Effect of spermidine on reproductive, seed quality and bio-physiological characteristics of chickpea (Cicer arietinum L.) genotypes under salt stress

The experiment aimed to investigate the impact of foliar application of spermidine on the physiological and reproductive aspects of chickpea genotypes subjected to salt stress, with a focus on its consequences for seed quality. The study involved treating chickpea genotypes (CSG 8962, HC 3, HC 5, RSG 931) with 4 and 8 dSm−1 Cl− dominate salinity during the seedling stage, and application of 0.5 and 1.0 mM spermidine at the flowering stage. Result revealed that salinity significantly reduced chlorophyll and membrane stability index by approximately 46.97% and 23.19%, respectively. Concurrently, pollen germination and viability decreased about 14.14% and 22.24%, leading to a substantial decline in seed protein content (37.70%) at 8 dSm−1 salinity. While there was an increase in antioxidant activity (45.83%), phenol content decreased in response to salinity stress. Foliar application of spermidine (0.5 and 1.0 mM) proved to be a promising intervention, enhancing chlorophyll stability and phenol content by approximately 24.35% and 36.05%, respectively, at 8 dSm−1 salinity. This improvement is associated with a notable 20.01% increase in pollen viability, resulting in a subsequent rise in protein content by about 20.73% at 1.0 mM spermidine. Additionally, the application of spermidine mitigated Na+ ion accumulation in chickpea seeds. The findings underscore the varying performance of chickpea genotypes under salinity stress, with CSG 8962 and RSG 931 exhibiting poorer outcomes compared to other genotypes. Notably, the positive impact of spermidine was more pronounced, especially with the use of 1.0 mM spermidine, which demonstrated a more significant positive effect in salt-sensitive chickpea genotypes. These results emphasize the potential of spermidine as a strategic tool in alleviating the adverse effects of salinity on chickpea crops, offering valuable insights for the development of sustainable practices to enhance chickpea resilience and seed quality under challenging environmental conditions.


Introduction
Salt stress is a critical abiotic factor affecting crop plants, exerting profound impacts on their morphological, physiological, and biochemical processes.This stress restricts crop growth, development, productivity globally, with approximately 85% of the world's land area facing salinity challenges across 118 countries.The widespread occurrence is attributed to poor agricultural practices, the use of saline water for irrigation, and a high evaporation rate coupled with low precipitation (Machado and Serralheiro 2017).Particularly alarming is the revelation that over 30% of the global topsoil and more than 6% of subsoil's are affected by salinity, predominantly in arid and semi arid climates (Kumar and Sharma 2020).This issue poses a significant threat to global agriculture, resulting in economic losses.Salinity primarily induces osmotic stress, specific ion toxicity, and oxidative stress in plants.The accumulation of sodium ions in the rhizosphere leads to osmotic stress, reducing water and nutrient absorption, causing K + ion efflux, Na + ion influx and nutrient imbalance (Nadeem et al 2019).Excessive Na + ion concentration in the cytoplasm cause ion toxicity, inactivating vital enzymes crucial for physiological processes, disrupting photosynthetic activity and the mitochondrial electro transport chain, leading to an imbalance in reactive oxygen species (ROS) production and degradation (Gill and Tuteja 2010).
Chickpea (Cicer arietinum L), a salt-sensitive rabi crop, ranks 2nd among legumes after common beans.Cultivated in 54 countries, including India, Australia, Mayanmar, Ethiopia, Turkey, and Pakistan, chickpea covers 15 million hectare globally, with a productivity of 10.57 kg/ha and an annual production of 15.87 million tonnes (FAO, 2021).Recognized for its nutritional value, chickpea is a good source of protein, vitamin B, and Potassium.In chickpea cultivation, Rhizobium bacteria are inoculated to enhance root nodulation, fixing 70% of soil nitrogen biologically and improving soil fertility (Shao et al 2022).Chickpea is particularly sensitive to Cl dominated salinity.Salinity adversely affects chickpea growth and development, diminishing, both the quality and quantity of production.It disrupts membrane integrity and chlorophyll stability, increasing the production of MDA and ROS (H 2 O 2 , 1 O 2 , O 2 ) within cells.ROS production inhibits cell signalling, proliferation, and membrane damage, leading to protein and nucleic acid degradation and ultimately, cell death (Hossain 2019).Pollen viability and germination are also affected, directly impacting yield.While some plants exhibit adaptations to survive under these conditions, salinity-tolerant cultivars bolster their antioxidant defence system, including phenols, to mitigate stress effects (Tiburcio et al 2014).Various plant growth regulators, when applied exogenously, prove effective under stress conditions.Polyamines, particularly spermidine (Spd), are recognized as potent regulators enhancing crop plant tolerance to abiotic stresses (Liu et al 2015).Acting as low molecular weight polycations, polyamines such as putrescine (Put), spermine (Spm), and spermidine (Spd) are ubiquitous in living organisms and play diverse roles in metabolic processes.They function in cell division, membrane stabilization, free radical scavenging, and modulation of ion channels, regulation of protein synthesis, DNA replication, and response to environmental stress (Li et al 2016).Among them, Spd is notably involved in plant stress response signalling and the initiation of tolerance mechanisms.Foliar application of Spd increases endogenous polyamine levels with Spm + Spd/Put ratio rising further under stress conditions (Shao et al 2022).Exogenous Spd application enhances non-enzymatic antioxidant phenols, stabilizing membranes chaperones, binding to negatively charged surfaces and safeguarding membranes and biomolecules (Chen et al 2018).It also stabilizes the chlorophyll molecule and increases the total chlorophyll content (Sawariya et al 2023).However, the mechanism through which exogenous spermidine application improves stress response in chickpea plants remains unclear.The present study aims to explore the impact of foliar polyamine application under salinity stress in chickpea by analyzing various plant reproductive, physiological, and biochemical parameters.

Plant material and treatments
The chickpea genotypes viz.CSG 8962, HC 3, HC 5, and RSG 8962 were procured from the pulses section at CCS Haryana Agricultural University, Hisar.Ensuring the health of the seeds, a meticulous process of surface sterilization with 0.2% HgCl 2 , followed by through washing with double distilled water, was undertaken.In polythene bag-lined pots filled with 7.0 kg of dune sand, seven seeds of each genotype were sown, placed under natural environmental conditions.Before sowing the chickpea seeds received inoculation with a Rhizobium strain to enhance nodule formation.
To produce Cl −1 dominated (4.0 and 8.0dSm −1 ) salinity, pots were saturated with desired levels of salt stress to maintain the stress and control pots were saturated with canal water.A central rubber pipe was inserted to maintain the circulation of salts so that ionic contents can be easily absorbed by the plant.For the 4 and 8.0dSm −1 salinity level, solutions were prepared by blending various salts, including MgSO4, CaCl 2 , NaCl, and MgCl 2 .The salt mixture ratios were formulated to achieve a Ca + Mg: Na ratio of 1:1, Mg: Ca ratio of 3:1, and Cl: SO 4 ratio of 7:3 on a milli-equivalent basis.At the flowering stage (approximately 50% flowering), exogenous spd was applied as a foliar spray in concentration of 0.5 and 1.0mM.The pots subjected to 4 dSm −1 salinity were categorized into three groups: one with 4 dSm −1 salinity alone, the second with 4 dSm −1 salinity and a 0.5 mM Spd foliar spray, and the third with 4 dSm −1 salinity alongside a 1.0 mM Spd foliar spray.A parallel grouping was applied to the pots experiencing 8 dSm −1 salinity.Sampling was conducted after 15 day interval post spermidine treatment to assess the specified parameters.The treatment groups, denoted as T0 to T6, correspond to distinct combinations of salinity levels with spermidine are as follows: T 0 = 0 dSm −1 + 0 mM spd T 1 = 4 dSm −1 + 0 mM spd T 2 = 4 dSm −1 + 0.5 mM spd T 3 = 4 dSm −1 + 1.0 mM spd T 4 = 8 dSm −1 + 0 mM spd T 5 = 8 dSm −1 + 0.5 mM spd T 6 = 8 dSm −1 + 1.0 mM spd

Statistical analysis
Results obtained from the experiment were analyzed using Complete Randomized Design (CRD) for two factors and compared using critical difference (CD) at 5% level of significance and graphical presentation were conducted using origin pro, 2022 and GGE biplot.CSI was measured using Koleyoreas (1958) method.Two sets of leaf tissue were prepared for experimentation.One set was placed under ambient room temperature conditions, while the other set underwent to a controlled temperature of 55 °C for one hour within a water bath, each immersed in 15 ml of distilled water contained in individual test tubes.Read the absorbance at 480, 645 and 665 nm to calculate total chlorophyll content of both the sets using the method given by Hiscox and Israelstam (1979).

CSI
Total chlorophyll under treated Total chlorophyll under untreated MSI analysis in fresh plant tissue followed the methodology outlined by Sairam et al (2002).Plants tissue was carefully placed in a test tube containing 15 ml of deionized distilled water, maintained at 25 °C.After four hour incubation period, the electrical conductivity (EC1) of the solution was measured.Subsequently, the same samples underwent a 15 min exposure to a boiling water bath, followed by another measurement of electrical conductivity (EC2) after cooling.MSI was calculated using the following equation:

Non-enzymatic antioxidant activity
The assessment of antioxidant activity in plant tissue was conducted using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay as described by (Blois 1958).Ethanol extract of the plant tissue were prepared and subsequently take supernatant, diluted it with methanol.DPPD was then introduced to the test tube, and the mixture was incubated in complete darkness for 30 min.Absorbance readings at 517 nm were recorded.A control solution without extract served as the baseline.Methanol served as the blank.Antioxidant activity was quantified using the following formula: Phenol content of plant tissue was estimated by Folin-Ciocalteu reagent method (Aiyegoro and Okoh 2010).To the methanolic plant extract add Na 2 CO 3 and FCR reagent.Heat the solution for 1-2 min at 100 °C and after cooling dilutes it using distilled water and read the absorbance at 650 nm.Prepare a standard curve using catechol and calculate the phenol content.

Reproductive parameters
At the anther dehiscence stage pollen germination and viability, number of ovule per pistil and ovule receptivity of chickpea genotypes was determined.

Pollen viability
Viability of pollen grains was assess by 2, 3, 5-triphenyl tetrazolium chloride (TTC) test (Hauser and Morrison 1964).A small amount of pollen grain was sprinkled on the drop of TTC solution taken on the clean and dry micro slide and cover immediately using cover slip.Incubate the slide at 35-40 °C for 5 min in dark.Record the viable pollen grain (bright red) under a light microscope.

Pollen germination and tube length
Prepare the semi solid medium consisted of sucrose, boric acid, calcium nitrate, and agar.Sprinkle the pollen grains with the help of brush on the semi solid medium in the petriplates and incubate it at 25 ± 2 °C for 3 h in dark.After incubation terminate the germination by flooding the medium with killing and fixing solution (Sass, 1951).Record the pollen germination and its tube length under a light microscope.

Ovule receptivity and number of ovules
Ovule receptivity was assessed by aniline test (Dumas and Knox 1983).Excised pistil a day before anthesis and softened it by treating with 8N NaOH for 24 h.After thorough wash in distilled water stain the pistil in aniline blue solution for 24 h at 25 ± 2 °C and then mount in 50% glycerin.The ovule receptivity and ovule number was measured under florescent microscope.

CSI
For each chickpea genotypes, exposure to salt stress (4 and 8 dSm ) led to a significant decrease in CSI compared to the control conditions (figure 1).The most pronounced reduction was observed in genotype RSG 931, declining from 74.65 to 39.58% followed by genotype HC 3, HC 5 and CSG 8962.However, the application of spd at concentrations of 0.5 and 1.0 mM resulted in a notable increase in CSI under both salinity levels.The most suitable improvement, recorded at 1.0 Mm spd was observed in genotype HC 3, increasing from 45.29 to 59.87% followed by genotype CSG 8962, RSG 931, and HC 5 at high salinity level.

MSI
In all chickpea genotypes, the MSI exhibit a significant decrease under salt stress conditions (4 and 8 dSm −1 ) compared to the control, impacting leaf, root and nodular tissue (figure 1).The most substantial reduction in MSI was recorded in genotype CSG 8962 decreasing from 74.65 to 39.58% in leaves and from 87.70%-50.45% in nodules at high salinity level.Conversely, in roots, the maximum decreases were noted in genotype RSG 8962.When compare to T4 (8 dSm −1 ), the application (0.5 and 1.0 mM) resulted in a significant increase in MSI (%) at both salinity levels.The most notable enhancement, particularly at 1.0 Mm spd, was documented in genotype CSG 8962, with MSI rising from 64.77 to 72.82% in leaves and from 50.43 to 70.13% in nodules.However in the case of roots, RSG 931 reported to have maximum increase under 8 dSm −1 salinity.

Phenol content
The phenol content in each chickpea genotype under salt stress conditions (4 and 8 dSm −1 ) showed a significant decrease in leaf, root, and nodules compared to their repective controls.Interestingly, in the leaf, there was an increase at 4 dSm −1 (figure 2).The maximum decrease was observed in genotype HC 5, with reductions of 56.3% and 21.90% in leaf and nodules, respectively.In contrast, CSG 8962 exhibited a significant decrease in root phenol content (66.18%) at the higher salinity level (8 dSm −1 ).When compared to T4 (8 dSm −1 ), the application of spd (0.5 and 1.0 mM) led to a significant increase in phenol content across all chickpea genotypes at both salinity levels.The highest increase, particularly at 1.0 mM spd application, was noted in genotype RSG 931, showing enhancements of 58.55% and 44.47% in root and nodules, respectively, under high salinity conditions.

Pollen viability, in vitro germination and tube length
In each chickpea genotypes, the pollen viability, germination and tube length experienced a significant decrease under salt stress (8 dSm −1 ) (figures 7 and 8).The most pronounced reduction in both pollen viability and germination was observed in genotype RSG 931 and CSG 8962, declining by 14.14% and 22.24% respectively, at 8 dSm −1 salinity level (figure 4).When compare to T4 (8 dSm −1 ), foliar application of spd (0.5 and 1.0 mM) resulted in a significant increase in both pollen viability and germination under salt stress (8 dSm −1 ).The maximum improvements for pollen viability and germination were documented in genotype HC 3 (10.95%)and HC 5 (20.01%).In contrast the pollen tube length experienced a notable decrease in genotype HC 3 by 61.92% at the 8 dSm −1 salinity level.However, with spd application, there was a significant recovery, showcasing a 60.61% increase in HC 3 under salt stress (8 dSm −1 ).

Ovule receptivity and number of ovule pistil −1
The presence of callose deposition on the ovule serves as an indicator of ovule non receptivity, as illustrated in figures 9 and 10.Salinity stress adversely impacts ovule receptivity across all chickpea genotypes at both salinity levels, with CSG 8962 displaying minimal callose deposition and RSG 931 exhibiting the maximum callose deposition on the ovule.Notably, exogenous applications of spd (0.5 and 1.0 mM) reveals a significant improvement in ovule receptivity by reducing callose deposition in all chickpea genotypes under high salinity conditions.The number of ovules experiences a decline under salt stress in all chickpea genotypes, with the exception of HC 3. Genotype RSG 931 witnesses the The maximum decrease, with a reduction of 22.23% followed by CSG 8962 and HC 5 (figure 4).Interestingly, the exogenous application of spermidine (0.5 and 1.0 mM) results  in a significant increase in the number of ovule pistil −1 in all chickpea genotypes under high salinity levels.The most substantial increase is observed in genotype CSG 8962 by 30.00% improvement at 1.0mM spermidine at 8 dSm −1 salinity level.

Seed Quality
Protein content of the seeds exhibited a significant decrease across all chickpea genotypes with the escalation of salinity levels from 4 to 8 dSm −1 , as illustrated in figure 5. Genotype CSG 8962 recorded the maximum reduction, showing a decline of 36.58%,31.50%, and 14.36% respectively, compared to their respective controls at the higher salinity level.Remarkably, foliar application of spermidine (0.5 and 1.0mM) resulted in a significant increase in the protein content of seed across all chickpea genotypes at both salinity levels.The most substantial improvement particularly at 1.0mM spermidine, was observed in genotype HC 5, with a remarkable increase of 20.73%, followed by RSG 931, CSG 8962, and HC 3 at 8 dSm −1 salinity.
Starch content of the seed demonstrated a significant increase as salinity levels rose from 4-8 dSm −1 in all chickpea genotypes, as depicted in figure 5. Genotype RSG 931 exhibited the maximum increment in starch content, recording a notable rise of 526%, followed by HC 3, HC 5, and CSG 8962, surpassing their respective controls at the high salinity level.Foliar application of spermidine (0.5 and 1.0mM) led to decrease significantly the protein content of seed in all chickpea genotypes at both salinity levels.The maximum decrease in starch content for 1.0mM spermidine was reported in genotype HC 3 by 2.88%, followed by RSG 931, CSG 8962 and HC 5, with decrease of 1.93%, 1.65%, and 0.87%, respectively, at the 8 dSm −1 salinity level.
Na + /K + ratio in seed The Na + ion content in seeds, induced by salt stress, led to an imbalance in the ionic ratio (Na + /K + ) within chickpea seeds.Relative to the (T0), salt stress (4 and 8 dSm −1 ) precipitated a substantial increase in Na + /K + ratio in all chickpea genotypes, as depicted in figure 5. Genotype HC 5 exhibited the maximum increase, with a notable rise of 30.19%.When compared to T4, the application of spd at concentrations of 0.5 and 1.0mM resulted in a decreases in the the Na + /K + ratio at both salinity levels.themost significant reduction, observed at 1.0 mM spd was reported in genotype RSG 931, demonstrating a decrease of 19.94% followed by CSG 8962, HC 3, and HC 5, with decreases of 12.92%, 11.17%, 5.87%, respectively, at the high salinity level.

Phosphorus ion content in seed
The phosphorus ion content in seeds was notably reduced under salt stress across all chickpea genotypes.Relative to control (T0), exposure to salt stress at 4 and 8 dSm −1 levels resulted in a significant decrease in phosphorus ion content in seeds, as depicted in figure 5.The maximum reduction was observed in genotype RSG 931, experiencing a decline of 45.45% followed by HC 3, CSG 8962, and HC 5.In compared to T4, the application of spd at concentrations of 0.5 and 1.0mM led to an increase in phosphorus ion content in seeds at both salinity levels.The most substantial improvement, observed at 1.0 mM spd was reported in genotype RSG 931, showing an increase of 16.3%, followed by HC 3, CSG 8962, and HC 5 with increases of 8.17%, 6.30%, 5.30%, respectively, particularly at high salinity level.

Response of various parameters to the treatments
Principle Component 1(PC 1) effectively segregates the treatments into two distinct categories on the biplot: the non stress (T0) conditions for each genotype are postioned on the right side, while the stress (T4) conditions for each genotype are on the left side (figure 6).Notably, the genotype HC 3 and HC 5 appear less differentiated under non-saline conditions, aligning closely on the biplot positioned farther apart.The genotype-treatment combinations are characterized by relatively high levels of Membrane Stability Index (MSI), Chlorophyll Stability Index (CSI), pollen germination (PG), pollen viability (PV), pollen tube length (PTL), filament length (FL), pistil length (PL), phenol content, and seed protein and phosphorus ion content.conversely, salt stress treatment (T4) exhibits relatively high levels for parameters located on the left side of the biplot, such as Na + /K + ratio, Starch content, and antioxidant activity (AA), and relatively low levels for those on the right side, including MSI, CSI, PG, PV, PTL, FL, PL, phenol content, and seed protein and phosphorus ion content.This indicates that the chickpea genotypes under saline and non-saline conditions manifest contrasting states for those variables.
Principle component 2 (PC 2) appears to further segregate the four genotypes under saline conditions on the biplot.CSG 8962 and RSG 931 under T4 are positioned on the upper left part, characterized by relatively high levels of phosphorus, Na + /K + ratio, and starch content.In contrast, genotype HC 5 and HC 3 are positioned on the upper left side of the biplot.Conversely, under T5 and T6, especially RSG 931 and HC 3 under T6, are located on the upper left characterized by relatively high levels of antioxidant activity (AA), PG, PV, PTL, FL, PL, and phenol content of the biplot.This suggest that the high levels of non-enzymatic antioxidant activity, phenol content, pistil and filament length, pollen germination, viability, and tube length, coupled with low levels of Na + /K + ratio and starch content, serve as indicators of spermidine-induced salt tolerance.

Discussion
Chickpea, a key crop aiding soil fertility through biological nitrogen fixation, faces challenges in growth and development under salinity stress.This stress disrupts chlorophyll structure and function, evident in a significant decrease in the CSI across chickpea genotypes.Lipid peroxidation of chloroplast membranes and increased chlorophyllase enzyme activity contribute to this decline (Kotula et al 2019).Additionally, Na + ion accumulation in mesophyll cells damages chloroplast structure, The results are similar to the findings of Arefian As shown in figures 7, 8, 9 and 10, salinity stress reduces pollen viability, in-vitro germination, tube length, pistil, filament length, ovule number, and ovule receptivity.Salinity stress induces the physiological drought condition in the chickpea plant which is a salt sensitive crop that ultimately leads to increase membrane damage, electrolyte leakage, and ion toxicity and imbalance (Khaleghi et al 2019).In the present study reduction in the chlorophyll stability indicate the reduction in photosynthetic production that affects the pollen development inside the anther.Also a mature leaf accumulates more ions in the reproductive organs during pod filling stage that ultimately affects the pollen development inside anther (ElSayed et al 2022).Callose deposition around the ovule indicates non-receptivity, affected by water stress, ionic and nutrient imbalance.Salinity also impacts seed quality, reducing protein and phosphorus ion content while increasing starch and Na+ ion content.Spd application improves reproductive organ growth and seed quality, aligning with previous research of Fang et al (2010) and Samineni et al (2011) in chickpea.
Polyamines, particularly endogenous Spd, play crucial roles in pollen growth, development, and stress signaling (Saleethong et al 2013).Their involvement in cell wall rigidity during pollen growth, maintenance of pollen viability, and pollen tube growth is well-documented (Aloisi et al 2016).Increased endogenous Spd levels enhance antioxidant activity, ion balance, flower development, and seed quality.Spd stabilizes chlorophyll structures, ensuring essential nutrient availability during pollen and pistil development under salt stress (Samineni et al 2011).The study suggests that pollen quality significantly influences chickpea seed quality, and exogenous Spd application improves seed quality by enhancing pollen growth and germination under salinity stress.

Conclusion
The experiment conducted on the chickpea genotypes was emphasized on physiological, reproductive aspects and quality of the seed under Cl − dominated salinity.The observed reduction in membrane stability index, pollen viability, germination and ovule number contribute to a substantial decline in seed protein content.Notably, both vegetative and reproductive stages manifest sensitivity to salt stress, with the latter stages particularly pollen development and pod filling, being even more susceptible.This heightened sensitivity occurs even in a nationally released salt-tolerant variety (CSG 8962), highlighting the urgency of addressing salt stress in chickpea cultivation.The study introduces the possible suggestion as exogenous application of 1.0 mM spermidine and is more effective under salinity.This treatment results in a improvement of the chickpea genotypes characterized by increased membrane stability, antioxidant activity and phenol content.Additionally, it positively influences pollen development, ultimately leading to improved seed quality across all chickpea genotypes.Furthermore, the study delves into underlying mechanisms of salt sensitivity, identifying not only a decrease in pollen quality but also affecting seed quality.This underscores the necessity of evaluating chickpea genotypes tolerance across both vegetative and reproductive stages to develop robust and comprehensive strategies for managing salinity stress in chickpea cultivation.

Figure 1 .
Figure 1.Effects of spermidine on Membrane Stability Index in leaf (A), root (B), nodules (C) and on Chlorophyll Stability Index of chickpea genotypes grown under salinity stress.Bars represent M ± SE.Different letters show significance at p 0.05 among the treatments according to Tukey's HSD Test.

Figure 2 .
Figure 2. Effects of spermidine on non-enzymatic antioxidant activity and phenol content in leaf (A) and (D), root (B) and (E), nodules (C) and (F) of chickpea genotypes grown under salinity stress.Bars represent M ± SE.Different letters show significance at p 0.05 among the treatments according to Tukey's HSD Test.

Figure 3 .
Figure 3. Effects of spermidine on pistil length (A) and filament length (B) of chickpea genotypes grown under salinity stress.Bars represent M ± SE.Different letters show significance at p 0.05 among the treatments according to Tukey's HSD Test.

Figure 5 .
Figure 5. Effects of spermidine on protein (A), starch (B), Na + /K + ratio (C), phosphorus ion (D) in seeds of chickpea genotypes grown under salinity stress.Bars represent M ± SE.Different letters show significance at p 0.05 among the treatments according to Tukey's HSD Test.

Figure 9 .
Figure 9.Effect of spermidine application on ovule receptivity in CSG 8962 (A) and HC 3 (B) genotypes under salt stress.Arrow shows the callose deposition on the ovule.

Figure 10 .
Figure 10.Effect of spermidine application on ovule receptivity in HC 5 (A) and RSG 931 (B) genotypes under salt stress.Arrow shows the callose deposition on the ovule.
et al 2014 in chickpea, Nahar et al 2016 in mung bean, Rathinapriya et al 2020 and Sun et al 2020 in foxtail.Salinity-induced disruption extends to the protein-pigment lipid complex through ROS production, degrading chlorophyll structure (Arefian et al 2014).However, foliar application of spermidine (Spd) enhances chlorophyll stability in chickpea leaves by protecting pigment-protein complexes and thylakoid membranes from oxidative stress.Spd also regulates chlorophyll biosynthesis and degradation pathways, contributing to overall plant resilience (Nahar et al 2016).Assessing oxidative stress involves examining membrane stability indices and antioxidant activity.A significant decrease in the membrane stability index occurs in leaf, root, and nodules of chickpea genotypes due to ROS imbalance and cell degradation under salt stress, consistent with previous studies of Chattopadhayay et al 2002 in rice, Arefian et al 2014 in chickpea, Sreenivasulu et al 2000 and Rathinapriya et al 2020 in foxtail millet.Salinity-induced water stress increases membrane permeability, causing ion imbalances.Spd application improves ion distribution, reduces Na + influx, and enhances Ca 2+ and K + absorption by activating reverse steering proteins (Hai et al 2022).Salinity stress induces ROS production within plant cells, prompting a nonenzymatic antioxidant defense mechanism.Phenol, with pro-oxidant and antioxidant activity (Amdouni and Ben 2016, Ahanger and Agarwal 2017), decreases significantly under salt stress in leaf and root but not in nodules.These observations support the finding of Taïbi et al (2016) in Phyaseolus vulgaris and Al-Mushhin 2022 in Vigna angularis.Exogenous Spd application further increases antioxidant activity in chickpea leaf, root, and nodules, alleviating oxidative stress.Spd acts as a free radical scavenger, enhancing the accumulation of free Spd and spm while decreasing put content in salt-sensitive cultivars, maintaining cell homeostasis (Li et al 2016).Spd application also increases phenolic content in leaf and root, potentially contributing to enhanced salt tolerance.The result coincides with the finding of Al-Mushhin 2022 in Vigna angularis; Chunthaburee et al 2015 in rice.