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Journal of Integrative Agriculture  2023, Vol. 22 Issue (11): 3364-3379    DOI: 10.1016/j.jia.2023.10.002
Special Focus: Germplasm and Molecular Breeding in Horticultural Crops Advanced Online Publication | Current Issue | Archive | Adv Search |
Genome-wide association studies reveal the genetic basis of amino acid content variation in tea plants

GUO Ya-fei*, LI Dai-li*, QIU Hai-ji, ZHANG Xiao-liang, LIU Lin, ZHAO Jing-jing, JIANG De-yuan#

National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, P.R.China
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摘要  

茶是世界上最受欢迎的非酒精饮料之一。游离氨基酸,尤其是茶氨酸,鲜味的主要组成。然而,关于茶树氨基酸含量变异的遗传基础仍不清晰此,基于靶向代谢组学的方法本研究连续两年检测了174份茶树种质嫩叶的游离氨基酸含量,并通过转录组分析获得了这些种质的基因型。基于代谢表型和基因型,通过全基因组关联研究研究影响茶树鲜叶游离氨基酸含量变异的位点。本研究鉴定到69-log10 (P-value) 大于 5位点。功能注释的结果分析显示支链氨基酸转移酶、谷氨酰胺合成酶、硝酸盐转运蛋白和谷氨酸脱羧酶可能在氨基酸代谢的过程发挥重要作用。因此,本研究从中选择了两个显著的位点:谷氨酰胺合成酶Glu1P=3.71×10-4Arg1P=4.61×10-5和支链氨基酸转移酶(Val1P=4.67×10-5I_Leu1P=3.56×10-6CsGSCsBCAT进行基因型分析,选择CsGS的两个等位基因CsGS-LCsGS-HCsBCAT的两个等位基因CsBCAT-LCsBCAT-H进行功能验证。CsGS-LCsGS-H过表达提高了转基因植株中谷氨酸和精氨酸含量CsBCAT-LCsBCAT-H过表达促进了缬氨酸、异亮氨酸和亮氨酸的积累。体外酶活性分析发现SNP1054CsGS催化谷氨酸生成谷氨酰胺的酶活性具有重要影响。此外,CsGS-LCsGS-H差异调控谷氨酰胺积累,CsBCAT-LCsBCAT-H差异调控支链氨基酸积累。综上所述,本研究结果将为茶树氨基酸含量变异的遗传基础解析提供新的认识,并为鉴定优质基因以提高茶树氨基酸含量提供理论依据。



Abstract  

Tea is one of the most popular non-alcoholic beverages in the world, and free amino acids, especially theanine, make a major contribution to the umami taste of tea.  However, the genetic basis of the variation in amino acid content in tea plants remains largely unknown.  Here, we measured the free amino acid content in fresh leaves of 174 tea accessions over two years using a targeted metabolomics approach and obtained genotype data via RNA sequencing.  Genome-wide association studies were conducted to investigate loci affecting the content of free amino acids.  A total of 69 quantitative trait loci (–log10(P-value)>5) were identified.  Functional annotation revealed that branched-chain amino acid aminotransferase, glutamine synthetase, nitrate transporter, and glutamate decarboxylase might be important for amino acid metabolism.  Two significant loci, glutamine synthetase (Glu1, P=3.71×10–4; Arg1, P=4.61×10–5) and branched-chain amino acid aminotransferase (Val1, P=4.67×10–5; I_Leu1, P=3.56×10–6), were identified, respectively.  Based on the genotyping result, two alleles of CsGS (CsGS-L and CsGS-H) and CsBCAT (CsBCAT-L and CsBCAT-H) were selected to perform function verification.  Overexpression of CsGS-L and CsGS-H enhanced the contents of glutamate and arginine in transgenic plants, and overexpression of CsBCAT-L and CsBCAT-H promoted the accumulation of valine, isoleucine and leucine.  Enzyme activity assay uncovered that SNP1054 is important for CsGS catalyzing glutamate into glutamine.  Furthermore, CsGS-L and CsGS-H differentially regulated the accumulation of glutamine, and CsBCAT-L and CsBCAT-H differentially regulated the accumulation of branched-chain amino acids.  In summary, the findings in our study would provide new insights into the genetic basis of amino acids contents variation in tea plants and facilitate the identification of elite genes to enhance amino acids content.

Keywords:  Camellia sinensis       amino acids        genetic variation        association studies        genotype analysis        functional verification  
Received: 16 January 2023   Accepted: 12 September 2023
Fund: This work was supported by the Huazhong Agricultural University Scientific & Technological Self-Innovation Foundation, China (2017RC002).
About author:  GUO Ya-fei, E-mail: yfguo@webmail.hzau.edu.cn; LI Dai-li, E-mail: dailili2333@163.com; #Correspondence JIANG De-yuan, E-mail: dyjiang@mail.hzau.edu.cn * These authors contributed equally to this study.

Cite this article: 

GUO Ya-fei, LI Dai-li, QIU Hai-ji, ZHANG Xiao-liang, LIU Lin, ZHAO Jing-jing, JIANG De-yuan. 2023. Genome-wide association studies reveal the genetic basis of amino acid content variation in tea plants. Journal of Integrative Agriculture, 22(11): 3364-3379.

Angelovici R, Lipka A E, Deason N, Gonzalez-Jorge S, Lin H, Cepela J, Buell R, Gore M, Dellapenna D. 2013. Genome-wide analysis of branched-chain amino acid levels in Arabidopsis seeds. Plant Cell25, 4827–4843.

Bai P X, Wei K, Wang L Y, Zhang F, Ruan L, Li H L, Wu L Y, Cheng H. 2019. Identification of a novel gene encoding the specialized alanine decarboxylase in tea (Camellia sinensis) plants. Molecules24, 540–554.

Besnard J, Pratelli R, Zhao C S, Sonawala U, Collakova E, Pilot G, Okumoto S. 2016. UMAMIT14 is an amino acid exporter involved in phloem unloading in Arabidopsis roots. Journal of Experimental Botany67, 6385–6397.

Binder S. 2010. Branched-chain amino acid metabolism in Arabidopsis thalianaThe Arabidopsis Book8, e0137.

Bolger A, Lohse M, Usadel B. 2014. Trimmomatic: A flexible trimmer for illumina sequence data. Bioinformatics30, 2114–2120.

Bradford M B. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry72, 248–254.

Cai H, Zhou Y, Xiao J, Li X, Zhang Q, Lian X. 2009. Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Reports28, 527–537.

Chen J, Hu X, Shi T T, Yin H R, Sun D F, Hao Y F, Xia X C, Luo J, Fernie A R, Chen W. 2020. Metabolite-based genome-wide association study enables dissection of the flavonoid decoration pathway of wheat kernels. Plant Biotechnology Journal18, 1722–1735.

Cheng S H, Fu X M, Wang X Q, Liao Y Y, Zeng L T, Dong F, Yang Z Y. 2017. Studies on the biochemical formation pathway of the amino acid l-theanine in tea (Camellia sinensis) and other plants. Journal of Agricultural and Food Chemistry65, 7210–7216.

Dong C X, Li F, Yang T Y, Feng L, Zhang S P, Li F D, Li W H, Xu G H, Bao S L, Wan X C, Lucas W J, Zhang Z L. 2019. Theanine transporters identified in tea plants (Camellia sinensis L.). Plant Journal101, 57–70.

Dullius A, Fassina P, Giroldi M, Goettert M I, Souza C F V. 2020. A biotechnological approach for the production of branched chain amino acid containing bioactive peptides to improve human health: A review. Food Research International131, 109002.

Fang K X, Xia Z Q, Li H J, Jiang X H, Qin D D, Wang Q, Pan C D, Li B, Wu H L. 2021. Genome-wide association analysis identified molecular markers associated with important tea flavor-related metabolites. Horticulture Research8, 42–58.

Filiault D, Maloof J A. 2012. Genome-wide association study identifies variants underlying the Arabidopsis thaliana shade avoidance response. PLoS Genetics8, e1002589.

Fischer W N, Andre B, Rentsch D, Krolkiewicz S, Tegeder M, Breitkreuz K, Frommer W B. 1998. Amino acid transport in plants. Trends in Plant Science3, 188–195.

Fu X M, Cheng S H, Liao Y Y, Xu X L, Wang X C, Hao X Y, Xu P, Dong F, Yang Z Y. 2020. Characterization of l-theanine hydrolase in vitro and subcellular distribution of its specific product ethylamine in tea (Camellia sinensis). Journal of Agricultural and Food Chemistry68, 10842–10851.

Fu X M, Liao Y Y, Cheng S H, Xu X L, Grierson D, Yang Z Y. 2021. Nonaqueous fractionation and overexpression of fluorescent-tagged enzymes reveals the subcellular sites of l-theanine biosynthesis in tea. Plant Biotechnology Journal19, 98–108.

Hanson A D, Gregory J F. 2011. Folate biosynthesis, turnover, and transport in plants. Annual Review of Plant Biology62, 105–125.

Hao D L, Zhou J Y, Yang S Y, Qi W, Yang K J, Su Y H. 2020. Function and regulation of ammonium transporters in plants. International Journal of Molecular Sciences21, 3557.

Huang R, Wang J, Yao M Z, Ma C L, Chen L. 2022. Quantitative trait loci mapping for free amino acid content using an albino population and SNP markers provides insight into the genetic improvement of tea plants. Horticulture Research, 9, uhab029.

Jin C, Sun Y Y, Shi Y H, Zhang Y Y, Chen K, Li Y, Liu G G, Yao F, Cheng D, Li J, Zhou J J, Qu L H, Liu X Q, Luo J. 2019. Branched-chain amino acids regulate plant growth by affecting the homeostasis of mineral elements in rice. Life Sciences62, 1107–1110.

Kang H M, Sul J H, Service S K, Zaitlen N A, Kong S, Freimer N B, Sabatti C, Eskin E. 2010. Variance component model to account for sample structure in genome-wide association studies. Nature Genetics42, 348–354.

Khandaker L, Akond M, Liu S M, Kantartzi S K, Meksem K, Bellaloui N, Lightfoot D A, Kassem M A. 2015. Mapping of QTL associated with seed amino acids content in “MD96-5722” by “spencer” RIL population of soybean using SNP markers. Polish Journal of Food and Nutrition Sciences6, 974–984.

Lam H M, Coschigano K, Schultz C, Melo-Oliveira R, Tjaden G, Oliveira I, Ngai N, Hsieh M H, Coruzzi G. 1995. Use of Arabidopsis mutants and genes to study amide amino acid biosynthesis. Plant Cell7, 887–898.

Lee J H, Kim Y C, Jung Y J, Han J H, Zhang C Y, Yun C W, Lee S. 2019. The overexpression of cucumber (Cucumis sativus L.) genes that encode the branched-chain amino acid transferase modulate flowering time in Arabidopsis thalianaPlant Cell Reports38, 25–35.

Li F, Dong C X, Yang T Y, Bao S L, Fang W P, Lucas W J, Zhang Z L. 2021. The tea plant CsLHT1 and CsLHT6 transporters take up amino acids, as a nitrogen source, from the soil of organic tea plantations. Horticulture Research8, 178–189.

Liu Z W, Li H, Wang W L, Wu Z J, Cui X, Zhuang J. 2017. CsGOGAT is important in dynamic changes of Theanine content in postharvest tea plant leaves under different temperature and shading spreadings. Journal of Agricultural and Food Chemistry65, 9693–9702.

Luan H M, Ji F F, Chen Y, Cai Z W. 2018. statTarget: A streamlined tool for signal drift correction and interpretations of quantitative mass spectrometry-based omics data. Analytica Chimica Acta1036, 66–72.

Ma J Q, Jin J Q, Yao M Z, Ma C L, Xu Y X, Hao W J, Chen L. 2018. Quantitative trait loci mapping for theobromine and caffeine contents in tea plant (Camellia sinensis). Journal of Agricultural and Food Chemistry66, 13321–13327.

Maeda H, Dudareva N. 2012. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annual Review of Plant Biology63, 73–105.

Maloney G S, Kochevenko A, Tieman D M, Tohge T, Krieger U, Zamir D, Taylor M G, Fernie A R, Klee H. 2010. Characterization of the branched-chain amino acid aminotransferase enzyme family in tomato. Plant Physiology153, 925–936.

Mei X, Lin C Y, Wan S, Chen B Y, Wu H L, Zhang L Y. 2021. A comparative metabolomic analysis reveals difference manufacture suitability in “Yinghong 9” and “Huangyu” teas (Camellia sinensis). Frontiers in Plant Science12, 767724.

Moison M, Marmagne A, Dinant S, Soulay F, Azzopardi M, Lothier J, Citerne S, Morin H, Legay N, Chardon F, Avice J C, Reisdorf-Cren M, Masclaux-Daubresse C. 2018. Three cytosolic glutamine synthetase isoforms localized in different-order veins act together for N remobilization and seed filling in Arabidopsis. Journal of Experimental Botany69, 4379–4393.

Moreira E, Coimbra S, Melo P. 2022. Glutamine synthetase: An unlikely case of functional redundancy in Arabidopsis thalianaPlant Biology24, 713–720.

Price A H. 2006. Believe it or not, QTLs are accurate. Trends in Plant Science11, 213–216.

Ruan J, Haerdter R, Gerendás J. 2010. Impact of nitrogen supply on carbon/nitrogen allocation: A case study on amino acids and catechins in green tea [Camellia sinensis (L.) O. Kuntze] plants. Plant Biology12, 724–734.

Srivastava A C, Ramos-Parra P A, Bedair M, Robledo-Hernández A L, Tang Y H, Sumner L W, Garza R I, Blancaflor E. 2011. The folylpolyglutamate synthetase plastidial isoform is required for postembryonic root development in ArabidopsisPlant Physiology155, 1237–1251.

Sun Y Y, Shi Y H, Liu G G, Yao F, Zhang Y Y, Yang C K, Guo H, Liu X Q, Luo J. 2020. Natural variation in the OsbZIP18 promoter contributes to branched-chain amino acid levels in rice. New Phytologist228, 1548–1558.

Taira M, Valtersson U, Burkhardt B, Ludwig R A. 2004. Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts. Plant Cell16, 2048–2058.

Wang J, Xian X H, Xu X F, Qu C M, Lu K, Li J N, Liu L Z. 2017. Genome-wide association mapping of seed coat color in Brassica napusJournal of Agricultural and Food Chemistry65, 5229–5237.

Wang M, Shen Q R, Xu G H, Guo S W. 2014. New insight into the strategy for nitrogen metabolism in plant cells. In: Kwang W J, ed., International Review of Cell and Molecular Biology. Vol. 310. Elsevier Academic Press, Burlington, The United States of America. pp. 1–37.

Wang R J, Gao X F, Yang J, Kong X. 2019. Genome-wide association study to identify favorable SNP allelic variations and candidate genes that control the timing of spring bud flush of tea (Camellia sinensis) using SLAF-seq. Journal of Agricultural and Food Chemistry67, 10380–10391.

Wei C L, Yang H, Wang S B, Zhao J, Liu C, Gao L P, Xia E H, Lu Y, Tai Y L, She G B, Sun J, Cao H S, Tong W, Gao Q, Li Y Y, Deng W W, Jiang X L, Wang W Z, Chen Q, Zhang S H, et al. 2018. Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality. Proceedings of the National Academy of Sciences of the United States of America115, E4151–E4158.

Wen B, Luo Y, Liu D M, Zhang X N, Peng Z, Wang K B, Li J, Huang J N, Liu Z. 2020. The R2R3-MYB transcription factor CsMYB73 negatively regulates l-theanine biosynthesis in tea plants (Camellia sinensis L.). Plant Science298, 110546–110555.

Wen W W, Li D, Li X, Gao Y Q, Li W Q, Li H H, Chen W, Luo J, Yan J B. 2014. Metabolome-based genome-wide association study of maize kernel leads to novel biochemical insights. Nature Communications5, 3438.

Wu Y L, Wang W Z, Li Y Z, Dai X L, Ma G L, Xing D W, Zhu M Q, Gao L P, Xia T. 2017. Six phenylalanine ammonia-lyases from Camellia sinensis: Evolution, expression, and kinetics. Plant Physiology and Biochemistry118, 413–421.

Xu J F, Long Y, Wu J G, Xu H M, Wen J, Meng J L, Shi C H. 2014. QTL mapping and analysis of the embryo and maternal plant for three limiting amino acids in rapeseed meal. European Food Research and Technology240, 147–158.

Xu L Y, Wang L Y, Wei K, Tan L Q, Su J J, Cheng H. 2018. High-density SNP linkage map construction and QTL mapping for flavonoid-related traits in a tea plant (Camellia sinensis) using 2b-RAD sequencing. BMC Genomics19, 955.

Ye J, Wang X, Hu T X, Zhang F X, Wang B, Li C X, Yang T X, Li H X, Lu Y G, Giovannoni J J, Zhang Y Y, Ye Z B. 2017. An indel in the promoter of Al-ACTIVATED MALATE TRANSPORTER9 selected during tomato domestication determines fruit malate contents and aluminum tolerance. Plant Cell29, 2249–2268.

Yu Z M, Yang Z Y. 2020. Understanding different regulatory mechanisms of proteinaceous and non-proteinaceous amino acid formation in tea (Camellia sinensis) provides new insights into the safe and effective alteration of tea flavor and function. Critical Reviews in Food Science and Nutrition60, 844–858.

Zhang D, Zhang H Y, Hu Z B, Chu S S, Yu K Y, Lv L L, Yang Y M, Zhang X Q, Chen X, Kan G Z, Tang Y, An Y Q, Yu D Y. 2019. Artificial selection on GmOLEO1 contributes to the increase in seed oil during soybean domestication. PLoS Genetics15, e1008267.

Zhang L, Cao Q Q, Granato D, Xu Y Q, Ho C T. 2020. Association between chemistry and taste of tea: A review. Trends in Food Science & Technology101, 139–149.

Zhang W Y, Zhang Y J, Qiu H J, Guo Y F, Wan H L, Zhang X L, Scossa F, Alseekh S, Zhang Q H, Wang P, Xu L, Schmidt M H W, Jia X X, Li D L, Zhu A T, Guo F, Chen W, Ni D J, Usadel B, Fernie A R, et al. 2020. Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties. Nature Communications11, 3719.

Zhong M, Wang L Q, Yuan D J, Luo L J, Xu C G, He Y Q. 2011. Identification of QTL affecting protein and amino acid contents in rice. Rice Science18, 187–195.

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