Silicon-Induced Changes in Antifungal Phenolic - Plant Physiology

12 Pages·2011·0.37 MB·English

Silicon-Induced Changes in Antifungal Phenolic Acids,
Flavonoids, and Key Phenylpropanoid Pathway Genes
during the Interaction between Miniature Roses and the
Biotrophic Pathogen Podosphaera pannosa1[W]
Radhakrishna Shetty*, Xavier Frette´, Birgit Jensen, Nandini Prasad Shetty, Jens Due Jensen,
Hans Jørgen Lyngs Jørgensen, Mari-Anne Newman, and Lars Porskjær Christensen
Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, DK–1871
Frederiksberg C, Denmark (R.S., B.J., N.P.S., J.D.J., H.J.L.J., M.-A.N.); Institute of Chemical Engineering,
Biotechnology, and Environmental Technology, Faculty of Engineering, University of Southern Denmark,
DK–5230 Odense M, Denmark (R.S., X.F., L.P.C.); and Plant Cell Biotechnology Department, Central Food
Technological Research Institute, Mysore 570 020, India (N.P.S.)
Application of 3.6 mM silicon (Si+) to the rose (Rosa hybrida) cultivar Smart increased the concentration of antimicrobial
phenolic acids and flavonoids in response to infection by rose powdery mildew (Podosphaera pannosa). Simultaneously, the
expression of genes coding for key enzymes in the phenylpropanoid pathway (phenylalanine ammonia lyase, cinnamyl
alcoholdehydrogenase,andchalconesynthase)wasup-regulated.Theincreaseinphenoliccompoundscorrelatedwitha46%
reduction in disease severity compared with inoculated leaves without Si application (Si2). Furthermore, Si application
withoutpathogeninoculationinducedgeneexpressionandprimedtheaccumulationofseveralphenolicscomparedwiththe
uninoculated Si2 control. Chlorogenic acid was the phenolic acid detected in the highest concentration, with an increase of
more than 80% in Si+ inoculated compared with Si2 uninoculated plants. Among the quantified flavonoids, rutin and
quercitrin were detected in the highest concentrations, and the rutin concentration increased more than 20-fold in Si+
inoculated compared with Si2 uninoculated plants. Both rutin and chlorogenic acid had antimicrobial effects on P. pannosa,
evidenced by reduced conidial germination and appressorium formation of the pathogen, both after spray application and
infiltration into leaves.The application ofrutinandchlorogenicacid reduced powderymildewseverity by40%to50%, and
observationofaneffectafterleafinfiltrationindicatedthatthesetwophenolicscanbetransportedtotheepidermalsurface.In
conclusion, we provide evidence that Si plays an active role in disease reduction in rose by inducing the production of
antifungal phenolic metabolites asa response topowderymildew infection.
Miniature potted roses (Rosa hybrida) have become 2005). However, environmental considerations have
anincreasinglypopularornamentalcropfortheflori- necessitated increasing restrictions on the use of pes-
culture industry (Pemberton et al., 2003). Powdery ticides;therefore,eco-friendlyproductionmethodsfor
mildew caused by Podosphaera pannosa is one of the plant disease suppression need to be developed. One
mostwidespreaddiseasesofpottedroses(Horst,1983; ofthemostpromisingmethodsistoincreasethelevel
Eken,2005),andthewhitecolonies,togetherwithleaf of silicon (Si) in the growth medium (or soil), as this
distortion, curling, and premature defoliation caused has been able to reduce the growth of a number of
by the pathogen, lead to poor marketing value (Eken, plant pathogens, such as Magnaporthe oryzae infecting
2005).Powderymildewistypicallymanagedthrough rice(Oryzasativa;Rodriguesetal., 2001,2004),Blume-
the use of synthetic fungicides (Horst, 1983; Eken, ria graminis f. sp. tritici infecting wheat (Triticum
aestivum; Be´langer et al., 2003; Re´mus-Borel et al.,
1This work was supported by Prydplantepakkens Projekt 2 2005), and Podosphaera fuliginea infecting cucumber
(DirektoratetforFødevareErhverv),theResearchSchoolforHorti- (Cucumis sativus; Fawe et al., 1998). Recently, we have
cultural Science, Department of Plant Biology and Biotechnology, shown that this effect is also seen in roses, since
FacultyofLifeSciences,UniversityofCopenhagen,Producentfore- application of 3.6 mM Si significantly reduced pow-
ningenforPrydplanter,DanskePrydplanter,andtheDanishInsti- dery mildew severity (Shettyet al., 2011).
tuteofAgriculturalSciences,DepartmentofHorticulture. Siisthesecondmostabundantelementinthecrust
*Correspondingauthor;[email protected].
oftheEarthandisregardedasasemiessentialnutrient
Theauthorresponsiblefordistributionofmaterialsintegraltothe
forplantgrowth(Epstein,1994).Siisreadilyabsorbed
findings presented in this article in accordance with the policy
by plant roots in the form of silicic acid [Si(OH) ; Ma
describedintheInstructionsforAuthors(www.plantphysiol.org)is: 4
and Yamaji, 2006]. The soluble silicic acid is trans-
RadhakrishnaShetty([email protected]).
[W]TheonlineversionofthisarticlecontainsWeb-onlydata. ported through the xylem to the vegetative tissues,
www.plantphysiol.org/cgi/doi/10.1104/pp.111.185215 concentrated through transpiration, polymerized as
2194 Plant Physiology(cid:1), December2011, Vol. 157, pp. 2194–2205,www.plantphysiol.org(cid:3) 2011AmericanSociety of Plant Biologists. AllRights Reserved.
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Silicon Induces Changes in Rose Polyphenols
amorphous Si, and deposited in intracellular and in- tomato (Solanum lycopersicum),andnoeffectwasseen
tercellular spaces (Ma and Takahashi, 2002). Wedem- for genes related to secondary metabolism (Fauteux
onstratedthatapplicationof3.6mMSiinroses,which et al., 2005; Chain et al., 2009; Ghareebet al., 2011).
isconsideredanonaccumulatorofSi,increasedleafSi Investigationsofmodernrosecultivarshaveshown
contentto14ppminthedrymattercomparedwithno that they are a rich source of polyphenols in both
application of Si (Si content of 3 ppm), and confocal leavesandpetals(Biolleyetal.,1994a,1994b;Helsper
microscopy showed that Si deposition mainly oc- et al., 2003). Our hypothesis is that secondary metabo-
curred in the apoplast, particularly in epidermal cell litesofthephenylpropanoidpathwaysuchasflavonoids
walls (Shetty et al., 2011). Earlier studies on Si have and phenolic acids are important defense com-
documented the ability of Si to alleviate abiotic and poundsinrosesagainstpowderymildew,actingeither
bioticstressbyactingasaphysicalbarriertoinfection as phytoanticipins or phytoalexins, and that appli-
andalsobyinducingactivedefensemechanisms(Ma, cation of Si in the form K SiO increases the produc-
2 3
2004; Fauteux et al., 2005). In accordance with this, tion of these phenolic compounds.
Shettyetal.(2011)foundthattheSi-inducedprotection
against P. pannosa in roses was accompanied by the
increased formation of papillae and fluorescent epi-
dermal cells (FEC) as well as the accumulation of RESULTS
callose and hydrogen peroxide, especially at the sites
Phenolic Acids and Flavonol Glycosides in Rose Leaves
of penetration and in FEC, which are believed to
representthe hypersensitive response. Several phenolic acids and flavonoids were identi-
Due to the threat of infection by pathogens, plants fied in extracts of rose leaves (Supplemental Fig. S1).
have evolved and developed a multitude of chemical TypicalHPLCchromatogramsat320and360nmofan
and structural barriers for their protection. Various aqueous methanol extract of the leaves are shown in
antimicrobial compounds, which are synthesized by Supplemental Figure S2 and Figure 1, respectively.
plantsafterinfection,havebeendiscovered(Osbourn, Based on the UV spectra, the compounds could be
1996). One group of compounds, phytoalexins, is grouped into (1) caffeic acid derivatives, with an
formed de novo after invasion, whereas others, phy- absorption band centered around 325 nm with a
toanticipins, are preformed compounds that may un- shoulder at around 300 nm, and (2) flavonol glyco-
dergo postinfection modifications in order to express sides,withl valuesbetween343to364nmand253
max
full toxicity (Barz et al., 1990). Secondary metabolites to 265 nm for peaks in bands I and II, respectively
of the phenylpropanoid pathway such as phenolic (Table I). The UVabsorptions of compounds 2 and 3
acids and flavonoids are well-known examples of clearly indicated that these compounds were deriva-
compounds that may be produced by plants as phy- tivesofcaffeicacid,whichwasalsoconfirmedbytheir
toanticipinsorphytoalexinsinordertofightinvading liquid chromatography-mass spectrometry (LC-MS)
microorganisms (Dixon and Paiva, 1995; Dixon et al., data.Compounds2and3hadapseudomolecularion
2002).Rapidandearlyaccumulationofphenoliccom- [M–H]– at mass-to-charge ratio (m/z) 353, compatible
poundsatinfectionsitesisacharacteristicofphenolic- with caffeoylquinic acids, and wereidentified as 3-O-
based defense responses. At the infection sites, the caffeoylquinic acid (neochlorogenic acid) and 5-O-
productionoftoxicphenoliccompoundsmayresultin caffeoylquinic acid (chlorogenic acid), respectively.
effective inhibition of the pathogen (de Ascensao and Compounds 4 to 14 showed typical UV spectra of
Dubery, 2003). Production of such metabolites may flavonol glycosides, which were also confirmed by
also be involved in the increased formation of FEC in theirLC-MSdata(TableI).Compounds4to12allgave
Si-treated roses after P. pannosa inoculation (Shetty an ion at m/z 301 corresponding to the aglycone
et al., 2011). Only a few secondary metabolites have quercetin.Compounds8and10showedapseudomo-
been implicated in Si-induced resistance against fun- lecular ion [M–H]– at m/z 463, clearly indicating that
gal diseases (i.e. flavonoid phytoalexins in cucumber thesecompoundsarequercetinhexosemolecules,and
aswellasditerpenoidphytoalexinsinrice;Faweetal., theywereidentifiedasquercetin-3-O-galactoside(hy-
1998; Rodrigues et al., 2004; Re´mus-Borel et al., 2005). peroside)andquercetin-3-O-glucoside(isoquercitrin),
Transcription analysis of genes encoding enzymes in respectively.Compounds4and9showed pseudomo-
the biosynthetic pathways of secondary metabolites, lecularions[M–H]–atm/z625and609,respectively,as
especially during the early stages of infection, could well as ions at m/z 463 and 301 corresponding to the
help explain the relationship between secondary me- lossoftwoGlcmoietiesincompound4andaRhaand
tabolites and Si-mediated resistance. Transcriptome Glcmoietyincompound9.Consequently,compounds
analysis in wheat and Arabidopsis (Arabidopsis thali- 4and9wereidentifiedasquercetin-3-O-gentiobioside
ana) showed that inoculation of both Si-treated and and quercetin-3-O-rutinoside (rutin), respectively
untreatedplantswithpowderymildewinducedalter- (TableI).Compound11wasidentifiedasquercetin-3-
ationsinexpressionlevelsofseveralhundredofgenes O-arabinoside (avicularin) based on its pseudomolec-
(Fauteux et al., 2005; Chain et al., 2009). On the other ular ion [M–H]– at m/z 433, which clearly indicated
hand,Siapplicationaloneonlyplayedalimitedrolein that it was a quercetin pentose.The pseudomolecular
thetranscriptomicchangesinwheat,Arabidopsis,and ion[M–H]–atm/z447forcompound12indicatedthat
Plant Physiol. Vol. 157, 2011 2195
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Shetty et al.
Figure 1. Typical HPLC-PDA chromatograms of aqueous 80% methanol extracts of leaves of rose at 360 nm for Si2
uninoculated(A),Si+uninoculated(B),Si2inoculated(C),andSi+inoculated(D).Compoundsareasfollows:1,unknown
phenolicacid;2,3-O-caffeoylquinicacid(neochlorogenicacid);3,5-O-caffeoylquinicacid(chlorogenicacid);4,quercetin-3-
O-gentiobioside; 5, quercetin diglycoside; 6, quercetin derivative; 7, quercetin pentoside; 8, quercetin-3-O-galactoside
(hyperoside); 9, quercetin-3-O-rutinoside (rutin); 10, quercetin-3-O-glucoside (isoquercitrin); 11, quercetin-3-O-arabinoside
(avicularin);12,quercetin-3-O-rhamnoside(quercitrin);13,kaempferol-3-O-pentoside;and14,kaempferol-3-O-rhamnoside
(afzelin).ThechromatographicconditionsandthevalidationoftheHPLCmethodaredescribedin“MaterialsandMethods.”
mAU,Milliabsorbanceunits.
it was a quercetin methyl-pentose; thus, it was iden- hai,exceptforanunknownphenolicacidat24and72
tified as quercetin-3-O-rhamnoside (quercitrin). hai. While the total concentration of total phenolic
Compounds 13 and 14 both gave an ion at m/z 285 acids, chlorogenic acid, and neochlorogenic acid in-
corresponding to the aglycone kaempferol. Com- creased at 24 to 120 hai in Si+ inoculated leaves, the
pound 14 showed a pseudomolecular ion [M–H] – at concentration of total phenolic acids and chlorogenic
m/z 431, clearly indicating that it was a kaempferol acidpeakedat24haiinSi2inoculatedleaves.Forthe
methyl-pentose;thus,itwasidentifiedaskaempferol- uninoculatedSi2andSi+leaves,theconcentrationsof
3-O-rhamnoside (afzelin). phenolic acids did not change over time, except for
chlorogenicacidandneochlorogenicacidat24and72
hai, respectively, with a higher level in Si+ uninocu-
Effects of Si Application and Inoculation on the lated than in Si2 uninoculated leaves. For all treat-
Concentration of Phenolic Acids
ments and time points, chlorogenic acid occurred at
The concentrations of all phenolic acids were unaf- the highest concentrations and made up more than
fected by Si treatment at 0 h after inoculation (hai). 80%ofthetotalphenolicacidsinmostcases(Supple-
However, at 24 to 120 hai, the concentrations of total mental Table S1).
phenolic acids,chlorogenic acid, neochlorogenic acid,
and an unknown phenolic acid increased in the inoc-
Effects of Si Application and Inoculation on the
ulated Si+ and Si2 compared with uninoculated Si2
Concentration of Flavonoids
and Si+ leaves (Fig. 2; Supplemental Table S1). Fur-
thermore, Si+ inoculated leaves had a higher level of The total concentrations ofallflavonoids weregen-
phenolicacidsthanSi2inoculatedleavesat24to120 erally unaffected by Si treatment at 0 hai (Fig. 3;
2196 Plant Physiol. Vol. 157, 2011
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Silicon Induces Changes in Rose Polyphenols
TableI. LC-PDA-MSanalysis(UVspectra,characteristicions,andmolecularmasses)ofphenolicacidsandflavonoidsinaqueous80%methanol
extractsofleavesofrose
Datarepresentresultsfromtheanalysisofsamplesfromtwoindependentexperiments,eachwiththreeindependentextractions.Compounds
listedhereweredetectedinallsamples.
Peak LC-MS(AtmosphericPressureChemical HPLC-PDA,UV
No.a Rtb Ionization,NegativeIonMode) Spectra,l Compound
max
min m/z(%basepeak) nm
1 10.5 347[M–H]–(56),301(52),139(100) 296sh,c323 Unknownphenolicacid
2 12.4 353[M–H]–(100),191(18),179(15) 302sh,329 3-O-Caffeoylquinicacid
(neochlorogenicacid)d
3 18.6 353[M–H]–(100),325(29),191(19) 298sh,325 5-O-Caffeoylquinicacid
(chlorogenicacid)d
4 25.6 625[M–H]–(100),463(28),301(9) 253,263sh,353 Quercetin-3-O-gentiobiosidee
5 31.3 609[M–H]–(100),447(21),301(8) 265,284sh,348 Quercetindiglycosidee,f
6 41.8 615[M–H]–(100),493(4),463(7),441(9),301(24) 262,291sh,352 Quercetinderivativef
7 42.9 433[M–H]–(7),301(100) 255,285sh,348sh,362 Quercetinpentosidef
8 44.7 463[M–H]–(100),301(6) 253,299sh,356sh,364 Quercetin-3-O-galactoside(hyperoside)d
9 47.2 609[M–H]–(100),463(67),301(19) 256,263sh,298sh,354 Quercetin-3-O-rutinoside(rutin)d
10 48.3 463[M–H]–(100),301(22) 256,263sh,299sh,354 Quercetin-3-O-glucoside(isoquercitrin)d
11 54.7 433[M–H]–(100),301(7) 256,263sh,301sh,352 Quercetin-3-O-arabinoside(avicularin)d
12 56.5 447[M–H]–(100),301(10) 256,262sh,305sh,348 Quercetin-3-O-rhamnoside(quercitrin)d
13 63.5 417[M–H]–(100),285(10) 264,294sh,331sh,345 Kaempferol-3-O-pentosidef
14 66.0 431[M–H]–(100),285(9) 263,296sh,324sh,343 Kaempferol-3-O-rhamnoside(afzelin)d
aPeak numbers correspond to the compound numbers in Figure 1 and Supplemental Figure S2. bR, Retention time on HPLC. csh,
t
Shoulder. dConclusivelyidentifiedbycomparisonwithauthenticstandard. eIdentificationbasedoncomparisonofretentiontime,UV,and
LC-MSdatawithdatafromtheliterature(Masadaetal.,2009). fTentativelyidentifiedbyUVandmassspectraldata.
Supplemental Table S2). However, at 24 to 120 hai, pared with Si2 uninoculated (Fig. 3F; Supplemental
significant changes in the concentrations of flavo- TableS2).
noids occurred, depending on treatment and time
point, except for a quercetin diglycoside (compound
5; Table I), where all treatments displayed a similar Disease Reduction Mediated by Si Application
low content at all time points (Supplemental Table
Assessment of disease on the remaining inoculated
S2). For the other detected flavonoids, four main
plants from the metabolite experiment showed that
patterns were observed. (1) From 24 to 120 hai, the
Si+ plants had a disease severity score of 48.1% com-
twoataslhciognhteerntthoafnflfaovroSnio2idisnofocurlSait+edinleoacvuelast,eidn lweahvicehs pared with 81.4% of Si2 plants (P , 0.001). The Si
application thus resulted in a 46% disease reduction
the concentration was higher than for both the unin-
(Supplemental Fig.S3).
oculatedtreatments.Thesamepatternwasseenfora
quercetin derivative (compound 6; Table I) at 24 hai
(SupplementalTableS2).(2)Forseveralofthedetected
Disease Reductions after Chlorogenic Acid and
flavonoids (e.g. avicularin and quercitrin), the inocu-
Rutin Treatment
lated leaves for both treatments had similar concen-
trations, especially at 24 and 72 hai, and these Treatmentofroseseitherbysprayapplicationorleaf
concentrations were higher than for the uninoculated infiltration with 1 mg mL21 chlorogenic acid or rutin
leaves. (3) In several cases, especially at 120 hai, the resulted in an overall reduction in powdery mildew
concentrationsinuninoculatedSi+andSi2leavesand severity (P , 0.001) compared with their respective
inoculated Si2 leaves did not differ, but the levels water-treatedcontrolsat9dafterinoculation(dai;Fig.
werelowerthanfortheinoculatedSi+leaves(e.g.for 4). The application of chlorogenic acid decreased the
hyperoside, rutin, and avicularin). (4) The levels diseaseseverityinbothsprayedandinfiltratedleaves
of quercetin derivative, avicularin, quercitrin, by 51%. Likewise, rutin also reduced the disease
and kaempferol-3-O-pentoside followed the order severity following both treatments, although at
Si+inoculated.Si2inoculated.Si+uninoculated. slightly lower levels (41% for spraying and 44% for
Si2 uninoculated. The concentration of total flavo- infiltration). Forbothtypes of treatments,chlorogenic
noidsandthe11quantifiedflavonoidsdidnotchange acid was moreeffectivethan rutin(P ,0.001).
over time (0–120 hai) for the uninoculated Si2 and Table II shows results from the quantitative bright-
Si+ leaves, except that the levels of avicularin and field and epifluorescence microscopy study of the
quercitrininSi+uninoculatedleaveswerehigherat24 interaction at 72 hai after spraying or infiltration
hai and that the quercetin derivatives, kaempferol- with chlorogenic acid, rutin, or water (control). The
3-O-pentosideandafzelin,werehigherat72haicom- different infection steps are calculated based on the
Plant Physiol. Vol. 157, 2011 2197
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Shetty et al.
Figure2. Contentsoftotalphenolicacids(A)and5-O-caffeoylquinicacid(chlorogenicacid;B)inleafextractsofrosefrom
plants either treated with (Si+) or without (Si2) Si followed by either inoculation with P. pannosa or no inoculation. Data
representresultsfromoneexperiment,andeachobservationrepresentsthemeanfromthreeextractions.Allvaluesarepresented
asmeans6SE.Meanswithineachtimepointarecomparable,andbarsmarkedbydifferentlettersaresignificantlydifferent.
FurtherinformationontheresultsfromthisexperimentisgiveninSupplementalTableS1.Thefindingsofthisexperimentwere
confirmedinasecondindependentexperiment.
number of germinated conidia. The percentages of DISCUSSION
germinated conidia (having a primary germ tube)
Active Role of Si in Disease Resistance of Rose:
and of conidia forming appressoria were reduced
Induction of the Production of Antifungal Phenolics
by both spray application and infiltration of the
against Powdery Mildew Infection
phenolicscomparedwiththewatercontrols.Onthe
otherhand,penetrationandformationofhaustoria This study provides evidence that root application
and elongating secondary hyphae (ESH) were not of3.6mMSi+totheminiaturerosecvSmartincreases
altered in plants treated with either phenolic com- the concentration of phenolic acids and flavonoids in
pound. None of the host responses examined (forma- responsetoP.pannosainfection,someofwhich,toour
tion of papillae and FEC) was affected by chlorogenic knowledge, have not been reported in roses before.
acidorrutinbyeitherapplicationmethod. This was accompanied by an increased expression of
genes encoding enzymes in the phenylpropanoid
pathway.ThiswasparticularlyprominentinSi+inoc-
Expression of Phenylpropanoid Pathway Genes
ulated plants, but there were also elevated transcript
The expression of genes encoding the key enzymes levelsinSi2inoculatedplants.Thus,accordingtothe
phenylalanineammonialyase(PAL),cinnamylalcohol definition of Ghareeb et al. (2011), most of the re-
dehydrogenase (CAD), and chalcone synthase (CHS) sponses observed representinduced resistance.How-
in the phenylpropanoid pathway were often affected ever,thecontentsofphenolicsandflavonoidsrepresent
both by Si application and powdery mildew inocula- priming,sinceSi+uninoculatedandSi2uninoculated
tion (Table III). Compared with Si2 uninoculated plants were not different. The level of potassium was
leaves, the transcript levels of PAL were elevated at different between the two nutrient solutions, due to
24 hai for the treatments Si2 inoculated, Si+ uninoc- the extra potassium present in SiKal compared with
ulated, and Si+ inoculated. However, at 72 hai, eleva- the control solution. It was only possible to partly
tion of PAL transcript was only found for Si+ compensate for the extra potassium present in the 3.6
inoculated plants, with a 39-fold increase followed mM Si treatment without affecting other nutrients.
byadecreasetoonlya3-foldup-regulationat120hai. Therefore, it cannot be ruled out that the extra potas-
For CHS, elevated transcript levels were seen for all sium potentially could have some influence on the
three treatments at 24 and 72 hai. In contrast, accu- level of disease, but this appears less important com-
mulation of CHS transcript was onlyseen for the two paredwiththeeffectofSi.Thus,therewasaveryclear
Si+ treatments at 120 hai. The transcription of CAD increaseinSicontentintheleaveswherethepathogen
followed a pattern differing markedly from the two was inhibited (Shetty et al., 2011). Furthermore, pre-
other genes. Thus, CAD only showed elevated tran- liminaryexperimentsshowedthatthecontentofother
script levels in the Si+ inoculated plants at 24 and 72 nutrientsinroseleaves,includingpotassium,wasnot
hai, while levels were elevated for all three treatment significantlydifferentbetweenplantsreceiving0or3.6
at 0 hai(i.e. immediately after inoculation). mM Si (data not shown).
2198 Plant Physiol. Vol. 157, 2011
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Silicon Induces Changes in Rose Polyphenols
Figure3. Contentsoftotalflavonoidsandselectedflavonoidsinleafextractsofrosefromplantseithertreatedwith(Si+)or
without (Si2) Si followed by either inoculation with P. pannosa or no inoculation. A, Total flavonoids. B, Quercetin-3-O-
galactoside (hyperoside). C, Quercetin-3-O-rutinoside (rutin). D, Quercetin-3-O-arabinoside (avicularin). E, Quercetin-3-O-
rhamnoside (quercitrin). F, Kaempferol-3-O-rhamnoside (afzelin). Data represent results from one experiment, and each
observationrepresentsthemeanfromthreeextractions.Allvaluesarepresentedasmeans6SE.Meanswithineachtimepointare
comparable, and bars marked by different letters are significantly different. Further information on the results from this
experiment is given in Supplemental Table S2. The findings of this experiment were confirmed in a second independent
experiment.
Plant Physiol. Vol. 157, 2011 2199
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Shetty et al.
Initial screening of methanol extracts by HPLC and
LC-MS from Si-treated rose leaves both with and
without powdery mildew inoculation revealed that
thecontentsofphenolicacidsandflavonolglycosides
wereclearlyaffected,whereasthecontentsofflavan-3-
ols (proanthocyanidins), which are known to play a
role in defense in some plants (Miranda et al., 2007;
Koskima¨kietal.,2009),werenotsignificantlyaffected.
Consequently, the focus in this investigation was on
the changes in the contents of phenolic acids and
flavonol glycosides. Chlorogenic acid, neochlorogenic
acid,andanunknownphenolicacidweredetectedin
roseleaves.Thetwoidentifiedphenolicacidsarewell-
known constituents in aerial parts of many plant
species (Christensen et al., 2008; Grevsen et al., 2008;
Schmitzer et al., 2009). However, neochlorogenic acid
has,tothebestofourknowledge,notpreviouslybeen
reported as a constituent in the aerial parts of roses.
Chlorogenicacidwasthephenolicacidpresentinthe
highest concentration, with an increase of more than
80%inSi+inoculatedcomparedwiththeSi2uninoc-
ulated leaves.
Figure4. Powderymildewseverityinleavesofroseaftersprayingor Flavonol glycosides like quercetin and kaempferol
leaf infiltration with chlorogenicacid and rutin. Controlplantswere are well-known constituents of rose species, and the
treatedwithwater.Diseaseseveritywasscored9dafterinoculation flavonoids identified in this investigation (Table I)
withP.pannosa.Datarepresentresultsfromoneexperiment,andeach
have all previously been detected in rose species
observation represents the mean from 22 leaves. All values are
(Biolleyetal.,1994a,1994b;Helsperetal.,2003;Kumar
presentedasmeans6SE.Meanswithineachapplicationmethodare
etal.,2009;Schmitzeretal.,2009),exceptforquercetin-
comparable, and bars marked with different letters are significantly
3-O-gentiobioside. Among the 11 quantified flavo-
different.Thefindingsofthisexperimentwereconfirmedinasecond
independentexperiment. noids, rutin and quercitrin occurred in the highest
concentrations, with rutin increasing more than 20-
fold in Si+ inoculated compared with Si2 uninocu-
All phenylpropanoids are derived from cinnamic lated plants (Fig.3C; Supplemental Table S2).
acid, which is formed from Phe by the action of PAL.
PAL is the branch-point enzyme between primary
Antimicrobial Activity of Major Phenolics in Rose
metabolism and the branch of secondary metabolism
leading to the phenylpropanoid pathway, which is The substantial increase in the contents of phenolic
consideredto beone of the most importantmetabolic acids and flavonoids in Si+ inoculated leaves corre-
pathwaysduetoitsresponsibilityforthesynthesisofa lated with a 46% reduction in disease severity com-
large range of secondary metabolites, including phe- pared with Si2 inoculated leaves (Supplemental Fig.
nolic acids and flavonoids (Dixon and Paiva, 1995). S3). In order to elucidate whether the identified phe-
CAD catalyzes the final step in a branch of phenyl- nolics could help explain the Si-mediated protection,
propanoid synthesis specific for the production of we tested the ability of chlorogenic acid and rutin to
lignin monomers, and an increased expression of this reduce powdery mildew development in roses, as
enzyme could indicate increased lignification (Walter these secondary metabolites were among the pheno-
et al., 1988), which,however, was not observed in the lics that were detected in the highest amounts in Si+
rose-P. pannosa interaction. A large number of stress- inoculatedleaves.Bothrutinandchlorogenicacidhad
induced phenylpropanoids are derived from the C an antimicrobial effect on P. pannosa when applied to
15
flavonoid skeleton, which is biosynthesized via CHS, leaves, reducing disease severity by 40% to 50% in
thekeyenzymeintheflavonoidbranchofthephenyl- planta. Interestingly, both spray application and leaf
propanoid pathway, catalyzing the production of infiltration gave comparable disease reductions (Fig.
tetrahydroxychalcone, the precursor of all flavonoids 4).Sincegerminationofconidiaaswellastheabilityof
(DixonandPaiva, 1995; Winkel-Shirley, 2001). conidiatoformappressoriawerereducedtothesame
Many plant phenolics can function as passive or extent by chlorogenic acid and rutin following both
inducible barriers against pathogens, and it is well applicationmethods,itappearsthatthesetwopheno-
knownthat,forexample,thecontentofflavonoidscan lics and perhapsother phenolics as well can betrans-
increase or the flavonoid composition can change in ported from the cell lumen to the epidermis to act as
response to pathogen attack. However, the involve- antimicrobialcompoundsagainstP.pannosa.Inaccor-
ment of flavonoids in plant defense depends on the dance with this, von Ro¨penack et al. (1998) also
species (Dixon and Paiva, 1995; Carlsen et al., 2008). suggested that the phenolic conjugate p-coumaroyl-
2200 Plant Physiol. Vol. 157, 2011
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Silicon Induces Changes in Rose Polyphenols
TableII. QuantitativerecordingsofinfectionbiologyofP.pannosaanddefenseresponsesinthefifthdevelopedleavesofrosecvSmart
Plants were treated with chlorogenic acid and rutin applied by spraying or leaf infiltration. Control plants were similarly treated with water.
Observationsweremadeat72hai,andvalueswerecalculatedonthebasisofthenumberofgerminatedconidia.Datarepresentresultsfromone
experiment,andeachobservationrepresentsthemeanfromthreeleaves.Allvaluesarepresentedasmeans6SE.Thefindingsofthisexperiment
wereconfirmedinasecondindependentexperiment.
Treatment OddsRatioa
Application
ChlorogenicAcid Rutin Control ChlorogenicAcid Rutin Control
Sprayingb
Germinated 15.360.33 14.760.33 25.360.33 0.53*** 0.51*** 1.00
Withappressoria 10.760.33 10.260.58 22.060.58 0.43*** 0.40*** 1.00
Withhaustoria 8.760.67 9.260.33 13.360.33 0.52NS 0.56NS 1.00
WithESH 8.760.67 9.260.33 13.360.33 0.52NS 0.56NS 1.00
WithFEC 8.060.00 8.160.00 7.960.67 1.00NS 1.02NS 1.00
Withpapillae 8.760.67 8.660.33 9.360.33 0.92NS 0.93NS 1.00
Infiltrationc
Germinated 15.360.33 16.760.33 24.760.33 0.55*** 0.60*** 1.00
Withappressoria 10.760.33 11.960.58 21.460.67 0.44*** 0.50*** 1.00
Withhaustoria 8.060.00 10.060.00 11.960.33 0.65NS 0.82NS 1.00
WithESH 8.060.00 10.060.00 11.960.33 0.65NS 0.82NS 1.00
WithFEC 9.360.33 10.760.33 9.460.33 0.99NS 1.15NS 1.00
Withpapillae 9.360.33 10.760.33 10.060.00 0.93NS 1.07NS 1.00
aOddsratioforcomparisonoftreatments(controlusedasareference;oddsratio=1.00).NS,Nonsignificantdifference;***significantatP,
0.001;*significantatP,0.05. bSprayedwithasolution(1mgmL21)ofchlorogenicacidorrutinuntilrunoff. cInfiltratedwithasolution(1
mgmL21)ofchlorogenicacidorrutin.
hydroxyagmatinewastransportedinvesiclesinbarley with this, application of the compounds reduced
(Hordeum vulgare) leaves to the sites of attempted prepenetration growth of P. pannosa but not the fre-
penetration by Blumeria graminis f. sp. hordei. An quencies of fungal developmental stages or host de-
antimicrobial effect of phenolics is also in accordance fense responsesafter penetration.
with the increased amounts of chlorogenic acid and
rutin as well as other phenolics observed in Si2
Expression of Key Phenylpropanoid Pathway Genes Is
inoculated compared with Si2 uninoculated rose
Altered by Si Application and by Powdery
leaves (Figs. 2 and 3; Supplemental Tables S1 and
Mildew Infection
S2). Phenolic acids and flavonol glycosides have also
been shown to play an important role in the defense Like the transcriptomic analysis of the wheat-B.
strategy of other plant species against pathogens. For graminisandtheArabidopsis-Golovinomycescichoracea-
example, in apple (Malus domestica) leaves and fruits rum pathosystems (Fauteux et al., 2005; Chain et al.,
infected with Venturia inaequalis, it has been shown 2009),wefoundanup-regulationofgenesinvolvedin
that the content of phenolic acids (e.g. chlorogenic secondary metabolism in the rose-P. pannosa patho-
acid), flavonol glycosides (e.g. rutin, quercitrin, and system. However, neither Chain et al. (2009) nor
isoquercitrin), and flavan-3-ols increased significantly Fauteux et al. (2005) were able to identify the key
in infected leaves compared with healthy tissues secondary metabolite genes or suggest a function of
(Petkovsˇek et al., 2008, 2009). Chlorogenic acid has secondary metabolite genes in their pathosystems.
alsobeenshowntoplayamajorroleinrelationtoscab Transcriptomic analysis of Si effects in wheat, Arabi-
resistance in potato (Solanum tuberosum) caused by dopsis,rice,andtomatoadditionallyindicatedthatSi
Streptomyces scabies (Johnson and Schaal, 1952), and hadalimitedroleonthetranscriptomeintheabsence
rutin and other flavonols showed significant antifun- of stress induced by pathogen inoculation (Watanabe
gal activity against the fungi Cylindrocarpon destruc- et al., 2004; Fauteux et al., 2005; Chain et al., 2009;
tans, Phytophthora megasperma, and Verticillium dahliae Ghareebetal.,2011).Incontrast,byusingquantitative
attacking olive trees (Olea europaea); therefore, rutin real-time reverse transcription (RT)-PCR, we demon-
andotherflavonolsarebelievedtoplayamajorrolein strated that the transcript levels for PAL, CAD, and
plantdefenseofoliveplants(Ba´idezetal.,2006,2007). CHS were often elevated by Si application compared
A characteristic for the most widespread phenolic withSi2uninoculatedplants(TableIII).Furthermore,
acids and flavonols is that they are not induced fol- in several cases, the Si+ uninoculated plants had an
lowing infection (i.e. they do not act as phytoalexins increased content of phenolics compared with the
but are considered phytoanticipins, which are pre- uninoculated controls, thus substantiating the gene
formed antifungal compounds, present in different expressionresultsthatSiprimarily inducesresistance
amounts, that may undergo postinfection increases in rose plants against powdery mildew. The discrep-
following infection; Harborne, 1999). In accordance ancy between our findings and those from the Arabi-
Plant Physiol. Vol. 157, 2011 2201
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Shetty et al.
TableIII. Quantitativereal-timeRT-PCRanalysisofPAL,CHS,andCADgeneexpressioninleavesofrosefromplantseithertreatedwith(Si+)
orwithout(Si2)SifollowedbyeitherinoculationwithP.pannosaornoinoculation
Valuesshownrepresentfoldup-ordown-regulationinSi2inoculated,Si+uninoculated,andSi+inoculatedplantsrelativetoSi2uninoculated
plants (relative expression ratio = 1) at each time point, after normalization of all treatments to 18S rRNA. Data represent results from one
experiment,andeachobservationrepresentsthemeanfromthreeextractions.Allvaluesarepresentedasmeans6SE.Thefindingsofthisexperiment
wereconfirmedinasecondindependentexperiment.*Significantchange;ns,nonsignificantchange.
FoldChange
Genes SiSupply Pathogen
0hai 24hai 72hai 120hai
PAL Si2 Uninoculated 1.0 1.0 1.0 1.0
Si2 Inoculated 21.760.17ns 3.761.25* 1.560.17ns 1.160.40ns
Si+ Uninoculated 21.660.14* 2.660.60* 1.260.27ns 21.060.25ns
Si+ Inoculated 21.460.11* 5.261.49* 39.3611.6* 3.060.58*
CHS Si2 Uninoculated 1.0 1.0 1.0 1.0
Si2 Inoculated 1.360.46ns 2.861.06* 1.560.30* 1.260.14ns
Si+ Uninoculated 21.460.24* 2.660.55* 2.660.30* 2.260.15*
Si+ Inoculated 21.060.32ns 4.560.45* 2.760.38* 2.160.36*
CAD Si2 Uninoculated 1.0 1.0 1.0 1.0
Si2 Inoculated 1.360.21* 1.660.59ns 21.860.04ns 21.260.23*
Si+ Uninoculated 2.160.40* 20.360.26ns 21.160.12ns 1.160.39ns
Si+ Inoculated 1.260.28* 2.060.17* 2.460.55* 2.560.40ns
dopsis and wheat transcriptomic analyses could re- starting unit in the biosynthesis of flavonoids (Dixon
flect differences in experimental design. Thus, in the andPaiva,1995;Winkel-Shirley,2001).Achangeinthe
investigations of Arabidopsis and wheat (Fauteux biosynthesis of flavonoids, therefore, may affect the
et al., 2005; Chain et al., 2009), leaves of different pool of phenolic acids and hence the content of phe-
physiological age were pooled for RNA extraction, nolic acids.
whereasweonlyanalyzedthe fifthdevelopedleaves. Theup-regulationofCHSat24to120haicompared
with0haiintheSi+inoculatedtreatmentisconsistent
PAL and CHS Gene Expression: Possible Correlations to with an increase in the amounts of most flavonol
glycosides in rose leaves at these time points (Fig. 3;
the Biosynthesis of Phenolic Acids and Flavonoids
Supplemental Table S2). However, in the Si2 inocu-
in Rose
lated rose leaves, there appeared to be a trend, al-
We found a clear correlation between the elevated thoughnotsignificant,toadecreaseintheamountsof
PAL and CHS transcript levels and an increased bio- flavonol glycosides at 24 to 120 hai (Fig. 3A; Supple-
synthesis of phenolics in rose leaves, especially for mentalTableS2),whichisalsoinaccordancewiththe
both the Si+ treatments. However, when comparing down-regulation of CHS observed at 24 to 120 hai
the up-regulation of PAL and CHS at specific time (Table III). Furthermore, the up-regulation of CHS in
points after inoculation (24, 72, and 120 hai) and the the Si+ uninoculated plants resulted in an increase in
amounts of phenolics produced, some discrepancies thecontentofindividualflavonoids,suchasavicularin
were revealed. In particular, the down-regulation of andquercitrin,butnotinthetotalamountsofflavonol
PAL at 120 hai compared with 72 hai for the Si+ glycosides (Fig. 3, D and E; Supplemental Table S2).
inoculated treatment (Table III) is not reflected in a Finally,itisinterestingthatrutin,isoquercitrin,avicu-
decreasedproductionofphenolicacidsfrom72to120 larin, and quercitrin have almost the same concentra-
hai. In fact, the total production of phenolic acids tion profiles after P. pannosa infection and in Si+ and
increasedfrom1.38mgg21sampleat72haito1.58mg Si2plantsatthedifferenttimepoints(Fig.3,CandD;
g21sampleat120hai(Fig.2A;SupplementalTableS1). Supplemental Table S2). This clearly indicates some
Therefore,a decreasein the concentration of phenolic correlation between the biosynthesis of these closely
acids at 72 to 120 hai would have been expected to relatedflavonoidsinresponsetoP.pannosainfectionof
result from the down-regulation of PAL during this rose leaves. Our results here also demonstrate that
timeinterval.Apossibleexplanationforthiscouldbe specific genes that encode flavonoid enzymes invol-
that the down-regulation of PAL only occurs at 72 to ved in the biosynthesis of specific flavonoids (Dixon
120 hai. Thus, the genes coding for the production of and Paiva, 1995; Winkel-Shirley, 2001) are expressed
specific enzymes involved in the biosynthesis (e.g. differently after Si application and infection with
cinnamate 4-hydroxylase, p-coumarate:CoA ligase) powdery mildew, which explains why some flavo-
are not affected or are down-regulated after 72 hai. noids are produced in much higher amounts com-
Alternatively,thelevelsofPALarekepthigh(i.e.there pared with others in the different treatments (Fig. 3;
isnoneedtokeeptranscribingthegeneiftheenzyme Supplemental Table S2). Therefore, it would be inter-
isstillpresent).Simplephenolicacidderivativessuch esting to investigate the expression of specific genes
as p-coumaroyl CoA is the shikimic acid-derived involvedinthebiosynthesisofantimicrobialphenolics
2202 Plant Physiol. Vol. 157, 2011
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Silicon Induces Changes in Rose Polyphenols
in more detail in order to understand the fundamental Extraction of Plant Material for Metabolite Analyses
mechanisms of disease resistance of rose plants against
Groundsamplesoffreeze-driedroseleaveswereextractedwith8mLof
powderymildewandantimicrobialdefensemechanisms
aqueous80%methanolinacentrifugetubewithlidandplacedinanorbital
in general. Hodson et al. (2005) found Si uptake in a shaker (200 rpm). From each of the two independent experiments, three
number of different plants, and the Si concentration independentextractionswerecarriedout(0.4g;0.1mmorlessparticlesize)in
varied among these species. The relationship between darkness for 90 min at room temperature (22(cid:4)C). After the extraction, the
sampleswerecentrifugedfor10minusingaSorvallSA-600head(maximum
functionandlevelofSiuptakeintheinvestigatedspecies
centrifugalforce=20.845;Buch&Holm),andthesupernatantwascollected
isnotfullyunderstood.Basedontheresultsofthisstudy, andstoredat220(cid:4)Cuntilanalysis.Thesampleswerefilteredthroughanylon
itcouldbeinterestingtoinvestigatewhetherSialsoplays 0.45-mmCameo25Psyringefilter(Bie&Berntsen)beforeanalysisbyHPLC
a role in the induced resistance of other horticultural and LC-electrospray ionization-MS/MS for phenolic acids and flavonoids.
species,butalsowild-typeplantsinnaturalsettings,and Theefficiencyandreproducibilityoftheextractionproceduredescribedabove
weredeterminedbyduplicateextractions(238mLof80%methanolor238
whether there is a correlation between Si uptake and mL of 90% methanol). This showed that extraction by 1 3 8 mL of 80%
defenseagainstpathogens. methanol was reproducible (coefficient of variation , 5%) and ensured
In conclusion, this study has demonstrated that the theextractionofmorethan95%ofthetotalflavonoidsandphenolicacidsin
accumulation of fungitoxic phenolic compounds, in thesamples.Fordeterminationoftheefficiencyoftheextractionmethod,the
extractsampleswerecentrifugedbetweeneachextractionandthesuperna-
particular chlorogenic acid and rutin, was stimulated
tantwascollectedandanalyzed.
by Si application. Exogenous application of these
phenolic compounds to rose plants enhanced resis-
Flavonoid and Phenolic Acid Standards
tance against powdery mildew. Thus, Si plays an
important and active role in stimulating the antimi- Quercetin-3-O-galactoside(hyperoside),quercetin-3-O-glucoside(isoquer-
crobial defense of roses. citrin), quercetin-3-O-rhamnoside (quercitrin), kaempferol-3-O-rhamnoside
(afzelin), and 3-O-caffeoylquinic acid were purchased from Extrasynthese,
and quercetin-3-O-arabinoside (avicularin) was purchased from Phytolab.
Quercetin-3-O-rutinoside (rutin) and 5-O-caffeoylquinic acid (chlorogenic
MATERIALS AND METHODS
acid)werepurchasedfromSigma-Aldrich.
Plants and Treatment with Si
Identification and Quantification of Phenolic Acids and
Theminiaturepottedrose(Rosahybrida‘Smart’),whichishighlysuscep-
Flavonoids in Extracts
tibletopowderymildew,wasobtainedfromAarhusUniversity,Department
ofHorticulture.Roseswerepropagated,maintained,andtreatedwithSias
Phenolicconstituents inextractsofroseleavesofcvSmartweredeter-
describedbyShettyetal.(2011).
minedbyHPLCcombinedwithphotodiodearray(PDA)detectionandLC-
SolubleSiwassuppliedinthenutrientsolutionataconcentrationof3.6
electrosprayionization-MS/MS(TableI).LC-MSdatawereobtainedusingan
mMSifromSiKal(9.1%Siand25.5%potassiumaspotassiummetasilicate
LTQXLLinearIonTrapMassSpectrometer(ThermoScientific)equippedwith
[KSiO]; Yara Industries), as described by Shetty et al. (2011). After the
2 3 a PDA detector and an evaporative light-scattering detector (ELSD; Sedex
propagationperiod(4weeks),plantsweremovedtothegrowthchamberand
wateredforthefirsttimewiththeSi+orSi2solution.Plantsweresubse- 80LT;SEDERE).SettingsfortheELSDwere50(cid:4)Cfortemperatureand3.7bar
for nitrogen pressure. Settings for the mass spectrometer fitted with the
quently watered with the two nutrient solutions every 72 h until disease
atmosphericpressurechemicalionizationsourceoperatedinnegativemode
scoring, a total of 10 times. After 3 weeks in the growth chamber, half of
theplantsineachofthetwogroups(Si+andSi2)wereinoculatedwiththe were40,10,and0(arbitraryunits)forsheath,auxiliary,andsweepgasflow
pathogenanddenotedSi+inoculatedandSi2inoculated,respectively.The rates,respectively.Dischargevoltageandcurrentwere1.13kVand5.38mA,
respectively. Vaporizer temperature was 400(cid:4)C, capillary temperature was
remaining plants from each group were not inoculated and denoted Si+
uninoculatedandSi2uninoculated,respectively. 250(cid:4)C,capillaryvoltagewas216.3V,tubelenswas100V,andautomaticgain
controltargetsettingswere33104forfullMS.Separationswereperformed
onaZorbaxEclipseXDB-C18column(5mm,15034.6mm;Agilent)withthe
Inoculation with Podosphaera pannosa
followingsolvents:solventA=0.1%formicacid(HPLCgrade,purityof99%;
Sigma-Aldrich)inwater,solventB=0.1%formicacidinacetonitrile(HPLC
Inoculum of P. pannosa was produced and inoculation took place as
grade;FisherScientific).Thesolventgradientwas0to5min,isocratic5%B;
describedbyShettyetal.(2011).Thefifthdevelopedleavesof7-week-old
5to65min,lineargradientfrom5%to20%B;65to75min,lineargradient
plantswereinoculatedanddenotedSi+inoculatedandSi2inoculated.Fifth
from 20% to 100% B; 75 to 80 min, isocratic 100% B; 80 to 85 min, linear
leaves of Si+ uninoculated and Si2 uninoculated plants were similarly gradientfrom100%to5%B;85to90min,isocratic5%B.Flowwas0.8Lmin21,
markedatthesametimepoint. columntemperaturewas35(cid:4)C,andinjectionvolumewas10mL.Theflowwas
Forallinvestigationsofmetabolitesandgeneexpression,twoindependent
split50:50(ELSDdetector:MSdetector)atexitofthePDAdetector.
experimentswerecarriedout,eachcomprisingatotalof168plants.Ineach
PhenolicacidsandflavonoidswerequantifiedinextractsbyHPLC-PDA
experiment,88plantswereinoculated(44Si+and44Si2)and80plantswere
onanAgilent1100HPLCsystem(AgilentTechnologies).Thephenolicacids
leftuninoculated(40Si+and40Si2).Leavesfrom160plants(80inoculated
and flavonoids were monitored at 320 and 360 nm, and UV spectra were
and80uninoculated)weresampledforfurtheranalysesasdescribedbelow,
recordedfrom210to600nm.Separationswereperformedunderthesame
andeightplantswereusedfordiseaseassessment9daiasdescribedbelow.
HPLCconditionsasusedforLC-MSanalyses,thusmakingcomparisonof
chromatograms and spectra completely reliable. Flavonoids and phenolic
SamplingofPlantMaterialforExtractionofMetabolites acidsweredeterminedinextractsfromexternalcalibrationcurvesofrutinand
and RNA chlorogenicacid,respectively.M correctionfactorsweretakenintoaccountin
r
thequantificationoftheindividual polyphenols. Meanrecoveryrates(ap-
Fromeachofthetwoexperiments,leavesofSi+andSi2plants,inoculated proximateaccuracy)forchlorogenicacidandrutinweremorethan98%,with
withP.pannosaorleftuninoculated,weresampledforextractionofpolyphe- arelativeSDoflessthan5%,andweredeterminedbyspikingaknownamount
nols(phenolicacidsandflavonoids).Markedleavesfrom20plantsofeachof ofauthenticstandardsofchlorogenicacidandrutin,respectively,toroseleaf
thefourtreatmentswereharvestedat0,24,48,and120h,groundinliquid extractsamples.TheprecisionoftheHPLCmethodwasdeterminedbyfour
nitrogen, and split into two portions. Approximately 0.2 g of the ground injectionsofaroseleafextractsampleonthesameday(intradayvariation)
materialwasimmediatelystoredat280(cid:4)CforRNAextraction.Another2gof and on four different days (interday variation). The overall intraday and
groundplantmaterialwasfreezedriedandstoredat280(cid:4)Cforextractionof interdayvariationswerefoundtobelessthan5%forbothflavonoidsand
polyphenols. phenolicacids.
Plant Physiol. Vol. 157, 2011 2203
Downloaded from on March 29, 2019 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.