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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. Some of the most fundamental decisions on fate and identity, as well as patterning and morphogenesis, are taken during the first days of plant life. Yet, embryos are diverse in structure and function, and embryogenesis in plants is by no means restricted to the zygote - the product of fertilization. In this Primer, we discuss the adventures of the young plant. We will consider what it means to be a plant embryo and how to become one. We will next highlight how the study of early embryogenesis can reveal principles underlying oriented cell division and developmental pattern formation in plants., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 Arabidopsis thaliana BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand binding receptors to prevent BAK1 receptor complex formation., (© 2017 American Society of Plant Biologists. All rights reserved.)

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. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation-fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction., Competing Interests: The authors have declared that no competing interests exist.

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. Like BRI1 itself, SERK3 is a leucine-rich repeat receptor kinase (LRR RK) with a single-pass transmembrane domain. The latest updated laboratory protocol is presented as well as examples of data analysis and typical results obtained. Potential drawbacks of the procedure employed for plant membrane proteins will be pointed out.

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. To decipher these effects, 4-d-old Arabidopsis (Arabidopsis thaliana) roots were studied using microarray analysis. A total of 698 genes, involved in multiple biological processes, were found to be differentially regulated in serk1-3serk3-2 double mutants. About half of these are related to brassinosteroid signaling. The remainder appear to be unlinked to brassinosteroids and related to primary and secondary metabolism. In addition, methionine-derived glucosinolate biosynthesis genes are up-regulated, which was verified by metabolite profiling. The results also show that the gene expression pattern in serk3-2 mutant roots is similar to that of the serk1-3serk3-2 double mutant roots. This confirms the existence of partial redundancy between SERK3 and SERK1 as well as the promoting or repressive activity of a single coreceptor in multiple simultaneously active pathways., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

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., (Copyright © 2015, American Association for the Advancement of Science.)

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. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development.

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. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., Results: Identification of in vivo BAK1 complex partners by LC/ESI-MS/MS uncovered two novel BAK1-interacting RLKs, BIR2 and BIR3. Phosphorylation studies revealed that BIR2 is unidirectionally phosphorylated by BAK1 and that the interaction between BAK1 and BIR2 is kinase-activity dependent. Functional analyses of bir2 mutants show differential impact on BAK1-regulated processes, such as hyperresponsiveness to pathogen-associated molecular patterns (PAMP), enhanced cell death, and resistance to bacterial pathogens, but have no effect on brassinosteroid-regulated growth. BIR2 interacts constitutively with BAK1, thereby preventing interaction with the ligand-binding LRR-RLK FLS2. PAMP perception leads to BIR2 release from the BAK1 complex and enables the recruitment of BAK1 into the FLS2 complex., Conclusions: Our results provide evidence for a new regulatory mechanism for innate immune receptors with BIR2 acting as a negative regulator of PAMP-triggered immunity by limiting BAK1-receptor complex formation in the absence of ligands., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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. Therefore, BRI1-mediated signalling is attractive for mathematical modelling approaches to understand and interpret the spatial and temporal dynamics of signal transduction cascades in planta. To establish such a model, quantitative data sets incorporating local protein concentration, binding affinity and phosphorylation state of the different pathway components are essential. Computational modelling is increasingly employed in studies of plant growth and development. In this section, we have focused on the use of quantitative imaging of fluorescently labelled proteins as an entry point in modelling studies., (© 2013 John Wiley & Sons Ltd.)

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. Recently, it was shown that the initiation of BR signal transduction requires the interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors. In addition, the resolved structure of the BRI1 ectodomain suggested that BRI1-ASSOCIATED KINASE1 [BAK1](SERK3) may constitute a component of the ligand-perceiving receptor complex. Therefore, we investigated the spatial correlation between BRI1 and BAK1(SERK3) in the natural habitat of both leucine-rich repeat receptor-like kinases using comparative colocalization analysis and fluorescence lifetime imaging microscopy. We show that activation of BR signaling by exogenous ligand application resulted in both elevated colocalization between BRI1 and BAK1(SERK3) and an about 50% increase of receptor heterooligomerization in the PM of live Arabidopsis root epidermal cells. However, large populations of BRI1 and BAK1(SERK3) colocalized independently of BRs. Moreover, we could visualize that approximately 7% of the BRI1 PM pool constitutively heterooligomerizes with BAK1(SERK3) in live root cells. We propose that only small populations of PM-located BRI1 and BAK1(SERK3) receptors participate in active BR signaling and that the initiation of downstream signal transduction involves preassembled BRI1-BAK1(SERK3) heterooligomers.

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. A computational approach was employed to predict root growth solely on the basis of BRI1 receptor activity. The developed mathematical model predicts that during normal root growth, few receptors are occupied with ligand. The model faithfully predicts root growth, as observed in bri1 loss-of-function mutants. For roots, it incorporates one stimulatory and two inhibitory modules, while for hypocotyls, a single inhibitory module is sufficient. Root growth as observed when BRI1 is overexpressed can only be predicted assuming that a decrease occurred in the BRI1 half-maximum response values. Root growth appears highly sensitive to variation in BR concentration and much less to reduction in BRI1 receptor level, suggesting that regulation occurs primarily by ligand availability and biochemical activity.

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. Here, we show a unidirectional inhibition of FLS2-mediated immune signaling by BR perception. Unexpectedly, this effect occurred downstream or independently of complex formation with BAK1 and associated downstream phosphorylation. Thus, BAK1 is not rate-limiting in these pathways. BRs also inhibited signaling triggered by the BAK1-independent recognition of the fungal PAMP chitin. Our results suggest a general mechanism operative in plants in which BR-mediated growth directly antagonizes innate immune signaling.

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. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein in vitro. Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state.

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. As a proof of principle, we quantified the Brassinosteroid Insensitive1 (BRI1) receptor fused to GFP, under control of its own promoter. The number of BRI1-GFP molecules per root epidermal cell ranges from 22,000 in the meristem and 130,000 in the elongation zone to 80,000 in the maturation zone, indicating that up to 6-fold differences in BRI1 receptor content exist. In contrast, when taking into account differences in cell size, BRI1-GFP receptor density in the plasma membrane is kept constant at 12 receptors μm⁻² in all cells throughout the meristem and elongation zone. Only the quiescent center and columella cells deviate from this pattern and have 5 to 6 receptors μm⁻². Remarkably, root cell sensitivity toward brassinosteroids appears to coincide with uniform meristem receptor density.

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. In this chapter, we describe two fluorescence fluctuation techniques, fluorescence cross-correlation spectroscopy and photon counting histogram analysis, to study the oligomerization state of receptor kinases in living plant cells in a quantitative manner.

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. This chapter describes two methods to study receptor mobility in the plasma membrane. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). Especially, the combination of FRAP and FCS provides a better insight into plasma membrane receptor mobility.

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. We found that GW7647 increased PPARalpha binding to 4220 binding regions. GW7647-induced binding regions showed a bias around the transcription start site and most contained a predicted PPAR binding motif. Several genes known to be regulated by PPARalpha, such as ACOX1, SULT2A1, ACADL, CD36, IGFBP1 and G0S2, showed GW7647-induced PPARalpha binding to their promoter. A GW7647-induced PPARalpha-binding region was also assigned to SREBP-targets HMGCS1, HMGCR, FDFT1, SC4MOL, and LPIN1, expression of which was induced by GW7647, suggesting cross-talk between PPARalpha and SREBP signaling. Our data furthermore demonstrate interaction between PPARalpha and STAT transcription factors in PPARalpha-mediated transcriptional repression, and suggest interaction between PPARalpha and TBP, and PPARalpha and C/EBPalpha in PPARalpha-mediated transcriptional activation. Overall, our analysis leads to important new insights into the mechanisms and impact of transcriptional regulation by PPARalpha in human liver and highlight the importance of cross-talk with other transcription factors.

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. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

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. SERK1 and SERK2 are essential for male microsporogenesis, again independent from BR. SERK5 does not appear to have any function under the conditions tested. Here, we show that the different SERK members are only redundant in pairs, whereas higher order mutant combinations only show additive phenotypes. Surprisingly, SERK members that are redundant within one are not redundant in another pathway. We also show that this evolution of functional pairs occurred by a change in protein function and not by differences in spatial expression. We propose that, in plants, closely related receptor kinases have a minimal homo- or heterodimeric configuration to achieve specificity.

The Arabidopsis thaliana primordia timing (pt) mutant was transformed with an AtSERK1::GUS construct. Liquid cultures of this line were used to study the relationship between somatic embryogenesis and the expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (AtSERK1) as a marker for cells competent to form embryos. In order to search for the expression of AtSERK1::GUS during early stages of somatic embryogenesis, histochemical as well as immunochemical approaches were used for the detection of beta-glucuronidase (GUS). Four sites of AtSERK1 expression were found in the embryogenic cultures: in embryogenic callus, where primary somatic embryos developed; in the basal parts of primary somatic embryos; in the outer layers of cotyledons of primary somatic embryos where secondary embryos were formed; and in provascular and vascular strands of developing somatic embryos. The in vitro expression of AtSERK1::GUS coincides with embryogenic development up to the heart-shaped stage. Prior to the expression in embryos, AtSERK1 was expressed in single cells and small cell clusters, indicating that AtSERK1 indeed marks embryogenic competence. Its expression in (pro)vascular strands, suggests that embryogenic cells in tissue culture retain at least in part their original identity.

Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes.

The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes.

Brassinosteroid (BR) signaling requires the BIN2 kinase-promoted interaction of 14-3-3 proteins with the transcriptional regulators BZR1 and BZR2, which are subsequently redistributed to the cytoplasm by BRs. In this issue of Developmental Cell, Gampala et al. show that this redistribution may fine-tune BR responses and serve to crosstalk with other signaling pathways.

Programmed cell death (PCD) is a common host response to microbial infection [1-3]. In plants, PCD is associated with immunity to biotrophic pathogens, but it can also promote disease upon infection by necrotrophic pathogens [4]. Therefore, plant cell-suicide programs must be strictly controlled. Here we demonstrate that the Arabidopsis thaliana Brassinosteroid Insensitive 1 (BRI1)-associated receptor Kinase 1 (BAK1), which operates as a coreceptor of BRI1 in brassinolide (BL)-dependent plant development, also regulates the containment of microbial infection-induced cell death. BAK1-deficient plants develop spreading necrosis upon infection. This is accompanied by production of reactive oxygen intermediates and results in enhanced susceptibility to necrotrophic fungal pathogens. The exogenous application of BL rescues growth defects of bak1 mutants but fails to restore immunity to fungal infection. Moreover, BL-insensitive and -deficient mutants do not exhibit spreading necrosis or enhanced susceptibility to fungal infections. Together, these findings suggest that plant steroid-hormone signaling is dispensable for the containment of infection-induced PCD. We propose a novel, BL-independent function of BAK1 in plant cell-death control that is distinct from its BL-dependent role in plant development.

The SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (SERK1) gene is expressed in the procambium of the vascular bundles in roots, hypocotyls, and inflorescence stems. In younger parts of roots and hypocotyls, SERK1 expression was less restricted and was also observed in protoxylem cells, immature metaxylem cells and phloem companion cells. In roots, SERK1 expression was first detected in root vascular stem cells and was notably absent from the QC. In general, the SERK1 protein level as visualized by expression of a SERK1-YFP fusion protein closely followed the pattern of gene expression. In hypocotyls, prolonged application of 2,4-D resulted in extensive unorganized proliferation of SERK1 expressing cells originating from the procambium and pericycle. In roots, 2,4-D treatment results in an increase in SERK1 transcription that results in a moderate increase in the amount of SERK1-YFP fusion protein. The restricted vascular pattern of SERK1 expression in roots remains unaffected after 2,4-D treatment.

Brassinosteroids (BRs) function as signaling molecules in plants and are involved in processes such as stem elongation, vascular differentiation, male fertility, timing of senescence and flowering, leaf development, and resistance to biotic and abiotic stresses. Unlike animal steroids that are perceived by nuclear receptors, BRs are perceived by transmembrane receptor kinase complexes that initiate a phosphorylation-mediated signaling cascade to transduce the steroid signal. BR binding to the extracellular domain of the receptor BRI1 induces kinase activation and hetero-oligomerization with the second transmembrane kinase BAK1. Activated BRI1 then dissociates from the BRI1-interacting protein BKI1, a newly identified negative regulator of BR signaling. In the presence of BR, the kinase BIN2, which is the Arabidopsis homolog of GSK3 (glycogen synthase kinase 3), is inhibited by an unknown mechanism, leading to dephosphorylation of BES1 and BZR1 inside the nucleus. This allows BES1 and BZR1 to homodimerize or combine with other transcription factors to bind to promoters of BR-responsive genes. These studies of BR signaling in plants have revealed signaling pathways that are distinctly different from related ones operating in animal cells.

Apomixis in plants is a form of clonal reproduction through seeds. A BAC clone linked to apomictic reproduction in Paspalum simplex was used to locate the apomixis locus on meiotic chromosome preparations. Fluorescent in situ hybridisation revealed the existence of a single locus embedded in a heterochromatin-poor region not adjacent to the centromere. We report here for the first time information regarding the sequencing of a large DNA clone from the apomixis locus. The presence of two genes whose rice homologs were mapped on the telomeric part of the long arm of rice chromosome 12 confirmed the strong synteny between the apomixis locus of P. simplex with the related area of the rice genome at the map level. Comparative analysis of this region with rice as representative of a sexual species revealed large-scale rearrangements due to transposable elements and small-scale rearrangements due to deletions and single point mutations. Both types of rearrangements induced the loss of coding capacity of large portions of the "apomictic" genes compared to their rice homologs. Our results are discussed in relation to the use of rice genome data for positional cloning of apomixis genes and to the possible role of rearranged supernumerary genes in the apomictic process of P. simplex.

MADS-box transcription factors are major regulators of development in flowering plants. The factors act in a combinatorial manner, either as homo- or heterodimers, and they control floral organ formation and identity and many other developmental processes through a complex network of protein-protein and protein-DNA interactions. Despite the fact that many studies have been carried out to elucidate MADS-box protein dimerization by yeast systems, very little information is available on the behaviour of these molecules in planta. Here, evidence for specific interactions between the petunia MADS-box proteins FBP2, FBP11, and FBP24 is provided in vivo. The dimers identified in yeast for the ovule-specific FBP24 protein have been confirmed in living plant cells by means of fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy and, in addition, some of the most likely, less stable homo- and heterodimers were identified. This in vivo approach revealed that particular dimers could only be detected in specific sub-nuclear domains. In addition, evidence for the in planta assembly of these ovule-specific MADS-box transcription factors into higher-order complexes is provided.

Leucine-rich repeat (LRR)-containing transmembrane receptor-like kinases (RLKs) are important components of plant signal transduction. The Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 (AtSERK1) is an LRR-RLK proposed to participate in a signal transduction cascade involved in embryo development. By yeast two-hybrid screening we identified AtCDC48, a homologue of the mammalian AAA-ATPase p97 and GF14lambda, a member of the Arabidopsis family of 14-3-3 proteins as AtSERK1 interactors. In vitro, the AtSERK1 kinase domain is able to transphosphorylate and bind both AtCDC48 and GF14lambda. In yeast, AtCDC48 interacts with GF14lambda and with the PP2C phosphatase KAPP. In plant protoplasts AtSERK1 interacts with GF14lambda.

In Arabidopsis thaliana brassinosteroid (BR), perception is mediated by two Leu-rich repeat receptor-like kinases, BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE3 [AtSERK3]). Genetic, biochemical, and yeast (Saccharomyces cerevisiae) interaction studies suggested that the BRI1-BAK1 receptor complex initiates BR signaling, but the role of the BAK1 receptor is still not clear. Using transient expression in protoplasts of BRI1 and AtSERK3 fused to cyan and yellow fluorescent green fluorescent protein variants allowed us to localize each receptor independently in vivo. We show that BRI1, but not AtSERK3, homodimerizes in the plasma membrane, whereas BRI1 and AtSERK3 preferentially heterodimerize in the endosomes. Coexpression of BRI1 and AtSERK3 results in a change of the steady state distribution of both receptors because of accelerated endocytosis. Endocytic vesicles contain either BRI1 or AtSERK3 alone or both. We propose that the AtSERK3 protein is involved in changing the equilibrium between plasma membrane-located BRI1 homodimers and endocytosed BRI1-AtSERK3 heterodimers.

The ABC model for floral development was proposed more than 10 years ago and since then many studies have been performed on model species, such as Arabidopsis thaliana, Antirrhinum majus, and many other species in order to confirm this hypothesis. This led to additional information on flower development and to more complex molecular models. AGAMOUS (AG) is the only C type gene in Arabidopsis and it is responsible for stamen and carpel development as well as floral determinacy. LLAG1, an AG homologue from lily (Lilium longiflorum Thunb.) was isolated by screening a cDNA library derived from developing floral buds. The deduced amino acid sequence revealed the MIKC structure and a high homology in the MADS-box among AG and other orthologues. Phylogenetic analysis indicated a close relationship between LLAG1 and AG orthologues from monocot species. Spatial expression data showed LLAG1 transcripts exclusively in stamens and carpels, constituting the C domain of the ABC model. Functional analysis was carried out in Arabidopsis by overexpression of LLAG1 driven by the CaMV35S promoter. Transformed plants showed homeotic changes in the two outer floral whorls with some plants presenting the second whorl completely converted into stamens. Altogether, these data strongly indicated the functional homology between LLAG1 and AG.

A seed marks the transition between two developmental states; a plant is an embryo during seed formation, whereas it is a seedling after emergence from the seed. Two factors have been identified in Arabidopsis that play a role in establishment of repression of the embryonic state: PKL (PICKLE), which codes for a putative CHD3 chromatin remodeling factor, and gibberellin (GA), a plant growth regulator. Previous observations have also suggested that PKL mediates some aspects of GA responsiveness in the adult plant. To investigate possible mechanisms by which PKL and GA might act to repress the embryonic state, we further characterized the ability of PKL and GA to repress embryonic traits and reexamined the role of PKL in mediating GA-dependent responses. We found that PKL acts throughout the seedling to repress expression of embryonic traits. Although the ability of pkl seedlings to express embryonic traits is strongly induced by inhibiting GA biosynthesis, it is only marginally responsive to abscisic acid and SPY (SPINDLY), factors that have previously been demonstrated to inhibit GA-dependent responses during germination. We also observed that pkl plants exhibit the phenotypic hallmarks of a mutation in a positive regulator of a GA response pathway including reduced GA responsiveness and increased synthesis of bioactive GAs. These observations indicate that PKL may mediate a subset of GA-dependent responses during shoot development.

In the postgenomic era it is essential that protein sequences are annotated correctly in order to help in the assignment of their putative functions. Over 1300 proteins in current protein sequence databases are predicted to contain a PAS domain based upon amino acid sequence alignments. One of the problems with the current annotation of the PAS domain is that this domain exhibits limited similarity at the amino acid sequence level. It is therefore essential, when using proteins with low-sequence similarities, to apply profile hidden Markov model searches for the PAS domain-containing proteins, as for the PFAM database. From recent 3D X-ray and NMR structures, however, PAS domains appear to have a conserved 3D fold as shown here by structural alignment of the six representative 3D-structures from the PDB database. Large-scale modelling of the PAS sequences from the PFAM database against the 3D-structures of these six structural prototypes was performed. All 3D models generated (> 5700) were evaluated using prosaii. We conclude from our large-scale modelling studies that the PAS and PAC motifs (which are separately defined in the PFAM database) are directly linked and that these two motifs form the PAS fold. The existing subdivision in PAS and PAC motifs, as used by the PFAM and SMART databases, appears to be caused by major differences in sequences in the region connecting these two motifs. This region, as has been shown by Gardner and coworkers for human PAS kinase (Amezcua, C.A., Harper, S.M., Rutter, J. & Gardner, K.H. (2002) Structure 10, 1349-1361, [1]), is very flexible and adopts different conformations depending on the bound ligand. Some PAS sequences present in the PFAM database did not produce a good structural model, even after realignment using a structure-based alignment method, suggesting that these representatives are unlikely to have a fold resembling any of the structural prototypes of the PAS domain superfamily.

From an enhancer trap screen for genes expressed in Arabidopsis embryos, we identified a gene expressed from the octant stage onward in the boundary between the two presumptive cotyledons and in a variety of postembryonic organ and meristem boundaries. This gene, CUP-SHAPED COTYLEDON3 (CUC3), encodes a putative NAC-domain transcription factor that is homologous with CUC1 and CUC2. Analysis of a CUC3 hypomorph and a putative cuc3 null mutant indicates that CUC3 function is partially redundant with that of CUC1 and CUC2 in the establishment of the cotyledon boundary and the shoot meristem, thus revealing an even higher degree of redundancy in this class of genes than was thought previously. The CUC3 expression pattern, the cuc3 phenotypes, and CUC3 expression in a series of shoot meristem mutants and transgenes suggest a primary role for CUC3 in the establishment of boundaries that contain cells with low proliferation and/or differentiation rates. The CUC-mediated establishment of such boundaries may be essential for the initiation of shoot meristems.

Seed formation in flowering plants requires meiosis of the megaspore mother cell (MMC) inside the ovule, selection of a megaspore that undergoes mitosis to form an embryo sac, and double fertilization to initiate embryo and endosperm formation. During apomixis, or asexual seed formation, in Hieracium ovules, a somatic aposporous initial (AI) cell divides to form a structurally variable aposporous embryo sac and embryo. This entire process, including endosperm development, is fertilization independent. Introduction of reproductive tissue marker genes into sexual and apomictic Hieracium showed that AI cells do not express a MMC marker. Spatial and temporal gene expression patterns of other introduced genes were conserved commencing with the first nuclear division of the AI cell in apomicts and the mitotic initiation of embryo sac formation in sexual plants. Conservation in expression patterns also occurred during embryo and endosperm development, indicating that sexuality and apomixis are interrelated pathways that share regulatory components. The induction of a modified sexual reproduction program in AI cells may enable the manifestation of apomixis in HIERACIUM:

The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does not interact with a catalytically inactive kinase mutant. Using mutant AtSERK1 proteins in which Thr 462, Thr 463, and Thr 468 in the A-loop of the AtSERK1 kinase domain were replaced by alanines, we show that phosphorylation status of the receptor is involved in interaction with KAPP. KAPP and AtSERK1 cDNAs were fused to two different variants of green fluorescent protein (GFP), the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP). Both KAPP and AtSERK1 proteins are found at the plasma membrane. Our results show that AtSERK1-CFP becomes sequestered into intracellular vesicles when transiently coexpressed with KAPP-YFP proteins. AtSERK1(T463A)-CFP and AtSERK1(3T-->A)-CFP proteins were partially sequestered intracellularly in the absence of KAPP-YFP protein, suggesting an active role for KAPP dephosphorylation of threonine residues in the AtSERK1 A-loop in receptor internalization. The interaction between the KAPP-CFP/YFP and AtSERK1-CFP/YFP fusion proteins was investigated with fluorescence spectral imaging microscopy (FSPIM). Our results show that AtSERK1-CFP and KAPP-YFP proteins are colocalized at the plasma membrane but only show fluorescence energy transfer (FRET) indicative of physical interaction in intracellular vesicles. These results suggest that KAPP is an integral part of the AtSERK1 endocytosis mechanism.

Arabinogalactan-protein (AGP) epitopes are known to display developmentally regulated patterns of expression in several plant tissues. Therefore, AGPs have been suggested to play a role in plant development. Somatic embryogenesis is regulated by AGPs as well as by EP3 endochitinases. Using four different methods we have analysed the composition of AGPs in immature carrot seeds. The results obtained show that: (1) the native electrophoretic mobility of such AGPs changes during development; (2) AGP epitopes in immature seeds are developmentally regulated; (3) enzymatically released fragments of AGPs show that the composition of these molecules changes as a function of development; and (4) the biological activity of AGPs on the formation of somatic embryos changes depending on the age of the seeds. Our results suggest that degradation of maternally derived AGPs occurs after fertilization, while cellularization of the endosperm leads to synthesis of a new set of AGPs. The presence of an endochitinase cleavage site as well as the capacity to increase somatic embryogenesis only occurred in AGPs that were isolated from seeds in which the endosperm had been cellularized. Apparently, both EP3 endochitinases and somatic embryogenesis-promoting AGPs are developmentally regulated in immature carrot seeds.

Zygotic embryos of three Arabidopsis thaliana (L.) Heynh. mutants lacking an embryonic shoot apical meristem (SAM), shoot meristemless (stm), wuschel (wus) and zwille/pinhead (zll/pnh) were used as explants to establish embryogenic cell cultures. Somatic embryos of all three mutants showed the same mutant phenotypes as their zygotic equivalents. These results provide genetic evidence that the developmental program of somatic and zygotic embryos is indistinguishable. They also suggest that a functional SAM is not required for somatic embryogenic cell formation in Arabidopsis.

Plant chitinases (EC 3.2.1.14) belong to relatively large gene families subdivided in classes that suggest class-specific functions. They are commonly induced upon the attack of pathogens and by various sources of stress, which led to associating them with plant defense in general. However, it is becoming apparent that most of them display several functions during the plant life cycle, including taking part in developmental processes such as pollination and embryo development. The number of chitinases combined with their multiple functions has been an obstacle to a better understanding of their role in plants. It is therefore important to identify and inventory all chitinase genes of a plant species to be able to dissect their function and understand the relations between the different classes. Complete sequencing of the Arabidopsis genome has made this task feasible and we present here a survey of all putative chitinase-encoding genes accompanied by a detailed analysis of their sequence. Based on their characteristics and on studies on other plant chitinases, we propose an overview of their possible functions as well as modified annotations for some of them.