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

China is one of the biggest apple output countries in the world,and the Loess Plateau is one of the main apple producing areas in China.However,the yield and quality of apple are affected seriously by low temperature stress.N~6-methyladenine(m~6A)is a widespread type of RNA epigenetic modification in eukaryotes.It is recognized by the m~6A reader and is involved in various stages of plant growth and development and stress response.Identifying the m~6A binding characteristics of apple YTH domain protein MhYTP2(Malus hupehensis YTH domain containing RNA binding protein)and studying its function and mechanism in apple resistance to low temperature stress can provide theoretical basis for low temperature resistance and growth regulation of apple.In this study,we further studied the binding properties of MhYTP2 to m~6A modified RNA,the impact of MhYTP2 on m~6A modification level,and its function and mechanism of action in low temperature stress based on the identification of RNA binding properties of apple YTH domain protein MhYTP2 and its function in stress.The main results obtained were as follows:1.MhYTP2 has m~6A binding properties,improves the m~6A modification level of m RNA.The apple RNA binding protein MhYTP2 was proved to be an m~6A reader by the EMSA assay of RNA.MhYTP2 affects the expression of m~6A methylase and demethylase genes,and improves the overall m~6A modification level in plants.2.MhYTP2 affects m RNA stability and translation.m~6A was highly enriched near coding region(CDS)and 3’untranslated region(UTR),and the proportion of m~6A peak in CDS region was lower in OE-2 than in WT,but higher in UTR region,indicating that MhYTP2 affects the stability and translation of m~6A modified m RNA.HOMER analysis shows that the m~6A domain of apple m RNA is"URUAY".Combined analysis of m~6A-seq and RNA-seq showed that m~6A modification in the exon region of apple appeared to reduce m RNA stability,while m~6A modification in UTR was positively correlated with m RNA abundance.By ribosome profiling(Ribo-seq)analysis,it was found that MhYTP2overexpression improved the translation efficiency of m RNA.GO analysis for the different ribosome occupancy transcripts showed that the transcripts were enriched in the antioxidation system,suggesting the effect of MhYTP2 on apple antioxidant system,and then affected the stress tolerance of plants.3.MhYTP2 enhances low temperature resistance of apple plants.The observation and statistics of the dormancy phenology of apple seedlings with 35S::MhYTP2 transgenic lines and WT showed that 35S::MhYTP2 transgenic lines entered the dormancy stage earlier than WT,broke dormancy later,fell leaves earlier in autumn,the photosynthetic system tended to be unstable earlier,and nitrogen reflux earlier in leaves,suggesting that MhYTP2plays a role in apple dormancy and nutrient reflux.Therefore,it may affect plant resistance to low temperature.35S::MhYTP2 transgenic lines and WT seedlings were treated at low temperature,and it was found that the electrolyte leakage of transgenic plants was lower than WT,the content of anthocyanin was higher than WT,and the antioxidant system was more stable.The survival rate of transgenic plants treated with freezing injury was higher than that of WT.These results indicated that MhYTP2 had a positive effect on apple resistance to low temperature stress.4.MhYTP2 enhances low temperature resistance of apple plants by regulating MdRH20 and MdGRP2.(1)MhYTP2 binds to MdRH20 and MdGRP2 m RNA,affecting the variable splicing and nucleus export of MdRH20.The results of EMSA showed that MhYTP2 binds to the m RNA of m~6A modified low-temperature response-related RNA helicase MdRH20(RNA helicase)and cold-shock protein MdGRP2(glycine-rich protein).MhYTP2 affected the variable splicing of MdRH20.Subsequent studies were conducted on MdRH20transcripts MdRH20X1 and MdRH20X2,which were expressed significantly different from35S::MhYTP2 transgenic lines and WT and were induced to be expressed at low temperature.MhYTP2 affects the nucleus export of MdRH20X1 and MdRH20X2 m RNA at low temperature.MdRH20X1 and MdRH20X2 were genetically transformed into tomato,and Sl GRP2 was silenced in tomato to obtain transgenic tomato materials for low temperature function verification.The results showed that MhYTP2,by combining MdRH20 and MdGRP2,affects the variable splicing and nucleus export of MdRH20,and thus affects the low temperature resistance of apple plants.(2)MdRH20X1 and MdRH20X2 enhance the low temperature resistance of apple plants by regulating MdGRP2.In addition,the EMSA experiment found that MdRH20X1and MdRH20X2 can bind to and uncoil MdGRP2 m RNA,and then MhYTP2 interacts with MdRH20X1 and MdRH20X2 to promote the uncoil activity of MdRH20X1 and MdRH20X2.MdRPS27A(ubiquitin-40S ribosomal protein S27a)was recruited to facilitate the translation of MdGRP2 after disintegration.Therefore,MhYTP2 not only affects MdGRP2 through direct binding,but also indirectly affects MdGRP2 through binding to MdRH20,thus affecting the low temperature resistance of apple plants.(3)MhYTP2 enhances m RNA stability and translation efficiency of antioxidant system genes.The m RNA stability of MdPER3(peroxidase),MdPER42,MdPER47,MdAPX2(L-ascorbate peroxidase 2)and MdGDH1L(glutamate dehydrogenase 1-like)in35S::MhYTP2 transgenic line OE-2 was higher than that in WT.MhYTP2 directly regulates the translation efficiency of m~6A-modified m RNA,such as MdGDH1L.MhYTP2 may also regulate non-MhYTP2 targets,such as MdPER3、MdPER42、MdPER47、MdAPX2 m RNA by regulating transcription extension factors and translation initiation factors with m~6A modifications.In addition,MhYTP2 not only binds to the m RNA of translation factor MdRPS27A,but also interacts with MdRPS27A.After interfering with MdRPS27A in apple callus with overexpression of MhYTP2,it is found that MdGDH1L is no longer accumulated.MdGDH1L in apple callus interfering with MdRPS27A with overexpression of MhYTP2recovered to WT level,suggesting that MhYTP2 also regulates m RNA translation by directly binding to MdRPS27A m RNA and interacting with MdRPS27A.In conclusion,this study preliminarily identified the regulatory function of m~6A reader MhYTP2 on apple low temperature stress,and analyzed the mechanism of MhYTP2response to low temperature stress.These results can deepen the understanding of the mechanism of apple response to low temperature stress and provide theoretical basis for precision molecular breeding of apple response to low temperature stress.