Introduction

Heavy metal contamination, particularly chromium (Cr), is a critical environmental stressor threatening global crop productivity, food security, and sustainable agriculture. The 2014 National Soil Pollution Survey Bulletin by China’s Ministry of Ecology and Environment reported that 1.1% of surveyed sites exceeded permissible Cr levels, affecting over 1.33 million hectares of farmland1. In soil, Cr exists primarily as trivalent Cr(III) or hexavalent Cr(VI), with Cr(VI) exhibiting higher mobility and toxicity, posing severe risks to agricultural ecosystems2,3. Thus, mitigating heavy metal stress to improve crop quality and yield is a pressing challenge for sustainable agricultural development4. The seed germination stage, a critical determinant of crop establishment, is highly susceptible to Cr(VI) stress and reduces field emergence rates, impairs nutrient and water uptake, and hinders subsequent plant development5. Enhancing rice seed germination and seedling resilience in Cr-contaminated soils is, therefore, vital for ensuring stable crop yields and safeguarding food security.

Proline (Pro) is a key osmoprotectant and stress-responsive metabolite with multifaceted roles in mitigating heavy metal toxicity in plants. It functions as an osmotic regulator, reactive oxygen species (ROS) scavenger, and membrane stabilizer while supplying energy under adverse conditions6,7. Previous studies demonstrate that exogenous Pro application mitigates heavy metal toxicity by enhancing antioxidant defenses. For example, foliar Pro reduced ROS accumulation and alleviated Pb toxicity in wheat8, while improving Cd tolerance in date palms by enhancing antioxidant activity and photosynthesis9. Furthermore, in the authors’ previous study, Pro dynamically regulated amino acid metabolism, particularly through glutamate-mediated synthesis and catabolism pathways by P5CS-catalyzed synthesis and PDH-mediated catabolism, to maintain cellular homeostasis under Cr(VI) stress10. Since 2020, additional studies have highlighted its role in modulating jasmonate signaling and repressing the MYB-bHLH-WDR complex to regulate anthocyanin accumulation, thus enhancing oxidative stress tolerance in rice seedlings exposed to Cr11.

Despite these advances, the majority of studies on the role of Pro in alleviating Cr stress consider mature plants12, neglecting the seed germination stage—the most vulnerable phase in the plant life cycle and a critical bottleneck for successful crop establishment under heavy metal stress13. Limited research exists on the mechanisms underlying the role of exogenous Pro application on germination and the physiological and biochemical processes of rice seeds under Cr(VI) stress. This study addresses this gap by investigating the effects of exogenous Pro pretreatment on rice seed germination and associated physiological and biochemical changes under varying concentrations of Cr(VI). By elucidating the regulatory mechanisms underlying the effect of Pro, this study aims to develop Pro-based strategies for improving rice establishment in Cr(VI)-contaminated soils, supporting sustainable agriculture, with molecular mechanisms reserved for future investigation.

Materials and methods

Materials and reagents

Assay kits for superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), Ascorbate Peroxidase (APX), Glutathione (GSH), Glutathione Reductase (GR), ascorbic acid (AsA), malondialdehyde (MDA), and soluble sugars (SS) were purchased from Nanjing Jiancheng Bioengineering Institute. Absolute ethanol, potassium chromate, and Pro were purchased from Sinopharm Chemical Reagent Co., Ltd. Rice seeds (Oryza sativa L. cv. XZX45) were obtained from Hunan Rice Research Institute.

Experimental design

  1. (1)

    Treatment of rice seeds: Approximately 1,200 rice seeds were washed and disinfected, then soaked for 12 h. Afterward, the seeds were divided into two groups. One group was pretreated with 1 mmol·L−1 Pro for 12 h [Pro + Cr(VI)], and the other group was the Cr(VI) stress group [Cr(VI)] (Supplementary Material S1, Figs. S1 & S2).

  2. (2)

    Treatment of seeds with Cr(VI): The cleaned and disinfected seeds were placed in Petri dishes lined with filter paper. Fifty seeds were placed in each Petri dish. Three Cr(VI) concentrations (2.0, 8.0, and 16.0 mg/L, representing low, medium, and high stress) were applied14, with a zero-concentration control. Each treatment had three replicates.

  3. (3)

    Observation of seed germination: The treated samples were placed in a constant-temperature incubator at 28 °C for dark culture. Rice seed germination was observed every 24 h. The germination rates (Grs) and germination potentials (GPs) of the seeds at 24 h and 48 h were calculated, respectively.

Determination of indicators

Determination of germination indexes

During the first 3 days, seed germination was observed every 24 h, and the Gr, GP, germination index (GI), and mean germination time (MGT) were calculated as follows15,16:

$$\text{Gr}=\:\frac{n}{N}\times\%$$
(1)

where n represents the number of germinated seeds and N represents the total number of seeds tested;

$$\text{GP}=\:\frac{a}{N}\times\%$$
(2)

where a represents the number of germinated seeds after 48 h;

$$\text{GI}= \:\sum\:\frac{Gt}{Dt}$$
(3)

where Gt is the number of seeds germinated on t days, and Dt represents the corresponding day of germination. The mean germination time (MGT) of rice seeds was calculated using the formulas16.

Determination of physiological and biochemical indicators

After 7 days of cultivation, rice seedlings (0.5 g fresh weight) were collected for analysis. After washing, grinding, vortex mixing, and centrifugation, the supernatant was taken to determine SOD, CAT, POD, MDA, AsA and SS17 according to the procedures in the instruction manual of Nanjing Jiancheng kits.

The photosynthetic pigments (chlorophyll and carotenoids) in rice seedlings were extracted and quantified using an ethanol-based method. Briefly, fresh leaf samples (0.2 g) were homogenized and soaked in 95% ethanol for 24 h in the dark. The absorbance of the extracts was measured at 470, 646, and 663 nm using 95% ethanol as the blank. The chlorophyll and carotenoids of rice seedlings were calculated according to the formulas18.

Quantitative real-time PCR analysis

Total RNA was extracted from liquid nitrogen-frozen rice seedlings (0.2 g) using an ultra-pure RNA extraction kit (CWBIO, CW0581), yielding RNA with A260/280 ratios of 1.8–2.0. cDNA was synthesized from 1 µg RNA using reverse transcriptase. qRT-PCR was performed on a CFX96 system (Bio-Rad) with SYBR® Premix Ex Taq™ II (Takara) in 20 µL reactions containing: 10 µL 2× SYBR Green Master Mix, 0.8 µL each primer (10 µM), 2 µL diluted cDNA (1:10), and 6.4 µL nuclease-free water.

Thermal cycling at 95 °C for 30 s (initial denaturation); 40 cycles of 95 °C for 5 s and 60 °C for 30 s; and melting curve analysis (65–95 °C, 0.5 °C/5 sec). OsGAPDH (Os08g03290.1) served as the internal reference. Primer sequences are listed in Table S1.

Data analysis

All data were expressed as mean ± standard deviation (SD) from three independent biological replicates. Statistical analyses were performed using SPSS 26.0 (IBM Corp., USA). Significant differences between treatment groups were evaluated by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test at a significance level of p < 0.05.

Pearson correlation analysis was performed to assess relationships between physiological, biochemical, and Cr distribution parameters. The Pearson correlation coefficient (r) and its significance level were calculated for each variable pair, with a statistical significance threshold set at p < 0.05.

Random forest (RF) modeling was conducted using the “Random Forest” package in R. The model was built with 500 decision trees (n tree = 500), and the number of variables randomly sampled as candidates at each split (mtry) was set to the square root of the total number of variables. Variable importance was evaluated based on the mean decrease in accuracy and the mean decrease in Gini impurity index. The overall accuracy and out-of-bag error rate were used to assess model performance.

Results

Effects of pro pretreatment on rice seed germination under Cr(VI) stress

Heavy metal stress significantly inhibits seed germination and early growth. As illustrated in Fig. 1, Pro pretreatment altered key germination parameters, including GP, Gr, GI, and MGT under Cr(VI) stress.

The 24-h and 48-h GP values were measured separately (Fig. S3 and Fig. 1a). The 48-h GP values were more obvious and thus selected for discussion in this section. In the Cr(VI)-stressed group, the 48-h GP values decreased progressively with an increase in the Cr(VI) concentration (75.3% at 2.0 mg Cr/L, 68.7% at 8.0 mg Cr/L, and 66.0% at 16.0 mg Cr/L; Fig. 1a). Pro pretreatment enhanced GP by 6.0% (2.0 mg Cr/L), 11.0% (8.0 mg Cr/L), and 28.0% (16.0 mg Cr/L), demonstrating dose-dependent effects. Similar trends were observed for Gr (Fig. 1b). The control Gr values under Cr(VI) stress were 89.3% (2.0 mg Cr/L), 86.7% (8.0 mg Cr/L), and 80.7% (16.0 mg Cr/L). Pro supplementation increased these Gr values by 0.8%, 6.9%, and 18.2%, respectively.

Under Cr(VI) stress, the GI values decreased to 28.7%, 26.3%, and 24.3% at 2.0, 8.0, and 16.0 mg Cr/L, respectively (Fig. 1c). Pro pretreatment reversed this decline, elevating the GIs to 104%, 124%, and 140% at low, medium, and high Cr(VI) levels, respectively. Notably, Pro pretreatment shortened MGTs across all treatments (Fig. 1d). At 2.0 mg Cr/L, MGT decreased significantly by 14.5% (p < 0.05), while reductions of 14.2% (8.0 mg Cr/L) and 6.9% (16.0 mg Cr/L) were observed. The control MGT values ranged between 1.8 and 1.9 d.

These results collectively indicate that Pro pretreatment enhances GP, Gr, and GI values and accelerates germination initiation, particularly under high hexavalent Cr stress (16.0 mg Cr/L). The concentration-dependent alleviation effects indicate the critical role of Pro in improving seed stress resilience to contaminated environments.

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Effects of exogenous Pro on seed GP, Gr, and GI under different concentrations of Cr(VI) stress (n = 50).

Pro pretreatment enhances antioxidant enzyme activities under Cr(VI) stress

Cr(VI) stress induces ROS accumulation during rice seed germination. Thus, elevated antioxidant enzyme activities are critical for ROS scavenging to mitigate oxidative damage. In Cr(VI)-stressed seedlings, SOD activities decreased with an increase in the Cr(VI) concentration, with measured values of 2509 U/mg protein (2.0 mg Cr/L), 2284 U/mg protein (8.0 mg Cr/L), and 730 U/mg protein (16.0 mg Cr/L) (Fig. 2a). Pro pretreatment significantly increased SOD activities by 87.8% (2.0 mg Cr/L) and 287% (16.0 mg Cr/L) compared with the Cr(VI)-stressed group (p < 0.05).

POD activities exhibited a non-linear response to Cr(VI) stress, with values of 623 U/mg protein (2.0 mg Cr/L), 232 U/mg protein (8.0 mg Cr/L), and 618 U/mg protein (16.0 mg Cr/L) (Fig. 2b). Pro supplementation elevated POD activities by 17.8% (2.0 mg Cr/L), 43.6% (8.0 mg Cr/L), and 67.3% (16.0 mg Cr/L) (p < 0.05).

CAT activities were severely suppressed under high Cr(VI) stress, declining to 28.5 U/mg protein (2.0 mg Cr/L), 18.5 U/mg protein (8.0 mg Cr/L), and 8.5 U/mg protein (16.0 mg Cr/L) (Fig. 2c). Pro pretreatment restored CAT activities, achieving 1.1-fold (2.0 mg Cr/L), 1.4-fold (8.0 Cr mg/L), and 4.6-fold (16.0 Cr mg/L) increases compared with the control (p < 0.05).

APX activity was significantly enhanced under Cr(VI) stress, increasing from 12.5 U/g protein (Control) to 19.5 U/g protein (2.0 mg Cr/L), 28.4 U/g protein (8.0 mg Cr/L), and 36.6 U/g protein (16.0 mg Cr/L) (Fig. 2d, p < 0.05). Pro pretreatment further amplified APX activity, reaching 22.5 U/g protein (2.0 mg Cr/L), 32.7 U/g protein (8.0 mg Cr/L), and 42.5 U/g protein (16.0 mg Cr/L)—representing 1.2, 1.1, and 1.2-fold increases over Cr(VI)-only treatments, respectively.

GR activity was significantly enhanced under Cr(VI) stress, increasing from 8.5 U/mg protein (Control) to 18.5 U/mg protein (2.0 mg Cr/L), 28.5 U/mg protein (8.0 mg Cr/L), and 38.4 U/mg protein (16.0 mg Cr/L) (Fig. S4, p < 0.05). Pro pretreatment further elevated GR activity, reaching 22.5 U/mg protein (2.0 mg Cr/L), 32.5 U/mg protein (8.0 mg Cr/L), and 42.6 U/mg protein (16.0 mg Cr/L), representing 1.22, 1.14, and 1.10-fold increases compared to their respective Cr(VI)-only treatments.

These findings demonstrate that exogenous Pro application counteracts the Cr(VI)-induced suppression of antioxidant enzymes and exerts the most potent protective effects under high Cr(VI) stress (16.0 mg Cr/L), where the SOD, CAT, and APX activities increased by over 250%, 350%, and 120%, respectively.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Effects of Pro on antioxidant enzymes in rice seedlings under different concentrations of Cr(VI) stress.

Pro pretreatment elevates GSH and AsA levels under Cr(VI) stress

GSH content significantly increased under Cr(VI) stress, rising from 3 µmol/g FW (Control) to 4.5 µmol/g FW (2.0 mg Cr/L), 6.2 µmol/g FW (8.0 mg Cr/L), and 8.0 µmol/g FW (16.0 mg Cr/L) (p < 0.05, Fig. 3a). Pro pretreatment further elevated GSH levels, reaching 5.0 µmol/g FW (2.0 mg Cr/L), 7.0 µmol/g FW (8.0 mg Cr/L), and 9.0 µmol/g FW (16.0 mg Cr/L)—representing 1.1, 1.1s, and 1.1-fold increases over Cr(VI)-only treatments, respectively.

AsA is an antioxidant that also plays a crucial role in plant growth and development. In the Cr(VI) stress group, when the concentrations of Cr(VI) were 2.0, 8.0, and 16.0 mg Cr/L, the AsA contents were 60.2, 21.4, and 32.0 mg/mg protein, respectively. After Pro pretreatment, when the concentration of Cr(VI) was 2.0 mg Cr/L, the AsA content increased by 1.3-fold (p < 0.05); at 8.0 mg Cr/L, the AsA content decreased by 2.8%; at 16.0 mg Cr/L, the AsA content increased by 1.1-fold (Fig. 3b). These results indicate that exogenous Pro effectively enhances AsA accumulation, with the most pronounced effect at low Cr(VI) concentrations. The elevated AsA content significantly synergizes with antioxidant enzymes (Sect. "Pro pretreatment enhances antioxidant enzyme activities under Cr(VI) stress") to strengthen ROS scavenging capacity within Cr(VI)-stressed seedlings.

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Effects of Pro on GSH and AsA content in rice seedlings under different concentrations of Cr(VI) stress.

Proline pretreatment alleviates membrane lipid peroxidation under Cr(VI) stress

Heavy metal-induced ROS accumulation triggers membrane lipid peroxidation, reflected by MDA production. Elevated MDA levels correlate with reduced seed viability, while its scavenging enhances stress resilience19. As shown in Fig. 4a, Cr(VI) stress progressively increased the MDA content in rice radicles. In the Cr(VI)-stressed groups, the MDA contents increased to 88.7 nmol/mg protein (2.0 mg Cr/L), 121.6 nmol/mg protein (8.0 mg Cr/L), and 123.5 nmol/mg protein (16.0 mg Cr/L). In contrast, in the Pro + Cr(VI) group, the MDA levels decreased to 13.3 nmol/mg protein (2.0 mg Cr/L), 14.4 nmol/mg protein (8.0 mg Cr/L), and 15.1 nmol/mg protein (16.0 mg Cr/L), representing reductions of 85.0%, 88.2%, and 87.8%, respectively (p < 0.001). This dramatic reduction in MDA accumulation (exceeding 85% across all treatments) demonstrates that Pro pretreatment substantially mitigates Cr(VI)-induced membrane damage, preserving cellular integrity in rice seedlings.

Pro pretreatment alleviates membrane lipid peroxidation under Cr(VI) stress

SS play a crucial role in mitigating oxidative damage in the early stages of seed germination. In the Cr(VI) stress group, when the concentrations of Cr(VI) were 2.0, 8.0, and 16.0 mg Cr/L, the SS contents in rice seedlings were 748.2, 847.9, and 836.4 µg/g (Fig. 4b), respectively. After Pro pretreatment, at 2.0 mg Cr/L, the SS content increased by 19.3%; at 8.0 mg Cr/L, the SS content increased by 6.2%; at 16.0 mg Cr/L, the SS content increased by 11.7%. Pro pretreatment increased the SS contents by 6.2–19.3% across treatments, with values rising from 748.2 µg/g (2.0 mg Cr/L) to 934.18 µg/g (16.0 mg Cr/L), indicating that Pro preserves existing sugar pools and stimulates de novo biosynthesis, likely through glyoxylate cycle activation, a key pathway linking lipid mobilization to carbohydrate synthesis during germination.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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Effects of Pro on MDA and SS contents in rice seedlings under the stress of different concentrations of Cr(VI).

Effects on photosynthetic pigments in rice seedlings

Photosynthetic pigments mainly include chlorophyll and carotenoids. The accumulation of ROS in plants disrupts chlorophyll synthesis, thus affecting photosynthesis20. As shown in Fig. 5a and b, the chlorophyll contents in rice seedlings exposed to Cr(VI) concentrations of 2.0, 8.0, and 16.0 mg Cr/L ranged from 0.44 to 0.47 mg/g, while the carotenoid contents ranged from 0.03 to 0.05 mg/g. Pro pretreatment significantly increased both the chlorophyll and carotenoid contents. At 2.0 mg Cr/L, the chlorophyll and carotenoid levels increased by 1.1- and 1.3-fold; at 8.0 mg Cr/L, the increases were 1.2- and 1.5-fold; at 16.0 mg Cr/L, the increases were 1.1- and 1.3-fold. These results indicate that Pro pretreatment enhances the accumulation of photosynthetic pigments (chlorophyll and carotenoids) in rice seedlings under Cr(VI) stress, thus mitigating the adverse effects associated with oxidative stress on photosynthesis.

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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Effects of exogenous Pro on photosynthetic pigments in rice seedlings under the stress of different concentrations of Cr(VI).

Pro modulates Cr partitioning in rice seedlings

As depicted in Fig. 6, the Cr content in plumules was positively correlated with Cr(VI) concentration (Pearson’s r, p < 0.05). Pro pretreatment reduced Cr in chaff and plumules by 12.4–78.5% while increasing Cr in grains, indicating altered metal partitioning. After Pro pretreatment, at 2.0 mg Cr/L, the Cr contents in the chaff and plumules decreased by 45.7% and 78.5%, respectively. At 8.0 mg Cr/L, the Cr contents in the chaff and plumules decreased by 34.3% and 17.5%, respectively; at 16.0 mg Cr/L, the Cr contents in the chaff and plumules decreased by 12.4% and 41.5%, respectively. However, the Cr contents in the grains of the Pro pretreatment groups were higher than those in the Cr(VI) stress groups. The results show that after pretreatment with exogenous Pro, the grains absorbed a large amount of Cr, effectively reducing the burden on plumules. Pro may enhance chelation or sequestration in grains, reducing plumule toxicity.

Fig. 6
Fig. 6The alternative text for this image may have been generated using AI.
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Cr contents in different parts of rice seedlings and the distribution coefficients of Cr under various concentrations of Cr(VI) stress with the effect of Pro.

According to the distribution coefficients (Table 1), the Cr contents in different parts of rice seedlings proceed as follows: plumules (58.4%) > chaff (32.1%) > grains (9.5%). Cr(VI) mainly accumulates in plumules. The distribution coefficients for chaff and grains are less than 40%, with minimum values of 10.2% and 14.6%, respectively, when the concentration of Cr(VI) is 8.0 mg/L. After Pro pretreatment, at 2.0 mg Cr/L, the distribution coefficient of Cr(VI) in seedlings decreases by 3.3%; at 8.0 mg Cr/L, it decreases by 4.6%. After Pro pretreatment, the Cr content in different parts of rice seedlings is reduced significantly (p < 0.05), and the antioxidant mechanism in the plumules becomes activated. The 41.5% reduction in the Cr content in plumules at 16.0 mg/L Cr(VI) correlates with SOD (3.4-fold) and CAT (4.6-fold) activation (Fig. 2a and c), indicating that Pro triggers antioxidant defenses concurrent with metal repartitioning.

Table 1 Distribution coefficients of Cr(VI) in different rice seedling tissues before and after pro treatment (%).
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Correlation analysis and random forest regression

According to the results of the correlation analysis in Fig. 7a and Fig. S5, in the Pro pretreatment group, Gr was speculated to be significantly and positively correlated with POD and CAT (p < 0.05), positively correlated with AsA and SOD, and negatively correlated with the MDA and chlorophyll contents. The GI is significantly and positively correlated with CAT (p < 0.05), positively correlated with SOD and POD, and significantly and negatively correlated with the chlorophyll content (p < 0.05). The MGT is significantly and positively correlated with the SS, chlorophyll, and carotenoid contents (p < 0.05), positively correlated with MDA, and negatively correlated with SOD and CAT. The significant positive correlation (p < 0.05) between MDA and POD in the Cr(VI) stress group becomes uncorrelated in the Pro pretreatment group. It is hypothesized that exogenous Pro can affect the MDA, POD, chlorophyll, and carotenoid contents.

Fig. 7
Fig. 7The alternative text for this image may have been generated using AI.
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Correlation analysis of physiological indicators and random forest analysis of rice seed germination under Cr(VI) stress with the effect of exogenous Pro.

According to the analysis of the feature importance ratios of the two groups (Fig. 6b and c) based on the random forest plot, Pro affects the GR of rice seeds by mediating factors such as chlorophyll, CAT, SOD, AsA, and carotenoids. After applying exogenous Pro, the feature importance ratio of chlorophyll increased from 8.5% to 14.6%, that of CAT rose from 13.6% to 16.7%, and that of SOD increased from 5.1% to 8.3% (Fig. 6b and c). Conversely, the feature importance ratio of AsA decreased from 15.3% to 10.4%, and that of carotenoids decreased from 20.3% to 16.7% (Fig. 6b and c). Based on the random forest regression, after treatment with exogenous Pro, the feature importance of chlorophyll, SOD, and CAT in rice seeds changed significantly. It is speculated that hydrogen peroxide (H2O2) was the main ROS that accumulated in rice seed cells under Cr(VI) stress. Pro mainly alleviates the toxicity of Cr(VI) by increasing the chlorophyll content and enhancing the activities of CAT and SOD to scavenge the ROS in cells.

Discussion

Pro-mediated mitigation of Cr(VI) toxicity in rice seedlings

Heavy metal ions, such as Cr(VI), inhibit seed germination by disrupting water imbibition, inducing oxidative damage to critical enzymes, and impairing membrane integrity21,22. This study elucidates the multifaceted protective mechanisms of Pro that significantly enhance rice seed germination under Cr(VI) stress. Pro mitigates Cr(VI)-induced membrane hyperpermeability, reducing electrolyte leakage by 85–88% (Fig. 4a), thus preserving cellular turgor, which is essential for radicle emergence. As a P5CS-catalyzed precursor of glutamate, Pro sustains carbon flux through the glyoxylate cycle, converting lipid reserves into carbohydrates during heterotrophic germination23. The 1.3-fold increase in AsA at 2.0 mg/L Cr(VI) (Fig. 3b) underscores enhanced redox balancing, critical for early seedling vigor. Furthermore, Pro-derived glutamate triggers Ca²⁺–H₂O₂ crosstalk, amplifying antioxidant enzyme production (Fig. S6, Supplementary Material S2). Random forest modeling identified chlorophyll (28.6%), CAT (22.1%), and SOD (19.8%) as key germination drivers, evidenced by a 14.6% increase in chlorophyll and 459% increase in CAT activation at 16.0 mg Cr/L.

A critical finding is the role of Pro in altering Cr(VI) partitioning within rice seedlings. As shown in Table 1, Cr(VI) predominantly accumulates in plumules (58.4%), followed by chaff (32.1%) and grains (9.5%). Pro pretreatment significantly reduces the Cr contents in plumules and chaff (12.4–78.5%) while increasing Cr in grains (Fig. 6), indicating enhanced chelation or sequestration in grains that alleviates plumule toxicity. This repartitioning correlates with a 41.5% reduction in plumule Cr content at 16.0 mg/L Cr(VI), accompanied by SOD (4.4-fold) and CAT (4.6-fold) activation (Fig. 2a and c). These results indicate that Pro bolsters antioxidant defenses and induces metal repartitioning, potentially by upregulating metal-chelating peptides such as phytochelatins or metallothioneins (Fig. S7, Supplementary Material S3). The positive correlation between the Cr content in plumules and Cr(VI) concentration (p < 0.05, Fig. 6) further highlights the role of plumules as a primary Cr sink that Pro mitigates through metabolic and compartmental adjustments. The MT induction aligns with our observed Cr redistribution data (reduced Cr in embryos by 41.5% with Pro), OsMT genes are likely to participate in Cr(III) sequestration. Pro’s amplification of OsMT3b suggests enhanced vacuolar compartmentalization, explaining lower embryonic Cr.

Pro-mediated physiological regulation under Cr(VI) stress

Cr(VI) stress disrupts energy metabolism during germination by impairing water imbibition, causing membrane rupture, and inducing organic acid leakage, leading to energy depletion22. The results demonstrate that exogenous Pro significantly restores germination indices, with GR, GP, and GI increasing by 18.2%, 28.3%, and 40.5%, respectively, at 16.0 mg Cr/L (Fig. 1). This recovery stems from the dual roles of Pro: (1) as an alternative carbon/nitrogen source via its derivatives (glutamate and aspartate), compensating for pyruvate and sugar losses, and (2) as an osmoprotectant, mitigating Cr(VI)-induced membrane permeability changes. At the molecular level, we observed that Pro triggers a self-reinforcing biosynthesis loop, with OsP5CS1 (the key proline synthesis gene) showing significant upregulation (peaking at 3.5-fold under 8 mg/L Pro, p < 0.01) while simultaneously suppressing proline catabolism (OsPDH2 downregulation by 40–60%, Fig. S6, Supplementary Material S2). This dual regulation ensures sustained high cellular proline pools under stress conditions.

Pro exhibited dose-dependent efficacy, with optimal effects at low-to-medium Cr(VI) concentrations (2.0–8.0 mg/L), but diminished protection at 16.0 mg/L Cr(VI). This limitation is evidenced by elevated SS accumulation (Fig. 4), indicating that severe oxidative stress overwhelms proline’s detoxification capacity while triggering stress-responsive energy conservation mechanisms22. These findings align with established reports of osmolyte-mediated stress tolerance in chitosan-coated seeds.

Pro orchestrates a robust antioxidant cascade, upregulating SOD (4.4-fold), POD (1.9-fold), and CAT (4.6-fold) activities (Fig. 2). SOD converts superoxide to H₂O₂, which CAT neutralizes to prevent hydroxyl radical formation by Fenton reactions21. Random forest modeling revealed strong correlations (p < 0.001) between antioxidant enzyme activities and germination indices, indicating Pro drives redox homeostasis through the transcriptional regulation of carotenoid biosynthesis and CAT-related genes20. The 18% increase in chlorophyll content further supports metabolic reprogramming toward stress resilience. Notably, Pro pretreatment reduces the Cr contents in chaff and plumules by up to 78.5% at 2.0 mg Cr/L while increasing Cr in grains (Fig. 6). This shift in metal distribution likely involves Pro-enhanced expression of vacuolar sequestration pathways in grains, reducing the Cr burden on metabolically active plumules23,24. The significant reduction in Cr contents (p < 0.05) across seedling parts, coupled with antioxidant activation, underscores the role of Pro in integrative stress mitigation. Pro improving germination and seedling vigor under Cr(VI) stress.

Previous studies have reported that other osmoprotectants, such as glycine betaine and SS, also enhance plant tolerance to heavy metal stress by maintaining osmotic balance and boosting antioxidant enzyme activities. For instance, glycine betaine has been shown to improve germination rates and reduce oxidative damage in rice and maize seedlings under cadmium and chromium stress by activating SOD, CAT, and increasing chlorophyll content. SS similarly contribute to osmoprotection and serve as alternative energy sources under stress conditions. Compared to these osmolytes, exogenous proline exhibits unique advantages. Besides its osmoprotectant role, proline participates in metal chelation, stabilizes subcellular structures, and functions as a molecular chaperone, which may explain the more pronounced increase in antioxidant enzyme activities and redistribution of Cr observed in our study. Additionally, proline acts as a metabolic precursor for glutamate and ascorbate biosynthesis, further enhancing redox homeostasis.

Conclusions

Exogenous Pro pretreatment significantly enhanced rice seed germination under Cr(VI) stress, as evidenced by increased germination potential (GP) and germination index (GI), along with reduced mean germination time (MGT). This improvement in seed viability was accompanied by Pro-mediated activation of antioxidant systems. Pro pretreatment elevated the activities of SOD (up to 287%), CAT (4.6-fold), and GR (1.22-fold at 16.0 mg/L Cr(VI)), with GR showing the more substantial induction under high Cr(VI) stress (16.0 mg/L). The synergistic upregulation of these enzymes correlated with a dramatic reduction in MDA levels (85–88%), indicating effective mitigation of oxidative damage. Pro increased chlorophyll (1.2-fold) and carotenoid (1.5-fold) contents, restoring photosynthetic efficiency disrupted by Cr(VI). Pro Elevated SS levels (6.2–19.3%) further supported energy metabolism during germination. While antioxidant and photosynthetic improvements were observed across all Cr(VI) concentrations, the most pronounced effects occurred at high Cr(VI) stress (16.0 mg/L), where GR activity and CAT induction peaked. Low-to-medium concentrations (2.0–8.0 mg/L) showed more substantial benefits in germination parameters (GP, GI) and chlorophyll accumulation. Pro redirected Cr accumulation from plumules to grains (reduction by 17.5–78.5%), reducing toxicity in sensitive tissues. Random forest analysis identified chlorophyll content, CAT, and SOD as key contributors to Pro-induced stress resilience, with their feature importance increasing by 71.8%, 22.8%, and 62.7%, respectively. These findings demonstrate that Pro pretreatment orchestrates a multi-layered defense against Cr(VI) toxicity, combining antioxidant activation and metal repartitioning to enhance rice establishment in contaminated soils.