Metal Based-oxide Nanoparticles Assisted the Uptake of the Essential Elements and Phytoremediation of Populus Alba : Essential Metabolic Processes and Genetic Stability

The present study evaluated the phytoremediation activities of Populus alba upon using nano metal-based-oxides (i.e., Fe 2 O 3 , ZnO, and Mn 2 O 3 -NPs) as analogues of three main heavy metals Fe, Zn and Mn exist in soil as micronutrients at three different concentrations (i.e., 20, 40, and 60 mg/L) compared to the control. The as-prepared nanoparticles have been prepared via co-precipitation method. In addition, the physico-chemical properties were investigated using transmission electron microscopy, Fourier transform infrared spectra, X-ray diffraction and dynamic light scattering techniques. Overall, a signicant difference in the biomass production-related parameters such as fresh weight, shoot length, root length, and root number compared to control upon the treatment with micronutrients-based nano-metal-oxides (i.e., Mn 2 O 3 > Fe 2 O 3 > ZnO NPs, respectively), except a signicant increase in the root number of Populus alba plant upon their treatment with ZnO NPs compared to other prepared nano-metal-oxides and control. Also, a remarkable increase in the chlorophyll index was monitored upon treatment with Fe 2 O 3 than other used Mn 2 O 3 and ZnO NPs, respectively. Moreover, RAPD-PCR bioassays were applied and the actual 6 primers showed a genetic variation percentage of 34.17% indicating that Populus alba is highly genetically stable even in a highly contaminated environment/soil. All these data enhance the idea of using the Populus alba plant in phytoremediation and heavy metal uptake as micronutrients to clean up the surroundings.


Introduction
Heavy metals are naturally occurring elements with a high atomic weight and a density around 5-fold that of water (Tchounwou et al. 2012). Because of their toxicity, endurance in the environment, and bio-accumulative nature, heavy metals are well-known environmental contaminants (Ali et al. 2019). In addition, their existence at concentrations higher than the environmental normal levels, which originated from human activities (i.e., mining, chemical, metal processing industries, and other allied industries) leads to be interference with the functioning of vital cellular components that exist in plant, animal, and human tissues through inhalation, diet and manual handling, which may affect the growth, development, and survival of the organisms (Ali et al. 2019, Asati et al. 2016, Jaishankar et al. 2014). Copper (Cu), manganese (Mn), lead (Pb), cadmium (Cd), mercury (Hg), nickel (Ni), cobalt (Co), iron (Fe), zinc (Zn), chromium (Cr), iron (Fe), arsenic (As), silver (Ag) and the platinum (Pt) as examples of naturally abundant heavy metals. These heavy metals except Pb, Cd and Hg may be essential in minute quantities by organisms, excessive amounts of these elements can become harmful to organisms (Briffa et al. 2020, Raskin et al. 1994. Whereas, some of heavy metals such as Fe, Mn and Zn were considered as essential micronutrients elements for plants, animals and other living organisms (Gomes et al. 2021, Rengel 2015, Singh &Schulze 2015; But toxic effect s could be monitored upon the uptake in excess to the requirements of plant, in such case it will have a negative effect on the nutritional equilibrium in soil (Djingova &Kuleff 2000, Reeves 2000, Wintz et al. 2002. Iron (Fe) is primarily involved in the process of photosynthesis in plants. The availability of micronutrients to plant roots is affected by soil pH, with iron being more readily available in soil with a low pH (Becker &Asch 2005, Morrissey &Guerinot 2009, Neina 2019, Rout &Sahoo 2015. Moreover, both of iron (Fe) and manganese (Mn) are essential micronutrients for plant growth and development, but they frequently compete for absorption because an abundance of one of these micronutrients makes the other less available plant roots. In addition, iron is existing as a basic component in the composition of heme proteins such cytochromes, catalase, peroxidase, and leghemoglobin, as well as iron Sulphur proteins like ferredoxin, aconitase, and superoxide dismutase (SOD) (Gautam &Dubey 2018). Whereas, zinc (Zn) aids in the production of chlorophyll in plants. When the soil is lacking in zinc, the leaves darken and plant growth is impeded (Hafeez et al. 2013). In addition, manganese (Mn) is a vital plant mineral nutrient that is involved in a variety of physiological activities, including photosynthesis. Manganese shortage is a common problem in sandy soils, organic soils with a pH above 6, and tropical soils that are highly weathered. Mn is easily transferred from root to shoot via the transpiration stream, but it is not easily remobilized to other organs via phloem once it reaches the leaves (Loneragan 1988).
Engineered nanomaterials have received great attention due to their unique properties, which provide an opportunity to be used in several applications especially in agricultural sector (Chemingui et al. 2019, Chhipa 2019, Naderi &Abedi 2012, Usman et al. 2020. Nanomaterials increase crop productivity and e ciently regulate micronutrients delivery to plants and targeted sites, ensuring that agrochemicals are used as little as possible (Mishra &Khare 2021). In addition, increase crop yield by increasing fertilizer nutrient availability in soil and nutrient uptake by plants (Sarkar et al. 2021). Nano-fertilizers or nano-encapsulated nutrients may have qualities that are bene cial to crops, such as on-demand release of nutrients, and controlled release of chemical fertilizers to regulate plant growth and accelerate target activity (DeRosa et al. 2010, Nair et al. 2010, Sampathkumar et al. 2020, Zhang et al. 2021 Phytoremediation is a renewable technology that restores contaminated soil and water sources by using either natural or genetically modi ed plant species. In various review articles examining the principles, types, and mechanisms of the phytoremediation process, the advantages of low cost, environmental friendliness, and safety are highlighted (Al-Khazan &Al-Zlabani, Arthur et al. 2005, Parmar &Singh 2015. Poplars are frequently used in phytoremediation because of their rapid growth, adaptability, a well-developed root system that reaches underground waters, and the ability to transpire large amounts of water (Aitchison et al. 2000). But they can't compete with hyper-accumulators in the heavy metal accumulation, but they didn't have a signi cant advantage in biomass production (Laghlimi et al. 2015, Liu et al. 2013, Pulford &Watson 2003 and a relatively high amount of extracted metal per plant (Di Lonardo et al. 2011). White poplars, in particular, are interesting due to their high tolerance to arid conditions and their use in horticulture and landscaping, especially genotypes with a pyramidal tree shape (Kovačević et al. 2010).
The in-vitro culture of tree species offers a rapid instrument to produce the clonal planting stock, but it may also facilitate studies on the effects of elevated levels of heavy metals on plant performance and the selection of metal-tolerant genotypes (Di Lonardo et al. 2011). Castiglione et al. observed that the in vitro model was a sensitive and reliable system for studying heavy metal stress responses in their study of white poplars based on developmental and molecular data (Castiglione et al. 2007). This is signi cant given the di culties of experimenting on large, long-lived organisms. The tolerance of white poplars to heavy metals, including lead, was tested under conditions, and genotype differences were encountered (Bojarczuk 2004, Kališová-Špirochová et al. 2003, Katanic et al. 2008).
Herein, we aimed to investigate the in-vitro assay of Populus alba upon the treatment with nano-oxide particles forms of each of Fe, Mn and Zn (e.g., Fe 2 O 3 , ZnO and Mn 2 O 3 NPs) as micronutrients through evaluation of growth parameters and photosynthetic index. In addition, testing their genetic stability using RAPD-PCR techniques. The obtained results will help us in tracking of phytoremediation activity of Populus alba (P. alba) via the tolerance of plant to grow in heavy metal contamination. and sodium hydroxide (NaOH, 98%), were used in the experiments without any further puri cation. All the chemicals used were of analytical reagent grade obtained from LOBA chemicals and deionized water is used for the preparation of solutions.

Materials And
2.2. Preparation of Nano-metal oxides for in-vitro phytoremediation 2.2.1. Preparation of Iron Oxide Nanoparticles Brie y, Iron oxide (Fe 2 O 3 -NPs) nanoparticles were synthesized via co-precipitation method as previously reported by Yang et al. (Yang et al. 2014) with slight modi cation. Brie y; iron salt precursor was mixed with deionized water under vigorous stirring for 10 min to ensure that all the ferrous salt was dissolved with a concentration of (1M). In a 100 ml beaker, sodium hydroxide was prepared with concentration of (2M). In this protocol, the sodium hydroxide solution was added to the iron solution drop-wise under constant stirring. After the complete addition of hydroxide solution, the reaction mixture was left for 2 h. Finally, the black solution was washed repeatedly with deionized water to remove the excess hydroxides keeping the pH of the slurry around 7 and dried at 50ºC overnight.

Synthesis of Zinc Oxide Nanoparticles
Zinc oxide nanoparticles were prepared via co-precipitation method according reported previously (Koutu et al. 2016, Kumar et al. 2013. Typically, sodium hydroxide aqueous solution was drop-wisely added with a slow rate to an aqueous solution of zinc sulfate with a molar ratio of 1:2 under vigorous stirring for 12 h. The obtained precipitate was centrifuged and washed with deionized water several time till pH (6.5 -7). The resultant precipitate was dried in an oven at 80°C and grinded to ne powder using a mortar. The powder obtained was calcined at 300°C for 2 h.

Synthesis of Manganese dioxide Nanoparticles:
Manganese oxide nanoparticles have been prepared using co-precipitation as reported by Kumar et al. (Kumar et al. 2013) and Cherian et al. (Cherian et al. 2016). Brie y; freshly prepared 100 ml aqueous solution of 2M NaOH was added drop by drop to 100 ml of 2M MnSO 4 . The solution was stirred continuously at 60ºC for 2h to precipitate the nanoparticles. The precipitate was then separated from the reaction mixture and washed several times with deionized water and dried in hot air oven at 100ºC overnight.

Characterization:
Page 4/17 The morphological structure of the as-prepared nano-metal oxides particles (e.g.; Fe 2 O 3 , ZnO and Mn 2 O 3 -NPs) using Transmission Electron Microscope (TEM) JOEL-JEM 2100 to investigate the micrograph of obtained samples under operating voltage 200 kV, and Xray diffraction (XRD) measurements have been carried out using a Bruker D8 advance X-Ray Powder Diffractometer operating with a Cu target with Kα1 = 1.54060 Å, Kα2 = 1.5444 Å, with 2θ step 4˚ and recorded at the range from 20˚ to 80˚. In addition; the size distribution of as-prepared nanoparticles was measured by Malvern zeta-sizer Nano ZS instrument with He/Ne laser (633 nm) at an angle of 173°c ollecting backscatter optics. Finally; Fourier-transform infrared spectroscopy (FT-IR) has been used for exploring the chemical groups functionalized their surfaces.

Plant Materials and Culture with nano-metal oxide
Explants of Populus alba nodal segments were cultured and acclimatized on Murashige and Skoog medium (MS medium) as reported by Ahmed and co-workers (Hewidy et al. 2019). Each nano-metal oxides of the heavy element (i.e., Fe, Zn, Mn) that used as micronutrients were prepared in three different concentrations of (i.e., 20, 40, 60 mg/L). These different concentrations were inoculated in MS jars separately. The nodal segments of Populus alba were cultured in these jars comparing to control. After that, these cultures were incubated at 25 o C for 4 weeks, then the measurements were estimated.

Morphological Measurements
The following seven morphological biometric parameters were measured for all these treatments: plantlet fresh weight, shoot length, shoot number, root length, leaf number, number of roots and rooting percentage.

Chlorophyll Estimation
Chlorophyll index was measured using OPTI-SCIENCES (CCM-200 plus) to indicate the photosynthesis rate in response to the different nano-heavy metals uptake.

Estimation of Nano-metal oxides particles in Populus Tissues
The nano-metal oxides particles (Fe, Zn and Mn) were estimated in the P. alba tissues. The plant tissues were dried in oven at 80 o C for 48h then grounded well to powder and xed known weight of samples (0.03 g) was digested with 5mL HNO 3 at 110 o C for 2 h, cooled, added 1mL of H 2 O 2 and boiled for 1 h. The clear digests were diluted to 50mL with triple-deionized water (Shirani Bidabadi 2020). The concentrations of the different nano-heavy metals were estimated using Agilent 4200 MP-AES according to manufacture instructions.

DNA Isolation and RAPD-PCR Bioassay
The genomic DNA of P. alba different treatments was isolated according to Lu et al. (Lu et al. 2011). RAPD-PCR bioassay was applied using 10 primers, only 6 of them gave clear reproducible bands. The reaction mixture was prepared as follow: 12.5 µl of master mix (Bioline®), 1.5 µl of both forward and reverse primers, 2 µl genomic DNA and diluted to 25 µl by adding 7.5 µl sterile ddH 2 O. The reaction program was performed in thermocycler Biometra® (Germany). The Ampli cation process was performed using 35 cycles of: 30 sec at 95°C; 30 sec at the melting temperature (Tm) of the speci c primer (Table 1), and 1 min at 72°C. The resulted PCR products were screened on a 1.4% (wt/vol) agarose gel (Agagel Mini; Biometra®). Gel images were analysis with BioRad Quantity One (4.6.2) software using the method described by Williamson and Campbell (Campbell 1997) and Bio-Rad Laboratories (2005). The statistical analysis of morphology, physiology and nano-heavy metals data in this work was performed using Minitab 19 with the following analysis: descriptive analysis, general linear model, grouping by Tukey pairwise comparison, correlation. A combined phylogenetic tree and correlation PCA blot was obtained from all data using Community Analysis Package (CAP software).

Synthesis and characterization of nano-metal oxides particles
Heavy metal-based oxides nanoparticles micronutrients including Fe 2 O 3 , ZnO and Mn 2 O 3 were prepared via co-precipitation methods as previously reported (Koutu et al. 2016, Kumar et al. 2013, Yang et al. 2014). In such cases, each of FeCl 2 , Zn(CH 3 COO) 2 and MnSO 4 have been used as metal precursors, which chemically hydrolysis in alkali medium forming metal-hydroxide. Then the metal-hydroxide precipitate was treated thermally to form metal-oxide nanoparticles. The formation mechanism for each of Fe 2 O 3 , ZnO and MnO 2 NPs was demonstrated as follow; The morphological structure has been con rmed via TEM and XRD measurements as shown in Figs. 1 and 2, respectively. TEM micrographs showed that Fe 2 O 3 NPs are quasi-spherical and relatively narrow size distribution. The average particle size was in a range of 35 ± 5 nm ( Figs. 1a and 1b). While in case of as-prepared ZnO NPs, the average particle size was in the range of 30 ± 5 nm with quasispherical shape (See Figs. 1c and 1d). Finally, the average particle size of Mn 2 O 3 was in range of 40 ± 10 nm, with agglomerated spherical nanoparticulate in vesicles (See Figs. 1e and 1f).
Furthermore, XRD measurements were carried out to investigate the crystallographic structure of as-prepared metal-based oxides nanoparticles of ZnO, Fe 2 O 3 and Mn 2 O 3 -NPs, as shown in Fig. 2. XRD patterns of ZnO NPs indicated to formation of hexagonal structure of ZnO nanoparticles (See Fig. 2a), where three strongest lines has been obtained at 31.28˚, 33.948˚ and 35.81˚ due to the (100), (002) and (101) Fig. 2c).
In addition, the colloidal properties based on DLS and zeta potential measurements for Fe 2 O 3 , ZnO and Mn 2 O 3 nanoparticles were investigated as shown in Fig. 3. It's clear that the hydrodynamic particle size of for as-prepared nanoparticles is remarkably increased, which is in agreement with their agglomeration presented in TEM micrographs as shown in Fig.1. This is due to their hydrophilicity nature. Furthermore, the strength of the steric forces of the functional groups on the surface of nanoparticles, as well as a steric interaction caused by the formation of a layer of water around the material (Marimón-Bolívar &González 2018). In this regard; Fe 2 O 3 nanoparticles was about 232 ± 29.24 nm (See Fig. 3a and Table 2 respectively See ( Fig. 3e and Table 2). The hydrodynamic particle size of ZnO NPs was 129.1 ± 16.6 nm, and the polydispersity index (PdI) is about 0.797 indicating to their fair dispersion in water. Moreover, Zeta potential ( ) of ZnO nanoparticles exhibits more electronegative was about -14 mV. Finally, the surface properties of the as-prepared metal-based oxides nanoparticles via exploring their FT-IR spectra, as shown in Fig. 4.  Fig. 4c).

Morphological Parameters and Chlorophyll Content
Seven morphological parameters included with a xed rooting percentage of (100%) have been investigated for all treatments. In this regard, the other six parameters varied and have signi cant differences upon their treatment with 3 different level of doses from metal oxide nanoparticles compared to control (Table 3). The chlorophyll index re ecting photosynthesis process varied but has no signi cant difference (Table 3). Moreover, the correlation relationship among all the parameters (Table 4) illustrating that nearly most of the parameter's range has positively affected each other, except rooting percentage which negatively affected the other parameters in response to metal oxide nanoparticles treatments.

Estimation of Metal oxide nanoparticles uptake in Populus alba
Populus alba treated with different nano-metal oxide. The metals uptake by P. alba was nearly the same with no signi cant differences (Table 5). This con rms the equivalent nano heavy metals-based oxides uptake by plants. Hence, the estimated morphological and chlorophyll parameters is related to the type of nano-metal oxides beside the concentration.

DNA Isolation and RAPD-PCR Bioassay
Genetic stability of Populus alba treatments by heavy metal-based oxides nanoparticles micronutrients have a great practical utility and commercial implications. In this study, we estimated the ngerprinting pro les of the regenerated culture by RAPD to con rm if the plantlets were genetically stable or not. A total of 10 random decamer-RAPD primers were tested for initial screening, among them only 6 primers gave clear and reproducible bands. The number of polymorphic bands ranged from 2 bands with Deca-7 primer, to 5 polymorphic bands with both Deca-11 and Deca-10 primers (Table 1). These 6 primers produced a total number of 23 bands with 8 polymorphic bands resulted in a total polymorphism percentage of 34.17% (See Fig. 5). The total molecular weights of all bands were ranged from 121-792 bp. Figure 6 showed a total phylogenetic tree resulted from all the combined morphological parameters, chlorophyll index and genetic variation. Figure 7 showed principal component analysis (PCA) blot which showed the correlation among the different nano-heavy metals effect on P. alba plant. The graph illustrated that the responses to nano-Mn concentrations are separated away, where control, nano-Fe and nano-Zn are grouped to each other.

Discussion
Recently, looking for economic and eco-friendly techniques for soil heavy metals remediation instead of conventionally used physicochemical techniques became urgent need. This is due to the biological techniques are based on phytoremediation not have any side effects on soil ecosystem; in addition, they are economically in contrast to other traditionally used techniques (Al-Khazan &Al-Zlabani , Gómez-Sagasti et al. 2012). The effect of Populus alba uptake to the nano-heavy metals micronutrients varied and re ected on morphological and chlorophyll parameters. As, some parameters were enhanced with exposure to heavy metal-based oxides nanoparticles micronutrients, where the others were inhibited comparing to control. The toxic effect of most heavy metals is caused by their bonding to -SH protein groups leading to the inhibition of enzyme activity and compromising their structure. They also substitute essential elements in biomolecules causing their de ciency (Van Assche &Clijsters 1990). High concentrations of metal ions in the soil limit the assimilation of important micro-and macronutrients by plants (Oleksyn et al. 1996).
The acclimatization of P. alba culture was performed by Ahmed et al. (Hewidy et al. 2019) and El-Sayed et al. (El-Sayed et al. 2019) to obtain the best growth conditions. Gomes and co-workers explored the morphological, anatomical parameters and chlorophyll content for affected vegetation area surrounding the mining company produced heavy metals such as Zn, Cd and Pb (Gomes et al. 2011).
Moreover, Sadak and Bakry suggested the using of ZnO as an example of metal oxide based nanomaterials than bulk form in the treatment of plant (Sadak &Bakry 2020). In such study; ZnO-NPs exhibited a good effect and improved each of biochemical, growth aspects, and consequent biomass yield.
Our ndings were in agreement with previous reports by Atteya et al. (Atteya et al. 2018) and Gheith et al. (Gheith et al. 2018), respectively. In their studies, a remarkable increase in the biomass yield and plant growth parameters observed upon the treatment of both of jojoba and maize with Zn precursors (Atteya et al. 2018, Gheith et al. 2018). However, Prasad and co-worker proved that ZnO-NPs has a remarkable effect on the enhancement of peanut plant growth, and biomass yield higher than ordinary Zn precursor such as ZnSO 4 at lower concentration. This is attributed to high adsorption of ZnO-NPs in the plant based on their smaller size than ZnSO 4 (Prasad et al. 2012). Finally, Taw k and co-worker reported that a signi cant increase in the growth parameters of Atriplex halimus upon treatment with ZnO-NPs in saline environments (Taw k et al. 2017).

Conclusion
This study evaluated the in vitro propagation ability, photosynthetic pigments contents and genetic variation of P. alba under pollution with heavy metal-based oxides nanoparticles causes variation in growth due to changes in their physiological and biochemical activities, which is especially true when the heavy metal involved plays no bene cial role in plant growth and development. Van Assche F, Clijsters H (1990): Effects of metals on enzyme activity in plants. Plant,Cell & Environment 13,[195][196][197][198][199][200][201][202][203][204][205][206] Wintz H, Fox T, Vulpe C (2002)