The DC1 domain protein BINUCLEATE POLLEN is required for pollen development in Arabidopsis

Abstract

Development of the male gametophyte is a tightly regulated process that requires precise control of cell division and gene expression. A relevant aspect to understand the events underlying pollen development regulation constitutes the identification and characterization of the genes required for this process. In this work we showed that the DC1 domain protein BINUCLEATE POLLEN (BNP) is essential for pollen development and germination. Pollen grains carrying the defective BNP allele failed to complete mitosis II and are impaired in pollen germination. By yeast two-hybrid analysis and bimolecular fluorescence complementation assays, we identified a set of BNP-interacting proteins. Among confirmed interactors we found NAC family transcriptional regulators Vascular Plant One-Zinc Finger 1 (VOZ1) and VOZ2. VOZ1 localization changes during pollen development, moving to the vegetative nucleus at the tricellular stage. We observed that this relocalization requires BNP, as in the absence of BNP in pollen from bnp/BNP plants, VOZ1 nuclear localization is impaired. As voz1voz2 double mutants showed the same developmental defect observed in bnp pollen grains, we propose that BNP requirement to complete microgametogenesis could be linked to its interaction with VOZ1/2 proteins. BNP could have a role of scaffold protein, recruiting VOZ1/2 to the endosomal system into assemblies that are required for their further translocation to the nucleus, where they act as transcriptional regulators. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/484815v3_fig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@8680b0org.highwire.dtl.DTLVardef@6ac6fcorg.highwire.dtl.DTLVardef@2ddf1eorg.highwire.dtl.DTLVardef@193693c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO BNP codes for a DC1 domain containing protein. (A) Schematic view of a region of Arabidopsis chromosome 2 (from 18,323,538 to 18,321,537 bp -TAIR10) showing BNP genomic location and bnp-1 and bnp-2 insertion sites. (B) BNP amino acid sequence with boxed DC1 domains. (C) Phylogenic tree of 140 DC1 domain containing proteins in Arabidopsis. Bootstrap test results (1000 replicates) of the major nodes are indicated. IDs of sequences grouped in clusters I to VIII are listed. (D) Alignment of BNP with its closest Arabidopsis homologs and CaDC1 (AEI52549), TaCHP (ACU80555), NtDC1a (BAF80452) and NtDC1b (BAF80453). Phylogenic tree with bootstrap test results (1000 replicates) for the alignment of the complete 18 sequences is shown. Amino acid region represented for each sequence is indicated. DC1 domains are framed, signature residues and binding loop regions are indicated (C=Cys, H=His, = loop). Higher intensity in grey scale denotes higher sequence similarity. C_FIG