Metabolome profiling and transcriptome analysis unveiling the crucial role of magnesium transport system for magnesium homeostasis in tea plants.

Magnesium (Mg2+) is a crucial nutrient for the growth and development of Camellia sinensis and is closely related to the quality of tea. However, the underlying mechanisms responding to low-Mg 2+ stress in tea plants remain largely unknown. In this study, photosynthetic parameters, metabolomics, and transcriptomics were utilized to explore the potential effects of low Mg2+ on the growth and metabolism of C. sinensis. Low-Mg2+ treatment increased the ratio of shoot dry weight to root dry weight but decreased the photosynthesis of C. sinensis. Forty and thirty metabolites were impacted by Mg2+ shortage in C. sinensis shoots and roots, respectively. Integrated transcriptome and metabolome analyses revealed the possible reasons for the decreased contents of chlorophyll and catechins and the increased theanine content in C. sinensis roots. Weighted gene co-expression network analysis indicated that the Mg2+ transport system was essential in the regulation of Mg2+ homeostasis in C. sinensis, in which CsMGT5 was identified to be the key regulator according to CsMGT5-overexpressing and complementary assays in Arabidopsis thaliana. Moreover, silencing of CsMGT5 in vivo reduced the content of chlorophyll in C. sinensis shoots. In addition, CsMGT5 might collaborate with ammonium transporters to keep the amino acid content steady, suggesting its potential application for tea quality improvement. All these findings demonstrate the key roles of CsMGTs for Mg2+ homeostasis in C. sinensis, providing a theoretical basis for Mg2+ efficient utilization in plants.

Dynamic changes and the effects of key procedures on the characteristic aroma compounds of Lu'an Guapian green tea during the manufacturing process.

As a kind of green tea with unique multiple baking processes, the flavor code of Lu'an Guapian (LAGP) has recently been revealed. To improve and stabilize the quality of LAGP, further insight into the dynamic changes in odorants during the whole processing is required. In this study, 50 odorants were identified in processing tea leaves, 14 of which were selected for absolute quantification to profile the effect of processes. The results showed that spreading is crucial for key aroma generation and accumulation, while these odorants undergo significant changes at the deep baking stage. By adjusting the conditions of the spreading and deep baking, it was found that low-temperature (4 °C) spreading for 6 h and low-temperature with long-time baking (final leaf temperature: 102 °C, 45 min) could improve the overall aroma quality. These results provide a new direction for enhancing the quality of LAGP green tea.

Csn-miR156d-CsSPL1 regulates flowering and anthocyanin metabolism.

MiR156 play important roles in regulation of plant growth and development, secondary metabolite synthesis, and other biological processes by targeting the SQUAMOSA promoter binding protein-like (SPL) family. Our previous sequencing data analysis suggested that Csn-miR156d may regulate flowering and anthocyanin accumulation by cleavage and degradation of the expression of the SPL in tea plant, but it remains to be elucidated. In this study, 5'RLM-RACE experiment, tobacco transient transformation, qRT-PCR, and antisense oligonucleotide (asODN) were used to verify that CsSPL1 is the target gene of Csn-miR156d. Stable transformation of Arabidopsis revealed that Csn-miR156d could delay flowering by negatively regulating the transcript levels of FT, AP1, FUL, and SOC1, while overexpression of CsSPL1 showed an opposite effect. Additionally, overexpression of Csn-miR156d in Arabidopsis could enhance the transcription of the anthocyanin biosynthesis-related structural genes DFR, ANS, F3H, UGT78D2, and LDOX, as well as regulatory genes PAP1, MYB113, GL3, MYB11, and MYB12, leading to anthocyanin accumulation. Moreover, asODN experiment revealed that Csn-miR156d could increase the anthocyanin content in tea plant. These results suggest that Csn-miR156d regulates flowering and anthocyanin accumulation in tea plant by suppressing the expression of CsSPL1. Our study provides new insights into the development and anthocyanin accumulation in tea plant and lays a theoretical foundation for further research on the molecular mechanism of miRNAs in regulating tea plant growth and secondary metabolism.

Metal tolerance protein CsMTP4 has dual functions in maintaining zinc homeostasis in tea plant.

Plants have evolved a series of zinc (Zn) homeostasis mechanisms to cope with the fluctuating Zn in the environment. How Zn is taken up, translocated and tolerate by tea plant remains unknown. In this study, on the basis of RNA-Sequencing, we isolated a plasma membrane-localized Metal Tolerance Protein (MTP) family member CsMTP4 from Zn-deficient tea plant roots and investigated its role in regulation of Zn homeostasis in tea plant. Heterologous expression of CsMTP4 specifically enhanced the tolerance of transgenic yeast to Zn excess. Moreover, overexpression of CsMTP4 in tea plant hairy roots stimulated Zn uptake under Zn deficiency. In addition, CsMTP4 promoted the growth of transgenic Arabidopsis plants by translocating Zn from roots to shoots under Zn deficiency and conferred the tolerance to Zn excess by enhancing the efflux of Zn from root cells. Transcriptome analysis of the CsMTP4 transgenic Arabidopsis found that the expression of Zn metabolism-related genes were differentially regulated compared with wild-type plants when exposed to Zn deficiency and excess conditions. This study provides a mechanistic understanding of Zn uptake and translocation in plants and a new strategy to improve phytoremediation efficiency.

Comprehensive comparison of aroma profiles and chiral free and glycosidically bound volatiles in Fujian and Yunnan white teas.

The Fujian and Yunnan provinces in China are the most representative origins of white tea. However, the key differences in the chemical constituents of the two white teas have rarely been revealed. In this study, a comprehensive comparison of the aroma profiles, chiral volatiles, and glycosidically bound volatiles (GBVs) in Fujian and Yunnan white teas was performed, and 174 volatiles and 28 enantiomers, including 22 volatiles and six GBVs, were identified. Linalool, linalyl-ß-primeveroside (LinPrim), and α-terpineol presented the opposite dominant configurations in Fujian and Yunnan white teas, and the chiral GBVs were firstly quantified with significant differences in the contents of R-LinPrim and ß-d-glucopyranosides of (2R, 5R)-linalool oxide A and (2R, 5S)-linalool oxide B. Moreover, discrimination functions for Fujian and Yunnan white teas were created using nine key variables with excellent reliability and efficiency. These results provide a new method for objectively distinguishing authentic white teas according to geographical origin.

Assessing the health risks of heavy metals and seasonal minerals fluctuations in Camellia sinensis cultivars during their growth seasons.

The risk assessment of heavy metals in tea is extremely imperative for the health of tea consumers. However, the effects of varietal variations and seasonal fluctuations on heavy metals and minerals in tea plants remain unclear. Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to evaluate the contents of aluminum (Al), manganese (Mn), magnesium (Mg), boron (B), calcium (Ca), copper (Cu), cobalt (Co), iron (Fe), sodium (Na), zinc (Zn), arsenic (As), cadmium (Cd), chromium (Cr), nickel (Ni), and antimony (Sb) in the two categories of young leaves (YL) and mature leaves (ML) of tea (Camellia sinensis) cultivars throughout the growing seasons. The results showed significant variations in the contents of the investigated nutrients both among the different cultivars and growing seasons as well. Furthermore, the average concentrations of Al, Mn, Mg, B, Ca, Cu, Co, Fe, Na, Zn, As, Cd, Cr, Ni, and Sb in YL ranged, from 671.58-2209.12, 1260.58-1902.21, 2290.56-2995.36, 91.18-164.68, 821.95-5708.20, 2.55-3.80, 3.96-25.22, 37.95-202.84, 81.79-205.05, 27.10-69.67, 0.028-0.053, 0.065-0.127, 2.40-3.73, 10.57-12.64, 0.11-0.14 mg kg-1, respectively. In ML, the concentrations were 2626.41-7834.60, 3980.82-6473.64, 3335.38-4537.48, 327.33-501.70, 9619.89-13153.68, 4.23-8.18, 17.23-34.20, 329.39-567.19, 145.36-248.69, 40.50-81.42, 0.089-0.169, 0.23-0.27, 5.24-7.89, 18.51-23.97, 0.15-0.19 mg kg-1, respectively. The contents of all analyzed nutrients were found to be higher in ML than in YL. Target hazard quotients (THQ) of As, Cd, Cr, Ni, and Sb, as well as the hazard index (HI), were all less than one, suggesting no risk to human health via tea consumption. This research might provide the groundwork for essential minerals recommendations, as well as a better understanding and management of heavy metal risks in tea.

Comprehensive dissection of variation and accumulation of free amino acids in tea accessions.

Free amino acids (FAAs) positively determine the tea quality, notably theanine (Thea), endowing umami taste of tea infusion, which is the profoundly prevalent research in albino tea genetic resources. Therefore, 339 tea accessions were collected to study FAAs level for deciphering its variation and accumulation mechanism. Interestingly, alanine (Ala) and Thea which had the highest diversity index (H') value among three varieties of Camellia sinensis (L.) O. Kuntze were significantly higher than wild relatives (P < 0.05). The intraspecific arginine (Arg) and glutamine (Gln) contents in C. sinensis var. assamica were significantly lower than sinensis and pubilimba varieties. Moreover, the importance of interdependencies operating across FAAs and chlorophyll levels were highlighted via the cell ultrastructure, metabolomics, and transcriptome analysis. We then determined that the association between phytochrome interacting factor 1 (CsPIF1) identified by weighted gene co-expression network analysis (WGCNA) and Thea content. Intriguingly, transient knock-down CsPIF1 expression increased Thea content in tea plant, and the function verification of CsPIF1 in Arabidopsis also indicated that CsPIF1 acts as a negative regulator of Thea content by mainly effecting the genes expression related to Thea biosynthesis, transport, and hydrolysis, especially glutamate synthase (CsGOGAT), which was validated to be associated with Thea content with a nonsynonymous SNP by Kompetitive Allele-Specific PCR (KASP). We also investigated the interspecific and geographical distribution of this SNP. Taken together, these results help us to understand and clarify the variation and profile of major FAAs in tea germplasms and promote efficient utilization in tea genetic improvement and breeding.

Non-Targeted Metabolomics Reveals the Effects of Different Rolling Methods on Black Tea Quality.

A non-targeted metabolomics approach and sensory evaluation, coupled with multivariate statistical analysis, systematically uncover the impact of the rolling time on the quality parameters of black tea. GC-MS analysis reveals that a moderate extension of rolling time favorably contributes to the accumulation of characteristic aroma components in black tea. The volatile components reach their highest concentration in black tea samples processed during an 80-min rolling period. UHPLC-Q-TOF/MS analysis demonstrates a substantial decrease in the contents of catechins and flavonoids with an increase in rolling time. Simultaneously, the production of theaflavins, coupled with the degradation of green bitterness volatiles (GBVs), significantly contributes to the formation of endogenous aroma components in black tea. These findings underscore the close relationship between rolling time control and black tea quality, emphasizing that a moderate extension of the rolling time fosters the development of improved black tea flavor quality. The comprehensive quality evaluation indicates that the optimal duration is 80 min. However, the initial 0 to 20 min of rolling is a crucial phase for the genesis and transformation of black tea quality. This study offers valuable insights into the influence of rolling time on black tea quality, potentially enhancing future studies of rolling technology. It provides theoretical guidelines for optimizing the processing of Gongfu black tea.

A highly effective SERS platform formed by the fabrication of Ag@ZIF-8@Au nanoparticles for rapid detection of acetamiprid in environment.

The unreasonable spraying and random migration of acetamiprid may cause pollution of crops, soil and water resources in the environment, resulting in threatening ecosystem and human health. However, the monitoring of acetamiprid using mass spectrum in the environment encounters challenges due to high-cost instruments and complex processing time. Herein, we fabricated a rapid and reliable SERS method based on Ag@ZIF-8@Au platforms for tracing acetamiprid residues in the environment. In this method, a MOF material named ZIF-8 is coated with silver nanoparticles and distributed internally between AgNPs and AuNPs to enhance Raman signal, which can enrich pesticide molecules into the hotspots area provided by noble material and helps avoid the oxidation of silver nanoparticles. High sensitivity (LOD of 9.027 × 10-10 M for acetamiprid, and SERS enhancement factor of 4.3 × 107), excellent reproducibility (6.496% or 7.198% RSD for 30 random points) and superior stability (3.127% RSD for 6 weeks) were achieved using the proposed method. Acetamiprid with concentrations from 10-4 to 10-9 M were successfully detected by SERS method. Furthermore, the linear detection models of acetamiprid in different environment matrices (lake water, tea leaves, tea garden soil, oranges and oranges orchard soil) were established and all the correlation coefficient (R2) were higher than or equal to 95%, indicating the excellent adaptability of Ag@ZIF-8@Au platform in environment. The randomly spiked concentrations of acetamiprid were also tested with good recovery values and low relative error values, further confirming the reliability of the detection method.

The key aroma components of steamed green tea decoded by sensomics and their changes under different withering degree.

Steamed green tea has a long history and unique aroma, but little is known about its key aroma components. In this study, 173 volatiles in steamed green tea were identified using solvent-assisted flavor evaporation and headspace-solid phase microextraction plus two chromatographic columns of different polarities. Aroma extract dilution analysis revealed 48 highly aroma-active compounds with flavor dilution factors 64-1024. Internal standards were used to calculate odorant active value (OAV), and 11 OAV > 1 key aroma compounds were determined. Omission test identified eight substances, including dimethyl sulfide, (E)-ß-ionone, cis-jasmone, linalool, nonanal, heptanal, isovaleraldehyde and (Z)-3-hexenol, as the key aroma active compounds of steamed green tea. With the increase of withering degree, the content of these substances increased first and then decreased except for heptanal and cis-jasmone. Moreover, the water content of 62 % was suggested to be an appropriate withering degree during the processing of steamed green tea.

Study on color, aroma, and taste formation mechanism of large-leaf yellow tea during an innovative manufacturing process.

This study used samples processed with an innovative manufacturing process to explore the dynamic changes of large-leaf yellow tea (LYT) in color, aroma, and taste substances, and the quality components were most significantly affected in the stages of first pile-yellowing (FP) and over-fired drying (TD). In this process, the moisture and temperature conditions caused chlorophyll degradation, Maillard reactions, caramelization reactions, and isomerization of phenolic substances, forming the quality of LYT. Specifically, chlorophyll degradation favored the formation of color quality; the taste quality was determined by the content of soluble sugars, amino acids, catechins, etc.; the aroma quality was dependent on the content changes of alcohols and aldehydes, as well as the increase of sweet and roasting aroma substances in the third drying stage. Additionally, twelve key aroma components, including linalool, (E)-ß-ionone, 2,3-diethyl-5-methyl-pyrazine, etc., were identified as contributors to revealing LYT rice crust-like and sweet aroma formation mechanism.

Mechanism of aroma enhancement methods in accelerating Congou black tea acidification subjected to room temperature storage.

Acidification of aroma-enhanced black tea during storage was studied. UPLC-Q-TOF/MS (Ultra Performance Liquid Chromatography and Quadrupole-Time of Flight Mass Spectrometer) and HPLC (High-Performance Liquid Chromatography) analysis of non-volatile substances and organic acids revealed a decrease of soluble sugars and amino acids in aroma-enhanced black tea, while an increase in organic acids such as oxalic acid, malic acid and quinic acid. Further in vitro experiments indicated that the acidification of aroma-enhanced tea during storage can be attributed to decomposition of sugars and amino acids by heating, oxidation of aromatic aldehydes. Meanwhile, the amino acids, catechins, soluble sugars and flavonoids that constitute the taste of black tea are further reduced, changing the taste composition of tea infusion and further increasing its acidity. This study revealed the reasons for black tea acidification during aroma enhancement and storage and provided a theoretical basis for improving black tea quality.

Non-targeted metabolomics characterization of flavor formation of Lichuan black tea processed from different cultivars in Enshi.

Nine tea cultivars planted in Enshi were selected and processed into "Lichuan black tea". Sensory evaluation showed that cultivar had the greatest influence on taste and aroma quality, including sweetness, umami and concentration of taste, as well as sweet and floral fragrances of aroma. The non-volatile and volatile components were identified by UPLC-Q-TOF/MS and GC-MS, and PCA analysis showed good separation between cultivars, which could cause the difference in quality. Baiyaqilan, Meizhan and Echa 10 had a floral aroma, with obvious difference in their aromatic composition from other cultivars. Moreover, Echa 10 also had a strong sweet aroma. The key aroma components in Echa 10 (with the largest cultivation area) were further investigated by GC-O-MS combined with odor activity value (OAV) analysis, included ß-damascenone, phenylethylaldehyde, nonenal, geraniol, linalool, jasmonone, (E)-2-nonenal, ß-cyclocitral, (E)-ß-ocimene, methyl salicylate, ß-ionone, 2,6,10,10-tetramethyl-1-oxaspiro[4.5]dec-6-ene, citral, ß-myrcene, nerol, phenethyl alcohol, benzaldehyde, hexanal, nonanoic acid, and jasmin lactone.

Compositions and Antioxidant Activity of Tea Polysaccharides Extracted from Different Tea (Camellia sinensis L.) Varieties.

Tea polysaccharide (TPS) is a bioactive compound extracted from tea. It has raised great interest among researchers due to its bioactivity. However, few studies focused on the diversity of TPS in its compositions and antioxidant activity. This study collected 140 different tea varieties from four tea germplasm gardens in China, and their TPSs in tea shoots were extracted. The extraction efficiency, composition contents, including neutral sugar, uronic acid, protein, and tea polyphenols, and the scavenging abilities of hydroxyl radical (·OH) and superoxide radical (O2-·) of 140 TPSs were determined and analyzed. The results showed significant differences in the compositions and antioxidant activities of TPS extracted from different tea varieties. By applying hierarchical clustering analysis (HCA), we selected nine tea varieties with high TPS extraction efficiency and 26 kinds of TPS with high antioxidant capacity.

Novel insights into the role of leaf in the cutting process of Camellia sinensis using physiological, biochemical and transcriptome analyses.

Cuttage is the preferred approach for rapid propagation of many species including tea plant (Camellia sinensis). Leaf serves as a key part of nodal cutting, but there is a lack of systematic research on its role in the cutting process. In this study, 24 tea cultivars were employed to prove the necessity of leaf and light during cuttage. Further leaf physiological parameters found that lower net photosynthesis rate probably promoted rooting. Phytohormone content detection showed that auxin content and composition pattern were related to rooting ability. Leaf transcriptome analyses of cuttings from a representative easy-to-root cultivar (cv. Echa 10) revealed that genes involved in carbohydrate metabolism, signal transduction, metabolite biosynthesis and transportation were differentially expressed during the rooting process. CsTSA1, CsYUC10, CsAUX1s, CsPIN3 and CsPIN5 were selected as the candidate genes, which possibly regulate the rooting of nodal cuttings. These results illustrate the necessity of the leaf in cuttage and provide molecular evidence that leaf is an important place for signal transduction, metabolite synthesis and transport during the rooting process.

Characterizing and Decoding the Effects of Different Fermentation Levels on Key Aroma Substances of Congou Black Tea by Sensomics.

Fermentation is the key technology for black tea aroma formation. The key aroma substances of black tea at different fermentation stages (unfermented (WDY), underfermented (F1H), fully fermented (F4H), and overfermented (F8H)) were characterized by the methodology of Sensomics. Aroma extract dilution analysis was performed on volatile fractions extracted by using solvent-assisted flavor evaporation and solid-phase microextraction, yielding 93 odor-active areas. Internal standard method plus stable isotope dilution analysis was used for quantitative analysis. The omission experiment identified 23 aroma substances. Further reduction and addition experiments revealed phenylacetaldehyde, (E,E)-2,4-heptadienal, geraniol, linalool, ß-damascenone, 2-methylbutyraldehyde, dimethyl sulfide, and isovaleraldehyde with odor activity values (OAV) > 100 as the characteristic aroma components of F4H and also as the main contributors to aroma differences between different fermentation degrees. The green odor of (E,E)-2,4-heptadienal was highlighted in WDY and F1H relative to that in F4H due to the lower contribution of phenylacetaldehyde and ß-damascenone in the former two samples. Additionally, excessive OAV increase of fatty aldehydes in F8H masked its similar floral and fruity aroma.

Study on taste quality formation and leaf conducting tissue changes in six types of tea during their manufacturing processes.

This study fristly investigated the taste quality formation and leaf conducting tissue changes in six types of Chinese tea (green, black, oolong, yellow, white, and dark) made from Mingke No.1 variety. Non-targeted metabolomics showed the vital manufacturing processes (green tea-de-enzyming, black tea-fermenting, oolong tea-turning-over, yellow tea-yellowing, white tea-withering, and dark tea-pile-fermenting) were highly related to their unique taste formation, due to different fermentation degree in these processes. After drying, the retained phenolics, theanine, caffeine, and other substances significantly impacted each tea taste quality formation. Meanwhile, the tea leaf conducting tissue structure was significantly influenced by high processing temperature, and the change of its inner diameter was related to moisture loss during tea processing, as indicated by its significant different Raman characteristic peaks (mainly cellulose and lignin) in each key process. This study provides a reference for process optimization to improve tea quality.

The aroma characteristics of oolong tea are jointly determined by processing mode and tea cultivars.

This study delved into the aroma characteristics of "Qingxiang" oolong tea, analyzing six different cultivars and their processing modes. The findings showed that both cultivars and processing modes have a significant impact on the oolong tea aroma system. The study identified 18 terpenoid volatiles (VTs), 11 amino-acid-derived volatiles (AADVs), 15 fatty-acid-derived volatiles (FADVs), 3 carotenoid-derived volatiles (CDVs), and 10 other compounds in oolong tea that differentiate it from green and black tea. The turn-over stage was found to be the primary processing stage for oolong tea aroma formation. Molecular sensory analysis revealed that the "fresh" odor attribute is the basis for its aroma, while "floral and fruity" fragrances are its aroma characteristics. The perception of oolong tea as "fresh" and "floral and fruity" is influenced by the interactions of its aroma components. These findings provide a new basis for breed improvement and process enhancement in oolong tea production.

Study on the Variation Law of the Main Mechanical Properties in the Processing of Longjing Tea.

Fresh tea leaves, both single bud and one leaf with a bud, were used as the test materials in this study. The variation in the main mechanical properties, such as texture and tensile properties, during the processing of Longjing tea was examined by using texture profile analysis (TPA) and stress-strain tensile tests. The plasticity showed a trend of first increasing and then decreasing during the processing, whereas the elasticity displayed the opposite tendency. The flexibility reached a maximum during the fixing stage and then slowly declined with a relatively small change. Initially, the maximum force dropped down and then gradually elevated later.Both the tensile strength and the fracture strain indicated an upward movement at the beginning and then a downfall afterward. The elastic modulus changed little before the final panning stage, then raised significantly. The correlation analysis revealed that the flexibility of tea leaves was highly positively correlated with water content. At water content of 30% and 50%, the plasticity and flexibility of tea leaves reached a clear peak and the maximum force was at a low level, which is suitable for the shaping of Longjing tea. The results also demonstrated that the main mechanical properties of different tender materials change differently during the processing. The research findings can provide parameters for optimizing the mechanical design and processing technology of Longjing tea.

The inhibitory effect and mechanism of theaflavins on fluoride transport and uptake in HIEC-6 cell model.

Fluoride (F-) is widely present in nature, while long-term excessive F- intake can lead to fluorosis. Theaflavins are an important bioactive ingredient of black and dark tea, and black and dark tea water extracts showed a significantly lower F- bioavailability than NaF solutions in previous studies. In this study, the effect and mechanism of four theaflavins (theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, theaflavin-3,3'-digallate) on F- bioavailability were investigated using normal human small intestinal epithelial cells (HIEC-6) as a model. The results showed that theaflavins could inhibit the absorptive (apical - basolateral) transport of F- while promote its secretory (basolateral - apical) transport in HIEC-6 cell monolayers in a time- and concentration-dependent (5-100 µg/mL) manner, and significantly reduce the cellular F- uptake. Moreover, the HIEC-6 cells treated with theaflavins showed a reduction in cell membrane fluidity and cell surface microvilli. Transcriptome, qRT-PCR and Western blot analysis revealed that theaflavin-3-gallate (TF3G) addition could significantly enhance the mRNA and protein expression levels of tight junction-related genes in HIEC-6 cells, such as claudin-1, occludin and zonula occludens-1 (ZO-1). Overall, theaflavins may reduce F- absorptive transport by regulating tight junction-related proteins, and decreasing intracellular F- accumulation by affecting the cell membrane structure and properties in HIEC-6 cells.