Introduction

Nowadays, all living organisms, such as plants, must deal with problems arising from urbanization, industry, overuse of phosphate fertilizers, traffic, mining resulting from human activities (Benakova et al. 2017). Such reasons bring about environmental stress by causing heavy metal accumulation in the soil. Heavy metals are composed of transition metals with some metalloids (Tamas et al. 2014; Emamverdian et al. 2015). Some heavy metals such as copper (Cu) are required for plant metabolism at low concentrations and are involved in a number of physiological processes and different metabolic pathways (Zengin and Kirbag 2007). In addition, Cu can reduce the toxic effects of metals by providing metal homeostasis. Other heavy metals, even in essential quantities, cause serious problems for all organisms in the atmosphere, water, and soil, affecting the bioaccumulation in the food chain (Sanita di Toppi and Gabbrielli 1999). For example, chromium (Cr+6) is not required for plant growth and development, but has various toxic effects on plants (Bahadur et al. 2017). Treatment of Cr in rice reduced chlorophyll content and plant growth (Zeng et al. 2012). Cr stress in Brassica napus L. caused reduction of stoma conductance, photosynthetic rate, and intercellular CO2 concentration. In addition, accumulation of heavy metals such as Cr at the cellular level leads to oxidative damage by producing reactive oxygen species (ROS) with malondialdehyde (MDA) formation (Jalmi et al. 2018). Although the amount of ROS, which produce signaling molecules, is less, their amounts under stress conditions are significantly increased. ROS, which have unpaired electrons such as superoxide, are highly reactive and thus damage plant cells at the molecular level. ROS, which occured with Cr stress, reacted with the structures of biomolecules such as DNA, RNA, and protein and adversely affected the vital functions of the cell (Gill et al. 2015). Antioxidant defense systems are intended to balance and maintain the production of ROS formed. This process is controlled by enzymatic [glutathione reductase (GR), superoxide dismutase (SOD), peroxidase (POD), ascorbic peroxidase (APX), catalase (CAT), etc.] and non-enzymatic [ascorbic acid (AsA), glutathione (GSH), etc.] antioxidants. These enzymatic antioxidants play as ROS scavengers. Thus, it protects plant cells from oxidative damage by controlling signaling pathways. AsA, which has high water solubility, has the ability to directly and indirectly reduce ROS. In addition, it acts as a cofactor for enzymes and regulates signal pathways and physiological processes through phytohormones (Aziz et al. 2018). Exogenously applied AsA increases the physiological parameters such as growth and development, ion transport, and photosynthesis rate by increasing the plant’s tolerance against abiotic stress (Akram et al. 2017). For example, in Hibiscus esculentus L. exposed to drought stress, the parameters such as proline content, sugar content, ion transport, protein content, and photosynthetic pigment content were observed to have increased. The plants have produced various strategies in order to cope with these stresses. The most important strategy for heavy metal detoxification at cellular level is the production of heavy metal binding proteins (Hossain and Komatsu 2012). Metallothionein (MT), a stress-responsive protein, binds to heavy metals with cysteine residues in its structure. MT has important roles such as detoxification of heavy metals and metal homeostasis and plays a role in the destruction of intracellular free radicals (Kisa et al. 2017). OsMT2b protein in the rice was found to have ROS clearing activities (Wong et al. 2004). Another strategy that helps to cope with heavy metal stress is the selection of plants that can accumulate these metals. Helianthus annuus L. has been investigated to have heavy metal tolerance and used in this study as it is an ideal plant to be used for phytotechnology in biomass productivity and removal of inorganic and organic pollutants (Rivelli et al. 2012).

Heavy metals stress has adverse effects on plants. The tolerance mechanisms of antioxidant enzymes ensure that plants cope with the stress caused by excessive heavy metal accumulation. In this study, it is aimed to understand the relationship between oxidative damage caused by Cr stress and antioxidant enzymes. For this reason, in this research, expression levels of MT genes and antioxidant enzyme activities (SOD, POD, and CAT) were determined in sunflower exposed to Cr stress. In addition, the protective effects of Cu and AsA on these parameters have been investigated.

Materials and Methods

Plant Materials

The Helianthus annuus L. seeds used in this study were provided by the Department of Field Crops, Faculty of Agriculture, Ataturk University (Erzurum, Turkey).

Growth Conditions and Treatments

Sunflower seeds were surface sterilized respectively with ethanol (70%; 1 min) and sodium hypochlorite (1%; 5 min) and then washed with sterile water. Sterilized seeds were germinated for 2 days and then transferred to a hydroponic system under a 16-h photoperiod at 25 ± 1 °C temperature, with a photon density of 120 μmol photons/m2s. Two weeks after germination [0 (control: distilled water) and 200 mg/L], AsA solution was sprayed to leaves. After 24 h of treatment, heavy metal solution at different doses [0 (control: distilled water); 1 mM Cr (K2Cr2O7); 0.25 mM Cu (CuSO4); 1 mM Cr and 0.25 mM Cu] was added. Sunflower plants were harvested after 2-week-long heavy metal treatment, then frozen in liquid nitrogen, and stored at − 80 °C for further assays.

Enzyme Assays

For the determination of SOD, CAT, and POT activities, fresh samples (0.5 g per sample) were separately homogenized in the presence of 2 mL phosphate buffer (pH 7.8; 50 mM) containing ethylenediaminetetraacetic acid (EDTA; 1 mM) and polyvinylpyrrolidone (PVP; 2%). Samples were centrifuged at 15,000×g for 20 min at 4 °C and the supernatant was used for enzyme assays.

200 µL of phosphate buffer (pH 7.8; 50 mM) mixture containing nitroblue tetrazolium chloride (NBT; 63 μM), riboflavin (50 μM), EDTA (0.1 mM), methionine (13 mM), and plant extract (50 μL) was prepared to determine SOD activity. The prepared mixture was incubated under fluorescent lamps for 10 min at room temperature. SOD activity unit was then measured at 560 nm wavelength and demonstrated to have the capacity to inhibit 50% of photoreduction of NBT.

To measure CAT activity, phosphate buffer (pH 7.0; 50 mM) mixture containing the plant extract (100 μL) with hydrogen peroxide (H2O2; 10 mM) was prepared and the absorbance value at 240 nm was measured.

Phosphate buffer (pH 5.5; 0.1 M) containing guaiacol (120 μL) and H2O2 (84 μL) was prepared to measure POD activity and the absorbance value at 470 nm was measured.

Lipid peroxidation level was measured by determining the MDA content by thiobarbituric acid method. Briefly, plant samples were thoroughly crushed in liquid nitrogen. After homogenization with 2 mL of the mixture containing thiobarbituric acid and trichloroacetic acid, the samples were incubated at 95 °C in water bath, and then the supernatants were removed. The absorbance values at 532 nm and 600 nm were measured.

Statistical Analysis for Enzyme Activity

Plant samples were independently evaluated over 3 replicates. Variance analysis (ANOVA) was performed to evaluate the obtained data statistically, and the data were tested by Duncan's multiple range test (P ≤ 0.05).

RNA Isolation and qRT-PCR Assays

To assess the expression of the genes, the frozen sunflower samples were milled in liquid nitrogen. Total RNA was obtained from plant samples using TRIzol reagent (Invitrogen, USA). Quantification of obtained RNAs, the A260/A280 value, was measured by Nanodrop spectrophotometer. Total RNAs were set at 100 ng/μL. cDNA synthesis was then performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer's instructions. The synthesized first-strand cDNA was used for qRT-PCR with a 25-μL reaction volume containing A Maxima SYBR Green/ROX qPCR Kit (Thermo Scientific) according to the manufacturer’s instructions. This reaction was conducted using a Rotor-Gene Q system (Qiagen, Germany). Applied PCR conditions were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The primers used in the experiment were the same as those reported in Tomas et al. (2015). The sunflower -Actin gene was used as the reference gene in qRT-PCR analysis and the expression of the genes was performed according to the reference gene expression.

qRT-PCR Data Analysis

The threshold (Ct) values of the samples were determined as a result of gene expression analysis. Standard deviations of Ct values of samples according to PCR analysis results were calculated with Microsoft Excel program. Each qRT-PCR experiments were evaluated over three independent replicates. Expression rates of sunflower genes were determined by the 2−ΔΔCt proportional calculation algorithm according to Livak and Schmittgen (2001). ANOVA was performed to evaluate the obtained data statistically, and the data were tested by Duncan's multiple range test (P ≤ 0.05).

Results

After exposure of the plants to heavy metal stress, there was a significant increase in MDA (lipid peroxidation product) levels in the roots and shoots compared to that in control (Fig. 1). MDA levels in all other treatments except control are higher in the roots than in the shoots. When compared with control in the roots, the highest increase in the MDA level was observed in the Cr treatment (954.09%). When compared with the Cr treatment, the addition of Cu (21.31%) and AsA (27.21%) separately in the Cr treatment reduced the MDA level.

Fig. 1
figure 1

Effect of MDA levels of Cr, Cu, and AsA treatments. Means with the same letter denote they are not significantly different by Duncan’s multiple range test at 0.05

Full size image

SOD enzyme increased in roots and shoots in response to Cr heavy metal compared to that of control (Fig. 2). It was determined that adding of Cu or AsA in Cr treatment cause a statistically non-significant change in enzyme activity. When the enzyme activity in roots and shoots is compared, it is observed that the amount of enzyme in the roots is higher than that in the shoots. When compared with the control, the activity of SOD enzyme increased in the Cr treatment (371.42%). When compared with the Cr treatment, the addition of Cu (5.45%) in the Cr treatment increased the activity of SOD enzyme. The highest increase in the activity of SOD enzyme was observed in the Cr + AsA treatment (55.75%).

Fig. 2
figure 2

Effect of SOD activity of Cr, Cu, and AsA treatments. Means with the same letter denote they are not significantly different by Duncan’s multiple range test at 0.05

Full size image

In the sunflower exposed to Cr stress, POD enzyme activity increased in both roots and shoots when compared to control (Fig. 3). This increase was observed to be more in the roots compared to the shoots. Cr treatment increased the activity of POD enzyme (117.49%) compared to control. It was determined that adding of Cu or AsA in Cr treatment cause a statistically non-significant change in enzyme activity.

Fig. 3
figure 3

Effect of POD activity of Cr, Cu, and AsA treatments. Means with the same letter denote they are not significantly different by Duncan’s multiple range test at 0.05

Full size image

The expression level of the MT2-1 gene in Cr, Cu, and AsA treatments is shown in Fig. 4. From the results of this study, Cr treatment in the roots significantly increased the expression level of MT2-1. The highest expression level was obtained in the roots compared to the shoots. When compared with control, the highest increase in the expression of the MT2-1 gene was observed in the Cr treatment, while the lowest level was observed in the Cu treatment in the roots. The highest increase in the expression of the MT2-1 gene was observed in the Cu treatment, while the lowest level was observed in the Cu + Cr + AsA treatment in the shoots.

Fig. 4
figure 4

Expression of MT2-1 gene in Cr, Cu, and AsA treatments in roots (a) and shoots (b) compared to control. Means with the same letter denote they are not significantly different by Duncan’s multiple range test at 0.05

Full size image

The expression of the MT4 gene in Cr, Cu, and AsA treatments is shown in Fig. 5. The expression of the MT4 gene changed according to the applied heavy metal and tissue. The expression level was observed to be the highest in the roots, but no significant change was observed in the shoots. When compared with the control, the highest expression level was observed in the Cr + AsA treatment, while the lowest expression level was observed in the AsA treatment in the roots. The highest expression levels were observed in the Cu + AsA treatment, while the lowest expression level was observed in the AsA treatment in the shoots.

Fig. 5
figure 5

Expression of MT4 gene in Cr, Cu, and AsA treatments in roots (a) and shoots (b) compared to control. Means with the same letter denote they are not significantly different by Duncan’s multiple range test at 0.05

Full size image

Discussion

Several studies have demonstrated ROS production by creating oxidative damage of heavy metal stress. MDA is an end product of lipid peroxidation and is often considered a marker for increased oxidative damage in plant tissues (Silambarasan et al. 2019). In the present study, there was an increase in MDA levels with Cr stress (Fig. 1). The treatments of Cu2+, Cr6+, and cadmium (Cd2+) in Trichosporon cutaneum increased ROS production (Lazarova et al. 2014). It has been observed that lipid peroxidation is increased in wheat seedlings exposed to lead (Pb) stress (Hasanuzzaman et al. 2018). In the control, AsA treatment increased the MDA level. However, when Cr and AsA were co-treated, MDA amount decreased compared to control. Similar to the findings obtained in our study, Zhang et al. (2019) observed that AsA in the Run Nong 35 genotype increased in MDA content at higher doses (0.3 mM and 0.5 mM) than 0.1 mM, whereas the Cd + AsA treatment decreased MDA levels compared to Cd alone. This finding suggests that AsA alone slightly increases MDA content and is not necessary for plant regulation, but it has an important function in stress detoxification.

Various strategies have been developed in heavy metal stress tolerance, but the AsA and Cu treatments are a new strategy against this stress. Indeed, the addition of Cu and AsA against Cr stress decreased the level of MDA. From the results of this study, the effect of Cu and AsA treatments against Cr stress is similar. Therefore, it was determined that the stress caused due to Cu and AsA treatments may be tolerated equally. SOD, POD, and CAT enzymes detoxify ROS and protect the cell membranes and play a role in the defense system (Rehman et al. 2019). In this study, Cr heavy metal increased the activity of SOD and POD enzymes involved in the defense of antioxidants (Figs. 2, 3). From the results of this study, it has been shown that plants can tolerate Cr stress to a certain level by increasing the antioxidant enzyme (SOD and POD) activities. It was determined that the Cu dose in the normal nutrient medium was sufficient for plant growth, but it could have a toxic effect even when extra non-toxic doses were added to the nutrient medium based on SOD and POD results. This confirms the fact that Cu is a micronutrient. Moreover, it was determined that Cu applied with Cr stress had antagonistic effect with this heavy metal. From the results of this study, Cu has a healing role only in plants exposed to Cr stress. In resistant plant species, SOD and POD activities were found to be high to enable the plants to protect themselves against the oxidative stress (Liu et al. 2004). However, it was found that the Cr + Cu and Cr + AsA treatments caused a slight increase in POD activity with respect to Cr, but this was not statistically significant in the study.

In regard to CAT activity, there was no significant change (data not shown). Enzymes were located at different parts of the cell, which had different resistances to heavy metal stress, and disruption of cellular system functions by heavy metal stress may have resulted in the ineffectiveness of enzyme activity (Hou et al. 2007). In the rice exposed to Cu stress, no significant change was observed in CAT enzyme (Thounaojam et al. 2012). In the Cu and AsA treatments against Cr stress, the amount of antioxidant enzyme increased. Bhaduri and Flukar (2012) report that APX is abundant in the cell, and especially in the presence of APX, the amount of AsA is remarkable increased. Because the affinity of APX to H2O2 is higher than that of CAT, ROS is more effective in scavenging.

MT plays an important role in protecting metal ion homeostasis, improving metal toxicity, and protecting against intracellular oxidative damage (Huang et al. 2018). In this study, the response of MT genes to Cr stress has changed according to the tissues. MT genes (MT2-1 and MT4) have been slightly modulated in the shoots, while they were upregulated in the roots along with Cr stress. This result may be related to the mechanism through which hyperaccumulatory plants are used to tolerate heavy metal stress because MT2 proteins have two cysteine-rich conserved domains responsible for metal binding and ROS clearing activities (Yamauchi et al. 2018). The upregulation of ScMT2-1-3 at Cu stress suggests that this gene plays a prominent role in Cu detoxification and storage in sugar cane cells (Guo et al. 2013a).

In the study, Cu + Cr treatment shows a decrease in the expression of MT2-1 gene, which is thought to be due to the antagonistic effect of Cu and Cr in the roots. As a matter of fact, Dube et al. (2003) reported an antagonistic effect between Cu and Cr. In this study, a high expression of the MT2-1 gene has been observed in the roots when the plants were exposed only to AsA. AsA, whose molecular mechanism is yet unknown because it functions as a signaling modulator in many physiological processes, may have increased the level of MT2-1 expression.

In this study, AsA treatment against Cr was observed to reduce the expression of MT2-1 gene, which is the probable cause; AsA is involved in the removal of ROS formed by Cr toxicity. Similar to the findings in the research, it has been reported that AsA treatment in Sebastes schlegelii under Pb stress considerably reduces the expression of MT gene (Kim and Kang 2017). The effect of both Cu and AsA applied against Cr stress was found to be similar in point of gene expression. The result of this study demonstrates that Cu and AsA are equally active against toxicity and that Cu acts as a micronutrient. It can be said that both Cu and AsA remove ROS formed by Cr stress and cause decrease of gene expression.

Based on the gene expression models in the shoot parts of the MT2-1 gene in the study, it is observed that it gives the most expression to Cu treatment compared to other treatments. The overexpression of this gene in Cu may be due to its role in Cu transport. Indeed, it has been reported that MT2b in Arabidopsis plays a major role in Cu homeostasis through the phloem and is involved in the transport of excess Cu to long distances between tissues (Guo et al. 2013b). On the other hand, MT2b has been reported to function as a Cu storage mechanism in the vascular tissue to provide Cu for lignification of cell walls (Guo et al. 2013b). For these reasons, Cu is thought to activate the MT2-1 gene. It is also possible that the finding that this gene expression is low in the treatment of Cr in the research is probably due to the fact that sunflower stores the heavy metal in the roots and prevents it from being transmitted to other organs.

In the research, it was determined that MT4 gene is more expressed in the Cr + AsA treatment when compared to the Cr treatment alone in the roots. From the study result, it can be said that the addition of AsA to the Cr treatment further increases the expression of the MT4 gene. As a matter of fact, studies on rat liver have shown that MT synthesis is induced by AsA treatment (Onosaka et al. 1987). On the other hand, it is expected to increase the expression of the gene because of the toxicity of Cr treatment. The down-regulation of this gene in the presence of AsA is due to the fact that Cr stress was absent in growth medium. Again in this research, given the expression of the MT4 gene in the shoots, it has been determined that there is no significant change in this gene expression when both the heavy metals are applied. This can be explained by the fact that the expression of the MT gene is tissue and organ specific (Kisa et al. 2017).

Conclusion

Sunflower, due to its high biomass productivity and phytoremediation efficiency, is a suitable plant to study heavy metal tolerance. Although the heavy metal stress has been studied in sunflower, the mechanisms contributing to the tolerance of these plants remain unclear. In this sense, this study may provide knowledge about the tolerance mechanisms of hyperaccumulatory plants. Further studies are needed to prove this. According to the results obtained in the study, overexpression of the MT2-1 gene, especially in the roots, has been found to have an important function in the maintenance of Cr and thus increased Cr tolerance. Therefore, MT2-1 genes can be used as a candidate gene to increase heavy metal tolerance by genetic transformation in plants. In addition, it was determined that AsA and Cu reduced oxidative stress caused by Cr.