Introduction

The rapid growth of industrialization and human activities has significantly escalated heavy metal contamination, a pressing environmental issue due to its toxicity, persistence, and bio accumulative properties1,2. Industrial discharges, agricultural practices, and improper waste management are major contributors to heavy metal release, thus disrupting ecosystems, lowering agricultural productivity, and posing severe risks to human and animal health through food chain bioaccumulation3,4. Some heavy metals serve as essential micronutrients at low concentrations but become toxic when thresholds are exceeded, thereby complicating their impact on plant metabolism and ecosystem sustainability5.

Lead is one of the most toxic heavy metals, capable of posing significant risks even at low concentrations, with exposure linked to serious health problems such as neurological, developmental, and cardiovascular issues, especially in children and pregnant women6,7. Its permissible limit in drinking water, as set by the World Health Organization (WHO), is 0.01 mg/L (10 µg/L). Contamination of lead arises from diverse sources, including soil, air, old lead-based paints, food, and water6. The widespread use of lead in industries such as manufacturing, construction, and chemicals is attributed to its versatile physical and chemical properties, with applications in batteries, pigments, cable sheathing, alloys, and petrol additives8,9.

Lead contamination in agricultural soils severely hampers crop productivity, as its accumulation disrupts essential plant biochemical pathways and growth processes10. Lead toxicity also triggers oxidative stress by inducing the overproduction of reactive oxygen species- hydroxyl ions, hydrogen peroxide, and superoxide, which damage cellular proteins, lipids, and nucleic acids. This oxidative stress disrupts ion balance, reduces leaf area, and increases metabolic costs, further affecting plant growth and functionality11.

Conventional physical and chemical methods for remediating lead-contaminated environments are expensive and require complex equipment and production facilities12. Furthermore, these methods often generate secondary pollution and additional waste13,14. In contrast, phytoremediation, an emerging green technology, has garnered significant attention11,15,16. Phytoremediation is an eco-friendly process that employs plants to remove, stabilize, transfer, or convert toxic substances, including heavy metals and organic pollutants, into less harmful forms11,16. Many plants can absorb and accumulate chemical elements from contaminated environments, either breaking them down or storing them in their tissues. Certain plant species, termed hyperaccumulators, can thrive in highly contaminated soils, absorbing large quantities of heavy metals and storing them in their shoots and leaves, thus detoxifying the soil13.

Several field trials have confirmed the feasibility of using plants for environmental cleanup13,17. For instance, Indian mustard effectively removes zinc, mercury, lead, and copper from contaminated soils18,19. Brassica juncea, Helianthus annuus, and Calotropis procera are known to accumulate substantial amounts of lead while maintaining growth20,21. Phytoremediation is appealing because it offers a sustainable, low-impact method to remediate contaminated areas while preserving the environment’s natural state11,16. It is also cost-effective and simpler to implement than conventional remediation techniques, with the added benefit that plants can be easily monitored. In addition, phytoremediation enables the recovery and reuse of valuable metals through phytomining, which helps preserve topsoil, maintain soil fertility, and reduce erosion and metal leaching22. While phytoremediation offers significant advantages, it also has limitations, including slow remediation rates, limited effectiveness in highly contaminated soils, and variability in plant uptake efficiency depending on species and environmental conditions. Despite these challenges, ongoing research into plant–microbe interactions and the use of stress-tolerant hyperaccumulators continues to expand its practical applications23. However, many hyperaccumulators struggle to thrive in environments with high levels of heavy metal contamination, particularly when lead concentrations are elevated. This is because lead is a highly phytotoxic metal, which can severely impair plant growth, damage root structures, and hinder essential physiological processes, including photosynthesis and nutrient uptake9,14,24.

To improve the efficiency of phytoremediation, it is essential to enhance the ability of hyperaccumulator plants to tolerate lead-induced stress. One promising approach is the utilization of plant-associated microorganisms, such as endophytic bacteria15,25. Endophytes, which include bacteria, actinobacteria, and fungi, form symbiotic relationships with plants by colonizing their healthy tissues10,26,27. Endophytes can enhance the phytoremediation potential of plants by aiding in nutrient uptake, producing phytohormones, and altering the plant’s metabolic pathways to improve tolerance to heavy metals28. Studies have demonstrated that endophytes can reduce lead phytotoxicity by promoting antioxidant enzyme activity, enhancing metal sequestration, and improving overall plant biomass under contaminated conditions26,27. Microbial-assisted phytoremediation has further enhanced lead uptake and stress tolerance, with endophytic bacteria such as Pseudomonas fluorescens, and Bacillus subtilis showed promising results29.

Plant growth-promoting bacteria such as Pantoea agglomerans enhance plant tolerance to heavy metal stress through mechanisms like phytohormone production, siderophore release, and metal biosorption30. Genome analyses of Pantoea have revealed the presence of metal-resistance determinants, including multidrug efflux systems and copper/silver transporters31. These features facilitate survival in metal-contaminated environments, indicating genetic mechanisms that mitigate heavy metal stress in plants.

The current study examines the role of P. agglomerans, an endophytic bacterium from Prosopis juliflora seeds, in improving the phytoremediation capacity of C. procera, a lead-accumulating plant adapted to extreme environments32,33,34,35. Pantoea species can enhance metal tolerance by producing chelating agents and enzymes that reduce metal toxicity36. This study evaluates the physiological and enzymatic responses of C. procera in a lead-treated hydroponic system to determine how P. agglomerans mitigates phytotoxicity and promotes plant growth. A hydroponic system offers precise control over variables, allowing a clearer understanding of plant–microbe-metal interactions, an essential step prior to field-level validation37,38.

We hypothesize that P. agglomerans enhance lead uptake, alleviates phytotoxicity by boosting antioxidant enzyme activity, and improves overall plant health. Although phytoremediation holds great promise, the combined physiological and biochemical effects of P. agglomerans on lead-stressed C. procera remain largely unexplored under controlled conditions. The present study aims to fill this gap and contribute to the development of scalable, sustainable strategies for remediating lead-contaminated environments.

Results

Isolation of lead-tolerant endophytic bacteria

A total of 25 endophytic bacterial strains were successfully isolated from the seeds of P. juliflora. A zone of inhibition assay was conducted to evaluate their lead tolerance using lead concentrations of 0, 20, 40, 60, and 80 mg/L. Among the isolates, strain 11 exhibited no inhibition zones at any tested concentration after 24 h of incubation at 37°C, indicating high lead tolerance (Fig. S1).

This strain was selected for further studies and was identified based on 16S rDNA nucleotide sequence analysis. BLAST analysis revealed 100% homology with P. agglomerans. A phylogenetic tree constructed using the partial 16S rDNA sequence of the isolate alongside related sequences from the NCBI database confirmed its identification (Fig. S2). In light microscopy studies on C. procera-inoculated roots at 23 days post inoculation, P. agglomerans cells were prevalent within the parenchyma cells of the cortex and in the xylem (Fig. S3).

Effect of lead treatment on plant growth parameters

Growth parameters, including shoot and root dry weight, shoot length, number of green and yellow leaves, and leaf dimensions, were assessed to determine C. procera performance under lead stress with and without Pantoea inoculation (Fig. 1, and Fig. S4). Significant effects were observed for Pantoea addition on both shoot and root dry weights, though no significant interaction was found between these factors. In the absence of lead, Pantoea-inoculated plants exhibited 23% higher shoot dry weight, and 5.7% higher root dry weight compared to non-inoculated plants (Fig. 1).

Fig. 1
figure 1

The effect of Pantoea agglomerans and lead concentration on the plant growth parameters of Calotropis procera.

Full size image

At higher lead concentrations, Pantoea-treated plants consistently outperformed non-inoculated plants, indicating enhanced growth even under stress. Similarly, shoot length and the number of green leaves were significantly higher in Pantoea-inoculated plants (Fig. 1). At 0 mg/L lead, Pantoea application reduced yellow leaves by 78.67%. At the highest lead concentration (80 mg/L), Pantoea increased green leaves by 5.85% and reduced yellow leaves by 40.29%. The number of leaves and their dimensions decreased as lead concentration increased, with non-inoculated plants showing a more pronounced increase in yellow leaves than inoculated plants. In the case of leaf length, both Pantoea inoculation and lead concentration had significant effects on the largest leaf in C. procera seedlings (P < 0.001), although their interaction was not significant (P > 0.05). Pantoea treatment consistently improved leaf length across all lead concentrations compared to untreated plants.

Effect of lead on photosynthetic pigment contents

Chlorophyll a, chlorophyll b, and carotenoids are essential photosynthetic pigments involved in light harvesting, energy absorption, and conversion in plants39. In this study, the effects of Pantoea treatment on these pigments were analyzed using analysis of variance (ANOVA). The results showed significant effects of lead treatment, Pantoea treatment, and their interaction on pigment concentrations (Fig. 2). Lead treatment had a highly significant effect (P < 0.0001) in chlorophyll a, with lower concentrations observed at higher lead doses. Although Pantoea treatment did not have a significant main effect (P = 0.36832), the interaction between lead and Pantoea was significant (P < 0.0001). Similarly, for carotenoids, both lead and Pantoea treatments had significant effects (P < 0.0001), with their interaction also proving significant (P = 0.00358).

Fig. 2
figure 2

The effect of Pantoea agglomerans and lead concentration on the photosynthetic pigments of Calotropis procera.

Full size image

In untreated plants, increasing lead concentrations caused a progressive decline in chlorophyll a, chlorophyll b, and carotenoid levels, reflecting lead’s adverse effect on the photosynthetic apparatus and antioxidant capacity (Fig. 2). In contrast, Pantoea-treated plants showed significantly mitigated declines in pigment levels. For instance, at 80 mg/L lead, Pantoea-treated plants exhibited higher chlorophyll a (0.315 mg/g FW) and chlorophyll b (0.222 mg/g FW) levels compared to untreated plants (0.288 mg/g FW for chlorophyll a, and 0.208 mg/g FW for chlorophyll b).

Similarly, carotenoid levels remained more stable in Pantoea-treated plants, reflecting enhanced tolerance to oxidative stress. At lower lead concentrations (20 and 40 mg/L), Pantoea-treated plants consistently exhibited higher pigment levels, highlighting the bacterium’s ability to alleviate initial lead toxicity (Fig. 2).

Chlorophyll fluorescence analysis

Chlorophyll fluorescence parameters FV/FM and PSII efficiency were assessed to determine the plant’s photosynthetic efficiency under lead stress with and without Pantoea inoculation. As the concentration of lead increased, FV/FM values decreased (Fig. 3). At 40 mg/L lead, the FV/FM ratio for plants with Pantoea (0.8214) remained higher than that of non-inoculated plants (0.765), indicating a protective effect of Pantoea against more severe lead toxicity (Fig. 3). The FV/FM values continued to decline at higher concentrations of lead (60 mg/L and 80 mg/L), with Pantoea-inoculated plants showing better maintenance of photosynthetic efficiency compared to non-inoculated plants (Fig. 3).

Fig. 3
figure 3

The effect of Pantoea agglomerans and lead concentration on the chlorophyll fluorescence of Calotropis procera.

Full size image

The PSII measurements supported these findings, with Pantoea-inoculated plants showing higher PSII efficiency at all lead concentrations. At 0 mg/L lead, the PSII value for Pantoea-treated plants (0.81) was higher than the non-inoculated plants (0.79), suggesting that Pantoea contributes to maintaining the integrity of the photosynthetic apparatus. At higher concentrations of lead, the PSII efficiency of the inoculated plants remained significantly higher than that of the non-inoculated plants. Pantoea species enhance the photosynthetic efficiency and PSII functionality of C. procera under lead stress, likely through mechanisms such as the reduction of oxidative stress or the enhancement of lead uptake and detoxification.

Quantification of hydrogen peroxide and malondialdehyde (MDA)

Hydrogen peroxide and MDA levels were measured as indicators of oxidative stress and lipid peroxidation. MDA levels, an indicator of lipid peroxidation and cellular damage, were notably elevated in treated plants exposed to higher lead concentrations, especially in shoots (Fig. 4). For instance, MDA content in shoots increased from an average of 8.87 nmol/g FW in untreated controls to as high as 16.13 nmol/g FW under 60 mg/L lead treatment with Pantoea inoculation (Fig. 4). In roots, MDA levels were comparatively lower, without significant variation across the treatments. Hydrogen peroxide levels, another marker of oxidative stress, also varied significantly with lead concentration and plant organ. In shoots, hydrogen peroxide content was generally higher in control plants than in Pantoea-treated plants at comparable lead concentrations. For instance, under 20 mg/L lead treatment, shoots of treated plants showed reduced hydrogen peroxide levels (9.57–10.58 µmol/g FW) compared to controls (11.73–15.31 µmol/g FW).

Fig. 4
figure 4

The effect of Pantoea agglomerans and lead on the concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in Calotropis procera.

Full size image

Antioxidant enzyme activity

The impact of Pantoea treatment on the activity of catalase (CAT) and guaiacol peroxidase (GPX) in shoots and roots of plants exposed to different concentrations of lead was analyzed. In the shoots, CAT activity decreased with increasing lead concentrations (Fig. 5). At 20 mg/L lead, CAT activity in Pantoea-treated plants was reduced by 42% compared to the 0 mg/L condition, while untreated plants exhibited a 27% decrease. At 40 mg/L lead, the reduction in CAT activity was smaller in Pantoea-treated plants (30%) compared to untreated plants (38%). However, at 80 mg/L lead, CAT activity in treated plants was 15% higher than that of the untreated control (Fig. 5). In the roots, Pantoea treatment increased CAT activity by 55% at 0 mg/L lead compared to untreated plants. As lead concentration increased, CAT activity declined in both treated and untreated plants. At 20 mg/L lead, treated plants showed a 46% decrease, while untreated plants had a 33% reduction. At 40 mg/L lead, treated plants retained 36% higher CAT activity than untreated plants, though both experienced a 69% decrease compared to their 0 mg/L levels. At 80 mg/L lead, the treated plants had a 75% decrease in CAT activity, while untreated plants showed a 93% decline (Fig. 5).

Fig. 5
figure 5

The effect of Pantoea agglomerans and lead on (a): catalase (CAT) and (b): guaiacol peroxidase (GPX), in Calotropis procera.

Full size image

In shoots, GPX activity in Pantoea-treated plants decreased with increasing lead concentration (Fig. 5). At 20 mg/L lead, enzyme activity decreased by approximately 35% compared to the 0 mg/L condition. At 40 mg/L lead, activity increased by 20% compared to the 20 mg/L treatment, but at 60 mg/L lead, activity decreased again by about 25%. At 80 mg/L lead, enzyme activity increased slightly by 5% compared to the 60 mg/L treatment. In roots, there was no significant change in GPX activity at 20 mg/L lead compared to the 0 mg/L condition. At 40 mg/L lead, activity increased by approximately 20%, and at 60 mg/L lead, it rose by 15%. At 80 mg/L lead, enzyme activity increased by around 10% compared to the 60 mg/L treatment (Fig. 5).

Proline and total protein content

The effect of P. agglomerans on proline accumulation in C. procera under varying lead concentrations in a hydroponic system revealed significant trends (Fig. 6). Proline, a critical osmolyte, demonstrated an adaptive response to lead-induced stress, with notable enhancement in treated plants compared to untreated controls.

Fig. 6
figure 6

The effect of Pantoea agglomerans and lead on (a): Proline and (b): total protein concentration, in Calotropis procera.

Full size image

In shoots, proline levels in treated plants increased progressively with lead concentrations, reaching a peak at 60 mg/L lead, where treated plants exhibited approximately 6.4 μmol/g FW, compared to 5.1 μmol/g FW in controls (Fig. 6). Similarly, roots of treated plants displayed elevated proline content, particularly at 80 mg/L lead, where values reached 6.6 μmol/g FW, slightly lower than the control at 7.6 μmol/g FW.

Protein content in shoots and roots was analyzed across lead treatments. In shoots, treated plants consistently exhibited higher protein levels than controls, highlighting the endophyte’s protective role in maintaining metabolic functions under stress. While protein content declined with increasing lead concentrations in both groups, the reduction was less pronounced in treated plants (Fig. 6). For instance, at 80 mg/L lead, treated shoots maintained 3.14 mg/g FW compared to 2.89 mg/g FW in controls. In roots, a similar trend was observed. Treated plants showed relatively stable protein levels, even at high lead concentrations (Fig. 6).

Pearson correlation matrix

The growth parameters demonstrated that shoot length exhibited significant positive correlations with shoot dry weight (r = 0.79), number of green leaves (r = 0.78), and photosynthetic efficiency (PSII; r = 0.77), indicating that Pantoea inoculation effectively mitigated lead-induced stress and enhanced shoot performance (Fig. 7). Root dry weight was moderately correlated with shoot length (r = 0.59) and PSII (r = 0.54), highlighting the interdependence between root health and photosynthetic efficiency under stress conditions. Similarly, the number of green leaves was strongly correlated with shoot length (r = 0.78) and shoot dry weight (r = 0.83), further underscoring the role of Pantoea in sustaining plant vigor. Antioxidant enzyme activities, including proline content, were inversely correlated with shoot length (r =  − 0.65) and shoot dry weight (r =  − 0.66), reflecting their role as stress indicators (Fig. 7).

Fig. 7
figure 7

Heatmap of Pearson’s correlation coefficients among growth parameters, antioxidant enzymes, and stress markers in Calotropis procera with Pantoea agglomerans inoculation.

Full size image

MDA, a marker of lipid peroxidation, showed negative correlations with shoot dry weight (r =  − 0.74) and shoot length (r =  − 0.65), indicating reduced oxidative damage due to inoculation. Protein content in shoots correlated positively with shoot length (r = 0.66) and dry weight (r = 0.53), indicative of improved metabolic activity, consistent with the principal component analysis PCA plot (Fig. S5). GPX activity in roots and shoots showed moderate positive correlations with root dry weight (r = 0.52) and shoot length (r = 0.22), pointing to enzymatic contributions in oxidative stress mitigation. Leaf dimensions were positively correlated with shoot length (r = 0.74, r = 0.77) and PSII (r = 0.67, r = 0.56), indicating improved photosynthetic capacity and overall plant health with Pantoea inoculation.

The positive correlations between shoot length, shoot dry weight, and PSII efficiency highlight the role of Pantoea inoculation in mitigating lead-induced stress by enhancing photosynthetic efficiency and biomass accumulation. The improved performance may be attributed to the symbiotic relationship promoting nutrient uptake and stress resilience. The significant relationship between root dry weight, protein content, and antioxidant enzyme activity suggests that Pantoea plays a critical role in maintaining root metabolic activity, which is vital for sustaining overall plant growth under lead stress.

The accumulation of proline, as indicated by its negative correlation with growth parameters, aligns with its known function as an osmoprotectant during abiotic stress. However, the reduction in lipid peroxidation (low MDA levels) and its association with decreased oxidative damage suggest that Pantoea inoculation effectively mitigates lead toxicity through enhanced antioxidant activity. These relationships validate the hypothesis that Pantoea enhances both growth and stress tolerance, supporting the expected outcomes of improved phytoremediation capabilities. The observed reduction in lead-induced damage and improved physiological performance in P. agglomerans treated plants further suggests that similar strains may employ conserved genomic mechanisms, such as metal-resistance systems, to mitigate stress and promote plant resilience.

Elemental analysis of roots, stems, and leaves of C. procera

X-ray fluorescence (XRF) spectroscopy was used to analyze heavy metal accumulation in the roots, stems, and leaves of C. procera exposed to 80 mg/L lead, with and without P. agglomerans (Table 1). In the leaves, the addition of Pantoea resulted in increased concentrations of magnesium (16%), phosphorus (11%), sulfur (8%), and potassium (13%). Interestingly, lead concentration in the leaves with Pantoea was slightly higher (0.53%) compared to the control (0.15%), suggesting that Pantoea may contribute to lead accumulation in leaves under hydroponic conditions. In the shoots, the presence of Pantoea led to a significant increase in magnesium (7%) and potassium (17%) concentrations compared to the control, while calcium and phosphorus remained relatively unchanged.

Table 1 Macronutrient concentrations in roots, stems, and leaves in Calotropis procera.
Full size table

No detectable lead accumulation was found in the shoots in both treatments. In the roots, the addition of Pantoea resulted in a marked increase in magnesium (10%) and calcium (13%) levels, while lead concentration was reduced by approximately 50% in the roots with P. agglomerans compared to those without (Table 1).

Discussion

The isolation of P. agglomerans suggests its potential to enhance plant resistance to heavy metal stress. Previous studies have shown that Pantoea species improve metal tolerance in plants through biosorption and enzymatic detoxification25,36,40. Results from plant growth parameters indicate that Pantoea inoculation significantly influences the growth of C. procera seedlings, enhancing both shoot and root dry weights even under non-stress conditions, likely through mechanisms such as improved nutrient uptake or enhanced phytohormones production. Under lead stress, Pantoea treatment mitigated the negative effects of lead on plant growth. Despite the consistent decline in growth parameters with increasing lead concentration, inoculated plants showed less reduction in shoot and root dry weights compared to non-inoculated plants, suggesting a protective effect of Pantoea against lead-induced oxidative stress, improved nutrient uptake, or enhanced plant defense mechanisms36,40.

Pantoea-treated plants exhibited more green leaves and fewer yellow leaves, indicating delayed senescence and maintained chlorophyll content, especially under higher lead stress. This aligns with Jan et al.41 who reported that endophytes enhance photosynthetic efficiency by reducing chlorophyll degradation in heavy metal-stressed plants. Such a result also aligns with our hypothesis that Pantoea enhances lead uptake and reduces phytotoxic effects.

Lead disrupts photosynthesis by interfering with chlorophyll biosynthesis and reducing iron uptake, which is essential for pigment production. The decline in pigment levels under high lead stress in untreated plants aligns with previous findings that lead disrupts chlorophyll biosynthesis and reduces iron uptake, which is essential for pigment production. Lead also induces lipid peroxidation and interacts with sulfhydryl (-SH) groups of biosynthetic enzymes, further inhibiting pigment formation synthesis42. Similar mechanisms have been observed in maize (Zea mays L.) under copper stress43. Pantoea inoculation appears to counteract these effects by promoting nutrient uptake, lead sequestration, and stress tolerance mechanisms, leading to stabilized pigment content, particularly at higher lead concentrations. The significant interaction between lead and Pantoea treatments indicates the bacterium’s role in mitigating oxidative stress and preserving photosynthetic capacity. The higher levels of chlorophyll and carotenoids in Pantoea-treated plants suggest that the bacterium enhances antioxidant defenses, reducing oxidative damage to the photosynthesis apparatus. These findings align with previous research highlighting the positive influence of endophytic bacteria like Pantoea on photosynthetic pigment content under abiotic stress27,44,45. The findings also support the expected outcome of improved photosynthetic efficiency and plant health.

The decrease in FV/FM ratio under lead stress suggests that the photosynthetic machinery was disrupted or damaged. Stress conditions can cause significant damage to plant leaf tissue and chloroplasts in various ways46. Due to its high reactivity, chlorophyll released from damaged chloroplasts must be quickly degraded to prevent further cellular injury47. Our findings are consistent with the work of Akinci et al.48 and Amanifar et al.49, who reported significantly reduced chlorophyll levels in tomato plants exposed to lead. However, Pantoea treatment improved the FV/FM ratio, suggesting its role in maintaining photosynthetic efficiency, possibly through enhanced nutrient uptake and oxidative stress mitigation.

The increase in MDA content in shoots reflects significant lipid peroxidation and cellular damage caused by lead exposure. Although Pantoea inoculation enhanced lead uptake, it did not fully mitigate oxidative stress at higher lead concentrations. The lower MDA levels observed in roots suggest that the protective effect of Pantoea was more pronounced in root tissues, possibly due to enhanced antioxidant activity. Hydrogen peroxide levels in treated plants were lower than controls at moderate lead concentrations (20 mg/L), indicating that Pantoea helped reduce oxidative stress through mechanisms like scavenging reactive oxygen species. However, at higher lead concentrations, hydrogen peroxide levels in treated shoots increased again, indicating that the protective effect of Pantoea might be overwhelmed under severe stress conditions. These results suggest that while Pantoea species can mitigate oxidative stress to some extent, particularly at moderate lead concentrations, their efficacy diminishes as lead levels increase. This highlights the importance of optimizing conditions for Pantoea-mediated phytoremediation, potentially through complementary antioxidants or other stress-modulating agents50. The differential responses between shoots and roots further underscore the complexity of plant–microbe interactions under heavy metal stress, supporting the expected outcomes that Pantoea enhances stress tolerance and mitigates lead toxicity. This is consistent with findings that Pantoea dispersa significantly reduces oxidative stress markers in plants under abiotic stress, such as salinity, by promoting the accumulation of key antioxidants like ascorbic acid and glutathione while reducing membrane damage and ion toxicity51.

Antioxidant enzymes play a crucial role in mitigating oxidative stress under heavy metal exposure52. The decrease in CAT activity in both treated and untreated plants at elevated lead concentrations indicates heightened oxidative stress, as lead disrupts the balance between reactive oxygen species production and neutralization53. Pantoea treatment, however, appeared to enhance the activity of antioxidant enzymes in the plants, particularly at lower lead concentrations, suggesting a potential benefit of endophyte inoculation in combating lead-induced toxicity. Similar findings have been reported for other heavy metals such as zinc and cadmium, where endophytes enhanced the antioxidant response of plants45,54. The mechanisms underlying this enhancement remain unclear, but it is hypothesized that traits of the Pantoea endophyte, such as antioxidant enzyme production and siderophore secretion, may contribute to this observed effect.

Proline accumulation is a well-known adaptive response to stress, functioning as an osmoprotectant that stabilizes proteins and membranes while mitigating oxidative damage. The disparity in proline accumulation between shoots and roots likely reflects differential tissue-specific responses and metabolic activity under lead stress. Proline accumulation is known to mitigate oxidative stress by stabilizing proteins and membranes, and its elevated levels in treated plants suggest a more robust defense mechanism induced by endophytic colonization. The higher proline levels in treated plants at lower lead concentrations indicate an early adaptive response facilitated by Pantoea, which may involve enhanced nutrient acquisition or modulation of stress-responsive genes. At higher lead concentrations, proline content in treated plants remained competitive with controls, suggesting that Pantoea played a critical role in maintaining homeostasis and mitigating lead toxicity. The interaction between Pantoea and C. procera may involve a combination of direct and indirect mechanisms, including the synthesis of phytohormones, improved nutrient assimilation, and activation of antioxidative pathways. Enhanced proline biosynthesis in treated plants confirms the potential of Pantoea and C. procera as a bioaugmentation agent for phytoremediation applications.

Protein content stability in treated plants further supports the role of Pantoea in mitigating lead-induced stress. The smaller reduction in protein levels under increasing lead concentrations indicates that Pantoea alleviates oxidative damage, by enhancing antioxidant enzyme activity and improving nutrient assimilation. The observed stability in protein content suggests that Pantoea helps maintain metabolic homeostasis, making it a promising candidate for phytoremediation strategies. Similar trends have been reported in other studies, where endophytic bacteria contributed to protein retention in plants under heavy metal stress33,34. These indicates Pantoea enhances stress tolerance mechanisms in C. procera, leading to improved metabolic stability under lead stress. Building on the observed correlations in the results, the positive relationships between shoot length, shoot dry weight, and PSII efficiency further emphasize the role of Pantoea inoculation in mitigating lead-induced stress by enhancing photosynthetic efficiency and biomass accumulation.

The improved performance may be attributed to the symbiotic relationship promoting nutrient uptake and stress resilience. The significant relationship between root dry weight, protein content, and antioxidant enzyme activity suggests that Pantoea plays a critical role in maintaining root metabolic activity, which is vital for sustaining overall plant growth under lead stress. The accumulation of proline, as indicated by its negative correlation with growth parameters, aligns with its known function as an osmoprotectant during abiotic stress. However, the reduction in lipid peroxidation (low MDA levels) and its association with decreased oxidative damage suggest that Pantoea inoculation effectively mitigates lead toxicity through enhanced antioxidant activity. These relationships validate the hypothesis that Pantoea enhances both growth and stress tolerance, supporting the expected outcomes of improved phytoremediation capabilities. The proposed mechanism is illustrated in Fig. 8.

Fig. 8
figure 8

Proposed mechanisms of lead detoxification and growth enhancement in Calotropis procera inoculated with Pantoea agglomerans.

Full size image

Furthermore, the origin of this P. agglomerans isolate from P. juliflora seeds adds ecological significance to its functional potential. P. juliflora is a highly resilient species that thrives in arid and saline environments, and its success is partly due to its association with stress-adapted endophytic microbiota55. Endophytes from Prosopis sp. have been shown to contribute to drought, salinity, and temperature stress tolerance in host plants through diverse mechanisms, including phytohormone modulation, osmoprotectant production, and improved nutrient acquisition56. To the best of our knowledge, there are no prior reports of P. agglomerans isolated from P. juliflora, emphasizing the novelty of this strain as well as its phytoremediation properties.

In addition to physiological and biochemical traits, the genomic features of Pantoea strains further support their role in heavy metal resistance. Previous studies have reported the presence of efflux systems, such as MdtABC-TolC and the CusA transporter, in Pantoea OB49, which are implicated in resistance to metals including lead, copper, and cadmium31. These transporters, part of the resistance-nodulation-cell division family, function in the active export of toxic ions from the cytoplasm, thereby contributing to cellular detoxification and survival under metal stress57. The co-existence of such systems with arsenite/antimonite antiporters and ATP-binding cassette transporters in related strains suggests that P. agglomerans may possess a similar genomic repertoire that facilitates heavy metal tolerance36. These insights provide a mechanistic basis for the observed reduction in lead toxicity and enhanced stress tolerance in inoculated C. procera plants. They indicate the potential of future genome-level investigations to elucidate the resistance pathways.

Previous research suggests that microbial-assisted phytoremediation can improve metal stabilization and reduce toxicity over time58,59,60. The presence of P. agglomerans in the hydroponic system positively influenced the ability of C. procera to manage lead uptake and distribution. Plants treated with Pantoea exhibited lower lead concentrations in their root tissues, suggesting that these endophytic bacteria reduce the bioavailability of lead, possibly through immobilization or transformation mechanisms. This enhancement in phytoremediation efficiency is accompanied by altered elemental composition in plant tissues, including increased levels of sulfur and magnesium in leaves and shoots. These changes indicate improved nutrient uptake, detoxification processes, and physiological stress tolerance in the plant.

Previous studies D’Souza et al.32; Kanwar et al.33; Usman et al.34; Almehdi et al.35; and Lottermoser61, have similarly highlighted the potential of C. procera as a phytoremediator of toxic metals such as cadmium and lead. The observed differences in element content between treated and untreated plants underscore the crucial role of Pantoea in modulating plant metabolism under metal stress, aligning with prior research on the influence of endophytic bacteria in enhancing plant growth and heavy metal uptake14,26.

Materials and methods

Isolation, screening, and molecular identification of endophytes

Seeds of P. juliflora were selected for isolating endophytic bacteria. To ensure sterility and prevent external microbial contamination, seeds underwent a thorough surface sterilization process. Initially, seeds were immersed in 70% ethanol for 1 min to remove surface contaminants, followed by treatment with 2.5% sodium hypochlorite for 10 min to eliminate residual microorganisms. After sterilization, seeds were rinsed multiple times with sterile distilled water to remove any traces of chemical agents. The outer seed coat was removed, and sterilized tissues were aseptically placed on Luria Bertani (LB) (Lab M Limited, Heywood, UK) agar plates and incubated at 37 ± 2°C for 7–10 days. Emerging bacterial colonies were characterized by morphology (shape, size, color), Gram staining, and microscopic examination. Identification was performed using the VITEK®2 Compact microbial identification system. Identified strains were preserved in 50% glycerol and stored at − 80°C.

Lead tolerance of the isolated strains was assessed using the well diffusion method with lead concentrations of 0, 20, 40, 60, and 80 mg/L. A bacterial inoculum was prepared to a 1.0–2.0 × 108 CFU/mL concentration using the McFarland 0.5 standard and spread evenly on Mueller–Hinton agar (Lab M) plates. Wells were aseptically created, and 20 µL of each lead concentration was added to the wells. The plates were incubated, and bacterial growth around the wells was monitored to assess lead tolerance.

For molecular identification, DNA was extracted from lead-tolerant strains using the ISOLATE II Genomic DNA Kit and quantified with a Nanodrop 2000. Polymerase chain reaction (PCR) amplification of the 16S rRNA region was performed with 60 ng/µL of DNA. PCR products were purified with the USB ExoSAP-IT Kit and sequenced using the Sanger method. Sequences were compared to the National Center for Biotechnology Information (NCBI) database using BLAST for species identification.

Germination of C. procera seeds

C. procera seeds were collected from a natural population near Cairo, Egypt. To prepare the seeds for germination, they were surface sterilized by soaking in 10% bleach for 5 min, followed by thorough rinsing with sterile distilled water. For stratification, seeds were submerged in 100 mL of sterile distilled water for 48 h, then transferred to a moist cotton cloth. The seeds were incubated in a Conviron growth chamber (CMP6010, Pembina, North Dakota, USA) at 30°C/20°C (12-h light/dark cycle) with 70–80% humidity for 5 days.

After germination, 190 seeds were inoculated with a freshly prepared bacterial suspension (treatment group), while another 190 seeds were treated with sterile distilled water as a control. Both groups were placed on moist cotton cloths in the growth chamber for 6 days, with moisture adjusted every 24 h. After 6 days, radicle lengths of the germinated seeds were measured.

Hydroponic cultivation and bacterial colonization

Seedlings from both control and treatment groups were prepared for hydroponic cultivation after radicle length measurement. To facilitate bacterial colonization, radicle tips were cut diagonally under sterile conditions, and seedlings were immersed in the bacterial suspension for 2 h. Treated seedlings were then transferred to a hydroponic setup consisting of sink strainers placed in beakers containing 10% Hoagland’s No. 2 basal salt mixture (Sigma-Aldrich Biochemie GmbH, Hamburg, Germany).

Each beaker held five seedlings with similar radicle lengths. For the treatment group, 1 mL of freshly prepared bacterial suspension was added to the hydroponic medium every 2 days. The system was maintained in a controlled growth chamber under the same light and temperature conditions used during germination. When signs of nutrient deficiency, such as yellowing leaves, were observed, the solution was replaced with a 20% Hoagland’s No. 2 mixture. Hydroponic media were stirred daily to ensure aeration, and solution levels were replenished as needed.

Endophytic colonization of plants was confirmed through bacterial isolation and identification. After 23 days of growth, representative plants from both control and treatment groups were harvested. Roots, shoots, and leaves were aseptically dissected into small pieces and plated on nutrient agar. Plates were incubated at 37°C for 24 h, and emerging bacterial colonies were examined for morphological characteristics. Colonies resembling Pantoea were isolated, purified via streak plating, and incubated under the same conditions. Identification of the isolated colonies was performed using the VITEK®2 microbial identification system, confirming successful colonization by the bacterial endophyte.

For light microscopy, roots of C. procera (3-week-old seedlings) inoculated with P. agglomerans were fixed in 2% glutaraldehyde in 0.17 M phosphate buffer (pH 7.2) under vacuum for 24 h at 25°C, followed by four washing cycles in the same buffer. Samples underwent post-fixation in 1% osmium tetroxide in 0.17 M phosphate buffer for 2 h, followed by rinsing with distilled water, dehydration using ethanol solutions, embedding in epoxy resin (Epon 812, Agar Scientific, UK), and polymerization for 24 h at 60°C. Semi-thin transverse slices (0.5 µm) were prepared and stained with 0.1% toluidine blue for examination under a light microscope. Sections were examined utilizing an Olympus BH-2 microscope (Olympus Optical Co. Ltd, Japan).

Exposure to lead and bacterial treatment

After 30 days of hydroponic cultivation, both treatment and control groups were exposed to varying concentrations of lead acetate (0, 20, 40, 60, and 80 mg/L) prepared in a 20% Hoagland’s No. 2 basal salt mixture. The experiment included six replicates per concentration, with each replicate consisting of five plants. Solution levels were monitored and maintained every two days, and fresh hydroponic media were provided weekly. The treatment group received 1 mL of freshly prepared bacterial suspension every 2 days.

Growth and morphological parameters

Plants were harvested to evaluate the effects of lead on growth and morphological characteristics after exposure to varying lead concentrations. Measured parameters included the longest shoot length (cm), shoot and root dry weight, total number of green leaves per plant, number of yellow leaves per plant, and the largest leaf length.

Plant pigment content

Plant pigments were quantified using a spectrophotometric approach with a microplate reader (BioTek Instruments, EPOCH2C, Vermont, USA). Ground leaf samples were mixed with 4 mL of cooled methanol and incubated at 4°C for 30 min in the dark. Samples were centrifuged at 21,000 x g for 5 min, and 200 µL of the supernatant was used to measure absorbances at 470, 653, and 666 nm for chlorophyll a, chlorophyll b, and carotenoids, respectively62.

Chlorophyll fluorescence analysis

To assess the photosynthetic performance of C. procera under lead stress, chlorophyll fluorescence parameters FV/FM and PSII efficiency were measured. Measurements were conducted using a pulse-modulated fluorescence monitoring system (FMS-2, Hansatech Instruments Ltd., Norfolk, UK). Seedlings were dark-adapted for 30 min to ensure all PSII reaction centers were fully open. The maximum quantum efficiency of PSII (Fv/Fm) was calculated as the ratio of variable fluorescence (Fv) to maximum fluorescence (Fm), where Fv = Fm − Fo, and Fo represents the minimum fluorescence after dark adaptation. PSII efficiency was measured under actinic light to stimulate photosynthesis, providing a real-time assessment of PSII performance under ambient conditions. All measurements were performed on fully expanded leaves, with data collected in triplicate for accuracy and reproducibility.

Estimation of hydrogen peroxide content and lipid peroxidation level

Hydrogen peroxide content was determined using a method adapted from Velikova et al.63. Shoot and root samples were ground in 0.1% trichloroacetic acid on ice. The homogenate was centrifuged, and 0.5 mL of the supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide. Absorbance was measured at 390 nm using a BioTek EPOCH2C microplate reader.

Lipid peroxidation, indicated by MDA production, was measured following Zhou and Leul64. Shoot and root samples were homogenized in 0.1% trichloroacetic acid (TCA) and centrifuged. The supernatant was mixed with an equal volume of TCA containing 0.5% thiobarbituric acid (TBA), heated at 95°C for 30 min, and then rapidly cooled on ice. The mixture was centrifuged at 12,000 x g for 15 min at 4°C to pellet any TBA precipitate. Absorbance of the supernatant was measured at 532 nm and 600 nm using a BioTek EPOCH2C microplate reader. MDA content was calculated and expressed as nmol/g FW.

Determination of antioxidant enzyme activities

CAT activity was determined by measuring the decrease in hydrogen peroxide absorbance at 240 nm65,66. The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.0) and 10.5 mM hydrogen peroxide. Enzyme extract was incubated with the reaction mixture at 25°C, and the decrease in absorbance at 240 nm was recorded.

GPX activity was measured in a 96-well plate. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 2 mM hydrogen peroxide, and 2.7 mM guaiacol. The formation of tetraguaiacol was monitored by an increase in absorbance at 470 nm65.

Determination of total protein and proline content

Total protein concentration in enzyme extracts was quantified using the Bradford method67. Proline content in shoot and root tissues was determined following the protocol by Bates et al.68. For analysis, 0.1 g of cryogenically ground tissue was homogenized in 3% sulfosalicylic acid and centrifuged. A 100 μL aliquot of the supernatant was mixed with 200 μL glacial acetic acid and 200 μL acid-ninhydrin, then incubated at 96°C for 1 h. After adding 1 mL toluene and vortexing, the toluene phase was transferred, and absorbance was measured at 520 nm. Proline content was determined using a standard curve of pure proline (Sigma-Aldrich) and expressed on a fresh weight basis.

XRF spectroscopy analysis of heavy metals in C. procera organs

XRF spectroscopy (Horiba XGT 7200, Kyoto, Japan) was used to analyze heavy metals in the roots, stems, and leaves of C. procera. The instrument, equipped with a 50-W rhodium X-ray tube, operated at three voltage levels with manual or auto current settings. A 50-μm X-ray beam at 50 kV and auto current mode was employed. Plant organs were crushed, milled, and placed in plastic sample holders sealed with X-ray transparent film. Micro-sized X-ray beams were focused on the sample, and emitted photons were detected by a silicon drift detector positioned at 45°. X-ray fluorescence spectra were collected from five points per sample with a 300-μA tube current and 1000-s analysis time per point. Element concentrations were calculated using Fundamental Parameter software and expressed as relative percentages of total element concentrations.

Statistical analysis

Two-way ANOVAs with Bonferroni post-hoc tests were used to assess the impact of Pantoea application on morphological parameters, photosynthetic pigments, and chlorophyll fluorescence. A three-way ANOVA with Tukey’s test was employed to evaluate the effects on hydrogen peroxide, MDA, antioxidant enzymes, total protein, and proline content. These statistical approaches are widely applied and validated in various phytoremediation studies69,70,71. In addition, Pearson’s correlations were calculated to assess relationships between variables. To explore multivariate patterns and treatment effects, PCA was performed, and the results were displayed in a biplot. All statistical analyses were performed using OriginPro 2025.

Conclusions

The present study demonstrates that P. agglomerans significantly alleviates lead stress in C. procera through multiple complementary mechanisms. Inoculated plants exhibited higher shoot and root biomass and healthier leaves, highlighting the bacterium’s role in promoting plant growth under both non-stress conditions and elevated lead concentrations. Treated plants maintained greater chlorophyll and carotenoid contents, alongside higher Fv/Fm and PSII efficiency, indicating Pantoea’s capacity to mitigate the detrimental effects of lead on the photosynthetic apparatus. Moreover, Pantoea inoculation led to lower levels of hydrogen peroxide and MDA in C. procera plants, signifying reduced oxidative stress. Enhanced activities of antioxidant enzymes, such as CAT and GPX, further confirm P. agglomerans’ role in strengthening C. procera’s defense mechanisms against lead-induced toxicity. In addition, elevated proline and total protein levels in inoculated plants reflect improved osmotic adjustment and metabolic homeostasis, suggesting a more robust resilience to lead-induced stress. Elemental analysis via XRF revealed that Pantoea inoculation influenced both lead distribution and nutrient profiles in plant tissues, pointing to possible lead sequestration or detoxification pathways. Overall, these findings underscore the promise of P. agglomerans as a bioinoculant in C. procera, contributing to improved growth, photosynthetic performance, and stress tolerance under lead exposure. Endophyte-assisted phytoremediation thus emerges as a sustainable, cost-effective approach for mitigating heavy metal contamination, offering the dual benefits of environmental remediation and plant biomass production.

This study highlights the role of P. agglomerans in alleviating lead-induced toxicity in C. procera. Future research should quantify lead bioaccumulation and translocation using inductively coupled plasma mass spectrometry (ICP-MS) and investigate the genetic mechanisms behind metal detoxification. Since this work was conducted in a hydroponic system, field-scale studies are needed to evaluate real-world applicability and ecological impact. Long-term studies, including microbial community profiling and metagenomic analysis, will be important for assessing the sustainability of microbial-assisted phytoremediation. Future work should also consider formulation and delivery strategies for P. agglomerans and explore synergistic interactions with other beneficial microorganisms like mycorrhizal fungi or other endophytes to enhance efficiency across varied environmental conditions.