## 4 Discussion
In our study, the distance between adjacent small vascular bundle (SVBs) in _OsVTC1_-_1 RNAi lines was also shorter than wild type, suggesting that cell size reduction of both parenchyma and vascular tissue may affect the length between adjacent SVBs. This result was supported by the distance between two adjacent vascular bundles decreased due to the reduction of mesophyll and bundle sheath cell thickness in the presence of NaCl [64]. Similarly, the thickness of cuticle and epidermis of the upper surface and lower cuticle thickness in _OsVTC1_-_1 RNAi leaf was significantly smaller than in wild type. A previous study also showed that the cell size in _vtc1_ and _vtc2_ were smaller than wild type in Arabidopsis [9]. AsA is necessary for many biological processes in plants, including cell expansion, cell division and cross-linking of the cell walls [2; 4]. Therefore, the reduction of AsA synthesis by knocking down _OsVTC1_-_1 gene expression likely disrupts the cell size and cellular organization in leaves. _VTC1_ catalyzes the synthesis of GDP-D-mannose, which is a substrate for cell wall polysaccharide and glycoprotein synthesis, and likewise GDP-L-galactose [3]. In grasses, xylose is the major component of hemicellulose monosaccharide (about 60%) [65]. Our study showed that xylose is abundantly present in leaf cell walls in both WT and _OsVTC1_RNAi lines. In addition, mannose and galactose composition as mole percentages of total sugars decreased in both RII-2 and RII-3 RNAi lines. Similar to a previous study in potato, the GMPase (VTC1) antisense plants exhibited a reduction in the mannose content of cell walls in leaves, while other monosaccharides content in cell walls were not different from the wild type. Reduction in the mannose content of cell walls in GMPase antisense plants was a result of a reduction in GDP-mannose production [44]. In the cell walls of Arabidopsis _vtc1_ mutant, mannose and fucose levels were also decreased [66]. Decreasing _VTC1_ expression led to a reduction in the mannose and galactose composition, suggesting that _VTC1_ may be involved in cell wall formation because sugars are components of plant cell wall polymers [67]. Sugars play a role in plant development, as well as plant signaling pathways [68]. Consistent with anatomical results, _OsVTC1-1_ RNAi lines showed a decrease in cell size. AsA is involved in the regulation of plant development processes, acts as a cofactor in cell wall hydroxyproline-rich glycoproteins post-translation for cell growth and is required for cell division [2; 5]. Transcriptome analysis revealed DEGs between wild type and _OsVTC1-1_ RNAi lines, and the 11 down-regulated differentially expressed genes (DEGs) were shared in both _OsVTC1-1_ RNAi lines. Several DEGs are involved in the signaling pathways, such as aspartokinase, CBL-interacting protein kinase 14 (CIPK14) and kinase. Consistent with our results, a previous study reported that 171 genes were differentially expressed between wild type and _vtc1_ mutants in Arabidopsis, including kinase and kinase receptors [69]. Aspartokinase catalyzes the phosphorylation of aspartic acid, which is the first step of amino acids synthesis, such as lysine, methionine and threonine [70]. Reduced aspartokinase activity in myxobacteria led to disruption of cell wall growth [71]. CIPK14 interacts with Calcineurin B-like (CBL) protein which responds to various abiotic stresses and associates with plant development [72]. In eukaryotic cells, kinases play a key role in cellular processes by adding phosphate groups and act as a major component which responds to abiotic and biotic stresses in plants [73]. Another identified DEG that involves in amino acids synthesis is aspartokinase/homoserine dehydrogenase 2. This enzyme is a bifunctional protein and catalyzes the synthesis of threonine, methionine and isoleucine [74]. The thionion-like peptide is a defense-related protein which has antimicrobial activity [75].