# Effects of different shading levels on the growth of Bornmuellera emarginata, a nickel hyper accumulator for use in agromining

Dimitrios Kyrkas, Nikolaos Mantzos, Paraskevi Beza, Vasileios Papantzikos, Chrysanthe Andreaki, Erini-evaggelia Konstantourou, Guillaume Echevarria, Maria Konstantinou, Panayiotis G Dimitrakopoulos

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Dimitrios Kyrkas, Nikolaos Mantzos, Paraskevi Beza, Vasileios Papantzikos, Chrysanthe Andreaki, et al.. Effects of different shading levels on the growth of Bornmuellera emarginata, a nickel hyper accumulator for use in agromining. Ecological Research, 2024, 39 (4), pp.460-470. 10.1111/1440-1703.12456. hal-04665040

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# Effects of different shading levels on the growth of *Bornmuellera emarginata*, a nickel hyperaccumulator for use in agromining

Dimitrios Kyrkas<fnref n="1" />,<fnref n="2" /> ID

Nikolaos Mantzos<fnref n="1" />

Paraskevi Beza<fnref n="1" />

Vasileios Papantzikos<fnref n="1" />

Chrysanthe Andreaki<fnref n="1" />

Erini-Evaggelia Konstantourou<fnref n="1" />

Guillaume Echevarria<fnref n="3" />,<fnref n="4" /> ID

Maria Konstantinou<fnref n="5" /> ID

Panayiotis G. Dimitrakopoulos<fnref n="2" /> ID

## Correspondence

Dimitrios Kyrkas, Department of Agriculture, University of Ioannina, Arta, Greece.

Email: dkyrkas@uoi.gr

[Correction added on 12 April 2024, after first online publication: The copyright line was changed.]

## Abstract

The Greek endemic *Bornmuellera emarginata*, known for its Ni hyperaccumulation capacity, is a promising candidate species for use in agromining. At the same time, the installation of photovoltaic systems on agricultural land has recently been increasing. Field observations that populations of this species usually occupy shaded places under shrubs or trees led us to study the effect of different shading levels on the growth of *B. emarginata*, with a view to combine the cultivation of the species with photovoltaic panels in the same area. The experiment was conducted under greenhouse conditions and lasted 210 days after sowing (DAS). Seeds were sown directly into 2-L pots filled with ultramafic soil collected from the field. Three plant samplings (81, 140, and 210 DAS) were conducted during the experiment to determine a set of morphological and physiological parameters of the species (fresh and dry plant biomass, plant height, number of lateral shoots, total number of leaves, proline and chlorophyll concentrations) alongside with its nickel yield at different levels of shading (0%, 20%, 35%, 60%, and 90%). Growth of *B. emarginata* was only significantly affected at the highest shading level. Nickel yield of the species increased over time and was maximized at the end of the experiment. The highest nickel yield per plant was observed at the intermediate shading level (35%). *Bornmuellera emarginata* can be considered as a candidate nickel hyperaccumulator for cultivation in ultramafic or nickel-polluted areas under photovoltaic panels with corresponding shading, providing important environmental benefits.

## KEYWORDS

Brassicaceae, chlorophyll, morphology, proline, ultramafic soil

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

© 2024 The Authors. *Ecological Research* published by John Wiley & Sons Australia, Ltd on behalf of The Ecological Society of Japan.

<footnote n="1">¹Department of Agriculture, University of Ioannina, Arta, Greece</footnote>

<footnote n="2">²Biodiversity Conservation Lab., Department of Environment, University of the Aegean, Mytilene, Greece</footnote>

<footnote n="3">³Université de Lorraine, INRAE, Laboratoire Sols et Environnement, Nancy, France</footnote>

<footnote n="4">⁴ECONICK, Lunéville, France</footnote>

<footnote n="5">⁵Department of Agriculture, International Hellenic University, Sindos (Thessaloniki), Greece</footnote>
# 1 | INTRODUCTION

Soils developed on ultramafic rocks usually have unique chemical characteristics, such as high concentrations of certain metals (Ni, Cr, Co, Fe, and sometimes Mn), low Ca/Mg ratio and lack of major macronutrients (N, K, P) (Echevarria, 2021; Kazakou et al., 2008; Proctor & Woodell, 1975). The geochemical peculiarities of ultramafic soils are reflected in the composition of the flora and vegetation, often becoming centers of great floristic diversity worldwide (Brooks, 1987; Jakovljević et al., 2022). Furthermore, the productivity of agricultural land found on ultramafic soils is often seriously affected, thus becoming unproductive and unattractive for conventional agriculture. The ultramafic flora is rich in species that hyperaccumulate Ni as the dominant element in this type of substrate. Hyperaccumulators are species that are able to take up certain elements from the substrate in concentrations 100–1000 times or more higher than nonaccumulator species from the same area (Bergmeier et al., 2009; Harrison & Inouye, 2002; van der Ent et al., 2013).

Nickel hyperaccumulators have come to the attention of applied research as plants suitable for phytomining/agromining activities (Baker & Brooks, 1989; Chaney et al., 1995, 2021; Nascimento et al., 2022; Nkrumah et al., 2016; van der Ent et al., 2015). Nickel agromining represents a new form of agriculture which can generate income from low-productivity agricultural land derived from ultramafic bedrock. In this case the final product is a metal, nickel, or pure nickel compounds, depending on the needs of the market. The process of nickel agromining consists of cultivating suitable hyperaccumulator species, the subsequent harvesting of the aerial biomass, its drying, incineration and processing, generating a bio-ore (ash) with up to 25% nickel (Chaney et al., 2007; van der Ent et al., 2015).

Europe has become a potential target for nickel agromining due to the extensive area of ultramafic rocks in the Balkan Peninsula. The first field trials, conducted in Albania focusing on the Mediterranean hyperaccumulator *Odontarrhena muralis* (syn. *Alyssum murale*), began in 2005 (Bani et al., 2010, 2021; Bani & Echevarria, 2019; Bani, Echevarria, Sulçe, & Morel, 2015; Bani, Echevarria, Zhang, et al., 2015). Nickel agromining field trials have recently been established in Greece as well, focusing on other native nickel hyperaccumulating species (e.g., *Bornmuellera emarginata*, *Bornmuellera tymphaea*; Kyrkas et al., 2023) in addition to *Odontarrhena muralis* (Kidd et al., 2018; Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Dimitrakopoulos, & Konstantinou, 2019; Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Tognacchini, et al., 2019).

*Bornmuellera emarginata* (Boiss.) Rešetnik is a Greek endemic, nickel hyperaccumulator of the Brassicaceae

family and an obligate serpentinophyte found on ultramafic substrates of the Pindus Mountain range (Dimopoulos et al., 2023; Jakovljević et al., 2022; Stevanović et al., 2003). Field observations showed that populations of this species usually occupy shaded places under shrubs or trees. Highest foliar Ni concentration observed up to date in *B. emarginata* when growing in their natural environment is 34.9 g kg⁻¹ DW (Jakovljević et al., 2022; Reeves et al., 1980, 1983). Recently, *B. emarginata* has been studied for its potential in Ni agromining in the Mediterranean area (Chardot et al., 2005; Kidd et al., 2018; Pardo et al., 2018; Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Dimitrakopoulos, & Konstantinou, 2019; Hipfinger et al., 2022). The highest nickel yield recorded for *B. emarginata* is 151 kg ha⁻¹, which was achieved from field experiments in its natural habitat in Greece (Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Dimitrakopoulos, & Konstantinou, 2019; Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Tognacchini, et al., 2019). The agronomic practices followed to date in Greek field trials showed that *B. emarginata* is a promising candidate for use in Ni agromining (Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Dimitrakopoulos, & Konstantinou, 2019; Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Tognacchini, et al., 2019).

Nowadays, as global energy demand is increased, renewable and environmental-friendly energies play an essential role. Among them, solar power is the most abundant and available source. Photovoltaics solar energy is an attractive strategy generating energy by converting solar to electrical energy (Dupraz et al., 2011; Touil et al., 2021). It is often observed that the installation of photovoltaics systems takes place on agricultural land which will result in a land-use conflict between energy and agricultural production (food, metal, etc.) (Weselek et al., 2019). Associating food or other crops such as “metal crops” and solar photovoltaics on the same land, which is referred as agrivoltaic systems or agrophotovoltaics, is one of the most promising emerging techniques in agriculture and is attracting significant research attention recently (Dinesh & Pearce, 2016; Dupraz et al., 2011; Li et al., 2023; Santra et al., 2017; Touil et al., 2021).

Greece is an area of interest for nickel agromining activities as about 3–4% of its territory is occupied by ultramafic substrates (Chatziliadou et al., 2023; Kidd et al., 2018). At the same time, the installation of photovoltaic systems on agricultural land (ultramafic land is included) has recently been increasing. Considering that *B. emarginata*, a promising candidate species for use in Ni agromining, inhabits shaded places under shrubs or trees, it would be interesting to test its potential for accumulation under the shading
conditions provided by photovoltaics. To achieve this objec-
tive, the growth of the species was measured through a set
of morphological and physiological parameters along with
determination of its nickel yield at different levels of shad-
ing under greenhouse conditions.

2 | MATERIALS AND METHODS

2.1 | Study site

The experiment was carried out from February 2022 to
August 2022 in the glass greenhouse of the Department of

Agriculture of the University of Ioannina, at the campus of
Arta. (39°07′15.7″ N 20°56′44.7″ E). Seeds of *B. emarginata*
used in the experiment were collected in July 2021 in Pefki,
Greece (39°47′50.1″N 21°23′09.4″ E, alt. 990 m a.s.l.).

2.2 | Preparation of seedlings

Seeds were sown directly into 2-L pots containing ultra-
mafic soil as a substrate (Figure 1). The soil had a sandy
clay texture with 18.7% clay, 29.1% silt, and 42.5%
sand, along with 4.34% organic matter, and was slightly
alkaline (pH = 7.4) (Table 1). Pots were placed on

<figure><img src="image_2.png" /></figure>

FIGURE 1 Individuals of *Bornmuellera emarginata* under different shading conditions 75 days after sowing: (a) control (full light); (b) shading level 20%; (c) shading level 35%; (d) shading level 60%; (e) shading level 90%; (f) an overview of the design of experiment.
<table><caption>TABLE 1 Physicochemical characterization of soil from the field site (mean ± 1SE).</caption><thead><tr><th colspan="2">Soil general properties</th></tr></thead><tbody><tr><td>Texture</td><td>Sandy clay</td></tr><tr><td>pH (H<sub>2</sub>O)</td><td>7.2 ± 0.0</td></tr><tr><td>EC (µS cm<sup>-1</sup>)</td><td>346.0 ± 32.0</td></tr><tr><td>CEC (cmol kg<sup>-1</sup>)</td><td>24.7 ± 2.4</td></tr><tr><td>TOC (g kg<sup>-1</sup>)</td><td>26.2 ± 5.4</td></tr><tr><td>TN (g kg<sup>-1</sup>)</td><td>2.1 ± 0.3</td></tr><tr><td>P Olsen (mg kg<sup>-1</sup>)</td><td>14.0 ± 1.1</td></tr><tr><td>Ca-Total (g kg<sup>-1</sup>)</td><td>7.0 ± 0.3</td></tr><tr><td>Mg-Total (g kg<sup>-1</sup>)</td><td>138.0 ± 3.0</td></tr><tr><td>Mn-Total (mg kg<sup>-1</sup>)</td><td>1500.0 ± 57.0</td></tr><tr><td>K-Total (mg kg<sup>-1</sup>)</td><td>1860.0 ± 34.0</td></tr><tr><td>Fe-Total (g kg<sup>-1</sup>)</td><td>92.9 ± 8.2</td></tr><tr><td>Ni-Total (mg kg<sup>-1</sup>)</td><td>2350.0 ± 37.0</td></tr><tr><td>Cr-Total (mg kg<sup>-1</sup>)</td><td>272.0 ± 41.0</td></tr><tr><td>Co-Total (mg kg<sup>-1</sup>)</td><td>17.0 ± 6.0</td></tr><tr><td colspan="2">Soil Ni availability</td></tr><tr><td>Ni-DTPA (mg kg<sup>-1</sup>)</td><td>71.1 ± 2.3</td></tr><tr><td>Ni-Sr(NO<sub>3</sub>)<sub>2</sub> (mg kg<sup>-1</sup>)</td><td>0.8 ± 0.0</td></tr></tbody></table>

Abbreviations: CEC, cation exchange capacity; EC, electrical conductivity; TOC, total organic C; TN, total N.

greenhouse tables with natural light and average recorded air temperature of 32°C. A drop system was set up for daily watering achieving 5% runoff, and complete fertilization was applied at 120 days after sowing (DAS).

## 2.3 | Shading treatments

For the shading treatments, standard shading nets (high-density polyethylene) reduced the amount of light by 20%, 35%, 60%, and 90%, respectively, according to manufacturer's specifications, while a treatment without a net was used as a control. Nets were placed 1.5 m above the plants in each treatment, forming a rectangular parallelogram (150 cm high, 80 cm wide × 200 cm long, Figure 1). Nine pots were used for each treatment to allow for three replicates for destructive measurements.

## 2.4 | Plant analysis

During the experiment, three plant samplings (81, 140, and 210 DAS) were conducted to determine the fresh and dry biomass of plants (g), plant height (cm), number of

lateral shoots, and total number of leaves per plant, nickel yield, and proline concentration. Three replicates for each shading level (five levels) at each time period (three periods) were used. The plant material collected at each of the three samplings was in the vegetative phase. Proline and chlorophyll concentrations were measured from the seventh to eighth well-developed leaf. Plants were dried in an oven at 70°C for 72 h for the determination of dry biomass of roots and shoots.

To determine Ni concentration in shoot tissues, 0.5 g samples of dried, ground plant material was acid-digested at 95°C with 2.5 mL of concentrated HNO<sub>3</sub> and 5 mL of H<sub>2</sub>O<sub>2</sub> (30%). The resulting solutions were filtered through a 0.45 µm DigiFILTER (SCP science, Canada) and diluted to 25 mL with deionized water. The Ni concentration of the final solutions was measured with an Atomic Absorption Spectrometer (PerkinElmer AAnalyst 600).

Proline determination of leaf tissue was calibrated against proline standards spectrophotometrically ($R^2$ = 0.9966) to obtain the metabolic response of plants to abiotic stress using the method described in Carillo and Gibon (2011) with modifications. Four milliliters of 70% ethanol were used as extraction solvent for 0.1 g of homogenized fresh leaf tissue, centrifuged at 4000g for 10 min and the final mixture was incubated at 95°C in water bath for 25 min. Absorbance was determined at 520 nm using a Jasco-V630 UV-VIS spectrophotometer and the results were reported in µmol of proline per g of fresh leaf. Results were presented as mean ± standard error.

The total chlorophyll content (a + b) was measured in nine samplings (64, 70, 81, 98, 104, 115, 140, and 210 DAS) by the nondestructive mode of SPAD-502 (Minolta Co., Ltd, Japan). Three replicates for each shading level (five levels) in each time (nine samplings) were used. To investigate the accuracy of the SPAD values for nondestructive estimation, readings were positively correlated ($R^2$ = 0.9445) with conventional destructive chlorophyll measurements, using the method described in Uddling et al. (2007). Ten milliliters of pure acetone were used as extraction solvent for 0.02 g of homogenized fresh leaf tissue and the samples were left overnight in the dark at 2-4°C. Absorbance was determined at 645 and 662 nm using a Jasco-V630 UV-VIS spectrophotometer and expressed in µg of fresh leaf per cm<sup>2</sup> of leaf area using the equations of Lichthenthaler and Buschmann (2001).

## 2.5 | Statistical analysis

Two-way analyses of variance (ANOVAs) were used to examine the main effects of “shading” (reduced
amount of light by 0%, 20%, 35%, 60%, and 90%), “time” (81, 140, and 210 DAS), and their interactions on fresh and dry plant biomass, plant height, number of lateral shoots per plant, total number of leaves per plant, Ni yield, and proline concentration. For total chlorophyll content, levels included in “time” were 64, 70, 81, 98, 104, 115, 140, and 210 DAS. Bonferroni multiple comparisons at the 0.05 level of significance were used to test for pairwise differences. All analyses were performed using the IBM SPSS Statistics Software (Version 28).

3 | RESULTS

Vertical plant growth was promoted during the experiment, as shown by the increase in plant height in all treatments with time (significant “shading × time” interaction, *p* < 0.001; Table 2). The number of lateral shoots remained constant over time (no significant effect of time; *p* > 0.05) but was reduced with shading (significant effect of “shading” (*F*<sub>4,30</sub> = 74.64, *p* < 0.001) yet no significant interaction between “shading” and “time”, *p* > 0.05; Table 2). A significantly higher number of leaves was produced over time (*F*<sub>2,30</sub> = 202.62, *p* < 0.001 for the effect of DAS), but leaf number decreased under conditions of increased shading without following a consistent pattern at different time levels (significant “shading × time” interaction; *F*<sub>8,30</sub> = 11.27, *p* < 0.001; Table 2).

Regarding plant biomass production, the two-way ANOVA revealed significant effects of “shading” (*F*<sub>4,30</sub> = 16.67; *p* < 0.001) and “time” (*F*<sub>2,30</sub> = 258; *p* < 0.001) on fresh plant biomass (Table 3). A significant “shading × time” interaction (*F*<sub>8,30</sub> = 3.05; *p* = 0.012) also emerged, indicating that the effect of shading on fresh mass varied across time (Table 3). Similar results were produced for dry mass (Table 3). The species reached its maximum biomass 210 days from sowing (Figures 2 and 3). At this time, maximum fresh or dry biomass did not differ between treatments (pairwise Bonferroni post hoc test; *p* > 0.05), except for 90% shading (*p* < 0.05) (Figures 2 and 3).

Proline concentration of leaf tissue of *B. emarginata* showed a decreasing trend with shading, but the pattern was different at each time level (significant “shading × time” interaction) (*F*<sub>8,30</sub> = 4.45; *p* = 0.001). At each time level, higher proline concentration was observed when plants were not exposed to shading stress (control), while lower concentrations were observed at intermediate levels of shading 210 DAS (no significant pairwise comparisons were observed among 20%, 35%, and 60% shading based on the Bonferroni test, *p* > 0.05) (Figure 4).

<table><caption>TABLE 2 Summary of analysis of variance (ANOVA) results for differences in (a) plant height, (b) total number of leaves, and (c) number of lateral shoots of *Boromuellera emarginata* under different shading conditions and time after sowing.</caption><thead><tr><th rowspan="2">Source</th><th rowspan="2">df</th><th colspan="3">(a) Plant height</th><th colspan="4">(b) Total number of leaves</th><th colspan="4">(c) Number of lateral shoots</th></tr><tr><th>SS</th><th>%SS</th><th>MS</th><th>F</th><th>p-value</th><th>SS</th><th>%SS</th><th>MS</th><th>F</th><th>p-value</th><th>SS</th><th>%SS</th><th>MS</th><th>F</th><th>p-value</th></tr></thead><tbody><tr><td>Shading</td><td>4</td><td>910</td><td>46.10</td><td>227</td><td>58.82</td><td>&lt;0.001</td><td>4957</td><td>29.66</td><td>1240</td><td>55.38</td><td>&lt;0.001</td><td>93</td><td>88.94</td><td>23.22</td><td>74.64</td><td>&lt;0.001</td></tr><tr><td>Time</td><td>2</td><td>831</td><td>42.12</td><td>416</td><td>107</td><td>&lt;0.001</td><td>9069</td><td>54.26</td><td>4530</td><td>202.62</td><td>&lt;0.001</td><td>1.24</td><td>1.19</td><td>0.62</td><td>2</td><td>0.153</td></tr><tr><td>Shading × Time</td><td>8</td><td>117</td><td>5.91</td><td>14.57</td><td>3.77</td><td>0.004</td><td>2018</td><td>12.07</td><td>252</td><td>11.27</td><td>&lt;0.001</td><td>0.98</td><td>0.94</td><td>0.12</td><td>0.39</td><td>0.916</td></tr><tr><td>Error</td><td>30</td><td>116</td><td>5.88</td><td>3.87</td><td></td><td></td><td>671</td><td>4.02</td><td>22.38</td><td></td><td></td><td>9.30</td><td>8.94</td><td>0.31</td><td></td><td></td></tr><tr><td>Total</td><td>44</td><td>1974</td><td></td><td></td><td></td><td></td><td>16,700</td><td></td><td></td><td></td><td></td><td>104</td><td></td><td></td><td></td><td></td></tr></tbody></table>

Note: Sum of squares (SS), %SS, degrees of freedom (df), mean squares (MS), F-ratio, and p-values for each variable are shown. Significant values (*p* < 0.05) are in bold.
<table><caption>TABLE 3 Summary of analysis of variance (ANOVA) results for differences in (a) fresh and (b) dry plant biomass of *Bornmuellera emarginata* under different shading conditions and time after sowing.</caption><thead><tr><th colspan="2">(a) Fresh biomass</th><th colspan="8">(b) Dry biomass</th></tr><tr><th>Source of variation</th><th>SS</th><th>%SS</th><th>df</th><th>MS</th><th>F</th><th>p-value</th><th>Source of variation</th><th>SS</th><th>%SS</th><th>df</th><th>MS</th><th>F</th><th>p-value</th></tr></thead><tbody><tr><td>Shading</td><td>1425</td><td>10.48</td><td>4</td><td>356</td><td>16.67</td><td><strong>&lt;0.001</strong></td><td>Shading</td><td>86.49</td><td>11.69</td><td>4</td><td>21.62</td><td>13.75</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Time</td><td>11010</td><td>80.98</td><td>2</td><td>5507</td><td>258</td><td><strong>&lt;0.001</strong></td><td>Time</td><td>571</td><td>77.20</td><td>2</td><td>286</td><td>181.52</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Shading × Time</td><td>521</td><td>3.83</td><td>8</td><td>65.13</td><td>3.05</td><td><strong>0.012</strong></td><td>Shading × Time</td><td>35.01</td><td>4.73</td><td>8</td><td>4.37</td><td>2.78</td><td><strong>0.02</strong></td></tr><tr><td>Error</td><td>641</td><td>4.71</td><td>30</td><td>21.37</td><td></td><td></td><td>Error</td><td>47.19</td><td>6.38</td><td>30</td><td>1.57</td><td></td><td></td></tr><tr><td>Total</td><td>13,600</td><td></td><td>44</td><td></td><td></td><td></td><td>Total</td><td>740</td><td></td><td>44</td><td></td><td></td><td></td></tr></tbody></table>

Note: Sum of squares (SS), %SS, degrees of freedom (df), mean squares (MS), F-ratio, and p-values for each variable are shown. Significant values (p < 0.05) are in bold.

<figure><img src="image_3.png" /><figcaption>FIGURE 2 Variation in fresh biomass (g/plant) of *Bornmuellera emarginata* under different shading conditions 81, 140, and 210 days after sowing. Three replicates (n = 3) for each shading level at each time level were used. Different lowercase letters show significant differences (at p < 0.05) among shading conditions at each time level.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>FIGURE 3 Variation in dry biomass (g/plant) of *Bornmuellera emarginata* under different shading conditions 81, 140, and 210 days after sowing. Three replicates (n = 3) for each shading level at each time level were used. Different lowercase letters show significant differences (at p < 0.05) among shading conditions at each time level.</figcaption></figure>

Leaf chlorophyll increased with time (p < 0.001), with the highest values reached 210 DAS for all treatments except the control and 20% shading (significant “shading × time” interaction) ($F_{8,30} = 55.7$; p < 0.001) (Figure 5).

Nickel yield differed across shading treatments ($F_{4,30} = 6.31$; p < 0.001) and increased with DAS
$(F_{2,30} = 84.5; p < 0.001)$. A marginally significant “shading × time” interaction $(F_{8,30} = 2.22; p = 0.055)$ also emerged, indicating that the effect of shading on nickel yield varied slightly at different time levels (Figure 6). Maximum nickel yield was achieved 210 DAS and at 35% shading (significantly different from the highest shading level, i.e., 90%; pairwise Bonferroni post hoc test, $p < 0.05$) (Figure 6).

<figure><img src="image_5.png" /></figure>

**FIGURE 4** Variation in proline concentration (µmol/g) in *Bornmuellera emarginata* individuals under different shading conditions, 81, 140, and 210 days after sowing. Three replicates ($n = 3$) for each shading level at each time level were used. Different lowercase letters show significant differences (at $p < 0.05$) among shading conditions at each time level.

<figure><img src="image_6.png" /></figure>

**FIGURE 5** Variation in chlorophyll concentration (µg/cm²) in *Bornmuellera emarginata* individuals under different shading conditions, 64, 70, 81, 98, 104, 115, 140, and 210 days after sowing. Three replicates ($n = 3$) for each shading level at each time level were used.

4 | DISCUSSION

This study examined for the first time the effect of shading on growth of a nickel hyperaccumulator, using *B. emarginata* as a model species. Our results showed that fresh and dry biomass of *B. emarginata* plants increased over time, were maximized at the end of the experiment (210 DAS), and were not affected by different shading levels except for the highest one (90%). Nickel yield followed a comparable pattern, showing a maximum in the same time frame, but at the intermediate shading level. Aspects of plant morphology were affected by shading, as expected: shading promoted growth of single-stemmed plants or plants with a small number of lateral shoots. Because the most important hyperaccumulator traits that make them suitable for use in agromining are high aboveground biomass production and high nickel yield (Nascimento et al., 2022; van der Ent et al., 2015), our results are promising for the coexistence of solar panels and nickel agromining crop systems on the same land.

The impact of solar panels on “typical” crop yield and fruit quality such as tomato, lettuce, pepper, cucumber, and strawberry has attracted much attention from researchers to date (Touil et al., 2021). Most studies on the growth of crops in agrivoltaic systems (photovoltaic greenhouse or ground-mounted systems) with a cover ratio equal to or less than 25% reported no significant impacts on plant growth and quality (average yield reduction of less than 25%), whereas inhibitory effects on the growth of crops (except for strawberry and spinach) with 50%-100% cover ratio were reported (Touil et al., 2021). Studies on plants that hyperaccumulate

<figure><img src="image_7.png" /></figure>

**FIGURE 6** Variation in nickel yield (mg/plant) in *Bornmuellera emarginata* individuals under different shading conditions, 81, 140, and 210 days after sowing. Three replicates ($n = 3$) for each shading level at each time level were used. Different lowercase letters show significant differences (at $p < 0.05$) among shading conditions at each time level.
metals or metalloids are rare. Li et al. (2023) found that a 58.4% reduction in annual solar radiation from photovoltaic panels had no inhibitory effect on growth of the Cd/Zn hyperaccumulator *Sedum plumbizincicola*. Our results are in accordance with previous studies, as the growth of *B. emarginata* plants expressed in terms of biomass production was not significantly affected, except at the highest shading level. Thus, because both hyperaccumulating species (Li et al., 2023) and crop species (e.g., Cossu et al., 2014) seem to perform well under reduced solar radiation conditions created underneath solar panels, possible reduction in density of the solar panels need not be considered. However, it should also be noted that the effect of shading on plant growth and thus on greenhouse or field crop productivity depends not only on the inherent characteristics of the crop species but also on other factors such as near-surface temperature and soil moisture.

Regarding the proline concentration of leaf tissue of *B. emarginata*, a decreasing pattern with shading was shown. At each time level, higher proline concentration was observed when plants were not exposed to shading stress, while lower concentrations were observed at intermediate levels of shading 210 DAS (Figure 4). This result is significant, meaning that shading stress does not affect plants as proline is an important osmolyte that plays a vital role in regulating plant cell osmotic pressure and alleviating damage caused by stress conditions (Haffani et al., 2014; Mahajan & Tuteja, 2005). This result is also consistent with the results of Chen et al. (2020) who investigated the effects of the interaction between shade and drought on physiological traits of *Calamus viminalis* seedlings (a climbing plant of the Palmae family). Regarding proline content, their results showed that it was lower under full light than in mild and moderate shading but increased significantly with deep shading. This suggests the damage caused by full light and deep shading triggers a large accumulation of proline in the plant (Arora & Saradhi, 2002; Chen et al., 2020).

Plants develop various adaptation strategies, including vertical growth, to optimize photosynthesis under limited-light conditions (Gruntman et al., 2017). An increase in plant height and a decrease in lateral shoots with increasing shading were observed. The increase in plant height under low-light conditions has been previously reported for sage species (Zervoudakis et al., 2012), paprika (Kim et al., 2022) and the hyperaccumulator *S. plumbizincicola* under photovoltaic panels (Li et al., 2023). Shading at high levels severely decreased branch number (and plant dry weight) in *Medicago* species (Gao et al., 2022).

Leaf chlorophyll concentrations were promoted by shading except for control and low shading (20%)

210 DAS. The decrease in chlorophyll content in *B. emarginata* leaves at 0% and 20% shading at the last sampling (210 DAS) may be the result of the plants' exposure to excessive summer light conditions of that period. Intense light inhibits chlorophyll synthesis, causing photoinhibition, in many plant species, when moderate shading can increase chlorophyll's density per unit area in order to optimize a plant's light absorption efficiency (Wittmann et al., 2001; Yue et al., 2021). Similar to our results, reduction in leaf chlorophyll content in plants growing under higher light intensity conditions was reported, among others, by Daia et al. (2009) for *Tetrastigma hemsleyanum* and Li et al. (2023) for the hyperaccumulating species *S. plumbizincicola*.

High metal yield is a very important determinant of suitability of a hyperaccumulator for use in agromining. *Bornmuellera emarginata* nickel yield increased with time after sowing, reaching maximum values 210 DAS and at the intermediate shading level (35%). This pattern was similar to that of biomass production, resulting in a significant decrease of nickel yield at 90% shading. In field trials carried out in Greece, nickel yield measured in *B. emarginata* during the flowering period after 18 months from sowing, ranged at similar levels (Kyrkas & Konstantinou, unpublished data). Li et al. (2023) found no inhibitory effects on plant growth or metal uptake by the Cd/Zn hyperaccumulator *Sedum plumbizincicola* when growing under photovoltaic panels.

Future studies should be carried out in field conditions to include variation of environmental conditions (climatic and soil parameters) between seasons and years, and the effect of different types of agrivoltaic systems on metal crops. Existing available knowledge on agronomic methods and techniques of cultivation of hyperaccumulators should be exploited in this direction (e.g., Bani, Echevarria, Zhang, et al., 2015; Bani & Echevarria, 2019; Kidd et al., 2018; Kyrkas et al., 2023), to confirm the results under greenhouse conditions and to validate the successful coexistence of metal crop-photovoltaics for the benefit of the environment.

5 | CONCLUSIONS

The results of the experimental study of *B. emarginata* growing under shading conditions similar to those provided by photovoltaic panels showed satisfactory plant growth under intermediate shading conditions while maximizing phytoextraction capacity. This justifies cultivation of the plant in nickel-rich areas where photovoltaic systems are installed and would provide an environmental benefit via both energy production and nickel extraction through agromining. Future studies should be carried out
in the field incorporating available knowledge on agro-
nomic methods of cultivation of the species.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ORCID

Dimitrios Kyrkas ID https://orcid.org/0000-0003-0267-3121

Guillaume Echevarria ID https://orcid.org/0000-0003-2124-1447

Maria Konstantinou ID https://orcid.org/0000-0002-4163-1736

Panayiotis G. Dimitrakopoulos ID https://orcid.org/0000-0002-8374-4392

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**How to cite this article:** Kyrkas, D., Mantzos, N., Beza, P., Papantzikos, V., Andreaki, C., Konstantourou, E.-E., Echevarria, G., Konstantinou, M., & Dimitrakopoulos, P. G. (2024). Effects of different shading levels on the growth of *Bornmuellera emarginata*, a nickel hyperaccumulator for use in agromining. *Ecological Research*, *1–11*. https://doi.org/10.1111/1440-1703.12456