Overall plant responses to Cd and Pb metal stress in

Overall plant responses to Cd and Pb metal stress in maize: Growth pattern, ultrastructure, and photosynthetic activity Francesca Figlioli, Maria Cristina Sorrentino, Valeria Memoli, Carmen Arena, Giulia Maisto, Simonetta Giordano, Fiore Capozzi, et al. Environmental Science and Pollution Research ISSN 0944-1344 Environ Sci Pollut Res DOI 10.1007/s11356-018-3743-y

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Author's personal copy Environmental Science and Pollution Research https://doi.org/10.1007/s11356-018-3743-y

RESEARCH ARTICLE

Overall plant responses to Cd and Pb metal stress in maize: Growth pattern, ultrastructure, and photosynthetic activity Francesca Figlioli 1 & Maria Cristina Sorrentino 1 & Valeria Memoli 1 & Carmen Arena 1 & Giulia Maisto 1 & Simonetta Giordano 1 & Fiore Capozzi 1 & Valeria Spagnuolo 1 Received: 10 July 2018 / Accepted: 12 November 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract This study provides a full description of the responses of the crop energy plant Zea mays to stress induced by Cd and Pb, in view of a possible extensive use in phytoattenuation of metal-polluted soils. In this perspective, (i) the uptake capability in root and shoot, (ii) the changes in growth pattern and cytological traits, and (iii) the photosynthetic efficiency based on photochemistry and the level of key proteins were investigated in hydroponic cultures. Both metals were uptaken by maize, with a translocation factor higher for Cd than Pb, but only Cd-treated plants showed a reduced growth compared to control (i.e., a lower leaf number and a reduced plant height), with a biomass loss up to 40%, at the highest concentration of metal (10−3 M). The observation of cytological traits highlighted ultrastructural damages in the chloroplasts of Cd-treated plants. A decline of Rubisco and D1 was observed in plants under Cd stress, while a relevant increase of the same proteins was found in Pb-treated plants, along with an increase of chlorophyll content. Fluorescent emission measurements indicated that both metals induced an increase of NPQ, but only Cd at the highest concentration determined a significant decline of Fv/Fm. These results indicate a different response of Z. mays to individual metals, with Pb triggering a compensative response and Cd inducing severe morphophysiological alterations at all investigated levels. Therefore, Z. mays could be successfully exploited in phytoattenuation of Pb-polluted soil, but only at very low concentrations of Cd to avoid severe plant damages and biomass loss. Keywords Chloroplast ultrastructure . Heavy metals . Photochemistry . Chlorophylls . Zea mays

Introduction Land use activities have transformed at a great extent planet surface, leading to an alarming decrease of non-renewable resources, including soil. Intensive agriculture and grazing, increasing farmland production, and expansion of urbanized areas implicate soil loss (Foley et al. 2005). In recent decades, industrial activities are playing a relevant role in soil loss; in fact, industrialized countries, despite the introduction of restrictive regulations and technological innovations, produce wastes increasing soil pollution by the release of potentially toxic elements all over the world (Fiorentino et al. 2016). Responsible editor: Elena Maestri * Fiore Capozzi [email protected] 1

Dipartimento di Biologia, Università degli Studi di Napoli Federico II, via Cinthia 4, 80126 Naples, Italy

Phytoremediation is an efficient, eco-friendly method, consisting of a suite of agricultural techniques aimed to clean up and restore contaminated soils by using plants and associated microbes, in order to reduce the concentration of the pollutants, or their diffusion in the environment (McIntyre 2003). Phytoremediation is particularly efficient for soils affected by low and diffuse level of contamination. It is receiving increasing attention because of the interest of the public opinion for green technologies, largely preferred to less conservative approaches; moreover, physical-chemical methods aimed at soil cleaning have higher costs and often give back poor soils, with altered properties, generally unsuitable for agriculture. Phytoremediation is specially used to remove metal contamination (Ali et al. 2013; Fiorentino et al. 2016; HernándezAllica et al. 2007; Llugany et al. 2012; Papazoglou 2011; Sorrentino et al. 2018). Soil pollution by heavy metals (HMs) is becoming a complex and challenging problem, since HMs cannot be degraded and can interfere with many metabolic processes, inactivating key enzymes. Cadmium (Cd) and

Author's personal copy Environ Sci Pollut Res

lead (Pb) are among the most hazardous non-nutrient, and their contamination results from many human activities including soil-applied chemicals, such as fertilizers (Alloway 1995; Sanità di Toppi and Gabbrielli 1999). Cadmium, released to the environment by several industrial and agricultural activities, is toxic for humans, animals, and plants even at low concentration (Jackson and Alloway 1992), although the mechanisms of its toxicity are not yet completely understood (Suzuki et al. 2001). In higher plants, Cd toxicity is usually accompanied by oxidative stress (Romero-Puertas et al. 1999; Dixit et al. 2001; León et al. 2002; Boominathan and Doran 2003); in fact, Cd causes transient depletion of glutathione and inhibition of antioxidative enzymes giving rise to H2O2 accumulation in the cell (Paradiso et al. 2008). Besides, in the absence of a prompt detoxification, it may trigger growth inhibition, stimulation of secondary metabolism, lignification, and finally cell death (Schützendübel and Polle 2002). Lead is present in nature only in small amount, but human activities have contributed to increase its levels worldwide. Lead is present in most soils and rocks at concentrations up to 50 mg kg−1 (Holmgren et al. 1993; Zitka et al. 2013) and generally shows relatively low mobility in soils and in vegetation, where typically reaches concentration within 10 mg kg−1 (Zitka et al. 2013). When Pb enters the plant roots from Pb-enriched soils, it shows small translocation to the aerial parts; nonetheless, increased concentrations of Pb in aboveground tissues can be caused by entering of metalbound dust and fine soil particles, or directly to leaves through stomata (Mojiri 2011). Lead affects different morphophysiological traits and biochemical functions: it inhibits root growth (Godbold and Kettner 1991; Gzyl et al. 1997) and photosynthetic pigment synthesis, it disturbs membrane permeability and the mineral nutrition balance by causing deficiencies or altered ion distribution within plant cells, influencing the catalytic activity of many enzymes (Trivedi and Erdei 1992). High biomass yield, root to shoot metal translocation, and high tolerance to metals are among the plant features valuable for phytoremediation. The main drawback of phytoextraction is the long-required remediation time, during which the treated soil remains unproductive. Although maize, similarly to other cereals like barley, is not considered a hyperaccumulator (Sarwar et al. 2017), the possibility to valorize its biomass for alternative uses (e.g., energy production, bioplastics) has increased maize culture on metal-polluted soils, unsuitable for agriculture. This practice, recently called phytoattenuation (Meers et al. 2010), provides the advantage of balancing the long required remediation time, by producing, in the while, goods other than typical agricultural products (food and feed). In addition, the use of this robust widespread crop in phytoextraction applied on soils with a limited metal pollution has relatively low costs, often preserving seed quality, due to

the scarce translocation observed in this plant (Meers et al. 2005; Wuana and Okieimen 2011). While metal uptake in maize was studied in deep (Ali et al. 2002; Chiu et al. 2005; Do Nascimento and Xing 2006; Lin et al. 2008; Poniedziałek et al. 2010; Mojiri 2011; Moosavi and Seghatoleslami 2013; Aliyu and Adamu 2014; Koptsik 2014), scarce information exists on morpho-physiological responses observed in maize under metal stress. However, these responses could influence biomass production and growth, compromising the efficacy of plant material for alternative uses. The present work is intended to cover this crucial gap; therefore, the aim of this study is to provide a full description of Z. mays response to stress induced by Cd and Pb experimentally supplied, by an integrated methodological approach, evaluating (i) the plant uptake capability in root and shoot, (ii) the changes in growth pattern and cytological traits, and (iii) the photosynthetic efficiency by measurements of photochemistry and photosynthetic key proteins.

Materials and methods Plant material and growth conditions Seeds of Z. mays DC were germinated to primary roots on wet filter paper at 25 °C in the dark until the development of plantlets. Plants were grown in a greenhouse under semicontrolled conditions as described in Arena et al. (2017a). Plants were watered every 2 days with Murashige-Skoog 1:2 liquid medium (Sigma Life Science) supplied with CdCl2 or Pb(NO3)2 at concentrations of 10−5, 10−4, and 10−3 M. For all the analyses, plant samples were harvested and processed after 35 days of culture.

Evaluation of Cd- and Pb-induced effects on plant growth At the end of the growth period (35 days), some morphological traits were evaluated to check the status of the plants and to determine different effects induced by exposure to heavy metals. The following growth parameters were examined: plant height and number of leaves, fresh weight, and tolerance index (TI, Amin et al. 2014). Each observation refers to 5 samples, if not specified otherwise.

Chemical analysis To determine the total concentration of the metals and their site of accumulation in the plant, we processed the plants treated with the highest metal concentration; the 10−3 M treated samples for both metals were separated in leaf and root, and treated and analyzed as described in Arena et al. (2017a).


Blanks and standard reference material (CTA-OTL 1, tobacco leaves) were also analyzed to check possible contamination, accuracy, and precision. The measurements were performed 3 times for each sample and the average values of accumulation and standard deviation were calculated. It was calculated also the translocation factor (TF) (Yoon et al. 2006), and assuming that the metals supplied in solution were 100% exchangeable, the exchangeable transfer factor (TFExc) (Esringü et al. 2014) as: [Metal concentration (plant shoot + root)/Exchangeable metal concentration in the soil at harvest time].

TEM observations Observations by electron microscopy were performed by a TEM Philips EM 208S on controls and samples treated with the three different concentrations of Cd and Pb (10−5 M, 10−4 M, and 10−3 M), at the 35th day of growth, following Sorrentino et al. (2018).

Pigment concentrations Chlorophylls and carotenoids were extracted from n = 5 leaves per treatment and determined following the procedure reported by Lichtenthaler (1987). Chlorophyll a, b and carotenoids were quantified by spectrophotometer (Cary 100 UVVIS, Agilent Technologies) at 662-, 630-, and 470-nm wavelengths.

Protein extraction and western blot analysis Protein extraction from leaves was carried out on 35-day-old plants according to Wang et al. (2006) and Bertolde et al. (2014) using 0.3 g of plant material for each sample. The procedure used for this analysis is described in Sorrentino et al. (2018). Primary antibodies (Agrisera) were used to reveal different proteins: Rubisco (anti-RbcL, rabbit polyclonal serum), D1 (anti-PsbA, hen polyclonal), and Actin (anti-ACT, rabbit polyclonal serum) as loading control. The immunorevelation was carried out by a chemiluminescence kit (Westar Supernova, Cyanagen) by ChemiDoc System (Bio-Rad). Densitometry analysis was made using the software ImageJ (Rasband, NIH) by normalizing each band to the corresponding actin band, and results were expressed as percentages of the control set to 100%.

Statistical analysis All data were processed using Microsoft Excel and STATISTICA ver. 8.0 (StatSoft, Inc. 2008). The normality and homogeneity of the variances of our datasets were assessed by the Shapiro–Wilk test and Levene’s test, respectively. The effects induced by the different treatments were statistically compared with one-way ANOVAs. In case of rejection of the null hypothesis, Tukey’s multiple pairwise comparison test was performed with a p < 0.05.

Results and discussion Fluorescence measurements Growth parameters Chlorophyll a fluorescence was determined on 35-day-old plants using a portable fluorometer equipped with a light sensor (FluorPen FP 100-MAX-LM, Photon System Instruments, Czech Republic) on full-expanded leaves of Z. mays DC. The ground fluorescence (Fo) was induced by an internal LED blue light (1–2 μmol photons m−2 s−1) on dark-adapted leaves. The maximal fluorescence in the dark (Fm) was induced by 1s saturating pulse of 3.000 μmol photons m−2 s−1. The maximal PSII photochemical efficiency (Fv/Fm) was calculated as the ratio of variable fluorescence to maximal fluorescence, where Fv is the difference between maximal and ground fluorescence (Fm − Fo). The measurements in the light were carried out in the morning under greenhouse at values of photosynthetic photon flux density (PPFD) ranging from 240 to 260 μmol photons m−2 s−1 at canopy level. The PSII quantum yield (QY) was determined by means of an open leaf-clip suitable for measurements under ambient light, according to Genty et al. (1989). QY was used to calculate the linear electron transport rate (ETR), according to Krall and Edwards (1992). Non-photochemical quenching (NPQ) was calculated following Bilger and Bjorkman (1990).

All growth parameters here considered are illustrated in Fig. 1 and Table 1. All treated plants (concentration from 10−5 to 10−3 M) had a number of leaves comparable to control plants (7–8 leaves per plant), except those treated with Cd 10−4 and 10−3 M, which had on average 6 leaves per plant (Fig. 1a). Lead-treated plants were significantly higher than control, but without any clear dose-dependent pattern, whereas Cd-treated plants were significantly shorter than control at higher concentrations (10−4 and 10−3 M), suggesting an opposite effect of Pb and Cd on growth pattern, with the first enhancing plant elongation (Fig. 1b). As for biomass production and tolerance index (Table 1), Pb 10−4 M increased plant weight of a good 30% (TI 1.29); by contrast, Cd decreased plant weight of 20 and 40% at concentrations of 10−4 and 10−3 M, respectively. Our data agree with previous literature reports on barley (Lentini et al. 2018), rice (Yu et al. 2006), and maize plants (Krantev et al. 2008) treated with Cd; a reduction in the weight, as well as in shoot and root length, was observed in these plants under Cd stress. These results suggest that


literature data on Z. mays (Huang et al. 1997). However, a different behavior was observed for the two metals: specifically, about 15% of the total Pb and over 30% of Cd were translocated to shoots (Table 2). In treated plants, the exchangeable translocation factor (TFexc) was also higher for Cd than Pb. These results agree with previous literature data (Alloway 1995; Chaney and Giordano 1977) indicating a high translocation for Cd and low for other elements, such as Cr, Hg, and Pb. In a previous paper (Arena et al. 2017a), higher accumulation and TF exc for both Cd and Pb were found in Cynara cardunculus; differences in plant uptake ability (cardoon is a hyper-accumulator), in metal concentrations (100 times higher in the present experiment), and bioavailability could explain the differences observed. The lower uptake and translocation ability of Z. mays compared to other species was already proved; Meers et al. (2005) found, indeed, that maize accumulated concentrations of Cd, Cu, Ni, Pb, and Zn lower than Brassica rapa L., Cannabis sativa L., and Helianthus annuus L., probably because maize has by far the highest biomass productions; these characteristics make this plant very tolerant to metal stress, and therefore similarly suited for phytoextraction and phytoattenuation despite the lower accumulation ability (Meers et al. 2005).

TEM observation Fig. 1 Number of leaves (a) and height of the plants (b), expressed as mean values (n = 5) and SE for the control and treated plants. Significant differences (according to one-way ANOVA and Tukey’s test with p < 0.05) among treatments are marked with different letters. Dark gray bars for Pb; light gray bars for Cd; white bar for control

phytoattenuation by maize could be an optimal choice for Pbpolluted soils, since Pb does not affect the growth pattern in a negative manner; by contrast, only low Cd contamination seems compatible with phytoattenuation by Z. mays. Although growth parameters were not investigated in previous studies to evaluate the efficiency of candidate species in phytoextraction or phytoattenuation, they could provide useful information for their selection; particularly, tolerance index (i.e., the ratio between the weight of treated and control plants), already used to estimate plant growth pattern under pollution condition in Glycine max (Amin et al. 2014,) and Boehmeria nivea L. (Chai et al. 2016) could give an estimation of biomass production in plants grown on polluted soil.

Chemical analysis In maize control plants, Cd and Pb concentrations were below detection limits. Cadmium and Pb mean concentrations measured in CTA-OTL1 standard showed an adequate recover for both metals, between 85 and 110%. The chemical analyses of Cd and Pb showed greater accumulation of both metals in the roots than in the shoots (Table 2), in agreement with the

Leaf control samples showed well-preserved cell traits with typical organelles as chloroplasts with some starch grains and well-packed grana (Fig. 2a, b). Chloroplasts of all Pb-treated samples had no morphological alterations, or damages to thylakoid organization; this is likely due to the limited translocation of this metal to the aerial parts (e.g., Alloway 1995; Mellem et al. 2009). However, a greater accumulation of starch granules appeared in the samples submitted to Pb treatment, in the bundle sheath cells (Fig. 2c). This increase is indicative of a maintained photosynthetic efficiency and Rubisco’s functionality. Accordingly, the analyses of the growth parameters showed a development of the plants exposed to Pb comparable to that observed in control plants (see Fig. 1a, b). Table 1 Mean value ± SE (n = 5) of plant weight and tolerance index for each treatment Treatments

Plant weight (g)

TI

Ctrl Pb 10−5 M Pb 10−4 M Pb 10−3 M Cd 10−5 M Cd 10−4 M Cd 10−3 M

37.5 39.7 48.3 40.0 41.8 30.9 21.6

1.0 1.1 1.3 1.1 1.1 0.8 0.6

± ± ± ± ± ± ±

5.0 3.3 1.4 2.5 1.9 4.3 1.5

Author's personal copy Environ Sci Pollut Res Table 2 Mean ± SE (n = 5) of Cd and Pb concentrations (μg g−1 d.w.) in the roots and shoots of Z. mays at 35 days, translocation factor (TF) and exchangeable transfer factor (TF exc)

Element

Metal provided (μg g−1 d.w. soil)

Root

Shoot

Control

Pb

0

50.6 ± 4.6

0.67 ± 0.29

Pb_treatment

Cd Pb

0 3200

0.96 ± 0.13 182.3 ± 9.9

Cd_treatment

Cd Cd

0 1800

Pb

0

In contrast, Cd-treated samples exhibited a noticeable state of stress, at 10−4 and 10−3 M, with altered chloroplast shape, poorly differentiated grana, sometimes with swollen Fig. 2 TEM microphotographs of leaves. Control samples showing a chloroplast in a mesophyll cell (a) and bundle sheath cells (b). c Bundle sheath cells with great accumulation of starch granules (upper left) and mesophyll cells (bottom right) both showing unaltered cytoplasm traits; plant treated with Pb 10−4 M. d Chloroplasts in mesophyll and bundle sheath cells; plant treated with Cd 10−4 M. e - Altered chloroplast shape, poorly differentiated grana in a bundle sheath cell; plant treated with Cd 10−3 M. f- Lobed chloroplast in a bundle sheath cell with poorly differentiated grana and swollen thylakoids; plant treated with Cd 10−3 M. (bs, bundle sheath cell; m, mesophyll cell)

TF

TF exc

0.14 ± 0.06 25.8 ± 4.4

15%

6.50%

1.34 ± 0.11 110.7 ± 13.1

0.31 ± 0.04 36.7 ± 9.9

33%

8.20%

3.33 ± 0.20

2.3 ± 0.5

thylakoids and a reduced number of starch granules (Fig. 2d, e, and f). However, well-developed grana were generally observed in mesophyll cells (Fig. 2d, e) even at


the highest Cd concentration (10−3 M). Given the limited exposure duration to Cd (35 days), the cells mostly affected by the metal-containing solution are those surrounding the vascular tissue, i.e., the bundle sheath cells. These results agree with a previous work carried out in Cynara cardunculus plants (Arena et al. 2017a), where Cd induced more severe ultrastructural damages than Pb. It is widely known that, as a result of translocation, metals reach the plant shoot where they affect the integrity of the photosynthetic apparatus by damaging the structure and functionality of the thylakoid membranes, as well as the capacity of lightharvesting complexes (Milone et al. 2003; Ciscato et al. 1997); particularly, Cd may also accumulate in chloroplasts impairing important plant physiological processes such as gas exchanges, water uptake (Van Assche and Clijsters 1990), and light reactions of photosynthesis (Ernst 1980). Ultrastructural observations are also consistent with the results obtained from photochemical analyses: indeed, Cd visibly damaged the plant, altering photosynthetic activity (see paragraphs below).

Pigment concentration In general, chlorophyll content was not negatively affected by the metals, as observed also in the other C4 plant Atriplex halimus (Manousaki and Kalogeraki 2009). Lead-treated samples (at concentration of 10−3 M) showed a significant increase of Chl a and b compared to control. Conversely, no significant difference was detected for Pb−5 and Pb−4 M (Table 3). A different behavior was observed for plants treated with Cd. More specifically, a significant increase was found only in Cd−5 M samples compared to control and only for Chl a. The ratio Chl a/b did not show substantial changes among treatments for both Cd and Pb; this result is consistent with that observed in cardoon grown on a metal-polluted soil enriched in Cd and Pb (Sorrentino et al. 2018) and indicates that Chl a and Chl b kept in general a similar trend. Table 3 Mean ± SE (n = 5) of chlorophyll a (Chl a) and b (Chl b) concentrations and chlorophyll a/b ratios in Z. mais leaves treated with different Cd and Pb concentrations. Different letters indicate statistical significant differences among metal treatments according to Tukey’s multiple comparison tests (at least p < 0.05) Treatment

Chl a (μg cm−2)

Control Cd 10−3 M Cd 10−4 M Cd 10−5 M Pb 10−3 M Pb 10−4 M Pb 10−5 M

18.38 17.68 16.31 23.13 27.59 19.50 20.88

± ± ± ± ± ± ±

0.32b 0.29b 0.24b 0.31a 0.35a 0.18b 0.27b

Chl b (μg cm−2) 5.96 6.67 5.14 6.90 8.41 5.22 6.07

± ± ± ± ± ± ±

0.19b 0.22b 0.21b 0.36b 0.23a 0.41b 0.31b

Chl a/Chl b 3.10 2.66 3.18 3.35 3.29 3.77 3.43

± ± ± ± ± ± ±

0.19 0.15 0.10 0.09 0.14 0.19 0.20

Nonetheless, maize leaves showed a different sensitivity to Pb and Cd. Both the lowest concentration of Cd (10−5 M) and the highest concentration of Pb (10−3 M) seemed to stimulate the synthesis of photosynthetic pigments. This effect, in the case of Pb 10−3 M, may be interpreted as a way to compensate for reduced photochemistry, as indicated by the photochemical indexes QY and ETR.

Chlorophyll fluorescence parameters Significant differences in photochemical behavior were evidenced at the highest concentration of both Cd and Pb, 10−3 M (Fig. 3). At these concentrations, in Cd-contaminated leaves, a significant decrease of QY, ETR was found compared to control. At the same time, a significant increase of NPQ was also observed for both Pb and Cd. These data suggest that photosynthetic apparatus is affected by elevated amount of both metals; however, the induced perturbations are more pronounced for Cd than Pb as evidenced also by the significant decrease of Fv/Fm in Cd 10−3 M leaves compared to control. These results are in agreement with Balakhnina et al. (2005) who found a significant decrease of Fv/Fm due to Cd toxicity. Differently from Cd-treated samples, in Pb 10−3 M treated leaves, the Fv/Fm ratio values were similar to control. The reason of the different photochemical behavior is likely due to the diverse mobility and bioavailability of these two metals in plant tissue, being these two properties higher for Cd than Pb (Arena et al. 2017a). The exposure of plants to higher concentrations of Cd caused a significant increase in thermal dissipation processes, more than with Pb, as indicated by the increase of the NPQ. The rise of non-photochemical quenching together with photochemical efficiency reduction may be interpreted as a safety mean to avoid the occurrence of irreversible damage to photosystems when photochemical reactions and/or carbon fixation are impaired (Müller et al. 2001; Lambrev et al. 2012; Arena et al. 2014, 2017b). However, it is noteworthy that both metals induced a significant increase in NPQ compared to leaves from uncontaminated medium, indicating the nonphotochemical dissipation processes as a fundamental mean in counteracting the damage risk to photosystems. These results are in agreement with previous studies in which has been demonstrated that Pb determine a decrease in ΦPSII in parallel with NPQ rise (Romanowska et al. 2006).

Western blot analysis of important photosynthetic enzymes A severe reduction in the level of RuBP and D1 was observed in Cd-treated maize samples, while both enzymes increased their concentration in Pb-treated ones (Fig. 4). Protein pattern observed in maize was consistent with plant growth pattern and chloroplast ultrastructure observed in Cd- and Pb-treated


b)

a) b

b

b

b

c

c

c)

d)

Fig. 3 Photosystem II quantum yield (QY) (a), electron transport rate (ETR), maximal photochemical efficiency (Fv /Fm ) (c), and nonphotochemical quenching (NPQ) (d) in control (Ctrl) and Pb- and Cd-

treated plants of Zea mays L. Each value represents the mean ± SE; n = 5. Different letters indicate significant differences among treatments at p < 0.05. Dark gray bars for Pb; light gray bars for Cd

samples. These latter showed higher biomass production, well-preserved ultrastructure, higher number of starch granules in the chloroplasts, and higher levels of D1 and Rubisco, all parameters indicating a multiple positive response of the maize under Pb stress; this could depend on the low amount of Pb reaching the leaves (Table 2). Cadmium instead, translocating for a noticeable fraction (33%, Table 2), induced

a detrimental growth pattern, leading to severe ultrastructural damage and decrease of Rubisco and D1. The results available in the literature, focused on metal stress and photosynthetic enzyme levels, evidenced two different trends, depending on plant sensitivity or tolerance to a specific metal. In sensitive plants, photosynthetic enzymes decrease in metal-treated samples, whereas c

c a

b

a b

a) Fig. 4 Western blot analysis (a) and densitometric analysis of D1 and Rubisco proteins in Z. mays in control (Ctrl) and treated plants (Cd and Pb 10−4 M). The bar diagrams represent pixel volumes of Rubisco and D1 proteins in samples. The bands were normalized to the relative actin band

b) and expressed as a fraction of the controls set to 1. Different letters indicate statistically significant differences among treatments (p < 0.05). Dark gray bars for Pb; light gray bars for Cd; white bars for control


tolerant species (accumulators) challenge the stress induced by heavy metals increasing enzyme production in roots and leaves (Tamás et al. 2010; Kosova et al. 2011). Nonetheless, our results indicate a different behavior in two tolerant plant species (i.e., maize and cardoon), even treated with the same metal. In fact, an opposite trend was observed in cardoon plants, where Pb decreased photosynthetic proteins and Cd increased them (Arena et al. 2017a). We hypothesized that in cardoon, the slight chloroplast damages induced by Cd 10−5 M could trigger a compensative response in photosynthetic proteins. In maize instead, the severe chloroplast damages, occurred in Cd 10−3 M treated plants, could have determined a parallel decrease of Rubisco and D1 levels. The different protein pattern observed in cardoon and maize under metal stress could be due to the different metal concentrations and mobility, and to the different metal-related metabolic responses of the two tolerant plant species.

Conclusions Zea mays is a highly biomass-producing plant able to accumulate Cd and Pb, especially in root tissue. The higher translocation of Cd to the shoot may explain the ultrastructural damages, especially observed in chloroplasts, induced by Cd. Chloroplast injuries directly affected photosynthesis, inducing a reduction of D1 and Rubisco levels and a significant decrease of Fv/Fm, with evident consequence on growth pattern. Cadmium-treated plants showed indeed significant growth deficits compared to Pb-treated samples, with severe decrease in biomass production. These results suggest a different response of Zea mays to individual metals. In fact, plants seem unable to cope with Cd-induced damages, showing a general decline at all the investigated levels, with loss of biomass up to 40%. By contrast, Pb likely activates acclimatization responses that successfully contribute to plant surviving. In fact, in Pb-treated plants, the absence of morphological and ultrastructural damages, as well as an overexpression of photosynthetic proteins (D1 and Rubisco) was found, along with no effect on photochemical performance, which remains stable compared to control. Overall, our results show a higher sensitivity of Zea mays to Cd than Pb, indicating maize as a suitable species for phytoattenuation of Pb-polluted soils, to be used with caution in the case of soils contaminated by Cd (i.e., to be used only for very low contamination level), to avoid severe plant damages leading to high loss of biomass, making detrimental any phytoattenuation plan.

Acknowledgements We would like to thank Dr. Sergio Sorbo for his helpful collaboration in TEM observations.

Funding information This work was financially supported by the Department of Biology of Federico II University.

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