Functional Analysis Of MdNAT1 And MdNAT7 Transporters In Malus Under Drought And Salinity Stresses

Nucleobase-Ascorbate transporters(NAT)exist extensively in fungal,protozoan,bacterial,plant and mammalian,most characterized NATs proteins were shown to transport xanthine.In plants,only the maize leaf permease 1(Lpe 1)and NAT3 and NAT12 from Arabidopsis have been reported to transport xanthine,adenine,guanine,hypoxanthine,uracil,and uric acid.Furthermore,little is known about NAT family in woody species such as Malus domestica(apple).By using the published apple genome database,we identified all the NAT genes in apple,overview of this gene family focused on chromosomal distribution,structure and localization,motifs and phylogenies.These genes displayed different expression patterns in various tissues.The expression of some MdNATs was up-regulated during the fruit maturation and some genes responded to drought or salt stress.Those suggested the function of NAT family in tissue development,fruit maturation,and plant adaptations to adverse growing environments.Then we conducted intensive studies on MdNAT1 and MdNAT7.The main results of this study are documented as follows:1.Fourteen members of NAT family in Malus were identified and cloned.They are distributed on 11 chromosomes of apple.MdNAT proteins contain NAT conservative domain structure.Phylogenetic analysis showed that NAT family of plants and animals can be divided into four Clusters,the same as other plant NAT genes,NAT genes in Malus mainly distributed in three Clusters.Tissue specific expression analysis showed that MdNATs gene were expressed in stem tip,mature leaves,young apple fruits and mature apple fruits.The expression analysis of MdNATs genes was up-regulated in fruit mature,drought and salt stresses,indicating that MdNATs may participate in apple fruit maturity and resistance against abiotic stresses.MdNAT1 and MdNAT7 tended to show an increasing expression profile during the fruits maturation,suggesting the two genes regulated the fruit development.Under drought stress,the transcription of MdNAT7 increased almost 16 folds,while MdNAT1 did not show obvious variation.MdNAT1 and MdNAT7 expression were significantly enhanced under salt stress.Therefore,we chose these two genes for our further study.2.MdNAT1 expresses in stem tip,leaves and fruits.The subcellular localization showed that it located to the cell membrane.There was no significant difference between wild-type plants and MdNAT1 transgenic GL3(‘Royal Gala’)apple under drought stress.But under salt stress,the resistance of Oe-transgenic plants showed stronger resistance than that of the WT.The transgenic apples showed lower leaf conductivity,less accumulation of malondialdehyde and the reactive oxygen species(ROS);higher chlorophyll content and enzyme activity in active oxygen removal activity.Transgenic plants showed less content of Na~+but more K~+,the expression of salt resistance related genes,such as MdSOS1,MdAKT1,MdKAE2 and MdNHX4,were higher than that in WT.Oe-MdNAT1 plants had higher salt resistance which may due to the improved ablity of removing ROS and maintaining ion homeostasis.3.The expression of MdNAT7 gene was induced by drought and salt stress.MdNAT7 was located to the cell membrane.MdNAT7 Oe-transgenic apple plants showed stronger resistance under drought stress,with smaller damage index,delaying the damage on chlorophyll and higher photosynthetic capacity than WT under drought stress.MdNAT7 transgenic plants improved the antioxidant enzyme activity to remove excess ROS.MdNAT7 interference plants showed the opposite results.Overexpression(Oe)and RNAi(Ri)of MdNAT7 improved and decreased apple tolerance to salinity stress,respectively.Under salinity stress,leaves from the Oe lines had higher ROS scavenging ability and contained less Na~+but more K~+than did WT samples,while the Ri lines showed an opposite trend.MdNAT7 positively regulated the expression of ascorbate?glutathione cycle-related and Na~+/K~+transporters genes under salt stress.Our results demonstrate that MdNAT7 enhanced apple salt tolerance by activating ascorbate?glutathione cycle and maintaining ion homeostasis.