Light control of porphyrin accumulation in acifluorfen-methyl-treated Lemna pausicostata

In Lemna pausicostata Hegelm. 6746, light is required for sufficient acifluorfenmethyl (AFM) stimulation of protoporphyrin IX (Proto IX) accumulation to cause significant herbicidal action. In darkness, AFM causes Proto IX levels to increase for about 2 h, after which Proto IX content is stable at levels significantly lower than those accumulated in light. In darkness, sucrose cannot increase levels of AFM-induced Proto IX. However, addition of δ-aminolevulinic acid (ALA) increases Proto IX levels in AFM-treated plants in darkness, demonstrating that the herbicide blocks the porphyrin pathway in darkness as it does in the light. Thus, Proto IX accumulation in darkness appears to be limited by ALA availability. This is supported by the finding that dioxoheptanoic acid caused more ALA to accumulate in light than in darkness. Heme is a feedback inhibitor of ALA synthesis, and heme synthesis is inhibited by AFM. However, total extractable heme levels were reduced by AFM by about the same amount in both light and darkness. Exogenously supplied hemin reduced AFM-caused Proto IX accumulation and herbicidal damage in the light and also reduced Proto IX accumulation caused by AFM or AFM plus ALA in darkness. AFM-stimulated Proto IX accumulation was inversely proportional to the log of the photon flux density between 5 and 500 μmol in m⁻² s⁻¹. Reduced effects of higher photon fluxes on AFM-stimulated Proto IX accumulation are probably due to both increased photobleaching of Proto IX and reduced porphyrin synthesis because of herbicidal damage. AFM-stimulated Proto IX accumulation in darkness could not be demonstrated to be under phytochrome control, but it appeared to be under the negative influence of protochlorophyllide levels.     

Protoporphyrin IX Content Correlates with Activity of Photobleaching Herbicides

Several laboratories have demonstrated recently that photobleaching herbicides such as acifluorfen and oxadiazon cause accumulation of protoporphyrin IX (PPIX), a photodynamic pigment capable of herbicidal activity. We investigated, in acifluorfen-treated tissues, the in vivo stability of PPIX, the kinetics of accumulation, and the correlation between concentration of PPIX and herbicidal damage. During a 20 hour dark period, PPIX levels rose from barely detectable concentrations to 1 to 2 nanomoles per 50 cucumber (Cucumis sativus L.) cotyledon discs treated with 10 micromolar acifluorfen. When placed in 500 micromoles per square meter per second PAR, PPIX levels decayed logarithmically, with an initial half-life of about 2.5 hours. PPIX levels at each time after exposure to light correlated positively with the cellular damage that occurred during the following 1 hour in both green and yellow (tentoxin-treated) cucumber cotyledon tissues. PPIX levels in discs incubated for 20 hours in darkness correlated positively with the acifluorfen concentration in which they were incubated. In cucumber, the level of herbicidal damage caused by several p-nitrodiphenyl other herbicides, a p-chlorodiphenylether herbicide, and oxadiazon correlated positively with the amount of PPIX induced to accumulate by each of the herbicide treatments. Similar results were obtained with acifluorfen-treated pigweed and velvetleaf primary leaf tissues. In cucumber, PPIX levels increased within 15 and 30 minutes after exposure of discs to 10 micromolar acifluorfen in the dark and light, respectively. These data strengthen the view that PPIX is responsible for all or a major part of the photobleaching activity of acifluorfen and related herbicides.     

Investigation of the porphyrins accumulation in barley leaves incubated with Mn cations and δ-aminolevulinic acid

In greening etiolated primary leaves of barley (Hordeum vulgare L.), Mn²⁺ ions have been shown to inhibit chlorophyll (Chl) accumulation in a dose dependent manner and to lead to an accumulation of protoporphyrin IX (Proto) and Mg-protoporphyrin IX monomethyl ester (MgPE). The amount of MgPE that accumulated, was 2 times higher than Proto. In the dark, Proto and MgPE were observed to have accumulated to high levels in seven-day old green and etiolated leaves in the presence of 5 mmol/L Mn²⁺, but only if 5 mmol/L δ-aminolevulinic acid (ALA) was present. The 24 hours of irradiation of the green barley leaves treated in this way, resulted in a photodynamic destruction of Proto and MgPE as well as of Chl and carotenoids (Car). The observed porphyrin accumulation caused by the Mn²⁺ ions was reversed in the presence of active iron (Fe²⁺). This effect was observed when the iron concentration in incubation solutions was half the Mn²⁺ concentration, most effective for porphyrin synthesis, i.e. 5 mmol/L. The action of Mn²⁺ on porphyrin accumulation is also discussed.     

Either soluble or plastidic expression of recombinant protoporphyrinogen oxidase modulates tetrapyrrole biosynthesis and photosynthetic efficiency in transgenic rice

Protoporphyrinogen oxidase (Protox) is the last shared enzyme of the porphyrin pathway. As a continuation of our previous work in which the transgenic rice plants expressing the Bacillus subtilis Protox in the cytoplasm or the plastid showed resistance to diphenyl ether herbicide, this study was undertaken to identify the effects of tertapyrrole biosynthesis in these transgenic rice plants. The transgenic plants either targeted into plastids or expressed in cytoplasm showed higher Protox activity than wild-type plants did. Photosynthetic activity, measured as a quantum yield of photosystem II, was slightly higher in transgenic plants than in wild-type plants, but chlorophyll contents were not significantly different between transgenic and wild-type plants. As for porphyrin biosynthesis, both cytoplasm-expressed and plastid-targeted transgenic plants showed increased synthesis of aminolevulinic acid, Mg-Proto IX, and protoheme in comparison to wild-type plants whereas synthesis of protoporphyrin IX was similar for wild-type and transgenic plants. These results indicate that either cytoplasm or plastid expression of B. subtilis Protox in rice can upregulate the porphyrin pathway leading to increase in photosynthetic efficiency in plants.     

Influence of photodynamic processes induced by 2,2′-dipyridyl on the enzymatic system of chlorophyll biosynthesis

The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl) biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time, the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent 4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized photosynthetic pigments.     

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.     

Increased expression of Fe-chelatase leads to increased metabolic flux into heme and confers protection against photodynamically induced oxidative stress

Fe-chelatase (FeCh, EC 4.99.1.1) inserts Fe²⁺ into protoporphyrin IX (Proto IX) to form heme, which influences the flux through the tetrapyrrole biosynthetic pathway as well as fundamental cellular processes. In transgenic rice (Oryza sativa), the ectopic expression of Bradyrhizobium japonicum FeCh protein in cytosol results in a substantial increase of FeCh activity compared to wild-type (WT) rice and an increasing level of heme. Interestingly, the transgenic rice plants showed resistance to oxidative stress caused not only by the peroxidizing herbicide acifluorfen (AF) as indicated by a reduced formation of leaf necrosis, a lower conductivity, lower malondialdehyde and H₂O₂ contents as well as sustained F_v/F_m compared to WT plants, but also by norflurazon, paraquat, salt, and polyethylene glycol. Moreover, the transgenic plants responded to AF treatment with markedly increasing FeCh activity. The accompanying increases in heme content and heme oxygenase activity demonstrate that increased heme metabolism attenuates effects of oxidative stress caused by accumulating porphyrins. These findings suggest that increases in heme levels and porphyrin scavenging capacity support a detoxification mechanism serving against porphyrin-induced oxidative stress. This study also implicates heme as possibly being a positive signal in plant stress responses.     

Growth Cycle-Dependent Overproduction and Accumulation of Protoporphyrin IX in Tetrahymena: Effect of Heavy Metals1

Cells of the ciliate Tetrahymena pyriformis GL overproduce and accumulate massive quantities of the heme intermediate, protoporphyrin IX. Protoporphyrin is localized intracellularly in discrete membranous compartments. The amount of porphyrin stored in the cell changes dramatically as cells progress through the growth cycle. Porphyrin overproduction is stimulated by δ-aminolevulinic acid, but only during the mid-stationary phase. Overproduction of protoporphyrin IX apparently results from an increase, late in the growth cycle, of activities subsequent to δ-aminolevulinic acid synthetase. Feedback inhibition in the pathway by accumulated protoporphyrin IX does not occur. The presence of Co²⁺ completely inhibits accumulation of protoporphyrin IX in a manner reversed by δ-aminolevulinic acid. Sn⁴⁺ stimulates protoporphyrin IX accumulation in the culture.     

Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen

The use of herbicides to control undesirable vegetation has become a universal practice. For the broad application of herbicides the risk of damage to crop plants has to be limited. We introduced a gene into the genome of tobacco (Nicotiana tabacum) plants encoding the plastid-located protoporphyrinogen oxidase of Arabidopsis, the last enzyme of the common tetrapyrrole biosynthetic pathway, under the control of the cauliflower mosaic virus 35S promoter. The transformants were screened for low protoporphyrin IX accumulation upon treatment with the diphenyl ether-type herbicide acifluorfen. Leaf disc incubation and foliar spraying with acifluorfen indicated the lower susceptibility of the transformants against the herbicide. The resistance to acifluorfen is conferred by overexpression of the plastidic isoform of protoporphyrinogen oxidase. The in vitro activity of this enzyme extracted from plastids of selected transgenic lines was at least five times higher than the control activity. Herbicide treatment that is normally inhibitory to protoporphyrinogen IX oxidase did not significantly impair the catalytic reaction in transgenic plants and, therefore, did not cause photodynamic damage in leaves. Therefore, overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporphyrinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen.     

Tetrapyrrole Accumulation in in vitro Selected Somaclones of Acifluorfen-Tolerant Solanum ptycanthum Dun

Acifluorfen-tolerant callus lines of Solanum ptycanthum were isolated by stepwise selection. Growth of unselected lines was completely inhibited at 0.5 µM acifluorfen, while some selected lines grew at 8 µM acifluorfen. Twenty-two of 25 acifluorfen-tolerant callus lines regenerated shoots. Acifluorfen-tolerant S. ptycanthum callus lines differed in protoporphyrin IX content ranging from 2.0 to 43.5 nmole per 100 mg protein. As the concentration of acifluorfen increased, the amount of protoporphyrin IX accumulated increased. These results indicated that the possible site of action of acifluorfen was protoporphyrinogen oxidase which might be the molecular target of the herbicide within plant cell.     

Native state,energetic interaction of chlorophyll precursors and intraplastid location of S-adenosyl-L-methionine: Mg-protoporphyrin IX methyltransferase in etiolated leaves

Low temperature fluorescence spectra (FS) and fluorescence excitation spectra (FES) of protoporphyrin IX (Proto), Mg-protoporphyrin IX and its monomethyl ester (MgProto-ME) and protochlorophyllide (Pchlide) in etiolated barley leaves treated with 5-aminolevulinic acid and/or 2,2'-dipyridyl were studied. The spectra of Proto and MgProto-ME showed a little dependence on temperature of registration and exhibited similarity to low temperature spectra in diluted organic and buffer solutions. However, a red wavelength shift for Soret bands of Proto and MgProto-ME was observed due to porphyrin interaction with bovine serum albumin in 0.05 M, Na2HPO4 solution at room temperature. Disaggregating treatments had no effect on Proto and MgProto-ME spectra in plants. These results suggested that in etiolated leaves Proto and MgProto-ME molecules were in a monomer state. The spectral properties of these molecules were determined by interaction of porphyrins with proteins and other plastid membrane components. The spectral analyses indicated an efficient energy migration from Proto and MgProto-ME molecules to active form of Pchlide which emitted at 656nm, and no energy transfer from carotenoids to porphyrins in vivo. These findings suggested that Proto and MgProto-ME from carotenoids, and close location of these porphyrins and photoactive Pchlide in etioplast membranes. The latter conclusion was strongly supported by an observation that in etiolated leaves, S-adenosyl-L-methionin:Mg-protoporphyrin IX methyltransferase, which converts MgProto into MgProtoME, were located not only in prothylakoids but also in prolamellar bodies containing photoactive Pchlide.     

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     

Relation between intracellular location and photodynamic efficacy of 5-aminolevulinic acid-induced protoporphyrin IX in vitro. Comparison between human glioblastoma cells and other cancer cell lines.

A promising clinical application of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PP IX) is fluorescence detection and photodynamic treatment of residual tumour tissue during surgical resection of high grade malignant glioma. U373 MG human glioblastoma cells were used as a model system to study the relation between intracellular location and photodynamic efficacy of 5-ALA-induced PP IX in more detail. Therefore, ultra-sensitive fluorescence microscopy, using either optical excitation of whole cells or selective excitation of the plasma membrane by an evanescent electromagnetic field, was combined with quantitative measurements of intracellular porphyrin amount and phototoxicity. Glioblastoma cells accumulated PP IX to a moderate extent as compared to T47D breast cancer cells (high accumulation) or OV2774 ovarian cancer cells (low accumulation). Although photodynamic inactivation of the different cell lines (decreasing in the order T47D > U373 MG > OV2774) seemed to be directly related to PP IX accumulation, examination of the data in more detail revealed that photodynamic efficacy per photosensitizer molecule (PE) was about two times higher in glioblastoma and ovarian cancer cells as compared to breast cancer cells. The different photodynamic efficacy of PP IX was related to the different intracellular location. In contrast to breast cancer cells where PP IX fluorescence was localized in small granules, PP IX fluorescence in glioblastoma cells and ovarian cancer cells originated mainly from cellular membranes. Thus, the intracellular location of PP IX in a predominantly lipophilic environment, characterized by a comparably high photostability (probed by photobleaching and photoproduct formation) and a lower degree of porphyrin aggregation (probed previously by fluorescence decay kinetics), seems to be the key factor for high photodynamic efficacy of 5-ALA-induced PP IX. In the case of OV2774 ovarian cancer cells, however, a low PP IX accumulation limited cell inactivation upon irradiation, whereas the results obtained for glioblastoma cells are encouraging to develop PDT to an additional therapeutic option for the treatment of tumour margins in patients who underwent fluorescence-guided resection of high grade malignant glioma after 5-ALA administration.     

Effects of the photobleaching herbicide, acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles, soybean root mitochondria, soybean root nodules, and bacteria

The photobleaching herbicide, acifluorfen-methyl (AFM), has been reported to be an inhibitor of the heme and chlorophyll biosynthetic enzyme protoporphyrinogen oxidase (Protox) in several plant species. However, AFM had no effect on the levels of Protox activity measured in a mitochondrial fraction from soybean roots. In contrast, AFM inhibited Protox activity in etioplasts from barley leaves and in mitochondria from barley roots, but the extent of inhibition varied depending upon the assay conditions and was maximal only in the presence of 5 mM dithiothreitol (DTT). AFM inhibition was enhanced by preincubation of barley organelle extract in the presence of DTT. Preincubation of barley extract with DTT and AFM together (but not with AFM alone) caused extensive enzyme inhibition which was not reversible by dialysis. These findings have implications for the mechanism of AFM action and for the differential effect of these herbicides on crop and weed species. AFM had no effect on the Protox activity of membranes from free-living bacterial cell of Bradyrhizobium japonicum or Escherichia coli, or on the high levels of Protox activity associated with the plant-derived membrane surrounding the symbiotic bacteria within the soybean root nodule.     

Photodynamics of Porphyric Insecticides

The discovery of porphyric insecticides was a direct fallout of the discovery and development of photodynamic herbicides. Tetrapyrrole-dependent photodynamic herbicides are compounds that force green plants to accumulate undesirable amounts of metabolic intermediates of the chlorophyll and heme metabolic pathways, namely, tetrapyrroles. In light, the accumulated tetrapyrroles photosensitize the formation of singlet oxygen that kills treated plants by oxidation of their cellular membranes. Demonstration of the potential for tetrapyrrole accumulation in insects was achieved by spraying T. ni larvae with δ-aminolevulinic acid (ALA) and 2,2-dipyridyl (Dpy). Treated larvae were placed overnight in darkness at 28°C in order to allow for tetrapyrrole accumulation. Extraction of treated, dark-incubated larvae with ammoniacal acetone, followed by spectrofluorometric examination of the larval extract, revealed the accumulation of massive amounts of protoporphyrin IX (Proto). A high degree of correlation was observed between Proto accumulation in darkness and larval death in the light. A few hours after exposure to light, the larvae became sluggish and flaccid due to loss of body fluids. Death was accompanied by extensive desiccation. Because control of insects by ingestion is as viable an option as control by spraying, and offers certain advantages under household conditions, studies were conducted to determine whether combinations of ALA and porphyric insecticide modulators would be effective if ingested with the food. The effect of ALA and 1,10-phenanthroline (Oph) were determined by incorporating them into the diet of T. ni larvae. After exposure to light, following 17 h of dark incubation, larvae underwent violent convulsions and vomiting and died within 20 to 40 s. Tetrapyrrole analysis of the treated larvae immediately after dark incubation revealed significant amounts of Proto and Zn-Proto accumulation. Correlation between tetrapyrrole accumulation and larval death was significant. Similar results were obtained when ALA and Dpy were administered to the larvae with the diet. The above results indicated that in addition to contact via spraying, porphyric insecticides had the potential to be very potent when ingested. For a more thorough understanding of the mode of action of porphyric insecticides, the phenomenology of tissue, cellular, and subcellular sites of tetrapyrrole accumulation in representative insect species was investigated. In T. ni larvae, on a unit protein basis, about 59% of the accumulated Proto was observed in the hemolymph, 35% in the gut, and 6% in the integument. Further understanding of the response of insect organs and tissues to porphyric insecticide treatment was obtained by investigating the response of isolated organs and tissues to incubation with ALA + Dpy or ALA + Oph in adult Blattella germanica (German cockroach), adult Anthonomus grandis (cotton boll weevil), fifth instar larvae of Heliothus zea (corn earworm), and fifth instar larvae of T. ni (cabbage looper). In T. ni, and H. zea, significant Proto accumulation was observed in incubated midgut and fat bodies. Proto accumulation occurred when tissues were incubated with Dpy, ALA + Dpy, Oph, and ALA + Oph (2). No response to treatment with ALA alone was observed. In cockroaches, more of the Proto appeared to accumulate in the male and female guts than in their abdomen. As in T. ni and H. zea, the response was elicited by each of the treatments that included Dpy or Oph. Cotton boll weevil abdomens appeared to be less responsive than the abdomens of the other three species. To determine whether Proto accumulation resulted in photodynamic damage of incubated tissues, T. ni midguts were incubated in darkness either in buffer, with ALA, or with Oph + ALA. Oxygen consumption of the tissue was monitored before and after exposure to 2-h of illumination. A 30% decrease in O₂ consumption was observed in midguts treated with Oph or with ALA + Oph after 2 h in the light. The decrease in oxygen consumption observed in isolated T. ni midguts was shown to be caused by photodynamic damage to mitochondrial enzymes. Finally, structure-function photodynamic insecticidal studies led to the identification of 36 compounds belonging to 10 different chemical families that were effective (>70% mortality) against at least one insect species. Of the 36 modulators, 10 exhibited potent activity toward cockroaches.     

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.     

The plastidic Arabidopsis protoporphyrinogen IX oxidase gene, with or without the transit sequence, confers resistance to the diphenyl ether herbicide in rice

Agrobacterium‐mediated gene transformation was used to introduce plastidic protoporphyrinogen IX oxidase (Protox) genes from Arabidopsis, with and without the transit sequence, into the rice genome. They were placed under the control of the constitutive and ubiquitous maize ubiquitin promoter, and their abilities to confer resistance to the diphenyl ether‐type herbicide, oxyfluorfen were compared. The integration and expression of the transgene in the T₁ generation was examined by Southern, northern and western blot analyses. Surprisingly, as judged by an in vivo seed germination assay and an in vitro cellular leakage assay, both lines were similarly resistant to oxyfluorfen. The tolerance to cellular damage (lipid peroxidation and electrolyte leakage) was higher in transgenic plants than in wild‐type plants. In transgenic plants, the degree of herbicide resistance varied directly with the absolute amount of Protox protein expression. Both the intact protein and the protein with the transit sequence deleted were accumulated in plastids.