Functional Analysis Of YTH Domain Containing RNA Binding Protein Genes MhYTP1 And MhYTP2 In Malus

RNA binding proteins participate in many plant stress responses and development processes.The YT521-B homology(YTH)domain containing RNA binding domain containing protein(YTP)was first found in rat(Rattus norvegicus)and was oxygen-deficient stress related.Unlike their characterizations in yeast and animals,the functions of YTPs in plants are less clear.The first plant YTP,cleavage and polyadenylation specificity factor 30(CPSF30),was discovered in Arabidopsis,where it participates in oxidative stress and other stress responses.Resesrchers also identified YTP gene family members in Arabidopsis and analyzed their functions in abiotic stress responses.Fruits of apple(Malus domestica Borkh.)are some of the most widely cultivated and consumed in the world.But its development and quality are effected by numerous stresses and developmental conditions.Additional researches on responses of apple YTPs to stresses and on their functions in apple development processes will help breeders improve apple resistance and fruit quality.Here,we firstly identified Malus YTPs family members and investigated their expression pattern to leaf senescence and various stresses.Then,we cloned two homologous YTP family members in M.hupehensis,i.e.MhYTP1 and MhYTP2.After that,functions of MhYTP1 and MhYTP2 are investigated in abiotic stress response,in biotic stress responses,in leaf senescence,and in fruit ripening.Finally,we discovered several significant results shown below:1.There are 15 members in Malus YTP family and we cloned two homologous YTP family members in M.hupehensis,i.e.MhYTP1 and MhYTP2.We were able to identify 15 apple YTPs that were expressed in various tissues,especially senescing leaves.Expression analysis showed that these YTPs also responded to several abiotic stresses.Besides,we cloned two homologous YTP family members in M.hupehensis,MhYTP1 and MhYTP2.MhYTP1 and MhYTP2 are localized to the entire cell and have RNA binding activities.Furthermore,we also cloned their promoter regions.There are many stress and development related cis-elements,indicating their functions in apple stress responses and development processes.According to the results of qRT-PCR and GUS staining experiments,MhYTP1 are mainly expressed in stems and MhYTP2 are mainly expressed in roots.Although MhYTP1 and MhYTP2 are homologous genes,they have difference functions in apple stressresponses and development processes because of different cis-elements in their peomoter regions and their different tissue localizations.2.Overexpressions of MhYTP1 or MhYTP2 in Arabidopsis and Malus can affect plant responses to exogenous applied phytohormones and various stresses.Both of MhYTP1 and MhYTP2 can be induced by exogenous applied abscisic acid(ABA).Moreover,MhYTP1 or MhYTP2 overexpression Arabidopsis and ‘GL-3’(‘Royal Gala’)plants are more resistant to ABA.ABA siganaling play important roles in drought responses and MhYTP1 and MhYTP2 can also be induced by water deficient.MhYTP1 or MhYTP2 overexpressions alter stomatal density and openings and make plants more resistant to drought.Then,molecular machians of their responses to drought were identified.According to results of yeast one hybrid and yeast two hybrid,we found that MhYTP1 and MhYTP2 participate in drought response in different ways.MhYTP1 are mainly expressed in stems and can be regulated by MhRD22,the drought marker gene,via interacting with its promoter;on the other hand,MhYTP2 are mainly expressied in roots and can interact with MhSCE1,ABA siganliang related protein,and then participate in drought responses.Besides,MhYTP1 and MhYTP2 also participate in responses to warter-logging,low/high temperature,high salinity and nutrition deficiency abiotic stresses and play roles in defences to D.mali infection.MhYTP1 or MhYTP2 overexpression make plants more resistant to water-logging,chilling,and nutrition deficiency and make plants more seneitive to D.mali infection via affecting JA and SA siganalings.But their functions can not be confirmed in high temperature and high salinity stress responses.3.Functions of MhYTP1 and MhYTP2 in leaf senscenc and fruit ripening.Expressions of most Malus YTP gene family memebers can be induced by leaf natural senescence,so are MhYTP1 and MhYTP2.Overexpression of MhYTP1 or MhYTP2 promotes detached leaf senescence in Arabidopsis.When MhYTP1 and MhYTP2 were over-expressed in GL-3 plants,those transgenic lines also showed earlier yellowing and lower chlorophyll concentrations when compared with the WT plants during dark-induced leaf senescence and leaf natural senscence.Fruit ripening can be regarded as a senescence process of fruit.When MhYTP1 and MhYTP2 were over-expressed in tomato plants,ripening periods were longer for WT plants than that for the overexpression plants.At each corresponding stage during maturation,carotenoid concentrations were higher and expression of carotenoid biosynthesis genes,ethylene biosynthesis genes,and ethylene-responsive genes were enhanced in the transgenic fruits when compared with the WT.All of these results suggest that overexpression of MhYTP1 or MhYTP2 could promote fruit ripening.In addition,we found that MhYTP2 interacts with an ethylene biosynthesis-related protein,acireductone dioxygenase 4(MhARD4),after yeast two hybrid screening.ARD4 participates in Met cycle and Met cycle has relationsinps with ethylene biosynthesis.Therefore,MhYTP1 and MhYTP2 affect ethylene biosynthesis and thus promote leaf senescence and shorten the time to maturity for tomato fruits.