Differentially expressed protein and gene analysis revealed the effects of temperature on changes in ascorbic acid metabolism in harvested tea leaves

Abstract

Tea is an important non-alcoholic beverage worldwide. Tea quality is determined by numerous secondary metabolites in harvested tea leaves, including tea polyphenols, theanine, caffeine, and ascorbic acid (AsA). AsA metabolism in harvested tea leaves is affected by storage and transportation temperature. However, the molecular mechanisms underlying AsA metabolism in harvested tea leaves exposed to different storage and transportation temperature conditions remain unclear. Here we performed RP-HPLC to detect dynamic changes in AsA content in tea leaves subjected to high- (38 °C), low- (4 °C), or room-temperature (25 °C) treatments. The AsA distribution and levels in the treated tea leaves were analyzed using cytological–anatomical characterization methods. The differentially expressed CsAPX1 and CsDHAR2 proteins, which are involved in the AsA recycling pathway, were identified from the corresponding proteomic data using iTRAQ. We also analyzed the expression profiles of 18 genes involved in AsA metabolism, including CsAPX1 and CsDHAR2. AsA was mainly distributed in tea leaf mesophyll cells. High- and low- temperature treatments upregulated the CsAPX1 and CsDHAR2 proteins and induced CsAPX and CsDHAR2 gene expression. These results indicated that the CsAPX1 and CsDHAR2 proteins might have critical roles in AsA recycling in tea leaves. Our results provide a foundation for the in-depth investigation of AsA metabolism in tea leaves during storage and transportation, and they will promote better tea flavor in tea production.

References

[1] Huang, J. et al. The anti-obesity effects of green tea in human intervention and basic molecular studies. Eur. J. Clin. Nutr. 68, 1075-1087 (2014).

[2] Lambert, J. D., Sang, S. & Yang, C. S. Biotransformation of green tea polyphenols and the biological activities of those metabolites. Mol. Pharm. 4, 819-825 (2007).

[3] Wolfram, S. Effects of green tea and EGCG on cardiovascular and metabolic health. J. Am. Coll. Nutr. 26, 373S-388S (2007).

[4] Mandel, S., Amit, T., Baram, O. & Youdim, M. B. Iron dysregulation in Alzheimer’s disease: multimodal brain permeable iron chelating drugs, possessing neuroprotective-neurorescue and amyloid precursor protein-processing regulatory activities as therapeutic agents. Prog. Neurobiol. 82, 348-360 (2007).

[5] Gallie, D. R. The role of L-ascorbic acid recycling in responding to environmental stress and in promoting plant growth. J. Exp. Bot. 64, 433-443 (2013).

[6] Linster, C. L. & Van, S. E. Vitamin C. Biosynthesis, recycling and degradation in mammals. FEBS J. 274, 1-22 (2007).

[7] Padayatty, S. J. et al. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J. Am. Coll. Nutr. 22, 18-35 (2003).

[8] Wang, J., Zhang, Z. & Huang, R. Regulation of ascorbic acid synthesis in plants. Plant Signal. Behav. 8, e24536- e24536 (2013).

[9] Conklin, P. L. & Barth, C. 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 (2004).

[10] Hasegawa, N., Niimi, N. & Odani, F. Vitamin C is one of the lipolytic substances in green tea. Phytother. Res. 16, 91-92 (2002).

[11] Ivanov, V., Roomi, M. W., Kalinovsky, T., Niedzwiecki, A. & Rath, M. Antiatherogenic effects of a mixture of ascorbic acid, lysine, proline, arginine, cysteine, and green tea phenolics in human aortic smooth muscle cells. J. Cardiovasc. Pharm. 49, 140-145 (2007).

[12] Sárközi, K. et al. Green tea and vitamin C ameliorate some neuro-functional and biochemical signs of arsenic toxicity in rats. Nutr. Neurosci. 19, 102-109 (2016).

[13] Son, Y. R., Park, T. S. & Shim, S. M. Pharmacokinetics and plasma cellular antioxidative effects of flavanols after oral Intake of green tea formulated with vitamin C and xylitol in healthy subjects. J. Med. Food 19, 211-217 (2016).

[14] Agius, F. et al. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat. Biotechnol. 21, 177-181 (2003).

[15] Lorence, A., Chevone, B. I., Mendes, P. & Nessler, C. L. myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol. 134, 1200-1205 (2004).

[16] Wheeler, G. L., Jones, M. A. & Smirnoff, N. The biosynthetic pathway of vitamin C in higher plants. Nature 393, 365-369 (1998).

[17] Wolucka, B. A. & Montagu, M. V. GDP-mannose 3′,5′-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J. Biol. Chem. 278, 47483-47490 (2003).

[18] Li, M., Ma, F., Guo, C. & Liu, J. Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages. Plant Physiol. Biochem. 48, 216-224 (2010).

[19] Davey, M. W. et al. Ascorbate biosynthesis in Arabidopsis cell suspension culture. Plant Physiol. 121, 535-543 (1999).

[20] Wolucka, B. A. & Van Montagu, M. The VTC2 cycle and the de novo biosynthesis pathways for vitamin C in plants: an opinion. Phytochemistry 68, 2602-2613 (2007).

[21] Zhang, W., Gruszewski, H. A., Chevone, B. I. & Nessler, C. L. An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate. Plant Physiol. 146, 431-440 (2008).

[22] Linster, C. L. & Clarke, S. G. L-Ascorbate biosynthesis in higher plants: the role of VTC2. Trends Plant Sci. 13, 567-573 (2008).

[23] Li, H. et al. Transcriptomic analysis of the biosynthesis, recycling, and distribution of ascorbic acid during leaf development in tea plant (Camellia sinensis (L.) O. Kuntze) . Sci. Rep. 7, 46212 (2017).

[24] Imai, T., Ban, Y., Terakami, S., Yamamoto, T. & Moriguchi, T. L-Ascorbate biosynthesis in peach: cloning of six L-galactose pathway-related genes and their expression during peach fruit development. Physiol. Plant. 136, 139-149 (2009).

[25] Dowdle, J., Ishikawa, T., Gatzek, S., Rolinski, S. & Smirnoff, N. Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability . Plant J. 52, 673-689 (2007).

[26] Wang, G. L. et al. Regulation of ascorbic acid biosynthesis and recycling during root development in carrot (Daucus carota L) . Plant Physiol. Biochem. 94, 10-18 (2015).

[27] Huang, W. et al. Transcriptional profiling of genes involved in ascorbic acid biosynthesis, recycling, and degradation during three leaf developmental stages in celery. Mol. Genet. Genomics 291, 1-13 (2016).

[28] Massot, C. et al. High temperature inhibits ascorbate recycling and light stimulation of the ascorbate pool in tomato despite increased expression of biosynthesis genes. PLoS ONE 8, e84474 (2013).

[29] Li, H., Huang, W., Wang, G., Wu, Z. & Zhuang, J. Expression profile analysis of ascorbic acid-related genes in response to temperature stress in the tea plant, Camellia sinensis (L) O. Kuntze . Genet. Mol. Res. 15, 1-10 (2016).

[30] Zhang, H., Wang, J., Nickel, U., Allen, R. D. & Goodman, H. M. Cloning and expression of an Arabidopsis gene encoding a putative peroxisomal ascorbate peroxidase. Plant Mol. Biol. 34, 967-971 (1997).

[31] Bonifacio, A. et al. Role of peroxidases in the compensation of cytosolic ascorbate peroxidase knockdown in rice plants under abiotic stress. Plant Cell Environ. 34, 1705-1722 (2011).

[32] Fryer, M. J. et al. Control of ascorbate peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves. Plant J. 33, 691-705 (2003).

[33] Shi, W. M., Muramoto, Y., Ueda, A. & Takabe, T. Cloning of peroxisomal ascorbate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana . Gene 273, 23-27 (2001).

[34] Sun, W. H. et al. Overexpression of tomato tAPX gene in tobacco improves tolerance to high or low temperature stress. Biol. Plant. 54, 614-620 (2010).

[35] Haroldsen, V. M., Chi-Ham, C. L., Kulkarni, S., Lorence, A. & Bennett, A. B. Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato. Plant Physiol. Biochem. 49, 1244-1249 (2011).

[36] Stevens, R. et al. Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress. Plant Cell Environ. 31, 1086-1096 (2008).

[37] Gest, N., Garchery, C., Gautier, H., Jimenez, A. & Stevens, R. Light-dependent regulation of ascorbate in tomato by a monodehydroascorbate reductase localized in peroxisomes and the cytosol. Plant Biotechnol. J. 11, 344-354 (2013).

[38] Yin, L. et al. Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco. Planta 231, 609-621 (2010).

[39] Ushimaru, T. et al. Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress. Plant Physiol. 163, 1179-1184 (2006).

[40] Wu, Z. J., Tian, C., Jiang, Q., Li, X. H. & Zhuang, J. Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant (Camellia sinensis) . Sci. Rep. 6, 19748 (2016).

[41] Wu, Z. J., Ma, H. Y. & Zhuang, J. iTRAQ-based proteomics monitors the withering dynamics in postharvest leaves of tea plant (Camellia sinensis) . Mol. Genet. Genomics 293, 1-15 (2017).

[42] Liu, Z. W. et al. CsGOGAT is important in dynamic changes of theanine content in post-harvest tea plant leaves under different temperature and shading spreadings . J. Agric. Food Chem. 65, 9693-9702 (2017).

[43] Wang, G. L. et al. Morphological characteristics, anatomical structure, and gene expression: novel insights into gibberellin biosynthesis and perception during carrot growth and development. Hortic. Res. 2, 15028 (2015).

[44] Chinoy, N. J. On the specificity of the alcoholic, acidic silver nitrate reagent for the histochemical localization of ascorbic acid. Histochemie 20, 105-107 (1969).

[45] Szklarczyk, D. et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 43, 87-105 (2015).

[46] Kuhn, M., von Mering, C., Campillos, M., Jensen, L. J. & Bork, P. STITCH: interaction networks of chemicals and proteins. Nucleic Acids Res. 36, 684-688 (2008).

[47] Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98 (1999).

[48] Wu, Z. J., Li, X. H., Liu, Z. W., Xu, Z. S. & Zhuang, J. De novo assembly and transcriptome characterization: novel insights into catechins biosynthesis in Camellia sinensis . BMC Plant. Biol. 14, 1-16 (2014).

[49] Wei, C. et al. Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality . Proc. Natl Acad. Sci. USA 115, E4151-E4158 (2018).

[50] Lee, T. C., Zhong, P. J. & Chang, P. T. The effects of preharvest shading and postharvest storage temperatures on the quality of ‘Ponkan’ (Citrus reticulata Blanco) mandarin fruits . Sci. Hortic. 188, 57-65 (2015).

[51] Vermaab, A. K., Agarwal, A. K. & Solomon, S. Influence of postharvest storage temperature, time, and invertase enzyme activity on sucrose and weight loss in sugarcane. Postharvest Biol. Technol. 73, 14-21 (2012).

[52] Martínez-Romero, D. et al. Postharvest sweet cherry quality and safety maintenance by Aloe vera treatment: a new edible coating. Postharvest Biol. Technol. 39, 93-100 (2006).

[53] Zhuang, H., Hildebrand, D. F. & Barth, M. M. Temperature influenced lipid peroxidation and deterioration in broccoli buds during postharvest storage. Postharvest Biol. Technol. 10, 49-58 (1997).

[54] Ristic, Z., Williams, G., Yang, G., Martin, B. & Fullerton, S. Dehydration, damage to cellular membranes, and heat-shock proteins in maize hybrids from different climates. Plant Physiol. 149, 424-432 (1996).

[55] Northcote, D. H. Structure and function of plant-cell membranes. Br. Med. Bull. 24, 107-112 (1968).

[56] Yadav, S. K. Cold stress tolerance mechanisms in plants. A review. Agron. Sustain. Dev. 30, 515-527 (2010).

[57] Gest, N., Gautier, H. & Stevens, R. Ascorbate as seen through plant evolution: the rise of a successful molecule?. J. Exp. Bot. 64, 33-53 (2013).

[58] Lee, S. K. & Kader, A. A. Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharvest Biol. Technol. 20, 207-220 (2000).

[59] Kanayama, Y. et al. Seasonal changes in abiotic stress tolerance and concentrations of tocopherol, sugar, and ascorbic acid in sea buckthorn leaves and stems. Sci. Hortic. 164, 232-237 (2013).

[60] Rosales, M. A. et al. Antioxidant content and ascorbate metabolism in cherry tomato exocarp in relation to temperature and solar radiation. J. Sci. Food Agric. 86, 1545-1551 (2006).

[61] Airaki, M. et al. Metabolism of reactive oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under low temperature stress . Plant Cell Environ. 35, 281-295 (2012).

[62] Munné-Bosch, S. & Lalueza, P. Age-related changes in oxidative stress markers and abscisic acid levels in a drought-tolerant shrub, Cistus clusii grown under Mediterranean field conditions . Planta 225, 1039-1049 (2007).

[63] Badejo, A. A. et al. 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. 63, 229-239 (2011).

[64] 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. 174, 606-612 (2008).

[65] Zechmann, B. Subcellular distribution of ascorbate in plants. Plant Signal. Behav. 6, 360-363 (2011).

[66] Zechmann, B., Stumpe, M. & Mauch, F. Immunocytochemical determination of the subcellular distribution of ascorbate in plants. Planta 233, 1-12 (2011).

[67] Shinozaki, K., Yamaguchi-Shinozaki, K. & Seki, M. Regulatory network of gene expression in the drought and cold stress responses. Curr. Opin. Plant Biol. 6, 410-417 (2003).

[68] Zhuang, J., Zhang, J., Hou, X. L., Wang, F. & Xiong, A. S. Transcriptomic, proteomic, metabolomic and functional genomic approaches for the study of abiotic stress in vegetable crops. Crit. Rev. Plant Sci. 33, 225-237 (2014).

[69] Tabata, K. & Takaoka, T. M. Gene expression of ascorbic acid-related enzymes in tobacco. Phytochemistry 61, 631-635 (2002).

[70] Tang, L. et al. Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature. Plant Cell Rep. 25, 1380-1386 (2006).

[71] Ma, Y. H. et al. Effects of high temperature on activities and gene expression of enzymes involved in ascorbate-glutathione cycle in apple leaves. Plant Sci. 175, 761-766 (2009).

[72] Li, J., Liang, D., Li, M. & Ma, F. Light and abiotic stresses regulate the expression of GDP-l-galactose phosphorylase and levels of ascorbic acid in two kiwifruit genotypes via light-responsive and stress-inducible cis-elements in their promoters. Planta 238, 535-547 (2013).

[73] Ioannidi, E. et al. Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. J. Exp. Bot. 60, 663-678 (2009).

[74] Massot, C., Stevens, R., Génard, M., Longuenesse, J. J. & Gautier, H. Light affects ascorbate content and ascorbate-related gene expression in tomato leaves more than in fruits. Planta 235, 153-163 (2012).

[75] Zhang, Y. Y. et al. Suppressed expression of ascorbate oxidase gene promotes ascorbic acid accumulation in tomato fruit. Plant Mol. Biol. Rep. 29, 638-645 (2011).

[76] Lee, S. H. et al. Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses. Plant Physiol. 164, 1626-1638 (2007).

[77] Kawakami, S., Matsumoto, Y., Matsunaga, A., Mayama, S. & Mizuno, M. Molecular cloning of ascorbate peroxidase in potato tubers and its response during storage at low temperature. Plant Sci. 163, 829-836 (2002).

[78] Fan, J. et al. Comparative iTRAQ proteome and transcriptome analyses of sweet orange infected by “Candidatus Liberibacter asiaticus”. Physiol. Plant. 143, 235-245 (2011).

[79] Shetty, V. et al. Quantitative immunoproteomics analysis reveals novel MHC class I presented peptides in cisplatin-resistant ovarian cancer cells. Proteomics 75, 3270-3290 (2012).

[80] Sui, W. et al. Differential proteomic analysis of renal tissue in mesangial proliferative glomerulonephritis using iTRAQ technology. Nephrology 26, 191-198 (2013).

[81] Aragüez, I., Cruzrus, E., Botella, M. Á., Medinaescobar, N. & Valpuesta, V. Proteomic analysis of strawberry achenes reveals active synthesis and recycling of L-ascorbic acid. Proteomics 83, 160-179 (2013).

[82] Lópezvidal, O. et al. Mitochondrial ascorbate-glutathione cycle and proteomic analysis of carbonylated proteins during tomato (Solanum lycopersicum) fruit ripening . Food Chem. 194, 1064-1072 (2016).

[83] Xie, H. et al. iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress . Biochem. Biophys. Res. Commun. 469, 768-775 (2016).

[84] Owiti, J. et al. iTRAQ-based analysis of changes in the cassava root proteome reveals pathways associated with post-harvest physiological deterioration. Plant J. 67, 145-156 (2011).

[85] Fercha, A. et al. Comparative analysis of metabolic proteome variation in ascorbate-primed and unprimed wheat seeds during germination under salt stress. Proteomics 108, 238-257 (2014).

[86] Alhagdow, M. et al. 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 (2007).

[87] Duan, M., Feng, H. L., Wang, L. Y., Li, D. & Meng, Q. W. Overexpression of thylakoidal ascorbate peroxidase shows enhanced resistance to chilling stress in tomato. Plant Physiol. 169, 867-877 (2012).

[88] Koussevitzky, S. et al. Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination . J. Biol. Chem. 283, 34197-34203 (2008).

[89] Tang, Z. X. & Yang, H. L. Functional divergence and catalytic properties of dehydroascorbate reductase family proteins from Populus tomentosa . Mol. Biol. Rep. 40, 5105-5114 (2013).

[90] Reumann, S. et al. In-depth proteome analysis of Arabidopsis leaf peroxisomes combined with in vivo subcellular targeting verification indicates novel metabolic and regulatory functions of peroxisomes. Plant Physiol. 150, 125-143 (2009).

[91] Murgia, I. et al. Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increased resistance to Paraquat-induced photooxidative stress and to nitric oxide-induced cell death . Plant J. 38, 940-953 (2004).

[92] Foyer, C., Rowell, J. & Walker, D. Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157, 239-244 (1983).

[93] Smirnoff, N. Ascorbate biosynthesis and function in photoprotection. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355, 1455-1464 (2000).

[94] Valero, E. et al. Modeling the ascorbate-glutathione cycle in chloroplasts under light/dark conditions. BMC Syst. Biol. 10, 11 (2016).

[95] Huang, G. & Shan, C. Lanthanum improves the antioxidant capacity in chloroplast of tomato seedlings through ascorbate-glutathione cycle under salt stress. Sci. Hortic. 232, 264-268 (2018).