Subscriber access provided by Northern Illinois University
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 29
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] 1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
ABSTRACT
2
Tea plant (Camellia sinensis) is one of the most economically valuable crops in
3
the world. Anthracnose can affect the growth of leaves and cause serious yield losses
4
of tea. Tea plants are rich in secondary metabolites, however, their roles in resistance
5
to anthracnose are unclear. Herein we compared the contents of total phenolic,
6
catechins and caffeine in two cultivars with different resistance to anthracnose during
7
Colletotrichum fructicola infection. The (-)-epigallocatechin-3-gallate (EGCG),
8
(+)-catechin (C), caffeine, and critical regulatory genes were induced in C.
9
fructicola-resistant tissues. In vitro antifungal tests showed that caffeine more strongly
10
inhibited mycelial growth than tea polyphenols and catechins. Both electron
11
microscopy and bioactivity analysis results showed that caffeine can affect mycelial
12
cell walls and plasma membranes. Through promoter sequences analysis, a number of
13
stress response-related cis-acting elements were identified in S-adenosylmethionine
14
synthetase and tea caffeine synthase. These results demonstrated that (-)-EGCG, (+)-C,
15
and caffeine may be involved in resistance of tea plants to anthracnose.
16
Keywords: Theaceae, disease resistance, tea ployphenols, catechins, Colletotrichum
17
2
ACS Paragon Plus Environment
Page 2 of 29
Page 3 of 29
Journal of Agricultural and Food Chemistry
18
INTRODUCTION
19
Tea plant (Camellia sinensis (L.) O. Kuntze) is a widely cultivated crop that plays
20
an important role in the economies of countries where it is planted, particularly in
21
China, the nation with the highest tea production. Therefore, increasing the production
22
and improving the quality of tea have many important economic implications.
23
Anthracnose, caused by Colletotrichum spp., is a disease that occurs commonly on the
24
leaves of tea plants, resulting in damage of tea leaves and ultimately influencing tea
25
yield and quality.1 Although some progress has been made in determining the
26
mechanisms by which plants become resistant to anthracnose,2,3 little is known about
27
the interaction between Colletotrichum and tea plants.
28
Tea plants are rich in various secondary metabolites that possess direct and
29
indirect antimicrobial activities in plants.4-6 Tea polyphenols (TP) is the most
30
important secondary metabolite in tea plants. Many in vitro studies have reported that
31
TP have a broad antimicrobial spectrum against various pathogens.7-9 In addition,
32
spraying TP on plants can improve their resistance to disease.10 Catechins are the
33
dominant flavonoids of tea plants, which are classified as ester type or non-ester type.
34
The major non-ester type catechins include (-)-epicatechin (EC), (-)-epigallocatechin
35
(EGC), (+)-catechin (C) and (+)-gallocatechin (GC), and the major ester type
36
catechins include (-)-epicatechin-3-gallate (ECG) and (-)-epigallocatechin-3-gallate
37
(EGCG).11 In tea plants, the concentration of ester type catechins is much greater than
38
that of non-ester type catechins.12,13 Previous in vitro studies have shown that
39
catechins extracted from tea have varying degrees of antibacterial effects, with 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
40
different catechins ordered according to their antibacterial effects, from strong to
41
weak, as follows: EGCG > EGC > ECG > EC > C.14
42
Catechins and caffeine are thought to exert their antimicrobial effects by multiple
43
mechanisms. The combination of EGCG and antimycotics has been shown to damage
44
biofilms of Candida species.15 EGCG may also enhance plant resistance to disease via
45
modulation of jasmonic acid signaling.16 Tea plants have abundant caffeine (1, 3,
46
7-trimethylxanthine).17 Biotic stress can increase caffeine content (CC) and enhance
47
hypersensitive responses in plants, an effect that may be mediated by the increase in
48
endogenous salicylic acid by caffeine.18 Furthermore, plants may depend on the toxic
49
effects of caffeine to help combat pathogens.19 Despite these findings, few studies
50
have been conducted on the secondary metabolites of tea plants as well as their in
51
vitro antifungal activity against Colletotrichum.
52
During pathogen-host interactions, plants activate multiple interconnected
53
defense signaling pathways and metabolic pathways.20 In tea plants, the non-ester
54
type catechins and caffeine biosynthesis pathways have been illustrated clearly
55
(Figure 1A and 2A).6,21 Many genes associated with the flavonoid pathway are
56
involved in the resistance of plants to disease. For example, in grapevine cultivars that
57
are resistant to anthracnose, the flavonoid pathway-associated genes, such as chalcone
58
synthase, chalcone isomerase, and dihydroflavonol-4-reductase, are expressed at
59
higher levels than in susceptible cultivars.22,23 Leucoanthocyanidin reductase (LAR)
60
and anthocyanidin reductase (ANR) play important roles in the flavonoid pathway of
61
tea plant. Leucoanthocyanidin can be transformed by LAR into flavan-3-ols (C, GC) 4
ACS Paragon Plus Environment
Page 4 of 29
Page 5 of 29
Journal of Agricultural and Food Chemistry
62
and by ANR into epi-flavan-3-ols (EC, EGC). 24 However, little is known about the
63
relationship between the two enzymes and disease resistance in tea plant.
64
The caffeine pathway is part of purine metabolism. S-adenosylmethionine
65
synthetase (SAMS) plays a crucial role in the caffeine pathway, as it is the only
66
methyl donor to xanthosine and is a major source of xanthosine in tea plant.25
67
Caffeine is synthesized through the conversion of theobromine by caffeine synthase.
68
In tea plant, all the identified tea caffeine synthase (TCS) are TCS1 type.26 It is
69
unclear whether these critical genes in the caffeine pathway are involved in the
70
response of tea plants to Colletotrichum.
71
Base on continuous field observation, we found that anthracnose is only diseased
72
on the mature leaves but not young leaves of tea plants. To determine the roles of
73
secondary metabolites in the tea plant response against Colletotrichum sp., using the
74
susceptible cultivar Longjing43 (LJ43) and the resistant cultivar Zhongcha108
75
(ZC108) as experiment materials, we determined the changes in the content of
76
secondary metabolites (total phenolic, catechins and caffeine) and the expression of
77
their related genes in plants at different levels of maturity after Colletotrichum sp.
78
inoculation. We estimated the antifungal effects of related reagents in vitro. We also
79
analyzed the promoters of critical genes associated with plant disease resistance.
80
Finally, we clarified the possible antifungal mechanism of caffeine in vitro.
81 82 83
MATERIALS AND METHODS Plants, Fungal Materials and Treatments. Five-year-old potted tea plants of the 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
84
resistant cultivar cv. Zhongcha108 (ZC108) and the susceptible cultivar cv.
85
Longjing43 (LJ43)27 with different levels of resistance to anthracnose were grown in
86
the greenhouse of the Tea Research Institute, Chinese Academy of Agricultural
87
Sciences (TRI, CAAS, N30°10', E120°5'), Hangzhou, China. The plants were
88
maintained in the greenhouse at 30°C during the day and 20°C at night with cycles of
89
14 h light and 10 h darkness and relative humidity of 80%-90%. Healthy plants were
90
selected at the one bud and 5th leaf stages for the inoculation experiment. The
91
pathogenic C. fructicola isolate L33 was originally isolated from the disease leaves of
92
LJ43 at the field of TRI, CAAS and identified by morphological characteristics and
93
partial sequencing of its genome as described by Weir, et al.28 Conidia suspension was
94
prepared by growing the L33 isolate on potato dextrose agar (PDA) for 7 days and
95
washing with sterile water. The spore concentration was determined using a
96
hemocytometer and adjusted to 106 spores/mL for inoculation.
97
Young and mature plant tissues were inoculated with conidia suspension (106
98
spores/mL). Young tissues (YT) were defined as a bud and 3rd leaves, and mature
99
tissues (MT) were defined as 4-5th leaves. In order to coat all tissue surfaces with
100
conidia, the spore suspension was sprayed onto the tissues until run-off. Control
101
plants were sprayed with sterile water. The YT and MT of two branches of a potted
102
plant were sampled at 0 (before inoculation), 24, and 72 hours post inoculation. Each
103
biological replicate was collected twice. The first group was prepared as fresh tissue
104
for analysis of secondary metabolites. The other groups were immediately transferred
105
to liquid nitrogen, stored at -80°C in a freezer, and subsequently used for quantitative 6
ACS Paragon Plus Environment
Page 6 of 29
Page 7 of 29
Journal of Agricultural and Food Chemistry
106
real time PCR (qRT-PCR) analysis.
107
Preparation of Sample Extracts. Extraction of secondary metabolites was
108
performed according to the methods described by Liang, et al.29 Fresh tea tissues
109
(0.25 g) were homogenized, and then ground and extracted with 10 mL 75% (v/v)
110
ethyl alcohol for 10 min. Samples were centrifuged at 4000 × g for 10 min (4°C) and
111
the supernatant was collected (Eppendorf, Germany) and diluted with 75% ethyl
112
alcohol to 10 mL.
113
Determination of Total Phenolic Content. Total Phenolic Content (TPC) was
114
determined using the Folin-Ciocalteu assay described by Lai, et al.30 Gallic acid
115
(Sigma-Aldrich Co., USA) was used as a standard. The concentration of TPC was
116
measured by spectrophotometry (UV 2550, Shimadzu, Japan).
117
Determination of Catechins and Caffeine Contents. Catechins and caffeine
118
contents were determined by high-performance liquid chromatography (HPLC)
119
according to the methods described by Wang, et al.31 The samples were filtered
120
through a 0.45-µm millipore filter and analyzed by a 2695-2489 HPLC System
121
(Waters alliance, USA) under the following conditions: inject volume: 10 µL; column:
122
C12, 4.6 mm × 250 mm (Phenomenex, USA); oven temperature: 40°C; phase A: 9%
123
(v/v) acetonitrile, 2 % (v/v) acetic acid, and 0.02% (m/v) EDTA; phase B: 80% (v/v)
124
acetonitrile, 2% (v/v) acetic acid, and 0.02% (m/v) EDTA; flow rate: 1 mL/min. The
125
absorbance at 280 nm was used to monitor peak intensities in real time. The HPLC
126
chemical standards were (−)-EGCG, (−)-EGC, (−)-ECG, (−)-EC, (−)-GCG, (+)-C,
127
(−)-GC, and caffeine (Sigma-Aldrich Co., USA). 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
128
qRT-PCR Analysis. Total RNA was extracted from all the samples using an
129
RNAprep Pure kit (Tiangen, China). An aliquot of 1 µg of total RNA was converted to
130
first-strand cDNA using a PrimeScript RT enzyme with a gDNA eraser (Takara,
131
Japan). qRT-PCR was performed on an Applied Biosystems 7500 Sequence Detection
132
System using SYBR® Premix Ex Taq™ II (Takara, Japan). In order to ensure the
133
accuracy of target genes, the genes that have been reported to have different
134
expressions in different tissues of tea plant were used for qRT-PCR analysis.
135
Gene-specific primers and GenBank numbers of target genes were listed in Table S1.
136
The polypyrimidine tract-binding protein (CsPTB1) gene was used as an internal
137
control.27
138
In vitro antifungal bioassay. The antifungal activities of TP, catechins, and
139
caffeine were assessed using the Poison Food Technique in solid media as described
140
by Zhang, et al.32 TP, catechins and caffeine (Aladdin, China) were dissolved in sterile
141
distilled water and then added to the sterilized PDA at 40-50°C to obtain final
142
concentrations ranging from 0.03125 to 16 mg/mL. Sterile distilled water was used as
143
control. Mixtures were poured into 9 cm petri plates and Mycelial discs (9 mm
144
diameter) taken from 5-day-old cultures were inoculated at the center of each petri
145
dish. The inoculated plates were incubated at 25°C overnight and mycelial growth
146
diameters were measured after 24 h and 48 h. The inhibition relative to the control
147
was calculated as previously described.32 The EC50 of each tested compound was
148
calculated using the mycelial growth inhibition obtained 24 h after treatment. The
149
minimum inhibitory concentration (MIC) of caffeine is the lowest concentration of 8
ACS Paragon Plus Environment
Page 8 of 29
Page 9 of 29
Journal of Agricultural and Food Chemistry
150
caffeine that resulted in no visible mycelial growth after 72 h of incubation.33
151
Determination of the Effect of Caffeine on Hyphae Bioactivity. The hyphae
152
bioactivity was analyzed using the method of Shao, et al.34 Mycelial discs (9 mm)
153
taken from 5-day-old cultures were inoculated into potato dextrose broth (PDB)
154
medium and allowed to grow at 25°C overnight at 160 rpm for 48 h. Caffeine (MIC: 4
155
mg/mL) was added to the PDB medium and the samples were collected from the
156
culture solution at 0, 12, and 24 h after treatment. The culture solution was
157
centrifuged at 4000 × g for 10 min and was used to determine the alkaline phosphatase
158
(ALP) activity, release of 260 nm absorbing material (A260), and the activity of
159
methane dicarboxylic aldehyde (MDA) and superoxide dismutase activity (SOD) by
160
assay kits (Nanjing Jiancheng Institute of Bioengineering, China). Absorbance was
161
determined using a spectrophotometer (UV 2550, Shimadzu, Japan). The untreated
162
PDB medium was used as control.
163
Effect of Caffeine on Hyphal Morphology and Ultrastructure. To observe the
164
effect of caffeine on hyphae morphology of C. fructicola, mycelial discs (4 mm
165
diameter) from 5-day-old colonies were cultured on PDA containing 0.869 mg/mL
166
(EC80) of caffeine and incubated at 25°C for 5 days. PDA without caffeine was used
167
as control. The hyphae samples were analyzed by scanning (SEM) and transmission
168
(TEM) electron microscopy as described by Yue, et al.35 using a Hitachi Model
169
TM-1000 SEM (Hitachi, Japan) and a Model H-7650 TEM (Hitachi, Japan)
170
respectively.
171
Cloning and Analysis of the Promoters. The promoter sequence was amplified 9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
172
by general PCR. The genomic DNA of the healthy leaves was extracted using a Plant
173
Genomic DNA Kit (Tiangen, China). Primers specific to promoter were designed by
174
Primer Premier 5 (Premier Biosoft International, CA) (Table S1) based on incomplete
175
genomic information (LJ43). The PCR amplification was performed in 50 µL reaction
176
mixtures by following the manufacturer’s instructions of PrimeSTAR® HS DNA
177
polymerase (TaKaRa, China). PCR product was gel-purified and cloned into the
178
pMD18-T vector (TaKaRa, China) for sequencing. The cis-element in the obtained
179
sequence was identified by PlantCARE.36
180
Statistical Analysis. SPSS 18 (SPSS Inc., USA) was used to conduct statistical
181
analyses. One-way analysis of variance (ANOVA) was used for statistical analyses
182
and p < 0.05 was considered significant. Differences between all tissues and LJ43-MT
183
were assessed as least significant difference (LSD) test. Values were expressed as
184
mean ± standard error. EC50 values were obtained using logistic and probit regression.
185
Graphs were generated using Prism 6 (GraphPad Software Inc., USA) and Illustrator
186
CS6 (Adobe Software Inc., USA).
187 188
RESULTS
189
Changes in TPC in Tea Plant after C. fructicola Infection
190
The TPC, total catechins content (TCC), and biosynthesis-related genes in MT
191
and YT of ZC108 and LJ43 were analyzed after C. fructicola inoculation. The levels
192
of TPC and TCC were enhanced in both cultivars after C. fructicola inoculation.
193
These changes were particularly salient in the ZC108 at 24 h compared to the LJ43. 10
ACS Paragon Plus Environment
Page 10 of 29
Page 11 of 29
Journal of Agricultural and Food Chemistry
194
Catechins are composed of ester type and non-ester type catechins. The content of
195
ester type catechins elevated in YT and MT of ZC108 as well as in YT of LJ43, but
196
was unchanged in MT of LJ43. The (-)-EGCG content in ZC108 was higher than that
197
in LJ43-MT (p < 0.05). In contrast, the (-)-ECG content in LJ43 was higher than that
198
in ZC108 (p < 0.05). The content of non-ester type catechins increased in the ZC108
199
tissues but decreased in the LJ43 tissues. The (+)-C content in both ZC108 and
200
LJ43-YT increased more than that in LJ43-MT (p < 0.05), while the initial content of
201
(-)-EC in LJ43 was higher than that in ZC108 (p < 0.05) (Figure 1B). LAR (GenBnak:
202
HP763707)21, a key gene in the biosynthesis of (+)-C and (+)-GC, was expressed
203
markedly in the YT of both cultivars relative to MT. ANR (c147477)6, a key gene in
204
the biosynthesis of (-)-EC and (-)-EGC, was increased slightly in LJ43-YT but was
205
down-regulated in LJ43-MT at 24 h after infection; and no significant change was
206
observed in ZC108 tissues (Figure 1C). These results suggest that polyphenols,
207
particularly (-)-EGCG and (+)-C, might involve in the tea plant defense to C.
208
fructicola.
209
Changes in CC in Tea Plant after C. fructicola Infection
210
The CC and biosynthesis-related genes in MT and YT of ZC108 and LJ43 were
211
analyzed after C. fructicola inoculation. After C. fructicola inoculation, the CC of the
212
ZC108 was increased markedly at 24 h, but was not significantly changed in LJ43
213
(Figure 2B). SAMS (AJ277206)25 and TCS1 (AB031280)37, the key genes in the
214
biosynthetic pathway of caffeine, were significantly up-regulated in the YT of both
215
tea cultivars in response to C. fructicola at 24 h after inoculation. Expression of TCS1 11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
216
in the resistant tissues (ZC108 tissues and LJ43 - YT) was significantly different from
217
that of diseased tissues (LJ43 - MT) (p < 0.05) (Figure 2C). These results indicated
218
that caffeine plays an important role in the resistance of tea plants to C. fructicola.
219
In Vitro Effects of Compounds on Mycelial Growth of C. fructicola
220
The mycelial growth of C. fructicola was examined after treatment with different
221
concentrations of TP, catechins and caffeine in vitro (Figure 3, Figure S1). All these
222
compounds inhibited mycelial growth in a dose-dependent manner. Caffeine
223
demonstrated significantly greater antifungal effects than TP and catechins with an
224
EC50 of 0.555 mg/mL, which was greater than that of TP (4.832 mg/mL) and catechins
225
(9.323 mg/mL) (Figure 3A, B). Moreover, caffeine had a long-lasting antifungal effect,
226
and the MIC value was only 4 mg/mL (Figure 3C). Since caffeine had effective
227
antifungal activity against C. furcticola, caffeine at the MIC was used in subsequent
228
experiments.
229
Effects of Caffeine on Cell Wall and Membrane of C. fructicola
230
In order to understand how caffeine inhibited C. fructicola, the integrity of cell
231
wall and plasma membrane of hyphae was analyzed (Figure 4). The bioactivities of
232
treated hyphae were significantly increased compared with control. ALP and release
233
of intracellular components have been shown to be good indicators of integrity of cell
234
wall and membrane.34,38 ALP was increased rapidly after treatment with caffeine (4
235
mg/mL) for 12 h (Figure 4A) and release of 260 nm absorbing material (A260 nm) was
236
enhanced after treatment with caffeine (4 mg/mL) for 24 h (Figure 4B), suggesting the
237
integrity of cell wall was affected and plasma membrane permeability increased. In 12
ACS Paragon Plus Environment
Page 12 of 29
Page 13 of 29
Journal of Agricultural and Food Chemistry
238
addition, the activities of MDA and SOD, which have relationships with
239
stress-tolerance,7 were significantly increased after treatment with caffeine (4 mg/mL)
240
(Figure 4C, D). These results indicated that the cell wall and plasma membrane were
241
damaged under caffeine treatment.
242
Effects of Caffeine on Hyphae Morphology and Ultrastructure
243
To further characterize the antifungal mechanism of caffeine against C. fructicola,
244
the hyphae morphology and ultrastructure after treatment with 0.869 mg/mL (EC80)
245
caffeine were observed by SEM (Figure 5) and TEM (Figure 6). SEM analysis
246
showed that the untreated hyphae exhibited characteristic morphology, with healthy,
247
robust, and uniform growth, and had plump cell bodies (Figure 5A-C). In contrast, the
248
treated hyphae displayed shriveling, curving, and collapse morphology (Figure 5D-F).
249
Figure 6 shows the ultrastructure of C. fructicola. The healthy hyphae have intact cell
250
walls and plasma membranes, and clear vision of organelles (Figure 6A, C). In
251
contrast, the treated hyphae exhibited that the cell wall was thickened; the organelles
252
and cytoplasm were degradation, especially mitochondria; the plasma membrane
253
became unclear and finally the hyphae appeared empty holes (Figure 6B, D, E).
254
Stress-Relevant Cis-Elements in the Promoters of Caffeine Pathway-Related
255
Genes
256
In an effort to explain the increased expression of the caffeine pathway-related
257
genes TCS1 and SAMS after C. fructicola inoculation, we cloned and analyzed their
258
promoter regions. The TCS1 promoter in ZC108 (-1094 bp upstream from start codon)
259
was 17 bp different from that in LJ43, and the SAMS (-1512 bp upstream from start 13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 14 of 29
260
codon) was 10 bp different from that in LJ43 (Figure S2). The genes had the same
261
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
263
abscisic acid-response elements, jasmonic acid methyl ester-responsive elements,
264
fungal elicitor-responsive elements, heat stress-responsive elements, defense and
265
stress-responsive elements, light-responsive elements and salicylic acid-response
266
elements
267
gibberellin-responsive elements, low-temperature responsive elements and the MYB
268
binding sites involved in drought-inducibility and light responsive elements (Figure
269
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
279
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
ACS Paragon Plus Environment
Page 15 of 29
Journal of Agricultural and Food Chemistry
282
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
294
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
302
Meanwhile, the antifungal activity was not determined for all catechins components,
303
and it is also unknown whether EGCG is involved in modulating signaling pathways 15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
304
in response to pathogens.16
305
Previous studies have shown that caffeine can effectively inhibit pathogens.5,18 In
306
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
ACS Paragon Plus Environment
Page 16 of 29
Page 17 of 29
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 18 of 29
Page 19 of 29
Journal of Agricultural and Food Chemistry
370
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
ACS Paragon Plus Environment
and
Technology
Journal of Agricultural and Food Chemistry
392
The authors declare no competing financial interest.
20
ACS Paragon Plus Environment
Page 20 of 29
Page 21 of 29
Journal of Agricultural and Food Chemistry
393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436
REFERENCES (1) Yoshida, K.; Takeda, Y. Evaluation of anthracnose resistance among tea genetic resources by wound-inoculation assay. JARQ 2006, 40, 379-386. (2) Quintin, J.; Cheng, S. C.; van der Meer, J. W. M.; Netea, M. G. Innate immune memory: towards a better understanding of host defense mechanisms. Curr. Opin. Immunol. 2014, 29, 1-7. (3) Cui, H.; Tsuda, K.; Parker, J. E. Effector-triggered immunity: from pathogen perception to robust defense. Annu. Rev. Plant Biol. 2015, 66, 487-511. (4) Daglia, M. Polyphenols as antimicrobial agents. Curr. Opin. Biotech. 2012, 23, 174-181. (5) Kim, Y. S.; Sano, H. Pathogen resistance of transgenic tobacco plants producing caffeine. Phytochemistry 2008, 69, 882-888. (6) Li, C. F.; Zhu, Y.; Yu, Y.; Zhao, Q. Y.; Wang, S. J.; Wang, X. C.; Yao, M. Z.; Luo, D.; Li, X.; Chen, L.; Yang, Y. J. Global transcriptome and gene regulation network for secondary metabolite biosynthesis of tea plant (Camellia sinensis). BMC Genomics 2015, 16, 560. DOI 10.1186/s12864-015-1773-0 (7) Jiang, X.; Feng, K.; Yang, X. In vitro antifungal activity and mechanism of action of tea polyphenols and tea saponin against Rhizopus stolonifer. J. Mol. Microbiol. Biotechnol. 2015, 25, 269-276. (8) Mikulic-Petkovsek, M.; Schmitzer, V.; Jakopic, J.; Cunja, V.; Veberic, R.; Munda, A.; Stampar, F. Phenolic compounds as defence response of pepper fruits to Colletotrichum coccodes. Physiol. Mol. Plant Pathol. 2013, 84, 138-145. (9) Yi, S. M.; Zhu, J. L.; Fu, L. L.; Li, J. R. Tea polyphenols inhibit Pseudomonas aeruginosa through damage to the cell membrane. Int. J. Food Microbiol. 2010, 144, 111-117. (10) Yang, X. P.; Jiang, X. D.; Chen, J. J.; Zhang, S. S. Control of postharvest grey mould decay of nectarine by tea polyphenol combined with tea saponin. Lett. Appl. Microbiol. 2013, 57, 502-509. (11) Cabrera, C.; Artacho, R.; Giménez, R. Beneficial effects of green tea-a review. J. Am. Coll. Nutr. 2006, 25, 79-99. (12) He, Q.; Yao, K.; Jia, D.; Fan, H.; Liao, X.; Shi, B. Determination of total catechins in tea extracts by HPLC and spectrophotometry. Nat. Prod. Res. 2009, 23, 93-100. (13) Kim, S. Y.; Ahn, B. H.; Min, K. J.; Lee, Y. H.; Joe, E. h. Phospholipase D isozymes mediate epigallocatechin gallate-induced cyclooxygenase-2 expression in astrocyte cells. J. Biol. Chem. 2004, 279, 38125-38133. (14) Sourabh, A.; Kanwar, S. S.; Sud, R. G.; Ghabru, A.; Sharma, O. P. Influence of phenolic compounds of Kangra tea [Camellia sinensis (L.) O. Kuntze] on bacterial pathogens and indigenous bacterial probiotics of Western Himalayas. Braz. J. Microbiol. 2013, 44, 709-715. (15) Ning, Y.; Ling, J.; Wu, C. D. Synergistic effects of tea catechin epigallocatechin gallate and antimycotics against oral Candida species. Arch. Oral Biol. 2015, 60, 1565-1570. (16) Hong, G.; Wang, J.; Hochstetter, D.; Gao, Y.; Xu, P.; Wang, Y. Epigallocatechin-3-gallate functions as a physiological regulator by modulating the jasmonic acid pathway. Physiol. Plant. 2015, 153, 432-439. (17) Ashihara, H.; Sano, H.; Crozier, A. Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry 2008, 69, 841-856. (18) Kim, Y. S.; Choi, Y. E.; Sano, H. Plant vaccination: stimulation of defense system by caffeine production in planta. Plant Signaling Behav. 2014, 5, 489-493. (19) Ashihara, H.; Crozier, A. Caffeine: a well known but little mentioned compound in plant science. Trends Plant Sci. 2001, 6, 407-413. 21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480
(20) Jones, J. D.; Dangl, J. L. The plant immune system. Nature 2006, 444, 323-329. (21) Shi, C. Y.; Yang, H.; Wei, C. L.; Yu, O.; Zhang, Z. Z.; Jiang, C. J.; Sun, J.; Li, Y. Y.; Chen, Q.; Xia, T.; Wan, X. C. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds. BMC Genomics 2011, 12, 131. DOI: 10.1186/1471-2164-12-131 (22) Gao, M.; Wang, Q.; Wan, R.; Fei, Z.; Wang, X. Identification of genes differentially expressed in grapevine associated with resistance to Elsinoe ampelina through suppressive subtraction hybridization. Plant Physiol. Biochem. 2012, 58, 253-268. (23) Louime, C.; Lu, J.; Onokpise, O.; Vasanthaiah, H. K.; Kambiranda, D.; Basha, S. M.; Yun, H. K. Resistance to Elsinoë ampelina and expression of related resistant genes in Vitis rotundifolia Michx. Grapes. Int. J. Mol. Sci. 2011, 12, 3473-3488. (24) Punyasiri, P.; Abeysinghe, I.; Kumar, V.; Treutter, D.; Duy, D.; Gosch, C.; Martens, S.; Forkmann, G.; Fischer, T. Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways. Arch. Biochem. Biophys. 2004, 431, 22-30. (25) Deng, W. W.; Li, M.; Gu, C. C.; Li, D. X.; Ma, L. L.; Jin, Y.; Wan, X. C. Low caffeine content in novel grafted tea with Camellia sinensis as scions and Camellia oleifera as stocks. Nat. Prod. Commun. 2015, 10, 789-792. (26) Li, Y.; Ogita, S.; Keya, C. A.; Ashihara, H. Expression of caffeine biosynthesis genes in tea (Camellia sinensis). Z.Naturforsch C. 2008, 63, 267-270. (27) Wang, L.; Wang, Y.; Cao, H.; Hao, X.; Zeng, J.; Yang, Y.; Wang, X. Transcriptome analysis of an anthracnose-resistant tea plant cultivar reveals genes associated with resistance to Colletotrichum camelliae. PloS One 2016, 11, e0148535. (28) Weir, B. S.; Johnston, P. R.; Damm, U. The Colletotrichum gloeosporioides species complex. Stud. Mycol. 2012, 73, 115-180. (29) Liang, H.; Liang, Y.; Dong, J.; Lu, J., Tea extraction methods in relation to control of epimerization of tea catechins. J. Sci. Food Agric. 2007, 87, 1748-1752. (30) Lai, Y. S.; Li, S.; Tang, Q.; Li, H. X.; Chen, S. X.; Li, P.-W.; Xu, J. Y.; Xu, Y.; Guo, X. The dark-purple tea cultivar ‘Ziyan’ accumulates a large amount of delphinidin-related anthocyanins. J. Agric. Food Chem. 2016, 64, 2719-2726. (31) Wang, W.; Xin, H.; Mingle Wang, Q. M.; Wang, L.; Kaleri, N. A.; Wang, Y.; Li, X. Transcriptomic analysis reveals the molecular mechanisms of drought-stress-induced decreases in Camellia sinensis leaf quality. Front. Plant Sci. 2016, 7, 385. DOI: 10.3389/fpls.2016.00385 (32) Zhang, J.; Yan, L. T.; Yuan, E. L.; Ding, H. X.; Ye, H. C.; Zhang, Z. K.; Yan, C.; Liu, Y. Q.; Feng, G. Antifungal activity of compounds extracted from Cortex Pseudolaricis against Colletotrichum gloeosporioides. J. Agric. Food Chem. 2014, 62, 4905-4910. (33) Yang, X.; Jiang, X. Antifungal activity and mechanism of tea polyphenols against Rhizopus stolonifer. Biotechnol. Lett. 2015, 37, 1463-1472. (34) Shao, X.; Cheng, S.; Wang, H.; Yu, D.; Mungai, C. The possible mechanism of antifungal action of tea tree oil on Botrytis cinerea. J. Appl. Microbiol. 2013, 114, 1642-1649. (35) Yue, C.; Cao, H. L.; Wang, L.; Zhou, Y. H.; Huang, Y. T.; Hao, X. Y.; Wang, Y. C.; Wang, B.; Yang, Y. J.; Wang, X. C. Effects of cold acclimation on sugar metabolism and sugar-related gene expression in tea plant during the winter season. Plant Mol. Biol. 2015, 88, 591-608. (36) Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico 22
ACS Paragon Plus Environment
Page 22 of 29
Page 23 of 29
Journal of Agricultural and Food Chemistry
481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510
analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325-327. (37) Kato, M.; Mizuno, K.; Crozier, A.; Fujimura, T.; Ashihara, H. Plant biotechnology: caffeine synthase gene from tea leaves. Nature 2000, 406, 956-957. (38) Yi, S. M.; Zhu, J. L.; Fu, L. L.; Li, J. R. Tea polyphenols inhibit Pseudomonas aeruginosa through damage to the cell membrane. Int. J. Food Microbiol. 2010, 144, 111-117. (39) Bennett, R. N.; Wallsgrove, R. M. Secondary metabolites in plant defence mechanisms. New Phytol. 1994, 127, 617-633. (40) Shalaby, S.; Horwitz, B. A. Plant phenolic compounds and oxidative stress: integrated signals in fungal-plant interactions. Curr. Genet. 2015, 61, 347-357. (41) Arici, S. E.; Kafkas, E.; Kaymak, S.; Koc, N. K. Phenolic compounds of apple cultivars resistant or susceptible to Venturia inaequalis. Pharm. Biol. 2014, 52, 904-908. (42) Gogoi, R.; Singh, D. V.; Srivastava, K. D. Phenols as a biochemical basis of resistance in wheat against Karnal bunt. Plant Pathol. 2001, 50, 470-476. (43) Yin, H.; Deng, Y.; Wang, H.; Liu, W.; Zhuang, X.; Chu, W. Tea polyphenols as an antivirulence compound disrupt quorum-sensing regulated pathogenicity of Pseudomonas aeruginosa. Sci. Rep. 2015, 5, 16158.DOI: 10.1038/srep17987. (44) Park, J. S.; Kim, J. B.; Hahn, B. S.; Kim, K. H.; Ha, S. H.; Kim, J. B.; Kim, Y. H. EST analysis of genes involved in secondary metabolism in Camellia sinensis (tea), using suppression subtractive hybridization. Plant Sci. 2004, 166, 953-961. (45) Zhang, X.; Liu, Y.; Gao, K.; Zhao, L.; Liu, L.; Wang, Y.; Sun, M.; Gao, L.; Xia, T. Characterisation of anthocyanidin reductase from Shuchazao green tea. J. Sci. Food Agric. 2012, 92, 1533-1539. (46) Zhang, H. Y.; Qi, L.; Zhang, Z. Z. Antifungal activity of caffeine against fungal pathogens of tea plant. Nanjing Nongye Daxue Xuebao 2010, 2, 63-67. (47) Yu, D.; Wang, J.; Shao, X.; Xu, F.; Wang, H. Antifungal modes of action of tea tree oil and its two characteristic components against Botrytis cinerea. J. Appl. Microbiol. 2015, 119, 1253-1262. (48) Borges, A. A.; Sandalio, L. M. Induced resistance for plant defense. Front. Plant Sci. 2015, 6, 109. DOI: 10.3389/fpls.2015.00109 (49) Boyd, L. A.; Ridout, C.; O'Sullivan, D. M.; Leach, J. E.; Leung, H. Plant-pathogen interactions: disease resistance in modern agriculture. Trends Genet. 2013, 29, 233-240.
23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 24 of 29
Page 25 of 29
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 26 of 29
Page 27 of 29
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 28 of 29
Page 29 of 29
Journal of Agricultural and Food Chemistry
553
TABLE OF CONTENTS GRAPHICS
554 555
29
ACS Paragon Plus Environment