A.Dezhban?A.Shirvany?P.Attarod?M.Delshad?M.Matinizadeh?M.Khoshnevis

Cadmium and lead effects on chlorophyll fluorescence, chlorophyll pigments and proline of Robinia pseudoacacia

A.Dezhban1?A.Shirvany1?P.Attarod1?M.Delshad2?M.Matinizadeh3?M.Khoshnevis3

Heavy metal contamination is one of the most important abiotic stresses affecting physiological activities of plants.We investigated the effects of cadmium(Cd)and lead(Pb)on chlorophyll fluorescence(Fv/Fm,Fo,and Fm), photosynthetic pigments(chlorophyll a and b),and proline in one-year-old seedlings of Robinia pseudoacacia.The seedlings were treated twice over a period of 10 days with Cd and Pb at concentrations of 0,250,500,1000 and 2000 mg L-1.Saline solution containing Cd and Pb was sprayed on the leaves.Chlorophyll and proline contents were measured after 10 days.Chlorophyll fluorescence of R.pseudoacacia was affected slightly by high concentrations(1000,2000 mg L-1)of Cd and Pb.Chlorophyll a and a/b increased at 1000 and 2000 mg L-1of Cd and proline contentof leaves was similar in alltreatments of Cd and Pb.Our results indicated thatphotosynthetic sensitivity of R.pseudoacacia to Cd and Pb contamination was weak. Photosystem II chlorophyll pigments were notdamaged by Pb and Cd stress.We conclude that chlorophyllfluorescence along with chlorophyll and proline contents are useful indicators of Cd and Pb stresses in R.pseudoacacia which widely planted in urban polluted regions in Iran.

Robinia pseudoacacia·Cadmium·Lead· Chlorophyll fluorescence·Chlorophyll·Proline

Introduction

Traffic generates particulate matter,aerosols and heavy metals in the urban roadway environment.Deposition of heavy metals on vegetation and soil can influence plant physiology by inhibiting enzyme systems and metabolic processes(Kramer and Kozlowski 1979;Kabata-Pendias and Pendias 1986).Black locust(Robinia pseudoacacia)is a heavy metalaccumulator(Sawidis etal.2001)and adapts to a wide range of environmental conditions(Rahmonov 2009).Gu¨lriz etal.(2006)reported concentrations of Pb in leaves of Robinia that were higher than in leaves of Acer, Populus,Ailanthus,Platanus or Fraxinus,the latter species being considered tolerant biomarker species for urban environments.Along most of Iran’s urban roadways and in parks R.pseudoacacia is planted to beautify landscapes because itis known to tolerate exposure to metals and other contaminant.Physiological processes such as photosynthesis have been shown to be very sensitive to heavy metals in higher plants(Tanyolac et al.2007).

In recent years,chlorophyll fluorescence analysis has been increasingly applied to ecophysiological studies. In vivo photosynthesis under field conditions can be assessed by chlorophyll fluorescence analysis(Schreiber et al.1994).Moreover,the effects of short-and long-term stress and damage to photosynthetic apparatus can bedefined,and once the stress is removed from the plant, chlorophyll fluorescence comes back to the initial state (Lichtenthaler and Rinderle 1988).A pulse amplitude modulated(PAM)system has recently been used to monitor the effects of potential pollutants of plants and algae by calculating the Fv/Fm(The photosystem II photochemical efficiency)parameter(Jones et al.1999;Lewis etal.2001;Frankartetal.2003;Nielsen etal.2003).Fv/Fmis a measurement of the light energy transfer in darkadapted samples or the photochemical quantum yield of open PSII centers(De Ell and Toivonen 2003).The Fv/Fmvariable can be a good indicator of the performance of the photosynthetic apparatus and shows the ability of the plants to tolerate environmental stresses(Maxwell and Johnson 2000).The increase in Fo(The minimum chlorophyll a fluorescence after the dark-adaptation)as an emission from the excited chlorophylls in PSII antenna subjected to Pb and Cd treatments can be related to an impact on the PSII reaction center,or to a reduction in the energy transfer from the chlorophyll a antennae attached with the PSII light–harvesting complex to the reaction center(Ralph and Burchett 1998).Fm(The maximum chlorophyll a fluorescence after the pulse of red light)emission indicates the state of PSII when all QAmolecules are in a reduced state in RCII(Mallick and Mohn 2003).

Based on chlorophyll fluorescence analyses,Ralph and Burchett(1998)reported that Pb was only mildly toxic to Halophila ovalis.The method of chlorophyll fluorescence measurement has many advantages.It can be done in a noninvasive and nondestructive way,continuously and rapidly,with no radioactivity,and without disrupting the integrity of the cell organism(Mallick and Mohn 2003). The pollutants of the air,such as heavy metals,affect the light harvesting complex,oxygen evolution complex,cytochrome complex,NADP+,and reduce the overall photosynthesis yield(Prasad and Strzalka 1999).

Other indicators of stress in plants are chlorophyll content and proline as an amino acid in the protein structure.Proline accumulates under a wide range of stress conditions such as high salinity,water shortage,high light intensity,high levels of heavy metals,and pollutants (Aspinall and Paleg 1981;Delauney and Verna 1993;Hare and Cress 1997).Measurements of chlorophyll a and b are important indices of the effects of air pollution and heavy metalstress on plants(Joshiand Swami2009).Some heavy metals,such as Hg2+,Cu2+,Cd2+,Zn2+and Ni2+,replace the central Mg2+atom in the chlorophyll molecule,lowering the fluorescence quantum yield and shifting the fluorescence spectrum(Ku¨pper etal.1996).Olivars(2003) investigated the effect of Pb on the phytochemistry of Tithonia diversifolia exposed to roadside automotive pollution,reporting that 120.79 mg kg-1of Pb concentration reduced chlorophyll from 9.32 to 9.11 mg g-1but the difference was not significant and T.diversifola growing wild on roadsides with heavy urban traffic presented levels of Pb in leaves and roots considered as typical of contaminated plants,but without visible symptoms of damage to the leaves or significant depletion of chlorophyll,carotenoids,proteins nor increases in lipid peroxidation and it was introduced as a resistant species to Pb.

The aim of this work was to evaluate changes in photosynthetic responses and chlorophyll and proline contents of R.pseudoacacia seedlings exposed to high concentrations of Cd and Pb to quantify tree species resistance used for afforestation in urban and industrial polluted areas.

Materials and methods

Plant material,growth,and treatment conditions

In June 2011 we evaluated responses to heavy metal exposure of one-year-old seedlings of R.pseudoacacia at Alborz research station on the southwest slope of Alborz Mountain in Iran(3548′N,5054′E and 1300 m a.s.l).The local climate is semi-arid.Plants were selected and assigned to five groups of treatments with solutions containing different concentrations of Cd and Pb:0(control), 250,500,1000,and 2000 mg L-1.Each treatment of Cd and Pb was applied to 9 individual plants.Heavy metal solutions were produced by dissolving chloride salts: (CdCl2·H2O)and(Pb2Cl2).The seedlings were treated with Cd and Pb concentrations two times at 5-day intervals. Saline solutions were sprayed on the leaves to determine the effects of pollutants without considering soil uptake, plant tissue concentrations of Cd and Pb or growth.

Measurements of chlorophyll fluorescence parameters

Chlorophyll fluorescence was determined using a portable fluorometer(PAM-2500,Heinz Walz Gmbh 2008).Plants were dark-adapted for 30 min to estimate the effect of treatments on photosystem II(PSII)efficiency.The following fluorescence parameters were measured:Fo,Fm, and Fv/Fm(Kitajima and Butler 1975;Genty et al.1989). Fluorescence measurements were made every other day after the initial exposure for the following 10 days.

Determination of chlorophyll pigments and proline contents

For pigment and proline analysis tree leaf samples were collected from each treatment after the final exposure on day 10.For chlorophyll pigment analysis,fresh plant material(100 mg)was homogenized with 10 mL acetone(80%),then homogenized leaves were centrifuged at 6000 rpm(15°C)for 10 min.Amounts of supernatants were then adjusted to 20 mL.The absorbance at 663 and 645 nm extracts was determined using a spectrophotometer (CAIHONG 722 UV/Spectrophotometer).Chlorophyll a and b were calculated on a fresh weight basis(mg g-1fw)(Arnon 1949).Proline content(mg g-1fw)of the collected leaves was determined spectrophotometrically by the method of Bates et al.(1973).

Statistics

We used a completely randomized design performed with nine replicates per treatment of Cd and Pb on the same plants.Data were subjected to analysis of variance (ANOVA)to examine the effects of time and treatment. Statistical analysis was carried out using the software SAS 9.1.Separation of means was performed on a given sampling day using Duncan test at 5%level of probability.

Result

Chlorophyll fluorescence

Photosynthesis of R.pseudoacacia was affected by exposure to high concentrations of Cd.Fo,increased with increasing Cd concentration,but was significant only at 2000 mg L-1treatment on the first day(day 0)(Table 1). Fmvalue decreased significantly on day 2 atconcentrations of 500,1000,and 2000 mg L-1and day 4 at the 250,500, 1000,and 2000 mg L-1concentrations of Cd.Also,Fv/Fmdeceased significantly in some concentrations of Cd such as 1000 and 2000 mg L-1on the first day(day 0)and on day 2,2000 mg L-1on day 4 and 500,1000,and 2000 mg L-1on days 6 and 8 compared to the control treatment (Table 1).

Foincreased significantly through the Pb treatments on the first day(day 0)by exposure to 500,1000, 2000 mg L-1and day 2 by exposure to 1000 and2000 mg L-1(Table 2).Fmincreased significantly only on the first day(day 0)at 500,1000,and 2000 mg L-1treatments of Pb.Fmwas not affected on the other days and there was no significant difference between treatments (Table 2).Fv/Fmdeclined significantly in Pb treatments at 1000 and 2000 mg L-1on the first day and on day 8 at 500,1000 and 2000 mg L-1on days 2 and 4 and at 2000 mg L-1on day 6(Table 2).

Table 1 Fo(minimum chlorophyll a fluorescence),Fm(maximum chlorophyll a fluorescence)and Fv/Fm(photosystem II photochemical efficiency)of R.pseudoacacia exposed to different concentrations of Cd

Table 2 Fo(minimum chlorophyll a fluorescence),Fm(maximum chlorophyll a fluorescence)and Fv/Fm(photosystem II photochemical efficiency)of R.pseudoacacia exposed to different concentrations of Pb

Chlorophyll content

Chlorophyll a content in R.pseudoacacia increased significantly at 1000 and 2000 mg L-1treatments of Cd compared to the control but chlorophyll b was not affected by Cd(Fig.1a,b).Chlorophyll a and b contents did not vary in responses to Pb treatments.(Fig.1a,b).Chlorophyll a/b tended to increase with increasing Cd and Pb concentrations,and the difference was significant at 1000 and 2000 mg L-1Cd and Pb concentrations(Fig.1c).This indicated that the increase of chlorophyll a content was more than chlorophyll b.

Proline content

Proline content of R.pseudoacacia was similar at all concentrations of Cd and Pb(Fig.2).

Discussion

Fig.1 Chlorophyll a(a),chlorophyll b(b)and chlorophyll a/b(c)of R.pseudoacacia leaves exposed to different concentrations of Cd and Pb.Data are expressed as mean±SD(n=3).Different letters on bars show significance differences at 0.05 level of probability according to Duncan’s multiple comparison test between treatments of Pb and Cd

Fv/Fmvalues for control treatment and for 250,500,1000, and 2000 mg L-1treatments of Pb and Cd were slightly lower than the optimalvalue of 0.83 reported by Bjo¨rkman and Demmig(1987)and Jones et al.(1999)(Tables 1,2). Previous studies showed chlorophyll fluorescence was reduced by exposure to heavy metals.For example Baumann et al.(2009)reported that chlorophyll fluorescence was reduced significantly at 10μmol mL-1of Cd added to algal cultures in Fucus vesiculosus,Cladophora rupestris, Palmaria palmata and Polysiphonia lanosa.Kopittke et al. (2010)reported toxic concentrations of Cd and Pb(0.3 and 5μM,respectively)that were lower than the concentrations of Cd and Pb applied in this research.In their study, the plants were grown in a dilute solution which mimicked the soil solution,whereas our study plants were exposed to saline solutions of Cd and Pb for a short-term period,and the solution was only sprayed on the leaves(similar to rainfall).In the present study,Fmand Fv/Fmdeclined and Foincreased at high concentrations of both pollutants, similar to findings of Ekmekci et al.(2008)who reported Fm,Fv/Fmdeclined and Foincreased athigh concentrations of Cd(0.6 and 0.9 mM,respectively).

Fig.2 Proline of R.pseudoacacia leaves exposed to different concentrations of Cd and Pb.Data are expressed as mean±SD (n=3).Different letters on bars show significance differences at 0.05 level of probability according to Duncan’s multiple comparison test between treatments of Pb and Cd

The increase in Fowith Pb and Cd treatments can be related to an impact on the PSII reaction center,or a reduction in the energy transfer from the chlorophyll a antennae attached with the PSII light-harvesting complex to the reaction center(Ralph and Burchett 1998).Therefore it is evident from our study that the efficiency of energy transfer from this complex to the PSII reaction center was affected by Pb and Cd stresses.

Fmas the maximal fluorescence yield was reduced by Cd and increased by Pb toxicity.In normal conditions,QAis kept oxidized by transferring electrons to NADP and finally to CO2via QB,the plastoquinone pool and PSI. Thus Fv/Fmremains fairly high.If reoxidation of QAis restricted by decreased or slight blockage of electron transport from PSII to PSI by any of the stress factors, Fv/Fmcan decline(Mallick and Mohn 2003).

Chlorophyll content was measured to evaluate whether R.pseudoacacia was affected by Cd and Pb treatments.In plants,the first visible symptom of Cd toxicity is the chlorosis of the leaves(Das etal.1997;Baryla etal.2001). Cd has been found to damage the structure of chloroplasts (Rascio et al.1993;Ouzounidou et al.1997).High Cd concentrations in leaf tissues have been suggested as indirectinfluences on the chlorophyllcontentvia metabolic disruption and premature senescence(Vassilev etal.1997). However,it has been reported in tolerant plants that chlorophyll content increases or does not change significantly in responses to metals treatments(Burzyn′ski and Buczek 1994;Stiborova et al.1986).When we measuredchlorophyll content after 10 days exposure to Cd and Pb concentrations,we found significant increases in chlorophyll a at 1000 and 2000 mg L-1of Cd and an increase in chlorophyll a/b at 1000 and 2000 mg L-1Cd and Pb treatments.Tripathi and Gautam(2007)reported an increase(12.8%)in the chlorophyll content of Mangifera indica leaves subjected to air pollution.Seyyednejad et al. (2009)reported increases in chlorophyll a,b,and total chlorophyll in Albizia lebbeck and Callistemon citrinus, under air pollution.In the present study,R.pseudoacacia apparently tolerated stresses from Cd and Pb by increasing the content of chlorophyll.

Increase of free proline content in response to various environmentalstresses in plants has been reported by many scientists(Levitt 1980).It has been reported that proline is one of the most universal poly-functional substances to protect plants under various stresses(Ashraf and Foolad 2007).El-Khatib and El-Swaf(2001)reported the accumulation of proline in urban trees subjected to suspended particulate pollution.In the present study,proline content of R.pseudoacacia leaves did not change at any concentration of Cd or Pb(Fig.2).Dinakar et al.(2008)reported that with increasing Cd concentrations,proline levels increased in Arachis hypogaea seedling tissues(leaves and roots)in response to 25 days of exposure.Schat et al. (1997)suggested that proline accumulation in plant tissues under Cd stress is due to the decrease in the plant water potential,and therefore,this accumulation could be related to the water equilibrium.

The resistance of trees to environmental stresses should be considered in afforestation programs.The weak physiological responses of R.pseudoacacia to Pb and Cd contamination reflect the tolerance of this species to Pb and Cd.Other physiological characteristics should be investigated in future studies and used in the selection of tree species for afforestation projects in urban polluted areas.

Conclusion

Pb and Cd contaminations weakly affected physiological characteristics of R.pseudoacacia.Responses of chlorophyll fluorescence of R.pseudoacacia to Pb and Cd were similar and Fv/Fmdeclined slightly in response to exposure to the higher concentrations of Cd and Pb(1000, 2000 mg L-1).The effects of different functions of heavy metals on plants were found to be attributed to the differences between concentrations of Pb and Cd.Detection of weak physiological responses of R.pseudoacacia to Cd and Pb confirmed that this species is tolerant to both pollutants.Therefore,high concentrations of Cd and Pb in dust and rainfallwould notbe expected to damage physiological functions of leaves such as PSII function and chlorophyll structure.Other physiological characteristics,however, should be considered in future studies and in the selection of tree species for afforestation in urban and industrial polluted areas.

AcknowledgementsWe thank Iran Research Institute of Forests and Rangelands as wellas the Laboratory of Horticultural Sciences of the University of Tehran,for their financial and technical supports. Suggestions offered by anonymous referees are highly appreciated.

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18 October 2012/Accepted:28 April 2014/Published online:25 April 2015

?Northeast Forestry University and Springer-Verlag Berlin Heidelberg 2015

Project funding:This paper was supported by Iran Research Institute of Forests and Rangelands and the Laboratory of Horticultural Sciences of the University of Tehran.

The online version is available at http://www.springerlink.com

Corresponding editor:Zhu Hong

?P.Attarod attarod@ut.ac.ir

1Department of Forestry and Forest Economics,Faculty of Natural Resources,University of Tehran,Karaj,Iran

2Departmentof Horticultural Sciences,Faculty of Agricultural Sciences and Engineering,University of Tehran,Karaj,Iran

3Research Institute of Forests and Rangelands,Tehran,Iran