Introduction

Date palm (Phoenix dactylifera L.) is one of the oldest cultivated trees in arid and semi-arid areas, where it is important for both socioeconomic and ecological reasons (Al-Alawi et al. 2017). Its fruits (dates) are high in carbohydrates, lipids, minerals, and amino acids, moreover they contain high levels of phenolic compounds, vitamins, and dietary fibers, which increase their nutritive and therapeutic values (Ghnimi et al. 2017; Al-Mssallem et al. 2020). From 1.25 million hectares of land, 9.61 million tons of dates are produced globally (FAO 2023). The top three countries for date palm production have always been Egypt, Iran, and Saudi Arabia (FAOSTAT 2018). Hababouk, Kimri, Bisr (or Khalal), Rutab, and Tamer are recognized as the various stages of date fruit development and ripening. These stages are cell division, unripe green (cell elongation), unripe full-colored, soft brown, and firm raisin-like fruit, respectively (Awad et al. 2011).

Plant growth regulators (PGRs) are signaling molecules that regulate plant cell division, flowering, and fruit development (El-Sharkawy et al. 2017; Talaat 2020; Shahsavar and Shahhosseini 2021; Talaat 2023). They interact with receptor proteins via signal transduction pathways to control a variety of metabolic processes (Todorova et al. 2016; Cutler and Nelson 2017; González-Hernández et al. 2022). Among these are GA3 and SA, which can stimulate cell elongation, cell division, and fruit development by controlling several activities within the plant cell, such as protein and carbohydrate synthesis, mineral uptake, phytohormonal balance, and antioxidant response (Talaat and Shawky 2022; Gill et al. 2023; Talaat et al. 2023).

The use of GA3 can promote the transition from vegetative to reproductive development, with key functions in flower development, fertilization, and fruit development (Prakash et al. 2022; Li et al. 2023). In tree fruit species, coordinated levels of gibberellin (GA) at different developmental stages are required for the proper establishment of reproductive growth (El-Sharkawy et al. 2017; Khalil 2020). Exogenous application of GA was shown to improve the fruit size and weight (Khan et al. 2020; Mosa et al. 2022). The positive effect of exogenous application of GA3 on fruit enlargement by promoting cell division and elongation was also demonstrated in a prior study by Zhang et al. (2020). Moreover, a lack of bioactive GA during plum fruit development resulted in serious developmental disorders, such as growth retardation, disrupted flower patterning, and constrained fruit characteristics (El-Sharkawy et al. 2012). 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 recognized, it is still not entirely clear how these effects are produced. Furthermore, as a simple phenolic phytohormone, SA plays a vital role in plant growth and development (Sharma et al. 2023; Talaat and Hanafy 2022,2023; Talaat et al. 2023). It can enhance the growth and quality of date fruit (Ahmed et al. 2021; Ahmed and Kaur 2022; Shareef et al. 2022). Numerous studies have shown that the exogenous application of SA improves the quality of date fruit by enhancing protein, carbohydrate, nutrient, soluble sugars, and amino acid content (Martínez-Esplá et al. 2018; Serrano et al. 2018; Elmenofy et al. 2021).

Polyamines (PAs) are growth regulators associated with numerous metabolic processes in plants including fruit maturation, ripening, softening, and senescence (González-Hernández et al. 2022; Jangra et al. 2022). Putrescine (Put), 1,4-diaminobutane, a type of polyamine, can regulate various physical, physiological, and biochemical processes of fruit (Todorova et al. 2016; Anwar et al. 2019; Kaur and Das 2022). Exogenous application of Put increased the bunch weight, fruit weight, pulp/seed ratio, total soluble solids and total sugars content, however titratable acidity and tannins values were decreased in ‘Amhat’ dates (Abd El-Migeed et al. 2013). Likewise, research on ‘Zaghloul’ dates revealed that Put treatment increases productivity and improves fruit quality via enhancing the content of amino acid, sugar, mineral (nitrogen, phosphorus, potassium), phytohormone (GA3, auxins, cytokinin), and photosynthetic pigment (Naser et al. 2016). Similar trend on fruit growth and yield by exogenous supply of Put was also detected by Ali et al. (2014), Hagagg et al. (2020), and Shanbehpour et al. (2020). Additionally, evidences suggest that PAs and GA3 work together to promote cell division (Krishnan and Merewitz 2017; Chen et al. 2019) and flower formation (Zhu et al. 2020).

To date, some studies have looked at date palm responses to plant growth regulators (GA3 or SA), but few have addressed the effect of diamine (Put) in improving date yield. Surprisingly, no study has yet examined the role of Put application in conjunction with GA3 and SA on improving the fruit growth and development. Therefore, the current study, as a first investigation, was carried out to assess the impact of the application of Put, alone and coupled with GA3 and/or SA, on growth, physiological, and metabolic processes of ‘Zaghloul’ fruits. Several physiological and biochemical parameters, including 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) profile, 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 in Phoenix dactylifera, cv. Zaghloul fruit to assess the impact of spraying Put, alone or in combination with GA3 and/or SA, on its yield and quality. Accordingly, it has been hypothesized that applying diamine (Put) and phytohormones (GA3 + SA) together can improve the yield of ‘Zaghloul’ fruit by encouraging the accumulation of protein and carbohydrate as well as enhancing the acquisition of minerals, amino acids, sugars, and phytohormones. The study paves the way to using diamine (Put) and phytohormones (GA3, SA) together to improve the fruit’s growth and quality.

Materials and Methods

Plant Material and Experimental Design

The present study was carried out during two successive growing seasons (2020 and 2021) in the experimental orchard of the Agricultural Research Centre, Ministry of Agriculture, Giza, Egypt. Zaghloul date palm cultivar of 15 years old was grown in clay loamy soil and planted at 8 m apart. Selected trees were healthy and as uniform as possible in age and growth. All the selected Zaghloul date palms received program of fertilization consists of 10 kg plant compost (2.5% N), 5.0 kg ammonium sulphate, (20.6% N), 1.5 kg triple calcium superphosphate (37.5% P2O5), and 1.5 kg potassium sulphate (48% K2O) per each palm. Plant compost fertilizer was added once at the middle of January. Ammonium sulphate was splitted into three equal batches and added at the first week of March, May, and July. Potassium sulphate was applied twice before pollination (last week of February) and just after fruit setting (last week of April). Other horticultural practices such as irrigation and pest management were carried out 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 five treatments. Each treatment was performed in four replications, three trees each, giving a total of sixty 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 (cell division) stage and Kimri [unripe green (cell elongation)] stage of date fruit development, bunches on the selected trees were subjected to the following spraying treatments: (i) control (distilled water spray); (ii) 1 mM Put; (iii) 1 mM Put + 100 mg L− 1 GA3; (iv) 1 mM Put + 100 mg L− 1 SA; and (v) 1 mM Put + 100 mg L− 1 GA3 + 100 mg L− 1 SA. The concentrations of 1 mM Put, 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 2 mM for Put and 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 fruit size (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.

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 (firm raisin-like fruit) stage were selected randomly, washed only with water, dried, and weighted. Moreover, fruits volume (using graduated cylinder; cm3) and dimensions (length and diameter) (using a vernier caliper; cm) were recorded. After weighting 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 were 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 (Sadler and Murphy 2010).

Sample Preparation for Chemical Analysis

Another 60 fruits from each replicate at the Tamar (firm raisin-like fruit) stage were selected randomly, washed only with water, dried, and used for the following chemical analysis.

Reducing, Non-Reducing, and Total Soluble Sugars Determination

One hundred mL of distilled water was used to homogenize three grams 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 and compared with the calibration curve, using pure glucose (Sigma). Additionally, the dinitrosalicylic acid (DNS) method was used to determine the reducing sugar (Miller 1959). Briefly, 0.2 mL of date extract were taken, the volume was made up to 1 mL, and 2 mL of DNS solution were 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 (Shimadzu UV-Visible 1800, Tokyo, Japan) was used to measure the absorbance at 575 nm. Non-reducing sugar was calculated by the difference between the total and reducing sugars. Sugar content was determined using a standard linear equation.

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 4000 g for 10 min. One mL of 5% distilled phenol was added to 1 mL of the extract. The absorbance was read at 490 nm, using a spectrophotometer (Shimadzu UV-Visible 1800, Tokyo, Japan) and compared with the calibration curve, using pure glucose. 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 (Shimadzu UV-Visible 1800, Tokyo, Japan).

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 ºC. 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 a sample buffer. The amino acid analyzer (Beckman 6300 system, New York, Valhalla, NY) was then used to analyze the samples and standards.

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 (Shimadzu UV-Visible 1800, Tokyo, Japan) 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 performed on a atomic absorption spectrometer (AAS) iCE 3300 AA of Thermo Scientific.

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 phenols content was determined. Briefly, 2 g of fresh pulp were 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. The equation of the standard curve was used to determine the total phenols and was expressed as milligram gallic acid equivalents (GAE) per 100 g of fresh weight (mg 100 g− 1 GAE). Additionally, the colorimetric technique described by Bentebba et al. (2020) was used to determine the amount of tannins 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 (Shimadzu UV-Visible 1800, Tokyo, Japan) was used to measure the absorbance at 500 nm. The total tannins content was expressed in milligram of catechin equivalents per 100 g of fresh weight (mg 100 g− 1 CE).

Enzyme Activity Determination

Two grams of date fresh pulp were 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.

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 (6 N); 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 25,000 g 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 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.

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).

Results

Put Application with GA3 and/or SA Promote Fruit Growth

The obtained results demonstrated that the growth and productivity of ‘Zaghloul’ dates were significantly increased by the exogenous application of 1 mM Put, either alone or in combination with 100 mg L− 1 GA3 and/or 100 mg L− 1 SA, compared to the control (Table 1; Fig. 1). Particularly, Put application in conjunction with GA3 and SA produced the highest values. It significantly (p < 0.05) improved the bunch weight, fruit weight, fruit volume, fruit length, fruit diameter, flesh weight, and flesh percentage by 136.2, 28.0, 102.3, 26.5, 38.1, 36.4, and 7.4%, respectively, as compared to the untreated control treatment. Additionally, the current findings also revealed that exogenous applications of Put, GA3, and SA resulted in a significant reduction in seed weight and seed weight/fruit weight ratio when compared to the control (Table 1). The Put application, in particular, achieved the lowest values when combined with GA3 and SA. In comparison to the untreated control treatment, it significantly reduced the seed weight and seed weight/fruit weight ratio by 36.4 and 50.0%, respectively.

Table 1 Changes in 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 as affected by exogenous applications of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) at Hababouk and Kimri fruit development stages
Full size table
Fig. 1
figure 1

Effect of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) spraying treatments at Hababouk and Kimri fruit development stages on the ‘Zaghloul’ dates at Tamar stage

Full size image

Put Application in Combination with GA3 and/or SA Modify Fruit Internal Characteristics

The exogenously applied treatments significantly changed the fruit internal characteristics compared to the control (Table 2). Put application in conjunction with GA3 and SA had the greatest effect and significantly (p < 0.05) improved the dry matter, crude fiber, ash, and TSS content, as well as TSS/TA ratio by 65.8, 60.0, 122.2, 75.8, and 313.2%, respectively, while decreasing moisture and TA content by 27.5 and 57.4%, respectively, as compared to the untreated control treatment.

Table 2 Changes in the percentage 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) of ‘Zaghloul’ dates as affected by exogenous applications of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) at Hababouk and Kimri fruit development stages
Full size table

Put Application with GA3 and/or SA Improve Fruit Sugar Content

Applying Put, either separately or combined with GA3 and/or SA, significantly increased the ‘Zaghloul’ Fruit sugar content than the untreated ones (Fig. 2A, B and C). Fruits treated with Put + GA3 + SA had the highest values and significantly (p < 0.05) improved the reducing, non-reducing, and total soluble sugars (54.5, 185.7, and 84.6%, respectively) as compared to the untreated control treatment.

Fig. 2
figure 2

Changes in the content of (A) reducing sugars, (B) non-reducing sugars, and (C) total soluble sugars of ‘Zaghloul’ dates as affected by exogenous applications of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) at Hababouk and Kimri fruit development stages. Values are mean ± standard error (n = 4). Letters above the bars indicate significant differences at p < 0.05 level following the Duncan’s test

Full size image

Put Application in Conjunction with GA3 and/or SA Trigger Carbohydrate and Protein Accumulation

The exogenous treatments significantly increased the amount of carbohydrate and protein compared to the control treatment (Fig. 3A and B). In comparison to the untreated control fruits, the date fruits treated with Put along with GA3 and SA as well as those treated with Put + GA3 showed the greatest improvement in carbohydrate content. In addition, all exogenous treatments significantly increased the total protein content in comparison to the control treatment, with a similar pattern. The application of Put coupled with GA3 and SA significantly (p < 0.05) improved the carbohydrate and protein content by 51.4 and 160.0%, respectively, as compared to the untreated control treatment.

Fig. 3
figure 3

Changes in the content of (A) carbohydrate and (B) total protein of ‘Zaghloul’ dates as affected by exogenous applications of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) at Hababouk and Kimri fruit development stages. Values are mean ± standard error (n = 4). Letters above the bars indicate significant differences at p < 0.05 level following the Duncan’s test

Full size image

Put Application with GA3 and/or SA Improve Fruit Amino Acid Content

Applying Put, alone or in conjugation with GA3 and/or SA, led to a significant increase in the concentration of essential amino acids (Fig. 4A-Q). When compared to the untreated control fruits, the date fruits treated with Put along with GA3 and SA showed the greatest effect and 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 49.4, 45.5, 99.8, 87.2, 133.3, 120.8, 165.7, 130.9, 86.6, 35.2, 88.6, 51.6, 75.5, 84.7, 95.3, 74.7, and 62.0%, respectively.

Fig. 4
figure 4

Changes in the content of (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 of ‘Zaghloul’ dates as affected by exogenous applications of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) at Hababouk and Kimri fruit development stages. Values are mean ± standard error (n = 4). Letters above the bars indicate significant differences at p < 0.05 level following the Duncan’s test

Full size image

Put Application in Conjugation with GA3 and/or SA Enhance Fruit Mineral Acquisition

The Put application, either separately or combined with GA3 and/or SA, significantly improved the mineral acquisition in ‘Zaghloul’ fruit compared to the control treatment (Fig. 5A-I). The highest improvement was recorded with the Put application in combination with GA3 and SA. It significantly (p < 0.05) improved the content of nitrogen, phosphorus, potassium, calcium, magnesium, sodium, zinc, iron, and manganese by 73.3, 100.0, 100.0, 100.0, 80.0, 50.0, 49.4, 71.7, and 48.3%, respectively, when compared to the control treatment.

Fig. 5
figure 5

Changes in the content of (A) nitrogen, (B) phosphorus, (C) potassium, (D) calcium, (E) magnesium, (F) sodium, (G) zinc, (H) iron, and (I) manganese of ‘Zaghloul’ dates as affected by exogenous applications of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) at Hababouk and Kimri fruit development stages. Values are mean ± standard error (n = 4). Letters above the bars indicate significant differences at p < 0.05 level following the Duncan’s test

Full size image

Put Application with GA3 and/or SA Alter Fruit Antioxidant Profile

Using Put, either separately or combined with GA3 and/or SA, significantly increased the activity of CAT and POD, while decreasing the content of total phenols and tannins when compared to the untreated control treatment (Fig. 6A-D). Put application in conjunction with GA3 and SA induced the lowest content of total phenols and tannins (30.3 and 40.0%, respectively) (Fig. 6A and B), while it resulted in the greatest increase in CAT and POD activity (113.5 and 90.1%, respectively) (Fig. 6C and D) compared with the control treatment.

Fig. 6
figure 6

Changes in the content of (A) total phenol and (B) tannins as well as the activity of (C) catalase and (D) peroxidase of ‘Zaghloul’ dates as affected by exogenous applications of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) at Hababouk and Kimri fruit development stages. Values are mean ± standard error (n = 4). Letters above the bars indicate significant differences at p < 0.05 level following the Duncan’s test

Full size image

Put Application in Conjugation with GA3 and/or SA Elevate Fruit Endogenous Hormonal Content

Spraying Put, either alone or in combination with GA3 and/or SA, significantly increased the endogenous phytohormone content when compared to the control (Fig. 7A-D). In comparison to the untreated control fruits, the date fruits treated with Put along with GA3 and SA showed the greatest effect and significantly (p < 0.05) improved the content of endogenous indole-3-acetic acid, cytokinins (trans-Zeatin and trans-Zeatin riboside), GA3, and SA by 244.8, 372.9, 275.2, and 121.0%, respectively.

Fig. 7
figure 7

Changes in the content of (A) indole-3-acetic acid, (B) cytokinins (trans-Zeatin and trans-Zeatin riboside), (C) gibberellic acid, and (D) salicylic acid of ‘Zaghloul’ dates as affected by exogenous applications of putrescine (Put; 1 mM), gibberellic acid (GA3; 100 mg L− 1), and salicylic acid (SA; 100 mg L− 1) at Hababouk and Kimri fruit development stages. Values are mean ± standard error (n = 4). Letters above the bars indicate significant differences at p < 0.05 level following the Duncan’s test

Full size image

Discussion

The date palm (Phoenix dactylifera L.) fruit is a staple food for millions of people in the Middle East, North Africa, Asia, and America due to its high nutritional value. Its fruits are high in dietary fibers, carbohydrate, protein, lipid, minerals, vitamins, and antioxidant compounds (Al-Mssallem et al. 2020; Bentrad and Hamida-Ferhat 2020). To maximize crop yield and improve quality attributes, exogenous application of growth-promoting phytohormones has been investigated (Talaat 2020,2021; Li et al. 2023) as an eco-friendly alternative to hazardous chemicals. Among them, GA3 and SA are two promising stimulators that have the potential to improve date fruit growth by enhancing cell size and division and altering differentiation patterns (Awad and Al-Qurashi 2012; Ahmed et al. 2021; Ahmed and Kaur 2022; Prakash et al. 2022; Shareef et al. 2022). Furthermore, Put application has been reported to confer certain complex beneficial changes on fruit growth as well as physiological and biochemical characters (Ali et al. 2014; Hagagg et al. 2020). Therefore, this investigation demonstrates, for the first time, the synergistic effect of the application of Put coupled with GA3 and SA on date fruit growth, physiological, and biochemical traits. Results from our experiment show that adding Put in combination with GA3 and SA improves the yield and quality of ‘Zaghloul’ fruit by up-regulating nutrient acquisition, sugar accumulation, amino acid profile, antioxidant response, and phytohormone performance. Our findings provide new insights into the role of Put application in conjunction with GA3 and SA in increasing fruit yield and quality.

Exogenous PGRs application is an effective way to increase fruit yield (Gill et al. 2023). In the current study, we found that exogenous application of Put, either alone or in combination with GA3 and/or SA, improved ‘Zaghloul’ productivity. This is attributed to the positive impact of these exogenous treatments on the accumulation of health-beneficial phytochemicals such as sugars, minerals, amino acids, and phytohormones (Cutler and Nelson 2017; Talaat 2021; Talaat et al. 2023). The results obtained from this study were consistent with previous studies which found that the GA3 application promotes fruit growth (Beerappa et al. 2019; Talat et al. 2020; Prakash et al. 2022; Mosa et al. 2022). Indeed, GA3 application increases fruit nutrient acquisition (Fortes et al. 2015), degrades the growth-repressing DELLA proteins (El-Sharkawy et al. 2017), and enhances cell division (Zhang et al. 2020), which improves fruit growth. Furthermore, our findings are in agreement with those of Ahmed et al. (2021), Gacnik et al. (2021), Ahmed and Kaur (2022), and Shareef et al. (2022), who found that exogenous SA application improved crop yield. This occurs mostly due to SA’s role in enhancing cell division and expansion (Brito et al. 2018a, b; Elmenofy et al. 2021) and improving the metabolite towards reproductive organs (Yusuf et al. 2013; Aghdam et al. 2016), which increases fruit growth. In the same line, exogenous application of Put was shown to promote fruit growth and development (Abd El-Migeed et al. 2013; Naser et al. 2016; Chen et al. 2019; Hagagg et al. 2020). This enhancement might be due to the fact that Put can play a role in many biological processes, including cell division and elongation, embryogenesis, root formation, floral initiation and development, fruit development and ripening, and pollen tube growth and senescence (Todorova et al. 2016; Jangra et al. 2022; Kaur and Das 2022). Moreover, it was suggested that PAs act as hormonal second-messengers of cell proliferation and differentiation in many processes or regulate plant sensitivity to auxins/cytokinins ratio (Anwar et al. 2015; González-Hernández et al. 2022).

Our findings exhibited a significant improvement in the content of dry matter, crude fiber, and ash in ‘Zaghloul’ fruits treated by Put, GA3, and SA, implying a direct impact on the fruit’s quality. This effect could be assigned to their roles in promoting fruit growth and nutritional status, which enhances the quality of date fruit. The experiment also displays that Put, GA3, and SA applications can produce better fruit flavor than the control by raising TSS and decreasing TA of date fruits. The increase in sugar accumulation was linked to this exogenous treatments’ positive effects. Our results are in line with previous studies that found an increase in TSS and a decrease in TA in fruits treated with Put (Abd El-Migeed et al. 2013; Naser et al. 2016; Kibar et al. 2021), GA3 (Beerappa et al. 2019; Talat et al. 2020; Mosa et al. 2022), and SA (García-Pastor et al. 2020; Fan et al. 2021; Hazarika and Marak 2022).

Our results also manifested a significant increase in sugar accumulation in ‘Zaghloul’ fruit treated with Put, GA3, and SA when compared to the untreated control fruits. This improvement could be explained by their effects on regulating several metabolic processes such as photosynthetic activity as well as photoassimilate loading, transport, and distribution (Abd El-Migeed et al. 2013; Naser et al. 2016; Beerappa et al. 2019; García-Pastor et al. 2020; Zhao et al. 2021; Mosa et al. 2022). Furthermore, evidences suggest that sucrose accumulation can be induced by SA application via increasing the expression of sucrose synthase, neutral invertase, and sucrose phosphate synthase (Yang et al. 2020; Zhao et al. 2021). Given the increase in sugar content, we believe that the use of Put in conjunction with GA3 and SA is a promising approach for improving fruit quality.

Regarding carbohydrate and protein accumulation, the obtained results demonstrated that exogenous Put, GA3, and SA treatments increased their content in ‘Zaghloul’ fruits. These findings are matched with those obtained by Naser et al. (2016), Shareef et al. (2022), Talaat (2021), and Talaat and Hanafy (2022). This effect could be assigned to the positive effect of these exogenous applications on the net photo-assimilate production and/or the sink strength of developing fruits (Suwandi et al. 2016; Brito et al. 2018b). Further evidence that Put has vital role in the regulation of sink strength of the developing organs, thereby enhancing fruit growth and quality (Kaur and Das 2022). In addition, SA plays an integrating role in regulating the source-to-sink relationship by inducing plant growth and development pathways and upregulating physiological responses related to carbon uptake and/or fixation (Brito et al. 2018a, b; Khan et al. 2022; Talaat and Hanafy 2023). Moreover, SA was shown to have a positive effect on nitrogen metabolism and protein accumulation (Talaat 2023). Like Put and SA, GA3 is also widely known to alter source-sink metabolism and sink formation (Suwandi et al. 2016). It is significant to note that increased protein and carbohydrate accumulation by Put, GA3, and SA applications may result in increased metabolite flow to developing organs, which enhances fruit growth and, consequently, the obtained yield.

Interestingly, dates contain essential amino acids that the human body is incapable of producing. According to Assirey (2015), dates contain 18 different amino acids, with the highest levels of glutamic acid, aspartic acid, proline, glycine, and alanine. Arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine were among the other amino acids found. With the exception of threonine, 17 of these 18 amino acids were also reported by Hamad et al. (2015). To our knowledge, no research has examined the impact of exogenous Put, GA3, and SA treatments on the amino acid profile of date fruit. Our findings display that spraying Put, GA3, and SA significantly improved amino acid performance. This is in line with their functions in activating a wide range of metabolic and physiological processes in plants (Suwandi et al. 2016; Koo et al. 2020). Our result is also supported by the findings of other studies (Arunadevi et al. 2019; Talat et al. 2020; Talaat 2023) that these exogenous applications can regulate amino acid biosynthesis. We can infer from the results that the use of Put coupled with GA3 and SA seems to be beneficial in encouraging the synthesis of assimilates such as sugar and amino acid and improving the efficiency of their transport to the developing fruit, leading to an increase in date fruit weight and improved fruit quality.

Essential elements like calcium, iron, magnesium, phosphorus, potassium, zinc, selenium, and manganese are abundant in date fruit and improve its nutritional value (USDA 2020). The mineral analysis profile in the present investigation revealed that the Put, GA3, and SA treatments increased nutrient (nitrogen, phosphorus, potassium, calcium, magnesium, sodium, zinc, iron, and manganese) acquisition in ‘Zaghloul’ fruit. This could be due to their effects on the source and sink relationship and nutrient allocation to reproductive organs (Naser et al. 2016; Alrashdi et al. 2017; Islam and Mohammad 2022; Mosa et al. 2022). It is worth noting that a growth-promoter effect of Put, GA3, and SA might have been the result of their functions in mineral uptake, which is necessary for various biochemical and physiological processes regulating plant growth and production.

Our study also revealed a significant fall in the content of total phenols and tannins with Put, GA3, and SA applications. This decrease with GA3 treatment is probably due to increased amino acid synthesis (Maudu et al. 2011; Ozkan et al. 2016) and/or the antagonistic relationship between ABA and GA3 (Prakash et al. 2022). Decreasing the content of total phenols and tannins with Put application has also been reported by (Abd El-Migeed et al. 2013). Furthermore, this trial’s findings also demonstrated that treated ‘Zaghloul’ fruits had significantly higher CAT and POD activities in comparison with the untreated ones. Our result is in corroboration with the findings of Shanbehpour et al. (2020) who showed that Put application increased CAT and POD activity, implying that Put could help to maintain antioxidant capacity by boosting the activity of antioxidant enzymes in fruits (Wannabussapawich and Seraypheap 2018; Zheng et al. 2019). Additionally, SA treatment had a similar effect, increasing CAT and POD activity, which is essential for plant cells’ antioxidant activity (Shareef et al. 2022; Talaat and Todorova 2022; Talaat et al. 2023). Actually, the application of SA can stimulate the expression of CAT genes (Xu and Tian 2008), which increases CAT activity. In addition, García-Pastor et al. (2020) revealed that SA preharvest treatment has the potential to delay the postharvest ripening process by increasing the activity of antioxidant enzymes. Our results suggest that Put, GA3, and SA applications may be beneficial in preserving the quality of ‘Zaghloul’ fruit.

Our research also manifested a significant rise in the endogenous indole-3-acetic acid, cytokinins (trans-Zeatin and trans-Zeatin riboside), GA3, and SA content in the pulp of ‘Zaghloul’ fruits treated with Put, GA3, and SA. Our results support El-Bassiouny et al. (2008) and Naser et al. (2016) findings, which demonstrated that exogenous application of Put raised indole acetic acid (IAA), GA, and cytokinins levels. In the same line, study by Anwar et al. (2015) suggested that Put can interact with a variety of phytohormones by upregulating the gene expression for IAA and SA. It was also shown that floral differentiation may be impacted by the crosstalk between PAs and GA3 (Kamiab et al. 2020). Additionally, prior study by Ding et al. (2015) revealed that exogenous SA application upregulated the expression of GA biosynthetic genes. Further evidence displayed that exogenous GA3 treatment improves Put accumulation (Shiozaki et al. 1998) and SA biosynthesis (Alonso-Ramírez et al. 2009; Khan et al. 2020). Therefore, it’s interesting to note that the enhancement in the internal hormone production could be explained by the effect of the exogenous Put, GA3, and SA applications, which can improve date fruit growth and development. In agreement with our findings, Naser et al. (2016) pointed out that increasing the cytokinin level could increase date palm production and improved fruit quality. Moreover, fruit growth and development can be divided into two stages: cell division stage, in which cytokinin plays an important role in facilitating cell number; and cell expansion stage, in which GA regulates cell expansion and elongation (Khan et al. 2020; Shahsavar and Shahhosseini 2021; González-Hernández et al. 2022).

Based on the findings of this study, the elevation of the endogenous hormonal content as well as the enhancement of the other essential inherent plant characteristics (sugar accumulation, amino acid performance, nutrient acquisition, antioxidant response, as well as carbohydrate and protein content) in response to the exogenous applications may discuss the synergistic effect of the dual treatment (diamine + phytohormones) tested in our study and its superiority over the individual one (diamine).

Conclusions

The findings of the present investigation revealed a significant impact of diamine (Put), either alone or in combination with phytohormones (GA3 or SA), on the yield and quality of ‘Zaghloul’ fruit. These exogenous applications not only improved fruit growth and production, but also enhanced fruit quality by upregulating sugar, amino acid, mineral, carbohydrate, protein, and endogenous hormone acquisition. Hence, the application of these plant bio-regulators improves the internal physiology of developing fruit by ensuring that they receive an adequate supply of nutrients and other compounds required for proper growth and development, resulting in increased size, quality, and ultimately better yield. Specifically, Put applied at 1 mM along with GA3 at 100 mg L-1 and SA at 100 mg L-1 proved to be the most effective in increasing the quantity and quality of ‘Zaghloul’ fruit. As a result, our findings highlight the synergistic effect of using both diamines (Put) and phytohormones (GA3 + SA) to promote date palm fruit yield and quality.