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Counteraction of Paraquat Toxicity at the Chloroplast Level*

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Counteraction of Paraquat Toxicity at the Chloroplast Level* Counteraction of Paraquat Toxicity at the Chloroplast Level* Brad L. Upham and Kriton K. Hatzios Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 24061, USA Z. Naturforsch. 42c, 824—828 (1987); received October 29, 1986 Paraquat, Hill Reaction, Parallel Electron Transport, NADP Reduction, Paraquat Reduction Six pyridyl derivatives [benzylviologen, 2-anilinopyridine, l,2-bis(4-pyridyl)ethane, l,2-bis(4- pyridyl)ethylene, 2-benzoylpyridine, and 2-benzylaminopyridine] and five heme-iron derivatives [hemoglobin, hemin, hematin, ferritin, and ferrocene] were screened for their potential to coun­ teract paraquat (l,l'-dimethyl-4,4'-bipyridinium ion) toxicity on pea ( Pisum sativum L.) isolated chloroplasts. The H:0 —» methylviologen(MV)/02 and H20 —> ferredoxin(Fd)/NADP+ were two Hill reactions assayed with these compounds. Antagonists of paraquat toxicity should inhibit the first Hill reaction but not the latter. All pyridyl derivatives examined did not inhibit the reaction H20 —» M V /02. Ferritin and ferrocene were also ineffective as inhibitors of this reaction. Hemo­ globin inhibited the reaction H20 —> MV/Oz without inhibiting the reaction H20 —> Fd/NADP+, providing protection to pea chloroplasts against paraquat. Hemin and hematin inhibited both Hill reactions examined. They also inhibited H20 —* diaminodurene(DAD)ox and durohydro- quinone —> M V /02 Hill reactions but not the dichlorophenol indophenolred —> M V /02 and D A D red —> M V /02 Hill reactions. These results suggest that hemin and hematin are inhibiting the photo­ synthetic electron transport in the plastoquinone-pool region. Introduction cide in tolerant plants; d) overproduction of a target Chemical and genetic manipulation of crop toler­ enzyme in tolerant plants; and e) oversynthesis of ance to herbicides has challenged herbicide tech­ substrates able to reverse the herbicide-induced in­ nologists for many years [1—3]. Recent advances in hibition of growth in tolerant plants. Specific exam­ agricultural biotechnology offer new options and ples for the involvement of these mechanisms in the alternative approaches to meet this challenge. Plant development of plant tolerance to herbicides have tolerance to nonselective herbicides can be a result of been reported [1,3]. Many of these mechanisms may one or more of the following mechanisms [1, 4]: be manipulated by genetic or chemical means. a) altered uptake and translocation or compartmen- Paraquat is a nonselective herbicide used exten­ tation of the herbicide in tolerant plants; b) extensive sively for total weed control in no-till crop produc­ metabolic detoxification of the herbicide in tolerant tion and as a harvest aid [5]. The active ingredient of plants; c) modification of the target site of the herbi- paraquat is methylviologen (MV) which is routinely used in studying selected photochemical reactions mediated by plant chloroplasts. Paraquat exerts its Abbreviations: DCMU, 3,4-dichlorophenyl-N,N'-di- phytotoxicity by accepting photosynthetic electrons methylurea; KFeCN, potassium ferricyanide; DCIP, di­ from PSl and transferring these electrons to oxygen chlorophenol indolphenol; DAD. 2,3,5,6-tetramethyl-/?- phenylenediamine (diaminodurene); DHQ, tetramethyl-/?- producing toxic oxygen species which cause lipid hydroquinone (durohydroquinone); MES, [2-(N-morpho- peroxidation and membrane breakdown [6]. During lino)-ethanesulfonic acid]; HEPES, [N-2-hydroxyethyl this process, paraquat is reduced to a cationic radical piperazine-N'-2-ethanesulfonic acid]; DBMIB, dibro- which is quickly reoxidized by molecular oxygen. mothymoquinone; PET, photosynthetic electron transport; PS I and PS II, photosystem I and II; Fd, ferredoxin; Chi, Superoxide radical (0 2_) is the product of this reac­ chlorophyll; Asc, ascorbate; SOD, superoxide dismutase; tion [7]. Disproportionation of superoxide radicals to MV, methylviologen (l,l'-dimethyl-4,4'-bipyridinium H20 2 is catalyzed enzymatically by superoxide dis­ dichloride). mutase (SOD) (EC 1.15.1.1), which is located in * Contribution number 572 from the Department of Plant plant chloroplasts [8]. Paraquat-mediated peroxide Pathology, Physiology and Weed Science, Virginia Poly­ technic Institute and State University, Blacksburg, Vir­ production is cytochemically located along the ginia, 24061. stroma lamellae and on the ends of the grana stack Reprint requests to Dr. K. K. Hatzios. [9]. Hydrogen peroxide readily accepts electrons Verlag der Zeitschrift für Naturforschung. D-7400 Tübingen from reduced transition metals [10—12] or from re­ 0341-0382/87/0600-0824 $ 01.30/0 duced paraquat [13] to form hydroxal radicals Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. B. L. Upham and K. K. Hatzios • Counteraction of Paraquat Toxicity 825 (O H ). Lipid peroxidation is most likely initiated by and oats from paraquat injury but the antidote to OH" [10, 14, 15]. The superoxide radical is neither a herbicide ratio (100:1) was very uneconomical [30]. strong oxidant nor reductant [16] and is probably not An alternative approach for the chemical manip­ directly involved in lipid peroxidation [17]. How­ ulation of plant tolerance to paraquat is the coun­ ever, superoxide is an efficient reductant of transi­ teraction of its activity at the chloroplast level. Until tion metals [11, 14, 18] and can play an indirect role recently, this approach was not apparently feasible in oxygen toxicity by maintaining transition metals in since it is traditionally accepted that paraquat is re­ a reduced state [10, 12, 19]. The plant is normally duced by the primary electron acceptor of PSI which protected from the harmful effects of 0 2~, H20 2 and is also responsible for the reduction of NADP+ [6]. OH' through the action of SOD, ascorbate peroxi­ Current work on the primary electron acceptor of dase (EC 1.11.1.7), catalase (EC 1.11.1.6), and a- PSI, however, shows that a series of electron accep­ tocopherol, respectively [8, 20]. Treatm ent of plants tors are involved before the reduction of ferredoxin with paraquat enhances the production of toxic oxy­ (Fd) [31]. The primary electron acceptor from P700 gen species to levels far exceeding those that can be may be a monomeric chlorophyll anion designated protected by the natural defense systems of plants. Ao~. Electron transfer probably proceeds to a semi- Interest in manipulating crop tolerance to para­ quinone (A[_) followed by three iron-sulfur (Fe-S) quat has been renewed recently, following the dis­ centers denoted as Fx, Fa, and Fb. It is still un­ covery of several weed biotypes that are tolerant to resolved whether these Fe-S centers act in series or in this herbicide. Biotypes of horseweed ( Conyza parallel. The experimental evidence tends to favor linefolia L. Cronq.) survive treatments with paraquat the concept that electron transfer to the Fe-S centers, by excluding the herbicide from its site of action. particularly Fa and Fb, is in parallel under physio­ Autoradiography and cytochemical, and biochemical logical conditions [31]. It is not known which one of studies showed that translocation of paraquat in tol­ these electron acceptors (Fx, Fa, and Fb) interacts erant Conyza biotypes was limited to the major leaf with Fd and paraquat in the reduction of 0 2. An veins and insufficient amounts of the herbicide unidentified inhibitor from hemolyzed rabbit sera is reached the mesophyll cells containing the target site capable of inhibiting paraquat- or Fd-mediated 0 2 (chloroplast) of this herbicide [21, 22]. The tolerance uptake without inhibiting NADP+-dependent 0 2 of perennial ryegrass ( Lolium perenne L.) biotypes evolution [32]. The physiological implication of this to paraquat, however, is not due to differential finding is that electron flow after PSI is branched uptake, translocation, or metabolism of paraquat (parallel). One branch is involved in NADP+ reduc­ [23]. It is the result of increased activity of the endo­ tion whereas another branch is involved in the para­ genous protectants of toxic oxygen species such as quat- or Fd-mediated 0 2 reduction. The branch for the enzymes catalase, SOD, and peroxidase [24], To­ reduction of 0 2 offers a potential target site for reg­ bacco ( Nicotiana tabacum L.) cell lines, tolerant to ulating paraquat toxicity since electron flow to para­ the herbicide paraquat, have been selected through quat could be inhibited without interfering with tissue culture procedures [25]. Increased levels of NADP’-reduction. peroxidase and catalase activities in these cell lines In this study, we report the results of our attempts were identified as the causes for the tolerance of to counteract paraquat toxicity at the chloroplast these lines to paraquat [26, 27]. level with several chemical compounds that could Attempts to chemically regulate plant tolerance to potentially inhibit electron flow to paraquat but not paraquat have also been reported. D-penicilamine, a to NADP+. Two major groups of compounds, non- copper chelate with superoxide dismutating activity, herbicidal pyridyl analogues
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