Synthesis And Metabolism Of L-ascorbic Acid In Chinese Jujube And Wild Jujube

L-ascorbic acid(As A), also named vitamin C, is a highly abundant and essential metabolite for plants and animals. As A is an essential component of human nutrition, but humans lack the ability to synthesize As A and and plants provide the major source of dietary vitamin C for humans. Chinese jujube(Ziziphus jujuba Mill.) and wild jujube belong to the genus Ziziphus(Rhamnaceae) and their fruits are rich in vitamin C. In this study, three jujube cultivars(‘Jinsixiaozao’, ‘Zanhuangdazao’, ‘Dongzao’) and one type of wild jujube(‘Xingtai0608’) were used as materials, the As A content, the related-genes’ expression and activity of enzymes involved in As A biosynthesis and recycling during fruit development were systematically investgated and analyzed. The results were as follows.1. As A contents in fruits of Chinese jujube and wild jujube were measured by HPLC.The results showed that changes of As A concentration in fruits of ‘Xingtai0608’,‘Jinsixiaozao’ and ‘Dongzao’ during fruit development were quite similar, i.e., As A content of their fruits decreased rapidly in white ripening stage or half-red ripening stage and then increased in whole-red ripening stage. As A concentration in ‘Zanghuangdazao’ gradually decline as fruit ripening. As A concentration in whole-red ripening stage of ‘Xingtai 0608’,‘Jinsixiaozao’, ‘Dongzao’ and ‘Zanhuangdazao’ was 1330 mg/l00 g FW, 705 mg/l00 g FW,481 mg/l00 g FW, 444 mg/l00 g FW, respectively.2. Eight genes related to ascorbic acid metabolism were isolated from jujube by homologous gene cloning method. GDP-D-mannose pyrophosphorylase(Zj GMP) has an open reading frame of 1086 bp, encoding 361 amino acids and its Gen Bank accession number KJ934995; GDP-Man-3’,5’-epimerase(Zj GME) has an ORF 1131 bp, encoding376 amino acids; GDP-L-galactose phosphorylase(Zj GGP) has an ORF of 1338 bp,encoding 445 amino acids; L-galactose-1-P phosphatase(Zj GPP, KJ739593) has an ORF of 813 bp, encoding 270 amino acids; L-galactose dehydrogenase(Zj GLDH) has an ORF of1785 bp, encoding 594 amino acids. Ascorbate oxidase(Zj AO) has an ORF of l776 bp,encoding 591 amino acids; Dhydroascorbate reductase(Zj DHAR) has an ORF 639 bp,encoding 212 amino acids; Monodehy droascorbate reductase(Zj MDHAR) has an open reading frame of 1086 bp, encoding 361 amino acids; Further analysis indicated that predicted amino acid sequences of these genes showed high homology with their respective gene in other plants.3. To investigate the molecular mechanisms of ascorbate accumulation in Chinese jujube and wild jujube. the expression of 9 related genes were analyzed by q RT- PCR. The expression pattern of GLDH showed the highest similarity with the variation of As A concentration during fruit ripening.4. The activities of GLDH, APX, AO, MDHAR and DHAR during fruit development were analyzed by spectrophotometry. The activities of these five enzymes in wild jujube were higher than that in jujube, and the higher activities of the enzymes invloved in As A synthesis and recycling in wild jujube is probably contribute to its higher As A content. The changes in GLDH activities among ‘Xingtai0608’, ‘Jinsixiaozao’ and ‘Zanhuangdazao’were quite similar during fruit development. The GLDH activities maintained a very low level in young-fruit stage and increased as fruit ripening. The highest activity appeared on pre-white stage and slightly decreases as fruit ripening. The GLDH activities in ‘Dongzao’reached the highest level in white stage, followed by a decrease in half-red ripening stage and then a slightly increase at the whole-ripening stage. APX and AO activities in four genotypes gradually increased as fruit ripening and APX activity was higher than AO activity. The MDHAR activity of ‘Xingtai 0608’ and ‘Dongzao’ exhibited ‘M’ growth trend during fruit development. The highest MDHAR activity of ‘Jinsixiaozao’ appeared on half-red stage and slightly decrease as fruit ripening. The MDHAR activity of‘Zanhuangdazao’ reached higher levels in young fruit stage and white ripening stage.Compared to MDHAR activity, the change of DHAR activity in four genotypes is relatively stable.In conclusion, the high level of As A accumulation in Chinese jujube and wild jujube during fruit development is probably due to a mixture contribution from As A biosynthesis and As A recycling. The L-galactose pathway takes the dominant role in As A biosynthesis,Zj GLDH and Zj MDHAR are probably the crucial genes for As A biosynthesis and recycling,respectively. The decreasing of As A content during the late fruit ripening stage in Chinese jujube and wild jujube was due to the higher activities of APX and AO.