Putrescine-functionalized carbon quantum dot nanoparticles (Put-CQD) effectively prime grape (Vitis vinifera cv. Sultana) against salt stress

Salinity is an important global problem with destructive impacts on plants leading to different biochemical and metabolic changes in plants through induced oxidative stress that disturbs metabolism, growth, performance and productivity of plants. Given that putrescine (Put) and carbon quantum dots (CQDs), individually, have promising effects on different plants processes, the idea of their combination in a nano-structure “Put-CQD” caused to its synthesizing for doubled positive effects. The current study aimed to investigate the application of newly-synthesized nanoparticles (NPs) consisting of CQDs and Put on grapevine (Vitis vinifera cv. Sultana) under salinity stress conditions. For this purpose, Put, CQDs and Put-CQD NPs at 5 and 10 mg L -1 concentrations were applied as chemical priming agents on ‘Sultana’ grapes 48 h prior salinity imposition (0 and 100 mM NaCl). Salinity signicantly decreased (P ≤ 0.05) morphological parameters, photosynthesis pigments, chlorophyll uorescence parameters and membrane stability index. In addition, salinity enhanced MDA, H 2 O 2 , proline and antioxidant enzymes activity. The results revealed that Put-CQD NPs, particularly at 10 mg L -1 concentration, alleviated the destructive impacts of salinity stress by improving leaf fresh and dry weights, K + content, photosynthetic pigments, chlorophyll uorescence and SPAD parameters, free proline content, total phenolics and antioxidant enzymatic activities (CAT, APX, GP and SOD), while decreasing Na + content, EL, MDA and H 2 O 2 levels. To conclude, Put-CQD NPs represent an innovative priming treatment that could be effectively applied on grapevine to improve plant performance under salinity stress conditions. putrescine-functionalized carbon quantum dots (Put-CQD NPs) at two (5 and 10 mg each treatment in three replications. Treatments were done in combination with Hoagland solution into the culture medium of pots. The last application of priming treatments was performed 48 h prior to imposition of salt two and through Hoagland and continued up to a month. All biochemical and enzymatic measurements were implemented three days after imposition of salt using fully expanded Sampled leaves were instantaneously 2 min and afterwards at °C freezer until measurements were carried out. Leaf samples were used to assay hydrogen peroxide (H 2 O 2 malondialdehyde (MDA), electrolyte leakage (EL), total phenolic compounds, proline, and antioxidant enzymatic activities. Other parameters including Na + /K + content, photosynthetic parameters and pigments were investigated a month after salinity application. Pigments were examined via the same above-mentioned sampling protocol, while leaf fresh and dry weights and photosynthetic parameters were assayed using fresh leaves. Three technical replications were used for each measurement. Control plants


Agronomic parameters
Salinity negatively affected leaf FW and DW. Leaf FW increased signi cantly (P≤0.05) by 10 mg L -1 Put and 5 and 10 mg L -1 Put-CQD NPs; however, the other treatments had no effect or decreased the weight under control conditions. All treatments enhanced leaf DW under control conditions. Under salt stress condition, 10 mg L -1 CQD NPs and 5 and 10 mg L -1 Put-CQD NPs increased leaf FW, while the other priming treatments exerted no effect compared with unprimed grapevines under salinity. All priming treatments enhanced leaf DW under salinity condition. Under both conditions, 10 mg L -1 Put-CQD NPs could be considered as the optimal priming treatment ( Table 1).

Ionic homeostasis
Salinity signi cantly decreased (P≤0.05) K + content and increased Na + content and Na + /K + ratio. Under control conditions, all treatments (except 5 mg L -1 CQDs) lowered Na + content compared with the control. K + content signi cantly increased by priming with 5 and 10 mg L -1 Put-CQD NPs, while the other treatments had no effect. Consequently, all priming treatments increased the Na + /K + ratio, with the exception of 5 mg L -1 put and 10 mg L -1 CQDs which had no effect compared with controls. Under salinity conditions, all treatments decreased Na + content and Na + /K + ratios, while Put and Put-CQD NPs treatments (5 and 10 mg L -1 ) increased K + content. Put-CQD NPs at 10 mg L -1 concentration demonstrated the best results for K + , Na + and Na + /K + ratio under both conditions (Table 1).

Physiological parameters
Chl a, b and carotenoid content were decreased by salinity. Signi cant increase (P≤0.05) in Chl a and b content was observed following Put and Put-CQD NPs priming treatments; all priming treatments enhanced carotenoid content under control conditions compared with the control. Under stress conditions, all treatments signi cantly increased Chl a,b and carotenoid content. Put-CQD NPs at 10 mg L -1 lead to the highest value for Chl a,b and carotenoid levels under both control and stress conditions. SPAD value was negatively affected by salinity. With the exception of CQDs at 10 mg L -1 which showed no signi cant difference to the control, other treatments enhanced SPAD values under non-stress conditions. Increase in SPAD values was additionally achieved by Put (5 and 10 mg L -1 ), CQDs (10 mg L -1 ) and Put-CQD NPs (5 and 10 mg L -1 ) priming treatments under salt stress conditions. The highest SPAD value was recorded at 10 mg L -1 Put-CQD NPs-primed grapevines ( Table 2).
Chlorophyll uorescence parameters were signi cantly negatively affected after imposing salinity. Fv / Fm was increased by all treatments, with the sole exception of 5 mg L -1 CQDs priming treatment which showed no difference to unprimed, unstressed samples. All priming treatments positively affected Fv / Fm under stress conditions. Put and Put-CQD NPs priming treatments increased Fv / Fo parameter under control and stress conditions. Regarding Y (II) parameter, Put-CQD NP treatments (5 and 10 mg L -1 ) leads to its signi cant increase (P≤0.05) under control conditions. Under salinity stress, all treatments enhanced Y (II) parameter. Considering all parameters, Put-CQD NPs at 10 mg L -1 concentration represented the optimal treatment under both control-stress and stress conditions (Table 3).

Cellular damage indicators
Salinity caused signi cant enhancement (P≤0.05) in Electrolyte leakage (EL) value. All priming treatments signi cantly decreased EL values under both control and stress conditions with optimal protection being achieved following 5 mg L -1 Put-CQD NPs priming treatment (Fig. 3).
As expected, MDA and H 2 O 2 contents increased after imposing salinity stress, while priming treatments signi cantly decreased (P≤0.05) MDA and H 2 O 2 contents under both control and stress conditions. In general, 10 mg L -1 Put-CQD NP priming treatment provided optimal results in terms of amelioration of MDA and H 2 O 2 increases under both conditions (Fig. 4a,b).
Proline and total phenolic compounds Salinity signi cantly enhanced (P≤0.05) proline content and total phenolic compounds of grape (Fig. 5). Under control and stress conditions, priming treatments including Put at 10 mg L -1 and Put-CQD NPs at 5 and 10 mg L -1 concentrations signi cantly increased proline content, while the other treatment exerted no signi cant effectin this regard. The highest proline content was recorded following 10 mg L -1 Put-CQD NP treatment under both conditions (Fig. 5a). Under control conditions, all priming treatments (with the exception of Put and CQDs at 5 mg L -1 concentrations) increased total phenolics. Under salinity conditions, 10 mg L -1 CQDs and 5 and 10 mg L -1 Put-CQD NP priming treatments enhanced phenolic content; the other treatments had no effect compared with unprimed grapevine under salinity. The highest proline content was recorded following 10 mg L -1 Put-CQD NP priming grapes under salt stress condition (Fig. 5b).

Antioxidant enzymatic activities
Antioxidant enzymatic activities (CAT, APX, GP and SOD) were enhanced after imposing salinity ( Figure 6). All priming treatments increased CAT enzyme activity under control and stress conditions, with the highest activity of the enzyme being recorded following 10 mg L -1 Put-CQD NP application, followed by Put at 10 mg L -1 concentration under salinity conditions (Fig. 6a).
Considering APX enzyme, all priming treatments enhanced its activity under control conditions with optimal results following 5 and 10 mg L -1 Put-CQD NP treatments. Under salinity conditions, only 5 and 10 mg L -1 Put-CQD NP priming signi cantly increased APX activity, while other priming treatments had no signi cant effect compared with unprimed grapevines under salinity (Fig. 6b).
Under control conditions, all treatments increased GP enzyme activity with optimal activity being recorded following 10 mg L -1 Put-CQD NP treatment. Most treatments, enhanced GP activity under salt stress, with the exception of 5 mg L -1 Put and 5 mg L -1 CQD treatments which demonstrated similar GP activity in unprimed grapevines under salinity. The highest activity was recorded following application of 5 mg L -1 Put-CQD NPs (Fig. 6c).
SOD enzymatic activity increased signi cantly (P≤0.05) following application of all priming treatments under control condition.
The highest activity was recorded following 10 mg L -1 Put-CQD NP treatment. Under salinity conditions, all treatments (except 5 mg L -1 Put) enhanced SOD activity, with optimal results being recorded following 5 and 10 mg L -1 Put-CQD NP priming treatments (Fig. 6d).

Discussion
Salinity negatively affects plant growth and its physiological, metabolic and biological routes through induced osmotic stress [2]. Therefore, salinity decreases leaf FW and DW (e.g., Zhang et al. [23]), in accordance with present ndings. The positive effect of some NPs on agronomic traits under salinity stress has been previously reported (e.g., Gohari et al. [12,13]), in line with Put-CQD NPs effects. Priming with Put also exerted positive effects regarding leaf FW and DW correlating with enhanced salt tolerance of grapevine. Similar ndings, supporting the protective role of Put or other PAs in plants under salinity stress, were previously reported by Saleethong et al. [26], Zhang et al. [23] and Khoshbakht et al. [28]. Put effects in improvement of growth parameters could be described via its polycationic nature and regulation of ion metabolism that enhance tolerance to salinity [28]. Therefore, positive effects of Put-CQD NPs could be attributed to the above-mentioned reasons.
In general, salinity enhances Na + and reduces K + ions in plants due to excessive amounts of Na + in soil or nutrient solution that imbalance ion uptake by plants [27,28,31]. Bene cial effect of Put or other PAs on reducing Na + and increasing K + contents in plants under salinity was reported [23,26]. This impact could be described through regulation of ion channel activity in root cells by PAs (e.g. Put) to repress Na + in ux into roots and enhance K + in ux from roots to shoots. In addition, reduction in the activity of plasma membrane-bound H + -ATPase via applied salinity could be alleviated by PA application, which then decreases Na + and increases K + contents in plants grown under salinity conditions [26]. Another possible reason for positive effect of Put in this regard might be through its role in stabilizing membranes and maintaining cation-anion balance [28]. Put acts as a signaling regulator responsible for inward recti cation of rectifying K + channels [28]. Mozafari et al. [31] reported positive effect of iron-NPs on decreasing Na + and increasing K + contents in plants under salinity conditions. To our knowledge, no previous report existed Page 6/16 showing the effect of carbon-based NPs on Na + and K + contents in plants under salt stress conditions. This is therefore the rst report demonstrating the positive effect of Put-CQD NPs on reducing Na + and enhancing K + contents in plants under salt stress conditions. Salinity causes a drop in photosynthetic pigment content including chl a, b and carotenoids. Chl content declines through decrease in chl biosynthesis and increase in its degradation/turnover. Breakdown of photosynthetic pigments could occur via the accumulation of toxic ions in chloroplasts and enhancing oxidative stress in plants after imposing salinity stress [33]. In addition, inhibition of photochemical reactions and down-regulation of chloroplast-encoded genes due to salinity cause lead to a decrease in chl content [26]. Reduction in chl a and b content of plants under salinity stress conditions was noticed by Hatami et al. [33] and Gohari et al. [11,12,13], in accordance with the current study. PAs could reverse these negative effects of salinity by stabilizing oligomeric photosynthetic proteins and in particular the chl a/b-binding proteins displaying protease action during stress [26]. Although CQDs treatments enhanced chl a, b and total chlorophyll, further increase in CQDs concentration subsequently lowered their values [34]. Besides, Gohari et al. [12] reported positive effect of modi ed-MWCNTs in chl a and b content. The current study reported positive effect of CQDs in chl a and b content under salinity conditions. Salinity stress is known to alter biosynthesis and accumulation of secondary metabolites like carotenoids [35]. Decrease in carotenoids of grapevine under salt stress might be due to induction of the pathway for abscisic acid production in order to modulate plant growth [19]. Increased carotenoids improve plant tolerance to stress condition due to quenching of ROS and preserving chloroplast from photo oxidation under stress conditions through their non-enzymatic antioxidant function [36]. Positive effect of some NPs such as TiO 2 and MWCNTs NPs on carotenoids has also been previously noticed in plants grown under stress conditions [12,13], thus supporting the encouraging impacts of Put-CQD NPs on carotenoid content in grapevine under salinity conditions.
Chlorophyll uorescence parameters are solid markers for the evaluation of physiological properties of plants and the detection of stress effects. A noteworthy decrease in chlorophyll uorescence parameters may be caused by the dissipation of a major proportion of light energy as heat under salt stress [37], previously reported by Netondo et al. [38] and Gohari et al. [12,13]. Some studies indicated chlorophyll uorescence adjustment following NP application [12,13]. Increase in chl a and b could occur through enhancement in light energy of PSI absorbed by chloroplast membrane to be transferred to PSII, promotion of light energy conversion to electron energy and electron transport and acceleration of water photolysis and oxygen evolution [39]. Another probable reason might be the increased absorption of carbon dioxide in plants and RuBisCO enzyme activity with an important role in photosynthesis and chlorophyll uorescence parameters [40].
Electrolyte leakage is a reliable cellular damage indicator that could identify any damage to cell membrane integrity [41]. Salinity increases EL value by disrupting cell membrane integrity. PA application lowered EL value of plants under salinity through their polycationic nature causing direct binding to negatively charged membrane phospholipid head groups that maintain membrane function and stabilized it under stress conditions [26,28]. Furthermore, SWCNT and MWCNT application at lower doses decreased EL value and increased cell membrane stability under salinity condition [12,33], while reduction in EL was also reported after SiO 2 NP application [42].
ROS generation at high concentrations has destructive impacts such as lipid peroxidation that disturbs membrane integrity and enhances MDA content [43]. Increased MDA content was reported in plants under salinity. Exogenous application of PAs decreased MDA levels in plants under salinity [24,26,27,44]. Furthermore, graphene QD application at low doses decreased MDA content [45]. Mozafari et al. [31] observed similar mitigating effect of iron-NPs on decreasing MDA content of grapevine under salinity. It is likely that stabilizing membrane integrity by Put and CQDs and their conjugated form (Put-CQD NPs) could (at least in part) justify the decrease in MDA content under salinity condition. H 2 O 2 , a key regulator for multiple processes linked with growth, development and stress protection [46], has binary effects depending on its concentration. At low concentration, it acts as a signaling molecule needed for initiation of resistance mechanisms to biotic and abiotic stresses, while it results in oxidative stress [47] and programmed cell death when at high concentrations. H 2 O 2 leads to lipid peroxidation through hydroxyl radical formation [48]. Increase in H 2 O 2 content of grapevine under salinity was previously reported [31]. The observed decrease in H 2 O 2 content following Put priming of plants under salinity comes in agreement with previous reports [24,26]. This ameliorative effect could be described through the scavenging of free radicals and protection of proteins by PAs [28]. In fact, PAs could reverse salinity effects like ROS generation (e.g., H 2 O 2 ), lipid peroxidation and corresponding MDA production [26]. Increase in proline by the treatments application could describe decrease in H 2 O 2 values as increased proline could reduce H 2 O 2 and other radicals either itself or by activating antioxidant enzymes activities (e.g., SOD, APX, GP and CAT) [49]. Mozafari et al. [31] reported that iron-NPs decrease H 2 O 2 content in grapevine plants under salinity via increasing the antioxidant enzyme activities.
Proline is an osmolyte, metal chelating, antioxidant and signaling molecule [50]. Proline accumulates under abiotic stresses due to its role as osmotic regulator and ROS detoxi er that preserves membrane integrity, subcellular structures, antioxidant enzymatic activities and protein structure [31,50]. Increase in proline content in salt-stressed grapevine has been previously recorded [31], likely due to a decrease in proline oxidation and increase in its biosynthesis [51]. Exogenous PA application including Put increased proline accumulation in plants under salinity stress [26,44]. This enhancement could be considered as a mechanism to protect plants against salinity since proline is an osmolyte, storage material for nitrogen through stress, ROS scavenger and a modulator for NADP + /NADPH redox state. Increased proline content following PA application leads to protection of intercellular macromolecules, osmotic adoptability and the scavenging of hydroxyl radicals resulting in salt tolerance [26].
Iron-NPs were also shown to enhance proline content of grapevine under control and salinity conditions [31]. This enhancement was reported following QD application, as well [45], in accordance with the current study.
Phenolics protect plant cells through their potential to act as non-enzymatic and water-soluble antioxidants. This property is achieved via quenching of ROS and free radicals [52]. Phenolics prevent ROS generation and accumulation, thus inhibiting oxidative stress and reducing its undesirable effects [16]. Most phenolics are stimulated under biotic and abiotic stresses (Lim et al., 2015), such as under salinity conditions [52]. Feng et al. [45] reported increased phenolics following application of low concentration of QDs. In addition, MWCNTs-COOH treatment enhanced plant phenolics under salinity conditions [12]. Current results demonstrated positive effect of CQDs at 10 mg L -1 and Put-CQDs at both concentrations probably via enhanced biosynthesis, as a line of antioxidant defense against oxidative stress imposed by NaCl. He et al. [53] demonstrated that PA application (spermidine) induced expression of genes related to enzymatic and non-enzymatic antioxidants (e.g. phenolics compound) in plants under salinity, in partial agreement with current ndings. SOD enzymatic activity eliminates superoxide radicals by dissimulating to H 2 O 2, which is then detoxi ed by several antioxidant enzymes (e.g. CAT, POD, APX, GP) [46]. CAT, as the main enzyme for H 2 O 2 quenching, removeσ superoxide radicals as the rst step of defense against ROS to reduce oxidative stress damage. APX removes H 2 O 2, similar to CAT, through the glutathioneascorbate cycle [54]. GP enzyme utilizes glutathione to detoxify H 2 O 2, reducing lipids and organic hydroperoxides [46]. As salinity causes oxidative stress via ROS generation and accumulation in plant cells, antioxidant enzymes (such as CAT, SOD, GP and APX) could act as a defense mechanism for ROS detoxi cation. Accordingly, ROS quenching via antioxidant enzymatic activities reduces stress impacts, as an essential strategy for enhanced tolerance to stress conditions [26]. Increase in major antioxidant enzymatic activities has been well recorded in plants after imposing salinity [26,54,55], in agreement with current ndings. PAs enhance the activity of antioxidant enzymes and non-enzymatic antioxidants (e.g. anthocyanins, avonoids; Zhang et al. [23], Saleethong et al. [26]). In addition, PAs increase plant tolerance to salinity stress by scavenging free radicals of cells and improving cell survivability [24], as well as by inducing the expression of genes encoding antioxidant enzymes. Interestingly, PAs also act as a direct free radical scavenger, due to PAs binding to antioxidant enzyme molecules [28]. Put prevents membrane peroxidation and denaturing of biomolecules under salinity through two mechanisms: First, extensive protonation of PAs at physiological pH enables them to scavenge free radicals directly or by conjugating to cell membrane; second, PAs by increasing antioxidant enzymatic activities and thus enhancing ROS detoxi cation and reducing oxidative damage. In total, these mechanisms lead to plant protection against salinity stress [23]. Such results were reported following exogenous application of PAs including Put on increased antioxidant enzymatic activities (SOD and CAT), leading to decreased ROS effects and membrane injuries [28]. In terms of the effect of nanomaterials, iron-NPs enhanced APX, SOD and POD enzymatic activities of grapevine under salinity conditions [31]. Feng et al. [45] reported enhanced CAT activity at lower graphene QD concentration, likely due to enhanced oxidative stress that reduces biosynthesis of antioxidant enzymes like CAT. Gohari et al. [12,13]  Hoagland solution and continued up to a month. All biochemical and enzymatic measurements were implemented three days after imposition of salt stress using fully expanded leaves. Sampled leaves were instantaneously kept into liquid nitrogen for 2 min and afterwards preserved at -80 °C freezer until measurements were carried out. Leaf samples were used to assay hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA), electrolyte leakage (EL), total phenolic compounds, proline, and antioxidant enzymatic activities. Other parameters including Na + /K + content, photosynthetic parameters and pigments were investigated a month after salinity application. Pigments were examined via the same above-mentioned sampling protocol, while leaf fresh and dry weights and photosynthetic parameters were assayed using fresh leaves. Three technical replications were used for each measurement.
Control plants were irrigated simply with ½-strength Hoagland solution.
Preparation of putrescine functionalized carbon quantum dots (Put-CQD NPs) In a 25 mL Te on-lined autoclave chamber containing 10 mL distilled water, 0.5 g putrescine and 2 g citric acid were added and heated at 200 °C for 12 h. After cooling the reaction temperature to room temperature, the pH value of resulted red-brown solution was set to 7 by NaOH before use and characterization. For comparison, the same procedure was used to synthesize bare CQDs.

Leaf fresh and dry weights
Five leaf samples were individually weighed for fresh weight (FW) and then kept in the oven (70 °C, 72 h) for dry weight (DW) measurements at the harvest stage. Na + and K + assay Leaf samples were randomly collected from each treatment, washed and air dried and then dried in hot-air oven at 60°C for 18 h.
Afterwards, the samples were ground in Willey mill and the powered samples were stored for the assay. The triacid digestion extract was used for estimation of Na + and K + by ame photometry as out-lined by Ghosh [56] and expressed in mmol kg -1 .

Quanti cation of photosynthetic pigments (chlorophyll a, b and carotenoids)
Fully expanded leaves (0.2 g) were extracted in 0.5 mL acetone (3% v/v) and then centrifuged (10000 rpm, 10 min) and the absorption of the obtained supernatant was recorded at 645 nm (Chl b), 663 nm (Chl a) and 470 nm (carotenoids) by UV-Vis spectrophotometry (UV-1800 Shimadzu, Japan). Chl a, b and carotenoids contents were calculated through the equations described by Sharma et al. [57].

Chlorophyll uorescence and SPAD assay
A dual-pam-100 chlorophyll uorometer (Heinz Walz, Effeltrich, Germany) was used to measure chlorophyll uorescence parameters including Fv / Fo , Fv / Fm and Y (II). The measurement was done after the plants were dark-adapted for 20 min [58].
Five randomly selected leaves of each pot were used to determine SPAD values (leaf chlorophyll concentrations) via a SPADmeter (502 Plus Chlorophyll Meter, Japan) [59].

Electrolyte leakage (EL) assay
For EL assay, 0.5-cm diameter discs of fully expanded leaves were cut; the discs were then washed thrice by deionized water and incubated in ambient temperate for 24 h. A conductivity meter (Hanna, HI98192) was used to measure the initial electrical

Proline quanti cation
To assay proline content, 0.5 g leaf samples were homogenized in 10 mL aqueous sulfosalicylic acid (3%) in an ice bath. After centrifuging (1000 rpm, 4°C), 2 mL ninhydrin acid and 2 mL g lacial acetic acid (a 1:1:1 solution) were added to 2 mL supernatant, nely mixed and incubated at 100 ºC for 1 h. The reaction was stopped in an ice bath and nally 4 mL toluene was added and mixed vigorously (20 s). The mixture absorbance was recorded at 520 nm using the spectrophotometer. Different concentration of L-proline was used for standard curve and nal calculation of proline values [63].
Quanti cation of total phenolic compounds Briefly, after digesting 0.1 g leaf sample with 5 mL 95% ethanol, the mixture was kept in dark (24 h) and then to 1 mL of supernatant, 1 mL 95% ethanol and 3 mL distilled water were added. Next step was adding 0.5 mL 50% Folin-Ciocalteu solution and 1 mL 5% sodium bicarbonate, and after 1 h in the dark, the absorbance was recorded at 725 nm using the spectrophotometer. The absorbance values were converted to total phenols through standard curve made by different concentrations of gallic acid and expressed as mg gallic acid (GAE) g -1 FW [64].

Assay of antioxidant enzymatic activities
Total soluble proteins and antioxidant enzymes activities were assayed through leaves formerly stored at -80 °C freezer. All steps of enzyme extraction were carried out at 4 °C as follows: leaves (0.5 g) were homogenized with potassium phosphate buffer (pH 6.8, 100 mM) containing 1 % polyvinylpyrrolidone (PVP) and EDTA (4 mM) using magnetic stirrer for 10 min. After centrifuging (6000 rpm, 20 min), the supernatant was collected to evaluate total soluble proteins, catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and guaiacol peroxidase (GP) enzymatic activities based on the same procedures described by Gohari et al. (2020b).

Statistical analysis
All obtained data analysis was performed by SAS software and the means of each treatment were analyzed by Duncan's multiple range test at the 95% level of probability (SAS Institute Inc., ver. 9.1, Cary, NC, USA).

Declarations
Ethics approval and consent to participate We con rm that our study does not involve human subjects.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request.
Competing interests 60. Nayyar H. Accumulation of osmolytes and osmotic adjustment in water-stressed wheat (Triticum aestivum) and maize (Zea mays) as affected by calcium and its antagonists.