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 .
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.
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 building the stem cell niche (SCN) of the RAM. The initials, after several rounds of divisions and differentiation, give rise to nearly all tissues necessary for root function. Hence, QC establishment, maintenance and function are key to produce the whole plant root system and are therefore at the foundation of plant growth and productivity. Although the concept of the QC is known since the 1950s, much of its molecular regulations and their intricate interconnections, especially in more complex root systems like cereal RAMs remain elusive. In Arabidopsis, molecular factors like phytohormones, small signaling peptides and their receptors, as well as key transcription factors (TFs) play important roles in a complex and intertwined regulatory network. In cereals, homologs of these factors are present, however, QC maintenance in these larger RAMs might also require a more complex control of QC cell regulation by a combination of asymmetric and symmetric divisions. Here, we summarise the current knowledge on QC maintenance in Arabidopsis and compare it with that of agriculturally relevant cereal crops.
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 understand the underlying complex and dynamic regulatory mechanisms in vivo. In this review we examine some of the applicable techniques to measure PPI 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 giving a comprehensive overview about how to measure in vivo PPI in plants.
Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compart-mentalisation 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 tha-liana, 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 con-densed 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 pres-ence of the polyQ sequence. This species undergoes LLPS in a pH and temperature-sensitive man-ner 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 mi-croscopies. Furthermore, we demonstrate that the hydrogel assumes a semi-ordered structure as determined by small angle X-ray scattering, electron microscopy and X-ray diffraction. These ex-periments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.
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, in situ complex formation of MADS-domain proteins cannot be detected. 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.
Total word count: 7260 20 of the PLT-WOX5 complexes to NBs, possibly by liquid-liquid phase separation, plays an 38 important role during determination of CSC fate. 39 differentiation 6,7 . WOX5 also represses QC divisions, maintaining the quiescence of the QC by 65 repressing CYCLIN D (CYCD) activity within the QC 8 . The auxin-induced PLTs form a clade 66 of six TFs, and act as master regulators of root development, as multiple plt mutants fail to 67 develop functional RAMs 5,9,10 . PLT1, 2, 3 and 4 are expressed mainly in and around the QC 68 and form an instructive gradient, which is required for maintaining the balance of stem cell fate 69 and differentiation. This PLT gradient is also necessary for separating auxin responses in the 70 SCN and for the correct positioning of the QC and the expression of QC markers 5,9,10 . 71Genetically, WOX5 and PLT1 were shown to play an interconnected role in auxin-regulated 72 CSC fate, whereas PLT1 and PLT3 were found to positively regulate WOX5 expression 11,12 . 73 Although the implication of PLTs and WOX5 in controlling stem cell regulation and 74 maintenance in the Arabidopsis RAM is well established and genetic evidence for cross 75 regulation exists, the underlying molecular mechanisms remain largely elusive. Here, we show 76 that the mutual regulation of expression, but also the ability of PLTs, especially PLT3, to recruit 77 WOX5 to NBs in CSCs controls stem cell homeostasis in the Arabidopsis RAM. We propose 78 a model in which the differential PLT/WOX5 complexes depending on subnuclear localisation 79 regulate stem cell fate in the RAM, possibly by phase separation of PLT3 to NBs. 80Suppl. Table 7). In the plt2 and plt3 single mutants, the frequency of roots lacking a CSC layer 130 increases to above 30 % (36 % and 32 %, respectively), and in the plt2, plt3 double mutant to 131 41% (see Fig. 3c,d,e,i, Suppl. Table 7). Interestingly, the wox5, plt3 double mutant as well as 132 the wox5, plt2, plt3 triple mutant show a frequency of differentiated CSCs comparable to the 133 wox5 single mutant (71 % and 77 %, respectively) ( Fig. 3g,h,i, Suppl. Table 7). This data 134suggests that PLTs and WOX5 may act together in the same pathway to maintain CSC 135 homeostasis, as there is no additive effect observable in the multiple mutant roots. To analyse 136
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.
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