Study On The Regulating Mechanism Of Ascorbate Concentration In Apple Leaves With Decreased Sorbitol Or Sucrose Synthesis And Its Relationship With GPP Expression

Ascorbic acid(AsA),also known as Vitamin C(Vitamin C),is synthesized by the derivation from hexose in plant cells.The photosynthetic carbohydrates exist mainly in the form of sorbitol and sucrose in the apple leaves.To explore the relationship between sorbitol/sucrose synthesis and ascorbate concentration in apple leaves.We investigated the changes of ascorbate concentration and its synthesis in transgenic apple leaves inhibited sorbitol or sucrose synthesis,and then a candidate gene GPP was transferred into Arabidopsis thaliana and apple to confirm its role in regulating ascorbate biosynthesis.The effect of GPP overexpression on ascorbate and sugar concentration was further analyzed.The main results are as follows:1.The A6RP interfered transgenic apple lines showed decreased concentration of sorbitol and increased concentration of sucrose in leaves.The SPS interfered transgenic apple lines showed the opposite situation.At the same time,both transgenic lines showed significantly increased inositol concentration.The determination of ascorbate and glutathione(GSH)concentration in leaves revealed that the total ascorbate and reduced ascorbate were significantly increased compared with the wild type,with an increase of 27%-36%.For GSH,the decrease of sorbitol synthesis increased the concentration of GSH in apple leaves,while the inhibition of sucrose synthesis had no significant influence.The two kind of transgenic apple lines showed higher gene expression level of GPP and lower gene expression level of APX.The enzyme activities of GLDH,a key enzyme in ascorbate synthesis,were also significantly increased in the transgenic apple lines.The enzyme activities of MDHAR,DHAR and GR were significantly reduced in the A6RP interfered transgenic apple lines.These results indicate that the increase of ascorbate concentration in transgenic apples may be related to upregulation of GPP genes and increased concentration of myo-inositol.2.In order to find the relationship of GPP expression with ascorbate concentration and myo-inositol,MdGPP was cloned from c DNA of “Gala” apple.By transforming this gene in wild-type and mutant Arabidopsis vtc4,we found that the expression of MdGPP in vtc4 restored the growth inhibition and ascorbate concentration.Overexpression of MdGPP in wild-type Arabidopsis increased the concentration of ascorbate by 30%.Transgenic Arabidopsis lines showed higher gene expression level of GME and Gal DH.Meanwhile,Overexpression of MdGPP in Arabidopsis reduced fructose and sucrose concentration in leaves,but without significant effect on inositol.These results indicated that the MdGPP gene can regulate ascorbate synthesis in Arabidopsis.3.In order to further verify the relationship between GPP and ascorbate concentration in apple,we obtained a GPP overexpression transformant apple line through Agrobacterium-mediated transformation method.The GPP gene expression level was increased by 27 times compared with wild type.The concentration of ascorbate in seedling leaves increased by about 10.5%.At the same time,the concentration of GSH in the transgenic apple leaves was significantly reduced,and the ratio of GSH/T-GSH was reduced by about 50%.Overexpression of GPP also increased GGP,MDHAR and DHAR expression levels in seedlings.The determination of sugar concentration in transgenic seedling leaves showed that the sucrose and sorbitol concentration in the transgenic lines decreased by 50%,while the fructose and inositol concentration increased.The above results showed that the synthesis of ascorbate regulated by the GPP gene in apple leaves is related to carbohydrate metabolism.