LOCALÎZATÎON OF LEAD ACCUMULATED BY BARLEY (Hordeum distichon L.) ROOT TIPS AND ITS EFFECTS

Barley roots were traeted for 1-24 hours vith 0.1-10 mMol /1 lead uitrate Solutions and examined by transmission eîectron ınicroscope. Lead deposits were observed in the middle lamella, celi walls and in the pinocytctically formed vesicle at the firs^ and third hours, at low conccntrations. The plasmalertıma acts as a barrier to the influx of lead intc the protoplasm. But this was not considered as a complete protection mechanism. With iucreasing concentra-tîons and time, lead has migrated to the cytopîasm, and even to the nucleus and the nucleolus. The epidermal and the cortical cells were highly susceptible to the lead. They were sericusiy injured exhibiting plasmolysis and disorganized celi structure. The endodermis has protected the Central cyîinder for a short time. The eîectron micropgraphs made after 6 and 24 hours of lead treatments in the ali experimental combinations have shown dense lead deposits in ali tissues. Lead has been taken up and has accumulated by barley rocts and caused structural damages in root cells.


INTRODUCTION
Hundreds of thousands of tons of lead are discharged annually into the earth's soil, atmosphere and water by various sources (NRIAG-U, 1979).From the atmosphere, this metal, being largely oxides and salts, is washed down by rain to the surface of the earth (SCHULZ-BALDES and LEWIN, 1976).The increasing problem of environmental poUution by heavy metals necessitates the study of toxicity of heavy metals in plants as well as in animals.The toxioity of heavy metals in crop plants varies from metal to metal and from crop to crop (HARA and SONODA, 1979).Lead and cadmium are not so much interesting because of the phytotoxicity, but rather because of its uptake and transportation into the food chain (FOY et al., 1978).
The analysis of pollutants in organisms has been shown to be a suitable method for the pollution monitoring.Plants, mosses and lichens clearly reflect deposition levels owing to their high accumulating rates of a number of poilutants (LAGERWERF, 1971; VAN STEV-ENICK et al., 1976;THOMAS et al., 1984;RODERER, 1984;VAS-QUEZ et al., 1987).The intake of lead is accumulated chiefly in the roots, occuring to a lesser degree in the upper parts of the plants (WONG et al., 1984).Plants absorb considerable quantity of lead through the roots, from where it is being transported to the parts above the ground (stem and leaves) with limited quantities (KLOKE and RIEBARTSCH, 1964;MARTEN and HAMMOND, 1966).
The researchs on lead uptake by plants done so far have generally been carried out on a practical agronomic hasis and have focused on the physiological character of this uptake (BAUMHARDT and WELCII, 1972;PETTERSSON, 1976;WONG et al., 1986;RAPPAPORT et al.. 1987).But any fraction or extraction procedure used will introduce the possibility of measuring only tbe existance of lead.Lead is an electron dense metal and it can be easily detected in the cells with electron microscope (MALONE et al., 1974;SHARPE and DENNY, 1976;WIERZBICKA, 1987;STOCKING and ONGUN, 1962).
The aim of present study is to determine the uptake and the physical localization of lead in barley root tip sections.

MATERIAL and METHODS
Barley (Hordeum distichon L. var cultuvar union) has been grown by hydroponics procedure in a clima chamber (TOKER, 1988).Oneweek-old seedlings have been used for the experiments and the electtron microscobic studies have been carried out in the root tip tissues.
Lead ions have been supplied ■with Pb(NO3)2 which is the highly water soluble compound of lead and the pH has been adjusted to 6.6 with 0.01 N tris buffer.The lead concentration have rauged from 0.1, 0.2, 1,5 to 10 mMol/1.The seedlings have been immersed in one üter of the lead Solutions for each concentration for the periods of 1-3-6 and 24 hours.The roots for the control have been cut out from the seedlings just before the lead treatment and have been fixed.
After the periods mentioned above, the roots have been rinsed in distilled water for 1 minute, then have been cut 2-3 mm in length from the root tips.They have been fixed in 2.5 % glutaraldehyde in 0.01 N NaOH buffer at pH 6.6 for 24 hours.The root tips have been rinsed witlı bidistilled water 2-3 times for one minute, then post-fixed with 1 % OSO4 (i % OSO4-1 % KMnO^-m / 200 CaCls) for 24 îıours.After ihe po8t-fixation, the root tips have been rinsed with bidistilled water 2-3 times for one minute and weıe stained with 2 •aranylacetate 0/ for 24 hours.After dehydratation in a graded series of ethylalcohol and propylene oxide, the root tips have been embedded in a 1 / 1 mixture of epon-spurr.
The blocks have been trimmed with the Reichert TM 60 specimen trimmer and the ultrathin cross sections of the root tips have been prepared with the Reichert OMU 3 ultramicrotome.The sections tvhich were not stained with lead have been examined in a Philips EM 301 electron microscope.

RESULTS
After 1 hour of incubation in 0.1 mMol Pb / 1 solution (iow concentration) lead was present in ali the root tip tissues examined.Numerous lead crystals were visible in the pinocytotic vacoules of the outher side of the epidermal celi (Fig. 1).There were many crystals seen in the inner vacuoles.The granular lead deposits were observed in the middle la-meUa of the celi walls.Large deposits were also occured on the plasmodesmatas.Though the sections were not stained with lead to obtain contrast, as it is done for regular preparations for electron microscopy, the epidermal cells showed strong contrast and the nucleolus and the chromatin structure in particular were very darkIy stained.These results showed that lead was accumulated in huge amounts by epidermal cells.The cortical cells showed the same appearance (Fig. 2).There were profuse small granular deposits in the celi wall.The same structures were observed in the protoplasm but they were denser than that of the plasmalemma.The mitochondria also exhibited the same de posits.
After 6 hours of incubation in 0.1 mMol Pb / 1 solution, the amount of lead in the root cells was high.A huge amount of lead crystals were observed in the celi ■wall and the intercellular spaces of the root's cörtical cells (Fig. 3).The accumulation of lead crystals was so abundant at the celi wall that the width of wall \vas enlarged.
After 24 hours incubation in 0.1 mMol Pb / 1 solution, there was a massive lead precipitation in the Central cylinder cells (Fig. 4).The organelles, nucleolus and protoplasm of these cells were stained dark ■ and the protoplasm in particular appeared sandy in aspect.The black accumulations accured between the celi walls and the protoplasm were heavy lead deposits.The cells developed pinocytotic vacuoles for the protection, but it was not successful.When the concentration was increased (1 mMol Pb / 1), a great amount of deposition was observed at the sections prepared after one hour (Fig. 5).Lead deposits were seen in intercellular spaces.The middle î 1 lamella, the celi organelles, the nucleus, the nucleolus and the proto plasm showed dense granular structure.The results of this concentration after 24 hours (1 mMol Pb / 1) showed that the epidermal cells and ali the cortical cells were completely damaged (Fig. 6-7).Behind the endodermis, the centıal cylinder cells by comparison with cortical cells look rather heallıty.But their dark coloring proves that lead has Within one lıour period of extreme concentration (10 mMol Pb / 1), lead deposits were seen in tbe intercellular spaees, the celi walls and especially along with plasmalemma (Fig. 8).After 24 hours of this ■r z w X' '^..  >'-e-Xf ■ concentration, lead deposits spoiled ali the celi structures and there was no sign of living organelles (Fig. 9).Ali the roots grown in lead uitrale Solutions showed that lead was taken up and then deposited at every kind of root cells, causing the celi deformation.
The control cells contained no electron-dense deposits (Fig. 10).

DISCUSSION
Although there have been many studies on the physiological path-•vvay of heavy metal and lead uptake by plants and animals (CANNON and BOWLES, 1962;BAUMHARDT and WELCH, 1972;JONES et al., 1973a;JONES et al., 1973b;SCHULZE and BRAND, 1978;CHAHAL et al., 1979;GODBOLD and HUTTERMANN, 1986) the cytological and the histological researches are insufficient.The localization of heavy metals in tissues and cells is easily made by using electron microscobic and autoradiographic technigues (WAISEL et al., 1970;MALONE et al., 1974;SHARPE and DENNA', 1976; VAN STEVENINCK et al., 1976;WIERZBICKA, 1987).
There are a few early Communications done on the subject.HA-VESY (1923) has suggested that laed was not translocated appreciably from the roots to the shoots even it was used as toxic guantities.HAMMETT (1928a) has reported that lead was absorbed from the so lution of Pb (NO3)2 PbCİ2, and Pb (C2H3O2)2 through the roots.Lead was accumulated largely at the areas showing maximum mitotic activity in roots, just behind the root cap of onions, beans and corns.He has also noted that the areas of the largest lead accumulation were blackened, but the black deposits were soluble in very dilute nitric acid.In addition to these results the root elongation was inhibited when the concentration had been increased (HAMMETT, 1928b).MILES and PARKER (1979a, b) have worked on the uptake and the effects of lead Solutions on plants.These researchers have reported that heavy metals are largely accumulated in the roots, while in the upper parts of the plants the concentration of the heavy metals was much lower (MARTEN and HAMMOND, 1966;JONES et al., 1973a).This shows that the transport of heavy metals from the roots to the shoots is limited.
Cadmium, a well-known environmental pollutant, is hound to the plant celi wall with a nonmetabolitic bond at first and then is transported to the protoplasm through the diffusion (CUTLER and RAINS, 1974).Lead is bound to the celi wall, especially to the middle lamella at the beginning of the treatment (MALONE et al., 1974;SHARPE and DENNY, 1976;FOY et al., 1978;WIERZBICKA, 1987).In the ceU wall there are many sites for lead binding.The middle lameUa incorporates many Chemical substances (i.e.pectates) for lead binding, and lead is readily bonded to the anionic parts of polyuronic acid in the celi wall (SHARPE and DENNY, 1976).In the present study, the lead treatment made at even lower concentrations and for a short periods of time has showed lead deposits in the ceU w ali in ali eîectron micrographs (Fig. 1-9).Lead was accumulated in the middle lamella and it was easily visible there due to its dark color.Also, smaU granular lead deposits were observed in the celi v/all of the epidermal cells (Fig. 1).
Lead precipitation in protoplasm first appeared between the plasmalemma and the celi wall since plasmalemma acts as a selective organelle in the celi (NISHIZAWA and MORI, 1977;VvIERZBICKA, 1987).In the leaf cells of Potamegoton ssp., the electrochemical potential gradients between celi vacuoles and the bathing solution range from -150 to -240 mV (SHARPE and DENNY, 1976) and this wouid favor a passive influx of lead into vacuoles in course of treatment.In the eîectron micrographs made by SHARPE and DENNY (1976) this influx was not observed.The reason for this was that the plasma lemma acted as a barrier to simple passive diffusion.This was said to be clearly the exclusion of lead.HIATT and LEGGET (1974) have argued that plasmalemma is a membrane which restricts the ion influx to the protoplasm.This effect has resulted from the celi wall precursors and PO4 anions.The dictyosomes closer to the celi wall, would have high phosphate content since they contain acidphosphatase.PO4, that forms insoluble precipitates with lead, could act as a sink for lead.At the outset of the treatment in the present study, the presence of lead deposits was shown in the celi wall and plasmalemma (Fig. 2).It was finely grained in the celi walls but the deposition was more more dense near the plasmalemma.After high concentrations and long periods of time much denser accumulation of lead were observed in the plasmalemma and between the plasmalemma and celi wall (Fig. 3).
Univalent and bivalent cations have induced pinocytosis (WHE-ELER and HANCKEY, 1971).SUTCLIFE (1962) has suggested that the pinocytosis ■was a mechanism of ion absorption by plant ceUs.But it has been shown that pinocytotic vacuoles inhibited the metal ions and saved the cells against their toxic effects ( WHEELER and HANCKEY, 1971;SHARPE and DENNY, 1976;NASSERY and JONES, 1976;NISHIZAWA and MORI, 1977).These researchers have said that this protection by inhibition was limited, since the increase at concentration and time could have caused deposition of lead in the protoplasm, even in the vacuoles.Furthermore, lead had penetrated into the chloroplast membrane and had been deposited along its grana (SHARPE and DENNY, 1976).Lead, like other toxic metals, caused membrane system deformation in the celi (VAZQUEZ, 1987).The ion fluxes through the plasmalemma in root cells must have been studied in short term (max.3-4 hrs.) experiments (PATTER-SON, 1976).Since pinocytosis was in action only for a short period and at low concentration and it was not regarded as a protection mechanism.The data obtained in the present study showed that lead ac cumulation has occured at first in the celi wall and then caused pino cytotic vacuoles in the plasmalemma in the periods of 1-3 hours (Fig. 3-4).After high doses of lead for long durations was applied the large and the uniform lead accumulation were observed in the inner structure of the cells.Toxic uranyl salts (0.1-1 mMol / 1) were absorbed by oat roots rapidly and were crystalized in the celi walls and in the interceîlular spaces.That, after 4-6 hours uranyl crystals had migrated pinocytotically into protoplast had been stated by WHEELER and HAN-CKEY (1971).Metal ions penetrating the protoplast have been concentrated inside it, due to the fact that they had found many binding sites there (cysteinyl, hystidyl side chain of proteins, purines, pteridines and porphyrins) (MATHYS, 1975).Our electron micrograplıs (Fig. 1-9) had a deep contrast that has come from lead which was absorbed by the protoplasm.
Protoplasm exhibits a granular structure because of lead.Dense lead accumulation has occured in the nuclear membrane and even in nucleolus (Fig. 9).OPHUS and GULVAG (1974), in their studies on lead uptake by moss Rhytidiadelphus sguarrosus using electron microscopy and X-ray microanalysis, have demostrated the presence of lead ■within the nuclear and chloroplast inciusions.According to SHARPE and DENNY (1976), after the long term treatment of Potamegoton pectinatus with lead, electron micrographs clearly have shown the accumulation of lead, mainly in the celi walls and also symptoms of senecens.observed.Biochemical studies autolysis and plasmolysis have been on the similariy treated tissue have showed increases in nucleic acid breakdown products and the free amino acids indicating that the nuclei had been effected by lead.Both the nucleus and the nucleolus have had metals and inos ( VAN STEVENINCK, 1976).a strong affinity for heavy MALONE et al. (1974), who have conducted a similar research on roots of Zea mays, have reported that lead accumulation occur first at the celi wall and then at the plasmalemma.These deposits were granular at the outset but later turned to large crystals.They had observed the same pinocytotic vacuoles as those seen in the barley roots in the present study.They had indicated that lead was taken up by the root system of corn, transported and precipitated throughout the plant.WIERZBICKA (1987) have investigated lead accumulation and translocation in AUium cepa roots using electron microscope and autoradiographic methods.Lead transport in the roots was possible.either through.the apoplast or symplast.He has suggested by cyto-Chemical estimations that lead probably migrates through the plasmalemma of the protoderm cells and was then symplasticaîly transpor- ted by the endoplasmic reticulum.After one hour of incubation lead had appeared in ali root tissues except for the Central cylinder.He has pointed out that lead was deposited in the celi wall, especially in the middle lamella, but endodermis cells had acted as a barrier against the lead.In the present study, after 24 hours incubation, the highest concentration of lead has been observed in ali root tissues.Also SUC-HODOLLER (1976) reported that the cortical cells were more sensitive than the Central cylinder.In our experimeııts of barley root tissues also have the same appearance with respect to the deposition of lead in these cells.The epidermis and the cortical cells of the root were seriously injured exhibiting plasmolysis and disorganized celi structure, with the exception of the celi of Central cylinder (Fig. 6).In electron micrographs made after 6 and 24 hours of lead treatment, however, ali root cells inciuding the Central cylinder ones showed dense lead depo sits for ali experimental combinations.
This study has shown that lead is taken through the roots of barley and precipitated throughout its cells.The results suggest that lead is available to plants under natural condition and that it would seriously affect organisms further down to the food chain.

Acknowledgment
ABBREVATIONS: cc el cr cw