In this study, we used rice seeds (Oryza sativa L. Jiafuzhan) obtained from the School of Life Sciences, Xiamen University, China. To prepare the seeds for experimental treatments, the following streamlined protocol was followed. Step 1, seed selection and sterilization: Fully matured rice seeds were selected to ensure consistent germination and growth. The seeds were first rinsed with ultrapure water to remove surface contaminants, then disinfected by immersion in 70% ethanol for 30 s. Subsequently, the seeds were soaked in 10% hydrogen peroxide (H₂O₂) for 10 min for enhanced sterilization (Miche and Balandreau, 2001). Finally, the seeds were rinsed with sterile ultrapure water to remove residual disinfectants. Step 2, seed germination: Sterilized seeds were placed in an illumination incubator (Ningbo Saifu, China) at 25 °C in complete darkness for 24 h to promote water absorption and initiate germination, facilitating uniform sprout development. Step 3, seedling establishment: Post-germination, seeds were evenly distributed on a sand net to support root development. Irrigation commenced with Hoagland solution (details in Supplementary Table S2) at 25% concentration. The nutrient solution was replaced every 3 days, with the concentration gradually increased until full-strength (100%) Hoagland solution was attained (Majidi et al., 2025; Mei et al., 2021). Step 4, seedling growth and preparation: Seedlings were cultivated in a greenhouse for 1 month under controlled conditions (25 ± 2°C with a 12-h light/12-h dark photoperiod) (Cui et al., 2023). Following cultivation, seedlings exhibiting uniform growth were selected for subsequent experimental treatments.

After transferring seedlings into hydroponic conditions, 1-month seedlings were subjected to varying concentrations of sodium arsenite (NaAsO₂) and ferrous sulfate (FeSO₄) to assess their combined effects over different exposure periods according to previous studies (Mei et al., 2021; Wang et al., 2021). The experimental design included three arsenite levels (Control: 0 μmol/L; Low As: 10 μmol/L; High As: 30 μmol/L) and amended ferrous iron concentrations (No additional Fe: 0 μmol/L; With additional Fe: 100 μmol/L). The following treatments were applied:

(1) CK+No-Fe: Control + No additional Fe; (4) CK+With-Fe: Control + Fe addition;

(2) LAs+No-Fe: Low As + No additional Fe; (5) LAs+With-Fe: Low As + Fe addition;

(3) HAs+No-Fe: High As + No additional Fe; (6) HAs+With-Fe: High As + Fe addition.

The study examined rice seedlings during the vegetative growth stage, prior to the booting phase. Seedlings underwent As exposures for 1 day, 1 week, and 1 month, with three replicates per treatment. All pots were randomly arranged in the illumination incubator under the same conditions described above.

To assess seedling growth, fresh weight biomass was measured using an electronic balance after collecting uniformly grown seedlings from each replicate. The dry weight of the seedlings was determined after oven-drying at 60 °C for 72 h until a constant weight was achieved. Fully expanded true leaves on plants were collected in triplicate for chlorophyll pigment analysis. Fresh leaves (~0.2 g) were ground, homogenized, and extracted using 90% acetone in darkness at 4 °C for 24 h. The extract was then centrifuged at 3000 rpm for 15 min, and the chlorophyll content in the supernatant was quantified using a UV–Vis spectrometer (Beijing Ruili, China) at wavelengths of 663 and 645 nm. Root morphology—including root length, diameter, surface area, and volume—was analyzed using a scanning apparatus (LA-S Plant Image Analyzer, Hangzhou Wseen, China).

To investigate the effects of As and Fe on plant root lignification, transverse root sections were prepared and analyzed. Fresh root segments (6–8 cm) were hand-sectioned using a rotary microtome. The sections were mordanted with ferric ammonium sulfate and stained with diluted hematoxylin to visualize lignified cell walls (Ji et al., 2006). Stained sections were subsequently examined and imaged under a photomicroscope.

Root porosity (POR) was determined using the pycnometer method. Fresh lateral roots (0.5 g) were cut into segments and wiped with air-laid paper prior to measurement (Mei et al., 2014). POR was calculated using Equation 1:

where PWVR (g) is the weight of the pycnometer with ultrapure water and vacuumed root; PWR (g) is the weight of the pycnometer with ultrapure water and fresh root; PW (g) is the weight of the pycnometer filled with ultrapure water; and FR (g) is the weight of the fresh root.

The rate of ROL in seedlings was measured using a modified titanium (Ti³⁺) citrate method (Mei et al., 2021, Mei et al., 2014). Roots were immersed in a deoxygenated titanium (Ti³⁺) citrate solution under dark conditions and sealed with a 2 cm paraffin oil layer to prevent atmospheric oxygen intrusion. The incubation was conducted for 24 h at room temperature under a 12-h light/12-h dark cycle. Afterward, the solution was gently agitated and sampled with syringes. The absorbance of the oxidized titanium (Ti³⁺) citrate was measured at 527 nm using a UV–Vis spectrometer (Beijing Ruili, China), corresponding to the absorption peak of its oxidized form. The ROL rate (mM O₂ day⁻¹ g⁻¹ DW) was calculated using Equation 2:

where V is the initial volume (L) of Ti³⁺-citrate; C_b is Ti³⁺ concentration (μmol/L) in the blank; C_i is the Ti³⁺ concentration (μmol/L) after 24 h incubation with the rice seedling; and W is the root dry weight (g DW).

Iron plaque was extracted from fresh roots with the dithionite–citrate–bicarbonate (DCB) solution, following established protocols to determine As and Fe contents in the plaque (Mei et al., 2021, Mei et al., 2014). The iron plaque extract was digested with H₂SO₄/H₂O₂ at 120°C for 6 h. The digested mixture was then centrifuged at 8000 rpm for 5 min (Hettich Universal 320R Centrifuge, Germany) and filtered through a 0.45 μm membrane. Fe concentration was measured via atomic absorption spectroscopy (AAS Vario 6, Thermo Fisher Scientific, USA), and As concentration was assessed by atomic fluorescence spectrometry (AFS-930, Beijing Jitian, China).

Roots (without iron plaque), stems, and leaves of all treated seedlings were freeze-dried at –20°C, weighed to determine dry weight and biomass, and analyzed for Fe and As content. Each plant part was ground using a ball mill and passed through an 80-mesh sieve to obtain a fine powder. Subsamples (~0.2 g) were digested overnight with 5 mL nitric acid at room temperature. The following day, 2 mL hydrogen peroxide was added and heated at 120 °C for 6 h until fully digested (Mei et al., 2021). The cooled digest solution was then diluted with 5% hydrochloric acid and stored at –20 °C for subsequent analysis. Fe and As concentrations were determined as described above.

The translocation factor (TFAs), defined as the ratio of As content in aerial parts (leaf and stem) to that in roots, was calculated using Equation 3:

where Aerail_As is the As content in the leaf and stem, Root_As is the As content in the root.

The significance of measured parameters was determined using one-way ANOVA. Differences among treatments were further analyzed with one-way ANOVA followed by Duncan’s multiple comparison test. Geisser–Greenhouse correction was applied to account for variance homogeneity. Different letters indicated significant differences among treatments at p < 0.05. Spearman’s correlation analysis was used to identify relationships among parameters. Redundancy analysis (RDA) was performed to explore the relationships between plant morphological variables and Fe/As distribution using R with the Vegan package. Data statistics and visualizations were performed using SPSS Statistics 23.0, GraphPad Prism 9, and the R statistics package (version 4.0.2, https://www.r-project.org).

As exposure significantly influenced chlorophyll content in rice seedlings, with both exposure duration and concentration playing critical roles. Chlorophyll levels exhibited an initial increase followed by a decrease over time, peaking at 263.84 mg·L⁻¹ after 1 week of treatment across all groups (Figure 1a). Although As exposure significantly affected chlorophyll content, plants under prolonged (1-month) low-level As (LAs) stress showed partial recovery, with chlorophyll content slightly exceeding that of the control group (CK), although this difference was not statistically significant (p > 0.05; Figure 1a). Importantly, Fe supplementation demonstrated a time-dependent mitigation effect. Although it did not alleviate As-induced chlorophyll loss after 1 month, high-concentration Fe application significantly enhanced chlorophyll content during the early stress phase (1 week; p < 0.05; Table 1).

As exposure significantly inhibited rice seedling growth, with root dry weight showing a clear time- and concentration-dependent response (Figure 1b). Although root biomass generally increased over the 1-month experimental period, reaching a maximum of 0.52 g in the control groups, it was substantially suppressed under As stress. After 1 month of exposure, root dry weight showed significant concentration-dependent inhibition (CK > LAs, HAs), declining to as low as 0.12 g in the HAs treatment. Notably, Fe supplementation effectively mitigated this suppression, particularly under prolonged stress. After 1 month, Fe-amended treatments produced plant biomass that was 1.18–1.28 times greater than their respective no-additional-Fe counterparts at equivalent As contents, demonstrating Fe’s crucial role in maintaining root growth under chronic As toxicity.

Root porosity increased in a time-dependent manner under As–Fe interactions, peaking at 9.84% after 1 month of treatment (Table 1). Acute As exposure (1 day) suppressed porosity (CK > LAs > HAs), whereas prolonged HAs exposure (1 week and 1 month) promoted it (HAs > CK > LAs). Counterintuitively, Fe amendment reduced porosity, with No-Fe groups exhibiting 1.32–1.91 times higher porosity than their Fe-treated counterparts at 1 week (p < 0.05).

Anatomically, aerenchyma formation initiated within 1 day (accounting for ~17% of the root cross-sectional area) and progressed to 46.3% under 1-month As–Fe co-treatment. The aerenchyma displayed radial, wheel-like structures (Figure 2) formed via lysigenous programmed cell death (PCD) of cortical parenchyma. Iron supplementation tended to increase the proportion of gas-filled tissue.

The ROL rate increased significantly with prolonged treatment duration, reaching 64.46 mM O₂·day⁻¹·g⁻¹ DW at 1 month (Figure 3). After 1-week treatment, the As content in the iron plaque on the root surface showed a significant positive correlation with the Fe content (R² = 0.92, p < 0.05). After 1 month, the Fe plaque content did not increase significantly (Figure 3e), yet As accumulation continued to rise.

Overall, ROL intensity increased with root growth in conjunction with iron plaque development (Figures 3a, d). As content negatively regulated ROL (CK > LAs > HAs), with HAs treatment suppressing the ROL rate to 50% of the control level after 1 month. Fe supplementation counteracted this As-induced inhibition, elevating ROL rates by 1.38–2.83 times in As-treated groups at 1 week.

The contents of As and Fe in plant tissues of O. sativa are shown in Figure 4. Fe treatment significantly affected Fe contents in roots, stems, and leaves, while exposure duration markedly influenced the redistribution of As and Fe within plant tissues (Supplementary Table S1). After 1 month of treatment, the Fe-treated group showed significantly higher root As content than the non-Fe-treated group under the same As exposure (Figure 4b).

As exposure for 1 week resulted in higher arsenic translocation factors (TF_As), with values of 0.22–0.73 at 1 day and 0.37–0.77 at 1 week. In contrast, 1-month treatment drastically reduced TF_As to 0.02–0.05 (Supplementary Figure S2). Notably, TF_As under 1-week As–Fe co-treatment were 36.5 times higher than under 1-month exposure, indicating a sharp decline in As translocation efficiency over time. As content inversely regulated TF_As during acute exposure (1 day: LAs > HAs) but exhibited the opposite trend at 1 week (HAs > LAs).

Fe amendment significantly altered As translocation patterns. Acute Fe supplementation significantly reduced TF_As by 38% (p < 0.001), indicating enhanced root sequestration via Fe-plaque formation. Meanwhile, Fe treatment increased As uptake by the roots, as the iron plaque acted as a sustained sink for As enrichment (Figure 4b). Root As content was positively correlated with root morphogenesis and chlorophyll content (Figure 5), suggesting that As stress promoted root architectural development. In Fe-treated groups, both root morphogenesis and chlorophyll content were significantly positively correlated with root Fe content (Figure 6), demonstrating that Fe amendment enhances the detoxification capacity of rice seedlings.

Our results demonstrate a biphasic response of chlorophyll content to As stress: an initial significant reduction under 1-week exposure, followed by a compensatory recovery under prolonged low-level As. This pattern suggests the activation of dynamic detoxification mechanisms, such as phytochelatin synthesis and antioxidant defenses, over time (Gaikwad et al., 2020; Mondal et al., 2022). The mitigation by Fe was both time- and concentration-dependent. Notably, because the baseline Hoagland solution contains Fe³⁺, all plants had access to Fe, enabling some plaque formation even without external Fe supplementation. As a result, the Fe and As contents in the iron plaque showed a significant positive correlation after both 1-week and 1-month exposure periods across all treatments (Figure 3e). The pronounced efficacy of high-dose Fe after a 1-week incubation underscores the role of rapid Fe-plaque formation in physically sequestering As at the root surface during the acute stress phase (Mei et al., 2021; Zulfiqar and Ashraf, 2022). Under prolonged exposure, sustained Fe supply likely served as a nutrient reservoir supporting ongoing chlorophyll synthesis and contributing to the observed recovery, as evidenced by differences between the With-Fe and No-Fe control groups.

The persistent biomass suppression under 1-month As exposure reflects the cumulative metabolic cost of detoxification. The modest biomass improvement with Fe supplementation—despite a more pronounced recovery in chlorophyll—implies a strategic reallocation of energy (Gaikwad et al., 2020). In this scenario, resources are prioritized for essential survival processes (e.g., maintaining detoxification pathways and antioxidant systems) over growth (Liu et al., 2011; Mei et al., 2012), a trade-off characteristic of metallophytes (Farooq et al., 2016; Maestri et al., 2010; Zulfiqar and Ashraf, 2022). This highlights the temporal complexity of As–Fe interactions: although Fe can rapidly mitigate As uptake and acutely protect photosynthesis, long-term growth benefits remain limited by the underlying metabolic burden associated with chronic stress.

Arsenic As content and exposure duration jointly shaped distinct root morphological patterns. The initial stimulatory effect of low As (LAs) on root growth aligns with the hormesis phenomenon, whereas chronic exposure led to suppression, particularly in root volume and diameter. The observed elongation of roots under 1-month HAs stress (HAs > LAs > CK for length/surface area) represents a compensatory adaptation to enhance nutrient foraging in a toxic environment (Gaikwad et al., 2020; Hossain et al., 2007), consistent with strategies reported in metallophytes (Eissenstat, 2000; Sobrino-Plata et al., 2013).

Fe amendment played a selective protective role, significantly restoring root volume and diameter but not influencing length and surface area. This selectivity likely stems from the mechanism of Fe-plaque formation, which immobilizes As in the root apoplast, thereby reducing its cytotoxicity and preserving the cellular integrity necessary for radial expansion (Paulelli et al., 2019). This hierarchical prioritization in root development underscores the plant’s sophisticated strategy for resource allocation under metal stress.

The treatment groups at different time points showed significant differences in root morphology-related parameters under the LSD test (p < 0.001; Table 1). The time-dependent increase in root porosity and aerenchyma formation under As stress indicates an adaptive response to potential hypoxia and oxidative stress. The counterintuitive reduction in porosity by Fe amendment suggests that Fe-plaque may physically impede gas diffusion or alter hormonal signals (e.g., ethylene) governing aerenchyma formation (Cheng et al., 2015; Rogers et al., 1992; Zhang et al., 1998).

The partial reversal of As-induced aerenchyma suppression by Fe is a critical finding (Cheng et al., 2015; He et al., 2004; Parsons et al., 2013). It correlates with Fe-plaque-mediated As sequestration, which may reduce As-triggered disruption of lysigenous programmed cell death (PCD) in the cortex (Robinson et al., 2003). The formation of radial, wheel-like aerenchyma structures highlights a tightly regulated anatomical adaptation. The interplay among As toxicity, Fe remediation, and aerenchyma development reflects the competing demands of oxygen transport (via aerenchyma) and detoxification (via Fe-plaque).

Redundancy analysis (RDA) revealed that plaque Fe content was negatively correlated with ROL in the control group, whereas under Fe amendment, both root Fe and As contents showed positive correlations with ROL (Figure 5). The negative correlation between plaque Fe and ROL in controls suggests that natural Fe deposition can slightly impede oxygen diffusion. Under As stress, however, Fe amendment reversed the As-induced suppression of ROL and correlated positively with both root Fe and As content (Mei et al., 2012). This shift indicates that Fe-plaque not only immobilizes As but also helps maintain a more oxidized rhizosphere, potentially mitigating the reductive mobilization of As(III). The observed decoupling of ROL from root porosity—contrary to some ROL models ROL (Mei et al., 2014), emphasizes the dominant role of fine, non-aerenchymatous lateral roots in oxygen release in rice (Cheng et al., 2010; Mei et al., 2012), and underscores that Fe’s primary mitigation function operates through chemical immobilization rather than through alterations in root anatomical traits (Abedin et al., 2002; Hu et al., 2019; Rebouillat et al., 2009).

The drastic decline in the arsenic translocation factor (TF_As) over time, particularly under Fe amendment, underscores a fundamental shift in plant strategy—from initial As distribution to robust root sequestration over the 1-month period. This process is initiated by the formation of Fe plaque formation (Liu et al., 2011), which acts as a primary barrier to reduce As accumulation within the root, as confirmed by the sharp reduction in TFAs following acute Fe treatment. However, Fe amendment increased As accumulation in the roots under prolonged stress, which may be attributed to Fe-plaque functioning as a sustained sink for As enrichment or to Fe-induced physiological changes that enhanced As immobilization (Mei et al., 2014).

In Fe-treated groups, both root morphogenesis and chlorophyll content were significantly positively correlated with root Fe content (Figure 6b). Fe amendment orchestrates a multifaceted protective response. Systemically, it enables the maintenance of higher chlorophyll content, revealing a direct positive correlation with Fe treatment that underscores the protection of the photosynthetic machinery (Wu et al., 2022; Zhao et al., 2010). At the root level, anatomical adaptations also occur. Root porosity showed an inverse correlation with As stress, suggesting structural modification toward a more resilient phenotype. PCA analysis indicated that root morphogenesis, ROL, and POR were major contributors to Principal Component 1 (PC1, Table 2).

These interconnected mechanisms (as shown in Figure 7)—rhizosphere sequestration, physiological maintenance, and anatomical adaptation—synergistically contribute to the observed improvement in biomass under Fe supplementation. This stands in contrast to the pronounced growth suppression in As-stressed groups without Fe. Thus, Fe amendment does not merely immobilize As passively; it fosters a more robust physiological state by protecting root meristems and photosynthetic centers, enhancing the intrinsic detoxification capacity of rice seedlings, and ultimately ensuring better growth under stress.