Silicon attenuates calcium deficiency by increasing ascorbic acid content, growth and quality of cabbage leaves

Calcium (Ca) deficiency in cabbage plants induces oxidative damage, hampering growth and decreasing quality, however, it is hypothesized that silicon (Si) added to the nutrient solution may alleviate crop losses. Therefore, this study aims at evaluating whether silicon supplied in the nutrient solution reduces, in fact, the calcium deficiency effects on cabbage plants. In a greenhouse, cabbage plants were grown using nutrient solutions with Ca sufficiency and Ca deficiency (5 mM) without and with added silicon (2.5 mM), arranged as a 2 × 2 factorial in randomized blocks, with five replications. At 91 days after transplanting, the plants were harvested for biological evaluations. In the treatment without added Si, Ca deficiency promoted oxidative stress, low antioxidant content, decreased dry matter, and lower quality leaf. On the other hand, added Si attenuated Ca deficiency in cabbage by decreasing cell extravasation while increasing both ascorbic acid content and fresh and dry matter, providing firmer leaves due to diminished leaf water loss after harvesting. We highlighted the agronomic importance of Si added to the nutrient solution, especially in crops at risk of Ca deficiency.

Plants and growing conditions. Cabbage (Brassica oleracea var. capitata) cv. Chato de Quintal seeds were sown in a 200-cell Styrofoam tray, filled with washed sand, and watered with distilled water twice a day until transplanting.
At 26 days after sowing (DAS), the seedlings were transplanted into 1.7 dm 3 polypropylene pots, filled with 1.5 dm 3 of washed sand. Every pot with a cabbage plant consisted of an experimental unit. The sand was placed in a fine sieve, washed in running water, then saturated with a 1 mol L −1 HCl solution over 24 h to eliminate the organic matter residue. Subsequently, the sand was washed with deionized water to remove excess HCl. After transplanting the seedlings, greenhouse temperature and relative humidity were recorded daily using a thermohygrometer (Fig. 1). The average maximum temperature varied widely, 38.7 ± 21.2 °C, during the experimental period, above the optimal temperature. Cultivation at temperatures above 27 °C is known to cause stress to the plants 18 .
After transplanting, the complete nutrient solution and the respective treatments were applied via root, twice a day in the early morning and late afternoon. The solutions were prepared using distilled water deionized twice and checked for Si in the laboratory. The absence of Si deemed the solution suitable for the experiment.
The iron source of the modified Hoagland and Arnon 17 nutritive solution was changed from Fe-EDTA to Fe-EDDHA, whereas Ca changed according to the studied treatments. The nutrient solution pH was adjusted daily to the target value of 5.5 ± 0.2, with HCL or NaOH accordingly.
In the first 10 days, the nutrient solution ionic strength was set at 25%, which was subsequently increased to 50%, remaining until the end of the experiment.  www.nature.com/scientificreports/ Analysis. Electrolyte leakage index. At 91 DAT, six leaf discs (7.61 mm each) were removed from the third youngest leaf in the newly formed cabbage head to determine the rate of cell electrolyte leakage following the methodology in Dionisio-Sese and Tobita 19 . Subsequently, the leaf discs were immersed in 20 mL deionized water in a beaker at room temperature for 2 h. The solution electrical conductivity (EC1) was measure in a benchtop conductivity meter (TDS-3 digital meter). The samples were then autoclaved at 121 °C for 20 min, and after cooling, a new reading was performed to obtain the final electrical conductivity (EC2). The electrolyte leakage was determined as follows EC1/EC2 × 100.
Ascorbic acid. The ascorbic acid (AsA) content was quantified by titration with a 2.6 dichloro-phenol-indophenol sodium solution (Tillman's reactive) and the results expressed as mg of ascorbic acid per 100 g FM (fresh matter) 20 . For extracting AsA, new and fully developed leaves from the cabbage middle region were mixed.
Leaf firmness index. Firmness was measured by inserting an 8 mm tip digital penetrometer to apply a force ranging from 5 to 200 N ± 1 N (Impac, Model IP-90DI, São Paulo, SP, Brazil) on the leaves of the cabbage third layer following the methodology proposed by Calbo et al. 21 . Three measurements were taken in the center of each leaf and expressed as Newton (N).
Accumulated fresh matter loss. The harvested cabbages were stored at 25 °C and weighed daily for five days on an electronic scale, ranging from 0.02 to 200 g ± 2 g (Marte® AL200C), to determine the accumulated fresh matter loss (AFML) in the evaluated period. The AFML was calculated based on the difference between the initial (first day) and final (fifth day) fresh matter weights, divided by the initial fresh matter (first day), and expressed as a percentage (%) as proposed by Chitarra and Chitarra 22 .
Fresh and dry matter of aerial parts and root dry matter. After harvesting, the aerial part was weighed to determine the fresh matter. Subsequently, cabbage and roots were washed in running water, then in a neutral detergent solution (0.1% v/v), HCl solution (0.3% v/v), and again in deionized water. After drying in a forced air circulation oven (65 ± 5 °C) to constant weight, the dry matter contents of aerial parts and roots were determined.
Ca and Si contents. The Ca content was determined using the dry samples previously ground in a Wiley mill following the methodology described by Bataglia et al. 23 whereas Si content was determined from wet digestion according to Kraska and Breitenbeck 24 . The readings were performed in an atomic absorption spectrophotometer as described by Korndörfer et al. 25 . Finally, Ca and Si accumulated in the plant aerial part and roots were calculated based on the Ca and Si contents and dry matter.
Statistical analysis. The data were submitted to analysis of variance by the F-test and, when significant, means were compared by the Student t test (LSD), at 5%, using SAS statistical software. Electrolyte leakage index and ascorbic acid content. The electrolyte leakage index and the AsA content of cabbage leaves were affected by the Ca and Si interaction (Fig. 3). The electrolyte leakage rates were significantly higher in plants grown under Ca deficiency, with or without Si (Fig. 3a). However, the added Si decreased the extravasation rate of electrolytes from cabbage leaves grown under Ca sufficiency or deficiency.

Results
The leaf AsA content is lower in cabbage grown under Ca deficiency, regardless of added Si. However, silicon supply increased vitamin C-AsA content in the leaf of cabbage under Ca deficiency on the harvesting day (Fig. 3b). Five days after harvesting, the treatment with added Si under Ca sufficiency resulted in cabbage leaves with the highest AsA content (Fig. 3c).
Fresh matter of the aerial part and dry matter of the aerial and root parts. The fresh matter of cabbage aerial parts was affected by the calcium and silicon interaction, decreasing up to 37 and 31% in plants under Ca deficiency and Ca sufficiency, respectively (Fig. 4a). Under Ca sufficiency, the supplied silicon did not affect cabbage fresh matter.
The dry matter of cabbage aerial part and root was significantly higher in plants under Ca sufficiency, with or without Si (Fig. 4b,c). But, the added Si promoted a greater dry matter accumulation in the aerial parts of cabbage under Ca deficiency (Fig. 4b). Indeed, the Ca and Si effects on the increasing dry matter of the aerial parts can be seen visually (Fig. 5) while highlighting that the added Si increased the root dry matter contents of cabbage plants grown under Ca sufficiency and deficiency. Accumulated fresh matter loss. AFML was affected by the Ca and Si interaction five days after harvesting (Fig. 6) Firmness index. The firmness index of cabbage leaves was affected by the calcium and silicon interaction.
The leaf firmness index was significantly lower in cabbage under Ca deficiency, with or without Si (Fig. 7). However, the added Si increased the leaf firmness index in plants grown under Ca deficiency.   www.nature.com/scientificreports/

Discussion
The Ca absorption rate decreased in cabbage grown under Ca deficiency but increased with added Si as demonstrated by the greater accumulation in the aerial part (Fig. 2a). This result indicates a synergistic effect of Si on the increase of Ca absorption, possibly because Si favors the growth rate of the root system 26 and the molecular effect of Si on the increasing ATPase activity in the membranes involved in Ca absorption 27,28 . To our knowledge, this report on the benefit of Si in cabbage grown under Ca deficiency is unprecedented since our literature review showed no reports on this subject. However, added Si also increased Ca absorption by the aerial parts of other plants grown under Ca sufficiency, as previously reported for corn by Kaya et al. 29 ; wheat, Mali and Aery 30 ; broccoli and cauliflower, Barreto et al. 13 ; and yellow passion fruit seedlings, Silva Júnior et al. 31 . These results show the importance of further studies aiming to understand the molecular mechanisms of the beneficial role Si plays in the absorption of other cations such as Ca.
The decreasing Ca absorption in Ca deficient plants compared to Ca sufficient, without Si, resulted in increased electrolyte leakage rate and decreased AsA content (Fig. 3). This result demonstrates that Ca deficiency increases the activity of pectolytic enzymes (polygalacturonase) 32 and lipid peroxidation due to increased malondialdehyde 10 , which aggravated by decreasing antioxidants (ascorbic acid), promoting degradation of membrane compounds and cell wall damage, increasing the electrolyte leakage in turn 33 .
The increasing stress observed in Ca deficient plant tissue, without Si, decreased plant growth and hampered the production of fresh and dry matter of aerial parts and roots (Fig. 4). Similarly, this behavior has been already reported for brassica cabbage 33 , cauliflower 34 , and other leafy vegetables such as lettuce 35 and basil 36 .
Besides, it was evident that Ca deficiency increased the AFML between the second and fifth days after harvesting (Fig. 6), and this increased water loss from the plant tissue can decrease vegetable shelf life. Likewise, this high water loss after harvesting through the leaves of plants grown under Ca deficiency has been previously reported for other leafy vegetables, more specifically in spinach leaves by Chao et al. 10 . The high AFML becomes evident as leaf wilts, and turgidity and firmness of plant tissue diminish as observed in the plants grown under Ca deficiency in this study. Ca plays an important structural role in plants by conferring rigidity to the plant tissue since it constitutes the middle lamella and cell wall Taiz et al. 37 while the increasing transpiration on the   (Fig. 2c,d). The Si content reached 1.6 g kg −1 (data not shown) in the aerial parts of plants under Ca sufficiency which (Fig. 2c), according to 39,40 , indicates a nonaccumulating plant since the Si content is less than 5 g kg −1 . Non-accumulating plants are those that accumulate Figure 6. Accumulated fresh matter loss of cabbage under Ca sufficiency and deficiency, with (− Si) and without Si (+ Si), determined on the second (a), third (b), fourth (c), and fifth (d) day after harvesting. F test was applied: *(p ≤ 0.05) and **(p ≤ 0.01). The student t means comparison test was applied. Lowercase letters (a,b) indicate significant differences to calcium within the same level as Si (p < 0.05). Uppercase letters (A,B) indicate significant differences for silicon within the same level as Ca (p < 0.05). The bars represent the mean standard error. n = 5.

Figure 7.
Leaf firmness index on the second day after harvesting of cabbage under Ca sufficiency and Ca deficiency, with (− Si) and without Si (+ Si). F test was applied: *(p ≤ 0.05) and **(p ≤ 0.01). The Student t means comparison test was applied. Lowercase letters (a,b) indicate significant differences to calcium within the same level as Si (p < 0.05). Uppercase letters (A,B) indicate significant differences for silicon within the same level as Ca (p < 0.05). The bars represent the mean standard error. n = 5. www.nature.com/scientificreports/ more Si in the roots than in the aerial parts, possibly due to the low activity of the Lsi1 and Lsi2 transporters in the membranes of the root cells 41 . Similar behavior was observed in this study since Si added to cabbage grown under Ca sufficiency increased Si accumulation in the roots (170 mg per plant) (Fig. 2d) compared to the aerial parts (30 mg per plant) (Fig. 2c). Clearly, Si added to cabbage under Ca deficiency exhibited decreased extravasation rate of cellular electrolytes and plant tissue with increased AsA content (Fig. 3). This Si benefit may be attributed to several factors such as the indirect effect of increasing Ca absorption (Fig. 2a) and the direct effects of increasing antioxidant compounds and ascorbic acid (Fig. 3b,c). There are reports in the literature on the decreasing leakage of cellular electrolytes ensuring the maintenance of tissue integrity for several species such as wheat Ma et al. 11 , chard and cabbage, via Si foliar spraying 12 , broccoli and cauliflower 13 , and corn 33 but no reports on cabbage.
The added Si lessened the effects of Ca deficiency by reducing plant stress as indicated by the decreased leakage of electrolytes (Fig. 3a), promoting cabbage growth given the increased fresh and dry matter of the aerial parts and roots (Fig. 4). This unprecedented finding shows that added Si reduces the nutritional stress of cabbage grown under Ca deficiency, becoming especially important in brassica since Ca deficiency is common in the culture, limiting its growth 5 .
Furthermore, added Si improved the quality of leaves of cabbage grown under Ca deficiency after harvesting since the AFML decreased between the second and fifth days post-harvesting (Fig. 6). On the second day after harvesting, the leaves of cabbage under Ca deficiency, with or without Si, were unsuitable for commercialization since AFML was greater than 10% (Fig. 6b). Further, Si added in the nutrition solution of plants under Ca sufficiency, resulted in 9% AFML, within the acceptable limits for commercialization, according to Chitarra and Chitarra 14 . This result shows the importance of Ca and Si for improving the quality of vegetables such as cabbage produced for human consumption.
On the fourth and fifth days post-harvesting, the AFML results indicate that Si added in plants under Ca sufficiency improved cabbage quality in the period (Fig. 6c,d). The post-harvest Si effect has already been reported for lettuce 14 , and chard and cabbage 12 grown without nutritional stress.
This reduced AFML (Fig. 6) and greater firmness of the plant tissue ( Fig. 7) with added Si is possibly associated with the pectin polymers as seen in rice plants [42][43][44] . Si acts on the hemicellulose and lignin synthesis, by increasing the components of the cell wall in rice plants 15,42 , reducing the activity of enzymes that degrade cell wall 33 and decreasing tissue degradation due to increased AsA content and antioxidant action 45 .
Also, the increased ascorbic acid content (Fig. 3b,c) is highly desirable due to its antioxidant action and for being a vitamin (C) source, essential for human health 46 , while indirectly promoting biofortification and better nutritional quality beyond the firmness.
Our research shows that added Si is beneficial to cabbage plants grown under nutritional stress, corroborating the literature reports that Si is more beneficial to plants grown under stress compared to stress-free crops 13,31,47 .

Conclusions
The Si added in the nutrient solution supplied to cabbage plants increased the contents of vitamin C and biomass while reducing leaf water loss and, thus, ensuring longer storage time post-harvest. This finding highlights the agronomic importance of adding Si to the nutrient solution used in the cabbage crop, especially those crops at risk of Ca deficiency. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.