Plant growth promoting rhizobacteria modulates the antioxidant defense and the expression of stress-responsive genes providing Pb accumulation and tolerance of grass pea

To ensure the success of phytoremediation, it is important to consider the appropriate combination of plants and microorganisms. This study was conducted to get a better insight into the underlying molecular and biochemical mechanism of grass pea (Lathyrus sativus L.) induced by plant growth promoting rhizobacteria (PGPR), when exposed for 3, 6, 9, and 14 days to 1 mM Pb in a hydroponic system. The significant positive effect of bacterial inoculation was reproduced in various parameters. Results indicated that inoculation of PGPR significantly increased the accumulation of Pb by 20%, 66%, 43%, and 36% in roots and by 46%, 55%, 37%, and 46% in shoots, respectively after 3, 6, 9, and 14 days of metal exposure compared to the uninoculated plants. The metal accumulation in grass pea plants triggered a significant elevation in the synthesis of non-protein thiols (NPT), particularly in inoculated plant leaves where it was about 3 and 2-fold higher than the uninoculated set on the 6th and the 9th day. Nevertheless, Pb treatment significantly increased oxidative stress and membrane damage in leaves with the highest hydrogen peroxide (H2O2) production and tissue malondialdehyde (MDA) concentration recorded in uninoculated plants. Furthermore, the PGPR inoculation alleviated the oxidative stress, improved significantly plant tolerance, and modulated the activities of antioxidant enzymes (SOD, CAT, APX, GR, DHAR, and MDHAR). Similarly, the expression patterns of LsPCS, LsGCN, LsCNGC, LsGR, and LsGST through qRT-PCR demonstrated that bacterial inoculation significantly induced gene expression levels in leaves 6 days after Pb treatment, indicating that PGPR act as regulators of stress-responsive genes. The findings suggest the key role of PGPR (R. leguminosarum (M5) + Pseudomonas fluorescens (K23) + Luteibacter sp. + Variovorax sp.) in enhancing Pb accumulation, reducing metal toxicity, strengthening of the antioxidant system, and conferring higher Pb tolerance to grass pea plants. Hence, the association Lathyrus sativus-PGPR is an effective tool to achieve the goal of remediation of Pb contaminated sites.


Introduction
Toxic heavy metals have been discharged in the environment through industrial activity, mining, and modern agricultural practices; it has been accumulated in the soils and becoming a major threat to food security and a prominent apprehension for human welfare (Khanna et al. 2019;Moon et al. 2019). Hence, there is undoubtedly an urgent need to remove harmful elements from the soil and prevent their transmission across the food chain, although some of these metals act as vital nutrients for plant growth. Conversely, they can be damaging when they exceed their optimal limits, while others (e.g., cadmium (Cd), lead (Pb), arsenic (As), or mercury (Hg)) have no physiological role in plants and can be deleterious even at low concentrations (Wilkins et al. 2016). Lead is one of the most abundant and toxic heavy metals in the environment whose widespread anthropogenic use has caused global contamination of biotopes and biocoenosis (Pourrut et al. 2013). Extensive research has found that Pb hampers normal growth and metabolic processes of plants and causes a range of damages that leads to severe falls in some vital plant processes and reduces agricultural productivity. Moreover, one of the most well-known effects of Pb exposure is free radical generation leading to oxidative stress which seems to play a central role in Pb toxicity (Lopes et al. 2016).
Various ex situ and in situ remediation techniques have been engaged by scientists to reduce the levels of contaminants in the soil and restore the ecosystem, but in situ phytoremediation approach remains one of the best alternatives to solve this issue. Indeed, phytoremediation is a environmentally friendly, cost-effective, and sustainable solution that provides a promising tool to clean up and revegetate heavy metal polluted sites with the restoration of the soil properties and plant health (Amin et al. 2018;Yan et al. 2020). Nonetheless, the phytoremediation process requires necessary adaptive measures. In metal-rich soils, to cope with metal toxicity, plants have developed certain efficient strategies at physiological, biochemical, and molecular levels to adapt and defend themselves (Devi et al. 2017;Yan et al. 2020). Furthermore, metal chelation, the synthesis of metal-binding proteins, exclusion, compartmentalization, and complexation are common among defense strategies evidenced in plants under metal stress (Anjum et al. 2015).
Various plant species have been successful in absorbing contaminants and considered potentially effective for remediation of metal contaminated area. Based on plants' ability to uptake heavy metals and metabolize them within tissues, phytoremediation approach has been classified into: phytostabilization, phytoextraction, phytovolatilization, rhizofiltration, phytodegradation, and rhizodegradation (Rai et al. 2021). However, high levels of metals in soil could reduce the effectiveness of remediation as it decreased plant growth and biomass due to the low nutrient content and biological activity of polluted soils, although this constraint can be overcome by leveraging ecological relationships between soil, microorganisms, and plants (He et al. 2020).
Recent evidence emphasizes that phytoremediation success is strongly dependent on plant-microorganisms interaction (Cicatelli et al. 2019). In fact, such cohesive interactions play a vital role in adapting to metalliferous environments besides the ability of metal resistant bacteria to immobilize, mobilize, or transform metals in soil as well as their capacity to extend the functional potential of the host plant (Ma et al. 2016;Gulzar and Mazumder 2022;Vocciante et al. 2022). Moreover, microorganisms are a key soil component that play an important role in maintaining the soil quality and fertility. Particulary, rhizospheric microbes have been shown a great help in protecting plants against heavy metal stress, promoting plant growth as well as in increasing their ability for metals uptake and accumulation (Abdelkrim et al. 2018c;Chiboub et al. 2018;He et al. 2020;Kurniawan et al. 2022). Likewise, it is noteworthy that plant growth promoting rhizobacteria (PGPR) inoculation reduce oxidative stress via scavenging over-produced reactive oxygen species (ROS), contribute towards the protection of the photosynthetic apparatus, and enhance the antioxidant system of the host plant thereby alleviating heavy metal stress (Abdelkrim et al. 2018a;Jebara et al. 2019;Chiboub et al. 2020;Shah et al. 2020).
On the other hand, it is argued that under heavy metal stress, plants are able to reprogram of the expression of various genes in stress-responsive pathways with specific expression patterns to counteract the toxicity of these elements. Nonetheless, stress-responsive genes still represent an important aspect of stress adaptation (Klsa 2017). A large number of genes and gene families have been identified in plants, which are playing key a role in metal uptake, transport, tolerance, and detoxification (Brunet et al. 2009;Wang et al. 2013;Manoj et al. 2020;Feng et al. 2021). However, there is very little research highlighting the role of PGPR inoculation for enhanced tolerance and accumulation of heavy metals in plants by altering the expression pattern of related genes, which play a predominant role in metal stress response. Therefore, to improve phytoremediation efficiency, an enhanced understanding of the plant and bacterial interactions and responses to contaminants are essential.
In our recent studies, grass pea is viewed as a potential candidate for removal of lead from contaminated sites (Abdelkrim et al. 2019). Furthermore, among a collection of efficient and Pb resistant bacteria isolated from root nodules of grass pea plants grown in heavy metals contaminated soils, four PGPR characterized as R. leguminosarum (M5), P. fluorescens (K23), Luteibacter sp., and Variovorax sp. were selected, mixed, and used as inoculum (Abdelkrim et al. 2019). After in vitro, greenhouse, and field experiments, the PGPR showed their ability to produce plant growth promoting substances and promoted the adaptability of grass pea to Pb-polluted soils, enhanced its phytoremediation potential, and protected the host plant from Pb toxicity (Abdelkrim et al. 2018b(Abdelkrim et al. , c, 2020. However, the molecular mechanism responsible for Pb uptake, tolerance, and detoxification in grass pea guided by PGPR inoculation is still poorly understood. Within this context, this study aims to provide a better understanding of the biochemical and molecular strategies whereby PGPR alleviates heavy metal toxicity, confers Pb tolerance, and promotes a higher metal accumulation in grass pea plants. Thus, (i) we analyzed the amount of Pb accumulated in inoculated and uninoculated plants; (ii) we studied the role of PGPR inoculation in cell 1 3 protection and acquisition of Pb tolerance; (iii) we explored the expression analysis of five selected genes (LsPCS, LsGCN, LsCNGC, LsGR, and LsGST) in the leaves and roots of grass pea exposed to 1 mM Pb in a hydroponic system.

Biological material and treatment
Seeds of grass pea obtained from Mahdia, Tunisia, were surface sterilized and germinated as previously described by Abdelkrim et al. (2018b). Afterward, germinated seeds were transferred into sterile perlite and inoculated with I5 consortium (R. leguminosarum (M5)+ P. fluorescens (K23)+ Luteibacter sp.+ Variovorax sp.) formed by mixing efficient and Pb-resistant bacterial strains (Abdelkrim et al. 2018b) ( Table 1 SM). In fact, each bacterial strain was cultured individually in YEM medium (Vincent 1970) for 48h at 28 °C under continuous stirring at 150 rpm to obtain a final concentration of 10 9 CFU ml -1 . For the co-inoculation treatment, the bacterial cultures of strains grown individually were mixed and added to the seeds in perlite. One week later, equal sized uniform seedlings were carefully transferred into sterilized plastic pots containing a nutrient solution (modified from Vadez et al. 1996) and the seedlings were then re-inoculated. The pots were left in a completely randomized block design in a greenhouse at 25 °C/19 °C (day/night), a 16 h light/8 h dark photoperiod, a relative humidity of 60%, and the nutrient solution was aerated with airflow. Lead treatment (concentration of 1 mM Pb) was applied at flowering stage (60 days after germination, which coincides with the optimum of functional activities of nodules (Faghire et al. 2013)) and plants were collected after 3, 6, 9, and 14 days for the subsequent experimental analyses. Pb was added as lead chloride (PbCl 2 ).
Roots and shoots were rinsed separately with deionized water and then divided into two portions: the first one was immediately frozen in liquid nitrogen, and stored at −80 °C; the second portion was dried at 70 °C for 72 h and ground to a fine powder to determine Pb accumulation in different plant tissues.

Metal accumulation in plants
Root and shoot samples (0.1 g of dry plant material) were digested with a nitric-perchloric acid mixture (HNO 3 / HClO 4 = 4/1 (v/v)) at 100 °C. After total evaporation, 0.5% HNO 3 was added and the mixture was filtered using Whatman filter paper (Zaier et al. 2010), and Pb concentration was determined using ICP-AES (Perkin Elmer).

Estimation of hydrogen peroxide and malondialdehyde levels
Level of H 2 O 2 in leaves was estimated following the method of Velikova et al. (2000). Leaf tissues (0.1 g) were homogenized in ice bath with 0.1% (w/v) trichloroacetic acid (TCA) and the mixture was centrifuged at 10000 rpm for 15 min. Then, 0.5 ml of 10 mM potassium phosphate buffer and 1 ml of 1 M KI were added to 0.5 ml of the supernatant. The absorbance was measured at 390 nm and H 2 O 2 content was quantified using a standard curve and expressed as μmol g -1 FW.
Lipid peroxidation was estimated by the level of malondialdehyde (MDA) production as described by Heath and Packer (1968). Fresh leaf samples (0.5 g) were homogenized in 5 ml of 0.1% TCA solution and centrifuged at 12000 rpm for 15 min. To 1 ml of the supernatant, 4 ml of 0.5% thiobarbituric acid (TBA) in 20% TCA was added and heated at 95 °C for 30 min before cooled in an ice-bath. After centrifugation for 10 min, the absorbance of the supernatant was recorded at 532 nm and 600 nm. The malondialdehyde (MDA) concentration was expressed in nmol g -1 FW and calculated according to the following equation: MDA equivalents (nmol ml −-1 ) = [(A 532 -A 600 )/155000]10 6 .
Homogenates were centrifuged at 12000 rpm for 20 min, and the supernatant was used for protein and enzyme determination. Protein concentration was measured according to the method of Bradford (1976), using bovine serum albumin as standard. For the analysis of ascorbate peroxidase (APX) 1 3 activity, the sample was extracted in the buffer supplemented with 5 mM ascorbate.

Total RNA extraction, cDNA synthesis, and quantitative real-time PCR analysis
Total RNA was extracted from the leaves and roots tissue (0.2 g) using a CTAB method (Chang et al. 1993). A total of 750 μl of extraction buffer (100 mM Tris-HCl pH 8; 25 mM EDTA pH 8; 2 M NaCl, 2% CTAB, 2% PVP) and 15 μl of β-mercaptoethanol were added to 200 mg of very thin powder sample. The mixture was incubated at 65 °C for 30 min, and was homogenized by vortexing 2-3 times during incubation. Then, an equal volume of chloroform/isoamyl alcohol (24:1) was added, vortexed, and centrifuged at 10000 rpm for 10 min at room temperature. The supernatant was collected and transferred to a new tube containing 1/4 volume of LiCl (10 M) and incubated overnight at 4 °C for total RNA precipitation. Next, the samples were centrifuged at 13800 rpm for 30 min at 4 °C. The pellets were resuspended in 200 μl of DEPC-treated water followed by adding of 600 μl of absolute ethanol and 100 μl of 3 M NaAc (pH 5.2) and further incubation at −80 °C for at least 30 min. The total RNA was recovered by centrifugation at 13800 rpm for 30 min at 4 °C. The RNA pellets were washed with 100% ethanol (500 μl), centrifuged during 5 min at 13800 rpm, dried on air, and finally resuspended in 50 μl of DEPC-treated water. Total RNA was quantified using micro-spectrophotometry (NanoDrop Technologies, Inc.). Then, RNA integrity and possible DNA contamination were checked by agarose gel electrophoresis (1.5%). Before cDNA synthesis, the total RNA samples were treated with 5 U of RNase-free DNase I (Thermo Fisher Scientific) for 30 min at 37 °C. cDNA was synthesized from 5 μg of total RNA using 200 U of RevertAid M-MuLV reverse transcriptase (Biomatik; Wilmington, Delaware, USA) as described by the manufacturer.
Each reaction mix was performed in 25 μl containing 5 μl of cDNA (100 ng), 12.5 μl of Maxima SYBR Green/  ROX qPCR Master Mix (2x) (Biomatik), and 0.3 μM of each gene-specific primer. The reaction mix was subjected to the following program: initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing/elongation at 60 °C for 1 min. The specificity of the PCR amplification was verified with a melt curve analysis (from 65 to 95 °C) following the final cycle of the PCR. Each RT-qPCR was replicated three times and the mean was used for RT-qPCR analysis. The gene coding for β-tubulin was used as a reference gene to normalise the data. The relative expression levels were calculated according to the comparative 2 -∆Ct method was used to analyze qRT-PCR data

Statistical analysis
Statistical analyses were performed using a one-way analysis of variance (ANOVA), followed by Tukey's HSD (p < 0.05) test using the statistical software SPSS. Data are reported as mean ± SD (standard deviation).

Pb accumulation and its translocation
In the time-course experiment, the accumulation of lead by plants was followed for 14 days. According to Fig. 1, the interaction between bacterial strains and grass pea exerted significant effects on lead accumulation in shoots and roots. In inoculated plants, Pb content increased significantly with increasing metal exposure; after 6 and 9 days in shoots and after 6 and 14 days in roots; in contrast, accumulation patterns differed in uninoculated plants, where metal concentration increased significantly only after 9 and 14 days in shoots and roots, respectively (Fig. 1). Nonetheless, after 3, 6, 9, and 14 days of metal exposure, Pb was accumulated at significantly higher concentrations in inoculated plants and was found to be enhanced by 20%, 66%, 43%, and 36%, in roots and by 46%, 55%, 37%, and 46% in shoots, respectively, compared to uninoculated plants. Furthermore, the treated plants showed that 6 days after metal exposure, roots accumulated more Pb than shoots, while from the 9 th day, day ratio of metal in plant shoots to roots presented remarkable increase leading to higher Pb accumulation in the aboveground part with the maximum occurring in inoculated plants on the 14 th day and was found to be 106.51 mg g -1 DW.

Non-protein thiols content
Non-protein thiols (NPT) content in L. satius roots and leaves is illustrated in Fig. 2a and b. An elevation in the accumulation of Pb in grass pea plants triggered a significant elevation in the synthesis of NPT after 6 and 9 days of metal exposure in leaves and after 3 days in roots. Moreover, it was noticed that PGPR inoculation further increased the NPT about 3-and 2-fold on the 6 th and 9 th day in leaves; and about 1.5-fold on 3 rd day in roots, greater than the uninoculated plants. Nonetheless, a decrease in non-protein thiols level was recorded after 14 days of metal exposure in leaves and after 6 days in roots but it was maintained significantly higher in inoculated plants by 61% and 86%, respectively, in comparison with uninoculated plants.

Hydrogen peroxide and malondialdehyde contents
Pb treatment significantly increased oxidative stress and membrane damage in leaves. The highest H 2 O 2 production (6.71± 0.46 μmol g -1 FW) and tissue MDA concentration (44.13 ± 3.37 nmol g -1 FW) were observed in uninoculated plants 9 and 14 days after metal exposure, respectively ( Fig. 2c and d). Nevertheless, inoculation alleviated the oxidative stress by reducing hydrogen peroxide and malondialdehyde contents. Indeed, PGPR inoculation significantly reduced H 2 O 2 level by 26%, 27%, and 27% at 6, 9, and 14 cultivation days, respectively, after Pb treatment compared with uninoculated plants. Furthermore, MDA production showed level significantly lower than uninoculated plants  Fig. 1 Lead accumulation (mg g −1 DW) in shoots (a) and roots (b) of uninoculated and PGPR-inoculated L. sativus treated with 1 mM Pb for 3, 6, 9, and 14 days. The values are means of three replicates. Means followed by a common lowercase letter are not significantly different at p < 0.05 according to Tukey's HSD test 1 3 by 25% and 30%, after 9 and 14 days of metal exposure, respectively.

Antioxidant enzymes
The change in the activities of SOD, CAT, GPOX, APX, MDHAR, DHAR, and GR in leaves of grass pea after adding 1 mM Pb to the nutrient solution is represented in Table 2.
Results revealed that superoxide dismutase and glutathione reductase activities increased with increasing exposure time and inoculation further causing a significant increase in their activities about 2-fold more than the uninoculated plants, after 14 days of metal treatment.
Catalase activity was inhibited under Pb stress after 3 days, slightly increased until 9 d and was significantly stimulated on the 14 th day. Likewise, it was 189% and 28% greater in inoculated plants than uninoculated ones, 6 and 14 days after Pb exposure.
GPOX activity was induced on the 3 rd day and was maintained at the same high level up to the 14 th day but did not show any significant difference between inoculated and uninoculated plants.
In the case of APX, enzyme activity was induced significantly on the 6 th day in response to Pb addition, while the highest level (934.52 ± 41.23 nmol ascorbate min -1 mg -1 protein) was achieved in the leaves of PGPR-inoculated plants which followed a significant increase on the 3 rd day where it was increased by 3.5-fold compared with uninoculated plants.
MDHAR activity was stimulated in uninoculated plants on the 3 rd day, maintaining the same level until the 6 th day and declined significantly at 9 and 14 days, whereas in inoculated plants, MDHAR activity continues to increase up to 6 days and followed the same trend of irreversible inhibition on the 9 th and the 14 th day. In addition, PGPR led to the increment in the activity of this enzyme (on the 3 rd and 6 th day) with the maximum activity registered on the 6 th day where it was enhanced about 2-fold than the uninoculated plants (Table 2).
Pb stress resulted in a considerable increase in DHAR activity on the 3 rd day followed by a decline on the 6 th day, an increase on the 9 th day, and another decrease on the 14 th day. Nevertheless, the greatest DHAR activity in leaves was recorded for inoculated plants after 6, 9, and 14 days of Pb exposure (1.59; 2.60; and 1.82 μmol AsA formed min -1 mg -1 protein, respectively).

Gene expression analysis
At leaf level, where large amounts of Pb were accumulated, a significant increase in the expression of the studied genes (LsPCS, LsGCN, LsCNGC, LsGR, and LsGST) was noted (Fig. 3). This induction being much greater in the leaves of the inoculated plants was detected 6 days after metal treatment. However, at the root level, a lower accumulation of LsCNGC, LsGR, LsGST, and LsPCS transcripts was found compared to leaf tissue, while their expression in the roots of the control plants was substantially enhanced by 2-, 3-, 5-, and 18-fold in PGPR-inoculated plants compared to those uninoculated.  Fig. 2 Effects of PGPR inoculation on non-protein thiol (NPT) content in leaves (a) and roots (b) of L. sativus as well as hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) accumulation at leaves level, before (control) and after 3, 6, 9, and 14 days of lead treatment (1 mM). Data are means of five replicates. Means followed by a common lowercase letter are not significantly different at p < 0.05 according to Tukey's HSD test On the other hand, Pb induced the accumulation of LsGCN transcripts, mainly in leaves of inoculated plants 6 days after metal exposure, where it was 3 fold higher than the uninoculated plants (Fig. 3a). In the roots, a significant decline in the expression of LsGCN was recorded but was maintained 40 times greater in inoculated plants at 9 days (Fig. 3b).
As shown in Fig. 3c, lead induced the expression of LsPCS gene in leaves on the 6 th and the 9 th day with the highest level registered after 6 days of Pb treatment in the inoculated plants (4-fold more than those uninoculated). However, metallic stress did not affect the expression of this gene in the roots of uninoculated plants against a decrease recorded in the inoculated ones, although the expression level of LsPCS was 9 times and 14 times higher than in uninoculated plants, respectively 9 and 14 days after metal exposure (Fig. 3d).
Results also revealed that the Pb treatment did not have any significant effect on the expression of the LsGR gene in uninoculated plants leaves (Fig. 3e) against an upregulation observed in those inoculated where the expression on the 6 th , 9 th , and 14 th day was respectively 8-, 7-, and 3-fold higher than that of uninoculated plants. At the root level, uninoculated plants treated with 1 mM Pb showed a significant increase in the expression of the LsGR gene after 6, 9, and 14 days of Pb treatment (Fig. 3f). By contrast, a modulation of the expression of this gene was noticed in PGPR-inoculated plants with the maximum relative expression registered 6 d after metal exposure.
Moreover, a significant accumulation of mRNA transcript of LsGST in leaves of PGPR-inoculated grass pea was emphasized 3 days after the metal treatment (3.5fold) compared to uninoculated plants (Fig. 3g). This stimulation continued for up to 6 days, even though the transcript expression did not reveal any significant differences between the plants studied after 6, 9, and 14 days of treatment. At the roots level, the transcript expression of LsGST was significantly increased in uninoculated plants under Pb stress on the 3 rd day, and was reduced on the 6 th , 9 th , and 14 th day; conversely, a downregulation was observed in inoculated plants (Fig. 3h).
Metallic stress induced the expression of LsCNGC after 3 days of treatment in uninoculated plants, although the expression level reached its maximum extent in inoculated plants at 6 days (Fig. 3i). However, RT-qPCR analysis showed no significant change in LsCNGC transcript expression in roots of all tested plants in response to Pb stress (Fig. 3j).

Effect of inoculation on shoot and root Pb concentrations
In the present study, the lead distribution in plant tissues was increased with with the rise of exposure duration, and high Pb concentrations in shoots and roots were recorded. Table 2 Activities of Superoxide dismutase (SOD, USOD mg −1 protein), catalase (CAT, μmol H 2 O 2 min −1 mg −1 protein), peroxidase (GPOX, μmol H 2 O 2 min −1 mg −1 protein), ascorbate peroxidase (APX, nmol ascorbate min −1 mg −1 protein), monodehydroascorbate reductase (MDHAR, μmol NADH oxidized min −1 mg −1 protein), dehydroascorbate reductase (DHAR, μmol AsA formed min -1 mg -1 protein), glutathione reductase (GR, nmol NADPH oxidized min −1 mg −1 protein) in leaves of the uninoculated (U), and inoculated grass pea plants (I) before (control) and after 3, 6, 9, and 14 days of Pb treatment Results are means (±SD) of five replicates. Within each line, means followed by a common lowercase letter are not significantly different at p < 0.05 according to Tukey's HSD test  Similarly, studies with elevated heavy metal concentrations in shoots and roots after long-term metal stress under hydroponic culture system were reported in Eichhornia crassipes under Pb stress (Malar et al. 2014), Petunia hybrida under Cd stress , and Solanum lycopersicum under Ni stress (Jahan et al. 2020). Indeed, the hydroponics system can provide an exact picture of the plant tolerance level for heavy metals due to the non-interference of micro and macro organisms, soil properties, and presence of other pollutants . Nevertheless, metal accumulation differs remarkably within plant species and in the tissues. Supplementation of bacterial strains leads to a statistically significant increase in the Pb concentrations in shoots and roots of grass pea plants. It seems likely that PGPR exhibits diverse effects on the levels of metal absorption by plants. This could possibly be due to a mechanism of action of PGPR on root functioning, maybe by stimulating the secretion of root exudates and other metabolites that form metal chelating complexes, therewith; their potential for absorption and accumulation by the plant is increased. In fact, the peculiar properties of PGPR are essential to support the plants used in phytoremediation (Vocciante et al. 2022).
The association of plant growth-promoting rhizobacteria with metal accumulator plants is considered to be an important component of phytoremediation technology (de Jing et al. 2007). Our findings are in accordance with previous studies in Brassica juncea (Ren et al. 2013), Salix integra (Niu et al. 2021), and Solanum nigrum (He et al. 2020), where Pb distributions in plant tissues were increased after bacterial inoculation. In contrast, a study conducted by Fang et al. (2020) demonstated that alfalfa when inoculated with S. meliloti reduced the Pb accumulation in plant organs.
The concentration of Pb was more in shoots compared to roots, which was associated with increased Pb translocation. In our previous study released with 0.5 mM Pb (30 d) and 1 mm Pb (24, 48, and 72 h) we found another response mechanism suggesting that plants' behavior depends on metal concentration in growth medium and exposure time. The translocation value greater than 1 indicated ability of plant to translocate metal from root to aboveground parts, via metal transporter system of plants (Anum et al. 2019). Furthermore, to regulate the concentration of metal ions inside the cell and to minimize the potential damage, plants initiate mechanisms to control metal retention, transportation, and homeostasis.

Effect of Pb treatments and PGPR inoculation on non-protein thiols
The present study revealed that non-protein thiols were increased in leaves and roots of grass pea under Pb stress. This fact is supported by increased levels of non-protein thiols in Sedum alfredii, Peganum harmalaa, and Brassica rapa in response to Pb exposure as reported in earlier studies (Gupta et al. 2010;Mahdavian et al. 2016;Akram et al. 2021). These metal chelators have a high affinity to bind to heavy metals therefore influencing their mobility, besides playing a crucial role as antioxidative metabolites and detoxicants in the plant defense mechanism against oxidative stress and imparts antioxidant properties to the plants (Gupta et al. 2020;Khanna et al. 2019). It has also been documented that tolerance of plants to heavy metals could be through chelating of metals to various thiol compounds in the cytosol and sequestering them into vacuoles (Yadav 2010). The present study also finds that PGPR inoculation stimulates the synthesis of NPT, and the highest levels were recorded in leaves after 9 d of metal exposure. The increment seems to be directly related to the increase of Pb concentration in shoots and may have played its part enhancing Pb uptake and accumulation and subsequent detoxification. In addition, it may also be suggested that enhancement in NPT concentration in inoculated plants would have further improved grass pea tolerance to Pb stress. Khanna et al. (2019) also observed that bacterial inoculation increased NPT content in Lycopersicon esculentum exposed to Cd stress.

Effect of Pb treatments and PGPR inoculation on MDA and H 2 O 2
In the present study, increased MDA and H 2 O 2 levels were registered upon the stress induced by lead. Indeed, plants are expected to undergo stressed condition when exposed to heavy metals that leads to an excess generation of reactive oxygen species which disrupts the cellular membrane integrity and alter the cell functions . The highest H 2 O 2 production was closely associated with a sharp rise in Pb concentration in the above ground part of plant after 9 days of Pb exposure followed by substantial increase in MDA level on the 14 th day, which suggests that the high Pb accumulation instigates excess production of ROS leading to a noticeable increase in membrane damage. Several researchers have documented that the production of ROS and MDA is commonly increased upon exposure to heavy metals (Al Mahmud et al. 2018;Dogan et al. 2018;Jahan et al. 2020). Nevertheless, cellular damages caused by lead stress were mitigated by PGPR inocultion, which is reflected in a noteworthy drop in H 2 O 2 and MDA contents. This mitigation effect, despite the high amount of metal accumulated by inoculated plants, is probably due to the elimination of free radicals through enzymatic and non-enzymatic antioxidant defense systems. These results are in agreement with the findings of Islam et al. (2014) who demonstrated that bacterial inoculation of metal-treated plants results in a considerable decline in H 2 O 2 and MDA levels.

Antioxidant enzyme activities
To restrict excessive production of ROS and duration of oxidative stress induced by toxic metal ions, plants are provided with a natural defense strategy including their non-enzymatic and enzymatic antioxidant systems that are activated according to specifc response mecanisms depeding on heavy metal concentrations and plant species (Hasan et al. 2017;Jahan et al. 2020). In this context, antioxidant enzymes such as catalase (CAT), guaiacol peroxidase (GPX), and superoxide dismutase (SOD) and enzymes of ascorbate-glutahione (AsA-GSH) cycle such as ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) are known to protect plants from devastating effects of heavy metal stress by scavenging ROS species (Sharma et al. 2012). In our experiment, the activity of all the enzymes mentioned above tended to increase after adding Pb to the growth medium and the increase of magnitude varied with the exposure time, which was due to the self-defense mechanism of grass pea. Additionally, increases in GPX, APX, and CAT activity should be induced by excessive H 2 O 2 production. Numerous studies have reported the enhancement in activities of different ROS-scavenging enzymes in plants under metal stress conditions (Rai et al. 2011;Gururani et al. 2013;Mahdavian et al. 2016;Khan et al. 2019;Awan et al. 2020). By contrast, Al Mahmud et al. (2018) showed a decrease in MDHAR, DHAR, GR, and CAT activity against a rise in SOD, GPX, and APX activity in Brassica juncea after exposure to severe metal stress. Nonetheless, during stress response, the majority of plants are unable to produce adequate amount of antioxidant enzymes to deal with destructive effects of ROS (Ali et al. 2021). Interestingly, PGPR were found to further improve the activities of SOD, CAT, APX, GR, MDHAR, and MDHAR indicating that PGPR inoculation alleviates the metal-induced oxidative stress through regulating the key antioxidant enzymes and subsequently protecting cells from damage caused by ROS. These findings are consistent with those of the previous research reporting the role of bacterial inoclation in conferring plant tolerance against heavy metals by boosting various antioxidative enzymes activity (Islam et al. 2014;Shah et al. 2020). This could be due to the production of metabolites by bacteria, including growth promoting hormones, which play an important role in the synthesis of antioxidant enzymes (Rahbari et al. 2021). In fact, SOD provides the first line of defense against ROS, involved in the dismutation of O 2 • − into H 2 O 2 and molecular oxygen. The CAT and POX enzymes are responsible for the conversion of H 2 O 2 to water and oxygen, and thus play necessary roles in providing tolerance to unfavorable conditions in plants (Hassan et al. 2020). Likewise, the efficient functions of enzymes Halliwell-Asada cycle contribute to overcoming oxidative damage as well as control cellular redox homeostasis (Sharma et al. 2012;Hasanuzzaman et al. 2017;Jahan et al. 2020).
Therefore, the inoculated plants had considerably lower reactive oxygen species accumulation and a higher level of antioxidants enzymes, hence, it can be concluded that under excess metallic condition, PGPR maintain plant health as well as Pb accumulation in plants at high concentrations, which are the key factors for efficient phytoremediation (Gulzar and Mazumder 2022).

Gene expression analysis
Plants have responded to various biotic and abiotic stress factors with a series of mechanisms through the transcriptional regulation of stress-responsive genes. To get a better insight into the underlying molecular mechanism of Lathyrus sativus under Pb stress induced by PGPR, gene expression profiling of LsPCS, LsGCN, LsCNGC, LsGR, and LsGST in leaves and roots have been studied through qRT-PCR. In the current study, we observed that grass pea plants exhibited a dynamic increase in the relative transcript level of LsPCS in leaves after metal exposure and reached to the maximum extent on the 6 th day and the expression further multiplied with bacterial inoculation, indicating that PGPR acts as a regulator of gene expression. Indeed, phytochelatin synthase (PCS) is a key enzyme involved in the synthesis of phytochelatins (PCs), which is thought to play an important role in heavy metal tolerance and detoxification (Liu et al. 2012). Similarly, Brunet et al. (2009) found that LsPCS transcript expression was improved in leaves of grass pea plants when grown in a medium supplemented with Pb for 96 h. Likewise, it was stated that the transcript level of PCS in the leaves of Solanum lycopersicum was stimulated due to the Pb treatment (Klsa 2019). Further studies on metal stress noted that Cd induced evident accumulation of mRNA transcript of PCS in the roots of Sulla coronaria (Chiboub et al. 2020). In another case, Begum et al. (2016) reported that PCS was significantly upregulated in roots of tolerant rice genotype in response to arsenic exposure but remained at the same level in the sensitive genotype. Thus, results suggest that increased LsPCS transcript accumulation in inoculated plants can reveal putative key role in Pb tolerance and may be linked with combating metal toxicity. Recently, Zhu et al. (2021) demonstrated that overexpression of BnPCS1 confers enhanced Cd tolerance, accumulation, and translocation in Arabidopsis, suggesting that enhancement of the expression of PCS in our experimentation was probably involved 1 3 in improving Pb accumulation in PGPR-inoculated plants. We further showed that bacterial inoculation significantly induced the accumultion of LsGST transcripts in leaves after 3 days of the metal treatment, which was followed by the increase in mRNA transcript of LsGR on the 6th day; however, LsGR remained unvaried in uninoculated plant leaves. This can be considered a strengthening of the antioxidative processes in inoculated plants as well as an increased potential for the sequestration of Pb ions to avoid lead-induced dammage to cell components (Brunet et al. 2009). In fact, one of the most important regulations in response to heavy metal is GR and GST, the key antioxidant defense enzymes that adjust the GSH pool besides the role of GR and GSH in detoxification of H 2 O 2 generated by Mehler reaction in chloroplast (Sharma et al. 2012;Klsa 2017). In addition, under metal stress, the role of GST may be related not only to the removal of toxic products of lipid peroxidation but also to its possible involvement in the transport of phytochelatin-metal complexes to the vacuole (Hossain et al. 2012;Hasanuzzaman et al. 2020). Moreover, both GST and GSH contribute in the accumulation of some flavonoids (anthocyanin), which also act as metal binders. Likewise, upregulating in the expression of GR and GST has been found to be related with improved adaptation and tolerance against different abiotic stresses including heavy metals (Trivedi et al. 2013;Klsa 2017;Gao et al. 2020;Horváth et al. 2020;Stavridou et al. 2021). Similarly, according to Raklami et al. (2019), the enhanced expression of the GR gene in roots of alfalfa inoculated with metal-resistant rhizobacteria, particularly at high metal concentrations, probably reflects the better management of the stress in inoculated plants. Omar et al. (2021) also reported that a significant increase in GST transcript amounts in plants inoculated with Rhizobium sp may improve the tolerance status of the sensitive rice genotype under drought condition. Thereby, it seems that inoculated plants further protect and defend themselves against ROS induced oxidative damage by the upregulation of antioxidant machinery. Thus, the overexpression of GST and GR in the current study might be involved in Pb stress tolerance in grass pea plants induced by PGPR. In addition, RT-qPCR analysis showed that the bacterial inoculation enhanced the transcript level of LsGCN under Pb stress. The ATPbinding cassette (ABC) F/GCN (general control non-derepressible) subfamily is a group of soluble ABC proteins (Li et al. 2018). Indeed, some ABC proteins serve as regulators instead of transporters, such as the regulation of protein synthesis and stability (Ha et al. 2021). Likewise, a previous study has indicated that GCN20/ABCF3 positively regulates stress adaptation in Arabidopsis by modulating endoplasmic reticulum stress responses and aquaporin gene expression and is involved for DNA damage repair in roots (Han et al. 2018;Li et al. 2018).
On the other hand, cyclic nucleotide gated ion channels (CNGCs) in plants are known to play multifaceted roles in pathways related to ion homeostasis, development, defense against biotic and abiotic challenges and have been implicated in providing stress tolerance (Duszyn et al. 2019;Barnham et al. 2021;Oranab et al. 2021). As non-selective cation transporters, some evidence suggests that plant CNGCs are not only involved in the nutrient uptake into cells but also represent a possible entry pathway for heavy metals (Jha et al. 2016). Notably, Moon et al. (2019) have recently provided functional evidence which supports the roles of some CNGCs in the uptake and transport of Pb 2+ ion in Arabidopsis thaliana. Therefore, our finding strongly suggests that the increases in the transcript levels of LsC-NGC results in better Pb uptake and transport in inoculated grass pea plants which leads to increased Pb concentration in plant tissues.

Conclusions
Our findings highlighted marked improvements of studied parameters at biochemical and molecular level after PGPR inoculation. In fact, beneficial influence of the plant growth promoting rhizobacterial strains R. leguminosarum, Pseudomonas fluorescens, Luteibacter sp., Variovorax sp. on Pb accumulation capacity of grass pea was found in this study compared to the uninoculated plants, when plants were exposed to 1 mM Pb. The metal accumulation in grass pea plants triggered a significant elevation in the synthesis of non-protein thiols, particularly in inoculated plant leaves. Nevertheless, Pb treatment significantly increased oxidative stress and membrane damage in leaves but the Pb-treated grass pea supplemented with PGPR reduced the oxidative stress as it lowered hydrogen peroxide and malondialdehyde levels and subsequently protected membranes from damage. Furthermore, bacterial inoculation helps plants to mitigate Pb toxicity and enhanced the activities of antioxidant enzymes, such as SOD, CAT, APX, GR, DHAR, and MDHAR to scavenge excess ROS, reflecting the better management of the stress in inoculated plants. Likewise, the upregulation of gene expression of LsPCS, LsGCN, LsC-NGC, LsGR, and LsGST suggests that inoculation with these strains triggered Pb stress-related defense pathways.
The study provides concise information about the active participation and the key role of PGPR which presents striking potential not only for increasing the metal removal efficiency of the grass pea plant but also in conferring Pb stress tolerance and detoxification mechanism.
Future research is still required to explore how complex metabolic networks interact and are modified in grass pea plants under Pb stress in the presence or absence of PGPR.