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A flux-based assessment of the effects of ozone on foliar injury, photosynthesis, and yield of bean (Phaseolus vulgaris L. cv. Borlotto Nano Lingua di Fuoco) in open-top chambers

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A flux-based assessment of the effects of ozone on foliar injury, photosynthesis, and yield of bean (Phaseolus vulgaris L. cv. Borlotto Nano Lingua di Fuoco) in open-top chambers
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  A flux-based assessment of the effects of ozone on foliar injury, photosynthesis,and yield of bean ( Phaseolus vulgaris  L. cv. Borlotto Nano Lingua di Fuoco)in open-top chambers Giacomo Gerosa a , Riccardo Marzuoli a , b , Micol Rossini c , Cinzia Panigada c , Michele Meroni c , RobertoColombo c , Franco Faoro d , Marcello Iriti d , * a Department of Mathematics and Physics, Universita` Cattolica del Sacro Cuore, via dei Musei 41, 20125 Brescia, Italy b Fondazione Lombardia per l’Ambiente, piazza Diaz 9, 20123 Milano, Italy c Remote Sensing of Environmental Dynamics Lab., DISAT, University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy d Plant Pathology Institute, Universita` di Milano, via Celoria 2, 20133 Milano, Italy Ozone stomatal fluxes affect leaf cell viability, photosynthetic performance, optical properties and crop yield of bean. a r t i c l e i n f o  Article history: Received 25 January 2008Received in revised form 7 June 2008Accepted 14 June 2008 Keywords: Stomatal conductance modelOzone fluxesLeaf symptomsChlorophyll fluorescenceField radiometryCrop yield a b s t r a c t Stomatal ozone uptake, determined with the Jarvis’ approach, was related to photosynthetic efficiencyassessed by chlorophyll fluorescence and reflectance measurements in open-top chamber experimentson  Phaseolus vulgaris . The effects of O 3  exposure were also evaluated in terms of visible and microscopicalleaf injury and plant productivity. Results showed that microscopical leaf symptoms, assessed as celldeath and H 2 O 2  accumulation, preceded by 3–4 days the appearance of visible symptoms. An effectivedose of ozone stomatal flux for visible leaf damages was found around 1.33 mmolO 3 m  2 . Significantlinear dose–response relationships were obtained between accumulated fluxes and optical indices (PRI,NDI,  D F  / F  m 0 ). The negative effects on photosynthesis reduced plant productivity, affecting the number of pods and seeds, but not seed weight. These results, besides contributing to the development of a flux-based ozone risk assessment for crops in Europe, highlight the potentiality of reflectance measurementsfor the early detection of ozone stress.   2008 Elsevier Ltd. All rights reserved. 1. Introduction High levels of tropospheric ozone in the Mediterranean regionsare well documented and represent a significant risk for naturaland semi-natural vegetation and for crop species (Fuhrer et al.,1997; Matyssek and Innes,1999; Pleijel et al., 2004; Paoletti, 2006).Current critical exposure levels for vegetation, established by themost recent European Directive (EC 2002/03), have been widelyand frequently exceeded in the last years, in several monitoringstations all over Italy(Gerosa et al.,1999). The toxic effects of ozoneon plants can be detected as visible alterations of leaf surface,including chlorosis (yellowing due to the chlorophyll breakdown,often distributed in spots over the leaf), bronzing (red-brown pig-mentation caused by phenylpropanoid accumulation), bleaching(small unpigmented necrotic spots), flecking (small brown necroticareas fading to grey or white), stippling (small punctuate spots,white, black or red in colour) and tipburn (dying tips, first reddish,later turning brown) especially in conifers (Innes et al., 2001).Despite the above mentioned visible injuries, in particularconditions and geographical areas, plants may experience theozone stress without manifesting any visible leaf symptoms, thussuffering from the so-called invisible damages (Bergmann et al.,1999). In some cases, this happens because cellular damages affectonly single cells scattered throughout the leaf tissues (Faoro andIriti, 2005). More generally, a complex physiopathologicalsyndrome is developed, which includes the decrease of photosyn-thetic activity and the senescence induction, resulting in reductionof dry matter production, detrimental effects on flowering andpollen tube extension, and finally yield losses (Drogoudi and Ash-more, 2000; Fumagalli et al., 2001; Salam and Soja,1995).Ozone effects on photosynthetic activity may be revealed bymeans of rapid and non-invasive optical measurements, as activechlorophyll  a  fluorescence (CF) and spectral reflectance, that havebeen proven effective for quantifying the effects of environmentalstresses on plant physiology, such as drought, extremetemperatures, nutrient deficiency or pollution (Carter, 1993, 1994; *  Corresponding author. Tel.:  þ 390250316766; fax:  þ 390250316781. E-mail address:  marcello.iriti@unimi.it (M. Iriti). Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol 0269-7491/$ – see front matter    2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.envpol.2008.06.028 Environmental Pollution 157 (2009) 1727–1736  Meroni et al., in press; Pen˜ uelas and Filella, 1998; Pen˜ uelas et al., 1995; Rossini et al., 2006).Spectral reflectance alterations at 531 nm are related to thede-epoxidation of xanthophyll pigments, which is a photo-protective process involved in excess energy dissipation as heat(Demming-Adams and Adams, 1996). Based on these observation,thePhotochemicalReflectanceIndex(PRI)wasproposedbyGamonet al. (1992), using reflectance at 531 nm (i.e. the xanthophyll af-fected wavelength) and 570 nm (i.e. a reference wavelength). Thisindex represents an indirect measure of heat dissipation by meansof remote optical measurements.CF and heat dissipation occur in competition with photochem-istry, hence, a variation of the yield of CF or heat dissipationprovides indirect information about a variation in the efficiency of the photosynthetic activity (Papageorgiou and Govindjee, 2004).Therefore, fluorescence based indices (e.g.  F  v / F  m ,  D F  / F  m 0 ) andreflectance based indices (e.g. PRI) are suitable indicators of pho-tosystemII(PSII)photochemicalefficiency,widelyusedtodiagnosestress conditions (Evain et al., 2004; Meroni et al., inpress; Strasseret al., 2000; The´not et al., 2002; Winkel et al., 2002).As the detrimental effect of ozone on photosynthetic activityinevitably influences plant productivity, it is widely accepted toconsiderthebiomassandtheyieldreductionasthemostimportanteffects for the evaluation of ozone impacts on vegetation (Karlssonet al., 2004). Many experiments have been performed in order todefine the relationships between ozone and plant productivity(Tonneijck and Van Dijk, 1997). It has been repeatedly shown thatozone impact is more strictly linked to ozone dose truly absorbedby plants through stomata, rather than to the mere exposure toa given concentration of the pollutant (Gru¨ nhage et al., 2004;Musselmann and Massman,1999; Pleijel et al., 2002,). This conceptis particularly relevant for Mediterranean areas, where recurrentdrought conditions can strongly reduce ozone uptake throughstomatal regulation, as well as influencing metabolic plant defencemechanisms(Alonsoetal.,2001;Herbingeretal.,2002).Estimationof ozone dose is based on the stomatal flux approach, which takesinto account the stomatal conductance of the vegetation. A clearunderstanding of stomatal conductance behaviours, in relation todifferent environmental conditions (especially in relation to wateravailability) becomes essential to quantify the amount of ozoneeffectively absorbed by plants (Gru¨ nhage et al., 2001).The objective of this paper was to investigate on how leaf tissues, photosynthetic performance and plant productivity of   P.vulgaris  are affected by ozone stomatal uptake, calculated by a sto-matal conductance model calibrated for Southern Europe condi-tions. A multidisciplinary approach was employed in order togathertherequiredinformationon:(1)visibleandmicroscopicleaf injury by means of histo-cytochemical analysis, (2) photosyntheticactivity by means of optical measurements (e.g. CF and spectralreflectance) and (3) productivity estimation of plants exposed todifferent ozone concentrations. The experiments were conductedin open-top chambers (OTCs) facilities of the Regional ForestNursery located at Curno (Northern Italy). 2. Materials and methods  2.1. Experimental facilities and plant material The research site is located within the Regional Forest Nursery of Curno (Lat.45  70 0 N, Long 9  62 0 E, elev. 242 masl) on the northern edge of the Po Valley, in thefoothillsofthefirstslopesofthealpinerangeinNorthernItaly.ThesoiltypeisTypicPaleudalf fine-silty, mixed, mesic according to the USDA classification, and theclimate is typically continental with quite dry and hot summers, rainy springs andautumns, cool and dry winters. Ozone levels in this region are among the highest inEurope and the critical levels for crops and forests are frequently exceeded (Gerosaand Ballarin-Denti, 2003; Sandroni et al., 1994).Four OTCs, as those described by Heagle et al. (1973), were used for theseexperiments in summer 2006. The OTCs were active from 11th July (sowing) to 5thOctober (harvest). Experiments were conducted using two ozone treatments: about50% ambient ozone in charcoal-filtered OTC (OTC-F) and about 95% ambient ozonein non-filtered OTC (OTC-NF).Within each plot, uniform seeds of common bean ( P. vulgaris  L.), cv. BorlottoNano Lingua di Fuoco, were manually sown in the ground, two at a time, in 2 cmdeep furrows, 20 cm apart and with 40 cm between rows. Seeds were coated withThiram (dimethyl dithiocarbamate) to prevent rotting. Seedlings were thinned justafter the emergence, and the plots were weeded as required. No pesticides wereappliedon the plants, with the exceptionof anacaricidesprayed twice, at the endof vegetativegrowthand after thepodfill, tocontrol Tetranychus urticae . Allplotsweremanually irrigated up to soil field capacity (at least twice a week in addition torainfall) and supplemented with a granular fertilizer (NPK, 5–10–10).Ozone concentrations, within each chamber, were continuously monitored byan ozone automatic analyzer model 1108 RS (Dasibi Italia s.r.l., Milano, I), via a so-lenoid valve switch system, which collects air from sampling points in the centre of each plot at 70 cma.g.l. The system was managed and controlled by a personalcomputer equipped with a NI-DAQ 6.9 I/O board and a Labview 6.1 program (Na-tional Instruments, Austin, TX) devised specifically for this experimentation. At thesame time, all the relevant agro-meteorological data, within two selected plots (onefor each O 3  treatment) and those collected from a two-level meteo tower (3 m and6 m), were continuously monitored and recorded (Fig. 1) by means of a CR10Xdatalogger (Campbell Scientific, Logan, UT) equipped with an AM16/32 relaymultiplexerand an SDM-SW8A switch closureinterface (Campbell Scientific, Logan,UT). Climate conditions and rainfall during the growing season are illustrated inFig.1.  2.2. Sampling scheme During each campaign, histo-cytochemical and optical measurements (i.e. CFand spectral reflectance) were made on fully expanded leaves. Histo-cytochemicalanalyses were done every 3 days onprimary, secondaryand tertiary leaves, in orderto detect invisible damages before the appearance of visible symptoms. Stomatalconductance for water vapour, CF and reflectance measurements were performedevery 3 days, on 30 leavesper treatment, afterthe complete expansion of secondaryleaves. The general conditions of plants under investigation were monitored dailyfortheappearanceofvisiblesymptoms.Attheendof theexperiment,140plantspertreatment were randomly selected and harvested in order to measure crop yieldparameters. Table 1 shows the scheduled campaigns and the collectedmeasurements. 1015202530350 10 20 30 40 50 60 70 80 DAS    T  m  e  a  n   (   °   C   ) 0102030405060 R ai  n (  mm )   RainT mean Fig. 1.  Mean daily temperature and rain during the growing season (11/07/2006–5/10/2006, DAS ¼ day after sowing). G. Gerosa et al. / Environmental Pollution 157 (2009) 1727–1736  1728   2.3. Stomatal conductance measurements Stomatal conductance for water vapour was measured by means of a cyclingdiffusiveporometerAP-4(Delta-TDevicesLtd.,Cambridge,UK).Theinstrumentwascalibrated prior to each series of measurements. Measurements were performedtwice aweek, from 13 to63 days aftersowing(DAS), attempting toinclude differentconditions of air temperature(T), photosynthetic photon flux density (PPFD) and airvapourpressuredeficit(VPD),inordertoensuretherepresentativityofthestomatalconductance model derived. Conductance was measured in the median abaxialportion of the leaf lamina, on top canopy leaves of 30 randomly selected plants.Some values of adaxial conductance were periodically acquired too, in order toassess the relative role of the upper leaf lamina in the leaf gas exchange process.Every 15 days, diurnal cycles of conductance measurements were performed bymeans of five series of measurements between 7 am and 5 pm. Overall, about 750measurements of stomatal conductance were performed.  2.4. Assessment of visible and invisible symptoms Visible leaf injuries were assessed during the first month of the experimentevery 2 days, in order to detect the onset time of visible symptoms throughout thedifferent ozone treatments.Samples of leaves were examined microscopically with histo-cytochemicaltechniques, in order to detect cell damages before the appearance of visiblesymptoms.TolocalizeH 2 O 2 accumulationsites,leafdiscs(1 cmindiameter)wererandomlypunched with a cork-bore from primary and secondary leaves, avoiding the mainveins. Discs were then dark infiltrated overnight with 1 mgml  1 3,3 0 -diaminobenzidine (DAB)–HCl, adjusted to pH 5.6 with 1 M NaOH. After DAB uptake,discs were cleared in 96% boiling ethanol and examined with a light microscope.H 2 O 2  deposits were visualized as a reddish-brown coloration. As negative control,DAB solution was supplemented with 10 mM ascorbic acid (Iriti and Faoro, 2003a).To assess cell death, Evans blue staining was carried out by boiling leaf discs for1 min in a mixture of phenol, lactic acid, glycerol, distilled water and 20 mgml  1 Evans blue (1:1:1:1), prepared immediately before use. Discs were then clarifiedovernightina solution of2.5 gml  1 chloralhydrate inwater. Deadcells stained blue,whereas the undamaged ones appeared unstained (Faoro and Iriti, 2005).All samples were examined with an Olympus BX50 light microscope (Olympus,Tokyo, Japan) equipped with differential interference contrast (DIC) and epipolari-zation filters.  2.5. Field optical measurements (fluorescence and reflectance) Chlorophyll fluorescence and spectral reflectance measurements in light-adaptedstatewereacquiredunderclear-skyconditions,withinapproximately2 hof solar noon. We attempted to standardize light conditions by selecting leavesexposed to saturating sunlight (photosynthetic photon flux density PPFD greaterthan 1300  m molm  2 s  1 ) from the top of the canopy.The actual photochemical efficiency of PSII ( D F  / F  m 0 ) was calculated with a PAM-2000 fluorometer (Walz, Effeltrich, Germany), according to Genty et al. (1989), as( F  m 0  F  t )/ F  m 0 , where  F  m 0 is the maximal fluorescence during light saturation and  F  t  isthe level of steady-state fluorescence of light-adapted leaves.Spectral measurements were acquired by a Fieldspec Handheld Prospectrometer (Analytical Spectral Devices, Boulder, CO,USA) within a spectral rangeof 325–1075 nm, a spectral resolution of 3.5 nm (FWHM, Full Width at Half Maxi-mum) and a sampling interval of 1.6 nm (1 nm resampling).Reflectance measurements were collected using a probe holder (RPH-1, OceanOptics, Dunedin, FL, USA), with a bifurcated fiberoptic attached to the spectroradi-ometer and to a tungsten halogen light source (HL-2000-FHSA, Ocean Optics,Dunedin, FL, USA). A circular spot of 6.35mm diameter was illuminated andobserved with a 45  angle relative to leaf normal (adaxial side upwards).Leaf reflectance was calculated by rationing the radiance reflected by the leaf with the radiance of a reflectance standard panel. Fifteen scans per sample wereaveraged. Two leaf reflectance indices were then calculated from the reflectancespectrum data, as follows:1. PRI (related to the de-epoxidation of the xanthophyll cycle pigments):PRI  ¼ ð R 531    R 570 Þ = ð R 531 þ R 570 Þ  (1)2. NDI (related to chlorophyll concentration):NDI  ¼ ð R 750    R 705 Þ = ð R 750  þ  R 705 Þ  (2)In the above indices,  R  refers to reflectance, and the subscript refers to the wave-bands in nanometres. The Photochemical Reflectance Index (PRI) was calculatedfrom leaf reflectance using 531 nm as the xanthophyll sensible band and 570 nm asthe reference band (Gamon et al.,1992). The Normalized Difference Index (NDI) wasdetermined from leaf reflectance in the near-infrared region (750 nm) and from theedge of the chlorophyll absorption feature (705 nm) (Gamon and Surfus, 1999;Gitelson and Merzlyak, 1994).  2.6. Crop yield analysis Harvest occurred at DAS 86, when 75% or more pods per plant completelysenesced. Seventy plants per plot (140 per treatment) were randomly selected anddestructively harvested. Yield ratios and productivity parameters considered were:(i) number and weight of seeds per plant; (ii) number and weight of seeds per pod;(iii)numberandweightofpodsperplant;(iv)weightof100seeds. Weightsofseedsand pods were measured after oven drying for 48–72 h at 70   C, until constantweight (Iriti and Faoro, 2003b).  2.7. Stomatal conductance modelling  In order to calculate stomatal fluxes, the stomatal conductance for bean plantswas modelled using the classic Jarvis multiplicative approach ( Jarvis,1976):  g  w  ¼  g  max ½  f  PHEN  f  T  f  LIGHT  f  VPD   (3)The  g  max  for water value was chosen by taking the 95th percentile of all theconductance measurements.The species-specific limiting functions  f  T ,  f  LIGHT  and  f  VPD , which give a valuebetween 0 and 1, were characterized by borderline analysis, using the pointscorresponding to the 98th percentile of   g  rel  (conductance value relative to the  g  max )plotted for each class of values of environmentalvariable considered (T, VPD, PPFD).It was thus possible to make the modelling less dependent on outliers representingmeasurements of doubtful quality.The  f  PHEN functionisatime-basedequationwhichdescribesthereductionof themaximum stomatal conductance due to leaf age and senescence. It was defined bymodelling the observed reduction of the maximum value of   g  max  measured at noonin different dates with similar environmental conditions (T, VPD, PPFD).  2.8. Ozone exposure and stomatal flux calculation Ozone exposure was calculated by means of the cumulative index AOT40 basedon the ozone mean hourly concentrations during sunlight hours:AOT40  ¼ X c i  :  ½ O 3  i  >  40 ppb c i  :  RadGlob  >  50 W = m 2  ½ O 3  i  40  D t   (4)Ozone stomatal fluxwas calculatedfora topcanopyleaf, accordingtoEmbersonet al. (2000) and Karlsson et al. (2000): F  st ; O 3  ¼ ½ O 3  =  R b ; O 3  þ 1 : 68 =  g  w   (5)where  g  w  is the stomatal conductance of the projected leaf area, which takes intoaccount the conductance of both upper and lower leaf lamina, obtained as:  g  w  ¼  g  w ; abaxial  þ  g  w ; adaxial  ¼  g  w ; abaxial ð 1 þ  r  Þ  (6)where r  istherelativecontributeoftheadaxialconductancetotheleafgasexchangewithrespecttotheabaxialconductance; 1.68accountsforthedifferentdiffusivityof waterinairwithrespecttotheozone.Theboundarylayerozoneresistance R b ; O 3  wascalculated using the Unsworth et al. (1984) equation: R b ; O 3  ¼  k ð d = u Þ 1 = 2 (7)where the empirical coefficient  k  is set to a value of 132, as supported by Thom(1975) measurements,  d  is the leaf mean width (10 cm), and  u  is the wind meanspeed at the top canopy level, set to the  u  value measured continuously byanemometers placed at canopy level (0.8 ms  1 on average).  Table 1 Summary of the field campaigns and measurements collected (DAS, day aftersowing)DATE 20 July 24 July 27 July 31 July 4 Aug 8 AugDAS 9 13 16 20 24 28Reflectance  U U U U U Fluorescence  U U U U U Microscopic symptoms  U U U U U Visible symptoms  U U U U U U Stomatal conductance Every 3 days up to 12 September G. Gerosa et al. / Environmental Pollution 157 (2009) 1727–1736   1729  Atmospheric resistance  R a  was not considered, since the ozone concentrationwas measured close to the top canopy level. In any case, within the OTCs the forcedventilationestablishesacontinuousturbulentmixingoftheairavoidingtheonsetof an O 3  height gradient.Two ozone stomatal flux indices were used for calculation of seasonal ozonedoses absorbed by plants: the first was a simple accumulated flux of ozone with noflux threshold (AF ST 0), while the second was an accumulated flux over a fluxthreshold of 6 nmolm  2 s  1 (AF ST 6), which has also been suggested by UN-ECE(2004) for agricultural crops experiments.  2.9. Statistical analysis To analyze the effects of different O 3  exposures on fluorescence and spectralreflectance measurements (i.e.  D F  / F  m 0 , PRI and NDI) we used general linear mixedmodels (GLMM) of the form: Y  ijkl  ¼  m  þ  DAS i  þ  T   j  þ OTC ð T  Þ k ð  j Þ þ DAS   T  ij  þ DAS  OTC ð T  Þ ik ð  j Þ þ 3 ijkl  (8)where  Y  ijkl  was the measured variable onplant  l , in the dayof measurement  i , in theOTC  k  receiving the treatment  j ,  m  was the grand mean and  3 ijkl  the error. The O 3 treatment ( T  ) and plant growing factor, expressed by the sampling date (DAS), weretreated as fixed factors, while the OTC nested within the treatment factor (OTC( T  ))was treated as random factor (Filion et al., 2000).Similarly, crop yield data, in terms of count and weight of pods and seeds, wereanalyzed by means of a following GLMM: Y   jkl  ¼  m  þ  T   j  þ OTC ð T  Þ k ð  j Þ þ 3  jkl  (9)The use of linear mixed model analysis avoided pseudoreplication, given by the factthatplantsgrowinginthe sameOTCshared thesameenvironmental conditionsandtherefore were not statistically independent. All the analyses were performed bymeans of PROC MIXED module in SAS (Littell et al., 1996), in the case of normalvariabledistribution,andwiththeGLIMMIXmacrointhecaseofPoissonornegativebinomial distributions (Littell et al., 1996). The effective denominator degrees of freedom in  F  -test were computed according to a general Satterthwaite approxi-mation (Satterthwaite,1946).Relationships between  D F  / F  m 0 , PRI and NDI and three O 3  indices (AF ST 0, AF ST 6and AOT40) were evaluated by ordinary least squares (OLS) regression analysis. Inorder to compare the regression accuracy the determination coefficients ( R 2 ) werecomputed, the significance of regressions were tested and the hypothesis of normaldistribution of the independent variable values were checked with the Shapiro–Wilk’s test (Shapiro and Wilk,1965). 3. Results  3.1. Stomatal conductance model The maximum abaxial stomatal conductance (  g  max ) observedwas 2.60 cm s  1 referred to the projected leaf area. The adaxialconductances were on average 31.8% of the abaxial ones, so thecoefficient  r   in Eq. (6) was set to 0.318.The functions that describe the dependence of the stomatalconductance on the considered environmental parameters,obtained by borderline analysis, are reported in Fig. 2.The function that links the stomatal conductance tothe PPFD is:  f  PPFD  ¼  1  e  a  PAR  (10) where  a  is equal to 0.004 and PPFD is expressed in  m mol photonsm  2 s  1 .The  f  T  function is described by:  f  T  ¼ ð T     T  min Þ  T  opt    T  min "  ð T  max    T  Þ  T  max    T  opt # b (11) where  T   represents the temperature of the air near the leaf;  T  opt , T  max ,  T  min  are three species-specific parameters representingoptimal,maximumandminimumtemperatures,allexpressedin  Cand whose values are 28   C, 34   C, 16   C, respectively;  b  is definedas: b  ¼  T  max    T  opt  T  opt    T  min   (12) If   T   is greater than T  max or smaller than T  min , the function  f  T  is set tothe minimum value of 0.1.The dependency of stomatal conductance from the evaporatingpower of the atmosphere is described by the following linearfunction:  f  VPD  ¼ ð 1  0 : 1 Þð c    VPD Þð c     d Þ þ 0 : 1 (13) where VPD is the water vapour pressure deficit expressed in KPa,  c  representsthevalueofVPDatwhichstomatalconductancereaches a 00.20.40.60.811.20 500 1000 1500 2000 PPFD [   mol m -2  s -1 ]     g     /    g     m    a    x     g     /    g     m    a    x     g     /    g     m    a    x b 00.20.40.60.811.25 10 15 20 25 30 35 40 45 T [°C] c 00.20.40.60.811.20 1 2 3 4 5 VPD [KPa] Fig. 2.  The functions describing the dependence of the stomatal conductance (  g  /  g  max )on photosynthetic photon flux density (PPFD) (a), air temperature ( T  ) (b), vapourpressure deficit (VPD) of the air (c). G. Gerosa et al. / Environmental Pollution 157 (2009) 1727–1736  1730  its lowest value, established at 0.1, and  d  represents the value of VPD beyond which stomatal conductance begins to suffer a limita-tion. In the case of VPD values lower than  d ,  f  VPD  is established at 1,whereas, when VPD exceeds the  c   value,  f  VPD  is established at 0.1.Parameters  c   and  d  are species-specific and their values are 1.7 kPaand 4.85 kPa, respectively.The phenology limiting function  f  PHEN  is a time-based equationwhich is defined as follows:  f  PHEN  ¼ 8>><>>: 0  c  DAS  <  a 0 : 1429 ð DAS  11 Þ  c  a  DAS  <  b 1  c  b  DAS  <  c   0 : 01 ð DAS  28 Þþ 1  c  DAS  c  (14) where  a ¼ 11 is the date when the first (trifoliate) leaves startedtheir expansion, expressed as DAS;  b ¼ 18 is the date when the a 050010001500200025003000350040004500500001/07/2006 01/08/2006 01/09/2006 01/10/2006 date    A   O   T   4   0   (   l   i  g   h   t   h  o  u  r  s   )   [  p  p   b   h   ] b 024681012141618202201/07/2006 01/08/2006 01/09/2006 01/10/2006 date    A   F    S   T    0   O    3    (  m  m  o   l  m   -   2    )   A   F    S   T    6   O    3    (  m  m  o   l  m   -   2    ) c 024681012141618202201/07/2006 01/08/2006 01/09/2006 01/10/2006 date FNFFNFFNF Fig. 3.  Ozone exposure as AOT40 (a), ozone dose as accumulated stomatal flux with noflux threshold (AF ST 0) (b) and accumulated stomatal flux over a flux threshold of 6 nmolm  2 s  1 of O 3  (AF ST 6) (c); the date 01/08/06 corresponds to 21 DAS. Fig. 4.  Phaseolus vulgaris  leaf fragments from plants grown in non-filtered (OTC-NF) (a,c, e, g, i) and filtered (b, d, f, h, l) open-top chambers (OTC-F); all bars ¼ 30  m m. Inprimary, still asymptomatic, leaves from OTC-NF, at 9 days after sowing (DAS), somedead mesophyll cells (a, stained in blue by Evans) are already visible in the substomatalcavity, and H 2 O 2  deposits (c, stained in brown by DAB) are localized in the wall andcytoplasm of adjacent epidermal cells. Neither dead cells (b) nor H 2 O 2  deposits (c) arepresent at the same time in primary leaves from OTC-F. At 13 DAS, typical bronzingappears in primary leaves from OTC-NF (e), but not in the same leaves from OTC-F (f).At the same time, still asymptomatic secondary leaves show some dead cells in thepalisade mesophyll (g, arrow) and many epidermal cell walls are stained in brown (i),indicating diffuse H 2 O 2  deposits. Neither dead cells (h), nor H 2 O 2  deposits are visible,at 13 DAS, in secondary leaves from OTC-F. G. Gerosa et al. / Environmental Pollution 157 (2009) 1727–1736   1731
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