Ragweed (Ambrosia artemisiifolia)

16 Pages·2014·0.79 MB·English

ElKelishetal.BMCPlantBiology2014,14:176
http://www.biomedcentral.com/1471-2229/14/176
RESEARCH ARTICLE Open Access
Ragweed (Ambrosia artemisiifolia) pollen
allergenicity: SuperSAGE transcriptomic analysis
upon elevated CO and drought stress
2
Amr El Kelish1,2, Feng Zhao1, Werner Heller1, Jörg Durner1,3, J Barbro Winkler4, Heidrun Behrendt5,6,
Claudia Traidl-Hoffmann6,9, Ralf Horres7, Matthias Pfeifer8, Ulrike Frank1,6* and Dieter Ernst1,6
Abstract
Background: Pollen ofcommon ragweed (Ambrosia artemisiifolia) is a main cause of allergic diseases inNorthern
America. The weed has recently become spreading as a neophyte inEurope,while climate change may also affect
thegrowth of the plant and additionally may also influence pollen allergenicity. To gain better insight in the
molecular mechanisms inthe development of ragweed pollen and its allergenic proteins under global change
scenarios, wegenerated SuperSAGE librariesto identify differentially expressed transcripts.
Results: Ragweed plants were grown in a greenhouse under 380ppm CO and under elevated level ofCO
2 2
(700ppm). In addition, drought experiments under both CO concentrations were performed. The pollen viability
2
was not altered under elevated CO ,whereas drought stressdecreased itsviability.Increased levels of individual
2
flavonoid metabolites were found under elevated CO and/or drought.Total RNA was isolated from ragweed
2
pollen, exposedto thefour mentioned scenarios and four SuperSAGE libraries were constructed.The library dataset
included 236,942 unique sequences, showingoverlappingas well as clear differently expressed sequence tags
(ESTs). The analysis targeted ESTs knownin Ambrosia, as well as in pollen of other plants. Among theidentified
ESTs, those encoding allergenic ragweed proteins (Amb a) increased under elevated CO and drought stress. In
2
addition, ESTs encoding allergenic proteins in other plants were also identified.
Conclusions: The analysis ofchanges inthe transcriptomeof ragweed pollen upon CO and drought stress using
2
SuperSAGE indicates that under global change scenarios the pollen transcriptomewas altered, and impacts the
allergenic potentialof ragweed pollen.
Keywords: Ambrosia artemisiifolia,Allergen,Allergy,CO ,Drought, Flavonoids, Pollen, Ragweed, Scanningelectron
2
microscopy, Transcriptome
Background began approximately 100 years ago and is currently pri-
Pollen ofthe commonragweed(Ambrosiaartemisiifolia) marily found in the Rhône valley, Hungary, Croatia,
is a main cause of allergic diseases in Northern America Bulgaria,NorthernItalyandEasternAustria,butitisalso
[1,2]. This species is the most widespread Ambrosia and spreading in Germany [4,5] (http://www.ambrosiainfo.de/
theweedhasbecomespreadingasaneophyteinEurope, 53223897640d5c602/index.html).
and will become a serious health problem in sensitized Sofar,theallergenicproteinsofragweedcanbearranged
populations [3]. The distribution of ragweed in Europe into six biological groups [3,6]. Approximately 48 allerge-
nic proteins are known for the genus Ambrosia, and 32
proteins, including multiple isoforms, are known for A.
*Correspondence:[email protected]
1InstituteofBiochemicalPlantPathology,HelmholtzZentrumMünchen, artemisiifolia (http://www.allergome.org). The major aller-
GermanResearchCenterforEnvironmentalHealth,85764Neuherberg, gen of ragweed is Amb a 1, an acidic non-glycosylated
Germany 38-kDa protein consisting of a 26-kDa α-chain and an
6CK-CARE,ChristineKühne–CenterforAllergyResearchandEducation,Davos,
associated 12-kDa β-chain [3].
Switzerland
Fulllistofauthorinformationisavailableattheendofthearticle
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Itishypothesizedthatclimate changeandairpollution [43,44].Inhuman health, IgE-bindingofallergens may be
will affect the allergenic potential of pollen, either by a influenced by attached flavonoids [45,46]. The patho-
changed pollen season, by a changed pollen amount, by genesis-related proteins (PRs) consist of a large group of
changes of the surface exine or by directly increasing the homologous proteins in different plant species and many
allergenic transcripts and proteins and interactions with PRs are expressed in pollen and can act as allergens [47].
biologically important ligands, e.g., flavonoids [2,7-11]. A direct interaction of birch PR-10c with biologically im-
Studiesoneffectsonclimatechangeonrespiratoryallergy portant molecules, including flavonoids, was shown by
arestilllackingandonlyafewepidemiological reportson Koistinen et al. [48]. Similarly, flavonoids bind to the
urbanization and air-pollution on pollen allergenicity are majorbirchallergenBetv1[9],whichalsobelongstothe
available [12]. An overview for risk factors on allergic PR-10family[49].Recentlyitwasshown,thataquercetin
disease discussing genetics aspects, indoor and outdoor derivativedirectlybindstotheC-terminalhelixofBetv1,
pollution,socio-economicfactors,climatechangeandmi- and that this binding plays an important role during the
gration has recently been published [12]. The proteomic inflammation response [50]. These results indicate
profiling of birch pollen isolated from different sites indi- that, in addition to allergenic proteins, additional aller-
cated differences between allergenic and non-allergenic genic carriersmay also be involved inpollen allergenicity,
proteins [13]. In contrast, birch pollen isolated from an which is not exclusively triggered by known allergenic
urban and rural site showed no difference in allergenic proteins[14,51,52].
protein expression, indicating that allergenicity is deter- These studies suggest that global change will affect the
minedbyadditionalallergencarriers[14].Aninvivostudy allergenic potential of pollen and play a role in human
on birch pollen also sampled from different sites could health diseases related to allergic rhinitis and asthma.
correlate elevated ozone levels to higher allergenicity as From this perspective, a transcriptome-wide analysis of
well as to an increased allergen content [15]. It was re- thehighly allergicpollenofragweedwould notonlyhelp
cently shown that twice ambient ozone levels resulted in in understanding climate impact on expressed pollen
anincreasedcontentofallergenicproteinsintworye cul- transcriptsbutalsogainadeeperinsightintotheexpected
tivars[16].Inragweed,elevatedozonefumigationresulted changes of pollen allergens. Flavonoids analysis will allow
in a changed transcriptional profile, including transcripts a better understanding of their possible function as add-
for allergenic proteins [17]. Elevated CO concentrations itional allergenic carriers and also contribute to the rele-
2
showed an increase growth of ragweed and its pollen vant UV-B-absorbing metabolites of pollen. In a previous
production [18-21], and an increased content of Amb a 1 study, we showed that twice the ambient level of ozone
allergenwasobserved[22]. resulted in a changed transcriptional profile of ragweed
In addition to increasing CO concentrations, future pollen, including encoded allergenic proteins [17]. In this
2
atmospheric warming, as well as hot and dry summer study,wemodifiedtheglobalclimatechangeapproachby
periods are also expected [23,24]; IPPC Report 2007. linking the transcriptional network changes of ragweed
Regulatory networks in cellular responses to drought, pollen to elevated CO concentrations and an extreme
2
including abscisic acid-dependent and -independent drought event. We highlight that the global change sce-
systems, are well known during plant growth and narios willaffect the transcriptomeof pollenand willalso
development [25-30]. Regarding transcriptomic and increase the abundance of allergen-related transcripts
proteomic analyses of pollen, literature reports have relevantforhumanhealth.
focused on different developmental stages of pollen,
mature pollen and pollen germination [31-36]. Regard- Results and discussion
ing temperature effects, differentially cold-regulated Morphologicalparametersandpollenviability
genes were detected in mature pollen of Arabidopsis Two main different leaf morphologies between the plants
thaliana [37]. were observed: plants with strong pinnate leaves (i) and
Flavonoids are ubiquitous plant secondary metabolites plants with only weak pinnate leaves (ii), as has been re-
andareimportantinplantdevelopmentandreproduction, ported for ragweed with the same genetic background in
as well as in protection against abiotic and biotic stress exposurechambers[21].
factors [38,39]. The yellow color of pollen can be traced Pollen viability was slightly reduced under elevated CO
2
backtoflavonoids,thusshieldingthepollengenomefrom levels; however, this result was not statistically significant
UV-B radiation [40]. In addition, flavonoids play a role in (Additional file 1). Similarly, it was shown that the pollen
male fertility, and quercetin is an important germination- performancedecreased in Raphanussativusinresponseto
inducing compound in maize and petunia but not in elevated CO levels [53]. Drought stress resulted in a re-
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Arabidopsis or parsley [41,42]. Flavonoids may be invol- duction of the pollen viability from approximately 46% to
vedinthemodulationofimmuneresponsesandthusmay 24%(Additionalfile1).Thedecreasedpollenviabilityunder
also be important in the allergenic response of pollen drought stress is in accordance with several literature
ElKelishetal.BMCPlantBiology2014,14:176 Page3of16
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reports also demonstrating a reduced viability and pollen derivative).Thischangeinindividualmetabolitesisincon-
grain production [54-57]. Interestingly, this drought effect trasttopollenofozone-fumigatedragweedthatshowedno
could be partially mitigated by elevated CO with a slight change of such individual metabolites. Flavonoids have
2
increase from 24% to 30% (Additional file1), indicating no been shown to accumulate under drought stress inseveral
additiveeffectsofelevatedCO anddrought. plants, thus playing a physiological role in water tolerance
2
and protection against oxidative stress [58-60]. Moreover,
Secondarymetabolites detailedanalysesshowedthatthelevelofquercetinderiva-
Typicalreverse-phasehigh-performanceliquidchromatog- tives also increased upon drought stress in different plants
raphy (RP-HPLC) diagrams for water soluble metabolite [60-62],clearlyindicatingthatinpollenofdrought-stressed
extractsrevealed17compounds,withthehighestamounts plants, the accumulation of individual flavonoid metabo-
in particular for metabolite 12 and 17, both are quercetin litesmayplayaprotectiveroleagainstoxidativestressand
derivatives and methanolic extracts showed 12 different damage of the pollen tissue. Elevated CO resulted in
2
metabolites, congruent to data given by Kanter et al. [17] increased levels of flavonoid metabolites in several plant
(Additional file 2). The total amounts of individual com- species[63-65].Inragweedpollen,themetabolitelevelwas
pounds for the final harvest are given in Figure 1. No sig- approximately at the same levels under drought, irrespec-
nificant changes could be observed between the control, tively of the CO concentration (Figure 1). Thus, drought
2
elevated CO , drought and elevated CO plus drought mightbe more important than elevated CO in increasing
2 2 2
samples, similar to what has been described for ozone- thelevelsoftheseindividualmetabolites.Asinglemetabol-
treated pollen. However, individual metabolites of the PBS ite (DA 5) was also increased under CO treatment alone
2
extract showed increased levels upon drought stress at (Figure 1), similar to theimpactof CO in soybean, where
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both CO concentrations (Figure 1a; DA1, DA3, DA5, theconcentrationofonlyoneflavonoid,aquercetinglyco-
2
DA10, DA13 (quercetin derivative) and DA16 (kaempferol side, was also increased [66]. This result indicates species-
(a)Watersolubleextracts (b)Methanolicextracts
34DA1 * 180 DA2 456DA3 * 22..0500 DA1 6DA2 1102DA3
6 4 8
2 3 1.50
4 6
ht[a.u.] 101024685DDAA47 102024680 DA5 * * * 110120500DDAA86+9 freshweight[a.u.] 001114...505005000000000 DDAA74+8 123400000200000DA5 123450000020240000005DDAA69
shweig 150 2468 ea/mg 123000000 1125050
ma/gfre 11005DA10 * 234000 DA11 2345000000000DA12 aPekar 4680000 DA10 0.046DA11 115000000Sum
are 5 10 100 20 2 500
ak 06DA13 * 400 DA14 205DA15 0 0 0
Pe 4 30 20
15
20
2 10
10 5
0 0 0
200DA16 * 250 DA17 1000Sum
150 200 800
150 600
100
100 400
50 50 200
0 0 0
Figure1AmountofPBS-soluble(a)andmethanolic-extractable(b)phenolicmetabolitesinragweedpollen.Theseparationwasperformed
byRP-HPLC.Thebars(N=5)indicateSDandsignificantdifferencesareindicatedbyanasterisk.
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specific CO responses in flavonoid content and compos-
2
a
ition[67,68].
control CO
2
69,150 tags 182,736 tags
4,303 40,782
SuperSAGEdataset
Thenumberofsequencedtagsrangedfromapproximately 11,220
4.5 × 106 to 17.2× 106 in the four libraries (Additional 4,532 43,533
file 3, Info). The tag frequencies are given in Additional
5,928 10,049
file 3 (All_Libs20101207). The SuperSAGE dataset inclu-
8,555 15,914
ded 236,942 different non redundant sequences (tags) of
26 bp in length (Additional file 3, All_Libs20101207). For 25,522
each of these sequences (tag), the tag amounts are pro- 21,635 4,731
vided and count how often a unique sequence was found 24,037 2,835
drought CO + drought
in each of the four libraries. One sequence (tag) can be 2
106,410 tags 13,336 139,987 tags
foundinone,two,threeorallfourofthelibraries,asindi-
cated in the overlappingregions in Figure 2a but, accord-
ingtothetranscriptexpression,indifferentquantities(tag
amounts). The sequenced tag counts for each unique b
control CO
sequence in all of the libraries ranged from≤50 (low), 27,570 tags 32,6218 tags
50–500 (mid), 500–5000 (high) and≥5000 (very high) 1,409 1,535
(Table 1). The normalized values of each tag in relation 1,229
to 106 tags (tpm) for each library resulted in approxi- 1,106 1,905
mately 99.5% of low- and mid-abundant unique tags,
1,874 2,702
while high- and veryhigh-abundant tagsrepresented only
1,243 1,062
approximately0.2%-0.4%(Table1).Asimilardistribution
of abundant classes has also been reported for other 17,457
SuperSAGE libraries [69-71]. The four libraries had 1,226 754
approximately the same unique sequences for the very 4,690 1,039
high-abundant class (31–37), the high abundant class drought CO2 + drought
(239–270) and the mid-abundant class (863–1129). In 29,625 tags 990 30,599 tags
contrast, the low-abundant class was more variable,
reflecting also the total number of unique sequences of
each library (Table 1). According to the cumulative fre- c
control CO
quencydistribution,onlythosetpmvaluesgreaterthan0.6 2
6,191 tags 443 421 6,534 tags
to 0.8canbe consideredexpressed [72] (Additional file 4).
291
Therefore, transcripts with a tpm threshold < 0.8 were
eliminated, resulting in more stringent values, coming up 411 325
with40,221uniquesequences(Figure2b).Finally,weelim-
inated all of the sequences with the description ‘no hits’ 352 338 475 270
and the score of the BLAST hit was set to≥40. These
3,557
parameters resulted then in 9,078 unique sequences and
287 276
an equal distribution in all 4 of the libraries (Figure 2c).
Thelow-abundancesequenceswerestronglyreducedinall 840 400
of the libraries to approximately 90.0%, whereas those drought 392 CO + drought
2
6,577 tags 6,535 tags
sequences in the mid- and high-abundant groups strongly
increased up to 10% (Table 2, Figure 3). Additionally,
Figure2Venndiagram.Numberofcommonandunique
MapMan was used to group the SuperSAGE tags into SuperSAGEsequencetags.Foreachsequence,thetagamountinthe
several functional categories (BIN-codes) [73]. For this individualsampleswasanalyzed.Sequenceswith≥1appearancesin
grouping, the SuperSAGE tags werematched to Ambrosia two,threeorallofthesamplesareshownbyindividualoverlapping
regions.Thetotalnumberofsequencetagsperlibraryisindicated.
454-transcriptome data (contigs + singletons) [17]. The
areflectsthedistributionofsequencetagsintheoriginaldataset.
data were then BLASTed against Arabidopsis (TAIR) to
bgivesthedistributionofsequencedtagsfilteredfortpm>0.8.c
identify Arabidopsis homologues, which then could be indicatesthesequencetagdistributionforastringentlyfiltereddataset
sorted to the BIN-codes (workflow: Additional file 5) and withthefollowingcriteria:tpm>0.8;scoreofBLASThit>40;and
onlylog -foldchangesofatleast1.5werefurtherexamined removalofsequencetagswithoutBLASTresult(“nohit”).
2
(Additional file 6). Interestingly, elevated CO + drought
2
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Table1Distributionoflow-toveryhigh-abundantsequencesdetectedinthefourSuperSAGElibrariesfromthecontrol
(380ppmCO ),CO (700ppmCO ),CO plusdroughtanddrought
2 2 2 2
Library Control(380ppm) CO (700ppm) CO +drought Drought
2 2
#detectedsequences 69,150 182,736 139,987 106,410
Abundanceclassesofdetectedsequences
#veryhigh-abundant: >5000tpm 37(0.05%) 31(0.02%) 34(0.02%) 34(0.03%)
#high-abundant: 500–5000tpm 239(0.35%) 252(0.14%) 270(0.19%) 263(0.25%)
#mid-abundant: 50–500tpm 863(1.25%) 1,092(0.60%) 1,126(0.81%) 1,005(0.95%)
#low-abundant: <50tpm 68,013(98.36%) 181,361(99.25%) 138,557(98.98%) 105,108(98.78%)
Totalamountofdetectedsequencespertreatmentareindicatedand%-valuesarerelatedtothesetotalnumberofsequences.
conditions resulted in higher log -fold changes compared differentially expressed genes were identified that were
2
to the single treatments, indicating additive effects. mainlyrelatedtoanAmbrosiatrifidapollencDNAlibrary.
Transcripts with homologies to abiotic stress were mainly All of the genes with a clear homologue and not only the
up-regulated under all three scenarios, including also description pollen cDNAlibrary arelistedinTable 3.The
dehydration-responsive transcripts, heat-shock proteins termragweed resulted in 48differentially expressed genes
andchaperones.Regardingdroughtstress,thisresultisnot that were also found in the Ambrosia search. The search
surprising and has also been reported in the literature term “pollen” showed 48 hits that were primarily related
[26,30,74,75]. For the BIN-name cell wall, a pectate lyase to an Ambrosia trifida pollen cDNA library and thus also
family member and expansin were clearly up-regulated. presentintheAmbrosiasearch.Forpollen,wealsocarried
Pectatelyasesareimportantforpollentubegrowthbypec- out a search for exine, intine, extensin, coat and cell wall.
tindegradation.However,inragweedpollen,pectatelyases However, no additional hits were found. Searching for
belongtothemajorallergenAmba1family(AllFamdata- “allergen” identified 4 Ambrosia genes, a calcium-binding
base; http://www.meduniwien.ac.at/allergens/allfam/chart. protein isoallergen 1, Amb a 1.1, Amb a 1.2 and Amb a
php?kingdom=Plants&exposure=Inhalation&list=10&- 1.3 that were all up-regulated under drought stress
page=0). Expansins are important for the pollen tube and (Table 3). In total, we could identify eight transcripts for
for cell wall changes and confer drought tolerance [76,77]. allergenic proteins from A. artemisiifolia: two calcium-
Moreover, expansins also belong to pollen allergens (All- binding proteins (EF hand domain, Amb a 9 and Amb a
Fam database). The most strongly up-regulated transcript 10), pectate lyases (Amb a 1.1, Amb a 1.2, Amb a 1.3
(CER1) in all three of the treatments is involved in wax and Amb a 1.2 precursor), an actin-binding protein
biosynthesis(log -fold5.3-9.2).CER1ismainlyexpressed (profilin-like) and a cystatin proteinase inhibitor (Amb
2
in inflorescences and siliques and is induced by osmotic a CPI) (shown bold in Table 3). Except for the transcript
stress [78]. This result demonstrates that wax biosynthesis of the Amb a 1.2 precursor protein, all of the other
isenhancedunderclimatechangescenarios. transcripts were up-regulated under drought. However,
Wealsoperformedapairwisecomparisonofthelibraries four of these transcripts were below the threshold of
according to possible global change scenarios: control vs. 1.5-fold (log =0.59). The transcript for a homologue
2
drought (AmK vs. AmT), control vs. elevated CO (AmK of a down-regulated ABA-responsive HVA22 protein
2
vs.AmC)andcontrolvs.CO +drought(AmKvs.AmCT). from A. trifida was found in very high abundance (more
2
Using the STDGE2GO-Tool kit, we first searched the than 10,000 tpm). In vegetative tissue, the HVA22 genes
AmK vs. the AmT library for the following parameters: are expressed in different tissues and show high levels of
Ambrosia, ragweed, pollen, extensin, exine, intine, cell expression in flowers and inflorescences [79]. Drought
wall, coat and allergen. Using the term Ambrosia, 50 stress suppressed HVA22a and HVA22c expression, had
Table2Distributionoflow-abundantsequencesfounduniquelyundercontrolconditions(380ppmCO ),under
2
elevatedCO (700ppmCO ),underelevatedCO plusdroughtanddrought(380ppmCO ),orfoundtobecommonin
2 2 2 2
allfourSuperSAGElibrariesatonetime
Library Control(380ppm) CO (700ppm) CO +drought Drought(380ppm) Alllibraries
2 2
#lowabundantuniquetags
Originaldataset 4,260(99.00%) 40,685(99.76%) 15,865(99.69%) 8,480(99.12%) 24,131(94.55%)
tpm>0.8 1,366(96.95%) 1,438(93.68%) 1,013(95.39%) 1,168(93.97%) 16,036(91.86%)
tpm>0.8;score>40;w/o“nohits” 429(96.84%) 374(88.84%) 254(94.07%) 334(94.89%) 3,207(90.16%)
Datawereanalyzedfordifferentfiltercriteria.
ElKelishetal.BMCPlantBiology2014,14:176 Page6of16
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cansensecellenergylevelsandregulateredoxhomeostasis
original tpm >0.8 tpm >0.8; score >40; w/o “no hits”
[82,83]. These proteins are important for stress regulation
100
and corresponding genes are up-regulated upon drought
%] stress[84].
s [90 Next, we searched the AllFam database of allergen
e
c families, restricted to plants and inhalation. This search
n
e included 59 allergen families with 233 allergens (http://
u10
seq 8 www.meduniwien.ac.at/allergens/allfam/chart.php?king-
# of 46 dthoims s=ePalracnht,st&heexpp-ovsauluree=wInahsasleattitoon&<lEis−t1=01,0e&xcpeapgtef=o0r).saIfn-
2 flower, which belongs also to the Asteraceae. In addition
tothe known allergens found under the Ambrosia search,
0000 0000 0000 0000 0000
5000 5000 5000 5000 5000 eight transcripts for putative allergenic proteins from
<500 <500 <500 <500 <500
50-0-5>5 50-0-5>5 50-0-5>5 50-0-5>5 50-0-5>5 other plants according to the Allfam database were iden-
0 0 0 0 0
5 5 5 5 5 tified (Table 3). Seven of these transcripts were clearly
common control CO CO+drought drought
in all only only2 2only only up-regulated under drought, at least by a three-fold
log change. In contrast, a homologue to an aspartic pro-
Figure3Distributionoflow-toveryhigh-abundantsequence 2
teinase precursor from maize was down-regulated. The
tags.Thetagswerefounduniquelyundercontrolconditions
(380ppmCO),underelevatedCO (700ppmCO),underCO plus highestabundanceswereseenforthetranscriptshomolo-
2 2 2 2
droughtorunderdrought,orfoundtobecommoninallfour goustoaprofilinofrubbertree,anacidicchitinaseofjelly
librariesatonetime.Thedatawereanalyzedforthedifferentfilter fig and a safflower carbonic anhydrase. As pathogenesis-
criteriaindicatedinthegraph.
related (PR) proteins are known to be allergenic, we also
searchedforthisterm,comingupwitha singlehitforPR
littleeffectonHVA22eexpressionandenhancedHVA22d 5–1 homologue of sunflower, which was up-regulated
expression in the inflorescent stems of Arabidopsis [79]. under drought(Table 3). However, it is important tonote
Nochangesoronlysmalleffectscouldbeobservedinthe that the abundances of all of these transcripts are low as
flowerbuds,exceptforaslightenhancementofexpression comparedtothe‘Amba’abundancesinragweedpollen.
under drought stress[79].In accordancewithourresults, The search of the AmK vs. the AmC library was per-
this result indicates that in addition to stress, the HVA22 formed for the terms given above. Under elevated CO
2
genes may also be important for the reproduction of concentration, the term Ambrosia resulted in 62 differen-
plants. A homologue for a putative pollen-specific tran- tiallyregulatedtranscriptsthatwerealsomainlyrelatedto
script from A. trifida was also found in high abundance anA.trifidapollencDNAlibraryandthespecifiedhomo-
andwasdown-regulatedbydrought.Otherpollen-specific logues are given in Tables 4 and 5. A search for ragweed
sequences were homologues to a pistil- and pollen-ex- resulted in 57 transcripts that were already present in the
pressedgenefromsunflower(SF21),apollencoatprotein Ambrosiasearch.Underthesearchforallergen,fivegenes
transcript from wild cabbage and a pollen-specific actin- ofA.artemisiifoliawereidentified: Amba1.1,Amba1.2,
depolymerisingfactorfromtobaccothatwerebothdown- Amba1.3andcalcium-bindingproteinisoallergen1were
regulated. SF21 belongs to a gene family expressed in up-regulatedunderelevatedCO ,whilethelow-abundant
2
pollen and pistil in angiosperms and the encoded protein profilinisoallergen1wasdown-regulated(Tables4and5).
isimportantforpollen-pistilinteractions[80,81];however, ThisincreaseinAmba1transcriptsisinaccordancewith
themolecularfunctionisstillunknown.Asearchwiththe anincreased levelofAmb a 1 protein content inragweed
term “drought” resulted in 38 transcripts to homologues pollengrownunderincreasedCO concentrations[22].In
2
of a drought-stress subtracted cDNA library of safflower, total,ninetranscriptsfor allergenicproteinsfromA.arte-
also belonging to the Asteraceae and 35 of these cDNAs misiifolia were identified: two calcium-binding proteins
were up-regulated in drought-stressed ragweed pollen. (Amb a 9, Amb a 10), pectate lyases (Amb a 1.1, Amb a
Among these cDNAs, homologues to a carbonic anhy- 1.2,Amba1.3andAmba1.2precursorprotein),acysta-
drase3,acyclophilinandaplastocyanin,proteinsthatare tin proteinase inhibitor (Amb a CPI), a profilin allergen
known to be allergenic, showed highly up-regulated (Amb a 8.1) (shown bold in Tables 4 and 5). Seven of
transcripts (AllFam database; (http://www.meduniwien.ac. these transcripts were up-regulated and two were down-
at/allergens/allfam/chart.php?kingdom=Plants&exposure= regulated (Amb a CPI and Amb a 8.1) under elevated
Inhalation&list=10&page=0).Interestingly,ahighlyup-re- CO . However, for two transcripts, the log fold change
2 2
gulated transcript for a CBS (cystathionine β-synthase) was below the threshold (Amb a CPI and Amb a 1.2
domain-containing protein homologue of A. trifida was precursor). Although at low abundance, the transcript
detected (log =9.01). CBS domain-containing proteins homologous to a lipid transfer protein (LTP) from A.
2
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Table3Up-anddown-regulatedtranscriptsinpollenofragweedfromcontrolanddroughtstressedplants
Database- Database Description Normalizedtagspermillion p-value Fold
id change
380ppmCO 380ppmCO +drought
2 2 (log )
2
TC52169 Asteraceae_TIGR A.artemisiifolia,calcium-binding,pollen 25.43 165.44 0 +2.70
allergenAmba9.1
296281908 Asteraceae A.trifida,putative60SribosomalproteinL34 685.5 1863.05 0 +1.44
255779233 Asteraceae A.trifida,ß-glucosidase 196.37 391.4 0 +1.00
255779153 Asteraceae A.trifida,SF16protein 796.31 1293.52 0 +0.70
296281890 Asteraceae A.trifida,conservedhypotheticalprotein 1463.21 2371.92 0 +0.70
302127809 GDB A.artemisiifolia,pollenallergenAmba1.1 615.23 852.94 0 +0.47
296281926 Asteraceae A.trifida,putativepollen-specificprotein 8292.9 4704.67 0 −0.82
166438 GDB A.artemisiifolia,Amba1.2precursor 372.17 184.79 0 −1.01
protein
255779319 Asteraceae A.trifida,abscisicacid-responsive 22129.39 10765.07 0 −1.04
HVA22familyprotein
255779264 Asteracea A.trifida,hypotheticalprotein 433.43 159.72 0 −1.44
283962764 Asteraceae A.trifida,conservedhypotheticalprotein 410.87 96.73 0 −2.09
296281756 Asteraceae A.trifida,putativeribokinase 71.21 0.05 0 −10.48
296281901 Asteraceae A.trifida,putativeCBSdomain-containing 0.05 25.69 6.64e-39 +9.01
protein
296281858 Asteraceae A.trifida,unnamedproteinproduct 18.8 62.53 1.70e-29 +1.73
296281775 Asteraceae A.trifida,putativegolgin-84-likeprotein 44.45 10.68 1.53e-28 −2.06
62249490 GDB A.artemisiifolia,calcium-binding,pollen 82.71 155.07 3.32e-27 +0.91
allergenAmba10
190607111 GDB A.trifida,gibberellin-regulatedprotein 24.99 2.79 1.27e-26 −3.17
255779170 Asteraceae A.trifida,photosystemIreactioncenter 1.11 18.42 1.32e-21 +4.06
subunitK
255779131 Asteraceae A.trifida,aminoacidtransporter 409.32 300.55 2.52e-21 −0.45
437311 Asteraceae A.artemisiifolia,cystatinproteinaseinhibitor 1459.28 1657.06 3.42e-16 +0.18
296281781 Asteraceae A.trifidaputativeepoxidehydrolase 0.05 9.13 2.99e-14 +7.51
302127815 GDB A.artemisiifolia,pollenallergenAmba1.3 113.89 168.07 1.21e-13 +0.56
296281905 Asteraceae A.trifida,clathrinassemblyprotein 1025.19 896.55 1.15e-11 −0.19
296281843 Asteraceae A.trifida,unnamedproteinproduct 1.99 11.61 1.03e-09 +2.54
296281917 Asteraceae A.trifida,DNA-directedRNApolymerasefamily 0.89 8.82 1.53e-09 +3.32
296281822 Asteraceae A.trifida,putativesignalpeptidase 1.55 8.82 1.45e-07 +2.51
255779252 Asteraceae A.trifida,60SribosomalproteinL38 1.77 9.13 2.23e-07 +2.37
302127811 GDB A.artemisiifolia,pollenallergenAmba1.2 30.08 47.36 6.73e-06 +0.66
34851181 GDB A.artemisiifolia,profilin-likeprotein(D03) 40.25 58.81 1.90e-05 +0.55
TC40290 Asteraceae_TIGR Pollen-specificproteinSF21(Helianthusannuus) 11.28 508.09 0 +5.49
TC52779 Asteraceae_TIGR Pollen-coatprotein(Brassicaoleracea) 710.51 80167 0 −3.15
DY921400 Asteraceae_TIGR Pollen-specificactin-depolymerizingfactor2 21.23 7.12 2.24e-10 −1.58
(Nicotianatabac.)
DC239985 Asteraceae_TIGR Profilin-6(Heveabrasiliensis) 0.66 33.12 7.01e-45 +5.64
TC8863 Asteraceae_TIGR α-Expansinprecursor(Nicotianatabacum) 1.77 17.02 3.82e-17 +3.27
33323054 GDB Acidicchitinase(Ficusawkeotsang) 0.05 46.89 0 +9.87
261291803 Asteraceae Cyclophilin(Carthamustinctorius) 0.05 18.11 1.39e-27 +8.50
195607463 GDB Asparticproteinasenepenthesin-2 24.33 0.05 3.32e-43 −8.93
precursor(Zeamays)
28959515 Asteraceae Carbonicanhydrase3(Carthamustinctorius) 0.05 194.23 0 +11.92
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Table3Up-anddown-regulatedtranscriptsinpollenofragweedfromcontrolanddroughtstressedplants(Continued)
FS486814 Asteraceae_TIGR 2-Cysperoxiredoxin-likeprotein 0.66 17.02 9.27e-22 +4.68
(Arabidopsisthaliana)
289595531 Asteraceae Plastocyanin(Carthamustinctorius) 0.05 5.42 1.01e-08 +6.76
TC5518 Asteraceae_TIGR Pathogenesis-relatedprotein5–1(Helianthusannuus) 7.96 29.87 1.31e-16 +1.91
Plantsweregrowninthegreenhouseundercontrol(380ppmCO)anddroughtstress(380ppmCO +drought).UsingtheSTDGE-toolkitfromGenXProdata
2 2
werefilteredforthetermsAmbrosia,ragweed,pollen,extensin,exine,intine,cellwall,coat,allergenandtheAllfamdatabase.KnownallergenicproteinsinAmbrosiaare
showninbold.
trifida was highly up-regulated (log =9.2) under elevated for an abscisic acid-responsive HVA22 transcript of A.
2
CO . LTPs are basic proteins that are abundant in higher trifida was found in high abundance and was down-
2
plants [85]. These proteins belong to the prolamin super- regulatedunderelevatedCO concentrations(Table4).The
2
family and their role in allergenicity has been reviewed transcript for the homologue of a putative pollen-specific
recently[86].Similartothedroughtlibrary,thehomologue protein from A. trifida waspresent inveryhighabundance
Table4Up-anddown-regulatedtranscriptsinpollenofragweedfrom380ppmCO (control)and700ppmCO
2 2
concentrationsfilteredforthetermsAmbrosia,ragweed,pollen,extensin,exine,intine,cellwall,coat,allergenand
theAllfamdatabase
Database-id Database Description Normalizedtagspermillion p-value Fold
change
380ppmCO 700ppmCO
2 2 (log )
2
296281908 Asteraceae A.trifida,putativeribosomalproteinL34 685.524 0.05 0 −13.74
255779233 Asteraceae A.trifida,ß-glucosidase 196.37 0.05 0 −11.94
296281756 Asteraceae A.trifida,putativeribokinase 71.21 0.17 0 −8.68
255779271 Asteraceae A.trifida,conservedhypotheticalprotein 410.97 115.33 0 −1.83
255779264 Asteraceae A.trifida,hypotheticalprotein 433.43 249.61 0 −0.8
255779319 Asteraceae A.trifida,abscisicacid-responsiveHVA22family 22129.3 13586.99 0 −0.7
296281926 Asteraceae A.trifida,putativepollen-specificprotein 8282.9 5596.37 0 −0.57
302127809 GDB A.artemisiifolia,pectatelyase,pollenallergenAmba1.1 615.2 1102.65 0 +0.84
166442 GDB A.artemisiifolia,pectatelyase,pollenallergenAmba1.3 113.89 233.1 0 +1.03
62249490 GDB A.artemisiifolia,calciumbinding,pollenallergenAmba10 82.71 218.2 0 +1.40
TC52169 Asteraceae_TIGR A.artemisiifolia,calciumbinding,pollenallergenAmba9.1 25.43 136.31 0 +2.42
255779240 Asteraceae A.trifida,lipidtransferprotein 0.05 29.38 0 +9.20
296281913 Asteraceae A.trifida,putative60SribosomalproteinL36 0.22 137.41 0 +9.28
296281836 Asteraceae A.trifida,putativeo-linkedn-acetylglucosaminetransferase 1.77 25.44 9.34e-34 +3.85
302127811 GDB A.artemisiifolia,pectatelyase,pollenallergenAmba1.2 30.08 71.62 6.40e-27 +1.25
437311 GDB A.artemisiifolia,cystatinproteinaseinhibitor 1459.28 1258.73 2.95e-25 −0.21
255779252 Asteraceae A.trifida,60SribosomalproteinL38 1.77 17.16 1.24e-20 +3.28
296281775 Asteraceae A.trifida,putativegolgin-84-likeprotein 44.45 19.82 3.03e-18 −1.66
34851181 GDB A.artemisiifolia,profilin-likeprotein(D03) 40.25 74.76 4.07e-17 +0.89
255779129 Asteraceae A.trifida,60Sribosomalprotein 1.99 13.56 2.14e-14 +2.77
190607080 GDB A.trifida,putativegalactan:galactangalactosyltransferase 303.84 378.62 3.49e-14 +0.32
166438 GDB A.artemisiifolia,pectatelyase,Amba1.2precursorprotein 372.17 451.88 1.85e-13 +0.28
296281810 Asteraceae A.trifida,putativeribosomalproteinL5 2.65 13.91 7.56e-13 +2.39
255779131 Asteraceae A.trifida,aminoacidtransporter 409.32 337.7 1.27e-12 −0.28
296282845 Asteraceae A.trifida,putativestellacyanin 25.87 49.49 1.30e-12 +0.94
296281843 Asteracea A.trifida,unnamedproteinproduct 0.89 9.22 7.99e-12 +3.38
190607111 Asteraceae A.trifida,gibberellin-regulatedprotein 24.99 10.78 1.28E-11 −1.22
Plantsweregrowninthegreenhouseundercontrol(380ppmCO)and700ppmCO concentrations.UsingtheSTDGE-toolkitfromGenXProdatawerefiltered
2 2
forthetermsAmbrosia,ragweed,pollen,extensin,exine,intine,cellwall,coat,allergenandtheAllfamdatabase.KnownallergenicproteinsinAmbrosiaareshown
inbold.
ElKelishetal.BMCPlantBiology2014,14:176 Page9of16
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Table5Up-anddown-regulatedtranscriptsinpollenofragweedfrom380ppmCO (control)and700ppmCO
2 2
concentrationsfilteredforthetermsAmbrosia,ragweed,pollen,extensin,exine,intine,cellwall,coat,allergenand
theAllfamdatabase
Database-id Database Description Normalizedtagspermillion p-value Foldchange
(log )
380ppmCO 700ppmCO 2
2 2
296281905 Asteraceae A.trifida,putativeclathrinassemblyprotein 1025.19 1139.63 5.92e-11 +0.15
296281822 Asteraceae A.trifida,putativesignalpeptidase 1.548 9.68 3.62e-10 +2.64
296281744 Asteraceae A.trifida,conservedhypotheticalprotein 1.77 9.97 6.62e-10 +2.49
296281890 Asteraceae A.trifida,conservedhypotheticalprotein 1463.71 1586.87 2.83e-09 +0.11
296281917 Asteraceae A.trifida,DNA-directedRNApolymerase 0.89 7.01 1.97e-08 +2.99
familyprotein
TC43769 Asteraceae_TIGR A.artemisiifolia,profilin,pollenallergen 4.64 0.64 3.27e-08 −2.86
Amba8.1
255779153 Asteraceae A.trifida,SF26protein 796.31 879.06 7.45e-08 +0.14
296281873 Asteraceae A.trifida,putativemitochondrialATPsynthase 9.29 20.23 1.94e-07 +1.12
6kDasubunit
255779194 Asteraceae A.trifida,putativecullin-1-protein 0.44 4.87 9.01e-07 +3.46
296281858 Asteraceae A.trifida,unnamedproteinproduct 18.8 32.11 1.20e-06 +0.77
255777293 Asteraceae A.trifida,mitochondrialoutermembrane 3.76 10.43 5.873-06 +1.37
membraneprotein
296281875 Asteraceae A.trifida,signalpeptidasesubunitfamilyprotein 0.05 2.9 1.40e-05 +5.87
296281777 Asetraceae A.trifida,calmodulin-likeprotein 8.85 17.1 2.93e-05 +0.95
255779292 Asteraceae A.trifida,60SribosomalproteinL35a 1.33 0.05 3.33e-05 −4.73
255779177 Asteraceae A.trifida,putativeCREG1 1.33 0.05 3.33e-05 −4.73
TC40290 Asteraceae_TIGR Pollen-specificproteinSF21(Helianthusannuus) 11.28 166.45 0 +3.88
TC52779 Asteraceae_TIGR Pollencoatprotein(Brassicaoleracea) 710.51 539.79 2.22e-39 −0.4
TC5878 Asteraceae_TIGR PollentubeRhoGDI2(Nicotianatabacum) 0.05 9.97 6.50e-18 +7.64
261291923 Asteraceae Pistil-specificextensin-likeprotein 0.89 4.93 2.31e-05 +2.47
(Carthamustinctorius)
126480015 GDB Seedcoat(Brassicanapus) 0.22 1800.66 0 +12.99
33323054 GDB Acidicchitinase(Ficusawkeotsang) 0.05 58.65 0 +10.20
TC7736 Asteraceae_TIGR Carbonicanhydrase(Solanumlycopersicum) 0.05 9.39 6.67e-17 +7.55
FS486814 Asteraceae_TIGR 2-Cysperoxiredoxin-likeprotein 0.66 114.17 0 +7.43
(Arabidopsisthaliana)
BU019358 Asteraceae_TIGR Thioredoxin(Medicagotrunculata) 305.83 600.70 0 +0.97
AI100454 All_TIGR_Plant.fa Serine/threonineproteinkinase(Brassicanapus) 152.14 34.25 0 −2.15
195607463 GDB Asparticproteinasenephentesin 24.33 3.01 2.99e-38 −3.01
precursor(Zeamays)
GR085079 Asteraceae Lipid-transferprotein(Salviamiltiorrhiza) 0.05 17.21 1.28-23 +9.20
TC5118 Asteraceae_TIGR Pathogenesis-relatedprotein5–1 7.96 33.21 1.16e-24 +2.06
(Helianthusannus)
KnownAmbrosiaartemisiifoliaallergensareshowninbold.
and was slightly down-regulated under the elevated homologue of a pollen coat protein from wild cabbage
CO regime (Table 4). In contrast, the transcript for the was down-regulated. However, this value was below the
2
pollen-specificproteinSF21homolog fromsunflowerwas threshold.Althoughnotdirectlylinkedtopollen,thetran-
clearly up-regulated (Table 5). Other up-regulated pollen script for a homologue of a seed coat protein from rape-
proteins included transcripts for a homologue of a pollen seed was extremely up-regulated (log =12.99) (Table 5).
2
tube protein from tobacco and a pistil-specific extensin- The general search for pollen showed 59 transcripts and
like protein from safflower, while the transcript for a 56out of these transcriptswerefrom thepollencDNAof
ElKelishetal.BMCPlantBiology2014,14:176 Page10of16
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A. trifida. Other highly regulated transcripts of Ambrosia The AllFam database indicated five additional tran-
included a ribokinase (log =−8.68) and a ribosomal scripts for allergenic proteins. Three of these transcripts
2
protein L36 (log =9.28). wereup-regulatedandtwoweredown-regulated(Table6).
2
The AllFam database search indicated seven transcripts
for putative allergenic proteins from other plants. Five of Quantitativereal-timeRT-PCR(qRT-PCR)
theseproteinswereup-regulatedunderelevatedCO con- qRT-PCR was performed for selected ‘Amb a’ transcripts
2
centrations, whereas the transcripts of a protein kinase (Figure4).Therelativeexpressionraterangedfrom1to4
and an aspartic proteinase were down-regulated, similar andincreasedforAmba1.1,Amba1.2,Amba1.3,Amb
as under drought stress (Table 5). Interestingly, the tran- a1.4,Amba8andAmba9,whiletheexpressionlevelsof
scriptofahomologueforanon-specificlipid-transferpro- Amb a 1.5, Amb a 5 and Amb a 6 were not influ-
tein of red sage was also strongly up-regulated, although enced or even reduced. The highest values were observed
at low abundance. As described for the drought stress fordroughtandCO +drought(Figure4)andAmba1.4,
2
conditions,thetranscriptlevelofPR5–1homologuefrom Amb a 8 and Amb a 9 showed the strongest increase. To
sunflowerwasalsoelevated(Table5). validate the results from the SuperSAGE, we compared
In a final step, we compared the Ambrosia control the log fold change of ‘Amb a’ transcripts found in the
2
library (AmK) vs. the elevated CO + drought-stressed SuperSAGE libraries and the qRT-PCR results. For the
2
library (AmCT). Under the search term Ambrosia, 55 Amb a 1 transcripts, a relatively good correlation was
transcripts and for ragweed 50 transcripts, mainly ho- found. The best correlation was observed for the drought
mologues from A. trifida, were identified. The homology treatment, whereas the elevated CO and elevated CO +
2 2
descriptionisgiveninTable6.Thesearchterm“allergen” droughtshowedthesameexpressiontrendbutnotidenti-
resulted in five trancripts from A. artemisiifolia and the cal absolute values. Using only the significantly changed
calcium-binding protein isoallergen 1, Amb a 1.1, Amb qRT-PCR ratios a significant correlation with the Super-
a 1.2 and Amb a 1.3 were up-regulated (Table 6). In SAGE data sets was found (Additional file 7). For Amb a
total, eight transcripts of up-regulated allergenic pro- 8,the qRT-PCR data contrasted the SuperSAGE data and
teins were identified for A. artemisiifolia: two calcium- for Amb a 9, the fold changes were much higher for the
bindingproteins(Amb a9andAmb a10),pectatelyases SuperSAGE data compared to the qRT-PCR values.
(Amb a 1.1, Amb a 1.2, Amb a 1.3 and Amb a 1.2 pre- However, this kind of result has also been reported in the
cursor), a profilin-like protein (Amb a 8) and a cystatin literature with coincident and contrasting data for Super-
proteinase inhibitor (Amb a CPI). However, the change SAGEandmicroarrays[87],aswellasfortheSuperSAGE
of Amb a 1.2 precursor and Amb a CPI were below the and qRT-PCR analyses [88]. In sheepgrass differences up
thresholdof1.5.AnLTPhomologuefromA.trifidawas toafactorof2.5betweendigitalgeneexpressiondataand
highly up-regulated (Table 6). The transcript of a low- RT-PCRratioandeveninconsistencieswerereported[89].
abundanceasparticproteasehomologuefromA.trifida, In poplar differences by factors of 4–16 between micro-
allergenic according to the AllFam database, was highly arrayandqRT-PCRdatawerereportedandinswitchgrass
up-regulated (Table 6). The transcript of the very high also factors up to 15 were found [90,91]. This result
abundant pollen-specific protein homologue from A. reflects a general problem when comparing transcript
trifida was slightly down-regulated, similar to the other abundance with different platforms and might be caused
two libraries, while the transcript of the pollen-specific by allele-specific gene expression [88,92]. Moreover, it is
proteinSF21homologuefromsunflowerwasup-regulated interesting tonotethat transcriptabundancesare import-
(Table 6). The transcript of a pollen coat protein ant when comparing different platforms and that good
homologue from wild cabbage was slightly down- correlations were found for high abundance transcripts
regulated and the seed coat protein transcripts homolo- andacorrelationdecreaseforlowerabundancetranscripts
gous to the one from rapeseed was extremely highly [93],asitwasalsogivenforAmba8inthisstudy.
up-regulated (log =14.71) (Table 6). The general search
2
for pollen resulted in 51 transcripts that were mainly Conclusions
related to the A. trifida pollen cDNA library. The search Our data on ragweed plants fumigated with elevated CO
2
input drought resulted in 33 differentially regulated tran- and drought stress conditions support the idea that pollen
scripts with homology to a safflower drought stress- transcripts related to allergenicity are influenced by such
subtracted library and 25 of these transcripts were global climate change factors. A strong up-regulation of
up-regulated. The homologue of an ABA-responsive ‘Amba’transcriptswasevidentunderelevatedCO ,drought
2
HVA22 transcript from A. trifida was down-regulated, as stress and elevated CO + drought stress conditions. Based
2
intheothertwolibraries.Althoughatlowabundance,the on normalized tags, Amb a 1.1 and Amb a CPI were
transcript for the CBS domain-containing protein was expressedatthehighestlevels.TheincreasedAmba1tran-
highlyup-regulated(Table6). script level is in accordance with an increased Amb a 1