Phase separation and molecular ordering of the prion-like domain of the Arabidopsis thermosensory protein EARLY FLOWERING 3.

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

Stem cell quiescence and dormancy in plant meristems.

Plants exhibit opportunistic developmental patterns, alternating between growth and dormancy in response to external cues. Moreover, quiescence plays a critical role in proper plant growth and development, particularly within the root apical meristem and the shoot apical meristem. In these meristematic tissues, cells with relatively slower mitotic activity are present in the quiescent center and the central zone, respectively. These centers form long-term reservoirs of stem cells maintaining the meristematic stem cell niche, and thus sustaining continuous plant development and adaptation to changing environments. This review explores early observations, structural characteristics, functions, and gene regulatory networks of the root and shoot apical meristems. It also highlights the intricate mechanism of dormancy within the shoot apical meristem. The aim is to contribute to a holistic understanding of quiescence in plants, which is fundamental for the proper growth and environmental response of plants.

PLETHORA-WOX5 interaction and subnuclear localization control Arabidopsis root stem cell maintenance.

Maintenance and homeostasis of the stem cell niche (SCN) in the Arabidopsis root is essential for growth and development of all root cell types. The SCN is organized around a quiescent center (QC) maintaining the stemness of cells in direct contact. The key transcription factors (TFs) WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and PLETHORAs (PLTs) are expressed in the SCN where they maintain the QC and regulate distal columella stem cell (CSC) fate. Here, we describe the concerted mutual regulation of the key TFs WOX5 and PLTs on a transcriptional and protein interaction level. Additionally, by applying a novel SCN staining method, we demonstrate that both WOX5 and PLTs regulate root SCN homeostasis as they control QC quiescence and CSC fate interdependently. Moreover, we uncover that some PLTs, especially PLT3, contain intrinsically disordered prion-like domains (PrDs) that are necessary for complex formation with WOX5 and its recruitment to subnuclear microdomains/nuclear bodies (NBs) in the CSCs. We propose that this partitioning of PLT-WOX5 complexes to NBs, possibly by phase separation, is important for CSC fate determination.

Visualization of in vivo protein-protein interactions in plants.

Molecular processes depend on the concerted and dynamic interactions of proteins, either by one-on-one interactions of the same or different proteins or by the assembly of larger protein complexes consisting of many different proteins. Here, not only the protein-protein interaction (PPI) itself, but also the localization and activity of the protein of interest (POI) within the cell is essential. Therefore, in all cell biological experiments, preserving the spatio-temporal state of one POI relative to another is key to understanding the underlying complex and dynamic regulatory mechanisms in vivo. In this review, we examine some of the applicable techniques to measure PPIs in planta as well as recent combinatorial advances of PPI methods to measure the formation of higher order complexes with an emphasis on in vivo imaging techniques. We compare the different methods and discuss their benefits and potential pitfalls to facilitate the selection of appropriate techniques by providing a comprehensive overview of how to measure in vivo PPIs in plants.

At the root of quiescence: function and regulation of the quiescent center.

The quiescent center (QC) of roots consists of a rarely dividing pool of stem cells within the root apical meristem (RAM). The QC maintains the surrounding more frequently dividing initials, together constituting the stem cell niche of the RAM. The initials, after several rounds of division and differentiation, give rise to nearly all tissues necessary for root function. Hence, QC establishment, maintenance, and function are key for producing the whole plant root system and are therefore at the foundation of plant growth and productivity. Although the concept of the QC has been known since the 1950s, much of its molecular regulations and their intricate interconnections, especially in more complex root systems such as cereal RAMs, remain elusive. In Arabidopsis, molecular factors such as phytohormones, small signaling peptides and their receptors, and key transcription factors play important roles in a complex and intertwined regulatory network. In cereals, homologs of these factors are present; however, QC maintenance in the larger RAMs of cereals might also require more complex control of QC cell regulation by a combination of asymmetric and symmetric divisions. Here, we summarize current knowledge on QC maintenance in Arabidopsis and compare it with that of agriculturally relevant cereal crops.

Unlocking nature's (sub)cellular symphony: Phase separation in plant meristems.

Plant development is based on the balance of stem cell maintenance and differentiation in the shoot and root meristems. The necessary cell fate decisions are regulated by intricate networks of proteins and biomolecules within plant cells and require robust and dynamic compartmentalization strategies, including liquid-liquid phase separation (LLPS), which allows the formation of membrane-less compartments. This review summarizes the current knowledge about the emerging field of LLPS in plant development, with a particular focus on the shoot and root meristems. LLPS regulates not only floral transition and flowering time while integrating environmental signals in the shoots but also influences auxin signalling and is putatively involved in maintaining the stem cell niche (SCN) in the roots. Therefore, LLPS has the potential to play a crucial role in the plasticity of plant development, necessitating further research for a comprehensive understanding.

One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells.

BACKGROUND: A commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, one pattern analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters. RESULTS: As a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, makes in situ complex formation of MADS-domain proteins barely detectable. CONCLUSIONS: We conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.

A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species.

Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.