Silicon Supplied via Root or Leaf Relieves Potassium Deciency Effects in Common Bean

Potassium (K) deciency affects physiological performance and decreasing vegetative growth in common bean plants. However, silicon (Si) supplied via nutrient solution or foliar application may relieve nutritional stress. Thus, two experiments were carried out: initially, a test was performed to determine the best source and concentration of leaf-applied Si. Subsequently, the chosen Si source was applied via nutrient solution or via leaf to verify if it is ecient in alleviating the effects caused by K deciency. To that end, a completely randomized 2 x 3 factorial design was used, with two levels of K: decient (0.2 mmol L − 1 of K) and sucient (6 mmol L − 1 of K); and Si: via nutrient solution (2 mmol L − 1 of Si) or foliar spray (5.4 mmol L − 1 of Si) and control (0 mmol L − 1 of Si). In the rst experiment, foliar spraying with sodium silicate and stabilized potassium at a concentration of 5.4 mmol L − 1 was better in favoring the physiology of bean plants. In the second experiment, K deciency without the addition of Si compromised the plant's growth. Si applied through nutrient solution or foliar spray relieved K deciency stress, increasing chlorophylls and carotenoids content, photosynthetic activity, water use eciency and vegetative growth.


Introduction
Potassium de ciency in bean plants (Phaseolus vulgaris L.) is common worldwide, inducing chlorosis on the edges of older leaves, evolving to necrosis [1]. At this stage, the increase in reactive oxygen species degrades chlorophyll, decreasing photosynthesis and increasing transpiration, with ine cient water use [1,2,3].
However, information about the relationship between Si and K de ciency stress in bean plants is nonexistent. A number of studies on other species indicate that supplying Si nutrient solution alleviates K de ciency in soybean [5], sorghum [2] and barley crops [7], but there are no reports on Si leaf spraying and this nutritional disorder in any species. Indeed, doubts remain regarding the best Si source and concentration for leaf spraying in bean plants.
As such, a number of questions must be answered. First and foremost is whether Si leaf spraying in bean plants is agronomically feasible depending on the source and concentration of the element. The hypothesis is that supplying Si alleviates K de ciency due to increase chlorophyll content, photosynthesis and water use e ciency of the bean plant. If so, mitigating K de ciency is more evident with the supply of Si via nutrient solution (roots) compared to foliar applications, although leaf spraying can also reduce de ciency in the plant.
The aim of this study was to determine the best Si source and concentration for foliar application, and whether supplying this source via nutrient solution is e cient in attenuating K de ciency stress in bean Page 3/20 plants.

Results
Leaf-applied Si and its effect on bean plants.
The rise in leaf-applied Si concentration increased accumulation of the element, total chlorophyll content, quantum e ciency of photosystem II and shoot dry matter of bean plants, irrespective of the source used ( Fig. 1a, b, c, d).
The leaf-applied Si concentrations in the form of SiK and SiNaK sources that resulted in the maximum Si accumulation, total chlorophyll content, quantum e ciency of photosystem II and dry matter were 8.93 and 8.57; 10.36 and 10.36; 8.57 and 9.64; and 9.29 and 8.57 mmol L − 1 of Si, respectively (Fig. 1).
Based on the results, SiNaK was similar to SiK in terms of increasing the element in the plant, but produced better results in total chlorophyll content and quantum e ciency of photosystem II, albeit not enough to affect dry matter (Fig. 1). The use of this source at I've always treated of L − 1 was associated with 90% of maximum dry matter production, a feasible option for leaf spraying bean plants.
Potassium and silicon.
Cultivating bean plants in a K-de cient nutrient solution resulted in less nutrient accumulation, regardless of Si treatments and control (-Si) (Fig. 2a). Only applying SiRO treatment in bean plants grown under K de ciency increased K accumulation compared to controls (Fig. 2a). In K-de cient plants, SiRO and SiLE treatment in relation to controls raised the e ciency of the macronutrient (Fig. 2b). This indicates the bene cial effect of Si in improving K absorption and Si use e ciency, contributing to alleviating nutritional stress in the bean plant.
Potassium-de cient beans plants when compared to K-su cient plants shows decreased Si accumulation only when the element was root supplied (Fig. 2c). The use of Si favored its accumulation in bean plants with and without K de ciency, particularly when the element was supplied via SiRO compared to SiLE (Fig. 2c).
Potassium de ciency in relation to su ciency decreased total chlorophyll and carotenoid content in control plants (-Si) and those that received leaf-applied Si (Fig. 3a, b). However, in K-de cient plants, SiRO treatment compared to the treatments SiLE and controls (-Si) resulted in higher total chlorophyll and carotenoid content (Fig. 3a, b). In plants with su cient K levels only SiRO treatment increased total chlorophyll and carotenoid content (Fig. 3a, b).
Bean plants cultivated in K-de cient nutrient solution exhibited chlorosis followed by necrosis on the edges of the oldest leaves. It was visually evident that this system was alleviated by supplying Si, especially in SiRO treatment (Fig. 3c).
Plants stressed by K de ciency increased electrolyte leakage (Fig. 4a), reduced photosynthesis rates ( Fig. 4b), raised transpiration rates (Fig. 4c) and lowered relative water content (Fig. 4d) and water use e ciency (Fig. 4e) compared to plants with su cient K levels. However, supplying Si, especially with SiRO treatment alleviated stress in K-de cient plants, since it raised photosynthesis, relative water content, and water use e ciency, in addition to minimizing transpiration rates and electrolyte leakage ( Fig. 4a, b, c, d, e).
However, K-de cient plants supplied with Si by the two application methods obtained an increase in leaf area (Fig. 5a), root length (Fig. 5b), root density (Fig. 5c), and root area (Fig. 5d), as well as shoot (Fig. 5e) and root dry matter (Fig. 5f), highlighting root over leaf application. On the other hand, in plants with su cient K levels, SiRO or SiLE only increased root density and root dry matter. These results were also obtained from photographic records (Fig. 6)

Discussion
Leaf-applied Si was agronomically feasible due to its increase in the plant, total chlorophyll content, QEPS II and consequent rise in dry matter production, irrespective of the source of the element (Fig. 1). A similar result was observed in bean plants by [8] when Si was supplied via leaves at concentrations of 2 and 4 ml L − 1 . Additionally, the two leaf-applied Si sources were e cient in raising accumulation of the element for the bean crop, as also veri ed in research by Jafarei et al. [9] who applied 3.6 g L − 1 of Si via leaf of bean plants.
The SiNaK source was better at raising total chlorophyll content and the QEPS II when compared to SiK, although not su cient to affect dry matter. This Si source stands out for exhibiting sorbitol in its composition, which provides greater stability in the solution, reducing the polymerization process of the element [10] on the leaf surface.
Bean plants cultivated in K-de cient nutrient solution (0.2 mmol L − 1 of K) showed lower accumulation of the nutrient when compared to plants with su cient levels, indicating the occurrence of nutritional stress ( Fig. 2a). However, when these K-de cient plants received Si, especially in SiRO treatment, they exhibited an increase in accumulated K compared to controls (-Si). This may have occurred because Si stimulated H + -ATPase activity, enzymes directly linked to K absorption by the plants [11]. This increased K accumulation in de cient plants was observed in soybean supplied with Si via nutrient solution [5]. Si supply to K-de cient plants, especially through the roots, enhanced K use e ciency by the bean plant compared to controls (Fig. 2b), due to the ability of Si to increase absorption of the element (Fig. 2a) and the physiological processes associated with biomass production.
Bean plants grown in potassium de ciency compared to su ciency showed reduced Si accumulation only in SiRO treatment (Fig. 2c). As such, K de ciency induced less Si accumulation even in plants that received the element via nutrient solution, a nding also reported for barley plants [7].
There was greater Si accumulation in bean plants when the element was supplied via SiRO compared to SiLE, irrespective of K supply (Fig. 2c). This occurred since the Si supplied in the nutrient solution makes the element available throughout the crop cycle, whereas leaf application was only performed in four stages. Leaf-applied Si promoted increased accumulation of this element in relation to controls (-Si), in plants with and without K de ciency (Fig. 2c). This indicates that leaf spraying raised Si absorption in the bean plants, a result also found in bean [9,12] and okra plants [13].
The K de ciency reduced total chlorophyll and carotenoid content in relation to su ciency of the macronutrient only in controls (-Si) and in SiLE treatment (Fig. 3a, b). The cultivation of plants with low K content in the nutrient solution decreased absorption of the element, causing a decline in total chlorophyll content (Fig. 2a), a nding also reported for sorghum [2]. This occurs because of the lack of this nutrient causes oxidative stress, given the increase in reactive oxygen species and putrescine content, a compound that, when in high concentrations, becomes toxic to plants [1,2].Thus, K de ciency resulted in chlorosis and necrosis on the edges of the oldest leaves ( Fig. 3c), as previously reported by Prado [1].
In K-de cient plants, both Si supply methods favored an increase in total chlorophyll and carotenoid content compared to controls (-Si), especially in SiRO treatment (Fig. 3a, b), a difference that is visibly apparent (Fig. 3c). The bene cial effect of Si supplied via nutrient solution in K-de cient plants on increasing chlorophyll content has also been reported for other species such as sorghum [2] and barley [7].
Potassium de ciency in bean plants, with no Si addition, increased electrolyte leakage in relation to plants with su cient levels (Fig. 4a). This occurred because K de ciency decreased intracellular pH, raising amine oxidase activity and stimulating reactive oxygen species accumulation, which oxidizes cell membrane compounds [2]. However, both Si application methods exhibited less electrolyte leakage in Kde cient plants than in controls (-Si) (Fig. 4a). This nding is corroborated Miao et al. by [5] in soybean plants that received Si via nutrient solution. This bene cial effect of Si in reducing electrolyte leakage occurred because the element induces greater plasma membrane protection [6], possibly since it increased carotenoid content (Fig. 3b). Carotenoid is a non-enzymatic antioxidant that eliminates singlet oxygen ( 1 O 2 ), especially toxic oxygen reactive species, which leads to lipid peroxidation, resulting in a loss of cell electrolytes [14,15] and lipid bilayer membrane stability [16].
Plants stressed by K de ciency decreased photosynthesis rates only in control plants (-Si) (Fig. 4b). This effect is due to the fact that K de ciency decreased total chlorophyll content (Fig. 3a) and raised electrolyte leakage (Fig. 4a), a nding also reported by other authors in sorghum plants [2,3].
The beans plants grown in K-de cient that received Si experienced a rise in photosynthesis rate in relation to controls (-Si), highlighting application of the SiRO (Fig. 4b), as observed in sorghum plants [2]. This effect is due to the fact that SiRO treatment increased K accumulation (Fig. 2a), as well as total chlorophyll (Fig. 3a) and carotenoid content (Fig. 3b).
Potassium de ciency without adding Si increased leaf transpiration rate (Fig. 4c) and decreased control plant (-Si) relative water content (Fig. 4d), since this nutrient regulates osmosis in the plant [1,3].
SiRO or SiLE treatment decreased foliar transpiration (Fig. 4c) and increased relative leaf water content ( Fig. 4d) only in K-de cient plants in relation to controls (-Si). The bene cial effect of Si on plant relative water content was reported in K-de cient sorghum [3]. This effect is due to the formation of a silica gel layer that links cellulose to epidermal cells, minimizing water loss [6], as well as the increase in aquaporin activity, a protein associated with enhancing water transport in the plant [3].
K-de ciency also decreased water use e ciency in control plants (-Si) (Fig. 4e). This is because de ciency reduces photosynthesis (Fig. 4b) and raises the leaf transpiration rate (Fig. 4c), resulting in low water use e ciency, a fact reported for other crops such as sorghum [3] and cotton [17].
However, both Si application methods increased the e ciency of water use in K-de cient plants, particularly SiRO treatment (Fig. 4e). This bene cial effect of Si in raising water use e ciency is due to the increase in photosynthesis (Fig. 4b) and decline in transpiration rate (Fig. 4c).
K-de ciency caused a decrease in plant growth (Fig. 5a, b, c, d, f and Fig. 6). This has been widely reported in the literature, given the functions of K in plants [1], where de ciency compromises biological variables, as previously mentioned. On the other hand, both Si application methods increased plant growth variables, especially via SiRO (Fig. 5a, b, c, d, f). A similar result of increased dry matter in Kde cient plants submitted to Si application via nutrient solution was obtained in other species such as soybean [5] and sorghum [2,3].
The bene t of Si in alleviating K de ciency can be explained by the different nutritional and physiological improvements, initiating with an increase in K accumulation (Fig. 2a) and another associated with the antioxidant action of the plant, evidenced by the rise in total chlorophyll (Fig. 3a) and carotenoid antioxidant compound content (Fig. 3b) due to the decline in electrolyte leakage (Fig. 4a), favoring the photosynthesis rate (Fig. 4b). Si also maintained water in the plant, given the decrease in transpiration rate (Fig. 4c), which favored a rise in water content (Fig. 4d) and, in turn, water use e ciency (Fig. 4e). Thus, the plant physiology and nutrition improvement with the Si supply to the K de cient plant increased the e ciency of the macronutrient use to convert it in biomass (Fig. 2b), hence in the plant growth. The bene cial effects of Si applied via root on physiology and growth in K-de cient plants were also reported [2,3,5].
It is important to underscore that SiLE improved the growth variables of K-de cient plants when compared to controls (-Si). This may be due to the effect of leaf Si in relation to controls (-Si) in raising total chlorophyll and carotenoid content as well as photosynthesis and water use e ciency. The bene ts of leaf-applied Si in alleviating K de ciency in bean plants have not been reported in the literature, since existing studies supplied the element only via nutrient solution. The present study demonstrates the mitigating effect of Si on K de ciency, especially supplied via nutrient solution, but leaf application is a feasible alternative in bean plants.
Finally, in K-su cient plants, SiRO or SiLE treatments had little effect on plant growth, since only root density and dry matter increased. As such, the present study showed that the most important role of Si is when plants are under nutritional stress in relation to those with su cient levels.

Materials And Methods
Local and growing conditions. Two experiments were conducted in a hydroponic growing system in the greenhouse at the School of Agricultural and Veterinarian Sciences (UNESP), Jaboticabal, Brazil.

Seeds of common beans (cv. BRS Estilo) were obtained from the Brazilian Agricultural Research
Corporation of the Ministry of Agriculture, Livestock and Food Supply, Brazil.
The use of plant parts in the present study complies with international, national, and/or institutional guidelines. This research was not conducted with endangered species and was conducted in accordance with the is in accordance with the Declaration of IUCN Policy on Research Involving Endangered Species.
The rst experiment was carried out to obtain the best Si concentration and source for Si leaf spraying, which occurred between August and the end of the crop cycle, which lasted 115 DAE (days after emergence). Based on results of the rst experiment, a second experiment was conducted to evaluate the effect of Si on the physiology and dry matter yield of common beans plants under K de ciency, starting in December and maintained until the emergence of K de ciency symptoms, corresponding to the phenological stage R5 (28 DAE).
The relative air humidity and maximum and minimum temperature were recorded throughout the experimental period. There was a high variation in the average relative humidity (34.39 ± 9% | 32.79 ± 8%), minimum temperature (17.9 ± 7°C | 19.47 ± 5°C) and maximum temperature (44.8 ± 8°C | 38.56 ± 7°C) to the rst and second experiment respectively. High temperatures may have induced plants to possible stresses, considering that the average temperature for optimum beans crop growth is between 18 and Growing conditions.
For the rst experiment, the seeds were sown in a trays. Then the seedlings at ve DAE were transplanted to 7 dm³ polypropylene pots (upper diameter: 16 cm; lower diameter: 11cm; height: 33 cm), lled with 6 dm³ medium texture sand, previously washed with water, 1% HCl solution and deionized water, maintaining two plants per pots. These were irrigated daily with nutritive solution applied in order to maintain 70% water-holding capacity in the substrate.
For the second experiment, the seeds were also sown in trays, and the seedlings at ve DAE were transplanted to polypropylene pots (length: 44 cm; width: 19 cm and height: 14 cm, with capacity for 10 liters), also lled with the nutritive solution.
The nutrient solution used in both experiments was proposed by Hoagland and Arnon [19]. The solution concentration during the rst and second week of the growing season was maintained at 10 and 25%, respectively. From the third week until the end of the experiments, the concentration was raised to 50%.
The pH value of nutritive solution was maintained between 5.5 and 6.5, adjusted using NaOH (1 mmol L − 1 ) and HCl (1 mmol L − 1 ) solution. In the second experiment, the hydroponic solution was modi ed with different levels of K, as per the treatment (Table 1), and renewed every week to replace the water, Si and nutrients absorbed by the plants. Table 1 Amount of K provided and adjustment between control (-Si), Si via roots (SiRO) and Si via leaf spraying (SiLE) treatments in the second experiment. Si application and K adjustment.
For the rst experiment, foliar Si applications (SiNaK and SiK) were performed in three stages of development: V4 (emergence of the 3rd trifoliate leaf), R6 ( owering -opening of the rst ower) and R7 (pod formation). The volume of the solution applied varied according to plant size and 8, 16 and 24 ml of the solution were sprayed in stages V4, R6 and R7 respectively.
For the second experiment, SiNaK was applied as a Si source. In the SiRO treatment, the Si supply via root was performed in a nutrient solution throughout the experiment.
To perform the foliar application in the second experiment (SiLE treatment), a solution with a concentration of 5.4 mmol L − 1 of Si (SiNaK) was made and, then, the application to the leaves was carried out manually. The volume of Si solution applied increased according to plant size, with 0.56; 0.84; 1.12 and 1.40 ml of the silicate solution per plant for the rst, second, third, and fourth spraying, respectively, at 8, 13, 18 and 23 DAE.
The solutions used for leaf spraying in both experiments were adjusted with a solution of NaOH and HCl to maintain a pH of 6.0 ± 0.2. Silicon was applied to the leaves immediately after solution preparation.
The SiNaK and SiK sources contains K in its composition, after Si sprayings, foliar applications were performed with potassium chloride (KCl) to balance the K of the treatments. In the second experiment, the K provided by the SiNaK source was also adjusted for the root supply (Table 1).
It is important to highlight that 0.7 mmol L − 1 of K from SiNaK does not meet the demand of 6 mmol L − 1 suggested by Hoagland and Arnon [19] to supply K to plants, and nutrient de ciency of this nutrient is expected.
Temperature ( o C) and relative humidity (%) in both experiments were measured during the foliar applications, obtaining values between 9 and 22 o C and 60 and 80% respectively.
Plant analysis.
In the rst experiment, assessments were conducted in stage R7, and at twenty ve DAE for the second experiment, both in the upper third of the trifoliate leaf.
Quantum e ciency of photosystem II and Gas exchange parameters. In the rst experiment, the quantum e ciency of photosystem II (QEPII) was measured with a uorimeter (Opti-Science®-Os30P+).
In the second experiment, Gas exchange parameters were determined between 9:00-11:00 a.m, using four replicates for each treatment. Total chlorophyll and carotenoid content. Total chlorophyll (a + b) and carotenoid content in the rst and second experiment were measured by an absorbance spectrophotometer at 663 nm for chlorophyll a, 647 nm for chlorophyll b, and 470 nm for carotenoids. Pigments concentrations were determined following the methodology of Lichtenthaler and Wellburn [20].
Electrolyte leakage and relative water content. In the second experiment, the electrolyte leakage index and relative water content (RWC) were measured according to the methodology proposed by Dionisio-Sese and Tobita [21] and González and González-Vilar [22], respectively.
Plant growth analysis and dry matter. Leaf area of the plant was measured with a LI -3100 Area Meter®. Moreover, the root system was analyzed using the Delta-TScan system and the length measured using the method developed by Harris and Campbell [23]. Root density was calculated by the ratio between root length and solution volume in the pot.
The plants were cut and separated into shoots and roots. Next, the samples were washed with deionized water, 0.1% detergent solution, 0.3% HCl solution and again with deionized water, and dried in a forced air oven at a temperature of 65°C ± 5, until reaching constant weight. After drying, root and shoot dry matter were obtained, followed by grinding in a Wiley mill.
Si accumulation and K use e ciency. To determine the Si content, shoot dry matter ( rst experiment) and shoot and root dry matter (second experiment) were used. For Si analysis, the samples were extracted following the methodology proposed by Kraska and Breitenbeck [24], and measured in a spectrophotometer at 410 nn to obtain Si content, following the methodology described by Korndörfer et al. [25].
In the second experiment, K content was analyzed by digestion in nitric perchloric acid solution, followed by atomic absorption spectrophotometer reading according to the methodology described by Zasoski and Burau [26]. K use e ciency was estimated considering the dry matter production and K content, according to the methodology described by Siddiqi and Glass [27]: (total dry matter production) 2 / (total nutrient content in the plant).
Based on Si, K and dry matter values, the accumulation of these elements in the entire plant (shoots and roots) was calculated following the formula: Element accumulation = ((element content g kg − 1 ) * (plant mass g per plant))/1000.

Statistical analysis.
Experimental data were submitted to analysis of variance applying the F-test, and when signi cant for qualitative variables, to Tukey's test (p < 0.05) to compare the means, using SAS statistical software 9.2 [28].

Conclusions
Leaf spraying with Si was agronomically feasible for bean plants, particularly silicate of sodium and potassium stabilized at a concentration of 5.4 mmol L − 1 .
Si supplied via nutrient solution or leaf application mitigated K-de ciency stress in the bean plant due to improvements in nutritional, physiological and growth variables, underscoring Si supply via nutrient solution compared to leaf application, although the latter also exhibited attenuating properties.    Leaf area (a), root length (b), root density (c), root area (d), shoot dry matter (e) and root dry matter (f) of bean plants cultivated in a hydroponic system under de ciency (-K) and su ciency (+K) of K, with Si supplied via nutrient solution (root) (SiRO), leaf spraying (SiLE), and control (-Si). The error bars in the gures represent standard error. Different letters, lower case between the supply of Si in the same concentration of K, and upper case between concentrations of K in the same form of supply of Si, indicate differences (P <0.05, Tukey's test) between treatments. Figure 6