1. Introduction
Irrigation water deficiency (IWD) is a substantial abiotic stress factor negatively affecting the growth and productivity of different crops. It is linked to the reduction of arable land and food production [
1], as well as livestock raising around the world. It causes changes in the indices of plant morphology, physio-biochemistry, including the antioxidant defense system, and molecular biology of plants [
2,
3,
4,
5,
6,
7]. Located in dry regions, more than 50% of the world’s agricultural sector lands face climate changes that increase the frequency of extreme water shortage conditions [
8,
9,
10]. Crop plants attempt to tolerate IWD by avoiding dehydration by retaining a higher amount of water through many plant strategies (e.g., reduced leaf area, stomatal closure, and older leaf senescence), so it rarely corresponds to high yield, and/or by tolerating dehydration by functioning under the IWD event [
11,
12]. An innate inconsistency between the accumulation of biomass and avoidance of stress has been demonstrated, because the low transpiration rates affect the acquisition of photo-assimilate that depends on the stomatal aperture and leaf area [
12,
13]. Additionally, increased output and large sinks of plants—especially in cereals, at least—add a burden to the shoot system regarding the status of plant water and maintenance of cell turgor under the adverse conditions of IWD [
11]. As a consequence, while the improved plant’s production potential may lead to a preferred performance with stress, it also monitors the increased demand for useful resources, including water. Thus, under IWD, a plant’s high water uptake ability may occasionally increase the frequency of the stress experience. Appropriately, the higher return potential should correlate with a boosted tolerance to stress [
12]. IWD causes pigment degradation, thus reducing the chlorophyll content as a result of excessive reactive oxygen species production [
14,
15]. It reduces carbon assimilation and the photosynthetic electron transport chain, thereby increasing photoinhibition [
16,
17,
18]. It also increases the NADPH and NADPH/NADP+ and decreases the photosynthetic efficiency and NADPH dissipation in chloroplasts, which also reduces the photosynthetic electron transport chain [
19,
20].
As a remarkable cereal crop, maize (
Zea mays L.) ranks first in terms of total production and second concerning the area planted, after wheat, worldwide [
21]. It is a sensitive crop to drought, and its production is affected destructively by IWD [
22]. With the expected rise of IWD in the coming time period due to climate change, this stress will primarily threaten the stability of the management and production of maize crops [
9,
10,
23,
24]. Therefore, it is very important to find approaches to elevate IWD tolerance in maize to enhance plant growth and production [
25,
26]. The most efficient approach to solve the problem is developing drought-tolerant maize hybrids, which needs a profound comprehension of the mechanisms in drought stress responses in maize. However, developing drought-tolerant hybrids require considerable effort, a long time, and high economical investments [
26,
27]. Therefore, some approaches can be helpful in this regard [
6,
26,
28,
29], including the use of plant hormones to enhance plant performance and output under IWD conditions [
30,
31,
32,
33,
34].
As perceived at the cell surface and maybe biosynthesized in the endoplasmic reticulum, brassinosteroids (BRs) are a class of plant hormones (polyhydric steroids) with a considerable growth-promoting effect. They exert their effect as multidimensional regulators of a plant response to various stresses, as well as the growth and development of different crops [
35]. As one of the most important BRs and a byproduct of brassinolide biosynthesis, 24-epibrassinolide (EBR
24) is a beneficial active molecule in plants. It has been suggested that EBR
24 is biosynthesized either through an independent or a dependent pathway, as both are compostanol-based [
36]. Although its accurate mechanisms are still mysterious [
36], it has the potential to improve growth and development and stimulate the different metabolic processes of plants, including CO
2 fixation, photosynthesis, and protein and nucleic acid biosynthesis [
37], through activating many enzymes related to photosynthesis and the components of the antioxidant defense system (e.g., free proline, CAT, POD, and SOD) [
35,
38] to help alleviate stress conditions, especially drought adversities in different plants [
36,
39,
40]. Several works have been reported stating that the exogenous use of EBR
24 can increase drought tolerance in plants by enhancing the morphological and physiological responses, including photosynthetic pigments biosynthesis, chlorophyll fluorescence, photosynthetic and photochemical activity, gas exchange indices (i.e., photosynthesis and stomatal conductance and transpiration rates), and the plant water status, which reflects positively in plant productivity [
33,
34,
38,
41]. Although they are widely distributed throughout plants, BRs are not transported over long distances among different tissues. However, BRs may have an indirect role in long-distance signals through their influence on other plant hormones. Besides, it has been reported that BRs regulate plant stress tolerance through modulation of the ROS signal, which is also involved in the systemic stress response [
42].
Numerous studies have indicated that EBR24 promoted the growth of drought-stressed plants. However, information on the mechanisms of growth and yield enhancement induced by EBR24 in maize hybrids is not available. Therefore, the current work aimed at determining the fruitful role of EBR24 used exogenously in boosting drought tolerance in six commercial maize hybrids (i.e., Giza-162, Giza-166, Giza-167, Giza-168, Giza-176, and Fine-276). This aim can be achieved under semi-arid conditions by exploring improvements in growth, yield, crop water productivity (CWP), physiological traits, and the plant antioxidant system with the application of EBR24 to these contrasting hybrids in drought tolerance.
4. Discussion
The sustainable production of maize crops is currently facing a great problem of environmental degradation due to many issues, including irrigation water shortage (IWD). IWD is one of the major problems facing crop productions (including maize), especially in dry (arid and semi-arid) regions, which limits crop productivity. Improving IWD tolerance in maize through ecofriendly sustainable strategies is the key to securing foods for the growing human population [
61].
The promotional influences of 24-epibrassinolide (EBR
24) on plant growth under drought stress have been reported; however, little information is available on the EBR
24-induced drought stress-conferring mechanisms for improving the growth of maize hybrids [
40,
41]. In this study, the potential improvements in maize hybrid yield-contributing traits under drought stress by improving the mechanisms in plant physio-biochemistry due to a foliar spray with EBR
24 are discussed.
IWD reduced the yield-contributing components of all maize hybrids (e.g., plant height, row and grain numbers on each ear, 1000-grain weight, grains, and biological yield per ha) to varying degrees based on the tolerance or sensitivity of the maize hybrid (
Figure 1). However, the foliar application of 5-μM EBR
24 enabled maize plants to perform well under IWD stress, especially under moderate stress. EBR
24 significantly reformed and awarded positive alterations in all indices of the plant morphology, physiology, and biochemistry and, consequently, the CWP and agronomic traits in all investigated maize hybrids growing under moderate (MDS) and severe drought stress (SDS) compared to the corresponding untreated controls. Photosynthetic traits, including photosynthetic pigments, are indicators of drought tolerance in plants. The results indicated that EBR
24 enhanced the drought tolerance in maize hybrids by improving the chlorophyll and carotenoids contents, photochemical activity, and photosynthetic efficiency in the tested hybrids, especially under MDS (
Table 2).
The promotional impacts of EBR
24 on the yield-contributing traits observed in
Figure 1 are potentially related to the improvements in the antioxidant system components (
Table 5). These improvements are reflected in the reduction of membrane damage (reduced MDA content and EL) (
Table 3 and
Table 4) and in the protection of the photosynthetic apparatus (
Table 2), which are attributed to the improvements in gas exchange, plant water status, and the osmo-protectant soluble sugars content (
Table 3 and
Table 4). This fact, coupled with the increase in chlorophyll content (
Table 2) and MSI (
Table 4) with the 5-µM EBR
24 treatment, ensured the maintenance of the net photosynthetic rate (
Table 3), reflecting an increase in the yield-contributing traits and final yields. Additionally, EBR
24 promoted the transpiration rate and stomatal conductance under IWD stress conditions potentially due to how EBR
24 acts in the stomatal closure in
Arabidopsis thaliana in an abscisic acid (ABA)-independent manner [
62]. Besides, EBR
24 may act by modulating ABA-mediated stomatal closure both positively and negatively, depending on its concentration. In fact, the process of stomatal opening and closure is not only ABA-dependent. In IWD stress conditions, a dose-dependent action has also been reported for other hormones, such as cytokinins and auxins [
62]. Therefore, it is possible that the dose-dependent action of EBR
24 on stomatal behavior is due to its crosstalk with other plant hormones [
32].
In the current study, due to oxidative damage to cell membranes under IWD stress, lipid peroxidation was identified as a level of malondialdehyde (MDA), an important biochemical marker of stress-inducing oxidative damage, as it minifies the production of the biomass, along with the plant’s acclimatization hypothesis [
63,
64]. Membrane lipid peroxidation is increased under drought stress, and as a consequence, the level of MDA is increased [
65,
66]. A low MDA level indicates a lower damage level to stressed cellular membranes, meaning that the plant is more stress-tolerant. When the peroxidase (POD) activity is associated with the level of membrane lipid peroxidation, a marked rise in the MDA level in stressed plants indicates an insufficient POD activity in the ROS collection to prevent damage to cellular membranes and minimize MDA production. In this study, the use of EBR
24 made the plants able to avoid IWD stress and reduce their MDA levels compared with the untreated plants (
Table 3), demonstrating the pivotal role of EBR
24 in reducing lipid peroxidation and maintaining plasma membrane stability and structure under IWD stress. The lipid peroxidation reduction that occurred by EBR
24 was connected with an increased enzymatic antioxidant activity, upregulating the membrane permeability in terms of the increased MSI (
Table 4). These findings are consistent with those reported in [
67,
68].
The status of water in plants is highly sensitive to drought stress and is thus predominant in assessing a plant’s response to stress. IWD reduces the hydraulic conductivity of plant roots and water flow from root system to shoot system, reducing the leaf water content and closing the stomata to maintain leaf water [
69,
70]. A low RWC of plant leaves causes a toxic effect, which leads to physiological and metabolic changes and the inhibition of plant growth. However, the use of EBR
24 markedly increased the leaf RWC and MSI in all IWD-stressed maize hybrids compared to the untreated controls (
Table 4). This positive finding could result from the improvement of the transpiration rate in the treated stressed plants (
Table 3). The same results were obtained by Shahid et al. [
71] and Lima and Lobato [
72]. EBR
24 maintains the plant RWC under IWD stress by improving the water, pressure, and osmotic (solute) potentials due to the beneficial role of EBR
24 in sustaining cellular membrane permeability and integrity under IWD stress conditions [
73]. Besides, Rady [
50] suggested that EBR
24 affects the protein and/or enzyme biosynthesis to enhance the plant metabolism by improving the expression of specific genes [
74].
In the current study, IWD considerably increased the leakage of electrolytes (EL). However, the use of EBR
24 significantly reduced the EL in all the maize hybrids evaluated under two levels (MDS and SDS) of drought (
Table 4). When the plants are exposed to drought, the leaf stomata are closed, causing a reduction in the fixation of CO
2, while the transfer of electrons and the light reaction remain naturally. These conditions restrict the acceptance of electrons by NADP; thereby, oxygen can perform as an electron acceptor, resulting in the overproduction of ROS (e.g., O
2•−, H
2O
2, and OH
−), which peroxidize the cell membranes and increase the EL [
75,
76]. In this regard, Rady [
50] reported a maximization of the EL and MDA in stressed plants. However, a follow-up treatment using 5-μM EBR
24 decreased the lipid peroxidation and ionic leakage. Likewise, Shakirova et al. [
30] and Mohammadi et al. [
34] disclosed that the lowest MDA content in EBR
24-treated plants exposed to SDS was associated with mitigating the stress-induced deleterious influences by enhancing the accumulation of osmolytes. This kept the cell membrane integrity, reduced the lipid peroxidation level, and produced various important metabolites. The use of EBR
24 induced physio-biochemical alterations, including increasing the root system size, nonenzymatic antioxidant content, and enzyme activity [
77].
In the present study, under IWD conditions, the soluble sugar content was significantly modified in all maize hybrids to contribute to osmotic modification and can, indirectly or directly, modify the gene expression implicated in plant metabolism and storage and defense functions [
78,
79]. Further, 5-μM EBR
24 highly increased the soluble sugar content in all maize hybrids evaluated under the three irrigation regimes (
Table 4). Like soluble sugars, the accumulation of free proline contributed to osmotic modification under IWD stress due to acclimatization to recompense for plant survival and, thus, helped in resisting drought stress [
80]. Free proline enhances plant tolerance by the detoxification of ROS and may quench the singlet oxygen (
1O
2) in a physical manner or react directly with OH
− radicals [
6]. Depending on the stress severity, the free cellular proline content is estimated to be approximately 20–80% of the total amino acid pool versus 5% under normal conditions, resulting from a decreased degradation and/or an increased biosynthesis of free proline in plants [
81,
82]. In this study, the total proline was increased by 5-μM EBR
24, and the maximum concentration was observed under SDS (
Table 5). Similarly, Talaat and Shawky [
67] and Chen et al. [
83] demonstrated the use of EBR
24 initiates and increased proline biosynthesis in plant cells to enhance the plant defense system to avoid oxidative damage stimulated by IWD stress [
4,
34].
Under MDS, the CAT, SOD, and POD activities were increased and further increased under SDS conditions (
Table 5). A strong correlation has been reported between oxidative stress tolerance and boosted enzymatic activities [
84,
85]. The plant tends to raise its antioxidant enzyme activities to withstand drought stress and eliminate ROS. These enzyme activities were markedly varied in the six maize hybrids assessed for their tolerance to IWD stress (
Table 5). Moreover, the application of EBR
24 boosted the CAT, POX, and SOD activities under IWD stress levels compared to the corresponding untreated control group (
Table 5), which can be attributed to the EBR
24 influence on transcription and/or translation of antioxidant genes [
39,
41,
86].
Crop water productivity (CWP) refers to the association between crop productivity and the water amount used in crop production [
56,
57]. Ameliorating the crop water productivity is critical to producing more food using less water, particularly in arid and semi-arid environments, to preserve the limited irrigation water. The obtained results revealed that the application of EBR
24 substantially increased the CWP
g and CWP
b by 9.9% and 12.4% under SDS and 6.6% and 7.4% under MDS compared to the untreated controls. The enhancement of CWP occurred through improving the photosynthetic efficiency, gas exchange indices, osmotic adjustment, water relations, and activities of antioxidant enzymes.
Identifying drought-tolerant maize hybrids is a pivotal approach to avoiding the destructive influences of drought stress, especially in arid environments, in light of the current climate changes. In the present study, the physiological parameters, CWP, and agronomic traits were used to assess the response of six hybrids of maize to IWD. Significantly, the examined hybrids demonstrated differences in their physiological and agronomic responses under IWD stress conditions. The hybrids introduced a significant alteration in the attributes of photosynthetic efficiency and gas exchange under three irrigation regimes (control; 9000 m
3 water ha
−1 versus 6000 and 3000 m
3 water ha
−1 applied as MDS and SDS, respectively). The hybrid pattern changed further under SDS compared to the MDS and well-watered conditions. The highest values of photosynthetic efficiency and gas exchange indices were assigned for Giza-168 under the MDS and well-watered conditions, followed by Fine-276, then Giza-167, while, under SDS, these indices exhibited the highest values by Fine-276, followed by Giza-167, then Giza-168 (
Table 2 and
Table 3). The rates of transpiration and gas exchange are related to the carbon uptake through opened stomata and the avoidance of dehydration, defined as the capability of plants to keep a high state of water. Therefore, these physiological behaviors helped the plants perform better under MDS and SDS (
Figure 1).
Osmotic adjustment is a principal plant adaptive reaction to IWD stress at the cellular level. It is one of the components of turgor maintenance and dehydration avoidance and, therefore, has positive effects on the grain yield and related traits under IWD. In response to IWD stress, plants tend to accumulate inorganic and organic substances such as free proline, soluble sugars, and metallic ions to lessen the osmotic potential and boost the cell water retention [
11,
87]. Accordingly, the osmotic adjustment maintains a high RWC under a low water potential to meet transpiration upon request, sustains cellular turgor, promotes cell expansion, and, hence, the yield-forming processes [
11,
13,
88]. In the current study, the Giza-167, Giza-168, and Fine-276 hybrids exhibited the highest free proline and soluble sugar contents under MDS and SDS compared to the other hybrids (
Table 4 and
Table 5). The large accumulation of free proline and soluble sugars contents provides an important adjusting role to postpone dehydration under unfavorable osmotic stress conditions and preserve a high RWC and MSI in these three hybrids (
Table 4).
Under normal metabolism, ROS are normally produced in low levels in plant cells; however, they are produced excessively under adverse conditions, including IWD [
89]. ROS production is controlled by the plant defense system, including CAT, SOD, POD, etc. and low-molecular-weight antioxidants [
90]. POD can positively modify the levels of ROS through scavenging/consuming H
2O
2. Moreover, CAT and SOD can restrain, or at least minimize, OH
− radical generation [
91,
92]. In the current study, the ROS levels in the maize hybrids may be eliminated due to the significant improvement in CAT, POD, and SOD activities under IWD conditions (
Table 5). In particular, Fine-276, Giza-167, and Giza-168 showed the highest antioxidant enzyme activities under MDS and SDS, which improved their agronomic performance and grain yield under IWD stress conditions.
The increment in gas exchange, photosynthetic efficiency, soluble sugar, proline contents, and antioxidant enzyme activities in maize hybrids may explain an increase in their agronomic traits under IWD stress conditions (
Figure 1). Accordingly, Fine-276 displayed the greatest ability to accumulate biomass in the shoot, followed by Giza-167 and Giza-168, as shown by the largest plant height, grain yield, and contributing traits. These hybrids demonstrated a mechanism to withstand dehydration by enhancing the efficiency of photosynthesis, gas exchange, osmotic adjustment, water relations, and enzymatic antioxidant activities. Furthermore, the drought-tolerant hybrids exhibited more grain and biological yields with higher CWP
g and CWP
b (
Table 6) compared to the drought-sensitive ones. The hybrids Giza-168, Fine-276, and Giza-167 displayed better CWP values associated with significantly greater growth and productivity—in particular, at low water amounts—compared to the sensitive ones. Therefore, using these drought-tolerant hybrids is preferred to improve the CWP and increase the grain and biological yields principally in arid environments [
93,
94,
95,
96].
The constituent plant traits have a crucial function in water use, dehydration avoidance, and producing an acceptable grain yield under IWD [
11]. Consequently, the yield potential can be defined as the traits that can boost the yield under IWD during the growth stages. The traits that confer drought tolerance can be divided into two types: firstly, improving the crop yield during water supply, and the second, which contributes to the survival of the plant during a very limited capacity of the soil to hold water [
13,
97].
Assessing the interrelationships between plant traits can provide useful information for screening maize hybrids under low available water conditions. A biplot of principal components is an appropriate statistical method for understanding the interrelationships among evaluated traits, which is estimated by the angle size of the trait vectors (
Figure 2). The results reflected that the agronomic traits were positively associated with the total chlorophyll and carotenoid contents, photochemical activity, rates of net photosynthesis and transpiration, photosynthetic efficiency, conductance of leafy stomata, RWC, and MSI. From this standpoint, it could be speculated that the high values of these physiological traits could illustrate more grain yields and contributing traits. Otherwise, the CWP
g and CWP
b proved to have highly positive associations with the levels of soluble sugars and free proline and activities of POD, CAT, and SOD. Additionally, the agronomic traits displayed highly negative associations with malondialdehyde and the electrolyte leakage. These results are in consonance with previous studies that have demonstrated the importance of physiological parameters as indicators for grain yield under abiotic stress [
98,
99,
100,
101,
102]. According to these findings, it is important to detect certain physiological traits that have a positive association with yield-related traits or CWP under drought stress.