Molecular Mechanism For The Accumulation Of High Content Of L-Ascorbic Acid In Chestnut Rose

L-ascorbic acid (AsA) is a highly abundant and essential metabolite for plants and animals. AsA is commonly called as vitamin C, since it performs the status of vitamin in some higher animals such as primates and human, who lack the capacity to synthesize AsA. So they have to absorb exogenous AsA from their foods. Among the dialy foods, fruits and vegetables contain relatively high concentration of AsA. Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop in Southwest China, and its fruits have a variety of phytochemicals that benefit for human health. Particularly, its fruits accumulate quite high content of AsA, which is remarkably higher than that of most fruit crops. However, the molecular mechanism for chestnut rose accumulating so high content of AsA is still almost blank. To understand it, based on three genotypes of chestnut rose, the changes in the contents of AsA and related metabolites, the cellular distribution and transportation of AsA and dehydroascorbate (DHA), the gene expression and activity of enzymes involved in AsA biosynthesis and recycling during fruit development were systematically investgated and analyzed; and the importance of crucial genes for AsA accumulation was confirmed in transgenic plants. This study obtained the following results:1. The changes of AsA concentration in chestnut rose fruit during fruit development were quite similar among three genotypes only with a certain diversity in fruit AsA concentration. AsA concentration in ripe fruit of chestnut rose MpF1was1542mg/100g FW. The fruit AsA concentration maintained a very low level before40days after anthesis (DAA), then quickly increased as fruit ripening. And the highest AsA accumulation rate appeared between65and80DAA with nearly a half of fruit AsA accumulated in this time. In contrast, AsA concentration in leaf maintained a low level throughout fruit development, which was averagely1%to3%of AsA concentration in ripe fruit for three genetypes.2. The changes in AsA cellular distribution in fruit reflected the changes of AsA concentration during fruit development. For intracellular distribution, AsA was mainly in the cytoplasm but barely in vacuoles. For intercellular distribution, AsA was mainly concentrated in the mesocarp, particularlly in cells nearby conducting tissue of fruit, other than in the exocarp, which became more and more obvious as fruit development and ripening.3. The changes in content of metablites related to AsA biosynthesis and catabolism also supported the changes of AsA concentration during fruit development. The changes in concentration of precursors and intermediate metabolite of L-galactose pathway for AsA biosynthesis all correlated well with the changes of AsA accumulation rates. While the concentrations of main products of AsA catabolism were quite low and nearly unchanged in both fruit and leaf throughout fruit development. Additionally, the changes in concentrations of main fruit organic acids also showed certain correlation with the changes of AsA concentration.4. Totally14genes were cloned in chestnut rose including nearly all important genes involved in AsA biosynthesis and recycling. And the changes in expression of these genes during fruit development were analyzed in three genotypes respectively. Among these genes, the expression pattern of recycling pathway gene DHAR (encoding dehydroascorbate reductase) correlated the best with the variation of AsA concentration. And the expression pattern of L-galactose pathway gene GME (encoding GDP-D-mannose-3,5-epimerase) showed the highest similarity with the variation of AsA concentration among all AsA biosynthetic genes. While the D-galacturonate pathway gene GalUR (encoding D-galacturonic acid reductase) highly expressed in the middle fruit developmental stages. But myo-inostiol pathway gene barely expressed during fruit development.5. The changes in activities of a part of enzymes involved in AsA biosynthesis and recycling during fruit development were analyzed. And the changes in activities of DHAR correlated well with the variation AsA accumulation. The changes in AsA redox states during fruit development also correlated well with the changes in gene expression and activity of DHAR.6. The changes in AsA and DHA concentration as well as AsA/T-AsA ratio in different tissues and organs during fruit development were quite distinct to that in fruit. In contrast to fruit, other tissues and organs all contained much more DHA than AsA. And leaflet had the highest concentration of DHA, followed by sepal, petiole, flesh, seed in turn, and branch the lowest. Histochemical location of DHA in different ogans all showed that DHA was mainly distributed in photo synthetic tissues and conducting tissues. And DHA cellular distribution existed ovbious connection with AsA cellular distribution in fruit. 7. The full length cDNA of DHAR and GME were cloned in chestnut rose by RACE, and named RrDHAR and RrGME respectively. Full length cDNA of strawberry (Fragaria x ananassa) DHAR was also cloned, which showed93%similarity with RrDHAR. There were some diffenences at several amino acid sites between their deduced protein sequences. The full length cDNA sequences of AO and APX were also obtained in chestnut rose.8. The eukaryotic expression vector of RrDHAR and RrGME were constructed respectively and transformed stably into Arabidopsis plants. Comparing with wild-type plants, over-expression of RrGME in Arabidopsis resulted in a1.8-fold increase in leaf T-AsA, and a7.2-fold increase in leaf AsA a3.3-fold increase in AsA/T-AsA ratio. While, over-expressiion of RrDHAR resulted in a1.4-fold increase in leaf T-AsA, a9.7-fold increase in leaf AsA, and the increase of AsA/T-As A ratio was twice higher than over-expressed lines of RrGME. It was similar for the results of detection in legume of transgenic plants.9. The full length gene sequences and partial promoter of RrDHAR and RrGME were obtained respectively. Sequence analysis indicated that they not only contained the core elements of pomoter, but also had a lot of light responsive elements, which were much more than that in the promoter of homologous genes in wild strawberry (Fragaria vesca).In conclusion, the high level of AsA accumulation during fruit development of chestnut rose is probably due to a mixture contribution from AsA biosynthesis and AsA recycling. The L-galactose pathway takes the dominant role in AsA biosynthesis, and RrGME probably is a crucial gene for controling AsA biosynthesis. More importantly, RrDHAR from recycling pathway should be responsible for the accumulation and maintenance of high content of fruit AsA during late fruit development. Additionally, long-distance transportation of DHA may also involve in the fruit AsA accumulation, but still depond upon the recycling role of RrDHAR. RrDHAR is a critical enzyme that exhibits not only a high efficiency of AsA regeneration, but also a trong capacity in regulating AsA content with fruit development in chestnut rose.