Characterization of CsUGT73AC15 as a Multifunctional Glycosyltransferase Impacting Flavonol Triglycoside Biosynthesis in Tea Plants.

Flavonol glycosides, contributing to the health benefits and distinctive flavors of tea (Camellia sinensis), accumulate predominantly as diglycosides and triglycosides in tea leaves. However, the UDP-glycosyltransferases (UGTs) mediating flavonol multiglycosylation remain largely uncharacterized. In this study, we employed an integrated proteomic and metabolomic strategy to identify and characterize key UGTs involved in flavonol triglycoside biosynthesis. The recombinant rCsUGT75AJ1 exhibited flavonoid 4'-O-glucosyltransferase activity, while rCsUGT75L72 preferentially catalyzed 3-OH glucosylation. Notably, rCsUGT73AC15 displayed substrate promiscuity and regioselectivity, enabling glucosylation of rutin at multiple sites and kaempferol 3-O-rutinoside (K3R) at the 7-OH position. Kinetic analysis revealed rCsUGT73AC15's high affinity for rutin (Km = 9.64 µM). Across cultivars, CsUGT73AC15 expression inversely correlated with rutin levels. Moreover, transient CsUGT73AC15 silencing increased rutin and K3R accumulation while decreasing their respective triglycosides in tea plants. This study offers new mechanistic insights into the key roles of UGTs in regulating flavonol triglycosylation in tea plants.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a ß-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

The Impact of Harvesting Mechanization on Oolong Tea Quality.

Mechanization is the inevitable future of tea harvesting, but its impact on tea chemistry and quality remains uncertain. Our study examines untargeted metabolomic data from 185 oolong tea products (Tieguanyin) made from leaves harvested by hand or machine based on UPLC-QToF-MS analysis. The data revealed a minimum 50% loss for over half of the chemicals in the machine-harvested group, including catechins, theaflavin, gallic acid, chlorogenic acid, and kaempferol-3-gluocside. Integrating sensory evaluation, OPLS-DA identified the six most important metabolites as significant contributors to sensory decline caused by harvesting mechanization. Furthermore, our research validates the possibility of using DD-SIMCA modelling with untargeted metabolomic data for distinguishing handpicked from machine-harvested tea products. The model was able to achieve 93% accuracy. This study provides crucial insights into the chemical and sensory shifts during mechanization, along with tools to manage and monitor these changes.

Metabolic Rewiring in Tea Plants in Response to Gray Blight Disease Unveiled by Multi-Omics Analysis.

Gray blight disease, which is caused by Pestalotiopsis-like species, poses significant challenges to global tea production. However, the comprehensive metabolic responses of tea plants during gray blight infection remain understudied. Here, we employed a multi-omics strategy to characterize the temporal transcriptomic and metabolomic changes in tea plants during infection by Pseudopestalotiopsis theae, the causal agent of gray blight. Untargeted metabolomic profiling with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) revealed extensive metabolic rewiring over the course of infection, particularly within 24 h post-inoculation. A total of 64 differentially accumulated metabolites were identified, including elevated levels of antimicrobial compounds such as caffeine and (-)-epigallocatechin 3-gallate, as well as oxidative catechin polymers like theaflavins, theasinensins and theacitrins. Conversely, the synthesis of (+)-catechin, (-)-epicatechin, oligomeric proanthocyanidins and flavonol glycosides decreased. Integrated omics analyses uncovered up-regulation of phenylpropanoid, flavonoid, lignin biosynthesis and down-regulation of photosynthesis in response to the pathogen stress. This study provides novel insights into the defense strategies of tea plants against gray blight disease, offering potential targets for disease control and crop improvement.

Integrative Metabolic and Transcriptomic Profiling in Camellia oleifera and Camellia meiocarpa Uncover Potential Mechanisms That Govern Triacylglycerol Degradation during Seed Desiccation.

Camellia seed oil is a top-end quality of cooking oil in China. The oil quality and quantity are formed during seed maturation and desiccation. So far, it remains largely unresolved whether lipid degradation occurs and contributes to Camellia oil traits. In this study, three different Camellia germplasms, C. oleifera cv. Min 43 (M43), C. meiocarpa var. Qingguo (QG), and C. meiocarpa cv Hongguo (HG) were selected, their seed oil contents and compositions were quantified across different stages of seed desiccation. We found that at the late stage of desiccation, M43 and QG lost a significant portion of seed oil, while such an event was not observed in HG. To explore the molecular bases for the oil loss In M43, the transcriptomic profiling of M43 and HG was performed at the early and the late seed desiccation, respectively, and differentially expressed genes (DEGs) from the lipid metabolic pathway were identified and analyzed. Our data demonstrated that different Camellia species have diverse mechanisms to regulate seed oil accumulation and degradation, and that triacylglycerol-to-terpenoid conversion could account for the oil loss in M43 during late seed desiccation.

Antifungal effects and biocontrol potential of lipopeptide-producing Streptomyces against banana Fusarium wilt fungus Fusarium oxysporum f. sp. cubense.

Fusarium wilt of banana (FWB), caused by Fusarium oxysporum f. sp. cubense (Foc), especially tropical race 4 (TR4), presents the foremost menace to the global banana production. Extensive efforts have been made to search for efficient biological control agents for disease management. Our previous study showed that Streptomyces sp. XY006 exhibited a strong inhibitory activity against several phytopathogenic fungi, including F. oxysporum. Here, the corresponding antifungal metabolites were purified and determined to be two cyclic lipopeptide homologs, lipopeptin A and lipopeptin B. Combined treatment with lipopeptin complex antagonized Foc TR4 by inhibiting mycelial growth and conidial sporulation, suppressing the synthesis of ergosterol and fatty acids and lowering the production of fusaric acid. Electron microscopy observation showed that lipopeptide treatment induced a severe disruption of the plasma membrane, leading to cell leakage. Lipopeptin A displayed a more pronounced antifungal activity against Foc TR4 than lipopeptin B. In pot experiments, strain XY006 successfully colonized banana plantlets and suppressed the incidence of FWB, with a biocontrol efficacy of up to 87.7%. Additionally, XY006 fermentation culture application improved plant growth parameters and induced peroxidase activity in treated plantlets, suggesting a possible role in induced resistance. Our findings highlight the potential of strain XY006 as a biological agent for FWB, and further research is needed to enhance its efficacy and mode of action in planta.

Metabolomics integrated with machine learning to discriminate the geographic origin of Rougui Wuyi rock tea.

The geographic origin of agri-food products contributes greatly to their quality and market value. Here, we developed a robust method combining metabolomics and machine learning (ML) to authenticate the geographic origin of Wuyi rock tea, a premium oolong tea. The volatiles of 333 tea samples (174 from the core region and 159 from the non-core region) were profiled using gas chromatography time-of-flight mass spectrometry and a series of ML algorithms were tested. Wuyi rock tea from the two regions featured distinct aroma profiles. Multilayer Perceptron achieved the best performance with an average accuracy of 92.7% on the training data using 176 volatile features. The model was benchmarked with two independent test sets, showing over 90% accuracy. Gradient Boosting algorithm yielded the best accuracy (89.6%) when using only 30 volatile features. The proposed methodology holds great promise for its broader applications in identifying the geographic origins of other valuable agri-food products.

Leaf Cuticular Transpiration Barrier Organization in Tea Tree Under Normal Growth Conditions.

The cuticle plays a major role in restricting nonstomatal water transpiration in plants. There is therefore a long-standing interest to understand the structure and function of the plant cuticle. Although many efforts have been devoted, it remains controversial to what degree the various cuticular parameters contribute to the water transpiration barrier. In this study, eight tea germplasms were grown under normal conditions; cuticle thickness, wax coverage, and compositions were analyzed from the epicuticular waxes and the intracuticular waxes of both leaf surfaces. The cuticular transpiration rates were measured from the individual leaf surface as well as the intracuticular wax layer. Epicuticular wax resistances were also calculated from both leaf surfaces. The correlation analysis between the cuticular transpiration rates (or resistances) and various cuticle parameters was conducted. We found that the abaxial cuticular transpiration rates accounted for 64-78% of total cuticular transpiration and were the dominant factor in the variations for the total cuticular transpiration. On the adaxial surface, the major cuticular transpiration barrier was located on the intracuticular waxes; however, on the abaxial surface, the major cuticular transpiration barrier was located on the epicuticular waxes. Cuticle thickness was not a factor affecting cuticular transpiration. However, the abaxial epicuticular wax coverage was found to be significantly and positively correlated with the abaxial epicuticular resistance. Correlation analysis suggested that the very-long-chain aliphatic compounds and glycol esters play major roles in the cuticular transpiration barrier in tea trees grown under normal conditions. Our results provided novel insights about the complex structure-functional relationships in the tea cuticle.

A Proposed Method for Simultaneous Measurement of Cuticular Transpiration From Different Leaf Surfaces in Camellia sinensis.

The plant cuticle is the major barrier that limits unrestricted water loss and hence plays a critical role in plant drought tolerance. Due to the presence of stomata on the leaf abaxial surface, it is technically challenging to measure abaxial cuticular transpiration. Most of the existing reports were only focused on leaf astomatous adaxial surface, and few data are available regarding abaxial cuticular transpiration. Developing a method that can measure cuticular transpiration from both leaf surfaces simultaneously will improve our understanding about leaf transpiration barrier organization. Here, we developed a new method that enabled the simultaneous measurement of cuticular transpiration rates from the adaxial and abaxial surfaces. The proposed method combined multi-step leaf pretreatments including water equilibration under dark and ABA treatment to close stomata, as well as gum arabic or vaseline application to remove or seal the epicuticular wax layer. Mathematical formulas were established and used to calculate the transpiration rates of individual leaf surfaces from observed experimental data. This method facilitates the simultaneous quantification of cuticular transpiration from adaxial and abaxial leaf surfaces. By applying this method, we demonstrated that the adaxial intracuticular waxes and the abaxial epicuticular waxes constitute the major transpiration barriers in Camellia sinensis. Wax analysis indicated that adaxial intracuticular waxes had higher coverage of very long chain fatty acids, 1-alkanol esters, and glycols, which may be attributed to its higher transpiration barrier than that of the abaxial intracuticular waxes.

Drought stress modify cuticle of tender tea leaf and mature leaf for transpiration barrier enhancement through common and distinct modes.

Cuticle is the major transpiration barrier that restricts non-stomatal water loss and is closely associated with plant drought tolerance. Although multiple efforts have been made, it remains controversial what factors shape up the cuticular transpiration barrier. Previously, we found that the cuticle from the tender tea leaf was mainly constituted by very-long-chain-fatty-acids and their derivatives while alicyclic compounds dominate the mature tea leaf cuticle. The presence of two contrasting cuticle within same branch offered a unique system to investigate this question. In this study, tea seedlings were subjected to water deprivation treatment, cuticle structures and wax compositions from the tender leaf and the mature leaf were extensively measured and compared. We found that cuticle wax coverage, thickness, and osmiophilicity were commonly increased from both leaves. New waxes species were specifically induced by drought; the composition of existing waxes was remodeled; the chain length distributions of alkanes, esters, glycols, and terpenoids were altered in complex manners. Drought treatment significantly reduced leaf water loss rates. Wax biosynthesis-related gene expression analysis revealed dynamic expression patterns dependent on leaf maturity and the severity of drought. These data suggested that drought stress-induced structural and compositional cuticular modifications improve cuticle water barrier property. In addition, we demonstrated that cuticle from the tender leaf and the mature leaf were modified through both common and distinct modes.

Location affects fatty acid composition in Camellia sinensis cv Tieguanyin fresh leaves.

Tieguanyin tea is a typical representative of oolong tea in China, and is famous for its orchid-like aroma. Fatty acids are one of the important precursors for aroma production. However, fatty acid contents and compositions in Tieguanyin largely remain undefined. In this study, we quantified the fatty acid composition in Tieguanyin and its offspring by gas chromatography-flame ionization detector, and compared the effects of growth sites and harvest time on the leaf fatty acid composition. The results showed that total fatty acid contents in Tieguanyin fresh leaves were higher than its offspring. Growth sites had significant impacts on fatty acid contents. Tieguanyin grown in Anxi County showed higher total fatty acid contents, and higher ratio of total unsaturated fatty acids to total saturated fatty acids. The fresh leaves in the morning showed higher total fatty acid contents compared to samples harvested in the afternoon or evening, suggesting a dynamic fatty acid degradation during day period. During tea processing, unsaturated fatty acids including linolenic acid, linoleic acid and oleic acid (18:1Δ9c) decreased 13.1%, 13.2% and 84.2%, respectively. The ratio of unsaturated fatty acids to saturated fatty acids still was above 300%. We found that Tieguanyin was a typical 18:3 plant, and the higher ratio of unsaturated fatty acids to saturated fatty acids of Tieguanyin grown in Anxi County may contribute to its characteristics aroma.

Plasticity of the Cuticular Transpiration Barrier in Response to Water Shortage and Resupply in Camellia sinensis: A Role of Cuticular Waxes.

The cuticle is regarded as a non-living tissue; it remains unknown whether the cuticle could be reversibly modified and what are the potential mechanisms. In this study, three tea germplasms (Wuniuzao, 0202-10, and 0306A) were subjected to water deprivation followed by rehydration. The epicuticular waxes and intracuticular waxes from both leaf surfaces were quantified from the mature 5th leaf. Cuticular transpiration rates were then measured from leaf drying curves, and the correlations between cuticular transpiration rates and cuticular wax coverage were analyzed. We found that the cuticular transpiration barriers were reinforced by drought and reversed by rehydration treatment; the initial weak cuticular transpiration barriers were preferentially reinforced by drought stress, while the original major cuticular transpiration barriers were either strengthened or unaltered. Correlation analysis suggests that cuticle modifications could be realized by selective deposition of specific wax compounds into individual cuticular compartments through multiple mechanisms, including in vivo wax synthesis or transport, dynamic phase separation between epicuticular waxes and the intracuticular waxes, in vitro polymerization, and retro transportation into epidermal cell wall or protoplast for further transformation. Our data suggest that modifications of a limited set of specific wax components from individual cuticular compartments are sufficient to alter cuticular transpiration barrier properties.

Establishing a System for Functional Characterization of Full-Length cDNAs of Camellia sinensis.

: Tea (Camellia sinensis) is enriched with bioactive secondary metabolites, and is one of the most popular nonalcoholic beverages globally. Two tea reference genomes have been reported; however, the functional analysis of tea genes has lagged, mainly due to tea's recalcitrance to genetic transformation and the absence of alternative high throughput heterologous expression systems. A full-length cDNA collection with a streamlined cloning system is needed in this economically important woody crop species. RNAs were isolated from nine different vegetative tea tissues, pooled, then used to construct a normalized full-length cDNA library. The titer of unamplified and amplified cDNA library was 6.89 × 106 and 1.8 × 1010 cfu/mL, respectively; the library recombinant rate was 87.2%. Preliminary characterization demonstrated that this collection can complement existing tea reference genomes and facilitate rare gene discovery. In addition, to streamline tea cDNA cloning and functional analysis, a binary vector (pBIG2113SF) was reengineered, seven tea cDNAs isolated from this library were successfully cloned into this vector, then transformed into Arabidopsis. One FL-cDNA, which encodes a putative P1B-type ATPase 5 (CsHMA5), was characterized further as a proof of concept. We demonstrated that overexpression of CsHMA5 in Arabidopsis resulted in copper hyposensitivity. Thus, our data demonstrated that this represents an efficient system for rare gene discovery and functional characterization of tea genes. The integration of a tea FL-cDNA collection with efficient cloning and a heterologous expression system would facilitate functional annotation and characterization of tea genes.

Terpenoid Esters Are the Major Constituents From Leaf Lipid Droplets of Camellia sinensis.

Lipid droplets (LDs) have been widely found from diverse species and exhibit diverse functions. It remains unexplored what potential roles they played in tea. To address this question, we analyzed the chemical composition and the dynamic changes of cytosolic LDs during leaf growth and diurnal cycle. Using TopFluor cholesterol and Nile Red staining we demonstrated that cytosolic LDs were heterogeneous in tea tree (Camellia sinensis cv. Tieguanyin); the size and number of LDs increased with leaf growth. Compositional analysis showed that terpenoid esters and diacylglycerol are the major components of cytosolic LDs. The contents of total sterol esters (SEs) and ß-amyrin esters increased with leaf expansion and growth; individual SE also showed diurnal changes. Our data suggest that cytosolic LDs from tea tree leave mainly serve as storage site for free sterols and triterpenoids in the form of esters. Cytosolic LDs were not the major contributors to the aroma quality of made tea.

Tender leaf and fully-expanded leaf exhibited distinct cuticle structure and wax lipid composition in Camellia sinensis cv Fuyun 6.

The goal of the present study was to compare the structural and compositional differences of cuticle between tender leaf and fully-expanded leaf in Camellia sinensis, and provide metabolic base for the further characterization of wax biosynthesis in this economically important crop species. The tender second leaf and the fully-expanded fifth leaf from new twig were demonstrated to represent two different developmental stages, their cuticle thickness were measured by transmission electron microscopy. The thickness of the adaxial cuticle on the second and fifth leaf was 1.15 µm and 2.48 µm, respectively; the thickness of the abaxial cuticle on the second and fifth leaf was 0.47 µm and 1.05 µm, respectively. The thickness of the epicuticular wax layer from different leaf position or different sides of same leaf were similar. However, the intracuticular wax layer of the fifth leaf was much thicker than that of the second leaf. Total wax lipids were isolated from the second leaf and the fifth leaf, respectively. Gas chromatography-mass spectrometry analysis identified 51 wax constituents belonging to 13 chemical classes, including esters, glycols, terpenoids, fatty acids and their derivatives. Wax coverage on the second and fifth leaf was 4.76 µg/cm2 and 15.38 µg/cm2, respectively. Primary alcohols dominated in the tender second leaf. However, triterpenoids were the major components from the fully-expanded fifth leaf. The predominant carbon chains varied depending on chemical class. These data showed that the wax profiles of Camellia sinensis leaves are development stage dependent, suggesting distinct developmental dependent metabolic pathways and regulatory mechanisms.