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Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine (Vitis vinifera L.)

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Abstract

Anthocyanins and tannins are two of the most abundant flavonoids found in grapevine, and their synthesis is derived from the phenylpropanoid pathway. As described for model species such as Arabidopsis thaliana, maize and petunia, the end-point branches of this pathway are tightly regulated by the combinatorial interaction of three families of regulatory factors; MYB, bHLH (also known as MYC) and WDR proteins. Among these, only MYB genes have been previously identified in grapes. Here, we report the isolation of the first members from the WDR and bHLH families found in Vitis vinifera, named WDR1, WDR2 and MYCA1. WDR1 contributed positively to the accumulation of anthocyanins when it was overexpressed in A. thaliana, although it was not possible to determine the function of WDR2 by ectopic expression. The sub-cellular localizations of WDR1 and MYCA1 were observed by means of GFP-fusion proteins, indicating both cytoplasm and nuclear localization, in contrast to the localization of a MYB factor exclusively in the nucleus. The expression patterns of these genes were quantified in coloured reproductive organs throughout development, and correlated with anthocyanin accumulation and the expression profiles of the flavonoid-related MYBA1-2, UFGT, and ANR genes. In vitro grapevine plantlets grown under high salt concentrations showed a cultivar-dependent response for anthocyanin accumulation, which correlated with the expression of MYBA1-2, MYCA1 and WDR1 genes. These results suggest that MYCA1 may regulate ANR and UFGT and that this last control is easier to distinguish whenever MYBA genes are absent or in low abundance. Future studies should address the specific interactions of these proteins and their quantitative contribution to flavonoid synthesis in grape berries.

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Abbreviations

bHLH:

Basic helix-loop-helix protein

WDR:

Tryptophan-Aspartic acid repeat protein

DFR:

Dihydroflavonol reductase

UFGT:

UDP-glucose:flavonoid 3-O-glucosyltransferase

PA:

Proanthocyanidin

ANR:

Anthocyanidin reductase

LAR:

Leucoanthocyanidin reductase

GFP:

Green fluorescent protein

WAA:

Weeks from/after anthesis

WAV:

Weeks after veraison

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Acknowledgments

We wish to thank Uely Grossniklaus (University of Zurich, SZ) for the p207-DONOR and pMDC84 GFP-fusion vectors, and Paula Salinas and Dave Jackson (Cold Spring Harbour Laboratory, NY) for their guidance on GFP visualization in tobacco and onion cells, respectively. Special thanks to Agnès Ageorges and Nancy Terrier (INRA Montpellier) for sharing bHLH and WDR QPCR primers. This work was supported by the Chilean Wine Consortium 05CTE01-03, the Fruit Consortium, 07Genoma01, Millennium Nucleus for Plant Functional Genomics (P06-009-F), FONDECYT 1100709, and by fellowships awarded to JTM (MECESUP and CONICYT AT24060171) and MJP (CONICYT). Finally, we thank Nancy Terrier and Michael Handford for critically reading the manuscript.

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Authors and Affiliations

  1. Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4560, Santiago, Chile

    J. T. Matus, E. Bordeu & J. A. Alcalde

  2. Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, PO Box 114-D, Santiago, Chile

    M. J. Poupin, P. Cañón & P. Arce-Johnson

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  1. J. T. Matus
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  2. M. J. Poupin
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  3. P. Cañón
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  4. E. Bordeu
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  5. J. A. Alcalde
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  6. P. Arce-Johnson
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Correspondence to P. Arce-Johnson.

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Supplementary material 1 (DOC 66 kb)

Figure S1

A. Alignment of deduced amino acid sequences of WDR1 and WDR2 and theirhomologues in plant species. The four repeats are boxed. B. Comparison between WDrepeats identified in WDR1 and WDR2 and the WD-repeat consensus sequencedescribed by Smith et al. (1999), which includes the a, b and c beta strands that form thebeta-propeller structure. Identical amino acid residues are shaded in yellow whilesimilar residues appear shaded in green or cyan. Dots represent gaps introduced toimprove the alignment (JPG 928 kb).

Figure S2

Best-fit alignment of the deduced amino acid sequences homologous to VvMYCA1.The MYC DNA-binding basic region is followed by two alpha-helices separated by avariable loop region. The positions of the interacting and bHLH domains are shown.Identical amino acid residues are shaded in yellow while similar residues appear shadedin green or cyan. Dots represent gaps introduced to improve the alignment (JPG 1027 kb).

Figure S3

Changes in berry weight (•), total soluble solids (º Brix) (□), and anthocyanins per gramfresh weight of berry skin (▲), measured at a two week interval during grapedevelopment (JPG 58 kb).

Figure S4

Affymetrix-based expression analyses of MYCA1,WDR1 and anthocyanin-relatedUFGT and MYBA genes in grapevine shoot tips under abiotic stress conditions. Days 8,12 and 16 after control, water stress or salt (NaCl) treatments are shown for each gene.Data taken from the grape Plant Expression (PLEX) database for microarray data(http://www.plexdb.org/plex.php?database=Grape) (JPG 140 kb).

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Matus, J.T., Poupin, M.J., Cañón, P. et al. Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine (Vitis vinifera L.). Plant Mol Biol 72, 607–620 (2010). https://doi.org/10.1007/s11103-010-9597-4

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