Plant embryogenesis.

Land plants are called 'embryophytes' and thus, their collective name is defined by their ability to form embryos. Indeed, embryogenesis is a widespread phenomenon in plants, and much of our diet is composed of embryos (just think of grains, beans or nuts; Figure 1). However, in addition to embryos as a source of nutrition, they are also a fascinating study object.

The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1.

BAK1 is a coreceptor and positive regulator of multiple ligand binding leucine-rich repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity, and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here, we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in BIR3 also has a strong negative impact on BR signaling.

Identification of Brassinosteroid Signaling Complexes by Coimmunoprecipitation and Mass Spectrometry.

A combination of coimmunoprecipitation (coIP) of tagged proteins followed by protein identification and quantitation using Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) has proven to be a reliable method to qualitatively characterize membrane-bound receptor complexes from plants. Success depends on a range of parameters, such as abundance and stability of the complex and functionality of the tagged receptors, efficiency of the protein complex isolation procedure, MS equipment, and analysis software in use. In this Chapter, we focus on the use of one of the green fluorescent protein-tagged receptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, of which SERK3, also known as BRASSINOSTEROID INSENSITIVE1 (BRI1) ASSOCIATED KINASE1 (BAK1), is a coreceptor of BRI1.

Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots.

Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters.

Transcriptional Analysis of serk1 and serk3 Coreceptor Mutants.

Somatic embryogenesis receptor kinases (SERKs) are ligand-binding coreceptors that are able to combine with different ligand-perceiving receptors such as BRASSINOSTEROID INSENSITIVE1 (BRI1) and FLAGELLIN-SENSITIVE2. Phenotypical analysis of serk single mutants is not straightforward because multiple pathways can be affected, while redundancy is observed for a single phenotype. For example, serk1serk3 double mutant roots are insensitive toward brassinosteroids but have a phenotype different from bri1 mutant roots.

Plant receptor complexes.

In this issue of Science Signaling, Somssich and co-workers use fluorescence techniques to show the dynamics that occur during the activation of two different receptor complexes in living plant cells.

Proteomics analysis of the zebrafish skeletal extracellular matrix.

The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species.

Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors.

Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function.

The leucine-rich repeat receptor kinase BIR2 is a negative regulator of BAK1 in plant immunity.

Background: Transmembrane leucine-rich repeat (LRR) receptors are commonly used innate immune receptors in plants and animals but can also sense endogenous signals to regulate development. BAK1 is a plant LRR-receptor-like kinase (RLK) that interacts with several ligand-binding LRR-RLKs to positively regulate their functions. BAK1 is involved in brassinosteroid-dependent growth and development, innate immunity, and cell-death control by interacting with the brassinosteroid receptor BRI1, immune receptors, such as FLS2 and EFR, and the small receptor kinase BIR1, respectively.

Visualization of BRI1 and BAK1(SERK3) membrane receptor heterooligomers during brassinosteroid signaling.

The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main ligand-perceiving receptor for brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana). Binding of BRs to the ectodomain of plasma membrane (PM)-located BRI1 receptors initiates an intracellular signal transduction cascade that influences various aspects of plant growth and development. Even though the major components of BR signaling have been revealed and the PM was identified as the main site of BRI1 signaling activity, the very first steps of signal transmission are still elusive.

Computational modelling of the BRI1 receptor system.

Computational models are useful tools to help understand signalling pathways in plant cells. A systems biology approach where models and experimental data are combined can provide experimentally verifiable predictions and novel insights. The brassinosteroid insensitive 1 (BRI1) receptor is one of the best-understood receptor systems in Arabidopsis with clearly described ligands, mutants and associated phenotypes.

The Arabidopsis thaliana SERK1 kinase domain spontaneously refolds to an active state in vitro.

Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK's) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain.

A mathematical model for BRASSINOSTEROID INSENSITIVE1-mediated signaling in root growth and hypocotyl elongation.

Brassinosteroid (BR) signaling is essential for plant growth and development. In Arabidopsis (Arabidopsis thaliana), BRs are perceived by the BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor. Root growth and hypocotyl elongation are convenient downstream physiological outputs of BR signaling.

Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1.

Plants and animals use innate immunity as a first defense against pathogens, a costly yet necessary tradeoff between growth and immunity. In Arabidopsis, the regulatory leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1 combines with the LRR-RLKs FLS2 and EFR in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and the LRR-RLK BRI1 in brassinosteroid (BR)-mediated growth. Therefore, a potential tradeoff between these pathways mediated by BAK1 is often postulated.

Fluorescence Correlation Spectroscopy and Fluorescence Recovery After Photobleaching to study receptor kinase mobility in planta.

Plasma-membrane-localized receptor kinases are essential for cell-cell communication and as sensors for the extracellular environment. Receptor function is dependent on their distribution in the membrane and interaction with other proteins that are either membrane-localized, present in the cytoplasm, or in the extracellular space. The organized distribution and mobility of receptor kinases is, therefore, thought to regulate the efficiency of downstream signaling.

Fluorescence fluctuation analysis of receptor kinase dimerization.

Receptor kinases are essential for the cellular perception of signals. The classical model for activation of the receptor kinase involves dimerization, induced by the binding of the ligand. The mechanisms by which plant receptors transduce signals across the cell surface are largely unknown but plant receptors seem to dimerize as well.

Quantification of the brassinosteroid insensitive1 receptor in planta.

In plants, green fluorescent protein (GFP) is routinely used to determine the subcellular location of fusion proteins. Here, we show that confocal imaging can be employed to approximate the number of GFP-labeled protein molecules present in living Arabidopsis (Arabidopsis thaliana) root cells. The technique involves calibration with soluble GFP to provide a usable protein concentration range within the confocal volume of the microscope.

Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle.

Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system.

Profiling of promoter occupancy by PPARalpha in human hepatoma cells via ChIP-chip analysis.

The transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) is an important regulator of hepatic lipid metabolism. While PPARalpha is known to activate transcription of numerous genes, no comprehensive picture of PPARalpha binding to endogenous genes has yet been reported. To fill this gap, we performed Chromatin immunoprecipitation (ChIP)-chip in combination with transcriptional profiling on HepG2 human hepatoma cells treated with the PPARalpha agonist GW7647.

Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE proteins serve brassinosteroid-dependent and -independent signaling pathways.

The Arabidopsis (Arabidopsis thaliana) SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes belong to a small family of five plant receptor kinases that are involved in at least five different signaling pathways. One member of this family, BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED KINASE1 (BAK1), also known as SERK3, is the coreceptor of the brassinolide (BR)-perceiving receptor BRI1, a function that is BR dependent and partially redundant with SERK1. BAK1 (SERK3) alone controls plant innate immunity, is also the coreceptor of the flagellin receptor FLS2, and, together with SERK4, can mediate cell death control, all three in a BR-independent fashion.