Proline-mediated redox regulation in wheat for mitigating nickel-induced stress and soil decontamination

Nickel (Ni) is known as a plant micronutrient and serves as a component of many significant enzymes, however, it can be extremely toxic to plants when present in excess concentration. Scientists are looking for natural compounds that can influence the development processes of plants. Therefore, it was decided to use proline as a protective agent against Ni toxicity. Proline (Pro) is a popularly known osmoprotectant to regulate the biomass and developmental processes of plants under a variety of environmental stresses, but its role in the modulation of Ni-induced toxicity in wheat is very little explored. This investigation indicated the role of exogenously applied proline (10 mM) on two wheat varieties (V1 = Punjab-11, V2 = Ghazi-11) exposed to Ni (100 mg/kg) stress. Proline mediated a positive rejoinder on morphological, photosynthetic indices, antioxidant enzymes, oxidative stress markers, ion uptake were analyzed with and without Ni stress. Proline alone and in combination with Ni improved the growth, photosynthetic performance, and antioxidant capacity of wheat plants. However, Ni application alone exhibited strong oxidative damage through increased H2O2 (V1 = 28.96, V2 = 55.20) accumulation, lipid peroxidation (V1 = 26.09, V2 = 38.26%), and reduced translocation of macronutrients from root to shoot. Application of Pro to Ni-stressed wheat plants enhanced actions of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and total soluble protein (TSP) contents by 45.70, 44.06, 43.40, and 25.11% in V1, and 39.32, 46.46, 42.22, 55.29% in V2, compared to control plants. The upregulation of antioxidant enzymes, proline accumulation, and uptake of essential mineral ions has maintained the equilibrium of Ni in both wheat cultivars, indicating Ni detoxification. This trial insight into an awareness that foliar application of proline can be utilized as a potent biochemical method in mitigating Ni-induced stress and might serve as a strong remedial technique for the decontamination of polluted soil particularly with metals.


Methodology
The experiment was executed at the experimental area, University of Agriculture, Faisalabad (Latitude: 32° 26´ 45.7046", Longitude: 74° 5´ 13.2811") to examine the influence of proline as foliar treatment on wheat under Ni stress.The experiment with completely randomized design had 4 replicates per treatment.The two wheat varieties were exploited namely Punjab-11 (VI) and Ghazi-11 (V2).Treatments for the experiment were T0 (0 mM Proline + 0 µM Ni), T1 (10 mM Proline + 0 µM Ni), T2 (0 mM Proline + 100 mg/kg Ni) and T3 (10 mM Proline + 100 mg/kg Ni).For sowing, each pot (28 cm × 24 cm × 21.5 cm) carrying 8 kg of soil was provided with twelve seeds.Plants were watered twice every week.After two weeks of seed sowing, thinning was done for maintaining 6 plants per pot.Nickel stress (100 mg kg −1 ) was imposed at early boot stage (BBCH identification code 41 40 and foliar application of proline was given as 0 and 10 mM concentration at end of heading stage (BCCH identification code 59) 40 .Weather data for crop duration November 2021 to April 2022 is presented in Fig. 1.Plants were harvested after 3 weeks of foliar application to record different parameters.

Plant sampling
Four healthy plants were uprooted from each pot.The two plants were used for recording morphological parameters (fresh and dry weights and length of root and shoot) and the leaves of other two plants were stored at − 20 °C to determine physiological and biochemical attributes.

Morphological parameters
Length and weight.The length of shoot and root were recorded in "cm" using measurement tape while fresh and dry weights were recorded in "g" using weighing balance.

Biochemical parameters
Photosynthetic pigments.Protocol devised by Arnon 41 was followed for determining the pigments involved in photosynthesis.Fresh leaves (0.1 g) were extracted in 80% acetone.The solution was kept overnight in the dark.The ultraviolet visible spectrophotometer (U2020 IRMECO) was utilized for taking the readings at 480, 645 and 663 nm.
Enzymatic antioxidants.Flag leaves of two wheat cultivars (0.25 g) were extracted in 5 mL of potassium phosphate buffer (pH 7.8) at 4 °C and centrifuged at 12,000 rpm for 15 min.Activities of enzymatic antioxidants were determined exploiting supernatant.
Catalase (CAT).The technique developed by Chance and Maehly 42 was tracked for investigating the CAT concentration in both stressed and non-stressed wheat cultivars.Each cuvette had phosphate buffer (1.9 mL), H 2 O 2 (1 mL) and plant extract (0.1 mL).Absorbance was noted for 2 min after every 30 s at 240 nm.
Peroxidase (POD).In accordance with the process developed by Chance and Maehly 42 , the accumulation pattern of POD was estimated.Each sample contains a measured and standard volume of phosphate buffer, guaiacol (20 mM), H 2 O 2 (40 mM) and 50 µL plant extract.The readings were taken at 470 nm after every 30 s for 2 min.Superoxide dismutase (SOD).The assessment of the activity of SOD activity was done by following Method by Spitz and Oberley 43 .The reaction mixture consisting of distilled water, phosphate buffer, L-Methionine, Triton-X, NBT, riboflavin and plant sample (50 µL), in a total volume of 2.5 mL was placed underneath a fluorescent lamp for 15-20 min.The readings were noted at 560 nm with a regular interval of 30 s interval for 2 min.
Total soluble proteins.The assessment of total soluble proteins was done by pursuing the protocol of Bradford 44 .The previously extracted antioxidant solution was used for estimating total soluble proteins (TSP).The Bradford reagent (5 mL) was mixed with enzyme extract and vortexed for 5 s.Absorbance was noted at 595 nm with UV-spectrophotometer.
Reactive oxygen species (ROS).1Hydrogen peroxide (H 2 O 2 ).H 2 O 2 determination followed Velikova et al. 45 .Frozen leaf tissue was extracted in 0.1% TCA, centrifuged at 12,000 rpm for 20 min.Equal volumes of supernatant, pH 7.00 potassium phosphate buffer, and 1 mL KI were mixed in a glass tube.Absorbance was measured at 390 nm after vortexing.Lipid peroxidation markers.Malondialdehyde (MDA).The concentration of MDA was estimated by following Cakmak and Horst 46 .The frozen leaves were ground in 0.1% TCA, followed by centrifugation at 13,000 rpm for 15 min.The upper transparent liquid was further diluted with 4 mL solution containing 20% thiobarbituric acid (TBA) fortified with 0.5% TCA, and boiled at 100 °C for 30 min.Reading for each sample was taken at 532 nm and 600 nm after being cooled in ice.
Anthocyanins.For the estimation of anthocyanins, Stark and Wray's 47 bioassay was employed.Leaf samples of each treatment and replicate was separately chopped in small pieces, supplemented with 2 mL of acidified methanol and boiled at 90 °C for 1 h and optical density was noted at 535 nm.www.nature.com/scientificreports/Total soluble sugars.Approximation of total soluble sugars was performed out by adopting protocol designed by Yoshida et al. 48.The leaf sample (0.1 g) was treated with deionized water (10 mL) and heated at 90 °C for 1 h.Dilution was done with deionized water to get desired volume.The anthrone reagent (5 mL) was mixed with this dilution (1.5 mL) and again heated 90 °C for 20 min.Absorbance was noted at 620 nm.
Flavonoids.The method developed by Zhishen et al. 49 for the evaluation of flavonoids was followed.The 0.1 g leaf sample was mixed with 80% acetone and kept overnight in dark.The 1 mL of this solution was supplemented with distilled water, 5% NaNO 2 , 10% AlCl 3 , and 1 M NaOH.Optical density was noted down at 510 nm.
Ascorbic acid (AsA).The AsA contents were recorded through subsequent procedure of Mukherjee and Choudhuri 50 .Leaf sample was ground in 6% TCA solution and centrifuged at 12,000 rpm for 10 min.The 2% dinitrophenyl hydrazine (1 mL) along with a tiny drop of 10% thiourea were added to the supernatant, subjected to heating in boiling water container at 90 °C for 15 min, later cooled down in ice.The freshly prepared sulphuric acid (80%) was added, and reading was taped at 530 nm.

Mineral nutrients
Na + , K + and Ca 2+ Dried shoot sample (0.1 g) was digested with 3 mL sulphuric acid and kept overnight to be digested at maximum.The reaction mixture for each sample was placed on hot plate and hydrogen peroxide (5 mL) was added.Hydrogen peroxide was added again until the solution became colorless.The solution was then allowed to cool after removing from hot plate and filtered.Dilution was done by adding distilled water until the final volume became 50 mL.The reading was taken on flame photometer (Sherwood-410) for the determining Na + , K + and Ca 2+ .

Statistical analysis
Three-way analysis of variance (ANOVA) was implicated and data significance was analysed by using Statistix 8.1 at significance level of 0.05.Principal component analysis (PCA), heat plot and correlation analysis were performed through R-studio (version R-4.3.1).

Plant guidelines
All the plant experiments were performed by relevant institutional, national, and international guidelines and legislations.

Proline reduced inhibitory effects of Ni by enhancing biomass production of wheat seedlings
Figure 2A-F describes some of the growth features of the two wheat varieties (V1 = Punjab-11, V2 = Ghazi-11), such as shoot fresh and dry weight (SF/DW), shoot length (SL), root fresh and dry weight (RF/DW) and root length (RL) when treated with Ni (100 mg kg −1 ) and proline (10 mM), along with no treatment (T 0 ) and proline (Pro) application alone (T 1 ).The application of Ni to both wheat varieties drops the growth parameters (Fig. 2).

Proline alleviates the photosynthesis reduction induced by Ni stress in wheat seedlings
In plants treated to Ni stress, chlorophyll a, chlorophyll b, total chlorophyll and carotenoids levels fell by 36.97,36.75, 35.78, and 40.65%, respectively, in relevance to control plants (Fig. 3A-D).In comparison to Ni-stressed plants, Pro treatment increased (P ≤ 0.01) the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents by 29.64%, 18.39%, 37.07%, and 32.55%.Proline also enhanced the amounts of chl.a, chl.b, total chl.and carotenoids in non-stressed plants by 14.46, 22.48, 17.38, and 23.33%, respectively (Fig. 3).All photosynthetic pigments showed a non-significant difference in both wheat varieties except carotenoids, where V2 showed higher accumulation of carotenoids with Pro treatment with (T 3 ) and without stress (T 1 ).

Proline elevates the endogenous antioxidant pooling Ni-stressed wheat seedlings
To verify the antioxidant role of exogenous 10 mM Pro under Ni stress, the contents of CAT, POD, SOD and TSP were recorded in wheat plants.Under  www.nature.com/scientificreports/

Proline alleviates the endogenous oxidative damage induced in Ni-stressed wheat seedlings
To foster the impact of Pro under Ni, reactive oxygen species (ROS) in non-radical form H 2 O 2 (Fig. 4E) and MDA (Fig. 4F) content were examined.The important findings specified that Ni toxicity has pointedly (P ≤ 0.001) increased endogenous H 2 O 2 , and MDA concentration by 28.96, 55.20% in V1, and 26.09, 38.26% in V2 wheat seedlings, respectively.However, oxidative damage was minimized (P ≤ 0.05) by the exogenous treatment of Pro (Fig. 4).In comparison to Ni treatment alone, Ni + Pro application appeared to reduce H 2 O 2 and MDA contents by 9.32-22.43%,and 15.25-28.42% in both wheat varieties, respectively (Fig. 4E,F).

Principal Component Analysis (PCA), Heat-plot and correlation analysis:
To reveal total variability, PCA analysis was performed and different variability dimensions were estimated.Among these, first two (Dim1 and Dim2) encompassed about 85.3% of the total variability explained by different morphological, agronomic and biochemical parameters.To comprehend the relationship among these variables under Proline and Nickel treatment, position of each variable was shown in PCA biplot based on both individual and variable projections (Fig. 7).Based on individual projections, factor analysis was performed in 2 × 2 factorials arrangements and was plotted for Nickel (Ni1 and Ni2) and Proline (Pr1 and Pr2) treatments.This led to four treatment combinations i.e., Ni1Pr1, Ni1Pr2, Ni2Pr2 and Ni2Pr1 which were distributed in II, I, III and IV quadrants respectively.To further comprehend the relationship with plant characteristics, these were plotted as vectors and degree of correlation as measure of angle in PCA biplot (Fig. 7).Our results showed that a group of plant variables including S Ca, RL, RDW, Chl a, Chl b, SFW, SL, SK, SDW and RFW were distributed in first quadrant showing positive correlation to Dim1 (67.5%) while biochemical parameters like Flavonoids, SOD, POD, CAT, AsA, TSS and TSP were positively correlated to Dim2 (17.8%).On contrary, variables like S Na, MDA and H 2 O 2 were negatively related to both Dim1 and Dim2 (Fig. 7).However, no clear pattern was observed for morphological, agronomic or biochemical parameters in PCA biplot.
To compare mean performance under different treatments (Variety, Nickel and Proline levels) in eight different treatment combinations (V1Ni1Pr1, V1Ni1Pr2, V1Ni2Pr1, V1Ni2Pr2, V2Ni1Pr1, V2Ni1Pr2, V2Ni2Pr1, V2Ni2Pr2) for all studied variable and log normalized data was plotted as heat map (Fig. 8).The results showed that based on mean performance of plant variables, eight treatment combinations could be divided into two distinct categories.All treatment combinations with Nickel level 1 (Ni1) like V1Ni1Pr1, V1Ni1Pr2, V2Ni1Pr1 and V2Ni1Pr2 were placed in one group which demonstrated higher mean performance for S Ca, SL, S K, SFW, RDW, RL, SDW, RFW, Seed number, 1000 GW, Spike length and few biochemical parameters like carotenoids, Anthocyanin, T Chl, Chl a and Chl b (Fig. 8).Similarly, all treatment combinations with Nickel level 2 (Ni2) including V1Ni2Pr1, V1Ni2Pr2, V2Ni2Pr1 and V2Ni2Pr2 were placed in same group which depicted higher mean values for majority of biochemical parameters like TSS, TSP, Flavonoids, SOD, AsA, CAT, POD, H 2 O 2 and MDA while all other variables showed lower mean values (Fig. 8).
The correlation analysis identified three main groups' correlations among the different variables.The first group includes higher positive and significant correlations between biochemical parameters like CAT, POD, AsA, SOD, TSP, Flavonoids and TSS along with lower positive non-significant correlation values for MDA and S Na and H 2 O 2 .The second larger group showed significant negative correlation values among Seed Number, 1000 GW, SDW, RFW, T Chl, Chl a, Chl b, Anthocyanins, Spike length, Carotenoids, RDW, SL SFW, S K, RL and S Ca (Fig. 9).

Plant growth and biomass
The risk for plant growth and development increased as a result of the unrestrained anthropogenic activities that added HMs to agricultural soils 51 .There have been reports on the negative effects of Ni at higher concentrations on the plant life cycle and its necessity for plants at lower concentrations [52][53][54] .Nevertheless, little research has been done on the negative effects of Ni toxicity and how plants can adapt to it.Growing wheat and other significant cereal crops in Ni-contaminated soil might pose substantial risks to the health of people, animals, and plants.Although Ni is a so-called vital microelement, too much of it can be hazardous 55 .Plant growth and photosynthetic capacity are also decreased as a result of Ni toxicity.In addition, it causes oxidative stress, impedes the intake of other metals, and suppresses the metabolism of nitrogen as well as enzymatic and mitotic processes.Arabidopsis thaliana (L.) was affected by a high Ni 2+ concentration in the growth medium, which caused geotropism deficiencies by inhibiting root cell elongation 56 .According to Wang and Zhou 57 , plant tolerance to heavy metals is frequently evaluated based on the degree of root or shoot growth restriction produced by the existence of undesirable metal/s in the growing media.The current findings also support the previous findings by exhibiting reduced biomass attributes wheat plants under Ni 2+ stress, as demonstrated by many scientists previously 37,58 .Nickel treated plants with a toxic concentration showed deprived root growth because of reticence of mitotic activity, thus restrict the overall growth of the plants 59 .Ni distribution in plant tissues is an indication of a change in chemical form resulting from complexation with plant-produced ligands.Studies on the uptake of HMs by plants have shown that HMs can also be transported passively from roots to shoots through the xylem vessels 60 .Nickel was also likely to have adverse effects on various metabolic and biochemical processes, as well as on plant cell proliferation, mitotic cell division, and cell wall flexibility 61,62 .These molecular disbalance resulted in arrested shoot and root weight as well as growth.To improve plant's adaptability against Ni 2+ stress, certain signal molecules could be applied exogenously to serve as a potent solution.Among many signal molecules, proline is a notable biological signal molecule in plant tissues to regulate numerous growth and development processes.In stressed plants, increased proline levels in the roots and shoots may lead to osmoregulation.Proline is essential for maintaining membrane integrity, controlling cytosolic pH, regulating osmosis, stabilizing enzymes, and scavenging free radicals 63 .Categorically, Pro performs three important functions under stress in addition to being a great osmolyte: it chelates metals, works as potential antioxidant, and signaling molecule.The stimulation of growth in response to exogenous proline treatment could be attributed to changes in net photosynthetic rate and gaseous exchange characteristics, which improves the osmoregulatory mechanism of plant with and without stress 64,65 .The Pro has considerably defeated the Ni and salinity-induced toxic effects at early growing stage, water status, and photosynthetic attributes.The Pro is efficient in regulating polyamine metabolism to create tolerance in pea against Ni 66 .
The toxic concentration of Ni caused a drastic reduction of dry biomass and yield occurred was found to be different for different plant species.The current study also demonstrated the significant reduction of yield, and yield-related components under Ni 2+ stress; while the exogenous application of Pro statistically stimulated the yield attributes, when compared with control.Previously, it has been documented that Ni 2+ toxicity imparts negative consequences on mung bean yield due to phenomenon reduction of its morpho-physiological and biochemical attributes 67 .

Lipid peroxidation and reactive oxygen species scavenging by proline under Ni stress
In this study, we found inhibition of gaseous, and photosynthetic attributes at 100 mg kg −1 concentration of Ni in wheat.Toxicity of Ni 2+ most likely impair photosynthetic process by interfering with membrane selectivity, CO 2 fixation, ultrastructure of chloroplast, and electron transport processes 68 .We recommended that higher transpiration rate indicated higher rate of translocation of Ni from roots to the shoot [69][70][71] .Proline mitigated the Ni 2+ toxicity in wheat seedlings by improving photosynthetic attributes 72 .
Nickel convincingly influences metabolic processes by accumulating higher rate ROS, which induce oxidative stress in plants 73 .Variety of plants has displayed this phenomenon of increased production of ROS under abiotic stress.The higher concentration of proline inhibits pyroline-5-carboxylate synthetase and generate ROS causing growth inhibition in plants 69 .Some experiments recommended that 20 mM endogenous proline can completely switch off singlet oxygen 74 .Proline functions as a protein compatible osmolyte, scavenging free toxic oxygen and nitrogen radicals, buffering cellular redox potential to stabilize compartmental structures, membranes, and other inclusions.Our results recommended the positive role of Pro in wheat facing Ni 2+ stress, might be attributed to upregulation of antioxidant enzymes against ROS in plants.Proline also has detrimental role in maintaining osmotic balance by keeping ionic homeostasis, successfully scavenging ROS, stabilizing antioxidant enzymes that minimize oxidative damage, and enhancing growth and yield 75 .The higher accumulation of proline in the plant is a response to the induction of stress that can cause the plant to show tolerance mechanism.Proline accumulated in response to metals stress in many plants particularly against cadmium in chickpea, and olive, Ni in pea, and aluminium in trifoliate orange 76,77 .The application of Ni inhibited essential elements (N, P, K) and showed higher accumulation of MDA under Ni 2+ stress 78

Micronutrient regulation by proline under Ni stress
Plants that are exposed to heavy metals excessively have lower concentrations of macro-and micronutrients.Plant growth and development require the correct levels of Ni and other minerals.Because Ni has the same ionic charge as Mg 2+ , Fe 2+ , and Zn 2+ , it exhibits properties comparable to those of these metals 79 .Nickel competes with both macronutrients (Ca, Mg) and micronutrients (Fe, Cu, and Zn) in plant sorption and transpiration because of their comparable properties 80 .Due to these similarities, a high Ni content prevents these nutrients from being absorbed, translocated, and sorption by plants 81 .Our findings support earlier research showing that Ni toxicity dramatically reduced the quantities of nutrients in wheat shoots.Proline has certainly improved the nutrient concentration both in stressed and non-stressed wheat seedlings.According to Zouari et al. 82 , exogenous proline also plays a protective role in osmotic adjustment maintenance, ionic homeostasis preservation, efficient ROS scavenging, antioxidant enzyme stabilization that reduces oxidative damage, plant photosynthetic rate enhancement, and growth and yield enhancement.

Conclusion
Our objective regarding current study was to examine the hypothesis that Pro can mitigate the deleterious effect of Ni toxicity in wheat seedling.Our findings show that Pro can upregulate both morphological and biochemical parameters in such a way that enhances the plant tolerance to Ni pollution.This regulatory mechanism was clearly embodied in improving the chlorophyll pigments that was associated with the concomitant increase in the plant biomass, higher ionic uptake, and yield of both wheat cultivars with and without Ni stress.This alleviation effects not only enhanced photosynthesis and biomass, but also regulated the biosynthesis and accumulation of
. The phytotoxicity induced by Ni on different growth and physiological parameters of wheat was mitigated by Pro

Figure 9 .
Figure 9. Correlation plot showing correlation matrix for various variables under Nickel and Proline treatments.Correlogram based on Pearson correlation.Only significant correlations (p < 0.05) are colored.Color gradient shows degree of relationship while positive and negative correlations were presented with different colors.(CAT: catalase, POD: peroxidase, AsA: Ascorbic acid, SOD: superoxide dismutase, TSP: total soluble proteins, TSS: total soluble sugars, S Na: shoot sodium, MDA: malondialdehyde, H 2 O 2 : hydrogen peroxide, 1000 GW: 1000 grain weight, SDW: shoot dry weight, RFW: root fresh weight, Chl a: chlorophyll a, T Chl: total chlorophyll, Chl b: chlorophyll b, RDW: root dry weight, SL: shoot length, SFW: shoot fresh weight, S K: shoot potassium, RL: root length, S Ca: shoot calcium).