Clade-D auxin response factors regulate auxin signaling and development in the moss Physcomitrium patens.

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. Δarfddub protonemata exhibit a delay in filament branching, as well as a delay in the chloronema to caulonema transition. Additionally, leafy gametophore development in Δarfddub 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.

Dual Role of Auxin in Regulating Plant Defense and Bacterial Virulence Gene Expression During Pseudomonas syringae PtoDC3000 Pathogenesis.

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen strain Pseudomonas syringae DC3000 (PtoDC3000) produces the plant hormone auxin (indole-3-acetic acid [IAA]) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth and that this phenotype was suppressed by introducing the sid2-2 mutation, which impairs SA synthesis. Thus, host auxin signaling is required for normal susceptibility to PtoDC3000 and is involved in suppressing SA-mediated defenses. Unexpectedly, tir1 afb1 afb4 afb5 quadruple-mutant plants lacking four of the six known auxin coreceptors that exhibit decreased auxin perception, supported increased levels of bacterial growth. This mutant exhibited elevated IAA levels and reduced SA-mediated defenses, providing additional evidence that auxin promotes disease by suppressing host defense. We also investigated the hypothesis that IAA promotes PtoDC3000 virulence through a direct effect on the pathogen and found that IAA modulates expression of virulence genes, both in culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop.

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.

The cyclophilin DIAGEOTROPICA has a conserved role in auxin signaling.

Auxin has a fundamental role throughout the life cycle of land plants. Previous studies showed that the tomato cyclophilin DIAGEOTROPICA (DGT) promotes auxin response, but its specific role in auxin signaling remains unknown. We sequenced candidate genes in auxin-insensitive mutants of Physcomitrella patens and identified mutations in highly conserved regions of the moss ortholog of tomato DGT. As P. patens and tomato diverged from a common ancestor more than 500 million years ago, this result suggests a conserved and central role for DGT in auxin signaling in land plants. In this study we characterize the P. patens dgt (Ppdgt) mutants and show that their response to auxin is altered, affecting the chloronema-to-caulonema transition and the development of rhizoids. To gain an understanding of PpDGT function we tested its interactions with the TIR1/AFB-dependent auxin signaling pathway. We did not observe a clear effect of the Ppdgt mutation on the degradation of Aux/IAA proteins. However, the induction of several auxin-regulated genes was reduced. Genetic analysis revealed that dgt can suppress the phenotype conferred by overexpression of an AFB auxin receptor. Our results indicate that the DGT protein affects auxin-induced transcription and has a conserved function in auxin regulation in land plants.

Mutations in the TIR1 auxin receptor that increase affinity for auxin/indole-3-acetic acid proteins result in auxin hypersensitivity.

The phytohormone auxin regulates virtually every aspect of plant development. The hormone directly mediates the interaction between the two members of the auxin coreceptor complex, a TRANSPORT INHIBITOR RESPONSE (TIR1)/AUXIN SIGNALING F-BOX protein and an AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressor. To learn more about the interaction between these proteins, a mutant screen was performed using the yeast (Saccharomyces cerevisiae) two-hybrid system in Arabidopsis (Arabidopsis thaliana). Two tir1 mutations were identified that increased interaction with Aux/IAAs. The D170E and M473L mutations increase affinity between TIR1 and the degron motif of Aux/IAAs and enhance the activity of the SCF(TIR1) complex. This resulted in faster degradation of Aux/IAAs and increased transcription of auxin-responsive genes in the plant. Plants carrying the pTIR1:tir1 D170E/M473L-Myc transgene exhibit diverse developmental defects during plant growth and display an auxin-hypersensitive phenotype. This work demonstrates that changes in the leucine-rich repeat domain of the TIR1 auxin coreceptor can alter the properties of SCF(TIR1).

Near telomere-to-telomere genome of the model plant Physcomitrium patens.

The model plant Physcomitrium patens has played a pivotal role in enhancing our comprehension of plant evolution and development. However, the current genome harbours numerous regions that remain unfinished and erroneous. To address these issues, we generated an assembly using Oxford Nanopore reads and Hi-C mapping. The assembly incorporates telomeric and centromeric regions, thereby establishing it as a near telomere-to-telomere genome except a region in chromosome 1 that is not fully assembled due to its highly repetitive nature. This near telomere-to-telomere genome resolves the chromosome number at 26 and provides a gap-free genome assembly as well as updated gene models to aid future studies using this model organism.

Hierarchical global and local auxin signals coordinate cellular interdigitation in Arabidopsis.

The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in tir1;afb1;afb2;afb4;afb5 mutant but not in tmk1;tmk2;tmk3;tmk4 mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.

Evolutionary crossroads in developmental biology: Physcomitrella patens.

The moss Physcomitrella patens has recently emerged as a powerful genetically tractable model plant system. As a member of the bryophytes, P. patens provides a unique opportunity to study the evolution of a myriad of plant traits, such as polarized cell growth, gametophyte-to-sporophyte transitions, and sperm-to-pollen transition. The availability of a complete genome sequence, together with the ability to perform gene targeting efficiently in P. patens has spurred a flurry of elegant reverse genetic studies in this plant model that address a variety of key questions in plant developmental biology.

The AFB1 auxin receptor controls the cytoplasmic auxin response pathway in Arabidopsis thaliana.

The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFB auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, is essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition by auxin. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx, which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response, whereas it regulates rapid changes in cell growth that contribute to root gravitropism.

The AFB1 auxin receptor controls the cytoplasmic auxin response pathway in Arabidopsis thaliana.

The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFBs auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, are essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response while it regulates rapid changes in cell growth that contribute to root gravitropism.

Comparative mutant analyses reveal a novel mechanism of ARF regulation in land plants.

A major challenge in plant biology is to understand how the plant hormone auxin regulates diverse transcriptional responses throughout development, in different environments, and in different species. The answer may lie in the specific complement of auxin signaling components in each cell. The balance between activators (class-A AUXIN RESPONSE FACTORS) and repressors (class-B ARFs) is particularly important. It is unclear how this balance is achieved. 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, 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.

Mutations in the Physcomitrium patens gene encoding Aminodeoxychorismate Synthase confer auxotrophic phenotypes.

To facilitate genetic mapping of developmental mutants of Physcomitrium patens, we produced a genetic marker that combines recessive auxotrophy with dominant positive selection. We first identified the gene affected by the pabB4 auxotrophic mutation and then replaced it with a cassette that confers antibiotic resistance. This strain may be used to produce bi-parental somatic hybrids with nearly any other strain.

Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions.

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.

Two cap-binding proteins CBP20 and CBP80 are involved in processing primary MicroRNAs.

MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in plants by mediating translational inhibition or cleavage of target transcripts. Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly pleiotropic phenotypes including serrated leaves and ABA hypersensitivity. Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II). Since Pol II transcripts are capped, we hypothesized that CBP (cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA) transcripts and play a role in their processing. Here, we show that cbp20 and cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels. Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and 172 could be associated with CBP20 and CBP80. We found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination. The lack of miR159 accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33 and MYB101 transcript levels, and presumably higher levels of these positive regulators result in ABA hypersensitivity. Genetic and molecular analyses show that CBP20 and 80 have overlapping function in the same developmental pathway as SE and HYL1. Our results identify new components in miRNA biogenesis.

Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins.

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.

The Arabidopsis Auxin Receptor F-Box Proteins AFB4 and AFB5 Are Required for Response to the Synthetic Auxin Picloram.

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.

Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response.

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.

Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway.

Auxin regulates most aspects of flowering-plant growth and development, including key developmental innovations that evolved within the vascular plant lineage after diverging from a bryophyte-like ancestor nearly 500 million years ago. Recent studies in Arabidopsis indicate that auxin acts by directly binding the TIR1 subunit of the SCF(TIR1) ubiquitin ligase; this binding results in degradation of the Aux/IAA transcriptional repressors and de-repression of auxin-responsive genes. Little is known, however, about the mechanism of auxin action in other plants. To characterize auxin signaling in a nonflowering plant, we utilized the genetically tractable moss Physcomitrella patens. We used a candidate-gene approach to show that previously identified auxin-resistant mutants of P. patens harbor mutations in Aux/IAA genes. Furthermore, we show that the moss Aux/IAA proteins interact with Arabidopsis TIR1 moss homologs called PpAFB and that a reduction in PpAFB levels results in a phenotype similar to that of the auxin-resistant mutants. Our results indicate that the molecular mechanism of auxin perception is conserved in land plants despite vast differences in the role auxin plays in different plant lineages.

The TRANSPORT INHIBITOR RESPONSE2 gene is required for auxin synthesis and diverse aspects of plant development.

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 Physcomitrella genome reveals evolutionary insights into the conquest of land by plants.

We report the draft genome sequence of the model moss Physcomitrella patens and compare its features with those of flowering plants, from which it is separated by more than 400 million years, and unicellular aquatic algae. This comparison reveals genomic changes concomitant with the evolutionary movement to land, including a general increase in gene family complexity; loss of genes associated with aquatic environments (e.g., flagellar arms); acquisition of genes for tolerating terrestrial stresses (e.g., variation in temperature and water availability); and the development of the auxin and abscisic acid signaling pathways for coordinating multicellular growth and dehydration response. The Physcomitrella genome provides a resource for phylogenetic inferences about gene function and for experimental analysis of plant processes through this plant's unique facility for reverse genetics.