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Extraction and Characterization of Cellulose from Jerusalem Artichoke Residue and Its Application in Blueberry Preservation

The utilization of industrial by-products is becoming more and more important for resource utilization. In this study, soluble dietary fiber (SDF) was extracted from Jerusalem artichoke residue, and a series of characterizations of SDF were carried out. The results showed that SDF had good properties. SDF (0%, 0.1%, 0.2%, 0.3%, and 0.4%) and chitosan (2%) were further used to prepare the coating that was used for the preservation of blueberry. The chemical structure of the film was obtained by FT-IR and XRD analysis. The microstructure of the film was analyzed by SEM, and the properties of the film were tested. The blueberry fresh-keeping test proved that the SDF-added film could effectively prolong the quality of blueberries in storage for 16 days. After 16 days of storage, compared with the control group, the decay rate of the coating group with 0.2% SDF decreased by 16.3%, the consumption of organic acids decreased by 43.7%, and the content of anthocyanin increased by 29.3%. SDF has a potential application in food preservation.

Introduction
The Jerusalem artichoke (Helianthus tuberosus L.) is a perennial sunflower herb in the Compositae family. It was introduced into China from Europe and is widely planted in north China. The Jerusalem artichoke is a potential energy crop with a high yield and rich nutrients, especially inulin [1]. Seeing its excellent physiological functions and wide application in food, the inulin industry has developed rapidly in recent years. Jerusalem artichoke residue is a by-product of inulin processing, which contains starch, cellulose, high quality protein, and other important available resources. Jerusalem artichoke residue is basically fed to livestock as a feed after drying, mostly without nutritional optimization. In addition, most Jerusalem artichoke residue is discarded as waste. This way has caused environmental pollution and a great waste of resources. At present, the research on Jerusalem artichoke and Jerusalem artichoke residues mainly focuses on the preparation of silage. For example, a study carried out by Piotr Koczocine et al. [2] studied the chemical changes that occur in Jerusalem artichoke silage. There are few reports on the comprehensive utilization of favorable components in Jerusalem artichoke residue.
Cellulose is a macromolecular polysaccharide composed of glucose. Dietary fiber is the general term for polysaccharides that cannot be digested by human body in food, which is divided into soluble dietary fiber and insoluble dietary fiber. Most dietary fibers are structural polysaccharides in plant cell walls. More and more studies have shown that dietary fiber has important physiological functions in the human body, especially in the human intestinal tract. In addition, based on the characteristics of dimensions, crystallinity, The thermal degradation processes of the SDF were measured using a Thermoanalyzer Systems instrument (Q1000DSC + LNCS + FACS Q600SDT, TA Instruments, New Castle, DE, USA). All samples were scanned from 50 to 600 • C at a heating rate of 10 • C/min and a nitrogen flow rate of 60 mL/min.
The appropriate amount of dried SDF was mixed with KBr and then ground to prepare the tablets. The infrared spectrum was analyzed by a Nicolet iS10 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) in the range of 400-4000 cm −1 .
The SDF was analyzed by X-ray diffraction (D8 Advance, Brock, Germany). The scanning speed was 5/min, and the range of 2θ was 5-50 • .
The SDF powder was fixed on the metal support with a double-sided conductive adhesive and coated with gold. The microstructures of the cellulose, such as cross-sectional and surface morphology, were characterized by SEM (FEI-Quanta 200, Hillsboro, OR, USA).

Preparation of Chitosan Composite Coating Solutions and Films
The chitosan composite coating solutions and films were prepared with reference to the method of Xueying Song et al. [11] and slightly modified. SDF was dissolved in 100 mL of 1% acetic acid solution according to Table 1. After the SDF was completely dissolved, 2 g of chitosan was added and stirred with a magnetic stirrer until the coating-forming solution was transparent. Then, 0.6 mL of glycerol was added as a plasticizer and stirred for 60 min. The coating solutions were degassed by an ultrasonic cleaning machine for 30 min. Then, 15 mL of coating solution was poured into a circular container with a diameter of 90 mm and dried. After drying, the film was placed in a vacuum dryer for standby. The initial film weight (M1) was dried to a constant weight (M2) at 105 • C. In the water solubility determination, the dry film was placed in a beaker containing distilled water and sealed with a plastic film for 24 h. Then, the film was removed for rapid drying and dried again at 105 • C to a constant weight (M3) [12].
Each film was fixed in a glass containing anhydrous calcium chloride. After weighing, the glass was placed in the dryer and weighed daily until the mass change was less than 0.001 g. W (g): increased weight, X (m): thickness, t (s): time required to reach the constant weight, A (m 2 ): penetration area, P: partial vapor pressure difference between dry atmosphere and pure water (2339 Pa at 20 • C) [13]. Water

SEM, TGA, FTIR, and XRD Analyses of the Films
The films were cut to the appropriate size, fixed on the metal support with a doublesided conductive adhesive, and coated with gold. The microstructures of the films, such as cross-sectional and surface morphology, were characterized by SEM (FEI-Quanta 200, Hillsboro, OR, USA).
The thermal degradation processes of the films were measured using a Thermoanalyzer Systems instrument (Q1000DSC + LNCS + FACS Q600SDT, TA Instruments, New Castle, DE, USA). All samples were scanned from 50 to 600 • C at a heating rate of 10 • C/min and a nitrogen flow rate of 60 mL/min.
Each film was attached to the metal support. The infrared spectrum was analyzed by a Nicolet iS10 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) in the range of 400-4000 cm −1 .

Application of Edible Coatings
Six groups of blueberry samples were prepared. The control group was not coated. The other five groups were immersed in chitosan coating solutions containing 0.00%, 0.10%, 0.20%, 0.30%, and 0.40% cellulose. The coating solution was evenly wrapped and dried. Each sample was stored for 16 days, and the corresponding indexes were measured every 4 days.

Determination of Weight Loss Rate, Decay Rate, and Frost Covering Rate
The mass of the blueberries was recorded the first time (m0) and each time (mf). The weight loss rate (W) of the blueberries was calculated according to the formula: The decay of blueberry fruit was mainly evaluated according to the softening degree of fruit, juice leakage caused by decay, and fungal infection. The corruption situation was divided into three levels: level 0, surface has no obvious change; level 1, obvious softening phenomenon; level 2, serious softening and juice leakage; and level 3, mold spot infection on the surface. The decay rate was calculated as follows: Decay rate (%) = ∑ Decay level × Numberof fruits at this level Highest decay level × Total fruit quantity × 100% According to the coverage score, blueberries were divided into four levels: level 0, fruitless cream; level 1, fruit frost covering area 0~1/3 of the fruit; level 2, fruit frost covering area is 1/3~2/3 of the fruit; level 3, fruit frost covering area is 2/3 of the fruit; and level 4, fruit frost covers the whole fruit and the fruit frost is thicker [16].
Frost covering index (%) = ∑ Fruit frost level × Number of fruit of this level Highest fruit frost level × Total fruit quantity × 100% (6) 2.6.3. Determination of pH, Titratable Acid, and Anthocyanin Content A juicer was used to break up the blueberries and filter them into juice. A pH meter was used to measure the pH of the juice. The titratable acid content in the blueberries was determined by reference to Carolina Medina-Jaramillo et al. [16]. Referring to Jungmin Lee's [17] method, the anthocyanin content in blueberries was determined by the pH differential method.

Determination of Texture of Blueberries
A texture analyzer (stable micro system-TA.XT. PLUS, UK was used to test the peel puncture strength and average flesh firmness of blueberries. The testing parameters were as follows: displacement of 5 mm and force of 10.0 g. In addition, the test was repeated six times in each group.

Statistical Analysis
All measurements were repeated three times and the results were averaged. The data analysis software used for the whole experiment was Microsoft Excel 2011, SPSS Statistics 26.0 (IBM, Armonk, New York, NY, USA), and RStudio 1.3. The differences between the different treatments were determined by a one-way ANOVA. The differences were considered significantly at the 95% confidence level (p < 0.05).

Characterization of SDF from Jerusalem Artichoke
Through the determination of polysaccharide content, the purity of SDF was 65%. The results of the thermal analysis are shown in Figure 1A. When the temperature is lower than 150 • C, the decrease in sample quality is attributed to the loss of free water and bound water. SDF began to decompose from 150 • C to 320 • C. Finally, at 600 • C, the mass of the sample was 60.45% of the original mass. The results showed that Jerusalem artichoke SDF had good thermal stability.  Figure 1B shows the results of Fourier transform-infrared spectroscopy (FT-IR). A strong peak appeared at 722 cm −1 , which proved the existence of -(CH2)n-(n ≥ 4). The stretching vibration of -OH appeared at 3600 cm −1 . The peak at 2600 cm −1 replaced S-H. The peak near 2350 cm −1 represents the characteristic absorption peak of P-H. The charac-  Figure 1B shows the results of Fourier transform-infrared spectroscopy (FT-IR). A strong peak appeared at 722 cm −1 , which proved the existence of -(CH 2 ) n -(n ≥ 4). The stretching vibration of -OH appeared at 3600 cm −1 . The peak at 2600 cm −1 replaced S-H. The peak near 2350 cm −1 represents the characteristic absorption peak of P-H. The characteristic absorption peak at 2100-2200 cm −1 is attributed to the C-H stretching vibration of alkynes, and the angular vibration absorption peak of C-H or C-O is in the range of 1300-1400 cm −1 [18].
The results of the XRD analysis are shown in Figure 1C. A typical cellulose diffraction peak signal appears near 2θ = 16 • (101 crystallographic plane), 23 • (002 crystallographic plane), and 35 • (040 crystallographic plane), and it can be judged as a cellulose I structure from the peak position. According to the formula, the crystallinity of the sample is 50.21% [19].
The microstructure of Jerusalem artichoke SDF was analyzed, and two scanning electron microscope photographs at 1000 times and 7000 times were obtained, as shown in Figure 1D,E. The typical crystal structure of a cellulose rod can be seen in the electron microscope pictures, which proves that the extracted soluble dietary fiber is in good condition [10].

Film Thickness Analysis
As shown in Figure 2A, the thickness of the films is closely related to the amount of SDF added. With the increase in SDF content from 0.1% to 0.4%, the thickness of the films gradually increased from 38.67 µm to 47.64 µm. Hydroxyl is an important bridge between chitosan molecules. Due to the presence of hydroxyl groups in the SDF, the spacing between chitosan molecules shortened, and the density of the SDF film increased to form a denser structure, so the thickness of the SDF film increased correspondingly.

Film Thickness Analysis
As shown in Figure 2A, the thickness of the films is closely related to the amount of SDF added. With the increase in SDF content from 0.1% to 0.4%, the thickness of the films gradually increased from 38.67 μm to 47.64 μm. Hydroxyl is an important bridge between chitosan molecules. Due to the presence of hydroxyl groups in the SDF, the spacing between chitosan molecules shortened, and the density of the SDF film increased to form a denser structure, so the thickness of the SDF film increased correspondingly. The moisture content of the films is shown in Figure 2B. The moisture content of the four composite films was about 23%, and the moisture content of the control group was the highest, which was 31.89%. A single factor analysis of variance showed that there was no significant difference among the four concentrations of SDF composite films, while the

Analysis of Moisture Content, Water Solubility, and Water Vapor Permeability (WVP)
The moisture content of the films is shown in Figure 2B. The moisture content of the four composite films was about 23%, and the moisture content of the control group was the highest, which was 31.89%. A single factor analysis of variance showed that there was no significant difference among the four concentrations of SDF composite films, while the difference was significant with the control group. It can be seen that the addition of SDF caused a decrease in the moisture content of the film.
It can be seen in Figure 2C that with the increase in SDF content from 0% to 4%, the water solubility of the film increased from 20.55% to 32.19%. With the addition of SDF, the proportion of soluble components in the film also increased [15]. Figure 2D shows the WVP of the film. The WVP of the group without SDF was 4.58 × 10 −11 g·m −1 ·s −1 ·Pa −1 , which was significantly lower than that of the four groups with SDF added. The WVP of the film increased first and then decreased with the increase in SDF content. When the SDF content was 0.3%, the WVP of the film was the highest, which was 9.16 × 10 −11 g·m −1 ·s −1 ·Pa −1 .

Morphology and Microstructure of Thin Films
The surface morphology of the films is shown in Figure 3A. The film without SDF was light yellow, smooth, and transparent. After the addition of SDF, the color of the film deepened, the transparency decreased, and the particle sense increased, which were confirmed by the results of the SEM, as shown in Figure 3B. The films with SDF addition levels less than 0.3% had smooth surfaces and no particles. The two kinds of films with SDF contents of 0.3% and 0.4% had granular surfaces. Due to the aggregation of cellulose, the higher the amount of SDF, the higher the roughness of the film surface. Figure 3C shows an SEM image of the cross section of the films. The structure of the films with SDF contents less than 0.4% was continuous and dense without voids and phase separation. Small pores appeared in the film with 0.4% SDF. In addition, with the increase in the amount of SDF, the film gradually appeared to have an uneven thickness [8]. The surface morphology of the films is shown in Figure 3A. The film without SDF was light yellow, smooth, and transparent. After the addition of SDF, the color of the film deepened, the transparency decreased, and the particle sense increased, which were confirmed by the results of the SEM, as shown in Figure 3B. The films with SDF addition levels less than 0.3% had smooth surfaces and no particles. The two kinds of films with SDF contents of 0.3% and 0.4% had granular surfaces. Due to the aggregation of cellulose, the higher the amount of SDF, the higher the roughness of the film surface. Figure 3C shows an SEM image of the cross section of the films. The structure of the films with SDF contents less than 0.4% was continuous and dense without voids and phase separation. Small pores appeared in the film with 0.4% SDF. In addition, with the increase in the amount of SDF, the film gradually appeared to have an uneven thickness [8].  Figure 4A shows the TGA curves of five films, indicating that the thermal degradation process of the film is divided into three stages. At 50-140 °C, the weight loss was attributed to the loss of free water. The bound water and glycerol in the molecule evaporated at 140-280 °C. At 280-500 °C, the polymer in the film gradually decomposed, and  Figure 4A shows the TGA curves of five films, indicating that the thermal degradation process of the film is divided into three stages. At 50-140 • C, the weight loss was attributed to the loss of free water. The bound water and glycerol in the molecule evaporated at 140-280 • C. At 280-500 • C, the polymer in the film gradually decomposed, and the functional-group-related chain broke. The weight tended to be stable above 500 • C. The residual masses of the five films after the TGA were 17.39%, 25.24%, 23.93%, 30.56%, and 27.00% of the original mass, respectively. It could be seen that the thermal stability of the films with SDF was better than the control group.

FT-IR Analysis
The FT-IR spectra reveal the molecular interactions between different film components, as shown in Figure 4B. The O-C-O stretching vibration appeared at 1640 cm −1 . The

FT-IR Analysis
The FT-IR spectra reveal the molecular interactions between different film components, as shown in Figure 4B. The O-C-O stretching vibration appeared at 1640 cm −1 . The peaks at 931 cm −1 and 790 cm −1 represent the α-1,6-glycosidic bond and α-1,4-glycosidic bond, which are the main chemical bonds of monosaccharide molecular linkage in cellulose. The stretching vibration of the hydrogen bond (O-H) appeared at 860 cm −1 in the fingerprint area, and the infrared absorption appeared at about 3600 cm −1 in the characteristic area, which confirmed the existence of a hydrogen bond. The stretching vibrations at 1170 cm −1 and 1245 cm −1 represent N-H and C-H, respectively [11].

XRD Analysis
As shown in Figure 4C, the XRD analysis revealed the crystalline properties of the films. The spectrum shows that diffraction peaks appeared at 7.99 • , 11.04 • , 15.34 • , 17.74 • , and 22.29 • , indicating that the films had certain crystallinity. The diffraction peak at 11.04 • was related to the 020 diffraction plane of the chitosan anhydrous crystal. However, with the increase in SDF content, the diffraction peak gradually weakened. This could be explained as the formation of hydrogen bonds, which destroys the close packaging between chitosan and cellulose molecules. The existence of hydrogen bonds is discussed in 3.2.5, which supports this view. In addition, the addition of glycerol in the coatingforming solution increased the fluidity of the polymer, with a reduction in the cohesion of the matrix, hindering the arrangement of long chains. The diffraction peaks at 29.41 • and 30.57 • appeared when the SDF content was 0.3% and 0.4%, which may be caused by the 040 crystal plane of cellulose [20].

Weight Loss Rate, Decay Rate, and Frost Coverage Index of Blueberries during Storage
The weight loss rates of blueberries with different treatments during storage are shown in Figure 5A. The weight loss rate of the control group was significantly higher than that of the other groups, and the weight loss rate of the control group reached 31.55% on day 16. It was proven that coating film treatment on blueberries could reduce the weight loss of blueberries. After 16 days of storage, the film with 0.1% SDF showed the best performance in preventing pulp weight loss, and the weight loss rate was 25.45%. With the increase in SDF content, the ability of the film to prevent weight loss decreased.

XRD Analysis
As shown in Figure 4C, the XRD analysis revealed the crystalline properties of the films. The spectrum shows that diffraction peaks appeared at 7.99°, 11.04°, 15.34°, 17.74°, and 22.29°, indicating that the films had certain crystallinity. The diffraction peak at 11.04° was related to the 020 diffraction plane of the chitosan anhydrous crystal. However, with the increase in SDF content, the diffraction peak gradually weakened. This could be explained as the formation of hydrogen bonds, which destroys the close packaging between chitosan and cellulose molecules. The existence of hydrogen bonds is discussed in 3.2.5, which supports this view. In addition, the addition of glycerol in the coating-forming solution increased the fluidity of the polymer, with a reduction in the cohesion of the matrix, hindering the arrangement of long chains. The diffraction peaks at 29.41° and 30.57° appeared when the SDF content was 0.3% and 0.4%, which may be caused by the 040 crystal plane of cellulose [20].

Weight Loss Rate, Decay Rate, and Frost Coverage Index of Blueberries during Storage
The weight loss rates of blueberries with different treatments during storage are shown in Figure 5A. The weight loss rate of the control group was significantly higher than that of the other groups, and the weight loss rate of the control group reached 31.55% on day 16. It was proven that coating film treatment on blueberries could reduce the weight loss of blueberries. After 16 days of storage, the film with 0.1% SDF showed the best performance in preventing pulp weight loss, and the weight loss rate was 25.45%. With the increase in SDF content, the ability of the film to prevent weight loss decreased.   Figure 5B shows the changes in the decay rates of the blueberries in each group. It can be seen that the decay rate of the blueberries in each group increased, and the decay rate accelerated after 8 days of storage. On the 16th day of storage at room temperature, the highest decay rate was 45.39% in the control group, followed by the no SDF group. The best protection against rot was the 0.3% SDF coating, which reduced the rot rate by 19.26% compared with the control group. The results showed that the coating treatment could slow down the corruption of blueberries, and the addition of SDF enhanced this ability of the coating. Figure 5C shows the changes in frost covering index of the blueberries in each group during storage. The results showed that, at the beginning of storage, the frost cover index of blueberries treated with a coating was lower than that of the control group. After four days of storage, the frost covering rate of the control group decreased rapidly, while the blueberries treated with a coating were relatively stable. In the treatment group with the 0.4% SDF coating, the change of the frost cover rate was only 26.66% during the whole storage period (41.67% in the control group). This shows that although the coating treatment would lead to the loss of blueberry surface frost, the cellulose composite membrane could play a good protective role on the frost in storage.

The pH, Titratable Acid (TA), and Anthocyanin Content of Blueberries during Storage
The changes in pH and titratable acid in blueberries during storage are shown in Figure 5D,E. With the progress of storage, organic acids in blueberries were gradually consumed, and the pH showed an upward trend, while titratable acid showed a downward trend. It can be seen in the diagram that the pH of blueberries treated with a coating was lower than that of the control group, and the titratable acid was higher than that of the control group. This shows that coating treatment could protect the organic acids in fruits and slow down the consumption of organic acids. Figure 5F shows the changes in anthocyanin content in blueberries during storage. It can be seen that the anthocyanin content of the blueberries in each group increased and then decreased. The peak of anthocyanin content in the control group and the 0.3% SDF coating group appeared on the fourth day of storage; the peak of anthocyanin content in the 0%, 0.1%, and 0.4% SDF coating groups was delayed to the eighth day of storage. The addition of 0.2% SDF delayed this peak to the 12th day of storage, and the change rate of anthocyanin content in this group was the smallest during the whole storage period. It indicated that SDF coating was beneficial to anthocyanin accumulation in blueberry fruit.

Changes in Blueberry Hardness during Storage
Pericarp puncture strength and average flesh firmness can accurately reflect fruit firmness. The pericarp puncture strength and average flesh firmness of blueberry were tested by a texture analyzer, and the results are shown in Figure 6B,C. During storage, the pericarp puncture strength and average flesh firmness of blueberries in the control group first increased and then decreased. On the 16th day of storage, compared with the control group and the group without SDF, the coating with SDF showed better performance in the maintenance of fruit hardness. Especially when the SDF content was 0.2%, the fruit could maintain good hardness during 16 days of storage.

Discussion
In this study, the soluble dietary fiber (SDF) extracted from Jerusalem artichoke was used to prepare a blueberry coating. First, the extracted SDF was characterized, and the results showed that it had a typical cellulose crystal structure. The properties and bioactivity of dietary fiber are related to its complex structure. As a polysaccharide, cellulose is very suitable for membrane materials, but most of the cellulose is insoluble, which increases the difficulty of preparing a coating-forming solution [21]. The FT-IR spectrum of SDF proved the existence of -OH in the sample, which gave Jerusalem artichoke dietary fiber good water solubility and was conducive to the preparation of a coating-forming solution. High-crystalline structures need to consume more energy during thermal decomposition, so materials with high crystallinity generally have higher thermal stability [22]. The results of the XRD analysis showed that the crystallinity of the extracted SDF was as high as 50.21%, and the TGA analysis also showed that the SDF had good thermal stability, which was the required performance of membrane materials [23].
The miscibility of each component in the coating-forming solution affects the microstructure of the film and further affects the mechanical properties and barrier properties of the film. The presence of -OH in SDF enhanced its hydrophilicity, but it was still difficult to dissolve with chitosan. The addition of acetic acid contributes to the dissolution of the two components [24]. In addition, glycerol is hydrophilic, which makes the membrane components form a closer connection by association. When an appropriate amount of SDF was added, the components in the coating-forming solution could effectively dissolve each other, and the prepared film had a dense and uniform microstructure, showing good

Discussion
In this study, the soluble dietary fiber (SDF) extracted from Jerusalem artichoke was used to prepare a blueberry coating. First, the extracted SDF was characterized, and the results showed that it had a typical cellulose crystal structure. The properties and bioactivity of dietary fiber are related to its complex structure. As a polysaccharide, cellulose is very suitable for membrane materials, but most of the cellulose is insoluble, which increases the difficulty of preparing a coating-forming solution [21]. The FT-IR spectrum of SDF proved the existence of -OH in the sample, which gave Jerusalem artichoke dietary fiber good water solubility and was conducive to the preparation of a coating-forming solution. High-crystalline structures need to consume more energy during thermal decomposition, so materials with high crystallinity generally have higher thermal stability [22]. The results of the XRD analysis showed that the crystallinity of the extracted SDF was as high as 50.21%, and the TGA analysis also showed that the SDF had good thermal stability, which was the required performance of membrane materials [23].
The miscibility of each component in the coating-forming solution affects the microstructure of the film and further affects the mechanical properties and barrier properties of the film. The presence of -OH in SDF enhanced its hydrophilicity, but it was still difficult to dissolve with chitosan. The addition of acetic acid contributes to the dissolution of the two components [24]. In addition, glycerol is hydrophilic, which makes the membrane components form a closer connection by association. When an appropriate amount of SDF was added, the components in the coating-forming solution could effectively dissolve each other, and the prepared film had a dense and uniform microstructure, showing good mechanical properties and barrier properties [25]. The barrier property is a very important performance, which can reflect the preservation ability of a coating. A coating with a good barrier property can effectively prevent microbial infection, reduce fruit moisture loss, and improve fruit storage resistance. If excessive SDF is added into the coating-forming solution, which exceeds the miscible ability of each component, it will cause the separation of membrane components, resulting in an uneven structure of the prepared film and a decrease in the barrier performance [26]. SDF has good thermal stability, and the addition of SDF improves the thermal stability of the film, especially when the SDF content is 0.3%, which is when the thermal stability of the film is the best and most suitable for food packaging [27].
The effect of the coating on blueberry preservation was further tested. The decline in blueberry quality during storage is mainly manifested as weight loss and softening. The loss of blueberry weight is mainly due to the loss of nutrition and water during storage. After weight loss, the skin of the fruit will wrinkle and the luster becomes dark [28]. The blueberry photos in Figure 6A show that the blueberry fruits of all groups were full and glossy on the 0th day. On the 16th day of storage, the blueberries appeared with different degrees of collapse, softening, and peel fold. The film without SDF or with a small amount of SDF (0.1%) had a good ability to prevent fruit weight loss, and this ability decreased with the increase of SDF content [29]. When the SDF content exceeds the mutual solubility of each component in the solution, the microstructure of the film will become uneven and even generate pores. This may be the cause of the increased weight losses of the high-SDF-content groups. Although the excessive addition of SDF resulted in an increase in the weight loss rate of blueberries during storage, it still had a good ability to prevent blueberry corruption, indicating that only water was volatilized through the pores of the film, without the loss of other nutrients in the blueberries [30]. The maturation of the fruit cell wall and the proliferation of microorganisms will cause the softening of blueberry fruit. In the early stage of storage, the peel and pulp of untreated blueberries increased in hardness due to dehydration. In the late stage of storage, the cell wall of the fruit gradually matured and damaged, and the microorganisms began to grow and reproduce, resulting in the softening of the fruit. The coating treatment can effectively delay the cell wall damage. The addition of SDF enhanced the viscosity and support of the coating on blueberries. On the one hand, the coating effectively blocked the invasion of microorganisms from the surface of fruit. On the other hand, it delayed the evaporation of water and the damage of the fruit cell wall, so the coating could maintain the hardness of blueberries and enhance the storage resistance of blueberries [31].
During the storage of blueberries, the consumption of organic acids in the fruit will lead to the growth and reproduction of microorganisms and accelerate corruption. The coating with SDF had a good protective effect on the organic acids in blueberries. Anthocyanins are secondary metabolites in blueberry metabolism [32]. Anthocyanins not only have high nutritional value but also have strong antioxidant capacity and are important nutrients in blueberries. At the beginning of storage, due to the postharvest ripening of blueberries, metabolism produced a certain amount of anthocyanin, and the anthocyanin content in the blueberries increased. With the extension of storage time, anthocyanins were oxidized and consumed. The coating could prevent blueberries from contacting with air and slow down the oxidation of anthocyanins. In addition, the coating treatment slowed down the consumption of organic acids, and the acidic environment was more conducive to the preservation of anthocyanins. In conclusion, the coating with SDF can effectively slow down the loss of nutrients in blueberries during storage [33].

Conclusions
In this study, soluble dietary fiber (SDF) with high purity and good state and properties was extracted from Jerusalem artichoke residue, and the extracted SDF was characterized. Five concentrations of SDF-chitosan composite coatings were prepared using the extracted SDF. The characterization of the coatings shows that the coatings have excellent properties to apply to blueberry preservation, and the results showed that the coating-added SDF could effectively improve the quality of blueberries during 16 days storage. The SDF coating has potential and broad prospects in application of the preservation of blueberries and other berries. The comprehensive utilization of Jerusalem artichoke residue, reducing the manufacturing cost of coating, and expanding the application range of coating need further research.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

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Alleviation of Ammonium Toxicity in Salvia splendens ‘Vista Red’ with Silicon Supplementation

Ammonium (NH4+) toxicity seriously hampers the yield and quality of salvia plants because most varieties or sub-species are highly sensitive to NH4+. Silicon (Si) is an alternative that is used to minimize these disturbances and maintain better growth under NH4+ toxicity. Nevertheless, the mitigatory effects of Si on NH4+-stressed salvia are unknown. Therefore, this study was carried out to determine how Si assists to alleviate the NH4+ toxicity degree in salvia. To this end, salvia plants were cultivated in a controlled environment supplied with a constant N (nitrogen) level (13 meq·L−1) in the form of three NH4+:NO3− ratios (0:100, 50:50, 100:0), each with (1.0 meq·L−1) or without Si. Physiological disorders and typical NH4+ toxicity symptoms, as well as interrupted photosynthesis, were observed in the 100% NH4+-treated plants. Furthermore, cation uptake inhibition and oxidative damage were also imposed by the 100% NH4+ supply. In contrast, in the presence of Si, the NH4+ toxicity degree was attenuated and plant growth was ensured. Accordingly, the NH4+ toxicity appearance ratio decreased significantly. Furthermore, Si-treated plants showed an ameliorated photosynthetic ability, elevated internal K and Ca levels, and enhanced antioxidative capacity, as reflected by improved major antioxidant enzyme activities, as well as diminished accumulation of ROS (reactive oxygen species) and MDA (malondialdehyde). Our findings enlightened the agronomic importance of additional Si to nutrient solutions, especially pertaining to bedding plants at risk of NH4+ toxicity.

Introduction
Nitrogen (N) is a fundamental nutrient that determines plant biomass production and crop gains. The absorption and assimilation of N by plants are predominately in the forms of ammonium (NH 4 + ) and nitrate (NO 3 − ) [1]. The acquisition processes of these two forms regarding the energetic and biochemical aspects are shown to be distinct [2]. NH 4 + assimilation is more metabolically efficient than NO 3 − assimilation because only NH 4 + can be incorporated into organic compounds, which should theoretically mean that NH 4 + is the preferred N form [3,4].
Paradoxically, it appears that most plants manifest poor growth performance when a high NH 4 + level is unintentionally presented or as the exclusive N source [5,6]. Indeed, an excessive supply of NH 4 + (usually millimolar level) for higher plants leads to the suppression of plant growth and development. Abundant ammonium assimilation causes acidic stress, which is considered the primary cause of NH 4 + toxicity [7]. This phenomenon can be developed and identified in many plant species, ranging from crops to vegetables [3,8,9]. Consequently, various characteristics concerning physiology and biochemistry are severely altered: inhibition of the regulation of intracellular pH, disruption of photosynthetic ability, impaired cation influxes (in particular K + , Ca 2+ , Mg 2+ ) and ion equilibrium, and interference with the overproduction of reactive oxygen species [10]. These summed factors during NH 4 + toxicity contribute to detrimental visual symptoms that are characterized by reduced plant growth, leaf chlorosis, and necrosis, as well as stunted roots [10,11].
Salvia (Salvia splendens F. Sellow ex Roemer & J.A. Schultes) is a widely cultivated herbaceous bedding plant in the Salvia genus. It is extensively utilized for culinary purposes and essential oil extraction [12]. Ornamentally, it is valued for the tender flowers and aromatic foliage [13]. However, according to pioneer reports, most of the salvia varieties or sub-species are determined to be extremely sensitive to NH 4 + [6,14,15]. Therefore, the breeding of salvia was highly hindered by the toxic levels of NH 4 + input. Thereafter, more optimal fertilization strategies regarding agronomic and agricultural attempts were warranted. Meanwhile, silicon supplementation benefits salvia growth in harsh environments [16]. Moreover, the supplementations of silicon nutrition can reverse or reduce the NH 4 + toxicity stress degree [17].
As the second-most abundant element after oxygen in the Earth's crust, silicon (Si) was recognized to be a 'quasi-essential' inorganic constituent for higher plants [18]. Outstandingly, the beneficial impacts of Si application were witnessed in plants under biotic [19,20] and abiotic stresses [21,22]. The improvement in photosynthetic capacity due to Si is correlated with the formation of a subcuticular double layer on the leaf epidermis, resulting in greater light interception and absorption abilities and lower water loss via transpiration [17,23]. Furthermore, Si is believed to overcome the depletion of ions caused by the NH 4 + toxicity because Si accumulated in roots promotes the hydraulic movement for the selective influx of cations [24]. On the one hand, Si is associated with the rigidity and integrity of cell walls, which contributes to the maintenance of chloroplast structures and diminishment of malondialdehyde (MDA), which is defined as the end product of lipid peroxidation [17,25]. On the other hand, Si assists in the modulation of activities of key antioxidant enzymes under oxidative stresses, accordingly detoxifying the over-accumulation of reactive oxygen species (ROS, i.e., O 2 ·− , H 2 O 2 ) [21,26,27]. Regarding the ameliorative role of Si against NH 4 + toxicity, relevant work in salvia is highly insufficient. Therefore, to determine the alleviatory effects of Si supplementation on NH 4 + -stressed salvia plants, the growth attributes, photosynthetic ability, key ion accumulations, antioxidative capacity (major antioxidant enzymes activities), ROS content, and lipid peroxidation level in response to an increasing NH 4 + nutrition supply were evaluated in the current study. To the best of the authors' knowledge, this is the first attempt to unveil the mitigator role of Si against NH 4 + toxicity in salvia. The seedlings with similar morphologies were screened out to new 200-cell plug trays (plug tray information can be found on https://www.amazon.com/Seedling-Starter-Trays-Extra-Strength/dp/B01GBBZU2Y accessed on 26 November 2021), which were subjected to running tap water for three days (21 DAS) to leach all the nutrition and maintained in a controlled alternating diurnal regime with 10 h light (10.8 daily light integral (DLI), white LED) and 14 h darkness at an air-conditioned temperature of 23 • C/18 • C and 60 ± 10% relative humidity. Subsequently, three NH 4 + :NO 3 − ratios (0:100, 50:50, and 100:0) at a constant N supply (13 me·L −1 ) were formulated based on the MNS, which corresponded with an optimized Si concentration (1.0 me·L −1 ) sourced from K 2 SiO 3 according to our lab's previous publication [28] or without Si (Table 1). Excessive potassium sourced from K 2 SiO 3 in 0:100, 50:50, and 100:0 solutions were reduced using KNO 3 , K 2 SO 4 , and K 2 SO 4 , respectively, and the resultant losses of nitrate and sulfate were balanced with the addition of nitric and sulfuric acid. Therefore, a total of six solutions consisting of 0:100 NH Si (+) were adopted for the treatments. For each treatment, three repetitions involving a total of 60 plants were laid out with a 2 × 3 factorial scheme in a completely randomized design. Moreover, blank columns of the plug trays between different treatments were intentionally kept.

Measurement of Plant Growth Parameters and Destructive Sampling
The treatment solutions were applied every two days for 15 days (36 DAS). Subsequently, the plants were demounted out of the plug tray and the medium was washed off (39 DAS). Whole-plant fresh weight (surface-blotted with absorbent paper) and dry weight (kept in an air-forced oven at 60 • C for 72 h) were measured with an electronic balance. Lengths of shoots and tap roots, as well as leaf lengths and widths, were measured with a metal ruler. The leaf area was determined with a leaf area analysis meter (Li-3000, Li Cor Inc., Lincoln, NE, USA). The two topmost true leaves from each plant were individually sampled, immediately frozen in liquid nitrogen, and preserved at −70 • C for further analysis.

Calculation of the Ammonium Toxicity Ratio (%)
The toxicity ratio per replicate was determined using the following equation: Ammonium toxicity ratio (%) = No. of plants with ammonium toxicity symptoms 20 × 100% where '20' is the number of plants per replicate.

Estimation of the Photosynthetic Capacity
The photosynthetic ability was estimated herein using the chlorophyll content and maximum photochemical quantum yield of PSII (Fv/Fm value). Specifically, the total chlorophyll concentration (sum of chlorophyll a and b) was determined following a pro-tocol proposed by Sims and Gamon [29]. Fv/Fm was detected using a FluorPen FP 100 (Instruments of Photon Systems, Drásov, Czech Republic).

Determinations of Si, K, Ca, and Mg Concentrations
Ion contents of Si, K, Ca, and Mg in the plants were determined with an inductively coupled plasma (ICP) spectrometer (Perkin Elmer, Rodgau, Germany).
Samples for the ICP analysis were prepared according to a slightly modified procedure [30]: Fresh whole plants were placed in an air-forced oven (Jeio Technology Co. Ltd., Daejeon, Korea) at 70 • C until they reached a constant weight. Afterward, 100 mg of dried samples were ashed using a Nabertherm muffle furnace programmed at 525 • C for 120 min. The ash specimen was digested with 5 mL 25% HCl and adjusted to a final volume of 30 mL by adding 25 mL of distilled water. These incubations were performed at ambient temperature.

Analysis of Antioxidant Enzyme Activities in Leaf Samples
Frozen leaf samples were finely ground in a pre-cooled mortar over an ice bath. Then, 100 mg of the fine powder was quickly weighed and homogenized in an extraction medium (50 mM of PBS, 1 mM of EDTA, 2% polyvinylpyrolidone, 0.05% triton-X at pH = 7.0). The mixture was subjected to centrifugation (13,000 rpm, 20 min, 4 • C) for the acquirement of the supernatant, which was used afterward for the total soluble protein quantification and enzymatic measurements.
The total protein content was quantified using Bradford's reagent [31]. Analysis of the enzyme activities was carried out over an ice bath following the method described by Biju [32]. Superoxide dismutase (SOD) activity was assayed based on NBT (nitroblue tetrazolium) reduction [33]. A method based on ascorbate oxidation was adopted for measuring the ascorbate peroxidase (APX) activity [34]. A rapid and sensitive procedure that utilized H 2 O 2 decomposition was employed for determining the catalase (CAT) activity [35]. Guaiacol peroxidase (GPX) activity was estimated based on the reaction of guaiacol oxidation [36]. Dehydroascorbate reductase (DHAR) activity was measured according to a methodology proposed by Nakano [34]. Glutathione reductase (GR) activity was determined as presented by Mavis [37].

Quantifications of O 2
·− , H 2 O 2 , MDA, and Carotenoids in Leaf Samples The O 2 ·− (superoxide) level was quantified following an approach proposed by Wu using hydroxylamine oxidization [38]. The H 2 O 2 (hydrogen peroxide) concentration was colorimetrically determined using a protocol according to Mukherjee [39]. The level of lipid peroxidation was monitored in terms of the MDA (malondialdehyde) content, which was measured based on the TBA (thiobarbituric acid) reaction [40]. The detailed procedure can be seen in Li's publication [41]. The carotenoid contents were assessed using the identical sample preparation methods as for chlorophyll [29] but the absorbance was spectrophotometrically read at 440 nm.

Statistical Analysis and Graphing
All the data presented are the means ± standard deviations (SDs) of at least three biological replicates. They were subjected to the Duncan's multiple range test or the two-sided Student's t-test to determine the significant differences at p = 0.05 using the SAS statistical software (V. 8.2, Cary, NC, USA) and GraphPad Prism 8.0 (GRAPH PAD software Inc., San Diego, CA, USA), respectively. And the F-test was performed to find the significance between treatments using SAS statistical software 8.2. The PCA graph was plotted using Origin 2022 software (Origin Lab Corp., Northampton, MA, USA) for the variability identification of antioxidative capacity results.

Effects of the Three NH 4 + :NO 3 − Ratios and Si Supplementation on the Plant Growth Attributes
In the absence of Si nutrition, the plant growth characteristics were shaped in contrasting manners by the different N forms. Similar growth was obtained between the plants cultivated with 0:100 and 50:50 NH 4 + :NO 3 − nutrition, whereas plant growth was remarkably slowed in solely NH 4 + -treated plants ( Figure 1A). Indeed, this observation was evidenced in terms of the whole plant weight, leaf area, and tap root length: the whole plant weight, leaf area, and tap root length were significantly reduced by 25, 63.4, and 22.4%, respectively, when the external NH 4 + supply increased from 50 to 100% ( Figure

NH4 + Toxicity Ratio as Influenced by Si Application
Notably, the salvia plants subjected to 100% NH4 + nutrition developed NH4 + toxicity symptoms, irrespective of the Si application, as physiologically characterized by visual chlorosis and foliage necrosis accompanied by burned tips (Figure 2A).
Concomitantly, we obtained strong interactions between the provided N forms and Si application regarding dry weight, shoot length, and leaf length and width, as depicted by the F-test results ( Table 2).

NH 4 + Toxicity Ratio as Influenced by Si Application
Notably, the salvia plants subjected to 100% NH 4 + nutrition developed NH 4 + toxicity symptoms, irrespective of the Si application, as physiologically characterized by visual chlorosis and foliage necrosis accompanied by burned tips (Figure 2A).

Photosynthetic Ability as Affected by the N Form and Si Supplementation
The photosynthetic capacity was visibly affected by the N form, which influenced the chlorophyll content and quantum efficiency of the photosystem II photochemistry, where the latter can be assessed using the Fv/Fm value. The salvia plants cultured with a mixture of NH4 + and NO3 − possessed a higher chlorophyll content. More chlorophyll degradation was observed when the external NH4 + supply increased from 50 to 100% ( Figure 3A). In a However, Si supplementation to the 100:0 NH 4 + :NO 3 − solution drastically ameliorated the plant growth status. Consequently, statistically, the NH 4 + toxicity appearance ratio after the Si addition significantly decreased (p = 0.0005) from 93.3 to 60% ( Figure 2B).

Photosynthetic Ability as Affected by the N Form and Si Supplementation
The photosynthetic capacity was visibly affected by the N form, which influenced the chlorophyll content and quantum efficiency of the photosystem II photochemistry, where the latter can be assessed using the Fv/Fm value. The salvia plants cultured with a mixture of NH 4 + and NO 3 − possessed a higher chlorophyll content. More chlorophyll degradation was observed when the external NH 4 + supply increased from 50 to 100% ( Figure 3A). In a similar manner to the Si-deficient plants, a progressively reduced Fv/Fm value was observed in response to an enhanced external NH 4 + supply. Specifically, salvia plants treated with 0:100 NH 4 + :NO 3 − solution showed 2.1 and 19.6% higher Fv/Fm values, respectively, compared with those cultured in 50:50 and 100:0 NH 4 + :NO 3 − regime ( Figure 3B).

The Accumulation of Silicon (Si), Potassium (K), Calcium (Ca), and Magnesium (Mg)
The Si supplementations for salvia plants cultivated under different treatments not only distinctly influenced the internal Si level, but also the uptake of certain NH4 + -associated cations, such as K + , Ca 2+ , and Mg 2+ .
Obviously, the external Si application significantly increased the accumulation of Si in plants, regardless of the NH4 + :NO3 -ratios. Specifically, the incorporation of Si in the 50:50 NH4 + :NO3 − solution provided a notably improved 4.3-fold higher amount of Si to the plants; likewise, the Si content in solely NO3 − -treated and NH4 + -treated salvia plants respectively enhanced the internal Si content by 3.4-and 3.3-fold after Si was applied ( Figure  4A). Notably, no statistical difference was found between the different N forms, regardless of the Si supply ( Figure 4A).
Thereafter, for the Si-deficient plants, we detected a gradual reduction in internal K and Ca levels in response to an increasing external level of NH4 + supply from 0 to 100%. However, as expected, except for the monitored Mg content in exclusively NH4 + -cultured plants, Si supplementation dramatically enhanced the K and Ca concentrations in salvia plants grown in both the 50:50 and 100:0 NH4 + :NO3 − regimes ( Figure 4B). Surprisingly, the K, Ca, and Mg contents did not increase for the solely NO3 − -treated plants in the presence of Si ( Figure 4B). More importantly, the plants upgraded their photosynthetic abilities to varying extents in response to Si supplementation, especially for the solely NH 4 + -treated plants. The addition of Si to the plants grown in the 100:0 NH 4 + :NO 3 − solution markedly enhanced the total chlorophyll content by 10% ( Figure 3A). In parallel, in comparison with the 100:0 NH 4 + :NO 3 − Si (−), the Fv/Fm value was significantly higher for the plants nourished with Si ( Figure 3B).

The Accumulation of Silicon (Si), Potassium (K), Calcium (Ca), and Magnesium (Mg)
The Si supplementations for salvia plants cultivated under different treatments not only distinctly influenced the internal Si level, but also the uptake of certain NH 4 +associated cations, such as K + , Ca 2+ , and Mg 2+ .
Obviously, the external Si application significantly increased the accumulation of Si in plants, regardless of the NH 4 + :NO 3 − ratios. Specifically, the incorporation of Si in the 50:50 NH 4 + :NO 3 − solution provided a notably improved 4.3-fold higher amount of Si to the plants; likewise, the Si content in solely NO 3 − -treated and NH 4 + -treated salvia plants respectively enhanced the internal Si content by 3.4-and 3.3-fold after Si was applied ( Figure 4A). Notably, no statistical difference was found between the different N forms, regardless of the Si supply ( Figure 4A).

Responses of Antioxidant Capacity to N Forms and Si Application
When the plants suffered from external stress, the oxidative protective system regarding antioxidant enzymes would be triggered to lower the stress degree. During this process, the activity of antioxidant enzymes was upregulated to produce enhanced antioxidant capacity. The activities of major antioxidant enzymes concerning SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase), GPX (guaiacol peroxidase), GR (glutathione reductase), and DHAR (dehydroascorbate reductase) were therefore quantified.
When the external NH4 + level increased from 50 to 100%, Si-deficient plants gradually improved the activity of antioxidant enzymes; similarly, the addition of 50% NH4 + to a solely NO3 − solution to varying extents decreased the enzyme activities, except for APX ( Figure 5B). By contrast, the added Si significantly boosted the enzyme activities, independent of the NH4 + :NO3 − ratios ( Figure 5). Outstandingly, the addition of Si to a 100:0 NH4 + :NO3 − solution produced a 1.4-fold higher SOD activity relative to 100:0 NH4 + :NO3 − Si (−) (***, p = 0.0003) ( Figure 5A). Concomitantly, and also significantly, less pronounced increases were observed for other enzyme activities in the presence of Si: supplementations of Si to the plants reinforced the activities of APX, CAT, GPX, GR, and DHAR, regardless of the NH4 + :NO3 − ratios ( Figure 5B-F). Thereafter, for the Si-deficient plants, we detected a gradual reduction in internal K and Ca levels in response to an increasing external level of NH 4 + supply from 0 to 100%. However, as expected, except for the monitored Mg content in exclusively NH 4 + -cultured plants, Si supplementation dramatically enhanced the K and Ca concentrations in salvia plants grown in both the 50:50 and 100:0 NH 4 + :NO 3 − regimes ( Figure 4B). Surprisingly, the K, Ca, and Mg contents did not increase for the solely NO 3 − -treated plants in the presence of Si ( Figure 4B).

Responses of Antioxidant Capacity to N Forms and Si Application
When the plants suffered from external stress, the oxidative protective system regarding antioxidant enzymes would be triggered to lower the stress degree. During this process, the activity of antioxidant enzymes was upregulated to produce enhanced antioxidant capacity. The activities of major antioxidant enzymes concerning SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase), GPX (guaiacol peroxidase), GR (glutathione reductase), and DHAR (dehydroascorbate reductase) were therefore quantified.
When the external NH 4 + level increased from 50 to 100%, Si-deficient plants gradually improved the activity of antioxidant enzymes; similarly, the addition of 50% NH 4 + to a solely NO 3 − solution to varying extents decreased the enzyme activities, except for APX ( Figure 5B). By contrast, the added Si significantly boosted the enzyme activities, independent of the NH 4 + :NO 3 − ratios ( Figure 5). Outstandingly, the addition of Si to a 100:0 NH 4 + :NO 3 − solution produced a 1.4-fold higher SOD activity relative to 100:0 NH 4 + :NO 3 − Si (−) (***, p = 0.0003) ( Figure 5A). Concomitantly, and also significantly, less pronounced increases were observed for other enzyme activities in the presence of Si: supplementations of Si to the plants reinforced the activities of APX, CAT, GPX, GR, and DHAR, regardless of the NH 4 + :NO 3 − ratios ( Figure 5B-F).

Oxidative Damage as Affected by the Three NH4 + :NO3 − Ratios and Si Supplementations
To better understand the responses of antioxidant capacity to the N form and Si application, the oxidative damage in terms of the ROS accumulation (O2 ·− and H2O2) and lipid peroxidation (MDA, chlorophyll, and carotenoids) were evaluated. NH4 + stress rapidly increased the O2 ·− (17.9%), H2O2 (37.4%), and MDA (26.0%) contents, while reducing the carotenoids (11.9%) in contrast to the plants cultivated with 50:50 NH4 + :NO3 − (Figure 6). In addition, a gradual enhancement was found regarding the O2 ·− and H2O2 accumulation as the external NH4 + increased from 0 to 100% ( Figure 6A,B,D).
As expected, the supplementation of Si to varying extents decreased the ROS accumulation and lipid peroxidation, regardless of the NH4 + :NO3 − solutions considered ( Figure  6). Outstandingly, after Si supplementation, the solely NH4 + -treated salvia remarkably diminished the accumulation of O2 ·− (22.2%), H2O2 (16.4%), and the production of MDA (22.7%); meanwhile, a significant increase was observed in the carotenoids content (6.3%). and Si (+) were determined using the two-tailed Student's t-test. Error bars denote mean ± standard deviation (SD).  (Figure 6). In addition, a gradual enhancement was found regarding the O 2 ·− and H 2 O 2 accumulation as the external NH 4 + increased from 0 to 100% ( Figure 6A,B,D).

Oxidative
As expected, the supplementation of Si to varying extents decreased the ROS accumulation and lipid peroxidation, regardless of the NH 4 + :NO 3 − solutions considered ( Figure 6). Outstandingly, after Si supplementation, the solely NH 4 + -treated salvia remarkably diminished the accumulation of O 2 ·− (22.2%), H 2 O 2 (16.4%), and the production of MDA (22.7%); meanwhile, a significant increase was observed in the carotenoids content (6.3%).

Responses between Antioxidant Capacity and Si Supplementation Are Supported by PCA Analysis
To visualize the influences of different NH4 + :NO3 − ratios with and without Si on the antioxidant enzymes and oxidative damage, as well as the relationship between antioxi dant capacity and Si supplementations, PCA (principal component analysis) based on the antioxidant enzyme activities, ROS accumulation (O2 ·− and H2O2), and lipid peroxidation data set was performed.
The computed model produced the related parameters along the first two principa components that captured 56.9% (PC1: 37.1%, PC2: 19.8%) of the total observed data var iability (Figure 7). The majority of the Si-deficient plants 'Si (−)' were distributed in the lef quadrants of the PC1 scatter plot, whereas Si-sufficient plants 'Si (+)' were separated to the right quadrants of the PC1 scatter plot, which indicated that Si (−) plants were nega tively correlated with the Si (+) plants on these mentioned traits (Figure 7 'PC1').

Responses between Antioxidant Capacity and Si Supplementation Are Supported by PCA Analysis
To visualize the influences of different NH 4 + :NO 3 − ratios with and without Si on the antioxidant enzymes and oxidative damage, as well as the relationship between antioxidant capacity and Si supplementations, PCA (principal component analysis) based on the antioxidant enzyme activities, ROS accumulation (O 2 ·− and H 2 O 2 ), and lipid peroxidation data set was performed.
The computed model produced the related parameters along the first two principal components that captured 56.9% (PC1: 37.1%, PC2: 19.8%) of the total observed data variability (Figure 7). The majority of the Si-deficient plants 'Si (−)' were distributed in the left quadrants of the PC1 scatter plot, whereas Si-sufficient plants 'Si (+)' were separated to the right quadrants of the PC1 scatter plot, which indicated that Si (−) plants were negatively correlated with the Si (+) plants on these mentioned traits (Figure 7 'PC1').

Discussion
The NH4 + -spiked supply inevitably instigated the over-assimilation of NH4 + by plants, resulting in plant tissue acidification, which is the primary cause of NH4 + toxicity [7]. It not only curtailed the shoot and root growth and the biomass production but also produced certain visual detrimental impacts [10,11,43,44]. The alleviatory effects of Si against NH4 + toxicity are well documented in many plant species, such as cauliflower [45], cucumber [46], tomato [47], and sugar beet [18], but the specific study on Si treatments in NH4 + -stressed salvia to date is still incipient. Therefore, the main purpose of the current study was to determine whether Si can attenuate the NH4 + toxicity degree in salvia.

Si Promoted Plant Growth and Alleviated the NH4 + Toxicity Degree
Si is able to promote several desirable plant physiological and morphological processes, consequently enhancing the growth and yield [48,49]. In our trials, salvia plants significantly improved in the shoot-and root-related parameters, as well as the whole dry matter mass, with Si supplementation (Figure 1, Table 2). These data were consistent with the findings of Barreto, who showed beneficial effects of Si in cauliflower and broccoli, regardless of the NH4 + :NO3 − ratio [45]. Likewise, the effect of added Si to a 50:50 NH4 + :NO3 − solution on the salvia plants improved these traits to varying extents ( Figure 1, Table 2). Thus, for the non-NH4 + -stressed plants, the strategy of adding Si to fertilizer can promote better plant growth, thereby contributing to a more sustainable production [17,18,22,50].
As stated above, 100% NH4 + nutrition given to the salvia plants showed not only a poor growth performance but also the development of typical NH4 + toxicity symptoms, as characterized by leaf necrosis, chlorosis, and stunted roots ( Figure 1, Table 2, Figure 2A), which agrees well with our previous reports that found that greenhouse-grown salvia was also sensitive to high NH4 + levels [6,15].

Discussion
The NH 4 + -spiked supply inevitably instigated the over-assimilation of NH 4 + by plants, resulting in plant tissue acidification, which is the primary cause of NH 4 + toxicity [7]. It not only curtailed the shoot and root growth and the biomass production but also produced certain visual detrimental impacts [10,11,43,44]. The alleviatory effects of Si against NH 4 + toxicity are well documented in many plant species, such as cauliflower [45], cucumber [46], tomato [47], and sugar beet [18], but the specific study on Si treatments in NH 4 + -stressed salvia to date is still incipient. Therefore, the main purpose of the current study was to determine whether Si can attenuate the NH 4 + toxicity degree in salvia.

Si Promoted Plant Growth and Alleviated the NH 4 + Toxicity Degree
Si is able to promote several desirable plant physiological and morphological processes, consequently enhancing the growth and yield [48,49]. In our trials, salvia plants significantly improved in the shoot-and root-related parameters, as well as the whole dry matter mass, with Si supplementation (Figure 1, Table 2). These data were consistent with the findings of Barreto, who showed beneficial effects of Si in cauliflower and broccoli, regardless of the NH 4 + :NO 3 − ratio [45]. Likewise, the effect of added Si to a 50:50 NH 4 + :NO 3 − solution on the salvia plants improved these traits to varying extents ( Figure 1, Table 2). Thus, for the non-NH 4 + -stressed plants, the strategy of adding Si to fertilizer can promote better plant growth, thereby contributing to a more sustainable production [17,18,22,50].
As stated above, 100% NH 4 + nutrition given to the salvia plants showed not only a poor growth performance but also the development of typical NH 4 + toxicity symptoms, as characterized by leaf necrosis, chlorosis, and stunted roots ( Figure 1, Table 2, Figure 2A), which agrees well with our previous reports that found that greenhouse-grown salvia was also sensitive to high NH 4 + levels [6,15]. Yet, as the most crucial part, Si is believed to reinforce the tolerance against abiotic stresses induced by NH 4 + toxicity [17,50]. As expected, the NH 4 + -stressed plant growth was ensured and the manifested NH 4 + toxicity degree was visibly reduced after the Si supplementation ( Figure 2A). Concomitantly, the injured leaves and roots were also drastically ameliorated ( Figure 1B-D). Consequently, the added Si significantly decreased the appearance of NH 4 + toxicity symptoms as compared with the Si-deprived plants ( Figure 2B). These data indicated that the NH 4 + toxicity degree in salvia could be alleviated by the added Si.

Si Ameliorated Damaged Photosynthetic Capacity Caused by NH 4 + Toxicity
Chlorophyll is an integral part of the photosynthetic reaction; it plays an important role in the absorption and transformation of light to chemical energy in support of the CO 2 fixation [51]. Plants that possess higher chlorophyll contents are usually susceptible to exhibiting a greater light reaction rate in photosynthesis [6,52,53]. Moreover, the ratio between the variable chlorophyll fluorescence (Fv) and maximum fluorescence (Fm), namely, 'Fv/Fm', is a key indicator of the PSII (photosystem II) capacity, which is positively correlated to the quantum yield efficiency [54]. Nutrient-stimulated stresses can limit the photosynthetic ability mainly by damaging the PSII, conferring that Fv/Fm can be considered as a necessary criterion of early dynamic photoinhibition stresses [55,56]. Thus, the determinations of chlorophyll level and fluorescence Fv/Fm indicated the overall photosynthetic capacity.
In the present study, salvia seedlings displayed notably declined photosynthesis under NH 4 + stresses, as characterized by the diminished chlorophyll content and Fv/Fm value ( Figure 3). Numerous pioneering reports in cabbage [9,50], lettuce [9], and tomato [57] also revealed impaired photosynthetic capacity under a high NH 4 + supply. Nevertheless, the Si-treated salvia plants had effectively enhanced chlorophyll contents and Fv/Fm values, irrespective of the NH 4 + :NO 3 − ratio (Figure 3). The elevated leaf area after Si supplementation possibly improved the light interception, presenting a higher photosynthetic rate and CO 2 assimilation [17,58]; augmented stomatal growth following Si supplementation, which can assist in the proper gas exchange and maintain the water status [17,18,45]; and the deposition of Si is able to enhance the protection of the photosynthetic pigments [17,59]. Succinctly, Si can help to improve the photosynthetic ability of salvia plants under NH 4 + stresses.

Si Alleviated the Inhibition of Key Cation Uptakes under NH 4 + Toxicity
To determine the uptake of Si and the quadratic effects on the regulations of other key ions, we further individually determined the K, Ca, Mg, and Si contents in the plant tissues from different treatments. It is evident that the external Si supply can significantly improve the internal Si level in plants ( Figure 4A), exhibiting a similar Si absorption rate to many species [18,[45][46][47], which indicated that salvia is a Si-accumulating species [60,61]. It is noteworthy that only minor fluctuations (no statistical differences) of Si content among different N treatments were observed, regardless of the Si-sufficient or -deficient plants ( Figure 4A), which showed a negligible effect of the N form on the Si uptake in salvia.
Excessive NH 4 + presented to the plants could instigate a nutritional disequilibrium due to the antagonistic impact of NH 4 + against the uptake of other inorganic cations, particularly K + , Ca 2+ , and Mg 2+ . Therefore, a large amount of cation extrusion is a key marker of NH 4 + toxicity [10,50,62]. Indeed, significant reductions in the contents of K and Ca in Si-deficient plants were observed when the external NH 4 + supply increased from 0 to 100% ( Figure 4B). Our data were in accordance with previous findings, displaying remarkably diminished K + , Ca 2+ , and Mg 2+ in cauliflower, broccoli, and cabbage cultivated with 100% NH 4 + [45,50]. Si supplementation of NH 4 + -stressed salvia plants distinctly reduced the extrusions of K and Ca ( Figure 4B). Furthermore, greater K, Ca, and Mg contents were found in the plants cultured with 50:50 NH 4 + :NO 3 − solutions with Si supplementation (Figure 4B), which may explain why the Si-sufficient plants had a better growth performance (Figure 1).
Si-treated salvia plants growing in the 100:0 NH 4 + :NO 3 − regime had higher contents of K relative to their Si-deprived counterparts, probably due to the finding that the inclusion of Si could improve the activity of membrane-located H-ATPase, which is a primary trans-porter in the K acquisition and regulation processes [63,64]. The uptake and distribution of NH 4 + were antagonistically modulated by K [15,65]. Accordingly, the alleviation of NH 4 + toxicity may stem from the modulation of Si on K.

Improved Antioxidative Enzyme Activities by Si Contributed to the Mitigation of NH 4 + Toxicity
Generally, the balance between the production and detoxification of ROS (mainly by O 2 ·− and H 2 O 2 ) can be disturbed when plants are under abiotic stresses [66]. NH 4 + toxicity-induced osmotic changes can boost the excessive accumulation of O 2 ·− and H 2 O 2 , accordingly resulting in higher lipid peroxidation and pigment degradation [11,17,62]. Plants have developed sophisticated strategies to tightly regulate the steady-state levels of ROS, such as antioxidant enzymes. As depicted in Figure 5, SOD was the most important and first line of defense that directly scavenged the O 2 ·− into H 2 O 2 , and subsequent rapid decomposition of H 2 O 2 to water was aided by APX, CAT, and GPX [67]. Concurrently, regarding the ascorbate-glutathione (ASA-GSH) metabolizing cycle, the synergistic action regarding APX and GPX interacting with DHAR and GR, respectively, further contributes to the conversion of H 2 O 2 to water [67,68] [67,69].
Consistently, in the absence of Si, the antioxidative enzyme activities were amplified to varying extents when the external NH 4 + supply increased from 50 to 100%, except for APX ( Figure 5), illustrating that NH 4 + -stressed salvia adopted a species-specific strategy in the lowering of NH 4 + toxicity [68]. Conspicuously, SOD, CAT, GPX, and DHAR activities in solely NH 4 + -treated plants were significantly enhanced as compared with the plants cultured with the 50:50 NH 4 + :NO 3 − solution ( Figure 5A,C,D,F). Numerous studies showed that Si improved the antioxidative defense capacity to reduce the excessively produced ROS under abiotic stresses [17,50,70,71]. Indeed, the added Si distinctly promoted the antioxidative ability as characterized by the activated activities of antioxidative enzymes in the presented metabolism route ( Figure 5). Outstandingly, SOD was the first sensing line of ROS, and thus, possessed the most reinforcements ( Figure 5A), meaning that SOD is extremely important in the rapid scavenging of O 2 ·− in salvia [72]. Therefore, Si can enhance the antioxidative capacity, as evidenced by boosting the activities of antioxidative enzymes, especially in NH 4 + -stressed plants, accordingly indicating a tight regulation by Si against NH 4 + toxicity-induced ROS metabolism. It can be assumed that the improved antioxidative enzyme activities by Si contributed to the mitigation of the NH 4 + toxicity degree. Previous reports regarding cabbage [50], cucumber [73], bamboo [74], and wheat [75] also advocated for the beneficial impacts of Si on the antioxidant machinery.

Si Decreased the ROS Accumulation, Lipid Peroxidation, and Pigment Degradation
As depicted above, a disproportionate accumulation of ROS could oxidize the membrane lipids and photosynthetic pigments, causing an upsurge in MDA and a decrease in chlorophyll and carotenoid contents [17,50,76]. Consistent with this, for Si-deficient plants, solely NH 4 + -treated salvia rapidly intensified the levels of O 2 ·− and H 2 O 2 while significantly decreasing the carotenoid and chlorophyll contents ( Figures 3A and 6), demonstrating that the integrity of the cellular membrane was damaged by NH 4 + toxicity. The MDA content can directly reflect the cell membrane injuries [25,77]. Similar findings were previously acquired in Hydrilla verticillata [78] and rice [79].
Meanwhile, in line with many previous studies [32,41,50,80], we also showed that the supplementation of Si decreased the ROS content and MDA level as a consequence of increased antioxidative enzyme activities ( Figure 6A-C). Moreover, degradation of chlorophyll and carotenoid contents were markedly truncated after the application of Si, except in solely NO 3 − -treated salvia plants ( Figures 3A and 6D). It is worth noting that the involvement of Si contributed to the decline of ROS accumulation, lipid peroxidation, and pigment degradation, which could be a modulation strategy that is involved in the mitigation of the NH 4 + toxicity mechanism. In addition, the PCA data further exhibited the distinguished differences in the antioxidative defense system between the Si-deficient and Si-sufficient salvia plants, regardless of the supplied N form (Figure 7). These results suggested that Si could also enhance the antioxidative enzyme activities and concomitantly diminish the antioxidative injuries, especially in NH 4 + -stressed salvia.

Conclusions
To sum up, the damage of Salvia splendens raised in a controlled environment that introduced NH 4 + toxicity manifested as seriously declined plant growth (biomass, shoot length, leaf length and width, leaf area, tap root length) and decreased photosynthetic ability (chlorophyll, quantum efficiency, and carotenoids). Similarly, NH 4 + -stressed salvia seedlings showed lower cation contents (K, Ca), together with increased synthesis of MDA and ROS. Conversely, the supplementation of Si significantly alleviated the NH 4 + toxicity degree. This alleviation potential conferred by Si may be ascribed to the ameliorated photosynthesis, increased cation uptake (especially K), diminished lipid peroxidation (MDA), and enhanced performance of the antioxidative machinery to scavenge the overproduction of ROS. It was accordingly concluded that the NH 4 + toxicity could be mitigated by Si in Salvia splendens.
In addition, acidic stress caused by excessive NH 4 + assimilation was elucidated as the primary cause of NH 4 + toxicity. Therefore, further molecular study on the interaction between Si and H + under high NH 4 + conditions appears mandatory to interpret how Si assists in the alleviation of NH 4 + toxicity.