Introduction

In the process of natural growth and development, plants inevitably encounter various adverse environmental conditions due to immobility (Liu et al. 2021). Thereinto, drought and salinity, two of the most widespread abiotic stress factors globally, adversely affect the establishment, growth, and development of plants, ultimately leading to reduced crop yield (Du et al. 2020; Zhuang et al. 2021). Moreover, these two abiotic stresses usually occur simultaneously in natural and agricultural ecosystems as the drought may lead to high soil salinity in the soil (Ma et al. 2020). Therefore, for the future development of saline cultivation in arid and semi-arid regions, it is necessary to understand plant stress responses and its response mechanisms under combined stresses, rather than considering only individual stress.

Plants undergo various changes in morphological, physiological, and biological processes to thrive in the harsh stress conditions. For instance, drought usually results in etiolation, atrophy, curling, senescence, and reduced size of leaves, slim and elongation of stem, and deep expansion of root system (Wu et al. 2022), and salinity causes dwarfism, reduced leaf area, and increased root/shoot ratio (R/T) (Ahanger et al. 2020; Shen et al. 2022). Recent studies have found that plants undergo a series of physiological, biochemical, and molecular changes, which ultimately disorder metabolic processes such as respiration in plants under stress conditions (Liu et al. 2020a). Stress-induced oxidative stress can result in the redistribution of glycolytic carbon flux into the PPP (Xu et al. 2022) and perturbation of the EMP pathway-TCA cycle by inhibiting related enzyme activities in the TCA cycle (Li et al. 2020). It is also reported that stress condition can shift TCA cycle to the GABA shunt (Che-Othman et al. 2020). However, maintaining respiration metabolism may play a vital role in plant adaptation to adverse environmental stress conditions (Liu et al. 2020a, b). One ideal solution would be to enhance plant stress tolerance by adding harmless exogenous substances to maintain normal life processes. In recent years, silicon (Si), an environment-friendly element beneficial to plants, has been effectively applied in the field of adversity stress to mitigate the inhibition of stress on plants (Jan et al. 2018).

Silicon plays a crucial role in sustainable agricultural development (Verma et al. 2020). Previous studies revealed that Si application could increase drought and salt tolerance of plants through either a direct or indirect mechanism (Wang et al. 2021; Dhiman et al. 2021). Specifically, Si alleviated the inhibitory effect of drought and salt stress on plant growth in various ways, such as by increasing leaf area and plant height (Shen et al. 2022), enhancing growth of roots (Ahire et al. 2021), and increasing biomass accumulation (Verma et al. 2020). In addition, Si could change the respiration and metabolism pathway of plants under drought and salt stress, including slowing down the TCA cycle by increasing the diversion of acetyl-CoA to lipid biosynthesis (Mundada et al. 2021) and inducing GABA shunt (Das et al. 2021), thus improving tolerance. Therefore, the regulation of respiratory metabolism in plants is one of the important mechanisms by which Si can improve plant tolerance.

As one of the main vegetation resources in arid and semiarid areas worldwide, Glycyrrhiza Uralensis Fisch. (G. uralensis) has strong drought and salt tolerance, and it plays not only an essential ecological role in soil and water conservation, wind prevention, and sand fixation but also is a traditional medicinal herb in Europe and Asia with great therapeutic value and potential for development (Zhong et al. 2022). Previous studies have shown that stress can increase the content of active ingredients in G. uralensis (Yao et al. 2022; Han et al. 2022). However, the seedling stage is extremely sensitive to stress tolerance (Zhang et al. 2022). Thus, it is of great scientific significance to explore the physiological stress process and response mechanism of G. uralensis seedlings under adverse conditions in alleviating stress. Previous studies have proved that Si can improve the stress tolerance of G. uralensis seedlings by regulating carbon and nitrogen metabolism (Cui et al. 2021). However, the comprehensive regulation mechanisms of Si on respiratory metabolism of G. uralensis seedlings under drought, salt, and combined stresses are still unclear. In the present study, the growth properties, respiratory metabolic pathways, and gene expression of G. uralensis seedlings were analyzed, which could provide a theoretical basis for using Si application in agriculture.

Materials and methods

Plant materials and growth conditions

The pot experiment was conducted in a constant temperature incubator at the Gansu Agricultural University, Lanzhou City, Gansu Province, China. Seeds were collected from wild G. uralensis plants in Urad Front Banner, Inner Mongolia, China, in September 2019, which was identified as G. uralensisFisch. by Professor Xinhui Zhang of Ningxia Medical University. Healthy seeds were selected and stored in a kraft paper bag at 4 °C  until use. Seeds were soaked in concentrated H2SO4 for 40 min to break the thick seed coat, surface-sterilized for 10 min by soaking in 10% H2O2, washed several times with distilled water, and then imbibed with distilled water for 7 h at 4 °C. Using the sand culture method, 60 seeds were sown in plastic pots (with a 9.5 cm base diameter, 11 cm height, and 13 cm diameter), which were filled with 1000 g of sterilized sand, and then covered with 200 g of sterilized sand. The sand in the pots was kept humid at 70 ± 5% of the field water-holding capacity until the second true leaf was fully developed (approximately 25 days later).

Treatments and experimental design

The experiments had a completely random design, and eight replications were conducted. The G. uralensis seedlings were subjected to the following eight treatments: soil relative water content of 70 ± 5% (control, CK); soil relative water content of 40 ± 5% (drought (D) stress); 75 mM NaCl (salt (S) stress); 75 mM NaCl + soil relative water content of 40 ± 5% (salt and drought (S+D) stress); soil relative water content of 70 ± 5% + 2 mM potassium silicate (K2SiO3) (CK+Si); soil relative water content of 40 ± 5%+ 2 mM K2SiO3 (D+Si); 75 mM NaCl + 2 mM K2SiO3 (S+Si); 75 mM NaCl + soil relative water content of 40 ± 5% + 2 mM K2SiO3 (S+D+Si). Soil moisture was controlled by weighing, and it was irrigated every two days. All pots were randomly arranged and periodically rotated to minimize the effects of environmental heterogeneity.

After 20 days of stress and Si treatment, plants were collected for subsequent experiments. Generally, eight pots were selected for each treatment. First, three pots were randomly selected for each treatment; all plants in the pots were removed from the soil, and aboveground growth indices were recorded. Next, the root samples were scanned using a scanner (Epson Perfection 4990 Photo type) with a resolution of 300 dpi, and the scanned images were analyzed using Win RHIZO root analysis software to measure characteristic parameters such as root length, surface area, and volume. The samples were then oven-dried at 60 °C for 48 h and weighed. The seedlings from the remaining five pots of each treatment were removed from the soil between 9 and 11 a.m., washed immediately with distilled water, and then a few leaves were randomly collected and mixed to determine chlorophyll (Chl) content, while the rest of the samples were immediately stored at − 80 °C for subsequent determination of enzyme activities.

Determination of key enzyme activities of respiratory metabolism

For the determination of various physiological indicators-associated respiratory metabolism, we used reagent kits (Sino Best Biological Technology Co., Ltd.), which included key enzymes in the EMP : hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK); key enzymes in the TCA cycle: pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (α-KGDH), mitochondrial isocitrate dehydrogenase (ICDHm), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) enzyme activities, as well as acetyl coenzyme A (acetyl-CoA) content; key enzymes in the PPP: glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconic dehydrogenase (6-PGDH).

The HK activity was determined using glucose as the substrate to record the increase in absorbance at 340 nm cause by the production of NADPH. PFK was determined using fructose-6-phosphate as the substrate to record the decrease in absorbance at 340 nm cause by the enzymatic oxidation of NADH. PK was determined using phosphoenolpyruvate (PEP) as the substrate to record the decrease in absorbance at 340 nm caused by the enzymatic oxidation of NADH. The PDH activity was determined using pyruvate and 2,6-dichlorophenol indophenol (2,6-DCPIP) as the substrate to record the decrease in absorbance at 605 nm. The content of acetyl-CoA was determined by recording the increase in absorbance at 340 nm cause by the production of NADPH using the coupling reaction of malate dehydrogenase and citrate synthase. The α-KGDH activity was determined using α-ketoglutarate as the substrate to record the increase in absorbance at 340 nm cause by the enzymatic reduction of NAD+, and ICDHm was determined by recording the increase in absorbance at 340 nm. SDH was determined using succinic acid as the substrate to record the reduction rate of 2,6-DCPIP at 600 nm. NAD-MDH was determined using oxaloacetate (OAA) as the substrate to record the decrease in absorbance at 340 nm. The G6PDH and 6-PGDH activity was determined by recording the increase in absorbance at 340 nm caused by the enzymatic reduction of NADP+.

RNA-Seq analysis

The work was conducted by Beijing Baimeike Company (Beijing, China) on an Illumina HiSeq 500 platform. Total RNA from the aerial tissue and underground tissue of G. uralensis seedlings collected from eight treatments (CK, CK+Si, S, S+Si, D, D+Si, S+D and S+D+Si) was used for cDNA library construction, with three replicates in each treatment. RNA concentration and purity was measured using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Sequencing libraries were constructed using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) and sequenced on Illumina platform. HISAT2 was used to map clean reads to reference genome. The read numbers were transformed into the fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) value for gene expression quantification (Florea et al. 2013). Differentially expressed genes (DEGs) were identified using DESeq2 package. Gene expression fold change ≥ 1.5 and false discovery rate (FDR) < 0.05 were set as the threshold values for subsequent analysis. GSEA was performed using BMKCloud (www.biocloud.net), pvalue  < 0.05 and FDR < 0.25 were set as the threshold values.

Statistical analyses

Statistical analysis was performed using SPSS Statistics 20.0. Differences between means were tested by one-way analysis of variance (ANOVA) using Duncan’s multiple range tests, and p < 0.05 was considered statistically significant. All treatments have three replication operations presented as the mean ± SD of each experiment. Principal component analysis (PCA) was performed with Canoco 5.0.

Results

Plant growth parameters and root morphological traits

The change in the morphological traits of stressed plant is the most intuitive representation of stresses threatening plant growth. Relative to the CK, salt stress inhibited the growth of G. uralensis seedlings by decreasing plant height, leaf number, leaf area, and aboveground biomass by 17.41%, 15.13%, 25.73% and 8.68%, respectively, but increased the main root diameter, underground biomass and R/T significantly by 21.10%, 15.05% and 25.87, respectively (Fig. 1,Table 1); drought stress remarkably declined the stem diameter by 7.36% but increased the lateral root number by 9.24%; combined stress significantly decreased plant height, stem diameter, leaf number, leaf area, total root volume, aboveground biomass, and total biomass by 21.03%, 9.81%, 23.45%, 33.81%, 16.37%, 30.13% and 19.99%, while increased R/T by 34.08%. The S+Si treatment increased leaf number by 15.13%, but decreased the total root length and lateral root number by 11.13% and 17.30%, relative to S alone. The D+Si treatment significantly increased underground biomass by 11.40% but decreased the leaf area and total root volume by 13.39% and 6.92% of D stressed-plants. Additionally, Si increased leaf number and aboveground biomass by 11.96% and 23.88%, but decreased R/T by 11.87% of G. uralensis subjected to S+D stress (Fig. 1, Table 1).

Fig. 1
figure 1

a A part of the experiment layout of G. uralensis seedlings grown under different stress without or with Si. Effect of exogenous Si on the growth parameters in aerial part (b)  and biomass (c) of G. uralensis seedlings grown under different stress. The different capital letters indicate the significant difference at p < 0.05 within the different treatments without Si, the different lowercase letters indicate the significant difference at p < 0.05 within the different treatments with Si addition. The “ * ” stands for the significant difference at p < 0.05 between two treatments under absence of Si and application of Si. Error bars represent the standard error

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Table 1 Effect of Si on the root morphological trait of G. uralensis seedlings under different stress
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EMP metabolism process

Both stress and Si had different effects on aerobic and anaerobic respiration in the EMP pathway. In the aerobic respiration phase, the S, D and S+D treatments significantly increased HK activity by 56.04%, 163.86% and 268.99% in the aerial part of G. uralensis seedlings, respectively, relative to CK alone (Figs. 2, 3). In addition, the PFK activity remarkably increased by 73.82% and 34.95% in the aerial part of D and S+D-treated plants (Figs. 2, 3). However, the transcriptional analysis showed no differences in the expression of genes encoding HK in their aerial parts between stress-treated plants and the CK (Fig. 5; Table 2). In addition, salt stress upregulated one gene encoding PFK and downregulated three genes encoding PFK, PK, and GAPDH, respectively. Drought stress upregulated three genes encoding PFK, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (PGAM), and PK, respectively. Combined stress upregulated six genes encoding diphosphate-dependent phosphofructokinase (PPi-PFK), GAPDH (NADP+), PGAM, PFK, and PK and downregulated twelve genes encoding PFK, PPi-PFK, GAPDH, PGAM, 2,3-bisphosphoglycerate 3-phosphatase (2,3-BPG 3-phosphatase), enolase, and PK. Silicon addition significantly increased the activity levels of PFK and PK by 18.14% and 44.70% in the aerial part of the seedlings exposed to S. However, the transcriptional analysis showed no differences in the expression of genes in the EMP pathway in the aerial part of G. uralensis seedlings between S and S+Si treatments. Compared with D treatment, Si application significantly increased the HK and PK by 84.57% and 27.32% but decreased PFK by 21.76% in their aerial parts. However, the addition of Si downregulated three genes encoding PFK, PGAM, and PK, respectively, in D-treated plants. In S+D +Si-treated seedlings, HK and PFK declined by 74.86% and 57.53%, respectively, relative to S+D-stressed seedlings. However, S+D+Si stress upregulated seven genes encoding HK, PPi-PFK, 2,3-BPG 3-phosphatase, enolase, and PK, and downregulated four genes encoding PFK, PPi-PFK, GAPDH (NADP+), and PK, respectively.

Fig. 2
figure 2

Effect of exogenous Si on key enzyme activities in EMP pathway in aerial and underground part of G. uralensis seedlings grown under different stress. HK hexokinase, PFK phosphofructokinase, PK pyruvate kinase. The different capital letters indicate the significant difference at p < 0.05 within the different treatments without Si, the different lowercase letters indicate the significant difference at p < 0.05 within the different treatments with Si addition. The “ * ” stands for the significant difference at p < 0.05 between two treatments under absence of Si and application of Si. Error bars represent the standard error

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Fig. 3
figure 3

Effect of exogenous Si on the enzyme activities in respiration metabolism of G. uralensis seedlings grown under different stress.HK hexokinase, G-6-P glucose-6-phosphate, F-6-P fructose-6-phosphate, PFK phosphofructokinase, FBP fructose-1,6-bisphosphate, PEP Phosphoenolpyruvate, PK pyruvate kinase, Pyr pyruvate, PDH pyruvate dehydrogenase, OAA oxaloacetic acid, ICDHm isocitrate dehydrogenase, 2-OG 2-oxoglutarate, α-KGDH 2-oxoglutarate dehydrogenase, SDH succinate dehydrogenase, NAD-MDH malate dehydrogenase, G6PDH glucose-6-phosphate dehydrogenase, D-G-6P D-glucose-6-phosphate, 6-PGDH 6-phosphogluconic dehydrogenase, Ru5P ribulose-5-phosphate, GAP Glyceraldehyde-3-phosphate, DHAP dihydroxyacetone-3-phosphate, Xu5P xylulose-5-phosphate

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Fig. 4
figure 4

Effect of exogenous Si on key enzyme activities in TCA cycle in aerial and underground part of G. uralensis seedlings grown under different stress. PDH, pyruvate dehydrogenase; ICDHm: isocitrate dehydrogenase; α-KGDH, 2-oxoglutarate dehydrogenase; SDH, succinate dehydrogenase, NAD-MDH, malate dehydrogenase;The different capital letters indicate the significant difference at p < 0.05 within the different treatments without Si, the different lowercase letters indicate the significant difference at p < 0.05 within the different treatments with Si addition. The “ * ” stands for the significant difference at p  < 0.05 between two treatments under absence of Si and application of Si. Error bars represent the standard error

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Fig. 5
figure 5

Effect of exogenous Si on the DEGs in respiration of G. uralensis seedlings grown under different stress. HK hexokinase, PFK phosphofructokinase, PK pyruvate kinase, TIM triosephosphate isomerase, GAPDH Glyceraldehyde-3-phosphate dehydrogenase, GAPDH(NADP+) glyceraldehyde-3-phosphate dehydrogenase (NADP+), PGAM phosphoglycerate mutase, 2,3-BPG 3-phosphatase 2,3-bisphosphoglycerate 3-phosphatase, PDC pyruvate decarboxylase, ALDH aldehyde dehydrogenase, ADH alcohol dehydrogenase, LDH L-lactate dehydrogenase, PDH pyruvate dehydrogenase, CS citrate synthetase, ICDHm isocitrate dehydrogenase, α-KGDH, 2-oxoglutarate dehydrogenase, SDH succinate dehydrogenase, MDH malate dehydrogenase, G6PDH glucose-6-phosphate dehydrogenase, FBPase fructose-1,6-bisphosphatase

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Table 2 Regulation of respiration metabolism-related DEGs in aerial part of G. uralensis seedlings by different stress or Si
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In addition to the genes mentioned above, in the anaerobic respiration phase, the genes encoding pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH) in the aerial part of G. uralensis were affected by stress or Si (Fig. 5; Table 2). Specifically, two genes encoding ADH upregulated in S_vs_CK comparison, four genes encoding PDC, ALDH, and ADH upregulated and one gene encoding ALDH down-regulated in D_vs_CK comparison, as well as three genes encoding ALDH and ADH downregulated and the other three genes encoding ALDH and ADH up-regulated in S+D_vs_CK comparison. However, Si addition had no effect on the genes related to the EMP pathway in the aerial part of G. uralensis seedlings under S stress. One gene encoding ALDH downregulated and two genes encoding ALDH and ADH, respectively, upregulated in D+Si_vs_D comparison, as well as five genes encoding PDC, ALDH, and ADH, upregulated and one gene encoding LDH downregulated in S+D+Si_vs_S+D comparison.

In the underground part of G. uralensis seedlings, salinity significantly increased the HK by 287.92% and decreased PK and PFK by 15.97% and 21.54% over the control (Figs. 2, 3). However, transcriptional analysis showed that S stress downregulated five genes encoding HK, PFK, and PK but upregulated one gene encoding GAPDH (NADP+) (Fig. 5; Table 3). Drought stress greatly increased HK and PK by 234.41% and 76.02%, respectively, but had no effect on genes encoding HK and PK. Besides, one gene encoding GAPDH was downregulated in the underground part of D-treated plants. Combined stress increased the PK by 17.12% and decreased the PFK by 34.75%, and downregulated four genes encoding HK, triosephosphate isomerase (TIM), PFK, and PK, respectively, and upregulated two genes encoding PFK and GAPDH (NADP+), respectively. With the Si addition, the variation trends of HK, PFK, and PK in the underground part were similar to those in the aerial part under S stress conditions, but the values of HK and PK were higher, while that of PFK was lower. Relative to drought-stressed plants, Si addition greatly increased the HK by 53.73% and decreased the activity levels of PK and PFK by 38.11% and 41.58% in the underground part. However, no related genes were differentially expressed in S+Si _vs_S and D+Si_vs_D comparison. The addition of Si increased HK and PK by 93.97% and 25.81% in the underground part of plants subjected to S+D stress and upregulated three genes encoding HK and PGAM.

Table 3 Regulation of respiration metabolism-related DEGs in underground part of G. uralensis seedlings by different stress or silicon
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In the anaerobic respiration phase, S stress upregulated two genes encoding ALDH and ADH, respectively, D stress downregulated one gene encoding PDC, and S+D stress downregulated three genes encoding PDC and ALDH and upregulated eight genes encoding PDC, ADH, and ALDH in the underground part (Fig. 5; Table 3). With the Si addition, one gene encoding PDC upregulated and one gene encoding ADH downregulated in S-treated plants. Si addition had no effect on the related genes in the underground part of G. uralensis seedlings under D and S+D stress conditions.

TCA cycle

Relative to control, ICDHm, SDH, and NAD-MDH and the content of acetyl-CoA in the aerial part decreased by 35.77%, 20.93%, 50.13% and 32.75% due to salt stress (Figs. 3, 4). Our transcriptome analysis results showed two genes encoding α-KGDH and SDH downregulated and one gene encoding MDH upregulated in S _vs_ CK comparison (Fig. 5, Table 2). D stress significantly decreased PDH, SDH, and NAD-MDH and the content of acetyl-CoA by 21.53%, 14.31%, 47.83% and 60.65%, respectively, but increased α-KGDH by 58.04% in the aerial part over the control. However, the transcriptional analysis showed that only one gene encoding ICDHm upregulated in D_vs_CK comparison. Under S+D stress, the patterns of variation for key enzymes in the TCA cycle in the aerial part were similar to those under D stress. However, the transcriptome results differed, with S+D stress downregulating eight genes encoding PDH, citrate synthase (CS), ICDHm, α-KGDH, SDH, aconitate hydratase, and upregulating one gene encoding MDH. Relative to salt stressed plants, Si addition decreased PDH and NAD-MDH by 27.50% and 67.64% but increased ICDHm, α-KGDH, and the content of acetyl-CoA by 108.71%, 19.71% and 39.97%. Relative to drought stressed plants, Si decreased SDH by 36.3% and increased NAD-MDH and the content of acetyl-CoA by 32.72% and 62.31%. However, transcriptome results showed no differences in S+ Si_vs_S and D+Si_vs_D comparison. Application of Si decreased SDH by 34.56% and increased PDH, α-KGDH, and NAD-MDH by 27.22%, 18.88% and 140.37%, respectively, and upregulated three genes encoding CS and α-KGDH in the aerial part of S+D-treated plants.

Relative to control, ICDHm decreased by 67.04% and PDH, α-KGDH, SDH, and NAD-MDH increased by 166.22%, 46.88%, 61.65% and 71.28%, respectively, in the underground part of salt stressed seedlings (Figs. 3, 4); drought stress decreased ICDHm by 78.73%, but increased PDH, SDH, and NAD-MDH by 50.02%, 116.94% and 44.87%, respectively; combined stress increased PDH, SDH, and NAD-MDH and the acetyl-CoA content 170.87%, 79.45%, 101.14% and 22.11%, while decreased ICDHm and α-KGDH by 70.39% and 38.26%, respectively. However, transcriptome results showed that salt stress downregulated one gene encoding CS and upregulated one gene encoding NAD-MDH; drought stress downregulated two genes encoding CS and α-KGDH, respectively; combined stress downregulated two genes encoding PDH and CS, respectively, and upregulated one gene encoding NAD-MDH (Fig. 5; Table 3). Relative to salt stressed plants, Si addition decreased PDH, ICDHm, and α-KGDH in the underground part by 20.41%, 49.25% and 50.71%, but increased NAD-MDH and the content of acetyl-CoA in the underground part by 24.16% and 24.86%, respectively. Relative to drought stressed plants, αD-stressed plants, adding Si decreased α-KGDH, SDH, and the acetyl-CoA content in the underground part by 19.89%, 73.54% and 17.93%, respectively. However, Si addition had no effect on genes related to the TCA cycle in their underground part under any stress.

Some intermediates of the TCA cycle are the synthetic precursors for many important organic compounds. The proper functioning of the TCA cycle can be compromised when its intermediates products are consumed in large quantities to synthesize organic compounds. The PEPC enzyme catalyzing PEP to replenish OAA is one of the important complementary mechanisms of the TCA cycle. Under stress conditions, the PEPC in the aerial part increased with S (2.78-fold), D (3.94-fold) and S+D (2.30-fold), respectively, relative to CK. (Fig. 6). However, the transcriptome results were different, with S and S + D stresses downregulating one gene encoding PEPC and D stress having no effect (Fig. 5; Table 2). Si addition greatly decreased PEPC by 42.37% in the aerial part of D-treated plants but had no effect on it of plants under S and S+D stresses. Transcriptome results showed that Si addition had no effect on the genes encoding PEPC in the aerial part of plants under any of the three stresses. As in the aerial part, PEPC in the underground part also increased under stress conditions to varying degrees (Fig. 6). Moreover, Si addition further increased the PEPC in the underground part by 69.07 with S+Si, 145.48% with D+Si and 31.19% with S+D+Si. However, either the stress nor Si addition affected the genes encoding PEPC in the underground part of G. uralensis seedlings (Fig. 5; Table 3).

Fig. 6
figure 6

Effect of exogenous Si on the PEPC activity in aerial and underground part of G. uralensis seedlings grown under different stress. The different capital letters indicate the significant difference at p < 0.05 within the different treatments without Si, the different lowercase letters indicate the significant difference at p < 0.05 within the different treatments with Si addition. The “ * ” stands for the significant difference at p  < 0.05 between two treatments under absence of Si and application of Si. Error bars represent the standard error

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PPP metabolism process

Relative to control, salt stress greatly increased G6PDH and 6-PGDH in the aerial part by 73.97% and 171.91% (Fig. 7); drought stress had less effect on these; combined stress increased G6PDH and 6-PGDH in the aerial part by 25.56% and 101.12%. Transcriptome results showed that S and D stresses did not affect genes related to the PPP, while S+D stresses upregulated three genes encoding G6PDH and ribose 5-phosphate isomerase (Fig. 5; Table 2). Si addition decreased G6PDH and 6-PGDH in the aerial part by 34.10% and 55.54% with S+Si, 13.22% and 72.93% with D+Si. However, transcriptome results showed that one gene encoding G6PDH was upregulated in S+Si-treated plants, but there was no difference in D+Si_vs_D comparison. Relative to S+D, Si significantly increased G6PDH by 25.77%, decreased 6-PGDH by 28.02%, and upregulated one gene encoding 6-phosphogluconolactonase in the aerial part of G. uralensis seedlings.

Fig. 7
figure 7

Effect of exogenous Si on key enzyme activities in PPP in aerial and underground part of G. uralensis seedlings grown under different stress. G6PDH, glucose-6-phosphate dehydrogenase; 6-PGDH, 6-phosphogluconic dehydrogenase. The different capital letters indicate the significant difference at p < 0.05 within the different treatments without Si, the different lowercase letters indicate the significant difference at p < 0.05 within the different treatments with Si addition. The “ * ” stands for the significant difference at p < 0.05 between two treatments under absence of Si and application of Si. Error bars represent the standard error

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Relative to control, salt stress increased G6PDH and 6-PGDH in the underground part by 72.88% and 159.47% (Fig. 7). However, the transcriptome results showed that one gene encoding 6-phosphogluconolactonase was upregulated and one gene encoding ribose 5-phosphate isomerase was downregulated in S_vs_CK comparison (Fig. 5; Table 3). Drought stress increased 6-PGDH in the underground part by 845.98% but had no effect on the genes related to the PPP. Combined stress increased G6PDH and 6-PGDH in the underground part by 24.15% and 208.21%. However, two genes encoding FBP and ribose 5-phosphate isomerase, respectively, were downregulated, while one gene encoding 6-phosphogluconolactonase was upregulated in S+D_vs_CK comparison. Si addition significantly decreased G6PDH in the underground part by 83.86% with S+Si, 77.00% with D+ i and 30.56% with S+D+Si. Additionally, 6-PGDH in the underground part decreased by 64.71% with D+Si and increased by 144.56% with S+D+Si. However, the transcriptome results showed that two genes encoding FBP and transketolase, respectively, were downregulated in S+Si_vs_S comparison, while there were no significant differences in D+Si_vs_D and S+D+Si_vs_S+D comparison.

Principal component analysis (PCA)

The results of PCA related to plant growth parameters and root morphological traits that we measured revealed a closer association between biological replicates than between stresses or Si treatments (Fig. 8a, b). PC1 explaining 45.97% of total variation uncovered differences between plants of CK and S treatments, CK and S+D treatments, while PC2, accounting for 30.14% of total variation, distinctly separated the plants provided with CK and D treatments, as well as S and S+Si treatments, D and D+Si treatments, and S+D and S+D+Si treatments. PC1 formation covered R/T, leaf area, plant height, leaf number, while PC2 formation covered total root length, lateral root number.

Fig. 8
figure 8

Principal component analysis of growth parameters and root morphological traits (a and b), enzyme activity in respiration metabolism (c and d) of G. uralensis seedlings under eight treatments (CK, CK+Si, S, S+Si, D, D+Si, S+D, S+D+Si). PH Plant height, SD Stem diameter, LN Leaf number, LA Leaf area, AGB Aboveground biomass, UGB Underground biomass, TB Total biomass, TRL Total root length, TSA Total surface aera, TRV Total root volume, MRD Main root diameter, ARD Average root diameter, LRD Lateral root number, HK-A HK activity in aerial part, PFK-A PFK activity in aerial part, PK-A PK activity in aerial part, PDH-A PDH activity in aerial part, AC-A Acetyl-CoA content in aerial part, ICDHm-A ICDHm activity in aerial part, α-K-A α-KGDH activity in aerial part, SDH-A SDH activity in aerial part, MDH-A NAD-MDH activity in aerial part, PEPC-A PEPC activity in aerial part, 6-P-A 6-PGDH activity in aerial part, G6PDH-A G6PDH activity in aerial part, HK-U HK activity in underground part, PFK-U PFK activity in underground part, PK-U PK activity in underground part, PDH-U PDH activity in underground part, AC-U Acetyl-CoA content in underground part, ICDHm-U ICDHm activity in underground part, α-K-U α-KGDH activity in underground part, SDH-U SDH activity in underground part, MDH-U NAD-MDH activity in underground part, PEPC-U PEPC activity in underground part, 6-P-U 6-PGDH activity in underground part, G6PDH-U G6PDH activity in underground part

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The results of PCA related to EMP pathway, TCA cycle and PPP indexs also revealed a relatively closer association between biological replicates than between stresses or Si treatments (Fig. 8c, d). PC1 explaining 32.49% of total variation revealed differences between plants of CK and S treatments, CK and D treatments, CK and S+D treatments, as well as S and S+Si treatments, and S+D and S+D+Si treatments, while PC2 accounting for 23.45% of total variation distinctly separated the plants provided with D and D+Si treatments. PC1 formation covered PEPC activity in both aerial and underground part, ICDHm activity in underground part, while PC2 formation covers the MDH activity in both aerial and underground part, 6-PGDH activity in aerial part and SDH activity in underground part.

Gene set enrichment analysis (GSEA)

The GSEA of respiration-associated pathways (ko00010: Glycolysis/Gluconeogenesis, ko00020: Citrate cycle, ko00030: Pentose phosphate pathway) were performed to ascertain the function changes in response to stress or Si addition (Fig. 9). In S-U_vs_CK-U comparison (Fig. 9a) and S+D-U_vs_CK-U comparison (Fig. 9b), the TCA cycle (KEGG pathway) were significantly enriched and downregulated. However, in S+Si_vs_S comparison, the TCA cycle were significantly enriched and upregulated in both aerial and underground parts (Fig. 9c, d). Besides, in the S+D+Si_vs_S+D comparison, both EMP and TCA pathways were significantly enriched in the aerial and underground parts, and the overall pathway was upregulated (Fig. 9e–h).

Fig. 9
figure 9

GSEA analysis of EMP, TCA cycle and PPP pathway in different treatments with p < 0.05, FDR < 0.25

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Discussion

Effect of exogenous Si on growth characteristics of G. uralensis seedlings grown under different stresses

As the climate continues to deteriorate, the agricultural ecosystems of the contemporary world are under severe stress and challenges. Drought and salt stresses are two major challenges for ecosystems, which hinder plant growth, development, and metabolic processes (Malik et al. 2021). Previous studies have reported that the growth of shoots and roots are significantly inhibited under salt stress (continuous irrigation with 50 mmol NaCl), drought stress (30–35% FWC), and combined stress, thus resulting in less biomass (Egamberdieva et al. 2021; Xie et al. 2019; Zhang et al. 2018). Interestingly, Si addition could ameliorate adverse effects of salt and drought stresses on G. uralensis seedlings (Zhang et al.,2020). In this study, salt stress severely inhibited the shoot growth but increased underground biomass (Fig. 1), the change in growth attributes was due to more assimilates allocated to the root system allowing seedlings to survive under S stress condition. The reduction in the stem diameter and increased number of lateral roots of D-stressed G. uralensis was observed, which could result in more water uptake and increase in stem hydraulic capacitance to resist stress (El-Katony et al. 2017; Verbeke et al. 2023). Combined stress significantly lowered biomass in both aerial and underground parts but increased R/T ratio due to the superposition effect. Based on the results of PCA, it can be judged that salt stress played a dominant role in combined stress. Si application significantly increased leaf number of S-stress plants, causing larger cover and abundant sunlight for growth, this is consistent with the results of Shen et al. (2022). As for D stress, Si addition decreased leaf area contributing to the reduction in transpiration water loss, which was also observed in soybean by Hussain et al. (2021). As with S stress, Si addition remarkably increased the leaf number of S+D-stress seedlings. In addition, Si addition improved the plant height, stem diameter, and leaf area to varying degrees. Surprisingly, these changes worked in concert to increase the aboveground biomass, thus alleviating the damage on the aerial part of plants under combined stresses.

Effect of exogenous Si on respiration of G. uralensis seedlings grown under different stresses

Plants have a variety of respiratory metabolic pathways to adapt to environmental changes, including EMP, TCA, and PPP. The EMP -TCA pathway serves as the primary pathway in the aerobic respiration of plants under normal conditions, providing energy and metabolites needed for plant growth and development (Li et al. 2020). However, the proportion of respiratory pathways in plants under stress conditions may change (Chen et al. 2021).

EMP pathway

HK, PFK, and PK catalyze rate-limiting steps in the EMP pathway, and their decreased activities can inhibit the EMP pathway (Zhong et al. 2016). HK catalyzes the conversion of glucose into glucose 6-phosphate (G-6-P), which is the intersection of the EMP and PPP (Fig. 3). Li et al. (2020) reported that the HK activity was decreased by salt stress in cucumber; however, Zhong et al. (2016) found the vice versa effect. In this study, S stress significantly increased HK enzyme activity in both aerial and underground parts of G. uralensis seedlings to accelerate catabolic metabolism of hexoses, which provided more G-6-P for the EMP pathway and PPP. However, salt stress had no effect on PFK and PK in the aerial part, but decreased them by downregulation of four genes encoding PFK and PK in underground part, to disorder the glycolysis metabolism (Zhong et al. 2016; Li et al. 2020), thereby the elevated G-6-P generated by increased HK might be principally used by the PPP. Drought stress significantly increased HK in both aerial and underground parts of G. uralensis seedlings, contributing to high levels of phosphorylation of hexose and the regulation of ROS production (Poór et al. 2019). Besides, drought stress significantly increased PFK and upregulated two genes encoding PFK and PGAM to accelerate the EMP pathway in aerial part, and enhanced PK in the underground part, thus producing more pyruvate for the TCA cycle, thereby improving drought tolerance (Li et al. 2020). Transcriptomic analysis showed 18 DEGs in aerobic respiration phase of the EMP pathway in the aerial part of S+D-treated plants, suggesting that combined stresses evoked a stronger response in the EMP process. Combined stress increased HK and PFK in the aerial part to facilitate the first half of the EMP process, but downregulated nine genes encoding enzymes involved in the reaction from FBP to PEP in the EMP pathway, including GAPDH, phosphoglycerate mutase (PGAM), 2,3-bisphosphoglycerate 3-phosphatase (2,3-BPG 3-phosphatase), and enolase, slowing down the second half of the EMP pathway. Combined stress increase PK in underground part to provide more substrate for the TCA cycle.

In this study, Si addition alleviated the effects of salt stress on HK, which indicated that Si addition diverted hexoses from catabolic reactions to osmotic adjustment, thereby improving the salt tolerance by promoting soluble sugar accumulation (Poór et al. 2011). Besides, Si addition significantly increased PFK and PK in both aerial and underground parts of S-treated seedlings, indicating that Si addition improved the salt tolerance of plants by promoting the EMP process and providing energy and substrate for subsequent life activities of plants (Xiao et al. 2022). However, this also implied that more G-6-P were preserved in the EMP pathway under S stress after Si addition, thus reducing the supply to the PPP. Unlike S stress, Si addition further increased HK in both organs of D-treated seedlings, which was important to ensure a more efficient metabolism of hexoses and that more energy was produced to combat stress. Meanwhile, Si addition significantly decreased PFK activity by downregulation of relative gene in the aerial part, and decreased PFK and PK activities in underground part of D-stressed seedlings to slow-down the EMP process. The results indicated that Si addition alleviated the effects of D stress on EMP process, it was a symbol that Si addition alleviated drought stress. In addition, Si mitigated the effect of S+D stress on the EMP pathway in aerial part by decreasing HK and PFK at physiological and biochemical level and regulating relative genes at the transcriptional level. The decrease in HK could result in soluble sugar accumulation, which was conducive to regulating the cellular osmotic pressure and membrane stabilization (Weiszmann et al. 2018). However, Si addition further increased HK and PK to accelerate the EMP pathway in the underground part of G. uralensis seedlings subjected to S+D stress, which is consistent with the GSEA results that the EMP pathway was significantly enriched in both aerial and underground parts, and the overall pathway was upregulated (Fig. 9e, g).

In the absence of oxygen, pyruvate is allocated to anaerobic metabolic pathways for energy production and recycling of NAD+, thus maintaining the EMP progress with sufficient NAD+ to provide ATP for plant life (Igamberdiev 2021). Pyruvate is catalyzed to lactate by lactate dehydrogenase (LDH) or acetaldehyde by pyruvate decarboxylase (PDC), both harmful to plants cells. The acetaldehyde is then transformed to acetate by aldehyde dehydrogenase (ALDH) or ethanol by alcohol dehydrogenase (ADH). Ethanol, which is harmless to plants, is easily diffused into the external environment through the lipid bilayer of the cell membrane (Zhou et al. 2017). Our transcriptomic analysis determined that stress and Si addition regulated the genes encoding enzymes related to anaerobic metabolic pathways in both parts of G. uralensis seedlings in varying degrees. Specifically, S stress upregulated relative genes encoding ADH or/and ALDH in both aerial and underground part to accelerate the transformation of harmful acetaldehyde to ethanol and acetate. D stress evoked anaerobic respiration to promote NAD+ regeneration to satisfy the demand for EMP pathway by upregulating relative genes encoding PDC ALDH, and ADH in the aerial parts, but inhibited anaerobic metabolism by downregulating one gene encoding PDC in the underground part. Combined stresses had a greater effect on the anaerobic respiration, wherein six and eleven DEGs were enriched in the anaerobic respiration pathway in the aerial and underground parts, respectively.

Si addition upregulated one gene encoding PDC to promote anaerobic respiration and downregulated one gene encoding ADH to promote more conversion of acetaldehyde to acetate to resist salt stress in the underground part of S-treated seedling (Xiao et al. 2022; Tagnon and Simeon 2017). Si addition further upregulated two genes encoding ALDH and ADH, respectively, to accelerate the transformation of acetaldehyde in the aerial parts of D-treated seedling, thus protecting cells from damage by excessive accumulation of acetaldehyde. In addition, Si addition also promoted anaerobic metabolism by upregulating the genes encoding PDC, ALDH, and ADH in the aerial parts of plants under S+D stresses. Interestingly, Si addition downregulated LDH in the aerial parts of S+D-treated plants, which may prevent cytoplasmic acidification and pH reduction resulting from excessive accumulation of lactate (Zhou et al. 2017) and promote acetate synthesis to withstand stress.

Overall, Si addition alleviated the effects of stress on the EMP of G. uralensis seedlings in different ways, with specific mitigation methods varying with stresses. Generally, under salt stress conditions, Si addition diverted hexoses from catabolic reactions to osmotic adaptation, thereby decreased HK activities, and accelerated EMP pathway due to increased PFK and PK activities and upregulation of EMP in transcriptional level. Under drought stress conditions, Si addition further increased HK activity, enhanced hexose catabolism to provide energy and inhibited ROS production. Si alleviated the elevated energy requirements of plants from exposure to drought, thereby decreased PFK and PK activities to slow-down EMP pathway. Also, Si accelerated acetaldehyde conversion by regulating related genes in aerial part to avoid its excessive accumulation induced by D stress. In addition, Si mitigated the effect of S+D stress on the EMP pathway at the transcriptional level by regulating genes and at the physiological and biochemical level by regulating HK and PK activities.

TCA cycle

Pyruvate enters the mitochondria under aerobic conditions and is catalyzed by PDH to generate acetyl-CoA, which links EMP and TCA. In this study, S stress decreased ICDHm, NAD-MDH, SDH and content of acetyl-CoA to slow down the TCA pathway in the aerial part, which was consistent with the results obtained from the study conducted on cucumber (Li et al. 2020). The reduced acetyl-CoA content might be due to a proportion of acetyl-CoA could be consumed to provide carbon skeletons for increased amino acid synthesis to resist stress (Kazachkova et al. 2013). S stress significantly increased PDH, α-KGDH, SDH and NAD-MDH but decreased ICDH in the underground part. However, GSEA analysis showed that the TCA pathway was significantly enriched and the whole pathway was downregulated at the transcriptional level in S-U_vs_CK-U comparison (Fig. 9a). Interestingly, PCA results showed that PC1 separated S + Si from S treatment with ICDHm-U being a top contributor (Fig. 8). That was, ICDHm-U may be an important indicator for regulating the TCA cycle in underground part of G. uralensis under salt stress. Transcriptome results show that S stress downregulated one gene encoding citrate synthase (CS) in the underground part, possibly reducing the production of energy and citrate by limiting the CS activity, thereby affected the subsequent reaction, which was supported by the results of Xiao et al. (2022). In this case, phosphoenolpyruvate carboxylase (PEPC) activity was enhanced to catalyze PEP to produce OAA in both organs of S-treated plants, thus replenishing intermediates for the TCA cycle, which was consistent with the results of Xiao et al. (2022).

D stress significantly increased α-KGDH and PEPC but decreased PDH, SDH, and NAD-MDH and acetyl-CoA content in aerial part. On the one hand, the increased PEPC could convert a large amount of PEP into OAA, resulting in a lower content of PEP to remain in the EMP pathway, thus decreasing the pyruvate production; on the other hand, D stress induced anaerobic respiration resulting in an increase of pyruvate consumption. Therefore, the TCA cycle was restricted at the substrate level, which further limited the PDH, SDH, and NAD-MDH activities, thus slowing down the TCA process in the aerial part of D-treated plants. However, D stress significantly increased α-KGDH activity, which might be because glutamic acid was regenerated into α-ketoglutaric acid and it entered the TCA cycle, thus stimulating the increased α-KGDH to catalyze α-ketoglutaric acid. This was also reported in Quercus ilex seedlings, wherein decreased glutamate levels were observed under drought stress (Rodríguez-Calcerrada et al. 2017). The change in pattern of the underground part in D-stressed plants was basically similar to that of S stress.

The changing trend of related enzymes in the TCA cycle in aerial part under S+D stress resembled that under D stress alone, which inhibited the TCA cycle in general. In addition, combined stress upregulated relative genes encoding PDH, CS, aconitase hydratase and SDH in aerial part, thus slowing down the TCA cycle at the transcriptional level. Combined stresses increased PDH to consume pyruvate caused by increased PK in underground part, producing more acetyl-CoA for TCA cycle. However, S+D stress downregulated one gene encoding CS, which might reduce the citrate synthesis, thereby affecting the subsequent reactions at the substrate level, leading to a decrease of ICDHm and α-KGDH. The GSEA analysis showed that the TCA pathway was significantly enriched and downregulated in S+D-U_vs_CK-U comparison (Fig. 9b), which was consistent with the result at the physiological and biochemical level. In this case, on the one hand, S+D stress might activate the GABA shunt, thereby bypassing the carbon flux from 2-oxoglutarate to succinate via succinyl-CoA. The succinate generated in the GABA shunt could enter the TCA cycle to participate in subsequent reactions, resulting in increase of SDH and NAD-MDH; on the other hand, S+D stress also increased PEPC activity to ensure the normal progress of the TCA cycle.

The GSEA analysis showed that TCA pathway were significantly enriched and upregulated in both aerial and underground parts in S+Si_vs_S comparison (Fig. 9c, d), which suggested that Si addition significantly alleviated the negative effect of salt stress on the TCA metabolism of G. uralensis seedlings from the transcriptional level. Si addition significantly decreased PDH and NAD-MDH but increased ICDHm, α-KGDH as well as acetyl-CoA content in the aerial part of S-treated plants. Based on these results, we proposed that, on the one hand, the increase acetyl-CoA might be due to the diversion of acetyl-CoA from secondary metabolites toward the TCA process on account of the mitigation effect of Si on S stress; on the other hand, decreased NAD-MDH result in less OAA reacting with acetyl-CoA, thus finally leading to the accumulation of acetyl-CoA in the aerial part of S-stressed seedlings, which might be responsible for the decrease of PDH because of the feedback inhibition of substrate. Si addition alleviated the inhibitory effect of S stress on ICDHm and increased ICDHm to regulate the C and N metabolism and antioxidant system to cope with salt stress (Xiao et al. 2022), which also led to an increase of α-KGDH to catalyze the extra α-ketoglutarate resulting from increased ICDHm. Si application significantly reduced PDH in the underground part of S-stressed seedlings, probably due to the substrate deficiency caused by Si-induced anaerobic respiration and increased PEPC. This also resulted in subsequent reactions being restricted at the substrate level, consequently declining ICDHm and α-KGDH. However, the increase of PEPC replenished the TCA cycle with amounts of malate, thereby stimulating an increase of NAD-MDH, thus maintaining the function of TCA cycle in the underground part of S-treated plants.

Si addition significantly decreased the SDH in aerial part of D-treated plants, which may contribute to reducing the production of mitochondrial ROS (Jardim-Messederet al. 2015). At the same time, it also resulted in a reduced NAD-MDH substrate, which lead to an insufficient amount of OAA synthesized by NAD-MDH reaction with acetyl CoA. In addition, the decreased PEPC also lead to a reduced replenishment of OAA in the TCA cycle., thus resulting in the accumulation of acetyl COA in aerial part of D-treated plants. Si application significantly decreased α-KGDH and NAD-MDH but increased SDH and PEPC in the underground part of D-stressed plants. This may be because Si addition activated GABA shunt, which plays a crucial role in preventing the ROS accumulation and regulating nitrogen metabolism (Mahmud et al. 2017; Che-Othman et al. 2020), thereby distributing α-ketoglutarate into GABA shunt and synthesize γ-aminobutyric acid to resist stress (Prabal et al. 2021), resulting in reduced α-KGDH at the substrate level. Subsequently, γ-aminobutyric acid may undergo a series of reactions to convert to succinic acid, which could enter the TCA cycle (Prabal et al. 2021), thus stimulating the SDH activity to catalyze the conversion of succinic acid to fumaric acid. However, Si addition decreased NAD-MDH and thus reduced the synthesis of OAA. As a result, the PEPC is increased to replenish intermediate products for the TCA cycle to ensure its normal progress.

Si addition significantly alleviated the inhibition effect of S+D stresses on the TCA cycle in the aerial part by increasing PDH, α-KGDH, and NAD-MDH and upregulating three genes encoding CS and α-KGDH in the aerial part of S+D-treated seedlings. Consistently, the EMP and TCA pathway were significantly enriched in both aerial and underground parts, and the overall pathway was upregulated in S+D+Si_vs_S+D comparison (Fig. 9f, h), which indicate that Si addition significantly alleviated the adverse effect of S+D stress on TCA cycle at the transcriptional level. However, Si addition decreased α-KGDH, SDH and content of acetyl-CoA in the underground part of S+D-treated seedlings. This might be because that a portion of acetyl-CoA was used in secondary metabolism to resist stress, thus affecting the subsequent process. In addition, Si addition significantly increased PEPC to replenish TCA cycle intermediates in the underground part of S+D-treated seedlings. Interestingly, PCA results showed that PC1 separated S+D+Si from S+D treatment with PEPC-U being a top contributor (Fig. 8). In other words, PEPC may be an important indicator of the TCA cycle regulation with S+D+Si.

Overall, Si addition alleviated the inhibitory effect of S stress on the TCA cycle by upregulating the TCA pathway at the transcriptional level and regulating acetyl CoA content and related enzyme activities at the physiological and biochemical levels. In addition, Si alleviated the effects of D stress on related enzyme activities and provoked GABA shunt in the underground parts of D-treated plants. Moreover, Si upregulated the TCA pathway at the transcriptional level, inhibited the GABA shunt and increased the PEPC enzyme activity to replenish the TCA cycle with intermediates, and finally alleviated the inhibitory effect of S+D stress on the TCA cycle.

PPP

PPP is another glucose degradation pathway that provides NADPH and substrates for nucleic acid synthesis and the shikimic acid pathway (Sharkey 2021), in which G6PDH and 6PGDH are the two key enzymes. In this study, S stress significantly increased G6PDH and 6-PGDH in both aerial and underground parts. Based on the above results, it was suggested that S stress transformed the EMP–TCA into PPP to enhance salt tolerance in G. uralensis seedlings, which has also been observed in salt-tolerant oats (Chen et al. 2021). D stress significantly increased 6-PGDH in the underground part of G. uralensis seedlings, which on the one hand promoted PPP and produced more energy to resist D stress, and on the other hand increased 6-PGDH promoted the synthesis of pentose phosphate, a substrate for nucleic acid synthesis, to meet the need for extra lateral roots to absorb more water and relieve stress. Combined stresses significantly increased G6PDH and 6-PGDH and upregulated relative genes in both aerial and underground part to promote PPP, which can provide enough energy for G. uralensis seedlings to resist stress.

Si addition decreased G6PDH and 6-PGDH in aerial part, G6PDH enzyme in the underground part, and downregulated two genes encoding FBP and transketolase, respectively, in underground part of S-treated plants to slow down PPP, which may be a symptom of stress relief and mitigation. Si weakened the PPP to avoid excessive carbohydrate consumption, which can probably be attributed to the fact that Si not only mitigated the inhibitory effect of salt stress on the TCA cycle but also accelerated the EMP pathway to provide sufficient energy to resist stress. Similarly, Si addition significantly reduced 6-PGDH in the aerial part and G6PDH and 6-PGDH in the underground part of D-stressed seedlings, indicating that Si significantly relieved D stress and slowed PPP to reduce carbohydrate consumption, resulting in biomass accumulation. However, Si addition increased G6PDH but decreased 6-PGDH in the aerial part, while it was the opposite trend in the underground part of S+D+Si-treated seedlings. In addition, Si addition upregulated a gene encoding 6-phosphogluconolactonase in the aerial part, which could further promote the PPP in S+D-stressed seedlings to provide more energy for resistance to stress.

Overall, Si mitigated the inhibitory effect of stress condition and decelerated PPP in G. uralensis seedlings exposed to S or D stress alone and perturbed PPP in those exposed to S+D stress by regulating G6PDH and 6-PGDH activities and related genes, thus providing more energy for subsequent stress- resistant activities in these plants.

Conclusion

Silicon addition effectively mitigated the inhibitory effect of stress on the respiratory metabolism in G. uralensis seedlings with different strategies, thus alleviating the inhibitory effect of stress on the growth of G. uralensis seedlings to varying degrees. Specifically, under salt stress conditions, Si addition increased leaf number by 15.13%, upregulated the TCA pathway at the transcriptional level, regulated related enzyme activities and increased acetyl CoA content in aerial and underground part by 39.97% and 24.86%, respectively, at the physiological and biochemical levels, thus relieving the inhibitory effect of salt stress on the EMP–TCA process, but weakening PPP by declining G6PDH and 6-PGDH in aerial part by 34.10% and 55.54%, and G6PDH in underground part by 83.86%. Under drought stress, Si application increased underground biomass by 11.40%, slowed the EMP by decreasing PFK in aerial part by 21.76%, PK and PFK in underground part by 38.11% and 64.71%, weakened PPP by reducing 6-PGDH in aerial part by 72.9%, G6PDH and 6-PGDH in underground part by 77.00% and 64.71%. Si increased SDH and PEPC by 28.66% and 145.48% with the TCA cycle and GABA shunt as the main respiratory pathways in the underground part of drought-stressed seedlings. Under combined stresses, Si addition increased leaf number and aboveground biomass by 11.96% and 23.88%, upregulated the EMP and TCA pathways at the transcriptional level, and alleviated the effects of combined stress on EMP–TCA metabolic processes at the physiological and biochemical levels by increasing PDH, α-KGDH and NAD-MDH in aerial part by 27.22%, 18.85% and 140.37%, HK, PK and PEPC in underground part by 93.97%, 25.81% and 31.19%, initiated the replenishment mechanism and inhibited GABA shunt in underground part of G. uralensis to provide energy for vital activities.