RAF-like protein kinases mediate a deeply conserved, rapid auxin response.

The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors.

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

ABP1-TMK auxin perception for global phosphorylation and auxin canalization.

The phytohormone auxin triggers transcriptional reprogramming through a well-characterized perception machinery in the nucleus. By contrast, mechanisms that underlie fast effects of auxin, such as the regulation of ion fluxes, rapid phosphorylation of proteins or auxin feedback on its transport, remain unclear1-3. Whether auxin-binding protein 1 (ABP1) is an auxin receptor has been a source of debate for decades1,4. Here we show that a fraction of Arabidopsis thaliana ABP1 is secreted and binds auxin specifically at an acidic pH that is typical of the apoplast. ABP1 and its plasma-membrane-localized partner, transmembrane kinase 1 (TMK1), are required for the auxin-induced ultrafast global phospho-response and for downstream processes that include the activation of H+-ATPase and accelerated cytoplasmic streaming. abp1 and tmk mutants cannot establish auxin-transporting channels and show defective auxin-induced vasculature formation and regeneration. An ABP1(M2X) variant that lacks the capacity to bind auxin is unable to complement these defects in abp1 mutants. These data indicate that ABP1 is the auxin receptor for TMK1-based cell-surface signalling, which mediates the global phospho-response and auxin canalization.

Highly Specific Protein Identification by Immunoprecipitation-Mass Spectrometry Using Antifouling Microbeads.

A common method to study protein complexes is immunoprecipitation (IP), followed by mass spectrometry (thus labeled: IP-MS). IP-MS has been shown to be a powerful tool to identify protein-protein interactions. It is, however, often challenging to discriminate true protein interactors from contaminating ones. Here, we describe the preparation of antifouling azide-functionalized polymer-coated beads that can be equipped with an antibody of choice via click chemistry. We show the preparation of generic immunoprecipitation beads that target the green fluorescent protein (GFP) and show how they can be used in IP-MS experiments targeting two different GFP-fusion proteins. Our antifouling beads were able to efficiently identify relevant protein-protein interactions but with a strong reduction in unwanted nonspecific protein binding compared to commercial anti-GFP beads.

Cell surface and intracellular auxin signalling for H+ fluxes in root growth.

Growth regulation tailors development in plants to their environment. A prominent example of this is the response to gravity, in which shoots bend up and roots bend down1. This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots while inhibiting it in roots via a yet unknown cellular mechanism2. Here, by combining microfluidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance understanding of how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on rapid regulation of apoplastic pH, a causative determinant of growth. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H+ influx, causing apoplast alkalinization. Simultaneous activation of these two counteracting mechanisms poises roots for rapid, fine-tuned growth modulation in navigating complex soil environments.

Specification and regulation of vascular tissue identity in the Arabidopsis embryo.

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.

Auxin Response Factors: output control in auxin biology.

The phytohormone auxin is involved in almost all developmental processes in land plants. Most, if not all, of these processes are mediated by changes in gene expression. Auxin acts on gene expression through a short nuclear pathway that converges upon the activation of a family of DNA-binding transcription factors. These AUXIN RESPONSE FACTORS (ARFs) are thus the effector of auxin response and translate the chemical signal into the regulation of a defined set of genes. Given the limited number of dedicated components in auxin signaling, distinct properties among the ARF family probably contribute to the establishment of multiple unique auxin responses in plant development. In the two decades following the identification of the first ARF in Arabidopsis, much has been learnt about how these transcription factors act, and how they generate unique auxin responses. Progress in genetics, biochemistry, genomics, and structural biology has helped to develop mechanistic models for ARF action. However, despite intensive efforts, many central questions are yet to be addressed. In this review, we highlight what has been learnt about ARF transcription factors, and identify outstanding questions and challenges for the near future.

Click-MS: Tagless Protein Enrichment Using Bioorthogonal Chemistry for Quantitative Proteomics.

Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts. By genetic encoding of p-azido-l-phenylalanine, the protein of interest can be selectively captured using copper-free click chemistry. We use Click-MS to enrich proteins that function in different cellular compartments, and we identify protein-protein interactions, showing the great potential of Click-MS for interaction proteomics workflows.

An in vivo model for analysis of developmental erythropoiesis and globin gene regulation.

Expression of fetal γ-globin in adulthood ameliorates symptoms of ß-hemoglobinopathies by compensating for the mutant ß-globin. Reactivation of the silenced γ-globin gene is therefore of substantial clinical interest. To study the regulation of γ-globin expression, we created the GG mice, which carry an intact 183-kb human ß-globin locus modified to express enhanced green fluorescent protein (eGFP) from the Gγ-globin promoter. GG embryos express eGFP first in the yolk sac blood islands and then in the aorta-gonad mesonephros and the fetal liver, the sites of normal embryonic hematopoiesis. eGFP expression in erythroid cells peaks at E9.5 and then is rapidly silenced (>95%) and maintained at low levels into adulthood, demonstrating appropriate developmental regulation of the human ß-globin locus. In vitro knockdown of the epigenetic regulator DNA methyltransferase-1 in GG primary erythroid cells increases the proportion of eGFP(+) cells in culture from 41.9 to 74.1%. Furthermore, eGFP fluorescence is induced >3-fold after treatment of erythroid precursors with epigenetic drugs known to induce γ-globin expression, demonstrating the suitability of the Gγ-globin eGFP reporter for evaluation of γ-globin inducers. The GG mouse model is therefore a valuable model system for genetic and pharmacologic studies of the regulation of the ß-globin locus and for discovery of novel therapies for the ß-hemoglobinopathies.

Transcriptional regulators Myb and BCL11A interplay with DNA methyltransferase 1 in developmental silencing of embryonic and fetal ß-like globin genes.

The clinical symptoms of hemoglobin disorders such as ß-thalassemia and sickle cell anemia are significantly ameliorated by the persistent expression of γ-globin after birth. This knowledge has driven the discovery of important regulators that silence γ-globin postnatally. Improved understanding of the γ- to ß-globin switching mechanism holds the key to devising targeted therapies for ß-hemoglobinopathies. To further investigate this mechanism, we used the murine erythroleukemic (MEL) cell line containing an intact 183-kb human ß-globin locus, in which the (G)γ- and ß-globin genes are replaced by DsRed and eGFP fluorescent reporters, respectively. Following RNA interference (RNAi)-mediated knockdown of two key transcriptional regulators, Myb and BCL11A, we observed a derepression of γ-globin, measured by DsRed fluorescence and qRT-PCR (P<0.001). Interestingly, double knockdown of Myb and DNA methyltransferase 1 (DNMT1) resulted in a robust induction of ε-globin, (up to 20% of total ß-like globin species) compared to single knockdowns (P<0.001). Conversely, double knockdowns of BCL11A and DNMT1 enhanced γ-globin expression (up to 90% of total ß-like globin species) compared to single knockdowns (P<0.001). Moreover, following RNAi treatment, expression of human ß-like globin genes mirrored the expression levels of their endogenous murine counterparts. These results demonstrate that Myb and BCL11A cooperate with DNMT1 to achieve developmental repression of embryonic and fetal ß-like globin genes in the adult erythroid environment.