Physiological And Molecular Mechanism Of Ascorbate Formation And Accumulation In Apple And Kiwifruit

L-Ascorbic acid (AsA), also known as vitamin C or ascorbate, is one of the most abundant antioxidants and cofactor for several enzymes. It has been reported that AsA is necessary for plant growth and development, and is also well known to have an important role in resistance to oxidative stress. Moreover, it is the main source of natural AsA to human because humans are incapable of synthesizing AsA and must secure it by means of dietary uptake to be healthy. However, results from the present study can't fully answer the reasons why there are clear differences in AsA content among different fruits. In the present study, to understand mechanism of AsA formation and accumulation, a systematical investigation on distribution, biosynthesis, recycling, transportation and accumulation of AsA as well as their light regulation was performed on physiological and molecular level in apple fruits (Malus domestica Borkh'Gala'.) and kiwifruits (Actinidia deliciosa cv. Qinmei), which has huge difference in AsA content. The main results were as follows.1. The full-length cDNA of MDHAR, DHAR2 and GME as well as partial cDNA of GalUR were cloned by RT-PCR from apple fruits. Homology analysis of these cDNA showed high homology with the cDNA from other plants. The full-length of MDHAR cDNA is 1400 bp and contains a complete open reading frame (ORF) of 1305 bp, which encodes a membrane bound MDHAR. The full-length of DHAR2 cDNA is 933 bp and contains an ORF of 798 bp, which encodes a GSH dependent-cytoplasmic DHAR. The full-length of GME cDNA is 1323 bp and contains an ORF of 1131, which encodes a NAD dependent-GME. The cDNA of GalUR is 668 bp. Homology analysis of GalUR cDNA showed 80% homology with the cDNA from strawberry, and it is a partial cDNA of apple GalUR contained 5'-terminal start codon.2. It was detected on gene expression and enzyme activities of GalLDH, GalDH and GalUR which were involved in AsA biosynthesis in apple fruits. Moreover, when different tissues of fruit were incubated with non-labeled putative substrates used to synthesize AsA, incubations with L-Gal and L-GL invoved in L-galactose panthway clearly increased AsA levels in peel and flesh, while D-GalUA was also able to stimulate AsA levels in peel. And the young fruits had stronger capability of AsA biosynthesis. These results suggest that apple fruit is able to synthesize AsA. In different tissues of fruit, the peel with higher AsA content showed higher gene expression level and enzyme activities of GalLDH, GalDH and GalUR as well as MDHAR and DHAR cpmpared with that in the flesh. Additionally, the sun-exposed peel with higher AsA concentration had stronger capability of AsA synthesizing and recycling than the shaded peel, while there was no difference in between the flesh of the sun-exposed side and the shaded side. In transportation, abundant AsA was found in vascular tissues of fruit, and the capability of AsA synthesis can be detected in pedicels of fruit and leaf, but there was no change in AsA level when exogenous AsA was added to fruit pedicels in vitro or vivo. It suggested that AsA could be transported to vascular tissues of fruit but could not well unload in fruits or the AsA in pedicels and vascular tissues might be synthesized by self to simply supply theses tissue's AsA requirement for ROS detoxification. Therefore, biosynthesis was a major pathway of AsA formation in apple fruits.3. Light directly influenced on capability of AsA biosynthesis and recycling in apple leaves and peel. After the whole trees were shaded for 20 days, AsA levels were significantly decreased in fruit peel and leaves, with clear decline of the mRNA expression levels and activities of GalLDH and GalDH as well as activities of recycling enzymes. Both of fruit and whole tree shading could not lead to clear changes of the mRNA expression levels and activities of GalLDH and GalDH as well as activities of recycling enzymes in the flesh, but the shading of whole tree could lead to decreasing of AsA content in the flesh but shading of fruit could not. Moreover, the fruit located on sun–exposed side of a tree top had higher AsA accumulation level compared with those for the inner shaded side of a tree. It was concluded that light directly affect AsA biosynthesis and recycling in the peel and leaves, but it indirectly affect AsA content via the leaves in the flesh of apple.4. During growth and development of apple fruits, T-AsA and AsA content based on per fresh weight in fruits were the highest in ovaries of 0 DAA, followed by a continuous decreasing and nearly reach a constant after 60 DAA. However, AsA accumulation level based on per fruit was continuous increased with growth of fruits, which demonstrated that AsA accumulation was occurred, and the rate of AsA synthesis in a fruit was higher than rate of DHA degradation through fruit growth and development in apple fruits. Expression levels of genes involved in AsA synthesis showed different patterns during growth and development of apple fruits. Markedly up-regulated mRNA expression level of GME, GGP and GalUR did not lead to changes of AsA content, which indicated that GME, GGP and GalUR did not play important roles in controlling AsA synthesis in apple fruits, while expression level of GPP was consistent with AsA content. And higher mRNA expression levels and enzyme activities of GalLDH and GalDH were agreeable with high capability of conversion from L-GL and L-Gal to AsA. In recycling system, mRNA expression levels and enzyme activities of MDHAR showed a decreased pattern with growth of fruits, and it was negative correlation with AsA/DHA.5. To understand the difference in controlling pattern of AsA synthesis and accumulation between fruit and leaves, it was investigated on AsA formation and its regulation in biosynthesis and recycling in apple leaves with different age. The results suggested that AsA accumulation in apple leaves mainly occurred during the transition phase from young to mature leaves with high rates of synthesis and recycling, while AsA content was clearly declined in senescent leaves with dropping of AsA synthesis and recycling. The datas also suggested that GPP could play an important role in controlling AsA biosynthesis via the L-galactose pathway in apple leaves, while patterns of GMP, GGP and GalLDH expression showed no roles in different age leaves of apple. Increased expression levels of GGP and GalUR were found in senescent leaves, which suggested that they might be correlative with leaf senescence. In recycling system, mRNA expression level and activity of MDHAR were relative to DHA content in leaves, which implied that MDHAR had important roles in maintaining redox state of AsA. Activity and the mRNA expression level of DHAR were clearly correlated with AsA content in leaves of different ages, which suggested that DHAR could play an important role in maintaining balance of AsA redox state, especially in mature leaves with low MDHAR activity.6. The full-length cDNA of DHAR2 and GGP as well as partial cDNA of MDHAR, DHAR1 and GME were cloned by RT-PCR from kiwi fruits. MDHAR cDNA is 1019 bp and partial cDNA of a membrane bound MDHAR. The full-length of DHAR1 cDNA is 821 bp and contains an ORF of 639 bp, while DHAR2 cDNA is 542 bp and partial cDNA of DHAR, both of which encode GSH dependent-cytoplasmic DHAR. The cDNA of GME is 846 bp and encodes a NAD dependent-GME. Homology analysis of GME cDNA showed high homology with the cDNA from other plants, and it is a partial cDNA of kiwi GME. The full-length of GGP cDNA is 1542 bp and contains an ORF of 1353 bp, which encodes a membrane bound GGP protein.7. It was detected on gene expression and enzyme activities of GalLDH, GalDH and GalUR in kiwifruit. When the flesh was incubated with non-labeled putative substrates used to synthesize AsA, not only incubations with L-Gal and L-GL could clearly increased AsA levels but also L-GulL and D-GalUA were also able to stimulate AsA levels. And the expression level of genes and enzyme activities as well as capability of AsA biosynthesis in young fruits were much higher than that in ripening fruits. Existing results indicated that not only kiwifruit could synthesize AsA by self via L-galactose but also alternative AsA biosynthetic pathways (e.g. D-galacturonic acid) pathway might be operated. In different tissues, the flesh and seed zone, which had higher gene expression level and enzyme activities of GalLDH, GalDH, MDHAR and DHAR, showed the highest AsA content, while the core was very low in AsA content with lower gene expression level and activities of these enzymes. Moreover, AsA distribution in kiwifruit showed cell diversities. AsA content was almost observed in peel cell. In cells of flesh, the big size of cell had abundant AsA not only in intracellular zone but also in cell wall while the AsA was hardly observed in small size of cell. Moreover, abundant AsA could be observed in wall of the core cell but not in intracellular, and AsA was also detected in ductal cell of vascular bundle. As in apple, AsA content and AsA synthesis can be detected in pedicels of fruit and leaf, but there was no influence on AsA levels when exogenous AsA, DHA and L-GL were added to pedicels of fruit in vitro and vivo. It also suggested that the AsA in pedicels and vascular tissue of kiwi might be synthesized by self and simply supplied AsA requirement for ROS detoxification in these tissues. However, AsA content in young fruit was clearly increased when exogenous sucrose was used to feed fruit pedicels at stage of young fruit, but not at stage of mature fruit. These results indicated that biosynthesis was a major pathway of AsA formation in kiwifruits, and supply of sugar source from leaves might influence on AsA synthesis rate in young fruit.8. Shading of fruit with bags also had no influence on AsA content and accumulation in kiwifruit, but AsA levels in young fruits were clearly regulated by diurnal variation, and the fruits at 6:00 had lower T-AsA and AsA content compared with that in noon or afternoon. Moreover, AsA content could be significantly decreased when the trees were shaded before 40 DAA. These results indicated that light indirectly influenced on AsA content via the leaves in kiwifruit but not directly. Shading of whole trees led to significant down-regulation of gene expression levels and enzyme activities related with AsA synthesis and recycling, which led to marked decrease of AsA content and increase of DHA content. When the trees were shaded at stage of young fruits (0-40 DAA), expression levels of genes (including GalLDH, GalDH, GPP, GME, GalUR, MDHAR and DHAR) and enzyme activities (including GalLDH, GalDH, MDHAR and DHAR) showed a huge decline in 40 DAA fruits, which led to significant decrease of AsA content and accumulation in 40 DAA fruits. But there was no influence on these indexes when the trees were shaded after 40 DAA caompared with the control. These results indicated that the capability of AsA biosynthesis was inhibited while the trees were shaded before 40 DAA. Although decreased sugar content did not result in the changes of AsA content as well as its synthesis and recycling in fruit after 40 DAA, given the fact that exogenous sucrose could increase AsA content in young fruits, decreased sugar content (especially sucrose) might be contributed to decline of AsA synthesis capability in young fruits while the trees were shaded before 40 DAA. These results suggested that light did not directly influence on AsA content and synthesis in kiwifruit, but indirectly controlled AsA synthesis and recycling with changes capability of sugar supply or other signal from leaf to fruit, which led to decline of AsA content in young fruits. And this decline of AsA content might be relative to growth of fruits after 40 DAA.9. During growth and development of kiwifruits, T-AsA and AsA content based on per fresh weight in fruits were rapidly increased after anthesis and reached the highest at 30 DAA, followed by a continuous decrease until it nearly maintained a constant after 60 DAA. Based on per fruit, T-AsA and AsA accumulation levels also showed significant increase after anthesis and reached the highest at 45 DAA, then it nearly maintained a constant until fruit maturate. These results demonstrated that AsA accumulation in kiwifruit mainly occurred at stage of young fruit (before 45 DAA). Expression pattern of GPP and GGP showed high agreement with AsA accumulation rate, which implied that they might have control roles in AsA biosynthesis and accumulation rate of kiwi fruits. But expression patterns of GalLDH, GalDH, GMP and GME showed that they did not play roles in regulating on AsA accumulation. In AsA recycling system, mRNA expression level and enzyme activities of MDHAR and DHAR had little role in accumulating at stage of young fruits, but higher mRNA expression and activities of MDHAR and DHAR implied that they play an important role in maintaining AsA level in fruits after 45 DAA. In carbohydrate, change patterns of AsA level were no clear correlative with total soluble sugar, reduced sugar, starch, fructose, sucrose and glucose content during growth and development of fruits, but changes patterns of sucrose and starch content were agreement with AsA content at stage of young fruit (before 45 DAA), which indicated that sucrose or starch might be contributed to AsA synthesis in young fruit of kiwi.In conclusion, biosynthesis is a main reason why AsA can be accumulated in apple and kiwi fruits. Compared with apple, kiwi fruits have much higher capability of AsA synthesis, and there are other pathways (e.g. D-galacturonate pathway) used to synthesize AsA in kiwifruit. Leaf could control AsA biosynthesis and recycling of fruits with changes of supply of sugar source or other substances to fruits, which regulates AsA content and accumulation in fruits. On patterns of AsA accumulation of each fruit, AsA accumulation is occurred through growth and development of apple fruits, but the rate is very slow compared with young kiwi fruits. However, AsA accumulation in kiwi fruits mainly occurs at stage of young fruit (before 45 DAA) with high rate before 30 DAA, and then it nearly maintains a constant until fruit maturate. In recycling system, MDHAR and DHAR do not play important roles in controlling accumulation levels of AsA in apple and kiwi fruits, but they have essential roles in maintaining redox state of AsA and balance between synthesis and degradation. Compared with apple fruits, kiwi fruits have much higher activities of MDHAR and DHAR during growth and development of fruits and lower rate of oxidized AsA, which is another reason of much higher AsA content in kiwi fruits. Based on analysis of relationship of expression levels of genes to AsA content and accumulation levels during growth and development of apple and kiwi fruits, among different age leaves of apple as well as under different light condition, it is indicated that GPP could play an important role in controlling AsA biosynthesis via L-galactose pathway.