the increase in respiratory diseases arising from allergies in industrialised countries in recent years is considered to be linked to changes in certain environmental factors . one such factor relates to the higher levels of atmospheric pollution and the greater presence and distribution of allergenic taxa . climate change has brought about large increases in the concentrations of some airborne allergens , with the resulting higher incidence and/or severity of allergic illnesses ( gilmour et  al . , 2006 ) . significant increases in allergic responses are often reported , even in individuals who have never had symptoms previously , and this leads to the belief that these allergic responses might be caused by new allergens that are released by plants used for ornamental purposes or reforestation , or by invasive plants . in europe the increases in pollinosis appear to be caused by pollen from birch , hornbeam and cypress trees , and above all , from ragweed , rather than by the classic allergenic pollens , such as grasses , pellitory , olive and mugwort ( corsico et  al . , 2000 ; asero , 2004 ; ridolo et  al . , 2006 ) . ambrosia artemisiifolia ( short or common ragweed ) is an annual , anemophilous , and extremely allergenic weed that can produce enormous amounts of pollen . as these pollen grains are small ( 1822  m ) , they can often be transported over long - distances ( mandrioli et  al . , 1998 ) . ragweed started its expansion in europe in the last decades of the 20th century , arriving from hungary , the country where it was most abundant . ragweed is now also found in italy ( damato et  al . , 1998 ) , and the two regions that have been most affected are lombardia and friuli venezia - giulia , which are located in the north - northeast of italy ( mandrioli et  al . , 1998 ; asero , 2002 ) . ragweed plants have been detected only sporadically in central - southern italy ( pignatti , 1997 ) . however , transboundary transport of ragweed pollen to central italy has been demonstrated , which could have a clinical effect on atopic patients ( corsico et  al . ragweed pollen concentrations have been reported to have increased over the last decade ( oswalt and marshall , 2008 ) , mainly because ragweed is invasive in europe and is an opportunistic and pioneer plant , invading field crops and open disturbed habitats or roadsides ( bassett and crompton , 1975 ) . however , the increase in allergic disease might be attributed not only to greater concentrations of ragweed pollen in the atmosphere , but also to modifications to the allergenicity this pollen can promote . although the role of atmospheric pollutants on the allergic sensitivity of airways is not yet completely clear , there is evidence to suggest that urbanisation increases allergic sensitization due to its high levels of exposure to ozone ( o3 ) , nitric oxides ( nox ) , sulphur oxides ( sox ) and particulate matter ( pm10 ) ( damato , 2002 ) . in addition to affecting the airways of allergic individuals , air pollutants can have direct effects on the aeroallergens in the atmosphere , which can result in changes in the antigenic characteristics of pollen . air pollutants , and especially o3 and respirable pm10 , can induce proinflammatory responses in the lung ( bernstein et  al . , 2004 ) , and can have immunological adjuvant effects on ige synthesis , as has been found with polyaromatic hydrocarbons in the particles of diesel exhaust ( nel et  al . , 1998 ) . ozone is the main component in the so - called summer smog comprised of photochemical oxidants and appears to account for up to 90% of the total oxidant levels in cities that have a mild sunny climate ( butkovic et  al . , ozone is generated at ground level by photochemical reactions that involve ultraviolet radiation of atmospheric mixtures of no2 and hydrocarbons that can derive from vehicle emissions . these o3 trends depend not only on the substrate supply ( no2 emitted by cars ) , but also on the sunny weather , because of the transformation of no2 into o3 during a photochemical smog . current safety standards for o3 levels are exceeded frequently in most mediterranean countries . indeed , the 8  h average levels of o3 for the period from 2000 to 2004 in different sites in italy showed that the background o3 pollution exceeded the european standards ( paoletti et  al . , 2007 ) that were fixed by the european union at 0.060  ppm ( directive 2002/3/ec , 2004 ) . its toxicity is due to the generation of reactive oxygen species ( ros ) , such as the superoxide anion radical ( o2 ) , hydrogen peroxide ( h2o2 ) , the  hydroxyl radical ( oh ) , and singlet oxygen ( o2 ) ( mudd , 1997 ) . the o3 effect on pollen grains has been considered in particular in studies of plant germination in  vitro . however , opposing effects have been reported : feder ( 1968 , 1981 ) observed a reduction in nicotiana tabacum pollen tube elongation , and similar results were reported for corn pollen by mumford et  al . ( 1983 ) reported that the ability of the pollen of pinus strobus to germinate was not significantly reduced by o3 fumigation . it has also been reported that o3 can influence allergen release from pollens grains , and masuch et  al . ( 1997 ) found that o3 increases the content of group 5 allergenic proteins of lolium perenne . when the pollen comes into contact with the airway mucosa , it releases not only allergenic proteins , but also lipid immuno - modulators , such as pollen - associated lipid mediators ( traidl - hoffmann et  al . , 2002 ) , and nicotinamide adenine dinucleotide phosphate ( nad[p]h ) oxidases ( boldogh et  al . , 2005 ) . these latter are enzymes that have an oxidase activity that can produce the o2 ion , which is converted into h2o2 through the action of the enzyme superoxide dismutase ( sod ) . therefore the nad(p)h oxidases have fundamental roles in inflammatory processes , as they can increase the levels of ros in the epithelium of the respiratory apparatus , and promote the flow of neutrophils towards the respiratory apparatus ( boldogh et  al . , 2005 ) . on the basis of the concomitance of episodes of high o3 levels ( gabusi and volta , 2005 ) and the high concentrations of ragweed pollen in the atmosphere during the summer months ( mandrioli et  al . , 1998 ) , the aim of the present study was to determine whether o3 affects mature ragweed pollen grains after their dispersal . in particular , we determine first whether o3 can increase the allergy potency of ragweed pollen by stimulating allergen and nad(p)h oxidase release , and secondly whether o3 can induce physiological alterations that affect pollen viability , which is the ability of the pollen to complete post - pollination events and to achieve fertilisation . for this purpose , under laboratory conditions , we exposed ragweed pollen to a high o3 concentration ( 100  nl  l ) during the day . this concentration corresponds to peak o3 levels measured during the summer in central italy ( http//www.arpa.umbria.it ) . we specifically evaluated : ( i ) pollen ros and no content , as no is considered to be a mediator of inflammatory responses ( moilanen and vapaatal , 1995 ) ; ( ii ) activity of the nad(p)h oxidase , which can generate ros ; ( iii ) expression of the major ragweed pollen allergens ; and ( iv ) pollen viability . ragweed ( ambrosia artemisiifolia ) pollen was purchased from greer laboratories ( lenoir , nc , usa ) . the pollen was aliquoted into 2  ml , sterile microcentrifuge tubes , and stored dry at 4  c until use . the o3 treatment was performed by exposure of 0.5  g pollen in petri dishes to 100  nl  l o3 for 5  h ( 08:00  h to 13:00  h ) per day for 7 consecutive days in a plexiglass chamber ( 0.32  m ) under light with a photosynthetic photon fluence of 120  mol  m  s , as previously described ( pasqualini et  al . , 2009 ) . a non - fumigated ( o3 ) pollen sample was maintained in a filtered - air plexiglass chamber under the same experimental conditions . after each daily o3 fumigation , the pollen samples were all left in a growth chamber under controlled conditions ( 14  h photoperiod , photosynthetic photon fluence rate of 120  mol  m  s , day / night air temperature 25  c/20  c , and relative humidity 60%75% ) until the next treatment . after the 7 days of this o3 fumigation , the pollen was either immediately analysed for viability and ros , h2o2 and no content , or frozen under liquid n2 and stored at  80  c for protein quantification , nad(p)h oxidase activity assay , and rna analysis . louis , mo , usa ) , as reported by heslop - harrison and heslop - harrison ( 1970 ) . pollen grains ( 2  mg ) were hydrated for 30  min in 2 then 20  l of these suspensions were placed on a microscope slide and stained with 4  m fda . the total number of pollen grains was visually counted using bright - field microscopy , while the fluorescent pollen grains in the same field of view were counted using a uv epifluorescence microscope ( dmlb ; leica , leica microsystems , wetzlar , germany ) with a 450  nm excitation filter and a 535  nm emission filter . pollen viability was determined as the percentage of fluorescing pollens relative to the total pollen grains . for each sample ( control and o3 treated ) , ten slides were prepared , and for each slide , at least 100 pollen grains were counted . ( 2009 ) with slight modifications using the fluorescent no indicator dye 4-amino-5-methylamino-2,7-difluorofluorescein diacetate ( daf - fm diacetate ; molecular probes , invitrogen , carlsbad , ca , usa ) ( kojima et  al . , 1998 ) . the pollen ( 1  mg ) was first incubated for 30  min at 4  c in 1  ml mes  kcl buffer containing 8% sucrose , 10  mm mes , 5  mm kcl , 50  mm cacl2 ( ph 6.8 ) , in the absence and presence of 200  m of the no - scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide ( cptio ; sigma daf - fm diacetate ( 10  m ) was then added , and the samples were incubated in the dark for 20  min , to allow the dye to enter the pollen . the pollen suspensions were then centrifuged at 6500    g for 1  min , and the resulting supernatant was discarded . fresh mes  kcl buffer was then added to the pellets , which were resuspended and left at room temperature for 20  min . finally , 50  l pollen suspension were placed onto a glass slide and covered with a glass coverslip before being examined with a uv epifluorescence microscope with a 450  nm excitation filter and a 535  nm emission filter . intracellular no was determined as the percentage of fluorescing pollens relative to the total pollen grains . for each sample ( control and o3 treated ) , six slides were prepared , and for each slide , at least 100 pollen grains were counted . ros detection was performed using the fluorescent ros indicator dye 2,7-dichlorodihydrofluorescein diacetate ( dcfh2-da ; molecular probes ) ( setsukinai et  al . , 2003 ) . pollen grains ( 2  mg aliquots ) were hydrated at 4  c in 2  ml 100  mm phosphate - buffered saline ( pbs ) for 30  min , and then transferred to 2  ml microcentrifuge tubes and centrifuged at 2000 g for 30  s. after centrifugation , the supernatant was discarded and fresh pbs containing 2.5  m dcfh2-da was added to the pellet . after an incubation in the dark at 25  c for 15  min , it was centrifuged at 2000    g for 30  s. after centrifugation , the supernatant was discarded and the pellet was immediately resuspended in fresh pbs , gently shaken , and centrifuged as before . finally , the pellet was resuspended in pbs , and 50  l of the pollen suspension was placed onto glass slides , covered with glass coverslips , and examined using a uv epifluorescence microscope with a 450  nm excitation filter and a 535  nm emission filter . content of ros was determined as the percentage of fluorescing pollens relative to the total pollen grains . for each sample ( control and o3 treated ) , six slides were prepared , and for each slide , at least 100 pollen grains were counted . pollen grains ( 100  mg ) were hydrated in 1  ml pbs for 25  min at room temperature , with gentle shaking . the pollen suspensions were then centrifuged at 14000    g for 10  min , and the supernatants were recovered ( s25 supernatants ) . the pollen pellets were resuspended with 1  ml pbs , shaken for 25  min and then centrifuged again at 14000    g for 10  min , to obtain the second supernatants ( s50 supernatants ) . this procedure was repeated once again to obtain the third supernatants ( s75 supernatants ) . the pellets were then resuspended in 0.5  ml pbs , sonicated , centrifuged at 14000    g for 10  min , and the supernatants were recovered ( sson supernatants ) . the soluble protein concentrations in the supernatants were measured using the dye - binding method of bradford ( 1976 ) , with bovine serum albumin as the standard . the nad(p)h oxidase activity was measured in the samples of these four supernatants obtained as above ( s25 , s50 , s75 and sson ) using the nitroblue tetrazolium ( nbt ) assay ( bacsi et  al . , this activity was only detectable in the s25 supernatants . here , 25  g protein was used for each assay and mixed with 2  mm nbt without or with 100  m reduced nicotinamide adenine dinucleotide ( nadh ) , or 100  m reduced nad phosphate ( nadph ) , and without or with the nad(p)h oxidase inhibitor diphenyleneiodonium ( dpi , 100  m ) or the o2-scavenging enzyme sod ( 100  u  ml ) . nbt was completely removed by repeated washing with fresh pbs , and the formazan precipitate was dissolved in 100% methanol . , 100  mg pollen grain aliquots were hydrated for 30  min at 4  c in 1  ml sterile distilled water ; they were then centrifuged at 14000    g for 10  min . the supernatants were recovered and immediately used for h2o2 quantification using the xylenol orange method ( jiang et  al . , 1990 ) , based on the peroxidase - mediated oxidation of fe followed by the reaction of fe with xylenol orange o - cresolsulfonephtalein 3-3-bis[methylimino ] diacetic acid , sodium salt . h2o2 was determined by adding 250  l of distilled h2o to 250  l of supernatant and 500  l of assay reagent ( 500  m ammonium ferrous sulphate , 50  mm h2so4 , 200  m xylenol orange , and 200  mm sorbitol ) . absorbance of the fe - xylenol orange complex ( a560 ) was detected after 45  min . the specificity for h2o2 was tested by eliminating h2o2 in the reaction mixture with catalase . standard h2o2 curves were obtained by adding increasing amounts of h2o2 from 0 to 100  nmoles . pollen grains ( 130  mg ) were incubated in 1  ml sterile distilled water and shaken gently for 30  min at 4  c . the pollen suspensions were then centrifuged at 14000    g for 10  min , and the soluble protein was measured as reported above . crude ragweed pollen extract ( 10  g total protein ) and 6  g natural amb a 1 ( namb a 1 ) , kindly provided by prof . t. p. king ( rockefeller university , ny , usa ) , were separated by denaturing sodium dodecyl sulphate  polyacrylamide gel electrophoresis ( sds - page ) and visualised by staining with coomassie brilliant blue r-250 ( laemmli , 1970 ) . for immunoblotting analysis , the protein was electroblotted onto nitrocellulose membranes ( protan , schleicher & schuell , dassel , germany ) and then incubated with mouse monoclonal anti - amb a 1 antibodies for 2  h. these mouse monoclonal anti - amb a 1 antibodies ( clone e10 ) were obtained by immunisation of mice with the purified recombinant amb a 1 protein ( ramb a 1 ) , as described by wopfner et  al . after washing , the membranes were incubated with alkaline - phosphatase - conjugated anti - mouse igg ( 1/1000 ; sigma labelling was detected using the nbt / bromo - chloro - indolyl phosphate substrate ( sigma  aldrich ) . for quantification , membranes were scanned and band intensities were determined with the image analysis software imagej ( http://rsb.info.nih.gov/ij ) . the expression of the amb a 1.1 , amb a 1.4 , amb a 2 , profilin 1 and profilin 2 allergens and of the house - keeping 18s rrna were analysed by reverse transcription - polymerase chain reaction ( rt - pcr ) . total rna was extracted from the control and o3-treated pollen grains using nucleospin rna plant ( macherey  nagel , dren , germany ) , with pollen samples ( 30  mg ) ground in a mortar under liquid nitrogen , and processed according to the manufacturer instructions . both the quality and concentration of the total rna were assessed by non - denaturing agarose gel electrophoresis and by measuring a260/a280 . the rna was immediately processed for rt - pcr or stored at  80  c . a given amount of total rna ( 1  g ) was reverse transcribed , as described in pasqualini et  al . images of the rt - pcr ethidium bromide - stained agarose gels were acquired with a digital kodak camera ( eastman kodak company , rochester , ny , usa ) and quantification of the bands was performed with kodak 1d image analysis software . the o3-fumigation experiment was replicated 3 times , and for each experiment , three replicates were used . one or two - way analysis of variance ( anova ) was applied to the data reported in the figures , as detailed in the figure  legends . the means were compared using duncan s multiple range tests , and the values followed by different letters are significantly different at p    0.01 . the data concerning the ragweed pollen viability after 7 days of o3 fumigation are shown in fig . the mean pollen viability in the non - fumigated pollen ( control ) was 55.9% , while fumigation with o3 significantly decreased this pollen viability ( 39% ; p  <  0.01 ) . in non - fumigated ragweed pollen , we measure a rather low viability ; nevertheless , that the pollen used for the experiments is a commercial pollen must be taken into account , as it was purchased from a company and kept at  + 4  c for more than 4 months before we carried out the viability assays . many reports have shown loss of viability of pollen and loss of membrane integrity during pollen storage ( jain and shivanna , 1989 ) . however , although the use of a commercial pollen did not allow us to work with pollen that had a high viability , it provided good homogeneity of the samples and the possibility for other groups to reproduce these experiments . in addition , the objective difficulty of the need to collect a sufficient amount of pollen for the analyses needs to be considered , particularly as ragweed grows only sporadically in central italy . as the toxicity of o3 is due to its ability to generate potentially lethal ros , we evaluated the release of h2o2 from pollen grains after this o3 fumigation as a possible cause of an oxidative burst in the pollen . the release of h2o2 from pollen grains was evaluated by analysing the liquid medium in which the pollen had been incubated for 30  min . about 0.9 mole h2o2  g fw were released from the non - fumigated pollen grains after their hydration , with similar levels detected in the o3-fumigated pollen , showing that o3 fumigation did not significantly influenced the pollen h2o2 content ( fig . using fluorescent microscopy and the ros - sensitive fluorescent probe dcfh2da , we were able to qualitatively detect ros inside the pollen . indeed , this probe lacks specificity among the ros and reacts only slowly with h2o2 or o2 , but very fast with the oh radical and peroxynitrite [ onoo ( setsukinai et  al . , 2003 ) ] ; it thus follows that this dcf fluorescence is an assay of generalised oxidative stress , rather than of any particular ros . our results show that the 7-days of o3 fumigation induced only a small , and non - significant , increase in ros inside the pollen grains ( fig . the nad(p)h oxidase in these ragweed pollen grains following the o3 fumigation was determined by nbt reduction to formazan , with this activity completely released after 25  min of hydration ( s25 supernatant ) ( fig . for the supernatants obtained from the longer hydrations ( s50 and s75 ) and from the sonication of the pollen pellets ( sson ) , there was no measurable nad(p)h oxidase activity . without nadph or nadh , nbt reduction by the pollen grains of ragweed was just detectable , with no significant difference seen between the non - fumigated and o3-fumigated pollen grains ( fig . on addition of nadh or nadph , the activities measured from the o3-treated pollen increased by 41% and 21% , respectively , with respect to the non - fumigated pollen ( fig . this reduction of nbt by the ragweed pollen extract was almost completely blocked by the addition of sod ( absorbance reduction , 82%    5% ) and of the nad(p)h oxidase inhibitor dpi ( absorbance reduction , 85%    3% ) , which suggests that nad(p)h oxidase is the major source of these ros . our data show here that high levels of constitutive no were detected in the ragweed pollen grains , and that the o3 fumigation induced a small , but significant , increase in no after 30  min of hydration ( fig .  2 a , b ;  +  10% , p  <  0.01 ) . to ensure that this fluorescence was no specific , the no - scavenger cptio was added to the probe . in this case , there was significant quenching of the green fluorescence , which indicates that the daf - fm probe was specific for no detection ( fig . there was a large release of soluble proteins after the first 25  min of pollen hydration ( 81% ) , with 14.85  mg  protein  g  fw and 14.71  mg  protein  g  fw detected in the s25 samples of the non - fumigated and o3-fumigated ragweed pollen , respectively ( fig . less protein was released from the pollen after 50  min of hydration ( s50 ; 19% ) . in the supernatants obtained from the further hydration ( s75 ) and from the sonication of the pellets ( sson ) the o3 treatment did not induce significant changes in protein release from the ragweed pollen . sds - page of the pollen extracts revealed similar profiles in both control and o3-fumigated pollen grains ( fig . the major ragweed allergens ranged from 8  kda to 43  kda and the migration band visible at about 38  kda was identified as amb a 1 . indeed , purified namb a 1 migrated at 38  kda , and also shows specific degradation into two fragments , of 26  kda ( namb a 1  ) and 12  kda ( namb a 1  ) ( fig . the crude protein released after 30  min hydration of the pollen was analysed by immunoblotting and probed with a monoclonal antibody against amb a 1 ( fig . here , it has been detected a 38  kda protein band that corresponded to amb a 1 , the major antigenic component of ragweed pollen , with no quantitative difference between non - fumigated and fumigated pollen grains . altogether , these data indicate that this 7-day o3 fumigation did not induce changes in the released protein pattern , nor in the content of the major allergen amb a 1 . the expression of the major ragweed allergens , amb a 1 , amb a 2 , profilin 1 and profilin 2 was analysed by rt - pcr using the primer pairs given in table  1 . fig . 4 shows that all of these allergens examined are expressed in these ragweed pollen grains , and that the o3 fumigation did not influence the transcript levels , substantially reflecting the unchanged amounts of the amb a 1 protein detected in the western blotting ( fig . in this study , we have sought to simulate the effects of acute o3 exposure on ragweed pollen after its dispersion in the atmosphere . for this purpose , under laboratory conditions , the ragweed pollen was exposed to 5  h of 100  nl  l o3 for 7 days , with some of the biochemical and molecular features of the pollen grains then analysed . however , it should also be considered that o3 can influence the pollen not only directly after its dispersion , but also indirectly by affecting the plant growth and the pollen maturation , since ragweed typically grows during the summer when the highest o3 peaks occur . indeed , it has been reported that in l.  perenne plants exposed to chronic o3 concentrations , the percentage of underdeveloped pollen was significantly increased compared to plants grown in filtered air ( schoene et  al . , 2004 ) . this effect was said to arise from an inhibition of pollen starch accumulation , which was responsible for this disturbance of the pollen development ( schoene et  al . , 2004 ) . also , many reports have shown that ros and no accumulate in the reproductive tissues , such as the stigma , anthers and pollen , where they have different biological roles , depending on the stage and tissue ( mcinnis et  al . , 2006 ; hiscock et  al . , 2007 ; however , whether o3 exposure of the plants during their growth can affect the ros and no contents of pollen grains is not at present known . a reduction in pollen viability by o3 has also been reported in other studies and in other species ( feder , 1968 , 1981 ; mumford et  al . , 1972 ) . the fluorochromatic reaction used to test pollen viability provides an indirect measurement of esterase activity and the intactness of the plasma membrane ( heslop - harrison and heslop - harrison , 1970 ) . as such , the reduction in pollen viability induced by this o3 fumigation is indicative of damage to the pollen membrane system . furthermore , the exposure of pollen to a high o3 concentration after its release from the plant can reduce its viability , resulting in decreased fertility , and consequently , in a reduction in seed production . as irreversible damage to cell membranes can be caused by an o3-induced oxidative burst , we evaluated the ros production in pollen grains . the results show the presence of constitutive ros accumulation in the hydrated ragweed pollen , which has been suggested to promote pollen germination and/or pollen - tube growth to the stigma ( potock et  al . , 2007 ) . nad(p)h oxidase is the major source of ros in pollen grains ( bacsi et  al . , 2006 ) , which produces the o2 ion ; this is converted into h2o2 through sod activity or by spontaneous dismutation . the superoxide ion can also act as a reducing agent for transition metals such as fe and cu , which leads to h2o2-dependent formation of oh , an extremely aggressive ros . hydroxyl radical accumulation in pollen grains can cleave polymers , including cell - wall polysaccharides ( fry , 1998 ) , which promotes the cell - wall loosening that is necessary to allow for the rapid cell elongation of growing pollen tubes ( eckardt , 2005 ) . in fumigated pollens , both the h2o2 released after hydration and ros localized in pollen grains by the fluorescent probe remained unchanged , indicating that 7 days of o3 fumigation did not induce oxidative stress in the pollen , at least when it was measured at the end of the o3 exposure . thus the damage to the membrane system that is seen here as decreased pollen viability might be due to other factors in addition to ros . as reported above we found that the nad(p)h oxidase was rapidly released ( in less than 30  min ) after pollen grain hydration , as indicated by bacsi et  al . ( 2006 ) , who showed that subpollen particles containing the amb a 1 allergen and nad(p)h oxidase activity are released from ragweed pollen grains after 35  min of hydration . when ragweed pollen grains were exposed to 7-day o3 fumigation , we saw significant induction of nad(p)h oxidase enzymatic activity . thus , as previously observed in leaves of o3-treated plants ( pellinen et  al . , 1999 ; ranieri et  al . , there is evidence that ros have prominent roles in the pathogenesis of allergic diseases ( bowler and crapo , 2002 ) . ( 2005 ) indicated that oxidative stress generated by nad(p)h oxidase in ragweed pollen grains can augment the immediate - type hypersensitivity reactions and pollen - antigen - driven allergic conjunctivitis . our data here indicate that when ragweed pollen grains are in the presence of o3 , there is an induction of nad(p)h oxidase activity which is released into the hydration medium , and may , under natural conditions , be released into the airway mucosa when pollen grains are inhaled . no is a key signalling molecule that controls a variety of physiological responses in plant and animal systems , and it has also been demonstrated to be a signal in plant defence responses against pathogens ( delledonne et  al . , 1998 ) and o3 ( ederli et  al . , 2006 ; meier et  al . , recently , it was shown that no is involved in plant reproductive processes , as it can modulate the growth of the pollen tube and the orientation of the pollen tube towards the stigma ( prado et  al . , 2004 , 2008 ) . it has been suggested that in interacting with h2o2 of the stigmatic papille , no can regulate the processes of recognition between pollen and stigma ( mcinnis et  al . , 2006 ) . the presence of no has been reported in hydrating pollen grains of various angiosperms ( bright et  al . , 2009 ) ; in that study , no and nitrites were shown to be released after hydration of the pollen , with greater levels of release seen for allergenic species compared to non - allergenic pollens in the limited number of taxa examined . ozone induces a weak , but significant increase in the no fluorescence signal , suggesting that the no acts as a signal molecule in response to stress also in the pollen . as no can produce onoo when in the presence of the o2 ion ( wendehenne et  al . , 2001 ) , we hypothesise that the fluorescent signal detected in the pollen grains loaded with dcfh2da was attributable to the oh radical , which was potentially derived through the fenton reaction from nad(p)h - oxidase - generated o2 , and to the onoo . the major allergenic proteins released from ragweed pollen ranged from 8  kda to 43  kda in all of the extracts examined , in agreement with results previously reported ( weber and nelson , 1985 ) . after o3 fumigation , the protein release by hydrated pollen grains was rapid , with similar protein pattern profiles in both control and o3-fumigated pollen . the major antigenic component of ragweed pollen , amb a 1 , was identified as a band visible at about 38  kda , and western blotting analysis revealed that its content did not change after o3 exposure . we also examined the expression profiles of the major ragweed pollen allergens : amb a 1 and amb a 2 , which are proteins belonging to the pectate lyase family ( rafnar et  al . , 1991 ) , and profilin 1 and profilin 2 , which are proteins involved in signal transduction from the outer cell membrane to the inside of the cell , and in the regulation of actin polymerisation ( goldschmidt - clermont et  al . , 1990 ) . the profilins are defined as panallergens , as they are widespread among the pollens of plants that are both closely and distantly related botanically ( van ree et  al . , our data show that o3 exposure did not affect the expression of the major ragweed pollen allergens . earlier studies concerning the effects of gaseous pollutants on the content of allergenic proteins provided no conclusive results . recently , it has been reported that pollen extracts from o3-fumigated plants showed increased allergen content and higher ige reactivity ( masuch et  al . 2010 ) . on the other hand , exposure of timothy grass pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts ( rogerieux et  al . , 2007 ) . ( 2010 ) used a proteomic approach to show that the major allergens of birch ( bet v 1 , 2 , 3 and 4 ) were not expressed differentially in their samples from rural and urban areas , but that the extracts collected in urban areas had higher chemotactic activities on human neutrophils . they then suggested that the greater allergenicity of pollen collected from polluted areas was determined by more than just the allergen content ( bryce et  al . , 2010 ) . our data support the concept that when ragweed pollen is exposed to o3 during its time in the atmosphere , it can suffer damage that is seen as decreased pollen viability , and it can become more allergenic through stimulation of inflammatory ros - generating nad(p)h oxidase , which would be released when pollen comes into contact with the cells of the respiratory apparatus .
air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries . 
 we investigated the impact of ozone ( o3 ) on reactive oxygen species ( ros ) and allergen content of ragweed pollen ( ambrosia artemisiifolia ) . 
 pollen was exposed to acute o3 fumigation , with analysis of pollen viability , ros and nitric oxide ( no ) content , activity of nicotinamide adenine dinucleotide phosphate ( nad[p]h ) oxidase , and expression of major allergens . 
 there was decreased pollen viability after o3 fumigation , which indicates damage to the pollen membrane system , although the ros and no contents were not changed or were only slightly induced , respectively . 
 ozone exposure induced a significant enhancement of the ros - generating enzyme nad(p)h oxidase . 
 the expression of the allergen amb a 1 was not affected by o3 , determined from the mrna levels of the major allergens . 
 we conclude that o3 can increase ragweed pollen allergenicity through stimulation of ros - generating nad(p)h oxidase .