Co-culture (Miscanthus x giganteus and Pelargonium x hortorum) phytoremediation of lead-contaminated soil, impact of this trace metal on plant physiology.

doi:10.21203/rs.3.rs-3392319/v1

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Co-culture (Miscanthus x giganteus and Pelargonium x hortorum) phytoremediation of lead-contaminated soil, impact of this trace metal on plant physiology.

https://doi.org/10.21203/rs.3.rs-3392319/v1

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The industrial past of most regions in Lorraine and the intensification of activities onsoils has increased the number of polluted sites. To rehabilitate these areas, several methods can be employed. The use of ornamental plants has been little studied, even if these species can be used to rehabilitate a site while improving its aesthetics. In this study, co-culture of Miscanthus x giganteus (MxG) and Pelargonium x hortorum (PxH) was used to clean up a soil mainly contaminated by Pb and trace metals. At the end of the experiment, lead concentrations were measured in the soil and plants. Furthermore, auxins, jasmonic acid, and salicylic acid were also measured to evaluate the defense mechanisms of the plants in front of pollutants. The results showed a reduction in lead concentrations following the phytoremediation process implemented and that PxH was able to extract lead from the soil. Results showed that co-culture was beneficial to the development of MxG. Concerning the molecules synthesized by the plants under stress conditions, only salicin was found in MxG roots and leaves in particular for plants grown in individual culture. According to the results obtained, it seems that co-culture can improve soil quality without their development being affected.

Soil

Lead

Phytoremediation

Miscanthus x giganteus

Pelargonium x hortorum

Phytohormones

Currently, soil pollution is mostly concentrated in industrial wastelands, which are formed from sites that were contaminated by previous industrial activities and are usually located near urban areas. They require intervention to re-enable their usage (“EUGRIS: the European Groundwater and Contaminated Land Information System,” n.d.). Due to its effectiveness and low cost of long-term application, phytoremediation seems to be the most beneficial remediation technique to mitigate these kinds of contaminations. The process of phytoremediation involves the use of plants for the degradation and removal of soil contaminants. The main benefit of this biological treatment is that it restores soil functions that may have been damaged by previous anthropogenic activities (Sterckeman et al., 2011). In addition, soil evolution influences the bioavailability and mobility since contamination is relatively recent in most cases (Froger et al., 2021). Lead is a mineral element naturally present at low concentrations in the soil, however, anthropogenic activities largely contribute to its presence in the environment (Cecchi, 2008). Lead’s behavior depends on several environmental factors, including the soil's pedological and physico-chemical characteristics (Baize, 2020), as well as the element's mobility (Juste, 1988). Generally, lead has low mobility in the soil, mainly accumulating in the superficial horizons (Sterckeman et al., 2000). However, plants can change the physico-chemical characteristics of soils, which can modify the speciation of metals and thus vary their mobility (Hinsinger, 2001). Indeed, roots are able to mobilize lead present in the soil by modifying the ionic concentrations in the rhizosphere (Hinsinger, 1998). Roots, aerial parts or both can absorb lead with increased mobility (Cecchi, 2008). The toxicity of lead depends on its concentration in the soil, the properties of the soil, and the plant species (Seregin and Ivanov, 2001). Miscanthus x giganteus (MxG) was chosen for its capacity to treat polycyclic aromatic hydrocarbons (PAHs) by phytodegradation and to stabilize metals (Wechtler et al., 2020), its low care requirements, and the possibility of using its biomass. However, the concentration of pollutants in the soil can affect the establishment of MxG rhizome. A second plant, Pelargonium x hortorum (PxH) was selected for its ability to accumulate heavy metals such as lead, zinc and cadmium by phytoextraction (Manzoor et al., 2020) which should reduce the concentration of lead, the main pollutant of our soil model. Using ornamental plants, such as MxG and PxH, has some advantages such as seed or rhizome availability or non-food use that reduces transfers in the food chain (Garcia et al., 2018; Liu et al., 2018). However, when confronted to biotic or abiotic stress, plants can synthesize certain defense molecules, such as salicylic acid or jasmonic acid. Salicylic acid (SA) is a conserved molecule in all plant species (Gomi, 2020). SA is implicated in several biochemical and physiological processes such as seed germination, growth regulation, and floral induction (Hayat et al., 2010). SA is also implicated in many defence mechanisms in plants, and in the regulation of responses against biotic and abiotic stress. The genotype of the plant, in addition to environmental factors, affects the efficiency of the plant defence responses (Gomi, 2020). For example, reducing damage caused by heavy metals is one of the adaptive strategies of plants against abiotic stress (Wójcik and Tukiendorf, 2005). Many studies have validated the protective role of SA (Guo et al., 2019) and in decreasing the toxicity of heavy metals (Hayat et al., 2010) such as lead (Mishra and Choudhuri, 1999). The jasmonate pathway is one of the main signalling pathways which allow plants to defend themselves against biotic or abiotic stress (Erb et al., 2012). Jasmonic acid (JA) contributes to plant resistance by acting as a precursor for defensive responses (Tretner et al., 2008). The conserved jasmonate pathway triggers a stress-adapted response facilitated by specific and non-specific stress recognition (Erb et al., 2012). This study aims to characterize the evolution of the quality of Pb-contaminated technosol after a co-culture phytoremediation (MxG – PxH), and more insight into the defense mechanisms potentially deployed by plants when they are confronted by these pollutants in mesocosms for a 6-month duration.

Soil preparation and experimental design

The experimental design includes two plant species: Miscanthus x giganteus (MxG) and Pelargonium x hortorum (PxH). Two different soil conditions are tested. For the first soil condition (Control), the technosol used contained 36.9 mg/kg of Pb. For the second condition (Pb-contaminated), the control technosol was spiked with lead by adding 1000 mg Pb/kg of soil. This concentration was chosen as ten-fold the natural concentration of Pb in the north of Moselle (France) (Darmendrail, 2000). Concentration in all pots after Pb-contamination was controlled at the beginning of the experiment to be sure that all contaminated conditions contained comparable concentration of Pb (Fig. 2a). All the experimental pots were filled with 9 kg of pre-sieved dried soil for each of the conditions tested.

Miscanthus x giganteus rhizomes were ordered from “Rhizosfer” while Pelargonium x hortorum underwent a germination stage of about 3 weeks before planting since plants must have 4 leaves before being introduced into the test soil.

For this test, one rhizome of MxG or four PxH were planted individually for simple culture pots; one rhizome of MxG and four PxH were planted in co-culture pots (called “CO") to verify the interest of their association in the remediation process. Pots were kept without planting (called “NP”) to compare the evolution of the soil composition with and without the action of the plants. Two pots were realized by condition. The mesocosm experiment was carried out under controlled conditions (70% humidity, 22°C and a photoperiod of 16h day − 8h night) in a phytotron during a total of 158 days.

Determination of trace metals concentration

This analysis aims to quantify the concentration of trace metals by spectrophotometric methods in the soil to evaluate the remediation process and in plants to determine Pb translocation which is a main important factor to valorize the biomass. Before mineralization, samples are dried and sieved to 200 µm. Then 0.5 g of soil sample and a mixture of hydrochloric (6 mL) and nitric acid (2 mL): "aqua regia", (or a mixture of hydrogen peroxide and nitric acid for plant samples), are added to the tubes and mineralization is carried out in microwave (MARSXpress5, CEM™). The mixture was heated at 170°C for 5 min and maintained for 13 min. After cooling, the samples were filtered and diluted to 50 mL with distilled water and stored at 4°C until the analysis. Analysis of Pb concentrations was carried out by inductively coupled plasma-mass spectrometry (ICP-MS; ICAP GUO 6000, ThermoFisher ™). The same protocol was applicated to plants using hydrogen peroxide (3 mL) and nitric acid (5mL) instead of aqua regia and the samples were filtered and diluted to 25 mL with distilled water.

Physiological monitoring

The flowering and chlorophyll content of both plants were monitored every fortnight. Chlorophyll content was measured by a non-intrusive method using a chlorophyll meter (SPAD-502 Plus, KONICA MINOLTA). The height and the number of Miscanthus x giganteus stems were also monitored every fortnight. All pots were weighed every week to optimize the plants' watering (70% of the water retention capacity of the soil).

Analysis of phytohormones

Several phytohormones, including jasmonids and salicylates, were analyzed by high-performance liquid chromatography (LC-MS) at the end of the mesocosm experiment. Before extraction, the plants were dried and ground to 2 mm and 5 g of the sample was mixed with methanol and toluene (70/30 ratio). The extraction was performed in the microwave (MARSXpress5, CEM™). The mixture was heated at 100°C for 15 min and maintained for 15 min. After cooling, the samples were filtered and evaporated to dryness and taken up in acetone before HPLC analysis.

Statistical analysis

Results obtained are analyzed with R Studio software version 1.2.1335. The normality of values distribution is checked by the Shapiro-Wilk test while the homogeneity of variances was tested by Bartlett’s test.

Non-normal data and data that was not homogenous were analyzed by the Kruskal-Wallis test followed by the Nemenyi posthoc test. Normal data with homogenous variances was analyzed by ANOVA followed by the Tukey HSD posthoc test. All tests are performed at the 5% significance level.

3.1. Lead concentrations

3.1.1. Soil

To determine the effectiveness of the selected plants for lead remediation, lead concentration in the soil was measured with ICP-MS. Figure 1 shows the results obtained for lead concentrations in soils of different tested conditions. In the case of the control technosols (a), no evolution of the lead concentration could be observed, and the final concentrations measured were similar in all conditions. Concerning the Pb-contaminated condition (b), at the beginning of the experiment, the control technosol was spiked with 1000 mg/kg of lead. The control measurements of initial concentrations in all pots show no statistical difference: all exposures are considered as similar. Lead concentrations measured after 6 months of cultivation were statistically lower than the initial time (T₀) in all culture conditions in technosol spiked with 1000 mg/kg. A loss of 200 mg Pb/kg was observed in the non-planted technosol indicating leaching of Pb. This could be explained by the technosol contamination process which does not include soil aging. The lowest lead concentration was found is soil cultivated with PxH (242.6 mg Pb/kg) followed by MxG (296.5 mg Pb/kg) and co-culture (476.15 mg Pb/kg). These results suggest that plants influenced the soil facilitating the Pb remediation. The differences observed between the modalities (PxH, MxG, CO) could be related to the different remediation strategies used by plants.

Thus, the lowest concentration of metal is measured for PxH which is classified as a Pb accumulator. MxG rather tends to stabilize the metals in the soil (Wechtler et al., 2020) and the technosol has higher Pb concentrations than for the culture with PxH alone. Finally, co-culture (CO) seems to be the least effective for the remediation of lead, probably because of the competition between the two plant strategies of remediation.

3.1.2. Plants

To verify the lead absorption capacity by the plants in individual or co-culture, an analysis of lead concentration in plants was performed by ICP-MS. Figure 2 represents the lead concentrations contained in each plant in mg/kg of dry matter (DM). Results of plants grown in the control technosol are shown in Figure 2 (a) and those of plants grown in the Pb-contaminated one are shown in Figure 2 (b). The results obtained for plants grown in the control technosol show that the lead concentrations in individual cultures are not significantly different between MxG (13.06 mg/kg) and PxH (7.114 mg/kg). However, when MxG and PxH are co-cultured, MxG accumulates more lead (18.256 mg/kg) than PxH (2.23 mg/kg). In MxG cultures, lead levels in plants grown on contaminated soil (396.51 mg/kg) and co-culture (396.813 mg/kg) were statistically similar. Thus, co-culture had no effect on lead accumulation by MxG. In contrast, with PxH, the lead concentration was higher in single cultures (143.192 mg/kg) than in co-cultures (57.693 mg/kg). Comparing the two plants under contaminated conditions, MxG accumulates more lead than PxH. Hence, these results underline the ability of both plants to accumulate lead in accordance with the results of Manzoor et al., (2020) concerning the ability of PxH to extract lead. On the other hand, it is necessary to measure lead in each part of MxG separately (roots, rhizome, and leaves) to determine whether the aerial parts of the plant can be reused.

To verify the possibility of valorization of MxG aerial parts, an analysis of lead concentration in each part of the plant (roots, rhizomes and leaves) was performed by ICP-MS. Figure 3 represents the lead concentrations contained in each part of MxG in mg/kg of dry matter (DM) for plants grown in the control technosol (Figure 3a) part and for plants grown in the Pb contaminated (Figure 3b). Concerning plants grown in control soil, in individual cultures, lead concentrations in MxG roots (8.9 mg/kg DM) show significant differences with rhizome (3.580 mg/kg) and leaves (0.530 mg/kg). In co-culture, results show that all lead concentrations are equivalent for rhizomes (2.011 mg/kg) and leaves (2.256 mg/kg) except for MxG roots (13.989 mg/kg). The results indicate that MxG roots accumulate more lead than other organs. Furthermore, the results display significant differences between culture conditions for MxG roots, showing that co-culture seems to improve lead accumulation.

As for plants grown in Pb-contaminated soil, in individual culture, results indicate that roots contain more lead (304,399 mg/kg) than rhizome (81.998 mg/kg) and (10.114 mg/kg) aerial parts. Lead concentrations in each measure part of the plant were significantly different in individual cultures. Moreover, the concentrations measured in rhizomes in co-culture (70.909 mg/kg) and in the aerial parts (37.507 mg/kg) are significantly lower than those of the roots (288.397 mg/kg). According to the results, the majority of Pb is stored in the subterranean parts of MxG regardless of cultural conditions. We can state that no Pb translocation took place to the aerial parts of the plant. These results confirm those of Laval-Gilly et al., (2017) and may facilitate valorization of the aboveground biomass. As PxH is a seasonal plant, the aerial parts can be harvested with the accumulated lead. This will lead to a gradual extraction of Pb from the soil, facilitating the establishment of MxG by decreasing the stress of the contaminated soil on the rhizomes.

Table 1. Bioconcentration factors of Miscanthus x giganteus and Pelargonium x hortorum under the different conditions tested.

Conditions	Plants	BCF
Control *Individual culture*	MxG	0.980 ± 0.141
Control *Individual culture*	PxH	0.521 ± 0.037
Control *Co-culture*	MxG	1.343 ± 0.141
Control *Co-culture*	PxH	0.164 ± 0.021
Pb contaminated *Individual culture*	MxG	1.356 ± 0.141
Pb contaminated *Individual culture*	PxH	0.351 ± 0.037
Pb contaminated *Co-culture*	MxG	0.834 ± 0.141
Pb contaminated *Co-culture*	PxH	0.121 ± 0.021

In addition, the bioconcentration factor (BCF) of the different modalities was calculated. The BCF represents the ratio between the concentration of lead in the medium (soil) and the concentration in the organism (plant). Results obtained for Miscanthus x giganteus and Pelargonium x hortorum are shown in Table 1.The results show that MxG has the highest BCF regardless the growing conditions whether in single cultures or co-cultures. In general, BCFs tend to be lower in plant co-cultures, except for the MxG in the control soil. Although the BCFs of Pelargonium x hortorum are lower than those of Miscanthus x giganteus, the results show that the plant can accumulate lead.

Table 2. Bioconcentration factors of different parts of Miscanthus x giganteus under the different culture conditions tested.

Conditions	Plants	BCF
Control *Individual culture*	MxG: Roots	0.671 ± 0.141
	MxG: Rhizomes	0.269 ± 0.081
	MxG: Leaves	0.039 ± 0.009
Pb contaminated *Individual culture*	MxG: Roots	1.040 ± 0.141
	MxG: Rhizomes	0.280 ± 0.021
	MxG: Leaves	0.035 ± 0.011
Control *Co-culture*	MxG: Roots	1.028 ± 0.093
	MxG: Rhizomes	0.148 ± 0.033
	MxG: Leaves	0.166 ± 0.011
Pb contaminated *Co-culture*	MxG: Roots	0.605 ± 0.043
	MxG: Rhizomes	0.149 ± 0.010
	MxG: Leaves	0.079 ± 0.009

Furthermore, Table 2 shows the BCF of the different parts of Miscanthus x giganteus (roots, rhizomes and leaves). The BCF was measured separately for each part to determine whether the aerial parts of the plant can be valorised.

Results showed that bioaccumulation is higher in the roots of Miscanthus x giganteus regardless of the culture conditions (0.671 for the control condition and 1.04 for the Pb-contaminated condition).

The BCF of rhizomes (0.148 in the control condition and 0.280 in the Pb contaminated condition) and leaves (0.039 in the control condition and 0.079 in the Pb contaminated condition). In general, those concentrations were lower than in the roots of MxG, confirming the possibility of valorization of the aboveground biomass of MxG. Finally, according to the Table 1, the bioaccumulation of Pelargonium x hortorum is slightly higher than that of MxG rhizomes, confirming the ability of PxH to extract lead.

3.2. Height and stems

Table 3. Number of height and stems of Miscanthus x giganteus under the different conditions tested.

Conditions	Height	Stems
*Control (MxG )*	79.5 cm ± 1.7	6 ± 1.4
*Control (Co-culture)*	78.5 cm ± 3.2	10 ± 4.2
*Pb contaminated (MxG)*	79.5 cm ± 1.4	17 ± 0.7
*Pb contaminated (Co-Culture)*	97.5 ± 5.1	12 ± 2.1

Only the aerial parts of MxG can be valorized for lead accumulation. We compared the heights of MxG in the different culture conditions and the number of stems to study the most favorable conditions for biomass production. In our experiments, MxG had the largest size in co-culture with Pelargonium x hortorum (97.5 cm). By contrast, MxG alone in the same soil conditions, showed the highest number of stems (17 stems). This increase of the number of MxG stems confirms that the co-culture is a certain way, beneficial for MxG development in lead-contaminated soils.

3.2. Physiological monitoring

3.2.1. Chlorophyll content

Figure 4 shows chlorophyll contents in plants grown on control soil (a) and in Pb-contaminated soil (b). In the control technosol, there was no statistical difference in chlorophyll content between plants at the initial stage of development corresponding to the beginning of the experiment. After 6 months of cultivation, chlorophyll levels were statistically lower for all conditions except the PxH single cultures. This may reflect stress related to the low available root space in the pots, which is lower in the case of co-cultures and culture of MxG alone compared to PxH alone. In the case of plants grown on Pb-contaminated technosol, chlorophyll contents profiles are equivalent to the control technosol modalities. Root space therefore seems to be a major parameter of chlorophyll stress for our model plants compared to the soil contamination.

3.2.2. Plant hormones

Following assays of the various phytohormones in our samples, the only molecule present was salicine. Table 3 present results obtained for salicin contents.

Table 3. Salicin content (in mg/kg) in plant extracts under the different conditions tested.

Conditions	Plants	Salicin
Control *Individual culture*	MxG: Roots	120.4 ± 4.7
	MxG: Rhizomes	< LQ
	MxG: Leaves	88.3 ± 10.3
	PxH	< LQ
Pb contaminated *Individual culture*	MxG: Roots	69.9 ± 4.3
	MxG: Rhizomes	< LQ
	MxG: Leaves	< LQ
	PxH	< LQ

Control *Co-culture*	MxG: Roots	< LQ
	MxG: Rhizomes	< LQ
	MxG: Leaves	59.5 ± 7.7
	PxH	< LQ
Pb contaminated *Co-culture*	MxG: Roots	< LQ
	MxG: Rhizomes	< LQ
	MxG: Leaves	< LQ
	PxH	< LQ

After performing an assay, salicin was detected in leaves of MxG under control conditions (120.4 mg/kg), in single culture (88.3 mg/kg), and in co-culture (59.7 mg/kg). The roots of MxG also contained salicin when the plant was grown alone, in both control (120.4 mg/kg) and Pb-contaminated (69.6 mg/kg) soil conditions. The size restrictions imposed on the plants culture pots could explain the presence of salicin, due to the stress it could induce.

Results indicate that plant physiology is not affected by the presence of lead, which is immobilized in underground parts of plants. But results highlight that MxG is more sensitive to growing conditions, and in particular to the place allocated to its roots.

4.1. Lead accumulation

Several studies, including those by Bastia, Al Souki & Pourrut, (2023) and Laval-Gilly et al., (2017) have highlighted the benefits of using Miscanthus x giganteus to stabilize trace metals in soils. High lead accumulation may be linked to the size of the culture pot, as shown by the study of Al Souki et al., (2022), the same MxG cultivar accumulated twice as much trace elements when grown in pots, with a limited volume and soil quantity compared to the field. Our results are in line with those obtained by (Wanat et al., 2013), who showed that roots are organs which accumulate the most metals, and leaves present the same lead concentrations as (Bastia et al., 2023) for MxG in individual culture. (Zgorelec et al., 2020; Pidlisnyuk et al., 2019) have also demonstrated the ability of MxG to stabilize soil contaminated by trace elements by accumulating them in their underground parts decreasing the accessibility in the aerial parts. Moreover, the results obtained for lead concentrations in MxG leaves coincide with those obtained by (Laval-Gilly et al., 2017) which showed that the metals were more particularly concentrated in the rhizome, allowing the plant’s biomass to be valorized without reintroducing the contaminants into the air. According to (Manzoor et al., 2020), P. hortorum is able to increase the available fraction of Pb and accumulate it into the plant for about 94 ± 5 mg/kg to 2000 mg/kg. These results are in good agreement with those obtained for our study as the accumulation of lead by PxH varies between 70 and 140 mg/kg which also confirms the plant's ability to accumulate lead (Khan et al., 2021; Arshad et al., 2020; Jost and Jost-Tse, 2018). About co-cultivation, our results show that lead concentrations in the soil are lower when plants are grown alone than when they are co-cultured. Similarly, when we look at lead concentrations in plants, on contaminated soil, there is less lead in PxH when it is co-cultured with MxG. On the other hand, there is no significant effect of co-culture on lead content in MxG, even when looking individually at roots, rhizome and leaves, there is no difference between the two culture conditions. Overall, co-culture did not increase the yield of plants to extract lead. In our study conditions and for this parameter, MxG and PxH appear to be more efficient when grown individually. This may be explained by the size of the experimental pots, as inter-species competition for different nutrients may have reduced at the same time the efficiency of MxG and PxH in extracting lead. These results can be compared with those of (Wechtler et al., 2020) where MxG was combined with white clover, resulting in better PAH removal. This comparison highlights that the effectiveness of plant associations depends not only on soil characteristics but also on species compatibility.

4.2. Physiological monitoring

Concerning chlorophyll content, no significant differences were observed between the initial and the final time. These results differ from those found by (Al Souki et al., 2022; Żurek et al., 2014; Técher, 2011) as they demonstrated a decrease in chlorophyll and carotenoids levels in Miscanthus x giganteus grown on soils also polluted with trace metals. Results obtained also in contrast to those of (Gul et al., 2020) in which the content of chlorophyll was the lowest at the highest trace metals concentrations. Nonetheless, our results are similar to those of (Bastia et al., 2023) which highlight that no significant differences were detected between the control and the highest lead concentration (700 mg/kg). In addition, the absence of impact in our experimental conditions is in accordance with studies of (Zeeshan et al., 2020; Zengin and Munzuroglu, 2005), which showed that lead is one of the trace metals that least affects these photosynthetic pigments. Furthermore, our results showed that the number of stems is not affected when the soil was contaminated with lead (1000 mg/kg) whether grown individually or in co-culture. Our results contrast with the results obtained by (Al Souki et al., 2022) who have also studied soil contaminated with trace metals (Cd, Pb, Zn) and showed that the number of stems decreased when MxG was grown on contaminated soil. But our results are also in accordance with those of (Figala et al., 2016), who showed that MxG is able to maintain growth rate even on industrially highly contaminated substrates. In our case, lead phytoextraction by PxH could reduce soil toxicity, inducing an increase in MxG size rather than having an impact on the number of stems. According to our results, the Miscanthus x giganteus is higher when co-cultured on contaminated soil, so co-culture seems to be beneficial for plant height. On the other hand, the number of shoots is increased in co-culture for plants in the control soil, but there are fewer shoots for plants in co-culture in the contaminated soil. There are several possible explanations for these results: firstly, in the control soil, stress on plants could be inter-species competition, and MxG would therefore has chosen to focus on the number of shoots to grow with PxH. Secondly, when the soil is contaminated, the plants are confronted with the stress of contamination in addition to co-cultivation, so MxG may prioritize height development over its number of shoots when confronted with several combined stresses. Thus, co-culture does not appear to have a beneficial effect on MxG biomass, since in our study conditions we observed either, for control soil, an increase in shoots but a decrease in height, or, for contaminated soil, an increase in height but a decrease in shoots. Then, concerning chlorophyll, no effect was observed, as co-culture had no effect on chlorophyll pigment levels.

4.3. Plant hormones

Several studies have shown that some defense molecules are induced by salicylic acid, while others are stimulated by jasmonic acid (Mikkelsen et al., 2003). Salicin, a molecule that induces the production of salicylic acid to protect the plant against aggressors was the only hormone detected in the plant samples. Space restrictions imposed on plants by the pot’s size can explain the presence of salicin in some conditions. Indeed, plants cannot grow as well in 9 kg of soil as they can in environmental conditions and this restriction prevents the root system to spread as much as in environmental conditions, which could explain the synthesis of molecules related to stress. According to the study of (Franko and Kobyletska, 2016), salicylic acid altered the cadmium accumulation by plants. For example, in corn cultures, cadmium content increased in roots while it decreased in leaves. These results are consistent with those obtained for Miscanthus x giganteus because lead levels are much higher in the roots than in the aerial parts. In addition, lead accumulation by plants correlates with cadmium and zinc accumulation, which have similar properties (Kramer and Bennett, 2006). The results obtained seem to indicate that Miscanthus x giganteus is more sensitive to growing conditions, and in particular to the space allocated to the roots, because in doped conditions MxG was able to develop without being affected by the presence of lead, which is immobilized in the underground parts. Focusing on the co-culture aspect, we note that salicin concentrations are higher in individual cultures than in co-culture. As salicin is at the origin of salicylic acid synthesis, co-culture seems to reduce the synthesis of stress molecules in both Miscanthus x giganteus and Pelargonium x hortorum.

The aim of this study was to characterize potential changes in the quality of a lead-contaminated technosol following phytoremediation treatment. The main objectives of the experiment were to study soil composition before and after plant action, and to ensure that a cultural association is possible and beneficial between Miscanthus x giganteus and Pelargonium x hortorum. The results show that both plants were able to grow without being affected by any stress that might have been induced by the presence of lead. Moreover, the results showed that co-culture did not improve lead extraction yield or MxG biomass production. On the other hand, the results of phytohormone assays seem to show that co-culture reduces salicin synthesis. It would therefore be interesting to study the ethylene pathway, which is the third plant defense pathway, as well as to analyze antioxidant enzymes to get an overview. This type of work can help to better understand the physico-chemical and biological mechanisms involved in environmental risk. Indeed, these experiments will enable better management of soils contaminated by TMEs, and PAHs. Finally, following these selection processes, larger-scale cultivation could be set up to remediate the pollutants on the study site.

Statements & Declarations

Acknowledgments: We would like to thank the Phytodia laboratory for phytohormones analyses.

Ethical Approval: Not applicable

Consent to Participate: Not applicable

Consent to Publish: All authors read and approved the final manuscript. All authors agreed with the content and that all gave explicit consent to submit.

Funding: The authors would like to thank Syndicat Mixte d’Études et d’Aménagement des Portes de l’Orne (SMEAPO) for their financial supports (Grant number : 2020-01115).

Competing Interest: The authors have no relevant financial or non-financial interest to disclose.

Author Contributions: Sarah Berns designed and conducted the study. Material preparation and data collection were performed by Sarah Berns, Antoine Bonnefoy and Lucas Charrois. Sarah Berns, Philippe Laval-Gilly and Jaïro Falla-Angel contributed to the data analysis. The first draft of the manuscript was written by Sarah Berns and Philippe Laval-Gilly and Jaïro Falla-Angel commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Co-culture (Miscanthus x giganteus and Pelargonium x hortorum) phytoremediation of lead-contaminated soil, impact of this trace metal on plant physiology.

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Abstract

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1. Introduction

2. Materials and Methods

3. Results

4. Discussion

4.1. Lead accumulation

4.2. Physiological monitoring

4.3. Plant hormones

5. Conclusion

Declarations

References

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