Integrated Metabolomic and Transcriptomic Analysis Reveals That Amino Acid Biosynthesis May Determine Differences in Cold-Tolerant and Cold-Sensitive Tea Cultivars.

Cold stress is one of the major abiotic stresses limiting tea production. The planting of cold-resistant tea cultivars is one of the most effective measures to prevent chilling injury. However, the differences in cold resistance between tea cultivars remain unclear. In the present study, we perform a transcriptomic and metabolomic profiling of Camellia sinensis var. "Shuchazao" (cold-tolerant, SCZ) and C. sinensis var. assamica "Yinghong 9" (cold-sensitive, YH9) during cold acclimation and analyze the correlation between gene expression and metabolite biosynthesis. Our results show that there were 51 differentially accumulated metabolites only up-regulated in SCZ in cold-acclimation (CA) and de-acclimation (DA) stages, of which amino acids accounted for 18%. The accumulation of L-arginine and lysine in SCZ in the CA stage was higher than that in YH9. A comparative transcriptomic analysis showed an enrichment of the amino acid biosynthesis pathway in SCZ in the CA stage, especially "arginine biosynthesis" pathways. In combining transcriptomic and metabolomic analyses, it was found that genes and metabolites associated with amino acid biosynthesis were significantly enriched in the CA stage of SCZ compared to CA stage of YH9. Under cold stress, arginine may improve the cold resistance of tea plants by activating the polyamine synthesis pathway and CBF (C-repeat-binding factor)-COR (cold-regulated genes) regulation pathway. Our results show that amino acid biosynthesis may play a positive regulatory role in the cold resistance of tea plants and assist in understanding the cold resistance mechanism differences among tea varieties.

A hierarchical model of ABA-mediated signal transduction in tea plant revealed by systematic genome mining analysis and interaction validation.

As a critical signaling molecule, ABA plays an important role in plant growth, development and stresses response. However, tea plant [Camellia sinensis (L.)], an important economical perennial woody plant, has not been systematically reported in response to ABA signal transduction in vivo. In this study, we mined and identified the gene structure of CsPYL/CsPP2C-A/CsSnRK gene families in the ABA signal transduction pathway through the genome-wide analysis of tea plants. Spatiotemporal expression and stress response (drought, salt, chilling) expression patterns were characterized. The results showed that most members of CsPYLs were conserved, and the gene structures of members of A-type CsPP2Cs were highly similar, whereas the gene structure of CsSnRK2s was highly variable. The transcription levels of different family members were differentially expressed with plant growth and development, and their response to stress signal patterns was highly correlated. The expression patterns of CsPYL/CsPP2C-A/CsSnRK2 gene family members in different tissues of tea plant cuttings after exogenous ABA treatment were detected by qRT-PCR, and the hierarchical model of ABA signaling was constructed by correlation analysis to preliminarily obtain three potential ABA-dependent signaling transduction pathways. Subsequently, the protein interaction of the CsPYL4/7-CsPP2C-A2-CsSnRK2.8 signaling pathway was verified by yeast two-hybrid and surface plasmon resonance experiments, indicating that there is specific selectivity in the ABA signaling pathway. Our results provided novel insights into the ABA-dependent signal transduction model in tea plant and information for future functional characterizations of stress tolerance genes in tea plant.

CsCBF5 depletion impairs cold tolerance in tea plants.

CBFs play important roles in tea plant cold tolerance. In our study, 16 tea varieties were used to investigate the relationship between the expression level of CsCBFs and cold tolerance in field experiments. A strong and positive correlation was found between cold stress-regulated CsCBF1, CsCBF3 and CsCBF5 expression levels (R2 > 0.8) in tea mesophyll cells and cold tolerance in 16 tea varieties. A previous study reported that CsCBF1 and CsCBF3 were important components associated with cold tolerance in tea plants; thus, the function of CsCBF5 in the CsCBF family was targeted. Our previous study reported that CsCBF5 was localized in the nucleus and exhibited transcriptional activity. In the current study, MDA content in leaves was significantly increased in CsCBF5-silenced leaves, which exhibited poor cold tolerance, compared with WT plants under cold stress. In contrast, increased germination rates and antioxidant enzyme activities under cold conditions compared with WT plants. Furthermore, CsCBF5 overexpression in Arabidopsis promoted the expression levels of the cold-regulated genes AtCOR15a, AtCOR78, AtERD4 and AtRD29B; however, the expression levels of downstream genes, including CsCOR47, CsCOR413, CsERD4 and CsRD29B, were significantly reduced in CsCBF5-silenced tea leaves. Taken together, our results indicated that CsCBF5 could function as a positive regulator in the cold stress response.

K-solubilizing bacteria (Bacillus) promote theanine synthesis in tea roots (Camellia sinensis) by activating CsTSI activity.

Theanine is an important quality parameter referring to tea quality. Applying nitrogen fertilizers is one strategy to improve the level of theanine; however, the effect of plant growth-promoting rhizobacteria on theanine synthesis in tea roots has been less studied. In this study, the bacteria isolated from Qimen County with the maximum potassium (K) solubilization were identified as Bacillus by biochemical and molecular analyses. We show that tartaric and pyruvic acids produced by Bacillus were important components related to K solubilization in vitro. Pot experiments and enzymatic assays in vitro showed that inoculation with Bacillus-secreted organic acids increased the level of available potassium in the soil. The increased K level activated recombinant CsTSI activity (theanine biosynthesis enzyme) and increased ethylamine content (the synthesis precursor of theanine), resulting in promoted theanine synthesis in tea roots. Therefore, our study indicates that Bacillus can be a potential bioinoculant for biofortification of tea.

Contribution of K solubilising bacteria (Burkholderia sp.) promotes tea plant growth (Camellia sinesis) and leaf polyphenols content by improving soil available K level.

K+ availability is important for growth and quality of tea (Camellia sine sis L.). K solubilising bacteria convert insoluble K to available K. This study was conducted to screen K solubilising bacteria isolated from tea rhizosphere soil in Qimen county, Anhui province, China. The maximum K solubilisation colony (the ratio of diameter halo/colony was 2.54) was identified as Burkholderia sp. (storage number: M2021105) by biochemistry and molecular analysis. Pot experiments (Laterite) showed that the inoculation of Burkholderia sp. significantly improved tea plant height (Zhongcha108, 1 year old) and total polyphenols content by 21.14% and 21.58% compared with the control, respectively. Higher polyphenol level promoted the formation of theaflavin in the fermentation experiments. Further experiments showed that tartaric acid and pryuvic acid produced by Burkholderia sp. are important components associated with K solubilisation in vitro . Burkholderia sp. significantly increased soil available K by 15.12%; however, there was no significant difference in available N and P, and Cu, Mg, Zn and Ca compared with the control. K content in inoculated tea roots and leaves was significantly higher (50% and 10%, respectively) than the control. Compared with the control, exogenous supply of 60mgkg-1 K significantly increased levels of polyphenol (53.97%), theaflavin (16.31%), theaflavin-3-gallate (20%), theaflavin 3'-gallic acid ester (32.24%) and theaflavin 3,3'-gallic acid ester (40.95%). Due to its ability to enable higher available soil K, ur study indicated that Burkholderia sp. have potential to increase total polyphenols content be a bio-inoculant for biofortification of tea.

Chloride and amino acids are associated with K+-alleviated drought stress in tea (Camellia sinesis).

Drought is one of the main limiting factors affecting tea plant yield and quality. Previous studies have reported that K+ (potassium) application significantly alleviated drought-induced damage in tea plants. However, the intrinsic mechanisms underlying K+-alleviated drought stress are still obscure. In our study, two contrasting varieties, Taicha12 (drought tolerant) and Fuyun6 (drought sensitive), were used to investigate the intrinsic mechanisms behind K+-alleviated drought stress in tea plants. In the present study, we compared with the case of tea plants under drought: higher water and chlorophyll contents were found in drought-stressed tea plants with an external K+ supply, confirming the role of externally supplied K+ in mitigating drought stress. We also found that an adequate K+ supply promoted Cl- accumulation in the mesophyll of Taicha12 (drought tolerant) over that of in Fuyun6 (drought sensitive). Moreover, Gly, Cys, Lys and Arg were not detected in Fuyun6 under 'Drought' or 'Drought + K+' conditions. Results showed that an exogenous supply of Arg and Val significantly alleviated drought-induced damage in Fuyun6, suggesting their role in K+-alleviated drought stress in tea plants. Collectively, our results show that chloride and amino acids are important components associated with K+-alleviated drought stress in tea plants.

Mesophyll cells' ability to maintain potassium is correlated with drought tolerance in tea (Camellia sinensis).

Tea plant is an important economic crop and is vulnerable to drought. A good understanding of tea drought tolerance mechanisms is required for breeding robust drought tolerant tea varieties. Previous studies showed mesophyll cells' ability to maintain K+ is associated with its stress tolerance. Here, in this study, 12 tea varieties were used to investigate the role of mesophyll K+ retention ability towards tea drought stress tolerance. A strong and negative correlation (R2 = 0.8239, P < 0.001) was found between PEG (mimic drought stress)-induced K+ efflux from tea mesophyll cells and overall drought tolerance in 12 tea varieties. In agreement with this, a significantly higher retained leaf K+ content was found in drought tolerant than the sensitive tea varieties. Furthermore, exogenous applied K+ (5 mM) significantly alleviated drought-induced symptom in tea plants, further supporting our finding that mesophyll K+ retention is an important component for drought tolerance mechanisms in tea plants. Moreover, pharmacological experiments showed that the contribution of K+ outward rectifying channels and non-selective cation channels in controlling PEG-induced K+ efflux from mesophylls cells are varied between drought tolerant and sensitive tea varieties.

Efficient iron plaque formation on tea (Camellia sinensis) roots contributes to acidic stress tolerance.

Tea plants grow in acidic soil, but to date, their intrinsic mechanisms of acidic stress tolerance have not been elucidated. Here, we assessed the tea plant response to growth on NH4 + nutrient media having different pH and iron levels. When grown in standard NH4 + nutrient solution (iron insufficient, 0.35 mg L-1 Fe2+ ), tea roots exhibited significantly lower nitrogen accumulation, plasma membrane H+ -ATPase activity, and protein levels; net H+ efflux was lower at pH 4.0 and 5.0 than at pH 6.0. Addition of 30 mg L-1 Fe2+ (iron sufficient, mimicking normal soil Fe2+ concentrations) to the NH4 + nutrient solution led to more efficient iron plaque formation on roots and increased root plasma membrane H+ -ATPase levels and activities at pH 4.0 and 5.0, compared to the pH 6.0 condition. Furthermore, plants grown at pH 4.0 and 5.0, with sufficient iron, exhibited significantly higher nitrogen accumulation than those grown at pH 6.0. Together, these results support the hypothesis that efficient iron plaque formation, on tea roots, is important for acidic stress tolerance. Furthermore, our findings establish that efficient iron plaque formation is linked to increased levels and activities of the tea root plasma membrane H+ -ATPase, under low pH conditions.

Metabolite profiling and transcriptomic analyses reveal an essential role of UVR8-mediated signal transduction pathway in regulating flavonoid biosynthesis in tea plants (Camellia sinensis) in response to shading.

BACKGROUND: Tea is the most popular nonalcoholic beverage worldwide for its pleasant characteristics and healthful properties. Catechins, theanine and caffeine are the major natural products in tea buds and leaves that determine tea qualities such as infusion colors, tastes and fragrances, as well as their health benefits. Shading is a traditional and effective practice to modify natural product accumulation and to enhance the tea quality in tea plantation. However, the mechanism underlying the shading effects is not fully understood. This study aims to explore the regulation of flavonoid biosynthesis in Camellia sinensis under shading by using both metabolomic and transcriptional analyses. RESULTS: While shading enhanced chlorophyll accumulation, major catechins, including C, EC, GC and EGC, decreased significantly in tea buds throughout the whole shading period. The reduction of catechins and flavonols were consistent with the simultaneous down-regulation of biosynthetic genes and TFs associated with flavonoid biosynthesis. Of 16 genes involved in the flavonoid biosynthetic pathway, F3'H and FLS significantly decreased throughout shading while the others (PAL, CHSs, DFR, ANS, ANR and LAR, etc.) temporally decreased in early or late shading stages. Gene co-expression cluster analysis suggested that a number of photoreceptors and potential genes involved in UV-B signal transductions (UVR8_L, HY5, COP1 and RUP1/2) showed decreasing expression patterns consistent with structural genes (F3'H, FLS, ANS, ANR, LAR, DFR and CHSs) and potential TFs (MYB4, MYB12, MYB14 and MYB111) involved in flavonoid biosynthesis, when compared with genes in the UV-A/blue and red/far-red light signal transductions. The KEGG enrichment and matrix correlation analyses also attributed the regulation of catechin biosynthesis to the UVR8-mediated signal transduction pathway. Further UV-B treatment in the controlled environment confirmed UV-B induction on flavonols and EGCG accumulation in tea leaves. CONCLUSIONS: We proposed that catechin biosynthesis in C. sinensis leaves is predominantly regulated by UV through the UVR8-mediated signal transduction pathway to MYB12/MYB4 downstream effectors, to modulate flavonoid accumulation. Our study provides new insights into our understanding of regulatory mechanisms for shading-enhanced tea quality.

Al(3+) -promoted fluoride accumulation in tea plants (Camellia sinensis) was inhibited by an anion channel inhibitor DIDS.

BACKGROUND: Generally, tea plants are grown in acid soil which is rich in aluminum (Al) and fluoride (F). A recent publication showed that pretreatment with Al(3+) promoted F accumulation in tea plants by increasing endogenous Ca(2+) and calmodulin (CaM). A high level of F in tea leaves not only impairs tea quality but also might pose a health risk for people drinking tea regularly. Therefore it is important to try to find some clues which might be beneficial in controlling F accumulation in tea plants grown in acid soil (Al(3+) ). RESULTS: It was found that diisothiocyanostilbene-2,2-disulfonic acid (DIDS) significantly reduced Al(3+) -promoted F accumulation in tea plants. Additionally, Al(3+) plus DIDS treatment stimulated significantly higher Ca(2+) efflux and decreased the CaM level in tea roots compared with Al(3+) treatment. Besides, significantly higher depolarization of membrane potential was shown in tea roots treated with Al(3+) plus DIDS than in those treated with Al(3+) , as well as higher net total H(+) efflux and plasma membrane H(+) -ATPase activity. CONCLUSION: Al(3+) -promoted F accumulation in tea plants was inhibited by an anion channel inhibitor DIDS. Ca(2+) /CaM and membrane potential depolarization may be the components involved in this process. © 2016 Society of Chemical Industry.

Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca²âº, CaM and Depolarization of Plasma Membrane Potential.

Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca(2+) efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca(2+) chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca(2+) fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca(2+) chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca(2+) and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca(2+)-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.

Localization of fluoride and aluminum in subcellular fractions of tea leaves and roots.

The tea plant is a fluoride (F) and aluminum (Al) hyperaccumulator. High concentrations of F and Al have always been found in tea leaves without symptoms of toxicity, which may be related to the special localization of F and Al in tea leaves. In this study, we for the first time determined the subcellular localization of F and Al in tea roots and leaves and provided evidence of the detoxification mechanisms of high concentrations of F and Al in tea plants. Results revealed that 52.3 and 71.8% of the total F accumulated in the soluble fraction of tea roots and leaves, and vacuoles contained 98.1% of the total F measured in the protoplasts of tea leaves. Cell walls contained 69.8 and 75.2% of the total Al detected in the tea roots and leaves, respectively, and 73.2% of Al sequestered in cell walls was immobilized by pectin and hemicellulose components. Meanwhile, 88.3% of the Al measured in protoplasts was stored in the vacuoles of tea leaves. Our results suggested that the subcellular distributions of F and Al in tea plants play two important roles in the detoxification of F and Al toxicities. First, most of the F and Al was sequestered in the vacuole fractions in tea leaves, which could reduce their toxicities to organelles. Second, Al can be immobilized in the pectin and hemicellulose components of cell walls, which could suppress the uptake of Al by tea roots.