enzyme activities and tetrapyrrole metabolites were analyzed for highly regulated metabolic steps including those of 5-aminolevulinic acid (ALA) biosynthesis and enzymes at the branch point for protoporphyrin IX distribution to Chl and heme. Although levels of the two enzymes of ALA synthesis, glutamyl-tRNA reductase and glutamate 1-semialdehyde aminotransferase, were elevated in dark grown kinetin-treated barley seedlings, the ALA synthesis rate was only significantly enhanced when plant were exposed to light. While cytokinin do not stimulatorily affect Fe-chelatase activity and heme content, it promotes activities of the first enzymes in the Mg branch, Mg protoporphyrin IX chelatase and Mg protoporphyrin IX methyltransferase, in etiolated seedlings up to the first 5 h of light exposure in comparison to control. This elevated activities result in stimulated Chl biosynthesis, which again parallels with enhanced photosynthetic activities indicated by the photosynthetic parameters F _V/F _M, J _CO2max and J _CO2 in the kinetin-treated greening seedlings during the first hours of illumination. Thus, cytokinin-driven acceleration of the tetrapyrrole metabolism supports functioning and assembly of the photosynthetic complexes in developing chloroplasts.     

Photodynamic processes and the synthesis of 5-aminolevulinic acid in chlorella cells treated with amino acids and 1,10-phenanthroline

Treatment of chlorella (Chlorella sp.) cells for 2 h in darkness with tetrapyrrole-dependent photodynamic herbicides (TDPH) derived on the basis of 0.3 mM 1,10-phenanthroline (Ph) combined with 0.6 mM Glu or 0.6 mM Gln induced the accumulation of sensitizers of photodynamic processes: magnesium protoporphyrin IX (MgPP) and MgPP monomethyl ester (MgPPE). Within the first day after chlorella cells treated with TDPH were illuminated, photodestruction of MgPP(E) was observed, and production of the first specific precursor of chlorophyll (Chl), 5-aminolevulinic acid (ALA), in the cells declined. Then the accumulation of ALA was stimulated, and the level of heme, which is a retroinhibitor of ALA synthesis, simultaneously fell. During the first two days of illumination, the content of Chl and carotenoids in the algae treated with TDPH did not differ from their levels in control culture, which suggests a high resistance of photosynthetic pigments to photodynamic process induced by porphyrins. Subsequently, a slight but rising in time accumulation of pheophytin (Pheo) was observed, as well as photodestruction of Chl and carotenoids. After five days of illumination, the difference in the content of Chl between the culture treated with TDPH and control material was 10–30% depending on the illuminance. Chlorella cells treated with TDPH remained capable of producing Chl from exogenous ALA in the dark for at least eight days. In the experiments simultaneously conducted with a higher plant, cucumber (Cucumis sativa L.), which accumulated in the dark essentially the same content of porphyrins in response to TDPH as algae did, the residual level of Chl after five days of illumination was only 10–20% of control plants. It was assumed that a high tolerance of the chlorella pigment pool to photooxidative stress induced by the accumulation of MgPP(E) and Pheo depended on a highly active state of the antioxidant protective system and the ability of ALA molecules additionally formed under the influence of TDPH to be converted into Chl, thereby participating in its de novo synthesis.     

THE PORPHYRIN REQUIREMENTS OF HAEMOPHILUS INFLUENZAE AND SOME FUNCTIONS OF THE VINYL AND PROPIONIC ACID SIDE CHAINS OF HEME

1. Iron protoporphyrin IX was required for the growth of H. influenzae. It could be replaced by protoporphyrin IX. When grown on protoporphyrin evidence was obtained for the presence of Fe porphyrin in the organism. It was concluded that the organism could insert iron into the protoporphyrin ring. 2. In the smooth strains, other porphyrins containing no iron such as deutero-, hemato-, meso-, and coproporphyrins could not replace protoporphyrin for growth. Since protoporphyrin has two vinyl groups which other porphyrins lack, it was concluded that the two vinyl groups were essential for growth. 3. When porphyrins lacking vinyl groups were converted chemically into iron porphyrins and then supplied to the organisms it was found that these iron porphyrins supported growth. It was concluded that the "smooth" organisms were able to insert iron only into the porphyrin containing the vinyl groups; i.e., protoporphyrin. One function of the vinyl groups then was to permit iron to be inserted biologically into the porphyrin ring. 4. An anomalous behavior in the rough Turner strain was observed and discussed. This organism was able to insert iron into mesoporphyrin at low concentrations but was inhibited by this compound at higher concentrations. In all other reactions with the porphyrins this rough strain behaved in the same was as did the smooth strains. 5. All strains which were grown on iron porphyrins lacking vinyl groups could not reduce nitrate to nitrite. When grown on protoporphyrin or Fe protoporphyrin reduction of nitrate occurred. It was concluded that the nitrate-reducing mechanism required the presence of the vinyl groups either for its formation or function. 6. The porphyrins lacking iron and lacking vinyl groups inhibited the growth of H. influenzae on Fe protoporphyrin. The inhibition between a porphyrin and Fe protoporphyrin was a competitive one. It was suggested that the porphyrin inhibited the growth-promoting properties of Fe protoporphyrin by attaching on to a particular apoprotein, thus preventing the formation of a heme catalyst. Likewise, competition between two growth-promoting Fe porphyrins for apoenzymes could be shown to occur. 7. Protoporphyrin and Fe protoporphyrin supported growth. When their propionic acid side chains were esterified they no longer supported growth. It was suggested that the esterified carboxyl groups could not attach to the specific apoproteins to form the heme enzymes and so could not act to support growth. For the same reason the inhibitory action of porphyrins lacking vinyl groups could be prevented by esterifying their propionic acid groups.     

Role of PUG1 in inducible porphyrin and heme transport in Saccharomyces cerevisiae

Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [(55)Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1-lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.     

Heme Mediates Cytotoxicity from Artemisinin and Serves as a General Anti-Proliferation Target

Heme (Fe2+ protoporphyrin IX) is an essential molecule that has been implicated the potent antimalarial action of artemisinin and its derivatives, although the source and nature of the heme remain controversial. Artemisinins also exhibit selective cytotoxicity against cancer cells in vitro and in vivo. We demonstrate that intracellular heme is the physiologically relevant mediator of the cytotoxic effects of artemisinins. Increasing intracellular heme synthesis through the addition of aminolevulinic acid, protoporphyrin IX, or transferrin-bound iron increased the cytotoxicity of dihydroartemisinin, while decreasing heme synthesis through the addition of succinyl acetone decreased its cytotoxic activity. A simple and robust high throughput assay was developed to screen chemical compounds that were capable of interacting with heme. A natural products library was screened which identified the compound coralyne, in addition to artemisinin, as a heme interacting compound with heme synthesis dependent cytotoxic activity. These results indicate that cellular heme may serve a general target for the development of both anti-parasitic and anti-cancer therapeutics.     

Nucleotide sequence of the Rhodobacter capsulatus hemH gene

The last step in heme synthesis is the insertion of iron into the ring of protoporphyrin IX. The enzyme which catalyzes this reaction, ferrochelatase (FC), is encoded by the hemH gene. A clone containing this gene from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, has been sequenced. A single open reading frame was found which could encode a protein of 351 amino acids. This putative protein is very similar to other FC and contains the FC signature sequence     

The C-terminal extension of ferrochelatase is critical for enzyme activity and for functioning of the tetrapyrrole pathway in Synechocystis strain PCC 6803

Heme and chlorophyll (Chl) share a common biosynthetic pathway up to the branch point where magnesium chelatase and ferrochelatase (FeCH) insert either magnesium for Chl biosynthesis or ferrous iron for heme biosynthesis. A distinctive feature of FeCHs in cyanobacteria is their C-terminal extension, which forms a putative transmembrane segment containing a Chl-binding motif. We analyzed the ΔH324 strain of Synechocystis sp. strain PCC 6803, which contains a truncated FeCH enzyme lacking this C-terminal domain. Truncated FeCH was localized to the membrane fraction, suggesting that the C-terminal domain is not necessary for membrane association of the enzyme. Measurements of enzyme activity and complementation experiments revealed that the ΔH324 mutation dramatically reduced activity of the FeCH, which resulted in highly upregulated 5-aminolevulinic acid synthesis in the ΔH324 mutant, implying a direct role for heme in the regulation of flux through the pathway. Moreover, the ΔH324 mutant accumulated a large amount of protoporphyrin IX, and levels of Chl precursors were also significantly increased, suggesting that some, but not all, of the “extra” flux can be diverted down the Chl branch. Analysis of the recombinant full-length and truncated FeCHs demonstrated that the C-terminal extension is critical for activity of the FeCH and that it is strictly required for oligomerization of this enzyme. The observed changes in tetrapyrrole trafficking and the role of the C terminus in the functioning of FeCH are discussed.     

Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.     

Characterization of a family of chlorophyll-deficient wheat (Triticum) and barley (Hordeum vulgare) mutants with defects in the magnesium-insertion step of chlorophyll biosynthesis.

During thylakoid membrane biogenesis, chlorophyll (Chl) biosynthesis and the accumulation of Chl-binding proteins are tightly linked, light-regulated processes. We have investigated the consequences faced by mutant plants with defects in Chl biosynthesis by studying a series of five homeologous allelic chlorina mutants in wheat (Triticum) and one phenotypically related barley (Hordeum vulgare) mutant that express the same pleiotropic mutant phenotype but to different extents. These mutants accumulate Chl at different rates, with the most severely affected plants having the slowest rate of Chl accumulation. Analysis of precursor pools in the Chl synthesis pathway indicates they have a partial block in Chl synthesis and accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The affected plants with the most severe phenotypes accumulate the most Proto. Chloroplasts isolated from these mutants exhibit a lower activity of the enzyme Mg-chelatase, which catalyzes the first committed step in Chl synthesis. The most severely affected plants exhibit the greatest reduction in Mg-chelatase activity. Heme levels and protoporphyrinogen oxidase activity were the same for mutant and wild-type plants. We suggest that a block in Mg-chelatase activity in these mutants could account for the other traits of their pleiotropic phenotype previously described in the literature.     

Zinc protoporphyrin IX binds heme crystals to inhibit the process of crystallization in Plasmodium falciparum

The intraerythrocytic Plasmodium falciparum parasite converts most of host hemoglobin heme into a nontoxic heme crystal. Erythrocyte zinc protoporphyrin IX, normally present at 0.5 microM, which is a ratio of 1:40,000 hemes, can elevate 10-fold in some of the anemias associated with malaria disease protection. This work examines a binding mechanism for zinc protoporphyrin IX inhibition of heme crystallization similar to the antimalarial quinolines. Zinc protoporphyrin IX neither forms crystals alone nor extends on preformed heme crystals. Inhibition of both seed heme crystal formation and crystal extension occurs with an inhibitory concentration (IC)50 of 5 microM. Field emission in-lens scanning electron microscopy depicts the transition and inhibition of heme monomer aggregates to heme crystals with and without seeding of preformed hemozoin templates. In vitro zinc protoporphyrin IX, like the quinolines, binds to heme crystals in a saturable, specific, pH, and time-dependent manner. The ratio at saturation is approximately 1 zinc protoporphyrin IX per 250 hemes of the crystal. Unlike the quinolines, zinc protoporphyrin IX binds measurably in the absence of heme. Isolated ring and trophozoite stage parasites have an elevated zinc protoporphyrin IX to heme ratio 6 to 10 times that in the erythrocyte cytosol, which also corresponds to elevated ratios found in heme crystals purified from Plasmodium parasites. This work implicates protection from malaria by a mechanism where elevated zinc protoporphyrin IX in anemic erythrocytes binds to heme crystals to inhibit further crystallization. In endemic malaria areas, severe iron deficiency anemia should be treated with antimalarials along with iron replenishment.     

Utilization of Heme as an Iron Source by Marine Alphaproteobacteria in the Roseobacter Clade

The bioavailability and utilization of porphyrin-bound iron, specifically heme, by marine microorganisms have rarely been examined. This study used Ruegeria sp. strain TrichCH4B as a model organism to study heme acquisition by a member of the Roseobacter clade. Analogs of known heme transporter proteins were found within the Ruegeria sp. TrichCH4B genome. The identified heme uptake and utilization system appears to be functional, as the heme genes were upregulated under iron stress, the bacterium could grow on ferric-porphyrin complexes as the sole iron source, and internalization of ⁵⁵ Fe from ferric protoporphyrin IX was observed. The potential ability to utilize heme in the Roseobacter clade appears to be common, as half of the isolates in the RoseoBase database were found to have a complete heme uptake system. A degenerate primer set was designed and successfully used to identify the putative heme oxygenase gene (hmus) in the roseobacter heme uptake system from diverse nonenriched marine environments. This study found that members of the Roseobacter clade are capable of utilizing heme as an iron source and that this capability may be present in all types of marine environments. The results of this study add a new perspective to the current picture of iron cycling in marine systems, whereby relatively refractory intracellular pools of heme-bound iron may be taken up quickly and directly reincorporated into living bacteria without previous degradation or the necessity of a siderophore intermediate.     

Ferrochelatase activity and protoporphyrin IX utilization in Haemophilus influenzae.

Previous research showed that the heme-requiring human pathogen Haemophilus influenzae lacks the first six of the seven enzymes required for heme synthesis, starting with the precursor, 5-amino levulinic acid. In this study, I demonstrated either directly or by reasonable inference that all 57 strains of H. influenzae examined, including 2 unable to grow on protoporphyrin IX, possess ferrochelatase, which catalyzes heme formation by insertion of Fe2+ into the protoporphyrin IX nucleus and which is the last enzyme in the heme synthetic pathway. Further, I showed that this enzyme can also function in the reverse direction, releasing Fe2+ from heme.     

Biosynthesis of delta-aminolevulinic acid and the regulation of heme formation by immature erythroid cells in man

Heme formation in the erythron is subject to end product regulation by negative feedback, but the exact point of metabolic control in human erythroid cells is unknown. To investigate the mode of action of heme on its own formation, the effects of micromolar concentrations of hemin on de novo synthesis of protoporphyrin IX and delta-aminolevulinate (delta-ALA) by intact human reticulocytes were examined in the presence of 1 mM alpha,alpha'-bipyridyl and 200 microM 4,6-dioxoheptanoate to block their further conversion by ferrochelatase or delta-ALA dehydrase, respectively. At final concentrations (25-40 microM), hemin, which is known to reduce incorporation of [2-14C]glycine into cellular heme, significantly inhibited formation of protoporphyrin IX and total delta-aminolevulinate in situ by these cells. Since synthesis of the first committed precursor, delta-aminolevulinate, as well as protoporphyrin (which is derived from it) were diminished, the effects of hemin on delta-aminolevulinate synthase (EC 2.3.1.37) were studied. Hemin, at concentrations up to 40 microM, had no direct effect on enzymatic activity, as measured with [5-14C] alpha-ketoglutarate (in hypotonically lysed cells) or [1,4-14C]succinyl coenzyme A (in deoxycholate lysates), even after preincubation. However, when intact human reticulocytes were incubated with hemin before assay for delta-ALA synthase, there was a rapid, concentration-dependent reduction in enzymatic activity (mean 42 and 23% inhibition after 60 min for these two substrates, respectively). Hemin had no effect on steady-state levels of delta-ALA synthase mRNA, as determined by Northern blot hybridization using an erythroid-specific human cDNA probe. Thus, a mechanism for inducing feedback inhibition of the tetrapyrrole pathway exists in human erythroid cells. It controls formation of the first committed precursor of protoporphyrin IX, delta-aminolevulinate, and hence regulates heme biosynthesis by limiting the availability of the porphyrin, rather than the metal substrate for the ferrochelatase reaction. Hemin interacts with constituents of the intact reticulocyte significantly to reduce delta-aminolevulinic acid synthase activity by an indirect cellular process that does not influence the abundance of erythroid-specific synthase mRNA but may either inhibit its ribosomal translation in an unknown manner or promote degradation of the enzyme itself by specific proteolysis.     

Porphyrin Accumulation and Export by Isolated Barley (Hordeum vulgare) Plastids (Effect of Diphenyl Ether Herbicides)

We have investigated the formation of porphyrin intermediates by isolated barley (Hordeum vulgare) plastids incubated for 40 min with the porphyrin precursor 5-aminolevulinate and in the presence and absence of a diphenylether herbicide that blocks protoporphyrinogen oxidase, the enzyme in chlorophyll and heme synthesis that oxidizes protoporphyrinogen IX to protoporphyrin IX. In the absence of herbicide, about 50% of the protoporphyrin IX formed was found in the extraplastidic medium, which was separated from intact plastids by centrifugation at the end of the incubation period. In contrast, uroporphyrinogen, an earlier intermediate, and magnesium protoporphyrin IX, a later intermediate, were located mainly within the plastid. When the incubation was carried out in the presence of a herbicide that inhibits protoporphyrinogen oxidase, protoporphyrin IX formation by the plastids was completely abolished, but large amounts of protoporphyrinogen accumulated in the extraplastidic medium. To detect extraplastidic protoporphyrinogen, it was necessary to first oxidize it to protoporphyrin IX with the use of a herbicide-resistant protoporphyrinogen oxidase enzyme present in Escherichia coli membranes. Protoporphyrinogen is not detected by some commonly used methods for porphyrin analysis unless it is first oxidized to protoporphyrin IX. Protoporphyrin IX and protoporphyrinogen found outside the plastid did not arise from plastid lysis, because the percentage of plastid lysis, measured with a stromal marker enzyme, was far less than the percentage of these porphyrins in the extraplastidic fraction. These findings suggest that of the tetrapyrrolic intermediates synthesized by the plastids, protoporphyrinogen and protoporphyrin IX, are the most likely to be exported from the plastid to the cytoplasm. These results help explain the extraplastidic accumulation of protoporphyrin IX in plants treated with photobleaching herbicides. In addition, these findings suggest that plastids may export protoporphyrinogen or protoporphyrin IX for mitochondrial heme synthesis.     

Glucose and δ-Aminolevulinic Acid Stimulate the Dark Chlorophyll Synthesis of Rice Seedlings

This research was to examine if rice (Oryza sativa L.), a monocotyledon of angiosperm, was able to synthesize chlorophyll (Chl) in complete darkness. Five-cm-tall etiolated seedlings of rice were used as starting materials and treated with or without various concentrations of glucose and/or δ-aminolevulinic acid (ALA) in the dark. Leaves harvested at the indicated time were determined for their contents of Chl, protoporphyrin Ⅸ(Proto), Mg-protoporphyrin Ⅸ(Mg-Proto) and protochlorophyllide (Pchlide). The mole percentage of porphyrin was calculated. The Chl content in the etiolated rice seedlings slightly increased from about 2.5 μg/g to 7.5 μg/g within 12 d in the dark, but the total Chl of dark-grown rice increased from 0.36 μg/g to 3.6 μg/g. While the mole percentages of Proto, Mg-Proto and Pchlide in the dark-grown seedlings without any treatment were about 65%, 27.5% and 7.5% at the beginning, respectively, those in the light-grown seedlings were about 42.5%, 35% and 22.5%, respectively. The mole percentage of porphyrin of etiolated seedlings resumed its normal ratio within 2 d after treatment with glucose. While the Chl content of etiolated seedlings grown in culture solution with 3% and 6% glucose increased 2.5 and 4.0 folds, respectively, those with 3% and 6% glucose and 1 mmol/L ALA increased 22 and 24 folds, respectively. It is concluded that angiosperm might be able to synthesize a small amount of Chl in complete darkness, that either glucose or ALA could stimulate dark Chl synthesis in angiosperm, and that a combination of glucose and ALA exhibited an additional effect. It is still unknown and remains to be further explored what is the mechanism of the effect of glucose and ALA on the Chl synthesis of rice in the dark. Key words: angiosperm; rice; dark chlorophyll synthesis; glucose; δ-aminolevulinic acid; protoporphyrin Ⅸ; Mg-protoporphyrin Ⅸ; protochlorophyllide