A key mutation in magnesium chelatase I subunit leads to a chlorophyll-deficient mutant of tea (Camellia sinensis).

Tea (Camellia sinensis) is a highly important beverage crop renowned for its unique flavour and health benefits. Chlorotic mutants of tea, known worldwide for their umami taste and economic value, have gained global popularity. However, the genetic basis of this chlorosis trait remains unclear. In this study, we identified a major-effect quantitative trait locus (QTL), qChl-3, responsible for the chlorosis trait in tea leaves, linked to a non-synonymous polymorphism (G1199A) in the magnesium chelatase I subunit (CsCHLI). Homozygous CsCHLIA plants exhibited an albino phenotype due to defects in magnesium protoporphyrin IX and chlorophylls in the leaves. Biochemical assays revealed that CsCHLI mutations did not affect subcellular localization or interactions with CsCHLIG and CsCHLD. However, combining CsCHLIA with CsCHLIG significantly reduced ATPase activity. RNA-seq analysis tentatively indicated that CsCHLI inhibited photosynthesis and enhanced photoinhibition, which in turn promoted protein degradation and increased the amino acid levels in chlorotic leaves. RT-qPCR and enzyme activity assays confirmed the crucial role of asparagine synthetase and arginase in asparagine and arginine accumulation, with levels increasing over 90-fold in chlorotic leaves. Therefore, this study provides insights into the genetic mechanism underlying tea chlorosis and the relationship between chlorophyll biosynthesis and amino acid metabolism.

Transcriptome and Biochemical Analyses of a Chlorophyll-Deficient Bud Mutant of Tea Plant (Camellia sinensis).

Tea leaf-color mutants have attracted increasing attention due to their accumulation of quality-related biochemical components. However, there is limited understanding of the molecular mechanisms behind leaf-color bud mutation in tea plants. In this study, a chlorina tea shoot (HY) and a green tea shoot (LY) from the same tea plant were investigated using transcriptome and biochemical analyses. The results showed that the chlorophyll a, chlorophyll b, and total chlorophyll contents in the HY were significantly lower than the LY's, which might have been caused by the activation of several genes related to chlorophyll degradation, such as SGR and CLH. The down-regulation of the CHS, DFR, and ANS involved in flavonoid biosynthesis might result in the reduction in catechins, and the up-regulated GDHA and GS2 might bring about the accumulation of glutamate in HY. RT-qPCR assays of nine DEGs confirmed the RNA-seq results. Collectively, these findings provide insights into the molecular mechanism of the chlorophyll deficient-induced metabolic change in tea plants.

Isolation and Characterization of CsWRKY7, a Subgroup IId WRKY Transcription Factor from Camellia sinensis, Linked to Development in Arabidopsis.

WRKY transcription factors (TFs) containing one or two WRKY domains are a class of plant TFs that respond to diverse abiotic stresses and are associated with developmental processes. However, little has been known about the function of WRKY gene in tea plant. In this study, a subgroup IId WRKY gene CsWRKY7 was isolated from Camellia sinensis, which displayed amino acid sequence homology with Arabidopsis AtWRKY7 and AtWRKY15. Subcellular localization prediction indicated that CsWRKY7 localized to nucleus. Cis-acting elements detected in the promotor region of CsWRKY7 are mainly involved in plant response to environmental stress and growth. Consistently, expression analysis showed that CsWRKY7 transcripts responded to NaCl, mannitol, PEG, and diverse hormones treatments. Additionally, CsWRKY7 exhibited a higher accumulation both in old leaves and roots compared to bud. Seed germination and root growth assay indicated that overexpressed CsWRKY7 in transgenic Arabidopsis was not sensitive to NaCl, mannitol, PEG, and low concentration of ABA treatments. CsWRKY7 overexpressing Arabidopsis showed a late-flowering phenotype under normal conditions compared to wild type. Furthermore, gene expression analysis showed that the transcription levels of the flowering time integrator gene FLOWERING LOCUS T (FT) and the floral meristem identity genes APETALA1 (AP1) and LEAFY (LFY) were lower in WRKY7-OE than in the WT. Taken together, these findings indicate that CsWRKY7 TF may participate in plant growth. This study provides a potential strategy to breed late-blooming tea cultivar.

Functional natural allelic variants of flavonoid 3',5'-hydroxylase gene governing catechin traits in tea plant and its relatives.

MAIN CONCLUSION: Functional allelic variants of the flavonoid 3',5'-hydroxylase (F3'5'H) gene provides new information of F3'5'H function of tea plant and its relatives. This insight may serve as the foundation upon which to advance molecular breeding in the tea plant. Catechins are the active components of tea that determine its quality and health attributes. This study established the first integrated genomic strategy for deciphering the genetic basis of catechin traits of tea plant. With the RNA-sequencing analysis of bulked segregants representing the tails of a F1 population segregated for total catechin content, we identified a flavonoid 3',5'-hydroxylase (F3'5'H) gene. F3'5'H had one copy in the genomic DNA of tea plant. Among 202 tea accessions, we identified 120 single nucleotide polymorphisms (SNPs) at F3'5'H locus. Seventeen significant marker-trait associations were identified by association mapping in multiple environments, which were involved in 10 SNP markers, and the traits including the ratio of di/tri-hydroxylated catechins and catechin contents. The associated individual and combination of SNPs explained 4.5-25.2 and 53.0-63.0% phenotypic variations, respectively. In the F1 population (validation population), the catechin trait variation percentages explained by F3'5'H diplotype were 6.9-74.3%. The genotype effects of ten functional SNPs in the F1 population were all consistent with the association population. Furthermore, the function of SNP-711/-655 within F3'5'H was validated by gene expression analysis. Altogether, our work indicated functional SNP allelic variants within F3'5'H governing the ratio of di/tri-hydroxylated catechins and catechin contents. The strong catechin-associated SNPs identified in this study can be used for future marker-assisted selection to improve tea quality.

Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in 'Anji Baicha' (Camellia sinensis).

BACKGROUND: The new shoots of the albino tea cultivar 'Anji Baicha' are yellow or white at low temperatures and turn green as the environmental temperatures increase during the early spring. 'Anji Baicha' metabolite profiles exhibit considerable variability over three color and developmental stages, especially regarding the carotenoid, chlorophyll, and theanine concentrations. Previous studies focused on physiological characteristics, gene expression differences, and variations in metabolite abundances in albino tea plant leaves at specific growth stages. However, the molecular mechanisms regulating metabolite biosynthesis in various color and developmental stages in albino tea leaves have not been fully characterized. RESULTS: We used RNA-sequencing to analyze 'Anji Baicha' leaves at the yellow-green, albescent, and re-greening stages. The leaf transcriptomes differed considerably among the three stages. Functional classifications based on Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed unigenes were mainly related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and carbon fixation in photosynthetic organisms. Chemical analyses revealed higher ß-carotene and theanine levels, but lower chlorophyll a levels, in the albescent stage than in the green stage. Furthermore, unigenes involved in carotenoid, chlorophyll, and theanine biosyntheses were identified, and the expression patterns of the differentially expressed unigenes in these biosynthesis pathways were characterized. Through co-expression analyses, we identified the key genes in these pathways. These genes may be responsible for the metabolite biosynthesis differences among the different leaf color and developmental stages of 'Anji Baicha' tea plants. CONCLUSIONS: Our study presents the results of transcriptomic and biochemical analyses of 'Anji Baicha' tea plants at various stages. The distinct transcriptome profiles for each color and developmental stage enabled us to identify changes to biosynthesis pathways and revealed the contributions of such variations to the albino phenotype of tea plants. Furthermore, comparisons of the transcriptomes and related metabolites helped clarify the molecular regulatory mechanisms underlying the secondary metabolic pathways in different stages.

Global transcriptome and gene regulation network for secondary metabolite biosynthesis of tea plant (Camellia sinensis).

BACKGROUND: Major secondary metabolites, including flavonoids, caffeine, and theanine, are important components of tea products and are closely related to the taste, flavor, and health benefits of tea. Secondary metabolite biosynthesis in Camellia sinensis is differentially regulated in different tissues during growth and development. Until now, little was known about the expression patterns of genes involved in secondary metabolic pathways or their regulatory mechanisms. This study aimed to generate expression profiles for C. sinensis tissues and to build a gene regulation model of the secondary metabolic pathways. RESULTS: RNA sequencing was performed on 13 different tissue samples from various organs and developmental stages of tea plants, including buds and leaves of different ages, stems, flowers, seeds, and roots. A total of 43.7 Gbp of raw sequencing data were generated, from which 347,827 unigenes were assembled and annotated. There were 46,693, 8446, 3814, 10,206, and 4948 unigenes specifically expressed in the buds and leaves, stems, flowers, seeds, and roots, respectively. In total, 1719 unigenes were identified as being involved in the secondary metabolic pathways in C. sinensis, and the expression patterns of the genes involved in flavonoid, caffeine, and theanine biosynthesis were characterized, revealing the dynamic nature of their regulation during plant growth and development. The possible transcription factor regulation network for the biosynthesis of flavonoid, caffeine, and theanine was built, encompassing 339 transcription factors from 35 families, namely bHLH, MYB, and NAC, among others. Remarkably, not only did the data reveal the possible critical check points in the flavonoid, caffeine, and theanine biosynthesis pathways, but also implicated the key transcription factors and related mechanisms in the regulation of secondary metabolite biosynthesis. CONCLUSIONS: Our study generated gene expression profiles for different tissues at different developmental stages in tea plants. The gene network responsible for the regulation of the secondary metabolic pathways was analyzed. Our work elucidated the possible cross talk in gene regulation between the secondary metabolite biosynthetic pathways in C. sinensis. The results increase our understanding of how secondary metabolic pathways are regulated during plant development and growth cycles, and help pave the way for genetic selection and engineering for germplasm improvement.

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

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.

CsEXL3 regulate mechanical harvest-related droopy leaves under the transcriptional activation of CsBES1.2 in tea plant.

Due to a labor shortage, the mechanical harvesting of tea plantations has become a focal point. However, mechanical harvest efficiency was hampered by droopy leaves, leading to a high rate of broken tea shoots and leaves. Here, we dissected the genetic structure of leaf droopiness in tea plants using genome-wide association studies (GWAS) on 146 accessions, combined with transcriptome from two accessions with contrasting droopy leaf phenotypes. A set of 16 quantitative trait loci (QTLs) containing 54 SNPs and 34 corresponding candidate genes associated with droopiness were then identified. Among these, CsEXL3 (EXORDIUM-LIKE 3) from Chromosome 1 emerged as a candidate gene. Further investigations revealed that silencing CsEXL3 in tea plants resulted in weaker vascular cell malformation and brassinosteroid-induced leaf droopiness. Additionally, brassinosteroid signal factor CsBES1.2 was proved to participate in CsEXL3-induced droopiness and vascular cell malformation via using the CsBES1.2-silencing tea plant. Notably, CsBES1.2 bound on the E-box of CsEXL3 promoter to transcriptionally activate CsEXL3 expression as CUT&TAG based ChIP-qPCR and ChIP-seq suggested in vivo as well as EMSA and Y1H indicated in vitro. Furthermore, CsEXL3 instead of CsBES1.2 decreased lignin content and the expressing levels of lignin biosynthesis genes. Overall, our findings suggest that CsEXL3 regulates droopy leaves, partially through the transcriptional activation of CsBES1.2, with the potential to improve mechanical harvest efficiency in tea plantations.

Simultaneous determination of protoporphyrin IX and magnesium protoporphyrin IX in Arabidopsis thaliana and Camellia sinensis using UPLC-MS/MS.

BACKGROUNDS: Insertion of Mg2+ into protoporphyrin IX (PPIX) to produce magnesium-protoporphyrin IX (Mg-PPIX) was the first step toward chlorophyll biosynthesis, which not only imparts plants green pigmentation but underpins photosynthesis. Plants that blocked the conversion of PPIX to Mg-PPIX displayed yellowish or albino-lethal phenotypes. However, the lack of systematic study of the detection method and the metabolic difference between species have caused the research on chloroplast retrograde signaling controversial for a long time. RESULTS: An advanced and sensitive UPLC-MS/MS strategy for determining PPIX and Mg-PPIX was established in two metabolic different plants, Arabidopsis thaliana (Columbia-0) and Camellia sinensis var. sinensis. Two metabolites could be extracted by 80% acetone (v/v) and 20% 0.1 M NH4OH (v/v) without hexane washing. Since the Mg-PPIX could be substantially de-metalized into PPIX in acidic conditions, analysis was carried out by UPLC-MS/MS with 0.1% ammonia (v/v) and 0.1% ammonium acetonitrile (v/v) as mobile phases using negative ion multiple reaction monitoring modes. Interestingly, it could be easier to monitor these two compounds in dehydrated samples rather than in fresh samples. Validation was performed in spiked samples and mean recoveries ranged from 70.5 to 916%, and the intra-day and inter-day variations were less than 7.5 and 10.9%, respectively. The limit of detection was 0.01 mg·kg- 1 and the limit of quantification was 0.05 mg·kg- 1. The contents of PPIX (1.67 ± 0.12 mg·kg- 1) and Mg-PPIX (3.37 ± 0.10 mg·kg- 1) in tea were significantly higher than in Arabidopsis (PPIX: 0.05 ± 0.02 mg·kg- 1; Mg-PPIX: 0.08 ± 0.01 mg·kg- 1) and they were only detected in the leaf. CONCLUSIONS: Our study establishes a universal and reliable method for determining PPIX and Mg-PPIX in two plants using UPLC-MS/MS. This procedure will facilitate studying chlorophyll metabolism and natural chlorophyll production.

Deeply functional identification of TCS1 alleles provides efficient technical paths for low-caffeine breeding of tea plants.

Caffeine is an important functional component in tea, which has the effect of excitement and nerve stimulation, but excessive intake can cause insomnia and dysphoria. Therefore, the production of tea with low-caffeine content can meet the consumption needs of certain people. Here, in addition to the previous alleles of the tea caffeine synthase (TCS1) gene, a new allele (TCS1h) from tea germplasms was identified. Results of in vitro activity analysis showed that TCS1h had both theobromine synthase (TS) and caffeine synthase (CS) activities. Site-directed mutagenesis experiments of TCS1a, TCS1c, and TCS1h demonstrated that apart from the 225th amino acid residue, the 269th amino acid also determined the CS activity. GUS histochemical analysis and dual-luciferase assay indicated the low promoter activity of TCS1e and TCS1f. In parallel, insertion and deletion mutations in large fragments of alleles and experiments of site-directed mutagenesis identified a key cis-acting element (G-box). Furthermore, it was found that the contents of purine alkaloids were related to the expression of corresponding functional genes and alleles, and the absence or presence and level of gene expression determined the content of purine alkaloids in tea plants to a certain extent. In summary, we concluded TCS1 alleles into three types with different functions and proposed a strategy to effectively enhance low-caffeine tea germplasms in breeding practices. This research provided an applicable technical avenue for accelerating the cultivation of specific low-caffeine tea plants.

The potential effects of chlorophyll-deficient mutation and tree_age on the accumulation of amino acid components in tea plants.

Albino tea has been receiving growing attention on the tea market due to its attractive appearance and fresh taste, mainly caused by high amino acid contents. Here, variations in the contents of five free amino acids in relation to pigment contents and tree age in two hybrid populations'Longjin 43'(♀) × 'Baijiguan'(♂) and 'Longjin 43'(♀) ×'Huangjinya'(♂) with 334 first filial generation individuals including chlorophyll-deficient and normal tea plants were investigated. The data showed that the contents of main amino acids in all filial generation gradually decreased as plant age increased. Principal component analysis indicated that the amino acid content of individual plant tended to be stable with the growth of plants. Correlation analysis clarified that several main amino acids were significantly negatively correlated with chlorophyll a, chlorophyll b and carotenoid contents. Our results showed that the accumulation of amino acids in tea plant was closely related to leaf color variation and the tree age during growing period.

Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant.

Tea is known for having a high catechin content, with the main component being (-)-epigallocatechin gallate (EGCG), which has significant bioactivities, including potential anti-cancer and anti-inflammatory activity. The poor intestinal stability and permeability of EGCG, however, undermine these health-improving benefits. O-methylated EGCG derivatives, found in a few tea cultivars in low levels, have attracted considerable interest due to their increased bioavailability. Here, we identify two O-methyltransferases from tea plant: CsFAOMT1 that has a specific O-methyltransferase activity on the 3''-position of EGCG to generate EGCG3''Me, and CsFAOMT2 that predominantly catalyzes the formation of EGCG4″Me. In different tea tissues and germplasms, the transcript levels of CsFAOMT1 and CsFAOMT2 are strongly correlated with the amounts of EGCG3''Me and EGCG4''Me, respectively. Furthermore, the crystal structures of CsFAOMT1 and CsFAOMT2 reveal the key residues necessary for 3''- and 4''-O-methylation. These findings may provide guidance for the future development of tea cultivars with high O-methylated catechin content.

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.

Free amino acids are one of the main chemical components in tea, and they contribute to the pleasant flavor, function, and quality of tea, notably the level of theanine. Here, a high-density genetic map was constructed to characterize quantitative trait loci (QTLs) for free amino acid content. A total of 2688 polymorphic SNP markers were obtained using genotyping-by-sequencing (GBS) based on 198 individuals derived from a pseudotestcross population of "Longjing 43" × "Baijiguan", which are elite and albino tea cultivars, respectively. The 1846.32 cM high-density map with an average interval of 0.69 cM was successfully divided into 15 linkage groups (LGs) ranging from 93.41 cM to 171.28 cM. Furthermore, a total of 4 QTLs related to free amino acid content (theanine, glutamate, glutamine, aspartic acid and arginine) identified over two years were mapped to LG03, LG06, LG11 and LG14. The phenotypic variation explained by these QTLs ranged from 11.8% to 23.7%, with an LOD score from 3.56 to 7.7. Furthermore, several important amino acid metabolic pathways were enriched based on the upregulated differentially expressed genes (DEGs) among the offspring. These results will be essential for fine mapping genes involved in amino acid pathways and diversity, thereby providing a promising avenue for the genetic improvement of tea plants.

A novel TcS allele conferring the high-theacrine and low-caffeine traits and having potential use in tea plant breeding.

Theacrine (1,3,7,9-tetramethyluric acid) is a natural product with remarkable pharmacological activities such as antidepressant, sedative and hypnotic activities, while caffeine (1,3,7-trimethylxanthine) has certain side effects to special populations. Hence, breeding tea plants with high theacrine and low caffeine will increase tea health benefits and promote consumption. In this study, we construct an F1 population by crossing 'Zhongcha 302' (theacrine-free) and a tea germplasm 'Ruyuan Kucha' (RY, theacrine-rich) to identify the causal gene for accumulating theacrine. The results showed that the content of theacrine was highly negatively correlated with caffeine (R2 > 0.9). Bulked segregant RNA sequencing analysis, molecular markers and gene expression analysis indicated that the theacrine synthase (TcS) gene was the candidate gene. The TcS was located in the nucleus and cytoplasm, and the theacrine can be detected in stably genetic transformed tobacco by feeding the substrate 1,3,7-trimethyluric acid. Moreover, an in vitro enzyme activity experiment revealed that the 241st amino acid residue was the key residue. Besides, we amplified the promoter region in several tea accessions with varied theacrine levels, and found a 234-bp deletion and a 271-bp insertion in RY. Both GUS histochemical analysis and dual-luciferase assay showed that TcS promoter activity in RY was relatively high. Lastly, we developed a molecular marker that is co-segregate with high-theacrine individuals in RY's offspring. These results demonstrate that the novel TcS allele in RY results in the high-theacrine and low-caffeine traits and the developed functional marker will facilitate the breeding of characteristic tea plants.

Transcriptomic and Metabolomic Analyses Provide Insights Into an Aberrant Tissue of Tea Plant (Camellia sinensis).

Tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most important economic crops with multiple mutants. Recently, we found a special tea germplasm that has an aberrant tissue on its branches. To figure out whether this aberrant tissue is associated with floral bud (FB) or dormant bud (DB), we performed tissue section, transcriptome sequencing, and metabolomic analysis of these tissues. Longitudinal sections indicated the aberrant tissue internal structure was more like a special bud (SB), but was similar to that of DB. Transcriptome data analysis showed that the number of heterozygous and homozygous SNPs was significantly different in the aberrant tissue compared with FB and DB. Further, by aligning the unmapped sequences of the aberrant tissue to the Non-Redundant Protein Sequences (NR) database, we observed that 36.13% of unmapped sequences were insect sequences, which suggested that the aberrant tissue might be a variation of dormant bud tissue influenced by the interaction of tea plants and insects or pathogens. Metabolomic analysis showed that the differentially expressed metabolites (DEMs) between the aberrant tissue and DB were significantly enriched in the metabolic pathways of biosynthesis of plant hormones and biosynthesis of phenylpropanoids. Subsequently, we analyzed the differentially expressed genes (DEGs) in the above mentioned two tissues, and the results indicated that photosynthetic capacity in the aberrant tissue was reduced, whereas the ethylene, salicylic acid and jasmonic acid signaling pathways were activated. We speculated that exogenous infection induced programmed cell death (PCD) and increased the lignin content in dormant buds of tea plants, leading to the formation of this aberrant tissue. This study advanced our understanding of the interaction between plants and insects or pathogens, providing important clues about biotic stress factors and key genes that lead to mutations and formation of the aberrant tissue.

Novel insight into theacrine metabolism revealed by transcriptome analysis in bitter tea (Kucha, Camellia sinensis).

Kucha (Camellia sinensis) is a kind of unique wild tea resources in southwest China, containing sizeable amounts of theacrine (1,3,7,9-tetramethyluric acid) and having a special bitter taste both in fresh leaves and made tea. Theacrine has good healthy function locally. But the molecular mechanism of theacrine metabolism in Kucha was still unclear. In order to illuminate the biosynthesis and catabolism of theacrine in Kucha plants, three tea cultivars, C. sinensis 'Shangyou Zhongye' (SY) with low-theacrine, 'Niedu Kucha 2' (ND2) with middle-theacrine and, 'Niedu Kucha 3' (ND3) with high-theacrine, were used for our research. Purine alkaloid analysis and transcriptome of those samples were performed by High Performance Liquid Chromatography (HPLC) and RNA-Seq, respectively. The related gene expression levels of purine alkaloid were correlated with the content of purine alkaloid, and the results of quantitative real-time (qRT) PCR were also confirmed the reliability of transcriptome. Based on the data, we found that theacrine biosynthesis is a relatively complex process, N-methyltransferase (NMT) encoded by TEA024443 may catalyze the methylation at 9-N position in Kucha plant. Our finding will assist to reveal the molecular mechanism of theacrine biosynthesis, and be applied to selection and breeding of Kucha tea cultivars in the future.

Baiyacha, a wild tea plant naturally occurring high contents of theacrine and 3″-methyl-epigallocatechin gallate from Fujian, China.

Baiyacha (BYC) is a kind of wild tea plant growing and utilizing in the remote mountain area of Fujian province, Southeastern China. However, scientific studies on this plant remain limited. Our results showed that BYC exhibits the typical morphological characteristics of Camellia gymnogyna Chang, a closely related species of C. sinensis (L.) O. Kuntze, which was not found in Fujian before. Chemical profiling revealed that parts of BYC plants are rich in purine alkaloids and catechins, especially featuring high levels of theacrine and 3″-methyl-epigallocatechin gallate (EGCG3″Me), chemical compounds with multiple biological activities that are rarely observed in regular tea plants. The contents of EGCG3″Me and theacrine in BYC both increased with the leaf maturity of tea shoots, whereas the caffeine content decreased significantly. The obtained results provide abundant information about the morphology and chemical compounds of BYC and may be used for tea production, breeding, and scientific research in the future.

Identifying Conserved Functional Gene Modules Underlying the Dynamic Regulation of Tea Plant Development and Secondary Metabolism.

Tea plants adjust development and metabolism by integrating environmental and endogenous signals in complex but poorly defined gene networks. Here, we present an integrative analysis framework for the identification of conserved modules controlling important agronomic traits using a comprehensive collection of RNA-seq datasets in Camellia plants including 189 samples. In total, 212 secondary metabolism-, 182 stress response-, and 182 tissue development-related coexpressed modules were revealed. Functional modules (e.g., drought response, theobromine biosynthesis, and new shoot development-related modules) and potential regulators that were highly conserved across diverse genetic backgrounds and/or environmental conditions were then identified by cross-experiment comparisons and consensus clustering. Moreover, we investigate the preservation of gene networks between Camellia sinensis and other Camellia species. This revealed that the coexpression patterns of several recently evolved modules related to secondary metabolism and environmental adaptation were rewired and showed higher connectivity in tea plants. These conserved modules are excellent candidates for modeling the core mechanism of tea plant development and secondary metabolism and should serve as a great resource for hypothesis generation and tea quality improvement.

The chromosome-scale genome reveals the evolution and diversification after the recent tetraploidization event in tea plant.

Tea is one of the most popular nonalcoholic beverages due to its characteristic secondary metabolites with numerous health benefits. Although two draft genomes of tea plant (Camellia sinensis) have been published recently, the lack of chromosome-scale assembly hampers the understanding of the fundamental genomic architecture of tea plant and potential improvement. Here, we performed a genome-wide chromosome conformation capture technique (Hi-C) to obtain a chromosome-scale assembly based on the draft genome of C. sinensis var. sinensis and successfully ordered 2984.7 Mb (94.7%) scaffolds into 15 chromosomes. The scaffold N50 of the improved genome was 218.1 Mb, ~157-fold higher than that of the draft genome. Collinearity comparison of genome sequences and two genetic maps validated the high contiguity and accuracy of the chromosome-scale assembly. We clarified that only one Camellia recent tetraploidization event (CRT, 58.9-61.7 million years ago (Mya)) occurred after the core-eudicot common hexaploidization event (146.6-152.7 Mya). Meanwhile, 9243 genes (28.6%) occurred in tandem duplication, and most of these expanded after the CRT event. These gene duplicates increased functionally divergent genes that play important roles in tea-specific biosynthesis or stress response. Sixty-four catechin- and caffeine-related quantitative trait loci (QTLs) were anchored to chromosome assembly. Of these, two catechin-related QTL hotspots were derived from the CRT event, which illustrated that polyploidy has played a dramatic role in the diversification of tea germplasms. The availability of a chromosome-scale genome of tea plant holds great promise for the understanding of genome evolution and the discovery of novel genes contributing to agronomically beneficial traits in future breeding programs.

Repressed Gene Expression of Photosynthetic Antenna Proteins Associated with Yellow Leaf Variation as Revealed by Bulked Segregant RNA-seq in Tea Plant Camellia sinensis.

The young leaves and shoots of albino tea cultivars are usually characterized as having a yellow or pale color, high amino acid, and low catechin. Increasing attention has been paid to albino tea cultivars in recent years because their tea generally shows high umami and reduced astringency. However, the genetic mechanism of yellow-leaf variation in albino tea cultivar has not been elucidated clearly. In this study, bulked segregant RNA-seq (BSR-seq) was performed on bulked yellow- and green-leaf hybrid progenies from a leaf color variation population. A total of 359 and 1134 differentially expressed genes (DEGs) were identified in the yellow and green hybrid bulked groups (Yf vs Gf) and parent plants (Yp vs Gp), respectively. The significantly smaller number of DEGs in Yf versus Gf than in Yp versus Gp indicated that individual differences could be reduced within the same hybrid progeny. Analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes revealed that the photosynthetic antenna protein was most significantly enriched in either the bulked groups or their parents. Interaction was found among light-harvesting chlorophyll a/b -binding proteins (LHC), heat shock proteins (HSPs), and enzymes involved in cuticle formation. Combined with the transcriptomic expression profile, results showed that the repressed genes encoding LHC were closely linked to aberrant chloroplast development in yellow-leaf tea plants. Furthermore, the photoprotection and light stress response possessed by genes involved in HSP protein interaction and cuticle formation were discussed. The expression profile of DEGs was verified via quantitative real-time PCR analysis of the bulked samples and other F1 individuals. In summary, using BSR-seq on a hybrid population eliminated certain disturbing effects of genetic background and individual discrepancy, thereby helping this study to intensively focus on the key genes controlling leaf color variation in yellow-leaf tea plants.