Journal of Tea Science

Genetic Diversity Analysis of Tea Genetic Resources in Sanjiang, Guangxi

MENG Rongjun, CHEN Liang, XU Yuan, LIN Wei, ZHOU Qiwei, XIE Yilin, LAI Dingqing, LAI Jiaye

Journal of Tea Science. 2023, 43(2): 147-158. doi:10.13305/j.cnki.jts.2023.02.009

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In order to clarify the genetic diversity of tea genetic resources in Sanjiang, Guangxi, two marker techniques, simple inter-repeat sequence amplification (ISSR) and correlated sequence amplification polymorphism (SRAP) were used to analyze the genetic diversity of 72 local tea genetic resources and five introduced cultivars in Sanjiang region. The results of both marker techniques show high levels of genetic diversity (H=0.30, I=0.44), high genetic differentiation between populations (Gst=0.056) and gene flow (Nm=8.64). The genetic variation observed within populations was much higher than that between populations. The cluster analysis shows that most of the tea genetic resources in Sanjiang were clustered into one group and the introduced cultivars were clustered into the other, which was consistent with the results of PCoA. This study provided evidence at the molecular level for the protection and utilization of tea germplasmin Sanjiang.

Identification and Expression Pattern Analysis of NUDIX Gene Family in Camellia sinensis

CHEN Zhenyan, ZHANG Xiangqin, CHEN Lan, XIE Siyi, LIU Shuoqian, TIAN Na

Journal of Tea Science. 2023, 43(2): 159-172. doi:10.13305/j.cnki.jts.2023.02.002

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NUDIX hydrolase belongs to pyrophosphatase, which plays an important role in information transmission, plant growth coordination and responses to adversity stresses. In this study, 43 NUDIX family genes were identified based on the tea (Camellia sinensis) genome, and analyzed by bioinformatics and fluorescence quantitative PCR. The results show that the protein molecular weight of 43 CsNUDXs rang from 11.8~89.2 kDa, with 102 to 342 amino acids. The theoretical isoelectric point was from 4.49 to 9.26, and 18.6% of them were stable proteins. According to the evolutionary relationship, CsNUDXs is divided into six subfamilies. Cis-acting element analysis of promoter shows that CsNUDXs have many functional elements related to hormone response, adversity stress, growth and development. The expression patterns of CsNUDXs in different organs were analyzed. It was found that the expression levels of CsNUDX3 and CsNUDX7 were high in fruits, while the expression levels of CsNUDX22 and CsNUDX25 were extremely low in fruits, and the expression level of CsNUDX30 was extremely low in old leaves. In addition, the results of real-time fluorescence quantitative PCR show that the expression levels of CsNUDXs, such as CsNUDX1, CsNUDX2 and CsNUDX33 increased first, then decreased and then increased under the treatment of 1 mmol·L^-1 MeJA. However, under the treatment of 1 mmol·L^-1 SA, the expression levels of CsNUDX4, CsNUDX12 and CsNUDX22 decreased first, then increased and then decreased. While under the treatment of 300 mmol·L^-1 NaCl, the expression levels of CsNUDX2, CsNUDX4 and CsNUDX22 increased first and then decreased. In summary, the basic characteristics and functions of CsNUDXs were preliminarily analyzed by bioinformatics technology, and it was found that CsNUDXs could respond to high salt stress, MeJA and SA treatments.

Study on the Characteristics of CsPHT1;3 and Its Response to Selenium in Tea Plants

GUO Lina, HAO Xinyuan, WANG Lu, QI Meng, LI Xiaoman, REN Hengze, ZHENG Qinghua, WANG Xinchao, ZENG Jianming

Journal of Tea Science. 2023, 43(2): 173-182. doi:10.13305/j.cnki.jts.2023.02.003

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Selenium (Se) is an essential microelement for human, and Se enriched products are important sources for Se intake in human. Tea plants (Camellia sinensis) have strong selenium enrichment ability. However, there is limited research on the molecular mechanism of phosphate transporters involved in Se absorption. In this study, CsPHT1;3 gene was cloned and its characteristics and responses to Se concentrations, valence, pH, time and expression in various Se-enriched tea resources were investigated. Gene characteristic analysis of CsPHT1;3 shows that CsPHT1;3 is grouped into phosphate transporter PHT1 subfamily and localized in the plasma membrane. The CsPHT1;3 protein contains the conserved domain GGDYPLSATIxSE, which belongs to the PHT1 protein. Expression pattern analysis of CsPHT1;3 in various tissues suggests that the expression levels of CsPHT1;3 in mature leaves and root tissues were significantly higher than those in other tissues. The induction results of different Se concentrations and valence states indicate that CsPHT1;3 was significantly induced by Se⁴⁺ at 1 d and 7 d after treatments. The expression of CsPHT1;3 in roots was obviously induced by Se⁶⁺ except for 3 d after treatment but largely unaffected by Se⁶⁺ concentration. The results of different pH and Se⁴⁺ treatments show that, at pH5, the highest expression of CsPHT1;3 in tea roots was observed at 24 h. While at pH3, the highest expression of CsPHT1;3 in tea roots was observed at 48 h. Moreover, at pH7, the highest expression of CsPHT1;3 in tea roots was observed at 72 h. The results of sodium selenate treatment on different Se-enriched tea resources indicate that the expression of CsPHT1;3 in the leaves and roots did not respond to sodium selenate treatment. However, the results of sodium selenite treatment on different Se-enriched tea resources suggest that CsPHT1;3 is significantly up-regulated in the leaves of Se-enriched tea resources. The above studies indicated that CsPHT1;3 may participate in the absorption and redistribution of selenite by roots in tea plants, which is important for the breeding of Se-enriched tea cultivars.

Identification of the Light-harvesting Chlorophyll-protein Complex Gene CsLhcb2 and Its Response to Low Temperature in Tea Plants

HU Zhihang, QIN Zhiyuan, LI Jingwen, YANG Ni, CHEN Yi, LI Tong, ZHUANG Jing

Journal of Tea Science. 2023, 43(2): 183-193. doi:10.13305/j.cnki.jts.2023.02.007

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Tea is one of the important cash crop in China. Its growth and development will be affected by different adversity, leading to the decline of tea quality and yield. Light-harvesting chlorophyll-protein complex mainly affects the photosynthetic efficiency of plants, and also plays important roles in adaptation to environmental stresses. In order to study the characteristics of the light-harvesting chlorophyll-protein complex in tea plants, the gene CsLhcb2 encoding the light-harvesting protein complex was cloned from tea cultivar ‘Longjing 43’, and the sequence characteristics, phylogenetic tree, physical and chemical properties, subcellular localization, secondary structure, tertiary structure and its expression profiles under low temperature treatment were analyzed. The results show that the open reading frame of CsLhcb2 gene is 798 bp, encoding 265 amino acids. This gene contains a typical of Chloroa-b-bind conservation domain. The similarity of CsLHCB2 amino acid sequence with 15 plant species was 91.32%. Phylogenetic tree analysis shows that the CsLHCB2 protein of tea plant was closely related to Datura stramonium, Sedum alfredii and Vitis vinifera, and far from Dendrocalamus latiflorus and Phyllostachys edulis. The relative molecular weight of CsLHCB2 protein is 28 662.77 and the theoretical isoelectric point is 5.69, which belongs to hydrophilic protein. Subcellular localization prediction results show that CsLHCB2 protein is mainly located in chloroplasts. Quantitative RT-PCR results show that CsLhcb2 gene may participate in the process of low temperature stress in tea plants. Under normal temperature treatment, the relative expression level of CsLhcb2 gene showed a trend of increasing first and then decreasing within a photoperiod (24 h). ‘Longjing 43’ and ‘Shuchazao’ reached their peak value at 1 h after light treatment, and 'Baiyeyihao' reached their peak value at 6 h after light treatment. Under low temperature of 4 ℃ treatment, the expression of Lhcb2 of the three tea cultivars all reached the peak at 12 h of light treatment, among which the expression level of CsLhcb2 in ‘Shuchazao’ was the highest, which was 1.18 and 1.98 times higher than that of ‘Longjing 43’ and ‘Baiyeyihao’, respectively. The results provided a reference for further research on the role of light-harvesting chlorophyll-protein complex response to low temperature in tea plants.

Isolation and Identification of Anthracnose Pathogen from Xihu Longjing Plantation and Screening of Its Plant-derived Fungicides

LI Hongli, ZHOU Tiefeng, MAO Yuxiao, HUANG Haitao, CUI Hongchun, ZHENG Xuxia, ZHAO Yun

Journal of Tea Science. 2023, 43(2): 194-204. doi:10.13305/j.cnki.jts.2023.02.010

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In order to screen out plant extracts with better antibacterial activity against anthracnose in Xihu Longjing tea producing area, the pathogens of tea anthracnose in this region were isolated and identified, and the inhibition activities of 12 plant extracts on the identified pathogen were determined using mycelial growth rate method. Concentration gradient test was further used to determine the antibacterial virulence of the top 3 plant extracts. The results show that Colletotrichum camelliae was the major pathogenic strain in the study region. At the concentration of 200 mg·L^-1, the antifungal activities of vitriol, oregano essential oil and perillal aldehyde were better, and their inhibition rates on the growth of anthrax were all above 68%, and their effective medium concentrations (EC₅₀) were 71.459, 77.155 mg·L^-1 and 110.753 mg·L^-1, respectively. Microscopic observation shows that the mycelium morphology of C. camelliae had obvious changes in the medium when supplemented with above plant extracts, showing deformation, distortion, shrinkage and shriveled. These results indicate that carvacrol, oregano essential oil and perill aldehyde have strong antibacterial activities against C. camelliae and possess potential for development as plant-derived fungicides.

Screening, Identification and Culture Condition Optimization of Antagonistic Endophytic Bacteria Against Gloeosporium theae-sinensis Miyake

ZHENG Shizhong, ZHOU Ziwei, CHEN Xiaohui, CAI Liewei, JIANG Shengtao, LIU Shengrong

Journal of Tea Science. 2023, 43(2): 205-215. doi:10.13305/j.cnki.jts.2023.02.006

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To screen the antagonistic endophytic bacteria against Gloeosporium theae-sinensis Miyake, the healthy leaves of tea plants were utilized as materials, and the plate antagonistic method was used. The isolated bacteria were identified and evaluated for antimicrobial efficacy. The culture conditions were optimized using response surface methodology. One antagonistic bacterium X13 was screened from 162 strains of endophytic bacteria isolated, which had a good inhibitory effect on Colletotrichum gloeosporioides. The strain X13 was identified as Bacillus subtilis through morphological observation, physiological and biochemical tests, and 16S rDNA phylogenetic analysis. The inhibitory rate of strain X13 on the mycelial growth of C. gloeosporioides reached 61.6%. The logarithmic growth was between 2-14 h. The optimal culture conditions were 4.0% (mass percentage concentration) corn flour, 1.0% (mass percentage concentration) NaNO₃, and 3.5% (volume fraction) inoculation. This study laid a key theoretical foundation for the prevention of the tea pathogen G. theae-sinensis and the development of its biocontrol agents.

Simultaneous Determination of Nicotinamide Adenine Dinucleotide and Its Four Precursors in Tea by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry

QIU Shiting, HOU Xue, LEI Shaorong, HAN Mei, HE Guangyun, LI Ying, QIN Shudi

Journal of Tea Science. 2023, 43(2): 216-226. doi:10.13305/j.cnki.jts.2023.02.012

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Nicotinamide ribosome (NR), nicotinamide mononucleotide (NMN), nicotinic acid (NA) and nicotinamide (NAM) are 4 precursor compounds of NAD⁺, which can be converted to NAD⁺ in vivo to work after oral administration. This study established a method for simultaneous determination of NAD⁺and its four precursors in tea by ultra-high performance liquid chromatography-tandem mass spectrometry. The tea samples were extracted by water, then diluted and directly analyzed by UPLC-MS/MS. Method validation shows this method has good linearity in their respective range with correlation coefficients (R²) higher than 0.99. The average recoveries ranged from 70.00 % to 120.00% with relative standard deviations (RSDs) of 2.09%-14.50%. The limits of quantitation (LOQs) were 0.10-0.50 μg·L^-1. The determination results of five target compounds in green tea, black tea and dark tea show that green tea and black tea were the good natural food sources of NR, NMN, NAD⁺. The results of principal component analysis show that the contents of five compounds could well distinguish black tea, green tea and dark tea. The cluster analysis shows the same type of tea from the same origin was of uneven quality, with high dispersion in samples, and it was difficult to identify each other.

Determination of Flavored Nucleotides in Oolong Tea by Ultra-High Performance Liquid Chromatography-Quadrupole/Orbitrap High Resolution Mass Spectrometry and Changes of Flavored Nucleotides in the Making Green Process of ‘Tieguanyin’ Tea

HUANG Yan, ZHOU Xiaosen, GAO Chenxi, ZHANG Lingzhi, RONG Jiefeng, SUN Weijiang

Journal of Tea Science. 2023, 43(2): 227-236. doi:10.13305/j.cnki.jts.2023.02.008

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Nucleotide is a kind of important substrate for the biosynthesis of nucleic acid and caffeine in tea, which is also one of the most important umami substances in tea infusion. Ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry was used for the determination of nucleotide contents. Five types of flavored nucleotides including cytidine 5'-monophosphate (CMP), uridine 5'-monophosphate (UMP), adenosine 5'-monophosphate (AMP), inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) in tea were determined within 4 min, and dynamic changes of the above nucleotide contents in the making green process of ‘Tieguanyin’ Oolong tea were analyzed. The results show that the detection limits of the five nucleotides were 0.87-1.47 μg·g^-1, with the limits of quantification of 2.62-4.41 μg·g^-1 and a linear range of 50-1 000 ng·mL^-1. The intra-day and inter-day precisions were 2.17%-4.17% and 2.95%-4.74%, with a recovery rate of 72.89%-91.98%. Three types of nucleotides, AMP, CMP and UMP, were detected in tea cultivar ‘Tieguanyin’, with the UMP content of 16.98-26.78 μg·g^-1, the CMP content of 14.31-17.56 μg·g^-1, and the AMP content of 7.80-9.91 μg·g^-1. The UMP, CMP, and AMP in ‘Tieguanyin’ were ranked in order from high to low and the CMP content increased slowly in late stages of making green process. The total amount of flavored nucleotides in the making green process varied from 40.78 to 56.69 μg·g^-1, showing the trend of mild and stable fluctuation. The method is rapid, accurate and sensitive with high stability and precision, which can be used as an analytical technique for the study of flavor chemistry in tea processing.

Identification of Key Volatile Components of “Peach Fragrance” in Blended Peach Oolong Tea

XU Lili, WANG Jiatong, ZHU Yin, SHI Jiang, LIN Zhi

Journal of Tea Science. 2023, 43(2): 237-249. doi:10.13305/j.cnki.jts.2023.02.005

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The combination of natural flowers and fruits with traditional tea can significantly enrich the flavor of blended tea. The blended peach oolong tea has the characteristic flavor quality of oolong tea and peach, which is popular in tea consumers. However, there is a lack of systematic research on the formation mechanism of the characteristic flavor quality of commercially available blended peach oolong tea. In this study, stir bar sorptive extraction (SBSE) combined with gas chromatography-mass spectrometry (GC-MS) was used to analyze 14 blended peach oolong tea samples with typical “peach fragrance” properties. A total of 14 alcohols, 3 phenols, 3 nitrogen heterocycles, 6 oxygen heterocycles, 8 lactones, 14 aldehydes, 16 ketones, 8 alkenes, 5 organic acids and 41 esters were identified. Among them, γ-decalactone (16.75%), γ-undecalactone (11.72%), linalool (10.31%), δ-decalactone (10.23%), phytol acetate (5.28%), γ-octanolactone (4.77%), trans-β-ionone (3.30%) were the most abundant volatile components. In addition, the results of sensory evaluation and relative odor activity value (ROAV) analysis show that trans-β-ionone, damascenone, linalool, δ-decalactone, isoamyl acetate, γ-undecanoic acid lactone, phytol acetate and ethyl caproate were the key flavor contributing compounds of blended peach oolong tea, and their contributions to “peach fragrance” were further analyzed by comparing with the aroma components of peach essential oil. The results of this study enriched the theoretical basis of aroma quality chemistry of blended tea, and provided scientific basis for cross-border utilization and quality evaluation of traditional tea and natural flowers and fruits.

Simultaneous Determination of 79 Pesticides Residues in Fresh Tea Leaves by Ultra-high Performance Liquid Chromatography-mass Spectrometry

PAN Yingjie, MENG Xianghe, QIAN Yuanfeng, NING Jianmei, WANG Yueyuan, LIU Qiming, TANG Shiqin, XU Xiaoan, YE Qin

Journal of Tea Science. 2023, 43(2): 250-262. doi:10.13305/j.cnki.jts.2023.02.004

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A method for simultaneous determination of 79 pesticides residues in fresh tea leaves by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was establish in this study. The fresh tea leaves were ground by high-speed wall breaking and then extracted by acetonitrile solution with NaCl. The liquid supernatant was centrifuged and purified by solid phase extraction of anhydrous magnesium sulfate, C₁₈, PSA and GCB. The scheduled multiple reaction monitoring mode (sMRM) was used for scanning and determination, and quantitative by external standard method. The results show that the 79 target compounds exhibited good linearity within the concentration range of 0.001~0.400 mg·L^-1 with a correlation coefficient (R²) of 0.990 6 to 0.999 9. The average recoveries at 5 spiked levels (0.01, 0.03, 0.05, 0.10, 0.20 mg·kg^-1) ranged from 70.7% to 119.0%, with the relative standard deviations (RSDs) ranging from 0.1% to 19.8%, and the limits of quantitation (LOQ) for the 79 pesticides ranging from 0.01 mg·kg^-1 to 0.05 mg·kg^-1. The proposed method is high sensitivity, good accuracy and quick, which is suitable for determination of 79 pesticides residues in fresh tea leaves.

Simultaneous Determination of Glyphosate, Glufosinate and Aminomethyl Phosphonic Acid Residues in Tea by Modified QuEChERS Method Coupled with UPLC-MS/MS

LI Ziqiang, YANG Mei, ZHANG Xinzhong, LUO Fengjian, LOU Zhengyun, LIANG Shuang

Journal of Tea Science. 2023, 43(2): 263-274. doi:10.13305/j.cnki.jts.2023.02.001

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An analytical method was developed for the simultaneous determination of glyphosate, glufosinate and aminomethyl phosphonic acid residues in black and green tea by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with a modified QuEChERS method. The samples were extracted twice with pure water, and the extraction solutions were purified by dispersive solid phase extraction with MWCNT, C₁₈, PVPP and CNT-OH adsorbents. The mass spectrometry was performed in multiple reaction monitoring (MRM) mode and quantified by external standard method. The results show that glyphosate, glufosinate and aminomethyl phosphonic acid had a good linear relationship within a certain concentration range with correlation coefficients (R²) ranging from 0.999 3 to 1.000 0 in black and green tea. The limits of detection ( LOD ) of the method were 0.005 0, 0.030 mg·kg^-1 and 0.030 mg·kg^-1, respectively. The limits of quantitation (LOQ) were 0.050, 0.10 mg·kg^-1 and 0.10 mg·kg^-1, respectively. The average spiked recovery rates and the relative standard deviations (RSD) of pesticides were in the range of 81.6%-120.0% and 0.6%-13.6%, respectively. The method is simple, inexpensive, reproducible, sensitive and suitable for the determination of glyphosate, glufosinate and aminomethyl phosphonic acid in black and green tea.

Application of Ranking and Napping-UFP in the Development of Standard Yellow Tea Samples from Western Anhui Province

ZHANG Ying, QIU Tong, HAN Ziyi, AN Qi, ZHAO Xiaoyi, JIANG Qing, LI Luqing, NING Jingming, WAN Xiaochun, DAI Qianying

Journal of Tea Science. 2023, 43(2): 275-286. doi:10.13305/j.cnki.jts.2023.02.011

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It is of great significance for the production and popularization of yellow tea with standard grade difference. In this paper, 9 yellow tea samples of 3 categories (yellow bud tea, small-leaf yellow tea, large-leaf yellow tea) from western Anhui province were selected. Then 80 consumers and 22 assessors were recruited to analyze these samples using ranking and Napping-UFP respectively. The results show that the 8 standard samples can be distinguished according to the appearance by ranking, except the super class and the first class of bud tea. In Napping-UFP, 22 assessors could distinguish different grades of samples by appearance. The categories (yellow bud tea, small-leaf yellow tea and large-leaf yellow tea) and the roasting degrees (light fire and sufficient fire) could be accurately distinguished based on their aroma or taste. At the same time, the assessors provided relatively rich descriptive terms (33 of appearance, 21 of aroma and 24 of taste), including "green", "yellow" and "fairly stalk" in the appearance attributes, "tend aroma", "sweet aroma" and "tobacco" in the aroma attributes, and "fresh" in the taste attributes. These terms could be used to accurately assess yellow tea from western Anhui province, which is conducive to consumers’ understanding and acceptance, as well as the market promotion of yellow tea from western Anhui province.

Inhibitory Effect and Mechanism of Theaflavin (TF₁) on Hepatoma Carcinoma Bel-7402 Cells

WANG Zhuan, WEI Wujun, REN Zhenzhen, HE Zhilong, FAN Yuchun, ZHOU Jie, GUO Guiyi, JIANG Lihe

Journal of Tea Science. 2023, 43(2): 287-296. doi:10.13305/j.cnki.jts.20230418.001

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To explore the inhibitory effect and mechanism of theaflavin (TF₁), human hepatoma carcinoma Bel-7402 cells were treated with different concentrations of TF₁. Cell viability was detected by CCK8 and cell proliferation was detected by colony formation assay. Cell migration was detected by cell wound healing experiment and transwell chamber assay. Cell apoptosis was detected by flow cytometry. Apoptosis-related proteins (Bax, Bcl-2, PARP), migration related proteins (E-cad, N-cad, Vimentin, MMP9) and signaling pathway related proteins (TGF-β, Smad3, p-smad3) were detected by western blot. The results show that different concentrations of TF₁ could inhibit the proliferation and migration of Bel-7402 cells and promote their apoptosis in a dose-dependent manner. The higher the dose, the stronger the inhibition effect (P<0.05). Compared with the control group, the expression levels of Bax and E-cad proteins in the experimental group were higher, while the expression levels of Bcl-2, PARP, N-cad, Vimentin, MMP9, TGF-β, Smad3 and p-smad3 were lower (P<0.05). In conclusion, TF₁ could inhibit the proliferation and migration of Bel-7402 cells and promote their apoptosis through TGF-β signaling pathway.

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