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Sequence differences in LcFGRT4 alleles are responsible for the diverse anthocyanin composition in the pericarp of Litchi chinensis

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Abstract

Glycosylation plays a major role in the diversity in the chemical compositions of flavonoids. In this study, we performed biochemical and molecular assays to identify a glucosyltransferase gene responsible for the anthocyanin composition in litchi. Cyanidin-3-glucoside and cyanidin-3-rutinoside were predominant anthocyanins in the red pericarp and young leaf of litchi. Anthocyanin composition varied among litchi varieties. Anthocyanin profile was primarily determined by genetic factors. Higher activities of UDP-rhamnose: cyanidin-3-glucoside rhamnosyltransferase (CGRT) were detected in the pericarps of the cyanidin-3-rutinoside predominant varieties. Three full-length putative UDP-rhamnose: flavonoid glycoside 2″-O-beta-l-rhamnosyltransferase-like genes were isolated and designated as LcFGRT2, LcFGRT4, and LcFGRT5. Phylogenetic analysis showed that these genes were clustered with other glucoside-glycosyltransferases. Notable activities in catalyzing cyanidin-3-rutinoside formation were observed in extracts of tobacco leaves and yeast with heterologous expression of LcFGRT4. However, the expression pattern of LcFGRT4 did not agree with the CGRT activity. This result suggests that the difference in CGRT among varieties occurred post-transcriptionally. Nucleotide variation in LcFGRT4 was surveyed by sequencing 30 litchi accessions with different anthocyanin profiles. Eight non-synonymous single-nucleotide polymorphisms were detected. Type A LcFGRT4 sequence (LcFGRT4A) was observed in cyanidin-3-glucoside-dominant varieties, whereas type B LcFGRT4 sequence (LcFGRT4B) was detected in cyanidin-3-glucoside-dominant varieties. A mutant in 343 C/G polymorphism was targeted as the critical point responsible for the CGRT activity. Results indicated that a single-point mutation in LcFGRT4 could alter the activity of CGRT and may contribute to the diverse anthocyanin profile of litchi.

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Acknowledgments

This study was supported by the China Litchi and Longan Industry Technology Research System (Project No. CARS-33), National Natural Science Fund of China (Project No. 30971985), and the Key Laboratory of Innovation and Utilization for Germplasm Resources in Horticultural Crops in Southern China of Guangdong Higher Education Institutes, South China Agricultural University (No. KBL11008). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Ruo Ouyang and Cheng-Ming Liu for providing information on the plant materials.

Author contributions

X.J.L. performed most of the experiments and data analysis and wrote the draft of the paper. B.L. and J.M.H. conducted part of the material collection and color measurement. J.T.Z, Y.H.Q. and X.M.H. participated in the data analysis and preparation of the manuscript. H.C.W. and G.B.H. conceived, designed, and coordinated the studies. All authors have read and approved the final manuscript.

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Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, 510642, People’s Republic of China

    Xiao-Jing Li, Biao Lai, Jie-Tang Zhao, Yong-Hua Qin & Gui-Bing Hu

  2. Guangdong Litchi Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, 510642, Guangdong, People’s Republic of China

    Xiao-Jing Li, Biao Lai, Jiang-Man He, Xu-Ming Huang, Hui-Cong Wang & Gui-Bing Hu

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  1. Xiao-Jing Li
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  2. Biao Lai
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Correspondence to Hui-Cong Wang or Gui-Bing Hu.

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Fig. S1

HPLC chromatogram of anthocyanin extracts from pericarp of cvs. ‘9911,’ ‘FZX,’ and standards captured at 520 nm (PDF 1041 kb)

Fig. S2

(A) HPLC chromatogram of anthocyanin extracts from young leaves of cvs. ‘9911’ and ‘NMC’; (B) relative levels of cyanidin-3-glucoside in the young leaves of six varieties. Vertical bars represent standard error of the mean (n = 3) (TIFF 1068 kb)

Fig. S3

Protein alignment of putative litchi flavonoids 3GTs and FGGTs, as well as UGT78D1 (PDF 752 kb)

Fig. S4

Phylogenetic analysis of selected plant GTs, putative litchi flavonoid GRTs (FGRTs) expressed in the pericarp. Bar = 0.1 amino acid substitution per site. Functional clusters (I, II, IIIa, IIIb, and IV) of flavonoid UGTs are shaded gray. Triangles and circles represent FGRTs in litchi. GenBank protein accession numbers for other plant GTs are listed in Li et al. (2016). Additional GenBank accession numbers for each gene are as follows: LcFGRT2, GenBank: KF954722; LcFGRT4, GenBank: KF954721 (TIFF 3127 kb)

Fig. S5

Structure-based sequence alignment of Litchi chinensis LcFGRT4, Vitis vinifera VvGT1, and Medicago truncatula UGT71G1 and UGT85H2 (PDF 164 kb)

Table S1

Thirty litchi accessions used in this study (DOC 40 kb)

Table S2

Primers for cloning of flavonoid GRTs in litchi pericarp (DOC 34 kb)

Table S3

Concentrations of anthocyanins and relative levels of cyanidin-3-glucoside in the pericarps of litchi cultivars ‘FZX’ and ‘HY’ sampled from different production areas (DOC 41 kb)

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Li, XJ., Lai, B., Zhao, JT. et al. Sequence differences in LcFGRT4 alleles are responsible for the diverse anthocyanin composition in the pericarp of Litchi chinensis . Mol Breeding 36, 93 (2016). https://doi.org/10.1007/s11032-016-0518-3

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