1 Introduction

Osmotic stress followed by specific ion toxicity is the major limiting factor for growth of plants cultivated on saline soils (Munns et al. 2016). Presence of excessive dissolved salts in saline soils reduces soil matric potential that alters plant root water uptake capacity (Sheldon et al. 2017; Tedeschi et al. 2017). Plants therefore experience water stress and ion-disequilibrium under hyper-osmolarity growth conditions (Alemán et al. 2009) which negatively affects turgor pressure and cellular expansion (Taiz and Zeiger 2010). Therefore, salinity-mediated physiological drought inhibits stomatal conductance directly affecting leaf photosynthetic activity (Tavakkoli et al. 2010).

One of the major factors affecting plant growth on saline soils is specific ion toxicity or Na+ toxicity (Zhu 2002). Higher soil Na+ trigger K+ deficiency primarily due to competition through high-affinity K+ transporters in plant root cells (Alemán et al. 2009; Hauser and Horie 2010; Pardo and Rubio 2011). The increased Na+ uptake and its higher concentration inside leaf mesophyll cells initiate specific ion toxicity (Taiz and Zeiger 2010; Hasegawa 2013) disturbing cellular ion homeostasis (Hasegawa et al. 2000; Munns and Tester 2008; Teakle and Tyerman 2010). In addition, redox reactions of photosynthesis and respiration are often impaired due to sodium-mediated enhanced production of reactive oxygen species (ROS) causing oxidative stress (Mittler 2002; Tavakkoli et al. 2010). Considering ion homeostasis, it has been suggested that K+ retention and higher Ca2+ inside cells is an important strategy in order to balance ion-disequilibrium to avoid Na+ build up (Marshner 1995; Shabala and Cuin 2007; Shahbala et al. 2016). Other than this, the ability of certain plants to exclude Na+ is also an important stress tolerance strategy (Colmer et al. 2006; Munns and Tester 2008). Nonetheless, plant responses to counter salinity stress vary greatly depending upon growth stages and stress severity.

Wheat (Triticum aestivum L.) is one of the dominant cereal crops and regarded as a moderately salt-tolerant glycophyte. On the other hand, PHF are carbon nanoparticles which exhibit remarkable electron-scavenging properties (Andrievsky et al. 2005; Sachkova et al. 2017). Recently involvement of PHF in drought tolerance of sugar beet (Borišev et al. 2016) and Brassica napus (Xiong et al. 2018) explained on the basis of improvements in oxidative stress tolerance and abcisic acid. Since high Na+ concentrations ultimately disturb cellular redox status, it will be interesting to investigate how the leaf tissue ion homeostasis responds to external supply of synthetic antioxidant. Accordingly, it was hypothesized that PHF application may improve tissue ion homeostasis through modulation of wheat antioxidant capacity and oxidative stress.

2 Materials and Methods

2.1 PHF Seed Invigoration and Seedling Growth

The PHF nanoparticles [C60(OH)20] were purchased from BuckyUSA (Houston Texas, USA). For seed priming, wheat seeds (cv. Ujala-2016) were soaked in different PHF concentrations viz. control (un-primed), hydro-priming, 10, 40, 80, and 120 nM PHF for 10 h with continuous aeration. After 10 h, the seeds were removed, the surface sterilized with a solution of sodium hypochlorite (0.1%) for 1 min, and then washed five times with de-ionized water. Finally, seeds were dried with blotting paper and sown in pots containing sand under control (0 mM NaCl) and salinity (150 mM NaCl) stress provided through Hoagland’s nutrient solution. The experiment was performed with four replications per treatment under control environmental conditions inside a plant growth chamber (Sanyo, Model MLR-351H) with following growth conditions (PAR, 370 μmol m−2 s−1; photoperiod, 10 h; day/night temperature, 18/23 °C).

2.2 Study of Growth and Biochemical Attributes

After 30 days of seed germination, seedlings were investigated for changes in various growth parameters and biochemical parameters. Photosynthetic pigments were quantified after extraction of fresh leaf tissue (100 mg) with acetone (80%) as described earlier (Arnon 1949; Kirk and Allen 1965). Concentration of oxidative stress indicators was determined after homogenization of fresh plant material in trichloroacetic acid (TCA) (10%) followed by centrifugation at 7000g. The concentration of malondialdehyde (MDA) was determined using thiobarbituric acid (Mukherjee and Choudhuri 1983) while hydrogen peroxide (H2O2) contents were determined using potassium iodide as described (Velikova et al. 2000).

For enzymatic antioxidant analyses, the homogenization of fresh plant material (200 mg) was performed in ice-cold potassium phosphate buffer (100 mM; pH 7.8). The homogenate was centrifuged at 12000g and supernatant was referred as a crude protein extract. The determination of antioxidant enzyme activities of SOD, POD, CAT, and APX were performed from this crude protein extract as described (Beauchamp and Fridovich 1971; Aebi 1984; Chance and Maehly 1955; Nakano and Asada 1981). Total soluble proteins were also quantified as described by Bradford (1976). The antioxidant activities were then presented as U mg−1 protein basis.

Furthermore, plant ethanol extracts were prepared for the determination of non-enzymatic antioxidants and some key osmolytes. For this purpose, 50 mg of plant dry material was homogenized with 10 mL ethanol (80%) and filtered through Whatman No. 41 filter paper. The residue was re-extracted with ethanol and the two extracts were pooled together to a final volume of 20 mL. The determination of flavonoids (Pękal and Pyrzynska 2014), phenolics (Bray and Thorpe 1954), free amino acids (Hamilton and Van Slyke 1943) and total sugars (Dubois et al. 1956) was performed from the extracts.

2.3 Study of Changes in Inorganic Ions

For the determination of various ions, dried plant material of shoot and root was digested using H2SO4/H2O2 mixture (Wolf 1982). Analyses of Na+, K+, and Ca2+ ions were carried out by using a flame photometer (Jenway PFP-7, UK). Moreover, the P contents were determined by using Barton’s reagent as described (Barton 1948). The inorganic ions were finally calculated by using respective calibration curves and ions presented in mg g−1 DW basis. In addition, the analyses of root and shoot Mg2+, Mn2+, Zn2+, and Fe2+ contents was performed using atomic absorption spectrophotometer (Supplementary file).

2.4 Statistical Analyses

Data collected for all the attributes was statistically analyzed by using CoStat software (CoHort Software, Monterey, CA, USA) using completely randomized layout. Differences among means were assessed by Duncan’s multiple range (DMR) test at 95% confidence level.

3 Results

3.1 PHF-Mediated Changes in Growth Attributes of Control and Salt-Stressed Wheat

Non-toxic and growth-promoting influence of PHF seed pre-treatment on shoot growth attributes of wheat seedlings was recorded both under control and salinity (Table 1). Seedlings exposed to 150 mM NaCl exhibited substantial reduction in shoot length, fresh weight and shoot dry weight (P < 0.01). On the other hand, seedlings germinated from PHF pre-treated seeds exhibited recovery in shoot growth attributes compared with untreated control seedlings (P < 0.05; Table-1). Similar to shoot, the root growth of salinity exhibited prominent reduction in terms of root length, root fresh and dry weight respectively (P < 0.05). In contrast, the PHF treated wheat seedlings exhibited better root growth attributes under salinity compared with un-treated ones (P < 0.05; Table-1). The PHF seed pre-treatment also improved the root growth attributes of plant grown without NaCl in the growth medium (P < 0.05).

Table 1 Polyhydroxy fullerene (PHF) seed pre-treatment mediated changes in the growth attributes of salinity-stressed wheat seedlings (means; n = 4 ± SE)
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3.2 PHF-Mediated Changes in Biochemical Attributes of Control and Salt-Stressed Wheat

3.2.1 Photosynthetic Pigments

Prominent reduction (P < 0.01) in chlorophyll a, b and total chlorophyll was evident under salinity. Similarly, reduction in carotenoids was evident among wheat seedlings exposed to salinity stress (P < 0.05; Table 2). However, in contrast, the wheat plants grown from PHF pre-treated seeds had higher chlorophyll a, b and total chlorophyll contents. Maximum improvements (P < 0.05) in the chlorophyll contents were evident in response to 40 and 80 nM PHF seed pre-treatments. Likewise, PHF mediated increase in the carotenoids was also recorded under salinity (Table 2). Overall, the Chl a b−1 and Car Chl−1 relative ratios increased under salinity (P < 0.05).

Table 2 Polyhydroxy fullerene (PHF) seed pre-treatment mediated changes in the photosynthetic pigments of salinity-stressed wheat seedlings (means; n = 4 ± SE)
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3.2.2 Oxidative Stress Markers, Antioxidants, and Osmolytes

The 30-day-old wheat plants exhibited prominent (P < 0.01) increase in MDA and H2O2 contents under salinity. Also, prominent increase in the antioxidant activities of SOD and APX was recorded under salinity (P < 0.05). Interestingly, the PHF-treated wheat plants exhibited substantial increase (P < 0.01) in the activities of CAT, POD, and APX antioxidant enzymes. While, PHF-mediated reduction in the SOD activity was also recorded (P < 0.05). In addition, the PHF seed pre-treatment resulted in substantial reduction in the MDA and H2O2 contents among wheat plants under salinity (Table 3; P < 0.05). Apart from this enzymatic antioxidants, wheat plants exposed to 150 mM NaCl exhibited higher values of ascorbic acid, flavonoids, total phenolics, and soluble sugars while reduced free amino acids (Table 4; P < 0.05). The PHF seed pre-treatment resulted in significant improvement (P < 0.01) in free amino acids and soluble sugars. The PHF treatment caused reduction (P < 0.05) in the total phenolics. Moreover, the influence of PHF on ascorbic acid and flavonoids under salinity stress was differential (Table 4).

Table 3 Polyhydroxy fullerene (PHF) seed pre-treatment mediated changes in the oxidative stress indicators and antioxidant activities of salinity-stressed wheat seedlings (means; n = 4 ± SE)
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Table 4 Polyhydroxy fullerene (PHF) seed pre-treatment mediated changes in the non-enzymatic antioxidants and osmolytes of salinity-stressed wheat seedlings (means; n = 4 ± SE)
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3.3 PHF-Mediated Changes in Inorganic Ions of Control and Salt-Stressed Wheat

3.3.1 Root Inorganic Ions

Wheat plants grown under salinity exhibited prominent (P < 0.01) increase in root Na+ concentration which ranged between 5.57 mg g−1 DW for control plants and 12.7 mg g−1 DW under salinity stress (Figure 1a). In contrast, salinity induced reduction (P < 0.05) in the root Ca2+ contents was recorded (Figure 1c), while root K+ and P contents exhibited insignificant differences. On the other hand, plants grown from PHF pre-treated seeds exhibited an increase (P < 0.01) in root K+ uptake under salinity (Fig. 1b). Furthermore, PHF-mediated improvements (P < 0.05) in the root P fraction were evident as well (Fig. 1d). Apart from this, the influence of PHF treatments on root Na+ and Ca2+ remained insignificant (P < 0.05). Reduction in the root Mn2+ contents was also recorded under salinity, while changes in Mg2+, Zn2+, and Fe were insignificant (Fig. S1A–D). Moreover, the influence of PHF on root Mg2+, Mn2+, Zn2+ and Fe contents was differential (Fig. S1A–D).

Fig. 1
figure 1

a–d Polyhydroxy fullerene (PHF) nanoparticles seed pre-treatment mediated changes in root Na+, K+, Ca2+, and P contents of control (0 mM NaCl) and salinity (150 mM NaCl) stressed wheat seedlings (n = 4; mean ± SE). Different lowercase letters in each attribute are significantly different at 95% confidence level

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3.3.2 Shoot Inorganic Ions

Similar to root, marked (P < 0.01) increase in the shoot Na+ contents was recorded under salinity (Fig. 2a). While seedlings grown from PHF-treated seeds exhibited considerable reduction in shoot Na+ (P < 0.05). Salinity also caused reduction (P < 0.05) in the shoot K+ and Ca2+ contents in comparison with control plants (Fig. 2b, c). The application of PHF nanoparticles at 40 nM concentrations improved (P < 0.05) shoot Ca2+ contents and also increased (P < 0.01) shoot P concentration at 40, 80, and 120 nM concentrations of wheat seedlings grown under salinity (Fig. 2d). Salinity induced changes in shoot P contents remained insignificant (P < 0.05). Apart from these, the influence of PHF on the shoot Mg2+, Mn2+, Zn2+, and Fe2+ contents remained insignificant (Fig. S2A–D).

Fig. 2
figure 2

a–d Polyhydroxy fullerene (PHF) nanoparticles seed pre-treatment mediated changes in shoot Na+, K+, Ca2+, and P contents of control (0 mM NaCl) and salinity (150 mM NaCl) stressed wheat seedlings (n = 4; mean ± SE). Different lowercase letters in each attribute are significantly different at 95% confidence level

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4 Discussion

Improvement in salinity tolerance of crops through various exogenous chemicals or biostimulants is an important area of research (Torbaghan et al. 2017; González-Pérez et al. 2018). Exposure of wheat seedlings to salinity reduced plant growth. In agreement, salinity inhibited plant growth through physio-biochemical disturbances (Hasegawa 2013; Geilfus 2018) and is also reported for wheat plants (Raza et al. 2007; Iqbal and Ashraf 2013; Shafiq et al. 2018). Wheat plants germinated from PHF-primed seeds exhibited prominent recovery in root and shoot growth under control and salinity. Consistent with these findings, PHF-application improved the growth attributes of Arabidopsis thaliana (Gao et al. 2011), Momordica charantia (Kole et al. 2013), and Hordeum vulgare (Panova et al. 2016). Moreover, a radio-labeled PHF compound improved wheat root growth under control growth conditions (Wang et al. 2016). In contrast, the evidence of PHF involvement in abiotic stress tolerance of plants is yet to be elucidated. So far, exogenous PHF-mediated drought tolerance in Beta vulgaris L. (Borišev et al. 2016) and Brassica napus L. (Xiong et al. 2018) is reported.

In this study, salinity caused reduction in Chl a, b and total Chl. Furthermore, a decrease in the carotenoid contents was also recorded for seedlings grown under salinity which caused an increase in the Car Chl−1 relative ratio under salinity stress. Salinity-mediated reduction in the photosynthetic pigments is reported earlier in wheat (Iqbal and Ashraf 2013) and is due to Na+ toxicity and oxidative stress (Shu et al. 2012; Raza et al. 2014; Shafiq et al. 2018). The wheat seedlings grown from PHF pre-treated seeds exhibited prominent recovery in the photosynthetic pigments under salinity stress. In this connection, PHF-treated control plants exhibited an increase in photosynthetic pigments (Wang et al. 2016). Our findings of PHF-mediated improvements in the photosynthetic pigments consistent with improvements in the growth attributes can be integrated with mitigation of oxidative stress.

Here, substantial increase in the MDA and H2O2 contents of wheat seedlings grown under salinity was recorded. Furthermore, an increase in the antioxidant activities of SOD, CAT, POD, and APX was also evident under salinity. While, PHF-treated plants exhibited marked reduction in oxidative stress under salinity, indicated by lesser contents of MDA and H2O2. The PHF-induced decrease in MDA and H2O2 contents in maize was attributed to better antioxidant capacity (Assemi et al. 2010). Here as well, PHF seed invigoration also improved the enzymatic antioxidant activities of CAT, POD, and APX. Consistent with these findings, the application of PHF as foliar spray increased the activities of CAT, APX, GPX, and GST antioxidant enzymes linked with H2O2 detoxification (Borišev et al. 2016). Further, PHF-mediated improvements in SOD and CAT activities among maize seedlings were also reported (Liu et al. 2016).

Interestingly, salinity stress increased the SOD activity among seedlings while the PHF application decreased SOD activity in comparison with untreated plants. The SOD serves as the first line of antioxidant defense system to dismutase O2− to H2O2 (Cavalcanti et al. 2007; Mittler 2006) which in turn is scavenged by H2O2 neutralizing enzymes (Apel and Hirt 2004). In this study, we recorded PHF-mediated increase in the activities of H2O2 neutralizing enzymes in the absence of any increase in the SOD activity. These findings can be explained on the basis of O2− radical scavenging ability of PHF molecule which has been regarded as radical sponges and O2− scavenging activity of PHF mimics SOD activity (Foley et al. 2002). Above all, PHF conferred oxidative stress tolerance in wheat plants under salinity through the upregulation of H2O2- neutralizing enzymes. The mitigation of oxidative stress in PHF-treated plants is consistent with the improvements in growth and recovery in the photosynthetic pigments.

Considering enzymatic antioxidants, PHF seed pre-treatment resulted in significant improvements in free amino acids, soluble sugars, and ascorbic acid contents under salinity. Salinity also caused increase in ascorbic acid and soluble sugars while reduced amino acids. Of limited reports, PHF-mediated accumulation of soluble sugars has been reported in Beta vulgaris under water-limited growth conditions (Borišev et al. 2016). The retention of compatible osmolytes like soluble sugars, proline, and other free amino acids is linked with better osmotic adjustments under salinity (Flowers and Colmer 2008; Shafiq et al. 2018). Other than osmolytes, another important component of osmoregulation is inorganic ions primarily Na+, K+, Ca2+, and P.

In the present study, marked increase in the root Na+ concentration while, a reduction in the K+ and Ca2+ contents was recorded. On the other hand, plants grown from PHF pre-treated seeds exhibited increased K+ uptake in the roots under salinity. Furthermore, PHF-mediated improvements in the root P contents were also recorded. In general, plants regulate cellular osmotic potential through effective adjustments of Na+/K+ ratio via increased K+ uptake and its retention (Cuin et al. 2009; Barragán et al. 2012). Both Ca2+ and K+ are actively involved in modulation of plant responses to salinity (Shabala 2017; Nedjimi 2017). The tissue-specific salinity tolerance in halophytes is also linked with K+ retention in mesophyll cells (Percey et al. 2016). Here PHF-mediated increase in the root K+ and P contents might have contributed to better cellular osmotic adjustments. For leaves, an increase in the shoot Na+ contents while reduction in K+ and Ca2+ contents was recorded. The increase in shoot Na+ fraction can be due to enhanced translocation of Na+ from root to shoot in order to achieve better osmotic adjustment through a process called xylem loading (Shabala et al. 2010; Zhang et al. 2017). Therefore, controlled Na+ transport effectively regulated Na+ accumulation in the metabolically active cells (Läuchli et al. 2008; Cuin et al. 2011). Other than Na+, PHF at 10 and 40 nM concentrations reduced Na+ uptake in the shoot while increased P at 40, 80, and 120 nM concentrations under salinity.

5 Conclusions

Salt stress reduced photosynthetic pigments and generated oxidative stress which negatively affected wheat growth parameters. In addition, salinity caused increased accumulation of sodium in root and shoot while it reduced potassium and calcium contents. By contrast, the plants grown from polyhydroxy fullerene nanoparticles–treated seeds exhibited salinity tolerance linked with increased antioxidant activities of catalase, peroxidase, and ascorbate peroxidase enzymes. Furthermore under salinity stress, increased root and shoot phosphorus was recorded for wheat plants in response to polyhydroxy fullerene. Above all, these biochemical modifications contributed to recovery in chlorophyll contents, osmolytes accumulation, and ultimately growth improvement under salt stress. Therefore, polyhydroxy fullerene nanoparticles seed pre-treatment promoted early seedling growth and establishment in wheat exposed to salt stress.