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 (RAM) and the shoot apical meristem (SAM). In these meristematic tissues, cells with relatively slower mitotic activity are present in the quiescent center (QC) and the central zone (CZ), respectively. These centers form long-term reservoirs of stem cells maintaining the meristematic stem cell niche (SCN), and thus sustaining continuous plant development and adaptation to changing environments. This review explores the early observations, structural characteristics, functions, and gene regulatory networks of the RAM and SAM. It also highlights the intricate mechanism of dormancy within the SAM. The aim is to contribute to a holistic understanding of quiescence in plants, which is fundamental for the plant proper growth and environmental response.

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.

In vivo FRET-FLIM reveals cell-type-specific protein interactions in Arabidopsis roots.

During multicellular development, specification of distinct cell fates is often regulated by the same transcription factors operating differently in distinct cis-regulatory modules, either through different protein complexes, conformational modification of protein complexes, or combinations of both. Direct visualization of different transcription factor complex states guiding specific gene expression programs has been challenging. Here we use in vivo FRET-FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to reveal spatial partitioning of protein interactions in relation to specification of cell fate. We show that, in Arabidopsis roots, three fully functional fluorescently tagged cell fate regulators establish cell-type-specific interactions at endogenous expression levels and can form higher order complexes. We reveal that cell-type-specific in vivo FRET-FLIM distributions reflect conformational changes of these complexes to differentially regulate target genes and specify distinct cell fates.

Precise transcriptional control of cellular quiescence by BRAVO/WOX5 complex in Arabidopsis roots.

Understanding stem cell regulatory circuits is the next challenge in plant biology, as these cells are essential for tissue growth and organ regeneration in response to stress. In the Arabidopsis primary root apex, stem cell-specific transcription factors BRAVO and WOX5 co-localize in the quiescent centre (QC) cells, where they commonly repress cell division so that these cells can act as a reservoir to replenish surrounding stem cells, yet their molecular connection remains unknown. Genetic and biochemical analysis indicates that BRAVO and WOX5 form a transcription factor complex that modulates gene expression in the QC cells to preserve overall root growth and architecture. Furthermore, by using mathematical modelling we establish that BRAVO uses the WOX5/BRAVO complex to promote WOX5 activity in the stem cells. Our results unveil the importance of transcriptional regulatory circuits in plant stem cell development.

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.

The Chromatin-Associated Protein PWO1 Interacts with Plant Nuclear Lamin-like Components to Regulate Nuclear Size.

Spatial organization of chromatin contributes to gene regulation of many cellular processes and includes a connection of chromatin with the nuclear lamina (NL). The NL is a protein mesh that resides underneath the inner nuclear membrane and consists of lamins and lamina-associated proteins. Chromatin regions associated with lamins in animals are characterized mostly by constitutive heterochromatin, but association with facultative heterochromatin mediated by Polycomb-group (PcG) proteins has been reported as well. In contrast with animals, plant NL components are largely not conserved and NL association with chromatin is poorly explored. Here, we present the connection between the lamin-like protein, CROWDED NUCLEI1 (CRWN1), and the chromatin- and PcG-associated component, PROLINE-TRYPTOPHANE-TRYPTOPHANE-PROLINE INTERACTOR OF POLYCOMBS1, in Arabidopsis (Arabidopsis thaliana). We show that PWO1 and CRWN1 proteins associate physically with each other, act in the same pathway to maintain nuclear morphology, and control expression of a similar set of target genes. Moreover, we demonstrate that transiently expressed PWO1 proteins form foci located partially at the subnuclear periphery. Ultimately, as CRWN1 and PWO1 are plant-specific, our results argue that plants might have developed an equivalent, rather than homologous, mechanism of linking chromatin repression and NL.

The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis.

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

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.

Green light for quantitative live-cell imaging in plants.

Plants exhibit an intriguing morphological and physiological plasticity that enables them to thrive in a wide range of environments. To understand the cell biological basis of this unparalleled competence, a number of methodologies have been adapted or developed over the last decades that allow minimal or non-invasive live-cell imaging in the context of tissues. Combined with the ease to generate transgenic reporter lines in specific genetic backgrounds or accessions, we are witnessing a blooming in plant cell biology. However, the imaging of plant cells entails a number of specific challenges, such as high levels of autofluorescence, light scattering that is caused by cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in plants therefore requires adapting or developing imaging techniques, as well as mounting and incubation systems, such as micro-fluidics. Here, we discuss some of these obstacles, and review a number of selected state-of-the-art techniques, such as two-photon imaging, light sheet microscopy and variable angle epifluorescence microscopy that allow high performance and minimal invasive live-cell imaging in plants.

Antagonistic peptide technology for functional dissection of CLE peptides revisited.

In the Arabidopsis thaliana genome, over 1000 putative genes encoding small, presumably secreted, signalling peptides can be recognized. However, a major obstacle in identifying the function of genes encoding small signalling peptides is the limited number of available loss-of-function mutants. To overcome this, a promising new tool, antagonistic peptide technology, was recently developed. Here, this antagonistic peptide technology was tested on selected CLE peptides and the related IDA peptide and its usefulness in the context of studies of peptide function discussed. Based on the analyses, it was concluded that the antagonistic peptide approach is not the ultimate means to overcome redundancy or lack of loss-of-function lines. However, information collected using antagonistic peptide approaches (in the broad sense) can be very useful, but these approaches do not work in all cases and require a deep insight on the interaction between the ligand and its receptor to be successful. This, as well as peptide ligand structure considerations, should be taken into account before ordering a wide range of synthetic peptide variants and/or generating transgenic plants.

Tackling drought stress: receptor-like kinases present new approaches.

Global climate change and a growing population require tackling the reduction in arable land and improving biomass production and seed yield per area under varying conditions. One of these conditions is suboptimal water availability. Here, we review some of the classical approaches to dealing with plant response to drought stress and we evaluate how research on RECEPTOR-LIKE KINASES (RLKs) can contribute to improving plant performance under drought stress. RLKs are considered as key regulators of plant architecture and growth behavior, but they also function in defense and stress responses. The available literature and analyses of available transcript profiling data indeed suggest that RLKs can play an important role in optimizing plant responses to drought stress. In addition, RLK pathways are ideal targets for nontransgenic approaches, such as synthetic molecules, providing a novel strategy to manipulate their activity and supporting translational studies from model species, such as Arabidopsis thaliana, to economically useful crops.

FER-like iron deficiency-induced transcription factor (FIT) accumulates in nuclear condensates.

The functional importance of nuclear protein condensation remains often unclear. The bHLH FER-like iron deficiency-induced transcription factor (FIT) controls iron acquisition and growth in plants. Previously described C-terminal serine residues allow FIT to interact and form active transcription factor complexes with subgroup Ib bHLH factors such as bHLH039. FIT has lower nuclear mobility than mutant FITmSS271AA. Here, we show that FIT undergoes a light-inducible subnuclear partitioning into FIT nuclear bodies (NBs). Using quantitative and qualitative microscopy-based approaches, we characterized FIT NBs as condensates that were reversible and likely formed by liquid-liquid phase separation. FIT accumulated preferentially in NBs versus nucleoplasm when engaged in protein complexes with itself and with bHLH039. FITmSS271AA, instead, localized to NBs with different dynamics. FIT colocalized with splicing and light signaling NB markers. The NB-inducing light conditions were linked with active FIT and elevated FIT target gene expression in roots. FIT condensation may affect nuclear mobility and be relevant for integrating environmental and Fe nutrition signals.

RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis.

The shoot apical meristem (SAM) is the fundamental structure that is located at the growing tip and gives rise to all aerial parts of plant tissues and organs, such as leaves, stems and flowers. In Arabidopsis thaliana, the CLAVATA3 (CLV3) pathway regulates the stem cell pool in the SAM, in which a small peptide ligand derived from CLV3 is perceived by two major receptor complexes, CLV1 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), to restrict WUSCHEL (WUS) expression. In this study, we used the functional, synthetic CLV3 peptide (MCLV3) to isolate CLV3-insensitive mutants and revealed that a receptor-like kinase, RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), also known as TOADSTOOL 2 (TOAD2), is another key regulator of meristem maintenance. Mutations in the RPK2 gene result in stem cell expansion and increased number of floral organs, as seen in the other clv mutants. These phenotypes are additive with both clv1 and clv2 mutations. Moreover, our biochemical analyses using Nicotiana benthamiana revealed that RPK2 forms homo-oligomers but does not associate with CLV1 or CLV2. These genetic and biochemical findings suggest that three major receptor complexes, RPK2 homomers, CLV1 homomers and CLV2-CRN/SOL2 heteromers, are likely to mediate three signalling pathways, mainly in parallel but with potential crosstalk, to regulate the SAM homeostasis.

Gated communities: apoplastic and symplastic signals converge at plasmodesmata to control cell fates.

Due to their rigid cell walls, plant cells can only communicate with each other either by symplastic transport of diverse non-cell autonomous signalling molecules via plasmodesmata (PDs) or by endo- and exocytosis of signalling molecules via the extracellular apoplastic space. PDs are plasma membrane-lined channels spanning the cell wall between neighbouring cells, allowing the exchange of molecules by symplastic movement through them. This review focuses on developmental decisions that are coordinated by short- and long-distance communication of cells via PDs. We propose a model combining both apoplastic and symplastic signalling events via secreted ligands and their PD-localized receptor kinases which gate the symplastic transport of information molecules through PDs. Cell communities can thus coordinate cell-fate decisions non-cell autonomously by connecting or disconnecting symplastic subdomains. Here we concentrate on the establishment of such subdomains in the plant's primary meristems that serve to maintain long-lasting stem cell populations in the shoot and root apical meristems, and discuss how apoplastic signalling via transport of information molecules through PDs is integrated with symplastic feedback signalling events.

The CURLY LEAF interacting protein BLISTER controls expression of polycomb-group target genes and cellular differentiation of Arabidopsis thaliana.

Polycomb-group (Pc-G) proteins are important regulators of many developmental processes in plants and animals and repress gene expression by imparting histone H3 lysine 27 trimethylation (H3K27me3). Here, we present the identification of the novel, plant-specific Arabidopsis thaliana protein BLISTER (BLI), which interacts with the Pc-G histone methyltransferase CURLY LEAF (CLF). We map the interaction of BLI with CLF to a predicted coiled-coil domain in BLI that shares similarity with STRUCTURAL MAINTENANCE OF CHROMOSOMES proteins. BLI colocalizes with CLF in the nucleus, shows an overlapping expression pattern with CLF throughout plant development that is strongest in dividing cells, and represses a subset of Pc-G target genes. Loss of BLI results in a pleiotropic developmental mutant phenotype, indicating that BLI prevents premature differentiation. Furthermore, bli mutants exhibit severe epidermal defects, including loss of cell adhesion, outgrowth of cells, and increased cotyledon cell size. As these phenotypes have not been observed in Pc-G mutants, we propose that BLI has functions related to Pc-G proteins but can also act independently in Arabidopsis development.

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.

The Arabidopsis SHORTROOT network coordinates shoot apical meristem development with auxin-dependent lateral organ initiation.

Plants produce new organs post-embryonically throughout their entire life cycle. This is due to stem cells present in the shoot and root apical meristems, the SAM and RAM, respectively. In the SAM, stem cells are located in the central zone where they divide slowly. Stem cell daughters are displaced laterally and enter the peripheral zone, where their mitotic activity increases and lateral organ primordia are formed. How the spatial arrangement of these different domains is initiated and controlled during SAM growth and development, and how sites of lateral organ primordia are determined in the peripheral zone is not yet completely understood. We found that the SHORTROOT (SHR) transcription factor together with its target transcription factors SCARECROW (SCR), SCARECROW-LIKE23 (SCL23) and JACKDAW (JKD), promotes formation of lateral organs and controls shoot meristem size. SHR, SCR, SCL23, and JKD are expressed in distinct, but partially overlapping patterns in the SAM. They can physically interact and activate expression of key cell cycle regulators such as CYCLIND6;1 (CYCD6;1) to promote the formation of new cell layers. In the peripheral zone, auxin accumulates at sites of lateral organ primordia initiation and activates SHR expression via the auxin response factor MONOPTEROS (MP) and auxin response elements in the SHR promoter. In the central zone, the SHR-target SCL23 physically interacts with the key stem cell regulator WUSCHEL (WUS) to promote stem cell fate. Both SCL23 and WUS expression are subject to negative feedback regulation from stem cells through the CLAVATA signaling pathway. Together, our findings illustrate how SHR-dependent transcription factor complexes act in different domains of the shoot meristem to mediate cell division and auxin dependent organ initiation in the peripheral zone, and coordinate this activity with stem cell maintenance in the central zone of the SAM.

Homo-FRET Imaging to Study Protein-Protein Interaction and Complex Formation in Plants.

Protein-protein interactions in living plant cells can be measured by changes in fluorescence anisotropy due to homo-FRET (Förster Resonance Energy Transfer). Here, the energy transfer between identical fluorophores, e.g., enhanced green fluorescent protein (EGFP) fused to a protein of interest, serves as a read-out for protein interaction and clustering. By applying homo-FRET imaging, not only dimeric complexes, but also bigger homomeric complex formation can be followed in vivo at high spatial and temporal resolution. Therefore, this method provides a powerful tool to investigate changes in complex formation over time in their natural environment with high precision at a subcellular level. Here, we describe the necessary theoretical background and how homo-FRET imaging is practically carried out. We also discuss potential pitfalls and points of consideration.