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.

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.

Comprehensive metabolic analyses provide new insights into primary and secondary metabolites in different tissues of Jianghua Kucha tea (Camellia sinensis var. assamica cv. Jianghua).

Background: Jianghua Kucha (JHKC) is a special tea germplasm with enriched specialized secondary metabolites, including theacrine, non-epimeric flavanols and methylated flavanols. Moreover, primary metabolites provide precursors and energy for the production of secondary metabolites. However, the accumulation patterns of primary and secondary metabolites in different tissues of JHKC are unclear. Methods: The changes of primary and secondary metabolites and related metabolic pathways (primary and secondary metabolism) in different JHKC tissues (the bud, 1st-4th leaves, and new stem) were investigated via metabolomics analysis with ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). Results: Significant differences were observed in 68 primary and 51 secondary metabolites mainly related with the pathways of starch and sucrose, amino acids, caffeine, and flavanols metabolism and TCA cycle. The bud exhibited higher levels of glucose-6-phosphate, citric acid, most amino acids, theobromine, catechin-gallate, epicatechin-gallate, procyanidins, and theasinensins; the 1st leaf showed higher levels of caffeine and epigallocatechin-3-gallate; and the 4th leaf contained higher levels of most monosaccharides, theacrine, and epigallocatechin-3-O-(3"-O-methyl)-gallate. In addition, primary metabolites and important secondary metabolites had certain correlations. Conclusion: This study provides comprehensive insight into primary and secondary metabolites in JHKC and offers guidelines for efficiently utilizing specialized metabolites of JHKC in the future.

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.

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.

Population sequencing enhances understanding of tea plant evolution.

Tea is an economically important plant characterized by a large genome, high heterozygosity, and high species diversity. In this study, we assemble a 3.26-Gb high-quality chromosome-scale genome for the 'Longjing 43' cultivar of Camellia sinensis var. sinensis. Genomic resequencing of 139 tea accessions from around the world is used to investigate the evolution and phylogenetic relationships of tea accessions. We find that hybridization has increased the heterozygosity and wide-ranging gene flow among tea populations with the spread of tea cultivation. Population genetic and transcriptomic analyses reveal that during domestication, selection for disease resistance and flavor in C. sinensis var. sinensis populations has been stronger than that in C. sinensis var. assamica populations. This study provides resources for marker-assisted breeding of tea and sets the foundation for further research on tea genetics and evolution.

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.

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.

The complete chloroplast genome sequence of Camellia tachangensis F. C. Zhang (Theaceae).

To understand genetic background and phylogenetic position of Camellia tachangensis, we determined its complete chloroplast genome sequence which is 157,026 bp in length with overall GC content of 36.7%. It has four sub regions: a large single-copy (LSC) region (86,669 bp) and a small single-copy (SSC) region (18,253 bp) are separated by two inverted repeat (IR) regions (26,052 bp each). A total of 129 genes were annotated, containing 86 protein-coding genes, 35 tRNA genes, and 8 rRNA genes. Phylogenetic trees showed C. tachangensis clustered with Camellia gymnogyna and Camellia taliensis and separated from Camellia sinensis and its two varieties, Camellia sinensis var. assamica and Camellia sinensis var. pubilimba.

Quantitative Trait Loci Mapping for Theobromine and Caffeine Contents in Tea Plant ( Camellia sinensis).

Understanding the genetic basis of theobromine and caffeine accumulation in the tea plant is important due to their contribution to tea flavor. Quantitative trait loci (QTL) analyses were carried out to identify genetic variants associated with theobromine and caffeine contents and ratio using a pseudo-testcross population derived from an intervarietal cross between two varieties of Camellia sinensis. A total of 10 QTL controlling caffeine content (CAF), theobromine content (TBR), sum of caffeine and theobromine (SCT), and caffeine-to-theobromine ratio (CTR) were identified over four measurement years. The major QTL controlling CAF, qCAF1, was mapped onto LG01 and validated across years, explaining an average of 20.1% of the phenotypic variance. The other QTL were detected in 1 or 2 years, and of them there were four, two, and three for TBR, SCT, and CTR, respectively. The present results provide valuable information for further fine mapping and cloning functional genes and for genetic improvement in tea plant.

Hongyacha, a Naturally Caffeine-Free Tea Plant from Fujian, China.

Hongyacha (HYC) is a type of new wild tea plant discovered in Fujian Province, China. This tea is helpful to the healing or prevention of disease in its original growing area. However, research on this tea is limited. Our results showed that HYC displayed obvious differences in its morphological characteristics compared with Cocoa tea ( Camellia ptilophylla Chang), a famous caffeine-free tea plant in China. Theobromine and trans-catechins, but not caffeine and cis-catechins, were the dominant purine alkaloids and catechins detected in HYC. HYC might contain abundant gallocatechin-(4 → 8)-gallocatechin gallate, 1,3,4,6-tetra- O-galloyl-ß-d-glucopyranose, and (-)-gallocatechin-3,5-di- O-gallate, which were not detected in regular tea. We also found that the TCS1 of HYC was distinct, and the responding recombinant protein exhibited only theobromine synthase activity. The obtained results showed that HYC is a new kind of caffeine-free tea plant and may be used for scientific protection and efficient utilization in the future.

A Novel F3' 5' H Allele with 14 bp Deletion Is Associated with High Catechin Index Trait of Wild Tea Plants and Has Potential Use in Enhancing Tea Quality.

Catechins are important chemical components determining the quality of tea. The catechin index (CI, ratio of dihydroxylated catechin (DIC)/trihydroxylated catechin (TRIC)) in the green leaf has a major influence on the amounts of theaflavins in black tea. In this work, the major catechin profiles of wild tea plants originating from Guizhou Province with high CI trait were investigated. We identified a novel flavonoid 3',5' hydroxylase gene ( F3' 5' H) allele with a 14 bp deletion in the upstream regulation region and developed an insertion/deletion (InDel) marker accordingly. The 14 bp deletion in the novel  F3' 5' H allele was associated with low F3' 5' H mRNA expression, thereby resulting in low TRIC content and high CI value. The allelic variant in the novel F3' 5' H allele associated with high CI values and DIC contents was confirmed by the introgression lines derived from a distant cross population. The novel F3' 5' H allele in wild tea plants is a valuable gene resource, which could be applied to breeding improvement on tea quality.

Comprehensive Dissection of Metabolic Changes in Albino and Green Tea Cultivars.

Albino tea cultivars are special mutants of tea plants with white or yellow leaf color. In this study, three albino tea cultivars, including 'Anji Baicha', 'Huangjinya', and 'Baijiguan', and two green tea cultivars, 'Longjing 43' and 'Fuding Dabaicha', were applied to metabolite profiling by gas chromatography-mass spectrometry and ultraperformance liquid chromatography-mass spectrometry. Multivariate analyses revealed significantly different metabolite phenotypes in leaves among albino cultivars and green cultivars. The differential metabolite-related pathways included galactose metabolism, tryptophan metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. For the young leaves of albino cultivars, the sugar (sorbitol and erythrose) and amino acid (mainly proline, isoleucine, ornithine, aspartic acid, threonine, and valine) concentrations increased, whereas gallocatechin and epigallocatechin gallate concentrations decreased. These results reveal the divergence in metabolic profiling between tea plant cultivars with different leaf colors. With the development of leaves, the concentrations of flavonoids increased largely in the older leaves of albino cultivars.

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.

Association mapping of caffeine content with TCS1 in tea plant and its related species.

Caffeine is the most abundant purine alkaloid in majority of tea plant and its related species. This purine alkaloid contributes to the important flavor and health attributes of tea. Tea caffeine synthase 1 (TCS1, EC 2.1.1.159/2.1.1.160) gene plays a crucial role in caffeine biosynthesis. The objective of this study was to investigate the genetic relationship between the TCS1 and caffeine content of tea plant and its related species using association mapping. We identified 87 single-nucleotide polymorphisms (SNPs, π = 0.00447) by resequencing the TCS1 locus of 44 tea accessions. Linkage disequilibrium (LD) analysis showed that LD did not extend over the entire gene (r(2) < 0.1, within 1000 bp). Two cleaved amplified polymorphism sequence (CAPS) markers were developed from sequence variations (SNP4318 and SNP6252). By association mapping, we identified SNP4318 associated with caffeine content in four environments, explaining 4.0%-7.7% of the phenotypic variance. We also validated the significant marker-trait associations in site-directed mutagenesis experiments. Examination of allelic variation and linkage disequilibrium by a candidate-gene-based approach can help to decipher the genetic basis of caffeine biosynthesis. Moreover, the SNP marker identified in this study can potentially be applied for future marker-assisted selection to improve tea quality.

Transcriptomic Analysis of Tea Plant Responding to Drought Stress and Recovery.

Tea plant (Camellia sinensis) is an economically important beverage crop. Drought stress (DS) seriously limits the growth and development of tea plant, thus affecting crop yield and quality. To elucidate the molecular mechanisms of tea plant responding to DS, we performed transcriptomic analysis of tea plant during the three stages [control (CK) and during DS, and recovery (RC) after DS] using RNA sequencing (RNA-Seq). Totally 378.08 million high-quality trimmed reads were obtained and assembled into 59,674 unigenes, which were extensively annotated. There were 5,955 differentially expressed genes (DEGs) among the three stages. Among them, 3,948 and 1,673 DEGs were up-regulated under DS and RC, respectively. RNA-Seq data were further confirmed by qRT-PCR analysis. Genes involved in abscisic acid (ABA), ethylene, and jasmonic acid biosynthesis and signaling were generally up-regulated under DS and down-regulated during RC. Tea plant potentially used an exchange pathway for biosynthesis of indole-3-acetic acid (IAA) and salicylic acid under DS. IAA signaling was possibly decreased under DS but increased after RC. Genes encoding enzymes involved in cytokinin synthesis were up-regulated under DS, but down-regulated during RC. It seemed probable that cytokinin signaling was slightly enhanced under DS. In total, 762 and 950 protein kinases belonging to 26 families were differentially expressed during DS and RC, respectively. Overall, 547 and 604 transcription factor (TF) genes belonging to 58 families were induced in the DS vs. CK and RC vs. DS libraries, respectively. Most members of the 12 TF families were up-regulated under DS. Under DS, genes related to starch synthesis were down-regulated, while those related to starch decomposition were up-regulated. Mannitol, trehalose and sucrose synthesis-related genes were up-regulated under DS. Proline was probably mainly biosynthesized from glutamate under DS and RC. The mechanism by which ABA regulated stomatal movement under DS and RC was partly clarified. These results document the global and novel responses of tea plant during DS and RC. These data will serve as a valuable resource for drought-tolerance research and will be useful for breeding drought-resistant tea cultivars.

Natural allelic variations of TCS1 play a crucial role in caffeine biosynthesis of tea plant and its related species.

Tea caffeine synthase 1 (TCS1) is an enzyme that catalyzes the methylation of N-3 and N-1 and considered to be the most critical enzyme in the caffeine biosynthetic pathway of tea plant. This study shows that TCS1 has six types of allelic variations, namely, TCS1a, TCS1b, TCS1c, TCS1d, TCS1e, and TCS1f, with a 252 bp insertion/deletion mutation in the 5'-untranslated region. Among tea plant and its related species, TCS1a is the predominant allele, and TCS1b-f are the rare alleles that mainly appear in few wild germplasms. The full-length cDNA sequences of three new alleles, namely, TCS1d, TCS1e, and TCS1f, were isolated from specific germplasms, and all of recombinant proteins have higher caffeine synthase (CS, EC 2.1.1.160) activity than theobromine synthase (TS, EC 2.1.1.159). Amino acid residue 269 is responsible for the difference in TCS activity and substrate recognition, which was demonstrated by using site-directed mutagenesis experiments. Furthermore, natural variations in TCS1 change the transcription levels. There are two molecular mechanisms controlling the caffeine biosynthesis in low-caffeine-accumulating tea germplasms, i.e., TCS1 allele with low transcription level or its encoded protein with only TS activity. Allelic variations of TCS1 play a crucial role in caffeine biosynthesis. Taken together, our work provides valuable foundation for a comprehensive understanding of the mechanism of caffeine biosynthesis in section Thea plants and useful guidance for effective breeding.

Differential Metabolic Profiles during the Albescent Stages of 'Anji Baicha' (Camellia sinensis).

'Anji Baicha' is an albino tea cultivar with white shoots at low air temperature and green shoots at high air temperature in early spring. The metabolite contents in the shoots dynamically vary with the color changes and with shoot development. To investigate the metabolomic variation during the albescent and re-greening stages, gas chromatography-mass spectrometry combined with multivariate analysis were applied to analyze the metabolite profiles in the different color stages during the development of 'Anji Baicha' leaves. The metabolite profiles of three albescent stages, including the yellow-green stage, the early albescent stage, and the late albescent stage, as well as the re-greening stage were distinguished using principal component analysis, revealing that the distinct developmental stages were likely responsible for the observed metabolic differences. Furthermore, a group classification and pairwise discrimination was revealed among the three albescent stages and re-greening stage by partial least squares discriminant analysis. A total of 65 differential metabolites were identified with a variable influence on projection greater than 1. The main differential metabolic pathways of the albescent stages compared with the re-greening stage included carbon fixation in photosynthetic organisms and the phenylpropanoid and flavonoid biosynthesis pathways. Compared with the re-greening stage, the carbohydrate and amino acid metabolic pathways were disturbed during the albescent stages. During the albescent stages, the sugar (fructofuranose), sugar derivative (glucose-1-phosphate) and epicatechin concentrations decreased, whereas the amino acid (mainly glycine, serine, tryptophan, citrulline, glutamine, proline, and valine) concentrations increased. These results reveal the changes in metabolic profiling that occur during the color changes associated with the development of the albino tea plant leaves.