Flagellin perception varies quantitatively in Arabidopsis thaliana and its relatives

Much is known about the evolution of plant immunity components directed against specific pathogen strains: They show pervasive functional variation and have the potential to coevolve with pathogen populations. However, plants are effectively protected against most microbes by generalist immunity components that detect conserved pathogen-associated molecular patterns (PAMPs) and control the onset of PAMP-triggered immunity. In Arabidopsis thaliana, the receptor kinase flagellin sensing 2 (FLS2) confers recognition of bacterial flagellin (flg22) and activates a manifold defense response. To decipher the evolution of this system, we performed functional assays across a large set of A. thaliana genotypes and Brassicaceae relatives. We reveal extensive variation in flg22 perception, most of which results from changes in protein abundance. The observed variation correlates with both the severity of elicited defense responses and bacterial proliferation. We analyzed nucleotide variation segregating at FLS2 in A. thaliana and detected a pattern of variation suggestive of the rapid fixation of a novel adaptive allele. However, our study also shows that evolution at the receptor locus alone does not explain the evolution of flagellin perception; instead, components common to pathways downstream of PAMP perception likely contribute to the observed quantitative variation. Within and among close relatives, PAMP perception evolves quantitatively, which contrasts with the changes in recognition typically associated with the evolution of R genes.     

Identification of a Disulfide Bridge Essential for Transport Function of the Human Proton-coupled Amino Acid Transporter hPAT1

The proton-coupled amino acid transporter 1 (PAT1, SLC36A1) mediates the uptake of small neutral amino acids at the apical membrane of intestinal epithelial cells after protein digestion. The transporter is currently under intense investigation, because it is a possible vehicle for oral drug delivery. Structural features of the protein such as the number of transmembrane domains, the substrate binding site, or essential amino acids are still unknown. In the present study we use mutagenesis experiments and biochemical approaches to determine the role of the three putative extracellular cysteine residues on transport function and their possible involvement in the formation of a disulfide bridge. As treatment with the reducing reagent dithiothreitol impaired transport function of hPAT1 wild type protein, substitution of putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 by alanine or serine was performed. Replacement of the two highly conserved cysteine residues Cys-180 and Cys-329 abolished the transport function of hPAT1 in Xenopus laevis oocytes. Studies of wild type and mutant transporters expressed in human retinal pigment epithelial (HRPE) cells suggested that the binding of the substrate was inhibited in these mutants. Substitution of the third putative extracellular nonconserved cysteine residue Cys-473 did not affect transport function. All mutants were expressed at the plasma membrane. Biotinylation of free sulfhydryl groups using maleimide-PEG₁₁-biotin and SDS-PAGE analysis under reducing and nonreducing conditions provided direct evidence for the existence of an essential disulfide bond between Cys-180 and Cys-329. This disulfide bridge is very likely involved in forming or stabilizing the substrate binding site.The solute carrier (SLC)² superfamily represents the second largest group of membrane proteins after the G-protein-coupled receptor (GPCR) superfamily in the human genome. Comprising 384 members, the 46 SLC families include transporters for inorganic ions, amino acids, neurotransmitters, sugars, purines, fatty acids, and other substances (1). Ten SLC families contain 47 known transporters for amino acids and 48 related orphan transporters. Phylogenetic analysis revealed four main clusters (α, β, γ, and δ). Together with members of the SLC32 and SLC38 families, the proton-coupled amino acid transporter 1 (PAT1) was placed into group β. PAT1 is a member of the SLC36 family (SLC36A1). It was originally identified as the lysosomal amino acid transporter (LYAAT1) in rat brain (2). Subsequently, mouse and human homologs were cloned from mouse intestine (3) and from Caco-2 cells (4), respectively. PAT1 is identical to the H⁺/amino acid cotransporter that has been functionally described in Caco-2 cells (5). It is localized mainly to the apical membrane of intestine epithelial cells and is also found in lysosomes in brain neurons (4) facilitating the transport of amino acids from luminal protein digestion or lysosomal proteolysis, respectively. The transport of substrates via PAT1 is driven by an inwardly directed H⁺ gradient. Recently we could identify the conserved His-55 as being responsible for binding and translocation of the proton (6).Prototypic substrates for PAT1 are small neutral amino acids (e.g. l-proline, glycine, β-alanine) and amino acid derivatives (e.g. γ-aminobutyric acid (GABA), α-(methylamino)-isobutyric acid) (3–5, 7–10). Recently, PAT1 gained much interest because it transports pharmaceutically relevant compounds such as d-cycloserine, l-azetidine-2-carboxylic acid, 3-amino-1-propanesulfonic acid, 3,4-dehydro-l-proline, vigabatrin, and other GABA analogs (8, 10, 11) rendering it an interesting target for the pharmaceutical industry. PAT1 seems to be one of the most important drug transporters in the intestine allowing oral availability of GABA-related and other drugs and prodrugs. Furthermore, a recent report shows involvement of this transporter family, namely the PAT2 subtype, in the autosomal dominant inherited disorder iminoglycinuria (12).Unfortunately, up to now the exact three-dimensional structure of PAT1, the transmembrane domain topology, and the substrate binding site are unknown. More structural information of PAT1 would allow a better understanding of the molecular mechanisms of its function and drug interaction, which is so far being investigated only in classic transport studies. Mutational analysis of putative extracellular regions is a suitable tool to get the first clue into transmembrane organization and relevant amino acid residues (6). This approach should also elucidate the spatial organization of the extracellular loops. The present study was performed to identify functionally important extracellular cysteine residues and their involvement in disulfide bridges. The relevance of disulfide bonds for membrane protein function is mainly based on the stabilization of a proper three-dimensional structure. The correct conformation in turn is essential for trafficking, surface expression, stability, and transport function. So far, intramolecular disulfide bonds have been identified for only very few SLCs, e.g. the serotonin transporter SERT and the dopamine transporter DAT (13–15). Native disulfide bonds are probably required for transporter function of the Na⁺/glucose cotransporter SGLT1 (16, 17). For the type IIa sodium/phosphate cotransporter, it was shown that cleavage of disulfide bonds results in conformational changes that lead to internalization and subsequent lysosomal degradation of the transport protein (18). A similar stabilizing effect of an intramolecular disulfide bridge was also reported for the human ATP-binding cassette (ABC) transporter ABCG2 (19).Linkage via cysteine residues can also be necessary for transporter oligomerization. For the rat serotonin transporter SERT (20) and for the human ABC transporter ABCG2 (21), intermolecular disulfide bridges could be identified. For the hexose transporter GLUT1, an intramolecular disulfide bond promotes tetramerization of the transporter (22, 23). On the other hand, removal of cysteine residues can also lead to an impaired trafficking and mislocalization of the transporter protein without a disulfide bridge being involved (13, 24, 25). In those cases, the cysteine residues themselves are assumed to play an important role for the trafficking and targeting of the transporter to the cell surface. Similarly, for several transporters, cysteine residues located in a transmembrane domain play a key role in substrate recognition. Single cysteines have been found to be essential for substrate binding of the rat organic cation transporters rOCT1 and rOCT2 (26) and the multidrug and toxin extrusion transporter MATE1 (27). The relevance of conserved cysteines for the integrity of a membrane protein has therefore to be investigated very thoroughly. Several earlier studies reported loss of function in cysteine mutants without testing membrane localization.After assessing a negative influence of the reducing reagent DTT on hPAT1 function, we performed systematic mutagenesis in this study. The three putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 were individually exchanged to either alanine or serine residues. The resulting mutants were analyzed for substrate binding and transport in human retinal pigment epithelial (HRPE) cells and electrogenic transport in Xenopus laevis oocytes. Biochemical approaches provided direct evidence for an essential disulfide bond between Cys-180 and Cys-329. A triple mutant was constructed and examined to exclude other juxtamembrane cysteine residues as potential partners for disulfide bridges. The data suggest that this disulfide bridge is involved in forming or stabilizing the putative substrate-binding pocket. In addition, our results strongly support the eleven transmembrane domain topology model of hPAT1. This is consistent with our recently published data on glycosylation of hPAT1 (28).     

A PCR-based method to distinguish the sibling species Stylonychia mytilus and Stylonychia lemnae (Ciliophora, Spirotrichea) using isocitrate dehydrogenase gene sequences

A differentiation, based on morphological characters, between Stylonychia mytilus and Stylonychia lemnae is very difficult, especially for non-specialists. These two sibling species were considered as one species, S. mytilus, until detailed cytological and genetic studies could show the existence of two genetically isolated varieties. Further morphological and biochemical analyses verified the separation and finally in 1983 a new species S. lemnae was described. The examination of several isoenzymes revealed unambiguous differences in the banding pattern of isocitrate dehydrogenase (IDH) between these two species. Therefore, the IDH gene of 30 isolates of S. lemnae and S. mytilus coming from various regions all over the world were amplified and sequenced. The sequence analyses revealed intraspecific as well as interspecific substitutions, which were used for the development of species-specific PCR primers for both species. Application of these species-specific primer pairs now allows a very easy and clear identification of both sibling species.     

Abscisic acid induces somatic embryogenesis and enables the capture of high-value genotypes in Douglas fir (Pseudotsuga menziesii [MIRB.] Franco)

Douglas fir (Pseudotsuga menziesii) is one of Europe’s most important non-native tree species due to its drought tolerance as well as timber quality and yield. To obtain superior seed from selected parental trees, breeding programs had been established in seed orchards. Douglas fir seed is used as source material for somatic embryogenesis with the aim to select elite genotypes invaluable for clonal mass propagation. To improve given protocols for somatic embryo initiation, we used immature Douglas fir zygotic embryos as explants and abscisic acid (ABA) as plant growth regulator in contrast to the application of auxins and cytokinins. With ABA supplementation, induction frequencies were slightly but in mean higher than with auxin/cytokinin, showing also a strong genotype effect. This offered the possibility to capture SE cultures from otherwise recalcitrant crosses. Furthermore, we observed remarkable differences between the two sets of plant growth regulators concerning the morphological development of the explants, including the absence of non-embryogenic callus by using ABA as inducer. This simplifies the detection of events and the handling of the obtained cultures. Nevertheless, a histological approach suggested, that the same competent cells are addressed by the different hormonal stimulation. Besides, we studied the influence of different points in time of cone harvest, two different basal media and different genetic backgrounds of the explants as well as the maturation ability of the induced embryogenic cultures. In sum, we were able to improve the first steps of somatic embryogenesis and to maintain a significantly higher number of high-value genotypes.

     

Metaorganismusforschung trifft Schule

The Darwin Day: modern science meets school During the Darwin Day at the University of Kiel high school students are introduced to recent research fields in modern biology. In 2018 the holistic view of a metaorganism offered the students new scientific ways of thinking and working, which are important for a profound understanding of biological interrelations as well as current clinical disorders. In order to bring new insights from biological research and modern science to school, different formats of science communication can be used. One possibility is the visit of extracurricular learning opportunities such as the Darwin Day at the University of Kiel. By linking current biological research with an out-of-school learning place, the importance of science communication can be illustrated. For this purpose, the Darwin Day and an accompanying teaching unit are presented in this article. Furthermore, insights into the evaluation of this outreach format are given.     

Cypriniform fish in running waters reduce hyporheic oxygen depletion in a eutrophic river

1. The hyporheic zone is an important habitat for benthic invertebrates and early-developmental stages of gravel spawning fish. However, the eutrophication of running waters and, in turn, the excessive periphyton biomass leads to its biological clogging. The result of these processes is oxygen depletion and a reduction in the habitat quality of the hyporheic zone.
2. This study assessed whether top-down effects of two important European river fish species, the large herbivorous cypriniform common nase (Chondrostoma nasus, L.) and the large omnivorous cypriniform European chub (Squalius cephalus, L.), can reduce eutrophication effects in the hyporheic zone. A 4-week mesocosm-based field experiment in a eutrophic river was conducted using cage enclosures stocked or not with either nase or chub.
3. The top-down control of periphyton was expected to reduce biological clogging and thereby increase oxygen availability in the hyporheic zone. Accordingly, we hypothesised that in enclosures stocked with either fish the concentrations of dissolved oxygen in the hyporheic zone would be higher and the periphyton biomass would be lower than in enclosures without fish stocking.
4. Hyporheic oxygen concentrations were significantly higher in enclosures stocked with either nase or chub than in enclosures without fish stocking. However, periphyton ash-free dry mass was significantly reduced only in enclosures stocked with nase, not in those stocked with chub. Thus, the positive effects of nase and chub on hyporheic oxygen availability were caused by different mechanisms.
5. Our results demonstrate that nase and chub can reduce eutrophication effects in the hyporheic zone of running waters. Hence, protecting and enhancing stocks of herbivorous and omnivorous fish will contribute to restoring the hyporheic zone in efforts to preserve biodiversity in eutrophic rivers.