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Formation of and changes in phytohormone levels in response to stress during the manufacturing process of oolong tea (Camellia sinensis)
Postharvest Biology and Technology 157 (2019) 110974
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Formation of and changes in phytohormone levels in response to stress during the manufacturing process of oolong tea (Camellia sinensis) Lanting Zenga,b, Xuewen Wanga,d, Yinyin Liaoa,d, Dachuan Gua, Fang Dongc, Ziyin Yanga,b,d,
T ⁎
a Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, No. 723 Xingke Road, Tianhe District, Guangzhou 510650, China b Center of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, No. 723 Xingke Road, Tianhe District, Guangzhou 510650, China c Guangdong Food and Drug Vocational College, No. 321 Longdongbei Road, Tianhe District, Guangzhou 510520, China d University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Abscisic acid Aroma Camellia sinensis Jasmonic acid Tea Volatile
As important upstream signals, phytohormones regulate the plant volatiles’ biosynthesis under various stresses. The formation of some characteristic aromas during the manufacturing process of oolong tea (postharvest stage) is due to the defense responses of tea leaves to stress. This study investigates the formation of phytohormone in response to stresses during the manufacturing process of oolong tea. Jasmonic acid (JA) and abscisic acid (ABA) levels enhanced during the manufacturing processes (enzyme-active stage) of oolong tea. Wounding from plucking activated JA synthetic gene expression, resulting in increased levels of JA (p ≤ 0.01), and continuous wounding from the turn over stage further enhanced JA synthesis (p ≤ 0.05). Dehydration stress during the withering stage activated ABA synthetic gene expression resulting in an increase of ABA (p ≤ 0.01). The study advances the understanding of key upstream signals, JA and ABA, during the manufacturing process of oolong tea.
1. Introduction Tea is made of the buds or tender leaves plucked from Camellia sinensis (L.) O. Kuntze plant. It is second in popularity to water because of health benefits and its characteristic flavor. The special properties of tea result from the contributions of specialized metabolites, including polyphenols, aroma compounds, and amino acids (eg. L-theanine) (Wan, 2003). Compared with polyphenols (18–36% in tea) and amino acids (1–4% in tea), aroma compounds take up under 0.03% of tea products (dry weight) (Wan, 2003). Aroma compounds affect tea flavor, so they act as key elements determining tea quality (Yang et al., 2013). Tea aroma compounds formed in both preharvest stage and postharvest stage (i.e., tea manufacture process). At the postharvest stage, tea can usually be classified into six kinds, and they are green, white, oolong, black, yellow, and dark tea (Baldermann et al., 2014; Wan, 2003). Enzymatic, thermophysical, and chemical reactions were involved in formation of tea aroma compound during manufacturing process (Zeng et al., 2018a). One of enzymatic reactions that forms tea aromas is linked to stresses from the manufacturing process of tea. Among the six tea kinds, tea leaves made oolong tea are subjected to the most stresses
(Zeng et al., 2018a). Apart from wounding, which is the most crucial stress and occurs during the plucking and turn over stages, other stresses, for instance drought (during the solar- and indoor-withering stages) and ultra-violet irradiation (during the solar-withering stage) are also related to the manufacturing process of oolong tea (Cho et al., 2007; Gui et al., 2015; Zeng et al., 2016, 2017b, 2018a, 2018b, Zhou et al., 2017b). Multiple stresses from the manufacturing process of oolong tea, especially wounding and low temperature, can enhance the formation of characteristic aroma compounds such as (E)-nerolidol, jasmine lactone, and indole (Zeng et al., 2016, 2018b; Zhou et al., 2017b). In addition, formation of these compounds results from activating the key synthetic genes of aroma compounds in response to the stresses (Zeng et al., 2016, 2018b; Zhou et al., 2017b). However, changes in the upstream signals, such as phytohormones, during the manufacturing process of oolong tea, are little-known. Phytohormones are the important links between environmental stresses and the formation of volatile compounds. In particular, many studies have reported that jasmonic acid (JA) participates in the interactions between vegetative part of plant and insects (Dong et al., 2016; Maffei et al., 2007; Wu et al., 2007). Similar to other plants, in
⁎ Corresponding authors at: Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, No. 723 Xingke Road, Tianhe District, Guangzhou 510650, China. E-mail address: [email protected] (Z. Yang).
https://doi.org/10.1016/j.postharvbio.2019.110974 Received 12 March 2019; Received in revised form 10 July 2019; Accepted 27 July 2019 Available online 09 August 2019 0925-5214/ © 2019 Elsevier B.V. All rights reserved.
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to extract phytohormones from powdered tea leaves (fresh weight, 300 mg) followed by vortexing for 30 s. Subsequently, the mixture was ultrasonic-extracted in ice-water bath for 20 min. During the extraction, the added [2H6] abscisic acid (ABA), [2H4] salicylic acid (SA), and [2H5] JA were used as inner-standards. After 5 min-centrifugation at 4 °C and 10,000×g, supernatants (2.5 mL) were collected, and gaseous N2 was applied to condense the supernatants. Two hundred microlitres of methanol were used to re-dissolve the extract. A membrane (0.22 μm) was used to filter the extract, which was then subjected to ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC–QTOF–MS) (Acquity UPLC I-Class/Xevo® G2-XS QTOF, Waters Corporation, MA, USA) analysis. Five microliters of extract were separated on a Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm). Water and acetonitrile, which both contained 0.1% formic acid (v/v), were selected as solvent A and B, respectively. Initially, gradient of the solvent was kept at 20% B, ramped to 35% B during 10 min in a linear fashion, followed ramped to 95% B in 0.1 min, and then remained at 95% B for 3 min. Finally, solvent B fell to 20% in 0.1 min and held for another 3 min. Temperature column and flow rate were 30 °C and 6.667 μL s−1. Negative mode was used for electrospray ionization. The details of MS condition were shown as follows: 0.167 L s−1 of desolvation gas flow, 0.014 L s−1 of cone gas flow, 300 °C of desolvation temperature, 100 °C of source temperature, and 1.5 kV of capillary voltage. The phytohormone standards were applied to make qualitative analysis, and quantitative analyses were performed based on the calibration curves of the standards. Concentrations are expressed as mg kg−1 on a dry weight basis. The dry weight was determined according to the weight of fresh and dried (acquired from drying) leaves.
preharvest tea leaves, insect attacks can increase JA, which can result in rapid synthesis and emission of volatile compounds, for instance indole, benzyl nitrile, α-farnesene, benzyl alcohol, (Z)-3-hexen-1-ol, linalool, and β-ocimene (Dong et al., 2011; Zeng et al., 2017a). Many emitted volatiles from tea leaves possess ecological functions, for example, attraction of the pests’ natural enemies (Han and Chen, 2002; Ishiwari et al., 2007). In contrast to the role of phytohormones in volatile compounds’ formation in tea leaves when exposed to stresses at preharvest stage, the same system in the postharvest setting is little-known. As the oolong tea process involves various stresses and contains abundant aroma compounds, it is an ideal model for studying tea postharvest biology (Zeng et al., 2017b, 2018a). To investigate how the phytohormones change in response to stresses from the manufacturing process of oolong tea, changes in phytohormones from plucking to turn over stages with enzyme-active were monitored. Effects of the key stresses from the manufacturing process of oolong tea on levels of phytohormones and phytohormone synthesis-related gene expression were studied. The purpose of present research was to study the formation of phytohormone under stresses from the manufacturing process of oolong tea and advance the understanding of key upstream signals during the manufacturing process of oolong tea. 2. Materials and methods 2.1. Plant materials and treatments The variety of C. sinensis used to conduct experiments was Jinxuan, which is popularly cultivated in southern China and usually selected to make oolong tea. In the study, the selected C. sinensis cv. Jinxuan plants were cultivated at ‘Yingde’ Tea Research Institute, Guangdong Academy of Agricultural Sciences (Yingde, Guangdong, China). Usually, one bud and two or three leaves are selected to process into oolong tea. Therefore, tea samples collected in September 2018 were selected to investigate the manufacturing process of oolong tea referred to our reported method (Zeng et al., 2016, 2017b). One bud and two or three leaves were freshly plucked (P) around at 9:00 am. The plucked samples were subjected to solar withering (SW), i.e. exposed to sunlight (about humidity of 58% and temperature of 35 °C) for 1 h. Subsequently, the samples were indoor-withered (IW) (about humidity of 80% and temperature of 28 °C) for 2 h, and then turned over for five times with 1.5 h-interval (T1–T5). The time of turn over were respectively 0.5, 1, 3, 5, and 8 min. Three independent biological samples were conducted, and they were collected at every stage (Fig. 1A) and quickly frozen in liquid N2 for future investigation. The same type of tea samples (one bud and two or three leaves) collected in September 2018 was selected to investigate wounding stress referred to partially modified method (Zeng et al., 2016; Zhou et al., 2017b). Plucked samples were continuous shaken at a shaking table with a temperature of 25 °C for 0, 1, 2, and 4 h. The samples in still state and kept at the above-mentioned conditions for the same times were set as control groups. Three independent biological samples were conducted, and they were quickly frozen in liquid N2 for future investigation. The same type of tea samples (one bud and two or three leaves) collected in October 2018 were selected to investigate dehydration stress. Plucked samples were kept at 25 °C for 0, 4, 8, and 12 h. Tea which had not been plucked, but had been cultivated in water at the above-mentioned conditions for the same times were set as control group. Three independent biological samples were conducted, and they were quickly frozen in liquid N2 for future investigation.
2.3. Expression level analysis of related genes The RNA was isolated from tea samples using Quick Plant Total RNA Kit (Huayueyang Biotechnology Co., Ltd., Beijing, China). Genomic DNA of the extracted RNA was removed using gDNA Eraser (Takara Bio Inc., Kyoto, Japan), and the purified RNA was reversely transcribed into complementary DNA (cDNA) by a PrimeScript RT Reagent Kit (Takara Bio Inc., Kyoto, Japan). The transcript expression level of the genes was analyzed using quantitative real time PCR (qRT–PCR) (Zeng et al., 2016, 2018b). Twenty microlitres mixture contained ddH2O (7.2 μL), cDNA (diluted into 20-fold, 2 μL), each specific primer (10 μM, 0.4 μL), and iTaq™ Universal SYBR® Green Supermix (10 μL, Bio-Rad, Hercules, CA, USA). A Roche LightCycle 480 (Roche Applied Science, Mannheim, Germany) was used to conduct qRT-PCR analysis. PCR conditions were a cycle for 60 s at 95 °C, and 40 cycles for 15 s at 95 °C and 30 s at 60 °C. A melt curve was performed to test the PCR product specificity at the end of PCR reaction. The expression level of genes was calculated based on the 2−△△ct method, and was normalized to mRNA level of encoding elongation factor1 (CsEF1), a reference gene. Evaluation of candidate reference genes of tea plants showed that CsEF1 was a most suitable reference gene in the present study (Hao et al., 2014). The primers of genes used for qRT–PCR analysis are provided in Table S1.
2.4. Statistical analysis Statistical analysis was carried out using a SPSS package (Version 23.0). One-way analysis of variance (ANOVA) with a probability level of 5% (p ≤ 0.05) indicated statistical significance among the different treatments, which depended on Duncan multiple-range test. All data are expressed as the mean ± standard deviation (S.D.).
2.2. Extraction and analysis of phytohormones in tea leaves Extraction and analysis of phytohormones in tea leaves were referred to the reported methods with many modifications (Wu et al., 2007; Zeng et al., 2017a). Three milliliter of ethyl acetate was applied 2
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Fig. 1. Effect of the manufacturing process of oolong tea on tea leaves. (A) Phenotypic changes in tea leaves during the manufacturing process of oolong tea. (B) Changes in phytohormone concentration during manufacturing process of oolong tea. JA, jasmonic acid; ABA, abscisic acid; SA, salicylic acid. Concentrations are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05).
3. Results and discussion
various environmental stresses have been partially studied (Zhang et al., 2018). Compared with phytohormone formation in tea leaves under stresses at preharvest stage, phytohormone formation in the response of postharvest tea leaves exposed to stresses during the tea manufacturing process is little-known. The tea classification is in accordance with the fermentation degree during the manufacturing process. Generally, six tea kinds are green tea (non-fermented), white tea (slightly fermented), oolong tea (semi-fermented), black tea (fully fermented), yellow tea (post-fermented), and dark tea (post-fermented) (Wan, 2003). Among these tea types, tea leaves made oolong tea maintain alive for the longest time, indicating that postharvest tea leaves can sense the stress from the manufacturing process even when they are detached from tea plants (Zeng et al., 2018a). In our study, JA concentrations drastically increased at the SW stage (Fig. 1B), suggesting that wounding from plucking may lead to high accumulation of JA. However, the wounding intensity increased at the turn over stage and the amount of JA did not significantly change (Fig. 1B). This means that JA is a fast responder to wounding. ABA concentration was significantly increased at the IW stage (Fig. 1B), suggesting that its accumulation may be related to dehydration stress from the withering process. Compared with JA and ABA, SA did not significantly change during the manufacturing process of oolong tea (enzyme-active stage) (Fig. 1B), suggesting that SA synthesis may not be formed in response to the stresses involved. Interestingly, the base level of SA even in nonstress conditions (once plucked, tea leaves were frozen in liquid N2) can
3.1. JA and ABA increased in tea leaves during the manufacturing process of oolong tea To monitor the change in phytohormone levels during the manufacturing process of oolong tea, C. sinensis cv. Jinxuan with one bud and two or three leaves were processed into oolong tea (from plucking to turn over stages; Fig. 1A), and the samples collected at every stage were analyzed using UPLC–QTOF–MS. In the study, JA increased quickly after the SW stage compared to the P stage, whereas the amount of ABA significantly increased at the IW stage, just after SW (Fig. 1B). Overall, both JA and ABA increased during the manufacturing process of oolong tea. However, the SA amount did not significantly alter during the manufacturing process, and had the highest level, compared with the other two phytohormones, JA and ABA (Fig. 1B). In plants, it is well known that phytohormone formation is a key response to external stress. Although not all cases are the same, JA is usually reported as being related to insect attack stress, SA is usually reported as being related to fungus infection stress, and ABA is usually reported as being related to drought stress (Davies, 2010). Similar to the model plants, these phytohormone formations in response to the stresses during the plant growth and development process have been intensively studied in many crop species. Referring to the knowledge on model plants, formation of phytohormone in tea plants as a response to 3
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Fig. 2. Effect of wounding stress on phytohormone levels. (A) Wounding treatment of tea leaves. (B) Changes in concentration of jasmonic acid (JA) and abscisic acid (ABA) during wounding treatment. Concentrations are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05).
wounding treatments (data not shown in the study), similar to the changes in SA during the manufacturing process of oolong tea with enzyme-active (Fig. 1B). This suggests that SA is relatively stable during the manufacturing process of oolong tea. To further investigate the changing pattern of JA levels under both plucking-induced single and continuous wounding treatments, we assessed effects of the wounding on the expression levels of JA biosynthetic genes, including lipoxygenases (LOXs), allene oxide synthases (AOSs), allene oxide cyclase (AOC), and 12-oxo-phytodienoic acid reductases (OPRs) (Fig. 3). Compared with the single wounding stress (induced by plucking), the expression levels of most CsLOXs, except CsLOX2, CsAOSs and CsOPRs were significantly increased in tea leaves under the longer continuous wounding treatment (Fig. 3). The results showed that the wounding intensity could prolong the increases in these genes expressions, thus resulting in the steady high level of JA (Fig. 2B). In addition, compared with other genes, CsLOX7, CsAOS1, CsAOS2, and CsOPR2 genes expression levels were quickly activated under the two treatments within 1 h, and were significantly increased by continuous wounding (Fig. 3), which was similar to the change pattern of JA (Fig. 2B). JA is ubiquitous in the plant species and is a part of the oxygenated fatty acid derivative family. Due to lacking of a comprehensive genetic transformation system, it is of difficulty to reveal the real metabolic pathways of tea plants in vivo. In tea plants, the current information on metabolite biosynthesis and phytohormone is partially referred to the published knowledge from other plant species. Recently, Zhu et al. (2018) have proposed the presumptive model for functions of CsLOX genes in tea plant, and they point out that CsLOX1, CsLOX6 and CsLOX7 are closely involved in JA synthesis. Indeed, the expression levels of these LOX genes were higher in continuous wounding stress than single wounding stress (Fig. 3), exhibiting a consistent changing pattern of JA between 2 h and 4 h treatment (Fig. 2B). In our previous study, CsAOS2 gene was reported to be the solely up-regulated gene related to JA synthesis in tea flowers after the insect (Thrips hawaiiensis (Morgan)) visited (Zhou et al., 2017a). Furthermore, over-expression of CsAOS2 in Nicotiana benthamiana leaves showed that this gene was closely related to JA synthesis in vivo (Peng et al., 2018). Subcellular localization
reach dozens of mg/kg dry weight, which was much higher than that in model plants, for instance, Nicotiana tabacum (Malamy et al., 1990) and Arabidopsis (Pan et al., 2010), and some crops such as Zea mays (Pál et al., 2005) under non-stress condition. In most cases, the amount of SA is lower in plants without stresses, and is significantly increased after stress stimuli. It would be interesting to study why tea leaves accumulate high levels of SA under non-stress conditions.
3.2. Wounding stress increased JA concentration in tea leaves After exposure to wounding from insect attacks, plants generally synthesize JA quickly to defend the plant against stresses (Maffei et al., 2007; Wu et al., 2007). Therefore, to confirm whether the phenomena also exist in tea plants, the tea leaves under the single and continuous wounding stress were investigated (Fig. 2A). Similar to other plants, tea leaves also quickly accumulate JA after exposure to wounding stress, even simple plucking-induced wounding (Fig. 2B). Continuous wounding intensity was higher than plucking-induced single wounding intensity; however, the JA accumulation levels were not significantly different at 1 h. This could explain why JA was mainly formed during the withering stage from the manufacturing process of oolong tea (Fig. 1B). Afterwards, the JA amount in the two treatments both gradually decreased at 2 h and 4 h, but the decrease degree of JA level in continuous wounding treatment was lower than the one in single wounding treatment (Fig. 2B). This pattern of changes in JA levels indicated that high accumulations of several characteristic aroma compounds including jasmine lactone, (E)-nerolidol, and indole due to continuous wounding stress during the manufacturing process of oolong tea may be closely related to JA formation (Zeng et al., 2016, 2018a, 2018b; Zhou et al., 2017b). ABA concentration increased with treatment time at both plucking-induced single wounding and continuous wounding treatments, which may be due to dehydration related to the two treatment times. However, compared with plucking-induced single wounding, continuous wounding did not significantly increase ABA concentration, suggesting that different wounding intensities did not influence ABA accumulation. SA concentration did not significantly change under either plucking-induced single wounding or continuous 4
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Fig. 3. Changes in expression level of jasmonic acid (JA) biosynthetic genes during wounding treatment. Expression levels are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05). AOC, allene oxide cyclase; AOS, allene oxide synthase; LOX, lipoxygenase; OPR, 12-oxo-phytodienoic acid reductase.
additional single wounding in this treatment, compared with the control group. This displayed the same pattern as the investigations on JA formation during wounding stress (Fig. 2B), further confirming that wounding stress was the key factor inducing JA formation in tea leaves. Considering the concentration effect due to water loss, the concentration of phytohormones was expressed as mg kg−1 on a dry weight basis. The results showed that the increase of these two phytohormones was above the concentration effect due to water loss (Fig. 4C). Expression level analysis of the genes related to the synthesis and signal transduction of ABA revealed that the ABA accumulation during dehydration stress was not only due to the activation of the ABA synthesis pathway, but also to the formation of the ABA receptor complexes protein phosphatase 2C (CsPP2C) and pyrabactin resistance-like protein 8 (CsPYL8) (Fig. 5). As an important signaling molecule, ABA regulates closure and opening of stomata to mediate the water loss of transpiration in response to many stress conditions (Bomke et al., 2008; Huerta et al., 2009; Xiao et al., 2010). Drought stress is ubiquitous in nature, so the role of ABA in controlling drought tolerance has been well established in many plant species. The regulation of this process is largely controlled by changes in the de novo synthesis of ABA (Schwartz and Zeevaart, 2010). In plants, when leaves are water-stressed, there is a striking increase in ABA levels (Schwartz and Zeevaart, 2010). The elevated ABA levels lead to mediation through a wide range of mechanisms to enhance drought tolerance (Kazan, 2015). The metabolism
analysis revealed that CsAOS2 was distributed in the membrane of chloroplast (Peng et al., 2018). As a stress response, up-regulation of LOXs in other plant species is generally more sensitive than other genes related to the JA biosynthesis (Wu et al., 2007). Our results showed that most CsLOXs gene was not significantly affected by short-time wounding stress, whilst CsLOX7, CsAOS1 and CsAOS2 were found to be fast responding genes that were strikingly up-regulated in 1 h (Fig. 3). This suggests that these three genes, especially the functionally characterized CsAOS2, may act as the key roles in the early JA-synthesis in tea leaves exposed to stress. 3.3. Dehydration stress increased ABA concentration in tea leaves To study the influence of dehydration stress on formation of ABA, one bud and two or three leaves were picked and then stacked for several hours (without a water supply), and the concentration of ABA was analyzed during the treatment (Fig. 4A). The same type picked from the tea branches cultivated in water (with a continuous water supply) were used as the control. Indeed, the water content of the tea leaves from treatment group was drastically lower than that in control one with longer dehydration stress (Fig. 4B). Similar to the change in ABA amount during the manufacturing process of oolong tea, ABA increased during dehydration stress, but did not vary drastically in tea leaves when continuously cultivated in water (Fig. 4C). The amount of JA also increased under dehydration stress (Fig. 4C) because there was 5
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Fig. 4. Effect of dehydration stress on phytohormone levels. (A) Dehydration treatment of tea leaves. (B) Water content of tea leaves during dehydration treatment. (C) Changes in concentration of jasmonic acid (JA) and abscisic acid (ABA) during dehydration treatment. Concentrations are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05).
leads to defoliation and even death (Upadhyaya and Panda, 2013), and it acts as a main stress limiting productivity. In tea plants, ABA can improve the drought tolerance by enhancing the expression of resistance proteins, protein transport, and carbon metabolism (Zhang et al., 2018). For instance, there is study confirming that exogenous ABA drastically altered proteome in tea plants under drought stress, leading to improvement of drought tolerance (Zhou et al., 2014). Without stress resistance, ABA has also been reported to affect bud dormancy of tea plants (Barua, 1969), and can induce somatic embryogenesis (globular and heart) without maturation of these embryos to shoot (Ghanati and Ishka, 2012). ABA is notoriously related to the control of tea plant growth (preharvest stage). Similarly, ABA can act an critical role in the tea leaves during the postharvest stage as well. Water loss is part of the manufacturing process, particularly at the withering stage in the production of six different tea kinds (Zeng et al., 2018a). There is a close relationship between water loss and ABA increase, and ABA was shown to increase during the manufacturing process of oolong tea (Cho et al., 2007), consistent with the results in our study (Fig. 1B). Because the tea leaves maintain alive during the manufacturing process of oolong tea, enzymes in tea leaves remain active (Zeng et al., 2018a). In this study, dehydration stress treatment further confirmed that the increase in ABA was due to the water loss during the withering stage (Fig. 4B). Possibly, tea leaves in the manufacturing process of oolong tea first accumulate ABA due to the activation of key biosynthetic genes of ABA, and the enhanced level of ABA results in diverse genes related to stress defense. The positive effect of postharvest dehydration on
and signaling of ABA have been widely studied in various plant species (Xue et al., 2008; Wang et al., 2016). Up to now, most studies only focus on the transcription regulation of ABA biosynthesis. For example, the gene expression of 9-cis-epoxycarotenoid dioxygenase (NCED), an important enzyme participating in ABA biosynthesis, is reported to be activated under environmental stresses (Qin and Zeevaart, 1999). In leaves and roots, accumulation of ABA induced by water stress is induced by substantial rises in both transcript and protein levels of PvNCED1 (Qin and Zeevaart, 1999). Only zeaxanthin epoxidase 2 (CsZEP2) and CsNCED1 were activated during dehydration stress in the present study, even though there are three CsZEPs and two CsNCEDs found in the tea plants (Fig. 5). Usually, multigene families encoded the mostly proteins related to ABA metabolism and signaling. For example, there are 5 NCEDs genes, which are the upstream genes, in Arabidopsis and 3 NCEDs genes in C. sinensis. However, only 1 abscisic aldehyde oxidase (AAO) gene and 1 short-chain dehydrogenase/reductase (SDR), which are the genes in the downstream pathway, are present in C. sinensis. The phenomena indicated that the genetic regulation of the downstream pathway of ABA metabolism may be more complicated than that of the upstream pathway. Furthermore, there are 80 PP2Cs in Arabidopsis (Xue et al., 2008), demonstrating that the genes are with redundant and partially dedicated functions. However, similar phenomena also occur in the tea plants, the precise mechanisms so far are still not clear. Consequently, more studies in tea are needed to elucidate the mechanism of drought tolerance under ABA-regulation. During the preharvest stage of tea plants, drought stress in long-time 6
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Fig. 5. Changes in expression levels of abscisic acid (ABA) biosynthetic genes during dehydration treatment. Expression levels are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05). ZEP, zeaxanthin epoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase; SDR, short-chain dehydrogenase/reductase; AAO, abscisic aldehyde oxidase; PP2C, protein phosphatase 2C; PYL8, pyrabactin resistance-like protein 8.
(Fig. 6). JA levels significantly enhanced during solar withering, as a result of wounding from plucking. CsAOS2 played important roles in JA synthesis in tea leaves under wounding stresses from the manufacturing process of oolong tea. ABA levels significantly enhanced at the indoor withering stage, due to dehydration stress from the withering process. Many genes, related to the synthetic pathway and signal transduction of ABA were activated by dehydration stress, simulating during the withering stage. This information will add to the knowledge of the key upstream signals which regulate the biosynthesis of important metabolites under stresses from the manufacturing process of oolong tea from the perspective of postharvest biology. Declaration of Competing Interests
Fig. 6. Activation of jasmonic acid and abscisic acid formation during the manufacturing process of oolong tea.
The authors declare no competing financial interests. qualitative attributes in grapes, including volatile compounds and phenolic compounds, has also been widely investigated. The studies showed that the postharvest dehydration is a critical process to make a high-quality wine (Constantinou et al., 2018; Noguerol-Pato et al., 2013). Therefore, in tea leaves, it can be speculated that enhanced ABA levels because of postharvest dehydration from the manufacturing process may also improve the volatile compounds formation (Cho et al., 2007). However, more researches are required to confirm this hypothesis proposed by Cho’s group. In addition, ABA is synthesized from a C40 carotenoid precursor that can also be catalyzed into carotenoidderived aroma compound, for instance, damascenone and β-ionone, significantly contributing to the tea flavor (Cutler and Krochko, 1999; Yang et al., 2013). Therefore, whether there is a negative correlation between ABA and carotenoid-derived aroma compounds will also attract the researchers’ attention.
Acknowledgments This study was supported by the financial support from the National Natural Science Foundation of China (31870684), the National Key Research and Development Program of China (2018YFD1000601), the China Postdoctoral Science Foundation (2018M640837), and the Guangdong Natural Science Foundation for Distinguished Young Scholar (2016A030306039). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.postharvbio.2019. 110974. References
4. Conclusions
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Formation of and changes in phytohormone levels in response to stress during the manufacturing process of oolong tea (Camellia sinensis) Lanting Zenga,b, Xuewen Wanga,d, Yinyin Liaoa,d, Dachuan Gua, Fang Dongc, Ziyin Yanga,b,d,
T ⁎
a Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, No. 723 Xingke Road, Tianhe District, Guangzhou 510650, China b Center of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, No. 723 Xingke Road, Tianhe District, Guangzhou 510650, China c Guangdong Food and Drug Vocational College, No. 321 Longdongbei Road, Tianhe District, Guangzhou 510520, China d University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Abscisic acid Aroma Camellia sinensis Jasmonic acid Tea Volatile
As important upstream signals, phytohormones regulate the plant volatiles’ biosynthesis under various stresses. The formation of some characteristic aromas during the manufacturing process of oolong tea (postharvest stage) is due to the defense responses of tea leaves to stress. This study investigates the formation of phytohormone in response to stresses during the manufacturing process of oolong tea. Jasmonic acid (JA) and abscisic acid (ABA) levels enhanced during the manufacturing processes (enzyme-active stage) of oolong tea. Wounding from plucking activated JA synthetic gene expression, resulting in increased levels of JA (p ≤ 0.01), and continuous wounding from the turn over stage further enhanced JA synthesis (p ≤ 0.05). Dehydration stress during the withering stage activated ABA synthetic gene expression resulting in an increase of ABA (p ≤ 0.01). The study advances the understanding of key upstream signals, JA and ABA, during the manufacturing process of oolong tea.
1. Introduction Tea is made of the buds or tender leaves plucked from Camellia sinensis (L.) O. Kuntze plant. It is second in popularity to water because of health benefits and its characteristic flavor. The special properties of tea result from the contributions of specialized metabolites, including polyphenols, aroma compounds, and amino acids (eg. L-theanine) (Wan, 2003). Compared with polyphenols (18–36% in tea) and amino acids (1–4% in tea), aroma compounds take up under 0.03% of tea products (dry weight) (Wan, 2003). Aroma compounds affect tea flavor, so they act as key elements determining tea quality (Yang et al., 2013). Tea aroma compounds formed in both preharvest stage and postharvest stage (i.e., tea manufacture process). At the postharvest stage, tea can usually be classified into six kinds, and they are green, white, oolong, black, yellow, and dark tea (Baldermann et al., 2014; Wan, 2003). Enzymatic, thermophysical, and chemical reactions were involved in formation of tea aroma compound during manufacturing process (Zeng et al., 2018a). One of enzymatic reactions that forms tea aromas is linked to stresses from the manufacturing process of tea. Among the six tea kinds, tea leaves made oolong tea are subjected to the most stresses
(Zeng et al., 2018a). Apart from wounding, which is the most crucial stress and occurs during the plucking and turn over stages, other stresses, for instance drought (during the solar- and indoor-withering stages) and ultra-violet irradiation (during the solar-withering stage) are also related to the manufacturing process of oolong tea (Cho et al., 2007; Gui et al., 2015; Zeng et al., 2016, 2017b, 2018a, 2018b, Zhou et al., 2017b). Multiple stresses from the manufacturing process of oolong tea, especially wounding and low temperature, can enhance the formation of characteristic aroma compounds such as (E)-nerolidol, jasmine lactone, and indole (Zeng et al., 2016, 2018b; Zhou et al., 2017b). In addition, formation of these compounds results from activating the key synthetic genes of aroma compounds in response to the stresses (Zeng et al., 2016, 2018b; Zhou et al., 2017b). However, changes in the upstream signals, such as phytohormones, during the manufacturing process of oolong tea, are little-known. Phytohormones are the important links between environmental stresses and the formation of volatile compounds. In particular, many studies have reported that jasmonic acid (JA) participates in the interactions between vegetative part of plant and insects (Dong et al., 2016; Maffei et al., 2007; Wu et al., 2007). Similar to other plants, in
⁎ Corresponding authors at: Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, No. 723 Xingke Road, Tianhe District, Guangzhou 510650, China. E-mail address: [email protected] (Z. Yang).
https://doi.org/10.1016/j.postharvbio.2019.110974 Received 12 March 2019; Received in revised form 10 July 2019; Accepted 27 July 2019 Available online 09 August 2019 0925-5214/ © 2019 Elsevier B.V. All rights reserved.
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to extract phytohormones from powdered tea leaves (fresh weight, 300 mg) followed by vortexing for 30 s. Subsequently, the mixture was ultrasonic-extracted in ice-water bath for 20 min. During the extraction, the added [2H6] abscisic acid (ABA), [2H4] salicylic acid (SA), and [2H5] JA were used as inner-standards. After 5 min-centrifugation at 4 °C and 10,000×g, supernatants (2.5 mL) were collected, and gaseous N2 was applied to condense the supernatants. Two hundred microlitres of methanol were used to re-dissolve the extract. A membrane (0.22 μm) was used to filter the extract, which was then subjected to ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC–QTOF–MS) (Acquity UPLC I-Class/Xevo® G2-XS QTOF, Waters Corporation, MA, USA) analysis. Five microliters of extract were separated on a Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm). Water and acetonitrile, which both contained 0.1% formic acid (v/v), were selected as solvent A and B, respectively. Initially, gradient of the solvent was kept at 20% B, ramped to 35% B during 10 min in a linear fashion, followed ramped to 95% B in 0.1 min, and then remained at 95% B for 3 min. Finally, solvent B fell to 20% in 0.1 min and held for another 3 min. Temperature column and flow rate were 30 °C and 6.667 μL s−1. Negative mode was used for electrospray ionization. The details of MS condition were shown as follows: 0.167 L s−1 of desolvation gas flow, 0.014 L s−1 of cone gas flow, 300 °C of desolvation temperature, 100 °C of source temperature, and 1.5 kV of capillary voltage. The phytohormone standards were applied to make qualitative analysis, and quantitative analyses were performed based on the calibration curves of the standards. Concentrations are expressed as mg kg−1 on a dry weight basis. The dry weight was determined according to the weight of fresh and dried (acquired from drying) leaves.
preharvest tea leaves, insect attacks can increase JA, which can result in rapid synthesis and emission of volatile compounds, for instance indole, benzyl nitrile, α-farnesene, benzyl alcohol, (Z)-3-hexen-1-ol, linalool, and β-ocimene (Dong et al., 2011; Zeng et al., 2017a). Many emitted volatiles from tea leaves possess ecological functions, for example, attraction of the pests’ natural enemies (Han and Chen, 2002; Ishiwari et al., 2007). In contrast to the role of phytohormones in volatile compounds’ formation in tea leaves when exposed to stresses at preharvest stage, the same system in the postharvest setting is little-known. As the oolong tea process involves various stresses and contains abundant aroma compounds, it is an ideal model for studying tea postharvest biology (Zeng et al., 2017b, 2018a). To investigate how the phytohormones change in response to stresses from the manufacturing process of oolong tea, changes in phytohormones from plucking to turn over stages with enzyme-active were monitored. Effects of the key stresses from the manufacturing process of oolong tea on levels of phytohormones and phytohormone synthesis-related gene expression were studied. The purpose of present research was to study the formation of phytohormone under stresses from the manufacturing process of oolong tea and advance the understanding of key upstream signals during the manufacturing process of oolong tea. 2. Materials and methods 2.1. Plant materials and treatments The variety of C. sinensis used to conduct experiments was Jinxuan, which is popularly cultivated in southern China and usually selected to make oolong tea. In the study, the selected C. sinensis cv. Jinxuan plants were cultivated at ‘Yingde’ Tea Research Institute, Guangdong Academy of Agricultural Sciences (Yingde, Guangdong, China). Usually, one bud and two or three leaves are selected to process into oolong tea. Therefore, tea samples collected in September 2018 were selected to investigate the manufacturing process of oolong tea referred to our reported method (Zeng et al., 2016, 2017b). One bud and two or three leaves were freshly plucked (P) around at 9:00 am. The plucked samples were subjected to solar withering (SW), i.e. exposed to sunlight (about humidity of 58% and temperature of 35 °C) for 1 h. Subsequently, the samples were indoor-withered (IW) (about humidity of 80% and temperature of 28 °C) for 2 h, and then turned over for five times with 1.5 h-interval (T1–T5). The time of turn over were respectively 0.5, 1, 3, 5, and 8 min. Three independent biological samples were conducted, and they were collected at every stage (Fig. 1A) and quickly frozen in liquid N2 for future investigation. The same type of tea samples (one bud and two or three leaves) collected in September 2018 was selected to investigate wounding stress referred to partially modified method (Zeng et al., 2016; Zhou et al., 2017b). Plucked samples were continuous shaken at a shaking table with a temperature of 25 °C for 0, 1, 2, and 4 h. The samples in still state and kept at the above-mentioned conditions for the same times were set as control groups. Three independent biological samples were conducted, and they were quickly frozen in liquid N2 for future investigation. The same type of tea samples (one bud and two or three leaves) collected in October 2018 were selected to investigate dehydration stress. Plucked samples were kept at 25 °C for 0, 4, 8, and 12 h. Tea which had not been plucked, but had been cultivated in water at the above-mentioned conditions for the same times were set as control group. Three independent biological samples were conducted, and they were quickly frozen in liquid N2 for future investigation.
2.3. Expression level analysis of related genes The RNA was isolated from tea samples using Quick Plant Total RNA Kit (Huayueyang Biotechnology Co., Ltd., Beijing, China). Genomic DNA of the extracted RNA was removed using gDNA Eraser (Takara Bio Inc., Kyoto, Japan), and the purified RNA was reversely transcribed into complementary DNA (cDNA) by a PrimeScript RT Reagent Kit (Takara Bio Inc., Kyoto, Japan). The transcript expression level of the genes was analyzed using quantitative real time PCR (qRT–PCR) (Zeng et al., 2016, 2018b). Twenty microlitres mixture contained ddH2O (7.2 μL), cDNA (diluted into 20-fold, 2 μL), each specific primer (10 μM, 0.4 μL), and iTaq™ Universal SYBR® Green Supermix (10 μL, Bio-Rad, Hercules, CA, USA). A Roche LightCycle 480 (Roche Applied Science, Mannheim, Germany) was used to conduct qRT-PCR analysis. PCR conditions were a cycle for 60 s at 95 °C, and 40 cycles for 15 s at 95 °C and 30 s at 60 °C. A melt curve was performed to test the PCR product specificity at the end of PCR reaction. The expression level of genes was calculated based on the 2−△△ct method, and was normalized to mRNA level of encoding elongation factor1 (CsEF1), a reference gene. Evaluation of candidate reference genes of tea plants showed that CsEF1 was a most suitable reference gene in the present study (Hao et al., 2014). The primers of genes used for qRT–PCR analysis are provided in Table S1.
2.4. Statistical analysis Statistical analysis was carried out using a SPSS package (Version 23.0). One-way analysis of variance (ANOVA) with a probability level of 5% (p ≤ 0.05) indicated statistical significance among the different treatments, which depended on Duncan multiple-range test. All data are expressed as the mean ± standard deviation (S.D.).
2.2. Extraction and analysis of phytohormones in tea leaves Extraction and analysis of phytohormones in tea leaves were referred to the reported methods with many modifications (Wu et al., 2007; Zeng et al., 2017a). Three milliliter of ethyl acetate was applied 2
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Fig. 1. Effect of the manufacturing process of oolong tea on tea leaves. (A) Phenotypic changes in tea leaves during the manufacturing process of oolong tea. (B) Changes in phytohormone concentration during manufacturing process of oolong tea. JA, jasmonic acid; ABA, abscisic acid; SA, salicylic acid. Concentrations are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05).
3. Results and discussion
various environmental stresses have been partially studied (Zhang et al., 2018). Compared with phytohormone formation in tea leaves under stresses at preharvest stage, phytohormone formation in the response of postharvest tea leaves exposed to stresses during the tea manufacturing process is little-known. The tea classification is in accordance with the fermentation degree during the manufacturing process. Generally, six tea kinds are green tea (non-fermented), white tea (slightly fermented), oolong tea (semi-fermented), black tea (fully fermented), yellow tea (post-fermented), and dark tea (post-fermented) (Wan, 2003). Among these tea types, tea leaves made oolong tea maintain alive for the longest time, indicating that postharvest tea leaves can sense the stress from the manufacturing process even when they are detached from tea plants (Zeng et al., 2018a). In our study, JA concentrations drastically increased at the SW stage (Fig. 1B), suggesting that wounding from plucking may lead to high accumulation of JA. However, the wounding intensity increased at the turn over stage and the amount of JA did not significantly change (Fig. 1B). This means that JA is a fast responder to wounding. ABA concentration was significantly increased at the IW stage (Fig. 1B), suggesting that its accumulation may be related to dehydration stress from the withering process. Compared with JA and ABA, SA did not significantly change during the manufacturing process of oolong tea (enzyme-active stage) (Fig. 1B), suggesting that SA synthesis may not be formed in response to the stresses involved. Interestingly, the base level of SA even in nonstress conditions (once plucked, tea leaves were frozen in liquid N2) can
3.1. JA and ABA increased in tea leaves during the manufacturing process of oolong tea To monitor the change in phytohormone levels during the manufacturing process of oolong tea, C. sinensis cv. Jinxuan with one bud and two or three leaves were processed into oolong tea (from plucking to turn over stages; Fig. 1A), and the samples collected at every stage were analyzed using UPLC–QTOF–MS. In the study, JA increased quickly after the SW stage compared to the P stage, whereas the amount of ABA significantly increased at the IW stage, just after SW (Fig. 1B). Overall, both JA and ABA increased during the manufacturing process of oolong tea. However, the SA amount did not significantly alter during the manufacturing process, and had the highest level, compared with the other two phytohormones, JA and ABA (Fig. 1B). In plants, it is well known that phytohormone formation is a key response to external stress. Although not all cases are the same, JA is usually reported as being related to insect attack stress, SA is usually reported as being related to fungus infection stress, and ABA is usually reported as being related to drought stress (Davies, 2010). Similar to the model plants, these phytohormone formations in response to the stresses during the plant growth and development process have been intensively studied in many crop species. Referring to the knowledge on model plants, formation of phytohormone in tea plants as a response to 3
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Fig. 2. Effect of wounding stress on phytohormone levels. (A) Wounding treatment of tea leaves. (B) Changes in concentration of jasmonic acid (JA) and abscisic acid (ABA) during wounding treatment. Concentrations are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05).
wounding treatments (data not shown in the study), similar to the changes in SA during the manufacturing process of oolong tea with enzyme-active (Fig. 1B). This suggests that SA is relatively stable during the manufacturing process of oolong tea. To further investigate the changing pattern of JA levels under both plucking-induced single and continuous wounding treatments, we assessed effects of the wounding on the expression levels of JA biosynthetic genes, including lipoxygenases (LOXs), allene oxide synthases (AOSs), allene oxide cyclase (AOC), and 12-oxo-phytodienoic acid reductases (OPRs) (Fig. 3). Compared with the single wounding stress (induced by plucking), the expression levels of most CsLOXs, except CsLOX2, CsAOSs and CsOPRs were significantly increased in tea leaves under the longer continuous wounding treatment (Fig. 3). The results showed that the wounding intensity could prolong the increases in these genes expressions, thus resulting in the steady high level of JA (Fig. 2B). In addition, compared with other genes, CsLOX7, CsAOS1, CsAOS2, and CsOPR2 genes expression levels were quickly activated under the two treatments within 1 h, and were significantly increased by continuous wounding (Fig. 3), which was similar to the change pattern of JA (Fig. 2B). JA is ubiquitous in the plant species and is a part of the oxygenated fatty acid derivative family. Due to lacking of a comprehensive genetic transformation system, it is of difficulty to reveal the real metabolic pathways of tea plants in vivo. In tea plants, the current information on metabolite biosynthesis and phytohormone is partially referred to the published knowledge from other plant species. Recently, Zhu et al. (2018) have proposed the presumptive model for functions of CsLOX genes in tea plant, and they point out that CsLOX1, CsLOX6 and CsLOX7 are closely involved in JA synthesis. Indeed, the expression levels of these LOX genes were higher in continuous wounding stress than single wounding stress (Fig. 3), exhibiting a consistent changing pattern of JA between 2 h and 4 h treatment (Fig. 2B). In our previous study, CsAOS2 gene was reported to be the solely up-regulated gene related to JA synthesis in tea flowers after the insect (Thrips hawaiiensis (Morgan)) visited (Zhou et al., 2017a). Furthermore, over-expression of CsAOS2 in Nicotiana benthamiana leaves showed that this gene was closely related to JA synthesis in vivo (Peng et al., 2018). Subcellular localization
reach dozens of mg/kg dry weight, which was much higher than that in model plants, for instance, Nicotiana tabacum (Malamy et al., 1990) and Arabidopsis (Pan et al., 2010), and some crops such as Zea mays (Pál et al., 2005) under non-stress condition. In most cases, the amount of SA is lower in plants without stresses, and is significantly increased after stress stimuli. It would be interesting to study why tea leaves accumulate high levels of SA under non-stress conditions.
3.2. Wounding stress increased JA concentration in tea leaves After exposure to wounding from insect attacks, plants generally synthesize JA quickly to defend the plant against stresses (Maffei et al., 2007; Wu et al., 2007). Therefore, to confirm whether the phenomena also exist in tea plants, the tea leaves under the single and continuous wounding stress were investigated (Fig. 2A). Similar to other plants, tea leaves also quickly accumulate JA after exposure to wounding stress, even simple plucking-induced wounding (Fig. 2B). Continuous wounding intensity was higher than plucking-induced single wounding intensity; however, the JA accumulation levels were not significantly different at 1 h. This could explain why JA was mainly formed during the withering stage from the manufacturing process of oolong tea (Fig. 1B). Afterwards, the JA amount in the two treatments both gradually decreased at 2 h and 4 h, but the decrease degree of JA level in continuous wounding treatment was lower than the one in single wounding treatment (Fig. 2B). This pattern of changes in JA levels indicated that high accumulations of several characteristic aroma compounds including jasmine lactone, (E)-nerolidol, and indole due to continuous wounding stress during the manufacturing process of oolong tea may be closely related to JA formation (Zeng et al., 2016, 2018a, 2018b; Zhou et al., 2017b). ABA concentration increased with treatment time at both plucking-induced single wounding and continuous wounding treatments, which may be due to dehydration related to the two treatment times. However, compared with plucking-induced single wounding, continuous wounding did not significantly increase ABA concentration, suggesting that different wounding intensities did not influence ABA accumulation. SA concentration did not significantly change under either plucking-induced single wounding or continuous 4
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Fig. 3. Changes in expression level of jasmonic acid (JA) biosynthetic genes during wounding treatment. Expression levels are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05). AOC, allene oxide cyclase; AOS, allene oxide synthase; LOX, lipoxygenase; OPR, 12-oxo-phytodienoic acid reductase.
additional single wounding in this treatment, compared with the control group. This displayed the same pattern as the investigations on JA formation during wounding stress (Fig. 2B), further confirming that wounding stress was the key factor inducing JA formation in tea leaves. Considering the concentration effect due to water loss, the concentration of phytohormones was expressed as mg kg−1 on a dry weight basis. The results showed that the increase of these two phytohormones was above the concentration effect due to water loss (Fig. 4C). Expression level analysis of the genes related to the synthesis and signal transduction of ABA revealed that the ABA accumulation during dehydration stress was not only due to the activation of the ABA synthesis pathway, but also to the formation of the ABA receptor complexes protein phosphatase 2C (CsPP2C) and pyrabactin resistance-like protein 8 (CsPYL8) (Fig. 5). As an important signaling molecule, ABA regulates closure and opening of stomata to mediate the water loss of transpiration in response to many stress conditions (Bomke et al., 2008; Huerta et al., 2009; Xiao et al., 2010). Drought stress is ubiquitous in nature, so the role of ABA in controlling drought tolerance has been well established in many plant species. The regulation of this process is largely controlled by changes in the de novo synthesis of ABA (Schwartz and Zeevaart, 2010). In plants, when leaves are water-stressed, there is a striking increase in ABA levels (Schwartz and Zeevaart, 2010). The elevated ABA levels lead to mediation through a wide range of mechanisms to enhance drought tolerance (Kazan, 2015). The metabolism
analysis revealed that CsAOS2 was distributed in the membrane of chloroplast (Peng et al., 2018). As a stress response, up-regulation of LOXs in other plant species is generally more sensitive than other genes related to the JA biosynthesis (Wu et al., 2007). Our results showed that most CsLOXs gene was not significantly affected by short-time wounding stress, whilst CsLOX7, CsAOS1 and CsAOS2 were found to be fast responding genes that were strikingly up-regulated in 1 h (Fig. 3). This suggests that these three genes, especially the functionally characterized CsAOS2, may act as the key roles in the early JA-synthesis in tea leaves exposed to stress. 3.3. Dehydration stress increased ABA concentration in tea leaves To study the influence of dehydration stress on formation of ABA, one bud and two or three leaves were picked and then stacked for several hours (without a water supply), and the concentration of ABA was analyzed during the treatment (Fig. 4A). The same type picked from the tea branches cultivated in water (with a continuous water supply) were used as the control. Indeed, the water content of the tea leaves from treatment group was drastically lower than that in control one with longer dehydration stress (Fig. 4B). Similar to the change in ABA amount during the manufacturing process of oolong tea, ABA increased during dehydration stress, but did not vary drastically in tea leaves when continuously cultivated in water (Fig. 4C). The amount of JA also increased under dehydration stress (Fig. 4C) because there was 5
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Fig. 4. Effect of dehydration stress on phytohormone levels. (A) Dehydration treatment of tea leaves. (B) Water content of tea leaves during dehydration treatment. (C) Changes in concentration of jasmonic acid (JA) and abscisic acid (ABA) during dehydration treatment. Concentrations are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05).
leads to defoliation and even death (Upadhyaya and Panda, 2013), and it acts as a main stress limiting productivity. In tea plants, ABA can improve the drought tolerance by enhancing the expression of resistance proteins, protein transport, and carbon metabolism (Zhang et al., 2018). For instance, there is study confirming that exogenous ABA drastically altered proteome in tea plants under drought stress, leading to improvement of drought tolerance (Zhou et al., 2014). Without stress resistance, ABA has also been reported to affect bud dormancy of tea plants (Barua, 1969), and can induce somatic embryogenesis (globular and heart) without maturation of these embryos to shoot (Ghanati and Ishka, 2012). ABA is notoriously related to the control of tea plant growth (preharvest stage). Similarly, ABA can act an critical role in the tea leaves during the postharvest stage as well. Water loss is part of the manufacturing process, particularly at the withering stage in the production of six different tea kinds (Zeng et al., 2018a). There is a close relationship between water loss and ABA increase, and ABA was shown to increase during the manufacturing process of oolong tea (Cho et al., 2007), consistent with the results in our study (Fig. 1B). Because the tea leaves maintain alive during the manufacturing process of oolong tea, enzymes in tea leaves remain active (Zeng et al., 2018a). In this study, dehydration stress treatment further confirmed that the increase in ABA was due to the water loss during the withering stage (Fig. 4B). Possibly, tea leaves in the manufacturing process of oolong tea first accumulate ABA due to the activation of key biosynthetic genes of ABA, and the enhanced level of ABA results in diverse genes related to stress defense. The positive effect of postharvest dehydration on
and signaling of ABA have been widely studied in various plant species (Xue et al., 2008; Wang et al., 2016). Up to now, most studies only focus on the transcription regulation of ABA biosynthesis. For example, the gene expression of 9-cis-epoxycarotenoid dioxygenase (NCED), an important enzyme participating in ABA biosynthesis, is reported to be activated under environmental stresses (Qin and Zeevaart, 1999). In leaves and roots, accumulation of ABA induced by water stress is induced by substantial rises in both transcript and protein levels of PvNCED1 (Qin and Zeevaart, 1999). Only zeaxanthin epoxidase 2 (CsZEP2) and CsNCED1 were activated during dehydration stress in the present study, even though there are three CsZEPs and two CsNCEDs found in the tea plants (Fig. 5). Usually, multigene families encoded the mostly proteins related to ABA metabolism and signaling. For example, there are 5 NCEDs genes, which are the upstream genes, in Arabidopsis and 3 NCEDs genes in C. sinensis. However, only 1 abscisic aldehyde oxidase (AAO) gene and 1 short-chain dehydrogenase/reductase (SDR), which are the genes in the downstream pathway, are present in C. sinensis. The phenomena indicated that the genetic regulation of the downstream pathway of ABA metabolism may be more complicated than that of the upstream pathway. Furthermore, there are 80 PP2Cs in Arabidopsis (Xue et al., 2008), demonstrating that the genes are with redundant and partially dedicated functions. However, similar phenomena also occur in the tea plants, the precise mechanisms so far are still not clear. Consequently, more studies in tea are needed to elucidate the mechanism of drought tolerance under ABA-regulation. During the preharvest stage of tea plants, drought stress in long-time 6
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Fig. 5. Changes in expression levels of abscisic acid (ABA) biosynthetic genes during dehydration treatment. Expression levels are expressed as mean ± S.D. (n = 3). Means with different letters are significantly different from each other (p ≤ 0.05). ZEP, zeaxanthin epoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase; SDR, short-chain dehydrogenase/reductase; AAO, abscisic aldehyde oxidase; PP2C, protein phosphatase 2C; PYL8, pyrabactin resistance-like protein 8.
(Fig. 6). JA levels significantly enhanced during solar withering, as a result of wounding from plucking. CsAOS2 played important roles in JA synthesis in tea leaves under wounding stresses from the manufacturing process of oolong tea. ABA levels significantly enhanced at the indoor withering stage, due to dehydration stress from the withering process. Many genes, related to the synthetic pathway and signal transduction of ABA were activated by dehydration stress, simulating during the withering stage. This information will add to the knowledge of the key upstream signals which regulate the biosynthesis of important metabolites under stresses from the manufacturing process of oolong tea from the perspective of postharvest biology. Declaration of Competing Interests
Fig. 6. Activation of jasmonic acid and abscisic acid formation during the manufacturing process of oolong tea.
The authors declare no competing financial interests. qualitative attributes in grapes, including volatile compounds and phenolic compounds, has also been widely investigated. The studies showed that the postharvest dehydration is a critical process to make a high-quality wine (Constantinou et al., 2018; Noguerol-Pato et al., 2013). Therefore, in tea leaves, it can be speculated that enhanced ABA levels because of postharvest dehydration from the manufacturing process may also improve the volatile compounds formation (Cho et al., 2007). However, more researches are required to confirm this hypothesis proposed by Cho’s group. In addition, ABA is synthesized from a C40 carotenoid precursor that can also be catalyzed into carotenoidderived aroma compound, for instance, damascenone and β-ionone, significantly contributing to the tea flavor (Cutler and Krochko, 1999; Yang et al., 2013). Therefore, whether there is a negative correlation between ABA and carotenoid-derived aroma compounds will also attract the researchers’ attention.
Acknowledgments This study was supported by the financial support from the National Natural Science Foundation of China (31870684), the National Key Research and Development Program of China (2018YFD1000601), the China Postdoctoral Science Foundation (2018M640837), and the Guangdong Natural Science Foundation for Distinguished Young Scholar (2016A030306039). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.postharvbio.2019. 110974. References
4. Conclusions
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