In order to establish the CNM regulatory module with statistical significance, all CNM-related genes were uploaded to the STRING (https://version-10-5.string-db.org/) software, and the protein–protein interaction (PPI) networks (combined score >0.4) were constructed. Then, the modules (resolution = 0.8) with higher visualization were performed by the program Gephi 0.9.2 (Lin et al., 2022b).

In order to identify the key module activated in CNM in rice seedlings exposed to SCN⁻, the results from PCR tests were used to calculate the possibility of the occurrence of genes grouped in four different modules. We first converted the data through the functions of “COMPUTE x_new=SQRT(X)” or “COMPUTE x_new = LN(x)” in the SPSS software since they were non-normally distributed. Then, the normcdf was calculated statistically.

As shown in Figure 2A , PEPC, upregulated genes in roots were Osppc4, Osppc2a, and Osppc2b, at all SCN⁻ treatments, while Osppc1, Osppc2a, and Osppc2b were upregulated in shoots. In roots of rice seedlings from the “SCN⁻ + 2-OG” treatments, Osppc4 and Osppc3 were upregulated, while Osppc4, Osppc1, and Osppc2a showed remarkable expression in shoots.

Expression of ERS-related genes is shown in Figure 2A , ERS, wherein OsERS1, OsERS2, and OsERS3 were generally upregulated in SCN⁻-exposed rice parts, i.e., in roots and shoots. However, the expression levels of OsERS1, OsERS2, and OsERS3 showed raised pattern in shoots of rice plants from the “SCN⁻ + 2-OG” treatments.

Figure 2A , TPS, depicts that higher expression levels of TPS-related genes of OSTPS5 and OSTPS1 were prominent in roots in SCN⁻ treatments, while OSTPS5 and OSTPS8 had greater expression in shoots. Interestingly, when rice seedlings were pretreated with 2-OG, OSTPS5, OSTPS8, and OSTPS9 were upregulated in roots. However, the expression levels of five TPS isogenes in shoots conferred a variance tendency, which reinforced at 0 mg SCN/L and then declined at 24 mg SCN/L.

Upregulation of three SPS isogenes (OsSPS1, OsSPS3, and OsSPS5) was observed in roots in all SCN⁻ treatments ( Figure 2A , SPS), while only OsSPS2 was upregulated in shoots. Differential expression patterns were found in SPS-related genes of roots in “SCN⁻ + 2-OG” treatments. The expression levels of OsSPS1, OsSPS3, and OsSPS5 in roots from the “SCN⁻ + 2-OG” treatments were higher than those of SCN⁻ treatments. OsSPS1 and OsSPS5 were upregulated in shoots of “SCN⁻ + 2-OG” treatments, which differed from SCN⁻ treatments.

Positive expressions of NR genes, i.e., OsNIA1, OsNIA2, and OsNR1, were observed in roots after SCN⁻ exposure ( Figure 2B , NR). However, the expression levels of OsNIA1, OsNIA2, and OsNR1 in shoots showed a disparate trend with an initial escalation from 24 mg SCN/L and then dropped at 96 mg SCN/L. In the case of rice seedlings from the “SCN⁻ + 2-OG” treatments, higher expression levels of OsNIA1, OsNIA2, and OsNR1 were observed in rice shoots.

Within NiR genes, only OsNiR-1 was upregulated in roots at all SCN⁻ treatments ( Figure 2B , NiR), while upregulation of OsNiR-1 and OsNiR-3 was detected in shoots. Interestingly, the expression levels of OsNiR-1, OsNiR-2, and OsNiR-3 conferred an accelerating pattern in both roots and shoots of the “SCN⁻ + 2-OG” treatments.

Differential expression of GS-related genes was observed between roots and shoots, with significant upregulation of OsGS2, OsGS1;2, and OsGS1;3 in roots and shoots (OsGS2 and OsGS1;3) ( Figure 2B , GS). However, the expression levels of OsGS2, OsGS1;2, OsGS1;1, and OsGS1;3 showed linear inclination with increasing SCN⁻ concentrations in both rice tissues by inoculation of 2-OG.

Transcriptional changes of NAD-IDH genes are shown in Figure 2C , IDH. As apparent from the figures, OsIDHa and OsIDHc;1 were upregulated in roots, while OsIDHc;2 and OsIDH1 were overexpressed in shoots. When rice seedlings were pretreated with 2-OG, upregulation of OsIDHc;1 was observed in roots, and OsIDHa, OsIDHc;2, and OsIDH1 were upregulated in shoots.

As presented in Figure 2C , ICDH, OsICDH2, OsICDH3, and OSICDH4 were significantly (p< 0.05) upregulated in roots after SCN⁻ exposure, while positive expressions (p< 0.05) of OsICDH1, OsICDH2, and OsICDH4 were observed in shoots. Interestingly, OsICDH1, OsICDH2, and OsICDH4 in roots presented an upregulated pattern in the “SCN⁻ + 2-OG” treatments and the expression levels of OsICDH2, OsICDH3, and OsICDH4 were remarkable in shoots.

Upregulation of OsGDH1, OsGDH2, and OsGDH4 was observed in both rice tissues after SCN⁻ exposure ( Figure 2C , GDH), while significantly (p< 0.05) higher expressions of OsGDH1, OsGDH2, and OsGDH3 were observed in roots, and significant upregulation of OsGDH1, OsGDH2, and OsGDH4 was detected in shoots of the “SCN⁻ + 2-OG” treatments.

The activities of CNM-related enzymes were assayed in SCN⁻ and “SCN⁻ + 2-OG” treatment plants ( Figure 3 ). The activity of PEPC in roots was affirmatively increased (p< 0.05) after SCN⁻ exposure compared with the control, while the activity of PEPC in shoots presented a downward tendency. The activity of ERS in roots was significantly inhibited (p< 0.05) after SCN⁻ exposure in comparison with the control, while the activity of ERS was increased in shoots. Activities of TSP and SPS presented significant increasing patterns in both roots and shoots in the presence of SCN⁻ stress (p< 0.05). Under “SCN⁻ + 2-OG” treatments, activities of PEPC, ERS, TSP, and SPS intensified in roots compared to control (p > 0.05), while activities of ERS, TSP, and SPS showed a decrement in shoots unlike control (p > 0.05).

Activities of NR and NiR in roots presented an exponential emulate compared with the control (p< 0.05), while activities of NR and NiR in shoots illustrated an inverted “U” shape curve under SCN⁻ stress ( Figure 3 ). The analogous activity of GS was noticed in both roots and shoots of SCN⁻ stress (p< 0.05), following linearity with stress concentration. Under “SCN⁻ + 2-OG” treatments, activities of NR, NiR, and GS showed a similar pattern of increment in both roots and shoots (p< 0.05).

Overall, the activities of all selected enzymes in “SCN⁻ + 2-OG” treatments were generally higher than those of SCN⁻ treatments. Exogenous 2-OG had a pronounced impact on enzyme activities in shoots, unlike its counterpart. To reveal the regulation mechanism of exogenous 2-OG on the CNM in rice plants under SCN⁻ exposure, we distinguished the effects of exogenous 2-OG on C and N metabolism in rice tissues.

The activity of NAD-IDH was inhibited significantly (p< 0.05) in both roots and shoots under SCN⁻ exposure compared with the control ( Figure 3 ). The activity of NADP-ICDH in SCN⁻-exposed roots was prominently enhanced; nevertheless, the scenario was reversed in the case of shoots (p< 0.05). Elevated activity of GDH was observed in both roots and shoots in the presence of SCN⁻ (p< 0.05). Under “SCN⁻ + 2-OG” treatments, the activities of NAD-IDH, NADP-ICDH, and GDH were generally increased in rice tissues, except for GDH in shoots of rice seedlings. These results indicated that the modification mechanism of exogenous 2-OG on CNM-related enzymes in rice seedlings under SCN⁻ exposure varied greatly.

Plants have evolved the coordinated actions responsible for their diverse physiological processes via either direct or indirect gene connections. In order to elucidate the functional module of genes activated in the CNM process, a co-expression network was performed by the STRING program, and four modules were obtained. Detailed information on gene interaction strengths in these four modules is given in Table S2 . Interestingly, all modules had similar interaction contributions, namely Module 1 (25.0%), Module 2 (25.0%), Module 3 (27.5%), and Module 4 (22.5%) ( Figure 4A ). We also noticed that the genes grouped in Modules 1 and 2 were involved in C metabolism (11 genes) and biosynthesis of 2-OG (10 genes), respectively; genes categorized in Module 3 were responsible for N metabolism (five genes) and biosynthesis of 2-OG (five genes). In addition, genes grouped in Module 4 were activated in C metabolism (one gene) and N metabolism (five genes) and biosynthesis of 2-OG (three genes) ( Figure 4B ).

The normcdf of rice shoots was quite different between under SCN⁻ and SCN−+2-OG treatments, based on the non-linear regression ( Figure 5 ). Herein, the threshold for the highest occurrence probability was set, p > 0.75. Therefore, Module 3 showed the highest occurrence probability, suggesting that the exogenous application of 2-OG mainly regulated the expression of genes activated in the N metabolism and 2-OG synthesis to modify the imbalance of CNM in rice plants imposed by SCN⁻ exposure. In fact, a similar conclusion was also reached in the analysis of C and N fractions in rice tissues. We noticed that the change of C fraction in rice shoots was almost constant ( Figures 1B, C ), while the change of N in rice shoots was evident between SCN⁻ treatments and “SCN⁻ + 2-OG” treatments ( Figures 1D, E ).

It is known that there are three innate routines for the biosynthesis of 2-OG in plants, in which the PEPC pathway and the NADP-ICDH/NAD-IDH pathway belong to C metabolism, and the GDH pathway is mainly involved in N metabolism. In this study, we focused on correlating and perceiving the most competent pathway in controlling the generation of 2-OG and regulating the imbalance of CNM in rice seedlings caused by SCN⁻ exposure.

The PEPC pathway in plants is an anaplerotic reaction to replenish the tricarboxylic acid (TCA) cycle with intermediates that are withdrawn for different biosynthesis pathways and N metabolism (Lemaitre et al., 2007). For example, PEPC is able to catalyze phosphoenolpyruvic acid (PEP) into 2-OG, and 2-OG synthesis from malate can be suppressed by the knockdown of Osppc4, therefore causing a decrease in plant growth and leaf area (Masumoto et al., 2010), suggesting that Osppc4 is crucial for the growth of rice plants. In the present study, significant downregulation of Osppc4 was observed in shoots of rice seedlings after SCN⁻ exposure ( Figure 2C ). The activity of PEPC was decreased in shoots after SCN⁻ treatments ( Figure 3 ), suggesting that the synthesis of 2-OG in shoots from the PEPC pathway was repressed by SCN⁻ exposure.

The second pathway of 2-OG generation is the NADP-ICDH/NAD-IDH pathway (Ferrario-Mery et al., 2001), in which citrate can be either exported from mitochondria to cytosol for 2-OG synthesis by cytosolic enzymes aconitase NADP-ICDH or transformed into 2-OG in mitochondria by TCA cycle enzyme aconitase NAD-IDH (Yuan et al., 2007). Indeed, NAD-IDH is often regarded as a major governing point in plants (Lemaitre et al., 2007), which is encoded with one single gene OsIDHa in rice plants (Lancien et al., 1998). Herein, significant downregulation of OsIDHa was detected in shoots after SCN⁻ exposure ( Figure 2C ), suggesting that SCN⁻ exposure could inhibit the expression of OsIDHa in shoots. A significant correlation was obtained in the enzymatic assay of NAD-IDH, wherein a decrease in the activity of NAD-IDH was observed in shoots of rice seedlings exposed to SCN⁻ ( Figure 3 ). Results from both C-related pathways indicated that SCN⁻ exposure significantly repressed both pathways to produce 2-OG, thereby causing a severe impact on the C metabolism and breaking the balance of CNM.

The third source to produce 2-OG is from the GDH pathway, in which the oxidative deamination of glutamate (Glu) into 2-OG is catalyzed by GDH. It has been reported that exogenous 2-OG increased the activities of GDH in wheat seedlings and promoted yield productivity under drought stress (Lancien et al., 1998). Additionally, GDH plays a unique role in the formation of NH₄ ⁺ and 2-OG during the assimilation of Glu (Lodwig et al., 2003). We found that the expression levels of OsGDH1 and OsGDH2 were significantly upregulated in shoots after SCN⁻ treatments ( Figure 2C ). The change of GDH activity in shoots was constructive against SCN⁻ exposure ( Figure 3 ). These results indicated that SCN⁻ exposure does not disturb the conversion of Glu into 2-OG through the activation of GDH. Combined with the results from C-related pathways of 2-OG, we have sufficient reasons to conclude that the imbalance of CNM in rice seedlings was evident due to SCN⁻ exposure through repressing the two C-related pathways.

Compared with SCN⁻ treatments ( Figure 2C ), the expression levels of Osppc4 in shoots of rice seedlings under “SCN⁻ + 2-OG” treatments were significantly upregulated, and the activity of PEPC in shoots was also positively responsive, suggesting that the application of 2-OG enhances the enzyme activity of PEPC and might stimulate the generation of 2-OG. A similar conclusion was also predicted in the second pathway of 2-OG generation due to the application of exogenous 2-OG, wherein a correlation between upregulated expression of OsIDHa and increases in NAD-IDH activity was obtained. Additionally, the expression levels of OsGDH1 and OsGDH2 in shoots of rice seedlings fed with 2-OG were significantly upregulated, and an increase of GDH activity in shoots was also detected ( Figure 3 ), suggesting that the conversion of Glu into 2-OG was independent of the application of 2-OG. Co-expression network analysis showed that the GDH-related genes in Module 1 had a higher connection degree with others ( Figure 4A ). Apparently, these results indicated that the two C-related pathways were significantly activated due to the application of 2-OG, in which sufficient 2-OG in plant cells was able to modify the imbalance of CNM in rice seedlings caused by SCN⁻ exposure, subsequently decreasing the negative impact on rice seedlings, which was judged by a measurable increase in biomass growth of rice seedlings from the SCN⁻-treated rice seedlings with 2-OG, compared with the SCN⁻-treated rice seedlings without 2-OG.

The TPS and SPS are primary targeted cytosolic enzymes involved in the C metabolism (Coruzzi and Zhou, 2001). Previous studies indicated that the expressions of OsTPS2, OsTPS5, and OsTPS6 in rice were negatively correlated with sucrose starvation (Wang et al., 2007). The expression of SPS genes was positively correlated with non-structure carbohydrate content in the leaf, wherein OsSPS1 expression and SPS activity were affirmatively corresponding to spike number and grain yield (Li and Cui, 2018). However, another study showed that mRNA levels of OsSPS1 and OsSPS6 were negatively correlated with sucrose concentrations (Yonekura et al., 2013). In the present study, upregulation of OsTPS5 was detected in both rice tissues under SCN⁻ stress ( Figure 2 ). In addition, upregulated OsSPS1 was also evident in both roots and shoots. Meanwhile, increases in TPS and SPS activities in rice tissues were detectable ( Figure 3 ). These results indicated that SCN⁻ exposure stimulated the expression of C metabolism-related master regulation genes, thus regulating the enzyme activities. Enzyme ERS has an important role in maintaining the physiological homeostasis of amino acids and C metabolism as well as redox status (Yang et al., 2018). Like TPS and SPS, upregulation of OsERS1 was distinguished in both rice tissues after SCN⁻ stress ( Figure 2 ). Also, we observed that responses of OsERS1 to SCN⁻ exposure were identical to the enzyme activity of ERS in rice tissues ( Figure 2 ).

NR, NiR, and GS are three key enzymes involved in N assimilation. The conversion of NO₃ ⁻ into NH₄ ⁺ is catalyzed by the enzymes NR and NiR, which is a rate-limiting step in NO₃ ⁻ assimilation. In the present study, three isogenes of NR (OsNIA1, OsNIA12, and OsNR1) and NiR (OsNiR-1, OsNiR-2, and OsNiR-3) showed a declining expression pattern in shoots after SCN⁻ exposure, of which SCN⁻ treatments at 24.0 and 96.0 mg/L positively regulated transcriptional changes in OsNR1, and 300.0 mg SCN/L treatment demonstrated a negative response ( Figure 2 ). Enzymatic assay showed that activities of NR and NiR had a positive correlation with gene expression ( Figure 3 ), suggesting that low-to-moderate concentrations of SCN⁻ exposure might stimulate the conversion of NO₃ ⁻, and higher SCN⁻ concentrations had a negative effect on this process, whereas a similar conclusion was also reached by Lin et al. (2022a). Another crucial enzyme in N metabolism is GS, which is responsible for converting NH₄ ⁺ into glutamine. In this study, almost all GS genes were upregulated in the SCN⁻ treatments ( Figure 2 ). It is established that GS in most plants occurs as GS2 in plastids and GS1 in the cytosol. The role of GS1 is to assimilate NH₄ ⁺ in roots and reassimilate NH₄ ⁺ in leaves, whereas GS2 is mainly responsible for assimilating NH₄ ⁺ derived from NO₃ ⁻ reduction in plastids (Lin et al., 2022a). The enzymatic assay also showed that activities of GS had a positive correlation to gene expression ( Figure 3 ), suggesting that SCN⁻ treatments do not inhibit the activity of GS and subsequently increase the conversion of NH₄ ⁺.

Compared with SCN⁻ treatments ( Figure 2 ), exogenous 2-OG decreased the expression of OsTPS5 in shoots of rice seedlings after SCN⁻ exposure. Similarly, the downregulation of OsSPS6 and OsERS1 was also detectable. Enzymatic assays indicated that the application of 2-OG decreased the activities of TPS, SPS, and ERS in shoots of rice seedlings ( Figure 3 ). A co-expression network analysis showed that the C metabolism-related genes in Module 2 and Module 4 had a lower connection degree with others ( Figure 4A ). However, we noticed that NR and NiR genes were upregulated in SCN⁻-treated rice seedlings inoculated with 2-OG, wherein enzymes of NR and NiR were positively responsive to 2-OG application, indicating that the application of 2-OG had a positive impact on the conversion of NO₃ ⁻. A previous study also reported that feeding of 2-OG increased transcripts of the NR gene in tobacco leaf (Ferrario-Mery et al., 2001). Additionally, under “SCN⁻ + 2-OG” treatments, the expression levels of OsGS1;2 were significantly higher than those under SCN⁻ treatments, suggesting that the application of 2-OG also increases the conversion of NH₄ ⁺ derived from the main N source of NO₃ ⁻ supplied. A co-expression network analysis showed that the N metabolism-related genes in Module 3 and Module 4 had a higher connection degree with others ( Figure 4A ). These results indicated that sufficient 2-OG in plant cells can modify the imbalance of N metabolism-related genes in rice seedlings caused by SCN⁻ exposure.