Synthesis, Antiviral Activity, and Induction of Plant Resistance of Indole Analogues Bearing Dithioacetal Moiety

Article abstract of DOI:10.1021/acs.jafc.9b05357

A series of compounds with potential activity to induce plant resistance was synthesized from indole and thiol compounds and methodically evaluated for antiviral activity. The results indicated that some of the synthesized compounds had high antipotato virus Y (PVY), anticucumber mosaic virus, and antitobacco mosaic virus activities. Notably, compound D21 exhibited the best activity against PVY among these compounds in vivo, and the 50% effective concentrations (EC₅₀) of protection activity is 122 μg/mL, which was distinctively better than the corresponding values for ribavirin (653 μg/mL), Ningnanmycin (464 μg/mL), and Xiangcaoliusuobingmi (279 μg/mL). Interestingly, we found that the protection activity of D21 was associated with improvement of chlorophyll content and defense-related enzyme activities. Moreover, D21 could trigger the malate dehydrogenase (MDH) signaling pathway, as further confirmed by the MDH activity evaluation. Hence, D21 can protect plants against viral activity and has potential as a novel activator for plant resistance induction.

Full text of DOI:10.1021/acs.jafc.9b05357

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Agricultural and Environmental Chemistry
Synthesis, Antiviral Activity and Induction of Plant
Resistance of Indole Analogues Bearing Dithioacetal Moiety
Baoan Song, Chunle Wei, Jian Zhang, Jing Shi, Xiuhai Gan, and De-Yu Hu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b05357 • Publication Date (Web): 13 Nov 2019
Downloaded from pubs.acs.org on November 16, 2019
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Figure 1. Chemical structures of Ningnanmycin, Ribavirin, and Indole.
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Figure 2. Design of target compounds.
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Figure 3. Synthetic route for target compounds D1-D25.
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Figure 4. Synthetic route for target compounds D26-D28.
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Figure 5. Effects of compound D21 on chlorophyll a (A), chlorophyll b (B) and chlorophyll total (C),
chlorophyll a/b (D) in tobacco leaves. Straight bars signify mean ± SD (n = 3).
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Figure 6. Effects of compound D21 on superoxide dismutase (SOD, A), peroxidase (POD, B), phenylalanine
ammonia lyase (PAL, C), and catalase (CAT, D) activity in tobacco leaves. Straight bars signify mean ± SD
(n= 3).
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Figure 7. Venn diagram shows that the proteome distribution between the D21+PVY and CK+PVY groups
changes uniquely and shares proteins.
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Figure 8. Volcano plot of the relative protein abundance changes between the D21+PVY and CK+PVY
treatments. The red points show significantly up-regulated proteins, whereas the blue points show
significantly down-regulated proteins.
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Figure 9. MDH signaling pathway in tobacco response to D21. Red color represents the up-regulated proteins
in this pathway. (Fd, Ferredoxin; FNR, ferredoxin-NADP reductase; FTR, Ferredoxin-thioredoxin reductase;
MDH, malate dehydrogenase; NTRC, chloroplast NADPH-thioredoxin reductase; OAA, oxaloacetate; OMT,
malate/OAA translocators; PS Ⅰ, photosystem Ⅰ; PS Ⅱ, photosystem Ⅰ; ROS, reactive oxygen species;
Trx, thioredoxin).
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Figure 10. D21 effects on the NAD-MDH and NADP-MDH activities of tobacco leaves at day 3. Straight bars
signify mean ± SD (n= 3).
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Synthesis, Antiviral Activity and Induction of Plant Resistance
of Indole Analogues Bearing Dithioacetal Moiety
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Chunle Wei, Jian Zhang, Jing Shi, Xiuhai Gan, Deyu Hu,* Baoan Song *
State Key Laboratory Breeding Base of Green Pesticide and Agricultural
Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, China.
*Corresponding author (Tel.: 86-851-88292170; Fax: 86-851-88292170; E-mail:
dyhu@gzu.edu.cn; songbaoan22@yahoo.com).
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ABSTRACT: A series of compounds with potential activity to induce plant resistance
was synthesized from indole and thiol compounds, and methodically evaluated for
antiviral activity. The results indicated that some of the synthesized compounds had
high anti-potato virus Y (PVY), anti-cucumber mosaic virus (CMV) and anti-tobacco
mosaic virus (TMV) activities. Notably, compound D21 exhibited the best activity
against PVY among these compounds in vivo, and the 50% effective concentrations
(EC50) of protection activity is 122 μg/mL, which was distinctively better than the
corresponding values for Ribavirin (653 μg/mL), Ningnanmycin (464 μg/mL), and
Xiangcaoliusuobingmi (279 μg/mL). Interestingly, we found the protection activity of
D21 was associated with improvement of chlorophyll content and defense-related
enzyme activities. Moreover, D21 could trigger the malate dehydrogenase (MDH)
signaling pathway, as further confirmed by the MDH activity evaluation. Hence, D21
can protected plants against viral activity, and has potential as a novel activator for
plant resistance induction.
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KEYWORDS: indole, dithioacetal, antiviral activity, plant resistance induction,
malate dehydrogenase.
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INTRODUCTION
Plant viruses cause heavy losses throughout the history of agricultural development.
Potato virus Y (PVY), cucumber mosaic virus (CMV), and tobacco mosaic virus
(TMV) are common plant viruses that affect crop quality and yield in many areas of
the world1. Especially, PVY causes significant problems in terms of economic losses
in a broad range of important crops. It has a wide host range and naturally causes
substantial damage in tobacco, tomato, and pepper2. The methods used to reduce the
incidence of viral disease are based on chemical agents, such as Ningnanmycin and
Ribavirin (Figure 1), but show unsatisfactory field efficacy3-5. Therefore, novel,
highly active, and environmentally friendly antiviral agents need to be developed to
manage the risk of a virus epidemic.
Figure 1
Natural products and their derivatives have a long history as important sources of
and inspiration for the discovery of novel agrochemicals. The demand for new
nature-derived agrochemicals and environmentally friendly sources has increased
owing to the increasingly stringent environmental, toxicological, and regulatory
requirements6-9. Indole (Figure 1) skeleton and its derivatives occur broadly in many
natural products, plants, animals, and marine organisms10. Indole derivatives comprise
an important class of active compounds in heterocyclic compounds, which are
associated with pharmacological activities11, such as antiviral4, 5, 12, 13, protein kinase
inhibitory14, antibacterial15, 16, and anticancer activities17-19. Considering the important
active structures, indoles may have the most important structure category in drug
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discovery20-22. Indole derivatives have a unique property that mimics the structure of
peptides, and it reversibly binds to enzymes23, 24, thus, they have attracted the attention
of pharmacologists.
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In our previous work, the novel dithioacetal derivative Xiangcaoliusuobingmi
(XCLSBM) (Figure 2) was discovered as an efficient antiviral agent25, 26. As an
extension to our research in dithioacetal and systematic study of dithioacetal
derivatives, a series of compounds with potential activator to induce plant resistance
was designed based on lead compound XCLSBM and synthesized (Figure 2) from
indole and 10 kinds thiol compounds. Their antiviral activities against PVY, CMV
and TMV and effect on defense-related enzyme activity, chlorophyll content, and
differentially expressed proteins (DEPs) were investigated in this study.
Figure 2
MATERIALS AND METHODS
Chemicals and Instruments. 1H, and 13C NMR spectra were obtained using a 500
or 400-MHz (100 MHz for 13C) instrument (Bruker, Germany) in acetone-d6,
DMSO-d6 or CDCl3 solution at room temperature. Tetramethylsilane was the internal
standard. HRMS was performed with Thermo Scientific Q Exactive (Thermo
Scientific, USA). The melting points were measured using the WRX-4 equipment
(Shanghai Yice Apparatus & Equipment Co., Ltd., Songjiang District, Shanghai,
China; uncorrected). Indolin-2-one (98% purity; CAS: 59-48-3) and other raw
material compounds were purchased from Aladdin Chemicals Co. (China). All
reagents were used without further purification and were dried before use unless
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otherwise stated.
General Procedure for Intermediates A1–A4, B, and C1–C4. The synthetic
route for a novel indole series containing dithioacetal moiety is shown in Figure 3.
The synthesis of intermediates A1–A4 was based on previously reported methods27.
Intermediate B has been widely reported28. Intermediates B (5 mmol) and substituted
1H-indole-3-carbaldehyde (5 mmol) were added into acetonitrile (30 mL) with
potassium carbonate (6 mmol) and were heated to reflux, until the reaction was
completion. Then, the solvent was concentrated in vacuo, and the solids were washed
with water and filtered. Intermediates C1−C4 were recrystallized from ethanol.
General Synthesis Procedure for the Indole Derivatives Containing
Dithioacetal Moiety D1−D27. Various types of thiol compounds (1.38 mmol) were
selectively converted to intermediates C1−C4 (0.519 mmol) in dichloromethane (25
mL) in the presence of NaHSO4/SiO2 (0.01 mmol) at room temperature. Upon
reaction completion, silica was removed via filtration, and the filtrate was evaporated.
Crude products were purified by using flash chromatography with EtOAc/n-hexane
(1:3, v/v) to obtain D1−D25 (Figure 3).
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Figure 3
General Synthetic Procedures for D26−D28. 1,2-Ethanedithiol (1.38 mmol) was
selectively converted to intermediates C1−C3 (0.519 mmol) in dichloromethane (25
mL) in the presence of NaHSO4/SiO2 (0.01 mmol) at room temperature. Upon
reaction completion, silica was removed by filtration, and the filtrate was evaporated.
The crude product was purified via flash chromatography with EtOAc/n-hexane (1:3,
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v/v) to obtain the target compounds D26−D28 (Figure 4).
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Figure 4
Antiviral Activity Assay. Extraction of PVY29 and CMV30. Viruses were
propagated in Nicotiana. tabacum cv. K326, ground in phosphate buffer and filtered
with a double-layer pledget. The extract was centrifuged for 5 min at 10000×g, and
the supernatant was used as the crude extract of the virus. The extraction process was
carried out at 4 ℃.
Extraction of TMV31. Viruses were propagated in Nicotiana. tabacum cv. K326,
ground in phosphate buffer and filtered with a double-layer pledget. The extract was
centrifuged for 5 min at 10000×g, treated twice through polyethylene glycol 6000,
and then centrifuged again. The extraction process was carried out at 4 ℃. The
absorbance values were tested using an ultraviolet spectrophotometer at 260 nm:
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where E represents the extinction coefficient for TMV; E0.1% 260nm is 3.1.
1cm
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Curative Activities of Target Compounds against PVY and CMV32 in vivo.
Chenopodium amaranticolor plants were used to evaluate the anti-PVY and
anti-CMV activities. The crude extracts of PVY (CMV) were dipped and inoculated
on the whole leaves, which were scattered with silicon carbide beforehand. The leaves
were washed with water after inoculation for 30 minutes and then dried. The target
compound solution was smeared on the right side of the leaves, and solvent was
smeared on the left side, which served as the control. All the plants were cultivated in
an incubator under an illumination of 10 000 lx at 28±2 ℃. The number of local
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lesions appearing 5 to 6 days after inoculation was counted. Measurements were
performed in triplicate.
Protective Activities of Target Compounds against PVY and CMV in vivo. The
target compound solution was smeared on the right side of the leaves, and solvent was
smeared on the left side, which served as the control. Chenopodium amaranticolor
plants were inoculated with PVY (CMV) after 24 hours. The leaves were washed with
water after inoculation for 30 minutes. All the plants were cultivated in an incubator
under an illumination of 10 000 lx at 28±2 ℃. The number of local lesions appearing
5 to 6 days after inoculation was counted. Measurements were performed in triplicate.
Measurements were performed in triplicate.
Curative Activities of Target Compounds against TMV in vivo. Nicotiana. tabacum
L. plants were used to evaluate the anti-TMV activities. TMV at a concentration of
6×10−3 mg mL−1 was dipped and inoculated on the whole leaves, which were
scattered with silicon carbide beforehand. The leaves were washed with water after
inoculation for 30 minutes and then dried. The target compound solution was smeared
on the right side of the leaves, and solvent was smeared on the left side, which served
as the control. All the plants were cultivated in an incubator under an illumination of
10 000 lx at 28±2 ℃. The number of local lesions appearing 3 to 4 days after
inoculation was counted. Measurements were performed in triplicate.
Protective Activities of Target Compounds against TMV in vivo. The target
compound solution was smeared on the right side of the leaves, and solvent was
smeared on the left side, which served as the control. Nicotiana. tabacum L. plants
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were inoculated with TMV after 24 hours. The leaves were washed with water after
inoculation for 30 minutes. All the plants were cultivated in an incubator under an
illumination of 10 000 lx at 28±2 ℃. The number of local lesions appearing 3 to 4
days after inoculation was counted. Measurements were performed in triplicate.
Measurements were performed in triplicate.
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The inhibitory rate of each compound was calculated as follows (‘av’ means
average):
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Physiological and Biochemical Analysis. Compound Treatments and Sampling.
Nicotiana. tabacum cv. K326 plants were selected until the four- to six-leaf stage.
Exactly 500 μg/mL of D21 solution was sprayed on the plants, comprising the
treatment group, whereas XCLSBM and water belonged to the positive and negative
(CK) controls, respectively. The five treatments were CK, D21, CK+PVY,
XCLSBM+PVY, and D21+PVY. Plant leaves were collected at 1, 3, 5, and 7 days
after infection with PVY for the defensive enzyme activity assay, and to determine the
chlorophyll content and DEPs. All the test plants were cultivated in an incubator
under an illumination of 10 000 lx at 28±2 ℃. Measurements were repeated in
triplicate.
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Determination of Enzyme Activities. The activities of catalase (CAT), phenylalanine
ammonia lyase (PAL), peroxidase (POD), and superoxide dismutase (SOD) were
tested using enzyme assay reagent kits (Suzhou Comin Bioengineering Institute,
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Jiangsu, China). MDH activity assay was performed based on the reagent kits from
Beijing Solarbio Science & Technology Co., Ltd.
Chlorophyll Content Test. Chlorophyll content assay was conducted as previously
described33. The absorption spectra of chlorophyll a (Ca) and b (Cb) were detected at
663 and 645 nm, respectively, and chlorophyll total (Ct) were calculated as: Ct = Ca+Cb.
Measurements were performed in triplicate.
DEPs Analysis. Extraction of Total Tobacco Protein. Total tobacco proteins were
extracted as previously described with slight modification31, 32. Approximately 1.5 g
of leaf sample was ground in liquid nitrogen to power, and homogenized in 5 mL
sucrose lysis buffer (0.04 M dithiothreitol, 0.05 M EDTA, 100 mM KCl, 500 mM
Tris-HCl, 700 mM sucrose, pH 7.5) at 25 ℃ for 30 minutes. 5 mL of Tris–phenol was
added, and then shaken for 30 minutes at 4 ℃. Centrifugation for 8 minutes at 6000
rpm, the upper phenol layer was collected. Five volumes of 100 mM ice-cold
ammonium acetate in methanol was added, and the sample was stored for 12 hours at
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-20 ℃. Then the sample was centrifuged at 4 ℃ and 6000 rpm for 15 minutes. The
precipitate was collected and washed thrice with cold acetone. The precipitate was
dried in Speed Vac (SIM International Group Co., Ltd., NJ, USA) for 5 hours and
solubilized in 1 mL of rehydration solution (PH 8.5, 0.04 M DTT, 0.1 M Tris, 8 M
urea) for 50 minutes at 37 °C. Protein concentration was measured by the Bradford
method. Afterward, solutions containing 100 μg proteins were collected and alkylated
with equal volume of 55 mM iodoacetamide. The mixture was incubated at 25 ℃ for
40 minutes in the dark, and then centrifuged with 3 kDa Millipore for 20 minutes at
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12000 rpm and 4 °C. Next, the protein was digested in 40 μL of 25 mM NH4HCO3
with 5 μg of trypsin (Promega, Madison, USA) at 37 °C and 800 rpm for 8 hours. The
suspension was centrifuged for 40 minutes at 12000 rpm and 4 ℃. Finally, peptide
solution was collected, dried, and solubilized in 45 μL of H2O (LC/MS grade)
containing 0.1 % formic acid (FA) for liquid chromatography-tandem MS
(LC-MS/MS) analysis.
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Protein Identification. Every peptide sample was resolved using a Nano
LC-1DTM plus system with triple-of-flight 5600 mass spectrometer (AB Sciex,
Foster City, CA, USA). Then, peptide (8 μL) was obtained via a full-loop injection
and then desalted on a ChromXP Trap column (Nano LC TRAP Column, 0.5
mm×350 μm, 3 μm C18-CL, 120 Å, AB Sciex, Foster City, CA, USA). The sample
was washed into a second analytical column (3C18-CL column, 15 cm ×75 μm, AB
Sciex, Foster City, CA, USA) by using a linear gradient composed of mobile phase
A (0.1 % FA, 95% ACN) and B (0.1 % FA, 5 % ACN) for 1.5 h at a flow rate of 5
nL/s. Three times TOF 5,600 MS was ran in data-dependent mode to conversion
between product ion acquisition and TOF-MS by using the Analyst (R) software
(TF1.6) (AB SCIEX, Foster City, CA, USA). Beta-galactosidase digestion was used
to calibrate each pair of samples by elution for 10 min and identification for 30
minutes.
Proteomics Data Analysis. The MaxQuant34 version 1.5.2.8 of the Andromeda
search engine was used, and the LC-MS/MS data was analyzed and quantified based
on the PVY proteome downloaded from UniProt (http://www.uniprot.org/). In
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Andromeda’s main research, the original mass tolerances for the debris and mass of
precursors are 20 and 6 ppm, respectively. The global false discovery rate (FDR)
cut-off was set to 0.01 in protein and peptide identification. Normalized protein
intensity was decided via label-free quantification35. The algorithm of the
intensity-based absolute quantification (iBAQ) was sorted with the absolute
abundance of DEPs36. Protein tables were filtered by eliminating identifications of
common contaminants and reverse database. Differentially accumulated proteins
were identified by Unpaired two-samples t-test of iBAQ data between control and
treatment groups.
Bioinformatics Analysis. For functional analyses, we used cluster samples to test
the increase/decrease in DEPs common for all three replicates. The Kyoto
Encyclopedia of Genes and Genomes (KEGG) annotation was retrieved from the
KEGG Pathway database (http://www.genome.jp/Pathway). The databases were
searched using the Uniprot software.
RESULTS AND DISCUSSION
Chemistry. Figure 3 shows the synthetic routes of indole derivatives containing a
dithioacetal group. 2-Chloro, 2-bromo, 5-bromo-2-chloro, and 2-methyl-1H-indole-3-
carbaldehyde A1–A4 were synthesized via Vilsmeier–Haack reaction to determine the
effects of electron-donating and electron-withdrawing groups on the antiviral activity.
C1–C4 were obtained by stirring and refluxing the corresponding A1–A4, B, and
potassium carbonate in acetonitrile for about 3 hours to obtain a yield of 87 % to 93 %.
To systematically study the effect of dithioacetal structure on the virus, we
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synthesized the target compounds D1–D25 via C1–C4 and nine different thiols
(mercaptoethanol, ethyl mercaptan, propanethiol, isopropyl mercaptan,
1,2-ethanedithiol, 4-chlorothiophenol, 2,4-dichlorothiophenol, butyl mercaptan,
n-butyl mercaptan, and dodecyl mercaptan) via Michael addition and achieved a yield
of 70 %–95 %. The dithioacetal moiety was further synthesized into a closed loop
(Figure 4). The chemical structures of key intermediates C1–C4 and target
compounds D1–D28 were identified by NMR and HRMS (Supporting Information
Ⅰ).
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Antiviral Activity. Anti-PVY Activity in Vivo. The anti-PVY activities of D1–D28
are summarized in Table 1. D21 displayed higher antiviral curative activities against
PVY with values of 61.6 %, than that of Ningnanmycin (50.1 %), Ribavirin (40.2 %),
and lead compound XCLSBM (55.2 %). D21 exhibited excellent protective activities
on PVY with values of 70.1 %, superior to Ningnanmycin (50.8 %), Ribavirin (41.2
%), and lead compound XCLSBM (57.7 %).
Table 1
To further confirm the anti-PVY activity of the target compounds, the EC50 of the
target compounds anti-PVY were tested and the results were listed in Table 2.
Evidently, D5, D21, and D24 exhibited good curative activity anti-PVY with EC50
values of 272, 217, and 288 μg/mL, respectively, which higher than that of Ribavirin
(686 μg/mL), Ningnanmycin (468 μg/mL), and lead compound XCLSBM (292
μg/mL). D2, D21, D23, and D24 exhibited higher protective activity anti-PVY with
EC50 of 212, 122, 268, and 244 μg/mL, respectively, than that of Ribavirin (653
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μg/mL), Ningnanmycin (464 μg/mL), and lead compound XCLSBM (279 μg/mL).
Anti-CMV Activity in Vivo. The anti-CMV activities of D1–D28 are displayed in
Tables 1 and 2. The protective activities of D2, D11, and D21 with EC50 of 187, 200,
and 156 μg/mL, respectively, which were higher than that of Ribavirin (690 μg/mL),
Ningnanmycin (439 μg/mL), and lead compound XCLSBM (240 μg/mL). The
curative effects of D2, D3, D5, D9, D11, D12, D19, D20, and D21 with EC50 levels of
199, 220, 202, 194, 225, 224, 217, 202, and 179 μg/mL, respectively, also surpassed
that of Ribavirin (704 μg/mL), Ningnanmycin (498 μg/mL), and lead compound
XCLSBM (275 μg/mL).
Anti-TMV Activity in Vivo. The anti-TMV activities of D1–D28 were measured
and shown in Tables 1 and 2. The protective activities anti-TMV EC50 values of D10,
D19, and D21 is 207, 212, and 200 μg/mL, respectively, which similar to
Ningnanmycin (198 μg/mL), but higher than that of Ribavirin (568 μg/mL) and lead
compound XCLSBM (497 μg/mL). D1-D3, D5, D10, D12, D14, D19–D23, and D25
had curative activities with EC50 levels of 225, 255, 209, 215, 322, 220, 364, 342, 212,
209, 293, 293, and 241 μg/mL, respectively, which were higher than that of Ribavirin
(668 μg/mL), Ningnanmycin 382 μg/mL), and lead compound XCLSBM (517
μg/mL).
Table 2
Structure−Activity Relationships (SARs). Results of preliminary SARs illustrated
that the presence of different thiol compounds influenced the anti-PVY activity of
indole derivatives containing dithioacetal moiety. This notion was proven by the
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following data, D21 (R2 = Propyl) > D17 (R2 = Tert-butyl), D24 (R2 = Propyl) > D8
(R2 = Tert-butyl). Thiol compounds in a linear chain had higher antiviral activity than
non-directly linked thiol compounds in which D3 (R2 = Propyl) > D4 (R2 = Isopropyl),
D12 (R2 = Propyl) > D13 (R2 = Isopropyl), D16 (R2 = Butyl) > D17 (R2 = Tert-butyl).
No change was observed in terms of the activity when electron-withdrawing (halogen)
or donating (methyl group) groups were introduced at the 2 position of the indole.
This phenomenon was verified by the following data, D10 (X = Br) ≈ D22 (X = CH3),
D2 (X = Cl) ≈ D23 (X = CH3), and D5 (X = Cl) ≈ D25 (X = CH3). The closed-loop
structure of dithioacetals were not contribute to antiviral activity (D26, D27, and D28
have weak antiviral activity). Hence, the dithioacetal structures are essential for
inducing plant resistance, and when R1 = Br, R2 = Propyl, and X = Cl, the target
compound was the best activator for plant resistance induction.
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Physiological and Biochemical Analysis. Plants’ immune system is activated
when infected with various aggressive pathogens. A range of defense responses can
cause strong physiological and biochemical changes in green plants, which can
significantly increase chlorophyll content and defense enzyme activity39,40
.
Effect on Chlorophyll Contents. Chlorophyll content is closely related to
photosynthesis and plays a major role in the growth process of plants. Ca, Cb, and Ct
content (Figure 5) decreased after PVY infected the Nicotiana tabacum cv. K326
plants in our study. In the D21 and D21+PVY groups, chlorophyll content gradually
increased from day 1 to day 5, and the maximum content was observed on day 5
(Figure 5A-C). So, D21 can increase chlorophyll content and promote photosynthesis,
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thereby enhancing defense responses and promoting plant host disease resistance.
Figure 5
Influences of Defense Enzyme Activity. Induced resistance is associated to enhance
activities of defensive enzymes, such as SOD, POD, PAL, and CAT. SOD is a
superoxide anion scavenging enzyme. POD can eliminate the toxicity of hydrogen
peroxide, phenols and amines to plants. PAL plays an important role in the normal
growth and development of plants and resists bacterial infection. And CAT can
scavenge reactive oxygen species. The antioxidant defense mechanisms of these
enzymes protect plants from oxidative stress induced by reactive oxygen species
(ROS)41. Hence, we systematically analyzed the defense enzyme activity of Nicotiana
tabacum cv. K326 after D21 treatment. After being infected by PVY, the SOD
activity of tobacco increased slightly on the first day and then decreased. After D21
treatment, the SOD activity of D21 group was remarkably higher than that of the
CK+PVY group, which reached the maximum on the 3rd day and increased by 46.6%
compared with CK group (Figure 6A). The POD activities of the CK+PVY,
D21+PVY, and XCLSBM+PVY groups notably increased after infecting the tobacco
plant with PVY. Simultaneously, the D21+PVY and XCLSBM+PVY groups reached
the maximum (731 and 622 U/mg prot) on the first day. The POD activity of the D21
and D21+PVY groups were higher than that of the CK and CK+PVY groups,
respectively, during the first to the 7th day (Figure 6B). The PAL activities of the D21,
D21+PVY and XCLSBM+PVY groups were higher than those of the CK group. The
D21+PVY group had the highest activity. The D21+PVY group reached maximum
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on the third day, which was 95.3% higher than the CK+PVY group (Figure 6C).
After PVY infection, the CAT activities of the D21 and D21+PVY groups increased
than those of the other treatment groups during the monitoring period. The CAT
activity of D21, D21+PVY and XCLSBM+PVY treatments first increased and then
decreased during the monitoring period, and reached the maximum on the third day
(Figure 6D). The results of defense enzyme activity evaluation indicated that D21 can
increase the activities of some defensive enzymes (SOD, POD, PAL, and CAT),
thereby enhancing defense responses and improving plant resistance.
Figure 6
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Proteomics Analysis. In the determination of physiological and biochemical
properties, the most remarkable change in tobacco after D21 treatment was observed
on the third day. Therefore, the total proteins of the CK+PVY and D21+PVY groups
on the third day were analyzed through label-free LC-MS/MS. The results displayed
that 844 proteins were identified (Supporting Information Ⅱ, Table 2). A total of 785
and 733 proteins were identified in the D21+PVY and CK+PVY groups, respectively
(Figure 7). In total, 111 (13.2 %) and 59 (7 %) proteins were discovered in the
CK+TMV and D21+TMV groups, respectively.
Figure 7
Figure 8 shows that the down-regulated (blue dots) and up-regulated (red dots) (p <
0.05, fold changes > 2.0) in the different treatment groups were 6 and 201 in the
CK+PVY and D21+PVY groups, respectively. To determine the expression levels of
these DEPs, a volcanic map was employed.
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Figure 8
Functional Classification by KEGG. The possible biological pathway of DEPs
between the D21+PVY and CK+PVY groups were identified via KEGG analysis.
The mode of action triggered by D21 was determined. The KEGG database categories
mapped DEPs at a level of P < 0.05. As shown in Table 3 and Figure 10, five MDHs
(EC 1.1.1.37), Malic enzyme, four Thioredoxin (Trxs), calvin cycle protein CP12-3,
Ferredoxin (Fd), two Ferredoxin-thioredoxin reductases (FTRs), four NADH
dehydrogenases, Peroxidase (EC 1.11.1.7) (POD), five Peroxiredoxins (EC 1.11.1.15)
(Prxs), and photosystem Ⅰ reaction center subunit IV B (PS Ⅰ) were upregulated.
These specific proteins played an important role in the MDH signaling pathway
(Figure 9). They can catalyze the reversible conversion between oxaloacetate (OAA)
and malate, which are involved in photosynthesis, C4 cycle, TCA cycle, and other
metabolic pathways43, 44. The electron transfer components from photosystem I to the
target enzyme are ferredoxin, FTRs and thioredoxin45. Calvin cycle protein CP12-3
acts as a linker and is essential in the assembly of a core complex of PRK/GAPDH.
The reversible inactivation of chloroplast enzymes GAPDH and PRK is coordinated
during darkness in photosynthetic tissues46. Trxs are widely present in plant
chloroplasts, and photosynthetic enzymes are photoregulated by FTRs. Trxs are
critical for the redox regulation of protein function and signaling through thiol redox
regulation47. NADH dehydrogenases are involved in the electron transport from
light-produced NADPH and Fd to the intersystem plastoquinone pool48. The
H2O2-decomposing antioxidant enzyme group consisted of Prxs. Prx proteins
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disintegrate peroxynitrites and alkyl hydroperoxides and lead to reduction in H2O2 .
PODs increase the plant defense against pathogens by reducing some aromatic
compounds (electron donors) and the toxicity of peroxides, and catalyzing the ability
of H2O2-dependent redox reduction50. In this study, D21 increased the protein to
regulate the expression level of the MDH and photoreaction system in the chloroplast.
Moreover, D21 increased the defense enzyme activity, thereby increasing plant
disease resistance.
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Table 3
Figure 9
MDH Activity. To verify the effect of D21 on the MDH signaling pathway, we
tested the NADP-MDH and NAD-MDH activities. Results (Figure 10) showed that
the NAD-MDH and NADP-MDH activities of the D21+PVY group increased by
8.4% and 85.5%, respectively, compared with the CK+PVY group on the third day
after PVY infection. D21 increased the MDH activity and acted on the MDH
signaling pathway.
Figure 10
In summary, Twenty-eight novel indole derivatives containing dithioacetal moiety
were designed, synthesized, and their activities against plant viruses were evaluated
methodically. Bioassay results indicated that D21 exhibited higher antiviral protective
activity than others target compounds, and superior to the commercial Ribavirin,
Ningnanmycin, and lead compound XCLSBM. The excellent antiviral protective
activity of D21 was attributed to potential activator for plant resistance induction,
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