Jan Willem Borst

Background: Mechanical signals are important regulators of cellular responses in plants. They guide plant development and can activate defense and repair mechanisms. Yet, the molecular mechanisms by which plants perceive, transduce, and interpret mechanical signals are still poorly understood. This is in part due to the lack of methods to apply local, precise and non-damaging mechanical forces to plant cells. Micro-indentation is highly suitable for this purpose, yet available instrumentation is often expensive and difficult to combine with high-resolution microscopy. Results: We designed an open-source and affordable modular indentation device, consisting of 3D printed elements, 3 commercially available piezo motors, and a variety of indentation needles. Due to its modularity, the setup can be readily adapted to meet experimental requirements and works on all microscopes with bespoke adaptors. We show that the setup can be used to explore both rapid and slower touch responses, exemplified by visualizing calcium waves and actin patches induced by touch. We also show that the setup is compatible with various plant species and tissues and can be combined with high-resolution functional imaging. Conclusions: The simple and flexible design of the indentation device presented in this paper ensures that any lab with a 3D printer can build their own setup at low cost and with minimal time investment. The system has a wide range of applications for live plant tissue, making indentation experiments and thereby plant mechanobiology studies, more accessible.

Transcription factors (TFs) are traditionally depicted as mono- or oligomeric units that recognize and bind specific DNA sequences to regulate transcription. Emerging evidence suggests that many TFs can undergo liquid phase separation, resulting in condensates that bind DNA through a different mechanism. For the auxin response factors (ARFs), canonical plant TFs, evidence for both scenarios exists, but which of these scenarios is operational in the plant nucleus is unclear. Here, we demonstrate using MpARF2 of Marchantia polymorpha that a third scenario is operational: MpARF2 forms nanoscopic clusters under physiological conditions. Nanoclusters combine DNA binding features that cannot be accessed by the other two scenarios: high-affinity, switch-like, and sequence-specific. Our results suggest nanoclustering as a mechanism that equips TFs with the DNA binding properties necessary for transcriptional regulation.

Eukaryotic cells compartmentalise to optimise their biochemistry. This occurs through membrane‐surrounded compartments, but also through membraneless organelles (MLOs). Many MLOs arise from Liquid–Liquid Phase Separation (LLPS) of proteins. To investigate proteins for their capacity to phase separate, as well as what properties this results in, purified protein is required for in vitro phase separation assays. We have observed that Auxin Response Factor 2 from the common liverwort Marchantia polymorpha (MpARF2) forms nuclear assemblies in vivo . Auxin Response Factors (ARFs) are transcription factors crucial for the regulation of gene expression in response to auxin, an essential signalling molecule in plants. It is currently unclear what causes the formation of MpARF2 assemblies and how they affect the protein's function. To decipher this, we present the procedures for purifying MpARF2 and preparing it for in vitro studies. The observed MpARF2 assemblies could arise by phase separation. The properties of phase‐separating proteins make their expression, purification and handling challenging; therefore, optimised procedures are required. Here, we show that the purification of soluble and monomeric full‐length MpARF2, as well as (truncated) variants, is possible using a three‐step purification procedure involving a protein solubility tag and buffers that suppress phase separation. We also describe the subsequent protein preparation that ensures reproducible phase separation of MpARF2 during in vitro experiments. These procedures pave the way for elucidating the phase separation properties of MpARF2 and the potential functional role of its assemblies while providing a protocol which can facilitate similar studies of other ARFs.

Transcription factors (TFs) are traditionally depicted as monomeric, or oligomeric, units that recognize and bind specific DNA sequences to regulate transcription. Emerging evidence suggests that many TFs can undergo liquid phase separation, resulting in condensates that bind DNA through a different mechanism. For the Auxin Response Factors (ARFs), canonical plant TFs, evidence for both scenarios exists. But which of these scenarios is operational in the plant nucleus is unclear. Here, we demonstrate using MpARF2 of Marchantia polymorpha that a third scenario is operational: MpARF2 forms nanoscopic clusters under physiological conditions. Nanoclusters combine DNA-binding features that cannot be accessed by the other two scenarios: high-affinity, switch-like, and sequence-specific. Our results suggest nanoclustering as a mechanism that equips TFs with the DNA-binding properties necessary for transcriptional regulation. Teaser: Transcription factor nanoclusters balance the DNA-binding trade-off between weakly binding oligomers and nonspecific condensates.

Auxin response critically depends on the concentrations and stoichiometry of competing A- and B-class AUXIN RESPONSE FACTOR (ARF) proteins. In Marchantia polymorpha , both A- and B-ARFs are unstable, and here we identify a minimal necessary and sufficient region for ARF degradation that is critical for development, and auxin response. Through comparative analysis, we find that ARF instability likely preceded the emergence of the auxin response system.

The signaling molecule auxin sits at the nexus of plant biology where it coordinates essentially all growth and developmental processes. Auxin molecules are transported throughout plant tissues and are capable of evoking highly specific physiological responses by inducing various molecular pathways. In many of these pathways, proteolysis plays a crucial role for correct physiological responses. This review provides a chronology of the discovery and characterization of the auxin receptor, which is a fascinating example of separate research trajectories ultimately converging on the discovery of a core auxin signaling hub that relies on degradation of a family of transcriptional inhibitor proteins—the Aux/IAAs. Beyond describing the “classical” proteolysis-driven auxin response system, we explore more recent examples of the interconnection of proteolytic systems, which target a range of other auxin signaling proteins, and auxin response. By highlighting these emerging concepts, we provide potential future directions to further investigate the role of protein degradation within the framework of auxin response.

The plant signaling molecule auxin controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of a ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and the DNA-binding ARF transcription factors. While extensive qualitative understanding of the pathway and its interactions has been obtained by studying the flowering plant Arabidopsis thaliana , it is so far unknown how these translate to quantitative system behaviour in vivo , a problem that is confounded by large NAP gene families in this species. Here we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knock-in fluorescent fusion proteins. Beyond revealing the native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs MpARF1 and MpARF2 are proteasomally degraded. This degradation serves two functions: it tunes the stoichiometry of auxin-responsive, positively acting MpARF1 and auxin-independent, negatively acting MpARF2, thereby permitting auxin response. Secondly, through mapping a minimal degradation motif, we found that degradation is likely selective for MpARF2 monomers and favours accumulation of dimers. Interfering with MpARF1:MpARF2 stoichiometry or preventing degradation of MpARF2 monomers caused strong growth defects associated with auxin response defects. Thus, quantitative analysis of the entire Marchantia NAP, allowed to identify a novel regulatory mechanism in auxin response, built on regulated ARF degradation.