Function Analysis Of M~6A Reader MhYTP2 In Response To Hypoxic Stress In Apple

China is the world’s largest producer of apples,but its production is often affected by waterlogging.The Loess Plateau is the largest apple producing region in China,and although this region is arid and water-scarce,seasonal rainfall is concentrated and predominant during the apple growth and harvest period,resulting in flooding and inundation,causing plant root hypoxia and severe losses.In addition,clay soils and soil slumping are also prone to soil hypoxia.m⁶A reader are involved in various stages of plant stress response and growth and development.Previous studies by our group have shown thatm⁶A reader MhYTP2 is involved in abiotic stress responses such as low oxygen,drought,high salt,high and low temperature.Therefore,in this paper,the overexpression transgenes of MhYTP2 and’GL3’wild-type apples were used as materials to study the function and mechanism of action of MhYTP2 in hypoxic stress to provide a theoretical basis for the improvement of apple resistance to hypoxia,which is of great significance for the development of apple industry in China.The main results obtained are as follows:1.Analysis of the resistance function of MhYTP2 under hypoxic stress.Overexpression of MhYTP2 increased the expression of genes related to ethylene synthesis and its response pathway,increased the activities of ADH,LDH and PDC,key enzymes of anaerobic respiration pathway,increased chlorophyll content of plant leaves,enhanced photosynthesis,enhanced root adaptation,reduced the accumulation of reactive oxygen species and damage to biofilm system,and thus improved the resistance of apple plants to hypoxic stress.2.Identification and analysis of the regulatory role of MhYTP2 hypoxia-related target m RNAs.The m RNA of a gene related to the ethylene signalling pathway was identified as MdERF54 in previous m⁶A-seq sequencing results in the laboratory,and RIP-Seq and EMSA results demonstrated that MhYTP2 binds to MdERF54 m RNA with m⁶A modification.to investigate the regulatory role of MhYTP2 on MdERF54,the overexpression of To investigate the regulatory effect of MhYTP2 on MdERF54,the degradation rate of MdERF54 m RNA was determined in MhYTP2 transgenic strain OE2and wild-type WT plants,and the results showed that MhYTP2 reduced the degradation rate of MdERF54 transcript.3.Function analysis of MdERF54 resistance under hypoxic stress.To investigate the mechanism of action of MhYTP2 under hypoxic stress,root transgenic apple materials WT-MdERF54^OE,WT-MdERF54^Ri,OE2,OE2-MdERF54^OE,OE2-MdERF54^Ri were obtained on the basis of WT and OE2 that overexpressed and interfered with MdERF54.The above transgenic materials,WT,and WT-2300 and WT-PK7 plants with transformed empty vectors were subjected to hypoxic treatment,and it was found that the roots of WT-MdERF54^OE plants exhibited higher ADH,LDH and PDC enzyme activities,as well as more ACC content and expression of ethylene response-related genes than the roots of WT plants,while the above indicators were restored to WT levels in OE2-MdERF54^Ri plants.This indicates that MdERF54 can improve the hypoxia resistance of apple plants and that MhYTP2 improves the resistance of apple to hypoxia stress by binding and regulating the stability of MdERF54 m RNA.