Silicon and Gibberellins: Synergistic Function in Harnessing ABA Signaling and Heat Stress Tolerance in Date Palm (Phoenix dactylifera L.)

Date palm is one of the most economically vital fruit crops in North African and Middle East countries, including Oman. A controlled experiment was conducted to investigate the integrative effects of silicon (Si) and gibberellic acid (GA3) on date palm growth and heat stress. The exogenous application of Si and GA3 significantly promoted plant growth attributes under heat stress (44 ± 1 °C). The hormonal modulation (abscisic acid [ABA] and salicylic acid [SA]), antioxidant accumulation, and the expression of abiotic stress-related genes were evaluated. Interestingly, heat-induced oxidative stress was markedly reduced by the integrative effects of Si and GA3 when compared to their sole application, with significant reductions in superoxide anions and lipid peroxidation. The reduction of oxidative stress was attributed to the enhancement of polyphenol oxidase, catalase, peroxidase, and ascorbate peroxidase activities as well as the upregulation of their synthesis related genes expression viz. GPX2, CAT, Cyt-Cu/Zn SOD, and glyceraldehyde3-phosphate dehydrogenase gene (GAPDH). The results showed the activation of heat shock factor related genes (especially HsfA3) during exogenous Si and GA3 as compared to the control. Furthermore, the transcript accumulation of ABA signaling-related genes (PYL4, PYL8, and PYR1) were significantly reduced with the combined treatment of Si and GA3, leading to reduced production of ABA and, subsequently, SA antagonism via its increased accumulation. These findings suggest that the combined application of Si and GA3 facilitate plant growth and metabolic regulation, impart tolerance against stress, and offers novel stress alleviating strategies for a green revolution in sustainable food security.


Introduction
Numerous environmental stresses that adversely influence plant growth and productivity have raised serious concerns in the context of global climate change. In the wake of climate change, high

Chlorophyll a and Chlorophyll b Quantification
Photosynthetic pigments, including Chl a, Chl b, and carotenoid, were extracted by grinding the leaves of the date palm seedlings (200 mg) in 80% acetone. The methodology described by Sumanta, et al. [20] was employed to estimate the Chl a and Chl b content. The absorbance values for Chl a, Chl b, and carotenoid were recorded at 663, 645, and 470 nm, respectively.

Leaf Relative Water Content (LRWC)
The protocol that was established by Cao, et al. [21] was used for the estimation of RWC. For each treatment, the second leaves of the plants were excised, and the fresh mass (FM) was immediately quantified. Thereafter, leaf discs were floated on 30 mL deionized water for 5 h in a petri dish and the saturated mass (SM) was determined. Subsequently, the leaves were dried at 80 • C to a constant weight and their dry mass (DM) was measured. The RWC was calculated with the following formula:

Quantification of Malondialdehyde (MDA)
The level of lipid peroxidation or formation of MDA was estimated using the methodology that was reported by Okaichi, et al. [22]. The tissue homogenates were extracted with 10 mM phosphate buffer (pH 7.0). For the quantification of MDA, 0.2 mL of tissue homogenate was combined with 0.2 mL of 8.1% sodium dodecyl sulfate (SDS), 1.5 mL of 20% acetic acid (pH 3.5), and 1.5 mL of 0.81% thiobarbituric aqueous acid (TBA) solution in a reaction tube. Thereafter, the mixture was heated in boiling water for 60 min. After cooling to room temperature, 5 mL butanol:pyridine (15:1 v/v) solution was added. The upper organic layer was separated and the optical density of the resulting pink solution was recorded at 532 nm while using a spectrophotometer. Tetramethoxypropane was used as an external standard. The level of O 2 •− was estimated using the method that was described by Gajewska and Skłodowska [23]. The homogenate for the reaction was prepared by immersing 1 g of fresh plant sample in phosphate buffer (pH 7.0) containing sodium phosphate (10 mM), nitrobluetetrazolium (NBT) (0.05%; w/v), and sodium azide (NaN 3 ) (10 mM). The mixture was placed at room temperature for 1 h. Afterwards, 5 mL of the mixture was taken in a new tube and heated for 15 min at 85 • C. Thereafter, the mixture was cooled and vacuum filtered. The absorbance of the sample was read at 580 nm with a spectrophotometer. The experiment was replicated three times.

Protein Quantification and Antioxidant Enzyme Assay
For protein quantification, the leaf samples from the date palm (100 mg) were ground using a chilled mortar pestle in 100 mM potassium phosphate buffer (pH 6.8) with 0.2 mM ethylenediaminetetraacetic acid (EDTA). The resulting mixture was centrifuged for 30 min at 12,000× g and the supernatant were used to determine the total protein. The total protein contents were estimated following the protocol that was reported by Bradford [24], with slight modifications. The assay was performed at 595 nm with a spectrophotometer.
The antioxidant enzymes catalase (CAT), ascorbate peroxidase (APX), and polyphenol peroxidase (PPO) were quantified with the methodology that was described by Manoranjan, et al. [25], with slight changes. In brief, date palm leaves (100 mg) were ground by using liquid nitrogen and Phosphate buffer (100 mM) was added to the sample to make a homogenous mixture of pH 7.0. The resulting homogenate were centrifuged for 30 min at 10,000 rpm and 4 • C.
For POD (Peroxidase) analysis, the reaction mixture consisted of 0.1 M potassium phosphate buffer (pH 6.8), 50 µL H 2 O 2 (50 µM), 50 µL pyrogallol (50 µM), and 100 µL crude extract sample. This mixture was incubated at 25 • C for 5 min, then 5% H 2 SO 4 (v/v) was added to stop the enzymatic reaction. The amount of purpurogallin produced was measured while using an optical density of 420 nm. The reaction mixture consisted of similar components as the POD assay, but without H 2 O 2 , and the final assay was calculated at 420 nm, in order to assess the polyphenol oxidase (PPO) activity. A single unit of PPO and POD was directly calculated using an increase of 0.1 units of absorbance. The CAT activity was assayed, as described by Aebi [26]. Briefly, the crude enzyme extract was added to 0.2 M H 2 O 2 in 10 mM phosphate buffer (pH 7.0), after which the CAT activity was determined as a decrease in absorbance at 240 nm and expressed as units (one unit of CAT was defined as the ng of H 2 O 2 released/mg protein/min).
For the quantification of APX (Ascorbate peroxidase), 1 mL phosphate buffer (50 mM; pH 7.0) containing 1 mM ascorbic acid and 1 mM EDTA was used for extraction, then homogenized at 50 Hz for 30 s, and the homogenate was centrifuged at 4,830× g at 4 • C for 15 min. Subsequently, the supernatant was mixed with a phosphate buffer solution (pH 7.0) containing 15 mM AsA and 0.3 mM H 2 O 2 . The reaction mixture was analyzed at 290 nm. One unit of APX was defined as a variable quantity of absorbance at 290 nm per min. All of the enzymatic assays were repeated three times and each time comprised of three replications.

RNA Extraction and cDNA Synthesis
RNA was extracted from date palm leaves while using an extraction buffer (0.25 M, NaCl; 0.05 M, Tris-HCl (pH = 7.5); 20 mM, EDTA; 1% (w/v) SDS; 4% polyvinylpyrrolidone (w/v)), as described by Liu, et al. [27]. Prior to the addition of the sample, 750 µL of the extraction buffer and chloroform: isoamyl alcohol (CI; 24:1 v/v) were added to a 2-mL RNase-free microcentrifuge tube followed by the addition of 40 µL β-mercaptoethanol. Thereafter, a fine powder (100 mg) of the sample was carefully transferred to a 2 mL tube containing the extraction buffer and CI. The mixture was vortexed and incubated at 20 • C for 15 min, followed by centrifugation at 4 • C for 10 min at 12,000× g. In the next step, 600 µL of the supernatant was transferred to a new 2 mL tube and the same volume of CI was added to the tube. The solutions were mixed gently and centrifuged at 4 • C for 10 min at 12,000× g. The upper layer was transferred to a new 1.5 mL micro centrifuge tube and 1/10 volume of 3 M sodium acetate (pH = 5.2) was added. For precipitation, two volumes of absolute ethanol were added, and after gently mixing, the tubes were incubated for 45 min at 4 • C. After incubation, the samples were centrifuged at 4 • C for 10 min. at 12,000× g and. The pellet was dissolved in 200 µL water (diethyl pyrocarbonate-treated) and 10 M LiCl was added (500 µL) to the solution. The solutions were mixed gently and then placed on ice for 60 min. In the final step, the samples were centrifuged at 4 • C for 10 min. at 12,000× g, and the pellet was washed with 70% ethanol. After removing the ethanol, the pellet was air dried and then dissolved in 50 µL of diethyl pyrocarbonate-treated water. The quality of RNA was checked with agarose gel electrophoresis and quantified while using the Qubit (3.0) RNA broad range kit. RNA was added to the Master Mix according to the concentration; for each 100 ng/µL RNA, 10 µL was taken for cDNA synthesis. Polymerase chain reaction was performed in a thermo-cycler at specific conditions (25 • C for 10 min, 37 • C for 2 h, and 85 • C for 5 min). The synthesized cDNA was refrigerated at −80 • C until further use.

Gene Expression Analysis
The synthesized cDNA was used for the amplification of genes (Table 1). Actin gene was used as a reference for all of the primers. Power up "SYBR" green Master Mix was used for the thermo-cycler (Quant studio 5 by Applied Bio Systems Life Technologies) PCR reaction. Primers (10 pM; forward and reverse) were used for all of the five genes. For each sample, the reaction was performed in triplicate to minimize errors and contamination. The reaction was performed at a specific condition such as 94 • C for 10 min in stage 1, 35 cycles of PCR reaction at 94 • C for 45 s, 60 • C for 45 s, 72 • C for 1 min, and finally, 72 • C for 10 min. A threshold level of 0.1 was set for gene amplifications. The experiment was repeated three times and each time comprised of three replications.

Abscisic Acid Extraction and Quantification
For the extraction and quantification of endogenous ABA levels in date palm leaves, the protocol that was reported by Qi, et al. [28] was used with slight modification, as described by Bilal, et al. [29]. Briefly, the extracted samples from the ground and freeze-dried plants were supplemented with [(±)−3,5,5,7,7,7-d6]-ABA as an internal standard and then further analyzed with GCMS (6890N network GC system) and a 5973 Network Mass Selective Detector (Agilent Technologies, Palo Alto, CA, USA). The spectra were recorded at selected ionization values of m/z 162 and 190 for Me-ABA and at m/z 166 and 194 for Me-[2H6]-ABA to expand the affectability of the method. The ABA was calculated from the value of the endo peak in comparison with their respective standards.

Salicylic Acid Extraction and Quantification
Salicylic acid (SA) was extracted and quantified from freeze dried samples of the date palm leaves according to the protocol that was described by Seskar, et al. [30] and Bilal, et al. [31]. The extracted samples were subjected to High Performance Liquid Chromatography (HPLC), which was performed while using a Shimadzu device outfitted with a fluorescence indicator (Shimadzu RF-10AxL) with excitation at 305 nm and emission at 365 nm and with a C18 reverse-phase HPLC column (HP Hypersil ODS, particle size 5 µm, pore size 120 Å, Waters). The flow rate was maintained at 1.0 mL/min.

Statistical Analysis
All of the experiments were repeated three times and the data were collected from each repetition were pooled together. All of the data present the mean values with standard error (SE). The means were analyzed for finding the significant differences among treatments by using one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (DMRT) in SAS software (V9.1, Cary, NC, USA; Figure S1).

Interactive Effects of GA and Si Promote Plant Growth Attributes under Heat Stress
The combined exogenous application of GA 3 and Si resulted in significant plant growth-promoting effects under the control conditions and rescued plant growth under heat stress compared to non-treated plants. The combined application of GA 3 and Si resulted in the maximum shoot length (35.1 ± 0.94 cm) and root length (14.1 ± 0.29 cm) in the absence of stress conditions. Further, the interactive effects of GA 3 and Si significantly mitigated the adverse impact of heat stress and resulted in the maximum shoot length (31.87 ± 0.59 cm) and root length (11.56 ± 0.38 cm), followed by the sole application of GA 3 and Si ( Figure 1A,B). Likewise, the fresh weight of shoot and root was detected to be maximum in both Sole GA 3 (Figure 2A,B). Under the control condition, the maximum carotenoids content was recorded with the combined application of GA 3 and Si followed by sole Si or GA 3 treatment and non-treatment. However, under stress conditions, combined GA 3 and Si-treated plants and sole Si-treated plants equally demonstrated higher levels of carotenoids content, followed by sole GA 3 -treated plants and non-treated plants ( Figure 2C). The heat stress-mitigating response of combined application of GA 3 and Si was further assessed by measuring the relative water potential of the plants. The current findings indicated that combined Si and GA 3 treatment as well as sole GA 3 treatment resulted in the maximum RWC under the control condition. Heat stress drastically reduced the RWC of non-treated plants, with a reduction of 1.73, 1.51, and 1.42 times when compared to the combined GA 3 and Si-treated plants and the sole GA 3 and Si-treated plants, respectively ( Figure 1D). equally demonstrated higher levels of carotenoids content, followed by sole GA3-treated plants and non-treated plants ( Figure 2C). The heat stress-mitigating response of combined application of GA3 and Si was further assessed by measuring the relative water potential of the plants. The current findings indicated that combined Si and GA3 treatment as well as sole GA3 treatment resulted in the maximum RWC under the control condition. Heat stress drastically reduced the RWC of non-treated plants, with a reduction of 1.73, 1.51, and 1.42 times when compared to the combined GA3 and Sitreated plants and the sole GA3 and Si-treated plants, respectively ( Figure 1D).

Interactive Effects of GA3 and Si Stimulate Plant Antioxidant System
The extent of O2 •-was investigated in the date palm plants to assess the generation of heatinduced reactive oxygen species. Heat-induced stress is known to trigger O2 •-accumulation in plants. The current findings indicated that exposure to heat stress resulted in the significant production of O2 •-in plants by showing maximum superoxide anion activity ( Figure 3A). However, the negative influence of heat stress was markedly mitigated after Si and GA3 treatment. The accumulation of O2 •was significantly retarded by the combined application of Si and GA3, with 2.3, 1.5, and 1.2 times less superoxide anion activity when compared to non-treated plants and sole GA3 and Si-treated plants, respectively. Similarly, the extent of lipid membrane peroxidation due to heat stress damages was investigated by measuring the MDA content. Under control conditions, no treatment was significantly different from the non-treated control; however, GA3 treatment differed from sole Si and combined Si and GA3 treatments. Moreover, the exposure to heat stress substantially increased the level of MDA in non-treated plants, whereas the combined application of GA3 and Si significantly decreased the MDA level, followed by sole Si and GA3 treatment ( Figure 3B). The antioxidant activities (CAT, POD, PPO, and APX) were measured to further characterize the combined effects of

Interactive Effects of GA 3 and Si Stimulate Plant Antioxidant System
The extent of O 2 •− was investigated in the date palm plants to assess the generation of heat-induced reactive oxygen species. Heat-induced stress is known to trigger O 2 •− accumulation in plants.
The current findings indicated that exposure to heat stress resulted in the significant production of O 2 •− in plants by showing maximum superoxide anion activity ( Figure 3A). However, the negative influence of heat stress was markedly mitigated after Si and GA 3 treatment. The accumulation of O2 •− was significantly retarded by the combined application of Si and GA 3 , with 2.3, 1.5, and 1.2 times less superoxide anion activity when compared to non-treated plants and sole GA 3 and Si-treated plants, respectively. Similarly, the extent of lipid membrane peroxidation due to heat stress damages was investigated by measuring the MDA content. Under control conditions, no treatment was significantly different from the non-treated control; however, GA 3 treatment differed from sole Si and combined Si and GA 3 treatments. Moreover, the exposure to heat stress substantially increased the level of MDA in non-treated plants, whereas the combined application of GA 3 and Si significantly decreased the MDA level, followed by sole Si and GA 3 treatment ( Figure 3B). The antioxidant activities (CAT, POD, PPO, and APX) were measured to further characterize the combined effects of GA 3 and Si on overcoming heat-induced oxidative stress ( Figure 3C-F). The results showed that the combined application of GA 3  GA3 and Si on overcoming heat-induced oxidative stress ( Figure 3C-F). The results showed that the combined application of GA3 and Si significantly triggered (p > 0.05) CAT activity under heat stress when compared to non-treatment. Similarly, PPO activities for all of the treatments were enhanced under heat stress; however, the combined application of GA3 and Si resulted in approximately 1.91, 1.43, and 1.16 times higher activity than non-treatment and sole GA3 and Si treatment, respectively. Furthermore, POD activity was significantly depressed in GA3 treatment, while non-treated plants and sole Si-treated plant exhibited insignificant levels of POD activity under control conditions. Whereas, the level of POD activity of combined Si and GA3 treatment was approximately comparable with sole Si treatment under control conditions. Whilst the maximum POD activity was recorded with combined application of GA3 and Si, followed by sole Si and GA3 treatment under heat stress. Under control conditions, the maximum APX activity was recorded in sole Si-treated plants, while non-treated plants, sole GA3-treated plants, and combined Si and GA-treated plants exhibited comparable APX activity. However, the combined application of Si and GA3 resulted in the maximum APX activity under heat stress, with approximately 1.91, 1.61, and 1.31 times higher activity than the non-treatment and sole GA3 and Si treatment ( Figure 3C-F).

Interactive Effects of Si and GA 3 Modulate Endogenous Hormonal Regulation
Endogenous ABA analysis revealed that the sole application of Si and combined application of Si and GA 3 considerably decreased free ABA content in date palm as compared to non-treatment and sole GA 3 treatment under control conditions ( Figure 4A). On the contrary, the level of ABA accumulation was significantly enhanced under heat stress with all of the treatments. However, a significantly low level of ABA was observed in the combined GA 3 and Si-treated plants when compared to the non-treated plants and the sole GA 3 or Si-treated plants. The combined GA 3 and Si-treated plants had approximately 2.06, 1.24, and 1.50 times lower ABA content under heat stress when compared to the non-treated and Si and GA 3 -treated plants, respectively. Similarly, analysis showed that the level of SA accumulation under the control condition was almost comparable in all treatments, except for the sole GA 3 -treated plants, which had the maximum (3.5 ± 0.31 ng/g) accumulation. On the other hand, heat stress significantly downregulated SA accumulation in non-treated plants, whereas the interactive effects of GA 3 and Si significantly upregulated SA content (5.3 ± 0.65 ng/g) in date palm, followed by sole GA 3 -treatment and Si treatment ( Figure 4B).

Interactive Effects of Si and GA3 Modulate Endogenous Hormonal Regulation
Endogenous ABA analysis revealed that the sole application of Si and combined application of Si and GA3 considerably decreased free ABA content in date palm as compared to non-treatment and sole GA3 treatment under control conditions ( Figure 4A). On the contrary, the level of ABA accumulation was significantly enhanced under heat stress with all of the treatments. However, a significantly low level of ABA was observed in the combined GA3 and Si-treated plants when compared to the non-treated plants and the sole GA3 or Si-treated plants. The combined GA3 and Sitreated plants had approximately 2.06, 1.24, and 1.50 times lower ABA content under heat stress when compared to the non-treated and Si and GA3-treated plants, respectively. Similarly, analysis showed that the level of SA accumulation under the control condition was almost comparable in all treatments, except for the sole GA3-treated plants, which had the maximum (3.5 ± 0.31 ng/g) accumulation. On the other hand, heat stress significantly downregulated SA accumulation in nontreated plants, whereas the interactive effects of GA3 and Si significantly upregulated SA content (5.3 ± 0.65 ng/g) in date palm, followed by sole GA3-treatment and Si treatment ( Figure 4B).

Modulation of Different Stress-Responsive Genes by Interactive Effects of Si and GA 3 Application
The interactive effects of Si and GA 3 application on the transcript levels of different abiotic stress-related genes were assessed. The expression level of the antioxidant-related gene glutathione peroxidase (GPX2) was significantly higher in sole GA 3 -treated plants under control conditions than in the combined GA 3 and Si-treated plants, sole Si-treated plants, and non-treated plants ( Figure 5A). The combined GA 3 and Si-treated plants exhibited a drastic enhancement in the GPX2 expression level under heat stress, followed by the sole GA 3 and Si-treated plants, and non-treated plants. Similarly, Cyt-Cu/Zn SOD transcript accumulation was significantly enhanced in all treatments under heat stress when compared to the control condition ( Figure 5B). However, the GA 3 and Si-treated plants demonstrated significant (p < 0.005) enhancement in the expression level of Cyt-Cu/Zn SOD as compared to sole Si and GA 3 -treated and non-treated plants. Likewise, the CAT expression level under control conditions was comparable among all treatments; however, combined GA 3 and Si-treated plants exhibited significantly enhanced levels under heat stress, followed by sole Si and GA3-treated plants and non-treated plants, respectively ( Figure 5C). Likewise, the transcript accumulation level of NADP-dependent glyceraldehyde3-phosphate dehydrogenase gene (GAPDH) under the control condition was non-significant in all treatments except in the combined GA 3 and Si-treated plants. However, GAPDH expression under heat stress was significantly up-regulated in all treatments when compared to the control condition ( Figure 5D). The combined GA 3 and Si-treated plants exhibited significant up-regulation, with 1.79, 1.48, and 1.16 times higher expression than the non-treated plants and the sole Si and GA 3 -treated plants, respectively. significantly different (p < 0.05). Means were analyzed for finding the significant differences among treatments by using one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (DMRT). Values represent mean (of four replicates) ± standard error.

Modulation of Different Stress-Responsive Genes by Interactive Effects of Si and GA3 Application
The interactive effects of Si and GA3 application on the transcript levels of different abiotic stressrelated genes were assessed. The expression level of the antioxidant-related gene glutathione peroxidase (GPX2) was significantly higher in sole GA3-treated plants under control conditions than in the combined GA3 and Si-treated plants, sole Si-treated plants, and non-treated plants ( Figure 5A). The combined GA3 and Si-treated plants exhibited a drastic enhancement in the GPX2 expression level under heat stress, followed by the sole GA3 and Si-treated plants, and non-treated plants. Similarly, Cyt-Cu/Zn SOD transcript accumulation was significantly enhanced in all treatments under heat stress when compared to the control condition ( Figure 5B). However, the GA3 and Si-treated plants demonstrated significant (p < 0.005) enhancement in the expression level of Cyt-Cu/Zn SOD as compared to sole Si and GA3-treated and non-treated plants. Likewise, the CAT expression level under control conditions was comparable among all treatments; however, combined GA3 and Sitreated plants exhibited significantly enhanced levels under heat stress, followed by sole Si and GA3treated plants and non-treated plants, respectively ( Figure 5C). Likewise, the transcript accumulation level of NADP-dependent glyceraldehyde3-phosphate dehydrogenase gene (GAPDH) under the control condition was non-significant in all treatments except in the combined GA3 and Si-treated plants. However, GAPDH expression under heat stress was significantly up-regulated in all treatments when compared to the control condition ( Figure 5D). The combined GA3 and Si-treated plants exhibited significant up-regulation, with 1.79, 1.48, and 1.16 times higher expression than the non-treated plants and the sole Si and GA3-treated plants, respectively.  The transcript accumulations of ABA receptor genes (PYL4, PYL8, and PYR1), which are known as core regulators of the ABA signaling pathway, were determined. The results showed that, under control conditions, the expression level of PYL4 for the combined GA 3 and Si-treated plants was significantly down-regulated, followed by that of the sole GA 3 -treated, Si-treated, and non-treated plants ( Figure 6A). Heat-induced stress significantly upregulated the expression level of PYL4 in the plants. However, the combined GA 3 -Si treated plants demonstrated the significantly reduced transcript accumulation of PYL4 under heat stress at levels 4.5, 1.75, and 2.08 times less than those of the non-treated and the sole Si and GA 3 -treated plants, respectively. The relative expression of PYL8 and PYR1 was significantly upregulated in non-treated plants under heat stress, whereas the combined GA 3 and Si-treated plants exhibited significant down-regulation ( Figure 6B,C).

Discussion
Exposure to high temperature limits plant growth and productivity by hampering morphological, biochemical, and physiological processes, in addition to favoring oxidative damage. In the current study, we observed the detrimental impact of high temperature on date palm seedlings, caused by the degradation of plant growth attributes, such as height, biomass, and chlorophyll (F) Heat shock factor protein HSF30-like. Total RNA was extracted from date palm seedlings grown under normal and heat stress conditions with/without exogenously applied silicon (Si) and gibberellic acid (GA 3 ) and their combination. Transcript levels were measured by real-time qPCR. Actin was used as an internal control. Bars represent mean (of four replicates) ± standard error. Different letters indicate the values are significantly different (p < 0.05). Means were analyzed for finding the significant differences among treatments by using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT).
The heat shock transcription factors are well known to be involved in the activation of stress-responsive genes for overcoming abiotic stresses, including heat-induced stress. In the current study, we analyzed the transcript accumulation of the heat stress transcription factor A−5−like gene (HSTF-A5), which exhibited the significant up-regulation in the combined GA 3 and Si-treated plants and significantly lowered expression in non-treated plants under the control condition. The relative expression of HSTF-A5 was significantly increased under heat stress with all treatments when compared to the control condition. However, the transcript accumulation of HSTF-A5 in non-treated plants was significantly lowered by 1.7, 1.16, and 1.19 times as compared to those of the combined treatment plants, sole GA 3 -treated plant, and sole Si-treated plants, respectively ( Figure 6D). Similarly, the heat stress transcription factors A−3 (HSTF-A3) was significantly up-regulated under control conditions in the combined GA 3 and Si-treated plants as well as in sole Si-treated plants, while the non-treated plants and sole GA 3 -treated plants equally exhibited the least expression ( Figure 6E). Under heat stress, the HSTF-A3 expression level was enhanced in all treatments, while the combined GA 3 and Si-treated plants demonstrated significantly higher expression, followed by the sole GA 3 -treated plants, sole Si-treated plants, and non-treated plants, respectively. In connection to this, the relative expression of HSF30 was significantly higher in the sole GA 3 -treated plants under the control condition ( Figure 6F). However, under stress condition, GA 3 and Si-treated plants displayed significantly higher expression, followed by sole Si-treated, sole GA 3 -treated, and non-treated plants.

Discussion
Exposure to high temperature limits plant growth and productivity by hampering morphological, biochemical, and physiological processes, in addition to favoring oxidative damage. In the current study, we observed the detrimental impact of high temperature on date palm seedlings, caused by the degradation of plant growth attributes, such as height, biomass, and chlorophyll content. The impact of exogenous application of GA 3 was more efficient in mitigating high temperature stress in date palm by significantly improving plant height and fresh, dry biomass weight when compared to Si application. However, the application of GA 3 coupled with Si markedly improved plant growth attributes and significantly minimized the adverse effects of high temperature when compared to their individual application. Such enhancement in the growth attributes of date palm seedlings under high temperature may be correlated with the combined effect of GA 3 and Si on positive regulation of chlorophyll content (a and b) and carotenoids augmentation under high temperature. The water status of the plant is considered to be vital under high temperature conditions, as the loss of water content in plant tissues is induced by high temperature stress and subsequently leads to reduced plant growth [32]. In the current study, GA 3 -treated plants exhibited higher relative water status when compared to Si-treated plants. However, their combined application significantly boosted the water status to nearly the same level as that of the control plants. This suggests that combined application of GA 3 and Si mitigated the adverse effects of high temperature; therefore, the plant leaves were able to hold higher water content for better growth and development. Previously, Luyckx, et al. [33] and Doaigey, et al. [34] reported that the application of silicon and GA 3 to date plam can lead to the to the development of a cuticle double layer under the leaf epidermis, which subsequently prevents water loss via transpiration under stress conditions. Moreover, the application of GA 3 and Si to plants is also reported for improving relative water content to induce better growth under hostile conditions [33,34].
The disruption of chlorophyll and the inhibition of photosynthetic activity due to high temperature stress can lead to the generation of a variety of ROS in the chloroplast [35]. The current findings indicated that the sole application of Si markedly alleviated heat-induced oxidative stress as compared to sole application of GA 3 . However, the combined application of GA 3 and Si significantly reduced the level of O 2 •− and that of MDA, which is known as the end product of lipid peroxidation in date palm, suggesting a strong protective role for their combined application against oxidative stress. The significant mitigation of O 2 •− and MDA content can be ascribed to the stress inhibitory effects of combined Si and GA 3 application, resulting in the upregulation of antioxidant defense system to encounter oxidative stress [36,37]. Plants have evolved a system of enzymatic and non-enzymatic antioxidants for managing ROS and thereby preventing oxidative stress damage. However, longer exposure and greater severity of heat stress have devastating effects on the antioxidant defense system of plants. On the contrary, Si application triggered antioxidant activities in date palm, while the addition of GA 3 in the presence of Si further elevated the antioxidative activities (CAT, PPO, POD, and APX). Such enhancement of enzymatic antioxidants can be linked to the scavenging of ROS and, therefore, a lower lipid peroxidation (MDA) and O 2 •− level was observed in combined Si and GA 3 -treated plants under stress conditions. This suggests that the combined application of Si and GA 3 to date palm is more efficient at imparting thermotolerance to date palm by augmenting their antioxidant defense system. Moreover, along with the up-regulation of APX activity, the transcription level of GPX2 was also significantly enhanced by the combined application of Si and GA 3 . This indicates that combined Si and GA 3 treatment simultaneously activated APX and GPX2 in preparation for the ROS encounter by suppressing toxic H 2 O 2 levels in plants under heat stress [38].
Heat stress can also lead to the disruption of GAPDH activity, which is crucial for carbon flux in the Calvin cycle and for regulating the carbon assimilation and photosynthesis rates. GAPDH aids in converting glycerate-3-phosphate to glyceraldehyde-3-phosphate through interactions with NADPH. This inhibits the ROS-induced breakdown of the photosystem II repair cycle by reducing ROS production, subsequently maintaining photosynthetic efficiency [39,40]. In the current study, the transcript accumulation of the NADP-dependent GAPDH gene was markedly reduced in only the heat stressed plants, whereas the combined application of Si and GA 3 led to significant expression of the gene under heat stress. The significant transcript accumulation of GAPDH implies that GA 3 and Si collectively provided ample energy to the date palm under heat stress for regulating ROS-induced metabolic responses for cellular adjustment by routing carbon away from glycerol and subsequently leading to glycolysis and ATP formation [41]. Moreover, the simultaneous co-expression of Cyt-Cu/Zn SOD and CAT indicate that the combined application of Si and GA3 efficiently enhanced the capability of date palm to cope with the heat-induced oxidative stress.
Plant hormone metabolism is closely interlinked with the plant abiotic stress coping potential. The findings from the current study illustrated that the endogenous ABA content of date palm is enhanced in response to heat stress. However, the combined application of Si and GA 3 drastically lowered the level of ABA accumulation in date palm under heat stress. The lower regulation of ABA following the combined application of Si and GA 3 might be correlated to the beneficial impact of GA 3 , which leads to better photosynthetic activity, stomatal regulation, and gas exchange, as well as the capability of Si to boost the antioxidant defense system. The synergist effects result in less ROS accumulation and a corresponding decrease in the level of ABA in the date palm. In line with this theory, the transcript levels of the ABA signaling-related genes (PYL4, PYL8, and PYR1) were down-regulated in the combined Si and GA 3 -treated plants under heat stress; therefore, a lower accumulation of ABA was recorded. These findings further highlight the effective role of the combined application of Si and GA 3 in ameliorating heat-induced stress in date palm. SA is a signaling molecule that is known to mitigate the adverse effects of heat stress in plants by regulating various physiological and biochemical processes to provide both basal and acquired thermotolerance [42]. In the current study, the combined application of Si and GA 3 resulted in higher accumulation of SA in date palm and successfully alleviated the adverse effects of heat stress. The higher accumulation of endogenous SA is reported to activate proline biosynthesis for the augmentation of osmotic potential, enabling plants to uptake water, and triggers antioxidant enzymes under heat stress to impart thermotolerance to plants [43]. The combined treatment of Si and GA 3 resulted in an antagonistic interaction of SA with ABA, which suggests that ABA signaling alleviates heat stress-induced leaf senescence, chlorophyll degradation, and redox modulation [44]. Plant heat shock transcription factors are known to participate in heat stress-related hormonal signaling pathways, such as SA. The activation of heat shock transcription factor genes, such as Hsf3, can boost plant defense against hostile conditions via the modulation of endogenous accumulation and signaling [42,45]. Therefore, the higher accumulation of endogenous SA in date palm under heat stress could be ascribed to the significant transcript accumulation of heat shock transcription factors Hsf3, HsfA5, and Hsf30 via the interactive effects of exogenous Si and GA 3 application, providing tolerance against heat stress.
Heat shock transcription factors are known to regulate the expression of HSPs to maintain homeostasis in plants against different stresses, including heat and chemical stresses [46]. The expression of HSPs by Hsfs genes is regulated via their interactions with a palindromic binding motif in the promoter region of heat-responsive genes, such as heat shock elements to counteract heat stress-induced ROS [47]. We found that the combined application of Si and GA 3 significantly triggered transcript accumulation of HsfA3, HsfA5, and Hsf30, suggesting that they play a heat stress ameliorative role in date palm by imparting protection from heat-induced ROS generation and boosting the antioxidative response. Therefore, the enhancement of the antioxidative activities (CAT, POD, PPO, and APX) and the expression of the corresponding genes can be correlated with the significant activation of Hsfs genes by the combined application of Si and GA3 in response to heat-induced stress. However, further transcriptomic-based studies are required in order to uncover the underlying mechanism behind the heat shock transcription factors of date palm by investigating the interactive effects of GA 3 and Si under heat stress.

Conclusions
In conclusion, the combined application of Si and GA 3 to the date palm successfully mitigated the adverse effects of heat stress in plants, directly improved plant growth and development, and induced physiological and biochemical modulation. Taken together, our data revealed that, when compared to sole application, combined application of GA 3 and Si significantly protected date palm plants from heat-induced stress. The effects of exogenous GA and Si significantly activated the heat shock transcription factors genes, particularly HsfA3, and the anti-oxidative system of date palm by up-regulating the GPX2, Cyt-Cu/Zn SOD, and CAT expression levels. Moreover, the interactive effects of GA and Si influenced the cross-talk between stress-related endogenous hormones (ABA and SA) by reducing endogenous ABA accumulation and down-regulating ABA signaling-related genes (PYL4, PYL8, and PYR1), which subsequently induced the antagonistic effects of SA. Therefore, the combined application of Si and GA 3 is efficient in enhancing date palm growth and development under heat stress condition.