Next Article in Journal
Impacts of Drought Stress on Water Use Efficiency and Grain Productivity of Rice and Utilization of Genotypic Variability to Combat Climate Change
Previous Article in Journal
Effect of Sodium Chloride Salt on Germination, Growth, and Elemental Composition of Alfalfa Cultivars with Different Tolerances to Salinity
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 12
Issue 10
10.3390/agronomy12102517
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Enhancement of the Expression of ZmBZR1 and ZmBES1 Regulatory Genes and Antioxidant Defense Genes Triggers Water Stress Mitigation in Maize (Zea mays L.) Plants Treated with 24-Epibrassinolide in Combination with Spermine

by
Neveen B. Talaat
1,*,
Ahmed S. Ibrahim
1 and
Bahaa T. Shawky
2
1
Department of Plant Physiology, Faculty of Agriculture, Cairo University, Giza 12613, Egypt
2
Department of Microbial Chemistry, Biotechnology Research Institute, National Research Centre, Giza 12311, Egypt
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(10), 2517; https://doi.org/10.3390/agronomy12102517
Submission received: 22 September 2022 / Revised: 8 October 2022 / Accepted: 12 October 2022 / Published: 15 October 2022
Browse Figures
Versions Notes

Abstract

:
Water shortages greatly threaten global food security and limit crop production. Hence, increasing crop water stress tolerance is a critical way to secure agricultural production. 24-Epibrassinolide (EBL) and spermine (Spm) are closely involved in plant growth and development, as well as stress tolerance. In this study, the potential role of 0.1 mg L−1 EBL and/or 25 mg L−1 Spm foliage applications in improving the tolerance of maize to water-deficit conditions (50% and 75% field capacity) was investigated. We found that EBL, either alone or in combination with Spm, plays a major role in maize drought tolerance through upregulating the expression of both regulatory genes (ZmBZR1 and ZmBES1) of the brassinosteroid signal transduction pathway and gene-encoding antioxidant defense enzymes ZmSOD, ZmCAT, ZmAPX, ZmMDHAR, ZmDHAR, and ZmGR. Moreover, exogenous treatments alleviated the inhibition of maize plant growth and productivity and mitigated drought-induced oxidative stress by improving antioxidant enzyme (superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase) activity, enhancing antioxidant molecule (ascorbate, glutathione) content, preventing reactive oxygen species accumulation, and maintaining cell membrane integrity. These findings reveal that the application of EBL, either individually or in combination with Spm, can be a good strategy for ameliorating water stress in sustainable agricultural systems.

1. Introduction

Environmental problems such as drought, salinity, high temperature, and low temperature cause morphological impairments and retard plant growth and productivity [1,2,3,4]. Water shortages greatly threaten global food security and limit crop production. Hence, increasing crop water stress tolerance is a critical way to secure agricultural production [5]. Drought induces oxidative stress through the production of reactive oxygen species (ROS) molecules [6]. A strict control of ROS levels is essential to prevent their toxicity and to ensure an accurate execution of their signaling functions. Therefore, plants have evolved an elaborate enzymatic and non-enzymatic antioxidant system which maintains ROS homeostasis in all cellular compartments [7]. The enzymatic components of the antioxidative defense system comprise several antioxidant enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2), while non-enzymatic antioxidants include glutathione (GSH), ascorbic acid (AsA), phenolic compounds, flavonoids, carotenoids, α-tocopherol, etc. [8,9].
Spermine (Spm) is a tetraamine that contributes to plant resistance to abiotic stress [10]. When applied exogenously, Spm can act as a potent plant defense activator [11]. By scavenging ROS radicals, increasing antioxidant enzyme activity, maintaining cationic-anionic stability, and involving gene transcription and protein translation, exogenously applied Spm lessens the detrimental impact of drought on plant development [12,13,14].
Plant hormones are critical for plant growth and development because they orchestrate intrinsic developmental programs as well as relay environmental stimuli [15,16,17]. Among them, brassinosteroids (BRs), a group of steroidal phytohormones, regulate a variety of plant physiological processes such as cell division, cell elongation, seed germination, stomata development, vascular differentiation, plant architecture, flowering, senescence, and abiotic stress responses [18,19,20]. Plants with defects in BR biosynthesis or signaling, in particular, have a dwarf phenotype, indicating that BR is important for normal plant growth and development [21]. Different studies have shown that the exogenous application of BRs increases the tolerance to abiotic stresses by stimulating the enzymatic and non-enzymatic antioxidant defense system [22,23,24,25,26,27].
BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMS-SUPPRESSOR 1 (BES1) are important transcription factors (TFs) in the BR signaling pathway [28]. Previous studies have shown that both BZR1 and BES1 can bind to the promoters of several genes involved in the regulation of plant growth and development [29]. Cell elongation, immune signaling, stress tolerance, cell division, flowering, and seed development are all regulated by BZR1 and BES1 [21,30,31]. To date, intensive studies have revealed that BES1 and BZR1 are regulated not only by endogenous BR levels, but also by exogenous BR levels [32]. When BR levels are high, the two proteins are dephosphorylated by a cooperative reaction of BRASSINOSTEROID INSENSITIVE2 (BIN2) kinase and protein phosphatase 2A (PP2A), leading to their nuclear accumulation and binding to the target gene promoters [33,34]. BES1/BZR1 play a key role in plant adaptation to environmental stresses such as high temperature [32], drought [35,36,37], freezing [38], and salinity [39]. To reduce drought responses, BES1 could interact with RESPONSIVE TO DESICCATION 26 (RD26) and inhibit its transcript level [36]. Several WRKY transcription factors also interacted directly with BES1 to boost BR-regulated plant development while inhibiting drought tolerance by silencing drought-inducible global genes [35]. Unlike Arabidopsis, where AtBES1 has a negative involvement in drought responses, wheat (Triticum aestivum) TaBZR2 has a positive role in drought tolerance [40], revealing a complicated regulation under abiotic stresses.
Maize (Zea mays L.) is one of the most prominent crops in many regions all over the world and drought becomes a serious problem for its production. Its average annual yield loss due to water deficit is around 15% of its potential yield [41]. Maize plants require large amounts of water to realize high yield during the ear development and grain-filling stages. However, the success of maize reproduction and the realization of potential yield under low water availability are dependent on the stress sensitivity of the reproductive and grain-filling stages and the overall plant growth and development [42].
The BES1 and BZR1 can play an important role in regulating plant growth and development, as well as abiotic stress responses [21,31]. However, there is little known about the potential impact of EBL and/or Spm on regulating BES1 and BZR1 expression, and alleviating the stress-induced inhibition of plant development. To fill this gap, we examined the effect of EBL and/or Spm foliar applications on the expression of the regulatory genes (ZmBZR1 and ZmBES1) of the brassinosteroid signal transduction pathway in maize plants grown under water-deficit conditions. Moreover, the expression of genes encoding the antioxidant defense enzymes ZmSOD, ZmCAT, ZmAPX, ZmMDHAR, ZmDHAR, and ZmGR along with hydrogen peroxide content, lipid peroxidation, and electrolyte leakage were also analyzed. We also investigated the effect of EBL and/or Spm exogenous applications on the activity of antioxidant enzymes (SOD, CAT, APX, DHAR, MDHAR, GR) and the content of antioxidant molecules (AsA, GSH). The purpose of this study was to verify the hypothesis that exogenous EBL and/or Spm could upregulate the antioxidant defense genes’ expression and modulate ROS homeostasis to improve maize growth and productivity under water-deficit environments. We found that the application of EBL, either individually or in combination with Spm, plays a major role in maize water stress tolerance by improving the activity of ZmBZR1 and ZmBES1, upregulating the activity ofthe genes encoding the antioxidant defense enzymes, preventing ROS accumulation, and maintaining cell membrane integrity. The results of this study could provide a comprehensive overview of EBL and Spm effectiveness in mediating drought tolerance.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Pot experiments were conducted in the greenhouse of the Department of Plant Physiology, Faculty of Agriculture, Cairo University, Egypt, during two successive seasons—2020 and 2021—under natural light and temperature conditions with an average day/night temperature of 35/25 ± 2 °C and average humidity of 65%. Grains of Giza 10 and Giza 129 maize hybrids were obtained from the Agriculture Research Center, Giza. These two genotypes were selected based on their high yield productivity and we tried to increase their drought tolerance by using EBL and/or Spm foliar applications.
The pots (30 cm in diameter and 35 cm in height) were filled with 15 kg of clay loamy soil (sand 37%, silt 28%, and clay 35%). Ammonium nitrate (33.5% N), calcium superphosphate (15.5% P2O5), and potassium sulfate (48% K2O) were applied at the rates of 2.0, 2.0, and 0.5 g pot−1, respectively, before planting. In addition, 30 days after planting, 2.0 g pot−1 ammonium nitrate was added. Table 1 shows the soil chemical analysis, which was performed according to Cottenie et al. [43]. For each pot, four grains thinned to two after germination were planted on 3 June in both seasons. All pots were irrigated to soil saturation before planting. After planting, irrigation was applied at the appropriate times with tap water to maintain soil moisture near maximum water-holding capacity for 30 days. Water treatments were carried out 30 days after seeding.
Water stress treatments were started one month after sowing through exposure of the plants to three soil water conditions: control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2). Soil water contents for the treatments with 100%, 75%, and 50% field capacity were 15.5%, 11.6%, and 7.7%, respectively. Soil water content (SWC) was calculated using the formula: SWC % = [(FW − DW)/DW] × 100, where FW was the fresh weight of a portion of the soil from the internal area of each pot and DW was the dry weight of the soil portion after oven-drying at 85 °C for 4 days [44].
For each drought stress treatment, 60-day-old maize plants were sprayed with four spraying treatments (0.00 (distilled water), 25 mg L−1 Spm, 0.1 mg L−1 EBL, and 25 mg L−1 Spm + 0.1 mg L−1 EBL). EBL (C28H48O6, MW = 480.7) was purchased from Sigma (Saint Louis, MO, USA) and was dissolved in sufficient quantity of ethanol. Spm (C10H26N4, MW = 202.3) was also purchased from Sigma (USA) and was dissolved in a sufficient quantity of autoclaved distilled water. Tween-20 (0.05%) was added as surfactant at the time of treatment. The concentrations of 0.1 mg L−1 EBL and 25 mg L−1 Spm were the most effective concentrations according to the preliminary experiment. The spraying was performed when the maize plants had six to eight leaves fully developed leaves (during the pre-female inflorescence emerging stage).
The experiment was performed in a completely randomized design with three factors: two maize hybrids, three soil water conditions, and four spraying treatments.

2.2. Samples Collection and RNA Isolation

After seven days of foliar applications, we collected six biological replicates of each treatment for RNA isolation, and each biological replicate had three plants. All samples were collected at the same time, ground into powder in liquid nitrogen, and stored at −80 °C for further use. Total RNA was extracted from the plant leaf samples (200 mg) using the RNA extraction kit (Agilent Plant RNA Isolation Mini Kit, 5188–2780). Total RNA concentration was determined using a spectrophotometer, and then intact mRNA was purified from total RNA using the NEBNext®Poly(A) mRNA Magnetic Isolation Module (NEB, E7490L) (New England Biolabs, Ipswich, MA, USA). The purified mRNA was reverse-transcribed (RT) into cDNA using an oligo (dT) 20 primer and the ProtoScript® First Strand cDNA Synthesis Kit (NEB ™ E6500S) following the manufacturer’s instructions.

2.3. Semi-Quantitative RT-PCR Analyses

Gene-specific primers for the semi-quantitative real-time PCR (RT- PCR) were designed based on the mRNA or EST sequences for the corresponding genes as follows: ZmSOD: superoxide dismutase; ZmCAT: catalase; ZmAPX: ascorbate peroxidase; ZmMDHAR: monodehydroascorbate reductase; ZmDHAR: dehydroascorbate reductase; ZmGR: glutathione reductase. Moreover, the two regulatory TFs genes encoding the Zea mays protein BZR1 homolog 1-like (ZmBZR1) and the Zea mays protein BES1 (ZmBES1) were used. Zea mays ubiquitin carrier proteingene (ZmUBCP) was used as an internal control. The gene-specific primers were used as shown in Table 2 and were designed using PRIMER3 (http://frodo.wi.mit.edu/primer3/input.htm accessed on 7 January 2020). Semi-quantitative reverse transcription–PCR was conducted in 20 μL reaction containing 3 μL of the template (diluted cDNA 1:5 H2O), 1 pmol of each primer of the target gene or the primers of ZmUBCP as an internal control, 200 μM of each dNTP, 2.5 mM MgCl2, and 1.5 unit of Hot Start Taq DNA Polymerase (NEB M0495S).

2.4. Gene Expression Analyses

Quantitative RT-PCR assays were performed using the same specific primers of semi-quantitative RT-PCR analyses. Samples were run in triplicate on each plate to validate the results of the quantitative RT-PCR; 6 biological replicates (representing 18 plants) for each sample were analyzed and repeated two times in two independent experiments (on two plates). qRT-PCR was performed using SYBRGreen (ABI-Invitrogen, CA, USA) on an ABI 7500 Real-Time PCR Detection System (Applied Biosystems, Carlsbad, CA, USA). Thereal-time RT-PCR reaction (20 μL) included 10 μL of 2x SYBR Green Master Mix, 3 μL of diluted cDNA (1:5 H2O), and 1 pmol of forward and reverse primers of the target gene or the primers of the ZmUBCP gene as an internal control. The thermal cycling conditions were: initial denaturation step at 94 °C for 10 min, followed by 40 cycles at 95 °C (30 s), 60 °C (30 s), and 72 °C (30 S). The relative expression levels of the selected genes were calculated using the 2−ΔΔCT method [45].
The physiological and biochemical traits, including antioxidant enzyme activity, antioxidant molecule content, lipid peroxidation, hydrogen peroxide content, electrolyte leakage, and growth parameters were measured in 67-day-old (after seven days of foliar application) maize plants. Data were collected from six replicates, each of which contained three plants.

2.5. Antioxidant Enzyme Activity

Fresh leaf samples (0.5 g) were ground with 5 mL of ice-cold 100 mM phosphate buffer (pH 7.4) containing 1% polyvinyl pyrrolidine and 1 mM EDTA, then centrifuged at 15,000× g for 10 min at 25 °C. The supernatant was collected and used for the determination of enzymatic activity. The SOD activity was determined by monitoring its inhibition of the photochemical reduction of nitroblue tetrazolium. One unit of enzyme activity was defined as the amount of enzyme bringing about a 50% inhibition in the reduction rate of nitroblue tetrazolium detected at 560 nm [46]. The CAT activity was determined by monitoring the decrease in absorbance at 240 nm due to the decomposition of H2O2 [47]. The APX activity was determined by measuring the decrease in absorbance at 290 nm caused by ascorbate oxidation [48]. NADH oxidation at 340 nm was used to determine MDHAR activity [49]. The DHAR activity was measured by examining ascorbate formation at 265 nm [50]. The oxidation of NADPH at 340 nm was used to measure GR activity [51].

2.6. Antioxidant Molecules (Glutathione (GSH) and Ascorbate (AsA)) Measurement

Fresh leaf samples (0.5 g) were homogenized in 5 mL of 5% (w/v) sulfosalicylic acid and centrifuged at 20,000× g for 20 min at 4 °C. The GSH was oxidized using 5,5′-dithio-bis-nitrobenzoic acid to give GSSG and TNB (5-thio-2-nitrobenzene). GSSG was reduced to GSH by the action of GR and NADPH. GSSG was assayed from the sample after the removal of GSH by 2-vinylpyridine and triethanolamine derivatizations. Changes in absorbance due to the rate of TNB formation were measured at 412 nm. The amount of GSH was the difference between total glutathione and GSSG [52].The assay of AsA is based on the reduction of Fe3+ to Fe2+ by ascorbic acid in acidic solution. The Fe2+ and dipyridine produce a colored complex that absorbs at 525 nm. DHA was reduced to AsA by pre-incubating the sample with dithiothreitol (DTT). The excess DTT was removed with N-ethylmaleimide, and the total ascorbate was determined. The amount of DHA was the difference between total ascorbate and the AsA [52].

2.7. Estimation of Stress Markers

For the estimation of the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, 0.1 g of fresh maize leaves were ground in a mortar with 900 µL buffer, following the instructions described in H2O2 and MDA kits, according to the method described by Nawaz et al. [53]. H2O2 and MDA contents were recorded at a wavelength of 405 nm and 532 nm, respectively.

2.8. Determination of Electrolyte Leakage (EL)

To assess the leakage of ions from membranes, leaves were collected and washed with distilled water. They were placed in test tubes with10 mL distilled water and kept in a water bath at 40 °C for 30 min and electrical conductivity (C1) was recorded. Later, the same samples were placed in a water bath for 10 min at 100 °C and electrical conductivity (C2) was noted. The electrolyte leakage was calculated by using the formula of Li et al. [54].
EL = [C1/C2] × 100

2.9. Plant Growth and Productivity Analysis

The leaf number, total leaf area, as well as shoot dry weight were measured. Total leaf area was estimated using a portable leaf area meter (LI-COR 3000, Lambda Instruments Corporation, Lincoln, NE, USA). For shoot dry weight determination, plants were dried at 75 °C for 48 h until a constant weight was obtained. At full maturity (plants at 120 days old), the number of grains and grain yield were recorded.

2.10. Statistical Analysis

Results were analyzed statistically according to complete randomized design, with a three-way factorial arrangement. Combined analysis was made for the two growing seasons, since their results followed a similar trend. All values are expressed as the mean ± standard error of mean (SE) of six replicates. The least significant difference (LSD) was calculated for the significant data at p < 0.05. SAS software (SAS Inc., Cary, NC, USA) was used for the statistical analysis.

3. Results

3.1. EBL and/or Spm Foliar Applications Upregulate the ZmBZR1 and ZmBES1 Genes and the Expression of Genes Encoding the Antioxidant Defense Enzymes under Water-Deficit Environments

To validate the new specific primers designed for qRT-PCR analyses in drought-stressed maize plants, semi-quantitative RT-PCR analyses were performed for each sample (n = 6); afterwards, 10 μL of 6 RT-PCR reactions representing each sample was mixed and 20 μL was used for gel electrophoresis analysis, as shown in Figure 1. The results of semi-quantitative RT-PCR analysis showed that ZmSOD, ZmCAT, ZmAPX, ZmMDHAR, ZmDHAR, ZmGR and the two regulatory TFs genes encoding the Zea mays protein BZR1 homolog 1-like (ZmBZR1) and the Zea mays protein BES1 (ZmBES1) in comparison with the ubiquitin carrier proteingene (ZmUBCP) as an internal control were differentially expressed in the leaves of the maize plants treated with EBL and/or Spm foliar applications and exposed to different drought stress conditions.

3.2. EBL and/or Spm Foliar Applications Enhance the Relative Expression Level of ZmBZR1 and ZmBES1 Genes in Maize Plants Grown under Water Deficiency

To gain insight into the contribution of the two major TFs in the BR pathway—BES1 and BZR1—to the water stress response, we analyzed the presence of BZR1 and BES1 (Figure 2). BZR1 was significantly (p < 0.05) increased in all EBL treatments of the highest level (9.64-fold and 8.35-fold) in Giza 129 and Giza 10 plants, respectively, sprayed with 0.1 mg L−1 EBL + 25 mg L−1 Spm under the highest water stress treatment (50% FC; WD2), in comparison with the unsprayed ones. BES1 was also significantly (p < 0.05) increased in all EBL applications with the highest occurrence (7.34-fold and 6.99-fold) in Giza 129 and Giza 10 plants, respectively, sprayed with the combined treatment under the water-deficit condition (50% FC; WD2) when compared with the unsprayed plants.

3.3. EBL and/or Spm Foliar Applications Enhance the Relative Expression Level of Genes Encoding SOD, CAT, APX, MDHAR, DHAR, and GR in the Antioxidant Machinery in Maize Plants Subjected to Water Stress Conditions

As shown in Figure 3, EBL treatments caused changes in the levels of expression of the genes encoding SOD, CAT, APX, MDHAR, DHAR, and GR in the antioxidant machinery of maize plants grown under water deficiency. Compared with the untreated plants, the EBL treatment alone significantly (p < 0.05) increased the relative levels of expression of ZmSOD, ZmCAT, ZmAPX, ZmMDHAR, ZmDHAR and ZmGR. Furthermore, spraying 0.1 mg L−1 EBL + 25 mg L−1 Spm further increased the levels of expression of these six genes under water stress, compared with the untreated plants, indicating a positive regulatory role of EBL in the transcription of those antioxidant-related genes.
The EBL treatment alone increased the relative levels of expression of ZmSOD in Giza 129 plants by 4.80-fold, 7.11-fold, and 8.89-fold, that of ZmCAT by 4.41-fold, 4.40-fold, and 4.25-fold, that of ZmAPX by 7.00-fold, 24.64-fold, and 22.28-fold, that of ZmMDHAR by 1.56-fold, 2.13-fold, and 2.49-fold, that of ZmDHAR by 1.49-fold, 2.02-fold, and 2.17-fold, and that of ZmGR by 5.07-fold, 5.35-fold, and 3.56-fold compared to the values of the unsprayed plants subjected to well-watered and drought (75% and 50% FC) conditions, respectively. Likewise, EBL application enhanced the relative levels of expression of ZmSOD in Giza 10 plants by 4.25-fold, 7.34-fold, and 8.18-fold, that of ZmCAT by 4.79-fold, 4.54-fold, and 4.60-fold, that of ZmAPX by 7.33-fold, 25.87-fold, and 22.19-fold, that of ZmMDHAR by 1.70-fold, 2.33-fold, and 2.60-fold, that of ZmDHAR by 1.56-fold, 2.01-fold, and 2.86-fold, and that of ZmGR by 5.79-fold, 7.19-fold, and 4.39-fold under well-watered and drought (75% and 50% FC) conditions, respectively, when compared with unsprayed plants.
Spraying 0.1 mg L−1 EBL + 25 mg L−1 Spm further increased the levels of expression of those genes in Giza 129 plants under water stress conditions. The relative levels of expression of ZmSOD were significantly (p < 0.05) increased by 6.39-fold, 11.28-fold, and 12.31-fold, the levels of ZmCAT by 5.65-fold, 5.55-fold, and 5.64-fold, of ZmAPX by 8.55-fold, 27.50-fold, and 23.61-fold, of ZmMDHAR by 1.93-fold, 2.31-fold, and 2.36-fold, of ZmDHAR by 1.72-fold, 2.28-fold, and 2.41-fold, and the levels of ZmGR by 5.43-fold, 5.98-fold, and 4.33-fold compared to the values of the unsprayed plants subjected to well-watered and drought (75% and 50% FC) conditions, respectively. Likewise, combined application significantly (p < 0.05) enhanced the relative levels of expression of ZmSOD in Giza 10 plants by 6.31-fold, 11.79-fold, and 12.15-fold, of ZmCAT by 5.17-fold, 4.94-fold, and 5.18-fold, of ZmAPX by 10.17-fold, 28.53-fold, and 23.29-fold, of ZmMDHAR by 2.01-fold, 2.37-fold, and 2.39-fold, of ZmDHAR by 1.64-fold, 2.09-fold, and 2.90-fold, and of ZmGR by 7.25-fold, 7.52-fold, and 4.55-fold under well-watered and drought (75% and 50% FC) conditions, respectively, when compared with unsprayed plants.

3.4. EBL and/or Spm Foliar Applications Improve the Activity of Antioxidant Enzymes (SOD, CAT, APX, MDHAR, DHAR, and GR) in Maize Plants Grown under Water Shortage Conditions

As shown in Figure 4, water stress treatments considerably increased the SOD, CAT, APX, and GR activities in maize plants, and decreased MDHAR and DHAR activities. The co-application of EBL and Spm led to an increase in SOD (70.7%), CAT (69.7%), APX (67.3%), MDHAR (92.3%), DHAR (92.3%), and GR (67.3%) activity in Giza 129 plants under drought (50% FC) conditions compared with the stressed untreated plants. Furthermore, the dual application of EBL and Spm improved the activity of SOD by 56.4%, CAT by 58.2%, APX by 64.3%, MDHAR by 90.0%, DHAR by 90.0%, and GR by 64.3% in Giza 10 plants grown under drought (50% FC) conditions compared with the stressed untreated ones.

3.5. EBL and/or Spm Foliar Applications Enhance the Content of Antioxidant Molecules (GSH and AsA) in Maize Plants Subjected to Water Stress Conditions

Furthermore, as shown in Figure 5, the GSH and AsA contents were significantly increased in maize plants grown under water stress conditions. The application of EBL and/or Spm further increased their accumulation in water-stressed plants. Maize plants grown under drought (50% FC) conditions and supplemented with the combined treatment accumulated much more GSH and AsA than the other treated plants. The combined treatment significantly enhanced the GSH and AsA contents in Giza 129 plants by 58.2% and 65.8%, respectively, as well as in Giza 10 plants by 51.6% and 52.5%, respectively, under drought (50% FC) conditions compared with the stressed untreated plants.

3.6. EBL and/or Spm Foliar Applications Alleviate Water Stress Impacts on Maize Growth and Productivity

Drought conditions suppressed maize growth and productivity, resulting in considerable reductions in the leaf number, leaf area, shoot dry weight, number of grains, and grain yield. Conversely, foliar applications of EBL and/or Spm enhanced these parameters, and the dual application conferred drought tolerance by significantly reducing the negative impact of water stress conditions (Table 3). The combined treatment significantly increased the leaf number (42.9%, 48.7%), leaf area (90.2%, 82.7%), shoot dry weight (102.6%, 97.6%), number of grains (108.0%, 98.0%), and grain yield (116.3%, 121.2%) in Giza 129 and Giza 10 plants, respectively, subjected to the highest water stress treatment (50% FC; WD2)compared with the stressed untreated plants.

3.7. EBL and/or Spm Foliar Applications Prevent H2O2 Accumulation and Maintain Cell Membrane Integrity in Maize Plants Grown under Water Deficiency

Both H2O2 and MDA are thought to be important markers of oxidative stress state in water-stressed plants. As shown in Table 4, water shortage environments significantly (p < 0.05) enhanced their content in maize leaves, whereas EBL and/or Spm applications significantly reduced their stress-induced buildup. Plants grown under drought (75% and 50% FC) conditions and treated with the combined EBL and Spm treatment showed a strong reduction in the H2O2 and MDA contents compared with plants subjected to individual applications. Exogenous EBL + Spm significantly reduced the H2O2 content (21.4%, 18.6%) and MDA content (32.6%, 22.3%) in Giza 129 and Giza 10 plants, respectively, grown under the highest water stress treatment (50% FC; WD2), compared with those in the stressed untreated plants.
Furthermore, as shown in Table 4, water stress increased EL, whereas this increase was significantly attenuated by exogenous EBL and/or Spm applications. EBL and Spm co-application had the greatest ameliorative effect. It significantly (p < 0.05) reduced the EL by 21.2% and 19.0% in Giza 129 and Giza 10 plants, respectively, when compared with the values of stressed untreated plants during the highest water stress treatment (50% FC; WD2).

4. Discussion

Drought is one of the most detrimental crop stresses that substantially impedes plant productivity globally [5]. Thus, the improvement of crop drought tolerance is essential for sustainable agriculture. Previous studies have indicated that EBL or Spm exogenous application can improve plant drought tolerance [12,14,18,23,55]. In the present study, we examined the impacts of exogenously applied EBL and/or Spm on the expression of BES1 and BZR1—two key TFs in the BR signal transduction pathway—the expression of genes encoding the antioxidant defense enzymes, and the ROS metabolism of maize plants subjected to water shortage environments.
BES1 and BZR1 have been shown to play important roles in the regulation of plant growth, development, and responses to environmental stresses [21,31]. In the current investigation, we found a significant rise in the expression of genes encoding ZmBZR1 and ZmBES1 transcription factors in water-stressed maize plants treated by EBL, either individually or in combination with Spm. The concerted induction of genes encoding these TFs suggests that EBL-induced stress tolerance is mediated by the transcriptional activation of genes involved in plant stress responses. Parallel to the presented results, previous reports have shown that BES1 and BZR1 are regulated by exogenous BR [32]. Similarly, EBL induced heat stress tolerance in Arabidopsis thaliana seedlings by upregulating the expression of two major TFs—BES1 and BZR1—in the BR signal transduction pathway [56].
BR is known as an important phytohormone involved in a wide spectrum of stress responses in plants. Several elements of BR signaling have been reported to mediate plant stress interactions, as well as plant growth and development [21]. Among them, the BZR1/BES1 family of TFs have critical roles in enhancing plant drought tolerance [35,36]. Our results showed that the application of EBL alone or in a combination with Spm improved the growth and productivity of maize plants exposed to water deficiency conditions. The obtained results postulate that the overexpression of ZmBZR1 and ZmBES1 could enhance the BR response and improve plant growth and the productivity of stressed treated maize plants. Our suggestion agrees with Zhang et al. [30], showing that the overexpression of ZmBZR1 in transgenic Arabidopsis plants increased the size of cotyledons, leaves, floral organs, and seeds through the BR signal pathway. ZmBZR1 can directly bind to the promoter region of organ-size-related genes to activate their expression. The function of the BZR and BES transcription factors in BR-regulated plant growth and stress responses has also been reported in Arabidopsis [28], soybean [57], tomato, and Arabidopsis [39]. BZR1/BES1 plays a key role in BR–drought crosstalk and serves as a critical switch for plants to convert between growth and drought tolerance [21]. BZR1/BES1 integrates different growth and development events via direct protein–protein interactions. For instance, DELLAs, PIFs, ARF6, and PKL all directly interact with BZR1/BES1, forming a BZR1/BES1-centered regulatory network to coordinate cell elongation [21,30]. Furthermore, a previous study has shown that BZR1/BES1 can act as an integrator or master regulator in multisignal-regulated plant growth and development by directly interacting with key elements from other pathways, including cell elongation, immune signaling, stress tolerance, cell division, and plant organ formation [31]. Overall, BZR1/BES1 play key roles in BR drought tolerance and havea vital role in plant growth and development under water deficiency.
BZR1 binds to the BR-response element (BRRE, CGTGT/CG) in the promoter of BR-repressed genes [58], whereas BES1 interacts with the BIM1 transcription factor, and together these proteins bind to the E-box element (CANNTG) of BR-induced genes [59], indicating that both BZR1 and BES1 can be activators for different target genes. Furthermore, the maize ZmBES1/BZR1-5 transcription factor conferred drought and salt tolerance in transgenic Arabidopsis by binding to the E-box to induce the expression of downstream stress-related genes [37]. Our results show that the application of EBL treatments may confer drought tolerance to maize plants by upregulating the expression levels of various antioxidant defense genes (ZmSOD, ZmCAT, ZmAPX, ZmMDHAR, ZmDHAR, and ZmGR). In line with our obtained data, previous reports have shown that exogenous EBL enhances plant stress tolerance by activating genes involved in the defense and antioxidant responses [24,60]. Our findings declare that this overexpression in genes encoding antioxidant enzymes could be positively correlated to the ZmBZR1 and ZmBES1 gene levels, indicating that the ZmBZR1 and ZmBES1 transcription factors could positively regulate maize drought tolerance by inducing the expression of the genes encoding the antioxidant defense enzymes.
In this investigation, our findings also revealed that Spm treatments upregulated the expression levels of various antioxidant defense genes, thus managing plant tolerance against water stress. A number of studies have demonstrated that the application of exogenous Spm can improve drought tolerance [12,14,18,55]. Spm has a vital role in the upregulation of cellular redox status, transcript expression, and the action of antioxidant enzymes [12,14]. It can regulate oxidative homeostasis during abiotic stresses [11]. Spm application may increase the expression levels of the stress-related genes that protect seedlings from stress damage [61]. It is fair to assume that EBL and Spm applications increase maize’s ability to withstand water stress, possibly by modulating antioxidant defense genes’ expression.
Plants have complex mechanisms that include enzyme antioxidant and non-enzyme antioxidant systems to scavenge excessive ROS during stressful conditions. The enzymatic antioxidant system includes SOD, CAT, APX, MDHAR, DHAR, and GR, which are the typical ROS scavenging enzymes in plants [8,9]. In the current study, their activities in maize plants treated by EBL and/or Spm were shown to be increased upon drought stress. Moreover, a concomitant decrease in H2O2 burst was also detected in treated plants. This observation is corroborated by other studies, which state that EBL is crucial in altering the performance of antioxidant enzymes, giving cells a better chance of surviving damage brought by stressful conditions [23,24,25,26]. In this study, it was shown that the expression of genes encoding ZmBZR1 and ZmBES1 transcription factors in water-stressed EBL-treated plants was increased, and there was also a concomitant decrease in H2O2 level in plants, indicating that these genes may have a function in ROS detoxification. Parallel to our results, a previous study has reported that the ROS accumulation level in the PtrBES1-7 overexpression line’s content was significantly lower than the control line, while the opposite was found in the PtrBES1 RNAi line, indicating that PtrBES1-7 could contribute to preventing ROS accumulation when P. trichocarpa is under drought stress [31]. Furthermore, this enhanced water stress tolerance by EBL is correlated with the plant defense system possessing activated antioxidant enzymes by elevation in the expression of their genes. Thus, we can conclude that EBL can assuage water stress damage by elevating the enzymatic antioxidant defense system in plants by upregulating the expression of the ZmBZR1 and ZmBES1 regulatory genes of the brassinosteroids signal transduction pathway, as well as the genes encoding the antioxidant defense enzymes ZmSOD, ZmCAT, ZmAPX, ZmMDHAR, ZmDHAR, and ZmGR. Additionally, studies have also proven that, in response to environmental challenges, Spm promotes the efficiency of antioxidant enzymes [12,13,18]. Spm application increased POD, CAT, and APX activities in plants grown under drought stress, and this facilitated the detoxification of ROS molecules [14]. Our results imply that upregulating antioxidant enzyme activation via EBL and Spm could be a plant’s way of dealing with water stress.
In addition to enhancing the activities of antioxidant enzymes, EBL and/or Spm foliage applications also contribute to AsA–GSH cycle modulation. Both AsA and GSH play a key role in ROS detoxification [62]. In the present study, when maize plants encounter water shortage environments, EBL and/or Spm treatments significantly meliorated the content of AsA and GSH, suggesting that treated plants could rely on antioxidants to maintain cellular redox homeostasis under stressful conditions. Our results reveal that EBL and/or Spm applications to stressed plants maintained AsA regeneration, which could be a result of increased APX, MDHAR, DHAR, and GR activities. Several researchers have reported that EBL and Spm improved AsA and GSH synthesis by activating APX, MDHAR, DHAR, and GR enzymes [63,64,65].Taken together, these results suggest that EBL and Spm resulted in a unique antioxidant profile. Antioxidant molecules and antioxidant enzymes are interconnected with a tight coordination to maintain an optimal cellular redox balance to trigger ROS production.
MDA is one of the products of cell membrane lipid peroxidation which damages cell membranes, finally leading to electrolyte extravasation and subsequent changes in the EL. The level of MDA and EL, therefore, reflect the degree of cell membrane integrity and are regarded as critical physiological indicators of plant senescence [62]. Our study shows that under drought stress, the application of EBL and/or Spm decreased the MDA and EL levels in the leaves. The mechanisms involved for improving maize drought tolerance with EBL and/or Spm might be due to overexpression in the ZmBZR1 and ZmBES1 genes, which could reduce the level of MDA and EL in stressed treated plants, indicating that ZmBZR1 and ZmBES1 play preventive roles against oxidative damage to the cell membrane. In P. trichocarpa, the overexpression of PtrBES1-7 positively decreases MDA and EL compared with the control line under drought stress [31]. The function of the BZR and BES transcription factors in BR-regulated MDA content has been studied in tomato and Arabidopsis [39]. It has been demonstrated by Setsungnern et al. [32] that BES1 under exogenous EBL application shows a transient increase in lipid hydroperoxides, which indicates a controlled generation of ROS that allows plant acclimation to stressful conditions. Moreover, it has been shown that the expression of ZmBES1/BZR1-5 in transgenic Arabidopsis facilitates shoot growth and root development, as well as enhances salt and drought tolerance with a lower level of MDA and EL under osmotic stress [37]. Taken together, BES1 and BZR1 could be drought stress-responsive TFs, whose overexpression could enhance ROS scavenging and decrease the degree of cell membrane damage, thereby improving the maize drought tolerance.
Additionally, the action of Spm in decreasing lipid peroxidation under drought stress is related to increased antioxidant enzyme activity leading to membrane stabilization and free radical scavenging [14]. Furthermore, considering that Spm contains four nitrogen groups, it could provide greater buffering capacity than spermidine (Spd) and putrescine (Put) [65]. This is in agreement with previous studies that revealed that exogenous Spm, unlike Spd and Put, has a potent anti-senescence effect on plants [66]. Studies have shown that Spm can act directly as a scavenger of free radicals against oxidative injury in plants or bind to antioxidant enzymes to break up the free radicals [67]. The spatial separation of positive charges in Spm at a physiological pH could enable it to bind negatively charged molecules such as nucleic acids, phospholipids and proteins, thereby protecting the structure and function of these macromolecules from degradation and modification [11]. This property would also enable the scavenging of free radicals and the stabilization of intracellular membranes under stress conditions. Our results clearly reveal that EBL and Spm facilitate ROS scavenging by increasing antioxidant enzyme activity and antioxidant molecule content in water-stressed maize leaves. These responses, in turn, resulted in the maintenance of membrane integrity and reduced cell membrane lipid peroxidation under drought conditions.
According to the results of this study, we can conclude that EBL and Spm might positively regulate maize drought tolerance and alleviate the stress-induced yield inhibition by upregulating the expression of the regulatory genes (ZmBZR1 and ZmBES1) of the BR signal transduction pathway, enhancing the expression of antioxidant defense-related genes, improving the enzymatic and non-enzymatic antioxidant machineries, preventing ROS accumulation, and maintaining cell membrane integrity (Figure 6). Additionally, although EBL or Spm alone were able to improve maize drought tolerance, their dual application was the most effective treatment in nullifying drought-induced damage;perhaps this could be due to their synergistic or additive effects. Both EBL and Spm play multiple functions in plant physiological processes under water stress environments [12,14,18,23,55]. In thissense, BR contributes to fix Spd, which is necessary for the production of Put required for stress tolerance [68]. Moreover, the positive effect of polyamines (PAs) has been associated with a cross-talk with other anti-stress hormones such BRs [69]. PAs induced the expression of genes involved in BR biosynthesis, supporting a cross-talk between PAs and BRs in stressed plants [70].

5. Conclusions

Overall, the co-application of EBL and Spm significantly increased the drought tolerance of maize by alleviating water stress-induced oxidative stress through modulating antioxidant defense gene expression and reprogramming the antioxidant defense machinery, which in turn facilitated the scavenging of excess ROS and thus increased the stability of the cellular membrane. Our study provides new insights into understanding the functions of EBL and Spm in plant growth and drought tolerance. We have elucidated the mechanism of how this combined treatment regulates plant growth, and the application of this treatment could alleviate water stress in plants and greatly accelerate processes of crop improvement and protection.

Author Contributions

N.B.T. conceived the idea, designed the experiment, carried out the experiments, generated and analyzed the data, and wrote the manuscript. A.S.I. carried out the experiments and generated the data. B.T.S. conceptualized the research. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Tester, M.; Langridge, P. Breeding technologies to increase crop production in a changing world. Science 2010, 327, 818–822. [Google Scholar] [CrossRef] [PubMed]
  2. Talaat, N.B.; Shawky, B.T. Influence of arbuscular mycorrhizae on root colonization, growth and productivity of two wheat cultivars under salt stress. Arch. Agron. Soil Sci. 2012, 58, 85–100. [Google Scholar] [CrossRef]
  3. Talaat, N.B. Effective microorganisms modify protein and polyamine pools in common bean (Phaseolus vulgaris L.) plants grown under saline conditions. Sci. Hortic. 2015, 190, 1–10. [Google Scholar] [CrossRef]
  4. Talaat, N.B. Effective Microorganisms: An innovative tool for inducing common bean (Phaseolus vulgaris L.) salt-tolerance by regulating photosynthetic rate and endogenous phytohormones production. Sci. Hortic. 2019, 250, 254–265. [Google Scholar] [CrossRef]
  5. Zampieri, M.; Ceglar, A.; Dentener, F.; Toreti, A. Wheat yield loss attributable to heat waves, drought and water excess at the global, national and subnational scales. Environ. Res. Lett. 2017, 12, 064008. [Google Scholar] [CrossRef]
  6. Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002, 7, 405–410. [Google Scholar] [CrossRef]
  7. Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004, 9, 490–498. [Google Scholar] [CrossRef] [PubMed]
  8. Apel, K.; Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004, 55, 373–399. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Noctor, G.; Foyer, C.H. Ascorbate and glutathione: The heart of the redox hub. Plant Physiol. 2011, 155, 2–18. [Google Scholar]
  10. Igarashi, K.; Kashiwagi, K. The functional role of polyamines in eukaryotic cells. Int. J. Biochem. Cell Biol. 2019, 107, 104e15. [Google Scholar] [CrossRef] [PubMed]
  11. Seifi, H.S.; Shelp, B.J. Spermine differentially refines plant defense responses against biotic and abiotic stresses. Front. Plant Sci. 2019, 10, 117. [Google Scholar] [CrossRef] [PubMed]
  12. Dawood, M.F.A.; Abeed, A.H.A. Spermine-priming restrained water relations and biochemical deteriorations prompted by water deficit on two soybean cultivars. Heliyon 2020, 5, e04038. [Google Scholar] [CrossRef] [PubMed]
  13. Geng, W.; Qiu, Y.; Peng, Y.; Zhang, Y.; Li, Z. Water and oxidative homeostasis, Na+/K+ transport, and stress-defensive proteins associated with spermine-induced salt tolerance in creeping bentgrass. Environ. Exp. Bot. 2021, 192, 104659. [Google Scholar] [CrossRef]
  14. da Silva, T.I.; Dias, M.G.; de Araújo, N.O.; Santos, M.N.; Ribeiro, W.S.; Filho, F.B.S.; Dias, T.J.; Barbosa, J.G.; Grossi, J.A.S. Spermine reduces the harmful effects of drought stress in Tropaeolum majus. Sci. Hort. 2022, 304, 111339. [Google Scholar] [CrossRef]
  15. Depuydt, S.; Hardtke, C.S. Hormone signalling crosstalk in plant growth regulation. Curr. Biol. 2011, 21, R365–R373. [Google Scholar] [CrossRef] [PubMed]
  16. Talaat, N.B.; Todorova, D. Antioxidant machinery and glyoxalase system regulation confers salt stress tolerance to wheat (Triticum aestivum L.) plants treated with melatonin and salicylic acid. J. Soil Sci. Plant Nutr. 2022, 22, 3527–3540. [Google Scholar] [CrossRef]
  17. Talaat, N.B.; Shawky, B.T. Synergistic effects of salicylic acid and melatonin on modulating ion homeostasis in salt-stressed wheat (Triticum aestivum L.) plants by enhancing root H+-pump activity. Plants 2022, 11, 416. [Google Scholar] [CrossRef]
  18. Todorova, D.; Talaat, N.B.; Katerova, Z.; Alexieva, V.; Shawky, B.T. Polyamines and brassinosteroids in drought stress responses and tolerance in plants. In Water Stress and Crop Plants: A Sustainable Approach; Ahmad, P., Ed.; John Wiley & Sons, Ltd.: Chichester, UK, 2016; Volume 2, pp. 608–627. [Google Scholar]
  19. Nawaz, F.; Naeem, M.; Zulfiqar, B.; Akram, A.; Ashraf, M.Y.; Raheel, M.; Shabbir, R.N.; Hussain, R.A.; Anwar, I.; Aurangzaib, M. Understanding brassinosteroid-regulated mechanisms to improve stress tolerance in plants: A critical review. Environ. Sci. Pollut. Res. Int. 2017, 24, 15959–15975. [Google Scholar] [CrossRef]
  20. Ackerman-Lavert, M.; Savaldi-Goldstein, S. Growth models from a brassinosteroid perspective. Curr. Opin. Plant Biol. 2020, 53, 90–97. [Google Scholar] [CrossRef]
  21. Li, Q.; Lu, J.; Yu, J.; Zhang, C.; He, J.; Liu, Q. The brassinosteroid-regulated transcription factors BZR1/BES1 function as a coordinator in multisignal-regulated plant growth. Biochim. Biophys. Acta Gene Regul. Mech. 2018, 1861, 561–571. [Google Scholar] [CrossRef]
  22. Talaat, N.B.; Shawky, B.T. 24-Epibrassinolide alleviates salt-induced inhibition of productivity by increasing nutrients and compatible solutes accumulation and enhancing antioxidant system in wheat (Triticum aestivum L.). Acta Physiol. Plant. 2013, 35, 729–740. [Google Scholar] [CrossRef]
  23. Vardhini, B.V.; Anjum, N.A. Brassinosteroids make plant life easier under abiotic stresses mainly by modulating major components of antioxidant defense system. Front. Environ. Sci. 2015, 2, 67. [Google Scholar] [CrossRef] [Green Version]
  24. Wu, X.X.; Chen, J.L.; Xu, S.; Zhu, Z.W.; Zha, D.S. Exogenous 24-epibrassinolide alleviates zinc-induced toxicity in eggplant (Solanum melongena L.) seedlings by regulating the glutathione-ascorbate-dependent detoxification pathway. J. Horti. Sci. Biotech. 2016, 91, 412–420. [Google Scholar] [CrossRef]
  25. Sharma, A.; Kumar, V.; Kanwar, M.K.; Thukral, A.K.; Bhardwaj, R. Ameliorating imidacloprid induced oxidative stress by 24-epibrassinolide in Brassica juncea L. Russ. J. Plant Physiol. 2017, 64, 509–517. [Google Scholar] [CrossRef]
  26. Shopova, E.; Katerova, Z.; Brankova, L.; Dimitrova, L.; Sergiev, I.; Todorova, D.; Talaat, N.B. Modulation of physiological stress response of Triticum aestivum L. to glyphosate by brassinosteroid application. Life 2021, 11, 1156. [Google Scholar] [CrossRef] [PubMed]
  27. Castañeda-Murillo, C.C.; Rojas-Ortiz, J.G.; Sánchez-Reinoso, A.D.; Chávez-Arias, C.C.; Restrepo-Díaz, H. Foliar brassinosteroid analogue (DI-31) sprays increase drought tolerance by improving plant growth and photosynthetic efficiency in lulo plants. Heliyon 2022, 8, e08977. [Google Scholar] [CrossRef]
  28. Zhang, D.; Ye, H.; Guo, H.; Johnson, A.; Lin, H.; Yin, Y. Transcription factors involved in brassinosteroid repressed gene expression and their regulation by BIN2 kinase. Plant Signal. Behav. 2014, 9, e27849. [Google Scholar] [CrossRef] [Green Version]
  29. Sun, Y.; Fan, X.Y.; Cao, D.M.; Tang, W.; He, K.; Zhu, J.Y.; He, J.X.; Bai, M.Y.; Zhu, S.; Oh, E.; et al. Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis. Dev. Cell 2010, 19, 765–777. [Google Scholar] [CrossRef] [Green Version]
  30. Zhang, X.; Guo, W.; Du, D.; Pu, L.; Zhang, C. Overexpression of a maize BR transcription factor ZmBZR1 in Arabidopsis enlarges organ and seed size of the transgenic plants. Plant Sci. 2020, 292, 110378. [Google Scholar] [CrossRef]
  31. Yang, J.; Wu, Y.; Li, L.; Li, C. Comprehensive analysis of the BES1 gene family and its expression under abiotic stress and hormone treatment in Populus trichocarpa. Plant Physiol. Biochem. 2022, 173, 1–13. [Google Scholar] [CrossRef]
  32. Setsungnern, A.; Muñoz, P.; Pérez-Llorca, M.; Müller, M.; Thiravetyan, P.; Munné-Bosch, S. A defect in BRI1-EMS-SUPPRESSOR 1 (bes1)-mediated brassinosteroid signaling increases photoinhibition and photo-oxidative stress during heat stress in Arabidopsis. Plant Sci. 2020, 296, 110470. [Google Scholar] [CrossRef] [PubMed]
  33. Tang, W.; Yuan, M.; Wang, R.; Yang, Y.; Wang, C.; Oses-Prieto, J.A.; Kim, T.-W.; Zhou, H.-W.; Deng, Z.; Gampala, S.S.; et al. PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat. Cell Biol. 2011, 13, 124–131. [Google Scholar] [CrossRef] [PubMed]
  34. Yu, X.; Li, L.; Zola, J.; Aluru, M.; Ye, H.; Foudree, A.; Guo, H.; Anderson, S.; Aluru, S.; Liu, P.; et al. A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana. Plant J. 2011, 65, 634–646. [Google Scholar] [CrossRef] [PubMed]
  35. Chen, J.; Yin, Y. WRKY transcription factors are involved in brassinosteroid signaling and mediate the crosstalk between plant growth and drought tolerance. Plant Signal. Behav. 2017, 12, e1365212. [Google Scholar] [CrossRef] [Green Version]
  36. Ye, H.; Liu, S.; Tang, B.; Chen, J.; Xie, Z.; Nolan, T.M.; Jiang, H.; Guo, H.; Lin, H.Y.; Li, L.; et al. RD26 mediates crosstalk between drought and brassinosteroid signaling pathways. Nat. Commun. 2017, 8, 14573. [Google Scholar] [CrossRef] [Green Version]
  37. Sun, F.; Yu, H.; Qu, J.; Cao, Y.; Ding, L.; Feng, W.; Khalid, M.H.B.; Li, W.; Fu, F. Maize ZmBES1/BZR1-5 decreases ABA sensitivity and confers tolerance to osmotic stress in transgenic Arabidopsis. Int. J. Mol. Sci. 2020, 21, 996. [Google Scholar] [CrossRef] [Green Version]
  38. Li, H.; Ye, K.; Shi, Y.; Cheng, J.; Zhang, X.; Yang, S. BZR1 positively regulates freezing tolerance via CBF-dependent and CBF-independent pathways in Arabidopsis. Mol. Plant 2017, 10, 545–559. [Google Scholar] [CrossRef] [Green Version]
  39. Jia, C.; Zhao, S.; Bao, T.; Zhao, P.; Peng, K.; Guo, Q.; Gao, X.; Qin, J. Tomato BZR/BES transcription factor SlBZR1 positively regulates BR signaling and salt stress tolerance in tomato and Arabidopsis. Plant Sci. 2021, 302, 110719. [Google Scholar] [CrossRef]
  40. Cui, X.-Y.; Gao, Y.; Guo, J.; Yu, T.-F.; Zheng, W.-J.; Liu, Y.-W.; Chen, J.; Xu, Z.-S.; Ma, Y.-Z. BES/BZR Transcription factor TaBZR2 positively regulates drought responses by activation of TaGST1. Plant Physiol. 2019, 180, 605–620. [Google Scholar] [CrossRef] [Green Version]
  41. Ziyomo, C.; Bernardo, R. Drought tolerance in maize: Indirect selection through secondary traits versus genomewide selection. Crop Sci. 2013, 53, 1269–1275. [Google Scholar] [CrossRef] [Green Version]
  42. Seebauer, J.R.; Singletary, G.W.; Krumpelman, P.M.; Ruffo, M.L.; Below, F.E. Relationship of source and sink in determining kernel composition of maize. J. Exp. Bot. 2010, 61, 511–519. [Google Scholar] [CrossRef] [PubMed]
  43. Cottenie, A.; Verloo, M.; Kiekens, L.; Camerlynck, R. Chemical Analysis of Plants and Soils. In Laboratory of Analytical and Agrochemistry; State University: Ghent, Belgium, 1982. [Google Scholar]
  44. Coombs, J.; Hall, D.O.; Long, S.P. Techniques in Bioproductivity and Photosynthesis; Pergamon Press: Oxford, UK, 1985. [Google Scholar]
  45. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  46. Rao, K.V.M.; Sresty, T.V.S. Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan L. Millspaugh) in response to Zn and Ni stresses. Plant Sci. 2000, 157, 113–128. [Google Scholar]
  47. Cakmak, I.; Marschner, H. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol. 1992, 98, 1222–1227. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Ramel, F.; Sulmon, C.; Bogard, M.; Couée, I.; Gouesbet, G. Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 2009, 9, 28. [Google Scholar] [CrossRef] [Green Version]
  49. Hossain, M.A.; Nakano, Y.; Asada, K. Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiol. 1984, 25, 385–395. [Google Scholar]
  50. Doulis, A.G.; Debian, N.; Kingston-Smith, A.H.; Foyer, C.H. Differential localization of antioxidants in maize. Plant Physiol. 1997, 114, 1031–1037. [Google Scholar] [CrossRef] [Green Version]
  51. Zhu, H.; Cao, Z.; Zhang, L.; Trush, M.A.; Li, Y. Glutathione and glutathione-linked enzymes in normal human aortic smooth muscle cells: Chemical inducibility and protection against reactive oxygen and nitrogen species-induced injury. Mol. Cell Biochem. 2007, 301, 47. [Google Scholar] [CrossRef]
  52. Hernandez, M.; Fernandez-Garcia, N.; Diaz-Vivancos, P.; Olmos, E. A different role for hydrogen peroxide and the antioxidative system under short and long salt stress in Brassica oleracea roots. J. Exp. Bot. 2010, 61, 521–535. [Google Scholar] [CrossRef] [Green Version]
  53. Nawaz, M.A.; Jiao, Y.; Chen, C.; Shireen, F.; Zheng, Z.; Imtia, M.; Bie, Z.; Huang, Y. Melatonin pretreatment improves vanadium stress tolerance of watermelon on seedlings by reducing vanadium concentration in the leaves and regulating melatonin biosynthesis and antioxidant-related gene expression. J. Plant Physiol. 2018, 220, 115–127. [Google Scholar] [CrossRef]
  54. Li, X.; Ahammed, G.J.; Zhang, Y.Q.; Zhang, G.Q.; Sun, Z.H.; Zhou, J.; Zhou, Y.H.; Xia, X.J.; Yu, J.Q.; Shi, K. Carbon dioxide enrichment alleviates heat stress by improving cellular redox homeostasis through an ABA-independent process in tomato plants. Plant Biol. 2015, 17, 81–89. [Google Scholar] [CrossRef]
  55. Talaat, N.B. 24-Epibrassinolide and spermine combined treatment sustains maize (Zea mays L.) drought-tolerance by improving photosynthetic efficiency and altering phytohormones profile. J. Soil Sci. Plant Nutr. 2020, 20, 516–529. [Google Scholar] [CrossRef]
  56. Divi, U.K.; Rahman, T.; Krishna, P. Gene expression and functional analyses in brassinosteroid-mediated stress tolerance. Plant Biotech. J. 2016, 14, 419–432. [Google Scholar] [CrossRef] [PubMed]
  57. Zhang, Y.; Zhang, Y.J.; Yang, B.J.; Yu, X.X.; Wang, D.; Zu, S.H.; Xue, H.W.; Lin, W.H. Functional characterization of GmBZL2 (AtBZR1 like gene) reveals the conserved BR signaling regulation in Glycine max. Sci. Rep. 2016, 6, 31134. [Google Scholar] [CrossRef] [Green Version]
  58. He, J.; Gendron, J.M.; Sun, Y.; Gampala, S.S.L.; Gendron, N.; Sun, C.Q.; Wang, Z. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science 2005, 307, 1634–1638. [Google Scholar] [CrossRef] [Green Version]
  59. Yin, Y.; Vafeados, D.; Tao, Y.; Yoshida, S.; Asami, T.; Chory, J. A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis. Cell 2005, 120, 249–259. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Sharma, P.; Kumar, A.; Bhardwaj, R. Plant steroidal hormone epibrassinolide regulate–Heavy metal stress tolerance in Oryza sativa L. by modulating antioxidant defense expression. Environ. Exp. Bot. 2016, 122, 1–9. [Google Scholar] [CrossRef]
  61. Pal, M.; Szalai, G.; Janda, T. Speculation: Polyamines are important in abiotic stress signaling. Plant Sci. 2015, 237, 16–23. [Google Scholar] [CrossRef] [Green Version]
  62. Talaat, N.B.; Mostafa, A.A.; El-Rahman, S.N.A. A novel plant growth–promoting agent mitigates salt toxicity in barley (Hordeum vulgare L.) by activating photosynthetic, antioxidant defense, and methylglyoxal detoxification machineries. J. Soil Sci. Plant Nutr. 2022. [Google Scholar] [CrossRef]
  63. Li, M.; Ahammed, G.J.; Li, C.; Bao, X.; Yu, J.; Huang, C.; Yin, H.; Zhou, J. Brassinosteroid ameliorates zinc oxide nanoparticles-induced oxidative stress by improving antioxidant potential and redox homeostasis in tomato seedling. Front. Plant Sci. 2016, 7, 615. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Alam, P.; Albalawi, T.H.; Altalayan, F.H.; Bakht, M.A.; Ahanger, M.A.; Raja, V.; Ashraf, M.; Ahmad, P. 24-Epibrassinolide (EBR) confers tolerance against NaCl stress in soybean plants by up-regulating antioxidant system, ascorbate-glutathione cycle, and glyoxalase system. Biomolecules 2019, 9, 640. [Google Scholar] [CrossRef] [Green Version]
  65. Shi, J.; Fu, X.Z.; Peng, T.; Huang, X.S.; Fan, Q.J.; Liu, J.H. Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response. Tree Physiol. 2010, 30, 914–922. [Google Scholar] [CrossRef] [PubMed]
  66. Serafini-Fracassini, D.; Di Sandro, A.; Del Duca, S. Spermine delays leaf senescence in Lactuca sativa and prevents the decay of chloroplast photosystems. Plant Physiol. Biochem. 2010, 48, 602–611. [Google Scholar] [CrossRef] [PubMed]
  67. Roychoudhury, A.; Ban, S.; Sengupta, D.N. Amelioration of salinity stress by exogenously applied spermidine and spermine in three varieties of indice rice differing in their level of salt tolerance. J. Plant Physiol. 2011, 168, 317–328. [Google Scholar] [CrossRef] [PubMed]
  68. Takahashi, T.; Kakehi, J. Polyamines: Ubiquitous polycations with unique roles in growth and stress responses. Ann. Bot. 2010, 105, 1–6. [Google Scholar] [CrossRef] [Green Version]
  69. Zheng, Q.; Liu, J.; Liu, R.; Wu, H.; Jiang, C.; Wang, C.; Guan, Y. Temporal and spatial distributions of sodium and polyamines regulated by brassinosteroids in enhancing tomato salt resistance. Plant Soil 2016, 400, 147–164. [Google Scholar] [CrossRef]
  70. López-Gómez, M.; Hidalgo-Castellanos, J.; Muñoz-Sánchez, J.R.; Marín-Peña, A.J.; Lluch, C.; Herrera-Cervera, J.A. Polyamines contribute to salinity tolerance in the symbiosis Medicago truncatula-Sinorhizobium meliloti by preventing oxidative damage. Plant Physiol. Biochem. 2017, 116, 9–17. [Google Scholar] [CrossRef] [PubMed]
Agronomy 12 02517 g001 550
Figure 1. Semi-quantitative RT-PCR analysis of ZmSOD: superoxide dismutase; ZmCAT: catalase; ZmAPX: ascorbate peroxidase; ZmMDHAR: monodehydroascorbate reductase; ZmDHAR: dehydroascorbate reductase; ZmGR: glutathione reductase; Zea mays protein BZR1 homolog 1-like (ZmBZR1);and Zea mays protein BES1 (ZmBES1) genes in comparison to the ubiquitin carrier protein (ZmUBCP) gene as an internal control for leaves of maize plants after seven days of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL to both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)).
Figure 1. Semi-quantitative RT-PCR analysis of ZmSOD: superoxide dismutase; ZmCAT: catalase; ZmAPX: ascorbate peroxidase; ZmMDHAR: monodehydroascorbate reductase; ZmDHAR: dehydroascorbate reductase; ZmGR: glutathione reductase; Zea mays protein BZR1 homolog 1-like (ZmBZR1);and Zea mays protein BES1 (ZmBES1) genes in comparison to the ubiquitin carrier protein (ZmUBCP) gene as an internal control for leaves of maize plants after seven days of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL to both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)).
Agronomy 12 02517 g001
Agronomy 12 02517 g002 550
Figure 2. Relative levels of expression of Zea mays protein BZR1 homolog 1-like (ZmBZR1) and Zea mays protein BES1 (ZmBES1) genes compared with the ubiquitin carrier protein (ZmUBCP) gene were analyzed in leaves of maize plants after seven days of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL to both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)). Values are the mean ± SE (n = 6). Different letters indicate significant differences at p < 0.05 level according to LSD test.
Figure 2. Relative levels of expression of Zea mays protein BZR1 homolog 1-like (ZmBZR1) and Zea mays protein BES1 (ZmBES1) genes compared with the ubiquitin carrier protein (ZmUBCP) gene were analyzed in leaves of maize plants after seven days of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL to both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)). Values are the mean ± SE (n = 6). Different letters indicate significant differences at p < 0.05 level according to LSD test.
Agronomy 12 02517 g002
Agronomy 12 02517 g003 550
Figure 3. Relative levels of expression of ZmSOD, superoxide dismutase; ZmCAT, catalase; ZmAPX, ascorbate peroxidase; ZmMDHAR,monodehydroascorbate reductase; ZmDHAR, dehydroascorbate reductase; and ZmGR, glutathione reductase genes, compared with the ubiquitin carrier protein (ZmUBCP) gene were analyzed in leaves of maize plants after seven days of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL to both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)). Values are the mean ± SE (n = 6). Different letters indicate significant differences at p < 0.05 level according to LSD test.
Figure 3. Relative levels of expression of ZmSOD, superoxide dismutase; ZmCAT, catalase; ZmAPX, ascorbate peroxidase; ZmMDHAR,monodehydroascorbate reductase; ZmDHAR, dehydroascorbate reductase; and ZmGR, glutathione reductase genes, compared with the ubiquitin carrier protein (ZmUBCP) gene were analyzed in leaves of maize plants after seven days of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL to both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)). Values are the mean ± SE (n = 6). Different letters indicate significant differences at p < 0.05 level according to LSD test.
Agronomy 12 02517 g003
Agronomy 12 02517 g004 550
Figure 4. Influence of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL on the activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) in leaves of maize plants of both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)). Values are the mean ± SE (n = 6). Different letters indicate significant differences at p < 0.05 level according to LSD test.
Figure 4. Influence of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL on the activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) in leaves of maize plants of both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)). Values are the mean ± SE (n = 6). Different letters indicate significant differences at p < 0.05 level according to LSD test.
Agronomy 12 02517 g004
Agronomy 12 02517 g005 550
Figure 5. Influence of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL on the content of reduced glutathione (GSH) and ascorbate (AsA) in leaves of maize plants of both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)). Values are the mean ± SE (n = 6). Different letters indicate significant differences at p < 0.05 level according to LSD test.
Figure 5. Influence of foliar application with distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL on the content of reduced glutathione (GSH) and ascorbate (AsA) in leaves of maize plants of both genotypes—Giza 129 and Giza 10—exposed to drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)). Values are the mean ± SE (n = 6). Different letters indicate significant differences at p < 0.05 level according to LSD test.
Agronomy 12 02517 g005
Agronomy 12 02517 g006 550
Figure 6. Foliar application of EBL and Spm alleviates water stress impact on maize growth and productivity by upregulating the genes encoding the antioxidant defense enzymes (ZmSOD, ZmCAT, ZmAPX, ZmMDHAR, ZmDHAR, ZmGR) via signaling through the regulatory genes BZR1 and BES1, which in turn improve the enzymatic and non-enzymatic antioxidant machineries, ROS detoxification, and cellular membrane stability.
Figure 6. Foliar application of EBL and Spm alleviates water stress impact on maize growth and productivity by upregulating the genes encoding the antioxidant defense enzymes (ZmSOD, ZmCAT, ZmAPX, ZmMDHAR, ZmDHAR, ZmGR) via signaling through the regulatory genes BZR1 and BES1, which in turn improve the enzymatic and non-enzymatic antioxidant machineries, ROS detoxification, and cellular membrane stability.
Agronomy 12 02517 g006
Table
Table 1. Chemical properties of the used soil.
Table 1. Chemical properties of the used soil.
pHHCO3− + CO32−
(mg kg−1)		Cl−
(mg kg−1)		SO42−
(mg kg−1)		Ca2+
(mg kg−1)		Mg2+
(mg kg−1)		Na+
(mg kg−1)		K+
(mg kg−1)		N
(mg kg−1)		P
(mg kg−1)
7.20200.8322.8447.095.142.43.929.418.73.0
Table
Table 2. Primer sequences used for gene expression analysis.
Table 2. Primer sequences used for gene expression analysis.
No.Gene
Accession No.		Primer NamePrimer SequenceFragment Size (bp)
1EU408345.1SOD_FTGC ATA TCG ACA GGA CCA CA159
2SOD_RTGG GCC AGT CAA AGG AAT CT
3J02976.1CAT_FACC GCA ACA TCG ACA ACT TC219
4CAT_RTCA TGG ATC CGT CGT AGT GG
5Z34934.1APX_FACA TTG TTG CGC TTT CTG GT175
6APX_RGAG AGG AGG GCT TTG TCA CT
7NM_001196274.1MDHAR_FCTG TAA AGG CGA TCA AGG GC199
8MDHAR_RACC TTG CCG TCC TTA ATC CA
9EU970663.1DHAR_FGCT GAT CTC TCT CTG GGT CC177
10DHAR_RGCG CCA TCC AGC AAT TAC AT
11NM_001148073.1GR_FATG GTG GGA CTT GCG TGA TA247
12GR_RGCA TCA ACT AGA CTG CCT GC
13NM_001157723.1BZR1_FGCC CCA CTA GTC GTT GTA GT194
14BZR1_RTTG TTG TTC TCC CGC TCC TT
15NM_001143314.1BES1_FAAC TAC GCG TCT CTT CCC AA190
16BES1_RCAG GGG TGC ACA TTC TTG AG
17NM_001111637.1UBCP_FACC GCC TGA CAC CCT ATA TG190
18UBCP_RTGC GAG CTC ATA ACC GTT TG
Table
Table 3. Variations in leaf number, total leaf area, shoot dry weight, grains number, and grain yield of two maize genotypes—Giza 129 and Giza 10—grown under different drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)) and exposure to foliar applications of distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL. Means ± SE (n = 6) with different letters within the same column are statistically different according to LSD test (p < 0.05).
Table 3. Variations in leaf number, total leaf area, shoot dry weight, grains number, and grain yield of two maize genotypes—Giza 129 and Giza 10—grown under different drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)) and exposure to foliar applications of distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL. Means ± SE (n = 6) with different letters within the same column are statistically different according to LSD test (p < 0.05).
Maize HybridsDrought Stress Treatments
+ Foliar Applications		Leaves Number
Plant−1		Total Leaf Area Plant−1 (cm2)Shoot Dry Weight Plant−1 (g)Grains Number Plant−1Grain Yield
Plant−1 (g)
Giza 129WW12.6 ± 0.66 cde3883 ± 95.1 de31.6 ± 0.95 ghi904 ± 10.3 efghi163.3 ± 4.1 def
WW + Spm14.1 ± 0.44 bc4712 ± 98.0 b39.2 ± 0.91 bcd1141 ± 11.1 bc202.8 ± 4.8 bc
WW + EBL15.3 ± 0.57 ab5081 ± 89.5 a41.3 ± 0.83 ab1199 ± 11.5 ab223.7 ± 5.1 ab
WW + Spm + EBL16.5 ± 0.65 a5298 ± 83.3 a44.2 ± 0.81 a1300 ± 10.9 a238.0 ± 5.8 a
WD110.8 ± 0.33 efg3418 ± 71.1 f25.8 ± 0.74 jk727 ± 7.3 klmn115.4 ± 3.9 hi
WD1 + Spm13.5 ± 0.39 bcd4415 ± 99.0 bc34.4 ± 0.79 efg955 ± 10.9 efgh158.9 ± 4.7 ef
WD1 + EBL14.3 ± 0.42 bc4633 ± 85.2 b36.3 ± 0.99 def1022 ± 9.3 cde178.0 ± 4.1 de
WD1 + Spm + EBL15.6 ± 0.64 ab5108 ± 88.6 a40.1 ± 1.03 abcd1172 ± 10.1 b197.7 ± 5.2 bc
WD2 9.1 ± 0.25 hi2343 ± 55.9 h15.3 ± 0.55 no425 ± 4.5 pq65.0 ± 1.9 lm
WD2 + Spm11.0 ± 0.35 efg3311 ± 79.1 f24.5 ± 0.66 kl680 ± 5.9 lmn103.0 ± 2.8 ij
WD2 + EBL11.6 ± 0.52 def3617 ± 61.1 ef28.3 ± 0.77 ijk733 ± 5.1 jklm110.0 ± 4.0 hi
WD2 + Spm + EBL13.0 ± 0.39 bcd4457 ± 91.9 bc31.0 ± 0.83 ghi884 ± 7.9 fghij140.6 ± 4.3 fg
Giza 10WW 12.2 ± 0.57 def3395 ± 77.5 f29.5 ± 1.06 hij831 ± 8.3 hijk131.9 ± 3.8 gh
WW + Spm13.5 ± 0.62 bcd3991 ± 94.8 d36.1 ± 0.88 def978 ± 9.1 def154.2 ± 3.7 def
WW + EBL14.1 ± 0.61 bc4223 ± 94.5 cd38.0 ± 0.73 bcd1055 ± 10.5 bcd180.6 ± 3.5 de
WW + Spm + EBL15.4 ± 0.57 ab4454 ± 72.9 bc40.8 ± 1.09 abc1144 ± 9.9 bc187.2 ± 5.0 cd
WD1 9.8 ± 0.35 gh2812 ± 61.1 g25.0 ± 0.77 kl627 ± 6.1 mno92.3 ± 2.1 jk
WD1 + Spm12.3 ± 0.59 def3412 ± 81.5 f29.4 ± 0.87 ghi812 ± 7.9 ijkl115.3 ± 3.3 hi
WD1 + EBL12.6 ± 0.62 cde3619 ± 82.8 ef32.5 ± 0.79 fgh873 ± 8.3 ghij126.3 ± 3.7 ghi
WD1 + Spm + EBL13.0 ± 0.40 bcd4007 ± 93.3 d36.1 ± 1.11 cde977 ± 9.1 defg143.2 ± 3.6 fg
WD2 7.8 ± 0.29 i1803 ± 49.0 i12.7 ± 0.37 o305 ± 2.5 q39.1 ± 1.4 n
WD2 + Spm10.8 ± 0.39 gh2512 ± 60.1 h19.3 ± 0.59 mn466 ± 3.9 p61.3 ± 1.5 mn
WD2 + EBL10.0 ± 0.42 fgh2634 ± 62.9 gh21.8 ± 0.67 lm531 ± 4.1 op70.3 ± 1.8 klm
WD2 + Spm + EBL11.6 ± 0.59 efg3294 ± 83.5 f25.1 ± 0.58 k604 ± 5.9 no86.5 ± 2.1 jkl
Table
Table 4. Variations in hydrogen peroxide (H2O2) content, lipid peroxidation, and electrolyte leakage of two maize genotypes—Giza 129 and Giza 10—grown under different drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)) and exposure to foliar applications of distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL. Means ± SE (n = 6) with different letters within the same column are statistically different according to LSD test (p < 0.05).
Table 4. Variations in hydrogen peroxide (H2O2) content, lipid peroxidation, and electrolyte leakage of two maize genotypes—Giza 129 and Giza 10—grown under different drought stress treatments (control (100% field capacity (FC); WW) and water deficit (75% FC and 50% FC; WD1 and WD2)) and exposure to foliar applications of distilled water (control), 25 mg L−1 Spm, 0.1 mg L−1 EBL, or 25 mg L−1 Spm + 0.1 mg L−1 EBL. Means ± SE (n = 6) with different letters within the same column are statistically different according to LSD test (p < 0.05).
Maize HybridsDrought Stress Treatments
+ Foliar Applications		H2O2 Content (nmol g−1 FW)Lipid Peroxidation
(nmol TBARS g−1 DW)		Electrolyte Leakage %
Giza 129WW 318 ± 6.1 kl133 ± 3.8 hijkl5.0 ± 0.11 mn
WW + Spm301 ± 6.5 lmn125 ± 4.0 ijkl5.0 ± 0.15 mn
WW + EBL284 ± 5.7 no114 ± 3.2 kl5.0 ± 0.13 mn
WW + Spm + EBL255 ± 4.5 o104 ± 3.3 l4.8 ± 0.11 n
WD1 351 ± 4.3 ghi154 ± 4.7 efg5.8 ± 0.14 hi
WD1 + Spm328 ± 5.3 ijk138 ± 3.3 ghi5.6 ± 0.16 ijk
WD1 + EBL314 ± 6.2 kl131 ± 4.2 hijk5.3 ± 0.09 klm
WD1 + Spm + EBL288 ± 5.6 mn111 ± 3.6 jkl5.0 ± 0.13 mn
WD2 415 ± 7.2 c184 ± 5.0 cd6.6 ± 0.20 de
WD2 + Spm380 ± 6.3 de155 ± 4.1 efg6.2 ± 0.23 fg
WD2 + EBL361 ± 5.5 efg136 ± 5.0 ghi5.7 ± 0.17 hij
WD2 + Spm + EBL326 ± 6.3 ijk124 ± 4.9 hijk5.2 ± 0.13 lm
Giza 10WW 352 ± 5.7 ghi160 ± 4.5 fgh5.5 ± 0.16 ijkl
WW + Spm340 ± 5.6 hij143 ± 4.8 ghij5.5 ± 0.18 ijkl
WW + EBL326 ± 5.1 jk131 ± 4.5 ghijk5.5 ± 0.13 ijkl
WW + Spm + EBL306 ± 4.7 klm114 ± 2.9 hijkl5.4 ± 0.09 jkl
WD1 404 ± 5.5 cd181 ± 6.0 bcd6.8 ± 0.27 cd
WD1 + Spm388 ± 6.5 de171 ± 5.5 cde6.6 ± 0.23 de
WD1 + EBL366 ± 6.2 ef163 ± 4.8 def6.3 ± 0.29 efg
WD1 + Spm + EBL343 ± 5.4 fgh142 ± 5.3 efg6.0 ± 0.19 gh
WD2 500 ± 8.2 a215 ± 6.0 a7.9 ± 0.27 a
WD2 + Spm466 ± 6.9 b184 ± 6.1 ab7.5 ± 0.29 b
WD2 + EBL443 ± 7.9 b177 ± 5.9 bc7.0 ± 0.27 c
WD2 + Spm + EBL407 ± 6.9 c167 ± 4.5 cde6.4 ± 0.28 ef
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Talaat, N.B.; Ibrahim, A.S.; Shawky, B.T. Enhancement of the Expression of ZmBZR1 and ZmBES1 Regulatory Genes and Antioxidant Defense Genes Triggers Water Stress Mitigation in Maize (Zea mays L.) Plants Treated with 24-Epibrassinolide in Combination with Spermine. Agronomy 2022, 12, 2517. https://doi.org/10.3390/agronomy12102517

AMA Style

Talaat NB, Ibrahim AS, Shawky BT. Enhancement of the Expression of ZmBZR1 and ZmBES1 Regulatory Genes and Antioxidant Defense Genes Triggers Water Stress Mitigation in Maize (Zea mays L.) Plants Treated with 24-Epibrassinolide in Combination with Spermine. Agronomy. 2022; 12(10):2517. https://doi.org/10.3390/agronomy12102517

Chicago/Turabian Style

Talaat, Neveen B., Ahmed S. Ibrahim, and Bahaa T. Shawky. 2022. "Enhancement of the Expression of ZmBZR1 and ZmBES1 Regulatory Genes and Antioxidant Defense Genes Triggers Water Stress Mitigation in Maize (Zea mays L.) Plants Treated with 24-Epibrassinolide in Combination with Spermine" Agronomy 12, no. 10: 2517. https://doi.org/10.3390/agronomy12102517

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop