Ragweed (Ambrosia artemisiifolia L.) Pollen after

12 Pages·2013·0.7 MB·English

Molecular and Immunological Characterization of
Ragweed (AmbrosiaartemisiifoliaL.) Pollen after
Exposure of the Plants to Elevated Ozone over a Whole
Growing Season
Ulrike Kanter1*, Werner Heller1, Jo¨rg Durner1, J. Barbro Winkler2, Marion Engel3, Heidrun Behrendt4,
Andreas Holzinger5, Paula Braun6, Michael Hauser7, Fatima Ferreira7, Klaus Mayer8, Matthias Pfeifer8,
Dieter Ernst1
1InstituteofBiochemicalPlantPathology,HelmholtzZentrumMu¨nchen,GermanResearchCenterforEnvironmentalHealth,Neuherberg,Germany,2ResearchUnitfor
EnvironmentalSimulation,HelmholtzZentrumMu¨nchen,GermanResearchCenterforEnvironmentalHealth,Neuherberg,Germany,3InstituteofSoilEcology,Helmholtz
ZentrumMu¨nchen,GermanResearchCenterforEnvironmentalHealth,Neuherberg,Germany,4CenterofAllergy&EnvironmentMu¨nchen,TechnischeUniversita¨tand
Helmholtz Zentrum Mu¨nchen, Neuherberg, Germany, 5Functional Plant Biology, Institute of Botany, Leopold-Franzens-University Innsbruck, Innsbruck, Austria,
6DepartmentofNaturalSciencesandMechatronics,HochschuleMu¨nchen,UniversityforAppliedScience,Munich,Germany,7DivisionofAllergyandImmunology,
DepartmentofMolecularBiology,UniversityofSalzburg,Salzburg,Austria,8InstituteofBioinformaticsandSystemsBiology,HelmholtzZentrumMu¨nchen,German
ResearchCenterforEnvironmentalHealth,Neuherberg,Germany
Abstract
Climatechangeandairpollution,includingozoneisknowntoaffectplantsandmightalsoinfluencetheragweedpollen,
knowntocarrystrongallergens.Wecomparedthetranscriptomeofragweedpollenproducedunderambientandelevated
ozoneby454-sequencing.Anenzyme-linkedimmunosorbentassay(ELISA)wascarriedoutforthemajorragweedallergen
Amba1.Pollensurfacewasexaminedbyscanningelectronmicroscopyandattenuatedtotalreflectance–Fouriertransform
infrared spectroscopy (ATR-FTIR), and phenolics were analysed by high-performance liquid chromatography. Elevated
ozonehadnoinfluenceonthepollensize,shape,surfacestructureoramountofphenolics.ATR-FTIRindicatedincreased
pectin-like material in the exine. Transcriptomic analyses showed changes in expressed-sequence tags (ESTs), including
allergens.However,ELISAindicatednosignificantly increasedamountsofAmba1underelevatedozoneconcentrations.
Thedatahighlightadirectinfluenceofozoneontheexinecomponentsandtranscriptlevelofallergens.Asthetotalprotein
amount of Amb a 1 was not altered, a direct correlation to an increased risk to human health could not be derived.
Additional,the454-sequencingcontributestotheidentificationofstress-relatedtranscriptsinmaturepollenthatcouldbe
grouped intodistinctgeneontology terms.
Citation:KanterU,HellerW,DurnerJ,WinklerJB,EngelM,etal.(2013)MolecularandImmunologicalCharacterizationofRagweed(AmbrosiaartemisiifoliaL.)
PollenafterExposureofthePlantstoElevatedOzoneoveraWholeGrowingSeason.PLoSONE8(4):e61518.doi:10.1371/journal.pone.0061518
Editor:GirdharKumarPandey,UniversityofDelhiSouthCampus,India
ReceivedOctober15,2012;AcceptedMarch11,2013;PublishedApril18,2013
Copyright: (cid:1) 2013 Kanter etal. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Thisworkwassupportedbythegrant3/09CK-Care,ChristineKu¨hneCenterforAllergyResearchandEducation.Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction Europeanpopulation[3].InEurope,commonragweedismainly
foundintheRhoˆnevalley,Hungary,Bulgaria,NorthernItalyand
Common ragweed (Ambrosia artemisiifolia L.) belongs to the EasternAustria,anditisnowspreadinginGermany[4](http://
familyAsteraceaeandisnativetoNorthAmerica.Ragweedisan www.Ambrosiainfo.de/53223897640d5c602/index.html).
important agronomic weed in the USA [1]. However, it causes
Climate change will alter plant growth and also influence the
problemsrelatedtohumanhealth,astheragweedpollenisknown
onset,periodandintensityofpollenproduction[5].Arelationship
to carry one of the strongest pollen allergens [2] and causes
between climate, pollen concentration and allergic rhinitis has
seasonal allergic rhinitis and asthmatic symptoms in North been reported [6]. Recent warming may be associated with the
America [1,3]. So far, ten different allergenic proteins consisting increased length of the ragweed pollen season in North America
of different isoforms could be identified in ragweed pollen and [7], and it has been shown that twice the ambient level of CO
2
were termed Amb a 1 to 10, with Amb a 1 the most important results in increased ragweed pollen production [8] and in
allergen [3]. Their molecular weights range from 9 kDa to significant increases in the Amb a 1 allergen [9]. However, there
38kDa, and they are grouped according to their biological remain lots of open questions about the link between climate
differences [3]. Ragweed was introduced from North America to change and elevated CO with public health [5]. Additional, the
2
Europe approximately 100 years ago through contaminated seed long-distance transport of pollen must also be taken into account
shipments, which has resulted in the allergic sensitisation of the [10].
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TranscriptomicAnalysisofRagweedPollen
Increasing air pollution, primarily caused by vehicle traffic, large scale analyses of ragweed pollen performed by deep
makes pollen allergens more aggressive [11]. The interactions of pyrosequencing technology. The data include transcripts that
airborneparticleswiththesurfaceofpollenorhighconcentrations weredifferentiallyexpressedunderambientandtwicetheambient
ofpollutantsisleadingtoachangedpollenmorphology[12],and level of ozone. An additional focus is the characterisation of
higher concentrations of allergenic proteins were found in the allergen-related transcripts and changed allergenic protein abun-
pollen of air-polluted regions [13]. However, there are also dances upon elevated ozone concentrations. Scanning electron
contrary reportsthat have indicated nodifferences between rural microscopy (SEM) analyses, attenuated total reflectance–Fourier
and urban regions or even a greater abundance of an allergenic transform infrared (ATR-FTIR) spectroscopy of the pollen wall
protein in rural regions [14]. Significant differences in allergenic and phenolic metabolite profiles of ozone-treated ragweed pollen
proteinsfrombirchpollenwerealsoobservedindifferentregions arealsopresented.Theexperiment,usingrealisticoutdoorozone
of Europe and in different years [15]. This indicates a complex fumigation, allowed the investigation of ragweed over a whole
interaction of different environmental factors on pollen allerge- growingseason,includingthedevelopmentofpollenandextends
nicity that isnot triggered byallergenic proteinsalone [14,16]. studies where only pollen was fumigated with ozone. It provided
Tropospheric ozone is one of the most harmful air pollutants a link between controlled chamber conditions and analyses of
and is also relevant to global change [17]. Chronic exposure of pollen inrural andurban sites.
vegetationtogroundlevelsofozonewilllikelyincreaseduringthe
upcomingdecades,andexposurewillstayatsuchlevels,increasing Materials and Methods
risksforvegetation[17].Withregardtopollen,ithasbeenshown
that elevated ozone results in decreased pollen germination and Ethics Statement
pollen tube growth [18]. In recent decades, the effects of ozone No specific permits were required for the described studies.
havebeenstudiedatthephysiological,biochemicalandmolecular Plantmaterialusedinthisstudywasgrowninexposurechambers
biologicallevels[17].Numeroustranscripts,up-regulatedinleaves under controlled conditions. Initial ragweed seeds were collected
by ozone, belong to the category ‘‘disease and defence’’, which inanoutdoorstand,forthisnospecificpermissionswererequired.
alsoincludespathogenesis-related(PR)proteintranscripts[17,19]. The sampling location was not privately-owned or protected in
TheexposureofryecultivarsandLoliumperennetoelevatedozone anyway.
concentrationsduringplantgrowthincreasedtheallergencontent
intheirpollen[20,21],whereasindifferentcultivarsofLolium,no Plant Growth Conditions
significant differences in group 5 allergen were evident between
Ragweedseedswerecollectedfromasingleplantatanoutdoor
control plants and plants grown in ozone concentrations up to
stand to avoid environmental-dependent epigenetic effects on
140ppb [22]. Ozone fumigation of ragweed plants, up to catkin
growthanddevelopment[36].Seedswereappliedtostandardsoil
initiation, showed no effects on growth parameters such as leaf
(FloradurH, Bayerische Ga¨rtnerei Genossenschaft, Mu¨nchen,
area, total biomass or catkin weight. It was concluded that
Germany) in pots, and plants were cultivated in eight Plexiglass
ragweed is insensitive to ozone levels up to 80ppb [1]. Similar
sub-chambers(1.1 m60.9m60.8m)placedwithintwophytotron
non-significant changes in plant size and pollen amount were
walk-in chambers [37] (Kanter et al., unpublished). (http://www.
found for ragweed grown under controlled conditions, fumigated
helmholtz-muenchen.de/en/eus/environmental-simulation-
with 80ppb (Kanter etal., unpublished). The direct invitro
facilities/phytotron/index.html). Thus, four sub-chambers served
fumigation of ragweed pollen with ozone resulted in a reduced
astechnicalrepetitionsofeachtreatment.Duringtheexperiment,
pollenviability,whereasnoinfluenceonAmba1proteincontent
theaverageseasonalcourseofclimaticconditionsbetweenMay1st
and theexpressionprofile ofmajor allergens wasobserved [23].
andSeptember15thweresimulatedonanhourlybasis(FigureS1).
Flavonoids, as well as hydroxycinnamic acid-derivatives, are
The light period was between 14.5 h and 16h per day (approx.
common metabolites of plant pollen, especially the glycosylated
500mmol m22sec21 PPFR with a realistic portion of UV-A
types. Flavonoids play a role in fertility, and quercetin has been
during the daily course). The day/night temperatures were 20–
demonstrated to be an important germination-inducing com-
30uC/10–20uC, and relative humidity was maintained at 30–
pound [24]. Additional, these metabolites are known to possess
50%/80–85% (day/night). Two sub-chambers in each phytotron
antioxidative and antimicrobial activities. Flavonoids also have
were fumigated with 40ppb (control) and 80ppb ozone for the
UV-B-absorbing properties, thus they protect the pollen, in
whole vegetation period, starting on June 19th. Watering was
addition to sporopollenin, during long-range transport [25].
carried out by an automated irrigation system. The number of
Flavonoids may be involved in the modulation of immune
seedlingswasreducedtooneperpotafterthreeweeks,plantswere
responses, which would also be a key role in the allergenic
further grown under normal air for four weeks to acclimate, and
response tothepollen [26,27].TheIgE-binding ofallergensmay
ozonetreatmentwasstartedonJune19th.Pollenwascontinuously
beinfluencedbyattachedflavonoids[28],andadirectinteraction
collected from July to August 30th, using a modified ARACON
of allergens with biologically important ligands, including
system (BETATECH, Ghent, Belgium) that covered the male
flavonoids, has been shown [29,30]. Air pollution resulted in an
inflorescences. On August 23rd and August 30th the collected
increasedamountofunknownflavonoidmetabolitesinthepollen
ofCupressaceae,andelevatedozoneconcentrationsresultedinthe pollen samples were storedat 280uCuntil use.
accumulation of apiin in parsley leaves [31,32]. Interestingly
apigenin, the aglycone of apiin, is able to immunomodulate Scanning Electron Microscopy (SEM)
dendritic cells [27]. SEMwasessentiallyperformedasdescribedbyHolzingeretal.
Pollen transcriptomic analyses have thus far only been carried (2009) [38]. Instead of the dehydration procedure, air-dry pollen
out primarily with model plant organisms such as Arabidopsis was gold surface-coated and examined with a Philips XL20
thalianaorGlycinemax[33,34].Furthermore,hierarchicalclustering scanningelectronmicroscope(PhilipsElectronics,Eindhoven,The
and principal component analysis indicated a clear separation of Netherlands) at 10kV.
pollen from other vegetative tissues and clearly defined pollen-
specifictranscripts[35].Inthisstudy,wedescribetheresultsfrom
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TranscriptomicAnalysisofRagweedPollen
Attenuated Total Reflectance–Fourier Transform Infrared Titanium Sequencing
Spectroscopy (ATR-FTIR) Total RNA was extracted from three pooled pollen samples
ATR-FTIR spectra of Ambrosia pollen fumigated with ambient (50 mgeach,grownunder40ppband80ppbozone),quantified
andtwiceambientozonewererecordedusingaBrukerTensor27 and analysed using a NanoDrop ND1000 at wavelengths of 230,
spectrometer equipped with the ATR accessory ZnSe crystal cell 260and280nm.Twonon-normalisedcDNAs,readyforGSFLX
attached to the spectrometer with a liquid nitrogen cooled TitaniumsequencingwerepreparedbyVertisBiotechnologyAG
mercury cadmium telluride detector and a KBr beam splitter. (Freising, Germany). 454-sequencing of both cDNAs was carried
ATR–FTIR spectra (3,050–900 cm21) were taken with 4 cm21 out using the Titanium Genome Sequence Systems according to
resolution and a sampling time of 32 scans [39]. Frozen pollen the manufacturer’s instruction (Roche Diagnostics GmbH,
samples were placed onto the crystal cell and gently compressed Mannheim, Germany).
during measurement.
Bioinformatic Analysis
Analyses of Phenolic Metabolites Theanalysisoftheoriginal454-readsetsaregiveninTableS1.
Frozen pollen was extracted with phosphate buffered saline Toavoidshortfragmentsduringtheassemblyprocess,thefraction
of small 454-reads (24%) was removed and excluded from the
(PBS),andtheresiduewasthenextractedwithmethanol.Reverse-
assembly process. For the assemblies, the Newbler v.2.5.3
phase high-performance liquid chromatography (RP-HPLC)
transcriptome assembly (-cDNA option) and default parameters
separationofthePBS-solubleandmethanol-extractablephenolics
were used. In order to quantify expression levels of Ambrosia
was performed asdescribed previously[40].
transcripts in the ozone and control treated plants, the available
454-readsoftheindividualsampleswerealignedtotheassembled
Enzyme-linked Immunosorbent Assay (ELISA)
isotigsbyusingvmatchv2.1.7(-l40-e1-identity98)(http://www.
Ambrosia pollen extracts were prepared from 100mg pollen by
vmatch.de/).Returnedalignmentswerefilteredandforeachread
shaking in 1ml 16 PBS buffer, pH 7.4 for 1.5h at room
only alignments with maximum e-value were considered for
temperature followed by centrifugation at 10,000 g for 15min.
furtheranalysis(workflow:FigureS2).Transcriptexpressionlevels
Thetotalproteinconcentrationoftheextractswasdeterminedin
were quantified in reads per kilobase of exon model per million
triplicate byaBradford assay.For directELISA,maxisorpplates
mappedreads(RPKM)thatmeasuresthereaddensitynormalized
(NUNC, Roskilde, Denmark) were coated with 50 mg ml21
forRNAlength andthetotal number ofreadsintheexperiment
Ambrosia pollen extract in 16 PBS buffer, 50ml per well and
[41].Thereby,individualreadsmappedtomultipleisoformswere
incubatedovernightat4uC.PlateswereblockedwithTrisbuffered
uniformlydividedtoallmappingpositions.ThematchedAmbrosia
saline(TBS),pH 7.4,0.05%(v/v)Tweenand0.5%(w/v)BSAfor
transcriptswerefurtheranalysedinordertodetectgenesthatwere
2 h at room temperature and incubated with a murine mono-
either matched by sequences of both groups or exclusively
clonal IgG1 anti-natural Amb a 1 antibody, diluted 1:1,000, for
matched onegroup.
1.5 hat 37uC.AfterwashingwithTBS,pH 7.4and0.05%(v/v) ForeachoftheNewblerisogroupwithtwoormorealternative
Tween, plates were incubated with an alkaline phosphatase-
splicingvariants,theisotigwithlongestpredictedproteinsequence
conjugated rabbit anti mouse IgG+IgM antibody (Jackson
was selected as gene representative. Then, putative orthologous
Immuno Research, PA, USA), diluted 1:1,000, for 1 h at 37uC gene pairs between assembled Ambrosia genes and Arabidopsis
and 1h at 4uC. 4-Nitrophenyl phosphate (Sigma-Aldrich, MO, (TAIR10) were identified based on a two level homology search
USA)at10mMwasusedassubstrate,andtheODwasmeasured against the Arabidopsis TAIR10 gene set (BLASTP, e-value
at 405/492nm. All measurements were performed in triplicate. #1e25) (workflow:Figure S2):
The resultsare presented as themeanODvalues.
i. bi-directional BLAST searches (Ambrosia vs. Arabidopsis and
RNA Isolation Arabidopsis vs.Ambrosia, respectively) wereperformed.
Total RNA was isolated from 50mg pollen using a modified ii. Arabidopsisgenes,whichwereexclusivelymatchedbythefirst
QiagenRNeasyMiniKitprotocol.Inbrief,pollen,togetherwith best BLAST hitof onedistinctAmbrosia isotig.
150ml of RLT buffer, was transferred to 2 ml tubes containing
1.4 mm ceramic spheres, 0.1mm silica spheres, and a single Moreover, sequence similarity searches against theNCBI non-
4 mm glass sphere. The pollen was homogenised eight times at redundant protein database (July 2011, BLASTP) excluding all
6.5 ms21foroneminuteeachondryiceinaFastPrep24machine Arabidopsisgenes,asetofknownAmbrosiaallergens(BLASTN)and
(MP Biomedicals, Eschwege, Germany). Then, another 600ml a set of known plant allergens (NCBI search with keywords
‘‘viridiplantae’’ and ‘‘allergen’’) (BLASTP) was performed. Only
RTL buffer was added, and the tube was shaken again. One
alignmentswithe-value#1e25wereconsideredassignificantand
volume of chloroform was added and incubated for 10min on
thefirst-best hit (fbh) againstthereference datasets extracted for
a shaker. After centrifugation, the supernatant was transferred to
each Ambrosia sequence. In total, 1,542 of 2,877 (54%) Ambrosia
a new reaction tube and mixed with 0.5 volumes of ethanol by
representativetranscriptsshowedconservedsequencehomologyto
gentlyinverting.ThesolutionwastransferredtoRNeasycolumns
at least onereference dataset (Table 1).
(RNeasyMiniKit,Qiagen,Hilden,Germany)andcentrifugedfor
Rawsequencingdataandassemblydataweresubmittedtothe
15s at 10,000 g. The column was incubated with 450ml RW1
European Nucleotide Archive (ENA) with the project accession:
buffer for 5 min and then centrifuged. The flow-through was
PRJEB1470.
discarded, and DNase digestion was performed following the
manufacturer’s instructions (RNase-Free DNase Set; Qiagen,
Results
Hilden,Germany).Subsequently,thecolumnwasincubatedtwice
with 500ml RPE buffer for 2–3min each. Drying and elution of
Scanning Electron Microscopy (SEM)
the RNA was performed according to the user manual (RNeasy
SEM showed the expected shape of ragweed pollen grain
Mini Kit,Qiagen).
(Figure1).Themeansizeoftheroundishpollenwasintherange
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Table1. Comparisonof 454transcriptome assembly against referenceprotein databases.
Arabidopsis(TAIR10) AmbrosiaAllergens NCBIPlantAllergens NCBInon.red. Total(non.red.)
Referenceproteins 35,386 47 7,384 399,458 –
Matchedreferenceproteins(anyhit) 7,831 30 587 22,949
Matchedreferenceproteins(fbh) 1,008 12 62 1,260 –
MappedAmbrosiatranscript 1,415 21 133 1,532 1,542
representatives
doi:10.1371/journal.pone.0061518.t001
of 15–24mm and exhibited the typical circular apertures. SEM ,2920,,2850and,1470weresignificantlydecreasedinpollen
analysesrevealedthattheexineofthepollenwascoatedwithsmall samplesthatweretreatedwithelevatedozone,indicatingreduced
cone-shaped needles. According to SEM photographs the pollen numbersofmethylenegroups,e.g.,inlipidandwaxcomponents.
shape appeared to be similar and no significant differences in Absorptionsrecordedbetween1660–1500 cm21correspondedto
pollensizebetweenthecontrolandozone-exposedplantscouldbe theso-calledaromaticdomainofthesamples,suchasinphenolic
detected (ozone: 20.5761.64mm, n=100; control: compounds, for example, in the aromatic rings of sporopollenin
21.1561.45mm,n=100). components (Figure 2B; d, d’). In addition, the proteins’ amid I
absorption was recorded between 1680–1620cm21, the exact
ATR-FTIR Analysis of Ragweed Pollen vibrational frequency of which depended on the CO and NH
TheATR-FTIRspectraofAmbrosiapollentreatedwithelevated conformational states (Figure 2B; c). The strong difference in
ozonecomparedtothecontrolareshowninFigure2andfurther absorptionbetweenozone-treatedandcontrolpollen,withapeak
describedinTable2.Thetwostrongabsorptionbandslocatedat intensityat,1512cm21andashoulderat,1603cm21,suggests
,2850 and ,2920cm21 originate from the asymmetric and a relative decrease of phenolic compounds of sporopollenin in
symmetric stretching vibrations of the methyl and methylene ozone-treated pollen [42]. Finally, a set of different absorption
groups present in hydrocarbon chains, for example, in glycer- bandswererecordedinthe1200–950cm21region(Figure2B;f’–
olipids and wax hydrocarbons (Figure 2B; e). The minor IR f’’’). The shape and relative intensities of the different shoulders
absorption band recorded at ,1470cm21 may be assigned to located at approximately 1100, 1048 and 1025cm21 can be
methylene deformation vibrations; its intensity is usually much clearly assigned to C-O and O-G deformations vibrations in
weakercomparedtotheCH -stretchingvibrations(Figure2B;e’). secondary alcohols and define the polysaccharide absorption
2
As shown by the IR difference spectra (Figure 2B), the bands at range. Specifically, the IR frequencies of ring and side group
Figure1.Scanningelectronmicroscopyofragweedpollenexposedtodifferentozoneconcentrations:(a–c)40ppbozone,(d–f)
80ppbozone.Bars:a,d50mm;b,e20mm;c,f5mm.
doi:10.1371/journal.pone.0061518.g001
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vibrations of pectin (,1100, ,1047, ,1017cm21) have been absorptionbandsofthelipidacylchaingroup)andanincreaseof
assigned by model studies to rhamnogalacturonic acid at absorption with maximum at ,1022cm21 (major absorption
,1043cm21, arabinan at ,1100cm21, glucan at ,1026cm21 band of secondary alcohols of pectin) as shown in the difference
andglucomannanat,1034cm21[43].Theabsorptionintensities spectrum(FigureS3,b).TheD-IR-absorptionobtainedbymerely
ofthepectinvibrationshaveapparentlyincreasedwhencompared raisingthepectinCcontentinthelipid/pectinmixtureissimilarto
tothelipidandproteinabsorptions,asisobviousfromboththeIR theD-IR-absorptionofozone-andcontrol-treatedAmbrosiapollen,
absorption anddifference spectra (Figure2B, f’–f’’’). namely an increase in the polysaccharide absorption range.
The absorption bands at ,1770 and ,1740cm21 may Naturally, in the complex mixture the resolution of the other
originate from the C=O stretching vibration of the carbonyl mediumandminorpectinabsorptionbandsisnotachieved.Thus,
groupofthepectin’sacetylesterbond(Figure2B;a)andeitherof the similarity of the difference spectra of the two component
the carbonyl of the pectin’s methylester or the wax and lipid’s mixture as compared to the complex mixture of residual
acylester(Figure2B;b),respectively[44].Acetylatedpectatesalso components in pollen walls further supports the notion of an
have a strong IR band at ,1250cm21 (Figure 2B; a’), which is increase inpolysaccharide, particularly pectin.
assigned to the (uCOC) of acetyl, whereas methylated PecAc has
been proposed to have three IR bands at ,1280, ,1250 and Secondary Metabolites
,1220cm21 (Figure 2A) [45]. Interestingly, the pectin’s acety- In PBS-soluble extracts, 17 prominent compounds were
lester absorption at ,1770cm21 is decreased in ozone-treated evaluated. The highest amounts were found for quercetin
pollen when compared to the pectin’s pyranoid ring group derivatives. Methanol-extractable phenolics showed additional 12
absorptions(at,1100,,1047,,1017cm21),perhapsindicating prominentcompounds,characterisedashydroxycinnamicamides
the deesterification of pectate [45]. It cannot be determined accordingtotheirtypicaldiode-arrayspectra.Onthebasisofpeak
whetherde-methylationofpectinoccurredduetotheoverlapping areasobtainedat280nm,totalamountsofindividualcompounds
of different IR active groups in the range of the pectin’s for the two harvest time points together with typical RP-HPLC
methylester.Nevertheless,elevatedozoneresultedintheapparent diagrams are given in Figure S4. No significant changes were
decrease of the lipid wax, protein and sporopollenin components observedbetweencontrol-andozone-treatedsamples.Summaris-
ascomparedtothepectinlayer.Itshouldbestated,however,that ing the amount of PBS-soluble as well as methanol-extractable
additionalanalyticsareneededtounequivocallyandquantitatively metabolites, a slight, but not significant ozone-dependent re-
determine the changes in pollen surface constituents upon duction,aswellasareductioninseveralmetaboliteswasobserved
exposuretoelevatedozone.Totestwhetheranincreaseofpectin overtime (Figure3, FigureS4).
compared to the lipid wax could be true in pollen, mixtures of
differentlipid/pectinCweightratioshavebeenanalysedbyATR- Roche Titanium Sequencing and Assembly of the 454-
FTIR (Figure S3). Thedoubling of pectin C relative tothelipids
Reads
resultedinalossofabsorptionat,2924and,2854cm21(major
Two sequencing runs (ozone and control) yielded a total of
1,242,132 raw reads. The length frequency distribution ranged
from40–772bp(FigureS5a).Toavoidshortfragmentsduringthe
assembly process, the fraction of small 454-reads (24%) were
removed and excluded from the assembly process (Table S1). A
total of 982,467 reads, equivalent to a total sequence length of
approximately 324Mb, was then used for the assembly process.
Using the cleaned sets of 454-reads, an ensemble transcriptome
assembly was performed using Newbler v2.5.3. This resulted in
5,720contigsand49,729singletons.Singletonswerenotusedfor
further analysis. Furthermore, the Newbler Transcriptome
Assembler combined the assembled contigs into 5,052 individual
transcripts (‘‘isotigs’’) that were collected in 2,938 ‘‘isogroups’’
based on shared branches in the underlying contig graph (Table
S2).Openreadingframes(ORFs)werepredictedfor4,950isotigs
(encoded by 100 or more nucleotides). The longest ORF was
chosenasmostprobableproteinsequenceofanisotigandusedfor
further analysis.Isotigs without protein sequence were discarded.
In order to quantify expression levels of Ambrosia transcripts in
theozoneandcontroltreatedplants,theavailable454-readsofthe
individual samples were aligned tothe assembled isotigs by using
vmatchv2.1.7(2l40-e1-identity98)(http://www.vmatch.de/).
Returned alignments were filtered and for each read only
alignments with maximum e-value were considered for further
analysis. AssummarisedinTableS3andvisualisedinFigureS2,
Figure 2. ATR-FTIR spectra of ozone- and control-treated atotalof299,051454-reads(52%)outof576,199rawreadsofthe
Ambrosiaartemisiifoliapollen. A shows the averaged absorption ozone-treatedsampleand349,759454-reads(53%)outof665,933
spectraofozone-treated(black;n=5)andcontrolpollen(grey;n=6)in raw reads of the control sample were matched against the 5,052
arangeofwavenumbersbetween900–3050cm21.Standarderrorsfor ensemble isotigs and aligned to 4,923 and 4,975 Ambrosia
these spectra are stated at the bottom of part A in black and grey,
transcripts, respectively. Thereby, the majority (81% and 82%)
respectively. B indicates the D-absorption of ozone spectra minus
of the matched 454-reads were unambiguously mapped to
control spectra. Small letters stated in part B are further described in
Table2. auniqueisotig.However,almostallmatchesofmultiplemapped
doi:10.1371/journal.pone.0061518.g002 454-reads(98%)were observed forisotigs ofthesame isogroup.
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TranscriptomicAnalysisofRagweedPollen
Table2. ATR-FTIRanalysis– explanation oflabelsfrom Figure 2.
LabelinFigure2 Bandorigin Wavenumberofabsorption[cm21] Changeduetoozone-treatment
Pectin:
a COO-R,R=Me,=Ac 1750–40,1770–60 2
a’ COC 1280,1250 2
Lipid:
b Acylester:COOR 1740–20 2
b’ COC 1170 2
Protein:amidI:
c CO,NH 1680–20 2
Sporopollenin:
d,d’ aromatic,phenolicstretches:CC,CO 1603,1512
Glycerolipid,wax:
e,e’ methylene:CH2 2920,2850,1465 22
Pectin:
f carboxyl:COO– 1410 +
f’,f’’,f’’’ pyranoidring:CC,COH,CH 1100,1048,1025 ++
ATR-FTIRanalysisofozone-treatedpollenofAmbrosiaartemisiifoliacomparedtothecontrol.Labelsstatedinthefigureareshown,aswellasthebandoriginand
wavenumbersofabsorption.Changesinabsorptionbetweenthetwotreatmentsarestated.Areductionintheozonesamplecomparedtothecontrolisindicatedby
aminussign(2or22),whereasa+or++reflectsanincreaseintheozone-treatedpollen.
doi:10.1371/journal.pone.0061518.t002
In order to filter low-abundance transcripts different RPKM low RPKM-values were further analysed by quantitative RT-
thresholdsbetween1RPKMand10RPKMwereapplied(Table PCR.Evensoaplanttoplantvariabilitywasdetectableapositive
S4).Morethan90%(2,607)oftheassembledAmbrosiatranscripts correlation of qRT-PCR results to RPKM-values could be
are commonly expressed with at least 7 RPKM (lower 5th detected withr=0.984(Pearson-correlation; Figure S6).
percentile of transcript abundances (Table S5), in the ozone and
controltreatment(Figure4)aswellas116ozonespecificgenesand Comparison of the 454-Transcriptome Assemblies to the
150control-specificgenes,respectively,couldbeidentified.Totest Arabidopsis Gene Set
the reliability of RPKM-values several transcripts with high and
ForeachoftheNewblerisogroupwithtwoormorealternative
splicingvariants,theisotigwithlongestpredictedproteinsequence
wasselectedasgenerepresentative.Then,a totalof957putative
orthologous gene pairs between assembled Ambrosia genes and
Arabidopsis (TAIR10) were identified based on a two level
homology search against the Arabidopsis TAIR10 gene set
(BLASTP, e-value #1e25) (workflow:Figure S2):
a) bi-directional BLAST searches (Ambrosia vs. Arabidopsis and
Arabidopsisvs.Ambrosia,respectively)wereperformedand774
best bidirectionalhits(bbh) identified.
b) The remaining hits 233 matched Arabidopsis genes were
further analysed and 183 reference genes were detected,
which were exclusively matched by the first best BLAST hit
of onedistinct Ambrosiaisotig.
Figure3.Accumulationoftotalphenolicsanalysed.Plantswere Figure 4. Venn diagram. Common and differently matched
fumigatedwith40ppb(control)or80ppbozone;barsindicate6SD; Arabidopsis genes. Number of Arabidopsis genes that matched by
n=7. eithersequencesofbothgroupsorsequencesofonegroupexclusively.
doi:10.1371/journal.pone.0061518.g003 doi:10.1371/journal.pone.0061518.g004
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TranscriptomicAnalysisofRagweedPollen
Adetailedtable,includingalltaggedArabidopsisgenes,thegene oneAmba4(defensin-like)andAmbaCPI(cystatin)werefound.
description,alignmentdetails,mappedAmbrosiareadsandRPKM- Changes in the transcript amounts upon ozone treatment are
values, is provided as a supplementary Excel file (Table S6). For giveninFigure6,representedbylog (RPKM /RPKM )-
2 ozone control
quantificationRPKM-valueswereusedandalog -fold-changeof values for each allergen annotated isotig. The expression of most
2
treatment against control was calculated. For genes with no Ambrosia allergen ESTs was higher in ozone-treated pollen, up to
expression in one group, the RPKM-value was set to 0.1 to 2.9-foldgreaterthanthecontrol,whereatanisotig-orspliceform-
calculatethefold-change.Toobtainstringentvaluesandtoavoid specificexpressionwasdetectable.OftheseAmbrosiaallergens,the
‘‘false positive’’ interpretation of log -fold-changes, all transcripts major allergen Amb a 1 and isotigs for the allergen Amb a CPI
2
with a RPKM-value less than 7 were considered to be not were expressed at the highest level represented by the high
expressed. Transcripts were then analysed via MapMan [46] to RPKM-values.
groupthemtoseveralfunctionalcategories(BIN-codes).Analysing
several BINs involved in plant stress showed a couple of genes ELISA
induced or repressed by ozone (Figure 5 and Table S7). Pollenproteinextractsfromragweedplantsgrowneitherunder
Transcriptswithhomologytogenesinvolvedinrespiratoryburst, controlconditionsorelevatedozonewereanalysedfortheirAmb
including a oxidoreductase acting on NAD(P)H, a monodehy-
a 1 content via direct ELISA. First, the antibody sensitivity in
droascorbate reductase from the term ‘‘redox’’ and a gene for detection of Amb a 1 was tested by immunoblot. Ambrosia pollen
a glutathione-S-transferase, were up-regulated in ozone-treated
extracts from 7 different groups were analysed via immunoblot
pollenuptoalog -foldchangeof2.23,whereastwoglutaredoxins
2 using polyclonal rabbit anti Amb a 1 sera, as well as murine
found within the BIN term ‘‘glutathione’’ were only slightly
monoclonal mAB anti Amb a 1 A39 (Figure S7). As the murine
induced by ozone (max. log -fold change: 0.67), which are
2 monoclonal antibody anti Amb a 1 A39 is more sensitive in
involvedincopingwiththeeliminationofreactiveoxygenspecies.
recognitiontoAmba1anditscleavageproductsalpha-chainand
Inaddition totranscripts withhomologytogenes involvedinthe
beta-chain, A39 was used for analyses of all Ambrosia pollen
redoxstateofthecell,alsotranscriptswithoxidoreductaseactivity
extractsvia directELISA.AsitisseeninFigure7,nosignificant
fromtheterm‘‘oxidasescopper,flavone’’(max.log -foldchange:
2 differences could be detected between the different treatments,
3.16) showed higher transcript levels in the ozone-treated pollen.
indicating that twice the ambient ozone might not have an
For the BIN term ‘‘secondary metabolites’’ a slight repression of
influenceontheproductionofthemajorragweedpollenallergen
transcripts involved in wax biosynthesis, such as the membrane
Amba 1.
boundO-acyltransferases(max.log -foldchange:20.99)andan
2
increase of transcripts involved in ‘‘glucosinolates’’ and were
Discussion
recognised due toelevated ozone (max.log -foldchange:2.81).
2
For theBINterm ‘‘cellwall’’,thepectinmethyl esterases were Onceozoneenterstheplantcell,ozonerapidlyreactswithcell
clearlyinducedduetotheozonetreatment(max.log2-foldchange: wallcomponentsandlipidstoformreactiveoxygenspecies(ROS),
3.39) and the expression of the pectate lyases was also elevated including singlet oxygen, hydrogen peroxides and hydroxyl
under ozone treatment (max. log2-fold change: 1.42). Beside this radicals [48]. This leads to oxidative stress and changes in the
also transcripts involved in ‘‘precursor synthesis’’ and ‘‘modifica- redox potential, with which the pollen has to cope. Genes
tion’’ were increased due to the ozone fumigation. Another encoding proteins associated with plant stress response were
interestinggroupinfluencedbyozonetreatmentwastheBINterm elevated, including enzymes involved in detoxifying ROS and
‘‘misc.nitrilase’’,whichincludedseveralberberinebridgeenzyme- pathogen-relatedproteins,similartoasitwasseeninbeech[19].
like proteins (max. log2-fold change: 2.33). This enzyme family is ESTs for a monodehydroascorbate reductase (MDAR) as well as
knowntoshowallergenaction,e.g.,inTimothygrass(Phlp4)or gluthatione-S-transferaseandathioredoxinwereactivateddueto
celery(Apig5)[47],andmightalsobeacandidateforapossible elevated ozone. He etal. (2006) reported an increased level of
allergen. MDAR after 10 days of elevated ozone in leaves of Ginkgo biloba,
followedbyadecreaseofMDARafter40daysoftreatment[49].
Comparison of the 454-Transcriptome Assemblies to the Dehydroascorbate reductase activity was lower than the control
NCBI Non-Redundant Protein Database aftertendays,followedbyanincreaseandthenbyadecreaseafter
A comparison of the Ambrosia transcriptome to the Arabidopsis 70daysoftreatment,leadingtotheconclusionthattheprotection
gene set will not reveal possible allergenic proteins. A BLAST system itself became affected after prolonged treatment and
comparison of all Ambrosia sequences against the NCBI non- resultedinloweredprotection[49],alsointhisstudysomeofthe
redundant protein database, against a set of known Ambrosia transcripts involved in the Bin-term ‘‘Redox’’ were found to be
allergens and against a set of known plant allergens (NCBI; repressed.
keywords ‘‘viridiplantae’’ and ‘‘allergen’’) was additionally per- With regards to secondary metabolites, ESTs involved in
formed, and only first best hits with e-value #1e25 were flavonoid biosynthesis were found to be slightly repressed. This
considered to be significant. Considering both groups, 23,566 observations for flavonoids was concordant with results found in
matches were found, taking only first best hits into account beechsaplings[19].Furthermore,anESTinvolvedinisoprenoid
1,322 fbh-matches were found. These matches were mapped to biosynthesis, of the mevalonate pathway, was repressed due to
1,517 Ambrosia transcripts from group1 and 1,521from group2. ozone treatment. This is interesting because isoprenoids such as
Withinthesepre-filtereddatasets,onlyallergenichitswerefurther isoprene and monoterpenes are known to protect leaves from
analysed. This resulted in 152 hits for group 1 and 151 hits for oxidative damage [50]. Moreover almost no changes were found
group2(Table3,TableS8).Theprimaryhomologieswerefound for ESTs involved in the biosynthesis of polyamins, such as
to the allergen families of polcalcin (calcium-binding protein, EF putrescineandspermidine,whichareknowntoplayvariousroles
hand domain), polygalacturonase, pectin methylesterase and in response to plant stress [51]. Only one arginine decarboxylase
pectatelyases.RegardingallergensfromAmbrosia,severalisoforms (ADC)homologousEST,thekeyenzymeinplantstresspolyamine
of Amb a 1 (pectate lyase), Amb a 8 (profilin) and Amb a 9 synthesis,waselevated[52].Astrongpositivecorrelationbetween
(calcium-binding protein), Amb a 10 (calcium-binding protein), ozone exposure and ADC induction was reported for barley and
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TranscriptomicAnalysisofRagweedPollen
Figure5.InterestingBINtermsdetectedbyMapMan[46].Arabidopsissequencematchesweregroupedduetotheirlog -fold-changevalueto
2
threegroups:black=inductionbyozone(log.1),grey=common(log#21;log $1),white=repressionbyozone(log,21).Forthesegroups,
2 2 2 2
thenumberoftranscriptsaccordingtotheBINtermisstated.Onlymatcheswithe-value#1e25andRPKM$7weretakenintoaccount.
doi:10.1371/journal.pone.0061518.g005
tobacco [53,54]. Additionally, ESTs involved in wax biosynthesis observed on the needles of Norway spruce [55]. Ozone stress
werereducedinpollenfumigatedwithelevatedozone.Thismight showed a clear influence on the cell structure, such as the
be concordant to structural degradations of epicuticular wax components of the cell wall. An EST for cellulose synthase was
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TranscriptomicAnalysisofRagweedPollen
Table3. Allergensdetected by454-sequencing inAmbrosia artemisiifolia pollen.
Proteinfunction AllergenAmbrosia Homologytootherplantallergensfoundin
Actinidin Actinidiadeliciosa
Aldoketomutase Oryzasativavar.japonica
Betv1like Vitisvinifera;Selaginellamoellendorffii
Bromelain Ananascomosus
Calcium-binding Amba9;Amba10 Arachishypogaea;Betulapendula;Glycinemax;Juniperusoxycedrus;
Medicagotruncatula;Oryzasativavar.japonica;Vitisvinifera
Cystatin AmbaCPI Actinidiadeliciosa
Defensin-like Amba4
Esterase Heveabrasiliensis;Oryzasativavar.japonica
Expansin Solanumlycopersicum
FAD-linkedoxidoreductaseBG60 Cynodondactylon
L-ascorbateoxidase-likeproteingrass Cynodondactylon
pollengroup4allergen
LTP Platanusorientalis
Olee1-like Arabidopsisthaliana;Ricinuscommunis
Papain Caricapapaya
Pectatelyase Amba1 Arabidopsisthaliana;Artemisiavulgaris
Pectinacetylesterase Arabidopsisthaliana
Pectinmethylesterase Elaeisguineensis;Salsolakali
Polygalacturonase Elaeisguineensis;Olatanusxacerifolia
PR-1 Artemisiavulgaris;Oryzasativavar.japonica
Profilin Amba8,AmbaD03
Proteinkinase Salsolakali
LEAIII-likepollenallergen Corylusavellana
Smallrubberparticleprotein Heveabrasiliensis
Thioredoxin;peroxiredoxin Arabidopsisthaliana;Brassicarapa
Vilin Nicotianatabacum
Xyloglucanendotransglycosylase Cryptomeriajaponica
Table3showstheproteinfunctionandcommonnamesofAmbrosiaallergensdetectedby454-sequencinginragweedpollen.Alsoplantswithhomologiestoother
knownallergensareindicated.Onlyfirstbesthitswithe-value#1e25wereconsideredassignificant.ForfurtherdetailseesupplementaryTableS8.
doi:10.1371/journal.pone.0061518.t003
repressed, whereas a glycosyl hydrolase found in the BIN ‘‘cell supposedly leads to a more rigid form of pectin [59].It isknown
wall.degradation.cellulase’’ was strongly induced due to ozone that the PME de-esterified homogalacturonan can bind class III
fumigation.Similartoresultsintheliterature,someESTsforthe peroxidasesthatmightinitiateligninpolymerisation[60]andthat
cell wall-modifying enzyme xyloglucan endotransglycosylase- PMEs react to several biotic and abiotic stresses summarised by
hydrolase (XTH), playing a role in plant development, were Pelloux etal. (2007) [61]. Because the carbon source is limited in
elevated [19,56]. With regard to XTH, it has been shown that it pollen, it appears to be that the only defence or antioxidative
reacts to a broad range of abiotic stresses, and it is influenced by mechanisms that are activated are not cost or energy intensive.
thestresshormoneethylene,suggestingaroleintheearlyeventsof Therefore,itispossiblethatthereconstructionorstiffeningofthe
the stress-related defensive response [56]. With ozone treatment, cell wall requires less in energy and carbon sources than the
several ESTs coding for enzymes involved in the biosynthesis of production of flavonoids, isoprenoids or waxes. Moreover,
cell wall precursors of the hemicelluloses and pectins were differences in the rigidity of the cell wall were not detectable by
induced. These might be involved in cell wall repair or in cell our SEM investigations and only a detailed chemical analysis
wall stiffening to provide better protection for the pollen. It has could elucidate thisquestion.
beenshownforseveralplants,suchaspoplar,birchandalder,that ATR-FTIR analysis of elevated ozone- and control-fumigated
cell wall thickening and increased pectin levels of mesophyll cells pollen provided insight for the components of the pollen surface.
occur underozone treatment [57].A large number of transcripts WedetectedaclearreductionintheFTIRbandsofozone-treated
for pectin modifying enzymes, such as pectate lyases and pectin pollen corresponding to glycerolipids or waxes, which is in line
methylesterases,wereincreasedintheozone-treatedpollen.Pectin with results obtained from the transcriptomic analysis. The
might also play a role in pollen protection because pectin reduced intensities of the FTIR peaks corresponding to the
methylesterases (PMEs) are involved in cell wall extension and acetylester of pectin were detected, in contrast to the increased
stiffening[58],andde-esterificationofpectinhasbeenpreviously absorptionofthepyranoidringgroupofpectin,indicatingthatde-
described as taking place during pollen development and esterification may have occurred due to ozone treatment. From
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TranscriptomicAnalysisofRagweedPollen
Figure7.Box-PlotofadirectELISAforthemajorallergenAmb
a 1 of Ambrosiapollen extracts. 50mg ml21 total protein were
coated.Crossindicatesthemeanvalue;ozonen=14,controln=19.
doi:10.1371/journal.pone.0061518.g007
Figure6.Log -foldchanges(RPKM /RPKM )ofknown
2 ozone control
Ambrosiaartemisiifolia allergens. All data presented are isotig Our dataonAmbrosiaplants thatwere fumigatedwithelevated
specificandnormalizedonRPKM. ozone over the entire vegetation period support the idea that
doi:10.1371/journal.pone.0061518.g006 ozoneexhibitsaninfluenceonstress-inducedtranscripts,cellwall
components and wax but no direct influence on the Amb a 1
theFTIR data,itisnot possibletodeterminewhether additional allergen content as tested by ELISA. As it is known from the
de-methylation of pectin took place because lipids are clearly literature that pollen from several plant species grown in urban
reducedduetoozonetreatment,andthecarbonylofthepectin’s habitats show higher allergenicity despite not showing higher
methylesterandthelipid’sacylesterbothcontributetheabsorption allergen or protein content [14,62], it is still possible that ozone-
band at ,1740–20 cm21 (Figure 2). However, as the transcrip- fumigated ragweed pollen shows higher allergenic potential. In
tomicdataalsoindicatede-methylation,itispossiblethatthispeak ozone-treated grass pollen an increase of damaged grain and
corresponds more to pectin than to the lipids. ESTs for wax areleaseofallergencontainingcytoplasmicgranuleswasobserved
biosynthesis are reduced in the ozone-fumigated pollen, and [65].AswedidnotseeanincreaseofdamagedpollenbySEM,an
areductioninwaxcompoundsisalsoseenbyFTIRanalysis.This ozone-induced release of such kind of particles is unlikely.
is concordant with studies on spruce needles that showed Allergenic potential then, has to be tested in a mouse model or
a structural degradation of epicuticular wax and a reduced wax by pricktests.
layeraroundthestomatacellsduetoincreasedozonefumigation
[55].
Supporting Information
Although the transcription level of some isotigs of the major
allergen Amba 1 were elevated inthe pollen fromozone-treated FigureS1 Light conditions (a) and mean temperature/
(80 ppb) plants, there was no significant difference seen in the relative humidity(b) inthechambers.
proteinlevel,astestedbydirectELISA.Thiswasalsoobservedby (PDF)
Pasqualini etal. (2011), who analysed mature pollen after seven
Figure S2 Workflow of the Ambrosia transcriptome
days of ozone fumigation, indicating that Amb a 1 itself is not
analysisfordefiningcommon/differentexpressedgenes
influenced by elevated ozone [23]. Prior studies on the allergen
in ragweed pollen; ozone treatment (80 ppb ozone),
contentofplantsgrownundertheinfluenceofgaseouspollutants
control (40ppbozone).
showed heterogeneous results. In polluted areas Zinnia pollen
(PDF)
showed an increased allergenicity and in Cupressus arizonica pollen
ahigherexpressionofCupa3,aPR-5protein,wasfound[13,62]. FigureS3 ATR-FTIR spectra of lipid/pectinC mixtures
In studies on rye and ryegrass an increased allergen content and at two different weight ratios. a) absorption spectra of the
higher IgE activity was measured, while a decrease amount of lipidpectinCmixturesinarangeofwavenumbersbetween900–
allergenwasdetectedintimothygrass[20,21,63].Studiesonbirch 3050cm21. Lipid/pectin mixture 1/500 w/w is given in black;
trees from urban and rural areas demonstrated no differences in themixture1/1000w/wisgiveningrey.b)differenceabsorption
theallergencontentofthemajorbirchallergen(Betv1,2,3and spectra of lipid/pectinC mixture (1/500w/w) minus lipid/
4), but showed higher chemotactic activity on human neutrophil pectinC (1/1000 w/w).
granulocytesfortheurbansamples,leadingtotheconclusionthat
(PDF)
greater allergenicity depends on more than just the allergen
content [14]. Recent results for ragweed pollen sampled along Figure S4 Typical RP-HPLC diagram of water-soluble
high-trafficroadsshowedpartiallyhigherIgEactivityagainstAmb and methanol-extractable metabolites (a–f) and accu-
a1,2,6and10.However,nocorrelationtothemeasuredozone mulation of individual metabolites (g–j) from ragweed
concentration in the different areas and the pollen allergenicity pollen.
was obvious[64]. (PDF)
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