Effect of Mechanical Damage in Green-Making Process on Aroma of Rougui Tea.

Rougui Tea (RGT) is a typical Wuyi Rock Tea (WRT) that is favored by consumers for its rich taste and varied aroma. The aroma of RGT is greatly affected by the process of green-making, but its mechanism is not clear. Therefore, in this study, fresh leaves of RGT in spring were picked, and green-making (including shaking and spreading) and spreading (unshaken) were, respectively, applied after sun withering. Then, they were analyzed by GC-TOF-MS, which showed that the abundance of volatile compounds with flowery and fruity aromas, such as nerolidol, jasmine lactone, jasmone, indole, hexyl hexanoate, (E)-3-hexenyl butyrate and 1-hexyl acetate, in green-making leaves, was significantly higher than that in spreading leaves. Transcriptomic and proteomic studies showed that long-term mechanical injury and dehydration could activate the upregulated expression of genes related to the formation pathways of the aroma, but the regulation of protein expression was not completely consistent. Mechanical injury in the process of green-making was more conducive to the positive regulation of the allene oxide synthase (AOS) branch of the α-linolenic acid metabolism pathway, followed by the mevalonate (MVA) pathway of terpenoid backbone biosynthesis, thus promoting the synthesis of jasmonic acid derivatives and sesquiterpene products. Protein interaction analysis revealed that the key proteins of the synthesis pathway of jasmonic acid derivatives were acyl-CoA oxidase (ACX), enoyl-CoA hydratase (MFP2), OPC-8:0 CoA ligase 1 (OPCL1) and so on. This study provides a theoretical basis for the further explanation of the formation mechanism of the aroma substances in WRT during the manufacturing process.

Genome-wide identification, expression profiling, and protein interaction analysis of the CCoAOMT gene family in the tea plant (Camellia sinensis).

BACKGROUND: The caffeoyl-CoA-O methyltransferase (CCoAOMT) family plays a crucial role in the oxidative methylation of phenolic substances and is involved in various plant processes, including growth, development, and stress response. However, there is a limited understanding of the interactions among CCoAOMT protein members in tea plants. RESULTS: In this study, we identified 10 members of the CsCCoAOMT family in the genome of Camellia sinensis (cultivar 'HuangDan'), characterized by conserved gene structures and motifs. These CsCCoAOMT members were located on six different chromosomes (1, 2, 3, 4, 6, and 14). Based on phylogenetic analysis, CsCCoAOMT can be divided into two groups: I and II. Notably, the CsCCoAOMT members of group Ia are likely to be candidate genes involved in lignin biosynthesis. Moreover, through the yeast two-hybrid (Y2H) assay, we established protein interaction networks for the CsCCoAOMT family, revealing 9 pairs of members with interaction relationships. CONCLUSIONS: We identified the CCoAOMT gene family in Camellia sinensis and conducted a comprehensive analysis of their classifications, phylogenetic and synteny relationships, gene structures, protein interactions, tissue-specific expression patterns, and responses to various stresses. Our findings shed light on the evolution and composition of CsCCoAOMT. Notably, the observed interaction among CCoAOMT proteins suggests the potential formation of the O-methyltransferase (OMT) complex during the methylation modification process, expanding our understanding of the functional roles of this gene family in diverse biological processes.

Aged oolong tea alleviates dextran sulfate sodium-induced colitis in mice by modulating the gut microbiota and its metabolites.

In this study, the mechanism of aged oolong tea (AOT) to alleviate colitis was investigated in terms of microbiome, metabolome, and fecal microbiota transplantation (FMT). AOT storage period could alleviate colitis in mice and there were some differences in AOT between storage periods, especially AOT-10. AOT improves UC by modulating oxidative stress and inflammatory factors and upregulating intestinal tight junction protein expression (Occludin, Claudin-1, ZO-1 and MUC2), which is associated with the recovery of gut microbiota. FMT and targeted metabolomics further demonstrate that the anti-inflammatory effects of AOT can reshape the gut microbiota through faecal bacterial transfer. Anti-inflammatory effects are exerted through the stimulation of metabolic pathways associated with amino acid, fatty acid and bile acid metabolites. Importantly, the study identified key bacteria (e.g., Sutterella, Clostridiaceae_Clostridium, Mucispirillum, Oscillospira and Ruminococcus) for the development and remission of inflammation. Conclusively, AOT may have great potential in the future adjuvant treatment of colitis.

The metabolic mechanism of flavonoid glycosides and their contribution to the flavor evolution of white tea during prolonged withering.

This study was the first to comprehensively investigate the metabolic mechanism of flavonoid glycosides (FGs) and their contribution to flavor evolution during white tea processing using quantitative descriptive analysis, metabolomics, dose-over-threshold factors and pseudo-first-order kinetics. A total of 223 flavonoids were identified. Total FGs decreased from 7.02 mg/g to 4.35 mg/g during processing, compared to fresh leaves. A total of 86 FGs had a significant impact on the flavor evolution and 9 key flavor FGs were identified. The FG biosynthesis pathway was inhibited during withering, while the degradation pathway was enhanced. This promoted the degradation of 9 key flavor FGs following pseudo-first-order kinetics during withering. The degradation of the FGs contributed to increase the taste acceptance of white tea from -4.18 to 1.32. These results demonstrated that water loss stress during withering induces the degradation of key flavor FGs, contributing to the formation of the unique flavor of white tea.

Gene mining and genomics-assisted breeding empowered by the pangenome of tea plant Camellia sinensis.

Tea is one of the world's oldest crops and is cultivated to produce beverages with various flavours. Despite advances in sequencing technologies, the genetic mechanisms underlying key agronomic traits of tea remain unclear. In this study, we present a high-quality pangenome of 22 elite cultivars, representing broad genetic diversity in the species. Our analysis reveals that a recent long terminal repeat burst contributed nearly 20% of gene copies, introducing functional genetic variants that affect phenotypes such as leaf colour. Our graphical pangenome improves the efficiency of genome-wide association studies and allows the identification of key genes controlling bud flush timing. We also identified strong correlations between allelic variants and flavour-related chemistries. These findings deepen our understanding of the genetic basis of tea quality and provide valuable genomic resources to facilitate its genomics-assisted breeding.

Widely targeted metabolomics analysis of different Wuyi Shuixian teas and association with taste attributes.

There are two major types of Wuyi Shuixian teas with distinct flavor properties in the market: regular Shuixian, and Laocong Shuixian that is produced from old tea plants with higher sale price. However, the chemical composition difference between these two types of Shuixian teas is still unclear. In this study, the widely targeted metabonomics and sensory evaluation were carried out to investigate the metabolite profiles and the flavor properties of Laocong and regular Shuixian tea samples. The results of organoleptic evaluation showed the Laocong Shuixian teas achieved dramatically higher total scores of sensory quality than that of regular Shuixian, and the sour palate of Laocong Shuixian tea was much lower than that of regular Shuxian. A total of 692 metabolites were identified by using metabolic determination, 43 of which were different metabolites in Laocong Shuixian teas discriminated from regular Shuixian. The contents of caffeic acid, kaemperfin, genistin, quercetin 3-glycosides and p-coumaric acid-O-glycoside were abundantly present in regular Shuixian tea. The analysis on different metabolites and taste attributes showed that phenolic acidic compounds were the major contributors to the sour taste of regular Shuixian. This study interpreted the chemicals underlying the different taste properties of Laocong and regular Shuixian teas.

Effects of Sun Withering Degree on Black Tea Quality Revealed via Non-Targeted Metabolomics.

In this study, the effects of different sun withering degrees (75% (CK), 69% (S69), 66% (S66), 63% (S63), and 60% (S60) water content in the withered leaves) on black tea sensory quality were investigated by means of sensory evaluation plus metabolomics analysis. Sensory evaluation results showed higher sensory quality scores for the black tea in S69-S66, due to better freshness, sweeter taste, and a sweet and even floral and fruity aroma. Additionally, 65 non-volatile components were identified using Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight-Mass Spectrometry (UPLC-Q-TOF/MS). Among them, the content increase of amino acids and theaflavins was found to promote the freshness and sweetness of black tea. The aroma of tea was analyzed using combined Solvent Assisted Flavor Evaporation-Gas Chromatography-Mass Spectrometry (SAFE-GC-MS) and Headspace-Solid Phase Micro Extract-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS), and 180 volatiles were identified, including 38 variable importance in projection (VIP) > 1 (p < 0.05) and 25 Odor Activity Value (OAV) > 1 volatiles. Statistical analysis revealed 11 volatiles as potential major aroma differential metabolites in black tea with a different sun withering degree, such as volatile terpenoids (linalool, geraniol, (E)-citral, and ß-myrcene), amino-acid-derived volatiles (benzeneethanol, benzeneacetaldehyde, and methyl salicylate), carotenoid-derived volatiles (jasmone and ß-damascenone), and fatty-acid-derived volatiles ((Z)-3-hexen-1-ol and (E)-2-hexenal). Among them, volatile terpenoids and amino acid derived volatiles mainly contributed to the floral and fruity aroma quality of sun-withered black tea.

Proteomic Analysis Reveals the Association between the Pathways of Glutathione and α-Linolenic Acid Metabolism and Lanthanum Accumulation in Tea Plants.

Lanthanum can affect the growth and development of the tea plant. Tieguanyin (TGY) and Shuixian (SX) cultivars of Camellia sinensis were selected to explore the mechanism underlying the accumulation of lanthanum (tea plants' most accumulated rare earth element) through proteomics. Roots and fresh leaves of TGY and SX with low- and high-accumulation potential for lanthanum, respectively, were studied; 845 differentially expressed proteins (DEPs) were identified. Gene ontology analysis showed that DEPs were involved in redox processes and related to molecular functions. Kyoto Encyclopedia of Genes and Genomes metabolic pathway analysis showed that DEPs were associated with glutathione (GSH) and α-linolenic acid metabolism, plant pathogen interaction, and oxidative phosphorylation. Thirty-seven proteins in the GSH metabolism pathway showed significant differences, wherein 18 GSH S-transferases showed differential expression patterns in the root system. Compared with the control, expression ratios of GST (TEA004130.1) and GST (TEA032216.1) in TGY leaves were 6.84 and 4.06, respectively, after lanthanum treatment; these were significantly higher than those in SX leaves. The LOX2.1 (TEA011765.1) and LOX2.1 (TEA011776.1) expression ratios in the α-linolenic acid metabolic pathway were 2.44 and 6.43, respectively, in TGY roots, which were significantly higher than those in SX roots. The synthesis of specific substances induces lanthanum-associated defense responses in TGY, which is of great significance for plant yield stability.

A Study on Origin Traceability of White Tea (White Peony) Based on Near-Infrared Spectroscopy and Machine Learning Algorithms.

Identifying the geographical origins of white tea is of significance because the quality and price of white tea from different production areas vary largely from different growing environment and climatic conditions. In this study, we used near-infrared spectroscopy (NIRS) with white tea (n = 579) to produce models to discriminate these origins under different conditions. Continuous wavelet transform (CWT), min-max normalization (Minmax), multiplicative scattering correction (MSC) and standard normal variables (SNV) were used to preprocess the original spectra (OS). The approaches of principal component analysis (PCA), linear discriminant analysis (LDA) and successive projection algorithm (SPA) were used for features extraction. Subsequently, identification models of white tea from different provinces of China (DPC), different districts of Fujian Province (DDFP) and authenticity of Fuding white tea (AFWT) were established by K-nearest neighbors (KNN), random forest (RF) and support vector machine (SVM) algorithms. Among the established models, DPC-CWT-LDA-KNN, DDFP-OS-LDA-KNN and AFWT-OS-LDA-KNN have the best performances, with recognition accuracies of 88.97%, 93.88% and 97.96%, respectively; the area under curve (AUC) values were 0.85, 0.93 and 0.98, respectively. The research revealed that NIRS with machine learning algorithms can be an effective tool for the geographical origin traceability of white tea.

High Light Intensity Triggered Abscisic Acid Biosynthesis Mediates Anthocyanin Accumulation in Young Leaves of Tea Plant (Camellia sinensis).

There is increasing interest in the production and consumption of tea (Camellia sinensis L.) processed from purple-leaved cultivar due to their high anthocyanin content and health benefits. However, how and why seasonal changes affect anthocyanin accumulation in young tea leaves still remains obscured. In this study, anthocyanin and abscisic acid (ABA) contents in young leaves of Zifuxing 1 (ZFX1), a cultivar with new shoots turning to purple in Wuyi Mountain, a key tea production region in China, were monitored over four seasons. Young leaves produced in September were highly purplish, which was accompanied with higher anthocyanin and ABA contents. Among the environmental factors, the light intensity in particular was closely correlated with anthocyanin and ABA contents. A shade experiment also indicated that anthocyanin content significantly decreased after 168 h growth under 75% shade, but ABA treatment under the shade conditions sustained anthocyanin content. To confirm the involvement of ABA in the modulation of anthocyanin accumulation, anthocyanin, carotenoids, chlorophyll, ABA, jasmonic acid (JA), and salicylic acid (SA) in the young leaves of four cultivars, including ZFX1, Zijuan (ZJ), wherein leaves are completely purple, Rougui (RG) and Fudingdabaicha (FDDB) wherein leaves are green, were analyzed, and antioxidant activities of the leaf extracts were tested. Results showed that ABA, not other tested hormones, was significantly correlated with anthocyanin accumulation in the purple-leaved cultivars. Cultivars with higher anthocyanin contents exhibited higher antioxidant activities. Subsequently, ZFX1 plants were grown under full sun and treated with ABA and fluridone (Flu), an ABA inhibitor. ABA treatment elevated anthocyanin level but decreased chlorophyll contents. The reverse was true to those treated with Flu. To pursue a better understanding of ABA involvement in anthocyanin accumulation, RNA-Seq was used to analyze transcript differences among ABA- or Flu-treated and untreated ZFX1 plants. Results indicated that the differentially expressed genes in ABA or Flu treatment were mainly ABA signal sensing and metabolism-related genes, anthocyanin accumulation-related genes, light-responsive genes, and key regulatory MYB transcription factors. Taking all the results into account, a model for anthocyanin accumulation in ZFX1 cultivar was proposed: high light intensity caused reactive oxygen stress, which triggered the biosynthesis of ABA; ABA interactions with transcription factors, such as MYB-enhanced anthocyanin biosynthesis limited chlorophyll and carotenoid accumulation; and transport of anthocyanin to vacuoles resulting in the young leaves of ZFX1 with purplish coloration. Further research is warranted to test this model.

Genome-Wide Identification of AMT2-Type Ammonium Transporters Reveal That CsAMT2.2 and CsAMT2.3 Potentially Regulate NH4+ Absorption among Three Different Cultivars of Camellia sinensis.

Ammonium (NH4+), as a major inorganic source of nitrogen (N) for tea plant growth, is transported and distributed across membranes by the proteins of ammonium transporters (AMTs). However, the AMT2-type AMTs from tea plants remain poorly understood. In this study, five CsAMT2 subfamily genes were identified in tea plant genomes, and their full-length coding sequences (CDS) were isolated from roots. Then, a NH4+ uptake kinetic comparison of Fudingdabaicha (FD), Huangdan (HD), and Maoxie (MX) showed that FD was a high N efficiency (HNE) cultivar that had a wide range of adaptability to NH4+, HD was a high N efficiency under high N conditions (HNEH) cultivar, in which it was easy to obtain higher yield in a high N environment, and MX was a high N efficiency under low N conditions (HNEL) cultivar, which had a higher affinity for NH4+ than the other two. Tissue-specific expression analysis suggested that CsAMT2.2 and CsAMT2.3 were highly expressed in the roots, indicating that these two members may be unique in the CsAMT2 subfamily. This is further supported by our findings from the temporal expression profiles in the roots among these three different N adaptation cultivars. Expression levels of CsAMT2.2 and CsAMT2.3 in FD and HD were upregulated by a short time (2 h) under high NH4+ treatment, while under low NH4+ treatment, CsAMT2.2 and CsAMT2.3 were highly expressed at 0 h and 2 h in the HNEL-type cultivar-MX. Furthermore, the functional analysis illustrated that CsAMT2.2 and CsAMT2.3 could make a functional complementation of NH4+-defective mutant yeast cells at low NH4+ levels, and the transport efficiency of CsAMT2.3 was higher than that of CsAMT2.2. Thus, we concluded that CsAMT2.2 and CsAMT2.3 might play roles in controlling the NH4+ uptake from the soil to the roots. These results will further the understanding of the NH4+ signal networks of AMT2-type proteins in tea plants.

Combined analysis of lipidomics and transcriptomics revealed the key pathways and genes of lipids in light-sensitive albino tea plant (Camellia sinensis cv. Baijiguan).

Currently, the mechanism by which light-sensitive albino tea plants respond to light to regulate pigment synthesis has been only partially elucidated. However, few studies have focused on the role of lipid metabolism in the whitening of tea leaves. Therefore, in our study, the leaves of the Baijiguan (BJG) tea tree under shade and light restoration conditions were analyzed by a combination of lipidomics and transcriptomics. The leaf color of BJG was regulated by light intensity and responded to light changes in light by altering the contents and proportions of lipids. According to the correlation analysis, we found three key lipid components that were significantly associated with the chlorophyll SPAD value, namely, MGDG (36:6), DGDG (36:6) and DGDG (34:3). Further weighted gene coexpression network analysis (WGCNA) showed that HY5 TF and GLIP genes may be hub genes involved lipid regulation in albino tea leaves. Our results lay a foundation for further exploration of the color changes in albino tea leaves.

Relationship between the Grade and the Characteristic Flavor of PCT (Panyong Congou Black Tea).

Panyong Congou black tea (PCT) is one of the most representative and historically famous Congou black teas in China and has been gaining more and more attention for its beneficial health properties. Currently, four grades of PCT are available, based on the raw leaf materials and consumer palatability. The chemical profiles distinguishing different grades of PCT are yet to be defined, nor has the relationship with grade been evaluated. In the present study, chemometric analysis showed that epigallocatechin (EGC), catechin (C), polyphenols, gallic acid (GA), and free amino acids are grade related bio-markers of PCT. These compounds are associated with the sweet and mellow aftertaste of PCT. A total of 34 volatile components were identified, of which the three component types with the highest relative percentages were alcohols (51.34-52.51%), ketones (27.31-30.28%), and aldehydes (12.70-13.18%). Additionally, our results revealed that sweet floral and fruity aromas were positively correlated with six volatile organic compounds (VOCs), 1-pentanol, propyl hexanoate, linalool, cyclohexanone, hexanal, and 2,5-dimethylpyrazine. Clear discrimination was achieved using orthogonal projections to latent structures discriminant analysis (OPLS-DA). The findings provide vital information on the characteristic flavor of each grade of PCT.

Exogenous stimulation-induced biosynthesis of volatile compounds: Aroma formation of oolong tea at postharvest stage.

Volatile organic compounds (VOCs) are produced by plants responding to biotic and abiotic stresses. According to their biosynthetic sources, induced VOCs are divided into three major classes: terpenoids, phenylpropanoid/benzenoid, and fatty acid derivatives. These compounds with specific aroma characteristics importantly contribute to the aroma quality of oolong tea. Shaking and rocking is the crucial procedure for the aroma formation of oolong tea by exerting mechanical damage to fresh tea leaves. Abundant studies have been carried out to investigate the formation mechanisms of VOCs during oolong tea processing in recent years. This review systematically introduces the biosynthesis of VOCs in plants, and the volatile changes due to biotic and abiotic stresses are summarized and expatiated, using oolong tea as an example.

Metabolomics Combined with Proteomics Provide a Novel Interpretation of the Changes in Flavonoid Glycosides during White Tea Processing.

In this study, nonvolatile metabolomics and proteomics were applied to investigate the change mechanism of flavonoid glycoside compounds during withering processing of white tea. With the extension of withering time, the content of the main flavonoid glycoside compounds significantly decreased, and then the flavonoid aglycones and water-soluble saccharides contents increased. However, the change trends of these compounds were inconsistent with the expression pattern of related biosynthesis pathway proteins, indicating that the degradation of flavonoid glycosides might exist in the withering process of white tea. One co-expression network that was highly correlated with variations in the flavonoid glycosides' component contents during the withering process was identified via WGCNA. Further analysis revealed that the degradation of flavonoid glycosides may be related to the antioxidant action of tea leaves undergoing the withering process. Our results provide a novel characterization of white tea taste formation during processing.

Widely Targeted Metabolomics Analysis Reveals the Differences of Nonvolatile Compounds in Oolong Tea in Different Production Areas.

The flavor differences in Oolong tea from different producing areas are caused by its complex differential compounds. In this study, representative samples of Oolong tea from four countries were collected, and their differential nonvolatile compounds were analyzed by a combination of widely targeted metabolomics, chemometrics, and quantitative taste evaluation. A total of 801 nonvolatile compounds were detected, which could be divided into 16 categories. We found that the difference in these compounds' content among Oolong teas from three producing areas in China was the largest. There were 370 differential compounds related to the producing areas of Oolong tea, which were mainly distributed in 67 Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. In total, 81 differential nonvolatile compounds made important contributions to the taste differences in Oolong tea from different producing areas, among which the number of flavonoids was the largest. Finally, the characteristic compounds of Oolong tea in six producing areas were screened. This study comprehensively identifies the nonvolatile compounds of Oolong tea in different producing areas for the first time, which provides a basis for the analysis of flavor characteristics, quality directional control, and the identification and protection of geographical landmark agricultural products of Oolong tea from different producing areas.

Identification of characteristic aroma and bacteria related to aroma evolution during long-term storage of compressed white tea.

Compressed white tea (CWT) is a reprocessed tea of white tea. Long-term storage has greatly changed its aroma characteristics, but the material basis and transformation mechanism of its unique aroma are still unclear. In this study, flavor wheel, headspace gas chromatography ion mobility spectroscopy, chemometrics, and microbiomics were applied to study the flavor evolution and important aroma components during long-term storage of CWT, and core functional bacteria were screened. During long-term storage, the aroma of CWT gradually changed from sweet, fruity and floral to stale flavor, woody and herbal. A total of 56 volatile organic compounds (VOCs) were identified, 54 of which were significantly differences during storage. The alcohols content was the highest during 1-5 years of storage, the esters content was the highest during 7-13 years of storage, and the aldehydes content was the highest during 16 years of storage. Twenty-nine VOCs were identified as important aroma components, which were significantly correlated with 6 aroma sub-attributes (P < 0.05). The functional prediction of bacterial community reminded that bacterial community could participate in the transformation of VOCs during storage of CWT. Twenty-four core functional bacteria were screened, which were significantly associated with 29 VOCs. Finally, 23 characteristic differential VOCs were excavated, which could be used to identify CWT in different storage years. Taken together, these findings provided new insights into the changes in aroma characteristics during storage of CWT and increased the understanding of the mechanism of characteristic aroma formation during storage.

Identification, Characterization and Expression Profiling of the RS Gene Family during the Withering Process of White Tea in the Tea Plant (Camellia sinensis) Reveal the Transcriptional Regulation of CsRS8.

Raffinose synthetase (RS) is a key enzyme in the process of raffinose (Raf) synthesis and is involved in plant development and stress responses through regulating Raf content. As a sweetener, Raf makes an important contribution to the sweet taste of white tea. However, studies on the identification, analysis and transcriptional regulation of CsRSs (Camellia sinensis RS genes) are still lacking. In this study, nine CsRSs were identified from the tea plant (Camellia sinensis) genome database. The CsRSs were classified into five groups in the phylogenetic tree. Expression level analysis showed that the CsRSs varied in different parts of the tea plant. Transcriptome data showed that CsRSs could respond to persistent drought and cold acclimation. Except for CsRS5 and CsRS9, the expression pattern of all CsRSs increased at 12 h and decreased at 30 h during the withering process of white tea, consistent with the change trend of the Raf content. Furthermore, combining yeast one-hybrid assays with expression analysis, we found that CsDBB could potentially regulate the expression of CsRS8. Our results provide a new perspective for further research into the characterization of CsRS genes and the formation of the white tea flavour.

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.