Potential objectives for gibberellic acid and paclobutrazol under salt stress in sweet sorghum (Sorghum bicolor [L.] Moench cv. Sofra)

Abstract

The phytohormones are important in plant adaptation to abiotic and biotic stresses by facilitating a wide range of adaptive responses. Application of gibberellic acid (GA₃) and paclobutrazol (PBZ) as GA₃ inhibitors have been shown to affect salinity tolerance through modulating phytohormones. The aim of this study was to find out the potential objectives for GA₃ and PBZ as affected by salinity through altering the phytohormones and biochemical parameters in sweet sorghum. Following seed germination, seedlings were cultured in Hoagland nutrient solution containing NaCl supplemented with GA₃ and PBZ for 12 days. The results were analyzed by principal component analysis to identify the best target(s) for salinity, GA₃, and PBZ in sweet sorghum. Paclobutrazol associated with salt improved root/shoot length, carotenoid, and total chlorophyll by modulating cytokinin (CK)/GA₃, indole acetic acid (IAA)/GA₃, and total polyamines/GA₃ ratios. Gibberellic acid-treated plants not exposed to salinity treatments notably improved phytohormones content such as cytokinin, auxin, abscisic acid (ABA), and polyamines resulting in increased stem growth. Moreover, the main objectives of GA₃ were ABA, spermidine, and ABA/GA₃ ratio in response to salinity. Though GA₃ and PBZ have different roles against salt stress, ABA/GA₃ ratio was a similar target of GA₃ and PBZ. This work suggests that altered levels of GA₃ resulting from PBZ- and GA₃-treated plants cause different allocation patterns in sweet sorghum by regulation of CK/GA₃, IAA/GA₃, and total polyamines/GA₃ ratio. Also, accumulation chlorophyll pigments, carotenoids, and water soluble carbohydrates of sorghum plants under salinity regulated by total polyamines/GA₃ and ABA/GA₃ ratios positively correlated with PBZ application.

Introduction

Salinity is a major problem in agriculture and crop production. High concentration of toxic ions creates salty soil regions around the world. Sodium and chloride are main ions in most of the salty areas, and NaCl is the most damaging compound for plant growth and development. The negative effects of salinity on plant growth and leaf senescence are related to osmotic stress and ion toxicity [1,2,3].

Sweet sorghum (Sorghum bicolor) has usually been grown in areas with relatively low rainfall, high temperatures, and saline soils. This plant, as a moderately salt tolerant crop, has been adapted to water limitation and is more tolerant to salinity at germination stage than in the later stages of growth [4, 5]. Moreover, sorghum is an important food and bioenergy source as well as a model plant for studying the mechanisms of drought and salt tolerance in cereals [3, 6]. A number of methods have been developed for screening salt tolerance in plants because majority of physiological processes (e.g., germination, photosynthesis, biomass production, and chlorophyll content) are highly sensitive to salt stress [3]. Plants can adjust their growth and development in response to salt stress by signaling molecules. Phytohormones are active members of signal cascade involved in the induction of salt stress responses [7]. Several phytohormones such as abscisic acid (ABA), cytokinin (CK), auxin (Aux), polyamines, and gibberellic acid (GA₃) have been used to reduce the negative effects of salt stress on seed germination, plant growth, and fruit setting [8]. Recently, GA₃ has been considered as a growth regulator to ameliorate salt stress. This hormone has been used to increase wheat and rice growth under salinity condition [8]. The function of phytohormones is complicated due to their interactions with other plant growth regulators. In case of GA₃ action, no specific target of GA-induced genes has been identified which may propose interaction with other hormones plays a key role in response to stress [7]. How gibberellins treatment may induce salt tolerance in plants has not been clearly answered yet [9]. It has been shown that GA₃ and ABA play antagonistic roles in many plants’ developmental processes such as germination, growth, and flowering. Moreover, the antagonistic effects of GA₃ and CK on some growth parameters have been previously reported [7]. In contrast, it has been indicated that GA₃ and auxin frequently act synergistically, especially in stem elongation [7]. Recently, it was reported that auxin is positively associated with GA₃ by promoting degradation of DELLA protein [7]. It has also been known that, salt stress reduces endogenous cytokinins and improves ABA and polyamines levels [7], especially spermine in sorghum [10]. Studies have shown that polyamines have positive effects in plants facing environmental stresses [10]. Although polyamines are essential for cell division and root initiation [10], little is known about signal transduction of polyamines and their interaction with GA₃. Triazoles such as paclobutrazol (PBZ) affect the isoprenoid pathway and alter the levels of certain plant hormones by inhibiting gibberellin synthesis, reducing ethylene evolution and increasing CK and ABA levels [11]. Triazole compounds also protect plants against various stresses, and they have been characterized as plant multi-protectants [12]. Application of PBZ has been reported to mitigate salt stress in some plant species by reducing the level of GA₃ via inhibition of monooxygenases which catalyze oxidative steps from ent-kaurene to ent-kaurenoic [13]. However, the actual mechanism of stress tolerance by PBZ remains unclear. It seems GA₃ and PBZ may increase salt tolerance by different mechanisms and they may have different physiological, biochemical, and hormonal objectives. To the best of our knowledge, no comparative study has been reported in plants using GA₃ and PBZ under salt stress. In the current study, we aim to understand: (1) How GA₃ and its inhibitor (PBZ) interact with other plant hormones under salt stress conditions; and (2) among carbohydrates, photosynthetic pigments, polyamines, and phytohormones, what are the best objectives for GA₃ and PBZ to ameliorate the negative effects of salt stress in sweet sorghum as a model plant.

Materials and methods

Hydroponic culture

Fresh seeds of sweet sorghum (Sorghum bicolor [L.] Moench cv. Sofra) were supplied from the seed stock University of Isfahan, Iran. Seeds were surface sterilized for 1–2 min in 95% ethanol, followed by 15% sodium hypochlorite (v/v) for 20 min. Seeds were then washed three times using sterilized distilled water and transferred into the plastic pots containing sterile perlite and watered with Hoagland nutrient solution. Pots were then kept in the culture room at 25 ± 1 °C with 16/8 h photoperiod under 100 µmol m⁻² s⁻¹ light intensity derived from white fluorescent lamps. After germination, five seedlings were transferred to the new pots and each pot was moved inside a 200 mL container filled with Hoagland nutrient solution [14]. After 4 days, the nutrient solution of pots was replaced by fresh Hoagland medium containing 0, 100, 150, 200, and 250 mM NaCl, supplemented with either PBZ (17 µM) or GA₃ (17 µM). Pots with neither salt nor PBZ or GA₃ were used as control. After 12 days, treated plants were harvested and their biochemical and physiological parameters were measured as described below.

Growth parameters

A number of leaves, stem and root lengths as well as fresh weight (FW) of plants were measured after 12 days. To determine the dry weight, samples were dried in a 70 °C oven for 48 h. Shoot water content was measured by calculating the difference between the fresh and dry weights of the plants.

Photosynthetic pigments

For photosynthetic pigments’ measurements, leaves (0.1 g FW) of plants were ground in 80% cold acetone and centrifuged at 5000 g for 10 min. The absorbance of the purified chlorophyll samples was measured at 470, 646, and 663 nm (Shimadzu UV-160, Kyoto, Japan). Chlorophyll and carotenoid contents were calculated according to Lichtenthaler and Wellburn [15].

Carbohydrate content

Water soluble carbohydrates (WSC) were determined based on the phenol–sulfuric acid method described by Dubois et al. [16]. To prepare carbohydrate extract, 10 mg of dry leaf was homogenized with 10 mL 80% ethanol. The extracts were centrifuged at 6000 rpm, and 0.5 mL of supernatant was mixed with 0.5 mL of 5% phenol and 2.5 mL 96% sulfuric acid. The samples were then vortexed gently for 30 min, and soluble carbohydrates were measured at 490 nm.

Measurement of phytohormones using HPLC

The level of indole acetic acid, cytokinin, and gibberellic acid was determined by method described below. Extraction: Approximately 2 g of tissue was ground in 30 mL cold 80% methanol and homogenized in darkness at 4 °C, then centrifuged at 5000 rpm at 4 °C for 15 min. The supernatant was filtered through 0.45 µm Whatman™ filter to remove suspended particles [17,18,19]. The filtered supernatant was treated sequentially with methanol–acetic acid (100:1, v/v), methanol–water–acetic acid (50:50:1, v/v/v), methanol–water–acetic acid (30:70:1, v/v/v), and finally with water before transferring 100 mL of filtrate to 10 C18 SPE columns (J.T. Baker, Phillipsburg, NJ, USA; 500 mg, 3 mL). The columns were washed with 10 mL water adjusted to pH 3 with acetic acid (acidified water). The phytohormones were eluted with 5 mL ethanol–water–acetic acid (80:20:1, v/v/v). The eluate was evaporated at room temperature under vacuum and finally, the evaporated eluate was dissolved in 1 mL methanol [17]. Prior to HPLC analysis, this reconstituted eluate was filtered using a 0.45 µm Whatman™ glass microfiber filter.

Evaluation by HPLC: Analysis of phytohormones was performed based on Ge et al. [20] and Ma et al. [17], using a HPLC system (Unicam, Crystal 200, Cambridgeshire, England) linked simultaneously to a photodiode array (PDA) system. Ten µL of extract was injected into a C18 reverse phase column (Zorbax SB-C18 100A°, 3.5 µm, 150 mm length, 2.1 mm diameter). The column thermostat was set at 25 °C. The initial HPLC column running conditions was initialized isocratically with methanol–formic acid buffer (10:90, v/v) for 5 min, then a linear gradient to methanol–formic acid buffer (30:70, v/v) in 5 min, which was later maintained isocratically for 10 min, before switching to a linear gradient toward a methanol–formic acid buffer (45:55, v/v) in 35 min and finally isocratically at methanol–formic acid buffer (45:55, v/v) for 15 min. Under the separation conditions, all compounds were successfully separated within 45 min. After each analysis, the column was washed with 95:5 methanol–formic acid buffer for 5 min. Then formic acid buffer–methanol 90:10 for 30 min was used to re-equilibrate. The peak area of the standard was considered for determination of sample concentration. Quantification of phytohormones was calculated based on Ge et al. [20], Ma et al. [17], and Tang et al. [19] methods using the peak areas with identified amounts of IAA, CK, and GA3.

For extraction and evaluation of ABA, 1 g of fresh leaves were ground in 10 mL of 80% methanol including 0.01 g of ascorbic acid and 0.01 g polyvinylpyrrolidone (PVP). The homogenate was stirred overnight at 4 °C. After centrifugation at 4000 g for 15 min, the supernatant was recovered and adjusted to pH 8.0. The aqueous methanol was evaporated under reduced pressure at 35 °C. The residue was dissolved in 5 mL of water. These samples were frozen and thawed for three cycles. After centrifugation at 4000 g for 15 min, the supernatant was recovered and adjusted to pH 2.5 and 10 mL ethyl acetate was added to collect free ABA in ethyl acetate. The ethyl acetate was then evaporated. The resulting dried precipitate was dissolved in 1 mL of 3% methanol containing 0.1 M acetic acid and was filtered through a 0.45 mm membrane filter [21]. Then, 10 µL of extract was injected into a C18 reverse phase column (4.6 × 250 mm Diamonsic C18, 5 μm). It was eluted with a linear gradient of methanol (3–97%) containing 0.01% acetic acid at a flow rate of 4 mL/min. The detection was run at 260 nm with a diode array detector. Quantification was obtained using the peak areas with known amounts of ABA (5–50 ng/ml) based on Li et al. [21].

The extraction and evaluation of polyamines were performed based on Walter and Geuns [22].

Statistical analysis

All experiments were conducted with three replicates, and the differences between treatments with different variables were tested by general linear model followed by Duncan test. Differences were considered as significant at p < 0.05. To determine the best target for PBZ and GA₃ and understand their roles under salinity, Principal component analysis (PCA) was applied, and the results were shown. Principal component analysis is a popular method to reduce a set of observations of possibly correlated variables into a set of principal components. In other words, each parameter in PCA analysis receives component score based on the quantified data which contain the same information but orthogonal to each other.

Results

Growth parameters

Increasing NaCl concentration negatively affected the fresh weight, dry weight, stem length, root length, and water content of sweet sorghum (Table 1). The results indicated that these characteristics were significantly altered by GA₃ and PBZ treatments. As a general pattern, GA₃ and PBZ treatments affected fresh weight, dry weight, stem length, and root length. The lengths of stem and roots as well as the fresh and dry weight of stems were significantly improved by GA₃ application. However, the impact of GA₃ was slightly reduced by increasing salinity in the medium. The stem length increased significantly by GA₃ either with or without salt as compared with control (no PBZ) and PBZ treatments. Though PBZ treatments reduced stem length and fresh and dry weights of stem, these characteristics did not change significantly in the medium supplemented with salt. However, the root length and fresh and dry weights of roots were significantly increased by PBZ, either with or without NaCl, compared to GA₃ treatments (Table 1). In our experiments, root/shoot length ratio and root/shoot dry weight ratio in sweet sorghum improved by increasing salinity. Looking at both ratios, it seemed GA₃ has a more negative effect compared to PBZ. Under salinity, the water content (WC) of sweet sorghum decreased significantly by increasing salinity for both GA₃ and PBZ treatments. Gibberellic acid increased WC in non-saline medium, but PBZ decreased it significantly compared to the control. Under salt stress, the reduction in WC in GA₃-treated plants was higher than PBZ-treated plants. For instance, the water content of plants treated with GA₃ and PBZ in 200 mM NaCl was reduced by 54% and 28%, respectively, compared to plants grown in the medium without salt.

Table 1 Effects of GA₃ and PBZ treatments on some growth parameters of sweet sorghum under salt stress

Photosynthetic Pigments

Our results indicated that chlorophyll a, chlorophyll b, the total chlorophyll as well as the carotenoid contents were increased significantly by higher salinity. Treated plants with PBZ and GA₃ under 200 mM salt increased total chlorophyll about 21, and 43%, respectively, compared with untreated plants. When 100 mM NaCl was added to the medium, chlorophyll content of plants treated with GA₃ decreased compared to PBZ-treated plants either with or without salt. In contrast, PBZ improved chlorophyll and carotenoid contents in response to 100 mM salt compared to the control and GA₃ (Table 2).

Table 2 Effect of GA₃ and PBZ on chlorophyll, carotenoid, and water soluble carbohydrate (WSC) of sweet sorghum under salinity

Water soluble carbohydrates

The level of water soluble carbohydrates (WSC) in the leaves of sweet sorghum was enhanced by increasing salinity, especially in 200 mM NaCl. Application of PBZ without salt improved WSC dramatically (around 100%) compared to control. However, the impact of PBZ was reduced slightly by increasing salinity in the medium. The highest WSC content was recorded in the medium containing PBZ and 200 mM salt, which was not significantly different from GA₃-treated plants at 200 mM NaCl (Table 2).

Polyamines

The results of GA₃, PBZ, and salinity on endogenous concentration of putrescine (PUT) are shown in Fig. 1A. Using GA₃ as a single (without salinity) treatment increased PUT content 70% more than untreated plants and 100% higher than PBZ treatment. The concentration of PUT in GA₃-treated plants decreased markedly by salinity; down to 60% in 200 mM salt. In contrast, PBZ as well as untreated plants with hormones increased PUT up to 100 mM NaCl and then decreased PUT to the lowest amount in 200 mM NaCl (Fig. 1A). Gibberellic acid and PBZ in combination with salt stress had different impact on spermidine content. The highest spermidine (SPD) content was observed in GA₃ treated plants. However, when NaCl was added to the medium, plants responded differently. For instance, in 100 mM NaCl spermidine concentration decreased in plants irrespective of the treatment (GA₃, PBZ, or control), while in 200 mM NaCl spermidine concentration increased (Fig. 1B). The overall pattern of spermine (SPM) content is shown in Fig. 1C. In this study, under 100 mM salt stress spermine content decreased, but it increased in 200 mM NaCl by PBZ. Salt stress without exogenous PBZ or GA₃ application increased spermine only in 200 mM NaCl. A similar pattern was observed when GA₃ was used.

Fig. 1

The effect of GA3 and PBZ treatments on leaf putrescine (A), spermidine (B), spermine (C), and total polyamines/GA₃ ratio (D) of sweet sorghum under salinity. The values are means of three replicates, ± standard error. Common letters are not significant (p < 0.05)

Because GA₃ content was affected by PBZ and GA₃ treatments, we were interested in knowing how and to what extend GA₃ variation may affect polyamine content. Therefore, we calculated the ratio of total polyamine/GA₃ which showed that the total polyamine/GA₃ in PBZ-treated plants was higher than GA₃ treated plants and untreated plants (control). This ratio was increased in 100 and 200 mM NaCl in PBZ and control plants, whereas in GA₃-treated plants it was increased only in 200 mM NaCl (Fig. 1D).

Phytohormones concentration

To understand the interaction of treatments including GA₃, PBZ, and NaCl, the endogenous concentrations of some key phytohormones were measured. As a general pattern, GA₃ content in plants decreased by increasing salinity. Our results indicated that the level of GA₃ content decreased significantly in 0, 100, and 200 mM NaCl, either with or without GA₃ or PBZ. Although, the plants treated by GA₃ showed higher GA₃ content than PBZ-treated plants, the reduction in GA₃ content was higher in GA₃-treated plants than PBZ in 200 mM NaCl. Our results show that GA₃ in 200 mM salt reduced GA₃ content by about 73%, but PBZ reduced GA₃ content by about 48% (Fig. 2A).

Fig. 2

The effect of GA₃ and PBZ on endogenous concentrations of leaf GA₃ (A), IAA (B), zeatin (C), and ABA (D) in sweet sorghum under salinity. The values are means of three replicates, ± standard error. Common letters are not significant (p < 0.05)

The level of indoleacetic acid (IAA) was much higher in GA₃-treated plants compared to PBZ under salt stress (Fig. 2B). The levels of IAA decreased in plants with or without GA₃ and PBZ as concentration of NaCl increased in the medium. The amount of IAA in PBZ-treated plants was significantly lower than GA₃-treated and -untreated plants. While large reduction in IAA concentration was observed by PBZ, GA₃, and untreated plants at 200 mM NaCl, these differences were not significant (Fig. 2B).

In GA₃-treated plants zeatin, concentration with or without NaCl application was the highest compared to untreated and PBZ-treated plants. Generally, zeatin content increased up to 100 mM NaCl, but significantly declined when salt concentration increased to 200 mM NaCl. Additionally, PBZ-treated plants had the lowest amount of zeatin, especially at 200 mM NaCl (Fig. 2C).

On the other hand, the concentration of ABA increased as the concentration of NaCl increased (Fig. 2D). The highest amount of ABA was observed in GA₃-treated plants with 200 mM NaCl.

To understand the role of different phytohormones in response to salt stress, we calculated their ratio to GA₃. In this regard, the ABA/GA₃ ratio (Fig. 3A) increased sharply in 200 mM NaCl under GA₃ or PBZ application. Interestingly, in severe salt stress (200 mM), this ratio was much higher in GA₃-treated plants than PBZ-treated ones.

Fig. 3

The effect of GA₃ and PBZ treatments on ABA/GA₃ (A), IAA/GA₃ (B), and zeatin/GA₃ (C) ratios, in leaves of sweet sorghum under salinity. The values are means of three replicates, ± standard error. Common letters are not significant (p < 0.05)

The IAA/GA₃ ratio in PBZ treatments with or without salt was higher than control and GA₃-treated plants. In GA₃-treated or control plants, this ratio was high when plants were exposed to 200 mM salt concentration. In PBZ-treated plants the IAA/GA₃ ratio declined under 100 mM NaCl and then increased when the salt concentration was increased to 200 mM (Fig. 3B).

Generally, the ratio of CK (in this case zeatin) to GA₃ was the highest in PBZ-treated plants irrespective of whether or not the plants were exposed to salinity treatment. When 100 mM NaCl was added to the medium, this ratio decreased slightly and remained constant in 200 mM salt concentration. In contrast, GA₃ treatment in response to salinity increased the CK/GA₃ ratio slightly (Fig. 3C).

Identification of NaCl, GA₃, and PBZ objectives

To identify the best objectives for salinity, GA₃ and PBZ in sweet sorghum, principal component analysis (PCA) was carried out. The principal components 1 and 2 (PCA1 and PCA2, respectively) obtained from the growth parameters as well as physiological and phytohormones parameters subjected to salinity are shown in Fig. 4A. Gibberellic acid and PBZ, as PCA1, described 61.1% and PCA2 described 20.5% (the sum equals to 81.6%) of all measured parameters. In fact, PCA visualized the main objectives of GA₃ and PBZ on sweet sorghum, under salt stress (Fig. 4A, B).

Fig. 4

Result of biplot Principal Components 1 and 2 analysis obtained from phytohormones and physiological parameters in sweet sorghum subjected to PBZ, salinity, GA₃ (A), and the main objectives (B)

The PCA results (Fig. 4) clearly indicated that growth parameters, that is physiological and phytohormones in this study, might be divided into three separate groups. Root length was a group associated with PBZ treatment (Fig. 4A). Whereas, root/shoot length, root/shoot dry weight, root fresh and dry weights, total chlorophyll, carotenoids, WSC, CK/GA₃, and IAA/GA₃ consisted a group linked with NaCl and PBZ + salt (Fig. 4A). Finally, auxin, cytokinin, PUT, SPD, ABA, water content (WC), shoot dry and fresh weights, and shoot length were found on the left side of the biplot strongly related to GA₃ (Fig. 4A). Figure 4B summarizes a simple illustration of the main objectives of the above-mentioned characteristics for GA₃ and PBZ under salinity. We found that the SPM, polyamines/GA₃, and ABA/GA₃ were the main objectives of salinity regulating total chlorophyll, carotenoid, and WS contents in sweet sorghum. In conclusion, the main objectives of PBZ under salinity were ABA/GA₃, CK/GA₃, and IAA/GA₃, while the levels of SPD, ABA, and ABA/GA₃ were the main objectives of GA₃ under salinity (Fig. 4B).

Discussion

As a general response to salt stress, plant growth is affected negatively by a variety of physiological changes, such as osmotic shock, ion toxicity, and nutritional imbalance [23]. This study showed that, although stem length, fresh and dry weight, and water content decreased the adverse effect of NaCl by GA₃ treatment, the impact of GA₃ was reduced slightly with rising salt level. It has been known that, GA₃ enhances plant growth by promoting cell division and elongation [24] under salinity, for instance in arabidopsis [25], wheat [26], and rice [8]. We found that application of GA₃ increased the levels of CK and IAA in leaves, perhaps due to the beneficial effects of hormonal homeostasis of GA₃ and its ion uptake and possibly the photosynthesis process in the salt-stressed plants [8]. Salt stress may reduce carbon movement to shoot [27], yet increase carbon transferring to roots. This will consequently increase the root/shoot ratio, which might be an adaptive response [8]. Previously, it was indicated that the root/shoot ratio was increased in PBZ-treated seedling of rice [28] and the adverse effect was observed by GA₃ [28]. We observed that, PBZ increased root length and subsequently resulted in improved water content under salt stress. The level of CK in sorghum was decreased in PBZ-treated plant. Interestingly, PCA showed that the root/shoot length ratio was positively correlated with CK/GA₃ and IAA/GA₃ ratios. In our study, shoot fresh weight of sorghum was correlated with changes in plant height as reported by Yim et al. [28]. However, we showed that shoot fresh weight was negatively associated with total polyamines/GA₃ ratio. Moreover, various studies have shown that high polyamines level enhances cell division, root initiation, and early growth [29]. Therefore, altered level of GA₃ resulting from PBZ and GA₃ treatment in sorghum plants leads to different allocation patterns. Consequently, it seems that, root/shoot ratio is regulated by CK/GA₃, IAA/GA₃, and total polyamines/GA₃ ratios.

Abiotic stress normally changes phytohormones level and photosynthesis process. It has been reported that chlorophyll content is decreased in salt susceptible plants such as tomato and pea [30], but it is increased or unchanged in relatively salt tolerant plants such as pearl millet, sugar beet, and cabbage [31]. Our data indicated that chlorophyll and carotenoid contents were increased significantly with salinity. This finding might be due to the nature of sorghum variety used in this study, which is a relatively salt tolerant sweet sorghum cultivar Sofra. However, the chlorophyll content in leaves of some varieties of sorghum reduced as a result of higher NaCl levels [32], indicating a genotype dependent response of sorghum cultivars to salinity [33]. It should be kept in mind that, chlorophyll content as a photosynthesis response of sorghum is also dependent on NaCl concentration. The positive effect of PBZ on chlorophyll accumulation was reported in some plant species [34], and a similar observation was seen in our study. This finding might be either due to decrease in leaf area by PBZ and condensation of chlorophyll [35] or increase in cytokinin levels in leaves [13]. In contrast, the decrease in chlorophyll resulted by GA₃ treatment might be due to the expansion of the leaves and dilution of chlorophyll [36]. It has also been shown that polyamines are involved in the delayed loss of chlorophyll [37]. Furthermore, the result of PCA analysis indicated that, the total chlorophyll and carotenoids contents were strongly correlated with the total polyamines/GA₃ ratio in sweet sorghum. Since the total amount of polyamines/GA₃ ratio was significantly enhanced by PBZ and salinity, it might be concluded that PBZ and salinity regulated chlorophyll and carotenoid accumulation by total polyamines/GA₃ ratio.

Some variations have been reported in soluble carbohydrates content in response to salinity in different varieties of sorghum [3, 38, 39]. For instance, salinity decreased the amount of glucose and fructose in sorghum cv. Keller, while increased them in cv. Sofra [38]. In this study, WSC was increased in response to salinity, but PBZ was effective only under non-salinity condition. In wheat, however, PBZ increased WSC in the leaves under salinity. On the other hand, in sorghum plants treated with salinity supplemented with either GA₃ or PBZ, WSC content correlated positively with the total amount of polyamines/GA₃ and ABA/GA₃ ratios. Interestingly, ABA/GA₃ is a common target of GA₃ and PBZ under salinity. Additionally, the increase in the total polyamines/GA₃ ratio led to improved chlorophyll content and consequently resulted in an increase in WSC in sorghum plants treated with PBZ.

Our results indicated that putrescine (PUT) levels decreased, while spermine (SPM) levels increased by salinity. Accumulation of SPM by raising salinity was reported by Maiale et al. [10] in rice. Chai et al. [40] provided strong evidence indicating that SPM improved growth of sweet sorghum under salinity. It can be suggested that SPM may act as a compatible solute, like proline, for homeostasis of the cells under NaCl treatment. Iqbal and Ashraf [26] reported that GA₃ affects plant ability to synthesize polyamines based on the concentration of GA₃ under salinity. Triazole compounds, including PBZ, reduce ethylene formation by blocking amino cyclopropane carboxylic acid (ACC) oxidase leading to an increase in polyamines level [13]. Low levels of putrescine detected in our study might be linked to the ABA biosynthesis and the inhibition of gibberellic acid biosynthesis [41]. Furthermore, accumulation of SPD in sweet sorghum is linked with ABA and is negatively correlated with IAA/GA₃ and CK/GA₃ ratios. Therefore, it seems that the reduction in GA₃ levels by PBZ together with the GA₃ addition to the medium regulates PUT concentration under salinity [26]. Seemingly, the alternation of PUT content might be modulated by ABA, IAA/GA, and CK/GA levels in sweet sorghum.

Application of paclobutrazol reduced GA₃ content in sorghum plants, possibly via the inhibition of monooxygenases that catalyze oxidative steps from ent-kaurene to ent-kaurenoic [13]. Accumulation of DELLA protein in GA signaling pathway or GA degradation in arabidopsis plants might be other reasons for sorghum plants lack of response to GA₃ as reported [24]. Interestingly, an increased level of DELLA expression was observed when sorghum treated with exogenous PBZ [42]. Therefore, the difference in sorghum plants response to GA₃ and PBZ treatments under salinity might be related to hormonal hemostasis, GA₃ metabolism, and the stability of DELLA proteins.

It was reported that auxin influences GA biosynthesis and interacts positively with GA in plant signaling [7]. For instance, increasing GA₃ levels leads to significant increase in IAA content [43]. This effect may be due to either increased IAA biosynthesis or increase in auxin transport from apical meristem. Björklund et al. [43] showed that, increasing IAA content in Populus sp. treated with GA₃ was correlated with auxin transport from apical meristem. Also, it is shown that the application of GA₃ during salt stress in rice improves auxin content, which is similar to our findings [44]. There is conflicting reports on PBZ interaction with auxin in plants under stress conditions. Some reports indicated that triazoles such as PBZ have no significant impact on auxin levels [13], but other investigations have reported that the IAA levels reduced by PBZ under abiotic stress [45]. In the present study, severe salinity dramatically reduced IAA, CK, and some polyamines levels in sorghum, as was reported by Björklund et al. [43] and Shao et al. [46] in other plant species. Salinity increased IAA/GA₃ ratio, which was also markedly increased by PBZ, a GA₃ antagonist. Björklund et al. [43] suggested that IAA/GA₃ ratio is established by complex cross-talk and self-control mechanisms and possibly by the expression of genes involved in auxin transport and GA₃ metabolism.

Since GA₃ can induce salt tolerance in plants by increasing CK and IAA, it seems that sorghum is able to survive under salinity with less damage through changes to CK and IAA levels. Moreover, triazole compounds can stimulate CK accumulation [13]. We found that the root/shoot ratio in PBZ-treated sorghum increased in response to NaCl. The decrease in CK content under salt stress might be due to the movement of CK from roots to shoots.

ABA has a critical role in abiotic and biotic stress and is a key hormone in response to salinity stress [47]. The detected trend of ABA accumulation under salinity in this study was consistent with many other works showing that either an improved ABA biosynthesis or an enhanced translocation from the roots to the shoots may happen, after a particular stress threshold, in response to salinity [48]. The antagonistic roles between ABA and GA₃ are presumed for numerous plant developmental processes such as germination and flowering [7]. However, there are conflicting data about the effect of GA₃ on ABA level. Although pretreatments of wheat seeds with GA₃ reduced ABA accumulation in response to seawater [49], Iqbal and Ashraf [26] reported that GA₃ improved the levels of free ABA in leaves in salt tolerant wheat cultivars, while reverse trends were observed for the salt intolerant cultivars. The PCA results indicated that ABA/GA₃ was a common target of GA₃ and PBZ under salinity. Therefore, the ABA/GA₃ ratio plays a pivotal role in response to salinity, explaining the levels of ABA in GA₃ and PBZ treatments.

Finally, our findings indicate that GA₃ can reduce the negative effects of salt by increasing some phytohormones in moderate salt stress. However, the main objectives of GA₃ under salinity were ABA and SPD contents as well as ABA/GA₃ ratio. Though GA₃ and PBZ have different roles against salt stress, ABA/GA₃ ratio was a similar target of GA₃ and PBZ. This study suggests that altered levels of GA₃ resulting from PBZ- and GA₃-treated plants cause different allocation patterns in sweet sorghum by regulation of CK/GA₃, IAA/GA₃, and total polyamines/GA₃ ratio. Also, accumulation of chlorophyll pigments, carotenoids, and water soluble carbohydrates in sorghum plants under salinity, that is regulated by the total polyamines/GA₃ and ABA/GA₃ ratios, is positively correlated with PBZ application.