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The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.

Auxin response factors (ARFs) are a family of transcription factors that are responsible for regulating gene expression in response to changes in auxin level. The analysis of ARF sequence and activity indicates that there are 2 major groups: activators and repressors. One clade of ARFs, clade-D, is sister to clade-A activating ARFs, but are unique in that they lack a DNA-binding domain. Clade-D ARFs are present in lycophytes and bryophytes but absent in other plant lineages. The transcriptional activity of clade-D ARFs, as well as how they regulate gene expression, is not well understood. Here, we report that clade-D ARFs are transcriptional activators in the model bryophyte Physcomitrium patens and have a major role in the development of this species. Δarfd dub protonemata exhibit a delay in filament branching, as well as a delay in the chloronema to caulonema transition. Additionally, leafy gametophore development in Δarfd dub lines lags behind wild type. We present evidence that ARFd1 interacts with activating ARFs via their PB1 domains, but not with repressing ARFs. Based on these results, we propose a model in which clade-D ARFs enhance gene expression by interacting with DNA bound clade-A ARFs. Further, we show that ARFd1 must form oligomers for full activity.

The coordinated action of the auxin-sensitive Aux/IAA transcriptional repressors and ARF transcription factors produces complex gene-regulatory networks in plants. Despite their importance, our knowledge of these two protein families is largely based on analysis of stabilized forms of the Aux/IAAs, and studies of a subgroup of ARFs that function as transcriptional activators. To understand how auxin regulates gene expression we generated a Physcomitrella patens line that completely lacks Aux/IAAs. Loss of the repressors causes massive changes in transcription with misregulation of over a third of the annotated genes. Further, we find that the aux/iaa mutant is blind to auxin indicating that auxin regulation of transcription occurs exclusively through Aux/IAA function. We used the aux/iaa mutant as a simplified platform for studies of ARF function and demonstrate that repressing ARFs regulate auxin-induced genes and fine-tune their expression. Further the repressing ARFs coordinate gene induction jointly with activating ARFs and the Aux/IAAs. Auxin is a plant hormone that regulates many aspects of growth and development. It does so by promoting the degradation of proteins called the Aux/IAAs. These proteins normally act to keep genes switched off by interacting with another family of proteins called ARFs that can bind directly to the genes. Some ARFs activate genes, while others repress gene activity. In most plants, large families of genes encode Aux/IAA and ARF proteins and individual members often have overlapping roles, which makes it more difficult to study how they work. To avoid this problem, Lavy et al. chose to study how these proteins work in a moss called Physcomitrella patens, which only has three genes that encode Aux/IAA proteins. The experiments show that the loss of all three Aux/IAA proteins results in the plants becoming completely insensitive to auxin. Furthermore, over a third of known moss genes had altered activity in the mutant plants compared to normal moss. These findings suggest that the Aux/IAA proteins have a bigger role in regulating the activities of genes than previously thought. Further experiments show that repressive ARFs act by directly competing with the activating ARFs for binding to auxin-regulated genes. However, repression of gene activity by the ARFs is weaker than the effect of the Aux/IAAs. Unexpectedly, the loss of repressive ARFs causes moss plants to become less sensitive to auxin, which Lavy et al. suggest is due to the recruitment of additional Aux/IAA proteins to the genes. Thus these findings demonstrate that control of gene activity by auxin involves the coordinated action of both types of ARFs and the Aux/IAAs. Future challenges are to find out which genes directly bind ARFs and the Aux/IAAs, and to use computational approaches to create models of how auxin regulates gene activity in the moss.

The Arabidopsis thaliana genome contains five class III homeodomain-leucine zipper genes. We have isolated loss-of-function alleles for each family member for use in genetic analysis. This gene family regulates apical embryo patterning, embryonic shoot meristem formation, organ polarity, vascular development, and meristem function. Genetic analyses revealed a complex pattern of overlapping functions, some of which are not readily inferred by phylogenetic relationships or by gene expression patterns. The PHABULOSA and PHAVOLUTA genes perform overlapping functions with REVOLUTA, whereas the PHABULOSA, PHAVOLUTA, and CORONA/ATHB15 genes perform overlapping functions distinct from REVOLUTA. Furthermore, ATHB8 and CORONA encode functions that are both antagonistic to those of REVOLUTA within certain tissues and overlapping with REVOLUTA in other tissues. Differences in expression patterns explain some of these genetic interactions, whereas other interactions are likely attributable to differences in protein function as indicated by cross-complementation studies.

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

The plant hormone auxin regulates a wide variety of transcriptional responses depending on the cell type, environment and species. How this diversity is achieved may be related to the specific complement of auxin-signalling components in each cell. The levels of activators (class-A AUXIN RESPONSE FACTORS) and repressors (class-B ARFs) are particularly important. Tight regulation of ARF protein levels is probably key in determining this balance. Through comparative analysis of novel, dominant mutants in maize and the moss Physcomitrium patens , we have discovered a ~500-million-year-old mechanism of class-B ARF protein-level regulation mediated by proteasome degradation, important in determining cell fate decisions across land plants. Thus, our results add a key piece to the puzzle of how auxin regulates plant development. Comparative analysis of maize and moss reveals a 500-million-year-old piece of the auxin-signalling puzzle that controls the protein level of ARFs, key transcriptional regulators.

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner.

Organogenesis at the shoot meristem requires a delicate balance between stem cell specification and differentiation. In Arabidopsis thaliana, WUSCHEL (WUS) is a key factor promoting stem cell identity, whereas the CLAVATA (CLV1, CLV2, and CLV3) loci appear to promote differentiation by repressing WUS expression. In a screen for mutations modifying clv1 mutants, we have identified a novel regulator of meristem development we term CORONA (CNA). Whereas cna single mutant plants exhibit subtle defects in meristem development, clv cna double mutants develop massively enlarged apices that display early loss of organogenesis, misexpression of WUS and CLV3, and eventual differentiation of the entire apex. The CNA gene was isolated by positional cloning and found to encode a class III homeodomain Leu zipper protein. A missense mutation resulting in the dominant-negative cna-1 allele was identified in a conserved domain of unknown function, and a likely null allele was shown to display a similar but weaker phenotype. CNA is expressed in developing vascular tissue, diffusely through shoot and flower meristems, and within developing stamens and carpels. Our analysis of WUS expression in wild-type, clv, and clv cna plants revealed that, contrary to current models, WUS is neither necessary nor sufficient for stem cell specification and that neither WUS nor CLV3 is a marker for stem cell identity. We propose that CNA functions in parallel to the CLV loci to promote organ formation.

Plant genomes encode large numbers of F-box proteins (FBPs), the substrate recognition subunit of SKP1–CULLIN–F-box (SCF) ubiquitin ligases. There are ∼700 FBPs in Arabidopsis , most of which are uncharacterized. TIR1 is among the best-studied plant FBPs and functions as a receptor for the plant hormone auxin. Here we use a yeast two-hybrid system to identify novel TIR1 mutants with altered properties. The analysis of these mutants reveals that TIR1 associates with the CULLIN1 (CUL1) subunit of the SCF through the N-terminal H1 helix of the F-box domain. Mutations that untether TIR1 from CUL1 stabilize the FBP and cause auxin resistance and associated growth defects, probably by protecting TIR1 substrates from degradation. Based on these results we propose that TIR1 is subject to autocatalytic degradation when assembled into an SCF. Further, our results suggest a general method for determining the physiological function of uncharacterized FBPs. Finally, we show that a key amino acid variation in the F-box domain of auxin signalling F-box (AFB1), a closely related FBP, reduces its ability to form an SCF, resulting in an increase in AFB1 levels. The plant auxin receptor TIR1 needs to associate with the SCF complex to be functional and target substrates. Newly discovered mutations that can block this interaction suggest that TIR1 is autocatalytically degraded once assembled in the SCF complex.