Redox Regulation of Plant Homeodomain Transcription Factors

Several families of plant transcription factors contain a conserved DNA binding motif known as the homeodomain. In two of these families, named Hd-Zip and glabra2, the homeodomain is associated with a leucine zipper-like dimerization motif. A group of Hd-Zip proteins, namely Hd-ZipII, contain a set of conserved cysteines within the dimerization motif and adjacent to it. Incubation of one of these proteins, Hahb-10, in the presence of thiol-reducing agents such as dithiothreitol or reduced glutathione produced a significant increase in DNA binding. Under such conditions, the protein migrated as a monomer in non-reducing SDS-polyacrylamide gels. Under oxidizing conditions, a significant proportion of the protein migrated as dimers, suggesting the formation of intermolecular disulfide bonds. A similar behavior was observed for the glabra2 protein HAHR1, which also contains two conserved cysteines within its dimerization domain. Site-directed mutagenesis of the cysteines to serines indicated that each of them has different roles in the activation of the proteins. Purified thioredoxin was able to direct the NADPH-dependent activation of Hahb-10 and HAHR1 in the presence of thioredoxin reductase. The results suggest that redox conditions may operate to regulate the activity of these groups of plant transcription factors within plant cells. Several families of plant transcription factors contain a conserved DNA binding motif known as the homeodomain. In two of these families, named Hd-Zip and glabra2, the homeodomain is associated with a leucine zipper-like dimerization motif. A group of Hd-Zip proteins, namely Hd-ZipII, contain a set of conserved cysteines within the dimerization motif and adjacent to it. Incubation of one of these proteins, Hahb-10, in the presence of thiol-reducing agents such as dithiothreitol or reduced glutathione produced a significant increase in DNA binding. Under such conditions, the protein migrated as a monomer in non-reducing SDS-polyacrylamide gels. Under oxidizing conditions, a significant proportion of the protein migrated as dimers, suggesting the formation of intermolecular disulfide bonds. A similar behavior was observed for the glabra2 protein HAHR1, which also contains two conserved cysteines within its dimerization domain. Site-directed mutagenesis of the cysteines to serines indicated that each of them has different roles in the activation of the proteins. Purified thioredoxin was able to direct the NADPH-dependent activation of Hahb-10 and HAHR1 in the presence of thioredoxin reductase. The results suggest that redox conditions may operate to regulate the activity of these groups of plant transcription factors within plant cells. The homeodomain (HD) 1The abbreviations used are: HD, homeodomain; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; GSSG, oxidized glutathione; GSH, reduced glutathione. is a 61-amino acid protein motif found in eukaryotic transcription factors generally involved in the regulation of developmental processes (1Gehring W.J. Affolter M. Bürglin T. Annu. Rev. Biochem. 1994; 63: 487-526Crossref PubMed Scopus (860) Google Scholar, 2Levine M. Hoey T. Cell. 1988; 55: 537-540Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 3Gehring W.J. Science. 1987; 236: 1245-1252Crossref PubMed Scopus (624) Google Scholar). It folds into a characteristic three-helix structure that interacts specifically with DNA (1Gehring W.J. Affolter M. Bürglin T. Annu. Rev. Biochem. 1994; 63: 487-526Crossref PubMed Scopus (860) Google Scholar, 4Qian Y.Q. Billeter M. Otting G. Müller M. Gehring W.J. Wüthrich K. 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In plants, several families of HD proteins have been described (13Chan R.L. Gago G.M. Palena C.M. Gonzalez D.H. Biochim. Biophys. Acta. 1998; 1442: 1-19Crossref PubMed Scopus (179) Google Scholar). One of these families, named Hd-Zip, comprises proteins with a typical leucine zipper motif adjacent to the C-terminal end of the HD (14Ruberti I. Sessa G. Lucchetti S. Morelli G. EMBO J. 1991; 10: 1787-1791Crossref PubMed Scopus (212) Google Scholar, 15Schena M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3894-3898Crossref PubMed Scopus (214) Google Scholar). As expected, these proteins bind DNA as dimers (16Sessa G. Morelli G. Ruberti I. EMBO J. 1993; 12: 3507-3517Crossref PubMed Scopus (220) Google Scholar). The removal of the leucine zipper or the introduction of extra amino acids between the HD and the zipper significantly reduces binding affinity, indicating that the leucine zipper is responsible for the correct positioning of the HD for efficient binding (16Sessa G. Morelli G. Ruberti I. EMBO J. 1993; 12: 3507-3517Crossref PubMed Scopus (220) Google Scholar). The analysis of binding at different protein concentrations suggests that dimer formation is a prerequisite for DNA binding (17Palena C.M. Gonzalez D.H. Chan R.L. Biochem. J. 1999; 341: 81-87Crossref PubMed Scopus (68) Google Scholar). It has been suggested that Hd-Zip proteins may be involved in regulating developmental processes associated with the response of plants to environmental conditions (13Chan R.L. Gago G.M. Palena C.M. Gonzalez D.H. Biochim. Biophys. Acta. 1998; 1442: 1-19Crossref PubMed Scopus (179) Google Scholar, 18Carabelli M. Sessa G. Baima S. Morelli G. Ruberti I. Plant J. 1993; 4: 469-479Crossref PubMed Scopus (144) Google Scholar, 19Schena M. Lloyd A.M. Davis R.W. Genes Dev. 1993; 7: 367-379Crossref PubMed Scopus (120) Google Scholar). A different family of plant HD proteins, named glabra2, consists of larger proteins with an N-terminal HD. These proteins also bind DNA as dimers and possess a dimerization motif that resembles a leucine zipper truncated by a loop (20Palena C.M. Chan R.L. Gonzalez D.H. Biochim. Biophys. Acta. 1997; 1352: 203-212Crossref PubMed Scopus (10) Google Scholar, 21Di Cristina M. Sessa G. Dolan L. Linstead P. Baima S. Ruberti I. Morelli G. Plant J. 1996; 10: 393-402Crossref PubMed Scopus (268) Google Scholar). Most members of the glabra2 family are expressed specifically in epidermal cells, and the first member to be identified (glabra2) is involved in the development of trichomes, root hairs, and the seed coat mucilage (22Lu P. Porat R. Nadeau J.A. O'Neill S.D. Plant Cell. 1996; 8: 2155-2168Crossref PubMed Scopus (277) Google Scholar, 23Masucci J.D. Rerie W.G. Foreman D.R. Zhang M. Galway M.E. Marks M.D. Schiefelbein J.W. Development. 1996; 122: 1253-1260PubMed Google Scholar, 24Hung C.-Y. Lin Y. Zhang M. Pollock S. Marks M.D. Schiefelbein J. Plant Physiol. 1998; 117: 73-84Crossref PubMed Scopus (126) Google Scholar, 25Ingram G.C. Magnard J.-L. Vergne P. Dumas C. Rogowsky P.M. Plant Mol. Biol. 1999; 40: 343-354Crossref PubMed Scopus (53) Google Scholar, 26Rerie W.G. Feldmann K.A. Marks M.D. Genes Dev. 1994; 8: 1388-1399Crossref PubMed Scopus (467) Google Scholar). The HD and dimerization motif constitute the most conserved part of the different members of each family. Sequence analysis also revealed the presence of conserved cysteine-containing motifs within variable regions (i.e. within the variable loop in glabra2 proteins and near the C terminus of the leucine zipper in Hd-ZipII proteins). Because conserved cysteines have been reported to be involved in the redox regulation of the properties of several transcription factors (27Akamatsu Y. Ohno T. Hirota K. Kagoshima H. Yodoi J. Shigesada K. J. Biol. Chem. 1997; 272: 14497-14500Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 28Arnér E.S.J. 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Our results indicate that redox conditions are a key factor determining the binding of these proteins to DNA and the formation of covalent oligomeric structures. We propose that a redox-dependent mechanism may operate in vivo to modulate the activity of these transcription factors in response to metabolic and/or environmental signals. An NheI/XbaI fragment containing sequences coding for the entire HD and dimerization motif plus additional amino acids (positions 86–325) from the sunflower glabra2 protein HAHR1 (34Valle E.M. Gonzalez D.H. Gago G. Chan R.L. Gene. 1997; 196: 61-68Crossref PubMed Scopus (16) Google Scholar) was excised from pHX1 (which contains this fragment in vector pUC119) with HincII and EcoRI and cloned into the SmaI and EcoRI sites of pGEX-3X as described (35Palena C.M. Gonzalez D.H. Guelman S. Chan R.L. Protein Expression Purif. 1998; 13: 97-103Crossref PubMed Scopus (21) Google Scholar). For the production of a protein with only the first half of the dimerization motif (amino acids 86–184), a KpnI deletion of this clone was used. Fragments encoding proteins with either Cys or at and by on HAHR1 and in with either or For HAHR1, and used with DNA from as with KpnI and cloned into similar sites in the These proteins the entire HD and dimerization motif (amino acids An fragment encoding amino acids from the sunflower Hd-ZipII protein Hahb-10 D.H. E.M. Gago G.M. Chan R.L. Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar), cloned into of was used as the for in and sites for and Fragments encoding the entire Hd-Zip plus the motif (amino acids and cloned into the and EcoRI sites of and for for or for and either or sequences as with Cys to within the leucine zipper and or and with and to N-terminal and C-terminal Hahb-10 The in containing and dithiothreitol at and by the to to in and of the fragment of DNA and was for at A of this was used to the and and in a similar of and DNA from as was by by DNA For E. the and as described (35Palena C.M. Gonzalez D.H. Guelman S. Chan R.L. Protein Expression Purif. 1998; 13: 97-103Crossref PubMed Scopus (21) Google Scholar). and of the as described by and Johnson Johnson Gene. 1988; 67: PubMed Scopus Google Scholar), with described by Palena (35Palena C.M. Gonzalez D.H. Guelman S. Chan R.L. Protein Expression Purif. 1998; 13: 97-103Crossref PubMed Scopus (21) Google Scholar). Purified proteins as by of used for the Protein as described by and J. S. Biochem. PubMed Scopus Google Scholar). E. thioredoxin in was expressed from this and by affinity E. thioredoxin was expressed from and as described by Protein Expression Purif. 1997; 9: PubMed Scopus Google Scholar). Purified proteins at in with redox agents in this for at in the For electrophoretic mobility shift of proteins with DNA by of the and or and and with by in the the fragment of E. DNA I. containing of of and of protein for at with and a in plus is The was in at for and indicated that of binding was For of binding affinity, was and from and by and of the into monomers and DNA the to the binding protein monomers and dimers and and DNA, (17Palena C.M. Gonzalez D.H. Chan R.L. Biochem. J. 1999; 341: 81-87Crossref PubMed Scopus (68) Google Scholar). SDS-polyacrylamide as described by PubMed Scopus Google Scholar), that was from the at in plus the indicated with and a with and from to the described in T. W. P. W. in Plant A Scholar). Protein by SDS-polyacrylamide and protein was as described J. S. Biochem. PubMed Scopus Google Scholar). For of of in reductants different conditions and non-reducing SDS-polyacrylamide gels. proteins to and (35Palena C.M. Gonzalez D.H. Guelman S. Chan R.L. Protein Expression Purif. 1998; 13: 97-103Crossref PubMed Scopus (21) Google Scholar) and We have that the sunflower HD protein HAHR1 a DNA as a dimer (20Palena C.M. Chan R.L. Gonzalez D.H. Biochim. Biophys. Acta. 1997; 1352: 203-212Crossref PubMed Scopus (10) Google Scholar, Palena C.M. Chan R.L. Gonzalez D.H. PubMed Scopus Google Scholar). acid of HAHR1 with proteins from the glabra2 family a high along the HD and dimerization with the of the loop that helix and II of the dimerization This loop is by the presence of and is variable in and are only suggesting that its is as a between the two this is that a that contains two cysteines is present within the loop of proteins described to This is an of an of this of the conserved cysteines within it. the of the of the conserved cysteines on the properties of HAHR1, we have the protein in the presence of the and the and the of DNA binding Incubation in the presence of produced a increase in DNA binding with to with suggesting that cysteines in the reduced are for efficient binding. A similar was the binding to and by This that the observed in DNA binding are to of the in binding activity in the and presence of the of the of dimers into monomers of and for the proteins in the presence of or indicating that the an increase in the affinity of the protein for analysis that the of of the is by with DTT, the of is significantly cysteines present within the dimerization motif to a protein redox conditions was the for the redox of HAHR1, we have tested the effect of the redox oxidized glutathione glutathione As in a significant activation was with GSH, indicating that this which has a DTT, is able to HAHR1 a or similar binding activity as with oxidants suggesting that the protein is oxidized or The of each of the two cysteines present in the loop of the dimerization motif was by proteins with to proteins to to a different was oxidizing conditions and was by was for activation on the significant binding only in the presence of The different behavior of the may in redox of the The fact that HAHR1 dimers in suggests that the cysteines present within the loop of the dimerization motif may form intermolecular disulfide This was by non-reducing conditions of proteins to different As in proteins with either one or cysteines with significantly reduced mobility to dimers in the of with as suggesting that are Incubation in the presence of oxidizing agents the of with reduced was as efficient as as It be that is an of the of monomers and DNA binding This may be to the different of (i.e. to be in SDS-polyacrylamide to to be by mobility shift at the formation of disulfide between adjacent monomers to be with the in DNA binding. The protein present in sunflower a similar of in the presence of reductants with a of to the monomer This was observed oxidizing conditions, most of the formation of larger which the In in the to the these conditions The response of HAHR1 from that of proteins from the as from a major group of proteins mobility only in the presence of DTT, several of conserved cysteines in plant HD proteins revealed that in proteins from the Hd-ZipII described to is a motif adjacent to the leucine zipper This motif contains two conserved cysteines and resembles the motif present in glabra2 proteins. Hd-ZipII proteins also redox-dependent we have expressed the sunflower Hd-ZipII protein Hahb-10 D.H. E.M. Gago G.M. Chan R.L. Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar) in E. and similar as described for as HAHR1, Hahb-10 DNA oxidizing conditions with produced a significant increase in DNA indicating that the of cysteines is for its also produced an increase in binding that produced by of the oxidizing and conditions indicated that an increase in binding analysis that the and of the (i.e. the was and the was in the presence of of cysteines to significantly reduced the in DNA binding the protein to oxidizing which is DNA binding is at protein concentrations This may be by the presence of additional cysteines within the leucine zipper of of cysteines in the motif of Hahb-10 produced proteins with DNA binding activity oxidizing conditions was that in the presence of oxidants and was significantly only in the presence of This that the behavior of the within the motif is similar for HAHR1 and indicated the presence of oxidizing conditions for the proteins Hahb-10 that migrated dimers, which in in oxidizing Full into monomers was only in the presence of DTT, was only This that is a between the formation of intermolecular covalent and the in DNA binding. these for the different proteins oxidizing conditions, that at part of the proteins that in covalent formation are in DNA binding. This suggests that the formation of disulfide binding activity in a different the formation of the of cysteines present in the leucine zipper of Hahb-10 was also investigated. The of Hahb-10 used for these has additional cysteines within the leucine zipper of them are present at a the dimer of the and and a the is at the of the adjacent and also near the to known leucine zipper J.D. T. Science. 1991; PubMed Scopus Google Scholar). and a are conserved in Hd-ZipII proteins, is present in most of The of these cysteines was by the analysis of the properties of proteins with different of The used for the different proteins is in A. the analysis of the DNA binding properties of the in which and a are to are to redox conditions in of binding activity proteins with at are to proteins in which is to a significant in binding These results indicate that adjacent cysteines are of the of DNA binding to and that cysteines within the motif also this of DNA binding. The formation of intermolecular disulfide was by non-reducing As expected, proteins, with the of the produced oxidizing conditions also a significant proportion of monomers which observed for the proteins. within the leucine zipper are responsible for the formation of dimers oxidizing conditions suggesting that from two adjacent monomers form covalent dimers by to be This that disulfide formation may to either or proteins, on the of the cysteines that are The fact that the of disulfide the activation of HAHR1 and Hahb-10 the that a similar is used conditions to regulate the properties of these transcription factors. in protein cysteines are usually in vivo by a protein that is in reduced by in the presence of thioredoxin E.S.J. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar, P. P. Biochem. Biophys. 1994; PubMed Scopus Google Scholar). We have the activation of HAHR1 and Hahb-10 and thioredoxin and thioredoxin from E. As in this was as efficient as in the activation of HAHR1 and This is with a of thioredoxin in in these transcription factors in are used by to modulate the activity of proteins. these are key of that to in redox conditions E.S.J. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2026) Google Scholar, P. P. Biochem. Biophys. 1994; PubMed Scopus Google Scholar, A. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, A. Annu. Rev. Biochem. PubMed Google Scholar). In the present we that two plant HD transcription factors that in affinity for DNA One of these HAHR1, to the glabra2 family members are to in epidermal development at the and plant (22Lu P. Porat R. Nadeau J.A. O'Neill S.D. Plant Cell. 1996; 8: 2155-2168Crossref PubMed Scopus (277) Google Scholar, 23Masucci J.D. Rerie W.G. Foreman D.R. Zhang M. Galway M.E. Marks M.D. Schiefelbein J.W. Development. 1996; 122: 1253-1260PubMed Google Scholar, 24Hung C.-Y. Lin Y. Zhang M. Pollock S. 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We B. and H. for the of thioredoxin and thioredoxin reductase.