Short-term changes of fructans in ryegrass (Lolium multiflorum ‘Lema’) in response to urban air pollutants and meteorological conditions

Ecotoxicology and Environmental Safety 96 (2013) 80–85

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Ecotoxicology and Environmental Safety

0147-65http://d

n CorrE-m

(R.d.C.L.mmingo

journal homepage: www.elsevier.com/locate/ecoenv

Short-term changes of fructans in ryegrass (Lolium multiflorum ‘Lema’)in response to urban air pollutants and meteorological conditions

Carla Zuliani Sandrin a, Rita de Cássia Leone Figueiredo-Ribeiro a,Welington Braz Carvalho Delitti b, Marisa Domingos a,n

a Instituto de Botânica, Caixa Postal 68041, 04045-972 São Paulo, SP, Brazilb Instituto de Biociências, Universidade de São Paulo, Departamento de Ecologia, Caixa Postal 11461, 05422-970 São Paulo, Brazil

a r t i c l e i n f o

Article history:Received 9 December 2012Received in revised form14 June 2013Accepted 21 June 2013Available online 18 July 2013

Keywords:Sulphur dioxideNitrogen dioxideTemperatureCarbohydrateBiomassRelative humidity

13/$ - see front matter & 2013 Elsevier Inc. Alx.doi.org/10.1016/j.ecoenv.2013.06.018

esponding author. Fax: +55 11 5073 3678.ail addresses: [email protected] (C.Z. SaFigueiredo-Ribeiro), [email protected] ([email protected], [email protected].

a b s t r a c t

We investigated whether the fructan content, a storage carbohydrate, of Lolium multiflorum ‘Lema’ plantsgrown in a subtropical urban environment characterized by typical diurnal profiles of air pollutants andmeteorological conditions changed over the course of a day during different seasons. Plants werecollected every 2 h on the last day of each two-month seasonal field experiment and separated into shoot(stubble or stubble+leaf blades) and roots for carbohydrate analyses and biomass determination. Diurnalcontents of total fructose in the stubbles increased with high temperatures. In the roots, fructoseaccumulation showed a positive relation with hourly variations of both temperature and particulatematter and a negative relation with irradiance and SO2. Seasonal variation in shoot and root biomassescoincided with the seasonal variation of total fructose and were negatively affected by relative humidityand SO2, respectively. We concluded that hourly changes of fructans over the course of a day mayincrease the ability of L. multiflorum to tolerate short-term oscillations in weather and air pollutioncommonly observed in the subtropical urban environment, increasing its efficiency in monitoring airquality.

& 2013 Elsevier Inc. All rights reserved.

1. Introduction

Second to starch, fructans (polyfructosylsucrose) are the prin-cipal storage carbohydrates of plants. In grasses, particularly inLolium species, fructans of the inulin series, the inulin neoseriesand the levan neoseries (Pavis et al., 2001a) are stored in thestems, tiller bases, leaf sheaths, elongating leaf bases and, to alesser extent, in the leaf blades and roots (Guerrand et al., 1996;Sandrin et al., 2006). Fructan synthesis in the leaves is closelylinked to photosynthetic sucrose synthesis and reflects transloca-tion and subsequent sucrose metabolism in the stems and roots(Housley and Pollock, 1993).

Fructans play important roles in plants growing under unfavor-able environmental conditions (Livingston et al., 2009), includingair pollution. In this case, fructans could be important actingdirectly as scavengers of reactive oxygen species (ROS) or indir-ectly by stimulating other specific antioxidative defense mechan-isms (Van den Ende and Valluru, 2009 and references therein).Indeed, a higher concentration of sugars has been observed in

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ndrin), [email protected]),br (M. Domingos).

species tolerant to air pollution in relation to sensitive ones(Seyyednejad et al., 2011), indicating that fructose polymers couldhelp tolerant species survive under unfavorable conditions. In ourprevious studies, for example, fructans have increased in ryegrass(Lolium multiflorum ssp. italicum cv. Lema) seasonally exposed tothe ambient air pollution of São Paulo (Brazil) (Moretto et al.,2009; Sandrin et al., 2008).

Taking in account that the carbohydrate metabolism in plants ispromptly affected by natural environmental oscillations (Kaganet al., 2011) our hypotheses were: (1) the seasonal responsesobserved in this ryegrass cultivar grown in a polluted environmentmay result from hour to hour changes in the fructan metabolism;and (2) these changes occur in response to typical variations ofstressful urban environmental conditions, generally registeredduring a single day in the subtropics. So we investigated in thepresent study whether the concentration of fructans in thestubbles and roots of L. multiflorum ssp. italicum cv. Lema waschanging over the course of a single day in each season in plantsgrown in an environment characterized by the typical diurnalprofiles of air pollutants from vehicular emissions and of climate.The growth of L. multiflorum plants under such conditions couldhelp to determine whether urban stress factors are involved inshort-term plant-atmosphere interactions, as mediated by changesin fructan metabolism.

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2. Materials and methods

2.1. Plant material and exposure

Seeds (0.8 g per pot) of L. multiflorum Lam. ssp. italicum Beck cv. Lema weregerminated and cultivated in plastic pots inside a greenhouse with filtered air atthe Institute of Botany (South Region of São Paulo City; 23138′S and 46137′W;805 m elevation). A commercial substrate (Eucatex Plantmax SFA) mixed with finevermiculite (3:1, v/v) was used. During the six weeks of cultivation, the plants weretrimmed weekly to a height of 4 cm from the substrate (VDI, 2003) and fertilized(40 cm3 per pot) with a nutrient solution containing only macronutrients (101.10 gKNO3, 236.16 g Ca(NO3)2 �4H2O, 115.08 g NH4H2PO4, 246. 49 g MgSO4 �7H2O in oneliter of deionized water). This procedure of growing the plants under controlledconditions was repeated before each field experiment.

The field experiments started with 25 pots containing approximately 20 plantseach and equipped with nylon wicks over deionized water basins to provide acontinuous water supply (VDI, 2003). The pots were exposed next to a street withintensive vehicular traffic (Congonhas—23136′S and 46139′W; 768 m elevation)(CETESB, 2010) for two months. Maximal accumulation of fructans could be foundin ryegrass plants during this period, as observed in a previous experimentperformed under similar subtropical condition (Sandrin, 2007).

Four field experiments were performed, one in each season of the year (spring:02nd October to 27th November/2002; summer: 30th December/2002 to 24thFebruary/2003; autumn: 19th March to 14th May/2003; winter: 01st July to 26thAugust/2003). Plants from three different pots (replicates) were collected every 2 h(at 8 h, 10 h, 12 h, 14 h, 16 h and 18 h) over the course of the last day of eachexperiment and separated into stubbles (composed of basal parts of stems+leafsheaths+expanding leaves) and roots for carbohydrate analyses. A total of 72

Fig. 1. Diurnal variation in temperature, relative humidity and irradiance next to the planthe sampling days ((D), (E) and (F)) of the different seasons. Distinct letters in parenthes(po0.05).

samples of stubbles (three replicates, six hourly samplings during the day in eachseason) and 36 samples of roots (three replicates, six hourly samplings during theday in the spring and autumn) were analysed. Additionally, plants from five potsexposed for two months at the polluted site during the spring, autumn and winterwere harvested in the morning of the sampling days, for the determination of theshoot (stubbles+leaf blades) and root biomasses. Hourly temperature and relativehumidity were measured during the sampling days using thermo-hygrographs. Theconcentrations of air pollutants (particulate material with diameter until 10 μm –

PM10, sulphur dioxide – SO2, nitrogen dioxide – NO2) were obtained from an airquality monitoring station of the State Company of Air Pollution Control (CETESB),installed next to the plant exposure frame. Hourly data of air pollutants concentra-tions from this monitoring station and hourly data of meteorological variables froma neighbor monitoring station (Ibirapuera) were used to draw the daily averagecurves (at 8 h, 10 h, 12 h, 14 h, 16 h, 18 h) during the exposure period in each season.We assumed that ozone was not a predictor of the fructan variations in the plantsduring the sampling days because it generally occurs in low concentrations next toNOx emission sources, which was the case of the exposure site according to CETESB(CETESB, 2000).

2.2. Extraction and analysis of soluble carbohydrates

The soluble carbohydrates (monosaccharides, sucrose and fructans) fromstubble and roots of L. multiflorum plants were extracted three times in boiling80% ethanol and once in hot distilled water (Pollock and Jones, 1979). Ethanol andwater extracts were combined, evaporated to dryness under vacuum and resus-pended in water. Total free and combined fructose were quantified by the anthronemethod (Jermyn, 1956), using fructose as standard. Aliquots of these extracts werepurified with polyvinylpolypyrrolidone (10%) (Pavis et al., 2001b) and passed

t exposure apparatus, during the two month-exposure period ((A), (B) and (C)) andis indicate significant differences in the diurnal profile observed among the seasons

C.Z. Sandrin et al. / Ecotoxicology and Environmental Safety 96 (2013) 80–8582

through a column containing cation (Amberlite, IR-120) and anion (Amberlite, IR-400) exchange resins and C18 modified silica (Waters) (Smouter and Simpson,1991). After elution with deionized water, the extracts were concentrated undervacuum. Component sugars were analyzed by high-performance anion exchangechromatography coupled with pulsed amperometric detection (HPAEC-PAD DX-300, Dionex) using an analytical Carbo-Pac PA1 column (4�250 mm) with asodium acetate gradient (1 cm3 min−1) in 150 mol m−3 NaOH. The elution programfollowed that of Itaya et al. (1997).

2.3. Statistics

Significant differences in the diurnal profiles of weather, air pollution and totalfructose of stubbles measured in each season were identified by means of nonparametric ANOVA on ranks, followed by the Tukey test for multiple comparisons.The non parametric Mann–Whitney test was used for comparing the diurnalprofiles of total fructose in roots harvested in the spring and autumn. In all cases,the average values obtained from 8 h to 18 h per sampling day comprised eachtreatment.

The significant differences in the shoot and root biomasses and root/shoot ratioamong seasons were indicated by ANOVA on ranks, followed by the Dunn´s Methodfor multiple comparisons.

A Principal Component Analysis (PCA) was performed for the abiotic (weatherand air pollutants) and biological (fructose contents of stubble and roots) datatransformed by ranging. FITOPAC software (Shepherd, 1996) was employed totransform the data, and PC-ORD version 3.0 for Windows (McCune and Mefford,1997) was used for the analysis.

A quantitative multivariate analysis was also performed to verify whether thevariations in total fructose and biomass of plants exposed at Congonhas could bepredicted from the meteorological (air temperature, relative humidity and irra-diance) or air pollution variables (atmospheric concentrations of PM10, NO2 and

Fig. 2. Diurnal variation in particulate matter, nitrogen dioxide and sulphur dioxide n(B) and (C)) and the sampling day ((D), (E) and (F)) of the different seasons. Distinct leamong the seasons (po0.05).

SO2). Data were transformed when necessary and analyzed with the stepwisemethod. The adjustment procedure started from the saturated model (with all ofthe variables present), removing the variable with the smallest participation toexplain the variations in total fructose, and new adjustments were successivelyperformed. Only those variables that significantly (po0.05) contributed to theexplanation of these variations remained in the final model.

3. Results

The temperature profiles were significantly lower during win-ter in both two month-period (Fig. 1A) and harvesting day(Fig. 1D). The average daily curves of relative humidity andirradiance did not vary significantly among the seasonal exposureperiods (Fig. 1B and C), but were significantly lower during thesampling day of winter, compared to the values monitored duringthe summer day (Fig. 1E and F).

Analyzing the diurnal profile of the meteorological variables,temperature tended to increase from 12 h to 16 h in the harvestingday of most seasons. In contrast, relative humidity tended todecrease around 12 h to 16 h in the days of spring, autumn, andsummer. Irradiance tended to increase from 8 h to 12 h, decreasingafter that in all the harvesting days (Fig. 1D to F).

On average, significant higher levels of particulate matter werefound during the two-month exposure period of spring, andsignificant higher levels of sulphur and nitrogen dioxides were

ext to the plant exposure apparatus, during the two month-exposure period ((A),tters in parenthesis indicate significant differences in the diurnal profile observed

C.Z. Sandrin et al. / Ecotoxicology and Environmental Safety 96 (2013) 80–85 83

found during the exposure periods of spring and winter (Fig. 2A toC). The atmospheric levels of particulate matter and nitrogendioxide were statistically similar during the four days of plantsampling (Fig. 2D and F). However, sulphur dioxide was signifi-cantly more concentrated during the harvesting day of winter(Fig. 2E). In general, the air pollutant levels tended to increasethroughout the two-month periods and during the harvestingdays in all of the seasons, except nitrogen dioxide during theharvesting day of winter (Fig. 2A to F).

The highest and lowest levels of fructose in the stubbles werefound during the days of summer and winter, respectively. Thecontent of fructose in the stubbles tended to increase in plantssampled at 12 h and at 14 h in the days of spring and winter, andat 14 h in the summer harvesting day. However, the levels offructose tended to decrease between 12 h and 14 h in the day ofautumn (Fig. 3).

Significantly higher fructose content was observed in the rootsduring the day of spring. Total fructose content was lower in theroots than in the stubbles, tending to increase in the afternoon inplants from experiments carried out in spring and autumn (Fig. 3).

The Principal Component Analysis (PCA) showed that the firsttwo axes assembled 78% of the variability of the diurnal profiles ofenvironmental conditions and contents of total fructose in thestubbles of plants harvested in all seasons. Total fructose, irradi-ance, temperature and sulphur dioxide were the variables moreclosely related with axis 1. However, as lower irradiance and airtemperature, as well as higher concentrations of air pollutantsfrom vehicular emissions characterize the sub-tropical winter, thevectors of both meteorological variables and sulphur dioxide wereassociated to opposite sides of axis 1. The units from the samplingday of winter were clearly associated with higher values of relativehumidity and sulphur dioxide, and lower values of irradiance,temperature e total fructose. The units of samplings performed in

Fig. 3. Diurnal content of total fructose in stubbles and roots of L. multiflorumplants during the sampling day of the different seasons. Distinct letters inparenthesis indicate significant differences in the diurnal profile observed amongthe seasons (po0.05). Bars indicate the standard error.

the afternoon (mainly at 16 h and 18 h) in the days of spring,summer and autumn were related with higher values of airpollutants. The units of samplings performed in the morning (at8 h to 12 h) were related with irradiance, temperature and totalfructose (Fig. 4).

The PCA also revealed that the first two axes assembled 82% ofthe variability of the profiles of environmental conditions andcontents of total fructose in the roots of plants harvested in thedays of spring and autumn. The air pollutants were the variablesmore closely related with axis 1, and total fructose, temperatureand relative humidity with axis 2. The units of samplingsperformed in the afternoon (mainly at 16 h and 18 h) of bothseasons were characterized by higher concentrations of air pollu-tants. Units of samplings performed at 10 h, 12 h and 14 h in theday of spring and at 12 h in the day of autumn were more relatedwith higher values of temperature, irradiance and total fructose(Fig. 4).

The multivariate stepwise analysis evidenced that 44% of thevariability in the content of total fructose in the stubble of plantsexposed at Congonhas could be significantly predicted by apositive relation with temperature. For the roots, 83% of thevariability in the fructose content could be significantly predicted

Fig. 4. Principal Component Analysis (PCA) for the diurnal values of the abiotic andbiological (stubbles and roots) variables during the sampling day of the differentseasons. Abbreviations: F¼total fructose; PM¼particulate material; SO2¼sulphurdioxide; NO2¼nitrogen dioxide; T¼temperature; RH¼relative humidity;I¼ irradiance.

C.Z. Sandrin et al. / Ecotoxicology and Environmental Safety 96 (2013) 80–8584

by a negative relation with sulphur dioxide and a positive relationwith temperature and particulate matter (Table 1).

Plants exposed during spring displayed significant higher shootbiomass and lower root biomass and root/shoot ratio than theplants exposed in the other seasons (Fig. 5). The variability ofbiomass of shoots (94%) and roots (70%) were significantlypredicted by a negative relation with relative humidity andsulphur dioxide, respectively (Table 1).

Fig. 5. Seasonal accumulation of shoot and root biomasses and root/shoot biomassratio of L. multiflorum plants. Distinct letters indicate significant differences amongthe seasons (po0.05). Bars indicate the standard error. Data of summer were notavailable due to vandalism.

4. Discussion

The diurnal variations of weather and air pollution during thesampling days mostly coincided with the average diurnal profilesmeasured during the two-month exposure period, indicating thatthe plants were sampled in one typical day of each season. Inconsequence, we may infer that our hypothesis that the fructanmetabolism of L. multiflorum ‘Lema’ was affected by a combinedeffect of typical variations of stressful urban environmental con-ditions generally registered over the course of a single day wasconfirmed. Our results from multivariate analyses indicated thattemperature, irradiance, SO2 and PM10 were the main environ-mental factors that influenced the diurnal profiles of fructancontents in stubbles and roots of plants exposed at thepolluted site.

The fructan contents in stubbles tended to increase in theafternoon (12–14 h) in all seasons, particularly during the summer,when the temperature reached the highest peak in a day. Underthis condition, photosynthesis might have been stimulated, result-ing in sugar accumulation. Our results differ from studies that haveshown fructan accumulation under low temperatures (Pollock andLloyd, 1987; Thorsteinsson et al., 2002). In contrast, they areconsistent with our previous findings, revealing that fructancontents of L. multiflorum plants from an urban environment isseasonally stimulated by increasing air temperatures (Sandrinet al., 2008). Adachi et al. (2000) also reported similar results inchrysanthemums plants.

Water soluble carbohydrates, including fructans, would beexpected to decrease overnight and to increase during the day, asphotosynthesis predominates (Kagan et al., 2011). In the presentwork, the diurnal profile of total fructose tended to vary in thestubble, but fructan composition (data not shown) remained fairlyconstant. In other grasses, diurnal changes of fructan concentrationand composition were very contrasting. Some studies pointedmarked diurnal variations (Kagan et al., 2011; Marais et al., 1993;Souza et al., 2005; Waite and Boyd, 1953) while others showedstable concentrations during the day in the aerial organs (Ciavarellaet al., 2000; Lechtenberg et al., 1972), indicating that fructanmetabolism is highly affected by several environmental factors.

In the roots of L. multiflorum, higher levels of total fructosewere also found in the afternoon, coinciding with high tempera-tures, low irradiance and the highest concentrations of air pollu-tants, reaffirming the possible influence of such environmentalvariables on the photosynthesis, already discussed. The pollutants,however, were related in different ways with the photosynthesisand carbohydrate accumulation in the roots. SO2, for example, that

Table 1Predictive linear model of total fructose contents (TFC) and biomass in L. multiflorum pPM10¼particulate material; RH¼relative humidity.

Linear predictive equation (po0.05) R2

[TFC]stubbles¼41.43+(7.59nT) 0.4[TFC]roots¼−20.01+(2.60nT)−(0.03nI)−(0.69nSO2)+(0.21nPM10) 0.8Biomassstubbles¼106.99−(1.28nRH) 0.9Biomassroots¼28.37−(0.91nSO2) 0.7

related negatively with the fructan contents in the roots, mighthave inhibited these physiological processes, similar to resultsfrom other studies (Sandhu et al., 1992; Sheu, 1994). This assump-tion is reasonable taking in account that many phytotoxic gasesare readily assimilated, causing direct injury to vegetation morerapidly than do the most common particulate materials (Grantzet al., 2003).

In contrast with SO2, after deposition on the soil surface androot uptake, toxic components adsorbed on the urban particlesmight have increased the sugar content in the roots, as alsoobserved by Frossard et al. (1989) in L. perenne. Although carbonexchange and carbon pools are generally reduced by particulatematter, its effects depend on the chemical composition of particles,including heavy metals, cations, sulfate and nitrate (Grantz et al.,2003). In L. multiflorum plants, the accumulation of fructans couldbe a response to protect plants against reactive oxygen speciesproduced by PM10 or a pathway to control osmoregulation, asproposed for other plants (Van den Ende and Valluru, 2009; Garciaet al., 2011).

Although the fructan content was included in the multivariateanalysis as one of the independent variables, it was not identifiedas a significant predictor of the variability of biomass. However,the seasonal variation in shoot and root biomasses of ryegrasscoincided with the seasonal variation of total fructose, asexampled by results obtained during autumn and winter sam-pling. Similar to other works (Pandey and Agrawal, 1994; Sheu,1994), SO2 seemed to cause not only a decrease in the levels offructose in the roots but also their biomass. As expected, thecarbohydrate pool was relevant to promote the biomass produc-tion of ryegrass.

L. multiflorum cultivar Lema is a standardized bioaccumulatorof heavy metals, sulphur and fluorine (VDI, 2003) and is exten-sively used in many biomonitoring programs of air quality. Itsability to grow and produce shoot biomass in an urban andpolluted environment with meteorological conditions differentfrom those where it was originated is crucial to confirm itsefficiency for such purposes. Additionally, if we consider that the

lants exposed at Congonhas. T¼temperature; I¼ irradiance; SO2¼sulphur dioxide;

Adjusted R2 Significance (p value)

4 0.42 o0.0013 0.73 0.0084 0.94 o0.0010 0.67 o0.001

C.Z. Sandrin et al. / Ecotoxicology and Environmental Safety 96 (2013) 80–85 85

level of accumulation of toxic elements in the shoot is inverselyproportional to the shoot biomass, its accumulating efficiency issupposedly higher during the autumn and winter seasons, whenshoot biomass was lower. By analogy, the accumulator efficiency isgreater to the extent that the content of fructans is smaller, anassumption that should be tested more properly forward.

In brief, the hour-to-hour changes in Congonhas over thecourse of a single day affected both fructose concentration andthe biomass of shoot (including stubbles) and roots of L. multi-florum ‘Lema’. Surprisingly, the plants were differently affected byour environmental conditions: while the shoot was affected bymeteorological factors (temperature and relative humidity), theroots were mainly affected by air pollutants (SO2 and PM10).

5. Conclusions

Our results showed that multiple stress factors may act togetherin L. multiflorum ‘Lema’ plants growing in an urban environment.These results evidenced the crucial importance of determining theenvironmental conditions at the time of the execution of biomoni-toring programs, to be able to interpret the results properly. Inaddition, they increase the still-limited knowledge of the capabilityof plants to synthesize carbohydrates in response to abiotic stress.Our findings indicate that L. multiflorum ‘Lema’ cultivar is able tomodify its carbohydrate metabolism to allow growth in the face ofshort-term oscillations in the weather and air pollution levels. Thisfeature improves the ability of this plant to tolerate the stressfulconditions observed in urban environments, increasing its effi-ciency in monitoring air quality.

Acknowledgments

This work is part of the PhD thesis of the first author and wassupported by FAPESP (Proc. 00/06422-4 and 2005/04139-7). C. Z.Sandrin thanks CAPES for the PhD fellowship. M. Domingos, W.C.Delitti and R.C.L. Figueiredo-Ribeiro are researchers associated toCNPq. The manuscript was edited for grammar, spelling andvocabulary and sentence structure by highly qualified nativeEnglish speaking editors at the American Journal Experts.

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