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.

Studying Protein-Protein Interactions at Plasmodesmata by Measuring Förster Resonance Energy Transfer.

Plasmodesmata (PD) provide interconnectivity between plant cells to enable the intercellular transport and communication that is requisite to multicellularity. Being at the interface of the apoplast, plasma membrane (PM), endoplasmic reticulum (ER), and symplast, PD are uniquely positioned to integrate exogenously and endogenously derived signals with plant developmental and physiological responses. The distinct membrane curvature and composition of PD allow them to function as microdomains to facilitate dynamic protein-protein interactions. Förster resonance energy transfer (FRET) combined with fluorescence lifetime imaging microscopy (FLIM) and fluorescence anisotropic decay measurements provides valuable tools to analyze these interactions in vivo and in planta. Here we describe a detailed methodology to perform FRET-FLIM and fluorescence anisotropy measurements to analyze protein-protein interactions at PD in a transient expression system using Nicotiana benthamiana; however this can be adapted to other plant species and subcellular compartments.

The boundary-expressed EPIDERMAL PATTERNING FACTOR-LIKE2 gene encoding a signaling peptide promotes cotyledon growth during Arabidopsis thaliana embryogenesis.

The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis suggests that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.

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.

Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies.

Signal perception and transmission of the plant hormone ethylene are mediated by a family of receptor histidine kinases located at the Golgi-ER network. Similar to bacterial and other plant receptor kinases, these receptors work as dimers or higher molecular weight oligomers at the membrane. Sequence analysis and functional studies of different isoforms suggest that the ethylene receptor family is classified into two subfamilies. In Arabidopsis, the type-I subfamily has two members (ETR1 and ERS1) and the type-II subfamily has three members (ETR2, ERS2, and EIN4). Whereas subfamily-I of the Arabidopsis receptors and their interactions with downstream elements in the ethylene pathway has been extensively studied in the past; related information on subfamily-II is sparse. In order to dissect the role of type-II receptors in the ethylene pathway and to decode processes associated with this receptor subfamily on a quantitative molecular level, we have applied biochemical and spectroscopic studies on purified recombinant receptors and downstream elements of the ethylene pathway. To this end, we have expressed purified ETR2 as a prototype of the type-II subfamily, ETR1 for the type-I subfamily and downstream ethylene pathway proteins CTR1 and EIN2. Functional folding of the purified receptors was demonstrated by CD spectroscopy and autokinase assays. Quantitative analysis of protein-protein interactions (PPIs) by microscale thermophoresis (MST) revealed that ETR2 has similar affinities for CTR1 and EIN2 as previously reported for the subfamily-I prototype ETR1 suggesting similar roles in PPI-mediated signal transfer for both subfamilies. We also used in planta fluorescence studies on transiently expressed proteins in Nicotiana benthamiana leaf cells to analyze homo- and heteromer formation of receptors. These studies show that type-II receptors as well as the type-I receptors form homo- and heteromeric complexes at these conditions. Notably, type-II receptor homomers and type-II:type-I heteromers are more stable than type-I homomers as indicated by their lower dissociation constants obtained in microscale thermophoresis studies. The enhanced stability of type-II complexes emphasizes the important role of type-II receptors in the ethylene pathway.

Studying Protein-Protein Interactions In Planta Using Advanced Fluorescence Microscopy.

The formation of protein complexes through direct protein-protein interaction is essential for virtually every biological process, and accordingly the ability to determine the interaction properties of specific proteins is important to understand these processes. Förster resonance energy transfer (FRET) measurements are state-of-the-art confocal fluorescence microscopy- and imaging-based techniques that allow the analysis of protein interactions in vivo and in planta, in specific compartments of single cells or tissues. Here we provide a step-by-step guide to perform FRET measurements by acceptor photobleaching (APB) and fluorescence lifetime imaging microscopy (FLIM) in the plant expression system Nicotiana benthamiana.

Q&A: How does peptide signaling direct plant development?

A significant part of the communication between plant cells is mediated by signaling peptides and their corresponding plasma membrane-localized receptor-like kinases. This communication mechanism serves as a key regulatory unit for coordination of plant growth and development. In the past years more peptide-receptor signaling pathways have been shown to regulate developmental processes, such as shoot and root meristem maintenance, seed formation, and floral abscission. More detailed understanding of the processes behind this regulation might also be helpful to increase the yield of crop plants.

Real-time dynamics of peptide ligand-dependent receptor complex formation in planta.

The CLAVATA (CLV) and flagellin (flg) signaling pathways act through peptide ligands and closely related plasma membrane-localized receptor-like kinases (RLKs). The plant peptide CLV3 regulates stem cell homeostasis, whereas the bacterial flg22 peptide elicits defense responses. We applied multiparameter fluorescence imaging spectroscopy (MFIS) to characterize the dynamics of RLK complexes in the presence of ligand in living plant cells expressing receptor proteins fused to fluorescent proteins. We found that the CLV and flg pathways represent two different principles of signal transduction: flg22 first triggered RLK heterodimerization and later assembly into larger complexes through homomerization. In contrast, CLV receptor complexes were preformed, and ligand binding stimulated their clustering. This different behavior likely reflects the nature of these signaling pathways. Pathogen-triggered flg signaling impedes plant growth and development; therefore, receptor complexes are formed only in the presence of ligand. In contrast, CLV3-dependent stem cell homeostasis continuously requires active signaling, and preformation of receptor complexes may facilitate this task.

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.

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis.

The MBW (for R2R3MYB, basic helix-loop-helix [bHLH], and WD40) genes comprise an evolutionarily conserved gene cassette that regulates several traits such as (pro)anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants. Trichome differentiation in Arabidopsis (Arabidopsis thaliana) is governed by GLABRA1 (GL1; R2R3MYB), GL3 (bHLH), and transparent TESTA GLABRA1 (TTG1; WD40). They are thought to form a trimeric complex that acts as a transcriptional activation complex. We provide evidence that these three MBW proteins form either GL1 GL3 or GL3 TTG1 dimers. The formation of each dimer is counteracted by the respective third protein in yeast three-hybrid assays, pulldown experiments (luminescence-based mammalian interactome), and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. We further show that two target promoters, Triptychon (TRY) and CAPRICE (CPC), are differentially regulated: GL1 represses the activation of the TRY promoter by GL3 and TTG1, and TTG1 suppresses the activation of the CPC promoter by GL1 and GL3. Our data suggest that the transcriptional activation by the MBW complex involves alternative complex formation and that the two dimers can differentially regulate downstream genes.

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.

TAF13 interacts with PRC2 members and is essential for Arabidopsis seed development.

TBP-Associated Factors (TAFs) are components of complexes like TFIID, TFTC, SAGA/STAGA and SMAT that are important for the activation of transcription, either by establishing the basic transcription machinery or by facilitating histone acetylation. However, in Drosophila embryos several TAFs were shown to be associated with the Polycomb Repressive Complex 1 (PRC1), even though the role of this interaction remains unclear. Here we show that in Arabidopsis TAF13 interacts with MEDEA and SWINGER, both members of a plant variant of Polycomb Repressive Complex 2 (PRC2). PRC2 variants play important roles during the plant life cycle, including seed development. The taf13 mutation causes seed defects, showing embryo arrest at the 8-16 cell stage and over-proliferation of the endosperm in the chalazal region, which is typical for Arabidopsis PRC2 mutants. Our data suggest that TAF13 functions together with PRC2 in transcriptional regulation during seed development.

Peptides and receptors controlling root development.

The growth of a plant's root system depends on the continued activity of the root meristem, and the generation of new meristems when lateral roots are initiated. Plants have developed intricate signalling systems that employ secreted peptides and plasma membrane-localized receptor kinases for short- and long-range communication. Studies on growth of the vascular system, the generation of lateral roots, the control of cell differentiation in the root meristem and the interaction with invading pathogens or symbionts has unravelled a network of peptides and receptor systems with occasionally shared functions. A common theme is the employment of conserved modules, consisting of a short signalling peptide, a receptor-like kinase and a target transcription factor, that control the fate and proliferation of stem cells during root development. This review intends to give an overview of the recent advances in receptor and peptide ligand-mediated signalling involved in root development.

Auxin-dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins.

Multicellular organisms depend on cell production, cell fate specification, and correct patterning to shape their adult body. In plants, auxin plays a prominent role in the timely coordination of these different cellular processes. A well-studied example is lateral root initiation, in which auxin triggers founder cell specification and cell cycle activation of xylem pole-positioned pericycle cells. Here, we report that the E2Fa transcription factor of Arabidopsis thaliana is an essential component that regulates the asymmetric cell division marking lateral root initiation. Moreover, we demonstrate that E2Fa expression is regulated by the LATERAL ORGAN BOUNDARY DOMAIN18/LATERAL ORGAN BOUNDARY DOMAIN33 (LBD18/LBD33) dimer that is, in turn, regulated by the auxin signaling pathway. LBD18/LBD33 mediates lateral root organogenesis through E2Fa transcriptional activation, whereas E2Fa expression under control of the LBD18 promoter eliminates the need for LBD18. Besides lateral root initiation, vascular patterning is disrupted in E2Fa knockout plants, similarly as it is affected in auxin signaling and lbd mutants, indicating that the transcriptional induction of E2Fa through LBDs represents a general mechanism for auxin-dependent cell cycle activation. Our data illustrate how a conserved mechanism driving cell cycle entry has been adapted evolutionarily to connect auxin signaling with control of processes determining plant architecture.

Nematode CLE signaling in Arabidopsis requires CLAVATA2 and CORYNE.

Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ESR (CLE)-like effector proteins. These proteins have been shown to act as ligand mimics of plant CLE peptides and are required for successful nematode infection; however, the receptors for nematode CLE-like peptides have not been identified. Here we demonstrate that CLV2 and CORYNE (CRN), members of the receptor kinase family, are required for nematode CLE signaling. Exogenous peptide assays and overexpression of nematode CLEs in Arabidopsis demonstrated that CLV2 and CRN are required for perception of nematode CLEs. In addition, promoter-reporter assays showed that both receptors are expressed in nematode-induced syncytia. Lastly, infection assays with receptor mutants revealed a decrease in both nematode infection and syncytium size. Taken together, our results indicate that perception of nematode CLEs by CLV2 and CRN is not only required for successful nematode infection but is also involved in the formation and/or maintenance of nematode-induced syncytia.

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.

Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane.

Stem cell number in shoot and floral meristems of Arabidopsis (Arabidopsis thaliana) is regulated by the CLAVATA3 (CLV3) signaling pathway. Perception of the CLV3 peptide requires the receptor kinase CLV1, the receptor-like protein CLV2, and the kinase CORYNE (CRN). Genetic analysis suggested that CLV2 and CRN act together and in parallel with CLV1. We studied the intracellular localization of receptor fusions with fluorescent protein tags and their capacities for interaction via efficiency of fluorescence resonance energy transfer. We found that CLV2 and CRN require each other for export from the endoplasmic reticulum and localization to the plasma membrane (PM). CRN readily forms homomers and interacts with CLV2 through the transmembrane domain and adjacent juxtamembrane sequences. CLV1 forms homomers independently of CLV2 and CRN at the PM. We propose that the CLV3 signal is perceived by a tetrameric CLV2/CRN complex and a CLV1 homodimer that localize to the PM and can interact via CRN.

Multiparameter fluorescence image spectroscopy to study molecular interactions.

Multiparameter Fluorescence Image Spectroscopy (MFIS) is used to monitor simultaneously a variety of fluorescence parameters in confocal fluorescence microscopy. As the photons are registered one by one, MFIS allows for fully parallel recording of Fluorescence Correlation/Cross Correlation Spectroscopy (FCS/FCCS), fluorescence lifetime and pixel/image information over time periods of hours with picosecond accuracy. The analysis of the pixel fluorescence information in higher-dimensional histograms maximizes the selectivity of fluorescence microscopic methods. Moreover it facilitates a statistically-relevant data analysis of the pixel information which makes an efficient detection of heterogeneities possible. The reliability of MFIS has been demonstrated for molecular interaction studies in different complex environments: (I) detecting the heterogeneity of diffusion properties of the dye Rhodamine 110 in a sepharose bead, (II) Förster Resonance Energy Transfer (FRET) studies in mammalian HEK293 cells, and (III) FRET study of the homodimerisation of the transcription factor BIM1 in plant cells. The multidimensional analysis of correlated changes of several parameters measured by FRET, FCS, fluorescence lifetime and anisotropy increases the robustness of the analysis significantly. The economic use of photon information allows one to keep the expression levels of fluorescent protein-fusion proteins as low as possible (down to the single-molecule level).

Randomized comparison of a titanium-nitride-oxide-coated stent with a stainless steel stent for coronary revascularization: the TiNOX trial.

BACKGROUND: Stent coating with titanium-nitride-oxide has been shown to reduce neointimal hyperplasia in the porcine restenosis model. We designed a prospective, randomized, clinical study to investigate the safety and efficacy of titanium-nitride-oxide-coated stents compared with stainless steel stents. METHODS AND RESULTS: Ninety-two patients with de novo lesions were randomly assigned to treatment with titanium-nitride-oxide-coated stents (n=45) or stainless steel stents of otherwise identical design (n=47; control). Baseline characteristics were similar in both groups. At 30 days, no stent thromboses or other adverse events had occurred in either group. Quantitative coronary angiography at 6 months revealed lower late loss (0.55+/-0.63 versus 0.90+/-0.76 mm, P=0.03) and percent diameter stenosis (26+/-17% versus 36+/-24%, P=0.04) in lesions treated with titanium-nitride oxide-coated than in control stents. Binary restenosis was reduced from 33% in the control group to 15% in the titanium-nitride oxide-coated stent group (P=0.07). Intravascular ultrasound studies at 6 months showed smaller neointimal volume in titanium-nitride-oxide-coated stents than in control stents (18+/-21 versus 48+/-28 mm3, P<0.0001). Major adverse cardiac events at 6 months were less frequent in titanium-nitride-oxide-coated stents than in control stent-treated patients (7% versus 27%, P=0.02), largely driven by a reduced need for target-lesion revascularization (7% versus 23%, P=0.07). CONCLUSIONS: Revascularization with titanium-nitride-oxide-coated stents is safe and effective in patients with de novo native coronary artery lesions. Titanium-nitride-oxide-coated stents reduce restenosis and major adverse cardiac events compared with stainless steel stents of otherwise identical design.