Gas exchange in castor bean cultivars in response to foliar application of potassium silicate

1 Department of Plant Science, Federal University of Viçosa (UFV), Viçosa, Brazil. 2 Agricultural Sciences, Embrapa Cotton, State University of Paraíba (UEPB/Embrapa), Campina Grande, Brazil. 3 Academic Unit of Agricultural Engineering, Federal University of Campina Grande, Campina Grande, CEP: 58.109-970, Campina Grande, Brazil. 4 Nucleus of Soil and Water Engineering, Federal University of Recôncavo of Bahia, Cruz das Almas, CEP: 44.380-00, Bahia, Brazil. 5 National Center for Cotton Research, Embrapa / Cotton, Oswaldo Cruz Street, 1143, Centenary, CEP: 58428-095, Campina Grande PB, Brazil. 6 Department of Biology Science, Federal University of Paraíba, Areia, CEP: 58.397-000, Paraiba, Brazil.


INTRODUCTION
The area planted with castor bean (Ricinus communis L.) has increased over time due to its importance both for industry and for the biofuel production.Currently, it has emerged as an alternative for the production of energy, representing an excellent possibility for the reduction in the use of non-renewable energy sources.
In this context, in the 2013/2014 season, the production estimate reached 64.4 thousand tons to be harvested in *Corresponding author.E-mail: sarabiologic@hotmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License an area of 105.9 thousand hectares, with a mean yield of 608 kg ha -1 (Conab, 2014).The stimulating prices, combined with good climatic conditions in most of the producing states, justify this increase.
Castor bean fertilization is a practice that, in the last crop seasons, has attracted a lot of interest, due to the increasing prices of fertilizers, because it is considered a nutrient-demanding crop (Brito Neto et al., 2014), which requires high amounts of fertilizers in order to obtain high yields.In this sense, the search for more efficient, lowcost alternatives, especially regarding the acquisition of fertilizers, has attracted the attention of research agencies, farmers and the scientific community.
As an alternative to improve plant nutritional status and its entire physiological apparatus, the use of siliconbased products has been considered, since this mineral is able to increase plant resistance against attacks of pests (Meena et al., 2014), nematodes (Marafon and Endes, 2013), bacteria and fungi (Guerra et al., 2014), minimizing production costs caused by the demand of pesticides.In addition, fertilization with potassium silicate can also influence some aspects of photosynthetic efficiency (Xie et al., 2014) and increase crop yield (Yogendra et al., 2014), among other factors.
According to the literature (Souza et al., 2010;Lalithya et al., 2014;Sarto et al., 2014), the beneficial effects of potassium silicate can be attributed to the joint action of Si and K on plant nutrition and production.According to Amaral et al. (2008) and Pinto et al. (2012), foliar application of potassium silicate in coffee and cocoa plants promoted greater covering of the leaf tissue because of Si precipitation and polymerization close to leaf cuticle, ensuring lower incidence of diseases and higher production and quality of the final product.For not being in the composition of organic compounds and not developing any structural function in plants (Hussein et al., 2014), K participates in the activation of enzymes such as synthetases, oxidoreductases, dehydrogenases, transferases, kinases, aldolases and rubisco (Catuchi et al., 2012), a key enzyme in the photosynthetic process.
This study aimed to evaluate the gas exchanges (net photosynthesis, stomatal conductance, transpiration, water use efficiency, carboxylation efficiency, Internal carbon concentration, chlorophyll a, b, total, carotenoids and the chlorophyll a/b ratio) of three castor bean cultivars in response to the foliar application of potassium silicate.

MATERIALS AND METHODS
The experiment was carried out under field conditions from June 2011 to December 2011, at the National Center for Research on Cotton of the Brazilian Agricultural Research Corporation (Embrapa -Cotton), located in Campina Grande, Paraíba, Brazil (7°13'1" S; 35°52'31" W; 551 m).According to Köppen climate classification, the climate of the study area is As, hot and humid with rains during autumn and winter.The rainy period occurs from April to July and the average annual rainfall is 800 mm .The maximum and minimum average annual temperatures are around 28.7 and 19.8°C, with small variation throughout the year (Coelho and Soncin, 1982).
During the conduction of the experiment, maximum and minimum temperatures and the rainfall of the studied area were daily recorded by a weather station (Figure 1).
The soil in the area is classified as dystrophic Regolithic Neosol, of sandy-loam texture (Embrapa, 2006).Soil samples were collected for the analysis of chemical attributes in the layer of 0-20 cm, under the canopy projection (Table 1).The samples were homogenized and analyzed in the Laboratory of Water and Soils of the Embrapa Cotton.
Crop fertilization followed the recommendations of the Laboratory of Soil Analysis of Embrapa Cotton, which indicated the application of 20 kg ha -1 of N and 30 kg ha -1 of P2O5, as urea and single superphosphate, respectively.Basal and topdressing fertilizations were performed 15 days after the emergence of the seedlings (DAE), by applying phosphate fertilizer directly into 30-cm depth in semicircular furrows.Nitrogen fertilization was divided into three doses; the first topdressing at 15 DAE and the second and the third applications at 30 and 45 DAE, respectively.
The experiment was set in a randomized block design and the treatments were distributed in a 5 × 3 factorial scheme, represented by five doses of potassium silicate (PS) (0, 222, 444, 665 and 836 mg L -1 ) and three castor bean cultivars (BRS Energia, BRS 149 Nordestina and BRS 188 Paraguaçu), in three replicates.A spacing of 0.5 m between plants and 1.0 m between rows was used, totaling 45 plants per plot.Only the central rows were considered for the analysis, disregarding border rows.The Si source was the commercial product Sifol ® , with the following characteristics: Si = 12%; K (K2O) = 15%; Saline index = 26; Electrical conductivity = 1.93 dS m -1 ; Density = 1.40 g L -1 ; pH = 10.96;Physical nature = Fluid.Si concentrations were obtained through the dilution of PS (Sifol ® ) in distilled water.Treatments were applied at 25 DAE, when plant height was 24.8 cm and leaf area 14.63 cm 2 , on an average.PS application was performed through foliar sprayings directed on the abaxial and adaxial surfaces of the leaves.For a better absorption efficiency of PS on leaf surface, a surfactant was used in the spray solution (Sávio et al., 2011).For the application, a pre-compressed manual sprayer with a 3-L tank, made of high-molecular-weight polyethylene and a piston pump with beak diameter of 34 mm were used.
Besides these variables, the photosynthetic pigments chlorophyll a (CLA), b (CLB), total chlorophyll (CLT), chlorophyll a/b ratio and carotenoids (CAR) in the leaves were also evaluated.For this, the leaves were collected and immediately placed in aluminum envelopes, stored in boxes with thermal insulation containing dry ice and transported to the laboratory.Then from the middle part circular fractions were taken without the midrib of the leaf tissue with size of 113 mm 2 .Fractions were macerated and tissue were placed in test tubes coated with aluminum foil, to which 5 mL of dimethylsulfoxide (DMSO) was added.The tubes were left in a dark environment at room temperature of 25 °C for a period of 48 h.After this time the solution containing DMSO + fraction of the plant tissue was filtered through a "filter paper" during the 5 min period.With the solution extracted absorbance readings were performed in a spectrophotometer (Biomate ® tm3 ) at respective wavelengths of 480, 649 and 665 nm (Wellburn, 1994).
For quantification of photosynthetic pigments the following equations were used according to the proposed by Wellburn (1994): The data were subjected to the analysis of variance, and regression, using the statistical software SISVAR (Ferreira, 2011).

RESULTS AND DISCUSSION
In general, foliar application of PS in castor bean plants promoted isolated effects only on internal carbon concentration (Ci).For the factor cultivar, there were significant effects for the variables net photosynthesis (A), stomatal conductance (gs), transpiration (E) and internal carbon concentration (Table 2), indicating that plant response to the application of PS depends more on the genotype than on the applied dose.For the interactions between PS doses and cultivars, the effects were more pronounced for the physiological variables transpiration (E), water use efficiency (WUE) and internal carbon concentration (Ci), suggesting that the effects of PS application on plants can vary widely as a function of the genotype.Possibly, these results point to the development of new studies focusing not only on the definition of doses and responsive genotypes, but also on plant age, form of application, conditions of temperature and rainfall, and crop phenological stage.
The foliar application of PS doses on the canopy of the castor bean cultivars BRS Energia, BRS 188 Paraguaçu and BRS 149 Nordestina promoted significant increases in net photosynthesis (A) and stomatal conductance (gs), and the best response was observed when using the cultivar BRS 149 Nordestina as the indicator plant (Figure 2A and B).
Although the cultural practices were identical for all the studied cultivars, as well as the form of fertilizer application, it is possible to infer that the response to fertilizer application, regardless of the dose, is dependent on the genotype.According to Ferraz et al. (2014), one of the explanations for these results is associated to the capacity of the cultivar to absorb and use the fertilizer.In addition, the genetic load of the cultivar is a characteristic that defines its degree of response to fertilization.In general, the cultivar BRS 149 Nordestina showed the highest mean values of net photosynthesis (19.41 µmol CO 2 m -2 s -1 ), stomatal conductance (0.49 mol CO 2 m -2 s -1 ) (Figure 2A and 2B).On the other hand, the cultivars BRS Energia and BRS 188 Paraguaçu showed similar responses and the lowest mean values of net photosynthesis (17.77 µmol CO 2 m -2 s -1 ) and stomatal conductance (0.35 mol CO 2 m -2 s -1 ) (Figure 2A and 2B).Although only a few research results claim that the use of PS-based foliar fertilizer is an excellent alternative to increase plant photosynthetic capacity, promote conditions to activate enzymes such as synthetases, oxidoreductase, dehydrogenase, transferases, kinases, aldolases and rubisco, a key enzyme in the photosynthetic process (Marschner et al., 2002), this mineral is of great importance for the metabolism, growth and yield of the cultivated species (Amaral et al., 2008).However, research results addressing the promising effects of the use of PS on the physiological aspects of castor bean plants are still scarce.Ferraz et al. ( 2014), evaluating the viability of foliar fertilization with PS in different cotton cultivars (BRS Topázio, BRS Safira and BRS Rubi) evidence that the response of this oil plant to PS fertilization is also dependent on the genotype.It is possible to infer, in part, that the extent of the response to the applied fertilizer depends on the capacity of the cultivar to absorb and metabolize the fertilizer.On the other hand, factors like irrigation, soil type, temperature, irrigation management, as well as the use of pesticides containing micronutrients, also influence the type of response of the genotype to a certain substance.
The transpiration (Figure 2C), water use efficiency (Figure 2D) and internal carbon concentration (Figure 2E) of the studied castor bean cultivars adjusted to quadratic and linear models in response to foliar fertilization with PS.According to Figure 2C, inside the interaction, the cultivars BRS Energia and BRS 149 Nordestina showed a similar quadratic adjustment, with reductions of up to 20.89 and 22.28% in transpiration (E), and critical values of 8.90 and 8.72 mmol H 2 O m -2 s -1 estimated with PS applications of 485 and 500 mg L -1 , in response to the foliar sprayings.On the other hand, the transpiration showed linear adjustment for the interaction between PS doses and the cultivar BRS 188 Paraguaçu, with a tendency of stabilization between the applied doses.
The transpiration of the cultivar BRS 188 Paraguaçu was not affected by the foliar fertilizer.Possibly, these results can be explained by the fact that Si minimizes water losses due to its deposition on the leaf cuticles of the plants.Studies conducted by Zanão Júnior et al. (2013) on roses also evidenced that plant transpiration is reduced with the increments in PS doses.Si might have improved water use efficiency, considerably reducing losses by transpiration, since it acts directly on water use efficiency.
The highest values of photosynthetic rate (A) and stomatal conductance (gs) were observed when the indicator plant was the cultivar BRS Nordestina, followed by BRS Energia and BRS Paraguaçu.On the other hand, water use efficiency (WUE), a variable that relates the amount of carbon fixed per unit of water lost in the transpiration process, showed the best response when the genotypes BRS Energia and BRS Paraguaçu were used, evidencing higher carbon assimilation per mole of transpired water in these genotypes.
In general, the differences between the highest and the lowest PS doses were small, evidencing that the use of high, intermediate or low doses of this fertilizer little influenced the WUE of the studied genotypes.On the other hand, PS application promoted a quadratic response of the cultivar BRS Energia, indicating that WUE is compromised from the dose of 500 mg L -1 onwards.
According to the literature (Marschner et al., 2002), Si is a mineral that associates with the cellulose from the epidermal walls, forming a film, thus reducing the movement of water through the wall, promoting higher water economy due to the decrease in the transpiration rate, as observed in Figure 2A.Therefore, plants sprayed with the PS dose of 836 mg L -1 show higher WUE compared with the control treatment, for the cultivar BRS 188 Paraguaçu.On the other hand, for the cultivar BRS 149 Nordestina, the treatment corresponding to the highest PS dose (836 mg L -1 ) promoted significant reduction in WUE, partially agreeing with the increase in the transpiration rate.According to the literature (Pinto et al., 2012), stomatal opening is directly related to the photosynthetic rates and transpiration, since plants lose water as they absorb CO 2 .In addition, the highest values of net photosynthesis (A) and stomatal conductance (gs) observed in this study allow higher availability of photoassimilates for plant growth and also for the defensive metabolic pathways, since this genotype is precocious and can be used in dense cultivation systems.According to Amaral (2008), knowing the stomatal dynamics of a certain crop is a very useful tool for the understanding of physiological processes, since stomata   are the main pathways for the gas exchanges occurring between the atmosphere and the interior of the plant photosynthetic apparatus.According to the results obtained in this study, the internal carbon concentration (Figure 2E) was also influenced by the foliar application of PS, with linear adjustments in the response of the cultivars BRS 188 Paraguaçu and BRS 149 Nordestina.
On the other hand, the cultivar BRS Energia responded quadratically, with an abrupt decrease in the internal carbon concentration from the dose of 665 mg L -1 onward.According to Shimazaki et al. (2007), the reduction in the internal CO 2 levels can be partially attributed to the decrease in carbon assimilation rates, since there is water loss during the processes of gas exchange and CO 2 absorption.In order to avoid water loss, the plant usually closes its stomata, resulting in lower rates of carbon assimilation and consequently lower internal carbon concentration.On the other hand, Taiz and Zeiger (2013) emphasize that the internal carbon concentration depends on climatic conditions and on the nutritional supply provided by pre-planting and post-planting fertilizations.In addition, for these authors, the genetic load of each cultivar weights the differences regarding the level of response to certain factor, in particular the internal carbon concentrations.The Internal carbon concentration remained stable and did not show significant differences, regardless of the genotype and the applied PS dose.For herbaceous cotton cultivars (BRS Topázio, BRS Safira and BRS Rubi), Ferraz et al. (2014) observed that foliar application of PS increased the Internal carbon concentration, unlike the results found in this study, and that plant response to this fertilizer also depends on the genetic load of the plant, particularly of the cultivar.
PS application promoted significant interactive effects on the contents of the photosynthetic pigments chlorophyll a (CLA), chlorophyll b (CLB), total chlorophyll (CLT), carotenoid (CAR) and the chlorophyll a/b ratio (CLa/b).On the other hand, isolated effects for the PS doses and the cultivar were only observed for the chlorophyll a/b ratio (Table 3).
According to Epstein (2006), Si usually increases chlorophyll leaf content and plant tolerance to biotic and abiotic stress.For Taiz and Zeiger (2013), chlorophylls are responsible for the absorption of light to stimulate photosynthesis.In this context, chlorophyll a is the pigment used for photochemistry, while the other ones help the absorption of light and the transfer of radiant energy to reaction centers, and are referred to as accessory pigments, which include other types of chlorophyll, such as a, b, c and d.On the other hand, carotenoids are also accessory pigments responsible for light reception and transfer to reaction centers, besides protecting chloroplasts against excessive heat.The determination of chlorophyll contents in the leaves is essential because plant photosynthetic activity partially depends on the leaf capacity to absorb light (Emrich et al., 2011).
The foliar application of PS in different castor bean cultivars promoted significant effects on the contents of chlorophylls and carotenoids.According to the Figure 3A, PS application promoted a quadratic response, regardless of the cultivar.On the other hand, the cultivars BRS 188 Paraguaçu and BRS 149 Nordestina showed the same tendency, with a slight decrease in the chlorophyll a contents, as the PS doses increased until 480.4 and 325.1 mg L -1 .Both cultivars showed an increasing tendency, showing that chlorophyll a contents increased significantly for both cultivars from that dose onward.The cultivar BRS Energia showed the opposite behavior; as the PS doses increased, chlorophyll a contents increased until the maximum at the dose of 325 mg L -1 , then reducing with the subsequent increments.Thus, the chlorophyll content was also affected by the application of the fertilizer.However, the level of response to the applied treatment was depended on the genotype.
All the studied cultivars showed a quadratic response for the chlorophyll b contents (Figure 3B) in response to the foliar application of PS.However, the cultivars BRS Energia and BRS 149 Nordestina showed the same tendency, decreasing as the doses increased until the maximum limit of 416.7 and 552 mg L -1 . Then, the contents of chlorophyll b increased until the maximum dose of 836 mg L -1 determined in this study.Chlorophyll b contents in the cultivar BRS 188 Paraguaçu were little affected by the foliar application of PS.
In general, chlorophyll b is considered an accessory pigment that helps the absorption of light and the transfer of radiant energy to reaction centers, which are located in the membranes of the thylakoids (Taiz and Zieger, 2013).Possibly, the increase in the contents of chlorophyll b has been favored by the action of the Si in plant tissues, since this mineral usually increases chlorophyll contents (Epstein, 2006).For Mebrahtu and Havolver (1991), the increment of chlorophyll b contents can be a consequence of the increase in the proportion of the lightharvesting chlorophyll a/b-protein complex, in relation to the P700-chlorophyll a-protein complex.Another important factor that must be taken into consideration is the better development of the "grana" structure in the chloroplasts, where the chlorophyll a/b-protein complex is found (Mebrahtu and Havolver, 1991).
According to Figure 3C, together with the increment of the contents of chlorophyll a and b, observed for the cultivar BRS 149 Nordestina, there was an increase in the content of total chlorophyll of the plants, which represents the sum of the contents of chlorophyll a and b.The total chlorophyll for this cultivar decreased initially, with a subsequent tendency of increase as the fertilizer doses increased.One of the possible explanations is the fact that the contents of chlorophyll a and b were directly correlated to the PS doses.
On the other hand, the contents of total chlorophyll for the cultivars BRS 188 Paraguaçu and BRS Energia showed opposite responses, that is, while total chlorophyll contents of the former increased in response to the increment in PS doses, for the latter they initially decreased and then increased for the subsequent doses (Figure 3C).In general, the studied castor bean cultivars showed different behaviors with respect to the contents of chlorophyll a, b and total chlorophyll, and only the cultivar BRS 149 Nordestina showed the same tendency for these variables.
Carotenoids are essential for plants, since they play a significant role in the protection of the photosynthetic apparatus against photodegradation of the photosystems, through the interconversions between the xanthophyll molecules (Cardoso, 1997).As for the content of carotenoids (Figure 3D) observed in this study, PS foliar sprayings promoted favorable effects for the cultivar BRS 149 Paraguaçu; as the doses increased, the content of carotenoids increased until the inflection point of 505.5 mg L -1 .On the other hand, PS foliar application promoted similar responses for the cultivars BRS Energia and BRS 149 Nordestina for this pigment.In general, the increment in PS doses initially reduced the carotenoid contents until the level of 307 and 407.75 mg L -1 . From this dose onward, the contents of this pigment started recovering slowly.Although the cultivars BRS Energia and BRS 149 Nordestina exhibit a decreasing tendency in the carotenoid contents in their tissues when sprayed with PS, the increments in this pigment were maintained.Thus, studies clarifying more precisely the response of different castor bean genotypes to PS application should be performed in order to provide farmers with efficient doses and the most adequate cultivars to be fertilized with PS-based products.
Chlorophyll a/b ratio was influenced by PS doses (Figure 3E), with different behaviors for the studied cultivars.However, chlorophyll a/b ratio decreased as the fertilizer doses increased, especially when the cultivars BRS Energia and BRS 188 Paraguaçu were used as indicator plants.On the other hand, the cultivar BRS 149 Nordestina showed a direct relation between chlorophyll a/b ratio and PS doses.The observed differences regarding the magnitude of response for this variable can be partially correlated with the increase in the content of chlorophyll b, which was directly proportional to the fertilizer doses, in relation to the content of chlorophyll a.On the other hand, an opposite response was observed for the cultivar BRS 149 Nordestina, in which the chlorophyll a/b ratio was favored by the increment of PS doses, despite the initial reductions in chlorophyll a contents, showing a recovery for the highest doses and significant reductions in the contents of chlorophyll b as the PS doses increased, with a tendency of increase in response to the application of higher doses.However, this is an atypical behavior with respect to the response of castor bean cultivars to the tested fertilizer, which suggests the conduction of new experimental studies under field conditions in order to confirm these results or present a new response to PS foliar fertilization.

Conclusions
1) The promising effects of foliar fertilization with potassium silicate in castor bean plants depend on the genotype; 2) The foliar application of potassium silicate increases photosynthetic capacity, stomatal conductance, water use efficiency, contents of chlorophyll a, b, total chlorophyll, carotenoids and the chlorophyll a/b ratio, besides reducing water losses by transpiration.

Figure 1 .
Figure 1.Maximum and minimum temperatures (°C) and rainfall (%) recorded during the conduction of the experiment, Embrapa Cotton, Campina Grande, PB.

Table 1 .
Soil chemical characteristics in the layer of 0-20 cm, Campina Grande, PB.

Table 2 .
Summary of the analysis of variance (mean squares) for the variables: net photosynthesis (A), stomatal conductance (gs), transpiration (E), water use efficiency (WUE) (A/E), carboxylation efficiency (CE) (A/Ci) and internal carbon concentration (Ci) in the castor bean cultivars BRS Energia, BRS 188 Paraguaçu and BRS 149 Nordestina, in response to the application of different doses of potassium silicate (PS), at 90 DAE, Campina Grande, PB.Significant at 0.05 of probability by F test; ** significant at 0.01 of probability, ns-not significant. *

Table 3 .
Summary of the analysis of variance (mean squares) for the variables: chlorophyll a (CLA), chlorophyll b (CLB), total chlorophyll (CLT), carotenoids (CAR) and the chlorophyll a/b ratio (CL a/b) in the castor bean cultivars BRS Energia, BRS 188 Paraguaçu and BRS 149 Nordestina, in response to the application of different doses of potassium silicate (PS) at 90 DAE, Campina Grande, PB.