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The BdGAMYB protein from Brachypodium distachyon interacts with BdDOF24 and regulates transcription of the BdCathB gene upon seed germination

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Abstract

Main conclusion

BdDOF24 interacting with BdGAMYB regulates the BdCathB gene upon germination.

During barley seed germination, hydrolytic enzymes (α-amylases, proteases, etc.) synthesized in the aleurone layer in response to gibberellins (GA), catalyse the mobilization of storage reserves accumulated in the endosperm during seed maturation. In Brachypodium distachyon, the BdCathB gene that encodes a Cathepsin B-like thiol-protease, orthologous to the wheat Al21 and barley HvCathB, is highly induced in germinating seeds and its expression is regulated by transcription factors (TFs) encoded by genes BdGamyb and BdDof24, orthologous to the barley HvGamyb and BPBF-HvDof24, respectively. Transcripts of both TF genes increase during germination and treatments with abscisic acid (ABA) or paclobutrazol (PAC, an inhibitor of GA biosynthesis) decrease mRNA expression of BdGamyb but do not affect that of BdDof24. Besides, proteins BdDOF24 and BdGAMYB interact in yeast-2 hybrid systems and in plant nuclei, and in transient expression assays in aleurone layers BdDOF24 is a transcriptional repressor and BdGAMYB is an activator of the BdCathB promoter, as occurs with the putative orthologous in barley BPBF-HvDOF24 and HvGAMYB. However, when both TFs are co-bombarded, BdDOF24 enhances the activation driven by BdGAMYB while BPBF-HvDOF24 strongly decreases the HvGAMYB-mediated activation of the BdCathB promoter. The different results obtained when BdDOF24 and BPBF-HvDOF24 interact with BdGAMYB and HvGAMYB are discussed.

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Abbreviations

GA:

Gibberellins

TF:

Transcription factor

ABA:

Abscisic acid

PAC:

Paclobutrazol

SSP:

Seed storage protein

GARC:

Gibberellin response complex

GARE:

Gibberellin response element

dap:

Days after pollination

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Acknowledgments

The authors thank Dr. David F. Garvin (University of Minnesota) for providing seeds of the inbred diploid Brachypodium distachyon line Bd21. Financial support from Ministerio de Ciencia e Innovación, Spain (Project BFU2009-11809; principal investigator PC) is gratefully acknowledged. CB-S has been financed by project BFU2009-11809 and RI-F holds a post-doctoral Juan de la Cierva contract (JCI-2010-07909). VG-C is the recipient of a FPU-UPM pre-doctoral fellowship from Universidad Politécnica de Madrid (UPM), Spain.

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  1. Cristina Barrero-Sicilia

    Present address: Biological Chemistry and Crop Protection Department, Rothamsted Research, Harpenden, AL5 2JQ, UK

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  1. Centro de Biotecnología y Genómica de Plantas (UPM-INIA). ETSI Agrónomos, Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón, 28223, Madrid, Spain

    Virginia González-Calle, Raquel Iglesias-Fernández, Pilar Carbonero & Cristina Barrero-Sicilia

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  1. Virginia González-Calle
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Electronic supplementary material

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Supplementary Fig. S1 Sequence analysis and comparison of PBF proteins of barley (BPBF-HvDOF24), wheat (WPBF), maize (MPBF), rice (RPBF) and Brachypodium (BdDOF24)

Supplementary Fig. S2 Rooted phylogenetic tree with Arabidopsis orthologous AtMYB65 (Gocal et al. 2001) and schematic distribution of conserved amino-acid motives (MEME; Bailey et al. 2006) among the deduced protein sequences encoded by the GAMYB genes of Hordeum vulgare, Triticum monococum, Brachypodium distachyon, Oryza sativa and Zea mays

Supplementary Fig. S3 Germination time course of Brachypodium distachyon seeds in the presence of different concentrations of abscisic acid (ABA) and paclobutrazol (PAC)

Supplementary Fig. S4 Comparison of HvGAMYB and BdGAMYB proteins

Supplementary Fig. S5 Polarity profiles of PBF proteins from barley (a), wheat (b), maize (c), Brachypodium (d) and rice (e) based on Zimmerman scale (Zimmerman et al. 1968; Gasteiger et al. 2005)

Supplementary Table S1 Oligonucleotide sequences of primers used for RT-qPCR analyses, amplicon length and PCR efficiency

Supplementary Table S2 List of primers used for cloning

Supplementary Table S3 Sequences of conserved amino-acid motives (MEME; Bailey et al. 2009) of the Cathepsin B-like proteins from Hordeum vulgare, Triticum aestivum, Brachypodium distachyon, Oryza sativa and Zea mays in Fig 1a

Supplementary Table S4 Sequences of conserved amino-acid motives (MEME; Bailey et al. 2009) of the PBF proteins from Brachypodium distachyon, Oryza sativa, Triticum aestivum, Hordeum vulgare and Zea mays in Fig. S1

Supplementary Table S5 Sequences of conserved amino-acid motives (MEME; Bailey et al. 2009) of the GAMYB proteins from Hordeum vulgare, Triticum monococum, Brachypodium distachyon, Oryza sativa and Zea mays in Fig S2

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González-Calle, V., Iglesias-Fernández, R., Carbonero, P. et al. The BdGAMYB protein from Brachypodium distachyon interacts with BdDOF24 and regulates transcription of the BdCathB gene upon seed germination. Planta 240, 539–552 (2014). https://doi.org/10.1007/s00425-014-2105-3

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