1 Introduction

Brinjal (Solanum melongena L.) of family Solanaceae has been originated in Indo-china domain, it is grown all over the world's tropical and subtropical regions (Rehman et al., 2019). In Pakistan, brinjal is grown over an area of 8,427 ha producing 84,255 tonnes annualy (Anonymous, 2019). Sindh and Baluchistan provinces of Pakistan follow Punjab in terms of area and production of this crop (Javed et al., 2017). Brinjal is Pakistan's most commonly used vegetable because of its unique flavor and high nutritional value. Its fruit is rich in vitamins, minerals and low-fat food. The brinjal fruit contains phenolic compounds, the most important being the chlorogenic acid which is beneficial for heart health and its bark has nasunin anthocyanin, which is causative agent of its specific black color. Nasunin is known as a powerful antioxidant and acts to combat free radicals and ultimately help to protect cell membranes.

Salinity, a global destructive environmental stress resulting in a notorious decrease in crop productivity. Globaly, 50 % of the agricultural land is disturbed by saltiness (Ahanger et al., 2020). Saline stress drastically decrease the availability of water by membrane instability, altering mineral distribution causing changes in turgor pressure, reduction in pigment synthesis, changes in gaseous exchange attributes, and subsequently decresead the crop yield (Ahanger et al., 2020; Kaya et al., 2020). Under the condition of stress different Reactive Oxygen Species (ROS) including hydroxyl, superoxides and hydrogen peroxide radicals produced which influences the damaging effects in the plants. In response to these ROS, plants activates stress resilience approaches including ion compartmentation, different antioxidant activites, and osmolytes production (Alam et al., 2019;). Moreover, manifestation of salt in soil exhibited the upregulation of antioxidant enzymes, osmolytes and metabolites including proline, flavonoids, glycine and other carbohydrates in plants (Ahanger et al., 2020; Kaya et al., 2020).

Brinjal plants are glycophytes and showed reduction in growth and productivity when exposed to salt stress as they are moderately sensitive to salinity as compared to other plants of the same family (Brenes et al., 2020a, 2020b). Brinjal is sensitive to salinity, a common factor responsible for reducing its biomass and yield components (Brenes et al., 2020a, 2020b). By these factors, understanding of intensity of salt amelioration in plants is important in increasing the yield of vegetable crops suplemented with saline solution. Organic acids expressed a substantial part in prolonging the physiological state of crops. The foliar application of citric acid (citrate or CA) has been developed as a practical method to enhance the plant resistance abil ity to conservational stresses and thus withstand crop yield. The (CA) is a six-carbon organic acid having a central role in the Krebs cycle. CA application can enhance the salt bearing abilily in plants and eventually improve the production (Mallhi et al., 2020). Plants have been displayed an improved tolerance under saline environment when supplemented with antioxidant solution (Ahmad et al., 2019; Kohli et al., 2019). Applying CA also modulates an antioxidant defense system, increase high photosynthetic pigment content affecting secondary metabolites enhancing plant productivity grown in stressed soil (Arif et al., 2021). CA treatment promoted essential oil components such as monoterpene hydrocarbons and oxygenated sesquiterpenes of Melissa officinalis grown in stress (salt) (Ahmed and Talaat, 2017). A study revealed that citrine fertilizers as a foliar application (0.3 %) improve maize plants' growth, yield, and chemical constituents (Mohamed and Yazal, 2019). Previous studies have revealed that the exogenous CA application can mitigates oxidative stress of plant by increasing the activity of antioxidant defense systems (Mallhi et al., 2020). There is non sufficient reports regarding the impacts of citric acid on the morphological, biochemical and physiological properties of brinjal plants in saline conditions has been put forward.

The functional characterization of the plants foliarly treated with citric acid especially the vegetables in salt stressed soil is not well described. Therefore, assessing the importance of citric acid in the brinjal plant's salt tolerance could be a valuable solution to mediate saline stress and increase the rate of crop production. It is postulated that appropriate doses of citric acid may enhance brinjal responses to saline stress by reducing the production of ROS thus, enhancing antioxidant enzymatic activities, ultimately stabilizing the plasma membrane. For that reason, the present study was designed to observe the physiological and biochemical effects of citric acid under salt-stressed brinjal plants reporting and an improved stress tolerance ability of the plant and mitigating the damages of plasma membrane caused by salinity via the synthesis of metabolites, osmoprotectants, and enzymatic as well as non-enzymatic antioxidants.

2 Methodology

A pot trial was executed to analyze the influence of citric acid (CA) on morphological and biochemical attributes in brinjal (Solanum melongena) under salt stress. The investigation was conceded in the field area of the University of Lahore, and lab work was performed in the General Botany Research Lab, Department of Institute of Molecular Biology and Biotechnology (IMBB), University of Lahore (UOL), Pakistan. The brinjal seeds of variety Puskranti were obtained from local market of Lahore, Pakistan and sown in pots containing 5 Kg of loam-sand-compost soil (w/w) in the ratio of 3:1:1. Seeds were disinfected by dipping in 5 % solution of sodium hypochlorite for 5 min before sowing and then rinsed meticulously thrice with distilled water. The pots were kept in a field under natural sun light and seasonal temperature conditions. The experiment was performed in a completely randomized design (CRD). Four replicates were taken for each treatment and it took 4.5 months from sowing of seeds to harvesting.

3 Results

3.3

3.3 Increase of leaf pigments by CA application

Salt stress significantly influences the leaf pigments, including chlorophyll a, b, total chlorophyll contents, and carotenoids of brinjal. A significant decrease was exhibited in chlorophyll a by 15 %, chlorophyll b by 15 %, total chlorophyll by 10 %, and carotenoid contents by 11 % under saline conditions over control plants. Exogenously applied CA at 200 ppm under salt stressed condition improved the chlorophyll a by 54 %, Chlorophyll b by 65 %, total chlorophyll by 61 % and carotenoids by 27 %, while, under control conditions, foliar application of CA increased the chlorophyll a, b total and carotenoids by 59 %, 69 %, 72 % by and 36 %, respectively, over control plants without the application of citric acid (Fig. 1a-d).

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Fig. 1

Impact of foliar application of differential CA concentrations on the (a) chlorophyll a, (b) chlorophyll b, (c) total chlorophyll, and (d) carotenoids of brinjal grown in salt stressed conditions. Letters on the bars signify different statistical means (p ≤ 0.05).

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3.4

3.4 Determination of biochemical attributes under CA application

A significant amount of reduction (8 %) in Total Free Aminoacids was recorded in specimens grown in saline soil as compared to non-saline soil. However, a 36 % increase in total soluble protein content was observed under stressed conditions in contrast to control plants. While the foliar spray of CA increased the total free aminoacids and soluble protein content. The exogenously applied citric acid (300 ppm) increased the total soluble proteins by 75 % and total free amino acids by 32 % under salt-stressed condition, while, 50 % and 53 % increase in total solube proteins and total free amino acids were observed by the foliar application of 200 ppm CA under non-stressed conditions, respectively, as compared to without citric acid (Fig. 2a-b).

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Fig. 2

Impact of foliar application of differential CA concentrations on the (a) Amino acid and (b) protein content of brinjal grown in salt stressed conditions. Letters on the bars signify different statistical means (p ≤ 0.05).

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Salt stress significantly increased the synthesis of phenolics and flavonoids. It causes a 31 % increase in total phenolics and a 96 % increase in total flavonoids under salt stress conditions over control plants. Moreover, exogenously applied citric acid at 200 ppm further promoted total phenolics up to 85 % and total flavonoids by 1.3-fold under saline condition. The concentration of 300 ppm CA under non saline environment increased the total phenolics by 82 % and total flavonoids by twofold (Fig. 3a-b).

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Fig. 3

Impact of foliar application of differential CA concentrations on the (a) total phenolics and (b) flavonoid content of brinjal grown in salt stressed conditions. Letters on the bars signify different statistical means (p ≤ 0.05).

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3.5

3.5 Determination of antioxidants

The effect of citric acid on antioxidant modulation, including CAT, POD, SOD, GPX, and POD, was measured under stressed environment. Salt stress increases the activities of CAT by 42 %, POD by 66 %, SOD by 44 %, GPX by 37 %, and PPO by 50 % than in non-saline conditions. Foliar application of citric acid causes more increases in antioxidant activities under salt-stressed as well as non-stressed conditions. Specifically, the foliar application of CA (300 ppm) increased the activities of CAT by 68 %, GPX by 1.6 fold PPO by 99 % and POD by 1.2 fold, but for SOD application of 200 ppm showed maximum value (2fold) than the plants without CA supplementation grown under saline condition. The plants grown under non saline environement and treated with 300 ppm CA showed an increase of 18 %, 20 % and 75 % in CAT, GPX and PPO as compared to the plants with no application of CA under non-saline environment. The POD and SOD activity was improved by 34 % and 43 %, respectively, in the plants grown under non-saline condition by treated with 200 ppm CA (Fig. 4a-e).

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Fig. 4

Impact of foliar application of differential CA concentrations on the (a) catalase, (b) peroxidase, (c) superoxide dismutase, (d) glutathione peroxidase, and (e) polyphenol oxidase activity of brinjal grown in salt stressed conditions. Letters on the bars signify different statistical means (p ≤ 0.05) (All enzymatic antioxidants are expressed as U/mg Protein).

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4 Discussion

Plants often bare unfavorable environmental conditions, such as abiotic stresses, which intensely limit the yields of crop species. Salt stress has been proved a severe issue for plant growth, reducing crop yield and production, worldwide. The strategy of foliarly applying citric acid is widely used all over the world and is essential for promoting sustainable agriculture. Citric acid, an important component of Krebs cycle for the production of compounds involved in the synthesis of amino acids and fatty acids. The cirtic acid cycle is very important for the production of ATP in providing precursors for various metabolic pathways. Salt stress affects the enzymatic activites involved in respiration thus disturbing the cycle. While, CA converted to 5 or 4-carbon compounds by splitting hydrogen atoms and cycle again converted to oxaloacetic acid (Wang et al., 2017). So, goal of the recent study was to determine how salinity-stressed brinjal plants' morphological, physiological, and biochemical contents were affected by the exogenous application of citric acid.

A damaging abiotic stress called salt stress restricts plant growth showing negative influence on a plant's morphological, biochemical, and physiological characteristics. Primary problems of plants to stress include stunted growth of roots and shoots, nutritional imbalance, sluggish rate of germination, closure of stomata, reduction in seedling growth, and deterioration of photosynthetic activity (Ahanger et al., 2020). Salinity considerably reduced the growth (18–27 %) and yield parameters(18–20 %), according to our study. These findings agree with accounts on a variety of plants from earlier times (Ahanger et al., 2020; Alam et al., 2020; Kaya et al., 2020). That could imply that plant tissues growing in a saline environment produced more proline. The decrease in cell water potential was also seen at the time, which in turn causes salt-stressed plants to develop more slowly and have less photosynthetic activity and plastid pigment. The results showed that when different concentrations of CA (100, 200, and 300 ppm) were applied topically to brinjal plants, 300 ppm of CA increased several growth parameters(40–80 %), including the number of leaves, the area of leaves, the height of the plant, and the weight of the plant, while 200 ppm of CA increased various yield attributes (70 %), including the number of fruits, their weight, diameter, and volume. According to Mohamed and Yazal (2019) who published comparable findings on maize plants, the conclusions of this study. The ability of CA to participate in nutrient absorption, water relations, stress signalling, and enhance photosynthesis and phytochelatin synthesis in plants may be responsible for improvements in biomass and plant growth. These actions ultimately have a positive influence on plant growth, yield, and quality of crop as well as on plant vegetative and reproductive development (Mallhi et al., 2020).

The primary components of light reactions in photosynthesis are photosynthetic pigments. The thylakoid membrane, the location where various types of photosynthetic pigments are deposited, is said to be damaged by practically all types of abiotic challenges, including salinity stress. To keep the photosynthetic system operating as it should, moderate salinity stress increases the production of total chlorophyll and carotenoid concentration (Hameed et al., 2021). Salinity considerably condensed the total chlorophyll (15 %) and carotenoid content (11 %) in the current study as compared to control plants. Our findings in this regard are consistent with earlier studies on different plants (Ahanger et al., 2020; Alam et al., 2020; Kaya et al., 2020). A comparison of means showed that the exogeniously applied concentrations (100,200, 300 ppm) of citric acid increased leaves' total chlorophyll content. Among them, 200 ppm CA resulted in significantly higher total chlorophyll (65 %) and chlorophyll a content of leaves in contrary to the control plants which follows the trend observed in chlorophyll b and carotenoid content where 200 ppm citric acid has a more positive effect under stressed or non-stressed conditions. Similar to another study reported by Arif et al. (2021) who found that a number of morphological and yield attributes, and physiological parameters (CHl a, Chl b, total chlorophyll, carotenoids and proline) were amplified by exogeneous treatment of CA in Gosspium barbadense, also indicated the better physiological parameters results in higher photosynthetic activity, reducing Reactive Oxygen Species, and also increase osmoregulation.

The current study also showed that salinity greatly influences the Total Free Aminoacids which were considerably decreased than that of control plants. Our findings are in accordance to the previous reports (Riaz et al., 2021) which indicated that salinity decreases the total free amino acids by increasing the formation of osmolytes such as proline, and GB in cluster bean which significantly drained the osmotic potential of the cell, maintaining the integrity of proteins and working as a molecular chaperone. Therefore, higher levels of osmolytes also serve as an antioxidant in their own right under saline conditions. However, a significant increasing trend was observed in total soluble protein content under salt stress conditions. In this regard, our results are in line with the findings of Aly et al. (2019) on the wheat cultivars and stated that these proteins could have an important part in signal transduction, antioxidative defense, anti-freezing, heat shock, metal binding, anti-pathogenesis and osmolyte synthesis, which is an important part in plant’s growth and physiological attributes. The current research also indicated that citric acid application at the rate of 300 ppm significantly improved the Total Soluble Proteins and free amino acids under salt stress conditions compared to control plants on which citric acid was not applied. Our results are in compliance with El- Beltagi et al. (2017) who foliarly sprayed citric acid on G. Barbadence and observed similar results, which showed that citric acid is deliberately taken as an important organic acids of respiration into the plant cell. Energey currency (ATP) of all the biochemical and physiological processes is provided by the citric acid cycle operated in mitochondria of the cell.

According to several studies, phenolics and flavonoids, non-enzymatic antioxidants with low molecular weights or secondary metabolites, exhibited various biochemical and molecular roles in plants, including maintaining redox-homeostasis, acting as signalling molecules, activating plant defence mechanisms, mediating auxin transport, and enhancing antioxidant free radical scavenging assays (Sirin and Aslam, 2019). According to the study's findings, salinity-stressed plants produced more total phenolics (30 %) and flavonoids (90 %) in their leaves than salinity-free control plants. In this regard, our findings are consistent with those of Kiani et al. (2021) who noted comparable outcomes in wheat varieties. However, the foliar application of citric acid at (200 ppm) under salt stress considerably boosted the total phenolics of leaves and flavonoids than the control plants. Related findings were found by Rangel et al. (2018) who reported that CA-treated wheat sprouts activated signal transduction pathways that resulted in a greater concentration of secondary metabolites. Another study in tea plants found that the CA supplementation stimulated the production of more proline and other metabolites in response to abiotic stressors (including phenolic compounds, flavonoids, tannins, and sugars) (Li et al., 2019). In stressful conditions, the hydrolysis and breakdown of cellular constituents in cell walls results in the buildup of phenolic compounds. Also, the use of CA decreased pH, which boosted the presence of flavonoids and anthocyanins in the plants (Kaur et al., 2017).

According to our findings, various antioxidants (CAT, POD, SOD, GPX, and PPO) were increased (50 %) by salt rather than control plants. These outcomes are in line with a number of earlier studies (Ahanger et al., 2020; Kaya et al., 2020) which noted comparable outcomes in various plants. In the current study, 300 ppm CA improved all antioxidant activities (CAT, POD, GPX, and PPO) under stressed plants rather than control plants among the various CA concentrations applied foliarly. In contrast to control plants, superoxide dismutase activity (SOD) was considerably higher at 200 ppm citric acid under salt stress. Accordingly, current results are in line with those of several earlier studies (Ahmad et al., 2017) which noted comparable results in sabdariffa, Beta vulgaris, and Oryza sativa, respectively, and came to the conclusion that CA supplementation is involved in ROS scavenging assays, enhancing antioxidant enzymes and secondary metabolites. During salt stress, antioxidants help to rummage ROS, prevent oxidative destruction to plants, and enhance cellular redox equilibrium. SOD defends plants from oxidative loss by transforming O₂^– (superoxide anion) to H₂O₂, whereas CAT and GPX directly reclaim ROS by converting H₂O₂ to water and oxygen (Kohli et al., 2019). Furthermore, peroxidase (POD) is known to shield cells from ROS by catalysing redox processes (Su et al., 2020). Inducing the oxidation of phenols to quinines, polyphenol oxidase (PPO) is widely present in plants and is thought to play an essential role in the plant's defence mechanism against environmental challenges (Akhtar and Mahmoo, 2017).

5 Conclusion

The goal of the experiment was to find out how citric acid helped brinjals to cope with salt stress. The amount of total free amino acids, photosynthetic pigments, and growth and yield characteristics are all dramatically decreased by salt stress, although total soluble proteins and secondary metabolites, such as phenolics and flavonoids, are increased. Moreover, plants produced under salt stress have three times more enzymatic antioxidant activity than control plants. Supplemental citric acid (200 and 300 ppm) markedly improved growth and yield-related traits, chlorophyll pigments, and antioxidant potential. The beneficial effects of exogenous citric acid treatment in brinjal under salt stress may have been seen in free radical scavenging tests, preservation of membrane stability, improved root functioning, and stimulation of antioxidant production. Based on these findings, commercial formulations for boosting the development and production of brinjal crops cultivated under stress could do well to consider citric acid doses of 200 and 300 ppm. To learn more about its impacts on metabolic and molecular pathways, more research is needed.

Funding

This work was supported by the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia (RSP-2023R350).

7 Author’s contributions

The research conceptualization was a collaborative effort involving all authors. NA, RR, and ZN were primarily responsible for drafting the experimental design, while NA and RR conducted the experiments. ZN and LW were involved in data analysis and validation. AA and PK contributed to the language editing of the manuscript. All authors participated in reviewing and approving the final version of the manuscript before its submission.