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Foliar application of a mixture of putrescine, melatonin, proline, and potassium fulvic acid alleviates high temperature stress of cucumber plants grown in the greenhouse

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  • Putrescine (Put), melatonin (MT), proline (Pro), and potassium fulvic acid (MFA) are widely used as plant growth regulators to enhance stress tolerance. However, the roles of their mixtures in response to stress are largely unknown. Here, we mixed Put with MT, Pro, and MFA (hereafter referred to as Put mixture) with different concentrations and foliar sprayed at different growth stages (seedling, flowering, and fruiting stage) of cucumber (Cucumis sativus L.) to investigate their roles on plant growth, fruit yield, and quality under high temperature stress. The foliar application of the Put mixture promoted cucumber growth, increased chlorophyll and Pro contents and net photosynthesis rate, and reduced the values of relative electrolyte leakage, H2O2 and malondialdehyde contents of cucumber leaves, indicating that treatment with Put mixture reduced the oxidative stress caused by high temperature. Furthermore, Put mixture-treated cucumber plants had lower fruit deformity rate and higher fruit yield compared with control. The contents of vitamin C and soluble solids of cucumber fruit significantly increased and the contents of tannin and organic acid decreased. The most profound effects were found in the plants treated with 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro and 0.3 g L−1 MFA every 7 d, three times at the seedling stage, indicating that cucumber seedlings treated with the mixture of Put, MT, Pro, and MFA significantly alleviated the negative effects of high temperature stress.
  • 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 Fig. S1 Temperature records registered in the greenhouse where the experiment was performed.
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  • Cite this article

    Wang Y, Liu H, Lin W, Jahan MS, Wang J, et al. 2022. Foliar application of a mixture of putrescine, melatonin, proline, and potassium fulvic acid alleviates high temperature stress of cucumber plants grown in the greenhouse. Technology in Horticulture 2:6 doi: 10.48130/TIH-2022-0006
    Wang Y, Liu H, Lin W, Jahan MS, Wang J, et al. 2022. Foliar application of a mixture of putrescine, melatonin, proline, and potassium fulvic acid alleviates high temperature stress of cucumber plants grown in the greenhouse. Technology in Horticulture 2:6 doi: 10.48130/TIH-2022-0006

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Foliar application of a mixture of putrescine, melatonin, proline, and potassium fulvic acid alleviates high temperature stress of cucumber plants grown in the greenhouse

Technology in Horticulture  2 Article number: 6  (2022)  |  Cite this article

Abstract: Putrescine (Put), melatonin (MT), proline (Pro), and potassium fulvic acid (MFA) are widely used as plant growth regulators to enhance stress tolerance. However, the roles of their mixtures in response to stress are largely unknown. Here, we mixed Put with MT, Pro, and MFA (hereafter referred to as Put mixture) with different concentrations and foliar sprayed at different growth stages (seedling, flowering, and fruiting stage) of cucumber (Cucumis sativus L.) to investigate their roles on plant growth, fruit yield, and quality under high temperature stress. The foliar application of the Put mixture promoted cucumber growth, increased chlorophyll and Pro contents and net photosynthesis rate, and reduced the values of relative electrolyte leakage, H2O2 and malondialdehyde contents of cucumber leaves, indicating that treatment with Put mixture reduced the oxidative stress caused by high temperature. Furthermore, Put mixture-treated cucumber plants had lower fruit deformity rate and higher fruit yield compared with control. The contents of vitamin C and soluble solids of cucumber fruit significantly increased and the contents of tannin and organic acid decreased. The most profound effects were found in the plants treated with 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro and 0.3 g L−1 MFA every 7 d, three times at the seedling stage, indicating that cucumber seedlings treated with the mixture of Put, MT, Pro, and MFA significantly alleviated the negative effects of high temperature stress.

    • Cucumber (Cucumis sativus L.) is one of the most important economic crops in China, and its protected cultivation area and yield are increasing year by year[1]. However, high temperature in late spring, summer, and autumn has become one of the main factors restricting protected cucumber production in the middle and lower reaches of the Yangtze River of China. High temperature stress leads to the decline in photosynthetic efficiency by affecting photosynthetic electron transport and related enzyme activities[2,3]. It is found that the contents of soluble protein and proline (Pro) in cucumber seedlings with different high temperature tolerance varieties increase with the enhancement of high temperature tolerance at 28, 38, and 42 °C[4]. Furthermore, the content of reactive oxygen species (ROS) and malondialdehyde (MDA) significantly increases under high temperature, which can damage cells and destroy the stability of the cell membrane[5]. Importantly, high temperature stress will damage the fruit and lead to fruit drop during the fruiting stage[6,7]. Therefore, it is imperative to alleviate the impact of high temperature on plants. Several studies have shown that exogenous spraying of growth regulators on plants can regulate their growth and improve their stress resistance.

      Polyamines (PAs) are aliphatic nitrogenous compounds with small molecular weight. In the physiological pH environment, they are generally positively charged to form polycations, and have high physiological activity[8]. Furthermore, they can covalently combine with other substances to form more stable compounds, which are not easily oxidized. PAs participate in physiological, biochemical, and molecular processes, such as resistance reaction and morphogenesis[9]. Putrescine (Put) is the core substance for the synthesis of other PAs. Exogenous application of Put increases plant stress tolerance through maintaining higher chlorophyll content and photosynthesis rate, enhancing antioxidant activity to scavenge excessive ROS, and inducing the expression of genes involved in stress response[911]. Put significantly increases the content of photosynthetic pigment and promotes the growth of cucumber seedlings under salt stress[12]. Short-term high temperature treatment increases the content of PAs, but long-term high temperature treatment inhibits the synthesis of endogenous PAs[13]. Exogenous application of Put increases high temperature stress tolerance through regulation of NO synthesis[14]. Furthermore, the foliar application of Put before a short-term high temperature stress improves the fruit quality of melon[15]. The role of Put in high temperature stress has been gradually revealed, but its functions in cucumber under high temperature stress are largely unknown.

      Melatonin (MT) is used as a biological regulator to enhance plant stress resistance through increasing chlorophyll content, accelerating photosynthetic carbon assimilation, activating ROS scavenging system, and delaying leaf senescence[1618]. High temperature stress induces the accumulation of MT, which regulates the activities of various downstream enzymes through a series of signal transductions to control the synthesis and decomposition of substances, resulting in improved tolerance of plants to high temperature stress[1923]. Our previous studies showed that exogenous foliar application of 100 µmol L−1 MT significantly enhances the high temperature tolerance of tomato[10,21,22,24].

      Pro is an effective osmoregulation substance, which provides sufficient free water and active substances for physiological and biochemical reactions by protecting the functional structure of biological macromolecules, resulting in improving the adaptability of plants to stress[25,26]. Pro also reduces the damage to the cell membrane caused by stress through maintaining the integrity of the membrane structure[27]. In crops, vegetables, flowers, and other plants, the content of Pro is found to reflect the stress resistance of plants to some extent[28,29]. Varieties with strong resistance to stress often accumulate more Pro[28,29]. Furthermore, the foliar application of 100 mg L-1 Pro inhibits the accumulation of H2O2 and MDA, increases water use efficiency and fruit total soluble solids in tomato under high temperature stress[30].

      Potassium fulvic acid (MFA) is a kind of fulvic acid fertilizer, in which fulvic acid accounts for more than 50%. It has the characteristics of low molecular weight, easy biological absorption and utilization, strong physiological activity, and easy solubility in water[31]. It can increase the content, absorption, and utilization rate of potassium fertilizer, improve crop yield and quality, and enhance crop resistance to environmental stresses[32,33]. Foliar application of MFA significantly promotes the growth and development of heading lettuce[34]. At present, the role of MFA on plants is mainly investigated under drought stress. MFA reduces stomatal opening, promotes root development, and increases chlorophyll content and antioxidant enzyme activity, resulting in enhanced plant drought stress resistance[3537]. Although the sole critical roles of Put, MT, Pro, and MFA have been identified in different plants, their combined functions in alleviation of high temperature stress are unclear. Here, the mixture of Put, MT, Pro, and MFA (hereafter referred to as Put mixture) with different concentrations was sprayed at different growth stages (seedling, flowering, and fruiting stage) of cucumber to investigate their roles on growth, fruit yield, and quality under high temperature stress. The results showed that the foliar application of the Put mixture promoted cucumber growth, and increased yield and quality. These effects were the most profound in the plants treated with the mixture of 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA every 7 d, three times at the seedling stage. Therefore, our results suggested that the foliar application of the Put mixture alleviated the damage caused by high temperature stress to cucumber.

    • As shown in Fig. 1, the foliar application of the Put mixture promoted the growth of cucumber plants under high temperature stress, especially in the treatment applied during the seedling stage. The plant height of S-1, S-2, and S-3 treatment at the seedling stage was significantly higher than that of the control (CK), with an increase of 21.4%, 16.9%, and 13.2%, respectively (Fig. 1a). The plant height of Fl-1, Fl-2 and Fl-6 treatment during the flowering stage was significantly higher than that observed in CK (Fig. 1a). However, only the plant height of Fr-5 treatment significantly increased by 8.3% compared with CK at the fruiting stage (Fig. 1a). The stem diameter of S-1, Fl-2, and Fr-6 was 17.1%, 16.4%, and 13.8%, respectively, higher than that in CK (Fig. 1b). Furthermore, the fresh and dry weight of cucumber plants at the seedling stage treatments increased significantly compared with CK (Fig. 1c & d).

      Figure 1. 

      Effects of the mixture of putrescine (Put), melatonin (MT), proline (Pro), and potassium fulvic acid (MFA) on the growth of cucumber plants under high temperature stress. (a) Plant height. (b) Stem diameter. (c) Fresh weight. (d) Dry weight. CK, cucumber plants without treatment with the mixture of 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA (Put mixture, original concentration). S, Fl, and Fr indicated cucumber plants-treated with the Put mixture at seedling stage, flowering stage, and fruiting stage, respectively. 1, 2, and 3 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 7 d, 3 times, respectively. 4, 5, and 6 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 14 d, 3 times, respectively. Data represent as the mean ± SD (n = 3). Different letters indicate significant differences according to Tukey’s test at P ≤ 0.05.

    • To investigate the role of the Put mixture on the cell membrane integrity of cucumber leaves, we analyzed the level of relative electrolyte leakage and MDA content of cucumber leaves after planting for 40 d. The level of relative electrolyte leakage in the Put mixture-treated plants was lower than that in CK (Fig. 2a). The level of relative electrolyte leakage in the treatment at flowering and fruiting stage every 7 d was significantly lower than those in every 14 d treatments (Fig. 2a). In addition, MDA content in all of the Put mixture-treated plants was lower than that in CK (Fig. 2b). The content of MDA in S-1 treatment was the lowest, which was 34.6% lower than that in CK (Fig. 2b). As shown in Fig. 2c, the content of Pro in cucumber leaves at the same treatment stage increased with the increase of spraying concentration. The content of Pro in S-1, Fl-1, Fl-2, and Fr-1 treatments increased by 29.1%, 22.8%, 18.2%, and 16.7%, respectively, compared with CK (Fig. 2c). There was no significant difference in the content of H2O2 between CK and Fl-6 and Fr-3 treatments, but the content of H2O2 in other treatments was significantly lower than that in CK (Fig. 2d).

      Figure 2. 

      Effects of the mixture of putrescine (Put), melatonin (MT), proline (Pro), and potassium fulvic acid (MFA) on relative electrolyte leakage, malondialdehyde, Pro, and H2O2 content of cucumber plants under high temperature stress. (a) Relative electrolyte leakage. (b) Malondialdehyde content. (c) Pro content. (d) H2O2 content. CK, cucumber plants without treatment with the mixture of 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA (Put mixture, original concentration). S, Fl, and Fr indicated cucumber plants-treated with the Put mixture at seedling stage, flowering stage, and fruiting stage, respectively. 1, 2, and 3 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 7 d, 3 times, respectively. 4, 5, and 6 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 14 d, 3 times, respectively. Data represent as the mean ± SD (n = 3). Different letters indicate significant differences according to Tukey’s test at P ≤ 0.05. FW, fresh weight.

    • As shown in Fig. 3, spraying different concentrations of Put mixture increased the total chlorophyll content of cucumber leaves in comparison to CK. Chlorophyll content in S-1 treatment was the highest, which was 28.2% higher than that in CK (Fig. 3).

      Figure 3. 

      Effects of the mixture of putrescine (Put), melatonin (MT), proline (Pro), and potassium fulvic acid (MFA) on chlorophyll content of cucumber leaves under high temperature stress. CK, cucumber plants without treatment with the mixture of 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA (Put mixture, original concentration). S, Fl, and Fr indicated cucumber plants-treated with the Put mixture at seedling stage, flowering stage, and fruiting stage, respectively. 1, 2, and 3 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 7 d, 3 times, respectively. 4, 5, and 6 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 14 d, 3 times, respectively. Data represent as the mean ± SD (n = 3). Different letters indicate significant differences according to Tukey’s test at P ≤ 0.05. FW, fresh weight.

      The net photosynthetic rate (Pn) of cucumber plants in the treatments at the seedling stage significantly increased compared with CK, and the effect was positively related to the spraying concentration, among which the plants of S-1 treatment had the highest Pn, and increased by 1.03-fold (Fig. 4a). Similarly, the Pn in the treatments at the flowering stage also decreased with the decrease of spraying concentration, and the effect of spraying mixture every 7 d was better than those in every 14 d at the same concentration (Fig. 4a). Except for Fr-6 treatment, the foliar application of the Put mixture significantly increased the transpiration rate (Tr) (Fig. 4b). The intercellular CO2 concentration (Ci) in the plants of S-1 and Fr-1 treatment increased by 28.6% and 32.6%, respectively, compared with CK (Fig. 4c). Except for Fr-5 and Fr-6 treatment, treatment with Put mixture significantly increased the value of stomatal conductance (Gs) in comparison to CK, especially in S-1 treatment, which was 1.85-fold higher than that in CK (Fig. 4d).

      Figure 4. 

      Effects of the mixture of putrescine (Put), melatonin (MT), proline (Pro), and potassium fulvic acid (MFA) on gas exchange parameters of cucumber under high temperature stress. (a) Net photosynthetic rate (Pn). (b) Transpiration rate (Tr). (c) Intercellular CO2 concentration (Ci). (d) Stomatal conductance (Gs). CK, cucumber plants without treatment with the mixture of 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA (Put mixture, original concentration). S, Fl, and Fr indicated cucumber plants-treated with the Put mixture at seedling stage, flowering stage, and fruiting stage, respectively. 1, 2, and 3 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 7 d, 3 times, respectively. 4, 5, and 6 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 14 d, 3 times, respectively. Data represent as the mean ± SD (n = 3). Different letters indicate significant differences according to Tukey’s test at P ≤ 0.05.

    • High temperature stress affects cucumber fruit and causes fruit deformity. Spraying Put mixture significantly reduced deformity rate (Fig. 5a). The deformity rate of cucumber fruit in CK was 16.2%, while it was only 6.3% in S-1 treatment, which decreased by 61.1% (Fig. 5a). Furthermore, the foliar application of the Put mixture also increased single fruit weight and fruit yield per plant, and the effect of S-1 treatment was the best, which increased by 38.1% and 18.1%, respectively, in comparison to CK (Fig. 5b & c).

      Figure 5. 

      Effects of the mixture of putrescine (Put), melatonin (MT), proline (Pro), and potassium fulvic acid (MFA) on fruit deformity rate and yield of cucumber. (a) Abnormal fruit ratio. (b) Single fruit weight. (c) Fruit yield per plant. CK, cucumber plants without treatment with the mixture of 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA (Put mixture, original concentration). S, Fl, and Fr indicated cucumber plants-treated with the Put mixture at seedling stage, flowering stage, and fruiting stage, respectively. 1, 2, and 3 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 7 d, 3 times, respectively. 4, 5, and 6 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 14 d, 3 times, respectively. Data represent as the mean ± SD (n = 3). Different letters indicate significant differences according to Tukey’s test at P ≤ 0.05.

      Tannin content of the fruit treated with Put mixture was significantly reduced compared with that in CK (Fig. 6a). At the seedling and flowering stage, tannin content decreased with the increase in spray concentration, and the effect of S-1 treatment was the most profound, which was reduced by 20.0% (Fig. 6a). Except for Fr-3 treatment, the content of organic acid decreased significantly compared to CK (Fig. 6b). The content of organic acid in S-1 treatment was the lowest, which decreased by 34.2% (Fig. 6b). During the fruiting stage, with the increase concentration of spray, the content of organic acid in fruit gradually decreased (Fig. 6b). Compared with CK, all treatments significantly increased the content of vitamin C in cucumber fruit (Fig. 6c). The contents of soluble solids in S-1, Fl-1, Fl-2, Fr-4, and Fr-5 treatments were significantly higher than that in CK (Fig. 6d).

      Figure 6. 

      Effects of the mixture of putrescine (Put), melatonin (MT), proline (Pro), and potassium fulvic acid (MFA) on fruit quality of cucumber. (a) Tannin content. (b) Organic acid content. (c) Vitamin C content. (d) Soluble solids content. CK, cucumber plants without treatment with the mixture of 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA (Put mixture, original concentration). S, Fl, and Fr indicated cucumber plants-treated with the Put mixture at seedling stage, flowering stage, and fruiting stage, respectively. 1, 2, and 3 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 7 d, 3 times, respectively. 4, 5, and 6 indicated cucumber plants-treated with the original, diluted five times, and diluted ten times concentration of the Put mixture every 14 d, 3 times, respectively. Data represent as the mean ± SD (n = 3). Different letters indicate significant differences according to Tukey’s test at P ≤ 0.05.

    • High temperature negatively affects plant growth and fruit quality, resulting in leaf wilting, and fruit deformity[38,39]. It has been demonstrated that the foliar application of a moderate concentration of plant growth regulator can ameliorate high temperature stress[2]. In agreement with previous studies, cucumber plants treated with the Put mixture significantly promoted growth under high temperature stress (Fig. 1). Among them, the effect of S-1 treatment was the most obvious on plant height, stem diameter, fresh and dry weight (Fig. 1). In addition, high temperature leads to plant water deficit and oxidative stress, which inhibits plant growth and reduces plant chlorophyll content[40]. The results of this experiment showed that the accumulation of cucumber seedling biomass was inhibited, and the chlorophyll content of CK was lower than that of the Put mixture treatment group under high temperature stress (Figs 1 & 3). Spraying the Put mixture on the leaves alleviated the inhibition of high temperature stress on the growth of cucumber plants.

      High temperature often leads to the accumulation of ROS in plants. ROS peroxide with unsaturated fatty acids on the cell membrane and produce a large amount of MDA. MDA further aggravates the oxidation reaction of cell biofilm and causes the destruction of the biofilm structure[41]. Therefore, the content of MDA in plant cells can indirectly represent the degree of oxidative stress. This study showed that treatment with the Put mixture reduced the levels of electrolyte leakage of cucumber leaves, decreased the contents of H2O2 and MDA (Fig. 2), indicating that application of Put mixture alleviated high temperature stress-induced oxidative stress. Similarly, the foliar application of Put removes free radicals and ROS, and reduces the degree of membrane lipid peroxidation under environmental stresses[9, 10]. In addition, Put enhances the scavenging capacity of ROS in chloroplasts by regulating the coordination of antioxidant enzymes and antioxidants in chloroplasts under environmental stress, alleviating the damage of environmental stress to chloroplast structure and function[4244]. Furthermore, MT is an antioxidant that can scavenge oxygen free radicals and repair its own oxidation products[21,45]. In addition to its direct reaction with ROS, it can enhance the resistance of plants to stress by improving the efficiency of the antioxidant system in plants[21,24,46,47]. Treatment with MFA also improves antioxidant enzyme activity and antioxidant content of plant seedlings under environmental stress to maintain the balance of ROS production and scavenging[48]. Furthermore, Pro maintains the osmotic balance between protoplast and environment through osmotic regulation, reduces water loss, and protects cell membrane structure[49]. This study showed that the foliar application of the Put mixture decreased H2O2 and MDA contents in cucumber leaves (Fig. 2). Therefore, treatment with the Put mixture reduced membrane damage, maintained osmotic balance, reduced oxidative stress, and improved plant high temperature resistance.

      Photosynthesis, one of the most important physiological processes in plants, is very sensitive to temperature changes. High temperature stress affects the activity of chlorophyll synthesis enzymes, reduces the content of photosynthetic pigment[50,51], resulting in a reduction of Pn. Studies have shown that single exogenous spraying of Put, MT, Pro, or MFA alleviates the decline of photosynthesis caused by environmental stress through maintaining the structural stability of chloroplasts, scavenging excessive ROS, inhibiting photosynthesis pigment degradation, and increasing the maximum quantum yield of photosystem II[16,27,29,43]. Similarly, this study showed that spraying Put mixture at the seedling stage significantly improved chlorophyll content, Pn, and Tr of plants under high temperature stress, alleviated the reduction of Gs caused by high temperature stress (Figs 3 & 4). The decrease of Pn under high temperature treatment was accompanied by the decrease of Gs and Ci (Fig. 4), indicating that the inhibition of stomatal factors might be the dominant reason for Pn decrease. However, the foliar application of the Put mixture alleviated the decrease of photosynthesis caused by stomatal restriction.

      High temperature stress seriously affects cucumber fruit, causing fruit deformity, and reducing fruit yield and quality[38,39]. The foliar application of the Put mixture significantly decreased the rate of deformity, improved fruit yield, the contents of vitamin C and soluble solids in cucumber fruit, and decreased the contents of tannins and organic acids (Figs 5 & 6).

    • The foliar application of the Put mixture promoted plant growth, reduced the level of relative electrolyte leakage and the content of H2O2 and MDA, increased the content of Pro and photosynthesis of cucumber plants. Furthermore, the fruit yield and content of vitamin C and soluble solids increased in Put mixture treated plants, but the content of tannins and organic acids decreased. Therefore, spraying Put mixture on the leaves, especially at the seedling stage with 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA every 7 d for three times, alleviated the inhibition of high temperature stress on the growth of cucumber plants, improved cucumber plant adaptability to high temperature stress, and increased the yield and quality.

    • Cucumber (Cucumis sativus L. cv Jinchun No. 4) was used as experimental material. Uniform seeds were sterilized with 10% NaClO for 10 min, followed by washing 5 times with deionized water and soaked in deionized water for 4 h, then the seeds were incubated for germination on moistened filter paper in an incubator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., Shanghai, China), which was maintained at 28 °C. The germinated seeds were sown in 32-well plastic trays filled with seedling substrates (Jiangsu Xingnong Substrate Technology Co., Ltd., China) and grown in the greenhouse of Baima Teaching and Research Base of Nanjing Agricultural University. The temperature in the greenhouse during the day was controlled at 25−28 °C, the temperature at night was 18−20°C, and the relative humidity was maintained at 75%−80%. When the fourth leaves were fully expanded, the seedlings were selected and planted in coconut coir substrates (Van der Knaap Group of Companies, Wateringen, Netherlands) on Apr. 17, 2021 in the same greenhouse. As shown in Supplemental Fig. S1, the maximum temperature was over 30 °C in the most of the cultivation period, indicating that cucumber plants suffered from high temperature stress.

    • Cucumber seedlings were randomly divided into 16 groups to treat with or without Put mixture, and each group contained 15 plants as one treatment. As shown in Table 1, cucumber plants without treatment with the mixture of 8 mmol L−1 Put, 50 µmol L−1 MT, 1.5 mmol L−1 Pro, and 0.3 g L−1 MFA (Put mixture, original concentration) were used as CK. S-1, S-2, and S-3 indicated cucumber plants treated with the original, diluted five times, and diluted ten times concentration of Put mixture at seedling stage every 7 d, 3 times, respectively. Fl-1, Fl-2, and Fl-3 indicated cucumber plants treated with the original, diluted five times, and diluted ten times concentration of Put mixture at flowering stage every 7 d, 3 times, respectively. Fl-4, Fl-5, and Fl-6 indicated cucumber plants treated with the original, diluted five times, and diluted ten times concentration of Put mixture at flowering stage every 14 d, 3 times, respectively. Fr-1, Fr-2, and Fr-3 indicated cucumber plants treated with the original, diluted five times, and diluted ten times concentration of Put mixture at fruiting stage every 7 d, 3 times, respectively. Fr-4, Fr-5, and Fr-6 indicated cucumber plants treated with the original, diluted five times, and diluted ten times concentration of Put mixture at fruiting stage every 14 d, 3 times, respectively.

      Table 1.  Spraying concentration and stage of cucumber with putrescine mixture.

      TreatmentPutrescine
      concentration
      (mmol L−1)
      Potassium fulvic acid concentration
      (g L−1)
      Proline
      concentration
      (mmol L−1)
      Melatonin
      concentration
      (µmol L−1)
      Spraying interval (d)Spraying stage
      CK
      S-180.31.5507Seedling stage
      S-21.60.060.3107Seedling stage
      S-30.80.030.1557Seedling stage
      Fl-180.31.5507Flowering stage
      Fl-21.60.060.3107Flowering stage
      Fl-30.80.030.1557Flowering stage
      Fl-480.31.55014Flowering stage
      Fl-51.60.060.31014Flowering stage
      Fl-60.80.030.15514Flowering stage
      Fr-180.31.5507Fruiting stage
      Fr-21.60.060.3107Fruiting stage
      Fr-30.80.030.1557Fruiting stage
      Fr-480.31.55014Fruiting stage
      Fr-51.60.060.31014Fruiting stage
      Fr-60.80.030.15514Fruiting stage
    • The plant growth parameters were measured after planting for 40 d. Plant height was measured from the stem base to the growth point with a ruler, and stem diameter was measured with a vernier caliper, 1 cm below the cotyledons. Fresh samples were washed with distilled water and dried with paper, and then fresh weight was weighed with an electronic scale. The samples were dried for 15 min at 105 °C in an oven (Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China), and the temperature was reduced to 75 °C until constant weight was obtained.

    • The Pn, Gs, Ci, and Tr of the fifth fully expanded leaf below the growth point were measured with a portable photosynthesis system (LI-6400; Li-COR, Lincoln, NE, USA) from 9:00 to 11:00 am after planting for 40 d. The measurement parameters were as follows: ambient CO2 concentration was 380 µmol mol−1, the leaf chamber temperature was maintained at 25 °C, and the photosynthetic photo flux density was 800 µmol m−2 s−1.

    • Relative electrolyte leakage was detected according to the method described previously[52]. The content of MDA was determined using the thiobarbituric acid method[53].

    • Fresh leaves were washed, cut into pieces and 0.2 g samples were placed in a 15-ml centrifuge tube. Five millilitres of 3% sulfosalicylic acid solution was added into the tubes, and extracted in a boiling water bath for 10 min (shaken every 5 min). After cooling, the tubes were centrifuged at 3000 r for 10 min and 1 ml of the supernatant was placed in a new tube, adding 1 ml of distilled water, 1 ml of glacial acetic acid, and 2 ml of acidic ninhydrin solution. Subsequently, the tubes were heated in a boiling water bath for 60 min. Four millilitres of toluene was added to the tube after cooling, and vortexed for 30 s. The upper layer of toluene Pro red solution was used to measure Pro concentration at 520 nm using a UV-1800 spectrophotometer (Shanghai Unico Instrument Co., Ltd., Shanghai, China) as previously described[54].

    • Cucumber leaves (0.2 g) were ground to homogenate in 1.6 ml of 0.1% TCA on ice and centrifuged at 12000 r for 20 min. The supernatant (0.2 ml) was added to 1 ml of 1 mol L−1 KI and 0.25 ml of 0.1 mol L−1 potassium phosphate buffer (pH = 7.8) for reaction for 1 h in the dark. The concentration of H2O2 was measured at 390 nm using a spectrophotometer and calculated as previously described[55].

      For the measurement of chlorophyll content, 20 ml of 95% ethanol was added to 0.2 g of fresh leaves and sealed. The tubes were placed in the dark for 24−36 h until the leaves turn white. The chlorophyll content was measured according to the method of Arnon[56].

    • Ten plants were labeled in each treatment for yield measurement. Fruit weight was measured each time after picking, and the yield per plant was calculated after harvest. Six fresh ripe cucumbers were collected from each treatment during the fruit stage, and the fruit soluble solids, tannins, organic acid, and vitamin C content were determined to evaluate the fruit quality.

      The content of soluble solids was detected as previously described[57]. Briefly, the content of soluble solids was determined using an Abbe refractometer (WZ-108, Beijing Wancheng Beizeng Precision Instrument Co., Ltd., Beijing, China). Before determination, the refractometer was calibrated with a standard sample and then the content of soluble solids was analyzed and determined.

      For measuring tannin content, cucumber fruit (5 g) was ground and transferred to a 150-ml conical flask, shaken and extracted for 15 min. Five millilitres of 1 mol L−1 zinc acetate standard solution and 3.5 ml of concentrated ammonia were added into a 100-ml volumetric flask, shaken, and the tannin extraction was slowly transferred into the volumetric flask, keeping it warm in a 35 °C water bath for 30 min with shaking. After cooling, the volume was adjusted to 100 ml with distilled water, fully mixed and filtered. Ten millilitres of filtrate was placed in a 150-ml conical flask, and 40 ml of distilled water, 12.5 ml of NH3-NH4Cl, and 10 drops of chrome black T indicator were added and mixed well. The mixture was titrated with 0.05 mol L−1 EDTA solution until the wine red changed to pure blue. The content of tannin was calculated as previously described[58].

      To measure the content of organic acid, cucumber fruits (5 g) were ground and washed into a 250-ml conical flask with distilled water to make the volume to 100 ml. Organic acids were extracted in a constant temperature water bath at 80 °C for 30 min and shaken continuously. After cooling, the extractions were filtered and the residues were washed with distilled water 3 times; the filtrate was mixed and fixed to 100 ml with distilled water. The organic acid content was titrated with 0.1 mol L−1 sodium hydroxide standard solution as previously described[59].

      The content of vitamin C was determined according to the method previously described[60].

    • All data were statistically analyzed using the SPSS 18.0 version (SPSS Inc., Chicago, IL, USA), and the results are presented as means ± SDs (n = 3). Analysis of variance (ANOVA) was used to test for significance, and the significance between treatments were analyzed with Tukey’s honestly significant difference test (HSD) at P < 0.05.

      • This work was supported by the National Key Research and Development Program of China (2019YFD1001902).

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

      • Supplemental Fig. S1 Temperature records registered in the greenhouse where the experiment was performed.
      • Copyright: © 2022 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 (6)  Table (1) References (60)
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    Wang Y, Liu H, Lin W, Jahan MS, Wang J, et al. 2022. Foliar application of a mixture of putrescine, melatonin, proline, and potassium fulvic acid alleviates high temperature stress of cucumber plants grown in the greenhouse. Technology in Horticulture 2:6 doi: 10.48130/TIH-2022-0006
    Wang Y, Liu H, Lin W, Jahan MS, Wang J, et al. 2022. Foliar application of a mixture of putrescine, melatonin, proline, and potassium fulvic acid alleviates high temperature stress of cucumber plants grown in the greenhouse. Technology in Horticulture 2:6 doi: 10.48130/TIH-2022-0006

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