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Functional Structure/Activity Relationships
Effects of different oligochitosans on isoflavone metabolites, antioxidant activity and isoflavone biosynthetic genes in soybean (Glycine max) seeds during germination Yijia Jia, YanLi Ma, Ping Zou, Gui-Guang Cheng, Jiexin Zhou, and Shengbao Cai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07300 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019
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Journal of Agricultural and Food Chemistry
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Effects of different oligochitosans on isoflavone metabolites, antioxidant activity
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and isoflavone biosynthetic genes in soybean (Glycine max) seeds during
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germination
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Yijia Jiaa, Yanli Mab, Ping Zouc, Guiguang Chenga, Jiexin Zhoua, Shengbao Caia*
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aYunnan
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Kunming, Yunnan Province, People’s Republic of China, 650500;
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b
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Hebei Province, People’s Republic of China, 071001;
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c Marine
Institute of Food Safety, Kunming University of Science and Technology,
College of Food Science and Technology, Hebei Agricultural University, Baoding,
Agriculture Research Center, Tobacco Research Institute of Chinese
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Academy of Agricultural Sciences, Qingdao, Shandong Province, People’s Republic
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of China, 266101
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* Corresponding author and proofs
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Shengbao Cai
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E-mail address:
[email protected]/
[email protected] 1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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Abstract
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Five oligochitosans with increasing degrees of polymerization (DPs), i.e., from
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chitotriose to chitoheptaose, were examined to clarify the structure–bioactivity
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relationship between the DPs of oligochitosans and their effects on the isoflavone
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metabolites, total phenolic and flavonoid contents (TPC and TFC, respectively), and
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antioxidant activity of soybean (Glycine max) seeds during germination.
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Oligochitosans of different DPs exhibited varying influences on the TPC, TFC, and
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antioxidant activities of soybean seeds. Chitohexaose exerted a strong effect and
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significantly increased the aforementioned parameters in soybean seeds 72 h after
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germination. Genistin, malonylgenistin, and genistein were the main isoflavones
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found, and the genistin and genistein contents were significantly enhanced by 67.32%
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and 131.38%, respectively, after chitohexaose treatment. Several critical genes
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involved in the isoflavone biosynthesis (i.e., PAL, CHS, CHI, IFS) of soybeans treated
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with and without chitohexaose were analyzed, and results suggested that chitohexaose
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application could dramatically stimulate the transcription of these genes.
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Keywords: Degree of polymerization, gene expression, germination, isoflavones,
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oligochitosan, soybean seeds
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INTRODUCTION In the natural environment, plants are constantly exposed to biological and
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non-biological factors, such as diseases, insects, ultraviolet light, saline-alkaline soil,
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and low temperature, during their growth. Plants also produce hydrogen peroxide
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during photosynthesis, which can generate hydroxyl radicals in the presence of
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transition metals, leading to oxidative stress and cell damage.1 To cope with these
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factors and ensure their survival and growth, plants have developed several defense
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mechanisms. 2 Among these innate defense measures, synthesis of secondary
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metabolites, such as alkaloids, terpenes, and phenolics, is considered an important
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means for plants to adapt to the environment.3 Plants can be protected from natural
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hazards by synthesizing secondary metabolites. Flavonoids belong to a large family of
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plant phenolics that are widely accumulated in plants as secondary metabolites. The
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flavonoid family encompasses thousands of compounds and can be classified into
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several groups, such as isoflavones, flavones, flavonols, and flavanols.4, 5 These
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secondary metabolites not only play extremely important roles in the plant host to
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prevent chemical and physical damage but also confer health benefits to its
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consumers.6 Therefore, many studies to increase plant flavonoid contents have been
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conducted. Research has found that a number of abiotic and biotic elicitors could
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effectively increase the flavonoid content of some crops.7-9
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Oligochitosan, a degradation product of chitosan, is mainly derived from the
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wastes of shellfish and crustaceans; it is composed of homo- or heterooligomers of
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D-glucosamine and N-acetyl-D-glucosamine.10 Oligochitosan has much better water 3 ACS Paragon Plus Environment
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solubility than chitosan and, thus, is, more suitable and convenient than the latter for
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practical applications. 11, 12 Extensive research to investigate the bioactivities of
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oligochitosan has been performed, and findings indicate that oligochitosans possess
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abundant functional properties, such as antitumor, antioxidant, and antimicrobial
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activities.13-16 These bioactivities allow the material to be applied to many fields, such
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as agriculture, biomedicine, and food.17 In agriculture, oligochitosans effectively elicit
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plant innate immunity, promote plant growth, and improve plant tolerance to adverse
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condition stress. Previous studies reported that oligochitosans could increase the
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photosynthesis of Dendrobium orchids by increasing their chlorophyll content and
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improve the chilling and salt stress tolerance of wheat seedlings.12, 18, 19 Zou et al.12
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and Zhang et al.19 revealed that the tolerance of wheat seedlings to chilling or salt
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stress closely depended on the degree of polymerization (DP) of the oligochitosan
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applied and that oligochitosans of DP = 6 or 7 showed better bioactivity than those of
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other DPs. Oligochitosans, when used as an elicitor, have been proven to promote the
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accumulation of secondary metabolites, such as stilbenes and isoflavones, in
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plants.20-23 Despite their helpful results, however, most previous reports use
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oligochitosan mixtures with different DPs; thus, the ability of individual
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oligochitosans to induce accumulation of secondary metabolites, especially
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flavonoids, in plants, remains unclear. Moreover, the underlying mechanisms of
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oligochitosan in flavonoid accumulation have yet to be illuminated.
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In the present work, an experiment was designed to clarify the structure– bioactivity relationship between the DPs of oligochitosans and their effects on the 4 ACS Paragon Plus Environment
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isoflavone metabolites, total phenolic and flavonoid contents (TPC and TFC,
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respectively), and antioxidant activity of soybean (Glycine max) seeds during
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germination. And the main isoflavones induced by five fully deacetylated single
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oligochitosans, i.e., from 3 (chitotriose) to 7 (chitoheptaose), in soybean sprouts were
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further identified and quantified. Finally, a series of isoflavone biosynthetic genes
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were analyzed to delineate the underlying mechanisms of oligochitosan on isoflavone
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metabolite accumulation in soybean seeds during germination.
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MATERIALS AND METHODS
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Chemicals and reagents
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Acetonitrile, methanol, and Folin - Ciocalteu reagent were obtained from Merck
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(Darmstadt, Germany). Standard samples of naringenin-7-O-glucoside(≥98.0%),
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genistein (≥98.0%), genistin (≥98.0%), daidzein (≥98.0%), daidzin (≥98.0%), glycitin
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(≥98.0%), glycitein (≥98.0%), and (-)-epigallocatechin (≥98.0%) were purchased
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from Chengdu Must Bio-technology Co., Ltd. (Chengdu, Sichuan, China). Chitotriose
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(≥95.0%), chitotetraose (≥95.0%), chitopentaose (≥95.0%), chitohexaose (≥95.0%),
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and chitoheptaose (≥95.0%) were obtained from Qingdao BZ Oligo Bio-technology
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Co., Ltd. (Qingdao, Shandong, China). Real-time fluorescence-based qRT–PCR
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reagents were purchased from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, China).
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Soybean seeds were purchased from a local market (Kunming, Yunnan, China). All
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other chemical reagents used in this work were of analytical grade.
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Soybean seed treatments
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Soybean seeds (G. max) were surface sterilized with distilled water for 3 min at 5 ACS Paragon Plus Environment
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75 °C and then wiped with sterilized gauze.24 The seeds were divided into six groups
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and separately immersed in distilled water containing 0.01% (m/v, 0.1 mg/mL)
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oligochitosans (i.e., chitotriose, chitotetraose, chitopentaose, chitohexaose, or
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chitoheptaose; treatment groups) or distilled water (control group) for 6 h. The
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concentrations of the oligochitosans were selected according to a previous study.12
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The seeds were then transferred to a germination device for germination at 25 °C for
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0, 24, 48, 72, or 96 h in the dark. The humidity was set to 85% ± 2%. After
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germination, some of the germinated seeds in each group were immediately frozen at
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–80 °C for further qRT–PCR assay; the other germinated seeds were lyophilized
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(Alpha 1-2 LD plus, Christ, Germany) and extracted with 80% methanol for
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identification and quantification of isoflavone metabolites and evaluation of TPC,
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TFC and antioxidant activity. Briefly, dried and powdered samples (0.30 g) were
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ultrasonically extracted with 15.0 mL of 80% methanol for 1 h at 40 °C. The extracted
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slurry was centrifuged at 4000 × g, and the supernatant was filtered by a syringe filter
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(0.2 μm) for further analysis.
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Determination of TPC
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The TPC of each group of soybean seeds (soybean sprouts) with and without
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oligochitosan treatment was measured by a previously described method.25 TPC was
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expressed as milligrams of gallic acid equivalents per 100 g of dry weight (DW).
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Determination of TFC
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The TFC of each group of soybean seeds (soybean sprouts) with and without
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oligochitosan treatment was measured as described earlier.26 TFC was expressed as 6 ACS Paragon Plus Environment
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milligrams of rutin equivalents per 100 g of DW.
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Evaluation of DPPH radical scavenging capacity
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The DPPH radical scavenging capacity of each sample was determined based on a
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previously reported method,26 and calculated using the following formula: DPPH
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scavenging capacity (%) = [(Acontrol – Asample)/Acontrol] × 100. All tests were conducted
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thrice.
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Evaluation of ABTS radical scavenging capacity
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To determine the ABTS radical scavenging capacity of each sample, a previously
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reported method was applied.26ABTS radical scavenging capacity was calculated
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using the following formula: ABTS scavenging capacity (%) = [(Acontrol –
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Asample)/Acontrol] × 100. All tests were conducted thrice.
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Identification and quantification of flavonoid metabolites by UHPLC–ESI–
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MS/MS
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The flavonoid metabolites of soybean seeds with and without chitohexaose
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treatment were identified and quantified at 72 h of germination by a Thermo Fisher
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Ultimate 3000 UHPLC System with a Q-Exactive Orbitrap mass spectrometer
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(Thermo Fisher Scientific, Bremen, Germany). An Agilent ZORBAX SB-C18 column
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(2.1 mm × 100 mm, 1.7 μm, USA) was used to separate the flavonoid metabolites in
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the filtrate of each sample at 30 °C. The gradients of the mobile phases, including
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water acidified with 0.5% (v/v) formic acid (phase A) and acetonitrile (phase B), were
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as follows: 0–1 min, 5% B; 1–5 min, 5%–15% B; 5–10 min, 15%–25% B; 10–15 min,
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25%–45% B; 15–16 min, 45%–5% B; 16–20 min, 5% B. The injection volume of the 7 ACS Paragon Plus Environment
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sample was 3.0 μL, and the flow rate was 0.2 mL/min. The mass spectrometer was
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operated in negative mode (3.3 kV). Other related parameters were fixed according to
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an earlier report,27,28 and the appropriate standards were used to identify and quantify
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flavonoid metabolites determined under the same conditions.
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Analysis of isoflavone biosynthetic gene expression by qRT–PCR
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Total RNA was extracted from soybean seeds with and without chitohexaose
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treatment at 72 h of germination using an RNAprep Pure Plant kit (TianGen)
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according to the manufacturer’s instructions. The cDNA was obtained from 600 ng of
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the total RNA using a Fast King RT kit (TianGen). Gene primers were designed by
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Sangon Biotech (Shanghai, China) based on a previous work7 and are summarized in
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Table S1. qRT–PCR was performed using the Talent qPCR PreMix SYBR Green kit
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(Tiangen) with an Applied Biosystems StepOnePlus™ Real-Time PCR system
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according to a method reported earlier with minor modifications.7 In the present work,
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the reactions were performed over 40 cycles of 95 °C/5 s and extension of 60 °C/15 s.
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Data were analyzed using ABI StepOnePlus™ software version 2.3. The transcript
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level of each gene was normalized against the soybean β-TUB gene, which was used
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as an internal control.
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Statistical analysis
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Each experiment was repeated thrice, and data are presented as mean ± standard
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deviation (SD). All data were analyzed by one-way ANOVA using Origin 8.5
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software (OriginLab, Northampton, MA, USA), and Tukey's test was used to
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determine significant differences (p < 0.05). 8 ACS Paragon Plus Environment
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RESULTS AND DISCUSSION
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TPC and TFC of soybean seeds during germination
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In the present study, the TPC and TFC of soybean seeds during germination
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increased significantly after oligochitosan treatment. The five oligochitosans (i.e.,
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chitotriose, chitotetraose, chitopentaose, chitohexaose, and chitoheptaose) showed
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different effects on the TPC and TFC of soybean seeds 96h after germination (Tables
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1 and 2). As shown in Table 1, in the control group, the TPC of soybean seeds did not
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significantly change over the first 2 d of germination (p > 0.05) but increased by
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8.05% and 10.10% at 72 and 96 h of germination (p < 0.05), respectively, when
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compared with that of soybean seeds at 0 h of germination. Compared with the
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control group, all five single oligochitosans did not dramatically increase the TPC of
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soybeans at 0 and 24 h of germination (p > 0.05). However, application of chitotriose,
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chitotetraose, chitopentaose, chitohexaose, and chitoheptaose significantly increased
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the TPC of soybeans by 7.09%, 11.96%, 7.51%, 11.34%, and 7.22% , respectively, at
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48 h of germination (p < 0.05).
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The TPCs in soybean seeds peaked at72 h of germination in all five oligochitosan
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treatment groups and then decreased at 96 h of germination. Some previous studies on
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the effects of elicitors on the TPC of plants have reported a similar phenomenon.21, 24
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Xu et al.,21 for example, reported that the TPCs of Vitis vinifera cell cultures treated
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with oligochitosan or sodium alginate peaked at 36 h and then decreased with further
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increases in treatment time. Liu et al.24 also found that mung bean sprouts treated with
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different concentrations of ethephon exhibited peak phenolic contents at 48 h, after 9 ACS Paragon Plus Environment
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which the TPCs of all sprouts dramatically decreased. In the present work, no
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statistically significant difference was observed in the TPCs of soybeans between the
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control, chitotriose, chitotetraose, chitopentaose, and chitoheptaose groups at 96 h of
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germination (p > 0.05), although the TPC of the chitohexaose treatment group
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remained significantly higher than those of the other groups (p < 0.05). In general, the
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TPC results indicate that, among the tested oligochitosans, chitohexaose exerts the
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strongest effects on the synthesis of phenolic compounds in soybean seeds during
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germination.
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The TFCs of soybean seeds treated by oligochitosans with different DPs during
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germination are summarized in Table 2. In the control group, TFCs significantly
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increased by 21.12%, 31.82%, and 43.68% at 48, 72, and 96 h of germination (p
3 was essential to improve
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the growth and photosynthesis of wheat seedlings and that chitoheptaose exhibited the
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strongest activity among oligochitosans of other DPs (DP: 2–8). Zou et al.12 found
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that both chitohexaose and chitoheptaose could markedly alleviate chilling stress in
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wheat seedlings. A previous study found that chitooctaose promoted the growth of
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wheat seedlings suffering from salt stress best when compared with oligochitosans of
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other DPs.19 The TPC and TFC results of the present work clearly indicate that DP
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plays a critical role in promoting the synthesis of phenolic compounds, especially that
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of flavonoids, in soybean seeds during germination and that chitohexaose possesses
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the strongest activity among the oligochitosans studied. According to the results of
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earlier studies and the current data, the optimal DP of oligochitosan varies among
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plants and their applications and could range from 5 to 8. Oligochitosans must first be 11 ACS Paragon Plus Environment
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recognized and then bound by their receptors on cell membranes to exert their
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bioactivity and trigger various defense responses in plants. However, the structures of
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receptors on cell membranes may vary among plants and functions, resulting in
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differences in the optimal DP. A previous study reported that the rice receptor
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chitin-elicitor binding protein preferably bound long-chain chitin oligosaccharides,
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such as heptamers or octamers, and then formed a unique sandwich-type dimer to
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activate defense signaling.33
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DPPH and ABTS radical scavenging activity of soybean seeds during
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germination
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Anti-oxidation is an important physiological function for biological organisms to
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cope with environmental stress. In the present study, the antioxidant activities of
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soybean seeds treated with oligochitosans of different DPs during germination were
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evaluated by the DPPH and ABTS radical scavenging methods; the results are
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summarized in Tables 3 and 4. A concentration of 20.0 mg of dry soybean seeds/mL
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was used in the DPPH radical scavenging test. In the control group, DPPH radical
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scavenging activity did not change significantly during germination (p > 0.05).
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Compared with the control, the five oligochitosans tested displayed different effects
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on the DPPH radical scavenging activity of soybean seeds during germination.
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Chitotriose and chitotetraose significantly increased the DPPH radical scavenging
249
activity of soybean seeds at 72 and 24 h of germination, respectively (p < 0.05). By
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contrast, chitopentaose did not significantly affect the DPPH radical scavenging
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activity of soybean seeds during the entire process of germination when compared 12 ACS Paragon Plus Environment
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with the control (p > 0.05). Among the five oligochitosans applied, chitohexaose and
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chitoheptaose showed the strongest effects on enhancing the DPPH radical
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scavenging activity of soybean seeds at 72 and 48 h of germination (p < 0.05),
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respectively. In particular, chitohexaose treatment increased the DPPH radical
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scavenging activity of the soybean seeds by approximately 20.72% at 72 h of
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germination.
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In the ABTS radical scavenging test, a concentration of 10.0 mg of dry soybean
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seeds/mL was used. In the control group, ABTS radical scavenging activity did not
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change significantly during germination (p > 0.05), except at 24 h of germination,
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during which ABTS radical scavenging activity unexpectedly significantly decreased
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(p < 0.05). In contrast to the results of the DPPH radical scavenging test, nearly all
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five oligochitosans significantly enhanced the ABTS radical scavenging activity of
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soybean seeds over 4 d of germination when compared with that of the control group
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(p < 0.05). Among the five oligochitosans tested, chitohexaose and chitoheptaose
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showed the strongest effects on increasing the ABTS radical scavenging activity of
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soybeans at 72 h of germination (p < 0.05); no significant difference between these
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two oligochitosans (p > 0.05) was noted. Some discrepancies in the effects of
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oligochitosans of different DPs on DPPH and ABTS radical scavenging activities
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during soybean seed germination may be attributed to the complicated compositions
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of antioxidant compounds in soybean seeds and/or differences in the evaluation
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methods. In general, regardless of the radical scavenging activity assessed, soybean
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seeds treated with chitohexaose showed the highest antioxidant activity after 72 h of 13 ACS Paragon Plus Environment
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germination (p < 0.05). According to the results in Tables1–4, good correlations between TPC and
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DPPH radical scavenging activity, as well as between TPC and ABTS radical
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scavenging activity, were observed (r = 0.383, p < 0.05; r = 0.572, p < 0.01,
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respectively). Moreover, Pearson’s correlation analyses between TFC and DPPH
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radical scavenging activity and between TFC and ABTS radical scavenging activity
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revealed that the TFCs of the samples were closely related to their antioxidant activity
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(r = 0.478, p < 0.01; r = 0.604, p < 0.01, respectively). All Pearson’s correlation
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analysis results indicated that phenolic compounds, especially flavonoids, may
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contribute significantly to the antioxidant activity of soybeans during germination,
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consistent with the findings of many previous studies reporting that phenolic
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compounds are the major antioxidants of the corresponding plant materials.34, 35
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Identification and quantification of flavonoid metabolites
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Since soybean seeds treated with chitohexaose showed the highest TPC and TFC
288
and the strongest antioxidant activity at 72 h of germination, the phenolic
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composition, especially that of flavonoids, of this sample and the corresponding
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control sample were comparatively investigated by UHPLC–ESI–HRMS/MS in
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negative mode. The related ion current chromatograms are illustrated in Figure 1, and
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Table 5 summarizes the mass data, including compound names, molecular formulas,
293
retention times (Rt), [M-H]- m/z, MS/MS ion fragments, and errors (ppm). As shown
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in Fig. 1 and Table 5, a total of 12 phenolic compounds, all of which were flavonoids,
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were tentatively or positively identified based on the mass data of available authentic 14 ACS Paragon Plus Environment
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standards or previous reports;36, 37, 38 among the flavonoids found, 10 were
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isoflavones. The ion current chromatograms of the control and chitohexaose-treated
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groups were very similar at 72 h of germination, although the intensity of some peaks
299
differed (Fig. 1). This result suggests that chitohexaose treatment does not change the
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phenolic composition of soybean seeds but affects their contents. Compounds 8, 9,
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and 12 showed high peak areas in the chromatograms of the control and
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chitohexaose-treated groups, thereby indicating that these phenolic compounds may
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be the main compounds in the two samples. Compound 8 ([M-H]- m/z = 431.0973)
304
was positively identified as genistin by an authentic standard; this compound
305
produced a characteristic ion fragment at m/z = 268.0372 due to the loss of glucose
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moiety (Fig. 2). Compound 9 was tentatively characterized as malonylgenistin
307
([M-H]- m/z = 517.0980); the characteristic ion fragment (m/z = 269.0450) of this
308
compound was produced by the loss of a malonyl–glucose moiety (Fig. 2).
309
Compound 12 ([M-H]- m/z = 269.0451) was positively characterized as genistein;
310
here, cleavage of ring C formed two characteristic ion fragments (m/z = 107.0123 and
311
m/z = 133.0281), as shown in Fig.2
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The quantitative results of the 12 flavonoid metabolites in the control and
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chitohexaose-treated groups are presented in Table 5. In the present work, eight of the
314
identified flavonoids (compounds 1, 2, 3, 4, 7, 8, 11, and 12) were quantified by their
315
corresponding commercial standards. Compounds 5 and 6, which were identified as
316
malonyldaidzin and acetyldaidzin, respectively, were quantified by daidzin, and
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compounds 9 and 10, which were identified as malonylgenistin and acetylgenistin, 15 ACS Paragon Plus Environment
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respectively, were quantified by genistin. As shown in Table 5, genistin,
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malonylgenistin, and genistein were the predominant flavonoids detected in both the
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control and chitohexaose-treated groups. In the control group, genistin,
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malonylgenistin and genistein respectively accounted for about 21.67%, 34.41%, and
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10.26% of the total contents of the 12 identified flavonoids; by comparison, the ratios
323
of these three flavonoids in the chitohexaose-treated group were approximately
324
26.55%, 23.20%, and 17.38%, respectively. After chitohexaose treatment, the
325
contents of nearly all flavonoids in the soybeans increased significantly, except that of
326
malonylgenistin (Table 5). The contents of genistin and genistein in the
327
chitohexaose-treated group increased by about 67.32% from 149.74 μg/g to 250.54
328
μg/g and by about 131.38% from 70.90 μg/g to 164.05 μg/g, respectively. By contrast,
329
the content of malonylgenistin decreased by about 7.92% from 237.76 μg/g to 218.94
330
μg/g. In general, the total contents of the 12 flavonoids in soybean seeds significantly
331
increased by about 36.57% at 72 h of germination after treatment with chitohexaose.
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Isoflavones, as a subgroup of plant flavonoids, are primarily synthesized in
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leguminous plants, especially in soybean seeds, and have structures similar to that of
334
17-β-estradiol. Isoflavones can bind to estrogen receptors to activate the estrogen
335
response, which is believed to exert health benefits when isoflavone-containing
336
products are consumed as a dietary supplement.7 Previous studies report that dietary
337
consumption of soy isoflavones exerts clear positive effects on the risk factors of
338
diseases associated with estrogen levels, such as hormone-dependent cancer and
339
osteoporosis.39 The main dietary isoflavones are glycitein, daidzein, genistein, and 16 ACS Paragon Plus Environment
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their glycosides, all of which were detected in the soybean seeds with and without
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chitohexaose treatment in the present work (Table 5). Yuk et al.7 reported that
342
ethylene could significantly induce the accumulation of daidzin, genistin,
343
malonyldaidzin, and malonylgenistin in soybean leaves; these isoflavones were
344
dramatically upgraded by chitohexaose treatment in soybean seeds in the present
345
work. Compared with the study of Yuk et al.,7 the oligochitosans used in the present
346
work as elicitors of isoflavone accumulation may be more suitable than ethylene to
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produce functional foods or agricultural products because they exert relatively fewer
348
side effects and, thus, could be considered safer. However, oligochitosan mixtures
349
rich in chitohexaose instead of pure chitohexaose should be used in practical
350
applications to address cost issues.
351
Expression of isoflavone biosynthetic genes
352
Oligochitosans are widely distributed in plant pathogen and often considered as a
353
signal of pathogen invasion in plants. The immune system of plant is activated when
354
the oligochitosan receptors in their cell membrane surface of plant was bound with
355
oligochitosans. Then, innate defense measures are taken. Synthesis of secondary
356
metabolites, such as phenolics and isoflavone metabolites, is considered an important
357
defense mechanism for plants to adapt to the environment (Fig. 3a). In the present
358
work, oligochitosans of different DPs, especially chitohexaose, could serve as
359
elicitors to improve the contents of flavonoid metabolites, which chiefly consist of
360
isoflavones, in soybean seeds. Isoflavones, such as genistein and daidzein, and their
361
glycosides are synthesized in a specific branch of the phenylpropanoid pathway (Fig. 17 ACS Paragon Plus Environment
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362
3b). Several key genes, including phenylalanine ammonia-lyase (PAL), isoflavone
363
synthase (IFS), chalcone synthase (CHS), and chalcone isomerase (CHI) are known to
364
be involved in the phenylpropanoid and isoflavone pathways during isoflavone
365
biosynthesis.40, 41 Thus, to illustrate the underlying mechanism of isoflavone
366
accumulation during chitohexaose application, the expressions of several critical
367
isoflavone biosynthetic genes in soybean seeds treated with and without chitohexaose
368
were analyzed by qRT–PCR. Results showed that all critical genes, including PAL,
369
CHS7, CHI1, CHI2, IFS1, and IFS2, were significantly up-regulated in soybean seeds
370
treated by chitohexaose by approximately 1.25-fold (CHS7) to 4-fold (IFS1) when
371
compared with those of the control (p < 0.05) (Fig. 4). Such findings indicate that
372
chitohexaose may induce the transcriptional expression of critical genes involved in
373
isoflavone biosynthesis, resulting in increases in the isoflavone content of soybean
374
seeds at 72 h of germination. The enzyme PAL catalyzes the first step of the
375
phenylpropanoid pathway, which transforms L-phenylalanine to produce cinnamate,
376
which, in turn, is used as a precursor for various secondary metabolites, such as
377
tannins, lignans, flavones, and isoflavones.40 The genes CHS6 and CHS7 catalyze
378
proteins with a particularly vital role in flavonoid and isoflavone biosynthesis. Several
379
reports have found a positive correlation between the expression of CHS genes and
380
the genistein and total isoflavone contents.42 The enzyme CHI converts naringenin
381
and isoliquiritigenin chalcones to their corresponding flavanones.43 As shown in Fig.
382
4, the expressions of CHI1 and CHI2 genes exhibited similarly sensitive responses to
383
chitohexaose treatment; only a slight difference in expression was observed between 18 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
384
this work and a previous study that reported that the CHI1 gene is more responsive to
385
ethephon treatment than the CHI2 gene.7 This discrepancy may be due to the different
386
elicitors and plant parts used in the previous and current studies. In the soybean
387
genome, IFS presents as two species,44 namely, IFS1 and IFS2, which synthesize the
388
corresponding enzymes belonging to cytochrome P450 monooxygenase and could
389
transform naringenin and liquiritigenin into genistein and daidzein, respectively. After
390
treatment with chitohexaose, the expressions of the IFS1 and IFS2 genes showed
391
significantly up-regulated tendencies (p < 0.05). The qRT–PCR results also revealed
392
that IFS1 is more sensitive to chitohexaose than IFS2, which suggests that IFS1
393
enzyme may play a key role in isoflavone biosynthesis in response to chitohexaose
394
signals; this finding differs from the results of a previous study that used ethylene as
395
an elicitor.7 The IFS gene expression results demonstrate that the phenylpropanoid
396
pathway, which produces isoflavones, is activated after chitohexaose treatment.
397
Chitohexaose treatment could improve the expressions of IFS1/2 and CHI1/2, which
398
are the main contributors to the formation of isoflavones, thereby resulting in
399
significant increments in TFC and TPC in soybean seeds. The expression of genes in
400
different isoforms showed differences in metabolite sensitivity and localization, which
401
may play differential roles in regulating isoflavone metabolism.45
402
In summary, this work demonstrated an effective and safe method of increasing
403
flavonoid contents in soybean seeds during germination. Oligochitosans of different
404
DPs exhibited different influences on the TPC, TFC, and antioxidant activities of
405
germinating soybean seeds, and chitohexaose could significantly increase the 19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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406
aforementioned parameters in soybean seeds at 72h of germination. Using UHPLC–
407
ESI–HRMS/MS, genistin, malonylgenistin, and genistein were identified as main
408
substances. The contents of genistin and genistein were significantly enhanced by
409
chitohexaose treatment. Moreover, a set of structural genes of soybean seeds treated
410
with and without chitohexaose were analyzed by qRT–PCR, and results suggested
411
that chitohexaose application could dramatically stimulate the transcription of genes
412
involved in isoflavone biosynthesis.
413
Supporting Information Available: Gene primers used for qRT-PCR in the present
414
work.
415
Funding
416
The present work was financially supported by the National Natural Science
417
Foundation of China (Grant No. 31660461) and the key lab of marine bioactive
418
substance and modern analytical technique, SOA (Grant No. MBSMAT-2016-06)
419
References
420
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Genistein production in rice seed via transformation with soybean IFS genes. 26 ACS Paragon Plus Environment
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(45)Kim, J. A.; Chung, I. M. Change in isoflavone concentration of soybean ( Glycine
562
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563
Figure captions
564
Fig.1 Negative ion current chromatograms of the control (a) and chitohexaose-treated
565
(b) soybean seeds at 72 h of germination.
566
Fig.2 MS/MS spectra and fragmentation patterns of three predominant isoflavones
567
detected in the control and chitohexaose-treated soybean seeds at 72 h of germination
568
by Q-Exactive Orbitrap Mass: genistin (a), malonylgenistin (b) and genistein (c).
569
Fig. 3 Schematic diagram of action mechanism. A diagram of a branch of
570
phenylpropanoid pathway (a). Several key genes used in present work: PAL:
571
phenylalanine ammonialyase; CHS: chalcone synthase; CHI: chalcone isomerase; and
572
IFS: isoflavone synthase; hypothetical model of the activation by oligochitosans of
573
different DPs on soybean seeds (b).
574
Fig. 4 Relative expression of isoflavonoid biosynthetic genes in control and
575
chitohexaose-treated soybean seeds at 72h of germination. (a): PAL, phenylalanine
576
ammonialyase; (b) and (c): CHS6 and CHS7, respectively (CHS, chalcone synthase);
577
(d) and (e): CHI1 and CHI2, respectively (CHI, chalcone isomerase); (f) and (g): IFS1
578
and IFS2, respectively (IFS, isoflavone synthase). *Significant difference between the
579
control and chitohexaose-treated soybean seeds (p < 0.05).
27 ACS Paragon Plus Environment
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Table 1Total phenolic contents (mg/100g of DW*) of soybean seeds (Glycine max) with different treatments during germination period 0h 24h 48h 72h 96h Control
108.06 ± 2.87aA
106.55 ± 3.63aA
109.03 ± 2.12aA
116.76 ± 3.41aB
118.97 ± 1.95aB
Chitotriose
113.15 ± 4.22aAB
109.14 ± 2.38aA
116.76 ± 3.33bB
118.28 ± 3.13aB
119.13 ± 2.67aB
Chitotetraose
111.91 ± 5.32aA
112.90 ± 2.26aA
122.07 ± 2.03cB
124.58 ± 2.16bB
119.05 ± 3.67aB
Chitopentaose
112.19 ± 1.86aA
109.79 ± 3.84aA
117.22 ± 3.87bB
120.30 ± 2.93bB
116.35 ± 4.38aB
Chitohexaose
113.34 ± 3.98aA
110.22 ± 4.11aA
121.40 ± 3.56cB
138.44 ± 1.95cC
129.19 ± 1.55bD
Chitoheptaose
112.29 ± 2.32aAB
109.37 ±2.94aA
116.90 ± 2.17bB
125.46 ± 2.32dC
119.43 ± 1.97aD
Values were expressed as the mean ± SD of three replicates; values with different lower case indicated significant differences between different samples at the same germination time(p < 0.05); values with different upper case letters indicated significant differences between the same sample at different germination time(p < 0.05);*DW: dry weight of soybean sprout.
28 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
Table 2 Total flavonoid contents (mg/100g of DW) of soybean seeds (Glycine max) with different treatments during germination period 0h 24h 48h 72h 96h Control
31.11 ± 0.89aA
31.50 ± 2.43aA
37.68 ± 2.64aB
41.01 ± 1.39aC
44.70 ± 1.54aD
Chitotriose
29.85 ± 0.79aA
36.89 ± 1.89bB
40.43 ± 3.64aC
43.44 ± 2.32aC
43.64 ± 1.94aC
Chitotetraose
29.95 ± 1.33aA
35.71 ± 0.64cB
38.83 ± 2.83aC
42.10 ± 1.34aC
41.10 ± 2.95aC
Chitopentaose
32.55 ± 0.55aA
37.43 ± 0.79cB
39.50 ± 1.14aB
47.33 ± 0.96bC
44.32 ± 1.42aD
Chitohexaose
44.71 ± 1.98bA
51.62 ± 1.63dA
53.34 ± 1.95bA
80.04 ± 2.08cB
62.29 ± 0.50bC
Chitoheptaose
36.97 ± 0.75cA
47.19 ± 1.75eB
53.86 ± 2.39bC
70.29 ± 1.77dD
57.54 ± 1.34cC
Values were expressed as the mean ± SD of three replicates; values with different lower case indicated significant differences between different samples at the same germination time(p < 0.05); values with different upper case letters indicated significant differences between the same sample at different germination time(p < 0.05); *DW: dry weight of soybean sprout.
29 ACS Paragon Plus Environment
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Table 3 DPPH radical scavenging activities of soybean seeds (Glycine max) with different treatments during germination period 0h 24h 48h 72h 96h DPPH radical scavenging activity (%) at 20.0 mg of dry soybean seeds/mL Control
58.90 ± 3.84aA
54.68 ± 4.22aA
56.60 ± 1.18aA
57.67 ± 0.93aA
55.26 ± 2.22aA
Chitotriose
60.03 ± 1.56aA
54.17 ± 2.19aB
56.48 ± 0.93aB
60.16 ± 0.87bA
55.05 ± 0.96aB
Chitotetraose
62.28 ± 3.52aA
60.32 ± 1.11bA
55.14 ± 2.29aB
56.89 ± 1.97aB
57.44 ± 3.28aB
Chitopentaose
60.25 ± 0.75aA
51.08 ± 0.88aB
54.97 ± 3.09aB
54.88 ± 2.27aB
53.79 ± 1.20aB
Chitohexaose
59.86 ± 2.12aA
59.31 ± 0.29bA
56.21 ± 2.72aA
69.62 ± 1.45cB
65.68 ± 2.01cC
Chitoheptaose
61.01 ± 2.92aA
63.67 ± 1.78cA
64.38 ± 3.26bA
61.22 ± 2.80bA
54.98 ± 1.43aB
Values were expressed as the mean ± SD of three replicates; values with different lower case indicated significant differences between different samples at the same germination time(p < 0.05); values with different upper case letters indicated significant differences between the same sample at different germination time(p < 0.05).
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Journal of Agricultural and Food Chemistry
Table 4 ABTS radical scavenging activities of soybean seeds (Glycine max) with different treatments during germination period 0h 24h 48h 72h 96h 3.17aA
ABTS radical scavenging activity (%) at 10.0 mg of dry soybean seeds/mL 62.93 ± 3.98aB 68.68 ± 1.60aA 67.26 ± 2.98aA 68.28 ± 1.92aA
Control
71.06 ±
Chitotriose
79.88 ± 1.01bA
68.93 ± 2.93bB
76.77 ± 2.45bA
70.84 ± 1.98aB
74.99 ± 2.28bA
Chitotetraose
77.80 ± 2.43bA
75.16 ± 1.78cA
77.68± 3.32bA
76.16 ± 2.48bA
69.02 ± 3.21aB
Chitopentaose
72.67 ± 1.52aA
63.67 ± 2.66aB
75.27 ± 1.54bA
79.39 ± 1.12bC
80.41 ± 2.60cD
Chitohexaose
80.39 ± 2.28bAC
76.74 ± 1.93cB
74.04 ± 2.35bB
85.71 ± 2.43cD
83.90 ± 1.17cC
Chitoheptaose
78.04 ± 1.39bA
77.95 ± 2.31cA
80.10 ± 1.06cA
83.16 ± 3.45cB
81.26 ± 2.48cA
Values were expressed as the mean ± SD of three replicates; values with different lower case indicated significant differences between different samples at the same germination time(p < 0.05); values with different upper case letters indicated significant differences between the same sample at different germination time(p < 0.05).
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Table 5 Identification and quantification of isoflavones in control (CG) and chitohexaose -treated (CHxG) groups at 72h of germination CG CHxG Rt* Error Peak No. Compounds Formula [M-H]Fragments References μg/g of D.W. (min) (ppm) 1
Naringenin-7-O-glucoside
C21H21O10
7.63
433.1138
151.0100(5), 270.0508(77)
1.955
Standard
15.56±0.89 a
25.87±1.27 b
2
(-)-epigallocatechin
C15H14O7
8.37
305.0696
165.0902(5), 305.0695(5)
9.819
Standard
0.60±0.03a
1.21±0.10b
3
Daidzin
C21H20O9
9.79
415.1027
252.0421(100), 415.1027(5)
0.798
Standard
34.20±0.68a
49.39±1.97b
4
Glycitin
C22H22O10
10.13
445.1131
283.0573(25), 445.1131(5)
0.330
Standard
0.22±0.01a
0.52±0.02b
1.272
36, 37
87.88±2.17a
105.71±3.86b
5
Malonyldaidzin#
C24H22O12
11.74
501.1034
253.0499(40), 295.0609(5), 457.1130(54), 501.1034(100)
6
Acetyldaidzin#
C23H22O10
12.01
457.1129
253.0492(80), 457.1129(5)
0.095
36, 37
0.92±0.05a
2.01±0.14b
C16H12O5
12.06
283.0608
268.0371(50)
2.332
Standard
13.58±0.84a
18.47±0.66b
7
Glycitein
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8
Genistin
C21H10O10
12.16
268.0372(100), 431.0973 269.0450(63), 431.0973(60)
0.317
Standard
1.329
37, 38
149.74±4.37a
250.54±7.67b
Malonylgenistin#
C24H22O13
13.75
268.0372(98), 269.0450(100), 517.0980 473.1082(8), 517.0980(30)
10
Acetylgenistin#
C23H22O11
13.91
473.1080
269.0451(100), 473.1080(10)
0.228
37, 38
1.07±0.07a
4.70±0.10b
11
Daidzein
C15H10O4
14.83
253.0500
117.0331(5), 253.0500(100)
1.678
Standard
78.48±1.85a
102.20±3.19b
12
Genistein
C15H10O5
16.73
269.0451
107.0123 (20), 133.0281(55), 269.0451(100)
2.305
Standard
70.90±2.02a
164.05±5.21b
690.91
943.61
9
Total content *Rt:
237.76±11.02 a
218.94±8.11b
Retention time; # daidzin was used for the semi-quantification of both acetyldaidzin and malonyldaidzin, and genistin was used for the
semi-quantification of both acetylgenistin and malonylgenistin; Values were expressed as the mean ± SD of three replicates; values with different lower case in the same row indicated significant differences between control and chitohexaose -treated groups (p < 0.05).
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Fig. 1
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Fig. 2
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Fig. 3
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