Abstract
We investigated the mechanism for conferring herbicide resistance in transgenic rice. Plants from Line M4 were resistant to PROTOX inhibitors and had yields similar to those from wild-type (WT) rice.Myxococcus xanthus PROTOX mRNA was abundantly expressed in the transgenic leaf tissues, and theM. xanthus PROTOX gene was stably transmitted into the T4 generation. We detected a protein with a predicted molecular mass of 50 kD, equal to the weight ofM. xanthus PROTOX, in M4 but not WT plants. Furthermore, several PROTOX-inhibitor herbicides — acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon — caused significant cellular leakage and lipid peroxidation in the WT, but not in the transgenics. Total PROTOX activity in untreated transformed rice was 17-fold higher than in the WT, with activity being inhibited in the latter genotype by 55%, 59%, 53%, or 60% as a result of treatment with acifluorfen, oxyfluorfen, carfentrazone-ethyl, or oxadiazon, respectively. However, PROTOX activities in transgenic rice were similar to their corresponding, untreated controls. The accumulation of Proto IX was 15-to 21-fold higher in the WT than in M4 when plants were treated with PROTOX inhibitors. In the former, their epicuticular wax and chloroplasts were severely damaged after oxyfluorfen treatment The lack of damage in transformed plants suggests that M4 does not accumulate Proto IX, probably due to the production of herbicide-resistant chloroplastic and mitochondria PROTOX.
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Literature cited
Becerril JM, Duke SO (1989) Protoporphyrin IX content correlates with activity of photobleaching herbicides. Plant Physiol90: 1175–1181
Buege JA, Ausl SD (1978) Microsomal lipid peroxidation. Meth Enzymol52: 302–310
Choi GJ, Lee HJ, Cho KY (1996) Lipid peroxidation and membrane disruption by vinclozolin in dicarboximide-susceptible and -resistant isolates ofBotrytis cinerea. Pestic Biochem Physiol55: 29–39
Choi KW, Han O, Lee HJ, Yun YC, Moon YH, Kim M, Kuk Yl, Han SU, Guh JO (1998) Generation of resistance to the diphenyl ether herbicide, oxyfluorfen, via expression of theBacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants. Biosci Biotechnol Biochem62: 558–560
Duke SO, Rebeiz CA (1994) Porphyrin biosynthesis as a tool in pest management: An overview,In SO Duke, CA Rebeiz, eds, Porphyric Pesticides: Chemistry, Toxicology, and Pharmaceutical Applications. American Chemical Society, Washington, DC, USA, pp 1–17
Duke SO, Lydon J, Becerril JM, Sherman TD, Lehnen LP, Malsumolo H (1991) Protoporphyrinogen oxidase-inhibiting herbicides. Weed Sci39: 465–473
Ferreira GC, Andrew TL, Karr SW, Dailey HA (1988). Organization of the terminal two enzymes of the heme biosynthetic pathway. Orientation of protoporphyrinogen oxidase and evidence for a membrane complex. J Biol Chem263: 3835–3839
Ha SB, Lee SB, Lee Y, Yang K, Lee N, Jang SM, Chung JS, Jung S, King YS, Wi SG, Back K (2004) The plastidicArabidopsis protoporphyrinogen IX oxidase gene, with or without the transit sequence, confers resistance to the diphenyl ether herbicide in rice. Plant Cell Environ27: 79–88
Ichinose K, Che FS, Kimura Y, Malsunobu A, Sato F, Yoshida S (1995) Selection and characterization of protoporphyrinogen oxidase inhibiting herbicide (S23142) resistant photomixotrophic cultured cells ofNicotiana tabacum. J Plant Physiol146: 693–698
Jacobs JM, Jacobs NJ, Sherman TD, Duke SO (1991) Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organella and plasma membrane enriched fractions of barley. Plant Physiol97: 197–203
Jung S, Lee Y, Yang K, Lee SB, Jang SM, Ha SB, Back K (2004) Dual targeting ofMyxococcus xanthus protoporphyrinogen oxidase into chloroplasts and mitochondria and high level oxyfluorfen resistance. Plant Cell Environ27: 1436–1446
Kouji H, Masuda T, Malsunaka S (1988) Action mechanism of diphenyl ether herbicides: Light-dependentO 2 consumption in diphenyl ether-treated tobacco cell homogenate. J Pestic Sci13:495–499
Lee HJ, Duke MV, Duke SO (1993) Cellular localization of protoporphyrinogen-oxidizing activities of etiolated barley (Hordeum vulgare L.) leaves. Plant Physiol102: 881–889
Lee HJ, Lee SB, Chung JS, Han SU, Guh JO, Jeon JS, An G, Back K (2000) Transgenic rice plants expressing aBacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen. Plant Cell Physiol41: 743–749
Lee KB (2006) Ultrastructure of mature embryos in the parasitic flowering plantCuscuta japonica. J Plant Biol49: 384–391
Lehnen LR, Sherman TD, Becerril JM, Duke SO (1990) Tissue and cellular localization of acifluorfen-induced porphyrins in cucumber cotyledons. Pestic Biochem Physiol37: 239–248
Lermonlova I, Grimm B (2000) Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenylether herbicide acifluorfen. Plant Physiol122: 75–83
LermonLova I, Kruse E, Mock HP, Grimm B (1997) Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase. Proc Natl Acad Sci USA94: 8895–8900
Li XS, Volrath L, Nicholl DBG, Chilcoll CE, Johnson MA, Ward ER, Law MD (2003) Development of protoporphyrinogen oxidase as an efficient selection marker forAgrobacterium tumefaciens-mediated transformation of maize. Plant Physiol133: 736–747
Malringe M, Camadro JM, Labbe P Scalla R (1989) Protoporphyrinogen oxidase the target for diphenyl ether herbicides. Biochem J260: 231–235
Morgan RR, Erringlon R, Elder GH (2004) Identification of sequences required for the import of human protoporphyrinogen oxidase to mitochondria. Biochem J377: 281–287
RDA (1998) Guidance methods for food crop cultivation. Rural Development Administration, Korea, pp 55–56
SAS (2000). SA/STAT User’s Guide, Version 7 (electronic), Statistical Analysis System. SAS Institute, Cary, NC, USA.
Scalla R, MaLringe M (1994) Inhibitors of protoporphyrinogen oxidase as herbicides: Diphenyl ethers and related photobleaching molecules. Rev Weed Sci6: 103–132
Spurr AR (1969) A low viscosity embedding medium for electron microscopy. J Ultrastruct Res26: 31–43
Warabi E, Usui K, Tanaka Y, Malsumolo H (2001) Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase. Pest Manage Sci57: 743–748
Walanabe N, Che FS, Iwano M, Takayama S, Nakano T, Yoshida S, Isogai A (1998) Molecular characterization of photomixotrophic tobacco cells resistant to protoporphyrinogen oxidase-inhibiting herbicides. Plant Physiol118: 751–758
Walanabe N, Che FS, Iwano M, Takayama S, Yoshida S, Isogai A (2001) Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons. J Biol Chem276: 20474–20481
Wilkowski DA, Halling BP (1989) Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen-methyl. Plant Physiol90: 1239–1242
Zhanyuan D, Bramlage WJ (1992) Modified thiobarbituric acid assay for measuring lipid oxidation in sugar-rich plant tissue extracts. J Agric Food Chem40: 1566–1570
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Jung, H.I., Kuk, Y.I. Resistance mechanisms in protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice. J. Plant Biol. 50, 586–594 (2007). https://doi.org/10.1007/BF03030713
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DOI: https://doi.org/10.1007/BF03030713