The Effects of Magnetic Treatment On Seedling Growth, Nitrogen Metabolism, And Mineral Nutrient Contents In Populus×Euramericana 'Neva' Under Cadmium Stress

Background: The potted experiment was carried out to investigate the mechanism of nutrient metabolism and seedling growth caused by magnetic treatment (MT) following exposure to cadmium (Cd) stress. One-year-old seedlings of Populus×euramericana 'Neva' were treated with different Cd(NO 3 ) 2 solutions for 30 days. Properties of seedling growth and root morphology were promoted by MT under Cd exposure. Results: Contents of ammonium (NH 4+ -N), nitrate (NO 3- -N), and total nitrogen (TN) in leaves, also NH 4+ -N and TN in roots, were increased by MT combined with Cd-stress, although NO 3- -N content was decreased. Activities of nitrate reductase (NR), nitrite reductase (NiR), glutathione reductase (GR), and glutamate synthase (GOGAT) in leaves and activities of NR, GS and GOGAT in roots were stimulated by MT; conversely, NiR activity in roots was inhibited. MT improved the synthesis of cysteine (Cys) and glutamine (Gln) in leaves and reduced the contents of glutamic acid (Glu) and glycine (Gly), while contents of Cys, Glu, Gln, and Gly were increased in roots. (4) The contents of Ca, Mg, Fe, Mn, Zn and Cu were increased by MT under Cd stress in leaves, whereas content of K was reduced. In roots, contents of K, Ca, and Fe were increased by MT under Cd-stress, but the contents of Na, Mg, Mn, Zn, and Cu were decreased. Conclusions: Magnetization could regulate the uptake of mineral nutrients by roots and translocation from the roots to aboveground parts by affecting the root morphology. MT could also improve nitrogen assimilation and the synthesis of free amino acids by stimulating the activities of key enzymes. respectively (all P<0.05), with Chl a showing the greatest decrease followed by Chl b. Compared with NM 0 , the contents of Chl a, Chl b, and Car in NM 100 decreased by 9.27%, 18.54%, and 20.57%, respectively (all P<0.05). In a comparison of NM 0 and NM 100 , we found that magnetic treatment stimulated the synthesis of photosynthetic pigments. Specically, compared with NM 0 , the contents of Chl a, Chl b, and Car in M 0 increased by 51.26%, 46.25%, and 35.48%, respectively (all P<0.05). Additionally, the photosynthetic pigment contents under M 100 improved by 2.28%, 17.17%, and 17.50% compared with NM 100 , respectively (all P<0.05).

compared to pure water [52,53]. The response of biological systems to magnetic treatment of water has been reported, and both the activation of ions and polarization of dipoles in living cells are in uenced by the magnetic eld. Some positive results have been documented with regard to the growth and output of crops, including irrigation with low-quality water treated by a magnetic device [52]. Accordingly, magnetic treatment of water, even the low-quality water used for agro-forestry, may be feasible because it is affordable, convenient, and non-polluting to the plants, soil and environment [3].
In recent years, many studies and applications have focused on the magnetic technique in agricultural elds, including seed treatment, crop stress resistance, magnetic treatment of irrigation water, and soil quality improvement. The results indicated that such treatment could promote seed germination, enhance stress resistance, and increase crop output and quality. First, magnetized water could enhance cellular metabolism and viability, thus promoting seed germination. For example, Morejón et al. [45] used magnetized water to irrigate the seeds of Pinus tropicalis, increasing the germination rate and signi cantly enhancing seedling growth. Maheshwari and Grewal [41] observed that crop yield and water use e ciency in snow peas (Pisum sativum L var. macrocarpon), peas (Pisum sativum L.), and celery (Apium graveolens L.) were improved following irrigation with regenerated water and saline water after magnetic treatment. Surendran et al. [51] found that the growth of cowpea (Vigna unguiculata variety Kanakamani) and brinjal (Solanum melongena variety Haritha) and their yield increased due to irrigation with magnetized saline water, and ul-Haq et al. [55] revealed that irrigation with magnetized water not only increased the germination rate, dry and fresh weights, and chlorophyll contents of rappini seeds (Brassica rapa L.) but also increased alpha-amylase and protease activities. Second, magnetized water can increase the permeability of biological membranes, the absorption of nutrients by plants, and the resistance of crops. Grewal and Maheshwari [22] showed that the magnetic treatment enhanced the biomass of snow peas (Pisum sativum L var. macrocarpon) and Cabrera chickpea (Cicer arietinum L.) by 20-25%, and absorption of N, K, Ca, Mg, S, Na, Zn, Fe, and Mn was also promoted. Yao et al. [64] reported that damage to cucumber (Cucumis sativus) seedlings subjected to UV-B (ultra-violet-B) stress was alleviated by magnetic treatment. Finally, magnetization changes the pH, conductivity, and hardness of water, thereby promoting the dissolution of inorganic salts. Khoshravesh et al. [33] revealed that the soil water content (7.5%) increased following drip irrigation with magnetized water, and Khoshravesh-Miangoleh and Kiani [32] demonstrated that soil density was effectively regulated by irrigation with magnetized water, whereas the cumulative in ltration and in ltration rates were increased. In addition, Mostafazadeh-Fard et al. [44] showed that the leaching of Cl -, HCO 3 -, and Na + was promoted by long-term irrigation with magnetized water, which signi cantly reduced secondary soil salinization. In conclusion, all these results could provide a theoretical basis for application of the magnetic treatment of water for improving stress tolerance in plants and promoting ecological restoration of the soil.
Previously, few reports have focused on the responses of plants to stress conditions under magnetization and the tolerance effects of the plants to Cd stress following exposure to the magnetic eld. Among the scant literature, Liu et al. [37] found that magnetized water reduced oxidative stress by increasing the activity of superoxide dismutase (SOD) and catalase (CAT) in maize (Zea mays) under Cd stress. Chen et al. [10] reported that the contents of H 2 O 2 , O 2-, and malondialdehyde (MDA), as well as electrolyte leakage, were reduced by magnetic treatment in Mung bean (Vigna radiate) seedlings under Cd exposure due to the increased nitric oxide synthase (NOS) activity and improved photosynthetic characteristics. Overall, these studies have preliminarily demonstrated the possibility of reducing Cd-induced damage to plants using magnetized water, thus improving seedling growth and growing conditions. Furthermore, magnetic techniques may be applied to study the ecological restoration of Cd-contaminated soil, aiming to elevate the e ciency of Cd enrichment in plants. Therefore, the hybrid species Populus×euramericana 'Neva' (Neva hereafter) was used as the experimental material in our study. We explored the possible mechanistic effects of the metabolic nutrition and biological characteristics affected by the toxicity of Cd in Neva underlying the in uence of magnetic treatment of irrigation water to study the feasibility of cultivating a tree species of Neva in Cd-contaminated soil.

Study area
A pot experiment was carried out in the greenhouse at the Forestry Research Station of Shandong Agricultural University (117°08'E, 36°11'N) from March to May in 2017. The project involved glasshouse experiments, which were conducted with natural light, day and night temperatures of 20-25°C, a relative humidity of 60%-70%, and a 12-hour photoperiod at 800-1000 μmol photons m -2 s -1 of photosynthetically active radiation per day of natural light throughout the study period.

Plant materials
The experimental materials consisted of one-year-old seedlings of Neva, that collecting from the seedling nursery garden.
The tree nursery locates in Gaoqiao state-owed forestry farm of Ningyang country, Shandong province. In late March, the mid-stems of seedlings with a diameter of 1.52 ± 0.11 cm and a length of 12.0 cm were cut and planted in a nutrition bag (20 cm × 22 cm). The cultivation substrate was perlite, and there were two cuttings per bag. During the early stage, uni ed water management was performed. After germination, a modi ed half-strength Hoagland nutrient solution was applied to irrigate the poplar seedlings. In early April, seedlings with similar development (15 cm in height with six leaves) were selected and further cultivated in half-strength modi ed Hoagland solution containing 0 or 100 μmol·L -1 Cd(NO 3 ) 2 for four weeks.

Experimental design
A magnetic treatment device supplied by Magnetic Technologies L.C.C. (Russia, United Arab Emirates branch) with a permanent magnet was used. The device (U050 mg, 0.5 inch, output 4-6 m 3 ·hr -1 ) supplying a magnetic induction of approximately 300 Gs was used for treating the water.
The main irrigation strategy was initiated in early April. The potted seedlings were irrigated with half-strength Hoagland solution containing 0 and 100 μmol·L -1 Cd(NO 3 ) 2 solution in the presence or absence of magnetic treatment. The pots were divided into four experimental groups: (1) ( MF, Cd(NO 3 ) 2 ) plants grown with half-strength modi ed Hoagland solution that was not subjected to magnetic treatment (NM 0 ).
Half of the pots were irrigated with magnetized nutrient solution, and both groups were maintained with optimal substrate watering, with irrigation every ve days. Young leaves and ne roots were collected after 30 days, rinsed with deionized water, and stored at -80°C for later testing.

Determination of biochemical and physiological characteristics
Growth characters analysis For the measurement of seedling growth, each sample was gently uprooted, carefully washed under running tap water and deionized water, and then divided into leaves and roots. The individual ground diameters of the seedlings were measured using Vernier calipers, and the data were recorded. The length, surface area, diameter of roots and number of root tips were determined using a root analysis system (WinRHIZO Pro 2007, Regent Instruments, Quebec, Canada). A portable leaf area meter (CI-202; CID Bio-Science, Inc., Camas, Washington, USA) was employed to analyze the leaf area.

Tissue nitrogen contents measurement
Fine roots and leaves were rinsed with deionized water, and 0.5 g of fresh sample was used to determine the ammonium nitrogen (NH 4 + -N) content using the salicylic acid-sulfuric acid (SA-H 2 O 2 ) method, as described by Zhao

Enzyme activities determination
The activities of nitrate reductase (NR) and nitrite reductase (NiR) were assessed in accordance with the method described by Kandeler et al. [31]. Glutamine synthetase (GS) activity was determined using hydroxylamine as a substrate, and γglutamylhydroxamate (γ-GHM) formation was evaluated using acidi ed ferric chloride in accordance with the assay described by Loyala-Vergas and De Jimenez [39]. Glutamate synthase (GOGAT) was assessed through NADH oxidation and spectrophotometric measurement at 340 nm, as described by Barbosa et al. [5].

Amino acid contents estimating
Fresh roots and leaves (0.5 g) were used to determine the contents of cysteine (Cys), glutamate (Glu), glutamine (Gln) and glycine (Gly) by high-performance liquid chromatography (HPLC) [27].

Photosynthetic pigments observation
The photosynthetic pigments chlorophyll a (Chl a), Chl b and carotenoids (Car) in 5 g of fresh leaves of each sample were extracted in 5 ml of 80% acetone, and absorbance was measured spectrophotometrically at 663, 646 and 450 nm using a TU-1900 according to the method described by Singh et al. [50].

Statistical analysis
The results for all treatments were obtained using at least three replicates. Data were statistically analyzed with SAS software (Version 8.0; SAS Institute, Cary, NC, USA). Homogeneity of variance among means was examined using one-way analysis of variance (ANOVA) of Duncan's multiple test at the level of 0.05.

Plant growth
A comparison was performed between the roots and leaves with regard to biomass (dry weight), leaf area, and the length of fresh treetop (Fig. 1) of 'Neva' seedlings under different treatments. The results showed that in comparison to the controls NM 0 treatments, the results for the M 100 and NM 100 treatments were as follows: leaf biomass decreased by 19.72% and 33.46% (Fig. 1A), respectively; root biomass decreased by 26.46% and 38.32% (Fig. 1B), respectively; leaf area decreased by 32.83% and 29.18% (Fig. 1C), respectively, and this difference was signi cant (P<0.05); and new-tip length decreased by 9.64% and 9.90% (Fig. 1D)

Activities of key enzymes in nitrogen metabolism
Different effects on the activities of nitrogen metabolism enzymes were found in the M 100 and NM 100 compared with the control treatments of M 0 and NM 0 (Fig. 4). Cd signi cantly stimulated NR activity (Fig. 4A The nitrogen metabolism enzymes also responded differently to Cd stress (Fig. 4), which resulted in increased NR, GS and GOGAT activities (Fig. 4A 5A, C, D), and the results for Gln and Gly were signi cantly different (P<0.05).
The Glu content in NM 100 was 81.06% higher than in NM 0 (Fig. 5B), and these differences were signi cant (P<0.05).
Comparison of NM 0 and NM 100 showed an increasing trend in Cys, Glu, Gln, and Gly contents under magnetic treatment.

Contents of the elements K, Ca, Na and Mg
Compared with the control treatments of M 0 and NM 0 , Cd stress treatments exerted different effects on the four mineral elements K, Ca, Na, and Mg in the leaves of 'Neva' (Fig. 6). In particular, M 100 and NM 100 induced an increase in K contents of 52.78% and 8.58% when comparing with M 0 and NM 0 , respectively (Fig. 6A), and the K contents between M 100 and M 0 were signi cantly different (P<0.05). Compared with M 0 , the Ca content in M 100 decreased by 9.93% (Fig. 6B). In contrast, the Ca content was signi cantly increased in NM 100 compared with that in NM 0 (P<0.05), with a percent increase of 17.12%.
M 100 treatment induced an increase in Na content by 9.05% when compared with the M 0 treatment (Fig. 6C), although compared with NM 0 , NM 100 inhibited Na absorption, which decreased by 9.29%; however, the differences in Na were not signi cant (P>0.05). Cd stress had a small effect on the Mg content in leaves (Fig. 6D), which increased in the range of 0.17-2.86%. Compared with NM 0 and NM 100 , M 0 and M 100 signi cantly inhibited the absorption of K by leaves (Fig. 6A, P<0.05), with decreases of 52.95% and 33.80%, respectively. Conversely, these treatments bene ted the accumulation of Ca and Mg in leaves (Fig. 6B, C), which increased by 32.33% and 0.09% in M 0 compared with that in NM 0 , respectively, and by 1.69% and 2.86% in M 100 compared with that in NM 100 , respectively. In addition, Ca contents in M 100 and M 0 showed signi cant differences, yet the difference in Mg content was not signi cant (P>0.05). Compared with NM 0 , the Na content in M 0 decreased by 8.33% (Fig. 6D); compared with NM 100 , the Na content in M 100 increased by 9.25%. Signi cant differences were not observed between these treatments (P>0.05).
A comparison of M 0 and NM 0 revealed that Cd induced an increase in K, Ca and Na contents in roots (Fig. 6A, B, D).
Speci cally, compared with M 0 , K, Ca and Na contents in M 100 increased by 3.84%, 104.64% and 12.37%, respectively, and compared with NM 0 , K, Ca and Na contents in NM 100 increased by 2.13%, 49.31% and 4.17%, respectively. Moreover, a signi cant difference in Ca contents between treatments was found (Fig. 6B, P<0.05). In contrast, the Mg content decreased by 1.11-4.53% under Cd stress, but these differences were not signi cant (Fig. 6C, P>0

Trace element contents
According to the results for the four trace elements of Fe, Mn, Zn, and Cu in leaves (Fig. 7), Cd stress promoted the absorption of Fe, Mn, and Zn compared (Fig. 7A, B

Discussion
Biomass is one of the most important indicators for evaluating plant resistance to stress. Cd stress in plants can affect cell division and tissue elongation by disturbing normal physiological metabolism, cell membrane functions, and related proton pump activity, thereby seriously inhibiting rootstock growth, dry matter accumulation, and even nutrient uptake and utilization [2]. This study found that 100 μmol·L -1 Cd stress inhibited biomass accumulation in leaves and roots of Neva, inhibited the growth of new treetop, and caused growth retardation, i.e., yellowing, crinkling, and defoliation in the blade, and a smaller leaf area. Our results are consistent with those of Liu et al. [38], who found that ≥1.0 kg·m -3 Cd stress suppressed biomass accumulation and leaf development and caused leaf chlorosis in Glycine soja Sieb. et Zucc. Our results are also consistent with those of Lagriffoul et al. [35], who demonstrated that 25.0 μmol·L -1 Cd stress led to decreases in new shoot growth and leaf biomass in Zea mays L., cv. Volga. Therefore, 100 μmol·L -1 Cd stress might induce an inhibition of seedling growth in Neva. In addition, magnetic treatment combined with Cd stress signi cantly promoted biomass accumulation in roots and leaves, as well as stalk growth and leaf area. These results are consistent with those of Ahmad et al. [1], who found that exogenous addition of nitric oxide (NO) promoted the growth of Solanum lycopersicum L. and increased the biological yield when exposed to Cd conditions; our ndings are also in accordance with those of Rahman et al. [48], who reported that the application of exogenous CaCl 2 maintained a high dry weight in 29 seedlings of Oryza sativa L. cv. BRRI.
The reasons for such differences in the results are as follows. The magnetization could alleviate the replaced metal elements that are essential for the metallothionein (MT) reaction center in the cells of Neva by Cd, which induced enhanced activities of lipoxygenase (LOX) and reduced activities of coenzyme II oxidase (NOX); these changes led to a lower accumulation of thiobarbituric acid reactive substances (TBARS), damaging biological macromolecules and blocking the growth and development of Neva [36]. Magnetic treatment stimulated the synthesis of photosynthetic pigments in response to Cd exposure, consistent with the results of Hasan et al. [25], who found that exogenous application of 28homobrassinolide (28-HBL) or 24-epibrassinolide (24-EBL) alleviated Cd-induced photosynthetic inhibition in tomato seedlings. Our results are also consistent with those of Christos et al. [13], who revealed that nitrogen application could alleviate chlorophyll reduction in the leaves of Zoysia japonica under Cd stress. Chlorophyll content is closely related to the photosynthetic rate, and magnetic treatment increases the contents of photosynthetic pigments in Neva leaves. These changes affect carbon xation, the Hill reaction, and PSII activity in Neva leaves, resulting in enhanced photochemical quenching and enhanced stomatal conductance in leaves [8], which can accelerate the photosynthetic capacity and Cd tolerance of Neva.
Leaf characteristics re ect survival strategies formed via plant adaptation to environmental changes [4]. Under Cd stress, magnetization induced an increase in the area of Neva leaves, indicating that Neva can survive by maximizing leaf length and width, thus increasing photosynthetic production [61]. Moreover, magnetic treatment was bene cial for root morphology (Table 1)  effectively maintained plasma membrane integrity, reduced absorption of Cd by alfalfa (Medicago truncatula) seedlings, and promoted root growth. The results of the present study are similar to these ndings and suggest that the limited resources could be distributed to the roots in poplar plants, thereby changing the morphological composition of its roots to adapt to the Cd stress environment [19]. Additionally, in poplar suffering Cd stress, the exchange sites in the walls of root cells to repel Cd 2+ can be stimulated by the magnetization and activated coordinated expression of genes in the glutathionedependent phytochelatin (PC) synthesis pathway, thereby enhancing Cd accumulation and tolerance in roots [11] to promote root growth and development. Furthermore, improved root morphology properties are bene cial for the absorption of water and nutrients, representing an important adaptation for poplar to Cd-contained environments and suggesting that the growth mechanism induced by Cd is mainly responsible for the observed root activity affected by the magnetic treatment.
Nitrogen is one of the most important essential nutrients for plants. Ammonium nitrogen and nitrate nitrogen are the main forms of nitrogen absorbed from soil by plants; the latter can be directly taken up and utilized by plants, and its capacity factor re ects the velocity of nitrogen metabolism [67]. After assimilation, a portion of the nitrate is acquired by leaves, whereas the remainder is reduced to nitrite by NR in roots. Nitrite enters plastids and is reduced by NiR to ammonium, which is ultimately assimilated into amino acids and proteins [46]. Thus, the activities of NR and NiR play a very important role in the regulation of primary nitrogen assimilation. The results of our study showed that 100 μmol·L -1 Cd promoted the activities of NR, GS, and GOGAT and restricted that of NiR. The effects on TN content in poplar seedlings included inhibition of TN absorption by roots and decreased TN contents in leaves. Furthermore, the changes in the NiR activity and TN content were consistent, with higher values observed in leaves than in roots. Higher leaf TN contents help to maintain leaf productivity and the maintenance of normal metabolism through the maximum carbon xation rate [7], which is also a type of adaptation of Neva to the Cd stress environment. Otherwise, we propose that NiR may be the key rate-limiting enzyme for nitrogen transport during the growth of Neva seedlings. However, further studies are required to understand the relationship between NiR metabolism and TN. Overall, magnetic treatment induced a signi cant increase in nitrate nitrogen in leaves but decreased this content in roots. Moreover, magnetization induced higher NR activity in leaves and roots, and NR activity in leaves was 4-5 times greater than in roots. Consistent with the ndings for nitrate nitrogen, NiR activity in leaves was enhanced and enzyme degradation was accelerated in roots; thus, leaf activity was increased more than two-fold compared with roots. The amount of ammonium nitrogen accumulated in roots was much higher than in leaves. Therefore, we conclude that the NH 4 + absorbed by roots cannot be translocated directly to leaves, which promotes preferential transport of NO 3 from roots to leaves and leads to a decrease in NO 3 content in the former and an increase in the latter. NO 3 is the main effector that induces NR and NiR, and within a certain range, the activities of NR and NiR depend on the concentration of NO 3 in the nutrient medium. Magnetization causes a su cient supply of NO 3 for poplar leaves, thereby promoting the activities of both NR and NiR [42]. This result is consistent with a previous nding that exogenous CaCl 2 and SA administration stimulate NR activity [6]. However, magnetization resulted in higher NR and NiR activities in leaves, which increased the e ciency of nitrate reduction to nitrite and facilitated the conversion of nitrate to nitrite. In addition, the enhanced NiR activity in leaves consumed a large amount of NH 4 + , which prevented the toxicity of nitrite accumulation in leaf tissues. NiR in roots promoted the reduction of NO 3 to NH 4 + and consumed an enormous amount of NO 2 -, which is the primary explanation for the decrease in NiR activity in roots. Moreover, the accumulation of NH 4 + in roots provided raw material for amino acid synthesis.
The GS-GOGAT cycle is one of the main pathways for ammonium ion assimilation, which not only relieves the toxic effects of ammonium ions on plant tissues but also helps meet the nitrogen nutrient requirements of plants [34]. GS is the key enzyme in the GS-GOGAT cycle and is involved in the regulation of various nitrogen metabolism pathways that have important effects on metabolic e ciency of the plants [58]. GS uses NH 4 + as a substrate and ATP as an energy source for amino acid synthesis and conversion, and an increase in GS activity enhances nitrogen metabolism [18]. Studies have found that Cd stress can inhibit the activity of GS and GOGAT and hence suppress the GS-GOGAT cycle e ciency, which leads to excessive NH 4 + accumulation. However, in our study, magnetization induced an increase in GS and GOGAT activities in leaves and roots and, based on the determination of total ATPase activity in cell membranes, signi cantly improved ATPase activity (Fig. S1 A). This nding indicates that GS induced by ATP could bind a large amount of NH 4 + to form Gln, which is then converted to two molecules of Glu by GOGAT, and the ammonium toxicity and supplementing glutamate storage can be alleviated by these transformations [16]. Moreover, magnetization induced a signi cant increase in the contents of Glu and Gln in root tissues, which were 3.62 to 8.93 times higher than those under nonmagnetic treatment, respectively. Glu and Gln are the main donors for nitrogen-containing organic compounds, such as amino acids, nucleic acids, chlorophyll, and polyamines, and increased Glu and Gln contents promote the synthesis of macromolecules, such as amino acids and proteins. We also found that the contents of Cys and particularly Gly synthesized in roots under magnetic treatment were approximately 18 times higher than under the nonmagnetic treatment in Neva, and these changes promoted the transport of NH 4 + from roots to leaves in the form of organic nitrogen, e.g., amino acids [47].
Under Cd stress, magnetic treatment resulted in signi cant increases in the contents of the four free amino acids Cys, Gln, Glu, and Gly in roots, which led to the following three ndings. Primarily, by regulating amino acid metabolism in tissues, magnetization alleviated physiological damage in Neva caused by high concentrations of Cd (Fig.S2 A) parts of the plant, which not only conserves energy used for NO 3 --N absorption, reduction, and the synthesis of amino acids, but it also allows 'Neva' to store additional nitrogen using only a small amount of energy [26].
When exposed to Cd conditions, the increased contents of Cys and Glu in leaves caused by the magnetic treatment provided the raw materials for protein synthesis. Under magnetization, higher GS activity increased the demand for Glu; more Glu was consumed to synthesize Gln, and a decrease in synthesized Gln was then observed. Glu exhibited a larger percent increase than did Gln, possibly because Gln has a stronger storage capacity for NH 4 + and thus plays a role in relieving the toxicity of free ammonium. Further analyses are needed to understand the magnetization-induced decrease in Gly synthesized in leaves, the relationship between Gly and other free amino acids, and their roles in nitrogen assimilation.
As a heavy metal, Cd affects the absorption and translocation of nutrients in plants, resulting in insu ciencies in nutrient elements or imbalances between nutrients and, thereby, inducing physiological disorder and inhibiting plant growth [69]. In this study, we found that Cd exposure promoted the absorption of K and Mn by roots and the transport of K and Mn from roots to leaves, causing an increase in K and Mn contents in leaves. However, Cd stress had less of an effect on the Mg content of roots and leaves, which showed no signi cant change. Hernández et al. [28] noted that the concentration of Cd in the medium was related to the absorption and transport of K by plants. Under Cd stress, the K concentration in roots increased with the accumulation of Cd and was higher than that in leaves, possibly due to some interaction between Cd and K in Neva roots [17]. Additionally, magnetization enhanced ATPase activity in cell membranes (Fig. S1 A), thus providing an explanation for the increase in K uptake. Under magnetization, the K content in roots increased with improving Cd content ( Fig. S2 A), and the translocation of K to aboveground parts decreased with an increasing biological transfer coe cient of Cd (Fig. S2 B). These results suggest that magnetic treatment stimulated the binding of excess Cd with ATP in roots, resulting in less available energy for the transmembrane transport system and thus reducing K uptake by aboveground parts of Neva [17]. Cd stress also promoted the accumulation of Mn in leaves. González et al. [20] found that excess Mn mainly damages the aboveground parts of plants, inducing the formation of leaf brown spots, chlorosis, and leaf shrinkage. We postulate that this phenomenon may be due to the Cd-induced rapid transport of Mn in plants [14], which also explains the observation of leaf chlorosis and shrinkage during Neva growth.
After addition of exogenous Cd, Ca and Na were absorbed by the roots of Neva, and the contents of Ca, Na, and Cu in leaves were greater than in roots. Ca 2+ and Cd 2+ have similar ionic radii and can be taken up by poplar roots simultaneously through a competitive relationship. Increased contents of Ca in leaves is the main reason for the prevention of Cd transfer from roots to the aboveground parts of Neva, thereby leading to Cd accumulation in roots. This nding was con rmed by Tripathi et al. (1995) [54], who found that the presence of Ca 2+ suppressed the uptake of Cd 2+ . After Cd stress, magnetization enhanced the ability of roots to internalize Ca and transfer it to leaves, leading to an increase in Ca in leaves.
Changes in the leaf Ca concentration promote Ca 2+ transporter regulation of the Ca concentration in Neva cells, inducing the initiation of calcium signaling [12] to activate calcium-dependent protein kinase (CDPK)-like proteins [23], which respond to the signal transduction generated by Cd stress and participate in the regulation of growth and development in Neva.
When subjected to Cd conditions, the magnetic treatment could alleviate the substitution effect of Cd 2+ for Fe 2+ , Zn 2+ , and Mg 2+ by regulating the absorption and transport of Fe, Zn, and Mg in roots and leaves. Cd and Zn have similar extranuclear electron con gurations and can be substituted for each other. The improvement in Zn content in poplar leaves reduced the damage caused by the inactivation of chlorophyll due to compositional changes in the central ion of chlorophyll, demonstrating the adaptation of poplar to Cd conditions. Moreover, this study showed that magnetization promoted the development of root morphology in Neva, which is an important basis for improving the absorption and accumulation of Fe in roots. Constitutive co-expression of FIT and bHLH genes can effectively induce the expression of IRT and FRO2 to regulate Fe contents in aboveground plant parts [65] and to avoid the accumulation of a large amount of Fe in the root cell cytoplasm, which would lead to iron poisoning. All the biochemical reactions involving Fe are completed in the chloroplast, which contains the largest iron pool in plant cells. After magnetic treatment, both the leaf Fe and Mg and K contents increased, which promoted maintenance of the chloroplast structure and the number of thylakoids, photosynthetic electron transfer in chloroplasts, and alleviation of photochemical damage caused by Cd stress [15]. Magnetization also stimulated the uptake of Mn, Zn, and Cu by poplar roots, promoted Mn, Zn, and Cu transport to aboveground parts, and enhanced the contents of these elements in leaves. These effects could reduce the substitution of Cd for the metal elements in the reaction center and generate the production of the heavy metal-binding protein Cu/Zn-SOD in poplar cells. Excessive heavy metal Cd can increase the activity of Cu/Zn-SOD, which in turn eliminates superoxide anions in plants and reduces reactive species (ROS) accumulation [49], thus maintaining the normal permeability and stability of 'Neva' cells. K and Na contents are important indicators of the ion balance and symptoms of ion damage in plant cells [40]. With magnetic treatment after addition of exogenous Cd, the Na content generally decreased, and the K/Na ratio was reduced in leaves and increased in roots (Fig. S1 B). These ndings indicate that Cd stress plays a certain role in the ability of leaves to regulate the ion balance in uenced by magnetization, thereby further affecting leaf-mediated ion regulation in the whole plant.

Conclusion
The results of our study indicated that magnetization could induce an improvement in seedling growth and accelerate the synthesis of photosynthetic pigments of Neva under Cd stress at a concentration of 100 μmol·L -1 . Additionally, it could contribute to root development. The tolerance against Cd stimulated the activity of NR and NiR in leaves, which seemed to reduce the toxicity of nitrite; otherwise, the accumulation of NH 4 + in roots could supply abundant raw materials for the synthesis of amino acids induced by irrigation with magnetic treatment of water. The enhancement of the GS-GOGAT cycle could promote the e ciency of nitrogen metabolism due to the stimulated activities of GS and GOGAT due to magnetization. The magnetization treatment could regulate the absorption and distribution of mineral nutrients in leaves and roots of Neva seedlings during exposure to Cd stress, and the enhanced concentration of ions, i.e., Ca, Fe, and K, could be helpful in improving adaptability to a Cd-contaminated environment.

Declarations Abbreviations
All the abrreviations were indicated in the manuscripts.
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Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information les].

Competing interests
The authors declare that they have no competing interests.      Contents of cysteine (Cys, A), glutamic acid (Glu, B), glutamine (Gln, C), and glycine (Gly, D) in leaves and roots of Neva irrigated with or without magnetized water following exposure to 0 or 100 μmol·L-1 cadmium stress. Values are the means ± SE of at least three replicates. Different lowercase letters indicate signi cant differences among the four treatments at the 0.05 level in leaves and roots.

Figure 6
Changes in the contents of potassium (K, A), calcium (Ca, B), magnesium (Mg, C), and sodium (Na, D) in leaves and roots of Neva irrigated with or without magnetized water following exposure to 0 or 100 μmol·L-1 cadmium stress. Values are the means ± SE of at least three replicates. Different lowercase letters indicate signi cant differences among the four treatments at the 0.05 level in leaves and roots.

Figure 7
Changes in the contents of iron (Fe, A), manganese (Mn, B), zinc (Zn, C), and copper (Cu, D) in leaves and roots of Neva irrigated with or without magnetized water following exposure to 0 or 100 μmol·L-1cadmium stress. Values are the means ± SE of at least three replicates. Different lowercase letters indicate signi cant differences among the four treatments at the 0.05 level in leaves and roots.

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