Quercetin induces pathogen resistance through the increase of salicylic acid biosynthesis in Arabidopsis

ABSTRACT Quercetin is a flavonol belonging to the flavonoid group of polyphenols. Quercetin is reported to have a variety of biological functions, including antioxidant, pigment, auxin transport inhibitor and root nodulation factor. Additionally, quercetin is known to be involved in bacterial pathogen resistance in Arabidopsis through the transcriptional increase of pathogenesis-related (PR) genes. However, the molecular mechanisms underlying how quercetin promotes pathogen resistance remain elusive. In this study, we showed that the transcriptional increases of PR genes were achieved by the monomerization and nuclear translocation of nonexpressor of pathogenesis-related proteins 1 (NPR1). Interestingly, salicylic acid (SA) was approximately 2-fold accumulated by the treatment with quercetin. Furthermore, we showed that the increase of SA biosynthesis by quercetin was induced by the transcriptional increases of typical SA biosynthesis-related genes. In conclusion, this study strongly suggests that quercetin induces bacterial pathogen resistance through the increase of SA biosynthesis in Arabidopsis.


Introduction
][10] Recent studies have reported that flavonoids such as naringenin, kaempferol, quercetin and rutin induce resistance to pathogens through the SA signaling pathway [11][12][13][14] .Notably, quercetin has also been found to participate in the defense response against pathogens such as Pseudomonas syringae and Pyricularia oryzae. 14,15Nevertheless, the molecular mechanisms to explain how quercetin induces pathogen resistance remain unclear.
7][18] The pathogens trigger the activation of SA production, which stimulates immune response.SA is produced by two distinct pathways, the isochorismate and the phenylalanine ammonialyase pathways. 17The key transcription factors, SARD1 and CBP60g, positively regulate the SA biosynthesis genes, including ICS1 and EDS5. 17,18SA promotes EDS1 and PAD4 expression, which initiates a positive feedback loop that further amplifies SA accumulation. 19,20PR1 functions as a major regulator in the SA signaling pathway. 16,17,21NPR1 is involved in the transcriptional activations of PR genes that are the representative marker genes of the disease resistance against biotrophic pathogens in plants. 22,23NPR1 is mainly located in the cytoplasm as an oligomer form in the absence of SA.However, NPR1 is monomerized by the increase of SA level in response to the pathogen infection. 24,25The monomeric NPR1 is then translocated into nucleus from cytoplasm, where it interacts with TGA transcription factors that lead to the transcriptional activation of PR genes. 17,24,26n our study, we discovered that quercetin triggers the upregulation of PR genes by the monomerization and nuclear translocation of NPR1.Additionally, we observed that the nuclear translocation of NPR1 is achieved by the accumulation of SA.Conclusively, we suggested that quercetin induces pathogen resistance by the activation of the SA-dependent signaling pathway through the increase of SA biosynthesis.

Plant materials and growth conditions
The following Arabidopsis lines were used in the study: Columbia-0 (Col-0) and 35S:NPR1-GFP in npr1-2.The seeds were washed with 70% EtOH for 1 min, followed by 10 min in 1/ 10-diluted commercial bleach (0.4% NaOCl) and five washes with sterile distilled water.The seedlings were grown on Murashige-Skoog (MS) salts and vitamins 27 , 2.0% sucrose and 0.8% agar.The plates were incubated for 3 d at 4°C in the dark and then grown in a growth chamber (16 h light/8 h dark cycle, light intensity of ~120 μmol m −2 s −1 ) at 22°C for 14 d.

Bacterial growth curve assays
Pseudomonas syringae pv.tomato DC3000 was used in this study.For observation of disease symptoms, four-week-old plant leaves were pre-infiltrated with 0.5 µM flg22 or 100 µM quercetin using a 1 mL needless syringe.After two days, mock or pre-infiltrated plants were inoculated with bacterial suspension of 5 × 10 5 colony-forming units CFU/mL in 10 mM MgCl 2 using a 1 mL needless syringe.After three days, leaf discs (total area 0.5 cm 2 ) were collected by cork borer.The samples were ground in 10 mM MgCl 2 and plated in serial dilution on Pseudomonas Agar F (MB cell, Korea).A two-tailed Student's t-test was used for statistical analysis.

Total RNA extraction and qPCR analysis
For transcription level analysis, Col-0 plants were treated with 0.5 µM flg22 or 100 µM quercetin for 24 h.All samples were immediately placed in liquid nitrogen and stored at −80°C.Total RNA was extracted using an RNA purification kit (Macherey-Nagel, Germany).cDNA was synthesized from 5 μg total RNA using a RevertAid Reverse Transcriptase (Thermo Scientific, USA).The expressions of PR1, PR2, ICS1, EDS1, PAL1 and CBP60g were quantified by qPCR (CFX384 Real-Time System, Bio-Rad, USA) as previously described 29 .The tubulin was used as an internal control.The primers used for qPCR are shown in Supplementary Table S1.

Fluorescence microscopy
The fluorescence imaging of leaves was generated using the confocal laser-scanning microscope (Olympus FV1000).Plants were treated with 200 μM SA or 100 μM quercetin for 3 h.488 nm laser line was used to detect GFP.GFP fluorescence was taken through images from 500 to 539 nm at a gain setting of 556.

Measurement of salicylic acid
The quantification of SA levels was accomplished through high-performance liquid chromatography (HPLC) employing a reverse-phase C18 Gemini column for subsequent HPLC -ESI-MS/MS analysis, utilizing an API 4000 instrument (SCIEX).This methodology closely followed a previously established protocol. 30About 50 mg of powdered, frozen sample was extracted with 500 μl of extraction solvent 2propanol/H 2 O/concentrated HCl (2:1:0.002,v/v/v) containing d6-SA, an internal standard for SA, respectively 24 h at 4°C.Dichloromethane (1 mL) was added to the supernatant and then centrifuged at 13,000 g for 5 min at 4°C.The lower phase was dried under nitrogen and resolved in pure methanol.The fully dissolved extract was transferred to a liquid chromatography vial with a reduced volume, following thorough vortexing and sonication.

Statistical analysis
Statistical analysis in this study was conducted using Prism 9 (GraphPad, USA).Each experiment was carried out in triplicate with three sets of biological samples.One-way analysis of variation (ANOVA) with Tukey's post hoc test was performed for multi-sample experiments with one variable.Two-way ANOVA with Tukey's post hoc test was performed for multi-variable analyses.All results are expressed as mean ± SD.

The physiological level of quercetin is an inducer of bacterial pathogen resistance
2][13][14] To test pathogen resistance, excessive concentrations of quercetin are used in the previous report. 14The concentration of quercetin can be increased up to approximately 100 µM by pathogen infection or laser irradiation. 31,32To test whether the physiological level of quercetin can induce bacterial pathogen resistance, we examined the pathogen resistance against Pseudomonas syringae pv.tomato DC3000 (Pst DC3000) after treatment with 100 µM quercetin.As a result, we found that the proliferation of Pst DC3000 was inhibited by the pre-treatment of quercetin (Figure 1a).This result suggests that quercetin induces pathogens resistance at the physiological concentration.To test whether quercetin-mediated pathogen resistance at the physiological level of concentrations is related to the expression of PR genes, we analyzed the transcript levels of PR genes after treatment with 100 µM quercetin.Interestingly, the gene expressions of PR1 and PR2 were 35.6-fold and 16.2-fold upregulated by the treatment with quercetin, respectively (Figure 1b, c).Taken together, these results suggest that quercetin induces pathogen resistance by transcriptional upregulation of PR genes at the physiological level of concentrations.

The monomerization and nuclear translocation of NPR1 is induced by quercetin
The activation of NPR1 is due to the conformational change from oligomeric form to monomeric form, which is induced by the direct binding to SA. 24,33 The monomerization of NPR1 is involved in the transcriptional up-regulation of PR genes. 21To investigate whether quercetin induces the monomerization of NPR1, we examined the level of NPR1 monomer by immunoblot.As previously reported, the oligomeric form of NPR1 is highly detected before the treatment with quercetin.However, the monomeric form of NPR1 is accumulated by the treatment with quercetin (Figure 2a).This result suggests that NPR1 is monomerized by quercetin.
The nuclear translocation of NPR1 after the monomerization is required for the induction of PR genes. 24,34Therefore, we examined whether quercetin increases the nuclear translocation of NPR1 by confocal microscopy.As a result, the fluorescence of NPR1-GFP was mostly visible in the cytoplasm of guard cells before the treatment with quercetin, whereas the fluorescence was highly detected in the nucleus after the treatment with quercetin (Figure 2b).This result showed that NPR1 is translocated from the cytoplasm to the nucleus by quercetin.Taken together, we conclude that quercetin triggers the transcriptions of PR genes through the monomerization and nuclear translocation of NPR1.

Quercetin induces the biosynthesis of SA by increasing the transcription level of SA biosynthesis-related genes
2][13] Therefore, we hypothesized that quercetin up-regulates the biosynthesis of SA.To test this hypothesis, we first examined the transcription levels of SA biosynthesis-related genes after the treatment with quercetin by qRT-PCR.As a result, we found that four SA biosynthesis-related genes, ICS1, PAL1, EDS1 and CBP60g, are about 2.5-fold, 1.6-fold, 2.1-fold and 2.7-fold up-regulated by quercetin, respectively (Figure 3).This result strongly indicates that quercetin induces SA biosynthesis through the induction of SA biosynthesis-related genes.Therefore, we measured the level of SA by HPLC-MS/MS after the treatment with quercetin.As a result, we found that the level of SA is about 2-fold increased by quercetin (Figure 4).The increased level of SA by quercetin is similar to the level in the case of treatment with an avirulence pathogen, the Pst DC3000 avrRpt2.This result suggests that quercetin induces the biosynthesis of SA by the transcriptional increase of SA biosynthesis-related genes.

Quercetin can induce resistance to pathogens at low concentrations
Flavonoids are major secondary metabolites found in many fruits, vegetables, seeds, grains tea and wine. 1,813][14]35 However, in the previous study, the excess concentration of quercetin was applied to examine the bacterial pathogen resistance. 14In this study, we showed that the pathogen resistance was obtained by the treatment with the physiological concentration of quercetin (Figure 1).Previous reports have shown that the physiological level of naringenin and kaempferol induced pathogen resistance against bacteria. 11,12This result suggests that the physiological concentration of quercetin is sufficient to induce pathogen resistance.[14]35,36 The synergistic effects of the different combinations of flavonoids, or the combinations of flavonoids with other defense compounds would be studied.Furthermore, it should be studied how flavonoid is recognized and transduced for disease resistance.

Quercetin induces resistance to pathogens through the accumulation of SA
SA is known to be accumulated by pathogen infection and stimulates plant immunity to the bacterial pathogen in local and systemic tissues. 37,38Previously, quercetin was able to activate pathogen resistance through ROS accumulation and induction of PAL1 gene. 14In this study, we showed that quercetin increased  the accumulation of SA by the transcriptional increase of SA biosynthesis-related genes (Figures 3 and 4).Similar results reported that SA biosynthesis was induced by naringenin, kaempferol, riboflavin and thiamine. 11,12,35,39These results suggest that flavonoid-induced pathogen resistance is achieved by the accumulation of SA.1][42][43] These results implicate that pathogen resistance can be commonly induced through the accumulation of SA in response to flavonoids in plants.Therefore, more detail experiments should be performed in different plants in near future.

Quercetin confers pathogen resistance through the activation of NPR1
The activation of NPR1 is known to be an essential process of SA-mediated immune responses. 38,44Since quercetin could not induce the pathogen resistance in npr1mutant, it was suggested that NPR1 is involved in quercetin-induced disease resistance. 14owever, the molecular mechanism to explain how quercetin activates NPR1 is not elucidated.In this study, we showed that quercetin activates NPR1 by the monomerization and nuclear translocation (Figure 2).Similarly, naringenin and kaempferol also induce the activation of NPR1. 11,12Meanwhile, because several kinases, such as PKS5 and SnRK2.8, have been associated with the phosphorylation of NPR1, 21,45,46 quercetin can be involved in the activations of these kinases.Therefore, the activities of these kinases by the treatment with quercetin should be tested in the future.
Based on this study, we proposed a working model to explain how quercetin induces pathogen resistance (Figure 5).In this model, we provide evidence that quercetin induces resistance to bacterial pathogens by the accumulation of SA.The accumulated SA by quercetin induces the expression of PR genes through the activation of NPR1.Taken together, we conclude that quercetin induces pathogen resistance through an SAdependent signaling pathway in Arabidopsis.These findings implicated that quercetin or similar flavonoids can be used to improve crop yield by increasing plant resistance against bacterial pathogens as natural compounds.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Quercetin induces an effective pathogen resistance against Pst DC3000.(a) the bacterial growth in Col-0 plants.Two days before the infiltration of Pst DC3000, four-week-old plants were infiltrated with 0.5 µM flg22 or 100 µM quercetin.Bars represent mean values (SD) of colony-forming units (CFU) per square centimeter from biological replicate samples derived from different plants.Different letters indicate significant differences (P < .001)tested by two-way ANOVA with Tukey's post hoc test.(b, c) qPCR analysis of PR1 and PR2 transcription levels in Col-0 plant treated with 0.5 µM flg22 or 100 µM quercetin for 24 h.The relative gene expression level was normalized to that of the tubulin internal control.Error bars indicate the SD of three biological replicates.Different letters indicate significant differences (P < .05)tested by one-way ANOVA with Tukey's post hoc test.

Figure 2 .
Figure 2. NPR1 is monomerized and translocated to the nucleus by quercetin.(a) NPR1 is monomerized by quercetin.Total proteins were extracted from two-week-old seedlings of 35S:NPR1-GFP/npr1-2 treated with 100 μM quercetin for 3 h and detected using anti-NPR1 antibodies.SDS-PAGE was performed with or without DTT in the sample buffer.(b) NPR1 is translocated to the nucleus by quercetin.35S:NPR1-GFP/npr1-2 were treated with 200 μM SA or 100 μM quercetin for 3 h.The fluorescence was observed by a confocal microscope.Scale bar = 20 μm.The experiment was repeated three times with similar results.

Figure 3 .
Figure 3. SA biosynthesis-related genes are up-regulated by quercetin.qPCR analysis ICS1, PAL1, EDS1 and CBP60g transcription levels in Col-0 plant treated with 0.5 µM flg22 or 100 µM quercetin for 24 h.The relative gene expression level was normalized to that of the tubulin internal control.Error bars represent the SD of three biological replicates.Different letters indicate significant differences (P <.05) tested by one-way ANOVA with Tukey's post hoc test.

Figure 4 .
Figure 4.The accumulation of SA is highly induced by quercetin.The level of SA in Pst DC3000-inoculated or 100 μM quercetin-treated Col-0 leaves at 24 hpi (hour post-infection) using HPLC-MS/MS.Error bars indicate the SD of three biological replicates.Different letters indicate significant differences (P <.001) tested by oneway ANOVA with Tukey's post hoc test.

Figure 5 .
Figure 5.The working model explains how quercetin induces pathogen resistance.Quercetin increases the biosynthesis of SA through the transcriptional increase of SA biosynthesis-related genes.The accumulation of SA by quercetin triggers the monomerization and nuclear translocation of NPR1, resulting in the induction of PR genes and pathogen resistance.