ARTICLE   Open Access    

Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system

  • # Authors contributed equally: Huan Yu, Rongrong Guo

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  • Received: 29 May 2024
    Revised: 11 July 2024
    Accepted: 31 July 2024
    Published online: 04 November 2024
    Fruit Research  4 Article number: e035 (2024)  |  Cite this article
  • In subtropical regions, the implementation of a two-crop-a-year cultivation system depends on local climatic conditions. Grape volatile compounds vary greatly with the season, due to climate differences, which lead to extreme differences between summer grape fruits (SF) and winter grape fruits (WF). In the present study, a gas chromatography-mass spectrometer (GC-MS) was used to analyze volatile compounds from 'Ruidu Kemei' grapes grown under the two-crop-a-year cultivation system. Results showed that fruits in summer and winter contained 620 volatile compounds in 15 categories. Among them, terpenoids constituted the largest group, with 122 metabolites, followed by 115 esters. This indicated that the main volatile characteristic substances of 'Ruidu Kemei' were terpenoids and esters. Higher volatile compounds in SF might be associated with higher active accumulated temperatures in the summer growing season. In addition, terpenoids, heterocyclic compounds, esters, and aromatics showed greater differences than other compounds between SF and WF. Regarding terpenoids, WF exhibited superior performance, while SF performed better in esters and aromatics. For WF, higher solar radiation intensity promoted the biosynthesis of terpenoids, which lead to more floral characteristics than SF. According to the flavor omics analysis, 'Ruidu Kemei' was primarily characterized by green, fruity, herbal, woody, sweet, floral, fresh, fatty, citrus, and earthy. In the SF, green and fruity flavors were more prominent, while floral was the dominant fruity aroma in WF. This work provides new insights into the metabolism of volatiles in summer and winter grapes and reference for the selection and promotion of varieties with suitable aromas for a two-crop-a-year cultivation system.
  • The tea plant (Camellia sinensis) was first discovered and utilized in China, where its tender leaves were processed into tea. Tea has become the second most popular beverage after water worldwide. Tea contains tea polyphenols, amino acids, vitamins, lipopolysaccharides and other nutrients, as well as potassium, calcium, magnesium, iron, fluorine and other trace elements, which have antioxidant, lipid-lowering, hypoglycaemic, anti-caries, enhance the body's immunity and other physiological regulatory functions[1]. Among them, fluorine is an element widely found in the earth's crust, mainly in the form of fluoride in the environment, is one of the essential trace elements for human beings, and is vital to the growth and development of human bones and teeth[2]. When the human body takes in an appropriate amount of fluorine, it can effectively prevent the formation of dental caries, enhance the absorption of calcium, phosphorus and other elements of the human body. Excessive intake of fluorine however will lead to chronic cumulative poisoning, damaged bone tissue, affect the function of various tissues and organs in the body, and cause harm to health[3]. Tea plants can absorb and accumulate fluoride from air, water and soil, mainly concentrated in the leaves, most of the fluoride in the leaves can be released into the tea soup and be absorbed by the human body, so the fluoride content in tea is closely related to human health[4]. Generally speaking, green tea, black tea, white tea, oolong tea and yellow tea are made from the young buds and shoots of the tea plant, and their fluorine content is low. Some dark tea made from leaves with lower maturity has lower fluorine content. However, the dark tea made from leaves with higher maturity has a high fluorine content, and therefore poses a risk of excessive fluorine[5]. Long-term drinking of dark tea with excessive fluorine content is a cause of tea-drinking fluorosis[6].

    Dark tea, with its smooth taste and digestive benefits, became an indispensable drink in the lives of the Chinese herders, who were mainly meat eaters[7]. Dark tea has also gained popularity in the wider population because of its important health-promoting effects, such as prevention of cardiovascular and cerebrovascular diseases, lowering blood pressure, and promoting weight loss and fat reduction[8]. Long-term consumption of dark tea is likely to cause fluorosis for two reasons: 1) Chinese border ethnic minorities generally use the boiling method to brew dark tea, which increases the leaching rate of fluoride[9] and leads to high fluoride levels in the human body; 2) most of the fresh tea leaves utilized to make dark tea are older and more mature leaves, which contain higher levels of fluoride than younger leaves[10]. The mature leaves of tea plants accumulate a large amount of fluoride, but can grow normally without fluoride poisoning, indicating that tea plants are able to accumulate and tolerate fluoride. Due to the problem of tea-drinking fluorosis, the excessive accumulation of fluoride in tea plants has attracted widespread attention[10]. It is important to investigate the mechanisms related to fluoride absorption, transportation, enrichment, and tolerance in tea plants to develop effective and practical management and control programs to reduce the fluoride content in tea and ensure its safety. Recent research on defluorination measures for tea has included preliminary screening of tea germplasm resources, management measures during tea plant cultivation, processing technologies, and tea brewing methods. In this review, we summarize the results of studies on how fluoride moves from the environment into tea plants and the factors affecting this process, how fluoride is transported in tea plants, the mechanisms of fluoride tolerance in tea plants, and current measures to reduce the fluoride content in tea.

    Tea plants are fluoride-accumulators with the ability to absorb and accumulate fluoride from the surrounding environment. The fluoride content in tea plants is significantly higher than that of other plants under similar growth conditions[11]. After fluoride is absorbed by the roots of tea plants, it is transferred to the above-ground parts, and is also transferred from the leaves downward (Fig. 1), but not from the above-ground parts to the below-ground parts (Fig. 2). External factors such as atmospheric, soil, and water conditions around the tea plant and internal factors can affect the fluoride content in different plant tissues.

    Figure 1.  Fluoride absorption by tea plants. Tea plants absorb fluoride from the atmosphere, soil, and water. Fluoride in the atmosphere is absorbed through the stomata or cuticle of the leaf epidermis. Fluoride ions and fluoride–aluminum complexes in soil and water are absorbed by the roots.
    Figure 2.  Fluoride transportation in tea plants. Fluoride absorbed by the leaves is transferred to the leaf tips and edges, chelates with metal ions, and the complexes are deposited in the leaves. Fluoride can be transferred from old leaves to new tips. Fluoride from soil and water can form complexes with organic acids and aluminum, which are stored in the leaves of tea plants. Fluoride from soil and water can also be individually absorbed and transported for storage in the leaves.

    Fluoride occurs in many forms in nature. Fluoride in the atmosphere mainly exists in the form of hydrogen fluoride, and that in the soil mainly exists in three forms: insoluble, exchangeable, and water-soluble fluoride. Atmospheric fluoride is mainly absorbed through the stomata of tea plant leaves or the cuticle of the epidermis, and its concentration is relatively low[12]. Insoluble fluoride and exchangeable fluoride in the soil cannot be absorbed by tea plants. Water-soluble fluoride is the main form absorbed by the root system of tea plants[13]. Several studies have detected a significant positive correlation between the fluoride content in tea and the water-soluble fluoride content in soil[14,15]. Water-soluble fluoride mainly refers to the fluoride ion (F) or fluoride complexes in soil and water solutions, including free F and fluoride complexed with ions. The water-soluble state has the strongest activity and the highest biological availability, so it is conducive to the migration of fluoride in the environment[16].

    The root system is the main organ responsible for fluoride absorption in tea plants. Water-soluble fluoride can enter the root system by passive or active absorption, depending on its concentration. Fluoride at lower concentrations in solution (0.1–10 mg/L) is mainly absorbed and enriched in the root system of tea plants via active absorption, with a kinetic curve following the Michalis–Menten kinetic model. At higher concentrations (50–100 mg/L), water-soluble fluoride is absorbed at a rate that increases with increasing concentration, and this is achieved via passive absorption[17]. Many studies have shown that the water-soluble fluoride content in soils in most tea-producing regions in China is below the threshold for passive absorption[1820], indicating that fluoride mainly enters tea plants via active absorption by the roots.

    The active uptake of fluoride by tea roots is mediated by ion pump carrier proteins and ABC transporter proteins. Ion pump carrier proteins can transport the substrate across the cell membrane against the electrochemical gradient, and use the energy of ATP hydrolysis to participate in the process of active transport of substances, mainly including proton pump H+-ATPase and calcium ion pump Ca2+-ATPase. ABC transporter proteins can transport ions or heavy metals to vesicles as chelated peptide complexes, thereby reducing toxicity to the cell and improving plant resistance to abiotic stress. Passive fluoride absorption by the root system of tea plants involves water channels and ion channels. Studies on the effects of applying external anion channel inhibitors, cation channel inhibitors, and water channel inhibitors showed that inhibition of external anion channels significantly reduced the absorption of fluoride by roots. This indicated that anion channels are an important pathway for the uptake and trans-membrane transport of fluoride in the root system of tea plants[17,21,22]. The homeostatic flow of ions through channels diffusing along a trans-membrane concentration gradient or potential gradient involves ion channel proteins[23]. Two phylogenetically independent ion channel proteins have recently been identified in tea plants: CLCF-type F/H+ reverse transporter proteins and the FEX (Fluoride export gene) family of small membrane proteins[24]. CLC proteins are involved in the transport of a variety of anions such as chlorine (Cl) and F into and out of the cell. The FEX proteins in tea plants are involved in fluoride absorption via thermodynamic passive electro-diffusion through transmembrane channels[25,26].

    After fluoride is absorbed, it is transported within the tea plant by several different pathways. These include transport after leaf absorption, transport after root absorption, and transfer inside tea plant cells. The fluoride absorbed by leaves can be transferred along the conduit to the leaf tips and edges, accumulating in the top and ipsilateral leaves, but not in the roots. Fluoride in the soil and water environment is taken up by the root system and transported to the xylem via intracellular and intercellular transport. It is then transported upwards via transpiration and eventually accumulates in the leaves[27,28]. Two transport mechanisms have been proposed for the translocation of fluoride via the xylem. One proposed mechanism is that it is translocated in the form of fluoride-aluminum complexes[29]. The other proposed mechanism is that aluminum and fluoride are transported separately and accumulate after reaching the leaves[30].

    The roots are the main organ responsible for fluoride absorption in tea plants. Therefore, most studies have focused on the transport process in roots, especially the transport of fluoride by fluoride-related transporters. It has been found that under acidic conditions, F preferentially forms complexes with Al3+ and these complexes are then absorbed by roots and transported upward in the same state[29,31]. Studies have shown that, compared with F, aluminum-fluoride complexes are more easily absorbed and transported to the new shoots by the root system. This may be related to the elimination of the separate toxic effects of F and Al3+ [29]. Fluoride can also be transported in tea plants by binding to aluminum-organic acid complexes, and then accumulate in the leaves[29]. Both tea plant fluoride transporter proteins, CsFEX1 and CsFEX2, are involved in fluoride transport, but their encoding genes can be differentially expressed among different varieties and depending on the concentration of fluoride. In one study, the expression level of CsFEX1 was consistent among different varieties, while the expression of CsFEX2 was induced under fluoride stress to increase fluoride efflux from tea plants, thereby reducing its accumulation in low-fluoride varieties[32]. In addition, the A–G subfamily of ABC transporters plays a carrier role in the transmembrane transport of F- and Cl- in tea plants[33]. It was found that the expression of the ABC transporter protein CsCL667 was up-regulated in response to fluoride treatment, and its ability to transport fluoride was enhanced, suggesting that CsCL667 functions in fluoride efflux[34]. Another study demonstrated that CsABCB9 localizes in chloroplasts and functions as a fluoride efflux transporter to reduce fluoride-induced damage in leaves and enhance chloroplast activity[35].

    Several factors affect the absorption and transport of fluoride in tea plants, including the absorbable fluoride concentration, soil pH, the presence of other ions, and the activity of ion channels[3638]. Fluoride in nature is present in the atmosphere and soil, and its concentration is the main factor affecting the fluoride content in tea plants. Under normal conditions, tea plants generally absorb fluoride from the soil through the roots, but when the hydrogen fluoride content in the atmosphere is high, tea plants can absorb it through the leaves. The fluoride content in tea plants growing in the same geographical area is similar, mainly because of the soil properties in that area. Tea plants grow in acidic environments, and fluoride in acidic soils is more easily absorbed. During the growth of tea plants, the roots secrete organic acids such as oxalic acid, citric acid, and malic acid, which promote the absorption of fluoride by the roots and its transport to above-ground parts[39]. Other ions such as Ca2+ and Mg2+ combine with F to form precipitates, resulting in lower concentrations of water-soluble fluoride in the soil, which also affects its absorption by tea plant roots[40]. Exogenously applied calcium at low concentrations can change the cell wall structure and membrane permeability in tea plant roots, ultimately leading to reduced fluoride content in tea leaves[36,41,42]. Meanwhile, Al3+ treatment can trigger Ca2+ signaling in tea plant roots, which in turn activates calmodulin and promotes fluoride absorption[43]. The H+ gradient generated by the plasma membrane H+-ATPase can also promote Ca2+ signaling in plants to regulate the transmembrane transport of ions, which affects fluoride absorption[43]. The abundance and activity of H+-ATPase in the plasma membrane of tea plant roots have been found to increase significantly under fluoride stress, and these increases result in improved absorption of fluoride, although this is also affected by the fluoride concentration and temperature[33]. Sodium fluoride was found to induce the expression of genes encoding ABC transporter proteins, resulting in the transmembrane absorption of large amounts of fluoride ions into cells[34]. ABC transporters also transport ions alone or in the form of chelated peptide complexes directly out of the cellular membrane, which improves cellular tolerance to these ions[44]. Some anions with the same valence state also affect the F content in tea plants. For example, the ion channel protein encoded by CLCF is more sensitive to F, more selective for F than for Cl, and functions to export F from the cytoplasm to protect against fluorosis[4547].

    It can be seen that reducing the absorption of fluorine by tea plants and changing the mechanism of fluorine transportation in tea plants can reduce the content of fluorine in different parts of the tea plants. From the mechanism of fluorine absorption, the most effective way is to directly change the form of soil fluorine to reduce the absorption of water-soluble fluorine by roots. On this basis, it is possible to further change the active absorption process of fluorine mediated by ion pump carrier protein and ABC transporter protein in tea roots by molecular techniques. From the perspective of fluorine transport mechanism, the toxic effect of fluorine on tea plants can be reduced mainly by promoting the function of transporter proteins to exclude fluorine from the cell or transport it to the vesicle. The comprehensive application of the above methods to limit fluorine absorption and promote fluorine transport in tea plants can limit the accumulation of fluorine in tea plants.

    The fluoride enrichment characteristics of tea plants are related to various factors, including the tea variety, the organ, and the season. Among different varieties of tea, differences in leaf structure and other physiological characteristics can lead to variations in fluoride absorption and enrichment[48]. Some studies have concluded that the variety is one of the main determinants of the fluoride content in tea leaves[10], and the differences in fluoride content among most varieties reached highly significant levels (Table 1), which can be divided into low-enriched, medium-enriched, and high-enriched germplasm[49]. Various organs of tea plants also show differences in fluoride accumulation. The fluoride content is much higher in leaves than in roots and stems, and significantly higher in old leaves than in new shoots[10,50,51]. The fluoride content can differ widely among tea plants at different developmental stages. In spring, the new leaves begin to accumulate fluoride from the environment, and the fluoride content increases as the leaves age. When the growth rate of tea leaves is slower, they absorb and accumulate more fluoride from the soil and air. When the temperature in summer and autumn is high, the growth rate of tea leaves is fast and the growth period is short, so less fluoride is absorbed and accumulated from the soil and air. This explains why the fluoride content in fresh tea leaves was higher in spring and relatively lower in summer and autumn[4]. Another study found that, in China, the fluoride content in tea leaves was higher in summer than in spring. This may have been related to the maturity level of the tea leaves at harvest and different patterns of fluoride transport[4].

    Table 1.  Fluoride content difference of different tea cultivars.
    Cultivars Province Parts Treatment Years Content (mg/kg) Ref.
    Liannandaye Sichuan Old leaves Drying at 80 °C and boiling water extraction 2006−2007 1,150.79 ± 4.86 [107]
    Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 1,296.66 ± 12.84 [110]
    Yuenandaye Old leaves Drying at 80 °C and boiling water extraction 2006−2007 1,352.89 ± 12.69 [107]
    Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 1,560.36 ± 27.10 [110]
    Chenxi NO.4 Old leaves Drying at 80 °C and boiling water extraction 2006−2007 1,865.61 ± 7.46 [107]
    Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 1,954.93 ± 10.96 [110]
    Meizhan Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,180.13 ± 14.42 [107]
    Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 1,732.2 ± 41.2 [110]
    Zhejiang Mature leaves Drying at 80 °C and nitric acid extraction 2002 2,015.48 ± 29.99 [106]
    Fudingdabai Sichuan Old leaves Drying at 80 °C and boiling water extraction 2006−2007 249.64 ± 24.3 [107]
    Guizhou One bud and
    five leaves
    Drying at 80 °C and hydrochloric acid extraction 2010 1,612.3 ± 43.1 [109]
    Fudingdabai Zhejiang Mature leaves Drying at 80 °C and nitric acid extraction 2002 137.1 ± 2.1 [106]
    Fujian Old leaves Drying at 80 °C and hydrochloric acid extraction 2010 282.1 [111]
    Hunan One bud and
    five leaves
    Steaming and boiling water extraction 2011 2,232.05 ± 85.52 [51]
    Zhuyeqi Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,750.16 ± 11.37 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 125.4 [110]
    Hunan One bud and
    five leaves
    Steaming and boiling water extraction 2011 2,330.74 ± 31.39 [51]
    Fujianshuixian Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,548.18 ± 40.97 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 103.7 ± 1.5 [110]
    Fujian Old leaves Drying at 80 °C and hydrochloric acid extraction 2010 1,150.79 ± 4.86 [111]
    Huangyeshuixian Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,424.70 ± 18.85 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 2,950.80 ± 27.73 [110]
    Qianmei 701 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,522.01 ± 45.33 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,693.09 ± 35.12 [110]
    Guizhou One bud and
    five leaves
    Drying at 80 °C and hydrochloric acid extraction 2010 389.95 ± 32.18 [109]
    Guizhou Old leaves Dry samples and hydrochloric acid extraction 2011 2,142.26 ± 16.30 [113]
    Mingshan 130 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,564.78 ± 51.22 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,036.13 ± 31.25 [110]
    Mengshan 9 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,647.31 ± 70.89 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,436.55 ± 20.21 [110]
    Yinghong NO. 2 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,669.02 ± 799.95 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,364.53 ± 51.72 [110]
    Mengshan 11 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,695.21 ± 59.89 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,582.83 ± 9.73 [110]
    Mingshan 311 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,716.22 ± 42.21 [107]
    Donghuzao 2,731.20 ± 20.78
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,107.27 ± 54.91 [110]
    Hainandaye Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,746.82 ± 39.71 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 2,961.53 ± 29.94 [110]
    Qianmei 502 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,878.23 ± 76.94 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,881.51 ± 16.48 [110]
    Guizhou One bud and
    five leaves
    Drying at 80 °C and hydrochloric acid extraction 2010 389.95 ± 30.2 [109]
    Old leaves Dry samples and hydrochloric acid extraction 2011 3,260.48 ± 32.12 [113]
    Zisun Ya'an and
    surroundings
    Drying at 80 °C and boiling water extraction 2006−2007 2,904.13 ± 35.40 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,140.80 ± 42.86 [110]
    Zhejiang Drying at 80 °C and nitric acid extraction 2002 1,742.7 ± 43.2 [106]
    Qianmei 303 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,918.13 ± 46.79 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 4,029.11 ± 81.86 [110]
    Guizhou One bud and
    five leaves
    Drying at 80 °C and hydrochloric acid extraction 2010 199.74 ± 16.6 [109]
    Old leaves Dry samples and hydrochloric acid extraction 2011 2,972.79 ± 169.82 [113]
    Anxishuixian Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 2,924.33 ± 41.39 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,454.68 ± 26.29 [110]
    Longjing 43 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 3,152.73 ± 27.70 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 4,437.79 ± 26.14 [110]
    Zhejiang Drying at 80 °C and nitric acid extraction 2002 1,377.1 ± 37.0 [106]
    Fujian Old leaves Drying at 80 °C and hydrochloric acid extraction 2010 116.2 ± 0.9 [111]
    Shuyong 307 Ya'an and
    surroundings
    Drying at 80 °C and boiling water extraction 2006−2007 3,223.55 ± 151.43 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 4,296.52 ± 54.98 [110]
    Zhenghedabaicha Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 3,295.74 ± 27.55 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 3,876.58 ± 21.09 [110]
    Zhejiang Drying at 80 °C and nitric acid extraction 2002 1,373.0 ± 41.9 [106]
    Taiwandaye Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 3,363.59 ± 456.39 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 4,739.89 ± 58.59 [110]
    Qianmei 419 Ya'an and
    surroundings
    Old leaves Drying at 80 °C and boiling water extraction 2006−2007 3,518.15 ± 76.19 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 4,541.43 ± 28.91 [110]
    Guizhou One bud and
    five leaves
    Drying at 80 °C and hydrochloric acid extraction 2010 133.70 ± 11.2 [109]
    Old leaves Dry samples and hydrochloric acid extraction 2011 2,370.47 ± 11.43 [113]
    Mengshan23 Ya'an and
    surroundings
    Drying at 80 °C and boiling water extraction 2006−2007 3,625.11 ± 86.07 [107]
    Ya'an Mature leaves Drying at 70 °C and hydrochloric acid extraction 2006 4,469.25 ± 40.85 [110]
    Sichuan group species 2,782.59 ± 146.46
    Meitantaicha Guizhou One bud and
    five leaves
    Drying at 80 °C and hydrochloric acid extraction 2010 272.93 ± 27.3 [109]
    Qianmei 101 Drying at 80 °C and hydrochloric acid extraction 2010 175.07 ± 13.5
    Dry samples and hydrochloric acid extraction 2011 3,218.33 ± 57.91 [113]
    Qianmei 601 Drying at 80 °C and hydrochloric acid extraction 2010 267.11 ± 26.31 [109]
    Dry samples and hydrochloric acid extraction 2011 2,823.02 ± 73.36 [113]
    Qianmei 809 Drying at 80 °C and hydrochloric acid extraction 2010 521.48 ± 50.32 [109]
    Dry samples and hydrochloric acid extraction 2011 2,327.91 ± 83.17 [113]
    Qianmei 308 Drying at 80 °C and hydrochloric acid extraction 2010 326.88 ± 29.3 [109]
    Dry samples and hydrochloric acid extraction 2011 3,432.86 ± 159.4 [113]
    Qianmei 415 Drying at 80 °C and hydrochloric acid extraction 2010 186.95 ± 14.2 [109]
    Dry samples and hydrochloric acid extraction 2011 5,090.83 ± 69.56 [113]
    Qiancha NO. 7 Drying at 80 °C and hydrochloric acid extraction 2010 218.81 ± 18.7 [109]
    Qianfu NO. 4 136.82 ± 11.6
    Dry samples and hydrochloric acid extraction 2011 3,066.49 ± 86.35 [113]
    Guiyucha NO. 8 Drying at 80 °C and hydrochloric acid extraction 2010 191.03 ± 18.6 [109]
    Dry samples and hydrochloric acid extraction 2011 2,882.94 ± 195.73 [113]
    Pingyangtezao Drying at 80 °C and hydrochloric acid extraction 2010 125.02 ± 12.1 [109]
    Yuanxiaolv 244.32 ± 20.5
    Nongkangzao 133.70 ± 12.3
    Mingshan 213 106.98 ± 6.74
    Mingke NO. 4 195.29 ± 16.8
    Maolv 174.73 ± 15.8
    Qianmei 412 Old leaves Dry samples and hydrochloric acid extraction 2011 3,396.92 ± 31.61 [113]
    Meitantaicha 2011 3,085.83 ± 101.9
    Zhenong 138 Zhejiang Mature leaves Drying at 80 °C and nitric acid extraction 2002 805.7 ± 6.0 [106]
    Zhenong 12 1,041.2 ± 23.3
    Shuigu 1,123.2 ± 33.5
    Hanlv 1,152.4 ± 2.4
    Zhuzhichun 1,248.2 ± 2.3
    Lvyafoshou 1,298.1 ± 2.0
    Zhenong 139 1,322.4 ± 40.7
    Shuixian 1,323.5 ± 36.1
    Biyun 1,400.9 ± 0.6
    Soubei 1,487.6 ± 29.7
    Maoxie 1,487.7 ± 31.0
    Anhui NO. 9 1,489.7 ± 40.0
    Zhenong 113 1,492.7 ± 43.5
    Yingshuang 1,509.9 ± 7.2
    Zhenong 25 1,521.2 ± 3.2
    Ribenzhong 1,543.8 ± 33.9
    Yunqi 1,549.3 ± 46.9
    Zhenong 23 1,576.7 ± 11.3
    Huangyezao 1,606.7 ± 40.5
    Jinshi 1,662.4 ± 42.4
    Pingyun 1,676.6 ± 44.6
    Zhenong 21 1,678.8 ± 49.6
    Jinfeng 1,705.2 ± 10.3
    Jiukeng 1,779.2 ± 5.0
    Juhuachun 1,993.4 ± 14.5
    Wuniuzao 2,163.2 ± 15.8
    Fujian Old leaves Drying at 80 °C and hydrochloric acid extraction 2010 98.0 ± 1.3 [111]
    Jinguanyin All leaves Drying at 80 °C and nitric acid extraction 2014−2015 536.49 ± 10.41 [112]
    Dangui 2,598.87 ± 24.12
    Old leaves Drying at 80 °C and hydrochloric acid extraction 2010 145.3 ± 0.2 [111]
    Jinmudan All leaves Drying at 80 °C and nitric acid extraction 2014−2015 1,030.21 ± 36.52 [112]
    Ruixiang Drying at 80 °C and nitric acid extraction 1,315.64 ± 21.56
    Xiapu yuanxiao Old leaves Drying at 80 °C and hydrochloric acid extraction 2010 124.6 ± 3.0 [111]
    Jinxuan 103.7 ± 3.5
    Fuandabai 103.0 ± 1.0
    Fuyun NO. 7 104.0 ± 1.1
    Fuyun NO. 6 131.6 ± 1.8
    Fudingdahao 118.0 ± 2.4
    Zaochunhao 107.7 ± 2.8
    Xiapu chunbolv 99.2 ± 1.2
    BaijiguanF1 102.1 ± 1.1
    Huanguanyin Old leaves Drying at 80 °C and hydrochloric acid extraction 99.2 ± 1.3
    Foxiang NO. 1 Yunnan One bud and
    four leaves
    Drying at 60 °C and hydrochloric acid extraction 2011 155.00 ± 6.94 [108]
    Foxiang NO. 2 214.30 ± 5. 94
    Foxiang NO. 4 219. 50 ± 7. 32
    Foxiang NO. 5 190.70 ± 4.09
    Yunkang NO. 10 121. 30 ± 5. 81
    Yunkang NO. 14 198.50 ± 8.49
    Yuncha NO. 1 135.10 ± 4.74
    Baihaozao Hunan One bud and
    five leaves
    Steaming and boiling water extraction 2011 113.2 [51]
    Bixiangzao 121.4
    Taoyuandaye 177.7
    Yulv 165.9
    Jianbohuang NO.13 168.5
    Gaoyaqi 162.8
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    In conclusion, selecting low-fluoride tea varieties and reducing the maturity of dark tea raw materials can be used as effective measures to reduce the fluoride content of dark tea.

    Plants can increase their resistance to fluoride through exocytosis and internal tolerance mechanisms. A series of reactions occurs in tea plants to reduce the toxic effects of fluoride and improve tolerance. The results of recent studies indicate that both external and internal factors are involved in fluoride resistance in tea plants. The external factors include the availability of cations that readily chelate fluoride, and the internal factors include the abundance and activity of certain transporters and the capacity of transporter and antioxidant systems (Fig. 3).

    Figure 3.  Mechanisms of fluoride tolerance in tea plants. (A) Metabolites in tea plants reduce the toxic effect of fluoride. Cell wall macromolecular components such as pectin, lignin, cellulose, hemicellulose, polysaccharide, and proteins chelate fluoride. The contents of some metabolites increase during adaptation of tea plants to fluoride. (B) Complexation of cations (Al3+, Ca2+, and Mg2+) with fluoride in tea plants. (C) Roles of the antioxidant system of tea plant in fluoride tolerance. Increases in the activity/abundance of antioxidant enzymes and in the ASA-GSH cycle reduce intracellular levels of reactive oxygen species, leading to increased fluoride tolerance. (D) Roles of transporters in fluoride tolerance. Transporter proteins transport fluoride into the vacuole, and this compartmentalization reduces damage to enzymes and organelles. The CsFEX, CsCLC, and CsABC transporters efflux fluoride from cells, thereby reducing its toxic effects. POD: Peroxidase, CAT: Catalase, SOD: Superoxide dismutase, APX: Ascorbate peroxidase, GR: Glutathione reductase, DHAR: Dehydroascorbate reductase, ABA: Abscisic acid, GA: Gibberellic acid, GHs: Glycoside hydrolases, ASA-GSH: Antioxidant system and the ascorbate-glutathione, ROS: Reactive oxygen species.

    Fluoride ions have a strong ability to form complexes with metal ions. Free F can form complexes with cations such as Al3+, Fe3+, and Ca2+, thereby altering ionic homeostasis and reducing its toxicity to tea plants[52]. Fluoride and aluminum ions form complexes and are enriched in leaves and other organs with a certain proportion. This reduces the toxicity of both Fl- and Al3+, and may be an important physiological mechanism of fluoride enrichment in tea plants[31,53]. Fluoride can also form complexes with Ca2+, so exogenous application of Ca2+ can effectively reduce the fluoride content and enhance the fluoride resistance of tea plants[42]. Fluoride combines with Mg2+, Al3+, and Ca2+ on the surface of tea leaves, and is present on the abaxial and adaxial leaf surfaces in the form of MgF2 and AlF3. The application of a small amount of MgF2 or CaF2 may be a means to reduce the toxicity of fluoride to tea seedlings[40]. Treatment with selenium was shown to reduce the fluoride content in tea, increase the accumulation of fluoride in roots, and reduce the proportion of water-soluble fluoride in tea beverages[54].

    The cell wall is widely involved in plant growth and development and in various stress responses. Fluoride ions can be chelated by the aldehyde, carboxyl, amino, and phosphate groups in polysaccharides, pectin, lignin, proteins, and other components as well as some metal ions adsorbed in the cell wall, which is usually called cell wall fixation[55,56]. Recent studies have shown that fluoride stress activates pathways related to cell wall metabolism, the stress response, signal transduction, and protein degradation, and all of these pathways may contribute to the accumulation/detoxification of fluoride in tea leaves[57,58]. After the application of exogenous fluoride, there are increase in the activities of key enzymes involved in the pectin biosynthetic pathway, in the transcript levels of their encoding genes, and in the pectin polysaccharide content, indicating that treatment with exogenous fluoride promotes pectin biosynthesis. In turn, it promotes the combination of absorbed fluoride with pectin[59]. Lignin is the main component of the plant cell wall, and its amount and the activity of its biosynthetic pathway increase in response to fluoride stress. The lignin content showed the same trend as the fluoride content in leaves, consistent with its important role in alleviating fluoride toxicity in tea plants[15]. Tea polysaccharides can also adsorb and bind fluoride. Compared with polysaccharides in other plants, tea polysaccharides have the highest fluoride content and the strongest fluoride complexation ability. The majority (80%) of fluoride in tea is bound with tea polysaccharides, and the formation of these complexes is one of the factors that enhances tea plants’ fluoride resistance[60]. Studies have found that with increasing fluoride concentration, the F content in the cell wall and its components, the metal ion content in the cell wall, and the contents in total cell wall materials, cellulose, and pectin increased with highly significant positive correlations[59,61].

    To maintain normal plant growth under adverse conditions, a series of metabolic reactions occur to activate defense responses. The formation and transformation of secondary metabolites under fluoride stress may be one way in which tea plants resist fluoride. One study found that as the fluoride concentration increased, catechin was catabolized to produce lignin, the polyphenol content decreased, and the lignin content increased. Thus, leaf lignification promotes stress resistance in tea plants[62]. Organic acids, carbohydrates, and amino acids also play important roles in the fluoride tolerance of tea plants[63]. The contents of free proline and citric acid were found to increase under fluoride stress, and the oxalic acid content in leaves first increased and then decreased as the fluoride concentration increased. These patterns of accumulation suggested that these metabolites were involved in a protective response against fluoride stress in tea plants[64,65]. Another study detected up-regulation of glycoside hydrolases (GHs), primary amine oxidase, and citrate synthetase under fluoride stress, indicating that these enzymes may be involved in the defense response[66]. Plant growth regulators such as abscisic acid and gibberellin play important roles in the response to fluoride stress and in signal transduction[67,68]. However, further studies are required to explore the roles of these and other plant growth regulators in the responses to fluoride stress and in fluoride enrichment in tea plants.

    Subcellular distribution analyses in tea plants have shown that F is concentrated in vacuoles in the cells of tea leaves, indicating that vacuoles are the main site of fluoride accumulation. Fluoride transporters are involved in the vacuole sequestration of fluoride[52]. The fluoride transporter gene FEX in tea plants is expressed in a tissue-specific manner and its product can enhance tolerance to fluoride by reducing the fluoride content in tissues[26]. Studies have shown that fluoride treatment activates the expression of genes encoding receptor-like kinases and MYB and MADS-box transcription factors, thereby regulating fluoride accumulation and fluoride tolerance in tea plants[6971]. Exploring the regulatory mechanism of fluoride transporters is the key to understanding the fluoride enrichment characteristics of tea plants, and is a new direction for molecular research.

    Under fluoride stress, tea plants can eliminate excess reactive oxygen species (ROS) within a certain concentration threshold by regulating their metabolism, thereby protecting themselves against oxidative damage. Under low-level or short-term fluoride treatments, the antioxidant system and the ascorbate-glutathione (ASA-GSH) cycle respond to fluoride stress, and there are increase in the activities of glutathione reductase, ascorbate peroxidase, dehydroascorbate reductase, peroxidase, catalase, and superoxide dismutase. Together, these enzymes remove ROS to reduce the toxicity of fluoride to tea plants. Tea plants that accumulate high levels of fluoride show a stronger ability to remove ROS[72]. Selenium treatment can also modulate fluoride-induced oxidative damage by increasing the activities of superoxide dismutase, peroxidase, and catalase, resulting in reduced malondialdehyde levels[54]. However, as the fluoride concentration increases beyond the detoxification capacity of protective enzymes and non-enzymatic antioxidants in both systems, ROS accumulate to excess levels and cause damage to tea plants[73].

    In conclusion, there are different forms of resistance to fluoride stress in the tea plants, so the adaptability of tea plants to fluorine stress can be improved by enhancing these resistance mechanisms. Appropriate agronomic measures in the tea gardens can enhance the expression level of stress resistance genes in the tea plants, and then increase the content of downstream metabolites to enhance stress resistance. At the same time, the necessary molecular technology can be used as an auxiliary means to carry out a certain aspect of the targeted improvement, and comprehensively enhance the fluorine tolerance of tea plants.

    Fluoride has dual effects on tea plant growth and metabolites related to tea quality. At low concentrations, fluoride has no obvious effect on growth and can promote the normal physiological metabolism of tea plants. High concentrations of fluoride adversely affect tea plants, inhibit growth, and exert toxic effects to reduce the yield. In addition to yield, metabolites related to tea quality are affected by fluoride. At high concentrations, fluoride reduces the synthesis of key secondary metabolites, free amino acids, polyphenols, and caffeine in tea plants, thereby reducing tea quality. In addition, leaf materials with a high fluoride content result in tea beverages with a high fluoride content (Fig. 4). In this way, excessive fluoride seriously affects the quality and safety of tea. Long-term drinking of tea with a high fluoride content can cause skeletal fluorosis, which endangers the health of consumers.

    Figure 4.  Effects of fluoride stress on tea plant growth and tea quality. Fluoride at low concentrations increases the chlorophyll content, photosynthetic rate, and quality-related metabolites in tea plants; and increases the activity of the antioxidant system and the ASA-GSH cycle to remove reactive oxygen species (ROS). Fluoride at high concentrations that exceed the tolerance limit of tea plants decreases the scavenging capacity of the antioxidant system and the ASA-GSH cycle, resulting in ROS accumulation. In addition, damage to chloroplast thylakoid membranes and decreases in chlorophyll content lead to decreases in the photosynthetic rate, stomatal conductance, and carbon assimilation capacity, resulting in decreased biomass and decreased content of quality-related metabolites, as well as leaf yellowing, leaf abscission, and even plant death. POD: Peroxidase, CAT: Catalase, SOD: Superoxide dismutase, APX: Ascorbate peroxidase, GR: Glutathione reductase, DHAR: Dehydroascorbate reductase, ASA-GSH: Antioxidant system and the ascorbate-glutathione, ROS: Reactive oxygen species.

    The growth responses of tea plants to fluoride depend on its concentration. When tea plants were treated with a low concentration of fluoride, the chlorophyll content and photosynthetic rate increased slightly, the initial respiration mode shifted from the glycolysis pathway to the pentose phosphate pathway, and respiration was enhanced. When tea plants were treated with a high concentration of fluoride, the toxic effect was mainly manifested as inhibition of metabolism and damage to cell structure. Excessive fluoride can damage the chloroplasts and cell membrane system of plants. Fluoride can also combine with Mg2+ in chlorophyll, resulting in damage to the chloroplast thylakoid membranes and significant decreases in the leaf photosynthetic rate, chlorophyll content, net photosynthetic rate, and stomatal conductance[7375]. Fluoride can also inhibit the carbon assimilation process by inhibiting the activity of rubisco, and inhibit the activity of ATP synthase on the thylakoid membrane of chloroplasts, thus hindering photophosphorylation[76]. Fluoride significantly inhibits the activities of enzymes involved in respiration, and causes the mitochondria of tea leaves to become vacuolated and degraded. In severe cases, it causes irreversible damage to mitochondria, which in turn leads to a smaller surface area for enzymes to attach to, resulting in weakened cellular respiration. Blocking of sugar metabolism in tea plants reduces respiration, and so ROS accumulate to excess levels[77,78]. Therefore, fluoride at high concentrations can lead to dwarfism, reduced growth, and leaf chlorosis[79,80]. However, few studies have explored the mechanism of fluoride’s effect on tea plant photosynthesis and respiration, and further research is needed.

    Metabolites that contribute to tea quality include polyphenols, amino acids, alkaloids, and aroma substances. The main class of polyphenols is catechins, followed by flavonoids and anthocyanins. Tea polyphenols confer astringency, an important taste quality character. In addition, the oxidation products of tea polyphenols such as theaflavins and thearubigins contribute to the infusion color of fermented teas such as black tea. Most of the flavonols of tea polyphenols are combined with a glycoside to form flavonoid glycosides, which are important contributors to the infusion color of non-fermented teas such as green tea[81]. Tea polyphenols are important antioxidants, and have tumor-inhibiting, anti-inflammatory, and antibacterial activities. Amino acids contribute to the freshness of tea infusions and are an important tea quality parameter. Amino acids can be divided into protein-source amino acids and non-protein-source amino acids. Theanine, a non-protein-source amino acid, is the main amino acid in tea. Theanine contributes to the freshness of tea infusions and offsets the astringency and bitterness of catechin and caffeine. It also has the effect of calming the nerves and promoting sleep in humans[82,83]. The main alkaloid in tea plants is caffeine, which is mainly synthesized and stored in the leaves, and is often stored in the vacuole as a complex with chlorogenic acid. Caffeine affects the quality of tea infusions, and contributes to the bitter taste. It also forms complexes with theaflavins and other substances with a refreshing taste. The quality of tea products is generally positively correlated with the caffeine content[84]. Caffeine has a stimulating effect and promotes blood circulation. Aroma substances in tea confer its unique scent and are important tea quality characters. The aroma of tea is not only an important and pleasant sensory character, but also an important factor in promoting human health.

    Previous studies have shown that fluoride treatments lead to changes in the types and abundance of metabolites such as minor polypeptides, carbohydrates, and amino acids in tea. However, depending on its concentration, fluoride can have dual effects on the physiological metabolism of tea plants. The contents of tea polyphenols, amino acids, caffeine, and water extracts were found to be enhanced by low concentrations of fluoride, but inhibited by fluoride at high concentrations[85]. Similarly, a low-concentration of fluoride was found to increase the contents of the main aroma components in tea and improve tea quality[86,87]. A high fluoride concentration can lead to significant decreases in the amounts of some tea polyphenols, total catechins, protein, theanine, and caffeine, resulting in decreased tea quality[8789]. Aroma is an important quality character of tea, and studies have shown that the amounts of aroma compounds in tea decrease as the fluoride concentration increases. Most aroma compounds show a trend of increasing and then decreasing as the fluoride concentration increases, and only alcohols show the opposite trend. Thus, a high concentration of fluoride adversely affects tea aroma and flavor quality[86,88]. In general, a high fluoride concentration decreases the abundance of important quality metabolites such as tea polyphenols, amino acids, caffeine, and aroma substances, resulting in weakened taste intensity, freshness, and aroma quality. The quality formation of tea is extremely unfavorable under high-fluoride conditions.

    In summary, fluorine stress affects the growth and metabolite content of tea plants, and then affect the safety and quality of tea products. It is necessary to find suitable measures to reduce fluorine in tea garden production, which can increase the content of tea quality metabolites while ensuring or promoting the growth and development of tea plants. On this basis, it is worth studying to further enhance the content of fluoride-tolerant metabolites of tea plants and is a worthwhile research direction.

    Although tea plants have characteristics of polyfluoride and fluoride resistance, excessive fluoride accumulation can still impair their growth and affect tea yield and quality. The long-term consumption of dark tea made from thick, mature leaves can cause tea-drinking fluorosis, and so dark tea has become an important target of tea safety risk research. On the whole, screening for low-fluoride tea varieties, improving soil management measures in tea plantations, and improving tea processing technologies will contribute to reducing the fluoride content in tea and ensuring its quality and safety (Fig. 5).

    Figure 5.  Defluoridation measures for tea plants. (A) Breeding low-fluoride varieties of tea plants. (B) Improving management measures during tea plant cultivation. (C) Improving tea processing technologies. (D) Appropriate brewing methods to prepare tea infusions.

    The fluoride accumulation characteristics vary among tea varieties and are mainly controlled by genotype. Different tea varieties have different fluoride accumulation capabilities. Selecting appropriate low-fluoride varieties is the primary measure to reduce the fluoride content in tea[10]. The differences in fluoride content among varieties are related to differences in leaf structure. Large and thin leaves with well-developed spongy tissue and large intercellular spaces are conducive to absorbing fluoride from the atmosphere, and accumulate a higher fluoride content[86]. Tea plants mainly absorb fluoride through their roots, and there is a significant correlation between root activity and fluoride content in tea plant roots. Therefore, differences in root activity among varieties may explain differences in fluoride uptake. Studies have also shown that fluoride accumulation in tea plants may be affected by the branching angle, a character that is under moderate to strong genetic control[65]. The fluoride content varies widely among different varieties of tea. Breeding and cultivating tea varieties with low fluoride content is an effective way to produce tea beverages with low fluoride concentrations.

    Tea plants can absorb fluoride from the environment. The origin of tea plants and environmental factors directly affect the accumulation of fluoride[90]. Areas where there is a low fluoride content in the soil should be selected for the cultivation of tea plants. The irrigation water should be low-fluoride water, and there should be no fluoride pollution in the air. At the same time, improving soil management measures can effectively reduce the fluoride content in tea leaves. The use of phosphorus fertilizers should be reduced during the planting process, and chemical or organic fertilizers with low fluoride contents should be used to prevent soil pollution. The application of nitrogen fertilizers at appropriate levels combined with root fertilization and foliar spraying can also affect fluoride enrichment in tea plants[91]. Calcium in different forms and concentrations can form CaF2 with fluoride or change the surface charge of soil particles, ion exchange capacity, and the stability of complexes. These changes can alter the soil pH and affect the soil exchangeable fluoride content[37,92]. Competitive adsorption and material chelation reactions in the soil can reduce the absorbable fluoride content. The addition of charcoal from bamboo and other materials can significantly reduce the water-soluble and available fluoride content in tea garden soil, as well as increasing the contents of organically bound fluoride and Fe/Mn-bound fluoride. This method can reduce the absorption and accumulation of fluoride in tea plants without adversely affecting the contents of major secondary metabolites[93,94]. Humic acid aluminum (HAA) adsorbents and low-molecular-weight organic acids can significantly reduce the fluoride content in the soil solution by chelating soluble fluoride, ultimately reducing its absorption by tea plants[95]. Soil defluoridation agents in tea gardens can decrease the soil fluoride content[96], although they do not necessarily decrease the fluoride content in fresh tea leaves.

    The fluoride content in tea mainly depends on the fluoride content in fresh tea leaves, which is affected by the tea genotype and the soil environment. The processing method has a smaller effect on the fluoride content in tea beverages. Compared to green tea, white tea, black tea, yellow tea and oolong tea, the processing of dark tea uses more mature leaves and old leaves, which affects the fluorine content of the finished tea, and there is a risk of excessive fluorine content, so it is necessary to improve dark tea processing technologies to reduce its fluoride content. One study found that appropriate blending of tea raw materials is an effective processing method to reduce the fluoride content in tea leaves. In this method, the fluoride content is measured when selecting raw materials, and fresh tea leaves with different fluoride contents are screened. Blending raw materials with high fluoride content, medium fluoride content, and low fluoride content can effectively control the final fluoride content[39]. During processing, the fluoride content in the tea leaves can be effectively reduced by washing the rolled tea leaves with room-temperature water for 1–2 min, a process that retains the effective components to the greatest extent[97]. Before the dark tea fermentation process, spraying microbial agents while stirring can effectively reduce the fluoride content and improve the quality, aroma, and taste of dark tea. In the processes of tea manufacturing and deep processing, adding different defluorination agents can effectively reduce the fluoride content in tea products without affecting the quality[94,98,99]. Studies have found that Eurotium cristatum is able to phagocytose fluoride. The fluoride content in black tea was effectively reduced using a E. cristatum strain mutagenized by ultraviolet radiation[100].

    Tea beverages are generally prepared by brewing or boiling. The leaching rate of fluoride from tea is affected by factors such as the extraction time, extraction method, and brewing time. Therefore, the brewing method can affect fluoride intake. The fluoride content in matcha depends, in part, on the brewing conditions[9]. The fluoride leaching rate of dark tea was found to be significantly correlated with the brewing method, and was significantly higher in tea prepared using the boiling method than in tea prepared using the ordinary brewing method. The rate of fluoride leaching from tea prepared using the boiling method was also higher with tap water than with pure water. A higher ratio of tea to water, increased water temperatures, and prolonged brewing time also increase the leaching rate of fluoride from tea[101-103]. Therefore, to significantly reduce the intake of fluoride by consumers and prevent fluorosis, it is recommended that tea should be prepared using pure water for brewing, an appropriate tea-water ratio and water temperature, and a shorter brewing time. Adding food-grade nutritional supplements to tea infusions can also reduce the fluoride content below the standard, and does not significantly affect the other bioactive components and quality factors[98].

    The issue of tea safety is an important concern in society. Research on the mechanisms of fluoride enrichment in tea plants and related research on fluoride control and defluorination technologies is of great significance to tea quality and safety, as well as tea genetics and breeding. Recently, some progress has been made in research on the fluoride enrichment and tolerance mechanisms of tea plants, and this has provided a theoretical basis for further research on methods to reduce the fluoride content in tea.

    (1) Although there has been some progress in research on how tea plants adsorb and transport fluoride, the specific mechanisms are still unclear. Further studies should focus on the molecular mechanisms of fluoride ion transport channel proteins (CLC, FEX, and ABC transporters), their interacting proteins, and how they are regulated to control fluoride enrichment. Studies have shown that the deposition of aluminum-fluoride complexes on the cell wall and compartmentalization in the vacuoles are important mechanisms for the detoxification of these ions in tea plants. However, it is still unclear which proteins regulate the absorption, efflux, transport, and storage of these complexes.

    (2) Tea is rich in secondary metabolites such as polyphenols, polysaccharides, and organic acid. Tea polysaccharides can combine with F to form complexes, thereby reducing the toxic effects of F ions[60]. Tea polyphenols contain multiple phenolic hydroxyl groups, have a strong acid-base buffering capacity, and can form complexes with various metal ions to generate ring-shaped chelates. The flavonol content of polyphenols was found to be significantly positively correlated with Al3+ accumulation, and their binding capacity was found to be higher than that of epigallocatechin gallate and proanthocyanidins in the root[104]. Whether polyphenols can further react with F after complexing with Al3+ is worthy of further study. It will be interesting to explore the roles and mechanisms of secondary metabolites in fluoride enrichment and tolerance in tea plants.

    (3) The degree of fluoride stress affects the growth and quality of tea[105]. How to maintain the balance between fluoride content and quality is a problem that needs to be solved in the industry. The reason why dark tea selects leaves with higher maturity is mainly because, in the same amount of leaves, the leaves with higher maturity contain more effective ingredients such as tea polyphenols, amino acids, trace elements and fiber required by the human body, and the finished dark tea with higher leaf maturity has a lower price and is more acceptable to consumers. If the fluorine content of dark tea is reduced by reducing the maturity of the raw materials, the taste will be inappropriate and difficult to be accepted by consumers. Therefore, it is necessary to reduce the fluorine content while maintaining the quality of dark tea, which needs further research .

    (4) There is still a lack of practical and effective fluoride reduction measures in the tea industry, and the development of such measures will be a key breakthrough. In terms of reducing fluoride levels in tea, the first step is to compare fluoride contents among different tea varieties and select varieties with relatively low fluoride content. The next steps are to improve the management of soil in tea plantations, improve tea processing technologies, and recommend appropriate brewing methods. However, there are still no systematic, efficient, and fully effective management measures for reducing the fluoride content in tea. Breeding new low-fluoride varieties of tea plants using traditional breeding methods is long and difficult, and has not yet been achieved using modern molecular breeding technologies. The use of a single defluoridation measure has certain limitations, so it is advisable to combine several strategies to reduce the fluoride content in tea leaves.

    The authors confirm contribution to the paper as follows: study conception and design: Zeng L; data collection: Yang J, Liu C; analysis and interpretation of results: Yang J, Liu C, Zeng L; draft manuscript preparation: Yang J, Liu C, Li J, Zhang Y, Zhu C, Gu D, Zeng L. All authors reviewed and approved the final version of the manuscript.

    The datasets generated during and/or analyzed during the current study are not publicly available due to management requests, but are available from the corresponding author on reasonable request.

    Part of the research aspects carried out by the authors are supported by the financial support from the Key-Area Research and Development Program of Guangdong Province (2023B0202120001), the Guangdong Natural Science Foundation for Distinguished Young Scholar (2023B1515020107), Tea garden standardized production and processing project of Yigong tea farm in Nyingchi City, the South China Botanical Garden, Chinese Academy of Sciences (QNXM-202302), the fund for China Agriculture Research System (CARS-19), Chinese Academy of Sciences Specific Research Assistant Funding Program (2021000064, 2023000030), the Science and Technology Project of Guangzhou (202206010185), the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2023KJ120), and the Science and Technology plan Project of Qingyuan (220804107510735).

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 The information of all detected metabolites.
    Supplemental Table S2 49 of the 620 metabolites arnotated to 20 KEGG pathways.
    Supplemental Table S3 143 differential volatile compounds amnotated to 159 sensory flavors.
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  • Cite this article

    Yu H, Guo R, Liu J, Shi X, Huang G, et al. 2024. Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system. Fruit Research 4: e035 doi: 10.48130/frures-0024-0029
    Yu H, Guo R, Liu J, Shi X, Huang G, et al. 2024. Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system. Fruit Research 4: e035 doi: 10.48130/frures-0024-0029

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Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system

Fruit Research  4 Article number: e035  (2024)  |  Cite this article

Abstract: In subtropical regions, the implementation of a two-crop-a-year cultivation system depends on local climatic conditions. Grape volatile compounds vary greatly with the season, due to climate differences, which lead to extreme differences between summer grape fruits (SF) and winter grape fruits (WF). In the present study, a gas chromatography-mass spectrometer (GC-MS) was used to analyze volatile compounds from 'Ruidu Kemei' grapes grown under the two-crop-a-year cultivation system. Results showed that fruits in summer and winter contained 620 volatile compounds in 15 categories. Among them, terpenoids constituted the largest group, with 122 metabolites, followed by 115 esters. This indicated that the main volatile characteristic substances of 'Ruidu Kemei' were terpenoids and esters. Higher volatile compounds in SF might be associated with higher active accumulated temperatures in the summer growing season. In addition, terpenoids, heterocyclic compounds, esters, and aromatics showed greater differences than other compounds between SF and WF. Regarding terpenoids, WF exhibited superior performance, while SF performed better in esters and aromatics. For WF, higher solar radiation intensity promoted the biosynthesis of terpenoids, which lead to more floral characteristics than SF. According to the flavor omics analysis, 'Ruidu Kemei' was primarily characterized by green, fruity, herbal, woody, sweet, floral, fresh, fatty, citrus, and earthy. In the SF, green and fruity flavors were more prominent, while floral was the dominant fruity aroma in WF. This work provides new insights into the metabolism of volatiles in summer and winter grapes and reference for the selection and promotion of varieties with suitable aromas for a two-crop-a-year cultivation system.

    • Grape (Vitis vinifera L.) is one of the most popular fruits in the world. Table grapes account for approximately 36% of global grape production[1]. In China, table grapes account for 80% of total grape production[2]. In light of this, it is very important to study the aroma of table grapes. The volatile compounds in grapes affect sensory evaluation, which could be the reason that consumers choose certain grapes over others[3,4]. Volatile compounds in fruits are responsible for defining their aroma and flavor. We can obtain grapes with distinct aromas and characteristics for the varying volatile combinations and concentrations[5]. Fruit volatile compounds are mainly comprised of esters, alcohols, aldehydes, ketones, lactones, terpenoids, and apocarotenoids[6]. Many factors affect volatile composition, including the genetic diversity[7,8], viticultural techniques[9,10], degree of maturity[4], climatic conditions[3,11,12], and postharvest storage conditions[4,13]. Among these factors, climate conditions (sunlight, temperature, water status, etc.) were often considered an important factor for grape volatile compounds for the same cultivar[11].

      Light is the primary climatic factor affecting volatile composition. As we all know, intensity, quality, and photoperiod are the main factors of light regulation[14]. Sunlight promotes the accumulation of terpenoids and monoterpene, which are the typical aroma components in Muscat grapes[3,12]. Modified canopy management (basal leaf removal) and exposure to appropriate proportions of blue and red light were effective strategies to improve the characteristic aroma[14,15]. Temperature is another important climate factor affecting volatile composition. Generally, excessively high temperature is deemed to have negative effects on fruit metabolism[16]. For example, high temperature in the winter season inhibited most VviCCDs expression than in summer grape berries, which was associated with norisoprenoid accumulation[3]. Temperate zones are more conducive to the formation of aroma substances[17]. Compared with grapes grown under cool conditions, the same grape variety presented a higher concentration of monoterpenes when cultivated under warm conditions[18]. The other factor that influences grape development is water availability. Proper water deficit has been proven to be available for increasing the characteristic aroma contents, especially terpenes and esters[19,20]. Therefore, improving water use efficiency can increase fruit flavor.

      Different terrains forms different aroma characteristics. The southern subtropical region of China was not a traditional viticultural area due to the sticky rainy weather and inadequate low-temperature accumulation[3,21]. With the application of grape two-crop-a-year cultivation technology, the above-mentioned problems have been conquered[22], and Guangxi (a province in southern China) has become a unique advantage viticulture area[23]. Due to the plentiful sunlight and temperature accumulation, grape berries could be harvested twice a year[3,22]. Summer grape fruits are the name of grapes harvested in the first growing season, while winter grape fruits are the name of grapes harvested in the second growing season[24]. 'Ruidu Kemei', breeding from a cross between 'Italy' and 'Muscat Louis', is a new table grape variety appropriate to two-crop-a-year cultivation[25,26]. At present, there are few reports about grape volatiles under two-crop-a-year cultivation systems. Recently, Lu et al. compared the volatile profiles of 'Riesling', 'Cabernet Sauvignon', 'Victoria', and 'Muscat Hamburg' grape berries under two-crop-a-year cultivation[3]. However, knowledge about the volatile profiles of two-crop grapes is still very rare. More work needs to be carried out to establish the aroma substance characteristics, and to provide a theoretical basis for improving aroma under two-crop-a-year cultivation systems.

      To distinguish grape volatiles under the two-crop-a-year cultivation system, the volatiles in summer and winter berries of 'Ruidu Kemei' were qualitatively and quantitatively analyzed by headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). Meanwhile, two crops' volatiles were also conducted in relation to climate factors in the present study.

    • This experiment was conducted during two growing seasons in 2022 on 3-year-old 'Ruidu Kemei' grapevines in the vineyards of the Grape and Wine Research Institute, Guangxi Academy of Agricultural Sciences, located in Nanning, Guangxi Province, China (22°36'39" N, 108°13'51" E). In this vineyard, the vines were managed on a canopy frame with a single trunk and were planted in north-south-oriented rows spaced 1.5 m (between vines) × 2.5 m (between rows). Nutrition, pest, water, and fertilizer management was carried out by uniform standards for two-crop-a-year as previously described[3].

      The key techniques of two-crop-a-year cultivation systems was described by Cheng et al.[24]. Summer grape fruits (SF) were harvested on July 15th, and winter grape fruits (WF) were harvested on December 31st.

    • Six vines for sampling were chosen based on their relatively consistent growth status. Six biological replicates were conducted in this study, and each biological replicate comprised 90 berries from six clusters of different vines, then sampled berries of each biological replicate were mixed and put into a 50 mL centrifuge tube, and immediately frozen in liquid nitrogen, and stored at −80 °C until needed.

      Temperature (°C), relative humidity (%), and solar radiation intensity (W/m2) were acquired according to Cheng et al.[24]. Growing degree days (base 10 °C) were calculated from bloom to harvest according to Bindi et al.[27].

    • Samples of each biological replicate were ground to powder in liquid nitrogen, and 500 mg powder was transferred immediately to a 20 mL head-space vial (Agilent, Palo Alto, CA, USA), containing NaCl-saturated solution, to inhibit any enzyme reaction. The vials were sealed using crimp-top caps with TFE-silicone headspace septa (Agilent). At the time of SPME analysis, each vial was placed at 60 °C for 5 min, then a 120 μm DVB/CWR/PDMS fiber (Agilent) was exposed to the headspace of the sample for 15 min at 60 °C.

    • After sampling, desorption of the VOCs from the fiber coating was carried out in the injection port of the GC apparatus (Model 8890; Agilent) at 250 °C for 5 min in the splitless mode. The identification and quantification of VOCs was carried out using an Agilent Model 8890 GC and a 7000D mass spectrometer (Agilent), equipped with a 30 m × 0.25 mm × 0.25 μm DB-5MS (5% phenyl-polymethylsiloxane) capillary column. Helium was used as the carrier gas at a linear velocity of 1.2 mL/min. The injector temperature was kept at 250 °C and the detector at 280 °C. The oven temperature was programmed from 40 °C (3.5 min), increasing at 10 °C/min to 100 °C, at 7 °C/min to 180 °C, at 25 °C/min to 280 °C, hold for 5 min. Mass spectra was recorded in electron impact (EI) ionization mode at 70 eV. The quadrupole mass detector, ion source, and transfer line temperatures were set, respectively, at 150, 230, and 280 °C. The MS with selected ion monitoring (SIM) mode was used for the identification and quantification of analytes.

    • Unsupervised PCA was performed by the statistics function prcomp within R (www.r-project.org). The data was unit variance scaled before unsupervised PCA.

    • The HCA results of samples and metabolites were presented as heatmaps with dendrograms, while Pearson correlation coefficients (PCC) between samples were calculated by the cor function in R and presented as only heatmaps. Both HCA and PCC were carried out by the R package ComplexHeatmap. For HCA, normalized signal intensities of metabolites (unit variance scaling) are visualized as a color spectrum.

    • For two-group analysis, differential metabolites were determined by VIP (VIP > 1) and absolute Log2FC (|Log2FC| ≥ 1.0). VIP values were extracted from OPLS-DA results, which also contain score plots and permutation plots, and was generated using R package MetaboAnalystR. The data was log transform (log) and mean centering before OPLS-DA. To avoid overfitting, a permutation test (200 permutations) was performed.

    • Identified metabolites were annotated using the KEGG Compound database (www.kegg.jp/kegg/compound, accessed on April 2nd, 2022), annotated metabolites were then mapped to the KEGG Pathway database (www.kegg.jpkegg/pathway.html, accessed on April 2nd, 2022). Pathways with significantly regulated metabolites mapped then fed into MSEA (metabolite sets enrichment analysis), their significance was determined by hypergeometric test's p-values.

    • Volatiles in grape berries were affected by meteorological parameters under the double cropping system[3]. Significant differences in meteorological parameters between the two crop growing seasons are shown in Table 1. The summer growing season was from 1 March to 15 July, and the winter growing season was from 1 September to 31 December. In the present study, the active accumulated temperatures for both growing seasons were greater than 3,100 °C (Table 1), meaning that the active accumulated temperatures were sufficient to guarantee normal grape maturity[28]. The active accumulated temperature, the effective accumulated temperature, and the daily average temperature for the summer growing season was higher than those of the winter growing season. However, there were 83.33 h of high temperatures over 35 °C during the summer growing season, which was less than the winter growing season (127.17 h). Moreover, the relative humidity during the summer growing season showed a higher value than the winter growing season. For the solar radiation intensity and cumulative solar radiation, the winter growing season was higher than the summer growing season.

      Table 1.  Phenology and climatic factors during the two crop-growing seasons in Nanning (China) in 2022.

      Meteorological data Summer Winter
      Phenology 1 Mar−15 Jul 15 Aug−31 Dec
      Active T (°C) 3,393.53 3,149.98
      Effective T (°C) 2,023.53 1,769.98
      Average daily temperature (°C) 24.78 21.92
      High temperature (> 35 °C) (°C) 83.33 127.17
      Relative humidity (%) 86.94 80.78
      Solar radiation Intensity (W/m2) 93.86 108.65
      Cumulative solar radiation (W/m2) 3,703,264.3 3,805,233.3
    • To figure out the difference between SF and WF, volatile metabolite analysis was applied in this study. A total of 620 metabolites in 15 categories were detected, including 122 terpenoids, 115 esters, 99 heterocyclic compounds, 60 hydrocarbons, 52 ketones, 48 alcohols, 47 aldehydes, 31 aromatics, 11 amines, 11 acids, eight phenols, seven nitrogen compounds, three halogenated hydrocarbons, two sulfur compounds, and four others (Fig. 1a, Supplementary Table S1). There was no difference between SF and WF for 12 categories (Fig. 1b). WF had more terpenoids and heterocyclic compounds than SF. Conversely, SF had more esters (Fig. 1b).

      Figure 1. 

      (a) Categorical all metabolite statistics. (b) Categorical metabolite statistics for SF & WF. (c) All metabolites for hierarchical cluster analysis (HCA). (d) The relative content of classified metabolites for SF & WF.

      For the relative metabolite contents, it was found that the metabolites were divided into two clusters, and significant differences could be observed in the substances between SF and WF. The metabolite relative contents in Cluster I were higher in SF, while WF exhibited higher relative contents in Cluster II metabolites (Fig. 1c). Phenols showed little difference between SF and WF, but SF was richer in the 14 other categories than that of WF (Fig. 1d).

      SF and WF were evidently distinguished by PCA (Fig. 2a), the explanation rate of the first five principal components reached 87.1% (Fig. 2b). The cluster dendrogram divided SF and WF into two groups, which was consistent with PCA (Fig. 2c). The results indicated that the volatile compounds differed greatly between SF and WF.

      Figure 2. 

      (a) Principal component analysis (PCA). (b) Grouped principal component analysis explanation rate plot. (c) Sample hierarchical clustering tree.

    • To better distinguish volatile compounds between summer fruits and winter fruits, metabolites with fold change ≥ 2 and fold change ≤ 0.5 were selected as significant differences. The comparison SF_vs_WF showed a total of 143 different metabolites accounted for 23.18 % of the total detected substances, including 85 up-regulated metabolites and 58 down-regulated metabolites (Fig. 3a). The metabolites with a higher number for up-regulated were terpenoids, ketone, hydrocarbons, ester, aldehyde, alcohol, halogenated hydrocarbons, acids, and others (Fig. 3b). The metabolites with a higher number for down-regulated were amine, aromatics, nitrogen compounds, phenol, and heterocyclic compounds. It's worth noting that no sulfur compounds showed a statistically significant difference between SF and WF (Fig. 3a & b). For relative content, terpenoids, heterocyclic compounds, esters, and aromatics showed greater difference than other compounds (Fig. 3c).

      Figure 3. 

      (a) Volcanic plot of differential volatile compounds. (b) Bar chart of the number of volatile compounds classified for up-regulation & down-regulation. (c) Scatter plot of differential volatile compounds. (d) Bar chart of the top 20 differential volatile compounds.

      To determine the metabolites with large differences for the SF_vs_WF comparison, a list of the top 20 substances using Log2FC was made, including 10 up-regulation substances and 10 down-regulation substances (Fig. 3d). There were obvious distinctions between the SF and WF. The top 20 substances using Log2FC contained five categories: terpenoids (8), aromatics (4), heterocyclic compounds (3), esters (3), and ketones (2). The top 10 up-regulation substances contained seven terpenoids, one aromatic, one heterocyclic compound, one ketone, while three esters, three aromatics, two heterocyclic compounds, and one ketone in the top 10 down-regulation substances. These results suggested that, for the top 20 substances using Log2FC, terpenoids were mainly up-regulated in WF, while esters and aromatics were up-regulated in SF. Specifically, the up-regulated and down-regulated substances with the largest Log2FC for SF_vs_WF comparison were [1α,4aα,8aα]-1,2,4a,5,6,8a-hexahydro-4-7-dimethyl-1-[1-methylethyl]naphthalene (terpenoid) and 3-Hexen-1-ol, acetate, (Z)-(ester).

    • Fourty-nine of the 620 metabolites were annotated to 20 KEGG pathways (Supplementary Table S2). Additionally, 13 differential volatile compounds out of 143 differential volatile compounds between SF and WF were primarily annotated and enriched in the following seven pathways: biosynthesis of secondary metabolites, metabolic pathways and sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, limonene and pinene degradation, terpenoid backbone biosynthesis, and α-Linolenic acid metabolism (Fig. 4a & b; Table 2). Among them, the top three KEGG pathway types were biosynthesis of secondary metabolites, metabolic pathways and sesquiterpenoid, and triterpenoid biosynthesis, accounting for 53.85%, 46.15%, and 38.46% of the total differential volatile compounds annotated in KEGG respectively (Fig. 4a). KEGG annotations and enrichment showed that sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, limonene and pinene degradation were the main KEGG pathways for the differential volatile compounds between SF and WF (Fig. 4b). Significantly except sesquiterpenoid and triterpenoid biosynthesis, the other six pathways were mainly down-regulated (Table 2). These 13 differential volatile compounds were nine terpenoids, three aldehydes, and one ester (Table 2). Only four terpenoids were more in WF when compared with SF, including (E)-β-Famesene, Naphthalene,1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-, (1S-cis)-, α-Farnesene, and (E)-1-Methyl-4-(6-methylhept-5-en-2-ylidene)cyclohex-1-ener (Table 2). All the remaining nine differential volatile compounds were less in WF than in SF (Table 2).

      Figure 4. 

      The classification of the KEGG enrichment pathway. (a) KEGG enrichment analysis of differential volatile compounds. (b) KEGG annotations and enrichment of differential volatile compounds for SF_vs_WF comparison.

      Table 2.  KEGG functional annotation and enrichment of differential volatile compounds between SF and WF.

      Formula Compounds KEGG_pathway Class Odor SF vs WF
      C10H18O L-α-Terpineol Metabolic pathways, Biosynthesis of secondary metabolites, Monoterpenoid biosynthesis Terpenoids Lilac, floral, terpenic Down
      C7H6O BenzAldehyde Metabolic pathways Aldehyde Sweet, bitter, almond, cherry Down
      C8H8O BenzAldehyde, 2-methyl- Metabolic pathways Aldehyde Mild floral, sweet Down
      C10H18O Bicyclo[3.1.0]hexan-2-ol,
      2-methyl-5-(1-methylethyl)-, (1α,2β,5α)-
      Metabolic pathways, Biosynthesis of secondary metabolites, Monoterpenoid biosynthesis Terpenoids Balsam Down
      C15H24 Naphthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-, (1S-cis)- Metabolic pathways, Biosynthesis of secondary metabolites, Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Thyme, herbal, woody, dry Up
      C7H6O2 2-hydroxy-BenzAldehyde Metabolic pathways Aldehyde Medical, spicy, cinmon, wintergreen, cooling Down
      C8H14O2 3-Hexen-1-ol, acetate, (Z)- Biosynthesis of secondary metabolites, α-Linolenic acid metabolism Ester Fresh, green, sweet, fruity, ba--, apple, grassy Down
      C15H24 α-Farnesene Biosynthesis of secondary metabolites, Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Citrus, herbal, lavender, bergamot, myrrh, neroli, green Up
      C15H24 (E)-1-Methyl-4-(6-methylhept-5-
      en-2-ylidene)cyclohex-1-ene
      Biosynthesis of secondary metabolites, Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Up
      C15H24O 2,6,10-Dodecatrienal,
      3,7,11-trimethyl-, (E,E)-
      Biosynthesis of secondary metabolites, Terpenoid backbone biosynthesis, Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Down
      C15H24 (E)-β-Famesene Sesquiterpenoid and triterpenoid biosynthesis Terpenoids Woody, citrus, herbal, sweet Up
      C10H16O Bicyclo[3.1.1]hept-2-ene-2-methanol, 6,6-dimethyl- Limonene and pinene degradation Terpenoids Woody, minty Down
      C10H16O 3-Oxatricyclo[4.1.1.0(2,4)]octane, 2,7,7-trimethyl- Limonene and pinene degradation Terpenoids Green Down
      − indicates no annotation of substance.
    • One hundred and fourty-three differential volatile compounds were annotated to 159 sensory flavors (Supplementary Table S3). The top 10 sensory flavors with the highest number of annotations were green (23), fruity (21), herbal (14), woody (14), sweet (13), floral (9), fresh (8), fatty (8), citrus (8), and earthy (7) (Fig. 5a), which were the most important sensory flavors for SF and WF. The top 10 differential volatile compounds with high numbers of sensory flavor features annotation were Hexanoic acid, propyl ester (Ester), 3-Hexen-1-ol,acetate,(Z)-(Ester), Butanoic acid,hexyl ester (Ester), Butanoic acid, octyl ester (Ester), Fenchone (Terpenoids), Isocyclocitral (Aldehyde), Pyrazine, 2-methyl-5-(1-methylethyl)-(Heterocyclic compound), Heptanal (Aldehyde), and Geranyl isobutyrate (Ester), which were the most important differential volatile compounds of sensory flavors for SF and WF (Fig. 5b).

      Figure 5. 

      (a) Radar map for analysis of differential metabolite sensory flavor characteristics. (b) Sankey diagram of flavor omics.

      Compared with WF, SF mainly showed green, fruity, herbal, woody, sweet, and earthy, the relevant substances were Hexanoic acid, propyl ester (Ester), 3-Hexen-1-ol,acetate,(Z)- (Ester), Butanoic acid,hexyl ester (Ester), Butanoic acid,octyl ester (Ester), Fenchone (Terpenoids), Isocyclocitral (Terpenoids), Pyrazine, 2-methyl-5-(1-methylethyl)-(Terpenoids), etc (Fig. 5b). WF mainly showed more floral, fresh, fatty, and citrus than SF, according to a higher number of up-regulated metabolites for SF_vs_WF comparison, including 2-Undecenal,E-(Aldehyde), 2-Octen-1-ol,(E)-(Alcohol), 2-Dodecenal,(E)-(Aldehyde), (E)-β-Famesene (Terpenoids), etc (Fig. 5b).

    • Meteorological data differ greatly between the two crop seasons in Guangxi (China). The active accumulated temperature, the effective accumulated temperature, the daily average temperature, and the relative humidity for the summer growing season were higher than those of the winter growing season[3,24,29], which were in line with the present research. However, there were more hours of high temperatures over 35 °C in the winter growing season than that in the summer growing season. This result is the opposite of other study findings[3,24]. Consistently with previous studies[24,29], the solar radiation intensity and cumulative solar radiation was higher in the winter growing season. According to the results of this research and literary references, meteorological data for two crop seasons varies by year.

      In the present research, 620 volatile compounds in 15 categories were detected in summer and winter fruits, including 122 terpenoids, 115 esters, 99 heterocyclic compounds, 60 hydrocarbons, 52 ketones, 48 alcohols, 47 aldehydes, 31 aromatics, 11 amines, 11 acids, eight phenols, seven nitrogen compounds, three halogenated hydrocarbons, two sulfur compounds, and four others. These results indicated that terpenoids were the main volatile characteristic substances of 'Ruidu Kemei', followed by esters. It has been confirmed that terpenoids were the characteristic aroma components of muscat flavored varieties, which was consistent with the present study[30].

      Grape cultivation in the field was greatly impacted by climate conditions. Berries were influenced greatly by their growing environment in terms of chemical composition. Due to variations in climate between the summer and winter growing seasons, the most important metabolites of grapes perform differently under a two-crop-a-year cultivation system, such as flavonoids[24,29], phenols, carotenoids[28], and volatiles[3]. The present study showed clear differences in the concentration of volatile compounds in response to meteorological data for two crop seasons, which verified the findings of previous research[3], while the compounds of volatiles mainly remained similar for the volatile compounds depending largely on the genotype of the grape cultivar rather than the growing environment[11]. However, when compared with WF, higher volatile compound concentration was observed in berries of summer, which would be caused by more hours of high temperatures over 35 °C during the winter growing season. Generally, lower temperatures were conducive to the accumulation of aromatic substances. Furthermore, this data appears to be related to the higher active accumulated temperatures in the summer growing season, which favored the grape ripening and volatile accumulation in the grape berries[10,31].

      To determine the distinction of volatile compounds between summer fruits and winter fruits, 143 significant different metabolites were selected. For number and relative content, terpenoids, heterocyclic compound, ester and aromatics showed greater differences than other compounds. In particular, for the top 20 substances using Log2FC, terpenoids (such as [1α,4aα,8aα]-1,2,4a,5,6,8a-hexahydro-4-7-dimethyl-1-[1-methylethyl]naphthalene) were mainly up-regulated in WF, while esters (such as 3-Hexen-1-ol, acetate, (Z)-) and aromatics (such as Benzene, (1-methoxypropyl)-) were up-regulated in SF, since heat and sunlight stress can reduce the aromatic content of grapes, while less solar radiation intensity favored the higher level of aromatics[9]. The most likely precursors for the esters were lipids and amino acids. Their metabolism during ripening will therefore play an important role in determining both the levels and types of esters formed[32]. It has been reported that cluster sunlight exposure in viticulture in dry-hot climates caused a notable decrease in esters, including ethyl hexanoate and hexyl acetate[15]. Sunlight was advantageous for accumulating terpenoids[3], the activation of terpene synthase genes (VvTPS54 and VvTPS56) and the synthesis of carotenoids in grapes, subsequently leading to the accumulation of terpenoids and norisoprenoids[33,34]. As reported, higher solar radiation intensity and cumulative solar radiation enhanced accumulation of terpenes[12]. Zhang et al. indicated that VvDXS2 and VvDXR were partially linked to differential terpene accumulation for different illumination conditions[35]. Sun et al. found that grape berries grown in rain shelters contain lower levels of terpenoids and norisoprenoids during development, possibly as a result of less light, inhibiting isoprenoids during development[10]. In this study, more hours of high temperatures over 35 °C in the winter growing season than that in the summer growing season, higher solar radiation intensity, and cumulative solar radiation still promoted sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, limonene, and pinene degradation (Fig. 4b). These results reconfirmed that the increased light exposure was beneficial for terpene accumulation, which could infer that the negative effect of the elevated berry temperature might be surpassed by the beneficial effect of increased synthesis of terpenes induced by light. However, Friedel et al. made an opposite judgment[33]. Thus, grape cultivars might respond differently to climate. In summary, for 'Ruidu Kemei', WF performed better in terpenoids, whereas SF displayed better in esters and aromatics. Based on previous studies of other grape varieties[3], it could be concluded that WF probably always forms higher concentrations of terpenes than SF under a two-crop-a-year cultivation system in the Guangxi region of South China, which has a typical subtropical humid monsoon climate.

      Among other qualities, aroma flavor contributes to consumers' acceptance of table grapes[30]. Table grapes' flavor was generally determined by their free volatiles since they were directly detectable and tasteable[36]. Flavors varied from different ingredients and different concentrations of volatile substances. In the present study, the different relative content of volatile substances was the reason for the different flavors of grapes in two growing seasons. Fruit aroma profile visually displayed that the most important sensory flavors for 'Ruidu Kemei' were green, fruity, herbal, woody, sweet, floral, fresh, fatty, citrus, and earthy. Green and fruity were the most critical aroma for 'Ruidu Kemei', due to most volatiles annotated. Green, fruity, herbal, woody, sweet, and earthy were more prominent in the SF, the relevant substances were Hexanoic acid, propyl ester, 3-Hexen-1-ol,acetate,(Z)-, Butanoic acid,hexyl ester, Butanoic acid, octyl ester, Fenchone, Isocyclocitral, Pyrazine, 2-methyl-5-(1-methylethyl)-, etc (Fig. 5b). Compared with SF, for WF, floral was the most prominent fruit smell, followed by fresh and fatty smell, and then citrus smell, the metabolites with the greatest contribution were 2-Undecenal,E-, 2-Octen-1-ol,(E)-, 2-Dodecenal,(E)-, (E)-β-Famesene, etc (Fig. 5b). In general, seasonal differences can be observed in the sensory properties of grape berries from the same variety[37]. Floral dominated in WF for performing better in terpenoids than SF. Due to their association with floral scents, terpenoids may attract appropriate pollinators and facilitate reproduction[37].

    • In the present research, 620 volatile compounds in 15 categories were detected in summer and winter fruits by a GC-MS/MS-based metabolomics approach. Terpenoids were the main volatile characteristic substances of 'Ruidu Kemei', followed by esters. Meteorological data for two crop seasons varied by years. The variational climatic factors in the summer and winter growing seasons were responsible for the difference in volatile metabolites between the two crops of grapes. Compared with the WF, higher active accumulated temperatures in the summer growing season contributed to higher volatile compound concentration in SF. Moreover, terpenoids, heterocyclic compounds, esters, and aromatics showed greater differences than other compounds between SF and WF. In addition, it was demonstrated that the winter cropping cycle promoted the biosynthesis of terpenoids by higher solar radiation intensity and cumulative solar radiation, which lead to more floral fruit smell than SF. On the contrary, more esters and aromatics were observed in SF in response to less solar radiation intensity, cumulative solar radiation and higher active accumulated temperatures in the summer growing season. Flavor omics analysis presented that the most important sensory flavors for 'Ruidu Kemei' were green, fruity, herbal, woody, sweet, floral, fresh, fatty, citrus, and earthy. Green and fruity were the most critical aroma for 'Ruidu Kemei', due to the most volatiles annotated. Green, fruity, herbal, woody, sweet, and earthy were more prominent in the SF. Floral was the most prominent fruit smell in WF. Clarification the characteristics of aroma substances of grape berries in two growing seasons can provide a basis for the scientific control of grape aroma, the improvement of grape quality, and the optimization of grape cultivation technology.

    • The authors confirm contribution to the paper as follows: study conception and design: Guo R, Lin L, Zhang Y; data collection: Lin L, Yu H, Liu J, Shi X; analysis and interpretation of results: Yu H, Huang G; draft manuscript preparation: Yu H; review & editing, project administration and funding acquisition: Guo R, Lin L. All authors reviewed the results and approved the final version of the manuscript.

    • All data generated or analyzed during this study are included in this published article and its supplementary information files.

      • This work was supported by grants from the Guangxi Key Research and Development Program (GuikeAB19245031), the special project for basic scientific research of Guangxi Academy of Agricultural Sciences (Guinongke2021YT127 & Guinongke2023YM111).

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Huan Yu, Rongrong Guo

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (5)  Table (2) References (37)
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    Yu H, Guo R, Liu J, Shi X, Huang G, et al. 2024. Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system. Fruit Research 4: e035 doi: 10.48130/frures-0024-0029
    Yu H, Guo R, Liu J, Shi X, Huang G, et al. 2024. Metabolome provides new insights into the volatile substances in 'Ruidu Kemei' grapes under the two-crop-a-year cultivation system. Fruit Research 4: e035 doi: 10.48130/frures-0024-0029

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