The Combination of Salicylic Acid, Nicotinamide, and Proline Mitigates the Damage Caused by Salt Stress in Nasturtium (Tropaeolum majus)

Academic Editors: Dayong Zhang and Pedro Diaz-Vivancos Received: 19 February 2025 Revised: 29 March 2025 Accepted: 7 April 2025 Published: 8 April 2025 Citation: Santos, T.S.d.; Correia, M.R.S.; Sena, L.S.; Santana, L.P.d.S.; Silva, G.B.G.d.; Lima, K.S.; Dutra, E.V.d.S.; Adas, M.E.; Ribeiro, M.C.B.d.O.; Ribeiro, J.E.d.S.; et al. The Combination of Salicylic Acid, Nicotinamide, and Proline Mitigates the Damage Caused by Salt Stress in Nasturtium (Tropaeolum majus). Plants 2025,14, 1156. https://doi.org/ 10.3390/plants14081156 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article The Combination of Salicylic Acid, Nicotinamide, and Proline Mitigates the Damage Caused by Salt Stress in Nasturtium (Tropaeolum majus) Thainan Sipriano dos Santos 1, Marcos Roberto Santos Correia 1, Luma Santos Sena 1, Laura Pereira dos Santos Santana 1, Geovanna Buique Gualberto da Silva 1, Keilane Silva Lima 1, Elienay Vinícius da Silva Dutra 1, Myriam El Adas 1, Maria Carolina Borges de Oliveira Ribeiro 1, João Everthon da Silva Ribeiro 2, Rogério Ferreira Ribas 1, Elania Freire da Silva 2,3, Alfredo Emilio Rubio-Casal 3 , Aurélio Paes Barros Júnior 2 , Xuguang Tang 4 , Thieres George Freire da Silva 5 , Alexandre Maniçoba da Rosa Ferraz Jardim 6,* and Toshik Iarley da Silva 1,* 1Center for Agrarian, Environmental, and Biological Sciences, Universidade Federal do Recôncavo da Bahia, Cruz das Almas 44380-000, BA, Brazil; [email protected] (T.S.d.S.); marcos_r[email protected] (M.R.S.C.); [email protected] (L.S.S.); [email protected] (L.P.d.S.S.); [email protected] (G.B.G.d.S.); [email protected] (K.S.L.); [email protected] (E.V.d.S.D.); [email protected] (M.E.A.); [email protected] (M.C.B.d.O.R.); [email protected] (R.F.R.) 2Department of Agricultural and Forestry Sciences, Federal Rural University of the Semi-Arid, Mossoró 59625-900, RN, Brazil; [email protected] (J.E.d.S.R.); [email protected] (E.F.d.S.); [email protected] (A.P.B.J.) 3 Department of Plant Biology and Ecology, University of Seville, Av. Reina Mercedes, s/n, 41012 Sevilla, Spain; [email protected] 4Institute of Remote Sensing and Geosciences, Hangzhou Normal University, Hangzhou 311121, China; [email protected] 5 Department of Agricultural Engineering, Federal Rural University of Pernambuco, Dom Manoel de Medeiros Avenue, s/n, Dois Irmãos, Recife 52171-900, PE, Brazil; [email protected] 6 Department of Biodiversity, Institute of Biosciences, São Paulo State University—UNESP, Rio Claro 13506-900, SP, Brazil *Correspondence: [email protected] (A.M.d.R.F.J.); [email protected] (T.I.d.S.) Abstract: Salinity represents a significant challenge for agriculture, especially in semi-arid regions, affecting the growth and productivity of plants such as nasturtium (Tropaeolum majus), which is valued for its ornamental, medicinal, and food uses. Salt stress disrupts biochemical, physiological, and anatomical processes, limiting plant development. This study investigated the application of attenuators, including salicylic acid, nicotinamide, and proline, to mitigate the effects of salt stress on nasturtium cultivated in a hydroponic system. The treatments involved different combinations of these compounds under saline conditions (40 mM NaCl). The attenuators reduced the negative impacts of salt stress, promoting improvements in gas exchange, such as increased net photosynthesis, water-use efficiency, and stomatal conductance. Additionally, the treatments enhanced vegetative and reproductive growth, increasing the dry biomass of leaves, stems, and flowers, as well as the number of flowers and flower buds. The combination of salicylic acid, nicotinamide, and proline stood out by providing greater efficiency in carbon assimilation, stability of photosynthetic pigments, and higher tolerance to salt stress. These findings reinforce the potential of using attenuators to optimize the cultivation of nasturtium in saline environments, promoting higher productivity and plant quality. Keywords: abiotic stress; edible flowers; plant hormones; vitamin B3; amino acids; gas exchange; inorganic and organic solutes Plants 2025,14, 1156 https://doi.org/10.3390/plants14081156 Plants 2025,14, 1156 2 of 21 1. Introduction Nasturtium (Tropaeolum majus L., Tropaeolaceae), also known in Brazil as “capuchinha”, “nastúrtio”, and “chaguinha”, is a non-conventional food plant, grown as an ornamental, medicinal, and edible plant, and widely cultivated worldwide due to its adaptability and diverse applications [ 1 ]. Its flowers exhibit a distinctive peppery flavor, a hallmark of the species, which has made them one of the most commercially valued edible flowers globally [ 2 ]. The leaves and flowers of nasturtium are highly versatile, frequently used in salads, sandwiches, and as decorative elements in dishes and desserts, enhancing both the aesthetic appeal and functional value of culinary creations [ 3 ]. The plant is characterized by rounded leaves, vibrant flowers in shades of yellow, orange, and red, often adorned with dark internal markings, and greenish fruits. All parts of the plant are considered edible, with the exception of the roots [ 4 ]. Furthermore, nasturtium is rich in fatty acids, glucosinolates, flavonoids, and tetracyclic triterpenes, and in Brazil it is widely employed for the treatment of ulcers, colds, cellulitis, and anemia [5,6]. The quality of nasturtium plants is strongly influenced by environmental conditions [ 1 ]. Under abiotic stress, the plants often experience reduced growth and diminished visual quality of their flowers [ 7 ]. This impact is particularly pronounced in arid and semi-arid regions, where stressors such as drought and salinity are the primary limiting factors for the cultivation of edible flowers [ 6 , 8 ]. Under these conditions, water deficit and salinity disrupt essential physiological processes, such as photosynthesis, and impair root development, leading to reduced productivity and overall plant quality [2]. Salinity, characterized by the accumulation of soluble salts such as sodium (Na + ) and chloride (Cl − ), is a significant environmental challenge that threatens agricultural sustainability. In addition to adversely affecting crop growth and productivity, salinity degrades soil structure, reduces water infiltration, diminishes biodiversity, and contaminates water resources [ 9 ]. The use of high-salinity water exacerbates these effects by subjecting plants to combined osmotic and ionic stresses. These stresses lead to a marked decline in vegetative growth due to reduced water and nutrient uptake, structural cell disorganization, and excessive production of reactive oxygen species (ROS), which cause oxidative damage [ 10 ]. Soil salinity impacts approximately 800 million hectares globally, accounting for around 20% of irrigated agricultural land, particularly in arid, semi-arid, and coastal areas, due to poor irrigation and drainage practices, limited rainfall, high evaporation rates, and the use of saline water for irrigation [ 8 , 11 ]. Excess Na + disrupts cellular metabolism and ionic balance, making it harmful to plants. For T. majus, salinity levels above 1 dS m −1 have been shown to inhibit rooting and seedling growth [ 12 ], although low salt concentrations may enhance biomass production [13]. In light of the challenges posed by soil salinization and increasing water scarcity, the adoption of sustainable agricultural practices is essential. In this context, hydroponics emerges as a promising alternative, offering greater water-use efficiency and enabling cultivation in regions with limited or low-quality water resources [ 14 ]. The exogenous application of stress alleviators has proven to be a promising strategy for mitigating the deleterious effects of salinity stress in nasturtium plants [ 6 , 8 , 15 – 18 ]. These compounds play a critical role in modulating various physiological and biochemical processes, enhancing the plants’ tolerance to adverse salinity conditions [ 19 ]. Among the most commonly used alleviators are phytohormones, vitamins, and amino acids, whose positive effects are widely documented in the literature. Salicylic acid (SA) is a well-studied phytohormone with proven effects in mitigating damage caused by salinity stress [ 17 ]. Its exogenous application reduces oxidative stress, Plants 2025,14, 1156 3 of 21 regulates ion uptake, promotes water balance, and positively influences stomatal behavior, gas exchange, chlorophyll fluorescence, and osmoregulation [ 20 ]. Nicotinamide (nicotinamide or niacin) functions as a growth regulator, influencing physiological processes such as enzyme, nucleic acid, and protein biosynthesis, while also serving as a critical coenzyme [ 21 ]. Proline, an amino acid involved in primary metabolism, plays a central role under stress conditions [ 18 ]. As one of the most accumulated compatible solutes in plants exposed to salinity, water deficit, or extreme temperatures, proline contributes to cellular protection and the maintenance of metabolic processes [22]. The innovative aspects of this research lie in its pioneering exploration of the combined use of salicylic acid, proline, and nicotinamide to mitigate salinity-induced damage in hydroponically grown nasturtium plants. Unlike previous studies that have focused individually on these compounds, this study evaluates their synergistic effects on physiological and biochemical processes, including gas exchange, photosynthetic pigments, organic and inorganic solutes, and overall plant growth. The novelty also extends to assessing the viability of hydroponic cultivation as a sustainable alternative for growing nasturtium under saline conditions, providing valuable insights into enhancing plant tolerance and productivity in challenging environments. The selection of SA, nicotinamide, and proline as stress alleviators was based on their complementary roles in enhancing plant resilience under salt stress through distinct mechanisms. SA is known for reducing oxidative stress, regulating ion uptake, improving water balance, and enhancing photosynthetic efficiency. Nicotinamide acts as a growth regulator, influencing enzyme, nucleic acid, and protein biosynthesis, while also serving as a crucial coenzyme. Proline functions as an osmoprotectant, contributing to osmotic balance, membrane stabilization, and ROS scavenging. Their combined application offers a synergistic effect, addressing multiple stress factors simultaneously, which is particularly relevant under saline conditions where plants face osmotic, ionic, and oxidative stress. Additionally, while these alleviators have been individually studied, their joint application in hydroponically grown nasturtium under salinity conditions is an innovative approach that aims to enhance plant tolerance and productivity more effectively than individual application. Although progress has been made in understanding the physiological and biochemical responses of nasturtium to salt stress, significant gaps remain, particularly concerning the use of attenuators such as salicylic acid, nicotinamide, and proline. These compounds have shown promising potential in mitigating the negative effects of salinity by regulating ion uptake, maintaining water balance, stabilizing membranes, modulating antioxidant metabolism, and improving photosynthetic efficiency. However, studies so far have mainly focused on the isolated application of these attenuators, without adequately exploring the combined effects that could provide a more robust approach to enhancing nasturtium’s tolerance to salt stress. Investigating the joint use of these compounds in hydroponic systems exposed to salinity represents an innovative strategy that could significantly improve the productivity and quality of nasturtium grown under adverse conditions. Despite advances in understanding the role of stress alleviators, studies on their use in nasturtium plants cultivated under salinity stress in hydroponic systems remain scarce. This study is a pioneering effort in exploring the combined use of salicylic acid, proline, and nicotinamide to mitigate salinity-induced damage in hydroponically grown nasturtium plants. Accordingly, the present research aimed to evaluate the effects of salicylic acid, nicotinamide, and proline application on gas exchange, photosynthetic pigments, organic and inorganic solutes, and the growth of Tropaeolum majus cultivated in a hydroponic system under salinity conditions. Plants 2025,14, 1156 4 of 21 2. Results 2.1. Effect of Salt Stress on Gas Exchange Salt stress and the application of attenuators influenced gas exchange in T. majus (Figure 1). The isolated application of nicotinamide (T4) reduced stomatal conductance (gs; Figure 1a), net photosynthesis (A; Figure 1b), transpiration rate (E; Figure 1c), intercellular CO 2 concentration (Ci; Figure 1d), intrinsic carboxylation efficiency (iCE; Figure 1e), and the ratio of intercellular to external CO 2 concentration (Ci/Ca; Figure 1h). On the other hand, the combination of nicotinamide with salicylic acid (T7) and with salicylic acid and proline (T8) increased stomatal conductance (0.13 and 0.14 mol H 2 O m −2 s −1 , respectively) and the transpiration rate (3.58 and 3.55 mmol H 2 O m −2 s −1 , respectively). Additionally, the combined application of all three attenuators increased the intercellular CO 2 concentration (278.33 µ mol CO 2 mol −1 air) and the Ci/Ca ratio (0.63), suggesting a possible improvement in the plant’s adaptive capacity under salt stress conditions. Plants 2025, 14, x FOR PEER REVIEW 4 of 21 organic and inorganic solutes, and the growth of Tropaeolum majus cultivated in a hydroponic system under salinity conditions. 2. Results 2.1. Effect of Salt Stress on Gas Exchange Salt stress and the application of attenuators influenced gas exchange in T. majus (Figure 1). The isolated application of nicotinamide (T4) reduced stomatal conductance (gs; Figure 1a), net photosynthesis (A; Figure 1b), transpiration rate (E; Figure 1c), intercellular CO 2 concentration (Ci; Figure 1d), intrinsic carboxylation efficiency (iCE; Figure 1e), and the ratio of intercellular to external CO 2 concentration (Ci/Ca; Figure 1h). On the other hand, the combination of nicotinamide with salicylic acid (T7) and with salicylic acid and proline (T8) increased stomatal conductance (0.13 and 0.14 mol H 2 O m −2 s −1 , respectively) and the transpiration rate (3.58 and 3.55 mmol H 2 O m −2 s −1 , respectively). Additionally, the combined application of all three attenuators increased the intercellular CO 2 concentration (278.33 µmol CO 2 mol −1 air) and the Ci/Ca ratio (0.63), suggesting a possible improvement in the plant’s adaptive capacity under salt stress conditions. Figure 1. Cont. Plants 2025,14, 1156 5 of 21 Plants 2025, 14, x FOR PEER REVIEW 5 of 21 Figure 1. (a) Stomatal conductance (gs), (b) net photosynthesis (A), (c) transpiration rate (E), (d) intercellular CO 2 concentration (Ci), (e) intrinsic carboxylation efficiency (iCE), (f) instantaneous water-use efficiency (WUE), (g) intrinsic water-use efficiency (iWUE), and (h) Ci/Ca ratio of Tropaeolum majus subjected to salt stress and the application of salicylic acid (SA), nicotinamide (NAM), and proline (Pro). T0 = control (0 mM NaCl); T1 = 40 mM NaCl; T2 = 40 mM NaCl + Pro; T3 = 40 mM NaCl + SA; T4 = 40 mM NaCl + NAM; T5 = 40 mM NaCl + Pro + SA; T6 = 40 mM NaCl + Pro + NAM; T7 = 40 mM NaCl + SA + NAM; and T8 = 40 mM NaCl + Pro + SA + NAM. Means followed by the same letter do not differ according to the Scott–Knott test (p ≤ 0.05). Bars are means ± standard error (n = 4). Under salt stress, in the absence of attenuators (T1), and with the application of nicotinamide combined with salicylic acid (T7) and with all three attenuators (T8), an increase in net photosynthesis (A, Figure 1b) was observed, reaching 10.54, 10.15, and 10.40 µmol CO 2 m −2 s −1 , respectively. Intrinsic carboxylation efficiency (iCE) improved with the application of salicylic acid combined with proline (T5) and with nicotinamide (T7), both resulting in 0.044. Water-use efficiency (WUE; Figure 1f) was elevated under salinity without attenuators (T1) and with the application of salicylic acid (T3), with values of 4.00 and 3.76 µmol CO 2 /mmol H 2 O m −2 s −1 , respectively. Additionally, intrinsic water-use efficiency (iWUE; Figure 1g) increased under salinity conditions with the application of nicotinamide (109.7 µmol CO 2 /mol H 2 O m −2 s −1 ). 2.2. Impact of Salt Stress on Photosynthetic Pigments The application of attenuators to nasturtium plants cultivated under salinity conditions influenced the levels of photosynthetic pigments (Figure 2). Salt stress reduced the contents of chlorophyll a (Figure 2a), chlorophyll b (Figure 2b), total chlorophyll (Figure 2c), and carotenoids (Figure 2d). However, the application of salicylic acid (T3) and the combination of all three attenuators (T8) contributed to a more stable maintenance of chlorophyll indices under salinity. On the other hand, the combined application of proline with salicylic acid (T5) resulted in a reduction in chlorophyll a (6.73 µg g −1 dry mass), chlorophyll b (6.11 µg g −1 DM), total chlorophyll (13.48 µg g −1 DM), and carotenoids (6.40 µg g −1 DM). Additionally, the combination of proline with nicotinamide (T6) also reduced chlorophyll b (6.20 µg g −1 DM) and carotenoid levels (6.35 µg g −1 DM), reinforcing the negative effects of these interactions on photosynthetic pigments. Figure 1. (a) Stomatal conductance (gs), (b) net photosynthesis (A), (c) transpiration rate (E), (d) intercellular CO 2 concentration (Ci), (e) intrinsic carboxylation efficiency (iCE), (f) instantaneous water-use efficiency (WUE), (g) intrinsic water-use efficiency (iWUE), and (h)Ci/Ca ratio of Tropaeolum majus subjected to salt stress and the application of salicylic acid (SA), nicotinamide (NAM), and proline (Pro). T0 = control (0 mM NaCl); T1 = 40 mM NaCl; T2 = 40 mM NaCl + Pro; T3 = 40 mM NaCl + SA ; T4 = 40 mM NaCl + NAM; T5 = 40 mM NaCl + Pro + SA; T6 = 40 mM NaCl + Pro + NAM; T7 = 40 mM NaCl + SA + NAM ; and T8 = 40 mM NaCl + Pro + SA + NAM. Means followed by the same letter do not differ according to the Scott–Knott test (p ≤ 0.05). Bars are means ± standard error (n= 4). Under salt stress, in the absence of attenuators (T1), and with the application of nicotinamide combined with salicylic acid (T7) and with all three attenuators (T8), an increase in net photosynthesis (A, Figure 1b) was observed, reaching 10.54, 10.15, and 10.40 µ mol CO 2 m −2 s −1 , respectively. Intrinsic carboxylation efficiency (iCE) improved with the application of salicylic acid combined with proline (T5) and with nicotinamide (T7), both resulting in 0.044. Water-use efficiency (WUE; Figure 1f) was elevated under salinity without attenuators (T1) and with the application of salicylic acid (T3), with values of 4.00 and 3.76 µ mol CO 2 /mmol H 2 O m −2 s −1 , respectively. Additionally, intrinsic water-use efficiency (iWUE; Figure 1g) increased under salinity conditions with the application of nicotinamide (109.7 µmol CO2/mol H2O m−2s−1). 2.2. Impact of Salt Stress on Photosynthetic Pigments The application of attenuators to nasturtium plants cultivated under salinity conditions influenced the levels of photosynthetic pigments (Figure 2). Salt stress reduced the contents of chlorophyll a (Figure 2a), chlorophyll b (Figure 2b), total chlorophyll (Figure 2c), and carotenoids (Figure 2d). However, the application of salicylic acid (T3) and the combination of all three attenuators (T8) contributed to a more stable maintenance of chlorophyll indices under salinity. On the other hand, the combined application of proline with salicylic acid (T5) resulted in a reduction in chlorophyll a (6.73 µ g g −1 dry mass), chlorophyll b (6.11 µ g g −1 DM), total chlorophyll (13.48 µ g g −1 DM), and carotenoids (6.40 µ g g −1 DM). Additionally, the combination of proline with nicotinamide (T6) also reduced chlorophyll b (6.20 µ g g −1 DM) and carotenoid levels (6.35 µ g g −1 DM), reinforcing the negative effects of these interactions on photosynthetic pigments. Plants 2025,14, 1156 6 of 21 Plants 2025, 14, x FOR PEER REVIEW 6 of 21 Figure 2. (a) Chlorophyll a, (b) chlorophyll b, (c) total chlorophyll, and (d) carotenoids of Tropaeolum majus subjected to salt stress and the application of salicylic acid (SA), nicotinamide (NAM), and proline (Pro). T0 = control (0 mM NaCl); T1 = 40 mM NaCl; T2 = 40 mM NaCl + Pro; T3 = 40 mM NaCl + SA; T4 = 40 mM NaCl + NAM; T5 = 40 mM NaCl + Pro + SA; T6 = 40 mM NaCl + Pro + NAM; T7 = 40 mM NaCl + SA + NAM; and T8 = 40 mM NaCl + Pro + SA + NAM. Means followed by the same letter do not differ according to the Scott–Knott test (p ≤ 0.05). Bars are means ± standard error (n = 4). 2.3. Inorganic Solutes of Leaves and Flowers The application of salt stress attenuators influenced the levels of inorganic solutes in the leaves and flowers of nasturtium (Figure 3). Under salt stress conditions, there was an increase in Na + , Cl − , and the Na + /K + ratio in the leaves (Figure 3a,e,g), differing from the control (T0). The treatment with salicylic acid (T3) resulted in the highest accumulation of these ions, with values of 99.8 mg g −1 DM, 24.0 µg g −1 DM, and 3.2, respectively. In the flowers, salt stress also led to an increase in Na + levels (Figure 3b). The treatment with salicylic acid (T3) resulted in the highest Na + accumulation in the flowers, reaching 9.4 mg g −1 DM. Conversely, the application of proline alone (T2) and its combinations with salicylic acid (T5) or with salicylic acid and nicotinamide (T8) maintained Na + levels in the flowers similar to the control (T0), with values of 6.68, 6.23, 6.34, and 6.52 mg g −1 DM, respectively. Figure 2. (a) Chlorophyll a, (b) chlorophyll b, (c) total chlorophyll, and (d) carotenoids of Tropaeolum majus subjected to salt stress and the application of salicylic acid (SA), nicotinamide (NAM), and proline (Pro). T0 = control (0 mM NaCl); T1 = 40 mM NaCl; T2 = 40 mM NaCl + Pro; T3 = 40 mM NaCl + SA; T4 = 40 mM NaCl + NAM; T5 = 40 mM NaCl + Pro + SA; T6 = 40 mM NaCl + Pro + NAM; T7 = 40 mM NaCl + SA + NAM; and T8 = 40 mM NaCl + Pro + SA + NAM. Means followed by the same letter do not differ according to the Scott–Knott test (p ≤ 0.05). Bars are means ± standard error (n= 4). 2.3. Inorganic Solutes of Leaves and Flowers The application of salt stress attenuators influenced the levels of inorganic solutes in the leaves and flowers of nasturtium (Figure 3). Under salt stress conditions, there was an increase in Na + , Cl − , and the Na + /K + ratio in the leaves (Figure 3a,e,g), differing from the control (T0). The treatment with salicylic acid (T3) resulted in the highest accumulation of these ions, with values of 99.8 mg g −1 DM, 24.0 µ g g −1 DM, and 3.2, respectively. In the flowers, salt stress also led to an increase in Na + levels (Figure 3b). The treatment with salicylic acid (T3) resulted in the highest Na + accumulation in the flowers, reaching 9.4 mg g −1 DM. Conversely, the application of proline alone (T2) and its combinations with salicylic acid (T5) or with salicylic acid and nicotinamide (T8) maintained Na + levels in the flowers similar to the control (T0), with values of 6.68, 6.23, 6.34, and 6.52 mg g −1 DM, respectively. Under salt stress conditions, the application of salicylic acid alone (T3) and in combination with all three attenuators (T8) resulted in the lowest K + levels in the leaves (Figure 3c), with values of 31.4 and 31.8 mg g −1 DM, respectively, while the control (T0) showed the highest accumulation, reaching 58.1 mg g −1 DM. Salinity also affected the levels of K + and Cl − in the flowers (Figure 3d,f). The combination of proline with nicotinamide (T6) significantly increased K + and Cl − levels, registering values of 58.1 mg g −1 DM and 16.0 µ g g −1 DM, respectively. In contrast, the combination of proline with salicylic acid (T5) reduced K + accumulation in the flowers, with a value of 29.7 mg g −1 DM. Additionally, the application of nicotinamide alone (T4), combined with salicylic acid (T7), and the combination of pro- Plants 2025,14, 1156 7 of 21 line with salicylic acid (T5) reduced Cl − levels in the flowers, showing values of 12.5, 12.9, and 12.4 µ g g −1 DM, respectively. Regarding the Na + /K + ratio in the leaves (Figure 3h), the combinations of proline with nicotinamide (T6) and all three attenuators (T8) under salt stress resulted in the lowest values (0.15, 0.14, and 0.14, respectively), similar to the control (T0). Conversely, nicotinamide alone (T4) and combined with salicylic acid (T7) showed the highest Na+/K+ratios, with values of 0.23 and 0.24, respectively. Plants 2025, 14, x FOR PEER REVIEW 7 of 21 Figure 3. (a,b) Sodium (Na + ), (c,d) potassium (K + ), (e,f) chloride (Cl − ), and (g,h) Na + /K + ratio in leaves and flowers, respectively, of Tropaeolum majus subjected to salt stress and the application of salicylic acid (SA), nicotinamide (NAM), and proline (Pro). T0 = control (0 mM NaCl); T1 = 40 mM NaCl; T2 = 40 mM NaCl + Pro; T3 = 40 mM NaCl + SA; T4 = 40 mM NaCl + NAM; T5 = 40 mM NaCl + Pro + SA; T6 = 40 mM NaCl + Pro + NAM; T7 = 40 mM NaCl + SA + NAM; and T8 = 40 mM NaCl + Pro + SA + NAM. Means followed by the same letter do not differ according to the Scott–Knott test (p ≤ 0.05). Bars are means ± standard error (n = 4). Figure 3. (a,b) Sodium (Na + ), (c,d) potassium (K + ), (e,f) chloride (Cl − ), and (g,h) Na + /K + ratio in leaves and flowers, respectively, of Tropaeolum majus subjected to salt stress and the application of Plants 2025,14, 1156 8 of 21 acid (SA), nicotinamide (NAM), and proline (Pro). T0 = control (0 mM NaCl); T1 = 40 mM NaCl; T2 = 40 mM NaCl + Pro; T3 = 40 mM NaCl + SA; T4 = 40 mM NaCl + NAM; T5 = 40 mM NaCl + Pro + SA; T6 = 40 mM NaCl + Pro + NAM; T7 = 40 mM NaCl + SA + NAM; and T8 = 40 mM NaCl + Pro + SA + NAM . Means followed by the same letter do not differ according to the Scott– Knott test (p≤0.05). Bars are means ±standard error (n= 4). 2.4. Organic Solutes of Leaves and Flowers Under salt stress conditions, the application of attenuators influenced the levels of organic solutes in nasturtium flowers (Figure 4). The levels of soluble carbohydrates, free amino acids, and soluble proteins in the leaves were not affected by the treatments. Soluble carbohydrate levels (Figure 4a) were reduced by the application of nicotinamide alone (T4), in combination with salicylic acid (T7), and in the interaction among all three attenuators (T8), showing values of 4.54, 4.55, and 4.58 µ mol g −1 DM, respectively. Conversely, the combination of proline with salicylic acid (T5) resulted in the highest levels of soluble carbohydrates, equivalent to those observed in the control (T0) and in the salt-stressed treatment without attenuators (T1), with values of 4.95, 5.01, and 5.00 µmol g−1DM, respectively. Plants 2025, 14, x FOR PEER REVIEW 9 of 22 Figure 4. (a) Soluble carbohydrates, (b) free amino acids, (c) soluble proteins, (d) free proline in flowers, and (e) free proline in leaves of Tropaeolum majus subjected to salt stress and the application of salicylic acid (SA), nicotinamide (NAM), and proline (Pro). T0 = control (0 mM NaCl); T1 = 40 mM NaCl; T2 = 40 mM NaCl + Pro; T3 = 40 mM NaCl + SA; T4 = 40 mM NaCl + NAM; T5 = 40 mM NaCl + Pro + SA; T6 = 40 mM NaCl + Pro + NAM; T7 = 40 mM NaCl + SA + NAM; and T8 = 40 mM NaCl + Pro + SA + NAM. Means followed by the same letter do not differ according to the Scott–Knott test (p ≤ 0.05). Bars are means ± standard error (n = 4). Salinity reduced the contents of free amino acids in the flowers of nasturtium (Figure 4b). The control (T0) showed the highest accumulation, with 15.74 µmol g −1 DM. Under salinity conditions, the highest contents of free amino acids were observed in treatments without attenuators (T1) and with the application of nicotinamide alone (T4), combined with proline (T6), salicylic acid (T7), and with both attenuators (T8), with values of 14.7 (T1), 14.7 (T4), 14.8 (T6), 14.1 (T7), and 14.7 µmol g −1 DM (T8), respectively. The application of nicotinamide combined with salicylic acid (T7) increased the levels of soluble proteins in the flowers, reaching 5.30 mg g −1 DM (Figure 4c). In contrast, under salinity without Figure 4. (a) Soluble carbohydrates, (b) free amino acids, (c) soluble proteins, (d) free proline in flowers, and (e) free proline in leaves of Tropaeolum majus subjected to salt stress and the application Plants 2025,14, 1156 9 of 21 of salicylic acid (SA), nicotinamide (NAM), and proline (Pro). T0 = control (0 mM NaCl); T1 = 40 mM NaCl; T2 = 40 mM NaCl + Pro; T3 = 40 mM NaCl + SA; T4 = 40 mM NaCl + NAM; T5 = 40 mM NaCl + Pro + SA; T6 = 40 mM NaCl + Pro + NAM; T7 = 40 mM NaCl + SA + NAM; and T8 = 40 mM NaCl + Pro + SA + NAM . Means followed by the same letter do not differ according to the Scott– Knott test (p≤0.05). Bars are means ±standard error (n= 4). Salinity reduced the contents of free amino acids in the flowers of nasturtium (Figure 4b) . The control (T0) showed the highest accumulation, with 15.74 µ mol g −1 DM. Under salinity conditions, the highest contents of free amino acids were observed in treatments without attenuators (T1) and with the application of nicotinamide alone (T4), combined with proline (T6), salicylic acid (T7), and with both attenuators (T8), with values of 14.7 (T1), 14.7 (T4), 14.8 (T6), 14.1 (T7), and 14.7 µ mol g −1 DM (T8), respectively. The application of nicotinamide combined with salicylic acid (T7) increased the levels of soluble proteins in the flowers, reaching 5.30 mg g −1 DM (Figure 4c). In contrast, under salinity without attenuators (T1) and with the application of proline alone (T2), the levels of soluble proteins decreased to 2.84 and 2.87 mg g−1DM, respectively. Free proline levels in the flowers (Figure 4d) were increased by the application of salicylic acid alone (T3) and in combination with nicotinamide (T7), reaching values of 1.87 and 1.89 µ mol g −1 DM, respectively. On the other hand, the lowest free proline level was observed under salinity without attenuators (T1), with 1.60 µ mol g −1 DM. Free proline levels in the leaves (Figure 4e) were also influenced by salinity and the application of attenuators. All treatments with attenuators increased free proline levels compared to the control (T0) and the salt-stressed treatment without attenuators (T1), which showed the lowest values at 1.16 and 1.25 µ mol g −1 DM, respectively. However, the treatments with attenuators did not differ significantly from each other. 2.5. Growth and Biomass Production The application of attenuators to nasturtium plants cultivated under salt stress influenced growth and biomass production (Figure 5). The tallest plants were observed with the application of salicylic acid (T3), reaching 51.1 cm, while the application of proline alone (T2) resulted in the shortest plants at 34.7 cm (Figure 5a). Salt stress reduced the number of leaves compared to the control, highlighting the negative impacts of this condition. However, the combined application of nicotinamide with salicylic acid (T7) resulted in the highest number of leaves (225.5) in salt-stressed plants, indicating a positive effect of this treatment in mitigating the damages caused by salinity (Figure 5b). Salinity and the application of attenuators influenced the number of flowers in nasturtium (Figure 2c). The application of salicylic acid (T3) and the combination of proline with nicotinamide (T6) increased the number of flowers, recording values of 25.5 and 23.25, respectively. The number of flower buds (Figure 5d) was also affected, with the highest value observed in the treatment with nicotinamide alone (T4), which reached 23.75 under salt stress. Conversely, the application of proline alone (T2) and its combination with salicylic acid (T5) resulted in reductions in the number of flower buds, with values of 10.25 and 10.75, respectively. The dry mass of the stems (Figure 5e), leaves (Figure 5f), and flowers (Figure 5g) was affected by salt stress and the application of attenuators. Under salinity conditions, the combination of proline with salicylic acid (T5) resulted in the greatest reduction in stem dry mass, recording 10.44 g. Conversely, the highest values of leaves dry mass were observed in the control (T0) and in the salt-stressed treatment without attenuators (T1), with 11.38 and 11.93 g, respectively. The application of proline alone (T2), combined with salicylic acid (T5), and nicotinamide alone (T4) reduced the leaves’ dry mass under salt stress, with values of 6.92, 7.04, and 7.29 g, respectively. Regarding flower dry mass, the application Plants 2025,14, 1156 16 of 21 for 15 min with 2 h intervals during both the daytime (6:00 a.m. to 6:00 p.m.) and nighttime (6:00 p.m. to 6:00 a.m.). 4.2. Growth Conditions In the present study, the seeds of T. majus var. ‘AnãSortida’ (ISLA Sementes, Porto Alegre, RS, Brazil) were sown in phenolic foam with 5 mm perforations, previously sterilized in a 5% sodium hypochlorite solution, and maintained under adequate humidity conditions for germination. The experiment began with the sowing of one seed per phenolic foam cell. Eight days after sowing (DAS), the seedlings were transferred to the nursery and kept in a nutrient solution (NS) formulated based on the recommendations of Furlani et al. [ 51 ] at 50% concentration for four days. At 12 DAS, the seedlings were transferred to the floating hydroponic system and subjected to the pre-defined salinity treatments. The nutrient solution was prepared following an adaptation of the recommendations by Furlani et al. [ 51 ] at 100% concentration, and salinity was adjusted to 40 mM by adding sodium chloride (NaCl). Preliminary tests (unpublished data) were conducted under the same experimental conditions prior to setting up the experiment described in this paper to determine an NaCl concentration that would cause moderate salt stress in the plants. The concentration of 40 mM NaCl was found to be the most suitable according to these preliminary tests. Nine days after transplantation (DAT), foliar application of the attenuators salicylic acid (SA—1 mM, Sigma Aldrich, São Paulo, Brazil), proline (Pro—10 mM, Synth, Diadema, SP, Brazil), and nicotinamide (NAM—300 mg L −1 , Synth, Diadema, SP, Brazil) was initiated. The foliar treatment was performed weekly, always in the early morning, using manual sprayers equipped with drop regulators. To enhance the adhesion of the attenuators to the leaf surface, Tween 20 (0.05%) was added to each solution applied. A total of 550 mL of each treatment was applied to each plant until the end of the experiment, with this amount being divided into six applications throughout the cultivation period. At 15 DAT, saline water replenishments (40 mM NaCl) were initiated, except for the control treatment. These replenishments were performed every five days, always in the late afternoon. At the same intervals, measurements of the electrical conductivity (EC) and pH of the nutrient solution were performed. 4.3. Variables Analyzed 4.3.1. Gas Exchange Gas exchange variables were measured using an infrared gas analyzer (IRGA, LICOR, Lincoln, NE, USA). Measurements were conducted between 8:00 and 10:00 a.m. under controlled conditions, with artificial light set to 1000 µ mol photons m −2 s −1 , ambient CO 2 concentration, and temperature. The evaluated variables included stomatal conductance (gs, mol H 2 O m −2 s −1 ), net photosynthesis rate (A, µ mol CO 2 m −2 s −1 ), transpiration rate (E, mmol H 2 O m −2 s −1 ), intercellular CO 2 concentration (Ci, µ mol CO 2 mol air −1 ), instantaneous water-use efficiency (WUE = A/E), intrinsic water-use efficiency ( iWUE = A/gs ), and Ci/Ca ratio. These measurements were performed on fully expanded leaves from the middle third of the plants. 4.3.2. Photosynthetic Pigments For the determination of the chlorophyll a, chlorophyll b, and carotenoid contents, 20 mg of lyophilized material was used. The material was transferred to test tubes, where leaf extracts were collected, and 3 mL of 80% acetone was added. The tubes were stored under refrigeration (4 ◦ C) in the dark for 24 h to ensure pigment stability. The samples were centrifuged at 14,000 × gfor 15 min, and the supernatants were collected. The contents of chlorophylls and carotenoids were determined by spectrophotometry, with readings Plants 2025,14, 1156 17 of 21 performed at wavelengths of 664.1 nm, 648.6 nm, and 470 nm, following the methodology described by Lichtenthaler and Buschmann [ 52 ]. Additionally, total chlorophyll was calculated as the sum of chlorophyll a and b. 4.3.3. Inorganic Solutes To prepare the samples for inorganic solute analysis, flowers and leaves previously dried in an oven were used. After drying, the material was ground, and 0.1 g of each sample was weighed and transferred to test tubes. Subsequently, 10 mL of deionized water was added to each tube. The samples were then placed in a thermostatic bath at 100 ◦ C for 1 h. After heating, the solutions were filtered and prepared for subsequent analyses. The contents of Na + and K + were determined using a flame photometer (model Q498M2, QUIMIS, Diadema, SP, Brazil) according to the method described by Silva et al. [ 53 ]. The Cl − content was determined by spectrophotometry using a spectrophotometer (model 2000 UV, BEL PHOTONICS, Piracicaba, SP, Brazil) based on the methodology of Silva et al. [54]. 4.3.4. Organic Solutes For the determination of organic solutes, three leaves and three flowers were collected, separately stored in bags, and subjected to lyophilization. After the lyophilization process, the samples underwent a two-step extraction protocol. Initially, 0.1 g of the lyophilized material was weighed into microcentrifuge tubes (Eppendorf), and 1.5 mL of 80% ethanol was added. The samples were centrifuged at 14,000 × gfor 15 min, and the supernatants were collected. Subsequently, 1.5 mL of 80% ethanol was added again to the residue, and the same procedure was repeated. The quantification of soluble carbohydrates was performed according to the method described by Dubois et al. [ 55 ], using 0.5 mL of the extract and the phenol–sulfuric acid method, with colorimetric reading at 490 nm and D-(+)-glucose as the standard. The determination of free proline was based on the method of Bates et al. [ 56 ], using 0.5 mL of the extract and ninhydrin reagent, with a colorimetric reading at 520 nm and pure proline as the standard. Free amino acids were quantified according to the method of Yemm and Cocking [ 57 ], using 0.5 mL of the extract and ninhydrin reagent, with a colorimetric reading at 570 nm and pure L-leucine as the standard. Soluble proteins were determined using the Bradford method [ 58 ], employing 0.1 mL of the extract and the dye-binding method, with a reading at 595 nm and bovine serum albumin as the standard. 4.3.5. Growth and Production The biometric analyses included measuring plant height (PH), counting the number of leaves, the number of flowers, and the number of flower buds. The production analyses involved determining the dry mass of the stem, leaves, roots, and flowers. All collections were carried out at the end of the experimental cycle, 51 days after transplantation (DAT). To obtain the data, the plants were harvested and separated into their different components. The materials were stored in paper bags and then dried in a forced-air circulation oven at 65 ◦C for 72 h to determine the dry masses. 4.4. Statistical Analysis The data obtained were subjected to analysis of variance (ANOVA) using the F-test. All data were tested for normality (Shapiro–Wilk test) and homogeneity of variance (Bartlett’s test). Treatment means were compared using the Scott–Knott test (p ≤ 0.05). All statistical analyses were performed using the R software version 4.3.1 [59]. Plants 2025,14, 1156 18 of 21 5. Conclusions The combination of nicotinamide, salicylic acid, and proline positively influenced Tropaeolum majus by improving physiological and biochemical responses under salt stress, including enhanced gas exchange parameters such as stomatal conductance, transpiration, and intercellular CO 2 concentration. This treatment also stabilized photosynthetic pigments, promoted osmotic balance by increasing proline and soluble sugar levels, and reduced the detrimental effects of ion accumulation and oxidative stress. Additionally, the combination enhanced the stability of chloroplasts and protected cellular structures, resulting in better overall plant growth and biomass allocation, particularly in reproductive tissues. Author Contributions: T.S.d.S., M.R.S.C., and T.I.d.S.: investigation, writing—original draft; T.S.d.S., A.E.R.-C., and X.T.: visualization and supervision; T.I.d.S. and A.M.d.R.F.J.: funding acquisition, validation, supervision, writing—review and editing; J.E.d.S.R., E.F.d.S., A.E.R.-C., and R.F.R.: investigation, writing—original draft; T.S.d.S., L.S.S., L.P.d.S.S., G.B.G.d.S., K.S.L., E.V.d.S.D., M.E.A., and M.C.B.d.O.R.: resources, investigation, validation, methodology, visualization, writing; E.F.d.S., A.P.B.J., X.T., and T.G.F.d.S.: writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES – Financing code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Financing code 001), Fundação de Amparo àPesquisa do Estado da Bahia (FAPESB), Instituto de Ciência, Inovação e Tecnologia do Estado da Bahia—Recursos Hídricos e Desenvolvimento Sustentável (INCITE Economia Verde), and Instituto Nacional de Ciência e Tecnologia em Agricultura Sustentável no Semiárido Tropical (INCT AgriS). Data Availability Statement: All data produced and/or analyzed in this study are included in the manuscript. The corresponding authors are available to provide additional data and materials upon reasonable request. Acknowledgments: The authors thank the CAPES, CNPq, FAPESB, INCITE Economia Verde, and INCT AgriS by support and funding. A.M.d.R.F.J. acknowledges support from the São Paulo Research Foundation (FAPESP-2023/05323–4). 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