Rainer Schwacke

ARAMEMNON, a Novel Database for Arabidopsis Integral Membrane Proteins

A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM spans and are possibly linked to transport functions. The ARAMEMNON DB enables direct comparison of the predictions of seven different TM span computation programs and the predictions of subcellular localization by eight signal peptide recognition programs. A special function displays the proteins related to the query and dynamically generates a protein family structure. As a first set of proteins from other organisms, all of the approximately 700 putative membrane proteins were extracted from the genome of the cyanobacterium Synechocystis sp. and incorporated in the ARAMEMNON DB. The ARAMEMNON DB is accessible at the URL http://aramemnon.botanik.uni-koeln.de.

Transcription analysis of Arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence

A comparative transcriptome analysis for successive stages of Arabidopsis (Arabidopsis thaliana) developmental leaf senescence (NS), darkening-induced senescence of individual leaves attached to the plant (DIS), and senescence in dark-incubated detached leaves (DET) revealed many novel senescence-associated genes with distinct expression profiles. The three senescence processes share a high number of regulated genes, although the overall number of regulated genes during DIS and DET is about 2 times lower than during NS. Consequently, the number of NS-specific genes is much higher than the number of DIS- or DET-specific genes. The expression profiles of transporters (TPs), receptor-like kinases, autophagy genes, and hormone pathways were analyzed in detail. The Arabidopsis TPs and other integral membrane proteins were systematically reclassified based on the Transporter Classification system. Coordinate activation or inactivation of several genes is observed in some TP families in all three or only in individual senescence types, indicating differences in the genetic programs for remobilization of catabolites. Characteristic senescence type-specific differences were also apparent in the expression profiles of (putative) signaling kinases. For eight hormones, the expression of biosynthesis, metabolism, signaling, and (partially) response genes was investigated. In most pathways, novel senescence-associated genes were identified. The expression profiles of hormone homeostasis and signaling genes reveal additional players in the senescence regulatory network.

Cyanophora paradoxa Genome Elucidates Origin of Photosynthesis in Algae and Plants

Plastid Origins The glaucophytes, represented by the alga Cyanophora paradoxa, are the putative sister group of red and green algae and plants, which together comprise the founding group of photosynthetic eukaryotes, the Plantae. In their analysis of the genome of C. paradoxa, Price et al. (p. 843; see the Perspective by Spiegel) demonstrate a unique origin for the plastid in the ancestor of this supergroup, which retains much of the ancestral diversity in genes involved in carbohydrate metabolism and fermentation, as well as in the gene content of the mitochondrial genome. Moreover, about 3.3% of nuclear genes in C. paradoxa seem to carry a signal of cyanobacterial ancestry, and key genes involved in starch biosynthesis are derived from energy parasites such as Chlamydiae. Rapid radiation, reticulate evolution via horizontal gene transfer, high rates of gene divergence, loss, and replacement, may have diffused the evolutionary signals within this supergroup, which perhaps explains previous difficulties in resolving its evolutionary history. An ancient algal genome suggests a unique origin of the plastid in the ancestor to plants, algae, and glaucophytes. The primary endosymbiotic origin of the plastid in eukaryotes more than 1 billion years ago led to the evolution of algae and plants. We analyzed draft genome and transcriptome data from the basally diverging alga Cyanophora paradoxa and provide evidence for a single origin of the primary plastid in the eukaryote supergroup Plantae. C. paradoxa retains ancestral features of starch biosynthesis, fermentation, and plastid protein translocation common to plants and algae but lacks typical eukaryotic light-harvesting complex proteins. Traces of an ancient link to parasites such as Chlamydiae were found in the genomes of C. paradoxa and other Plantae. Apparently, Chlamydia-like bacteria donated genes that allow export of photosynthate from the plastid and its polymerization into storage polysaccharide in the cytosol.

Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca2+ and protein-kinase activity

Cell-wall components from the ectomycorrhizal fungi Amanita muscaria and Hebeloma crustuliniforme and from the spruce pathogen Heterobasidion annosum elicited a transient release of active oxygen species from cultured spruce cells (Picea abies (L.) Karst.). Since the detection of active oxygen was suppressed by catalase, H2O2 was assumed to be the prevailing O2 species. On the other hand, superoxide dismutase enhanced the concentration of detectable H2O2 indicating that the superoxide anion was formed before dismutating to H2O2. The elicitors induced the formation of active oxygen in a dose-dependent manner. Interestingly, elicitors from mycorrhizal fungi had a lower H2O2-inducing activity than equal amounts of cell-wall preparations from the pathogen H. annosum. In Ca2+-depleted medium the production of active oxygen by elicitor-treated spruce cells was suppressed. Additionally, the ionophore A 23187 induced active oxygen formation in a medium with Ca2+ but not in a Ca2+-depleted medium. Furthermore, the protein-kinase inhibitor staurosporine inhibited the oxidative burst. At a concentration of 34 nM the effect was diminished to 50%. From these results it is suggested that the release of active oxygen species from cultured spruce cells triggered by cell-wall-derived fungal elicitors depends on external Ca2+ and a protein-kinase activity. In these respects the effect shows similarities with the well-studied respiratory burst of mammalian neutrophils.

LeProT1, a Transporter for Proline, Glycine Betaine, and γ-Amino Butyric Acid in Tomato Pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein–protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r2 = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Comparative survey of plastid and mitochondrial targeting properties of transcription factors in Arabidopsis and rice

A group of nuclear transcription factors, the Whirly proteins, were recently shown to be targeted also to chloroplasts and mitochondria. In order to find out whether other proteins might share this feature, an in silico-based screening of transcription factors from Arabidopsis and rice was carried out with the aim of identifying putative N-terminal chloroplast and mitochondrial targeting sequences. For this, the individual predictions of several independent programs were combined to a consensus prediction using a naïve Bayes method. This consensus prediction shows a higher specificity at a given sensitivity value than each of the single programs. In both species, transcription factors from a variety of protein families that possess putative N-terminal plastid or mitochondrial target peptides as well as nuclear localization sequences, were found. A search for homologues within members of the AP2/EREBP protein family revealed that target peptide-containing proteins are conserved among monocotyledonous and dicotyledonous species. Fusion of one of these proteins to GFP revealed, indeed, a dual targeting activity of this protein. We propose that dually targeted transcription factors might be involved in the communication between the nucleus and the organelles in plant cells. We further discuss how recent results on the physical interaction between the organelles and the nucleus could have significance for the regulation of the localization of these proteins.

Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana

Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake‐deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9±1.7 and 1.7±0.3 mM at pH 4, respectively. The simultaneous transport of [1‐14C]GABA and [2,3‐3H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2‐mediated [1‐14C]GABA transport was inhibited by proline and quaternary ammonium compounds.

Identification of an Arabidopsis mitochondrial succinate–fumarate translocator

Complementation of a yeast acr1 mutant carrying a deletion of the succinate/fumarate carrier gene enabled functional identification of a mitochondrial succinate translocator from Arabidopsis thaliana (AtmSFC1). Thus complementation of yeast mutants is applicable also for identification and characterization of organellar transporters. Reverse transcription polymerase chain reaction and promoter‐GUS fusion showed expression of AtmSFC1 in 2 day old dark grown seedlings, which declined in cotyledons during further development, consistent with a role in export of fumarate for gluconeogenesis during lipid mobilization at early germination of Arabidopsis seeds. In mature plants, expression was found in developing and germinating pollen, suggesting a role in ethanolic fermentation.

GabiPD – The GABI Primary Database integrates plant proteomic data with gene-centric information

GabiPD is an integrative plant “omics” database that has been established as part of the German initiative for Genome Analysis of the Plant Biological System (GABI). Data from different “omics” disciplines are integrated and interactively visualized. Proteomics is represented by data and tools aiding studies on the identification of post-translational modification and function of proteins. Annotated 2D electrophoresis-gel images are offered to inspect protein sets expressed in different tissues of Arabidopsis thaliana and Brassica napus. From a given protein spot, a link will direct the user to the related GreenCard Gene entry where detailed gene-centric information will support the functional annotation. Beside MapMan- and GO-classification, information on conserved protein domains and on orthologs is integrated in this GreenCard service. Moreover, all other GabiPD data related to the gene, including transcriptomic data, as well as gene-specific links to external resources are provided. Researches interested in plant protein phosphorylation will find information on potential MAP kinase substrates identified in different protein microarray studies integrated in GabiPD’s Phosphoproteomics page. These data can be easily compared to experimentally identified or predicted phosphorylation sites in PhosPhAt via the related Gene GreenCard. This will allow the selection of interesting candidates for further experimental validation of their phosphorylation.