Metal accumulation and damage in rice (cv. Vialone nano) seedlings exposed to cadmium

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Environmental and Experimental Botany 62 (2008) 267–278

Metal accumulation and damage in rice (cv. Vialone nano) seedlings exposed to cadmium Nicoletta Rascio a,∗ , Francesca Dalla Vecchia a , Nicoletta La Rocca a , Roberto Barbato b , Cristina Pagliano b , Marco Raviolo b , Cristina Gonnelli c , Roberto Gabbrielli c a

Dipartimento di Biologia, Universit`a di Padova, I-35131 Padova, Italy Dipartimento di Scienze Ambientali e della Vita, Universit`a del Piemonte Orientale ‘Amedeo Avogadro’, I-15100 Alessandria, Italy c Dipartimento di Biologia Vegetale, Universit` a di Firenze, I-50100 Firenze, Italy b

Received 24 November 2006; received in revised form 21 August 2007; accepted 30 September 2007

Abstract The effect of exposure to increasing cadmium concentrations was analyzed in rice seedlings (cv. Vialone nano). The highest Cd accumulation was found in roots, mostly in the apoplastic environment. Cd taken up in cells led to an increase in sulfhydryl groups, the appearance of phytochelatins, and formation of electron-dense vacuolar inclusions. The metal-exposure inhibited root growth and also interfered with correct root morphogenesis, causing disordered division and abnormal and forward enlargement of epidermal and cortical cell layers in the apical region. Cd accumulation in shoots was lower than in roots. In leaf cells, there was neither a substantial increase in sulfhydryl groups nor the appearance of phytochelatins. Shoot growth was reduced and, differently from in roots, leaf cell enlargement was inhibited. Chloroplasts had lowered contents of chlorophyll and a reduced number of thylakoids, but underwent structural alterations only at the highest Cd concentration tested (250 ␮M). Photosynthetic activity was limited due to the curtailment of CO2 availability caused by the greater resistance of Cd-exposed leaves. The damage suffered by seedlings worsened with the increase in Cd concentration, but was already evident at the lowest concentration examined (50 ␮M), showing that the cv. Vialone nano has a Cd-sensitivity higher than other rice cultivars. © 2007 Elsevier B.V. All rights reserved. Keywords: Oryza sativa L.; Cadmium pollution; Cd Accumulation; Seedling responses; Root damage; Shoot damage

1. Introduction Cadmium is one of the most toxic heavy metals and its environmental concentration is increasing due to industrial and agro-chemical usages and anthropogenic activities. This very dangerous pollutant exerts its toxic effects on both plants and animals, arriving through the food chain (Wagner, 1993). Although Cd is a non-essential element, it is readily absorbed by plant roots, probably in competition with other bivalent ions (Clemens, 2001), and is then transported from roots to aerial organs. The phytotoxicity of cadmium is well established, even though the mechanisms involved are still not completely

∗

Corresponding author. Tel.: +39 049 8276278; fax: +39 049 8276278. E-mail address: [email protected] (N. Rascio).

0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2007.09.002

understood. This is essentially due to the manifold and interlacing effects that this heavy metal can exert on basic events of plant growth, development and physiology and to the fact that the responses to Cd, as regards a given event, can be different, and sometimes opposite, according to the species. For instance, in leaves of Brassica napus Cd leads to a reduction of mesophyll cell size (Baryla et al., 2001), while leaf thickness and cell size increase in Pisum sativum exposed to the heavy metal (Sandalio et al., 2001). In this latter species Cd inhibits photosynthesis by strongly affecting the PSII activity (Chugh and Sawhney, 1999), whereas no direct Cd effects on the photosystem are found in either Brassica juncea (Haag-Kerwer et al., 1999) or Arabidopsis thaliana (Perfus-Barbeoch et al., 2002). Furthermore, it has been noticed that Cd uptake and distribution inside the plant, as well as degree of tolerance and physiological responses to this heavy metal, can vary in the same species even among cultivars and populations, depending

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on genotypic as well as ecotypic differences (Hart et al., 1998; Zhao et al., 2002; Arao et al., 2003; Lux et al., 2004). Two main research lines have been followed in studying plants exposed to Cd pollution, namely (i) identification and characterization of hyperaccumulating species that are potentially useful for phytoremediation (Escarr´e et al., 2000; Zhao et al., 2003; Klang-Westin and Eriksson, 2003; Cosio et al., 2004); and (ii) studies on Cd accumulation and effects in crops of significant economic and alimentary interest. Among the latter, rice is undoubtedly one of most important and widespread species, being the staple food crop for the majority of the world’s populations. Interestingly, in rice, as in other species, it has been found that Cd uptake, distribution and tolerance can differ according to the cultivar (Morishita et al., 1987). To date, most studies dealing with the effects of Cdcontamination on rice have been performed on oriental (indica and japonica) cultivars (Moya et al., 1993; Shah and Dubey, 1995; Shah et al., 2001; Kim et al., 2002; Hsu and Kao, 2003). Vialone nano is a rice cultivar of great economic interest in Western countries such as Italy. In a first recent work on this rice cultivar we noticed some effects of low Cd concentrations (7.5–75 ␮M) on the photosynthetic apparatus (Pagliano et al., 2006). Since Cd can affects several events of plant growth, metabolism and physiology, we deemed of interest to carry out a research on Vialone nano cv. using a wider range of Cd concentrations (50–250 ␮M) to define its degree of tolerance and to analyze the response to Cd through different developmental and functional parameters. Accordingly, we studied seed germination and plantlet growth, Cd accumulation and distribution in roots and shoots, and damage suffered by roots and leaves in cv. Vialone nano treated with different heavy metal concentrations. Morphological and ultrastructural observations were integrated with functional, biochemical and molecular analyses. As with the majority of previous studies on other rice cultivars, we used seedlings, because in the life cycle of rice plants the seedling stage is considered critical for damage due to environmental stresses (Shah and Dubey, 1995). 2. Materials and methods 2.1. Plant material Rice (Oryza sativa L., cv. Vialone nano) caryopses were surface sterilized with 1% sodium hypochlorite, rinsed and then imbibed overnight in water. Seeds were germinated and plantlets raised hydroponically in a series of pots (30 plantlets per pot) on a half-strength Johnson nutrient solution (Johnson et al., 1957) either devoid of cadmium (control) or supplemented with increasing concentrations (50, 100, 250 ␮M) of Cd(NO3 )2 . The solutions were renewed every 3 days. Plantlets were maintained in a growth chamber at 30 ◦ C/25 ◦ C day/night temperature and 80% relative humidity, with a photoperiod of 16h and an irradiance of 80 ␮mol photons m−2 s−1 PAR. All the analyses were carried out in four independent experiments run in triplicate on the shoots and roots of 10-day-old plantlets, except where specified using 20-day-old seedlings.

2.2. Seed germination analysis The percentage of germination was measured on sets of 100 seeds, maintained in the growth chamber at the above described environmental conditions and placed in Petri dishes on paper soaked with nutrient solution devoid of cadmium or supplemented with increasing concentrations of the heavy metal. The count of germinated seeds was carried out after 7 days from the beginning of treatment. 2.3. Measurement of plant growth Root and shoot length was measured on sets of 30 plantlets grown either on the control nutrient solution or on solutions supplied with different Cd concentrations. The measurements were made from the collette to the tip of the longest root and from the collette to the tip of the longest leaf. 2.4. Cd determination Roots and shoots from control and Cd-treated plantlets were carefully washed either with distilled water or with 1M HCl followed by 1M Na2 EDTA to remove the heavy metal bound to cell wall components (Shah and Dubey, 1998). Samples were then dried at 80 ◦ C until constant weight, ground to a fine powder and digested in concentrated HNO3 in a microwave oven (MDS2000, CEM, Buckingam,UK). Cd amount was determined using a flame atomic absorption spectrophotometer (Perkin-Elmer 4000, Norwalk, CT, USA) and expressed on the basis of dry weight. 2.5. Determination of A276 and total SH groups Samples cultivated and collected as described above were weighted, frozen in liquid nitrogen, homogenized with a mortar and pestle in a 1:2 ratio (w/v) in cold 20 mM Tris–HCl, 100 mM sodium ascorbate, pH 8, and filtered through Miracloth tissue. The homogenates were centrifuged at 10,000 × g for 25 min at 4 ◦ C and the resulting extracts were applied onto a 2.6 × 52 cm Sephadex G-50 Superfine column (Amersham Pharmacia GE Healthcare, Buckinghanshire, UK), equilibrated and eluted at 4 ◦ C under nitrogen with 10 mM Tris–HCl, 100 mM NaCl, pH 8 at a flow rate of 10 ml h−1 . Column was calibrated with known molecular weight standard proteins. Fractions of 5 ml were assayed for A276 , Cd and SH concentration. Total SH groups in extracts and fractions were assayed spectrophotometrically at 412 nm, using 4 mg ml−1 5-5 -dithiobis-2-nitrobenzoic acid in 0.1N sodium phosphate buffer, pH 8 (Ellman, 1959). Fresh solutions of cysteine were used for calibration. 2.6. Determination of dry weight/fresh weight ratio Ten shoots from control plants or plants treated with different Cd concentrations were weighed and then dried at 100 ◦ C until constant weight, which was recorded.

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Table 1 Cadmium supplied in growth medium and content of Cd, –SH groups and –SH/Cd ratio inside the cells of root tissues Cd supply (␮M)

[Cd] (nmol g−1 DW)

[SH] (nmol g−1 DW)

0 50 100 250

nd 1270.9 ± 154.2 a 2848.0 ± 182.1 b 4024.3 ± 102.3 c

307.5 1461.5 3846.2 6153.9

± ± ± ±

42.6 a 127.7 b 151.3 c 220.8 d

nmol SH/nmol Cd 1.15 1.35 1.53

Data are means ± standard deviations of four replicates. Values followed by the same letters were not significantly different (P < 0.05) as determined by DMRT.

Fig. 1. Root and shoot length of plants exposed to different Cd concentrations, as a percentage of control plants (0) (the length of control roots was 5.95 ± 0.68 cm; control shoots 17.2 ± 1.2 cm). Data are means ± standard deviations of four replicates. Values followed by the same letter on root and shoot histograms were not significantly different (P < 0.05) as determined by DMRT.

2.7. Chlorophyll analysis Chlorophylls from leaf samples were analyzed in a doublebeam spectrophotometer (GBC UV/VIS 918, GBC Scientific equipment Pty Ltd., Victoria, Australia) after extraction with N,N-dimethylformamide and pigment concentrations were calculated using the extinction coefficients proposed by Porra et al. (1989). 2.8. Light and electron microscopy Samples of the second leaf subapical region and the tip segment of roots from control and Cd-treated plants were fixed

Fig. 2. Cadmium content in roots of control plants (0) and plants treated with different Cd concentrations, after washing with H2 O or with EDTA (DW = dry weight). Data are means ± standard deviations of four replicates. Values followed by the same letter are not significantly different (P < 0.05) as determined by DMRT.

overnight at 4 ◦ C in 3% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 6.9), post-fixed for 2 h in 1% osmium tetroxide in the same buffer and then processed as described previously by Rascio et al. (1991). For light microscopy thin sections (1 ␮m) cut with an ultramicrotome (Ultracut, Reichert-Jung, Wien, Austria) were stained with equal volumes of 1% toluidine blue and 1% sodium tetraborate and examined (Ortholux, Leitz, Wetzlar, ˚ Germany). For electron microscopy ultrathin sections (600 A) were cut with the same ultramicrotome, stained with lead citrate and observed with a transmission electron microscope (TEM 300, Hitachi, Tokyo, Japan) operating at 75 kV. 2.9. Isolation of thylakoid membranes Thylakoid membranes were isolated from plastids of control leaves and leaves treated with different Cd concentrations by grinding the tissues in ice-cold buffer containing 100 mM Tricine–NaOH (pH 7.8), 5 mM MgCl2 , 15 mM NaCl and 330 mM sorbitol. After centrifugation at 10,000 g for 20 min, the pellet was resuspended in 50 mM HEPES–NaOH (pH 7.2), 5 mM MgCl2 and 15 mM NaCl and centrifuged as before. Thylakoids were then resuspended in the same buffer containing 100 mM sorbitol, recentrifuged at 10,000 × g for 10 min and finally resuspended in a solution containing 50 mM Na2 CO3 , 50 mM dithiothreitol and 20% sucrose. Thylakoid

Fig. 3. Relationship between gel filtration profiles, Cd and SH concentrations in root extract from plants treated with 250 ␮M Cd.

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anti-D1 (Barbato et al., 1991) was used. The colored band was observed upon addition of nitro blue tetrazolium and 4-chloro5-bromo-p-indolylphosphate. Each lane was loaded with 50 ␮g of protein. 2.11. Determination of transpiration and CO2 assimilation rates Both transpiration and CO2 assimilation rates were measured in the blades of second leaves of control plants and plants exposed to the different Cd concentrations by a differential CO2 /H2 O Infra-Red Gas Analyzer (CIRAS-1, PP Systems, Hitchin Herts, UK) The analyses were carried out at constant temperature (20 ◦ C) and relative humidity (100%) and at different photon flux density (from 0 to 1300 ␮mol photons m−2 s−1 PAR). 2.12. Assays of in vivo oxygen release Photosynthetic oxygen release was measured in vivo from leaf samples with an oxygen monitor (ISY Model 53; Yellow Spring Instrument Co., Ohio, USA) according to the method of Ishii et al. (1977). The suspension medium was 50 mM HEPES (pH 7.4) containing 0.5 mM CaSO4 , 20 mM KHCO3 as an inorganic carbon source and 35 ␮g/ml of carbonic anhydrase. The analyses were carried out under saturating light (1400 photons m−2 s−1 PAR, at the level of the closed test tube containing the sample) using a slide projector (Ikolux; Zeiss Ikon, Oberkochen, Germany) with a 24 V, 150 W lamp (Philips, Aachen, Germany) as the light source. During the analyses the suspension medium, isolated from the air, was stirred and kept at a constant temperature of 20 ◦ C. Fig. 4. Micrographs of thin longitudinal sections of root tips. (A) Root from a control plant with very regularly arranged cell rows. (B) Root from a plant treated with 50 ␮M Cd. Note: The less ordered cell rows and the misshapen and enlarged epidermal (arrow) and cortical (double arrow) cells. Some cells show uncorrected division plans (arrow head). Numerous dark inclusions can be distinguished in the cells. (C) Root from a plant treated with 250 ␮M Cd. Note: The disordered, voluminous and misshapen epidermal and cortical cells. Very numerous dark inclusions are recognizable in the cells (bar = 50 ␮m; longitudinal bars = 500 ␮m).

extracts were analyzed for protein content by the Bio-Rad Protein Assay method (Bio-Rad, Hercules, CA, USA) (Bradford, 1976).

2.13. Statistical analysis A one-way analysis of variance (ANOVA) was applied to the data. Statistical analyses were performed with SPSS 10.0 (Noruˇsis, 1993). All probabilities are two tailed. Data were checked for normality and homogeneity of variance (Levene test). Differences between means were evaluated for significance by using Duncan’s multiple range test (DMRT) (P < 0.05). 3. Results

2.10. SDS-PAGE and immunoblotting

3.1. Seed germination and seedling growth

Gel electrophoresis and immunoblotting of thylakoid extracts were carried out as described by Laemmli (1970) and Dunn (1986), respectively. Prior to immunodetection, proteins were visualized by staining the filter with 0.2% Ponceau S in 3% trichloracetic acid. Filters, blocked with 10% skimmed milk in Tris buffer saline (TBS), were incubated with appropriate rabbit primary antibody, followed by biotinylated goat-antirabbit IgG and then with conjugated streptavidin alkaline phosphatase. For detection of the D1 thylakoid protein, a polyclonal antibody

Cadmium-contamination did not affect germination, which at 7 days after seed moistening, reached values around 90% in control grains as well as in grains treated with the different concentrations of Cd (data not shown). In contrast, the heavy metal greatly interfered with seedling growth. Ten days after germination two leaves had been produced by both the control seedlings and those grown on the different Cd-concentrations. However, the cadmium-exposed plantlets showed a reduced length of roots and shoots (Fig. 1). The growth inhibition at 50 ␮M Cd was about

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Fig. 5. Micrographs of ultrathin sections of root cells. (A) Meristematic cells of a control root without dark inclusions in the small vacuoles. (B) Cortical cells of a control root with more developed vacuoles devoid of dark inclusions. (C) Meristematic cells of a 50 ␮M Cd-exposed root. Numerous dark inclusions (arrow) are present in the small vacuoles. (D) Cortical cells of a 50 ␮M Cd-exposed root, showing large dark inclusions (arrow) in the vacuoles. (E) Meristematic cells from a root treated with 250 ␮M Cd. Numerous large dark inclusions can be seen in the vacuoles (bar = 2 ␮m).

50% and 40% for root and shoot, respectively, with a further reduction in organ length at the higher Cd concentrations. 3.2. Roots 3.2.1. Cd concentration Quantitative analyses (Fig. 2) revealed that roots of Cdtreated plants accumulated a large amount of cadmium and that the metal concentration increased as Cd concentrations rose in the growth medium. However, after root washing with EDTA to remove the heavy metal bound in the apoplast to cell wall components, the Cd quantity underwent a sharp fall (Fig. 2). Additionally, the concentrations of Cd taken up in the root symplast gradually increased at the rise of Cd in the growth medium, although remaining under 20% of the total amount of heavy metal even in roots exposed to the highest Cd concentration.

3.2.2. Total –SH group amount and gel-filtration elution profiles Compared to the constitutive amount of –SH groups of control roots, the –SH group concentration sharply rose in Cdtreated roots, proportionally to the metal increase inside the tissues (Table 1) This led to nmol –SH/nmol Cd ratios from 1.15 to 1.53 in the range of Cd concentrations tested. The gel filtration elution profile of 250 ␮M Cd-treated root extracts (Fig. 3) showed that the presence of Cd in the culture medium induced the appearance of two peaks of Cd with apparent MWs around 13,500 Da and <1500 Da. The fractions containing the Cd peak with the higher apparent MW also showed significant amounts of total –SH groups. No Cd peaks were detectable in the gel filtration elution profile of control roots (data not shown).

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N. Rascio et al. / Environmental and Experimental Botany 62 (2008) 267–278 Table 2 Cadmium supplied in growth medium and content of Cd, –SH groups and –SH/Cd ratio inside the cells of shoot tissues Cd supply (␮M) 50 100 250

[Cd] (nmol g−1 DW)

[SH] (nmol g−1 DW)

nd 279.0 ± 33.8 a 618.2 ± 38.2 b 860.4 ± 29.8 c

1967.2 2032.8 2295.1 2535.8

± ± ± ±

36.8 a 55.4 a 40.3 b 70.2 c

nmol SH/nmol Cd 7.3 3.7 2.9

Data are means ± standard deviations of four replicates. Values followed by the same letters were not significantly different (P < 0.05) as determined by DMRT.

Fig. 6. Cadmium contents in shoots of control plants (0) and plants treated with different Cd concentrations, after washing with H2 O or with EDTA (DW = dry weight). Data are means ± standard deviations of four replicates. Values followed by the same letters are not significantly different (P < 0.05) as determined by DMRT.

3.2.3. Morphological and ultrastructural features of root tips The morphological analyses demonstrated that the Cdtreatment strongly affected the root differentiation pattern. Comparison using light microscopy of longitudinal tip sections from a control root (Fig. 4A) and a root treated with 50 ␮M Cd (Fig. 4B) clearly showed less ordered cell rows in those treated with Cd. Moreover, the epidermis and the outer cortical layers showed misshapen cells with a greatly anticipated and faster growth, so that, at the same distance from the apical meristem (500 ␮m), they appeared hugely enlarged compared to the corresponding control cells. Furthermore, the enlarged cells were not lengthened, but remained rather isodiametric with incorrect division plans. These root alterations were worsened in plants exposed to higher concentrations of Cd (Fig. 4C). In all Cd-treated roots numerous dark inclusions were distinguishable inside most cells (Fig. 4B and C). By electron microscopy these electron-dense bodies, never found in control roots (Figs. 4 and 5Figs. 4A and 5A, B), were already present in the small vacuoles of meristematic cells from 50 ␮M Cdtreated roots (Fig. 5C), with a frequency and size increasing towards more differentiated cortical cells (Fig. 5D). Moreover, these vacuolar inclusions became more abundant and voluminous in roots exposed to higher concentrations of the heavy metal (Fig. 5E).

in plants exposed to the highest Cd concentration (Fig. 6). Furthermore, a significant decrease in Cd concentrations was found in shoot tissues after washing with EDTA (Fig. 6). Comparison of Cd levels in roots and shoots deprived of the metal bound to apoplastic components showed that the Cd quantities taken up in shoot cells were more than four times lower than those found inside root cells. It could be calculated that only 3–4% of the total Cd accumulated in roots reached the cytoplasm of shoot cells. 3.3.2. Total –SH group amount and gel-filtration elution profiles Conversely to what observed in roots, rather modest increases in total –SH groups were induced in shoot tissues by exposure to increasing Cd concentrations (Table 2). Consequently, the nmol –SH/nmol Cd ratios were greatly lowered inside the cells, as Cd concentrations rose in simplasts. In shoots of plants treated with 250 ␮M Cd (Fig. 7), gel filtration chromatography showed a very low Cd concentration in fractions with an apparent MWs of 13,500 Da and above 50,000 Da. Compared to control plants (not shown), no significant increase in –SH concentration was detectable in the eluted fractions.

3.3. Shoots 3.3.1. Cd content Total Cd amounts measured in shoots of plants treated with the heavy metal were much lower than those in roots. Although the amounts increased with higher Cd concentrations in growth medium, they remained below 10% of that found in roots, even

Fig. 7. Relationship between gel filtration profiles, Cd and SH concentrations in shoot extract from plants treated with 250 ␮M Cd.

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Fig. 9. Transpiration rates at different photon flux densities in second leaves from 20-day-old control plants and plants exposed to different Cd concentrations. Data are means ± standard deviations of three replicates.

Fig. 8. Thin transversal sections of second leaf blades. (A) Leaf from a control plant. (B) Leaf from a plant treated with 250 ␮M Cd. Note: The reduced thickness and the smaller mesophyll cells. No dark vacuolar inclusions are visible (bars = 25 ␮m).

3.3.3. Morphological, ultrastructural and functional features of leaves Morphological observations using light microscopy of the second leaf revealed that the Cd-treatment, in addition to hampering elongation, led to a decrease in leaf blade thickness which, however, maintained the same number of cell layers as control leaves. This was particularly evident when comparing a leaf section from a control plant (Fig. 8A) to that treated with 250 ␮M Cd (Fig. 8B). No dark inclusions were found in the cell vacuoles of Cd-treated leaves. Leaf blade thinning, which transverse sections showed to be due to a reduced enlargement of mesophyll cells, could be coupled with a gradual rise of the tissue dry weight/fresh weight ratio. Referred to the value of control shoots (118.4 ± 7.5 mg dry weight/g fresh weight), this increased from 11% to 27% and 48%, respectively, in plants exposed to 50, 100, and 250 ␮M Cd. Another feature of Cd-treated plants emerged from assay of the leaf transpiration rate. Due to the slowed growth rate, analyses were carried out on 20-day-old plants whose leaves were large enough to permit the measurement. Compared to controls, leaves exposed to 50 and 250 ␮M Cd exhibited a very slow transpirational water loss that was insensitive to changes in light intensity (Fig. 9). In Cd-treated plants, leaf tissues were rather bleached, as confirmed by analyses of the amount of chlorophyll (Fig. 10), which demonstrated a gradual pigment decrease at higher metal concentrations. Electron microscopy revealed that in mesophyll cells of Cd-exposed leaves, chloroplasts (Fig. 11A) had a reduced thylakoid system compared to control plants. This feature, already visible in chloroplasts of 50 ␮M Cd-treated leaves (Fig. 11B), was confirmed in leaves treated with 100 ␮M Cd (data not shown), whereas in chloroplasts of leaves treated with 250 ␮M Cd, in addition to the membrane decrease, some alterations and thylakoid swelling could also be seen (Fig. 11C).

However, Western blot analysis carried out on the D1 protein (Fig. 12) showed that this component of the PSII reaction center did not undergo evident changes in chloroplasts from plants exposed to cadmium. Oxygen release, measured in vivo as a photosynthetic activity parameter, slowed in Cd-treated leaves. This decline (Fig. 13), which was very high when referred to dry weight, was substantial even if measured on the basis of the chlorophyll content of leaf tissues. Moreover the rate of net CO2 assimilation analyzed on leaves of 20-day-old plants, revealed a sharp decrease in leaves treated with 50 and 250 ␮M Cd over a range of photon flux densities from 100 to 1300 ␮mol m−2 s−1 (Fig. 14). 4. Discussion Inhibition of seed germination can be one of the manifold Cd effects on events of plant growth and differentiation (Iqbal et al., 1991). However, in our rice cultivar seed germination was not affected in the range of Cd concentrations tested, which brought about inhibition of subsequent

Fig. 10. Chlorophyll [Chl (a + b)] amounts in second leaves of control plants (0) and plants treated with different Cd concentrations. Data are means ± standard deviations of four replicates. Values followed by the same letters are not significantly different (P < 0.05) as determined by DMRT.

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Fig. 13. Photosynthetic oxygen emission from second leaves of plants treated with different Cd concentrations, referred both to dry weight and to chlorophyll content, as a percentage of control leaves (0) (the values of oxygen release from control leaves are 213.5 ± 15.7 ␮mol O2 h−1 g−1 DW and 18.3 ± 1.9 ␮mol O2 h−1 mg−1 Chl (a + b)). Data are means ± standard deviations of four replicates. Values followed by the same letters on histograms referred to DW or Chl (a + b), respectively, are not significantly different (P < 0.05) as determined by DMRT.

Fig. 11. Micrographs of chloroplasts from the sub-apical region of second leaves. (A) Chloroplast of a control leaf, exhibiting a well organized thylakoid system (gt = granal thylakoids; st = stromal thylakoids). (B) Leaf chloroplast of a plant treated with 50 ␮M Cd. Note the reduced thylakoid system. (C) Leaf chloroplast of a plant treated with 250 ␮M. Some thylakoids are altered and swollen (arrows) (bars = 1 ␮m).

Fig. 12. Immunoblot with anti-D1 of thylakoid membranes isolated from control leaves (lane 0) and leaves of plants exposed to 50 ␮M Cd (lane 50), 100 ␮M Cd (lane 100) and 250 ␮M Cd (lane 250). The analysis was repeated three times and showed the same result.

plantlet growth. This suggests that in rice, as reported in barley and wheat (Titov et al., 1996), the two events are differently responsive to Cd and that particularly high metal concentrations are required for inhibition of germination. The first phenotypic evidence of Cd-treatment was reduced elongation of roots and shoots, which was already apparent at the lowest Cd concentration. Inhibition of seedling growth is a common effect of many heavy metals and is used as a parameter for phytotoxicity (Ernst et al., 1992). The growth responses of Vialone nano to the lowest Cd concentrations show that this rice cultivar is more Cd-sensitive than cv. Bah´ıa (Moya et al., 1993), cvs.Ratna and Jaya (Shah et al., 2001) and cv. Tainung 67 (Hsu and Kao, 2003). In investigations carried out on these

Fig. 14. CO2 assimilation rates at different photon flux densities in the second leaves from 20-day-old control plants and plants exposed to different Cd concentrations. Data are means ± standard deviations of three replicates.

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oriental cultivars, a range of 100–500 ␮M Cd has been reported as moderately toxic. Our rice seedlings accumulated amounts of Cd that were higher as the Cd concentration increased in the growth medium. However, the metal distribution was different in roots and shoots. Roots accumulated the highest Cd quantities, retaining most of the heavy metal outside the cells in the apoplastic environment. This system of Cd exclusion from protoplasts, which relies on its binding to anionic groups of cell walls (Ernst et al., 1992), is part of the defense strategies of many plants against metal excess. However this is not a generalized system. In maize roots, for instance, Cd accumulates in both cell wall and soluble fraction of cells, while in pea roots it accumulates mainly in the soluble fraction (Lozano-Rodriguez et al., 1997). Moreover, only a negligible accumulation of Cd occurs in cell walls of Silene vulgaris roots (Verkleij et al., 1990). Cd sequestration outside protoplasts was not the only detoxification system in our rice roots and in fact Cd-exposure brought about a large increase in sulfhydryl groups, which was higher as the amount of the metal rose in protoplasts. The synthesis of –SH groups inside the cells was related to the concomitant appearance of Cd-complexing compounds that had different molecular weights. Plausibly, the Cd-complexant at higher MW might represent a polymerized form of phytochelatins (PCs) (Zenk, 1996), while that at lower MW might comprise more simple PCs or glutathione (GSH) (Sanit`a di Toppi et al., 1998). An additional response seen in Cd-exposed roots of cv. Vialone nano was the formation of electron-dense vacuolar inclusions that were more numerous and larger as the Cd concentration became higher inside cells. Correlation of this event with Cd detoxification was suggested by the absence of these inclusions in control plants, and thus they cannot be considered a characteristic root feature of this rice cultivar. Electron-dense vacuolar inclusions were noticed in root cells of Cd-treated Agrostis gigantea and interpreted to be intracellular sites of metal accumulation (Rauser and Ackerley, 1987). Similar electron-dense deposits of Cd inside vacuoles were also observed in roots and leaves of the hyperaccumulator Thlaspi caerulescens (W´ojcik et al., 2005). Moreover, the vacuoles were found to be the preferential site of the heavy metal storage in leaves of oilseed rape (B. napus) grown on Cd-contaminated soil (Carrier et al., 2003). In reality, Cd can be sequestered into vacuoles by importing Cd–PC complexes from the cytoplasm (Sanit`a di Toppi and Gabbrielli, 1999). This is regarded as a cell strategy to diverted Cd from cytoplasm and other metabolic active compartments (Carrier et al., 2003). The effect of Cd-exposure on the initial morphogenesis of cv. Vialone nano roots was very interesting. It should be pointed out that controversial information exists on how Cd negatively affects root growth and differentiation. The genotoxic effects of Cd on meristematic cells have been reported in Allium cepa (Borboa and De la Torre, 1996) and Hordeum vulgare (Zhang and Xiao, 1998), whereas it was recently shown that in Scots pine Cd accelerates differentiation and xylogenesis without causing injury to root tips (Sch¨utzend¨ubel et al., 2001). In our case, the effects of Cd were found in the first 500 ␮m from the root apical meristem at the lowest metal concentra-

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tion tested (50 ␮M). Cd interfered with formation of regular cell rows and caused, in the most external layers, hastened and huge enlargement of cells which also looked misshapen with anomalous division planes. To our knowledge, these damaging effects have not been previously reported and might be correlated with the inhibition of growth of Cd-exposed roots. In roots, in fact, above the apical meristem there is a so-called “transition postmitotic region” (about 400–500 ␮m in length) (Baluˇska et al., 1996) whose cells undergo a cytological and physiological “preparatory phase” that is critical for the subsequent root growth. Stress conditions, to which the cells of this region are particularly sensitive, can affect the root developmental program, unsettling the correct growth pattern (Ishikawa and Evans, 1995). An early and substantial cell enlargement in the postmitotic region with inhibition of the root growth, for instance, has previously been noticed in onion roots experimentally altered in the ascorbate system (De Tullio et al., 1999). It cannot be excluded that the altered root morphogenesis might also depend on an interaction of Cd with calcium. Cd enters the cells in competition with Ca (Clemens, 2001) leading to a lowered concentration of this essential micronutrient. Moreover, it was shown that inside the cells Cd can bind calmodulin (Rivetta et al., 1997). This prevents the formation of the Ca–calmodulin complex which acts as second messenger in events of plant growth and development (Hepler and Wayne, 1985). The different extra- and intracellular strategies to retain Cd in roots could account for the low percentage of metal that moved to the rice shoot. Moreover, in leaf tissues a substantial fraction of Cd, which arrived via xylem, also remained in the apoplast and bound to cell wall anionic components. Indeed, the Cd amount taken up in leaf protoplasts was more than 40 times lower than total Cd accumulated in roots and about 4.5 times lower than the amount of Cd that entered in root protoplasts. This might explain why only a small increase in –SH groups was seen as additional detoxification mechanism in leaf cells. Furthermore, this might also account for the lack of the electrondense Cd deposits inside cell vacuoles. Thlaspi caerulescens, which shows the Cdcontaining granules in the leaf vacuoles, indeed, translocates a greater quantity of the heavy metal to this organ (W´ojcik et al., 2005). However, the Cd that reached the rice leaves brought about severe damage. Leaf growth was inhibited and blade thickness was diminished due to the reduced enlargement of mesophyll cells, with a consequent increase in the tissue dry weight/fresh weight ratio. Moreover, leaf conductance sharply lowered and became insensitive to light, suggesting that stomatal functionality was compromised (Perfus-Barbeoch et al., 2002). Changes in plant water relations, with a decline in the transpiration rate, have been observed in other Cd-polluted species (Costa and Morel, 1994; Baryla et al., 2001; Pandey and Sharma, 2002) and were related to the decreased leaf blade expansion (Haag-Kerwer et al., 1999). Although a reduction in leaf surface area is a rather common consequence of exposure to Cd, the information about the Cd effects on leaf cell enlargement is somewhat contradictory. In Phaseolus vulgaris, for example, Cd causes a decrease in cell size (Barcel´o et al., 1986), whereas in both B. napus and

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P. sativum it leads to an increase of mesophyll cell dimensions (Baryla et al., 2001; Sandalio et al., 2001). Moreover, in the aquatic plant Elodea canadensis the Cd-exposure of submerged leaves inhibits cell division but induces a significant enlargement of only one of the two cell layers forming the leaf blade (Dalla Vecchia et al., 2005). Finally, in young Cd-treated wheat plants, leaves become thinner and have less enlarged mesophyll cells (Kovaˇcevi´c et al., 1999), as was found for the leaves of our rice plantlets. This suggests that the change in leaf cell enlargement caused by Cd may actually be due to specific morphogenetic effects rather than just impaired water balance. Along these lines, it is interesting to point out that Cd causes a reduced cell enlargement in leaf tissues, while it induces cell enlargement in tissues of the root apical region. Lower amounts of chlorophyll were found in Cd-treated leaves, whose chloroplasts exhibited a reduced number of thylakoids. A bleaching effect of Cd on leaf tissues is known, although the mechanism by which the metal brings about chlorosis is still unclear. This event has been attributed to a number of effects including inhibition of chlorophyll biosynthesis (Stobart et al., 1985), chlorophyll degradation (Somashekaraia et al., 1992), hastened senescence and disorganization of chloroplasts (Rascio et al., 1993; Ouzounidou et al., 1997; McCarthy et al., 2001), and oxidative stress (Gallego et al., 1999; Sandalio et al., 2001). In our case a reduction in chlorophyll already occurring in rice leaves exposed to the lowest Cd concentration was associated with a decreased number of photosynthetic membranes, but not with symptoms of chloroplast senescence or thylakoid alterations, which were observed only at the highest Cd concentrations. Moreover, the amount of D1, which is a highly oxidative stress-sensitive thylakoid polypeptide (Giardi et al., 1997), did not change over the range of Cd concentration used, indicating that there is not accumulation of damaged PSII reaction centres. These data strongly suggest that chlorosis found in cv. Vialone nano leaves does not come from degradative events, but principally from the inhibitory effects of Cd on chlorophyll biosynthesis and thylakoid synthesis. Cd-polluted leaves also underwent a decline of photosynthesis, as shown by the in vivo drop of both CO2 assimilation and O2 emission. This might be due to the reduced amount of photosynthetic pigments and the number of thylakoids. However, oxygen evolution slowed even when measured on the basis of chlorophyll concentration, showing that the decrease in photosynthesis could not be all ascribed to pigment shortage. Cd can negatively affect different steps of the photosynthetic process (Pagliano et al., 2006). In our case, an event involved in photosynthesis inhibition is likely to be the decreased availability of CO2 , owing to the increase in leaf resistance, seen in Cd-exposed plants. In contrast to what observed in other species, the deleterious effects of Cd on photosynthesis do not depend on ultrastructural alterations or oxidative damage of chloroplasts. However, it has to be mentioned that a reduced photosynthesis can also be caused by an impaired mineral nutrition, and it is well known that Cd greatly affects uptake and accumulation of essential microelements (Gussarson et al., 1996; Hern´andez et al., 1996; Ramos et al., 2002). In particular, Cd can inhibit

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