Auxin-mediated modulation of root architecture enhances cadmium tolerance and root retention in Typha latifolia seedlings

Auxin-mediated modulation of root architecture enhances cadmium tolerance and root retention in Typha latifolia seedlings | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Auxin-mediated modulation of root architecture enhances cadmium tolerance and root retention in Typha latifolia seedlings Stephanie Rosales-Loredo, Gisela Adelina Rolón-Cárdenas, Candy Carranza-Álvarez, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9440581/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Typha latifolia is a wetland macrophyte widely used for Cd phytoremediation. However, the role of auxins in enhancing Cd tolerance and metal retention in this species remains poorly understood. This study evaluated the effects of exogenous indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA) on seed germination, root morphology, and Cd tolerance in T. latifolia . IAA and NAA accelerated early seed germination and significantly increased the density of root hairs in a concentration-dependent manner; however, high NAA concentrations induced root surface oxidation. In plants exposed to 40 mg/L Cd, treatment with IAA (1 mg/L) and NAA (0.5 mg/L) mitigated the stress by preserving root biomass and morphology. Antioxidant responses were activated, as evidenced by increased catalase (CAT) activity and glutathione (GSH) content. Auxin treatments promoted Cd retention in root tissues while limiting its translocation to shoots, suggesting localized metal detoxification. Histological analysis revealed increased Schiff reagent staining in auxin-treated roots, indicating that alterations in carbohydrate distribution within root tissues may contribute to Cd immobilization. These findings suggest that exogenous auxins improve Cd stress tolerance in T. latifolia through modulation of root development, activation of antioxidant defenses, and promotion of localized metal immobilization within the root system. This study underscores the potential applicability of auxin supplementation as a biotechnological tool to enhance phytoremediation efficiency in Cd-contaminated environments. Typha latifolia cadmium IAA NAA phytoremediation antioxidant response root morphology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Cadmium (Cd) contamination significantly threatens plant growth and agricultural sustainability due to its high toxicity and mobility from soil and water systems to plant tissues (Gallego et al., 2012 ; Li et al., 2019 ). Cd disrupts essential plant processes, including photosynthesis, nutrient uptake, and cellular integrity, inhibiting growth and reducing biomass production (Awa & Hadibarata, 2020 ; Li et al., 2019 ). However, some plant species can grow in the presence of Cd and other heavy metals. These metal-tolerant plants are used in phytoremediation strategies to remove heavy metal contaminants from polluted environments. Phytoremediation is a sustainable, non-invasive, ecological, and low-cost technology that uses plant species, either in situ or ex-situ , to reduce, remove, or immobilize heavy metals present in water, soils, sludge, and sediments (Ali et al. 2013 ; Yan et al. 2020 ). Typha latifolia is widely used owing to its great ability to tolerate and remove heavy metals such as Zn, Ni, Cu, Pb, Co, Mn, and Cd (Bonanno and Cirelli 2017 ; Amare and Workagegn 2022 ). In recent years, the use of plant growth regulators has gained considerable attention as a sustainable approach in phytoremediation strategies for heavy metals contamination (Rolón-Cárdenas et al. 2022a ). Plant growth regulators, such as auxins, modulate plant development and enhance stress resilience. Indole-3-acetic acid (IAA), a naturally occurring auxin, and 1-naphthaleneacetic acid (NAA), its synthetic analog, regulate numerous aspects of plant physiology, including cell elongation, root formation, vascular differentiation, and responses to abiotic stress (Zhang et al. 2022 ; Rolón-Cárdenas et al. 2022a ). Auxins have also been implicated in improving plant tolerance to heavy metals by modulating root architecture, stimulating antioxidant defense systems, and altering metal uptake and compartmentalization (Li et al. 2018 ; Mathur et al. 2022 ; Ejaz et al. 2023 ). Previous studies have reported that Cd exposure can disrupt endogenous IAA biosynthesis, reduce root growth, and alter hormonal homeostasis, ultimately impairing plant development (Ronzan et al. 2018 ; Rolón-Cárdenas et al. 2022b ; Araniti et al. 2023 ). In contrast, the exogenous application of auxins has been shown to alleviate heavy metal stress by enhancing antioxidant enzyme activity, preserving root integrity, and promoting metal sequestration in root tissues (Zhu et al. 2013 ; Liu et al. 2024 ). Therefore, auxin supplementation may represent a practical strategy for improving the phytoremediation potential of tolerant species (Rolón-Cárdenas et al. 2022a ). However, little is known about the effects of exogenous auxins on T. latifolia . This study evaluated the effects of exogenous IAA and NAA on seed germination, early seedling development, root architecture, and Cd tolerance in T. latifolia . The findings improve our understanding of the role of auxins in stress adaptation and offer insights into their potential application in phytoremediation strategies. Materials and Methods In vitro germination of T. latifolia seeds supplemented with auxins Mature seeds of T. latifolia were surface-sterilized using 50% sodium hypochlorite with 0.02% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and rinsed six times with sterile distilled water. The seeds were then placed in flasks with 20 ml of 0.5% (w/v) water-agar supplemented with indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA) (Sigma-Aldrich) at 0.1, 0.5, and 1 mg/L. The culture flasks were incubated at 28°C for 15 days under fluorescent light with a 16 h light/8 h dark photoperiod. Finally, seedlings were collected to determine germination percentage, number, and length of shoots and roots. Effect of indole acetic acid and naphthalene acetic acid in T. latifolia seedlings exposed and non-exposed to Cd Surface-sterilized seeds of T. latifolia were sown in culture flasks containing 25 ml of Murashige and Skoog 0.2 X agar (MS; Sigma-Aldrich, St. Louis, MO, USA), 1.0% (w/v) glucose, and 1.5% (w/v) phytagar (Sigma Aldrich, St. Louis, MO, USA). Culture flasks were incubated at 28°C, under fluorescent light with a photoperiod of 16 h/8 h light/dark, for 15 days. Germinated seeds were transferred to hydroponic systems (3 seedlings per system) containing 150 ml 0.2X MS medium, 1.0% (w/v) glucose, and MES buffer (3.5 mM, pH 5.7), and incubated at 28°C for 1 month under fluorescent light with photoperiod of 16 h/8 h light/dark. After a month of incubation, the effect of IAA and NAA was tested in seedlings with and without cadmium exposure for ten days. To determine the effect of IAA and NAA in T. latifolia development, seedlings were transferred to 0.2X MS medium without glucose at pH 5.7, supplemented with 0.1, 0.5, and 1 mg/L of either IAA or NAA. On the other hand, to determine the role of IAA and NAA in T. latifolia exposed to Cd, seedlings were transferred to 0.2X MS without glucose at pH 5.7, containing 0.1, 0.5, and 1 mg/L of either IAA or NAA and exposed to 40 mg/L Cd (CdCl 2 ) (Fermont, Monterrey, Mexico). All hydroponic cultures were incubated at 28°C under fluorescent light with a photoperiod of 16 h light/8 h dark for ten days. Quantification of photosynthetic pigments Total chlorophyll and carotenoid amounts were determined in the aerial tissue according to the methodology proposed by (Lichtenthaler and Buschmann 2001 ). Fresh tissue (50 mg) was homogenized with 2 ml of 95% ethanol and stored in the dark for 24 h at 4°C. The extract was filtered, and absorbance was assessed using a UV-Vis spectrophotometer (Evolution 221, Thermo Scientific, Waltham, MA, USA) at 664.1 nm (chlorophyll a), 648.6 nm (chlorophyll b), and 470 nm (carotenoids). Photosynthetic pigments were calculated according to the equations. Chlorophyll a: \(\:Cha=\frac{\left[\left(13.36*{A}_{664.1}\right)\:-\:(5.19*{A}_{648.6}\right]\:*\:solvent\:volume}{Sample\:}\) (1) Chlorophyll b: \(\:Chb=\frac{\left[\left(27.43*{A}_{648.6}\right)\:-\:(8.12*{A}_{664.1}\right]\:*\:solvent\:volume}{Sample\:}\) (2) Total Chlorophyll: \(\:Chtotal=Cha+Chb\) (3) Carotenoids: \(\:Cx+c=\frac{\left[\frac{\left(1000*{A}_{470}\right)\:-\:\left(2.13*{Ch}_{a}\right)\:-\:\left(97.64*{Ch}_{b}\right)}{209}\right]\:*\:solvent\:volume}{Sample}\) (4) Determination of electrolyte leakage The electrolyte leakage was determined on roots as described by (Umnajkitikorn et al. 2021 ). 100 mg of fresh tissue was immersed in 10 mL of deionized water at 20°C for 24 h. The initial electrical conductivity (ECi) was determined using a conductivity meter (PC2700 Oakton, Cole-Parmer, Vernon Hills, IL, USA). Subsequently, the samples were heated at 120°C in an autoclave for 15 min, and the final electrical conductivity (ECf) was determined as above. Deionized water was used as a blank. The electrical conductivity of roots was calculated according to the equation: $$\:\text{%}\:\text{E}\text{L}=\left[\frac{ECi\:-ECi\:blank}{ECf-ECf\:blank}\right]*100$$ 5 Determination of glutathione content Total glutathione (GSH) determination was performed on root tissue as described by (Rahman et al. 2006 ). Fresh tissue (250 mg) was powdered in liquid nitrogen and homogenized with 500 µl of 100 mM potassium phosphate buffer pH 7.5 with 5 mM EDTA (KPE). Samples were centrifuged at 12000 xg for 10 min at 4°C, 150 µl of the supernatant was then mixed with 150 µl of extraction buffer (0.1% Triton X-100 and 0.6% sulfosalicylic acid in KPE). Subsequently, 100 µl of the sample was treated with 700 µl KPE buffer, 60 µl DTNB 1.68 mM, 60 µl glutathione reductase (50 U/ml; GR, Sigma-Aldrich, St. Louis, MO, USA) and 60 µl 0.9 mM NADPH. The reaction mixture was incubated at room temperature for 10 min in the darkness, and the absorbance was determined at 412 nm. The glutathione content was calculated using the GSH standard curve (Sigma-Aldrich, St. Louis, MO, USA). Determination of catalase activity Catalase activity (CAT) was determined in leaves. 150 mg of fresh tissue was homogenized with 1.5 ml of KPE buffer (100 mM phosphate buffer pH 7.5 with 5 mM EDTA). The samples were centrifuged at 10°C for 15 min at 13000 xg. The total protein content of the supernatant was determined using the Bradford method. Samples were then adjusted to 50 µg/ml protein (using KPE buffer), and finally, catalase activity was determined spectrophotometrically at 240 nm, using the method of (Aebi 1984 ). Determination of Cadmium content Cd content in plant tissues was determined using the methodology according to (Carranza-Álvarez et al. 2008 ). Root tissue was washed with deionized water and then with 0.01 M EDTA. The seedling was sectioned into leaves and roots and dried at 70°C for 48 h. The dry roots were digested with concentrated HNO 3 plus 30% H 2 O 2 , while the shoots were digested with a mixture of HCl:HNO 3 (3:1 v/v). Samples of the hydroponic medium were diluted to 1:10 and acidified with trace grade HNO 3 to 5%. Cd determination in the plant samples and the hydroponic medium was carried out by air-acetylene flame atomic absorption spectrophotometry (AAS) (iCE 3000 series from Thermo Scientific™) at a wavelength of 228.8 nm, using a hollow cathode Cd lamp (Catalogue No.: 942339030481, Thermo Scientific™). The metal concentration was estimated using a Cd standard curve, normalizing the Cd content with the dry weight in the plant samples and expressing it in mg/Kg. Finally, the translocation factor (TF), bioconcentration factor (BCF), and bioaccumulation coefficient (BAC) were calculated. Root structure of T. latifolia seedlings To determine structural changes in the roots of T. latifolia seedlings exposed to the treatments, root cuttings were obtained according to the methodology described by Chen et al. ( 2017 ). Fresh root samples were taken and fixed in neutral formalin (4%) for 48 h. They were then dehydrated in a 25 ml ethanol series at 60°C as follows: 30%, 50%, 70%, 85%, 95% and 100%, for 30 min. Subsequently, the samples were infiltrated in paraffin and cut on a microtome (ECOSHEL) to obtain 5 µm thick root samples, which were fixed on salinized slides. Periodic acid Schiff (PAS) staining was used to stain the fixed tissue as described by (Rincón-Barón et al. 2023 ). PAS stains were observed under an inverted microscope (Zeiss axiovert 135) and analyzed with ImageJ version 1.53e software. Results Effect of IAA and NAA on seed germination and development of T. latifolia seedlings Initially, the germination percentage of T. latifolia seeds was assessed with and without IAA and NAA supplementation. The results showed that both IAA and NAA induced early germination in 25% of the seeds within 24 h, whereas seeds in the control group exhibited delayed germination. By 48 h, seeds treated with IAA or NAA reached a germination rate of 98%, which was statistically comparable to that of the control (Fig. 1). Although final germination rates were similar between auxin-treated and control seeds, auxin treatment promoted earlier germination, indicating that IAA and NAA accelerate the onset of germination. Fifteen days after germination, the effects of IAA and NAA on the early developmental stages of T. latifolia seedlings were evaluated. The results showed that the application of IAA at 0.5 and 1 mg/L significantly increased the number of roots per seedling compared to the untreated control (p ≤ 0.05). However, a dose-dependent reduction in the root length was observed with increasing IAA concentrations (Fig. 2). In addition, the highest concentration (1 mg/L) significantly reduced shoot length and leaf number compared with the control. In contrast, treatment with 0.1 mg/L NAA significantly enhanced seedling growth compared to the 0.5 and 1 mg/L treatments. Specifically, 0.1 mg/L NAA increased shoot length and leaf number and maintained root length at levels comparable to the control. Higher NAA concentrations produced contrasting effects: while 0.5 mg/L reduced shoot growth but increased the number of roots, 1 mg/L markedly inhibited both root elongation and root formation (Fig. 3). Overall, these results indicate that exogenous application of IAA and NAA modulates the early growth and development of T. latifolia , particularly by increasing the number of roots during the post-germination stage (15 days after germination). Due to the marked effects observed on root development, a more detailed analysis of structural and morphological changes was subsequently conducted using hydroponically cultivated plants. Effects of IAA and NAA on the root morphology of T. latifolia One-month-old T. latifolia plants grown in hydroponic systems were used in dose-response experiments to evaluate the morphological effects of IAA and NAA on the root system over a ten-day period. The results showed an increase in root hair formation in T. latifolia , which was positively correlated with auxin concentration. For IAA, auxin-treated plants showed increased root hair formation compared with the control. Although differences were not statistically significant, auxin-treated plants tended to exhibit higher root biomass, with fresh and dry weights of 0.655 and 0.030 g/plant, respectively. In contrast, control plants developed fewer root hairs and showed fresh and dry weights of 0.382 and 0.021 g/plant, respectively (Fig. 4). On the other hand, seedlings treated with NAA showed responses similar to those observed with IAA at lower concentrations, although adverse morphological effects became evident at higher doses. All tested concentrations produced visible alterations in the surface morphology of the primary root, characterized by increased surface roughness that intensified with auxin concentration. Additionally, treatment with 1 mg/L NAA caused visible tissue oxidation in the root compared with the untreated control. In terms of biomass, NAA-treated plants tended to show higher fresh and dry root weights than control plants. Although differences in root biomass were not statistically significant among treatments, a trend toward increased root biomass with NAA application was observed. For example, seedlings treated with 0.5 mg/L NAA exhibited fresh and dry weights of 0.754 and 0.044 g/plant, respectively, whereas control plants showed values of 0.574 and 0.029 g/plant (Fig. 5). Furthermore, microscopic changes induced by IAA and NAA treatments in the root tissues of T. latifolia were examined in 5 µm-thick root sections. Plants treated with IAA exhibited structural alterations in the root tissues compared with the control. Based on comparisons with the scale bar, treated plants showed an apparent increase in root diameter relative to the untreated control. Similar structural changes were observed in the root tissues of plants treated with NAA (Fig. 6). Overall, these observations indicate that both IAA and NAA induce morphological modifications in the root tissues of T. latifolia . Influence of IAA and NAA on the root morphological response of T. latifolia under Cd stress The effects of IAA and NAA during Cd exposure were evaluated using the concentrations that showed the most favorable responses in T. latifolia plants: 1 mg/L IAA and 0.5 mg/L NAA. T. latifolia plants were exposed to 40 mg/L Cd, with or without supplementation of IAA or NAA, under hydroponic conditions. Cd exposure caused a significant reduction in both fresh and dry weights of T. latifolia shoots, along with visible wilting of aerial tissues compared to control plants (Fig. 7a). In Cd-exposed plants, no significant changes in shoot fresh or dry weight were observed in response to NAA treatment. In contrast, IAA treatment significantly increased shoot biomass compared with Cd-treated plants, partially mitigating the inhibitory effect of Cd on shoot growth. Regarding the root system, Cd stress significantly reduced both the fresh and dry weights of roots compared with the non-exposed control. However, IAA and NAA treatments did not show a significant effect on root biomass production in T. latifolia plants exposed to Cd (Fig. 7b, c). Morphological observations showed that Cd exposure also decreased the number of root hairs and caused browning and thinning of the roots relative to control plants. However, supplementation with IAA and NAA maintained root hair development and density, and reduced browning of root tissues compared to Cd-treated roots without auxin supplementation. Additionally, Schiff staining indicated stronger staining intensity in auxin-treated roots, suggesting possible alterations in carbohydrate distribution (Fig. 8). Biochemical response of T. latifolia plants treated with IAA and NAA under Cd stress The biochemical response of T. latifolia plants to Cd exposure, with or without auxins supplementation, was also evaluated. In this study, we evaluated total chlorophyll content, electrolyte leakage (EL), catalase (CAT) activity and glutathione (GSH) content. Cd exposure did not cause statistically significant changes in total chlorophyll content compared with the control without Cd (Fig. 9a). Similarly, CAT activity showed only a slight increase under Cd exposure, although differences were not statistically significant relative to the control (Fig. 9c). However, Cd exposure significantly increased electrolyte leakage and glutathione content in roots (Fig. 9b, d). Application of NAA in Cd-exposed plants did not significantly affect total chlorophyll content, CAT activity, or GSH levels (Fig. 9a, c, d). However, NAA treatment significantly increased electrolyte leakage (by 38%) compared with Cd-treated plants (Fig. 9b). Similarly, IAA treatment had no significant effect on chlorophyll content or CAT activity (Fig. 9a, c). In contrast, IAA supplementation significantly increased both electrolyte leakage (44%) and glutathione content (100%) in roots compared with the Cd-only treatment (Fig. 9b, d). Influence of IAA and NAA on Cd uptake in T. latifolia Table 1 shows the Cd content and accumulation in both shoots and roots of T. latifolia plants treated with auxins. The results indicate that Cd content was consistently higher in roots than in shoots across all treatments. A significant increase in Cd concentration was observed in the roots of plants supplemented with NAA (3595 mg kg⁻¹) compared with the Cd-only treatment (2880 mg kg⁻¹); however, this treatment did not promote Cd accumulation in shoots. In contrast, IAA supplementation resulted in intermediate Cd concentrations in roots (3200 mg kg⁻¹), which were not significantly different from either the Cd treatment or the NAA treatment. Likewise, the application of IAA or NAA did not produce statistically significant differences in the translocation factor (TF), bioconcentration factor (BCF), or bioaccumulation coefficient (BAC) compared with plants exposed to Cd alone. Table 1 Cadmium (Cd) content and accumulation in T. latifolia plants treated with NAA and IAA. Treatment Cd content (mg/Kg) Cd removal (%) TF BCF BAC Shoots Roots Control ND ND ND ND ND ND Cd 1387.40 \(\:\pm\:\:131.53\) a 2880.71 \(\:\pm\:\:\) 161.88 b 15.76 \(\:\pm\:\:\) 8.27 a 0.481 \(\:\pm\:\:\) 0.02 a 68.99 \(\:\pm\:\:\) 3.59 a 33.23 \(\:\pm\:\) 3.04 a Cd + NAA 1410.12 \(\:\pm\:\:\) 92 a 3595.44 \(\:\pm\:\:276.09\:\) a 14.53 \(\:\pm\:\) 2.33 a 0.394 \(\:\pm\:\) 0.04 a 85.11 \(\:\pm\:\) 9.19 a 33.33 \(\:\pm\:\) 2.62 a Cd + IAA 1442.36 \(\:\pm\:\) \(\:212.51\) a 3200.77 \(\:\pm\:\:255.54\) ab 14.81 \(\:\pm\:\) 0.92 a 0.450 \(\:\pm\:\) 0.04 a 76.62 \(\:\pm\:\:\) 6.35 a 34.54 \(\:\pm\:\) 5.29 a TF: translocation factor; BCF: bioconcentration factor; BAC: bioaccumulation coefficient; ND: not detected. Values represent the mean \(\:\pm\:\) SD (n = 3). Different letters represent statistically different means (p ˂ 0.05, Tukey´s test). Discussion Auxins are phytohormones that regulate plant organogenesis, vascular tissue development, and differential plant growth (Tan et al. 2021 ). IAA is the most prevalent auxin, which is synthesized endogenously by actively growing tissues, root tips, leaf apices, coleoptiles, young leaves, and developing fruits, where it coordinates cambial growth and vascular development (Flasiński and Hąc-Wydro 2014 ). IAA synthesis in plants exposed to Cd has been reported to decrease; however, this response varies depending on the plant species, Cd concentration, and the specific plant tissue analyzed (Rolón-Cárdenas et al. 2022b ). Despite the well-established role of auxins in plant development, their influence on germination, root architecture, and cadmium tolerance in T. latifolia remains poorly understood. Therefore, this study evaluated the effects of IAA and NAA on T. latifolia seedlings with and without Cd. The results indicated that IAA and NAA improved T. latifolia seed germination. Although final germination percentages were similar between auxin-treated and control seeds, the earlier onset of germination observed in this study suggests that exogenous auxin application accelerates the initiation of germination rather than increasing the final germination capacity. Similar results have been reported in Zea mays , where supplementation with IAA and indole butyric acid (IBA) increased germination by 10% after both 48 h and 72 h compared with non-supplemented seeds (Ullah et al. 2023 ). Likewise, IAA has been shown to enhance germination in Nicotiana tabacum , Araucaria angustifolia , and Helianthus annuus (Pieruzzi et al. 2011 ), further supporting the role of exogenous auxin application in promoting germination processes. Auxins exert many of their physiological effects in roots, where they regulate root system architecture and development. Therefore, understanding their influence on root morphology is essential to elucidate how auxin supplementation affects early root development in T. latifolia . In this context, the present study evaluated the effects of IAA and NAA on the root morphology of T. latifolia . The results showed that IAA increased the number of roots in T. latifolia seedlings in a dose-dependent manner. Conversely, the synthetic auxin, NAA, exhibits its positive effect only at the lowest concentration (0.1 mg/L). Although positive effects depend on type and auxin concentration, IAA and NAA increased the number of roots per seedling. Similarly, exogenous IAA increased the number of roots in two-month-old T. latifolia seedlings (Rolón-Cárdenas et al. 2022b ). In addition, exogenous applications of NAA and IBA have been reported to produce similar effects on root development in Parthenocissus quinquefolia and Allium cepa (Abu-Zahra et al. 2012 ; Sun et al. 2020 ). Furthermore, IAA increased the number of root hairs and tended to increase fresh and dry weight in the roots of T. latifolia seedlings in a dose-dependent manner. Similarly, IAA and IBA enhance root biomass from 13.10% − 35.60% in Pinus yunnanensis Franch species (Xu et al. 2012 ), while in Triticum aestivum and Solanum melongena IAA increases the root length (Agami and Mohamed 2013 ; Singh and Prasad 2015 ). On the other hand, even though NAA increased the number of root hairs, it caused visible surface alterations characterized by increased root surface roughness and tissue oxidation in the primary root of T. latifolia seedlings in correlation with its concentration. This effect may be attributed to the synthetic auxin NAA, which at higher concentrations disrupts root architecture and normal root development (Rosier et al. 2004 ; Rahman et al. 2006 ). Regarding the microscopic evaluation of seedling root architecture, the results suggest that IAA and NAA treatments increased root hair diameter in a dose-dependent manner. Moreover, the enhanced affinity for the Schiff reagent, evidenced by the formation of a pink-colored Schiff reaction product, may indicate alterations in carbohydrate distribution within root tissues, potentially reflecting modifications in cell wall components such as pectin, cellulose, and hemicellulose involved in root structural organization (Majda and Robert 2018 ; Šípošová et al. 2023 ). Other significant components of the root cell wall that may be influenced by auxin signaling include cellulose, hemicellulose, and structural glycoproteins, which contribute to mechanical strength, porosity, and wall remodeling (Showalter 2001 ; Cosgrove 2005 ). Auxin-induced regulation of these molecules facilitates cell elongation and root hair differentiation, as demonstrated by their essential roles in root development (Velasquez et al. 2011 ). Given the pronounced effects of auxins on root development observed in this study, we next evaluated whether these phytohormones could also modulate plant responses to cadmium-induced stress. Our results showed that IAA supplementation significantly increased shoot biomass relative to Cd-treated plants, partially mitigating the inhibitory effects of the metal. In contrast, NAA supplementation did not produce statistically significant changes in plant biomass under Cd exposure. This is consistent with findings in Brassica juncea , where IAA increased biomass even under Cd exposure (Chen et al. 2020 ) and Solanum nigrum (Ji et al. 2015 ), which demonstrate that IAA favors the growth of plants exposed to Cd. IAA has also been shown to protect the photosynthetic apparatus from deleterious Cd effects by restoring carotenoids and total chlorophyll content in Solanum melongena , Solanum nigrum (Ran et al. 2020 ), Amaranthus hypochondriacus , and Sedum alfredii (Chen et al. 2017 ; Sun et al. 2020 ). However, in T. latifolia seedlings, the auxin supplementation did not show evident effects. Although IAA tended to mitigate pigment loss, the effect was not statistically significant in T. latifolia , suggesting species-specific responses. Concerning the root architecture, both IAA and NAA preserved root integrity by increasing the number of roots even under Cd-induced stress conditions. Similar results have been reported in Medicago sativa (Wang et al. 2016 ) and Oryza sativa (Wang et al. 2021 ) following exposure to heavy metals. In addition, IAA and NAA maintained root hair diameter in Cd-exposed plants and enhanced affinity to the Schiff reagent, suggesting possible alterations in carbohydrate distribution within root tissues. These changes may reflect modifications in cell wall carbohydrates involved in root structural organization, potentially contributing to Cd retention or stabilization within the root system. However, further studies are required to quantify carbohydrate content and clarify the mechanisms underlying these responses. Cd has been shown to induce lipid peroxidation of cell membranes, thereby altering root cell membrane integrity. In this study, electrolyte leakage increased in T. latifolia roots, suggesting a loss of membrane integrity after Cd exposure. Similar results have been reported in T. latifolia (Rolón-Cárdenas et al. 2022b ), T. angustifolia (Ren et al. 2020 ), Trifolium repens (Wu et al. 2022 ), and Brassica oleracea (Jia et al. 2023 ) plants exposed to Cd. Furthermore, auxin supplementation further increased electrolyte leakage, similar to that reported by (Rolón-Cárdenas et al. 2022b ) in two-month-old T. latifolia seedlings. These findings indicate that auxin applications may enhance certain stress responses but may also contribute to increased membrane destabilization under Cd exposure, particularly in developing root tissues (one-month-old seedlings). Reactive oxygen species (ROS) are released upon metal stress. However, plants activate enzymatic and non-enzymatic antioxidant systems to mitigate ROS damage (Raihan et al. 2022 ). In the present study, catalase (CAT) activity showed a slight increase after Cd exposure; however, this change was not statistically significant compared with control plants. Catalase activity has been reported to increase after Cd exposure in wheat varieties, indicating increased ROS production caused by the metal (Guo et al. 2019 ). Likewise, both IAA and NAA increased CAT activity in T. latifolia plants similarly to responses reported in Solanum lycopersicum , Sorghum bicolor , and Camellia sinensis exposed to Cd (Zhan et al. 2017 ; Zhang et al. 2022 ; Guan et al. 2022 ). These results suggest that auxins do not suppress, but rather enhance catalase activity under Cd stress, possibly as part of a broader antioxidant response mechanism (Zhan et al. 2017 ; Guan et al. 2022 ). Moreover, glutathione (GSH) increased in plants exposed to Cd, similar to that observed in T. angustifolia , where exposure to Cd promoted the synthesis of glutathione and phytochelatins (Liu et al. 2016 ; Ren et al. 2020 ). Likewise, GSH content increased in plants exposed to IAA. (Khan et al. 2019 ) and (Bashri and Prasad 2016 ) report a similar effect in Lycopersicum esculantum and Trigonella foenum-graecum with the addition of IAA under Cd stress. This reinforces that IAA can stimulate glutathione biosynthesis under cadmium exposure, improving cellular detoxification and metal tolerance. The observed increase in GSH and CAT likely contributed to root integrity preservation and improved biomass under Cd stress. Finally, IAA and NAA increased Cd content in the roots of T. latifolia seedlings. NAA has been shown to increase Cd accumulation in Arabidopsis thaliana , by inducing hemicellulose synthesis in the roots, thereby increasing Cd 2+ binding to the cell wall (Zhu et al. 2013 ). Similarly, IAA has been reported to promote heavy metal accumulation in the roots of Solanum nigrum and Helianthus annuus (Ran et al. 2020 ; Chen et al. 2021 ). Overall, the results of this study indicate that auxin supplementation primarily promotes Cd retention in root tissues rather than enhancing metal translocation to aerial organs. This localized accumulation may contribute to improved Cd tolerance by restricting metal mobility and reducing systemic toxicity. Taken together, these findings demonstrate that exogenous IAA and NAA modulate root development and physiological responses in T. latifolia , particularly through the maintenance of root architecture, activation of antioxidant mechanisms, and enhanced Cd immobilization within the root system. Future studies should explore the molecular mechanisms underlying these responses, including auxin-responsive gene regulation and Cd transporter activity. Conclusion This study demonstrated that exogenous application of IAA and NAA modulates the germination and early development of T. latifolia seedlings. Both auxins accelerated seed germination and promoted root architecture development by increasing the number of root hairs in a concentration-dependent manner. However, NAA at higher concentrations caused visible alterations in root surface integrity, suggesting auxin-specific and dose-dependent effects on root architecture. Under Cd exposure, applying 1 mg/L IAA and 0.5 mg/L NAA partially mitigated the adverse effects of Cd, preserving root morphology and biomass and activating antioxidant defense mechanisms. Auxin treatments also enhanced root hair development and were associated with alterations in carbohydrate distribution in root tissues, potentially contributing to Cd retention within the root system without promoting translocation to aerial parts. Overall, these findings indicate that auxin-mediated modulation of root architecture and antioxidant responses may contribute to improved Cd tolerance in T. latifolia by promoting localized metal immobilization within the root system. This physiological response suggests the potential applicability of auxin supplementation as a strategy to support phytoremediation processes in Cd-contaminated environments. Declarations Conflict of interest . The authors declare that they have no conflict of interest. Consent to participate . This study does not contain any personal data requiring your consent. Consent to publish . This study does not contain any personal data requiring your consent. Funding The work reported was supported by grants from CONACYT, Programa Presupuestario F003 (Formerly Fondo Sectorial de Investigación para la Educación CB2017-2018), Project number A1-S-40454 to Alejandro Hernández-Morales. Author Contribution SRL, GARC, and AHM conceived and designed the study. SRL performed the research, CCA and JRMP analyzed the data and supervision. SRL wrote the paper. AHM edited and revised the paper. AHM received funding and edited the final version of the paper. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9440581","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627721118,"identity":"5ee351be-9f79-4362-aa58-99df24152465","order_by":0,"name":"Stephanie Rosales-Loredo","email":"","orcid":"","institution":"Autonomous University of San Luis Potosí","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Rosales-Loredo","suffix":""},{"id":627721119,"identity":"59446896-1cee-4a93-a854-2e48de1e9114","order_by":1,"name":"Gisela Adelina Rolón-Cárdenas","email":"","orcid":"","institution":"Autonomous University of San Luis Potosí","correspondingAuthor":false,"prefix":"","firstName":"Gisela","middleName":"Adelina","lastName":"Rolón-Cárdenas","suffix":""},{"id":627721120,"identity":"08a694cb-4348-422b-86fd-49f229e23e39","order_by":2,"name":"Candy Carranza-Álvarez","email":"","orcid":"","institution":"Autonomous University of San Luis Potosí","correspondingAuthor":false,"prefix":"","firstName":"Candy","middleName":"","lastName":"Carranza-Álvarez","suffix":""},{"id":627721122,"identity":"68b3f224-7571-4cc8-82b2-10ee5d5486ec","order_by":3,"name":"José Roberto Macías-Pérez","email":"","orcid":"","institution":"Autonomous University of San Luis Potosí","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Roberto","lastName":"Macías-Pérez","suffix":""},{"id":627721123,"identity":"5dcba360-7cfe-4092-90cd-f10fffd6b9ab","order_by":4,"name":"Alejandro Hernández-Morales","email":"data:image/png;base64,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","orcid":"","institution":"Autonomous University of San Luis Potosí","correspondingAuthor":true,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Hernández-Morales","suffix":""}],"badges":[],"createdAt":"2026-04-16 16:25:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9440581/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9440581/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108225468,"identity":"710ea1a8-a72e-463f-8afe-4b868cb2344c","added_by":"auto","created_at":"2026-04-30 16:17:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":842933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e germination percentage of \u003cem\u003eT. latifolia\u003c/em\u003e after 15 days supplemented with auxins. 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Different letters indicate a significant difference, using Dunn's test (\u003cem\u003ep\u003c/em\u003e ≤ 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9440581/v1/83c5a86308d5bc36678b7337.png"},{"id":108225470,"identity":"814a88d5-8911-4bad-b463-6641394a9ea9","added_by":"auto","created_at":"2026-04-30 16:17:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":862458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eT. latifolia \u003c/em\u003eafter 15 days of \u003cem\u003ein vitro \u003c/em\u003egermination supplemented with 1-naphthaleneacetic acid. (\u003cstrong\u003ea\u003c/strong\u003e) plant morphology (\u003cstrong\u003eb\u003c/strong\u003e)number (\u003cstrong\u003ec\u003c/strong\u003e) length of shoots and roots per plant. Data represent the mean ± SD (n = 3). 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Data represent the mean ± SD (n = 3). \u0026nbsp;Different letters indicate a significant difference at \u003cem\u003ep\u003c/em\u003e≤ 0.05 (Tukey's post hoc test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9440581/v1/8778a560dd83b410c4a2b1af.png"},{"id":108491062,"identity":"147764fa-1da4-4d7f-b66f-492c33e28af4","added_by":"auto","created_at":"2026-05-05 09:51:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":421613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eT. latifolia \u003c/em\u003eroots, one-month-old, after exposure to 1-naphthaleneacetic acid(10 days). Data represent the mean ± SD (n = 3). \u0026nbsp;Different letters indicate a significant difference at \u003cem\u003ep\u003c/em\u003e≤ 0.05 (Tukey's post hoc test).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9440581/v1/83c2963d59e21b3db9650356.png"},{"id":108225472,"identity":"f01a5881-73fb-421c-b7d1-00b9ced59e3f","added_by":"auto","created_at":"2026-04-30 16:17:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9875845,"visible":true,"origin":"","legend":"\u003cp\u003eHistology, one-month-old T. \u003cem\u003elatifolia\u003c/em\u003e, after auxins exposure (10 days).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9440581/v1/99daa8eb40fb4fbec778a42c.png"},{"id":108225474,"identity":"821d47ee-6c0a-4d82-9afc-1e58eafefef0","added_by":"auto","created_at":"2026-04-30 16:17:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4361004,"visible":true,"origin":"","legend":"\u003cp\u003eOne-month-old\u003cem\u003e T. latifolia \u003c/em\u003eplants exposed to 40 mg/L ofCd + auxins \u003cstrong\u003e(a) \u003c/strong\u003elateral and top view of\u003cem\u003e \u003c/em\u003eshoots (\u003cstrong\u003eb\u003c/strong\u003e) fresh weight (\u003cstrong\u003ec\u003c/strong\u003e) dry weight of aerial tissue and roots. Datarepresent the mean ± SD (n = 3). Different letters indicate a significant difference at \u003cem\u003ep \u003c/em\u003e≤ 0.05 (Tukey's post hoc test).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9440581/v1/41cf58bea5c40daa2235de71.png"},{"id":109081150,"identity":"6bbec93f-f868-460a-82b8-e7de5e309c6e","added_by":"auto","created_at":"2026-05-12 12:02:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5762438,"visible":true,"origin":"","legend":"\u003cp\u003eOne-month-old \u003cem\u003eT. latifolia \u003c/em\u003eroot histology, exposed to 40 mg/L Cd (10 days).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9440581/v1/ae3a1a151ff185664af4070b.png"},{"id":108225476,"identity":"9a8666f3-f508-44fd-86c3-b2ff936e47e1","added_by":"auto","created_at":"2026-04-30 16:17:19","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":422315,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of auxins (NAA and IAA) on \u003cem\u003eT.\u003c/em\u003e \u003cem\u003elatifolia \u003c/em\u003eplants exposed to 40 mg/L of Cd (10 days)\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003ea\u003c/strong\u003e) total chlorophyll (\u003cstrong\u003eb\u003c/strong\u003e) electrolyte leakage (\u003cstrong\u003ec\u003c/strong\u003e) CAT activity and (\u003cstrong\u003ed\u003c/strong\u003e) glutathione. Data represent the mean ± SD (n = 3). Different letters indicate a significant difference at \u003cem\u003ep\u003c/em\u003e≤ 0.05 (Tukey's post hoc test).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9440581/v1/1f42705ba8fa583d8fcc63e0.png"},{"id":109082595,"identity":"64a81511-810a-49cc-8d7a-d8cc4103a27b","added_by":"auto","created_at":"2026-05-12 12:41:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26754440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9440581/v1/ec2388c4-00f1-41ea-a5d5-251ca0c391dd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAuxin-mediated modulation of root architecture enhances cadmium tolerance and root retention in \u003cem\u003eTypha latifolia\u003c/em\u003e seedlings\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCadmium (Cd) contamination significantly threatens plant growth and agricultural sustainability due to its high toxicity and mobility from soil and water systems to plant tissues (Gallego et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cd disrupts essential plant processes, including photosynthesis, nutrient uptake, and cellular integrity, inhibiting growth and reducing biomass production (Awa \u0026amp; Hadibarata, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, some plant species can grow in the presence of Cd and other heavy metals. These metal-tolerant plants are used in phytoremediation strategies to remove heavy metal contaminants from polluted environments.\u003c/p\u003e \u003cp\u003ePhytoremediation is a sustainable, non-invasive, ecological, and low-cost technology that uses plant species, either \u003cem\u003ein situ\u003c/em\u003e or \u003cem\u003eex-situ\u003c/em\u003e, to reduce, remove, or immobilize heavy metals present in water, soils, sludge, and sediments (Ali et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eTypha latifolia\u003c/em\u003e is widely used owing to its great ability to tolerate and remove heavy metals such as Zn, Ni, Cu, Pb, Co, Mn, and Cd (Bonanno and Cirelli \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Amare and Workagegn \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn recent years, the use of plant growth regulators has gained considerable attention as a sustainable approach in phytoremediation strategies for heavy metals contamination (Rol\u0026oacute;n-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Plant growth regulators, such as auxins, modulate plant development and enhance stress resilience. Indole-3-acetic acid (IAA), a naturally occurring auxin, and 1-naphthaleneacetic acid (NAA), its synthetic analog, regulate numerous aspects of plant physiology, including cell elongation, root formation, vascular differentiation, and responses to abiotic stress (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rol\u0026oacute;n-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Auxins have also been implicated in improving plant tolerance to heavy metals by modulating root architecture, stimulating antioxidant defense systems, and altering metal uptake and compartmentalization (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mathur et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ejaz et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies have reported that Cd exposure can disrupt endogenous IAA biosynthesis, reduce root growth, and alter hormonal homeostasis, ultimately impairing plant development (Ronzan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rol\u0026oacute;n-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Araniti et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, the exogenous application of auxins has been shown to alleviate heavy metal stress by enhancing antioxidant enzyme activity, preserving root integrity, and promoting metal sequestration in root tissues (Zhu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, auxin supplementation may represent a practical strategy for improving the phytoremediation potential of tolerant species (Rol\u0026oacute;n-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). However, little is known about the effects of exogenous auxins on \u003cem\u003eT. latifolia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThis study evaluated the effects of exogenous IAA and NAA on seed germination, early seedling development, root architecture, and Cd tolerance in \u003cem\u003eT. latifolia\u003c/em\u003e. The findings improve our understanding of the role of auxins in stress adaptation and offer insights into their potential application in phytoremediation strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003egermination of\u003c/b\u003e \u003cb\u003eT. latifolia\u003c/b\u003e \u003cb\u003eseeds supplemented with auxins\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMature seeds of \u003cem\u003eT. latifolia\u003c/em\u003e were surface-sterilized using 50% sodium hypochlorite with 0.02% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and rinsed six times with sterile distilled water. The seeds were then placed in flasks with 20 ml of 0.5% (w/v) water-agar supplemented with indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA) (Sigma-Aldrich) at 0.1, 0.5, and 1 mg/L. The culture flasks were incubated at 28\u0026deg;C for 15 days under fluorescent light with a 16 h light/8 h dark photoperiod. Finally, seedlings were collected to determine germination percentage, number, and length of shoots and roots.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of indole acetic acid and naphthalene acetic acid in\u003c/b\u003e \u003cb\u003eT. latifolia\u003c/b\u003e \u003cb\u003eseedlings exposed and non-exposed to Cd\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSurface-sterilized seeds of \u003cem\u003eT. latifolia\u003c/em\u003e were sown in culture flasks containing 25 ml of Murashige and Skoog 0.2 X agar (MS; Sigma-Aldrich, St. Louis, MO, USA), 1.0% (w/v) glucose, and 1.5% (w/v) phytagar (Sigma Aldrich, St. Louis, MO, USA). Culture flasks were incubated at 28\u0026deg;C, under fluorescent light with a photoperiod of 16 h/8 h light/dark, for 15 days. Germinated seeds were transferred to hydroponic systems (3 seedlings per system) containing 150 ml 0.2X MS medium, 1.0% (w/v) glucose, and MES buffer (3.5 mM, pH 5.7), and incubated at 28\u0026deg;C for 1 month under fluorescent light with photoperiod of 16 h/8 h light/dark.\u003c/p\u003e \u003cp\u003eAfter a month of incubation, the effect of IAA and NAA was tested in seedlings with and without cadmium exposure for ten days. To determine the effect of IAA and NAA in \u003cem\u003eT. latifolia\u003c/em\u003e development, seedlings were transferred to 0.2X MS medium without glucose at pH 5.7, supplemented with 0.1, 0.5, and 1 mg/L of either IAA or NAA. On the other hand, to determine the role of IAA and NAA in \u003cem\u003eT. latifolia\u003c/em\u003e exposed to Cd, seedlings were transferred to 0.2X MS without glucose at pH 5.7, containing 0.1, 0.5, and 1 mg/L of either IAA or NAA and exposed to 40 mg/L Cd (CdCl\u003csub\u003e2\u003c/sub\u003e) (Fermont, Monterrey, Mexico). All hydroponic cultures were incubated at 28\u0026deg;C under fluorescent light with a photoperiod of 16 h light/8 h dark for ten days.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of photosynthetic pigments\u003c/h2\u003e \u003cp\u003eTotal chlorophyll and carotenoid amounts were determined in the aerial tissue according to the methodology proposed by (Lichtenthaler and Buschmann \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Fresh tissue (50 mg) was homogenized with 2 ml of 95% ethanol and stored in the dark for 24 h at 4\u0026deg;C. The extract was filtered, and absorbance was assessed using a UV-Vis spectrophotometer (Evolution 221, Thermo Scientific, Waltham, MA, USA) at 664.1 nm (chlorophyll a), 648.6 nm (chlorophyll b), and 470 nm (carotenoids). Photosynthetic pigments were calculated according to the equations.\u003c/p\u003e \u003cp\u003eChlorophyll a: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Cha=\\frac{\\left[\\left(13.36*{A}_{664.1}\\right)\\:-\\:(5.19*{A}_{648.6}\\right]\\:*\\:solvent\\:volume}{Sample\\:}\\)\u003c/span\u003e\u003c/span\u003e (1)\u003c/p\u003e \u003cp\u003eChlorophyll b: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Chb=\\frac{\\left[\\left(27.43*{A}_{648.6}\\right)\\:-\\:(8.12*{A}_{664.1}\\right]\\:*\\:solvent\\:volume}{Sample\\:}\\)\u003c/span\u003e\u003c/span\u003e (2)\u003c/p\u003e \u003cp\u003eTotal Chlorophyll: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Chtotal=Cha+Chb\\)\u003c/span\u003e\u003c/span\u003e (3)\u003c/p\u003e \u003cp\u003eCarotenoids: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Cx+c=\\frac{\\left[\\frac{\\left(1000*{A}_{470}\\right)\\:-\\:\\left(2.13*{Ch}_{a}\\right)\\:-\\:\\left(97.64*{Ch}_{b}\\right)}{209}\\right]\\:*\\:solvent\\:volume}{Sample}\\)\u003c/span\u003e\u003c/span\u003e (4)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of electrolyte leakage\u003c/h3\u003e\n\u003cp\u003eThe electrolyte leakage was determined on roots as described by (Umnajkitikorn et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 100 mg of fresh tissue was immersed in 10 mL of deionized water at 20\u0026deg;C for 24 h. The initial electrical conductivity (ECi) was determined using a conductivity meter (PC2700 Oakton, Cole-Parmer, Vernon Hills, IL, USA). Subsequently, the samples were heated at 120\u0026deg;C in an autoclave for 15 min, and the final electrical conductivity (ECf) was determined as above. Deionized water was used as a blank. The electrical conductivity of roots was calculated according to the equation:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{%}\\:\\text{E}\\text{L}=\\left[\\frac{ECi\\:-ECi\\:blank}{ECf-ECf\\:blank}\\right]*100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eDetermination of glutathione content\u003c/h3\u003e\n\u003cp\u003eTotal glutathione (GSH) determination was performed on root tissue as described by (Rahman et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Fresh tissue (250 mg) was powdered in liquid nitrogen and homogenized with 500 \u0026micro;l of 100 mM potassium phosphate buffer pH 7.5 with 5 mM EDTA (KPE). Samples were centrifuged at 12000 xg for 10 min at 4\u0026deg;C, 150 \u0026micro;l of the supernatant was then mixed with 150 \u0026micro;l of extraction buffer (0.1% Triton X-100 and 0.6% sulfosalicylic acid in KPE). Subsequently, 100 \u0026micro;l of the sample was treated with 700 \u0026micro;l KPE buffer, 60 \u0026micro;l DTNB 1.68 mM, 60 \u0026micro;l glutathione reductase (50 U/ml; GR, Sigma-Aldrich, St. Louis, MO, USA) and 60 \u0026micro;l 0.9 mM NADPH. The reaction mixture was incubated at room temperature for 10 min in the darkness, and the absorbance was determined at 412 nm. The glutathione content was calculated using the GSH standard curve (Sigma-Aldrich, St. Louis, MO, USA).\u003c/p\u003e\n\u003ch3\u003eDetermination of catalase activity\u003c/h3\u003e\n\u003cp\u003eCatalase activity (CAT) was determined in leaves. 150 mg of fresh tissue was homogenized with 1.5 ml of KPE buffer (100 mM phosphate buffer pH 7.5 with 5 mM EDTA). The samples were centrifuged at 10\u0026deg;C for 15 min at 13000 xg. The total protein content of the supernatant was determined using the Bradford method. Samples were then adjusted to 50 \u0026micro;g/ml protein (using KPE buffer), and finally, catalase activity was determined spectrophotometrically at 240 nm, using the method of (Aebi \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1984\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDetermination of Cadmium content\u003c/h3\u003e\n\u003cp\u003e Cd content in plant tissues was determined using the methodology according to (Carranza-\u0026Aacute;lvarez et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Root tissue was washed with deionized water and then with 0.01 M EDTA. The seedling was sectioned into leaves and roots and dried at 70\u0026deg;C for 48 h. The dry roots were digested with concentrated HNO\u003csub\u003e3\u003c/sub\u003e plus 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, while the shoots were digested with a mixture of HCl:HNO\u003csub\u003e3\u003c/sub\u003e (3:1 v/v). Samples of the hydroponic medium were diluted to 1:10 and acidified with trace grade HNO\u003csub\u003e3\u003c/sub\u003e to 5%. Cd determination in the plant samples and the hydroponic medium was carried out by air-acetylene flame atomic absorption spectrophotometry (AAS) (iCE 3000 series from Thermo Scientific\u0026trade;) at a wavelength of 228.8 nm, using a hollow cathode Cd lamp (Catalogue No.: 942339030481, Thermo Scientific\u0026trade;). The metal concentration was estimated using a Cd standard curve, normalizing the Cd content with the dry weight in the plant samples and expressing it in mg/Kg. Finally, the translocation factor (TF), bioconcentration factor (BCF), and bioaccumulation coefficient (BAC) were calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRoot structure of\u003c/b\u003e \u003cb\u003eT. latifolia\u003c/b\u003e \u003cb\u003eseedlings\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine structural changes in the roots of \u003cem\u003eT. latifolia\u003c/em\u003e seedlings exposed to the treatments, root cuttings were obtained according to the methodology described by Chen et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Fresh root samples were taken and fixed in neutral formalin (4%) for 48 h. They were then dehydrated in a 25 ml ethanol series at 60\u0026deg;C as follows: 30%, 50%, 70%, 85%, 95% and 100%, for 30 min. Subsequently, the samples were infiltrated in paraffin and cut on a microtome (ECOSHEL) to obtain 5 \u0026micro;m thick root samples, which were fixed on salinized slides. Periodic acid Schiff (PAS) staining was used to stain the fixed tissue as described by (Rinc\u0026oacute;n-Bar\u0026oacute;n et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). PAS stains were observed under an inverted microscope (Zeiss axiovert 135) and analyzed with ImageJ version 1.53e software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of IAA and NAA on seed germination and development of\u003c/b\u003e \u003cb\u003eT. latifolia\u003c/b\u003e \u003cb\u003eseedlings\u003c/b\u003e\u003c/p\u003e \u003cp\u003eInitially, the germination percentage of \u003cem\u003eT. latifolia\u003c/em\u003e seeds was assessed with and without IAA and NAA supplementation. The results showed that both IAA and NAA induced early germination in 25% of the seeds within 24 h, whereas seeds in the control group exhibited delayed germination. By 48 h, seeds treated with IAA or NAA reached a germination rate of 98%, which was statistically comparable to that of the control (Fig.\u0026nbsp;1). Although final germination rates were similar between auxin-treated and control seeds, auxin treatment promoted earlier germination, indicating that IAA and NAA accelerate the onset of germination.\u003c/p\u003e \u003cp\u003eFifteen days after germination, the effects of IAA and NAA on the early developmental stages of \u003cem\u003eT. latifolia\u003c/em\u003e seedlings were evaluated. The results showed that the application of IAA at 0.5 and 1 mg/L significantly increased the number of roots per seedling compared to the untreated control (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). However, a dose-dependent reduction in the root length was observed with increasing IAA concentrations (Fig.\u0026nbsp;2). In addition, the highest concentration (1 mg/L) significantly reduced shoot length and leaf number compared with the control.\u003c/p\u003e \u003cp\u003eIn contrast, treatment with 0.1 mg/L NAA significantly enhanced seedling growth compared to the 0.5 and 1 mg/L treatments. Specifically, 0.1 mg/L NAA increased shoot length and leaf number and maintained root length at levels comparable to the control. Higher NAA concentrations produced contrasting effects: while 0.5 mg/L reduced shoot growth but increased the number of roots, 1 mg/L markedly inhibited both root elongation and root formation (Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eOverall, these results indicate that exogenous application of IAA and NAA modulates the early growth and development of \u003cem\u003eT. latifolia\u003c/em\u003e, particularly by increasing the number of roots during the post-germination stage (15 days after germination).\u003c/p\u003e \u003cp\u003eDue to the marked effects observed on root development, a more detailed analysis of structural and morphological changes was subsequently conducted using hydroponically cultivated plants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of IAA and NAA on the root morphology of\u003c/b\u003e \u003cb\u003eT. latifolia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOne-month-old \u003cem\u003eT. latifolia\u003c/em\u003e plants grown in hydroponic systems were used in dose-response experiments to evaluate the morphological effects of IAA and NAA on the root system over a ten-day period. The results showed an increase in root hair formation in \u003cem\u003eT. latifolia\u003c/em\u003e, which was positively correlated with auxin concentration.\u003c/p\u003e \u003cp\u003eFor IAA, auxin-treated plants showed increased root hair formation compared with the control. Although differences were not statistically significant, auxin-treated plants tended to exhibit higher root biomass, with fresh and dry weights of 0.655 and 0.030 g/plant, respectively. In contrast, control plants developed fewer root hairs and showed fresh and dry weights of 0.382 and 0.021 g/plant, respectively (Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eOn the other hand, seedlings treated with NAA showed responses similar to those observed with IAA at lower concentrations, although adverse morphological effects became evident at higher doses. All tested concentrations produced visible alterations in the surface morphology of the primary root, characterized by increased surface roughness that intensified with auxin concentration. Additionally, treatment with 1 mg/L NAA caused visible tissue oxidation in the root compared with the untreated control. In terms of biomass, NAA-treated plants tended to show higher fresh and dry root weights than control plants. Although differences in root biomass were not statistically significant among treatments, a trend toward increased root biomass with NAA application was observed. For example, seedlings treated with 0.5 mg/L NAA exhibited fresh and dry weights of 0.754 and 0.044 g/plant, respectively, whereas control plants showed values of 0.574 and 0.029 g/plant (Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003eFurthermore, microscopic changes induced by IAA and NAA treatments in the root tissues of \u003cem\u003eT. latifolia\u003c/em\u003e were examined in 5 \u0026micro;m-thick root sections. Plants treated with IAA exhibited structural alterations in the root tissues compared with the control. Based on comparisons with the scale bar, treated plants showed an apparent increase in root diameter relative to the untreated control. Similar structural changes were observed in the root tissues of plants treated with NAA (Fig.\u0026nbsp;6). Overall, these observations indicate that both IAA and NAA induce morphological modifications in the root tissues of \u003cem\u003eT. latifolia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInfluence of IAA and NAA on the root morphological response of\u003c/b\u003e \u003cb\u003eT. latifolia\u003c/b\u003e \u003cb\u003eunder Cd stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effects of IAA and NAA during Cd exposure were evaluated using the concentrations that showed the most favorable responses in \u003cem\u003eT. latifolia\u003c/em\u003e plants: 1 mg/L IAA and 0.5 mg/L NAA. \u003cem\u003eT. latifolia\u003c/em\u003e plants were exposed to 40 mg/L Cd, with or without supplementation of IAA or NAA, under hydroponic conditions.\u003c/p\u003e \u003cp\u003eCd exposure caused a significant reduction in both fresh and dry weights of \u003cem\u003eT. latifolia\u003c/em\u003e shoots, along with visible wilting of aerial tissues compared to control plants (Fig.\u0026nbsp;7a). In Cd-exposed plants, no significant changes in shoot fresh or dry weight were observed in response to NAA treatment. In contrast, IAA treatment significantly increased shoot biomass compared with Cd-treated plants, partially mitigating the inhibitory effect of Cd on shoot growth.\u003c/p\u003e \u003cp\u003eRegarding the root system, Cd stress significantly reduced both the fresh and dry weights of roots compared with the non-exposed control. However, IAA and NAA treatments did not show a significant effect on root biomass production in \u003cem\u003eT. latifolia\u003c/em\u003e plants exposed to Cd (Fig.\u0026nbsp;7b, c).\u003c/p\u003e \u003cp\u003eMorphological observations showed that Cd exposure also decreased the number of root hairs and caused browning and thinning of the roots relative to control plants. However, supplementation with IAA and NAA maintained root hair development and density, and reduced browning of root tissues compared to Cd-treated roots without auxin supplementation. Additionally, Schiff staining indicated stronger staining intensity in auxin-treated roots, suggesting possible alterations in carbohydrate distribution (Fig.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiochemical response of\u003c/b\u003e \u003cb\u003eT. latifolia\u003c/b\u003e \u003cb\u003eplants treated with IAA and NAA under Cd stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe biochemical response of \u003cem\u003eT. latifolia\u003c/em\u003e plants to Cd exposure, with or without auxins supplementation, was also evaluated. In this study, we evaluated total chlorophyll content, electrolyte leakage (EL), catalase (CAT) activity and glutathione (GSH) content.\u003c/p\u003e \u003cp\u003eCd exposure did not cause statistically significant changes in total chlorophyll content compared with the control without Cd (Fig.\u0026nbsp;9a). Similarly, CAT activity showed only a slight increase under Cd exposure, although differences were not statistically significant relative to the control (Fig.\u0026nbsp;9c). However, Cd exposure significantly increased electrolyte leakage and glutathione content in roots (Fig.\u0026nbsp;9b, d).\u003c/p\u003e \u003cp\u003eApplication of NAA in Cd-exposed plants did not significantly affect total chlorophyll content, CAT activity, or GSH levels (Fig.\u0026nbsp;9a, c, d). However, NAA treatment significantly increased electrolyte leakage (by 38%) compared with Cd-treated plants (Fig.\u0026nbsp;9b). Similarly, IAA treatment had no significant effect on chlorophyll content or CAT activity (Fig.\u0026nbsp;9a, c). In contrast, IAA supplementation significantly increased both electrolyte leakage (44%) and glutathione content (100%) in roots compared with the Cd-only treatment (Fig.\u0026nbsp;9b, d).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInfluence of IAA and NAA on Cd uptake in\u003c/b\u003e \u003cb\u003eT. latifolia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the Cd content and accumulation in both shoots and roots of \u003cem\u003eT. latifolia\u003c/em\u003e plants treated with auxins. The results indicate that Cd content was consistently higher in roots than in shoots across all treatments. A significant increase in Cd concentration was observed in the roots of plants supplemented with NAA (3595 mg kg⁻\u0026sup1;) compared with the Cd-only treatment (2880 mg kg⁻\u0026sup1;); however, this treatment did not promote Cd accumulation in shoots. In contrast, IAA supplementation resulted in intermediate Cd concentrations in roots (3200 mg kg⁻\u0026sup1;), which were not significantly different from either the Cd treatment or the NAA treatment. Likewise, the application of IAA or NAA did not produce statistically significant differences in the translocation factor (TF), bioconcentration factor (BCF), or bioaccumulation coefficient (BAC) compared with plants exposed to Cd alone.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCadmium (Cd) content and accumulation in \u003cem\u003eT. latifolia\u003c/em\u003e plants treated with NAA and IAA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCd content (mg/Kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCd removal (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBCF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBAC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eShoots\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eRoots\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCd\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1387.40 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:131.53\\)\u003c/span\u003e\u003c/span\u003e \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2880.71 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e161.88 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.76 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e8.27 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.481 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.99 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e3.59 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33.23 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 3.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCd\u0026thinsp;+\u0026thinsp;NAA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1410.12 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e92 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3595.44 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:276.09\\:\\)\u003c/span\u003e\u003c/span\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.53 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 2.33 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.394 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85.11 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 9.19 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33.33 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 2.62 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCd\u0026thinsp;+\u0026thinsp;IAA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1442.36 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:212.51\\)\u003c/span\u003e\u003c/span\u003e \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3200.77 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:255.54\\)\u003c/span\u003e\u003c/span\u003e \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.81 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.92 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.450 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e76.62 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e6.35 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.54 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 5.29 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eTF: translocation factor; BCF: bioconcentration factor; BAC: bioaccumulation coefficient; ND: not detected. Values represent the mean \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e SD (n\u0026thinsp;=\u0026thinsp;3). Different letters represent statistically different means (p ˂ 0.05, Tukey\u0026acute;s test).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAuxins are phytohormones that regulate plant organogenesis, vascular tissue development, and differential plant growth (Tan et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). IAA is the most prevalent auxin, which is synthesized endogenously by actively growing tissues, root tips, leaf apices, coleoptiles, young leaves, and developing fruits, where it coordinates cambial growth and vascular development (Flasiński and Hąc-Wydro \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). IAA synthesis in plants exposed to Cd has been reported to decrease; however, this response varies depending on the plant species, Cd concentration, and the specific plant tissue analyzed (Rol\u0026oacute;n-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Despite the well-established role of auxins in plant development, their influence on germination, root architecture, and cadmium tolerance in \u003cem\u003eT. latifolia\u003c/em\u003e remains poorly understood. Therefore, this study evaluated the effects of IAA and NAA on \u003cem\u003eT. latifolia\u003c/em\u003e seedlings with and without Cd.\u003c/p\u003e \u003cp\u003eThe results indicated that IAA and NAA improved \u003cem\u003eT. latifolia\u003c/em\u003e seed germination. Although final germination percentages were similar between auxin-treated and control seeds, the earlier onset of germination observed in this study suggests that exogenous auxin application accelerates the initiation of germination rather than increasing the final germination capacity. Similar results have been reported in \u003cem\u003eZea mays\u003c/em\u003e, where supplementation with IAA and indole butyric acid (IBA) increased germination by 10% after both 48 h and 72 h compared with non-supplemented seeds (Ullah et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Likewise, IAA has been shown to enhance germination in \u003cem\u003eNicotiana tabacum\u003c/em\u003e, \u003cem\u003eAraucaria angustifolia\u003c/em\u003e, and \u003cem\u003eHelianthus annuus\u003c/em\u003e (Pieruzzi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), further supporting the role of exogenous auxin application in promoting germination processes.\u003c/p\u003e \u003cp\u003eAuxins exert many of their physiological effects in roots, where they regulate root system architecture and development. Therefore, understanding their influence on root morphology is essential to elucidate how auxin supplementation affects early root development in \u003cem\u003eT. latifolia\u003c/em\u003e. In this context, the present study evaluated the effects of IAA and NAA on the root morphology of \u003cem\u003eT. latifolia\u003c/em\u003e. The results showed that IAA increased the number of roots in \u003cem\u003eT. latifolia\u003c/em\u003e seedlings in a dose-dependent manner. Conversely, the synthetic auxin, NAA, exhibits its positive effect only at the lowest concentration (0.1 mg/L). Although positive effects depend on type and auxin concentration, IAA and NAA increased the number of roots per seedling. Similarly, exogenous IAA increased the number of roots in two-month-old \u003cem\u003eT. latifolia\u003c/em\u003e seedlings (Rol\u0026oacute;n-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). In addition, exogenous applications of NAA and IBA have been reported to produce similar effects on root development in \u003cem\u003eParthenocissus quinquefolia\u003c/em\u003e and \u003cem\u003eAllium cepa\u003c/em\u003e (Abu-Zahra et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, IAA increased the number of root hairs and tended to increase fresh and dry weight in the roots of \u003cem\u003eT. latifolia\u003c/em\u003e seedlings in a dose-dependent manner. Similarly, IAA and IBA enhance root biomass from 13.10% \u0026minus;\u0026thinsp;35.60% in \u003cem\u003ePinus yunnanensis\u003c/em\u003e Franch species (Xu et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), while in \u003cem\u003eTriticum aestivum\u003c/em\u003e and \u003cem\u003eSolanum melongena\u003c/em\u003e IAA increases the root length (Agami and Mohamed \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Singh and Prasad \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). On the other hand, even though NAA increased the number of root hairs, it caused visible surface alterations characterized by increased root surface roughness and tissue oxidation in the primary root of \u003cem\u003eT. latifolia\u003c/em\u003e seedlings in correlation with its concentration. This effect may be attributed to the synthetic auxin NAA, which at higher concentrations disrupts root architecture and normal root development (Rosier et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Rahman et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Regarding the microscopic evaluation of seedling root architecture, the results suggest that IAA and NAA treatments increased root hair diameter in a dose-dependent manner. Moreover, the enhanced affinity for the Schiff reagent, evidenced by the formation of a pink-colored Schiff reaction product, may indicate alterations in carbohydrate distribution within root tissues, potentially reflecting modifications in cell wall components such as pectin, cellulose, and hemicellulose involved in root structural organization (Majda and Robert \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Š\u0026iacute;pošov\u0026aacute; et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Other significant components of the root cell wall that may be influenced by auxin signaling include cellulose, hemicellulose, and structural glycoproteins, which contribute to mechanical strength, porosity, and wall remodeling (Showalter \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Cosgrove \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Auxin-induced regulation of these molecules facilitates cell elongation and root hair differentiation, as demonstrated by their essential roles in root development (Velasquez et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the pronounced effects of auxins on root development observed in this study, we next evaluated whether these phytohormones could also modulate plant responses to cadmium-induced stress. Our results showed that IAA supplementation significantly increased shoot biomass relative to Cd-treated plants, partially mitigating the inhibitory effects of the metal. In contrast, NAA supplementation did not produce statistically significant changes in plant biomass under Cd exposure. This is consistent with findings in \u003cem\u003eBrassica juncea\u003c/em\u003e, where IAA increased biomass even under Cd exposure (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and \u003cem\u003eSolanum nigrum\u003c/em\u003e (Ji et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which demonstrate that IAA favors the growth of plants exposed to Cd. IAA has also been shown to protect the photosynthetic apparatus from deleterious Cd effects by restoring carotenoids and total chlorophyll content in \u003cem\u003eSolanum melongena\u003c/em\u003e, \u003cem\u003eSolanum nigrum\u003c/em\u003e (Ran et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eAmaranthus hypochondriacus\u003c/em\u003e, and \u003cem\u003eSedum alfredii\u003c/em\u003e (Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, in \u003cem\u003eT. latifolia\u003c/em\u003e seedlings, the auxin supplementation did not show evident effects. Although IAA tended to mitigate pigment loss, the effect was not statistically significant in \u003cem\u003eT. latifolia\u003c/em\u003e, suggesting species-specific responses.\u003c/p\u003e \u003cp\u003eConcerning the root architecture, both IAA and NAA preserved root integrity by increasing the number of roots even under Cd-induced stress conditions. Similar results have been reported in \u003cem\u003eMedicago sativa\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and \u003cem\u003eOryza sativa\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) following exposure to heavy metals. In addition, IAA and NAA maintained root hair diameter in Cd-exposed plants and enhanced affinity to the Schiff reagent, suggesting possible alterations in carbohydrate distribution within root tissues. These changes may reflect modifications in cell wall carbohydrates involved in root structural organization, potentially contributing to Cd retention or stabilization within the root system. However, further studies are required to quantify carbohydrate content and clarify the mechanisms underlying these responses.\u003c/p\u003e \u003cp\u003eCd has been shown to induce lipid peroxidation of cell membranes, thereby altering root cell membrane integrity. In this study, electrolyte leakage increased in \u003cem\u003eT. latifolia\u003c/em\u003e roots, suggesting a loss of membrane integrity after Cd exposure. Similar results have been reported in \u003cem\u003eT. latifolia\u003c/em\u003e (Rol\u0026oacute;n-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e), T. \u003cem\u003eangustifolia\u003c/em\u003e (Ren et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eTrifolium repens\u003c/em\u003e (Wu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and \u003cem\u003eBrassica oleracea\u003c/em\u003e (Jia et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) plants exposed to Cd. Furthermore, auxin supplementation further increased electrolyte leakage, similar to that reported by (Rol\u0026oacute;n-C\u0026aacute;rdenas et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e) in two-month-old \u003cem\u003eT. latifolia\u003c/em\u003e seedlings. These findings indicate that auxin applications may enhance certain stress responses but may also contribute to increased membrane destabilization under Cd exposure, particularly in developing root tissues (one-month-old seedlings).\u003c/p\u003e \u003cp\u003eReactive oxygen species (ROS) are released upon metal stress. However, plants activate enzymatic and non-enzymatic antioxidant systems to mitigate ROS damage (Raihan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, catalase (CAT) activity showed a slight increase after Cd exposure; however, this change was not statistically significant compared with control plants. Catalase activity has been reported to increase after Cd exposure in wheat varieties, indicating increased ROS production caused by the metal (Guo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Likewise, both IAA and NAA increased CAT activity in \u003cem\u003eT. latifolia\u003c/em\u003e plants similarly to responses reported in \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, \u003cem\u003eSorghum bicolor\u003c/em\u003e, and \u003cem\u003eCamellia sinensis\u003c/em\u003e exposed to Cd (Zhan et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These results suggest that auxins do not suppress, but rather enhance catalase activity under Cd stress, possibly as part of a broader antioxidant response mechanism (Zhan et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, glutathione (GSH) increased in plants exposed to Cd, similar to that observed in \u003cem\u003eT. angustifolia\u003c/em\u003e, where exposure to Cd promoted the synthesis of glutathione and phytochelatins (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ren et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Likewise, GSH content increased in plants exposed to IAA. (Khan et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and (Bashri and Prasad \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) report a similar effect in \u003cem\u003eLycopersicum esculantum\u003c/em\u003e and \u003cem\u003eTrigonella foenum-graecum\u003c/em\u003e with the addition of IAA under Cd stress. This reinforces that IAA can stimulate glutathione biosynthesis under cadmium exposure, improving cellular detoxification and metal tolerance. The observed increase in GSH and CAT likely contributed to root integrity preservation and improved biomass under Cd stress.\u003c/p\u003e \u003cp\u003eFinally, IAA and NAA increased Cd content in the roots of \u003cem\u003eT. latifolia\u003c/em\u003e seedlings. NAA has been shown to increase Cd accumulation in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, by inducing hemicellulose synthesis in the roots, thereby increasing Cd\u003csup\u003e2+\u003c/sup\u003e binding to the cell wall (Zhu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similarly, IAA has been reported to promote heavy metal accumulation in the roots of \u003cem\u003eSolanum nigrum\u003c/em\u003e and \u003cem\u003eHelianthus annuus\u003c/em\u003e (Ran et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, the results of this study indicate that auxin supplementation primarily promotes Cd retention in root tissues rather than enhancing metal translocation to aerial organs. This localized accumulation may contribute to improved Cd tolerance by restricting metal mobility and reducing systemic toxicity.\u003c/p\u003e \u003cp\u003eTaken together, these findings demonstrate that exogenous IAA and NAA modulate root development and physiological responses in \u003cem\u003eT. latifolia\u003c/em\u003e, particularly through the maintenance of root architecture, activation of antioxidant mechanisms, and enhanced Cd immobilization within the root system. Future studies should explore the molecular mechanisms underlying these responses, including auxin-responsive gene regulation and Cd transporter activity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated that exogenous application of IAA and NAA modulates the germination and early development of \u003cem\u003eT. latifolia\u003c/em\u003e seedlings. Both auxins accelerated seed germination and promoted root architecture development by increasing the number of root hairs in a concentration-dependent manner. However, NAA at higher concentrations caused visible alterations in root surface integrity, suggesting auxin-specific and dose-dependent effects on root architecture.\u003c/p\u003e \u003cp\u003eUnder Cd exposure, applying 1 mg/L IAA and 0.5 mg/L NAA partially mitigated the adverse effects of Cd, preserving root morphology and biomass and activating antioxidant defense mechanisms. Auxin treatments also enhanced root hair development and were associated with alterations in carbohydrate distribution in root tissues, potentially contributing to Cd retention within the root system without promoting translocation to aerial parts.\u003c/p\u003e \u003cp\u003eOverall, these findings indicate that auxin-mediated modulation of root architecture and antioxidant responses may contribute to improved Cd tolerance in \u003cem\u003eT. latifolia\u003c/em\u003e by promoting localized metal immobilization within the root system. This physiological response suggests the potential applicability of auxin supplementation as a strategy to support phytoremediation processes in Cd-contaminated environments.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003e \u003cb\u003eConflict of interest\u003c/b\u003e.\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e \u003cb\u003eConsent to participate\u003c/b\u003e.\u003c/strong\u003e \u003cp\u003eThis study does not contain any personal data requiring your consent.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e \u003cb\u003eConsent to publish\u003c/b\u003e.\u003c/strong\u003e \u003cp\u003eThis study does not contain any personal data requiring your consent.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe work reported was supported by grants from CONACYT, Programa Presupuestario F003 (Formerly Fondo Sectorial de Investigaci\u0026oacute;n para la Educaci\u0026oacute;n CB2017-2018), Project number A1-S-40454 to Alejandro Hern\u0026aacute;ndez-Morales.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSRL, GARC, and AHM conceived and designed the study. SRL performed the research, CCA and JRMP analyzed the data and supervision. SRL wrote the paper. AHM edited and revised the paper. AHM received funding and edited the final version of the paper. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eStephanie Rosales-Loredo thanks SECIHTI for the scholarship to study Biochemical Master Sciences (CVU 805117).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbu-Zahra TR, Hasan MK, Hasan HS (2012) Effect of Different Auxin Concentrations on Virginia Creeper (Parthenocissus quinquefolia) Rooting\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAebi H (1984) [13] Catalase in vitro. In: Methods in Enzymology. 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Journal of Hazardous Materials 263:398\u0026ndash;403. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhazmat.2013.09.018\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2013.09.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-biosystems","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Plant Biosystems](https://link.springer.com/journal/44473)","snPcode":"44473","submissionUrl":"https://submission.springernature.com/new-submission/44473/3?","title":"Plant Biosystems","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Typha latifolia, cadmium, IAA, NAA, phytoremediation, antioxidant response, root morphology","lastPublishedDoi":"10.21203/rs.3.rs-9440581/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9440581/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eTypha latifolia\u003c/em\u003e is a wetland macrophyte widely used for Cd phytoremediation. However, the role of auxins in enhancing Cd tolerance and metal retention in this species remains poorly understood. This study evaluated the effects of exogenous indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA) on seed germination, root morphology, and Cd tolerance in \u003cem\u003eT. latifolia\u003c/em\u003e. IAA and NAA accelerated early seed germination and significantly increased the density of root hairs in a concentration-dependent manner; however, high NAA concentrations induced root surface oxidation.\u003c/p\u003e \u003cp\u003eIn plants exposed to 40 mg/L Cd, treatment with IAA (1 mg/L) and NAA (0.5 mg/L) mitigated the stress by preserving root biomass and morphology. Antioxidant responses were activated, as evidenced by increased catalase (CAT) activity and glutathione (GSH) content. Auxin treatments promoted Cd retention in root tissues while limiting its translocation to shoots, suggesting localized metal detoxification. Histological analysis revealed increased Schiff reagent staining in auxin-treated roots, indicating that alterations in carbohydrate distribution within root tissues may contribute to Cd immobilization.\u003c/p\u003e \u003cp\u003eThese findings suggest that exogenous auxins improve Cd stress tolerance in \u003cem\u003eT. latifolia\u003c/em\u003e through modulation of root development, activation of antioxidant defenses, and promotion of localized metal immobilization within the root system. This study underscores the potential applicability of auxin supplementation as a biotechnological tool to enhance phytoremediation efficiency in Cd-contaminated environments.\u003c/p\u003e","manuscriptTitle":"Auxin-mediated modulation of root architecture enhances cadmium tolerance and root retention in Typha latifolia seedlings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 16:17:13","doi":"10.21203/rs.3.rs-9440581/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-22T10:51:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-18T05:03:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-18T05:03:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Biosystems","date":"2026-04-16T16:11:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-biosystems","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Plant Biosystems](https://link.springer.com/journal/44473)","snPcode":"44473","submissionUrl":"https://submission.springernature.com/new-submission/44473/3?","title":"Plant Biosystems","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"91bcfdf6-65af-4865-8ff2-528957c59f4c","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T16:17:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 16:17:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9440581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9440581","identity":"rs-9440581","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}