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Cloning and characterization of soybean gene Fg1 encoding flavonol 3-O-glucoside/galactoside (1→6) glucosyltransferase

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Abstract

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Flavonoids are important secondary metabolites in plants. Sugar–sugar glycosyltransferases are involved in the final step of flavonoid biosynthesis and contribute to the structural diversity of flavonoids. This manuscript describes the first cloning of a sugar–sugar glucosyltransferase gene in the UGT family that attaches glucose to the 6″-position of sugar bound to a flavonol. The results provide a glimpse on the possible evolution of sugar–sugar glycosyltransferase genes and identify putative amino acids responsible for the recognition of the hydroxyl group of the sugar moiety and specification of sugar. A scheme for the genetic control of flavonol glycoside biosynthesis is proposed.

Abstract

Flavonol glycosides (FGs) are predominant in soybean leaves and they show substantial differences among genotypes. In previous studies, we identified two flavonoid glycoside glycosyltransferase genes that segregated in recombinant inbred lines developed from a cross between cultivars Nezumisaya and Harosoy; one was responsible for the attachment of glucose to the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol and the other was involved in the attachment of glucose to the 6″-position. This study was conducted to clone and characterize the 6″-glucosyltransferase gene. Linkage mapping indicated that the gene was located in the molecular linkage group I (chromosome 20). Based on the genome sequence, we cloned a candidate cDNA, GmF3G6"Gt from Harosoy but the corresponding cDNA could not be amplified by PCR from Nezumisaya. The coding region of GmF3G6″Gt in Harosoy is 1386 bp long encoding 462 amino acids. This gene was not expressed in leaves of Nezumisaya. The GmF3G6″Gt recombinant protein converted UDP-glucose and kaempferol 3-O-glucoside or kaempferol 3-O-galactoside to kaempferol 3-O-glucosyl-(1→6)-glucoside or kaempferol 3-O-glucosyl-(1→6)-galactoside, respectively. These results indicate that GmF3G6″Gt encodes a flavonol 3-O-glucoside/galactoside (1→6) glucosyltransferase and corresponds to the Fg1 gene. GmF3G6″Gt had an amino acid similarity of 82 % with GmF3G6″Rt encoding flavonol 3-O-glucoside/galactoside (1→6) rhamnosyltransferase, suggesting a recent evolutionary divergence of the two genes. This may be the first cloning of a sugar-sugar glucosyltransferase gene in the UGT family that attaches glucose to the 6″-position of sugar bound to a flavonol. A scheme for the control of FG biosynthesis is proposed.

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Acknowledgments

The authors are grateful to Dr. Joseph G. Dubouzet (New Zealand) for critical reading of the manuscript. This study was partially supported by the Japanese Government (MEXT) Scholarship to F.R.R. a scholarship grant from the China Scholarship Council to S.D. and the JSPS KAKENHI Grant Number 25440148 to KY-S.

Author Contributions

R.T. designed experiments and together with F.R.R. and S.D. carried out genetic analysis, linkage mapping, molecular cloning and gene expression assays. Y.M. and T.I. carried out chemical analysis. S.S., T.M., R.N., K.Y.-S. and K.S. performed functional analysis.

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Author notes

  1. Felipe Rojas Rodas

    Present address: Universidad Católica de Oriente, Rionegro, Antioquia, Colombia

  2. Shaokang Di

    Present address: Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People’s Republic of China

Authors and Affiliations

  1. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan

    Felipe Rojas Rodas, Shaokang Di & Ryoji Takahashi

  2. Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki, 305-0005, Japan

    Yoshinori Murai & Tsukasa Iwashina

  3. The RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan

    Satoko Sugawara, Tetsuya Mori, Ryo Nakabayashi, Keiko Yonekura-Sakakibara & Kazuki Saito

  4. NARO Institute of Crop Science, Tsukuba, Ibaraki, 305-8518, Japan

    Ryoji Takahashi

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  1. Felipe Rojas Rodas
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Correspondence to Ryoji Takahashi.

Electronic supplementary material

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11103_2016_523_MOESM1_ESM.doc

Figure S1 Alignment of the 5′ upstream region of GmF3G6″Gt gene in soybean cultivars Harosoy and Nezumisaya. Polymorphic nucleotides are shown in red font. The coding sequence is underlined. (DOC 32 KB)

11103_2016_523_MOESM2_ESM.pdf

Figure S2 Multiple alignment of flavonoid glycoside glycosyltransferases (FGGs). The PSPG-box is underlined. Amino acid residues conserved in flavonoid FGGs are in white font highlighted in black. Amino acid residues conserved in flavonoid FG6″Gs and FG2″Gs are in white font highlighted in red and blue, respectively. Amino acid residues specific to glucosyltransferases and rhamnosyltransferases are in white font highlighted in orange and green, respectively. The position of amino acid responsible for sugar specificity in an FGG gene involved in saponin biosynthesis is shown by white triangle. (PDF 42 KB)

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Rojas Rodas, F., Di, S., Murai, Y. et al. Cloning and characterization of soybean gene Fg1 encoding flavonol 3-O-glucoside/galactoside (1→6) glucosyltransferase. Plant Mol Biol 92, 445–456 (2016). https://doi.org/10.1007/s11103-016-0523-2

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  • DOI: https://doi.org/10.1007/s11103-016-0523-2

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