Comprehensive dissection of variation and accumulation of free amino acids in tea accessions

Abstract

Free amino acids (FAAs) positively determine the tea quality, notably theanine (Thea), endowing umami taste of tea infusion, which is the profoundly prevalent research in albino tea genetic resources. Therefore, 339 tea accessions were collected to study FAAs level for deciphering its variation and accumulation mechanism. Interestingly, alanine (Ala) and Thea which had the highest diversity index (H′) value among three varieties of Camellia sinensis (L.) O. Kuntze were significantly higher than wild relatives (P < 0.05). The intraspecific arginine (Arg) and glutamine (Gln) contents in C. sinensis var. assamica were significantly lower than sinensis and pubilimba varieties. Moreover, the importance of interdependencies operating across FAAs and chlorophyll levels were highlighted via the cell ultrastructure, metabolomics, and transcriptome analysis. We then determined that the association between phytochrome interacting factor 1 (CsPIF1) identified by weighted gene co-expression network analysis (WGCNA) and Thea content. Intriguingly, transient knock-down CsPIF1 expression increased Thea content in tea plant, and the function verification of CsPIF1 in Arabidopsis also indicated that CsPIF1 acts as a negative regulator of Thea content by mainly effecting the genes expression related to Thea biosynthesis, transport, and hydrolysis, especially glutamate synthase (CsGOGAT), which was validated to be associated with Thea content with a nonsynonymous SNP by Kompetitive Allele-Specific PCR (KASP). We also investigated the interspecific and geographical distribution of this SNP. Taken together, these results help us to understand and clarify the variation and profile of major FAAs in tea germplasms and promote efficient utilization in tea genetic improvement and breeding.

References

[1.] Wei C, Yang H, Wang S. 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 U S A. 2018;115: E4151-8

[2.] Zhang X, Chen S, Shi L. et al. Haplotype-resolved genome assem-bly provides insights into evolutionary history of the tea plant Camellia sinensis. Nat Genet. 2021;53:1250-9

[3.] Chen L, Zhou ZX, Yang YJ. Genetic improvement and breeding of tea plant (Camellia sinensis) in China: from individual selection to hybridization and molecular breeding. Euphytica. 2007;154: 239-248

[4.] Zhang W, Zhang Y, Qiu H. et al. Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties. Nat Commun. 2020;11:3719

[5.] Pang Y, Abeysinghe IS, He J. et al. Functional characterization of proanthocyanidin pathway enzymes from tea and their applica-tion for metabolic engineering. Plant Physiol. 2013;161:1103-16

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

[7.] McCarthy AA, McCarthy JG. The structure of two N-methyltransferases from the caffeine biosynthetic pathway. Plant Physiol. 2007;144:879-89

[8.] Pang D, Liu Y, Sun Y. et al. Menghai Huangye, a novel albino tea germplasm with high theanine content and a high catechin index. Plant Sci. 2021;311:110997

[9.] da Silva TBV, Castilho PA, Sá-Nakanishi AB. et al. The inhibitory action of purple tea on in vivo starch digestion compared to other Camellia sinensis teas. Food Res Int. 2021;150:110781

[10.] Prasanth MI, Sivamaruthi BS, Chaiyasut C. et al. Areview of the role of green tea (Camellia sinensis) in antiphotoaging, stress resistance, neuroprotection, and autophagy. Nutrients. 2019;11:474

[11.] Guo J, Zhu B, Chen Y. et al. Potential ‘accelerator’ and ‘brake’ reg-ulation of theanine biosynthesis in tea plant (Camellia sinensis). Hortic Res. 2022;9:uhac169

[12.] Chen T, Ma J, Li H. et al. CsGDH2.1 negatively regulates theanine accumulation in the late-spring tea plants (Camellia sinensis var. sinensis). Hortic Res. 2023;10:uhac245

[13.] Lin S, Chen Z, Chen T. et al. Theanine metabolism and transport in tea plants (Camellia sinensis L.): advances and perspectives. Crit Rev Biotechnol. 2022;17:1-15

[14.] Cheng S, Fu X, Wang X. et al. Studies on the biochemical for-mation pathway of the amino acid L-theanine in tea (Camellia sinensis) and other plants. J Agric Food Chem. 2017;65:7210-6

[15.] Masclaux-Daubresse C, Reisdorf-Cren M, Pageau K. et al. Glu-tamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco. Plant Physiol. 2006;140:444-56

[16.] Ma Q, Li H, Zou Z. et al. Transcriptomic analyses identify albino-associated genes of a novel albino tea germplasm ‘Huabai 1’. Hortic Res. 2018;5:54

[17.] Xie N, Zhang C, Zhou P. et al. Transcriptomic analyses reveal variegation-induced metabolic changes leading to high L-theanine levels in albino sectors of variegated tea (Camellia sinensis). Plant Physiol Biochem. 2021;169:29-39

[18.] Feng L, Gao MJ, Hou RY. et al. Determination of quality con-stituents in the young leaves of albino tea cultivars. Food Chem. 2014;155:98-104

[19.] Wang JY, Chen JD, Wang SL. et al. Repressed gene expression of photosynthetic antenna proteins associated with yellow leaf variation as revealed by bulked segregant RNA-seq in tea plant Camellia sinensis. J Agric Food Chem. 2020;68:8068-79

[20.] Al-Sady B, Ni W, Kircher S. et al. Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol Cell. 2006;23:439-46

[21.] Jiao Y, Lau OS, Deng XW. Light-regulated transcriptional net-works in higher plants. Nat Rev Genet. 2007;8:217-30

[22.] Liebers M, Gillet FX, Israel A. et al. Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis. EMBO J 2020;39:e104941

[23.] Cheng S, Fu X, Liao Y. et al. Differential accumulation of special-ized metabolite L-theanine in green and albino-induced yellow tea (Camellia sinensis)leaves. Food Chem. 2019;276:93-100

[24.] Gao X, Zhang C, Lu C. et al. Disruption of photomorphogenesis leads to abnormal chloroplast development and leaf variegation in Camellia sinensis. Front Plant Sci. 2021;12:720800

[25.] Zheng Y, Wang P, Chen X. et al. Integrated transcriptomics and metabolomics provide novel insight into changes in specialized metabolites in an albino tea cultivar (Camellia sinensis (L.) O. Kuntze). Plant Physiol Biochem. 2021;160:27-36

[26.] Dong C, Li F, Yang T. et al. Theanine transporters identified in tea plants (Camellia sinensis L). The Plant J. 2020;101:57-70

[27.] Feng L, Yang T, Zhang Z. et al. Identification and characterization of cationic amino acid transporters (CATs) in tea plant (Camellia sinensis). Plant Growth Regul. 2018;84:57-69

[28.] He Y, Wang Z, Ge H. et al. Weighted gene co-expression network analysis identifies genes related to anthocyanin biosynthesis and functional verification of hub gene SmWRKY44. Plant Sci. 2021;309:110935

[29.] Lin CT, Xu T, Xing SL. et al. Weighted gene co-expression network analysis (WGCNA) reveals the hub role of protein ubiquitination in the acquisition of desiccation tolerance in boea hygrometrica. Plant Cell Physiol. 2019;60:2707-19

[30.] Cheng H, Wu W, Liu X. et al. Transcription factor CsWRKY40 regulates L-theanine hydrolysis by activating the CsPDX2.1 promoter in tea leaves during withering. Hortic Res. 2022;9: uhac025

[31.] Jiang CK, Ma JQ, Liu YF. et al. Identification and distribution of a single nucleotide polymorphism responsible for the catechin content in tea plants. Hortic Res. 2020;7:24

[32.] Huang R, Wang JY, Yao MZ. et al. 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. Hortic Res. 2022;9:uhab029

[33.] Kfoury N, Morimoto J, Kern A. et al. Striking changes in tea metabolites due to elevational effects. Food Chem. 2018;264: 334-41

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

[35.] Ying C, Chen J, Chen J. et al. Differential accumulation mecha-nisms of purine alkaloids and catechins in Camellia ptilophylla,a natural theobromine-rich tea. Beverage Plant Res. 2023;3:15

[36.] Xia E, Tong W, Hou Y. et al. The reference genome of tea plant and resequencing of 81 diverse accessions provide insights into its genome evolution and adaptation. Mol Plant. 2020;13:1013-26

[37.] Jiang A, Guo Z, Pan J. et al. The PIF1-miR408-PLANTACYANIN repression cascade regulates light-dependent seed germination. Plant Cell. 2021;33:1506-29

[38.] Kato Y, Hyodo K, Sakamoto W. The photosystem II repair cycle requires FtsH turnover through the EngA GTPase. Plant Physiol. 2018;178:596-611

[39.] Li F, Lv C, Zou Z. et al. CsAAP7.2 is involved in the uptake of amino acids from soil and the long-distance transport of theanine in tea plants (Camellia sinensis L.). Tree Physiol. 2022;42:2369-81

[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.] Miyashita Y, Dolferus R, Ismond KP. et al. Alanine aminotrans-ferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana. Plant J. 2007;49:1108-21

[42.] Chen Z, Lin S, Chen T. et al. Haem Oxygenase 1 is a potential target for creating etiolated/albino tea plants (Camellia sinensis) with high theanine accumulation. Hortic Res. 2023;10:uhac269

[43.] Fu X, Cheng S, Liao Y. et al. Characterization of L-theanine hydrolase in vitro and subcellular distribution of its specific product ethylamine in tea (Camellia sinensis). J Agric Food Chem. 2020;68:10842-51

[44.] Chang M, Ma J, Sun Y. et al. γ -Glutamyl-transpeptidase CsGGT2 functions as light-activated theanine hydrolase in tea plant (Camellia sinensis L.). Plant Cell Environ. 2023;46:1596-609

[45.] Yu Y, Kou X, Gao R. et al. Glutamine synthetases play a vital role in high accumulation of theanine in tender shoots of albino tea germplasm “Huabai 1”. J Agric Food Chem. 2021;69:13904-15

[46.] Jiang CK, Moon DG, Ma JQ. et al. Characteristics of non-volatile metabolites in fresh shoots from tea plant (Camellia sinensis)and its closely related species and varieties. Beverage Plant Res. 2022;2: 1-8

[47.] Fang K, Xia Z, Li H. et al. Genome-wide association analysis identified molecular markers associated with important tea flavor-related metabolites. Hortic Res. 2021;8:42

[48.] Hermans C, Bourgis F, Faucher M. et al. Magnesium deficiency in sugar beets alters sugar partitioning and phloem loading in young mature leaves. Planta. 2005;220:541-9

[49.] Zhang Q, Shi Y, Hu H. et al. Magnesium promotes tea plant growth via enhanced glutamine synthetase-mediated nitrogen assimilation. Plant Physiol. 2023;192:1321-37

[50.] Peng YY, Liao LL, Liu S. et al. Magnesium deficiency triggers SGR-mediated chlorophyll degradation for magnesium remobiliza-tion. Plant Physiol. 2019;181:262-75

[51.] Castal F, Nelson CJ. Nitrogen use within the growing leaf blade of tall fescue. Plant Physiol. 1994;105:191-7

[52.] Otegui MS, Noh YS, Martínez DE. et al. Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean. Plant J. 2005;41:831-44

[53.] Wang L, Yue C, Cao H. et al. Biochemical and transcriptome anal-yses of a novel chlorophyll-deficient chlorina tea plant cultivar. BMC Plant Biol. 2014;14:352

[54.] Qian J, Liao Y, Jian G. et al. Light induces an increasing release of benzyl nitrile against diurnal herbivore Ectropis grisescens Warren attack in tea (Camellia sinensis) plants. Plant Cell Environ. 2023;46: 3464-80

[55.] Chen S, Fang J, Wang Y. et al. Evolutionary genomics of structural variation in the tea plant, Camellia sinensis. Tropical Plants. 2022;1: 1-11

[56.] Fu X, Liao Y, Cheng S. et al. Nonaqueous fractionation and overexpression of fluorescent-tagged enzymes reveals the sub-cellular sites of L-theanine biosynthesis in tea. Plant Biotechnol J. 2021;19:98-108

[57.] Jin JQ, Ma JQ, Ma CL. et al. Determination of catechin content in representative Chinese tea germplasms. J Agric Food Chem. 2014;62:9436-41

[58.] Liu H, Liu L, Liang D. et al. SlBES1 promotes tomato fruit softening through transcriptional inhibition of PMEU1. iScience. 2021;24:102926

[59.] Clough SJ, Bent AF. Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16:735-43