Priority impacts of plant growth promoting fungi and proline under NaCl stress: boosting chickpea plants tolerance and performance
Rabab A. Metwally, Maha A. Azb, Marwa M. El-Demerdash, Reda E. Abdelhameed
TL;DR
This study shows that using fungi and proline can help chickpea plants tolerate salt stress, improving their growth and health in salty soils.
Contribution
The study introduces AMF inoculation and proline application as innovative methods to reverse salinity-induced damage in chickpea plants.
Findings
AMF and proline reduced H2O2 levels and membrane leakage in salt-stressed chickpea plants.
AMF and proline improved growth, pigments, and carbohydrates in chickpea under NaCl stress.
AMF and proline helped maintain leaf anatomy and ion balance in salt-stressed chickpea.
Abstract
Soil salinity threatens global agriculture by impairing plant growth, crop productivity, and soil health. This study was conducted to assess the impact of salinity on chickpea performance at the vegetative stage and the possible ameliorating role of arbuscular mycorrhizal fungi (AMF) and proline applications. A greenhouse experiment with 30 pots (5 replicates × 6 treatments) subjected half the treatments to 200 mM NaCl, AMF was applied at sowing, and proline was sprayed two weeks post-planting. Total pigments dramatically decreased [49.18%] in salt-stressed chickpea. Biomass, protein and carbohydrate metabolism were also affected. For instance, plant height and total fresh weight (TFW) showed inhibitions of 37.83% and 72.19% as compared to control. Conversely, chickpea under salt stress had an increased accumulation of H2O2 (13.12 mg/g DW) and higher electrolyte leakage (54.72%),…
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Figure 7- —Zagazig University
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Taxonomy
TopicsMycorrhizal Fungi and Plant Interactions · Plant-Microbe Interactions and Immunity · Plant Stress Responses and Tolerance
Introduction
Abiotic stresses along with climate change are key players in crop losses worldwide. Salinity, one of these stresses, is a prevalent soil hazard that affects 30% of Egypt’s arable land and more than 800 million hectares worldwide [1]. Changing climatic patterns and improper farming practices, such as the overuse of synthetic chemical fertilizers, groundwater irrigation, and flood irrigation accompanied by poor drainage, exacerbate this problem by turning fertile soil into saline soil and reducing crop productivity [2]. It is worrisome to note that the area of salinized land is expected to rise by up to 10% a year [3]. Salinity reduces plant productivity because of the excessive buildup of NaCl which has a negative impact on plants for a couple of main reasons: the poisonous effects of Na^+^ and Cl^−^ ions on plants, as well as the decreased water availability brought on by salt buildup in the soil, which lowers soil water potential [4, 5]. Plants endure direct cellular damage from Na^+^ ions, and an increased concentration of Na^+^ in the root zone also prevents K^+^ uptake due to their antagonistic effect [6]. Since K^+^ is essential for maintaining membrane potential, enzyme activity, and cell turgor, this lack of K^+^ within the cell inevitably results in an interruption in plant development. Moreover, Na^+^ influx reduces the photosynthetic surface available to sustain the ongoing growth of salt-affected plants by causing chlorosis, necrosis, and premature senescence of adult leaves [7]. Salt stress also causes anatomical abnormalities, reportedly resulting in shrinkage of the laminar, spongy, and woody tissues of leaves [8]. Furthermore, plants that are exposed to high salinity levels suffer oxidative damage due to the production of reactive oxygen species (ROS), which inhibits plant growth [9–12].
Plants exhibit adaptive mechanisms to deal with this stress by exhibiting growth plasticity, controlling the cytoplasmic ion content which includes ion homeostasis, osmotic adjustment, and upregulation of hormones and antioxidants to preserve the cellular structure and maintain osmotic equilibrium within the cell by continuous water input; but these defenses are often overwhelmed under intense stress [13–16]. Among salt-sensitive crops, legumes are particularly vulnerable, posing a serious threat to global food security as arable land becomes increasingly salinized [17, 18]. Chickpea (Cicer arietinum L.), a nutritionally vital legume high in protein and micronutrients, suffers significantly under salinity [19–21], according to estimates salinity causes an 8–10% loss of their total global production [22, 23]. To enhance chickpea salt tolerance, exogenous proline application has been widely studied due to its role in osmotic regulation, membrane protection, and redox balance. Additionally, arbuscular mycorrhizal fungi (AMF) improve water and nutrient uptake, ionic balance, and antioxidant capacity under stress.
Proline, a crucial amino acid, is the most prevalent endogenous osmolyte that has a role in cell osmotic adjustment, membrane stabilization, and detoxification of harmful substances in plants under salt stress [24–26]. Several investigations have demonstrated that the exogenous application of proline can increase plant tolerance to salt by enhancing their enzymatic antioxidant defense mechanism [27, 28]. The beneficial effects of exogenous proline on salt tolerance have been evaluated considering many factors, including growth stage, plant tolerance and Na^+^/K^+^ [29]. Under salinity stress, its administration affects plant growth and physiological processes [30]. Proline-treated plants increase their K^+^ content and decrease their Na^+^ accumulation, ensuring a better K^+^/Na^+^. Moreover, the application of proline enhanced the growth of Lupinus termis L. plants under salt stress by improving the morphoanatomical characteristics like the plant’s overall stem diameter, pith layer, cortex thickness, and xylem vessel diameter [31, 32].
Another biological green approach that aims at improving the tolerance of plants to abiotic stresses is the application of beneficial soil microorganisms. A dominant group of soil microbes is arbuscular mycorrhizal fungi (AMF), which are widely distributed in ecosystems [33, 34]. They effectively improve the growth, development, and plant fitness, thanks to their ability to form symbiotic associations with the roots of most terrestrial plants, allowing better nutrient and water absorption to promote plant growth and yield [35, 36]. Many plants have demonstrated indications of AMF symbiosis, which increases their adaptation to soil salinity; these plants include chickpea, maize, clover, tomato, sorghum, beans, pea, olive, rice, cucumber, and lettuce [37, 38]. The benefits associated with AMF inoculation include enhancing the rhizospheric soil properties, stimulating and altering the architecture of the root system, improving root hydraulic conductivity at low water potential in plants, superior water use efficiency and osmoprotection, preservation of cell ultrastructure, greater leaf area and photosynthetic efficiency, enhanced antioxidant metabolism, and ionic homeostasis maintenance [39, 40]. These effects act in coordination to improve plant’s resilience. As well, AMF colonization may have benefits for improving the plant performance by preventing possible plant diseases from colonizing the roots [41].
Since chickpeas are highly sensitive to salt stress and the morphological, physiological and biochemical characteristics are all impacted by this stress, it matters to examine the survival tactics in salt-affected environments. In addition, developing environmentally friendly practices is the primary challenge at the forefront of sustainable agriculture and environmental issue mitigation. Therefore, the purpose of this study was to explore the hypothesis that exogenous proline application and arbuscular mycorrhizal fungi (AMF) inoculation independently improve chickpea tolerance to salinity stress during the vegetative stage through coordinated regulation of ion balance, osmotic adjustment, antioxidant defense, and anatomical integrity.
Materials and methods
Seeds, soil and mycorrhizal inoculum
Seeds of chickpea (Cicer arietinum L.) “Giza 531” were bought from Agricultural Research Center (ARC), Giza, Egypt. Seeds were surface sterilized with sodium hypochlorite (5%) for 10 min, followed by three rinses with distilled water. Soil was collected from the top layer (0–20 cm) from El-Sharkia Governorate, Egypt and was sterilized to eliminate native AMF by autoclaving at 121 °C for 1 h for 3 successive days. According to Gerdemann and Nicolson [42], the mycorrhizal inoculum was first isolated from El-Sharkia Governorate soil, in Egypt, using wet sieving and decanting processes. For two cycles of five months, the spore combination was grown in pots with sterile sandy clay soil and mounted with roots of Sudan grass (Sorghum sudanenses Pers.) as a trap plant. The AMF spores were Rhizophagus irregularis, Gigaspora margarita, Funneliformis mosseae and F. constrictum.
Experimental design and treatment application
The experiment was carried out under controlled conditions [Temp. 23 °C/14°C (day/night) and relative humidity of 50–75%] at the Botany and Microbiology Department, Faculty of Science, Zagazig University, Egypt (30°58′N Latitude, 31°48′E Longitude). Plastic pots of 30 cm in height and 24 cm in diam. were used and each pot was filled with 2.5 kg of autoclaved sandy clay soil (pH 7.4) then sown with 10 seeds and irrigated with water. After seedling emergence, seedlings were thinned out (6 seedlings/ pot) to maintain uniform seedlings. The experiment included 30 pots (5 replicates × 6 treatments), as follows: Control, Salt, Proline (Pro.), Pro.+Salt, AMF, AMF + Salt. Firstly, the pots were divided into 2 equal groups (AMF and non-AMF inoculated). The AMF-inoculum was added as 25 g/pot at the sowing stage that contained spores (90 spore/g soil), hyphae, colonized root fragments (100%, colonization index), and soil.
The salt stress was applied as 200 mM of NaCl in the irrigation water to 15 pots “3 treatments: Salt, Pro.+Salt and AMF + Salt after 20 days of sowing. The unstressed pots were only irrigated with tap water. After 2 weeks, proline was supplied in the irrigation water at a conc. of 100 mg/L, 3 times over 2 weeks to Pro. and Pro.+Salt pots. The plants were then harvested after 45 days of sowing for further analyses.
AMF estimation
At the end of the experiment, randomly selected roots of chickpea plants were investigated for AMF growth and abundance. These roots are firstly washed with water and then cut into 0.5–1 cm segments. Using Phillips and Hayman [43] technique, the segments were cleared using 10% KOH, acidified in 1 N HCl and stained with 0.05% (w/v) trypan blue to be investigated under the light microscope.
Harvesting and analyses
After 45 days of sowing, plants were harvested and their roots were washed with tap water carefully to wipe out the adhered soil particles. Then morphological, physiological and biochemical analyses were performed. Also, leaf samples were collected for further anatomical studies.
Morphological analyses
Five plants per treatment were randomly taken for the evaluation of growth parameters. The total plant height was measured with the help of a scale in centimeters. A digital weighing machine was used to determine the total fresh (TFW) and total dry (TDW) weights of plant samples. Drying samples was at 60 °C in the oven for 48 h. Branches and leaves were counted per plant and their average number was added.
Na+, K+ and K+/Na+ determination
Na^+^ and K^+^ (mg/g DW) in chickpea root and shoot samples were measured in the oven-dried tissues. The samples were digested by a mixture of acids (perchloric acids, nitric and sulphuric [1:2:3]) until a transparent mixture was obtained. Double-distilled water was used and the volume of the resulting sample was adjusted. The Agilent 4210 MP-AES (Microwave Plasma Atomic Emission Spectrometer, Agilent Inc.) at Helwan University’s Ecology Laboratory, Faculty of Science, performed this analysis. Also, K^+^/Na^+^ was calculated for shoots and roots.
Anatomical investigation
For leaf structure evaluation, chickpea leaves were gathered, cleaned, and then stored in a solution of formalin, glacial acetic acid and 70% ethyl alcohol (5:5:90 v/v) [44, 45]. Subsequently, sectioning was performed at 12–15 μm with a microtome, followed by embedding in paraffin wax and subsequently, affixed to slides utilizing egg albumin as an adhesive agent. The slides were exposed to a decreasing series of ethyl alcohol solutions, varying from 100% to 50% concentrations and staining with double stained (safranin and light green) and mounted in Canada balsam [46]. All light microscopic histological evaluations were conducted in the imaging section of the Department of Human Anatomy and Embryology, Faculty of Medicine, Zagazig University, utilizing the Leica Q Win Plus Image Analysis System (Leica Micros Imaging Solutions Ltd, Cambridge, UK). The selected sections (five from each treatment) were examined in order to visualize the following microscopic features: including upper and lower epidermis, midrib, number of vessels and main vascular bundle thickness of leaves (µm). In addition mesophyll thickness (µm) which includes the length of the palisade and spongy cells was also evaluated.
Physiological analyses
Relative water content (RWC)
The RWC of chickpea leaves was measured according to Schonfeld et al. [47]. Leaves were collected and cut into small segments and FW measurements were taken immediately. The leaves were then soaked in deionized water for 4 h at room temperature. Their turgid weights (TW) were measured after drying the leaves using tissue paper. Their DWs were then obtained by placing the leaves in a hot air oven at 60 °C for 24 h, and RWC was calculated using the following formula:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$RWC\;(\%)=\frac{FW-DW}{TW-DW}\times100$$\end{document}Photosynthetic pigments
According to Metzner et al. [48], chickpea leaf samples (0.25 g FW) were extracted in 85% acetone and then centrifuged at 8000 rpm for 10 min. Using a UV–vis spectrophotometer, the absorbance was read at 452.5, 644, and 663 nm; 85% acetone served as the blank. The conc. of pigments (mg/g FW) was calculated using specific formulas developed by Lichtenthaler and Wellburn’s [49].
Electrolyte leakage
To properly perform electrolyte leakage studies; the chickpea leaves were rinsed with deionized water to eliminate any surface contaminants. Then, leaves were cut into homogeneous leaf pieces that weighed nearly one gram each using a steel cylinder with a diameter of 1 cm. Each of these leaf segments was placed into a test tube with 20 mL of deionized water. The test tubes were kept for 2.5 h at 25 °C to facilitate the electrolytes to permeate from the leaf tissues into the surrounding water. The electrical conductivity (EC1) of the water solution following the incubation period was determined using a pre-calibrated EC meter. The test tubes were then heated for 20 min at 120 °C in a water bath to measure the second electrical conductivity (EC2) [50].
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Electrolyters\;leakage\;(\%)=\left(EC1/EC2\right)\times100$$\end{document}Biochemical analyses
Hydrogen peroxide (H2O2) content
H_2_O_2_ content in chickpea leaves was measured at 390 nm after extracting with 0.1% TCA and centrifuged at 10,000 rpm for 15 min. The extracts were then mixed with a 10 mM phosphate buffer solution (pH 7.0) and KI (1 M) [51].
Proline content
Proline content was assessed calorimetrically employing the previously reported approach of Bates et al. [52]. Leaf tissues (500 mg) were homogenized in 5 mL of 3% sulphosalicylic acid. Equal volumes (2 mL) of the extract, glacial acetic acid and ninhydrin reagent were mixed and incubated for 30 min at 100 °C in a boiling water bath. The reaction mixture was cooled, and then 4 mL of toluene was added to the fume hood to elute it. The mixture was then vortexed, and a UV-visible spectrophotometer was used to measure the absorbance of toluene-containing chromophore at 520 nm.
Carbohydrate content
Carbohydrates (simple sugars, oligosaccharides, polysaccharides, and their derivatives) were determined following the protocol of Nielsen [53] using the phenol sulfuric acid method. In the presence of strong sulfuric acid and heat, carbohydrates react to produce furan derivatives, which condense with phenol to form persistent yellow-gold molecules that are spectrophotometrically measurable.
Total soluble proteins and antioxidant enzymes
To estimate the protein and various kinds of antioxidant enzymes, a known FW of leaf material (1 g) was ground in 5 mL of 50 mM of potassium phosphate buffer (pH 7.0), and 1 mM EDTA. This homogenate was centrifuged for 10 min at 15,000 rpm at 5 °C and the supernatant was then utilized for the following analyses. Protein was assessed by Lowry et al. [54] using an alkaline reagent solution and bovine serum albumin was the standard. With minor modifications, the Aebi ’s method [55] was used to measure catalase (CAT) activity by tracking the decrease in absorbance at 240 nm. Peroxidase (POX) activity was measured using pyrogallol as the substrate at 470 nm using Chance and Maehly [56] technique. The ascorbate peroxidase (APX) and polyphenol oxidase (PPO) activities were measured at 290 nm and 430 nm, respectively, and their activities were expressed as U g^− 1^ FW, following Nakano and Asada [57] and Beyer and Fridovich [58].
Statistical analysis
The treatment effects were evaluated by analysis of variance using SPSS (version: 14). The data presented are the means ± standard error of five replicates (n = 5). The mean values were compared and the significant difference between the control and the treated groups was determined using Duncan’s multiple range test at p ≤ 0.05. Figures were performed using Origin.
Results
Effect of salinity on AMF components and abundance
At the end of the experiment, the whole plant was properly lifted out of the pot by flipping it over. The plant roots were thoroughly cleaned under running water to get rid of any soil particles. The different structural colonization items (hyphae, arbuscules and vesicles) were investigated (Suppl. Fig. 1) in the chickpea root segments and results are presented in (Table 1). Given the soil was sterilized before the experiment; there was no fungal colonization in the non-inoculated plants. The highest root colonization by fungal hyphae (HC%) was recorded under unstressed conditions (98.44%) versus (90%) under salinity. The negative salinity impact is more pronounced in the percentages of AMF arbuscules (ARC%) and vesicles (VC%), as their values were 48.6% and 53.2% lower than their unstressed counterparts, respectively.
Table 1. Impact of salt (200 mM NaCl) stress and exogenous proline (100 mg/L) application on the mycorrhizal features of chickpea (C. arietinum L.) plant roots expressed by hyphal (HC), vesicular (VC) and arbuscular colonization (ARC) percentages in the root systemTreatmentsHC (%)VC (%)ARC (%)Control0b0c0cSalt0b0c0cPro.0b0c0cPro. + Salt0b0c0cAMF98.44 ± 5.20a55.55 ± 2.93a23.43 ± 1.23aAMF + Salt90.00 ± 4.76a26.00 ± 1.37b12.04 ± 0.63bData presented represent the mean of 5 replicates with standard error. Different letters indicate significant differences among treatments using a one-way ANOVA followed by the Duncan’s multiple range test (p ≤ 0.05)
Effect of salinity, AMF and proline on chickpea morphological characteristic
The morphology (plant height, TFW, TDW, branching and leaves number) of chickpeas at the vegetative phase is significantly attenuated by salinity (Fig. 1; Table 2). However, this reduction was alleviated in plants treated with exogenous proline or AMF. A significant variation in plant heights was observed among the different treatments with the shortest (34.5 ± 0.913^f^ cm) plant height under the salt stress and the higher record of unstressed treatments of proline (60 ± 1.587^b^ cm) and AMF (66.5 ± 1.759^a^ cm). Statistical analysis also showed corresponding variation among the results for TFW where the highest fresh biomass (7.96 ± 0.211^a^ g) was observed in the unstressed treatments inoculated by AMF versus (1.66 ± 0.044^f^ g) in salt-stressed plants. A similar trend was observed in all tested morphological parameters, including TDW, number of leaves, and branches. However, the AMF or proline treatments showed significantly better performance with the privilege to AMF inoculation. Under unstressed conditions, the best vegetative growth was also achieved by the AMF treatment followed by proline application as compared to control.
Fig. 1. Effects of AMF colonization and exogenous proline (100 mg/L) addition on morphological appearance (shoots and roots) of chickpea (C. arietinum L.) under salt stress (200 mM NaCl) conditions
Table 2. Effect of AMF colonization and exogenous proline (100 mg/L) application on selected plant growth related parameters of chickpea (C. arietinum L.) under controlled and salt (200 mM NaCl) stressed conditionsTreatmentsPlant height (cm/plant)Total FW(g/plant)Total DW(g/plant)No. Leaves (No./plant)No. Branches (No./plant)Control55.5 ± 1.458c5.97 ± 0.158c0.763 ± 0.021c16 ± 0.423b2 ± 0.053bSalt34.5 ± 0.913f1.66 ± 0.044f0.3415 ± 0.009e12 ± 0.317d0 ± 0.0dPro.60 ± 1.587b7 ± 0.185b0.897 ± 0.024b19 ± 0.503a2 ± 0.053bPro. + Salt41 ± 1.085e3.5 ± 0.093e0.529 ± 0.014d14 ± 0.37c1 ± 0.026cAMF66.5±1.759a7.96 ± 0.211a0.987 ± 0.026a19 ± 0.503a3 ± 0.079aAMF + Salt48 ± 1.27d4.05 ± 0.107d0.5704 ± 0.015d15 ± 0.396bc1 ± 0.026cData represent the mean of 5 replicates with standard error. Different letters indicate significant differences among treatments using a one-way ANOVA followed by the Duncan’s multiple range test (p ≤ 0.05)
Na+ and K+ contents and K+/Na+ in chickpea plants exposed to 200 mM NaCl, AMF and proline
Table 3 showed that the shoots and roots of stressed chickpea plants not given proline or AMF treatment had the greatest Na^+^ concentration with more Na^+^ in roots (25.16 mg/g DW) than shoots (15.2 mg/g DW). Table 3’s results also demonstrated that stressed plants treated with either proline or AMF had much lower Na^+^ contents in their roots and shoots compared to salt-stressed plants alone, with AMF having a superior effect. Exogenous proline decreased the amount of Na^+^ in the shoots by 50.65% and in the roots by 27.96%, while AMF decreased its amount by 52.17% and 29.30%, respectively. Consequently, the greatest K^+^/Na^+^ (Fig. 2) is found in unstressed mycorrhizal plants (13.47 in shoots, 3.03 in roots) followed by proline application (11.12 in shoots, 2.46 in roots) in contrast to those of NaCl-stressed plants (0.84 and 0.43).
Table 3. Effect of AMF colonization and exogenous proline application (100 mg/L) on Na^+^ and K^+^ (mg/g DW) content in shoots and roots of chickpea (C. arietinum L.) under controlled and salt (200 mM NaCl) stressed conditionsTreatmentsNa^+^ content (mg/g DW)K^+^contentShootsRootsShootsRootsControl3.61 ± 0.095c10.18 ± 0.269c31.47 ± 0.833c23.08 ± 0.611bSalt15.2 ± 0.402a25.16 ± 0.666a12.78 ± 0.338e11.01 ± 0.291dPro.3.17 ± 0.084c10 ± 0.26c35.26 ± 0.932b24.55 ± 0.65bPro. + Salt7.5 ± 0.198b18.125 ± 0.48b21.58 ± 0.571d16.49 ± 0.436AMF3.01 ± 0.079c9.25 ± 0.245c40.55 ± 1.07a28 ± 0.741aAMF + Salt7.27 ± 0.19b17.788 ± 0.471b23.07 ± 0.61d18.11 ± 0.479cData represent the mean of 5 replicates with standard error. Different letters indicate significant differences among treatments using a one-way ANOVA followed by the Duncan’s multiple range test (p ≤ 0.05)
Fig. 2. Impact of AMF colonization and exogenous proline (100 mg/L) application on potassium/sodium (K+/Na+) in shoots (a) and roots (b) of chickpea (C. arietinum L.) grown under controlled and salt (200 mM NaCl) stressed conditions. Different letters indicate significant differences among treatments using a one-way ANOVA followed by the Duncan’s multiple range test (p ≤ 0.05)
Chickpea leaf anatomy in response to salinity, AMF and proline
Results regarding the effect of NaCl, AMF and proline on chickpea leaf anatomy were illustrated in Table 4; Figs. 3, 4 and 5. Our findings revealed that the mesophyll of untreated control chickpea plants is composed of one layer of palisade parenchyma and 3–4 layers of sponge parenchyma with intercellular space and the midrib has a collateral vascular bundle. In contrast, salt (200 mM NaCl) stress led to a decrease in the thickness of the upper and lower epidermis, phloem and xylem tissues with lower vessel numbers. The present results (Table 4) revealed that the vascular bundle’s thickness reduced at salinity as it reported a 25.33 μm lower value compared with 42.116 μm in untreated control plants. There is a significant effect of the amino acid “proline” treatment where this treatment recorded the highest values in the thickness of mid bundle, upper and lower epidermis, thickness of midrib as well as increase in layers of palisade mesophyll tissue as become three layers and reaching 60.357, 18.987, 10.878, 255.775, 112.975, 55.549 μm, respectively when compared with control treatment which their recoded values in these studied characteristics were 42.116, 17.025, 10.782, 177.524, 53.619, 55.549 μm, respectively (Table 4). Regarding the effect of AMF on chickpea leaf anatomy under salt stress, AMF caused a slight increase in leaf thickness, which is accompanied by an increase in the thickness of the spongy and palisade tissues. Additionally, this treatment increased the thickness of the vascular bundle as compared to salt-stressed plants.
Table 4. Impact of AMF colonization and exogenous proline (100 mg/L) application on anatomical characteristics of flag leaf of chickpea (C. arietinum L.) exposed to salt stress conditionsTreatmentsThickness of mid bundleVessels No.Thickness of upper epidermisThickness of lower epidermisThickness of midribThickness of Palisade tissueThickness of spongy tissueControl42.116 ± 2.228b13 ± 0.687b17.025 ± 0.900a10.782 ± 0.570b177.524 ± 9.393bc53.619 ± 2.837c61.013 ± 3.228abSalt25.330 ± 1.340c6 ± 0.317d7.420 ± 0.392c6.805 ± 0.360c148.552 ± 7.860c73.428 ± 3.885b57.532 ± 3.044abPro.60.357 ± 3.193a16 ± 0.846a18.987 ± 1.004a10.878 ± 0.575b255.775 ± 13.534a112.975 ± 5.978a55.549 ± 2.939abPro. + Salt42.180 ± 2.231b10 ± 0.529c17.786 ± 0.941a10.546 ± 0.558bc230.767 ± 12.211a74.112 ± 3.921b51.565 ± 2.728bcAMF53.770 ± 2.845a17 ± 0.899a16.825 ± 0.890a9.239 ± 0.488bc181.345 ± 9.595bc70.224 ± 3.715b45.928 ± 3.430cAMF + Salt37.183 ± 1.967b11 ± 0.582bc10.328 ± 0.546b50.610 ± 2.678a193.607 ± 10.244b71.768 ± 3.797b64.203 ± 3.397aData represent the mean of 5 replicates with standard error. Different letters indicate significant differences among treatments using a one-way ANOVA followed by the Duncan’s multiple range test (p ≤ 0.05)
Fig. 3. Impact of AMF colonization and exogenous proline (100 mg/L) application on whole leaflet of chickpea (C. arietinum L.) under controlled and salt (200 mM NaCl) stressed conditions
Fig. 4. Impact of AMF colonization and exogenous proline (100 mg/L) application on midrib of chickpea (C. arietinum L.) under controlled and salt (200 mM NaCl) stressed conditions
Fig. 5. Impact of AMF colonization and exogenous proline (100 mg/L) application on mesophyll of chickpea (C. arietinum L.) under controlled and salt (200 mM NaCl) stressed conditions. UE = upper epidermis, LE = lower epidermis, PL = Palisade tissue, SP = spongy tissue
Physiological and biochemical response of chickpea plants to salinity, AMF and proline
Relative water content (RWC)
As one crucial indicator of plant stress, NaCl (200 mM) significantly reduced RWC (65.24 ± 1.73^d^ %) as compared to the control treatment (81.64 ± 2.16^bc^ %) (Table 5). However, AMF inoculation followed by proline noticeably maintained the highest RWC (90.49 ± 2.39^a^, 85.87 ± 2.27^ab^) in unstressed conditions (Table 5), while both treatments showed close RWC results under stress (77.07 ± 2.039^c^, 76.27 ± 2.02^c^; respectively).
Table 5. Impact of AMF colonization and exogenous proline (100 mg/L) application on pigment fractions (mg/g FW), relative water content (RWC, %), electrolyte leakage (%) and hydrogen peroxide (H_2_O_2_, mg/g FW) of chickpea (C. arietinum L.) plant leaves exposed salt (200 mM NaCl) stressTreatmentsChlorophyll a + b (mg/g FW)Total pigments (mg/g FW)RWC(%)Electrolyte leakage (%)H_2_O_2_(mg/g FW)Control2.884 ± 0.076b4.083 ± 0.108b81.64 ± 2.16bc28.321 ± 0.749d9.37 ± 0.247cSalt1.413 ± 0.037e2.075 ± 0.055e65.24 ± 1.73d54.720 ± 1.45a13.12 ± 0.347aPro.3.061 ± 0.081b4.310 ± 0.114b85.87 ± 2.27ab25.050 ± 0.663e8.55 ± 0.226cPro. + Salt1.700 ± 0.045d2.497 ± 0.066d76.27 ± 2.02c41.032 ± 1.08b11.04 ± 0.292bAMF3.440 ± 0.091a4.842 ± 0.128a90.49 ± 2.39a22.727 ± 0.601e7.03 ± 0.186dAMF + Salt2.331 ± 0.062c3.299 ± 0.087c77.07 ± 2.039c34.199 ± 0.905c10.68 ± 0.283bData represent the mean of 5 replicates with standard error. Different letters indicate significant differences among treatments using a one-way ANOVA followed by the Duncan’s multiple range test (p ≤ 0.05)
Photosynthetic pigments
All salt-stressed treatments showed a decrease in chlorophyll a + b content and total pigments (Table 5). The NaCl treatment showed the highest loss, their contents decreased by 51% and 49.18%, respectively, compared to the control plants. However, exogenous proline treatment enhanced the photosynthetic capacity and reduced this percentage to 41.05% and 38.84%. This reduction percent was noticeably diminished in the AMF-treated plants to 19.17% and 19.20%. Contrariwise, the unstressed plants recorded higher contents than the control by 19.27% and 18.59% in AMF-treated plants, followed by proline (6.14% and 5.56%).
Electrolyte leakage and H2O2
The present data in Table 5 showed that chickpea leaves under salt stress had an increased accumulation of H_2_O_2_ (13.12 mg/g DW) and higher electrolyte leakage (54.72%). Conversely, the proline or AMF supplementation decreased the H_2_O_2_ and electrolyte leakage (Table 5), indicating that proline and AMF were essential in lowering ROS-induced oxidative stress and protecting cell membrane strength from salt-induced damage.
Compatible solutes and osmoregulators (proline, carbohydrate and protein contents)
Proline, carbohydrate and protein contents were evaluated in response to salt stress (Fig. 6). These osmolytes help maintain cell turgor and protect cellular structures from the damaging effects of high salt concentrations. The NaCl-stressed chickpea plants have significantly higher proline content (41.97%) than control. Also, the accumulation of proline in chickpea leaves was enhanced by its exogenous administration (154.80 µmols/g FW) or AMF inoculation (108.46 µmols/g FW) relative to control (61.13 µmols/g FW). A further enhancement in its content was observed under salt stress (Fig. 6) recording its highest value (174.61 µmols/g FW) in proline-treated samples.
Fig. 6. Impact of AMF colonization and exogenous proline (100 mg/L) application on osmoregulatory substances [a: Proline (µmols/g FW), b: Carbohydrates (mg/g DW) and c: Protein (mg/g FW) contents of chickpea (C. arietinum L.) plant leaves grown under salt (200 mM NaCl) stress. Different letters indicate significant differences among treatments using a one-way ANOVA followed by the Duncan’s multiple range test (p ≤ 0.05)
The total carbohydrate contents in chickpea leaves were significantly enhanced by AMF inoculation and proline application. They increased total carbohydrates by 23.44% and 19.43% under stress and by 39.01% and 36.56% without stress; respectively, when compared to the control (Fig. 6). Protein content significantly varied among different treatments (Fig. 6), the highest content (13.647 ± 0.361 mg/g FW) was found in the stressed plant treated with AMF among all treatments. However, the control plants have the lowest content (7.617 ± 0.201 mg/g FW), which reflects the positive impact of either proline or AMF on plant proteins under normal and stressed conditions.
Antioxidant enzymes (CAT, APX, PPO and POX)
At the biochemical level, chickpea plants employ various mechanisms to counter the oxidative damage caused by salt stress. Our results (Fig. 7) indicating elevated APX, PPO, POX, and CAT enzyme activity under salinity stress. Moreover, the present finding revealed that AMF and proline-treated chickpea plants exhibited a significant increase in enzymatic antioxidant activity in both saline and non-saline environments (Fig. 7).
Fig. 7. Impact of AMF colonization and exogenous proline (100 mg/L) application on antioxidant enzymes [a: CAT (Catalase, U/g FW), b: APX (Ascorbate peroxidase, U/g FW), c: POX (Peroxidase, U/g FW) and d: PPO (Polyphenol oxidase, U/g FW)] of chickpea (C. arietinum L.) plant leaves grown under salt (200 mM NaCl) stress conditions. Different letters indicate significant differences among treatments using a one-way ANOVA followed by the Duncan’s multiple range test (p 0.05)
Discussion
With the shrinkage of arable land globally, an estimated more than 50% of cultivated land will be rendered unproductive by 2050 due to salt stress and the increasing demand for agricultural products as a primary source of food. It is commonly known that during a plant lifecycle, seedlings and the first phases of vegetative growth are the most vulnerable to salinity [59]. Therefore, innovative alternative methods are needed to allow crops to thrive and endure salt stress. So, this work was, undertaken to explore the possible remedial measures by using the mutual association of AMF or the aid of the proline exogenous application.
According to the current findings, it is obvious that salinity impacted the mycorrhizal colonization percentages and AMF development. Although AMF can adapt in a salty environment, the presence of salt lowered the colonization percentages of AMF components (hyphae, vesicles, and arbuscules). The lower mycorrhizal colonization may be due to the growth inhibition of the mycelium, vesicles, and arbuscules under salt stress [60–62]. Furthermore, AMF is directly harmed by Na^+^ suggesting that salinity suppresses the AMF symbiotic effect [63, 64] and this could be due to the fungal inoculum originated from unstressed soil. According to reports, native AMF species that have been isolated from salty environments continue to exhibit increased colonization traits [65, 66]. Salinity stress affects the growth, development, and productivity of crops. All tested morphological criteria (plant height, TFW, TDW, branching and leaves number) are inhibited, which negatively influences the overall functioning of the plant. Nonetheless, the growth of AMF or proline-treated plants was noticeably better, as compared to control. Salt stress decreased plant length, leaf count, and DW of the shoot and root [67, 68]. This reduction may be due to nutritional imbalances, ionic toxicity, which inhibits cell elongation, oxidative damage and ROS generation [69, 70]. Moreover, Pooja et al. [71] discussed potential causes of decreased DW of root and shoot including decreased photosynthetic pigments, nutritional unavailability, and energy expenditure to offset the harmful effects of salt.
It is well known that AMF can adapt to salty environments and are critical to the natural ecology [72]. Consistent with our findings, Huang et al. [73] and Kakabouki et al. [74] found that in legume crops, AMF colonization increases FWs and DWs, and plant height. The root biomass is directly impacted by AMF hyphae, which improves nutrition and water uptake. Most especially, AMF facilitates host NPK and Mg^2+^ uptake through the extensive extraradical hyphal network that allows mycorrhizal plants to explore more soil volume than non-mycorrhizal plants [33, 75]. The results align with those of Abd-Alla et al. [76] and Nouraldinvand et al. [77]. Also, it was found that proline applied exogenously can successfully increase plant’s resistance to salt stress by controlling their endogenous proline metabolism as its exogenous application modulates several aspects of plant growth and development, such as increases in biomass and productivity [78, 79].
Plant salt tolerance relies on minimizing Na⁺ toxicity while maintaining sufficient K⁺ uptake, as salt stress disrupts the cytoplasmic K⁺/Na⁺ balance and increases the Na⁺/K⁺ ratio, impairing osmotic regulation and enzyme function [6, 9, 80, 81]. Under salt stress, chickpea accumulated high Na⁺ levels, particularly in roots, where concentrations were approximately twice those in shoots. This root-based Na⁺ retention likely reflects an adaptive mechanism that limits Na⁺ transport to shoots via xylem exclusion, thereby protecting metabolic processes and supporting improved shoot growth [82]. In this study, treating chickpea plants with either proline or AMF renders lower Na^+^ contents in their roots and shoots. These findings support previous studies on soybeans [83] and faba beans [84], where they suggested that a higher K^+^/Na^+^ is linked to salt tolerance. AMF inoculation enhances K⁺ translocation to shoots by upregulating the SKOR gene, which promotes K⁺ secretion into the xylem, thereby increasing K⁺ accumulation and improving the K⁺/Na⁺ ratio [39, 85]. In addition to preventing disruptions in cellular enzymatic processes and inhibiting protein synthesis, a notable increase in K^+^ concentration in mycorrhizal plants’ roots and leaves also improves stomatal conductance, which raises the amount of water needed for transpiration [22, 86]. Furthermore, mycorrhizal plants can control Na^+^ transport to the upper parts of plant, as well as regulate the Na^+^ internal concentration [39, 87] via their capacity to either exclude Na^+^ from the cytosol or sequester it into the vacuoles, protecting the photosynthetic tissues from harming of Na^+^ ions [39, 88].
Exogenous proline application under saline and non-saline conditions similarly increased K⁺ content and K⁺/Na⁺ ratio while reducing Na⁺ levels Table 3; Fig. 2). These effects align with previous findings showing that proline enhances K⁺ uptake, limits Na⁺ and Cl⁻ accumulation, and alleviates salinity stress in several crops, including Zea mays, Helianthus annuus, and others [28, 89, 90]. The proline-mediated improvement in ionic balance is likely linked to reduced membrane damage and improved water status under salt stress. Most especially, de Freitas et al. [91] reported that external application of proline decreased both Na^+^ and Cl^−^ contents, but increased the K^+^ content and the K^+^/Na^+^ ratio in salt-stressed Zea mays L. Khan et al. [92] demonstrated that exogenous proline alleviated the negative effect of 120 mM salt, and enhanced K^+^ content, and reduced Na^+^ concentration in Helianthus annuus L. Reduced membrane damage and increased water contents during salinity stress are two signs that proline may be involved in altering K^+^/Na^+^.
Results of leaf anatomy revealed a deformity of the mesophyll of salt-stressed chickpea leaf and a decrease in the thickness of the upper and lower epidermis and the vascular bundles which accompanied by degradation of the palisade and spongy parenchyma with large intercellular space in the mesophyll as compared to control plants (Table 4; Figs. 3, 4 and 5). Additionally, a reduction in the size of the vascular bundles was appeared that fully agree with Taie et al.[93] and El-Taher et al. [94]. As, osmotic stress and ionic toxicity from excessive intake of Cl^−^ and Na^+^ cause deficiencies in Ca^2+^ and K^+^ as well as other nutritional imbalances leading to the harmful impact on plant cells [95, 96]. Also, the decrease in the thickness of the vascular bundle might result from procambial activity being hampered and cell division and growth being restricted leading reduction in water transfer [97] as in the mature leaves of Jojoba plants [98] and Vitis vinifera L [99].
The use of proline and AMF treatments revealed an increase in the thickness of the upper and lower epidermis and the vascular bundle relative to the control treatment. The noteworthy ameliorative effect of exogenous proline treatment may result from its influence in enhancing growth, expansion of cells, and chlorophyll pigment levels in the plant so it enhances respiration, resulting in the synthesis of energy molecules (ATP), essential for cell division and growth [40, 100, 101]. Concurrent with the present results, the application of proline improved every histological feature of the lupine plants’ salinity-stressed which implies that the detrimental effects of soil salinity on the stem anatomy are reversed by exogenous proline [31]. AMF further improved leaf anatomy under salt stress by enhancing nutrient uptake, photosynthetic pigments, and biomass through their extensive mycelial networks and stress-adaptive effects, resulting in increased thickness of leaf tissues such as the midrib, epidermis, and mesophyll [95, 102, 103]. This anatomical protection provides a direct, structural explanation for the observed physiological improvements in water relations and photosynthesis. The link between biochemical mitigation (ion balance, antioxidant boost) and anatomical preservation is a significant contribution of this work, supporting similar observations in lupine [31] and highlighting the holistic benefit of these treatments.
As one crucial indicator of salt stress is RWC that decreased in salt-treated chickpea leaves than control (Fig. 6). This is possibly caused by their diminished ability to absorb water, reduced energy and ion imbalance and decreased hydraulic conductivity of the roots at higher salt concentrations [71, 104]. Plants use osmoregulation as a defense mechanism including proline, betaine, polyamines, sugars, organic acids, amino acids, and trehalose against salt stress to counteract this effect [92]. Likewise, the AMF inoculation and proline effectively sustained the highest RWC as in polpar and Capsicum annum as conducted by Han et al. [105] and Pal et al. [106]. AMF colonization has a distinct impact on the expression of tonoplast and plasma membrane aquaporins (PIPs and TIPs), which improves the plant water status [39]. Proline application, a predominant osmolyte, aids in the stability of proteins and enzymes, the quenching of ROS, and the preservation of membrane integrity [28, 107] and subsequently improve the water content of plants.
Into the bargain, salinity has a detrimental effect on photosynthesis because it causes the loss of photosynthetic pigments (Table 5) due to an excess of Na^+^ and Cl^–^ or oxidative stress caused by salinity resulted in a decrease in photosynthesis in a variety of legumes [108, 109]. This response limits the proper CO_2_ utilization, lowering the photosynthetic rate and retards the plant growth and productivity due to the direct effects of salt on stomatal closure [109]. A worth mentioned result was that AMF inoculation and proline application caused significant enhancement in pigments in chickpea plants under control and salt-stressed conditions. Concurrent with our results, Dadasoglu et al. [110] demonstrated a notable decrease in the chlorophyll content of chickpea plants under salt stress while this effect was ameliorated by AMF inoculation that might be achieved by enhanced nutrient uptake, efficient water relation, promoted antioxidative capacity and osmolyte build up [71, 111]. Also, under stress, exogenous proline positively increased the photosynthetic capacity by restoring the water use efficiency, increasing nutrient assimilation and stabilizing the overall photosynthetic processes [84, 112, 113].
One of the most crucial criteria for identifying plants that resist salt is electrolyte leakage from plasma membranes. There is a link between the development of oxidative damage brought on by increased H_2_O_2_ levels and the suppression of growth caused by salt [114]. According to the current study, chickpea leaves under salt stress expressed more electrolyte leakage and accumulated more H_2_O_2_ (Table 5). In contrast, proline or AMF supplementation reduced H_2_O_2_ and electrolyte leakage, suggesting their crucial role in reducing ROS-induced oxidative stress and shielding cell membrane strength from damage. Electrolyte leakage is mostly associated with K^+^ efflux, where its content enhanced in AMF plant cells (Table 2), maintaining lower leakage [115]. Additionally, to keep cells from being dehydrated, plants respond by lowering their water potential through accumulating more osmolytes in mycorrhizal plants than in non-mycorrhizal plants [116]. Consistently, proline application alleviated the oxidative damage by decreasing the stress markers (e.g.: H_2_O_2_ and electrolyte leakage) and scavenging ROS in Brassica juncea L [117].
One of the primary physiological adaptations in chickpea plants is osmotic adjustment by accumulating osmolytes such as proline and soluble carbohydrates [9, 15]. In this study, salinity-stressed chickpea plants elevated their endogenous proline content (Fig. 6), a response also documented in maize and orange under salt stress [91, 118]. Exogenous proline application further increased this accumulation, consistent with findings in chickpeas and peas [29, 119]. The applied proline likely enhanced osmotic potential, supported antioxidant activity, and helped stabilize membranes [25, 120]. AMF inoculation similarly elevated proline levels, a phenomenon observed in mycorrhizal plants that may be linked to enhanced biosynthesis and reduced degradation of proline [121].
Conversely, salt stress reduced leaf carbohydrate content, consistent with earlier findings [118]. This may be due to metabolic disruption, increased respiratory demand, and oxidative damage to carbohydrate metabolism [122]. Interestingly, the total carbohydrate contents in chickpea leaves were significantly enhanced by AMF inoculation and proline application (Fig. 6). AMF-treated plants had the highest carbohydrate content which was linked with the higher increase in photosynthetic pigments (Table 5). Similarly, AMF increased the amount of total carbohydrates by improved photosynthetic quality, reduced oxidative damage, and enhanced uptake of carbon and nutrients [123]. Additionally, proline’s beneficial effect on carbohydrate content was attributed by Abbas et al. [118] and Khan et al. [92] to its function in promoting plant growth and reducing oxidative stress, which causes the accumulation of carbohydrates in the leaves.
Protein content varied significantly among treatments, with the highest level detected in AMF-treated, salt-stressed plants (Fig. 6). This increase may involve the synthesis of stress-responsive proteins, which play vital roles in metabolism and osmoregulation [124, 125]. Nevertheless, the control plants had the lowest concentration, indicating that both proline and AMF have a beneficial effect on plant proteins under both normal and stressful circumstances. Similarly, Gao et al. [126] previously informed the higher protein content in proline-amended stressed celery. Additionally, AMF has been demonstrated to increase the proline, proteins, and K^+^ ions concentration in Ocimum basilicum L. shoots hence improving salinity tolerance [127].
The present results indicating elevated APX, PPO, POX, and CAT enzyme activity under salinity stress which are consistent with Kaur et al. [68] and Yusuf et al. [128] studies on chickpeas and peas. It is well known that these enzymes work together to protect plants from membrane damage caused by H_2_O_2_, converting it H_2_O. Moreover, the present finding revealed that AMF and proline-treated chickpea plants exhibited a significant increase in enzymatic antioxidant activity in both saline and non-saline environments. Several researches has demonstrated that AMF uses an effective ROS scavenging system as one of its ways to mitigate salt stress [22, 39] by increasing the synthesis of antioxidant molecules and boosting the activities of antioxidant enzymes through better uptake of micronutrients [39]. According to Hosseinifard et al. [129] and Rahman et al. [130], proline supplementation significantly raised the levels of antioxidant enzymes under NaCl stress, which is supported by the current study. During salt stress, proline stimulates the expression of genes linked to ion homeostasis and antioxidant biosynthesis [89].
Conclusion
Salinity stress significantly impairs chickpea growth by disrupting key physiological, and biochemical processes and leaf anatomy. Most clearly AMF and proline application improved chickpea performance to salinity via promoting growth, improving total pigments, carbohydrate and protein contents, and regulating antioxidants such as proline, POX, PPO, CAT, and APX. Also, both treatments modulate the leaf structure and anatomy of chickpeas. The success of using AMF and proline can pave the way for sustainable agriculture in salt-affected areas and ensure the continued availability of this important crop for global food security. Beyond assessing individual treatments, this study’s framework supports future investigation into the synergistic potential of combined proline and AMF application, and employ a salinity gradient to define tolerance thresholds. Such dual-treatment approaches may offer enhanced and integrated resilience under salinity. Molecular analyses would further elucidate the specific genes and pathways involved in the observed physiological and anatomical protections.
Supplementary Information
Supplementary Material 1.
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