Abstract
The strawberry (Fragaria × ananassa) is an economically important perennial crop plant, and its fruits are rich in vitamin C (l-ascorbic acid [AsA]) and other nutrients. l-galactono-1,4-lactone dehydrogenase (GalLDH) is a key enzyme in the terminal step of AsA biosynthesis pathway in plants. Here, the GalLDH gene (FaGalLDH) was cloned from ‘Benihoppe’ strawberries. AsA content increased during fruit development and peaked at the red-ripening stage, and AsA concentrations in different tissues were correlated with enzyme activity and transcription level of FaGalLDH. Transient over-expression of FaGalLDH in strawberry fruit increased its overall expression and AsA production significantly, whereas transient RNAi of FaGalLDH decreased its expression and AsA content. Furthermore, the optimum pH and temperature for FaGalLDH activity were 8.0 and 25 °C, respectively. Ectopic expression of the FaGalLDH gene in Arabidopsis resulted in higher AsA content and enzyme activity in transgenic lines than in wild-type plants. FaGalLDH over-expression resulted in enhanced tolerance to salt stress due to reduced accumulation of malondialdehyde, H2O2, and O2.−, as well as higher survival rates, root length, proline content, and superoxide dismutase, peroxidase, and catalase activities. These results provide useful information regarding AsA biosynthesis and salt tolerance, which may help to improve strawberry fruit quality and productivity.
Similar content being viewed by others
References
Agius F, González-Lamothe R, Caballero JL, Muñoz-Blanco J, Botell MA, Valpuesta V (2003) Engineering increased vitamin C levels in plants by overexpression of a d-galacturonic acid reductase. Nat Biotechnol 21:177–181
Akram NA, Shafiq F, Ashraf M (2017) Ascorbic acid-a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Front Plant Sci 8:613
Alhagdow M, Mounet F, Gilbert L, Nunes-Nesi A, Garcia V, Just D, Petit J, Beauvoit B, Fernie AR, Rothan C, Baldetet P (2007) Silencing of the mitochondrial ascorbate synthesizing enzyme l-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato. Plant Physiol 145:1408–1422
Barth C, De Tullio M, Conklin PL (2006) The role of ascorbic acid in the control of flowering time and the onset of senescence. J Exp Bot 57(8):1657–1665
Bartoli CG, Guiamet JJ, Kiddle G, Pastori GM, Cagno RD, Theodoulou FL, Foyer CH (2005) Ascorbate content of wheat leaves is not determined by maximal l-galactono-1, 4-lactone dehydrogenase (GalLDH) activity under drought stress. Plant Cell Environ 28:1073–1081
Carvalho RF, Carvalho SD, O’Grady K, Folta KM (2016) Agroinfiltration of strawberry fruit-a powerful transient expression system for gene validation. Curr Plant Biol 6:19–37
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743
Conklin PL, Barth C (2004) Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant Cell Environ 27:959–970
Cruz-Rus E, Amaya I, Sánchez-Sevilla JF, Botella MA, Valpuesta V (2011) Regulation of l-ascorbic acid content in strawberry fruits. J Exp Bot 62(12):4191–4201
Davey MW, Montagu MV, Inzé D, Sanmartín M, Kanellis AK, Smirnoff N, Benzie IJ, Strain JJ, Favell D, Fletcher J (2000) Plant l-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J Food Sci Agr 80:825–860
Giovannoni JJ (2007) Completing a pathway to plant vitamin C synthesis. Proc Natl Acad Sci USA 104:9109–9110
Guo X, Liu RH, Fu X, Sun X, Tang K (2013) Over-expression of l-galactono-γ-lactone dehydrogenase increases vitamin C, total phenolics and antioxidant activity in lettuce through bio-fortification. Plant Cell Tiss Organ Cult 114:225–236
Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends Plant Sci 6:431–438
Imai T, Karita S, Shiratori G, Hattori M, Nunome T, Oba K, Hirai M (1998) l-galactono-γ-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis. Plant Cell Physiol 39:1350–1358
Ioannidi E, Kalamaki MS, Engineer C, Pateraki I, Alexandrou D, Mellidou I, Giovannonni J, Kanellis AK (2009) Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. J Exp Bot 60(2):663–678
Ishikawa T, Shigeoka S (2008) Recent advances in ascorbate biosynthesis and the physiological significance of ascorbate peroxidase in photosynthesizing organisms. Biosci Biotechnol Biochem 72(5):1143–1154
Kakan X, Yu Y, Li S, Li X, Huang R, Wang J (2021) Ascorbic acid modulation by ABI4 transcriptional repression of VTC2 in the salt tolerance of Arabidopsis. BMC Plant Biol 21(1):112
Kong F, Deng Y, Zhou B, Wang G, Wang Y, Meng Q (2014) A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress. J Exp Bot 65(1):143–158
Landi M, Fambrini M, Basile A, Salvini M, Guidi L, Pugliesi C (2015) Overexpression of L-galactono-1,4-lactone dehydrogenase (L-GalLDH) gene correlates with increased ascorbate concentration and reduced browning in leaves of Lactuca sativa L. after cutting. Plant Cell Tiss Organ Cult 123:109–120
Leferink NG, van den Berg WA, van Berkel WJ (2008) L-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis. FEBS J 275(4):713–726
Li M, Liang D, Pu F, Ma F, Hou C, Lu T (2009) Ascorbate levels and the activity of key enzymes in ascorbate biosynthesis and recycling in the leaves of 22 Chinese persimmon cultivars. Sci Hortic 120:250–256
Li MJ, Gao J, Ma FW, Liang D, Hou CM (2010) Relationship between expressions of GalDH and GalLDH and ascorbate content in apple fruits. Scientia Agricultura Sinica 43(2):351–357
Linster CL, Clarke SG (2008) l-Ascorbate biosynthesis in higher plants: the role of VTC2. Trends Plant Sci 13(11):567–573
Liu W, An HM, Yang M (2013) Overexpression of Rosa roxburghii L-galactono-1,4-lactone dehydrogenase in tobacco plant enhances ascorbate accumulation and abiotic stress tolerance. Acta Physiol Plant 35:1617–1624
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−△△CT method. Methods 25:402–408
Loewus FA (1999) Biosynthesis and metabolism of ascorbic acid in plants and of analogs of ascorbic acid in fungi. Phytochemistry 52:193–210
Lorence A, Chevone BI, Mendes P, Nessler C (2004) myo-Inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol 134:1200–1205
Ma CH, Ma FW, Li MJ, Han MY, Shu HR (2006) Effects of exogenous ascorbic acid on senescence of detached apple leaves. Acta Horticulturae Sinica 33:1179–1184
Maruta T, Yonemitsu M, Yabuta Y, Tamoi M, Ishikawa T, Shigeoka S (2008) Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis. J Biol Chem 283(43):28842–28851
Matamoros MA, Dalton DA, Ramos J, Clemente MR, Rubio MC, Becana M (2003) Biochemistry and molecular biology of antioxidants in the rhizobia-legume symbiosis. Plant Physiol 133:499–509
Nascimento JR, Higuchi BK, Gomez ML, Oshiro RA, Lajolo FM (2005) l-Ascorbate biosynthesis in strawberries: l-Galactono-1,4-lactone dehydrogenase expression during fruit development and ripening. Postharvest Biol Technol 38:34–42
Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–279
Ôba K, Ishikawa S, Nishikawa M, Mizuno H, Yamamoto T (1995) Purification and properties of l-galactono-γ-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots. J Biochem 117(1):120–124
Østergaard J, Persiau G, Davey MW, Bauw G, Van Montagu M (1997) Isolation of a cDNA coding for l-galactono-γ-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants: purification, characterization, cDNA cloning, and expression in yeast. J Biol Chem 272(48):30009–30016
Pateraki I, Sanmartin M, Kalamaki MS, Gerasopoulos D, Kanellis AK (2004) Molecular characterization and expression studies during melon fruit development and ripening of l-galactono-1,4-lactone dehydrogenase. J Exp Bot 55(403):1623–1633
Rice-Evans C, Miller NJ (1995) Antioxidants-the case for fruit and vegetables in the diet. Br Food J 97:35–40
Rodríguez-Ruiz M, Mateos RM, Codesido V, Corpas FJ, Palma JM (2017) Characterization of the galactono-1,4-lactone dehydrogenase from pepper fruits and its modulation in the ascorbate biosynthesis. Role of Nitric Oxide. Redox Biol 12:171–181
Schimmeyer J, Bock R, Meyer EH (2016) l-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis. Plant Mol Biol 90:117–126
Shi SG, Ma FW, Li YH, Feng FJ, Shang ZZ (2012) Overexpression of L-galactono-1, 4-lactone dehydrogenase (GLDH) in Lanzhou lily (Lilium davidii var. unicolor) via particle bombardment-mediated transformation. In Vitro Cell Dev Biol-Plant 48:1–6
Smirnoff N (1996) The function and metabolism of ascorbic acid in plants. Ann Bot 78:661–669
Smirnoff N, Conklin PL, Loewus FA (2001) Biosynthesis of ascorbic acid in plants: a renaissance. Annu Rev Plant Physiol Plant Mol Biol 52:437–467
Tabata K, Ôba K, Suzuki K, Esaka M (2001) Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for L-galactono-1,4-lactone dehydrogenase. Plant J 27:139–148
Tokunaga T, Miyahara K, Tabata K, Esaka M (2005) Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for L-galactono-1,4-lactone dehydrogenase. Planta 220:854–863
Valpuesta V, Botella MA (2004) Biosynthesis of l-ascorbic acid in plants: new pathways for an old antioxidant. Trends Plant Sci 9:573–577
Wei Q, Luo Q, Wang R, Zhang F, He Y, Zhang Y, Qiu D, Li K, Chang J, Yang G, He G (2017) A wheat R2R3-type MYB transcription factor TaODORANT1 positively regulates drought and salt stress responses in transgenic tobacco plants. Front Plant Sci 8:1374
Wang S, Shi M, Zhang Y, Pan Z, Xie X, Zhang L, Sun P, Feng H, Xue H, Fang C, Zhao J (2022) The R2R3-MYB transcription factor FaMYB63 participates in regulation of eugenol production in strawberry. Plant Physiol 188(4):2146–2165
Wheeler GL, Jones MA, Smirnoff N (1998) The biosynthetic pathway of vitamin C in higher plants. Nature 393:365–369
Yabuta Y, Yoshimura K, Takeda T, Shigeoka S (2000) Molecular characterization of tobacco mitochondrial L-galactono-γ-lactone dehydrogenase and its expression in Escherichia coli. Plant Cell Physiol 41:666–675
Yang A, Dai X, Zhang WH (2012) A R2R3-type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice. J Exp Bot 63(7):2541–2556
Yao PF, Li CL, Zhao XR, Li MF, Zhao HX, Guo JY, Cai Y, Chen H, Wu Q (2017) Overexpression of a Tartary buckwheat gene, FtbHLH3, enhances drought/oxidative stress tolerance in transgenic Arabidopsis. Front Plant Sci 8:625
Zhang Z, Wang J, Zhang R, Huang R (2012) The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis. Plant J 71:273–287
Zhang J, Li B, Yang Y, Mu P, Qian W, Dong L, Zhang K, Liu X, Qin H, Ling H, Wang D (2016) A novel allele of L-galactono-1,4-lactone dehydrogenase is associated with enhanced drought tolerance through affecting stomatal aperture in common wheat. Sci Rep 6:30177
Zhang X, Chen L, Shi Q, Ren Z (2020) SlMYB102, an R2R3-type MYB gene, confers salt tolerance in transgenic tomato. Plant Sci 291:110356
Zhao Y, Yang Z, Ding Y, Liu L, Han X, Zhan J, Wei X, Diao Y, Qin W, Wang P, Liu P, Sajjad M, Zhang X, Ge X (2019) Over-expression of an R2R3 MYB Gene, GhMYB73, increases tolerance to salt stress in transgenic Arabidopsis. Plant Sci 286:28–36
Acknowledgements
This research was supported by the National Key R&D Program of China (2018YFD1000200) and Anhui Provincial Natural Science Foundation (2108085MC105).
Author information
Authors and Affiliations
Contributions
XBX and CBF conceived and designed the experiments and wrote the paper. WWD, XW, LW, and JJL performed the experiments and analyzed the data. JZ and PPS participated in the preparation of the manuscript. All authors have read and approved the final manuscript.
Corresponding authors
Ethics declarations
Conflict of Interest
The authors declare no conflict of interest.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Dun, W., Wei, X., Wang, L. et al. Over-expression of FaGalLDH Increases Ascorbic Acid Concentrations and Enhances Salt Stress Tolerance in Arabidopsis thaliana. J. Plant Biol. 66, 35–46 (2023). https://doi.org/10.1007/s12374-022-09376-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12374-022-09376-z