Introduction

Barley (Hordeum vulgare L.) belongs to Poaceae family and it is the fourth major cereal crop in terms of World and Egyptian production after maize, wheat, and rice crops (Hafez et al. 2014, Hafez et al. 2016). Abiotic stresses such as water deficit, waterlogging, salinity, and high or low temperatures have been reported to significantly reduce photosynthesis and other biochemical processes related to plant growth and crop productivity (Samarah 2005; Tiwari et al. 2010; Abdelaal et al. 2017; Abdelaal et al. 2018). Drought is a climatic term that means the absence of rain for more than 14 days. Water deficit is a crop management term used to describe production under less than optimum water requirements or when irrigation is not sufficient for reaching field capacity. Water scarcity is the most serious abiotic stress restricting plant productivity throughout the globe (Kotob et al. 2009; Saleem et al. 2016) including areas of Egypt. Under stressful growth conditions, an increment of electrolyte leakage and respiration rate is expected due to oxidative stress, which results in generation of reactive oxygen species (ROS) (Asada 2006). The detrimental effects of abiotic stresses such as water deficit is mainly due to oxidation of cellular biomolecules (i.e., proteins), which is initiated by ROS and free radicals driving the cell to death (Mittler 2002; Helaly et al. 2017). The balance between ROS generation and its scavenging is strongly controlled by the plant antioxidant defense system, particularly under optimal growth conditions (Hameed et al. 2011). Water deficit stress is one of the most common ecological factors which threatens crop plant productivity (Wang et al. 2003) mainly during the flowering and grain-filling phases. Water deficit stress causes significant reduction in stem length, number of branches and leaves of plant as well as leaf area (Abdelaal 2015a). Plants possess many mechanisms to counter the injurious effects of water deficit stress (Vaseva et al. 2012). Various chemicals such as osmoprotectants, growth regulators, and stress signaling molecules are being successfully used against several biotic and abiotic stresses to induce the tolerance (Farooq et al. 2010). Potassium is a macronutrient which plays an important role in growth development of plants and activates more than 60 enzymes (Tisdale et al. 1990). Also, potassium improves water deficit tolerance in plants by maintaining water balance and it is crucial for osmoregulation, photosynthesis, transpiration, stomatal opening and closing as well as protein synthesis (Milford and Johnston 2007; Behairy et al. 2015). The product actosol® is a soil conditioner and fertilizer which contains a mixture of natural humic and fulvic acids. It improves soil structure, enhances plant growth, regulates the carbon cycle and releasing of nutrients, leads to positive effects on plant growth, and induces stress tolerance (Stevenson 1994; Ekinci et al. 2015; Abdelaal 2015b). Application of actosol® and potassium dihydrogen phosphate, respectively, significantly improves growth parameters and chlorophyll concentration (Mahmoud and Youssif 2015). Amino acids, the building blocks of proteins are important for cell growth stimulation and have positive roles in increasing the concentration of osmotic components due to their cell internal function as osmoregulatory (Treichel 1975; Rai 2002; Abd El-Samad et al. 2010). The foliar application of nutrients is readily absorbed by leaves and enhancing the physiological processes and cell growth as well (Robredo et al. 2007) to face the great needs of nutrients during some growth stages particularly at grain-filling stage. However, several experiments have previously conducted to study the harmful effects of water deficit stress on plants; nevertheless, a little information is available concerning water deficit stress around physiological characters and reproductive stage of barley plants. Therefore, the aim of this current research is to study the effect of select osmoregulators, i.e., simple and compound inorganic fertilizers, and complex organic fertilizers that are supposed help plants maintain water balance and for that reason are called osmoregulators, such as potassium dihydrogen phosphate, Compound fertilizer, actosol® (humates), as well as Amino more (amino acids) on morpho-physiological, biochemical, and yield characters of barley plants (Hordeum vulgare L. Giza123) under water deficit stress.

Materials and methods

Field experiments

Field experiments during two successive growing seasons 2014/2015 and 2015/2016 were installed at the Experimental Farm, Faculty of Agriculture, Kafrelsheikh University, Egypt, with the aim to study the effects of four different osmoregulators on growth, morpho-physiological, biochemical characters, and productivity of barley plants (Hordeum vulgare L. var. Giza123) under water deficit stress. Meteorological data of the experimental site are presented in Table 1. The used osmoregulators were commercial products with the following names: (1) Potassium dihydrogen phosphate; (2) actosol®; (3) Amino more; and (4) Compound fertilizer. More details about description, concentrations, and application rate and frequency of the osmoregulators used are presented in Table 2. Barley plants were exposed to water deficit stress by not irrigating them beyond sowing and the one-time surface irrigation flooding and draining after germination. Control plots were well watered receiving four more irrigations during the entire growing season, where five irrigations after germination are the standard water requirement for barley in the region. Barley plants under water deficit stress were treated with one of the four above-mentioned osmoregulators to alleviate such abiotic stress by spraying plants twice (Table 1). Barley seeds were sown (120 kg ha−1) on 8th December 2014 (first season) and 11th December 2015 (second season) in a typical clay soil with the following physiochemical characteristics measured according to Horwitz (2005): pH (water) 8.2, electrical conductivity (water extract) 1.8 dS m−1, available N 32.4 ppm, available P 10.5 ppm, available K 289 ppm, soil organic matter (SOM) 1.9%, sand 17.3%, silt 35.5%, clay 47.2%, and clayey texture. The experimental plot area was 2.5 × 2.5 m2 and the experimental layout was a randomized complete block design with four replicates. Recommended NP were added at the rate of 110 kg N ha−1 as ammonium sulfate (21%) and 125 kg P2O5 ha−1 as superphosphate (15.5%). Plants were harvested on 4th May 2015 (first season) and 9th May 2016 (second season).

Table 1 Meteorological data of experimental location during both growing seasons (2014/2015 and 2015/2016) as monthly average
Full size table
Table 2 Description of used osmoregulators and their application rates
Full size table

Morpho-physiological characters

Ten plants from each plot were randomly taken at anthesis date to measure stem length, plant fresh and dry mass, number of leaves, chlorophyll a and b, nitrogen (N), phosphorus (P), and potassium (K) contents. A sample of 0.5 g of fresh barley leaves was homogenized with acetone (90% v/v), filtered, and made up to a final volume of 50 mL. The absorbance was measured spectrophotometrically at 663 and 648 nm according to (Lichtenthaler 1987) to estimate chl a and b content. For N, P, and K determination, plant samples were washed with diluted hydrochloric acid (HCl) solution to remove any adhered substances, then immersed three times in deionized water to remove any residue of HCl. Later, plant samples were dried at ambient temperature in aerated rooms before thermal drying at 70 °C for 48 h. The dried samples were powdered using a grinder and kept in plastic bags for further analysis. For N, P, and K determination, powdered plant samples were digested according to Peterburgski (1968), and N was determined after Block (1968). P was calorimetrically measured according to Jackson (1967), and K was measured by flame photometer using Corning 400 flame photometer (Peterburgski 1968).

Histobiochemical analysis and yield characters

Superoxide (O2·−) and hydrogen peroxide (H2O2) were visualized in leaf samples as a purple coloration of nitro blue tetrazolium (NBT) and a reddish-brown coloration of 3,3-diaminobenzidine (DAB), respectively. The first seven leaves of barley plants from the top were collected for histobiochemical analysis at 6th March 2015 (for first season) and 17th March 2016 (for second season). The collected leaf blades were vacuum infiltrated with 10 mM potassium phosphate buffer (pH 7.8) containing 0.1 w/v % NBT (Sigma-Aldrich, Steinheim, Germany) according to Ádám et al. (1989) or 0.1 w/v % DAB (Fluka, Buchs, Switzerland). NBT- and DAB-treated samples were incubated under day light for 20 min and 2 h, respectively, and subsequently cleared in 0.15 w/v % trichloroacetic acid in ethanol: chloroform 4:1 v/v for 1 day (Hueckelhoven et al. 1999; Kiraly et al. 2008). Cleared samples were washed with water and placed in 50% glycerol prior to evaluation. Discoloration of leaf discs resulted by NBT or DAB staining was quantified using a ChemiImager 4000 digital imaging system (Alpha Innotech Corp., San Leandro, USA) in integrated density value units (IDV).

The antioxidant enzyme activities were measured in plant samples as follows: 0.5 g fresh mass of leaf material was homogenized at 0–4 °C in 3 ml of 50 mM TRIS buffer (pH 7.8), containing 1 mM EDTA-Na2, and 7.5% polyvinylpyrrolidone. The homogenates were centrifuged (12,000 rpm, 20 min, 4 °C), and the total soluble enzyme activities were measured spectrophotometrically in the supernatant (Hafez et al. 2012). The measurements were carried out at 25 °C, using the model UV-160A spectrophotometer (Shimadzu, Japan). Activity of catalase (CAT) was determined spectrophotometrically according to Aebi (1983). Polyphenol oxidase (PPO) activity was determined according to Malik and Singh (1980); the absorbance was recorded at 495 nm. Enzymes activity was expressed as the increase in absorbance min−1 g−1 fresh mass. Peroxidase (POX) activity was directly determined in the crude enzyme extract according to a typical procedure proposed by (Hammerschmidt et al. 1982). The absorbance was recorded at 470 nm at every 30 s interval for 3 min.

Electrolyte leakage was determined according to Szalai et al. (1996) as follows: 20 discs (1 cm2) of barely leaves were taken randomly and placed individually into flasks each containing 25 mL deionized water. The samples were shaken for 20 h at ambient temperature to facilitate electrolyte leakage from tissues. Initial electrical conductivity measurements were recorded for each vial using an Acromet AR20 electrical conductivity meter (Fisher Scientific, Chicago, IL). Flasks were then immersed in a hot water bath (Fisher Isotemp, Indiana, PA) at 80 °C (176 °F) for 1 h to induce cell rupture. The vials were again placed on the Innova 2100 platform shaker for 20 h at 21 °C (70 °F). Final conductivity was measured for each flask. Electrolyte leakage percentage was calculated as follows: initial conductivity/final conductivity × 100 according to Szalai et al. (1996), Abdelaal et al. (2014), and Hafez et al. (2014).

Lipid peroxidation was assayed according to Davenport et al. (2003) by measuring the amount of malondialdehyde (MDA). The MDA concentration was estimated by the following formula: MDA (nmol g−1fm) = [6.45 × (A532 − A600) − (0.56 × A450)] × V − 1 W, where V = volume (mL) and W = mass (g).

At harvest (4nd May 2015 and 9th May 2016), the yield components (spike length, number of grains per spike, biological yield (biomass), grain yield/ha, and 1000-grain weight) were determined (Donald and Hamblin 1976). The laboratory analyses were carried out at the (EPCRS) Excellence Center (ISO 9001, ISO 14001 and OHSAS 18001 certified) and the Plant Pathology and Biotechnology Lab (ISO 17025 certified), Department of Agricultural Botany, Kafrelsheikh University.

Statistical analysis

The data were subjected to analysis of variance (ANOVA) procedures according to Gómez et al. (1984) using the MSTAT-C Statistical Software package. The significant difference between the means of treatments was determined at the p ≤ 0.05 level by Duncan’s new multiple range test (Duncan 1955).

Results

Vegetative characters of barley plants under water deficit stress and osmoregulators

Water deficit stress significantly (p ≤ 0.05) reduced the development of barley plants in both seasons compared to control plants (well watered) (Table 3). In the first growing season, average stem length, number of leaves, and fresh and dry mass of barley plants suffered from water deficit stress—when giving plants only one irrigation during the entire growing season—and were 47.5 cm, 4.2 (plant−1), 4.08 (g plant−1), and 1.45 (g plant−1), respectively, while in the irrigated control, they were 61.0 cm, 5.2 (plant−1), 7.07 (g plant−1), and 2.60 (g plant−1), respectively. Data for the second growing season were consistent with those in first season (Table 3). Each of the four osmoregulators tested significantly enhanced the growth of water deficit-stressed barley plants in both seasons. The osmoregulators were applied as a foliar spray. The plants were sprayed twice during their entire life cycle. Data from first growing season showed that osmotic-stressed barley plants supplied with actosol® had higher average values for stem length (68.5 cm), number of leaves (5.8), and fresh and dry mass (8.16 and 3.03 g plant−1, respectively) than the water deficit-stressed control in both growing seasons. Stem length was also significantly higher even than that in the well-watered control in the first season and so was the number of leaves for the second season. Potassium dihydrogen phosphate caused significant increases compared to the water deficit-stressed control in all parameters in both seasons except the number of leaves in season one. Amino more and the Compound fertilizer caused significant increases compared to the water deficit-stressed control in all parameters in both seasons except the number of leaves. Compared to the other three osmoregulators, treatment with actosol® caused higher mean values for all parameters in both seasons except for stem length in season two with potassium dihydrogen phosphate (statistically significant and shorter), and these higher values were statistically significant except for stem length in season one with potassium dihydrogen phosphate, and in season two with Amino more, for the number of leaves in both seasons for potassium dihydrogen phosphate, for fresh mass in season one and dry mass in both seasons with potassium dihydrogen phosphate, and dry mass in the first season with Amino more (Table 3).

Table 3 Vegetative parameters of barley plants treated with different osmoregulators, i.e., potassium dihydrogen phosphate, actosol®, Amino more, and Compound fertilizer under water deficit stress during two growing seasons (2014/2015–2015/2016) compared to the irrigated control
Full size table

Photosynthetic pigments of barley plants under water deficit stress and after treatment with osmoregulators

Photosynthetic pigments estimated by chlorophyll a (Chl a) and b (Chl b) concentrations were statistically significantly lower in osmotic-stressed plants in both seasons compared to the well-watered control (Table 4). Chlorophyll a and b concentrations increased statistically significantly in both seasons after application of each osmoregulator, respectively. Amino more application gave the highest Chl a concentration (1.114 and 1.103 mg g−1 FM) in both seasons. actosol® application gave the highest Chl a concentration (identical to that caused by Amino more, 1.114 mg g−1 FM) in the first season. Meanwhile, Chl b concentrations decreased in all treatments compared to control plants (well watered) in the first season. The highest Chl b concentrations were obtained with potassium dihydrogen phosphate (0.486 mg g−1 FM) and actosol® (0.498 mg g−1 FM) treatments in the first and second seasons (Table 4), and these values were elevated compared to both controls.

Table 4 Chlorophyll a and b, as well as N, P, and K contents of barley plants treated with different osmoregulators, i.e., potassium dihydrogen phosphate, actosol®, Amino more, and Compound fertilizer under water deficit stress during two growing seasons (2014/2015–2015/2016) compared to the well-watered control
Full size table

NPK contents of barley plants under water deficit stress and osmoregulators

Nitrogen phosphorous and potassium (NPK) levels in the biomass were influenced by water deficit stress and foliar sprays of different osmoregulators, especially actosol® and potassium dihydrogen phosphate. NPK levels were lower in water deficit-stressed plants in both seasons. Application of the osmoregulators increased NPK contents in barley plants compared to osmotic-stressed plants. The highest concentrations of N (2.8% and 2.7% in seasons 1 and 2, respectively) and K (2.5% and 2.6%, in seasons one and two, respectively) were recorded with actosol® treatment, and these values are higher than those of the controls and the treatments with other osmoregulators. As for P, the highest levels were found with potassium dihydrogen phosphate treatment (0.66%) in the first season and with actosol® treatment (0.60%) in the second season (Table 4). The above differences were all statistically significant.

Histochemical and biochemical characters of barley plants under water deficit stress and with osmoregulators

Histochemical analysis of reactive oxygen species

The reactive oxygen species (ROS), superoxide (O2·−), and hydrogen peroxide (H2O2), were visualized (purple and brown discoloration, respectively) in barley leaves by histochemical staining. The discoloration increased in osmotic-stressed barley plants in comparison with control plants (well watered) and plants treated with osmoregulators (Fig. 1). The levels of O2·− and H2O2 were expressed as integrated density values (IDV) and ROS levels significantly increased in water deficit-stressed barley plants. Treatment with osmoregulators greatly reduced the concentration of ROS (Fig. 2). Potassium dihydrogen phosphate and actosol® application diminished production of O2·− (29.9 and 31.6 in both seasons) and (34.8 and 30.7 in both seasons) respectively, compared to the osmotic-stressed plants (48.1 and 54.9 in both seasons). The same treatments reduced H2O2 production (23.9 and 22.5 in both seasons) and (27.8 and 26.5 in both seasons) respectively, compared to the osmotic-stressed plants (39.2 and 36.5 in both seasons).

Fig. 1
figure 1

Histochemical staining for superoxide, O2·− (nitro blue tetrazolium, upper row) and hydrogen peroxide, H2O2 (diaminobenzidine, lower row) in leaves of barley plants treated with different osmoregulators, i.e., potassium dihydrogen phosphate, actosol®, Amino more, and Compound fertilizer under water deficit stress during second growing season (2015/2016) compared to the control plants (well watered).*Potassium dihydrogen phosphate

Full size image
Fig. 2
figure 2

Quantification of staining for superoxide, O2·− (a) and hydrogen peroxide, H2O2 (b) in leaves of osmotic-stressed barley plants treated with different osmoregulators, i.e., potassium dihydrogen phosphate, actosol®, Amino more, and Compound fertilizer under water deficit stress during growing seasons (2014/2015–2015/2016) compared to control plants (well watered). (*Potassium dihydrogen phosphate; **Compound fertilizer)

Full size image

Activities of antioxidant enzymes

Exposing barley plants to water deficit stress caused significant increases in both seasons in the activities of antioxidant enzymes CAT (+ 79%, + 64%), POX (+ 88%, + 65%), and PPO (+ 109%, + 101%), compared to the well-watered control. Plants treated with different osmoregulators under water deficit stress had significantly lower levels of the antioxidant enzyme activities than the water deficit-stressed plants, but the levels were still significantly higher than in the well-watered control, except for POX in response to Amino more in season 2 (Fig. 3). Foliar application of the potassium dihydrogen phosphate, actosol®, Amino more, and Compound fertilizer, respectively, reduced the activity of CAT, POX, and PPO in leaves of osmotic-stressed plants, but the changes in enzyme activities were not statistically significant between every osmoregulator. actosol® reduced CAT activity the least (189.2 and 193.4 mM H2O2 g−1 FM min−1) in water deficit-stressed plants compared to well-watered plants (105.6 and 117.6 mM H2O2 g−1 FM min−1) in two seasons. The same trend in changes in response to treatments with different osmoregulators was observed for all three enzymes, i.e., CAT, POX, and PPO (Fig. 3). The lowest activities, i.e., strongest reduction in any treatment were recorded in the Amino more and Compound fertilizer treatments, and these values were statistically significantly different from each other for CAT in year 2, and for PPO in year 1 (Fig. 3). For example, Compound fertilizer reduced, in both seasons, CAT (− 24%, − 19%), PPO (− 33%, − 38%), and POX (− 29%, − 30%), while Amino more reduced CAT in season 2 by 17% and this value is not significantly different from that for Compound fertilizer.

Fig. 3
figure 3

Activities of catalase, CAT (a); peroxidase, POX (b); and polyphenol oxidase, PPO (c) enzymes in leaves of water deficit-stressed barley plants treated with different osmoregulators, i.e., potassium dihydrogen phosphate, actosol®, Amino more, and Compound fertilizer under osmotic stress during growing seasons (2014/2015–2015/2016) against control plants (well watered). (*Potassium dihydrogen phosphate; **Compound fertilizer). Means in the same year with the same letter over the bar are not significantly different according to Duncan’s test at 0.05 level. FM, fresh mass

Full size image

Electrolyte leakage and lipid peroxidation

Electrolyte leakage (EL) significantly increased in plants when growing under water deficit stress (+ 69%, + 44%, in seasons 1 and 2) (Fig. 4). All osmoregulators tested significantly decreased electrolyte leakage from detached barley leaves compared to well-watered plants and water deficit-stressed plants, the latter showing the highest electrolyte leakage values (43.2 and 41.3 μS cm−2 in year 1 and 2, respectively). All four treatments reduced EL compared to the water deficit-stressed control and to or below the level found in well-watered control. The best treatment was potassium dihydrogen phosphate, where reduction in electrolyte leakage was − 51% and − 43% in seasons 1 and 2, respectively, compared to the water deficit-stressed control, and by − 15% and − 17% compared even to the well-watered control (25.6 and 28.6 μS cm−2) in seasons 1 and 2, respectively. The other three osmoregulators were less effective in the order Compound fertilizer < Amino more < actosol® (Fig. 4).

Fig. 4
figure 4

Electrolyte leakage (a) and malondialdehyde (b) in leaves of osmotic-stressed barley plants treated with different osmoregulators, i.e., potassium dihydrogen phosphate, actosol®, Amino more, and Compound fertilizer under water deficit stress during growing seasons (2014/2015–2015/2016) compared to control plants (well watered). (*Potassium dihydrogen phosphate; **Compound fertilizer). Means in the same year with the same letter over the bar are not significantly different according to Duncan’s test at 0.05 level. FM, fresh mass

Full size image

Malondialdehyde (MDA) concentration significantly increased in barley plants growing under water deficit stress. MDA content was 7.1 and 6.9 μmol g−1 FM in two seasons, respectively, for osmotic-stressed plants, while control plants had an MDA content of 4.4 and 4.6 μmol g−1 FM, in both seasons, respectively; this represents a + 59% and + 50% change for seasons 1 and 2, respectively. All four treatments caused significant reduction of MDA compared to the water deficit-stressed control. However, MDA content in leaves of osmotic-stressed barley plants, when supplied with potassium dihydrogen phosphate and actosol© were similar to or restored to values those measured in control plants (not significantly different from well-watered control). MDA content was (4.9 and 4.6 μmol g−1 FM) in the treatment with potassium dihydrogen phosphate and (4.8 and 4.7 μmol g−1 FM) in treatment of actosol® in seasons 1 and 2, respectively (Fig. 4). However, plants receiving Compound fertilizer still had statistically significantly higher MDA contents (6.13 and 5.63 μmol g−1 FM in years 1 and 2, respectively) than the well-watered control.

Yield of barley plants under water deficit stress and osmoregulators

Water deficit stress significantly (p ≤ 0.05) reduced the spike length, number of grains per spike, biological yield, grain yield, as well as weight of 1000 grains (Table 5). Application of osmoregulators caused a recovery or even an increase in yield components of osmotic-stressed barley plants in both seasons. The highest values in spike length were obtained with actosol® treatment (9.00 and 8.75 cm, in two seasons) and with potassium dihydrogen phosphate treatment (9.00 cm) in the second season only. Moreover, the highest values in grains per spike were obtained with actosol® treatments (66.00 and 64.50) in both seasons. Also, actosol® treatment caused the highest biological yield (6.8 and 6.3 ton/ha, in both seasons) and grain yield (3.4 and 3.3 ton/ha, in both seasons) compared to control (well watered) and water deficit stress treatments. However, all tested osmoregulators caused higher values for barley yield and yield components, not only in comparison with osmotic-stressed plants but also the well-watered control plants. Furthermore, the highest values of 1000 grains weight were recorded with actosol® treatment (64.51 and 63.01 g) and potassium dihydrogen phosphate (63.61 and 62.04 g) in seasons 1 and 2, respectively (Table 5).

Table 5 Yield parameters of barley treated with different osmoregulators, i.e., potassium dihydrogen phosphate, actosol®, Amino more, and Compound fertilizer under water deficit stress during two growing seasons (2014/2015–2015/2016) compared to the well-watered control
Full size table

Discussion

Water deficit resulted in stress as estimated by stress indicators and decreased growth and poor health and development as reflected in decreased chlorophyll and b content and major nutrient (NPK) levels in tissues, stem length, number of leaves, and fresh and dry mass as well yield components such as spike length, grains per spike, biological yield, grain yield, and 1000-grain weight in barley. As a response to water deficit stress, reactive oxygen species (superoxide and hydrogen peroxide) levels increased resulting in lipid peroxidation as estimated by malondialdehyde concentration and decreased membrane integrity and increases in antioxidant enzymes such as CAT, PPO, and POX.

The four treatments to alleviate the water deficit stress included simple and complex inorganic fertilizers and their combinations with organic fertilizers and biostimulants. They all act, in part, as osmoregulators, helping plants maintain turgor and take up nutrients under lower water potential conditions. They were applied as a spray that reached both the foliage and the soil.

Water deficit stress can be mitigated by treatments that supply osmoprotectants, stimulate the uptake and/or production of osmoprotectants, and improve water use efficiency (Barati et al. 2015; Singh et al. 2015). Furthermore, such treatments may indirectly help plants by suppressing their pests and diseases (Kesba and Al-Shalaby 2008). The positive effect of actosol® to enhance stem length and number of leaves may be due to that it contains certain necessary nutrients for growth and promote soil structure and increases the metabolic activity of microorganisms. These results are in agreement with those reported by (Kazemi 2013). The decrease in fresh and dry mass of water deficit-stressed barley plants could be a result to the effect of water on stimulating and regulating the photosynthetic enzymes which influences both fresh and dry matter production (Abdalla and El-Khoshiban 2007). Potassium is an essential macronutrient with roles in transpiration, water movement, and translocation of other nutrients and organics in xylem and phloem tissues (Cao and Tibbitts 1992). Potassium dihydrogen phosphate application enhances photosynthesis and consequently increases carbohydrate content, which increases carboxylation efficiency under water deficit stress conditions leading to an increase of dry mass of plants (Marschner 2012). The detrimental impacts of water deficit stress could be attributed to the decrease in photo-assimilation and oxidative damage to the chloroplasts as well as disorganization of thylakoid membranes and chlorophyll degradation under water deficiency conditions (Siddiqui et al. 2015).

The positive effects of osmoregulators, especially actosol®, may be due to increasing the availability of nutrient elements for plant growth and production (Abd El-Aal et al. 2005; Kotob et al. 2009; Kazemi 2013). Furthermore, actosol® may improve nutrients uptake and their mobility in soil through the stimulation of microbiological activity (Denre et al. 2014). Humates regulate the osmotic pressure and thus assisting movement of the water from soil to the plant (Alobaidy 2008; ARCTECH Inc. 2015). These results are in agreement with the findings of others (Albayrak and Camas 2005; Ali and Elbordiny 2009; Shahryari et al. 2009; Mahmoud and Youssif 2015; Bettoni et al. 2016; Bettoni et al. 2017; Al-Fraihat et al. 2018). The increase in potassium content can prevent photosynthesis inhibition (Egilla et al. 2005), enhancing the total dry mass accumulation of osmotic-stressed plants; this may be related to stomatal regulation by K+ and corresponding higher rates of photosynthesis (Marschner 2012). Potassium can also alleviate the damage caused by ROS under water deficit stress (Abd El-Aal et al. 2005; Cakmak 2005). Correspondingly, actosol® inhibition of ROS accumulation may be mediated by enhancement of the enzymatic antioxidants and non-enzymatic antioxidants (Laspina et al. 2005; Fallahi et al. 2017). The beneficial effects of potassium dihydrogen phosphate on enzyme activities could result from enhancing CO2 fixation and transport of photosynthates throughout plant organs (Cakmak 2005). The harmful effects of water deficit stress also derive from cellular dehydration. The beneficial effect of potassium dihydrogen phosphate on reduction of electrolyte leakage maybe attributed to potassium, which plays an important role in improving the function and stability of cell membrane and regulating water deficit stress. Our results with applications of potassium dihydrogen phosphate were consistent with those reported by others (Kant and Kafkafi 2002; Kaya et al. 2007). At the genetic and molecular levels, potassium transporters play a role in ion homeostasis in barley and related grain crops (Song et al. 2014; Shahzad et al. 2015; Wu et al. 2015; Zhang et al. 2018). A high-affinity potassium transporter not only plays important roles in K acquisition and translocation but also positively regulates responses to drought stress in rice (Chen et al. 2017) and has been studied in high salt tolerant barley accessions (Qiu et al. 2011; Han et al. 2018).

Also, the adverse effect of water deficit stress and the resulting of dehydration of protoplasm, decreasing relative water content (Hussain et al. 2008), causes oxidative stress and membrane damage. The beneficial role of potassium dihydrogen phosphate and actosol® may be mainly attributed to improving cell membrane stability and water deficit adjustment ability and consequently decreased malondialdehyde concentration. Electrolyte leakage was alleviated by humic acid and potassium application as was found for bean as well (Aydin et al. 2012).

Intracellular free amino acid levels are part of the water deficit, draught, and salinity stress response (Rai 2002; Sarwat and EI-Sherif 2007; Alobaidy 2008; Abd El-Samad et al. 2010; Singh et al. 2015) and external application of amino acids can support the stressed plant, especially in combination with mineral nutrients alleviates the stress as we have found.

Water deficit stress can reduce yield components by causing reduction in leaf area expansion, which in turn, will lead to reductions in all other yield components (Samarah 2005; McGranahan and Poling 2018). Each one of the four biostimulant osmoregulators contributes to osmotic balance by supplying low molecular weight osmolytes such as ions or amino acids, and by supporting photosynthesis that can add to the osmotically active metabolite pool in the sap (Rai 2002). In agreement with our findings, it was reported that osmoregulators improve growth characters and relative water content correspondingly, increase yield and yield components (Clarke and McCaig 1982). Similar results were recorded in some crops under drought and salinity stresses by (Ekinci et al. 2015; Mahmoud and Youssif 2015; Helaly et al. 2017).

Conclusion

The current study aimed to evaluate the role of select osmoregulators, i.e., actosol®, potassium dihydrogen phosphate, Amino more, and Compound fertilizer to mitigate the detrimental impacts of water deficit stress in order to help crop production where water scarcity is a problem around the world. When barley plants grew with only one irrigation after germination, the growth dynamics and yield were significantly affected compared to control (well watered).

Water deficit resulted in stress as indicated decreased growth and poor health and development as reflected in decreased chlorophyll and b content, and major nutrient (NPK) levels in tissues, stem length, number of leaves, and fresh and dry mass as well yield components such as spike length, grains per spike, biological yield, grain yield, and 1000-grain weight in barley. As a response to water deficit stress, reactive oxygen species (superoxide and hydrogen peroxide) levels increased resulting in increased malondialdehyde levels and decreased membrane integrity and increases in antioxidant enzymes such as CAT, PPO, and POX.

In addition, levels of reactive oxygen species, i.e., superoxide and hydrogen peroxide considerably increased causing increased malondialdehyde levels from lipid peroxidation of membranes, electrolyte leakage, and elevation in the activities of antioxidant enzymes (catalase, peroxidase and polyphenol oxidase). Spraying barley plants twice during the entire growth season with any of the tested osmoregulators substantially enhanced the barley plants’ growth by diminishing the generation of superoxide and hydrogen peroxide and also reduced electrolyte leakage and malondialdehyde concentration, thus maintaining homeostasis in plant cells resulting in better growth, and the yield in certain treatments was even better than in the well-watered control plants. actosol®, followed by potassium dihydrogen phosphate, was the best treatment overall that alleviated the adverse effects of water deficit in barley (Hordeum vulgare L. Giza123) at the Experimental Farm, Faculty of Agriculture, Kafrelsheikh University, Egypt.