Functional Analysis Of Two Apple Sucrose Nonfermenting-1-Related Kinase Genes MpSnRK2.10 And MdCIPK03 In Response To Abiotic Stress

In eukaryotic organisms,one of the major mechanisms for signal transduction is reversible protein phosphorylation,which is catalyzed by protein kinases and phosphatases.Sucrose nonfermenting-1-related kinase(SnRK)2 and SnRK3 subfamily members are plant-specific serine/threonine protein kinases.SnRK2 are the essential components and major positive regulators in ABA signaling pathway,and they also play critical functions in signaling and tolerance of osmotic stress in plants.SnRK3,also referred to as CBL-interacting protein kinases(CIPK),interacting with calcineurin B-like proteins(CBL),are key regulators of ion homeostasis required to cope with salt and nutrient stress.Apple(Malus domestica)is one of the most economically important fruit tree species in the world.The northwest Loess Plateau is the main apple producing area in China,but frequently occurred environmental stresses such as drought,salt and freezing limit the tree growth as well as fruit yield and quality.Functional study of apple SnRK2 and SnRK3 genes in response to abiotic stresses will be helpful for improving stress tolerance breeding of apple.In this study,we first performed a genome-wide identification and characterization of SnRK2 and SnRK3 gene family in apple,then examined their expression pattern under various abiotic stress treatments.After that,two representative genes from each family,MpSnRK2.10 and MdCIPK03 were selected for further investigation of their functions in response to abiotic stress.The main results are documented as follows.1.Genome-wide identification and expression profiling of the SnRK2 gene family in MalusThere are 14 complete putative nucleotide sequences encoding 12 deduced SnRK2 proteins within the apple genome.Chromosomal location and synteny analysis of the apple SnRK2 genes indicated that tandem and segmental duplications have likely contributed to the expansion and evolution of these genes.All 12 full-length coding sequences were confirmed by cloning from Malus prunifolia.Phylogenetic analysis showed that MpSnRK2 could be classified into four groups,and the sequences within each group present similar gene structure and motif compositions.Expression profiling of these genes presented differential patterns in various tissues.The transcript levels of some MpSnRK2 were up-regulated in leaves under drought,salinity,or ABA treatments.This result suggested their possible roles in plant response to abiotic stress.2.Overexpression of MpSnRK2.10 confers tolerance to drought and salt stress in transgenic plantsThe coding sequence of MpSnRK2.10 was overexpressed in Arabidopsis and apple to further investigate its potential function in abiotic stress response.The results showed that overexpression of this gene did not affect plant growth and development under normal conditions.However,the transgenic plants showed enhanced tolerance to drought and salt,as indicated by the amelioration in phenotype appearance and physiological indices related to stress damage.Additionally,MpSnRK2.10-overexpressed apple plants exhibited greater sensitivity to ABA in terms of ABA-induced growth inhibition.Moreover,the expression of stress marker genes including MdRAB18,MdRD22 and MdRD29 B was more strongly induced in transgenic apple plants than in the WT when they were subjected to ABA,salt and mannitol treatments.Taken together,these results indicated that overexpression of MpSnRK2.10 may result in an enhanced ABA signal output in response to stress,thus allow tolerance to drought and salt stress in transgenic plants.3.Genome-wide identification and expression profiling of the CIPK/SnRK3 gene family in appleA total of 44 complete putative nucleotide sequences encoding 32 deduced CIPK proteins were identified within the apple genome,and they were termed MdCIPK01 to MdCIPK32 according to the orders of chromosome location.Among these putative sequences,19 full-length coding sequences were verified by cloning from Malus domestica cv.Golden Delicious.Gene chromosomal location and synteny analysis showed that 10 pairs of MdCIPK are tandemly duplicated gens,while 11 pairs of MdCIPK are segmentally duplicated genes,indicating that tandem and segmental duplications have likely contributed to the expansion and evolution of MdCIPK gene family.Protein sequence characterization showed that the ATP binding site,activation loop,NAF and PPI motif are highly conserved in MdCIPK.Phylogenetic analysis showed that 12 and 20 MdCIPK belong to intron-rich and intron-less clade,respectively.The transcript levels of many MdCIPK were down-regulated in leaves in response to drought,salinity,ABA and cold treatments.However,one member of the family,MdCIPK03,was exclusively up-regulated under all current stress treatments,indicating its potential positive function in response to abiotic stress.4.Overexpression of MdCIPK03 confers tolerance to drought,salt and freezing stress in transgenic ArabidopsisThe coding sequence of MdCIPK03 was overexpressed in Arabidopsis to further investigate its potential function in response to abiotic stress.Under normal conditions,MdCIPK03-overexpressed Arabidopsis showed no difference with the WT in plant growth and development.However,the transgenic plants showed enhanced tolerance to drought,salt and freezing,as indicated by the amelioration in phenotype appearance and physiological indices related to stress damage.These results suggested that MdCIPK03 plays as a positive regulator in response to abiotic stress in apple.In addition,MdCIPK03 interacted with MdCBL1,MdCBL3,MdCBL4 and MdCBL5 in a yeast two-hybrid assay,indicating that the activity and function of MdCIPK03 were mediated by these calcium-binding proteins in vivo.