Mechanism Of MdAGO1.2 And MdAGO4.1 In Regulating Apple Salt And Drought Stress

Apple is one of the main fruits in the world,and China is the largest apple producer with the largest cultivation area and production.The Loess Plateau is the largest and best apple producing area in China.However,the annual precipitation in this region is low and unevenly distributed throughout the annual cycle,therefore long-term moderate drought is the main factor that restricts apple production and the sustainable development of the industry.In addition,soil salinization is one of the major environmental factors in some apple production areas.The Argonaute（AGO）family proteins are core effectors of RNAi pathways in eukaryotes,involved in plant growth and stress response widely.In previous research,we found most AGO family members could response to drought,salt,ABA,and low temperature stresses.MdAGO1.2 from cluster I and MdAGO4.1 from cluster III could be significantly induced by salt and drought.This research focused on exploring the function and mechanisms of MdAGO1.2 and MdAGO4.1 under salt and drought stress.The main results are as follows:1.MdAGO1.2 positively regulated salt stress tolerance in apple.Leaves exhibited the highest expression levels and its expression was significantly induced by salt stress.We created MdAGO1.2-overexpressing（OE）Arabidopsis,MdAGO1.2-overexpressing（OE）apple calli and MdAGO1.2-RNAi apple plants.Overexpression of MdAGO1.2 enhanced the salt tolerance of transgenic Arabidopsis and apple calli.MdAGO1.2-RNAi apple plants were more sensitive to salt stress.Stress-related indices showed that MdAGO1.2 positively regulates salt tolerance by maintaining photosynthesis,improving antioxidant enzyme activities,inhibiting ROS accumulation and Na⁺accumulation,promoting the accumulation of K⁺and polyamines.2.MdAGO1.2 positively regulated drought stress tolerance and water use efficiency in apple.MdAGO1.2 overexpression improved drought tolerance in transgenic Arabidopsis.Overexpression of MdAGO1.2 in apple calli enhanced the tolerance of osmotic stress.MdAGO1.2-RNAi apple plants displayed reduced drought tolerance.After short-term drought treatment,the REL,MDA content,accumulation of O₂^-and H₂O₂ in MdAGO1.2-RNAi plants were significantly higher than those in GL-3,while antioxidant enzyme activities,chlorophyll content,photosynthetic rate were lower than GL-3.Chloroplast damage was also more severe in MdAGO1.2-RNAi plants.After long-term moderate drought treatment,the water use efficiency was significantly decresed in MdAGO1.2-RNAi apple plants.The result of physiological indexes showed that the photosynthetic capacity of MdAGO1.2-RNAi apple plants was lower than GL-3,and the content of starch,glucose and soluble sugar were also lower,which resulted in the less biomass accumulation in MdAGO1.2-RNAi apple plants.MdAGO1.2 could interact with ferredoxins family gene（Md Fd1）by protein interaction screening.Further investigation showed that Md Fd1 participates in photosynthetic electron transfer,and MdAGO1.2 could enhance its electron transfer ability.These results indicated that MdAGO1.2 can affect photosynthesis,biomass accumulation and water use efficiency by interacting with Md Fd1.3.MdAGO4.1 positively regulated salt stress tolerance by improving root development in apple.The expression of MdAGO4.1 was significantly induced by salt stress.We created MdAGO4.1-OE Arabidopsis,MdAGO4.1-OE apple calli,MdAGO4.1-RNAi apple plants and MdAGO4.1-OE apple roots.Overexpression of MdAGO4.1 enhanced the salt tolerance in Arabidopsis and apple calli.MdAGO4.1-RNAi apple plants were more sensitive to salt stress,which mainly showed the higher REL,MDA content,ROS accumulation and Na⁺content,the lower photosynthetic capacity,activities of antioxidant enzymes,and K⁺content.Further salt treatment carried on MdAGO4.1-OE apple roots,The results showed that less accumulation of O₂^-and H₂O₂,higher antioxidant enzymes activity in MdAGO4.1-OE apple roots.Analysis of root growth showed that the development of MdAGO4.1-RNAi roots was more severely inhibited by salt stress,while MdAGO4.1-OE roots development was better than wild type.Based on the transcriptional activation activity of MdAGO4.1,we screened for downstream target genes of MdAGO4.1.The result showed that MdAGO4.1 could directly bind to the promoter of Md YUCCA8 and Md YUCCA10 to promote their transcription,which involved in auxin synthesis.These results indicate that MdAGO4.1 may modulate root architecture and salt tolerance by regulating Md YUCCA8 and Md YUCCA10 expression.4.MdAGO4.1 regulated the expression of antioxidant enzyme genes and autophagy gene to enhance drought resistance in apple.MdAGO4.1 overexpression improved drought tolerance in transgenic Arabidopsis.Overexpression of MdAGO4.1 in apple calli also enhanced the tolerance of osmotic stress.After short-term drought treatment,the REL and accumulation of ROS were higher in MdAGO4.1-RNAi plants,while the RWC,net photosynthetic rate and antioxidant enzyme activities were significantly decreased.These results indicated that interference with MdAGO4.1 reduced drought tolerance in apple plants.Expression analysis showed that the transcription level of antioxidant enzyme encoding genes were significantly decreased in MdAGO4.1-RNAi plants after drought treatment.MdAGO4.1could directly bind to the promoters of Md POD1,Md APX1,and Md GPX4 by transcription regulation analysis,and MdAGO4.1 promotes their expression.We obtained Md GPX4-OE Arabidopsis and apple calli.After drought and mannitol treatment with transgenic Arabidopsis and apple calli,it was found that overexpression of Md GPX4 enhanced the tolerance of transgenic materials.These results indicate that MdAGO4.1 can improve antioxidant enzyme activity and drought tolerance by promoting the expression of antioxidant enzyme genes.After long-term moderate drought treatment,it was found that the stomatal aperture and photosynthetic rate decreased,root development and root activity were inhibited,resulting in significantly lower biomass accumulation and water use efficiency in MdAGO4.1-RNAi plants than in wild type.In addition,MdAGO4.1 could bind to the promoters of Md ATG8i by transcription regulation analysis,and MdAGO4.1 promotes its expression.The autophagic activity of MdAGO4.1-RNAi plants was significantly lower than wild type under drought stress.These results indicate that MdAGO4.1 could affect drought tolerance and water use efficiency through autophagy pathway.