Effect of salt stress on growth, physio-chemical mechanisms and anatomical alterations of Nigella sativa L

doi:10.21203/rs.3.rs-2979057/v1

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Effect of salt stress on growth, physio-chemical mechanisms and anatomical alterations of Nigella sativa L

https://doi.org/10.21203/rs.3.rs-2979057/v1

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Nigella sativa L. (Black cumin ) is a medicinal plant, well known for its uses as herbal medicine for the treatment of many health conditions like asthma, inflammation and bronchitis. It is an enriched source of protein, antioxidants and oil contents. In this study, Black cumin was studied for the estimation of osmoprotectants and anatomical features under salt stress. Black cumin seeds were obtained from the Ayub Agricultural Research Institute, Faisalabad (AARI). The experiment was planned in the Botanic Research area of Government College Women University Faisalabad. The experimental design was completely Randomized Design (CRD) with three replicates per treatment. After 4 weeks of germination, plants were treated with different concentrations of salt (control, 50, 100, 150 and 200 mM NaCl). After 6 weeks of salt treatment once in week plants were harvested and analyzed for different morpho-physiological, biochemical and anatomical attributes. Results showed that biomass production (fresh and dry weight) and plant height were decreased in Black cumin under salt stress. However, Plants with high to moderate (50 to 200 mM NaCl) salt concentration reduced root epidermal thickness depicting their adaptation strategy under salt stress. Furthermore, the area of the root and stem cortical cells was reduced in all salt-treated plants which avoid water loss during osmotic stress. Increasing salt stress in the growth-medium of Black cumin showed stunted growth and anatomical modifications.

Nigella sativa

osmoprotectants

antioxidants

anatomical adaptations

salt stress

Extreme environmental conditions, such as salinity, extreme temperatures, drought and waterlogging, overwhelmingly affect agricultural production. Salinity is a significant constraint that leads to lower the agriculture production and poor yield quality. As a result of soil salinity, which causes soil erosion and desertification, climate change is regarded as one of the main contributing reasons (Rogel et al. 2000). The significant buildup of salt in soil and water is a problem in dry and semi-dry regions. A high concentration of soluble salts present in the water of the root system is referred to as salinity in agricultural soils. Due to the strong osmotic pressures caused by these soluble salinity levels, the roots' ability to absorb moisture has an impact on plant growth. Because the excessive salt content in the soil solution inhibits plants from properly absorbing vital nutritional elements (Tester and Devenport 2003).Detrimental impacts are caused by the excessive salt content on 5% of agricultural land and 7% of the overall earth's surface (Rubio et al. 2009). Across the world, salinity is the main issue (nearly 1 billion ha of land). In addition, it damages almost 50% of the world's irrigated agricultural land, or about 900 million ha, or approximately 20% of the total area of land (Lee et al. 2020). Agriculture is harmed by salinity stress because it reduces productivity globally. therefore, Salinity is crucial for achieving sustainable agricultural methods and meeting global food demand today and in the future (Zorb et al. 2019).

The structural as well as biochemical mechanisms in plants suffer from salinity stress. Salt concentration prevents plant growth, expansion, and productivity (Zhang and Dai 2019). Hindering photosynthesis reduces the number of resources available and suppresses the division of cells, which inhibits crop production and produces many different physiological and biological alterations. Salinity influences the progression of light-harvesting complexes and controls the conversion of photosynthesis into its active stage (Van Zelm et al. 2020; Chen 2015). Salt stress reduces the number of photosynthetic pigments, modifies the PSII system and chloroplast extrinsic membrane, and decreases stomatal conductance, which all affect transpiration and gaseous exchange. Abscisic acid is generated in response to sodium stress and when delivered to guard cells, it closes the stomata. Inhibiting cell elongation, Osmotic pressure impacts plant development immediately, either directly or indirectly through abscisic acid (Pan et al. 2020). Due to salt, stomatal closure and a decrease in CO₂ assimilation rate may directly affect leaves. Additionally, it causes oxidative stress, which might harm the leaf's photosynthetic apparatus. In addition, intracellular Na⁺ buildup modifies the potassium-to-sodium ratio, causing an osmotic imbalance and an increase in reactive oxygen species, which has an impact on several enzymatic activities.

Pakistan is located in a region with a semi-arid climate. Approximately, 80 million hectares of Pakistan's total land area are covered by a well-developed irrigation infrastructure that consists of 62,400 kilometres of canals, form these19.3 mha is suitable for cultivation and irrigation agriculture. The greatest canal network in the world, which is connected by dams, is principally responsible for supplying agricultural water. Sindh and Southern Punjab are more severely impacted than other regions of the country. The amount of salt that ruined agricultural land at a rate of about 40.000 hectares per year can be used to gauge the scope of the issue (Rehmman et al. 2021).

Black cumin an annual oilseed and medicinal crop native to the Mediterranean region and a member of the Ranunculaceae family is well-known for its therapeutic properties. Different nations, including Syria, India, South Europe, Saudi Arabia, Turkey, and Pakistan cultivate this crop (Datta et al. 2012). Short-lived annual Black cumin grows in normal soils and is a part of a semiarid natural community where therophytes predominate (Kara et al. 2015). It is utilized for nutritional and therapeutic purpose due to its rich protein, antioxidant and oil contents, around the world (Ramadan 2007).

The main abiotic stress that Black cumin and many other crops endure is salinity. Although black cumin is a crucial crop for nutrition and medicine, soil salinity has a significant impact on its productivity (Rizwan et al. 2015). Anatomical modifications were interconnected with physiological adaptations in Black cumin under stress conditions. Consequently, the primary goal of the research was to evaluate how the anatomical properties of Nigella under a saline environment changed in connection to morphological physiological traits.

The seed of Black cumin was taken from Ayub Agricultural Research Institute (AARI), Faisalabad. The present study was planned to investigate anatomical modifications of Black cumin to salinity stress. A pot experiment was carried out in the Botanical Research area of Government College Women University, Faisalabad (GCWUF) under natural sunlight during the last week of October with 34–36^○C set as the day temperature. Similarly, 32^oC as the night temperature and the day length from 13–16 hours. Black cumin seeds were germinated to assess the tolerance and sensitivity under salinity. Plastic pots were used for sowing. All of the pots were given a moderate quantity of water. Seeds began to sprout within 2 weeks of seeding. 2 months old plants were given treatments for salinity, such as 0, 50, 100, 150, and 200 mM NaCl for 6 weeks once time in a week.

Leaf chlorophylls and carotenoid contents

In an 80% acetone solution, leaf carotenoid and photosynthetic pigments concentrations were calculated for all ecotypes (Arnon 1949; Kirk and Allen 1965). Using a spectrophotometer (Hitachi U-2001, Japan, Tokyo), the absorbance was measured at wavelengths 663, 645 and 480 nm. Then, these values were determined using particular following formulas.

Chl a = [12.7(ΔA 663) − 2.69 (ΔA 645)] Χ V/1000 Χ W

Chl. b = [22.9 (ΔA 645) − 4.68 (ΔA 663)] Χ V/1000 Χ W

Carotenoids (mg ml^− 1) = A._car / Em 100% Χ 100

Where. Em (Emission) = Em 100% = 2500.

A._car (µg/g FW) = [(ΔA 480) + 0.114 (ΔA 663) – 0.638 (ΔA 645)]

ΔA = Absorbance at respective wavelength.

V = Volume of extract (ml). W = Fresh weight of the sample (g).

Estimation of antioxidants enzymes

0.5 g of leaf was thoroughly mixed with 10 mL of buffered phosphate solution (50 mM) at a pH of 7.8 to ascertain the antioxidant enzymes. The plant material was ground up into a homogeneous mixture using a mortar and pestle, and the finished product was centrifuged at 20,000 g for 20 minutes at 4°C. Total soluble sugars, free amino acids and antioxidant activity is measured in the separated supernatant.

Estimation of SOD

The activity of SOD to prevent the photochemical degradation of nitro blue tetrazolium was assessed using the method developed by Giannopolitis and Ries in 1977. 50 M NBT, 1.3 M riboflavin, 75 nM EDTA, 13 mM methionine, 50 mM phosphate buffer (pH 7.8) and 20–50 L enzyme extract were all present in the reaction solution, which was 3 mL in size. The reaction mixture in test tubes was exposed to bright light (15-W fluorescent bulbs) while being irradiated at 78 mol m-2 s-1 for 15 minutes. The wavelength of the irradiated solution at 560 nm was determined by a spectrophotometer (Hitachi U-2100, Japan, Tokyo). The amount of enzyme that induced a 50% inhibition of the photochemical NBT decrease was used to define one unit of SOD activity.

Estimation of Catalase and POD

The method of Chance and Maehly (1955) was modified to a certain extent to be able to assess the activities of CAT and POD, 5.9 mM H₂O₂, 0.1 ml of enzyme extract, 50 mM phosphate buffer (pH 7.0), and 3 mL of the CAT reaction mixture were also included. The enzyme extract was added to start the procedure. Every 20 seconds, the reaction solutions at 240 nm absorbance were measured for variations. A change in absorbance of 0.01 units per minute was used to define one unit of CAT activity. The POD reaction solution (3 mL) contains 0.1 mL of enzyme extract, 20 mM guaiacol, 40 mM H2O2, and 50 mM phosphate buffer (pH 5.0). Every 20 seconds, the reaction solution's 470 nm absorbance was measured for changes. One unit POD activity was defined as an absorbance change of 0.01 units/min. Each enzyme's activity was described in terms of proteins. Using the Bradford method (1976), the crude extract's protein concentration was determined.

Approximation of lipid peroxidation

Malondialdehyde (MDA) level was assessed using a TBA reaction combination (Heath and Packer 1968). Using a polytron ultra-mixer, trichloroacetic acid (TCA) at 0.1% was used to thoroughly homogenize the leaves (PT, 2000; Switzerland). The homogenous mixture was centrifuged at 10,000 g for 5 minutes. Supernatants were boiled at 95°C for 30 minutes by adding TCA (20%) and TBA (0.5%). After cooling down test tubes with ice, the sample was once more centrifuged at 10,000 g for 5 minutes. Using an Ultrospec 3000 (Biochrom Ltd., Cambridge, England), the reading was obtained at 532 nm and then set once more for general absorption at 600 nm (the absorption/extinction coefficient was 156 mM cm^− 1).

Assessment of ascorbic acid content

According to Mukherjee and Choudhuri (1983) protocol ascorbic acid content was measured. Ten milliliters of 6% trichloroacetic acid were used to extract leaf material (0.25 g of the third leaf). Four milliliters of the extraction were combined with two milliliters of 2% dinitro-phenyl hydrazine (in an acidic medium), one drop of 10% thiourea (in 70% ethanol), and four milliliters of the mixture. Five milliliters of 80% (v/v) H₂SO₄ were added to the mixture at 0°C after the mixture had boiled for 15 minutes in a water bath and cooled to room temperature. At 530 nm, the absorbance was measured. From a standard curve plotted with known ascorbic acid concentrations, the concentration of ascorbic acid was estimated. After 58 days, two plants from each replicate were sown.

Estimation of phenolics

The Folin-Ciocalteu reagent was used to measure the total phenolic content (Singleton and Rossi 1965). 200 mL of diluted samples were combined with Folin-Ciocalteu reagent in 1 mL of a 1:10 diluted solution. 800 mL of a 7% Na₂CO₃ solution was then added after the reaction mixture had been properly mixed and allowed to sit for 6 minutes After 90 minutes of incubation, the mixture's absorbance at 760 nm was assessed at 23°C using a Microplate Reader (model BioTek ELx 808). The standard curves were created using gallic acid solutions (0–500 mg/L).

Estimation of total soluble sugars

Using a technique developed by Dubois et al. 1956, the total amount of soluble sugar found in leaves was determined. During analysis, the sample (0.2 g) was combined with distilled water, pushed through the mesh and after that kept in dark, airtight containers at room temperature. The mixture was then promptly treated with 2.5 mL of concentrated H₂SO₄ after receiving 0.5 mL of a 5% phenol solution. After 10 minutes, the absorbance at 485, 490 and 480 nanometers were measured in order to estimate the amounts of Xylose-Arabinose, Glucose and Mannose, respectively. Glucose has already created standard solutions.

Estimation of free amino acids

The total quantity of free amino acids was calculated using the Moore and Stein method (1948). One milliliter of the extract and one milliliter of the ninhydrin solution was added to a 20-millilitre test tube. The sample bottles were heated for 20 minutes in a water bath while being covered in aluminium foil. The liquid in the tubes was then mixed with 5 mL of the diluent was added, chilled in cold water and then incubated once more for 15 minutes at room temperature. Using a UV-Visible spectrophotometer, at 570 nm, the optical density was observed (Hitachi 220, Japan).

Anatomical attributes

The plants used for various morpho-physiological features had their biggest tiller and thickest base adventitious root cut from the root-shoot junction. Before being put into an acetic alcohol solution (v/v acetic acid 25%, ethanol 75%) for long-term storage, the sample was fixed first for 48 hours in a formalin acetic alcohol fixative (v/v formaline 10%, acetic acid 5%, ethyl alcohol 50%, and distilled water 35%). Using ethanol serial dehydration and the use the safranine and quick green stains in a double-stained method, permanent free-hand sectioning slides were created. using a light microscope and camera to calibrate an ocular micrometre (Nikon 104, Japan), measurements and images were taken with a stage micrometre. We used a light microscope with a camera to obtain measurements and pictures (Nikon 104, Japan).

Statistical analysis

Data were subjected to an analysis of variance (ANOVA) using the statistical programme Costat version 6.2, Cohorts Software, 2003, Monterey, CA, USA. Two factors and three replications were used in the Completely Randomized Design (CRD) of the experiment. R Development Core Team for 2017 Using the statistical software Minitab, the data were analyzed statistically using ANOVA (analysis of variance) and LSD for the comparison of mean values (Version: Minitab 19.1.1). (Kim 2014; Steel et al. 1997). PCA and heatmap were constructed by using R. Different letters show significant differences (P < 0.05). Similar while similar letters did not show significant differences., while different letters show significant differences.

Plant Morphological attributes

Data related to morphological parameters are given in Fig. 1. When exposed to salinity, it was noticed that plant height was significantly reduced in all plants of Black cumin as the salt level increased in the external growth medium. In comparison to the control, plants showed an 8% reduction in plant height at 50mM NaCl treatment level. Plants showed a progressive decrease of 14%, 26% and 38% in plant height at 100, 150 and 200mM NaCl levels as compared to untreated plants, respectively. Shoot fresh weight was reduced significantly in all Black cumin plants under various amounts of salt stress treatment. A maximum reduction of 57% was recorded at 200 mM NaCl in contrast to untreated plants. Compared to the control, plants showed a 26% reduction in shoot fresh weight at 50mM NaCl treatment level.

In the current study root fresh weight of Black cumin under different salt levels was determined. According to the result, it was observed that the root fresh weight was significantly and gradually decreased in Black cumin plants under salt stress. A maximum reduction of 42.8% was noticed at 200 mM NaCl as compared to control plants.Data about shoot dry weight revealed that gradual and significant reduction in this parameter was recorded at varying treatment levels of salt. Applications of 150 mM and 200 mM NaCl significantly reduce the shoot dry weight from 30.43%, and 56.67% respectively as compared to control plants. Root dry weight analysis showed that this parameter decreased progressively in Black cumin under different treatment levels of salinity. Maximum reduction of 70% was recorded at 200mM NaCl as compared to 0 mM NaCl control.

Photosynthetic Pigments:

Data regarding the photosynthetic pigments is presented in Fig. 2. Application of salt stress decreased the Chl a (58%, 72.8%, 76.6% and 88%), Chl b (60%, 68.6%, 73.6% and 78.5%) value in Black cumin at 50 mM, 100 mM, 150 mM, 200 mM salt stress respectively as compared to non-treated 0 mM NaCl plants. A significant decline in carotenoid content was observed in Black cumin under salt stress. A greater reduction of 76% was recorded at 200 mM NaCl level as compared to 0 mM NaCl.

Osmoprotectants

The osmoprotectant response in Black cumin leaves exposed to salinity stress are given in Fig. 3. Salinity stress induced the progressive rise in overall soluble sugars. A significant enhancement of 48% and 81% was noticed at 150 mM NaCl, 200 mM NaCl respectively when compared with absolute control plants. However, data related to total free amino acid depicted a significant enhancement of 9%, and 20%. 38% was recorded as compared to non-treated plants with 100, 150, and 200 mM NaCl added in the growing medium respectively. Similarly, a remarkable decline in the content of protein (28.6%, 44.13%) was noticed in Black cumin grown under 150 mM and 200 mM NaCl levels respectively as compared to the corresponding non-saline treatment level (0 Mm NaCl). The result related to proline contents suggested that this parameter was increased significantly by 10.4%, 17.8%, 19.7% and 27.6% when exposed to salinity toxicity of 50, 100, 150, and 200 mM NaCl respectively compared to control plants. Application of salinity stress significantly enhanced the glycine betaine contents in leaves of Black cumin A gradual enhancement of 122%, 168%, and 231% in glycine betaine value was recorded at 100, 150 and 200 mM NaCl respectively in comparison to absolute control.

Antioxidants activity:

The antioxidants activities in Black cuminunder the influence of salt stress ispresented in Fig. 4. Results revealed that POD activity was declined gradually and significantly under differential levels of salt. The more reduction 23% was noticed under 200 mM NaCl as compared to untreated plants. In comparison to the control plants, catalase value was significantly and progressively enhanced at 11.47%, 21%, 24% and 28% under 50 mM, 100 mM, 150 mM and 200 mM respectively. The greater reduction was observed at 200 mM NaCl. The level of MDA was increased by 25.85and %, 50%, 73%, and 186% as compared to 0 mM NaCl. The level of MDA was maximum and showed an increase of 186% at 200 mM NaCl stress. Data regarding the H₂O₂ revealed a notable rise in H₂O₂ concentration under different salt stresses. A remarkable increase of 44.44%, 82.84%, 129.59%, and 165.11% in H₂O₂ were recorded with the addition of 50, 100, 150 and 200 mM NaCl accordingly in the growth medium. The application of salt in an external growth medium decreased the values of total phenolics. A significant increase of 1.66% in total phenolics was observed at 200 mM NaCl as compared to the absolute control. However, results related to ascorbic acid showed that ascorbic acid value declined significantly and progressively under different concentrations of salt stress. A remarkable decrease of 30%, 46.76%, 58%, and 65.5% in ascorbic acid content was noticed at 50, 100, 150, and 200 mM NaCl respectively as compared to 0 mM NaCl.

Anatomical features

Stem anatomy

The result regarding the stem anatomy under salt stress were shown in Fig. 5. Stem thickness reflected the continuous and significant decrease in epidermal thickness under varying levels of salt stress in external growth medium. Plants displayed epidermal thickness reduction of 33%, 50%, 63.33%, and 73.33% concentrations under 50, 100, 150, and 200 mM NaCl, respectively compared to absolute 0 mM NaCl. A steady and substantial deterioration in the epidermal cell area was noted under the influence of different levels of salt stress in external growing media. A profound reduction of 84.52% was noticed at 200 mM NaCl in comparison to the control plants. The cortical cell area of the stem declines significantly and progressively from 50 mM NaCl to 200 mM NaCl. A greater reduction of 66.67% was observed at 200 mM NaCl as compared to the absolute control. The supplementation of salt decreases vascular bundle cell area in Black cumin The percentage of reduction rate was 30.76%, 53.84%, and 69.23% at 100, 150 and 200 mM NaCl respectively in comparison to non-treated plants. The imposition of salinity in the external medium reduced the metaxylem cell area by 33.33%, 44.44%, and 66.67% at 100, 150, and 200 mM salt stress accordingly in comparison to control plants.

Plate 1. Stem transverse sections of Nigella sativa L. under changing root zone salinity levels (0, 50,100, 150, and 200 mM NaCl).

Root anatomy

The experimental data regarding the effect of salt stresson root anatomical features of Black cumin shown in Fig. 6. The resultsrevealed that the epidermal thickness of the root was significantly reduced under different levels of salt stress. However, a profound decline was observed in root epidermal thickness by 69.23% at 200 mM NaCl in nutrient solution as compared to control plants. It was recorded that root epidermal cell area decrease significantly under salinity stress (50, 100, 150 and 200 mM NaCl) in growth medium. The highest reduction of 78.79% was noted at 200 mM NaCl respectively as compared to control plants. Number of root cortical cells was decreased significantly by 20.69%, 34.48%, 48.28% and 55.17% under differential levels 50, 100, 150 and 200 mM of NaCl, respectively in comparison to non-saline control plants. Similarly, the root vascular bundle cell area declined with the application of salt stress in the nutrient solution. At 200 mM NaCl maximum reduction was recorded as compared to 0 mM NaCl. The area of the root metaxylem cells declined slowly and significantly under different levels of salinity. The greatest diminution of 68.81% was noticed at 200 mM NaCl as compared to control plants of Black cumin

Plate 2

Root transverse sections of Nigella sativa L. under changing root zone salinity levels (0, 50, 100, 150, and 200 mM NaCl).

Pearson and Heatmap

Pearson correlation for growth and physiological parameters (Fig. 6A) showed strong negative relation with RFWT, SDWT, Protein, POD, PH, Caro, RDWT, SFWT, ASA, Chlb and Chla, while linear positive correlation among RFWT, SDWT, Protein, POD, PH, Caro, RDWT, SFWT, ASA, Chlb and Chla. Moreover, a positive correlation was also shown among H₂O₂, GB, SS, CAT, SOD, Proline, TFAA, MDA and Pheno. Furthermore, anatomical analysis (Fig. 6B) showed a strong linear correlation among RMCA, RET, RVBCA, RECA, SMCA, SVBCA, SCCA, SEPICA, SEPIT and RCCA. Heatmap (Fig. 7 ) showed that SMACA, POD, SVBCA, RVBCA, RECA, plant height, RMCA, proteins, RET, SDWT, RFWT, carotenoids, SCCA, SEPICA, SEPIT, ASA, RCCA, ASA, RCCA, RDWT, SFWT, chlb and chla content were higher at 0 mM NaCl, followed by 50 mM and 100 mM NaCl, while lower at 150 and 200 mM salt stress, respectively. In contrast, H₂O₂, GB, SS, Catalase, SOD, Proline, TFAA, MDA and phenolics content was lower at non-stressful conditions, and higher with increasing concentrations of salt stress.

The current study clearly outlines the negative impacts of salt stress applied to Black cumin root regions. In this investigation, it was found that higher salinity level induces changes in values of osmoprotectants, antioxidant activities and photosynthetic pigments of Black cumin There are many adaptive mechanisms for plants to survive under saline stress (Akhter et al. 2021). Different physiological pathways have been thought to contribute to growth and inhibition of growth following salt treatment has been extensively discussed in many literary works (Munns 2002). The stability in respective tissues broadly contributed to Na⁺ and Cl⁻ transportation and accumulation.

The study depicted that root and shoot fresh weights decrease in response to saline stress in a nutrient medium. Our stance is backed up by the findings of Akhter et al. 2022. In most halophytes, plant biomass is a reliable measure of stress resistance and may even rise at low salinities. Salinity stress lowered the amount of chlorophyll in the present study that are similar to the results of Sharma 1991. This study highlighted that saline stress causes carotene degradation, which causes a decrease in the carotenoids that are integrated constituents of thylakoid membranes (Thaiz and Zeiger 2004). Degradation in the production of carotenoids may therefore indicate that chlorophylls are deteriorated (Lima et al. 2004). Leaf chlorosis caused by saline stress finally led to considerable photoinhibition and the photodegradation of chlorophyll pigments.

Osmoprotectants increase stress tolerance and maintain ion homeostasis in many salt plant species (Slama et al. 2015). Total free amino acid, proline and glycine betaine accumulation may have been increased in Nigella sativa under saline stress. The enhancement in these parameters production improved osmotic adjustments under stressful environmental conditions (Parida et al. 2008). The significance of soluble sugars in plant ability to tolerate salt is also well known (Gil et al. 2013). In the current study total soluble sugars increase under stress in a growth medium. Depending on the degree of salt stress, the length of exposure, and the plant organs where it has been measured, total soluble sugars provide defense mechanism to tolerate salt stress (NeMati et al. 2011). However, soluble proteins decreased in our investigation using Black cumin subjected to 0 mM, 50 mM, 100 mM, 150 mM, and 200 mM of NaCl. With varying salt concentrations.

It is now clear that a more effective antioxidant system is linked to salt tolerance in the majority of agricultural plants (Gossett et al. 1994, Noctor and Foyer 1998, Mittova et al. 2002, Bor et al. 2003). In the current study ascorbic acid level decreased due to saline stress in Nigella plants. It have been directly destroyed by ROS in this salt-induced decrease, as has previously been shown in peas under drought-stress circumstances (Iturbe-Ormaetxe et al. 1998). The H₂O₂ level was increased in Nigella sativa plants under salt stress in the present experiment. Superoxide radicals are created during photosynthesis as a result of directly transferring electrons to oxygen, which leads to the production of H₂O₂ (Mittler 2002). However, several enzymes control the intercellular amount of H₂O₂. In the present research, salinity stress increases the levels of total phenolics, SOD and CAT in Black cumin This suggests that Nigella plants up-regulate the SOD-CAT-Phenolics antioxidative system to scavenge ROS. These results of the current study's analysis of the actions of antioxidant enzymes suggest that an increase in catalase (CAT) activity may have prevented an increase in ROS as a result of salt stress. The level of MDA was increased in Nigella plants under saline conditions. According to Kesh et al. 2022, MDA is an essential parameter of plant that was increased by NaCl stress to overcome the negative effects of salinity on different plant tissues.

Plants are more susceptible to environmental abiotic stress like salt and show different anatomical alterations in stem of black cumin The plants of Nigella sativa depicted maximum reduction in epidermal thickness in response of salt levels. A consistent reduction of 33–73% was noticed in Nigella plants under salinity levels. Our stance is backed up by the findings of Akhter et al. (2022) who reported that under saline condition stem epidermal thickness was decrease under salt stress condition. However, Akcin et al. 2017 give findings that salt stress increase stem epidermal thickness in salicornia plant species. Epidermal thickness of stem could be very effective tissue that show resistance to water lose in response to salt stress (Akhter et al. 2021). As in our study maximum reduction in stem epidermal (84%), cortical (66%), vascular bundle (69%) and metaxylem cell area (66%) were decrease in Nigella plants under salinity stress (200 mM NaCl) conditions. Our findings were in accordance to the results of Khare et al. 2023 that describe that in glycophytes cortical cell area decrease due to much crumpled cortical cells in root. An increase in these parameters under osmotic stress may aid in storing more water within the plant while monitoring water storage and loss. When exposed to saline, Kallar grass displayed a decrease in stem vascular bundles, indicating an adaptation for increasing water use effectiveness and preventing plants from bursting during physiological drought (Alnefaie et al. 2023). Similarly many plants decrease water and mineral permeability due to the width of xylem channels (Hajihashemi et al. 2020). Our findings were consistent with those seen in the stem of S. persica, where increased salinity reduced the cell area of the cortex. Cortical cell breakdown with a return to very salinity is the main cause of the reduction in cortical cell thickness in the stem (Karlova et al. 2021).

Root anatomical structure is an important part to study the changes in plants as it is directly in connection with the soil particles and manage the different abiotic stresses. We depicted a constant decrease in root anatomical characteristics at all levels of salinity stress. Root epidermal thickness is crucial part to measure the extent of environmental stress. Root thickness is usually due to parenchymatous cells that function as storage (Akhter et al. 2022) to conserve more water in spacious cells and hence considered as stress tolerance tool. Root epidermal thickness consistently decrease (69%) in black cumin plants in accordance with increase salinity. However, reduction in root epidermal thickness in fact demonstrate deterioration in number and size of cell of vascular bundles in root that may decline the capacity to transport less water under stress condition (Akhter et al. 2023). The reduction in vascular bundle in Nigella was 62% at the highest stress level (200 mM) of salt. Similarly, in the current study, the cortical cell area of the root also decreases (from 20–55%) in response to a saline environment. Good conduction in such stressed climatic conditions might be of great ecological importance to deal with high saltiness. A gradual decline in metaxylem cell area i.e., from 17–60% in Black cumin showed shortened water interactions because large channels in various Plants are productive for transporting nutrients, water and the metaxylem channel (Dolatabadian et al. 2011). cell area may be closely associated to the active conduction of nutrients and water (Grattan and Grieve 1998). Vessel bundles greater in size under salinity (Atabayeva et al. 2013), maintained the truth that hydraulic conductivity exists because large-area vessels are more prone to implosion under conditions of physiological drought (water stress) brought on by salt contamination in the nutrient medium (Dolatabadian et al. 2011). Similar findings were also confirmed during correlation analysis related to growth, physiochemical and anatomical alterations (Fig. 7–8).

On the whole, it is revealed that varying levels of salt stress resulted in injurious changes in growth and metabolism of Nigella plants. Root zone salinity caused decrease in morpho-physiological attributes in Nigella with increase in antioxidant enzyme activity. Plant responses were linked to levels of salinity that mainly induced oxidative stress thereby reducing different parameters and cellular damages.Changes in endogenous levels of AsA, POD, SOD and osmolytes had a protecting influence on growth against salt-induced oxidative stress. Therefore, tolerance of Nigella plants to salinity was entirely dependent upon level of salinity and better antioxidants defense system along with osmo-protectants and cellular adjustments. These attributes collectively worked for effective removal of ROS and maintenance of cellular activities. We recommend extensive molecular experimentation to unravel genetic basis of tolerance and survival in Nigella for its breeding and use in phytoremediation. It is also suggested that use of additional means e.g. chemical application must be used to improve response of these plants as it could offer extraordinary aid to crop production problems under salinity.

Acknowledgment

Authors are highly thankful to Government College for Women University, Faisalabad Pakistan for providing experimental station and lab facilities.

Author contribution

NA, AN: Conceived the Idea and designed the experiment; NA, MA, MMS, and AK: Writing, Review, Editing; MA,RB,IH, OMA: performed experiment, gathered literature; OMA, NZ, , REN, MH: analyzed the data, and helps in interpretation of results; MKA and AN: Critically revised the manuscript; All authors approved the final version of the manuscript.

Conflict of interest

The authors declare no conflict of interest.

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Plate 1 and 2 are available in Supplementary Files section.

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Plate1.png
Plate 1. Stem transverse sections of Nigella sativa L. under changing root zone salinity levels (0, 50,100, 150, and 200 mM NaCl).
Plate2.png
Plate 2: Root transverse sections of Nigella sativa L. under changing root zone salinity levels (0, 50, 100, 150, and 200 mM NaCl).

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Effect of salt stress on growth, physio-chemical mechanisms and anatomical alterations of Nigella sativa L

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Abstract

Figures

INTRODUCTION

Materials and Methods

Leaf chlorophylls and carotenoid contents

Estimation of antioxidants enzymes

Estimation of SOD

Estimation of Catalase and POD

Approximation of lipid peroxidation

Assessment of ascorbic acid content

Estimation of phenolics

Estimation of total soluble sugars

Estimation of free amino acids

Anatomical attributes

Statistical analysis

Results

Plant Morphological attributes

Photosynthetic Pigments:

Osmoprotectants

Antioxidants activity:

Anatomical features

Stem anatomy

Root anatomy

Pearson and Heatmap

Discussion

Conclusion

Declarations

References

Plate 1 and 2

Supplementary Files

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