Trihelix DNA-binding Protein with Specificities for Two Distinctcis-Elements

Abstract

The DE1 sequence is acis-regulatory element necessary and sufficient for light down-regulated and dark-inducible expression of the pea GTPasepra2 gene. This sequence does not show any sequence similarity to the previously reported ones involved in light-regulated gene expression. A one-hybrid screen isolated a cDNA encoding a DNA-binding protein, named DF1, with specificity for the DE1 sequence 5′-TACAGT. DF1 has domains similar to the trihelix DNA-binding domain found in the GT-1 and GT-2 proteins, which are plant transcription factors. The DE1-binding domain of DF1 is most similar to the carboxyl-terminal trihelix domain of the rice GT-2 protein with specificity for the GT2 sequence 5′-GGTAATT, which is also necessary for dark-inducible expression of the rice phyA gene. An electrophoretic mobility shift assay showed that this DNA-binding domain specifically binds to two types of DNA sequences, DE1 and GT2. Additionally, using DF1/GT-1 chimeras, we show that the second and third helices of the trihelix DNA-binding domain of DF1 are responsible for this dual DNA binding specificity. Our results show that DF1 has specificity for the two distinct cis-regulatory elements, both important for light down-regulated and dark-inducible gene expression in higher plants. The DE1 sequence is acis-regulatory element necessary and sufficient for light down-regulated and dark-inducible expression of the pea GTPasepra2 gene. This sequence does not show any sequence similarity to the previously reported ones involved in light-regulated gene expression. A one-hybrid screen isolated a cDNA encoding a DNA-binding protein, named DF1, with specificity for the DE1 sequence 5′-TACAGT. DF1 has domains similar to the trihelix DNA-binding domain found in the GT-1 and GT-2 proteins, which are plant transcription factors. The DE1-binding domain of DF1 is most similar to the carboxyl-terminal trihelix domain of the rice GT-2 protein with specificity for the GT2 sequence 5′-GGTAATT, which is also necessary for dark-inducible expression of the rice phyA gene. An electrophoretic mobility shift assay showed that this DNA-binding domain specifically binds to two types of DNA sequences, DE1 and GT2. Additionally, using DF1/GT-1 chimeras, we show that the second and third helices of the trihelix DNA-binding domain of DF1 are responsible for this dual DNA binding specificity. Our results show that DF1 has specificity for the two distinct cis-regulatory elements, both important for light down-regulated and dark-inducible gene expression in higher plants. base pair 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside electrophoretic mobility shift assay wild type glutathione S-transferase decahistidine-tagged Light is one of the most important signals affecting plant gene expression (1Terzaghi W.B. Cashmore A.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 445-474Crossref Scopus (460) Google Scholar). Light signals regulate the transcription of a number of genes in positive and negative manners. The mechanisms of light-enhanced transcription have been extensively studied (1Terzaghi W.B. Cashmore A.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 445-474Crossref Scopus (460) Google Scholar). Variouscis-regulatory elements and trans-acting factors responsible for light-enhanced gene expression have been characterized (1Terzaghi W.B. Cashmore A.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 445-474Crossref Scopus (460) Google Scholar). In contrast, the mechanism of light down-regulated transcription is less clear. Only a few cis-regulatory elements and trans-acting factors responsible for down-regulation have been reported. For example, the GT2 (5′-GGTAATT) sequence in the light down-regulated ricephyA gene is necessary for dark-activation of this gene (2Dehesh K. Bruce W.B. Quail P.H. Science. 1990; 250: 1397-1399Crossref PubMed Scopus (92) Google Scholar). Affinity screening using GT2 sequences has been used to isolate the DNA-binding protein, named GT-2 (2Dehesh K. Bruce W.B. Quail P.H. Science. 1990; 250: 1397-1399Crossref PubMed Scopus (92) Google Scholar). The GT-2 protein is a member of the trihelix DNA-binding proteins (3Zhou D.-X. Trends Plant Sci. 1999; 4: 210-214Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), which are considered to be specific to plants and have domain(s) containing three α helices involved in DNA binding. Although the GT-2 protein is one of the well characterized plant DNA-binding proteins, the function of its binding site, the GT2 sequence, is less clear because there has been no report concerning a gain-of-function analysis using the GT2 sequence fused to the minimal promoter. Therefore, combinatorial interaction between the GT2 element and the other element may be required for dark activation. Except for the GT2 protein, we do not know of a DNA-binding protein with specificity for the cis-regulatory element involved in light down-regulated gene expression. Previously, we reported that a small GTPase gene in pea,pra2, which belongs to the YPT/rab family, is one of the genes whose expression is down-regulated by photoreceptor phytochrome (4Yoshida K. Nagano Y. Murai N. Sasaki Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6636-6640Crossref PubMed Scopus (33) Google Scholar). The pra2 gene is mainly expressed in the growing zone of etiolated epicotyls, and its expression is repressed when the plant is illuminated (5Nagano Y. Okada Y. Narita H. Asaka Y. Sasaki Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6314-6318Crossref PubMed Scopus (43) Google Scholar). The DE1 sequence (5′-GGATTTTACAGT) in thepra2 gene is another cis-regulatory element necessary for light down-regulated and dark-enhanced expression in plants (6Inaba T. Nagano Y. Sakakibara T. Sasaki Y. Plant Physiol. 1999; 120: 491-500Crossref PubMed Scopus (24) Google Scholar). Gain-of-function analysis has shown that the DE1 sequence is sufficient to confer dark-inducible and light down-regulated expression to a minimal promoter (7Inaba T. Nagano Y. Reid J.B. Sasaki Y. J. Biol. Chem. 2000; 275: 10723-19727Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). This sequence does not show any sequence similarity to the previously reported ones involved in light-regulated gene expression. In addition, to our knowledge, DE1 is the first discovered cis-regulatory element that is both necessary and sufficient for light regulation. Physiological and genetic analyses have shown that the DE1 sequence receives the signals from phytochrome A, phytochrome B, and blue-light photoreceptors (7Inaba T. Nagano Y. Reid J.B. Sasaki Y. J. Biol. Chem. 2000; 275: 10723-19727Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Therefore, the DE1 sequence should be a useful tool for studying the molecular mechanism involved in light down-regulated gene expression in plants. In this study, we identified a cDNA whose product binds to the DE1 sequence. We found that this protein is a member of the trihelix DNA-binding proteins and binds to two distinctcis-regulatory elements, DE1 and GT2, both important for light down-regulated and dark-inducible expression in plants. The methods for the creation of reporter constructs were described previously (7Inaba T. Nagano Y. Reid J.B. Sasaki Y. J. Biol. Chem. 2000; 275: 10723-19727Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). For the first screening, the pHISi reporter plasmid (CLONTECH, Palo Alto, CA) containing five tandem copies of the 18-bp1sequence, which contains the DE1 sequence with neighboring 3 bp at both ends, was used. For the second screening, the pLacZi reporter plasmid (CLONTECH) containing nine tandem copies of the 18-bp sequence, which contains the DE1 sequence with neighboring 3-bp at both ends, was used. For the third screening, the pLacZi reporter plasmid containing three tandem copies of the 20-bp sequence, which contains the DE1 sequence with neighboring 4 bp at both ends, was used. The reporter constructs were integrated into the yeastSaccharomyces cerevisiae YM4271 (CLONTECH). Total RNA was prepared from the growing zones of pea (Pisum sativum cv. Alaska) stems (between 0 and 1 cm from the top of the hook) that had been grown in the dark for 7 days at 25 °C as described elsewhere (8Nagano Y. Murai N. Matsuno R. Sasaki Y. Plant Cell Physiol. 1993; 34: 447-455PubMed Google Scholar). Poly(A)+-RNA was extracted by the Poly(A)Tract mRNA isolation system (Promega, Madison, WI). Complementary DNA was synthesized with the HybriZAP-2.1 two-hybrid cDNA synthesis kit (Stratagene, La Jolla, CA), cloned into the EcoRI andXhoI sites of HybriZAP-2.1 or λZapII, and packaged using the MaxPlax packaging extract (Epicentre Technologies, Madison, WI). The plasmid library was constructed by excising from the HybriZAP library by infection with the helper phage ExAssist (Stratagene). The resulting HybriZAP cDNA library contained ∼8 × 106 independent cDNAs. The histidine yeast reporter strain was transformed with a pea cDNA library by the LiAc/polyethylene glycerol method. Approximately 1.5 × 107 cDNA plasmids were screened. Based on their large colony size and rapid growth, about 500 histidine-positive clones were selected. Plasmids were recovered and electroporated into theEscherichia coli strain DH10B (Life Technologies, Inc.). Plasmids were rescreened by transforming the second lacZreporter strain. The filter-replica method using X-gal was used to confirm β-galactosidase activities. Three plasmids showed the blue color. These plasmids were rescreened by transforming the thirdlacZ reporter strain. One plasmid showed the blue color. To isolate the full-length cDNA, a λZapII cDNA library was screened using a part of this cDNA insert as a probe. The gene was named DF1. The accession number for the sequence reported in this article is AB052729. The protein expression vector used in this study was pGEX-4T-3 (Amersham Pharmacia Biotech) or the pET-16b vector (Novagene). Procedures for the production and purification of a fusion polypeptide were carried out as suggested by the manufacturer. The polypeptide containing the trihelix DNA-binding domain (from 519 to 651) of the pea DF1 protein was expressed in E. coli as a decahistidine-tagged (His10 tag) protein or a glutathioneS-transferase (GST)-fused protein. The GST-fused GT-1 protein contains the Arabidopsis GT-1 sequence (from 65 to 195). DF1/GT-1 chimeras were created by the overlap extension method (9Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 51-59Crossref PubMed Scopus (6825) Google Scholar). The GST-fused chimera protein, GT-1helix 1-DF1helices 2–3, contains theArabidopsis GT-1 sequence (from 65 to 108) and the pea DF1 sequence (from 563 to 651). Another GST-fused chimera protein, DF1helices 1–2-GT-1helix 3, contains the pea DF1 sequence (from 519 to 578) and the Arabidopsis GT-1 sequence (from 125 to 195). The electrophoretic mobility shift assay (EMSA) was performed by the method previously described (6Inaba T. Nagano Y. Sakakibara T. Sasaki Y. Plant Physiol. 1999; 120: 491-500Crossref PubMed Scopus (24) Google Scholar). The probes used in this study, DE1 and GT2, are shown in Figs. 3 A and 4 A, respectively. The recombinant protein (5 pmol) was mixed in 20 μl of the binding buffer (6Inaba T. Nagano Y. Sakakibara T. Sasaki Y. Plant Physiol. 1999; 120: 491-500Crossref PubMed Scopus (24) Google Scholar) containing 2 μg of poly(dI-dC)-poly(dI-dC), bovine serum albumin (500 μg/μl) and competitor DNA. The competitor DNAs used in this study are listed in Figs. 3 A and 4 A. The protein-DNA complex was formed by incubating at 25 °C for 20 min with 10,000 cpm of 32P-labeled probe (4 fmol). Electrophoresis was conducted at 4 °C in a 5% polyacrylamide Tris/borate/EDTA gel containing 10% glycerol. The gel was dried and subjected to autoradiography.Figure 4Determination of the DNA sequence bound to the DF1 protein using the GT2 probe. A, nucleotide sequences of various competitors used to define the DF1-binding site. The positions of the mutated nucleotide are shaded. The core-binding site of the DF1 protein is underlined. B, EMSA using various competitors. The competitorsWT and MT are shown in Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The yeast one-hybrid strategy (10Wang M.-M. Reed R.R. Nature. 1993; 364: 121-126Crossref PubMed Scopus (369) Google Scholar) was used to screen for pea cDNA encoding for protein that binds to the DE1 sequence. Tandem copies of the 18-bp sequence containing the 12-bp DE1 sequence with a neighboring 3 bp at each end were subcloned into the upstream region of HIS3 andlacZ reporter genes. About 500 histidine-positive clones were selected from 1.5 × 107 transformants (Fig.1 A). We rescreened plasmids recovered from histidine-positive clones by transforming thelacZ reporter strain and obtained three plasmids showinglacZ-positive phenotypes (Fig. 1 B). To exclude the cDNAs whose products bind to the joint sequence between the 18 bp sequences, we rescreened the plasmids using the reporter constructs having tandem copies of the 20-bp sequence containing the DE1 sequence with a neighboring 4 bp at each end. Finally, one plasmid showed alacZ-positive phenotype (Fig. 1 C). To isolate longer cDNAs, this cDNA was used to screen a λZAPII cDNA library. We further characterized the longest cDNA of the isolated clones. The gene, designated as DF1, contains an open reading frame of 682 amino A sequence similarity that DF1 has domains similar to the trihelix DNA-binding domain (3Zhou D.-X. Trends Plant Sci. 1999; 4: 210-214Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) found in GT-1 E. Plant 4: PubMed Scopus Google J. K. Plant 4: PubMed Scopus Google Scholar), GT-2 (2Dehesh K. Bruce W.B. Quail P.H. Science. 1990; 250: 1397-1399Crossref PubMed Scopus (92) Google K. H. Quail P.H. J. PubMed Scopus Google Scholar), and J. J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) GT-1 and GT-2 are DNA-binding proteins that elements and are trihelix DNA-binding which three α helices involved in DNA binding. Fig. 2 A the of DF1 and other trihelix DNA-binding proteins, DF1 is the most similar to proteins having trihelix DNA-binding as GT-2 and These trihelix DNA-binding proteins from GT-1 because GT-1 has one trihelix DNA-binding The DF1 protein also has trihelix In to having two DNA-binding and DF1 proteins each have an a which is between two DNA-binding for the three the amino sequences of trihelix DNA-binding proteins are not Fig. 2 the trihelix DNA-binding in analysis has that the trihelix domain is to the DNA-binding domain Y. Plant Physiol. 2000; PubMed Scopus Google Scholar), the the DNA-binding proteins have well amino the amino sequence of DF1 is most similar to that of the protein from the (Fig. 2 C). DF1 is not to GT-2 and proteins having trihelix DNA-binding as DF1, and may have because a of The Arabidopsis gene is to the GT-2 gene, in the on To the DNA-binding specificity of the DF1 protein, we conducted an EMSA using a DNA probe containing the DE1 sequence A, We on the carboxyl-terminal trihelix domain of DF1 because a one-hybrid screen first isolated this DNA-binding and of both domains have been We expressed the carboxyl-terminal trihelix DNA-binding domain of DF1 in E. coli as a or a GST-fused protein. The of the DF1 protein to the binding showed the of the (Fig. 3 B, The of the GST-fused DF1 protein to the binding also showed the of (Fig. 3 is to sufficient of recombinant GST-fused protein and is to the DF1 protein in we used the GST-fused DF1 protein in the The results were the as of the using the protein not To the binding was specific to the DE1 sequence, we carried out The competitor sequences are shown in Fig. 3 A. The of a competitor the (Fig. 3 B, The MT competitor contained the three nucleotide which not bind to the factors with specificity for the DE1 sequence (6Inaba T. Nagano Y. Sakakibara T. Sasaki Y. Plant Physiol. 1999; 120: 491-500Crossref PubMed Scopus (24) Google Scholar). A gain-of-function analysis using the 3-bp mutated constructs fused to the minimal promoter has shown that this does not have the for light down-regulation (7Inaba T. Nagano Y. Reid J.B. Sasaki Y. J. Biol. Chem. 2000; 275: 10723-19727Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The EMSA showed that the MT competitor not the B, These that the DNA binding specificity is with the results of the EMSA using (6Inaba T. Nagano Y. Sakakibara T. Sasaki Y. Plant Physiol. 1999; 120: 491-500Crossref PubMed Scopus (24) Google Scholar) and the gain-of-function analysis (7Inaba T. Nagano Y. Reid J.B. Sasaki Y. J. Biol. Chem. 2000; 275: 10723-19727Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To the core-binding site of the DF1 protein, we carried out using the mutated competitors A, In our (6Inaba T. Nagano Y. Sakakibara T. Sasaki Y. Plant Physiol. 1999; 120: 491-500Crossref PubMed Scopus (24) Google Scholar), we identified the DE1 element using the mutated constructs by assay The (Fig. 3 not show The This analysis that the 12-bp sequence, designated DE1 phytochrome down-regulation and that the sequence is the region for The of a competitor not the B, The of an competitor the (Fig. 3 B, the of an competitor the (Fig. 3 B, These results show that recombinant DF1 binds the DE1 sequence and the core-binding site is the sequence 5′-TACAGT. results are with of the assay analysis (6Inaba T. Nagano Y. Sakakibara T. Sasaki Y. Plant Physiol. 1999; 120: 491-500Crossref PubMed Scopus (24) Google Scholar). To the DF1-binding site we carried out using the competitors A, The of and which to the region of the DE1 not the (Fig. 3 B, The of the and competitors the (Fig. 3 B, and the of other competitors the (Fig. 3 B, and These results show that the sequence is the core-binding site of the DF1 protein, and its sequence is involved in the binding of the DF1 protein. The DE1-binding domain sequence to trihelix DNA-binding the binding sequences of GT-1 E. Plant 4: PubMed Scopus Google J. K. Plant 4: PubMed Scopus Google Scholar) and GT-2 (2Dehesh K. Bruce W.B. Quail P.H. Science. 1990; 250: 1397-1399Crossref PubMed Scopus (92) Google K. H. Quail P.H. J. PubMed Scopus Google Scholar) proteins have been The element of pea the binding site of core-binding sequence is The GT2 and the sequences of rice phyA are the binding sites of carboxyl-terminal and trihelix DNA-binding of the rice GT-2 protein K. H. Quail P.H. J. PubMed Scopus Google Scholar). the DF1 binding sequence is from the binding sequences of GT-1 and GT-2 The DE1-binding domain of the DF1 protein is most similar to the GT2 domain of the rice GT-2 protein. To DF1 has the to bind to the binding sites of trihelix DNA-binding proteins, we carried out using competitors containing the binding sequences (Fig. 3 A, The of and competitors not the (Fig. 3 B, 18 and the of a GT2 competitor the B, results that DF1 has the to bind to the GT2 The sequence of the GT2 probe is In this sequence, the sequence is similar to the DE1 sequence 5′-TACAGT. Therefore, the DF1 protein bind to this sequence and not to the GT2 sequence. To the DNA binding specificity to the GT2 sequence, we also conducted the EMSA using a DNA probe containing the GT2 sequence A, and the competitors (Fig. 4 A). The of the GST-fused DF1 protein to the binding showed the of the complex (Fig. 4 The of MT and which to the region of the GT2 not the (Fig. 4 B, The of the competitors the (Fig. 4 B, and the of the GT2, and the DE1 competitors the (Fig. 4 B, This analysis showed that the core-binding site of DF1 is This specificity of the DF1 protein is similar to that of the GT-2 protein (2Dehesh K. Bruce W.B. Quail P.H. Science. 1990; 250: 1397-1399Crossref PubMed Scopus (92) Google Scholar). results show that DF1 has the to bind specifically to the GT2 sequence, which is from the DE1 sequence. We are in which helices of the trihelix DNA-binding domain of DF1 are responsible for the dual DNA binding specificity. In addition, has not been which helices of the trihelix DNA-binding domain of the other protein DNA sequences, and analyses have shown that the trihelix DNA-binding domain of Arabidopsis GT-1 is for DNA binding K. H. Plant PubMed Scopus Google Scholar). To the of for two distinct DNA sequences, we conducted the EMSA using DF1/GT-1 chimeras Fig. A the recombinant proteins used in this DF1 and GT-1helix 1-DF1helices proteins were to bind to two types of sequences, the GT2 and DE1 sequences B, 3, and the DF1 protein bound to sequences of two types of proteins have similar to the GT2 and DE1 In contrast, the DF1helices 1–2-GT-1helix 3 protein bound to of two types of sequences (Fig. B, 4 and The GT-1 protein bound to both sequences (Fig. B, and the second and third helices of the trihelix domain are responsible for the binding to the DE1 and GT2 The creation of the chimera protein may the DNA binding specificity. To the DNA binding specificity of the DF1helices 1–2-GT-1helix 3 protein to both sequences, we conducted the EMSA using the competitors. The of and competitors to the binding containing the GT2 probe not the (Fig. the other the of GT2, and competitors the (Fig. 3, and the specificity to the sequence was the GT2 sequence, be the core-binding site of the DF1helices 1–2-GT-1helix 3 protein. the of and competitors to the binding containing the DE1 probe not the (Fig. the other the of and competitors the (Fig. and These results show that the DE1 sequence is the core-binding site of the DF1helices 1–2-GT-1helix 3 protein. this chimera protein two distinct DNA sequences, DE1 and GT2, that second and third helices of the DNA-binding domain are responsible for the dual DNA binding specificity. The carboxyl-terminal DNA-binding domain of DF1 two distinct cis-regulatory elements, DE1 and GT2, both important for light down-regulated and dark-inducible gene expression in higher plants. This is an mechanism as DF1 protein from the light to the two DE1 and GT2. have this mechanism the of One for this mechanism is that DF1 protein bind to one sequence and the in the amino sequence of the DNA-binding domain in the dual DNA binding specificity. Another is that in both the DNA-binding domain and its sequences in the dual DNA binding specificity. In the study, we have described a DNA-binding protein having a domain that two distinct DNA sequences, both elements having similar two sequences show no or sequence To in which one DNA-binding protein has the specificity for sequences have been reported. For example, the DNA-binding protein a member of the proteins in binds to two types of is A, or A as the and R. A. A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, binds to the sequence. A in a from the dual DNA binding to the specificity R. A. A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, with the specificity R. A. A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the trihelix DNA-binding domain may be to the DNA-binding domain Y. Plant Physiol. 2000; PubMed Scopus Google Scholar). or in the trihelix DNA-binding domain from the protein dual DNA binding specificity for two distinct and cis-regulatory The dual DNA binding specificity be the of the trihelix DNA-binding protein. Arabidopsis GT-1 binds not to the sequence also to the sequences K. H. Plant PubMed Scopus Google Scholar). the of the protein have not been because the specificity to the sequence has not been and two sequences may be Affinity screening using GT-1 binding sites has isolated the protein E. Plant 4: PubMed Scopus Google J. K. Plant 4: PubMed Scopus Google Scholar). These have that three α helices in GT-1 be involved in DNA binding. analyses of GT-1 have shown that the trihelix DNA-binding domain of GT-1 is involved in DNA binding K. H. Plant PubMed Scopus Google Scholar). has not been which helices are responsible for DNA binding. DF1/GT-1 chimeras, the study showed that second and third helices of the trihelix DNA-binding domain of DF1 are responsible for specific DNA binding. This of is similar to of the other DNA-binding proteins containing three as the DNA-binding domain and the A in study has that the trihelix DNA-binding domain is to the DNA-binding domain Y. Plant Physiol. 2000; PubMed Scopus Google Scholar). this is with our results using DF1/GT-1 In the study, we a characterized member of trihelix DNA-binding The carboxyl-terminal DNA-binding domain of DF1 bind to two distinct cis-regulatory elements, DE1 and GT2, both responsible for light down-regulated and dark-inducible gene expression in higher plants. The rice GT-2 protein binds to the GT2 sequence, which is necessary for dark-inducible expression of the ricephyA gene. the GT-2 protein a of similarity to DF1, the GT-2 protein also bind to the DE1 sequence. Another trihelix DNA-binding protein, bind to the sequence, which is necessary not sufficient for expression of the pea gene E. Plant 4: PubMed Scopus Google J. K. Plant 4: PubMed Scopus Google Scholar). the trihelix DNA-binding proteins be important for light-regulated gene expression. In addition, the study the of trihelix DNA-binding proteins for light down-regulated and dark-inducible gene expression in the of which was Arabidopsis Scholar), contains trihelix DNA-binding proteins of which are listed in K. Nagano Y. Murai N. Sasaki Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6636-6640Crossref PubMed Scopus (33) Google and K. H. Plant PubMed Scopus Google Scholar). of the trihelix DNA-binding proteins, of trihelix DNA-binding proteins DF1 protein, should be an important for the of light-regulated gene expression in plants. We K. for useful