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. 2023 Oct 29;29(9):1339–1352. doi: 10.1007/s12298-023-01370-0

Sage leaf rock rose water extract: a bio-solution for enhancing the growth and salt stress resistance of sorghum plants

Zoulfa Roussi 1,✉, Abdelhamid Ennoury 1, Azzouz Krid 2, Mohamed Nhiri 1
PMCID: PMC10678872  PMID: 38024950

Abstract

Sorghum bicolor, a versatile cereal grain, holds significant agronomic importance globally and plays a crucial role in addressing food insecurity. However, salinity, a major abiotic stress, poses a threat to food production by reducing soil fertility and hindering plant growth and yield. In this study, we investigated the potential of Cistus salviifolius water extract (CSE) in mitigating salt stress in sorghum plants. Salt stress severely impacted plant growth, biomass, and chlorophyll production, and reduced indole-3-acetic acid (IAA) levels, which negatively affected plant development. Salt stress also led to the buildup of reactive oxygen species (ROS), hence, resulting in oxidative harm to sorghum plants and also affecting their carbon and nitrogen metabolism. On the other hand, CSE treatments increased IAA and chlorophyll content which promoted growth under stress. Furthermore, this extract exhibited strong ROS scavenging capacity and safeguarded plants against oxidative stress by enhancing the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase) and increasing the production of osmolytes. Additionally, CSE treatments enhanced the activities of carbon/nitrogen enzymes (phosphoenolpyruvate carboxylase, malate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and glutamine synthase), promoting energy synthesis and crop growth. This led to a significant increase in sorghum growth in salted soil with the highest rise recorded for 5 mg/L of CSE (an increase of 48.23% and 158.36% in length and weight compared to the salt control), which highlights this extract’s potential as a biostimulant to enhance crop tolerance to salinity and contribute to sustainable agriculture.

Keywords: cistus salviifolius, Salt stress, Antioxidant enzymes, Reactive oxygen species, Osmolytes, Carbon-nitrogen metabolism

Introduction

Sorghum bicolor is a cereal grain cultivated for various purposes, including food, forage, fiber, and energy production (Wang et al. 2016). Traditionally grown in semi-arid and subtropical regions, sorghum exhibits exceptional water use efficiency (Roone  2014). However, its maturity, growth, and composition can be profoundly influenced by the surrounding environment (Roone 2014). In the battle against food insecurity, sorghum plays a crucial role and holds significant agronomic importance globally with an annual production of over 54 million tons in 2021 (“FAOSTAT”). It is ranked as the fifth most economically significant crop worldwide and the second most important in the African continent, where its cultivation is vital for addressing the needs of food-insecure populations (Taylo 2003; Hariprasanna and Rakshi  2016).

Sorghum bicolor (L.) Moench, a well-recognized cultivar of sorghum bicolor, has gained prominence for its potential suitability as a crop in saline soils owing to its notable tolerance to high salt and alkali levels (Nie et al. 2023). Moreover, it exhibits exceptional photosynthetic efficiency and displays adaptability to conditions characterized by drought and soil with low fertility. It is worth emphasizing, however, that sorghum, including this particular variety, demonstrates limited resistance when exposed to elevated salt stress levels, typically withstanding concentrations up to 150 mM (Nie et al. 2023).

Salinity is a significant abiotic stressor reported to severely damage food production. To date, 8.7% of the land worldwide are affected by salinity which threatens soil fertility (“World map of salt-affected soils launched at virtual conference”). Salinity affects water availability, which causes osmotic stress (Zhao et al. 2021). Furthermore, it generates ionic imbalance and triggers the overproduction of reactive oxygen species (ROS), ultimately resulting in oxidative damage (Hasanuzzaman and Fujit 2022). In addition, salt stress can hinder the uptake of essential nutrients, reduce photosynthesis, and impede stomatal conductance, eventually resulting in diminished growth and yield (Loudari et al.  2022).

Under the pressure on food production caused by the increasing world population, the loss of farmable lands due to salinity presents a serious risk to food’s security. Discovering innovative and sustainable practices that effectively support crop growth under salt stress and enable the utilization of already affected soil can significantly contribute to tackling this issue. The use of biostimulants has emerged as a leading solution in sustainable agriculture to stimulate nutrient uptake and enhance plant tolerance to biotic and abiotic stresses, thereby improving crop yield and quality (Calvo et al. 2014). The application of plant extracts has shown beneficial effect on enhancing crops growth and improving their resilience against stress (Van Oosten et al. 2017; Arif et al.  2023). For instance, Lorenzo et al. (2019) reported that the bark extract of Acacia dealbata effectively attenuated salt stress in onion plants. This was achieved through an increase in sugar and protein contents, leading to significant improvements in plants’ length and weight, as well as the stimulation of root growth in soil irrigated with 120 mmol/L of NaCl (Lorenzo et al. 2019). Moringa oleifera leaf extract (MLE) was reported to have successfully ameliorated the growth and yield of many plants affected by salinity such as damask rose, Silybum marianum, Phaseolus vulgaris L., and Ocimum basilicum (Safi-naz and Rad  2015; Alkuwayti et al.  2020; Hassan et al.  2020; Yap et al.  2021). These reports mentioned the ability of MLE to improve the physiochemical parameters and the antioxidant defense system, in addition to scavenging ROS and attenuating MDA accumulation thus providing a shield against oxidative damage caused by salinity. Other plant extracts, such as chaste plant extract and Crataegus oxyacantha, has been reported to be effective in mitigating salt stress in tomato crop (Abdelhamid et al. 2022; Naboulsi et al.  2022).

In light of the above information, we aim to explore the potential of an aqueous plant extract from Cistus salviifolius in alleviating the adverse impacts of salinity stress on sorghum bicolor plants. Cistus salviifolius, commonly known as sage-leaved rockrose, is a shrub belonging to the Cistaceae family (El Euch et al. 2015). It can grow up to 60 cm and exhibits strong resistance to both drought and salt stress (Bartoli et al. 2014). This plant originates from the Mediterranean region and is known for its richness in biologically active compounds (Gürbüz et al. 2015). Previous studies have reported its strong antioxidant activity (Sayah et al. 2017), which could be helpful in mitigating oxidative stress caused by salinity. We previously reported the effect of Cistus salviifolius water extract (CSE) in alleviating cadmium toxicity in sorghum plants, which appears to be the only report on the biostimulant effect of this plant to date (Roussi et al. 2022).

To verify the potential role of CSE treatments (5, 20, and 30 mg/L) against salt stress (150 mM) in sorghum plants, we investigated multiple morphological and physiological parameters that influence plant growth and development. Additionally, we examined the impact on oxidative stress markers such as hydrogen peroxide (H2O2), superoxide ion (O2−), and malondialdehyde (MDA). We also studied the effect of salinity and CSE on enzymatic (superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR)) and non-enzymatic mechanisms essential for scavenging ROS. Moreover, we surveyed the activities of multiple carbon-nitrogen enzymes, namely phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (NAD-MDH), glutamate dehydrogenase (GDH), aspartate aminotransferase (AAT), and glutamine synthase (GS).

Materials and methods

Extracts preparation

The Cistus salviifolius (CS) plant was sourced from the “Jbel Lahbib” mountain in northern Morocco, with precise geographical coordinates (latitude: 35.4664997, longitude: −5.7971542). Its leaves were air-dried and subsequently ground into a fine powder at room temperature. Each 100 gram portion of the powder was combined with 1 L of distilled water, boiled for 2 h at 100 °C, and transferred into petri dishes. The mixture was then dried at 37 °C in an incubator until complete evaporation of the water. The resulting dried extract was further utilized to create three different concentrations by mixing it with distilled water: 5, 20, and 30 mg/L. Given the limited understanding of biostimulants’ operational mechanisms, in this study, we opted for a broad extraction approach using water as a solvent in order to explore the potential synergistic impact of compounds present in Cistus salviifolius extract. The water extraction also guarantees the absence of solvent remnants that might affect plants or the treated soil and it is a simple, quick, and cost-effective extraction method.

Plant material, experimental treatments, growth conditions, and agronomic traits measurements

Sorghum bicolor (l.) Moench seeds, obtained from the National Research Institute for Agriculture (INRA), were disinfected with 5% of sodium hypochlorite solution for three minutes and then thoroughly washed with distilled water. Every six seeds were planted in soil-filled pots. A week later, sorghum plants were divided into five groups as follows: plants irrigated with water only (C), plants irrigated with 150 mM of NaCl (C+), plants irrigated with 5 mg/L of CSE + 150 mM of NaCl (CS5), plants irrigated with 20 mg/L of CSE + 150 mM of NaCl (CS20), and plants treated with 30 mg/L of CSE + 150 mM of NaCl (CS30). For each condition, four repetitions were made. Sorghum plants were cultivated under controlled conditions of 12 h of light and 12 h of darkness at 29 °C. Plants were harvested four weeks after the first treatment and their height and weight were registered.

Cistus salviifolius chemical composition analysis

The flavonoid content of Cistus salviifolius water extract was estimated according to the protocol of Bouargalne et al. ( 2022) and expressed as a milligram of Quercetin equivalence (QE) per gram of extract.

The total phenolic content was determined according to Ben Mrid et al. (2019) using the method of Folin–Ciocalteu and expressed as mg gallic acid/g DW.

Indole-3-acetic Acid, free amino acid, soluble sugar, and protein contents protocols are presented in the materials and methods sections that follow.

Determination of chlorophyll content

To determine the chlorophyll content, the method of Armon (1949) was used as follows: 200 µl of plant’s soluble pellets was homogenized in water then added to 800 µL of acetone (80%). The solution was incubated at 4 °C for 72 h, and then the chlorophyll content was measured in four independent replicates and calculated using Armon (1949) formula:

Total Chlorophyll (mg.ml- 1) = Chlorophyll a content + Chlorophyll b content.

Where: Chlorophyll a (mg.ml−1) = (0.0127×OD663) - (0.00269×OD645)

Chlorophyll b (mg.ml- 1) = (0.0229×OD645)-(0.00468×OD663)

OD645nm and OD663nm are the optical densities at 645 and 663 nm, respectively.

Preparation of plant extracts and estimation of indole acetic acid, amino acid, and soluble sugar contents

The plant leaves were collected and subjected to drying in an incubator at 37 °C for 48 h. Subsequently, 200 mg of the dried material was crushed with a mortar in the presence of 80% ethanol at 4 °C. The resulting mixture was then centrifuged at 5000 g for 15 min at 4 °C. The supernatant obtained after centrifugation was examined for its content of indole acetic acid, amino acids, and soluble sugars.

Estimation of indole acetic acid content

The content of indole acetic acid (IAA) was determined using Salkowski’s reagent technique and commercial IAA as the standard (Ehman 1977). 1.5 ml of supernatant was combined with 500 ml of Salkowski’s reagent (FeCl3 in 36% perchloric acid) and incubated for 30 min at 25 °C. Following this procedure, the optical density at 530 nm was determined by comparing it to a blank sample comprising 1.5 ml of distilled water and 500 µl of Salkowski’s reagent.

Estimation of amino acid content

To assess amino acid content, 200 µl of the remaining supernatant from centrifugation was mixed with 1.8 ml of a solution containing 2% ninhydrin. This ninhydrin solution was prepared by dissolving it in a mixture of 0.2 M citrate buffer with a pH of 5 and ethylene glycol. After thorough mixing, the mixture was heated in a water bath for 15 min. Following cooling to room temperature, the optical density was recorded at 570 nm. Amino acid levels were determined using a calibration curve constructed with various concentrations of glycine (Ennoury et al. 2022).

Estimation of soluble sugar content

The quantity of soluble sugar was determined using the Yemm and Willis (1954) method. Plant leaf extract (1 ml) was combined with 2 ml of a solution containing anthrone reagent (dissolved at a concentration of 1 g/L in 95% sulfuric acid) to create a mixture. After thorough mixing, the mixture was heated in a water bath for 10 min. Following heating, the mixture was cooled on ice, and its spectrophotometric absorbance was measured at 630 nm. The sugar content was calculated utilizing a standard curve generated by varying amounts of anhydrous glucose.

Estimation of proline content

The measurement of proline content followed the protocol outlined by Bates et al. (1973).

Estimation of MDA, H2O2 and O2- contents

A modified version of the Bouchmaa et al. (2019) method was employed to measure the malondialdehyde content. The plant cell homogenate was mixed with trichloroacetic acid (20%) and tetrabutylammonium hydroxide (TBA) (0.67%). The mixture was then heated at 95 °C for 1 h. After cooling, 1 mL of n-butanol was added, and the mixture was centrifuged at 12,000 rpm for 12 min. The resulting supernatant was collected and the absorbance was measured at 532 and 600 nm to determine the MDA content.

The hydrogen peroxide content was determined by mixing 250 µL of plant homogenate in water with 0.1% trichloroacetic acid. After centrifugation at 12,000 rpm and 4 °C for 15 min, the resulting supernatant was combined with 1 mL of 10 mM phosphate buffer (pH 7) and 2 mL of 1 M potassium iodate. This mixture was left in the dark for an hour and then measured for absorbance at 390 nm. The H2O2 concentrations were calculated using a standard curve, as outlined in the research conducted by Roussi et al. (2022).

The method outlined by Kubiś (2008) was employed to determine the concentration of superoxide ions. Initially, 100 mg of fresh plant leaves were finely cut into 1 mm fragments and placed in a solution composed of 10 mM K-phosphate buffer (pH 7.8), 0.05% nitroblue tetrazolium (NBT), and 10 mM sodium azide. This mixture was allowed to incubate at room temperature for one hour. Subsequently, 2 mL of the incubated solution underwent heating at 85 °C for 15 min, followed by rapid cooling. The optical density was then measured at 580 nm, and the O2− content was quantified as the increase in absorbance per gram of fresh weight (A580 g−1 FW).

Extraction and assay of SOD, GPx, GST, GR, and GDH enzymes activities

To prepare the enzyme extract, 200 mg of fresh leaves were crushed in a cold mortar with a solution consisting of 100 mM HEPES-KOH, 20 µM FAD, 10 mM MgCl2, 1 mM PMSF, and 14 mM β-Mercaptoethanol. After centrifugation at 20,000 g and 4 °C for 20 min, the resulting supernatant was utilized for subsequent enzyme activity analysis. Superoxide dismutase (SOD) was determined using a method outlined by Beauchamp and Fridovich (1971), which relies on its capacity to hinder the photochemical reduction of nitroblue tetrazolium chloride (NBT). In this procedure, the reaction mixture contained 50 mM phosphate buffer (pH 7.8), 2 mM methionine, 75 µM NBT, 1 µM EDTA, 2 µM riboflavin, and the enzyme extract. After 30 min under a flat light panel, the absorbance at 560 nm was measured. The enzyme activity was quantified as the amount necessary to cause a 50% reduction in absorbance compared to the control, representing the point where maximum color development occurred.

The assessment of glutathione peroxidase (GPx) activity was determined with adaptations from the method outlined by Bouchmaa et al. (2018). In this procedure, the reaction mixture comprised 50 mM potassium phosphate buffer (pH 7.4), 1 mM EDTA, 1 mM sodium azide, 1 mM GSH, glutathione reductase (GR) at a concentration of 4 µg/mL, 0.2 mM NADPH, 0.25 mM H2O2, and the enzyme extract. The rate of NADPH oxidation was observed at 340 nm to measure GPx activity.

A revised approach based on the method developed by Habig et al. (1974) was employed to assess glutathione S-transferase (GST) activity. In this modified technique, the assay mixture included the enzyme extract, 5 mM GSH, 2.5 mM 1-chloro-2,4-dinitrobenzene (CDNB), and 0.1 M phosphate buffer (pH 5.5). The reaction progress was spectrophotometrically monitored at 340 nm and 30 °C. The concentrations of the reaction product were determined using a molar extinction coefficient of 9.6 mM−1 cm−1.

Glutathione reductase (GR) activity was determined by monitoring the oxidation of NADPH at 340 nm, using a technique based on the method outlined by Latique et al. (2021) with certain adjustments. The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.8), 0.2 mM NADPH, 1 mM GSSG, and the appropriate amount of enzyme extract. The reaction was initiated by adding NADPH at 30 °C.

Glutamate dehydrogenase (GDH) activity was assessed as outlined by Ben Mrid et al. (2017). In the reaction mixture, the enzyme extract was combined with 100 mM Tris-HCl buffer (pH 8), 1 mM CaCl2, 13 mM α-ketoglutarate, 50 mM (NH4)2SO4, and 0.25 mM NADH. Spectrophotometric monitoring at 340 nm was conducted over a 30 min duration to measure the enzyme activity.

Extraction and assay of PEPC, GS, AAT, and NADH‑MDH enzymes activities

Fresh plant leaves were crushed in a cooled mortar using Tris-HCl buffer (100 mM, pH 8) containing various additives such as 10 mM MgCl2, 1.4 mM glycerol, 1.4 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, 1 mM EGTA, 9.4 µM leupeptin, and 16.5 µM chymostatin. The resulting homogenate was then centrifuged at 12,000 g for 15 min at 4 °C. Subsequently, the supernatant was subjected to a saturation process using solid ammonium sulfate (60%) for 30 min. After another round of centrifugation under the same conditions, the pellet obtained was resuspended in the extraction buffer and used for enzyme activity analysis.

To assess the activity of phosphoenolpyruvate carboxylase (PEPC), the method described by El Omari et al. (2018) was employed. This method involves monitoring the oxidation of NADH at 340 nm. The assay mixture consisted of 100 mM Hepes-KOH (pH 7.3), 5 mM MgCl2, 5 mM NaHCO3, 2.5 mM PEP, 0.25 mM NADH, 3 units of malate dehydrogenase (MDH), and the enzyme extract.

The activity of glutamine synthetase (GS) was estimated using the procedure outlined by Ben Mrid et al. (2016). The reaction mixture contained 90 mM imidazole-HCl (pH 7.0), 120 mM L-glutamine, 3 mM MnCl2, 0.4 mM ADP, 20 mM potassium arsenate, 60 mM hydroxylamine, and the enzyme extract. L-glutamine and ADP were excluded in the blank test. The mixture was incubated at 37 °C for 20 min and then stopped by adding a solution of 10% FeCl3·6H2O (in 0.2 N HCl), 24% trichloroacetic acid (TCA), and 5% HCl (in a 1:1:1 ratio). The formation of γ-glutamyl hydroxamate was measured at 540 nm.

The activity of aspartate aminotransferase (AAT) was measured using the method described by Ben Mrid et al. (2018).The assay mixture included Tris-HCl (50 mM, pH 7.8), L-aspartate (50 mM), 2-oxoglutarate (10 mM), NADH (0.1 mM), and 2 units of MDH. The reaction was initiated by adding 2-oxoglutarate, and the activity was monitored spectrophotometrically at 340 nm for 30 min.

The activity of malate dehydrogenase (NADH-MDH) was determined using the protocol outlined by Setién et al. (2014). The reaction buffer contained 100 mM Hepes-KOH (pH 7.5), 5 mM MgCl2, 2 mM oxaloacetate, and 0.2 mM NADH. The enzyme extracts were added, and the activity was monitored spectrophotometrically at 340 nm for 30 min. MDH activity was determined by examining the oxidation of NADH and the reduction kinetics of NAD+.

Protein content determination

The protein content of the samples was quantified using the Bradford method with Bovine serum albumin (BSA) as a standard (Bradfor 1976).

 Statistical analysis

IBM SPSS Statistics, Version 25.0, for Windows, developed by IBM Corp in Armonk, NY, was utilized for conducting all statistical analyses. One-way analysis of variance (ANOVA), followed by the Student Newman-Keuls post hoc test, was employed to assess differences in means (p < 0.05). Significant differences are indicated by different letters. The presented data represent mean values ± standard deviation.

In this research, we included 24 plants for each specific condition, distributing them evenly into four pots with six plants in each pot, thus creating technical replicates. As a result, there were a total of four pots per condition. To carry out the physiological and biochemical analysis, we collected samples twice from each pot, considering them as biological replicates. Within each of these extracts, we conducted two technical replicates, resulting in a total of four replicates for each condition.

Results

Cistus salviifolius extract composition

The analysis of Cistus salviifolius extract revealed the presence of several metabolites. At a concentration of 1 mg/ml, the extract contained 0.877 ± 0.015 mg. g−1 DW of soluble sugars, 0.274 ± 0.012 mg. g−1 DW indole acetic acid content, 357.50 ± 0.160 mg. g−1 DW free amino acid content 65.117 ± 0.001 mg. g−1 DW flavonoid content, 305.350 ± 0.005 mg. g−1 DW polyphenol content, and 147.585 ± 3.795 mg. g−1 DW protein content (Table 1).

Table 1.

Cistus salviifolius (CSE) extract composition

CSE composition Flavonoid Polyphenol Indole acetic acid Free amino acid Soluble Sugar Protein content
Concentration (mg. g−1 DW) 65.117 ± 0.001 305.350 ± 0.005 0.274 ± 0.012 357.50 ± 0.160 0.877 ± 0.015 147.585 ± 3.795
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Cistus salviifolius extract promoted the growth and biomass accumulation and prevented the loss of chlorophyll pigment under salt stress

Salt stress exerted detrimental effects on the morphological traits of sorghum plants (Fig. 1). The height and weight of sorghum plants were severely reduced under salinity conditions, with a noticeable decrease of 38.51% and 60.30% in length and biomass, respectively, compared to the control plants treated with water only (Table 2). Additionally, the chlorophyll pigment content in sorghum plants experienced a significant decline of 25.22% (Fig. 2). In the face of salt stress CSE treatments effectively enhanced the morphological traits of sorghum plants (Fig. 1), including their length and weight. All concentrations of CSE used in the experiment exhibited positive effects on sorghum’s growth, but the concentration of 5 mg/L showed the most promising results, leading to a remarkable increase of 48.23% and 158.36% in length and weight, respectively, compared to the salt control plants (Table 2). Furthermore, CSE treatments positively influenced the chlorophyll content, with significant improvements of 17.55%, 40.25%, and 16.10% observed at concentrations of 5 mg/L, 20 mg/L, and 30 mg/L, respectively, compared to the salt control (Fig. 2).

Fig. 1.

Fig. 1

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Effect of CSE on the growth of sorghum under 150 mM of NaCl. From left to right: C: treated only with water; C+: treated with 150 mM of NaCl; CS5: treated with 5 mg/L of CSE + 150 mM of NaCl; CS20: treated with 20 mg/l of CSE + 150 mM of NaCl; CS30: 30 mg/l of CSE + 150 mM of NaCl

Table 2.

Effect of CSE on the growth and biomass of sorghum plants under salt stress

Conditions Control NaCl (150mM) CS5 + NaCl (150mM) CS20 + NaCl (150mM) CS30 + NaCl (150mM)
Height (cm) 55.3 ± 1.78a 34 ± 3.6b 50.4 ± 4.48a 45 ± 1.8c 40.1 ± 2.14ac
Weight (g) 5.87 ± 0.70a 2.33 ± 0.44b 6.02 ± 0.82a 4.51 ± 0.71c 5.16 ± 0.75ac
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Control: irrigated with water; CS5: irrigated with 5 mg/L of CSE + 150 mM of NaCl; CS20: irrigated with 20 mg/l of CSE + 150 mM of NaCl; CS30: irrigated with 30 mg/l of CSE + 150 mM of NaCl. Each value represents the mean of twelve independent observations ± SD. Means with the same letters are not significantly different at the 5% probability

Fig. 2.

Fig. 2

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Chlorophyll content in sorghum plants treated by different concentrations of Cistus salviifolius extract (CSE) under salt stress. C: treated only with water; C+: treated with 150 mM of NaCl; CS5: treated with 5 mg/L of CSE + 150 mM of NaCl; CS20: treated with 20 mg/l of CSE + 150 mM of NaCl; CS30: 30 mg/l of CSE + 150 mM of NaCl. Each value represents the mean of four independent observations ± SD. Means with the same letter are not significantly different at the 5% probability

Cistus salviifolius extract reduced oxidative stress markers buildup in salt stress exposed sorghum plants

Salt stress led to a considerable increase in MDA content (by 112.5%) and triggered elevated levels of reactive oxygen species (ROS), including O2− and H2O2, by 91.43% and 13.96%, respectively (Fig. 3). On the other hand, CSE treatments successfully reduced the MDA content in salt-stressed sorghum plants, with the concentration of 5 mg/L demonstrating the greatest efficacy in reducing MDA levels by 64.75% (Fig. 3a). Additionally, CSE treatments showed the ability to alleviate the elevated levels of ROS induced by salt stress, with reductions of 40.37% in O2− content at 5 mg/L (Fig. 3b) and 71.97%, 53.20%, and 54.34% in H2O2 content achieved at concentrations of 5 mg/L, 20 mg/L, and 60 mg/L, respectively, compared to the salt control plants (Fig. 3c).

Fig. 3.

Fig. 3

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Malondialdehyde (MDA) (a), superoxide ion (O2−) (b), and hydrogen peroxide (H2O2) (c) contents in sorghum plants treated with different concentrations of Cistus salviifolius (CSE) under salt stress. C: treated only with water; C+: treated with 150 mM of NaCl; CS5: treated with 5 mg/L of CSE + 150 mM of NaCl; CS20: treated with 20 mg/l of CSE + 150 mM of NaCl; CS30: 30 mg/l of CSE + 150 mM of NaCl. Each value represents the mean of four independent observations ± SD. Means with the same letter are not significantly different at the 5% probability

Cistus salviifolius extract affected indole-3-acetic acid and osmolytes content under salt stress

Salinity had a notable impact on the IAA content in sorghum leaves, resulting in a decrease of 20.70%. The elevated salinity levels caused a reduction in the natural levels of IAA in the plants. However, when supplemented with 5 mg/L of CSE, a significant increase in IAA content by 22.07% was observed compared to the salt-stressed plants (Fig. 4a). Salt stress conditions led to an increase in amino acid, soluble sugar, and proline content in sorghum plants, with a substantial rise of 47.35%, 54.16%, and 3.067% respectively, compared to the control plants treated with water alone (Fig. 4). The supplementation of CSE further increased the amino acid levels. The addition of 5 mg/L, 20 mg/L, and 30 mg/L of CSE resulted in additional enhancements of amino acid content by 65.77%, 66.41%, and 64.89%, respectively, compared to the salt-stressed plants (Fig. 4b). In contrast, CSE treatments effectively decreased the soluble sugar content (Fig. 4c). Treatment with 5 mg/L, 20 mg/L, and 30 mg/L of CSE led to reductions in soluble sugar content by 26.48%, 35.85%, and 30.73%, respectively, compared to the salt-stressed plants (Fig. 4c). In addition, CSE treatment using 5 mg/L increased proline content in stressed sorghum plants by 3.63% (Fig. 4d).

Fig. 4.

Fig. 4

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Indole acetic acid (IAA) (a), amino acid (b), soluble sugar (c) and proline (d) contents in sorghum plants treated with different concentrations of CSE under salt stress. C: treated only with water; C+: treated with 150 mM of NaCl; CS5: treated with 5 mg/L of CSE + 150 mM of NaCl; CS20: treated with 20 mg/l of CSE + 150 mM of NaCl; CS30: 30 mg/l of CSE + 150 mM of NaCl. Each value represents the mean of four independent observations ± SD. Means with the same letter are not significantly different at the 5% probability

Cistus salviifolius extract induced antioxidant enzymes activities under salt stress

Salinity upregulated the activities of the antioxidant enzymes. Specifically, compared to the water control, there was a notable rise in GR, GST, GPx, and SOD activities by 74.10%, 20.32%, 6.42%, and 47.09% (Fig. 5). Additionally, CSE further upregulated GST, GPx, and SOD activities, with the most prominent increase observed in plants treated with 30 mg/L in the three activities. A rise of 56.57%, 32.63%, and 100.26% was recorded for GST, GPx, and SOD activities, respectively, compared to their respective salt control (Fig. 5b, c, d). On the other hand, CSE irrigation decreased GR activity by 23.66%, 8.33%, and 40.79%, respectively, compared to the salt control (Fig. 5a).

Fig. 5.

Fig. 5

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Activities of glutathione reductase (GR) (a), glutathione-s-transferase (GST) (b), superoxide dismutase (SOD) (c), and glutathione-peroxidase (GPx) (d) in sorghum plants treated by different concentrations of Cistus salviifolius extract (CSE) under salt stress. C: treated only with water; C+: treated with 150 mM of NaCl; CS5: treated with 5 mg/L of CSE + 150 mM of NaCl; CS20: treated with 20 mg/l of CSE + 150 mM of NaCl; CS30: 30 mg/l of CSE + 150 mM of NaCl. Each value represents the mean of four independent observations ± SD. Means with the same letter are not significantly different at the 5% probability

Cistus salviifolius extract regulated carbon nitrogen enzymes activities under salt stress

Carbon and nitrogen fixation play crucial roles in plant development. This study explored the impact of salt stress and CSE treatment on sorghum enzyme activities. The findings revealed that the application of 150 mM NaCl markedly reduced the activities of enzymes involved in carbon fixation. Specifically, PEPC activity decreased by 96.37% compared to the control group. In contrast, the induction of three concentrations of CSE significantly increased PEPC activity. The highest activity was observed in plants treated with 5 mg/L of CSE, resulting in a 255.95% increase in PEPC activity compared to the control (Fig. 6a).

Fig. 6.

Fig. 6

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Activities of phosphoenolpyruvate carboxylase (PEPC) (a), malate dehydrogenase (MDH) (b), glutamine synthetase (GS) (c), aspartate aminotransferase (AAT) (d), glutamate dehydrogenase (GDH) (e) in sorghum plants treated with different concentrations of Cistus salviifolius extract (CSE) under salt stress. C: treated only with water; C+: treated with 150 mM of NaCl; CS5: treated with 5 mg/L of CSE + 150 mM of NaCl; CS20: treated with 20 mg/l of CSE + 150 mM of NaCl; CS30: 30 mg/l of CSE + 150 mM of NaCl. Each value represents the mean of four independent observations ± SD. Means with the same letter are not significantly different at the 5% probability

MDH activity, another carbon-nitrogen activity, exhibited a reduction in stressed plants compared to non-stressed plants, as depicted in Fig. 6b. The results showed that MDH activity decreased by 75.01% compared to the control.

Furthermore, the activities of GS and AAT enzymes were negatively affected. The application of 150 mM NaCl reduced GS activity by 5.37% and AAT activity by 71.5% (Fig. 6c, d). However, both activities were stimulated by CSE treatment. Plants treated with 30 mg/L of CSE exhibited the highest GS activity, which increased GS activity by 84.81% compared to the activity in stressed plants (Fig. 6c). AAT activity, conversely, was significantly stimulated by the treatment with 5 mg/L of CSE, resulting in a notable 180.27% increase (Fig. 6d). In contrast, GDH activity was the only activity that showed an increase in stressed plants. A significant increase of 10.37% was recorded in plants under salt stress compared to the control (Fig. 6e). The application of CSE ameliorated GDH activity, particularly at a concentration of 30 mg/L, which increased GDH activity by 63.14% compared to that found in salt-stressed plants (Fig. 6e).

Discussion

Salinity greatly impacted the growth of sorghum plants, diminished their biomass and length, and reduced chlorophyll production, a pigment essential for capturing sunlight for the photosynthetic system which is the basis of plant growth (Naboulsi et al. 2022). It has been shown that salt toxicity causes osmotic perturbation and reduces stomatal conductance, thereby limiting the supply of CO2 and consequently hampering the photosynthetic process and chloroplast activity (Safdar et al. 2019). Nevertheless, the implementation of CSE had a substantial impact on improving the chlorophyll content of the salt-stressed sorghum plants and improving their morphological parameters, resulting in a better growth pattern. Many studies reported the effectiveness of plant extracts in improving crop morpho-physiological traits under salt stress; for example, applying onion extract at a concentration of 25% to soybean plants in the presence of 150 Mm NaCl notably stimulated the development of this crop and its photosynthetic pigments content (Saravanan et al. 2023). Similarly, the use of Arthrocnemum macrostachyum extract sprayed on the leaves proved to be beneficial in enhancing the growth traits and photosynthetic pigments levels in soybean plants subjected to salinity (Osman et al. 2021).

Additionally, salt stress also decreased the amount of IAA, the most common auxin form in plant tissue, in sorghum leaves. Auxin is a phytohormone that regulates vegetation growth through affecting various plant development processes including cell division and elongation (Di et al. 2016). Furthermore, auxin can be involved in regulating stress tolerance and phytohormone signal crosstalk (Li et al. 2023). The diminished levels of IAA hampered the development of salt-stressed sorghum. However, the addition of CSE treatments increased IAA amount, which positively influenced the growth of plants afflicted by salt stress. The beneficial effect of IAA on stressed plants has been previously reported in multiple crops (Kaya et al. 2013; Husen et al. 2016). The addition of IAA externally increased yield, weight, relative water, and chlorophyll a and b contents in salt-stressed maize and pea plants, consequently promoting healthier growth under stressful conditions (Kaya et al.  2013; Husen et al.  2016).

The examination of markers for oxidative stress in sorghum plants subjected to salinity stress revealed a surge in reactive oxygen species content; H2O2 and O2−, in addition to lipid peroxidation index; MDA. ROS are oxidizing species that, in low concentrations, can provide signals to induce protective mechanisms that can help in the fight against stress induced by biotic and abiotic agents, however, they can cause significant irreversible damage to proteins, lipids, and nucleic acids when present in excess in plant cells (Del Rí 2015). This excessive production of ROS results in oxidative stress, which can ultimately trigger cellular death (Khan et al. 2023). Abiotic stress has been consistently linked to the overproduction of ROS and oxidative damage (Awan et al. 2023). In salt-stressed soil, ROS overproduction is regarded as the most frequently occurring phenomenon and has previously been observed in maize, sweet pepper, mung beans, sunflower, tomato, and sesame (Kesawat et al. 2023). The overproduction of ROS reflects the venerability of the antioxidant system against the toxicity caused by salt stress. Nevertheless, the CSE extract used in the present work demonstrated an important ROS scavenging capacity, proven by the significant decrease in H2O2, O2−, and MDA contents, especially when applying 5 mg/L of CSE. This capacity allowed the safeguarding of sorghum plants from the harm caused by salt stress.

To detoxify this overly produced ROS, both enzymatic and non-enzymatic antioxidant defensive mechanisms interfere to mitigate the oxidative damage resulting from salinity. Osmolytes are essential components of the non-enzymatic defense system and can help with stress tolerance, ROS reduction, and removal of MDA toxic effects (Chen et al. 2023). The increase in osmolytes generation was demonstrated to have a notable impact on regulating stomatal conductance and transpiration rate during abiotic stress conditions (Fu et al. 2019), explaining the elevated production of soluble sugars, amino acids, and proline noticed in salt-stressed sorghum plants. The notable rise in osmolyte content, especially when subjected to a 5 mg/L concentration of CSE, can greatly contribute to maintaining the balance of osmotic pressure in saline soils.

Apart from examining the non-enzymatic defense system, SOD, GPx, GR, and GST enzymes activities were studied to understand how NaCl impacts sorghum plants. SOD is a vital enzyme in the plant’s defense system; it works collectively with GPx to neutralize ROS in cells. SOD converts O2− into H2O2, which is then neutralized by GPx to water and oxygen (Hasanuzzaman et al. 2021). GPx has another role of reducing hydroperoxides to alcohol, which can be extremely beneficial in minimizing lipid peroxidation, thus, avoiding cell membrane damage (Nadaraja 2020). GR is also essential for lowering ROS content; it interferes by transforming the oxidized glutathione (GSSG) to its reduced form (GSH) which is a strong non-enzymatic antioxidant capable of protecting plant cells from oxidative damage through detoxifying ROS and averting protein denaturation caused by oxidation (Abdelhamid et al. 2022). GSH also interferes in many mechanisms within the plants such as cell proliferation and gene expression and can help maintain a balanced redox status in plant cells under salt stress which renders it important for normal plant growth (Hasanuzzaman et al.  2017). Along with these enzymes, GST interferes in the detoxification reactions and helps shield the plant from oxidative stress. Reports suggest that GST provides cell protection against chemical toxicity through xenobiotics excretion (Kumar and Trived  2018), which are compounds that can be very toxic to plants if persisted in cells (Bártíková et al 2015). In sorghum crop experiencing salt stress, we registered a serious upregulation of all four enzyme activities in reaction to the surge in ROS content. The addition of CSE further enhanced the antioxidant system activities which lowered ROS and MDA content and provided protection from oxidative damage. The improvement of the defense system thanks to the application of CSE, attenuated oxidative injuries and ameliorated the photosynthetic behavior of sorghum plants, enabling them to better withstand salt toxicity and ultimately improve their growth.

In a similar manner to CSE extract, several phytoextracts have recently been reported as natural protective agents for crops against abiotic stress (Shahid et al. 2015; Desoky et al. 2019). Although they may differ in potential, mode of application, or composition, many plant extracts have proven efficient in enhancing resistance to salt-induced stress in different crops by reducing ROS and MDA content while boosting the antioxidant defense system (Akram et al. 2022).For instance, the supplementation of salt-stressed wheat plants with licorice root extract in combination with lipoic acid increased the activities of SOD, Ascorbate peroxidase (APX), catalase (CAT), peroxidase (POX), and GR enzymes, thereby reducing ROS levels (Elrys et al. 2020). This treatment also improved the non-enzymatic antioxidant activities and nutrient uptake, as well as photosynthetic pigments, leading to improved growth characteristics in salt-affected wheat plants (Elrys et al. 2020). Abdelhamid et al. (2022) achieved comparable outcomes in tomato plants cultivated in saline environments and treated with chaste plant extract (CPE); they reported a substantial upregulation in antioxidant enzyme and carbon-nitrogen enzyme activities in plants irrigated with CPE, alongside a significant improvement in plant morpho-physiological parameters.

Carbon and nitrogen fixation are key processes in organic matter production, and these mechanisms are facilitated by several enzymes. Some enzymes, such as PEPC and MDH are responsible for carbon fixation (Ben Mrid et al. 2017). However, stressed plants exhibit lower activity of these enzymes compared to non-stressed ones. This reduction in activity may be a contributing factor to the deterioration of morphological and physiological parameters in stressed plants (Jeanneau et al. 2002). It is well-known that the decrease in PEPC activity leads to a reduction in carbonic substances (Ben Mansour et al. 2019). PEPC’s primary role is to synthesize oxaloacetate using HCO3−and phosphoenolpyruvate as substrates. The synthesized oxaloacetate is then converted to malate by MDH (Kchikich et al.  2021). MDH also shows reduced activity when exposed to 150 mM NaCl.

Several studies have verified the detrimental effect of salt stress on the activity of PEPC and MDH enzymes. Naboulsi et al. (2022) and Abdelhamid et al. (2022) demonstrated that the use of 75 mM NaCl significantly decreased PEPC and MDH activity in tomato plants. This reduction in enzyme activity impairs the production of oxaloacetate and malate, which are crucial for plant growth. Both of these molecules enable the TCA cycle to continue (Setién et al. 2014). Consequently, the decrease in these activities can adversely affect the production of energy and structural compounds, such as sugars, amino acids, and proteins (Kchikich et al.  2021).

The reduction in GS activity was discovered to be associated with a rise in GDH activity. This discovery corresponds with the findings of Ennoury et al. (2023), who observed similar changes in tomato plants exposed to 75 mM NaCl-induced stress. The contradictory activities of GS and GDH can be explained by Kumar et al. (2000), who suggested that GDH becomes an alternative enzyme to manage nitrogen flow in cases of abiotic stress when GS activity is limited. It’s known that the non-elimination of ammonium can harm plants. Ammonium enters cells through potassium channels, triggering the release of protons and leading to acidification in the extracellular milieu and causing issues in cellular homeostasis (Hoopen et al. 2010). The substitution in GDH and GS activity under salt stress helps minimize the accumulation of ammonium (Ennoury et al. 2022).

During this study, we observed that CSE increased the activities of PEPC and MDH, which had a positive impact on crop growth. The availability of carbon substances can provide plants with a better source for the synthesis of soluble sugars, which are known to be an alternative source of carbon when the quantity of carbonic substances is low (Mohsen et al. 2011). Additionally, these carbon substances play an important role as antioxidant molecules (Hoopen et al. 2010). Furthermore, the heightened enzymes activities of GS and GDH can stimulate the accumulation of amino acids through ammonium assimilation (Ennoury et al. 2023). The stimulation of AAT enzyme, responsible for amino acid synthesis by converting glutamate into other forms of amino acids, provides the plant with the capacity to synthesize various phytohormones that utilize amino acids as precursors. Additionally, AAT activity promotes the production of antioxidant compounds such as glycine betaine and proline (Hanana et al. 2011).

The application of CSE has yielded remarkable results in regard to mitigating salt stress in sorghum plants. CSE, rich in amino acids, soluble sugars, and secondary metabolites, operated through multiple mechanisms, enhancing chlorophyll content crucial for photosynthesis, and regulating auxin levels, hence, facilitating better growth in salt-stressed plants. Notably, CSE demonstrated robust scavenging abilities against ROS, effectively reducing oxidative damage induced by salt stress. It activated enzymatic defense enzymes like SOD, GPx, and GST, while also increasing osmolytes, thus, aiding in stress tolerance. Furthermore, CSE positively influenced essential enzymatic activities involved in carbon and nitrogen fixation, including PEPC, MDH, GS, GDH, and AAT. By enhancing these processes, CSE provided vital building blocks for growth and stress response. In essence, CSE not only reduced salt-induced stress but also enhanced physiological processes, enabling sorghum plants to withstand salt toxicity and promoting healthier growth.

Conclusion

The results obtained indicated that exposure to salt stress severely damages the growth of sorghum bicolor. This deleterious effect can be ascribed to the excessive accumulation of reactive oxygen species (ROS) and a decline in the activities of enzymes crucial for carbon and nitrogen fixation in the face of salt stress. However, the introduction of CSE in an irrigation medium to sorghum bicolor plants exhibited a remarkable ability to mitigate the harmful effects of salt stress by fortifying the antioxidant defense system and revitalizing carbon and nitrogen metabolism. As a result, the introduction of CSE led to a substantial reduction in ROS levels while concurrently augmenting the availability of vital carbon and nitrogen compounds essential for metabolite synthesis. Furthermore, the inclusion of CSE proved advantageous in promoting the synthesis of indole-3-acetic acid (IAA) and enhancing chlorophyll content in stressed sorghum plants, thereby propelling their growth in salt-laden conditions. Collectively, the integration of CSE significantly amplified the salt stress tolerance of sorghum, exerting a profoundly positive influence on its overall growth and development.

Author contribution

Conceptualization: ZR; Methodology: ZR, AE; Formal analysis and investigation: ZR; Writing - original draft preparation: ZR, AE; Writing - review and editing: ZR, AE, AK; Supervision: MN.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this work.

Data availability

The data used to support the findings of this study are included in the article. 

Declarations

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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