Preliminary Study On The Mechanisms Of Resistance To Acetolactate Synthase-inhibiting Herbicide In Brassica Napus Mutant M342

Mechanisms of resistance to acetolactate synthase(ALS)-inhibiting herbicides in plants can be broadly classified into target-site resistance and non-target-site resistance.At present,there are many researches on target-site resistance,while the mechanisms of non-target resistance remain unknown.Herbicide non-target-site resistance is related to plant detoxification metabolism,transport,immune defense response and amino acid homeostasis,etc.The aim of this study is to study the effects of ALS herbicides on plant growth and development and explore the non-target resistance mechanisms of M342.The cytological,physiological,and proteomical changes of the ALS herbicide-resistant Brassica napus mutant M342 and its wild-type N131 after tribenuron-methyl treatment were analyzed.This study may provide a new clue for understanding the mechanism of non-target resistance to ALS herbicides in plants and laid a theoretical foundation for the herbicide-resistant crops breeding.The main results are as follows:1.Cytological,physiological and biochemical analysis.The results showed that:(1)After tribenuron-methyl treatment,the growth of N131 plants retarded,meanwhile the leaves and roots turned yellowed,and the plant biomass and root activity decreased in N131.However,no significant difference was observed in the mutant plants,which suggested that tribenuron-methyl inhibits the growth and development of N131,while the growth of the mutant remain unaffected.(2)After tribenuron-methyl treatment,the chloroplast structure of the N131 was damaged,the chlorophyll content and photosynthetic rate decreased,which indicated that the chloroplast structure of the N131 was destroyed and its photosynthetic function was impaired.In contrast,no difference was found in the mutant,suggesting unimpaired photosynthetic function in the mutant after the herbicide treatment.(3)After tribenuron-methyl treatment,the ALS activity and the protein content of N131 decreased,and no significant change was found in the protein content of the mutant.The activity of ALS decreased slightly in the early stage of treatment and then returned to the normal level quickly.These results indicated that tribenuron-methyl could inhibit ALS activity in N131 and thus affect the synthesis of protein,but only has a weak inhibitory effect on the ALS activity of the mutant,and had no effect on its protein content.(4)After tribenuron-methyl treatment,the SOD,CAT,GR activity of N131 changes more than the mutant,the activity of APX decreased,the content of reactive oxygen(ROS)and MDA increased in N131,while the content of ROS and MDA decreased slightly in the mutant,suggesting that the N131 was more sensitive to tribenuron-methyl treatment,and the redox balance of N131 was disrupted,and induced oxidative stress.(5)The activity of GST decreased in N131 and increased slightly in the mutant after tribenuron-methyl treatment,indicating that the detoxification metabolic ability of N131 decreased,which may be one of the the toxicity effects of tribenuron-methyl on N131 plants,and the enhancement of detoxification metabolic ability of mutant may be related to its herbicide resistance.2.Proteomic results:(1)the number of differentially accumulanted proteins identified in the comparison group of N131 and mutant at 1 day after tribenuron-methyl treatment was the most(443),and the number of down-accumulated proteins in the N131 after 1 day tribenuron-methyl treatment was the highest(300).(2)After tribenuron-methyl treatment,the down-accumulated proteins in N131 were significantly enriched in detoxification metabolism,stress and immune defense response,ribosomal protein,photosynthesis,carbohydrate and energy metabolism.In contrast,the up-accumulated proteins of mutant were significantly enriched in stress and immune defense response,while the proteins related to other metabolic pathways remained relatively stable.After tribenuron-methyl treatment,proteins involved in stress and immune defense response was up-accumulated in the mutants compared to that in the N131 plants,suggesting that these up-accumulated proteins in the mutants might account for its herbicide resistance.(3)Some genes encoding the differentially accumulanted proteins were selected for fluorescence quantitative analysis,and the results showed that the transcriptional level of most genes was consistent with the expression trend at the protein level.