The light-dependent generation of active oxygen species is termed photooxidative stress. This can occur in two ways: (1) the donation of energy or electrons directly to oxygen as a result of photosynthetic activity; (2) exposure of tissues to ultraviolet irradiation. The light-dependent destruction of catalase compounds the problem. Although generally detrimental to metabolism, superoxide and hydrogen peroxide may serve useful functions if rigorously controlled and compartmentalised. During photosynthesis the formation of active oxygen species is minimised by a number of complex and refined regulatory mechanisms. When produced, active oxygen species are eliminated rapidly by efficient antioxidative systems. The chloroplast is able to use the production and destruction of hydrogen peroxide to regulate the thermal dissipation of excess excitation energy. This is an intrinsic feature of the regulation of photosynthetic electron transport. Photoinhibition and photooxidation only usually occur when plants are exposed to stress. Active oxygen species are part of the alarm-signalling processes in plants. These serve to modify metabolism and gene expression so that the plant can respond to adverse environmental conditions, invading organisms and ultraviolet irradiation. The capacity of the antioxidative defense system is often increased at such times but if the response is not sufficient, radical production will exceed scavenging and ultimately lead to the disruption of metabolism. Oxidative damage arises in high light principally when the latter is in synergy with additional stress factors such as chilling temperatures or pollution. Environmental stress can modify the photooxidative processes in various ways ranging from direct involvement in light-induced free radical formation to the inhibition of metabolism that renders previously optimal light levels excessive. It is in just such situations that the capacity for the production of active oxygen species can exceed that for scavenging by the antioxidative defense systems. The advent of plant transformation, however, may have placed within our grasp the possibility of engineering greater stress tolerance in plants by enhancement of the antioxidative defence system.

Photooxidative stress in plants

Magnesium (Mg) deficiency exerts a major influence on the partitioning of dry matter and carbohydrates between shoots and roots. One of the very early reactions of plants to Mg deficiency stress is the marked increase in the shoot-to-root dry weight ratio, which is associated with a massive accumulation of carbohydrates in source leaves, especially of sucrose and starch. These higher concentrations of carbohydrates in Mg-deficient leaves together with the accompanying increase in shoot-to-root dry weight ratio are indicative of a severe impairment in phloem export of photoassimilates from source leaves. Studies with common bean and sugar beet plants have shown that Mg plays a fundamental role in phloem loading of sucrose. At a very early stage of Mg deficiency, phloem export of sucrose is severely impaired, an effect that occurs before any noticeable changes in shoot growth, Chl concentration or photosynthetic activity. These findings suggest that accumulation of carbohydrates in Mg-deficient leaves is caused directly by Mg deficiency stress and not as a consequence of reduced sink activity. The role of Mg in the phloem-loading process seems to be specific; resupplying Mg for 12 or 24 h to Mg-deficient plants resulted in a very rapid recovery of sucrose export. It appears that the massive accumulation of carbohydrates and related impairment in photosynthetic CO2 fixation in Mg-deficient leaves cause an over-reduction in the photosynthetic electron transport chain that potentiates the generation of highly reactive O2 species (ROS). Plants respond to Mg deficiency stress by marked increases in antioxidative capacity of leaves, especially under high light intensity, suggesting that ROS generation is stimulated by Mg deficiency in chloroplasts. Accordingly, it has been found that Mg-deficient plants are very susceptible to high light intensity. Exposure of Mg-deficient plants to high light intensity rapidly induced leaf chlorosis and necrosis, an outcome that was effectively delayed by partial shading of the leaf blade, although the Mg concentrations in different parts of the leaf blade were unaffected by shading. The results indicate that photooxidative damage contributes to development of leaf chlorosis under Mg deficiency, suggesting that plants under high-light conditions have a higher physiological requirement for Mg. Maintenance of a high Mg nutritional status of plants is, thus, essential in the avoidance of ROS generation, which occurs at the expense of inhibited phloem export of sugars and impairment of CO2 fixation, particularly under high-light conditions.

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Role of magnesium in carbon partitioning and alleviating photooxidative damage

Activities of a surprisingly large number of herbicide families are directly or indirectly influenced by light. Herbicide mechanisms that are dependent on light or are enhanced by light fall into several different categories. These herbicides inhibit electron flow in photosystem II in the photosynthetic light reaction (e.g., triazines, phenyl ureas, and uracils), capture electrons in photosystem I in the photosynthetic light reaction (e.g., bipyridiniums such as paraquat), inhibit glutamine synthetase in the nitrogen assimilation pathway (e.g., glufosinate), inhibit protoporphyrinogen oxidase during chlorophyll biosynthesis (e.g., diphenyl ethers [DPEs] and N-phenyl heterocycles), directly inhibit carotenoid biosynthesis by inhibiting one of the desaturase enzymes (e.g., norflurazon and fluridone), or indirectly inhibit carotenoid biosynthesis by inhibiting a quinone cofactor involved in the desaturase reaction (e.g., triketones and isoxazoles). Surprisingly, in all instances the involvement of light in herbicide action is not a direct interaction of light and herbicide at the primary site of action. In addition, the events that cause tissue damage (necrosis) are not associated with the primary target and are always due to membrane damage caused by lipid peroxidation of polyunsaturated fatty acids.

Light-dependent herbicides: an overview.

Publisher Summary
Protoporphyrinogen oxidase-inhibiting herbicides, also referred to as Protox- or PPO-inhibiting herbicides, were commercialized in the 1960s. Nitrofen was the first Protox-inhibiting herbicide to be introduced for commercial use in 1964. This diphenyl ether (DPE) herbicide was eventually recognized as a relatively weak inhibitor of Protox, but it was a lead compound of an entire class of structurally related herbicides that were much more active. Several DPE herbicides have been successfully commercialized. Numerous nonoxygen-bridged compounds with this same site of action have also been discovered. Before glyphosate-resistant crops, there were several reasons for the high level of interest among agrochemical companies in the discovery and development of new Protox inhibitors. Protox is a particularly good target that can be inhibited by structurally diverse classes of herbicides, allowing for the development of a new chemistry not yet patented by other companies. These compounds are effective at very low application rates and have generally good ecotoxicology and human toxicology profiles at recommended application rates. Many are highly compatible with the trend toward no-tillage agriculture. Furthermore, unlike with some of the other herbicide classes, weeds do not readily evolve resistance at this particular site of action, although the discovery of a Protox inhibitor-resistant Amaranthus rudis biotype suggests that resistance can and will occur if the selection pressure is sufficient. The relatively low dose rates required for herbicidal activity may be far below the dose needed to adversely affect porphyrin metabolism in animals in field situations or in humans as a result of exposures in food, water, air, or during application.

Protoporphyrinogen Oxidase-Inhibiting Herbicides

The present invention provides novel eukaryotic DNA sequences coding for native protoporphyrinogen oxidase (protox) or modified forms of the enzyme which are herbicide tolerant Plants having altered protox activity which confers tolerance to herbicides are also provided These plants may be bred or engineered for resistance to protox inhibitors via mutation of the native protox gene to a resistant form or through increased levels of expression of the native protox gene, or they may be transformed with modified eukaryotic or prokaryotic protox coding sequences or wild type prokaryotic protox sequences which are herbicide tolerant Diagnostic and other uses for the novel eukaryotic protox sequence are also described Plant genes encoding wild-type and altered protox, purified plant protox, methods of isolating protox from plants, and methods of using protox-encoding genes are also disclosed

Manipulation of protoporphyrinogen oxidase enzyme activity in eukaryotic organisms

The effect of acifluorfen-methyl on tetrapyrrole synthesis in greening chloroplasts of Cucumis sativus was examined. Formation of Mg-proto-porphyrin IX from delta-aminolevulinate was reduced 98% by 10 micromolar acifluorfen-methyl. Conversion of protoporphyrin IX to Mg-protoporphyrin IX was unaffected, but protoporphyrin IX synthesis from delta-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. ((c) 1989 FMC Corporation. All rights reserved.).

Inhibition of Plant Protoporphyrinogen Oxidase by the Herbicide Acifluorfen-Methyl

Treatment with acifluorfen-methyl (AFM), methyl 5-(2-chloro-4-[tri-fluoromethyl] phenoxy)-2-nitrobenzoate, inhibited protochlorophyllide synthesis in dark-held, δ-amino levulinic acid-fed, excised cotyledons of cucumber (Cucumis sativus L.). Protochlorophyllide and protoporphyrin IX levels in AFM-treated cotyledons were inversely related and dependent on AFM concentration; as the herbicide dose increased, protoporphyrin IX levels also increased with a concomitant loss of protochlorophyllide. Significant protoporphyrin IX accumulation was induced by concentrations of AFM from the linear region of the membrane disruption dose response curve. The pattern of precursor accumulation seen in HPLC chromatograms from extracts of AFM-treated tissue indicate that Mg insertion into the tetrapyrrole ring is inhibited, suggesting interference with Mg-chelatase. An inhibitor of δ-amino levulinic acid synthesis, gabaculine (3-amino-2,3-dihydrobenzoic acid), completely blocked the membrane disruption activity of AFM in illuminated cotyledons. Protoporphyrin IX accumulating in AFM-treated tissues may serve as the primary photosensitizer for initiating lipid peroxidation.

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Accumulation of photodynamic tetrapyrroles induced by acifluorfen-methyl.

Disclosed is a method and a pharmaceutical composition for killing mammalian tumor cells by subjecting said cells to light in the presence of a light-activatable tetrapyrrole, in which the improvement comprises treating said cells with a compound which inhibits the enzymatic conversion of protoporphyrinogen to protoporphyrin IX by protoporphyrinogen oxidase in said cells thereby causing a buildup of protoporphyrin IX in said cells. Also disclosed is a method for the production of protoporphyrin IX which comprises growing eukaryotic microalgae in the presence of a protoporphyrinogen oxidase inhibitor.

Method and composition for photodynamic treatment and detection of tumors

Treatment of polycythemia vera or hyperbilirubinaemia by administering a compound which is a specific inhibitor of the enzymatic conversion of protoporphyrinogen to heme in mammalian cells.

Treatment of disease

A random source PSI electron-accepting herbicide lead, ethyl 1,2,4-benzotriazine-3-acetate 1, was optimized using molecular probing, bioisosteric replacement, Free-Wilson analysis, Sequential Simplex Optimization, and ultimately linear regression analysis. Some variables in the initial regression equations were found to be cross correlated, representing the same factors as revealed by factor analysis. The technique of prefiltering potential variables for linear regression analysis utilizing factor analysis is exemplified. For the set of 1 analogs, eleven independent factors were identified which were then submitted to linear regression analysis. The resulting equations are statistically more secure, all terms being independent. These equations are discussed in the context of known SAR for herbicidal PSI electron acceptors, especially the bipyridiniums.

Debra A. Witkowski

Papers

Inhibition of Plant Protoporphyrinogen Oxidase by the Herbicide Acifluorfen-Methyl

Accumulation of photodynamic tetrapyrroles induced by acifluorfen-methyl.

Method and composition for photodynamic treatment and detection of tumors

Treatment of disease

Discovery and Optimization of a PSI Electron‐Accepting 1,2,4‐Benzotriazine Herbicide