CsMYBL2 homologs modulate the light and temperature stress-regulated anthocyanin and catechins biosynthesis in tea plants (Camellia sinensis).

Anthocyanin and catechin production in tea (Camellia sinensis) leaves can positively affect tea quality; however, their regulatory mechanisms are not fully understood. Here we report that, while the CsMYB75- or CsMYB86-directed MYB-bHLH-WD40 (MBW) complexes differentially activate anthocyanin or catechin biosynthesis in tea leaves, respectively, CsMYBL2a and CsMYBL2b homologs negatively modified the light- and temperature-induced anthocyanin and catechin production in both Arabidopsis and tea plants. The MBW complexes activated both anthocyanin synthesis genes and the downstream repressor genes CsMYBL2a and CsMYBL2b. Overexpression of CsMYBL2b, but not CsMYBL2a, repressed Arabidopsis leaf anthocyanin accumulation and seed coat proanthocyanin production. CsMYBL2b strongly and CsMYBL2a weakly repressed the activating effects of CsMYB75/CsMYB86 on CsDFR and CsANS, due to their different EAR and TLLLFR domains and interactions with CsTT8/CsGL3, interfering with the functions of activating MBW complexes. CsMYBL2b and CsMYBL2a in tea leaves play different roles in fine-tuning CsMYB75/CsMYB86-MBW activation of biosynthesis of anthocyanins and catechins, respectively. The CsbZIP1-CsmiR858a-CsMYBL2 module mediated the UV-B- or cold-activated CsMYB75/CsMYB86 regulation of anthocyanin/catechin biosynthesis by repressing CsMYBL2a and CsMYBL2b. Similarly, the CsCOP1-CsbZIP1-CsPIF3 module, and BR signaling as well, mediated the high temperature repression of anthocyanin and catechin biosynthesis through differentially upregulating CsMYBL2b and CsMYBL2a, respectively. The present study provides new insights into the complex regulatory networks in environmental stress-modified flavonoid production in tea plant leaves.

Aluminum and Fluoride Stresses Altered Organic Acid and Secondary Metabolism in Tea (Camellia sinensis) Plants: Influences on Plant Tolerance, Tea Quality and Safety.

Tea plants have adapted to grow in tropical acidic soils containing high concentrations of aluminum (Al) and fluoride (F) (as Al/F hyperaccumulators) and use secret organic acids (OAs) to acidify the rhizosphere for acquiring phosphorous and element nutrients. The self-enhanced rhizosphere acidification under Al/F stress and acid rain also render tea plants prone to accumulate more heavy metals and F, which raises significant food safety and health concerns. However, the mechanism behind this is not fully understood. Here, we report that tea plants responded to Al and F stresses by synthesizing and secreting OAs and altering profiles of amino acids, catechins, and caffeine in their roots. These organic compounds could form tea-plant mechanisms to tolerate lower pH and higher Al and F concentrations. Furthermore, high concentrations of Al and F stresses negatively affected the accumulation of tea secondary metabolites in young leaves, and thereby tea nutrient value. The young leaves of tea seedlings under Al and F stresses also tended to increase Al and F accumulation in young leaves but lower essential tea secondary metabolites, which challenged tea quality and safety. Comparisons of transcriptome data combined with metabolite profiling revealed that the corresponding metabolic gene expression supported and explained the metabolism changes in tea roots and young leaves via stresses from high concentrations of Al and F. The study provides new insight into Al- and F-stressed tea plants with regard to responsive metabolism changes and tolerance strategy establishment in tea plants and the impacts of Al/F stresses on metabolite compositions in young leaves used for making teas, which could influence tea nutritional value and food safety.

Single-cell transcriptome atlas reveals developmental trajectories and a novel metabolic pathway of catechin esters in tea leaves.

The tea plant is an economically important woody beverage crop. The unique taste of tea is evoked by certain metabolites, especially catechin esters, whereas their precise formation mechanism in different cell types remains unclear. Here, a fast protoplast isolation method was established and the transcriptional profiles of 16 977 single cells from 1st and 3rd leaves were investigated. We first identified 79 marker genes based on six isolated tissues and constructed a transcriptome atlas, mapped developmental trajectories and further delineated the distribution of different cell types during leaf differentiation and genes associated with cell fate transformation. Interestingly, eight differently expressed genes were found to co-exist at four branch points. Genes involved in the biosynthesis of certain metabolites showed cell- and development-specific characteristics. An unexpected catechin ester glycosyltransferase was characterized for the first time in plants by a gene co-expression network in mesophyll cells. Thus, the first single-cell transcriptional landscape in woody crop leave was reported and a novel metabolism pathway of catechin esters in plants was discovered.

Tea plant roots respond to aluminum-induced mineral nutrient imbalances by transcriptional regulation of multiple cation and anion transporters.

BACKGROUND: Tea is one of the most popular non-alcoholic beverages in the world for its flavors and numerous health benefits. The tea tree (Camellia sinensis L.) is a well-known aluminum (Al) hyperaccumulator. However, it is not fully understood how tea plants have adapted to tolerate high concentrations of Al, which causes an imbalance of mineral nutrition in the roots. RESULTS: Here, we combined ionomic and transcriptomic profiling alongside biochemical characterization, to probe the changes of metal nutrients and Al responsive genes in tea roots grown under increasing concentrations of Al. It was found that a low level of Al (~ 0.4 mM) maintains proper nutrient balance, whereas a higher Al concentration (2.5 mM) compromised tea plants by altering micro- and macro-nutrient accumulation into roots, including a decrease in calcium (Ca), manganese (Mn), and magnesium (Mg) and an increase in iron (Fe), which corresponded with oxidative stress, cellular damage, and retarded root growth. Transcriptome analysis revealed more than 1000 transporter genes that were significantly changed in expression upon Al exposure compared to control (no Al) treatments. These included transporters related to Ca and Fe uptake and translocation, while genes required for N, P, and S nutrition in roots did not significantly alter. Transporters related to organic acid secretion, together with other putative Al-tolerance genes also significantly changed in response to Al. Two of these transporters, CsALMT1 and CsALS8, were functionally tested by yeast heterologous expression and confirmed to provide Al tolerance. CONCLUSION: This study shows that tea plant roots respond to high Al-induced mineral nutrient imbalances by transcriptional regulation of both cation and anion transporters, and therefore provides new insights into Al tolerance mechanism of tea plants. The altered transporter gene expression profiles partly explain the imbalanced metal ion accumulation that occurred in the Al-stressed roots, while increases to organic acid and Al tolerance gene expression partly explains the ability of tea plants to be able to grow in high Al containing soils. The improved transcriptomic understanding of Al exposure gained here has highlighted potential gene targets for breeding or genetic engineering approaches to develop safer tea products.

Characterization of CsTSI in the Biosynthesis of Theanine in Tea Plants (Camellia sinensis).

Theanine is a unique major amino acid in tea plants responsible for umami taste and mental health benefits of tea. However, theanine biosynthesis and physiological role in tea plants are not fully understood. Here, we demonstrate that tea plant theanine synthetase is encoded by a glutamine synthetase gene CsTSI. The expression pattern of CsTSI is closely correlated with theanine and glutamine levels in various tissues. CsTSI transcripts were accumulated in root tip epidermal cells, pericycle and procambial cells, where CsTSI presents as a cytosolic protein. Ectopic expression of the gene in Arabidopsis led to greater glutamine and theanine production than controls when fed with ethylamine (EA). RNAi knockdown or overexpression of CsTSI in tea plant hairy roots reduced or enhanced theanine and glutamine contents, respectively, compared with controls. The CsTSI recombinant enzymes used glutamate as an acceptor and ammonium or EA as a donor to synthesize glutamine and theanine, respectively. CsTSI expression in tea roots responded to nitrogen supply and deprivation and was correlated with theanine contents. This study provides fresh insights into the molecular basis for the biosynthesis of theanine, which may facilitate the breeding of high-theanine tea plants for improving the nutritional benefit of tea.

Molecular Basis of the Distinct Metabolic Features in Shoot Tips and Roots of Tea Plants (Camellia sinensis): Characterization of MYB Regulator for Root Theanine Synthesis.

The physiological and metabolic differences between shoot tips and roots of tea plants are significant, and understanding them is required for improvement of tea quality and plant growth. A high-quality full-length transcriptome sequencing on tea plant roots and shoot tips by PacBio SMRT technology was done to gain a further understanding. Approximately 160699 and 166120 full-length transcripts were recovered in roots and shoots, respectively, including 31232 and 41068 novel isoforms and 16960 and 26029 alternative splicing (AS) isoforms. These supported 21699 full-length reads and 31232 and 41068 novel transcripts from root and shoot, respectively, including 1679 long noncoding RNAs (lncRNAs) and 2772 fusion transcripts, which significantly upgrade the Camellia sinensis genome annotation. The respective 6475 and 6981 transcripts in roots and shoots differ in 3'-untranslated regions. Meanwhile, extensive analyses of novel transcripts, ASs, and lncRNAs also revealed a large number of ASs and lincRNAs closely related to the regulation of characteristic secondary metabolites including catechins, theanine, and caffeine. Finally, a root-specific CsMYB6 was characterized to regulate theanine biosynthesis by genetic and molecular analyses. CsMYB6 directly bound to and activate the promoter of theanine synthetase gene (CsTSI). The study lays a foundation for the further investigation of metabolic genomics and regulation in tea plants.