A plastidial methylerythritol 4-phosphate (MEP) pathway functions on producing isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) as the building blocks (C5) of isoprenoid metabolites. The MEP pathway is composed of seven steps, and in the first step, a 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) produce a 1-Deoxy-D-xylulose 5-phosphate (DXP) using a pyruvate and a glyceraldehyde-3-phosphate, and a DXP reductase (DXR) biosynthesize a MEP. After that, four kinds of structural enzymes (IspD, IspE, IspF, and IspG) function sequentially in biosynthesis of (E)-4-Hydroxy-3-methylbut-2-enyl Diphosphate (HMBPP), and in the last step, IPP/DMAPP are biosynthesized by a HMBPP reductase (IspH). In this study, of those, three core enzymes (DXS, DXR, and IspH) are selected for functional analysis on carotenoid metabolism in rice (Oryza sativa). In the first step, the expression patterns of those genes were analyzed through quantitative Real-time PCRs on tissues and developmental stages of rice. The results showed that those genes were mainly expressed in leaves of seedling stages to reproductive stages with different patterns. To investigate the function of each OsDXS, OsDXR or OsIspH in rice, two kinds of transgenic plants were constructed using p600-PGD1::OsMEPs (PGD1::OsMEPs) for the constitutive expression of OsMEPs in the whole plant tissues, and pMJ103-PGD1::OsMEPs_Glb::stPAC (OsMEPs-stPAC) for additional expression of a stPAC (a β-carotene biosynthetic gene) in seed endosperms. After the homozygosity of their T4 generation plants was determined using TaqMan PCRs, carotenoid contents in their seeds were analyzed and compared with the carotenoid contents of the homozygous Glb::stPAC transgenic plants overexpressing only stPAC, to elucidate the functions of three genes on a rice carotenoid metabolism. The results showed that of them, the carotenoid content of OsDXS-stPAC seeds was analyzed to be four to nine times higher than it of OsDXR-stPAC and OsIspH-stPAC seeds, suggesting that the DXS step of MEP pathway could play an important role as the rate limiting step for supplying isoprene (C5) building blocks to rice carotenoid metabolism. In parallel, a total carotenoid content of OsDXS-stPAC seeds was analyzed to be three times higher than it of Glb::stPAC seeds, as well as, the β-carotene content of OsDXS-stPAC seeds was seven times higher than it. Conversely, the carotenoid contents of OsDXR-stPAC and OsIspH-stPAC seeds were not much different, compared to it of Glb::stPAC seeds.
