ZINC TOXICITY AND TOLERANCE-RELATED RESPONSES IN Inga marginata AND Allophylus edulis SEEDLINGS

Although zinc (Zn) is a micronutrient, excessive amounts in the soil can have toxic effects on plants. Fertilizers, limestone materials, pesticides, and fungicides added with Zn have contributed to increasing the concentration of this element in agricultural soils. Accordingly, it is necessary to find Zn-tolerant plant species to be properly used in degraded soil restoration programs. Thus, the current study aims to investigate the influence of different Zn concentrations on photosynthetic variables, antioxidant activity, and growth of I. marginata and A. edulis seedlings to determine their potential to be used as phytoremediation species. The experiment was installed in a 2×5 factorial scheme, with the first factor being two species ( Allophylus edulis and Inga marginata ), and the second factor: five concentrations of Zn (2, 75, 150, 225, and 300 μM), with three replications per treatment. Each sampling unit consisted of a pot with five plants. Photosynthetic, morphological variables of the shoot and root systems, chlorophyll a fluorescence, photosynthetic pigments, antioxidant enzyme activity, lipid peroxidation, hydrogen peroxide concentration, and Zn accumulated in the roots and shoot were evaluated. Zn stress has activated an efficient antioxidant system, which reduced oxidative damage in the leaves of both species; consequently, it did not decrease shoot biomass production in Inga marginata and Allophylus edulis seedlings. High Zn accumulation in plant tissues and lack of negative effects on Inga marginata and Allophylus edulis shoot have suggested that these plant species are tolerant to Zn and may be indicated for Zn-polluted soil phytoremediation purposes


INTRODUCTION
Environmental pollution caused by heavy metals poses a serious threat to living organisms (Aguilar et al., 2023).The long-term deposition of these materials in the soil can lead to their accumulation, transport, and toxicity in plants 2 Revista Árvore 2024;48:e4801

TOXICIDADE DO ZINCO E RESPOSTAS RELACIONADAS À TOLERÂNCIA EM MUDAS DE
Inga marginata E Allophylus edulis (Hammerschmit et al., 2020).Activities such as mining and smelting of metallic ores, industrial emissions, insecticide applications, fertilizers, and sewage sludge disposal have contributed to increasing heavy metals levels in the soil (Kuinchtner et al., 2021).In addition, plant micronutrients such as zinc (Zn), copper (Cu), and manganese (Mn) have been added to agricultural systems, a fact that increases the risk of toxicity in plants (Tiecher et al., 2016).
Zinc is an essential micronutrient for plants since it acts as a cofactor for several metalloproteins such as superoxide dismutase, carbonic anhydrase, dehydrogenases, proteases, peptidase, and phosphohydrolases (Brunetto et al., 2018;Somavilla et al., 2018).Despite the key role played by Zn in plant growth and development, this micronutrient can easily become toxic when its concentration reaches high levels in the soil and, consequently, in plant tissues (Bernardy et al., 2016).
Zinc accumulation level in plants depends on differences between plant species.Moreover, it can be affected by varied factors, such as plant growth stage and Zn absorption, accumulation, and translocation control in plant tissues (Hammerschmit et al., 2020).Plants grown in soils subjected to Zn excess can significantly accumulate this heavy metal in their tissues and develop toxicity symptoms.In general, changes such as reduced growth, increased mean root diameter, and reduced root length can be observed in plants subjected to high Zn concentrations (Zhang et al., 2019).In addition, plants can present negative biochemical and physiological responses due to such disorders, which can change depending on the plant species and organ affected, on element concentration, and on tissue tolerance to high Zn levels (Tiecher et al., 2018;Aguilar et al., 2023).
However, plants may adopt different strategies to deal with high levels of metals, such as Zn, in their growth environment.Tolerance mechanisms mainly comprise detoxification processes based on complexation by organic chelators, Zn sequestration in vacuoles (Zalamena et al., 2015), and antioxidant system activation.These strategies help protect the metabolism of plants subjected to Zn excess.
Despite the great interest of the scientific community in ecological and environmental issues caused by excess heavy metals in the soil-plant system, little emphasis has been given to the impact of these metals on tree species (Marques et al., 2018).Therefore, it is necessary to improve knowledge about forest species with phytoremediation potential, mainly those presenting a fast growth rate, extensive geographic distribution, and phenotypic plasticity that can be used to reforest contaminated areas (Aguilar et al., 2023).
The Allophylus edulis (A. St.-Hil., A. Juss. & Cambess.)Radlk species belongs to the Sapindaceae family (Pereira et al., 2016); it was described as an early secondary species (Ballestreri et al., 2021).It is popularly known as "chal-chal" or "cocu"; moreover, it can be found in different South American countries such as Brazil, Bolivia, Paraguay, Argentina, and Uruguay. A. edulis is a medium-sized tree species capable of growing in soils presenting medium-to-poor fertility, so it is widely used to help to recover degraded ecosystems (Pereira et al., 2016).
Inga marginata is a pioneer species belonging to the Fabaceae family.It is known as "ingazinho", "inga", and "ingá-feijão", and is widely distributed in South America (Turchetto et al., 2020).Since I. marginata is a leguminous species, it can establish symbiotic associations with nitrogen-fixing bacteria, making it an ideal species for nutrient-poor environments and regeneration areas.In addition, I. marginata has the potential to be used for phytoremediation purposes; therefore, it is useful for ecological restoration (Cabral et al., 2017).
Thus, the present study aimed to investigate the influence of different Zn concentrations on the photosynthetic variables, antioxidant activity, and growth of I. marginata and A. edulis seedlings to determine their potential to be used as Zn phytoremediation species.The current hypothesis is that the investigated species can accumulate and tolerate high Zn concentrations by maintaining biomass production and activating antioxidant mechanisms.

Study site and plant material
The study was conducted in a greenhouse at the Biology Department of the Federal University of Santa Maria (UFSM) -Santa Maria Campus, Rio Grande do Sul State (RS), Brazil.The average temperature inside the greenhouse was 25ºC, and relative air humidity was 60%.
I. marginata and A. edulis seedlings were produced at UFSM Forest Nursery -Santa Maria Campus.Seeds were collected from parent trees grown in forest fragments in the investigated region, and sowing was carried out right in non-toxic polypropylene plastic tubes (180 cm3 in volume) filled with substrate.The substrate used for seedlings' production was Carolina Soil ® , composed of Sphagnum sp., vermiculite, and added with 30% carbonized rice husk.Sowing fertilization was carried out with controlled-release fertilizer of the Osmocote® type (CRF), which comprised 15% of N, 9% P2O5, 12% KCl, 1% Mg, 2.3% S, 0.05% Cu, 0.06% Mn, 0.45% Fe, and 0.2% Mo, and six-month nutrient release.
Seedlings at the age of 120 days and approximately 25 cm in height and 2.0±0.2mm in stem diameter were sent to the greenhouse of the Biology Department at UFSM -Campus of Santa Maria for acclimation purposes until the experiment was carried out.

Conducting the experiment
The experiment was carried out in a completely randomized design arranged in a 2×5 factorial scheme with three replications.The first factor was composed of two tree species (Allophylus edulis and Inga marginata).The second factor was composed of five Zn concentrations (2, 75, 150, 225, and 300 μM).Each replication consisted of a pot containing five plants, totaling fifteen plants per treatment.The standard Zn concentration in the nutrient solution of Hoagland and Arnon (1950) is 2 μM, which was used as a control treatment, and for the other concentrations, 75, 150, 225, and 300 μM of Zn were added into the nutrient solution complete.
I. marginata and A. edulis seedlings were removed from the tubes and had their roots carefully washed with tap water to remove the substrate from their surroundings.Each seedling was placed in a 6 L pot filled with a complete nutrient solution, based on Hoagland and Arnon (1950).A Styrofoam sheet was added to the surface of each pot, with five holes to allow the plant to pass through.Besides allowing plants to be fixed, the Styrofoam sheet has also contributed to reducing nutrient solution evaporation in each pot.Treatments were applied after the acclimation period was over.Seedlings were subjected to different Zn concentrations for 21 days, which totaled 36 days in the hydroponic system.The nutrient solution in each pot was replaced twice a week, and its pH was adjusted to 5.5±0.2daily by using 1.0 mol L -1 HCl or 1.0 mol L -1 NaOH.

Evaluated morphological variables and Tissue Zn analysis
Shoot height, and the length of the main root of the investigated plants were measured with a millimeter ruler before the experiment installation and at the end of it.Shoot height (SHI) and main root (RI) increase values were calculated based on measurement results.
The morphological featuring of the roots was conducted based on digitized images in the WinRhizo Pro 2013 software, coupled with EPSON Expression 11000 scanner, equipped with additional light (TPU), at 600-DPI resolution.After the images' scanning procedure was over, root surface area (cm2 plant -1), total root length (cm plant -1), root volume (cm3 plant -1), and average root diameter (mm) were determined.
Shoot and roots were washed in running water and, subsequently, in distilled water.They were dried in an air-forced circulation oven at 65 ºC until they reached constant weight.Shoot (SDW) and root (RDW) dry weight were calculated based on these results.Afterward, shoot and root dry weight samples were ground in a Wiley mill and sieved in 2 mm mesh.Plant tissues (roots and shoot) were subjected to nitric-perchloric digestion (3.0 mL of HNO 65% PA and 1 mL of HClO 70% PA) (Embrapa, 2009).Total Zn concentration in tissues was analyzed in an atomic absorption spectrophotometer (AAS, Perkin Elmer Analyst 200, USA).

Photosynthetic variables
The net CO2 assimilation rate (A), transpiration rate (E), stomatal conductance (Gs), and Rubisco instantaneous carboxylation efficiency (A/Ci -obtained based on net CO2 assimilation: intercellular CO2 concentration ratio were evaluated in the third fully expanded leaf by using the infrared gas analyzer [(IRGA), Mod.Li-COR ® 6400 XT], at photosynthetic radiation of 1,500 μmol m -2 s -1 and CO2 concentration of 400 μmol mol -1.Measurements were taken in the morning, between 8:00 a.m. and 10:00 a.m., 20 days after the beginning exposure of plants to the treatments, before plants were collected for growth analysis.

Chlorophyll a fluorescence
Analyses were performed on fully expanded leaves grown in the middle third of plants in the morning of a sunny day, between 8:00 a.m. and 11:30 a.m., with the aid of a portable light-modulated fluorometer (Junior-Pam Chlorophyll Fluorometer WalzMess-und-Regeltechnik, Germany).Leaves were preadapted to the dark for 30 min before measurements to determine initial fluorescence (Fo).Subsequently, samples were subjected to a saturating light pulse (10,000 µmol m -2 s -1) for 0.6 s to determine maximum fluorescence (Fm) and electron transport rate (ETRm).PSII (Fv/Fm) maximum potential quantum yield was obtained based on the ratio between variable fluorescence and maximum fluorescence (Fv = Fm-Fo).

Biochemical variables
Three plants from each experimental unit were collected for biochemical analysis, totaling 90 plants.They were evaluated, sectioned, and washed their leaves and roots in distilled water, added to aluminum foil envelopes, and frozen in liquid nitrogen (N) immediately to avoid sample degradation.These samples were kept in an ultra-freezer at -80°C until analysis time, when they were macerated in liquid N, homogenized in a specific buffer, and analyzed later.
Chlorophyll a, chlorophyll b, and carotenoid concentrations in the shoot were extracted based on the method by Hiscox and Israelstan (1979) and estimated based on the equation by Lichtenthaler (1987).The hydrogen peroxide (H2O2) concentration was determined based on Loreto and Velikova (2001), and results were expressed as µmol g -1 fresh weight.Superoxide dismutase (SOD) activity was determined based on the spectrophotometric method described by Giannopolitis and Ries (1977), whereas guaiacol peroxidase activity was determined based on Zeraik et al. (2008).The membrane lipid peroxidation degree was estimated based on the method by El-Moshaty et al. (1993).Lipid peroxidation results were expressed as nmol MDA mg -1 of protein.

Statistical analysis
Error distribution normality was checked through the Shapiro-Wilk test, whereas error variance homogeneity was checked through the Bartlett test; these tests were applied to all experimental variables.Whenever these assumptions were met, the data were subjected to analysis of variance and the Scott-Knott test (p < 0.05) in the Sisvar statistical software (Ferreira, 2019).

Morphological variables
Based on the results of the analysis of variance, the factors evaluated (two plant species and Zn concentrations) had a significant effect (p ≤ 0.05) on the morphological growth variables.Thus, the highest mean values of taproot increase were observed for the control and 75 µM Zn in A. edulis and only for the control in I. marginata (Figure 1A).When comparing species, it was observed that I. marginata seedlings grown in the control showed the highest increment  values in the main root, significantly differing from A. edulis (Figure 1A).Zn concentrations did not negatively affect the shoot dry weight of both species, compared to the control, and similar behavior was observed for the root dry weight of I. marginata (Figures 1B and 1C).Furthermore, the lowest value recorded for the root dry weight of A. edulis seedlings was only evident at the highest Zn concentration (300 µM) (Figure 1C).Root length in both species significantly reduced as Zn concentrations in the nutrient solution increased (Figure 1D).However, the lowest average seedling root volume for A. edulis and the lowest root surface area for both species were observed at 300 µM Zn (Figures 1F and 1E).However, it was observed that higher average shoot and root dry weight, root length, root surface area, and root volume were evident in I. marginata seedlings compared to A. edulis species at all Zn concentrations (Figure 1).

Physiological variables
There was a significant effect (p ≤ 0.  (Gs), and transpiration rate (E) in A. edulis seedlings were found at Zn concentrations of 150 and 300 µM (Figure 2).However, I. marginata seedlings significantly decreased in the abovementioned variables as the addition of Zn to the nutrient solution increased.The lowest values recorded for instantaneous carboxylation efficiency (A/Ci) of Rubisco in both species were observed at the highest concentration of Zn (300 µM) (Figure 2D).
When comparing species, it was observed that concentrations of 2 µM Zn (control) and 225 µM Zn promoted lower values of photosynthetic rate, transpiration rate, and A/Ci in A. edulis seedlings than in I. marginata (Figure 2).On the other hand, the photosynthetic rate was higher in A. edulis at the highest Zn concentration compared to I. marginata.Furthermore, the highest stomatal conductance values were observed in I. marginata seedlings compared to the A. edulis, except at 300 µM Zn, where no significant difference was observed between the species (Figure 2B).
The highest electron transport rates (ETRm) were observed for A. edulis seedlings in the control group and those subjected to 75 µM Zn, differing significantly from I. marginata (Figure 2E).At the same time, the highest ETRm values recorded for I. marginata seedlings were observed in plants subjected to 150 μM Zn (Figure 2E).However, the significant effect (p ≤ 0.05) of different Zn concentrations was not observed for Fv/ Fm, and there was no significant difference between species (Figure 2F).0.05) on the biochemical variables investigated in the present study.The lower levels of total chlorophyll in A. edulis seedlings were related to increasing Zn concentration in the nutrient solution (Figure 3A).There was no significant difference in the concentration of carotenoids in I. marginata seedlings, regardless of Zn concentrations.In contrast, the highest carotenoid concentration in A. edulis seedlings was observed at 75 and 150 µM Zn (Figure 3B).When comparing species, it was observed that concentrations of 75 and 225 µM Zn promoted the lowest total Chl values for A. edulis, differing significantly from I. marginata (Figure 3A), while in control, A. edulis presented higher total Chl content, compared to I. marginata.Furthermore, it was noted that concentrations of 150 and 225 µM Zn resulted in the lowest carotenoid content values for I. marginata and A. edulis, respectively (Figure 3B).

Biochemical variables and Zn in tissues
The highest values of superoxide dismutase (SOD) activity in the shoot and roots of A. edulis were observed at 150 µM Zn.However, they were statistically equal at Zn concentrations of 75 and 225 µM in the shoot (Figures 3C and 3D).However, the highest values observed for SOD activity in the shoot of I. marginata seedlings were found at 225 µM Zn and differed from those recorded for other Zn concentrations (Figure 3C), while Zn reduced SOD activity at roots of I. marginata seedlings at concentrations of 150 and 225 μM (Figure 3D).However, when comparing species, it was observed that concentrations of 75 and 150 µM Zn resulted in the lowest values for SOD activity in the shoot of I. marginata.In contrast, the concentration of 225 µM Zn resulted in the highest values for SOD in the shoot of I. marginata, differing from A. edulis (Figure 3C).Furthermore, it was noted that the lowest SOD values in the roots were observed in I. marginata seedlings at 150 and 225 µM Zn (Figure 3D) and in A. edulis in the control treatment.
The highest values recorded for POD activity in the shoot of seedlings were evident from 150 µM Zn and at 75, 225, and 300 µM Zn in I. marginata seedlings (Figure 3E).POD activity in the roots of A. edulis seedlings had the highest values at 75 and 150 μM Zn, while POD activity in the roots of I. marginata seedlings had the highest values at 75 and 300 μM Zn (Figure 3F).In the comparison between species, it was possible to observe that the concentration of 150 µM Zn resulted in the lowest POD activity in the shoot and roots in I. marginata, while the concentrations of 225 and 300 µM Zn resulted in the lowest POD activity in the roots and shoot of A. edulis seedlings (Figures 3E and 3F).
The significant effect (p ≤ 0.05) of different Zn concentrations was not observed for the H2O2 content of the roots, and between species, there was also no significant difference (Figure 4B).The highest MDA concentrations in the shoot and roots of A. edulis seedlings were observed at 225 and 300 µM Zn, and similar behavior was observed for MDA levels in the shoot of I. marginata (Figures 4C and 4D).In the comparison between the species, it was observed that the lowest averages of H2O2 content in the shoot and MDA contents in roots were evident in the seedlings of I. marginata compared to A. edulis at all Zn concentrations, and similar behavior was observed in A. edulis for MDA contents in the shoot (Figure 4).
The zinc concentration in the shoot and roots tissues of seedlings belonging to both species recorded the lowest averages in the control group (Figures 4E and 4F).The significant effect between species was not observed for Zn content in shoot tissues.For Zn content in roots tissues, in general, the species I. marginata accumulated lower concentrations of Zn (Figures 4E and 4F).

DISCUSSION
The lowest values recorded for taproot increase in I. marginata and A. edulis seedlings were observed after Zn addition to the nutrient solution (Figure 1A).It may have happened because root elongation inhibition is plants' most evident physiological response to heavy metal-related stress since roots have direct contact with contaminants (Zhang et al., 2019).
The lowest values found for total root length were also observed as Zn concentration in the nutrient solution increased, and the lowest values recorded for root surface area were observed at 300 µM of Zn in both species, compared to control (Figures 1D  and 1E).Zn excess can lead to changes in root morphological parameters (e.g., lateral root formation inhibition and reduced root length), as well as to anatomical changes (increased cortex area, vascular cylinder, and number of cortical layers), which result in roots deformations (Ambrosini et al., 2015).However, it is important to notice that, despite differences observed in roots morphological variables, there was no significant difference in roots dry weight in I. marginata seedlings (Figure 1C).In other words, negative Zn effects on roots morphological variables did not affect final biomass production in I. marginata plants; this outcome may indicate species' tolerance to Zn.
In the comparison between species, it was observed that the highest shoot and roots dry weight, root length, root surface area, and root volume were evident in seedlings of I. marginata compared to A. edulis at all Zn concentrations (Figure 1).This may have occurred because I. marginata is a pioneer species, which presents rapid growth and consequently generates greater biomass production in a shorter period compared to A. edulis, which is considered an initial secondary and presents a slower growing rate.
The negative Zn effect on roots morphological variables has negatively affected roots biomass production in species A. edulis, mainly at the highest Zn concentration (300 µM) (Figure 1C).This response was due to higher Zn availability in the nutrient solution and, consequently, higher Zn uptake rate by plants can cause cell division and elongation inhibition, a process capable of explaining the observed roots biomass production reduction (Tiecher et al., 2016).
On the other hand, Zn concentrations did not negatively affect the shoot dry weight of both species compared to the control (Figure 1B).This response was evidenced because Zn toxicity often leads to significant roots growth decrease, mainly of lateral roots, although it has little influence on shoot growth (Zalamena et al., 2015).Thus, it is likely that Zn application and its increasing concentration did not reduce nutrient absorption to the point of decreasing shoot dry weight production in the plants evaluated, although these variables had some negative effects on roots morphological variables.Results in the current study meet those reported by Stoláriková-Vaculíková et al. (2015), who observed that high Zn levels did not reduce leaf dry weight production in Populus deltoides.Similarly, Bernardy et al. (2016) reported that the BRA accession of Pfaffia glomerata (Spreng.),grown in a hydroponic system presenting high Zn concentration, did not show reduced total and shoot dry weight production.
The lowest values recorded for stomatal conductance, photosynthetic, transpiration, and electron transport (ETRm) rates were observed in both species after Zn application, compared to control (Figure 2).Such a photosynthetic rate reduction occurred because heavy metals can decrease Rubisco enzymatic activity and reduce transpiration and stomatal conductance in the mesophyll (Tiecher et al., 2018).The ETRm value may have decreased due to increased non-photochemical dissipation, indicating that plants have dissipated light in the form of heat to protect leaves from light-induced damage (Singh and Reddy, 2015).However, despite the decrease in the photosynthetic rate, there was no significant difference in the Fv/Fm value recorded for A. edulis and I. marginata seedlings (Figure 2F).This outcome has indicated a lack of shoot dry weight decrease in plants belonging to both species, which showed high tolerance to Zn.

Concentrations of 75 and 225 µM Zn
promoted the lowest total Chl values for A. edulis, differing significantly from I. marginata (Figure 3A).Such a decrease in chlorophyll synthesis or an increase in its degradation associated with Zn excess can cause negative effects on the electron transport rate during photosynthesis (Figure 2E) (Tiecher et al., 2018).Carotenoid concentration in I. marginata seedlings did not show a significant difference with the application of Zn (Figure 3B).However, compared to the control, the highest carotenoid concentration was observed in A. edulis at 75 and 150 µM Zn (Figure 3B).Such an increase in carotenoid concentration may have happened due to plants' response to oxidative stress since carotenoids play a key role in controlling free radicals and protecting thylakoid membranes (Kuinchtner et al., 2021).
Although Zn cannot directly generate reactive oxygen species (ROS) through the Fenton reaction, high concentrations can generate oxidative stress and interfere with plants' antioxidant defense system (Bernardy et al., 2016).Thus, excessive Zn uptake by plants can lead to oxidative stress due to an imbalance between antioxidant responses and increased ROS production (Brunetto et al., 2018).ROS can damage plants' membrane lipids, proteins, pigments, and nucleic acids and in extreme cases, it can lead to plant death.SOD is one of the main antioxidant enzymes involved in ROS elimination processes, and it also helps maintain homeostasis in plant cells (Zhang et al., 2019).Thus, increased SOD activity was observed in the shoot of both investigated species, as well as in A. edulis roots subjected to Zn application, compared to I. marginata (Figure 3C).This response may have occurred because SOD plays a crucial role in removing O2-• from compartments where radicals are formed.The O2-• decomposition is always followed by H2O2 production, which acts as an oxidant and reductant.H2O2 is less harmful and reactive than superoxide anion when it accumulates in plant tissues, and it can be eliminated by catalases and peroxidases (Aguilar et al., 2023).Zinc, in general, triggered an increase in POD enzyme activity in the shoot in both species compared to the control (Figure 3E).The highest values of guaiacol peroxidase (POD) enzyme activity and the lowest values of H2O2 concentration were observed after Zn application in both species, compared to the control (Figures 3 and 4).Increased POD enzyme activity has suggested that H2O2 was mainly degraded in the cells.POD plays an important role in cellular detoxification processes since it can catalyze many oxidative reactions in plants by using H2O2 as a substrate (Bernardy et al., 2016).
Despite the reduction in H2O2 concentration, malondialdehyde (MDA) increase was observed in the shoot of both investigated species, as well as in the roots of A. edulis seedlings at the highest Zn concentration, compared to the control (Figures 4c and 4d).Such a membrane lipid peroxidation increase is likely associated with an increase in other ROS produced from Zn excess, which contributed to increased MDA values.MDA is an oxidized membrane lipid product that accumulates in plants exposed to oxidative stress.Increased MDA concentration may be associated with Zn toxicity, indicating oxidative stress in the seedlings.Consequently, it may cause irreversible damage to long-term plant tissue development and function.However, the greatest damage to membrane lipids caused by Zn was not manifested by the shoot biomass reduction of I. margina and A. edulis plants.This response emphasizes these species' high tolerance to Zn excess.
The highest Zn concentration in tissues was mainly observed in the roots of both species (Figure 4F).Such higher Zn accumulation in the root may have been a strategy adopted by plants to mitigate the impact of this micronutrient on their growth since Zn can cause more severe damage when it is translocated to the shoot (Somavilla et al., 2018) -which is the most metabolically active tissue in the plant.This result may also have been attributed to high Zn levels added to the solution, as well as to the similarity between ionic radii of divalent cations, such as copper (Cu), manganese (Mn), and iron (Fe) (Hammerschmit et al., 2020).Thus, Zn ions can replace any of these divalent cations and be absorbed by roots.Therefore, when these ions enter plants, Zn excess can change their physiological balance due to competition with other cations at primary absorption sites or in nutrient transport zones in the roots (Tiecher et al., 2018).High Zn concentrations in the root system of I. marginata and A. edulis plants have indicated that these species can be used for Zn phytostabilization in soils contaminated with this metal.Thus, high metal concentrations lead to increased nonspecific membrane permeability, Revista Árvore 2024;48:e4801 11 and they may cause nutritional imbalance in plants grown in soils subjected to high concentrations of heavy metals, such as Zn.However, despite the high Zn concentrations found in roots, they may not have been high enough to impair plants' biochemical and physiological processes; consequently, they did not affect shoot dry weight production, as observed for I. margina and A. edulis seedlings, compared to the control, a fact that confirms our initial hypothesis.It may have happened because plants' tolerance to heavy metals results from the combination of biochemical, physiological, and anatomical responses, as well as depends on the chemical element plants are exposed to, their ability to translocate it, and the time they remained exposed to it (Ambrosini et al., 2015).Furthermore, other tolerance mechanisms may also be operating in the species, such as, for example, Zn compartmentalization in the cellular vacuole, Zn complexation/chelation, and accumulation in less sensitive plant tissues, such as roots, decreasing Zn bioavailability (Zalamena et al., 2015).
In general, the seedlings of I. marginata and A. edulis showed high Zn accumulation in the roots tissues, and the dry mass production of the shoot of both species was not affected by Zn, being considered suitable for phytoremediation of contaminated soils by zinc.However, I. marginata is a pioneer species, which presents fast growth and tolerance to light and is therefore usually cultivated under full sun conditions, as the species grows very well under these conditions.On the other hand, A. edulis is a non-pioneer species with a slower growth rate, requiring a shadier environment, and does not adapt very well under full sun conditions, as the species grows better in a subtropical environment forest.Therefore, when choosing species for phytoremediation, these factors must be considered.

CONCLUSION
Toxic effects caused by Zn excess were significant in both plant species investigated in the current study, and these effects were mainly evidenced by changes in chlorophyll concentration and decreased photosynthetic rates.On the other hand, Zn stress has activated an efficient antioxidant system, reducing H2O2 levels.Consequently, there was no reduction in biomass production in the shoot of Inga marginata and Allophylus edulis, even with the increase in MDA content.High Zn accumulation in plant tissues and lack of negative effects on shoot of Inga marginata and Allophylus edulis have suggested that these plants are tolerant to Zn and can be used for the phytoremediation of Zn-polluted soils.

Figure 1 -
Figure 1 -Mean values recorded for root increase (RI) (A), shoot dry weight (B), root dry weight (C) total root length (D), root surface area (E) and root volume (F) in Allophylus edulis (A.edulis) and Inga marginata (I.marginata) seedlings grown under different Zn concentrations.Different letters between treatments represent statistically significant difference in the Scott-Knott test.Bars represent the mean ± standard deviation.
05) of species and Zn concentrations on the physiological variables observed in the present study.The lowest means of net CO2 assimilation rate (A), stomatal conductance 6 Revista Árvore 2024;48:e4801

Figure 3 -
Figure 3 -Mean values recorded for total chlorophyll (A), carotenoids (B), superoxide dismutase (SOD) enzyme activity in shoots (C) and roots (D) and guaiacol peroxidase enzyme (POD) in shoots (E) and roots (F) of Allophylus edulis and Inga marginata seedlings grown under different Zn concentrations.Different letters between treatments represent statistically significant difference in the Scott-Knott test.Bars represent the mean ± standard deviation.