Function Study Of Apple Fructokinase Gene MdFRK2 In Regulating Sugar Metabolism

In plants,soluble sugars,including sucrose(Suc),glucose(Glu)and fructose(Fru),not only play as signaling molecule regulating the expression of tens of thousands of genes,affecting plant growth,development and resistance to adversity,but also regulate intracellular osmotic potential,turgor and redox potential,and the types and composition of soluble sugars also affect the quality of fruits such as apple fruit.Unlike most higher plant species where sucrose is the end product of photosynthesis,in Rosaceae apple trees,sorbitol is the primary end-product of photosynthesis,and almost all of sorbitol is converted to fructose in sink cells.Therefore,apples have higher fructose utilization than other plants.It is of great significance to elucidate the regulation mechanism of fructose metabolism and utilization in apple for improving fruit quality and regulating growth and development.In this paper,fructokinase gene MdFRK2,a highly expressed fructose metabolism-related gene in sink cells,was cloned from the apple genome,and its expression and catalytic properties were studied.We also generated MdFRK2-overexpression lines and investigate its function in carbohydrate metabolism.Some main results have been achieved as follows:1.Twelve FRK homologous genes were identified in apple genome,and phylogenetic analysis confirmed that MdFRK2 clusters with tomato high affinity fructokinase gene LeFRK2.The ORF of MdFRK2 is a complete 990 bp of nucleotides encoding a fructokinase gene of 330 amino acids.It contains the characteristics of pfkB family and FRK-specific domain,including sugar-binding domain and ATP-binding domain.Expression analysis showed MdFRK2 gene was mainly localized in cytoplasm,and it’s transcript was extremely high in metabolically active tissues such as shoot tips and young fruit,negatively correlated with fructose content during fruit development.In order to study its catalytic properties,MdFRK1 and MdFRK2 proteins expressed heterologously in E.coli were extracted and purified.Activity analysis showed that they had high substrate specificity for fructose and their activity was inhibited by feedback of high concentration of fructose.Analyses of heterologously expressed proteins revealed that MdFRK2 protein had a higher affinity for fructose than MdFRK1 protein with Km being 0.1 and 0.62 mM,respectively.The two proteins,however,exhibited similar Vmax values with their activities significantly inhibited by high concentrations of fructose.MdFRK2 protein has much higher affinity for fructose than MdFRK1 protein.These results indicate that the high utilization of fructose in apple is related to the high expression and catalytic activity of MdFRK2.2.To investigate the functions of MdFRK2 gene in apple(Malus domestica)fructose concentration and carbohydrate metabolism,we constructed plant overexpression vector and transformed ‘gala’ apple by Agrobacterium tumefaciens-mediated method.We identified three over-expression transgenic lines through molecular and enzyme acticity characterizations,which exhibited evident increase in the MdFRK2 transcript and FRK2 protein expression compared to the WT plants.The growth performance and net photosynthetic rate did not differ between the transgenic and WT plants grown in incubator condition.Increased FRK activity not only resulted in decreased fructose concentration,but also in a decrease in sucrose and glucose content.Enzyme activity analysis showed that enzyme involved in sorbitol synthesis(A6PR)and the degradation pathway(SDH)were significantly up-regulated in transgenic lines whereas those involved in sucrose synthesis(SPS)and other degradation processes(SUSY/NINV)were down-regulated.These results support the view that MdFRK2 plays an important role in regulating sugar metabolism in Rosaceae plants.At the same time,studies on MdFRK2 overexpression transgenic tomato plants also showed that both MdFRK2 transcript and FRK2 protein expression increased significantly,and fructose,glucose and sucrose concentrations also decreased.3.Changes of sugar concentration in MdFRK2 overexpression transgenic apple plants were contrary to those of plants treated with high concentrations of melatonin.Thus,it seems to be a link between FRK2 expression and melatonin application.To test this hypothesis,transgenic apple lines overexpressing MdFRK2 were used to investigate the role of MdFRK2 in regulating sugar concentrations and plant growth under exogenous melatonin application.In our study,high concentration melatonin inhibited MdFRK2 transcript and FRK activity,resulted in significantly increased fructose,glucose and sucrose concentration and stunted growth phenotype in wide-type apple plants.MdFRK2 promoter transiently expressed in tobacco leaves further supported that the expression of MdFRK2 could be inhibited by high concentrations exogenous melatonin.However,soluble fructose,glucose and sucrose concentrations increased less in transgenic apple plants after applying high does exogenous melatonin;MdFRK2-overexpressing plants exhibited slightly inhibited growth as compared to wild-type apple plants under melatonin treatment.Taken together,these results demonstrate the involvement of MdFRK2 gene in melatonin-induced sugar accumulation and growth inhibition.4.Soluble sugar concentrations were determined again after the transgenic apple plant were grown in the greenhouse for one year.Interestingly,the concentrations of fructose,sucrose and glucose in mature leaves were significantly increased when compared with the wild type plants.Further analysis of molecular and enzyme acticity characterizations shows that MdFRK2 expression increased by 24-45 times while the activity of FRK decreased in the mature leaves of these one-year old transgenic plants.Alao,the expression of key genes(such as MdSDH1,MdSPS1,MdSUSY2)and activities of enzyme(such as SDH,SPS,SUSY)involved in sugar metabolism showed a completely opposite tendency to that of three-month apple plants grown in chamber.These results indicated that MdFRK2 protein may have post-translational modification,and further confirmed that FRK activity determined by MdFRK2 gene regulates intracellular sugar signaling and metabolism.We studied the differential gene expression analysis using RNA-sequencing technology.477 DEGs were observed in the transgenic apple plant were grown in the greenhouse,41 genes were functionally annotated in the protein pathway.These information will provide a set of fundamental data about the potential regulation mechanism of fructose signal in apple.