Introduction

Salinity-alkalinity stress represents a significant abiotic stressor that hinders plant growth and agricultural productivity on a global scale 1. In particular, plants exposed to saline-alkaline stress encounter disruptions in their metabolic activities due to increased osmotic pressure and the accumulation of reactive oxygen species (ROS) 2,3. Consequently, plants respond by upregulating a cascade of antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), as well as increasing the activities of enzymes that eliminate ROS and maintain the structural integrity of cellular membranes by decreasing in lipid peroxidation rates 4,5.

Elevated levels of alkaline salts in the plant growth medium decrease the potassium (K+) concentration and increase sodium (Na+) uptake and accumulation, resulting in the efflux of K+ ions and the promotion of K+ leakage from plant cells. Furthermore, under alkaline stress conditions, the Na+ concentration exceeds the K+ concentration, leading to poor nutrient absorption and disruption of the Na+/K+ balance. A relatively high pH also causes aberrant root morphology, resulting in leaf chlorosis and necrotic lesions, all of which impede plant growth and development, ultimately reducing crop yield 6. Although saline-alkaline stresses seldom occur together, their mechanisms are intricately interwind. Salinity stress is primarily caused by the accumulation of neutral salts, exacerbated by high pH levels exceeding 8.5, which compromise root vigor, photosynthesis, and cell membrane integrity 7. Alkaline stress also effects plant metabolism at the physiological and biochemical levels, inducing variations in phytohormone levels and altering gene expression networks 5.

Several studies regarding geographical and significant temporal variations in phytohormones in response to different abiotic stress conditions have been reported in literature. Among the phytohormones, cytokinins (CK), salicylic acid (SA), auxins, ethylene (ET), gibberellins (GA), abscisic acid (ABA), jasmonates (JA), and brassinosteroids (BR) play important roles in abiotic stress signaling pathways. In general, ABA modulates osmotic stress, SA boosts important enzymes of the antioxidant system, and IAA and CK support the plant in sustaining development in high-pH growth media 1,8.

Naphthalene acetic acid (NAA) has been demonstrated to increase root development in various plant species, including maize, citrus, rice, and malus, under adverse environmental conditions 9,10. Studies have indicated that the auxin gradient induced by stress plays a pivotal role in regulating a wide array of physiological and biochemical processes, such as the synthesis of aquaporins, control of the stomatal aperture, positioning of lateral roots, and accumulation of crucial osmolytes such as soluble sugars and proline. Auxin has been shown to increase plant stress tolerance by effectively scavenging reactive oxygen species (ROS), reducing the accumulation of Na+ ions, and safeguarding photosystem II (PSII) against stress-induced damage 6.

Summer squash [Lagenaria siceraria (Molina) Stndl], a member of the Cucurbitaceae family, is widely distributed in the Indo-Pak region and is commonly consumed as a vegetable worldwide. Research efforts have resulted in the identification of various bioactive compounds in L. siceraria, including phenolic compounds such as chlorogenic acid, benzoic acid, quercetin, luteolin, and kaempferol, as well as p-coumaric acid and sinapic acid. Number of biochemical and physiological changes have been linked to abiotic stresses, such as alkaline stress, yet limited information is available in literatures regarding alkaline tolerance in squash. Additionally, there is a little data to support the evidence of the protective effects of growth hormones in enhancing crop yield under stressful conditions. The effect of exogenously applied NAA on squash cultivation under alkaline stress conditions is not well understood form the literature. This study addresses a significant gap in the research by exploring the potential of foliar applied NAA to mitigate alkaline stress in squash. Moreover, this study highlights the novel effect of NAA in the growth performace, biochemical and physiological resilience under alkaline stress, an areae with limited prior reported studies. The findings of the current study suggest that "the application of naphthalene acetic acid foliarly may improve the growth and yield of squash under alkaline stress."

Materials and methods

The research study took place in the wire house facility at the prestigious Institute of Molecular Biology and Biotechnology (IMBB) at the University of Lahore, Pakistan. This study aimed to investigate the impact of foliar NAA application on squash plants subjected to alkaline stress. All the experimental procedures were carried out at the state-of-the-art General Botany Research Laboratory at IMBB, University of Lahore.

Procurement of the plant material and experimental setup

The seeds of squash were collected from the local seed market and sown in clay pots containing 7 kg of soil in each pot. Five seeds were sown in each pot and let to germinate under natural conditions until the two-leaf stage. One week after seed germination, the pots were divided into two sets: one was considered as control, while the other set of pots was irrigated with 40 mM NaHCO3 causing alkine stress to the plants at an interval of 10 days for 4 times throughout the experimental period ensuring the sustained presence of stress (Fig. 1).

Fig. 1
figure 1

(a, b) The effect of foliar application of different concentrations of NAA on the morphology of squash plant under (a) control and (b) alkaline stress conditions.

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Exogenous application of NAA

After two weeks of application of stress at the appearance of visible stress signs, the plants were treated with 0, 25, 50 or 75 mg/L NAA. A surfactant (Tween-20) was added to the solution of NAA at a ratio of 5% per liter. The plants were sprayed NAA thrice at an intervals of 5 days, while the control plants were sprayed with distilled water. The plants were harvested one month after the onset of fruit, making sure that the roots and shoots were undamaged, and the growth parameters were recorded. The plants were grown for total 60 days from sowing to harvesting

Growth performance and yield evaluation

The plants were harvested after the onset fruiting stage, and the fresh biomass of the plants were measured on a weight scale. Root and shoot samples from each treatment were taken and dried in a laboratory oven at 65°C to measure the dry biomass of the plants.

The root and shoot lengths of the harvested plants were recorded with a measuring tape. Fruits were separated from the petiole, and the number of fruits was recorded and their weight was also measured. Leaves from each plant in each treatment group were collected, and the number of leaves per pot was recorded. The volume displacement method was used to measure the fruit volume. The volume was calculated via the following formula:

$${\text{Fruit volume }}\left( {{\text{ml}}} \right) \, = {\text{initialwater level }} - {\text{finalwater level}}$$

Analysis of stress induced physiological changes

The plant leave extract was prepared by taking 0.5 g of fresh leaf from four replications of each treatment in 10 ml of phosphate buffer solution by storing it in a freezer for further analysis.

Analysis of photosynthetic pigments

Photosynthetic pigments, including chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, were determined by the methods proposed by Arnon (1949) 11. For tgese analysis, fresh leaves (0.5 g) from each pot of each treatment were chopped and immersed in 80% acetone. The tubes were incubated at room temerature overnight, and the absorbance was measured with a spectrophotometer at three different wavelengths of 480, 645 and 663 nm, separately taking acetone (80%) alone as a blank.

Analysis of primary and secondary metabolites

The total free amino acid were determined spectrophotometrily following the protocol proposed by Hamilton and Slyke (1943), taking the absorbance at 570 nm. The Bradford method (Bradford, 1976) was used to determine the total soluble protein content in the plant leaves and the absorbance of the BSA solution was measured at 595 nm by using spectrophotometer (UV/V spectrophotometer, HALO SB-10). The total phenol content was determined according to the proposed protocol of Julkunen-Titto (1985) by taking the absorbance at 760 nm at spectrophotometer.

Analysis of antioxidant enzyme assays

Evaluation of antioxidant enzymes was performed by taking fresh leaves (500 mg) homogenized in 5 mL of phosphate buffer (50 mM, pH 7.8) at 4°C. For 15 minutes, this extract was centrifuged at 12,000 rpm. The supernatant of this centrifugate was collected in autoclaved Eppendorf tubes for the determination of peroxidase (POD) and catalase (CAT) activity 12.

Statistical analysis

The experimental data were analyzed via computer-based software (STATISTIX 8.1) ver. 10.1 using analysis of variance (ANOVA). Pairwise comparisons between the groups were performed via Tukey’s HSD test.

Results

Foliar application of NAA promoted growth indices in squash plants under alkaline stress

The impact of foliar- applied of NAA on growth and biomass of squash was studied under both normal and alkaline stress conditions. Alkalinity in the soil reduced the root and shoot lengths by 42.1% and 33.8%, respectively, compared to the control plants. Similarly, the fresh biomass of a plant comprising root and shoot fresh weights decreased by 53.4% and 29.3%, respectively, in saline-stressed plants. The foliar application of NAA solution accelerated the growth in both stressed and nonstressed plants. Foliar application of 75 mg/L NAA increased shoot length by 53% and 46%, root length by 51% and 4%, and fresh plant biomass by 68% and 62% in stressed and nonstressed plants, respectively. Furthermore, NAA foliar application also increased the number of leaves on squash plants under both conditions (Table 1). Notably, compared with control plants, alkali-treated plants exhibited a 43% reduction in leaf count. However, 75 mg/L NAA increased the leaf count by 65% under stressed conditions and by 47% under control conditions demonstrating the growth promoting effects of NAA.

Table 1 Treatment details of pot experiment under different concentration of NAA.
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Foliar application of NAA improved photosynthetic pigments in squash plants under alkaline stress

The photosynthetic pigments of squash plants including chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were adversely reduced by 2% (Fig. 2a), 78% (Fig. 2b), 43% (Fig. 2c) and the 54% in alkaline-stressed plants as compared to control plants (Fig. 2d). However, an exogenously applied NAA solution increased the photosynthetic pigments, although more concentrated solution presented greater increase in pigments, as 75 mg/L NAA solution increased the chlorophyll a content by 54% and 43%, the chlorophyll b content by 53% and 87%, the total chlorophyll by 64% and 55%, and the carotenoid content by 45% and 66% in nonstressed and stressed plants, respectively (Fig. 2a–d). These findings underscore the role of NAA in enhancing photosynthetic efficiency under stress conditions.

Fig. 2
figure 2

(a–d) The effect of naphthalene acetic acid (NAA) under alkaline stress on photosynthetic pigments of squash plant. (a) Chlorophyll a, (b) Chlorophyll b, (c) total chlorophyll, and (d) caratenoids. Bars sharing similar alphabetic letters do not differ significantly (P ≤ 0.05).

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Foliar application of NAA modulates the concentrations of plant metabolites in squash plants under alkaline stress

The effects of NAA application on plant primary metabolites such as total soluble proteins (TSPs) and total free amino acids (TFAs) revealed that alkaline stress led to an 8% decrease in both analysis in stressed squash plants. However, foliar application of NAA reversed this decline and a significant improvement was observed with increasing concentrations of NAA (0, 25, 50 and 75 mg/L) solution. The foliar application of 75 mg/L NAA resulted in an increase of 27% in total soluble proteins under both stressed and nonstressed conditions, while, the same concentration of NAA, exhibited an increases of total free amino acids by 38% and 46% noted under stressed and unstressed conditions, respectively (Fig. 3a, b).

Fig. 3
figure 3

(a–d) The effect of naphthalene acetic acid (NAA) under alkaline stress on metabolites of squash plant. (a) Total soluble proteins (TSP), (b) total free amino acids (TFA), (c) total phenols, (d) falvanoids. Bars sharing similar alphabetic letters do not differ significantly (P ≤ 0.05).

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The plant secondary metabolites were also adversely affected by alkaline stress and in turm modulated by the application of NAA. Compared with the control condition, alkaline stress increased the total phenolic and flavonoid contents by 12% and 45%, respectively. However, 75 mg/L NAA application modulate the phenolic content by 28% and 24% and the flavonoid content by 27% and 20% in nonstressed and stressed plants, respectively (Fig. 3c, d).

Foliar application of NAA improved the antioxidant defense system in squash plants under alkaline stress

The antioxidant activities of squash plants grown in alkaline soil were also investigated under the effect of foliar application of NAA. The data revealed that, alkaline stress boost the enzyme activities in squash plants with catalase (CAT) and peroxidase (POD) activities increasing by 26% and 40%, respectively. However, the application of 75 mg/L NAA amplified these defense mechanisms by increasing CAT activity by 42% and 32%, whereas POD activity by 51% and 34% in nonstressed and stressed plants, respectively. The results revealed the NAA efficacy in strengthening plant defense combating stress effectively (Fig. 4a, b).

Fig. 4
figure 4

(a, b) The effect of naphthalene acetic acid (NAA) under alkaline stress on antioxidant enzymes of squash plant. (a) Catalase; (b) peroxidase. Bars sharing similar alphabetic letters do not differ significantly (P ≤ 0.05).

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These comprehensive analyses showed that application of NAA mitigate the worse effects of alkalinity and alsi enhances plant resilience, growth and metabolic functions under both stressed and non-stressed conditions

Discussion

Plant growth regulators (PGRs) are pivotal in regulating the life cycle of plants by orchestrating and overseeing numerous physiological processes that govern the plant growth and crop productivity. At lower concentrations, PGRs have the potential to enhance the physiological aspects of crops, contributing significantly in an increase crop yield 13.

Alkaline stress, an abiotic stress, typically induced by Na2CO3 and NaHCO3 which elevates soil pH levels inhibiting root growth, impair physiological functions, and disrupt cellular integrity. This stress disrupts ion homeostasis in roots, leading to the precipitation of Ca2+, Mg2+, and H2PO4−, while, the production of reactive oxygen species (ROS) is accelerated, which, interferes with metabolic pathways (Fan et al., 2021). High alkalinity caused by salts such as Na2CO3 and NaHCO3 pose significant harm to plant growth and nutrient absorption as evidenced in various crops 13,14.

In this study, foliar application of different concentrations of NAA (0, 50, 75, 100 ppm) showed significant improvement in growth parameters of squash plants under alkaline condition (Table 2). Alkalinity led to a decrease in plant length, fresh biomass, and leaf count, whereas the application of NAA ameliorated these reductions exhibiting its growth promoting effects. Similar reports have also been found for wheat, periwinkle, and tomato plants under alkaline stress conditions 15,16.

Table 2 Interactive effect of different concentrations of naphthalene acetic acid (NAA) under alkaline stress on growth indices of squash plant.
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Alkaline stress also reduced the photosynthetic pigments, including chlorophyll a, b, total chlorophyll and carotenoids (Fig. 1). Photosynthetic pigments, key components of light reactions in photosynthesis, are sensitive to almost all types of stresses, including alkaline stress causing damage in the thylakoid membrane, the site where all different types of photosynthetic pigments accumulate and hence suppressing the pigment synthesis enzymes 17. Additionally, stress impairs the enzymes involved in carotenoid biosynthesis such as phytoene synthase causing reduction in carotenoids in fruits and vegetables 18,19. However the foliar application of NAA notably increased the pigment level restoring the photosynthetic effeicenct which initiates the synthesis of important signaling molecules to support plant growth and development 20. On the other hand, the findings of this study (Fig. 2) align with previous studies who showed that exogenous plant hormone like NAA alleviate abiotic stress by improving photosynthetic pigment retention 21,22.

The total free amino acid and total soluble protein, the primary metabolites of inorganic assimilation, decreased in squash plants under alkaline conditions, whereas exogenously applied NAA reversed these declines (Fig. 3a, b). Secondary metabolites (phenolics and flavonoids) increased under stress as a positive response (Fig. 3c, d). Foliar application of NAA enhanced these metabolites, corroborating findings in cotton under same stress conditions as studied in the current experiment 23.

The scavenging of reactive oxygen species (ROS) is carried out in plants via the antioxidant enzyme system 24. Antioxidant production in response to abiotic stress, such as salinity and water stress, is an effective strategy for combating oxidative stress 25. Plants generate antioxidants in response to stress, which operates as a protective mechanism against the detrimental effects of ROS. In response to stress-triggered ROS accumulation, plants activate antioxidant defense mechanisms to eliminate excess ROS from their cells 26.

Antioxidant enzyme activity including catalase (CAT) and peroxidase (POD) were clearly increased by alkaline stress (Table 2), as plant activated its defense systems to combat ROS accumulation, these results are in consistent with those of 27, who suggested that root cell damage, and consequently growth inhibition, in rice seedlings under alkaline stress is closely associated with ROS accumulation. Analysis of variance (ANOVA) also showed variations in morphological parameters (Table 3). Exogenous NAA application played its role in mitigating oxidative stress supporting plant defense mechanism. Mechanistically, NAA improved CAT and POD activities reducing oxidative damage under stress, moreover, the role of NAA in maintaining ionic homeostasis by improved ion transportation and reduced precipitation of Ca2+ and Mg2+ helps in plant growth and nutrient assimilation. These impacts may highlight the auxin-mediated homonal crosstalk to optimize signaling and metabolic programming 28,29.

Table 3 ANOVA values of different concentrations of naphthalene acetic acid (NAA) under alkaline stress on growth indices of squash plant.
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Overall, the tested concentrations of NAA (0, 50, 75, and 100 ppm) applied exogenously improved the morphological, physiological and biochemical attributes of the squash plants grown in both the control and alkaline environments demonstrating its potential as a practical tool for managing crop productivity in stressed environments.

Conclusion

The current study demonstrates that foliar application of naphthalene acetic acid (NAA) effectively alleviates the adverse effects of alkaline stress on squash (Lagenaria siceraria), enhancing plant growth, yield, and biochemical attributes. Alkalinity-induced reductions in growth, photosynthetic pigments, and primary metabolites were significantly mitigated, with the foliar application of 75 mg/L NAA showing the most pronounced improvements. NAA not only boosted chlorophyll and carotenoid levels but also enhanced the synthesis of secondary metabolites like phenolics and flavonoids, essential for stress adaptation. Furthermore, it amplified the activities of antioxidant enzymes catalase (CAT) and peroxidase (POD), underscoring its role in fortifying plant defense mechanisms. These findings highlight the potential of NAA as a promising plant growth regulator to improve crop performance under both normal and stress-prone conditions, offering a practical approach to sustain agricultural productivity in alkaline soils. Further studies on molecular pathways and gene expression analysis will help to find out the precise mechanism underlying these responses