Mechanisms Of Penoxsulam Resistance In AXXZ-6 And AXXZ-8 Populations Of Barnyardgrass In Rice Fields From Xuancheng City,Anhui Province

Barnyardgrass（Echinochloa crus-galli）is one of the most malignant weeds in rice fields worldwide and the chemical herbicides were the mainly tools to control this weed.Penoxsulam,which targets on the acetolactate synthase（ALS）,is one of the most common herbicides used to control barnyardgrass and various annual weeds.With the increase in usage time,the penoxsulam resistance in barnyardgrass began to appear and developed severely.Our previously study reported the penoxsulam resistance level and target-site resistance mechanism of several resistant barnyardgrass populations.In these studies,it was found that the expression of target enzyme ALS gene was not responsible for the penoxsulam resistance in AXXZ-8 population collected from Xuancheng City in Anhui Province.Only a point mutation（Phe-206-Leu）of ALS at position 206 was detected.The mutation was first identified in organisms and its role in penoxsulam resistance was not clear.Meanwhile,AXXZ-6 population collected from Xuancheng City in Anhui Province displayed high level resistance to penoxsulam,and the mutation of Trp-574-Arg was the target-site based mechanisms.At the same time,the AXXZ-6 population also developed severely cross-resistance and multiple-herbicide resistance to ACCase inhibitors and auxin-like herbicides.It was predicted that the complex resistance files of AXXZ-6 population were caused by non-target-site resistance mechanisms.Based on these two issues remaining,the penoxsulam resistance mechanisms were explored from the target-site based and non-target-site based aspects,taking the AXXZ-8 and AXXZ-6 population collected from Xuancheng City of Anhui Province as cases.The results were listed as followed.1.The amino acid mutation of ALS gene at position 206 conferred penoxsulam resistance in AXXZ-8 population barnyardgrass.The relationship between amino acid mutation at position 206 and penoxsulam resistance was studied from three aspects.Firstly,homology modeling and molecular docking was used for confirmation.The obtained high-quality ALS model was used to carry out the molecular docking with penoxsulam.Theπ-πstacking between the benzene ring of Phe-179（equivalent to Phe-206 in Arabidopsis）and penoxsulam was detected.When the Phe-179 was transformed to Leu-179,the docking conformation of the penoxsulam and ALS protein did not significantly change.However,π-πstacking between position 179 and the ALS protein disappeared,and the docking score decreased 9.78%.These results indicated that theπ-πstacking provided by Phe-206 and penoxsualm stabilized the combination of ALS and penoxsulam.Based on the conformational change and the docking score,the Phe-206-Leu mutation could weaken the binding between ALS and penoxsulam and then cause penoxsulam resistance.The molecular docking experiment provides theoretical evidence that Phe-206-Leu confers resistance to penoxsulam.The insect cell SF9 were used to obtain the recombinant ALS protein and the ALS proteins were purified sequentially.Western-blot revealed a single band near 70 k Da,which was close to the expected size,indicating that the mutant and sensitive recombinant protein was successfully expressed.ALS catalytic activity in controls（without penoxsulam treatment）showed that the Phe-206-Leu mutation decreased the catalytic activity of ALS.The IC₅₀ values for the mutant and sensitive ALS proteins were 6.56μM and 0.81μM,respectively.Therefore,the in vitro sensitivity of the mutant ALS protein to penoxsulam was～eight-fold lower than that of the sensitive ALS protein.These results showed that the mutation（Phe-206-Leu）caused lower sensitivity of the ALS protein to penoxsulam,which explained the penoxsulam resistance of AXXZ-8 population.Sensitive and mutant ALS sequences from barnyardgrass were transferred into Arabidopsis by flower dipping method for sensitivity verification,wild type Arabidopsis thaliana（Col-0）was used as control.The results of Petri dish method showed that three genotypes Arabidopsis displayed varying degrees penoxsulam resistance levels:mutant Arabidopsis>sensitive Arabidopsis>Col-0.This indicated that the transformation of ALS gene from barnyardgrass would improve the penoxsulam resistance of Arabidopsis.More importantly,mutant Arabidopsis showed higher resistance to penoxsulam than sensitive Arabidopsis,indicating that amino acid mutation at position 206 of ALS gene led to penoxsulam resistance in Arabidopsis.The whole-plant bioassay under multiple doses was carried out by soil culture method,and it was further concluded that mutant Arabidopsis had about 4 times penoxsulam resistance higher compared with sensitive Arabidopsis.These results proved that the amino acid mutation at position 206 would lead to the resistance to penoxsulam at the whole plant level,and the new mutation was the target-site basis mechanisms for the penoxsulam resistance in AXXZ-8 population.Therefore,it was confirmed that the Phe-206-Leu mutation confers penoxsulam resistance.The Phe-206 is the ninth amino acid residue reported to confer resistance to ALS herbicides in weeds.2.Potential genes that might be involved in the metabolic resistance to penoxsulam in AXXZ-6 population were excavatedCytochrome P450 inhibitors（PBO and malathion）and glutathione S-transferases（GST）inhibitor NBD-Cl could significantly decrease the penoxsulam resistance of AXXZ-6population,indicating that metabolic resistance mechanism mediated by P450 and GST might also be involved in the penoxsulam resistance in the AXXZ-6 population.Further,through transcriptome sequencing,802 genes expressed after penoxsulam treatment were excavated.These genes belong to eight reported metabolic resistance related families,of which 696 genes were differential expression genes.By q RT-PCR verification,it was found that the expression of 12 P450s,1 esterase,6 peroxidases,3 oxidases,2 hydrolases,3 GSTs and 3 ABC transporters genes in AXXZ-6 population were still significantly higher than that in sensitive population.These genes might be involved in the metabolic resistance of penoxsulam in barnyardgrass.The expression patterns of these 30 genes under the treatment of three other herbicides（pyroxsulam,metamifop and quinclorac）were measured.Under the pyroxsulam,metamifop and quinclorac treatment,14,11 and 5 genes were overexpressed,respectively.Of note,two P450s,EC＿v6.g088422（CYP74B2）and EC＿v6.g045480（CYP78A9）,1esterase,EC＿v6.g099076（palmitoyl-protein thioesterase 1）,and 1 hydrolase EC＿v6.g096321（hydroxyacylglutathione hydrolase）were overexpressed in all herbicide treatments.In addition,the expression level of EC＿v6.g098075（probable L-ascorbate peroxidase,APX6）was significantly increased by 27 and 19 times under the treatment of pyroxsulam and metamifop,respectively,and increased 5 times under the treatment of quinclorac without significant difference.These results suggested that these genes might participate in some common progresses in resistance to these four herbicides,but the detailed resistance mechanism needs to be further studied.We made function confirmations of four selected genes（EC＿v6.g024973,EC＿v6.g073605,EC＿v6.g099076 and EC＿v6.g096321）that might be related to the metabolic resistance of penoxsulam.The knockout and overexpression vectors of four metabolic enzyme genes in Nipponbare were constructed,and the overexpression and knockout mutants of the corresponding genes were obtained by rice tissue culture technology.However,the growth phenotypes of the knockout and overexpression mutants of the four genes were consistent under the treatment of penoxsulam,indicating that the four genes were not involved in the metabolic resistance of barnyardgrass to penoxsulam.The remaining genes need to be verified in subsequent studies.3.The APX6 gene in AXXZ-6 population was identified and proved to be involved in the non-target-site based mechanism of resistance to penoxsulam by removing hydrogen peroxideThe EC＿v6.g098075 in AXXZ-6 population was found to be overexpressed compared with the sensitive population under the treatment of four herbicides.Through the evolutionary tree analysis of APX6 gene in gramineous plants,the EC＿v6.g098075 was determined to be the APX6 gene in barnyardgrass.Further,the functional verification of the APX6 in herbicide sensitivity were made by the same methods as previous described.The herbicide phenotype of T2 generation rice mutants were verified,and the Nipponbare rice was used as the control.The results showed that the APX6 overexpression mutant and knockout mutant displayed different sensitivities to penoxsulam.The GR₅₀ of the three genotypes were 416.17,147.23,and 71.72 g a.i.ha^-1for the overexpression mutant,Nipponbare,and knockout mutant,respectively.The overexpression mutant and knockout mutant showed 2.83-fold and 0.49-fold penoxsulam resistance,respectively,compared with Nipponbare,indicating that the APX6 could change the penoxsulam resistance.After treatment with 480 g a.i.ha^-1 penoxsualm for 72 hours,penoxsulam residues in the three genotypes were close and displayed no significant differences(349.18,318.41,and 336.50μg m L^-1 for Nipponbare,overexpression mutant,and knockout mutant,respectively).The results showed that APX6 was not involved in the metabolism of penoxsulam in rice.According to the function of APX family that removing hydrogen peroxide（H₂O₂）,the H₂O₂ content was measured.Without herbicide treatment,H₂O₂ content displayed no difference at a low level(approximately 28μmol L^-1 g fresh weight),whereas at 72 h after herbicide treatment,H₂O₂ content in the three rice genotypes was induced to a high level.After herbicide treatment,it was found that H₂O₂ content in the overexpression mutant was the lowest(62.49μmol L^-1 g^-1),and in Nipponbare was moderate(95.97μmol L^-1 g^-1)in the knockout mutant was the highest(142.65μmol L^-1 g^-1)respectively,with significant difference.This showed that overexpression of APX6 could accelerate the removal of H₂O₂ induced by penoxsulam treatment,protecting rice from oxidative stress and causing resistance to herbicide,while the knockout of APX6 decreased the ability of removal of H₂O₂,causing rice sufferring oxidative stress and being sensitive to penoxsulam.Simultaneously,APX6 was also generally involved in the resistance to pyroxsulam,metamifop,quinclorac and florpyrauxifen-benzyl,however,APX6 had little effect on the sensitivity to cyhalofop-butyl.These results showed that APX6 could scavenge H₂O₂ produced by plants under herbicide stress at a varying degree and participate in herbicide resistance,explaining the non-target-site resistance of AXXZ-6 population to penoxsulam from the perspective of antioxidative stress.