Activation of antioxidative and detoxificative systems in Brassica juncea L. plants against the toxicity of heavy metals

Plant metal hyperaccumulators, to which Brassica juncea belongs, must have very efficient defence mechanisms that enable growth and development in an environment polluted with various heavy metals. B. juncea (Indiana mustard) v. Małopolska was exposed to the activity of trace elements such as cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) in combinations: CuPb, CuCd, CuZn, PbCd, PbZn, and ZnCd in a concentration of 25 μM each for 96 h during control cultivation. We observed a clear tendency for metal uptake and accumulation in above-ground parts which is characteristic of hyperaccumulators. The combinations of CuCd, CuZn, and PbCd inhibited the development of the seedlings the most. The used metal combinations increased the levels of reactive oxygen species (ROS) such as: hydrogen peroxide (H2O2), superoxide anion (O2.−) and oxidized proteins in B. juncea organs, generating oxidative stress conditions in the cells. We determined the level of transcription of the respective defence proteins of the detoxification and antioxidant systems. We have shown that in the first 24 h of stress condiction, activation of glutamylcysteine-γ synthetase (yECS) and glutathione reductase (GR1) enzymes related to the detoxification of heavy metals is important for B. juncea plants. In addition, the data provide important information on how plants respond to the presence of heavy metals in the first days of stress conditions.


Results
Levels of metal accumulation. The levels of metal accumulation in the roots, stems, and leaves of B. juncea were determined using the inductively coupled plasma mass spectrometry (ICP-MS), (Fig. 1). The highest accumulation of Pb was observed in the roots, especially in the case of the PbZn combination and it reached over 70% of all metals taken, while to the above-ground parts, Pb was transported in the amount-20% in stems, 6% in leaves. Cd ions were transported to the above-ground parts in plants treated with ZnCd the most: roots 39%, 15% shoots, and 46% leaves. An equally high accumulation of Pb and Cd metals was observed in the shoots of plants treated with PbCd, amounting to 45% for Cd and 39% for Pb.

Effect of the metal uptake on micronutrient accumulation in plants B. juncea. Our research on
the effect of treating mustard seedlings with trace metals on the plant's ability to uptake micronutrients yielded results (Fig. 2). We expected that the presence of high concentrations of exogenous metals in the medium would lower the content of copper and zinc. Meanwhile, during the treatment with metals, zinc uptake increases and copper decreases in whole plants, and significant differences are observed at the level of individual organs. Under the tested conditions, there is a clear tendency for microelements to accumulate in the stems, both for Cu and Zn. This may be partly explained by an increase in apoplastic transport to the conducting bundles and the opening of nonspecific ion channels in the presence of trace element ions competing for specific transporters. The metal accumulation profile is similar in roots and leaves, where the metal uptake into cells from the solutions is observed-soil in the roots and apoplastic transport in leaves. Transport to the conductive bundles requires different transporters (Fig. 3).
Biomass and morphological changes. The highest inhibition of seedling biomass growth was observed for B. juncea plants treated with combinations of CuCd, CuZn, and PbCd (Fig. 4). After 96 h of cultivation, the biomass of seedlings was about 43-59% lower than at the beginning of cultivation and 68% -76% lighter than in the control plants. The weakest inhibitory effect was observed for the ZnCd-treated seedlings, because after 96 h of treatment, the seedlings were 38% heavier than at the beginning of the cultivation and only 20% lighter than the control plants. We observed morphological changes in the area of the leaf blade, which were manifested by the presence of necrotic spots on the leaves and the inhibition of the growth of the leaf surface in relation to the control seedlings. Moreover, in the case of seedlings treated with CuZn and CuCd, the leaves were slightly twisted, and with CuPb, PbCd, and PbZn, there was chlorosis. The ZnCd combination caused the smallest morphological changes.
Higher levels of O 2 .− were observed in roots exposed to constant bimetal stress than in the above-ground parts, reaching maximum values between the 24 and 72 h of cultivation (Fig. 5). We observed the highest values of O 2 .− in seedlings treated with the ZnCd combination, both in the roots and in the above-ground parts. A high level of O 2 .− in mustard roots was also observed for the combination of CuPb (after 24 h) and PbZn (after 72 h). In the above-ground parts, the highest level of O 2 .− was observed after 24 h especially for ZnCd, and then in the following days of cultivation it decreased by about 50%.
The profile of changes and the level of H 2 O 2 values were similar in the roots and in the above-ground parts (Fig. 5 .− , was observed in the roots of B. juncea treated with CuPb, CuCd, and ZnCd, while the highest generation of H 2 O 2 was observed in the roots exposed to CuPb, CuZn, PbZn, and ZnCd after 24 h of cultivation (Fig. 6).
Level of protein oxidation. The levels of protein oxidative modification imposed by the bimetal treatment were 2-4 times higher in the roots and the above-ground parts compared to the control plants (Fig. 7). In the roots, the level of oxidized proteins reached its maximum after 48 h, while in the above-ground parts after 72 h of exposure to combination bimetals.

Levels of gene transcripts.
To estimate the changes at the level of transcripts of genes encoding CuZ-nSOD, MnSOD, yECS, and GR1, we used the electrophoretic separation technique and the CpAtlas program (Fig. 8).
An almost double increase in the expression of the gene encoding CuZnSOD was observed in the roots after 4 and 8 h for plants treated with the CuPb combination, while in the above-ground parts, mainly for combinations with Cu, i.e. CuPb, CuZn and CuCd, there was an increase of about 50% after 4 h. In the early hours of the stress factors activity, that is, after 2 and 4 h, no significant changes in the transcription levels of the gene encoding MnSOD in the roots were observed as compared to the control plants. Only after 8 h, an induction of the MnSOD gene expression in the roots by the ZnPb combination was observed. On the other hand, in the above-ground parts, a significant increase in the level of transcription was noted, over 2 times, for the combination of CdPb after 4 and 8 h of cultivation. When analyzing the changes in the expression of the gene encoding the γECS a 2-3 times, an increase in the level of transcription was observed in roots after 4 h and 8 h of exposure to combinations of CuPb and CuZn.
In the above-ground parts, a clear gene induction was observed, expressed by more than a twofold increase in the transcript level after 8 h for the samples treated with combinations of CuCd, CuPb, ZnPb, ZnCd. Plants treated with CuCd, CuPb, ZnPb still showed a very high level of transcription, three-fivefold also after 24 h.

Discussion
Abiotic stresses, including the presence of trace metals in soil, are estimated to be the main cause of global crop yield reduction by ca. 70% and thus are considered a great constraint to crop production 5 . B. juncea is the oldest cultivated crop for eating, oil food, and biodiesel production 17 . Therefore, it is very important to understand its defence mechanisms against the toxic effects of trace metals. Plants, especially hyperaccumulating plants, have developed very efficient defence mechanisms, both in terms of metal detoxification and removing damage resulting from the presence of metals, including oxidative damage 18 . Thus, it is important to learn about these mechanisms and determine which one whether detoxifying or antioxidant, is activated first at the level of transcription of genes encoding suitable enzymes. Understanding these mechanisms will make it possible to determine the role of both the detoxification and antioxidant systems under the conditions of abiotic factors. In our study, we found that in the case of B. juncea v. Malopolska, all the mentioned combinations of metals used in the concentrations of 25 μM each displayed moderate phytotoxic properties. The greatest reduction in biomass growth was caused by the mixtures of CuCd, CuZn and PbCd, only the ZnCd combination caused no its significant reduction. At the same time, we observed an intense uptake and accumulation of Cd in the above-ground parts of plants treated with ZnCd. These results demonstrate a synergistic interaction of these metals in B. juncea during translocation in shoots. Other researchers have also noted that B. juncea accumulates metals such as: Cd, As, Pb and Cu in the aboveground parts 15 , whereas different authors 19 reported that Zn induced a decrease in the Cd uptake and a simultaneous increase in the Zn accumulation in tomato plants. This suggested strong competition between Zn and Cd for the same membrane transporters. Using the ICP-MS method, we recorded higher translocation and accumulation of Pb, Cd, and Zn in the above-ground parts of Brassica juncea than in roots. Other authors 20 have also noted levels of heavy metals in B. juncea shoots and the order of bioaccumulation was as follows Cu > Cd > Pb > Cr. Similarly, Rajput et al. 21 found, that the ability to hyperaccumulate different pollutants can be correlated either positively (Cd-Zn, Pb-Zn, Co-Cu, Cd-Pb) or negatively (Cu-PAHs, Co-Cd, It was found that Cd, Pb, and Zn are carried into vacuoles by the same P-type ATPase HMA3, suggesting that these metals are hyperaccumulated by the same mechanisms 21 . Trace metals can compete with each other for metal transporters, e.g. Cd 2+ ions have physicochemical properties similar to essential divalent metal ions and use their transporters (HMA2/4, ZIP4, etc.). This is an example of direct competition between metals for pathways to  www.nature.com/scientificreports/ enter cells. Lead ions are transported mainly apoplastically or through channels for bivalent ions. Calcium ions enter the plant in a similar way; therefore, the increased concentration of lead ions may destabilize the calcium metabolism 1,22,23 . We observed an increase in zinc uptake and a decrease in copper in individual organs. The inhibition of biomass and plant growth by trace metals may be caused by many factors, such as water stress, which leads to a reduction in stomatal conductivity, lower transpiration and relative water content in leaves 5,7 . Plants, through the root cortex tissues, take metals from the soil solution due to their similarity with the same essential micronutrients such as Zn or Cu, and then symplastically or apoplastically they pass to the xylem and are transported to the shoots and stored in vacuoles 7,20 . Milner and Kochian 24 found that Zn was bound to histidine and organic acids inside the roots and in the free ionic form of Zn 2+ in xylem juice. In contrast, the Zn in shoots was found mainly in combination with organic acids, and only some parts of it were in the free ionic form. This suggests that the metal ions are transported from the roots in the chelated form as free ions and then stored again in the shoots, where they are bound by the chelates 9 . Other authors have also noted that Zn and, to a lesser extent, Cd are mobile and can be transferred by phloem to the above-ground parts of plants and accumulate in meristems (root tips, shoot tips, axillary buds) 23 . Israr et al. 25 concluded that the co-presence of metals (Pb + Cu, Pb + Ni, Pb + Zn, Cu + Ni, Cu + Zn, Zn + Ni, Pb + Ni + Cu + Zn) results in a greater reduction in plant biomass www.nature.com/scientificreports/ than exposure to single metals because of the occurrence of a synergistic or additive response. We observed the accumulation of the metals Cd, Zn, and Pb in the above-ground parts of plants in an amount 50% greater than in the roots. These results indicate an increase in apoplastic transport to the conducting bundles and the opening of nonspecific ion channels in the presence of trace element ions competing for specific transporters. There was a synergistic response between metals in the combinations PbZn and ZnCd, resulting in an increased accumulation of the two metals in the above-ground parts. B. juncea is a good accumulator of trace metals, especially Cd and could be used for the remediation of Cd from contaminated soils, because of its ability to translocate large amounts of this metal into its stems and leaves 15 . Trace metals can cause plant growth reduction by decreasing photosynthetic rates, as they disturb mineral nutrition and water balance, change hormonal status, and affect the membrane structure and permeability and chlorophyll content 26,27 . We noted that the presence of other metals in the medium changes the mineral balance of the whole B. juncea plant. Heavy metals may cause severe toxicity in plants by disturbing essential groups such as -SH of enzymes leading to their inactivation, destroying  www.nature.com/scientificreports/ the integrity of important biomolecules, modifying some macromolecules, replacing essential metal ions from structural formulae of biomolecules, and enhancing the generation of ROS 7,25 .
We observed a significant increase in ROS levels compared to the control plants in all plants treated with the combination of heavy metals, both in the roots and in the aerial parts. The highest values were observed in the organs of plants treated with ZnCd, CuPb, CuCd and PbZn. Therefore, it can be concluded that the most prooxidative are the combinations containing the metals Cu and Zn.
Toxic metals/metalloids are categorized into redox-active (Fe, Cu, Cr, etc.) and redox inactive (Cd, Pb, Zn, Ni, Al, etc.). Metals with higher redox potential than biological molecules (such as Fe 2+ and Cu 2+ ) participate in biological redox reactions and ultimately induced the ROS directly through Haber-Weiss and Fenton reactions. Other metals/metalloids such as Cd, Pb, Al, Ni, Zn, etc. cause the ROS generation by impairing the antioxidant defence system (mainly glutathione pool), inducing NADPH oxidase, displacing obligatory cations from binding sites of enzymes and activating the Ca-dependent system 28,29 . Other authors have also reported higher H 2 O 2 production in B. juncea: under the influence of Cd it was twofold higher after 7 days 18 , for Cr, Si and Cr + Si it was about 1.5-fold higher 30 , for Cd about tenfold after 5 days 31 and for As about 2-fold higher 32 . Also, an over threefold increase in O 2 .− was observed in 10-day-old B. juncea seedlings treated with Pb, and with Cd over twofold 33 . Similarly, a two-fold increase in O 2 .− and H 2 O 2 was observed in B. juncea seedlings treated with Cd at a concentration of 100 mg/kg for 45 and 60 days 34 . The fluorescence signal indicating the presence of O 2 .− and H 2 O 2 was observed mainly in the meristematic and elongation zones of B. juncea roots, which was also confirmed by other authors in B. juncea roots exposed to 50 μM Cu 35 . This is probably due to the fact that these zones are most exposed to the toxic effects of metals. An increasing histochemical detection of O 2 .− and H 2 O 2 was observed in the leaves of B. juncea seedlings treated with Cd 36 .
The increase in ROS in the roots and above-ground parts of B. juncea resulted in an oxidative stress condition. Under these conditions, pro-oxidative-antioxidant reactions towards oxidation are disturbed 37 . Consequently, this led to a two-fourfold increase in the level of oxidized proteins in B. juncea organs. ROS plays a key role as a signaling molecule in plants and controls the physiological functions of plants under stress. Therefore, the balance between ROS production and their elimination is of great importance for the proper functioning of plant cells 28 .
The rapid increase in oxidative stress conditions in cells requires the rapid activation of the antioxidant and the detoxification system, both at the level of activity and the expression of key enzyme genes. In our study, we observed differences in the induction of gene expression encoding enzymes depending on exposure time and metal combination. On the basis of our research, induction of gene expression was observed: CuZnSOD mainly by combinations with copper, MnSOD mainly by combinations of PbZn, and GR1, and yECS mainly by combination containing Cu and Pb ions. Other authors have also 38 observed an increase in the transcript amounts of γ-glutamylcysteine synthetase (γ-ECS), an enzyme which catalyses the first step of the glutathione (GSH) synthesis, in B. juncea plants under Cu stress. For the genes encoding CuZnSOD, GR1 and y-ECS, there was a greater increase in expression in shoots than in roots. This is probably due to the fact that hyperaccumulators are characterized by a higher accumulation of metals in the above-ground parts of the plants. Plants respond to abiotic factors such as heavy metal stress via immediate change in expression of the stress responsive genes at the transcriptional level 39 . The superoxide radical dismutation reaction is one of the first stages of the cascade of processes leading to the detoxification of ROS 5,37 . Therefore, the induction of genes encoding SOD, including CuZnSOD and MnSOD, is a very important factor determining the acclimatization of plants to stressful conditions 5,40 . However, it seems that B. juncea plants, which are hyperaccumulators, tolerate oxidative stress conditions well and do not show the need to induce the antioxidant system at the level of protein transcription. Our results indicate that during the first 24 h of exposure to metal stress, gene expression is induced, mainly the detoxification enzymes GR1 and yECS. Naturally, under metal stress, ROS are generated-hence the need to induce the expression of genes encoding antioxidant proteins. In our previous research, we observed an increase in the level of the gene encoding CuZnSOD in B. juncea plants treated with single copper, zinc, and lead ions the highest after 4 h in the roots and after 8 h in the above-ground parts, and MnSOD in the roots after 8 h exposure to Zn and Pb ions 5 . Other authors 41 using microarray results found that the Cd stress response in Brassica nigra plants after 72 h was much stronger in roots (88 genes showing increased or decreased mRNA levels) than in shoots (24 responding genes). They detected an increase in the expression of genes encoding y-ECS and PCS (phytochelatin synthase) in the roots and leaves of the tolerant and nontolerant ecotypes of B. nigra. CuZn superoxide dismutase (CSD1), catalase (CAT3 and CAT1), and peroxidase genes were downregulated, indicating regulation of hydrogen peroxide contents. Also, a significant effect of metal Cd stress on the induction of gene expression related to GSH (glutathione) metabolism in B. juncea using Comparative Transcriptome Profiling was noted by Thakur et al. 42 . These authors observed that among the transcripts mapping to GSH metabolism, the gene expression of a total of ten important components including glutathione S-transferase (GST), glutathione synthase (GSS), and gamma-glutamyltranspeptidase (GGT) was found to be modulated by cadmium. A total of 66 different transcripts encoding GST were found to be significantly upregulated. Additionally, other authors 43 observed in Brassica chinensis L. cultivar Aikangqing (a Cd-tolerant cultivar) an increase in expression of genes encoding y-ECS and GR in shoots after exposure to Cd ions. The authors postulate that in response to Cd stress in plants, there is overexpression of enzymes related to sulphur assimilation, thus increasing the tolerance to heavy metals. The addition of S in the experiment identified an increase in expression of defence enzyme genes. S uptake results in the initial formation of the first solid product, cysteine, which is needed for the synthesis of GSH, a low molecular weight antioxidant. GSH, in turn, is involved in both the ASA-GSH cycle and the biosynthesis of phytochelatins to regulate heavy metal detoxification. Therefore, whether the regulation of S assimilation related to heavy metal toxicities is essential in plant defence mechanisms still requires research 42,43 . Undoubtedly, however, exposure to toxic metal ions or high concentrations of non-toxic ions triggers stress reactions in plants that require adaptation at various levels: structural, physiological, biochemical and molecular. Based on our present results, it seems that triggering defence mechanisms related to detoxification of heavy metals is Scientific Reports | (2021) 11:22345 | https://doi.org/10.1038/s41598-021-01827-w www.nature.com/scientificreports/ crucial for B. juncea plants. However, we have previously reported 6 that plants are not adequately protected by the detoxification system because trace metals penetrate areas with high metabolic activity, such as the cytoplasm, mitochondria, or cell membrane-hence, the strong need for the mutual cooperation of two defence systems of plants: detoxicative and antioxidative.

Materials and methods
Plant material. The plant material was grown according to Malecka et al. 5 . Seeds of B. juncea v. Malopolska were grown in Petri dishes for 7 days under optimal conditions. Next, young seedlings were cultivated hydroponically on Hoagland's medium for one week in a growth room with a 16/8 h photoperiod, day/night at room temperature and light intensity of 82 μmol m 2 s −1 . After that, we changed the medium to 100 × -diluted Hoagland's solutions. Next, on 14 day old seedlings a trace metal solution was applied in combination: Measurements of the level of reactive oxygen species. Superoxide anion content was determined according to Doke 44 at 580 nm. The plant roots and above-ground parts (0.5 g) were placed in the test tubes and filled with 7 mL of mixture containing 50 mM phosphate buffer (pH 7.8), 0.05% NBT (nitro blue tetrazolium) and 10 mM of NaN 3 . Next, the test tubes were incubated in the dark for 5 min, and then 2 mL of the solution were taken from the tubes heated at 85 °C for 10-15 min, cooled in the ice for 5 min and the absorbance was measured spectrophotometrically (SHIMADZU UV-1800, Japan) at 580 nm against the control.
Hydrogen peroxide content was determined according to Patterson et al. 45 . The plant roots and above-ground parts were homogenized in 5% TCA (Trichloroacetic acid). The homogenate was centrifuged twice at 13,000g for 20 min. The level of hydrogen peroxide was determined in the supernatant by the spectrophotometric method at 508 nm. The reaction mixture contained: 50 mM phosphate buffer (pH 8.4), reagent containing 0.6 mM 4-(-2 pyridylazo) resorcinol, 0.6 mM potassium-titanium oxalate in (1:1). The corresponding concentration of H 2 O 2 was determined against the standard curve of H 2 O 2.
Determination of the level of protein oxidation. The method of Levine et al. was used to determine the level of oxidized proteins 48 . Roots and above-ground parts (0.5 g) were incubated with isolation buffer containing 0.1 M Na phosphate buffer, 0.2% (v / v) Triton X-100, 1 mM EDTA and 1 mM PMSF. After centrifugation at 13,000 × g for 15 min, supernatants (200 µl) were mixed with 300 µl of 10 mM DNPH in 2 M HCl. The blank was incubated in 2 M HCl. After 1 h of incubation at room temperature, the proteins were precipitated with 10% (w/v) trichloroacetic acid (TCA) and the pellets were washed three times with 500 µl of ethanol / ethyl acetate (1: 1). The pellets were finally dissolved in 6 M guanidine hydrochloride in 20 mM potassium phosphate buffer (pH Isolation of total RNA and RT-PCR. Isolation of Total RNA and RT-PCR was performed using the method described by Małecka et al. 5 . Roots and above-ground parts (100 mg) of B. juncea plants in the presence of trace metals and under control conditions were collected for total RNA isolation. The RNA was isolated with TRIzol reagent and tested spectrophotometrically for purity at 260 and 280 nm. Then, RNA was reversetranscribed with oligo (dT) primers using the RevertAid Reverse Transcriptase Kit (Thermo Science, Lithuania, European Union) after DNA was treated with DNase I (Thermo Science). The primers used for qPCR are listed in Table 1.

Statistical analyses.
All experiments were performed in three biological and technical replicates. Calculated the mean values ± SD are provided in figures. Obtained the data were analysed statistically using IBM SPSS Statistics (Version 22 for Windows). Significant differences between treatments were analysed using ANOVA, assuming p < 0.05 as the significance threshold.

Statement.
In this study we used B. juncea seeds from the Palikije Plant Breeding Station in Poland and our experimental research on plants was complied with institutional, national, or international guidelines.

Conclusions
The current study demonstrates the influence of Pb, Cu, Cd, and Zn in binary combinations on the metal uptake, biomass growth, level of oxidative stress, and the level of transcriptions of detoxicative and antioxidative enzyme systems of B. juncea. Plants accumulated high amounts of trace metals in the above-ground parts, especially Cd using combination of ZnCd. The presence of metals resulted in a considerable reduction in B. juncea biomass; the highest reduction was caused by binary combinations containing Cu and PbCd. Trace metals lead to the production of O 2 .− and H 2 O 2 and increase the level of oxidized proteins. We noticed that under the conditions of metal stress, transcript level of enzymes: yECS and GR, which are associated with metal detoxification, increase, especially in shoots. No significant differences in the levels of CuZnSOD and MnSOD transcripts both in roots and in shoots were observed. The obtained results indicate that in the organs of B. juncea v. Malopolska, in the first hours of stress, the expression of genes encoding proteins related to the detoxicative system is induced.   TUB1  GTG ATT GCT TGC AGG GTT TT  CAG AAT ACG GAA GCA AAT GTCA X54844.1   GR1  AGG GTT GGA GAA TGT TGG TG CAA TAG GTG GCT GGG AGA AA  X98274.1   CSD1  GGA ACT GCC ACC TTC ACA AT  CGT TTT CAA CCA CGT CCT CT  U30841.1   MSD1  CCT TGC TCC TGT CAA GGA AG TTC AGA TAA TCC GGC CTC AC  M63003.1   γ-ECS:  TGT GGC TGA AGA TGT CCT GA  CAC ACA TCG AAA TCG TCC AG  AF128455.1