首页 抗嗪草酮羽扇豆突变体的抗性机理研究 精灵论文

抗嗪草酮羽扇豆突变体的抗性机理研究 精灵论文

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抗嗪草酮羽扇豆突变体的抗性机理研究 精灵论文抗嗪草酮羽扇豆突变体的抗性机理研究 精灵论文 Non-target-site based metribuzin tolerance in two induced mutants of narrow-leafed lupin (Lupinus angustifolius L.) 12331PAN Gang, SI Ping, YU Qin, POWLES Steve, TU Jumin 5 (1. Agronomy Department, College of Agriculture and Biotechn...

抗嗪草酮羽扇豆突变体的抗性机理研究 精灵论文
抗嗪草酮羽扇豆突变体的抗性机理研究 精灵论文 Non-target-site based metribuzin tolerance in two induced mutants of narrow-leafed lupin (Lupinus angustifolius L.) 12331PAN Gang, SI Ping, YU Qin, POWLES Steve, TU Jumin 5 (1. Agronomy Department, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, 310029, China; 2. Centre for Legumes in Mediterranean Agriculture (CLIMA), Faculty of Natural and Agricultural Sciences, the University of Western Australia, 35 Stirling Highway, Crawly, 6009, Australia; 10 3. Australian Herbicide Resistance Initiative (AHRI), School of Plant Biology, the University of Western Australia, 35 Stirling Highway, Crawly, 6009, Australia) Abstract: Lupin (Lupinus angustifolius L.) is the most important grain legume crop in Australia. Metribuzin is widely used to control weeds in lupin crops. This study investigates metribuzin tolerance mechanism in narrow-leafed lupin (Lupinus angustifolius L.) using two tolerant mutants Tanjil-AZ-33 15 and Tanjil-AZ-55 in comparison to the susceptible wild type cv Tanjil. Sequencing of the highly conserved region of the chloroplast psbA gene that covers all the potential herbicide resistance-endowing mutations revealed that the sequences between the wild type and mutants are identical and no known resistance psbA mutations were identified. This lack of resistance mutations in the psbA gene, together with the nuclear inherited tolerance pattern determined in our previous study, 20 indicate that tolerance in the two mutants is likely non-target site based. Additionally, in contrast to the susceptible plants, the photosynthetic rates of the two tolerant mutants were reduced to 70% of the control 0.5 days after metribuzin treatment, but recovered to the level of the control within 2.5 days of the treatment. The initial reduction and later recovery in photosynthetic rate of tolerant mutants further indicates that target site chloroplast is susceptible and mechanism of tolerance is non-target site 25 based. Investigation of cytochrome P450 inhibitors on plant survival revealed that tolerance in the two mutants was reversed by all three P450 inhibitors (omethoate, malathion and phorate), suggesting P450-mediated metabolism likely to be the mechanism endowing metribuzin tolerance. Interestingly, the GST inhibitor tridiphane reversed tolerance of only one mutant Tanjil-AZ-55, not Tanjil-AZ-33. 30 The differences of the two mutants in response to tridiphane suggest that different metabolic resistance mechanisms are likely involved in tolerance of the two mutants. Key words: Metribuzin resistance, Cytochrome P450 inhibitor, Lupinus angustifolius L. 0 Introduction 35 Weed control in grain crops in Australian dryland agricultural farming systems remains a high priority and herbicides prove to be the most effective management strategy. Metribuzin,a ne herbicide, has been widely used either as pre- or post-emergence to control a wide triazino range of monocot and dicot weeds during crop production. Hence, metribuizn-tolerant wheat, narrow-leafed lupin and other grain legumes have been developed to provide effective 40 management of weeds (Kleeman and Jill 2007; Si et al 2006; Day et al, 2006). Narrow-leafed lupin (Lupinus angustifolius L.) is the main grain legumes in Australia and metribuzin is the main post-emergent herbicide used for selective control of the difficult weed wild radish. Improving metribuzin tolerance is one of main objectives of the lupin breeding program. Two tolerant mutants have been developed to assist the breeding for higher herbicide tolerance in lupin (Si et al, 45 2009). However, the mechanisms of tolerance in these mutants are unknown. Knowledge of Foundations: Financial support from the Grains Research and Development Corporation (GRDC) of Australia,and Fund for the Doctoral Program of Higher Education of China (20070335109), PG was financially supported in part by Australia Endeavour Pos-Doc Fellowship Brief author introduction:PAN Gang(1973-),male, Associate Prof.,Plant breeding Correspondance author: SI Ping (1962-),Female, Research Fellow, Plant breeding. E-mail: pangang12@hotmail.com metribuzin tolerance mechanisms in lupin plants would assist breeding of tolerant cultivars and weed management in the field. Mechanisms of herbicide resistance/tolerance in plants can be target-site based (e.g. target site mutation or over-expression) or non-target-site based (e.g. reduced herbicide uptake, translocation and enhanced metabolism). Metribuzin (like triazines) is 50 the photosynthesis system II-inhibiting herbicide. It competes with plastoquinone (PQ) at the PQ binding site on the D1 protein within the photosystem two (PSII) complexes. A number of point mutations in the chloroplast psbA gene encoding the D1 protein confer herbicides resistance (Goloubinoff et al, 1984; Park and Mallory-Smith, 2006; Cseh et al, 2009). For example, atrazine tolerance in canola is associated with the mutation of psbA gene (Hirschberg et al, 1987). 55 It is known that psbA resistance mutations often result in a lower photosynthetic rate and fitness penalty (see Powles and Yu 2010 and references therein). However, metribuzin tolerance in soybean and tomato was found to be associated with higher rates of metribuzine metabolism (Park et al. 1988; Frear et al. 1985). Cytochrome P450 and glutathione –S transferase (GST) have been reported to be involved in the PSII herbicide resistance (please pick up some reference from 60 Powles and Yu 2020), and the involvement of P450 and GST in resistance can be demonstrated by the in vivo synergism of P450 or GST inhibitors (e.g. piperonyl butoxide and tridiphane) on metribuzin (Bleeke et al., 1985; Gaul et al., 1995). The present study investigate the basic underlying mechanisms of metribuzin tolerance in the lupin mutants by investigating target site psbA gene mutations and and non-target-site in vivo 65 effects of P450 and GST inhibitors on metribuzin tolerance. The knowledge obtained will be useful for the further identification and cloning of the tolerance genes and explain the difference between sites and environments. 1 Materials and Methods 1.1 Plant materials 70 Two induced metribuzin-tolerant mutants Tanjil-AZ-33 and Tanjil-AZ-55 (Si et al, 2009), and the susceptible wild type cv. Tanjil were used in this study. Seeds were sown in 17.5 cm pots containing 5 kg standard potting soil and seedlings were grown outdoor in the normal winter growing season (from Sept. to Nov., 2009) for P450 inhibitor experiments, or in the 20/12 ? (day/night) glasshouse for the GST inhibitor experiment and net photosynthetic rate measurement. 75 The plants were watered and fertilized as required. Mortality and dry weight was determined 2 weeks after treatment. 1.2 Genomic DNA extraction, PCR amplification and partial sequencing of psbA gene A highly conserved region of the chloroplast psbA gene containing potential mutation sites 80 was amplified, sequenced, and compared between the tolerant and the susceptible. Bulked shoot material from the S (Tanjil) and the two T (Tanjil-AZ-33 and Tanjil-AZ-55) genotypes without herbicide treatment was used for genomic DNA extraction using a Nucleon Phytopure DNA extraction kit (Amersham Biosciences). A pair of primers was designed based on homologous regions of psbA sequences from arabidopsis (GenBank accession number X79898.1), alfalfa 85 (X04973.1), rapeseed (M36720.1), soybean (X00152.1), and faba bean (X17694.2). The forward primer 5’-CGTGAGTGGGAACTTAGTTT-3’ and reverse primer 5’-TGAGCATTACGTTCATGCAT-3’ were used to amplify a 633 bp fragment encompassing the highly conserved region and potential resistance mutation sites of the psbA gene (Oettmeier 1999). The PCR was conducted in a 25 µL volume that consisted of about 300 ng of genomic DNA, 0.5 90 µM of each primer, and 12.5 µL of 2 x GoTag Green Master Mix? (Promega). The PCR was run in a Mastercycler (Eppendorf, Germany) with the following profile: 94? 4 min, 35 cycles of 94 ? 30 s, 58? 30 s, and 72? 30 s , followed by a final extension step of 5 min at 72?. The PCR product was purified from agarose gel with Wizard? SV Gel and PCR Clean-up System (Promega) and sequenced from both ends with the AB-Big Dye Terminator system using a commercial sequencing service. Bulked DNA samples from each R biotype were used for initial 95 sequence analysis and subsequently three single plants from each tolerant mutant were analyzed. 1.3 Metribuzin treatment in combination with P450 or GST inhibitors Our preliminary experiments with P450 inhibitors malathion, omethoate and phorate showed no effect on seedling growth and shoot dry weight in lupin at a rate of 1500, 1500, and 600 g ai/ha, respectively. So these rates were used to examine the interactions between inhibitors and 100 metribuzin in this study. The GST inhibitor tridiphane at 50 g ai/ha was selected for this study based on the research of Gaul et al (1995). Seedlings of the three genotypes were treated at the four-leaf stage with P450 or GST inhibitors about 2 h prior to the treatment of metribuzin (0, 200, 400, and 800 g ai/ha). All inhibitors and metribuzin treatments were applied onto 4-leaf -1 plants using a cabinet sprayer delivering 112 L hawater at a pressure of 200 kPa. Plants were 105 arranged in a completed randomized design with 4 replicates with 7-11 plants per replicate per herbicide treatment. Plant survival and shoot dry weight for each treatment were recorded 2 weeks after treatment. Shoots were cut above ground and the dry weight for each pot measured after 72 hr at 80 ?C in a fan-forced oven. 1.4 Photosynthesis measurement 110 Single-leaflet net photosynthetic rates were measured with a LI-6400 portable photosynthesis system (LI-COR, USA) before and after metribuzin treatment (0, 200, 400, and 800 g ai/ha) over a course of 6 days at 24 hour-interval, but the initial point was undertaken at 12 hours after treatment. Measurements were carried out on the middle leaflet of the first and second treated leaf 2under 1500 μmol/ms photosynthetic photon flux density provided by the red blue light source of 115 the equipment. External air was scrubbed of COand mixed with a supply of pure COto result in 2 2 a reference concentration of 380 μmol/mol. Flow rate was 500 μmol/s. Relative humidity of the air in the leaf chamber was controlled at 50%~60% and leaf temperature at 22 ?C. The constant values of photosynthetic rate and intercellular COconcentration of each sample leaf were 2 recorded after 200 s. All measurements were performed between 9:00 and 12:00 am. 120 1.5 Statistical analysis Regression analysis was conducted for dose response curves using SigmaPlot 11.0 (Systat Software Inc., Oint Richmond, CA, USA) and LDwas calculated using four-parameter logistic 50 curve regression (Seefeldt et al, 1995). Analysis of variance was performed for cross tolerance data. 125 2 Results 2.1 Target site 2.1.1 psbA gene sequencing revealed no known mutations PCR with the aforementioned primer pair produced a single band of expected length of 633 bp in both the susceptible and the tolerant genotypes. The nucleotide sequence of the clearly 130 identified region (about 620 bp) showed 96% homology with psbA genes from Vicia faba. This amplified sequence flanks the known PSII resistance-conferring mutation sites (amino acid codon 211-266) of the chloroplast psbA gene. Sequence alignment showed no difference between the susceptible and the two tolerant mutants (data not shown). So the known mutations in the psbA 135gene conferring the metribuzin resistance in the weeds were not present in the two mutants. 2.1.2 Photosynthetic rate revealed initial reduction but recovery in mutants In absence of metribuzin, net photosynthetic rates for the susceptible wild type and the two 2tolerant mutants were similar with the values around 35 μmol/ms (Fig. 1), indicating no inherent photosynthetic differences between them. However, large differences existed when they were treated with metribuzin (Fig.1). Photosynthetic rates of the susceptible Tanjil decreased to 140 2 2μmol/ms 0.5 days after application of metribuzin rates and remained undetectable (close to zero) till 5.5 days even at the lowest rate of 100 g/ha metribuzin (Fig. 1a). For the two tolerant mutants, the net photosynthetic rates were reduced 0.5 days after metribuzin application, but started to recover 1.5 days after application (Fig. 1b and 1c). At 100 g ai/ha metribuzin, the net photosynthetic rates of the two mutants (Tanjil-AZ-33 and Tanjil-AZ-55) reached the control level 145 at the 1.5 days and 2.5 days after treatment, respectively (Fig 1b and 1c). The initial reduction and in photosynthetic rate of tolerant mutants indicates that target site chloroplast is later recovery susceptible and mechanism of tolerance is non-target site based. (a) 50 40 Net photosythetic rate 30s) (μmol/m2 20 10 0Net photosythetic rate (μmol/m -10 0 1 2 3 4 5 6 150 (b) 50 40 s) 302 20 10 0 -10 0 1 2 3 4 5 6 (c) 50 40 s) 302 20 10 0Net photosythetic rate (μmol/m -10 0 1 2 3 4 5 6 Days after treatment (day) Figure 1 Net photosynthetic rate of the treated leaves of the susceptible wild-type Tanjil (a), and the tolerant mutants Tanjil-AZ-33 (b) and Tanjil-AZ-55 (c) at 0 (?), 100 (?), 200 (?), 400 (?) and 800 g ai/ha metribuzin. 155 Higher metribuzin rates had more severe impact on photosynthesis. At 800 g ai/ha metribuzin, the two mutants Tanjil-AZ-55 and Tanjil-AZ-33 just recovered their photosynthesis by 30% and 60% of the control. The net photosynthetic rates of the mutant Tanjil-AZ-55 was also lower than that of the mutant Tanjil-AZ-33 (Fig. 1b and 1c). This indicated that the tolerance for the mutant Tanjil-AZ-33 was higher than that of Tanjil-AZ-55 mutant. 160 Chlorophyll contents were also measured for each point of the photosynthetic rates as above. No differences were observed between the susceptible and the tolerance at various rates of metribuzin (data not shown), confirming that metribuzin affected photosynthetic rates, but not chlorophyll. 2.2 Non-target site165 2.2.1 P450 inhibitors reverse tolerance in the two mutants Plant survival of tolerant mutants (Tanjil-AZ-33 and Tanjil-AZ-55) was not affected by metribuzin as high 800 g/ha, but all the susceptible plants were killed by 400 g/ha metribuizn (Fig 2). However, metribuzin plus omethoate reduced the tolerant mutants to susceptible with all mutant plants killed at 400 g/ha metribuzin + omethoate (Fig 2), 170 120 (a) 100 80 60 40 20 0 100 200 300 400 500 600 700 8000 120(b) 100 80 60 40 20Plant survival (%control ) Plant survival (%control ) Plant survival (%control ) 0 0 100 200 300 400 500 600 700 800 (c) 120 100 80 60 40 20 0 0 100 200 300 400 500 600 700 800 Rate of metribuzin (g/ha) Figure 2 Plant survival (% control) of the susceptible wild-type Tanjil (a), and the tolerant mutants Tanjil-AZ-33 (b) and Tanjil-AZ-55 (c) in response to metribuzin in the absence (?) or presence (?) of 1500 g ai/ha omethoate 175 at 2 weeks after treatment. although the P450 inhibitor omethoate alone had no effects on lupin seedling growth. Plant survival of the susceptible was also reduced by the treatment of metribuzin plus omethoate. The reduction in metribuzin LDvalues in the presence of omethoate was 20.4 -fold for mutant 50 180 Tanjil-AZ-33; 7.4 -fold for Tanji-AZ-55 and 3.6-fold for the susceptible Tanjil (Table 1). The LDvalues of metribzin plus omethoate for the tolerant mutants were even lower than that for the 50 susceptible wild type in the absence of omethoate (Table 1). Omethoate plus metribuzin completely reversed metribuzin tolerance in the tolerant mutants. 185 Table 1. Metribuzin LDvalues (mean+s.e.) of the susceptible cv Tanjil and the two tolerant mutants Tanjil-50 AZ-33 and Tanjil-AZ-55 in the absence or presence of 1500g/hamalathion, 1500 g/ha omethoate and 600 g/ha phorate. To add LD50 for tridiphane Metribuzin + Metribuzin + Metribuzin + Genotypes Metribuzin alone Omethoate Phorate Malathion a Tanjil-AZ33 2496+14.5274.3+0.2 122.1+2.8 260.5+35.9 a Tanjil-AZ55 1717+2.1571.7+15.0 231.0+3.6 320.6+16.4 Tanjil 299.0+5.5 46.4+6.0 83.0+0.0 220.6+15.8 a The data were cited from Si et al (2009). 190 Similar results were obtained by the treatment of metribuzin plus the other two P450 inhibitors, malathion and phorate (Table 1). Presence of malathion increased metribuzin sensitivity of both the tolerant and the susceptible, with LD50 values reduced in Tanjil-AZ-33 by 9 fold and in Tanjil by 6 fold. Presence of phorate also increased metribuzin sensitivity of the tolerant. These consistent results of P450 inhibitor synergism on metribuzine indicate that 195 metribuzin tolerance in the two mutants likely to be P450-mediated enhanced herbicide metabolism. 2.2.2 GST inhibitor reverses tolerance in only one mutant GST inhibitor tridiphane alone showed no effect on the plant growth of the tolerant and the 200 susceptible (Fig. 3). However, plant survival of the tolerant Tanjil-AZ-55 decreased to 25.82% when treated with 400 g ai/ha metribuzin plus tridiphane (Fig 3c). The survival of the other tolerant mutant Tanjil-AZ-33 remained at about 90% regardless of metribuzin rates (from 200 to 800 g ai/ha) plus tridiphane (Fig 3b). The metribuzin LDin the presence of tridiphane of the 50 susceptible decreased to 118 g ai/ha, compared with 435 g ai/ha in the absence of tridiphane (Fig 205 3a). The metribuzin LDin the presence of tridiphane for Tanjil-AZ-55 decreased to 260 g ai/ha., 50 lower than that for the susceptible. Tolerance in Tanjil-AZ-55 was clearly reversed by tridiphane. The different responses of the two mutants to tridiphane indicate that different herbicide detoxifying enzymes are likely involved in mertibuzine tolerance in the mutants. 3 Discussion 210 3.1 Metribuzin tolerance in mutants is non-target site based A range of herbicides, including triazines, triazinones, ureas and uracils, block the photosynthetic electron transport chain on the reducing side of photosystem II (PS II) in many plant species. Herbicides that target PSII compete with Qfor binding to the D1 protein of PS II B and inhibit electron transport from Qto Qby acting as nonreducible analogs of plastoquinone. A B 215 The D1 protein is a subunit of the PS II core complex and is encoded by the chloroplast psbA gene. Mutations in positions 184, 219, 251, 264 and 266 are known to confer resistance to metribuzin in weed species 120 (a) 100 80 60 40 20 Plant survival (%control ) 0 0 100 200 300 400 500 600 700 800 120(b) ) Plant survival (%control) 100 80 (%control 60 40 20Plant survival 0 0 100 200 300 400 500 600 700 800 220 120 (c) 100 80 60 40 20 0 0 100 200 300 400 500 600 700 800 Rate of metribuzin (g/ha) Figure 3 Plant survival (% control) of the susceptible wild-type Tanjil (a) and the tolerant mutants Tanjil-AZ-33 (b) and Tanjil-AZ-55 (c) in response to metribuzin in the absence (?) or presence (?) of 50 g/ha tridiphane at 2 weeks after treatment. 225 (Schwenger-Erger et al, 1993 and 1999; Mengistu et al, 2000 and 2005; Park and Mallory-Smith, 2006; Vass et al, 2000; Mechant et al, 2008, Powles and Yu 2010;). Triazine tolerant canola has the psbA gene mutated in position 264 (Reith and Straus, 1987). Because the psbA gene is encoded in the chloroplast genome, a characteristic of PSII target site resistance is its maternal inheritance. However, in the two metribuzin tolerant mutants studied here, we did not 230 find any known resistance mutations in the highly conserved psbA gene. In addition, our inheritance study confirms that metribuzin tolerance in the two mutants (Tanjil-AZ-33 and Tanjil-AZ-55) is controlled by nuclear genes (Si et al. 2010). Therefore, target site mutation in the chloroplast psbA gene is not the tolerance mechanism in the two tolerant mutants. 235 Reduced photosynthetic capacity is associated with target site mutation in the psbA gene encoding for the D1 protein of photosystem II. In the case of triazine tolerant canola, mutation in the psbA gene results in a inherent lower rate of electron transfer which limits leaf photosynthetic 1988). This reduced photosynthetic capacity in absence of the herbicide (Jursinic and Pearcy capacity leads to 15-20% seed yield penalties in triazine tolerance canola cultivars when compared with triazine sensitive cultivars with similar genetic background (Auld et al. 1991; Robertson et 240 al. 2002). In contrast to triazine tolerant canola, no difference in photosynthetic rates of lupin leaves between tolerant mutants and the susceptible was found in this study. Furthermore, no seed yield penalties were observed in the absence of metribuzin between the two mutants Tanjil-AZ-33 and Tanjil-AZ-55 and the susceptible wild type cv. Tanjil (Si et al. 2009b). The initial reduction and later recovery in photosynthetic rate of the tolerant mutants after metribuzin 245 application suggests that the tolerance mechanism is likely to be of herbicide metabolism. Recovery of photosynthetic rate to the control level within 1.5 days after herbicide application suggests that metribuzin had been metabolized and photo-toxic damage been removed. 3.2 Cytochrome P450 and GST are likely involved in metribuzine tolerance Detoxification of metribuzin in resistant plants occurs by several pathways including 250 reductive deamination, diketo (Klamroth et al, 1989), homoglutathione conjugation following sulphoxidation, and N-glycosidation (Frear et al, 1985) (Fig 4). However, the main detoxification in soybeans and tomato is N-glucosidation and homoglutathione conjugation to form non-toxic metabolites (Frear et al, 1983 and 1985; Davis et al, 1991). Therefore, metribuzin tolerance in the two mutants studied is likely caused by detoxification involving cytochrome P450 255 monooxygenases, GSTs, glycosyltransferases or ABC transporters (Yuan et al, 2006; Siminszky, 2006, reviewed by Powles and Yu, 2010). The organophosphate insecticides such as omethoate, malathion and phorate have been shown to synergize the effects of clomazone and metribuzin by inhibiting the activity of microsomal P450s mediating the oxidative metabolism of these herbicides in soybean and cotton 260 (Ferhatoglu et al, 2005; Hammond, 1996; Christianson, 1991). In this study, we demonstrated that the organophosphate insecticides malathion, omethoate and phorate can synergise metribuzin and reverses tolerance in the mutants Tanjil-AZ-33 and Tanjil-AZ-55 (Fig 2 & Table 1). These results indicate the possible role of P450-mediated metabolism of metribuzin in the mutants. The proposed pathway for metribuzin metabolism (Fig. 4) shows that plant GSTs are another 265 important enzyme family catalyzed the conjugation of glutathione or homoglutathione (in legumes) to various substrates to form a polar S-glutothionylated product (Yuan et al, 2006; Hall et al, 2001). The previous studies also indicated that metribuzin resistance in soybean was likely to be due to formation of homoglutathione conjugates by GSTs (Frear et al, 1985; Brown and Neighbors, 1987). MZH2091 and tridiphane are the effective synergists for metribuzin and have been 270 demonstrated as the GST inhibitors in ivyleaf moringglory (Klamroth et al, 1989) and soybean (Gaul et al, 1995). In this study, metribuzin metabolism in the mutant Tanjil-AZ-55 is strongly inhibited by tridiphane, (Fig. 3b), suggesting that the possible involvement of GST-based metabolism in endowing metribuzin tolerance in Tanjil-AZ-55. However, there is evidence showing that tridiphane inhibits P450 activities in wheat microsomes (Mougin et al. 1991). 275 Therefore, it is also possible that different P450 enzymes may be involved in tolerance in the two mutants. Figure 4 Proposed metabolic pathway of metribuzin metabolism in plants 280 Interestingly, cross tolerance to field rates of other PSII herbicides and herbicides of different modes of action was not observed in the two metribuzin tolerant mutants . This may suggest that P450 or GST enzymes involved in metribuzin metabolism in the tolerant mutants has higher substrate specificity. 285 3.3 Metribuzin plus P450 or GST inhibitors increases the phototoxicity of the mutants and the wild type As a follow-up to the successful commercialization of certain P450 inhibitors such as sesamex and PBO, a variety of chemical compounds such as 1-aminobenzotriazole and oranophosphate insecticides have been tested over the years as potential P450 inhibitors in plants (Roberts, 2000). Furthermore, GST inhibitors such as MZH-2091 and tridiphane have also been 290 reported to interact with metribuzin (Roberts, 2000; Klamroth et al, 1989; Gaul et al, 1995). In this study, high dose of metribuzin (no more than 800 g ai/ha) just decreases plant growth in the tolerance mutants such as the leaves, plant height and the shoot dry weight (data not shown) and the net photosynthetic rate (Fig. 1), but the effect on the plant survival is very small (Fig. 1b &1c). However, metribuzin plus P450 inhibitors significantly decrease plant survival (Fig. 2b & 2c) and 295 the shoot dry weight (data not shown). In addition, the GST inhibitor tridiphane just specifically synergise metribuzin in the wild type and the mutant Tanjil-AZ-55 (Fig 3a & 3c), but not the mutant Tanjil-AZ-33 (Fig. 3b). Therefore, due to increase the phototoxicity of the lupin plants (Fig. 1), the P450 inhibitors including piperonyl butoxide and oranophosphate insecticides, and the GST inhibitor tridiphane can not be used in the lupin, especially for the mutant Tanjil-AZ-55. 300 3.4 Possible different mechanisms for the two mutants The previous study revealed that the mutant Tanjil-AZ-33 was six times and Tanjil-AZ-55 was just four times more tolerant than the wide-type Tanjil (Si et al, 2009a). In the present study, the leaflet net photosynthetic rates of the mutants were significantly affected after spraying metribuzin, but the mutant Tanjil-AZ-33 and Tanjil-AZ-55 can recover by 60-100% and 30-100%, 305 respectively, compared with the control (Fig. 1b and 1c). This suggested that detoxification of metribuzin was faster in the mutant Tanjil-AZ-33 than in the mutant Tanjil-AZ-55. Tolerance in Tanjil-AZ-33 is controlled by a single nuclear gene Mt3 whilst Tanjil-AZ-55 by the Mt5 gene. These two genes are of non-allelic nature and of additive effects (Si et al, 2010). This study revealed that Tanjil-AZ-33 and Tanjil-AZ-55 responded differently to omethoate and tridiphane 310 inhibitors. Given that omethoate is a P450 inhibitor, tolerance in Tanjil-AZ-33 is likely to be mediated by P450. Metabolism in Tanjil-AZ-55 is likely to be mediated by GST and some P450. The metabolic mechanisms in these two mutants are likely to be complementary to each other as 2010). plants containing the two tolerance genes increases tolerance further by 5-fold (Si et al, 315 In summary, this study revealed that the tolerance mechanisms in two tolerant mutants are non-target site based, likely involving P450 or GST – mediated metribuzin metabolism. Metribuzine metabolism in the two mutants may involve different P450s or GSTs. Tanjil-AZ-33 likely has a different metabolic pathway than Tanjil-AZ-55. 320 References [1] Bleeke MS, Smith MT and Casida JE. 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Australian Herbicide Resistance Initiative (AHRI), School of Plant Biology, the University of Western Australia, 35 Stirling Highway, Crawly, 6009, Australia) 摘要:羽扇豆是澳大利亚最重410 要的豆科作物。为了控制杂草的生长及危害,在羽扇豆田里除 草剂嗪草酮被广泛应用。尽 管在过去的研究中,已针对高抗嗪草酮的羽扇豆突变体 Tanjil-AZ-33 和 Tanjil-AZ-55 进 行了不同方面的研究,但有关其抗性机理却一无所知。通过 对嗪草酮的靶标基因 psbA 基因 进行测序分析 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 明,突变体中的该基因并没有发生任何突变。 进一步研究表明,P450 抑制剂 (包括 omethoate, malathion 和 phorate)能够显著提高除草剂 嗪草酮的活性,迅速杀死两个 突变体,从而推测 P450 基因是该除草剂降解的重要途径。然 而,GST 抑制剂 tridiphane 却仅415 能提高除草剂在 Tanjil-AZ-55 中的活性,说明在 Tanjil-AZ-55 中,GST 也参与了除草剂的脱毒。 关键词:嗪草 酮;细胞色素 P450 抑制剂;羽扇豆 中图分类 号:S51 420
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