The R2R3-MYB transcription factor CsMYB42 regulates theanine biosynthesis in albino tea leaves.

Theanine is a unique secondary metabolite in tea plants and contributes to the umami taste and health benefits of tea. However, theanine biosynthesis in tea plants is not fully understood, and its mechanism of transcriptional regulation remains poorly reported. Theanine content was significantly correlated with the expression of theanine biosynthesis-related gene CsGS1c and transcription factor CsMYB42 in different leaf positions and picking times, but there was no significant correlation in different tissues of albino tea plant 'Anjibaicha'. This suggests that CsMYB42 may regulate CsGS1c to synthesize theanine in albino tea leaves, and the regulation is tissue specific. CsMYB42 is a nuclear-localized R2R3-MYB transcription factor gene with transcriptional activation activity. Yeast one-hybrid assay and electrophoretic mobility shift assay confirmed the direct binding of CsMYB42 to the promoter of CsGS1c. Luciferase assay showed that CsMYB42 activates the CsGS1c expression. Furthermore, the inhibition of CsMYB42 using an antisense oligonucleotide in tea leaves decreased CsGS1c expression and theanine content. These results indicate that CsMYB42 plays a crucial role in activating the expression of CsGS1c and may be involved in the biosynthesis of theanine in albino tea leaves. This study provides fresh insights into the tissue-specific regulation of theanine biosynthesis, which laid a foundation for breeding high-theanine tea plants.

Effect of Leaf Grade on Taste and Aroma of Shaken Hunan Black Tea.

Shaken Hunan black tea is an innovative Hunan black tea processed by adding shaking to the traditional Hunan black tea. The quality of shaken black tea is influenced by leaf grades of different maturity. In this study, the taste and aroma quality of shaken Hunan black tea processed with different grades were analyzed by sensory evaluation (SP, HPLC, and HS-SPME/GC-MS). The results showed that shaken Hunan black tea processed with one bud and two leaves has the best quality, which has a sweet, mellow, and slightly floral taste, as well as a floral, honey, and sweet aroma. Moreover, caffeine and EGCG were identified as the most important bitter and astringent substances in shaken Hunan black. Combined with the analysis of GC-MS and OAV analysis, geraniol, jasmone, ß-myrcene, citral, and trans-ß-ocimene might be the most important components that affect the sweet aroma, while methyl jasmonate, indole, and nerolidol were the key components that affect the floral aroma of shaken Hunan black tea. This study lays a foundation for this study of the taste and aroma characteristics of shaken Hunan black tea and guides enterprises to improve shaken black tea processing technology.

Identification of a WRKY transcriptional activator from Camellia sinensis that regulates methylated EGCG biosynthesis.

Naturally occurring methylated catechins, especially methylated EGCG in tea leaves are known to have many health benefits. Although the genes involved in methylated EGCG biosynthesis have been studied extensively, the transcriptional factors controlling methylated EGCG biosynthesis are still poorly understood. In the present study, a WRKY domain-containing protein termed CsWRKY57like was identified, which belongs to group IIc of the WRKY family, and contains one conserved WRKY motif. CsWRKY57like was found to localize in the nucleus, function as a transcriptional activator, and its expression positively correlated with methylated EGCG level. In addition, CsWRKY57like activated the transcription of three genes related to methylated EGCG biosynthesis, including CCoAOMT, CsLAR, and CsDFR by specifically interacting with their promoters via binding to the cis-acting element (C/T)TGAC(T/C). Further assays revealed that CsWRKY57like physically interacts with CsVQ4, and participates in the metabolic regulation of O-methylated catechin biosynthesis. Collectively, we conclude that CsWRKY57like may positively impact the biosynthesis of methylated EGCG in the tea plant, which comprehensively enriches the regulatory network of WRKY TFs associated with methylated EGCG and provide a potential strategy for the breeding of specific tea plant cultivars with high methylated EGCG .

Transcriptomic analyses reveal variegation-induced metabolic changes leading to high L-theanine levels in albino sectors of variegated tea (Camellia sinensis).

Camellia sinensis cv. 'Yanling Huayecha' (YHC) is an albino-green chimaeric tea mutant with stable genetic traits. Here, we analysed the cell ultrastructure, photosynthetic pigments, amino acids, and transcriptomes of the albino, mosaic, and green zones of YHC. Well-organized thylakoids were found in chloroplasts in mesophyll cells of the green zone but not the albino zone. The albino zone of the leaves contained almost no photosynthetic pigment. However, the levels of total amino acids and theanine were higher in the albino zone than in the mosaic and green zones. A transcriptomic analysis showed that carbon metabolism, nitrogen metabolism and amino acid biosynthesis showed differences among the different zones. Metabolite and transcriptomic analyses revealed that (1) downregulation of CsPPOX1 and damage to thylakoids in the albino zone may block chlorophyll synthesis; (2) downregulation of CsLHCB6, CsFdC2 and CsSCY1 influences chloroplast biogenesis and thylakoid membrane formation, which may contribute to the appearance of variegated tea leaves; and (3) tea plant variegation disrupts the balance between carbon and nitrogen metabolism and promotes the accumulation of amino acids, and upregulation of CsTSⅠ and CsAlaDC may enhance L-theanine synthesis. In summary, our study provides a theoretical basis and valuable insights for elucidating the molecular mechanisms and promoting the economic utilization of variegation in tea.

Disruption of Photomorphogenesis Leads to Abnormal Chloroplast Development and Leaf Variegation in Camellia sinensis.

Camellia sinensis cv. 'Yanlingyinbiancha' is a leaf-variegated mutant with stable genetic traits. The current study aimed to reveal the differences between its albino and green tissues, and the molecular mechanism underlying the variegation. Anatomic analysis showed the chloroplasts of albino tissues to have no intact lamellar structure. Photosynthetic pigment in albino tissues was significantly lower than that in green tissues, whereas all catechin components were more abundant in the former. Transcriptome analysis revealed most differentially expressed genes involved in the biosynthesis of photosynthetic pigment, photosynthesis, and energy metabolism to be downregulated in albino tissues while most of those participating in flavonoid metabolism were upregulated. In addition, it was found cryptochrome 1 (CRY1) and phytochrome B (PHYB) genes that encode blue and red light photoreceptors to be downregulated. These photoreceptors mediate chloroplast protein gene expression, chloroplast protein import and photosynthetic pigment biosynthesis. Simultaneously, SUS gene, which was upregulated in albino tissues, encodes sucrose synthase considered a biochemical marker for sink strength. Collectively, we arrived to the following conclusions: (1) repression of the biosynthesis of photosynthetic pigment causes albinism; (2) destruction of photoreceptors in albino tissues suppresses photomorphogenesis, leading to abnormal chloroplast development; (3) albino tissues receive sucrose from the green tissues and decompose their own storage substances to obtain the energy needed for survival; and (4) UV-B signal and brassinosteroids promote flavonoid biosynthesis.