Vitamin C in fleshy fruits: biosynthesis, recycling, genes, and enzymes

L-ascorbic acid (vitamin C) is a plant secondary metabolite that has a variety of functions both in plant tissues and in the human body. Plants are the main source of vitamin C in human nutrition, especially citrus, rose hip, tomato, strawberry, pepper, papaya, kiwi, and currant fruits. However, in spite of the biological significance of L-ascorbic acid, the pathways of its biosynthesis in plants were fully understood only in 2007 by the example of a model plant Arabidopsis thaliana. In the present review, the main biosynthetic pathways of vitamin C are described: the L-galactose pathway, L-gulose pathway, galacturonic and myo-inositol pathway. To date, the best studied is the L-galactose pathway (Smyrnoff–Wheeler pathway). Only for this pathway all the enzymes and the entire cascade of reactions have been described. For other pathways, only hypothetical metabolites are proposed and not all the catalyzing enzymes have been identified. The key genes participating in ascorbic acid biosynthesis and accumulation in fleshy fruits are highlighted. Among them are L-galactose pathway proteins (GDP-mannose phosphorylase (GMP, VTC1), GDP-D-mannose epimerase (GME), GDP-L-galactose phosphorylase (GGP, VTC2/VTC5), L-galactose-1-phosphate phosphatase (GPP/VTC4), L-galactose-1-dehydrogenase (GalDH), and L-galactono1,4-lactone dehydrogenase (GalLDH)); D-galacturonic pathway enzymes (NADPH-dependent D-galacturonate reductase (GalUR)); and proteins, controlling the recycling of ascorbic acid (dehydroascorbate reductase (DHAR1) and monodehydroascorbate reductase (MDHAR)). Until now, there is no clear and unequivocal evidence for the existence of one predominant pathway of vitamin C biosynthesis in fleshy fruits. For example, the L-galactose pathway is predominant in peach and kiwi fruits, whereas the D-galacturonic pathway seems to be the most essential in grape and strawberry fruits. However, in some plants, such as citrus and tomato fruits, there is a switch between different pathways during ripening. It is noted that the final ascorbic acid content in fruits depends not only on biosynthesis but also on the rate of its oxidation and recirculation.

1. Agius F., Gonzalez-Lamothe R., Caballero J., Munoz-Blanco J., Botella M., Valpuesta V. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. NatBiotechnol. 2003;21:177-181. DOI 10.1038/nbt777.

2. Akram N., Shafiq F., Ashraf M. Ascorbic acid – a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Front. Plant Sci. 2017;8:613. DOI 10.3389/ fpls.2017.00613.

3. Alhagdow M., Mounet F., Gilbert L., Nunes-Nesi A., Garcia V., Just D., Petit J., Beauvoit B., Fernie A.R., Rothan C., Baldet P. Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato. Plant Physiol. 2007;145:1408-1422. DOI 10.1104/pp.107.106500.

4. Alós E., Rodrigo M.J., Zacarías L. Differential transcriptional regulation of L-ascorbic acid content in peel and pulp of citrus fruits during development and maturation. Planta. 2014;239:1113- 1128. DOI 10.1007/s00425-014-2044-z.

5. Badejo A.A., Wada K., Gao Y.S., Maruta T., Sawa Y., Shigeoka S., Ishikawa T. Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway. J. Exp. Bot. 2012;63:229-239. DOI 10.1093/jxb/err275.

6. Baydoun E.A.H., Fry S.C. [2-3H]Mannose incorporation in cultured plant cells: investigation of L-galactose residues of the primary cell wall. J. Plant Physiol. 1988;132:484-490. DOI 10.1016/S0176-1617(88)80068-3.

7. Bulley S.M., Rassam M., Hoser D., Otto W., Schünemann N., Wright M., MacRae E., Gleave A., Laing W. Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyltransferase is a major control point of vitamin C biosynthesis. J. Exp. Bot. 2009;60(3):765- 778. DOI 10.1093/jxb/ern327.

8. Bulley S., Wright M., Rommens C., Yan H., Rassam M., LinWang K., Andre C., Brewster D., Karunairetnam S., Allan A.C., Laing W.A. Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. Plant Biotechnol. J. 2012;10:390-397. DOI 10.1111/j.1467-7652. 2011.00668.x.

9. Cholet C., Claverol S., Claisse O., Rabot A., Osowsky A., Dumot V., Ferrari G., Gény L. Tartaric acid pathways in Vitis vinifera L. (cv. Ugni blanc): a comparative study of two vintages with contrasted climatic conditions. BMC Plant Biol. 2016;16:144. DOI 10.1186/s12870-016-0833-1.

10. Conklin P.L., Gatzek S., Wheeler G.L., Dowdle J., Raymond M.J., Rolinski S., Isupov M., Littlechild J.A., Smirnoff N. Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme. J. Biol. Chem. 2006;281:15662-15670. DOI 10.1074/jbc.M601409200.

11. Conklin P.L., Norris S.R., Wheeler G., Williams E.H., Smirnoff N., Last R.L. Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc. Natl. Acad. Sci. USA. 1999;96:4198-4203. DOI 10.1073/pnas.96.7.4198.

12. Cruz-Rus E., Amaya I., Sanchez-Sevilla J.F., Botella M.A., Valpuesta V. Regulation of L-ascorbic acid content in strawberry fruits. J. Exp. Bot. 2011;62:4191-4201. DOI 10.1093/jxb/ err122.

13. Cruz-Rus E., Botella M.A., Valpuesta V., Gomez-Jimenez M.C. Analysis of genes involved in L-ascorbic acid biosynthesis during growth and ripening of grape berries. J. Plant Physiol. 2010;167:739-748. DOI 10.1016/j.jplph.2009.12.017.

14. Davey M.W., Gilot C., Persiau G., Østergaard J., Han Y., Bauw G.C., Van Montagu M.C. Ascorbate biosynthesis in Arabidopsis cell suspension culture. Plant Physiol. 1999;121:535-543. DOI 10.1104/pp.121.2.535.

15. Davey M.W., Van Montagu M., Inze D., Sanmartin M., Kanellis A., Smirnoff N., Benzie I.J.J., Strain J.J., Favell D., Fletcher J. Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J. Sci. Food Agric. 2000;80:825-860. DOI 10.1002/(SICI)1097-0010(20000515)80:7 3.0.CO;2-6.

16. Di Matteo A., Sacco A., Anacleria M., Pezzotti M., Delledonne M., Ferrarini A., Frusciante L., Barone A. The ascorbic acid content of tomato fruits is associated with the expression of genes involved in pectin degradation. BMC Plant. Biol. 2010;10:163. DOI 10.1186/ 1471-2229-10-163.

17. Diplock A.T., Charleux J.L., Crozier-Willi G., Kok F.J., Rice-Evans C., Roberfroid M., Stahl W., Vina-Ribes J. Functional food science and defence againstreactive oxidative species. Br. J. Nutr. 1998;80:77-112. DOI 10.1079/BJN19980106.

18. do Nascimento J.R.O., Higuchi B.K., Gomez M.L.P.A., Oshiro R.A., Lajolo F.M. L-ascorbate biosynthesis in strawberries: L-galactono-1,4-lactone dehydrogenase expression during fruit development and ripening. Postharvest Biol. Technol. 2005;38:34-42. DOI 10.1016/j.postharvbio.2005.05.014.

19. Dowdle J., Ishikawa T., Gatzek S., RolinskiS., SmirnoffN. Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J. 2007;52:673-689. DOI 10.1111/j.1365-313X.2007. 03266.x.

20. El Airaj H., Gest N., Truffaul V., Garchery C., Riqueau G., Gouble B., Page D., Stevens R. Decreased monodehydroascorbate reductase activity reduces tolerance to cold storage in tomato and affects fruit antioxidant levels. Postharvest Biol. Technol. 2013;86:502-510. DOI 10.1016/j.postharvbio.2013.07.035.

21. Figueroa-Méndez R., Rivas-Arancibia S. Vitamin C in health and disease: its role in the metabolism of cells and redox state in the brain. Front. Physiol. 2015;6:397. DOI 10.3389/ fphys.2015.00397.

22. Gatzek S., Wheeler G.L., Smirnoff N. Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis. Plant J. 2002;30(5):541-553. DOI 10.1046/j.1365-313X.2002.01315.x.

23. Gest N., Garchery C., Gautier H., Jiménez A., Stevens R. Lightdependent regulation of ascorbate in tomato by a monodehydroascorbate reductase localized in peroxisomes and the cytosol. Plant Biotechnol. J. 2013;11:344-354. DOI 10.1111/pbi.12020.

24. Gilbert L., Alhagdow M., Nunes-Nesi A., Quemener B., Guillon F., Bouchet B., Faurobert M., Gouble B., Page D., Garcia V., Petit J., Stevens R., Causse M., Fernie A.R., Lahaye M., Rothan C., Baldet P. GDP-D-mannose 3,5-epimerase (GME) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynthesis in tomato. Plant J. 2009;60:499-508. DOI 10.1111/j.1365- 313X.2009.03972.x.

25. Hancock R.D., Walker P.G., Pont S.D.A., Marquis N., Vivera S., Gordon S.L., Brennan R.M., Viola R. L-Ascorbic acid accumulation in fruit of Ribes nigrum occurs by in situ biosynthesis via the L-galactose pathway. Funct. Plant Biol. 2007;34:1080-1091. DOI 10.1071/FP07221.

26. Huang M., Xu Q., Deng X.X. L-Ascorbic acid metabolism during fruit development in an ascorbate-rich fruit crop chestnut rose (Rosa roxburghii Tratt). J. Plant Physiol. 2014;171(14):1205- 1216. DOI 10.1016/j. jplph.2014.03.010.

27. Huang W., Qing G., Zhang H., Wu J., Zhang S. Distribution and metabolism of ascorbic acid in pear fruit (Pyrus pyrifolia Nakai cv. Aikansui). Afr. J. Biotechnol. 2013;12:1952-1961. DOI 10.5897/AJB11.4048.

28. ImaiT., BanY., Terakami S., YamamotoT., MoriguchiT. L-ascorbate biosynthesis in peach: cloning of six L-galactose pathway-related genes and their expression during peach fruit development. Physiol. Plant. 2009;136:139-149. DOI 10.1111/j.1399-3054.2009. 01213.x.

29. Imai T., Karita S., Shiratori G., Hattori M., Nunome T., Oba K., Hirai M. L-galactono-γ-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis. Plant Cell Physiol. 1998;39:1350-1358. DOI 10.1093/oxfordjournals. pcp.a029341.

30. Ioannidi E., Kalamaki M.S., Engineer C., Pateraki I., Alexandrou D., Mellidou I., Giovannonni J., Kanellis A.K. Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. J. Exp. Bot. 2009;60:663-678. DOI 10.1093/jxb/ern322.

31. Iqbal Y., Ihsanullah I., Shaheen N., Hussain I. Significance of vitamin C in plants. J. Chem. Soc. Pakistan. 2009;31:169-170.

32. Jain A.K., Nessler C.L. Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol. Breed. 2000;6(1): 73-78. DOI 10.1023/A:1009680818138.

33. Keller R., Springer F., Renz A., Kossmann J. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. Plant J. 1999;91:131-141. DOI 10.1046/j.1365-313X.1999.00507.x.

34. Laing W.A., Frearson N., Bulley S., MacRae E. Kiwifruit L-galactose dehydrogenase: molecular, biochemical and physiological aspects of the enzyme. Funct. Plant Biol. 2004;31:1015-1025. DOI 10.1071/FP04090.

35. Laing W.A., Martinez-Sanchez M., Wright M.A., Bulley S.M., Brewster D., Dare A.P., Rassam M., Wang D., Storey R., Macknight R.C., Hellens R.P. An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis. Plant Cell. 2015;27:772-786. DOI 10.1105/ tpc.114.133777.

36. Laing W.A., Wright M.A., Cooney J., Bulley S.M. The missing step of the L-galactose pathway of ascorbate biosynthesis in plants, an L-galactose guanyltransferase, increases leaf ascorbate content. Proc. Natl. Acad. Sci. USA. 2007;104:9534-9539. DOI 10.1073/pnas.0701625104.

37. Leferink N.G., van den Berg W.A., van Berkel W.J. L-Galactonoγ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis. FEBS J. 2008;275:713- 726. DOI 10.1111/j.1742-4658.2007.06233.x.

38. Li J., Li M., Liang D., Cui M., Ma F. Expression patterns and promoter characteristics of the gene encoding Actinidia deliciosa L-galactose-1-phosphate phosphatase involved in the response to light and abiotic stresses. Mol. Biol. Rep. 2013;40:1473-1485. DOI 10.1007/s11033-012-2190-y.

39. Li M., Chen X., Wang P., Ma F. Ascorbic acid accumulation and expression of genes involved in its biosynthesis and recycling in developing apple fruit. J. Am. Soc. Hortic. Sci. 2011;136:231-238.

40. Li M., Ma F., Liang D., Li J., Wang Y. Ascorbate biosynthesis during early fruit development is the main reason for its accumulation in kiwi. PLoS One. 2010;5(12):e14281. DOI 10.1371/journal.pone.0014281.

41. Li M.J., Ma F.W., Zhang M., Pu F. Distribution and metabolism of ascorbic acid in apple fruits (Malus domestica Borkh cv. Gala). Plant Sci. 2008;174:606-612. DOI 10.1016/j. plantsci.2008.03.008.

42. Liang D., Zhu T., Ni Z., Lin L., Tang Y., Wang Z., Wang X., Wang J., Lv X., Xia H. Ascorbic acid metabolism during sweet cherry (Prunus avium) fruit development. PLoS One. 2017;12(2):e0172818. DOI 10.1371/journal.pone.0172818.

43. Linster C.L., Adler L.N., Webb K., Christensen K.C., Brenner C., Clarke S.G. A second GDP-L-galactose phosphorylase in Arabidopsis en route to vitamin C: covalent intermediate and substrate requirements for the conserved reaction. J. Biol. Chem. 2008;283:18483-18492. DOI 10.1074/jbc.M802594200.

44. Linster C.L., Gomez T.A., Christensen K.C., Adler L.N., Young B.D., Brenner C., Clarke S.G. Arabidopsis VTC2 encodes a GDP-L-galactose phosphorylase, the last unknown enzyme in the Smirnoff–Wheeler pathway to ascorbic acid in plants. J. Biol. Chem. 2007; 282:18879-18885. DOI 10.1074/ jbc.M702094200.

45. Liu F., Wang L., Gu L., Zhao W., Su H., Cheng X. Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry. Food Chem. 2015;188:399-405. DOI 10.1016/j.foodchem.2015. 05.036.

46. Loewus F.A. Biosynthesis and metabolism of ascorbic acid in plants and of analogs of ascorbic acid in fungi. Phytochemistry. 1999;52: 193-210. DOI 10.1016/S0031-9422(99)00145-4.

47. Loewus F.A., Kelly S. The metabolism of D-galacturonic acid and its methyl ester in the detached ripening strawberry. Arch. Biochem. Biophys. 1961;95:483-493. DOI 10.1016/0003- 9861(61)90180-1.

48. Loewus F.A., Loewus M.W. Biosynthesis and metabolism of ascorbic acid in plants. Crit. Rev. Plant Sci. 1987;5:101-119. DOI 10.1080/ 07352688709382235.

49. LorenceA., ChevoneB.I., MendesP., NesslerC.L. Myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol. 2004;134:1200-1205. DOI 10.1104/pp.103. 033936.

50. Lukowitz W., Nickle T.C., Meinke D.W., Last R.L., Conklin P.L., Somerville C.R. Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis. Proc. Natl. Acad. Sci. USA. 2001;98(5):2262-2267. DOI 10.1073/ pnas.051625798.

51. Maruta T., Ichikawa Y., Mieda T., Takeda T., Tamoi M., Yabuta Y., Ishikawa T., Shigeoka S. The contribution of Arabidopsis homologs of L-gulono-1,4-lactone oxidase to the biosynthesis of ascorbic acid. Biosci. Biotechnol. Biochem. 2010;74:1494- 1497. DOI 10.1271/bbb.100157.

52. Mellidou I., Chagné D., Laing W., Keulemans J., Davey M.W. Allelic variation in paralogs of GDP-L-galactose phosphorylase is a major determinant of vitamin C concentrations in apple fruit. Plant Physiol. 2012;160:1613-1629. DOI 10.1104/ pp.112.203786.

53. Mellidou I., Kanellis A.K. Genetic control of ascorbic acid biosynthesis and recycling in horticultural crops. Front. Chem. 2017;5:50. DOI 10.3389/fchem.2017.00050.

54. Michell R.H. Evolution of the diverse biological roles of inositols. Biochem. Soc. Symp. 2007;74:223-246. DOI 10.1042/ BSS0740223.

55. Mieda T., Yabuta Y., Rapolu M., Motoki T., Takeda T., Yoshimura K., Ishikawa T., Shigeoka S. Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate. Plant Cell Physiol. 2004;45:1271-1279. DOI 10.1093/pcp/pch152.

56. Mounet-Gilbert L., Dumont M., Ferrand C., Bournonville C., Monier A., Jorly J., Lemaire-Chamley M., Mori K., Atienza I., Hernould M., Stevens R., Lehner A., Mollet J.C., Rothan C., Lerouge P., Baldet P. Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development. J. Exp. Bot. 2016;67:4767-4777. DOI 10.1093/jxb/erw260.

57. Mutsuda M., Ishikawa T., Takeda T., Shigeoka S. Subcellular-localization and properties of L-galactono-γ-lactone dehydrogenase in spinach leaves. Biosci. Biotechnol. Biochem. 1995;59:1983- 1984. DOI 10.1271/bbb.59.1983.

58. Nishikimi M., Yagi K. Biochemistry and Molecular Biology of Ascorbic Acid Biosynthesis. In: Harris J.R. (Ed.). Subcellular Biochemistry (Ascorbic Acid: Biochemistry and Biochemical Cell Biology). Springer, Boston, MA, 1996;25:17-39. DOI 10.1007/978-1-4613-0325-1_2.

59. Oesterhelt C., Schnarrenberger C., Gross W. The reaction mechanism of phosphomannomutase in plants. FEBS Lett. 1997;401:35-37. DOI 10.1016/S0014-5793(96)01425-1.

60. Pastori G.M., Kiddle G., Antoniw J., Bernard S., Veljovic-Jovanovic S., Verrier P.J., Noctor G., Foyer C.H. Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling. Plant Cell. 2003;15:939-951. DOI 10.1105/tpc.010538.

61. Pateraki I., Sanmartin M., Kalamaki M.S., Gerasopoulos B., Kanellis A.K. Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4- lactone dehydrogenase. J. Exp. Bot. 2004;55:1623-1633. DOI 10.1093/jxb/erh186.

62. Radzio J.A., Lorence A., Chevone B.I., Nessler C.L. L-Gulono-1,4- lactone oxidase expression rescues vitamin C-deficient Arabidopsis (vtc) mutants. Plant Mol. Biol. 2003;53(6):837-844. DOI 10.1023/B:PLAN.0000023671.99451.1d.

63. Roberts R.M. The metabolism of D-mannose-14C to polysaccharide in corn roots. Specific labelling of L-galactose, D-mannose, and L- fucose. Arch. Biochem. Biophys. 1971;145:685-692. DOI 10.1016/S0003-9861(71)80029-2.

64. Rodríguez-Ruiz M., Mateos R.M., Codesido V., Corpas F.J., Palma J.M. Characterization of the galactono-1,4-lactone dehydrogenase from pepper fruits and its modulation in the ascorbate biosynthesis. Role of nitric oxide. Redox Biol. 2017;12:171-181. DOI 10.1016/j.redox. 2017.02.009.

65. Sauvage C., Segura V., Bauchet G., Stevens R., Do P.T., Nikoloski Z., Fernie A.R., Causse M. Genome-wide association in tomato reveals 44 candidate loci for fruit metabolic traits. Plant Physiol. 2014;165: 1120-1132. DOI 10.1104/pp.114.241521.

66. Shigeoka S., Nakano Y., Kitaoka S. The biosynthetic pathway of L-ascorbic acid in Euglena gracilis z. J. Nutr. Sci. Vitaminol. 1979;25: 299-307. DOI 10.3177/jnsv.25.299.

67. Smirnoff N. Ascorbic acid metabolism and functions: a comparison of plants and mammals. Free Radic. Biol. Med. 2018;122:116- 129. DOI 10.1016/j.freeradbiomed.2018.03.033.

68. Streltsina S.A., Burmistrov L.A., Nikitina E.V. Nutritious and biologically active substances of mountain ash fruits (Sorbus L.) in the conditions of the northwestern zone of horticulture in Russia. Agrarnaya Rossiya = Agricultural Russia. 2010;3:10-17. (in Russian)

69. Suekawa M., Kondo T., Fujikawa Y., Esaka M. Regulation of Ascorbic Acid Biosynthesis in Plants. In: Hossain M.A. et al. (Eds.). Ascorbic Acid in Plant Growth, Development and Stress Tolerance. Springer, 2017;157-176. DOI 10.1007/978-3-319- 74057-7_6.

70. Torabinejad J., Donahue J.L., Gunesekera B.N., Allen-Daniels M.J., Gillaspy G.E. VTC4 is a bifunctional enzyme that affects myoinositol and ascorbate biosynthesis in plants. Plant Physiol. 2009;150: 951-961. DOI 10.1104/pp.108.135129.

71. Truffault V., Fry S.C., Stevens R.G., Gautier H. Ascorbate degradation in tomato leads to accumulation of oxalate, threonate and oxalyl threonate. Plant J. 2017;89(5):996-1008. DOI 10.1111/ tpj.13439.

72. Walker P.G., Viola R., Woodhead M., Jorgensen L., Gordon S., Brennan R., Hancock R. Ascorbic acid content of blackcurrant fruit is influenced by both genetic and environmental factors. Func. Plant Sci. Biotech. 2010;1:40-52.

73. Wang L.Y., Meng X., Yang D.Y., Wang G.D., Meng Q.W. Overexpression of tomato GDP-L-galactose phosphorylase gene in tobacco improves tolerance to chilling stress. Plant Cell Rep. 2014;33(9): 1441e1451. DOI 10.1007/s00299-014-1627-2.

74. Watanabe K., Suzuki K., Kitamura S. Characterization of a GDP-D-mannose 3″,5″-epimerase from rice. Phytochemistry. 2006;67:338-346. DOI 10.1016/j.phytochem.2005.12.003.

75. Wheeler G.L., Jones M.A., Smirnoff N. The biosynthetic pathway of vitamin C in higher plants. Nature. 1998;393:365-369. DOI 10.1038/30728.

76. Wolucka B.A., Persiau G., Van Doorsselaere J., Davey M.W., Demol H., Vandekerckhove J., Van Montagu M., Zabeau M., Boerjan W. Partial purification and identification of GDP-mannose 3”,5”-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc. Natl. Acad. Sci. USA. 2001;98:14843-14848. DOI 10.1073/pnas.011578198.

77. Wolucka B.A., Van Montagu M. GDP-mannos 3,5-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J. Biol. Chem. 2003;278:47483- 47490. DOI 10.1074/jbc.M309135200.

78. Xu Q., Chen L., Ruan X., … Nagarajan N., Deng X., Ruan Y. The draft genome of sweet orange (Citrus sinensis). Nat. Genet. 2013;45:59-66. DOI 10.1038/ng.2472.

79. Yabuta Y., Yoshimura K., Takeda T., Shigeoka S. Molecular characterization of tobacco mitochondrial L-galactono-γ-lactone dehydrogenase and its expression in Escherichia coli. Plant Cell Physiol. 2000;41:666-675. DOI 10.1093/pcp/41.6.666.

80. Yang X.Y., Xie J.X., Wang F.F., Zhong J., Liu Y.Z., Li G.H., Peng S.A. Comparison of ascorbate metabolism in fruits of two citrus species with obvious difference in ascorbate content in pulp. J. Plant Physiol. 2011;168:2196-2205. DOI 10.1016/j. jplph.2011. 07.015.

81. Yoshimura K., Nakane T., Kume S., Shiomi Y., Maruta T., Ishikawa T., Shigeoka S. Transient expression analysis revealed the importance of VTC2 expression level in light/dark regulation of ascorbate biosynthesis in Arabidopsis. Biosci. Biotechnol. Biochem. 2014;78:60-66. DOI 10.1080/09168451.2014.877831.

82. Zhang C.J., Liu J.X., Zhang Y.Y., Cai X.F., Gong P.J., Zhang J.H., Wang T.T., Li H.X., Ye Z.B. Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato. Plant Cell Rep. 2011;30:389-398. DOI 10.1007/s00299-010-0939-0.

83. Zhang Y.Y., Li H.X., Shu W.B., Zhang C.J., Zhang W., Ye Z.B. Suppressed expression of ascorbate oxidase gene promotes ascorbic acid accumulation in tomato fruit. Plant Mol. Biol. Rep. 2011;29:638-645. DOI 10.1007/s11105-010-0271-4.

84. Zhou Y., Tao Q.C., Wang Z.N., Fan R., Li Y., Sun X.F., Tang K.X. Engineering ascorbic acid biosynthetic pathway in Arabidopsis leaves by single and double gene transformation. Biol. Plant. 2012;56:451-457. DOI 10.1007/s10535-012-0119-x.