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.

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.

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.

The high yield mutants require a high-throughput screening method to obtain them quickly. Here, we developed an L-arginine biosensor (ARG-Select) to obtain increased L-arginine producers among a large number of mutant strains. This biosensor was constructed by ArgR protein and argC promoter, and could provide the strain with the output of bacterial growth via the reporter gene sacB; strains with high Larginine production could survive in 10% sucrose screening. To extend the screening limitation of 10% sucrose, the sensitivity of ArgR protein to L-arginine was decreased. Corynebacterium crenatum SYPA5-5 and its systems pathway engineered strain Cc6 were chosen as the original strains. This biosensor was employed, and L-arginine hyperproducing mutants were screened. Finally, the HArg1 and DArg36 mutants of C. crenatum SYPA5-5 and Cc6 could produce 56.7 and 95.5 g L −1 of L-arginine, respectively, which represent increases of 35.0 and 13.5%. These results demonstrate that the transcription factor-based biosensor could be applied in high yield strains selection as an effective high-throughput screening method.

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.