The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors.

In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.

Identical amino acid substitutions in the repression domain of auxin/indole-3-acetic acid proteins have contrasting effects on auxin signaling.

Auxin/indole-3-acetic acid (Aux/IAA) proteins function as repressors of auxin response gene expression when auxin concentrations in a cell are low. At elevated auxin concentrations, these repressors are destroyed via the ubiquitin-proteasome pathway, resulting in derepression/activation of auxin response genes. Most Aux/IAA repressors contain four conserved domains, with one of these being an active, portable repression domain (domain I) and a second being an auxin-dependent instability domain (domain II). Here, we have analyzed the effects of amino acid substitutions in the repression domain of selected Aux/IAA proteins. We show that stabilized versions of Aux/IAA proteins with amino acid substitutions in domain I display contrasting phenotypes when expressed in transformed Arabidopsis (Arabidopsis thaliana) plants. An alanine-for-leucine substitution in the LxLxL (where L is leucine and x is another amino acid) repression domain of IAA3, IAA6, or IAA19 confers enhanced auxin response gene expression and "high-auxin" phenotypes when expressed from the 35S or IAA19 promoter (as tested with IAA19) in transformed Arabidopsis plants. In marked contrast, a single alanine-for-leucine substitution in domain I of IAA12 or IAA17 confers repression of auxin response genes and "low-auxin" phenotypes. These results point to intrinsic differences in the repression domain(s) of IAA proteins and suggest that some IAA proteins have stronger or more complex repression domains than others.

The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element.

CONSTANS is an evolutionarily-conserved central component of the genetic pathway that controls the onset of flowering in response to daylength. However, the specific biochemical mechanism by which the CONSTANS protein regulates the expression of its target genes remains largely unknown. *By using a combination of cell-based expression analysis and in vitro DNA binding studies, we have demonstrated that CONSTANS possesses transcriptional activation potential and is capable of directly binding to DNA. *CONSTANS was found to bind DNA via a unique sequence element containing a consensus TGTG(N2-3)ATG motif. This element is present in tandem within the FLOWERING LOCUS T promoter and is sufficient for CO binding and activity. The conserved CCT (CONSTANS, CONSTANS-like and TOC1) domain of CONSTANS was shown to be required for its recruitment to the DNA motif and other CCT-containing proteins were also found to have the ability to regulate gene expression via this element. *The CCAAT box, which has been previously hypothesized as a recruitment site for complexes containing the CONSTANS protein, potentiated CONSTANS-mediated activation but was not essential for CONSTANS recruitment to a target promoter or for its activity as a transcriptional factor.

The Arabidopsis transcription factor MYB77 modulates auxin signal transduction.

Auxin is a key plant hormone that regulates plant development, apical dominance, and growth-related tropisms, such as phototropism and gravitropism. In this study, we report a new Arabidopsis thaliana transcription factor, MYB77, that is involved in auxin response. In MYB77 knockout plants, we found that auxin-responsive gene expression was greatly attenuated. Lateral root density in the MYB77 knockout was lower than the wild type at low concentrations of indole-3-acetic acid (IAA) and also under low nutrient conditions. MYB77 interacts with auxin response factors (ARFs) in vitro through the C terminus (domains III and IV) of ARFs and the activation domain of MYB77. A synergistic genetic interaction was demonstrated between MYB77 and ARF7 that resulted in a strong reduction in lateral root numbers. Experiments with protoplasts confirmed that the coexpression of MYB77 and an ARF C terminus enhance reporter gene expression. R2R3 MYB transcription factors have not been previously implicated in regulating the expression of auxin-inducible genes. Also it was previously unknown that ARFs interact with proteins other than those in the Aux/IAA family via conserved domains. The interaction between MYB77 and ARFs defines a new type of combinatorial transcriptional control in plants. This newly defined transcription factor interaction is part of the plant cells' repertoire for modulating response to auxin, thereby controlling lateral root growth and development under changing environmental conditions.

NPH4/ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root formation.

Auxin response factors (ARFs) bind auxin response promoter elements and mediate transcriptional responses to auxin. Five of the 22 ARF genes in Arabidopsis thaliana encode ARFs with glutamine-rich middle domains. Four of these can activate transcription and have been ascribed developmental functions. We show that ARF19, the fifth Q-rich ARF, also activates transcription. Mutations in ARF19 have little effect on their own, but in combination with mutations in NPH4/ARF7, encoding the most closely related ARF, they cause several phenotypes including a drastic decrease in lateral and adventitious root formation and a decrease in leaf cell expansion. These results indicate that auxin induces lateral roots and leaf expansion by activating NPH4/ARF7 and ARF19. Auxin induces the ARF19 gene, and NPH4/ARF7 and ARF19 together are required for expression of one of the arf19 mutant alleles, suggesting that a positive feedback loop regulates leaf expansion and/or lateral root induction.

AUXIN RESPONSE FACTOR7 restores the expression of auxin-responsive genes in mutant Arabidopsis leaf mesophyll protoplasts.

AUXIN RESPONSE FACTOR7 (ARF7) is one of five ARF transcriptional activators in Arabidopsis thaliana that is proposed to regulate auxin-responsive expression of genes containing TGTCTC auxin response elements in their promoters. An Arabidopsis mutant (nonphototropic hypocotyl4-1 [nph4-1]) that is a null for ARF7 showed strongly reduced expression of integrated auxin-responsive reporter genes and natural genes that were monitored in Arabidopsis leaf mesophyll protoplasts. Expression of the reporter and natural genes was restored in an auxin-dependent manner when protoplasts were transfected with a 35S:ARF7 effector gene, encoding a full-length ARF7 protein. Transfection of effector genes encoding other ARF activators restored auxin-responsive gene expression to varying degrees, but less than that observed with the ARF7 effector gene. Arabidopsis lines that were null for ARF6, ARF8, or ARF19 were not defective in expression of the reporter and natural auxin response genes assayed in mesophyll protoplasts, suggesting that ARF7 plays a major role in regulating expression of a subset of auxin response genes in leaf mesophyll cells. Auxin-responsive gene expression was induced in wild-type protoplasts and restored in nph4-1 protoplasts only with auxin and not with other hormones, including brassinolide. In the presence of auxin, however, brassinolide modestly enhanced auxin-responsive gene expression.

Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4.

Transcription factors of the auxin response factor (ARF) family have been implicated in auxin-dependent gene regulation, but little is known about the functions of individual ARFs in plants. Here, interaction assays, expression studies and combinations of multiple loss- and gain-of-function mutants were used to assess the roles of two ARFs, NONPHOTOTROPIC HYPOCOTYL 4 (NPH4/ARF7) and MONOPTEROS (MP/ARF5), in Arabidopsis development. Both MP and NPH4 interact strongly and selectively with themselves and with each other, and are expressed in vastly overlapping domains. We show that the regulatory properties of both genes are far more related than suggested by their single mutant phenotypes. NPH4 and MP are capable of controlling both axis formation in the embryo and auxin-dependent cell expansion. Interaction of MP and NPH4 in Arabidopsis plants is indicated by their joint requirement in a number of auxin responses and by synergistic effects associated with the co-overexpression of both genes. Finally, we demonstrate antagonistic interaction between ARF and Aux/IAA gene functions in Arabidopsis development. Overexpression of MP suppresses numerous defects associated with a gain-of-function mutation in BODENLOS (BDL)/IAA12. Together these results provide evidence for the biological relevance of ARF-ARF and ARF-Aux/IAA interaction in Arabidopsis plants and demonstrate that an individual ARF can act in both invariantly programmed pattern formation as well as in conditional responses to external signals.

Aux/IAA proteins contain a potent transcriptional repression domain.

Aux/IAA proteins are short-lived nuclear proteins that repress expression of primary/early auxin response genes in protoplast transfection assays. Repression is thought to result from Aux/IAA proteins dimerizing with auxin response factor (ARF) transcriptional activators that reside on auxin-responsive promoter elements, referred to as AuxREs. Most Aux/IAA proteins contain four conserved domains, designated domains I, II, III, and IV. Domain II and domains III and IV play roles in protein stability and dimerization, respectively. A clear function for domain I had not been established. Results reported here indicate that domain I in Aux/IAA proteins is an active repression domain that is transferable and dominant over activation domains. An LxLxL motif within domain I is important for conferring repression. The dominance of Aux/IAA repression domains over activation domains in ARF transcriptional activators provides a plausible explanation for the repression of auxin response genes via ARF-Aux/IAA dimerization on auxin-responsive promoters.

The roles of auxin response factor domains in auxin-responsive transcription.

Auxin response factors (ARFs) are transcription factors that bind to TGTCTC auxin response elements in promoters of early auxin response genes. ARFs have a conserved N-terminal DNA binding domain (DBD) and in most cases a conserved C-terminal dimerization domain (CTD). The ARF CTD is related in amino acid sequence to motifs III and IV found in Aux/IAA proteins. Just C terminal to the DBD, ARFs contain a nonconserved region referred to as the middle region (MR), which has been proposed to function as a transcriptional repression or activation domain. Results with transfected protoplasts reported here show that ARFs with Q-rich MRs function as activators, whereas most, if not all other ARFs, function as repressors. ARF DBDs alone are sufficient to recruit ARFs to their DNA target sites, and auxin does not influence this recruitment. ARF MRs alone function as activation or repression domains when targeted to reporter genes via a yeast Gal4 DBD, and auxin does not influence the potency of activation or repression. ARF CTDs, along with a Q-rich MR, are required for an auxin response whether the MRs plus CTDs are recruited to a promoter by an ARF DBD or by a Gal4 DBD. The auxin response is mediated by the recruitment of Aux/IAA proteins to promoters that contain a DNA binding protein with a Q-rich MR and an attached CTD.

A Ca2+/CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter.

An immuno-homologue of maize Ca2+/calmodulin (CaM)-dependent protein kinase with a molecular mass of 72 kDa was identified in pea. The pea kinase (PsCCaMK) was upregulated in roots in response to low temperature and increased salinity. Exogenous Ca2+ application increased the kinase level and the response was faster than that obtained following stress application. Low temperature-mediated, but not salinity-mediated stress kinase increase was inhibited by the application of EGTA and W7, a CaM inhibitor. The purification of PsCCaMK using immuno-affinity chromatography resulted in coelution of the kinase with another polypeptide of molecular mass 40 kDa (p40). Western blot revealed the presence of PsCCaMK in nuclear protein extracts and was found to phosphorylate p40 in vitro. Gel mobility shift and South-Western analysis showed that p40 is a DNA-binding protein and it interacted specifically with one of the cis acting elements of the Arabidopsis CaM5 gene (AtCaM5) promoter. The binding of p40 to the specific elements in the AtCaM5 promoter was dependent of its dephosphorylated state. Our results suggest that p40 could be an upstream signal component of the stress responses.