Novel trans-Ferulic Acid Derivatives Containing a Chalcone Moiety as Potential Activator for Plant Resistance Induction

Article abstract of DOI:10.1021/acs.jafc.7b00958

A series of novel trans-ferulic acid derivatives containing a chalcone moiety were designed and synthesized to induce plant resistance. Antiviral activities of the compounds were evaluated. Bioassay results demonstrated that compounds F3, F6, F17, and F27 showed remarkable curative, protective, and inactivating activities against tobacco mosaic virus (TMV). With a 50% effective concentration (EC₅₀) value of 98.78 μg mL^-1, compound F27 exhibited the best protective activity compared with trans-ferulic acid (328.6 μg mL^-1), dufulin (385.6 μg mL^-1), and ningnanmycin (241.3 μg mL^-1). This protective ability was associated with potentiation of defense-related enzyme activity and activation of photosynthesis of tobacco at an early stage. This notion was confirmed by up-regulated expression of stress responses and photosynthesis regulating proteins. This work revealed that F27 can induce resistance and enhance plant tolerance to TMV infection. Hence, F27 can be considered as a novel activator for inducing plant resistance.

Full text of DOI:10.1021/acs.jafc.7b00958

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Article
Novel Trans-Ferulic Acid Derivatives Containing a Chalcone
Moiety as Potential Activator for Plant Resistance Induction
Xiuhai Gan, De-Yu Hu, Yanjiao Wang, Lu Yu, and Bao-An Song
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00958 • Publication Date (Web): 03 Apr 2017
Downloaded from http://pubs.acs.org on April 4, 2017
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Novel Trans-Ferulic Acid Derivatives Containing a Chalcone Moiety as
Potential Activator for Plant Resistance Induction
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Xiuhai Gan,†‡ Deyu Hu,† Yanjiao Wang,† Lu Yu,† Baoan Song* †
CORRESPONDING AUTHOR FOOT NOTE
* To whom correspondence should be addressed. Tel.: +86ꢀ851ꢀ83620521. Fax: +86ꢀ851ꢀ83622211.
Eꢀmail: songbaoan22@yahoo.com
Current address:
†State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
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Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Center for
Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, PR China
‡College of Chemistry and Life Science, Guizhou Education University, Guiyang 550018, PR China
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ABSTRACT: A series of novel transꢀferulic acid derivatives containing a chalcone
moiety were designed, and synthesized to induce plant resistance. Antiviral activities of
the compounds were evaluated. Bioassay results demonstrated that compounds F3, F6,
F17, and F27 showed remarkable curative, protective and inactivating activities against
tobacco mosaic virus (TMV). With an 50% effective concentration (EC50) value of 98.78
ꢀg mLꢀ1, compound F27 exhibited the best protective activity compared with
transꢀferulic acid (328.6 ꢀg mLꢀ1), dufulin (385.6 ꢀg mLꢀ1), and ningnanmycin (241.3 ꢀg
mLꢀ1). This protective ability was associated with potentiation of defenseꢀrelated enzyme
activity and activation of photosynthesis of tobacco at an early stage. This notion was
confirmed by upꢀregulated expression of stress responses and photosynthesis regulating
proteins. This work revealed that F27 can induce resistance and enhance plant tolerance
to TMV infection. Hence, F27 can be considered as a novel activator for inducing plant
resistance.
KEYWORDS: transꢁferulic acid, chalcone, tobacco mosaic virus, antiviral activity,
plant resistance
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INTRODUCTION
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Tobacco mosaic virus (TMV) is a wellꢀstudied plant virus worldwide and causes massive
crop loss; this virus infects more than 400 plant species of 36 families, including tobacco,
tomato, potato, pepper, and cucumber.1 Viral infection is extremely difficult to control
under field conditions. Ningnanmycin (Figure 1) is the most effective plant virus
inhibitor and is used to prevent TMV disease, however, this antiviral is not widely used in
field trial because of the agent’s photosensitivity and water stickiness.2 In fact, no
chemical treatment can thus far absolutely inhibit TMV once it has infected a plant.3 Thus,
experts face the challenge of fully protecting plants from TMV infection.
In plants, resistance induced by plant activators can provide defense against pathogens.
Such resistance manifests as various defensive responses, such as oxidative burst,
cellꢀwall reinforcement, and phytoalexin synthesis.4–6 Some studies reported about plant
activators,
including
salicylic
acid
(SA)
and
its
derivatives,7–9
benzo[d][1,2,3]ꢀthiadiazoleꢀ7ꢀcarboxylic acid (CGA210007) and its derivatives,6,10 and
jasmonic acid (JA)11 (Figure 1). These activators are metabolic products during plant
growth. Plant metabolic products are highly efficient and environmentꢀfriendly and
involve unique modes of action.12,13 As such, these metabolic products are considered
rich source of plant activators that help protect plants from TMV infection. Some
metabolic products exhibit good inhibitory activities against TMV, these metabolic
products include βꢀcarbolines,14,15 quassinoids,16 limonoids,17 phenanthroindolizidine and
its analogues.18 However, only few products were applied in field trials because these
compounds difficult to be obtained from plants and are too complex to be synthesized.19
Our previous research showed that the aminophosphonate derivative, dufulin (DFL)
(Figure 1), displays good inhibitory activity against TMV, cucumber mosaic virus, and
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other plant viruses. This derivative can activate SA signaling pathway to induce host
plants to generate antiviral responses.20 This property was widely exploited in China to
prevent the spread of plant viral diseases. We also discovered a number of
α,βꢀunsaturated carbonyl derivatives that exhibit good antiviral activities.21–24
Transꢀferulic acid is an important α,βꢀunsaturated carbonyl metabolic product of plant
(Figure 1); it is derived from cinnamic acid and can produce phytoalexins and SA through
biosynthesis for defense against pathogens.25 Some transꢀferulic acid derivatives were
reported as antiꢀTMV agents.26–28 Aside from transꢀferulic acid, chalcones also exhibit
antiꢀTMV29,30 activities. However, these compounds present low antiviral activities, and
no study can explain their mechanism of action for plant resistance induction.
To further develop highly effective, fullꢀscale antiviral agents, a series of novel
transꢀferulic acid derivatives containing a chalcone moiety (Figure 2 and Scheme 1) were
designed and synthesized to induce plant resistance in tobacco. Then, halfꢀleaf method in
vivo31 was used to evaluate inhibitory activities of these agents against TMV. The present
work investigated plant defense response mechanisms of compound F27; such
mechanisms include enzyme activities, chlorophyll content, photosynthesis, and
differentially expressed proteins (DEPs). To the best of our knowledge, this study first
demonstrated that compound F27 can enhance resistance in tobacco and can be
considered as novel activator for plant resistance induction.
MATERIALS AND METHODS
Instruments and Chemicals. Melting points were determined on an XTꢀ4 binocular
microscope melting point apparatus (Beijing Tech Instrument Co., China; uncorrected).
Nuclear magnetic resonance (NMR) spectroscopy was performed on solvents CDCl3 and
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dimethyl sulfoxideꢀd6 (DMSOꢀd6) at 500 and 125 MHz using a JEOLꢀECX 500 NMR
spectrometer (JEOL Ltd., Japan) and tetramethylsilane as internal standard.
Highꢀresolution mass spectrometer (HRMS) was conducted using a Thermo Scientific Q
Exactive (Thermo, USA). Transꢀferulic acid (99% purity; CAS: 537ꢀ98ꢀ4) and other
reagents were purchased from Aladdin Company. All solvents were of analytical reagent
grade and were dried and purified in accordance with standard procedures before use.
General Procedures for Preparing Intermediates. Transꢀferulic acid ester
intermediates 1a–1d were synthesized with transꢀferulic acid and various alcohols
(Scheme 1). Then, potassium carbonate (12.0 mmol) was added to a solution of
corresponding intermediate 1a–1d (10 mmol) in butanone (30.0 mL), and resulting
mixture stirred for 1 h at room temperature. Next, 1,2ꢀdibromoethane (15.0 mmol) was
added to the mixture, which was then warmed to 80 °C and stirred for 2–4 h. Upon
reaction completion (as indicated by thinꢀlayer chromatography (TLC)), solids were
removed by filtration, and solvent was removed under reduced pressure. Residue was
purified by silicaꢀgel column chromatography using petroleum ether/ethyl acetate (4:1,
v:v) to obtain intermediates 2a–2d. Intermediate 3 was synthesized with
4ꢀhydroxyacetophenone and various aromatic aldehydes in accordance with reported
procedures.30
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General Synthetic Procedures for Title Compounds F1–F26. Reaction mixture was
added to a solution of intermediate 3 (2.0 mmol) and potassium carbonate (2.4 mmol) in
dimethylformamide (DMF) (5.0 mL), and stirred at room temperature for 1 h. A solution
of corresponding intermediate 2 (1.9 mmol) in DMF (5.0 mL) was added to the mixture,
which was warmed to 60 °C afterward. Stirring was continued for 2 h to 4 h. Upon
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completion of reaction (indicated by TLC), cold saturated salt solution was dropwise
added. Solids were filtered and washed with cold water. Crude product was recrystallized
from CH2Cl2/CH3OH (1:1, v:v) to yield 72.2% to 93.3% of title compounds F1–F26.
General Synthetic Procedures for Title Compounds F27–F30. Reaction mixture
was added to a solution of corresponding acrylate (1.0 mmol) and sodium hydroxide (2.0
mmol) in H2O (10.0 mL) and stirred at 60 °C until completion of reaction (indicated by
TLC). Mixture was then acidified by dropwise addition of aqueous HCl, filtered, and then
washed with cold water. Crude product was recrystallized from CH3OH to obtain acrylic
acid compounds F27–F30.
General Synthetic Procedures for Title Compounds F31–F35. Reaction mixture
was added to a solution of corresponding acrylic acid (0.5 mmol) and potassium
carbonate (1.0 mmol) in CH3CN (10.0 mL) and stirred at room temperature for 0.5 h,
Corresponding substituent benzyl chloride intermediate (0.5 mmol) was added to the
mixture, which was then warmed to 80 °C and stirred until completion of reaction. Then,
resultant mixture was filtered. Solvent was removed under reduced pressure, and crude
product was recrystallized from CH2Cl2/CH3OH (1:1, v:v) to obtain the compounds
F31–F35. Section on Supporting Information list physical, NMR, and HRMS data of title
compounds.
Antiviral Biological Assay. Nicotiana. tabacum cv. K326 and Nicotiana. tabacum L.
plants were cultivated in a greenhouse. N. tabacum cv. K326 was used to determine
systemic TMV infection and N. tabacum L. was used as local lesion host when plants
grew to 5−6 leaf stage. TMV was purified by Gooding method,32 and in vivo modes of
compounds were determined through a reported technique; in vivo modes include
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curative, protective, and inactivating activities.31 Positive controls included transꢀferulic
acid, commercial antiviral agent ribavirin, DFL, and ningnanmycin. Measurements were
performed in triplicates.
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Physiological and Biochemical Analysis. Plant growth and compound treatments.
Similarly grown N. tabacum cv. K326 were selected at the seventh leaf stage, and 500
ꢀg·mLꢀ1 F27 solution was smeared on whole leaves. Solvent and DFL were used as
negative (CK) and positive controls, respectively. Plant leaves were inoculated with the
virus after 12 h and cultivated in a greenhouse. Four treatments were adopted: CK,
CK+TMV, DFL+TMV, and F27+TMV. Tissue samples were collected at 1, 3, 5, and 7
days after inoculation treatment for assays on chlorophyll content, photosynthetic
characteristics, and defensive enzyme activities assay. Measurements were performed in
triplicates.
Chlorophyll Content. Using a modified reported method,33 Chlorophyll contents of
samples were measured every two days. Test samples in triplicates of each treatment
were sliced into small uniform pieces by
a hole puncher while avoiding the midrib,
Samples weighed 50 mg and were placed in 5 mL cold solution of 1:1 mixture of 85%
acetone and 85% ethanol (v/v). Samples were homogenized, incubated for 0.5 h at 35 °C
and centrifuged for 15 min at 6500 rpm. Absorbance spectra were recorded at 663 and
645 nm for chlorophyll a (Ca) and chlorophyll b (Cb), respectively, against a solution as
reference. Ca, Cb, and total chlorophyll content (Ct) were calculated as follows:
Ca (mg Lꢀ1) = 9.784OD663 – 0.990OD645
Cb (mg Lꢀ1) = 21.426OD645 – 4.650OD663
Ct (mg Lꢀ1) = Ca + Cb = 5.134OD663 + 20.643OD645
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Quantification (mg gꢀ1 fresh weight) was performed using the following equation:
Q= CV/1000W
where C is concentration (mg Lꢀ1); V is volume of solvent (mL); W refers to sample
fresh weight (g).
Measurement of Photosynthetic Characteristics. With slight modifications of a
described method,34 photosynthetic characteristics of fully expanded leaves were
recorded using infrared gas analyzer (Li–6400, Li–COR, Lincoln, NE, USA); these
characteristics included net photosynthetic rate (Pn), stomatal conductance (Gs),
intercellular CO2 concentration (Ci), transpiration rate (Tr), and chlorophyll fluorescence
(Fv/Fm) of plants treated with F27. Recording was conducted between 9:00 and 11:00
a.m. every two days for six plants covered by each treatment. During measurements,
photosynthetic active radiation, temperature, and CO2 concentration were 300 ꢀmol mꢀ2
sꢀ1, 30 °C, and 400 ꢀmol molꢀ1, respectively. Chlorophyll fluorescence was measured
after adaptation to dark for 30 min to ensure complete relaxation of all reaction centers.
Minimum chlorophyll fluorescence (F0) was determined by a measuring beam, whereas
maximum chlorophyll fluorescence (Fm) was measured after 0.8 s of saturation with
light pulse (6000 ꢀmol mꢀ2 sꢀ1). Actinic light (300 ꢀmol mꢀ2 sꢀ1) was applied for 1 min to
drive photosynthesis. Maximum quantum yield of photosystem II (PSII) (Fv/Fm) was
calculated as (FmꢀF0)/Fm.
Determination of Defensive Enzyme Activities. Activities of superoxide dismutase
(SOD), peroxidase (POD), catalase (CAT), and phenylalanine ammonia lyase (PAL)
were measured and calculated with enzyme assay reagent kits in accordance with
manufacturer’s instructions (Suzhou Comin Bioengineering Institute, China).
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DEP Analysis. Protein Extraction. Total tobacco proteins were extracted in
accordance with reported methods with slight modifications.35,36 Leaf sample (1.0 g)
was ground to power in liquid nitrogen, homogenized, and suspended in 5 mL of
iceꢀcold extraction buffer (0.7 M sucrose, 0.5 M TrisꢀHCl, 0.1 M KCl, 50 mM ethylene
diamine tetraacetic acid, 40 mM dithiothreitol (DTT), pH 7.5) at room temperature for
10 min. An equal volume of phenol saturated with TrisꢀHCl pH 7.5 was added. Mixture
was shaken at 4 °C for 30 min. After centrifugation at 5000 rpm for 10 min, the upper
phenol layer was transferred to a new tube. Five volumes of 100 mM ammonium acetate
in methanol were added, and resulting mixture was stored at −20 °C for 12 h. Mixture
was then centrifuged at 5000 rpm and 4 °C for 20 min. Precipitate was collected and
washed thrice with iceꢀcold 80% acetone at −20 °C. Then, precipitate was airꢀdried and
solubilized in 150 ꢀL rehydration solution (8 M urea, 0.1 M Tris, 10 mM DTT, PH 8.5)
at 37 °C for 1 h. Protein concentration was determined using the Bradford method.
Afterward, solutions containing 100 ꢀg protein were collected. An equal volume of 55
mM iodoacetamide was added, and mixture was incubated for 30 min at room
temperature in the dark. Mixture was then centrifuged at 12000 rpm and 4 °C for 20 min
with 3 kDa Millipore. Protein extract was washed six times with diluent rehydration
solution and dissolved in 100 ꢀL MilliꢀQ water. Then, sample was incubated with 12.5
ꢀg trypsin (Sigma, USA) at 4 °C for 30 min and then at 37 °C for at least 12 h. Mixture
was centrifuged at 12000 rpm for 20 min at 4 °C. Peptide solution was collected,
airꢀdried and solubilized in 50 ꢀL highꢀperformance liquid chromatography grade H2O
containing 0.1% formic acid (FA) for liquid chromatography−tandem MS (LC−MS/MS)
analysis.
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LC−MS/MS Analysis. Peptides were analyzed by NanoꢀLC−MS/MS using a Triple
Timeꢀofꢀflight (TOF) 5600 mass spectrometer (Foster City, CA, USA). For each sample,
8 ꢀL peptide solution was injected using fullꢀloop injection, and was desalted on a
ChromXP Trap column (Nano LC TRAP Column, 3 ꢀm C18ꢀCL, 120 Å, 350 ꢀm×0.5
mm, Foster City, CA, USA) with equilibration of 1% acetonitrile (ACN) and 0.1% FA
in water. Next, each sample was washed for 10 min at 300 nL min−1 flow rate. Then,
each sample was eluted by reverseꢀphase column chromatography (Nano LC C18, 3 ꢀm
C18ꢀCL, 75 ꢀm×15 cm, Foster City, CA, USA) using linear gradient formed by mobile
phase A (5% ACN, 0.1% FA) and mobile phase B (95% ACN, 0.1% FA) over 120 min
at a flow rate of 300 nL minꢀ1. Eluted peptides were directed by Triple TOF 5600 MS in
dataꢀdependent mode to automatically switch between TOF–MS and product ion
acquisition using Analyst (R) Software (TF1.6). βꢀGalactosidase digest was used to
calibrate each pair of samples by 10 min of elution and 30 min of identification.
Proteomics Data Analysis. LCꢀMS/MS data were analyzed and quantified using
MaxQuant37 version 1.5.2.8 by Andromeda search engine and based on tobacco
proteome downloaded from UniProt. Results of MaxQuant analysis included an initial
search at a precursor mass tolerance of 20 ppm, which is also used for mass
recalibration.38 The search comprised variable modifications of methionine oxidation,
Nꢀterminal acetylation, and cysteine carbamidomethylation. Precursor mass and
fragment mass presented initial mass tolerance values of 6 and 20 ppm, respectively, in
the main Andromeda search. Peptide length ranged from seven amino acids to two
miscleavages, and false discovery rate was set to 0.01. Normalized protein intensity was
determined using labelꢀfree quantification with minimum of two ratio counts.38 iBAQ
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algorithm was used to rank absolute abundance of DEPs within a single sample.39
Protein tables were filtered by eliminating identifications of common contaminants and
reverse database. Unpaired tꢀtest of iBAQ data of twoꢀsamples was used to identify
differentially accumulated proteins between treatment groups and control.
Bioinformatics and annotations. Classification of DEPs was conducted with gene
ontology (GO) annotation on Kyoto Encyclopedia of Genes and Genomes (KEGG) by
Uniprot software (http://www.uniprot.org/). GO items lacking corresponding
annotations were first removed from protein list. Subsequently, ID of listed proteins
were plotted at levels of biological process, cellular component, and molecular
function.40 DEPs (expression level > 1.5 fold) were mapped to GO database
(http://www.geneontology.org/), and the number of proteins at each GO term was
computed. Results that came from labelꢀfree proteomics were used as the target list.
Background list was generated by downloading the GO database.
Statistical analysis. Each measurement was performed in triplicates and arranged
with completely randomized design. Experimental data were analyzed using SPSS
Version 16.0 (SPSS, Chicago. IL, USA). Multiple comparisons were performed among
different samples with Duncan’s multipleꢀrange tests (P < 0.05).
RESULTS AND DISCUSSION
Chemistry. Scheme 1 displays synthetic routes of transꢀferulic acid derivatives
containing a chalcone group. Transꢀferulic acid methyl, ethyl, propyl, and isopropyl
esters 1a–1d were synthesized to determine the effects of different esters on antiviral
activity. Afterward, key intermediates 2a–2d were obtained from corresponding
intermediates 1a–1d and reacted with potassium carbonate and 1,2ꢀdibromoethane in
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butanone with a yield of 52.5% to 63.5%. Acetone, acetonitrile, tetrahydrofuran, and
DMF were each used as solvents and were investigated. Low yields were acquired from
the first three solvents. By contrast, compound 2 was obtained at 50% yield in DMF, but
was difficult to purify with excess amounts of the chemical. Thus, butanone was
considered as suitable solvent, and ratio of reactant to solvent was optimized as 1:10.
Using a similar method from our previous work,41 title compounds F1–F26 were
synthesized with yield of 72.2% to 93.3%. Most plant activators are carboxylic acid and
their derivatives. Hence, carboxylic acid compounds F27–F30 were prepared by
hydrolyzing corresponding acrylates. To explore the effect of bulky groups on antiviral
activity, acrylates F31–F35 containing bulky groups were synthesized with
corresponding acrylic acid and substituent benzyl chloride intermediates. Compounds
1
structures were confirmed by H NMR, 13C NMR, and HRMS; data are reported in
Supporting Information. In the present study, we considered F1 as representative
example. Accordingly, the main characteristic of 1H NMR spectra of title compound was
presence of four lowꢀfrequency downfield doublets at δ 7.93 (d, 1H, J = 16.0 Hz), 7.68 (d,
1H, J = 15.5 Hz), 7.58 (d, 1H, J = 16.0 Hz), and 6.56 (d, 1H, J = 16.0 Hz) ppm. These
results reveal the presence of four trans =C–H protons. Two broad singlets at 4.42 and
4.35 ppm indicate that F1 contains two O–CH2– protons. Two singlets at 3.77 and 3.68
ppm signify that the structure contains two –OCH3. Typical chemical shifts near δ 187.78
13
and 167.52 ppm of C NMR spectra indicate the presence of C=O. Meanwhile, shifts
near δ 67.43 and 67.23 ppm confirm the presence of –OCH2–. Typical shifts near δ 56.12
and 51.87 ppm also verify the presence of –OCH3. Electrospray ionization–HRMS
showed [M+Na]+ mass to be 499.15277; this value is consistent with calculated value for
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C28H25O6FNa [M+Na]+ 499.15329.
Antiviral Activity against TMV in Vivo. Antiviral activities of title compounds
F1–F35 against TMV were tested and are reported in Table 1. Some title compounds
exhibited remarkable antiviral activities against TMV at 500 ꢀg mLꢀ1. F3, F6, F12, F17,
and F19 exhibited superior curative activities with values of 63.9%, 61.9%, 56.7%,
56.3%, and 57.2%, respectively, compared with those of transꢀferulic acid (41.5%), DFL
(43.8%), and ningnanmycin (54.5%). F3, F6, F12, F13, F16, F17, and F19 exhibited
significantly greater protective activities against TMV with values of 64.6%, 64.4%,
68.1%, 66.8, 69.4%, 68.0%, and 66.5%, respectively, than transꢀferulic acid (53.5%),
DFL (51.4%), and ningnanmycin (62.8%). F3, F6, F9, F13, and F17 showed superior
inactivating activities with values of 92.3%, 95.3%, 92.1%, 92.6%, and 93.6%,
respectively, compared with transꢀferulic acid (78.6%), DFL (76.5%), and ningnanmycin
(91.5%). Among these compounds, F3 (Ar = 2ꢀFꢀPh), F6 (Ar = 4ꢀFꢀPh), and F17 (Ar =
4ꢀClꢀPh) exhibited greater curative, protective and inactivating activities than
transꢀferulic acid, DFL, and ningnanmycin. These findings implied that
electronꢀwithdrawing groups of aromatic ring can enhance antiviral activity. Further
antiviral assay of carboxylic acids containing electronꢀwithdrawing groups of aromatic
rings showed that compounds F27–F30 displayed better protective activities against
TMV than ningnanmycin. Notably, F27 exhibited excellent curative, protective, and
inactivating activities with values of 55.6%, 71.2%, and 92.4%, respectively, which were
superior to ningnanmycin. However, F31–F35 demonstrated lower antiviral activities
against TMV compared with ningnanmycin. These results reveal that bulk groups are
unfavorable for antiviral activity of acrylate. EC50 values of protective activities suggest
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that compounds F3, F6, F12, F13, F16, F17, F19, and F25–F30 hold remarkable
protective activities against TMV. EC50 values ranged from 98.7 ꢀg mLꢀ1 to 224.2 ꢀg
mLꢀ1. In particular, F27 displayed the best protective activity with EC50 value of 97.8 ꢀg
mLꢀ1. This value was superior to those of transꢀferulic acid (328.6 ꢀg mLꢀ1), DFL (385.6
ꢀg mLꢀ1) and ningnanmycin (241.3 ꢀg mLꢀ1).
Results
of
preliminary
structureꢀactivity
relationships
showed
that
electronꢀwithdrawing groups of aromatic rings favor antiviral activity at the same
position. These findings were proven by the following activity order: F1 (Ar = 4ꢀFꢀPh) >
F5 (Ar = 4ꢀOCH3ꢀPh), F6 (Ar = 4ꢀFꢀPh) > F10 (4ꢀOCH3ꢀPh) and F12 (Ar = 4ꢀClꢀPh) >
F23 (Ar = 4ꢀ(CH3)3ꢀPh). Bulky group of aromatic rings does not favor antiviral activity.
This notion was supported by the activity order, F6 (Ar = 4ꢀFꢀPh) > F7 (Ar = 4ꢀClꢀPh),
F27 (Ar = 4ꢀFꢀPh) > F28 (Ar = 4ꢀClꢀPh) and F12 (Ar = 4ꢀClꢀPh) > F10 (Ar =
4ꢀOCH3ꢀPh) > F23 (Ar = 4ꢀ(CH3)3ꢀPh). Introducing an aromatic heterocycle also
disfavors for antiviral activity. This finding was confirmed by the following activity order:
F4 (Ar = benzene) > F11 (Ar = thiophen) > F15 (Ar = furan). Acrylates involving
suitable steric hindrance of their groups favor antiviral activity, as shown by the
following activity order: F16 (R= iꢀPr) > F6 (R = Et) > F1 (R = Me) > F32 (R =
CH2Ph) > F34 (R = CH2(3ꢀCH3ꢀPh)). Results also revealed that carboxylic acid
compounds hold greater protective activities against TMV than those of their
corresponding acrylates. This notion explains why most plant activators contain carboxyl
and indicates that transꢀferulic acid derivatives containing a chalcone group may serve as
potential antiꢀTMV agents.
Physiological and Biochemical Responses of Tobacco. Plants face infection by
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various aggressive pathogens over their lifetime; to protect themselves during pathogenic
infections, plants evolved unique immune systems and developed various defense
responses; such defense mechanisms include those involving chlorophyll content, defense
enzyme activity,42 and photosynthetic rate.43 That is, virusꢀinfected plants develop strong
physiological and biochemical alterations.
Effect on Chlorophyll Contents. As a special and the most basic life process of green
plants, photosynthesis can provide necessary plant growth and energy. Chlorophylls make
up core components of chloroplasts, which play a major role in photosynthesis.44 In our
study, chlorophyll content of tobacco plant (Figure 3) decreased gradually with TMV
inoculation; these chlorophylls included Ca, Cb, and Ct (Figure 3A, 3B, 3C). However, in
TMVꢀinoculated tobacco leaves treated with F27, chlorophyll content increased from day
1 to day 5 and reached the highest value at day 5. Variation in trends of F27+TMV
treatment surpassed that of DFL+TMV treatment. Chlorophyll content of F27ꢀtreated
tobacco decreased gradually. When chlorophyll content was measured, similar variation
tendencies were observed in chlorophyll a/b ratios of all treatments (Figure 3D). That is,
Ca and Cb, were altered simultaneously. Hence, F27 may increase chlorophyll content,
improve photosynthesis, and thereby enhance plant host resistance to diseases.
Effect on Photosynthesis. Photosynthesis is a highꢀrate redox metabolic process and
causes reactive oxygen species (ROS) to be released in plants.45 This process can avoid
damage to plants due to excessive oxygen. In this study, we found that TMV and
compound F27 affected photosynthetic functions at various levels. Pn and Tr significantly
reduced in TMVꢀinoculated tobacco leaves (Figure 4 A, 4C). However, responses of Pn
and Tr increased in F27+TMV treatment group and reached their highest values on days 5
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and 3, respectively. Afterward, Pn and Tr significantly declined. Pn and Tr in F27+TMV
treatment group increased by 33.4% and 35.1%, respectively, relative to those of CK. Gs
increased gradually and then decreased after treatments (Figure 4 B). Ci decreased in
F27+TMV treatment at day 1 to day 5 and then significantly increased afterward (Figure
4 D). Fv/Fm measurement is a frequently used parameter in studies of plant ecophysiology.
No significant change in Fv/Fm was noted among treatments. Notably, Fv/Fm increased
after F27+TMV treatment (Figure 5). Photosynthesis assay results indicate that F27 may
enhance photosynthesis.
Effect on Defensive Enzyme Activities. Induced resistance is significantly related to
enhanced activities of defensive enzymes, such as SOD, POD, and PAL.42,45 Antioxidant
defense machinery protects plant cells from oxidative damage induced by ROS.
Furthermore, ROS generation can reflect the state of photosynthesis.45 Therefore, we
analyzed defensive enzyme activities of F27ꢀtreated tobacco. SOD activity of F27+TMV
treatment group increased at day 1 to day 3 and reached the highest value on the third day.
SOD activity of F27+TMV treatment group was higher than that of TMVꢀinoculated
group. Then, activity decreased at day 3 to day 7 (Figure 6 A). POD activities of
CK+TMV, DFL+TMV, and F27+TMV treatment groups were higher than that of CK.
Notably, POD activity of F27+TMV treatment group was visibly higher than that of CK
treatment group and reached the highest value (58.7%) at day 5 (Figure 6 B). As a
defense gene, POD can induce biosynthetic pathway of SA, lignin, and phytoalexins,
which can activate SAR46 and promote cellꢀwall reinforcement and pathogen inhibition.47
CAT can catalyze decomposition of hydrogen peroxide to water and oxygen. This process
can protect cells against oxidative damage from ROS. CAT activities increased
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significantly in DFL+TMV and F27+TMV treatment groups and reached their maximum
values at day 3 (Figure 6 C). Activities (90.4% and 78.5%, respectively) were higher than
that of CK treatment. In F27 and TMV treatment groups, PAL activity of tobacco leaves
increased at day 1 to day 5, reached maximum on the fifth day, and then dropped at day 5
to day 7 (Figure 6 D). PAL activity of F27+TMV treatment group was higher (80.0%)
than that of CK group on the fifth day. PAL is a catalytic enzyme involved in biosynthesis
of phenylpropanoids to cinnamic acid. Such process can produce SA for defense against
pathogens.25 Results of defensive enzyme activity assay demonstrated that F27 can
improve disease resistance of tobacco through induction defensive responses in the form
of enzymes.
Identification of DEPs of Tobacco in Response to F27
In physiological and biochemical assays, the highest values of most parameters were
observed on day 3. To determine the effect of proteins on TMVꢀinoculated tobacco after
F27 treatment, total proteins of samples on day 3 were extracted and identified through
label−free LC−MS/MS. Label−free analysis revealed significant changes in proteins of
treatment groups with respect to those of the control group. In total, 270 and 283
proteins were identified in CK+TMV and F27+TMV treatment groups, respectively
(Supporting Information Table S1). A total of 244 proteins (79.0%) were common in
both groups. A total of 26 and 39 proteins were unique in CK+TMV and F27+TMV
treatment groups, respectively (Figure 7), 25 and 6 proteins were upꢀ (F27+TMV versus
CK+TMV ratio > 1.5, P < 0.05) and downꢀregulation (F27+TMV versus CK+TMV
ratio < 0.7, P < 0.05), respectively (Supporting Information Table S2).
Functional classification by GO
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To assign functional information to DEPs between TMV plants and those treated with
F27+TMV, GO analysis was carried out; this analysis can provide hierarchical
relationships for representation of information on cellular components, molecular
function, and biological processes.48 As shown in Figure 8, Go term enrichment analysis
of DEPs (P < 0.05) showed that main cellular components involved “membrane”,
“membrane part”, “thylakoid”, “plastid thylakoid”, “plastid thylakoid membrane”,
“chloroplast thylakoid”, “chloroplast thylakoid membrane”, “chloroplast envelope”,
“photosystem”, “photosystem II”, and “photosynthetic membrane”. Molecular function
included “cation binding”, “metalꢀion binding”, “ion binding”, “calciumꢀion binding”,
and “electron carrier activity”. Main biological process were “electron transport chain”,
“cation transport”, “regulation of defense response”, “regulation of response to stress”,
“response to toxin”, and “regulation of response to stimulus”. Protein ratio of regulating
immune response accounted for 22.6% of total DEPs, including heat shock protein 26,
constitutive plastidꢀlipidꢀassociated protein, SOD (CuꢀZn), putative glutathione
Sꢀtransferase, and dehydroascorbate reductase. Results of GO analysis indicated that
compound F27 can strongly reshape tobacco proteome by influencing many aspects of
plant physiology; some of these aspects include stress responses and photosynthesis.
Functional classification by KEGG
To further investigate the relationship between DEPs and biological functions, KEGG
was used to identify potential biological pathways of DEPs between CK+TMV and
F27+TMV treatment groups. DEPs were mapped to KEGG database categories at P <
0.05. Only two pathways were enriched: photosynthesis (pathway ID, ko00195) and
amyotrophic lateral sclerosis (pathway ID, ko00195). Enriched pathways included 10 and
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2 DEPs, respectively. These results revealed that photosynthesis is a significant pathway
for DEPs, as shown in Table 2 and Figure 9. Photosynthesis regulated by seven upꢀ and
three downꢀregulated expressed proteins, respectively.
Identification of proteins related to photoreaction system
Photosynthesis is one of the most important metabolic processes in plants; it requires
four protein components of photosynthetic electron transport chain, which is responsible
for electron transfer from water to oxidized form of nicotinamide adenine dinucleotide
phosphate, including PSII, PSI, cytochrome complex, and adenosine triphosphate
synthase. As shown in Figure 9 and Table 2, 10 DEPs were observed in F27+TMV versus
CK+TMV treatment groups. These DEPs included ETR12 (2.28ꢀfold), PsbP2 (2.10ꢀfold),
PsbQ (2.07ꢀfold), PsbP3 (1.64ꢀfold), PetC2 (1.59ꢀfold), PsbO (1.54ꢀfold), and PsaEB
(1.51ꢀfold); they were distinctly upꢀregulated in F27+TMV than in CK+TMV treatment
group. However, only three downꢀregulated proteins were found in F27+TMV versus
CK+TMV treatment groups; this downꢀregulation caused changes in structure and
function of the four protein components in photosynthetic organism pathway. As extrinsic
proteins, PsbO, PsbP, and PsbQ are subunits of oxygenꢀevolving complex, which is a
core of PSII in high plants. These extrinsic proteins are located at luminal surface of PSII
and are attached to its intrinsic subunits.49 PsbO protein is termed as
manganeseꢀstabilizing protein and is involved in regulation of PSII affinity for Mn. PsbO
protein is also required for PSII assembly/stability in higher plants and protects CP43 and
CP47 from proteolytic attack.50 PsbP and PsbQ are involved in increasing binding
affinities for both calcium and chloride, which are essential cofactors for oxygen
evolution.51 Results indicated that compound F27 increased expression quantity of
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proteinꢀregulated photoreaction system. F27 also activated immunity system and
enhanced plant tolerance to TMV infection.
In summary, 35 novel transꢀferulic acid derivatives containing a chalcone moiety were
designed and synthesized to induce plant resistance. Results showed that compounds F3,
F6, F17, and F27 exhibited better curative, protective and inactivating activities than
those of ningnanmycin. Especially, compound F27 exhibited the best protective activity
among title compounds. This protective ability was associated with potentiation of
defensive enzyme activity, chlorophyll content, and photosynthesis of tobacco after
treatment with F27. This finding was confirmed by upꢀregulated expression of stress
responses and photosynthesis regulatory proteins. This study demonstrated that F27 can
induce resistance and enhance plant tolerance to TMV infection. F27 can also be
considered as novel potential activator in field of plant protection.
ASSOCIATED CONTENT
Supporting Information
Supporting information illustrates synthesis, characterizations, physical, and analytical
data of intermediate 2a–2d and 3, target compounds F1–F35, and DEPs of F27+TMV
versus CK+TMV. This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86ꢀ851ꢀ83620521. Fax: +86ꢀ851ꢀ83622211. Eꢀmail: songbaoan22@yahoo.com.
ACKNOWLEDGMENTS
We gratefully acknowledge the assistance from the National Natural Science Foundation
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of China (21362004).
Notes
The authors declare no competing financial interest.
REFERENCES
(1) Craeger, A. N.; Scholthof, K. B.; Citovsky, V.; Scholthof, H. B. Tobacco mosaic virus:
pioneering research for a century. Plant cell 1999, 11, 301–308.
(2) Chen, M. H.; Chen, Z.; Song, B. A.; Bhadury, P. S.; Yang, S.; Cai, X. J.; Hu, D. Y.;
Xue, W.; Zeng, S. Synthesis and antiviral activities of chiral thiourea derivatives
containing an Rꢀaminophosphonatemoiety. J. Agric. Food Chem. 2009, 57, 1383–1388.
(3) Reichman, M.; Devash, Y.; Suhadolnik, R. J.; Sela, I. Human leukocyte interferon and
the antiviral factor (AVF) from virusꢀinfected plants stimulate plant tissues to produce
nucleotides with antiviral activity. Virology 1983, 128, 240–244.
(4) Kim, Y.; Komoda, E.; Miyashita, M.; Miyagawa, H. Continuous stimulation of the
plant immune system by the peptide elicitor PIP 1 is required for phytoalexin
biosynthesis in tobacco cells. J. Agric. Food Chem. 2014, 62, 5781−5788.
(5) Jones, J. D.; Dangl, J. L. The plant immune system. Nature 2006, 444, 323–329.
(6) Du, Q. S.; Zhu, W. P.; Zhao, Z. J.; Qian, X. H.; Xu, Y. F. Novel benzoꢀ
1,2,3ꢀthiadiazoleꢀ7ꢀcarboxylate derivatives as plant activators and the development of
their agricultural applications. J. Agric. Food Chem. 2012, 60, 346−353.
(7) Silverman, F. P.; Petracek, P. D.; Ju, Z.; Fledderman, C. M.; Heiman, D. F.; Warrior, P.
Salicylate activity. 1. Protection of plants from paraquat injury. J. Agric. Food Chem.
2005, 53, 9764–9768.
(8) Silverman, F. P.; Petracek, P. D.; Ju, Z.; Fledderman, C. M.; Heiman, D. F.; Warrior, P.
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 43
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
Salicylate activity. 2. Potentiation of atrazine. J. Agric. Food Chem. 2005, 53,
9769–9774.
(9) Silverman, F. P.; Petracek, P. D.; Heiman, D. F.; Fledderman, C. M.; Warrior, P.
Salicylate activity. 3. Structure relationship to systemic acquired resistance. J. Agric.
Food Chem. 2005, 53, 9775–9780.
(10) Fan, Z. J.; Shi, Z. G.; Zhang, H. K.; Liu, X. F.; Bao, L. L.; Ma, L.; Zuo, X.; Zheng, Q.
X.; Mi, N. Synthesis and biological activity evaluation of 1,2,3ꢀthiadiazole derivatives as
potential elicitors with highly systemic acquired resistance. J. Agric. Food Chem. 2009,
57, 4279–4286.
(11) Kauss, H.; Krause, K.; Jeblick, W. Methyl jasmonate conditions parsley suspension
cells for increased elicitation of phenylpropanoid defense responses. Biochem. Biophys.
Res. Commun. 1992, 189, 304–308.
(12) Qian, X. H.; Lee, P. W.; Cao, S. China: forward to the green pesticides via a basic
research program. J. Agric. Food Chem. 2010, 58, 2613–2623.
(13) Seiber, J. N. Sustainability and agricultural and food chemistry. J. Agric. Food Chem.
2011, 59, 1–21.
(14) Chen, J.; Yan, X. H.; Dong, J. H.; Sang, P.; Fang, X.; Di, Y. T.; Zhang, Z. K.; Hao, X.
J. Tobacco mosaic virus (TMV) inhibitors from Picrasma quassioides Benn. J. Agric.
Food Chem. 2009, 57, 6590−6595.
(15) Song, H. J.; Liu, Y. X.; Liu, Y. X.; Wang, L. Z.; Wang, Q. M. Synthesis and antiviral
and
fungicidal
activity
evaluation
of
βꢀcarboline,
dihydroꢀβꢀcarboline,
tetrahydroꢀβꢀcarboline alkaloids, and their derivatives. J. Agric. Food Chem. 2014, 62,
1010–1018.
22
ACS Paragon Plus Environment

Page 23 of 43
Journal of Agricultural and Food Chemistry
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
(16) Yan, X. H.; Chen, J.; Di, Y. T.; Fang, X.; Dong, J. H.; Sang, P.; Wang, Y. H.; He, H.
P.; Zhang, Z. K.; Hao, X. J. Antiꢀtobacco mosaic virus (TMV) quassinoids from Brucea
javanica (L.) Merr. J. Agric. Food Chem. 2010, 58, 1572−1577.
(17) Ge, Y. H.; Liu, K. X.; Zhang, J. X.; Mu, S. Z.; Hao, X. J. The limonoids and their
antitobacco mosaic virus (TMV) activities from Munronia unifoliolata Oliv. J. Agric.
Food Chem. 2012, 60, 4289−4295.
(18) Wang, Z. W.; Wei, P.; Wang, L. Z.; Wang, Q. M. Design, synthesis, and antiꢀtobacco
mosaic virus (TMV) activity of phenanthroindolizidines and their analogues. J. Agric.
Food Chem. 2012, 60, 10212–10219.
(19) Isman, M. B. Botanical insecticides: For richer, for poorer. Pest Manage. Sci. 2008,
64, 8−11.
(20) Chen, Z.; Zeng, M. J.; Song, B. A.; Hou, C. R.; Hu, D. Y.; Li, X. Y.; Wang, Z. C.;
Fan, H. T.; Bi, L.; Liu, J. J.; Yu, D. D.; Jin, L. H.; Yang, S. Dufulin activates HrBP1 to
produce antiviral responses in tobacco. PLoS one 2012, 7, e37944.
(21) Luo, H.; Liu, J. J.; Jin, L. H.; Hu, D. Y.; Chen, Z.; Yang, S.; Wu, J.; Song, B. A.
Synthesis and antiviral bioactivity of novel (1E,4E)ꢀ1ꢀarylꢀ5ꢀ(2ꢀ(quinazolinꢀ4ꢀ
yloxy)phenyl)ꢀ1,4ꢀpentadienꢀ3ꢀone derivatives. Eur. J. Med. Chem. 2013, 63, 662–669.
(22) Ma, J.; Li, P.; Li, X. Y.; Shi, Q. C.; Wan, Z. H.; Hu, D.Y.; Jin, L. H.; Song, B. A.
Synthesis and antiviral bioactivity of novel 3ꢀ((2ꢀ((1E,4E)ꢀ3ꢀoxoꢀ5ꢀarylpentaꢀ1,4
ꢀdienꢀ1ꢀyl)phenoxy)methyl)ꢀ4(3H)ꢀquinazolinone derivatives. J. Agric. Food Chem. 2014,
62, 8928–8934.
(23) Han, Y.; Ding, Y.; Xie, D. D.; Hu, D. Y.; Li, P.; Li, X. Y.; Xue, W.; Jin, L. H.; Song,
B. A. Design, synthesis and antiviral activity of novel rutin derivatives containing
23
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Journal of Agricultural and Food Chemistry
Page 24 of 43
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
1,4ꢀpentadienꢀ3ꢀone moiety. Eur. J. Med. Chem. 2015, 92, 732–737.
(24) Long, C. W.; Li, P.; Chen, M. H; Dong, L. R.; Hu, D. Y.; Song, B. A. Synthesis,
antiꢀtobacco mosaic virus and cucumber mosaic virus activity, and 3DꢀQSAR study of
novel 1,4ꢀpentadienꢀ3ꢀone derivatives containing 4ꢀthioquinazoline moiety. Eur. J. Med.
Chem. 2015, 102, 639–647.
(25) Gozzo, F. Systemic acquired resistance in crop protection: from nature to a chemical
approach. J. Agric. Food Chem. 2003, 51, 4487–4503.
(26) Ouyang, M. A.; Wein, Y. S.; Zhang, Z. K.; Kuo, Y. H. Inhibitory activity against
tobacco mosaic virus (TMV) replication of pinoresinol and syringaresinol lignans and
their glycosides from the root of Rhus javanica var. roxburghiana. J. Agric. Food Chem.
2007, 55, 6460–6465.
(27) Wu, M.; Wang, Z. W.; Meng, C. S.; Wang, K. L.; Hu, Y. N.; Wang, L. Z.; Wang, Q.
M. Discovery and SARs of transꢀ3ꢀaryl acrylic acids and their analogs as novel antiꢀ
tobacco mosaic virus (TMV) agents. PLoS one. 2013, 8, e56475.
(28) Huang, G. Y.; Cui, C.; Wang, Z. P.; Li, Y. Q.; Xiong, L. X.; Wang, L. Z.; Yu, S. J.; Li,
Z. M.; Zhao, W. G. Synthesis and characteristics of (Hydrogenated) ferulic acid
derivatives as potential antiviral agents with insecticidal activity. Chem. Cent. J. 2013, 7,
33–44.
(29) Du, G.; Han, J. M.; Kong, W. S.; Zhao, W.; Yang, H. Y.; Yang, G. Y. Chalcones from
the flowers of rosa rugosa and their antiꢀtobacco mosaic virus activities. B. Kor. Chem.
Soc. 2013, 34, 1263–1265.
(30) Wan, Z. H.; Hu, D. Y.; Li, P.; Xie, D. D.; Gan, X. H. Synthesis, antiviral bioactivity
of novel 4ꢀthioquinazolinederivatives containing chalcone moiety. Molecules 2015, 20,
24
ACS Paragon Plus Environment

Page 25 of 43
Journal of Agricultural and Food Chemistry
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
11861–11874.
(31) Song, B. A.; Zhang, H. P.; Wang, H.; Yang, S.; Jin, L. H.; Hu, D. Y.; Pang, L. L.;
Xue, W. Synthesis and antiviral activity of novel chiral cyanoacrylate derivatives. J.
Agric. Food. Chem. 2005, 53, 7886–7891.
(32) Gooding, G. V. Jr.; Hebert, T. T. A simple technique for purification of tobacco
mosaic virus in large quantities. Phytopathology 1967, 57, 1285–1287.
(33) Zhao, J.; Zhou, J. J.; Wang, Y. Y.; Gu, J. W.; Xie, X. Z. Positive regulation of
phytochrome B on chlorophyll biosynthesis and chloroplast development in rice. Rice
Science 2013. 20, 243–248.
(34) Yi, X. P.; Zhang, Y. L.; Yao, H. S.; Luo, H. H.; Gou, L.; Chow, W. S.; Zhang, W. F.
Different strategies of acclimation of photosynthesis, electron transport and antioxidative
activity in leaves of two cotton species to water deficit. Functional Plant Biology 2016,
43, 448–460.
(35) Tahara, S. T.; Mehta, A.; Rosato, Y. B. Proteins induced by Xanthomonas
axonopodis pv. passiflorae with leaf extract of the host plant (Passiflorae edulis).
Proteomics 2003, 3, 95–102.
(36) Qian, G. L.; Zhou, Y. J.; Zhao, Y. C.; Song, Z. W.; Wang, S. Y.; Fan, J. Q.; Hu, B. S.;
Venturi, V.; Liu, F. Q. Proteomic analysis reveals novel extracellular virulenceꢀassociated
proteins and functions regulated by the diffusible signal factor (DSF) in Xanthomonas
oryzae pv. oryzicola. J. Proteome Res. 2013, 12, 3327−3341.
(37) Cox, J.; Neuhauser, N.; Michalski, A.; Scheltema, R. A.; Olsen, J. V.; Mann, M.
Andromeda: a peptide search engine integrated into the MaxQuant environment. J
Proteome Res. 2011, 10, 1794–1805.
25
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Journal of Agricultural and Food Chemistry
Page 26 of 43
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
(38) Cox, J.; Michalski, A.; Mann, M. Software lock mass by twoꢀdimensional
minimization of peptide mass errors. J. Am. Soc. Mass Spectr. 2011, 22, 1373–1380.
(39) Luber, C. A.; Cox, J.; Lauterbach, H.; Fancke, B.; Selbach, M.; Tschopp, J.; Akira, S.;
Wiegand, M.; Hochrein, H.; O’Keeffe, M.; Mann, M. Quantitative proteomics reveals
subsetꢀspecific viral recognition in dendritic cells. Immunity 2010, 32, 279–289.
(40) Gene Ontology Consortium. The gene ontology (GO) database and informatics
resource. Nucleic. Acids Res. 2004, 32, 258–261.
(41) Gan, X. H.; Hu, D. Y.; Li, P.; Wu, J.; Chen, X. W.; Xue, W.; Song, B. A. Design,
synthesis, antiviral activity and threeꢀdimensional quantitative structureꢀactivity
relationship study of novel 1,4ꢀpentadienꢀ3ꢀone derivatives containing the
1,3,4ꢀoxadiazole moiety. Pest Manag. Sci. 2016, 72, 534–543.
(42) Fan, H. T.; Song, B. A.; Bhadury, P. S.; Jin, L. H.; Hu, D. Y.; Yang, S. Antiviral
activity and mechanism of action of novel thiourea containing chiral phosphonate on
tobacco mosaic virus. Int. J. Mol. Sci. 2011, 12, 4522–4535.
(43) Rahoutei, J.; GarcíaꢀLuque, I.; Barón, M. Inhibition of photosynthesis by viral
infection: effect on PSII structure and function. Physiol. Plant. 2000, 110, 286–292.
(44) Ahmad, I.; Cheng, Z. H.; Meng, H. W.; Liu, T. J.; Nan, W. C.; Khan, M. A.; Wasila,
H.; Khan, A. R. Effect of intercropped garlic (Allium sativum) on chlorophyll contents,
photosynthesis and antioxidant enzymes in pepper. Pak. J. Bot. 2013, 45, 1889–1896.
(45) Dietz, K. J.; Turkan, I.; KriegerꢀLiszkay, A. Redoxꢀ and reactive oxygen
speciesꢀdependent signaling into and out of the photosynthesizing chloroplast. Plant
Physiol. 2016, 171, 1541–1550.
(46) Nyström, L.; Mäkinen, M.; Lampi, A. M.; Piironen, V. Antioxidant activity of steryl
26
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595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
ferulate extract from rye and wheat bran. J. Agric. Food Chem. 2005, 53, 2503–2510.
(47) Nicholson, R. L.; Hammerschmidt, R. Phenolic compounds and their role in disease
resistance. Annu. Rev. Phytopathol. 1992, 30, 369−389.
(48) Ashburner, M.; Ball, C. A.; Blake, J. A.; Botstein, D.; Butler, H.; Cherry, J. M; Davis,
A. P.; Dolinski, K.; Dwight, S. S.; Eppig, J. T.; Harris, M. A.; Hill, D. P.; IsselꢀTarver, L.;
Kasarskis, A.; Lewis, S.; Matese, J. C.; Richardson, J. E.; Ringwald, M.; Rubin, G. M.;
Sherlock, G. Gene ontology: tool for the unification of biology. Nat. Genet. 2000, 25,
25–29.
(49) Chen, L. B.; Jia, H. Y.; Du, L. B.; Tian, Q.; Gao, Y. L.; Liu, Y. Release of the
oxygenꢀevolving complex subunits from photosystem II membranes in phosphorylation
condition under light stress. Chin. J. Chem. 2011, 29, 2631–2636.
(50) Popelkova, H.; Commet, A.; Kuntzleman, T.; Yocum, C. F. Inorganic cofactor
stabilization and retention: the unique functions of the two PsbO subunits of eukaryotic
photosystem II. Biochemistry 2008, 47, 12593–12600.
(51) Mummadisetti, M. P.; Frankel, L. K.; Bellamy, H. D.; Sallans, L.; Goettertb, J. S.;
Brylinski, M.; Limbachc, P. A.; Bricker, T. M. Use of protein crossꢀlinking and radiolytic
footprinting to elucidate PsbP and PsbQ interactions within higher plant Photosystem II.
Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 16178–16183.
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Table, Scheme and Figure Captions
Table 1. In Vivo Antiviral Activities of Test Compounds against TMV
Table 2. DEPs Involved in Photoreaction Systems
618
Scheme 1. Synthetic Route for Title Compounds
619
620
621
Figure 1. Structures of some plant activators and antiviral compounds.
Figure 2. Design of title compounds.
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
Figure 3. Effects of compound F27 on Ca (A), Cb (B), Ct (C), and Chlorophyll a/b (D) in
tobacco leaves. Vertical bars refer to mean ± SD (n = 3).
Figure 4. Effects of compound F27 on Pn (A), Gs (B), Tr (C), and Ci (D) in tobacco leaves.
Vertical bars refer to mean ± SD (n = 3).
Figure 5. Effects of compound F27 on Fv/Fm in tobacco leaves. Vertical bars refer to
mean ± SD (n = 3).
Figure 6. Effects of compound F27 on SOD (A), POD (B), CAT (C), and PAL (D)
activity in tobacco leaves. Vertical bars refer to mean ± SD (n = 3).
Figure 7. Changed proteome distribution between F27+TMV and CK+TMV, Venn
diagram showing unique and shared proteins.
Figure 8. Cellular components, molecular functions, and biological processes involving
DEPs in F27+TMV versus CK+TMV.
Figure 9. KEGG map of photosynthetic pathway of DEPs in F27+TMV versus CK+TMV.
Boxes with red frame indicate the corresponding DEPs were upꢀregulated in F27+TMV
samples. Boxes with green frame indicate that corresponding DEPs were downꢀregulated
in F27+TMV samples.
638
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Table 1. In Vivo Antiviral Activities of Test Compounds against TMVa
640
Curative
activityb
(%)
Protective Inactivating
EC50 of protective
activityb
(%)
activityb
(%)
Compd.
activity (ꢀg mLꢀ1)
R
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
Me
nꢀpr
nꢀpr
nꢀpr
Me
iꢀPr
iꢀPr
iꢀPr
Me
Me
Me
nꢀpr
nꢀpr
iꢀPr
Me
Me
H
H
H
H
Ar
4ꢀFꢀPh
4ꢀClꢀPh
2ꢀFꢀPh
ꢀPh
4ꢀOCH3ꢀPh
4ꢀFꢀPh
4ꢀClꢀPh
2ꢀFꢀPh
ꢀPh
4ꢀOCH3ꢀPh
thiophen
4ꢀClꢀPh
2ꢀFꢀPh
40.0±2.1
50.4±3.9
63.9±3.0
52.4±2.3
21.5±1.8
61.9±3.4
43.5±4.1
39.8±3.5
30.0±3.9
32.7±2.3
46.6±1.9
56.7±2.7
31.0±2.3
43.8±5.1
43.8±2.8
32.9±4.6
56.3±1.2
40.3±3.9
57.2±1.9
45.6±2.1
41.3±1.7
33.1±3.2
18.7±2.8
27.8±2.1
45.2±1.5
48.5±3.1
55.6±3.3
51.2±2.6
52.6±4.1
48.5±2.2
49.2±3.7
51.1±3.2
53.4±5.1
46.3±4.5
52.8±2.4
41.5±2.7
43.8±2.5
54.5±2.3
58.2±2.4
51.4±0.6
64.6±1.1
61.1±1.8
56.4±2.0
64.4±3.1
49.7±3.3
55.9±1.9
58.5±3.8
56.7±3.1
49.8±3.6
68.1±3.5
66.8±6.7
54.7±1.3
45.8±2.3
69.4±1.9
68.0±2.5
57.7±5.0
66.5±2.6
53.7±2.7
51.4±1.9
56.5±2.9
54.3±3.4
62.3±2.7
64.3±2.5
65.7±2.4
71.2±2.9
68.5±2.8
68.7±1.5
65.7±3.1
62.5±3.3
60.5±1.1
54.8±3.1
49.5±1.6
60.1±0.6
53.5±1.8
54.1±1.6
62.8±1.9
89.3±2.3
89.0±1.5
92.3±1.9
88.9±3.7
87.5±2.3
95.3±1.1
61.7±2.3
81.4±1.9
92.1±0.4
79.6±2.6
80.7±1.4
85.8±4.5
92.6±1.6
91.3±2.9
84.7±2.1
87.6±2.7
93.6±1.0
90.4±2.5
87.9±1.9
85.8±1.7
89.9±0.7
89.5±2.3
78.8±1.9
90.2±2.8
87.2±2.9
79.2±2.2
92.4±0.8
81.7±1.6
74.3±2.4
75.8±3.5
93.5±1.5
90.1±0.3
87.1±0.9
88.3±1.7
89.4±0.5
78.6±2.5
76.5±2.8
91.5±2.1
304.7±3.1
442.5±3.6
214.2±1.9
281.1±1.4
311.4±5.1
217.6±2.1
487.9±3.7
333.3±2.8
330.5±1.9
279.1±2.1
549.1±2.5
208.3±1.6
210.1±2.9
346.0±3.2
534.4±3.1
193.5±2.2
162.2±1.9
269.4±4.2
224.2±1.2
414.4±4.0
403.1±3.2
369.3±1.9
405.4±4.0
292.5±3.2
201.5±2.2
189.6±3.5
98.7±1.5
185.4±1.2
164.9±3.7
183.4±5.1
248.5±1.7
265.6±3.1
312.2±3.1
458.2±3.2
298.8±2.6
328.6±3.1
385.6±3.9
241.3±1.2
F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
F11
F12
F13
F14
F15
F16
F17
F18
F19
F20
F21
F22
F23
F24
F25
F26
F27
F28
F29
F30
F31
F32
F33
F34
F35
ꢀPh
furan
4ꢀFꢀPh
4ꢀClꢀPh
2ꢀFꢀPh
3ꢀFꢀPh
2,4ꢀdiFꢀPh
2,6ꢀdiFꢀPh
2,4ꢀdiOCH3ꢀPh
4ꢀC(CH3)3ꢀPh
2,4ꢀdiClꢀPh
4ꢀBrꢀPh
2ꢀClꢀPh
4ꢀFꢀPh
4ꢀClꢀPh
2ꢀFꢀPh
2ꢀClꢀPh
2ꢀFꢀPh
4ꢀFꢀPh
4ꢀFꢀPh
CH2Ph
CH2Ph
CH2(2,4ꢀdiClꢀPh)
CH2(3ꢀCH3ꢀPh)
CH2(2,4ꢀdiClꢀPh)
4ꢀFꢀPh
4ꢀClꢀPh
transꢀferulic acidc
dufulind
ningnanmycind
641
642
643
aAverage of three replicates. bConcentration of compounds is 500 ꢀg·mLꢀ1. cPurity ≥ 99%.
dCommercial antiviral agent ribavirin and ningnanmycin as positive control.
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