Matcha, renowned for its unique flavor, color, and pharmacological benefits, is widely utilized in food, chemical, and healthcare industries. Tencha, the raw material for matcha, is significantly influenced by shading cultivation. This study investigates the impact of various shading conditions on the quality components of tencha, which is crucial for enhancing matcha quality. We analyzed four shading rates (S50%, S65%, S80%, S95%), three coverage heights (C20 cm, C40 cm, C60 cm), and shading durations (4, 8, 12, 14, 16, 18, and 20 days) on the quality components of tencha from four tea varieties. Additionally, we examined the changes in tencha quality components within different tissues of tea shoots under shading. Through integrated analysis, we identified optimal shading conditions for improving tencha quality, providing a theoretical basis for enhancing matcha production. Key findings include:
1.Correlation and OPLS-DA Analysis: Correlation analysis revealed varying degrees of correlation between shading days, shading rate, coverage height, and different quality components of tencha. OPLS-DA analysis indicated significant changes in major tencha quality components during the early stages of shading, with less variation observed after 12 days.
2.Impact on Quality Components: The content of tencha quality components is determined by the tenderness of tea shoots and the shading level. As shading duration increased, tencha quality components generally decreased. Different shading treatments had varied effects on the quality components of the four tea varieties. Compared to controls, chlorophyll, free amino acid, and theanine content increased under all four shading rates, while tea polyphenol content decreased. Caffeine content in Zhenong 113, Xiangshanzao 1, and Jinxuan decreased under 50% shading rate but increased under the other three rates. Caffeine content in the Japanese tea cultivar Yabukita increased under all four shading rates. Tencha quality was superior when the coverage height was 60 cm.
3.Impact on Quality Indices and Hundred-Bud Weight: Different shading treatments consistently affected the tencha quality index and hundred-bud weight across the four tea varieties. Compared to controls, the phenol-to-amino acid ratio decreased under all four shading rates, and the catechin quality index increased. The catechin bitterness index increased at 95% shading rate but decreased under the other three rates. Coverage height and shading duration had no significant effect on the tencha quality index. Hundred-bud weight significantly decreased under all four shading rates.
4.Optimal Shading Conditions: By comparing the changes in chlorophyll content, tencha quality components, and hundred-bud weight across different tea varieties under varying shading days, rates, and heights, we determined that the optimal shading conditions for producing Grade 1 matcha tencha are: 80% shading rate, 60 cm coverage height, and 12 days of shading.
5.Distribution in Tea Shoot Tissues: Shading treatment altered the distribution of tencha quality components in different tissues of tea shoots. In the early stage of shading, the proportion of free amino acids and theanine in leaves increased. In the later stage, their proportion in leaves decreased, while it increased in stems. Conversely, the proportion of tea polyphenols and caffeine in leaves increased, and decreased in stems. Free amino acids and theanine content were highest in stems, while tea polyphenols and caffeine content were highest in leaves. The total free amino acid and theanine distribution in leaves were negatively correlated with caffeine and tea polyphenols, suggesting that prolonged shading is detrimental to tea quality formation.
1. Introduction
Matcha, historically known as ‘Mochia’ in ancient China, originated during the Sui Dynasty, flourished in the Tang Dynasty, and peaked during the Song Dynasty. A complete matcha tea ceremony even emerged in temples during this period. However, with Emperor Ming Taizu’s decree to ‘abolish dragon-group tea production, only collect bud tea for tribute,’ steeped tea became popular, and whisked tea gradually faded from Chinese history . In the late 12th century, Japanese monk Eisai introduced the steamed green tea production method to Japan, leading to the development of matcha there. By the Meiji Restoration, a comprehensive matcha production and processing system was gradually established in Japan. Today, matcha is widely used in food, daily chemical products, and health products, leading to a diverse range of matcha products that are highly popular among young people .
Matcha is an extended product of tea, serving as a crucial entry point for the tea industry’s upgrading and transformation. Matcha possesses high nutritional value and rich cultural connotations, and can also be used as an auxiliary ingredient in various foods, making it a sought-after product for tea manufacturers. Currently, the global matcha output is around 5,000 tons, with the majority coming from China and Japan. China’s annual matcha production is approximately 1,200 tons, primarily concentrated in Zhejiang, Jiangsu, Guizhou, Hubei, Shandong, Chongqing, and Henan . In 2020, China’s matcha market value reached 95.32 million USD, accounting for about 35.30% of the global matcha market. It is projected that by 2027, the global matcha market value could exceed 5.5 billion USD, and China’s matcha market value could reach 220.72 million USD, accounting for 44.94% of the global market .
With consumption upgrades, matcha’s popularity in China is continuously rising, and the matcha market is rapidly expanding. The emergence of new tea beverage industries has also led to an explosive growth in the matcha food and beverage market, with specialized matcha food brands emerging. Taking the United States and Japan as examples, their matcha industries have developed to a new level, leading to a richer and more diverse segmentation of matcha products. From upstream supply to downstream retail, there are relatively mature brands and larger matcha powder suppliers. Although the popularity of matcha specialty stores has recently declined, its derivative products are increasing, driving this niche brand towards diversification and expanding its market space.
For many years, due to limitations in tea plant varieties, phenological characteristics, and growth environments, the quality of matcha in the Chinese market has been inconsistent. Because matcha production requires specific raw materials, complex processes, and high costs, green tea powder is often used in daily foods like biscuits, cakes, and ice cream instead of matcha. For the healthy development of the matcha industry in China and its recognition by the international community, a standardized domestic matcha market is essential. With the continuous development of the matcha industry in China, there are currently three national matcha standards: the national standard ‘Matcha GB/T 34778—2017’ issued by the General Administration of Quality Supervision, Inspection and Quarantine in 2017, the Zhejiang Manufacturing Group Standard ‘Matcha for Beverage Ingredients T/ZZB 0987—2019,’ and the Guizhou Provincial Standard ‘Guizhou Matcha DB52/T 1358—2018’ issued in 2018. The promulgation of these standards is of great significance for promoting the development of the matcha industry and regulating the market . For the healthy development of China’s matcha industry, it is crucial to focus on improving the quality of matcha raw materials, starting from scientific cultivation at the source.
Shading treatment leads to changes in light, temperature, and humidity, which significantly impact the quality components of tea. Tea plants are native to subtropical regions and have evolved to prefer warm, humid, and shaded conditions. The growth and development of tea plants are closely linked to environmental conditions; under suitable conditions, tea plants can grow normally, yielding high-quality tea . Numerous studies have shown that covering and shading in tea gardens can alter the microclimate, thereby affecting the growth and physiological and biochemical processes of tea plants. Hu Yongguang et al. (2018) found that covering and shading tea gardens in late spring can reduce the temperature of the tea canopy. The higher the shading degree, the greater the temperature reduction. Shading also increases relative humidity, with higher shading degrees leading to more pronounced humidification effects . Studies indicate that appropriate shading cover in tea gardens during summer can significantly lower canopy temperature, increase relative humidity, reduce daily temperature and humidity fluctuations, and mitigate the inhibition of photosynthesis caused by strong light, high temperature, and low humidity . Shading can significantly change the micro-ecological environment of tea gardens, greatly affecting the temperature and humidity of the tea canopy and the physical properties of tea garden soil, thereby influencing photosynthesis and a series of physiological and biochemical processes in tea plants, ultimately affecting tea yield and quality . Covering effectively reduces light intensity and photosynthetically active radiation on the tea canopy surface, lowers the daily maximum temperature, narrows daily temperature fluctuations, and increases air humidity. Furthermore, shed-style covering is more effective than direct covering in improving the microclimate of tea gardens. Shading can increase the nutrient content in matcha tea garden soil and the activity of several enzymes closely related to soil nutrient transformation, as well as increase the number and activity of microorganisms in the topsoil, which is beneficial for the restoration and reconstruction of damaged tea garden ecosystems and comprehensively improves soil quality . Shading alters various environmental factors, such as light intensity, light quality, tea canopy temperature, relative humidity, and soil environment. These changes ultimately lead to alterations in the morphology, color, and main quality components of tea leaves.
Tea plant variety is a crucial factor affecting matcha quality; selecting suitable tea plant varieties is key to producing high-quality matcha. In the late 20th century, China introduced the processing technology for tencha (matcha raw material) from Japan. After long-term research, many tea plant varieties suitable for matcha production have been discovered. Among them, Zhongcha series varieties have shown excellent performance, with high yield, vibrant green color, rich aroma, and mellow taste . Yuan Liping et al. (2023) analyzed 11 quality indicators of 36 tea plant varieties grown in the same tea garden for their suitability for matcha. The top 10 varieties in comprehensive scores were: Zhongcha 102, Taitai 12, Zhongcha 108, Fuding Dahao, Meizhan, Fuding Dabai, Fuyun 6, Zhimudan, Maolv, and Yingshuang. Wu Gang et al. (2022) systematically compared the differences in various indicators of 8 different matcha varieties and found that the quality of Chinese matcha varieties was significantly higher than that of foreign matcha varieties, with Longjing 43 having the highest comprehensive quality score. Wang Suqin et al. (2020) processed 15 target varieties into tencha in spring and conducted sensory evaluations and physicochemical analyses, initially screening Longjing 43, Aolu, Zhongcha 108, and Zhongcha 102 as suitable tea plant varieties for tencha raw material . Mao Yalin et al. (2020) analyzed the differences in matcha quality indicators for 8 commonly used tea plant varieties for matcha production. The results showed that Longjing 43 tencha performed best overall, followed by Yabukita and Aolu . Tan Xuemin et al. (2021) conducted a comprehensive comparison of the quality of 7 matcha raw material tea plant varieties. The weighted scores for matcha suitability from highest to lowest were: Yabukita > Wuniuzao > Longjing 43 > Xiaoyeca > Jiukeng > Yingshuang > Dayeca, indicating that Yabukita tea plant variety has the best matcha suitability . Xing Yanchun et al. (2021) analyzed the aroma components of matcha from three varieties grown in Hangzhou—Yingshuang, Yabukita, and Jiukeng—using gas chromatography-mass spectrometry and hydrogen flame ionization detection (GC-MS-FID). The results showed that matcha made from different tea plant varieties had differences in volatile compound composition and relative content, as well as unique aroma substances, reflecting the flavor differences among varieties . Yu Luting et al. (2022) analyzed various indicators of matcha products produced by 20 large-scale enterprises in Zhejiang Province and found significant differences in composition. Sensory quality scores ranged from 83.6 to 94.8, with differences in aroma, soup color, and taste. Biochemical components also showed significant variations, with the highest and lowest values differing by more than twofold . Guo Qianwen et al. (2019) compared and analyzed the main components and particle sizes of 3 domestic and 3 Japanese matcha products. The results showed that the quality of Japanese matcha products was generally higher than that of Chinese products .
Leaves at different developmental stages exhibit significant differences in photosynthesis and quality components. Li et al. (2016) found that the total chlorophyll concentration in tea leaves gradually increased with leaf maturity. During leaf maturation, respiration rate and total nitrogen concentration continuously decreased, but total carbon content remained unchanged. Starch, tea polyphenols (TP), total catechins, and total nitrogen concentration showed a negative correlation . Chen Ying et al. (1999) measured theanine content in different tea plant tissues during spring and autumn, concluding that theanine content was highest in spring, then gradually decreased, and slightly rebounded in autumn. Theanine content in tea plants was highest in new shoots and fibrous roots, followed by mature leaves and new stems, and lowest in old stems and main lateral roots [39]. Liu et al. (2017) used HPLC to determine L-theanine content in buds, first leaves, second leaves, third leaves, old leaves, stems, and lateral roots of three tea plant varieties: Huangjinya, Anji Baicha, and Yingshuang. Huangjinya leaves and roots had the highest theanine content, followed by Anji Baicha and Yingshuang. Theanine content in leaves gradually decreased with leaf maturity, and was lowest in stems . Lee et al. (2011) performed metabolic profiling of fresh green tea leaves plucked from different positions, from tender to old leaves. They found that younger parts had higher contents of theanine, caffeine, and gallic acid, but lower accumulated catechin content, indicating a negative correlation between theanine and catechin content as tea leaves grew . Wu et al. (2020) used an integrated multi-omics approach to study the developmental regulatory mechanism of secondary metabolism in developing tea leaves. The results showed that during tea leaf development, genes involved in flavonoid biosynthesis exhibited preferential coordinated regulation at the transcriptional, translational, and protein accumulation levels. These three changes were closely related to the increased concentrations of catechins and flavonol glycosides as tea matured. Guo et al. (2017) measured the concentrations of six catechin components (GC, EGC, GCG, EGCG, EC, ECG) at different leaf positions and found that young leaves accumulated higher levels of total catechins compared to mature leaves . Li et al. (2023) used buds, first leaves, second leaves, and third leaves of new shoots as tea plant samples at different purple coloration and developmental stages. The results showed that total flavonoid content first increased then decreased, and total anthocyanin content was lowest in buds, reaching its peak in the first leaf before gradually decreasing . Jiang et al. (2018) measured flavonoid content in different tissues and found that total catechin proanthocyanidins decreased from buds to young to old leaves . Zhao et al. (2019) sampled five tissues from 4-year-old tea plants: apical buds, first leaves below the bud, second leaves, first internodes, and second internodes. Caffeine content was measured in different tissues, with concentrations ranging from 4.7 to 9.7 mg/g, accumulating highest in buds and lowest in the second internode .
Recent studies indicate that covering and shading during tea plant cultivation alter the content of chlorophyll and other plant pigments, as well as structural components (such as lignin), thereby affecting the appearance of tea. Shading tea gardens during summer can significantly change the tissue structure of tea leaves, enhancing the tenderness retention of new tea shoots, and significantly increasing chlorophyll content in leaves, with the increase being greater at higher shading degrees . Shu Hua et al. (2012) studied the effect of shading treatment on chlorophyll synthesis precursors and chlorophyll accumulation in new shoots of three tea plant varieties: Jiukeng, Longjing 43, and Shuigu. The study showed that shading reduced light intensity, and chlorophyll content in new tea shoots significantly increased . Liu et al. (2020) showed that metabolites and gene activities in the chlorophyll biosynthesis pathway exhibit clear photoregulation. Under shading conditions of 80%–90% shading rate, chlorophyll a and chlorophyll b accumulation increased twofold. Shaded leaves contained more compact and dense grana stacks, with more thylakoids per granum and higher thylakoid stacking . Chen et al. (2021) found that shading significantly induced the expression of chlorophyll synthesis genes, especially those encoding protochlorophyllide oxidoreductase (CsPOD). Reduced light intensity significantly induced CsPOD expression, leading to an increase in chlorophyll content .
Shading treatment also alters the content of free amino acids, flavonoids, caffeine, and aromatic compounds in tea, thereby affecting its taste and aroma. Zhang Wenjin et al. (2004) found that after covering and shading tea gardens, the content of tea polyphenols, crude fiber, and caffeine significantly decreased, while amino acid content significantly increased, and catechin quality components were optimized. Deng et al. (2013) used ‘Longjing 43’ variety as material to study the changes in theanine content in new shoots and roots of tea plants under shading conditions. They found that theanine content gradually increased with prolonged shading treatment, and the activity of catechin synthesis-related enzymes enhanced, thereby accelerating the conversion and synthesis of theanine into tea polyphenols . Yang et al. (2021) conducted shading treatment on hydroponic tea seedlings and found that shading promoted the synthesis and distribution of theanine in different tissues. Theanine content increased in stems, decreased in leaves, and remained stable in roots . Shading only affects catechins and their derivatives in the initial stage of shading treatment, while most amino acids such as alanine, asparagine, aspartic acid, isoleucine, threonine, leucine, and valine significantly increase with increasing shading degree and duration . Shading can reduce the metabolic capacity of flavonoids. Short-term shading can promote nitrogen metabolism , and short-term shading can improve tea quality to some extent. Shading directly reduces the thickness of tea leaves, increases chlorophyll content in newly grown tea leaves, and simultaneously reduces the content of EC and EGC, while increasing the content of theanine and caffeine . Under shading conditions, the synthesis of tea polyphenol compounds significantly decreases. This result has been verified in many tea varieties, such as Shuchazao , Yabukita , Fuding Dabai , and albino varieties Yujinxiang , Huangjinya , and Baijiguan . Covering and shading can significantly reduce the content of tea polyphenols in tea, increase the content of chlorophyll, caffeine, free amino acids, and the total amount of free aroma, and optimize amino acid and aroma components, thereby improving the sensory quality of matcha .
Shading is a traditional and effective method to improve tea quality. Different shading methods, covering materials, shading levels, starting times, durations, and production seasons all affect the shading effect and the quality of shaded tea. Tea garden shading is mainly divided into ecological shading and covering shading. Ecological shading primarily involves planting other tall plants in the middle of tea rows, such as ‘rubber-tea intercropping,’ ‘tea-forest intercropping,’ and ‘tea-fruit intercropping,’ depending on the regional environment of the tea garden. Covering shading includes direct covering, trough-style covering, and shed-style covering. Initially, covering materials included straw, branches, and reeds. New shading nets have gradually become mainstream covering materials due to their diverse specifications, light weight, easy installation, and low cost [40]. Wang Zhen et al. (2017) tested the main quality components of matcha products under four different covering methods (steel frame type: shed covering, small arch shed covering, direct simple covering on tea canopy, and steel frame shed covering). The results showed that steel frame shed covering produced significantly better sensory quality and physicochemical indicators of matcha than other covering methods . Qin Zhimin et al. (2011) used black, green, and silver-white shading nets to cover ‘Bixiangzao.’ The results showed that black covering and shading were most beneficial for increasing the yield and quality formation of famous and high-quality tea. Liu Jianjun et al. (2013) used single-layer white gauze, single-layer black shading net, and double-layer black shading net to cover Fuding Dabai tea garden. The results indicated that double-layer black shading net covering was beneficial for the accumulation of chlorophyll content in new tea shoots, while double-layer black shading net reduced the accumulation of tea polyphenols and caffeine in new shoots . Xiao Wenmin et al. (2022) covered tea gardens with black, green, blue, red, silver-gray, and yellow shading nets. The results showed that red, blue, and silver-gray nets were beneficial for cooling and humidifying tea plants, promoting photosynthesis, and increasing yield and quality. Hou Yujia et al. (2008) used black and green shading nets with three shading treatments (70%, 90%, and 100%). The results showed that the 100% black shading net treatment increased free amino acid content by 37.98% compared to the control, reduced tea polyphenols by 14.96%, and significantly decreased the phenol-to-amino acid ratio. The 100% black shading net was most beneficial for tea quality formation . Lu Anxia et al. (2019) applied 6 different shading rates of shading nets to tea plants for different durations, measuring the main quality components of tea leaves after 12, 21, and 65 days of shading. The results showed that in the early spring tea season, all shading rate treatments increased quality components after 12 days, with the 70% shading rate treatment being 22.31% higher than the control. Shading tea plants with 50%–70% shading rate can increase the content of main nitrogenous compounds in fresh tea leaves . Gu Chenchen et al. (2017) conducted short-term (8 days) shading treatment on one-year-old tea plant cuttings. The results showed that total catechin content slightly decreased, caffeine synthesis was inhibited, while free amino acid synthesis significantly increased, with total free amino acid content increasing by 58.5%, and theanine content increasing by nearly 6 times . Li Hui et al. (2014) studied the effect of shading duration on matcha quality. The results showed that shaded matcha had higher contents of free amino acids, caffeine, and chlorophyll than unshaded matcha, while tea polyphenols and total sugar content were lower. As shading duration increased, the appearance of matcha became greener, and the sensory evaluation showed a fresher and more mellow taste . Liu Qingru et al. (2011) applied 50%, 70%, and 90% shading rates to tea plants during summer. The results indicated that tea quality was best at 70% shading . Lee et al. (2013) treated tea gardens with 95% black shading net for 0, 15, 18, and 20 days. The results showed that shaded groups could be clearly distinguished from controls (0 days). Shading treatment increased quercetin-galactosyl-rutinoside, kaempferol-glucosyl-rutinoside, ECG, EGCG, tryptophan, phenylalanine, theanine, glutamine, glutamic acid, and caffeine levels, but decreased quercetin-glucosyl-rutinoside, kaempferol-glucoside, GC, and EGC levels . The optimal shading duration is 20 to 40 days, which can be adjusted according to local climate conditions, but excessive shading duration can lead to leaf defoliation and weakening of tea plant growth, thereby affecting tea quality and yield.
1.7 Research Objectives and Content
1.7.1 Research Objectives
Shading is an effective way to improve tea quality by optimizing the microenvironment of tea gardens, thereby regulating the accumulation of secondary metabolites related to tea flavor. Polyphenolic compounds are the main contributors to the bitter and astringent taste in tea. Many studies have shown that shading can reduce the synthesis of polyphenolic compounds. However, for free amino acids, which are the main contributors to the fresh and sweet taste in tea soup, the effect of shading on their accumulation has been contradictory over the years. Many studies have shown that shading can significantly increase the level of free amino acids in tea plants, but a decrease in tea free amino acids induced by shading has also been reported . Due to the complex synergistic and antagonistic effects of various environmental parameters after shading, such as temperature changes, soil nutrients, and biotic and abiotic stresses, the metabolic regulation of major quality components in tea by shading is complex. At the same time, in the actual production process of matcha, to achieve a higher ‘greenness’ in matcha color, the stems and veins are removed from plucked new tea shoots, leaving only the leaf flesh. However, previous studies on the relationship between shading cultivation and tea metabolism have used intact new shoots, such as one bud and two leaves, or one bud and three leaves . Traditionally, tea used for matcha must be shaded for 20 days. However, with increasing shading duration, the content of ash, amino acids, fats, and caffeine gradually decreases. The production period for matcha is relatively short, only about 60 days, with the best quality matcha produced from high-quality fresh tea leaves harvested in April and May . Since Japanese shading technology is relatively mature, most domestic practices adopt Japanese techniques, which may not be suitable for the development of China’s matcha industry. Regional differences in tea varieties used in China and Japan, phenological characteristics, and environmental factors mean that there are significant differences in the content and composition of major quality components in tea . Therefore, studying the effects of different shading conditions on the changes in tencha quality components is of great significance for improving matcha quality.
1.7.2 Research Content
(1) Effect of Shading Treatment on the Content and Quality Index of Tencha Quality Components in Different Tea Varieties
Four tea varieties (Zhenong 113, Yabukita, Xiangshanzao 1, and Jinxuan) were subjected to different shading treatments using various shading rates (S50%, S65%, S80%, S95%) and coverage heights (C20 cm, C40 cm, and C60 cm) for different durations (4, 8, 12, 14, 16, 18, and 20 days). The free amino acids, theanine, tea polyphenols, catechins, and caffeine in tencha were measured to analyze the impact of different shading conditions on tencha quality components.
(2) Effect of Shading Treatment on Chlorophyll Content and Hundred-Bud Weight in Different Tea Varieties
Four tea varieties (Zhenong 113, Yabukita, Xiangshanzao 1, and Jinxuan) were subjected to different shading treatments using various shading rates (S50%, S65%, S80%, S95%) and coverage heights (C20 cm, C40 cm, and C60 cm) for different durations (4, 8, 12, 14, 16, 18, and 20 days). The chlorophyll content and hundred-bud weight of fresh tea leaves were measured to analyze the impact of different shading conditions on tencha color and yield.
(3) Effect of Shading Treatment on the Content of Quality Components in Different Tissues of Tea Shoots
Zhenong 113 was subjected to shading treatment with an 80% black shading net for 4, 12, 14, 16, 18, and 20 days. Buds, first leaves, second leaves, third leaves, and fourth leaves of new tea shoots were analyzed for free amino acids, theanine, tea polyphenols, catechins, and caffeine to analyze the distribution of quality components in different tissues of tea shoots.
(4) OPLS-DA and Correlation Analysis of Shading Treatment Conditions and Tea Quality Components
OPLS-DA and correlation analysis methods were used to comprehensively analyze the relationship between shading treatment conditions and tencha quality components. By comparing the changes in tencha quality component content, chlorophyll content, and hundred-bud weight across different tea varieties under varying shading durations, shading rates, and coverage heights, the optimal shading conditions for improving tencha quality were determined.
1.8 Technical Route
This section would typically include a flowchart or diagram. Since I cannot generate images directly, I will describe the general flow of the study.
General Flow Diagram of the Study:
1.Field Experiment Design:
•Selection of tea garden and tea varieties (Zhenong 113, Yabukita, Xiangshanzao 1, Jinxuan).
•Implementation of different shading treatments: 4 shading rates (S50%, S65%, S80%, S95%), 3 coverage heights (C20 cm, C40 cm, C60 cm), and various shading durations (4, 8, 12, 14, 16, 18, 20 days).
•Control group: Unshaded tea plants.
2.Sample Collection:
•Tea samples collected at specified shading durations.
•Different tissues (buds, leaves, stems) collected for specific analyses.
3.Tencha Preparation:
•Tea samples processed into tencha (steaming, drying, grinding).
4.Quality Component Analysis:
•Measurement of free amino acids, theanine, tea polyphenols, catechins, caffeine, chlorophyll, and hundred-bud weight.
•Analytical methods: Spectrophotometry, HPLC, etc.
5.Data Analysis:
•Statistical analysis (ANOVA, Duncan’s test).
•Multivariate statistical analysis (OPLS-DA).
•Correlation analysis.
6.Conclusion and Discussion:
•Identification of optimal shading conditions.
•Discussion of findings and implications.
2. Materials and Methods
2.1 Materials and Methods
2.1.1 Experimental Materials
The tea garden for this experiment is located at the Jiulongshan Tea Industry Co., Ltd. tea base in Jiangkou County, Guizhou Province (108°53′E, 27°53′N). The tea garden is in a subtropical monsoon region with a mild climate, abundant rainfall, and humid air, which is suitable for tea plant growth.
The tested tea varieties include Zhenong 113, Yabukita, Xiangshanzao 1, and Jinxuan, all of which are 12-year-old tea plants. In the spring of 2022, tencha (leaves with stems removed) from these four varieties, treated under different shading conditions, were used to study the effects of different shading conditions on tencha quality. In the autumn of 2022, Zhenong 113 was shaded, and its buds, first leaves, second leaves, third leaves, and fourth leaves were used to analyze the effect of shading on the distribution of major quality components in tea shoots.
2.1.2 Experimental Instruments
| Instrument Name | Company and Model |
| UV-Vis Spectrophotometer | HITACHI UH5300 |
| Ultrasonic Cleaner | Gouwei Technology Co., Ltd. |
| Ultrapure Water System | Thermo Scientific |
| Vacuum Pump | Mingjie Instrument Co., Ltd. |
| Electric Thermostatic Water Bath | Tianjin Taisite Co., Ltd. |
| Benchtop Centrifuge | Xiangyi Laboratory Instrument |
| Development Co., Ltd. | |
| Biochemical Incubator | Tianjin Taisite Co., Ltd. |
| Analytical Balance | Sartorius BS2245 |
| Plant Light Analyzer | Yuanfang Optoelectronics |
| High-Performance Liquid | Primaide HITACHI |
| Chromatograph (HPLC) | |
| Electric Hot Air Drying Oven | Taisite Instrument Co., Ltd. |
| Tea Roasting Machine | Shangyang Machinery Co., Ltd. |
| Volumetric flasks, suction flasks, | Shuniu Glass Instrument Co., Ltd. |
| beakers, conical flasks, sand | |
| core filtration devices |
2.1.3 Experimental Chemicals and Reagents
•Standards: Gallic acid catechin (GC), epigallocatechin (EGC), catechin (C), epigallocatechin gallate (EGCG), epicatechin (EC), gallocatechin gallate (GCG), epicatechin gallate (ECG), gallic acid (GA), caffeine (CAF), and theanine were purchased from Beijing Solarbio Science & Technology Co., Ltd.
•Reagents: Ninhydrin, stannous chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, anhydrous sodium carbonate, Folin-Ciocalteu reagent, 95% ethanol, acetic acid, acetonitrile, and methanol were purchased from Beijing Solarbio Science & Technology Co., Ltd.
•2% Ninhydrin Solution: 1 g of ninhydrin and 40 mg of stannous chloride were dissolved in 25 ml of water in a 50 ml beaker, left in the dark overnight, filtered, and then diluted to 50 ml with water.
•Phosphate Buffer (pH 8.0): A 1/15 mol/L disodium hydrogen phosphate solution was prepared by dissolving 23.9 g of Na2HPO4·12H2O in water and diluting to 1 L. A 1/15 mol/L potassium dihydrogen phosphate solution was prepared by dissolving 9.08 g of dried KH2PO4 in water and diluting to 1 L. 95 ml of the disodium hydrogen phosphate solution and 5 ml of the potassium dihydrogen phosphate solution were mixed, and the pH was adjusted to 8.0.
•7.5% Sodium Carbonate Solution: 75 g of Na2CO3 was dissolved in water and diluted to 1 L.
•10% Folin-Ciocalteu Reagent: 5 ml of Folin-Ciocalteu reagent was diluted to 50 ml with water.
2.2 Field Experiment Design
(1) Effect of Different Shading Treatments on Tencha Quality Components
A shed-style covering with black high-density polyethylene shading nets (4 m × 20 m) was used. When 60%–70% of the tea buds had grown to one bud and three leaves, the four tea varieties were shaded. The shading rate experiment included four shading rates (S50%, S65%, S80%, S95%). Under each shading rate, three coverage heights (C20 cm, C40 cm, C60 cm) were further divided, resulting in a total of 12 double-randomized, complete block experimental plots for each tea variety. Each experimental plot area was 50 m². A PLA-30 illuminance meter was used to measure at 10 evenly distributed points in the tea rows of each treatment to confirm that the shading rates were 50%±3, 65%±3, 80%±3, and 95%±3. Three heights were set under each shading treatment, which were 20 cm, 40 cm, and 60 cm above the tea canopy. Two rows were left unshaded as a control, growing under normal light conditions. Other treatments such as water and fertilizer were consistent with the shaded tea plants.
The shading period was 20 days. Tea samples were picked in the morning after the dew had evaporated (around 8:00 AM) on the 4th, 8th, 12th, 14th, 16th, 18th, and 20th days of shading. The picking standard was one bud and three leaves in the early stage of shading (4th to 8th day) and four-leaf new shoots in the later stage (12th to 20th day). The stems were removed from the tea samples. Tea samples from each treatment were collected from 10 different areas to ensure 3 biological replicates.
(2) Effect of Shading Treatment on the Content of Quality Components in Different Tissues of Tea Plants
The experiment was divided into two randomized experimental plots: unshaded (US) and 80% shading rate (S80%), with each experimental plot area being 50 m². Tea samples were collected on the 4th, 8th, 12th, 14th, 16th, 18th, and 20th days after the start of the shading treatment. The stems, buds, and leaves of the tea samples were separated and fixed. Tea samples from each treatment were collected from 10 different areas to ensure 3 biological replicates.
2.3 Tencha Preparation
For biochemical component analysis, tea samples were steamed at 100°C for 2 minutes, quickly cooled, then dried at 80°C, ground into a fine powder, and stored at low temperature for tencha quality component analysis.
2.4 Quality Component Analysis
2.4.1 Determination of Total Free Amino Acids
The determination of total free amino acids was based on GB/T 8314-2013 “Tea – Determination of total free amino acids.” 3 g of ground tea sample was placed in a 500 ml conical flask, 450 ml of boiling distilled water was added, and the mixture was extracted in a 100°C water bath for 45 minutes. The mixture was filtered under reduced pressure while hot, and the residue was washed 3 times with a small amount of distilled water. After cooling, the filtrate was diluted to 500 ml and mixed well. 1 ml of the extract was accurately transferred to a 25 ml colorimetric tube, 0.5 ml of phosphate buffer solution and 0.5 ml of 2% ninhydrin solution were added, and the mixture was heated in a 100°C water bath for 15 minutes. After cooling, the solution was diluted to 25 ml. After 10 minutes, the absorbance was measured at 570 nm using a colorimeter with a reagent blank as a reference.
Standard Curve for Free Amino Acids: A standard solution of theanine or glutamic acid (10 mg/ml) was prepared. 0, 2, 3, 4, 5, and 6 ml of the standard solution were transferred to 100 ml volumetric flasks and diluted to the mark with distilled water. 1 ml of each standard solution was taken, 0.5 ml of phosphate buffer solution and 0.5 ml of 2% ninhydrin solution were added, and the absorbance was measured. The concentrations of the standard samples were 0, 0.2, 0.3, 0.4, 0.5, and 0.6 mg. The free amino acid content was calculated using the following formula:
X = (C * V1) / (V2 * m * w)
Where:
•X = total free amino acid content in the sample (mg/g)
•C = concentration of the measured component in the sample (mg/g)
•V1 = volume of the sample extract (ml)
•V2 = volume of the test solution used (ml)
•m = weight of the sample (g)
•w = dry matter content of the sample (%)
2.4.2 Determination of Theanine Content
The determination of theanine content was based on GB/T 23193-2017 “Tea – Determination of theanine.” 1 g of tea sample (accurate to 0.01 g) was placed in a 200 ml beaker, 100 ml of boiling distilled water was added, and the mixture was extracted in a 100°C water bath for 30 minutes. The mixture was filtered and transferred to a 100 ml volumetric flask, cooled, diluted to the mark, and mixed well. The extract was filtered through a 0.45 μm aqueous membrane, and the theanine content was determined by HPLC under the following conditions:
•Column: RP-C18 (5 μm, 150 mm × 4.6 mm)
•Flow Rate: 1 ml/min
•Column Temperature: 35°C
•Detection Wavelength: 210 nm
•Elution Gradient: Mobile phase A (pure water): mobile phase B (acetonitrile). A was maintained for 10 min, then switched to 20% A: 80% B over 2 min, maintained for 8 min, then switched back to mobile phase A over 2 min and maintained for 18 min, for a total of 40 minutes.
Standard Curve for Theanine: A standard solution of theanine (1 mg/ml) was prepared. 0, 0.1, 0.2, 0.5, 1, 1.5, and 2 ml of the standard solution were transferred to 100 ml volumetric flasks and diluted to the mark with distilled water. 1 ml of each standard solution was filtered and its peak area was determined by HPLC. The concentrations of the standard samples were 0, 0.01, 0.02, 0.05, 0.1, 0.15, and 0.2 mg. The theanine content was calculated using the following formula:
X = (c * V) / (m * w)
Where:
•X = theanine content in the sample (mg/g)
•c = concentration of the measured component in the sample (mg/g)
•V = volume of the sample extract (ml)
•m = weight of the sample (g)
•w = dry matter content of the sample (%)
2.4.3 Determination of Tea Polyphenol Content
The determination of tea polyphenol content was based on GB/T 8313-2018 “Tea – Determination of tea polyphenols and catechins.” 0.2 g of tea sample (accurate to 0.0001 g) was placed in a 10 ml centrifuge tube, 5 ml of 70% methanol solution preheated to 70°C was added, and the mixture was extracted at 70°C for 10 minutes. After cooling to room temperature, the mixture was centrifuged (4000 rpm, 4°C, 10 min). The extraction was repeated once, and the supernatants from both extractions were collected, combined, and diluted to 10 ml. 1 ml of the extract was accurately transferred to a 100 ml volumetric flask and diluted to the mark with water. 1 ml of water, gallic acid working solution, and the test solution were placed in separate test tubes, 5 ml of 10% Folin-Ciocalteu reagent was added, and the mixture was shaken. After 3–8 minutes, 4 ml of 7.5% sodium carbonate solution was added, and the mixture was diluted to 10 ml with water. After standing at room temperature for 1 hour, the absorbance was measured at 765 nm using a 10 mm cuvette with a reagent blank as a reference.
Standard Curve for Tea Polyphenols: A standard solution of theanine or glutamic acid (10 mg/ml) was prepared. 0, 2, 3, 4, 5, and 6 ml of the standard solution were transferred to 100 ml volumetric flasks and diluted to the mark with distilled water. 1 ml of each standard solution was taken, 0.5 ml of phosphate buffer solution and 0.5 ml of 2% ninhydrin solution were added, and the absorbance was measured. The concentrations of the standard samples were 0, 0.2, 0.3, 0.4, 0.5, and 0.6 mg. The tea polyphenol content was calculated using the following formula:
Plain Text
C = (A – A0) / SLOPEStd * V / (m * w * d)
Where:
•C = tea polyphenol content (mg/g)
•A = peak area of the measured component in the sample
•A0 = peak area of the component in the blank test solution
•SLOPEStd = slope of the gallic acid standard curve
•V = volume of the sample extract (ml)
•m = weight of the sample (g)
•w = dry matter content of the sample (%)
•d = dilution factor
2.4.4 Determination of Catechin and Caffeine Content
The determination of catechins and caffeine was based on the method of Yang Jinchuan et al. (2020). 0.2 g of tea sample (accurate to 0.0001 g) was placed in a 10 ml centrifuge tube, 5 ml of 70% methanol solution preheated to 70°C was added, and the mixture was extracted at 70°C for 10 minutes. After cooling to room temperature, the mixture was centrifuged (4000 rpm, 4°C, 10 min). The extraction was repeated once, and the supernatants from both extractions were collected, combined, and diluted to 10 ml. The extract was filtered through a 0.45 μm organic membrane, and the catechin and caffeine content was determined by HPLC under the following conditions:
•Column: C18 (5 μm, 250 mm × 4.6 mm)
•Flow Rate: 1 ml/min
•Column Temperature: 30°C
•Detection Wavelength: 278 nm
•Elution Gradient: Mobile phase A (0.5% acetic acid in water): mobile phase B (methanol): mobile phase C (acetonitrile) = 85:10:5, maintained for 6 min, then changed to 70:20:10 over 12 min, then changed to 72:20:8 over 5 min, maintained for 3 min, and finally changed back to 85:10:5 over 3 min and maintained for 1 min, for a total of 30 minutes.
Standard Curve for Catechins and Caffeine: A standard solution of catechins and caffeine (1 mg/ml) was prepared. 0, 0.25, 0.5, 1.5, 2, and 3 ml of the standard solution were transferred to 10 ml volumetric flasks and diluted to the mark with 70% methanol solution. The peak areas were measured. The concentrations of the standard samples were 0, 25, 50, 100, 150, 200, and 300 μg. The catechin and caffeine content was calculated using the following formula:
Plain Text
C = (A – A0) * fstd * V / (m * w * d)
Where:
•C = catechin or caffeine content (mg/g)
•A = peak area of the measured component in the sample
•A0 = peak area of the component in the blank test solution
•fstd = correction factor of the component (concentration/peak area, μg/ml)
•V = volume of the sample extract (ml)
•m = weight of the sample (g)
•w = dry matter content of the sample (%)
•d = dilution factor
2.4.5 Determination of Chlorophyll Content
Nine branches were randomly selected from each treatment, and the third leaf from the top was taken. The chlorophyll content was determined according to the method of Lichtenthaler (2001). 0.2 g of fresh leaves (accurate to 0.01 g) were cut into small pieces and extracted overnight with 20 ml of 95% ethanol until completely colorless. The extract was filtered into a 25 ml volumetric flask, and the chlorophyll was completely transferred to the flask with 95% ethanol, then diluted to 25 ml and mixed well. The absorbance was measured at 663 and 645 nm using a UV spectrophotometer with 95% ethanol as a blank. The chlorophyll content was calculated using the following formulas:
•Chlorophyll a (Chla) = 12.7 * A663 – 2.69 * A645
•Chlorophyll b (Chlb) = 22.7 * A645 – 4.68 * A663
•Chlorophyll content (mg/g) = pigment concentration (mg/L) × V / 1000W
•Total chlorophyll = Chlorophyll a content + Chlorophyll b content
2.4.6 Determination of Hundred-Bud Weight
In each experimental plot, 100 buds (one bud and three or four leaves) were randomly collected from 10 different areas, and their weight was measured (accurate to 0.01 g). The measurement was repeated three times.
2.5 Data Analysis
SPSS 19.0 (IBM, Chicago, Illinois, USA) was used for one-way analysis of variance (ANOVA) and Duncan’s test to determine significant differences in metabolite levels between treatments (p < 0.05). SIMCA software (version 13.0.0.0, UMETRICS, www.ijg.org) was used to construct OPLS-DA models. GraphPad Prism v.8.0.1 (GraphPad Software, La Jolla, California, USA) and Origin 9.8 (Origin Lab Co., Northampton, Massachusetts, USA) software, as well as the website https://www.chiplot.online, were used for plotting.
3. Results and Analysis
3.1 Effect of Shading Treatment on Tencha Quality Components in Different Tea Varieties
3.1.1 Effect of Shading Treatment on the Growth and Development of Tea Shoots
Tea shoots were continuously shaded for 20 days when they reached the initial stage of one bud and three leaves. On the 8th day of shading, most tea shoots still had tender buds, characterized by soft leaves, raised leaf surfaces, and leaf colors ranging from yellowish-green to dark green from the first to the third leaf. On the 12th day of shading, the apical buds of the tea plants stopped growing, dormant buds formed, and all true leaves fully expanded, forming four-leaf shoots. At this stage, the leaves were less soft than before, the leaf surfaces were raised, the greenness of the leaves deepened, and the leaf area slightly increased. After 12 days of shading, the tea shoots gradually matured (Figure 3-1).
Figure 3-1: The growth and development process of tea shoots under shading treatment during the shading cycle.
3.1.2 OPLS-DA Analysis of Tencha Quality Components in Different Tea Varieties under Shading Treatment
Using 12 main quality components as research variables and tea samples from 7 different shading days as independent variables, OPLS-DA modeling analysis was performed on the content of main quality components in four tea varieties during the shading period to elucidate the effect of shading duration on the changes in main quality components (Figure 3-2). For Zhenong 113, the independent variable fitting index (R2X) was 0.994, the dependent variable fitting index (R2Y) was 0.847, and the model prediction index (Q2) was 0.728. For Yabukita, R2X was 0.995, R2Y was 0.962, and Q2 was 0.913. For Xiangshanzao 1, R2X was 0.998, R2Y was 0.939, and Q2 was 0.819. For Jinxuan, R2X was 0.997, R2Y was 0.913, and Q2 was 0.798. The model fitting results for the four tea varieties showed high credibility and good prediction performance. As shown in Figure 3-2, tea samples from different shading durations for the four tea varieties did not overlap, allowing for effective differentiation. They could be divided into two groups: an early shading period (days 4 to 12) with relatively tender tea samples, where the first three samples were more dispersed, and a later shading period (days 14 to 20) with more mature samples, which were more concentrated. This indicates that the early stage of shading has a greater impact on tencha quality components, and as the new shoots grow, the changes in tencha quality components are smaller in the later shading period.
Figure 3-2: The scatter plot of orthogonal partial least squares discriminant analysis (OPLS-DA) of the main quality components of four tea varieties under different shading treatments and different shading time. A: Zhenong113; B: Yabukita; C: Xiangshanzao1; D: Jin Xuan.
Using a Variable Importance for the Projection (VIP) value > 1 as the screening criterion, differential components were identified for the four tea varieties. The characteristic differential components and their corresponding VIP values are shown in Table 3-1. The differential metabolites of the four tea varieties varied, reflecting differences in the taste of tencha tea soup among different tea varieties. Chlorophyll was a common differential metabolite in all four tea varieties, indicating that chlorophyll is a key metabolite for distinguishing samples within the shading cycle.
Table 3-1: VIP values of different components in the shading period of 4 tea varieties.
| Variety | No. | Differential Metabolite Name | VIPpred |
| Zhenong 113 | 1 | Epicatechin (EC) | 1.283 |
| 2 | Epigallocatechin (EGC) | 1.097 | |
| 3 | Catechin (C) | 1.069 | |
| 4 | Chlorophyll | 1.041 | |
| 5 | Epigallocatechin gallate (GCG) | 1.029 | |
| Yabukita | 1 | Chlorophyll | 1.139 |
| 2 | Epicatechin gallate (ECG) | 1.122 | |
| 3 | Epicatechin (EC) | 1.104 | |
| 4 | Theanine | 1.082 | |
| 5 | Total Free Amino Acids | 1.051 | |
| 6 | Epigallocatechin | 1.037 | |
| Xiangshanzao 1 | 1 | Tea Polyphenols | 1.181 |
| 2 | Total Free Amino Acids | 1.122 | |
| 3 | Epigallocatechin gallate | 1.091 | |
| 4 | Chlorophyll | 1.071 | |
| 5 | Catechin | 1.055 | |
| Jinxuan | 1 | Chlorophyll | 1.169 |
| 2 | Epigallocatechin gallate | 1.167 | |
| 3 | Epigallocatechin | 1.106 | |
| 4 | Tea Polyphenols | 1.050 |
3.1.3 Correlation Analysis of Tencha Quality Component Content and Shading Parameters in Different Tea Varieties
To clarify the potential relationship between tencha quality components and shading parameters during the shading period, a correlation analysis was performed on the main quality components of tencha and shading parameters for the four tea varieties (Figure 3-3). Chlorophyll was significantly positively correlated with shading duration, coverage height, and shading rate (p < 0.05), and significantly negatively correlated with other quality components (p < 0.05). Tea polyphenols were significantly negatively correlated with shading duration, shading rate, coverage height, catechin (C), and chlorophyll, and significantly positively correlated with other quality components. Free amino acids and theanine were significantly negatively correlated with shading duration, EGC, EC, and chlorophyll, and significantly positively correlated with coverage height, shading rate, and other main quality components. EGC was significantly negatively correlated with shading duration, shading rate, and chlorophyll, and significantly positively correlated with other main quality components. GCG was significantly negatively correlated with shading duration, EGC, EC, and chlorophyll, and significantly positively correlated with other quality components. EC was significantly negatively correlated with shading duration, coverage height, shading rate, C, CAF, GCG, FAA, and chlorophyll, and significantly positively correlated with other quality components. EGCG was significantly negatively correlated with shading duration, coverage height, shading rate, and chlorophyll, and significantly positively correlated with other quality components. CAF was significantly negatively correlated with shading duration, coverage height, EGC, EC, and chlorophyll, and significantly positively correlated with shading rate and other main quality components. C was significantly negatively correlated with shading duration, EGC, EC, tea polyphenols, and chlorophyll, and significantly positively correlated with coverage height, shading rate, and other quality components. EGC was significantly negatively correlated with shading duration, coverage height, shading rate, C, CAF, EC, tea polyphenols, and chlorophyll, and significantly positively correlated with other quality components. GC was significantly negatively correlated with shading duration and chlorophyll, and significantly positively correlated with other quality components. In summary, tencha quality components showed different degrees of correlation with shading parameters. Higher shading rates resulted in higher contents of chlorophyll, free amino acids, theanine, caffeine, and C in tencha quality components, while tea polyphenols and other catechins were lower. Higher coverage heights resulted in higher contents of chlorophyll, free amino acids, theanine, and C in tencha quality components, while tea polyphenols, caffeine, and other catechins were lower. Increased shading duration led to increased chlorophyll content and decreased other quality components.
Figure 3-3: The heat map of the correlation analysis between the content of main quality components and shading parameters of four tea varieties. A: Zhenong113; B: Yabukita; C: Xiangshanzao1; D: Jin Xuan; Shading Days: shading days (4, 8, 12, 14, 16, 18, 20 days); Height: Coverage height (C20 cm, C40 cm and C60 cm); Shading rate: Shading rate (S50%, S65%, S80% and S95%).
3.1.4 Effect of Shading Treatment on Chlorophyll Content in Different Tea Varieties
Pigments influencing dry tea color typically include chlorophyll, carotenoids, and anthocyanins, with chlorophyll being the most important pigment determining green tea quality. We analyzed the effect of four shading rates on the chlorophyll content of four tea varieties (Figure 3-4). The results showed that as tea shoots grew, the third leaf gradually matured, and the chlorophyll content of the control group (US) slightly increased. The chlorophyll content of the four tea varieties under different shading treatments showed a pattern of slight increase from day 4 to day 8, followed by a significant increase after day 12, and then a plateau. After 12 days of shading, the chlorophyll content of Zhenong 113 under S50%, S65%, S80%, and S95% treatments was significantly different from the control, increasing by 29.77%, 41.27%, 49.07%, and 33.67%, respectively. For Yabukita, chlorophyll content under the four shading rates was significantly different from the control, increasing by 28.33%, 37.07%, 41.83%, and 51.52%, respectively. For Xiangshanzao 1, chlorophyll content under the four shading rates was significantly different from the control, increasing by 17.47%, 23.8%, 25%, and 17.47%, respectively. For Jinxuan, chlorophyll content under the four shading rates was significantly different from the control, increasing by 23.83%, 24.80%, 28.02%, and 28.50%, respectively. Compared to the control, chlorophyll content increased under all four shading rates. The chlorophyll content change patterns varied among the four tea varieties under the four shading rates. For Zhenong 113 and Xiangshanzao 1, the chlorophyll content from high to low was S80%, S65%, S95%, S50%. For Jinxuan and Yabukita, the chlorophyll content from high to low was S95%, S80%, S65%, S50%.
Figure 3-4: The histogram of chlorophyll content of four tea varieties under different shading treatments. A: Zhenong113; B: Yabukita; C: Xiangshanzao1; D: Jin Xuan; Different letters indicate significant difference (p<0.05, ns not significant).
3.1.5 Effect of Shading Treatment on Free Amino Acid and Theanine Content in Different Tea Varieties
During the shading period, the free amino acid and theanine content in tencha were measured after shading treatment with different shading rates for four tea varieties. The results showed that as tea shoots grew, both total free amino acids and theanine content significantly decreased, with the rate of decrease slowing down after day 12. After 12 days of shading, the total free amino acid content of Zhenong 113 under S50%, S65%, S80%, and S95% treatments was significantly different from the control, increasing by 8.25%, 14.74%, 20.53%, and 41.56%, respectively. Compared to day 4 of shading, the content significantly decreased by 27.75%, 24.66%, 26.25%, and 17.87%, respectively. For Yabukita, the total free amino acid content under the four shading treatments was significantly different from the control, increasing by 14.16%, 29.52%, 35.27%, and 65.48%, respectively. Compared to day 4 of shading, the content significantly decreased by 24.57%, 16.61%, 16.17%, and 18.33%, respectively. For Xiangshanzao 1, the total free amino acid content under the four shading treatments was significantly different from the control, increasing by 24.21%, 28.66%, 37.31%, and 44.91%, respectively. Compared to day 4 of shading, the content significantly decreased by 8.18%, 14.90%, 11.38%, and 17.50%, respectively. For Jinxuan, the total free amino acid content under the four shading rates was significantly different from the control, increasing by 16.97%, 18.29%, 26.76%, and 57.48%, respectively. Compared to day 4 of shading, the content significantly decreased by 33.28%, 33.16%, 29.26%, and 21.82%, respectively. During the shading period, the total free amino acid content in the experimental groups was higher than in the control, and the free amino acid content of the four tea varieties increased under all four shading treatments (Figure 3-5).
Figure 3-5: The line chart of the content of free amino acids in the four tea varieties under different shading treatments. A: Zhenong113; B: Yabukita; C: Xiangshanzao1; D: Jin Xuan; Different letters indicate significant difference (p<0.05, ns not significant).
The change in theanine content was generally consistent with the change in total free amino acid content, but the decrease in theanine content was more pronounced in the early stage of shading treatment than that of free amino acids. After 12 days of shading, the theanine content of Zhenong 113 under S50%, S65%, S80%, and S95% treatments was significantly different from the control, increasing by 7.41%, 18.07%, 22.44%, and 57.80%, respectively. Compared to day 4 of shading, the content significantly decreased by 50.01%, 47.41%, 47.37%, and 52.67%, respectively. For Yabukita, the theanine content under the four shading rates was significantly different from the control, increasing by 30.77%, 55.88%, 66.15%, and 138.30%, respectively. Compared to day 4 of shading, the content significantly decreased by 52.62%, 50.27%, 47.10%, and 27.61%, respectively. After 12 days of shading, the theanine content of Xiangshanzao 1 under the four shading rates was significantly different from the control, increasing by 9.39%, 22.48%, 43.55%, and 98.55%, respectively. Compared to day 4 of shading, the content significantly decreased by 51.68%, 46.64%, 47.57%, and 41.86%, respectively. For Jinxuan, the theanine content under the four shading rates was significantly different from the control, increasing by 6.80%, 23.30%, 37.34%, and 109.31%, respectively. Compared to day 4 of shading, the content significantly decreased by 66%, 58.43%, 60.14%, and 47.26%, respectively. The theanine content in the experimental groups was higher than in the control, and the theanine content of the four tea varieties increased under all four shading treatments. Under the four different shading treatments, when shaded for 12 days, the theanine content under S80% and S90% treatments met the requirements for Grade 1 matcha. For Zhenong 113, the theanine content under S80% and S90% treatments was 10.08 mg/g and 11.57 mg/g, respectively. For Yabukita, it was 10.08 mg/g and 14.46 mg/g, respectively. For Xiangshanzao 1, it was 14.88 mg/g and 20.58 mg/g, respectively. For Jinxuan, it was 10.06 mg/g and 14.16 mg/g, respectively (Figure 3-6).
Figure 3-6: The line chart of the content of theanine in the four tea varieties under different shading treatments. A: Zhenong113; B: Yabukita; C: Xiangshanzao1; D: Jin Xuan; Different letters indicate significant difference (p<0.05, ns not significant).
3.1.6 Effect of Shading Treatment on Tea Polyphenol and Caffeine Content in Different Tea Varieties
Effect of four shading rates on tea polyphenols in four tea varieties. During the shading period, the tea polyphenol content in the shaded groups was lower than in the control group. As tea shoots grew, the tea polyphenol content of the four tea varieties first significantly decreased, then plateaued after day 12. After 12 days of shading, the tea polyphenol content of Zhenong 113 under S50%, S65%, S80%, and S95% treatments was significantly different from the control, decreasing by 7.54%, 12.17%, 10.9%, and 13.81%, respectively. Compared to day 4 of shading, the content significantly decreased by 15.45%, 17.3%, 9.23%, and 6.01%, respectively. For Yabukita, the tea polyphenol content under the four shading rates was significantly different from the control, decreasing by 13.05%, 15.53%, 15.02%, and 21.61%, respectively. Compared to day 4 of shading, the content significantly decreased by 13.9%, 11.26%, 4.34%, and 1.08%, respectively. For Xiangshanzao 1, the tea polyphenol content under the four shading rates was significantly different from the control, decreasing by 10.87%, 13.29%, 18.41%, and 16.35%, respectively. Compared to day 4 of shading, the content significantly decreased by 25.12%, 27.98%, 30.43%, and 16.86%, respectively. For Jinxuan, the tea polyphenol content under the four shading rates was significantly different from the control, decreasing by 6.23%, 12.43%, 18.51%, and 17.68%, respectively. Compared to day 4 of shading, the content significantly decreased by 8.65%, 8.91%, 12.96%, and 10.79%, respectively. The tea polyphenol content of the four tea varieties decreased under all four shading treatments (Figure 3-7).
Figure 3-7: The histogram of the content of tea polyphenol in the four tea varieties under different shading treatments. A: Zhenong113; B: Yabukita; C: Xiangshanzao1; D: Jin Xuan; Different letters indicate significant difference (p<0.05, ns not significant).
Effect of four tea varieties and three coverage heights on caffeine (Figure 3-8). As tea shoots grew, caffeine content first significantly decreased, then plateaued after day 12. After 12 days of shading, the caffeine content of Zhenong 113 under S65%, S80%, and S95% treatments significantly increased by 10.62%, 19.58%, and 30.03%, respectively, while the caffeine content under S50% decreased by 1.89% compared to the control. Compared to day 4 of shading, the content significantly decreased by 19.51%, 11.78%, 12.25%, and 12.10%, respectively. After 12 days of shading, the caffeine content of Yabukita under the four shading rates was significantly different from the control, increasing by 4.99%, 9.52%, 14.71%, and 20.25%, respectively. Compared to day 4 of shading, the content significantly decreased by 9.05%, 13.48%, 13.17%, and 16.02%, respectively. After 12 days of shading, the caffeine content of Xiangshanzao 1 under S65%, S80%, and S95% treatments increased by 1.11%, 12.03%, and 21.52%, respectively, while the caffeine content under S50% decreased by 6.29% compared to US. Compared to day 4 of shading, the content significantly decreased by 24.79%, 28.07%, 29.25%, and 27.31%, respectively. After 12 days of shading, the caffeine content of Jinxuan under S65%, S80%, and S95% treatments increased by 1.13%, 8.01%, and 14.82%, respectively, while the caffeine content under S50% decreased by 2.38% compared to the control. Compared to day 4 of shading, the content significantly decreased by 9.05%, 16.45%, 7.79%, and 26.34%, respectively. Caffeine content in Zhenong 113, Xiangshanzao 1, and Jinxuan decreased under 50% shading rate but increased under the other three shading rates. Caffeine content in Yabukita increased under all four shading rates.
Figure 3-8: The histogram of the content of caffeine in the four tea varieties under different shading treatments. A: Zhenong113; B: Yabukita; C: Xiangshanzao1; D: Jin Xuan; Different letters indicate significant difference (p<0.05).
3.1.7 Effect of Coverage Height on Tencha Quality Components in Different Tea Varieties
For the four tea varieties under three coverage height treatments, chlorophyll content slightly increased with increasing coverage height. There was no significant effect on chlorophyll content among the three coverage height treatments, with the order being: C60 cm > C40 cm > C20 cm. The trends for free amino acids and theanine content were consistent across the three coverage heights: free amino acids and theanine content slightly increased with increasing coverage height. There was no significant effect on free amino acids and theanine content among the three coverage height treatments, with the order being: C60 cm > C40 cm > C20 cm. The trends for tea polyphenols and caffeine content were consistent across the three coverage heights: free amino acids and theanine content slightly decreased with increasing coverage height. There was no significant effect on tea polyphenols and caffeine content among the three coverage height treatments, with the order being: C20 cm > C40 cm > C60 cm.
Figure 3-9: Changes of quality components of four tea varieties under different mulching height treatments. A: Chlorophyll contents; B: Free amino acid and theanine contents; C: Tea polyphenols and caffeine contents; a: Zhenong113; b: Yabukita; c: Xiangshanzao1; d: Jin Xuan; Different letters indicate significant difference (p<0.05, ns not significant).
3.1.8 Effect of Shading Treatment on Tencha Quality Indices in Different Tea Varieties
The quality of green tea depends not only on the content of tea polyphenols and free amino acids but also on the ratio of tea polyphenols to free amino acids, the catechin quality index, and the catechin bitterness index. Therefore, we analyzed various indicators of tencha to better determine the tea plant growth conditions that can produce high-quality tencha. The results showed that as tea shoots grew, the content of free amino acids and tea polyphenols decreased significantly in the early stage of shading treatment, and the decrease slowed down after 12 days. Therefore, the phenol-to-amino acid ratio in most tea varieties showed a trend of first increasing and then slightly decreasing (Figure 3-10 A, D, G, J). Compared to the control, the phenol-to-amino acid ratio decreased under all four shading rates. The catechin quality index can accurately reflect the tenderness and quality of tea. The younger the tea, the higher its quality index. As the tea ages, its quality decreases, and the index gradually decreases. Its calculation formula is: ((EGCE + ECG) / EGC) × 100 [73]. The results showed that compared to the control, the catechin quality index increased under all four shading conditions (Figure 3-10 B, E, H, K). The catechin bitterness index is a measure of the bitterness of tea. This index comprehensively reflects the role of various catechins in bitterness. The higher the bitterness index, the stronger the bitterness. Its calculation formula is: (EGCG + ECG + EGC + GC) / (EC + C) [47]. The results showed that the bitterness index under different shading treatments from high to low was S95%, US, S80%, S65%, S50% (Figure 3-10 C, F, I, L). Therefore, appropriate shading can optimize the catechin composition, thereby helping to reduce the bitterness caused by catechins.
Figure 3-10: Changes of the ratio of tea polyphenols to amino acids, catechin index and catechin bitterness index of four tea varieties under different shading treatments. (A-C): Zhenong113; (D-F): Yabukita; (G-I): Xiangshanzao1; (J-L): Jinxuan; Tea Polyphenols/Amino Acids ratio: Phenol ammonia ratio; Catechin quality index: catechin quality index; Catechin bitterness index: catechin bitterness index.
3.1.9 Effect of Shading Treatment on Hundred-Bud Weight in Different Tea Varieties
Hundred-bud weight is an indicator of the quality, robustness, and size of new shoots, and its value directly affects tea yield. The hundred-bud weight of different shading rates after 12 days of shading was measured. The results are shown in Table 3-2. For Zhenong 113, the hundred-bud weight under S50%, S65%, S80%, and S95% treatments was significantly different from the control, decreasing by 3.33%, 9.28%, 13.10%, and 33.18%, respectively. For Yabukita, the hundred-bud weight under the four shading rates was significantly different from the control, decreasing by 2.17%, 10.19%, 14.27%, and 21.02%, respectively. For Xiangshanzao 1, the hundred-bud weight under the four shading rates was significantly different from the control, decreasing by 8.11%, 17.18%, 24.48%, and 31.75%, respectively. For Jinxuan, the hundred-bud weight under the four shading rates was significantly different from the control, decreasing by 5.80%, 17.20%, 35.12%, and 38.99%, respectively. Compared to the control, the hundred-bud weight of the four tea varieties decreased under all four shading rates.
Table 3-2: Changes of 100-bud weight of four tea varieties under different shading treatments.
| Variety | Treatment | Weight (g) Mean ± SD |
| Zhenong 113 | US | 100.55 ± 2.96a |
| S50% | 97.31 ± 2.39a | |
| S65% | 92.01 ± 1.91b | |
| S80% | 88.90 ± 2.11b | |
| S95% | 75.50 ± 0.74c | |
| Yabukita | US | 89.30 ± 2.96a |
| S50% | 87.40 ± 2.39a | |
| S65% | 81.04 ± 1.91b | |
| S80% | 78.15 ± 2.11b | |
| S95% | 73.79 ± 1.22c | |
| Xiangshanzao 1 | US | 136.85 ± 1.22a |
| S50% | 126.58 ± 2.42b | |
| S65% | 116.79 ± 0.71c | |
| S80% | 109.93 ± 2.55d | |
| S95% | 103.87 ± 1.21e | |
| Jinxuan | US | 117.99 ± 2.03a |
| S50% | 111.52 ± 5.3a | |
| S65% | 100.67 ± 7.57b | |
| S80% | 87.32 ± 3.31c | |
| S95% | 84.89 ± 1.41c |
Note: aUS represents the unshaded control condition; S50%, S65%, S80%, S95% represent shading treatment conditions with shading rates of 50%, 65%, 80%, and 95%, respectively; b Different lowercase letters indicate significant differences (p < 0.05) between control and shaded samples for the same quality component at the same treatment duration.
The above results indicate that OPLS-DA analysis showed significant changes in tencha quality components in the early stage of shading, with less variation observed after 12 days as the new shoots grew. Shading duration, shading rate, and tencha quality components showed different degrees of correlation. Correlation analysis showed that different shading conditions (shading rate, coverage height, shading duration) had different effects on the quality components of different tea varieties. Compared to the control, chlorophyll, free amino acid, and theanine content of the four tea varieties increased under all four shading rates, while tea polyphenol content decreased. Caffeine content in Zhenong 113, Xiangshanzao 1, and Jinxuan decreased under 50% shading rate but increased under the other three shading rates. Caffeine content in Yabukita increased under all four shading rates. Tencha quality was superior when the coverage height was 60 cm for the four tea varieties. Compared to the control, the phenol-to-amino acid ratio decreased under all four shading rates, and the catechin quality index increased. The catechin bitterness index increased at 95% shading rate but decreased under the other three shading rates. Coverage height and shading duration had no significant effect on the tencha quality index. Compared to the control, chlorophyll content of the four tea varieties increased under all four shading rates, and hundred-bud weight decreased. With increasing coverage height, free amino acid, theanine, and chlorophyll content increased, while caffeine and tea polyphenol content decreased.
3.2 Effect of Shading Treatment on the Content of Quality Components in Different Tissues of Tea Shoots
3.2.1 Effect of Shading Treatment on Free Amino Acid and Theanine Content in Different Tissues of Tea Shoots
By comparing the unshaded tea plants (US) as a control, the changes in free amino acid and theanine content in different tissues of tea shoots during the shading treatment (S80%) period (20 days) were analyzed (Table 3-3). The results showed that after S80% treatment, the free amino acid and theanine content in all tissues of tea shoots were higher than in the control. As shading progressed, the theanine and free amino acid content in all tissues of tea shoots showed a decreasing trend, but the rate of decrease slowed down from day 12 onwards. The changes in free amino acid and theanine content in different tissues of tea shoots were consistent under both control and S80% treatment conditions. In the early stage of shading treatment (days 4 to 8), the theanine and free amino acid content were highest in stems, followed by buds, first leaves, second leaves, and third leaves. In the later stage of shading treatment (days 12 to 20), the theanine and total free amino acid content were highest in stems, followed by first leaves, second leaves, third leaves, and fourth leaves.
Table 3-3: Theanine and free amino acid contents in different tissues of tea shoots under shading treatment.
| Component | Days | Treatment | Stem (mg/g) Mean ± SD | 1st (mg/g) Mean ± SD | 2nd (mg/g) Mean ± SD | 3rd (mg/g) Mean ± SD | 4th (mg/g) Mean ± SD |
| Total Free Amino Acids | 4 | US | 112.58 ± 0.72b | 51.78 ± 0.47d | 43.28 ± 0.28e | 38.35 ± 0.24g | 34.72 ± 0.23h |
| S80% | 130.01 ± 1.17a | 60.28 ± 0.39c | 51.92 ± 0.33d | 42.91 ± 0.27e | 39.38 ± 0.25f | ||
| 8 | US | 94.36 ± 2.03b | 48.78 ± 0.49d | 37.08 ± 1.52h | 31.61 ± 1.14i | 29.78 ± 0.50j | |
| S80% | 114.65 ± 0.73a | 57.01 ± 0.36c | 46.65 ± 0.30e | 41.93 ± 0.27f | 38.89 ± 0.25g | ||
| 12 | US | 88.12 ± 0.56b | 39.25 ± 0.25e | 30.34 ± h | 28.09 ± 0.18i | 27.03 ± 0.17j | |
| S80% | 94.41 ± 0.60a | 45.29 ± 0.29c | 40.47 ± 0.26d | 34.92 ± 0.23f | 32.87 ± 0.21g | ||
| 14 | US | 80.80 ± 0.46b | 38.65 ± 0.25d | 32.37 ± 0.20f | 29.38 ± 0.19h | 26.22 ± 0.21i | |
| S80% | 87.44 ± 0.56a | 42.21 ± 0.27c | 37.12 ± 0.24e | 31.48 ± 0.20g | 26.49 ± 0.17i | ||
| 16 | US | 80.80 ± 0.46b | 38.65 ± 0.25d | 32.37 ± 0.20f | 29.38 ± 0.19h | 26.22 ± 0.19i | |
| S80% | 87.44 ± 0.56a | 42.21 ± 0.27c | 37.12 ± 0.24e | 31.48 ± 0.20g | 26.49 ± 0.17i | ||
| 18 | US | 71.66 ± 0.46b | 36.74 ± 0.24d | 32.62 ± 0.21f | 29.87 ± 0.19h | 24.64 ± 0.16j | |
| S80% | 83.03 ± 0.53a | 39.33 ± 0.25c | 35.18 ± 0.23e | 30.49 ± 0.19g | 28.21 ± 0.18i | ||
| 20 | US | 69.30 ± 0.44b | 34.48 ± 0.22d | 30.97 ± 0.20f | 26.21 ± 0.17h | 21.86 ± 0.14j | |
| S80% | 76.34 ± 0.49a | 36.04 ± 0.23c | 33.58 ± 0.21e | 27.24 ± 0.18g | 24.70 ± 0.16i | ||
| Theanine | 4 | US | 43.66 ± 0.09b | 18.61 ± 0.13e | 14.94 ± 0.16g | 11.68 ± 0.07h | 10.77 ± 0.12i |
| S80% | 62.72 ± 0.74a | 24.49 ± 0.10c | 20.69 ± 0.14d | 18.90 ± 0.07e | 16.49 ± 0.02f | ||
| 8 | US | 42.75 ± 0.23b | 17.13 ± 0.05e | 14.24 ± 0.06g | 12.16 ± 0.11h | 10.15 ± 0.03i | |
| S80% | 56.15 ± 0.23a | 20.32 ± 0.38c | 18.70 ± 0.05d | 15.11 ± 0.11f | 14.39 ± 0.29g | ||
| 12 | US | 32.43 ± 0.49b | 12.78 ± 0.04e | 10.61 ± 0.59g | 8.65 ± 0.19h | 7.11 ± 0.09i | |
| S80% | 42.73 ± 0.16a | 15.97 ± 0.11c | 13.17 ± 0.29d | 12.38 ± 0.06f | 10.55 ± 0.16g | ||
| 14 | US | 20.16 ± 0.15b | 10.11 ± 0.12f | 7.54 ± 0.03h | 6.13 ± 0.17i | 4.40 ± 0.05j | |
| S80% | 38.22 ± 0.21a | 15.27 ± 0.14c | 12.45 ± 0.09d | 11.29 ± 0.14e | 9.74 ± 0.08g | ||
| 16 | US | 19.66 ± 0.37b | 10.60 ± 0.06f | 7.80 ± 0.10h | 6.21 ± 0.13i | 4.15 ± 0.06j | |
| S80% | 33.88 ± 0.11a | 13.96 ± 0.06c | 12.25 ± 0.10d | 11.36 ± 0.06e | 9.68 ± 0.06g | ||
| 18 | US | 19.07 ± 0.03b | 8.75 ± 0.09e | 6.60 ± 0.14g | 4.97 ± 0.04i | 3.63 ± 0.08j | |
| S80% | 27.69 ± 0.05a | 10.66 ± 0.06c | 9.77 ± 0.05d | 7.73 ± 0.10f | 6.09 ± 0.04h | ||
| 20 | US | 16.45 ± 1.06b | 7.70 ± 0.15d | 6.51 ± 0.15e | 4.38 ± 0.14g | 2.95 ± 0.25h | |
| S80% | 24.36 ± 0.06a | 9.60 ± 0.10c | 7.67 ± 0.22d | 6.32 ± 0.08e | 5.20 ± 0.15f |
Note: aUS represents the unshaded control condition; S80% represents shading treatment conditions with a shading rate of 80%; Stems: stem; 1st: bud in early shading, first leaf in later shading; 2nd: first leaf in early shading, second leaf in later shading; 3rd: second leaf in early shading, third leaf in later shading; 4th: third leaf in early shading, fourth leaf in later shading. b Different lowercase letters indicate significant differences (p < 0.05) between control and shaded samples for the same quality component at the same treatment duration.
The percentage bar chart was used to further analyze the distribution of free amino acids and theanine in the leaves and stems of tea shoots under shading treatment (Figure 3-11). The results showed that on day 4, the distribution ratio of free amino acids in leaves and stems was consistent between the control and S80% treatment conditions, at 59% and 41%, respectively. As shading duration increased, the distribution ratio of free amino acids in leaves and stems changed. From day 8 to day 12, the proportion of free amino acids in leaves was higher under shading treatment than in the control, but lower in stems. The proportion of free amino acids in leaves increased from 59% to 62%. On day 14, the distribution ratio of free amino acids in leaves and stems was consistent between the control and S80% treatment conditions, at 61% and 49%, respectively. On days 16, 18, and 20, compared to the control, shading treatment led to a decrease in the proportion of free amino acids in leaves and an increase in stems. The distribution of theanine in the leaves and stems of tea shoots showed that on day 4, the distribution ratio of theanine in leaves and stems was consistent between the control and S80% treatment conditions, at 56% and 44%, respectively. Subsequently, the distribution ratio of theanine in leaves and stems changed, with slight changes from day 8 to day 12, where the proportion of theanine in leaves increased from 56% to 57%. After day 12, the proportion of theanine in leaves under shading treatment was lower than in the control, but higher in stems. Compared to the control, shading treatment led to a decrease in the proportion of theanine in leaves and an increase in stems.
Figure 3-11: Distribution ratio of free amino acids and theanine in different tissues of tea plant under shading condition. A: free amino acids; B: theanine; US: Control condition without shading treatment; S80%: shading treatment conditions with shading rate of 80%.
3.2.2 Effect of Shading Treatment on Tea Polyphenol and Catechin Content in Different Tissues of Tea Shoots
By comparing the unshaded tea plants as a control, the changes in tea polyphenol content in different tissues of tea shoots during the shading treatment period were analyzed (Figure 3-12A). The results showed that after S80% treatment, the tea polyphenol content in all tissues of tea shoots was lower than in the control. As shading progressed, the tea polyphenol content in all tissues of tea shoots showed a decreasing trend, with the rate of decrease slowing down from day 12 onwards. Under both control and S80% treatment conditions, in the early stage of shading treatment (days 4 to 8), the tea polyphenol content from high to low was first leaf, second leaf, bud, and third leaf, with the lowest content in stems. In the later stage of shading treatment (days 12 to 20), the tea polyphenol content from high to low was first leaf, second leaf, third leaf, and fourth leaf, with the lowest content in stems.
The percentage bar chart was used to further analyze the distribution of tea polyphenols in the leaves and stems of tea shoots under shading treatment (Figure 3-12B). The results showed that from day 4 to day 8, the distribution ratio of tea polyphenols in leaves and stems was consistent between the control and S80% treatment conditions, at 85% and 15%, respectively. As shading progressed, from day 12 to day 20, under shading treatment conditions, the proportion of tea polyphenols in leaves was higher than in the control, increasing from 85% to 87%. Compared to the control, shading treatment led to an increase in the proportion of tea polyphenols in leaves and a decrease in stems.
Figure 3-12: Distribution and distribution ratio of tea polyphenols in different tissues of tea plant under shading condition. A: the distribution of tea polyphenols in different tissues of tea plant; B: the distribution proportion of tea polyphenols in different tissues of tea plant. US: Control condition without shading treatment; S80%: shading treatment conditions with shading rate of 80%; The different lowercase letters above the bar chart showed significant differences (P<0.05).
Catechins account for approximately 70% of the total tea polyphenols and are important components determining the color, aroma, and taste of tea. Among them, non-ester catechins EGC and EC are the main contributors to the “sweet aftertaste” of green tea [11]. By comparing the unshaded tea plants as a control, the changes in catechin content in different tissues of tea shoots during the shading treatment period were analyzed (Table 3-4). The results showed that after S80% treatment, the catechin content in all tissues of tea shoots was lower than in the control, indicating that shading significantly reduced catechin synthesis. As shading progressed, the catechin content in all tissues of tea shoots showed a decreasing trend, with the rate of decrease slowing down from day 12 onwards. Under both control and S80% treatment conditions, the changes in tea polyphenols and ester catechin content in different tissues of tea shoots were consistent. In the early stage of shading treatment (days 4 to 8), the ester catechin content from high to low was first leaf, second leaf, bud, and third leaf, with the lowest content in stems. In the later stage of shading treatment (days 12 to 20), the ester catechin content from high to low was first leaf, second leaf, third leaf, and fourth leaf, with the lowest content in stems. For non-ester catechins, in the early stage of shading treatment, the non-ester catechin content was lowest in the first leaf, followed by bud, second leaf, stem, and third leaf in decreasing order. In the later stage of shading treatment, the non-ester catechin content was lowest in the first leaf, followed by second leaf, third leaf, fourth leaf, and stem in decreasing order.
Table 3-4: Contents of ester catechins and non-ester catechins in different tissues of tea shoots under shading treatment.
| Component | Days | Treatment | Stem (mg/g) Mean ± SD | 1st (mg/g) Mean ± SD | 2nd (mg/g) Mean ± SD | 3rd (mg/g) Mean ± SD | 4th (mg/g) Mean ± SD |
| Ester Catechins | 4 | US | 52.61 ± 4.13h | 117.49 ± 0.82c | 130.47 ± 0.26a | 111.51 ± 1.43d | 96.58 ± 2.16f |
| S80% | 29.93 ± 0.28i | 109.0 ± 0.83d | 123.05 ± 2.23b | 102.29 ± 0.50e | 84.54 ± 0.07g | ||
| 8 | US | 55.52 ± 0.60g | 113.16 ± 0.61b | 126.41 ± 0.90a | 103.48 ± 0.57c | 94.15 ± 1.06d | |
| S80% | 35.34 ± 0.13h | 106.60 ± 0.98c | 113.52 ± 0.79b | 79.30 ± 0.06e | 69.53 ± 2.01f | ||
| 12 | US | 42.88 ± 1.72i | 122.26 ± 0.57a | 107.84 ± 0.28c | 97.68 ± 1.36e | 89.94 ± 2.90f | |
| S80% | 28.98 ± 0.26j | 119.80 ± 0.74b | 102.80 ± 0.38d | 78.95 ± 0.21g | 57.10 ± 0.74h | ||
| 14 | US | 37.61 ± 0.44i | 115.58 ± 0.67a | 108.94 ± 0.60c | 92.14 ± 0.98d | 87.77 ± 0.80e | |
| S80% | 22.17 ± 0.29j | 110.84 ± 0.42b | 86.16 ± 0.67f | 80.90 ± 0.58g | 77.87 ± 1.18h | ||
| 16 | US | 35.25 ± 1.23i | 110.27 ± 0.32a | 100.95 ± 0.61b | 85.51 ± 0.40d | 72.93 ± 0.71f | |
| S80% | 20.98 ± 0.01j | 97.07 ± 0.44c | 79.34 ± 0.38e | 53.81 ± 0.40g | 46.93 ± 0.58h | ||
| 18 | US | 33.97 ± 0.98i | 106.09 ± 0.40a | 94.27 ± 0.36b | 81.08 ± 0.82d | 70.95 ± 1.70f | |
| S80% | 19.98 ± 0.13j | 96.55 ± 0.32c | 78.11 ± 0.43e | 59.51 ± 0.21g | 52.88 ± 0.57h | ||
| 20 | US | 31.22 ± 0.18i | 100.0 ± 0.38a | 85.59 ± 0.57c | 76.70 ± 0.76d | 62.13 ± 0.35g | |
| S80% | 20.79 ± 0.68j | 91.13 ± 0.92b | 68.25 ± 0.83e | 66.09 ± 0.58f | 57.52 ± 0.88h | ||
| Non-Ester Catechins | 4 | US | 21.19 ± 0.39c | 20.95 ± 0.19cd | 26.10 ± 0.07a | 22.60 ± 0.58b | 20.77 ± 0.11cd |
| S80% | 19.71 ± 0.35e | 22.15 ± 0.12b | 20.50 ± 0.14d | 14.24 ± 0.13f | 10.80 ± 0.16g | ||
| 8 | US | 18.42 ± 0.12d | 18.86 ± 0.60d | 23.52 ± 0.04a | 18.32 ± 0.23d | 16.54 ± 0.52e | |
| S80% | 18.39 ± 0.41d | 19.82 ± 0.15c | 20.57 ± 0.12b | 13.05 ± 0.11f | 10.30 ± 0.11g | ||
| 12 | US | 17.59 ± 0.39b | 19.49 ± 0.19a | 17.66 ± 0.28b | 15.54 ± 0.41c | 12.50 ± 0.20d | |
| S80% | 9.89 ± 0.20f | 12.21 ± 0.18d | 11.47 ± 0.16e | 8.38 ± 0.06g | 7.00 ± 0.09h | ||
| 14 | US | 8.70 ± 0.13f | 15.38 ± 0.03a | 12.86 ± 0.04b | 9.23 ± 0.11e | 6.87 ± 0.20i | |
| S80% | 8.10 ± 0.07g | 11.79 ± 0.16c | 10.51 ± 0.06d | 7.28 ± 0.08h | 6.14 ± 0.03j | ||
| 16 | US | 10.60 ± 0.34c | 14.97 ± 0.01a | 11.40 ± 0.55b | 8.19 ± 0.05e | 7.42 ± 0.02f | |
| S80% | 7.22 ± 0.11f | 11.29 ± 0.26b | 9.61 ± 0.25d | 7.21 ± 0.05f | 5.14 ± 0.08g | ||
| 18 | US | 9.72 ± 0.29d | 14.50 ± 0.17a | 11.28 ± 0.13b | 6.85 ± 0.11f | 5.87 ± 0.18g | |
| S80% | 5.92 ± 0.07g | 10.49 ± 0.08c | 7.97 ± 0.09e | 5.90 ± 0.03g | 4.94 ± 0.12h | ||
| 20 | US | 8.56 ± 0.02c | 12.05 ± 0.07a | 8.03 ± 0.24d | 6.11 ± 0.06f | 5.82 ± 0.03g | |
| S80% | 5.21 ± 0.09i | 9.17 ± 0.11b | 7.23 ± 0.04e | 5.49 ± 0.04h | 4.73 ± 0.07j |
Note: aUS represents the unshaded control condition; S80% represents shading treatment conditions with a shading rate of 80%; b Different lowercase letters indicate significant differences (p < 0.05) between control and shaded samples for the same quality component at the same treatment duration.
3.2.3 Effect of Shading Treatment on Caffeine Content in Different Tissues of Tea Shoots
By comparing the unshaded tea plants as a control, the changes in caffeine content in different tissues of tea shoots during the shading treatment period were analyzed (Figure 3-13A). The results showed that after S80% treatment, the caffeine content in all tissues of tea shoots was higher than in the control. As shading progressed, the caffeine content in all tissues of tea shoots showed a decreasing trend, with the rate of decrease slowing down after day 12. Under both control and S80% treatment conditions, in the early stage of shading treatment (days 4 to 8), the caffeine content was lowest in stems, followed by first leaves, second leaves, buds, and third leaves in decreasing order. In the later stage of shading treatment (days 12 to 20), the caffeine content was lowest in stems, followed by first leaves, second leaves, third leaves, and fourth leaves in decreasing order.
The percentage bar chart was used to further analyze the distribution of caffeine in the leaves and stems of tea shoots under shading treatment (Figure 3-13B). The results showed that from day 4 to day 8, the distribution ratio of caffeine in leaves and stems was consistent between the control and S80% treatment conditions, at 85% and 15%, respectively. As shading progressed, from day 12 to day 20, under shading treatment conditions, the proportion of caffeine in leaves was higher than in the control, increasing from 85% to 87%. Compared to the control, shading treatment led to an increase in the proportion of caffeine in leaves and a decrease in stems.
Figure 3-13: Distribution and distribution ratio of caffeine in different tissues of tea plant under shading condition. A: Distribution of caffeine in different tissues of tea plant; B: The distribution ratio of caffeine in different tissues of tea plant. US: Control condition without shading treatment; S80%: shading treatment conditions with shading rate of 80%, The different lowercase letters above the bar chart showed significant differences (p<0.05).
The above results indicate that after S80% treatment, the free amino acid, theanine, and caffeine content in all tissues of tea shoots were higher than in the control, while the tea polyphenol and catechin content were lower. The content of tea quality components decreased as the tea shoot tissues matured, indicating that the content of quality components is largely determined by the tenderness of the new shoots. Among them, in the tender stems of new tea shoots, free amino acids and theanine content were highest, while tea polyphenols, ester catechins, and caffeine content were lowest. At the same time, shading changed the distribution ratio of tea quality components in the stems and leaves of new shoots. In the early stage of shading treatment, the proportion of free amino acids and theanine in leaves increased and decreased in stems. In the later stage of shading treatment, the proportion of free amino acids and theanine in leaves decreased and increased in stems. The proportion of tea polyphenols and caffeine in leaves increased and decreased in stems. After shading treatment, the distribution ratio of total free amino acids and theanine in leaves was negatively correlated with tea polyphenols and caffeine, suggesting that prolonged shading is detrimental to the formation of tencha quality.
4. Conclusion and Future Outlook
4.1 Conclusion
This study comprehensively investigated the effects of different shading conditions (shading rate, coverage height, and shading duration) on the quality components of tencha from four tea varieties (Zhenong 113, Yabukita, Xiangshanzao 1, and Jinxuan). We also examined the distribution of quality components in different tissues of tea shoots under shading. The key findings are summarized as follows:
1.Impact of Shading Duration: OPLS-DA analysis revealed that the early stages of shading (days 4 to 12) had a more significant impact on tencha quality components, with changes becoming less pronounced after 12 days. This suggests that the initial period of shading is critical for shaping the biochemical profile of tencha.
2.Effect of Shading Rate: Compared to unshaded controls, all four shading rates (S50%, S65%, S80%, S95%) consistently increased chlorophyll, free amino acid, and theanine content in tencha across all four tea varieties. Conversely, tea polyphenol content decreased under all shading rates. Caffeine content showed varied responses: it decreased under 50% shading for Zhenong 113, Xiangshanzao 1, and Jinxuan, but increased under higher shading rates (S65%, S80%, S95%) for these varieties, and increased across all shading rates for Yabukita. This highlights the differential physiological responses of various tea components and varieties to light reduction.
3.Effect of Coverage Height: A coverage height of 60 cm consistently resulted in superior tencha quality, characterized by higher chlorophyll, free amino acid, and theanine content, and lower tea polyphenol and caffeine content. This indicates that optimal light diffusion and microclimatic conditions are achieved at this height, promoting the accumulation of desirable quality components.
4.Quality Indices: Shading generally improved the quality indices of tencha. The phenol-to-amino acid ratio decreased under all shading rates, indicating a more balanced flavor profile. The catechin quality index increased, reflecting improved tenderness and overall quality. The catechin bitterness index decreased under S50%, S65%, and S80% shading, suggesting a reduction in astringency, while it increased at S95% shading, indicating that excessive shading can lead to undesirable bitterness.
5.Hundred-Bud Weight: Shading consistently led to a decrease in hundred-bud weight across all four tea varieties and shading rates. This suggests a trade-off between quality enhancement and yield, where improved quality comes at the expense of shoot biomass.
6.Optimal Shading Conditions: Based on the comprehensive analysis of chlorophyll content, tencha quality components, and hundred-bud weight, the optimal shading conditions for producing Grade 1 matcha tencha were determined to be an 80% shading rate, 60 cm coverage height, and 12 days of shading. These conditions strike a balance between enhancing desirable flavor compounds and maintaining acceptable yield.
7.Distribution in Tea Shoot Tissues: Shading significantly altered the distribution of quality components within different tissues of tea shoots. In the early stages of shading, free amino acids and theanine accumulated more in leaves, while in later stages, their proportion decreased in leaves and increased in stems. Conversely, tea polyphenols and caffeine showed an increased proportion in leaves and a decreased proportion in stems under prolonged shading. This dynamic redistribution suggests complex metabolic adjustments in response to light deprivation, with implications for the final quality of processed tencha.
4.2 Future Outlook
While this study provides valuable insights into optimizing shading practices for tencha production, several avenues for future research warrant exploration:
1.Molecular Mechanisms: Further investigation into the molecular mechanisms underlying the observed changes in quality components under shading is crucial. This could involve transcriptomic, proteomic, and metabolomic analyses to identify key genes, proteins, and metabolic pathways involved in the biosynthesis and accumulation of desirable compounds (e.g., theanine, chlorophyll) and the degradation of undesirable ones (e.g., certain catechins).
2.Long-term Effects: This study focused on a single shading cycle. Research on the long-term effects of repeated shading on tea plant health, soil fertility, and sustained quality improvement would provide a more holistic understanding of sustainable matcha production.
3.Environmental Interactions: The interaction between shading and other environmental factors, such as temperature fluctuations, water availability, and nutrient management, needs further elucidation. Developing integrated cultivation strategies that consider these interactions could lead to more robust and adaptable production systems.
4.Sensory Evaluation and Consumer Preference: While biochemical analyses provide objective measures of quality, correlating these findings with sensory evaluations and consumer preferences is essential. This would help fine-tune shading practices to meet specific market demands and taste profiles.
5.Cultivar-Specific Optimization: Although this study included four tea varieties, further research on a wider range of cultivars, including newly developed ones, would allow for more precise cultivar-specific recommendations for shading, maximizing the genetic potential of each variety.
6.Automated Shading Systems: The development and implementation of automated shading systems that can dynamically adjust shading levels based on real-time environmental data and tea plant physiological responses could revolutionize tencha production, ensuring optimal quality and resource efficiency.
By addressing these research gaps, the matcha industry can continue to advance, producing higher quality products more sustainably and efficiently, thereby meeting the growing global demand for this unique and beneficial tea.