*3.1. Amino Acid Metabolism*

The GABA shunt is comprised of three steps, including the α-decarboxylation of glutamate to GABA by glutamate decarboxylase (*GAD*), the conversion of GABA to succinic semialdehyde by GABA transaminase (*GABA-T*), and the oxidization of succinic semialdehyde to succinate catalyzed by succinic semialdehyde dehydrogenase (*SSADH*) [30,31]. There are reported connections between GABA shunt and plant dwarfism. The suppression of *GABA-T* induces prominent GABA accumulation, dwarfism, and infertility in the tomato [32]. Rice plants overexpressing *OsGAD2*Δ*C* have dwarf phenotypes [33]. In the present study, there was significant increase of GABA and decrease of glutamate levels in both M and ECE. Besides, the succinate level and the *GAD4* gene expression was down-regulated in M. Although both glutamate and *GAD4* were down-regulated in M, other metabolic pathways (e.g., arginine and proline metabolism) may contribute to the increase of GABA level. It indicates that *OsCYP96B4* functions affect rice GABA shunt, which may be connected with the dwarfism phenotype [32,33].

The glutamine synthetase/glutamine:2-oxoglutarate amidotransferase (i.e., GS/GOGAT) cycle plays a regulatory role in the nitrogen assimilation process in plants [34]. While glutamine synthetase (*GS*) catalyzes the conversion of glutamate and ammonia into glutamine, glutamate synthase (i.e., *GOGAT*, or glutamine:2-oxoglutarate amidotransferase) catalyzes the formation of glutamate from glutamine and 2-oxoglutarate. Here, the significant decrease of glutamate was observed in both M and ECE, together with the up-regulated *GS*, *GOGAT2* gene expression, and glutamine level in ECE. It suggests the function of *OsCYP96B4* on rice glutamate and glutamine metabolism, along with GS/GOGAT cycle, which may be connected with the nitrogen assimilation process [34] and the dwarfism phenotype.

Isoleucine, leucine, and valine are three branched-chain amino acids (BCAAs). Here, significant decrease of isoleucine and valine were observed in both M and ECE, along with the significantly reduced leucine level in M. As both valine and isoleucine possess the glucogenic property, their level decrease and the increase of glucose concentration may indicate the possible gluconeogenesis process in the mutant rice. Although at present there are no relevant reports, it suggests the effect of *OsCYP96B4* mutation on rice branched-chain amino acid metabolism, which may be connected with the dwarfism phenotype.

Phosphoethanolamine/phosphocholine phosphatase (*PHOSPHO1*) catalyzes the conversion of phosphoethanolamine to ethanolamine and the conversion of phosphocholine to choline. Choline can be further converted to betaine by the action of choline monooxygenase (*CMO*) and betaine aldehyde dehydrogenase 2 (*BADH2*). It was shown that simultaneous expression of *Spinacia oleracea* chloroplast *CMO* and *BADH* genes contribute to dwarfism in transgenic *Lolium perenne* [35]. In the current study, there were significantly decreased ethanolamine level and *BADH2* gene expression level, along with significantly increased choline level in M. While, there were significantly decreased ethanolamine and phosphocholine levels and significantly increased *PHOSPHO1* gene expression level in ECE. These observations suggest the influence of *OsCYP96B4* function on rice choline metabolism and dwarfism [35].

In addition, *OsCYP96B4* mutation showed potential effects on the metabolism of other amino acids, including the significant decrease of threonine, aspartate, histidine levels, and *LL-DAP-AT* gene expression in both M and ECE, the down-regulation of *AAT* expression level in M, and the significantly decreased levels of asparagine, arginine, and *ALT* expression along with the significantly increased levels of alanine and *AK* expression in ECE.

### *3.2. Carbohydrate Metabolism*

Sucrose synthase (*SUS*) is a key enzyme for the regulation of carbon partitioning in plants by providing UDP-glucose as a substrate for the biosynthesis of cellulose and other polysaccharides [36]. It has been shown that AtCesA8::SUS3 transgenic rice plants exhibited largely improved biomass saccharification and lodging resistance by reducing cellulose crystallinity and increasing cell wall thickness [36]. *OsIDD2* overexpression leads to severely dwarfed rice plants and *OsIDD2* negatively

regulates the transcription of genes involved in lignin biosynthesis, cinnamyl alcohol dehydrogenase 2 and 3 (*CAD2* and *3*), and sucrose metabolism sucrose synthase 5 (*SUS5*) [37]. In the present study, although there were significant increases of glucose, fructose, and uridine levels in M, the significant decrease of *SUS1* gene expression may lead to the unchanged level of sucrose. While for ECE, the significantly increased levels of glucose, fructose, and *HXK8* and *PGM* gene expression and the decreased uridine level may result in the decrease of sucrose level, in spite of the unchanged *SUS1* gene expression. It suggests the influence of *OsCYP96B4* on sucrose metabolism, which may be connected with dwarfism [37]. Besides, the significantly decreased *FBA*, *GAPDH*, and *iPGM* gene expression levels in M further supported the effect of *OsCYP96B4* gene mutation on rice carbohydrate metabolism.

The TCA cycle is carried out through a variety of interconnected enzymatic reactions, e.g., the conversion of succinate to fumarate by succinate dehydrogenase (*SDH*), the oxidation of malate to oxaloacetate via malate dehydrogenase (*MDH*), and the transformation of citrate to *cis*-aconitate and then isocitrate by aconitase (*ACO*). *OsAPX2* (rice ascorbate peroxidase 2) knock-out leads to shoot dwarfing and up-regulation of enzymes linked to glycolysis and TCA cycle in rice flag leaves [38]. *Arabidopsis thaliana* mutants lacking plastidial NAD-dependent MDH (*pdnad-mdh*) are embryo-lethal, and constitutive silencing (*miR-mdh-1*) leads to a dwarfed phenotype [39]. In the present study, there were significant decreases of TCA cycle intermediates (i.e., succinate, fumarate, malate, and citrate) and *ACO* gene expression level in M, along with the significant up-regulation of *SDH* and the down-regulation of *MDH* gene expression levels in ECE. It is inferred that the perturbation in TCA cycle is related to *OsCYP96B4* gene function and may be connected with the rice dwarfism phenotype [38,39].

#### *3.3. Nucleotide Metabolism*

As an important intermediate in the purine metabolism, adenosine monophosphate (AMP) is formed from adenine by adenine phosphoribosyltransferase (*APRT*) [40] and is interconvertible with adenosine through hydroxylation and phosphorylation reactions. Allantoin, an intermediary metabolite of purine catabolism, is hydrolyzed to allantoate under the catalysis of allantoinase (*ALN*) [41]. Similarly, uridine and UMP are two interconvertible intermediates in the pyrimidine metabolism. *APRT* was shown to be potentially involved in thermo-sensitive genic male sterility (TGMS) in the rice line 'Annong S-1- [42] and associated with growth retardation and male sterility in Arabidopsis [43]. In the current study, there was significant increase of adenosine and *APRT* gene expression, but significant decrease of AMP, uridine, and *ALN* gene expression level in ECE, along with significant increase of uridine and the significant decrease of *ALN* gene expression level in M. It suggests the potential function of *OsCYP96B4* on rice nucleotide metabolism, which may be associated with the rice dwarfism phenotype. However, to the best of our knowledge, currently, there are no reports on such association.

#### *3.4. Secondary Metabolism*

The shikimate and aromatic amino acids (AAA) biosynthesis pathways represent a link between primary and secondary metabolism in plants [44]. The catabolism of AAAs results in a variety of secondary metabolites, e.g., phenylpropanoids and flavonoids from phenylalanine, tocochromanols and phenylpropanoids from tyrosine, and indole-containing metabolites from tryptophan [45]. Cytochrome P450 monooxygenases (*CYP450s*) play important roles in the biosynthesis of plant secondary metabolites, including phenylpropanoids, terpenes, and alkaloids [46]. As one of the major phenylpropanoid pathway end-products, lignin is a major component of the secondary cell wall and is vital for providing mechanical strength to reduce lodging stress in plants [47,48]. Phenylalanine ammonia-lyase (*PAL*) catalyzes the conversion of phenylalanine to *trans*-cinnamic acid and plays an important role in the biosynthetic pathway of lignin [49]. In the dwarf rice mutant Fukei 71, elevated levels of p-coumaric acid (PCA), ferulic acid (FA), and *PAL* were observed in the abnormal parenchyma tissue, indicating that the abnormal activation of phenylpropanoid pathway leads to the biosynthesis of polysaccharide-linked

FA and PCA [50]. The AAAs metabolism is also connected with dwarfism. The overexpression of a rice tyrosine decarboxylase (*TyDC*) leads to tyramine accumulation in rice cells and causes a dwarf phenotype via reduced cell division [51]. The suppression of serotonin N-acetyltransferase 2 (*SNAT2*) leads to melatonin-deficient rice with a semidwarf phenotype [52]. Our present study showed that there was significant decrease in levels of phenylalanine, tyrosine, and tryptophan in both M and ECE, along with the significant decrease of *TAT*, *PRAI*, *IGPS*, and *ASB2* expression levels in M and the significant decrease of *PRAI* expression level in ECE. It may indicate the declined synthesis of phenylalanine, tyrosine, and tryptophan and reflect the function of *OsCYP96B4* on shikimate-mediated secondary metabolism in rice, which may be possibly connected with the dwarfism phenotype [51,52].
