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. 1993 Jul;102(3):881–889. doi: 10.1104/pp.102.3.881

Cellular Localization of Protoporphyrinogen-Oxidizing Activities of Etiolated Barley (Hordeum vulgare L.) Leaves (Relationship to Mechanism of Action of Protoporphyrinogen Oxidase-Inhibiting Herbicides).

H J Lee 1, M V Duke 1, S O Duke 1
PMCID: PMC158860  PMID: 12231874

Abstract

Seven-day-old, etiolated barley (Hordeum vulgare L. var Post) leaves were fractionated into crude and purified etioplast, microsomal, and plasma membrane (PM) fractions. Protoporphyrinogen oxidase (Protox) specific activities of crude etioplast, purified etioplast, microsome, and PM fractions were approximately 29, 26, 23, and 12 nmol h-1 mg-1 of protein, respectively. The herbicide acifluorfen-methyl (AFM), at 1 [mu]M, inhibited Protox activity from crude etioplasts, purified etioplasts, microsomes, and PM by 58, 59, 23, and 0% in the absence of reductants. Reductants (ascorbate, glutathione [GSH], dithiothreitol [DTT], and NADPH) individually reduced the Protox activity of all fractions, except that microsomal Protox activity was slightly stimulated by NADPH. Ascorbate, GSH, or a combination of the two reductants enhanced Protox inhibition by AFM, and AFM inhibition of Protox was greatest in all fractions with DTT. NADPH enhanced AFM inhibition significantly only in etioplast fractions. Uroporphyrinogen I (Urogen I) and coproporphyrinogen I (Coprogen I) oxidase activities were found in all fractions; however, etioplast fractions had significantly more substrate specificity for protoporphyrinogen IX (Protogen IX) than the other fractions. Urogen I and Coprogen I oxidase activities were unaffected by AFM in all fractions, and 2 mM DTT almost completely inhibited these activities from all fractions. Diethyldithiocarbamate inhibited PM Protox activity by 62% but had less effect on microsome and little or no effect on etioplast Protox. Juglone and duroquinone stimulated microsomal and PM Protox activity, whereas the lesser effect of these quinones on etioplast Protox activity was judged to be due to PM and/or microsomal contaminants. These data indicate that there are microsomal and PM Protogen IX-oxidizing activities that are not the same as those associated with the etioplast and that these activities are not inhibited in vivo by AFM. In summary, these data support the view that the primary source of high protoporphyrin IX concentrations in AFM-treated plant tissues is from Protogen IX exported by plastids and oxidized by AFM-resistant extraorganellar oxidases.

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Selected References

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  1. Bovarnick J. G., Chang S. W., Schiff J. A., Schwartzbach S. D. Events surrounding the early development of Euglena chloroplasts: experiments with streptomycin in non-dividing cells. J Gen Microbiol. 1974 Jul;83(0):51–62. doi: 10.1099/00221287-83-1-51. [DOI] [PubMed] [Google Scholar]
  2. Camadro J. M., Matringe M., Scalla R., Labbe P. Kinetic studies on protoporphyrinogen oxidase inhibition by diphenyl ether herbicides. Biochem J. 1991 Jul 1;277(Pt 1):17–21. doi: 10.1042/bj2770017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Camadro J. M., Urban-Grimal D., Labbe P. A new assay for protoporphyrinogen oxidase - evidence for a total deficiency in that activity in a heme-less mutant of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1982 Jun 15;106(3):724–730. doi: 10.1016/0006-291x(82)91771-5. [DOI] [PubMed] [Google Scholar]
  4. Chanson A., McNaughton E., Taiz L. Evidence for a KCl-Stimulated, Mg-ATPase on the Golgi of Corn Coleoptiles. Plant Physiol. 1984 Oct;76(2):498–507. doi: 10.1104/pp.76.2.498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davies D. D., Davies S. Purification and properties of L(+)-lactate dehydrogenase from potato tubers. Biochem J. 1972 Oct;129(4):831–839. doi: 10.1042/bj1290831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Michelis M. I., Spanswick R. M. H-pumping driven by the vanadate-sensitive ATPase in membrane vesicles from corn roots. Plant Physiol. 1986 Jun;81(2):542–547. doi: 10.1104/pp.81.2.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duine J. A. Quinoproteins: enzymes containing the quinonoid cofactor pyrroloquinoline quinone, topaquinone or tryptophan-tryptophan quinone. Eur J Biochem. 1991 Sep 1;200(2):271–284. doi: 10.1111/j.1432-1033.1991.tb16183.x. [DOI] [PubMed] [Google Scholar]
  8. Gallagher S. R., Leonard R. T. Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots. Plant Physiol. 1982 Nov;70(5):1335–1340. doi: 10.1104/pp.70.5.1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hodges T. K., Leonard R. T. Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol. 1974;32:392–406. doi: 10.1016/0076-6879(74)32039-3. [DOI] [PubMed] [Google Scholar]
  10. Jacobs J. M., Jacobs N. J. Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles. Biochem J. 1987 May 15;244(1):219–224. doi: 10.1042/bj2440219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jacobs J. M., Jacobs N. J. Porphyrin Accumulation and Export by Isolated Barley (Hordeum vulgare) Plastids (Effect of Diphenyl Ether Herbicides). Plant Physiol. 1993 Apr;101(4):1181–1187. doi: 10.1104/pp.101.4.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobs J. M., Jacobs N. J., Sherman T. D., Duke S. O. Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley. Plant Physiol. 1991 Sep;97(1):197–203. doi: 10.1104/pp.97.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jacobs N. J., Jacobs J. M. Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis. Enzyme. 1982;28(2-3):206–219. doi: 10.1159/000459103. [DOI] [PubMed] [Google Scholar]
  14. Kenyon W. H., Duke S. O. Effects of Acifluorfen on Endogenous Antioxidants and Protective Enzymes in Cucumber (Cucumis sativus L.) Cotyledons. Plant Physiol. 1985 Nov;79(3):862–866. doi: 10.1104/pp.79.3.862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Law M. Y., Charles S. A., Halliwell B. Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of Paraquat. Biochem J. 1983 Mar 15;210(3):899–903. doi: 10.1042/bj2100899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee H. J., Ball M. D., Parham R., Rebeiz C. A. Chloroplast Biogenesis 65 : Enzymic Conversion of Protoporphyrin IX to Mg-Protoporphyrin IX in a Subplastidic Membrane Fraction of Cucumber Etiochloroplasts. Plant Physiol. 1992 Jul;99(3):1134–1140. doi: 10.1104/pp.99.3.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matringe M., Camadro J. M., Block M. A., Joyard J., Scalla R., Labbe P., Douce R. Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides. J Biol Chem. 1992 Mar 5;267(7):4646–4651. [PubMed] [Google Scholar]
  18. Matringe M., Camadro J. M., Labbe P., Scalla R. Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides. Biochem J. 1989 May 15;260(1):231–235. doi: 10.1042/bj2600231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matringe M., Camadro J. M., Labbe P., Scalla R. Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: oxadiazon, LS 82-556 and M&B 39279. FEBS Lett. 1989 Mar 13;245(1-2):35–38. doi: 10.1016/0014-5793(89)80186-3. [DOI] [PubMed] [Google Scholar]
  20. Matringe M., Mornet R., Scalla R. Characterization of [3H]acifluorfen binding to purified pea etioplasts, and evidence that protoporphyrinogen oxidase specifically binds acifluorfen. Eur J Biochem. 1992 Nov 1;209(3):861–868. doi: 10.1111/j.1432-1033.1992.tb17358.x. [DOI] [PubMed] [Google Scholar]
  21. Mortimer P. P. Human B19 parvovirus infection: an example of multiple pathogenic effects determined by differences in host susceptibility. Mem Inst Oswaldo Cruz. 1992;87 (Suppl 5):129–131. doi: 10.1590/s0074-02761992000900020. [DOI] [PubMed] [Google Scholar]
  22. Varsano R., Matringe M., Magnin N., Mornet R., Scalla R. Competitive interaction of three peroxidizing herbicides with the binding of [3H]acifluorfen to corn etioplast membranes. FEBS Lett. 1990 Oct 15;272(1-2):106–108. doi: 10.1016/0014-5793(90)80459-v. [DOI] [PubMed] [Google Scholar]

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