Transformation of catechins into theaflavins by upregulation of CsPPO3 in preharvest tea (Camellia sinensis) leaves exposed to shading treatment

Journal Pre-proofs Transformation of catechins into theaflavins by upregulation of CsPPO3 in preharvest tea (Camellia sinensis) leaves exposed to shading treatment Zhenming Yu, Yinyin Liao, Lanting Zeng, Fang Dong, Naoharu Watanabe, Ziyin Yang PII: DOI: Reference:

S0963-9969(19)30728-8 https://doi.org/10.1016/j.foodres.2019.108842 FRIN 108842

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Food Research International

Received Date: Revised Date: Accepted Date:

14 August 2019 15 November 2019 18 November 2019

Please cite this article as: Yu, Z., Liao, Y., Zeng, L., Dong, F., Watanabe, N., Yang, Z., Transformation of catechins into theaflavins by upregulation of CsPPO3 in preharvest tea (Camellia sinensis) leaves exposed to shading treatment, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108842

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Running title: Shading and theaflavins in tea

Title: Transformation of catechins into theaflavins by upregulation of CsPPO3 in preharvest tea (Camellia sinensis) leaves exposed to shading treatment

Authors: Zhenming Yu a, †, Yinyin Liao a, b, †, Lanting Zeng a, c, Fang Dong d, Naoharu Watanabe e, Ziyin Yang a, b, c, *

Affiliation: 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, Xingke Road 723, Tianhe District, Guangzhou 510650, China b University c Center

of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China

of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, Xingke

Road 723, Tianhe District, Guangzhou 510650, China d

Guangdong Food and Drug Vocational College, Longdongbei Road 321, Tianhe District,

Guangzhou 510520, China e

Graduate School of Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku,

Hamamatsu 432-8561, Japan

*

Corresponding author:

Ziyin Yang, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China; Tel: +86-20-38072989; Email address: [email protected]. 1

†

These authors equally contributed to this work.

Abstract Catechins and theaflavins are important metabolites contributing to tea function and quality. Catechins are known to transform into theaflavins during the tea manufacturing process, but the same transformation in preharvest tea leaves is unknown. Herein, we determined that shade treatment (dark), an agronomic practise widely used in tea cultivation, reduced the contents of most catechins, but increased the theaflavin contents, in preharvest tea leaves (cv. Yinghong No.9). This was attributed to the activation of polyphenoloxidase (PPO) activity in darkness. Furthermore, CsPPO3 was highly expressed under darkness, and thus CsPPO3 had been cloned, sequenced, and characterization. The CsPPO3 recombinant protein exhibited PPO function. Furthermore, shade treatment also reduced the catechin contents and increased the theaflavin contents in Yabukita and Hoshinomidori, suggesting that this phenomenon might not be specific to certain tea cultivars. This information will aid in understanding of theaflavin formation and its response to environmental factors at the preharvest tea stage.

Keywords: Biosynthesis; Camellia sinensis; Catechin; Polyphenoloxidase; Shading; Tea; Theaflavin

Chemical compounds studied in this article Catechin (PubChem CID: 9064); (-)-Catechin Gallate (PubChem CID: 6419835); (-)Epicatechin (PubChem CID: 72276); (-)-Epicatechin Gallate (PubChem CID: 107905); (-)Epigallocatechin (PubChem CID: 72277); (-)-Epigallocatechin Gallate (PubChem CID: 65064); Gallic acid (PubChem CID: 370); (-)-Gallocatechin (PubChem CID: 9882981); (-)2

Gallocatechin Gallate (PubChem CID: 199472); (-)-Theaflavin (PubChem CID: 114777); Theaflavin-3-Gallate (PubChem CID: 102115506); Theaflavin-3′-Gallate (PubChem CID: 102115505); Theaflavin-3,3′-Digallate (PubChem CID: 467320).

Abbreviations C, Catechin; CG, Catechin gallate; EC, Epicatechin; ECG, Epicatechin gallate; EGC, Epigallocatechin; EGCG, Epigallocatechin gallate; GA, Gallic gcid; GC, Gallocatechin; GCG, Gallocatechin gallate; PPO, Polyphenoloxidase; TF1, Theaflavin; TF2A, Theaflavin-3gallate; TF2B, Theaflavin-3′-gallate; TF3, Theaflavin-3,3′-digallate; UPLC-QTOF-MS, Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry.

1. Introduction

Tea (Camellia sinensis) leaves contain multiple specialized metabolites such as polyphenols, caffeine, amino acids, and aroma compounds, which make contribution to the special functions, taste, and fragrance of tea (Bushman, 1998; Trevisanato, & Kim, 2000; Wan, 2003; Yang, Baldermann, & Watanabe, 2013; Yu, & Yang, 2019; Zeng, Watanabe, & Yang, 2019). Drinking tea has been linked to a reduced risk of cardiovascular and cancer disease based on some experimental and epidemiological studies (Yang et al., 2000). These beneficial effects are proposed to mainly result from the occurrences of polyphenols in tea. The major polyphenols in green tea (or fresh tea leaves) are catechins. The products from the oxidation of catechins during fermentation including theaflavins, thearubigins, and theabrownins are characteristic polyphenols in black tea (Wan, 2003). In fresh tea leaves, there are eight commonly reported catechins, namely, catechin (C), catechin gallate (CG), gallocatechin (GC), gallocatechin gallate (GCG), epicatechin (EC), epicatechin gallate (ECG), 3

epigallocatechin (EGC), and epigallocatechin gallate (EGCG). In general, EGCG, ECG, and EGC are reported to be present with relatively high contents in leaves of many tea cultivars (Jiang et al., 2013; Wan, & Xia, 2015; Chen et al., 2018). Many studies have validated catechins’ contributions to tea taste and function (Wan, & Xia, 2015). Under the action of polyphenoloxidase (PPO), catechins can be oxidized to form high-molecular-weight polyphenols, such as theabrownins, thearubigins, and theaflavins during the black tea fermentation process (Wan, 2003; Wan, & Xia, 2015). However, at present only theaflavins’ chemical structures having been identified and elucidated (Yang et al., 2007; Yang et al., 2008). More than 20 theaflavin derivatives have been reported, but the main reported theaflavins in black tea are theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B), and theaflavin-3,3'-digallate (TF3) (Yang et al., 2008). Theaflavins possess a benzotropolone skeleton and their color are orange or orange-red (Roberts, 1958), and make an important contribution to tea properties, including the ‘mouthfeel’, color, and tea cream formation extent (Roberts, 1962; Millin, Crispin, & Swaine, 1969; Powell et al., 1993). Many studies have shown the biological functions of theaflavins, including anti-inflammatory and cancer chemopreventive (Pan et al., 2000), antimutagenic (Apostolides et al., 1997), and antioxidant activities (Yang et al., 2008). Due to theaflavins’ roles in tea quality and function, much attention has been paid to theaflavin formation from catechins at the tea postharvest stage (during the tea manufacturing process) (Wan, 2003; Wan, & Xia, 2015). However, it is unknown concerning theaflavin formation at the tea preharvest stage (during the tea plant growth process). Shade management (dark treatment) of tea plants is an agronomic practise widely used in tea cultivation at the preharvest tea stage. Although prolonged shading/darkness can potentially lead to a reduction in tea leaf biomass (Fu et al., 2015), it can also effectively enhance free amino acids (Yang et al., 2012; Chen et al., 2017) and reduce catechin contents 4

in preharvest tea leaves (Yang et al., 2012). This led us question whether a part of catechins were transformed into theaflavins in tea leaves exposed to shade treatment. Thus, here we investigated the effect of shade treatment on the catechin and theaflavin contents, and the activity of the enzyme involved, PPO, in preharvest tea leaves (cv. Yinghong No.9 cultivated in Guangdong, China). Furthermore, the upregulated CsPPO gene under shade treatment was isolated, cloned, sequenced, and functionally characterized. Finally, to exclude the effects of different tea cultivars and cultivation environments, we investigated the effect of shade treatment on the catechin and theaflavin contents in preharvest tea leaves of two cultivars in Japan (cv. Yabukita and cv. Hoshinomidori, Shizuoka, Japan). This information will aid understanding of theaflavin formation and its response to environmental factors at the preharvest tea stage.

2. Materials and methods 2.1. Chemicals and regents Catechin (C), catechin gallate (CG), epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG), gallocatechin (GC) and gallocatechin gallate (GCG) were bought from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China. Theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3′-gallate (TF2B), and theaflavin-3,3′-digallate (TF3) were bought from Chengdu PuRuiFa Technology Development Co., Ltd. Sichuan, China. The purities of all standards were above 98%.

2.2. Plant materials and illumination system C. sinensis cv. Yinghong No.9 tea plants (that is a normal tea cultivar developed by Tea Research Institute of Guangdong Academy of Agricultural Science in 1988 and generally used for making black tea in south China (Li et al., 1999)) were widely cultivated in the 5

experimental station at the Tea Research Institute, Guangdong Academy of Agricultural Sciences (Yinghong town, Guangdong, China). Tea plants were maintained in an illumination incubator at 25°C, with 60% relative humidity under shade treatments (dark) and light (12-h photoperiod with a light intensity of 1000 μmol m-2 s-1) conditions for 15 days, respectively. The C. sinensis cv. Yabukita and cv. Hoshinomidori tea leaves under shade treatments were obtained from the Tea Research Center, Shizuoka Prefectural Research Institute of Agriculture and Forestry, Japan. Tea plants were shaded by canopies (150 cm × 160 cm, purchased from Dio Chemicals, Ltd.) excluding 98% of the light for 3 weeks. The average photosynthetic photon flux density per day in the 98% shade conditions were 2% of PPFD in the 0% shade condition. The leaves harvested from five individual plants were considered as one biological replicate, and three biological replicates were performed. The sampled leaves were immediately frozen in liquid nitrogen after harvest and stored at -80°C until the RNA extraction, the determination of PPO enzyme activity, and the analyses of metabolites.

2.3. Extraction and analysis of catechins and theaflavins in tea samples Finely powdered samples of nonshaded and shade-treated tea leaves (100 mg, fresh weight) were blended with methanol (1 mL) by vortexing for 1 min, followed by ultrasonic extraction in ice-cold water for 5 min, centrifugation (12,000 g, 4 C, 5 min), and filtration through a 0.22-m membrane. For catechin determination, the filtrate (200 L) was diluted with 1 mM ascorbic acid (600 L) and subjected to HPLC using a Waters system (Waters, Milford, MA, USA). For theaflavin determination, the filtrate (60 L) was diluted with 1 mM ascorbic acid (60 L) and subjected to UPLC-QTOF-MS) analysis. Detailed steps are provided in the supplementary material.

2.4. Extraction and analysis of PPO enzyme activity in tea examples 6

Tea leaves (100 mg, fresh weight) from nonshaded and shade-treated plants were homogenized on ice and extracted using a Polyphenoloxidase Assay Kit (Beijing Leagene Biotech. Co., Ltd., Beijing, China) and a Dounce homogenizer. The extract was centrifuged at 12,000 g and 4 °C for 15 min, and the supernatant was transferred to a new tube. PPO enzyme activity was assayed spectrophotometrically by measuring the light absorbance of the oxidation products at 420 nm using catechol as substrate (Kumar et al., 2008), because the brown O-quinone formed led to an increase in the absorbance value. The mixed solution was boiled for 5 min and used as the blank control. The unit for quantifying the enzyme activity was described individually as an increase in absorbance of 0.001 per minute (∆A min1), expressed as micromoles of quinone formed per minute per milligram of protein.

2.5. Phylogenetic analysis of CsPPOs from various plant species The candidate genes of C. sinensis PPO were mined from the tea genome database (Wei et al., 2018) with two conserved domains, namely, PPO1_DWL (PF12142) and PPO1_KFDV (PF12143). Multiple alignment of amino acid sequences was conducted using CLUSTALX version 2.0 software (Thompson et al., 1997). Phylogenetic analysis was conducted by Molecular Evolutionary Genetics Analysis (MEGA) version 5.05 software (Lynnon Biosoft, Foster City, CA, USA) using the neighbor-joining statistical method (Saitou, & Nei, 1987) and bootstrapping with 1000 replicates. Amino acid sequences of the CsPPO proteins used for phylogenetic analysis are listed in Table S1.

2.6. Transcript expression analysis of CsPPOs in tea samples Total RNA was extracted from the frozen powder of each tea leaf sample (50 mg) separately using a Quick RNA Isolation Kit (Huayueyang Biotechnology Co., Ltd., Beijing,

7

China) following the manufacturer protocol. Detailed steps are provided in the supplementary material. The gene-specific primers are shown in Table S2.

2.7. CsPPO3 recombinant expression in Escherichia coli and Western blot analysis The recombinant pMD18-T-CsPPO3 plasmid (removing the chloroplast transit peptide at position 1-94 aa) was digested with Sal I and Xho I and recombined into expression vector pET32a containing an N-terminal (6×His)-tag (Novagen, Madison, WI, USA) using an InFusion HD Cloning Kit (Takara Bio Inc., Kyoto, Japan). After checking the ligation product via TSINGKE Biological Technology (Beijing, China), the plasmid was transferred into competent E. coli BL21 (DE3). Positive colonies were selected on Luria–Bertani (LB) agar plates containing 100 g mL1 ampicillin and reevaluated by PCR analysis. When the recombinant E. coli BL21 (DE3) cells expressing pET32a-CsPPO3 in LB medium containing 100 g mL1 ampicillin reached the mid-logarithmic phase (optical density at 600 nm was 0.6), 1.0 mmol L1 isopropyl β-D-thiogalactopyranoside (IPTG) was added, followed by culturing at 18 C with shaking at 200 rpm for an additional 8 h. The collected bacterial protein (10 g) was subjected to 12% SDS-PAGE. The protein concentration was calculated using the BCA Protein Assay Kit (Waltham, MA, USA). Following SDS-PAGE analysis, the target proteins were transferred from the unstained gel onto polyvinylidine fluoride (PVDF) using a semi-dry transfer apparatus (BioRad, Hercules, CA, USA). Precision Plus Protein Standards (10–250 kDa; Bio-Rad Laboratories, Hercules, CA, USA) and EasySee Western Marker (25–90 kDa; TransGen Biotech Co., Beijing, China) were used as molecular markers. Two antibodies, namely, His-Tag mouse monoclonal antibody (Pearland, TX, USA) and HRP-conjugated goat anti-mouse IgG (Pearland, TX, USA) were applied for Western blot analysis. Immunoreactive protein bands were analyzed using an ultrasensitive sandwich-type electrochemiluminescent (ECL) protocol according to 8

the manufacturer description.

2.8. CsPPO3 recombinant enzyme assay CsPPO3 recombinant enzyme assay was conducted following the method of Liu et al. (2010) with slight modifications. CsPPO3 recombinant enzyme was incubated with 0.1 mol L1 citrate buffer, 1.0 g L1 L-proline (pH 6.5), and 10 g L1 catechol in a ratio of 10:2:3 (v/v/v) at 37 C for 20 min. The assay was terminated by adding 1.0 mol L1 metaphosphoric acid (50 L). Consistent with the above PPO activity assay, CsPPO3 activity was spectrophotometrically detected at 460 nm by measuring the light absorbance of the catechol oxidation products. Furthermore, E. coli BL21-expressed protein CsPPO3 (10 g) was tested for the transformation from catechins to theaflavins. The reaction mixture containing 0.2 mmol L1 phosphate buffer (pH 7.4) and the mixed solution of EC+EGC, EC+ EGCG, ECG+EGC, and ECG+EGCG (100 L, 100 ng mL1 of each) was incubated at 37 C for 30 min. After the reaction, the final products were filtered through 0.22-m millipore express polyethersulfone (PES) membrane and subjected to UPLC-QTOF analysis as described above for theaflavins determination.

2.9. Statistical analysis Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Student’s t-test. A two-tailed Student’s t test was used to determine differences between tea leaves with and without shade treatment. A probability level of 5% (p ≤ 0.05) indicated a significant difference.

3. Results and discussion 9

3.1. Effect of shade treatment on catechin and theaflavin contents in preharvest tea leaves

To understand the effects of shade treatment on catechins (flavan-3-ols) and theaflavins in preharvest tea leaves, the contents and compositions of catechins (Figure 1A) and theaflavins (Figure 1B) were quantified. Compared with nonshaded leaves, shade treatment continuously consumed the total catechins content (from 36.88 to 31.37 mg/g FW), while the amount of total theaflavins in preharvest tea (C. sinensis cv. Yinghong No.9) leaves was significantly increased. Catechins from preharvest tea leaves mainly comprised C, CG, GC, GCG, EC, ECG, EGC, and EGCG, especially galloylated catechins (ECG and EGCG), as wellrecognized secondary metabolites, which are fundamental to their function and quality (Jiang et al., 2013). Except for GCG, the contents of seven individual catechins, namely, C, CG, EC, ECG, EGC, EGCG, and GC, were reduced to different degrees, with GC, EGC, C, and EC showing dramatic declines (Figure 1A). The primary theaflavin derivatives in black tea include TF1, TF2A, TF2B, and TF3 (Yang et al., 2008). Four theaflavin monomers containing TF1, TF2A, TF2B, and TF3 were substantially increased (Figure 1B). These findings indicated that the total catechin content decreased continuously, while the theaflavin content increased under shading treatment in preharvest tea (C. sinensis cv. Yinghong No.9) leaves. Shade management (dark treatments) of tea plants is a general method to enhancing the amount of quality-related metabolites in preharvest tea leaves (Yang et al., 2012; Chen et al., 2017; Yu, & Yang, 2019). However, prolonged shading/dark might lead to reduced tea leaf biomass (Fu et al. 2015). Notably, shade treatment stimulated the transformation of catechins into theaflavins in preharvest tea (C. sinensis cv. Yinghong No.9) leaves in the present study. To exclude the effects of different tea cultivars and tea cultivation environments, we also 10

investigated the effect of shade treatment on the catechin and theaflavin contents in preharvest tea leaves of two cultivars from Japan (cv. Yabukita and cv. Hoshinomidori). These two tea cultivars showed similar results to those of cv. Yinghong No. 9 grown in China, with shade treatment reducing the catechin contents (Figure 2A) and increasing the theaflavin contents (Figure 2B). These suggest that the transformation of catechins into theaflavins in tea leaves exposed to shade treatment might not be specific to certain tea cultivars, but a common phenomenon in tea leaves in response to dark conditions. Previously, the contents of tea catechins and polyphenols decreased significantly under shade treatment in preharvest tea leaves (Wan, 2003), which was attributed to the synthesis of catechins and polyphenols being inhibited (Wan, 2003; Wan, & Xia, 2015). Leucocyanidin reductase (LAR, EC 1.17.1.3) and anthocyanidin reductase (ANR, EC 1.3.1.77) might play critical roles in determining the catechin compositions (Wan, & Xia, 2015; Liu et al., 2018). ANR catalyzes the production of flavan-3-ol monomers (such as C, EC, and EGC), while LAR is essential for converting leucocyanidin or leucodelphinidin into the corresponding 2,3trans-flavan-3-ols (such as C, EC, EGC, and GC) (Zhang et al., 2016). Shade treatment significantly inhibited the expression of CsLAR and CsANR (Figure S1), which was a ratelimiting step in the production of ECG and EGCG. Notably, the theaflavins content after shading treatment had not been investigated in the previous studies. The catechins content was significantly affected by shading treatment, which might be largely due to the biodegradation or biotransformation of catechins in preharvest tea leaves (Figures 1 and 2), because catechins can be converted into theaflavins under catalysis by tea PPO (Liu et al., 2010; Teng et al., 2017). This suggested that catechin degradation might lead to the rapid increase in theaflavin content. Theaflavins are generated from catechins under the action of PPO during black tea manufacturing process and contribute to orange color of black tea infusion. In fresh tea 11

leaves, the colored compounds are mainly attributed to chlorophyll (Wan, 2003; Chen et al., 2017). The amount of chlorophyll was significantly reduced under darkness (Chen et al., 2017), while the chlorophyll level was accumulated after moderate shading treatment. Major catechins, including C, EC, GC and EGC, decreased significantly in tea leaves after shading treatment, as well as the amount of catechins and flavonols (Song et al., 2017; Liu et al., 2018). The above phenotypic features were consistent with the simultaneous down-regulation of biosynthetic genes (such as F3’H, FLS, ANS, ANR, LAR, DFR and CHSs) and transcription factors (such as MYB4, MYB12, MYB14 and MYB111) associated with flavonoid biosynthesis (Song et al., 2017; Liu et al., 2018).

3.2. Effect of shade treatment on PPO enzyme activity and CsPPO3 expression level in preharvest tea leaves (cv. Yinghong No.9)

To investigate whether theaflavins were derived from catechins in tea leaves, the critical enzyme that catalyzes this reaction, PPO, was determined in preharvest tea (C. sinensis cv. Yinghong No.9) leaves. PPO enzyme activity was significantly increased under shade treatment (2.97 U mg1 min1) compared with that in nonshaded leaves (1.84 U mg1 min1) (Figure 3A). This result suggested that catechins could be converted into theaflavins by tea PPO in vivo. This was consistent with the reported literature, which shows that PPO catalyzes the oxidization of catechin derivatives into theaflavins in vitro (Subramanian et al., 1999; Wan, & Xia, 2015; Teng et al., 2017). To further explore the key genes involved in the biosynthesis of theaflavins from catechins under shading treatment, three candidate genes encoding PPO, namely, CsPPO1, CsPPO2, and CsPPO3, were screened based on functional annotation of the tea genome (Wei et al., 2018). CsPPO1, CsPPO2 and CsPPO3 were generated with nested PCR and submitted to 12

GenBank under accession numbers MK977642, MK977643, and MK977644 (Table S1 and S2), corresponding to TEA005488, TEA032324, and TEA026892 in the tea genome (Wei et al., 2018), respectively. CsPPO1 contained a 1740-bp ORF that encoded for 579 amino acids with a predicted molecular mass of 64.6 kDa. CsPPO2 included an ORF of 1800-bp encoding 599 amino acids with a native molecular weight of 67.2 kDa. The full-length cDNA of CsPPO3 contained a 1788-bp ORF encoding a 595-amino-acid protein with a native molecular mass of 65.9 kDa. Two critical motifs, PPO1_DWL (pfam12142) and PPO1_KFDV (pfam12143), were highly conserved in tea CsPPO1, CsPPO2, and CsPPO3 (Figure S2), which was almost identical to observations reported for multiple plants, including Malus domestica (Kampatsikas et al., 2017), Oryza sativa (Yu et al., 2008), Pyrus pyrifolia (Liu et al., 2019), and Sorghum bicolor (Yan et al., 2017). Two conserved copper-binding domains, CuA and CuB, were observed in tea CsPPO1, CsPPO2, and CsPPO3 (Figure S2), because PPOs are bicopper metalloenzymes coordinated by three histidine residues (Demeke, & Morris, 2002). Phylogenetic analysis suggested that PPOs from C. sinensis cv. Yinghong No.9 and other organisms were divided into two clusters. Among these, CsPPO1, CsPPO2, and CsPPO3 were evolutionarily closer to dicotyledonous plants, such as Actinidia chinensis, Camellia nitidissima, Camellia ptilophylla, and Vitis vinifera, than to four monocotyledonous plants, namely, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, and Zea mays (Figure 3B). Overall, as phylogenetic analysis within the same branch is likely to share similar roles, we hypothesized that CsPPO1, CsPPO2, and CsPPO3 might be involved in converting catechins into theaflavins. We investigated the effect of shade treatment on the transcript levels of CsPPO1, CsPPO2, and CsPPO3 in tea leaves using qRT-PCR technology. CsPPO1 and CsPPO2 were significantly downregulated, while CsPPO3 was obviously upregulated under shade treatment (Figure 3A, p < 0.01), indicating that CsPPO3 might primarily be involved in PPO activation 13

during the shading period, and was a suitable candidate PPO for further investigation. The result suggested that CsPPO3 ( that originated from C. sinensis cv. Yinghong No.9) displayed a shading-inducible expression, which was significantly upregulated under shade treatment.

3.3. PPO function of CsPPO3 recombinant enzyme

Further research is urgently needed to understand the catalytic function of CsPPO3. CsPPO3 was introduced into prokaryotic expression vector pET32a to generate pET32aCsPPO3, and then transformed into E. coli BL21 (DE3) stain. Recombinant plasmid pET32aCsPPO3 was successfully constructed and overexpressed under the control of T7 promoter in E. coli BL21 competent cells (Figure 4A). Furthermore, Western blot analysis confirmed that the highly expressed product was in a fused form with the His-tag (Figure 4B). The purified CsPPO3 protein was reacted with catechol as substrate to measure enzyme activity. Recombinant CsPPO3 catalyzed the oxidation of catechol with an activity of 35.17 U mg1 min1 (Figure 5A), which was consistent with that reported for PPO in vitro (Liu et al., 2010; Teng et al., 2017; Liu et al., 2019). Previous studies have shown that TF1, TF2A, TF2B, and TF3 can be biosynthesized by EC+EGC, EC+EGCG, ECG+EGC, and ECG+EGCG, respectively (Wan, 2003), but this required verification. Therefore, assays of the oxidization polymerizing reaction with different catechin combinations (namely, EC+EGC, EC+EGCG, ECG+EGC, and ECG+EGCG) and polyphenol oxidase CsPPO3 as catalyst were performed. The results showed that recombinant CsPPO3 could catalyze the transformation of EC+EGC into TF1 as a main product, and samller amounts of TF2A and TF2B, in vitro, the conversion of EC+EGCG to TF1 and TF2A as main products and small amount of TF2B, the conversion of ECG+EGC to large quantity of TF1 and small amounts of TF2A and TF2B, and the conversion of ECG+EGCG to TF1, TF2A, TF2B, and TF3 in vitro (Figure 5B). CsPPO3 was 14

capable of extensively producing TF1 with three different catechin combinations (EC+EGC, EC+EGCG and ECG+EGC, see the y axis values of Figure 5B), and was responsible for TF3 generation from ECG and EGCG,. These results suggest that CsPPO3 was the key enzyme catalyzing the conversion of catechins into theaflavins. Generally, based on the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), PPO (EC 1.10.3.1) belongs to a multigene family, is composed of monophenol monooxygenases (such as tyrosinase, E.C. 1.14.18.1), catechol oxidases (E.C. 1.10.3.1) and laccases (E.C. 1.10.3.2). PPO is a group of Cu-binding proteins that is widely distributed among plants and can catalyze the oxidization of phenolics into o-quinones (catechins into theaflavins in tea leaves). Catechin, epicatechin and caffeic acid derivatives are major substrates of PPOs in many food and agricultural productions (Taranto et al., 2017). PPO-mediated browning affects the commercial value of agricultural productions including apple, banana, eggplant, grape, mango, pear, peach, lettuce and potato, while it is of great importance in the production of black tea, coffee and cocoa (Taranto et al., 2017), and its physiological reactions are catalyzed by PPOs. Up to now, PPO has been identified in more than 26 plant species, such as apple, chestnut kernel, C. sinensis, rice, Malpighia glabra, Pyrus pyrifolia, Salvia miltiorrhiza, tomato and wheat (Kar and Mishra, 1976; Demeke and Morris, 2002; Kumar et al., 2008; Liu et al., 2009; Tran et al., 2012; Gong et al., 2015; Kampatsikas et al., 2017; Huang et al., 2018). However, only two complete coding sequences of PPO had been cloned from C. sinensis (NCBI accession no.: AY659975 and EF635860), which were functionally characterized and prokaryotic expressed in Escherichia coli (Liu et al., 2010; Wu et al., 2010; Singh et al., 2017), converting epicatechins to theaflavins and thearubigins in vitro. Herein, CsPPO3 was able to yield massive TF1, and small amounts of TF2A, TF2B and TF3 (Figure 5B). Due to the economic importance of browning, the physico-chemical properties of PPOs 15

have been largely investigated. However, their role in plant physiology is less clear. Multiple studies reveal a positive correlation between PPO transcription and resistance/tolerance to abiotic and biotic stresses. Herbivores, mechanical damage, pathogens and plant hormone (such as ethylene (ET), methyl jasmonate (MeJA), jasmonic acid (JA), and salicylic acid (SA)) treatment, or even the light-inducible regulation (Tegelberg et al., 2008), can upregulate PPO activity in multiple plants, such as apple, pineapple, poplar, strawberry, tea and tomato (Shetty et al., 2012; Jia et al., 2016; Li et al., 2017; Huang et al., 2019; Liao et al., 2019), Unfortunately, the targeted genes are responsible for the upregulated PPO activity induced by the shading treatment remains unknown. Previous studies have shown that oxidation of catechins into TFs occurred in the broken cells of tea leaves (Zhang et al., 2019). PPOs of tea leaves are found to be localized in the chloroplasts while the phenolic substrates are stored in the vacuole. The disruption of plant cell compartmentalization, due to handling, interactions with pests and pathogens, senescence and wounding during postharvest processing and storage, results in contact between PPOs and phenolic substrates (Tran et al., 2012). These procedures have a considerable impact on the quality of black tea, particularly color, flavor, and fragrance (Wan, & Xia, 2015; Zeng et al., 2016). Amazingly, present study seems to indicate that PPO happens in whole unbroken tea leaves. The involvement of PPOs is not associated with the loss of sub-cellular compartmentation, leading to the contact of PPOs with their phenolic substrates, is seemingly counter-intuitive. However, the up-regulation of PPO can be inspired by the abiotic stresses, which may be result from the development of spontaneous necrotic lesions. The up-regulated expression of PPO was detected in Juglans regia subjected to water deficit. Besides, PPOsilenced plants (that PPO activity was suppressed) had displayed a higher water-resistance ability (Araji et al., 2014). It is presumed that PPO may be linked to photosynthesis as an oxygen buffer or interacting with the Mehler-peroxidase reaction, but chloroplast phenolic 16

substrate has not yet been identified (Boeckx et al., 2015). Possibly, PPO may be associated with its metastatic properties. Given that all plant PPO genes encode mature proteins of 52-62 kDa, with 8-12 kDa transit peptides targeting to the thylakoid lumen (Mayer, 2006). Transit peptides are N-terminal extensions that serve to route nuclear encoded proteins into chloroplasts via a post-translational mechanism (Lee et al., 2008). The reported PPO1 contained a 288-bp fragment with the signal sequence, which could not be recognized by the E. coli expression (Wu et al., 2010). Tea CsPPO3 also consisted of an N-terminal 84-aa transit peptide (Figure S2), which were likely to be employed to transport the needed proteins. Further studies are required to validate this hypothesis. Growing evidence has been reported from systematic research on PPO regarding subcellular localization, physico-chemical properties, extraction and purification, enzymatic oxidation into theaflavins, and immobilized enzymes (Kumar et al., 2008; Liu et al., 2010; Gong et al., 2015; Teng et al., 2017). However, PPO enzymes from preharvest tea leaves and the enzymatic biosynthesis of theaflavin monomers, such as TF1, TF2A, TF2B, and TF3, have rarely been reported. In our study, we molecularly identified three PPO genes from tea (C. sinensis cv. Yinghong No.9) based on a genome-wide investigation, which possessed several conserved motifs from the reported PPO (Figure 3B, S2). CsPPO3 was significantly upregulated, in agreement with the increased PPO activity under shade treatment (Figure 3A). Furthermore, the 5' non-coding region of the PPO is relatively low, especially the promoter region, which may result in the differential transcription levels in plant (Mayer, 2006; Shetty et al., 2012). Five light-responsive cis-elements, namely, AE-box, Box 4, G-box, GT1 motif, and MRE motif (Guan et al., 2018), were widespread in the 1000-bp putative CsPPO3 promoter region (Figure S3). Two transcript factors, elongated hypocotyl 5 (HY5, positive regulation factor) and phytochrome-interacting factor 3 (PIF3, negative regulation factor), were stably regulated by light in plants (Toledo-Ortiz et al., 2014; Chenge-Espinosa et al., 17

2018). CsHY5 was notably downregulated, while CsPIF3 was significantly upregulated under shade treatment (Figure S1), in accordance with CsPPO3 (Figure 3A). UVR8-mediated signaling genes are transcriptionally correlated with the shading reduced flavonoid biosynthesis, leading to a reduction of HY5, but an increase of PIF3 stabilization (Liu et al., 2018), and thus stimulates the reduction of major tea catechins and the accumulation of theaflavins. Therefore, tea CsPPO3 was responsible for PPO activation during the shading period, might result from the light-inducible elements in CsPPO3 promoter regions.

4. Conclusions

The present study has shown that the increased theaflavin contents in preharvest tea (C. sinensis cv. Yinghong No.9) leaves originate from the decrease in catechins catalyzed by CsPPO3 (Figure 6). Catechins have been observed to convert into theaflavins under PPO catalysis in vitro, such as in continuous fermentation, and our results provided similar information regarding polyphenol oxidase CsPPO3, similar to the reported PPO in vitro, which regulated theaflavin accumulation in preharvest tea leaves grown in the shade. The results obtained in this study will improve understanding of theaflavins formation and its response to environmental factors at the preharvest tea stage.

Acknowledgments This study was supported by the financial support from the National Key Research and Development Program of China (2018YFD1000601), the Guangdong Natural Science Foundation for Distinguished Young Scholar (2016A030306039), the Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar (50117G25002), and the Foundation of Key Laboratory of South China Agricultural Plant Molecular Analysis and 18

Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences (KF201906). The authors thank Prof. Dr. Kunbo Wang at the Hunan Agricultural University, China for kindly providing the sequence of CsPPO.

Competing financial interests

The authors declare no competing financial interests.

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Figure captions

Figure 1 Effect of shade treatment on catechins (A) and theaflavins (B) in tea (C. sinensis cv. Yinghong No.9) leaves. (A) C, catechin. CG, catechin gallate. CK, without shade treatment. EC, epicatechin. ECG, epicatechin gallate. EGC, epigallocatechin. EGCG, epigallocatechin gallate. FW, fresh weight. GC, gallocatechin. GCG, gallocatechin gallate. TR, with shade treatment. All data are expressed as mean ± S.D. (n=9). Significant differences between tea leaves without shade treatment and tea leaves with shade treatment are indicated (* p ≤ 0.05, and ** p ≤ 0.01). (B) CK, without shade treatment. FW, fresh weight. TF1, theaflavin. TF2A, theaflavin-3-gallate. TF2B, theaflavin-3′-gallate. TF3, theaflavin-3,3′-digallate. TR, with shade treatment. All data are expressed as mean ± S.D. (n=9). Significant differences between tea leaves without shade treatment and tea leaves with shade treatment are indicated (* p ≤ 0.05, and ** p ≤ 0.01).

Figure 2 Effect of shade treatment on catechins (A) and theaflavins (B) in tea leaves of the two cultivars (cv. Yabukita and cv. Hoshinomidori) grown in Japan. (A) C, catechin. CG, catechin gallate. EC, epicatechin. ECG, epicatechin gallate. EGC, epigallocatechin. EGCG, epigallocatechin gallate. FW, fresh weight. GC, gallocatechin. GCG, gallocatechin gallate. All data are expressed as mean ± S.D. (n=3). Significant differences between tea leaves without shade treatment and tea leaves with shade treatment are indicated (* p ≤ 0.05, and ** p ≤ 0.01). (B) FW, fresh weight. ND, not detected. TF1, theaflavin. TF2A, theaflavin-3-gallate. TF2B, theaflavin-3′-gallate. TF3, theaflavin-3,3′-digallate. All data are expressed as mean ± S.D. (n=3). Significant differences between tea leaves without shade treatment and tea leaves with shade treatment are indicated (* p ≤ 0.05, and ** p ≤ 0.01).

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Figure 3 Effect of shade treatment on the PPO enzyme activity and the transcript levels of three PPO genes in pre-harvest tea (C. sinensis cv. Yinghong No.9) leaves. (A) Analysis of the PPO activity and the mRNA levels of corresponding coded PPO genes in tea (C. sinensis cv. Yinghong No.9) leaves. CK, without shade treatment. TR, with shade treatment. All data are expressed as mean ± S.D. (n=9). Significant differences between tea leaves without shade treatment and tea leaves with shade treatment are indicated (* p ≤ 0.05, and ** p ≤ 0.01). (B) Molecular phylogenetic trees of CsPPOs and PPOs from other plants. Multiple alignments were carried out using the neighbor-joining method with MEGA 6.06 software. Values at branch-points of tree topology indicate percentage frequencies after 1000 iterations. The NCBI accession numbers are as follows: AcPPO (PSR98570), BdPPO (Bradi2g52260), CnPPO (ACM43505), CpPPO (ABF19601), DiPPO (AKJ70977), DzPPO (XP_022720501), MdPPO (BAA21676), OsPPO (LOC_Os01g58100), SbPPO1 (Sb10g024220), SbPPO2 (Sb03g035850), TcPPO (XP_017978714), VvPPO (CAN61652) and ZmPPO (ACG28948), which represent PPO proteins from Actinidia chinensis, Brachypodium distachyon, Camellia nitidissima, Camellia ptilophylla, Dimocarpus longan, Durio zibethinus, Malus domestica, Oryza sativa, Sorghum bicolor, Theobroma cacao, Vitis vinifera and Zea mays, respectively.

Figure 4 Identification of CsPPO3 recombinant protein expressed in Escherichia coli BL21. (A) SDS-PAGE analysis of synthetically constructed CsPPO3 in E. coli BL21 (DE3) cells. Black arrow indicates target protein. pET32a is the prokaryotic expression vector, which is used for the CsPPO3 recombinant protein. (B) Western blot analysis of E. coli-expressed CsPPO3.

Figure 5 Functional identification of CsPPO3 from C. sinensis cv. Yinghong No.9. 26

(A) Enzyme activities of CsPPO3 expressed in E. coli BL21 (DE3) cells. N.D., not detected. All data are expressed as mean ± S.D. (n=9). Significant differences between tea leaves without shade treatment and tea leaves with shade treatment are indicated (** p ≤ 0.01). (B) Analysis of the theaflavins content in products of CsPPO3 enzyme assay. N.D., not detected. EC, epicatechin. ECG, epicatechin gallate. EGC, epigallocatechin. EGCG, epigallocatechin gallate. TF1, theaflavin. TF2A, theaflavin-3-gallate. TF2B, theaflavin-3′-gallate. TF3, theaflavin-3,3′-digallate. All data are expressed as mean ± S.D. (n=9). Significant differences between pET32a with catechins and pET32a-CsPPO3 recombinant protein with catechins are indicated (** p ≤ 0.01). Figure 6 A summary of CsPPO3 stimulates the transformation from catechins to theaflavins in preharvest tea leaves under shade treatment. Here, the transcription level of CsPPO3 gene is obviously upregulated in vivo under shade treatment, due to the activation of the PPO enzyme activity. In parallel experiment, CsPPO3 is responsible for catalyzing the conversion of catechins into theaflavins in E. coli (in vitro).

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Figure 1 28

Figure 2 29

Figure 3 30

Figure 4 31

Figure 5 32

Figure 6 33

Declaration of interests  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Author Contributions:

Ziyin Yang conceived and designed the experiments; Zhenming Yu, Yinyin Liao, Lanting Zeng, and Fang Dong conducted the experiments; Zhenming Yu and Yinyin Liao analyzed the results; Zhenming Yu and Ziyin Yang wrote the manuscript; Ziyin Yang and Naoharu Watanabe revised the manuscript. all authors read and approved the final manuscript.

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Graphical Abstract

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Highlights ► Shade treatment reduced the contents of most catechins in preharvest tea leaves. ► Shade treatment increased the theaflavin contents in preharvest tea leaves. ► Shade treatment increased polyphenoloxidase (PPO) activity in preharvest tea leaves. ► CsPPO3 was highly expressed under shade treatment. ►CsPPO3 recombinant protein exhibited PPO function.

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