Synergism between RPBF Dof and RISBZ1 bZIP activators in the regulation of rice seed expression genes

Abstract

The Dof (DNA binding with one finger) transcriptional activator rice (Oryza sativa) prolamin box binding factor (RPBF), which is involved in gene regulation of rice seed storage proteins, has been isolated from rice cDNA expressed sequence tag clones containing the conserved Dof. RPBF is found as a single gene per haploid genome. Comparison of RPBF genomic and cDNA sequences revealed that the genomic copy is interrupted by one long intron of 1,892 bp in the 5' noncoding region. We demonstrated by transient expression in rice callus protoplasts that the isolated RPBF trans-activated several storage protein genes via an AAAG target sequence located within their promoters, and with methylation interference experiments the additional AAAG-like sequences in promoters of genes expressed in maturing seeds were recognized by the RPBF protein. Binding was sequence specific, since mutation of the AAAG motif or its derivatives decreased both binding and trans-activation by RPBF. Synergism between RPBF and RISBZ1 recognizing the GCN4 motif [TGA(G/C)TCA] was observed in the expression of many storage protein genes. Overexpression of both transcription factors gave rise to much higher levels of expression than the sum of individual activities elicited by either RPBF or RISBZ1 alone. Furthermore, mutation of recognition sites suppressed reciprocal trans-activation ability, indicating that there are mutual interactions between RISBZ1 and RPBF. The RPBF gene is predominantly expressed in maturing endosperm and coordinately expressed with seed storage protein genes, and is involved in the quantitative regulation of genes expressed in the endosperm in cooperation with RISBZ1.

Figure 1.

A, Organization of the RPBF protein and the RPBF locus. Schematic diagram of the primary structure of the RPBF protein (top section). The deduced length of the amino acid sequence encoded by the RPBF cDNA is shown. The shaded region represents the Dof domain. The location of the Cys residues is probably essential for formation of a zinc finger where indicated by arrows. Genomic structure of the RPBF gene (bottom section). Numbers indicate the positions from the putative transcription initiation site. The gray bar and the black box represent an intron and a coding region. The vertical arrow indicates the transcription initiation site as determined by primer extension analysis. The triangle indicates the putative transcription termination site. B, Comparison of the PBF protein primary sequences and Dof class proteins. Shaded boxes indicate identical amino acids. The vertical arrows indicate locations of the Cys residues likely to be essential for formation of a zinc finger. BPBF is from barley, WPBF is from wheat (Mena et al., 1998), MPBF is from maize (Vicente-Carbajosa et al., 1997), and OsDOF3 is from rice.

Figure 2.

Expression pattern of RPBF. A, Total RNA (30 μg) from roots, seedlings, and seeds during seed development (5, 10, 15, 20, and 30 DAF) was analyzed by northern blot using gene-specific sequences downstream of the DOF domain. To compare expression patterns of the RPBF gene with GluB-1 and RISBZ1 (Onodera et al., 2001) genes, the GluB-1 coding sequence and the RISBZ1-specific sequence corresponding to the region downstream of the Leu zipper were also used as probes. 25S rRNAs are shown as a loading control. B, Total protein from embryo and endosperm of 15 DAF seeds, roots, and seedlings was analyzed by western blot with anti-RPBF, anti-RISBZ1, and anti-glutelin GluB. Protein samples were separated on a 12.5% SDS-PAGE gel and stained with Coomassie Brilliant Blue (CBB). C, Total RNAs (7.5 μg) from imbibed seeds (top section) and from deembryonated half seeds incubated in the presence or absence of GA₃ (bottom section) were subjected to northern-blot analysis. The blot was probed with the RPBF-specific sequence. Times of imbibition and GA treatment are at the top of the section.

Figure 3.

Methylation interference experiments of RISBZ1 and RPBF binding to NRP33 promoter. DNA fragments corresponding to NRP33 (−386 to −276 and −295 to −172) were labeled on both strands (top and bottom). Both strands were partially methylated and incubated with GST-RISBZ1 or GST-RPBF. Free (F) and retarded (S) protein-DNA complex fragments were separated by PAGE. Fragments were eluted from the gel, chemically digested with piperidine, and separated on a sequencing gel in parallel with the sequencing ladder of this fragment.

Figure 4.

Sequences of the proximal GluB-1, NRP33, and Glb-1 promoters, and summary of RISBZ1 and RPBF binding sites. Nucleotide positions relative to the transcriptional (GluB-1 and Glb-1) or translational (NRP33) start sites are indicated. The shaded boxes indicate ATG start codons. G residues, which are protected from methylation by RISBZ1 (larger circle) and RPBF (larger arrowhead), are indicated. G residues partially protected by RISBZ1 (smaller circle) and RPBF (smaller arrowhead) are also indicated. Mutant nucleotides introduced for loss-of-function analyses are shown in lowercase letters below each of the corresponding RISBZ1 and RPBF binding sites.

Figure 5.

EMSA of RPBF protein with a P box in the GluB-1 promoter. Nucleotide sequences of oligonucleotides used as probe and competitors are depicted. WILD, 29 bp sequence containing a P box from the GluB-1 promoter (−152 to −123); M1 to M6, 29 bp sequence with successive dinucleotide mutations. The P-box and AAAG motif are underlined and boxed, respectively. The GST-RPBF fusion protein was used for EMSA with the 29 bp sequence containing the P box. Competitors were added in 100-fold molar excess. Lane 1, No protein; lane 2, no competitor; lanes 3 to 9, with competitors (wild type [W] and M1 to M6).

Figure 6.

Functional analysis of RISBZ1 and RPBF binding sites using a transient expression assay. Schematic diagrams of the proximal GluB-1 (−199 to +23), NRP33 (−386 to −13), and Glb-1 (−340 to +73) promoters are shown. The mutagenized promoters, each carrying one mutated site (marked with an X), were designed for loss-of-function assays. These promoters were linked to the GUS reporter gene and electroporated into protoplasts in the presence of RISBZ1, RPBF, or RISBZ1 + RPBF. Expression of a GUS reporter gene in the presence of the RISBZ1 was used as a control (100%). Relative GUS activities with reference to each wild-type promoter activity are shown in parentheses. RISBZ1 binding sites are the GCN4 motif, GbR1, GbR2, and GbR3. RPBF binding sites are the P box and GbP.

Figure 7.

EMSA of RISBZ1 and RPBF with the GluB-1 promoter and endosperm box in the NRP33 promoter. DNA fragments, corresponding to the intact GluB-1 promoter (positions −199 and +23; WT) and to the GluB-1 promoter containing a mutation in either the GCN4 motif or P box (MG or MP), were used as probes. The oligonucleotides, corresponding to the intact endosperm box in the NRP33 promoter (WT) and to the endosperm box containing a mutation in either P-box (M1) or GCN4 motif (M2), were used as probes. The 6xHis tagged RISBZ1 and 6xHis tagged RPBF fusion proteins were used for EMSA with the NRP33 promoter.