*2.2. OsCYP96B4 Gene Mutation Induced Metabolic Changes in Mutant Rice*

Clear differentiation in the metabolic profiles among the three rice phenotypes was shown in the PCA scores plots (Figure S1) and the corresponding OPLS-DA scores plots (Figure 2). The OPLS-DA model quality was evaluated by the sevenfold internal cross-validation, with R2(X) = 0.368, R2(Y) = 0.988, Q<sup>2</sup> = 0.940 for the comparison between WT and M (Figure 2a), R2(X) = 0.537, R2(Y) = 0.985, Q2 = 0.959 for the comparison between WT and ECE (Figure 2b), and R2(X) = 0.464, R2(Y) = 0.972, Q2 = 0.915 for the comparison between M and ECE (Figure 2c). The model validities were demonstrated by CV-ANOVA of OPLS-DA models (Figure 2) and permutation tests on the corresponding PLS-DA models (Figure S2). Detailed metabolic profile difference among the three phenotypes were shown in the color-coded loadings plots of OPLS-DA models (Figure 2) and further summarized in Figure 3 and Table S2.

**Figure 2.** Cross-validated OPLS-DA scores plots (**left**) and the corresponding loadings plots (**right**) derived from the comparison of 1H NMR spectra for (**a**) the wild type (WT, -) and the *oscyp96b4* semi-dwarf mutant (M, •), (**b**) the wild type (WT, -) and the *OsCYP96B4* ectopic expression (ECE, ◆), (**c**) the *oscyp96b4* semi-dwarf mutant (M, •) and the *OsCYP96B4* ectopic expression (ECE, ◆). Metabolite keys are shown in Figure 1 and Table S1.

**Figure 3.** Heat map showing metabolites with significant level changes (*p* < 0.05) for (**a**) the *oscyp96b4* semi-dwarf mutant (M) vs. the wild type (WT), (**b**) the *OsCYP96B4* ectopic expression (ECE) vs. the wild type (WT), and (**c**) the *OsCYP96B4* ectopic expression (ECE) vs. the *oscyp96b4* semi-dwarf mutant (M). It was color-coded with the Pearson correlation coefficients from the corresponding OPLS-DA models, where a warm color (e.g., red) indicates significant increase of metabolites in M (**a**) or ECE (**b**,**c**) as compared to the counterpart, a cool color (e.g., blue), indicating significant decrease, and the grey color indicates no significant difference.

Compared to WT, M showed higher levels of γ-aminobutyrate, choline, carbohydrates (glucose, fructose), uridine, N-methylnicotinate, lipid, and mono-methyl phosphate, but lower levels of most amino acids (isoleucine, leucine, valine, threonine, glutamate, aspartate, tyrosine, histidine, tryptophan, phenylalanine), ethanolamine, and TCA cycle intermediates (malate, succinate, citrate, and fumarate) (*p* < 0.05, Figures 2a and 3a, Table S2). ECE presented more alanine, γ-aminobutyrate, glutamine, carbohydrates (glucose, fructose), adenosine, N-methylnicotinate, lipid, and formate, but less amino acids (isoleucine, valine, threonine, arginine, glutamate, aspartate, asparagine, tyrosine, histidine, tryptophan, phenylalanine), choline metabolites (ethanolamine, phosphocholine), sucrose, nucleotide metabolites (uridine, adenosine monophosphate), and mono-methyl phosphate than WT (*p* < 0.05, Figures 2b and 3b, Table S2). Compared with M, there were increases of γ-aminobutyrate, glutamine, alanine, carbohydrates (glucose, fructose), TCA cycle intermediates (succinate, fumarate and malate), lipid, and formate, but decreases of amino acids (asparagine, arginine, histidine, phenylalanine, tyrosine, tryptophan), choline metabolites (ethanolamine, phosphocholine), sucrose, uridine, and mono-methyl phosphate in ECE (*p* < 0.05, Figures 2c and 3c, Table S2).

#### *2.3. Gene Expression Analysis*

The qRT-PCR analysis was performed to acquire supporting information for the aforementioned metabolic changes induced by *OsCYP96B4* gene mutation. Compared with WT, significant alterations (*p* < 0.05) in M or ECE were observed for the expression levels of key enzyme-encoding genes, responsible for the regulation of GABA shunt, glutamate/glutamine metabolism, choline metabolism, carbohydrate metabolism, nucleotide metabolism, and secondary metabolism (Figure 4).

Along with the metabolite level changes, *OsCYP96B4* gene mutation induced significant alterations in the expression levels of key genes in GABA shunt and glutamate/glutamine metabolism, including the down-regulation of glutamate decarboxylase 4 gene (*GAD4*) in M and the up-regulation of glutamate synthase 2 gene (*GOGAT2*) and glutamine synthetase gene (*GS*) in ECE. *OsCYP96B4* gene mutation also resulted in significant changes of gene expression related to choline metabolism, i.e., down-regulation of betaine aldehyde dehydrogenase 2 gene (*BADH2*) in M and up-regulation of phosphoethanolamine/phosphocholine phosphatase gene (*PHOSPHO1*) in ECE. In addition, significant alterations were also observed for the gene expression pertaining to the metabolism of other amino acids, including the down-regulation of aspartate aminotransferase gene (*AAT*) and LL-diaminopimelate aminotransferase gene (*LL-DAP-AT*) in M, along with the up-regulation of *AAT* and aspartate kinase gene (*AK*) and down-regulation of alanine transaminase gene (*ALT*) and *LL-DAP-AT* in ECE (Figure 4a).

There were significant changes in the gene expression involved in carbohydrate metabolism, including significant down-regulation of genes encoding sucrose synthase 1 (*SUS1*), fructose-bisphosphate aldolase (*FBA*), glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (*iPGM*) and aconitase (*ACO*) in M, along with the up-regulation of genes encoding hexokinase-8 (*HXK8*), phosphoglucomutase (*PGM*), and succinate dehydrogenase (*SDH*) and down-regulation of genes encoding malate dehydrogenase (*MDH*) in ECE (Figure 4b).

Significant changes also occurred in the gene expression related to nucleotide metabolism, i.e., the up-regulation of adenine phosphoribosyltransferase gene (*APRT*) in ECE, and down-regulation of allantoinase (*ALN*) gene in both M and ECE (Figure 4c). In addition to the significant alterations of gene expression in primary metabolism, *OsCYP96B4* gene mutation also led to significant changes of gene expression in secondary metabolism, i.e., the down-regulation of tyrosine aminotransferase gene (*TAT*), phosphoribosylanthranilate isomerase gene (*PRAI*), indole-3-glycerol phosphate synthase gene (*IGPS*), anthranilate synthase beta subunit 2 gene (*ASB2*) in M, and the down-regulation of *PRAI* in ECE (Figure 4c).

For the comparison between ECE and M, the gene expression alterations were nearly the same as the comparison between ECE and WT, except for the significant increase of *ACO* and no significant change of *PRAI* in ECE when compared with M (at the significance level of 0.05, Figure 4a–c).

**Figure 4.** Gene expression levels in the wild-type (WT, blue bars), the *oscyp96b4* semi-dwarf mutant (M, orange bars), and the *OsCYP96B4* ectopic expression (ECE, grey bars) rice lines measured by qRT-PCR. Data shown are means ± SE of three biological replicates each with three technical replicates (\* *p* < 0.05, #*<sup>P</sup>* <sup>&</sup>lt; 0.01, as compared to WT; † *<sup>p</sup>* <sup>&</sup>lt; 0.05, ‡ *<sup>p</sup>* <sup>&</sup>lt; 0.01, as compared to M).

#### **3. Discussion**

The aforementioned metabolomics and gene expression data showed that *OsCYP96B4* gene mutation resulted in comprehensive metabolic responses in rice plants. The responses were summarized in Figure 5, mainly including alterations in amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, and secondary metabolism. In general, such changes were more comprehensive in ECE than in M when compared with WT, especially on the gene expression levels. Moreover, significant differences were observed in the metabolic and gene expression levels between ECE and M. These differences may be related to the developmental disparities in the two mutants. The *oscyp96b4* mutant displayed a semi-dwarf phenotype, but with the development of panicles and seeds (i.e., complete mature plant formation). However, the ECE plants remained severely dwarf, without any panicle formation, and normally died after about 4 weeks in the pot. Taken together, the results shed light on the *OsCYP96B4* gene function and may be associated with the plant phenotype (i.e., dwarfism).

**Figure 5.** Systems metabolic reprogramming induced by *OsCYP96B4* gene mutation in rice. Symbols: boxes () represent metabolite levels; circles (-) represent gene expression levels; both the solid and dashed arrows indicate the enzyme catalyzed metabolic reactions, where the dashed arrows were intentionally used to prevent the intersection between solid ones. Colors: red indicates significant increase (*p* < 0.05), blue indicates significant decrease (*p* < 0.05), grey indicates no significant change; purple letters denote identified metabolites; italic brown letters denote genes with examined transcript levels. Metabolite abbreviations: AMP, adenosine monophosphate; GABA, γ-aminobutyrate; NMN, nicotinamide mononucleotide; NMNA, N-methylnicotinate; PPi, diphosphate; UDP, uridine diphosphate; UMP, uridine 5'-monophosphate; UTP, uridine triphosphate. Gene abbreviations: *SUS1*, sucrose synthase 1; *PGM*, phosphoglucomutase; *HXK8*, hexokinase-8; *FBA*, fructose-bisphosphate aldolase; *GAPDH*, glyceraldehyde 3-phosphate dehydrogenase; *iPGM*, 2,3-bisphosphoglycerate-independent phosphoglyceratemutase;*SDH*, succinate dehydrogenase;*MDH*,malate dehydrogenase;*ACO*, aconitase;*PAL*, phenylalanine ammonia-lyase; *TAT*, tyrosine aminotransferase; *PRAI*, phosphoribosylanthranilate isomerase; *IGPS*, indole-3-glycerol phosphate synthase; *ASB2*, anthranilate synthase beta subunit 2; *LL-DAP-AT*, LL-diaminopimelate aminotransferase; *AK*, aspartate kinase; *ALT*, alanine transaminase; *AAT*, aspartate aminotransferase; *BADH2*, betaine aldehyde dehydrogenase 2; *CMO*, choline monooxygenase; *PHOSPHO1*, phosphoethanolamine/phosphocholine phosphatase; *GABA-T*, GABA transaminase; *GAD4*, glutamate decarboxylase 4; *GOGAT2*, glutamine:2-oxoglutarate amidotransferase 2; *GS*, glutamine synthetase; *APRT*, adenine phosphoribosyltransferase; *ALN*, allantoinase.
