Mercury uptake, distribution and DNA affinity in durum wheat (Triticum durum Desf.) plants

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The Science of the Total Environment 243/244 (1999) 119-127

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Mercury uptake, distribution and DNA affinity in durum wheat ( Triticum durum Desf.) plants Andrea Cavallini"?",Lucia Natali", Mauro Durante", Biancaelena Masertib aDipartimento di Biologia delle Piante Agrarie della Universit;, Sezione di Genetica, Ir,a Matteotti 1 / B , 1.56124 Pira, Italy bC.N.R.,Istituto di Biojisica, Ir,a S. Lorenzo 26, 1-56100 Pisa, Italy Received 21 July 1999; accepted 7 September 1999

Abstract

Mercury uptake, distribution and translocation mechanisms in plants of Tn'ticurn dururn were studied after treatments with 203Hg(N03),(mercuric nitrate). Results show that the highest mercury levels are observed in the roots or in the leaves depending on the way by which mercury was subministrated to the plants (i.e. by water or air, respectively). Autoradiographic experiments on histological sections show that mercury is preferentially bound to the cell walls of the outer layers of the root cortical cylinder; moreover, labeling is also observed in the outer layer of the central cylinder and in the parenchyma cell nuclei. In the leaf, mercury is incorporated on epidermal and stomata1 cell walls and on parenchyma cell nuclei. DNA extracted from '03Hg-treated leaves was analyzed in Cs,SO, gradients and it was observed that mercury is preferentially linked to certain DNA families. 0 1999 Elsevier Science B.V. All rights reserved. Keywords: Mercury; Wheat; Uptake; DNA

1. Introduction

The effects of certain heavy metals on cellular systems has received a great deal of attention in

* Corresponding author. Tel.: + 39-50-571567; fax: + 39050-576750. E-mail address: [email protected] (A. Cavallini)

recent decades due to the increasing exposure of living organisms to these metals in the environment. Human-related activities, such as smelting and combustion of fossil fuels, have increased the release of metals, while processes like precipitation, gravitational Settling and adsorption are responsible for the transport of such metals to great distances from the site of introduction to aquatic

0048-9697/99/$ - see front matter 0 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 ( 9 9 ) 0 0 3 6 7 - 8

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and terrestrial environments (Oftedal and Brogger, 1986; Sharma and Talukder, 1989). Metal actions may affect cell and/or mitochondrial membrane permeability, lysosome membrane stability, protein denaturation and/or precipitation, enzyme inhibition, irreversible conformational changes and mutations in nucleic acids (Sharma and Talukder, 1989). In plants, mercury ions may substitute metal ions in photosynthetic pigments causing a decrease in photosynthesis rates (Xylander et al., 1996; Kupper et al., 1998). In terms of genotoxicity, mercuric ions tend to form covalent bonds, because of their easily deformable outer electron shells. A number of potentially reactive sites for mercury bonding are present in DNA, depending on external conditions such as ionic strength, presence of different competing ions, and base composition (Sharma and Talukder, 1989). It has been observed that Hg2+ may induce sister chromatid exchanges in plant nuclei (Panda et al., 1996). The interaction between mercury and plant systems is of particular importance because this metal has largely been employed in seed disinfectants, in fertilizers and in herbicides (Rao et al., 1966; McLaughlin et al., 1996). Ross and Stewart (1960, 1962) have shown that some mercury compounds used on tree foliage as fungicides can be translocated and redistributed in plants. The ability of plants to absorb mercury vapor from the atmosphere is well established (Hitchcock and Zimmermann, 1957; Waldron and Terry, 1975; Goren and Siegel, 19761, though, to our knowledge, studies on histology and DNA biochemistry of mercury-treated plants do not exist. In our previous field experiments, we had assessed mercury concentration values in the abiotic and biotic compartments of some of the cinnabar mineralized areas of the Mediterranean region. In the Monte Amiata (Italy) area (Ferrara et al., 1991) we had measured very high metal concentration in the wild Poa spp., sampled very close to an abandoned mine, both in the leaves (86 pg/g dry wt.) and even in the roots (13.7 pg/g dry wt.) The mercury levels in the soil had been ranging 350-2000 pg/g dry wt. and the air at the ground level was approximately 300-500

ng/m3. The capability of this gramineous plant to uptake mercury has been confirmed by the mercury levels found in the mineralized area of Alm a d h (Spain) (Maserti et al., 1996). We observed high concentrations of mercury, in the leaves (2.6 pg/g dry wt.) and in the roots (4.5 pg/g dry wt.), when metal concentrations of the soil ranged from 15 to 200 pg/g dry wt. and the mercury levels in the layer of the air close to the ground were also very high (300 ng/m3). The high mercury concentrations values generally measured in the atmosphere up to the ground are probably due to soil degassing mechanisms (Ferrara et al., 1992a, 1997). Elevated mercury levels both in the soil and air did not permit an identification of the contribution of each of these sources to the mercury concentrations found in plants of these areas. The aim of this paper was to evaluate how plants may face the presence of mercury in the environment. In our experiments we studied the uptake and distribution of mercury added as radioactive mercuric nitrate in roots of durum wheat (Triticum durum Desf. cv. Creso) and evaluated histologically the presence of a translocation mechanism from the root to the leaf and vice versa. During these experiments, mercury concentrations in the air of culture room were measured. Higher mercury levels than those observed as background values in the same culture room were found very close to glass trays where the plants had been growing. This indicated that a volatile mercury species, collected by a gold absorber, was released by the radioactive mercuric nitrate solution. We then used the same radioactive mercuric nitrate solution as a volatile mercury generator to study the mercury uptake by the aerial portion of the plant and performed experiments in a climate room keeping mercury uptake from the air separated from mercury uptake from the soil. Moreover, since mercury labeling was observed on nuclei, biochemical analyses of genomic DNA were performed. Considering the large concern on mercury as a global pollutant and the role played by higher plant in the food chain, the laboratory experimental data reported in this paper may be useful in the compre-

A. Cavallini et al. /The Science of the Total Environment 243/244 (1999)119-127

hension of the mechanisms of mercury uptake and tolerance in higher plants growing on naturally and/or anthropogenic mercury-enriched soil. 2. Materials and methods

2.1. Plant material Triticum durum cv. Creso seeds were germinated in Petri dishes on wet 'perlite' at 20°C under continuous illumination (35 pE/m/s) in a culture room. Three- to four-day-old-seedlings were used for two different experiments. 2.1.1. Mercury uptake by the roots To evaluate the uptake of mercury from the roots, 100 seedlings were cultured in a water solution to which 203Hg(N03),was added at a concentration of 50 pg Hg2+/1 (5 mCi/mg Hg). This concentration had been chosen after considering that, in our field experiments, carried out in cinnabar rich areas such as Monte Amiata (Italy) (Ferrara et al., 19911, and Almadh (Spain) (Maserti et al., 19961, we found healthy plants of diffcrcnt spccics, growing on soils with mcrcury concentrations ranging from 15 to 300 pg/g dry wt. The mercury concentration used in our experiment might be rather high for an aqueous medium, where the rate of mercury uptake is higher than that in the soil, but it is worth noting that in our laboratory experiment, the exposure time is much shorter than that in the natural environment. Plantlets were placed in glass trays covered with a perforated parafilm lid such that the shoots were above the parafilm and the roots below. In this experimental condition the atmospheric mercury concentration was monitored every day and was approximately 250 ng/m3 around the leaves and 40 ng/m3 in the culture room. Five samples of 10 plantlets each were collected after 12 h and 7 days of incubation and then stored at -20°C. 2.1.2. Mercury uptake by the leaves To evaluate the uptake of mercury from the leaves, 150 seedlings were cultured in tap water (mercury concentration 10 ng/l) in glass trays

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under a plastic plant chamber (10 1). The air inside the chamber was enriched with mercury released from 10 pg/ml of 203Hg(N03),mercuric nitrate solution contained in a vessel. The internal mercury concentration in the chamber was monitored every day and was approximately 5000 ng/m3. The glass trays were covered with a perforated parafilm lid, as described above, to reduce mercury deposition from the air into the growing medium (at the end of the experiment, mercury concentration in the medium was 45 f 5 ng/l). Five samples of 10 plantlets each were collected after 12 h and 7 days of incubation and then stored at -20°C. Fifty plants were left in the same culture conditions for 15 days and then analyzed to evaluate mercury uptake.

2.1.3. Control plants Fifty seedlings were cultured in tap water in glass trays, collected after 12 h and 7 days in the culture room and then stored at -20°C. 2.2. Analytical procedures Mercury concentration was determined as follows.

2.2.1. Mercury in tap water A 400-ml tap water sample was acidified with 400 p l of a solution of potassium dichromate (0.4% K,Cr,O, in 9 M H,SO,) and then photooxidized for 15 min using a UV immersion lamp (90 W). The Hg2+ was reduced with 10 ml of tin chloride solution (10% SnCl, in 4 M H,SO,) as described elsewhere (Seritti et al., 1980). Mercury was measured by cold vapor Atomic Absorption Spectroscopy (AAS) after preconcentration on a gold absorber (Seritti et al., 1980). 2.2.2. Mercury in spiked water Mercury concentration was determined by adding 10 ml of tin chloride solution to 50 ml of the spiked water and measuring by AAS. 2.2.3. Mercury in culture room The background concentration in the culture

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room was monitored every day by sucking the air on a gold trap by means of a membrane pump (flow rate 1 l/min) for 10 min. The electrothermally desorbed mercury was determined by a modified AAS as described elsewhere (Ferrara et al., 1992b). 2.2.4. Mercury in plant chamber To determine mercury levels in the air in the plant chamber, 100 ml of air was sampled by a syringe and injected onto a gold trap. Mercury content was determined as described previously. 2.2.5. Mercury in plantlets For each experiment, 50 wheat plantlets were divided into five samples of 10 leaves or roots and then dried at 35°C for 60 h. A 300-mg dried sample was mineralized with 3 ml of HNO, (Merck Selectipur) in a Teflon bomb at 160°C for 1 h. The mercury content of each sample was determined independently using AAS and was the mean of three replicates; every mercury concentration value reported is the mean of mercury concentration in each group of five samples of 10 leaves or roots. The typical contribution of the blank was approximately 1-2 ng of mercury. The results of the analyses on the reference standard materiel (BCR 62, Olea europea) issued by the Commission of the European Community was 0.28 f 0.02 pg/g of mercury with respect to the certified value of 0.28 f 0.03 pg/g of the metal. 2.3. Histological and autoradiographic analyses

Root and vegetative apices and leaf fragments from '03Hg-treated plantlets were fixed in 1.6% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.41, for 15 min at 4°C. The tissue fragments were washed overnight in the same buffer and then embedded in Technovit 7100 (Kulzer) plastic historesin. Semi-thin sections (3 pm) were obtained by a rotatory microtome and placed on gelatin-coated slides. For autoradiography, Ilford L4 emulsion was applied on the slides. After different exposure times (25-50 days), slides were developed in D19 (Kodak) developer, washed in tap water and

stained in 2% Giemsa solution in Sorensen buffer. 2.4. DNA extraction and purification

DNA extraction and purification were performed according to standard protocols for plant DNA studies: these methods may determine loss of unbound metals, while the DNA-bound metals should be maintained (Durante et al., 1986). DNA extractions were carried out according to Doyle and Doyle (1990). DNA was purified in a solution of 0.8 g/ml CsCl plus 300 pg/ml ethidium bromide, centrifuged at 44000 rev./min in a Beckman rotor 60 Ti for 18 h at 20°C; the DNA band was visualized under long-wave UV illumination and collected. After removal of ethidium bromide and CsCl, DNA was ethanol-precipitated and solubilized in water. Preparative density gradient ultracentrifugations of the DNAs were performed in Na,SO, (0.1 M) containing Cs,SO, at a final refractive index of 1.3745. Centrifugations were carried out at 35000 rev./min in the same rotor for 67 h at 20°C. The gradient was fractionated (200 p l per fraction) and absorbance was continuously recorded at 257 nm by an LKB Uvicord 11. Radioactivity was counted in a Packard Tricarb scintillation spectrophotometer after adding 10 ml of Insta-gel (Packard) to 50 p l of each fraction. Net disintegrations per minute (d.p.m.) were calculated by subtracting the mean background levels. Each experiment was repeated twice. 3. Results

Mercuric nitrate is not known to be a volatile compound. However, preliminary experiments made in the absence of plants demonstrated that mercury was somehow released from a mercuric nitrate solution. This finding was confirmed in the first experiments when a radioactive mercuric nitrate solution was used to study mercury uptake from roots. It may be hypothesized that mercuric ions are naturally reduced to elemental mercury in the tap water by bacterial activity, as found for Elbe river water (Ebinghaus and Wilken, 1993) or

A. Cavallini et al. /The Science of the Total Environment 243/244 (1999)119-127

by photochemical mechanisms at the water surface (Xiao et al., 1991). Another possible explanation is that the mercuric nitrate solutions (both radioactive or not) contained and/or released mercury species that can be collected by a gold absorber. For this reason, the radioactive mercuric nitrate solution was used to evaluate mercury uptake by either roots and leaves, monitoring the mercury concentrations in the water and in the air. 3.1. Uptake of mercury by roots In Table 1, the uptake of mercury in roots of plantlets cultured in '03Hg-enriched water is reported. Very high mercury concentrations (25 pg/g dry wt.) were observed in the root after only 12 h of treatment; mercury concentration in roots did not increase with the incubation time. Lower mercury levels (0.14 pg/g dry wt.) were measured in the leaf after 12 h, with increasing values during the experiment (Table 1). At the beginning of the experiment, mercury background values were 0.02 pg/g dry wt. for the leaves and 0.04 pg/g dry wt. for the roots (Table 1). In Table 2, the results of analyses after 58 days of autoradiographic exposure are summarized. These results display very good correspondence to the mercury uptake pattern (Table 1) both in roots and leaves; heavy labeling is already found in the root after 12 h of culture, while leaves are labeled only after 7 days. In the root, most labeling (observed after 25 days of exposition) is found on cell walls in the outer layers of the cortical cylinder (Fig. la) but silver grains are also deposited on the outer layer

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Table 1 Mercury uptake (pg/g dry wt.) in root and shoots of plantlets of Tiiticum duium cultured in the presence of '03Hg in the water and in air, respectivelya Air

Treatment duration

Water Root

Leaf

Root

Leaf

Oh 12 h 7 days

0.04 (a) 25.00 (d) 24.15 (d)

0.02 (a) 0.14 (a) 0.50 (b)

0.04 (a) 0.07 (a) 0.48 (b)

0.02 (a) 0.39 (b) 1.90 (c)

"Values followed by the same letter are not significantly 0.01 according to Tukey's ranking test. different for P I

of the central cylinder, as observed after 58 days of exposure (Fig. lb). Besides cell walls, labeling is evidenced on some cell nuclei (Fig. lc), in different root tissues. No difference in labeling intensity and localization is observed between 12 h and 7 days of culture. After a 12-h treatment, light labeling is observed in the shoot meristem, sometimes on nuclei. In the leaves, no labeling is observed after 12 h of treatment; slight labeling, without specific localization, is shown after 7 days of treatment. 3.2. Uptake of mercury by the leaves When plantlets were exposed to high levels of mercury in the air (5000 ng/m3) higher mercury concentrations were observed in the leaves than in the roots (Table 1). These concentrations in roots and in leaves increased during the experiment. After 7 days of treatment, mercury levels in the leaves (1.90 pg/g dry wt.) were approximately 100 times higher than at the beginning of the

Table 2 Labeling intensity (after 58 days of exposure) on histological sections of different organs from plantlets of Tiiticumduium cultured in the presence of '03Hg in the water and in air, respectivelya Treatment duration

Water Root

Shoot meristem

Leaf

Air Root

Shoot meristem

Leaf

12h 7 days

65.9 f 4.2 71.1 f 5.0

29.1 f 1.6 30.0 f 1.5

0.9 f 0.3 15.7 f 0.7

0.5 f 0.3 12.4 f 0.6

1.1f 0.4 32.1 f 2.0

13.8 f 1.0 49.3 f 2.2

"Values are reported as number of silver grains in an area of 40 pm'. Twenty areas per five different plants per treatment were scored (mean f S.E.).

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Fig. 3. Cs,SO, density gradient of DNA extracted from leaves of wheat treated with 203Hg(N03), (degassed in the air) for 7 days. The main peak of wheat DNA is indicated (density 1.423 g/ml). Letters indicate the labeled peaks; their densities are reported in Table 3.

4. Discussion

The ability of plants to absorb mercury is well established (Hitchcock and Zimmermann, 1957). Previous studies indicated that, in pea plants, when mercury is taken up by roots, it remains mostly confined to roots (Beauford et al., 1977). Other studies, performed on wheat plants, showed that mercury vapors determine accumulation of mercury especially into the leaves (Browne and Fang, 1978). In our experiments, when mercury is added to the water, a fast uptake of the metal by the roots is evident even after 12 h of treatment. The fast binding of mercury to the root could be due to an initial passive mechanism of adsorption to the cell walls and a diffusion through intercellular spaces, followed by an active mechanism of absorption in the pericycle, as demonstrated by the presence of labeling in the outer layer of the central cylinder (Fig. lb). Our results confirm those reported by other authors (Beauford et al., 1977) that the root is an important absorption site for mercury and creates

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a barrier to further transport of this element to the leaves. Increasing mercury concentration was observed also in the leaves of the plants that had their roots exposed to mercury. This might be related to a mercury translocation from the root to the leaf as indicated by labeling on autoradiographed sections of the shoot meristem and of the leaf. When plantlets grew in the presence of high levels of mercury in the air (5000 ng/m3), uptake by the leaves is observed. The increasing rate of the concentration values during the experiment indicates that the uptake mechanism by the leaves is slow (Table 1). Mercury concentration values in plants grown in 5000 ng/m3 of mercury for 15 days increased to 4.95 pg/g (dry wt.) confirming the above mentioned hypothesis. It is to be noted that both the increasing value of the ratio of mercury in the root/mercury in the leaf during the experiment (see Table 1) and the presence of a slight labeling on the root after 7 days (Table 2) indicate a possible translocation of the metal from the leaf to the root, though a passage of mercury from the air in the chamber to the culture medium may not be excluded. Autoradiographic experiments, after 7 days of mercury treatment, also showed a significant incorporation on some nuclei (Table 2, Fig. lc). Because of the high energy of radioactivity, Ag precipitation in the emulsion does not permit us to determine whether or not precise nuclear portions are preferentially labelled. Nuclear incorporation of mercury is probably occurring in particular DNA families. Biochemical analyses (Figs. 2 and 3) indicate that some DNA sequences show a particular affinity to mercury: the fractionation of genomic DNA in Cs,SO, gradients allow the separation of DNA fractions depending on their density and hence on their base content. When heavy metals are added to DNA, they may increase DNA density (Durante et al., 1986) and hence change its position in the gradient. The presence of one discrete peak of radioactivity in the DNA extracted after 12 h of treatment indicates that one ‘family’ of DNA sequences is probably linked more specifically to mercury than others (Fig. 2). Since it is

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A. Cavallini et al. / T h e Science of the Total Environment 243/244 (1999)119-127

known that mercury preferentially binds in vitro to A-T-rich DNA (Sissoeff et al., 1976; Sletten and Nerdal, 1997) it is conceivable that, in our materials, the radioactivity peak is due to mercury bonds to A-T-rich regions within G-C-rich DNA families, and/or to the nuclear localization of the DNA sequences (i.e. in the nuclear periphery) so that they are the first to bind the metal. After 7 days of treatment, three peaks of radioactivity are observed (Fig. 31, which should correspond to three DNA families bound to mercury; it is conceivable that peaks C and D represent two DNA families different from that observed after 12 h of treatment. On the contrary, peak B might correspond to the DNA family already bound to mercury after 12 h of treatment (peak A in Fig. 2): more mercury would be linked to this DNA family, thus determining a further increase of its density. To test these hypotheses, we are cloning the DNA families with the aim of determining their DNA base composition and base sequence. Concerning the nature and function of mercury-linked DNA sequences, it may be hypothesized that they belong to the heterochromatin that is localized in the periphery of the nucleus. This heterochromatin should have a protective role from action of mutagens for the internal nuclear portions that should contain structural genes (Hsu, 1975; Durante et al., 1989). In situ hybridization experiments using mercury bonded DNA families as probes will allow us to clarify this point. In conclusion, our data indicate that the root cell wall plays a role in capturing mercury and sequestering away elements such that they do not enter other plant organs. This should be a key element in the tolerance to this metal, as observed for mercury (Coquery and Welbourn, 1994) and for other trace elements such as zinc, lead and copper in other grass plants (Reilly and Reilly, 1973). However, experiments (Table 1) show that, at high mercury concentrations, translocation of mercury from root to leaves and vice versa do occur. Autoradiographic analyses confirm the presence of mercury in every plant structure, including cell nuclei. Labeling on nuclei, probably related to particular genome fractions (Figs. 2

and 31, might indicate that nuclear capturing of mercury may be a further mechanism to increase, even at high mercury concentrations, the tolerance of the plant to this metal. Obviously, to demonstrate this hypothesis, it will be necessary to isolate and sequence these DNA families to establish their nature and to localize them in the nucleus. Acknowledgements

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