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The maize Dof protein PBF activates transcription of γ-zein during maize seed development

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Abstract

Maize PBF (prolamin-box binding factor) belongs to the Dof class of plant specific transcription factors containing one highly conserved zinc finger DNA-binding domain, called Dof (DNA binding with one finger) domain. Maize PBF trans-activates the γ-zein gene (γZ) promoter in developing maize seeds as shown by transient expression in maize endosperms. Co-transfection of a γZ:GUS construct with 35S:PBF resulted in a sevenfold increase in GUS expression, however, PBF mutation in Cys residues within the Dof domain abolishes both, binding to DNA and the capacity to activate γZ promoter. We present two pieces of evidence that PBF transactivates γZ promoter by binding to the Pb3 motif (TGTAAAG). First, recombinant Dof domain of PBF (bdPBF) specifically recognized Pb3 site as shown by gel mobility shift assays and second, co-expression of PBF with γZ promoter mutated in Pb3 motif suppressed PBF trans-activation capacity. Immunocytochemical analysis on developing endosperm sections shows that PBF is localized in the nuclei of the peripheral layer cells of starchy endosperm, the tissue in which the initial accumulation of γ-zein protein occurs. By contrast, PBF is detected in the cytosol of the starchy endosperm cells newly differentiated from aleurone daughter cells, where γ-zein was absent. Taken together these data indicate that maize PBF plays an essential role in the regulation of the temporal and spatial expression of γZ gene.

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Acknowledgements

This work was supported by the MCYT-FEDER (BIO 2004-03202) and Generalitat de Calalunya (CeRBa and 2005 SGR00182). P.M. was recipient of a fellowship from Spanish Ministerio de Educación y Ciencia. E.G was recipient of a fellowship from AGAUR (Generalitat de Catalunya). We thank Robert Schmidt (UCSD, USA) for kindly providing PBF cDNA. We are grateful to Maite Galiñánez for the technical assistance in the antibody production and to Monica Pons for her assistance in immuno microscopy.

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  1. Pau Marzábal

    Present address: ERA Biotech, S.A. Josep Samitier 1-5, 08028, Barcelona, Spain

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  1. Consorci CSIC-IRTA, Jordi Girona 18-26, 08034, Barcelona, Spain

    Pau Marzábal, Elisabet Gas, Pilar Fontanet, Margarita Torrent & M. Dolores Ludevid

  2. Centro de Biotecnología y Genómica de plantas, ETS Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28040, Madrid, Spain

    Jesús Vicente-Carbajosa

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Fig. A

The opaque 2 transcription factor (O2) interacts in vitro with the GZM motif. (A1) Oligonucleotides oBB, oBBDPb3 and oBBDGZM used in EMSA assays showing the bifactorial box motifs Pb3 and GZM as well as the nucleotide substitutions introduced in these cis-elements (arrows over lower case). (A2) EMSA assays with the O2 protein produced by in vitro transcription and translation, using as probes the oligonucleotides detailed in A. As it can be seen in the figure, the incubation of O2 with the wild type probe (oBB; lane 2) produced several band shifts. Only the upper band (arrow) corresponds to O2 binding because the rest of the shifts are also observed in the negative control (lane 7). The interaction of the O2 to the oligonucleotide oBB takes place specifically at the GZM motif. The specific band shift observed in lane 2 (arrow) is abolished when GZM motif was mutated (oBBDGZM; lane 4), but not when Pb3 motif was mutated (oBBDPb3; lane 6). Lanes 1, 3 and 5 correspond to the probe incubated in the absence of O2 (TIF 949 kb)

Fig. B

The over-expression of PBF does not affect the transcriptional rate of the αZ promoter of the 22 kD α-zein gene on transiently transformed 15 DAP maize endosperms. (B1) Schematic representation of the reporter (22αZ:LUC) and the effector genes used in functional studies. The O2 (Z1, Z2 and Z3) and Pb (prolamin-box) ciselements present in the reporter gene promoter are shown. (B2) Representation of the folds of activation of the 22αZ promoter when co-expressed with PBF and O2 used as effectors. The values are expressed as folds of activation in relation to the basal 22αZ:LUC activity (lane C) obtained by using an empty vector as effector. The overexpression of O2 increased significantly the expression of the 22αZ promoter (more than 3-folds, lane O2). No effect on the expression of this promoter was observed when PBF was over-expressed (lane PBF). Even when both, O2 and PBF were overexpressed (lane PBF + O2) the slight increase observed on 22αZ:LUC activity as compared to that observed in lane O2 was not significant (TIF 299 kb)

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Marzábal, P., Gas, E., Fontanet, P. et al. The maize Dof protein PBF activates transcription of γ-zein during maize seed development. Plant Mol Biol 67, 441–454 (2008). https://doi.org/10.1007/s11103-008-9325-5

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