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.

A schematic biosynthetic pathway of the resveratrol and piceid in the developing seeds of I.526 is shown in Fig 1. In this pathway, phenylalanine is converted to p-coumaroyl CoA in a three-step process by the action of phenylalanine ammonia-lyase (PAL) [38], cinnamate 4-hydroxylase (C4H) [39] and 4-coumarate: CoA ligase (4CL) [40]. Then, p-coumaroyl CoA is converted to resveratrol by AhRS3 upon the addition of malonyl-CoA [24,29]. 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 [26,28,33-37]; however, 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).
In this study, we investigated the number of UGT genes whose expression levels were affected by the introduction of AhRS3, with the aim to identify candidate UGT gene(s) involved in piceid biosynthesis. In addition, 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.

Growth of resveratrol-producing transgenic rice lines
To develop resveratrol-producing transgenic rice lines, AhRS3 (DQ124938) was cloned from groundnut (A. hypogaea) and inserted into pSB2220 vector carrying the maize Ubi1 promoter for overexpression of a target gene [29]. Agrobacterium tumefaciens strain LBA4404 carrying pSB2220 vector with AhRS3, was cocultivated with calli derived from seeds of Dongjin, a japonica rice cultivar (O. sativa L. subsp. japonica cv. Dongjin), to generate transgenic calli. Transgenic rice plants were regenerated from transgenic calli selected. These works were performed by Dr. Baek's research team at the NICS, Republic of Korea [29].
Seedlings of wild-type rice (Oryza sativa subsp. japonica cv. Dongjin) and inbred lines of its transgenic counterpart, I.515 and I.526, were transplanted in the genetically modified organism (GMO) greenhouse (30 cm × 15 cm) of the National Institute of Crop Science (NICS), Republic of Korea, using the standard rice cultivation method of the NICS. A completely randomized design (CRD) was used, with three replications. Developing seeds were harvested from one panicle per plant (total four plants per replication) at 9 days after heading (DAH).
The seedlings of wild-type rice and I.526 were transplanted in the GMO paddy field (30 cm × 15 cm) of the NICS, Republic of Korea, using the standard rice cultivation method of the NICS. As mentioned above, a CRD was used, with three replications. Developing seeds were harvested from one panicle per plant (total four plants per replication) at 6, 13, 20, 31 and 41 days after heading (DAH).

Quantification of resveratrol and piceid by UPLC
Seed extracts were prepared from each sample, as described previously [29], and 1-μl aliquots of each sample were analyzed using the ACQUITY UPLC system (Waters Corporation, Milford, MA, USA) with ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA). Resveratrol was analyzed over a total run time of 30 min using acetonitrile (solvent A) and water (solvent B) as the mobile phase. The column was initially equilibrated with 10% A and 90% B. The ratio of A:B was gradually changed to 25:75 by 20 min from the initial time with curve7 and then to 100:0 (curve6) by 21 min. The A:B ratio was kept at 100:0 by 25 min with curve6, changed to 10:90 (curve6) by 26 min and then kept at 10:90 by 30 min with curve6. To analyze piceid, the column was initially equilibrated with 10% A and 90% B. The A:B ratio was gradually changed to 50:50 with curve9 by 20 min and then transformed to 100:0 with curve6 by 21 min. The UPLC analytical method for piceid from 21-30 min was same as that used for resveratrol, as described above. Both resveratrol and piceid were detected at 308 nm using the ACQUITY UPLC Tunable UV detector (Waters Corporation, Milford, MA, USA), and the flow rate was maintained at 0.2 ml/min.
Resveratrol and piceid standards were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The UPLC solvents, water and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Total RNA extraction and RNA-seq
Total RNA was extracted from frozen and milled samples of developing seeds, including 9 DAH seeds of Dongjin, I.515 and I.526, and 6, 13, 20, 31 and 41 DAH seeds of Dongjin and I.526, using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. The total RNA samples of Dongjin and I.526 collected at 20 DAH seeds were sent to Macrogen, Inc. (Seoul, Republic of Korea) for RNA-seq using the Illumina technology.
Raw sequence reads were cleaned by removing low-quality nucleotides (Phred score < 20) and short sequence reads (read length < 20 nt). The cleaned reads were mapped onto the Nipponbare reference genome sequence retrieved from the Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu, version 7.0) using the HISAT2 software with default parameters [42]. The featureCounts software [43] was used to quantify the raw read counts from the BAM file, and normalized read counts were calculated by dividing the read counts of all genes with those of the OsUBI1 gene. Raw data for RNA-seq are available at https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9695.

Gene expression analysis by RT-qPCR
The isolated total RNA was quantified by NanoVue Plus (GE Healthcare Life Sciences, Chicago, IL, USA), and cDNA was synthesized from 1 μg of total RNA using the iScript TM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The primer sets used in the quantitative real time-polymerase chain reaction (RT-qPCR) are shown in S1 and S2 Tables. The rice ACTIN1 gene (OsACT1; LOC_Os03g50885) was chosen as a reference (S1 Table). The RT-qPCR was carried out on the CFX96 TM Real-Time Detection System (Bio-Rad, Hercules, CA, USA) using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). The relative expression of AhRS3, UGT genes, and genes acting upstream of AhRS3, including PAL, C4H and 4CL, was determined using the Pfaffl method [44].

Resveratrol and piceid biosynthesis in the developing seeds of resveratrolproducing rice inbred lines, Iksan 515 and Iksan526
The expression of AhRS3 was confirmed in seeds of both I.515 and I.526 at 9 DAH, and the expression level of AhRS3 was higher in 9 DAH seeds of I.515 than that in I.516 (Fig 2A).
However, significantly higher quantities of resveratrol and piceid, respectively, were detected in 9 DAH seeds of I.526, compared to those of I.515 (Fig 2B-2D). Based on biosynthesis of resveratrol and piceid in developing seeds, I.526 was chosen for paddy field experiments to investigate biosynthesis of resveratrol and piceid, and gene expression analysis.
The expression level of AhRS3 in I.526 seeds increased with development, reaching a peak at 41 days after heading (DAH) (Fig 3A). 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 then decreased with development (Fig 3B-3E); however, both resveratrol and piceid reached maximal biosynthesis at different time points (41 and 20 DAH, respectively) (Fig 3B and 3C).
In addition, the expression of genes acting upstream of AhRS3 in the resveratrol biosynthesis pathway was investigated by quantitative real-time PCR (RT-qPCR) analysis (S1 Fig). The relative expression levels of PAL, C4H and 4CL were altered in developing seeds of I.526 compared with those in Dongjin. In I.526 seeds, all genes tested in this study, including PAL, C4H and 4CL, showed typical expression patterns with significantly higher expression at 20, 31 and 41 DAH than those in Dongjin (S1 Fig).

RNA-seq analysis of Dongjin and I.526 seeds
To analyze the transcriptome of the developing seeds of Dongjin and I.526, we performed RNA-seq analysis. Because UPLC analysis revealed that the glycosylation of resveratrol in I.526 seeds was the highest at 20 DAH (Fig 3C), we chose this time point for RNA-seq analysis. Raw sequence data of Dongjin and I.526 were mapped to the Nipponbare reference genome, and read counts of each gene were normalized relative to those of the rice UBIQUITIN 1 (OsUBI1; LOC_Os03g13170) gene. A total of 245 UGT genes were selected from the normalized data (S2 Fig and S3 Table). Functional annotation of these genes using the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu) and the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) databases revealed their putative roles in the glycosylation of several secondary metabolites, including cytokinin, anthocyanidin, indole-3-acetate, flavonoids, hydroquinone, cis-zeatin, limonoid, betanidin, cyclo-DOPA and N-hydroxythioamide. In I.526 seeds at 20 DAH, 43 out of 245 UGT genes were upregulated by more than 2-fold compared with Dongjin seeds, whereas 95 UGT genes showed extremely low expression (S2 Fig and S3 Table). To verify the RNA-seq data, 15 out of 43 UGT genes upregulated in I.526 seeds were selected for RT-qPCR analysis (S2 and S3 Tables). The expression of all 15 UGT genes were first investigated by RT-qPCR analysis in seeds of Dongjin, I.515 and I.526 at 9 DAH, thereby resulting in consistency with the RNA-seq data, except for LOC_Os03g55010 (Fig 4). In seeds of Dongjin and I.526 at 20 DAH, the results of RT-qPCR analysis of all 15 UGT genes were consistent with the RNA-seq data (Fig 5). Taken together, these results indicate that our RNAseq data were highly reliable (Figs 4 and 5).

Discussion
The AhRS3 [21,24] gene, a missing link in the resveratrol biosynthesis pathway in rice, was successfully transformed into rice, resulting in the production of resveratrol [29] via the phenylpropanoid pathway, which produces p-coumaroyl CoA, a precursor of resveratrol [47] (Fig 1). In this study, RT-qPCR and UPLC analyses showed that AhRS3 expression and resveratrol and piceid biosynthesis, respectively, increased in I.526 seeds with maturation, indicating that the biosynthesis of resveratrol is closely related to seed development (Fig 3). However, the expression of AhRS3 and quantity of piceid in I.526 seeds were the highest at 41 and 20 DAH, respectively (Fig 3). This molecular phenotype might be related to expression patterns of UGTs (data not known) and availability of UDP-glucose in developing rice seeds [48]. Moreover, in seeds of black rice cultivars, the maximal biosynthesis  In previous studies conducted on the metabolic engineering of resveratrol in tomato, piceid was detected along with resveratrol [26,28]; however, in genetically engineered plants of alfalfa, Arabidopsis, apple, poplar and B. napus, only piceid was detected [33]. This difference is probably related to endogenous UGTs, which are involved in the modification of secondary metabolites (to improve their stability and water solubility), inactivation and detoxification of xenobiotics, and regulation of hormones, including auxin, abscisic acid, cytokinins, brassinosteroids and salicylic acid [49].
Overall, we showed that in the developing seeds of I.526, the time points of maximal biosynthesis of resveratrol and piceid were distinct, and AhRS3 altered the expression pattern of PAL, C4H, 4CL, and UGTs, resulting in the biosynthesis of resveratrol and piceid. Based on the expression data of target genes obtained by RNA-seq and RT-qPCR, we propose a