Metabolic Changes of Caffeine in Tea Plant (Camellia sinensis (L.) O

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Metabolic Changes of caffeine in tea plant (Camellia sinensis (L.) O. Kuntze) as denfence response to Colletotrichum fructicola yuchun Wang, Wen-Jun Qian, Na-Na Li, Xinyuan Hao, Lu Wang, Bin Xiao, Xinchao Wang, and Yajun Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02044 • Publication Date (Web): 19 Aug 2016 Downloaded from http://pubs.acs.org on August 19, 2016

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Journal of Agricultural and Food Chemistry

Metabolic Changes of caffeine in tea plant (Camellia sinensis (L.) O. Kuntze) as denfence response to Colletotrichum fructicola Yu-Chun Wang†,§, Wen-Jun Qian†,§, Na-Na Li§, Xin-Yuan Hao§, Lu Wang§, Bin Xiao†*, Xin-Chao Wang§*, Ya-Jun Yang†,§* †

College of Horticulture, Northwest A & F University, Yangling 712100, People’s

Republic of China §

Tea Research Institute, Chinese Academy of Agricultural Sciences/National Center

for Tea Improvement/Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Hangzhou 310008, People’s Republic of China

*Corresponding Authors: Xin-Chao Wang Tel/Fax: +86 571-8665 3162. Email: [email protected] Ya-Jun Yang Tel/Fax: +86 571-8665 0226. Email: [email protected] Bin Xiao Tel/Fax: +86 29-8708 1195. Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Tea plant (Camellia sinensis) is one of the most economically valuable crops in

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the world. Anthracnose can affect the growth of leaves and cause serious yield losses

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of tea. Tea plants are rich in secondary metabolites, however, their roles in resistance

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to anthracnose are unclear. Herein we compared the contents of total phenolic,

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catechins and caffeine in two cultivars with different resistance to anthracnose during

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Colletotrichum fructicola infection. The (-)-epigallocatechin-3-gallate (EGCG),

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(+)-catechin (C), caffeine, and critical regulatory genes were induced in C.

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fructicola-resistant tissues. In vitro antifungal tests showed that caffeine more strongly

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inhibited mycelial growth than tea polyphenols and catechins. Both electron

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microscopy and bioactivity analysis results showed that caffeine can affect mycelial

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cell walls and plasma membranes. Through promoter sequences analysis, a number of

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stress response-related cis-acting elements were identified in S-adenosylmethionine

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synthetase and tea caffeine synthase. These results demonstrated that (-)-EGCG, (+)-C,

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and caffeine may be involved in resistance of tea plants to anthracnose.

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Keywords: Theaceae, disease resistance, tea ployphenols, catechins, Colletotrichum

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INTRODUCTION

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Tea plant (Camellia sinensis (L.) O. Kuntze) is a widely cultivated crop that plays

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an important role in the economies of countries where it is planted, particularly in

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China, the nation with the highest tea production. Therefore, increasing the production

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and improving the quality of tea have many important economic implications.

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Anthracnose, caused by Colletotrichum spp., is a disease that occurs commonly on the

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leaves of tea plants, resulting in damage of tea leaves and ultimately influencing tea

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yield and quality.1 Although some progress has been made in determining the

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mechanisms by which plants become resistant to anthracnose,2,3 little is known about

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the interaction between Colletotrichum and tea plants.

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Tea plants are rich in various secondary metabolites that possess direct and

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indirect antimicrobial activities in plants.4-6 Tea polyphenols (TP) is the most

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important secondary metabolite in tea plants. Many in vitro studies have reported that

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TP have a broad antimicrobial spectrum against various pathogens.7-9 In addition,

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spraying TP on plants can improve their resistance to disease.10 Catechins are the

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dominant flavonoids of tea plants, which are classified as ester type or non-ester type.

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The major non-ester type catechins include (-)-epicatechin (EC), (-)-epigallocatechin

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(EGC), (+)-catechin (C) and (+)-gallocatechin (GC), and the major ester type

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catechins include (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate

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(EGCG).11 In tea plants, the concentration of ester type catechins is much greater than

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that of non-ester type catechins.12,13 Previous in vitro studies have shown that

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catechins extracted from tea have varying degrees of antibacterial effects, with 3



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different catechins ordered according to their antibacterial effects, from strong to

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weak, as follows: EGCG > EGC > ECG > EC > C.14

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Catechins and caffeine are thought to exert their antimicrobial effects by multiple

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mechanisms. The combination of EGCG and antimycotics has been shown to damage

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biofilms of Candida species.15 EGCG may also enhance plant resistance to disease via

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modulation of jasmonic acid signaling.16 Tea plants have abundant caffeine (1, 3,

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7-trimethylxanthine).17 Biotic stress can increase caffeine content (CC) and enhance

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hypersensitive responses in plants, an effect that may be mediated by the increase in

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endogenous salicylic acid by caffeine.18 Furthermore, plants may depend on the toxic

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effects of caffeine to help combat pathogens.19 Despite these findings, few studies

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have been conducted on the secondary metabolites of tea plants as well as their in

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vitro antifungal activity against Colletotrichum.

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During pathogen-host interactions, plants activate multiple interconnected

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defense signaling pathways and metabolic pathways.20 In tea plants, the non-ester

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type catechins and caffeine biosynthesis pathways have been illustrated clearly

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(Figure 1A and 2A).6,21 Many genes associated with the flavonoid pathway are

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involved in the resistance of plants to disease. For example, in grapevine cultivars that

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are resistant to anthracnose, the flavonoid pathway-associated genes, such as chalcone

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synthase, chalcone isomerase, and dihydroflavonol-4-reductase, are expressed at

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higher levels than in susceptible cultivars.22,23 Leucoanthocyanidin reductase (LAR)

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and anthocyanidin reductase (ANR) play important roles in the flavonoid pathway of

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tea plant. Leucoanthocyanidin can be transformed by LAR into flavan-3-ols (C, GC) 4


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and by ANR into epi-flavan-3-ols (EC, EGC). 24 However, little is known about the

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relationship between the two enzymes and disease resistance in tea plant.

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The caffeine pathway is part of purine metabolism. S-adenosylmethionine

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synthetase (SAMS) plays a crucial role in the caffeine pathway, as it is the only

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methyl donor to xanthosine and is a major source of xanthosine in tea plant.25

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Caffeine is synthesized through the conversion of theobromine by caffeine synthase.

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In tea plant, all the identified tea caffeine synthase (TCS) are TCS1 type.26 It is

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unclear whether these critical genes in the caffeine pathway are involved in the

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response of tea plants to Colletotrichum.

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Base on continuous field observation, we found that anthracnose is only diseased

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on the mature leaves but not young leaves of tea plants. To determine the roles of

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secondary metabolites in the tea plant response against Colletotrichum sp., using the

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susceptible cultivar Longjing43 (LJ43) and the resistant cultivar Zhongcha108

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(ZC108) as experiment materials, we determined the changes in the content of

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secondary metabolites (total phenolic, catechins and caffeine) and the expression of

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their related genes in plants at different levels of maturity after Colletotrichum sp.

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inoculation. We estimated the antifungal effects of related reagents in vitro. We also

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analyzed the promoters of critical genes associated with plant disease resistance.

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Finally, we clarified the possible antifungal mechanism of caffeine in vitro.

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MATERIALS AND METHODS Plants, Fungal Materials and Treatments. Five-year-old potted tea plants of the 5



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resistant cultivar cv. Zhongcha108 (ZC108) and the susceptible cultivar cv.

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Longjing43 (LJ43)27 with different levels of resistance to anthracnose were grown in

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the greenhouse of the Tea Research Institute, Chinese Academy of Agricultural

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Sciences (TRI, CAAS, N30°10', E120°5'), Hangzhou, China. The plants were

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maintained in the greenhouse at 30°C during the day and 20°C at night with cycles of

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14 h light and 10 h darkness and relative humidity of 80%-90%. Healthy plants were

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selected at the one bud and 5th leaf stages for the inoculation experiment. The

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pathogenic C. fructicola isolate L33 was originally isolated from the disease leaves of

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LJ43 at the field of TRI, CAAS and identified by morphological characteristics and

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partial sequencing of its genome as described by Weir, et al.28 Conidia suspension was

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prepared by growing the L33 isolate on potato dextrose agar (PDA) for 7 days and

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washing with sterile water. The spore concentration was determined using a

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hemocytometer and adjusted to 106 spores/mL for inoculation.

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Young and mature plant tissues were inoculated with conidia suspension (106

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spores/mL). Young tissues (YT) were defined as a bud and 3rd leaves, and mature

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tissues (MT) were defined as 4-5th leaves. In order to coat all tissue surfaces with

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conidia, the spore suspension was sprayed onto the tissues until run-off. Control

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plants were sprayed with sterile water. The YT and MT of two branches of a potted

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plant were sampled at 0 (before inoculation), 24, and 72 hours post inoculation. Each

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biological replicate was collected twice. The first group was prepared as fresh tissue

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for analysis of secondary metabolites. The other groups were immediately transferred

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to liquid nitrogen, stored at -80°C in a freezer, and subsequently used for quantitative 6


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real time PCR (qRT-PCR) analysis.

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Preparation of Sample Extracts. Extraction of secondary metabolites was

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performed according to the methods described by Liang, et al.29 Fresh tea tissues

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(0.25 g) were homogenized, and then ground and extracted with 10 mL 75% (v/v)

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ethyl alcohol for 10 min. Samples were centrifuged at 4000 × g for 10 min (4°C) and

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the supernatant was collected (Eppendorf, Germany) and diluted with 75% ethyl

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alcohol to 10 mL.

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Determination of Total Phenolic Content. Total Phenolic Content (TPC) was

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determined using the Folin-Ciocalteu assay described by Lai, et al.30 Gallic acid

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(Sigma-Aldrich Co., USA) was used as a standard. The concentration of TPC was

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measured by spectrophotometry (UV 2550, Shimadzu, Japan).

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Determination of Catechins and Caffeine Contents. Catechins and caffeine

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contents were determined by high-performance liquid chromatography (HPLC)

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according to the methods described by Wang, et al.31 The samples were filtered

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through a 0.45-µm millipore filter and analyzed by a 2695-2489 HPLC System

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(Waters alliance, USA) under the following conditions: inject volume: 10 µL; column:

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C12, 4.6 mm × 250 mm (Phenomenex, USA); oven temperature: 40°C; phase A: 9%

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(v/v) acetonitrile, 2 % (v/v) acetic acid, and 0.02% (m/v) EDTA; phase B: 80% (v/v)

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acetonitrile, 2% (v/v) acetic acid, and 0.02% (m/v) EDTA; flow rate: 1 mL/min. The

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absorbance at 280 nm was used to monitor peak intensities in real time. The HPLC

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chemical standards were (−)-EGCG, (−)-EGC, (−)-ECG, (−)-EC, (−)-GCG, (+)-C,

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(−)-GC, and caffeine (Sigma-Aldrich Co., USA). 7



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qRT-PCR Analysis. Total RNA was extracted from all the samples using an

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RNAprep Pure kit (Tiangen, China). An aliquot of 1 µg of total RNA was converted to

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first-strand cDNA using a PrimeScript RT enzyme with a gDNA eraser (Takara,

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Japan). qRT-PCR was performed on an Applied Biosystems 7500 Sequence Detection

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System using SYBR® Premix Ex Taq™ II (Takara, Japan). In order to ensure the

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accuracy of target genes, the genes that have been reported to have different

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expressions in different tissues of tea plant were used for qRT-PCR analysis.

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Gene-specific primers and GenBank numbers of target genes were listed in Table S1.

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The polypyrimidine tract-binding protein (CsPTB1) gene was used as an internal

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control.27

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In vitro antifungal bioassay. The antifungal activities of TP, catechins, and

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caffeine were assessed using the Poison Food Technique in solid media as described

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by Zhang, et al.32 TP, catechins and caffeine (Aladdin, China) were dissolved in sterile

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distilled water and then added to the sterilized PDA at 40-50°C to obtain final

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concentrations ranging from 0.03125 to 16 mg/mL. Sterile distilled water was used as

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control. Mixtures were poured into 9 cm petri plates and Mycelial discs (9 mm

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diameter) taken from 5-day-old cultures were inoculated at the center of each petri

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dish. The inoculated plates were incubated at 25°C overnight and mycelial growth

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diameters were measured after 24 h and 48 h. The inhibition relative to the control

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was calculated as previously described.32 The EC50 of each tested compound was

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calculated using the mycelial growth inhibition obtained 24 h after treatment. The

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minimum inhibitory concentration (MIC) of caffeine is the lowest concentration of 8


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caffeine that resulted in no visible mycelial growth after 72 h of incubation.33

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Determination of the Effect of Caffeine on Hyphae Bioactivity. The hyphae

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bioactivity was analyzed using the method of Shao, et al.34 Mycelial discs (9 mm)

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taken from 5-day-old cultures were inoculated into potato dextrose broth (PDB)

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medium and allowed to grow at 25°C overnight at 160 rpm for 48 h. Caffeine (MIC: 4

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mg/mL) was added to the PDB medium and the samples were collected from the

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culture solution at 0, 12, and 24 h after treatment. The culture solution was

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centrifuged at 4000 × g for 10 min and was used to determine the alkaline phosphatase

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(ALP) activity, release of 260 nm absorbing material (A260), and the activity of

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methane dicarboxylic aldehyde (MDA) and superoxide dismutase activity (SOD) by

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assay kits (Nanjing Jiancheng Institute of Bioengineering, China). Absorbance was

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determined using a spectrophotometer (UV 2550, Shimadzu, Japan). The untreated

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PDB medium was used as control.

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Effect of Caffeine on Hyphal Morphology and Ultrastructure. To observe the

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effect of caffeine on hyphae morphology of C. fructicola, mycelial discs (4 mm

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diameter) from 5-day-old colonies were cultured on PDA containing 0.869 mg/mL

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(EC80) of caffeine and incubated at 25°C for 5 days. PDA without caffeine was used

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as control. The hyphae samples were analyzed by scanning (SEM) and transmission

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(TEM) electron microscopy as described by Yue, et al.35 using a Hitachi Model

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TM-1000 SEM (Hitachi, Japan) and a Model H-7650 TEM (Hitachi, Japan)

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respectively.

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Cloning and Analysis of the Promoters. The promoter sequence was amplified 9



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by general PCR. The genomic DNA of the healthy leaves was extracted using a Plant

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Genomic DNA Kit (Tiangen, China). Primers specific to promoter were designed by

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Primer Premier 5 (Premier Biosoft International, CA) (Table S1) based on incomplete

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genomic information (LJ43). The PCR amplification was performed in 50 µL reaction

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mixtures by following the manufacturer’s instructions of PrimeSTAR® HS DNA

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polymerase (TaKaRa, China). PCR product was gel-purified and cloned into the

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pMD18-T vector (TaKaRa, China) for sequencing. The cis-element in the obtained

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sequence was identified by PlantCARE.36

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Statistical Analysis. SPSS 18 (SPSS Inc., USA) was used to conduct statistical

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analyses. One-way analysis of variance (ANOVA) was used for statistical analyses

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and p < 0.05 was considered significant. Differences between all tissues and LJ43-MT

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were assessed as least significant difference (LSD) test. Values were expressed as

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mean ± standard error. EC50 values were obtained using logistic and probit regression.

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Graphs were generated using Prism 6 (GraphPad Software Inc., USA) and Illustrator

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CS6 (Adobe Software Inc., USA).

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RESULTS

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Changes in TPC in Tea Plant after C. fructicola Infection

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The TPC, total catechins content (TCC), and biosynthesis-related genes in MT

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and YT of ZC108 and LJ43 were analyzed after C. fructicola inoculation. The levels

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of TPC and TCC were enhanced in both cultivars after C. fructicola inoculation.

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These changes were particularly salient in the ZC108 at 24 h compared to the LJ43. 10


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Catechins are composed of ester type and non-ester type catechins. The content of

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ester type catechins elevated in YT and MT of ZC108 as well as in YT of LJ43, but

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was unchanged in MT of LJ43. The (-)-EGCG content in ZC108 was higher than that

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in LJ43-MT (p < 0.05). In contrast, the (-)-ECG content in LJ43 was higher than that

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in ZC108 (p < 0.05). The content of non-ester type catechins increased in the ZC108

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tissues but decreased in the LJ43 tissues. The (+)-C content in both ZC108 and

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LJ43-YT increased more than that in LJ43-MT (p < 0.05), while the initial content of

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(-)-EC in LJ43 was higher than that in ZC108 (p < 0.05) (Figure 1B). LAR (GenBnak:

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HP763707)21, a key gene in the biosynthesis of (+)-C and (+)-GC, was expressed

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markedly in the YT of both cultivars relative to MT. ANR (c147477)6, a key gene in

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the biosynthesis of (-)-EC and (-)-EGC, was increased slightly in LJ43-YT but was

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down-regulated in LJ43-MT at 24 h after infection; and no significant change was

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observed in ZC108 tissues (Figure 1C). These results suggest that polyphenols,

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particularly (-)-EGCG and (+)-C, might involve in the tea plant defense to C.

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fructicola.

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Changes in CC in Tea Plant after C. fructicola Infection

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The CC and biosynthesis-related genes in MT and YT of ZC108 and LJ43 were

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analyzed after C. fructicola inoculation. After C. fructicola inoculation, the CC of the

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ZC108 was increased markedly at 24 h, but was not significantly changed in LJ43

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(Figure 2B). SAMS (AJ277206)25 and TCS1 (AB031280)37, the key genes in the

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biosynthetic pathway of caffeine, were significantly up-regulated in the YT of both

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tea cultivars in response to C. fructicola at 24 h after inoculation. Expression of TCS1 11



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in the resistant tissues (ZC108 tissues and LJ43 - YT) was significantly different from

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that of diseased tissues (LJ43 - MT) (p < 0.05) (Figure 2C). These results indicated

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that caffeine plays an important role in the resistance of tea plants to C. fructicola.

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In Vitro Effects of Compounds on Mycelial Growth of C. fructicola

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The mycelial growth of C. fructicola was examined after treatment with different

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concentrations of TP, catechins and caffeine in vitro (Figure 3, Figure S1). All these

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compounds inhibited mycelial growth in a dose-dependent manner. Caffeine

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demonstrated significantly greater antifungal effects than TP and catechins with an

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EC50 of 0.555 mg/mL, which was greater than that of TP (4.832 mg/mL) and catechins

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(9.323 mg/mL) (Figure 3A, B). Moreover, caffeine had a long-lasting antifungal effect,

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and the MIC value was only 4 mg/mL (Figure 3C). Since caffeine had effective

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antifungal activity against C. furcticola, caffeine at the MIC was used in subsequent

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experiments.

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Effects of Caffeine on Cell Wall and Membrane of C. fructicola

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In order to understand how caffeine inhibited C. fructicola, the integrity of cell

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wall and plasma membrane of hyphae was analyzed (Figure 4). The bioactivities of

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treated hyphae were significantly increased compared with control. ALP and release

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of intracellular components have been shown to be good indicators of integrity of cell

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wall and membrane.34,38 ALP was increased rapidly after treatment with caffeine (4

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mg/mL) for 12 h (Figure 4A) and release of 260 nm absorbing material (A260 nm) was

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enhanced after treatment with caffeine (4 mg/mL) for 24 h (Figure 4B), suggesting the

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integrity of cell wall was affected and plasma membrane permeability increased. In 12


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addition, the activities of MDA and SOD, which have relationships with

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stress-tolerance,7 were significantly increased after treatment with caffeine (4 mg/mL)

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(Figure 4C, D). These results indicated that the cell wall and plasma membrane were

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damaged under caffeine treatment.

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Effects of Caffeine on Hyphae Morphology and Ultrastructure

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To further characterize the antifungal mechanism of caffeine against C. fructicola,

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the hyphae morphology and ultrastructure after treatment with 0.869 mg/mL (EC80)

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caffeine were observed by SEM (Figure 5) and TEM (Figure 6). SEM analysis

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showed that the untreated hyphae exhibited characteristic morphology, with healthy,

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robust, and uniform growth, and had plump cell bodies (Figure 5A-C). In contrast, the

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treated hyphae displayed shriveling, curving, and collapse morphology (Figure 5D-F).

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Figure 6 shows the ultrastructure of C. fructicola. The healthy hyphae have intact cell

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walls and plasma membranes, and clear vision of organelles (Figure 6A, C). In

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contrast, the treated hyphae exhibited that the cell wall was thickened; the organelles

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and cytoplasm were degradation, especially mitochondria; the plasma membrane

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became unclear and finally the hyphae appeared empty holes (Figure 6B, D, E).

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Stress-Relevant Cis-Elements in the Promoters of Caffeine Pathway-Related

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Genes

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In an effort to explain the increased expression of the caffeine pathway-related

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genes TCS1 and SAMS after C. fructicola inoculation, we cloned and analyzed their

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promoter regions. The TCS1 promoter in ZC108 (-1094 bp upstream from start codon)

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was 17 bp different from that in LJ43, and the SAMS (-1512 bp upstream from start 13



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codon) was 10 bp different from that in LJ43 (Figure S2). The genes had the same

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cis-acting elements or MYB binding site in both cultivars. A number of stress

262

response-related cis-acting elements were identified in the TCS1 promoter, such as

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abscisic acid-response elements, jasmonic acid methyl ester-responsive elements,

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fungal elicitor-responsive elements, heat stress-responsive elements, defense and

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stress-responsive elements, light-responsive elements and salicylic acid-response

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elements

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gibberellin-responsive elements, low-temperature responsive elements and the MYB

268

binding sites involved in drought-inducibility and light responsive elements (Figure

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7B).

270

Discussion

(Figure

7A).

In

addition,

SAMS

has

auxin-response

elements,

271

Secondary metabolites, especially polyphenols and caffeine, play important roles

272

in plant defense against biotic stress.18,39 Tea plant leaves are rich in polyphenols and

273

caffeine, however information about the role of secondary metabolites in resistance

274

against Colletotrichum is limited. In this study, we examined the changes of the TPC,

275

TCC and CC, as well as their biosynthetic genes in tea plant in response to C.

276

fructicola.

277

In fungal-plant interactions, plant phenolic compounds are one of the major

278

chemical defense materials in plant.40 When hosts are infected with pathogens, plant

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phenolic compounds could be significantly accumulation,41 especially in the

280

bordering zone between the healthy and infected plant tissues and stimulated the

281

production of reactive oxygen species,8 which might inhibit or restrict the pathogen in 14


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the infected site.42 In addition, in vitro antifungal test results also demonstrated that

283

tea polyphenols could play an antivirulent role in controlling bacterial infection.43 In

284

our study, TPC was markedly increased in resistant cultivars (ZC108) but remained

285

unchanged in susceptible cultivars (LJ43) during the infection period, suggesting that

286

phenolic compounds may be involved in resistance of tea plants to anthracnose

287

(Figure 1B).

288

Catechins are the most important phenolic compounds in tea plant, which are

289

mainly synthesized in fresh leaves.44 In this study, the TCC in tea plants was generally

290

increased, and the contents in the YT were higher than those in the MT (Figure 1B).

291

These results indicated that TCC was induced with C. fructicola, which is consistent

292

with its distribution in tea plant. EGCG, which comprises 76% of catechins,12 plays a

293

crucial role in plant defense against pathogens,14 and more strongly inhibits pathogens

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than other catechins components in vitro.14,15 In the present study, the contents of each

295

compound were different in the two cultivars after inoculation. Specifically, the

296

contents of (+)-C, (-)-EGC, and (-)-EGCG in ZC108 tissues were greater than or

297

equal to those in LJ43-YT. In contrast, the contents of (-)-ECG and (-)-EC, which are

298

precursors of ECG biosynthesis (Figure 1A), in LJ43 tissues were higher than those in

299

ZC108 (Figure 1B). These results suggest that the (-)-EGCG and (+)-C play an

300

important role in the resistance of tea plants to anthracnose. Unfortunately, the GC

301

content was not determined in our study due to the low GC content in fresh leaves.44

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Meanwhile, the antifungal activity was not determined for all catechins components,

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and it is also unknown whether EGCG is involved in modulating signaling pathways 15



304

in response to pathogens.16

305

Previous studies have shown that caffeine can effectively inhibit pathogens.5,18 In

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this study, the caffeine content was markedly increased in YT of resistant cultivars

307

(Figure 2B), suggesting that caffeine is important in C. fructicola defense. The higher

308

caffeine content in the YT could be explained by greater caffeine synthesis in young

309

sprouts or that C. fructicola may affect diseased leaves of plant.

310

It was also shown in this study that expression of genes related to caffeine and

311

flavonoid biosynthesis, SAMS, TCS1 and LAR, were increased markedly in tea plants

312

after C. fructicola infection (Figure 1C, 2C). Their expressions, meanwhile, were

313

higher in YT than in MT and were higher in resistant cultivars than in susceptible

314

cultivars. These results were consistent with changes in the production of these

315

compounds. In contrast, there was no correlation between (-)-EC and (-)-EGC content

316

and expression levels of the flavonoid biosynthesis-related gene ANR. This is in

317

agreement with previous findings showing no significant correlation between

318

enzymatic activity of ANR and content of (-)-EC and (-)-EGC.45 The ester type

319

pathway and flavan-3-ol gallate synthase (FGS), which are related to synthesis of

320

(-)-EGCG and (-)-ECG, were not examined in this study due to the lack of genomic

321

information and low enzymatic activities.21 Therefore, it is unclear whether these

322

related genes are involved in plant resistance to pathogens.

323

Previous studies have shown that TP, catechins, and caffeine can inhibit growth of

324

pathogens in vitro.7,10,32,34 To further understand the effects of the three metabolic

325

pathways against C. fructicola and in particular, the antifungal mechanism of caffeine, 16


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326

the present study evaluated their in vitro antifungal activity. Our results indicated that

327

the inhibitory activity of caffeine on the growth of C. fructicola was better than the

328

other two compounds (EC50 of TP and catechins was 4.832 and 9.323 mg/mL,

329

respectively) (Figure 3). In addition, caffeine had a stronger inhibitory effect on C.

330

fructicola than on Pestalotiopsis theae, which could cause grey blight disease in tea

331

plant (0.917 mg/mL).46 The inhibitory activity of TP against C. fructicola was weaker

332

than that against Rhizopus stolonifer (2.9 ± 0.05 mg/mL)33 but stronger than that

333

against Botrytis cinerea (8.7 mg/mL).10 The effects of individual catechins against

334

pathogens were not examined, but it has been demonstrated that EGCG has good

335

inhibitory activity.14,15

336

In order to characterize the antifungal mechanisms of caffeine, its effects on

337

mycelium morphology, cell wall and plasma membrane were analyzed in vitro.

338

Previous studies indicated that extracted plants may exert antifungal effects through

339

damage to pathogen cell walls and membranes, ultimately leading to pathogen death

340

or inhibition of growth.47 Our results showed that after caffeine treatment, the cell

341

walls and plasma membranes of C. fructicola were affected (Figure 4A, B). Moreover,

342

the MDA and SOD activities of the hyphae were also consistent with the results

343

mentioned above (Figure 4C, D). In addition, both SEM and TEM analysis results

344

demonstrated that the hyphae structures of C. fructicola were damaged after caffeine

345

treatment (Figure 5, 6). These results are consistent with previous reports.7,33,38,47

346

Invading pathogens induce the generation of plant hormones which trigger

347

expression of various genes involved in plant defenses, such as salicylic acid, 17



348

jasmonates, ethylene, abscisic acid, auxin, cytokinin, and gibberellin.48 Elicitor of

349

pathogen can be recognized by pattern recognition receptors located in plasma

350

membrane of plant and triggers the plant immune system.49 Various stress

351

response-related cis-acting elements or motifs were present in the promoters of TCS1

352

and SAMS in our study (Figure 7). In particular, fungal elicitor responsive elements in

353

TCS1 are known to regulate the level of TCS1 expression in response to C. fructicola

354

(Figure 7A). We speculated C. fructicola may induce the hormone secretion of tea

355

plant. The phytohormones activate stress response-related cis-acting elements in the

356

promoters of TCS1 and SAMS, and may further invoke expression of both genes.

357

Meanwhile, C. fructicola elicitor directly combined with fungal elicitor responsive

358

elements in promoter of TCS1, which could also induce the expression of both genes.

359

The results described above indicate that (-)-EGCG, (+)-C, and caffeine play

360

important roles in the tea plant response to C. fructicola. Caffeine may exert its

361

antimicrobial effects by damaging the cell wall and membrane of pathogens. The

362

fungal elicitor responsive elements on the TCS1 promoter also indirectly suggested

363

that caffeine pathway in tea plant may be involved in disease resistance via

364

modulation of plant hormones. As the contribution of each component in flavonoid

365

antifungal activity was unknown, future studies should examine their individual roles

366

and potential synergistic antifungal interactions.

367

ABBREVIATIONS USED

368

ALP, alkaline phosphatase; ANR, anthocyanidin reductase; C, catechin; CC, caffeine

369

content; EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, 18


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epigallocatechin-3-gallate; FGS, flavan-3-ol gallate synthase; GC, gallocatechin; LAR,

371

leucoanthocyanidin reductase; LJ43, Longjing43; MDA, methane dicarboxylic

372

aldehyde; MIC, minimum inhibitory concentration; MT, mature tissues; SAMS,

373

S-adenosylmethionine synthetase; SOD, superoxide dismutase; TCC, total catechins

374

content; TCS, tea caffeine synthase; TP, tea polyphenols; TPC, total phenolic content;

375

YT, young tissues; ZC108, Zhongcha108.

376

ASSOCIATED CONTENT

377

Supporting Information

378

[Primers were used in this study. The colonies of C. fructicola on PDA medium with

379

different treatments after two days. (A) The colonies of C. fructicola treated with TP;

380

(B) The colonies of C. fructicola treated with catechins; (C) The colonies of C.

381

fructicola treated with caffeine. The promoter sequences alignment of TCS1 and

382

SAMS in ZC108 and LJ43. (A) The promoter sequences alignment of TCS1 in two

383

cultivars; (B) The promoter sequences alignment of TCS1 in two cultivars.]

384

This material is available free of charge via the Internet at http://pubs.acs.org.

385

ACKNOWLEDGMENTS

386

This work was supported by the Earmarked Fund for China Agriculture Research

387

System (CARS-23), the Chinese Academy of Agricultural Sciences through an

388

Innovation

389

(CAAS-ASTIP-2014-TRICAAS), and the Major Project for New Agricultural

390

Varieties Breeding of Zhejiang Province (2012C2905-3).

391

Notes

Project

for

Agricultural

Sciences

19


and

Technology


392

The authors declare no competing financial interest.

20


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511

Figure captions

512 513

Figure 1. Changes in TPC and TCC (B) and relative expression of LAR and ANR (C)

514

in flavonoid biosynthetic pathway (A) of ZC108 and LJ43 after C. fructicola

515

inoculation at 0, 24, and 72 h. F3′5′H: flavonoid 3′, 5′-hydroxylase; F3′H: flavonoid

516

3′-hydroxylase; DFR: dihydroflavonol 4-reductase; ANS: anthocyanidin synthase;

517

ANR: anthocyanidin reductase; LAR: leucoanthocyanidin reductase; FGS: flavan-3-ol

518

gallate synthase. Asterisks show statistically significant differences between

519

treatments.

520

24


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521 522

Figure 2. Changes in caffeine content (B) and relative expression levels of the

523

caffeine biosynthesis (A) genes SAMS and TCS1 (C) in ZC108 and LJ43 after C.

524

fructicola inoculation at 0, 24, and 72 h. 7-NMT: 7-methylxanthosine synthase;

525

N-MeNase: N-methylnucleotidase; SAMS: S-adenosylmethionine synthetase; TCS:

526

tea caffeine synthase. The asterisks show statistically significant differences between

527

treatments.

528

25



529 530

Figure 3. In vitro effect of three compounds on mycelial growth rates of C. fructicola.

531

(A) The mycelial growth inhibition of C. fructicola by TP; (B) The mycelial growth

532

inhibition of C. fructicola by catechins; (C) The mycelial growth inhibition of C.

533

fructicola by caffeine. EC50 was determined by probit-log analysis.

534

26


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535 536

Figure 4. Effects of caffeine on ALP activity in vitro (A), A260 absorbing material (B),

537

MDA content (C), and SOD activity (D) of C. fructicola.

538

539 540

Figure 5. SEM of the hyphae of C. fructicola treated with or without caffeine

541

(EC80=0.869 mg/mL). (A-C) Morphology of healthy hyphae. (D-F) Morphology of 27



542

hyphae treated with caffeine.

543 544

Figure 6. TEM of the hyphal ultrastructure of C. fructicola treated with or without

545

caffeine (EC80=0.869 mg/mL). (A, C) The ultrastructure of healthy hyphae. (B, D, E)

546

The ultrastructure of hyphae treated with caffeine.

547

548 549

Figure 7. Promoter elements of TCS1 and SAMS in ZC108. (A) Distribution of

550

stress-related cis-elements in promoter region (-1096 bp) of TCS1. (B) Distribution of

551

stress-related cis-elements and MYB binding site in the promoter region (-1512 bp) of

552

SAMS. 28


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TABLE OF CONTENTS GRAPHICS

554 555

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Metabolic Changes of Caffeine in Tea Plant (Camellia sinensis (L.) O

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