Accumulation Of Proto Research Articles

The function of PROTOPORPHYRINOGEN IX OXIDASE in chlorophyll biosynthesis requires oxidised plastoquinone in Chlamydomonas reinhardtii

In the last common enzymatic step of tetrapyrrole biosynthesis, prior to the branching point leading to the biosynthesis of heme and chlorophyll, protoporphyrinogen IX (Protogen) is oxidised to protoporphyrin IX (Proto) by protoporphyrinogen IX oxidase (PPX). The absence of thylakoid-localised plastid terminal oxidase 2 (PTOX2) and cytochrome b6f complex in the ptox2 petB mutant, results in almost complete reduction of the plastoquinone pool (PQ pool) in light. Here we show that the lack of oxidised PQ impairs PPX function, leading to accumulation and subsequently uncontrolled oxidation of Protogen to non-metabolised Proto. Addition of 3(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) prevents the over-reduction of the PQ pool in ptox2 petB and decreases Proto accumulation. This observation strongly indicates the need of oxidised PQ as the electron acceptor for the PPX reaction in Chlamydomonas reinhardtii. The PPX-PQ pool interaction is proposed to function as a feedback loop between photosynthetic electron transport and chlorophyll biosynthesis.

A protoporphyrinogen oxidase gene expression influences responses of transgenic rice to oxyfluorfen

We compared the defense against photoinhibitions and oxidative stress in transgenic rice expressing a modified Myxococcus xanthus protoporphyrinogen oxidase (MxProtox) gene and in wild-type (WT) rice. Although the MxProtox transgenic lines had higher content of protoporphyrin IX (Proto IX) than the untreated controls, they did not exhibit a drastic accumulation of Proto IX as in the WT after 2 d of 50 μM oxyfluorfen (OF) treatment. In the transgenic lines S4 and S11, the transcriptions of OsProtox and modified MxProtox genes were almost sustained in response to OF, although transcription of OsProtox was greatly down-regulated in the WT. The excess Proto IX in the WT plants treated with OF generated a severe stress mediated by singlet oxygen (1O2), leading to a prominent increases in electrolyte leakage and malondialdehyde production. This stress in the WT necessitated not only a substantial accumulation of zeaxanthin and antheraxanthin, but also strong increases in activities of superoxide dismutase, catalase, and peroxidase as well as transcriptions of CatalaseB, Ascorbate Peroxidase, and Heme Oxygenase2 genes. By contrast, the transgenic plants did not result in any noticeable increase in these parameters. Our results demonstrate that the transgenic rice expressing modified MxProtox efficiently prevented accumulation of photosensitizing Proto IX through sustaining higher transcriptions of porphyrin biosynthetic genes, thereby reducing the stress imposed by OF.

Resistance mechanisms in protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice

We investigated the mechanism for conferring herbicide resistance in transgenic rice. Plants from Line M4 were resistant to PROTOX inhibitors and had yields similar to those from wild-type (WT) rice.Myxococcus xanthus PROTOX mRNA was abundantly expressed in the transgenic leaf tissues, and theM. xanthus PROTOX gene was stably transmitted into the T4 generation. We detected a protein with a predicted molecular mass of 50 kD, equal to the weight ofM. xanthus PROTOX, in M4 but not WT plants. Furthermore, several PROTOX-inhibitor herbicides — acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon — caused significant cellular leakage and lipid peroxidation in the WT, but not in the transgenics. Total PROTOX activity in untreated transformed rice was 17-fold higher than in the WT, with activity being inhibited in the latter genotype by 55%, 59%, 53%, or 60% as a result of treatment with acifluorfen, oxyfluorfen, carfentrazone-ethyl, or oxadiazon, respectively. However, PROTOX activities in transgenic rice were similar to their corresponding, untreated controls. The accumulation of Proto IX was 15-to 21-fold higher in the WT than in M4 when plants were treated with PROTOX inhibitors. In the former, their epicuticular wax and chloroplasts were severely damaged after oxyfluorfen treatment The lack of damage in transformed plants suggests that M4 does not accumulate Proto IX, probably due to the production of herbicide-resistant chloroplastic and mitochondria PROTOX.

Physiological basis for resistance to diphenyl ether herbicides in common waterhemp (Amaranthus rudis)

Common waterhemp seeds were collected from two Missouri soybean fields where biotypes were not controlled by acifluorfen. Plants grown from these seeds were tested for resistance to the diphenyl ether herbicides acifluorfen and lactofen. Resistance to susceptibility (R/S) ratios, calculated as the ratio of the dose required to inhibit dry weight accumulation by 50% (GR50) in resistant plants to that for susceptible plants, were 9.5 and 11 for the Meadville biotype and 28 and 44 for the Bethel biotype exposed to acifluorfen and lactofen, respectively. Electrolyte leakage assays determined that light-induced lipid peroxidation by acifluorfen was greatest on a control population (Bradford), intermediate for the Meadville biotype, and lowest for the Bethel biotype. Levels of the photodynamic pigment protoporphyrin IX (Proto) accumulating in leaf disks exposed to acifluorfen were much lower in the resistant biotypes than in the susceptible wild type, and the level of Proto accumulation was significantly correlated to the degree of membrane disruption. Although the binding of acifluorfen to protoporphyrinogen oxidase in chloroplasts may have been altered in the resistant biotypes, the molecular and biochemical factors involved in the mechanism of resistance remain to be fully characterized. However, this study establishes that the physiological basis for the evolved resistance to diphenyl ethers in common waterhemp rests on the reduction of Proto accumulation.

Modulator of heme biosynthesis induces apoptosis in leukemia cells.

The purpose of this research is the investigation of the possible cause(s) of the dark-cell death phenomenon induced by 1,10-phenanthroline (Oph), a porphyrin biosynthesis modulator. We have previously shown that porphyrin biosynthesis modulators, such as Oph, which is also an iron-chelating agent, enhance protoporphyrin IX (Proto) accumulation in mammalian neoplastic cells treated with delta-aminolevulinic acid (ALA). As a result of the enhanced Proto accumulation, a significant increase in photodynamic damage was observed under illumination. Also tetrapyrrole and heme-biosynthesis modulators have been shown to cause death in treated insect larvae in darkness, a phenomenon referred to as dark-cell death. Dark-cell death was also observed in Oph + ALA-treated transformed mammalian cells. Neoplastic cells were treated with ALA, Oph, and ALA + Oph, and the following cell properties were investigated: growth arrest, membrane permeability, cell survival, nucleosomal cleavage, and cell cycle alterations. It was observed that Oph but not ALA induced growth arrest, in a T-cell leukemia line (MLA 144) as assessed by reduction in DNA synthesis. Exogenous Proto and isomers of Oph lacking the iron-chelating property of Oph also caused a dose-dependent inhibition of proliferation in MLA 144 cells. Although the plasma membrane of Oph-treated cells remained intact following 3 h of dark-incubation, the cells exhibited DNA internucleosomal cleavage, characteristic of cells undergoing apoptosis. Cell cycle analysis using the DNA intercalating dye propidium iodide (PI) coupled to flow cytometry, indicated that 81 +/- 5.6% of Oph-treated MLA 144 cells were apoptotic, with the majority of the cells arrested in the early S phase. On the other hand, treatment with either ALA or Proto did not alter the cell cycle. Also, using a double-labeling protocol with Hoechst 33342, and PI, and analysis by flow cytometry, Oph-treated cells were found to be 82% apoptotic after 3 h of dark-incubation. Apoptosis was reduced by 75% (p < 0.05) by the cytoplasmic protein synthesis inhibitor cycloheximide. These results indicate that in addition to enhancing Proto accumulation, the heme biosynthesis modulator Oph also induces growth arrest and apoptosis in transformed cells in darkness.

Determination of Porphyrin-Induced Peroxidation of Plant Lipids by Oxygen Electrode.

Protoporphyrinogen oxidase-inhibiting herbicides cause massive accumulation of protoporphyrin IX (Proto IX) in treated plant cells. Reactive oxygen species (ROS) induced by the accumulated Proto IX is thought to cause peroxidative damage, however, the process of peroxidation is still unclear. To investigate the mechanism of the peroxidation of lipids by Proto IX-induced ROS, we used an oxygen electrode. By measuring dissolved oxygen consumption in the reaction mixture, the rate of peroxidation was evaluated in vitro. Production of lipid hydroperoxide was confirmed by measuring thiobarbituric acid reacting substances (TBARS). The linear decrease of dissolved oxygen and linear increase of lipid hydroperoxides occurred immediately after light irradiation, suggesting that the method used in this study is applicable as a model system to investigate lipid peroxidation by Proto IX-induced ROS. Determination of the antioxidants effect on the reduction of dissolved oxygen indicated that both superoxide and hydrogen peroxide scavenging enzymes as well as singlet oxygen scavengers remarkably suppressed the peroxidative reaction. These results suggest that in addition to singlet oxygen, other ROS are involved in the Proto IX-induced peroxidation processes in plant cells. Furthermore, comparison of porphyrin intermediates derived from chlorophyll synthesis showed that the peroxidizing ability of Proto IX was not higher than other porphyries. This suggests that greater accumulation of Proto IX is necessary to initiate lipid peroxidation in plant cells.

Protoporphyrin IX and oxidative stress

The short- and long-term pro-oxidant effect of protoporphyrin IX (PROTO) administration to mice was studied in liver. A peak of liver porphyrin accumulation was found 2 h after the injection of PROTO (3.5 mg/kg, i.p.); then the amount of porphyrins diminished due to biliar excretion. After several doses of PROTO (1 dose every 24 h up to 5 doses) a sustained enhancement of liver porphyrins was observed. The activity of δ-amino-levulinic acid synthetase was induced 70–90% over the control values 4 h after the first injection of PROTO and stayed at these high levels throughout the period of the assay. Administration of PROTO induced rapid liver damage, involving lipid peroxidation. Hepatic GSH content was increased 2 h after the first injection of PROTO, but then decreased below the control values which were maintained after several doses of porphyrin. After a single dose of PROTO, Cu-Zn superoxide dismutase (SOD) was rapidly induced, suggesting that superoxide radicals had been generated. Increased levels of hydrogen peroxide coming from the reaction catalyzed by SOD and lipid peroxides as a consequence of membrane peroxidation, induced the activity of catalase and glutathione peroxidase (GPx), while decreased GSH levels induced glutathione reductase (GRed) activity. However after 5 doses of PROTO, the activity of SOD was reduced reaching control values. GPx and catalase activities slowly went down, while GRed continued increasing as long as the levels of GSH were kept very low. TBARS values, although lower than those observed after a single dose of PROTO, remained above control values; Glutathione S-transferase activity was instead greatly diminished, indicating sustained liver damage.Our findings would indicate that accumulation of PROTO in liver induces oxidative stress, leading to rapid increase in the activity of the antioxidant enzymes to avoid or revert liver damage. However, constant accumulation of porphyrins provokes a liver damage so severe that the antioxidant system is compromised.

Selectivity and mode of action of carfentrazone-ethyl, a novel phenyl triazolinone herbicide

Post-emergence application of carfentrazone-ethyl at rates as low as 2.2 g ha -1 caused greater leaf injury and growth reduction in ivyleaf morningglory (Ipomoea hederacea) and velvetleaf (Abutilon theophrasti) than in soybean (Glycine max). The herbicide was more rapidly metabolized in the crop than in the weed species, with 26.7, 54.3 and 60.6% of the parent compound remaining in soybean, ivyleaf morningglory and velvetleaf, respectively, 24 h after exposure. The free acid metabolite, carfentrazone, was present in all species and accounted for 21.2-27.4% of the total radioactivity. Unknown metabolites (R f 0 and 0.22) were four to five times more abundant in soybean than in the weed species. Carfentrazone-ethyl induced more leakage from leaf discs from the weeds than those from soybean and the degree of injury correlated with the amount of protoporphyrin IX (Proto IX) present in the treated tissues. Both carfentrazone-ethyl and carfentrazone were potent inhibitors of protoporphyrinogen oxidase (Protox). Therefore, the selectivity of this herbicide may, at least in part, be attributed to the lower accumulation of Proto IX in soybean than in the weeds, probably because of the ability of soybean to metabolize more carfentrazone into unknown metabolites than the weeds.

Selection and Characterization of Protoporphyrinogen Oxidase Inhibiting Herbicide (S23142) Resistant Photomixotrophic Cultured Cells of Nicotiana tabacum

Summary S23142 and Acifluorfen-ethyl (AFE) inhibit protoporphyrinogen oxidase (Protox) and induce accumulation of protoporphyrin IX (Proto IX) which is a strong phytotoxic photosensitizer. A S23142-resistant cell line, YZI-1 S, of photomixotrophically cultured tobacco was selected and its resistance mechanism was characterized. While growth rates of wild-type and YZI-1 S cells were similar in the absence of the herbicide, S23142 concentrations that reduced the chlorophyll contents by 50 % were 2 and 250 nM for wild-type and YZI-1 S cell lines, respectively. The YZI-1 S cells also exhibited resistance for other types of Protox inhibiting herbicides (acifluorfenethyl, acif luorf en, bif enox, oxadiazon, chlomethoxynil, nitrof en and chloronitrofen), but were sensitive to atrazine and DCMU, which inhibit photosynthetic electron transport. YZI-1S cells did not accumulate Proto IX, even at 100nM S23142 in which the wild-type cells accumulated large amounts of Proto IX. Protox isolated from YZI-1S cells showed a 2-fold higher activity than that of wild-type cells and also exhibited a 20-fold increase in tolerance to S23142. On the other hand, treatment with 1 mM 6-Aminolevulinic acid (ALA), a tetrapyrrole precursor, induced photobleaching by accumulation of Proto IX in both YZI-1 S and wild-type cells under high light irradiation. From these results, we conclude that the resistance of YZI-1S cells to S23142 is due mainly to the increase of Protox activity.

Accumulation of protoporphyrin IX in light-sensitive mutants of Escherichia coli

The accumulation of protoporphyrin IX (Proto IX) in light-sensitive mutants of Escherichia coli was detected by spectrofluorimetry. Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells. Proto IX clearly accumulated in cells with mutations in the visA ( hemH) gene but not in the wild-type strain CA274 or in visA mutants that had been rendered light-resistant by introduction of the wild-type visA + gene. Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene. These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.

Photodestruction of tumor cells by induction of endogenous accumulation of protoporphyrin IX: enhancement by 1,10-phenanthroline.

Rapidly proliferating transformed mammalian cells can be photodestroyed in vitro upon inducing the accumulation of endogenous protoporphyrin IX (Proto). Proto biosynthesis and accumulation were triggered by manipulation of the porphyrin-heme biosynthetic pathway. Proto accumulation in cultured cells was induced by treatment with 1.0 mM delta-aminolevulinic acid (ALA), a naturally occurring 5-carbon amino acid, for 3.5 h. In darkness, significant Proto accumulation became evident within 3.5 h of incubation. In the light, the accumulated tetrapyrroles triggered destruction of treated cells within the first 30 min of illumination, probably via the rapid oxidation of cellular constituents by singlet oxygen. Protoporphyrin IX accumulation and specific cell lysis increased significantly by inclusion of 0.75 mM 1,10-phenanthroline (Oph), a tetrapyrrole biosynthesis modulator. Slower growing untransformed cells did not accumulate significant amounts of Proto following ALA and Oph treatment unless stimulated to proliferate with the mitogenic lectin Concanavalin A.

Protoporphyrinogen Oxidase-Inhibiting Herbicides

Several commercial and experimental herbicides such asp-nitrodiphenyl ethers, oxadiazoles, and cyclic imides inhibit protoporphyrinogen IX oxidase (Protox), the enzyme that converts protoporphyrinogen IX to protoporphyrin IX (Proto). This leads to uncontrolled autooxidation of the substrate and results in accumulation of Proto. Blockage of the porphyrin pathway at this site inhibits synthesis of both chlorophylls and heme. Heme is a feedback regulator of the porphyrin pathway. Thus, inhibition of Protox also deregulates the pathway, causing increased carbon flow to the accumulating pool of Proto. Proto is a potent photosensitizer that generates high levels of singlet oxygen in the presence of molecular oxygen and light. In plants treated with these herbicides, damage is light dependent and closely correlated with the level of Proto that accumulates. Proto accumulation is apparently largely extraplastidic, resulting in rapid photodynamic damage to the plasmalemma and tonoplast. After high levels of Proto accumulate in response to these herbicides, protochlorophyllide (PChlide) levels can increase also; however, Proto appears to be the primary photodynamic pigment responsible for the herbicidal activity.

Protoporphyrin IX Accumulation in Lemna paucicostata Hegelm. Caused by Diphenyl Ether Herbicides and Their Herbicidal Activity.

The relationship between protoporphyrin IX (Proto IX) levels and herbicidal activity of diphenyl ether herbicides in intact higher plant Lemna paucicostata Hegelm. strain 441 was determined. Rapid accumulation of Proto IX in the plant was observed after simultaneous exposure to light and oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl) benzene]. At 4hr, the accumulation reached the maximum and this was followed by a rapid decrease. The rate of decrease was slowed when the plants were transferred to darkness after 4hr light irradiation, although some Proto IX degradation continued in the dark. This suggests that not only photo-decomposition but degradation and/or metabolism which occurred in darkness are involved in the decrease. Proto IX levels accumulated in the plants and electrolyte leakage from the plants were increased with increasing concentration of oxyfluorfen. A positive correlation was found between Proto IX content and electrolyte leakage. Diphenyl ether herbicides used in the study had differential activity in the accumulation of Proto IX. A positive correlation was also found between Proto IX levels and their herbicidal activity to the plant.These data suggest that Proto IX is the principal compound responsible for the rapid electrolyte leakage from plants, and that variation in the herbicidal activity of the diphenyl ethers observed in the study is caused by differential activity in accumulation of Proto IX.

The role of protoporphyrin IX in the mechanism of action of diphenyl ether herbicides

AbstractPhotobleaching diphenyl ether, cyclic imide and oxadiazole herbicides inhibit protoporphyrinogen oxidase (Protox), resulting in uncontrolled, non‐enzymic oxidation of accumulated protoporphyrinogen to protoporphyrin IX (Proto). Proto accumulation is stimulated by deregulation of the porphoyrin pathway due to blockage of heme synthesis. Heme is a Proto product and a feedback inhibitor of δ‐aminolevulinic acid synthesis. Strong correlations were found in several species under various conditions (e.g. sprayed on intact plants or taken up by leaf dises) between the accumulation of Proto and the degree of herbicidal damage caused by these herbicides. Levels of protochlorophyllide, Mg‐Proto, or Mg‐Proto monomethyl ester did not correlate significantly with herbicidal damage. Kinetic data indicated that elevated levels of protochlorophyllide in tissues treated with these herbicides only occurred after Proto had accumulated to high levels. Thus, Proto apparently re‐enters the porphyrin pathway to a significant extent after other cellular sites are saturated. Fluorescence microscopy indicated that considerable amounts of Proto accumulated outside the plastid in herbicide‐treated cells. All of the data were consistent with the hypothesis that Proto plays a critical role as a photosensitizing pigment in the mode of action of Protox‐inhibiting herbicides and that chlorophyll precursors that are derivatives of Proto are not significantly involved in the mode of action of these herbicides.