Not So Dry After All: DRY Mutants of the AT1A Receptor and H1 Receptor Can Induce G-Protein-Dependent Signaling.

G-protein-coupled receptors (GPCRs) are seven transmembrane spanning receptors that regulate a wide array of intracellular signaling cascades in response to various stimuli. To do so, they couple to different heterotrimeric G proteins and adaptor proteins, including arrestins. Importantly, arrestins were shown to regulate GPCR signaling through G proteins, as well as promote G protein-independent signaling events. Several research groups have reported successful isolation of exclusively G protein-dependent and arrestin-dependent signaling downstream of GPCR activation using biased agonists or receptor mutants incapable of coupling to either arrestins or G proteins. In the latter category, the DRY mutant of the angiotensin II type 1 receptor was extensively used to characterize the functional selectivity downstream of AT1AR. In an attempt to understand histamine 1 receptor signaling, we characterized the signaling capacity of the H1R DRY mutant in a panel of dynamic, live cell biosensor assays, including arrestin recruitment, heterotrimeric G protein activation, Ca2+ signaling, protein kinase C activity, GTP binding of RhoA, and activation of ERK1/2. Here, we show that both H1R DRY mutant and the AT1AR DRY mutant are capable of efficient activation of G protein-mediated signaling. Therefore, contrary to the common belief, they do not constitute suitable tools for the dissection of the arrestin-mediated, G protein-independent signaling downstream of these receptors.

Nod factor receptors form heteromeric complexes and are essential for intracellular infection in medicago nodules.

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.

Interaction of Medicago truncatula lysin motif receptor-like kinases, NFP and LYK3, produced in Nicotiana benthamiana induces defence-like responses.

Receptor(-like) kinases with Lysin Motif (LysM) domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP and LYK3 mediate signalling upon perception of bacterial lipo-chitooligosaccharides, termed Nod factors, during the establishment of mutualism with nitrogen-fixing rhizobia. However, little is still known about the exact activation and signalling mechanisms of MtNFP and MtLYK3. We aimed at investigating putative molecular interactions of MtNFP and MtLYK3 produced in Nicotiana benthamiana. Surprisingly, heterologous co-production of these proteins resulted in an induction of defence-like responses, which included defence-related gene expression, accumulation of phenolic compounds, and cell death. Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves. Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like) kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response. Importantly, structure-function studies revealed that the MtNFP intracellular region, specific features of the MtLYK3 intracellular region (including several putative phosphorylation sites), and MtLYK3 and AtCERK1 kinase activity were indispensable for cell death induction, thereby mimicking the structural requirements of nodulation or chitin-induced signalling. The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like) kinases. Notably, the conserved structural requirements for MtNFP and MtLYK3 biological activity in M. truncatula (nodulation) and in N. benthamiana (cell death induction) indicates the relevance of the latter system for studies on these, and potentially other symbiotic LysM receptor-like kinases.

Gamete fertility and ovule number variation in selfed reciprocal F1 hybrid triploid plants are heritable and display epigenetic parent-of-origin effects.

Polyploidy and hybridization play major roles in plant evolution and reproduction. To investigate the reproductive effects of polyploidy and hybridization in Arabidopsis thaliana, we analyzed fertility of reciprocal pairs of F1 hybrid triploids, generated by reciprocally crossing 89 diploid accessions to a tetraploid Ler-0 line. All F1 hybrid triploid genotypes exhibited dramatically reduced ovule fertility, while variation in ovule number per silique was observed across different F1 triploid genotypes. These two reproductive traits were negatively correlated suggesting a trade-off between increased ovule number and ovule fertility. Furthermore, the ovule fertility of the F1 hybrid triploids displayed both hybrid dysgenesis and hybrid advantage (heterosis) effects. Strikingly, both reproductive traits (ovule fertility, ovule number) displayed epigenetic parent-of-origin effects between genetically identical reciprocal F1 hybrid triploid pairs. In some F1 triploid genotypes, the maternal genome excess F1 hybrid triploid was more fertile, whilst for other accessions the paternal genome excess F1 hybrid triploid was more fertile. Male gametogenesis was not significantly disrupted in F1 triploids. Fertility variation in the F1 triploid A. thaliana is mainly the result of disrupted ovule development. Overall, we demonstrate that in F1 triploid plants both ovule fertility and ovule number are subject to parent-of-origin effects that are genome dosage-dependent.

Role of N-glycosylation sites and CXC motifs in trafficking of medicago truncatula Nod factor perception protein to plasma membrane.

The lysin motif receptor-like kinase, NFP (Nod factor perception), is a key protein in the legume Medicago truncatula for the perception of lipochitooligosaccharidic Nod factors, which are secreted bacterial signals essential for establishing the nitrogen-fixing legume-rhizobia symbiosis. Predicted structural and genetic analyses strongly suggest that NFP is at least part of a Nod factor receptor, but few data are available about this protein. Characterization of a variant encoded by the mutant allele nfp-2 revealed the sensitivity of this protein to the endoplasmic reticulum quality control mechanisms, affecting its trafficking to the plasma membrane. Further analysis revealed that the extensive N-glycosylation of the protein is not essential for biological activity. In the NFP extracellular region, two CXC motifs and two other Cys residues were found to be involved in disulfide bridges, and these are necessary for correct folding and localization of the protein. Analysis of the intracellular region revealed its importance for biological activity but suggests that it does not rely on kinase activity. This work shows that NFP trafficking to the plasma membrane is highly sensitive to regulation in the endoplasmic reticulum and has identified structural features of the protein, particularly disulfide bridges involving CXC motifs in the extracellular region that are required for its biological function.

Identification of genes up-regulated during somatic embryogenesis of cucumber.

Somatic embryogenesis is a method of plant regeneration, but it can also be used as a model to study plant development. A normalized library of cDNA fragments representing genes up-regulated after the induction of somatic embryogenesis in cucumber suspension cultures was constructed using the suppression subtractive hybridization technique. Candidate cDNA fragments (119) were classified according to their similarity to genes encoding known proteins and the presence of potential functional domains. Of the translation products with homology to known proteins, about 23% were possibly involved in metabolism, 13% represented proteins with a probable role in cellular communication and signal transduction, about 12% were likely to participate in protein synthesis, while around 10% were potential transcription factors. The genes corresponding to four of the cDNAs were subsequently analyzed in more detail: CsSEF2, CsSEM1 and CsSESTK1 encoding putative transcription factors or co-activators, and CsSECAD1 encoding cinnamyl alcohol dehydrogenase. Full-length cDNAs were isolated and analyzed. RT-PCR confirmed the up-regulation of these genes after the induction of somatic embryogenesis and showed the presence of their transcripts in other tissues. The in situ localization of transcripts of the CsSEF2 and CsSEM1 genes demonstrated that signalling in somatic embryo tissues involving these factors is concentrated in the cotyledon primordia and roots.

Förster resonance energy transfer demonstrates a flavonoid metabolon in living plant cells that displays competitive interactions between enzymes.

We have used Förster resonance energy transfer detected by fluorescence lifetime imaging microscopy (FLIM-FRET) to provide the first evidence from living plants cells for the existence of a flavonoid metabolon. The distribution of flux within this system may be regulated by the direct competition of enzymes that catalyze key branch-point reactions, flavonol synthase 1 and dihydroflavonol 4-reductase, for association with the entry-point enzyme, chalcone synthase. Because the flavonoid enzymes were likely recruited from pathways of primary metabolism, our findings suggest a new general working model for the regulation of dynamic pathways in their native cellular context.

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.