Silicon alleviates cadmium toxicity by enhanced photosynthetic rate and modified bundle sheath's cell chloroplasts ultrastructure in maize

Ecotoxicology and Environmental Safety 120 (2015) 66–73

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Silicon alleviates cadmium toxicity by enhanced photosynthetic rate and modified bundle sheath's cell chloroplasts ultrastructure in maize Marek Vaculík a,n, Andrej Pavlovič a,b, Alexander Lux a a

Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina B2, SK-842 15 Bratislava, Slovakia Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University in Olomouc, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic b

art ic l e i nf o

a b s t r a c t

Article history: Received 27 February 2015 Received in revised form 15 May 2015 Accepted 16 May 2015

Silicon was shown to alleviate the negative effects of various biotic and abiotic stresses on plant growth. Although the positive role of Si on toxic and heavy metal Cd has been already described, the mechanisms have been explained only partially and still remain unclear. In the present study we investigated the effect of Si on photosynthetic-related processes in maize exposed to two different levels of Cd via measurements of net photosynthetic rate (AN), chlorophyll a fluorescence and pigment analysis, as well as studies of leaf tissue anatomy and cell ultrastructure using bright-field and transmission electron microscopy. We found that Si actively alleviated the toxic syndromes of Cd by increasing AN, effective photochemical quantum yield of photosystem II (ϕPSII) and content of assimilation pigments, although did not decrease the concentration of Cd in leaf tissues. Cadmium did not affect the leaf anatomy and ultrastructure of leaf mesophyll’s cell chloroplasts; however, Cd negatively affected thylakoid formation in chloroplasts of bundle sheath cells, and this was alleviated by Si. Improved thylakoid formation in bundle sheath’s cell chloroplasts may contribute to Si-induced enhancement of photosynthesis and related increase in biomass production in C4 plant maize. & 2015 Elsevier Inc. All rights reserved.

Keywords: C4 plant anatomy and physiology Cadmium (Cd) Chloroplast ultrastructure Chlorophyll a fluorescence Photosynthesis Silicon (Si)

1. Introduction Silicon (Si) and its importance for plant growth and development is documented by several research papers published during the last years. Although this second most abundant element in the earth crust is not easily accessible for plants, some of them, mostly from the family Poaceae, take up this element in relatively high amount (Wiese et al., 2007). In addition to the importance of Si in plant nutrition, optimal growth and development (Guntzer et al., 2012), it plays a significant role in the alleviation of various symptoms of biotic as well as abiotic stresses (Ma, 2004). It was also shown that Si can alleviate iron deficiency (Pavlovic et al., 2013). On the other hand, it is known that Si can decrease the negative effects of elements, including some heavy metals, when present in excess (Liang et al., 2007; Balakhina and Borkowska, 2013; Wu et al., 2013). Formation of aluminosilicates in the presence of Si was suggested as mechanism of detoxification of aluminum (Al) excess in some plants (e.g. Pragabar et al., 2011). Similarly, the mitigation role of Si has been recently described for several other metals or dangerous toxic elements, including n

Corresponding author. Fax: þ421 265429064. E-mail address: [email protected] (M. Vaculík).

http://dx.doi.org/10.1016/j.ecoenv.2015.05.026 0147-6513/& 2015 Elsevier Inc. All rights reserved.

chromium (Ali et al., 2013), lead (Li et al., 2012), antimony (Huang et al., 2012; Vaculíková et al., 2014) or arsenic (Fleck et al., 2013; Tripathi et al., 2013). Previously, it has been reported that Si can mitigate negative influence of dangerous heavy metal cadmium (Cd) on growth of various plants. In maize, the alleviation of Cd toxicity was partially attributed to Si-enhanced cell wall elasticity and plasticity (Vaculík et al., 2009) as well as increased deposition of Cd in the cell walls (Vaculík et al., 2012; Lukačová et al., 2013). Decrease in plant Cd uptake and translocation from root to shoot was suggested as other beneficial role of Si in many species, including maize (Liang et al., 2005, Da Cunha et al., 2008), Solanum nigrum (Liu et al., 2013b) or mangrove seedlings (Zhang et al., 2013), although this feature seems to be species and/or cultivar specific and also depends on the concentration of used chemicals (Liang et al., 2005; Vaculík et al., 2009; Lukačová et al., 2013). Additionally, the alleviative effect of Si on Cd toxicity was partially attributed to the changes in the activity of important antioxidative enzymes involved in scavenging of free radicals formed by presence of Cd in pakchoi (Song et al., 2009); maize (Lukačová et al., 2013), S. nigrum (Liu et al., 2013b) and other species. There are also some reports that Si influences the development of apoplasmic barriers, root suberization and lignification, as well as modifies leaf and root anatomy in plants exposed to Cd (Da Cunha and do Nascimento

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2009; Vaculík et al., 2012; Vatehová et al., 2012; Zhang et al., 2013). Relatively less is known about the influence of Si on the assimilation tissues and processes related with photosynthesis in Cd-treated plants. It was already reported that Si can ameliorate the negative effects of Cd on the content of photosynthetic pigments, gas exchange parameters and photosynthesis in rice (Nwugo and Huerta, 2008), cucumber (Feng et al., 2010) or cotton (Farooq et al., 2013), however the mechanisms are still unclear. Therefore, to get a better insight in the mechanisms behind the positive effect of Si on the growth of maize-economically important agricultural plant suffered by Cd toxicity, we conducted experiments to investigate the role of Si in relation to the photosynthesis, shoot biomass production and leaf tissue and cell structure, that are shown in this paper.

2. Material and methods 2.1. Cultivation of plants Caryopses of maize (Zea mays L., hybrid Jozefina) were sterilized for 20 min in 5% Savo (Biochemie, Czech Republic) and washed carefully several times with distilled water before the germination. Thereafter, they were imbibed in water for four hours at room temperature and germinated in rolls of wet filter paper for 72 hours at 25 °C in a dark. Seedlings were transferred to 3 L glass containers (10 plants per container) filled with half strength modified Hoagland solution (Hoagland and Arnon, 1950) with or without Cd and/or Si. After two days of the cultivation the medium was changed to full strength Hoagland solution. The solutions were changed every second day. In total, the plants in each treatment were cultivated for 10 days. Six different treatments were applied:

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2.3. Determination of Cd concentration in above-ground plant parts The concentration of Cd was determined in finely ground dried shoot tissue using atomic absorption spectrometry (AAS) in the Geoanalytical Laboratories of Institute of Geomaterials, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia. 2.4. Determination of changes in photosynthesis and chlorophyll fluorescence Rates of net photosynthesis (AN) and chlorophyll a fluorescence were measured simultaneously on the 2nd fully developed maize leaves with a CIRAS-2 (PP-Systems, Hitchin, UK) and a fluorcam FC1000-LC (Photon Systems Instruments, Brno, Czech Republic) attached to the infrared gas analyser. The actinic light of 75 μmol m  2 s  1 PAR was switched on for induction of photosynthesis. Then the light intensity was increased stepwise with irradiation periods of 3.5 min, steady state fluorescence (Ft) was recorded and subsequent saturation pulses (4000 μmol m  2 s  1 PAR, 800 ms duration, λ ¼620 nm) were applied for estimation of ‵ ) maximum chlorophyll fluorescence in the light-adapted state (Fm at each light intensity, until 1700 μmol m  2 s  1 PAR was reached. Then the actinic light was switched off and after 10 min, rate of respiration in the dark (RD) was recorded. The light was provided by blue (λ ¼455 nm) and red (λ ¼620 nm) LED diodes. Effective photochemical quantum yield of photosystem II (ΦPSII) was cal‵ −Ft Fm ‵ according to Maxwell and Johnson (2000) culated as Fm

(

)

and Roháček (2002). Simultaneously, the rate of net photosynthesis (AN) was recorded at CO2 concentration 380 μmol mol  1, leaf temperature 25 71 °C and relative air humidity 65–70%. 2.5. Analyses of chlorophyll and carotenoid content

1. Control (C)-Hoagland solution without Cd and Si; 2. Cadmium 5 (Cd5)-Hoagland solution with 5 μM Cd(NO3)  4H2O; 3. Cadmium 50 (Cd50)-Hoagland solution with 50 μM Cd(NO3)  4H2O; 4. Silicon (Si)-Hoagland solution with 5 mM Si in the form of sodium silicate solution (27% SiO2 dissolved in 14% NaOH); 5. Cadmium 5 plus silicon (Cd5 þSi)-Hoagland solution with addition of both Cd and Si in the same concentrations as in the Cd5 and Si treatments; and 6. Cadmium 50 plus silicon (Cd50 þSi)-Hoagland solution with addition of both Cd and Si in the same concentrations as in the Cd50 and Si treatments.

Approx. 50 mg of fresh leaf tissue samples from the mid part of the 2nd fully developed maize leaves were grinded in a mortar with bit of sand and MgCO3, and photosynthetic pigments were extracted with chilled 80% acetone. The suspension was centrifuged at 4 °C for 5 min at 5000g. The concentration of chlorophylls and carotenoids in supernatant was determined spectrophotometrically (Jenway 6400, London, UK) and total content of chlorophyll a, b and carotenoids was calculated according to Lichtenthaler and Wellburn (1983).

The pH of each cultivation solution was adjusted to 6.2 using HCl. The Si concentration used in our experiments was based on our previous experiments with the same maize cultivar. It should be noted that no precipitation of Si in the solution was observed. Young maize plants were cultivated in hydroponics till the second fully developed leaf in a growth chamber with a 12-h photoperiod, a temperature 25/23 °C (day/night), 75% humidity and 200 mmol m  2 s  1 PAR.

For the investigation of anatomical differences of leaf tissues, tissue segments, approx. 2  1 mm large, were collected from the mid part of the 2nd fully developed leaf and fixed in 2% glutaraldehyde and 0.2% osmium tetroxide. After dehydration with ethanol and propylene oxide the samples were embedded in Spurr resin (Serva). Approximately 2 μm thick semi-thin sections were prepared using microtome Tesla BS 490 and stained with 0.5% toluidine blue and 0.1% basic fuchsine according to Lux (1981). The sections were analyzed with Zeiss Axioskop 2 plus epifluorescence microscope (Zeiss, Germany), equipped with Olympus DP72 camera. Quantitative anatomical analysis of leaf tissues was performed with the image analysis software Lucia G 4.80 (LIM, Czech Republic). For the investigation of ultrastructural changes of leaf cells, ultra-thin sections, approx. 80 nm thick, were prepared from previously embedded samples using ultramicrotome ReichertJung (Vienna, Austria) and stained with 2% uranium acetate and 2% lead citrate. Sections were analyzed with transmission electron microscope JEM 2000FX (JEOL, Japan). For quantitative

2.2. Evaluation of plant's growth and biomass production Plant material was harvested at the fully developed second leaf stage at the end of the cultivation (13th day after imbibition, or 10th day of hydroponic cultivation) and processed in followed experiments. The plants were divided into below- and aboveground parts. Fresh weights of below- and above-ground parts of plants were determined. Root and shoot material was dried at 70 °C for 72 h, and the dry weights of below- and above-ground parts were determined.

2.6. Determination of quantitative changes in the leaf anatomy and cell ultrastructure

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ultrastructural analysis, average number of well developed and clearly visible thylakoids was evaluated per certain area of at least five different chloroplasts of mesophyll cells (MCHL) and bundle sheath cells (BSCHL) (the area of 100  100 nm and 400  400 nm for granum of MCHL and stroma of BSCHL, respectively). The total number, average length and width of starch grains in five different BSCHL from five various plant samples of each treatment were also evaluated. 2.7. Statistical analysis Statistical significance was assessed with Student's t-test using the Statgraphics Centurion XV v. 15.2.05 (StatPoint, Inc., Warrenton, VA, USA) and Excel (Microsoft Office 2007) programs and a single-step multiple comparisons of means was performed via Tukey test. A P-value o0.05 was defined as significant. The data presented (biomass) are from six different replicates. For determination of changes in photosynthesis, chlorophyll a fluorescence and pigments, four different plant samples from each treatment were evaluated. For determination of changes in leaf anatomy and cell ultrastructure five different plant samples from each treatment were evaluated.

3. Results 3.1. Influence of Cd and/or Si on plant growth and morphology Cadmium at both applied concentrations (Cd5 and Cd50) decreased the growth of bellow- and above-ground biomass, the reduction in growth was more pronounced when higher dose of Cd was applied. We already reported that addition of Si ameliorated the negative effect of Cd on maize root length and root fresh and dry biomass (Vaculík et al., 2012). Similarly, shoot fresh and dry weight significantly decreased with increased concentration of Cd in the medium. However, addition of Si to Cd treated plants alleviated the negative influence of Cd, and fresh and dry biomass of Cd5 þSi and Cd50 þSi treated plants were significantly greater when compared with Cd5 and Cd50 plants, respectively. Silicon application to Cd non-treated plants did not influence the fresh and dry weight of aboveground plant parts when compared with the control (Table 1). The negative influence of Cd resulted in a decrease of the 1st and the 2nd fully developed maize leaves area. Addition of Si increased the area of the 1st and the 2nd leaves in Cd5þSi when compared with Cd5 treatment. In Cd50 þ Si plants, the addition of Si did not influence the area of the 1st leaf, and fully ameliorated the negative influence of Cd on the 2nd leaf (Table 1). The DW/FW Table 1 The average fresh (FW) and dry (DW) weight of shoot, dry/fresh weight quotient (DW/FW) and the area of the 1st (Area L1) and the 2nd (Area L2) fully developed leaves of young maize plants grown for 10 days in hydroponics and exposed to various concentrations of Cd and/or Si; six treatments were used (C – Control, Cd5 – 5 μM Cd, Cd50 – 50 μM Cd, Cd5 þSi – 5 μM Cd þ5 mM Si, Cd50 þ Si – 50 μM Cd þ 5 mM Si, Si – 5 mM Si). Values are means 7 SD (n ¼6). Different letters in columns indicate significant differences between the treatments at Po 0.05 according to Tukey’s test. Treatment Shoot FW Shoot DW (mg) (mg) C Cd5 Cd5 þ Si Cd50 Cd50 þ Si Si

950 751a 769 749b 940 779a 530 752d 639 732c 969 770a

64.37 4.6a 54.2 7 4.7b 71.5 7 8.9a 47.0 7 2.8c 57.17 2.2b 69.7 7 6.2a

Area L1 (cm2) Area L2 (cm2) DW/FW

7.28 70.82b 6.3370.63d 6.99 70.37c 6.0770.76d 6.18 70.54d 7.59 70.75a

15.007 1.51a 12.487 1.25c 13.29 7 0.61b 11.337 0.70d 12.487 0.74c 15.147 1.70a

0.068 0.070 0.076 0.089 0.089 0.072

Fig. 1. Net photosynthetic rate (AN) (a), and effective photochemical quantum yield of photosystem II (ΦPSII) (b) in response to irradiance in the 2nd fully developed leaf of young maize plants grown for 10 days in hydroponics and exposed to various concentrations of Cd and/or Si; six treatments were used (C – Control, Cd5–5 μM Cd, Cd50 – 50 μM Cd, Cd5 þSi – 5 μM Cd þ5 mM Si, Cd50 þSi – 50 μM Cd þ 5 mM Si, Si – 5 mM Si). Values are means 7SE (n¼ 4). Abbreviations: PAR – photosynthetically active radiation.

quotient increased with increasing concentration of Cd. Addition of Si increased the DW/FW quotient in Cd5þSi plants when compared with Cd5, and no differences have been observed between Cd50 and Cd50 þSi plants (Table 1). 3.2. Influence of Cd and/or Si on Cd concentration in above-ground plant parts We found that with increasing concentration of Cd the concentration of this metal also increased in the shoot. No difference was observed between control and Si treated plants. However, the concentration of Cd was higher in Cd5þ Si than in Cd5 treated plants about 23%. Addition of Si to higher Cd treatment (Cd50 þSi) did not modify the Cd concentration in above-ground tissues when compared with single Cd50 treatment (Fig. S1). 3.3. Influence of Cd and/or Si on the photosynthesis The rate of net photosynthesis (AN) in the young maize plants decreased with increasing Cd. In general, the differences between treatments were more obvious with increasing irradiance (Fig. 1a).

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Addition of Si to Cd treated plants alleviated the negative effect of Cd on the photosynthesis. Silicon in Cd5þ Si treatment evidently increased the AN when compared with Cd5 up to the values similar to control plants. Similarly, the same mitigation effect of Si was observed at higher applied level of Cd. No differences in AN were observed between control and Si treated plants (Fig. 1a). 3.4. Influence of Cd and/or Si on chlorophyll fluorescence A decrease in the effective photochemical quantum yield of photosystem II (ΦPSiI) was recorded in Cd treated plants when compared with the control. However, the decrease was more evident when higher Cd level was applied (Cd50). In general, the differences between treatments were more obvious with increasing irradiance (Fig. 1b). Addition of Si to Cd treated plants alleviated the negative effect of Cd on the ΦPSiI. Silicon in Cd50 þ Si treatment increased the ΦPSiI when compared with Cd50 up to the levels comparable with Cd5 treatment. Similarly, Si addition in Cd5þ Si treatment increased the ΦPSiI when compared with Cd5 treatment without Si. No differences in ΦPSiI were observed between control and Si treated plants (Fig. 1b). The negative effect of Cd resulted in the visible changes on the leaf tissues, as chlorophyll a fluorescence imaging indicates. Cadmium at lower concentration (Cd5) caused a decrease in ΦPSiI along the leaf veins, and higher Cd concentration (Cd50) resulted in spatially separated area with decrease of ΦPSiI. Addition of Si reduced the negative effects of Cd on the growth of maize by increasing ΦPSiI in leaves (Fig. 2). 3.5. Influence of Cd and/or Si on the content of chlorophylls and carotenoids Cadmium at both applied concentrations (Cd5 and Cd50) decreased the content of chlorophyll a (chla) and chlorophyll b (chlb), which resulted in the significant decrease of total chlorophylls content in the 2nd maize leaf when compared with control plants. The addition of Si mitigated the negative influence of Cd on

Fig. 2. Changes of effective photochemical quantum yield of photosystem II (ΦPSII) in the 2nd fully developed leaf of young maize plants grown for 10 days in hydroponics and exposed to various concentration of Cd and/or Si; six treatments were used (C – Control, Cd5 – 5 μM Cd, Cd50 – 50 μM Cd, Cd5 þ Si – 5 μM Cd þ 5 mM Si, Cd50 þ Si – 50 μM Cd þ 5 mM Si, Si – 5 mM Si). Representative images from 4 different plants are shown.

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Table 2 The total content of chlorophyll a and b (chla þb), chlorophyll a/b (chla/b) ratio and content of carotenoids (car) in the 2nd fully developed leaf of young maize plants grown for 10 days in hydroponics and exposed to various concentrations of Cd and/ or Si; six treatments were used (C – Control, Cd5 – 5 μM Cd, Cd50 – 50 μM Cd, Cd5 þSi – 5 μM Cd þ5 mM Si, Cd50 þ Si – 50 μM Cd þ 5 mM Si, Si – 5 mM Si). Values are means 7 SD (n ¼4). Different letters indicate significant differences between the treatments at Po 0.05 according to Tukey's test. Treatment

chlaþ b (mg g  1FW)

chla/b

Car (mg g  1FW)

C Cd5 Cd5 þSi Cd50 Cd50 þSi Si

2.39 7 0.14a 2.127 0.14b 2.38 7 0.2a 2.007 0.2b 2.38 7 0.12a 2.357 0.06a

2.23 2.11 2.43 2.16 2.17 2.31

0.0385 70.006a 0.035870.005a 0.0362 70.003a 0.0306 70.004b 0.0360 70.006a 0.0360 70.004a

the chlorophylls content. In Cd5 þSi and Cd50 þSi treatments the total chlorophylls content was not significantly different when compared with the control plants. Silicon, when applied alone (Si treatment) did not influence the content of chlorophylls compared with control plants (Table 2). Similarly, Cd decreased the chlorophyll a/b ratio in Cd5 and Cd50 compared to control. In Cd5þSi the chla/b ratio was increased when compared with Cd5 treatment, however no difference was observed between Cd50 and Cd50 þSi plants. Silicon, when applied alone slightly increased the chla/b ratio when compared with control plants (Table 2). Cadmium also decreased the content of carotenoids in the 2nd maize leaf when compared with the control; however, the decrease was significant only in the treatment with higher applied Cd level (Cd50). This negative effect of Cd was alleviated by addition of Si in Cd50 þSi treatment. No differences between Cd5 and Cd5 þSi plants were found. Similarly, no differences were observed between plants treated with addition of Si (Si treatment) and control (Table 2). 3.6. Influence of Cd and/or Si on the leaf anatomy and cell ultrastructure Differences in the thickness of maize leaves (Fig. 3a) were observed between the treatments. Cadmium at both applied concentrations (Cd5 and Cd50) increased the thickness of the 2nd fully developed maize leaf when compared with the control. Similarly, the leaf thickness was increased when comparing Cd5 þSi and Cd50 þ Si with control plants. However, no differences were observed between Cd treated (Cd5 and Cd50) and Cd þSi treated (Cd5þ Si and Cd50 þ Si) plants. Silicon when applied alone (Si treatment) increased the thickness of the maize leaf when compared with the control. Moreover, this increase was significantly higher than in Cd5, Cd50, Cd5þSi and Cd50 þSi treatments (Fig. 3b). As maize is plant with C4 type of metabolism, structural and functional heterogeneity between chloroplasts of mesophyll cells (MCHL) and bundle sheath cells (BSCHL) can be recognized (Fig. 4a–d). Ultrastructural analysis revealed no significant differences in MCHL between control and Cd treatment. In all treatments the chloroplasts contained well differentiated thylakoid system with individual thylakoids of stroma and also thylakoids forming granas, and no difference between numbers of thylakoids between all treatments was observed. An increased number of plastoglobuli has been, however, observed in MCHL from Cd treated plants than in control (Fig. 4a,b,e). On the other hand, significant differences were observed in BSCHL. In control and Si treatment chloroplasts contained well differentiated long individual thylakoids. Increasing concentration of Cd caused disorganization of chloroplasts internal thylakoid structure (Fig. 4c,d), and resulted in increased number and size of starch grains in

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Fig. 3. Semi-thin cross section of the 2nd fully developed maize leaf in control treatment (a), and thickness of the same leaves of maize plants grown for 10 days in hydroponics and exposed to various concentrations of Cd and/or Si (b); six treatments were used (C – Control, Cd5 – 5 μM Cd, Cd50 – 50 μM Cd, Cd5 þSi – 5 μM Cd þ 5 mM Si, Cd50 þ Si – 50 μM Cd þ 5 mM Si, Si – 5 mM Si). Values are means 7 SD (n ¼5). Different letters indicate significant differences between the treatments at Po 0.05. Abbreviations: ade – adaxial epidermis, abe – abaxial epidermis, s – sclerenchyma, lm – leaf mesophyll, bs – bundle sheath cell, vb – vascular bundle, arrow pointed on chloroplast in mesophyll cell. Bar 50 μm.

BSCHL (Table S3). This was partially mitigated by Si in Cd5 þSi treatment; number of thylakoids increased to the same level as in the control (Fig. 4f), and starch grains were thinner, although the total number and length of starch grains was not affected (Table S3). In the higher Cd treatment, no clearly visible thylakoids in BSCHL were observed, and no effects of Si on thylakoid formation and starch grain size and number between BSCHL have been detected (Fig. 4d and f; Table S3).

4. Discussion Negative effect of Cd on plant growth and development is well documented. Along with the entire group of unfavorable aspects of Cd on the rhizosphere (Lux et al., 2011), several toxicity reports of Cd on the cell level (Benavides et al., 2005; Martinka et al., 2014), as well as negative influence of this heavy metal on photosynthetic-related processes in leaf mesophyll tissues are known (Hasan et al., 2009). In maize, Cd was shown to decrease the chlorophyll synthesis (Lagriffoul et al., 1998; Chaneva et al., 2010), effective photochemical quantum yield of photosystem II (ΦPSiI), as well as rate of CO2 fixation (Ekmekçi et al., 2008; Wang et al., 2009; Da Silva et al., 2012). In our previous studies, mostly focused on roots, we found that plants simultaneously growing in the presence of different concentrations of Cd and Si produced higher amount of biomass then plants treated only with Cd, although the concentration of Cd, as well as total content of Cd in both roots and shoots of Cd þSi treated plants was higher than in Cd treated plants (Vaculík et al., 2009; 2012). Similarly, in the present study we found that addition of Si alleviated the negative effects of Cd on shoot appearance and biomass production, although the concentration of Cd was either increased in Cd5þSi or not influenced in Cd50 þ Si, when compared with respective treatments without Si. To better understand this phenomenon, we investigated photosynthesis as one of the processes closely related with plant growth and biomass

production. We found that the rate of net photosynthesis (AN) in the young maize plants decreased with increasing Cd. However, addition of Si ameliorated the negative influence of Cd in Cd5þSi as well as in Cd50 þSi plants. Enhancement of photosynthesis due to Si addition was observed also in cucumber (Feng et al., 2010), cotton (Farooq et al., 2013) or maize (Mihaličová Malčovská et al., 2014) exposed to Cd as well as in bean and zucchini plants exposed to higher salinity (Zuccarini, 2008; Savvas et al., 2009). Shen et al. (2010b) reported that addition of Si increased the rate of net photosynthesis in soybean seedlings also under UV-B radiation stress. Hattori et al. (2005) observed improvement of photosynthetic rate after Si application in drought stressed sorghum plants. Decrease in photosynthetic activity has been often connected with decreased assimilation pigments synthesis and lower chlorophylls content in plants, including maize (Pal et al., 2006; Krantev et al., 2008). In our recent experiments we found that Cd applied at lower (Cd5), as well as higher (Cd50) concentration decreased the content of chla and chlb, which resulted in the significant decrease of total chlorophylls content when compared with control plants. However, the addition of Si suppressed the negative effect of both lower and higher Cd stress. Additionally, lower level of Cd did not influence the content of carotenoids while higher Cd significantly decreased the content of carotenoids, which was alleviated by Si addition. This is in agreement with other authors that observed the alleviating effect of Si on chlorophylls and carotenoids content in Cd stressed plants (Feng et al., 2010; Farooq et al., 2013). The positive effect of Si on the chlorophyll content, decreased by other types of abiotic stresses, was recently described in various plants. Doncheva et al. (2009) found Si induced increase in chla, chlb and carotenoid content in maize grown under Mn toxicity. Shen et al. (2010a) observed that Si increased the content of chlorophylls in UV-B-stressed soybean, and Tale Ahmad and Haddad (2011) observed the same effect on drought-stressed wheat. Several studies described that Si alleviated chlorophylls content decrease in various plant species exposed to salinity stress (tomato, Al-Aghabary et al., 2004; wheat, Levent Tuna et al., 2008; spinach, Eraslan et al., 2008; soybean, Lee et al., 2010; or canola, Hashemi et al., 2010). The chlorophyll a fluorescence measurement is a non-destructive technique that allows determining sensitive changes primarily observed in photosystem II (Da Silva et al., 2012). To further investigate the alleviation effect of Si, we used this method and found that ΦPSiI decreased with increasing Cd concentration, and addition of Si to Cd treated maize alleviated this negative effect. Although similar results were obtained by Feng et al. (2010) in Cd treated cucumber, Doncheva et al. (2009) found, that in maize Si ameliorated the Mn induced negative effect on ΦPSiI at relatively high Mn concentration (0.5 mM), and this was not observed at lower Mn concentration (0.2 mM). Chlorophyll a fluorescence imaging in our experiments also shown that at lower Cd stress lower values of ΦPSiI were detected especially along the bundle sheet cells and at higher Cd stress a specific spatially separated areas were detected. Both effects were significantly reduced in treatments with applied Si. This might partially result from Si induced decrease in cell free-available Cd and increase in cell wallbound portion of Cd, as was observed in our foregoing experiments with maize (Vaculík et al., 2012), as well as possible inhibited Cd cell uptake caused by Si-modified cell wall properties (Liu et al., 2013a). We found that addition of both levels of Cd (Cd5 and Cd50), as well as combined Cd þSi treatments (Cd5 þ Si and Cd50 þSi) increased the thickness of maize leaves when compared with the control. Moreover, the thickness of leaves was significantly increased in Si treatment when compared with all other treatments. Doncheva et al. (2009) also observed that leaf of maize increased

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Fig. 4. Chloroplasts in mesophyll cells of maize leaf in control (a) and Cd50 (b) treatment, and chloroplasts in bundle sheath cells of maize leaf in Cd5 (c) and Cd5 þ Si (d) treatment; total amount of well developed and clearly visible thylakoids per certain area of chloroplasts of mesophyll cells (MCHL) (e) and bundle sheath cells (BSCHL) (f) of each treatment (C – Control, Cd5 – 5 μM Cd, Cd50 – 50 μM Cd, Cd5 þ Si – 5 μM Cd þ5 mM Si, Cd50 þSi – 50 μM Cd þ 5 mM Si, Si – 5 mM Si). Evaluated area was 100  100 nm 2and 400  400 nm2 for granum of MCHL and stroma of BSCHL, respectively. Values are means 7 SD (n¼ 5). Different letters indicate significant differences between the treatments at P o 0.05; ND¼ not determined. Abbreviations: CW – cell wall, G – granum, SG – starch grain, V – vacuole; n indicates plasmodesmata, arrows pointed on plastoglobuli. Bar 200 nm.

in its thickness after Si application; and that excess of Mn also increased the leaf thickness of tolerant maize variety and it became even thicker after Si addition. Oppositely, Mn decreased the leaf thickness of sensitive maize variety, and addition of Si alleviated this (Doncheva et al., 2009). Da Cunha and do Nascimento (2009) found Si induced increase in mesophyll thickness in maize grown in Cdþ Zn contaminated soil. However, our present results showed no differences in leaf thickness between Cd and Cdþ Si treated plants. We suggest that Si increased the thickness of leaves when applied alone, and this is probably related with lower tissue water capacity and increased DW/FW quotient and leaf biomass. The effect of heavy metals, including Cd, on the photosynthetic apparatus of plants can be recognized at the ultrastructural level. Cadmium has been found to alter chloroplast ultrastructure, especially thylakoid system arrangement in several species

(Vittória et al., 2001; Dalla Vecchia et al., 2005; Tkalec et al., 2008). Our recent results indicate that application of Cd even at higher concentration (Cd50) did not modify the thylakoid internal structure of mesophyll's cell chloroplasts. We observed only Cdinduced increase in the number of plastoglobuli that was alleviated by Si addition. The number and size of plastoglobuli was also increased in pea exposed to Cd (McCarthy et al., 2001), and it seems to be a common feature of chloroplasts that have been exposed to heavy metals (Solymosi and Bertrand, 2012). Previous studies reported that Si could alleviate negative effect of Cd and some other metals, like Mn and Cr on the mesophyll's cell chloroplasts ultrastructure (Doncheva et al., 2009; Feng et al., 2010; Ali et al., 2013). However, to our knowledge no study has been conducted to describe the differences between the structure of mesophyll's and bundle sheath's cell chloroplasts, that are

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characteristic for C4 plants. In our current experiments a visible symptoms of Cd toxicity in the form of disorganization of thylakoid structure and increase in number and size of starch grains have been observed especially in bundle's sheath cell chloroplasts. As these specific cells are in direct contact with vascular tissues that distribute ions, including Cd, from root to shoot, higher concentration of Cd and related toxicity effects in bundle sheath cells are therefore expected. The thylakoid formation in BSCHL was partially enhanced by Si at lower (Cd5) but not at higher (Cd50) stress. These observation correspond with the lower values of ΦPSiI detected especially along the bundle sheet cells of Cd5 treated plants, which was alleviated by Si. Moreover, improved thylakoid formation might be a consequence of decreased portion of soluble Cd and increased portion of cell wall-bound Cd in the leaves of Si treated plants as we have previously shown (Vaculík et al., 2012). We therefore suggest that Si-induced improvement of thylakoid formation in BSCHL may contribute to efficiency of photosynthesis in Si treated plants which is followed by enhanced biomass production when compared with plants treated with Cd without Si. This represents a novel finding how Si in plants with C4 metabolism, such as maize, might improve the photosynthesis and in this way alleviate metal toxicity.

5. Conclusions We found that exposure of maize plants to Cd reduced the growth and shoot biomass production and negatively influenced photosynthesis. Application of Si increased chlorophylls and carotenoids content, and improved ΦPSiI and AN, although did not decrease Cd concentration in leaf tissues. Although no differences in the leaf thickness and ultrastructure of mesophyll’s cell chloroplasts have been observed between Cd and Cd þSi treated plants, Cd negatively affected the thylakoid formation in bundle sheath's cell chloroplasts, which was alleviated by Si. Silicon enhanced thylakoid formation in bundle sheath's cell chloroplast might be responsible for increased rate of photosynthesis and biomass production which partially explains the way how Si improve the growth of plants exposed to Cd.

Acknowledgments The work was supported by Slovak Research and Development Agency under the Contract APVV SK-SRB 2013-0021, and by Grant Agency VEGA1/0817/12. The authors declare that they have no conflict of interest.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.05. 026.

References Al-Aghabary, K., Zhu, Z., Shi, Q.H., 2004. Influence of silicon supply on chlorophyll content, chlorophyll fluorescence, and antioxidative enzyme activities in tomato plants under salt stress. J. Plant Nutr. 27, 2101–2115. Ali, S., Farooq, M.A., Yasmeen, T., Hussain, S., Arif, M.S., Abbas, F., Bharwana, S.A., Zhang, G., 2013. The influence of silicon on barley growth, photosynthesis and ultra-structure under chromium stress. Ecotoxicol. Environ. Saf. 89, 66–72. Balakhina, T., Borkowska, A., 2013. Effects of silicon on plant resistance to environmental stresses: a review. Int. Agrophys. 27, 225–232. Benavides, M.P., Gallego, S., Tomaro, M.L., 2005. Cadmium toxicity in plants. Braz. J. Plant Physiol. 17, 21–34.

Chaneva, G., Parvanova, P., Tzvetkova, N., Uzunova, A., 2010. Photosynthetic response of maize plants against cadmium and paraquat impact. Water Air Soil Pollut. 208, 287–293. Da Cunha, K.P.V., do Nascimento, C.W.A., 2009. Silicon effects on metal tolerance and structural changes in maize (Zea mays L.) gown on a cadmium and zinc enriched soil. Water Air Soil Pollut. 197, 323–330. Da Cunha, K.P.V., do Nascimento, C.W.A., Silva, A.J., 2008. Silicon alleviates the toxicity of cadmium and zinc for maize (Zea mays L.) grown on a contaminated soil. J. Plant Soil Sci. 171, 849–853. Dalla Vecchia, F., La Rocca, N., Moro, I., De Faveri, S., Andreoli, C., Rascio, N., 2005. Morphogenetic, ultrastructural and physiological damages suffered by submerged leaves of Elodea canadenesis exposed to cadmium. Plant Sci. 168, 329–338. Da Silva, A.J., do Nascimento, C.W.A., Gouveia-Neto, A.D., da Silva, E.A., 2012. LEDinduced chlorophyll fluorescence spectral analysis for the early detection and monitoring of cadmium toxicity in maize plants. Water Air Soil Pollut. 223, 3527–3533. Doncheva, Sn, Poschenrieder, C., Stoyanova, Z., Georgieva, K., Velichkova, M., Barceló, J., 2009. Silicon amelioration of manganese toxicity in Mn-sensitive and Mn-tolerant maize varieties. Environ. Exp. Bot. 65, 189–197. Ekmekçi, Y., Tanyolaç, D., Ayhan, B., 2008. Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars. J. Plant Physiol. 165, 600–611. Eraslan, F., Inal, A., Pilbeam, D.J., Gunes, A., 2008. Interactive effects of salicylic acid and silicon on oxidative damage and antioxidant activity in spinach (Spinacia oleracea L. cv. Matador) grown under boron toxicity and salinity. Plant Growth Regul. 55, 207–219. Farooq, M.A., Ali, S., Hameed, A., Ishaque, W., Mahmood, K., Iqbal, Z., 2013. Alleviation of cadmium toxicity by silicon is related to elevated photosynthesis, antioxidant enzymes; suppressed cadmium uptake and oxidative stress in cotton. Ecotoxicol. Environ. Saf. 96, 242–249. Feng, J., Shi, Q., Wang, X., Wei, M., Yang, F., Xu, H., 2010. Silicon supplementation ameliorated the inhibition of photosynthesis and nitrate metabolism by cadmium (Cd) toxicity in Cucumis sativus L. Sci. Hortic. 123, 521–530. Fleck, A.T., Mattusch, J., Schenk, M.K., 2013. Silicon decreases the arsenic level in rice grain by limiting arsenite transport. J. Plant Nutr. Soil Sci. 176, 785–794. Guntzer, F., Keller, C., Meunier, J.-D., 2012. Benefits of plant silicon for crops: a review. Agron. Sustain. Dev. 32, 201–213. Hasan, S.A., Fariduddin, Q., Ali, B., Hayat, S., Ahmad, A., 2009. Cadmium: toxicity and tolerance in plants. J. Environ. Biol. 30, 165–174. Hashemi, A., Abdolzadeh, A., Sadeghipour, H.R., 2010. Beneficial effects of silicon nutrition in alleviating salinity stress in hydroponically grown canola, Brassica napus L., plants. Soil Sci. Plant Nutr. 56, 244–253. Hattori, T., Inanaga, S., Araki, H., An, P., Morita, S., Luxová, M., Lux, A., 2005. Application of silicon enhanced drought tolerance in Sorghum bicolor (L.) Moench. Physiol. Plant 123, 459–466. Hoagland, D.R., Arnon, D.I., 1950. The Water-culture Method for Growing Plants Without Soil. Agricultural Experiment Station, Circular, Berkeley, CA: California, p. 347. Huang, Y.Z., Zhang, W.Q., Zhao, L.J., 2012. Silicon enhances resistance to antimony toxicity in the low-silica rice mutant, lsi1. Chem. Ecol. 28, 341–354. Krantev, A., Yordanova, R., Janda, T., Szalai, G., Popova, L., 2008. Treatment with salicylic acid decreases the effect of cadmium on photosynthesis in maize plants. J. Plant Physiol. 165, 920–931. Lagriffoul, A., Mocquot, B., Mench, M., Vangronsveld, J., 1998. Cadmium toxicity effects on growth, mineral and chlorophyll contents, and activities of stress related enzymes in young maize plants (Zea mays L.). Plant Soil 200, 241–250. Lee, S.K., Sohn, E.Y., Hamayun, M., Yoon, J.Y., Lee, I.J., 2010. Effect of silicon on growth and salinity stress of soybean plant grown under hydroponic system. Agrofor. Syst. 80, 333–340. Levent Tuna, A.L., Kaya, C., Higgs, D., Murillo-Amador, B., Aydemir, S., Girgin, A.R., 2008. Silicon improves salinity tolerance in wheat plants. Environ. Exp. Bot. 62, 10–16. Li, L., Zheng Ch, Fu. Y., Wu, D., Yang, X., Shen, H., 2012. Silicate-mediated alleviation of Pb toxicity in banana grown in Pb-contaminated soil. Biol. Trace Elem. Res. 145, 101–108. Liang, Y., Sun, W., Zhu, Y.G., Christie, P., 2007. Mechanisms of silicon-mediated alleviation of abiotic stresses in higher plants: a review. Environ. Pollut. 147, 422–428. Liang, Y., Wong, J.W.C., Wei, L., 2005. Silicon-mediated enhancement of cadmium tolerance in maize (Zea mays L.) grown in cadmium contaminated soil. Chemosphere 58, 475–483. Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11, 591–592. Liu, J., Ma, J., He, C., Li, X., Zhang, W., Xu, F., Lin, Y., Wang, L., 2013a. Inhibition of cadmium ion uptake in rice (Oryza sativa) cells by a wall-bound form of silicon. New Phytol. 200, 691–699. Liu, J., Zhang, H., Zhang, Y., Chai, T., 2013b. Silicon attenuates cadmium toxicity in Solanum nigrum L. by reducing cadmium uptake and oxidative stress. Plant Physiol. Biochem. 68, 1–7. Lukačová, Z., Švubová, R., Kohanová, J., Lux, A., 2013. Silicon mitigates the Cd toxicity in maize in relation to cadmium translocation, cell distribution, antioxidant enzymes stimulation and enhanced endodermal apoplasmic barrier development. Plant Growth Regul. 70, 89–103.

M. Vaculík et al. / Ecotoxicology and Environmental Safety 120 (2015) 66–73

Lux, A., 1981. Rapid method for staining of semithin section from plant material. Biol. Bratisl. 36, 753–757. Lux, A., Martinka, M., Vaculík, M., White, P.J., 2011. Root responses to cadmium in the rhizosphere: a review. J. Exp. Bot. 62, 21–37. Ma, J.F., 2004. Role of silicon in enhancing the resistance of plants to biotic and abiotic stresses. Soil Sci. Plant Nutr. 50, 11–18. Martinka, M., Vaculík, M., Lux, A., 2014. Plant cell responses to cadmium and zinc. In: Nick, P., Opatrný, Z. (Eds.), Applied Plant Cell Biology. Plant Cell Monographs 22. Springer, Berlin, pp. 209–246. Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence – a practical guide. J. Exp. Bot. 51, 659–668. McCarthy, I., Romero-Puertas, M.C., Palma, J.M., Sandalio, L.M., Corpas, F.J., Gómez, M., Del Río, L.A., 2001. Cadmium induced senescence symptoms in leaf peroxisomes of pea plants. Plant Cell Environ. 24, 1065–1073. Mihaličová Malčovská, S., Dučaiová, Z., Maslaňáková, I., Bačkor, M., 2014. Effect of silicon on growth, photosynthesis, oxidative status and phenolic compounds in maize (Zea mays L.) grown in cadmium excess. Water Air Soil Pollut. 225, 2056. Nwugo, Ch.C., Huerta, A.J., 2008. Effects of silicon nutrition on cadmium uptake, growth and photosynthesis of rice exposed to low-level cadmium. Plant Soil 311, 73–86. Pal, M., Horvath, E., Janda, T., Paldi, E., Szalai, G., 2006. Physiological changes and defense mechanisms induced by cadmium stress in maize. J. Plant Nutr. Soil Sci. – Z. Pflanzenernähr. Bodenkd. 169, 239–246. Pavlovic, J., Samardzic, J., Maksimovic, V., Timotijevic, G., Stevic, N., Laursen, K.H., Hansen, T.H., Husted, S., Schjoerring, J.K., Liang, Y., Nikolic, M., 2013. Silicon alleviates iron deficiency in cucumber by promoting mobilization of iron in the root apoplast. New Phytol. 198, 1096–1107. Pragabar, S., Hodson, M.J., Evans, D.E., 2011. Silicon amelioration of alumunium toxicity and cell death in suspension cultures of Norway spruce (Picea abies (L.) Karst.). Environ. Exp. Bot. 70, 266–276. Roháček, K., 2002. Chlorophyll fluorescence parameters: the definitions, photosynthetic meaning, and mutual relationships. Photosynthetica 40, 13–29. Savvas, D., Giotis, D., Chatzieustratiou, E., Bakea, M., Patakioutas, G., 2009. Silicon supply in soilles cultivation of zucchini alleviates stress induced by salinity and powdery mildew infections. Environ. Exp. Bot. 65, 11–17. Shen, X., Li, X., Li, Z., Li, J., Duan, L., Eneji, A.E., 2010a. Growth, physiological attributes and antioxidant enzyme activities in soybean seedlings treated with or without silicon under UV-B radiation stress. J. Agric. Crop Sci. 196, 431–439. Shen, X., Zhou, Y., Duan, L., Li, Z., Eneji, E., Li, J., 2010b. Silicon effects on photosynthesis and antioxidant parameters of soybean seedlings under drought and ultraviolet radiation. J. Plant Physiol. 167, 1248–1252. Solymosi, K., Bertrand, M., 2012. Soil metals, chloroplasts, and secure crop production: a review. Agron. Sustain. Dev. 32, 245–272.

73

Song, A., Li, Z., Zhang, J., Xue, G., Fan, F., Liang, Y., 2009. Si-enhanced resistance to cadmium toxicity in Brassica chinensis L. is attributed to Si-suppressed cadmium uptake and transport and Si-mediated antioxidant defence capacity. J. Hazard. Mat. 172, 74–83. Tale Ahmad, S., Haddad, R., 2011. Study of silicon effects on antioxidant enzyme activities and osmotic adjustment of wheat under drought stress. Czech J. Genet. Plant 47, 17–27. Tkalec, M., Prebeg, T., Roje, V., Pevalak-Kozlina, B., Ljubesic, N., 2008. Cadmiuminduced responses in duckweed Lemna minor L. Acta Physiol. Plant 30, 881–890. Tripathi, P., Tripathi, R.D., Singh, R.P., Dwivedi, S., Goutam, D., Shri, M., Trivedi, P.K., Chakrabarty, D., 2013. Silicon mediates arsenic tolerance in rice (Oryza sativa L.) through lowering of arsenic uptake and improved antioxidant defence system. Ecol. Eng. 52, 96–103. Vaculík, M., Landberg, T., Greger, M., Luxová, M., Stoláriková, M., Lux, A., 2012. Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants. Ann. Bot. 110, 433–443. Vaculík, M., Lux, A., Luxová, M., Tanimoto, E., Lichtscheidl, I., 2009. Silicon mitigates cadmium inhibitory effects in young maize plants. Environ. Exp. Bot. 67, 52–58. Vaculíková, M., Vaculík, M., Šimková, L., Fialová, I., Kochanová, Z., Sedláková, B., Luxová, M., 2014. Influence of silicon on maize roots exposed to antimony – Growth and antioxidative response. Plant Physiol. Biochem. 83, 279–284. Vatehová, Z., Kollárová, K., Zelko, I., Richterová-Kučerová, D., Bujdoš, M., Lišková, D., 2012. Interaction of silicon and cadmium in Brassica juncea and Brassica napus. Biologia 67, 498–504. Vittória, A.P., Lea, P.J., Azevedo, R.A., 2001. Antioxidant enzymes responses to cadmium in radish tissues. Phytochemistry 57, 701–710. Wang, H., Zhao, S.C., Liu, R.L., Zhou, W., Jin, J.Y., 2009. Changes of photosynthetic activities of maize (Zea mays L.) seedlings in response to cadmium stress. Photosynthetica 47, 277–283. Wiese, H., Nikolic, M., Römheld, V., 2007. Silicon in plant nutrition. In: Sattelmacher, B., Horst, W.J. (Eds.), The Apoplast of Higher Plants: Compartment of Storage, Transport and Reactions. Springer, pp. 33–47. Wu, J.W., Shi, Y., Zhu, Y.X., Wang, Y.C., Gong, H.J., 2013. Mechanisms of enhanced heavy metal tolerance in plants by silicon: a review. Pedosphere 23, 815–825. Zhang, Q., Ch, Yan, Liu, J., Lu, H., Wang, W., Du, J., Duan, H., 2013. Silicon alleviates cadmium toxicity in Avicennia marina (Forsk.) Vierh. seedlings in relation to root anatomy and radial oxygen loss. Mar. Pollut. Bull. 76, 187–193. Zuccarini, P., 2008. Effects of silicon on photosynthesis, water relations and nutrient uptake of Phaseolus vulgaris under NaCl stress. Biol. Plant 52, 157–160.