1 Introduction

The date palm (Phoenix dactylifera L.) belongs to the Arecaceae family, which contains over 2500 species and about 200 genera (Eoin 2016). Dates are a staple food for many people in the Middle East, North Africa, Asia, and America due to their high sugar, fat, mineral, amino acid, vitamin, and dietary fiber content (Al-Mssallem et al. 2020; Ahmed et al. 2022a; Alyafei et al. 2022). They also have anti-mutagenic and anti-carcinogenic properties due to the high content of bioactive components such as flavonoids, tannins, and other phenolic compounds (Khalid et al. 2017; Al Shaibani et al. 2022; Alkaabi et al. 2023). According to data from the UN’s Food and Agriculture Organization, Egypt is the world’s leading date producer, with a total production of around 1.7 million tons in 2021, followed by Saudi Arabia with 1.6 million tons and Iran with 1.3 million tons (Fleck 2023). The developmental and maturational stages of date fruit are Hababouk, Kimri, Bisr (or Khalal), Rutab, and Tamer. These stages characterize cell division, immature green (cell elongation), mature hard full colored, soft brown, and firm raisin-like fruit, respectively (Awad et al. 2011; Ahmed et al. 2021).

Plant growth regulators (PGRs) are signaling molecules that regulate plant cell division, flowering, and fruit development (Cutler and Nelson 2017; Talaat 2020). They interact with receptor proteins via signal transduction pathways to control a variety of metabolic processes (Todorova et al. 2016; Shah et al. 2023). By enhancing photo-assimilates partitioning, nutrient acquisition, carbon and nitrogen metabolism, biomass accumulation, and secondary metabolite production, their exogenous application has been recognized as an environmentally friendly method to boost plant productivity (Martínez-Esplá et al. 2018; Khalil et al. 2022; Talaat and Hanafy 2022; Talaat 2023). Among these are GA3 and SA, which can stimulate cell elongation, cell division, seed germination, and fruit development by controlling several activities within the plant cell, such as protein synthesis, photosynthetic efficiency, mineral uptake, phytohormonal balance, and antioxidant response (Serrano et al. 2018; Gacnik et al. 2021; Talaat et al. 2023).

Dates marketing is impacted by the weight and size of the fruit because larger sizes are typically preferred over smaller ones (Al-Qurashi and Awad 2011). Exogenous application of GA3 is one of the methods that has been used to increase date palm bunch weight and improve fruit growth, yield, and quality (Beerappa et al. 2019; Khalil 2020; Mosa et al. 2022). Some studies have supported the use of GA3 to transition from vegetative to reproductive development, which is essential for flower development, fertilization, and fruit development (Plackett and Wilson 2016; Prakash et al. 2022; Li et al. 2023). Moreover, a lack of GA3 transmission and production prevents the development of anthers and pollen grains (Chhun et al. 2007). Both pollen grain germination and pollen tube growth, two vital plant reproductive processes, have been seen to be impacted by GA3 (Ozkan et al. 2016). Evidence has also emerged demonstrating that GA3 can initiate and maintain fruit development by its precise effect on promoting cell elongation (Zhu et al. 2019; Khan et al. 2020; Zhang et al. 2020). For the proper establishment of reproductive growth in tree fruit species, coordinated levels of gibberellin (GA) at the various developmental stages are required (Ozkan et al. 2016; Khalil 2020; Mosa et al. 2022). Similar to this, a deficiency in bioactive GA during the development of plum fruit resulted in severe developmental disorders like growth retardation, disrupted flower patterning, and constrained fruit characteristics (El-Sharkawy et al. 2012; Khan et al. 2020). In agreement with its function as a growth-promoter, GA3 can influence source-sink metabolism and sink formation (Suwandi et al. 2016). Although the potential impact of GA3 on fruit development processes has been acknowledged, it is still not entirely clear how these effects are produced.

Furthermore, as a broad-spectrum plant growth regulator, SA plays a vital role in controlling the cell division and expansion (Sharma et al. 2023; Talaat 2023). It can control the growth and development of plant organs by influencing various biological properties (Ahmed et al. 2022b; Sharma et al. 2023; Talaat and Hanafy 2023; Talaat et al. 2023). The fruit size and single-fruit quality of palms can be enhanced by spraying SA (Martínez-Esplá et al. 2018; Ahmed et al. 2022b; Shareef et al. 2022). The advantages of its use are linked to an increase in date fruit quality parameters, storage life, and bioactive compounds content (Mohamed et al. 2017; Ahmed et al. 2021; Shareef et al. 2022). However, the exact mechanism by which SA works is still not completely known.

To the best of our knowledge, little research on the “Zaghloul” date palm cultivar has been conducted, and no information on the synergistic effect of GA3 and SA on the growth and development of its fruit is currently available. Therefore, this study, as a first investigation, aims to investigate the efficacy of exogenous application of GA3 and SA, either as single or combined treatment, in promoting “Zaghloul” fruit growth and development by controlling some essential inherent plant characteristics such as nutrient, sugar, amino acid, and phytohormone content. Accordingly, it has been hypothesized that combining GA3 and SA can improve the yield of “Zaghloul” fruit by enhancing the synthesis of protein and carbohydrate as well as the acquisition of minerals, amino acids, sugars, and phytohormones. To verify this hypothesis, the changes in reducing, non-reducing, and total soluble sugar accumulation, amino acid (glutamic acid, aspartic acid, proline, glycine, alanine, arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, valine) content, nutrient (nitrogen, phosphorus, potassium, calcium, magnesium, sodium, zinc, iron, manganese) acquisition, endogenous phytohormonal (indole-3-acetic acid, cytokinins, GA3, SA) alteration, antioxidant (total phenols and tannins content, as well as peroxidase and catalase activity) performance, along with certain other physiological responses such as dry matter, crude fiber, ash, moisture, total soluble solids, total acidity, carbohydrate, and protein contents, were elucidated to assess the impact of spraying GA3 and SA, alone or in combination, on the yield and quality of Phoenix dactylifera, cv. Zaghloul fruit. The study paves the way for using GA3 in conjunction with SA to improve the fruit’s growth and quality.

2 Materials and Methods

2.1 Plant Materials and Experimental Procedure

During 2020 and 2021 growing seasons, 48-uniform “Zaghloul” date palm trees of 15-years-old were randomly selected in the experimental orchard of the Agricultural Research Centre, Ministry of Agriculture, Giza, Egypt. Selected trees were healthy and as uniform as possible in age and growth. All trees received the same horticulture practices as recommended by the Ministry of Agriculture. At the beginning of each season, the number of inflorescences was adjusted to 10 per tree. Bunches of each date palm tree were thinned to the same number of strands. All trees uniformly received the same artificial pollination practices.

The experiment was designed as a randomized complete block design with four treatments. Each treatment was performed in four replications, three trees each, giving a total of forty-eight females of the “Zaghloul” trees. The experiment continued in the second year on the same date palms selected in the first year. Bunches were harvested about 190–200 days after pollination in both years, and two bunches were harvested from each tree.

At the Hababouk and Kimri stages of date fruit development, bunches on the selected trees were subjected to the following spraying treatments: (i) control (distilled water spray); (ii) 100 mg L−1 GA3; (iii) 100 mg L−1 SA; and (iv) 100 mg L−1 GA3 + 100 mg L−1 SA. The concentrations of 100 mg L−1 GA3 and 100 mg L−1 SA were the most effective concentrations according to preliminary experiment within a range from 0 to 200 mg L−1 for GA3 and SA. Indeed, in date palm, the efficiency of growth regulators in increasing fruit size is dependent on the stage of development at the time of growth regulators application. A physiologically active stage known as the “depressed period” during the early second sub-stage of Kimri stage (about 15–16 weeks following pollination) was defined as the most responsive stage for growth regulators application in increasing bunch and fruit weight (Awad and Al-Qurashi 2012). A non-ionic wetting agent (Tween 20 surfactant) at 0.01% was included in all spray applications. The spray solution was applied by a hand sprayer and each bunch received about 250 mL.

2.2 Physical Characteristics, Moisture, Dry Matter, Crude Fiber, Ash, Total Soluble Solids (TSS), and Total Acidity (TA) Determination

At lab, yield was evaluated by weighting the bunches (kg). A sample of 60 fruits from each replicate at the Tamar stage were selected randomly, washed only with water, dried, and weighed. Moreover, fruits volume (using graduated cylinder; cm3) and dimensions (length and diameter) (using a vernier caliper; cm) were recorded. After weighing the fruits and seeds, flesh weight was obtained and fruit flesh pulp (%) was calculated. Date fruit and seed weight were determined using an electronic balance. Fruit volume was obtained by water displacement procedure. Fruit flesh weight was calculated as fruit weight minus seed weight. Additionally, seed/fruit ratio was also recorded.

The determination of moisture, dry matter, crude fiber, and ash contents was performed following the official methods ascribed by Association of Official Analytical Chemistry (AOAC 2005).

The TSS were measured as Brix (%) in fruit juice by using a digital refractometer (DR 6000, A. Kruss Optronic GmbH, Hamburg, Germany). The TA was determined in juice by titrating with 0.1 N sodium hydroxide in the presence of phenolphthalein as indicator and the results were expressed as a percentage of citric acid.

Another 60 fruits were selected from each replicate, washed, and used for the following measurements.

2.3 Reducing, Non-reducing, and Total Soluble Sugar Determination

One hundred milliliters of distilled water was used to homogenize 3 g of fresh date pulp. After centrifuging the mixture for 5 min at 3000 rpm, the supernatant was used. The phenol-sulfuric acid method was used to determine the amount of total sugar (Nielsen 2010). In brief, 0.05 mL of 80% phenol and 5 mL of sulfuric acid were mixed with 2 mL of the supernatant. The mixture was stirred on a vortex mixer for 1 min, then stood for 10 min before being submerged for 10 min in a water bath at 25 °C. Using a spectrophotometer (Shimadzu UV-Visible 1800, Tokyo, Japan), the absorbance was measured at 490 nm. Additionally, the dinitrosalicylic acid (DNS) method was used to determine the reducing sugar (Miller 1959). Briefly, 0.2 mL of date extract was taken, the volume was made up to 1 mL, and 2 mL of DNS solution was added. The mixture was stirred and heated for 30 min in a water bath at 80 °C. Subsequently, the mixture was then given 20 min to cool. The spectrophotometer was used to measure the absorbance at 575 nm. Non-reducing sugar was calculated by the difference between the total and reducing sugars.

2.4 Carbohydrate and Total Protein Determination

According to Yih and Clark (1965) and Dubois et al. (1956), total carbohydrate was extracted and estimated. After being extracted with 1.5 N H2SO4, the samples were centrifuged at 4000g for 10 min. One milliliter of 5% distilled phenol was added to 1 mL of the extract, and the absorbance at 490 nm was measured using a spectrophotometer. In addition, the Lowry et al. (1951) method was used to determine the total protein content. To precipitate the proteins, 5% trichloroacetic acid was added to the samples after they had been homogenized in double-distilled water. In a solution of 1% NaOH, the precipitate was dissolved. The blue color was developed using Folin phenol reagent, and the absorbance was read at 660 nm, using a spectrophotometer.

2.5 Amino Acid Analysis

Different amino acids (glutamic acid, aspartic acid, proline, glycine, alanine, arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, and valine) were determined according to the method described by Laurey (1997). Samples and standards of date fruit were hydrolyzed by vapors HCl for 20 h at 110 °C. Following hydrolysis, samples were extracted three times using 100 μL of 40% acetonitrile and 0.5% trifluoroacetic acid. After that, the extracts were completely dried in a Speed Vac before being re-dissolved in sample buffer. The amino acid analyzer (Beckman 6300 system, New York, Valhalla, NY, USA) was then used to analyze the samples and standards.

2.6 Mineral’s Content

Three grams of fresh pulp were dried in an oven at 70 °C until a consistent weight and crushed. The samples were digested with H2SO4 and H2O2. The nitrogen content was determined using the micro-Kjeldahl method, as described by Wang et al. (2016). Using a spectrophotometer at a wavelength of 405 nm, the phosphorus content was determined using the vanadomolybdate method, as mentioned by Wieczorek et al. (2022). A flame photometer (SKZ International Co., Ltd., Jinan Shandong, China) was used to measure the potassium and sodium (Asch et al. 2022). According to Stafilov and Karadjova (2009), calcium at 422.8 nm, magnesium at 285.2 nm, iron at 248.3 nm, zinc at 213.9 nm, and manganese at 279.5 nm were determined using atomic absorption (3300).

2.7 Determination of Total Phenols and Tannins

According to the Folin–Ciocalteu assay as described by Singleton et al. (1999) and Eberhardt et al. (2000), the total phenol content was determined. Briefly, 2 g of fresh pulp was extracted for 24 h with 10 mL of methanol. The Folin–Ciocalteu reagent (125 μL) was added after the extract (125 μL) had been diluted 1/5 (v/v) with distilled water. The mixture was then left to stand for 3 min. After that, 1.25 mL of 70 g L−1 sodium carbonate solution was added for a final volume of 3 mL with distilled water. The mixture was incubated at room temperature for 90 min. The absorbance at 760 nm was measured using a UV-visible spectrophotometer. A standard curve of total phenols was prepared using gallic acid. Additionally, the colorimetric technique described by Bentebba et al. (2020) was used to determine the amount of tannin in the date extracts. First, 0.4 mL of extract or catechin as standard was mixed with 1 mL of 4% vanillin solution made in absolute ethanol and 0.2 mL of 37% HCl. After the mixture had been shaken and had been reacting at room temperature in the dark for 15 min, a spectrophotometer was used to measure the absorbance at 500 nm.

2.8 Enzyme Activity Determination

Two grams of date fresh pulp was homogenized in 5 mL of ice-cold 100 mM phosphate buffer (pH 7.4) containing 1% polyvinyl pyrrolidine and 1 mM EDTA. The homogenate was centrifuged at 15,000 g for 10 min at 25 °C. The obtained supernatant was collected and used to measure the catalase (CAT, EC 1.11.1.6) and peroxidase (POD; EC 1.11.1.7) activities. Monitoring the decrease in absorbance at 240 nm caused by H2O2 decomposition was used to measure the activity of CAT (Aebi 1984). By analyzing the guaiacol oxidation at 470 nm using the method of (Hemeda and Klein 1990), the POD activity was determined.

2.9 Extraction and GC-MS Analysis of Endogenous Phytohormones

With some modifications, the endogenous fruit hormones, including indole-3-acetic acid, cytokinins, GA3, and SA, were measured in accordance with Nehela et al. (2016). Briefly, 2 mL of ice-cold extraction solvent (methanol/water/HCl (6N); 80/19.9/0.1; v/v/v) was used to extract 5 g of fruit tissues after they had been homogenized and ground in liquid nitrogen. The supernatant was collected and used to determine the presence of phytohormones after the extract had been centrifuged at 25,000g for 5 min at 4 °C. For indole-3-acetic acid and SA, 50 μL of the supernatant was derivatized with 40 μL of methyl chloroformate (MCF) and dried by adding sodium sulfate. For cytokinins and GA3, 50 μL of the supernatant was derivatized and dried with 100 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) by heating at 85 °C for 45 min. For GC-MS analysis, 1 μL was injected into the GC-MS running in the selective ion mode (SIM-mode). A Clarus 680 GC with SQ8-T Mass Spectrometer system (Perkin Elmer, Waltham, MA, USA) fitted with an Elite-5MS capillary column (low bleed, 30 m × 0.25 mm × 0.025-μm film thickness; Perkin Elmer, Waltham, MA, USA) was used to analyze all samples and phytohormone standards. Helium was the carrier gas with flow rate 1 mL min–1. For indole-3-acetic acid and SA, the temperature program was as follows: the column was maintained at 50 °C for 3 min, and then was raised to 200 °C at a rate of 4 °C min–1, held for 5 min. The procedure for GA3 and cytokinins was as follows: the column was kept at 60 °C for 2 min, increased to 160 °C at 20 °C min–1, and then increased to 290 °C at 5 °C min–1. Temperatures for the injector and detector were set at 250 °C and 260 °C, respectively. The TurboMass software version 6.1 (Perkin Elmer, Waltham, MA, USA) was used to analyze chromatograms. Identification of auxins, cytokinins, GAs, and SA was performed by comparing their retention time, linear retention indices (LRIs), and the selected ions with those of authentic standards.

2.10 Statistical Analysis

One-way variance analysis (ANOVA) was used to analyze the data that was obtained. The data were analyzed based on a randomized complete block design with four replications. Given that the results of the two seasons followed a similar trend, a combined analysis was conducted for them. Duncan’s multiple range test was used to determine the statistical significance of the means at p < 0.05. The SAS software (SAS Inc., Cary NC) was used for data analysis. The data are presented as means ± standard error (SE).

3 Results

3.1 Treatments of GA3 and/or SA Promote “Zaghloul” Fruit Growth

The obtained results indicated that spraying GA3 and SA, alone or in combination, increased the fruit growth significantly more than the control treatment (Table 1, Fig. 1). In particular, the combined treatment significantly improved the bunch weight more than the individual ones, compared with the control treatment. In addition, the date fruits treated with GA3 in combination with SA, as well as those treated with GA3 alone, showed the greatest improvement in the fruit weight, volume, length, and diameter, as well as the flesh weight and percentage when compared to the untreated control fruits. The combination of GA3 and SA significantly (p < 0.05) improved the bunch weight, fruit weight, fruit volume, fruit length, fruit diameter, flesh weight, and flesh percentage by 119.1%, 60.9%, 121.1%, 18.4%, 28.6%, 76.7%, and 9.8%, respectively, in comparison to the control treatment.

Table 1 Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the bunch weight (kg), fruit weight (g), fruit volume (cm3), fruit length (cm), fruit diameter (cm), flesh weight (g), flesh (%), seed weight (g), and seed weight/fruit weight (%) of “Zaghloul” dates. Values are mean ± standard error (n = 4). Letters within the same column are statistically different at p < 0.05 level following Duncan’s test. Where, * reflects p < 0.05
Full size table
Fig. 1
figure 1

Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the growth and production of “Zaghloul” dates

Full size image

The current findings also showed that the GA3 and SA treatments, either separately or in combination, decreased the seed weight and seed weight/fruit weight ratio compared with the control treatment (Table 1). The lowest values were recorded for the dual treatment and the single application of GA3 when compared to the solely SA-treated plants. Using GA3 in conjunction with SA significantly decreased the seed weight and seed weight/fruit weight ratio by 31.8% and 57.5%, respectively, compared with the untreated control treatment.

3.2 Spraying of GA3 and/or SA Alter Fruit Internal Characteristics

Data from Table 2 show that the dry matter, crude fiber, ash, and TSS contents, as well as TSS/TA ratio were increased with all exogenous treatments, compared with the control one. Particularly, the dual treatment significantly increased TSS more than the individual ones, compared to the control treatment. Moreover, spraying date fruits with GA3 either alone or in combination with SA increased dry matter, crude fiber, and ash contents, as well as TSS/TA ratio more than SA alone. The dual application significantly (p < 0.05) improved the content of dry matter, crude fiber, ash, and TSS, as well as the TSS/TA ratio by 57.3%, 80.0%, 100.0%, 82.3%, and 264.6%, respectively, compared with the untreated control treatment.

Table 2 Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the content of moisture, dry matter, crude fiber, ash, total soluble solids (TSS), and total acidity (TA) as well as the ratio of total soluble solids/total acidity (TSS/TA) in “Zaghloul” dates. Values are mean ± standard error (n = 4). Letters within the same column are statistically different at p < 0.05 level following Duncan’s test. Where, * reflects p < 0.05
Full size table

However, the content of moisture and TA was significantly reduced with the application of all tested treatments, compared with the untreated control fruits (Table 2). Spraying GA3 either separately or in combination with SA reduced their content more than with the SA treatment. In comparison to the control treatment, applying GA3 along with SA significantly diminished the content of moisture and TA by 24.0% and 50.0%, respectively.

3.3 Exogenously Applied GA3 and/or SA Modulate Fruit Sugar Profile

Our study found that the date fruits treated with GA3 in combination with SA, as well as those treated with GA3 alone, showed the greatest improvement in the amount of reducing and total soluble sugars when compared to those treated with SA alone (Fig. 2a–c). In comparison to the untreated control treatment, the GA3 and SA combination treatment significantly (p < 0.05) improved the content of reducing, non-reducing, and total soluble sugars by 80.0%, 167.3%, and 100.0%, respectively.

Fig. 2
figure 2

Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the a reducing sugars, b non-reducing sugars, and c total soluble sugars in “Zaghloul” dates. Values are the mean ± standard error (n = 4). Different letters on the top of the bars indicate significant differences at p < 0.05 level following Duncan’s test

Full size image

3.4 Exogenous Application of GA3 and/or SA Trigger Fruit Quality

Data from Fig. 3a and b illustrate that the date fruits treated with GA3 either alone or in combination with SA showed a greatest improvement in the protein and carbohydrate content when compared to the untreated control fruits. Using GA3 in conjunction with SA significantly (p < 0.05) improved the carbohydrate and protein content by 56.3% and 68.6%, respectively, compared with the untreated control treatment.

Fig. 3
figure 3

Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the (a) carbohydrate and (b) total protein in “Zaghloul” dates. Values are the mean ± standard error (n = 4). Different letters on the top of the bars indicate significant differences at p < 0.05 level following Duncan’s test

Full size image

3.5 Exogenous Treatments of GA3 and/or SA Improve Fruit Amino Acid Profile

The obtained results indicated that fruits treated with exogenous treatments maintained the highest levels of amino acids when compared to the untreated ones (Fig. 4a–q). In general, combining GA3 with SA has been proved to be the most effective treatment. It significantly (p < 0.05) improved the concentration of glutamic acid, aspartic acid, proline, glycine, alanine, arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, and valine by 57.0%, 36.7%, 56.7%, 48.2%, 108.3%, 53.2%, 89.5%, 92.9%, 55.6%, 13.2%, 62.0%, 59.2%, 87.0%, 29.6%, 31.1%, 29.0%, and 31.3%, respectively, compared with the untreated control treatment.

Fig. 4
figure 4

Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the a glutamic acid, b aspartic acid, c proline, d glycine, e alanine, f arginine, g cysteine, h histidine, i isoleucine, j leucine, k lysine, l methionine, m phenylalanine, n serine, o threonine, p tyrosine, and q valine in “Zaghloul” dates. Values are the mean ± standard error (n = 4). Different letters on the top of the bars indicate significant differences at p < 0.05 level following Duncan’s test

Full size image

3.6 Spraying of GA3 and/or SA Stimulate Fruit Mineral Acquisition

The application of GA3 coupled with SA presented the most effective treatment to increase nutrient amount compared to the control treatment (Fig. 5a–i). In this study, no significant change in sodium content was observed after the single application of GA3 or SA (Fig. 5f). In comparison to the untreated control treatment, the GA3 and SA combination treatment significantly (p < 0.05) improved the content of nitrogen, phosphorus, potassium, calcium, magnesium, sodium, zinc, iron, and manganese by 46.7%, 100.0%, 116.7%, 71.4%, 77.8%, 20.8%, 38.8%, 60.7%, and 26.3%, respectively.

Fig. 5
figure 5

Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the a nitrogen, b phosphorus, c potassium, d calcium, e magnesium, f sodium, g zinc, h iron, and i manganese in “Zaghloul” dates. Values are the mean ± standard error (n = 4). Different letters on the top of the bars indicate significant differences at p < 0.05 level following Duncan’s test

Full size image

3.7 Exogenously Applied GA3 and/or SA Modulate Fruit Antioxidant Profile

The data obtained revealed that spraying “Zaghloul” fruits with GA3 and/or SA significantly increased the antioxidant enzyme activity, while significantly reducing the total phenols and tannins content compared to the untreated control treatment (Fig. 6a–d). Particularly, combining GA3 with SA has been shown to be the most effective treatment. When compared to the untreated control treatment, it significantly (p <0.05) enhanced the activity of CAT and POD by 23.1% and 38.5%, respectively, while decreasing the percentage of total phenols and tannins by 27.3% and 40.0%, respectively.

Fig. 6
figure 6

Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the content of a total phenols and b tannins as well as the activity of c catalase and d peroxidase in “Zaghloul” dates. Values are the mean ± standard error (n = 4). Different letters on the top of the bars indicate significant differences at p < 0.05 level following Duncan’s test

Full size image

3.8 Exogenous Treatments of GA3 and/or SA Regulate Fruit Phytohormone Performance

As shown in Fig. 7a–d, the endogenous phytohormone analysis revealed that the sole application of GA3 or SA, as well as their combined application, significantly increased the phytohormone content compared to control ones. In general, combining GA3 with SA has been proved to be the most effective treatment. It significantly (p < 0.05) improved the endogenous content of indole-3-acetic acid, cytokinins (trans-Zeatin and trans-Zeatin riboside), GA3, and SA by 202.4%, 200.0%, 200.9%, and 111.8%, respectively, compared with the untreated control treatment.

Fig. 7
figure 7

Effect of exogenous applications of gibberellic acid (GA3; 100 mg L−1), salicylic acid (SA; 100 mg L−1), and their combination at Hababouk and Kimri fruit development stages on the a indole-3-acetic acid, b cytokinins (trans-Zeatin and trans-Zeatin riboside), c gibberellic acid, and d salicylic acid in “Zaghloul” dates. Values are the mean ± standard error (n = 4). Different letters on the top of the bars indicate significant differences at p < 0.05 level following Duncan’s test

Full size image

From all the above obtained results, we showed that the sole application of GA3 markedly increased as compared to the sole application of SA. However, the combined application of GA3 and SA gave the best results.

4 Discussion

In particular, larger and heavier date fruits are preferred in fresh consumption cultivars like “Zaghloul.” Therefore, it is crucial to find ways to enhance the growth and development of date fruit. Phytohormones like GA3 and SA, which are newly emerging players, are essential for maintaining plants’ physiological and developmental processes (Talaat 2021a, 2021b; Prakash et al. 2022; Talaat and Shawky 2022; Li et al. 2023). Consequently, this investigation demonstrates, for the first time, the synergistic effect of applying GA3 in combination with SA on the physiological and biochemical characteristics of “Zaghloul” fruit. Our study’s findings demonstrate that adding GA3 coupled with SA increases the yield and quality of “Zaghloul” fruit by upregulating the sugar accumulation, amino acid performance, nutrient acquisition, endogenous phytohormonal profile, and antioxidant capacity (Fig. 8). Our findings highlight the possibility of using GA3 in conjunction with SA as a tool to enhance fruit’s yield and quality.

Fig. 8
figure 8

Different mechanisms played by gibberellic acid and salicylic acid in improving date palm fruit growth and quality

Full size image

Exogenous PGRs supplementation is an effective way to increase plant productivity (Talaat 2020; Shah et al. 2023). Our findings indicate that the sole and integrative application of GA3 and SA improved date fruit growth. This is attributed to the synergistic action of GA3 and SA in maintaining cellular integrity and enhancing the strength of carbohydrate sink, which in turn improves date fruit size and weight (Cutler and Nelson 2017). The results obtained from this study were compatible with previous studies which found that the GA3 application stimulates fruit growth (Awad and Al-Qurashi 2012; Plackett and Wilson 2016; Khalil 2020; Talat et al. 2020; Prakash et al. 2022; Mosa et al. 2022). Actually, the application of GA3 can improve cell elongation (Erogul and Sen 2015), enhance fruit nutrient acquisition (Fortes et al. 2015), degrade the growth-repressing DELLA proteins (El-Sharkawy et al. 2017), and increase cell division (Zhang et al. 2020), which in turn increases fruit weight and size. Moreover, our findings are in line with those of Mohamed et al. (2017), Ahmed et al. (2021), Gacnik et al. (2021), Ahmed et al. (2022b), and Shareef et al. (2022), who demonstrated that the exogenous SA application can be crucial in coordinating date fruit growth. This occurs mostly because of the role played by SA in enhancing the cell division and expansion (Mohamed et al. 2014; Brito et al. 2018a, 2018b; Elmenofy et al. 2021) and improving the metabolite flow to reproductive organs (Yusuf et al. 2013; Aghdam et al. 2016), which cause significant increases in fruit growth.

Our results manifested a significant rise in the content of dry matter, crude fiber, and ash by applying GA3 and/or SA. This is attributed to their functions in stimulating fruit growth and nutritional status, which in turn improves date fruit quality. The current study also showed that GA3 and/or SA applications can produce better fruit flavor than control by increasing TSS and lowering TA of date fruits. This beneficial effect of the exogenous treatments was associated with an increase in the sugar accumulation. These results are in corroboration with the findings of Beerappa et al. (2019), Moradinezhad et al. (2019), García-Pastor et al. (2020), Talat et al. (2020), Fan et al. (2021), Hazarika and Marak (2022), and Mosa et al. (2022). Our research revealed that the GA3 and/or SA treatments significantly increased the total soluble sugars in “Zaghloul” fruits when compared to untreated control. The result is identical to those of Suwandi et al. (2016), Brito et al. (2018b), Beerappa et al. (2019), García-Pastor et al. (2020), Zhao et al. (2021), and Mosa et al. (2022) who concluded that the exogenous GA3 or SA application can enhance leaf photosynthetic capacity, control photo-assimilate loading, transport, and distribution, and promote sink strength, thereby improving fruit quality. According to Yang et al. (2020) and Zhao et al. (2021), the upregulation in the expression of sucrose synthase, neutral invertase, and sucrose phosphate synthase, as well as the downregulation in the expression of sucrose transporter may be related to the increase in sugar accumulation caused by SA treatment. It is interesting to note that applying GA3 along with SA may be a treatment that actively contributes to improving the fruit’s quality.

One of the interesting results of this study is the encouraging effect of GA3 and/or SA treatments on the carbohydrate and protein content. Similar to our findings, a significant increase in carbohydrate and protein content by GA3 or SA application was also documented by Alrashdi et al. (2017), Hafez et al. (2021), Shareef et al. (2022), and Talaat (2021a, 2021b). In this concern, Suwandi et al. (2016) and Brito et al. (2018b) stated that GA3 or SA treatment improved fruits quality via increasing the sink strength of developing fruits and/or the net photo-assimilate production. According to Brito et al. (2018a, 2018b), Khan et al. (2022), and Talaat and Hanafy (2023), SA application enhances source-to-sink relationship and carbon uptake and/or fixation. GA3, like SA, has been shown to influence sink formation and source-sink metabolism (Suwandi et al. 2016). It is important to note that increased protein and carbohydrate accumulation by GA3 and/or SA may result in increased metabolite flow to developing organs, which in turn enhances fruit growth and, consequently, the obtained yield.

Dates contain essential amino acids that the human body is unable to produce (Assirey 2015; Hamad et al. 2015). To the best of our knowledge, no studies have looked into how adding GA3 and/or SA affects the amino acid profile in “Zaghloul” fruit. Our results demonstrated that applying GA3 and/or SA significantly increased amino acid acquisition. This is consistent with their role in triggering a variety of metabolic and physiological activities in plants (Suwandi et al. 2016; Kamiab et al. 2020). The finding is supported by other investigations mentioning that applied GA3 or SA increased the amino acid biosynthesis (Arunadevi et al. 2019; Talat et al. 2020). Based on the obtained results, we can draw the conclusion that the simultaneous application of GA3 and SA appears to be beneficial in promoting the synthesis of assimilates like sugar and amino acid and improving the efficiency of these assimilates’ transport to the developing fruit, resulting in an increase in date fruit weight and improved fruit quality.

The results of the current study show that one of the major effects of applying GA3 and/or SA is an improvement in date nutritional quality by increasing the nutrient acquisition in treated fruits. This could be due to their effects on the source-sink relationship and nutrient distribution to reproductive organs (Alrashdi et al. 2017; Islam and Mohammad 2022; Mosa et al. 2022). As a result, treated fruits can provide a good source of macro- and micro-nutrients required for bone growth, red blood cell production, and energy metabolism. It is worth noting that the combination of GA3 and SA may have had a growth-promoting effect due to their roles in mineral uptake, which is necessary for many biochemical and physiological processes regulating growth and development.

In comparison to the untreated fruits, treated ones contained less total phenol and tannins. This finding concurs with those of Maudu et al. (2011) and Ozkan et al. (2016), who reported that the application of GA3 increased amino acid synthesis, which may have reduced the accumulation of polyphenols. Another explanation for the decrease in the total phenol content after treatment with GA3 is the antagonistic interaction between ABA and GAs (Prakash et al. 2022). In addition, according to our study, the fruits treated with GA3 and SA, either separately or together, had significantly higher CAT and POD activities than the control. The result is identical to those of Shareef et al. (2022), Talaat and Todorova (2022), and Talaat et al. (2023) who concluded that SA treatment increased CAT and POD activities, which are essential for plant cell antioxidant activity. Actually, the application of SA can stimulate the expression of CAT genes (Xu and Tian 2008), which in turn increases CAT activity. Furthermore, García-Pastor et al. (2020) demonstrated that SA preharvest treatment has the potential to delay the postharvest ripening process by increasing the activity of antioxidant enzymes, allowing them to effectively scavenge reactive oxygen species produced during fruit ripening, a process that is thought to be a functionally modified protracted form of senescence. Our findings imply that increasing antioxidant enzyme activity with GA3 and SA, either separately or together, may activate a defense mechanism.

Fruit growth and the phytohormone profile are closely related. Previous studies by Sotelo-Silveira et al. (2014) and Khan et al. (2020) revealed that the exogenous growth regulator application has a positive effect on the fruit growth and development by promoting fruit setting and improving fruit shape and size. In the current study, applications of GA3 and/or SA significantly increased the content of endogenous indole-3-acetic acid, cytokinins (trans-Zeatin and trans-Zeatin riboside), GA3, and SA, which in turn improves fruit growth. Our results imply that upregulating the internal hormone production may be the mechanism by which GA3 and SA applications improve date fruit growth and development. In this concern, Naser et al. (2016) reported that raising the cytokinin level could increase date palm productivity and enhance the quality of its fruit. Collectively, fruit growth and development can be divided into two stages: cell division stage, where cytokinin promotes the increase in cell number; and cell expansion stage, where GA regulates the cell expansion and elongation (Hassan et al. 2023). According to Shahsavar and Shahhosseini (2021) and González-Hernández et al. (2022), both phytohormones play a significant role in determining the size of the fruit.

Our findings are consistent with earlier studies using GA3 and SA to regulate endogenous hormonal acquisition. Evidence demonstrates that SA application induces the overexpression of GA biosynthetic genes (Ding et al. 2015). In addition, exogenous GA3 application enhances SA biosynthesis (Alonso-Ramírez et al. 2009; Khan et al. 2020). Therefore, we suggest that the increased accumulation of endogenous hormones in “Zaghloul” fruit may be attributed to the crosstalk between internal phytohormones caused by the application of exogenous GA3 and SA, providing better growth and yield performance as well as better nutrient, amino acid, and sugar acquisition.

In sum, our findings highlight the different mechanisms played by GA3 and SA in improving date palm fruit growth and quality. This improvement could display the synergistic effect of the dual treatment (GA3 and SA) and its superiority over the individual one.

5 Conclusions

The findings of this study demonstrate that the growth and development of date fruits can be successfully enhanced by applying exogenous gibberellic acid and/or salicylic acid to the bunch of “Zaghloul” at the Hababouk and Kimri stages. Specifically, the application of gibberellic acid in conjunction with salicylic acid significantly increased the production of date fruits via regulating the nutrient (nitrogen, phosphorus, potassium, calcium, magnesium, sodium, zinc, iron, and manganese) acquisition, sugar (reducing, non-reducing, and total) accumulation, amino acid (glutamic acid, aspartic acid, proline, glycine, alanine, arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, and valine) profile, antioxidant (total phenols and tannins content as well as peroxidase and catalase activity) response, and phytohormone (indole-3-acetic acid, cytokinins, gibberellic acid, and salicylic acid) performance. It was concluded that the application of 100 mg L−1 gibberellic acid along with 100 mg L−1 salicylic acid can be an effective, cost-effective, and natural strategy for improving “Zaghloul” fruit’s yield and quality. Our findings contribute to a better understanding of how gibberellic acid or salicylic acid affect date palm productivity and endorse the potential of their combined use as a tactic to improve fruit growth and development.