Physiological and Ultrastructural Effects of Cadmium on Wheat(Triticum aestivumL.) Leaves

G. Ouzounidou, M. Moustakas, E. P. Eleftheriou

Department of Botany, Aristotle University of Thessaloniki, P.O. Box 109, Thessaloniki 540 06, Greece

Received: 15 March 1996/Revised: 4 June 1996

Abstract. The effects of a 7-day exposure of 3-day-old wheatplants to increasing Cd concentrations are described, withspecial attention being given to chloroplast ultrastructuralchanges, chlorophyll fluorescence responses, chlorophyll andnutrient concentration changes as well as growth changes of thewhole plant. The plants treated with 1 mM Cd showedsymptoms of heavy metal toxicity. The root, shoot-leaf lengthand the root, shoot-leaf biomass progressively decreased withincreasing Cd in nutrient solution and in 1 mM of Cd an almostcomplete inhibition of growth was found. Shoot-leaf Cdaccumulation increased under Cd-treatments, while a Fe, Mg,Ca, and K decline in the above ground parts was observed. Thegrowth reduction and the inhibition of chlorophyll content andphotosynthesis observed in the upper plant parts seemedprincipally due to indirect Cd effects on the content of essentialnutrients. Cadmium treatment was shown to damage thestructure of chloroplasts, as manifested by the disturbed shapeand the dilation of the thylakoid membranes. These ultrastruc-tural changes suggest that Cd probably induced prematuresenescence.

Among the pollutants of the environment, cadmium (Cd) hasbecome increasingly hazardous and its toxicity to men andanimals is well documented (Adriano 1986; Wagner 1993).Kidney and liver represent the principal target organs for Cdaccumulation in mammals, while a wide range of pathologicaleffects on fish and other aquatic organisms have been reported(Igeret al.1994). Cadmium has received considerable attentionover the past years as a result of increased environmentalburdens from industrial, agricultural, energy and municipalsources (Adriano 1986). A general increase in the levels ofcadmium threatens the health of terrestrial and aquatic organ-isms and therefore has become a major topic of toxicologicalresearch. At least 70% of the Cd intake by humans originatesfrom plant foods (Wagner 1993). Thus, plant tissues may serveas indicators of environmental concentrations of contaminants.

Although not essential for plant growth, cadmium ions arereadily taken up by roots and translocated into the leaves inmany plant species (Marshner 1983). Foliar absorption anddirect stem uptake also represent potentials modes of entry(Haghiri 1973; Gregeret al.1993). While Cd toxicity for plantshas been proven to be a major environmental problem, themechanism of its action has not been fully investigated.Cadmium generally inhibits germination of seeds (Rascioet

al. 1993), plant growth (Stiborovaet al. 1987; Gregeret al.1991), nutrient distribution (Moralet al. 1994) photosynthesis(Baszynskiet al.1980; Clijsters and VanAssche 1985; Krupaetal. 1993), increases several enzymes activity,e.g.,glucose-6-phosphate-dehydrogenase (Van Asscheet al. 1988) whereasother enzyme activities are influenced differently (Karataglisetal. 1991). Since Cd21 ions accumulate at higher levels in leavesthan in other parts of plants (Marschner 1983) most researchinto the phytotoxic effects of Cd has been focused on theinhibition of photosynthesis. Experiments have shown effectsof Cd on stomatal function (Barceloet al. 1988; Costa andMorel 1994), on electron transport (Baszynskiet al. 1980;Siedlecka and Baszynski 1993) and on Calvin cycle (Weigel1985; Sheoranet al. 1990). Little information is available onthe effects of Cd on chloroplast organization which is animportant factor in understanding the physiological alterationsinduced by the metal, because of the relationship betweenstructure and function of the thylakoid system (Baszynskiet al.1980; Krupaet al. 1987; Barceloet al. 1988; Ghoshroy andNadakovukaren 1990; Rascioet al.1993). Long-term exposureof whole plants to Cd, may affect chlorophyll synthesis and thushave an important role in both the chloroplast development inyoung leaves and the inhibition of photosynthesis (Stobartet al.1985; Barceloet al. 1988; Padmajaet al. 1990; Boddiet al.1995). Ultrastructural studies report disorganization of granaand increased number and size of plastoglobuli in chloroplastsas well as increased cell and vacuole size and inducedvesiculation in the cytoplasm (Baszynskiet al.1980; Reeseetal. 1986; Barceloet al.1988; Rascioet al.1993).In the present work we focused on the effects of Cd on

growth, essential mineral nutrients and chlorophyll content,photosynthesis and chloroplast structure of wheat leaves, inorder to establish an overall picture of the Cd toxicity syndromeat the structural and functional level.Correspondence to:M. Moustakas

Arch. Environ. Contam. Toxicol. 32, 154–160 (1997) A R C H I V E S O F

EnvironmentalContaminationa n d Toxicologyr 1997 Springer-Verlag New York Inc.

Materials and Methods

Plant Culture

Seeds of wheat (Triticum aestivumL., cv Dio) provided by the CerealInstitute of Thessaloniki, Greece were germinated in a growth chamberon moist filter paper in petri dishes for 3 days. The seedlings wererandomly placed in polyethylene pots and filled with a modifiedHoagland nutrient solution containing: KNO3 (0.6 mM), Ca(NO3)2 (0.4mM), NH4H2PO4 (0.2 mM), MgSO4 (0.1 mM), KCl (50 µM), H3BO4

(25 µM) FeNaEDTA (20 µM) MnSO4 (2 µM), ZnSO4 (2 µM), CuSO4(0.5 µM) and (NH4)6Mo7O24 (0.5 µM).A randomized block, factorial design with four Cd treatments (0,

265, 530 µM, and 1 mM Cd) and three replicates was used. Cadmiumwas supplied as Cd(NO3)2. The control solution and cadmium treat-ments were replaced twice a week. The plants were grown for 7 days ina growth chamber (16 h light/dark) under light intensity of 15 W m22

(produced by neon lamps, Philips TL 40W/55) at plant level with aday/night temperature regime of 226 1/186 1°C and relative humidity of65 6 2/756 2%. After 7 days of cadmium treatments, root, shoot-leaflength and root, shoot-leaf biomass (fresh weight) were measured.

Metal Content

Seven days after exposure to Cd, shoot-leaves were washed in distilledwater and dried for 24 h at 80°C. Dry plant material was wet digested incylinders filled with HNO3-HClO4 (4:1) at 120–130°C for 5 h. Aftercooling, Cd, Ca, Fe, Mg, and K were determined by a Perkin Elmer2380 atomic absorption spectrophotometer (Ouzounidouet al.1992).

Chlorophyll (a1 b) Determination

Chlorophyll (a1 b) of the second wheat leaf was extracted in 80%acetone. Absorbance was measured at 663 and 645 nm, using an LKBUltraspec II spectrophotometer (Ouzounidouet al.1993).

Chlorophyll Fluorescence

In vivo chlorophyll fluorescence was measured after 7 days ofCd-treatment on the upper surface of the second leaf with a commer-cially available fluorometer Plant Stress Meter (BioMonitor SCI AB,Umea, Sweden). Prior to measurement of fluorescence, plants were leftfor at least 30 min to dark adaptation at room temperature (Moustakaset al. 1994). Chlorophyll was excited for 10s by actinic light with aphoton flux density of 400 µmol m22s21.

Electron Microscopy

Leaves samples of the control and of the 1mMCd-treated plants (3 mmlength) were fixed immediately in a 3% glutaraldehyde solutionbuffered in 0.05 M sodium cacodylate, pH 7.2, for 3 h at roomtemperature (about 25°C). The samples were further post-fixed for 3 hin 1% OsO4 similarly buffered, dehydrated in graded ethanol, andembedded in Spurr’s low-viscosity epoxy resin. Ultrathin sections(60–90 nm), were taken at 1.5 mm behind the root tip, stained withuranyl acetate and lead citrate and finally examined and photographedwith a Zeiss 9 S-2 Electron microscope (Ouzounidouet al.1992).

Statistics

The experiment was set up in a completely randomized design with 3replications. The significant difference between the treated and thecontrol samples was analysed by Student’s t-test. The values of root,shoot-leaf length, and biomass are means of 30 measurements, whileshoot-leaf chlorophyll and mineral content values are means pooledover three measurements. Chlorophyll fluorescence parameters aremeans of five independent measurements.

Results

Plant Growth

The response of root, shoot-leaf length and root, shoot-leafbiomass to different concentrations of Cd is given in Table 1.Root and shoot-leaf length growth revealed a drastic reductioneven from the lower Cd-treatment by 53 and 40%, respectively(P, 0.05). Plants grown on the nutrient solution containing 1mM Cd showed more evident effect of toxicity, since an almostcomplete inhibition of root and above ground part elongation by72 and 60%, respectively, was observed (P, 0.05, Table 1).The EC50, representing the concentration in which Cd inhibitsgrowth by 50%, of Cd for root and shoot-leaf length growth wasfound around 265 µM and 530 µM, respectively.Root and shoot-leaf biomass, expressed as mg FW per plant,

progressively decreased with increasing Cd in nutrient solution(Table 1). Exposure to 1 mM Cd resulted in a significant loss ofplant biomass by 71 and 45% for root and, shoot-leaf biomass,respectively (P, 0.05, Table 1). The EC50of Cd for root and aboveground part biomass was found at 530 µM and 1 mM, respectively.

Leaf Mineral Content

The concentrations of Cd, Fe, Mg, Ca, and K in shoot-leaves(on a dry weight basis) after 7 days Cd-treatments are shown inTable 2.Cadmium accumulation showed a sharp increase in 265 µM

Cd treatment by about 18 times of the Cd concentration of thecontrol (Table 2). In the higher Cd-treatment (1 mM) Cdcontent in shoot-leaves was by 40-times higher than that of thecontrol (P, 0.05). Cadmium concentrations resulted in animportant decline in nutrients in plant tissues. Iron, Mg, Ca, andK accumulation in shoot-leaf displayed a gradual decrease withincreasing Cd in plant and nutrient solution (Table 2). The mostvisible negative correlation of metals to Cd toxicity wasobserved at 1 mMCd.An almost complete Fe and Ca loss by 85and 78%, respectively (P, 0.05) and a significant decline ofMg and K by 33 and 53% (P, 0.05), respectively, was foundunder 1 mM Cd treatment (Table 2).

Chlorophyll Content—Chlorophyll FluorescenceParameters

Data of chlorophyll (a1 b) content from 7-day Cd-treatedwheat plants showed that on a fresh weight basis total chloro-phyll decreased with increasing Cd in the medium (Table 3).Under 265 µM of Cd a reduction of 28% in leaf chlorophyll

Effects of Cadmium on Wheat 155

content compared with the control was observed, that at 1 mMof Cd was more severe (75%) (P, 0.05, Table 3). In Table 3,the effect of Cd stress on some fluorescence parameters is alsoshown.A slight reduction of Fv/Fm values under the various Cdconcentrations was observed that in the higher concentration (1mM) was equal to 7% of the control. With increase inconcentration of Cd applied to intact plants, the initial fluores-cence (Fo) decreased, with a total decrease of 35% in the case oftreatment with 1 mM. The half time rise (t1/2) tends to increaseby 35% of control at 265 µM of Cd (P, 0.05), while in 1 mMCd-treatment t1/2 values was three times greater than that of thecontrol (Table 3). Fm and Fv parameters depressed slightly (16and 19%, respectively) under 265 µM of Cd, but decreaseddramatically by 50 and 53%, respectively (P, 0.05) under 1mM of Cd (Table 3).

Chloroplast Ultrastructure

The chloroplasts from untreated plants were typical mesophyllchloroplasts (Figure 1); they show a well-organized internalmembrane structure with normally developed grana and stromathylakoids (Figure 2). Cadmium treated plastids show a dis-turbed shape (Figure 3), with a wavy appearance of the granaand stroma thylakoids and the intrathylakoidal space swollenbut the envelope intact (Figure 4). Dilation of the thylakoidmembranes was apparent in all chloroplasts.In the ultrastructure of the chloroplasts in both untreated and

Cd-treated plants, aggregations of microtubule-like structureswere observed (Figures 2, 4). Bundles of microtubule-likestructures have been observed on electron micrographs ofdeveloping and transforming plastids in algae and higher. Theirchemical composition and function are not known, but probablyare not composed of tubulin (Artuset al.1990).

Discussion

Parameters, such as biomass and shoot-leaf, root length havebeen used as indicators of metal toxicity in plants (Baker andWalker 1989). The inhibition of growth produced by Cd-treatments mainly affected the root than the shoot-leaf. Rootand shoot-leaf fresh weight inhibited less than their length,while no third leaf under 1 mM of Cd in wheat plants wasobserved; the second one was fully expanded at all Cdconcentrations. Alia and Saradhi (1991) and Moyaet al.(1993)also found a reduction of both shoot and root length and anincrease of the DW/FW ratio that was mainly due to a decreaseof FW in rice and wheat plants treated with Cd.

The reduction in growth could be a consequence of Cdinterference with a number of metabolic processes associatedwith normal development, especially: (1) synthesis of proteins(Stiborova et al. 1987); (2) activities of some importantenzymes by binding to free amino, carboxylate or side groups,and/or replace some important metal ions associated with suchgroups (VanAssche and Clijsters 1990; Alia and Saradhi 1991);and (3) various photosynthetic processes such as chlorophyllbiosynthesis (Stobartet al. 1985), photosystems activity, orelectron transport (Murthyet al.1990).According to our results and those of Greger and Lindberg

(1987), excess of Cd in nutrient solution can cause ionsdeficiency with visible symptoms and be deleterious to theplants. However, according toAlloway (1990), it is not possibleto rely on the onset of visible symptoms of Cd toxicity to act asa warning when food crops have accumulated excessiveamounts of metals, such as Cd, which could be hazardous tohealth. Relatively, large concentrations of Cd can accumulate inedible portions without the plant showing symptoms of stress,although the Plastochron Index, which is a numerical index ofthe developmental age of plants, may indicate a reduced rate ofdevelopment (Alloway 1990). Visible signs of Cd toxicityinclude brown margin to leaves, chlorosis, curled leaves, brownstunted roots, redish veins and petioles and general reduction ingrowth.The concentration of Cd in exposed shoot-leaves increased

with increasing external Cd concentration. Cadmium alsocaused severe decrease in Ca concentration of the leaves, andsince Ca is necessary for the development of the cell wall andthe maintenance of membrane structure, Cd may also indirectlyaffect plant growth (Greger and Lindberg 1987; Greger andBertell 1992). Trivedi and Erdei (1992) reported also thedecreased Ca21 in wheat shoots during Cd treatment, whileVerbost et al. (1988) suggested that Cd21-ions displacedCa21-ions from the ligands in membrane preparation of animalorigin. Excess of the metal in plant tissues resulted in adepressed K, Fe and Mg accumulation.In plants, Fe21 and Mg21 are necessary for the proper

functioning of the light-induced electron transport chain and forCO2 assimilation and chlorophyll formation (Bazzaz and Go-vindjee 1974; Greger and Lindberg 1987). In Fe-deficientplants, destruction of the lamellar system of thylakoids andchanges in lipid and protein composition of these membranesare observed (Abadiaet al.1989). These phenomena are alwaysaccompanied by diminished photochemical activities of thephotosynthetic apparatus. Similar symptoms are also found inchloroplasts of Cd-treated plants (Baszynski 1986; Siedleckaand Baszynski 1993). The strong inhibition of chlorophyllcontent in wheat leaves under Cd stress was in accordance withthe finding of Sheoranet al. (1990). In barley and maizeseedlings, Cd has been shown to inhibit the chlorophyllsynthesis by affecting the activity of photochlorophyllidereductase (Stobartet al. 1985; Rascioet al. 1993). Thereduction in photosynthesis found in wheat leaves, therefore,could be explained also on the basis of the chlorophyllreduction. Therefore, it is postulated that the inhibition ofchlorophyll content and photosynthesis observed in Cd-stressedwheat plants resulted from an indirect effect of Cd. This effectcan be related to Fe and Mg decreased concentrations in leavesdue to excess of Cd in nutrient medium (Ouzounidou 1994).According to Stobartet al.(1985) and Sheoranet al.(1990), the

Table 1. Effect of Cd stress on root and shoot-leaf growth ofTriticumaestivumcv Dio plants. Data are means of 15 measurements (6se)

CdTreatments

RootLength(cm)

Shoot-leafLength(cm)

RootBiomass F.W.Plant21 (mg)

Shoot-leafBiomass F.W.Plant21 (mg)

0 µM 15.66 1.1 24.86 2.8 776 16 2256 16265 µM 7.36 0.7 15.06 1.0 456 9 1866 20530 µM 6.06 0.7 13.56 0.5 356 3 1306 111 mM 4.36 0.3 10.06 0.6 226 1.9 1236 5

G. Ouzounidouet al.156

inhibition of photosynthesis seems to occur indirectly eitherthrough the decreased chlorophyll content or as a result ofdecreased stomatal conductance. Yet, it is likely that Cd21 ionsinhibit PS II by displacing the Ca21 ions that are also requiredfor electron flow. However Krupaet al. (1987) have proposedthat an alteration in the lipid environment around PS II (i.e.,achange in the lipid composition of the thylakoids) may be themain cause of the loss of PS II activity that follows treatmentwith Cd21 ions.The growth reduction and chlorosis observed in the upper

plant parts clearly have to be considered as consequences of thetoxic Cd effects in roots. These indirect effects are principallycaused by alteration in the content of essential mineral nutri-ents, decrease photosynthesis as a consequence of declinedchlorophyll content and stomatal closure. Nutrient deficienciesand heavy metal toxicities are known to produce starchaccumulation within leaves (Vaszquezet al.1987). This may bedue to both a decrease of the sink force, because of reduced rootgrowth, and an inhibitory effect on vein loading (Rauser andSamarakoon 1980). In our experiment no significant increase ofstarch accumulation in leaves of Cd-treated plants occured.Primary events in electron transport appeared not to be

affected as judged from an analysis of the early phases offluorescence kinetics; the ratio of Fv/Fm remained almostconstant. Similar results have been found by Greger and Ogren(1991) in sugar beet and by Krupaet al. (1993) in Cd-stressedbeans. The ratio Fv/Fm represents the maximum efficiency ofthe primary photochemistry of PSII, thus, there was a slightdecline in efficient use of the radiant energy captured by thealready lost chlorophylls during Cd-stress. Moreover, thehalf-rise time (t1/2) of the fluorescence induction was increasedin Cd-treated plants. This result suggests that the amount ofactive pigments associated with the photochemical apparatusdecreased under Cd-stress (Greger and Ogren 1991). Comparedwith the controls, the functional chlorophyll antennae size ofthe photosynthetic apparatus was smaller in Cd-treated wheatplants, as indicated by the increased value of t1/2 in thefluorescence induction curve. In addition, variable fluorescence

(Fv5 Fm2 Fo) severely inhibited by Cd mainly because ofthe strong decline of Fm rather than the decline of Fo. Thedecreased Fv suggests inhibition on the photooxidizing side ofPSII as well as important alteration to thylakoid structureaffecting electron transfer via PSII (Ouzounidou 1993). Analteration of the PSII reaction centre upon Cd treatment mayalso be involved (Li andMiles 1975). The drop in Fo, suggests areduction in the fluorescence that originates from the antennachlorophyll of PS II.Simola (1977) did not observe any great changes in the fine

structure under the effect of Cd on moss (Spagnummemoreum).In tomato, however, Cd caused disorganization of grana andincreased the number and size of plastoglobuli (Baszynskiet al.1980). The thylakoid membrane system of chloroplasts inCd-treated plants resembles that described by Heet al. (1994)for ultraviolet-B exposure and by Hurkman (1979) for chloro-plast senescence. The ultrastructural changes observed inchloroplasts of Cd-treated plants suggests that Cd probablyinduced premature senescence.The effect of Cd on chloroplasts differ from those induced by

other heavy metals. Nickel, cobalt, and copper induce starch,probably by inhibition of vein loading (Rauser and Samarakoon1980). In Cd-treated plants no starch accumulation was ob-served in the chloroplasts either in our experiment or by others(Barceloet al. 1988), which indicates that Cd either had noeffect on vein loading or, more probably, Cd inhibited photosyn-thesis more intensively than the translocation of photoassimi-lates (Barceloet al.1988).Cadmium as opposed to other heavy metals like Al (Elefthe-

riou et al.1993; Moustakaset al.1996) or Cr (Vaszquezet al.1987) appears to cause more marked ultrastructural changes inthe aerial parts of plants than in roots (Barceloet al.1988). Thismay be explained by both the capacity of roots to store Cd in amore inactive form, and the relatively high mobility of Cdwithin plants (Barceloet al.1988).Long-term exposure to Cd resulted in growth retardation,

decreased mineral concentrations, ultrastructural alterations ofchloroplasts and lowering of photosynthetic activity of wheat

Table 2. Metal concentrations inTriticum aestivumcv Dio shoot-leaf after 7 days exposure to various Cd treatments. Each value is the mean6 sebased on three replicates of three independent series

Cdtreatments

Metal concentrations in wheat shoot-leaf (µg g21 D. W)

Cd Fe Mg Ca K

0 µM 2.76 0.8 2506 38 1496 13 4506 17 58606 210265 µM 48 6 9 646 10 1216 9 4266 8 55706 90530 µM 73 6 12 556 5 1206 5 2406 20 51036 651 mM 107 6 15 376 4 1006 2 976 9 27606 30

Table 3. Effect of Cd treatments on chlorophyll content and some chlorophyll fluorescence parameters of intact second leaves ofTriticum aestivumcv Dio. Chlorophyll content values are means6 se of three measurements, while chlorophyll fluorescence parameters are means6 se of fivemeasurements

Cdtreatments

Chlorophyll content(mg g21F. W.) Fv/Fm t1/2 Fo Fm Fv

0 µM 1.526 0.15 0.813 40 0.406 0.04 2.136 0.07 1.736 0.05265 µM 1.106 0.09 0.781 54 0.396 0.02 1.786 0.08 1.406 0.06530 µM 0.756 0.10 0.777 61 0.386 0.03 1.736 0.03 1.346 0.021 mM 0.386 0.03 0.757 124 0.266 0.02 1.076 0.05 0.816 0.03

Effects of Cadmium on Wheat 157

leaves. The phytotoxic effect of Cd could probably be aconsequence of its interference with a number of metabolic pro-cesses associated with normal development. It must be pointed outthat another noticed action of Cd consists of stimulating thebiosynthesis of ethylene (Rascioet al.1993), a phytohormonewith a well-known role in plant senescence induction.In conclusion,Triticum aestivumchloroplasts undergo both

structural and functional changes when plants exposed to Cd.

Acknowledgments.This work was supported by a grant from theGreek General Secretariat for Research and Technology.

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