Characteristics of galacturonate reductase (GalUR) genes in garlic (Allium sativum L.) and changes in their expression in response to abiotic stressors

In plants, the synthesis of L-ascorbic acid (Aa), in addition to the main L-galactose pathway, is carried out by three known alternative pathways. One of them, the D-galacturonic acid pathway, is thought to be specific for tissues with excess D-galacturonate, the substrate of D-galacturonate reductase (GalUR), which belongs to the Aldo-Keto Reductase (AKR) superfamily. In this study, the AKR gene family of garlic Allium sativum L. was identified and seven genes, AsGalUR1–7, presumably encoding GalUR enzymes, were determined. The structure and phylogeny of the AsGalUR1–7 genes and the proteins they encode, as well as the AsGalUR1–7 expression pattern in different organs of the garlic plant (in silico and qRT-PCR), were characterized. Based on the obtained data, the genes were conditionally divided into root (AsGalUR1–4) and leaf (AsGalUR5–7) groups depending on the highest expression level in the underground and aboveground parts of the plant, respectively. The AsGalUR expression in leaves and roots was analyzed in response to drought, salt and cold stresses, as well as exogenous phytohormones (abscisic acid, methyl jasmonate), accompanied by the AsA content measurement. It was shown that hormone treatment suppresses the expression of all analyzed genes in both organ types. Cold conditions stimulate the expression of root group genes and suppress that of leaf group genes in roots, and have the opposite effect in leaves. Osmotic stressors (NaCl, PEG) suppress the transcription of all genes in leaves, but do not change (NaCl) or stimulate (PEG) it in roots, which is accompanied by an increase in AsA accumulation in organs of both types. A positive correlation between the expression of the AsGalUR1 and 4 genes and the AsA content is found in leaves under stress conditions. The data obtained can form the basis for further study of the mechanisms regulating AsA synthesis in garlic and other Allium species.

1. Agius F., González-Lamothe R., Caballero J.L., Muñoz-Blanco J., Botella M.A., Valpuesta V. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat Biotechnol. 2003;21(2):177-181. doi 10.1038/nbt777

2. Anisimova O.K., Shchennikova A.V., Kochieva E.Z., Filyushin M.A. Identification and variability of the GDP-L-galactose phosphosphorylase gene ApGGP1 in leek cultivars. Russ J Genet. 2021a;57(3): 311-318. doi 10.1134/S1022795421030030

3. Anisimova O.K., Seredin T.M., Shchennikova A.V., Kochieva E.Z., Filyushin M.A. Estimation of the vitamin C content and GDP-L-galactose phosphorylase gene (VTC2) expression level in leek (Allium porrum L.) cultivars. Russ J Plant Physiol. 2021b;68(1):85-93. doi 10.1134/S1021443720060023

4. Anisimova O.K., Shchennikova A.V., Kochieva E.Z., Filyushin M.A. Identification of monodehydroascorbate reductase (MDHAR) genes in garlic (Allium sativum L.) and their role in the response to Fusarium proliferatum infection. Russ J Genet. 2022;58(7):773-782. doi 10.1134/S1022795422070031

5. Badejo A.A., Wada K., Gao Y., 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(1):229-239. doi 10.1093/jxb/err275

6. Broad R.C., Bonneau J.P., Hellens R.P., Johnson A.A.T. Manipulation of ascorbate biosynthetic, recycling, and regulatory pathways for improved abiotic stress tolerance in plants. Int J Mol Sci. 2020; 21(5):1790. doi 10.3390/ijms21051790

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

8. Duan W., Huang Z., Li Y., Song X., Sun X., Jin C., Wang Y., Wang J. Molecular evolutionary and expression pattern analysis of AKR genes shed new light on GalUR functional characteristics in Brassica rapa. Int J Mol Sci. 2020;21(17):5987. doi 10.3390/ijms21175987

9. Fattorini L., Falasca G., Kevers C., Rocca L.M., Zadra C., Altamura M.M. Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers. Planta. 2009;231(1):155-168. doi 10.1007/s00425-009-1035-y

10. Filyushin M.A., Anisimova O.K., Kochieva E.Z., Shchennikova A.V. Correlation of ascorbic acid content and the pattern of monodehydroascorbate reductases (MDHARs) gene expression in leek (Allium porrum L.). Russ J Plant Physiol. 2021;68(5):849-856. doi 10.1134/S1021443721050034

11. Filyushin M.A., Seredin T.M., Shchennikova A.V., Kochieva E.Z. Vitamin C content and profile of ascorbate metabolism gene expression in green leaves and bleached parts of the pseudostem of leek (Allium porrum L.) F1 hybrids. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J Genet Breed. 2025;29(2):200-209. doi 10.18699/vjgb-25-23

12. Franceschi V.R., Tarlyn N.M. L-Ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants. Plant Physiol. 2002;130(2):649-656. doi 10.1104/pp.007062

13. Fu Q., Cao H., Wang L., Lei L., Di T., Ye Y., Ding C., Li N., Hao X., Zeng J., Yang Y., Wang X., Ye M., Huang J. Transcriptome analysis reveals that ascorbic acid treatment enhances the cold tolerance of tea plants through cell wall remodeling. Int J Mol Sci. 2023; 24(12):10059. doi 10.3390/ijms241210059

14. Ha J., Kang Y.G., Lee T., Kim M., Yoon M.Y., Lee E., Yang X., Kim D., Kim Y.J., Lee T.R., Kim M.Y., Lee S.H. Comprehensive RNA sequencing and co-expression network analysis to complete the biosynthetic pathway of coumestrol, a phytoestrogen. Sci Rep. 2019; 9(1):1934. doi 10.1038/s41598-018-38219-6

15. Huang J., Wu H., Gao R., Wu L., Wang M., Chu Y., Shi Y., Xiang L., Yin Q. Integrated multi-omics analysis reveals glycosylation involving 2-O-β-D-Glucopyranosyl-L-ascorbic acid biosynthesis in Lycium barbarum. Int J Mol Sci. 2025;26(4):1558. doi 10.3390/ijms26041558

16. Isherwood F.A., Mapson L.W. Biological synthesis of ascorbic acid: the conversion of derivatives of D-galacturonic acid into L-ascorbic acid by plant extracts. Biochem J. 1956;64(1):13-22. doi 10.1042/bj0640013

17. Ishikawa T., Nishikawa H., Gao Y., Sawa Y., Shibata H., Yabuta Y., Maruta T., Shigeoka S. The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase. J Biol Chem. 2008;283(45):31133-31141. doi 10.1074/jbc.M803930200

18. Li H., Huang W., Wang G.L., Wang W.L., Cui X., Zhuang J. Transcriptomic analysis of the biosynthesis, recycling, and distribution of ascorbic acid during leaf development in tea plant (Camellia sinensis (L.) O. Kuntze). Sci Rep. 2017;7:46212. doi 10.1038/srep46212

19. Liu H., Wei L., Ni Y., Chang L., Dong J., Zhong C., Sun R., Li S., Xiong R., Wang G., Sun J., Zhang Y., Gao Y. Genome-wide analysis of ascorbic acid metabolism related genes in Fragaria×ananassa and its expression pattern analysis in strawberry fruits. Front Plant Sci. 2022;13:954505. doi 10.3389/fpls.2022.954505

20. Mohnen D. Pectin structure and biosynthesis. Curr Opin Plant Biol. 2008;11(3):266-277. doi 10.1016/j.pbi.2008.03.006

21. Mølhøj M., Verma R., Reiter W.D. The biosynthesis of D-Galacturonate in plants. functional cloning and characterization of a membraneanchored UDP-D-Glucuronate 4-epimerase from Arabidopsis. Plant Physiol. 2004;135(3):1221-1230. doi 10.1104/pp.104.043745

22. Peltonen K.E., Richard P. Identification of a D-galacturonate reductase efficiently using NADH as a cofactor. Biotechnol Rep (Amst). 2022; 35:e00744. doi 10.1016/j.btre.2022.e00744

23. Penning T.M. The aldo-keto reductases (AKRs): overview. Chem Biol Interact. 2015;234:236-246. doi 10.1016/j.cbi.2014.09.024

24. Popa P.-M., Băbeanu C., Cosmulescu S.-N. Evaluation of chemical compounds in local garlic genotypes from southwestern Romania. Appl Sci. 2024;14(16):6899. doi 10.3390/app14166899

25. Quiñones C.O., Gesto-Borroto R., Wilson R.V., Hernández-Madrigal S.V., Lorence A. Alternative pathways leading to ascorbate biosynthesis in plants: lessons from the last 25 years. J Exp Bot. 2024;75(9):2644-2663. doi 10.1093/jxb/erae120

26. Richardson A.T., McGhie T.K., Cordiner S.B., Stephens T.T.H., Larsen D.S., Laing W.A., Perry N.B. 2-O-β-d-Glucopyranosyl l-ascorbic acid, a stable form of vitamin C, is widespread in crop plants. J Agric Food Chem. 2021;69(3):966-973. doi 10.1021/acs.jafc.0c06330

27. Ruggieri V., Bostan H., Barone A., Frusciante L., Chiusano M.L. Integrated bioinformatics to decipher the ascorbic acid metabolic network in tomato. Plant Mol Biol. 2016;91(4-5):397-412. doi 10.1007/s11103-016-0469-4

28. Sengupta D., Naik D., Reddy A.R. Plant aldo-keto reductases (AKRs) as multi-tasking soldiers involved in diverse plant metabolic processes and stress defense: a structure-function update. J Plant Physiol. 2015;179:40-55. doi 10.1016/j.jplph.2015.03.004

29. Shchennikova A.V., Kochieva E.Z., Filyushin M.A. Ascorbate biosynthesis and recycling genes are involved in the responses of garlic Allium sativum L. plants to Fusarium proliferatum infection. Dokl Biochem Biophys. 2025;520(1):49-52. doi 10.1134/S1607672924601057

30. Skoczylas J., Jędrszczyk E., Dziadek K., Dacewicz E., Kopeć A. Basic chemical composition, antioxidant activity and selected polyphenolic compounds profile in garlic leaves and bulbs collected at various stages of development. Molecules. 2023;28(18):6653. doi 10.3390/molecules28186653

31. Smirnoff N. Ascorbic acid metabolism and functions: a comparison of plants and mammals. Free Radic Biol Med. 2018;22:116-129. doi 10.1016/j.freeradbiomed.2018.03.033

32. Smirnoff N., Wheeler G.L. The ascorbate biosynthesis pathway in plants is known, but there is a way to go with understanding control and functions. J Exp Bot. 2024;75(9):2604-2630. doi 10.1093/jxb/erad505

33. Smirnoff N., Conklin P.L., Loewus F.A. biosynthesis of ascorbic acid in plants: a renaissance. Annu Rev Plant Physiol Plant Mol Biol. 2001; 52:437-467. doi 10.1146/annurev.arplant.52.1.437

34. Šnirc M., Lidiková J., Čeryová N., Pintér E., Ivanišová E., Musilová J., Vollmannová A., Rybnikár S. Mineral and phytochemical profiles of selected garlic (Allium sativum L.) cultivars. South Afr J Bot. 2023;158:319-325. doi 10.1016/j.sajb.2023.05.024

35. Suekawa M., Fujikawa Y., Inada S., Murano A., Esaka M. Gene expression and promoter analysis of a novel tomato aldo-keto reductase in response to environmental stresses. J Plant Physiol. 2016;200:35-44. doi 10.1016/j.jplph.2016.05.015

36. Sun X., Zhu S., Li N., Cheng Y., Zhao J., Qiao X., Lu L., … Zhao X., Tian S., Su J., Cheng Z., Liu T. A chromosome-level genome assembly of garlic (Allium sativum) provides insights into genome evolution and allicin biosynthesis. Mol Plant. 2020;13(9):1328-1339. doi 10.1016/j.molp.2020.07.019

37. Teper-Bamnolker P., Buskila Y., Lopesco Y., Ben-Dor S., Saad I., Holdengreber V., Belausov E., Zemach H., Ori N., Lers A., Eshel D. Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem. Plant Physiol. 2012; 158(4):2053-2067. doi 10.1104/pp.112.194076

38. Vargas J.A., Leonardo D.A., D’Muniz Pereira H., Lopes A.R., Rodriguez H.N., Cobos M., Marapara J.L., Castro J.C., Garratt R.C. Structural characterization of L-galactose dehydrogenase: an essential enzyme for vitamin C biosynthesis. Plant Cell Physiol. 2022;63(8): 1140-1155. doi 10.1093/pcp/pcac090

39. Xie Q., Essemine J., Pang X., Chen H., Cai W. Exogenous application of abscisic acid to shoots promotes primary root cell division and elongation. Plant Sci. 2020;292:110385. doi 10.1016/j.plantsci.2019.110385

40. Xu X., Zhang Q., Gao X., Wu G., Wu M., Yuan Y., Zheng X., … Qi T., Li H., Luo Z., Li Z., Deng W. Auxin and abscisic acid antagonistically regulate ascorbic acid production via the SlMAPK8-SlARF4- SlMYB11 module in tomato. Plant Cell. 2022;34(11):4409-4427. doi 10.1093/plcell/koac262

41. Yenealem D., Eyayu D., Tibebe D., Mulugeta M., Kassa Y., Moges Z., Kerebih F., Fentie T., Amare A., Ayalew M. Electrochemical characterization and detection of ascorbic acid in garlic using activated glassy carbon electrode: a comprehensive study. Food Anal Methods. 2024;17:1473-1483. doi 10.1007/s12161-024-02660-3

42. Younis M.E., Hasaneen M.N., Kazamel A.M. Exogenously applied ascorbic acid ameliorates detrimental effects of NaCl and mannitol stress in Vicia faba seedlings. Protoplasma. 2010;239(1-4):39-48. doi 10.1007/s00709-009-0080-5