Exogenous Si Mitigates the Effects of Cinnamic-Acid-Induced Stress by Regulating Carbon Metabolism and Photosynthetic Pigments in Cucumber Seedlings

Abstract

(1) Background: Cinnamic acid (CA) is a harmful substance secreted by the roots of continuous-cropping crops. (2) Methods: This study aimed to investigate how exogenous Si affects chlorophyll content and carbon metabolism in cucumber seedlings under CA-induced stress. (3) Results: The levels of chlorophyll a, chlorophyll b, chlorophyll a+b, and carotenoids were significantly reduced due to CA-induced stress. The addition of exogenous Si significantly alleviated this reduction. Under CA-induced stress, exogenous Si significantly increased the activities of ribulose-1,5-bisphosphate carboxylase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, fructose-1,6-bisphosphate aldolase, and transketolase. CA-induced stress significantly increased the fructose, glucose, and sucrose contents and reduced the starch content in the leaves and roots of seedlings. Similarly, the sucrose phosphate synthase, sucrose synthase, acid invertase, and neutral invertase activities were significantly reduced in plants under CA-induced stress. Overall, exogenous Si significantly reduced the soluble sugar content, increased the starch content, and promoted sucrose metabolism-related enzymatic activity in seedlings. (4) Conclusion: Exogenous Si can effectively increase the content of photosynthetic pigments in leaves of seedlings and maintain the balance of osmotic potential in the plant by reducing the accumulation of carbon assimilation products, which ultimately promotes tolerance to CA-induced autotoxicity stress.

1. Introduction

Cucumber (Cucumis sativus L.) is an economically important crop and one of the most widely cultivated vegetables in facilities in China. Intensive cultivation in facilities causes leaching from above-ground plant parts, which causes the accumulation of autotoxins and plant root secretions in the soil and leads to the occurrence of continuous cropping obstacles [1]. Cucumber roots are weaker and more susceptible to injury from autotoxic substances because of their sensitive response. Most autotoxins and root secretions are phenolic chemosensitizers, such as cinnamic acid (CA) [2]. The presence of CA in the soil affects seed germination, which has adverse effects on seedling morphogenesis and subsequent plant growth and development, resulting in reduced crop yield and quality [3]. High concentrations of CA (>200 mg/L) affect the morphology and growth of the soil mycelium, destroy microbial activity and soil genetic diversity, and cause imbalances in soil microbiology [4,5]. CA can also exacerbate the occurrence of soil-borne diseases [6]. In addition, CA-induced autotoxicity can reduce photosynthetic pigment content, inhibit the activities of key enzymes for photosynthesis, and cause the accumulation of osmoregulatory substances in plants [7,8,9].

Si—the second-most abundant element in Earth’s crust—is present in plants at levels equal to, or exceeding those, of other common elements (such as N, P, and K) that are taken up by plants through fertilization. Si is more readily taken up by plant roots when it is present in the soil as a monomer or monosilicate (H₄SiO₄) [10]. Under normal cultivation conditions, Si (nano-SiO₂) application promotes both seed germination and seedling growth. Several monocots and some dicots actively absorb and accumulate large amounts of Si in their organs, which promotes crop growth, development, yield, and quality [11,12]. Si has also been shown to positively regulate plant resistance to biotic and abiotic stresses. Moreover, the addition of exogenous Na₂SiO₃ can significantly increase pigment content in crop leaves, stimulate the phenylpropane pathway to promote the production of phenols and flavonoids, and effectively alleviate Al³⁺ ion toxicity in crops [13]. Si (H₂SiO₃) application can also regulate hormone levels in plants [14], establish protective barriers, regulate ion homeostasis [15], reduce lipid peroxidation, mitigate oxidative damage [16], alter photosynthetic parameters, up-regulate the expression of photosynthesis-related genes, and promote photosynthesis [17], thus allowing crops to resist stresses caused by adverse external conditions. The use of Si as a fertilizer in agriculture is of great ecological and environmental benefit due to its non-corrosive and low-polluting nature, and research on the role of Si in this regard has been popular in recent years.

Carbohydrate metabolism is a crucial component of whole-plant metabolic processes [18,19]. A plant under stress conditions inevitably depends on endogenous carbon metabolism [20]. As the main osmoregulatory substance, carbohydrates regulate the osmotic potential of cells through anabolic and catabolic metabolic processes, thus helping plants resist the effects of environmental stress [21]. Studies have shown that foliar spraying of Na₂SiO₃·9H₂O can significantly increase the accumulation of certain carbohydrates, such as soluble sugar and sucrose, in stems and leaves, increasing stem strength and lodging resistance under stress [22]. In addition, the application of K₂SiO₃ to plants under salt stress can increase sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities, promote carbon metabolism, and improve crop growth and yield [23].

In the current study, we investigated whether Si application is beneficial for cucurbits. The application of Si (H₂SiO₃) fertilizer has previously been shown to improve fruit quality, extend fruit storage time, and increase the yield of cucurbit crops [24,25,26]. In addition, Si application improves osmoregulatory potential and increases osmotic pressure, nutrient transport, water conduction, and stomatal opening and closing in plants [27]. However, it is not known whether Si can mitigate the effects of CA-induced stress by regulating carbon metabolism in cucumber seedlings. Therefore, we investigated the effects of Si on the light-trapping capacity of antenna pigments, key enzyme activities in the Calvin cycle, the levels of carbohydrates (such as fructose, glucose, sucrose, and starch) associated with carbon metabolism, and related enzyme activities in cucumber seedlings under CA-induced stress. The aim of this study was to determine the mitigating effect of Si on the growth of cucumber seedlings under CA-induced stress and to analyze the underlying mechanism. Our results provide a theoretical reference for future studies on the mitigation of autotoxicity stress by Si.

2. Materials and Methods

2.1. Cultivation of Test Materials

The experimental materials used in this study were selected and cultured according to the methods described by previous authors [28]. Cucumber seeds of the “Xinchun No. 4” cultivar were purchased from the seed market of the Gansu Academy of Agricultural Sciences. Neat and full cucumber seeds were soaked for 8 h, disinfected with 0.03% potassium permanganate solution for 15 min, and placed in a tray lined with two layers of filter paper. An appropriate amount of tap water was added to ensure wetness without dripping. The trays were covered with cling film and placed in an artificial climate chamber, and the seeds were allowed to germinate under dark conditions at 28 °C. Light incubation was performed when the proportion of germinating seeds exceeded 80%. During the incubation period, the diurnal temperature was maintained at 28 °C/18 °C, the light intensity was 30,000 lx, the photoperiod was 12 h, and the seedlings were provided with sufficient water. After 5–6 d, cucumber seedlings of uniform growth were transplanted into hydroponic boxes (4 seedlings per box), fixed with sponges, and cultured in an artificial climate chamber. The nutrient solution used was the Yamazaki cucumber special formula (pH = 6.0 ± 0.1; 3.5 mM Ca (NO₃)₂·4H₂O, 6 mM KNO₃, 1 mM NH₄H₂PO₄, 2 mM MgSO₄·7H₂O, 70 μM EDTA-Fe, 10 μM MnSO₄·4H₂O, and 50 μM H₃BO₃), which was changed every 2 d. When the cucumber plants grew to have two cotyledons and one true leaf, intact seedlings with uniform growth were selected for different treatments.

2.2. Experimental Design

Cucumber seedlings with uniform growth were selected after 20 d of incubation in nutrient solution. For the experiment, we used a moderate CA concentration (0.8 mM) for the stress treatment and the optimal exogenous Si (Na₂SiO₃·9H₂O) concentration (1.0 mM) for stress mitigation, as reported by a previous study [28]. The treatment groups were as follows:

(1)

CK: 1/2 Yamazaki nutrient solution;

(2)

Si: 1/2 Yamazaki nutrient solution + 1.0 mM Si;

(3)

CA: 1/2 Yamazaki nutrient solution + 0.8 mM CA;

(4)

CA + Si: 1/2 Yamazaki nutrient solution + 0.8 mM CA + 1.0 mM Si.

2.3. Measurement Index and Method

2.3.1. Pigment Content

The photosynthetic pigment content was determined using the acetone method [29]. After 10 d of treatment, 6 leaves were randomly selected from cucumber seedlings in the different treatment groups. The freshly collected leaves were wiped clean, cut, and mixed. A 0.1 g sample of the leaves was placed in a test tube, and 10 mL of 80% acetone solution was added. Pigments were extracted in the dark for 48 h (during which the test tubes were shaken periodically) until the leaves turned white, and each treatment was performed in triplicate. The extracts were filtered, and their levels of absorbance at 440, 663, and 645 nm were measured by spectrophotometry. The levels of chlorophyll a, chlorophyll b, chlorophyll a+b, and carotenoids were calculated per gram of leaves.

2.3.2. Activity of Key Enzymes of Photosynthesis

The activities of ribulose-1, 5-bisphosphate carboxylase (Rubisco), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1, 6-bisphosphatase (FBPase), fructose-1, 6-bisphosphate aldolase (FBA), and transketolase (TK) were determined using a plant enzyme immunoassay kit from Shanghai Yaji Biotechnology Co., Ltd. (Shanghai, China). For this process, 1 g of leaf tissue was cut into pieces and ground into a powder in a mortar with liquid nitrogen. Subsequently, 9 mL of homogenate (0.01 mol/l PBS, pH= 7.2–7.4) was added, and the mixture was centrifuged. The reagent in the kit was added to the resulting supernatant in accordance with the instructions of the kit. Finally, the absorbance value at 450 nm was measured using an HBS-1096A microplate reader (Detie, Nanjing, China), from which the concentration was calculated. The unit of enzyme activity is expressed in IU/L.

2.3.3. Carbohydrate Content of Cucumber Leaves and Roots

Carbohydrates were measured using the method of Buysse and Merckx [30] as follows: 0.3 g of fresh leaves were placed in a mortar, ground into powder with liquid nitrogen, and loaded into a centrifuge tube. Then, 8 mL of 80% ethanol solution (v/v) was added to it. The centrifuge tube was placed in a water bath at 85 °C for 30 min and then centrifuged at 3500× g for 5 min at 25 °C. The supernatant was discarded, and the remaining residue was extracted three times as described above. The extracted supernatants were combined, and activated carbon (0.01 g) was added to adsorb the pigment. The resulting solution was used to measure the levels of fructose, glucose, and sucrose, and the precipitate was used to measure the starch content.

2.3.4. Enzyme Activities Related to Carbon Metabolism in Cucumber Leaves and Roots

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SS), acid invertase (AI), and neutral invertase (NI) were determined using plant enzyme immunoassay kits from Shanghai Yaji Biotechnology Co., Ltd. For this process, 1 g of leaf tissue was cut into pieces and ground into a powder in a mortar with liquid nitrogen. Subsequently, 9 mL of homogenate (0.01 mol/l PBS, pH= 7.2–7.4) was added, and the mixture was centrifuged. The reagent in the kit was added to the resulting supernatant in accordance with the instructions of the kit. Finally, the absorbance value at 450 nm was measured using an HBS-1096A microplate reader (Detie, Nanjing, China), from which the concentration was calculated. The unit of enzyme activity is expressed in IU/L.

2.4. Statistical Analysis

Excel 2016 (Microsoft Corp., Washington, WA, USA) and SPSS statistical software (IBM SPSS Statistics for Windows, version 19.0; IBM Corp., New York, NY, USA) were used for the statistical analysis of the data. One-way analysis of variance (ANOVA) and Duncan’s test were used for multiple comparisons (p < 0.05).

3. Results

3.1. Effect of Si on the Photosynthetic Pigment Content of Cucumber Leaves under CA-Induced Stress

The levels of chlorophyll a (Figure 1A), chlorophyll b (Figure 1B), chlorophyll a+b (Figure 1C), and carotenoids (Figure 1D) in cucumber leaves showed consistent trends in each treatment group. Compared with the control (CK) group, the levels of chlorophyll a, chlorophyll b, chlorophyll a+b, and carotenoids were significantly reduced by 47.91%, 55.24%, 49.47%, and 33.57%, respectively, in the CA-induced-stress treatment group. However, the exogenous addition of Si under conditions of CA-induced stress significantly increased the chlorophyll and carotenoid contents of the cucumber leaves by 19.72% (chlorophyll a), 17.06% (chlorophyll b), 19.25% (chlorophyll a+b), and 14.76% (carotenoids), compared to those in the CA treatment group. Compared with those of the CK, the chlorophyll a, chlorophyll b, chlorophyll a+b, and carotenoid contents decreased significantly in the Si-only treatment; however, the addition of Si increased the contents of these pigments under CA stress. These results indicate that CA-induced stress either blocked chlorophyll synthesis or accelerated chlorophyll degradation in cucumber leaves, whereas exogenous Si alleviated this stress.

3.2. Effect of Si on the Activity of the Key Enzymes of Photosynthesis in Cucumber Leaves under CA-Induced Stress

The addition of exogenous Si under normal conditions reduced the activities of Rubisco (Figure 2A) and GAPDH (Figure 2B) in cucumber leaves; however, these enzyme activities were higher than those in the CA and CA + Si treatment groups. Compared with the enzyme activities in the CK group, CA-induced stress significantly reduced the activities of Rubisco and GAPDH by 1.45× and 2.53×, respectively. In contrast, the exogenous application of Si increased the enzyme activities of Rubisco and GAPDH in cucumber leaves under CA-induced stress. This showed that Si could alleviate the inhibitory effect of autotoxicity on the enzyme activities of Rubisco and GAPDH.

The enzyme activities of FBPase (Figure 2C), FBA (Figure 2D), and TK (Figure 2E) were not significantly different between the Si and CK groups. Compared to the CK group, CA-induced autotoxicity stress significantly reduced the enzyme activities of FBPase (1.64×), FBA (2.02×), and TK (1.85×). However, the exogenous addition of Si significantly increased the activities of FBPase, FBA, and TK by 16.48%, 27.86%, and 36.62%, respectively (as compared to the CA treatment group). This indicates that exogenous Si alleviated the inhibitory effects of CA-induced autotoxicity stress on the enzyme activities of FBPase, FBA, and TK.

3.3. Effect of Si on the Carbohydrate Contents of Cucumber Leaves and Roots under CA-Induced Stress

The fructose (Figure 3A), glucose (Figure 3B), and sucrose (Figure 3C) contents of cucumber leaves and roots were significantly higher under CA-induced autotoxicity stress than in the CK group, whereas the opposite was true for starch (Figure 3D). The fructose content of leaves was significantly higher in Si than in CK. In contrast, the glucose, sucrose, and starch contents of leaves were significantly lower in Si than in CK. Treatment with Si alone significantly elevated the fructose content of roots. However, the glucose content of roots was not significantly different between Si and CK, and sucrose and starch levels were significantly lower in Si than in CK. The fructose, glucose, and sucrose contents of leaves were significantly lower in CA + Si (by 41.15%, 20.91%, and 24.5%, respectively) than in CA, whereas the starch content was significantly higher (by 50%). The fructose, glucose, and sucrose contents of roots increased by 49.29%, 6.52%, and 30.71%, respectively, in CA compared to those in CK. However, the starch content of roots was significantly reduced (5-fold) in CA than in CK. The addition of exogenous Si significantly reduced the fructose, glucose, and sucrose contents of roots (by 62.3%, 20.5%, and 24.52%, respectively) and significantly increased the starch content (3-fold) compared to those in the CA treatment group. Compared with those in CK, the glucose, sucrose, and starch contents in the Si-only treatment decreased, but the fructose content was significantly higher than that in the CK and CA + Si treatments.

3.4. Effect of Si on the Activities of SPS and SS in Cucumber Leaves and Roots under CA-Induced Stress

The activity trends of SPS (Figure 4A) and SS (Figure 4B) in cucumber leaves and roots were similar trends all treatments. Treatment with Si alone significantly reduced the activities of SPS and SS compared to those in CK; however, these activities were still higher than those in the other two treatment groups. Compared with CK, CA-induced autotoxicity stress significantly reduced the activities of these enzymes in cucumber leaves and roots by 2.75× and 1.71× (SPS) and by 3.71× and 1.57× (SS), respectively. However, the activities of SPS and SS in cucumber leaves and roots increased after the exogenous addition of Si.

3.5. Effects of Si on the Activities of AI and NI in Cucumber Leaves and Roots under CA-Induced Stress

The activities of AI (Figure 5A) and NI (Figure 5B) in cucumber leaves and roots were slightly lower in Si than in CK, but these activities were higher than those in the CA and CA + Si treatment groups. Compared with CK, CA-induced autotoxicity stress significantly reduced the activities of these enzymes in cucumber leaves and roots by 3.35× and 1.31× (AI) and by 1.87× and 1.07× (NI), respectively. The addition of Si under conditions of autotoxicity stress increased the enzyme activities of cucumber leaves and roots by 20.59% and 14.63% (AI) and by 23.23% and 1.5% (NI), respectively. These results indicate that the addition of exogenous Si alleviated the effects of CA-induced autotoxicity stress on the activities of AI and NI in cucumber leaves and roots.

4. Discussion

Continuous crop cultivation causes the accumulation of root-secreted autotoxic substances in the soil, which changes the physicochemical properties of soil and hinders crop growth and development. Autotoxic substances can significantly affect the decomposition and synthesis of photosynthetic pigments in leaves and disrupt PSII reaction centers and electron transfer, thus causing a series of physiological and metabolic disorders [31]. The results of this study show that the chlorophyll a, chlorophyll b, chlorophyll a+b, and carotenoid contents of cucumber seedlings (Figure 1A–D) were significantly reduced under CA-induced autotoxicity stress. The exogenous addition of high concentrations (>0.5 mM) of CA can affect most major physiological processes in plants, including seed germination [32], root growth [33], mineral uptake [34], photosynthesis [35], membrane peroxidation, and oxidative stress [36]. In the present study, the chlorophyll and carotenoid contents of cucumber leaves (Figure 1A–D) increased significantly after the addition of exogenous Si. Previous studies have also reported that Si can increase the size and number of chloroplasts, improve chlorophyll content and photosynthesis, and promote dry-matter accumulation in plants [37,38]. Under abiotic stress, the application of appropriate amounts of Si to different crops can also change the leaf structure, increase the chlorophyll content, and improve the photosynthetic rate [17,39].

The Calvin cycle plays a key role in carbon fixation during photosynthesis, and it consists mainly of light-independent redox reactions that occur in the chloroplast stroma. Rubisco is mainly responsible for the catalytic-carboxylation phase of the Calvin cycle, and the photosynthetic rate of plants is limited by the carboxylation reaction of Rubisco and the regeneration capacity of ribulose 1,5 -diphosphate (RuBP) [40]. The GAPDH in the chloroplast isomer participates in the Calvin cycle by catalyzing the reduction of 1,3-diphosphoglycerate [41]. Usually, the activities of photosynthetic enzymes—including Rubisco and GAPDH—in plant leaves are susceptible to adverse conditions, which reduce the rate of CO₂ assimilation and hinder normal photosynthesis in plants [42]. In this experiment, the activities of Rubisco and GAPDH (Figure 2A,B) were significantly reduced under CA-induced stress. However, the addition of exogenous Si increased the activities of both enzymes to some extent (Figure 2A,B). Previous studies have also demonstrated that Si improves plant photosynthesis by increasing stomatal conductance, improving the rate of photosynthetic electron transfer, and regulating Rubisco activity [17,43].

FBPase, FBA, and TK mainly function during the RuBP regeneration reaction in the Calvin cycle and convert glyceraldehyde-3-phosphate and dihydroxyacetone phosphate into the CO₂ receptor molecule, RuBP [44]. A decrease in the activities of FBPase, FBA, and TK in transgenic potato plants was found to inhibit RuBP regeneration, and hence photosynthesis [43]. In our study, CA-induced autotoxicity stress significantly reduced the activities of FBPase, FBA, and TK (Figure 2C–E). This result is consistent with the findings of Yang et al. [45], who reported that autotoxicity stress inhibits the photosynthetic capacity of crop leaves by significantly reducing the activities of key enzymes in the Calvin cycle. The addition of exogenous Si significantly increased the activities of FBPase, FBA, and TK in the leaves of cucumber seedlings, which promoted the regeneration of RuBP in the Calvin cycle, and thus accelerated CO₂ fixation.

Carbohydrates are not only a major source of energy for plant metabolic activities, but they also function as protective agents for regulating cellular osmotic pressure. Under adverse conditions, plants increase metabolically compatible solutes (including soluble sugars) in order to maintain their internal water and osmotic potentials [46,47]. Under normal growth conditions, some small molecules involved in osmoregulation (such as sucrose, glucose, and fructose) are maintained at low concentrations in plants, and their accumulation and transport are in dynamic equilibrium. However, when the external growth environment is abnormal, these small molecules accumulate in large quantities in order to regulate the balance of osmotic potential between the cell and the outside world, which helps the organism resist the adversity [48]. In our study, when cucumber seedlings were grown in a nutrient solution containing 0.8 mM CA, the fructose, glucose, and sucrose contents of the leaves (Figure 3A–C) were significantly higher than those of the leaves in the control and Si-treated groups. In addition, the fructose, glucose, and sucrose contents of the roots showed a similar trend in each treatment group. This suggests that cucumber seedlings can avoid excessive toxicity from exogenous CA by increasing the soluble sugar content (fructose, glucose, and sucrose) of the whole plant. The levels of starch under CA-induced autotoxicity stress showed opposite trends to those of fructose, glucose, and sucrose. In particular, the starch content was significantly lower under CA-induced stress than in the control (Figure 3D). There are two possible explanations for this pattern. One is that the plant was in an adverse environment where the activity of starch synthase was reduced, whereas hydrolase activity was increased. This imbalance of enzymatic reactions led to an accelerated breakdown of starch, thus reducing the starch content. The second explanation is that, in cucumber seedlings under autotoxicity stress, the carbon fixed in the leaves through photosynthesis was converted from starch to small molecules involved in osmoregulation (fructose, glucose, and sucrose, among others) [48,49]. Our results suggest that, when subjected to CA-induced autotoxicity stress, cucumber leaves may convert starch into soluble sugars with osmoregulatory functions and promote their accumulation in the plant. This is achieved by regulating the carbon metabolism and improving the osmoregulatory capacity of cells, which helps the plant resist the damage caused by autotoxicity. However, the levels of fructose, glucose, and sucrose in the leaves and roots of the cucumber seedlings were significantly reduced after the exogenous addition of Si, and some of these were even restored to normal levels. Exogenous Si also significantly increased the starch content of cucumber seedlings. This may have occurred because Si application improved plant tolerance to external stresses by altering the levels of solutes, such as proline, carbohydrates, and total soluble sugars [50,51,52].

In phytosomes, sucrose is synthesized after fructose-6-phosphate and UDP-glucose are catalyzed by SPS [53]. In the present study, SPS (Figure 4A) activity was reduced in the leaves and roots of cucumber seedlings under CA-induced stress, inhibiting the conversion of fructose and glucose to sucrose. This allowed the accumulation of fructose and glucose in the plant, which helped the plant counteract autotoxicity-related stress. SS (Figure 4B) is another key enzyme in sucrose metabolism. SS catalyzes reversible reactions related to sucrose catabolism and synthesis, but it is mainly involved in sucrose catabolism. In addition, SS is also involved in the synthesis of starch and fiber [54]. In our study, the SS activity of cucumber seedlings was significantly reduced under CA-induced stress, and the normal breakdown of sucrose and starch synthesis were hindered. This significantly increased the sucrose content and significantly reduced the starch content of the leaves and roots of cucumber seedlings. However, the addition of exogenous Si increased the activities of SPS and SS. Moreover, when treated with Si alone, the SPS and SS activities of seedlings (leaves and roots) were second only to those of plants in the control group. These results suggest that Si can alleviate the effects of CA-induced stress on the activities of key enzymes related to sucrose metabolism in cucumber seedlings.

AI and NI are present in the vacuole and cytoplasm of plants, respectively. These are key enzymes in sucrose catabolism, in which they catalyze the conversion of sucrose to fructose and glucose [55]. In this study, CA-induced stress significantly reduced the activities of AI and NI (Figure 5A,B) in cucumber plants. This inhibited the hydrolysis of sucrose to fructose and glucose, thus allowing sucrose accumulation in the plants. A decrease in sucrose-converting enzyme activity is accompanied by an increase in malondialdehyde (MDA) content. Previous studies have reported that CA-induced stress increased the levels of MDA and reactive oxygen species in cucumber seedlings, causing membrane lipid peroxidation and inhibiting sucrose convertase activity [28,56]. However, the addition of exogenous Si significantly increased the activities of AI and NI. This effectively alleviated the inhibition of sucrose-converting-enzyme activity by CA-induced stress and allowed sucrose metabolism to normalize.

In this study, the measured indexes under Si treatment alone were lower than those of CK, which may be due to the fact that the Si concentrations used in this experiment were screened under CA stress. Therefore, it is more conducive to promote the growth and development of cucumber seedlings, but it cannot promote the growth of plants under normal conditions. In different test varieties, developmental stages, and measurement indicators, the effects of silicon may be different. Zhang et al. [57] found that after 70 days of treatment, chlorophyll b, leaf area, leaf dry biomass, photosynthetic rate, and transmission rate were lower than those under normal conditions. Another study also showed that the shoot/root ratio, total root water content, and the chlorophyll a/b ratio under silicon treatment alone were lower than those under normal conditions [58]. Furthermore, it has also been reported that silicon is pressure-dependent in some measurement indicators (such as chlorophyll). In other words, the same silicon concentration will show better mitigation effects under pressure [57].

Impairment of the photosynthetic machinery results in a reduction in the energy available for subsequent Calvin cycle reactions, and changes in carbohydrate content and related enzymatic activities will inevitably lead to related metabolic disorders, thereby affecting normal plant growth and development [59]. CA stress reduced the contents of photosynthetic pigments in cucumber seedlings (Figure 1), resulting in reduced photosynthesis and insufficient ATP. The addition of exogenous silicon increased photosynthesis and the activity of key enzymes in the Calvin cycle (Figure 2), facilitating the normalization of the Calvin cycle. Additionally, autotoxicity stress induced by CA caused small molecules involved in osmoregulation (fructose, glucose, and sucrose; Figure 3) to accumulate in large quantities in the organism so as to regulate the osmotic potential between the cell and the outside world, such that it could withstand adversity [48]. In addition, CA stress reduced the activity of key enzymes for sucrose metabolism (SPS, SS, AI, and NI; Figure 4 and Figure 5), which disrupted carbon metabolism and allowed further accumulation of carbohydrates in the plant. However, the addition of exogenous silicon regulated the carbohydrate content and key enzymatic activities, allowing carbon metabolism to function properly.

5. Conclusions

In the present study, we investigated the effects of exogenous Si on chlorophyll content, key enzyme activities in the Calvin cycle, and carbon metabolism in cucumber seedlings under CA-induced autotoxicity stress. We found that the addition of exogenous Si increased the chlorophyll content and consolidated the light-trapping capacity of leaves in cucumber seedlings under CA-induced autotoxicity stress. The activities of key enzymes in the Calvin cycle also showed an increasing trend after the addition of exogenous Si, which promoted the rate of CO₂ assimilation. In addition, cucumber seedlings under CA-induced autotoxicity stress alleviated the effects of CA-induced toxicity by increasing the fructose, glucose, and sucrose contents of the leaves and roots and reducing their starch content to increase the osmotic potential. After exogenous Si treatment, the levels of fructose, glucose, and sucrose decreased, the starch content increased, and the osmotic potential tended to normal levels (Figure 6). Moreover, the application of exogenous Si inhibited the negative effects of CA on cucumber seedlings by improving the activities of key enzymes related to sucrose metabolism. This study and others show that Si use is a sustainable strategy for mitigating future biotic and abiotic stresses in agriculture. Therefore, to further elucidate the mechanisms of Si application for mitigating autotoxicity stress, gene expression analysis and genomic transcriptome analysis should be considered to investigate the interactions between Si and plants. According to the results of this study, additions of exogenous Si promoted carbon metabolism and photosynthetic pigment accumulation in cucumber seedlings under CA-induced autotoxicity stress.