## 4 Discussion
### Fatty Acid and Phospholipid Biosynthesis  
Stearic acid, palmitic acid, and oleic acid are constituents of phosphatidylcholine, phosphatidylcholine, and phosphatidylethanolamine [14]. Metabolome analysis revealed an increase in inositol and 2-ethanolamine levels in tomato fruits under drought stress, suggesting that under these conditions, fatty acid biosynthesis is activated, along with phospholipid biosynthesis.  
Palmitic acid, stearic acid, and oleic acid are used to synthesize phosphatidic acid (PA) via conjugation to glycerol 3P through acyl-bond formation [14]. The PA pathway separates into the 1,2-Diacyl-sn-glycerol or CDP-1,2-Diacyl-sn-glycerol pathway: the 1,2-Diacyl-sn-glycerol pathway synthesizes phosphatidylethanolamine, and the CDP-1,2-Diacyl-sn-glycerol pathway synthesizes phosphatidylinositol [14].  
The levels of 2-ethanolamine and inositol in the ripened fruits increased approximately 2.7-fold compared to the control group. The metabolism of 2-ethanolamine generates two metabolites: phosphoethanolamine and CDP-ethanolamine. These metabolites are metabolized to phosphatidylcholine or phosphatidylethanolamine, respectively [14].  
Figure 3: Mass spectrometry imaging of functional ingredients in tomato fruits during ripening under drought stress. (**a**) Bright-field images of control or drought-stressed fruits. (**b**,**c**) Mass spectrometry imaging of \(\beta\)-Alanine +Na \(m/z\) 112 (**b**) and tomatidine +H \(m/z\) 416 (**c**).  
The level of phosphoethanolamine N-methyltransferase transferase 1 (NMT1), which converts phosphoethanolamine to phosphocholine, increased in the drought-stressed group versus control group [9].  
We previously determined that phosphatidylcholine and dipalmitoyl-phosphatidylcholine (DPPC) levels increased with the activation of fatty acid biosynthesis in tomato fruits at the mature stage, especially in seeds and the gels around seeds [9]. We also detected an increase in the levels of fatty acids, ethanolamine, and inositol, suggesting that the accumulation of phosphatidylinositol and phosphatidylethanolamine increases under drought stress. Based on these findings, phospholipid levels must also increase in tomato fruits during the ripening period under drought conditions.  
Phospholipids likely accumulate in the outer pericarp to prevent water loss during drought stress. In addition, seeds are likely protected from dehydration by accumulating phospholipids during the mature period.  
Figure 4: Model of the metabolic pathways in tomato fruits under drought stress. Genes or components upregulated or downregulated by drought-stressed conditions are shown in red and blue, respectively. The enzyme-encoding genes that were extracted as differentially expressed genes (DEGs) based on enrichment analysis of microarray data are shown in italics. The metabolites identified as accumulating by MSI are marked by bold font. The reaction in the white box shows desaturation of the PC constituent fatty acids in the ER. G6P, Glucose-6-phosphate; F6P, Glucose-6-phosphate; PA, phosphatidic acid; FabB, 3-oxoacyl-(acyl-carrier-protein) synthase I, chloroplastic; FabH, 3-oxoacyl-ACP synthase3; FatA, oleoyl-acyl carrier protein thioesterase, chloroplastic; NM,; phosphoethanolamine N-methyltransferasearse; ROD1, phosphatidylcholine:diacylglycerolylycerolcholine phosphotransferase 1; IMP, inositol monophosphate; MOO, inositol oxygenase; PME, pectin esterase; PL, pectate lyase; FAD2, delta-12 fatty acid desaturase; FAD6, omega-6 fatty acid desaturase.