Oxidative Stress Response and Metal Transport in Roots of Macleaya cordata Exposed to Lead and Zinc

Heavy metal pollution possesses potential hazards to plant, animal and human health, which has become the focus of recent attention. Hence, phytoremediation has been regarded as one of the most important remediation technologies for heavy-metal-contaminated soils. In this research, a dominant mine tailing plant, Macleaya cordata, was used as the experimental material to compare the metal transport and oxidative stress response in its roots under lead (Pb) and zinc (Zn) treatments. The result showed that Pb was mainly accumulated in the roots of M. cordata under the Pb treatment; less than 1% Pb was transported to the parts above. An analysis of the Zn content demonstrated a 39% accumulation in the shoots. The production of reactive oxygen species was detected using the in situ histological staining of roots, which showed that hydrogen peroxide in the root tips was observed to increase with the increase in both Pb and Zn concentrations. No significant superoxide anion changes were noted in the root tips under the Pb treatment. An analysis of the root enzyme activity showed that increase in NADPH oxidase activity can be responsible for the production of superoxide anions, subsequent the inhibition of root growth and decrease in antioxidant enzyme activities in the roots of M. cordata exposed to excess Zn. In total, this research provides evidence that the root of M. cordata has a high antioxidant capacity for Pb stress, so it can accumulate more Pb without oxidative damage. On the other hand, the Zn accumulated in the roots of M. cordata causes oxidative damage to the root tips, which can stimulate more Zn transport to the shoots to reduce the damage to the roots. This result will provide a basis for the application of M. cordata in the phytoremediation of soil polluted by Pb-Zn compounds.


Introduction
With increase in mining activities as well as the use of pesticides and fertilizers on a large scale, heavy metal soil pollution has expanded rapidly. In particular, the heavy metal pollution of regional cultivated land has seriously affected the safety and quality of agricultural products [1]. In recent decades, phytoremediation had been widely used to alleviate heavy metal soil contamination, and it has attracted more and more attention due to its low cost, high efficiency, safety and environmental friendliness [1,2].
Plant tolerance to heavy metals is the primary consideration for phytoremediation technology [3]. Plants have evolved the mechanisms to tolerate and accumulate heavy metal in the process of adapting to their environment. Firstly, plants can control their heavy metal uptake and transport through the transporters located in their plasma membrane or they can sequester the heavy metals in vacuoles by tonoplast transporters [1,4]. Secondly, plants can induce the synthesis of specific organic compounds, including organic acids, amino acids, phytochelatins and metallothioneins, to directly bind with excess metal ions in cells or export them to the apoplast to act as barriers against heavy metals, thereby protecting the cell protoplasm [5]. Thirdly, the tolerance of most plants to heavy metals is Plants 2023, 12, 516 2 of 12 significantly related to the activity of antioxidant enzymes [6][7][8], since heavy metals cause plant oxidative stress by replacing essential elements binding with proteins [9]. However, little is known on the tolerance mechanisms involved in the toxicity of lead (Pb) and excess zinc (Zn).
Pb is one of the most toxic environmental pollutants for plants, and Zn is an essential micronutrient associated with proteins and enzymes; however, excess Zn, like Pb, can interfere with numerous biochemical and physiological processes, including metal uptake and transport in plants [4]. The toxicities of Pb and excess Zn are mediated by the formation of reactive oxygen species (ROS), such as superoxide anions (O 2 − ) and hydrogen peroxide (H 2 O 2 ). ROS are toxic and can oxidize lipids, proteins and nucleic acids, thus causing lipid peroxidation, membrane damage, and enzyme inactivation [10,11]. Plants have antioxidant defense mechanisms that eliminate ROS, especially by enzymatic components such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and others. On the other hand, plasma-membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is believed to be responsible for O 2 − production in plants under heavy metal stress [10][11][12][13].
Macleaya cordata, a perennial medicinal plant in the family Papaveraceae, is a dominant plant with a large biomass in various mining areas, popular for its high tolerance and ability to accumulate various heavy metals; thus, it can be a good material for phytoremediation. Moreover, there are various bioactive alkaloids in this plant [14]; therefore M. cordata was approved by the European Food Safety Authority (EFSA) as a safe plant for the manufacture of feed additives [15]. Li et al. (2015) reported that M. cordata accumulated extremely high amounts of uranium, up to 278 times that of ordinary plants, and it can be used as a hyperaccumulator of uranium [16]. Wang et al. (2018) found that the molybdenum content of the aboveground parts of M. cordata reached 1207.4 mg·kg −1 in solution, and can be used as the hyperaccumulator of molybdenum [17]. Nie et al. (2016) also reported that M. cordata has a strong cadmium transport capacity [18]. In addition, the ability of M. cordata to successively accumulate lead, zinc and manganese has also been reported [19][20][21]. In a previous study, we investigated the accumulation of lead, zinc, cadmium and copper in M. cordata under hydroponic conditions, and found that M. cordata has a high tolerance and accumulation capacity for Pb and Zn [21]. This study aims to find out the different tolerance involved in oxidative stress and antioxidant protection in the roots of M. cordata exposed to Pb and excess Zn.

Effects of Pb and Zn on Shoot Height and Root Length of M. cordata
The shoot height and root length of M. cordata was measured after seven days of the Pb and Zn treatments. Compared with that of the control, the shoot height of M. cordata was not significantly affected by the treatment except for 100 and 200 µmol·L −1 Zn concentrations (Figure 1a,b). However, 100 µmol·L −1 Pb and the three Zn concentrations significantly inhibited the elongation growth of the roots of M. cordata (Figure 1c,d).

Content of Pb and Zn in Roots of M. cordata
After seven days of the Pb and Zn treatments, the contents of Pb and Zn in the roots and shoots of M. cordata were detected, respectively. The results found that no Pb was detected in the control. Moreover, with the increase in the Pb treatment concentration, the Pb content in roots increased sharply, but the Pb content in shoots did not significantly increase until the treatment of 100 µmol·L −1 Pb, which was up to 0.7% of that in the roots (Figure 2a). This indicated that most of the Pb is retained in the M. cordata roots and is rarely transported to the plant's aboveground parts. In contrast, the Zn content increased significantly in the roots and shoots of M. cordata under the Zn treatment, and the Zn content in the shoots was about 38% of that in the roots under the 100 and 200 µmol·L −1 Zn treatments (Figure 2b). This showed that the roots of M. cordata have a stronger ability to transport Zn to the shoots as compared to Pb. It is worth mentioning that the Zn content

Content of Pb and Zn in Roots of M. cordata
After seven days of the Pb and Zn treatments, the contents of Pb and Zn in the roots and shoots of M. cordata were detected, respectively. The results found that no Pb was detected in the control. Moreover, with the increase in the Pb treatment concentration, the Pb content in roots increased sharply, but the Pb content in shoots did not significantly increase until the treatment of 100 μmol·L −1 Pb, which was up to 0.7% of that in the roots (Figure 2a). This indicated that most of the Pb is retained in the M. cordata roots and is rarely transported to the plant's aboveground parts. In contrast, the Zn content increased significantly in the roots and shoots of M. cordata under the Zn treatment, and the Zn content in the shoots was about 38% of that in the roots under the 100 and 200 μmol·L −1 Zn treatments (Figure 2b). This showed that the roots of M. cordata have a stronger ability to transport Zn to the shoots as compared to Pb. It is worth mentioning that the Zn content in the roots and shoots hardly changed under the Pb treatment, except when it significantly increased the under 100 μmol·L −1 Pb treatment (Figure 2c).

Content of Pb and Zn in Roots of M. cordata
After seven days of the Pb and Zn treatments, the contents of Pb and Zn in the roots and shoots of M. cordata were detected, respectively. The results found that no Pb was detected in the control. Moreover, with the increase in the Pb treatment concentration, the Pb content in roots increased sharply, but the Pb content in shoots did not significantly increase until the treatment of 100 μmol·L −1 Pb, which was up to 0.7% of that in the roots ( Figure 2a). This indicated that most of the Pb is retained in the M. cordata roots and is rarely transported to the plant's aboveground parts. In contrast, the Zn content increased significantly in the roots and shoots of M. cordata under the Zn treatment, and the Zn content in the shoots was about 38% of that in the roots under the 100 and 200 μmol·L −1 Zn treatments (Figure 2b). This showed that the roots of M. cordata have a stronger ability to transport Zn to the shoots as compared to Pb. It is worth mentioning that the Zn content in the roots and shoots hardly changed under the Pb treatment, except when it significantly increased the under 100 μmol·L −1 Pb treatment ( Figure 2c).

Effects of Pb and Zn on Production of ROS in Root Tips of M. cordata
3,3 -diaminobenzidine (DAB) staining in situ is based on the rapid reaction of H 2 O 2 with DAB to form brownish-red spots. The amount of H 2 O 2 in roots can be detected by observing the location of the colored spots. As shown in Figure 3a,b, the production of H 2 O 2 in the root tips of M. cordata gradually increased with the increase of the Pb or Zn concentration, and a small amount of damage was observed on the root tips under the 200 µmol·L −1 Zn treatment. Nitroblue tetrazolium chloride (NBT) staining in situ relies on the reaction of O 2 − with NBT to produce a blue product. Likewise, the amount of O 2 − production in roots can be detected by observing the location of the blue spots. As shown in Figure 3c,d, blue-black precipitates appeared in the root tips of the control and treatment groups with different degrees of coloration. The O 2 − production was not significantly increased in the root tips of the Pb treatment; however, the blue-black spots in the root tips were significantly increased under the 100 and 200 µmol·L −1 Zn treatments. Meanwhile, excess Zn, especially 100 µmol·L −1 Zn, evidently promoted root hair development. Furthermore, the H 2 O 2 content and O 2 − -producing rate of the roots were basically consistent with the histochemical staining of the root tips of M. cordata ( Figure 4).
with DAB to form brownish-red spots. The amount of H2O2 in roots can be detected by observing the location of the colored spots. As shown in Figure 3a,b, the production of H2O2 in the root tips of M. cordata gradually increased with the increase of the Pb or Zn concentration, and a small amount of damage was observed on the root tips under the 200 μmol·L −1 Zn treatment. Nitroblue tetrazolium chloride (NBT) staining in situ relies on the reaction of O2 − with NBT to produce a blue product. Likewise, the amount of O2 − production in roots can be detected by observing the location of the blue spots. As shown in Figure 3c,d, blue-black precipitates appeared in the root tips of the control and treatment groups with different degrees of coloration. The O2 − production was not significantly increased in the root tips of the Pb treatment; however, the blue-black spots in the root tips were significantly increased under the 100 and 200 μmol·L −1 Zn treatments. Meanwhile, excess Zn, especially 100 μmol·L −1 Zn, evidently promoted root hair development. Furthermore, the H2O2 content and O2 − -producing rate of the roots were basically consistent with the histochemical staining of the root tips of M. cordata (Figure 4).

Effects of Pb and Zn on Antioxidant Enzyme Activity in Roots of M. cordata
In order to explore the source of the ROS in the M. cordata roots, the activity of the plasma membrane NADPH oxidase was analyzed. Compared with the control, the activity of NADPH oxidase in the roots of M. cordata significantly increased with 100 µmol·L −1 Zn, but not under the Pb treatment ( Figure 5). Additionally, the enzyme activities of SOD, CAT, APX and POD significantly increased in the roots of M. cordata under 50 and 100 µmol·L −1 Pb; in particular, the POD activity increased more than twice than that of the control ( Figure 6). In contrast, the roots exposed to the 100 and 200 µmol·L −1 Zn treatments had significantly lower enzyme activities of SOD, APX and POD compared to the control, while those in CAT, APX and POD showed an increase in activity with 50 µmol·L −1 Zn (Figure 7).   Values are means ± SE (n = 3) with three separate replicates. Means denoted by different letters refer to the significant differences (p < 0.05, Duncan's test).

Effects of Pb and Zn on Antioxidant Enzyme Activity in Roots of M. cordata
In order to explore the source of the ROS in the M. cordata roots, the activity of the plasma membrane NADPH oxidase was analyzed. Compared with the control, the activity of NADPH oxidase in the roots of M. cordata significantly increased with 100 μmol·L −1 Zn, but not under the Pb treatment ( Figure 5). Additionally, the enzyme activities of SOD, CAT, APX and POD significantly increased in the roots of M. cordata under 50 and 100 μmol·L −1 Pb; in particular, the POD activity increased more than twice than that of the control ( Figure 6). In contrast, the roots exposed to the 100 and 200 μmol·L −1 Zn treatments had significantly lower enzyme activities of SOD, APX and POD compared to the control, while those in CAT, APX and POD showed an increase in activity with 50 μmol·L −1 Zn (Figure 7).

Discussion
When plants are subjected to heavy metal stress, the elongation growth of their roots is firstly inhibited, followed by their shoot height and dry weight [4]. In this research, the Pb treatment had no significant effect on the shoot height of M. cordata, while 100 and 200

Discussion
When plants are subjected to heavy metal stress, the elongation growth of their roots is firstly inhibited, followed by their shoot height and dry weight [4]. In this research, the Pb treatment had no significant effect on the shoot height of M. cordata, while 100 and 200 µmol·L −1 Zn caused a significant decrease in the shoot height. These findings were consistent with those of a previous study on M. cordata treated with Pb and Zn for 20 d [21]. In addition, we showed that either 100 µmol·L −1 Pb or the three Zn concentrations significantly inhibit the elongation of a plant's roots (Figure 1), showing M. cordata may have a stronger tolerance to Pb than Zn in the present study. Interestingly, the inhibition of root growth did not increase with a higher Zn concentration, which can be attributed to a good intracellular Zn homeostasis system in M. cordata; there was similar effect in the roots of Elsholtzia haichowensis under excess Cu [22].
One of the mechanisms of plant tolerance to heavy metals is to limit the transport of heavy metals to the aboveground parts or to the cells [5]. Our findings demonstrated that the amount of Pb accumulated in the roots of M. cordata was very high, reaching 5000 µg·g −1 , but the transport ability to the shoots was less than 1% (Figure 2a). With the same concentration of Zn, the Zn content in the roots of M. cordata was lower than that of Pb, but the transport ability to the shoots was up to 39% (Figure 2b). In a previous study, when the treatment was increased to 1000 µmol·L −1 Pb and Zn for 20 d, the Zn content of the roots of M. cordata exceeded that of Pb [21], which can be attributed to the serious damage of the roots caused by the high concentration Pb. Pb could easily form Pb-binding compounds with cysteine, non-protein thiols, which caused Pb deposition in vacuoles or cell walls, and decreased the Pb transport in the plants [23]. It was reported that the levels of cysteine and glutathione were higher in the Pb-accumulating ecotypes of Sedum alfredii than those of the Pb non-accumulating ecotypes [24]. In the majority of cases, only a small part of the Pb absorbed by the roots can be transported to the shoots [24]. For the whole plant, the accumulation capacity of M. cordata for Zn is higher than that for Pb; for example, the Zn content in the shoots can reach to 1072 µg·g −1 (Figure 2b). Taking into consideration the large biomass of M. cordata, the Zn content of a single plant is more than some reported hyperaccumulators of Zn [25][26][27], so M. cordata can be considered as a Zn accumulator for phytoremediation [21]. Curiously, the Zn content of the roots and shoots of M. cordata was hardly affected by the Pb treatment, except when the Zn content significantly increased in the roots with 100 µmol·L −1 Pb (Figure 2c). Zn is an essential element; plants have many zinc transporters or zinc-binding proteins to control their Zn uptake and transport under heavy metal stress. This may be the reason why M. cordata roots have a high tolerance to excess Zn [28].
Large amounts of ROS are produced under heavy metal stress, which ultimately causes serious damage to membrane systems and proteins, and can even lead to the death of the plant [5,22,29]. The amount of H 2 O 2 and O 2 − production in plant tissues can be detected by histochemical staining of DAB and NBT, or measuring the H 2 O 2 content and O 2 − -producing rate [10]. In the present study, the O 2 − production had a significant increase in the roots of M. cordata with the Zn treatment; however, there was no significant change with the Pb treatment (Figures 3 and 4), which was consistent with the activity change of NADPH oxidase under the Pb and Zn treatments ( Figure 5). NADPH oxidases can use cytosolic NADPH to produce O 2 − , which is quickly transformed to H 2 O 2 by SOD or spontaneous action [10,30]. Besides NADPH oxidase, pH-dependent cell wall peroxidases have been proposed as sources of H 2 O 2 in the apoplast [31]. In a previous study, we also found that an increase in NADPH oxidase activity was involved in the ROS production of Cd-induced Solanum nigrum [11], Cu-induced E. haichowensis [10], and Zn-induced S. alfredii [13], and the increase in [Ca 2+ ] cyt and root hair development in the roots of S. alfredii [13]. The Pb-induced H 2 O 2 increase in the roots of M. cordata was similar to that of the Zn-induced increase in the present study, except a small amount of damage was observed on the root tips with the 200 µmol·L −1 Zn treatment (Figure 3b), denoting the root tips of M. cordata were damaged to some extent by Zn, but not by the Pb treatment. SOD, CAT, APX and POD constitute the important defense enzyme systems to various stresses in plants [11,32,33]. SOD mainly reduces toxicity by scavenging O 2 − . CAT and APX can scavenge excessive H 2 O 2 to maintain normal levels in the cells of plant. POD can not only catalyze the oxidation reaction of H 2 O 2 , but can also catalyze the conversion of H 2 O 2 and O 2 − into hydroxyl radicals to intensify peroxidation [34]. In this study, the enzyme activities of SOD, CAT, APX and POD in the roots significantly increased under Pb stress compared with those of the control ( Figure 6). Nonetheless, the antioxidant enzyme activities in the roots significantly decreased with the Zn treatment except for 50 µmol·L −1 Zn (Figure 7). The difference in antioxidant enzyme activity may be related to the damage of the roots of M. cordata under the Pb and Zn treatments. This finding showed that the O 2 − produced by NADPH oxidase in the roots of M. cordata with excess Zn was not been eliminated owing to the decrease in antioxidant enzyme activity; however, the ROS induced by Pb can be removed by the antioxidant enzyme system in the roots of M. cordata. Zn-induced ROS were produced and antioxidant enzymes decreased in the roots of the hyperaccumulating ecotype of S. alfredii [13]. It was also reported that the translocation of heavy metals from the roots to shoots was a detoxification pathway to reduce the stress toxicity to the plant roots [4].

Hydroponic Culture
The seeds of M. cordata were collected from mine tailing of Huaguoshan in Luoyang, China. After the seeds were germinated in vermiculite, the seedlings with uniform growth were transferred to 2.5 L plastic vessels containing Hoagland nutrient solution (1 mM for the Pb treatment, and each treatment had three replicate vessels. After 7 days of exposure, the roots and shoots were then removed and separated for metal content determination, and the fresh roots were removed for detection of in situ staining, H 2 O 2 content, O 2 − -producing rate and enzymatic activity analysis.

Metal Content Analysis
Shoots and roots of M. cordata were collected after washing them with distilled water, and the whole roots were immersed in 25 mmol·L −1 EDTA-Na 2 solution for 10 min and then washed with distilled water again. The samples were then oven-dried at 80 • C and weighed to obtain dry weight. About 0.2 g of the dried samples were digested by the mixed acid of HNO 3 :HClO 4 (87:13, v/v) following the procedure described by Zhang et al. [21]. The ICP-OES (Optima 8000, PerkinElmer, Waltham, MA, USA) was used to analyze the content of Zn and Pb.

Histochemical Detection of Metals, H 2 O 2 and O 2 − In Situ
The H 2 O 2 and O 2 − formation in situ in roots was detected separately by DAB and NBT staining as reported by Zhang et al. [22]. The roots were immersed in 25 mmol·L −1 EDTA-Na 2 solution for 10 min and then washed with distilled water before detection of metals in situ. Subsequently, the cut roots were immersed in a staining solution for 15 min. DAB staining solution: 1mg·mL −1 solution of DAB with a pH of 3.8. NBT staining solution: 0.1% NBT in 50 mM of K-phosphate buffer containing 10 mM sodium azide with a pH of 6.4. After thoroughly washing them with distilled water, the well-stained root tips Plants 2023, 12, 516 9 of 12 were immediately observed under an Olympus microscope fitted with a Nikon D7100 digital camera.

Determination of H 2 O 2 , and O 2 − in the Root Extracts
The content of H 2 O 2 was measured by monitoring the absorbance at 415 nm of the titanium-peroxide complex following the method described by Jiang and Zhang [30]. The production rate of O 2 − was measured by monitoring the absorbance at 530 nm of the nitrite formation from hydroxylamine hydrochloride in the presence of O 2 − , as described by Jiang and Zhang [30].

Isolation of the Plasma Membrane and Determination of NADPH Oxidase Activity
Plasma-membrane-enriched fractions were isolated as described by Zhang et al., with some modifications [10]. Briefly, the roots were homogenized in a pre-cooled mortar with two volumes of freshly prepared media containing 250 mM sucrose, 50 mM Tris-HCl buffer (pH 7.5), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1.5% (w/v) polyvinylpolypyrrolidone (PVPP). The homogenate was filtered through four layers of cheesecloth and then centrifuged at 12,000× g for 30 min at 4 • C, then the supernatant was centrifuged at 100,000× g for 40 min at 4 • C to obtain a microsomal membrane pellet. The pellet was gently resuspended in medium with 5 mM phosphate buffer (pH 7.8) containing 0.33 mM sucrose and 2 mM dithiothreitol (DTT), and isolated by an aqueous two-phase polymer system to give a final composition of 6.2% (w/w) dextran T500, 6.2% (w/w) PEG 3350, 0.33 M sucrose, 3 mM KCl, and 5 mM phosphate buffer (pH 7.8). Three successive rounds of partitioning followed. The upper phase was diluted five-fold in Tris-HCl buffer (10 mM, pH 7.5) containing 0.25 M sucrose, 1 mM EDTA, 1 mM DTT and 1 mM PMSF. All procedures were carried out at 4 •C. The plasma membrane pellets were quickly frozen and stored at −80 • C until they were used for the enzyme assays. To check the degree of enrichment of the plasma membrane, vanadate-sensitive (100 mM vanadate), nitratesensitive (100 mM KNO 3 ) and azide-sensitive (1 mM NaN 3 ) ATPase activities (plasma membrane, tonoplast, and mitochondrial enzyme markers, respectively) were determined in plasma membrane pellets as described by Zhang et al. [35].

Enzyme Activity Assay
Fresh roots were extracted with precooling extraction buffer (including 50 mM potassium phosphate with 1 mM EDTA and 1% PVPP (w/v) with a pH of 7.0) on the ice, as described by Zhang et al. [10]. CAT activity was determined by measuring the consumption of H 2 O 2 at 240 nm according to the method of Aebi [37]. APX activity was measured by monitoring the decrease in the absorbance at 290 nm as ascorbate was oxidized, as described by Nakano and Asada [38]. POD activity was determined by measuring the change in the absorption at 470 nm according to Zheng and Van Huystee [39]. SOD activity was determined by its ability to inhibit the formation of nitroblueformazan from NBT according to the method of Giannopolitis and Ries [40]. Protein content was estimated with the same root extracts according to Bradford [41].

Statistical Analysis
Data were analyzed using one-way variance of SPSS 22.0. Values were presented as means ± SE (n = 3) with three separate replicates, and means denoted by different letters refer to the significant differences (p < 0.05, Duncan's test). The staining experiments were repeated at least five times with similar results.

Conclusions
In this study, Pb was mainly accumulated in the roots of M. cordata with the Pb treatment, and less than 1% Pb was transported to the parts above; however, our analysis of the Zn content demonstrated a 39% accumulation in the shoots. The high tolerance of M. cordata to Pb can be realized by fixing the Pb in the apoplast of the roots and limiting its transport to the aboveground parts. Moreover, the Pb induced the H 2 O 2 production in the roots, and a significant increase in the activities of the antioxidant enzymes, which further improved the Pb tolerance of the roots of M. cordata. On the other hand, the Zn stress induced large amounts of H 2 O 2 and O 2 − in the roots, but was accompanied by a significant decrease in the activities of antioxidant enzymes, which caused oxidative damage to the root tips. In order to reduce the root damage, the excess Zn in the roots of M. cordata can be transported to the shoots. The different mechanisms of tolerance to the Pb and Zn in the roots of M. cordata may be beneficial as an excellent remediation material for soil polluted with Pb-Zn-compounds. Pb-Zn-contaminated soil as a culture medium will be tested in the future to support the conclusions of the study.