Analysis of ascorbic acid in jujube/sour jujube and identification of key function genes

Abstract：【Objective】Ascorbic acid (AsA) plays a positive role in cell activity and antioxidant function, and is also necessary for human body. Jujube and sour jujube fruits are rich in AsA, but they differ in AsA content. In this paper, we measured and compared ascorbic acid content in jujube and sour jujube, clarified AsA accumulation pattern in jujube and sour jujube, analyzed the transcriptome data of different varieties of jujube and sour jujube fruits and drought-treated jujube fruits, and identified the key functional genes affecting AsA accumulation.【Methods】The AsA content at young fruit stage, white ripening stage, half red stage and full red stage was measured in Jinsi No. 4 and Taishan Sour Jujube as the representative variety of jujube and sour jujube respectively, and their AsA accumulation patterns at different developmental stages were compared. The AsA content in 26 jujube and 42 sour jujube varieties was determined, and the differences in AsA content among the genotypes were compared. AsAwas determined with a high performance liquid chromatography (HPLC) equipped with an Agilnet C18 column and an UV DetectorL-2400. With the transcriptome data of fruits of 5 jujube and 5 sour jujube varieties obtained previously as reference, the key genes affecting the accumulation of AsA in jujube and sour jujube fruits were analyzed. The contents of organic acids, ascorbic acid and soluble sugars determined previously were used as quality traits, and the weighted gene co-expression network analysis (WGCNA) was performed on 30 transcriptome data of the 10 different samples, so as to investigate the relationship between AsA accumulation and organic acid content. Based on the transcriptome data previously obtained from jujube fruit at white ripening stage and half red stage exposed to moderate drought stress and severe stress, the key genes involving response of AsA metabolism to drought stress were studied.【Results】The accumulation trend of AsA in jujube and sour jujube fruits was the same. AsA was lowest at the young fruit stage, gradually increased during the development process and reached the highest at the white ripening stage, and then decreased slightly as the fruit matured. AsA content in sour jujube was higher than in jujube in all periods except for the young fruit stage. The average AsA content of cultivated jujube was significantly lower than that of sour jujube, and the range of AsA content in cultivated jujubes was relatively narrow, while that in sour jujubes population was relatively large. Cluster analysis of transcriptome data of fruits of the 10 jujube and sour jujube varieties showed that the expression levels of LGalDH, MIOX4, GME-2 and VTC2 were higher in sour jujubes than in cultivated jujubes, while the expression levels of genes GME-1 and MDAR5 and AsA oxidation genes APX1, APX2 and APXT were higher in cultivated jujubes. WGCNA showed that the ascorbic acid content and organic acid content were positively correlated with the gene expression in the bright green module. Further analysis of the genes in the module showed that they included calcium-transporting ATPase 1, calciumtransporting ATPase 3, one vacuole membrane proton pump called ATPase 10, and one NADP-dependent malolase. It was speculated that acidity influenced the stability of AsA. Transcriptome data from drought stress experiment showed that in the control fruits the expression levels of l-galactose pathway genes LGalDH, GME-2 and LGalLDH and recycle genes MDAR4 and DHAR1 were higher in the white ripening stage than in the half red stage. Although the key genes that affect AsA accumulation in the white ripening stage and the half red stages were not exactly the same, the expression levels of MDAR5, MIOX4 and MIOX1 were significantly increased after drought stress. It was speculated that the myo-inositol pathway and recycling pathway played an important role in drought stress.【Conclusion】AsA was the lowest in the young fruit stage, reached the highest level in the white ripening stage, and gradually declined in the ripening stage. The average AsA content in sour jujubes was significantly higher than in cultivated jujubes. The key genes associated with the difference in AsA accumulation between jujubes and sour jujubes included LGalDH, MIOX4, GME-2 and VTC2. It was hypothesized that acidic environment affects AsA accumulation, and the key metabolic pathways in response to drought stress might be myo-inositol pathway and recycling pathway.