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Article

Arbuscular Mycorrhizal Fungi Enhance Antioxidant Defense Systems in Sugarcane Under Soil Cadmium Stress

by
Gloria Magaly Paladines-Beltrán
1,2,*,
Nathalia Alejandra Venegas
2 and
Juan Carlos Suárez
3,4
1
Programa de Doctorado Ciencias Naturales y Desarrollo, Facultad de Ciencias Agropecuarias, Universidad de la Amazonia, Florencia 180001, Colombia
2
Centro de Investigaciones Amazónicas CIMAZ Macagual César Augusto Estrada González, Grupo de Investigación en Productos Naturales Amazónicos GIPRONAZ, Universidad de la Amazonia, Florencia 180001, Colombia
3
Programa de Ingeniería Agroecológica, Facultad de Ingeniería, Universidad de la Amazonia, Florencia 180001, Colombia
4
Centro de Investigaciones Amazónicas CIMAZ Macagual César Augusto Estrada González, Grupo de Investigaciones Agroecosistemas y Conservación en Bosques Amazónicos-GAIA, Florencia 180001, Colombia
*
Author to whom correspondence should be addressed.
Plants 2025, 14(18), 2916; https://doi.org/10.3390/plants14182916
Submission received: 25 July 2025 / Revised: 22 August 2025 / Accepted: 25 August 2025 / Published: 19 September 2025
(This article belongs to the Section Plant Protection and Biotic Interactions)
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Abstract

Cadmium (Cd) is a toxic metal that affects living organisms even at low concentrations, causing physiological alterations and biomass reduction in plants. Arbuscular mycorrhizal fungi (AMF) represent a biological strategy that increases tolerance to heavy metals, although their specific mechanisms in sugarcane remain poorly understood. To address this knowledge gap, an open-field experiment was conducted to evaluate the effects of AMF on Cd accumulation, oxidative stress, photosynthetic pigments, enzymatic antioxidant system, and non-enzymatic antioxidant compounds in sugarcane variety CC 01-1940, using a randomized block design. Results showed that AMF established symbiosis with plants, retaining Cd in the roots and reducing its translocation to leaves. Additionally, they decreased Cd-induced oxidative stress by reducing lipid peroxidation (MDA) and proline content. Although an initial decrease in photosynthetic capacity was observed, AMF helped maintain stable levels of photosynthetic pigments, preserving photosynthetic efficiency. They also activated antioxidant enzymes and increased antioxidant compounds such as reduced glutathione (GSH), non-protein thiols (NP-SH), ascorbic acid (AA), and phytochelatins (PC). These findings demonstrate that symbiosis with AMF protects sugarcane plants from cellular oxidative damage and reduces Cd concentrations in leaves. Therefore, the use of AMF represents an effective strategy to improve the antioxidant defense and resistance of sugarcane plants to cadmium stress.

1. Introduction

Sugarcane (Saccharum officinarum) is a crop of high economic value, particularly in tropical and subtropical regions characterized by acidic soils [1]. Its importance lies in its multiple industrial, economic, medicinal, and food applications, on which a significant portion of the world’s population depends. For the year 2023, South America recorded a production exceeding 867 million tons, with Colombia contributing 32.4 million tons [2]. This energy crop provides 70% of the global sugar supply, in addition to generating raw materials for various industries through its byproducts. However, its extensive cultivation has raised concerns regarding potential contamination of the human food chain. Studies have shown that sugarcane can accumulate considerable amounts of cadmium (Cd) in its tissues [3,4], tolerating concentrations of up to 500 μM Cd2+ in solution, with an accumulation of 451 mg kg−1 dry weight in shoots [5,6].
Cadmium shows increased solubility and mobility in acidic soils, facilitating its transport from root to stem and leaves in the soil-plant system, which increases its incorporation into the food chain [7]. This element is a highly toxic contaminant that, even in trace concentrations, significantly affects plants, animals, and other living organisms [8]. In humans, it can trigger various types of cancer, affect the skeletal, respiratory, renal, and reproductive systems, as well as cause cardiovascular diseases [9,10]. In plants, Cd interferes with essential metabolic processes, altering growth and development, causing significant physiological, ultramorphological, and biochemical anomalies, resulting in a substantial reduction in biomass production [11,12,13].
The Cd phytotoxicity induces oxidative damage through the overproduction of reactive oxygen species (ROS) in the cytosol, a process initiated by free metal ions [14]. This damage affects multiple cellular components, including mitochondrial proteins, lipid membranes, and DNA, while also inhibiting antioxidant enzymes, which can lead to cell death [15,16]. As a defense mechanism, plants have developed both enzymatic and non-enzymatic antioxidant systems to regulate and reduce excessive ROS levels [16,17]. The antioxidant systems include Enzymatic: Catalase (CAT, E.C.1.11.1.6), Ascorbate Peroxidase (APX, E.C. 11.1.11.1), Glutathione Reductase (GR, EC 1.6.4.2), Superoxide Dismutase (SOD, E.C.1.15.1.1), Peroxidase (POD, E.C.1.11.1.7), Non-enzymatic: Reduced Glutathione (GSH), Total Non-protein Thiols (NP-SH). The latter strengthens the defensive system through the activation of phytochelatins via phytochelatin synthase [13,16].
Plants have developed sophisticated mechanisms for heavy metal detoxification, among which the formation of complexes with phytochelatins (PC) stands out [18]. These PCs respond to the presence of toxic metals by forming PC-metal and PC-metalloid complexes, characterized by their extraordinary stability due to their high cysteine and glutathione content. These complexes, synthesized through an enzymatic reaction catalyzed by PC synthase (PCS), are subsequently stored in cellular vacuoles, where the harmful impact of Cd is significantly reduced [19]. In this context, arbuscular mycorrhizal fungi (AMF) emerge as natural enhancers of these defense mechanisms. The AMF not only increases the exploration area of the root system and improves metal ion solubility through chelator production [20,21,22] but also promotes PC formation and activation of the antioxidant system. This capability positions them as a promising biological solution for increasing plant resistance to stress from toxic metals such as Cd [23].
The benefits of AMF extend beyond protection against heavy metals. These organisms establish a “living bridge” between soil and plant [24], facilitating bidirectional nutrient translocation: from soil to roots and carbon transport in the opposite direction. This symbiosis results in improved growth, overall nutritional status of the plant [25], phytohormone level, photosynthetic activity, real quantum yield, and heat dissipation level [26,27,28]. Considering this background, it is fundamental to investigate the potential of the symbiosis between AMF and sugarcane to strengthen defense mechanisms against Cd stress. This study aims to evaluate the protective role of arbuscular mycorrhizal fungi (AMF) in sugarcane plants under cadmium stress conditions by assessing the colonization efficiency and its impact on cadmium translocation patterns, analyzing the modulation of enzymatic and non-enzymatic antioxidant defense mechanisms, determining the effectiveness of AMF symbiosis in mitigating oxidative stress and maintaining photosynthetic capacity, as well as investigating the temporal dynamics of antioxidant responses in AMF-colonized sugarcane exposed to cadmium stress. This research seeks to provide insights into the potential use of AMF as a biological strategy for enhancing sugarcane resilience in cadmium-contaminated soils and developing sustainable phytoremediation approaches. Symbiosis between arbuscular mycorrhizal fungi (AMF) and sugarcane is expected to enhance antioxidant defense mechanisms and increase tolerance to cadmium stress by activating enzymatic and non-enzymatic defense systems.

2. Results

2.1. Colonization of Arbuscular Mycorrhizal Fungi and Cadmium Content

Treatments with inoculum demonstrated substantial colonization levels in sugarcane roots, evidenced by the abundant presence of hyphae, vesicles, and arbuscules. This colonization was significantly higher than that observed in native AMF (arbuscular mycorrhizal fungi) from the region’s soil (Figure 1). The S+F-Cd and S+F+Cd treatments achieved colonization rates of 64%, while in treatments without inoculum, colonization was below 35%. These results demonstrate the effectiveness of commercial inoculum in enhancing mycorrhizal symbiosis. Adequate colonization levels were established before the addition of the metal, which was fundamental to ensure the validity of the experimental process.
The Cd concentration was determined in leaves and rhizosphere soil to assess its distribution (Table 1). The results revealed a statistically significant increase in Cd accumulation in both leaves and soil in treatments with CdCl2 addition. The soil Cd content in S-F+Cd exceeded the treatment concentration (50 mg kg−1), probably due to the initial soil Cd content (0.16 mg Kg−1) and the residual from the pretreatment of the experimental soil with phosphate-based fertilizers, which are a known source of Cd [29]. Higher Cd accumulation was observed in leaves from the S-F+Cd treatment compared to S+F+Cd, while the latter showed lower Cd retention in soil. These results demonstrate that Cd underwent translocation between soil and sugarcane leaves in metal-containing treatments, with this translocation being reduced in the presence of stronger colonization by AMF (Table 1).

2.2. Analysis of Photosynthetic Pigments and Biochemical Stress Markers

Photosynthetic pigment fractions were significantly affected by Cd and AMF inoculation (Figure 2). In all treatments, the contents of chlorophyll a, b, total chlorophyll, carotenoids, and the ratios of Chl a/b and total Chl/Car decreased significantly from initial hours to 48 h, showing an increase at 15 days. The Chl a/b and total Chl/Car ratios (Figure 2e,f) in inoculated plants showed no statistically significant differences throughout the experimental period up to 120 days, with a decrease observed in the Chl a/b ratio and an increase in total Chl/Car. Cd addition significantly reduced carotenoid content in plants. Treatments with AMF and without metal showed higher carotenoid and chlorophyll b content between 12 and 48 h.
Lipid peroxidation, estimated through malondialdehyde (MDA) content, increased significantly in treatments containing Cd (Figure 3a). However, during the initial hours, the S+F+Cd treatment showed significantly lower levels than S-F+Cd. A significant increase in lipid peroxidation was observed at 96 h, 15 days, and 30 days. At 120 days, MDA levels decreased in all treatments without significant differences between them, indicating a reduction in oxidative stress. Proline content, a compatible solute that increases under stress conditions, was used as a stress indicator. It showed an increase in the S-F+Cd treatment at 0, 12, 96 h, and 30 days (Figure 3b). Treatments with AMF inoculum tended to show lower proline values at several time points, suggesting a potential role of AMF in modulating stress responses, although the high variability observed prevents drawing definitive conclusions.

2.3. Enzymatic Antioxidant Defense System

The enzymatic antioxidant response in sugarcane leaves is shown in Figure 4. The CAT activity showed a higher response during initial hours in AMF treatments, significantly decreasing to 120 days (Figure 4a). APX enzyme showed inhibition in the initial hours, significantly increasing at 120 days (Figure 4b). The GR enzyme demonstrated a higher active response in the presence of AMF compared to the control; however, the most significant activation was observed at 24 h in plants exposed to cadmium without AMF (Figure 4c). The SOD activity was found active from hour 0 when sugarcane plants were in symbiosis with AMF, but as time progressed, enzyme inhibition occurred relative to the control; additionally, plants with AMF and metal showed activation at 120 days (Figure 4d). The POD enzyme showed an active response in the first hours in treatments with metal and AMF, significantly decreasing at 120 days (Figure 4e). The GPX enzyme showed activity in initial hours up to 24 h in plants exposed to Cd; however, the addition of AMF did not show significant effects during the experimental period (Figure 4f).

2.4. Non-Enzymatic Antioxidant Defense System

The non-enzymatic antioxidant compounds in sugarcane leaves are shown in Figure 5. At 3 and 24 h, the highest concentrations of reduced Glutathione (GSH) and non-protein thiols (NP-SH) were observed in the S-F+Cd and S+F-Cd treatments, showing statistically significant differences compared to other treatments (Figure 5a,b). However, at 96 h, a decrease in concentrations was observed, followed by an increase at 120 days. The phytochelatin (PC) content (Figure 5c) throughout the experimental period showed no statistically significant differences between treatments, which may be related to the activation of enzymes that generate plant tolerance. The highest Ascorbic Acid (AA) content (Figure 5d) was observed in the initial hours in plants with AMF (Arbuscular Mycorrhizal Fungi). However, after 12 h, there was a significant decrease in treatments with metal and AMF.

2.5. Principal Component Analysis of Photosynthetic Pigments and Antioxidant Systems in AMF-Colonized Sugarcane Under Cd Stress

The principal component analysis performed on the matrix of photosynthetic pigments, oxidative damage, enzymatic and non-enzymatic antioxidant systems, considering treatments and experimental time as factors, explained 39.7% of the total data variability (Dim 1: 27.1%; Dim 2: 12.6%). According to the Monte Carlo test, a significant effect of the variables in relation to the factors was found (p-value: 0.001). Dim 1 related treatments S+F+Cd, S+F-Cd, and S-F+Cd at 9, 12 h, and 120 days with activation of enzymatic and non-enzymatic antioxidant systems, MDA, and the ratios of Chl a/b and Chl T/Car (Figure 6a,b), while Dim 2 associated treatments S-F-Cd and S-F+Cd at 3, 24 h, and 30 days with proline content, AA, and photosynthetic pigments Chl a, Chl b, total Chl, and carotenoids (Figure 6a,b).
The correlations observed between variables in the PCA were confirmed by Pearson’s linear correlation coefficients, shown in Figure 7 with a 95% confidence level. Chlorophyll contents Chl a, b, and Car showed a highly significant linear correlation with Chl T content. GSH positively correlated with NP-SH, while enzymes CAT, APX, GR, GPX, and POD positively correlated with GSH and negatively with Chl T. AA, APX, GSH, NP-SH, and GR negatively correlated with photosynthetic pigments. SOD negatively correlated with the Chl a/b ratio. Additionally, proline contents correlated with Chl a, b, Car, T contents, Chl T/Car ratio, and MDA (Figure 7).

3. Discussion

AMF showed a substantial colonization level in sugarcane plants, which allowed the analysis of metal translocation from soil to aerial tissues. This adaptability of AMF is attributed to the edaphoclimatic conditions produced by the mutualistic symbiosis with the plant, which releases exudates and organic compounds such as sugars, organic acids, and amino acids, promoting hyphal morphogenesis with the development of arbuscules and vesicles [30,31]. The symbiosis with plant roots generated extraradical fungal mycelium (EFM) development, helping to intercept and chelate heavy metals by restricting metal movement through hydroxyl and carboxyl groups [32,33,34], contributing to reduced translocation to leaves. This behavior was similar to that reported by Song et al. [35], where 98% of Cd was retained in the soil-root system, while only 2% was translocated to leaves, attributed to the plant’s active response through AMF stimulation of defense mechanisms and adaptability in regulating metal ions through chelation and retention [36].
Cd exposure was associated with increases in lipid peroxidation (MDA) and proline content, although the magnitude of these changes varied over time. AMF tended to reduce MDA and proline levels compared to non-inoculated treatments, suggesting an improvement in plant tolerance to Cd stress through modulation of antioxidant system activity [37]. AMF presence exerted a protective effect by promoting a favorable redox balance, thus improving plant resistance to Cd stress [38]. Similar results were reported by Li et al. [26] in Medicago truncatula seedlings with AMF exposed to Cd, where AMF inoculation decreased metal and MDA content in leaf samples while increasing plant biomass. This effect is attributed to the increase in various amino acids and organic acids, which facilitate a metal remediation mechanism by AMF.
The Cd exposure affected photosynthetic efficiency through foliar chlorosis, as observed in the studied plants. Both mycorrhizal and non-mycorrhizal plants showed decreased photosynthetic content due to metal exposure, consistent with findings reported in studies on Sesbania drummondii and Robinia pseudoacacia L. [39,40]. The Chl a/b and Chl T/Car ratios demonstrated light adaptation and absorption patterns, with a decrease in carotenoids (Car), leaving plants vulnerable to excess light exposure [41]. This could lead to chloroplast membrane peroxidation, chlorophyll degradation, and photosynthetic disruption due to metal interaction with -SH groups of biosynthesis-responsible enzymes [41,42,43]. However, AMF-colonized plants maintained relatively more stable pigment levels throughout the experiment, which may indicate an additional contribution of AMF to Cd detoxification.
Multiple studies have shown that AMF symbiosis can enhance antioxidant enzymatic activities, helping to reduce oxidative damage generated by ROS production when metabolic balance is altered by Cd presence [7,40,43]. Increased MDA levels likely resulted from Cd-induced damage to chloroplasts and electron transport chains, leading to protein decomposition through oxidative reactions [44]. Antioxidant enzymes play a crucial role in ROS elimination, minimizing cellular oxidation [40]. Specifically, SOD enzyme activation in the presence of AMF during initial hours suggests more effective superoxide radical (O2•−) elimination in sugarcane cells. This process catalyzes the conversion of superoxide radicals (O2•−) to H2O2, activating CAT, POD, and GPX enzymes, which convert H2O2 to H2O and O2, protecting various cellular compartments from oxidative damage [45,46].
The POD enzyme represents an alternative mechanism for H2O2 elimination, showing higher activation in response to this compound compared to CAT. However, this affinity can vary depending on the type of stress, whether biotic or abiotic [41]. In this study, the highest POD activity was observed in Cd treatment without AMF, indicating low enzyme affinity for AMF (Figure 4e). This behavior aligns with reports by Shi et al. [47] and Yang et al. [41], who demonstrated that POD response was not significant in Carthamus tinctorius L. exposed to Cd, nor in Robinia pseudoacacia L. with AMF under Pb stress. The CAT, an oxidoreductase present in peroxisomes and mitochondria, decomposes two H2O2 molecules [45,46]. Recent studies have shown that AMF stimulates CAT enzyme activity in plant leaves grown in Cd-contaminated soils [48,49], potentially generating more O2•− dismutation reactions and H2O2 accumulation [50]. This H2O2 increase promotes CAT activation without requiring a reducing agent. However, when H2O2 levels decrease, its activity reduces, activating enzymes like APX that eliminate residual H2O2 using ascorbate as a reducing agent, thus protecting cells from oxidative damage [45,50], as observed at 120 days.
This behavior corresponds with Yousefi et al. [3] findings in sugarcane leaf samples exposed to Cd, where APX enzyme decreased as CAT response increased, demonstrating sugarcane’s high capacity to eliminate metal-induced ROS. Additionally, APX and GR enzymes participate in the ascorbate-glutathione (ASA-GSH) cycle, contributing to reactive oxygen species elimination in plants [51]. APX and GPX, present in the cytosol, cell wall, vacuole, and extracellular spaces [40], showed significant GPX activation in the early hours of sugarcane plants exposed only to Cd. According to Yang et al. [40], this is attributed to H2O2 catalysis and phenolic compounds for cell wall lignification processes, as well as possible GSH oxidation to GSSG under abiotic stress. This behavior might have reduced GSH and NP-SH concentrations, inhibiting GPX enzymatic activity after 48 h, and activating GR at 24 h to regulate the GSH/GSSG ratio [51]. The increase in APX at 120 days contributed to H2O2 reduction to water, using initial AA reserves as a reducing agent; GSH regenerates AA from its oxidized form DHA [51]. GSH is used by POD and GPX to reduce H2O2, oxidizing to GSSG; the transition back to the reduced form occurs through GR activation in the presence of GSSG and NADPH as a reducing agent [51,52].
GSH is essential for APX functioning and maintaining AA in its active form [50,51]. Additionally, in the presence of Cd and NP-SH, it promotes PC synthesis. The AMF contributed to GSH increase, generating metal tolerance over time (Figure 5a), induced by continuous Cd chelation through PC compound [3,7,51]. Therefore, AMF symbiosis helped sugarcane plants reduce oxidative stress and maintain active antioxidant enzyme response under Cd stress conditions (Figure 2). These results are achieved through metabolic pathways involving various antioxidant enzymes and electron donors. However, detailed information about the relationship between AMF and enzymes in sugarcane leaves remains limited, requiring further research. It is important to note that other antioxidant enzymes and/or non-enzymatic antioxidants might also be involved and should be considered for future studies. Although the presence of symbiosis with arbuscular mycorrhizal fungi (AMF) can modify the antioxidant defense systems of host plants, thereby reducing the proliferation of reactive oxygen species (ROS), the mechanisms involved may vary depending on the fungal species and the availability of micronutrients [26]. Several morphological studies have demonstrated a direct relationship with ROS, identifying the presence of H2O2 in the apoplastic space around the tips of the hyphae and in the final stages of the arbuscule’s life cycle, so the elimination of H2O2 is highly positively correlated with arbuscule richness [53].
Overall, multivariate analyses supported these findings, with the PCA showing a clear association of these enzymes with inoculated treatments. Pearson’s analysis confirmed these observations, revealing strong positive correlations between GSH, NP-SH, and the antioxidant enzymes CAT, APX, GR, GPX, and POD. These results highlight the integrative role of AMF in jointly regulating enzymatic activity, non-enzymatic antioxidants, and pigment dynamics, making a decisive contribution to stress mitigation.

4. Materials and Methods

4.1. Crop Establishment and Treatments

This study was conducted at the Center for Amazonian Research, CIMAZ Macagual César Augusto Estrada González, located in Florencia, Caquetá, Colombia (1°37′ N and 75°36′ W), an area characterized by a Tropical climate (AF) according to Köppen’s classification. For the experimental development, sugarcane (Saccharum officinarum) bud variety CC 01-1940 was used, provided by the CIMAZ-Macagual germplasm bank. The experimental substrate had been previously treated with a commercial phosphate-based fertilizer (special formula 2-4-6 + micronutrients) and was subsequently subjected to a disinfection process aimed at reducing the number of arbuscular mycorrhizal fungi. This process consisted of thermal treatment at 70–95 °C for 2 h, followed by exposure to solar radiation for 2 weeks [54]. Soil analyses revealed the following physicochemical characteristics: pH 4.74, organic matter 76.49 g kg−1, oxidizable carbon 33.69 g kg−1, phosphorus (P-Bray II) 86.35 mg kg−1, total bases 2.32 cmol kg−1, sulfur 70.55 mg kg−1, nickel 60.7 mg kg−1, and cadmium 0.16 mg kg−1.
The experimental design was implemented using 480 sugarcane plants, individually planted in 5 kg capacity bags. At planting time, 240 plants were inoculated with 500 g of commercial arbuscular mycorrhizal fungi (AMF) (SAFER MICORRIZAS M.A ® to promote fungal colonization in the roots. After three months, four treatments were established under a completely randomized block design: a control treatment without AMF and without addition of CdCl2·H2O (S-F-Cd), a second treatment with AMF and 50 mg kg−1 CdCl2·H2O (S+F+Cd), a third treatment with AMF and without CdCl2·H2O (S+F-Cd), and a fourth treatment without AMF and with 50 mg kg−1 CdCl2·H2O (S-F+Cd). Each treatment had 40 experimental units, and the experiment was conducted under open field conditions, allowing for a realistic evaluation of the interactions between AMF and sugarcane plants under cadmium stress.

4.2. Determination of AMF Colonization and Cadmium Content Analysis

The assessment of arbuscular mycorrhizal colonization percentage was conducted three days prior to Cd treatment application, following the modified Phillips and Hayman method [55]. Root samples were thoroughly washed with distilled water and cut into 1 cm segments, then cleared by boiling in a KOH solution for 60 min. Samples were neutralized with HCl and stained with 0.05% trypan blue in lactoglycerol. Microscopic examination was performed at 10× and 40× magnification using a biological microscope. A “colonized field” was defined as a microscopic field in which at least one arbuscule, vesicle, or hyphal segment of arbuscular mycorrhizal fungi was present. Each field corresponded to the area visible within the eyepiece at the given magnification (0.18 mm2 at 40×). For each root sample, a minimum of 50 fields was randomly examined. The colonization percentage was calculated by dividing the number of colonized fields by the total observed fields and multiplying by 100 [56,57]. For cadmium content analysis, foliar and soil samples were collected both before and after the experiment (0 and 120 days). These samples were dried at 60 °C, ground, and sieved to 0.5 mm. Analysis was performed using Energy Dispersive X-ray Fluorescence Spectroscopy (HD XRF®) (Z-Spec E-max, East Greenbush, NY, USA), with cadmium concentration reported in mg kg−1.

4.3. Collection and Pretreatment of Samples for Biochemical Parameters

Foliar tissue sampling was conducted at multiple time points following Cd treatment application, starting at 7 a.m.: 0, 3, 6, 9, 12, 24, 48, 96 h, and at 15, 30, and 120 days. For each sampling event, three plants were randomly selected from each treatment group. The collected tissue samples were immediately macerated with liquid nitrogen. The macerated samples were stored at −80 °C in an ultra-freezer (Infrico Medcare, Lucena, Spain) until further analysis.

4.4. Analysis of Photosynthetic Pigments and Biochemical Stress Markers

Chlorophyll a, b, total chlorophyll, and carotenoids were determined following the method of Nishiyama et al. [58]. A sample of 0.5 g of macerated sugarcane leaves underwent three extractions with 80% acetone, followed by filtration. The absorbance was measured at 663, 642, and 470 nm using a Multiskan SkyHigh microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) [59]. Malondialdehyde (MDA) content was determined by preparing an extract using 0.1 g of macerated tissue with 2 mL of 5% w/v trichloroacetic acid (TCA). The mixture was centrifuged at 4000 rpm at 25 °C for 10 min. The analysis was performed by combining 1 mL of the extract supernatant with 1 mL of 0.6% w/v thiobarbituric acid (TBA). This mixture was heated in a water bath at 98 °C for 10 min, and absorbance was measured at 532, 600, and 450 nm using a Multiskan SkyHigh microplate reader [60]. Proline content was analyzed following Bates’ method [61] with modifications. A 0.2 g sample of macerated sugarcane leaves was homogenized with 2 mL of 3% sulfosalicylic acid and centrifuged at 4000 rpm for 10 min; 1 mL of supernatant was combined with 1 mL of acetic acid-ninhydrin mixture and 1 mL of glacial acetic acid. The mixture was incubated at 98 °C for 30 min in a water bath, followed by extraction with 3 mL of toluene. Absorbance was measured at 520 nm using a Multiskan Go microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) [62].

4.5. Enzymatic Antioxidant Defense System

Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured based on its ability to inhibit the photochemical reduction of Nitroblue tetrazolium (NBT) following the Beauchamp and Fridovich method [63] with modifications. NBT photoreduction was monitored at 560 nm using a Multiskan SkyHigh microplate reader [64]. Catalase activity (CAT, EC 1.11.1.6) was determined by monitoring the decrease in absorbance at 240 nm using a Multiskan Go microplate reader, following Aebi’s method [65], which measures hydrogen peroxide (H2O2) consumption [66]. Ascorbate peroxidase activity (APX, EC 1.11.1.11) was measured according to the Ascorbate/peroxide method by Nakano and Asada [67], tracking the conversion of ascorbate to monodehydroascorbate and dehydroascorbate at 290 nm using a Multiskan Go microplate reader [68].
Glutathione reductase activity (GR, EC 1.8.1.7) was evaluated based on NADPH consumption during GSSG reduction, following the Foyer and Halliwal method [69] with modifications. The reduction was monitored at 340 nm using a Multiskan SkyHigh microplate reader [70]. Peroxidase activity (POD, EC 1.11.1.6) was determined using Kireyko et al.’s modified method [71], measuring the catalytic process of H2O2 with o-dianisidine, which forms a colored compound detectable at 436 nm using a Multiskan SkyHigh microplate reader [4]. Guaiacol peroxidase activity (GPX, EC 1.11.1.7) was determined using the Neves and Lourenço method [72] with modifications, measuring guaiacol oxidation in the presence of H2O2 at 470 nm using a Multiskan SkyHigh microplate reader [68]. All results were expressed per mg of protein content, determined according to Bradford’s method [73].

4.6. Non-Enzymatic Antioxidant Defense System

Phytochelatin (PC) concentrations were quantified by subtracting reduced glutathione (GSH) concentrations from non-protein thiols (NP-SH) [74]. An extract was prepared using 0.2 g of macerate and 2 mL of 15% w/v trichloroacetic acid (TCA), then centrifuged at 10,000 rpm at 4 °C for 10 min. Reduced glutathione (GSH) was determined using the Tawfik method [75], and non-protein thiols (NP-SH) were measured using Ellman’s method [76] with modifications. The production of NTB (2-nitro-5-thiobenzoic acid) was quantified due to the reduction of DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] at 412 nm using a Multiskan SkyHigh microplate reader. GSH was used as a standard for GSH measurements, and Cysteine was used as a standard for NP-SH measurements [77,78]
Ascorbic acid (AA) content was determined by preparing an extraction mixture of formic acid/methanol/water (1:24:25) with 0.1 g of macerate using ultrasound. The solution was filtered directly into vials using 0.22 µm filters (13 mm diameter) and analyzed by HPLC-UV (Shimadzu LC-2010A HT, Kyoto, Japan). Analysis was performed using a Luna C18 column (250 mm × 4.6 mm, 5 µm; Luna Phenomenex, Macclesfield, UK) with a binary mobile phase of water/formic acid (99:1, v/v) and acetonitrile, at wavelengths of 254 and 280 nm under isocratic gradient conditions, with a flow rate of 1 mL min−1. AA identification was based on chromatogram analysis and retention times. Ascorbic acid was used as a standard [79].

4.7. Statistical Analysis

A linear mixed model (LMM) was used to detect significant differences between the level of colonization, Cd content, and different variables of enzymatic and non-enzymatic antioxidant activity. Treatments were considered as fixed factors, and blocks as a random factor. The assumptions of normality and homogeneity of variance were verified through an exploratory analysis of residuals. Differences in the means of fixed factors were verified using the DGC test (p < 0.05). Additionally, principal component analyses (PCA) were performed with the different variables. The LMM was performed using the lme function from the nlme package. The PCA was performed using the fviz_dend and fviz_pca_ind functions, respectively, from the factoextra package. Statistical analyses were performed in the statistical software InfoStat version 2020 [80] and in the R language version 4.3.3 (R Development Core Team 2025) [81].

5. Conclusions

The study results demonstrated that arbuscular mycorrhizal fungi (AMF) established an effective symbiosis with sugarcane plants, achieving colonization rates of 64% in S+F-Cd and S+F+Cd treatments, significantly higher than the 35% observed in treatments without inoculum. This symbiosis resulted in effective cadmium retention in the roots, evidenced by a significant reduction in metal translocation to the leaves, where a concentration of 0.47 mg kg−1 was recorded in colonized plants (S+F+Cd) compared to 1.21 mg kg−1 in non-colonized plants (S-F+Cd). The presence of AMF demonstrated a protective effect against cadmium-induced oxidative stress. This was manifested in a significant decrease in lipid peroxidation (MDA) and proline content in AMF treatments. Although an initial reduction in photosynthetic capacity was observed, AMF-colonized plants maintained more stable levels of photosynthetic pigments throughout the experimental period, preserving Chl a/b and Chl T/Car ratios. The antioxidant defense system was enhanced by the presence of AMF through multiple mechanisms. Significant activation of key antioxidant enzymes was observed, including Catalase (CAT), Ascorbate Peroxidase (APX), Glutathione Reductase (GR), Superoxide Dismutase (SOD), and Peroxidase (POD). Particularly notable was the activation of SOD during initial hours in the presence of AMF, suggesting more effective superoxide radical elimination. Additionally, an increase in non-enzymatic antioxidant compounds was recorded, including Reduced Glutathione (GSH), non-protein thiols (NP-SH), and ascorbic acid (AA), with the highest concentrations of GSH and NP-SH observed at 3 and 24 h in S-F+Cd and S+F-Cd treatments. The AMF-sugarcane symbiosis proved to be an effective strategy for protection against cellular oxidative damage and reduction of cadmium toxicity. Principal component analysis explained 39.7% of the total data variability (Dim 1: 27.1%; Dim 2: 12.6%), confirming the significant correlation between AMF presence and activation of antioxidant defense systems. These results suggest that AMF application represents a viable biological strategy for phytoremediation and sustainable sugarcane cultivation in cadmium-contaminated soils, providing an ecological solution for crop management in areas affected by heavy metals.

Author Contributions

Conceptualization, G.M.P.-B., N.A.V. and J.C.S.; methodology, G.M.P.-B., N.A.V. and J.C.S.; software, G.M.P.-B. and N.A.V.; validation, G.M.P.-B., N.A.V. and J.C.S.; formal analysis, G.M.P.-B., N.A.V. and J.C.S.; investigation, G.M.P.-B. and N.A.V.; resources, G.M.P.-B.; data curation, G.M.P.-B., N.A.V. and J.C.S.; writing—original draft preparation, G.M.P.-B.; N.A.V.; and J.C.S.; writing—reviewing and editing, G.M.P.-B., N.A.V. and J.C.S.; visualization, G.M.P.-B., N.A.V. and J.C.S.; supervision, G.M.P.-B. and J.C.S.; project administration, G.M.P.-B.; funding acquisition, G.M.P.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the doctoral training call 933-2023 of the Ministry of Science, Technology and Innovation-Minciencias for the project entitled “Evaluation of the physiological, biochemical, molecular and ultrastructural responses of sugarcane (Saccharum officinarum) to stress caused by aluminum and cadmium” with code 100387.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Acknowledgments

The authors are grateful for the support provided by the Universidad de la Amazonia for the realization of this study at the Centro de Investigaciones Amazonicas CIMAZ Macagual. The authors would like to thank the different students who helped during sample collection and preparation.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Musa, Y.; Ridwan, I.; Ponto, H.; Ala, A.; Farid, B.M.; Widiayani, N.; Yayank, A.R. Application of Arbuscular Mycorrhizal Fungus (AMF) improves the growth of single-bud sugarcane (Saccharum officinarum L.) seedlings from different bud location. IOP Conf. Ser. Earth Envion. Sci. 2020, 486, 012122. [Google Scholar] [CrossRef]
  2. FAO FAOSTAT 2025. Available online: https://www.fao.org/faostat/en/#data/QCL (accessed on 12 July 2025).
  3. Yousefi, Z.; Kolahi, M.; Majd, A.; Jonoubi, P. Effect of cadmium on morphometric traits, antioxidant enzyme activity and phytochelatin synthase gene expression (SoPCS) of Saccharum officinarum var. cp48-103 in vitro. Ecotoxicol. Envion. Saf. 2018, 157, 472–481. [Google Scholar] [CrossRef] [PubMed]
  4. Zeng, Q.; Ling, Q.; Hu, F.; Wu, J.; Yang, Z.; Qi, Y.; Li, Q. Genotypic Differences in Growth and Antioxidant Enzyme Activities Under Cadmium Stress in Sugarcane. Bull. Envion. Contam. Toxicol. 2017, 99, 607–613. [Google Scholar] [CrossRef]
  5. Guo, J.; Xu, L.; Su, Y.; Wang, H.; Gao, S.; Xu, J.; Que, Y. ScMT2-1-3, a metallothionein gene of sugarcane, plays an important role in the regulation of heavy metal tolerance/accumulation. Biomed. Res. Int. 2013, 2013, 904769. [Google Scholar] [CrossRef] [PubMed]
  6. Sereno, M.L.; Almeida, R.S.; Nishimura, D.S.; Figueira, A. Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes. J. Plant Physiol. 2007, 164, 1499–1515. [Google Scholar] [CrossRef] [PubMed]
  7. Zhang, X.F.; Hu, Z.H.; Yan, T.X.; Lu, R.R.; Peng, C.L.; Li, S.S.; Jing, Y.-X. Arbuscular mycorrhizal fungi alleviate Cd phytotoxicity by altering Cd subcellular distribution and chemical forms in Zea mays. Ecotoxicol. Envion. Saf. 2019, 171, 352–360. [Google Scholar] [CrossRef]
  8. Zhang, C.; Lin, C.; Li, L.; Mohsen, M.; Wang, T.; Wang, X.; Zhang, L.; Huang, W. Single and combined effects of microplastics and cadmium on the sea cucumber Apostichopus japonicus. Mar. Envion. Res. 2023, 186, 105927. [Google Scholar] [CrossRef]
  9. Bora, M.S.; Sarma, K.P. Anatomical and ultrastructural alterations in Ceratopteris pteridoides under cadmium stress: A mechanism of cadmium tolerance. Ecotoxicol. Envion. Saf. 2021, 218, 112285. [Google Scholar] [CrossRef]
  10. Liu, J.; Zhang, J.; Kim, S.H.; Lee, H.S.; Marinoia, E.; Song, W.Y. Characterization of Brassica rapa metallothionein and phytochelatin synthase genes potentially involved in heavy metal detoxification. PLoS ONE 2021, 16, e0252899. [Google Scholar] [CrossRef]
  11. Carvalho, M.E.A.; Castro, P.R.C.; Azevedo, R.A. Hormesis in plants under Cd exposure: From toxic to beneficial element? J. Hazard. Mater. 2020, 384, 121434. [Google Scholar] [CrossRef]
  12. Daud, M.K.; Ali, S.; Variath, M.T.; Zhu, S.J. Differential physiological, ultramorphological and metabolic responses of cotton cultivars under cadmium stress. Chemosphere 2013, 93, 2593–2602. [Google Scholar] [CrossRef] [PubMed]
  13. Garg, N.; Kaur, H. Response of Antioxidant Enzymes, Phytochelatins and Glutathione Production Towards Cd and Zn Stresses in Cajanus cajan (L.) Millsp. Genotypes Colonized by Arbuscular Mycorrhizal Fungi. J. Agron. Crop Sci. 2013, 199, 118–133. [Google Scholar] [CrossRef]
  14. Nualla-ong, A.; Phongdara, A.; Buapet, P. Copper and zinc differentially affect root glutathione accumulation and phytochelatin synthase gene expression of Rhizophora mucronata seedlings: Implications for mechanisms underlying trace metal tolerance. Ecotoxicol. Envion. Saf. 2020, 205, 111175. [Google Scholar] [CrossRef]
  15. Franchina, D.G.; Dostert, C.; Brenner, D. Reactive Oxygen Species: Involvement in T Cell Signaling and Metabolism. Trends Immunol. 2018, 39, 489–502. [Google Scholar] [CrossRef]
  16. Riaz, M.; Kamran, M.; Fang, Y.; Wang, Q.; Cao, H.; Yang, G.; Deng, L.; Wang, Y.; Zhou, Y.; Anastopoulos, I.; et al. Arbuscular mycorrhizal fungi-induced mitigation of heavy metal phytotoxicity in metal contaminated soils: A critical review. J. Hazard. Mater. 2021, 402, 123919. [Google Scholar] [CrossRef]
  17. Luo, P.; Wu, J.; Li, T.T.; Shi, P.; Ma, Q.; Di, D.W. An Overview of the Mechanisms through Which Plants Regulate ROS Homeostasis under Cadmium Stress. Antioxidants 2024, 13, 1174. [Google Scholar] [CrossRef]
  18. Bellini, E.; Maresca, V.; Betti, C.; Castiglione, M.R.; Fontanini, D.; Capocchi, A.; Sorce, C.; Borsò, M.; Bruno, L.; Sorbo, S.; et al. The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins. Int. J. Mol. Sci. 2020, 21, 1583. [Google Scholar] [CrossRef] [PubMed]
  19. Faizan, M.; Alam, P.; Hussain, A.; Karabulut, F.; Tonny, S.H.; Cheng, S.H.; Yusuf, M.; Adil, M.F.; Sehar, S.; Alomrani, S.O.; et al. Phytochelatins: Key regulator against heavy metal toxicity in plants. Plant Stress. 2024, 11, 100355. [Google Scholar] [CrossRef]
  20. Bolchi, A.; Ruotolo, R.; Marchini, G.; Vurro, E.; di Toppi, L.S.; Kohler, A.; Tisserant, E.; Martin, F.; Ottonello, S. Genome-wide inventory of metal homeostasis-related gene products including a functional phytochelatin synthase in the hypogeous mycorrhizal fungus Tuber melanosporum. Fungal Genet. Biol. 2011, 48, 573–584. [Google Scholar] [CrossRef]
  21. Haselwandter, K.; Winkelmann, G. Ferricrocin—An ectomycorrhizal siderophore of Cenococcum geophilum. BioMetals 2002, 15, 73–77. [Google Scholar] [CrossRef]
  22. Morel, M.; Kohler, A.; Martin, F.; Gelhaye, E.; Rouhier, N. Comparison of the thiol-dependent antioxidant systems in the ectomycorrhizal Laccaria bicolor and the saprotrophic Phanerochaete chrysosporium. New Phytol. 2008, 180, 391–407. [Google Scholar] [CrossRef] [PubMed]
  23. Sakamoto, K.; Iijima, T.; Higuchi, R. Use of specific phospholipid fatty acids for identifying and quantifying the external hyphae of the arbuscular mycorrhizal fungus Gigaspora rosea. Soil. Biol. Biochem. 2004, 36, 1827–1834. [Google Scholar] [CrossRef]
  24. Xu, Z.; Ban, Y.; Li, Z.; Chen, H.; Yang, R.; Tang, M. Arbuscular mycorrhizal fungi play a role in protecting roots of Sophora viciifolia Hance. from Pb damage associated with increased phytochelatin synthase gene expression. Environ. Sci. Pollut. Res. 2014, 21, 12671–12683. [Google Scholar] [CrossRef] [PubMed]
  25. Garg, N.; Bhandari, P. Cadmium toxicity in crop plants and its alleviation by arbuscular mycorrhizal (AM) fungi: An overview. Plant Biosyst. 2014, 148, 609–621. [Google Scholar] [CrossRef]
  26. Li, W.; Chen, K.; Li, Q.; Tang, Y.; Jiang, Y.; Su, Y. Effects of Arbuscular Mycorrhizal Fungi on Alleviating Cadmium Stress in Medicago truncatula Gaertn. Plants 2023, 12, 547. [Google Scholar] [CrossRef]
  27. Kuang, Y.; Li, X.; Wang, Z.; Wang, X.; Wei, H.; Chen, H.; Hu, W.; Tang, M. Effects of Arbuscular Mycorrhizal Fungi on the Growth and Root Cell Ultrastructure of Eucalyptus grandis under Cadmium Stress. J. Fungi 2023, 9, 140. [Google Scholar] [CrossRef] [PubMed]
  28. Zhuang, X.; Liu, S.; Xu, S.; Qin, S.; Lyu, D.; He, J.; Zhou, J. Arbuscular Mycorrhizal Fungi Alleviate Cadmium Phytotoxicity by Regulating Cadmium Mobility, Physiological Responses, and Gene Expression Patterns in Malus hupehensis Rehd. Int. J. Mol. Sci. 2025, 26, 1418. [Google Scholar] [CrossRef]
  29. Römkens, P.; Rietra, R.; Kros, H.; Cees Voogd, J.; De Vries, W. Impact of Cadmium Levels in Fertilisers on Cadmium Accumulation in Soil and Uptake by Food Crops; CABI Databases: Wageningen, The Netherlands, 2018. [Google Scholar]
  30. Atakan, A.; Özkaya, H.Ö. Arbuscular Mycorrhizal Fungi and Glomalin. Turk. J. Agric.—Food Sci. Technol. 2021, 9, 2371–2375. [Google Scholar] [CrossRef]
  31. Likus-Cieślik, J.; Józefowska, A.; Frouz, J.; Vicena, J.; Pietrzykowski, M. Relationships between soil properties, vegetation and soil biota in extremely sulfurized mine soils. Ecol. Eng. 2023, 186, 106836. [Google Scholar] [CrossRef]
  32. Giovannini, L.; Palla, M.; Agnolucci, M.; Avio, L.; Sbrana, C.; Turrini, A.; Giovannetti, M. Arbuscular Mycorrhizal Fungi and Associated Microbiota as Plant Biostimulants: Research Strategies for the Selection of the Best Performing Inocula. Agronomy 2020, 10, 106. [Google Scholar] [CrossRef]
  33. Liu, C.L.; Gao, Z.Y.; Shang, L.G.; Yang, C.H.; Ruan, B.P.; Zeng, D.L.; Guo, L.; Zhao, F.; Huang, C.; Qian, Q. Natural variation in the promoter of OsHMA3 contributes to differential grain cadmium accumulation between Indica and Japonica rice. J. Integr. Plant Biol. 2020, 62, 314–329. [Google Scholar] [CrossRef]
  34. Zhang, W.; Guan, M.; Chen, M.; Lin, X.; Xu, P.; Cao, Z. Mutation of OsNRAMP5 reduces cadmium xylem and phloem transport in rice plants and its physiological mechanism. Environ. Pollut. 2024, 341, 122928. [Google Scholar] [CrossRef]
  35. Song, Y.; Jin, L.; Wang, X. Cadmium absorption and transportation pathways in plants. Int. J. Phytoremed. 2017, 19, 133–141. [Google Scholar] [CrossRef] [PubMed]
  36. Liu, X.; Guo, Y.; Li, Y.; Li, Q.; Yao, L.; Yu, J.; Chen, H.; Wu, K.; Qiu, D.; Wu, Z.; et al. Mitigating sediment cadmium contamination through combining PGPR Enterobacter ludwigii with the submerged macrophyte Vallisneria natans. J. Hazard. Mater. 2024, 473, 134662. [Google Scholar] [CrossRef]
  37. Li, X.; Liang, J.; Wei, H.; Kuang, Y.; Chen, H.; Tang, M.; Hu, W. Arbuscular mycorrhizal fungi inoculation and exogenous indole-3-acetic acid application induce antioxidant defense response to alleviate cadmium toxicity in Broussonetia papyrifera. Pedosphere 2024, 34, 447–459. [Google Scholar] [CrossRef]
  38. Garg, N.; Singh, S. Arbuscular Mycorrhiza Rhizophagus irregularis and Silicon Modulate Growth, Proline Biosynthesis and Yield in Cajanus cajan L. Millsp. (pigeonpea) Genotypes Under Cadmium and Zinc Stress. J. Plant Growth Regul. 2018, 37, 46–63. [Google Scholar] [CrossRef]
  39. Ruley, A.T.; Sharma, N.C.; Sahi, S.V.; Singh, S.R.; Sajwan, K.S. Effects of lead and chelators on growth, photosynthetic activity and Pb uptake in Sesbania drummondii grown in soil. Environ. Pollut. 2006, 144, 11–18. [Google Scholar] [CrossRef] [PubMed]
  40. Yang, Y.; Han, X.; Liang, Y.; Ghosh, A.; Chen, J.; Tang, M. The Combined Effects of Arbuscular Mycorrhizal Fungi (AMF) and Lead (Pb) Stress on Pb Accumulation, Plant Growth Parameters, Photosynthesis, and Antioxidant Enzymes in Robinia pseudoacacia L. PLoS ONE 2015, 10, e0145726. [Google Scholar] [CrossRef]
  41. Zhang, Q.; Yang, Y.; Wu, J.; Li, H.; Li, Y.; Li, Z.; He, Y. Enhanced Ultraviolet B Radiation Suppresses Magnaporthe oryzae Infection and Alleviates Its Damage to the Photosynthesis of Rice Leaves. Phyton-Int. J. Exp. Bot. 2024, 93, 2613–2628. [Google Scholar] [CrossRef]
  42. Singh, S.; Eapen, S.; D’Souza, S.F. Cadmium accumulation and its influence on lipid peroxidation and antioxidative system in an aquatic plant, Bacopa monnieri L. Chemosphere 2006, 62, 233–246. [Google Scholar] [CrossRef]
  43. Chen, L.; Hu, X.; Yang, W.; Xu, Z.; Zhang, D.; Gao, S. The effects of arbuscular mycorrhizal fungi on sex-specific responses to Pb pollution in Populus cathayana. Ecotoxicol. Envion. Saf. 2015, 113, 460–468. [Google Scholar] [CrossRef]
  44. Fan, P.; Wu, L.; Wang, Q.; Wang, Y.; Luo, H.; Song, J.; Yang, M.; Yao, H.; Chen, S. Physiological and molecular mechanisms of medicinal plants in response to cadmium stress: Current status and future perspective. J. Hazard. Mater. 2023, 450, 131008. [Google Scholar] [CrossRef]
  45. Černý, M.; Habánová, H.; Berka, M.; Luklová, M.; Brzobohatý, B. Hydrogen Peroxide: Its Role in Plant Biology and Crosstalk with Signalling Networks. Int. J. Mol. Sci. 2018, 19, 2812. [Google Scholar] [CrossRef]
  46. Yang, T.; Poovaiah, B.W. Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin. Proc. Natl. Acad. Sci. USA 2002, 99, 4097–4102. [Google Scholar] [CrossRef]
  47. Shi, G.; Liu, C.; Cai, Q.; Liu, Q.; Hou, C. Cadmium accumulation and tolerance of two safflower cultivars in relation to photosynthesis and antioxidantive enzymes. Bull. Envion. Contam. Toxicol. 2010, 85, 256–263. [Google Scholar] [CrossRef] [PubMed]
  48. Hashem, A.; Abd Allah, E.F.; Alqarawi, A.A.; Al Huqail, A.A.; Egamberdieva, D.; Wirth, S. Alleviation of cadmium stress in Solanum lycopersicum L. by arbuscular mycorrhizal fungi via induction of acquired systemic tolerance. Saudi J. Biol. Sci. 2016, 23, 272–281. [Google Scholar] [CrossRef]
  49. Wang, L.; Jia, X.; Zhao, Y.; Zhang, C.Y.; Zhao, J. Effect of arbuscular mycorrhizal fungi in roots on antioxidant enzyme activity in leaves of Robinia pseudoacacia L. seedlings under elevated CO2 and Cd exposure. Environ. Pollut. 2022, 294, 118652. [Google Scholar] [CrossRef] [PubMed]
  50. Mhamdi, A.; Queval, G.; Chaouch, S.; Vanderauwera, S.; Van Breusegem, F.; Noctor, G. Catalase function in plants: A focus on Arabidopsis mutants as stress-mimic models. J. Exp. Bot. 2010, 61, 4197–4220. [Google Scholar] [CrossRef] [PubMed]
  51. Lv, J.; Tai, R.; Cao, Y.; Ge, Y.; Chen, J.; Li, J. Genome-wide identification and comparative expression analysis of ascorbate peroxidase (APX) gene family in apple fruit under 1-methylcyclopropene (1-MCP) and ethephon treatments during ripening. Sci. Hortic. 2024, 329, 113016. [Google Scholar] [CrossRef]
  52. Janeczko, A.; Przywara, M.; Maslanka, R.; Raś, B.; Ziaja, K.; Kwolek-Mirek, M.; Zadrag-Tecza, R.; Bednarska, S. Redox perturbations in yeast cells lacking glutathione reductase. Fungal Genet. Biol. 2023, 167, 103810. [Google Scholar] [CrossRef]
  53. Wu, Q.S.; Zou, Y.N.; Fathi Abd-Allah, E. Mycorrhizal Association and ROS in Plants. In Oxidative Damage to Plants Antioxidant Networks and Signaling; Academic Press: Cambridge, MA, USA, 2014; pp. 453–475. [Google Scholar] [CrossRef]
  54. Wood, J.P. Review of techniques for the in-situ sterilization of soil contaminated with Bacillus anthracis spores or other pathogens. Res. Microbiol. 2024, 175, 104175. [Google Scholar] [CrossRef]
  55. Phillips, J.M.; Hayman, D.S. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 1970, 55, 158-IN18. [Google Scholar] [CrossRef]
  56. Aguilar-Ulloa, W.; Arce-Acuña, P.; Galiano-Murillo, F.; Torres-Cruz, T.J. Spore isolation and evaluation of inoculation methods in the production of mycorrhizae in trap crops. Rev. Tecnol. Marcha 2016, 29, 5–14. [Google Scholar] [CrossRef]
  57. Garg, N.; Chandel, S. Role of Arbuscular Mycorrhizal (AM) Fungi on Growth, Cadmium Uptake, Osmolyte, and Phytochelatin Synthesis in Cajanus cajan (L.) Millsp. Under NaCl and Cd Stresses. J. Plant Growth Regul. 2012, 31, 292–308. [Google Scholar] [CrossRef]
  58. Nishiyama, I.; Fukuda, T.; Oota, T. Genotypic Differences in Chlorophyll, Lutein, and β-Carotene Contents in the Fruits of Actinidia Species. J. Agric. Food Chem. 2005, 53, 6403–6407. [Google Scholar] [CrossRef]
  59. Zhang, Y.; Chen, Y.; Guo, Y.; Ma, Y.; Yang, M.; Fu, R.; Sun, Y. Elevated CO2 delayed yellowing by maintaining chlorophyll biosynthesis and inhibiting chlorophyll degradation and carotenoid accumulation of postharvest broccoli. Postharvest Biol. Technol. 2022, 194, 112089. [Google Scholar] [CrossRef]
  60. Abdel Latef, A.A.; Tran, L.S.P. Impacts of priming with silicon on the growth and tolerance of maize plants to alkaline stress. Front. Plant Sci. 2016, 7, 182842. [Google Scholar] [CrossRef]
  61. Bates, L.S.; Waldren, R.P.; Teare, I.D. Rapid determination of free proline for water-stress studies. Plant Soil. 1973, 39, 205–207. [Google Scholar] [CrossRef]
  62. Anitha, R.; Mary, P.C.N.; Joseph, M.A.; Savery, R.; Sritharan, N.; Purushothaman, R.S. Differential responses of sugarcane (Saccharum officinarum L.) genotypes under salt stress condition. Plant Arch. 2015, 15, 1055–1060. [Google Scholar]
  63. Beauchamp, C.; Fridovich, I. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 1971, 44, 276–287. [Google Scholar] [CrossRef]
  64. Kumar, A.; Prasad, A.; Sedlářová, M.; Pospíšil, P. Malondialdehyde enhances PsbP protein release during heat stress in Arabidopsis. Plant Physiol. Biochem. 2023, 202, 107984. [Google Scholar] [CrossRef]
  65. Aebi, H. Catalase. In Methods of Enzymatic Analysis; Elsevier: Amsterdam, The Netherlands, 1974; pp. 673–684. [Google Scholar] [CrossRef]
  66. Azizollahi, Z.; Ghaderian, S.M.; Ghotbi-Ravandi, A.A. Cadmium accumulation and its effects on physiological and biochemical characters of summer savory (Satureja hortensis L.). Int. J. Phytoremed. 2019, 21, 1241–1253. [Google Scholar] [CrossRef]
  67. Nakano, Y.; Asada, K. Purification of Ascorbate Peroxidase in Spinach Chloroplasts; Its Inactivation in Ascorbate-Depleted Medium and Reactivation by Monodehydroascorbate Radical. Plant Cell Physiol. 1987, 28, 131–140. [Google Scholar] [CrossRef]
  68. dos Santos, M.L.S.; de Almeida, A.A.F.; da Silva, N.M.; Oliveira, B.R.M.; Silva, J.V.S.; Junior, J.O.S.; Ahnert, D.; Baligar, V.C. Mitigation of cadmium toxicity by zinc in juvenile cacao: Physiological, biochemical, molecular and micromorphological responses. Envion. Exp. Bot. 2020, 179, 104201. [Google Scholar] [CrossRef]
  69. Foyer, C.H.; Halliwell, B. The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism. Planta 1976, 133, 21–25. [Google Scholar] [CrossRef] [PubMed]
  70. Fijalkowski, K.L.; Kwarciak-Kozlowska, A. Phytotoxicity assay to assess sewage sludge phytoremediation rate using guaiacol peroxidase activity (GPX): A comparison of four growth substrates. J. Envion. Manag. 2020, 263, 110413. [Google Scholar] [CrossRef]
  71. Kireyko, A.V.; Veselova, I.A.; Shekhovtsova, T.N. Mechanisms of peroxidase oxidation of o-dianisidine, 3,3′,5,5′- tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate. Russ. J. Bioorg Chem. 2006, 32, 71–77. [Google Scholar] [CrossRef]
  72. Neves, V.A.; Lourenço, E.J. Peroxidase from peach fruit: Thermal Stability. Braz. Arch. Biol. Technol. 1998, 41, 179–186. [Google Scholar] [CrossRef]
  73. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
  74. Alsiary, W.A.; Madany, M.M.Y.; AbdElgawad, H. The pleiotropic role of Salinicoccus bacteria in enhancing ROS homeostasis and detoxification metabolism in soybean and oat to cope with pollution of triclosan. Plant Physiol. Biochem. 2024, 207, 108327. [Google Scholar] [CrossRef]
  75. Tawfik, D.S. Modification of Sulfhydryl Groups with DTNB; Springer: Berlin/Heidelberg, Germany, 2002. [Google Scholar] [CrossRef]
  76. Ellman, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70–77. [Google Scholar] [CrossRef]
  77. Jinadasa, N.; Collins, D.; Holford, P.; Milham, P.J.; Conroy, J.P. Reactions to cadmium stress in a cadmium-tolerant variety of cabbage (Brassica oleracea L.): Is cadmium tolerance necessarily desirable in food crops? Environ. Sci. Pollut. Res. 2016, 23, 5296–5306. [Google Scholar] [CrossRef] [PubMed]
  78. Berthou, M.; Clarot, I.; Gouyon, J.; Steyer, D.; Monat, M.A.; Boudier, A.; Pallotta, A. Thiol sensing: From current methods to nanoscale contribution. Microchem. J. 2022, 183, 107994. [Google Scholar] [CrossRef]
  79. Castillo-Israel, K.A.T.; Flandez, L.E.L.; Tuaño, A.P.P.; Sartagoda, K.J.D.; Compendio, M.C.M. Vitamin C levels of selected Philippine indigenous berries as affected by fruit maturity and processing treatment. Food Prod. Process. Nutr. 2023, 5, 33. [Google Scholar] [CrossRef]
  80. Di Rienzo Julio Macchiavelli, R.; Casanoves, F. Modelos Mixtos en Infostat. In Manual de Usuario; Editorial Brujas: Córdoba, Argentina, 2012; p. 193. [Google Scholar]
  81. Pinheiro, J.; Bates, D.; DebRoy, S.; Sarkar, D.; EISPACK; Heisterkamp, S.; Willigen, B.; Van Ranke, J.; The Comprehensive R. Linear and Nonlinear Mixed Effects Models (Package ’nlme’). 2025. Available online: https://cran.r-project.org/web/packages/nlme/nlme.pdf (accessed on 18 December 2024).
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Figure 1. (a) Colonization percentage by arbuscular mycorrhizal fungi (AMF) in sugarcane plants exposed to Cd (b,c) root sample hyphae enriched with AMF, observed at 40×. (d) Control root sample without AMF. Different letters indicate significant differences according to the DGC test (p < 0.05). Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition).
Figure 1. (a) Colonization percentage by arbuscular mycorrhizal fungi (AMF) in sugarcane plants exposed to Cd (b,c) root sample hyphae enriched with AMF, observed at 40×. (d) Control root sample without AMF. Different letters indicate significant differences according to the DGC test (p < 0.05). Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition).
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Figure 2. Effect of AMF on photosynthetic pigment content in sugarcane subjected to Cd. (a) Chl a: Chlorophyll a; (b) Chl b: Chlorophyll b; (c) Chl t: Total chlorophyll; (d) Car: Carotenoids; (e) Chl a/b: Chlorophyll a and b ratio; (f) Chl T/car: Total chlorophyll and carotenoids ratio. Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, SF+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition); * 15, 30 and 120 correspond to days.
Figure 2. Effect of AMF on photosynthetic pigment content in sugarcane subjected to Cd. (a) Chl a: Chlorophyll a; (b) Chl b: Chlorophyll b; (c) Chl t: Total chlorophyll; (d) Car: Carotenoids; (e) Chl a/b: Chlorophyll a and b ratio; (f) Chl T/car: Total chlorophyll and carotenoids ratio. Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, SF+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition); * 15, 30 and 120 correspond to days.
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Figure 3. Effect of AMF on oxidative damage indicators and proline in sugarcane subjected to Cd (a) MDA: Malondialdehyde (b) Proline. Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition); * 15, 30 and 120 correspond to days.
Figure 3. Effect of AMF on oxidative damage indicators and proline in sugarcane subjected to Cd (a) MDA: Malondialdehyde (b) Proline. Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition); * 15, 30 and 120 correspond to days.
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Figure 4. Effect of AMF on enzymatic antioxidants in sugarcane exposed to Cd (a) CAT: Catalase; (b) APX: Ascorbate peroxidase; (c) GR: Glutathione Reductase; (d) SOD: Superoxide Dismutase; (e) POD: Peroxidase; (f) GPX: Guaiacol Peroxidase. Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition); * 15, 30 and 120 correspond to days.
Figure 4. Effect of AMF on enzymatic antioxidants in sugarcane exposed to Cd (a) CAT: Catalase; (b) APX: Ascorbate peroxidase; (c) GR: Glutathione Reductase; (d) SOD: Superoxide Dismutase; (e) POD: Peroxidase; (f) GPX: Guaiacol Peroxidase. Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition); * 15, 30 and 120 correspond to days.
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Figure 5. Impact of AMF on non-enzymatic antioxidant compounds in sugarcane exposed to Cd (a) GSH: Reduced Glutathione; (b) NP-SH: Non-protein thiols; (c) PC: Phytochelatins; (d) AA: Ascorbic acid. Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: Soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition); * 15, 30 and 120 correspond to days.
Figure 5. Impact of AMF on non-enzymatic antioxidant compounds in sugarcane exposed to Cd (a) GSH: Reduced Glutathione; (b) NP-SH: Non-protein thiols; (c) PC: Phytochelatins; (d) AA: Ascorbic acid. Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: Soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition); * 15, 30 and 120 correspond to days.
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Figure 6. (a) Principal components by treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: No addition) and (b) Principal components by time, * 15, 30, and 120 corresponds to days.
Figure 6. (a) Principal components by treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: No addition) and (b) Principal components by time, * 15, 30, and 120 corresponds to days.
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Figure 7. Pearson correlation coefficients between photosynthetic pigments, oxidative damage, the enzymatic antioxidant system, and non-enzymatic antioxidant compounds of sugarcane exposed to Cd with the addition of AMF p ≤ 0.05.
Figure 7. Pearson correlation coefficients between photosynthetic pigments, oxidative damage, the enzymatic antioxidant system, and non-enzymatic antioxidant compounds of sugarcane exposed to Cd with the addition of AMF p ≤ 0.05.
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Table 1. Effect of AMF on Cadmium (Cd) concentrations (mg kg−1) in rhizosphere soil and sugarcane leaves over 120 days.
Table 1. Effect of AMF on Cadmium (Cd) concentrations (mg kg−1) in rhizosphere soil and sugarcane leaves over 120 days.
TreatmentsSoilLeaves
S-F+Cd68.38 ± 4.55 A1.21 ± 0.10 A
S+F+Cd48.73 ± 4.55 B0.47 ± 0.10 B
S-F-Cd0.18 ± 4.55 C0.15 ± 0.10 B
S+F-Cd0.18 ± 4.55 C0.15 ± 0.10 B
Data are represented as mean value ± S.E. Different letters indicate significant difference according to the DGC test at p < 0.05. Treatments S-F-Cd, S-F+Cd, S+F-Cd, S+F+Cd, (S: soil, F: Fungi; Cd: Cadmium 50 mg kg−1; +: Addition; -: Without addition).
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MDPI and ACS Style

Paladines-Beltrán, G.M.; Venegas, N.A.; Suárez, J.C. Arbuscular Mycorrhizal Fungi Enhance Antioxidant Defense Systems in Sugarcane Under Soil Cadmium Stress. Plants 2025, 14, 2916. https://doi.org/10.3390/plants14182916

AMA Style

Paladines-Beltrán GM, Venegas NA, Suárez JC. Arbuscular Mycorrhizal Fungi Enhance Antioxidant Defense Systems in Sugarcane Under Soil Cadmium Stress. Plants. 2025; 14(18):2916. https://doi.org/10.3390/plants14182916

Chicago/Turabian Style

Paladines-Beltrán, Gloria Magaly, Nathalia Alejandra Venegas, and Juan Carlos Suárez. 2025. "Arbuscular Mycorrhizal Fungi Enhance Antioxidant Defense Systems in Sugarcane Under Soil Cadmium Stress" Plants 14, no. 18: 2916. https://doi.org/10.3390/plants14182916

APA Style

Paladines-Beltrán, G. M., Venegas, N. A., & Suárez, J. C. (2025). Arbuscular Mycorrhizal Fungi Enhance Antioxidant Defense Systems in Sugarcane Under Soil Cadmium Stress. Plants, 14(18), 2916. https://doi.org/10.3390/plants14182916

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