Unveiling the potential of A. fabrum and γ-aminobutyric acid for mitigation of nickel toxicity in fenugreek

Nickel (Ni) is a heavy metal that adversely affects the growth of different crops by inducing oxidative stress and nutrient imbalance. The role of rhizobacteria (RB) is vital to resolve this issue. They can promote root growth and facilitate the uptake of water and nutrients, resulting in better crop growth. On the other hand, γ-aminobutyric acid (GABA) can maintain the osmotic balance and scavenge the reactive oxygen species under stress conditions. However, the combined effect of GABA and RB has not been thoroughly explored to alleviate Ni toxicity, especially in fenugreek plants. Therefore, in the current pot study, four treatments, i.e., control, A. fabrum (RB), 0.40 mM GABA, and 0.40 mM GABA + RB, were applied under 0Ni and 80 mg Ni/kg soil (80Ni) stress. Results showed that RB + 0.40 mM GABA caused significant improvements in shoot length (~ 13%), shoot fresh weight (~ 47%), shoot dry weight (~ 47%), root length (~ 13%), root fresh weight (~ 60%), and root dry weight (~ 15%) over control under 80 Ni toxicity. A significant enhancement in total chlorophyll (~ 14%), photosynthetic rate (~ 17%), stomatal CO2 concentration (~ 19%), leaves and roots N (~ 10 and ~ 37%), P (~ 18 and ~ 7%) and K (~ 11 and ~ 30%) concentrations, while a decrease in Ni (~ 83 and ~ 49%) concentration also confirmed the effectiveness of RB + 0.40 mM GABA than control under 80Ni. In conclusion, fabrum + 0.40 mM GABA can potentially alleviate the Ni toxicity in fenugreek plants. The implications of these findings extend to agricultural practices, environmental remediation efforts, nutritional security, and ecological impact. Further research is recommended to elucidate the underlying mechanisms, assess long-term effects, and determine the practical feasibility of using A. fabrum + 0.40GABA to improve growth in different crops under Ni toxicity.

acid Among different biotic and abiotic stresses 1-5 nickel (Ni) accumulation in agricultural areas is a serious issue.The intentional and unintentional discharge of untreated Ni-contaminated sewage sludge and industrial effluents resulted in its accumulation in soil [6][7][8] .Its toxicity in plants can hinder seed germination and growth, thus decreasing crop biomass production 9,10 .Furthermore, Ni toxicity leads to chlorosis, necrosis, and interference with vital physiological processes such as photosynthesis and transpiration, resulting in oxidative damage in plants [11][12][13] .Rhizobacteria inoculation can play a crucial role in overcoming this critical problem.
The rhizosphere has various soil microbes, i.e., rhizobacteria and fungi, which benefit plant growth [14][15][16] .These rhizobacteria and fungi play a direct or indirect role in enhancing both plant growth and soil health 14,15,[17][18][19][20] .In soil, rhizobacteria secrete enzymes that aid in mobilizing fixed nutrients, thus improving their uptake by the plant 21 .Numerous studies reported that rhizobacteria, which promote plant growth, can also mitigate the toxicity

Seeds sterilization
Fenugreek seeds (Qasuri Methi) were purchased from a certified seed dealer authorized by the Government of Punjab, Pakistan.The seeds were sterilized with a 5% sodium hypochlorite solution, followed by three washes using 95% ethanol.Subsequently, the seeds underwent three additional washes with sterilized deionized water to eliminate any residual sterilizing agents.Each pot containing 5 kg of soil was sown with 20 seeds.After germination, thinning was performed to maintain five seedlings in each pot.

Seeds inoculation with rhizobacteria and GABA foliar
Inoculating fenugreek seeds with A. fabrum (RB) was conducted using peat and 10% sugar solution as sticky material.For 50 g seeds, 10 g peat was used while 10 ml of sugar solution was added.After inoculation, the seeds were allowed to dry under controlled conditions to ensure proper adhesion of the inoculum to the seed surface.The characteristics of rhizobacteria are provided in Table 1.The solution was made in deionized, sterilized water.The solution of 0.40 mM GABA was applied as foliar at a rate of 25 mL per pot five times (at 7, 14, 21, 28, and 35 days after transplantation).

Statistical analysis
The collected data underwent standard statistical analysis for comparison.A two-way ANOVA was applied to assess the significance of treatments.The Tukey test was utilized for pairwise comparison of treatments with a significance level set at p ≤ 0.05.Additionally, cluster plot convex hull, hierarchical cluster plot, and Pearson correlation were performed using OriginPro software 57 to further explore patterns and relationships in the data.

Ethics approval and consent to participate
We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue.So, it is not applicable.

Study protocol must comply with relevant institutional, national, and international guidelines and legislation
Our experiment follows the with relevant institutional, national, and international guidelines and legislation.
In the case of 0Ni, inoculation of RB led to a 10.35% increase in shoot dry weight compared to control.Applying 0.40GABA resulted in 22.47%, while RB + 0.40GABA showed a 33.92% increase in shoot dry weight compared to the control under 0Ni.At 80Ni stress, over control, applying RB showed a 12.20% improvement in shoot dry weight.Adding 0.40GABA and RB and 0.40GABA resulted in a 22.05% and 46.85% increase from the control at 80Ni stress (Fig. 1B).

Root attributes
Applying RB resulted in 7.09%, 0.40GABA caused 16.04%, while RB + 0.40GABA showed a 38.81% increase in root fresh weight than the control at 0Ni.In the case of 80Ni, RB showed a 14.89% increase in root fresh weight than control.Applying 0.40GABA resulted in 31.21%, while RB + 0.40GABA had a 60.28% increase in root fresh weight compared to control under 80Ni stress (Fig. 2A).
For root dry weight, an increase of 2.65, 5.29 and 11.64% was observed in RB, 0.40GABA and RB + 0.40GABA over control under 0Ni.Under 80Ni stress, compared to control, a 5.66% increase in root dry weight was noted in RB, 10.69% in 0.40GABA and 14.47% in RB + 0.40GABA (Fig. 2B).www.nature.com/scientificreports/Adding RB caused 2.95%, while 0.40GABA resulted in a 5.03% increase in root length over the control group under 0Ni.Treatment RB + 0.40GABA showed an 8.60% enhancement in root length compared to the control at 0Ni.Under 80Ni stress, an increase was noted in root length where RB (3.28%), 0.40GABA (6.23%) and RB + 0.40GABA (12.47%) were applied over control (Fig. 2C).

Chlorophyll a, b, and total
Results showed that RB increased chlorophyll a content by 4.19% while 0.40GABA by 7.87% compared to control at 0Ni.Treatment RB + 0.40GABA showed a 9.88% improvement in chlorophyll a content than control under 0Ni.Inoculation of RB increased chlorophyll a content (1.36%) than control under 80 Ni stress.However, 0.40GABA showed 7.71%, and RB + 0.40GABA showed a 9.77% improvement in chlorophyll a content compared to the control under 80Ni stress (Fig. 3A).

Gas exchange attributes
Inoculating RB increased the photosynthetic rate by 3.65% while 0.40GABA treatment by 7.13% compared to control under 0Ni.It was noted that RB + 0.40GABA caused a 14.41% enhancement in photosynthetic rate over control at no Ni toxicity.RB caused 7.55%, 0.40 GABA 11.98% and RB + 0.40GABA 16.78% increase in photosynthetic rate from control under 80 Ni stress (Fig. 4A).In the case of stomatal CO 2 concentration, a 4.79% increase was found in RB, 9.89% in 0.40 GABA and 18.09% in RB + 0.40 GABA compared to control under 0Ni.In 80Ni stress, RB caused 6.39%, 0.40 GABA 10.86%, and RB + 0.40 GABA 18.54% increase in stomatal CO 2 concentration than control (Fig. 4B).
At 0Ni, RB increased stomatal conductance by 4.43% compared to the control.On the other hand, 0.40 GABA caused an enhancement of 5.95%, while RB with 0.40 GABA showed an 8.40% increase over the control under 0Ni.Inoculating RB increased stomatal conductance by 4.52%, 0.40 GABA by 11.03% and RB + 0.40 GABA by 16.62% over the control under 80Ni stress (Fig. 4C).
It was noted that applying RB resulted in 5.20%, 0.40GABA 11.08% and RB + 0.40GABA 14.64% increase in the transpiration rate than control under 0Ni.In the case of 80Ni stress, RB resulted in 1.98%, 0.40GABA 3.87% and RB + 0.40GABA 5.19% increase in transpiration rate compared to control under 80Ni stress (Fig. 4D).

Antioxidants
Treatments RB caused 16.47% while 0.40 GABA showed a 37.44% decline in SOD activity under 0Ni.It was noted that RB + 0.40 GABA resulted in a 60.22% decrease in SOD activity where 0Ni was present.Under 80 Ni stress, RB reduced SOD activity by 9.07%, 0.40 GABA by 15.27% and RB + 0.40 GABA by 23.40% over control (Fig. 5A).
In the case of Apx activity, RB showed a 6.83% decrease, while 0.40 GABA showed a 19.64% decrease over control under 0Ni.Applying RB with 0.40 GABA resulted in a 39.79% decrease compared to the control at 0Ni.At 80Ni stress, compared to control Apx activity, it decreased by 4.98%, 9.08%, and 16.27%, where RB, 0.40 GABA, and RB + 0.40 GABA were applied, respectively (Fig. 6B).
Under 0Ni, RB decreased 11.11% in POD activity over control.Adding 0.40GABA and RB + 0.40GABA, treatments also reduced the POD activity by 28.00% and 33.33% compared to the control at 0Ni.In the case of 80Ni stress, RB showed a 3.29%, 0.40GABA 7.14%, and RB + 0.40GABA 15.38% decline in POD activity from control (Fig. 5C).Applying RB resulted in 6.95%, while 0.40 GABA showed an 8.21% decline in CAT activity compared to the control group in 0Ni.Treatment RB + 0.40 GABA showed an 11.17% decrease in CAT activity compared to the control under 0Ni.Results showed that RB caused 3.53% 0.40 GABA 7.00% and RB + 0.40 GABA 9.48% decrease in CAT activity over control under 80Ni stress (Fig. 5D).

Malondialdehyde (MDA), Ascorbate peroxidase (Apx) and Electrolyte leakage
Inoculation of RB reduced 5.06%, and 0.40 GABA caused an 11.07%decline in MDA compared to the control under 0Ni.However, RB + 0.40 GABA showed a 17.81% decrease in MDA than the control at 0Ni.On the other hand, RB resulted in 5.97%, 0.40 GABA showed 6.85%, and RB + 0.40 GABA caused a 13.67% decrease in MDA than control under 80Ni stress (Fig. 6A).

Shoot N, P, K and Ni concentration
Under 0Ni and 80 Ni stress, RB and 0.40 GABA increased shoot N compared to the control.The RB + 0.40 GABA improved by 10.34 and 9.62% shoot N over control under 0Ni and 80 Ni stress, respectively.Similar results were also noted in shoot P, where RB and 0.40 GABA caused a significant enhancement in shoot P compared to the control group at 0Ni and 80 Ni stress.However, RB + 0.40 GABA showed 14.29 and 17.65% enhancement in shoot P compared to control at 0Ni and 80 Ni stress, respectively.In the case of shoot K, treatment RB + 0.40  2).

Root N, P, K and Ni concentration
Results showed a significant improvement in root N, P, and K, while a decline in Ni concentration was observed where RB and 0.40 GABA were treated under 0Ni and 80 Ni stress.It was noted that RB + 0.40 GABA showed an increase in root N (9.64 and 37.10%), P (11.76 and 6.56%), and K (17.39 and 25.00%) while a decrease in Ni compared to control at 0Ni and 80 Ni stress respectively.The first two principal components accounted for substantial variance in the data, capturing 98.08% and 0.64%, respectively.Most data points, characterized by 0Ni, form a tight cluster in the lower-right quadrant of the plot.These data points are represented by scores on PC1 ranging from approximately 0.25 to 7.39 and scores on PC2 ranging from approximately −0.38 to 0.12.
In contrast, a smaller group of data points, labeled as 80Ni, form a separate cluster in the upper-left quadrant of the plot.These data points exhibit scores on PC1 ranging from approximately −7.25 to −0.12 and scores on PC2 ranging from approximately −0.70 to 0.39 (Fig. 7A).A smaller cluster labeled 0.40GABA is also located in the plot's lower center.The data points in this cluster have scores on PC1 ranging from approximately −3.57 to 5.59 and scores on PC2 ranging from approximately −0.38 to 0.39.Lastly, a cluster labeled RB + 0.40GABA is in the upper-right quadrant of the plot.The data points in this cluster display scores on PC1 ranging from approximately −1.90 to 7.39 and scores on PC2 ranging from approximately −0.40 to 0.53 (Fig. 7B).
Chlorophyll b (mg/g) and total chlorophyll (mg/g) showed a high similarity of 0.22735, suggesting a close relationship between these two chlorophyll-related variables.Similarly, shoot P (%) and chlorophyll a (mg/g) demonstrated a significant similarity of 0.29596, indicating a potential association between shoot phosphorus content and chlorophyll a level.Shoot length (cm) and root length (cm) exhibited a similarity of 0.34924, indicating their linkage in terms of plant morphology.Similarly, root fresh weight (g) and shoot nitrogen (%) showed a similarity of 0.35047, suggesting a possible correlation between root weight and shoot nitrogen content.On the other hand, some variables appeared to be significantly dissimilar to others.Carotenoid (mg/g) and stomatal conductance (mol H 2 O m −2 s −1 ) displayed a dissimilarity of 1.074, indicating they might be unrelated in this context (Fig. 7C).

Ni toxicity
Ni contamination affected the nutrient uptake, growth, and physiology of Fengureek in our study 41 .This was obvious due to higher Ni concentration in the root and leaf (Table 2).Ashraf et al. 58 found a noticeable decrease in growth, biomass, photosynthetic pigments, and nutrient acquisition in response to nickel toxicity 59 .An overabundance of Ni in plants can disturb various physiological processes, adversely impacting growth and development 60 .Despite its role as a crucial micronutrient in enzyme activation and nitrogen metabolism, Ni can turn toxic when present in elevated concentrations 61 .It can also disrupt enzyme functions, trigger oxidative stress, and create nutrient imbalances by interfering with the assimilation of vital elements 62 .The impairment of root growth further hampered water and nutrient absorption, leading to stunted growth, diminished biomass, and reduced crop yields 63 .

GABA
The uptake of Ni in roots and leaves was reduced with the application of GABA in our study (Table 2) 42 .This might be because GABA is known to modulate stress responses and stimulate antioxidant systems in plants, www.nature.com/scientificreports/which could mitigate the impact of nickel-induced oxidative stress and, in turn, influence the plant's ability to cope with nickel uptake 64,65 .In response to abiotic stresses, signaling triggered by GABAs is linked to stimulating the antioxidative defense system, regulating osmotic balance, maintaining levels, and acting as a buffering agent for carbon and nitrogen metabolism 66 .The low electrolyte leakage and MDA in the current study also validated the positive functioning of GABA regarding the alleviation of Ni stress (Fig. 6).GABA can bind and sequester Ni ions, which decreases their mobility.This procedure reduces Ni buildup by stopping the transport of Ni to delicate plant organs 67 .

A. fabrum
A. fabrum inoculation decreased the uptake of Ni and enhanced N, P, and K nutrients in the root and leaf (Table 2).This was mainly attributed to this rhizobacteria's P and K solubilizing ability (Table 1).Enhanced uptake of essential nutrients such as nitrogen (N), phosphorus (P), and potassium (K) plays a pivotal role in promoting plant growth.Improved nutrient absorption facilitates robust metabolic processes, supports energy transfer, and enhances cell division 68 .This heightened nutrient availability synthesizes vital biomolecules, increasing photosynthetic efficiency, healthier root development, and ultimately fostering overall plant growth and productivity 69 .Matile et al. 70 proposed that elevated ethylene concentrations due to abiotic stress, i.e., heavy metal toxicity 71 , lead to the breakdown of lipids in the cell wall.They posited that when ethylene meets the chlorophyllase (chlase) gene, it induces chlorophyll degradation, resulting in chlorosis.In the current study, improvement in the chlorophyll contents validated the effectiveness of RB as the rhizobacteria could secrete ACC deaminase, which was imperative in decreasing the stress ethylene 16,72 .Furthermore, phytohormones, i.e., indole-3-acetic acid (IAA), also have essential biological functions in improving the growth of plants 73 .

Combined effects of A. fabrum and GABA
GABA can potentially improve nutrient absorption and translocation inside the plant, whereas rhizobacteria increase nutrient availability in the rhizosphere 74 .Even when Ni stress is present, this cooperative effort provides an adequate delivery of vital nutrients.It functions as a stress reliever, lowering the adverse effects of Ni stress on fenugreek, while rhizobacteria prime the plant's defense systems and improve stress tolerance 75 .Rhizobacteria and GABA together, the plant's adaptive responses may be enhanced through overexpression of stress tolerance and growth promotion genes 76 .

Conclusions
In conclusion, the combination of A. fabrum + 0.40 mM foliar GABA (0.40GABA) proved highly effective in improving shoot and root growth parameters, chlorophyll content, and gas exchange attributes.Moreover, applying A. fabrum + 0.40GABA can also improve antioxidants, N, P, and K concentration in leaves and roots while reducing Ni concentration.These findings collectively confirmed the efficacy of A. fabrum + 0.40GABA in alleviating Ni stress and enhancing the growth and health of fenugreek plants exposed to Ni toxicity.

Figure 1 .
Figure 1.Impact of A. fabrum (RB) and GABA on fenugreek shoot fresh weight (A), dry weight (B), and shoot length (C) under 0Ni and 80 mg Ni/kg soil.The bars (n = 4) ± SE showed different letters for treatments significance at p ≤ 0.05; Tukey test.

Figure 2 .
Figure 2. Impact of A. fabrum (RB) and GABA on fenugreek root fresh weight (A), dry weight (B) and root length (C) under 0Ni and 80 mg Ni/kg soil.The bars (n = 4) ± SE showed different letters for treatments significance at p ≤ 0.05; Tukey test.

Figure 3 .
Figure 3. Impact of A. fabrum (RB) and GABA on fenugreek chlorophyll a (A), chlorophyll b (B), and total chlorophyll (C) under 0Ni and 80 mg Ni/kg soil.The bars (n = 4) ± SE showed different letters for treatments significance at p ≤ 0.05; Tukey test.

Table 2 .
Effect of treatment on N, P, K, and uptake of Ni concentration of shoot and root.Values are an average of 4 replicates.Different letters showed significant changes at p ≤ 0.05, Tukey Test. A. fabrum = RB.