Reinterpretation of anthocyanins biosynthesis in developing black rice seeds through gene expression analysis.

The biosynthesis of anthocyanins is still questionable in regulating the quantities of anthocyanins biosynthesized in rice seeds and the expression levels of transcription factors and the structural genes involved in the biosynthetic pathway of anthocyanins. We herein investigated the relationship between the accumulated anthocyanin contents and the expression levels of genes related to the biosynthesis of anthocyanins in rice seeds. Liquid chromatography/mass spectrometry-mass spectrometry analysis of cyanidin 3-glucoside (C3G) in rice seeds showed no accumulation of C3G in white and red rice cultivars, and the differential accumulation of C3G among black rice cultivars. RNA-seq analysis in rice seeds, including white, red, and black rice cultivars, at twenty days after heading (DAH) further exhibited that the genes involved in the biosynthesis of anthocyanins were differentially upregulated in developing seeds of black rice. We further verified these RNA-seq results through gene expression analysis by a quantitative real-time polymerase chain reaction in developing seeds of white, red, and black rice cultivars at 20 DAH. Of these genes related to the biosynthesis of anthocyanins, bHLHs, MYBs, and WD40, which are regulators, and the structural genes, including chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3´-hydroxylase (F3´H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), were differentially upregulated in black rice seeds. The correlation analysis revealed that the quantities of C3G biosynthesized in black rice seeds were positively correlated to the expression levels of bHLHs, MYBs and WD40, CHS, F3H, F3´H, DFR, and ANS. In addition, we present bHLH2 (LOC_Os04g47040) and MYBs (LOC_Os01g49160, LOC_Os01g74410, and LOC_Os03g29614) as new putative transcription factor genes for the biosynthesis of anthocyanins in black rice seeds. It is expected that this study will help to improve the understanding of the molecular levels involved in the biosynthesis of anthocyanins in black rice seeds.

Arachis hypogaea resveratrol synthase 3 alters the expression pattern of UDP-glycosyltransferase genes in developing rice seeds.

The resveratrol-producing rice (Oryza sativa L.) inbred lines, Iksan 515 (I.515) and Iksan 526 (I.526), developed by the expression of the groundnut (Arachis hypogaea) resveratrol synthase 3 (AhRS3) gene in the japonica rice cultivar Dongjin, accumulated both resveratrol and its glucoside, piceid, in seeds. Here, we investigated the effect of the AhRS3 transgene on the expression of endogenous piceid biosynthesis genes (UGTs) in the developing seeds of the resveratrol-producing rice inbred lines. Ultra-performance liquid chromatography (UPLC) analysis revealed that I.526 accumulates significantly higher resveratrol and piceid in seeds than those in I.515 seeds and, in I.526 seeds, the biosynthesis of resveratrol and piceid reached peak levels at 41 days after heading (DAH) and 20 DAH, respectively. Furthermore, RNA-seq analysis showed that the expression patterns of UGT genes differed significantly between the 20 DAH seeds of I.526 and those of Dongjin. Quantitative real-time PCR (RT-qPCR) analyses confirmed the data from RNA-seq analysis in seeds of Dongjin, I.515 and I.526, respectively, at 9 DAH, and in seeds of Dongjin and I.526, respectively, at 20 DAH. A total of 245 UGTs, classified into 31 UGT families, showed differential expression between Dongjin and I.526 seeds at 20 DAH. Of these, 43 UGTs showed more than 2-fold higher expression in I.526 seeds than in Dongjin seeds. In addition, the expression of resveratrol biosynthesis genes (PAL, C4H and 4CL) was also differentially expressed between Dongjin and I.526 developing seeds. Collectively, these data suggest that AhRS3 altered the expression pattern of UGT genes, and PAL, C4H and 4CL in developing rice seeds.