Subscriber access provided by UNIVERSITY OF THE SUNSHINE COAST
Article
Enhanced Production of Two Bioactive Isoflavone Aglycones in Astragalus membranaceus Hairy Root Cultures by Combining Deglycosylation and Elicitation of Immobilized Edible Aspergillus niger Jiao Jiao, Qing-Yan Gai, Li-Li Niu, Xi-Qing Wang, Na Guo, Yu-Ping Zang, and Yu-Jie Fu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03148 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017
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 35
Journal of Agricultural and Food Chemistry
1 2 3
Enhanced Production of Two Bioactive Isoflavone Aglycones in Astragalus
4
membranaceus Hairy Root Cultures by Combining Deglycosylation and
5
Elicitation of Immobilized Edible Aspergillus niger
6 7 8
Jiao Jiao, †,‡ Qing-Yan Gai, †,‡ Li-Li Niu, ‡ Xi-Qing Wang, ‡ Na Guo, ‡ Yu-Ping Zang, ‡
9
and Yu-Jie Fu, *,§
10 11 12
‡
13
University, Harbin 150040, People’s Republic of China
14
§
15
Forestry University, Beijing 100083, People’s Republic of China
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry
Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing
16 17 18 19 20
*Corresponding authors: Y.-J. Fu
21
Tel./Fax: +86-451-82190535
22
E-mail:
[email protected],
[email protected].
23 24
†
These authors contributed equally to this work
25
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
26 27
ABSTRACT A co-cultivation system of Astragalus membranaceus hairy root cultures
28
(AMHRCs) and immobilized food-grade fungi was established for the enhanced
29
production of calycosin (CA) and formononetin (FO). The highest accumulations of
30
CA (730.88 ± 63.72 µg/g DW) and FO (1119.42 ± 95.85 µg/g DW) were achieved in
31
34 day-old AMHRCs co-cultured with immobilized A. niger (IAN) for 54 h, which
32
were 7.72- and 18.78-fold higher than CA and FO in non-treated control, respectively.
33
IAN deglycosylation could promote the formation of CA and FO by conversion of
34
their glycoside precursors. IAN elicitation could intensify the generation of
35
endogenous signal molecules involved in plant defense response, which contributed to
36
the significantly up-regulated expression of genes in CA and FO biosynthetic pathway.
37
Overall, the coupled culture of IAN and AMHRCs offered a promising and effective in
38
vitro approach to enhance the production of two health-promoting isoflavone
39
aglycones for possible nutraceutical and pharmaceutical uses.
40 41 42
KEY WORDS: edible fungi, elicitation, deglycosylation, hairy root cultures,
43
isoflavone aglycones, antioxidant activity
44 45 46 47 48 49 50
2
ACS Paragon Plus Environment
Page 2 of 35
Page 3 of 35
Journal of Agricultural and Food Chemistry
51
INTRODUCTION
52
Astragalus membranaceus is an economically important leguminous crop widely
53
cultivated in China. Its roots have been acknowledged as traditional folk medicines in
54
East Asian areas, and as functional foods/nutraceuticals in the United States and
55
European countries 1–3. Like most of leguminous plants, A. membranaceus contains
56
diverse isoflavones, among which calycosin (CA), formononetin (FO),
57
calycosin-7-O-β-D-glucoside (CAG) and formononetin-7-O-β-D-glucoside (FOG)
58
possess various health-enhancing benefits including antioxidant, anti-inflammatory,
59
antiviral, anti-fatigue, hematopoietic, neuroprotective and estrogenic activities 4, 5.
60
However, it is well documented that isoflavones in their aglycone forms are more
61
metabolically active in intestines resulting in better bioavailability than their
62
glycosides 6–9, which suggested that the intake of isoflavone aglycone-rich foodstuffs
63
might be more beneficial for human health. In this respect, it is not surprising that the
64
two isoflavone aglycones, CA and FO, have frequently been used as indices for the
65
quality evaluation of A. membranaceus roots 10.
66
In recent years, the production of bioactive secondary metabolites by plant
67
cell/organ cultures is an attractive alternative to the classical extraction of whole plant
68
materials, in which the quality of phytochemicals is often fluctuating due to
69
environmental, ecological and climatic variations 11, 12. Moreover, Food and
70
Agriculture Organization of the United Nations (FAO) endorses plant cell/organ
71
culture techniques as feasible tools to produce high-value natural compounds for food
72
purposes 13, 14. In this context, we have established a reliable in vitro culture platform,
73
i.e. A. membranaceus hairy root cultures (AMHRCs), that could supersede field
74
cultivated plants for the efficient production of CA and FO 15. Generally, isoflavones
75
can function as phytoalexins or phytoanticipins that are strongly inducible and
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
76
sensitive to environmental stresses 16. Thus, application of external elicitors can further
77
trigger the biosynthesis of CA and FO in AMHRCs by inducing plant defense
78
responses. In view of the bio-safety of products, it is recommendable to use elicitors of
79
biological origin for the enhancement of CA and FO accumulation in AMHRCs. Aspergillus niger and Aspergillus oryzae are widely used in the food fermentation
80 81
industry for the production of wine, soy sauce, vinegar, soybean paste, etc., and they
82
are approved as GRAS (Generally Recognized as Safe) fungi by the United States
83
Food and Drug Administration 17, 18. The both food-grade fungi have proven to be
84
promising and effective elicitors for the enhanced production of health-promoting
85
compounds (resveratrol, ginsenosides, rosmarinic acid, glycyrrhizic acid, etc.) in plant
86
seedlings or plant cell/organ cultures 19–22. However, in these reports, extracts of fungal
87
mycelia and culture filtrates are always prepared as elicitors rather than the direct
88
utilization of live fungi. Factually, A. niger and A. oryzae are capable of secreting
89
extracellular glucosidases, and this characteristic can make them act as effective
90
biocatalysts for the deglycosylation of glycosides to their aglycones 23. It is worth
91
mentioning that CA and FO can be obtained by the hydrolysis of glucosyl residues in
92
their precursors (CAG and FOG) 24. Recently, immobilization of fungal spores has
93
opened a new avenue for the bio-production of various bioactive compounds, which is
94
associated with advantages of operational stability, reusability and scale-up feasibility
95
25, 26
96
immobilized Aspergillus (elicitation and deglycosylation) for promoting CA and FO
97
production in AMHRCs.
98
. Based on the foregoing, an interesting possibility is to exploit the dual abilities of
In the present study, the coupled culture of AMHRCs and immobilized food-grade
99
Aspergillus was established in an attempt to enhance the production of CA and FO by
100
fungal elicitation and deglycosylation. Initially, the feasibility of immobilized A. niger
4
ACS Paragon Plus Environment
Page 4 of 35
Page 5 of 35
Journal of Agricultural and Food Chemistry
101
(IAN) and immobilized A. oryzae (IAO) for promoting FO and CA accumulation in
102
AMHRCs was investigated. Subsequently, IAN and IAO were compared by
103
monitoring the levels of CA and FO as well as their precursors (CAG and FOG) in
104
AMHRCs along a time course from 0 to 72 h. Moreover, the generation of endogenous
105
defense signal molecules in AMHRCs upon the selected IAN treatment was
106
investigated. Meanwhile, the expression of associated genes involved in CA and FO
107
biosynthetic pathway was determined. Additionally, antioxidant activities of enzymes
108
and extracts from AMHRCs before and after IAN treatment were also evaluated. To
109
the best of our knowledge, there is no report on the combination of elicitation and
110
deglycosylation by immobilized food-grade fungi to enhance isoflavone aglycone
111
production in plant in vitro cultures.
112
MATERIALS AND METHODS
113
Hairy Root Line and Aspergillus Strains
114
All experiments were conducted using an A. membranaceus hairy root line II
115
(AMHRL II, Figure 1A) with the high productivity of isoflavones established
116
previously by our laboratory 15. AMHRCs (Figure 1B) were initiated by culturing
117
AMHRL II under the optimal conditions as described previously 15. Two food-grade
118
Aspergillus strains (A. niger 3.3883 and A. oryzae 3.951) were purchased from the
119
Institute of Microbiology, Heilongjiang province, China. Pure plate cultures of both
120
fungi were grown on potato dextrose agar (PDA) medium, and incubated at 30 ± 1 °C
121
till sporulation (Figure 1C and Figure 1D). Fungal spores were collected and counted
122
for the following immobilization experiment.
123
Co-cultivation of AMHRCs and Immobilized Edible Aspergillus
124 125
IAN or IAO were prepared by immobilization of their spores in Ca-alginate gel (CG) beads. The detailed operation procedures were performed according to our
5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
126
previous report 25, 26. For co-cultivation, IAN or IAO beads were transferred into a
127
series of 250 mL Erlenmeyer’s flasks containing AMHRCs (34 day-old) with 100 mL
128
of fresh culture medium (Figure 1E), and these flask cultures were then incubated on
129
an orbital shaker at 120 rpm and maintained under continuous darkness. On the basis
130
of preliminary studies (data not shown), the incubation temperature (30 °C), spore
131
amount of IAN or IAO load (ca.104 spores/ flask), and initial pH value of media (7.0)
132
were determined as the appropriate co-cultivation parameters. For control, non-treated
133
AMHRCs and CG-treated AMHRCs (addition of CG beads without fungal spores)
134
underwent the same culture conditions. To evaluate the feasibility of the co-cultivation
135
system, 34 day-old AMHRCs were initially treated with IAN and IAO for 2 days
136
under the pre-determined parameters. Additionally, a 72 h time course of non-, IAN-
137
and IAO-treated AMHRCs was conducted at a series of time points (0, 6, 12, 18, 24,
138
30, 36, 42, 48, 54, 60 and 72, h). After co-cultivation (Figure 1F), the harvested hairy
139
roots were rinsed by distilled water, and divided into three parts for the respective
140
extraction of isoflavones, endogenous signal molecules and total RNA. Meanwhile,
141
media were also collected for the liquid-liquid extraction of isoflavones. Moreover, the
142
immobilized fungus beads could be simply recovered by filtration, washed with sterile
143
water, and used for the next cycle to evaluate their reusability.
144
LC-MS/MS Analysis
145
Isoflavones extraction from AMHRCs and sample preparation for LC-MS/MS
146
analysis were performed as previously described 27. The simultaneous determination of
147
four target isoflavones (CAG, FOG, CA and FO) was conducted by a LC-MS/MS
148
method with selected reaction monitoring (SRM) mode as established before 15. The
149
precursor ion–product ion combinations of m/z 445.2 → 283.0, m/z 428.8 → 266.9,
150
m/z 283.0 → 268.0 and m/z 267.0 → 252.0 were adopted for the identification and
6
ACS Paragon Plus Environment
Page 6 of 35
Page 7 of 35
Journal of Agricultural and Food Chemistry
151
quantification of CAG, FOG, CA and FO, respectively. The content of each analyte was
152
calculated by the corresponding calibration curve, and expressed as microgram per
153
gram based on the dry weight (DW) of root samples.
154
Quantification of Endogenous Signal Molecules
155
Fresh hairy root samples were homogenized thoroughly by an Ultra turrax system
156
(IKA Co., Germany). The extraction and determination of endogenous nitric oxide
157
(NO) from the resulting homogenates was conducted by an established method
158
described by Zhou et al. 28. Quantification of NO was expressed as micromole per
159
gram based on the FW of root samples. Additionally, fresh hairy root samples were
160
ground under liquid nitrogen using a mortar and pestle until fine powders were
161
obtained. The extraction and determination of endogenous salicylic acid (SA) and
162
jasmonic acid (JA) from the resulting powders was performed according to an
163
established method reported by Segarra et al. 29. Quantification of SA and JA was
164
expressed as nanogram per gram based on the fresh weight (FW) of root samples.
165
Quantitative Real-time PCR (qRT-PCR) Analysis
166
Total RNA was extracted from frozen hairy root samples using a MiniBEST Plant
167
RNA Extraction Kit (TaKaRa, Dalian, China), and RNA was reverse-transcribed to
168
cDNA using a PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China). Specific
169
primers of genes involved in CA and FO biosynthetic pathway were designed
170
according to our previous report 27. The reaction solution for qRT-PCR assay was
171
prepared with a SYBR Premix Ex Taq™ II Kit (TaKaRa, Dalian, China) following the
172
manufacturer’s guidelines. The qRT-PCR amplification procedure was performed as
173
previously described 27. 18S was used as the internal reference gene, and the relative
174
expression level of each target gene was quantified by the ∆∆CT method 30.
175
Determination of Antioxidant Activity
7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
176
Activities of two antioxidant enzymes including superoxide dismutases (SOD)
177
and catalase (CAT) in fresh hairy root samples were measured following the methods
178
described by Arbona et al. 31, and activities of SOD and CAT were expressed as units
179
per mg of protein that was detected in enzyme extracts. Additionally, non-enzymatic
180
antioxidant properties of extracts (NEAPE) from AMHRCs was determined by the
181
β-carotene/linoleic acid oxidation method reported by Simic et al. 32, and NEAPE
182
activity was calculated as the β-carotene protection ratio of the tested sample relative
183
to the control.
184
Statistical Analysis
185
All experiments were conducted in triplicate, and results were given as averages ±
186
standard deviations. All statistical analyses were carried out using the SPSS statistical
187
software 17.0 (SPSS Inc, Chicago, USA). One-way analysis of variance with Tukey’s
188
test was used to determine significant differences between multiple groups of data at P
189
values < 0.05.
190
RESULTS AND DISCUSSION
191
Feasibility of IAN/IAO Treatment for Enhancing FO and CA Yield in AMHRCs
192
In our previous report, AMHRCs initiated by culturing a high-productive A.
193
membranaceus hairy root line (AMHRL II) at day 34 exhibited the maximum
194
productivity of isoflavone derivatives (CAG, FOG, CA and FO), independent of the
195
elicitor used 15. Accordingly, 34 day-old AMHRCs was adopted as the ideal system
196
processed by immobilized Aspergillus to further promote the production of two target
197
isoflavone aglycones (CA and FO) in this work. Moreover, in most of the studies done
198
to date to boost phytochemicals production in plant cell/organ cultures treated by fungi,
199
extracts of fungal mycelia and culture filtrates have always been utilized 33. However,
200
there are very few studies available where live fungus cells are used as elicitor. Thus,
8
ACS Paragon Plus Environment
Page 8 of 35
Page 9 of 35
Journal of Agricultural and Food Chemistry
201
34 day-old AMHRCs were initially co-cultured with IAN and IAO for 2 days under
202
the pre-determined parameters to evaluate whether IAN and IAO treatment can
203
enhance CA and FO accumulation in this work.
204
As shown in Figure 2, the levels of two target isoflavone aglycones in AMHRCs
205
challenged by IAN (658.69 ± 54.51 µg/g DW of CA and 998.45 ± 86.92 µg/g DW of
206
FO) and IAO (466.77 ± 33.02 µg/g DW of CA and 613.78 ± 51.14 µg/g DW of FO)
207
were much higher as compared to those in non-treated AMHRCs (96.44 ± 1.83 µg/g
208
DW of CA and 61.29 ± 0.91 µg/g DW of FO) and CG-treated AMHRCs (95.71 ± 2.60
209
µg/g DW of CA and 62.55 ± 3.28 µg/g DW of FO). Moreover, there were no
210
significant differences in CA and FO yield between non-treated AMHRCs and CG
211
-treated AMHRCs, which clearly eliminated the interference of the immobilization
212
matrix (CG) on phytochemicals production in AMHRCs. As expected, the application
213
of IAN and IAO treatment was feasible for the augmented production of CA and FO in
214
AMHRCs, which also opened a gate to perform the following studies.
215
Time-course of Isoflavone Yield in AMHRCs Co-cultured With IAN/IAO
216
For the accurate comparison of IAN and IAO treatment on isoflavone profiles in
217
AMHRCs, yields of CA and FO as well as their glycosides (CAG and FOG) were
218
monitored along a time course from 0 to 72 h. Moreover, the dynamic study was also
219
favorable to investigate and understand the dual effects (elicitation and deglycosylation)
220
of immobilized Aspergillus in AMHRCs.
221
In comparison with non-treated control, the accumulation pattern of all analytes in
222
AMHRCs challenged by IAN and IAO was similar at 6−24 h post fungal treatment,
223
exhibiting a gradual increase tendency (Figure 3). Due to the natural pathogenicity of
224
microbes to host plants, fungi are capable of producing various pathogenesis-related
225
substances (proteins and carbohydrates as well as their derivatives) that can induce
9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
226
hypersensitive responses in plant cells, thus quickly prompting the phytoalexin
227
production during the initial stages of fungal infection 33. In this regard, the present
228
results were consistent with the aforementioned description. Interestingly, the yields of
229
two isoflavone glycosides (CAG and FOG) were observed to decrease synchronously
230
from 24 to 36 h in IAN-treated AMHRCs and from 24 to 42 h in IAO-treated
231
AMHRCs, and kept at low levels afterwards (Figure 3). Under the constant attacks of
232
potential pathogens, plants cells are able to transfer the secondary metabolites
233
excessively accumulated in cytoplasm to the extracellular region, which can diminish
234
the toxicity expected when the intracellular secondary metabolites are present at high
235
levels, and also is conducive to defense the pathogen invasion 34. It is worth
236
mentioning that A. niger and A. oryzae are capable of secreting extracellular
237
β-glucosidase that can hydrolyze glucoside moieties, whereby isoflavones glycosides
238
can be effectively conversed into their aglycones 23. As inferred, the β-glucosidase
239
secreted from IAN and IAO could exercise their deglycosylation function for the
240
hydrolysis of glucoside residues in CAG and FOG to produce CA and FO in the
241
culture media of AMHRCs, which might be the reason for the continuous decrease of
242
CAG and FOG over the periods of 24 to 42 h in this study. In contrast to isoflavones
243
glycosides, the yields of two target isoflavone aglycones (CA and FO) still maintained
244
an increasing trend from 24 to 54 h in IAN-treated AMHRCs and from 24 to 48 h in
245
IAO-treated AMHRCs, and kept high levels toward the end of the time course (Figure
246
3). Indeed, the increased yields of CA and FO could be ascribed to the deglycosylation
247
effect of IAN and IAO. However, the actual yields of CA and FO at each time point in
248
the conversion process (24 to 42 h) were much higher than their forecasted values that
249
were calculated from the deglycosylation of CAG and FOG (data not shown), which
250
suggested that the elicitation effect of IAN and IAO mainly contributed to the
10
ACS Paragon Plus Environment
Page 10 of 35
Page 11 of 35
Journal of Agricultural and Food Chemistry
251
significant enhancement in yields of CA and FO.
252
Additionally, it is clearly observed from Figure 3 that IAN was superior as against
253
IAO for promoting CA and FO production in AMHRCs during the overall time course.
254
More specifically, the highest yields of CA (730.88 ± 63.72 µg/g DW) and FO
255
(1119.42 ± 95.85 µg/g DW) in IAN-treated AMHRCs was achieved at the time point of
256
54 h, which were significantly higher than the maximum levels of CA (481.03 ± 41.22
257
µg/g DW) and FO (625.60 ± 38.77 µg/g DW) in IAO-treated cultures obtained at 48 h.
258
This can be explained by the fact that different species of microbes have significantly
259
different abilities to induce phytoalexins synthesis in plants. Moreover, the optimal
260
accumulations of CA and FO in IAN-treated AMHRCs increased 7.72- and 18.78-fold
261
as against CA (94.71 ± 3.66 µg/g DW) and FO (59.62 ± 2.64 µg/g) in non-treated
262
control (54 h), respectively. The remarkable enhancement of CA and FO could be
263
appreciated by comparing LC-MS/MS chromatograms of extracts from non- and
264
IAN-treated AMHRCs (Figure 4).
265
Signal Molecule Generation in AMHRCs in Response to IAN Treatment
266
Generally, the invasion from pathogens (fungi, bacteria, viruses, etc.) can be
267
recognized by specific receptors localized to plasma membranes of plant cells, which
268
will activate signaling cascades and prompt the release of endogenous signal molecules
269
(terming as danger-associated molecular patterns) in cytosol that ultimately trigger
270
defensive secondary metabolism and thereby enhance phytoalexin biosynthesis 35. NO,
271
SA and JA are vital endogenous signal molecules involved in plant defense regulatory
272
systems against pathogen attack 36. To verify whether the generation of signal
273
molecules would be enhanced in AMHRCs in response to IAN treatment, the contents
274
of NO, SA and JA in fresh root samples harvested at 0 h, 6 h, 12 h, 18 h, 24 h, 36 h,
275
and 42 h post-treatment were determined in this work.
11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
276
As shown in Figure 5A, NO generated immediately in IAN-treated AMHRCs,
277
reached its peak value (229.41 ± 35.66 µmol/g FW) at 6 h, and declined rapidly to the
278
control level afterwards. As exhibited in Figure 5B, the SA content in IAN-treated
279
AMHRCs was observed to be highest (481.22 ± 57.93 ng/g FW) at 12 h, and reverted
280
to the control level after 36 h. As displayed in Figure 5C, the JA generation in
281
IAN-treated AMHRCs began to increase at 18 h, achieved the maximum level (28.38 ±
282
3.04 ng/g FW) at 36 h, and decreased gradually afterwards. Obviously, these signal
283
molecules accumulated transiently after IAN elicitation, and the enhanced generation
284
of NO, SA and JA occurred sequentially.
285
In most case, NO burst is considered to be an early response of plant cells to
286
fungal pathogens, which can also mobilize the generation of other signal molecules
287
such as SA, JA, ethylene, etc. 36. Additionally, SA can accumulate rapidly at the
288
infection site following fungal invasion, due to its function as an inducer of systematic
289
acquired resistance in plant-pathogen interactions 35. Moreover, SA is reported to
290
antagonize JA biosynthesis in plant responses to pathogen/pest attacks, and SA
291
reaching a certain level often suppresses the endogenous production of JA, thus
292
leading to JA accumulation being later than SA 37. Overall, the results presented here
293
concluded that the sequentially transient accumulation of endogenous NO, SA and JA
294
constituted an important line in the defense responses of IAN treatment, which might
295
contribute to the up-regulated expression of associated genes in CA and FO
296
biosynthetic pathway.
297
Biosynthetic Gene Expression in AMHRCs Underlying IAN Treatment
298
To further investigate the molecular events following the aforementioned signal
299
transduction underlying IAN treatment, the transcriptional profiles of eight genes
300
encoding enzymes that are involved in CA and FO biosynthetic pathway (Figure 6), i.e.
12
ACS Paragon Plus Environment
Page 12 of 35
Page 13 of 35
Journal of Agricultural and Food Chemistry
301
phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate
302
coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR),
303
chalcone isomerase (CHI), isoflavone synthase (IFS) and isoflavone 3’-hydroxylase
304
(I3’H), were determined by qRT-PCR. Hairy root samples collected from IAN-treated
305
AMHRCs at different time intervals (18 h, 36 h, 54 h and 72 h) were applied for
306
qRT-PCR analysis in this work.
307
As shown in Figure 6, all investigated genes were significantly up-regulated in
308
IAN-treated AMHRCs during the period from 18 h to 54 h, which indicated that the
309
elevated production of CA and FO might be achieved through the enhanced
310
transcription of these biosynthetic genes. Interestingly, the highest expression levels of
311
upstream genes in the biosynthetic pathway, i.e. PAL, C4H, 4CL, CHS, CHR and CHI,
312
were found at the time points before 54 h that was necessary for the optimal
313
accumulation of CA and FO. This was ascribed to a typical metabolic phenomenon
314
that a time lag exists between the upstream gene expression and the downstream
315
product synthesis 38. However, the transcriptional profiles of two downstream genes
316
(IFS and I3’H) were consistent with the contents of CA and FO over the time from 18
317
h to 54 h. Particularly, the expression level of IFS gene in IAN-treated AMHRCs at 54
318
h was found to be highest among all tested genes, i.e. 32.35-fold higher relative to
319
control.
320
Factually, IFS is the critical checkpoint that can direct metabolic entry to
321
isoflavonoid biosynthesis 39. The significant induction of IFS transcription here
322
suggested that it might play a vital role in the up-regulation of CA and FO production
323
in AMHRCs following IAN treatment. This was consistent with our previous report
324
that IFS was a crucial regulatory gene controlling isoflavonoid biosynthesis in
325
AMHRCs elicited by methyl jasmonate 40. Additionally, the expression levels of all
13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
326
biosynthetic genes were observed to be considerably repressed at 72 h in comparison
327
with control, which can be explained that the prolonged exposure of plant cells/organs
328
to pathogens would lead to the excessive hypersensitive responses that were
329
characterized by the rapid metabolic damage or cell death in extreme cases 36.
330
Antioxidant Activity in AMHRCs Following IAN Treatment
331
A significant characteristic of reactions in plant cells attacked by fungal
332
pathogens is the sudden overproduction of reactive oxygen species (ROS), which can
333
be toxic to invading pathogens but also cause damages to nucleic acids, proteins and
334
lipids in host cells viz. oxidative stress 34. In this work, the hairy root tissues of
335
IAN-treated AMHRCs exhibited a dark yellow color (Figure 7A) in comparison with
336
the white root tissues observed in non-treated control cultures (Figure 7B), which
337
indicated a conclusive evidence of oxidative stress following the fungal attack. Plant
338
cells are equipped with an efficient antioxidant defense system comprised of
339
antioxidant enzymes and non-enzymatic antioxidant metabolites that can work in
340
concert to detoxify the detrimental effects of ROS mediated oxidative stress 41.
341
Accordingly, activities of two representative antioxidant enzymes (SOD and CAT) as
342
well as non-enzymatic antioxidant properties of extracts (NEAPE) from hairy root
343
samples were evaluated in this work at 0 h, 18 h, 36 h, 54 h and 72 h post-treatment to
344
clarify the antioxidant response of AMHRCs following IAN treatment.
345
As shown in Figure 7C, the SOD activity in IAN-treated AMHRCs was noticed to
346
increase strongly during the first 18 h, while it decreased rapidly afterwards. As
347
exhibited in Figure 7D, the CAT activity increased gradually in IAN-treated AMHRCs,
348
achieved its maximum value at 36 h, and decreased gradually afterwards. As displayed
349
in Figure 7E, the NEAPE level in IAN-treated AMHRCs was not significantly changed
350
during the early elicitation period, began to increase at 36 h, and entered a platform
14
ACS Paragon Plus Environment
Page 14 of 35
Page 15 of 35
Journal of Agricultural and Food Chemistry
351
phase after 54 h. Comparison of antioxidant enzyme activities showed the higher
352
activity of SOD as against CAT in IAN-treated AMHRCs during the early phase of
353
post-elicitation (0-18 h). Factually, SOD can directly dismutate O2•– into H2O2, which
354
is considered to be the first line of defense against ROS overproduction in plants
355
suffering from environmental stresses, while CAT is an indispensable enzyme
356
responsible for the subsequent dismutation of H2O2 into H2O and O2 41. Additionally,
357
the results in this work indicated an early up-regulation of SOD and CAT activity (0-36
358
h), while NEAPE level were enhanced at the end of post-elicitation (54-72 h).
359
Generally, plants utilize antioxidant enzymes as the premier contributors for ROS
360
detoxification during stressed conditions. However, the depletion of antioxidant
361
enzyme activity needs to be compensated by the action of non-enzymatic antioxidant
362
metabolites 41.
363
It is noteworthy that IAN-treated AMHRCs showed the highest NEAPE level
364
(3.31-fold higher relative to control) at 54 h post-treatment where the accumulation of
365
CA and FO was maximum. Accordingly, it was inferred that the increase in the content
366
of CA and FO might contribute to the enhancement in antioxidant activity of
367
IAN-treated samples. Factually, CA and FO as two representative isoflavone algycones
368
in A. membranaceu exhibit much better antioxidant activity than their glycosides
369
(CAG and FOG), which is ascribed to the presence of more phenolic hydroxyls that
370
can act as hydrogen/electron donors to neutralize ROS or peroxyl free radicals 42.
371
Moreover, it is reported that leguminous plants challenged by fungal pathogens can
372
drastically induce isoflavone aglycone biosynthesis, thus leading to the significant
373
increase in antioxidant activity 43. Furthermore, stress-induced ROS overproduction
374
can be counteracted by other antioxidant metabolites as diverse as ascorbic acid,
375
glutathione, praline, tocopherol, carotenoid, etc. 41. Therefore, the higher antioxidant
15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
376
property of extracts from IAN-treated AMHRCs was likely attributed to the synergistic
377
effects and redox interactions among the aforementioned antioxidants.
378
Overall, the elevation of SOD and CAT activity and NEAPE level within
379
IAN-treated AMHRCs not only suggested a positive-feedback response to fight the
380
oxidative stress, but also indicated an attempt to maintain the cellular redox status to
381
minimize the destructive consequences of oxidative stress. Also, this study
382
demonstrated the potential role of AMHRC extracts rich in CA and FO as natural
383
antioxidant additives for possible nutraceutical and pharmaceutical uses.
384
Reusability of IAN Beads in AMHRCs
385
Investigation of the reusability of immobilized microorganisms is necessary for
386
the evaluation of their practical application potential. In this work, the reusability of
387
IAN beads in AMHRCs was checked by the determination of CA and FO yield during
388
10 successive batches. After 5 cycles, 74.06% and 81.87% of the initial CA and FO
389
yield was still achieved in IAN-treated AMHRCs (Figure 8A), respectively, which
390
indicated the acceptable reusability performance of IAN beads. Comparison of the
391
photographs of IAN beads before (Figure 8B) and after 5 cycles (Figure 8C) revealed
392
that the shape of the recovered beads was nearly unchanged. However, the beads after
393
multiple uses exhibited a light yellow color, which was likely to be ascribed to the
394
inherent adsorption properties of the matrix of IAN beads, i.e. CG that might adsorb
395
the colored metabolites form AMHRCs. For further reducing costs of the overall
396
process, the improvement in the reusability of IAN beads through other immobilization
397
methods will be the next challenge.
398
In conclusion, this work provided a safe and efficient method for the high-level
399
production of two bioactive isoflavone aglycones (CA and FO) using a well-controlled
400
co-cultivation system of food-grade IAN and AMHRCs. During the co-cultivation
16
ACS Paragon Plus Environment
Page 16 of 35
Page 17 of 35
Journal of Agricultural and Food Chemistry
401
process, the enhanced yield of CA and FO might be partly due to the IAN
402
deglycosylation, but mainly owing to the IAN elicitation. In detail, IAN elicitation
403
could trigger the sequentially transient accumulation of endogenous signal molecules
404
NO, SA and JA, thus leading to the transcriptional activation of genes involved in CA
405
and FO biosynthetic pathway, which ultimately boosted the accumulation of two target
406
isoflavone aglycones in AMHRCs. Moreover, the up-regulation of SOD and CAT
407
activity followed by the enhancement of NEAPE level suggested a positive-feedback
408
response to detoxify the harmful ROS within AMHRCs challenged by IAN elicitation.
409
Furthermore, the satisfactory reusability of IAN beads indicated that the proposed
410
approach could offer an economic way for industrial applications. Thus, it is highly
411
expected to utilize this promising co-cultivation system for the scale-up production of
412
CA and FO by the aid of bioreactor technology in the future.
413 414 415 416 417 418 419 420 421 422 423 424 425
17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
426 427
ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial supports by National Key R&D
428
Program of China (2017YFD0600205), Fundamental Research Funds for the Central
429
Universities (2572017DA04), Heilongjiang Province Science Foundation for Youths
430
(QC2017012), Scientific Research Start-up Funds for Talents Introduction of Northeast
431
Forestry University (1020160010), and Double First-rate Special Funds (41112432).
432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450
18
ACS Paragon Plus Environment
Page 18 of 35
Page 19 of 35
Journal of Agricultural and Food Chemistry
451
REFERENCES
452
(1) Napolitano, A.; Akay, S.; Mari, A.; Bedir, E.; Pizza, C.; Piacente, S. An analytical
453
approach based on ESI-MS, LC-MS and PCA for the quail-quantitative analysis of
454
cycloartane derivatives in Astragalus spp. J. Pharm. Biomed. Anal. 2013, 85, 46–54.
455
(2) Zhang, L. J.; Liu, H. K.; Hsiao, P. C.; Kuo, L. M. Y.; Lee, I. J.; Wu, T. S.; Chiou,
456
W. F.; Kuo, Y. H. New isoflavonoid glycosides and related constituents from astragali
457
radix (Astragalus membranaceus) and their inhibitory activity on nitric oxide
458
production. J. Agric. Food Chem. 2011, 59, 1131–1137.
459
(3) Zheng, K. Y. Z.; Choi, R. C. Y.; Cheung, A. W. H.; Guo, A. J. Y.; Bi, C. W. C.;
460
Zhu, K. Y.; Fu, Q.; Du, Y.; Zhang, W. L.; Zhan, J. Y. X.; Duan, R.; Lau, D. T. W.;
461
Dong, T. T. X.; Tsim, K. W. K. Flavonoids from Radix Astragali induce the
462
expression of erythropoietin in cultured cells: a signaling mediated via the
463
accumulation of hypoxia-inducible factor-1α. J. Agric. Food Chem. 2011, 59,
464
1697–1704.
465
(4) Fu, J.; Wang, Z.; Huang, L.; Zheng, S.; Wang, D.; Chen, S.; Zhang, H.; Yang, S.
466
Review of the botanical characteristics, phytochemistry, and pharmacology of
467
Astragalus membranaceus (Huangqi). Phytother. Res. 2014, 28, 1275–1283.
468
(5) Li, X.; Qu, L.; Dong, Y.; Han, L.; Liu, E.; Fang, S.; Zhang Y.; Wang, T. A review
469
of recent research progress on the astragalus genus. Molecules 2014, 19, 18850–18880.
470
(6) Chen, K. I.; Lo, Y. C.; Su, N. W.; Chou, C. C.; Cheng, K. C. Enrichment of two
471
isoflavone aglycones in black soymilk by immobilized β-glucosidase on solid carriers.
472
J. Agric. Food Chem. 2012, 60, 12540–12546.
473
(7) Zheng, Y.; Hu, J.; Murphy, P. A.; Alekel, D. L.; Franke, W. D.; Hendrich, S. Rapid
474
gut transit time and slow fecal isoflavone disappearance phenotype are associated with
475
greater genistein bioavailability in women. J. Nutr. 2003, 133, 3110−3116.
19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
476
(8) Zubik, L.; Meydani, M. Bioavailability of soybean isoflavones from aglycone and
477
glucoside forms in American women. Am. J. Clin. Nutr. 2003, 77, 1459−1465.
478
(9) Kawakami, Y.; Tsurugasaki, W.; Nakamura, S.; Osada, K. Comparison of
479
regulative functions between dietary soy isoflavones aglycone and glucoside on lipid
480
metabolism in rats fed cholesterol. J. Nutr. Biochem. 2005, 16, 205−212.
481
(10) Wen, X. D.; Qi, L. W.; Li, B.; Li, P.; Yi, L.; Wang, Y. Q.; Liu, E. H.; Yang, X. L.
482
Microsomal metabolism of calycosin, formononetin and drug–drug interactions by
483
dynamic microdialysis sampling and HPLC–DAD–MS analysis. J. Pharm. Biomed.
484
Anal. 2009, 50, 100−105.
485
(11) Rimando, A. M.; Duke, S. O. Human health and transgenic crops symposium
486
introduction. J. Agric. Food Chem. 2013, 61, 11693−11694
487
(12) Murthy, H. N.; Lee, E. J.; Paek, K. Y. Production of secondary metabolites from
488
cell and organ cultures: strategies and approaches for biomass improvement and
489
metabolite accumulation. Plant Cell Tissue Organ Cult. 2014, 118, 1–16.
490
(13) Dias, M. I.; Sousa, M. J.; Alves, R. C.; Ferreira, I. C. Exploring plant tissue
491
culture to improve the production of phenolic compounds: A review. Ind. Crop. Prod.
492
2016, 82, 9–22.
493
(14) Murthy, H. N.; Georgiev, M. I.; Park, S. Y.; Dandin, V. S.; Paek, K. Y. The safety
494
assessment of food ingredients derived from plant cell, tissue and organ cultures: a
495
review. Food Chem. 2015, 176, 426–432.
496
(15) Jiao, J.; Gai, Q. Y.; Fu, Y. J.; Ma, W.; Peng, X.; Tan, S. N.; Efferth, T. Efficient
497
production of isoflavonoids by Astragalus membranaceus hairy root cultures and
498
evaluation of antioxidant activities of extracts. J. Agric. Food Chem. 2014, 62,
499
12649–12658.
500
(16) Yi, J.; Derynck, M. R.; Li, X.; Telmer, P.; Marsolais, F.; Dhaubhadel, S. A
20
ACS Paragon Plus Environment
Page 20 of 35
Page 21 of 35
Journal of Agricultural and Food Chemistry
501
single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression
502
and affects isoflavonoid biosynthesis in soybean. Plant J. 2010, 62, 1019–1034.
503
(17) Schuster, E.; Dunn-Coleman, N.; Frisvad, J. C.; van Dijck, P. W. M. On the safety
504
of Aspergillus niger–a review. Appl. Microbiol. Biotechnol. 2002, 59, 426–435
505
(18) Taylor, M.J., Richardson. T. Applications of microbial enzymes in food systems
506
and in biotechnology. Adv. Appl. Microbiol. 1979, 25, 7–35.
507
(19) Li, J.; Wang, J.; Li, J.; Liu, D.; Li, H.; Gao, W.; Li, J.; Liu, S. Aspergillus niger
508
enhance bioactive compounds biosynthesis as well as expression of functional genes in
509
adventitious roots of Glycyrrhiza uralensis Fisch. Appl. Biochem. Biotech. 2016, 178,
510
576–593.
511
(20) Li, J.; Liu, S.; Wang, J.; Li, J.; Liu, D.; Li, J.; Gao, W. Fungal elicitors enhance
512
ginsenosides biosynthesis, expression of functional genes as well as signal molecules
513
accumulation in adventitious roots of Panax ginseng CA Mey. J. Biotechnol. 2016,
514
239, 106–114.
515
(21) Kümmritz, S.; Louis, M.; Haas, C.; Oehmichen, F.; Gantz, S.; Delenk, H.;
516
Steudler, S.; Bley, T.; Steingroewer, J. Fungal elicitors combined with a sucrose feed
517
significantly enhance triterpene production of a Salvia fruticosa cell suspension. Appl.
518
Microbiol. Biot. 2016, 100, 7071–7082.
519
(22) Aisyah, S.; Gruppen, H.; Slager, M.; Helmink, B.; Vincken, J. P. Modification of
520
prenylated stilbenoids in peanut (Arachis hypogaea) seedlings by the same Fungi that
521
elicited them: the fungus strikes back. J. Agric. Food Chem. 2015, 63, 9260–9268.
522
(23) Cao, H.; Chen, X.; Jassbi, A. R.; Xiao, J. Microbial biotransformation of bioactive
523
flavonoids. Biotechnol. Adv. 2015, 33, 214–223.
524
(24) Zhao, B. S.; Fu, Y. J.; Wang, W.; Zu, Y. G.; Gu, C. B.; Luo, M.; Efferth, T.
525
Enhanced extraction of isoflavonoids from Radix Astragali by incubation pretreatment
21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
526
combined with negative pressure cavitation and its antioxidant activity. Innov. Food.
527
Sci. Emerg. 2011, 12, 577–585.
528
(25) Jin, S.; Luo, M.; Wang, W.; Zhao, C. J.; Gu, C. B.; Li, C. Y.; Zu, Y. G.; Guan, Y.
529
Biotransformation of polydatin to resveratrol in Polygonum cuspidatum roots by
530
highly immobilized edible Aspergillus niger and Yeast. Bioresource Technol. 2013,
531
136, 766–770.
532
(26) Gai, Q. Y.; Jiao, J.; Luo, M.; Wang, W.; Yao, L. P.; Fu, Y. J. Deacetylation
533
biocatalysis and elicitation by immobilized Penicillium canescens in Astragalus
534
membranaceus hairy root cultures: towards the enhanced and sustainable production of
535
astragaloside IV. Plant Biotechnol. J. 2017, 15, 297–305
536
(27) Jiao, J.; Gai, Q. Y.; Wang, W.; Luo, M.; Gu, C. B.; Fu, Y. J.; Ma, W. Ultraviolet
537
radiation-elicited enhancement of isoflavonoid accumulation, biosynthetic gene
538
expression, and antioxidant activity in Astragalus membranaceus hairy root cultures. J.
539
Agric. Food Chem. 2015, 63, 8216–8224.
540
(28) Zhou, B.; Guo, Z.; Xing, J.; Huang, B. Nitric oxide is involved in abscisic
541
acid-induced antioxidant activities in Stylosanthes guianensis. J. Expe. Bot. 2005, 56,
542
3223–3228.
543
(29) Segarra, G.; Jáuregui, O.; Casanova, E.; Trillas, I. Simultaneous quantitative
544
LC–ESI-MS/MS analyses of salicylic acid and jasmonic acid in crude extracts of
545
Cucumis sativus under biotic stress. Phytochemistry 2006, 67, 395–401.
546
(30) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using
547
real-time quantitative PCR and the 2− ∆∆CT method. Methods 2001, 25, 402–408.
548
(31) Arbona, V.; Flors, V.; Jacas, J.; García-Agustín, P.; Gómez-Cadenas, A.
549
Enzymatic and non-enzymatic antioxidant responses of Carrizo citrange, a
550
salt-sensitive citrus rootstock, to different levels of salinity. Plant Cell Physiol. 2003,
22
ACS Paragon Plus Environment
Page 22 of 35
Page 23 of 35
Journal of Agricultural and Food Chemistry
551
44, 388–394.
552
(32) Simic, S. G.; Tusevski, O.; Maury, S.; Hano, C.; Delaunay, A.; Chabbert, B.;
553
Lamblin, F.; Lainé, E.; Joseph, C.; Hagège, D. Fungal elicitor-mediated enhancement
554
in phenylpropanoid and naphtodianthrone contents of Hypericum perforatum L. cell
555
cultures. Plant Cell Tissue Organ Cult. 2015, 122, 213–226.
556
(33) Baldi, A.; Srivastava, A. K.; Bisaria, V. S. Fungal elicitors for enhanced
557
production of secondary metabolites in plant cell suspension cultures. In Symbiotic
558
Fungi; Varma, A., Kharkwal, A. C., eds.; Springer-Verlag: Berlin Heidelberg,
559
Germany, 2009, pp. 373–380.
560
(34) Pusztahelyi, T.; Holb, I. J.; Pócsi, I. Plant-fungal interactions: special secondary
561
metabolites of the biotrophic, necrotrophic, and other specific interactions. In Fungal
562
Metabolites; Mérillon, J. M., Ramawat, K. G., eds.; Springer-Verlag: Berlin
563
Heidelberg, Germany, 2017, pp. 133–190.
564
(35) Zhao, J.; Davis, L. C.; Verpoorte, R. Elicitor signal transduction leading to
565
production of plant secondary metabolites. Biotechnol. Adv. 2005, 23, 283–333.
566
(36) Abdin, M. Z.; Khan, M. A.; Ali, A.; Alam, P.; Ahmad, A.; Sarwat, M. Signal
567
transduction and regulatory networks in plant-pathogen interaction: a proteomics
568
perspective. In Stress Signaling in Plants: Genomics and Proteomics Perspective;
569
Sarwat, M., Ahmad, A., Abdin, M. Z., eds.; Springer-Verlag: Berlin Heidelberg,
570
Germany, 2013, pp. 69–90.
571
(37) Mur, L. A.; Kenton, P.; Atzorn, R.; Miersch, O.; Wasternack, C. The outcomes of
572
concentration-specific interactions between salicylate and jasmonate signaling include
573
synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol. 2006,
574
140, 249–262.
575
(38) Expósito, O.; Bonfill, M.; Onrubia, M.; Jané, A.; Moyano, E.; Cusidó, R. M.;
23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
576
Palazón, J.; Piñol, M. T. Effect of taxol feeding on taxol and related taxane production
577
in Taxus baccata suspension cultures. New Biotechnol. 2009, 25, 252–259.
578
(39) Shih, C. H.; Chen, Y.; Wang, M.; Chu, I. K.; Lo, C. Accumulation of isoflavone
579
genistin in transgenic tomato plants overexpressing a soybean isoflavone synthase
580
gene. J. Agric. Food Chem. 2008, 56, 5655–5661.
581
(40) Gai, Q. Y.; Jiao, J.; Luo, M.; Wang, W.; Gu, C. B.; Fu, Y. J.; Ma, W. Tremendous
582
enhancements of isoflavonoid biosynthesis, associated gene expression and antioxidant
583
capacity in Astragalus membranaceus hairy root cultures elicited by methyl jasmonate.
584
Process Biochem. 2016, 51, 642–649.
585
(41) Gill, S. S.; Tuteja, N. Reactive oxygen species and antioxidant machinery in
586
abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 2010, 48, 909–930.
587
(42) Yu, D.; Duan, Y.; Bao, Y.; Wei, C.; An, L. Isoflavonoids from Astragalus
588
mongholicus protect PC12 cells from toxicity induced by L-glutamate. J.
589
Ethnopharmacol. 2005, 98, 89–94.
590
(43) Ahuja, I.; Kissen, R.; Bones, A. M. Phytoalexins in defense against pathogens.
591
Trends Plant Sci. 2012, 17, 73–90.
592 593 594 595 596 597 598 599 600
24
ACS Paragon Plus Environment
Page 24 of 35
Page 25 of 35
Journal of Agricultural and Food Chemistry
601
FIGURE CAPTIONS
602 603
Figure 1. (A) Cultivation of the high-productive AMHRL II on MS solid medium; (B)
604
AMHRCs obtained by culturing AMHRL II for 34 days in MS liquid medium; (C) A.
605
niger 3.3883 colony on PDA medium; (D) A. oryzae 3.951 colony on PDA medium; (E)
606
co-cultivation of IAN beads with 34 day-old AMHRCs at the initial stage; (F)
607
co-cultivation of IAN beads with 34 day-old AMHRCs for 54 h.
608 609
Figure 2. Effects of non-, CG, IAN- and IAO-treatments on yields of CA and FO in
610
34-day-old AMHRCs (spores loaded per flask ca.104, incubation temperature 30 °C,
611
initial pH value 7.0 and time 48 h). Non, non-treated AMHRCs; CG, CG-treated
612
AMHRCs; IAN, IAN-treated AMHRCs; IAO, IAO-treated AMHRCs. Mean ± SD
613
values not sharing the same lowercase letters are significantly different (P < 0.05).
614 615
Figure 3. A 72 h time course of isoflavone profile in 34-day-old AMHRCs under non-,
616
IAN- and IAO-treatments (spores loaded per flask ca.104, incubation temperature
617
30 °C and initial pH value 7.0). Mean ± SD values not sharing the same lowercase
618
letters are significantly different (P < 0.05).
619 620
Figure 4. Representative LC–MS/MS with SRM total ion chromatograms of extracts
621
from non- and IAN-treated AMHRCs.
622 623
Figure 5. Accumulation of endogenous signal molecules including NO (A), SA (B)
624
and JA (C) in non- and IAN-treated AMHRCs at different time-points (0, 6, 12, 18, 24,
625
36 and 42, h). Mean ± SD values not sharing the same lowercase letters are
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
626
significantly different (P < 0.05).
627 628
Figure 6. Transcriptional profiles of eight enzymatic genes involved in CA and FO
629
biosynthetic pathway in IAN-treated AMHRCs at different time-points (18, 36, 54 and
630
72, h). PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL,
631
4-coumarate coenzyme A ligase; CHS, chalcone synthase; CHR, chalcone reductase;
632
CHI, chalcone isomerase; IFS, isoflavone synthase; I3’H, isoflavone 3’-hydroxylase.
633
Mean ± SD values not sharing the same lowercase letters are significantly different (P
634
< 0.05).
635 636
Figure 7. Phenotypes of AMHRCs before (A) and after (B) IAN-treatment; activities
637
of SOD (C), CAT (D) and NEAPE (E) in non- and IAN-treated AMHRCs at different
638
time-points (0, 18, 36, 54 and 72, h). Mean ± SD values not sharing the same
639
lowercase letters are significantly different (P < 0.05).
640 641
Figure 8. (A) Reusability of the recovered IAN beads during 10 successive batches;
642
photographs of IAN beads (B) before and (C) after 5 cycles. Mean ± SD values not
643
sharing the same lowercase letters are significantly different (P < 0.05).
644 645 646 647 648 649 650 651
26
ACS Paragon Plus Environment
Page 26 of 35
Page 27 of 35
Journal of Agricultural and Food Chemistry
Figure 1
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 2
28
ACS Paragon Plus Environment
Page 28 of 35
Page 29 of 35
Journal of Agricultural and Food Chemistry
Figure 3
29
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 4
30
ACS Paragon Plus Environment
Page 30 of 35
Page 31 of 35
Journal of Agricultural and Food Chemistry
Figure 5
31
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 6
32
ACS Paragon Plus Environment
Page 32 of 35
Page 33 of 35
Journal of Agricultural and Food Chemistry
Figure 7
33
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 8
34
ACS Paragon Plus Environment
Page 34 of 35
Page 35 of 35
Journal of Agricultural and Food Chemistry
Graphic for table of contents
35
ACS Paragon Plus Environment