ABSTRACT
In many flowering plants, asymmetric division of the zygote generates apical and basal cells with different fates. In Arabidopsis thaliana, the apical cell generates the embryo while the basal cell divides anticlinally, leading to a suspensor of six to nine cells that remain extra-embryonic and eventually senesce. In some genetic backgrounds, or upon ablation of the embryo, suspensor cells can undergo periclinal cell divisions and eventually form a second twin embryo. Likewise, embryogenesis can be induced from somatic cells by various genes, but the relationship with suspensor-derived embryos is unclear. Here, we addressed the nature of the suspensor to embryo fate transformation and its genetic triggers. We expressed most known embryogenesis-inducing genes specifically in suspensor cells. We next analyzed morphology and fate-marker expression in embryos in which suspensor division was activated by different triggers to address the developmental paths towards reprogramming. Our results show that reprogramming of Arabidopsis suspensor cells towards embryonic identity is a specific cellular response that is triggered by defined regulators, follows a conserved developmental trajectory and shares similarity to the process of somatic embryogenesis from post-embryonic tissues.
Subject(s)
Arabidopsis Proteins/metabolism , Seeds/metabolism , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Cellular Reprogramming/genetics , Cellular Reprogramming/physiology , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Seeds/geneticsABSTRACT
Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.
Subject(s)
Arabidopsis/chemistry , Arabidopsis/cytology , Cell Polarity/physiology , Plant Cells/physiology , Polymerization , Protein Domains , Animals , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Axin Protein/chemistry , Axin Protein/metabolism , Bryopsida/chemistry , Bryopsida/cytology , Bryopsida/genetics , Bryopsida/growth & development , COS Cells , Chlorocebus aethiops , Dishevelled Proteins/metabolism , HEK293 Cells , Humans , Marchantia/chemistry , Marchantia/cytology , Marchantia/genetics , Marchantia/growth & development , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Plants, Genetically Modified , Repressor Proteins/metabolism , Wnt Signaling PathwayABSTRACT
Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases involved in several, seemingly unrelated, plant-signaling pathways. In Arabidopsis thaliana, functional and genetic analysis of four SERK proteins has indicated that they are only partly redundant; their functions overlap but each performs a specific subset of signaling roles. The molecular basis for the functional specificity within this highly homologous protein family is currently not known. Sequence analysis of SERK proteins from different plant species indicates that the SERKs are a highly conserved protein family present in monocots, dicots, and non-vascular plants. Residues in the extracellular domain that are important for interaction with other receptor kinases are highly conserved, even among SERK members without a function in the corresponding pathways. SERK2, for instance, does not function in the brassinosteroid pathway, does not interact with BRI1, but is conserved in its BRI1-interacting domain. Further sequence analysis indicates that SERK3/BAK1 and SERK4/BKK1 have diverged from the original SERK protein in both their extracellular and cytoplasmic domains. Functional analysis of chimeric SERK proteins shows that different domains provide the SERK proteins with different functional specificity. For instance, the SERK1 or SERK2 extracellular domains are essential for SERK function in male sporogenesis, while the SERK3 extracellular and cytoplasmic domains are essential for SERK3 activity in brassinosteroid and flagellin signaling. The emerging picture is that SERKs are ancient genes, whose products have been recruited as co-receptors in the newly evolved signaling pathways. The SERK ligand-binding and protein-protein interaction domains are highly conserved, allowing all SERKs to form complexes, albeit with different affinity. However, specific functional residues must have been altered, in both the extracellular and intracellular domains, to allow for the observed differences in functionality.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/chemistry , Arabidopsis/enzymology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Arabidopsis/embryology , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Computational Biology , Germ Cells, Plant/enzymology , Germ Cells, Plant/growth & development , Molecular Sequence Data , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Sequence AlignmentABSTRACT
Brassinosteroids (BRs) are key regulators in plant growth and development. The main BR-perceiving receptor in Arabidopsis (Arabidopsis thaliana) is BRASSINOSTEROID INSENSITIVE1 (BRI1). Seedling root growth and hypocotyl elongation can be accurately predicted using a model for BRI1 receptor activity. Genetic evidence shows that non-ligand-binding coreceptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family are essential for BRI1 signal transduction. A relatively simple biochemical model based on the properties of SERK loss-of-function alleles explains complex physiological responses of the BRI1-mediated BR pathway. The model uses BRI1-BR occupancy as the central estimated parameter and includes BRI1-SERK interaction based on mass action kinetics and accurately describes wild-type root growth and hypocotyl elongation. Simulation studies suggest that the SERK coreceptors primarily act to increase the magnitude of the BRI1 signal. The model predicts that only a small number of active BRI1-SERK complexes are required to carry out BR signaling at physiological ligand concentration. Finally, when calibrated with single mutants, the model predicts that roots of the serk1serk3 double mutant are almost completely brassinolide (BL) insensitive, while the double mutant hypocotyls remain sensitive. This points to residual BRI1 signaling or to a different coreceptor requirement in shoots.
Subject(s)
Arabidopsis Proteins/metabolism , Models, Theoretical , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Algorithms , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Brassinosteroids/pharmacology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hypocotyl/drug effects , Hypocotyl/genetics , Hypocotyl/growth & development , Microscopy, Confocal , Mutation , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/growth & development , Plants, Genetically Modified , Protein Binding , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Steroids, Heterocyclic/pharmacologyABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Brassinosteroids/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Brefeldin A/metabolism , Brefeldin A/pharmacology , Cell Membrane/metabolism , Microscopy, Fluorescence/methods , Plant Roots/cytology , Plant Roots/metabolism , Plants, Genetically Modified , Protein Kinases/genetics , Protein Multimerization , Protein Serine-Threonine Kinases/genetics , Signal Transduction , Triazoles/pharmacologyABSTRACT
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.
Subject(s)
Arabidopsis/immunology , Arabidopsis/microbiology , Brassinosteroids/pharmacology , Plant Immunity/drug effects , Pseudomonas/metabolism , Receptors, Pattern Recognition/metabolism , Signal Transduction/drug effects , Arabidopsis/drug effects , Arabidopsis/enzymology , Arabidopsis Proteins/metabolism , Enzyme Activation/drug effects , Flagellin/pharmacology , Plant Immunity/immunology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Pseudomonas/drug effects , Signal Transduction/immunology , Steroids, Heterocyclic/pharmacologyABSTRACT
Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.
Subject(s)
Arabidopsis Proteins/immunology , Arabidopsis/enzymology , Arabidopsis/immunology , Immunity, Innate , Oomycetes/pathogenicity , Plant Diseases/immunology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/immunology , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Ligands , Molecular Sequence Data , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Oomycetes/immunology , Peptides/genetics , Peptides/metabolism , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Pseudomonas syringae/immunology , Pseudomonas syringae/pathogenicity , Recombinant Fusion Proteins/metabolism , Signal TransductionABSTRACT
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⻲ 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⻲. Remarkably, root cell sensitivity toward brassinosteroids appears to coincide with uniform meristem receptor density.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Protein Kinases/metabolism , Arabidopsis/cytology , Blotting, Western , Cell Size , Green Fluorescent Proteins/metabolism , Meristem/cytology , Meristem/metabolism , Microscopy, Confocal , Organ Specificity , Plant Epidermis/cytology , Plant Epidermis/metabolism , Recombinant Fusion Proteins/metabolism , Reproducibility of Results , Seedlings/metabolismABSTRACT
The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.
Subject(s)
DNA, Mitochondrial/genetics , Mammals/classification , Animals , DNA, Mitochondrial/analysis , DNA, Mitochondrial/blood , Electrophoresis, Capillary , Humans , Mammals/genetics , Polymerase Chain Reaction , Polymorphism, Genetic , Sequence Analysis, DNA , Species SpecificityABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Brassinosteroids , Cell Death , Cholestanols/metabolism , Mutation , Phenotype , Plant Diseases , Plant Infertility , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Steroids, Heterocyclic/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/immunology , Protein Serine-Threonine Kinases/physiology , Pseudomonas syringae/immunology , Arabidopsis/microbiology , Arabidopsis/physiology , Brassinosteroids , Cell Death/physiology , Cholestanols/metabolism , Gene Expression Profiling , Plant Diseases , Plant Growth Regulators/metabolism , Pseudomonas syringae/physiology , Signal Transduction/physiology , Steroids, Heterocyclic/metabolismABSTRACT
The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family of plasma membrane receptors consists of five closely related members. The SERK1 and SERK2 genes show a complex expression pattern throughout development. Both are expressed in anther primordia up to the second parietal division. After this point, expression ceases in the sporocytes and is continued in the tapetum and middle layer precursors. Single knockout mutants of SERK1 and SERK2 show no obvious phenotypes. Double mutants of SERK1 and SERK2 are completely male sterile due to a failure in tapetum specification. Fertility can be restored by a single copy of either gene. The SERK1 and SERK2 proteins can form homodimers or heterodimers in vivo, suggesting they are interchangeable in the SERK1/SERK2 signaling complex.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/physiology , Protein Serine-Threonine Kinases/physiology , Spores/growth & development , Alleles , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Base Sequence , DNA Primers , Genetic Complementation Test , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Plasmids , Protein Kinases , Protein Serine-Threonine Kinases/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We report here that the pea early nodulin genes PsENOD5 and PsENOD12A are induced during the interaction of pea roots and the endomycorrhizal fungus Gigaspora margarita. Using the pea nodulation mutant Sparkle-R25, which is mutated in SYM8, it is shown that SYM8 is essential for the induction of PsENOD5 and PsENOD12Ain pea roots interacting either with Rhizobium or the endomycorrhizal fungus Gigaspora margarita. Our results suggest that mycorrhizal signals activate a signal transduction cascade sharing at least one common step with the Nod factor-activated signal transduction cascade.