No abstract

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

Summary• 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 CCTcontaining 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 CONSTANSmediated activation but was not essential for CONSTANS recruitment to a target promoter or for its activity as a transcriptional factor.

Aux/IAA genes are early auxin response genes that encode short-lived nuclear proteins with four conserved domains, referred to as I, II, III, and IV. Arabidopsis Aux/IAA proteins repressed transcription on auxin-responsive reporter genes in protoplast transfection assays. Mutations in domain II resulted in increased repression, whereas mutations in domains I and III partially relieved repression. Aux/IAA proteins fused to a heterologous DNA binding domain were targeted to promoters of constitutively expressed reporter genes and actively repressed transcription in an auxinresponsive and dose-dependent manner. In comparison with an unfused luciferase protein, luciferase fused to Aux/IAA proteins displayed less luciferase activity, which further decreased in the presence of auxin in transfected protoplasts. Domain II mutations increased and domain I mutations decreased luciferase activity with the fusion proteins. These results suggested that Aux/IAA proteins function as active repressors by dimerizing with auxin response factors bound to auxin response elements and that early auxin response genes are regulated by auxin-modulated stabilities of Aux/ IAA proteins.

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.

SummaryAuxin 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.

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/IAAgene 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.

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.