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Over-expression of the transcription factor HlMYB3 in transgenic hop (Humulus lupulus L. cv. Tettnanger) modulates the expression of genes involved in the biosynthesis of flavonoids and phloroglucinols

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Abstract

Genes belonging to the R2R3 MYB transcription factor family play an important role in the regulation of the expression of structural genes involved in the biosynthesis of flavonoids. In this study, the regulatory gene s-HlMYB3 under the control of the CaMV 35S promoter was genetically transformed into hop cv. Tettnanger. This work was aimed at investigating the effect of s-HlMYB3 on the expression of genes involved in the biosynthesis of flavonoids and phloroglucinols. The transgene s-HlMYB3 was stably integrated and expressed in transgenic plants. Quantitative real-time PCR indicated that HlMYB3 was highly over-expressed in transgenic events compared to wildtype plants. The effect of the over-expressed transgene on the expression rate of structural genes of flavonoid and phloroglucinol biosynthesis, such as PAL, C4H, 4CL, CHS_H1, CHI, F3H, F3′H, FLS, F3′5′H, OMT1, HlPT1, and VPS, as well as on regulatory genes of hop like, HlMYB1, HlMYB2, HlMYB7, HlbZip1, HlbZip2, HlbHLH2, and WDR1 was examined. Transgenic events with an elevated expression of genes of flavonoids and phloroglucinols were identified. Chemical analysis revealed higher levels of cohumulone and colupulone in transgenic plants. In hop, flavonoids and phloroglucinols play an important role as flavoring compounds of beer. Moreover, prenylated chalcones (e.g. xanthohumol and desmethylxanthohumol) have received considerable attention because of their pharmacological properties. Metabolic engineering using genes of homologous MYB transcription factors may open the possibility for increasing the production of important secondary metabolites in hop.

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Abbreviations

4CL:

4-Coumarate-CoA ligase

C4H:

Cinnamate 4-hydroxylase

CHI:

Chalcone isomerase

CHS_H1:

Chalcone synthase_H1

DFR:

Dihydroflavonol reductase

DMX:

Desmethylxanthohumol

F3′5′H:

Flavonoid 3′, 5′-hydroxylase

F3′H:

Flavonoid 3′-hydroxylase

F3H:

Flavanone-3-hydroxylase

FLS:

Flavonol synthase

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GPPS.LSU:

Geranylgeranyldiphosphate synthase large subunit

HPLC:

High performance liquid chromatography

IAA:

Indole-3-acetic acid

LDOX:

Leucoanthocyanidin dioxygenase

OMT1:

O-Methyltransferase 1

PAL:

Phenylalanine ammonia-lyase

RT-qPCR:

Reverse transcriptase quantitative real-time PCR

TDZ:

1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea

UF3GT:

UDP-Glucose flavonoid 3-glucosyltransferase

VPS:

Valerophenone synthase

X:

Xanthohumol

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Acknowledgments

The generous financial support of Hop Growers Cooperative (Tettnang, Germany), Hopfenverwertungsgenossenschaft e.G. (HVG, Wolnzach, Germany) and Czech Science Foundation (project GACR 521/08/0740) is gratefully acknowledged. The scholarship from the German Academic Exchange Service (DAAD) and support from the University of Costa Rica are gratefully acknowledged by A. Gatica-Arias.

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Authors and Affiliations

  1. Plant Breeding and Biotechnology, Institute of Plant Breeding, Seed Science and Population Genetics, University of Hohenheim, Stuttgart, Germany

    A. Gatica-Arias, M. Stanke, K. R. Häntzschel & G. Weber

  2. Biology Centre v.v.i. ASCR, Institute of Plant Molecular Biology, Ceske Budejovice, Czech Republic

    J. Matoušek

Authors
  1. A. Gatica-Arias
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  2. M. Stanke
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  3. K. R. Häntzschel
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  4. J. Matoušek
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  5. G. Weber
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Corresponding author

Correspondence to G. Weber.

Electronic supplementary material

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Supplementary material 1 (DOC 103 kb)

11240_2012_268_MOESM2_ESM.tif

Supplementary material 2 (TIFF 158 kb). Partial sequences and BLAST search output of the PCR amplification products obtained using the primer set Hlmyb3-2-F/Hlmyb3-2-R and DNA from plasmid pLV-71 (P+), wildtype (WT), and transgenic plant no. 2. Lanes N−: negative control (PCR reaction mix template), M: molecular weight marker (100 bp DNA Ladder)

11240_2012_268_MOESM3_ESM.tif

Supplementary material 3 (TIFF 165 kb) Phylogenetic analysis for nucleotide sequences of selected plant MYB transcription factors involved in the regulation of flavonoids biosynthesis. The trees were generated using the PhyML v. 3.0. The generated data were visualized using TreeDyn (Dereeper et al. 2008). Accession number of each sequence are indicated. The partial sequence of HlMYB3 obtained in this study is indicated by an arrow

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Gatica-Arias, A., Stanke, M., Häntzschel, K.R. et al. Over-expression of the transcription factor HlMYB3 in transgenic hop (Humulus lupulus L. cv. Tettnanger) modulates the expression of genes involved in the biosynthesis of flavonoids and phloroglucinols. Plant Cell Tiss Organ Cult 113, 279–289 (2013). https://doi.org/10.1007/s11240-012-0268-9

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  • DOI: https://doi.org/10.1007/s11240-012-0268-9

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