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Article

Characterization of Physiological and Biochemical Attributes of Neem (Azadirachta indica A. Juss) under Salinity Stress

by
Muhammad Akram
1,
Zunera Sajid
1,
Abu Bakr Umer Farooq
2,
Iftikhar Ahmad
1,*,
Aftab Jamal
3,
Humaira Rizwana
4,
Bandar M. Almunqedhi
4 and
Domenico Ronga
5,*
1
Department of Environmental Sciences, COMSATS University Islamabad, Vehari Campus, Vehari 61100, Pakistan
2
Department of Biotechnology, COMSATS University Islamabad, Vehari Campus, Vehari 61100, Pakistan
3
Department of Soil and Environmental Sciences, Faculty of Crop Production Sciences, The University of Agriculture, Peshawar 25130, Pakistan
4
Department of Botany and Microbiology, College of Science, King Saud University, P.O. 2455, Riyadh 11451, Saudi Arabia
5
Pharmacy Department, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(7), 702; https://doi.org/10.3390/horticulturae10070702
Submission received: 23 May 2024 / Revised: 5 June 2024 / Accepted: 28 June 2024 / Published: 3 July 2024
Browse Figures
Versions Notes

Abstract

:
Salinity poses a significant threat to agricultural productivity worldwide, with its detrimental effects on plant growth and physiological processes. Understanding the mechanisms by which plants respond to salt stress is crucial for developing strategies to mitigate its impact on crop yield and sustainability. To address this issue, a pot study was conducted to determine the effect of salt stress on the physiological and biochemical attributes of neem (Azdiarchta indica A. Juss). Plants were grown for 10 months in pots filled with soil having different salinity levels of 3, 6, 9, 12, 15, and 18 dS m−1 and compared with a control of 1.7 dS m−1. The results showed that plant growth and chlorophyll contents declined as salinity levels increased. Due to oxidative stress, the contents of H2O2 increased under higher salt levels. The mitigation of oxidative stress was achieved through the activation of antioxidant enzymes (catalase, ascorbate peroxidase, peroxidase, and superoxide dismutase). Multivariate analysis indicated that Na+ accumulation in plants was positively related to H2O2 production and enzymatic activities, and negatively related to plant biomass, chlorophyll contents, root and shoot K+ concentration, and root K+/Na+. The experimental results suggest that neem plants can be grown in moderate saline soils.

1. Introduction

Soil salinity is of major environmental stress and seriously affects plant growth and development in arid and semi-arid regions [1,2]. Salinity is a severe abiotic stress that comprises all the problems caused by salts, mainly by the sodium chloride accumulated from natural sources or irrigation [3]. Due to an increase in soil salinity, millions of hectares of cultivated land have become unsuitable for cultivation. It has been estimated that nearly half of the irrigated area is directly or indirectly affected by salinity and/or sodicity [4]. Nearly 7% of the world’s total land area is salt-affected, whereas, in Pakistan, nearly 10 Mha of area has the problem of salinity stress and it contributes to 12.9% of the total country land [5].
Salt-affected lands are prevalent across nearly all climatic regions, spanning from the humid tropics to polar areas. Saline soils are distributed in vast areas with altitudes that range from below sea level, such as the Dead Sea environments, to elevations of more than 5000 m, such as the Tibetan Plateau and the Rocky Mountains. Additionally, saline soils are not limited to desert conditions [6]. Soil salinity is rising in irrigated areas as a result of ineffective irrigation methods, insufficient rainfall, and elevated transpiration rates [2]. When the salt amount increases in soil, it affects plant growth [7]. In plants, high salt levels cause osmotic stress and ionic imbalance which cause numerous biochemical processes [1].
Salinity stress causes many morphological, physiological, and chemical changes in plants [1,8,9]. During the dry season, the poor quality of irrigation water increases the level of soil salinity which results in the reduction in seedling growth and plant development [10]. Salinity has various effects on plants, depending on factors such as the type and concentrations of salts, the duration of time to which the plant is exposed to the stress, the species of the plant, and the stage of growth of the plant [11].
The toxic impacts of salinity on plant growth include the (i) osmotic effect, (ii) ion toxicity and production of reactive oxygen species, and (iii) imbalance in nutrition [10,12]. All the main functions of a plant, including photosynthesis, protein synthesis, and energy and lipid metabolism, are impacted when salt stress first appears and persists [1,13]. Ionic stress causes the early aging of older leaves and often results in toxicity symptoms such as chlorosis and necrosis in mature leaves. This is because the high levels of Na+ interfere with protein synthesis and affect enzyme activity adversely to the plant [14]. Ion toxicity is mainly due to Na+ and Cl− ions which can be rapidly absorbed and taken up by plant roots [15]. The elevated absorption of Na+ and Cl− ions leads to the restricted uptake of vital nutrients like K+, Ca2+,, and NO3− [16,17].
Salinity stress triggers an excess production of intracellular reactive oxygen species (ROS) in plants, including superoxide radicals, singlet oxygen, hydrogen peroxide, and hydroxyl radicals [14]. The excessive production of ROS in plants subjected to salinity stress results in oxidative damage [1]. These excessively produced ROS are detoxified by enzymes and co-enzymes [8], and their activities varied greatly in plant species grown under saline conditions [18]. There is a sequential process of antioxidant enzymes for the scavenging of ROS, e.g., SOD catalyzes the conversion of O2− into H2O2 and O2 which can cleave H2O2 and H2O and O2 [19,20]. Plants having a higher activity of antioxidants maintain a better ionic balance, resulting in the production of higher biomass under saline conditions [21]. A perfect equilibrium between the production of ROS and the initiation of the antioxidant defense mechanisms indicates the potential of the plant to tolerate and survive under adverse environmental stress [22].
Under changing climatic conditions, it is important to explore the salt tolerance potential of valuable medicinal plants. Azadirachta indica A. Juss, also known as neem, is a member of the family meliaceae and is an extremely reputable tree, with many properties and applications, particularly for its incredible therapeutic and ethnomedicinal values for humans [23]. The neem is an evergreen robust tree and is predominantly distributed in tropical and subtropical regions across the globe. It is medium to large in size and has a brown to dark grey trunk and a dense curved crown of pinnate leaves [24]. The plant is reported to live up to two centuries [25]. The neem plant possesses a wide deep root system for its survival in the dry regions of the world.
The neem plant has more than 140 compounds of a very diverse and complex nature. The important classes are triterpenoids and limonoids: saladucin, geducin, valassin, meliacin, nimbin nimbicin, azadirachtin, etc. [26]. Consequently, every part of the neem plant holds significant value and has been utilized in treating various ailments such as tooth decay, ulcers, liver swelling, malaria, dysentery, diarrhea, and more [27]. It is considered a precious raw material for the development of herbal industrial products [28]. However, despite its ethnomedicinal significance, the biochemical mechanisms underlying neem’s salt tolerance remain underexplored.
Neem is classified as moderately salt-tolerant, which means it can withstand moderate levels of soil salinity without severe adverse effects on growth and productivity. Existing studies on neem have shown some mechanisms for salt tolerance. For instance, it has been demonstrated that neem trees can survive and grow in saline conditions through adjusting the osmotic balance by enhancing the activities of antioxidant enzymes and maintaining ion homeostasis [29,30]. Such mechanisms allow neem trees to succeed in saline environments and make them very valuable crops for arid regions. However, the detailed mechanisms of the biochemical pathways and genetic mechanisms in neem’s response to salt stress are still not fully understood.
The studies performed on salt stress in other tree species like Eucalyptus, Acacia, and Prosopis give an idea which can be implemented in neem. For instance, Eucalyptus species showed considerable salt tolerance through ion exclusion, osmotic adjustment, and the enhancement of activities of antioxidants [31,32]. Similarly, Acacia and Prosopis species adapt to saline environments by modulating their physiological and biochemical processes, including the production of osmoprotectants and the activation of antioxidant defense systems [33,34]. Understanding these mechanisms in other species helps in drawing parallels and hypothesizing similar adaptive responses in neem.
Hence, this study hypothesizes that neem possesses specific biochemical attributes enabling it to withstand and ameliorate the adverse effects of soil salinity, thus exhibiting resilience and potential for cultivation in saline environments. To test this hypothesis, our research objectives are twofold: (1) to explore the biochemical properties of neem contributing to its salt tolerance potential and (2) to evaluate the suitability of neem for cultivation in saline areas based on its biochemical responses to salinity stress. By addressing these objectives, we aim to provide valuable insights into harnessing neem’s adaptive characteristics for sustainable agriculture practices in salinized landscapes.

2. Materials and Methods

2.1. Soil Properties and Treatments

A pot experiment was conducted in the Department of Environmental Sciences, COMSATS University Islamabad, Vehari Campus, to explore the salt tolerance and growth performance of neem (Azadirachta indica A. Juss) under salinity stress conditions. The sandy loam soil (7% clay, 49% silt, and 44% sand) used in the experimental pot had 0.4% of organic matter with electrical conductivity of saturated extract (ECe), i.e., 1.7 dS m−1, pH of 8.0, total nitrogen 7.83 ppm, available phosphorus 5.7 ppm, exchangeable potassium 118.3 ppm, and the saturation percentage of the soil was 35%. Before filling the soil in the pots, the calculated amount of sodium chloride (NaCl) salt was mixed with the soil of each pot to achieve the desired salinity levels (3, 6, 9, 12, 15, and 18 dS m−1). Three months old seedlings were collected from a nursery of District Khanewal, Pakistan. The seedlings were then transplanted in pots having soil of various salinity levels, as described before. The surface area of each pot was 0.035 m2 and every pot was loaded with 9 kg of soil. The pots were randomly arranged with four replications using a completely randomized design (CRD).

2.2. Growth Conditions

The current experiment was performed in a greenhouse. The temperature was controlled to mitigate the effects of fluctuations on the plants. Also, a relative humidity of 70% was maintained inside the greenhouse. Throughout daytime hours, from 09:00 to 17:00, the temperature was maintained at 29 °C, whereas during the night, it was regulated at 21 °C. The length of the day followed natural conditions. The pots were irrigated with tap water at the same time as and when required. Fertilizer was applied with a 20-10-10 NPK ratio, calculated at a rate of 453.6 g of fertilizer for every inch of trunk diameter. Urea (46% N), diammonium phosphate containing 18% of N, and 46% of P2O5 and sulphate of potash having 50% of K2O were used as a source of nitrogen, phosphorus, and potassium, respectively. Hoeing was performed to remove the weeds. However, no fungicides and pesticide were used during the period of experimentation.

2.3. Harvesting and Growth Measurements

The plants were harvested after a period of ten months and were separated into root and shoot. After harvesting, first the samples were air-dried under shade and then dried in an oven at 65 °C until the constant weight was attained. After that, dry root and shoot dry weight were determined by using a digital weighing balance.

2.4. Determination of Antioxidants

For the determining of enzymatic antioxidant activities, i.e., catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and superoxide dismutase (SOD), 0.5 g of fresh leaves of the neem plant was grinded to a fine powder under liquid nitrogen with a mortar and pestle. After that, the fine ground powder was homogenized in the potassium phosphate buffer solution (100 mM) of 6 mL with a pH of 7.0, supplemented with 1 mM of EDTA-Na2 and 2% (w/v) polyvinylpyrrolidone-40 (PVP-40). After homogenization, the mixture underwent centrifugation at 11,000 rpm for 15 min at the temperature of 4 °C. Following centrifugation, the supernatant (sample) was collected for the determination of the antioxidant enzyme.
The procedure outlined by Aebi [35] was adopted for the determination of catalase activity. The reaction mixture had potassium phosphate buffer (50 mM) of pH 7.0, H2O2, (10 mM), and enzyme extract of 50 µL. The decomposition of H2O2 was measured at a 240 nm wavelength using a UV–visible spectrophotometer, and the absorbance was recorded. The enzymatic activity was quantified as U mg−1 of protein (units per milligram of protein).
The activity of ascorbate peroxidase (APX) was measured as outlined by Amako et al. [36]. The assay mixture (3 mL) comprised ascorbate (0.25 mM), potassium phosphate buffer (50 mM) of pH 7.0, H2O2, (0.25 mM), and an enzyme extract of 50 µL. The absorbance was recorded at 290 nm using a UV–visible spectrophotometer. The extinction coefficient composed of 2.8 mM−1 cm−1 was used to determine the ascorbate peroxidase activity (U mg−1 of protein).
The assay of peroxidase (POD) activity was determined by following the protocol of Hemeda and Klein [37]. The assay mixture consists of phosphate buffer (50 mM) of pH 6, H2O2 (15 mM), guaiacol (12 mM), and enzyme extract (200 μL). The reaction mixture was run on a UV–visible spectrophotometer at 470 nm to determine the POD activity and it was presented as the units per mg of protein (U mg−1 of protein).
Superoxide dismutase (SOD) activity was measured according to the method as described by Dhindsa et al. [38]. The reaction was performed in test tubes that contained a 3 mL reaction mixture containing EDTA (0.1 mM), riboflavin (60 µM), NBT (75 µM), methionine (13 mM), phosphate buffer (50 mM) with a pH of 7.8, and an enzymatic extract of 50 µL. After that, tubes were illuminated for 15 min by two fluorescent lamps to start the reaction. The absorbance was determined at a wavelength of 560 nm with a UV–visible spectrophotometer. One unit of SOD activity is calculated on the basis of quantity that reduced the 50% absorbance as compared to the control.

2.5. Total Chlorophyll, Protein Contents, and Reactive Oxygen Species (H2O2)

The method of Lichtenthaler [39] was used for the determination of leaf chlorophyll contents. The samples of fresh leaves (0.5 g) were ground in 80% acetone at 4 °C. The centrifugation of the extracts was conducted at 10,000 g for 5 min and the sample was run at a wavelength of 480 nm, 646 nm, and 663 nm, respectively, on a UV–visible spectrophotometer.
Total Chl = [(20.2(OD645) + 8.02(OD663)] V/1000 × W
where V is considered as the volume of the extract (mL) and W is considered as the weight of the fresh leaf tissue (g).
Reactive oxygen species (H2O2) were determined according to the procedure of Islam et al. [40]. The reaction mixture contained phosphate buffer solution (10 mM) of pH 07 and its volume was made to 1000 mL. Then, 132.8 g of acetone ground sample was added to buffer solution (10 mM) of 1 mL and potassium iodide of 2 mL. After being left for 15 min, the mixture was analyzed using a spectrophotometer set to measure absorbance at 390 nm.
The total protein contents of the fresh leaves extract was determined by using the Bradford method [41]. The frozen fresh leaves samples (0.25 g) were ground with a mortar and pestle in 3 mL of potassium phosphate buffer (0.1 M) of pH 7. After centrifugation at 10,000× g for 20 min at a temperature of 4 °C, the supernatant obtained was kept frozen for protein assays. The sample absorbance was measured at a wavelength of 595 nm on a UV–vis spectrophotometer (Lambda 25, PerkinElmer, Inc., Waltham, MA, USA).

2.6. Determination of Ion Contents

The oven-dried samples of root and shoot were ground to a fine powder and then passed through a 2 mm sieve. These samples were digested using HNO3 and H2O2 in a 2:1 fraction [42]. The concentrations of the Na+ and K+ ions in digested samples were measured on a BWB-XP5 flame photometer.

2.7. Statistical Analysis

The data collected were tabulated and prepared for analysis using Statistix version 8.1 (Analytical Software, Tallahassee, FL, USA). Furthermore, the treatment means having significant differences were compared and separated by using the least significant difference (LSD) test at a level of significance of 5%. Principal component analysis of the evaluated attributes and the visual representation of the data were created using OriginPro Software (Origin Lab Corporation, Northampton, MA, USA), version 2024.

3. Results

3.1. Plant Growth

The impact of salinity stress on the root and shoot dry weights of the neem plant was prominent and with the rise in salinity levels, the root and shoot dry weight (DW) was decreased. Although the effect of salinity stress on root dry weight was found to be non-significant, the maximum values of root and shoot DW were recorded at control treatment and minimum values were recorded at the highest salinity level, i.e., 18 dS m−1 (Figure 1a,b). Salinity stress had a more pronounced effect on shoot DW compared to root DW (Figure 1b).

3.2. Antioxidant Activity

The analyzed data (Figure 2a) show that catalase activity was remarkably increased along with the increase in the salinity levels in the soil. Although, the highest activity of catalase was recorded at 18 dS m−1 and was statistically at par to the activity resulted at 9 dS m−1 treatment. The non-significant difference of catalase activity was observed below the salinity level of 9 dS m−1, except the control treatment (Figure 2a).
Ascorbate activity (APX) was recorded at its maximum at the highest salinity level, i.e., 18 dS m−1, and the minimum level was observed at the lowest salinity levels (control) (Figure 2b). Peroxidase activity (POD) was recorded at its maximum at a higher salinity level (18 dS m−1) and minimum at a lower salinity level (control), which shows that POD activity was remarkably increased by the increase in salinity level. Statistically non-significant differences regarding POD activity were observed among higher salinity treatments (Figure 2c). Superoxide dismutase (SOD) activity also increased with an increase in the salinity level in the soil and the maximum was found at an elevated salinity level, i.e., 18 d S m−1 (Figure 2d) and minimum in the control treatment.

3.3. Total Chlorophyll, Protein, and H2O2 Contents

In the present study, the total chlorophyll contents decreased with increasing the salinity level; however, the decrease was statistically significant particularly at higher salinity levels compared to the lower salinity treatments in the soil (Figure 3a). The protein contents increased by increasing the salinity levels and the maximum was recorded at 18 dS m−1 (Figure 3b). The increasing salinity levels in the soil caused an increase in H2O2 activity. The activity of H2O2 was maximum at the salinity level of 18 and 15 dS m−1 and the minimum was recorded at a lower salinity level, i.e., the control treatment (Figure 3c).

3.4. Root and Shoot Ion Contents

The analyzed data presented in Figure 4a,b showed that sodium (Na+) concentration increased both in root and shoot with increasing the salinity levels in the soil. In root and shoot, the minimum value of Na+ concentration was found at the control treatment. The increase in Na+ concentration was more pronounced in the root as compared to the shoot of the neem plant (Figure 4a).
Figure 4c,d show the potassium (K+) concentration in the root and shoot, respectively. The analyzed data also indicate that with increasing the soil salinity level, the K+ concentration of both the root and shoot of the plant starts decreasing. The maximum concentration of K+ was observed in the control while the minimum was recorded at the highest salinity levels (18 dS m−1). By elevating the salinity levels, there was a notable reduction in K+ concentration, particularly in the shoots compared to the roots (Figure 4d).
Salt stress in soil reduces significantly the ratio of K+/Na+ both in the root and shoot (Figure 5a,b) of the neem plant. It was observed that both in root and shoot, the maximum value of the K+/Na+ ratio was found in the control and after that, it started sharply decreasing and the minimum value was found at the highest salinity level (18 dS m−1). The shoot maintained the higher K+/Na+ ratio as compared to the root (Figure 5b).

3.5. Correlation Analysis

Pearson’s correlation coefficient indicated a noteworthy positive correlation of H2O2 with CAT, APX, and SOD and a negative correlation with chlorophyll contents. A significant positive (r = 0.62 *) correlation between SDW and chlorophyll contents was also observed. There was a highly significant negative correlation between root and shoot Na+ and K+ concentrations (Table 1).

3.6. Principal Component Analysis (PCA)

To estimate the correlation between different variables, we performed Principal Component Analysis (PCA) (Figure 6). The contributions of different components in the PCA are shown on the x-axis (PC1) and y-axis (PC2). PC1 explains 87.4% of the variance, and PC2 explains 6.8%, together accounting for 94.2% of the total variance in the dataset. The results indicated the strong positive correlation between Na+ uptake by plant parts (root and shoot) and H2O2, POX, CAT, APX, SOD, and total protein. This strong positive correlation was further evident by the combined clustering of these parameters in PCA. Furthermore, on comparison, the separate clustering of treatments was observed in PCA (Figure 6).

4. Discussion

Biomass production by root and shoot was decreased in neem with increasing salt levels. This reduction in growth under salinity is attributed to the osmotic effect, ion toxicity, and oxidative stress, which have been widely reported in previous studies [1,8,14]. Salinity stress caused a gradual decrease in the dry weight (DW) of both the root and shoot of the neem plant. Shahid et al. [43] also found that salinity stress is a major cause of reduction in plants’ DW, which may be due to a reduction in tissue water contents (as a result of the osmotic pressure of soil), which affects the cell division and differentiation as it reduces the uptake of water in plants and eventually causes a decrease in plant growth and the DW of root and shoot [1].
In the present investigation, H2O2 activity was reduced under low salinity levels (control) and enhanced significantly with higher salinity levels. This trend agreed with previous work that indicated that plants under stressful conditions increase the activities of antioxidant enzymes, including catalase (CAT) and peroxidase (POD), for the detoxification of ROS and the alleviation of oxidative damage [44,45]. The higher activity of H2O2 under 18 and 15 dS m−1 salinity might indicate that these salinity levels are the most stressful for the plants and, hence, they require more antioxidant defenses. This is in line with the concept that, if not properly managed, salt stress may drastically deteriorate plant cells and membranes [46]. Reactive oxygen species levels increase when plants are exposed to abiotic and biotic stress. The oxidation of fatty acids and other cellular activities like photorespiration are caused by these ROS [47].
In the present study, the activities of enzymes increased in the neem plant with an increase in salinity levels. It was observed that at the lower level of salinity, the activities of CAT, APX, and SOD were lower and then they increased gradually with an increase in the salinity level because plants produce antioxidants to remove and detoxify ROS [44].
The plants produce a high level of antioxidant enzymes including CAT, APX, and SOD in the intracellular environment under salinity stress for maintaining a balance between the formation and removal of H2O2 [9]. The sensitivity of plants to salinity stress is determined by their level of antioxidant enzymes such as CAT, POD, and SOD [48]. The results regarding SOD activity indicated that it remarkably increased at a higher salinity level (18 dS m−1) as compared to the control. Ryang et al. [49] also stated that an increase in SOD activity is related to the higher ability of plants to tolerate salinity. The CAT activity under the salinity stress condition increases with an increase in salinity levels which may be for the elimination of H2O2 [50].
In the present study, APX activity was also increased with an increase in the salinity level. To determine the action of antioxidants, it is important to find out the affinity of enzymes for their substrate. Peroxidase showed a better mechanism for defense against salinity stress [51]. The increase in POD activity by increasing the salinity level in the growth medium may be due to the ability of plants having high POD levels to protect themselves from oxidative stress [52].
The chlorophyll content gradually declined with an increase in salinity levels, and it was maximum at a lower salinity level and minimum at a higher salinity level. These results are similar to Iqbal et al. [1], who found that chlorophyll contents decrease with an increase in the salinity level, which may be due to chlorophyll enzyme degradation due to salt-induced acceleration [14]. Similarly, another study reported that the chlorophyll content in neem leaves was significantly lowered under salt stress conditions. The greatest reduction occurred at the highest salinity level of 12 dS m−1, where total dry matter decreased by 72% and relative water content by 40% compared to control plants [53]. In the present study, the total protein contents were increased slightly by increasing the salinity stress levels in the soil. This might be due to the fact that salt-tolerant cultivars have evolved mechanisms to cope with salt stress by accumulating more proteins [54]. These proteins help protect cells from stress effects and maintain physiological processes under challenging conditions [55]. Numerous research studies have reported similar results for various crops [56,57,58]. The proteins are also involved in Na+ extrusion, also reducing the cytosolic Na+ accumulation and its toxic effects [59,60]. Some proteins play an integral role in Na+, Cl−, and K+ transport from roots to shoots and are mediated by Na+ loading and unloading in the xylem as well as exclusion from roots to soil [61].
The transport of ions from soil to plant and also within the plant is altered by salt stress [62]. Plants have different mechanisms for salt tolerance depending on their types; for example, some plants remove their Na+ ion at the root level while some transport it to leaves where it is accumulated in the vacuole in order to protect from the toxicity of Na+ and Cl− [8,44]. Ionic imbalance can occur due to the high concentration of Na+ and Cl− [63]. The concentration of sodium (Na+) in both the root and shoot of neem plants increased with rising levels of salinity. This increase in Na+ was more obvious in the root compared to that of the shoot, indicating a differential distribution pattern of Na+ within the plant under salinity stress [64]. Salinity is a major cause of this ionic imbalance because an increase in Na+ concentration results in a corresponding decrease in K+ concentration [65]. This result aligns with the research conducted by Omar et al. [29], which shows greater Na+ accumulation in the roots of neem plants compared to the shoots under salinity stress.
In the present study, a decrease in K+ concentration was found with increasing salinity levels in the growth medium. It has been reported that due to an increase in NaCl concentration in the soil, the root and shoot K+ concentration is decreased [66], which may be due to the reason that K+ channels are occupied by Na+ under high salt stress [65].
The concentrations of root and shoot K+/Na+ in neem declined with a rise in the salinity level, and the lowest value was observed at the most elevated salinity level. Salt diminishes the K+/Na+ ratio in plants, and typically, the plant’s salt tolerance is evaluated by assessing the cytosolic ratio of K+/Na+ along with the cellular metabolic state [2,43]. Ionic toxicity can occur due to a reduction in the K+ content and K+/Na ratio under saline conditions, and thus plant growth is greatly affected [67,68].

5. Conclusions

The neem plant has moderate salt tolerance capacity as the dry weight (DW) of the plant significantly decreases with increasing the salinity level. Although the increasing levels of salinity in the soil caused the maintenance of antioxidants’ enzymes, it is due to the activation of the antioxidant defense system, such as CAT, APX, and SOD, which significantly increased in salinity stress to mitigate the stress conditions in neem plants. The reduction in chlorophyll contents, which increase in salinity level, conferred adverse effects of salinity on the chlorophyll content of neem plants. Salinity also has a significant effect on the Na+ as well as K+ concentration of the plant. Correlation and principal component analyses offered a deeper understanding of the interconnections among various parameters and highlighted the strong positive correlation between Na+ uptake and oxidative stress-related parameters, as well as the total protein content. It is worth noting to recognize the limitations of this study; notably, open field trials have not been conducted, and salinity effects on the quality of neem plants are assessed. These are major limitations that need further consideration. Future research initiatives should incorporate these elements to improve the comprehensive understanding of salinity’s impact on the forest ecosystem. Moreover, further investigation into the molecular aspects of salinity stress across various growth stages of the neem plants is crucial for gaining deeper insights into its metabolic and physiological processes.

Author Contributions

Conceptualization, M.A. and I.A.; methodology, Z.S.; software, Z.S.; validation, I.A., M.A. and A.B.U.F.; formal analysis, A.J.; investigation, Z.S. and A.B.U.F.; resources, I.A.; data curation, A.J., H.R. and B.M.A.; writing—original draft preparation, M.A., Z.S. and A.B.U.F.; writing—review and editing, I.A., D.R., A.J., H.R. and B.M.A.; visualization, I.A.; supervision, D.R.; project administration, H.R.; funding acquisition, D.R. and H.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Researchers Supporting Project number (RSPD2024R1048) at King Saud University, Riyadh, Saudi Arabia.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors due to institutional data protection.

Acknowledgments

The authors extend their appreciation to the Researchers Supporting Project number (RSPD2024R1048), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effect of salinity levels on root DW per plant (a) and shoot DW per plant (b) of neem plant grown under different levels of soil salinity. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation.
Figure 1. Effect of salinity levels on root DW per plant (a) and shoot DW per plant (b) of neem plant grown under different levels of soil salinity. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation.
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Figure 2. Effect of salinity levels on catalase (CAT) activity (a), ascorbate peroxidase (APX) activity (b), peroxidase (POD) activity (c), superoxide dismutase (SOD) activity (d) of neem plant. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation.
Figure 2. Effect of salinity levels on catalase (CAT) activity (a), ascorbate peroxidase (APX) activity (b), peroxidase (POD) activity (c), superoxide dismutase (SOD) activity (d) of neem plant. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation.
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Figure 3. Effect of salinity levels on total chlorophyll (a), total protein (b), and H2O2 (c) of neem plant. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation. FW = Fresh weight of leaf sample.
Figure 3. Effect of salinity levels on total chlorophyll (a), total protein (b), and H2O2 (c) of neem plant. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation. FW = Fresh weight of leaf sample.
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Figure 4. Effect of salinity levels on root Na+ (a), shoot Na+ concentration (b), root K+ (c), shoot K+ concentration (d) of neem plant. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation.
Figure 4. Effect of salinity levels on root Na+ (a), shoot Na+ concentration (b), root K+ (c), shoot K+ concentration (d) of neem plant. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation.
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Figure 5. Effect of salinity levels on root K+/Na+ ratio (a) and shoot K+/Na+ ratio (b) of neem plants. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation.
Figure 5. Effect of salinity levels on root K+/Na+ ratio (a) and shoot K+/Na+ ratio (b) of neem plants. The data are shown as the averages of three replicates. Different letters following the means denote statistical significance at p < 0.05. The error bars represent the standard deviation.
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Figure 6. Comparisons of different parameters of neem plant grown under salt stress using PCA. T0–T6: salinity levels (3, 6, 9, 12, 15, and 18 dS m−1), POD: peroxidase, CAT: catalase activity, H2O2: reactive oxygen species, SOD: superoxide dismutase, APX: ascorbate peroxidase, Na-Root: root sodium concentration, Na-Shoot: shoot sodium concentration, K/Na-shoot: ratio of potassium to sodium concentration in the shoot, K/Na-root: ratio of potassium to sodium concentration in the root, K-shoot: shoot potassium concentration, K-root: root potassium concentration, Root-D.W: root dry weight, Shoot-D.W: shoot dry weight.
Figure 6. Comparisons of different parameters of neem plant grown under salt stress using PCA. T0–T6: salinity levels (3, 6, 9, 12, 15, and 18 dS m−1), POD: peroxidase, CAT: catalase activity, H2O2: reactive oxygen species, SOD: superoxide dismutase, APX: ascorbate peroxidase, Na-Root: root sodium concentration, Na-Shoot: shoot sodium concentration, K/Na-shoot: ratio of potassium to sodium concentration in the shoot, K/Na-root: ratio of potassium to sodium concentration in the root, K-shoot: shoot potassium concentration, K-root: root potassium concentration, Root-D.W: root dry weight, Shoot-D.W: shoot dry weight.
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Table 1. Correlation coefficient analysis of different parameters of neem plant under salinity stress.
Table 1. Correlation coefficient analysis of different parameters of neem plant under salinity stress.
ParametersCATAPXPODSODChlorophyllH2O2Root
DW		Shoot
DW		Na+
Root		Na+
Shoot		K+
Root		K+
Shoot		K+/Na+ RootK+/Na+
Shoot
CAT1
APX0.65 **1
POD0.54 *0.36 NS1
SOD0.79 **0.67 *0.23 NS1
Chlorophyll−0.52 *−0.39 NS−0.26 NS−0.491
H2O20.67 *0.55 *0.480.59 *−0.441
Root DW−0.40−0.52 *−0.11 NS−0.39 NS0.29 NS−0.451
Shoot DW−0.42−0.36 NS−0.13 NS−0.21 NS0.62 *−0.440.421
Na+ Root0.88 **0.83 **0.52 *0.89 **−0.59 *071 **−055 *−0.381
Na+ Shoot0.89 **0.78 **0.440.91 **−0.64 *0.67 *−0.47 NS−0.41 NS0.96 **1
K+ Root−0.86 **−0.81 **−0.36 NS−0.90 **0.51 *−0.62 *0.490.35 NS−0.94 **−0.96 **1
K+ Shoot−0.67 *−0.74 **−0.30 NS−0.70 **0.34 NS−0.56 *0.22 NS0.15 NS−0.76 **−0.76 **0.76 **1
K+/Na+
Root		−0.34 NS−0.21 NS0.07 NS−0.480.36 NS−0.27 NS0.30 NS0.24 NS−0.77 **−0.42 NS0.35 NS0.15 NS1
K+/Na+
Shoot		−0.53 *−0.52 *−0.12 NS−0.55 *0.44−0.430.490.36 NS−0.63 *−0.63 *0.63 *0.410.401
** = Correlation is significant at the 0.01 level. * = Correlation is significant at the 0.05 level. CAT = Catalase, APX = Ascorbate peroxidase, POD = Per oxidase, SOD = Superoxide dismutase, DW = Dry weight, Na+ = Sodium, K+ = Potassium. NS = non-significant.
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Akram, M.; Sajid, Z.; Farooq, A.B.U.; Ahmad, I.; Jamal, A.; Rizwana, H.; Almunqedhi, B.M.; Ronga, D. Characterization of Physiological and Biochemical Attributes of Neem (Azadirachta indica A. Juss) under Salinity Stress. Horticulturae 2024, 10, 702. https://doi.org/10.3390/horticulturae10070702

AMA Style

Akram M, Sajid Z, Farooq ABU, Ahmad I, Jamal A, Rizwana H, Almunqedhi BM, Ronga D. Characterization of Physiological and Biochemical Attributes of Neem (Azadirachta indica A. Juss) under Salinity Stress. Horticulturae. 2024; 10(7):702. https://doi.org/10.3390/horticulturae10070702

Chicago/Turabian Style

Akram, Muhammad, Zunera Sajid, Abu Bakr Umer Farooq, Iftikhar Ahmad, Aftab Jamal, Humaira Rizwana, Bandar M. Almunqedhi, and Domenico Ronga. 2024. "Characterization of Physiological and Biochemical Attributes of Neem (Azadirachta indica A. Juss) under Salinity Stress" Horticulturae 10, no. 7: 702. https://doi.org/10.3390/horticulturae10070702

APA Style

Akram, M., Sajid, Z., Farooq, A. B. U., Ahmad, I., Jamal, A., Rizwana, H., Almunqedhi, B. M., & Ronga, D. (2024). Characterization of Physiological and Biochemical Attributes of Neem (Azadirachta indica A. Juss) under Salinity Stress. Horticulturae, 10(7), 702. https://doi.org/10.3390/horticulturae10070702

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