Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZIP proteins (original) (raw)
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Nucleic Acids Research, 1994
The G-box Is a cls-acting DNA sequence present in several plant promoters that are regulated by diverse signals such as UV Irradiation, anaerobiosis, absclssic acid and light. Several baslc/leuclne zipper (bZIP) proteins from different plant species have been identified as high affinity G-box binding proteins. Although their capability to enhance transcription has been demonstrated, their precise function In transcrlptional activation Is still unknown. We have isolated three cDNAs from young tomato fruit that encode bZIP G-box binding proteins (GBF4, GBF9 and GBF12). They bind to the G-box sequence In the tomato rbcS1, rbcS2 and rbcS3A promoters. GBF9 binding resulted in a DNase I footprint Identical to that obtained with tomato nuclear extract and different from the DNase I protection obtained with GBF4 and GBF12. The mRNAs of all three GBFs were most abundant in tomato fruit and seeds, moderately abundant in root and least abundant in leaves. Protein sequences outside of the bZIP domains were compared with the known GBFs from other plants and seven conserved motifs of seven to 35 amlno acids length have been Identified. Based on the presence of these motifs, three classes of GBFs can be defined that are conserved among plant species. GBF9, the predominantly expressed tomato GBF, is the first member of its class isolated from dlcot plants. Three conserved motifs from two of the classes are highly hydrophilic and are predicted to be exposed on the surface of the proteins. These motifs likely define novel interactive domains in the different classes of GBFs that could provide a new tool to determine how distinct regulatory signals are transmitted through GBFs to activate transcription.
The EMBO journal, 1992
The G-box is a cis-acting element found within the promoters of many plant genes where it mediates expression in response to a variety of different stimuli. This palindromic DNA motif (CCACGTGG) is composed of two identical half sites, the base pairs of which we have numbered -4 to +4 (numbering from 5' to 3'). Both half sites are involved in the binding of the bZIP protein GBF1, a member of the GBF family of Arabidopsis thaliana. Here we demonstrate using the random binding site selection method that GBF1 interacts with, in addition to the palindromic G-box, other DNA motifs that fall into seven distinct groups. All groups share the ACGT core sequence, common to most DNA motifs bound by plant bZIP proteins so far characterized. Our studies demonstrate that a high affinity GBF1 binding site is further defined by the following two parameters: first, all sites contain a G residue at position +3 (as in ACGTG) and secondly, only certain base pair combinations are allowed at posi...
Proceedings of the National Academy of Sciences, 1994
A fourth member of the Arabidopsis G-box-binding factor (GBF) family of bZIP proteins, GBF4, has been isolated and characterized. In a manner reminiscent of the Fos-related oncoproteins of mammalian systems, GBF4 cannot bind to DNA as a homodimer, although it contains a basic region capable of specifically recognizing the G-box and G-box-like elements. However, GBF4 can interact with GBF2 and GBF3 to bind DNA as heterodimers. Mutagenesis of the leucine zipper of GBF4 indicates that the mutation of a single amino acid confers upon the protein the ability to recognize the G-box as a homodimer, apparently by altering the charge distribution within the leucine zipper.
The EMBO Journal, 1990
Communicated by M.van Montagu G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-IA promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively. We show here that a-390 to-60 rbcS-IA promoter fragment containing the G box and two I boxes activates transcription from a tnmcated iso-l-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae. Mutagenesis of either the rbcS-IA G box or both I box sequences eliminated the expression mediated by this fragment. When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYCl promoter. Single-copy G box sequences from the Arabidopsis rbcS-JA, Anabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF. In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts.
A Maize Protein Associated with the G-Box Binding Complex Has Homology to Brain Regulatory Proteins
The Plant Cell, 1992
The G-box element is a moderately conserved component of the promoter of many inducible genes, including the alcohol dehydrogenase genes of Arabidopsis and maize. We used monoclonal antibodies generated against partially purified G-box binding factor (GBF) activity to characterize maize proteins that are part of the DNA binding complex. Antibodies interacted with partially purified maize GBF complexes to produce a slower migrating complex in the gel retardation assay. lmmunoprecipitation experiments suggested that the protein recognized by the antibody is nota DNA binding protein in and of itself, but rather is associated with the DNA binding complex. These monoclonal antibodies were used to isolate cDNA clones encoding a protein that we have designated GF14. Maize GF14 contains a region resembling a leucine zipper and acidic carboxy and amino termini, of which the latter can form an amphipathic a-helix similar to known transcriptional activators such as VP16 and GALA. Protein gel blot analysis of cell culture extract showed that a single, major protein of approximately 30 kD is recognized by anti-GFl4; the protein is also present predominantly in the kernel and root. The deduced amino acid sequence of maize GF14 is more than 80% identical to Arabidopsis GF14 and Oenothera PHP-O, and is more than 60% identical to a class of mammalian brain proteins described as both protein kinase C inhibitors and activators of tyrosine and tryptophan hydroxylases. GF14 is found in a variety of monocotyledons and dicotyledons, gymnosperms, and yeast. This suggests a deep evolutionary conservation of a potential regulatory protein associated with a core sequence found in the promoter region of many genes.
Genome, 2002
A heterotrimeric GTP-binding protein (G protein) plays a number of important roles in the signaltransduction pathways of eukaryotic cells. The allotetraploid tobacco genome has two α-subunit genes, NtGA1 and NtGA2, of the heterotrimeric G protein. In this study, we determined the nucleotide sequences and the exon-intron structures of the NtGA loci in tobacco and its ancestral diploid species. The genomic sequences of the NtGA loci were interrupted by 13 introns. The sizes of most exons (12 of 14) were completely conserved among the NtGA genes and the Arabidopsis α-subunit gene (GPA1), but most introns (11 of 13) in the NtGA genes were longer than those in GPA1. In comparison with the genomic sequences of the NtGA orthologues of ancestral Nicotiana sylvestris and Nicotiana tomentosiformis, the tobacco NtGA1 and NtGA2 were concluded to be homoeologous and assigned to the S and T genomes, respectively. More than 300 mutations including insertions-deletions (indels) and nucleotide substitutions were found in the intron regions between the NtGA1 and NtGA2 loci, whereas the exon sequences were highly conserved among these and GPA1. The structural comparison revealed larger divergence at the NtGA2 locus than at NtGA1.
Transcription Factor Veracity: Is GBF3 Responsible for ABA-Regulated Expression of Arabidopsis Adh?
The Plant Cell, 1996
Assignment of particular transcription factors to specific roles in promoter elements can be problematic, especially in systems such as the G-box, where multiple factors of overlapping specificity exist. In the Arabidopsis alcohol dehydfogenase (Adh) promoter, the G-box regulates expression in response to cold and dehydration, presumably through the action of abscisic acid (ABA), and is bound by a nuclear protein complex in vivo during expression in cell cultures. In this report, we test the conventional wisdom of biochemical approaches used to identify DNA binding proteins and a s s e s their specific interactions by using the G-box and a nearby half G-box element of the Arabidopsis Adh promoter as a model system. Typical in vitro assays demonstrated specific interaction of G-box factor 3 (GBF3) with both the G-box and the half G-box element. Dimethyl sulfate footprint analysis confirmed that the in vitro binding signature of GBFB essentially matches the footprint signature detected in vivo at the G-box. Because RNA gel blot data indicated that GBFB is itself induced by ABA, we might have concluded that GBFB is indeed the GBF responsible in cell cultures for binding to the Adh G-box and is therefore responsible for ABA-regulated expression of Adh. Potential limitations of this conclusion are exposed by the fact that other GBFs bind the G-box with the same signature as GBF3, and subtle differences between in vivo and in vitro footprint signatures indicate that factors other than or in addition to GBF3 interact with the half G-box element.
Plant Cell, 2007
The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Ga and Gb subunit genes and two Gg subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Gg genes revealed specialized roles for each Gg subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1-and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbg1 and Gbg2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Gg subunit in this root response differs, with Gbg1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbg2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbg signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Gg subunits form functional Gbg dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.
THE PLANT CELL ONLINE, 2007
A tetraploidy left Arabidopsis thaliana with 6358 pairs of homoeologs that, when aligned, generated 14,944 intragenomic conserved noncoding sequences (CNSs). Our previous work assembled these phylogenetic footprints into a database. We show that known transcription factor (TF) binding motifs, including the G-box, are overrepresented in these CNSs. A total of 254 genes spanning long lengths of CNS-rich chromosomes (Bigfoot) dominate this database. Therefore, we made subdatabases: one containing Bigfoot genes and the other containing genes with three to five CNSs (Smallfoot). Bigfoot genes are generally TFs that respond to signals, with their modal CNS positioned 3.1 kb 59 from the ATG. Smallfoot genes encode components of signal transduction machinery, the cytoskeleton, or involve transcription. We queried each subdatabase with each possible 7-nucleotide sequence. Among hundreds of hits, most were purified from CNSs, and almost all of those significantly enriched in CNSs had no experimental history. The 7-mers in CNSs are not 59-to 39-oriented in Bigfoot genes but are often oriented in Smallfoot genes. CNSs with one G-box tend to have two G-boxes. CNSs were shared with the homoeolog only and with no other gene, suggesting that binding site turnover impedes detection. Bigfoot genes may function in adaptation to environmental change.
The Plant Cell, 1992
To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBFl), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBFl was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylating activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low K, value for GBFl (220 nM compared to-10 pM for casein) was in the range observed for identified physiological substrates of casein kinase 11. Phosphorylation of GBFl resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.