Chapter 8
Factors Affecting Protoporphyrin Accumulation in Plants Treated with Diphenyl Ether Herbicides
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Judith M. Jacobs and Nicholas J. Jacobs Department of Microbiology, Dartmouth Medical School, Hanover, NH 03755-3842
The toxic action of photobleaching diphenylether herbicides is dependent upon their ability to cause accumulation of the phototoxic tetrapyrrole intermediate, protoporphyrin. Although these herbicides inhibit the plastid enzyme which converts protoporphyrinogen to protoporphyrin, the mechanism by which accumulated protoporphyrinogen is converted to protoporphyrin remains unknown. It has recently been demonstrated that this conversion can be mediated by herbicide-resistant mechanisms associated with extraorganellar membranes such as the plasma membrane. In addition, we have reported that protoporphyrinogen is preferentially exported from isolated intact plastids incubated in the presence of porphyrin precursors and herbicide. In this report, we summarize our findings that protoporphyrinogen can also be decomposed to non-porphyrin compounds by cytosolic factors under conditions where protoporphyrin is quite stable. We also summarize our recent findings on the distribution of the terminal enzymes of heme biosynthesis in mitochondria and plastids. A l l these results are discussed in terms of their role in explaining the accumulation of protoporphyrin in herbicide treated plants. It is concluded that this is best explained by the export of protoporphyrinogen from the plastid, followed by its oxidation to protoporphyrin through the mediation of herbicide-resistant mechanisms associated with extraorganellar membranes.
The purpose of this chapter is to discuss factors affecting the accumulation of the porphyrin intermediate, protoporphyrin, which occurs when plants are treated with diphenylether (DPE) herbicides. The toxic action of photobleaching DPE herbicides is dependent upon their ability to cause the accumulation of excessive levels of protoporphyrin. In the presence of light, protoporphyrin catalyzes photooxidative destruction of plant cell membranes (1-13). Thus, a complete understanding of the mechanism of herbicide action requires elucidation of the factors which govern the levels of protoporphyrin which accumulate upon treatment with herbicide.
0097-6156/94/0559-0105$08.00/0 © 1994 American Chemical Society
Duke and Rebeiz; Porphyric Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 6, 2017 | http://pubs.acs.org Publication Date: April 15, 1994 | doi: 10.1021/bk-1994-0559.ch008
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PORPHYRIC PESTICIDES
The primary mechanism by which these herbicides cause protoporphyrin accumulation is their ability to strongly inhibit the porphyrin biosynthetic enzyme protoporphyrinogen oxidase (15-20). This enzyme converts protoporphyrinogen to protoporphyrin and is present in the plastid which contains all of the enzymes necessary for heme and chlorophyll synthesis from early precursors (see Figure 1 for this biosynthetic pathway). Herbicide inhibition of protoporphyrinogen oxidase would be expected to cause accumulation of protoporphyrinogen, the substrate of the inhibited enzyme. The question remains unanswered as to how this accumulated protoporphyrinogen is converted to protoporphyrin in herbicide-treated plants. The assumption had been made that this accumulated protoporphyrinogen was oxidized spontaneously to protoporphyrin by chemical autooxidation. However, we will discuss data which suggest that the oxidation of protoporphyrinogen to protoporphyrin occurs through the mediation of herbicide-resistant oxidizing factors present in the plasma membrane or other membranes located outside of plastids or mitochondria. We will also discuss some recent data concerning the nature of this extraplastidic oxidizing mechanism and how it differs from the biosynthetic protoporphyrinogen oxidase present in the plastid. Our recent findings that protoporphyrinogen can be preferentially exported from isolated intact plastids incubated with early porphyrin precursors in the presence of herbicide lends support to the importance of this extraplastidic protoporphyrinogen oxidation. Protoporphyrin accumulation would also be influenced by reactions which utilize accumulated protoporphyrinogen without giving rise to protoporphyrin. For instance, we will mention recent findings from our laboratory that protoporphyrinogen can be decomposed to non-porphyrin products in the presence of plant cytoplasm. We will also present and discuss recent evidence that protoporphyrin itself can be converted to later intermediates in the biosynthetic pathway even in the presence of herbicide. Our recent findings on the interorganellar localization of the three terminal enzymes of heme synthesis in plants will also be summarized. These findings have implications for protoporphyrin and protoporphyrinogen accumulation in the plant cytoplasm, since a recent hypothesis has proposed that the plastid synthesizes protoporphyrinogen for export and use by the plant mitochondria for mitochondrial heme synthesis (to be discussed in a later section). Formation of Protoporphyrin from Accumulated Protoporphyrinogen Demonstration of Extraplastidic, Herbicide-Resistant Protoporphyrinogen Oxidation. The question of why protoporphyrin accumulates when protoporphyrinogen oxidase is inhibited first arose when it was discovered that the human hereditary disease variagate porphyria was a result of a partial deficiency of the mitochondrial protoporphyrinogen oxidase enzyme (21, 22). Protoporphyrin accumulates in the tissue of these patients and causes photosensitivity. The assumption was that protoporphyrinogen is spontaneously oxidized to protoporphyrin. Chemical studies indicate that most porphyrinogens are spontaneously oxidized to the porphyrin form, a process which is accelerated by low pH, light, and aeration. In fact, in other porphyria diseases involving deficiencies in the enzymes utilizing other porphyrinogens, such as uroporphyrinogen or coproporphyrinogen, uroporphyrin and coproporphyrin are found in urine and feces. However, the spontaneous oxidation of protoporphyrinogen has been less well studied, and there were early indications that it was difficult to chemically
Duke and Rebeiz; Porphyric Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 6, 2017 | http://pubs.acs.org Publication Date: April 15, 1994 | doi: 10.1021/bk-1994-0559.ch008
8. J A C O B S A N D J A C O B S
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Protoporphyrin Accumulation in Plants
5 - Aminolevulinate
Porphobilinogen —Uroporphyrinogen -4C0 -2C0
-4H Protoporphyrinogen -
