Genome-wide identification of RNA editing sites in chloroplast transcripts and multiple organellar RNA editing factors in tea plant (Camellia sinensis L.): Insights into the albinism mechanism of tea leaves.

RNA editing is a post-transcriptional modification process, the chloroplast genes of which are involved in the process of chloroplast development in plant. However, the RNA editing sites of chloroplast genes remains unknown. In this study, we identified 39 RNA editing sites in 18 chloroplast genes from chloroplast genome of C. sinensis. Furthermore, the feature, structures and specificity of RNA editing sites were systematic analyzed. The differential editing efficiency were examined at 11 RNA editing sites among C. sinensis var. sinensis 'Huabai 1', 'Baiye 1' and 'Longjing 43'. Meanwhile, we identified 10 C. sinensis MORFs from five subgroups and performed comparative analyses of chromosome locations, duplication model and expression profiles. Expression analysis showed that the expression level of CsMORF9.2 was down-regulated significantly in 'Huabai 1' albino tea cultivar. This study provides a foundation for further reveal in the role of chloroplast RNA editing in albinism process of tea leaves.

Glutamine Synthetases Play a Vital Role in High Accumulation of Theanine in Tender Shoots of Albino Tea Germplasm "Huabai 1".

Theanine (N-ethyl-γ-l-glutamine) is a special nonprotein amino acid that contributes to the umami taste and health function of tea. Although recent studies on tea breeding have focused on albino tea because of its umami taste, a factor of higher theanine concentration, the mechanism of biosynthesis of l-theanine is still unclear. In this study, four glutamine synthetase genes (CsGSs) were obtained and functionally characterized by overexpressing them in Arabidopsis. The enzyme activities of the purified CsGS proteins from Escherichia coli were detected. The results showed that CsGSs have a dual function in the synthesis of glutamine and theanine in vivo and in vitro. Interestingly, l-theanine was abundantly synthesized in the tender shoots of "Huabai 1". In the white tender shoots, the cytosol CsGS1.2 might exhibit increased expression to compensate for decreasing levels of chloroplast CsGS2, which plays a vital role in high accumulation of theanine in "Huabai 1". In addition, CsGS2 was most likely the key l-theanine synthases in green tissues of tea. The present findings will provide basis for and considerably broaden the scope of understanding the function of CsGSs and the mechanism of l-theanine accumulation in the tender shoots of "Huabai 1", and will be useful for breeding and screening tea with high l-theanine content.

Metabolomic Analyses Provide New Insights into Signaling Mechanisms for Nutrient Uptake by Lateral Roots of Pruned Tea Plant (Camellia sinensis).

Pruning is an important plant management practice in tea cultivation. However, the mechanism underlying the dynamics of nutrient uptake by roots of pruned tea is unknown. This study investigated the metabolic alterations in lateral roots of pruned tea to unveil the mechanism of nutrient uptake. Elemental analysis revealed that pruning significantly increases the uptake of nutrients by lateral roots. Metabolic profiling showed significant metabolic variations in lateral roots of pruned tea. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that flavonoid biosynthesis, phenylpropanoid biosynthesis, and amino acid metabolism were differentially regulated in lateral roots. Caffeine metabolism was significantly hindered, while ethylene signaling was significantly induced in lateral roots of pruned plants. In addition, intermediates in the tricarboxylic acid (TCA) cycle were upregulated, indicating high rates of the TCA cycle. Therefore, pathways related to phenylpropanoid biosynthesis, TCA cycle, ethylene biosynthesis, and metabolism of amino acids contribute to higher nutrient uptake by lateral roots of the tea plant.

A novel insight into nitrogen and auxin signaling in lateral root formation in tea plant [Camellia sinensis (L.) O. Kuntze].

BACKGROUND: Tea plant (Camellia sinensis) is one of the most popular non-alcoholic beverages worldwide. In tea, lateral roots (LRs) are the main organ responsible for the absorption of moisture and mineral nutrients from the soil. Lateral roots formation and development are regulated by the nitrogen and auxin signaling pathways. In order to understand the role of auxin and nitrogen signaling in LRs formation and development, transcriptome analysis was employed to investigate the differentially expressed genes involved in lateral roots of tea plants treated with indole-3-butyric acid (IBA), N-1-naphthylphthalamic acid (NPA), low and high concentrations of nitrogen. RESULTS: A total of 296 common differentially expressed genes were identified and annotated to four signaling pathways, including nitrogen metabolism, plant hormone signal transduction, glutathione metabolism and transcription factors. RNA-sequencing results revealed that majority of differentially expressed genes play important roles in nitrogen metabolism and hormonal signal transduction. Low nitrogen condition induced the biosynthesis of auxin and accumulation of transcripts, thereby, regulating lateral roots formation. Furthermore, metabolism of cytokinin and ethylene biosynthesis were also involved in lateral roots development. Transcription factors like MYB genes also contributed to lateral roots formation of tea plants through secondary cell wall biosynthesis. Reversed phase ultra performance liquid chromatography (RP-UPLC) results showed that the auxin concentration increased with the decreased nitrogen level in lateral roots. Thus, tea plant lateral roots formation could be induced by low nitrogen concentration via auxin biosynthesis and accumulation. CONCLUSION: This study provided insights into the mechanisms associated with nitrogen and auxin signaling pathways in LRs formation and provides information on the efficient utilization of nitrogen in tea plant at the genetic level.

Prediction of suitable brewing cuppages of Dahongpao tea based on chemical composition, liquor colour and sensory quality in different brewing.

Oolong tea is famous for its characteristic of durably brewing. To explore suitable brewing cuppages and the scientific methods to brew Oolong tea in multiple steeping process. Dahongpao tea (Zhengyan, Banyan and Zhouyan tea) is well known Oolong tea variety, brewed at 14 times and assessed its chemical composition, infusion colour and sensory quality in different brewing intervals. The results showed that Zhengyan tea (A3) had the best quality of steeping among the chosen tea. It could be brewed up to 10 cuppages with 80% sensory score. The chemical composition and tea infusion colour strength were higher in Zhengyan tea. Though, 70% caffeine leached within first three steeping. The Forest regression model revealed that the suitable brewing time ranges between 4 and 10 in the chosen Dahongpao tea variety. This study provides a scientific method and suitable steeping times for the drinking of different Dahongpao tea through dynamic analysis of quantity of chemical composition, infusion colour strength and sensory quality.

Isolation and expression analysis of CsCML genes in response to abiotic stresses in the tea plant (Camellia sinensis).

Calmodulin-like (CML) proteins are a class of important Ca2+ sensors in plants, which play vital roles in regulating plant growth and development and response to abiotic stress. Tea plant (Camellia sinensis L.) is the most popular non-alcoholic economic beverage crop around the world. However, the potential functions of CMLs in either tea plants growth or in the response to environmental stresses are still unclear. In the present study, five CsCML genes (CsCML16, CsCML18-1, CsCML18-2, CsCML38, and CsCML42) were isolated from tea plant, and functionally characterized. The CsCML genes showed diverse expression patterns in leaves, roots, old stems, immature stems and flowers of tea plants. To investigate the expression changes of the genes under various abiotic stresses and ABA treatment, time-course experiments were also performed, the results indicated that the expression levels of CsCML16, 18-2 and 42 were significantly induced under low temperature and salt condition, while CsCML38 was induced distinctly under drought stress and ABA treatment. Overall, CsCML genes showed diverse function in tea plant under various stimuli. These results will increase our knowledge of the significance of CsCML genes in tea plant in response to abiotic stresses and hormone treatments.

Transcriptomic analyses identify albino-associated genes of a novel albino tea germplasm 'Huabai 1'.

Albinism in shoots of tea plants is a common phenotypic expression which gives the tea infusion a pleasant umami taste. A novel natural albino mutant tea germplasm containing high amino acids content was found and named as 'Huabai 1'. 'Huabai 1' has white jade tender shoots under low temperature and turns green with increased temperature. In order to understand the molecular mechanism of color change in leaf of 'Huabai 1', transcriptome analysis was performed to identify albino-associated differentially expressed genes (DEGs). A total of 483 DEGs were identified from white shoots of 'Huabai 1' compared to its green shoots. There were 15 DEGs identified to be involved in phenylpropanoid biosynthesis, which account for the majority of characterized DEGs. The metabolites related to phenylpropanoid biosynthesis revealed similar expression pattern of DEGs. Furthermore, metabolic pathways such as ubiquonone, tyrosine, and flavonoid biosynthesis associated with phenylpropanoid biosynthesis could also contribute to the color change in 'Huabai 1' tender shoots. Protein-protein interaction analysis revealed a hub protein NEDD8 (CSA009575) which interacted with many regulated genes in spliceosome, nitrogen metabolism, phenylpropanoid biosynthesis, and other pathways. In conclusion, the findings in this study indicate that the color change of 'Huabai 1' tender shoots is a combined effect of phenylpropanoid biosynthesis pathway and other metabolic pathways including flavonoid biosynthesis in tea plants. Chlorophyll biosynthesis-related genes LHCII and SGR may also play some roles in color change of 'Huabai 1'.

Benzothiadiazole and B-Aminobutyricacid Induce Resistance to Ectropis Obliqua in Tea Plants (Camellia Sinensis (L.) O. Kuntz).

In order to investigate the effect of benzothiadiazole (BTH) and ß-aminobutyric acid (BABA) on the resistance of tea plants (Camellia sinensis) to tea geometrid (Ectropis obliqua), three levels each of benzothiadiazole (BTH) and ß-aminobutyric acid (BABA) were sprayed on 10-year-old tea plants. Generally PPO and PAL activities increased with low concentrations of BTH and BABA treatments. Quantitative RT-PCR revealed a 1.43 and 2.72-fold increase in PPO gene expression, and 3.26 and 3.99-fold increase in PAL gene expression with 75 mg/L BTH and 400 mg/L BABA respectively. Analysis of hydrolysis of synthetic substrates also revealed that chymotrypsin-like enzyme activity present in larval midgut extracts was not significantly inhibited by BTH and BABA. However, proteinase activity was found to be inversely proportional to the age of tea geometrid. Larvae pupation rate decreased by 8.10, 10.81 and 21.62% when tea geometrid were fed with leaves treated with 25, 50 and 75 mg/L BTH solutions, while 100, 200 and 400 mg/L BABA solutions decreased same by 8.10, 16.21 and 13.51% respectively. Also, larvae development period delayed to 23.33 and 26.33 days with 75 mg/L BTH and 400 mg/L BABA treatments respectively. The results in this study; therefore, suggest that benzothiadiazole (BTH) and ß-aminobutyric acid (BABA) play a role in inducing resistance in tea plants to tea geometrid, with the optimal effect achieved at BTH-3 (75 mg/L) and BABA-3 (400 mg/L), respectively.