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Identification and characterization of AcUFGT6b, a xylosyltransferase involved in anthocyanin modification in red-fleshed kiwifruit (Actinidia chinensis)

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Abstract

Anthocyanins are responsible for the red color in red-fleshed kiwifruits. In most of these cyanidin 3-O-xyl-galactoside is the predominant anthocyanin. This requires a second glycosylation modification which attaches the UDP-xylose to the cyanidin 3-O-galactoside. We report here the functional characterization of two second glycosyltransferases from ‘Hongyang’ kiwifruit, AcUFGT6b and AcUFGT7c. These were found to cluster with other plant GGTs. In vitro, recombinant AcUFGT6b recognized 3-O-glycosylated anthocyanins and UDP-xylose, while AcUFGT7c showed no or negligible activity. In tobacco leaves, co-expression of AcMYBF110, AcUFGT3a and AcUFGT6b resulted in the accumulation of three new products: cyanidin 3-O-xyl-galactoside, cyanidin 3-O-xyl-glucoside and cyanidin 3-O-xyl-rutoside. RNAi assays showed that a lack of AcUFGT6b and AcUFGT7c in immature fruits of Actinidia arguta prevents the formation of cyanidin 3-O-xyl-galactoside. Also, the content of cyanidin 3-O-xyl-galactoside was obviously increased when AcUFGT6b was injected together with AcMYBF110 and AcUFGT6b7c-RNAi. In contrast, for AcUFGT7c, as well as in vitro, we show no activity in either tobacco leaves or kiwifruit. Both AcUFGT6b and AcUFGT7c are located in the ER and their promoters can be positively activated by AcMYBF110, indicating the functional differences of them are due neither to their cell localization nor to the upstream regulation of AcMYBF110. Interestingly, the deletion in the C-terminal of AcUFGT7c is likely to change its 3D protein structure compared with AcUFGT6b protein, resulting in it having no capacity to attract the UDP sugar moiety. These results suggest that AcUFGT6b, but not AcUFGT7c, is responsible for the end-product of the anthocyanins biosynthesis pathway in red-fleshed kiwifruit.

Key message

AcUFGT6b attracts the UDP-xylose to the cyanidin 3-O-galactoside generating cyanidin 3-O-xyl-galactoside, and positively regulated by the AcMYBF110.

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Acknowledgements

This work was supported by the National Seeds Innovation Engineering: Kiwifruit Breeding Innovation (Grant No: C000082). The authors thank professor Wan Jianmin for kindly providing the ER-located marker labeled with mCherry. The authors also thank help of Qianqian Shi for the experiments.

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Authors and Affiliations

  1. College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Yanfei Liu, Jia Liu, Yingwei Qi, Aling Zhang, Zhande Liu & Xiaolin Ren

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  1. Yanfei Liu
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  2. Jia Liu
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  3. Yingwei Qi
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  4. Aling Zhang
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  5. Zhande Liu
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Contributions

YL designed the experiments, performed the research, wrote and revised this manuscript; JL, YQ and AZ performed the subcellular localization assay and helped revised manuscript. ZL designed the experiments and provided all samples tested; XR designed the experiments, discussed results, and revised this manuscript. All authors have participated in this research and approved the final manuscript.

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Correspondence to Zhande Liu or Xiaolin Ren.

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11240_2019_1622_MOESM1_ESM.xlsx

Supplementary material 1 (XLSX 29 kb) Table S1 Primers used in this study. Table S2 Substrate specificity of AcUFGT6b and AcUFGT7c.

11240_2019_1622_MOESM2_ESM.tif

Supplementary material 2 (TIFF 6974 kb) Figure S1 The predicted 3D structures of AcUFGT6b (a) and AcUFGT7c (b) and their corresponding distribution of α-helix and β-sheet (c). The magenta boxed region indicates the PSPG motif.

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Liu, Y., Liu, J., Qi, Y. et al. Identification and characterization of AcUFGT6b, a xylosyltransferase involved in anthocyanin modification in red-fleshed kiwifruit (Actinidia chinensis). Plant Cell Tiss Organ Cult 138, 257–271 (2019). https://doi.org/10.1007/s11240-019-01622-6

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