CsXDH1 gene promotes caffeine catabolism induced by continuous strong light in tea plant

Abstract

Tea plant (Camellia sinensis) is an important cash crop with extensive adaptability in the world. However, complex environmental factors force a large variation of tea quality-related components. Caffeine is essential for the formation of bitter and fresh flavors in tea, and is the main compound of tea that improves human alertness. Continuous strong light stimulation was observed to cause caffeine reduction in tea leaves, but the mechanism is not clear. In this study, the response of tea plant to light intensity was analysed mainly by multi-omics association, antisense oligodeoxynucleotide (asODN) silencing technique, and in vitro enzyme activity assay. The results revealed multiple strategies for light intensity adaptation in tea plant, among which the regulation of chloroplasts, photosynthesis, porphyrin metabolism, and resistance to oxidative stress were prominent. Caffeine catabolism was enhanced in continuous strong light, which may be a light-adapted strategy due to strict regulation by xanthine dehydrogenase (XDH). asODN silencing and enzymatic activity assays confirmed that CsXDH1 is a protein induced by light intensity to catalyze the substrate xanthine. CsXDH1 asODN silencing resulted in significant up-regulation of both caffeine and theobromine in in vitro enzyme activity assay, but not in vivo. CsXDH1 may act as a coordinator in light intensity adaptation, thus disrupting this balance of caffeine catabolism.

Acknowledgements

This work was supported by the Chongqing Technology Innovation and Application Demonstration Project (cstc2021jscx-gksbX0016) and the Germplasm Creation Research Program of Southwest University.

Author contributions

Z.W., L.Z., L.L., and C.Y. designed and supervised this research; Q.T. and K.L. performed this research; Q.T. and Z.W. analysed data; Q.T. drafted the manuscript; Z.W. and L.Z. revised and finalized the manuscript.

Data availability

All relevant data in this study are provided in the article and its supplementary file.

Conflict of interest

All authors declare that they have no conflict of interest.

References

[1] Ashihara H, Mizuno K, Yokota T et al. Xanthine Alkaloids: Occurrence, Biosynthesis, and Function in Plants. In: Kinghorn A, Falk H, Gibbons S, Kobayashi J, eds. Progress in the Chemistry of Organic Natural Products, Vol. 105. Springer: Cham, 2017; 105: 1-88.

[2] Yan J, Tong H . An overview of bitter compounds in foodstuffs: classifications, evaluation methods for sensory contribution, separation and identification techniques, and mechanism of bitter taste transduction. Compr Rev Food Sci Food Saf. 2023; 22: 187-232.

[3] Guo C, Li J, Chen Y et al. Complexation of caffeine and theophylline with epigallocatechin gallate in aqueous solution: nuclear magnetic resonance, molecular docking and thermodynamics studies. Food Res Int. 2021; 148: 110587.

[4] McLellan TM, Caldwell JA, Lieberman HR . A review of caffeine’s effects on cognitive, physical and occupational performance. Neurosci Biobehav Rev. 2016; 71: 294-312.

[5] Smith A . Effects of caffeine on human behavior. Food Chem Toxicol. 2002; 40: 1243-55.

[6] Turnbull D, Rodricks JV, Mariano GF . Neurobehavioral hazard identification and characterization for caffeine. Regul Toxicol Pharmacol. 2016; 74: 81-92.

[7] Wikoff D, Welsh BT, Henderson R et al. Systematic review of the potential adverse effects of caffeine consumption in healthy adults, pregnant women, adolescents, and children. Food Chem Toxicol. 2017; 109: 585-648.

[8] Zhang S, Jin J, Chen J et al. Purine alkaloids in tea plants: component, biosynthetic mechanism and genetic variation. Beverage Plant Res. 2022; 2: 1-9.

[9] Ashihara H, Kubota H . Patterns of adenine metabolism and caffeine biosynthesis in different parts of tea seedlings. Physiol Plant. 1986; 68: 275-81.

[10] Meyer A, Ngiruwonsanga T, Henze G . Determination of adenine, caffeine, theophylline and theobromine by HPLC with amperometric detection. Fresenius J Anal Chem. 1996; 356: 284-7.

[11] Chen L, Zhou ZX . Variations of main quality components of tea genetic resources [ Camellia sinensis (L.) O. Kuntze] preserved in the China national germplasm tea repository . Plant Foods Hum Nutr. 2005; 60: 31-5.

[12] Ashihara H, Sano H, Crozier A . Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry. 2008; 69: 841-56.

[13] Ashihara H, Crozier A . Biosynthesis and metabolism of caffeine and related purine alkaloids in plants. Adv Bot Res. 1999; 30: 117-205.

[14] Kato M, Mizuno K, Fujimura T et al. Purification and characterization of caffeine synthase from tea leaves. Plant Physiol. 1999; 120: 579-86.

[15] Kato M, Mizuno K, Crozier A et al. Caffeine synthase gene from tea leaves. Nature. 2000; 406: 956-7.

[16] Ma JQ, Jin JQ, Yao MZ et al. Quantitative trait loci mapping for theobromine and caffeine contents in tea plant (Camellia sinensis). J Agric Food Chem. 2018; 66: 13321-7.

[17] Suzuki T . The participation of S-adenosylmethionine in the biosynthesis of caffeine in the tea plant . FEBS Lett. 1972; 24: 18-20.

[18] Koshiishi C, Kato A, Yama S et al. A new caffeine biosynthetic pathway in tea leaves: utilisation of adenosine released from the S-adenosyl-L-methionine cycle . FEBS Lett. 2001; 499: 50-4.

[19] Ashihara H, Gillies FM, Crozier A . Metabolism of caffeine and related purine alkaloids in leaves of tea (Camellia sinensis L.). Plant Cell Physiol. 1997; 38: 413-9.

[20] Wang W, Zhu BY, Wang P et al. Enantiomeric trimethylallantoin monomers, dimers, and trimethyltriuret: evidence for an alternative catabolic pathway of caffeine in tea plant. Org Lett. 2019; 21: 5147-51.

[21] Deng WW, Ashihara H . Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings . Plant Cell Physiol. 2010; 51: 2105-18.

[22] Deng C, Ku X, Cheng LL et al. Metabolite and transcriptome profiling on xanthine alkaloids-fed tea plant (Camellia sinensis) shoot tips and roots reveal the complex metabolic network for caffeine biosynthesis and degradation . Front Plant Sci. 2020; 11: 551288.

[23] Ashihara H, Stasolla C, Fujimura T et al. Purine salvage in plants. Phytochemistry. 2018; 147: 89-124.

[24] Kottawa-Arachchi JD, Gunasekare MTK, Ranatunga MAB . Biochemical diversity of global tea [ Camellia sinensis (L.) O. Kuntze] germplasm and its exploitation: a review . Genet Resour Crop Evol. 2019; 66: 259-73.

[25] Zeng L, Zhou X, Liao Y et al. Roles of specialized metabolites in biological function and environmental adaptability of tea plant (Camellia sinensis) as a metabolite studying model . J Adv Res. 2021; 34: 159-71.

[26] Wang W, Xin H, Wang M et al. Transcriptomic analysis reveals the molecular mechanisms of drought-stress-induced decreases in Camellia sinensis leaf quality . Front Plant Sci. 2016; 7: 385.

[27] Safaei Chaeikar S, Marzvan S, Jahangirzadeh Khiavi S et al. Changes in growth, biochemical, and chemical characteristics and alteration of the antioxidant defense system in the leaves of tea clones (Camellia sinensis L.) under drought stress . Sci Hortic. 2020; 265: 109257.

[28] Yang YZ, Li T, Teng RM et al. Low temperature effects on carotenoids biosynthesis in the leaves of green and albino tea plant (Camellia sinensis (L.) O. Kuntze). Sci Hortic. 2021; 285: 110164.

[29] Li X, Wei JP, Ahammed GJ et al. Brassinosteroids attenuate moderate high temperature-caused decline in tea quality by enhancing theanine biosynthesis in Camellia sinensis L. Front Plant Sci. 2018; 9: 1016.

[30] Song R, Kelman D, Johns KL et al. Correlation between leaf age, shade levels, and characteristic beneficial natural constituents of tea (Camellia sinensis) grown in Hawaii . Food Chem. 2012; 133: 707-14.

[31] Lee LS, Choi JH, Son N et al. Metabolomic analysis of the effect of shade treatment on the nutritional and sensory qualities of green tea. J Agric Food Chem. 2013; 61: 332-8.

[32] Zhang Q, Shi Y, Ma L et al. Metabolomic analysis using ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS) uncovers the effects of light intensity and temperature under shading treatments on the metabolites in tea. PLoS One. 2014; 9: e112572.

[33] Liu L, Li Y, She G et al. Metabolite profiling and transcriptomic analyses reveal an essential role of UVR8-mediated signal transduction pathway in regulating flavonoid biosynthesis in tea plants (Camellia sinensis) in response to shading . BMC Plant Biol. 2018; 18: 233.

[34] Teng J, Yan C, Zeng W et al. Purification and characterization of theobromine synthase in a theobromine-enriched wild tea plant (Camellia gymnogyna Chang) from Dayao Mountain, China . Food Chem. 2020; 311: 125875.

[35] Zhu B, Chen LB, Lu M et al. Caffeine content and related gene expression: novel insight into caffeine metabolism in Camellia plants containing low, normal, and high caffeine concentrations . J Agric Food Chem. 2019; 67: 3400-11.

[36] Saccenti E, Timmerman ME . Approaches to sample size determination for multivariate data: applications to PCA and PLS-DA of omics data. J Proteome Res. 2016; 15: 2379-93.

[37] Westerhuis JA, van Velzen EJJ, Hoefsloot HCJ et al. Multivariate paired data analysis: multilevel PLSDA versus OPLSDA. Metabolomics. 2010; 6: 119-28.

[38] Zheng C, Ma JQ, Ma CL et al. Regulation of growth and flavonoid formation of tea plants (Camellia sinensis) by blue and green light . J Agric Food Chem. 2019; 67: 2408-19.

[39] Xiang P, Zhu Q, Tukhvatshin M et al. Light control of catechin accumulation is mediated by photosynthetic capacity in tea plant (Camellia sinensis). BMC Plant Biol. 2021; 21: 478.

[40] Liu ZW, Li H, Wang WL et al. CsGOGAT is important in dynamic changes of theanine content in postharvest tea plant leaves under different temperature and shading spreadings. J Agric Food Chem. 2017; 65: 9693-702.

[41] Kumar D, Bansal G, Narang A et al. Integrating transcriptome and proteome profiling: strategies and applications. Proteomics. 2016; 16: 2533-44.

[42] Wang C, Liu N, Geng Z et al. Integrated transcriptome and proteome analysis reveals brassinosteroid-mediated regulation of cambium initiation and patterning in woody stem. Hortic Res. 2022; 9: uhab048.

[43] Li Z, Liu N, Zhang W et al. Integrated transcriptome and proteome analysis provides insight into chilling-induced dormancy breaking in Chimonanthus praecox. Hortic Res. 2020; 7: 198.

[44] Alseekh S, Zhu F, Vallarino JG et al. Autophagy modulates the metabolism and growth of tomato fruit during development. Hortic Res. 2022; 9: uhac129.

[45] Szymańska R, Ślesak I, Orzechowska A et al. Physiological and biochemical responses to high light and temperature stress in plants. Environ Exp Bot. 2017; 139: 165-77.

[46] Phua SY, De Smet B, Remacle C et al. Reactive oxygen species and organellar signaling. J Exp Bot. 2021; 72: 5807-24.

[47] Roeber VM, Bajaj I, Rohde M et al. Light acts as a stressor and influences abiotic and biotic stress responses in plants. Plant Cell Environ. 2021; 44: 645-64.

[48] Agati G, Azzarello E, Pollastri S et al. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 2012; 196: 67-76.

[49] Smirnoff N. Ascorbic acid metabolism and functions: a comparison of plants and mammals. Free Radic Biol Med. 2018; 122: 116-29.

[50] Noctor G, Mhamdi A, Chaouch S et al. Glutathione in plants: an integrated overview. Plant Cell Environ. 2012; 35: 454-84.

[51] Suzuki T, Waller GR . Effects of light on the production and degradation of caffeine in Camellia sinensis L. seedlings . Plant Cell Physiol. 1985; 26: 765-8.

[52] Brychkova G, Fluhr R, Sagi M . Formation of xanthine and the use of purine metabolites as a nitrogen source in Arabidopsis plants . Plant Signal Behav. 2008; 3: 999-1001.

[53] Ma X, Wang W, Bittner F et al. Dual and opposing roles of xanthine dehydrogenase in defense-associated reactive oxygen species metabolism in Arabidopsis. Plant Cell. 2016; 28: 1108-26.

[54] Takagi H, Ishiga Y, Watanabe S et al. Allantoin, a stress-related purine metabolite, can activate jasmonate signaling in a MYC2-regulated and abscisic acid-dependent manner. J Exp Bot. 2016; 67: 2519-32.

[55] Chen D, Yuan K, Zhang J et al. Response analysis of fluorescence parameters of tomato seedlings oriented to vertical light environment adaptation. Plant Sci. 2022; 314: 111118.

[56] Lazzarin M, Meisenburg M, Meijer D et al. LEDs make it resilient: effects on plant growth and defense. Trends Plant Sci. 2021; 26: 496-508.

[57] Fu W, Li P, Wu Y . Effects of different light intensities on chlorophyll fluorescence characteristics and yield in lettuce. Sci Hortic. 2012; 135: 45-51.

[58] Liu Y, Luo L, Liao C et al. Effects of brewing conditions on the phytochemical composition, sensory qualities and antioxidant activity of green tea infusion: a study using response surface methodology. Food Chem. 2018; 269: 24-34.

[59] Wang J, Liu L, Tang Q et al. Evaluation and selection of suitable qRT-PCR reference genes for light responses in tea plant (Camellia sinensis). Sci Hortic. 2021; 289: 110488.

[60] Pfaffl MW . A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29: e45.