Magnesium Leaf Application as a Rapid Tool for Salt Stress Resistance in Faba Beans (Vicia faba L.)

TL;DR

This study shows that applying magnesium to the leaves of faba beans helps them resist salt stress by restoring ionic balance and improving plant health.

Contribution

The study reveals magnesium's critical role in ionic homeostasis under salt stress, beyond potassium's influence.

Findings

Mg2+ deficiency under salt stress caused a 14-fold increase in leaf Na+ concentration.

Foliar Mg2+ application reduced the toxic Na+/Mg2+ ratio by 90% and restored plant growth and gas exchange.

Mg2+ is a unique regulator of ion balance that cannot be replaced by K+ supplementation alone.

Abstract

Potassium (K+) is often the primary target for research on salinity stress. However, the role of magnesium (Mg2+) under salinity stress has not been properly investigated. We aimed to answer the following question: could magnesium (Mg2+), often neglected, be the real protector of ionic balance under salt stress? It is known that a deficiency in Mg2+ increases K+ uptake. Based on this understanding, we hypothesized that Mg2+ starvation could worsen salinity defenses compared to K+ starvation. The nutrient concentration of 0.02 mM Mg2+ was maintained in the nutrient solution to induce Mg2+ deficiency in Vicia faba plants. Mg2+ foliar application was carried out five times, at an interval of two times a week, over two weeks of induced salinity stress. Harvesting was carried out 45 days after transplanting, i.e., 2 weeks after salinity stress (50 mM NaCl) was initiated at 4 weeks of transplanting. Salinity had the highest impact on Mg2+-deficient plants, resulting in a major alteration in ionic composition. Mg2+ deficiency under salt stress resulted in a 14-fold increase in the leaf Na+ concentration compared to controls. Application of foliar Mg2+ was very effective in reversing the loss of Mg2+-deficient plants, renewing growth, gas exchange, and stomatal activity, and reducing the toxic Na+/Mg2+ ratio (by 90%). Mg2+ is a unique regulator of ion homeostasis in salinity stress, and its deficiency causes an imbalanced ionic medium that cannot be normalized through K+ supplementation alone. Therefore, the strategic method of selective foliar application of Mg2+ is a necessary approach for achieving high-quality yields in salt-affected croplands.

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Full text

1. Introduction

Salinity stress is one of the most widespread issues and poses a significant challenge to crop cultivation in most countries. It affects plant growth and reduces crop yields in various regions across the globe [1]. Salinity stress leads to physiological disturbances, such as osmotic stress [2], ion toxicity [3], and oxidative damage [4], which inhibit photosynthesis, nutrient homeostasis, and water regulation. Salinity is a significant threat to the faba bean (Vicia faba), endangering the cultivation of this crop in affected areas [5]. High amounts of sodium (Na^+^) in saline soils interfere with nutrient balance because they hinder the uptake of important nutrients such as potassium (K^+^) and magnesium (Mg^2+^). Mg^2+^ deficiency (MGD) is commonly caused by salinity stress; ions are replaced by soil colloids or there is competition between the roots in the uptake process, intensifying metabolic and structural dysfunctions [6]. These include macronutrients, especially Mg^2+^ and K^+^, which enable plants to endure stress conditions. Emerging evidence suggests an antagonism between K^+^ and Mg^2+^ under saline conditions [7]. Mg^2+^ plays a vital role in many physiological and biochemical pathways that are crucial for plant growth and development [8]. Foliar application of Mg^2+^ can adequately regulate chlorophyll content and promote positive growth [9]. Mg^2+^ foliar application (FA) has been demonstrated as an alternate option for root supply [10] during drought [8], salinity stress [11], and even different levels of K^+^ application [12], where abiotic stress coupled with nutrient antagonism may be reduced by leaf application effectively.

Our previous study identified salinity as one of the key threats to the faba beans. A deficiency in Mg^2+^ in the root zone interferes adversely with ion homeostasis compared to K^+^ deficiency. We found that Mg^2+^-deficient plants experience a significant increase in the leaf and root Na^+^/Mg^2+^ ratio, which is linked to retarded growth and impaired physiology [7]. This emphasizes the major influence of Mg^2+^, rather than K^+^, on Na^+^ accumulation and ionic balance during root uptake under saline conditions. This study identified an additional challenge, i.e., the antagonistic relationship between K^+^ and Mg^2+^, which inhibits Mg^2+^ acquisition through the roots, particularly in saline soils, where ion competition and functional impairment of the root increase Mg^2+^ deficiency. This limits Mg^2+^ fertilization under salinity conditions. FA of nutrients is an alternative approach that has the potential to avoid soil–root barriers and provide essential elements to shoots, bypassing the uptake limitations of saline substrates [11]. Although FA of Mg^2+^ has proved to be effective in reducing Mg^2+^ deficiency in crops like maize under optimal conditions [12], little is known about the influence of FA on Mg^2+^. This could be applied to the re-programming of ion homeostasis and physiological adaptation to the combined pressure of salinity and induced Mg^2+^ deficiency in plants such as faba beans. Thus, the main hypothesis of this study is that the FA of Mg^2+^ is an effective method for alleviating salinity stress in faba beans by directly providing adequate Mg^2+^ at the leaf level. Thus, ion homeostasis, photosynthetic activity, and the growth rate can improve regardless of the antagonistic soil–root uptake pathway. This work provides information on precision nutrient management practices under salinity by examining the efficacy and physiological consequences of foliar Mg^2+^ in agricultural landscapes affected by salinity.

2. Results

2.1. Biomass

Figure 1 represents the biomass of plants experiencing salinity stress under varied concentrations of Mg^2+^. The control group (+Mg+K) had the highest shoot fresh weight (SFW). MGD (−Mg+K) inhibited SFW by 51%, and this reduction was intensified to 65% when combined with salinity stress (−Mg+K+S). However, this effect was alleviated by FA of Mg^2+^, which resulted in a slight increase in SFW even under salinity stress.

The same effect was observed in root biomass. MGD alone led to a 67% reduction in the fresh weight of roots (RFWs), which increased to an 83% reduction when MGD was combined with salinity stress. In the case of dry weight, foliar Mg^2+^ application increased the shoot dry weight (SDW) by 31% compared to Mg^2+^-deficient plants subjected to salt stress. The root dry weight (RDW) decreased by 67% and 84% during MGD and salinity stress, respectively (Figure 1 and Figure 2). Importantly, foliar Mg^2+^ application was able to restore RDW to slightly higher than that of MGD plants under salinity stress (−Mg+K+S).

Plants cultivated with adequate Mg^2+^ levels (+Mg+K) had a low and consistent root–shoot (R:S) ratio. This ratio reflects a balanced weight distribution between the roots and shoots in favorable non-stressful nutritional conditions. Comparatively, MGD (−Mg+K) caused an excessive redistribution of biomass, increasing the R:S ratio more than twice. Salt stress in MGD plants (−Mg+K+S) resulted in a much lower R:S ratio than the treatment without Mg^2+^ (−Mg+K). Salinity also acted against the allocation of biomass to the roots induced by MGD. FA of Mg^2+^ (−Mg+K+S+FA) did not cause a significant difference in the R:S ratio for non-treated plants subjected to stress under the same conditions. The treatment (−Mg+K+S+FA) was statistically insignificant compared to −Mg+K+S plants but remained significantly higher than the control (+Mg+K) (Figure 1C).

2.2. SPAD Index, Gas Exchange, Osmolytes, and Stomatal Movements

Limited Mg^2+^ in faba bean plants resulted in interveinal chlorosis on old leaves three weeks after transplanting (WAT). In line with these visual symptoms, there was a 22% reduction in chlorophyll content in MGD plants compared to the control. MGD under salinity (−Mg+K+S) resulted in the most severe chlorosis, as shown in Figure 3A and Figure 4a. A further 50% reduction was observed compared to the control. FA of Mg^2+^ resulted in a major recovery in terms of chlorophyll concentration. Gas exchange measurements revealed significant differences in net photosynthesis and transpiration rate measured in the fourth leaf across the treatments (Figure 4b,c). MGD coupled with salinity (−Mg+K+S) experienced 47% and 57% decreases in the net assimilation rate and transpiration rate, respectively. The rate of transpiration reached near zero during saline conditions, whereas the FA of Mg^2+^ restored both the net assimilation and transpiration rates.

The concentration of leaf sucrose increased by 63% in MGD under salinity (−Mg+K+S). The content of sucrose decreased upon the FA of Mg^2+^. In comparison to the control (+Mg+K), MGD reduced malate by 37% and 68% in −Mg+K and −Mg+K+S, respectively. FA of Mg^2+^ also led to a significant increase in the malate content of the leaves (Figure 5b). In agreement with reduced rates of transpiration, stomatal apertures of plants after two weeks of salinity stress (−Mg+K+S) under light conditions showed stomatal apertures that were reduced in size compared to the control plants (Figure 6). Regulation of stomatal pore size was also different in treatment groups since the salt stress at various levels of Mg^2+^ and K^+^ decreased the stomatal pore size. The plants that received FA of Mg^2+^ (−Mg+K+S+FA) had large stomatal apertures, as opposed to the plants to which salinity stress was applied (−Mg+K+S). Salinity severely suppressed aperture widths in the case of the opening stimulus under light conditions by 76% in −Mg+K+S plants, and the FA of Mg^2+^ increased the opening of stomata. Stomatal sensitivity to the dark-enforced closure was also higher in the −Mg+K+S group, which showed a 45% more significant aperture width reduction compared to the control (Figure 6).

2.3. Ion Homeostasis and Nutrient Interactions

MGD coupled with salinity stress (−Mg+K+S) resulted in a significant reduction in K^+^ (by 32%). FA of Mg^2+^ also led to a reduction in the uptake of K^+^ (by 43%) (Figure 7a). Plants resupplied with foliar Mg^2+^ had nearly the same concentration of Mg^2+^ as that of the control plants. Ca^2+^ content significantly declined in MGD plants. Conversely, Ca^2+^ uptake was found to be greater under salinity stress than in the negative control (−Mg+K). FA of Mg^2+^ also led to an increase in the uptake of Ca^2+^ (Figure 7c).

Na^+^ and Cl^−^ ions in leaves were enhanced by the moderate level of salinity stress induced (50 mM NaCl). Na^+^ and Cl^−^ ions in the leaves were significantly high in −Mg+K+S plants. FA of Mg^2+^ (−Mg+K+S+FA) resulted in a reduction in Na^+^ and Cl^−^ ions (Figure 7d,e). The ratio of Na^+^/K^+^ was observed to be high under MGD and salinity stress (−Mg+K+S). The antagonistic effect of Mg^2+^ and K^+^ was also observed as the FA of Mg^2+^ decreased this ratio by 14%. The Na^+^/Mg^2+^ ratio was observed to be greater in MGD plants under salinity stress (−Mg+K+S). This antagonistic interaction between K^+^ and Mg^2+^ considerably increased the ratio of Na^+^/Mg^2+^; the K^+^/Mg^2+^ ratio was similarly impacted, which implies the role of Mg^2+^ in maintaining ion homeostasis (Figure 8).

3. Discussion

Our results reveal that the FA of Mg^2+^ is an effective intervention that can reverse the ionic imbalance caused by salinity stress and MGD. Salt stress causes extreme damage to the photosynthetic machinery of the faba bean, mostly through the degradation of chlorophyll under ion toxicity. This confirms the fact that chlorophyll is a direct deleterious factor, as it causes ionic imbalance in the chloroplast. The loss of chlorophyll resulted in a multitude of impaired downstream physiological processes in this study, including a 50% reduction in chlorophyll concentration (Figure 4a), 47% reduction in the net assimilation rate (NAR) (Figure 4b), a 57% reduction in transpiration rate (E) (Figure 4c), and an inhibition of biomass accumulation (65% SFW and 83% RFW) (Figure 1A,B). This aligns with the consequences of Mg^2+^ deficiency reported in maize, including a drastic reduction in the protein content of RuBisCO, the most important protein in photosynthesis [13]. The known sequence of events, whereby Na^+^/Cl^−^ accumulation interferes with the synthesis of chlorophyll and stomatal closure due to the dysfunction of guard cell osmotic gradients, restricting the diffusion of CO_2_, agrees with these known deficits. Instant recovery of CO_2_ assimilation in the fourth leaf was observed when foliar Mg^2+^ was applied, which contrasts the SPAD values and agrees with the findings in [9,13,14]. Notably, this is a phenomenon of foliar Mg^2+^ supply in maize, which was uniquely efficient in restoring contents of RuBisCO proteins and, by extension, the photosynthetic apparatus, compared to nutrients provided via the root [13]. The accumulation of sucrose (Figure 5a) in the presence of MGD signifies a two-fold reaction: it is a competent compatible solute to osmotic regulation, and, presumably, it suggests the malfunctioning of phloem loading and translocation under MGD [15].

Although foliar Mg^2+^ application significantly reversed ionic disturbances and rejuvenated photosynthetic gas exchange in Mg^2+^-deficient, salt-stressed plants (−Mg+K+S+FA), shoot and root biomass did not match that of the control (+Mg+K). This is not only informative but also expected. After four weeks of Mg^2+^ deficiency, salinity stress was initiated, and, by this time, the plants had already suffered severe chlorosis, near-zero transpiration, and ceased growth. The purpose of foliar Mg^2+^ was to prevent this degenerative process and re-initiate metabolism based on the restoration of physiological parameters and ion homeostasis. However, it was incapable of compensating for the irreversible loss of biomass experienced during the initial stress period. The recovery of biomass was only partial, which indicates a shortcoming in the foliar strategy itself; on the other hand, it highlights the critical importance of timely intervention.

While salinity causes the stomata to close due to the imbalance of ion concentrations (as shown by 76% reductions in the aperture in −Mg+K+S plants), application of foliar Mg^2+^ during salinity stress (−Mg+K+S+FA) allowed the apertures to remain open (Figure 6ii(d)). This paradox results from the known accumulation of sucrose solutes and other organic cytosols in the leaves, which replace inorganic ions during the process of turgor generation in guard cells [16,17]. A reduction in the amount of sucrose occurs after feeding foliar Mg^2+^, which indicates a critical change. Mg^2+^ facilitates the movement of this stored carbon [18], as observed by the reduced levels of sucrose in the leaves. Instead of being trapped in osmotic functions, sucrose is used as a source of energy or transported to stimulate growth, repair mechanisms, and improve the overall adaptation of the plant to salinity stress. The recovery of malate levels due to foliar Mg^2+^ application is mechanistically important (Figure 5b). Mg^2+^ is a divalent cation needed for PEP carboxylase, which plays a central role in the production of organic anions such as malate. It is proposed that recovery of malate levels in response to foliar application of Mg^2+^ is due to re-energization of the metabolic pathway. This recovery is necessary to facilitate Na^+^ detoxification, possibly via Mg^2+^, which is used as a cofactor in PEP carboxylase (Figure 9).

Importantly, foliar Mg^2+^ treatment (−Mg+K+S+FA) stimulated a phenomenal 10-fold decrease in Na^+^ uptake by the leaves (Figure 7d), thus restoring ion homeostasis. The steep decrease in Na^+^ accumulation in the leaves may be attributed to the fact that Mg^2+^ is a known ion channel modulator; Mg^2+^ has been reported to block Na^+^ channels as a voltage-dependent channel blocker. FA of Mg^2+^ (−Mg+K+S+FA) acted as a master regulator to inhibit the harmful Na^+^/Mg^+^ ratio. Equally, the K^+^/Mg^2+^ ratio increased in response to MGD and decreased greatly when Mg^2+^ was applied to the leaves (Figure 8). This antagonism between the two cations has been reported in maize and oat [19,20]. The cationic homeostatic effect of Mg^2+^ is similar to the effects of Mg^2+^ in acidic soils, where foliar Mg^2+^ reduced Al^3+^ toxicity in wheat [21], suggesting a potentially conserved Mg^2+^ homeostatic process.

Na^+^ at high concentrations competitively blocks Mg^2+^ absorption by replacing Mg^2+^ in membrane transport sites. The amount of Mg^2+^ in the leaves exceeds the capacity of the cell to sequester it. It is this loss of cellular control that results in ionic imbalance and salt toxicity [6]. The initial defense is Na^+^ exclusion and regulation, which prevent excessive Na^+^ entry in the plant in the first place. Mg^2+^ also plays an important role by indirectly controlling this phenomenon. SOS1 transports Na^+^ out of the cells and back into the soil with the energy of the proton gradient (H^+^) provided by H^+^-ATPases. This is one of the major mechanisms of excluding Na^+^. Na^+^ efflux and K^+^ retention are known to be observed with the reactivation of proton pumps, e.g., H^+^-ATPases, the activity of which is known to be dependent on Mg-ATP [22,23,24]. We therefore propose that foliar Mg^2+^ enhances the proton force required to drive Na^+^ exclusion by SOS1 [25]. Research on sugar beet provides information on this regulation; foliar Mg^2+^ has been found to regulate plasma membrane H^+^ dynamics by reducing active ATP-based H^+^ pumping and simultaneously increasing passive H^+^ efflux [26]. This rebalancing of proton export can maintain the apoplastic acidification required to promote SOS1 activity and even conserve cellular energy under stress.

The dramatic rise in the Mg^2+^ content in the leaves upon FA of Mg^2+^ even under salinity stress conditions suggests that the uptake of Mg^2+^ is a priority for plant survival (Figure 7b). This vital cation is provided directly by FA. Mg^2+^ also competes with Na^+^ for binding sites on membranes and transporters, which can decrease Na^+^ influx. Mg^2+^ has a concentration-sensitive regulatory action in Na^+^/Ca^2+^, and Na^+^/K^+^ exchange pathways, thereby inducing the salt release pathway of SOS [27,28,29]. It increases the activity of H^+^ pumps at low concentrations and inhibits the given transport system at higher concentrations. Moreover, sufficient cytosolic Mg^2+^ is essential in the preservation of the negative membrane potential, which is an important barrier against the entry of Na^+^ [30].

4. Materials and Methods

4.1. Plant Material and Cultivation Conditions

Faba bean plants were grown hydroponically to study the effects of magnesium and salt stress. The research was carried out at the Institute of Plant Nutrition and Soil Science, University of Kiel, Germany. Seedlings were placed in 10 L plastic containers, with four plants per pot. The nutrient solution was at full strength by day 3, increasing from one-quarter of the solution. Growth conditions were 20/16 °C day/night temperature and a photosynthetically active radiation (PAR) level of 250 µmol m^−2^ s^−1^. The nutrient solution was as follows: 2.0 mM Ca(NO_3_)2, 2.0 mM K_2_SO_4_, 0.1 mM NH_4_H_2_PO_4_, 0.2 mM KCl, 0.5 mM MgSO_4_, 10 µM H_3_BO_3_, 2.0 µM MnSO_4_, 0.5 µM ZnSO_4_, 0.2 µM CuSO_4_, 0.05 µM (NH_4_)6_Mo_7_O_24, and 60 µM Fe-EDTA. Mg^2+^-deficient treatments contained 0.02 mM MgSO_4_·7H_2_O. All the chemicals were purchased from Merck (Darmstadt, Germany) unless otherwise stated. Four distinct treatments were applied: +Mg+K (0.5 mM MgSO_4_ × 2 mM K_2_SO_4_); −Mg+K (0.02 mM MgSO_4_ × 2 mM K_2_SO_4_); −Mg+K+S (0.02 mM MgSO_4_ × 2 mM K_2_SO_4_ × 50 mM NaCl); and −Mg+K+S+FA (0.02 mM MgSO_4_ × 2 mM K_2_SO_4_ × 50 mM NaCl × 250 mM MgSO_4_).

The foliar MgSO_4_ concentration (250 mM) was many-fold greater than the level of sufficiency in the root zone (0.5 mM). This is normal regarding foliar nutrition uptake dynamics. Root systems are very effective at extracting nutrients in an intermittent, diluted solution. The foliar uptake, on the other hand, is restricted by the nonporous cuticle of the leaf; a high concentration is needed to generate a sufficient diffusion gradient upon the entry of Mg^2+^ during the short period of droplet drying. Foliar sprays were implemented five times across two weeks of stress. Each of the four treatments was replicated four times. Salt stress was induced 4 weeks after transplanting; harvesting was performed at 45 DAT, which equates to 2 weeks of salinity stress. The fresh weight (FW) of the separate shoot and root tissues was noted immediately after harvest. Dry weight (DW) was determined by placing plant material in a forced-air drying oven at a temperature of 70 °C for a total of 72 h or until a constant mass was attained. The root-to-shoot ratio (R:S) was then calculated for each biological replicate in the following way:

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4.2. Analysis of Ion Content in Leaf Tissue

To analyze the elements, the dried leaf samples were first ground into a homogeneous powder. Each of the powdered samples (200 mg) was then subjected to microwave-assisted acid digestion in 10 mL of 69% nitric acid at 1800 W. The reagents were purchased from Merck (Darmstadt, Germany). The heating of the digestion was programmed through a stage-by-stage increase in temperature: the initial increase was made to 100 °C and held for 2 min; it was then increased to 120 °C and held for 1 min; and finally increased to 180 °C and maintained for 20 min before cooling for 20 min. The samples were digested and subsequently diluted with deionized water (18.2 MΩ cm), brought to a final volume of 100 mL, and maintained at 4 °C. The concentrations of different ions in the prepared solutions were then determined using the inductively coupled plasma optical emission spectrometry (ICP-OES, Optima 3000, PerkinElma, Waltham, MA, USA) [7].

4.3. Measurement of Sucrose, Chloride, and Malate

To analyze the sucrose and organic compounds, 20 mg of the pulverized leaf samples was extracted in aqueous solution as follows [31]. The powder was dried at 100 °C for 5 min in 1 mL of deionized water. The samples were centrifuged after cooling on ice (12,000 rpm, 10 min). Afterward, the aqueous supernatant was subjected to purification. It was diluted 10-fold, combined with chloroform, and used to remove other substances that interfere with the purity. Another round of centrifugation (12,000 rpm, 5 min, 4 °C) was performed, and finally, solid-phase extraction was carried out using C18 columns. All analytes were quantified using an ion chromatography Dionex ICS-5000 system (Thermo Scientific, Waltham, MA, USA).

4.4. Assessment of Stomatal Behavior

To investigate stomatal dynamics, leaf epidermal peels from the abaxial surface of six-week-old plants were used. The experimental design included separate procedures to track opening and closure. The opening protocol began with pre-dawn collection of peels, which were then submerged in a buffer (10 mM Mes-KOH, 10 mM KCl, 50 µM CaCl_2_, and pH 6.5). These samples were kept in darkness for 2 h before being transferred to light (250 µmol m^−2^ s^−1^) for an additional 2 h at 22 °C to stimulate opening. The closure protocol reversed this sequence: peels were first exposed to light for 2 h to ensure stomata were open, and then their closure was recorded over the next 2 h. For all samples, we captured images with a Zeiss Axioskop microscope and determined the stomatal aperture (internal pore width) using ImageJ Fiji version v1.54f. The sample size consisted of four plants per treatment (one leaf each), with measurements taken from no fewer than 40 individual stomata [32,33].

4.5. Data Analysis

Data were analyzed using STATISTIX 10 software version 10.1. Differences in treatments were evaluated by one-way ANOVA followed by Tukey’s HSD test. Results p ≤ 0.05 were considered statistically significant. Figure 9 was created using Figure Labs (https://www.figurelabs.ai).

5. Conclusions

This study is informative as it tests the recovery of plant performance after reductions in Mg^2+^ levels under salt stress. The foliar application of Mg^2+^ was assessed, which has hitherto not been studied. The effects of Mg^2+^ foliar application on salt-sensitive crops such as faba beans have also not been investigated under conditions of salinity stress. Based on this, foliar application of magnesium has become an extremely important intervention to prevent the effects of salinity stress that undermine the ability of roots to take up magnesium. Foliar application follows the path of diffusion through stomata or the cuticle into the apoplastic space of the plant and into the cytosol of the mesophyll cells. This resulted in improved photosynthesis and carbohydrate synthesis, uptake, and transport of important nutrients such as potassium (K^+^) and calcium (Ca^2+^), as well as decreased toxicity of sodium (Na^+^) during salinity stress. Mg^2+^, a phloem-mobile nutrient, was readily transported across the growing plant sections, and this contributed to the conservation of the Na^+^/Mg^2+^ ratio, which is a novel physiological characteristic of salinity tolerance in faba bean plants.