riboflavin-induced priming for pathogen defense in arabidopsis thaliana

Journal of Integrative Plant Biology 2009, 51 (2): 167–174

Riboflavin-induced Priming for Pathogen Defense inArabidopsis thaliana

Shujian Zhang†, Xue Yang†, Maowu Sun, Feng Sun, Sheng Deng and Hansong Dong∗

(Plant Growth and Defense Signaling Laboratory, Group of Key Laboratory of Monitoring and Management of Plant Pathogens and Insect Pests,

Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China)

Abstract

Riboflavin (vitamin B2) participates in a variety of redox processes that affect plant defense responses. Previously wehave shown that riboflavin induces pathogen resistance in the absence of hypersensitive cell death (HCD) in plants.Herein, we report that riboflavin induces priming of defense responses in Arabidopsis thaliana toward infection by virulentPseudomonas syringae pv. tomato DC3000 (Pst). Induced resistance was mechanistically connected with the expressionof defense response genes and cellular defense events, including H2O2 burst, HCD, and callose deposition in the plant.Riboflavin treatment and inoculation of plants with Pst were neither active but both synergized to induce defense responses.The priming process needed NPR1 (essential regulator of systemic acquired resistance) and maintenance of H2O2 burst butwas independent of salicylic acid, jasmonic acid, ethylene, and abscisic acid. Our results suggest that the role of riboflavinin priming defenses is subject to a signaling process distinct from the known pathways of hormone signal transduction.

Key words: callose deposition; defense responses; hypersensitive cell death; hydrogen peroxide; priming; riboflavin.

Zhang S, Yang X, Sun M, Sun F, Deng S, Dong H (2009). Riboflavin-induced priming for pathogen defense in Arabidopsis thaliana. J. Integr. PlantBiol. 51(2), 167–174.

Available online at www.jipb.net

Riboflavin (vitamin B2) biosynthetic and functional pathwaysaffect plant growth, development, and defense responses bymultiple mechanisms. Riboflavin is involved in anti-oxidation(Upreti et al. 1991; Packer et al. 1996) and peroxidation (Zubay1998), which both affect the production of reactive oxygenspecies (ROS) during oxidative burst. H2O2 is an importantform of ROS and is essential to the induction of hypersensitivecell death (HCD) and pathogen defense (Levine et al. 1994;Lamb and Dixon 1997; Alvarez et al. 1998). Levels of riboflavinin plants are believed to be important to these processes (Dong

Received 3 Dec. 2007 Accepted 12 Jun. 2008

Supported by the National Science Fund for Distinguished Young Schol-

ars (30525088), the State Key Basic Research and Development Plan of

China (2006CB101902), the National Natural Science Foundation of China

(30771441), and the Hi-Tech Research and Development Program of China

(2006AA10Z430).

†These authors contributed equally to this work.∗Author for correspondence.

Tel: +86 25 8439 9006;

Fax: +86 25 8439 5325;

E-mail: <[email protected]>.

C© 2008 Institute of Botany, the Chinese Academy of Sciences

doi: 10.1111/j.1744-7909.2008.00763.x

and Beer 2000). Exogenous application of riboflavin promotesplant growth (Peng et al. 2002) and induces resistance to fungal,bacterial, and viral pathogens (Aver’yanov et al. 2000; Dong andBeer 2000; Taheri and Hofte 2006). Plant resistance can also beinduced when riboflavin is applied as an ingredient of sprayingmixture (Wang and Tzeng 1998).

Some mechanistic connections have been established be-tween stimulation of plants by riboflavin treatment and severalknown signaling pathways. The ethylene signaling pathway isactivated to facilitate plant growth (Peng et al. 2002). NPR1(nonexpressor of PR genes1), essential regulator of systemicacquired resistance (SAR) (Cao et al. 1994; Cao et al. 1997;Dong 1998), and undefined protein kinases are recruited (Dongand Beer 2000) during the induction of pathogen resistanceby riboflavin. Salicylic acid (SA), which usually mediates theSAR pathway (Delaney et al. 1994; Ryals et al. 1996), is notrequired (Dong and Beer 2000). Pathogen defenses are oftenassociated with HCD, which, however, is not necessary todefense responses induced by riboflavin (Dong and Beer 2000).Moreover, riboflavin does not directly affect pathogens, butinstead, it induces defensive responses in plants (Aver’yanovet al. 2000; Dong and Beer 2000; Taheri and Hofte 2006).It is supposed that riboflavin-induced defense has functionallinkages with particular signaling pathways in plants (Dong andBeer 2000). A possible intermediate tache may be priming of

168 Journal of Integrative Plant Biology Vol. 51 No. 2 2009

plant defensive mechanisms towards infection by pathogens(Conrath et al. 2002, 2006; Kohler et al. 2002).

In many plants, defense priming can be induced by biotic andabiotic factors to increase capacity of resistance to pathogens(Conrath et al. 2002, 2006; Beckers and Conrath 2007). In-ducers of defense priming tested thus far include natural andsynthetic compounds, such as β-aminobutyric acid (Zimmerliet al. 2000; Ton and Mauch-Mani 2004; Hamiduzzaman et al.2005; Ton et al. 2005), vitamin B1 (Ahn et al. 2007) and SAor its structural analogs (Ryals et al. 1996; Sticher et al. 1997;Dempsey et al. 1999; Kohler et al. 2002). In general, molecularmechanisms that underlie the priming process is instrumental inimproving plants’ ability to perceive stress stimuli more rapidly,thereby coping with different forms of stress more efficiently(Conrath et al. 2002). Under priming conditions, specific molec-ular and cellular defense responses are activated quicker andgreater than those during regular infection (Ton et al. 2005;Ahn et al. 2007). An important response is the expression ofdefense response genes, like those encoding pathogenesis-related protein (PR) and phenylalanine ammonia-lyase (PAL)(Friedrich et al. 1996; Guo et al. 1998; Reuber et al. 1998). H2O2

burst (Levine et al. 1994; Lamb and Dixon 1997; Alvarez et al.1998), HCD (Peng et al. 2003; Ahn et al. 2007), and callosedeposition (Kohler et al. 2002; Ton and Mauch-Mani 2004;Hamiduzzaman et al. 2005) are often induced coordinatelyduring the priming process. Hence, priming is an importantmechanism of induced resistance in plants (Pieterse et al. 2006;Beckers and Conrath 2007). It is unclear whether these primedevents contribute to plant resistance induced by riboflavin.

This study is aimed at the mechanistic connection betweenriboflavin-induced priming and pathogen resistance in Arabidop-sis. We describe that riboflavin induces priming of molecular andcellular defense responses to cope with infection by the bacterialpathogen Pseudomonas syringae pv. tomato DC3000 (Pst). Wealso show evidence that riboflavin-induced priming is dependenton H2O2 burst and NPR1 but is not affected by several knownpathways of hormone signal transduction.

Results

Riboflavin and Pst synergize to induce priming of cellularand molecular defense responses

Previously we have shown that riboflavin induces plant resis-tance to Pst (Dong and Beer 2000) but omitted the modeof riboflavin action. To study whether riboflavin plays a rolein priming of defense responses, we tested H2O2 burst, cal-lose deposition, and HCD in plants pretreated with riboflavinor water and then maintained either no inoculation, or wereinoculated with Pst at 5 d post-treatment (dpt). H2O2 burst,callose deposition, and HCD were determined at 6, 12, and12 h postinoculation (hpi), respectively, when the responses

become evident based on previous studies (Peng et al. 2003;Ahn et al. 2007). The three responses were induced only whenriboflavin treatment and subsequent inoculation with Pst wereapplied to plants (Figure 1A). H2O2 burst, callose deposition,and HCD were not evident in plants treated with only riboflavinand in water-treated plants with and without Pst inoculation. Incontrast, conspicuous H2O2 burst, callose deposition, and HCDwere observed in plants pretreated with riboflavin and inoculatedwith Pst. In these plants, the extents of H2O2 burst and HCDwere significantly greater (ANOVA tests, P < 0.01) than thosein plants treated with only riboflavin and those in water-treatedplants with and without inoculation (Figure 1B).

PR and PAL gene transcripts are molecular indicators for theactivation of plant defensive pathways (Friedrich et al. 1996).Our previous study showed that PR-1 and PR-2 were expressedin Arabidopsis plants treated with riboflavin (Dong and Beer2000). Here, it was found that the expression of PR-1, PR-2,and PAL1 in leaves of plants observed 6 hpi is a part of defensepriming (Figure 1C). Transcription of the three genes was notevident in water-treated plants inoculated with Pst or thosethat were not inoculated. Conversely, profuse amounts of thethree transcripts were observed in riboflavin-pretreated and Pst-inoculated plants. In these plants, transcript levels were muchgreater compared with those in plants treated with riboflavin butnot inoculated. Clearly, riboflavin treatment primes Arabidopsisplants for augmented induction of defense responses whenchallenged by the pathogen.

Catalase nullifies priming induced by riboflavin

We used catalase in a pharmacological study of the role ofH2O2 in riboflavin-induced priming of plant defense responses.Catalase acts to scavenge H2O2 but does not inhibit pathogenproliferation (van Wees and Glazebrook 2003). We confirmedthat catalase applied alone to plants did not induce any evidentresponses, whereas, riboflavin-induced priming for defenseresponses was arrested by the enzyme present in Pst inoculum(Figure 2). When catalase was present in Pst suspension, leafsymptoms became severe (Figure 2A, top) and bacterial num-bers increased (Figure 2B) relative to those in plants treated withonly riboflavin. The enzymes also caused great compromiseson combinative effects of riboflavin and Pst in eliciting H2O2

burst, callose deposition, and HCD (Figure 2A, lower threephoto panels). Induced expression of the defense responsegenes were also depressed by the application of catalase(Figure 2C). Therefore, riboflavin treatment requires H2O2 burstas an essential signal to prime the defense responses.

Riboflavin-induced priming is dependent on NPR1 butindependent of the basal defense signals

To reveal possible crosstalk between riboflavin-primed de-fenses and basal defense pathways of SA, jasmonic acid (JA)

Riboflavin Signaling in Pathogen Defense 169

Figure 1. Combinative effects of riboflavin and Pseudomonas syringae

pv. tomato (Pst) on defense priming in Arabidopsis.

The defense responses were tested in accordance with treatments

noted in the lower caption panels. Arabidopsis plants were sprayed with

a riboflavin solution (Rib+) or water (Rib−). Plants were incubated 5 d

post-treatment (dpt) with Pst (Pst+) or remained free from the pathogen

(Pst−).

and ethylene signal transduction (Dong 1998; Dangl and Jones2001), Arabidopsis mutants that have defects in basal defenseswere compared with wild-type (WT) plants in the induction ofdefense priming responses. H2O2 burst differed in the plantgenotypes (Figure 3A). NahG (a transgenic plant that doesnot accumulate SA), etr1-1 (a mutant insensitive to ethylene),and jar1-1 (a mutant insensitive to JA) were similar to WT. Inthese plants, H2O2 burst was induced evidently subsequent toriboflavin treatment followed by Pst inoculation in comparisonwith only riboflavin treatment. In contrast, the induction of H2O2

burst was eliminated in npr1-1, which carries a deficient NPR1gene (Cao et al. 1994; Cao et al. 1997). In assays with thesame set of plants, the induction of HCD and callose depositionwas impaired in npr1-1 but not in the other three genotypescompared with WT (Figure 3B). Despite pretreatment with wateror riboflavin, leaf symptoms were more severe (Figure 3C) andin planta Pst cell numbers were greater (Figure 3D) in npr1-1plants than in other genotypes. These genotypes, in contrastto npr1-1, had fewer symptoms and Pst cell numbers, whichare functions of riboflavin versus water. Differences in bacterialpopulation were significant (ANOVA tests, P < 0.01) betweennpr1-1 and other genotypes following treatment with riboflavinand inoculation with Pst (Figure 3D). Thus, a functional NPR1 isrequired for riboflavin to induce priming of defense responses.

ABA signaling does not affect riboflavin-induced primingof pathogen defense

To gain more information about the relationship betweenriboflavin-primed defense and hormone signaling pathways, wetested the effects of ABA signaling on the function of riboflavin.In Arabidopsis, transcription factor ABI4 acts to repress ABA-dependent gene expression during oxidative stress (Staneloniet al. 2007), which is affected by riboflavin (Zubay 1998). Wechose to assay the Arabidopsis ABA-insensitive mutant abi4-1 since it dominates other abi mutants in blocking pathogenresistance in plants (Pourtau et al. 2004; Ton and Mauch-Mani2004; Kaliff et al. 2007). We found that abi4-1 did not affectriboflavin-induced priming of Arabidopsis defense responses

←

(A) Microscopic observations on H2O2 burst, callose deposition, and

cell death in leaves of plants 6, 12 and 12 h postinoculation (hpi). Scale

bars, 300 μm.

(B) Quantification of H2O2 and cell death in plants from (A), tested at

the same times. Bars across histogram tops refer to standard deviation

(SD).

(C) Expression of representative defense response genes. RNA was

isolated at 6 hpi from the untreated top two leaves of plants. Reverse

transcription-polymerase chain reaction (RT-PCR) was carried out using

EF1α as a standard.


Figure 2. Inhibitory effects of catalase on riboflavin-induced priming of

Arabidopsis defense responses.

Plant treatments (inoculation) are shown at the bottoms.

(A) Leaf symptoms and the defensive responses. Leaf symptoms were

observed 3 d postinoculation (dpi). H2O2 burst, callose deposition, and

cell death were determined at 6, 12 and 12 h postinoculation (hpi),

respectively. Scale bars, 300 μm.

(B) In planta Pst population. Logarithmic numbers of bacteria recovered

from leaves of plants 3 dpi were quantified versus fresh weight (FW) of

leaves and are shown as mean ± SD (n = 3 repeats).

(C) Expression of defense response genes in untreated leaves of plants

tested 6 hpi.

toward infection by Pst. As shown in Figure 3A, cellular defenseresponses evaluated were all induced in abi4-1, similar to WTwhen riboflavin was applied before inoculation with Pst. Leafsymptoms were alleviated (Figure 3B) and in planta bacterialpopulation (Figure 3C) was decreased at close extents in abi4-1 and WT plants pretreated with riboflavin. Consistent with thedispensability of ABI1 and ABI2 in pathogen defense elicited bya biotic elicitor (Dong et al. 2005), neither abi1-1 nor abi2-1 wereinvolved in the riboflavin action (data not shown). Overall, theseresults suggest that ABA signaling is not engaged in riboflavin-induced priming.

Discussion

We have studied defense priming by riboflavin in Arabidopsis.The molecular basis for priming was characterized. Riboflavininduces plant resistance to Pst, while Pst grows well in thepresence of riboflavin (Dong and Beer 2000). Enhanced diseaseperturbation without a direct effect on the causal pathogen

Figure 3. Comparison of Arabidopsis wild type (WT) and abscisic acid

(ABA)-insensitive abi4-1 mutant in riboflavin-induced defense priming.

Experiments were similar to those in Figure 1.

(A) Assays of H2O2 burst (top two photo panels), cell death (middle two

photo panels), and callose deposition (lower two panels). Scale bars,

300 μm.

(B) Leaf symptoms at 3 d postinoculation (dpi).

(C) Pst population in leaves. Histograms represent means ± SD bars

(n = 3 repeats).

confirms the alternative role of riboflavin as an inducer of plantdefense priming. Systemic expression of defense responsegenes as an indicator of activated SAR (Uknes et al. 1992,1993) was observed in the leaves of riboflavin-treated plants(Dong and Beer 2000; the present study). The gene expres-sion in riboflavin-treated plants was augmented subsequent toplant inoculation with Pst. Augmented resistance provides clearevidence that riboflavin treatment primes Arabidopsis defense.

We have established a mechanistic connection betweenriboflavin-primed defenses with plant cellular responses. Neitherriboflavin treatment nor inoculation of plants with Pst wereactive, but both synergized to trigger H2O2 burst, HCD, andcallose deposition (Figure 1A,B). HCD and callose depositionfrequently occurred as a consequence of ROS burst (Levineet al. 1994) and the three cytological events are recognized


as typical responses to pathogen infection in primed plants.Our findings indicate that riboflavin-primed Arabidopsis in asurveillance state was extremely sensitive to pathogen infection.Riboflavin and pathogen challenge were required for H2O2

production (Figure 1A), and rapid H2O2 production is thusconsidered as one of the mechanisms of defense primingby riboflavin. Catalase scavenges H2O2 but did not inhibitpathogen proliferation (van Wees and Glazebrook 2003). Cata-lase present in Pst inoculum eliminated a significant portion ofH2O2 accumulation as combinative effects of Pst and riboflavin.The inhibitory role of catalase accompanied abolition of diseaseresistance, callose deposition, HCD, and expression of defense

Figure 4. Comparison of the specific genotypes of Arabidopsis in riboflavin-induced defense priming.

The plant mutants NahG and npr1 (defect at salicylic acid (SA) signaling), etr1 (defective in ethylene perception), and jar1 (insensitive to jasmonic

acid (JA)) were compared with wild type (WT) for the defensive responses. Experiments were similar as in Figure 1. Scale bars, 300 μm in (A) and

(B). In (C), histograms represent means ± SD bars (n = 3 repeats).

(A) Assays of H2O2 burst.

(B) Assays of cell death and callose deposition.

(C) Differences in leaf symptoms.

(D) Pst population in leaves.

response genes (Figure 2A–C). These observations suggestthat H2O2 is critical for riboflavin-induced defense priming.

Defense priming by riboflavin seems distinct from severalknown pathways of hormone signal transduction but is de-pendent on NPR1 (Figures 3,4). Primed defense responses incytology were absent in npr1-1 (Figure 4A), which carries adeficient NPR1 (Cao et al. 1994; Delaney et al. 1995; Cao et al.1997). The NPR1-regulated SAR pathway needs SA signaling(Ryals et al. 1996; Dong 1998), which, however, did not affectpriming (Figure 4). The roles of signaling by JA and ethylenewere ruled out similarly (Figure 4). ABA signaling is also notlikely to play a role in the priming process based on the behavior


of abi4-1 (Figure 3). However, abi4-1 dominates over other abimutants to block plant defenses towards infection by pathogensand environmental cues (Pourtau et al. 2004; Ton and Mauch-Mani 2004; Jakab et al. 2005; Kaliff et al. 2007). These resultssupport the notion that modes of priming are largely influencedby the types of stimuli (Ahn et al. 2007).

In this study, we have demonstrated that priming and itsassociated cellular and molecular defense mechanisms wereinduced by riboflavin. Figure 5 depicts a working model of theprocess. Schematic linkages between the events are mech-anistic. The physiological connection remains to be studied.Protein kinases have been implicated in riboflavin-induced plantresistance to pathogens (Dong and Beer 2000) and whetherthey donate to priming also requires further study. Moreover,whether riboflavin-mediated redox is related to redox statusfor NPR1 activation and whether riboflavin signaling uses thescheme similar to that used by NPR1 to regulate PR genetranscription (Mou et al. 2003) are of great interest.

Figure 5. Model of plant defense priming by riboflavin.

Only the components that are relevant to this study are shown. Arrows

point to event fluids. Bars indicate inhibition. The two question marks

indicate unknown factors that are supposed to act at those places.

Materials and Methods

Plants and pathogens

The Arabidopsis genotypes tested were the ecotype Columbia(Col-0), npr1-1, jar1-1, etr1-1 and abi4-1 mutants (seedstock nos.: CS1092, CS3726, CS8072, CS237, CS8104 athttp://www.arabidopsis.org) as well as transgenic plant NahG.NahG seeds were a gift from Dr Zuhua He. Seeds were chilledat 4 ◦C for 5 d and sown in 60-mL pots containing a mixture of

sand and potting soil. Plants were incubated in chambers witha 12:12 h light : dark cycle (light 200 μE/m2 per s at 24 ◦C)(night, 20 ◦C) for 40 d before use. Pseudomonas syringae pv.tomato DC3000 (Pst) without any foreign avirulence genes waslyophilized, stored at −80 ◦C, and cultured on Luria Bertani Agarmedium (Gerhardt et al. 1981) before inoculation.

Plant treatment and resistance scoring

Plants were sprayed with water (CK) or 0.5 mmol/L riboflavin(EMD Biosciences, Darmstadt, Germany) in the presence ofthe surfactant Silwet L-77 (0.03%). Plants were inoculated 5 dptby spraying plant tops with a Pst suspension (1 × 108 cfu/mL).For use of catalase (Sigma-Aldrich Chemical Co., St. Louis,MO, USA), a Pst suspension (5 × 106 cfu/mL) with and withoutcatalase (5 000 units/mL) was infiltrated into leaf intercellularspaces of the top two expanded leaves of a plant. The methodwas chosen to allow efficient function of catalase in intercellularleaf spaces. Symptoms caused by Pst on leaves were observedand bacterial population in leaves were determined 3 dpi usinga previous method (Dong et al. 1999). Induced resistance inriboflavin-treated plants was defined based on decreases insymptom severity and in planta bacterial numbers relative tothose in CK plants.

Gene expression analysis

Reverse transcription-polymerase chain reaction (RT-PCR) wascarried out using the kit RT-PCR Beads as per the manufac-turer’s protocol (Amersham Pharmacia Biotech, Piscataway,NJ, USA). RNA was isolated from leaves as described (Clark1997; Dong and Beer 2000). The EF1α gene that is highly con-served and constitutively expressed in eukaryotes (Berberichet al. 1995; Gallie et al. 1998) was used as a loading control.Each gene was amplified for 25–30 cycles. PR-1 and PR-2gene specific primers were the same as used in a previ-ous study (Peng et al. 2003). Primers specific for the PAL1gene were as follows: 5′-GGAGTGGACGCTATGTTTG-3′ and5′-CATTGACGCCATTCCAGATC-3′. Sequences of RT-PCRproducts were confirmed by sequencing and comparison usingthe Blast program (Liu et al. 2006). RT-PCR products wereresolved by electrophoresis with agarose gel and then capturedwith Bio-Rad Molecular Imager Gel Doc XR System (BIO-RADLaboratories-Segrates, Milan, Italy) after staining with ethidiumbromide.

Studies of cellular defense responses

The concentration of H2O2 in leaves was determined at 6 hpiwith Pst 5 d after water or riboflavin treatment by monitoring theA415 of the titanium-peroxide complex (Jiang and Zhang 2001).H2O2 accumulation was visualized by microscopic observa-tions on detached leaves stained with 3,3′-diaminobenzidinetetrachloride (DAB) (Amresco, Solon, OH, USA) as previouslydescribed (Rea et al. 2004). Leaves were immersed with


1 mg/mL solution of DAB (pH 3.8) for 8 h under light at 25 ◦Cand decolorized by immersion of leaves in ethanol (96%) for8–12 h.

Hypersensitive cell death that had occurred in the form ofmicro-HR was observed by microscopy of leaves stained withtrypan blue (Peng et al. 2003). Levels of cell death weredetermined based on Evan’s blue uptake by dead cells (Andiet al. 2001). Micro-HR was monitored at 12 hpi with Pst bymicroscopic observation of the treated leaves after staining withtrypan blue, a protocol that can visualize cell death that hasoccurred at low frequency and is undetectable by eye (Penget al. 2003). Levels of cell death that had occurred in leaf tissueswere estimated at 12 hpi by Evan’s blue uptake assay (Andiet al. 2001). In the assay, leaf discs were incubated for 20 minwith 0.05% Evan’s blue and washed extensively with highlypure water to remove unbound dye. Dye bound to dead cellswas solubilized by incubating for 45 min at 50 ◦C in an aqueoussolution of 50% methanol and 1% sodium dodecyl sulfate (SDS).The absorbance of the solubilized dye was determined byspectrophotometry at 600 nm.

Callose deposition in plants was visualized at 12 hpi withPst by staining with aniline blue (Reuber et al. 1998). The toptwo leaves were infiltrated with 5 mL of a solution made ofphenol, glycerol, lactic acid, water, and 95% ethanol (1:1:1:1:2,v/v). Leaves in solution were incubated in a 65 ◦C bath untilthey were judged clear and then stained with aniline blue. Thestaining reaction was held in the dark for 4 h for leaves. Sampleswere observed by microscopy under ultraviolet light.

Data treatment

All experiments were carried out in three replicates with sim-ilar results; each replicate contained 10 plants unless other-wise noted. Quantitative data were subjected to ANOVA tests(P < 0.05 and P < 0.01) to evaluate significance in differencesbetween CK and other individuals of treatments.

Acknowledgements

We thank Dr Zuhua He (Shanghai Institutes of Biological Sci-ences, the Chinese Academy of Sciences, Shanghai, China) forthe donation of NahG Arabidopsis seeds.

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