Influence of Exogenous 28-Homobrassinolide Optimized Dosage and EDAH Application on Hormone Status, Grain Filling, and Maize Production

Abstract

Exogenously applied phytohormones improve the endosperm cells and establish greater kernel sink capacity and grain filling, improving grain yield. In this study, 28-Homobrassinolide (HBR) dosages (20, 25, and 30 mg a. i. ha⁻¹) were applied separately at the silking stage under controlled conditions, and EDAH (a mixture of ethephon and diethyl aminoethyl hexanoate) dosage of 90 g a. i. ha⁻¹ was sprayed at the jointing stage to enhance the lodging resistance. Our objective was to investigate whether the application of HBR under controlled conditions or with EDAH could enhance the grain filling rate by regulating endogenous hormones. The results showed that HBR at the silking stage significantly increased endogenous hormones (ABA, IAA, Z+ZR), hampered leaf senescence, enhanced photosynthetic, improved dry matter accumulation in grains, and increased the grain-filling period, filling rate, and thousand-grains weight. Additionally, HBR 25 and 30 mg a. i. ha⁻¹ increased the final yield by 9.9% and 19.5% compared to the control (CK) in 2020 and 14.1% and 18.95% in 2021, respectively. There was no significant difference between the results obtained from HBR-controlled and EDAH treatments at the jointing stage. Thus, we conclude that spraying HBR 25~30 mg a. i. ha⁻¹ under controlled conditions may increase the grain yield under normal weather conditions. In adverse weather conditions and heavy wind, spraying EDAH 90 g a. i. ha⁻¹ at the jointing stage and HBR 30 mg a. i. ha⁻¹ at the silking stage can enhance maize production.

1. Introduction

Maize (Zea mays L.) is the world’s most important cereal crop and is widely grown for multi-purposes such as food, feed, and raw materials for industrial applications [1]. Maize grain contains protein, oils, starch, fats, minerals, and vitamins A and B. It is cultivated on over 197 million hectares worldwide, and the global production of maize is 1137 million metric tons, which is 39 percent of the world’s total cereals production [2,3]. According to FAO’s 2017 report, maize production in China contributes 20 percent of the global maize production and accounts for more than one-third of China’s total cereal production [1].

Maize is C4 thermophilic and originates from tropical regions. All stages of maize growth and development are susceptible to low (5–15 °C) and high temperatures [4,5] and other abiotic factors limiting the plant growth in different geographical distributions [6]. Abiotic stresses such as high and low temperatures, drought, salinity, nutrient deficiency, heavy metals, and waterlogging are the main environmental factors that affect maize growth [7,8]. In particular, recent extreme temperatures and uneven precipitation frequency distribution are the growth-limiting abiotic factors that severely affect plant physiology by disturbing the leaf photosynthesis, which causes plant growth inhibition and decreased biomass, yield, and maize final yield production [9,10,11,12]. Moreover, Anderson et al. [13,14] reported that in long growing season areas, each °C of increased temperature resulted in a 10 percent maize yield reduction under optimum irrigation, while in regions of short growing periods, each °C increased temperature caused a 27 percent decrease in yield in low-water-availability conditions.

Furthermore, the variation in temperatures and unpredicted distribution of heavy winds and rainfall, especially in the grain-filling stage, have the worst lodging impact on maize crops [15]. Abiotic factors such as insects, pests, pathogens, bacteria, and viruses also cause yield reductions worldwide. With the growing global population, it is a big challenge to meet the food needs. There is an urgent need to increase crop production by adopting modern agricultural technologies and better agronomic practices, i.e., changing cropping patterns, adjusting crops sowing time, water management, minimizing yield gaps, and mitigating the biotic and abiotic stresses by applying plant growth regulators (PGRs), which are low toxicity and environmentally safe.

PGRs are the chemical substances that control the plant metabolic processes at low concentrations and regulate plant growth and development, such as stem elongation, cell division, breaking seed dormancy, altering fruit maturity, suppressing shoot growth, and increasing branching and other functions [16,17]. EDAH (mixture of 27% ethephon and 3% diethyl aminoethyl hexanoate (DA-6)) is an essential plant growth regulator to increase the grain yield by regulating plant growth and lodging resistance [18]. Spraying EDAH on maize in the jointing stage can significantly reduce the maize lodging and increase grain yield [19]. It can maintain source and sink balance and regulate the activity of the root and shoot. As EDAH is a mixture of ethephon (ETH) and DA-6, ETH plays an important role in altering the internode elongation [20], while DA-6 regulates photosynthesis and biomass production, improving germination seedling establishment [21,22]. The application of EDAH to maize in the jointing stage improved lodging resistance by increasing mechanical strength, photosynthesis, and grain yield in summer maize [23], and it is safe for consumers and the environment [24,25].

Brassinolides (BRs) are novel plant growth-promoting steroid hormones extracted from rape pollens [26], which play a vital role in plant growth stimulation and inducing hormonal responses to environmental stimuli [27]. The adaptation and survival of plants in adverse environmental conditions are due to the changes in molecular and cellular levels. Plant endogenous hormones govern these changes, such as the cell membrane composition and morpho-physiological modification [28]. BRs are directly and indirectly involved in resistance against abiotic stress by inducing different molecular changes such as higher photosynthetic efficiency [29,30], the overexpression and maintenance of stress-responsive genes [31,32], osmoprotectant accumulation, and antioxidant enzyme induction [33,34]. Furthermore, phytohormones combat the stress against heavy metals such as soil acidity [35], heat stress [36], and drought stress [28], by inducing different stress-responsive genes. It regulates the photosynthetic activity, chlorophyll, and stomatal activity by ensuring the availability of magnesium (Mg) content in several plant species [37], which is very important during the grain-filling period. The maintenance of photosynthetic pigments and chlorophyll during the reproduction stage accelerates the grain filling rate, improves grain size, and increases yield production [38].

In China, arid and semi-arid regions are very critical for the production of maize [39]. To increase maize production in these regions, improving the filling rate is essential [40]. The final stage of plant growth is the grain filling stage, in which the ovary converts into caryopses [41]. The grain filling rate and filling duration are the main factors determining the grain size and weight; improving these factors can improve the final yield [42,43]. After applying plant growth regulators, changes and improvements in the morphology and physiology of plants are due to the response of endogenous hormones.

Abscisic acid (ABA) is a plant hormone that regulates plant growth, plant stress response, vegetative growth, seed development, seed germination, and dormancy. ABA is sesquiterpene (C₁₅) synthesized from carotenoids via oxidative cleavage [44,45]. In cereal crops, ABA contents and the grain filling rate are positively correlated [46]. A higher seed filling rate means higher ABA content in grains [47]. Previous studies showed that indole-3-acetic acid (IAA), ABA, and zeatin riboside (ZR) contents positively correlate with the grain filling rate except for ETH and gibberellic acid (GA), which showed a negative correlation [48,49]. It is reported that GA1 and GA4 act as bioactive hormones in GA biosynthetic pathways [50], and produce hydrolases such as α-amylase, which are not conducive to starch synthesis [51]. Higher contents of ZR increase the grain filling rate and are significantly positively correlated to grain filling in maize and wheat [52,53]. It has been demonstrated in previous studies that an optimum concentration of endogenous hormones is required to regulate grain filling and increase yield production [42,43].

28-Homobrassinolide (HBR) is a naturally occurring polyhydroxylated steroidal hormone that regulates plant growth and productivity. It involves a wide range of physiological processes such as stem elongation, accelerated photosynthesis, and amino acid and protein synthesis [54]. HBR plays an essential role under biotic and abiotic stresses [55]. It is evident in previous studies that HBR has an ameliorative effect on heavy metals in wheat crops [56], improved the fruit quality of fruit crops [57], and improved photosynthesis and the antioxidant system under salt stress in maize [58] and vigna radiate [59]. However, there are relatively few studies to test HBR dosages and EDAH effects on different crops, particularly maize. In our study, different HBR dosages were exogenously applied in the silking stage to regulate the growth and development of maize. EDAH was exogenously applied at the jointing stage to avoid lodging. We hypothesized that the application of HBR in the silking stage would increase the photosynthesis of functional leaves and increase the grain filling rate and final yield of maize. The objective of this study was to (1) obtain the optimum HBR dosage for maize to spray at the silking stage; (2) evaluate the endogenous hormonal changes that occurred by spraying various HBR dosages; (3) investigate the effect of HBR alone or with EDAH association on crop growth, the grain filling process, and final yield.

2. Material and Methods

2.1. Experimental Site

The present experiment was performed during the summer of 2020 and 2021 at the Jiyang research station of Shandong Academy of Agricultural Sciences (SAAS), Jinan, China (36°58′ N, 116°58′ E). The soil of the field was sandy clay loam with PH 8.5. The soil chemical analysis was performed at the beginning of field experiments in 2020 and 2021. The data measured showed that the soil layer (0–40 cm) comprised 15.3 g kg⁻¹ of organic matter, 1.12 g kg⁻¹ of total nitrogen (N), 42.7 mg kg⁻¹ of available phosphorus (P), and 135.12 mg kg⁻¹ of available potassium (K), respectively. The annual crop rotation of the land used for the experiment was wheat–maize, and the last cultivated crop was wheat. The experimental site region was classified as semi-arid with yearly temperature, precipitation, humidity, and wind speed of 35–37.5 °C, 703 mm, 37.5–42.5%, and 0.5–1.3 m s⁻¹, respectively. At the time of spraying HBR at the silking stage, the mean temperature, wind speed, and humidity were 28.5 °C, 0.17 m s⁻¹, and 26.5%, respectively. The current weather data at the spraying time were measured by PM6252B digital anemometer. The detailed annual weather conditions are shown in Figure 1.

2.2. Experimental Design and Field Management

Summer maize Deng hai 605 (♀DH351 × ♂DH382 by Shandong Denghai Seeds Co., Ltd., Laizhou, China) was manually sown at a planting density of 75,000 plants ha⁻¹ on 15 June and 10 June and harvested on 10 October and 5 October in 2020 and 2021, respectively. Irrigation (755 mm) was applied by using the flood method immediately after sowing to ensure seed germination. A split-plot design with three replications was used in the experiments. Main plot treatments were separately sprayed with EDAH (90 g a.i. ha⁻¹) or distilled water (Control) at the jointing stage, and subplot treatments were sprayed with HBR (20, 25, 30 mg a. i. ha⁻¹) or distilled water CK at the silking stage as shown in

Table 1. Experiment treatments of maize in 2020 and 2021.

Main Plots	Treatments	HBR Dosage (mg a. i. ha−1)	Spray Method	Spray Volume (L ha−1)
Control	CK	0	KMS	450
	HBR1	20
	HBR2	25
	HBR3	30
EDAH	CK	0
	HBR1	20
	HBR2	25
	HBR3	30

. All treatments were sprayed by a knapsack manual sprayer (KMS) 3WBD-16L (Taizhou Gufeng Sprayer Co., Ltd., Taizhou, China) with a spray volume (SV) of 450 L ha⁻¹ in the afternoon between 2:00 PM to 5:00 PM over two years. The net plot size was 78 m⁻², i.e., 10 m long, 7.8 m wide, with 12 rows spaced 60 cm apart. At the sowing time, 240 kg ha⁻¹ of N, 150 kg ha⁻¹ of P, and 150 kg ha⁻¹ of K were incorporated into the soil. All P and K and 60% of N were incorporated one day before sowing. The remaining 40% of N was applied at the jointing stage. The experiments were conducted under no water stress; pests and weeds were controlled adequately during experiments.

2.3. Measurement and Sample Collection

2.3.1. The Photosynthetic Attributes and Relative Chlorophyll Content

Photosynthetic parameters, including the net photosynthetic rate (Pn), transpiration rate (Tr), intercellular carbon dioxide (Ci), and stomatal conductance (Gs) of the maize ear leaf were determined using Photosynthesis System Infrared Gas Analyzer (IRGA) (Li-6400XT Lincoln, New York, NY, USA). Fifteen representative plants (five plants per replication) were selected per treatment at 10 and 20 days after silking (DAS) in 2020 and only on 40 DAS in 2021 because, after the application of the chemical on 10th of August, the mean temperature and precipitation were almost the same. Due to differences in precipitation in October, photosynthetic parameters were measured on 40 DAA. During the measurement, the atmospheric temperature of the ear leaf was 25 ± 1 °C, and PAR was 1200 μmol m⁻² s⁻¹ over two years. To determine the greenness of maize ear leaves, a chlorophyll meter SPAD-502 Plus (Konica Minolta Sensing Inc., Sakai, Japan) was used at 20 and 40 DAS in 2020 and 2021. The same fifteen plant ear leaves were selected from each treatment, by which photosynthetic parameters were measured.

2.3.2. Leaf Senescence

Fifteen representative plants (five plants per replication) were selected from each treatment to determine the leaf area of green leaves at 10-day intervals from silking to maturity. Yellow leaves were considered senesced, and the leaf senescence rate was calculated as follows:

Leaf senescence rate = ΔS/Δd

where ΔS is the difference of leaf area values between two different periods per plant, and Δd is the difference in the interval of days at which leaf area was taken.

2.3.3. Grain Filling Rate

The grain filling rate was measured by the dry matter accumulation of grains. Before chemical application, 65 plants tasseled on the same day with the same ear diameter and height were tagged. Grain-filling samples were collected by removing nine ears per treatment from a 7-day interval from silking to maturity. After collecting ears, 100 grains were removed from different positions of each ear (300 grains per replication), and the fresh weight was taken. Grains were first oven-dried at 110 °C for 10 min and then at 70 °C until a constant weight was reached. The logistic growth equation was fitted by following [60], as previously described by [61];

$$W=A/1+B{e}^{-Kt}{)}^{-1/N}$$

(1)

The grains filling rate (R) was calculated by using the derivative of Equation (1).

$$R=AKB{e}^{-Kt}/1+B{e}^{-Kt}{)}^{N+1/N}$$

(2)

where W is the grain weight (mg), A is the final grain weight at physiological maturity (mg), t is the time after silking (d), and B, K, and N are to be measured by the regression.

2.3.4. Endogenous Hormones

The method used for the extraction and quantification of ABA, Z+ZR, GA, and IAA content was described by [62]. Grain samples were taken at 7-day intervals from 7 DAS to physiological maturity. Grains were frozen in liquid nitrogen and stored at −80 °C. Grain samples (0.5 g) were ground in a mortar on ice containing 5 mL of an 80% (v/v) methanol extraction medium and 1 mM butylated hydroxytoluene as an antioxidant. The methanolic extract was incubated for 4 h and then centrifuged for 15 min at 4500 rmp at 4 °C. The supernatant was passed through Chromosep C18 columns and pre-washed with 5 mL and 10 mL of 80% and 100% methanol, respectively. The hormone fractions were dried with N2 and dissolved in 1 mL phosphate buffer saline containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin (pH 7.5), before use in enzyme-linked immunosorbent assays (ELISA).

2.3.5. Yield and Yield Components

To determine the final yield and components at 12% moisture, 6 rows (2 rows per replication) of 5 m in length were harvested at maize physiological maturity. After removing the husk, the ears were counted to estimate the ear numbers ha⁻¹, sun-dried for one week, and then threshed to determine the final grain yield ha⁻¹. Ten ears were randomly selected from each replication to count the grain number per ear and thousand-grains weight after drying at 75 °C.

2.3.6. Statistical Analysis

Statistical analysis was conducted by SPSS 16.0 (SPSS, Chicago, IL, USA). The differences between treatments were determined by full-factorial ANOVA at p < 0.05 followed by the least significant difference (LSD) test. Growth equations were fitted by Origin 2019 software (OriginLab Co., Northampton, MA, USA).

3. Results

3.1. Photosynthetic Attributes and Relative Chlorophyll Content

The results in Figure 2 showed that HBR improved the photosynthetic parameters. Pn and Tr increased gradually with a medium and high dosage of HBR, and it was the maximum at 10 and 20 DAS in 2020 under both silking and jointing treatments. The highest values of Pn and Tr in HBR3 were observed in silking treatments at 20 DAS (Figure 2B,E), which were 20.29% and 25.32% higher than their CK. However, Pn of silking treatments with different HBR dosages was higher than jointing treatments, but statistically, the differences were insignificant. The same trend was observed at 40 DAS in the growing season of 2021. Due to leaf senescence, Pn and Tr values of silking and jointing treatments decreased at 40 DAS. While HBR2 and HBR3 of silking treatments were 19.22% and 16.3% and jointing treatments were 18.4% and 14.2% significantly higher than their CK, there was no significant difference between CK and HBR1 under both conditions (Figure 2C,F). Although medium- and high-dosage silking treatments were higher than jointing treatments, they were not statistically different (Figure 2A–F).

The Ci of maize ear leaf was increased with an increased dosage of HBR at 10 and 20 DAS under silking and jointing treatments in 2020 (Figure 3A,B,D,E). The highest values of Ci were observed in HBR3 at 20 DAS, which were 8.45% and 9.92% and 7.40% and 8.51% higher than CK and HBR1 under silking and jointing treatments, respectively. At 40 DAS in 2021, the Ci of HBR3 and HBR2 under the silking and jointing treatments had higher values as compared to the remaining treatments. Overall, no significant difference was observed between Ci values of silking and jointing treatments. Stomatal conductance is the plant water relation, and Gs also increased with the same trend, but the increase in values of Gs was in a higher ratio than Ci and other parameters. Higher Gs values were observed in medium and high dosages of HBR under the EDAH treatment. This is because ethephon application at the jointing stage makes the root stronger and strengthens the plant water relation. Although HBR2 and HBR3 under control conditions also have better values than their CK, the maximum values of Gs were observed at 20 DAS under silking treatments. HBR2 and HBR3 under silking treatments were 20.6% and 14.8% higher than their CK, while statistically, there was no significant difference between the higher dosage of silking and jointing treatments. At 40 DAS in 2021, low, medium, and high HBR dosages decreased with the same trend as other photosynthetic parameters due to leaf senescence. During this condition, HBR3 under both conditions was well maintained as compared to other HBR dosages (Figure 3F).

Soil plant analysis development (SPAD) of maize ear leaf followed the same trend as gaseous exchange parameters (Figure 4). Maximum SPAD values of ear leaf increased along with increased HBR dosage levels in silking and jointing treatments. At 20 DAS, SPAD values of HBR3 were higher than other HBR treatments in 2020 and 2021 (Figure 4A,C), while there was no significant difference between the values of all the treatments in the two years. SPAD values of HBR3 under jointing and silking treatments were 12.9% and 14.2%, and 11.16% and 9.53% higher than their CK in 2020 and 2021, respectively. At 40 DAS, SPAD values of all the treatments decreased, while HBR3 under silking and jointing treatments maintained a significant difference of 14.9% and 15.8% higher compared to CK. The difference between the SPAD values of all treatments was non-significant over two years.

3.2. Leaf Senescence

Leaf senescence was low at 10 DAS in all the treatments. As shown in Figure 5, leaf senescence was elevated at 30 DAS in all the treatments. Because of aging, the green leaf area of different canopy plants started to decrease in all treatments, but the leaf senescence rate under HBR1 and CK was higher as compared to HBR2 and HBR3 treatments. The application of HBR alone in the silking stage and the combination with EDAH significantly maintained the leaf area and hampered the chlorophyll breakdown and continued assimilation transport to kernels to promote grain filling. HBR2 and HBR3 had a better leaf area than HBR1 and CK under jointing and silking treatments. Although the leaf senescence of HBR treatments in combination with EDAH treatments was less than that of HBR-alone treatments, the difference in values of EDAH and controlled treatments were not statistically different.

3.3. Endogenous Hormonal Changes

3.3.1. ABA Contents

ABA plays a critical role in regulating organ size, protein synthesis, and grain filling. ABA contents were affected by the application of HBR alone or in combination with EDAH (Figure 6). The application of HBR in the silking stage under control and with EDAH combination significantly increased ABA contents. The maximum ABA contents were acquired at 28 DAS in average parts of the maize ear. In HBR application without EDAH treatment, the ABA contents were maximum at 28 DAS over two years, while in EDAH treatments, it reached the maximum at 28 DAS in 2020 and 2021, and the maximum contents were detected at 21 DAS (Figure 6D); after that, a gradual decrease was observed in all the treatments until physiological maturity. With the application of HBR alone or in combination with EDAH, the ABA contents were higher than CK in both conditions. There was no significant difference between HBR-controlled treatments and EDAH treatments. The average contents of ABA in seven stages (7–49 DAS) showed that HBR1, HBR2, and HBR3 without EDAH increased by 2.3%, 5.7%, and 4.24% in 2020 and 3.5%, 6.7%, and 8.1% in 2020 as compared to HBR with the combination of EDAH. Medium and high dosages of HBR had higher ABA contents as compared to low dosages and CK, and there was no significant difference between HBR1 and in HBR-controlled or EDAH treatment conditions.

3.3.2. Z+ZR Contents

ZR is a ubiquitous form of cytokinins that plays a vital role in cell division, slows down the aging process, and increases the grain filling in cereals. Z+ZR contents were increased in HBR application under controlled conditions or with EDAH application at the jointing stage (Figure 7). Z+ZR contents increased from 7 DAS to 28 DAS in all the treatments and gradually decreased. The Z+ZR contents were significantly higher in HBR application alone as compared to EDAH application at the jointing stage and HBR application at the silking stage. HBR2 and HBR3 applications at the silking stage had higher ZR contents than HBR1 and CK in both conditions, either applied alone or combined with EDAH. The average contents of Z+ZR of the seven stages showed that the HBR1, HB2, and HBR3 treatments without EDAH application were 7.49%, 5.34%, and 9.73% higher in 2020 and 0.8%, 0.97%, and 1.41% higher in 2021 as compared to the combination of HBR and EDAH treatments. The difference in Z+ZR contents within HBR dosages was highly significant with HBR application alone at the silking stage or with HBR and EDAH application. At the same time, the difference between HBR-alone treatments at the silking stage and the combination of HBR and EDAH was statistically non-significant.

3.3.3. IAA Contents

IAA acts as the main auxin, which plays an essential role in plant growth and development regulation, such as cell division, elongation, and response to pathogens. HBR application significantly increased IAA contents under control and combined with EDAH treatments (Figure 8). IAA contents gradually increased from 7 DAS to 28 DAS under HBR-alone application in 2020 and combined application of HBR with EDAH in 2021 (Figure 8A,D), while IAA contents of HBR alone increased gradually from 7 DAS to 21 DAS in 2021 and HBR combined with EDAH in 2020 and then decreased gradually (Figure 8B,C). The average results of seven intervals of IAA contents in HBR-alone treatments were 6%, −3.3%, and 1.5% higher in 2020 and 7.07%, 9.2%, and 6.3% higher in 2021 as compared to combined treatment of HBR at the silking stage and EDAH application at the jointing stage. The difference between the dosage of HBR within controlled HBR treatments and combined treatment of HBR and EDAH was highly significant compared to their CK. At the same time, there was no significant difference between the two main plot treatments.

3.3.4. GA3 Contents

GA3 is familiar because of its role in plant reproductive growth, delay in leaf senescence, cell elongation, and tolerance against environmental stress. The application of EDAH at the jointing stage and HBR at the silking stage significantly increased GA3 contents (Figure 9). GA3 contents were significantly increased with HBR and EDAH, and HBR applications alone. The contents of GA3 increased from 7 DAS to 21 DAS in all the treatments and then gradually decreased in HBR under controlled treatments (Figure 9A,C), while in EDAH and HBR combined treatments, GA3 was maintained at 21 DAS to 28 DAS and after that started to decrease gradually (Figure 9B,D). Unlike other hormones, GA3 contents were higher in EDAH and HBR combined treatments compared to HBR-alone treatment at the silking stage. The average results of seven intervals of GA3 contents under EDAH treatments were 2.8%, 1.5%, and 3.9% higher in 2020 and 1.4%, 1.29%, and 2.23% higher in 2022 compared to HBR-alone treatments at the silking stage. The difference in GA3 contents of HBR dosages within EDAH combined with HBR and HBR-alone treatments was also highly significant over two years. HBR1 and CK of HBR combined with EDAH treatment and HBR-alone treatments were not significantly different. HBR2 and HBR3 had better results than other treatments.

3.4. Grain Filling Rate

Dry matter accumulation of maize grains was significantly increased by applying HBR at the silking stage, alone and in combination with EDAH. The grain weight increased gradually from 7 DAS to 42 DAS in all the treatments, and then it was constant (Figure 10). The maximum dry grain weight was observed in HBR application under the controlled conditions, which was 1.11%, −2.29%, and 1.73% higher in 2020 and 2.25%, 3.6%, and 6.11% higher in 2021 as compared to EDAH and HBR combined treatments. Furthermore, the difference in HBR dosage under controlled conditions and EDAH combined treatments was also significant, in which HBR2 and HBR3 were 9.18% and 15.94% higher and 15.78% and 19.21% higher than CK of HBR under controlled conditions and the combination of HBR and EDAH treatments, respectively.

The grain filling rate was positively influenced by different HBR dosages and durations. Early (R1) and late-stage (R3) grain filling was the same, and the highest grain filling rate was observed in the potential increase period (R2). During the potential grain filling period, HBR3 had the highest filling rate of 16.75 mg grain⁻¹ day⁻¹, which was 33.25% higher than their CK under the controlled conditions without EDAH application at the jointing stage. While under the EDAH treatment, HBR3 gave a 25.39% higher grain filling rate than its CK. Comparing the difference in the highest filling rate between controlled and EDAH treatments, HBR3 of the controlled treatment had a 1.71% higher grain filling rate than EDAH treatment at the jointing state during the peak grain filling period, but it was not statistically different. Meanwhile, in the case of the overall maximum grain filling rate (Rmax), HBR3 under no EDAH application was 23.95% higher than their CK, while EDAH treatment was 28.3% higher than its CK. However, the data obtained from two years of maximum grain filling rate were not statistically different.

The total grain filling period is crucial in seed development and grain weight. Medium and higher dosages had better performance in continuing the filling until the late stage compared to CK and a low HBR dosage under both controlled and EDAH applications at the jointing stage. A maximum filling period was observed in HBR 3 and HBR2, which was 36.8, 34.1, and 36.8, 35.6 under controlled and EDAH applications in 2020 and 34.8 and 34.7, and 34.9 and 33.8 in 2021. The time to reach the maximum grain filling (Tmax) was highest in CK under EDAH and non-EDAH application treatments, and a high dosage of HBR took the least time to reach the maximum grain filling in two years (

Table 2. Maize grain filling affected by HBR dosage under controlled conditions at silking stage and combination with EDAH sprayed at jointing stage.

Year	Jointing Treatment	Silking Treatment	R1	R2	R3	Rmax	Wmax	Tmax	D (Days)
2020	Control	CK	2.72 c	10.42 b	3.06 c	10.62 c	102.21 c	18.50 a	27.12 b
		HBR1	2.97 bc	10.76 b	3.30 bc	12.42 b	137.93 bc	17.21 a	29.07 b
		HBR2	3.45 a	12.46 a	3.50 ab	12.08 b	190.32 a	15.46 b	34.19 a
		HBR3	3.52 a	12.83 a	3.77 a	13.97 a	193.14 a	15.28 b	36.83 a
	EDAH	CK	2.70 c	10.61 b	3.12 c	9.97 c	116.27 c	19.16 a	27.14 b
		HBR1	2.75 c	10.89 b	3.27 bc	12.42 b	102.49 c	18.47 a	29.00 b
		HBR2	3.42 a	12.46 a	3.61 ab	13.65 a	187.39 ab	15.52 b	35.69 a
		HBR3	3.31 ab	12.61 a	3.70 a	13.97 a	189.66 ab	15.28 b	36.83 a
2021	Control	CK	2.62 d	11.18 c	3.25 c	9.13 c	101.56 c	23.33 a	28.40 c
		HBR1	3.05 bc	11.18 c	3.50 bc	9.50 c	102.49 c	21.00 ab	28.11 c
		HBR2	3.39 a	16.09 ab	3.72 ab	12.54 a	167.50 ab	17.36 b	33.71 b
		HBR3	3.52 a	16.75 a	4.00 a	14.34 a	174.78 a	16.88 b	34.83 a
	EDAH	CtK	2.70 cd	11.34 c	3.31 c	10.92 c	104.05 c	23.33 a	27.14 c
		HBR1	2.95 cd	10.95 c	3.46 bc	11.02 bc	103.67 c	20.01 ab	27.71 c
		HBR2	3.30 ab	15.20 b	3.84 ab	11.49 ab	158.70 b	16.86 b	33.81 b
		HBR3	3.27 ab	14.74 b	3.92 a	12.68 a	174.78 a	17.46 b	34.93 a
	ANOVA	Dosage	**	*	*	***	**	*	**
		EDAH	NS	NS	NS	NS	NS	NS	NS
		Year	NS	*	NS	*	**	NS	**
		D×E	NS	NS	NS	NS	NS	NS	NS
		D×Y	NS	*	NS	*	NS	NS	NS
		E×Y	NS	NS	NS	NS	NS	NS	NS
		D×E×Y	NS	NS	NS	NS	NS	NS	NS

NS is non-significant; *, **, and *** show the level of significant results at p < 0.05, 0.01, and 0.001, respectively; D refers to HBR dosage, E is EDAH, and Y refers to year; R1, R2, and R3 grain filling rate at gradual (mg grain⁻¹ day⁻¹), peak, and slight increase period; Rmax: Maximum grain filling; Wmax: Grains weight at maximum grain filling (mg grain⁻¹ day⁻¹); Tmax: Time to reach maximum grain filling (d); D: Total number of active days during which grain filling process was continued (d); values with the same letters in columns are not significantly different at probability level < 0.05 followed by LSD test.

), which showed that higher and middle dosages of HBR performed better during the overall grain filling. The application of EDAH-controlled lodging occurred because no noticeable difference was found in grain filling dynamics between controlled and EDAH treatment conditions.

3.5. Grain Yield and Yield Components

The effect of different HBR dosages positively influenced yield and yield components. Final grain yield is characterized by the yield components. Higher and medium dosages of HBR showed a positive impact on grain yield components, including thousand-grains weight (TGW) and grain number per ear with or without EDAH application at the jointing stage. HBR3 showed 531 and 528 grains in 2019 under non-EDAH and EDAH treatments, which were 9.22% and 6.06% higher than their CK in 2020 and 7.73% higher in 2021, respectively. Thousand-grains weight (TGW) also showed the same trend as the grain number, while TGW of high dosage under non-EDAH treatment was 15.9% higher than their CK and 12.9% higher than CK under EDAH treatment in 2020 and was 17.4% and 19.12% higher in 2021. TGW of EDAH treatment was higher than the controlled treatment of HBR at the silking stage, but the difference was statistically insignificant. There was a noticeable change in grain yield with different HBR dosages under EDAH and non-EDAH treatments. A higher dosage of HBR performed better as compared to CK and a low dosage of HBR. Although the medium dosage had a lower yield as compared to HBR3, its effect on grain yield was far better than HBR1 and CK. A 19.5% increased yield was observed in HBR3 compared with CK under non-EDAH treatment, a 19.37% increased yield was observed under the EDAH treatment in 2020, while 18.95% and 13.7% increases were noticed under non-EDAH and EDAH treatments in 2021 (

Table 3. Effect of HBR dosages under controlled conditions at silking stage and combination with EDAH sprayed at jointing stage on yield and yield components.

Year	Jointing Treatment	Silking Treatment	Grains Number (Ear−1)	Ear Number (m−2)	TGW (g)	Grain Yield (t ha−1)
2020	Control	BK	482.0 c	7.47 a	290.0 c	9.82 c
		HBR1	498.0 bc	7.48 a	299.3 bc	9.66 bc
		HBR2	518.0 ab	7.51 a	319.3 ab	10.90 ab
		HBR3	531.0 a	7.49 a	345.0 a	12.20 a
	EDAH	EK	496.0 c	7.46 a	295.0 c	9.78 c
		HBR1	502.0 b	7.48 a	296.0 c	9.54 bc
		HBR2	509.0 ab	7.49 a	326.6 ab	11.53 ab
		HBR3	528.0 a	7.49 a	339.0 ab	12.13 a
2021	Control	BK	505.6 c	7.47 a	288.3 c	10.05 bc
		HBR1	513.0 c	7.50 a	292.6 bc	10.05 bc
		HBR2	534.0 b	7.53 a	330.0 ab	11.71 ab
		HBR3	548.0 a	7.51 a	349.3 a	12.40 a
	EDAH	EK	501.0 c	7.46 a	276.6 c	9.97 c
		HBR1	505.7 c	7.47 a	288.2 bc	10.39 bc
		HBR2	536.0 ab	7.52 a	321.6 ab	11.59 ab
		HBR3	543.0 ab	7.49 a	342.0 ab	12.23 a
	ANOVA	Dosage	***	NS	***	***
		EDAH	NS	NS	NS	NS
		Year	**	NS	*	*
		D×E	NS	NS	NS	NS
		D×Y	NS	NS	NS	*
		E×Y	NS	NS	NS	NS
		D×E×Y	NS	NS	NS	NS

NS is non-significant; *, **, and *** show the level of significant results at p < 0.05, 0.01, and 0.001, respectively; TGW refers to thousand-grains weight (g); values with the same letters in columns are significantly not different at probability level < 0.05 followed by LSD test.

).

4. Discussion

BRs are the endogenous plant hormones required in small concentrations for multiple physiological processes essential for normal plant growth and development [30,63]. Photosynthesis is a primary physiological process closely associated with plant health and yield. The application of BRS helps increase and maintain photosynthesis and chlorophyll to ensure the availability of necessary compounds to the plant sink and increase the grain filling rate [37,64]. The application of HBR in the silking stage maintains the functional leaf greenness and delays the leaf senescence, maintains the chlorophyll content, and supports the gas exchange parameters [65]. It is also reported that photosynthetic parameters tend to decrease from 25–30 DAS [66]. In this experiment, the application of HBR significantly increased the SPAD values. Medium and high dosages of HBR (HBR2, HBR3) had the highest SPAD values on 20 DAS and decreased at 40 DAS. HBR application under controlled conditions and in combination with EDAH had similar results, and the trend of increased and decreased chlorophyll was also the same under both conditions (Figure 4). This increase in chlorophyll is in agreement with [67,68], who explained that hormones alter the biothensis of genes, which results in changing the chlorophyll biosynthesis.

Assessment of chlorophyll is an essential factor that estimates photosynthesis [69]. HBR application increased photosynthetic parameters such as GS, stomatal aperture size, and transpiration rate (Tr), essential for transporting inorganic material through the xylem. Therefore, intercellular CO₂ increases with an increase in GS and Tr [70], which start the Calvin cycle by binding with rubisco in the presence of CA [71]. Hence, HBR increases NR and CA activity, which is already proven in previous studies [72]. In the presence of BR, an increase in NR activity is due to the result of enhanced translation/transcription of NR genes [54]. The observed increase in Gs, Tr, Ci, NA, and CA activity continuously improved the Pn. Our results also followed previous studies wherein HBR application alone or combined with EDAH significantly increased photosynthetic parameters at 10–20 DAS, and then decreased at 40 DAS because of leaf aging. Application of EDAH and HBR significantly reduced the leaf senescence (Figure 5), hampered the chlorophyll breakdown, maintained the leaf functions by maintaining chlorophyll, and led to enhanced photosynthesis (Figure 2 and Figure 3). The enhancement of the photosynthetic rate improved the source and sink relation at the grain filling stage.

Furthermore, it is evident in previous studies that HBR improves the photosynthesis and grain filling process by regulating plant hormones [42,73]. The application of PGRs better regulates plant hormones and improves the grain filling process [46,74]. Our study revealed that the application of HBR alone or in combination with EDAH significantly increased the seed filling rate and grain dry matter accumulation (Figure 10). The changes in endogenous hormones regulate the grain filling rate in maize [40], and PGRs can significantly increase the endogenous hormones in plants and increase the grain filling rate. Our study found that exogenous application of HBR in silking stages alone or in combination with EDAH significantly increased the hormone contents. The increase in ABA, IAA, and Z+ZR contents was lower in HBR and EDAH combination treatments than in HBR treatment alone (Figure 6, Figure 7 and Figure 8), which may be because EDAH application at the jointing stage had remaining traces of ethephon in plants, which decrease hormone synthesis. This is also confirmed by [40,49], who demonstrated that ethephon leaves traces of ethylene in plants, which are negatively correlated with grain filling and the grain filling rate in cereal crops. Although all the endogenous hormones increased by applying HBR alone at the silking stage, GA3 contents were lower under the HBR control treatments than EDAH and HBR combined treatments. This is because ethephon increased the GA3 contents, as shown in Figure 9, and GA3 contents were increased from 7 DAS to 21 DAS and then gradually decreased in all the treatments. The decrease rate of GA3 in the HBR3-alone application was higher than EDAH treatments. The increase in GA3 content delayed the leaf senescence in EDAH treatments (Figure 5), which maintained photosynthesis and finally increased the grain filling rate (Table 2). It was also confirmed in previous studies that ethephon negatively correlates with grain filling and grain weight [40,41,49]. In this study, HBR-alone treatments had higher grains number per ear, grain weight, and final yield as compared to EDAH and HBR applications (Table 3). In our study, we found that the application of HBR alone or in combination with EDAH significantly enhanced chlorophyll, delayed leaf senescence, and regulated photosynthesis. HBR and EDAH increase the hormone contents in plants, enhanced plant growth and development by enhancing the filling rate, and increased the final grain yield.

5. Conclusions

In conclusion, the application of HBR at the silking stage maintained the leaf area index and delayed leaf senescence. An HBR dosage of 25 and 30 mg ha⁻¹ significantly enhanced photosynthetic parameters, including Pn, Gs, Tr, and Ci. The increase in gas exchange parameters was mainly due to the improvement in chlorophyll contents and the changes in endogenous hormones. HBR and EDAH significantly changed hormone status, either sprayed alone or in a combined application at different growth stages. HBR-alone application at the silking stage significantly increased ABA, IAA, and Z+ZR contents in maize grains. This increase in hormone content was more than when using HBR with the combination of EDAH.

In contrast, the EDAH application could better control the lodging in maize. The improvement in endogenous hormones enhanced the grain filling rate and ultimately increased the thousand-grains weight, number of grains per ear, and final grain yield. Based on our results, we concluded that spraying HBR 25 mg a. i. ha⁻¹ at the silking stage could increase the grain yield in normal weather conditions, while if there is a risk of lodging and harsh weather conditions, spraying EDAH 90 mg a. i. ha⁻¹ at the jointing stage and HBR 30 mg a. i. ha⁻¹ at the silking stage can help to decrease lodging and increase the final yield.