Citric Acid Alleviated Salt Stress by Modulating Photosynthetic Pigments, Plant Water Status, Yield and Nutritional Quality of Black gram [Vigna mungo (L.) Hepper]

Grain legumes are a vital source of protein, a wide range of minerals and numerous vitamins which are essential for robust human health (Iqbal et al., 2019). These are the cheapest source of protein and hence referred to as poor man’s meat (Abbas et al., 2021; Iqbal et al., 2018). Among pulses, black gram [Vigna mungo (L.) Hepper] has attained strategic pertinence in ensuring the food and nutritional security of a rapidly increasing population (Iqbal et al., 2024; Ramamoorthy and Ariraman, 2023). Various traits including self-pollination, well-grown root network, drought tolerance, short life cycle (Hema et al., 2024) and unprecedented adaptability under varying agro-climatic conditions favor its cultivation (Sadiq et al., 2023). Despite significant genetic improvement of field crops using genetic engineering approaches (Li and Iqbal, 2024), legumes yield has remained stagnant owing to frequent incidence of abiotic stresses (soil salinity, heat stress, drought, etc.) (Iqbal and Iqbal, 2015) and biotic stresses (Arthanari, 2024).
       
Recently, soil salinity (SS) has emerged as one of the most challenging abiotic stresses for sustainable production of field crops including black gram (Ahmad et al., 2023). The SS directly imposes physiological disruptions of vital metabolic processes like photosynthesis in crop plants, which eventually causes stunted growth and even crop failure becomes imminent (EL Sabagh et al., 2021). The saline conditions led to the imposition of ionic imbalance and osmotic stress which ultimately induces oxidative stress (Sagar et al., 2023; Yasir et al., 2021). Although, black gram is reported to be a moderately saline-tolerant crop, however, its productivity suffers under SS, which necessitates conducting fresh studies to find out biologically viable SS mitigation strategies. Citric acid (CA) is a 6-carbon tricarboxylic acid compound that is primarily synthesized by acetyl-CoA and oxaloacetate using citrate synthase catalyzed condensation process (Khatun et al., 2019). Moreover, CA modulated the physiological response of common bean plants (El-Tohamy et al., 2013). Likewise, CA applied in conjunction with ascorbic acid and salicylic acid ameliorated the adverse effects of SS in maize by improving photosynthesis rates and osmoregulation along with reduced biosynthesis of reactive oxygen species (ROS) (El-Hawary and Nashed, 2019).
       
Although, SS tends to hamper black gram productivity, however, very few research studies have previously aimed to mitigate the deleterious effects of SS on black gram by foliar application organic acids like CA. There have been noticeable research gaps pertaining to the optimization of CA doses for boosting yield attributes, grain yield and quality of black gram. Therefore, this study was undertaken to assess the adverse effects of different levels of salinity on the growth, physiology, nutritional status and yield of black gram and to sort out the most superior dose of foliar applied CA for boosting the yield and nutritional quality of black gram sustainably in an eco-friendly manner.

An experiment was carried out in shade-house conditions at Department of Agronomy, Hajee Mohammad Danesh Science and Technology University (HSTU), Dinajpur, Bangladesh (25°37'N latitude and 88°39'E longitude and the elevation was 37.5 m) during 2022. A two factorial experiment was carried out entailing three levels of salt stress viz. S₀ = 0 mM, S₁ = 50 mM and S₂ = 100 mM NaCl and three levels of citric acid (CA) viz., CA₀ = 0 µM, CA₁ = 50 µM and CA₂= 100 µM applied as sodium nitroprusside (SNP) for black gram (cv. BARI Mash-3). The experiment was laid out with completely randomized design (CRD) in factorial arrangement with three replications (three additional replications for recording response variables involving destructive protocols such as root and shoot length and weight, etc).
       
For filling pots, soil was thoroughly mixed and pots were filled with 10 kg soil. Nitrogen (N), phosphorus (P), potassium (K) and sulphur (S) were applied as urea, triple super phosphate (TSP), muriate of potash (MOP) and gypsum, respectively, at the rate of 45, 90, 40 and 50 kg ha^-1, respectively, as a basal dose at the time of pots preparation. The seeds were treated with Bavistin-200 (0.3%) and sown on 6 March 2022 in 2-3 cm depth, wherein 20 seeds were sown per pot.
       
For measuring grain yield (GY), the grains were separated from each plant manually and sun-dried. The grain and stover stover yields (StY) were converted into yield (g) plant^-1. The biological yield (BY) of black gram was estimated by using the Eq 1 as described by Iqbal et al., (2019).
   
The harvest index (HI) was determined by using the Eq. 2 as described by Iqbal et al., (2019).
   
Photosynthetic pigments measurements
 
From each treatment, third trifoliate leaves of black gram were separated carefully for determining the chlorophyll content (Witham et al., 1986). Fresh weight of leaves (0.10 g) was taken, then leaves were kept immersed in acetone (10 ml) for 48 hours in dark condition at room temperature. For estimating the chlorophyll a, b and total chlorophyll of black gram, the absorbance of the supernatant was noted using an UV-visible spectrophotometer (663 nm wavelength). Thereafter, their contents were measured (mg g^-1) using Eq 3-5 as described by Ahmad et al., (2023).
   
Where,
D= Chlorophyll extract’s absorbance reading at a specific wavelength.
V= 80% acetone-chlorophyll extract final volume.
W= Tissue extracts fresh weight.
       
The terminal leaflets of the fully expanded leaves of black gram plants from each treatment were used to record fresh, dry and turgid weights of leaf segments for estimating the relative water content (RWC), water retention capacity (WRC), water saturation deficit (WSD) and water uptake capacity (WUC) (Islam et al., 2021b).
 
The oven dried grains were subjected to mechanical grinding with the help of a Willy grinding machine for estimation of protein (P) and nitrogen (N) content. Subsequently, 1.0 g of finely ground grains were taken into a 250 ml conical flask and 10 ml if di-acid mixture (HNO₃:HClO₄ = 2:1) by following wet oxidation method (Jackson 1973), whereas the N content was determined by using the Kjeldhal method.
 
Statistical analysis
 
The recorded data were statistically analyzed to determine the significance among employed treatments of salinity levels and citric acid doses using MSTAT-C statistical package. For this purpose, two-way analysis of variance (ANOVA) was performed to determine the overall significance of employed treatment factors (salinity levels and citric acid doses) and thereafter, significance among treatment means was estimated by employing the least significant difference (LSD) test at 5% probability level (Gomez and Gomez, 1984).

Plant height and number of leaves per plant
 
The plant height and number of leaves plant^-1 showed significant variation in saline environment at all sampling stages (Table 1). The results indicated that these traits of black gram decreased with increasing levels of SS. However, application of CA (100 µM) significantly increased plant height, which were statistically similar to 50 µM CA. The tallest plants of 37.83, 69.00 and 71.67 cm were observed from S₀CA₂ (0 mM NaCl + 100 µM CA) at 35, 55 DAS and FH, respectively, while the minimum plant height (13.83, 23.33 and 23.83 cm) values were observed for S₂CA₀ (100 mM NaCl + 0 µM CA) treatment at 35, 55 DAS and FH, respectively. The results also showed that the highest number of leaves per plant (6.00, 8.33 and 8.67) was exhibited by S₀CA₂ (0 mM NaCl + 100 µM CA) and the lowest number of leaves per plant (3.00, 5.00 and 5.33) was observed in S₂CA₀ (100 mM NaCl + 0 µM CA) i.e. without CA at 35, 55 DAS and FH, respectively. Nevertheless, addition of CA enhanced the number of leaves per plant and the treatment S₁CA₂ (50 mM NaCl + 100 µM CA) remarkably increased the number of leaves per plant (4.33, 7.67 and 8.33 at 35, 55 DAS and FH, respectively).
 

 
Roots fresh and dry weights and chlorophyll a, b and total contents
 
The SS significantly diminished the root fresh and weights, along with chlorophyll a, b and total contents of black gram (Table 2). The CA application enhanced root fresh weight and the highest values (0.074 and 0.427 g plant^-1) were recorded by CA₂ treatment which was statistically at par with CA₁ (0.063 and 0.356 g plant^-1) at 35 and 55 DAS, respectively. Regarding root dry weight, the highest root dry weight (0.024, 0.130 and 0.204 g plant^-1 at 35, 55 DAS and FH, respectively) was observed in S₁CA₂ (50 mM NaCl + 100 µM CA), while the lowest dry weight of root (0.010, 0.097 and 0.098 g plant^-1, respectively) was found in S₂CA₀ (100 mM NaCl + 0 µM CA) treatment. Combined effect of SS and CA on the Chl a was statistically significant and SS-induced reduction of Chl a was improved due to CA. Under higher SS, the highest Chl a (1.39 mg g^-1 FW) was recorded with the application of CA₂ (S₂CA₂) which was alike (1.35 mg g^-1 FW) to CA₁ (S₂CA₁). Additionally, CA application increased Chl b concentrations as the highest Chl b (0.77 mg g^-1 FW) was recorded in S₂CA₂ (100 mM NaCl + 100 µM CA) treatment which was similar to S₂CA₁ (0.73 mg g^-1 FW). The results revealed that TChl content showed a significant increase as the level of CA was increased as due to combined effect of salt and CA, the highest TChl (2.44 mg g^-1 FW) was noted for S₁CA₂ (50 mM NaCl + 100 µM CA) treatment (Table 2).
 

 
Yield contributing traits and water status
 
The interaction effect of SS and CA showed that the maximum pods number per plant (11.33) was found in S₀CA₂ (0 mM NaCl + 100 µM CA) (Table 3). It was also observed that the highest pod length (4.68 cm) was recorded from S₀CA₂ (0 mM + 100 µM) and the lowest corresponding value (3.03 cm) was found in S₂CA₀ (100 mM NaCl + 0 µM CA). Moreover, the highest grains number per pod (5.12) was recorded from without SS and CA condition i.e. S₀CA₀ (0 mM NaCl NaCl + 0 µM CA) and the lowest corresponding value (3.39) was found in higher salt without CA i.e. S₂CA₀ (100 mM NaCl + 0 µM CA). Under higher SS condition, the highest grains number per pod (4.20) was recorded in CA₂ (S₂CA₂) which significantly differed with CA₁ (S₂CA₁). The SS (100 mM) reduced the grains number per pod, while CA application increased its corresponding value (7.67 and 23.89%, respectively). Furthermore, Application of CA increased the 100-grains weight over without CA. The combined effect revealed that the treatment combination S₀CA₀ (0 mM NaCl + 0 µM CA) produced the highest grain weight (3.63 g), whereas the treatment combination S₂CA₀ (100 mM NaCl + 0 µM) showed the lowest corresponding grain weight (1.54 g). Application of CA (100 µM CA) increased the HGW by 72.72% under 100 mM NaCl-induced saline stress in (S₂CA₂).
 

       
Regarding water relations, S₂CA₂ combination exhibited the highest relative water content of 69.16% which was statistically equal to S₂CA₁ and the lowest value of 62.98% was given by S₂CA₀. Likewise, the interaction effect of salinity levels and CA doses revealed that the highest water saturation deficit (37.02) was recorded for S₂CA₀ combination and the lowest corresponding value (24.17) was observed in S₁CA₂ combination. Additionally, the CA application increased the water retention capacity in plants as the highest value of 7.05 was observed in plants treated with 100 µM CA (CA₂), indicating a better ability to retain water. In contrast, the lowest water retention capacity of 6.34 was recorded in plants treated with 0 µM CA (CA₀). Moreover, spraying of CA relieved the plants from SS and decreased the water uptake capacity. The highest water uptake capacity (2.67) was observed without CA and the lowest (1.65) was in CA₂ which was statistically dissimilar to CA₁ (2.14).
 
Grain, stover and biological yields, harvest index, nitrogen and protein contents
 
Under varying salinity levels, the highest and lowest grain yields (0.64 and 0.35 g plant^-1) were observed for S₂CA₂ (100 mM NaCl NaCl + 100 µM CA) and S₂CA₀ (100 mM NaCl NaCl + 0 µM CA), respectively (Table 4). Nonetheless, the maximum stover yield of 1.90 g plant^-1 under severe SS was found with spraying CA₂ (S₂CA₂) (100 mM NaCl + 100 µM CA) which was statistically alike to S₂CA₁. Interestingly, the results revealed that the maximum biological yield (4.69 g plant^-1) was recorded by S₀CA₂ (0 mM NaCl +100 µM CA). Additionally, the highest biological yield (2.69 g plant^-1) was found in S₁CA₂ (50 mM + 100 µM CA). Contrastingly, the lowest biological yield of 1.54 g plant^-1 was observed for S₂CA₀ (100 mM + 0 µM CA). Furthermore, the CA application increased the harvest index from 31.66% (CA₀) to 34.11 and 35.54% at 50 and 100 µM CA, respectively. Moreover, the results showed that CA remarkably increased nitrogen content from 3.06 to 3.54 mg g^-1 DW accounting by 15.69% (S₂CA₂) which was statistically similar to S₂CA₁. Moreover, the CA application significantly increased protein content as the treatment combination S₂CA₂ (100 mM + 100 µM) showed the highest value of protein content (22.13%) under severe SS (100 mM) (Table 4).
 

 
Correlation analysis
 
The results showed that stem dry weight at final harvest had positive non-significant correlation with plant height at harvest, PL, grain weight, stover yield, biological yield, harvest index and grain yield of black gram sown in saline environment. However, rest of the traits showed positive significant associated with one to another indicating p=0.05, *; P=0.01, ** and p=0.001, *** probability levels (Fig 1). On the contrary, positive significant associations were found among the photosythetic pigments and plant water relation traits with grain yield (Fig 1). In this case, water uptake capacity and water saturation deficit showed significant negative associated with relative water content, water retention capacity, Chl a, Chl b, Tchl and grain yield following different probability levels (p=0.05, *; P=0.01, ** and p=0.001, ***). Other traits recorded significant positive associated with one to another as water retention capacity with relative water content, Chl a, Chl b, Tchl, grain yield, water uptake capacity and water saturation deficit.
 

       
The obtained results indicated that the SS decreased the yield attributes of black gram, while CA application remained effective in boosting these traits of black gram plants by alleviating the adverse effects of SS. The CA foliar spraying stimulated the photosynthesis process which led to increased synthesis and accumulation of carbohydrates (Abd El-al and Faten, 2009). Likewise, CA triggered the biosynthesis of antioxidants that stimulated cell division which led to taller plants (Chakrobortty et al., (2022). In another study, CA improved the root system of wheat under saline-sodic soils by decreasing sodium absorption ratio (SAR), soil pH, exchangeable sodium percentage (ESP) and electrical conductivity (ECe) which increased plant height significantly (Aslam et al., 2022). These findings are in agreement with the previous results whereby plant fresh weight of tomato was increased under SS by organic acid sprays (Ghoohestani et al., 2012). CA could counteract the harmful effect of SS on growth by improving soil properties, photosynthetic pigments, photosynthetic rate, transpiration rate, stomatal conductance and activity of enzymes like catalase (CAT), proline dehydrogenase (ProDH), Ascorbate oxidase (AO) (Behairy et al., 2017) leading to increase yield attributes.
       
The chlorophyll content of leaves determines plant’s photosynthesis capability and growth rate, while the SS declines the chlorophyll content in plants by inhibiting translocation and assimilation of photosynthetic products, contributing to early leaf senescence and photosynthesis inhibition. The SS damaged the chloroplast structure and inhibited photosynthetic activity (Fidalgo et al., 2004). However, it could be attributed that CA exogenous application effectively reduced the adverse effects of SS on photosynthetic machinery by improving the chlorophyll biosynthesis and the mobilization of internal tissue nitrate (Behairy et al., 2017).
       
The plant cell’s capacity to retain water is termed as relative water content which demonstrates cellular water deficit caused by SS (Islam et al., 2021a). The highest relative water content (76.47%) was observed in plants treated with 100 µM CA, indicating efficient water uptake and retention and the value was statistically identical to 50 µM CA. It was also concluded that CA application in saline-stressed plants mitigated the adverse effects of SS and increased the relative water content in plants (Sun and Hong, 2011). The CA might have neutralized the NaCl-induced toxic effects in plants which promoted water use efficiency (WUE) under SS along with reducing water saturation deficit under SS by osmotic adjustment (Chakrobortty et al., 2022).
       
The results of the present findings demonstrated that SS reduced the pods number per plant, while CA application alleviated SS and increased the pod number plant^-1. These results remained in agreement with Khan et al., (2010), who reported that plant growth hormones significantly increased the pod length of mungbean under SS. The pod length of bean (Phaseolus vulgaris L.) is increased with CA under drought stress as reported by El-Tohamy et al., (2013).
       
Yield is the final manifestation of the growth and photosynthetic processes and the results of the current study demonstrated that SS declined the grain yield, but application of CA increased the grain yield under non-stress and NaCl-induced SS conditions by alleviating the adverse effect of stress. The increased grain yield by the application of CA might be attributed to an increase in yield attributes especially number of pods and grains per pod and 1000 grain weight. Previously, it was reported that CA application significantly increased the grain yield by neutralizing the deleterious effects of saline environment and promoted the yield attributes in different field crops (Dadrwal et al., 2022).
       
The N content was declined due to the incidence of SS, whereas its content was enhanced significantly by the foliar applied CA in different doses. Previously, it was depicted that SS reduced the N content in wheat while application of ascobin (ascorbic acid and citric acid in 2:1 ratio) significantly increased the N content (Elhamid et al., 2014)). In this study, various levels of CA application in saline soil had a significant effect on the protein content of black gram grains. The SS impaired the nitrogen accumulation and reduced protein content, whereas CA exogenous application enhanced the accumulation of nitrogen, consequently increasing the value of protein in soybean (Sheteaw, 2007). This study also assessed the relative performance of yield variables under SS by employing the correlation analysis as reported by previous studies (Islam et al., 2021b; Islam et al., 2023). We noted a strong positive correlation between the grain yield and plant growth, yield-related traits as well as photosynthetic and water status owing to CA application under SS and non-stress conditions. Furthermore, these results remained in agreement with those of Islam et al., (2023), who recorded that SS significantly reduced vital yield attributes that had linear association with grain yield and ultimatley grain yield was reduced drastically in saline environment.

The recorded findings were in accordance with the research hypothesis as saline environment negatively affected blakgram yield contributing characteristics and grain yield, whereas citric acid application significantly alleviated the deleterious effects of varying levels of salt stress. According to recorded findings, citric acid (100 µM) remained unmatched in terms of morphology, growth, physiology, yield contributing traits, yield and grain quality of black gram, whereas it was statistically identical with 50 µM concentration for different yield attributes and grain yield. Therefore, black gram (cv. BARI Mash-3) could be sustainably grown under mild salt stress (50 mM stress) by using citric acid (50 µM) foliar application. These encouraging findings necessitate conducting further in-depth studies for evaluating higher doses of citric acid in terms of their efficacy for boosting black gram yield by alleviating the deleterious effects of a saline environment. Moreover, nutritional quality assessment of black gram grain must be studied in response to foliage applied citric acid doses and varying levels of salinity along with exploring the underlying citric acid associated mechanisms which neutralize the adverse effects of salinity.

The authors extend their appreciation to the Researchers Supporting Project number (RSP-2024R298), King Saud University, Riyadh, Saudi Arabia.
 
Funding
 
The authors extend their appreciation to the Researchers Supporting Project number (RSP-2024R298), King Saud University, Riyadh, Saudi Arabia.

The authors declare no conflict of interest.