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. 2022 Nov 20;28(10):1889–1903. doi: 10.1007/s12298-022-01250-z

Exogenous menadione sodium bisulphite alleviates detrimental effects of alkaline stress on wheat (Triticum aestivum L.)

Ali Akbar 1, Muhammad Arslan Ashraf 1,✉, Rizwan Rasheed 1, Iqbal Hussain 1, Shafaqat Ali 2,3, Abida Parveen 1
PMCID: PMC9723007  PMID: 36484028

Abstract

Menadione sodium bisulphite (MSB) is known to augment plant defense responses against abiotic and biotic stresses. Wheat is an essential cereal with significant sensitivity to alkaline stress. The present study investigated the effects of MSB seed priming (5 and 10 mM) in alleviating the damaging effects of alkaline stress on hydroponically grown wheat cultivars (salt-sensitive cv. MH-97 and salt-tolerant cv. Millat-2011). Our findings revealed a significant reduction in growth, chlorophyll contents, total soluble proteins, free amino acids, K+, Ca2+, P, and K+/Na+ in wheat cultivars under alkaline stress. In contrast, a noteworthy accretion in lipid peroxidation, H2O2 production, proline levels, antioxidant enzyme activities, soluble sugars, antioxidant compounds, and Na+ levels was noticed in wheat plants grown in alkaline hydroponic medium. MSB priming significantly lowered chlorophyll degradation, Na+ levels, and osmolyte accumulation. Further, K+/Na+ ratio, antioxidant compounds, and antioxidant enzyme activities were higher in plants primed with MSB. Therefore, seed priming eminently protected plants by regulating osmotic adjustment and strengthening oxidative defense under alkaline stress. Plants administered 5 mM MSB as seed priming manifested better tolerance to alkaline stress.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-022-01250-z.

Keywords: Alakline salts, Oxidative injury, Nutrient uptake, Hydopronic culture, Antioxidant compounds

Introduction

Menadione sodium bisulphite (MSB) is a derivative of vitamin K3 and is easily soluble in water. It possesses 2-methyl-1,4-naphthoquinone core (menadione). Previously, MSB was considered synthetic, but now it has been isolated from fungi and phanerogams (Rasheed et al. 2018; Jiménez-Arias et al. 2019). Exogenous supplementation of MSB induces a slight oxidative burst that initiates ROS-related stress signaling networks. Therefore, it is widely used to monitor oxidative stress responses in plants (Ashraf et al. 2019). MSB mediates different physiological functions in plants due to its redox properties. Vitamin K-induced oxidative stress regulates the production of different stress-related proteins and enzyme activities, contributing to stress tolerance (Borges et al. 2014). Vitamin K acts as an electron carrier in thylakoid membrane and is recognized as an essential compound of photosystem I redox chain. MSB shields plants from oxidative damage, nutrient deficiency, specific ions toxicity and osmotic stress under saline conditions. Further, MSB regulates osmotic adjustment of salinity-stressed plants to combat osmotic stress (Rasheed et al. 2018; Ashraf et al. 2019). It has been reported that MSB application improved growth characteristics, photosynthetic pigments and maintains ion homeostasis in Arabidopsis under salinity stress (Jiménez-Arias et al. 2015). Akbar et al. (2021) also documented that MSB lessened chlorophyll degradation and improved water potential and photosynthesis in wheat under saline conditions.

Plants growing in soil with a high amount of alkaline salts such as Na2CO3 and NaHCO3 experience alkaline stress modulating growth, physiological and biochemical processes (Latef and Tran 2016). Alkaline conditions cause osmotic stress, specific ion toxicity and high pH to undermine the important plant defense mechanisms to abridge yield production (Nie et al. 2018). The damaging effects of alkalinity on plants are more severe than salinity due to the high pH of the growth medium, besides osmotic stress, oxidative stress and ion excess toxicity. Alkaline stress significantly diminishes yield, nutrient acquisition, root development, and seed germination (Chuamnakthong et al. 2019). The literature displayed more comprehensive studies on salinity effects on plants, while plant defense responses to alkaline stress lagged behind (Latef and Tran 2016). Furthermore, there is scarce information that examines the modulations in critical plant defense mechanisms, including osmotic adjustment, antioxidant defense, secondary metabolism, and ion homeostasis in plants under alkaline stress (Abd-Alla et al. 2014). Researchers have not thoroughly studied alkalinity effects on plants concerning essential physiological and biochemical processes (Latef and Tran 2016).

The inherent redox characteristics of alkaline salts such as Na2CO3 also generate toxicity through the enhanced production of reactive oxygen species (ROS), such as superoxide radicals (O2·‒), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radical (OH·) (Liu et al. 2019). Plants overcome ROS-generated oxidative injury through an antioxidant defense system that comprises enzyme and non-enzyme antioxidants (Qureshi et al. 2020; Hossain et al. 2022). Besides, plants growing on soils rich in alkaline salts also manifest growth reductions due to osmotic stress and excess ion toxicity (Fu et al. 2021). However, the accumulation of organic and inorganic compounds set up the osmotic adjustment mechanism in plants under alkaline stress (Jia et al. 2019). The most widely reported organic compounds accumulated by plants include proline, glycine betaine, trehalose, soluble proteins, and free amino acids. Whereas inorganic ions such as Na+, K+, Ca2+, and Cl‒ eminently mediate plant osmotic adjustment under alkaline stress (Wang et al. 2021).

The nutrient acquisition is severely impeded in plants under alkaline stress. The poor nutrient acquisition may lead to limited chlorophyll biosynthesis, which reduces photosynthesis and crop yield (Kaiwen et al. 2020). However, fewer studies examined the effects of alkaline stress on nutrient acquisition in plants. Therefore, understanding the plant defense responses becomes central to protect the damaging effects of alkalinity on growth and yield production. The high amount of salts in the soil creates ionic imbalance generating ions toxicity (Latef and Tran 2016). The elevated Na+ in the soil is the primary stress factor that inhibits the uptake of essential nutrients, particularly K+, due to Na+ competition with K+ uptake creating K+ deficiency in plants. Further, researchers also reported the significance of K+/Na+ ratio in defining plant tolerance to alkaline stress (Wang et al. 2022).

Wheat (Triticum aestivum L.) is the most widely grown cereal after maize and rice (Kusale et al. 2021). Wheat is a staple food in many Asian countries, including Pakistan. However, Pakistani soils are alkaline with a pH closer to 7, and further addition of alkaline salts in the soil due to anthropogenic activities creates alkalinity stress for plants. Wheat is sensitive to alkaline stress and shows significant depression in growth and yield when it encounters alkalinity (Ramadan et al. 2022). This research examines the emerging role of vitamin K3 derivative MSB on growth, osmolyte accumulation, secondary metabolism, antioxidant defense system, and nutrient acquisition in hydroponically grown wheat under alkaline stress.

Materials and methods

Seeds of two wheat cultivars with differential salinity tolerance (sensitive cv. MH-97 and tolerant cv. Millat-2011) were sterilized in 1% (v/v) ethanol and 1% sodium hypochlorite for 2 min. Afterward, seeds were washed thoroughly with distilled water (Khan et al. 2020). Seeds were primed in different doses of MSB (5 and 10 mM) for 12 h. Seeds were dried for 6 h in the shade. Seeds were planted in sand-filled 1 L plastic containers. Plants were given 250 mL of full-strength Hoagland’s nutrient solution after a 5-day interval for providing nutrition to plants in the sand medium (Hoagland and Arnon 1950). After 16 days of germination, uniform healthy wheat seedlings were transplanted to the nutrient solution. Plants were allowed to grow in the hydroponic medium for 20 days before stress imposition. Alkaline stress as 20 mM Na2CO3 was added to the nutrient solution. The different treatments were (1) Unprimed (not primed with MSB) + 0 mM Na2CO3 (2) Hydroprimed (primed with distilled water) + 0 mM Na2CO3 (3) 5 mM MSB primed + 0 mM Na2CO3 (4) 10 mM MSB primed + 0 mM Na2CO3 (5) Unprimed (not primed with MSB) + 20 mM Na2CO3 (6) Hydroprimed (primed with distilled water) + 20 mM Na2CO3 (7) 5 mM MSB primed + 20 mM Na2CO3 (8) 10 mM MSB primed + 20 mM Na2CO3. The hydroponic medium was continuously aerated with the help of an electric pump. The nutrient solution was replaced after every 3 days intervals. The pH of the nutrient solution after the addition of Na2CO3 reached 10.8, while the control plants were at pH 6.5. Plants were harvested 2 weeks following stress imposition at the vegetative stage (Feeke: 2; Zadoks: 21), and data were recorded. The experiments were carried out in a completely randomized design using four treatment replications.

Growth attributes

Plants were harvested and data was taken in terms of shoot length, shoot and root fresh and dry weight, leaf area, and number of tillers per plant. Shoot length was measured with the help of measuring tape. Shoot and root fresh and dry weights were taken with the help of electrical weighing balance. Gradner's methodology was used to calculate leaf area following the given formula: Leaf area: leaf length × leaf width × 0.75.

Chlorophyll pigments

Chlorophyll a, b and carotenoids were calculated by the method of Arnon (1949). Leaf sample 0.5 g was homogenized in 80% acetone. Homogenized samples were centrifuged at 12,000 × g for 5 min. Absorption of the samples were taken at 663, 645 and 480 with the help of spectrophotometer (Hitachi U-1800).

Non-enzyme antioxidants

Phenolics were measured following the protocol of Wolfe et al. (2003) with minor modifications. Fresh leaf (0.1 g) was homogenized in 5 mL of 80% methanol. The homogenate was then centrifuged at 12,000 × g for 5 min. The supernatant (0.1 mL) was mixed with 1 mL Folin–Ciocalteu’s phenol reagent, followed by vigorous shaking. Afterwards, 5 mL of 20% sodium carbonate (Na2CO3) was added to the reaction solution. The volume of the reaction solution was made to 10 mL using distilled water. Absorbance of the samples were recorded at 750 nm wavelength using a spectrophotometer. A standard curve of gallic acid was prepared to calculate phenolic content. Flavonoids were determined following the method of Zhishen et al. (1999). The supernatant (1 mL) was mixed with 5% NaNO2 and incubated at 25 ℃ for 5 min. This reaction mixture was treated with 1 mL of 10% AlCl3. Kept the samples for 5 min at room temperature. Then the reaction mixture was mixed with 2 mL of 1 M NaOH. The volume of reaction solution was made to 10 mL with distilled water. Absorbance of the mixture was red at 510 nm with spectrophotometer. The standard curve of quercetin was made to calculate flavonoid contents. Anthocyanin contents were measured by the protocol of Mita et al. (1997). Leaf tissues were ground in 1% acidic methanol and absorbance was read at 657 and 530 nm. Ascorbic acid was measured following the methodology of Mukherjee and Choudhuri (1983). For this purpose, leaf tissue 0.5 g was triturated in 6% trichloroacetic acid. Samples were centrifuged at 12,000 × g for 5 min. The supernatant (1.5 mL) was reacted with 1 mL of 2% dinitrophenyl hydrazine and added one drop of 10% thiourea. Samples were then incubated at 95 ℃ for 40 min. Afterward, 1.5 mL of sulfuric acid (80%) was added in reaction mixture and absorbance of the samples were taken at 530 nm. The standard curve of ascorbic acid was prepared for the estimation of ascorbic acid contents in leaf samples.

Soluble sugars

Fresh leaf material (0.5 g) was homogenized in 80% methanol and centrifuged at 12,000 × g for 5 min. The supernatant taken was used to measure total soluble sugars and reducing sugar.

Total soluble sugar was determined following the method of Yemm and Willis (1954). The supernatant (100 µL) was reacted with 3 mL anthrone reagent. The reaction solution was incubated in a water bath at 95 ℃ for 10 min. The absorbance was read at 520 nm using spectrophotometer. Whereas, reducing sugar was determined employing Henson and Stone (1988) protocol. The supernatant (200 µL) reacted with 1 mL DNSA (3,5-dinitrosalicylic acid) reagent. The DNSA reagent was prepared by dissolving 1 g of 3,5 dinitrosalicylic acid in 20 mL of 2 M NaOH followed by the addition of 30 g sodium potassium tartrate. The volume was made to 100 mL with distilled water. Absorbance was read at 540 nm on spectrophotometer. Non-reducing sugar was calculated by taking difference of total soluble sugars and reducing sugar.

Total soluble proteins

Total soluble proteins were determined following the method of Bradford (1976). Fresh leaf (0.5 g) was homogenized in 10 mL of potassium phosphate buffer (50 mM, pH 7.5) and centrifuged samples at 10,000 × g for 20 min at 4 ℃. The supernatant (100 µL) of the samples reacted with Bradford reagent (3 mL) and absorbance was taken at 595 nm. Bovine serum albumin (BSA) was used to make standard curve.

Total free amino acids

Total free amino acids were determined by the procedure of Hamilton et al. (1943). The supernatant (1 mL) used in total soluble protein assay was reacted with 1 mL of both 10% of pyridine and 2% of ninhydrin. The reaction mixture was kept in a water bath at 95 ℃ for 30 min and absorbance was measured at 570 nm.

Proline

Proline was determined by the procedure of Sarker and Oba (2018a). Fresh leaf (0.5 g) was crushed in 10 mL of 3% sulfosalicylic acid. Homogenate was filtered and 2 mL filtrate was reacted with equal volume of ninhydrin and galacial acetic acid. Samples were incubated at 95 ℃ for 45 min. After incubation, 5 mL toluene was added in each sample and absorbance was taken at 520 nm.

Malondialdehyde (MDA) and hydrogen peroxide (H2O2)

Fresh leaf (0.5 g) was homogenized in 10 mL of 6% trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000 × g for 5 min. The supernatant was extracted to measure MDA and H2O2 content.

MDA was measured by the method of Cakmak and Horst (1991). The supernatant (0.5 mL) was reacted with 2 mL of 5% thiobarbituric acid (TBA). The reaction mixture was placed in water bath at 95 ℃ for 45 min. Afterward, absorbance was read at 600 and 532 nm. Whereas H2O2 was determined following the protocol of Velikova et al. (2000). For this purpose, supernatant 0.5 mL (the one used for MDA determination) was reacted with 1 mL of 1 M (potassium iodide) KI and 0.5 mL 50 mM phosphate buffer pH (7.5). After incubation of the samples for 50 min at 25 ℃, OD of the samples was taken at 390 nm.

Enzyme antioxidants

Fresh leaf tissue (0.5 g) was frozen in liquid nitrogen and finely ground with the help of pestle and mortar. Potassium phosphate buffer (10 mL, pH 7.5) was added in the powdered leaf material. Samples were centrifuged at 10,000 × g for 10 min at 4 ℃. The approach described by Sarker and Oba (2018b) was used to determine the activity of SOD utilizing photoreduction of nitroblue tetrazolium (NBT). Briefly, reaction mixture (3 mL) contained phosphate buffer (50 mM, pH 7.5), riboflavin (1.3 µM), NBT (50 µM), methionine (13 mM) and enzyme extract (50 µL). The reaction mixture was kept in florescent bulb for 15 min until the color of reaction changes. Absorbance was read at 560 nm with the help of spectrophotometer. CAT activity was measured by the method of Sarker and Oba (2020). The reaction mixture consisted of phosphate buffer (50 mM), supernatant (100 µL), and H2O2 (20 mM). The optical density of the reaction mixture was read at 240 nm for 120 s. APX activity was determined by the method of Nakano and Asada (1981). Briefly, 100 µL enzyme extract was mixed with 0.5 mM ascorbic acid and potassium phosphate buffer (50 mM, pH 7.5). The reaction was initiated by the addition of 0.1 mL H2O2 (30 mM). The drop in absorbance was read at 290 nm for 120 s. Activity of POD was determined following the method of Polle et al. (1994) with minor modifications. The reaction mixture (3 mL) contained guaiacol (20 mM), H2O2 (10 mM) and enzyme extract (100 µL). The increase in absorbance was read at 470 nm for 120 s.

Ion analysis

Ions were determined followed by the protocol of Allen et al. (1986). Dried plant material (0.1 g) was digested in concentrated H2SO4. For this purpose, 2 mL H2SO4 was added in each sample and placed it for overnight. Later, the digested mixture was heated at 150 ℃ and H2O2 was added slowly. Aforementioned step was repeated until transparent solution appeared in the samples. Na+, K+ and Ca2+ were determined from digested solution using flame photometer (Sherwood, Model 360). Phosphorus was determined from the digested material following the protocol of Jackson (1969).

Statistical analysis

The experiment was carried out in completely randomized design with four replicates. Data were subjected to analysis of variance (ANOVA) using COSTAT statistical software. Means were compared for significance at 95% confidence level. Origin pro 2019 software was employed for graphical presentation of the data. Correlation plot and principle component analysis were generated in R studio.

Results

Plant growth

A significant decline (P ≤ 0.001) in fresh shoot weight and shoot length was evident in two wheat cultivars grown in hydroponic culture containing 20 mM Na2CO3. The depression in shoot fresh and dry weights and length was more visible in salt-sensitive cultivar MH-97, while cv. Millat-2011 displayed better shoot growth characteristics under alkaline conditions. Seed priming treatment with 5 and 10 mM menadione sodium bisulphite (MSB) manifested a remarkable (P ≤ 0.001) improvement in fresh shoot and dry weight and shoot length of hydroponically grown wheat cultivars under alkaline conditions (Fig. 1; Supplementary Table S1).

Fig. 1.

Fig. 1

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Effect of exogenous MSB on growth and photosynthetic pigments in hydroponically grown wheat cultivars under alkaline stress. The different lower case letters on bars represent difference among means at 95% confidence level

Similarly, root fresh and dry weight dropped significantly (P ≤ 0.001) in both wheat cultivars under alkaline conditions. The fall in the root growth attributes was more conspicuous in sensitive wheat cultivar MH-97. MSB administration as pre-sowing seed treatment (5 and 10 mM) showed a substantial upsurge in root growth characteristics. Higher root fresh and dry weight was seen in plants pretreated with 5 mM MSB under alkaline conditions (Fig. 1; Supplementary Table S1).

Likewise, alkaline conditions diminished (P ≤ 0.001) the leaf area of hydroponically grown wheat cultivars. Salt-sensitive wheat cv. MH-97 exhibited a more significant drop in leaf area compared to salt-tolerant cv. Millat-2011. Seed priming with 5 and 10 mM MSB caused a substantial rise (P ≤ 0.001) in the leaf area of wheat plants grown in alkaline hydroponic medium. Besides, plants administered 5 mM MSB as seed pretreatment showed higher leaf area under alkaline conditions (Fig. 1; Supplementary Table S1). Alkaline conditions also significantly diminished (P ≤ 0.01) the number of tillers in wheat plants grown in hydroponic medium. Further, MSB seed priming treatment (5 and 10 mM) resulted in a conspicuous rise (P ≤ 0.001) in the number of tillers in wheat plants under alkaline conditions (Fig. 1; Supplementary Table S1).

Photosynthetic pigments

Alkaline conditions significantly abridged chlorophyll a and b (P ≤ 0.001) in wheat cultivars grown in hydroponic culture. Likewise, total chlorophyll content also diminished (P ≤ 0.001) in wheat plants subjected to alkaline conditions in the hydroponic medium. Seed priming treatment with MSB (5 and 10 mM) considerably enhanced total chlorophyll and chlorophyll a and b values in wheat plants under alkaline conditions. Additionally, 5 mM MSB priming resulted in higher chlorophyll content under alkaline conditions. Further, Chlorophyll a/b dropped significantly (P ≤ 0.01) in wheat plants under alkaline conditions. Seed pretreatment with MSB (5 mM) resulted in further decrease in chlorophyll a/b ratio in two wheat cultivars (Fig. 1; Supplementary Table S1).

Carotenoid content diminished significantly (P ≤ 0.001) in wheat cultivars grown in alkaline hydroponic culture. Higher carotenoids were seen in salt tolerant cv. Millat-2011, while salt sensitive cv. MH-97 was inferior in this context. Besides, seed priming (5 and 10 mM) substantially enhanced (P ≤ 0.001) carotenoid contents in wheat plants grown in alkaline hydroponic medium. Further, plants pretreated with 5 mM displayed greater values of carotenoids in wheat cultivars under alkaline conditions (Fig. 2; Supplementary Table S1).

Fig. 2.

Fig. 2

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Effect of exogenous MSB on cytosolutes accumulation and antioxidant compounds in hydroponically grown wheat cultivars under alkaline stress. TSS, total soluble sugars; RS, reducing sugars; NRS, non-reducing sugars; TSP, total soluble sugars. The different lower case letters on bars represent difference among means at 95% confidence level

Non-enzyme antioxidants

Flavonoid level increased significantly (P ≤ 0.001) in two wheat cultivars grown in alkaline hydroponic medium. Additionally, cv. MH-97 had higher values for flavonoids under alkaline stress. MSB seed priming treatment 5 and 10 mM produced a substantial increase in flavonoids in wheat plants under alkaline conditions. Higher flavonoid values were present in plants fed with 5 mM MSB seed priming treatment (Fig. 2; Supplementary Table S1).

Phenolic contents diminished significantly in salt-sensitive wheat cultivar MH-97, while phenolics increased substantially in salt-tolerant cultivar Millat-2011 under alkaline conditions. Further, plants pretreated with 5 and 10 mM manifested a more significant increase (P ≤ 0.001) in phenolic contents of hydroponically grown wheat cultivars in alkaline conditions. Plants pretreated with 5 mM MSB showed a more visible increase in phenolic contents under alkaline conditions (Fig. 2; Supplementary Table S1).

Ascorbic acid content increased substantially (P ≤ 0.001) in two wheat cultivars grown in alkaline hydroponic culture. Salt-sensitive wheat cv. MH-97 had lesser values of ascorbic acid compared with salt-tolerant cv. Millat-2011. Seed priming with MSB (5 and 10 mM) manifested a remarkable increase in ascorbic acid values in two wheat cultivars under alkaline conditions (Fig. 2; Supplementary Table S1).

Anthocyanins decreased in sensitive wheat cultivars MH-97, while salt-tolerant wheat cultivar Millat-2011 manifested a rise in anthocyanins under alkaline conditions. Furter, plants pretreated with MSB as seed priming (5 and 10 mM) displayed a significant (P ≤ 0.01) upsurge in this variable under alkaline conditions. MSB 5 mM pretreatment manifested a more substantial increase in anthocyanin level in two wheat cultivars grown in alkaline conditions (Fig. 2; Supplementary Table S1).

Enzyme antioxidants

SOD activity increased significantly (P ≤ 0.001) in hydroponically grown plants under alkaline conditions. Higher SOD activity was present in salt-tolerant wheat cv. Millat-2011 compared to salt-sensitive cv. MH-97 under alkaline conditions. Plants supplemented with 5 and 10 mM MSB as seed priming treatment manifested more excellent SOD activity. In this context, plants pretreated with 5 mM MSB had highest values of SOD activity under alkaline conditions (Fig. 3; Supplementary Table S1).

Fig. 3.

Fig. 3

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Effect of exogenous MSB on oxidative stress markers, antioxidant enzymes and phosphorus uptake in hydroponically grown wheat cultivars under alkaline stress. TFAA, total free amino acids; MDA, malondialdehyde; H2O2, hydrogen peroxide; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; APX, ascorbate peroxidase; P, phosphorus. The different lower case letters on bars represent difference among means at 95% confidence level

Alkaline stress resulted in a decrease in CAT activity of hydroponically grown wheat cultivars. Higher CAT activity was evident in salt-tolerant cv. Millat-2011 compared to cv. MH-97 under alkaline conditions. Besides, MSB seed priming (5 and 10 mM) induced a conspicuous increase in CAT activity of wheat plants grown in alkaline hydroponics. Exogenous MSB administration as seed priming (5 and 10 mM) remarkably (P ≤ 0.001) enhanced CAT activity under alkaline conditions. Further, plants with 5 mM MSB priming manifested more significant CAT activity under alkaline conditions (Fig. 3; Supplementary Table S1).

Alkaline stress induced a significant (P ≤ 0.001) accretion in APX activity in hydroponically grown wheat cultivars. Salt-tolerant cv. Millat-2011 showed greater APX activity compared with salt-sensitive cv. MH-97 grown in alkaline hydroponic medium. MSB seed priming (5 and 10 mM) exhibited a conspicuous improvement in APX activity under alkaline conditions. Further, plants pretreated with 5 mM MSB had higher APX activity under alkaline conditions (Fig. 2; Supplementary Table S1).

Alkaline stress produced a substantial rise (P ≤ 0.001) in POD activity in two wheat cultivars grown in alkaline hydroponic medium. Wheat cultivars did not differ for this variable under alkaline conditions. MSB seed priming (5 and 10 mM) exhibited a prominent accretion in POD activity in wheat cultivars under alkaline conditions. In this context, MSB priming with 5 mM improved POD activity of hydroponically grown wheat cultivars under alkaline conditions (Fig. 3; Supplementary Table S1).

Accumulation of osmoprotectants

Total soluble sugars (P ≤ 0.001) increased significantly in hydroponically grown wheat plants under alkaline conditions. Total soluble sugars were higher in salt-tolerant wheat cv. Millat-2011 compared to salt-sensitive wheat cv. MH-97 under alkaline conditions. Seed priming treatments (5 and 10 mM) MSB substantially improved total soluble sugars in wheat plants under alkaline conditions. Seed priming treatments with 5 mM MSB produced conspicuous accretion in total soluble sugar values of wheat cultivars under alkaline conditions. Further, priming with 5 mM MSB showed a significant rise in total soluble sugar content than other priming treatments under alkaline conditions (Fig. 2; Supplementary Table S1).

The wheat cultivars did not vary for reducing sugar accumulation under alkaline stress. However, alkaline conditions produced a significant increase in reducing sugar content of hydroponically grown wheat cultivars. Seed priming treatments with 5 and 10 mM MSB displayed a substantial accretion in reducing sugar content under alkaline stress. Further, seed priming with 5 mM MSB had higher reducing sugar level than other treatments under alkaline stress.

Non-reducing sugars increased markedly (P ≤ 0.001) in wheat cultivar under alkaline conditions. Salt-tolerant cv. Millat-2011 had higher non-reducing sugar content compared with salt-sensitive wheat cv. MH-97. Furthermore, MSB seed priming (5 and 10 mM) significantly enhanced non-reducing sugar level in wheat plants under alkaline conditions. Plants administered 5 mM MSB showed greater non-reducing sugar content compared with 10 mM MSB dose under alkaline conditions (Supplementary Table S1).

Alkaline stress prominently abridged (P ≤ 0.01) total soluble protein in two wheat cultivars. Further, the cultivars did not vary concerning this variable under alkaline conditions. MSB seed priming (5 and 10 mM) treatments resulted in a further increase in total soluble protein of hydroponically grown wheat cultivars under alkaline conditions (Fig. 2; Supplementary Table S1).

Total free amino acids diminished significantly in two wheat cultivars under alkaline conditions. Higher total free amino acid levels were present in salt-tolerant cv. Millat-2011 compared with salt-sensitive wheat cv. MH-97 under alkaline conditions. Plants administered MSB as seed priming (5 and 10 mM) manifested greater total free amino acids content. In this context, 5 mM MSB seed priming displayed greater total free amino acids in wheat cultivars under alkaline conditions (Fig. 3; Supplementary Table S1).

Alkaline stress produced a significant increase (P ≤ 0.001) in proline content in hydroponically grown wheat cultivars. Salt-sensitive wheat cv. MH-97 manifested substantial accumulation of proline than salt-tolerant wheat cv. Millat-2011. Administration of MSB as pre-sowing seed treatment (5 and 10 mM) showed a significant increase in proline accumulation. Additionally, plants administered 5 mM MSB had maximal proline values under alkaline conditions (Fig. 3; Supplementary Table S1).

Oxidative stress indicators

The production of lipidsas byproduct was increased in the form of MDA accumulation. The results indicated a significant increase in MDA levels in two wheat cultivars under alkaline conditions. The MDA level was higher in cv. MH-97 compared with salt-tolerant cv. Millat-2011. Administration of MSB as seed priming (5 and 10 mM) significantly diminished MDA accumulation in hydroponically grown wheat plants under alkaline conditions. The fall in MDA content was more visible in plants with 5 mM MSB priming (Fig. 3; Supplementary Table S1).

The generation of H2O2 was markedly higher (P ≤ 0.001) in wheat plants under alkaline conditions. Salt-sensitive wheat cultivar MH-97 displayed greater endogenous levels of H2O2 than tolerant wheat cv. Millat-2011. MSB seed priming treatments (5 and 10 mM) showed greater decrease (P ≤ 0.001) in H2O2 accumulation in hydroponically wheat cultivars under alkaline conditions. Further, Plants administered 5 mM MSB had lesser H2O2 accumulation in hydroponically grown wheat under alkaline conditions (Fig. 3; Supplementary Table S1).

Nutrient uptake

Root and leaf phosphorus (P) content decreased significantly (P ≤ 0.001) in hydroponically grown wheat cultivars under alkaline conditions. Higher P values were seen in salt-tolerant cv. Millat-2011 compared with salt-sensitive cv. MH-97. Plants provided with MSB as seed priming (5 and 10 mM) manifested a substantial improvement in P contents under alkaline conditions. In this context, plants pretreated with 5 mM MSB had higher P values under alkaline conditions (Fig. 3; Supplementary Table S1).

Our results revealed higher root Na+ accumulation in salt-tolerant wheat cv. Millat-2011 compared with salt-sensitive cv. MH-97 under alkaline conditions. Besides, MSB seed priming (5 and 10 mM) remarkedly (P ≤ 0.001) diminished Na+ accumulation in roots of hydroponically grown wheat cultivars under alkaline conditions. Furthermore, plants fed with 5 mM MSB exhibited prominent fall in root Na+ accumulation under alkaline conditions (Fig. 4; Supplementary Table S1).

Fig. 4.

Fig. 4

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Effect of exogenous MSB on elemental uptake in hydroponically grown wheat cultivars under alkaline stress. K+, potassium; Ca2+, calcium: Na+, sodium. The different lower case letters on bars represent difference among means at 95% confidence level

Leaf Na+ accumulation was higher in salt-sensitive wheat cv. MH-97 than salt-tolerant wheat cv. Millat-2011 in hydroponically grown wheat cultivars under alkaline conditions (Fig. 4; Supplementary Table S1).

The endogenous levels of root and leaves K+ diminished markedly (P ≤ 0.001) in wheat cultivars under alkaline conditions. In this context, wheat cv. MH-97 had lesser K+ concentration than salt-tolerant cv. Millat-2011. Furthermore, MSB seed priming (5 and 10 mM) resulted in a conspicuous increase in K+ accumulation of wheat plants grown in alkaline hydroponic conditions. Plants fed with 5 mM MSB had higher K+ levels under alkaline conditions (Fig. 4; Supplementary Table S1).

The accumulation of Ca2+ in root and leaves diminished (P ≤ 0.001) remarkably in two wheat cultivars grown in alkaline hydroponic medium. The wheat cultivars did not differ for Ca2+ accumulation under alkaline conditions. Besides, plants pretreated with 5 and 10 mM MSB displayed a conspicuous increase in Ca2+ accumulation in roots and leaves of wheat plants under alkaline conditions. Plants administered 5 mM MSB had higher Ca2+ levels under alkaline conditions (Fig. 4; Supplementary Table S1).

K+/Na+ and Ca2+/Na+ in roots and leaves diminished significantly in hydroponically grown wheat cultivars under alkaline conditions. Salt-tolerant wheat cv. Millat-2011 had higher root and leaf K+/Na+ and Ca2+/Na+ accumulation under alkaline stress. Furthermore, plants pretreated with MSB displayed a remarkable increase in root and leave K+/Na+ and Ca2+/Na+, particularly in plants with 5 mM MSB pretreatment under alkaline stress conditions (Fig. 4; Supplementary Table S1).

Discussion

Higher pH in alkaline conditions creates a nutrient imbalance in soil, depleting phosphorus and iron that severely impedes plant growth and yield potential. Alkaline stress is more damaging for plants because plants encounter both excess ion toxicity and cellular damage due to high pH (Chuamnakthong et al. 2019; Yang et al. 2022). In our study, alkaline stress notably diminished growth characteristics, chlorophyll content, and nutrient acquisition in hydroponically grown wheat cultivars with different salinity tolerance. Alkaline stress pared growth in plants due to massive damage to roots and inhibited nutrient acquisition (Ahmad et al. 2018; Rasheed et al. 2022). Besides, the results revealed a significant accretion in root and shoot Na+ accumulation in wheat plants under alkaline conditions. Javed et al. (2022) reported that excess Na+ accumulation deterred growth in maize. Further, the detrimental effects of alkaline stress on various growth characteristics of wheat plants might occur due to metabolic disorders and impaired cell division and elongation (Mohsenian and Roosta 2015; Latef and Tran 2016). In salinity, the decline in plant growth is ascribed to osmotic stress and ion excess toxicity (Sarker and Oba 2019). In contrast, besides osmotic and ion excess injury in the case of alkaline conditions, plants also face additional stress in the form of high pH (Liu et al. 2010; Msimbira and Smith 2020). The growth characteristics and photosynthetic pigments displayed a negative correlation with lipid peroxidation and ROS production (Figs. 5, 6). The oxidative damage is reported to diminish growth in plants under alkaline conditions (Liu et al. 2019). In our study, MSB remarkably pared the detrimental effects of alkaline stress on wheat plants by improving nutrient uptake, ROS metabolism, and secondary metabolite accumulation. Consequently, MSB priming improved plant growth under alkaline stress. In previous reports, MSB improved chlorophyll, ion homeostasis, and oxidative defense in wheat under Cr toxicity (Askari et al. 2021) and okra under Cr and Cd stress (Rasheed et al. 2018; Ashraf et al. 2021).

Fig. 5.

Fig. 5

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Pearson correlation among different growth and biochemical attributes. Abbreviations: SFW, shoot fresh weight; T.Chl., total chlorophyll; MDA, malondialdehyde; H2O2, hydrogen peroxide; TSS, total soluble sugars; TSP, total soluble proteins; TFAA, total free amino acids; P, phosphorus; K, potassium; Ca, calcium

Fig. 6.

Fig. 6

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Principal component analysis displaying association of oxidative stress indicators with shoot dry weight and antioxidant enzymes. POD, peroxidase; MDA, malondialdehyde; CAT, catalase; SOD, superoxide dismutase; SDW, shoot dry weight; TSP, total soluble proteins; H2O2, hydrogen peroxide

Chlorophyll a is a crucial pigment in photosynthesis, transforming solar energy into chemical energy. Light energy conversion efficiency is strongly related to the amount of chlorophyll a content. Besides, chlorophyll b pigments function as light-harvesting, mediating light absorption and transmission. Further, the reduction in chlorophyll molecules indicates oxidative injury due to excess ion toxicity (Hashem et al. 2019). The decline in chlorophyll content could be due to nutrient imbalance that leads to chlorophyll degradation (Sarker and Oba 2018a, 2018c). Further, enhanced chlorophyllase activity is also responsible for chlorophyll degradation (Latef and Tran 2016). Our results also revealed a significant decline in chlorophyll content under alkaline conditions. The decline in oxidative damage also lessened the chlorophyll degradation in plants under alkaline conditions (Liu et al. 2018). In the present investigation, MSB priming significantly protected plants from oxidative injury mirrored as lowered H2O2 and MDA levels in hydroponically grown wheat plants under alkaline conditions. The minimal oxidative damage improved chlorophyll accumulation in hydroponically grown wheat plants under stress. The accumulation of higher Na+ levels in plants leads to chlorophyll degradation (Wu et al. 2020). Our results also exhibited a negative correlation of K+/Na+ and Ca2+/Na+ ratios with chlorophyll content. MSB administration as seed priming diminished Na+ accumulation alongside improved K+/Na+ and Ca2+/Na+ that might have enhanced chlorophyll content in wheat under alkaline stress. The decrease in carotenoid content has been reported previously in wheat under alkaline conditions. However, the antioxidant function of alpha lipoic acid diminished oxidative injury and enhanced carotenoids (Ramadan et al. 2022). Our results also manifested improvement in carotenoids due to exogenous MSB that is reported to improve the oxidative defense system in plants. The aggrandized ABA production in plants under alkaline conditions might diminish carotenoids because ABA and carotenoids are produced from the same precursors, violaxanthin and neoxanthin (Ramadan et al. 2022). Intriguingly, ABA priming notably ameliorated alkaline stress tolerance in rice seedlings (Wei et al. 2015).

To neutralize the effects of ROS, plants contain an antioxidant system comprising non-enzyme (AsA, anthocyanins, flavonoids, and phenolics) and enzyme antioxidants (SOD, POD, APX, CAT) (Sarker et al. 2018; Sarker and Oba 2018d; Dawood et al. 2021). Our results on antioxidant compounds also revealed improvement in flavonoids, phenolics, AsA, and anthocyanins in wheat plants under alkaline stress. MSB administration eminently enhanced the concentration of antioxidant compounds, thereby increasing plant capacity to detoxify ROS effects. In this context, Dawood et al. (2021) reported non-enzyme antioxidants as an essential tool for ROS detoxification in artichoke seedlings. Our results indicated a significant positive association of antioxidant compounds with H2O2, displaying greater H2O2 instigating a profound increase in concentration of antioxidant compounds. Likewise, antioxidant enzymes also played a critical part in detoxifying ROS effects and abridged oxidative damage (Liu et al. 2015; Sarker and Oba 2018b). Our results revealed MSB-mediated strengthening of the antioxidant system both in the form of higher antioxidant compound level and improved antioxidant enzyme activities in wheat under alkaline conditions. The better scavenging of ROS by antioxidant compounds improved growth and diminished lipid peroxidation reflected as lowered MDA contents. Likewise, higher alkaline stress tolerance was reported in maize due to improved antioxidant enzyme activities (Sriramachandrasekharan et al. 2021). The accumulation of MDA content is also documented in maize under alkaline stress (Latef and Tran 2016). The present study manifested the increase in MDA levels in hydroponically wheat cultivar grown in alkaline conditions. MSB-mediated strengthened antioxidant defense system significantly lowered lipid peroxidation through active detoxification of ROS.

Plants accumulate soluble proteins under stress conditions, and these soluble proteins are used up in osmotic adjustment rather than in plant growth under alkaline stress (Latef and Tran 2016). Plants overcome the detrimental effects of alkaline stress by activating dual defense mechanisms involving improved antioxidant capacitance and enhanced osmolyte accumulation (Zou et al. 2018). The immediate impact of alkaline stress on plants is the decreased water uptake due to the higher osmolarity of the soil solution (Munns and Tester 2008). Plants accumulate osmolytes in a substantial amount to maintain water balance (Tang et al. 2019). In the present investigation, we noticed a significant accumulation of sugars, total free amino acids, total soluble proteins, and proline in wheat plants under alkaline conditions. Further, the results also manifested a more significant accumulation of these osmolytes in plants primed with MSB. Proline helps maintain cell turgor, stabilizes membranes, detoxifies ROS, and diminishes electrolyte leakage (Shafi et al. 2019). Besides, proline, soluble proteins, sugars, and total free amino acids also substantially improved osmotic adjustment, which enhanced plant growth under alkaline conditions (Rasheed et al. 2022).

Alkaline stress significantly increased Na+ content alongside the eminent decrease in K+ values in hydroponically grown wheat cultivars (Fig. 4). The increment in Na+ lowered K+ content in turn, significantly abridged K+/Na+ ratio. The fall in K+ values of plants is due to competition with Na+ on the absorption site in plants under alkaline conditions (Azooz et al. 2015; Latef and Tran 2016). MSB-primed wheat plants showed diminished Na+ level and improved K+ concentration, resulting in a greater K+/Na+ ratio (Fig. 4). MSB notably protects the plasma membranes by actively detoxifying ROS through antioxidant compounds and enzymes, remarkably improving K+ uptake in wheat under alkaline stress. In this context, oxidative damage due to excess Na+ substantially aggrandizes membrane damage that might deteriorate nutrient and water uptake in wheat under salinity (Shafiq et al. 2021). The rise in K+/Na+ indicated the balancing effect of MSB on Na+ and K+ levels under alkaline conditions, encouraging the application of MSB as seed priming for better plant growth on alkaline soils.

Conclusion

Seed treatment with MSB mediated tissue-specific management of toxic Na+ under alkaline stress associated with improved Ca2+, K+ and P uptake. Further, MSB priming enhanced K+/Na+ and Ca2+/Na+ ratios essential in establishing plant tolerance under alkaline stress. MSB priming increased the accumulation of proline, amino acids, proteins and sugars that shielded plants from the damaging effects of osmotic stress, excess ion toxicity and high pH under alkaline conditions. Seed pretreatment with MSB notably pared oxidative damage reflected as minimal H2O2 and MDA levels through the augmented oxidative defense. Plants primed with 5 mM MSB depicted ebbed oxidative injury due to improved antioxidant system and enhanced osmolyte accumulation. Further research involving advanced molecular approaches is needed to comprehend underlying mechanisms in MSB-mediated signaling pathways which improve plant survival under alkaline conditions.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 26 kb) (26.1KB, docx)

Acknowledgements

The authors are thankful to Government College University Faisalabad, Pakistan for providing essential equipment and labortory space for the execution of the experiment.

Authors contribution

MAA conceived and designed the experiment; AA, IH and RR performed the experiment; MAA and AP wrote the manuscript; MAA contributed to reagents/analysis/materials; SA assisted in review, editing and software use for graphical representation of data.

Funding

This work was supported by Higher Education Commission of Pakistan (8345/Punjab/NRPU/R&D/HEC/2017).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Consent for publication

Not applicable.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 26 kb) (26.1KB, docx)

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Not applicable.

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