Comparative adaptations of high-tolerant species and broccoli cultivars to salinity stress during germination and early development stages

Comparative adaptations of high-tolerant species and broccoli cultivars to salinity stress during germination and early development stages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative adaptations of high-tolerant species and broccoli cultivars to salinity stress during germination and early development stages Angel Almagro-Lopez, Juan Nicolas- Espinosa, Jose M. Mulet, Micaela Carvajal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6112599/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 May, 2025 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Salinity imposes significant physiological and biochemical challenges on plants, disrupting key processes such as germination, involving growth, and water balance. Under saline conditions, plants activate various defense mechanisms to mitigate salinity-induced damage. While many of these mechanisms are well-characterized in mature plants, their role during germination and early seed development remains largely unexplored. In this work, we studied four pre-commercial broccoli ( Brassica oleracea L. var. italica) cultivars previously selected for their enhanced salinity tolerance and compared to the high tolerant Eruca vesicaria subsp. sativa . The results provide insights into key mechanisms involved in salinity tolerance, including osmotic potential regulation, mineral homeostasis, and antioxidant enzymatic activity and ATP concentration. The ATP availability and utilization emerged as critical determinants of the stress response profiles of the seeds during germination. Notably, the BQ1 cultivar demonstrated the most efficient ATP utilization, probably enabling a broader, more sustained, and effective response under saline conditions. These findings highlight ATP as a crucial factor in salinity tolerance during early seeds development. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Salinity is one of the largest global challenges in arid and semi-arid regions, severely affecting agricultural production (El Sabagh et al., 2020). Salinity affects approximately 20% of the total cultivated land, and 33% of the irrigated agricultural lands worldwide [ 1 , 2 ], a situation expected to worsen and accelerate by 2050, due to climate change. Factors such as rising sea levels, which lead to seawater intrusion into aquifers due to excessive groundwater extraction in dry regions, exacerbate the problem [ 3 ]. Salinity refers to the high concentration of salt ions in the soil solution, especially Na + and Cl − [ 4 ], and it impacts plant development through two distinct mechanisms: osmotic stress and ionic toxicity [ 5 ]. Osmotic stress occurs within first minutes of salt accumulation in the root area. The presence of salt in the soil solution reduces the osmotic potential of soil solution thus reducing the ability of plant to absorb water, which leads to reductions in the growth rate [ 3 , 4 ]. Ionic stress, on the other hand, manifests from a few minutes to few hours and is associated with prolonged ion accumulation, especially Na+, in the shoot tissues, which induces tissue toxicity. Excessive salt uptake via transpiration can result in cellular damage within transpiring leaves, potentially causing further reductions in growth and, in severe cases, plant death [ 3 , 4 ]. Numerous studies have been published on the effects of salinity on plant species; however, the majority primarily focus on sprouts and adult plants [ 6 , 7 ]. While these studies are valuable, it is equally important to consider the impact of salinity through first developmental stages. Salt stress affects key physiological and biochemical processes, including germination, growth, photosynthesis, water relations, nutrient homeostasis, oxidative stress management, and overall yield [ 8 ]. Seed germination is one of the most fundamental and vital stages in the growth cycle of a plant [ 3 ]. Germination is particularly important in plant species of socioeconomic importance for sprout production, as they are extensively utilized as food sources. Broccoli ( Brassica oleracea L. var. italica ) has gained popularity due to its excellent nutritional and biochemical properties [ 9 ]. Recently, the importance of broccoli sprouts as nutrient source has also been studied. Broccoli sprouts are health-promoting vegetables that are even more nutritious than the mature inflorescence due to their significantly higher levels of bioactive compounds such as glucosinolates and phenolic compounds [ 10 ]. Particularly, ion toxicity interferes with enzyme activity, damages membranes, and disrupts metabolic pathways, causing increased production of reactive oxygen species (ROS) such as superoxide radicals (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH-) potentially affecting the different development stages of the plant, including germination [ 11 ]. ROS are highly reactive molecules that can damage proteins, lipids, DNA, and other cellular components if the plant antioxidant defense mechanisms are not activated [ 12 ]. This has been associated to the increased production of malondialdehyde (MDA) as an indicator of membrane damage at a cellular level under salt stress [ 13 ]. To prevent oxidative damage to cellular component caused by ROS, plants have developed a complex antioxidant system. The primary components of this system include ascorbate and glutathione in addition to enzymes such as catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) [ 14 ]. The functionality of this complex antioxidant network is required of ascorbate and glutathione pools, whose biosynthesis is directly related to NADPH and ATP levels in the cell [ 15 ]. This is even more important during germination under salinity as energy levels are directly related to ATP reservoirs within the seed. Likewise, osmotic stress affects the water uptake process during seed imbibition, as reported by Khan and Weber (2006), while ion toxicity disrupts the enzymatic activities associated with nucleic acid and protein metabolism [ 17 , 18 ]. Additionally, salinity interferes with metabolic equilibrium [ 19 ], impairing the mobilization and utilization of seed reserves [ 20 ]. Under salinity stress, efficient mechanisms to store in vacuoles the toxic concentrations of Na⁺ are essential [ 21 ]. The majority of these mechanisms are ATP-dependent, underscoring the critical role of intracellular energy levels in maintaining ion homeostasis [ 22 ]. Under salt stress conditions, various strategies have been described to enhance plant growth and yield. In this way, investigations have been pointed the critical need of identifying suitable cultivars to improve crop performance and germination under salt stress [ 23 ]. Comparative studies of physiological salinity responses between varieties within the same family provide valuable insights into differences in growth patterns and salt tolerance levels [ 24 , 25 ]. Also, the investigation of the different genotypes according to their salinity resilience in all development stages is a challenge [ 24 , 26 ]. Moreover, the response to salinity during germination is essential, as both germination and sprout growth are highly susceptible stages in the life cycle of the plants. In this sense, an investigation of the diverse responses exhibited by salt-tolerant species during germination can facilitate the identification of key resistance traits active during early development. These traits can then be used as focal points for breeding programs aimed at developing new and more resilient cultivars [ 27 ]. In this study, we investigate the effect of salinity on seed germination, by analyzing the germination rate, the mineral nutrient composition, the antioxidant metabolism (enzymes and lipid peroxidation) in relation to ATP availability, of four different pre-commercial broccoli cultivars, comparing these to those observed in a species from the Brassicaceae family, Eruca vesicaria subsp. Sativa ( E. vesicaria ), known for its salt-tolerant [ 28 , 29 ]. This methodology enables the assessment of the salt tolerance of the cultivars and the various adaptive strategies employed by different cultivars and species in response to salt stress. Materials and methods Germination and growth conditions Seeds of the four broccoli cultivars (BG1, BH1, BX1 and BQ1) were provided by Sakata (SAKATA SEED IBÉRICA SLU) and E. vesicaria seeds were purchased in CANTUESO (CANTUESO Natural Seeds). To ensure an antiseptic environment, 25 seeds from each species and cultivar were sterilized for 10 minutes in a 1:1 water-sodium hypochlorite solution. Following disinfection, the seeds were rinsed with distilled water and placed in Petri dishes containing two layers of filter paper. Four experimental conditions were established: a control (distilled water), and treatments with 50 mM, 100 mM, and 150 mM NaCl. Each treatment was replicated four times for each cultivar and species reaching a total of 100 seeds analyzed for each condition. A total of 7 mL of the respective solution was added to each Petri dish. Plates were completely covered with aluminum paper to assure darkness and then placed in an incubator at 28°C. The duration of the experiment was 5 days (including day 0 when the seeds were placed and day 4 when sprouts were removed). The humidity of the Petri dish was monitored daily during these 5 days. At the end of the assay, the samples were placed immediately in liquid nitrogen and later stored in the ultra-freezer. The freeze samples were grinded using pestle and mortar in constant presence of liquid nitrogen. The obtained powder was again store in the ultra-freezer for further analyses. Germination rate, length, weight and osmotic potential The number of germinated seeds was counted every day and the length of the sprouts was measure thought image analysis using the program ImageJ [ 30 ]. Total weight from sprouts on each petri plate was measure on the last day of the essay before storage. Growth rate (GR) was calculated with the following formula. GR = (Final length/days). The osmotic potential (Ѱµ) was measured using 100 mg of fresh powder from each treatment using a freezing-point depression osmometer (Digital Osmometer, Roebling, Berlin) at 25 ± 1°C [ 31 ]. Analysis of ATP concentrations Determination of ATP concentrations were performed following [ 32 ], adapting the protocol because of the different kinds of samples used. ATP was extracted using 200 mg of fresh powder from the sprouts of the four treatments with 100 µl (1–1, v/v) pre-cooled methanol-water and 50 µl of the internal standard (IS) solution (35 ng/mL N-acetylglucosamine SIGMA). After vortexing for 2 min, samples were centrifuged at 12500 x g for 15 minutes. 100 µL of the supernatant were analyzed using High-Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS) at the Metabolomic and Proteomic Laboratory (ACTI - Murcia University, Spain). ATP was measured using an ATP patron (SIGMA). ATP per sprout was calculated for a visually enhanced and more accurate measure. Analysis of Mineral Nutrients Macro- and micronutrient concentrations were determined using the dry-freeze powder obtained for each treatment. The analyses were conducted using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) on a Thermo ICAP 6500 Duo instrument (Thermo Fisher Scientific, Waltham, MA, USA) as described by Nicolas-Espinosa et al. (2024). The nutrient concentrations were expressed as mg 100 g − 1 DW for macronutrients and mg g − 1 DW for micronutrient. Preparation of extracts and enzyme assay Antioxidant enzymes were extracted from 300 mg of fresh powder from sprouts in control and 100 mM conditions using 50 mM potassium phosphate buffer (pH 7.8), containing 0.1 mM Na 2 –EDTA and 1% insoluble polyvinylpolypyrrolidone (PVPP). This extract was used to determine the activity of the enzyme catalase (CAT) and oxidative damage to lipids [ 33 ]. The same extract plus 10 mM β-mercaptoethanol was used for glutathione reductase (GR), and with 4 mM ascorbic acid for ascorbate peroxidase (APX) [ 34 ]. The supernatants were stored at − 20°C for subsequent enzymatic assays. Catalase (CAT) activity was measured by the disappearance of H 2 O 2 as described by Aebi (1984). The extract was mixed with 50 mM potassium phosphate buffer (pH 7.0) and the reaction was initiated by adding 10.6 mM H 2 O 2 to the sample and monitoring the change in absorbance at 240 nm every 30 sec for 3 min. The oxidation rate of ascorbate was measured using ascorbate peroxidase (APX) activity as described by Amako et al. (1994) and Nakano and Asada (1981). The extract was mixed with 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM Na 2 –EDTA, and 0.5 mM ascorbic acid. The addition of H 2 O 2 was used to start the reaction and the decrease in absorbance at 290 nm was recorded every 30 sec for 3 min. Glutathione reductase (GR) activity was determined as described by Carlberg and Mannervik (1985) and Foyer and Halliwell (1976). The reaction mixture contained 0.1 M HEPES (pH 7.8), 1 mM Na 2 –EDTA, 3 mM MgCl 2 , 0.5 mM oxidized glutathione, and the enzyme extract. Finally, 0.2 mM NADPH was added. The rate of NADPH oxidation was monitored by the decrease in absorbance at 340 nm every 30 sec for 2 min. The data of the enzymatic activities were normalized by the mg of protein, determined by the Bradford method [ 40 ]. Then, the results of antioxidant enzymes (APX, GR and CAT) were expressed as µmol reactive/min mg protein. Three biological and three technical replicates of the control and 100 mM treatments samples were analyzed. Lipid Oxidative damage Lipid peroxidation (LP) was determined using the previous extract and was determined as described by Minotti and Aust (1987). The reaction was performed by mixing the extract with a reaction mixture containing 20% trichloroacetic acid (TCA), 0.5% 2-thiobarbituric acid (TBA), 0.01% butyl hydroxytoluene (HBT) and 0.25 N HCl, and incubating the mixture at 95ºC for 30 min. After stopping the reaction in ice and centrifuging the samples, the supernatant was used for spectrophotometric reading at 535 nm. The calibration curve was made using malondialdehyde (MDA) in the range of 0 to 10 µM. The results were expressed in µmol MDA/min mg protein. Statistical analyses Statistical analyses and data presentation were conducted using Origin (Pro) (Version 2021 software package by OriginLab Corporation, Northampton, MA, USA). Significant differences among the values of all the parameters were determined at p ≤ 0.05, according to Tukey’s test. Results Physiological parameters Germination rates were above 90% in all broccoli cultivars across all treatments (Fig. 1 A). No significant differences in germination rates were observed between treatments in the broccoli cultivars, except for BQ1, where a significant decrease was observed in the 150 mM treatment (Fig. 1 A). E. vesicaria showed a significant decrease in the 100 mM and 150 mM treatments (Fig. 1 A), with these values being significantly lower compared to the rest of cultivars. A direct correlation between an increase in salinity and a reduction in sprout length, weight and growth rate was observed in all varieties (Fig. 1 B, C, D and Fig. sup.1). In the case of BX1, BQ1, and E. vesicaria , no significant differences were observed between the control and 50 mM treatments in length and growth rate (Fig. 1 B, D). Similarly, only BQ1 showed no weight differences between the control and 50 mM treatments (Fig. 1 C). Furthermore, BQ1 exhibited the highest values of length, weight, and growth rate under all treatment conditions. In addition, when analyzed osmotic potential of sprouts, a significant decrease was observed in all species and cultivars across treatments (Fig. 2 ). In BH1 and BQ1, no differences were observed between the 100 mM and 150 mM treatments, but both were lower compared to the control. In BX1, the lowest value was observed at 100 mM, but osmotic potential at 150 was similar to the control. Significant differences between cultivars and species were observed only under 150 mM salinity treatment, where BG1 and E. vesicaria exhibited the lowest values, while BX1 has the least negative value in this condition. ATP concentration per sprout showed a significant reduction in some of the broccoli cultivars (BG1, BX1 and BQ1) but no differences were observed in BH1 and E. vesicaria (Fig. 3 ). These two species were also the ones with the lowest values of ATP concentration in control conditions. The largest difference between treatments was observed in the BX1 cultivar, which exhibited the biggest value in control condition and the lowest in the 150 mM treatment. Analysis of Mineral Nutrients Alterations in mineral composition were observed across species, cultivars, and specific minerals analyzed. Calcium (Ca) showed a significant decrease in BQ1 with the progressive increase of salinity concentration, but its values were similar to those obtained in the rest of cultivars (Fig. 4 A). E. vesicaria exhibited the same significant decrease under salinity, with the lowest values across all treatments. Magnesium (Mg) concentration changed only in E. vesicaria where a significant increase was observed under salinity. Phosphorous (P) showed a decrease in BG1 under salinity (Fig. 4 B) while E. vesicaria had the lowest values under all conditions. Notably, sulfur (S) concentration did not change with increasing salinity; however, each cultivar appeared to have different intrinsic S level. BG1 and BQ1 were grouped together with higher S concentrations, while BH1 and BX1 exhibited the lowest levels. A decrease in potassium (K) concentration was observed in BX1 and BQ1 cultivars under salinity (Fig. 4 E) but the highest K levels in all treatments were observed in BX1, E. vesicaria , and BQ1 under control conditions respectively. Na concentrations increased under salinity (Fig. 4 F) and were higher in BX1, BQ1 and E. vesicaria in control conditions; however, their concentrations remained unchanged with increasing salinity. The Na/K ratio increased under salinity treatments in all cultivars and species (Fig. 5 A); nevertheless, no differences were observed between the different cultivars and E. vesicaria . PCA analysis showed a clear differentiation between the broccoli varieties and E. vesicaria on the horizontal axis when the macro- and micronutrient sprout compositions were analyzed (Fig. 5 B), particularly dominated by the concentration of Na and K, under the different conditions. Additionally, a distinction was observed between the broccoli varieties, specifically forming two groups: BG1 and BQ1 clustered together, while BH1 and BX1 formed a separate group. Enzyme activity and lipid oxidative damage For enzyme activity and lipid oxidative damage, the 100 mM and control treatments were selected to simplify the interpretation and management of the results. Ascorbate peroxidase (APX) activity showed a significant increase in BG1, BX1 and E. vesicaria under salinity (Fig. 6 A). The highest activity was observed in E. vesicaria under both control and salinity conditions. Glutathione reductase (GR) activity increased in BG1 and BX1 and E. vesicaria under salinity conditions (Fig. 6 B). All broccoli cultivars presented a low catalase (CAT) activity, which was not altered by salinity. E. vesicaria showed a notably high basal CAT activity under control conditions and was the only species to exhibit a significant increase in CAT activity under salinity conditions (Fig. 6 C). Lipid oxidative damage showed a significant decrease in all broccoli cultivars, except for BH1 and E. vesicaria , where no changes were observed under salinity conditions (Fig. 6 D). Discussion Seeds are complex structures equipped with the necessary machinery and stored compounds to support germination and early seed growth. These intrinsic characteristics, however, vary significantly among species and cultivars, influencing their ability to withstand environmental stresses, such as salinity. To determine salt tolerance, our study compared physiological, biochemical, and molecular parameters between control and salinity conditions during seed germination of four different broccoli cultivars. Additionally, these results were compared with those obtained from the salt-tolerant species E. vesicaria , which was selected as a reference. Our results showed that germination rates were primarily dependent on the cultivar and species, and to a lesser extent, on the salinity concentration. Salinity did not affect germination rates in any of the broccoli cultivars (except for BQ1 under 150 mM) but remarkably E. vesicaria showed a significant decrease in germination rates when treated with 100 mM and 150 mM of NaCl. This significant decrease observed in E. vesicaria has also been observed in the germination rate of different species under salinity stress within the Brassicaceae family. Different studies have shown that there may be a great difference among abiotic stress tolerance at the germination stage, and in the adult stage. For instance tomato is considered to be less salt sensitive than broccoli but the germination rates under salt stress are similar [ 42 ].Bybordi (2010) observed how Brassica napus exhibited significant germination decline at 150–200 mM NaCl, consistent with findings in radish and beetroot [ 44 , 45 ]. Different studies have shown that there may be a great difference among abiotic stress tolerance at the germination stage, and in the adult stage. For instance tomato is considered to be less salt sensitive than broccoli but the germination rates under salt stress are similar. Three cultivars of broccoli has been observed to decline germination similarly under 80–100 mM NaCl [ 46 ]. Similarly, 100 mM NaCl significantly decreased germination rates in broccoli sprouts [ 47 ]. Although previous studies indicated that salinity concentrations above 80 mM negatively affected seed germination in Brassicas, our results did not show significant differences in germination rates under salinity conditions, mainly due to the prior selection of salt-tolerant pre-commercial broccoli cultivars. Despite the lack of a significant effect of salinity on seed germination, we observed a decline in physiological parameters under salinity conditions. All broccoli cultivars exhibited reduced length and growth rate with increasing salinity. However, BX1, BQ1, and E. vesicaria demonstrated greater tolerance under low salt concentration, as their growth rates and length were unaffected by the 50 mM salinity treatment. A similar decrease was observed in weight under salinity conditions, but only BQ1 showed no differences between the control and 50 mM treatment. This reduction in sprout length and weight under salinity was also observed by Tian et al. (2016). Nevertheless, our cultivars showed higher values, suggesting enhanced tolerance to salinity. The bottom line is that all our broccoli cultivars exhibited better overall performance in terms of germination rate, length, and weight, not only under salinity conditions but also in general when compared with previous studies, demonstrating a high level of salinity tolerance at the germination stage, with BQ1 showing outstanding performance. This observation is consistent with previous findings, where certain varieties were identified as salinity-tolerant in a study on adult plants, suggesting that their resilience to salinity extends to the germination stage [ 49 ]. Although Eruca vesicaria subsp. sativa is widely recognized as a highly salt-tolerant species, our study indicates that this tolerance is not fully expressed during the germination stage, in contrast to the higher tolerance observed in broccoli during germination. E. vesicaria exhibited reduced germination rates under increasing salinity, with lower physiological performance compared to broccoli cultivars. This does not mean low salinity tolerance but it suggests that its underlying mechanisms may not be active during germination. A similar phenomenon has been observed in other halophyte species, such as Cakile maritima , which fails to germinate under high salinity (200 mM) despite its ability to grow at much higher concentrations [ 50 ]. This pattern may represent an adaptive strategy to prevent germination in unfavorable environments, such as seawater-exposed conditions, where high salinity levels could compromise sprout establishment and survival. As previously stated, salinity is a major abiotic stress that disrupts germination and sprout development by imposing osmotic stress, ion toxicity, and nutrient imbalances. To mitigate the damage caused by salinity, plant cells typically synthesize and accumulate compatible organic molecules, which help lower the osmotic potential of the cells, thus enhancing water absorption under these conditions. The reduction in osmotic potential is a common adaptive response of plants to cope with salt stress and has been previously reported in broccoli, where the accumulation of compatible osmolytes, has been associated with salinity tolerance in broccoli cultivars [ 51 ]. When the osmotic potential of the different cultivars and species was analysed, a lower osmotic potential under salinity was observed, indicating active osmolyte accumulation in response to this stress. A progressive accumulation of osmolytes appeared to be associated with the decrease in osmotic potential caused by the increase in salt concentration, particularly in BG1 and E. vesicaria . In contrast, in BH1, BX1, and BQ1, osmolyte accumulation was triggered when the seeds were exposed to 100 mM salinity and maintained at a similar concentration when salinity increased to 150 mM, except for BX1, which exhibited a value similar to the control condition. This seemed to indicate that BH1 and BQ1 employ a threshold-dependent response to salinity, where osmolyte accumulation is triggered only when stress reaches a critical level (100–150 mM). This reduces unnecessary energy expenditure during mild stress (e.g., 50 mM), preserving resources for higher stress levels. Furthermore, BX1 seems to struggle ATP reserve management under salt stress, exhibiting a marked reduction in sprout ATP content at higher salt concentrations (100 mM and 150 mM). This could be related to its inability to synthesize compatible osmolytes due to an energy-saving mechanism, leading to a lack of osmotic potential adjustments in these plants. Moreover, this specific cultivar demonstrated the poorest performance under salinity stress conditions, indicating the essential role of osmotic potential adjustments in salinity tolerance, which is directly related to energy management and ATP usage. Changes in Ca concentration was only observed in BQ1 in salinity condition although levels were similar to those observed in the rest of the cultivars and species. This reduction was also observed in E. vesicaria but in this case, the Ca levels were the lowest in salinity condition indicating a direct effect of salinity in this species. Calcium has been reported to mitigate the adverse effects of salinity in plants, as salinity conditions reduce Ca uptake by displacing it from the cell membrane or disrupting membrane function, leading to increased Na accumulation in leaves and impairing K/Na selectivity [ 52 ]. Similar findings have been observed in halophyte species, where calcium supplementation alleviated salinity-induced damage, as Na directly affects cell wall properties and plasma membrane function through Na/Ca displacement [ 53 ]. In this context, maintaining adequate Ca levels is critical for salinity tolerance, which was observed exclusively in the broccoli cultivars under salinity conditions, suggesting enhanced mechanisms to prevent Ca displacement. BQ1 exhibited higher calcium levels under control conditions, suggesting this cultivar has inherently elevated seed calcium content. This trait may serve as an adaptive mechanism to mitigate salinity-induced stress without expending energy to transport calcium in order to maintain membrane stability. P reduction under salinity was observed exclusively in the BG1 cultivar, with E. vesicaria again exhibiting the lowest values. This suggests these two species show reduced absorption or impaired mobility, as certain phosphate transporters are sensitive to salt, hindering P uptake and its mobilization from internal reservoirs [ 54 ]. A reduction in K levels under salinity conditions was observed only in the BX1 and BQ1 cultivars, which, along with E. vesicaria , exhibited the highest K concentrations under salinity. This reduction is understandable as Na and K show similar chemical properties so they can share channels reducing K transport or absorption [ 55 ]. BQ1 showed the highest growth rate and maintained the highest K concentration at 150 mM NaCl, suggesting a potential correlation between growth performance, salinity tolerance, and K accumulation. Given the importance of K homeostasis in salinity tolerance, seed K levels may serve as an indicator of salt stress resilience. Notably, BQ1 exhibited the highest K concentration in seeds. Regarding Na, the broccoli cultivars with the highest physiological measures in salinity (BX1 and BQ1) along with E. vesicaria showed the highest concentration of Na in control conditions, indicating that the levels of Na in these seeds are the highest. A clear relation between seed exposure to higher Na levels in seed and a better tolerance to salinity is evident, but also supported by previous studies where high Na concentrations in seed has been observed in halophytes species [ 56 , 57 ]. Another significant phenomenon is that all sprouts exhibited an increase in Na concentration upon exposure to 50 mM NaCl; however, this intracellular Na concentration remained stable despite further increases in external salinity (100 mM and 150 mM). This suggests that seeds have a threshold for intracellular Na accumulation, likely regulated by Na compartmentalization within the vacuole. This threshold appeared to be reached at 50 mM, which, which could be reasonable considering that these seeds are only five days old and still undergoing development. At this stage, vacuolar structures and their associated transport mechanisms may be predominantly functional in the cotyledons, as these tissues are already differentiated. This compartmentalization mechanism effectively mitigates ionic toxicity, stress and cellular damage. However, as salinity increases, vacuolar sequestration becomes insufficient, suggesting the activation of alternative adaptive mechanisms. Given the absence of significant changes in overall Na concentration within the sprouts, these compensatory mechanisms may involve Na efflux through ATP-dependent transporters, highlighting the critical role of energy allocation in salinity tolerance. If we observe all the changes in mineral composition, BQ1 is the one showing a distinct pattern. BQ1 displays the highest values of Ca, K and Na in seed, all of which are key elements associated with salinity stress tolerance. An early exposure to Na may preventively activate the mechanisms related to salinity tolerance. This along with an enhanced concentration of Ca and K can generate the perfect environment to trigger mechanism to prevent excessive oxidative damage under salinity. Some of these mechanisms have already been discussed such as Na sequestration within the vacuole, exclusion of excess Na via selective ion transporters as SOS1 or HKT transporters as well as K retention trough efficient K channels to maintain enzymatic function and osmotic balance [ 58 ]. The combined ability of BQ1 to manage Na and K levels minimizes ion toxicity and osmotic imbalance supporting sustained growth and metabolism under salinity. However, E. vesicaria might rely on more limited or less efficient mechanisms, such as early vacuolar sequestration of Na, which could limit its overall tolerance to saline environments. Antioxidants, including ascorbate and glutathione, play a critical role in enhancing plant defense mechanisms against oxidative stress. These antioxidant levels are directly related to antioxidant enzymatic activities such as APX, GR and CAT activity that are part of the oxidative stress response. In the APX measure, almost all the sprouts showed an increase in APX activity under salinity conditions only significant in BG1, BX1, and E. vesicaria . The last one showed the highest value in both conditions. GR activity showed a significant increase in BG1, BX1 and E. vesicaria under salinity conditions. Regarding CAT activity, broccoli cultivars showed low activity in both treatments and was E. vesicaria the one with the highest values being 4 times higher in control and 10 times higher in salinity. These results indicate how E. vesicaria exhibited the highest and strongest antioxidant enzymatic response. BQ1 and BH1 are the ones that showed no differences between treatments indicating no oxidative damage response through antioxidant enzymes routes. Exposure to salinity conditions is reported to increase antioxidant enzymes activity such as APX, GR and CAT in plants as observed in mungbean [ 59 ] under 100 mM. The same has been reported in other species such as Djulis [ 60 ]. Therefore, the absence of changes in these activities under salinity conditions may indicate an alternative response pathway that does not involve antioxidant enzymes or suggest that the sprout is not experiencing oxidative stress. This likely indicates that the ion homeostasis mechanisms in these cultivars exhibit superior functionality compared to others, thereby mitigating oxidative stress since the Na/K ratio remains consistent across all broccoli cultivars. BQ1 and BH1 appear to exhibit more efficient sodium compartmentalization. Consequently, the SOS1-NHX1 system in these two cultivars may have higher expression levels or more effective isoforms. To gain a clearer understanding of this process, lipid oxidative damage is a key parameter to consider. Lipid oxidative damage is another measure that can provide insight of how badly oxidative stress is affecting a plant as it measures actual damage. All broccoli cultivars except for BH1 showed a decrease in lipid oxidation, indicating that the various antioxidant mechanisms at play are effectively preventing oxidative damage under salinity conditions. E. vesicaria did not show this reduction; instead, an increase was observed. This implies how BQ1 and BH1, which showed no significant increase in antioxidant enzyme activity under salinity, displayed both low lipid oxidative damage, while E. vesicaria , despite its high antioxidant activity, failed to prevent oxidative damage effectively. BQ1 and BH1 may depend on non-enzymatic antioxidant systems or more efficient sodium compartmentalization mechanisms to mitigate oxidative damage, reducing the necessity for enzymatic pathway activation. This could be facilitated by pre-synthesized antioxidant reservoirs stored in seeds or the presence of upregulated Na transporters, as previously mentioned. Plants significantly activates the aspartate-glutathione pathways to synthesis these non-enzymatic antioxidants involved in detoxify ROS up to tolerable levels under salinity conditions [ 61 ]. Moreover, observing that even the cultivars that did not show changes in enzyme activity still exhibited no increase in lipid oxidative damage supports the idea of an alternative mechanism at play. These alternative mechanisms may be closely related to cellular energy balance. The antioxidant system requires energy in order to function properly and to synthesize antioxidant molecules such as ascorbate and glutathione, so does the solute accumulation within the cell. ATP values were the lowest in BH1 and no changes were observed under salinity conditions, something we also observe in E. vesicaria , which showed increase in these antioxidant activities but no changes in ATP. This suggests that all mobilized or synthesized ATP is rapidly utilized in antioxidant defense mechanisms or in the activation of membrane ATPases, enhancing the capacity for Na and K transport. This facilitates the sequestration of Na into the vacuole. In BH1, the ATP mobilization and usage can be more related to antioxidant molecules biosynthesis and Na sequestration to prevent ROS damages, as no changes in enzymatic activity was observed. In the rest of the cultivars, we observe higher ATP pools in the seed and higher ATP use, indicating that a larger ATP availability can allow multiple responses. BQ1 is a clear example of that being the cultivar with the best performance during germination and sprout growth under high levels or salinity but no changes in enzymatic activity were observed. This indicates that BQ1 may be using its large ATP pool to trigger alternative mechanism, some already described such as ion efflux mechanisms to alleviate ion toxicity, with Na⁺ toxicity being particularly significant and the maintenance of ion homeostasis, particularly K⁺ and Ca 2+ homeostasis. This is important because calcium plays an important role in processes that preserve the structural and functional integrity of plant membranes avoiding ROS damage [ 62 ]. The threshold-triggered osmolyte accumulation strategy is equally important as it minimise energy wastage under low stress conditions (50 mM) while enabling a robust response under severe stress (100–150 mM). The majority of these mechanisms are ATP-dependent, underscoring the critical role of intracellular energy levels in maintaining optimal performance of these mechanisms [ 22 ]. Therefore, ATP levels may represent a key determinant of the differential responses profiles observed in sprouts. A larger ATP pool, and more importantly, a well-regulated ATP utilization, can be considered critical tolerance traits in early-stage sprouts, as they enable prolonged and more diverse physiological responses over time. Conclusion The results revealed that all broccoli cultivars exhibited higher germination rates and superior physiological performance compared to E. vesicaria , with the highest performance observed in the BQ1 cultivar. These findings suggest that different mechanisms are activated in each development stage across cultivars and species to mitigate salinity-induced stress. These mechanisms include solute accumulation, enhancement of antioxidant activity alongside alternative strategies such as Na⁺ sequestration and exclusion, K⁺ transport, and overall mineral homeostasis. All these processes are energy-dependent, relying on ATP availability. Thus, ATP availability emerges as a key factor defining the salinity response profile of each cultivar. These profiles differentiate cultivars in terms of salinity tolerance, revealing specific physiological strategies. BG1 efficiently utilizes a moderate ATP pool to maintain performance under moderate salinity (50–100 mM). BH1 exhibits limited stress responses due to low ATP availability. Although BX1 possesses the largest ATP reservoir, it fails to regulate ATP allocation effectively, compromising osmotic regulation. In contrast, BQ1 maintains a balanced ATP utilization strategy, enabling the activation of a broader range of adaptive mechanisms. Meanwhile, E. vesicaria struggles to prevent oxidative damage despite its physiological responses. Overall, BQ1 integrates multiple protective strategies to mitigate, prevent, and tolerate salinity-induced stress in sprouts, achieving the highest physiological performance and outperforming the enzymatic responses observed in E. vesicaria. Declarations Acknowledgements The authors acknowledge SAKATA S.L.U Company for providing the broccoli seeds. Author Contributions MCA, JMM and JNE contributed to the conception and design of this work. AAL carried out the experiments and JNE the statistical analytical work. AAL prepared figures and tables, and prepared the first draft of the manuscript. MCA, JMM and JNE contributed to manuscript revisions, reads and approved the submitted version. MCA obtained the funding. All authors have read and approved the manuscript. Funding This research was funded by Spanish Ministerio de Ciencia e Innovación (CPP2022-009860). Ethics approval and consent to participate Nothing to declare. Consent for publication Nothing to declare. Competing interests The authors declare no competing interests Author information Authors and Affiliations Aquaporins Group. Centro de Edafologia y Biologia Aplicada del Segura. CEBAS-CSIC. Campus Universitario de Espinardo - 25. E-30100. Murcia. Spain. Angel Almagro-Lopez, Juan Nicolas-Espinosa and Micaela Carvajal. Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universitat Politècnica de València-Consejo Superior de Investigaciones Científicas, 46022 Valencia, Spain. JM Mulet. Email: [email protected] Corresponding author Correspondence to Micaela Carvajal. Email: [email protected] References Mukhopadhyay R, Sarkar B, Jat HS, Sharma PC, Bolan NS. Soil salinity under climate change: Challenges for sustainable agriculture and food security. J Environ Manage. 2021;280:111736. Shrivastava P, Kumar R. Soil salinity: A serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation. Saudi J Biol Sci. 2015;22:123–31. Parihar P, Singh S, Singh R, Singh VP, Prasad SM. Effect of salinity stress on plants and its tolerance strategies: a review. Environ Sci Pollut Res. 2015;22:4056–75. Corwin DL. Climate change impacts on soil salinity in agricultural areas. Eur J Soil Sci. 2021;72:842–62. Munns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59 Volume 59, 2008:651–81. Nicolas-Espinosa J, Yepes-Molina L, Martinez-Bernal F, Fernandez-Pozurama M, Carvajal M. Deciphering the effect of salinity and boron stress on broccoli plants reveals that membranes phytosterols and PIP aquaporins facilitate stress adaptation. Plant Sci. 2024;338:111923. Zaghdoud C, Alcaraz-López C, Mota-Cadenas C, Martnez-Ballesta MDC, Moreno DA, Ferchichi A, et al. Differential Responses of Two Broccoli (Brassica oleracea L. var Italica) Cultivars to Salinity and Nutritional Quality Improvement. Sci World J. 2012;2012:291435. Läuchli A, Grattan SR. Plant Growth And Development Under Salinity Stress. Advances in Molecular Breeding Toward Drought and Salt. Tolerant Crops. 2007;:1–32. Li H, Xia Y, Liu HY, Guo H, He XQ, Liu Y, et al. Nutritional values, beneficial effects, and food applications of broccoli (Brassica oleracea var. italica Plenck). Trends Food Sci Technol. 2022;119:288–308. Wang P, Li X, Tian L, Gu Z, Yang R. Low salinity promotes the growth of broccoli sprouts by regulating hormonal homeostasis and photosynthesis. Hortic Environ Biotechnol. 2019;60:19–30. Poschenrieder C, Cabot C, Martos S, Gallego B, Barceló J. Do toxic ions induce hormesis in plants? Plant Sci. 2013;212:15–25. Das K, Roychoudhury A. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Front Environ Sci. 2014;2 DEC:121942. Ibrahim EA. Seed priming to alleviate salinity stress in germinating seeds. J Plant Physiol. 2016;192:38–46. Gupta DK, Palma JM, Corpas FJ. Antioxidants and antioxidant enzymes in higher plants. Antioxid Antioxid Enzymes High Plants. 2018;:1–300. Latowski D, Surówka E, Strzałka K. Regulatory role of components of ascorbate-glutathione pathway in plant stress tolerance. Ascorbate-Glutathione Pathw Stress Tolerance Plants. 2010;:1–53. Khan MAjmal, Weber DJ. Ecophysiology of High Salinity Tolerant Plants (Tasks for Vegetation Science). Ecophysiology of High Salinity Tolerant Plants. 2006;:XVIII–404. Dantas BF, De Sá Ribeiro L, Aragão CA. Germination, initial growth and cotyledon protein content of bean cultivars under salinity stress. Revista Brasileira de Sementes. 2007;29:106–10. Gomes-Filho E, Lima CRFM, Costa JH, Da Silva ACM, Da Guia Silva Lima M, De Lacerda CF, et al. Cowpea ribonuclease: Properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment. Plant Cell Rep. 2008;27:147–57. Khan MA, Rizvi Y. Effect of salinity, temperature, and growth regulators on the germination and early seedling growth of Atriplex griffithii var. stocksii. Can J Bot. 1994;72:475–9. Othman Y, Al-Karaki G, Al-Tawaha A, Al-Horani A. Variation in germination and ion uptake in barley genotypes under salinity conditions. 2006. Hamaji K, Nagira M, Yoshida K, Ohnishi M, Oda Y, Uemura T, et al. Dynamic Aspects of Ion Accumulation by Vesicle Traffic Under Salt Stress in Arabidopsis. Plant Cell Physiol. 2009;50:2023–33. Brini F, Masmoudi K. Ion Transporters and Abiotic Stress Tolerance in Plants. Int Sch Res Notices. 2012;2012:927436. Jiang X, Zhang W, Fernie AR, Wen W. Combining novel technologies with interdisciplinary basic research to enhance horticultural crops. Plant J. 2022;109:35–46. Denaxa NK, Nomikou A, Malamos N, Liveri E, Roussos PA, Papasotiropoulos V. Salinity Effect on Plant Growth Parameters and Fruit Bioactive Compounds of Two Strawberry Cultivars, Coupled with Environmental Conditions Monitoring. Agronomy 2022, Vol 12, Page 2279. 2022;12:2279. Shahzad B, Rehman A, Tanveer M, Wang L, Park SK, Ali A. Salt Stress in Brassica: Effects, Tolerance Mechanisms, and Management. J Plant Growth Regul. 2022;41:781–95. Ahmad I, Zhu G, Zhou G, Younas MU, Suliman MSE, Liu J, et al. Integrated approaches for increasing plant yield under salt stress. Front Plant Sci. 2023;14:1215343. Singh M, Nara U, Kumar A, Choudhary A, Singh H, Thapa S. Salinity tolerance mechanisms and their breeding implications. J Genetic Eng Biotechnol. 2021;19:173. Afsar S, Bibi G, Ahmad R, Bilal M, Naqvi TA, Baig A, et al. Evaluation of salt tolerance in Eruca sativa accessions based on morpho-physiological traits. PeerJ. 2020;8:e9749. Ashraf M. Organic substances responsible for salt tolerance in Eruca sativa. Biol Plant. 1994;36:255–9. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012. 2012;9:7. Navarro JM, Garrido C, Martínez V, Carvajal M. Water relations and xylem transport of nutrients in pepper plants grown under two different salts stress regimes. Plant Growth Regul. 2003;41:237–45. Wang Jmei, Chu Y, Li W, Wang X, yang, Guo Jhua, Yan L, lu, et al. Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC–MS/MS. J Chromatogr B. 2014;958:96–101. Gogorcena Y, Iturbe-Ormaetxe I, Escuredo PR, Becana M. Antioxidant Defenses against Activated Oxygen in Pea Nodules Subjected to Water Stress. Plant Physiol. 1995;108:753–9. Moran JF, Becana M, Iturbe-Ormaetxe I, Frechilla S, Klucas RV, Aparicio-Tejo P. Drought induces oxidative stress in pea plants. Planta. 1994;194:346–52. Aebi H. Oxygen Radicals in Biological Systems - Catalase in Vitro. Methods Enzymol. 1984;105:121–6. Amako K, Chen GX, Asada K. Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants. Plant Cell Physiol. 1994;35:497–504. Nakano Y, Asada K. Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts. Plant Cell Physiol. 1981;22:867–80. Carlberg I, Mannervik B. [59] Glutathione reductase. Methods Enzymol. 1985;113 C:484–90. Foyer CH, Halliwell B. The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism. Planta. 1976;133:21–5. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. Minotti G, Aust SD. The requirement for iron (III) in the initiation of lipid peroxidation by iron (II) and hydrogen peroxide. J Biol Chem. 1987;262:1098–104. Benito P, Ligorio D, Bellón J, Yenush L, Mulet JM. Use of Yucca (Yucca schidigera) Extracts as Biostimulants to Promote Germination and Early Vigor and as Natural Fungicides. Plants 2023, Vol 12, Page 274. 2023;12:274. Bybordi A. The Influence of Salt Stress on Seed Germination, Growth and Yield of Canola Cultivars. Not Bot Horti Agrobot Cluj Napoca. 2010;38:128–33. Yuan G, Wang X, Guo R, Wang Q. Effect of salt stress on phenolic compounds, glucosinolates, myrosinase and antioxidant activity in radish sprouts. Food Chem. 2010;121:1014–9. Zapata PJ, Serrano M, Pretel MT, Amorós A, Botella MÁ. Polyamines and ethylene changes during germination of different plant species under salinity. Plant Sci. 2004;167:781–8. Guo L, Yang R, Wang Z, Guo Q, Gu Z. Effect of NaCl stress on health-promoting compounds and antioxidant activity in the sprouts of three broccoli cultivars. Int J Food Sci Nutr. 2014;65:476–81. Guo RF, Yuan GF, Wang QM. Effect of NaCl treatments on glucosinolate metabolism in broccoli sprouts. J Zhejiang Univ Sci B. 2013;14:124–31. Tian L, Li X, Yang R, Gu Z. NaCl treatment improves reactive oxygen metabolism and antioxidant capacity in broccoli sprouts. Hortic Environ Biotechnol. 2016;57:640–8. Chevilly S, Dolz-Edo L, Morcillo L, Vilagrosa A, López-Nicolás JM, Yenush L, et al. Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica). BMC Plant Biol. 2021;21:1–16. Yuan F, Guo J, Shabala S, Wang B. Reproductive physiology of halophytes: Current standing. Front Plant Sci. 2019;9:432034. Muries B, Carvajal M, Martínez-Ballesta M. del C. Response of three broccoli cultivars to salt stress, in relation to water status and expression of two leaf aquaporins. Planta. 2013;237:1297–310. Volkmar KM, Hu Y, Steppuhn H. Physiological responses of plants to salinity: A review. https://doi.org/104141/P97-020. 2011;78:19–27. Rygol J, Zimmermann U. Radial and axial turgor pressure measurements in individual root cells of Mesembryanthemum crystallinum grown under various saline conditions. Plant Cell Environ. 1990;13:15–26. Miura K. Nitrogen and phosphorus nutrition under salinity stress. Ecophysiology Responses Plants under Salt Stress. 2013;9781461447474:425–41. Kumari S, Chhillar H, Chopra P, Khanna RR, Khan MIR. Potassium: A track to develop salinity tolerant plants. Plant Physiol Biochem. 2021;167:1011–23. De Araújo SAM, Silveira JAG, Almeida TD, Rocha IMA, Morais DL, Viégas RA. Salinity tolerance of halophyte Atriplex nummularia L. grown under increasing NaCl levels. Revista Brasileira de Engenharia Agrícola e Ambiental. 2006;10:848–54. Debez A, Ben Hamed K, Grignon C, Abdelly C. Salinity effects on germination, growth, and seed production of the halophyte Cakile maritima. Plant Soil. 2004;262:179–89. Joshi S, Nath J, Singh AK, Pareek A, Joshi R. Ion transporters and their regulatory signal transduction mechanisms for salinity tolerance in plants. Physiol Plant. 2022;174:e13702. Ghosh S, Mitra S, Paul A. Physiochemical Studies of Sodium Chloride on Mungbean (Vigna radiata L. Wilczek) and Its Possible Recovery with Spermine and Gibberellic Acid. Sci World J. 2015;2015:858016. Chao YY, Hsueh IE. Insights into Physiological Mechanisms of Salt Stress Tolerance in Djulis (Chenopodium formosanum Koidz.) Sprouts. J Plant Biology. 2019;62:263–73. Hasanuzzaman M, Borhannuddin Bhuyan MHM, Anee TI, Parvin K, Nahar K, Al Mahmud J et al. Regulation of Ascorbate-Glutathione Pathway in Mitigating Oxidative Damage in Plants under Abiotic Stress. Antioxidants 2019, Vol 8, Page 384. 2019;8:384. Hadi MR, Karimi N. The role of calcium in plants’ salt tolerance. J Plant Nutr. 2012;35:2037–54. Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1.pdf Cite Share Download PDF Status: Published Journal Publication published 27 May, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 08 Apr, 2025 Reviews received at journal 08 Apr, 2025 Reviews received at journal 05 Apr, 2025 Reviewers agreed at journal 24 Mar, 2025 Reviewers agreed at journal 23 Mar, 2025 Reviewers agreed at journal 03 Mar, 2025 Reviewers agreed at journal 02 Mar, 2025 Reviewers invited by journal 01 Mar, 2025 Editor invited by journal 27 Feb, 2025 Editor assigned by journal 27 Feb, 2025 Submission checks completed at journal 27 Feb, 2025 First submitted to journal 26 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6112599","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":422866488,"identity":"9b078dd0-139f-49a2-b55e-6e5d07bb0bee","order_by":0,"name":"Angel Almagro-Lopez","email":"","orcid":"","institution":"Centro de Edafología y Biología Aplicada del Segura","correspondingAuthor":false,"prefix":"","firstName":"Angel","middleName":"","lastName":"Almagro-Lopez","suffix":""},{"id":422866489,"identity":"7e62c7c4-cdc3-40cd-90a3-610b8b848b87","order_by":1,"name":"Juan Nicolas- Espinosa","email":"","orcid":"","institution":"Centro de Edafología y Biología Aplicada del Segura","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"Nicolas-","lastName":"Espinosa","suffix":""},{"id":422866490,"identity":"051cb9e1-5258-4585-b6f0-1fe83f39d1ce","order_by":2,"name":"Jose M. Mulet","email":"","orcid":"","institution":"Universitat Politècnica de València-Consejo Superior de Investigaciones Científicas","correspondingAuthor":false,"prefix":"","firstName":"Jose","middleName":"M.","lastName":"Mulet","suffix":""},{"id":422866491,"identity":"29a56765-e9a9-48d0-9e92-a03bc1a67c03","order_by":3,"name":"Micaela Carvajal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYPACNgZ+EAEGzMRqkWwgUQsDg8EBmBZCQLf98LPPPDV88sbHDx978OMPg7x8O/MDhg9/cGsxO5NmPJvnGJvhtjNp6Ya9bQyGGw6zGTDObMOj5QaDMeMMNjbGbTd4zCR4GxgSDJh5GJiBDDxa2D8zzvjHZr95Bv83yT9/GBLkm4Fa/uBz2A0eY4aPbWyJGyR42KR52BgSGA4DteALCrMzOcUMH/vYkmecSTM3lm2TAPvlYC8+vxw/vpkh4dsx235g0D1888dGXr7/8ENQ0BECx2AMCTB5gKAGBoYaItSMglEwCkbBiAUAnKVLFR51MUUAAAAASUVORK5CYII=","orcid":"","institution":"Centro de Edafología y Biología Aplicada del Segura","correspondingAuthor":true,"prefix":"","firstName":"Micaela","middleName":"","lastName":"Carvajal","suffix":""}],"badges":[],"createdAt":"2025-02-26 11:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6112599/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6112599/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-06723-3","type":"published","date":"2025-05-27T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77611972,"identity":"c3171cd0-a376-4fb0-8762-a2a425b4536d","added_by":"auto","created_at":"2025-03-03 14:33:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44088,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological parameters in broccoli cultivars BG1, BH1, BX1 and BQ1, as well as \u003cem\u003eE. vesicaria \u003c/em\u003eunder control, 50 mM, 100 mM, and 150 mM NaCl conditions. A) Germination rates (n=100). B) Sprout length (n=6). C) Sprout weight (n=6). D) Growth rate (n=6). Different letters indicate significant differences according to ANOVA followed by a \u003cem\u003epost hoc\u003c/em\u003e Tukey´s test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between treatments within the same species. Each bar represents the mean ± SE.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6112599/v1/f06ce9862f791782e08777b8.png"},{"id":77611970,"identity":"17f41e5d-8543-4941-ba4d-8ec3ac0dc745","added_by":"auto","created_at":"2025-03-03 14:33:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10981,"visible":true,"origin":"","legend":"\u003cp\u003eOsmotic potential value measured in BG1, BH1, BX1 and BQ1 broccoli cultivars and \u003cem\u003eE. vesicaria\u003c/em\u003e under control, 50 mM, 100 mM, and 150 mM NaCl conditions. Different letters indicate significant differences determined by ANOVA followed by Tukey´s \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between treatments within the same species. Each bar represents the mean ± SE (n=3).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6112599/v1/0641a824ec6dc0cc76f83372.png"},{"id":77611977,"identity":"641a4650-186a-4983-9071-b4a7c83f0452","added_by":"auto","created_at":"2025-03-03 14:33:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10326,"visible":true,"origin":"","legend":"\u003cp\u003eATP concentration in ng per sprouts in BG1, BH1, BX1 and BQ1 broccoli cultivars and \u003cem\u003eE. vesicaria \u003c/em\u003eunder control, 50 mM, 100 mM, and 150 mM NaCl conditions. Different letters indicate significant differences according to ANOVA followed by Tukey´s \u003cem\u003epost hoc\u003c/em\u003e test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between treatments within the same species. Each bar represents the mean ± SE (n=3).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6112599/v1/5b2647df97f232af37eb4f74.png"},{"id":77611975,"identity":"9115dfcb-ba86-4833-8508-195f2b49e0ab","added_by":"auto","created_at":"2025-03-03 14:33:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61618,"visible":true,"origin":"","legend":"\u003cp\u003eMacronutrient concentration (g/100g) measure in BG1, BH1, BX1 and BQ1 broccoli cultivars and \u003cem\u003eE. vesicaria\u003c/em\u003e under control, 50 mM, 100 mM, and 150 mM conditions. \u0026nbsp;Different letters indicate significant differences according to ANOVA followed by a \u003cem\u003epost hoc\u003c/em\u003e Tukey´s test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between treatments within the same species. Each bar represents the mean ± SE (n=3).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6112599/v1/d933ecdc1f36c4786c4c2a54.png"},{"id":77611974,"identity":"853fff35-3040-4265-8bb1-a388db78cc7c","added_by":"auto","created_at":"2025-03-03 14:33:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33487,"visible":true,"origin":"","legend":"\u003cp\u003eA) Na/K ratio in BG1, BH1, BX1 and BQ1 broccoli cultivars and \u003cem\u003eE. vesicaria\u003c/em\u003e expressed in molar values under control, 50 mM, 100 mM, and 150 mM conditions. B) Principal Component Analysis (PCA) of macro- and micronutrients in BG1, BH1, BX1 and BQ1 broccoli cultivars and \u003cem\u003eE. vesicaria \u003c/em\u003eunder control, 50mM, 100mM, and 150mM conditions. Different letters indicate significant differences according to ANOVA followed by a \u003cem\u003epost hoc\u003c/em\u003eTukey´s test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between treatments within the same species. Each bar represents the mean ± SE (n=3).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6112599/v1/08ecf17f944daf54050115e3.png"},{"id":77613413,"identity":"c1bff50f-b4b0-4a43-a350-16e39b482718","added_by":"auto","created_at":"2025-03-03 14:41:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":44854,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic activity and lipid damage measure in BG1, BH1, BX1 and BQ1 broccoli cultivars and \u003cem\u003eE. vesicaria\u003c/em\u003eunder control and 100 mM conditions. A) Ascorbate peroxidase activity. B) Glutathione reductase activity. C) Catalase activity. D) Lipid oxidative damage. Different letters indicate significant differences according to ANOVA followed by a \u003cem\u003epost hoc\u003c/em\u003e Tukey´s test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between species within same treatment. Asterisks (*) indicate significant differences between treatments within the same cultivar or species according to ANOVA followed by a \u003cem\u003epost hoc\u003c/em\u003e Tukey´s test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Each bar represents the mean ± SE (n=3).\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6112599/v1/b8f79f4a82c55ed14b07dc70.png"},{"id":83782858,"identity":"65e69005-0110-48ed-bc26-291840b2ca83","added_by":"auto","created_at":"2025-06-02 16:07:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1065708,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6112599/v1/7835558a-9483-4aac-ada7-ba5c967685bb.pdf"},{"id":77611989,"identity":"5879fe51-fb5c-4e4a-896d-430ab092580e","added_by":"auto","created_at":"2025-03-03 14:33:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":51038986,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6112599/v1/f905c1d76eeecd6326371202.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative adaptations of high-tolerant species and broccoli cultivars to salinity stress during germination and early development stages","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSalinity is one of the largest global challenges in arid and semi-arid regions, severely affecting agricultural production (El Sabagh et al., 2020). Salinity affects approximately 20% of the total cultivated land, and 33% of the irrigated agricultural lands worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], a situation expected to worsen and accelerate by 2050, due to climate change. Factors such as rising sea levels, which lead to seawater intrusion into aquifers due to excessive groundwater extraction in dry regions, exacerbate the problem [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSalinity refers to the high concentration of salt ions in the soil solution, especially Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and it impacts plant development through two distinct mechanisms: osmotic stress and ionic toxicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Osmotic stress occurs within first minutes of salt accumulation in the root area. The presence of salt in the soil solution reduces the osmotic potential of soil solution thus reducing the ability of plant to absorb water, which leads to reductions in the growth rate [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Ionic stress, on the other hand, manifests from a few minutes to few hours and is associated with prolonged ion accumulation, especially Na+, in the shoot tissues, which induces tissue toxicity. Excessive salt uptake via transpiration can result in cellular damage within transpiring leaves, potentially causing further reductions in growth and, in severe cases, plant death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous studies have been published on the effects of salinity on plant species; however, the majority primarily focus on sprouts and adult plants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. While these studies are valuable, it is equally important to consider the impact of salinity through first developmental stages. Salt stress affects key physiological and biochemical processes, including germination, growth, photosynthesis, water relations, nutrient homeostasis, oxidative stress management, and overall yield [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Seed germination is one of the most fundamental and vital stages in the growth cycle of a plant [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Germination is particularly important in plant species of socioeconomic importance for sprout production, as they are extensively utilized as food sources. Broccoli (\u003cem\u003eBrassica oleracea L. var. italica\u003c/em\u003e) has gained popularity due to its excellent nutritional and biochemical properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, the importance of broccoli sprouts as nutrient source has also been studied. Broccoli sprouts are health-promoting vegetables that are even more nutritious than the mature inflorescence due to their significantly higher levels of bioactive compounds such as glucosinolates and phenolic compounds [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eParticularly, ion toxicity interferes with enzyme activity, damages membranes, and disrupts metabolic pathways, causing increased production of reactive oxygen species (ROS) such as superoxide radicals (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH-) potentially affecting the different development stages of the plant, including germination [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. ROS are highly reactive molecules that can damage proteins, lipids, DNA, and other cellular components if the plant antioxidant defense mechanisms are not activated [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This has been associated to the increased production of malondialdehyde (MDA) as an indicator of membrane damage at a cellular level under salt stress [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To prevent oxidative damage to cellular component caused by ROS, plants have developed a complex antioxidant system. The primary components of this system include ascorbate and glutathione in addition to enzymes such as catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The functionality of this complex antioxidant network is required of ascorbate and glutathione pools, whose biosynthesis is directly related to NADPH and ATP levels in the cell [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This is even more important during germination under salinity as energy levels are directly related to ATP reservoirs within the seed.\u003c/p\u003e \u003cp\u003eLikewise, osmotic stress affects the water uptake process during seed imbibition, as reported by Khan and Weber (2006), while ion toxicity disrupts the enzymatic activities associated with nucleic acid and protein metabolism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, salinity interferes with metabolic equilibrium [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], impairing the mobilization and utilization of seed reserves [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Under salinity stress, efficient mechanisms to store in vacuoles the toxic concentrations of Na⁺ are essential [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The majority of these mechanisms are ATP-dependent, underscoring the critical role of intracellular energy levels in maintaining ion homeostasis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder salt stress conditions, various strategies have been described to enhance plant growth and yield. In this way, investigations have been pointed the critical need of identifying suitable cultivars to improve crop performance and germination under salt stress [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Comparative studies of physiological salinity responses between varieties within the same family provide valuable insights into differences in growth patterns and salt tolerance levels [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Also, the investigation of the different genotypes according to their salinity resilience in all development stages is a challenge [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, the response to salinity during germination is essential, as both germination and sprout growth are highly susceptible stages in the life cycle of the plants. In this sense, an investigation of the diverse responses exhibited by salt-tolerant species during germination can facilitate the identification of key resistance traits active during early development. These traits can then be used as focal points for breeding programs aimed at developing new and more resilient cultivars [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we investigate the effect of salinity on seed germination, by analyzing the germination rate, the mineral nutrient composition, the antioxidant metabolism (enzymes and lipid peroxidation) in relation to ATP availability, of four different pre-commercial broccoli cultivars, comparing these to those observed in a species from the Brassicaceae family, \u003cem\u003eEruca vesicaria subsp. Sativa\u003c/em\u003e (\u003cem\u003eE. vesicaria\u003c/em\u003e), known for its salt-tolerant [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This methodology enables the assessment of the salt tolerance of the cultivars and the various adaptive strategies employed by different cultivars and species in response to salt stress.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGermination and growth conditions\u003c/h2\u003e \u003cp\u003eSeeds of the four broccoli cultivars (BG1, BH1, BX1 and BQ1) were provided by Sakata (SAKATA SEED IB\u0026Eacute;RICA SLU) and \u003cem\u003eE. vesicaria\u003c/em\u003e seeds were purchased in CANTUESO (CANTUESO Natural Seeds). To ensure an antiseptic environment, 25 seeds from each species and cultivar were sterilized for 10 minutes in a 1:1 water-sodium hypochlorite solution. Following disinfection, the seeds were rinsed with distilled water and placed in Petri dishes containing two layers of filter paper. Four experimental conditions were established: a control (distilled water), and treatments with 50 mM, 100 mM, and 150 mM NaCl. Each treatment was replicated four times for each cultivar and species reaching a total of 100 seeds analyzed for each condition. A total of 7 mL of the respective solution was added to each Petri dish. Plates were completely covered with aluminum paper to assure darkness and then placed in an incubator at 28\u0026deg;C. The duration of the experiment was 5 days (including day 0 when the seeds were placed and day 4 when sprouts were removed). The humidity of the Petri dish was monitored daily during these 5 days. At the end of the assay, the samples were placed immediately in liquid nitrogen and later stored in the ultra-freezer. The freeze samples were grinded using pestle and mortar in constant presence of liquid nitrogen. The obtained powder was again store in the ultra-freezer for further analyses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGermination rate, length, weight and osmotic potential\u003c/h3\u003e\n\u003cp\u003eThe number of germinated seeds was counted every day and the length of the sprouts was measure thought image analysis using the program ImageJ [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Total weight from sprouts on each petri plate was measure on the last day of the essay before storage. Growth rate (GR) was calculated with the following formula.\u003c/p\u003e \u003cp\u003eGR = (Final length/days).\u003c/p\u003e \u003cp\u003eThe osmotic potential (Ѱ\u0026micro;) was measured using 100 mg of fresh powder from each treatment using a freezing-point depression osmometer (Digital Osmometer, Roebling, Berlin) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnalysis of ATP concentrations\u003c/h3\u003e\n\u003cp\u003eDetermination of ATP concentrations were performed following [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], adapting the protocol because of the different kinds of samples used. ATP was extracted using 200 mg of fresh powder from the sprouts of the four treatments with 100 \u0026micro;l (1\u0026ndash;1, v/v) pre-cooled methanol-water and 50 \u0026micro;l of the internal standard (IS) solution (35 ng/mL N-acetylglucosamine SIGMA). After vortexing for 2 min, samples were centrifuged at 12500 x g for 15 minutes. 100 \u0026micro;L of the supernatant were analyzed using High-Performance Liquid Chromatography \u0026ndash; Mass Spectrometry (HPLC-MS) at the Metabolomic and Proteomic Laboratory (ACTI - Murcia University, Spain). ATP was measured using an ATP patron (SIGMA). ATP per sprout was calculated for a visually enhanced and more accurate measure.\u003c/p\u003e\n\u003ch3\u003eAnalysis of Mineral Nutrients\u003c/h3\u003e\n\u003cp\u003eMacro- and micronutrient concentrations were determined using the dry-freeze powder obtained for each treatment. The analyses were conducted using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) on a Thermo ICAP 6500 Duo instrument (Thermo Fisher Scientific, Waltham, MA, USA) as described by Nicolas-Espinosa et al. (2024). The nutrient concentrations were expressed as mg 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW for macronutrients and mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW for micronutrient.\u003c/p\u003e\n\u003ch3\u003ePreparation of extracts and enzyme assay\u003c/h3\u003e\n\u003cp\u003eAntioxidant enzymes were extracted from 300 mg of fresh powder from sprouts in control and 100 mM conditions using 50 mM potassium phosphate buffer (pH 7.8), containing 0.1 mM Na\u003csub\u003e2\u003c/sub\u003e\u0026ndash;EDTA and 1% insoluble polyvinylpolypyrrolidone (PVPP). This extract was used to determine the activity of the enzyme catalase (CAT) and oxidative damage to lipids [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The same extract plus 10 mM β-mercaptoethanol was used for glutathione reductase (GR), and with 4 mM ascorbic acid for ascorbate peroxidase (APX) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The supernatants were stored at \u0026minus;\u0026thinsp;20\u0026deg;C for subsequent enzymatic assays.\u003c/p\u003e \u003cp\u003eCatalase (CAT) activity was measured by the disappearance of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as described by Aebi (1984). The extract was mixed with 50 mM potassium phosphate buffer (pH 7.0) and the reaction was initiated by adding 10.6 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to the sample and monitoring the change in absorbance at 240 nm every 30 sec for 3 min.\u003c/p\u003e \u003cp\u003eThe oxidation rate of ascorbate was measured using ascorbate peroxidase (APX) activity as described by Amako et al. (1994) and Nakano and Asada (1981). The extract was mixed with 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM Na\u003csub\u003e2\u003c/sub\u003e\u0026ndash;EDTA, and 0.5 mM ascorbic acid. The addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used to start the reaction and the decrease in absorbance at 290 nm was recorded every 30 sec for 3 min.\u003c/p\u003e \u003cp\u003eGlutathione reductase (GR) activity was determined as described by Carlberg and Mannervik (1985) and Foyer and Halliwell (1976). The reaction mixture contained 0.1 M HEPES (pH 7.8), 1 mM Na\u003csub\u003e2\u003c/sub\u003e\u0026ndash;EDTA, 3 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 mM oxidized glutathione, and the enzyme extract. Finally, 0.2 mM NADPH was added. The rate of NADPH oxidation was monitored by the decrease in absorbance at 340 nm every 30 sec for 2 min.\u003c/p\u003e \u003cp\u003eThe data of the enzymatic activities were normalized by the mg of protein, determined by the Bradford method [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Then, the results of antioxidant enzymes (APX, GR and CAT) were expressed as \u0026micro;mol reactive/min mg protein. Three biological and three technical replicates of the control and 100 mM treatments samples were analyzed.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLipid Oxidative damage\u003c/h2\u003e \u003cp\u003eLipid peroxidation (LP) was determined using the previous extract and was determined as described by Minotti and Aust (1987). The reaction was performed by mixing the extract with a reaction mixture containing 20% trichloroacetic acid (TCA), 0.5% 2-thiobarbituric acid (TBA), 0.01% butyl hydroxytoluene (HBT) and 0.25 N HCl, and incubating the mixture at 95\u0026ordm;C for 30 min. After stopping the reaction in ice and centrifuging the samples, the supernatant was used for spectrophotometric reading at 535 nm. The calibration curve was made using malondialdehyde (MDA) in the range of 0 to 10 \u0026micro;M. The results were expressed in \u0026micro;mol MDA/min mg protein.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eStatistical analyses and data presentation were conducted using Origin (Pro) (Version 2021 software package by OriginLab Corporation, Northampton, MA, USA). Significant differences among the values of all the parameters were determined at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, according to Tukey\u0026rsquo;s test.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological parameters\u003c/h2\u003e \u003cp\u003eGermination rates were above 90% in all broccoli cultivars across all treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). No significant differences in germination rates were observed between treatments in the broccoli cultivars, except for BQ1, where a significant decrease was observed in the 150 mM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). \u003cem\u003eE. vesicaria\u003c/em\u003e showed a significant decrease in the 100 mM and 150 mM treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), with these values being significantly lower compared to the rest of cultivars. A direct correlation between an increase in salinity and a reduction in sprout length, weight and growth rate was observed in all varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, D and Fig. sup.1). In the case of BX1, BQ1, and \u003cem\u003eE. vesicaria\u003c/em\u003e, no significant differences were observed between the control and 50 mM treatments in length and growth rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, D). Similarly, only BQ1 showed no weight differences between the control and 50 mM treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, BQ1 exhibited the highest values of length, weight, and growth rate under all treatment conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, when analyzed osmotic potential of sprouts, a significant decrease was observed in all species and cultivars across treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In BH1 and BQ1, no differences were observed between the 100 mM and 150 mM treatments, but both were lower compared to the control. In BX1, the lowest value was observed at 100 mM, but osmotic potential at 150 was similar to the control. Significant differences between cultivars and species were observed only under 150 mM salinity treatment, where BG1 and \u003cem\u003eE. vesicaria\u003c/em\u003e exhibited the lowest values, while BX1 has the least negative value in this condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eATP concentration per sprout showed a significant reduction in some of the broccoli cultivars (BG1, BX1 and BQ1) but no differences were observed in BH1 and \u003cem\u003eE. vesicaria\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These two species were also the ones with the lowest values of ATP concentration in control conditions. The largest difference between treatments was observed in the BX1 cultivar, which exhibited the biggest value in control condition and the lowest in the 150 mM treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Mineral Nutrients\u003c/h2\u003e \u003cp\u003eAlterations in mineral composition were observed across species, cultivars, and specific minerals analyzed. Calcium (Ca) showed a significant decrease in BQ1 with the progressive increase of salinity concentration, but its values were similar to those obtained in the rest of cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). \u003cem\u003eE. vesicaria\u003c/em\u003e exhibited the same significant decrease under salinity, with the lowest values across all treatments. Magnesium (Mg) concentration changed only in \u003cem\u003eE. vesicaria\u003c/em\u003e where a significant increase was observed under salinity. Phosphorous (P) showed a decrease in BG1 under salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) while \u003cem\u003eE. vesicaria\u003c/em\u003e had the lowest values under all conditions. Notably, sulfur (S) concentration did not change with increasing salinity; however, each cultivar appeared to have different intrinsic S level. BG1 and BQ1 were grouped together with higher S concentrations, while BH1 and BX1 exhibited the lowest levels. A decrease in potassium (K) concentration was observed in BX1 and BQ1 cultivars under salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) but the highest K levels in all treatments were observed in BX1, \u003cem\u003eE. vesicaria\u003c/em\u003e, and BQ1 under control conditions respectively. Na concentrations increased under salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) and were higher in BX1, BQ1 and \u003cem\u003eE. vesicaria\u003c/em\u003e in control conditions; however, their concentrations remained unchanged with increasing salinity. The Na/K ratio increased under salinity treatments in all cultivars and species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA); nevertheless, no differences were observed between the different cultivars and \u003cem\u003eE. vesicaria\u003c/em\u003e. PCA analysis showed a clear differentiation between the broccoli varieties and \u003cem\u003eE. vesicaria\u003c/em\u003e on the horizontal axis when the macro- and micronutrient sprout compositions were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), particularly dominated by the concentration of Na and K, under the different conditions. Additionally, a distinction was observed between the broccoli varieties, specifically forming two groups: BG1 and BQ1 clustered together, while BH1 and BX1 formed a separate group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme activity and lipid oxidative damage\u003c/h2\u003e \u003cp\u003eFor enzyme activity and lipid oxidative damage, the 100 mM and control treatments were selected to simplify the interpretation and management of the results. Ascorbate peroxidase (APX) activity showed a significant increase in BG1, BX1 and \u003cem\u003eE. vesicaria\u003c/em\u003e under salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The highest activity was observed in \u003cem\u003eE. vesicaria\u003c/em\u003e under both control and salinity conditions. Glutathione reductase (GR) activity increased in BG1 and BX1 and \u003cem\u003eE. vesicaria\u003c/em\u003e under salinity conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). All broccoli cultivars presented a low catalase (CAT) activity, which was not altered by salinity. \u003cem\u003eE. vesicaria\u003c/em\u003e showed a notably high basal CAT activity under control conditions and was the only species to exhibit a significant increase in CAT activity under salinity conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Lipid oxidative damage showed a significant decrease in all broccoli cultivars, except for BH1 and \u003cem\u003eE. vesicaria\u003c/em\u003e, where no changes were observed under salinity conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeeds are complex structures equipped with the necessary machinery and stored compounds to support germination and early seed growth. These intrinsic characteristics, however, vary significantly among species and cultivars, influencing their ability to withstand environmental stresses, such as salinity. To determine salt tolerance, our study compared physiological, biochemical, and molecular parameters between control and salinity conditions during seed germination of four different broccoli cultivars. Additionally, these results were compared with those obtained from the salt-tolerant species \u003cem\u003eE. vesicaria\u003c/em\u003e, which was selected as a reference. Our results showed that germination rates were primarily dependent on the cultivar and species, and to a lesser extent, on the salinity concentration. Salinity did not affect germination rates in any of the broccoli cultivars (except for BQ1 under 150 mM) but remarkably \u003cem\u003eE. vesicaria\u003c/em\u003e showed a significant decrease in germination rates when treated with 100 mM and 150 mM of NaCl. This significant decrease observed in E. vesicaria has also been observed in the germination rate of different species under salinity stress within the Brassicaceae family. Different studies have shown that there may be a great difference among abiotic stress tolerance at the germination stage, and in the adult stage. For instance tomato is considered to be less salt sensitive than broccoli but the germination rates under salt stress are similar [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].Bybordi (2010) observed how \u003cem\u003eBrassica napus\u003c/em\u003e exhibited significant germination decline at 150\u0026ndash;200 mM NaCl, consistent with findings in radish and beetroot [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Different studies have shown that there may be a great difference among abiotic stress tolerance at the germination stage, and in the adult stage. For instance tomato is considered to be less salt sensitive than broccoli but the germination rates under salt stress are similar. Three cultivars of broccoli has been observed to decline germination similarly under 80\u0026ndash;100 mM NaCl [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Similarly, 100 mM NaCl significantly decreased germination rates in broccoli sprouts [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Although previous studies indicated that salinity concentrations above 80 mM negatively affected seed germination in Brassicas, our results did not show significant differences in germination rates under salinity conditions, mainly due to the prior selection of salt-tolerant pre-commercial broccoli cultivars. Despite the lack of a significant effect of salinity on seed germination, we observed a decline in physiological parameters under salinity conditions. All broccoli cultivars exhibited reduced length and growth rate with increasing salinity. However, BX1, BQ1, \u003cem\u003eand E. vesicaria\u003c/em\u003e demonstrated greater tolerance under low salt concentration, as their growth rates and length were unaffected by the 50 mM salinity treatment. A similar decrease was observed in weight under salinity conditions, but only BQ1 showed no differences between the control and 50 mM treatment. This reduction in sprout length and weight under salinity was also observed by Tian et al. (2016). Nevertheless, our cultivars showed higher values, suggesting enhanced tolerance to salinity. The bottom line is that all our broccoli cultivars exhibited better overall performance in terms of germination rate, length, and weight, not only under salinity conditions but also in general when compared with previous studies, demonstrating a high level of salinity tolerance at the germination stage, with BQ1 showing outstanding performance. This observation is consistent with previous findings, where certain varieties were identified as salinity-tolerant in a study on adult plants, suggesting that their resilience to salinity extends to the germination stage [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eEruca vesicaria\u003c/em\u003e subsp. \u003cem\u003esativa\u003c/em\u003e is widely recognized as a highly salt-tolerant species, our study indicates that this tolerance is not fully expressed during the germination stage, in contrast to the higher tolerance observed in broccoli during germination. \u003cem\u003eE. vesicaria\u003c/em\u003e exhibited reduced germination rates under increasing salinity, with lower physiological performance compared to broccoli cultivars. This does not mean low salinity tolerance but it suggests that its underlying mechanisms may not be active during germination. A similar phenomenon has been observed in other halophyte species, such as \u003cem\u003eCakile maritima\u003c/em\u003e, which fails to germinate under high salinity (200 mM) despite its ability to grow at much higher concentrations [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This pattern may represent an adaptive strategy to prevent germination in unfavorable environments, such as seawater-exposed conditions, where high salinity levels could compromise sprout establishment and survival.\u003c/p\u003e \u003cp\u003eAs previously stated, salinity is a major abiotic stress that disrupts germination and sprout development by imposing osmotic stress, ion toxicity, and nutrient imbalances. To mitigate the damage caused by salinity, plant cells typically synthesize and accumulate compatible organic molecules, which help lower the osmotic potential of the cells, thus enhancing water absorption under these conditions. The reduction in osmotic potential is a common adaptive response of plants to cope with salt stress and has been previously reported in broccoli, where the accumulation of compatible osmolytes, has been associated with salinity tolerance in broccoli cultivars [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. When the osmotic potential of the different cultivars and species was analysed, a lower osmotic potential under salinity was observed, indicating active osmolyte accumulation in response to this stress. A progressive accumulation of osmolytes appeared to be associated with the decrease in osmotic potential caused by the increase in salt concentration, particularly in BG1 and \u003cem\u003eE. vesicaria\u003c/em\u003e. In contrast, in BH1, BX1, and BQ1, osmolyte accumulation was triggered when the seeds were exposed to 100 mM salinity and maintained at a similar concentration when salinity increased to 150 mM, except for BX1, which exhibited a value similar to the control condition. This seemed to indicate that BH1 and BQ1 employ a threshold-dependent response to salinity, where osmolyte accumulation is triggered only when stress reaches a critical level (100\u0026ndash;150 mM). This reduces unnecessary energy expenditure during mild stress (e.g., 50 mM), preserving resources for higher stress levels. Furthermore, BX1 seems to struggle ATP reserve management under salt stress, exhibiting a marked reduction in sprout ATP content at higher salt concentrations (100 mM and 150 mM). This could be related to its inability to synthesize compatible osmolytes due to an energy-saving mechanism, leading to a lack of osmotic potential adjustments in these plants. Moreover, this specific cultivar demonstrated the poorest performance under salinity stress conditions, indicating the essential role of osmotic potential adjustments in salinity tolerance, which is directly related to energy management and ATP usage.\u003c/p\u003e \u003cp\u003eChanges in Ca concentration was only observed in BQ1 in salinity condition although levels were similar to those observed in the rest of the cultivars and species. This reduction was also observed in \u003cem\u003eE. vesicaria\u003c/em\u003e but in this case, the Ca levels were the lowest in salinity condition indicating a direct effect of salinity in this species. Calcium has been reported to mitigate the adverse effects of salinity in plants, as salinity conditions reduce Ca uptake by displacing it from the cell membrane or disrupting membrane function, leading to increased Na accumulation in leaves and impairing K/Na selectivity [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Similar findings have been observed in halophyte species, where calcium supplementation alleviated salinity-induced damage, as Na directly affects cell wall properties and plasma membrane function through Na/Ca displacement [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In this context, maintaining adequate Ca levels is critical for salinity tolerance, which was observed exclusively in the broccoli cultivars under salinity conditions, suggesting enhanced mechanisms to prevent Ca displacement. BQ1 exhibited higher calcium levels under control conditions, suggesting this cultivar has inherently elevated seed calcium content. This trait may serve as an adaptive mechanism to mitigate salinity-induced stress without expending energy to transport calcium in order to maintain membrane stability. P reduction under salinity was observed exclusively in the BG1 cultivar, with \u003cem\u003eE. vesicaria\u003c/em\u003e again exhibiting the lowest values. This suggests these two species show reduced absorption or impaired mobility, as certain phosphate transporters are sensitive to salt, hindering P uptake and its mobilization from internal reservoirs [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. A reduction in K levels under salinity conditions was observed only in the BX1 and BQ1 cultivars, which, along with \u003cem\u003eE. vesicaria\u003c/em\u003e, exhibited the highest K concentrations under salinity. This reduction is understandable as Na and K show similar chemical properties so they can share channels reducing K transport or absorption [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. BQ1 showed the highest growth rate and maintained the highest K concentration at 150 mM NaCl, suggesting a potential correlation between growth performance, salinity tolerance, and K accumulation. Given the importance of K homeostasis in salinity tolerance, seed K levels may serve as an indicator of salt stress resilience. Notably, BQ1 exhibited the highest K concentration in seeds. Regarding Na, the broccoli cultivars with the highest physiological measures in salinity (BX1 and BQ1) along with \u003cem\u003eE. vesicaria\u003c/em\u003e showed the highest concentration of Na in control conditions, indicating that the levels of Na in these seeds are the highest. A clear relation between seed exposure to higher Na levels in seed and a better tolerance to salinity is evident, but also supported by previous studies where high Na concentrations in seed has been observed in halophytes species [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Another significant phenomenon is that all sprouts exhibited an increase in Na concentration upon exposure to 50 mM NaCl; however, this intracellular Na concentration remained stable despite further increases in external salinity (100 mM and 150 mM). This suggests that seeds have a threshold for intracellular Na accumulation, likely regulated by Na compartmentalization within the vacuole. This threshold appeared to be reached at 50 mM, which, which could be reasonable considering that these seeds are only five days old and still undergoing development. At this stage, vacuolar structures and their associated transport mechanisms may be predominantly functional in the cotyledons, as these tissues are already differentiated. This compartmentalization mechanism effectively mitigates ionic toxicity, stress and cellular damage. However, as salinity increases, vacuolar sequestration becomes insufficient, suggesting the activation of alternative adaptive mechanisms. Given the absence of significant changes in overall Na concentration within the sprouts, these compensatory mechanisms may involve Na efflux through ATP-dependent transporters, highlighting the critical role of energy allocation in salinity tolerance.\u003c/p\u003e \u003cp\u003eIf we observe all the changes in mineral composition, BQ1 is the one showing a distinct pattern. BQ1 displays the highest values of Ca, K and Na in seed, all of which are key elements associated with salinity stress tolerance. An early exposure to Na may preventively activate the mechanisms related to salinity tolerance. This along with an enhanced concentration of Ca and K can generate the perfect environment to trigger mechanism to prevent excessive oxidative damage under salinity. Some of these mechanisms have already been discussed such as Na sequestration within the vacuole, exclusion of excess Na via selective ion transporters as SOS1 or HKT transporters as well as K retention trough efficient K channels to maintain enzymatic function and osmotic balance [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The combined ability of BQ1 to manage Na and K levels minimizes ion toxicity and osmotic imbalance supporting sustained growth and metabolism under salinity. However, \u003cem\u003eE. vesicaria\u003c/em\u003e might rely on more limited or less efficient mechanisms, such as early vacuolar sequestration of Na, which could limit its overall tolerance to saline environments.\u003c/p\u003e \u003cp\u003eAntioxidants, including ascorbate and glutathione, play a critical role in enhancing plant defense mechanisms against oxidative stress. These antioxidant levels are directly related to antioxidant enzymatic activities such as APX, GR and CAT activity that are part of the oxidative stress response. In the APX measure, almost all the sprouts showed an increase in APX activity under salinity conditions only significant in BG1, BX1, and \u003cem\u003eE. vesicaria\u003c/em\u003e. The last one showed the highest value in both conditions. GR activity showed a significant increase in BG1, BX1 and \u003cem\u003eE. vesicaria\u003c/em\u003e under salinity conditions. Regarding CAT activity, broccoli cultivars showed low activity in both treatments and was \u003cem\u003eE. vesicaria\u003c/em\u003e the one with the highest values being 4 times higher in control and 10 times higher in salinity. These results indicate how \u003cem\u003eE. vesicaria\u003c/em\u003e exhibited the highest and strongest antioxidant enzymatic response. BQ1 and BH1 are the ones that showed no differences between treatments indicating no oxidative damage response through antioxidant enzymes routes. Exposure to salinity conditions is reported to increase antioxidant enzymes activity such as APX, GR and CAT in plants as observed in mungbean [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] under 100 mM. The same has been reported in other species such as Djulis [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Therefore, the absence of changes in these activities under salinity conditions may indicate an alternative response pathway that does not involve antioxidant enzymes or suggest that the sprout is not experiencing oxidative stress. This likely indicates that the ion homeostasis mechanisms in these cultivars exhibit superior functionality compared to others, thereby mitigating oxidative stress since the Na/K ratio remains consistent across all broccoli cultivars. BQ1 and BH1 appear to exhibit more efficient sodium compartmentalization. Consequently, the SOS1-NHX1 system in these two cultivars may have higher expression levels or more effective isoforms. To gain a clearer understanding of this process, lipid oxidative damage is a key parameter to consider. Lipid oxidative damage is another measure that can provide insight of how badly oxidative stress is affecting a plant as it measures actual damage. All broccoli cultivars except for BH1 showed a decrease in lipid oxidation, indicating that the various antioxidant mechanisms at play are effectively preventing oxidative damage under salinity conditions. \u003cem\u003eE. vesicaria\u003c/em\u003e did not show this reduction; instead, an increase was observed. This implies how BQ1 and BH1, which showed no significant increase in antioxidant enzyme activity under salinity, displayed both low lipid oxidative damage, while \u003cem\u003eE. vesicaria\u003c/em\u003e, despite its high antioxidant activity, failed to prevent oxidative damage effectively. BQ1 and BH1 may depend on non-enzymatic antioxidant systems or more efficient sodium compartmentalization mechanisms to mitigate oxidative damage, reducing the necessity for enzymatic pathway activation. This could be facilitated by pre-synthesized antioxidant reservoirs stored in seeds or the presence of upregulated Na transporters, as previously mentioned. Plants significantly activates the aspartate-glutathione pathways to synthesis these non-enzymatic antioxidants involved in detoxify ROS up to tolerable levels under salinity conditions [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Moreover, observing that even the cultivars that did not show changes in enzyme activity still exhibited no increase in lipid oxidative damage supports the idea of an alternative mechanism at play. These alternative mechanisms may be closely related to cellular energy balance. The antioxidant system requires energy in order to function properly and to synthesize antioxidant molecules such as ascorbate and glutathione, so does the solute accumulation within the cell. ATP values were the lowest in BH1 and no changes were observed under salinity conditions, something we also observe in \u003cem\u003eE. vesicaria\u003c/em\u003e, which showed increase in these antioxidant activities but no changes in ATP. This suggests that all mobilized or synthesized ATP is rapidly utilized in antioxidant defense mechanisms or in the activation of membrane ATPases, enhancing the capacity for Na and K transport. This facilitates the sequestration of Na into the vacuole. In BH1, the ATP mobilization and usage can be more related to antioxidant molecules biosynthesis and Na sequestration to prevent ROS damages, as no changes in enzymatic activity was observed. In the rest of the cultivars, we observe higher ATP pools in the seed and higher ATP use, indicating that a larger ATP availability can allow multiple responses. BQ1 is a clear example of that being the cultivar with the best performance during germination and sprout growth under high levels or salinity but no changes in enzymatic activity were observed. This indicates that BQ1 may be using its large ATP pool to trigger alternative mechanism, some already described such as ion efflux mechanisms to alleviate ion toxicity, with Na⁺ toxicity being particularly significant and the maintenance of ion homeostasis, particularly K⁺ and Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis. This is important because calcium plays an important role in processes that preserve the structural and functional integrity of plant membranes avoiding ROS damage [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The threshold-triggered osmolyte accumulation strategy is equally important as it minimise energy wastage under low stress conditions (50 mM) while enabling a robust response under severe stress (100\u0026ndash;150 mM). The majority of these mechanisms are ATP-dependent, underscoring the critical role of intracellular energy levels in maintaining optimal performance of these mechanisms [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, ATP levels may represent a key determinant of the differential responses profiles observed in sprouts. A larger ATP pool, and more importantly, a well-regulated ATP utilization, can be considered critical tolerance traits in early-stage sprouts, as they enable prolonged and more diverse physiological responses over time.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results revealed that all broccoli cultivars exhibited higher germination rates and superior physiological performance compared to \u003cem\u003eE. vesicaria\u003c/em\u003e, with the highest performance observed in the BQ1 cultivar. These findings suggest that different mechanisms are activated in each development stage across cultivars and species to mitigate salinity-induced stress. These mechanisms include solute accumulation, enhancement of antioxidant activity alongside alternative strategies such as Na⁺ sequestration and exclusion, K⁺ transport, and overall mineral homeostasis. All these processes are energy-dependent, relying on ATP availability. Thus, ATP availability emerges as a key factor defining the salinity response profile of each cultivar. These profiles differentiate cultivars in terms of salinity tolerance, revealing specific physiological strategies. BG1 efficiently utilizes a moderate ATP pool to maintain performance under moderate salinity (50\u0026ndash;100 mM). BH1 exhibits limited stress responses due to low ATP availability. Although BX1 possesses the largest ATP reservoir, it fails to regulate ATP allocation effectively, compromising osmotic regulation. In contrast, BQ1 maintains a balanced ATP utilization strategy, enabling the activation of a broader range of adaptive mechanisms. Meanwhile, \u003cem\u003eE. vesicaria\u003c/em\u003e struggles to prevent oxidative damage despite its physiological responses. Overall, BQ1 integrates multiple protective strategies to mitigate, prevent, and tolerate salinity-induced stress in sprouts, achieving the highest physiological performance and outperforming the enzymatic responses observed in \u003cem\u003eE. vesicaria.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge SAKATA S.L.U Company for providing the broccoli seeds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMCA, JMM and JNE contributed to the conception and design of this work. AAL carried out the experiments and JNE the statistical analytical work. AAL prepared figures and tables, and prepared the first draft of the manuscript. MCA, JMM and JNE contributed to manuscript revisions, reads and approved the submitted version. MCA obtained the funding. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Spanish Ministerio de Ciencia e Innovaci\u0026oacute;n (CPP2022-009860).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNothing to declare.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNothing to declare.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe authors declare no competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor information\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAquaporins Group. Centro de Edafologia y Biologia Aplicada del Segura. CEBAS-CSIC. Campus Universitario de Espinardo - 25. E-30100. Murcia. Spain.\u003c/p\u003e\n\u003cp\u003eAngel Almagro-Lopez, Juan Nicolas-Espinosa and Micaela Carvajal.\u003c/p\u003e\n\u003cp\u003eInstituto de Biolog\u0026iacute;a Molecular y Celular de Plantas (IBMCP), Universitat Polit\u0026egrave;cnica de Val\u0026egrave;ncia-Consejo Superior de Investigaciones Cient\u0026iacute;ficas, 46022 Valencia, Spain.\u003c/p\u003e\n\u003cp\u003eJM Mulet. Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Micaela Carvajal. Email: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMukhopadhyay R, Sarkar B, Jat HS, Sharma PC, Bolan NS. Soil salinity under climate change: Challenges for sustainable agriculture and food security. J Environ Manage. 2021;280:111736.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShrivastava P, Kumar R. Soil salinity: A serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation. Saudi J Biol Sci. 2015;22:123\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParihar P, Singh S, Singh R, Singh VP, Prasad SM. Effect of salinity stress on plants and its tolerance strategies: a review. Environ Sci Pollut Res. 2015;22:4056\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorwin DL. Climate change impacts on soil salinity in agricultural areas. Eur J Soil Sci. 2021;72:842\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59 Volume 59, 2008:651\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicolas-Espinosa J, Yepes-Molina L, Martinez-Bernal F, Fernandez-Pozurama M, Carvajal M. Deciphering the effect of salinity and boron stress on broccoli plants reveals that membranes phytosterols and PIP aquaporins facilitate stress adaptation. Plant Sci. 2024;338:111923.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaghdoud C, Alcaraz-L\u0026oacute;pez C, Mota-Cadenas C, Martnez-Ballesta MDC, Moreno DA, Ferchichi A, et al. Differential Responses of Two Broccoli (Brassica oleracea L. var Italica) Cultivars to Salinity and Nutritional Quality Improvement. Sci World J. 2012;2012:291435.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026auml;uchli A, Grattan SR. Plant Growth And Development Under Salinity Stress. Advances in Molecular Breeding Toward Drought and Salt. Tolerant Crops. 2007;:1\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Xia Y, Liu HY, Guo H, He XQ, Liu Y, et al. Nutritional values, beneficial effects, and food applications of broccoli (Brassica oleracea var. italica Plenck). Trends Food Sci Technol. 2022;119:288\u0026ndash;308.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang P, Li X, Tian L, Gu Z, Yang R. Low salinity promotes the growth of broccoli sprouts by regulating hormonal homeostasis and photosynthesis. Hortic Environ Biotechnol. 2019;60:19\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoschenrieder C, Cabot C, Martos S, Gallego B, Barcel\u0026oacute; J. Do toxic ions induce hormesis in plants? Plant Sci. 2013;212:15\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas K, Roychoudhury A. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Front Environ Sci. 2014;2 DEC:121942.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIbrahim EA. Seed priming to alleviate salinity stress in germinating seeds. J Plant Physiol. 2016;192:38\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta DK, Palma JM, Corpas FJ. Antioxidants and antioxidant enzymes in higher plants. Antioxid Antioxid Enzymes High Plants. 2018;:1\u0026ndash;300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLatowski D, Sur\u0026oacute;wka E, Strzałka K. Regulatory role of components of ascorbate-glutathione pathway in plant stress tolerance. Ascorbate-Glutathione Pathw Stress Tolerance Plants. 2010;:1\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan MAjmal, Weber DJ. Ecophysiology of High Salinity Tolerant Plants (Tasks for Vegetation Science). Ecophysiology of High Salinity Tolerant Plants. 2006;:XVIII\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDantas BF, De S\u0026aacute; Ribeiro L, Arag\u0026atilde;o CA. Germination, initial growth and cotyledon protein content of bean cultivars under salinity stress. Revista Brasileira de Sementes. 2007;29:106\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomes-Filho E, Lima CRFM, Costa JH, Da Silva ACM, Da Guia Silva Lima M, De Lacerda CF, et al. Cowpea ribonuclease: Properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment. Plant Cell Rep. 2008;27:147\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan MA, Rizvi Y. Effect of salinity, temperature, and growth regulators on the germination and early seedling growth of Atriplex griffithii var. stocksii. Can J Bot. 1994;72:475\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOthman Y, Al-Karaki G, Al-Tawaha A, Al-Horani A. Variation in germination and ion uptake in barley genotypes under salinity conditions. 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamaji K, Nagira M, Yoshida K, Ohnishi M, Oda Y, Uemura T, et al. Dynamic Aspects of Ion Accumulation by Vesicle Traffic Under Salt Stress in Arabidopsis. Plant Cell Physiol. 2009;50:2023\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrini F, Masmoudi K. Ion Transporters and Abiotic Stress Tolerance in Plants. Int Sch Res Notices. 2012;2012:927436.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang X, Zhang W, Fernie AR, Wen W. Combining novel technologies with interdisciplinary basic research to enhance horticultural crops. Plant J. 2022;109:35\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenaxa NK, Nomikou A, Malamos N, Liveri E, Roussos PA, Papasotiropoulos V. Salinity Effect on Plant Growth Parameters and Fruit Bioactive Compounds of Two Strawberry Cultivars, Coupled with Environmental Conditions Monitoring. Agronomy 2022, Vol 12, Page 2279. 2022;12:2279.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahzad B, Rehman A, Tanveer M, Wang L, Park SK, Ali A. Salt Stress in Brassica: Effects, Tolerance Mechanisms, and Management. J Plant Growth Regul. 2022;41:781\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad I, Zhu G, Zhou G, Younas MU, Suliman MSE, Liu J, et al. Integrated approaches for increasing plant yield under salt stress. Front Plant Sci. 2023;14:1215343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh M, Nara U, Kumar A, Choudhary A, Singh H, Thapa S. Salinity tolerance mechanisms and their breeding implications. J Genetic Eng Biotechnol. 2021;19:173.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfsar S, Bibi G, Ahmad R, Bilal M, Naqvi TA, Baig A, et al. Evaluation of salt tolerance in Eruca sativa accessions based on morpho-physiological traits. PeerJ. 2020;8:e9749.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshraf M. Organic substances responsible for salt tolerance in Eruca sativa. Biol Plant. 1994;36:255\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012. 2012;9:7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavarro JM, Garrido C, Mart\u0026iacute;nez V, Carvajal M. Water relations and xylem transport of nutrients in pepper plants grown under two different salts stress regimes. Plant Growth Regul. 2003;41:237\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Jmei, Chu Y, Li W, Wang X, yang, Guo Jhua, Yan L, lu, et al. Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC\u0026ndash;MS/MS. J Chromatogr B. 2014;958:96\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGogorcena Y, Iturbe-Ormaetxe I, Escuredo PR, Becana M. Antioxidant Defenses against Activated Oxygen in Pea Nodules Subjected to Water Stress. Plant Physiol. 1995;108:753\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoran JF, Becana M, Iturbe-Ormaetxe I, Frechilla S, Klucas RV, Aparicio-Tejo P. Drought induces oxidative stress in pea plants. Planta. 1994;194:346\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAebi H. Oxygen Radicals in Biological Systems - Catalase in Vitro. Methods Enzymol. 1984;105:121\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmako K, Chen GX, Asada K. Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants. Plant Cell Physiol. 1994;35:497\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakano Y, Asada K. Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts. Plant Cell Physiol. 1981;22:867\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarlberg I, Mannervik B. [59] Glutathione reductase. Methods Enzymol. 1985;113 C:484\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoyer CH, Halliwell B. The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism. Planta. 1976;133:21\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinotti G, Aust SD. The requirement for iron (III) in the initiation of lipid peroxidation by iron (II) and hydrogen peroxide. J Biol Chem. 1987;262:1098\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenito P, Ligorio D, Bell\u0026oacute;n J, Yenush L, Mulet JM. Use of Yucca (Yucca schidigera) Extracts as Biostimulants to Promote Germination and Early Vigor and as Natural Fungicides. Plants 2023, Vol 12, Page 274. 2023;12:274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBybordi A. The Influence of Salt Stress on Seed Germination, Growth and Yield of Canola Cultivars. Not Bot Horti Agrobot Cluj Napoca. 2010;38:128\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan G, Wang X, Guo R, Wang Q. Effect of salt stress on phenolic compounds, glucosinolates, myrosinase and antioxidant activity in radish sprouts. Food Chem. 2010;121:1014\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZapata PJ, Serrano M, Pretel MT, Amor\u0026oacute;s A, Botella M\u0026Aacute;. Polyamines and ethylene changes during germination of different plant species under salinity. Plant Sci. 2004;167:781\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo L, Yang R, Wang Z, Guo Q, Gu Z. Effect of NaCl stress on health-promoting compounds and antioxidant activity in the sprouts of three broccoli cultivars. Int J Food Sci Nutr. 2014;65:476\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo RF, Yuan GF, Wang QM. Effect of NaCl treatments on glucosinolate metabolism in broccoli sprouts. J Zhejiang Univ Sci B. 2013;14:124\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian L, Li X, Yang R, Gu Z. NaCl treatment improves reactive oxygen metabolism and antioxidant capacity in broccoli sprouts. Hortic Environ Biotechnol. 2016;57:640\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChevilly S, Dolz-Edo L, Morcillo L, Vilagrosa A, L\u0026oacute;pez-Nicol\u0026aacute;s JM, Yenush L, et al. Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica). BMC Plant Biol. 2021;21:1\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan F, Guo J, Shabala S, Wang B. Reproductive physiology of halophytes: Current standing. Front Plant Sci. 2019;9:432034.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuries B, Carvajal M, Mart\u0026iacute;nez-Ballesta M. del C. Response of three broccoli cultivars to salt stress, in relation to water status and expression of two leaf aquaporins. Planta. 2013;237:1297\u0026ndash;310.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVolkmar KM, Hu Y, Steppuhn H. Physiological responses of plants to salinity: A review. https://doi.org/104141/P97-020. 2011;78:19\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRygol J, Zimmermann U. Radial and axial turgor pressure measurements in individual root cells of Mesembryanthemum crystallinum grown under various saline conditions. Plant Cell Environ. 1990;13:15\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiura K. Nitrogen and phosphorus nutrition under salinity stress. Ecophysiology Responses Plants under Salt Stress. 2013;9781461447474:425\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumari S, Chhillar H, Chopra P, Khanna RR, Khan MIR. Potassium: A track to develop salinity tolerant plants. Plant Physiol Biochem. 2021;167:1011\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Ara\u0026uacute;jo SAM, Silveira JAG, Almeida TD, Rocha IMA, Morais DL, Vi\u0026eacute;gas RA. Salinity tolerance of halophyte Atriplex nummularia L. grown under increasing NaCl levels. Revista Brasileira de Engenharia Agr\u0026iacute;cola e Ambiental. 2006;10:848\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDebez A, Ben Hamed K, Grignon C, Abdelly C. Salinity effects on germination, growth, and seed production of the halophyte Cakile maritima. Plant Soil. 2004;262:179\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoshi S, Nath J, Singh AK, Pareek A, Joshi R. Ion transporters and their regulatory signal transduction mechanisms for salinity tolerance in plants. Physiol Plant. 2022;174:e13702.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhosh S, Mitra S, Paul A. Physiochemical Studies of Sodium Chloride on Mungbean (Vigna radiata L. Wilczek) and Its Possible Recovery with Spermine and Gibberellic Acid. Sci World J. 2015;2015:858016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChao YY, Hsueh IE. Insights into Physiological Mechanisms of Salt Stress Tolerance in Djulis (Chenopodium formosanum Koidz.) Sprouts. J Plant Biology. 2019;62:263\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHasanuzzaman M, Borhannuddin Bhuyan MHM, Anee TI, Parvin K, Nahar K, Al Mahmud J et al. Regulation of Ascorbate-Glutathione Pathway in Mitigating Oxidative Damage in Plants under Abiotic Stress. Antioxidants 2019, Vol 8, Page 384. 2019;8:384.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHadi MR, Karimi N. The role of calcium in plants\u0026rsquo; salt tolerance. J Plant Nutr. 2012;35:2037\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6112599/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6112599/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSalinity imposes significant physiological and biochemical challenges on plants, disrupting key processes such as germination, involving growth, and water balance. Under saline conditions, plants activate various defense mechanisms to mitigate salinity-induced damage. While many of these mechanisms are well-characterized in mature plants, their role during germination and early seed development remains largely unexplored. In this work, we studied four pre-commercial broccoli (\u003cem\u003eBrassica oleracea\u003c/em\u003e L. var. italica) cultivars previously selected for their enhanced salinity tolerance and compared to the high tolerant \u003cem\u003eEruca vesicaria subsp. sativa\u003c/em\u003e. The results provide insights into key mechanisms involved in salinity tolerance, including osmotic potential regulation, mineral homeostasis, and antioxidant enzymatic activity and ATP concentration. The ATP availability and utilization emerged as critical determinants of the stress response profiles of the seeds during germination. Notably, the BQ1 cultivar demonstrated the most efficient ATP utilization, probably enabling a broader, more sustained, and effective response under saline conditions. These findings highlight ATP as a crucial factor in salinity tolerance during early seeds development.\u003c/p\u003e","manuscriptTitle":"Comparative adaptations of high-tolerant species and broccoli cultivars to salinity stress during germination and early development stages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 14:33:04","doi":"10.21203/rs.3.rs-6112599/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-08T07:32:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-08T04:20:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-05T14:55:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337428566323704794296656842145881682999","date":"2025-03-24T10:43:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145545453190308593747571824353258684180","date":"2025-03-24T00:58:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147246932018463449692517711840263601005","date":"2025-03-03T09:13:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18045159997827332711749641127242759878","date":"2025-03-02T09:40:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-01T08:10:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-27T09:16:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-27T07:26:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-27T07:25:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-02-26T11:01:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a490dfbc-e29c-4494-a495-1b24c42e0414","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:00:47+00:00","versionOfRecord":{"articleIdentity":"rs-6112599","link":"https://doi.org/10.1186/s12870-025-06723-3","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-05-27 15:57:24","publishedOnDateReadable":"May 27th, 2025"},"versionCreatedAt":"2025-03-03 14:33:04","video":"","vorDoi":"10.1186/s12870-025-06723-3","vorDoiUrl":"https://doi.org/10.1186/s12870-025-06723-3","workflowStages":[]},"version":"v1","identity":"rs-6112599","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6112599","identity":"rs-6112599","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}