不同黄化茶树种质中咖啡碱合成部位的研究

doi:10.13305/j.cnki.jts.2024.04.011

摘要/Abstract

摘要： 咖啡碱作为茶树中的主要特征代谢物,是茶叶品质风味形成的重要组分和天然功能性成分。目前,茶树中咖啡碱的功能作用、分布规律、合成途径及关键基因已基本探明,但在亚细胞水平上,咖啡碱的合成部位有待进一步明确。以白鸡冠茶树及其自然杂交后代的不同黄化单株为材料,通过透射电镜对叶片细胞超微结构的观察,发现黄化叶片中叶绿体结构均有不同程度的受损,且与叶片SPAD值及叶色表型紧密相关;通过高效液相色谱法测定咖啡碱含量,发现黄化叶片中仍有大量咖啡碱积累,甚至超过正常绿色叶片。通过咖啡碱合成关键基因CsTCS1表达量测定、原位杂交及亚细胞定位发现,CsTCS1在不同黄化茶树种质叶片中的表达信号强度存在差异,但表达部位基本相同,主要分布在栅栏组织的细胞核和细胞质中;在亚细胞水平上,茶树叶片中咖啡碱的合成部位主要是细胞核和细胞质。

关键词: 茶树, 黄化叶片, 咖啡碱, 合成部位, 亚细胞水平

Abstract: As the main characteristic metabolite in tea plants, caffeine contributes to tea quality and flavor formation and is a natural functional component. Its function, distribution, biosynthetic pathway and related key genes in tea plants have been basically identified, but its synthetic site at subcellular level needs to be further clarified. In this study, ‘Baijiguan’ and its half-sib offsprings with different etiolated leaves were used as materials. The results of transmission electron microscopy show that the chloroplast structures in etiolated leaves were damaged or destroyed, which was closely related to the SPAD value and leaf phenotype. High performance liquid chromatography (HPLC) was used to determine the caffeine content. It was found that there was still a large amount of caffeine accumulation in etiolated leaves, even more than in normal green leaves. Then, the expression and location of CsTCS1, a key gene involved in caffeine synthesis, were studied by real-time PCR, in-situ hybridization and subcellular localization. It was shown that the expression level of CsTCS1 in different etiolated leaves varied obviously. But the expression site was basically consistent, mainly distributed in the nucleus and cytoplasm of palisade tissues. These results reveal that the synthetic site of caffeine at subcellular level in tea leaves were mainly nucleus and cytoplasm, but not chloroplasts.

Key words: tea plants, etiolated leaf, caffeine, synthetic site, subcellular level

中图分类号:

S571.1
Q52

张亚真, 钟思彤, 陈志辉, 孔祥瑞, 单睿阳, 郑士琴, 余文权, 陈常颂. 不同黄化茶树种质中咖啡碱合成部位的研究[J]. 茶叶科学, 2024, 44(4): 575-584. doi: 10.13305/j.cnki.jts.2024.04.011.

ZHANG Yazhen, ZHONG Sitong, CHEN Zhihui, KONG Xiangrui, SHAN Ruiyang, ZHENG Shiqin, YU Wenquan, CHEN Changsong. Study on the Synthetic Site of Caffeine in Different Etiolated Tea Germplasms[J]. Journal of Tea Science, 2024, 44(4): 575-584. doi: 10.13305/j.cnki.jts.2024.04.011.

参考文献

[1] Gramza-Michalowska A.Caffeine in tea camellia sinensis: content, absorption, benefits and risks of consumption[J]. The Journal of Nutrition, Health& Aging, 2014, 18(2): 143-149.
[2] 宛晓春. 茶叶生物化学[M]. 3版. 北京: 中国农业出版社, 2003.
Wan X C.Tea biochemistry [M]. 3rd ed. Beijing: China Agriculture Press, 2003.
[3] Mohanpuria P, Kumar V, Yadav S K.Tea caffeine: metabolism, functions, and reduction strategies[J]. Food Science and Biotechnology, 2010, 19(2): 275-287.
[4] Ashihara H, Suzuki T.Distribution and biosynthesis of caffeine in plants[J]. Frontiers in Bioscience, 2004, 9: 1864-1876.
[5] Ashihara H, Sano H, Crozier A.Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering[J]. Phytochemistry, 2008, 69: 841-856.
[6] 吴华玲, 陈栋, 李家贤. 茶树咖啡碱代谢及低咖啡碱茶树育种研究进展[J]. 热带作物学报, 2011, 32(9): 1780-1785.
Wu H L, Chen D, Li J X.Research progress in caffeine metabolism and low caffeine content germplasm breeding of tea plants (Camellia sinensis (L.) O. Kuntze)[J]. Chinese Journal of Tropical Crops, 2011, 32(9): 1780-1785.
[7] 晏嫦妤, 任秋婧, 陈小芳, 等. 咖啡碱合成N-甲基转移酶研究进展[J]. 茶叶科学, 2014, 34(6): 531-540.
Yan C Y, Ren Q J, Chen X F, et al.Research progress of N-methyltransferases involved in caffeine biosynthesis[J]. Journal of Tea Science, 2014, 34(6): 531-540.
[8] 刘玉飞, 金基强, 姚明哲, 等. 茶树咖啡碱合成酶基因稀有等位变异TCS1g的筛选、克隆及功能[J]. 中国农业科学, 2019, 52(10): 1772-1783.
Liu Y F, Jin J Q, Yao M Z, et al.Screening, cloning and functional research of the rare allelic variation of caffeine synthase gene (TCS1g) in tea plant[J]. Scientia Agricultura Sinica, 2019, 52(10): 1772-1783.
[9] Zhou M, Yan C, Zeng Z, et al.N-methyltransferases of caffeine biosynthetic pathway in plants[J]. Journal of Agricultural and Food Chemistry, 2020, 68: 15359-15372.
[10] Wang Y, Liu Y F, Wei M Y, et al. Deeply functional identification of TCS1 alleles provides efficient technical paths for low-caffeine breeding of tea plants [J]. Horticulture Research, 2023, 10(2): uhac279. doi: 10.1093/hr/uhac279.
[11] Wang Q, Wu Y, Peng A, et al.Single-cell transcriptome atlas reveals developmental trajectories and a novel metabolic pathway of catechin esters in tea leaves[J]. Plant Biotechnology Journal, 2022, 20: 2089-2106.
[12] 关智晶, 孙超. 植物次生代谢的区室化研究进展[J]. 生物技术通报, 2024, 40(1): 1-11.
Guan Z J, Sun C.Research progress in the compartmentalization of plant specialized metabolism[J]. Biotechnology Bulletin, 2024, 40(1): 1-11.
[13] Ashihara H, Yokota T, Crozier A.Biosynthesis and catabolism of purine alkaloids[J]. Advances in Botanical Research, 2013, 68: 111-138.
[14] 韦康, 王丽鸳, 王新超, 等. 黄茶“中黄2号”的亚细胞结构透射电镜观察[J]. 食品与生物技术学报, 2017, 36(12): 1246-1250.
Wei K, Wang L Y, Wang X C, et al.Transmission electron microscopic study of subcellular structure of yellow tea cultivar ‘Zhonghuang 2’[J]. Journal of Food Science and Biotechnology, 2017, 36(12): 1246-1250.
[15] 张晨禹, 王铭涵, 高羲之, 等. 茶树‘湘妃翠’黄化枝光合生理与组织学[J]. 分子植物育种, 2019, 17(23): 7892-7900.
Zhang C Y, Wang M H, Gao X Z, et al.Photosynthetic physiological and histology in novel etiolated branch of the ‘Xiangfeicui’ tea plant (Camellia sinensis)[J]. Molecular Plant Breeding, 2019, 17(23): 7892-7900.
[16] 王丽鸳, 赵容波, 成浩, 等. 叶色特异茶树品种选育现状[J]. 中国茶叶, 2020, 42(1): 15-19.
Wang L Y, Zhao R B, Cheng H, et al.Current situation on breeding of tea cultivars with special leaf colors[J]. China Tea, 2020, 42(1): 15-19
[17] Zhang Y Z, Wei K, Guo L L, et al.Functional identification of purine permeases reveals their roles in caffeine transport in tea plants(Camellia sinensis)[J]. Frontiers in Plant Science, 2022(13): 1033316. doi:10.3389/fpls.2022.1033316.
[18] Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J]. Methods, 2001, 25(4): 402-408.
[19] Kato A, Crozier A, Ashihara H.Subcellular localization of the N-3 methyltransferase involved in caffeine biosynthesis in tea[J]. Phytochemistry, 1998, 48(5): 777-779.
[20] Breda S V, Merwe C F, Robbertse H.Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves[J]. Planta, 2013, 237: 849-858.
[21] 李娜娜. 新梢白化茶树生理生化特征及白化分子机理研究[D]. 杭州: 浙江大学, 2015.
Li N N.Physiological, biochemical characteristics and molecular albinism of the albino tea (Camellia sinensis) plant [D]. Hangzhou: Zhejiang University, 2015.
[22] Chen X, Li J, Yu Y, et al.STAY-GREEN and light-harvesting complex II chlorophyll a/b binding protein are involved in albinism of a novel albino tea germplasm ‘Huabai 1’[J]. Scientia Horticultuare, 2022, 293: 110653. doi: 10.1016/j.scienta.2021.110653.
[23] 田月月. 黄金芽茶树叶色响应光质的生理特性及机制研究[D]. 泰安: 山东农业大学, 2020.
Tian Y Y.Mechanism of physiological characteristics of leaf color in Camellia sinensis cv. Huangjinya response to light quality [D]. Tai′an: Shandong Agricultural University, 2020.
[24] 林馨颖, 王鹏杰, 杨如兴, 等. 高茶氨酸茶树新品系‘福黄1号’黄化变异机理[J]. 中国农业科学, 2022, 55(9): 1831-1845.
Lin X Y, Wang P J, Yang R X, et al.The albino mechanism of a new high theanine tea cultivar Fuhuang 1[J]. Scientia Agricultura Sinica, 2022, 55(9): 1831-1845.
[25] Li Q, Huang J A, Liu S Q, et al.Proteomic analysis of young leaves at three developmental stages in an albino tea cultivar[J]. Proteome Science, 2011, 9(31): 1-12. doi: 10.1186/1477-5956-9-44
[26] 娄艳华, 何卫中, 刘瑜, 等. 14个黄化、白化变异茶树品种(系)综合性状评价与分析[J]. 茶叶, 2020, 46(2): 84-90.
Lou Y H, He W Z, Liu Y, et al.Comprehensive assessment of quality traits of 14 etiolated and albino tea cultivars[J]. Journal of Tea, 2020, 46(2): 84-90.
[27] Mohanpuria P, Kumar V, Joshi R, et al.Caffeine biosynthesis and degradation in tea [Camellia sinensis (L.) O. Kuntze] is under developmental and seasonal regulation[J]. Molecular Biotechnology, 2009, 43: 104-111.
[28] 李金, 魏艳丽, 庞磊, 等. 茶树咖啡碱合成途径中TCS1、TIDH、SAMS的基因表达量差异及其与咖啡碱含量的相关性[J]. 江苏农业科学, 2013, 41(10): 21-24.
Li J, Wei Y L, Pang L, et al.Different gene expression levels of TCS1, TIDH, and SAMS in caffeine synthesis pathway and their correlation with caffeine content in tea plants[J]. Jiangsu Agricultural Sciences, 2013, 41(10): 21-24.
[29] 李远华, 江昌俊, 宛晓春. 茶树咖啡碱合成酶基因mRNA表达的研究[J]. 茶叶科学, 2004, 24(1): 23-28.
Li Y H, Jiang C J, Wan X C.Study on the expression of caffeine synthase gene mRNA in tea plant[J]. Journal of Tea Science, 2004, 24(1): 23-28.
[30] Li Y, Gu W, Ye S.Expression and location of caffeine synthase in tea plants[J]. Russian Journal of Plant Physiology, 2007, 54(5): 698-701.
[31] Zhong H, Wang Y, Qu F R, et al. A novel TcS allele conferring the high-theacrine and low-caffeine traits and having potential use in tea plant breeding [J]. Horticulture Research, 2022, 9: uhac191. doi: 10.1093/hr/uhac191.
[32] Teng J, Yan C Y, Zeng W, et al.Purification and characterization of Theobromine Synthase in a Theobromine-Enriched Wild Tea Plant(Camellia gymnogyna Chang) from Dayao Mountain, China[J]. Food Chemistry, 2020(311): 125875. doi: 10.1016/j.foodchem.2019.125875.
[33] Ogawa M, Herai Y, Koizumi N, et al.7-Methylxanthine methyltransferase of coffee plants[J]. The Journal of Biological Chemistry, 2001, 276: 8213-8218.
[34] Kodama Y, Shinya T, Sano H.Dimerization of N-methyltransferases involved in caffeine biosynthesis[J]. Biochimie, 2008, 90(3): 547-551.