The PB1 domain in auxin response factor and Aux/IAA proteins: a versatile protein interaction module in the auxin response.

An integral part of auxin-regulated gene expression involves the interplay of two types of transcription factors, the DNA binding auxin response factor (ARF) activators and the interacting auxin/indole acetic acid (Aux/IAA) repressors. Insight into the mechanism of how these transcription factors interact with one another has recently been revealed from crystallographic information on ARF5 and ARF7 C-terminal domains (i.e., a protein-protein interaction domain referred to as domain III/IV that is related to domain III/IV in Aux/IAA proteins). Three-dimensional structures showed that this domain in ARF5 and ARF7 conforms to a well-known PB1 (Phox and Bem1) domain that confers protein-protein interactions with other PB1 domain proteins through electrostatic contacts. Experiments verifying the importance of charged amino acids in conferring ARF and Aux/IAA interactions have confirmed the PB1 domain structure. Some in planta experiments designed to test the validity of PB1 interactions in the auxin response have led to updated models for auxin-regulated gene expression and raised many questions that will require further investigation. In addition to the PB1 domain, a second protein interaction module that functions in ARF-ARF dimerization and facilitates DNA binding has recently been revealed from crystallography studies on the ARF1 and ARF5 DNA binding domains.

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.

Auxin-related gene families in abiotic stress response in Sorghum bicolor.

Sorghum, a C4 model plant, has been studied to develop an understanding of the molecular mechanism of resistance to stress. The auxin-response genes, auxin/indole-3-acetic acid (Aux/IAA), auxin-response factor (ARF), Gretchen Hagen3 (GH3), small auxin-up RNAs, and lateral organ boundaries (LBD), are involved in growth/development and stress/defense responses in Arabidopsis and rice, but they have not been studied in sorghum. In the present paper, the chromosome distribution, gene duplication, promoters, intron/exon, and phylogenic relationships of Aux/IAA, ARF, GH3, and LBD genes in sorghum are presented. Furthermore, real-time PCR analysis demonstrated these genes are differently expressed in leaf/root of sorghum and indicated the expression profile of these gene families under IAA, brassinosteroid (BR), salt, and drought treatments. The SbGH3 and SbLBD genes, expressed in low level under natural condition, were highly induced by salt and drought stress consistent with their products being involved in both abiotic stresses. Three genes, SbIAA1, SbGH3-13, and SbLBD32, were highly induced under all the four treatments, IAA, BR, salt, and drought. The analysis provided new evidence for role of auxin in stress response, implied there are cross talk between auxin, BR and abiotic stress signaling pathways.

Functional analysis of the structural domain of ARF proteins in rice (Oryza sativa L.).

Auxin response factors (ARFs) are key regulators of plant growth and development. Through interaction with auxin/indole acetic acid (Aux/IAA) proteins, they influence the expression of auxin response genes. An ARF gene family has been predicted in rice, but the functions of the individual structural domains of the OsARFs remain obscure. Bioinformatics was used to analyse the position of the DNA-binding domain (DBD), middle region (MR), and C-terminal dimerization domain (CTD) of OsARFs, and experimentally confirmed the presence of a classical monopartite nuclear localization signal (NLS) in the DBD. The DBD was shown to contribute to nuclear localization of OsARF proteins in addition to its known DNA-binding function. Interactions between 14 integrated OsARFs and 15 OsIAA proteins were tested using yeast two-hybrid assays. It was found that eight OsARF activators interacted with the 15 OsIAA proteins, while six OsARF repressors did not. The interactions between the MR+CTD or CTD of 10 OsARFs and 15 OsIAA proteins were also tested and the results were consistent with those of each intact OsARF, although some slight differences in interaction intensity were observed by α-galactosidase quantitative assays. The truncated CTD of OsARF11 did not interact with any OsIAA, implying that the CTD is required for ARF-IAA dimerization, and that the MR influences the interaction intensity in yeast. A subset of the interactions in yeast were also observed in tobacco plants using firefly luciferase complementation imaging assays, indicating that these interactions are specific in plants, and might have a special role in the auxin signalling response. This study provides new insight into the structure of OsARF proteins and ARF-Aux/IAA interactions.

Auxin response factors.

Auxin signaling is key to many plant growth and developmental processes from embryogenesis to senescence. Most, if not all, of these processes are initiated and/or mediated through auxin-regulated gene expression. Two types of transcription factor families are required for controlling expression of auxin response genes. One of these, the auxin response factor (ARF) family, functions by binding to auxin response elements (AuxREs) on promoters of auxin response genes, activating or repressing the auxin response genes, and recruiting a second family of transcription factors, the Aux/IAA repressors, that confer an auxin response to the genes. Recent advances have provided information on regulation of ARF gene expression, ARF roles in growth and developmental processes, and target genes regulated by ARFs.

Transfection assays with protoplasts containing integrated reporter genes.

Transient expression assays with protoplasts that utilize stably integrated reporter genes along with transfected effector genes provide several advantages over assays in which both the reporter gene and effector gene(s) are transfected into protoplasts. A protocol for carrying out transient expression assays with Arabidopsis leaf mesophyll protoplasts containing single-copy integrated reporter genes is described.

ARF-Aux/IAA interactions through domain III/IV are not strictly required for auxin-responsive gene expression.

Auxin response factors (ARFs), together with auxin/indole acetic acid proteins (Aux/IAAs), are transcription factors that play key roles in regulating auxin-responsive transcription in plants. Current models for auxin signaling predict that auxin response is dependent on ARF-Aux/IAA interactions mediated by the related protein-protein interaction domain (i.e., referred to as the CTD) found in the ARF and Aux/IAA C-terminal regions. When auxin concentrations in a cell are low, ARF activators residing on the promoters of auxin response genes are thought to be inactive because of the association with dominant Aux/IAA repressors. When auxin concentrations are elevated, the Aux/IAA repressors are recruited to auxin receptors and degraded via the ubiquitin-proteasome pathway. Destruction of the Aux/IAA repressors allows the ARF activators to function in derepressing/activating auxin response genes. While this auxin signaling pathway is simple and attractive, it is unclear whether auxin-regulated gene expression is solely dependent on ARF-Aux/IAA interactions. Here we show that auxin can affect the expression of auxin response genes in a manner that is independent of the ARF activator CTD.

Getting a grasp on domain III/IV responsible for Auxin Response Factor-IAA protein interactions.

Auxin Response Factors (ARFs) and Indole Acetic Acid (IAA) proteins contain a similar carboxyl-terminal domain (domain III/IV) that facilitates interactions among these transcription factors as well as other proteins. The specificity of these interactions is controversial, and the mechanisms involved in these interactions have not been investigated. Here, we review some of the controversies about the specificities and requirements for ARF and IAA interactions and discuss some of the technical problems that might contribute to differences reported for these interactions. We make some preliminary conclusions that ARF activator-IAA, ARF activator-ARF activator, and ARF repressor-ARF repressor interactions are favored over ARF repressor-IAA and ARF repressor-ARF activator interactions, and we suggest that IAA-IAA interactions are largely indiscriminant. Based upon the predicted secondary structure of domain III/IV, we introduce a model for how ARF and IAA proteins might interact with one another through a ubiquitin-like ß-grasp fold.

Do some IAA proteins have two repression domains?

The plant hormone auxin regulates the transcription of specific genes through the interplay of Auxin Response Factors (ARFs) and Aux/IAA (IAA) repressors. We have recently shown that stabilized IAA repressors with identical amino acid substitutions in their conserved repression domains (i.e., domain I) confer either "low auxin" or "high auxin" phenotypes when the IAA proteins are constitutively expressed in transformed Arabidopsis plants. We have suggested that when domain I loses its capacity to repress, "high auxin" phenotypes generally result, but a subset of IAA proteins (e.g., IAA17) appear to contain a second repression domain resulting in the maintenance of "low auxin" phenotypes. Here we provide evidence for a second repression domain that lies between domains I and II in IAA7, an IAA repressor within the same clade as IAA17.

Constitutive repression and activation of auxin signaling in Arabidopsis.

Aux/IAA proteins are proposed to be transcriptional repressors that play a crucial role in auxin signaling by interacting with auxin response factors and repressing early/primary auxin response gene expression. In assays with transfected protoplasts, this repression was previously shown to occur when auxin concentrations in a cell are low, and derepression/activation was observed when auxin concentrations are elevated. Here we show that a stabilized version of the Arabidopsis (Arabidopsis thaliana) IAA17 repressor, when expressed constitutively or in a specific cell type in Arabidopsis plants, confers phenotypes similar to plants with decreased auxin levels. In contrast, a stabilized version of IAA17 that was converted to a transcriptional activator confers phenotypes similar to plants with increased auxin levels, when expressed under the same conditions in Arabidopsis plants. Free auxin levels were unchanged compared to control (DR5:beta-glucuronidase), however, in the seedlings expressing the IAA17 repressor and activator. These results together with our previous results carried out in transfected protoplasts suggest that the hormone auxin can be bypassed to regulate auxin signaling in a cell-autonomous manner in plants.

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.

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.

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.

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.