The results indicate a notable improvement in growth parameters after applying MnSO₄ and salicylic acid to the leaves, including the leaf area index and the fresh and dry weight of the leaves, stem, and root. An important element for plant metabolism is manganese. In plants, it functions as a co-factor and activator for hundreds of metalloenzymes. Mn is essential for a wide variety of enzyme-catalyzed processes, such as phosphorylation, hydrolysis, decarboxylation, and redox reactions, due to its propensity to quickly alter oxidation state in biological systems26. Mn functions as a metalloenzyme in the plant system, including manganese superoxide dismutase (Mn-SOD) and oxygen-evolving complexes (OEC) of photosystem II (PS II)27. Mn is especially accumulated in the spongy and palisade parenchyma cells and the periphery cells of the leaf petiole and petiolule28. The isochorismate (IC) route and the phenylalanine ammonia-lyase (PAL) pathway are the two separate processes involved in SA production in plants. These processes contribute to the development of plants, thermogenesis, and ion absorption29. In our study, the leaf area index was significantly affected by the simultaneous foliar application of Mn and SA compared to separate applications. When SA is used with MnSO₄, it significantly impacts leaf size and growth. Research has revealed that salicylic acid promotes the growth and division of cells, which is essential for increasing the size of leaves and improving the leaf area index as the concentration of the acid increases. Applying salicylic acid from an external source has been proven to improve growth characteristics and net photosynthesis without stress while reducing drought's negative impacts30. Similar findings found in mustard by Nazar et al., 201530. It is well recognized that SA functions as a signaling molecule in plants, inducing a range of physiological and morphological reactions. It controls the generation of reactive oxygen species (ROS), which are essential to the metabolic activities of plants31. In addition, the combination of higher levels of salicylic acid and Mn improves crop growth by regulating different physiological processes in plants, including thermogenesis, flower induction, nutrient uptake, ethylene production, stomatal movement, photosynthesis, and antioxidative enzyme activity32. These factors likely contribute to the observed increase in growth attributes in mustard. The promotion of cell growth by salicylic acid can result in the development of larger leaves, which in turn increases the surface area of the leaves. This leads to an augmentation of the leaf area index and enhances physiological activities. In addition, the application of micronutrients to the leaves of plants activates around 35 enzymes involved in metabolic pathways, thereby improving the process of photosynthesis33. The application of MnSO₄ on leaves has been shown to increase the overall chlorophyll content in leaves due to its positive impact on cellular metabolic processes34. In the photosynthetic oxygen-evolving complex (OEC) of photosystem II (PSII), manganese is an essential component that helps convert light energy into chemical energy during photosynthesis, which is used for the synthesis of organic compounds, leading to plant growth and biomass accumulation35. In addition, MnSO₄ promotes dry matter accumulation by increasing photosynthesis through higher chlorophyll levels, although not as much as when salicylic acid is applied. The activities of many enzymes involved in the growth and development of plants are influenced by SA. For instance, it has been seen to boost the activity of the enzyme nitrate reductase, which is essential for plant growth and biomass accumulation and is involved in nitrogen metabolism36. The results of our study show that the application of 150 parts per million (ppm) of salicylic acid combined with 0.5 millimolar (mM) of manganese sulphate (MnSO₄) resulted in increased fresh and dry leaf weights. The augmentation in leaf area and photosynthetic rate results in a more substantial accumulation of dry matter, thereby contributing to an increase in fresh and dry stem weight. These results align with the earlier discoveries in Brassica juncea by Fariduddin et al. (2003)16. Previous studies have documented comparable impacts of salicylic acid treatment on dry matter accumulation and plant development in rapeseed37,38,39 and sugarcane40. Salicylic acid has attracted attention due to its ability to enhance root development in different plant species. It promotes the growth of roots by increasing their length and the number of branches, increasing the overall mass and weight of the roots. In addition, salicylic acid assists plants in reacting to both living and non-living factors that cause stress, such as pathogen invasion, lack of water, and high salt levels. Salicylic acid induces stress responses in plants, allowing them to adapt to unfavourable environmental conditions while promoting root development and biomass41.
Research examining the impact of salicylic acid has shown that higher concentrations stimulate the growth of longer radicle or seminal roots42 (Bahrani and Pourreza, 2012). Monocots respond to salicylic acid concentration in a manner that depends on the concentration. Low levels of salicylic acid enhance the growth of the radicle, while high levels of salicylic acid diminish it. This response is similar to that observed in dicots43. The study found that higher concentrations of salicylic acid increased root growth. Manganese, an essential micronutrient for plants, affects various physiological processes, such as the development of roots. Sufficient manganese levels promote root growth, increasing the weight of both fresh and dry roots. Applying MnSO₄ directly to plants lacking manganese can improve these symptoms and stimulate root growth, increasing fresh and dry root mass. In addition, the application of manganese increases the effectiveness of auxin, leading to the development of longer roots and an increase in both fresh and dry weight of plants44. Prior studies have shown that treating crops like grapes45 and barley44 with manganese enhances root metrics, specifically fresh and dry weight. In our study, we applied a concentration of 150 parts per million (ppm) of salicylic acid along with a concentration of 0.5 millimolar (mM) of MnSO₄.The use of H₂O led to increased fresh and dry root weights, with the application of 300 ppm salicylic acid and 0.25 mM MnSO₄ following closely behind. Salicylic acid is crucial in controlling stomatal behaviour, affecting the water lost through transpiration. Studies have shown that the application of salicylic acid prompts the closure of stomata in plants, resulting in a decrease in water loss and an improvement in water usage efficiency46. Stomatal cell conductance's regulatory role helps maintain an optimal level of relative water content in leaves. In addition, the application of salicylic acid has been shown to enhance the stability and integrity of plant membranes, which may lead to improved water retention47. The rise in relative water content can be attributed to an increase in cytoplasmic osmotic pressure caused by the production of higher amounts of proline (osmolytes), which helps in the absorption of water in unfavourable conditions48. In addition, applying salicylic acid can increase the ability of plant tissues to transport water by increasing the expression of aquaporin genes and improving water absorption efficiency49. The increased water transport capacity helps plants maintain water content and reduce water stress50. Similarly, manganese is vital in numerous plant physiological processes, such as regulating stomatal conductance and water absorption. Optimal manganese levels can enhance roots' growth and function by improving water absorption from the soil, which may increase the relative water content in plant tissues51. It has consistently found that the application of manganese increases the leaf relative water content in mustard and rapeseed52,53,54,55,56, rice11 and barley57. There is a direct correlation between the increase in salicylic acid and MnSO₄ concentration and the increment in leaf relative water content. Nevertheless, salicylic acid exhibits superior effects on the relative water content when compared to the application of only MnSO₄. The synergistic use of Salicylic Acid and MnSO₄ produces superior outcomes compared to using either compound individually. Our study's results demonstrate that applying MnSO₄ and salicylic acid to the leaves has a significant positive effect on leaf relative water content. Specifically, the combination of 150 ppm salicylic acid with 0.5 mM MnSO₄ significantly increases leaf relative water content compared to other combinations. Salicylic acid has been shown to increase plant chlorophyll production by activating gene expression in the chlorophyll biosynthesis pathway. The upregulation results in an elevated production of chlorophyll a, b, and chlorophyll ab concentrations in plant tissues58. By promoting defensive photosynthetic activities, SA regulates the amount of chlorophyll and carotenoid content, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, stomatal conductance, and carbon dioxide (CO2) absorption31. In addition, salicylic acid has antioxidant properties that allow it to remove reactive oxygen species (ROS) produced during photosynthesis. Salicylic acid helps reduce oxidative stress and protects chloroplasts from damage caused by light, resulting in higher levels of chlorophyll in leaves59. In addition, salicylic acid improves the process of photosynthesis in plants and increases their efficiency in absorbing carbon. This leads to higher levels of chlorophyll, as chlorophyll molecules are constantly produced and replaced to support photosynthesis30,60. Furthermore, salicylic acid impacts the expression of various genes that break down chlorophyll, such as chlorophyllase and pheophytinase. As a result, it hinders chlorophyll degradation and helps maintain higher concentrations of chlorophyll molecules in plant tissues61. Research has shown that higher concentrations of salicylic acid in Brassica juncea lead to increased chlorophyll content, as reported by Sharma et al. (2017)62; Parashar et al. (2014)63 and Alam et al. (2013)52. Similar findings found in rapeseed by Hasanuzzaman et al. (2019)55. Manganese plays a critical role as a micronutrient in the process of photosynthesis. It is essential for increasing the production of chlorophyll by activating enzymes involved in the biosynthesis pathways of chlorophyll51. As a result, administering MnSO₄ increases the concentrations of chlorophyll a, chlorophyll b, and total chlorophyll in plant tissues. Similar results were reported for Brassica rapa by Jannah et al. (2022)64, Brassica juncea by Khan et al. (2016)65, and Brassica cultivars by Khodabin et al. (2021)66. Chlorophylls a and b are essential pigments that capture light energy in photosynthesis and play a crucial role in forming and functioning photosystem II (PSII) complexes, including chlorophyll molecules. Optimal manganese levels are necessary for the efficient functioning of PSII, which allows for the adequate absorption of light and conversion of sunlight into chemical energy67. The levels of salicylic acid and MnSO₄ have a direct proportional relationship with the increase in chlorophyll a, b, and total chlorophyll content. Nevertheless, salicylic acid demonstrates superior efficacy when compared to the application of MnSO₄ alone. The combined application of salicylic acid and MnSO₄ leads to better results compared to using each treatment separately, as shown by the increase in the content of chlorophyll a, chlorophyll b, and chlorophyll ab after applying them to the leaves. Our study demonstrates that the utilisation of 150 parts per million (ppm) salicylic acid combined with 0.5 millimolar (mM) MnSO₄ leads to an increase in the levels of chlorophyll a, b, and total chlorophyll content.
Salicylic acid has a notable impact on improving the membrane stability index (MSI) and decreasing the membrane injury index (MII) in mustard leaves68. The enhancement in MSI can be attributed to a significant reduction in lipid peroxidation, resulting in a substantial decrease in MII. Salicylic acid reduces the production of free radicals, which helps to prevent damage to cellular membranes caused by lipid peroxidation. In addition, it helps to decrease the leakage of electrolytes from the leaves, indicating a lower membrane injury index69. Godara et al. (2016)53 found similar results in Brassica juncea, while Aldesuquy and Ghanem (2015)69 observed comparable outcomes in wheat. On the other hand, Khan et al. (2016) 65 conducted a study on the effect of foliar application of zinc and manganese on Brassica juncea under water stress condition and reported that plants treated with different doses of MnSO4 significantly decrease the membrane injury index. Similarly, manganese has a similar effect by reducing the membrane injury index and decreasing electrolyte leakage. According to Ghorbani et al. (2019)34, an increase in manganese concentration leads to a decrease in the membrane injury index and an increase in the membrane stability index. The membrane stability index was significantly enhanced when salicylic acid and manganese were applied together, compared to when either compound was applied individually. More precisely, we noticed the external administration of 150 parts per million (ppm) of salicylic acid and 0.5 millimolar (mM) of MnSO₄.The presence of H₂O resulted in an increased membrane stability index and a decreased membrane injury index, followed by the application of 300 ppm salicylic acid and 0.25 mM MnSO₄.
Salicylic acid regulates gene expression and metabolic pathways, potentially affecting protein synthesis and accumulation. The influence of this phenomenon reaches the expression of genes related to different cellular functions, such as protein metabolism. This can occur by increasing the expression of genes that encode enzymes involved in protein synthesis or by modifying the activity of transcription factors that control gene expression in this pathway30,70. The use of salicylic acid has shown notable enhancements in the protein content of grains by controlling photosynthesis and antioxidant enzymes like superoxide dismutase (SOD)71. The use of salicylic acid in our study significantly affected the nitrogen content in the grains, indicating an increase in protein content. These results are consistent with previous findings in different Brassica species72 and Triticum aestivum71. On the contrary, manganese does not directly affect the amount of protein in grains. However, it does have essential functions in photosynthesis, enzyme activation, and nitrogen metabolism, which indirectly affect the synthesis of proteins34. Ensuring sufficient levels of manganese is essential for plants' overall well-being and strength, which may lead to increased protein content in grains. According to research on soybean (Glycine max), foliar spraying of Mn and Si together enhanced the grain's protein content, resulting in a 7% increase in protein content73. The results of our study showed that the simultaneous use of salicylic acid and manganese had a more substantial impact on the protein content of grains than using either compound alone. More precisely, the external application of 150 parts per million (ppm) of salicylic acid combined with 0.5 millimolar (mM) of MnSO₄ led to an increase in the protein content of the grains.
Applying salicylic acid (SA) and manganese (Mn) dramatically increases the oil content of mustard grains. Although there is no clear and established connection between salicylic acid and oil content in mustard grains, research has demonstrated that salicylic acid can enhance the effectiveness of enzymes involved in sulphur assimilation. This, in turn, leads to an improvement in the oil content of Brassica juncea grains30. Research has shown a direct relationship between salicylic acid and the oil level in Brassica juncea38. Similarly, the use of MnSO₄ as a foliar application has been shown to increase the amount of oil in rapeseed grains by improving cellular processes like photosynthesis. Manganese (Mn) impacts the activity of enzymes crucial for the metabolism of fatty acids, which could potentially increase the oil content of grains66,74. Moreover, genetic factors substantially influence the proportion of mustard oil generated. In addition, the element Mn enhances cellular processes and plant growth, increasing oil production in Brassica napus 33, Brassica juncea62 and various Brassica species by Muhal et al. (2014)72. Our study found that the simultaneous use of SA and Mn had a more significant effect on grain oil content than using only SA or Mn individually. More precisely, the external application of 150 parts per million (ppm) of salicylic acid combined with 0.5 millimolar (mM) of MnSO₄ resulted in an enhancement of the oil content in the grains.