Abstract
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.
Highlights
The expression of AhRS3 was confirmed in seeds of both I.515 and I.526 at 9 days after heading (DAH), and the expression level of AhRS3 was higher in 9 DAH seeds of I.515 than that in I.516 (Fig 2A)
Ultra-performance liquid chromatography (UPLC; Waters Corp., Milford, MA, USA) analysis revealed that the biosynthesis of resveratrol and piceid in I.526 seeds increased initially and decreased with development (Fig 3B–3E); both resveratrol and piceid reached maximal biosynthesis at different time points (41 and 20 DAH, respectively) (Fig 3B and 3C)
In I.526 seeds, all genes tested in this study, including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL), showed typical expression patterns with significantly higher expression at 20, 31 and 41 DAH than those in Dongjin (S1 Fig)
Summary
Resveratrol (3,5,4’-trihydroxystilbene), a stilbene compound, occurs in many plant species including grape (Vitis vinifera), groundnut (Arachis hypogaea), Eucalyptus spp., Texas fescue. Dongjin) carrying the groundnut resveratrol synthase 3 (AhRS3; DQ124938) gene [29,31,32] These inbred lines were generated by Dr Baek’s research team at the National Institute of Crop Science (NICS), Republic of Korea, using the Agrobacterium-mediated transformation method [29,31,32]. Piceid is readily generated from resveratrol by the action of UDP-glycosyltransferase(s) (UGTs) This is consistent with previous reports on the metabolic engineering of resveratrol in other plant species [26,28,33,34,35,36,37]; the role of UGT genes in resveratrol biosynthesis has not been reported previously, except for the bi-functional resveratrol/hydroxycinnamic acid glucosyltransferase gene reported in Concord grape (Vitis labrusca) [41] (Fig 1). We examined whether the expression patterns of genes acting upstream of AhRS3, including PAL, C4H and 4CL, were altered by the introduction of AhRS3 in the developing seeds of I.526
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