Biochemical and molecular characterization of orange- and tangerine-colored rice calli

We established an orange (L) and a tangerine (D) colored callus line by repeatedly subculturing rice callus line (N). The L line accumulated lycopene, beta-carotene, and two lycopene isomers, while the D line accumulated much higher concentration of lycopene as well as a lycopene isomer than the L line. Genomic sequencing revealed the presence of SNP that results in the incomplete lycopene beta-cyclase (beta LCY) enzyme in the D line. We analyzed expression levels of carotenoid biosynthetic genes by qRT-PCR. Regarding the 2-C-methyl-D-erythritol-4-phosphate pathway genes, expression of the gene encoding geranylgeranyl diphosphate synthase 1 (GGPS1) was enhanced in both L and D lines as compared to the N line. Regarding the carotenoid pathway genes, the transcript amounts of the genes encoding phytoene synthase 2, phytoene desaturase, and zeta-carotene desaturase, were larger in the L line than in the N line; however, this pattern was not found in the D line. The expression levels of beta LCY and lycopene epsilon-cyclase (epsilon LCY) gene in the D line and epsilon LCY in the L line were lower than in the N line. The transcript amounts of two carotenoid oxygenase genes were decreased both in L and D lines. We concluded that increased expression of GGPS1 and reduced expression of epsilon LCY, beta LCY, and carotenoids oxygenase genes might allow an excess accumulation of carotenoids in L and D lines, and that the mutation in beta LCY is responsible for the conspicuous phenotype in the D line as compared to the L line.