Dirk Roeland Boer

Apical hook development is an ideal model for studying differential growth in plants and is controlled by complex phytohormonal crosstalk, with auxin being the major player. Here, we identified a bioactive small molecule that decelerates apical hook opening in Arabidopsis thaliana . Our genetic studies suggest that this molecule enhances or maintains the auxin maximum found in the inner hook side and requires certain auxin signaling components to modulate apical hook opening. Using biochemical approaches, we then revealed the WD40 repeat scaffold protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A) as a direct target of this compound. We present data in support of RACK1A playing a positive role in apical hook opening by activating specific auxin signaling mechanisms and negatively regulating the differential auxin response gradient across the hook, thereby adjusting differential cell growth, an essential process for organ structure and function in plants.

The Auxin Response Factors (ARFs) family of transcription factors are the central mediators of auxin-triggered transcriptional regulation. Functionally different classes of extant ARFs operate as antagonistic auxin-dependent and -independent regulators. While part of the evolutionary trajectory to the present auxin response functions has been reconstructed, it is unclear how ARFs emerged, and how early diversification led to functionally different proteins. Here, we use in silico and in vivo analyses to revisit the molecular events that led to the origin and subsequent evolution of the ARFs. We reveal the shared origin of ARFs from preexisting domains, uncovering a protein fold homologous to the ARF DNA-binding fold in a conserved eukaryotic chromatin regulator. Building on this, we reconstruct the complete evolutionary history of ARFs, including the divergence events leading to the appearance of the ARF classes and defining the main molecular targets for their functional diversification. We derive a complete evolutionary trajectory that led to the emergence of the nuclear auxin signalling pathway.

To cope with environments with high levels of radiation, non-silicon semiconductors such as silicon carbide detectors are being proposed for instrumentation. 4H-SiC diodes for radiation detection have been fabricated in the IMB-CNM Clean Room, for which different strategies to define the electrical contact of the implants had been implemented, in an attempt to optimise the technology for, e.g., medical applications or low energy radiation detection, as the material choice can affect the sensitivity of the device. Among these technologies, it is included an epitaxially-grown graphene layer as part of the electrical contact. In this paper, a selection of four configurations of the IMB-CNM SiC diodes are characterised in terms of radiation detector response. Photodiode performance under 20 keV X-rays irradiation in the XALOC beam line at ALBA Synchrotron is presented. Over-responses in the range of 12–19% linked to the interaction of the radiation with the metallic layers are observed. A good uniformity response as well as a good linearity at 0 V bias is reported, even in the under-depleted devices. This work exemplifies the good performance of SiC detectors fabricated at IMB-CNM specifically for low-energy X ray characterization at high X-ray intensities.

The Auxin Response Factors (ARFs) family of transcription factors are the central mediators of auxin-triggered transcriptional regulation. Functionally different classes of extant ARFs operate as antagonistic auxin-dependent and -independent regulators. While part of the evolutionary trajectory to the present auxin response functions has been reconstructed, it is unclear how ARFs emerged, and how early diversification led to functionally different proteins. Here, we use in silico and in vivo analyses to revisit the molecular events that led to the origin and subsequent evolution of the ARFs. We reveal the shared origin of ARFs from preexisting domains, uncovering a protein fold homologous to the ARF DNA-binding fold in a conserved eukaryotic chromatin regulator. Building on this, we reconstruct the complete evolutionary history of ARFs, including the divergence events leading to the appearance of the ARF classes and defining the main molecular targets for their functional diversification. We derive a complete evolutionary trajectory that led to the emergence of the nuclear auxin signalling pathway.

Apical hook development is an ideal model for studying differential growth in plants, and is controlled by complex hormonal crosstalk, with auxin and ethylene being the major players. Here, we identified a bioactive small molecule that decelerates apical hook opening in Arabidopsis thaliana . Our genetic studies suggest that this molecule enhances or maintains the auxin maximum found in the inner hook side and requires certain auxin and ethylene signaling components to modulate apical hook opening. Using biochemical approaches, we then revealed the WD40 repeat scaffold protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A) as a direct target of this compound. We present data in support of RACK1A playing a positive role in apical hook opening by negatively regulating the differential auxin response gradient across the hook via specific auxin and ethylene signaling mechanisms and thereby adjusting differential cell growth, an essential process for organ structure and function in plants. We have thus identified a role for RACK1A and auxin-ethylene crosstalk in negatively regulating differential cell growth to promote apical hook opening. Significance Statement: Differential growth, or the growth of cells at different rates across tissues, is essential for providing shape and structure during plant development. The apical hook is a transient structure formed by differential cell growth across the hypocotyl tip in dark-grown seedlings, which protects the underlying tissues, and which opens during seedling development. We identified a small molecule that decelerates hook opening and discovered that it targets the protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A). We then showed that RACK1A promotes apical hook opening at the level of crosstalk between the plant hormones auxin and ethylene, by adjusting differential cell growth. Our work paves the way to a better understanding of how plants regulate and adapt their growth during development.

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.

Transcriptional regulation usually requires the action of several proteins that either repress or activate a promotor of an open reading frame. These proteins can counteract each other, thus allowing tight regulation of the transcription of the corresponding genes, where tight repression is often linked to DNA looping or cross-linking. Here, the tetramerization domain of the bacterial gene repressor Rco from Bacillus subtilis plasmid pLS20 (Rco_pLS20) has been identified and its structure is shown to share high similarity to the tetramerization domain of the well known p53 family of human tumor suppressors, despite lacking clear sequence homology. In Rco_pLS20, this tetramerization domain is responsible for inducing DNA looping, a process that involves multiple tetramers. In accordance, it is shown that Rco_pLS20 can form octamers. This domain was named TetD_loop and its occurrence was identified in other Bacillus species. The TetD_loop fold was also found in the structure of a transcriptional repressor from Salmonella phage SPC32H. It is proposed that the TetD_loop fold has evolved through divergent evolution and that the TetD_loop originates from a common ancestor predating the occurrence of multicellular life.

DNA replication is essential to all living organisms as it ensures the fidelity of genetic material for the next generation of dividing cells. One of the simplest replication initiation mechanisms is the rolling circle replication. In the streptococcal plasmid pMV158, which confers antibiotic resistance to tetracycline, replication initiation is catalysed by RepB protein. The RepB N-terminal domain or origin binding domain binds to the recognition sequence (bind locus) of the double-strand origin of replication and cleaves one DNA strand at a specific site within the nic locus. Using biochemical and crystallographic analyses, here we show how the origin binding domain recognises and binds to the bind locus using structural elements removed from the active site, namely the recognition α helix, and a β-strand that organises upon binding. A new hexameric structure of full-length RepB that highlights the great flexibility of this protein is presented, which could account for its ability to perform different tasks, namely bind to two distinct loci and cleave one strand of DNA at the plasmid origin.

The signaling molecule auxin is pivotal in coordinating many growth and development processes in plants mainly through the modulation of gene expression. The transcriptional response to auxin is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif (TGTC[TC]/[GG]) called the auxin-response element (AuxRE). ARFs can homodimerize through their DNA binding domains (DBD) thereby enabling cooperative binding for a bipartite inverted AuxRE (IR7). In addition to the DBD, most ARFs contain a C-terminal Phox and Bem1p (PB1) domain both 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 each of these domains contributes to conferring 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 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 affinity and kinetics seen for the DBDs of different 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 towards composite DNA response elements can be tuned by small changes of their dimerization equilibrium suggesting that this effect has major implications for ARF-mediated transcriptional activity.