Chapter 5
The Porphyrin Pathway as a Herbicide Target Site 1
2
Ujjana B. Nandihalli and Stephen O. Duke 1
Center for Alluvial Plains Studies, Delta State University, Cleveland, MS 38733 Southern Weed Science Laboratory, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 350, Stoneville, MS 38776 2
The porphyrin pathway is one of the most important metabolic pathways in plants. Nevertheless, this pathway has been virtually overlooked as a herbicide target site in biorational design strategies. For over a decade, several pyridine and phenanthroline analogues were known to interfere with the porphyrin pathway by inducing porphyrin accumulation in treated plants. The potential of δ-aminolevulinic acid (ALA), a porphyrin precursor, as an environmentally safe herbicide has been studied extensively. Plants treated with ALA either alone or in combination with the chemical modulators accumulate large amounts of different types of porphyrin compounds. Excessive amounts of porphyrins injure plants by type II photoperoxidation. Recently, numerous commercial herbicides such as diphenyl ethers have been found to cause accumulation of protoporphyrin IX by inhibiting protoporphyrinogen oxidase, the last enzyme common to both heme and chlorophyll synthesis. In this article, we review the modes of action of herbicides that interfere with the porphyrin pathway and analyze the potential of the porphyrin pathway for biorational herbicide design. In the past, the reduction of chlorophyll (Chi) content in herbicide-treated plants has been used as a quantitative measurement of herbicide phytotoxicity. The reduced Chi levels were thought mostly to be due to "secondary effects." The porphyrin pathway as a direct herbicide target site has been of strong interest only during the last 8 years due to two major findings. First, 5-aminolevulinic acid (ALA) was found to act as potent herbicide in combination with synthetic modulators of the porphyrin pathway (7). Second, several major classes of herbicides were found to act through inhibition of protoporphyrinogen oxidase (Protox), resulting in the accumulation of large amounts of photodynamic protoporphyrin I X (Proto IX) (2-4). The widespread use of herbicides for controlling weeds has raised serious environmental concerns. The porphyrin pathway is much more active in plants than in animals because of the requirement for continued Chi synthesis. Furthermore, those parts of the pathway exclusively committed to Chi synthesis are not found on animals. A L A is a natural compound 0097-6156/93/0524-0062S06.00/0 © 1993 American Chemical Society
5. NANDIHALLI & DUKE
The Porphyrin Pathway as a Herbicide Target Site
present in both paints and animals. A L A and its products (porphyrins) degrade rapidly in the environment, thus causing no deleterious effects on ecosystems. Thus, the porphyrin pathway is a potential site for biorational design of toxicologically benign herbicides. In this review, we discuss ALA-based as well as Protox-inhibiting synthetic herbicides and the potential of other enzymes in the porphyrin pathway as herbicide target sites. The Porphyrin Pathway The enzyme properties and catalytic reactions of the porphyrin pathway have been reviewed recently (5) (Figure 1). A L A is the first committed precursor of the porphyrin pathway which provides all the carbon and nitrogen required for porphyrin synthesis. The synthesis of A L A is also considered to be the rate-limiting step in the biosynthesis of Chi. Porphobilinogen (PBG) is formed by the condensation of two A L A molecules by A L A dehydratase. Uroporphyrinogen DDL, coproporphyrinogen ID, and protoporphyrinogen I X (Protogen) are "porphyrinogens" which are non-fluorescent and non-photosensitizing compounds. Their macrocycles are non-aromatic and non-planar. The compounds from Proto IX to monovinyl-protochlorophyllide (MV Pchlide) are called porphyrins. These are fluorescent and photosensitizing structures, containing aromatic and planar macrocycles. The formation of chlorophyllide (Chlide) from either M V or divinyl (DV) Pchlide requires light; therefore, the porphyrin pathway halts at the Pchlide level in darkness. The biochemical aspects of the enzymes of the porphyrin pathway are poorly understood. Most of the steps in the porphyrin pathway are catalyzed by multiple enzyme systems.
p
ALA B G deaminase Uro m dehydratase Uro HI cosynthase decarboxylase A L A —•Porphobilinogen—•Uroporphyrinogen HI—•Coproporphyrinogen HI
ICoproin Mg-Chelatase JMU^^og^ J Mg-Protoporhyrin E X ^ Protoporphyrin I X ^ Protoporphyrinogen I X Fe-Chelatase S AM-Mg-Proto I X Me transferase
Heme
Cyclase system 4-Vinyl reductase Mg-Protoporphyrin IX—•DV-Protochlorophyllide—•MV-Protochlorophyllide monomethylester \ / \ / ^ N k /NADPH:Pchlide , „ \r / oxidoreductase Chlorophyllases \ / Chlorophylls ^ Chlorophyllides n
M
Figure 1. The porphyrin biosynthetic pathway in higher plants (5). Porphyric Herbicides To be classified as a porphyric herbicide (PH), the chemical must be able to cause the accumulation of large amounts of one or more porphyrin compounds in treated
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PEST CONTROL WITH ENHANCED ENVIRONMENTAL SAFETY
plants either in darkness or in the light. The porphyrins are photosensitizers and excessive levels of porphyrins in plant cells lead to the production of reactive singlet oxygen ( 0 ) under light. 0 reacts with the membrane lipids by a type II photodynamic process, resulting in membrane lipid peroxidation, membrane breakdown and ultimately, tissue death. Several reviews have appeared recently on both the natural ALA-based P H (6-8) and synthetic PHs that exclusively inhibit plant Protox (9-12). 1
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A L A as a Natural Porphyric Herbicide In untreated plants kept in darkness, only a small amount of Pchlide accumulates due to 'he limited availability of A L A in darkness. When these plants are irradiated, Pchlide is rapidly and almost fully photoconverted to Chlide which is then utilized in the synthesis of Chi, causing no photodynamic injury to the plant. Long before the introduction of the P H concept by Rebeiz et al. (7), it was known that plant leaves incubated with exogenous A L A accumulated large amounts of Pchlide in the dark (73). These leaves were bleached when irradiated with white light. Furthermore, Proto DC and Mg-Proto DC Me were accumulated by barley leaves that were simultaneously treated with a,a'-dipyridyl (DPY) and A L A . Duggan and Gassman (14) found that several iron-complexing compounds induce porphyrin buildup in treated plant tissue. These compounds were collectively termed "modulators" by Rebeiz et al. (7). Porphyrin Photodegradation and Herbicidal Effects. The porphyrins that accumulate in the dark in response to A L A or ALA+modulator treatments are not rapidly utilized in the formation of Chi when plants are exposed to the light. It is believed that a large proportion of the loss of accumulated porphyrins during exposure to light in these treatments is due to photodegradation rather than movement to Chi. In ALA+DPY-treated cucumber plants kept in darkness for 17 h, both Pchlide and Mg-Proto DC equivalents (MPE) were degraded differently when irradiated (7). Sixty-five percent of the dark-accumulated Pchlide was lost within 30 min of irradiation and it took 4 h to reach the levels found in untreated plants. The dark-accumulated M P E level decreased rapidly within 30 min of irradiation, followed by an increase for up to 2 h and then decreased to undetectable levels after 4 h. Recently, Chakraborty and Tripathy (75) demonstrated that the chloroplasts isolated from plants treated with A L A and kept in darkness for 14 h lost 40% of dark-accumulated Pchlide within 15 min of irradiation and further exposure for up to 1 h did not affect the Pchlide concentration. These high levels of dark-accumulated non-phototransferable porphyrins in plants are responsible for the peroxidation of the membrane lipids. Herbicidal injury occurs rapidly, usually being detectable within the first 20 min of irradiation of dark-incubated plants. Initially, isolated water soaked spots are seen on tissue, followed by loss of leaf turgidity and desiccation. Herbicidal Synergism Between A L A and Modulators. In the A L A alone treatment, Pchlide is the only porphyrin that accumulates, but in A L A + modulator treatments several other types of porphyrins are accumulated, depending upon the target site of the modulator. The total porphyrin content present in plant tissue before exposure to light and the eventual tissue damage in the light are correlated (Table I). Also, the modulators typically interact with A L A in a synergistic fashion with respect to both porphyrin accumulation and herbicidal damage.
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The Porphyrin Pathway as a Herbicide Target Site
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Table I. Synergistic interaction of A L A (5 mM) and picolinic acid (20 mM) on porphyrin production and herbicidal damage in cucumber. Five-day-old seedlings were sprayed with the chemicals and incubated in darkness for 20 h before determination of porphyrins and exposure to light. Herbicidal injury was evaluated 24 h after exposure to the light (Nandihalli and Rebeiz, unpublished data) Treatment
Control ALA Picolinic acid A L A + Picolinic acid
Mg-Proto IX Me
Pchlide
Total porphyrins
—(nmoles/100 mg protein)— 54 0.7 53 383 2.6 380 84 8.0 76 586 78.5 507
Herbicide damage (%) 0 39 0 95
Classification of Modulators. Based on the types of porphyrins that accumulate in response to modulator treatment, modulators have been classified into four groups (6): (a) inducers of porphyrins, which cause porphyrin accumulation in treated plants alone, (b) enhancers of conversion of exogenous A L A into M V Pchlide, (c) enhancers of conversion of exogenous A L A into D V Pchlide, and (d) inhibitors of M V Pchlide accumulation. In the case of (b) and (c), porphyrin contents of ALA+Modulator treatments are significantly greater than that of A L A alone treatment. Mechanism of Action of Inducer-Type Modulators. Even though chelating agents such as 1,10-phenanthroline (o-P) and D P Y have been utilized in the investigations of control mechanisms of Chi biosynthesis since the late 1950s, their biochemical mode of action in plants is not completely understood. The most obvious mechanism is that the chelators inhibit heme synthesis by chelating free F e ^ , thus diverting Proto DC solely into the magnesium porphyrin pathway. However, Duggan and Gassman (14) found this reasoning to be invalid because heme levels in the untreated bean leaves were not high enough (
