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Regulation of flavonoid metabolism in ginkgo leaves in response to different day-night temperature combinations
Journal Pre-proof Regulation of flavonoid metabolism in ginkgo leaves in response to different day-night temperature combinations Jing Guo, Xin Zhou, Tongli Wang, Guibin Wang, Fuliang Cao PII:
S0981-9428(19)30517-0
DOI:
https://doi.org/10.1016/j.plaphy.2019.12.009
Reference:
PLAPHY 5966
To appear in:
Plant Physiology and Biochemistry
Received Date: 24 June 2019 Revised Date:
7 December 2019
Accepted Date: 9 December 2019
Please cite this article as: J. Guo, X. Zhou, T. Wang, G. Wang, F. Cao, Regulation of flavonoid metabolism in ginkgo leaves in response to different day-night temperature combinations, Plant Physiology et Biochemistry (2020), doi: https://doi.org/10.1016/j.plaphy.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.
1
Regulation of Flavonoid Metabolism in Ginkgo Leaves in Response to Different Day-Night
2
Temperature Combinations
3 4
Jing Guoa, b, Xin Zhoua, Tongli Wangb, Guibin Wanga,* and Fuliang Caoa
5 6
a
7
159 Longpan Road, Nanjing 210037, China
8
b
9
Columbia, Vancouver V6T 1Z4, Canada
Nanjing Forestry University, Co-Innovation Centre for Sustainable Forestry in Southern China,
Department of Forest and Conservation Sciences, Faculty of Forestry, the University of British
10 11
*Corresponding Author
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Guibin Wang
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Tel: +86-85427201
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Email: [email protected]
15
1
16
Abstract
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Flavonoids are the most important secondary metabolites in ginkgo (Ginkgo biloba L.) leaves that
18
determine its medicinal quality. Studies have suggested that secondary metabolism is strongly
19
affected by temperature in other plant species, but little is known about ginkgo. In this study, we
20
investigated the effects of different day-night temperature combinations (15/10, 25/20, and 35/30
21
°C (day/night)) on key enzyme activity, growth regulator concentrations, and flavonoid
22
accumulation in ginkgo leaves. We found that phenylalanine ammonia-lyase (PAL) activity was
23
enhanced and inhibited at 15/10 and 35/30 °C, respectively. Cinnamate-4-hydroxylase (C4H)
24
activity was relatively stable under the three temperature conditions, and the p-coumarate CoA
25
ligase (4CL) activity showed different trends under the three temperature conditions. The
26
concentrations of flavonoid constituents (quercetin, kaempferol, and isorhamnetin) were
27
decreased and increased under the 35/30 and 15/10 °C conditions, respectively. Low temperature
28
promoted soluble sugar accumulation, while temperature had a limited impact on the accumulation
29
of soluble protein. The pattern of change in the total flavonoid concentration was not always in
30
agreement with PAL activity due to its complex pathway. Indoleacetic acid (IAA) and gibberellin
31
(GA) changes shared similar patterns and had limited effects on flavonoid accumulation, while
32
abscisic acid (ABA) acted as a promotor of flavonoid accumulation under high-temperature
33
conditions. The total flavonoids achieved the highest content under the 15/10 °C treatment on the
34
40th day. Therefore, the lower temperature (15/10 °C) is more favorable for flavonoid
35
accumulation and will provide a theoretical basis for further study. 2
36 37
Keywords: Ginkgo biloba L., temperature combination, flavonoids, enzyme activity, growth
38
regulators
39 40
Abbreviations
41
GBE, Ginkgo biloba extracts; ROS, reactive oxygen species; DW, dry weight; FW, fresh weight.
42 43
3
44 45
1 Introduction Ginkgo (Ginkgo biloba L.) is an ancient living tree species that originated in the Jurassic
46
period and commonly known as a “living fossil” (Stromgaard and Nakanishi, 2004). Due to its
47
multiple purposes, Ginkgo has been introduced from China to many countries and regions
48
worldwide (Xu et al., 2015). The most common utilization purposes of ginkgo are the collection of
49
leaves for pharmaceutical applications and the planting of trees for landscaping (Wu et al., 2019).
50
In the pharmaceutical industry, flavonoids and terpene lactones are the most important secondary
51
metabolites of ginkgo and have many functions in medicinal and health care, including lowering
52
blood pressure (Xiao et al., 2014), anti-tumor, anti-inflammatory (Van Beek and Montoro, 2009),
53
antioxidant (Wang et al., 2016), and radical scavenging activities (Ellnain-Wojtaszek et al., 2003).
54
Ginkgo biloba extracts (GBE) are the most popular product for the clinical treatment of cognitive
55
disorders (Le Bars et al., 1997). The chemical components of GBE mainly include 24%
56
flavonoids, 6% terpene lactones, and 5-10% organic acids, and medical effects are mainly
57
achieved via flavonoid constituents (Bastianetto et al., 2000). Flavonoids also play key roles in the
58
survival and normal growth of plants, such as the production of pigmentation for ultraviolet
59
radiation protection and resistance against microbial pathogens (Harborne and Williams, 2000;
60
Mierziak et al., 2014).
61
Instead of participating in the construction of plants directly, secondary metabolites are
62
generally involved in the protection process to reduce the damaging effects of different biotic and
63
abiotic stresses (Liu et al., 2015a). Numerous studies have shown increasing concentrations of 4
64
secondary metabolites when plants are exposed to extreme temperature, high salinity, water
65
deficiency, and nitrogen deficiency situations (Dai et al., 2015; Liu et al., 2015a, 2017).
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Flavonoids are present in many types of plants and exist as many active compounds, such as
67
quercetin, kaempferol and isorhamnetin (Liu et al., 2015b). The important features of flavonoids
68
are to filter out ultraviolet radiation, regulate auxin transport, promote pollen germination, and act
69
as antioxidants (Ni et al., 2017). Additionally, plants generate high levels of reactive oxygen
70
species (ROS) in response to stress, and quercetin has the highest radical-scavenging capacity
71
(Baek et al., 2015).
72
Flavonoid biosynthesis is regulated by multiple factors, among which temperature is
73
known to be the most important environmental factor. Temperature influences plant growth,
74
organogenesis, and primary and secondary metabolism (Gouot et al., 2019). For instance, adverse
75
temperature conditions will affect the plant physiological status and then cause metabolic
76
alterations, which lead to changes in the composition of plant metabolites (Goh et al., 2016).
77
Isoflavonoid concentrations were altered in Glycine max seedlings growing under different
78
temperature regimes (Posmyk et al., 2005). When suffering heat stress, plants will be protected by
79
antioxidant systems through the production of natural antioxidant metabolites, such as flavonoids
80
and phenolics (Cheng et al., 2018; Ghasemzadeh et al., 2010; Gooding et al., 2001).
81
Primary metabolism refers to a series of reactions and pathways that are essential for plant
82
survival, while secondary metabolism involves secondary metabolites that participate in
83
protection and development processes (Kroymann, 2011). Photosynthesis, glycolysis, the 5
84
tricarboxylic acid (TCA) cycle, and the shikimate pathway are vital series of reactions and
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pathways to provide energy and products for the synthesis of secondary metabolites. A negative
86
correlation was detected between total flavonoid production and soluble protein contents in
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Labisia pumila Blume under lower fertilization conditions, while total flavonoid biosynthesis is
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positively correlated with soluble sugar concentrations (Ibrahim et al., 2011). Flavonoids are
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synthesized via the phenylpropanoid pathway and share the same precursors and common genes as
90
other phenolic compounds. Many enzyme families act as catalysts through the phenylpropanoid
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pathway, such as phenylalanine ammonia-lyase (PAL; the first enzyme in the phenylpropanoid
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pathway), p-coumaroyl: CoA ligase (4CL), cinnamate-4-hydroxylase (C4H), chalcone reductase
93
(CHS) and other enzymes (Gouot et al., 2019). Additionally, flavonoid synthesis is also affected
94
by plant growth regulators, such as auxin, cytokinin (CK), gibberellins (GAs), and abscisic acid
95
(ABA). Growth regulators are also affected by abiotic stress, which contributes to signaling
96
mechanisms in cells.
97
Knowledge regarding the effect of temperature on flavonoid concentration, flavonoid
98
metabolism, and growth regulator synthesis is scarce. Consequently, the impact of global warming
99
on the growth of ginkgo and the production of flavonoids remains a big concern. Thus, the main
100
objectives of this study were to understand the relationships between temperature and flavonoid
101
contents, the activity of key enzymes (PAL, 4CL, and C4H) and the concentrations of growth
102
regulators (indoleacetic acid (IAA), GAs, and ABA) in ginkgo leaves. Additionally, we explored
103
the optimum temperature for flavonoid accumulation, which will benefit the utilization efficiency 6
104
of ginkgo leaves. Our study was conducted under controlled growing conditions, including three
105
alternating day/night temperatures (15/10, 25/20, and 35/30 °C).
106 107
2 Materials and Methods
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2.1 Plant Material and Experimental Conditions
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The experiment was carried out in the artificial chambers of Nanjing Forestry University,
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Jiangsu Province, China. One-year-old ginkgo seedlings were cultivated in plastic pots (diameter
112
× height: 19 × 16 cm), with two seedlings per pot. The seedlings were obtained from an
113
experimental nursery in Pizhou city. The seedlings were from the same variety, and the average
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height and ground diameter were 43.9 and 8.66 mm, respectively. The pots were filled with a
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mixture of loam, sand, and peat (2:1:1, v/v). The nutrient contents of the mixture were as follows:
116
organic matter, 12.0 g·kg-1; hydrolyzed nitrogen, 121 mg·kg-1; available phosphorus (P2O5), 12.7
117
mg·kg-1; and available potassium (K2O), 11.7 mg·kg-1. The mixture was characterized by a field
118
water-holding capacity of 22.0% and a pH value of 8.0.
119
The experiments were performed in July with initial acclimatization temperatures of 25
120
and 15 °C (day/night) and relative humidity (RH) of 75% for two weeks. After preculture,
121
seedlings were transferred to three artificial chambers with temperature conditions of 15/10,
122
25/20, and 35/30 °C (day/night), the light density was 800 µm·m-2·s-1, the photoperiod cycle was
123
16 h light–8 h dark, and the relative humidity (RH) was 75%. A total of 50 pots and 100 seedlings 7
124
were contained in each chamber; 45 pots were divided into 3 groups; and 5 pots were cultured in
125
the case of accidents. The top fourth and fifth leaves from 5 randomly selected seedlings in each
126
group were collected and pooled. The leaves were collected at 10 d, 20 d, 30 d, and 40 d after being
127
transferred to the respective chambers. The samples were immediately frozen in liquid nitrogen
128
and stored at -70 °C until further determination.
129 130 131
2.2 Extraction and Determination of Enzyme Activity The extraction and determination of PAL activity were performed according to the method
132
of Ortega-García and Peragón (2009). Briefly, the enzyme extraction was carried out in an ice
133
bath. The samples were pulverized, and the resulting powder was homogenized. The homogenates
134
were centrifuged at 20,000×g for 15 min at 4 °C. The supernatant was then collected for PAL
135
activity determination as a crude enzyme extract. The reaction mixture was incubated at 37 °C for
136
30 min under darkness and was terminated by adding 0.5 mL HCl (6 M). The absorbance of the
137
reaction mixture was measured at 290 nm using a spectrophotometer versus a blank. One unit of
138
PAL activity was defined as the amount of enzyme needed to catalyze the formation of 1 µmol
139
trans-cinnamic acid in a 1 cm cuvette under the present determination conditions.
140
C4H activity was determined according to the method of Koopmann et al. (1999). Fresh
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leaves (0.2 g) were homogenized in 5 mL of 0.1 mol·L-1 precooled potassium phosphate buffer.
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The supernatant that underwent filtration and centrifugation was used for further analysis. The
143
reaction system contained 0.2 mL enzyme extract, 3 mL 4.0 mg·10 mL-1 NADPH, 0.2 mL 50 8
144
mmol·L-1 EDTA, and 3 mL 0.1 M potassium phosphate buffer (pH 7.6). The reaction mixture was
145
incubated at 30 °C for 30 min under darkness and was terminated by adding 0.5 mL HCl (6 M).
146
The absorbance of the reaction mixture was measured at 340 nm using a spectrophotometer versus
147
a blank.
148
4CL activity was determined according to the method of Knobloch and Hahlbrock (1977).
149
Briefly, fresh leaves (0.5 g) were homogenized in 5 mL of Tris-HCl buffer. The supernatant that
150
underwent filtration and centrifugation was used for further analysis. The reaction system
151
contained 150 µL enzyme extract and 3 mL Tris-HCl buffer. The reaction mixture was incubated
152
at 40 °C for 10 min under darkness and was terminated by adding 0.5 mL HCl (6 M). The
153
absorbance of the reaction mixture was measured at 333 nm using a spectrophotometer versus a
154
blank.
155 156 157
2.3 Analysis of Soluble Sugar, Soluble Protein, and Flavonoid Contents The soluble sugar content was determined according to the protocol of Wang et al. (2014).
158
The leaves were cut into pieces and extracted in boiling water for 30 min after 10 mL distilled
159
water was added. The extraction solution was transferred to a 25 mL volumetric flask and fixed to
160
a volume of 25 mL. Then, 0.5 mL extract, 0.5 mL ethyl anthrone acetate reagent, 1.5 mL distilled
161
water and 5 mL 95% (v/v) sulfuric acid were mixed together in the test tube. The tube was placed
162
in boiling water for 10 min. Distilled water replaced the extract and acted as a blank control. The
163
absorbance of the reaction mixture was measured at 620 nm using a spectrophotometer. The 9
164
soluble sugar content was calculated from a standard curve. The soluble protein content was
165
determined according to the protocol of Wang et al. (2014). Fresh leaves (0.3 g) were
166
homogenized in 5 mL distilled water and centrifuged for 10 min at 4 °C. One milliliter of extract
167
and 5 mL of Coomassie brilliant blue reagent were mixed together. The absorbance of the reaction
168
mixture was measured at 595 nm using a spectrophotometer. The soluble protein content was
169
calculated from a standard curve.
170
Flavonoid contents were determined according to the protocol proposed by the Chinese
171
Pharmacopoeia Commission (2010). The samples were oven-dried and ground into powder (0.15
172
mm sieve). A total of 0.500 g of each sample was placed in Soxhlet extractors, extracted in 100 mL
173
petroleum ether for 1 h, and then evaporated to dryness to obtain the residue. The residue was
174
added to 100 mL methanol, refluxed for 4 h, and then evaporated to dryness again with rotary
175
evaporation. A mixed liquor of methanol and 25% (v/v) muriatic acid (4:1, v/v) was added, and
176
reflux was performed for 30 min. The obtained extraction was filtered through a membrane filter
177
(0.2 µm), transferred to a 50 mL flask, and then diluted with methanol solution to 50 mL.
178
Flavonoid contents were determined using an HPLC system with a 1.0 mL/min flow rate. HPLC
179
was performed on a Diamonsil C18 column (4.6 mm×250 mm, 5 µm). The detection wavelength
180
was set at 360 nm, and the column temperature was maintained at 25 °C. The mobile phase
181
consisted of methanol-1.0% acetic acid (50:50, v/v). Peaks were identified using retention time
182
standards from the National Institute for the Control of Pharmaceutical and Biological Products.
183
Reference solutions containing 30 µg quercetin, 30 µg kaempferol, 20 µg isorhamnetin and 1 mL 10
184
methanol were precisely prepared. A 10 µL reference solution and extract solution were
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transferred into a liquid chromatograph (Waters 2695, USA), and the contents of quercetin,
186
kaempferol, and isorhamnetin were calculated according to the calibration curves of standard
187
solutions. The total flavonoid content = (quercetin content + kaempferol content + isorhamnetin
188
content) × 2.51 (Chinese Pharmacopoeia Commission, 2010). The injection volume of the sample
189
solution was 10 µL, and the experiment was repeated three times.
190 191
2.4 Determination of IAA, GAs, and ABA
192
The determination of IAA, GAs, and ABA in leaves was conducted using indirect ELISA
193
techniques according to the methods of Yang et al. (2001) and He (1993). Samples of 0.5 g were
194
homogenized in liquid nitrogen and extracted in cold 80% (v/v) methanol containing 1 mM
195
butylated hydroxytoluene overnight at 4 °C. The extracts were centrifuged at 10,000xg (4 °C) for
196
20 min and then passed through a C18 Sep-Pak cartridge (Waters) and dried in N2. The residues
197
were dissolved in PBS (0.01 M, pH 7.4) to determine the IAA, GA, and ABA levels. Microtitration
198
plates were coated with IAA, GAs, or ABA-ovalbumin conjugates in NaHCO3 buffer (50 M, pH
199
9.6) and left overnight at 37 °C. Ovalbumin solution (10 mg/mL) was then added to each well to
200
block nonspecific binding. After incubation for 30 min at 37 °C, standard IAA, GA, and ABA
201
samples and antibodies were added and incubated for 45 min at 37 °C. Antibodies were provided
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by Dr. Baomin Wang (Chinese Agricultural University). Then, horseradish POD-labelled goat
203
antirabbit immunoglobulin was added to each well and incubated for 1 h at 37 °C. Finally, the 11
204
buffered enzyme substrate (orthophenylenediamino) was added, and the reaction was performed in
205
the dark at 37 °C for 15 min and then terminated using 3 M H2SO4. The absorbance was recorded
206
at 490 nm. Calculations of the enzyme-immunoassay data were performed according to Weiler
207
and Spanier (1981). The percentage recovery of each hormone was calculated by adding known
208
amounts of standard hormone to a split extract. The percentage recoveries were all above 90%, and
209
all sample extract dilution curves paralleled the standard curves, indicating the absence of
210
nonspecific inhibitors in the extracts.
211 212 213
2.5 Statistical Analyses Data analyses were performed using SPSS software version 20.0 (IBM Inc., Chicago,
214
Illinois, USA). The collected data are presented as the means ± standard deviations (n = 3).
215
One-way analysis of variance and Duncan’s range test were used to distinguish the differences
216
among treatments during the same period; P<0.05 was considered statistically significant.
217
Pearson’s correlation among growth regulator concentrations and metabolite contents was also
218
detected using SPSS.
219 220
3 Results
221 222
3.1 Secondary Metabolism-Related Enzyme Activities
12
223
The effect of temperature conditions on three secondary metabolism-related key enzyme
224
activities during the 40-day incubation is summarized in Fig. 1. The PAL activities under the three
225
temperature conditions decreased over time, with a modest rebound under 15/10 and 25/20 °C
226
conditions at the 40-day sampling point (Fig. 1). Specifically, significantly higher PAL activities
227
were detected under 15/10 °C conditions at the 10-day, 30-day, and 40-day sampling points
228
(P<0.0001, P<0.00001, and P<0.0001, respectively). Among these three treatments, the highest
229
average PAL activity (19.77 µmol·min-1·g-1) was recorded under the 15/10 °C condition, and the
230
lowest average PAL activity (10.15 µmol·min-1·g-1) was recorded under the 35/30 °C condition.
231
Unlike the PAL activities, the 4CL activities were relatively stable, and no significant differences
232
were observed among the treatments for all sampling points (P>0.05). The average 4CL activities
233
under the 15/10, 25/20, and 35/30 °C conditions were 14.34, 14.13, and 16.84 µmol·min-1·g-1,
234
respectively. Additionally, the C4H activities were significantly higher under 15/10 °C conditions
235
than under the other temperature conditions at all sampling points except the 30-day sampling
236
point (P<0.01). The average C4H activities under the 15/10, 25/20, and 35/30 °C conditions were
237
20.65, 15.03, and 14.93 µmol·min-1·g-1, respectively. Positive stimulations of PAL and C4H
238
activities were obtained under relative low temperature, and the 4CL activities were relatively
239
stable under the three temperature conditions.
13
240 241
Fig. 1. Effects of temperature on PAL, 4CL, and C4H activities in ginkgo leaves during the
242
treatment periods (mean ± SD). The vertical bars indicate the standard deviations, and the asterisks
243
represent significant differences between temperature treatments at the same sampling point
244
(P<0.05).
245 246 247 248 249
3.2 IAA, GA and ABA Concentrations Dynamic changes in the contents of the three growth regulators subjected to different
250
temperature treatments were detected (Fig. 2). The ABA concentration increased under the 15/10
251
and 35/30 °C conditions in the first 30-day treatment period and decreased at the 40-day sampling
252
point. The dynamic pattern of ABA contents under the 25/20 °C condition differed from that of
253
other treatments. Specifically, significantly higher ABA contents were observed under the 25/20
254
°C condition at the 10-day sampling point (P<0.0001), and significantly higher ABA contents
255
were observed under the 35/30 °C condition at the 20-, 30-, and 40-day sampling points (P<0.001, 14
256
P<0.0001, and P<0.00001, respectively). The IAA and GA contents of the temperature treatments
257
appeared to fluctuate and showed similar patterns during the whole treatment period. The highest
258
contents of IAA and GAs were observed at the 20- and 30-day sampling points under the 35/30
259
and 15/10 °C conditions, respectively. The IAA and GA contents showed significant differences
260
among the temperature treatments at the 10-, 20-, 30-, and 40-day sampling points (P<0.05).
261 262
Fig. 2. Effects of temperature on the ABA, IAA, and GA concentrations in ginkgo leaves during
263
the treatment periods (mean ± SD). The vertical bars indicate the standard deviations, and the
264
asterisks represent significant differences among temperature treatments at the same sampling
265
point (P<0.05).
266 267 268
3.3 Primary and Secondary Metabolites The contents of important primary and secondary metabolites during the 40-day incubation
269
are summarized in Fig. 3. Generally, the soluble sugar contents of the 15/10 °C treatment were
270
higher than those of the other treatments except at the 10- and 20-day sampling points, and
271
significant differences were only obtained at the 40-day sampling point (P<0.01). The highest and 15
272
lowest soluble sugar contents were observed under the 15/10 °C condition (3.91 mg·g-1) and under
273
the 35/30 °C condition (1.84 mg·g-1) at the 40-day sampling point, respectively. The soluble
274
protein contents of the 35/30 °C treatment were higher than those of the other treatments except at
275
the 30-day sampling point, and significant differences were only obtained at the 10-day sampling
276
point (P<0.05). The highest soluble protein content was observed at the 10-day sampling point
277
under the 35/30 °C condition (0.68 mg·g-1). The contents of quercetin, kaempferol, isorhamnetin,
278
and total flavonoids shared the same trend along the treatment period. The 35/30 °C and 25/20 °C
279
treatments had higher secondary metabolite contents at the 10- and 20-day sampling points,
280
respectively, and no significant differences were observed (P>0.05). The 35/30 °C and 15/10 °C
281
treatments had significantly higher secondary metabolite contents at the 30- and 40-day sampling
282
points (P<0.01 and P<0.05, respectively). The highest total flavonoid, quercetin, kaempferol, and
283
isorhamnetin contents were observed at the 40-day sampling point under the 15/10 °C treatment
284
(20.21, 4.65, 3.81, and 1.10 mg·g-1 DW, respectively).
16
285 286
Fig. 3. Effects of temperature on the soluble sugar, soluble protein, quercetin, kaempferol,
287
isorhamnetin, and total flavonoid contents under the different temperatures during the treatment
17
288
periods (mean ± SD). The different lowercase letters represent significant differences between the
289
temperature treatments at the same sampling point at the P<0.05 level.
290 291 292
3.4 Correlation Analysis The Pearson correlations among growth regulator concentrations and metabolite contents
293
under different temperature treatments are presented in Table 1. Different correlation patterns
294
were observed among soluble sugar, soluble protein, and total flavonoids under different
295
temperature treatments. Total flavonoids were positively correlated with soluble sugar and
296
negatively correlated with soluble protein under the 15/10 °C and 35/30 °C treatments, and
297
completely opposite results were observed under the 25/20 °C treatment. Total flavonoids were
298
significantly positively correlated with ABA under the 35/30 °C treatment (P<0.05), while
299
negative correlations were observed between total flavonoids and ABA under the 15/10 °C and
300
25/20 °C treatments. Significant positive correlations were observed among soluble sugar, IAA,
301
and GAs under the 35/30 °C treatment (P<0.05). The growth regulators were mutually positively
302
correlated.
303
Table 1. Pearson correlations between the primary and secondary metabolites and growth
304
regulators under the different temperature treatments. Temperature treatment 15/10 °C
Total flavonoids Soluble sugar Soluble protein
Soluble sugar
Soluble protein
ABA
IAA
0.373 -0.073
0.099
18
ABA IAA GAs
25/20 °C
35/30 °C
Soluble sugar Soluble protein ABA IAA GAs Soluble sugar Soluble protein ABA IAA GAs
-0.538 0.130 -0.283
-0.44 0.395 0.077
0.223 0.397 0.434
0.401 0.775**
0.857**
-0.229 0.052 -0.298
0.741** 0.879**
0.563
-0.423 -0.120 -0.085
0.699* 0.336
0.778**
-0.165 0.492
0.121
-0.396 -0.181 -0.389
-0.188 0.007 0.002
0.104 -0.223
-0.198
0.767** 0.506 -0.051
0.432 0.725** 0.816**
305 306 307
4 Discussion Temperature is considered one of the key environmental factors influencing plant growth
308
and metabolism (Dusenge et al., 2019). The present study demonstrated that temperature had a
309
profound impact on secondary metabolite-related key enzyme activities. PAL is the first enzyme
310
of the biosynthetic pathway of phenylpropanoids and is important for the synthesis of many
311
secondary compounds, such as flavonoids, lignin (Olsen et al., 2009) and phenolic compounds
312
(Christopoulos and Tsantili, 2015). The PAL activity was higher under the low-temperature
313
treatment than at room temperature or under the high-temperature treatment, with similar
314
observations reported by Wang et al. (2014). Studies have confirmed that plants induce the
315
synthesis and accumulation of anthocyanin through the phenylpropanoid pathway when exposed
316
to low temperature (Janas et al., 2002). Coupled with this process, transcripts of flavonoid 19
317
biosynthetic genes also increased, especially PAL (Yang et al., 2018). These observations
318
confirmed the induction effect of low temperature on the PAL activity and thus on the flavonoid
319
accumulation (Leyva et al., 1995). C4H and PAL are also key enzymes in the phenylpropanoid
320
pathway, where C4H catalyzes the hydroxylation of cinnamic acid to produce p-coumaric acid and
321
4CL plays a key role in the conversion process of 4-coumaric acid to CoA thiol esters (Lillo et al.,
322
2008). We found that the 4CL activity was relatively stable, and slightly higher values were found
323
under the high-temperature treatment. The C4H activity trend differed among the three
324
temperature treatments, which was in accordance with the trend of the flavonoid content over time
325
(Wang et al., 2014).
326
The coordination of several growth regulators has been shown to help plants deal with
327
many types of adversities (Shakirova et al., 2016). It is well documented that both auxin (IAA, the
328
main type of auxin present in higher plants) and GA are required for the promotion of plant growth
329
and that ABA represents a stress growth regulator. Delayed growth is a strategy to cope with the
330
environmental stress in plants (Scottin et al., 2004). Hence, inhibition of growth-promoting GAs
331
and IAA synthesis is a common way to improve resistance to stress (Achard et al., 2006). In this
332
study, we found that IAA and GAs shared similar variation patterns under each temperature
333
condition. A previous study focusing on Arabidopsis reported that root growth was controlled by
334
auxin through the regulation of cell responses to the phytohormone GA (Fu and Harberd, 2003),
335
which showed a synergistic effect. Similarly, the GA1 content in tobacco leaves significantly
336
decreased following a decrease in temperature (Niu et al., 2013). Growth regulators affect the 20
337
accumulation of secondary metabolites; namely, IAA and GAs can inhibit flavonoid accumulation
338
and GAs can inhibit the phenylpropanoid pathway at the PAL activity level (Li et al., 2003; Yuan
339
et al., 2012). ABA has been proposed to serve as a mediator when the plant responds to abiotic and
340
biotic stresses. The higher ABA concentration under the high-temperature treatment could be
341
explained by ABA being involved in the heat signaling mechanisms in cells (Gouot et al., 2019).
342
As reported by Carbonell-Bejerano et al. (2013), the expression of ABA biosynthetic genes was
343
upregulated under the high-temperature conditions, and the ABA content was increased; however
344
a reduction in anthocyanin content was observed. Our results confirmed these findings. Moreover,
345
the levels of the key enzymes involved in flavonoid metabolism were not consistent with the
346
flavonoid contents, possibly because the growth regulator has a certain regulatory effect on the
347
metabolism of ginkgo flavonoids.
348
Compared with other studies, we found that soluble sugar and soluble protein showed no
349
significant correlations with the flavonoid constituents. The accumulation of soluble sugar was
350
promoted by low temperature, which is in accordance with previous studies reporting that plants
351
under low-temperature treatment accumulated more soluble sugar (Atkin et al., 2000). Previous
352
studies have indicated that the enhanced degradation of chlorophyll and the subsequent reduced
353
photosynthesis induced by high temperature (36 °C) were considered to be primary factors for the
354
decrease in soluble sugar contents (Zhao et al., 2011). Soluble proteins and flavonoids require the
355
same substrate. Quercetin, kaempferol, and isorhamnetin are flavonols and are the three main
356
flavonoid constituents in ginkgo leaves. Quercetin possessed the highest content (ranging from 21
357
1.665 to 4.646 mg·g-1), followed by kaempferol (0.399 to 1.100 mg·g-1). In this study, quercetin,
358
kaempferol, isorhamnetin, and total flavonoids shared the same variation patterns with the
359
processing time. The room- and high-temperature treatments resulted in higher flavonoid contents
360
during the first 30 d, but the low-temperature treatment resulted in relatively high flavonoid
361
contents at the 40-day sampling point. The light intensity and quality of the three artificial
362
chambers were completely consistent, and the strong temperature effect should be highlighted
363
from the results of our study. Generally, higher flavonol contents were observed when the
364
temperature was reduced by 8 °C compared with the control temperature (Pastore et al., 2017). In
365
addition, low temperatures can induce an increase in the synthesis of flavonols in Arabidopsis, and
366
conversely, flavonols act as antioxidants to defend plants and avoid damage from chilling stress
367
(Petridis et al., 2016). Among these constituents, quercetin was considered the most effective
368
cryoprotectant for plant leaves (Orthen and Popp, 2000). As reported by Solfanelli et al. (2006),
369
the increased soluble sugar content under low-temperature conditions acted as a signaling agent
370
for stimulating flavonoid biosynthesis. At the end of our experiment, we found that high
371
temperature (35/30 °C) decreased the contents of flavonoid components (quercetin, kaempferol,
372
and isorhamnetin). The lower flavonol contents under the high-temperature treatment were
373
probably due to enzymatic or chemical degradation and not simply the inhibition of secondary
374
metabolism (Mori et al., 2007). A high-temperature inhibitory effect on flavonols was also
375
observed by Spayd et al. (2002). High temperatures also repress anthocyanin accumulation in
376
various plants. For instance, high temperature reduced the total anthocyanin content in the 22
377
‘Cabernet Sauvignon’ grape (Mori et al., 2005). Similar to the flavonols, the anthocyanin
378
concentration of Dodonaea ‘Dana’ fruit growing in the lower temperature regime was four times
379
higher than that in fruit growing at an elevated temperature (Nissim-Levi et al., 2013). Taken
380
together, the results of our study suggested that the inhibition of flavonoid accumulation in ginkgo
381
leaves under high-temperature conditions is caused by the combined effects of various pathways.
382 383 384
5 Conclusions Different temperatures affected the enzyme activity, flavonoid accumulation, and growth
385
regulator metabolism. Compared with ginkgo grown at 25/20 °C and 35/30 °C, treatment under
386
15/10 °C condition significantly promoted total flavonoids and the accumulation of their
387
constituents. The results of the present study showed that IAA and GAs have negative effects on
388
flavonoid accumulation, while ABA had the opposite effect on flavonoid accumulation under the
389
35/30 °C condition. Positive correlations were observed between soluble sugars, IAA, and GAs,
390
which may directly promote the growth of plants. The high flavonoid concentration was not
391
always parallel to high PAL activity, which indicated that flavonoid biosynthesis is a complex
392
pathway that has many regulators and is not limited to only one or a few factors. Generally, high
393
temperature decreased flavonoid concentrations by regulating enzyme activity and growth
394
regulator metabolism in ginkgo. Therefore, the relatively lower temperature (15/10 °C) is more
395
favorable for flavonoid accumulation and will provide a theoretical basis for further study and
396
practical applications. 23
397 398
Acknowledgments
399
This work was supported by the Agricultural Science and Technology Independent Innovation
400
Funds of Jiangsu Province (CX(16)1005), the National Key Research and Development Program
401
of China (2017YFD0600700), the Priority Academy Program Development of Jiangsu Higher
402
Education Institution (PAPD), the Postgraduate Research & Practice Innovation Program of
403
Jiangsu Province (KYCX18_0955) and the Doctorate Fellowship Foundation of Nanjing Forestry
404
University.
405 406
Declarations of Interest: none.
407
24
408 409
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1. The content of flavonoid components increased under 15/10 °C condition. 2. PAL activity was promoted under low temperature condition. 3. Low temperature promoted soluble sugar accumulation.
4. Abscisic acid promoted flavonoid accumulation under high temperature condition.
Author Contributions: study design, Xin Zhou and Guibin Wang; samples collection and analysis, Xin Zhou and Jing Guo; statistical analyses, Jing Guo; first draft, Xin Zhou and Jing Guo; review and editing, Tongli Wang, Fuliang Cao and Guibin Wang.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0981-9428(19)30517-0
DOI:
https://doi.org/10.1016/j.plaphy.2019.12.009
Reference:
PLAPHY 5966
To appear in:
Plant Physiology and Biochemistry
Received Date: 24 June 2019 Revised Date:
7 December 2019
Accepted Date: 9 December 2019
Please cite this article as: J. Guo, X. Zhou, T. Wang, G. Wang, F. Cao, Regulation of flavonoid metabolism in ginkgo leaves in response to different day-night temperature combinations, Plant Physiology et Biochemistry (2020), doi: https://doi.org/10.1016/j.plaphy.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.
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Regulation of Flavonoid Metabolism in Ginkgo Leaves in Response to Different Day-Night
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Temperature Combinations
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Jing Guoa, b, Xin Zhoua, Tongli Wangb, Guibin Wanga,* and Fuliang Caoa
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a
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159 Longpan Road, Nanjing 210037, China
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b
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Columbia, Vancouver V6T 1Z4, Canada
Nanjing Forestry University, Co-Innovation Centre for Sustainable Forestry in Southern China,
Department of Forest and Conservation Sciences, Faculty of Forestry, the University of British
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*Corresponding Author
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Guibin Wang
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Tel: +86-85427201
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Email: [email protected]
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1
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Abstract
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Flavonoids are the most important secondary metabolites in ginkgo (Ginkgo biloba L.) leaves that
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determine its medicinal quality. Studies have suggested that secondary metabolism is strongly
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affected by temperature in other plant species, but little is known about ginkgo. In this study, we
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investigated the effects of different day-night temperature combinations (15/10, 25/20, and 35/30
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°C (day/night)) on key enzyme activity, growth regulator concentrations, and flavonoid
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accumulation in ginkgo leaves. We found that phenylalanine ammonia-lyase (PAL) activity was
23
enhanced and inhibited at 15/10 and 35/30 °C, respectively. Cinnamate-4-hydroxylase (C4H)
24
activity was relatively stable under the three temperature conditions, and the p-coumarate CoA
25
ligase (4CL) activity showed different trends under the three temperature conditions. The
26
concentrations of flavonoid constituents (quercetin, kaempferol, and isorhamnetin) were
27
decreased and increased under the 35/30 and 15/10 °C conditions, respectively. Low temperature
28
promoted soluble sugar accumulation, while temperature had a limited impact on the accumulation
29
of soluble protein. The pattern of change in the total flavonoid concentration was not always in
30
agreement with PAL activity due to its complex pathway. Indoleacetic acid (IAA) and gibberellin
31
(GA) changes shared similar patterns and had limited effects on flavonoid accumulation, while
32
abscisic acid (ABA) acted as a promotor of flavonoid accumulation under high-temperature
33
conditions. The total flavonoids achieved the highest content under the 15/10 °C treatment on the
34
40th day. Therefore, the lower temperature (15/10 °C) is more favorable for flavonoid
35
accumulation and will provide a theoretical basis for further study. 2
36 37
Keywords: Ginkgo biloba L., temperature combination, flavonoids, enzyme activity, growth
38
regulators
39 40
Abbreviations
41
GBE, Ginkgo biloba extracts; ROS, reactive oxygen species; DW, dry weight; FW, fresh weight.
42 43
3
44 45
1 Introduction Ginkgo (Ginkgo biloba L.) is an ancient living tree species that originated in the Jurassic
46
period and commonly known as a “living fossil” (Stromgaard and Nakanishi, 2004). Due to its
47
multiple purposes, Ginkgo has been introduced from China to many countries and regions
48
worldwide (Xu et al., 2015). The most common utilization purposes of ginkgo are the collection of
49
leaves for pharmaceutical applications and the planting of trees for landscaping (Wu et al., 2019).
50
In the pharmaceutical industry, flavonoids and terpene lactones are the most important secondary
51
metabolites of ginkgo and have many functions in medicinal and health care, including lowering
52
blood pressure (Xiao et al., 2014), anti-tumor, anti-inflammatory (Van Beek and Montoro, 2009),
53
antioxidant (Wang et al., 2016), and radical scavenging activities (Ellnain-Wojtaszek et al., 2003).
54
Ginkgo biloba extracts (GBE) are the most popular product for the clinical treatment of cognitive
55
disorders (Le Bars et al., 1997). The chemical components of GBE mainly include 24%
56
flavonoids, 6% terpene lactones, and 5-10% organic acids, and medical effects are mainly
57
achieved via flavonoid constituents (Bastianetto et al., 2000). Flavonoids also play key roles in the
58
survival and normal growth of plants, such as the production of pigmentation for ultraviolet
59
radiation protection and resistance against microbial pathogens (Harborne and Williams, 2000;
60
Mierziak et al., 2014).
61
Instead of participating in the construction of plants directly, secondary metabolites are
62
generally involved in the protection process to reduce the damaging effects of different biotic and
63
abiotic stresses (Liu et al., 2015a). Numerous studies have shown increasing concentrations of 4
64
secondary metabolites when plants are exposed to extreme temperature, high salinity, water
65
deficiency, and nitrogen deficiency situations (Dai et al., 2015; Liu et al., 2015a, 2017).
66
Flavonoids are present in many types of plants and exist as many active compounds, such as
67
quercetin, kaempferol and isorhamnetin (Liu et al., 2015b). The important features of flavonoids
68
are to filter out ultraviolet radiation, regulate auxin transport, promote pollen germination, and act
69
as antioxidants (Ni et al., 2017). Additionally, plants generate high levels of reactive oxygen
70
species (ROS) in response to stress, and quercetin has the highest radical-scavenging capacity
71
(Baek et al., 2015).
72
Flavonoid biosynthesis is regulated by multiple factors, among which temperature is
73
known to be the most important environmental factor. Temperature influences plant growth,
74
organogenesis, and primary and secondary metabolism (Gouot et al., 2019). For instance, adverse
75
temperature conditions will affect the plant physiological status and then cause metabolic
76
alterations, which lead to changes in the composition of plant metabolites (Goh et al., 2016).
77
Isoflavonoid concentrations were altered in Glycine max seedlings growing under different
78
temperature regimes (Posmyk et al., 2005). When suffering heat stress, plants will be protected by
79
antioxidant systems through the production of natural antioxidant metabolites, such as flavonoids
80
and phenolics (Cheng et al., 2018; Ghasemzadeh et al., 2010; Gooding et al., 2001).
81
Primary metabolism refers to a series of reactions and pathways that are essential for plant
82
survival, while secondary metabolism involves secondary metabolites that participate in
83
protection and development processes (Kroymann, 2011). Photosynthesis, glycolysis, the 5
84
tricarboxylic acid (TCA) cycle, and the shikimate pathway are vital series of reactions and
85
pathways to provide energy and products for the synthesis of secondary metabolites. A negative
86
correlation was detected between total flavonoid production and soluble protein contents in
87
Labisia pumila Blume under lower fertilization conditions, while total flavonoid biosynthesis is
88
positively correlated with soluble sugar concentrations (Ibrahim et al., 2011). Flavonoids are
89
synthesized via the phenylpropanoid pathway and share the same precursors and common genes as
90
other phenolic compounds. Many enzyme families act as catalysts through the phenylpropanoid
91
pathway, such as phenylalanine ammonia-lyase (PAL; the first enzyme in the phenylpropanoid
92
pathway), p-coumaroyl: CoA ligase (4CL), cinnamate-4-hydroxylase (C4H), chalcone reductase
93
(CHS) and other enzymes (Gouot et al., 2019). Additionally, flavonoid synthesis is also affected
94
by plant growth regulators, such as auxin, cytokinin (CK), gibberellins (GAs), and abscisic acid
95
(ABA). Growth regulators are also affected by abiotic stress, which contributes to signaling
96
mechanisms in cells.
97
Knowledge regarding the effect of temperature on flavonoid concentration, flavonoid
98
metabolism, and growth regulator synthesis is scarce. Consequently, the impact of global warming
99
on the growth of ginkgo and the production of flavonoids remains a big concern. Thus, the main
100
objectives of this study were to understand the relationships between temperature and flavonoid
101
contents, the activity of key enzymes (PAL, 4CL, and C4H) and the concentrations of growth
102
regulators (indoleacetic acid (IAA), GAs, and ABA) in ginkgo leaves. Additionally, we explored
103
the optimum temperature for flavonoid accumulation, which will benefit the utilization efficiency 6
104
of ginkgo leaves. Our study was conducted under controlled growing conditions, including three
105
alternating day/night temperatures (15/10, 25/20, and 35/30 °C).
106 107
2 Materials and Methods
108 109
2.1 Plant Material and Experimental Conditions
110
The experiment was carried out in the artificial chambers of Nanjing Forestry University,
111
Jiangsu Province, China. One-year-old ginkgo seedlings were cultivated in plastic pots (diameter
112
× height: 19 × 16 cm), with two seedlings per pot. The seedlings were obtained from an
113
experimental nursery in Pizhou city. The seedlings were from the same variety, and the average
114
height and ground diameter were 43.9 and 8.66 mm, respectively. The pots were filled with a
115
mixture of loam, sand, and peat (2:1:1, v/v). The nutrient contents of the mixture were as follows:
116
organic matter, 12.0 g·kg-1; hydrolyzed nitrogen, 121 mg·kg-1; available phosphorus (P2O5), 12.7
117
mg·kg-1; and available potassium (K2O), 11.7 mg·kg-1. The mixture was characterized by a field
118
water-holding capacity of 22.0% and a pH value of 8.0.
119
The experiments were performed in July with initial acclimatization temperatures of 25
120
and 15 °C (day/night) and relative humidity (RH) of 75% for two weeks. After preculture,
121
seedlings were transferred to three artificial chambers with temperature conditions of 15/10,
122
25/20, and 35/30 °C (day/night), the light density was 800 µm·m-2·s-1, the photoperiod cycle was
123
16 h light–8 h dark, and the relative humidity (RH) was 75%. A total of 50 pots and 100 seedlings 7
124
were contained in each chamber; 45 pots were divided into 3 groups; and 5 pots were cultured in
125
the case of accidents. The top fourth and fifth leaves from 5 randomly selected seedlings in each
126
group were collected and pooled. The leaves were collected at 10 d, 20 d, 30 d, and 40 d after being
127
transferred to the respective chambers. The samples were immediately frozen in liquid nitrogen
128
and stored at -70 °C until further determination.
129 130 131
2.2 Extraction and Determination of Enzyme Activity The extraction and determination of PAL activity were performed according to the method
132
of Ortega-García and Peragón (2009). Briefly, the enzyme extraction was carried out in an ice
133
bath. The samples were pulverized, and the resulting powder was homogenized. The homogenates
134
were centrifuged at 20,000×g for 15 min at 4 °C. The supernatant was then collected for PAL
135
activity determination as a crude enzyme extract. The reaction mixture was incubated at 37 °C for
136
30 min under darkness and was terminated by adding 0.5 mL HCl (6 M). The absorbance of the
137
reaction mixture was measured at 290 nm using a spectrophotometer versus a blank. One unit of
138
PAL activity was defined as the amount of enzyme needed to catalyze the formation of 1 µmol
139
trans-cinnamic acid in a 1 cm cuvette under the present determination conditions.
140
C4H activity was determined according to the method of Koopmann et al. (1999). Fresh
141
leaves (0.2 g) were homogenized in 5 mL of 0.1 mol·L-1 precooled potassium phosphate buffer.
142
The supernatant that underwent filtration and centrifugation was used for further analysis. The
143
reaction system contained 0.2 mL enzyme extract, 3 mL 4.0 mg·10 mL-1 NADPH, 0.2 mL 50 8
144
mmol·L-1 EDTA, and 3 mL 0.1 M potassium phosphate buffer (pH 7.6). The reaction mixture was
145
incubated at 30 °C for 30 min under darkness and was terminated by adding 0.5 mL HCl (6 M).
146
The absorbance of the reaction mixture was measured at 340 nm using a spectrophotometer versus
147
a blank.
148
4CL activity was determined according to the method of Knobloch and Hahlbrock (1977).
149
Briefly, fresh leaves (0.5 g) were homogenized in 5 mL of Tris-HCl buffer. The supernatant that
150
underwent filtration and centrifugation was used for further analysis. The reaction system
151
contained 150 µL enzyme extract and 3 mL Tris-HCl buffer. The reaction mixture was incubated
152
at 40 °C for 10 min under darkness and was terminated by adding 0.5 mL HCl (6 M). The
153
absorbance of the reaction mixture was measured at 333 nm using a spectrophotometer versus a
154
blank.
155 156 157
2.3 Analysis of Soluble Sugar, Soluble Protein, and Flavonoid Contents The soluble sugar content was determined according to the protocol of Wang et al. (2014).
158
The leaves were cut into pieces and extracted in boiling water for 30 min after 10 mL distilled
159
water was added. The extraction solution was transferred to a 25 mL volumetric flask and fixed to
160
a volume of 25 mL. Then, 0.5 mL extract, 0.5 mL ethyl anthrone acetate reagent, 1.5 mL distilled
161
water and 5 mL 95% (v/v) sulfuric acid were mixed together in the test tube. The tube was placed
162
in boiling water for 10 min. Distilled water replaced the extract and acted as a blank control. The
163
absorbance of the reaction mixture was measured at 620 nm using a spectrophotometer. The 9
164
soluble sugar content was calculated from a standard curve. The soluble protein content was
165
determined according to the protocol of Wang et al. (2014). Fresh leaves (0.3 g) were
166
homogenized in 5 mL distilled water and centrifuged for 10 min at 4 °C. One milliliter of extract
167
and 5 mL of Coomassie brilliant blue reagent were mixed together. The absorbance of the reaction
168
mixture was measured at 595 nm using a spectrophotometer. The soluble protein content was
169
calculated from a standard curve.
170
Flavonoid contents were determined according to the protocol proposed by the Chinese
171
Pharmacopoeia Commission (2010). The samples were oven-dried and ground into powder (0.15
172
mm sieve). A total of 0.500 g of each sample was placed in Soxhlet extractors, extracted in 100 mL
173
petroleum ether for 1 h, and then evaporated to dryness to obtain the residue. The residue was
174
added to 100 mL methanol, refluxed for 4 h, and then evaporated to dryness again with rotary
175
evaporation. A mixed liquor of methanol and 25% (v/v) muriatic acid (4:1, v/v) was added, and
176
reflux was performed for 30 min. The obtained extraction was filtered through a membrane filter
177
(0.2 µm), transferred to a 50 mL flask, and then diluted with methanol solution to 50 mL.
178
Flavonoid contents were determined using an HPLC system with a 1.0 mL/min flow rate. HPLC
179
was performed on a Diamonsil C18 column (4.6 mm×250 mm, 5 µm). The detection wavelength
180
was set at 360 nm, and the column temperature was maintained at 25 °C. The mobile phase
181
consisted of methanol-1.0% acetic acid (50:50, v/v). Peaks were identified using retention time
182
standards from the National Institute for the Control of Pharmaceutical and Biological Products.
183
Reference solutions containing 30 µg quercetin, 30 µg kaempferol, 20 µg isorhamnetin and 1 mL 10
184
methanol were precisely prepared. A 10 µL reference solution and extract solution were
185
transferred into a liquid chromatograph (Waters 2695, USA), and the contents of quercetin,
186
kaempferol, and isorhamnetin were calculated according to the calibration curves of standard
187
solutions. The total flavonoid content = (quercetin content + kaempferol content + isorhamnetin
188
content) × 2.51 (Chinese Pharmacopoeia Commission, 2010). The injection volume of the sample
189
solution was 10 µL, and the experiment was repeated three times.
190 191
2.4 Determination of IAA, GAs, and ABA
192
The determination of IAA, GAs, and ABA in leaves was conducted using indirect ELISA
193
techniques according to the methods of Yang et al. (2001) and He (1993). Samples of 0.5 g were
194
homogenized in liquid nitrogen and extracted in cold 80% (v/v) methanol containing 1 mM
195
butylated hydroxytoluene overnight at 4 °C. The extracts were centrifuged at 10,000xg (4 °C) for
196
20 min and then passed through a C18 Sep-Pak cartridge (Waters) and dried in N2. The residues
197
were dissolved in PBS (0.01 M, pH 7.4) to determine the IAA, GA, and ABA levels. Microtitration
198
plates were coated with IAA, GAs, or ABA-ovalbumin conjugates in NaHCO3 buffer (50 M, pH
199
9.6) and left overnight at 37 °C. Ovalbumin solution (10 mg/mL) was then added to each well to
200
block nonspecific binding. After incubation for 30 min at 37 °C, standard IAA, GA, and ABA
201
samples and antibodies were added and incubated for 45 min at 37 °C. Antibodies were provided
202
by Dr. Baomin Wang (Chinese Agricultural University). Then, horseradish POD-labelled goat
203
antirabbit immunoglobulin was added to each well and incubated for 1 h at 37 °C. Finally, the 11
204
buffered enzyme substrate (orthophenylenediamino) was added, and the reaction was performed in
205
the dark at 37 °C for 15 min and then terminated using 3 M H2SO4. The absorbance was recorded
206
at 490 nm. Calculations of the enzyme-immunoassay data were performed according to Weiler
207
and Spanier (1981). The percentage recovery of each hormone was calculated by adding known
208
amounts of standard hormone to a split extract. The percentage recoveries were all above 90%, and
209
all sample extract dilution curves paralleled the standard curves, indicating the absence of
210
nonspecific inhibitors in the extracts.
211 212 213
2.5 Statistical Analyses Data analyses were performed using SPSS software version 20.0 (IBM Inc., Chicago,
214
Illinois, USA). The collected data are presented as the means ± standard deviations (n = 3).
215
One-way analysis of variance and Duncan’s range test were used to distinguish the differences
216
among treatments during the same period; P<0.05 was considered statistically significant.
217
Pearson’s correlation among growth regulator concentrations and metabolite contents was also
218
detected using SPSS.
219 220
3 Results
221 222
3.1 Secondary Metabolism-Related Enzyme Activities
12
223
The effect of temperature conditions on three secondary metabolism-related key enzyme
224
activities during the 40-day incubation is summarized in Fig. 1. The PAL activities under the three
225
temperature conditions decreased over time, with a modest rebound under 15/10 and 25/20 °C
226
conditions at the 40-day sampling point (Fig. 1). Specifically, significantly higher PAL activities
227
were detected under 15/10 °C conditions at the 10-day, 30-day, and 40-day sampling points
228
(P<0.0001, P<0.00001, and P<0.0001, respectively). Among these three treatments, the highest
229
average PAL activity (19.77 µmol·min-1·g-1) was recorded under the 15/10 °C condition, and the
230
lowest average PAL activity (10.15 µmol·min-1·g-1) was recorded under the 35/30 °C condition.
231
Unlike the PAL activities, the 4CL activities were relatively stable, and no significant differences
232
were observed among the treatments for all sampling points (P>0.05). The average 4CL activities
233
under the 15/10, 25/20, and 35/30 °C conditions were 14.34, 14.13, and 16.84 µmol·min-1·g-1,
234
respectively. Additionally, the C4H activities were significantly higher under 15/10 °C conditions
235
than under the other temperature conditions at all sampling points except the 30-day sampling
236
point (P<0.01). The average C4H activities under the 15/10, 25/20, and 35/30 °C conditions were
237
20.65, 15.03, and 14.93 µmol·min-1·g-1, respectively. Positive stimulations of PAL and C4H
238
activities were obtained under relative low temperature, and the 4CL activities were relatively
239
stable under the three temperature conditions.
13
240 241
Fig. 1. Effects of temperature on PAL, 4CL, and C4H activities in ginkgo leaves during the
242
treatment periods (mean ± SD). The vertical bars indicate the standard deviations, and the asterisks
243
represent significant differences between temperature treatments at the same sampling point
244
(P<0.05).
245 246 247 248 249
3.2 IAA, GA and ABA Concentrations Dynamic changes in the contents of the three growth regulators subjected to different
250
temperature treatments were detected (Fig. 2). The ABA concentration increased under the 15/10
251
and 35/30 °C conditions in the first 30-day treatment period and decreased at the 40-day sampling
252
point. The dynamic pattern of ABA contents under the 25/20 °C condition differed from that of
253
other treatments. Specifically, significantly higher ABA contents were observed under the 25/20
254
°C condition at the 10-day sampling point (P<0.0001), and significantly higher ABA contents
255
were observed under the 35/30 °C condition at the 20-, 30-, and 40-day sampling points (P<0.001, 14
256
P<0.0001, and P<0.00001, respectively). The IAA and GA contents of the temperature treatments
257
appeared to fluctuate and showed similar patterns during the whole treatment period. The highest
258
contents of IAA and GAs were observed at the 20- and 30-day sampling points under the 35/30
259
and 15/10 °C conditions, respectively. The IAA and GA contents showed significant differences
260
among the temperature treatments at the 10-, 20-, 30-, and 40-day sampling points (P<0.05).
261 262
Fig. 2. Effects of temperature on the ABA, IAA, and GA concentrations in ginkgo leaves during
263
the treatment periods (mean ± SD). The vertical bars indicate the standard deviations, and the
264
asterisks represent significant differences among temperature treatments at the same sampling
265
point (P<0.05).
266 267 268
3.3 Primary and Secondary Metabolites The contents of important primary and secondary metabolites during the 40-day incubation
269
are summarized in Fig. 3. Generally, the soluble sugar contents of the 15/10 °C treatment were
270
higher than those of the other treatments except at the 10- and 20-day sampling points, and
271
significant differences were only obtained at the 40-day sampling point (P<0.01). The highest and 15
272
lowest soluble sugar contents were observed under the 15/10 °C condition (3.91 mg·g-1) and under
273
the 35/30 °C condition (1.84 mg·g-1) at the 40-day sampling point, respectively. The soluble
274
protein contents of the 35/30 °C treatment were higher than those of the other treatments except at
275
the 30-day sampling point, and significant differences were only obtained at the 10-day sampling
276
point (P<0.05). The highest soluble protein content was observed at the 10-day sampling point
277
under the 35/30 °C condition (0.68 mg·g-1). The contents of quercetin, kaempferol, isorhamnetin,
278
and total flavonoids shared the same trend along the treatment period. The 35/30 °C and 25/20 °C
279
treatments had higher secondary metabolite contents at the 10- and 20-day sampling points,
280
respectively, and no significant differences were observed (P>0.05). The 35/30 °C and 15/10 °C
281
treatments had significantly higher secondary metabolite contents at the 30- and 40-day sampling
282
points (P<0.01 and P<0.05, respectively). The highest total flavonoid, quercetin, kaempferol, and
283
isorhamnetin contents were observed at the 40-day sampling point under the 15/10 °C treatment
284
(20.21, 4.65, 3.81, and 1.10 mg·g-1 DW, respectively).
16
285 286
Fig. 3. Effects of temperature on the soluble sugar, soluble protein, quercetin, kaempferol,
287
isorhamnetin, and total flavonoid contents under the different temperatures during the treatment
17
288
periods (mean ± SD). The different lowercase letters represent significant differences between the
289
temperature treatments at the same sampling point at the P<0.05 level.
290 291 292
3.4 Correlation Analysis The Pearson correlations among growth regulator concentrations and metabolite contents
293
under different temperature treatments are presented in Table 1. Different correlation patterns
294
were observed among soluble sugar, soluble protein, and total flavonoids under different
295
temperature treatments. Total flavonoids were positively correlated with soluble sugar and
296
negatively correlated with soluble protein under the 15/10 °C and 35/30 °C treatments, and
297
completely opposite results were observed under the 25/20 °C treatment. Total flavonoids were
298
significantly positively correlated with ABA under the 35/30 °C treatment (P<0.05), while
299
negative correlations were observed between total flavonoids and ABA under the 15/10 °C and
300
25/20 °C treatments. Significant positive correlations were observed among soluble sugar, IAA,
301
and GAs under the 35/30 °C treatment (P<0.05). The growth regulators were mutually positively
302
correlated.
303
Table 1. Pearson correlations between the primary and secondary metabolites and growth
304
regulators under the different temperature treatments. Temperature treatment 15/10 °C
Total flavonoids Soluble sugar Soluble protein
Soluble sugar
Soluble protein
ABA
IAA
0.373 -0.073
0.099
18
ABA IAA GAs
25/20 °C
35/30 °C
Soluble sugar Soluble protein ABA IAA GAs Soluble sugar Soluble protein ABA IAA GAs
-0.538 0.130 -0.283
-0.44 0.395 0.077
0.223 0.397 0.434
0.401 0.775**
0.857**
-0.229 0.052 -0.298
0.741** 0.879**
0.563
-0.423 -0.120 -0.085
0.699* 0.336
0.778**
-0.165 0.492
0.121
-0.396 -0.181 -0.389
-0.188 0.007 0.002
0.104 -0.223
-0.198
0.767** 0.506 -0.051
0.432 0.725** 0.816**
305 306 307
4 Discussion Temperature is considered one of the key environmental factors influencing plant growth
308
and metabolism (Dusenge et al., 2019). The present study demonstrated that temperature had a
309
profound impact on secondary metabolite-related key enzyme activities. PAL is the first enzyme
310
of the biosynthetic pathway of phenylpropanoids and is important for the synthesis of many
311
secondary compounds, such as flavonoids, lignin (Olsen et al., 2009) and phenolic compounds
312
(Christopoulos and Tsantili, 2015). The PAL activity was higher under the low-temperature
313
treatment than at room temperature or under the high-temperature treatment, with similar
314
observations reported by Wang et al. (2014). Studies have confirmed that plants induce the
315
synthesis and accumulation of anthocyanin through the phenylpropanoid pathway when exposed
316
to low temperature (Janas et al., 2002). Coupled with this process, transcripts of flavonoid 19
317
biosynthetic genes also increased, especially PAL (Yang et al., 2018). These observations
318
confirmed the induction effect of low temperature on the PAL activity and thus on the flavonoid
319
accumulation (Leyva et al., 1995). C4H and PAL are also key enzymes in the phenylpropanoid
320
pathway, where C4H catalyzes the hydroxylation of cinnamic acid to produce p-coumaric acid and
321
4CL plays a key role in the conversion process of 4-coumaric acid to CoA thiol esters (Lillo et al.,
322
2008). We found that the 4CL activity was relatively stable, and slightly higher values were found
323
under the high-temperature treatment. The C4H activity trend differed among the three
324
temperature treatments, which was in accordance with the trend of the flavonoid content over time
325
(Wang et al., 2014).
326
The coordination of several growth regulators has been shown to help plants deal with
327
many types of adversities (Shakirova et al., 2016). It is well documented that both auxin (IAA, the
328
main type of auxin present in higher plants) and GA are required for the promotion of plant growth
329
and that ABA represents a stress growth regulator. Delayed growth is a strategy to cope with the
330
environmental stress in plants (Scottin et al., 2004). Hence, inhibition of growth-promoting GAs
331
and IAA synthesis is a common way to improve resistance to stress (Achard et al., 2006). In this
332
study, we found that IAA and GAs shared similar variation patterns under each temperature
333
condition. A previous study focusing on Arabidopsis reported that root growth was controlled by
334
auxin through the regulation of cell responses to the phytohormone GA (Fu and Harberd, 2003),
335
which showed a synergistic effect. Similarly, the GA1 content in tobacco leaves significantly
336
decreased following a decrease in temperature (Niu et al., 2013). Growth regulators affect the 20
337
accumulation of secondary metabolites; namely, IAA and GAs can inhibit flavonoid accumulation
338
and GAs can inhibit the phenylpropanoid pathway at the PAL activity level (Li et al., 2003; Yuan
339
et al., 2012). ABA has been proposed to serve as a mediator when the plant responds to abiotic and
340
biotic stresses. The higher ABA concentration under the high-temperature treatment could be
341
explained by ABA being involved in the heat signaling mechanisms in cells (Gouot et al., 2019).
342
As reported by Carbonell-Bejerano et al. (2013), the expression of ABA biosynthetic genes was
343
upregulated under the high-temperature conditions, and the ABA content was increased; however
344
a reduction in anthocyanin content was observed. Our results confirmed these findings. Moreover,
345
the levels of the key enzymes involved in flavonoid metabolism were not consistent with the
346
flavonoid contents, possibly because the growth regulator has a certain regulatory effect on the
347
metabolism of ginkgo flavonoids.
348
Compared with other studies, we found that soluble sugar and soluble protein showed no
349
significant correlations with the flavonoid constituents. The accumulation of soluble sugar was
350
promoted by low temperature, which is in accordance with previous studies reporting that plants
351
under low-temperature treatment accumulated more soluble sugar (Atkin et al., 2000). Previous
352
studies have indicated that the enhanced degradation of chlorophyll and the subsequent reduced
353
photosynthesis induced by high temperature (36 °C) were considered to be primary factors for the
354
decrease in soluble sugar contents (Zhao et al., 2011). Soluble proteins and flavonoids require the
355
same substrate. Quercetin, kaempferol, and isorhamnetin are flavonols and are the three main
356
flavonoid constituents in ginkgo leaves. Quercetin possessed the highest content (ranging from 21
357
1.665 to 4.646 mg·g-1), followed by kaempferol (0.399 to 1.100 mg·g-1). In this study, quercetin,
358
kaempferol, isorhamnetin, and total flavonoids shared the same variation patterns with the
359
processing time. The room- and high-temperature treatments resulted in higher flavonoid contents
360
during the first 30 d, but the low-temperature treatment resulted in relatively high flavonoid
361
contents at the 40-day sampling point. The light intensity and quality of the three artificial
362
chambers were completely consistent, and the strong temperature effect should be highlighted
363
from the results of our study. Generally, higher flavonol contents were observed when the
364
temperature was reduced by 8 °C compared with the control temperature (Pastore et al., 2017). In
365
addition, low temperatures can induce an increase in the synthesis of flavonols in Arabidopsis, and
366
conversely, flavonols act as antioxidants to defend plants and avoid damage from chilling stress
367
(Petridis et al., 2016). Among these constituents, quercetin was considered the most effective
368
cryoprotectant for plant leaves (Orthen and Popp, 2000). As reported by Solfanelli et al. (2006),
369
the increased soluble sugar content under low-temperature conditions acted as a signaling agent
370
for stimulating flavonoid biosynthesis. At the end of our experiment, we found that high
371
temperature (35/30 °C) decreased the contents of flavonoid components (quercetin, kaempferol,
372
and isorhamnetin). The lower flavonol contents under the high-temperature treatment were
373
probably due to enzymatic or chemical degradation and not simply the inhibition of secondary
374
metabolism (Mori et al., 2007). A high-temperature inhibitory effect on flavonols was also
375
observed by Spayd et al. (2002). High temperatures also repress anthocyanin accumulation in
376
various plants. For instance, high temperature reduced the total anthocyanin content in the 22
377
‘Cabernet Sauvignon’ grape (Mori et al., 2005). Similar to the flavonols, the anthocyanin
378
concentration of Dodonaea ‘Dana’ fruit growing in the lower temperature regime was four times
379
higher than that in fruit growing at an elevated temperature (Nissim-Levi et al., 2013). Taken
380
together, the results of our study suggested that the inhibition of flavonoid accumulation in ginkgo
381
leaves under high-temperature conditions is caused by the combined effects of various pathways.
382 383 384
5 Conclusions Different temperatures affected the enzyme activity, flavonoid accumulation, and growth
385
regulator metabolism. Compared with ginkgo grown at 25/20 °C and 35/30 °C, treatment under
386
15/10 °C condition significantly promoted total flavonoids and the accumulation of their
387
constituents. The results of the present study showed that IAA and GAs have negative effects on
388
flavonoid accumulation, while ABA had the opposite effect on flavonoid accumulation under the
389
35/30 °C condition. Positive correlations were observed between soluble sugars, IAA, and GAs,
390
which may directly promote the growth of plants. The high flavonoid concentration was not
391
always parallel to high PAL activity, which indicated that flavonoid biosynthesis is a complex
392
pathway that has many regulators and is not limited to only one or a few factors. Generally, high
393
temperature decreased flavonoid concentrations by regulating enzyme activity and growth
394
regulator metabolism in ginkgo. Therefore, the relatively lower temperature (15/10 °C) is more
395
favorable for flavonoid accumulation and will provide a theoretical basis for further study and
396
practical applications. 23
397 398
Acknowledgments
399
This work was supported by the Agricultural Science and Technology Independent Innovation
400
Funds of Jiangsu Province (CX(16)1005), the National Key Research and Development Program
401
of China (2017YFD0600700), the Priority Academy Program Development of Jiangsu Higher
402
Education Institution (PAPD), the Postgraduate Research & Practice Innovation Program of
403
Jiangsu Province (KYCX18_0955) and the Doctorate Fellowship Foundation of Nanjing Forestry
404
University.
405 406
Declarations of Interest: none.
407
24
408 409
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1. The content of flavonoid components increased under 15/10 °C condition. 2. PAL activity was promoted under low temperature condition. 3. Low temperature promoted soluble sugar accumulation.
4. Abscisic acid promoted flavonoid accumulation under high temperature condition.
Author Contributions: study design, Xin Zhou and Guibin Wang; samples collection and analysis, Xin Zhou and Jing Guo; statistical analyses, Jing Guo; first draft, Xin Zhou and Jing Guo; review and editing, Tongli Wang, Fuliang Cao and Guibin Wang.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: