Overexpression of Plastidic Protoporphyrinogen IX Oxidase Leads to Resistance to the Diphenyl-Ether Herbicide Acifluorfen

Abstract

The use of herbicides to control undesirable vegetation has become a universal practice. For the broad application of herbicides the risk of damage to crop plants has to be limited. We introduced a gene into the genome of tobacco (Nicotiana tabacum) plants encoding the plastid-located protoporphyrinogen oxidase of Arabidopsis, the last enzyme of the common tetrapyrrole biosynthetic pathway, under the control of the cauliflower mosaic virus 35S promoter. The transformants were screened for low protoporphyrin IX accumulation upon treatment with the diphenyl ether-type herbicide acifluorfen. Leaf disc incubation and foliar spraying with acifluorfen indicated the lower susceptibility of the transformants against the herbicide. The resistance to acifluorfen is conferred by overexpression of the plastidic isoform of protoporphyrinogen oxidase. The in vitro activity of this enzyme extracted from plastids of selected transgenic lines was at least five times higher than the control activity. Herbicide treatment that is normally inhibitory to protoporphyrinogen IX oxidase did not significantly impair the catalytic reaction in transgenic plants and, therefore, did not cause photodynamic damage in leaves. Therefore, overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporphyrinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen.Protoporphyrinogen oxidase (EC 1.3.3.4) (PPOX) is the last enzyme in the common tetrapyrrole biosynthesis pathway before the pathway branches toward chlorophyll and heme synthesis. PPOX catalyzes the oxygen-dependent oxidation of non-fluorescent protoporphyrinogen IX to fluorescent protoporphyrin IX and is associated with the chloroplast envelope and the thylakoid membrane (Matringe et al., 1992a). Parallel to the plastidic tetrapyrrolic pathway, activities of the last two enzymes of the heme synthesizing pathway, PPOX and ferrochelatase, were found in mitochondria (Jacobs et al., 1991; Smith et al., 1993) Thus, protoporphyrinogen IX is distributed between the plastidic pathway and the mitochondrial heme synthesis pathway. The control mechanism of the intercompartmental allocation is still not known. We recently identified two different cDNA sequences for PPOX in tobacco (Nicotiana tabacum).The deduced protein sequences designated as PPOX I and II have molecular masses of 60 and 55 kD, respectively, and share only 30% similarity. Translocation studies and immunological analysis proved that the proteins are imported either exclusively into plastids (PPOX I) or into mitochondria (PPOX II) (Lermontova et al., 1997).PPOX I and PPOX II closely resemble known PPOX sequences, e.g.

Level of protoporphyrinogen oxidase activity tightly correlates with photodynamic and defense responses in oxyfluorfen-treated transgenic rice

“…To measure the Proto IX content, plant tissue (0.1 g) was ground in 1 ml methanol : acetone : 0.1 N NaOH (9 : 10 : 1) and the homogenate was centrifuged at 10,000 g for 10 min to remove cell debris. 19) Proto IX was separated by HPLC as described above.…”

“…Protox activity was determined following the method of Lermontova and Grimm. 19) The Protogen IX substrate was prepared by chemical reduction of Proto IX with sodium mercury amalgam (Sigma-Aldrich, St. Louis, MO, USA). The assay contained 70 ml suspension of crude chloroplasts and mitochondria (0.5 mg protein) and 140 ml assay buffer.…”

“…18) The overexpression of Protox in either chloroplasts or mitochondria diminishes the action of Protox-inhibiting herbicides by preventing the accumulation of Protogen IX, and decreases light-dependent phytotoxicity. [19][20][21][22][23] The present paper investigates whether the level of Protox activity tightly correlates to peroxidizing-herbicide resistance in the two transgenic rice systems involving the chloroplastidic expression of Arabidopsis Protox (AP line) 21) and the dual expression of Myxococcus xanthus Protox in chloroplasts and mitochondria (TTS line). 23) The relation between the levels of Protox activity and the oxidative status was monitored in the two transgenic and WT lines imposed by oxifluorfen compared with controls.…”

cor Gene Expression in Barley Mutants Affected in Chloroplast Development and Photosynthetic Electron Transport

“…Previous work has suggested that nuclear gene expression might be controlled by the levels of the chlorophyll precursor protoporphyrin IX (Lermontova and Grimm, 2000). To verify whether the differences in cor14b accumulation among albino and xantha mutants could be correlated with protoporphyrin IX levels, we have determined protoporphyrin IX levels in xantha f17 and xantha-s46 before and after cold stress treatment by using the method of Lermontova and Grimm (2000).…”

Current Status and Perspective of Weed Management in Herbicide-Resistant Crops

“…Glyphosate 저항성 콩은 glyphosate 저항성 클론 cp4 EPSPS를 콩에 삽입시켜 개발되었으며, 면화, 옥 수수, 캐놀라, 알팔파, 벤트그라스(Agrostis stolonifera), 사 탕무 등에서도 glyphosate 저항성 형질을 도입한 형질전환 체들이 개발되었다 (Pasgette et al, 1996). 그리고 엽록소 생합성을 저해하는 acifluorfen의 형질전환작물은 엽록소 생합성과 관련된 목표 효소의 과대발현을 통하여 개발되 었다 (Lermontova and Grimm, 2000). 두 번째 시도는 glufosinate와 bromoxynil 저항성으로, 이 들의 제초활성 물질이 작물에서 무독성인 물질로 전환하 는 제초제 대사를 증진하는 유전자의 도입에 의하여 이루 어졌다 (Haumann, 1997).…”