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Overexpression of PPOX II in tobacco results only in slight resistance against acifluorfen

5.2 Different mechanisms of herbicide resistance

5.2.2 Overexpression of PPOX II in tobacco results only in slight resistance against acifluorfen

Discussion 88

overexpressing transgenic lines. Photodynamic symptoms caused by reactive oxygen species could not be observed on leaves of these lines in comparison to the wild type (Fig. 22). The light dosage (= light intensity x exposure time) and the time of application play a major role in the efficiency of the herbicide and, inversely, in the protective response of the plant.

Application of peroxidizing herbicides before dark causes an improved efficiency on the following day when plants are exposed to sunlight (Wakabayashi and Böger, 1999).

Apart from environmental factors the dosage effects of herbicide action depends on the physiological state of the plants. Plants show a natural variation in susceptibility to peroxidizing herbicides (Sherman et al., 1991). The resistance mechanisms are very complex and not completely understood. They can include enzymatic resistance, increased degradation of the herbicide or the accumulating porphyrins, and improved natural adaptive capacity of the antioxidative pathway for detoxification of reactive oxygen species generated during herbicide action (Knörzer and Böger, 1999). We are currently investigating in transgenic tobacco plants expressing Arabidopsis PPOX I whether other resistance mechanisms support the effects of PPOX overexpression in transgenic tobacco. Future exploitation of these plant properties is required to engineer higher resistance against peroxidizing herbicides.

5.2.2 Overexpression of PPOX II in tobacco results only in slight resistance against

transgenic plants. We were not able to measure heme content in tobacco plants due to experimental difficulties, but it has been demonstrated that heme inhibits ALA synthesis in intact plastids (Chereskin and Castelfranco, 1982). ALA formed in chloroplast is the common precursor of chlorophyll and heme synthesizing in chloroplast and mitochondria. Heme which is synthesized in mitochondria may therefore also be involved in feedback regulation of ALA synthesis. Strong reduction of ALA synthesizing capacity relative to the control was observed in the leaves of transgenic line S36 under high light conditions, while LL-grown transformants had the same ALA synthesizing capacity as the wild type. We can speculate that under high light conditions increased heme content leads to inhibition of ALA synthesis with consequent reduction of chlorophyll content.

Northern and Western blot analysis revealed an increase in the levels of mRNA and protein in PPOX II sense plants relative to the wild type. Expression of mRNA for PPOX I is not effected in PPOX II overexpressing plants (Fig. 39).

As described above, the resistance against peroxidizing herbicides can be achieved by the generation of tobacco plants overexpressing the plastidal isoform of PPOX. Previously Matringe et al. (1989) have demonstrated that PPOX-inhibiting herbicides such as S23142 and acifluorfen inhibit PPOX activity in both chloroplasts and mitochondria in vitro.

Enzymological studies with the recombinant PPOX II, overproduced in E. coli, confirmed that this isoform, as well as PPOX I, is sensitive to the peroxidizing herbicides. (Lermontova et al., 1997). It was predicted that the overexpression of the mitochondrial isoform also could lead to a reduced sensitivity of plants to peroxidizing herbicides. To test the herbicide resistance of PPOX II overexpressing plants, we performed nearly the same experiments as it was described above for PPOX I sense plants.

Germination of seeds from PPOX II transformants on MS medium containing acifluorfen showed that transgenic seeds are sensitive to the herbicide like the wild type (data not shown).

After spraying with 10 µM of acifluorfen, PPOX II sense plants, similar to the wild type, developed necrotic lesions within two days. Only leaf discs of LL-and HL-grown PPOX II overexpressing plants after incubation with 500 nM and 1 µM of acifluorfen accumulated ca.

50 % less of protoporphyrin IX than the control (Fig. 40). Therefore, it can be concluded that the concentration of applied herbicides and the way of their application are very important factors for the investigation of herbicide action in plants and for the comparative analysis of the herbicide sensitivity of the wild type and transgenic plants.

Discussion 90

Comparison of the data obtained from analysis of PPOX II sense plants with the data for PPOX I sense plants suggested that overexpression of PPOX II leads to a lower resistance of transformants against peroxidizing herbicides. This could be due to several reasons.

Expression of the homologous gene very often leads to cosuppression or to low overexpression of the protein encoded by transgene. Comparison of transgenic tobacco plants carrying the PPOX I transgene from Arabidopsis and tobacco plants carrying the tobacco PPOX II transgene confirm this observation. In the first case, the protein level of PPOX I was increased up to 6 fold, in the second case the level of PPOX II protein increased about 2 fold in transgenic plants in comparison to the wild type. Another explanation for this difference may be the different impacts of PPOX I and PPOX II in tetrapyrrole biosynthesis. PPOX II contributes to the biosynthesis of mitochondrial heme, while PPOX I is the common enzyme for the plastidal heme and chlorophyll biosynthetic pathways. As was discussed above, inhibition of heme synthesis in mitochondria via inhibition of PPOX II could be overcome in PPOX I overexpressing plants by a compensatory exchange of heme between plastids and mitochondria. It is not known if increased amount of mitochondrial PPOX could compensate inhibition of heme and chlorophyll biosynthesis in plastids. Previously Watanabe et al. (1998) have suggested that resistance of a photomixotrophic tobacco cell culture, mentioned in section 5.2.1, was due to an increasing activity of protoporphyrinogen oxidase and 10 fold increase in the level of mitochondrial PPOX mRNA. They proposed that in mutant cells the excessive Protogen, accumulated due to the inhibition of PPOX I, is metabolized in mitochondria because of higher levels of mitochondrial PPOX. The growth of photomixotrophic tobacco cells depends on both photosynthesis and catabolism of sugar, therefore inhibition of chlorophyll biosynthesis in these cells can not be as critical as in plants.

These tissue culture cells are hardly comparable with tobacco plants.

To establish conclusively the role of mitochondrial PPOX in biosynthesis of tetrapyrroles and to elucidate the mechanism of PPOX II inhibition by peroxidizing herbicide it would be useful to overexpress the heterologus gene encoding PPOX II in tobacco. It will make these plants comparable with PPOX I overexpressing plants. Comparative analysis of PPOX I and II overexpressing plants may help to understand the mechanism of the interaction between the two isoforms and the mechanism of herbicide action.

Theoretically, the overexpression of both isoforms of PPOX must lead to a higher tolerance against peroxidizing herbicide (Fig. 43B) than overexpression of either PPOX I or PPOX II.

Therefore, the generation of transgenic plants overexpressing two isoforms of PPOX could lead to a significant increase in herbicide resistance.

5.2.3 Mutagenesis of cDNA encoding PPOX is another way to obtain herbicide resistance