Ethylene (ET) controls many facets of plant growth and development under abiotic and biotic stresses. MtEIN2, as a critical element of the ET signaling pathway, is essential in biotic interactions. However, the role of MtEIN2 in responding to abiotic stress, such as combined nutrient deficiency, is less known. To assess the role of ethylene signaling in nutrient uptake, we manipulated nitrate (NO3−) and phosphate (Pi) availability for wild-type (WT) and the ethylene-insensitive (MtEIN2-defective) mutant, sickle, in Medicago truncatula. We measured leaf biomass and photosynthetic pigments in WT and sickle to identify conditions leading to different responses in both genotypes. Under combined NO3− and Pi deficiency, sickle plants had higher chlorophyll and carotenoid contents than WT plants. Under these conditions, nitrate content and gene expression levels of nitrate transporters were higher in the sickle mutant than in the WT. This led to the conclusion that MtEIN2 is associated with nitrate uptake and the content of photosynthetic pigments under combined Pi and NO3−deficiency in M. truncatula. We conclude that ethylene perception plays a critical role in regulating the nutrient status of plants.

Surfactant proteins are well known from the human lung where they are responsible for the stability and flexibility of the pulmonary surfactant system. They are able to influence the surface tension of the gas–liquid interface specifically by directly interacting with single lipids. This work describes the generation of reliable protein structure models to support the experimental characterization of two novel putative surfactant proteins called SP-G and SP-H. The obtained protein models were complemented by predicted posttranslational modifications and placed in a lipid model system mimicking the pulmonary surface. Molecular dynamics simulations of these protein-lipid systems showed the stability of the protein models and the formation of interactions between protein surface and lipid head groups on an atomic scale. Thereby, interaction interface and strength seem to be dependent on orientation and posttranslational modification of the protein. The here presented modeling was fundamental for experimental localization studies and the simulations showed that SP-G and SP-H are theoretically able to interact with lipid systems and thus are members of the surfactant protein family.

Under the auspices of the European Training and Networking Activity programme of the European Union, a ‘Metabolic Profiling and Data Analysis’ Plant Genomics and Bioinformatics Summer School was hosted in Potsdam, Germany between 20 and 29 September 2006. Sixteen early career researchers were invited from the European Union partner nations and the so‐called developing nations (Appendix). Lectures from invited leading European researchers provided an overview of the state of the art of these fields and seeded discussion regarding major challenges for their future advancement. Hands‐on experience was provided by an example experiment – that of defining the metabolic response of Arabidopsis to treatment of a commercial herbicide of defined mode of action. This experiment was performed throughout the duration of the course in order to teach the concepts underlying extraction and machine handling as well as to provide a rich data set with which the required computation and statistical skills could be illustrated. Here we review the state of the field by describing both key lectures given at and practical aspects taught at the summer school. In addition, we disclose results that were obtained using the four distinct technical platforms at the different participating institutes. While the effects of the chosen herbicide are well documented, this study looks at a broader number of metabolites than in previous investigations. This allowed, on the one hand, not only to characterise further effects of the herbicide than previously observed but also to detect molecules other than the herbicide that were obviously present in the commercial formulation. These data and the workshop in general are all discussed in the context of the teaching of metabolomics.

4-Hydroxybenzoate oligoprenyltransferase of E. coli, encoded in the gene ubiA, is an important key enzyme in the biosynthetic pathway to ubiquinone. It catalyzes the prenylation of 4-hydroxybenzoic acid in position 3 using an oligoprenyl diphosphate as a second substrate. Up to now, no X-ray structure of this oligoprenyltransferase or any structurally related enzyme is known. Knowledge of the tertiary structure and possible active sites is, however, essential for understanding the catalysis mechanism and the substrate specificity.With homology modeling techniques, secondary structure prediction tools, molecular dynamics simulations, and energy optimizations, a model with two putative active sites could be created and refined. One active site selected to be the most likely one for the docking of oligoprenyl diphosphate and 4-hydroxybenzoic acid is located near the N-terminus of the enzyme. It is widely accepted that residues forming an active site are usually evolutionary conserved within a family of enzymes. Multiple alignments of a multitude of related proteins clearly showed 100% conservation of the amino acid residues that form the first putative active site and therefore strongly support this hypothesis. However, an additional highly conserved region in the amino acid sequence of the ubiA enzyme could be detected, which also can be considered a putative (or rudimentary) active site. This site is characterized by a high sequence similarity to the aforementioned site and may give some hints regarding the evolutionary origin of the ubiA enzyme.Semiempirical quantum mechanical PM3 calculations have been performed to investigate the thermodynamics and kinetics of the catalysis mechanism. These results suggest a near SN1 mechanism for the cleavage of the diphosphate ion from the isoprenyl unit. The 4-hydroxybenzoic acid interestingly appears not to be activated as benzoate anion but rather as phenolate anion to allow attack of the isoprenyl cation to the phenolate, which appeared to be the rate limiting step of the whole process according to our quantum chemical calculations. Our models are a basis for developing inhibitors of this enzyme, which is crucial for bacterial aerobic metabolism.

In this study, we report the cloning of the three‐member LePS2 gene family of acid phosphatases via subtractive screening of a cDNA library of Pi‐starved cultivated tomato cells (Lycopersicon esculentum Mill. cv. Lukullus). As members of the plant Pi‐starvation response, LePS2 genes were tightly regulated in cultivated cells and tomato seedlings by Pi availability. The LePS2 enzymes which are most likely expressed in the cytoplasma could be involved in processes that are accompanied by degradation of phosphorylated organic substrates. Independently from exogenous phosphate supply LePS2 expression was detected in tomato endosperm during germination. LePS2 genes were differentially induced after infection with the bacterial pathogen Pseudomonas syringae and in the early stages of flower development. Using RT–PCR it was found that the gene LePS2B was the most abundant transcript in phosphate‐depleted cells, but a reduced expression was determined in floral buds and it was not found during pathogen interaction. In this respect, it is interesting that the promoter sequences of the LePS2 genes are also divergent. LePS2 gene products may have functions in developmental processes which are restricted to distinct plant tissues or cell types.

[17‐2H2]GA20‐13‐O‐[6′‐2H2]glucoside was synthesized and applied to seedlings of Phaseolus coccineus L. After incubation for 72 h the conjugate metabolites were purified and shown by LC‐ESI‐tandem‐MS and GC‐MS to be [17‐2H2]GA1‐13‐O‐[6′‐2H2]glucoside and [17‐2H2]GA29‐13‐O‐[6′‐2H2]glucoside. This is the first evidence for the conversion of intact GA‐O‐glucosides, and represents an additional metabolic pathway of the gibberellin metabolism in P. coccineus L. The results indicate that intact GA‐O‐glucosides are accepted by 2‐ and 3‐oxidases in the plant.

Phosphate (Pi) plays a central role as reactant and effector molecule in plant cell metabolism. However, Pi is the least accessible macronutrient in many ecosystems and its low availability often limits plant growth. Plants have evolved an array of molecular and morphological adaptations to cope with Pi limitation, which include dramatic changes in gene expression and root development to facilitate Pi acquisition and recycling. Although physiological responses to Pi starvation have been increasingly studied and understood, the initial molecular events that monitor and transmit information on external and internal Pi status remain to be elucidated in plants. This review summarizes molecular and developmental Pi starvation responses of higher plants and the evidence for coordinated regulation of gene expression, followed by a discussion of the potential involvement of plant hormones in Pi sensing and of molecular genetic approaches to elucidate plant signalling of low Pi availability. Complementary genetic strategies in Arabidopsis thaliana have been developed that are expected to identify components of plant signal transduction pathways involved in Pi sensing. Innovative screening methods utilize reporter gene constructs, conditional growth on organophosphates and the inhibitory properties of the Pi analogue phosphite, which hold the promise for significant advances in our understanding of the complex mechanisms by which plants regulate Pi‐starvation responses.

Brassinosteroids are a class of steroidal phytohormones with high growth-promoting properties. The preferred side-chain conformations of 10 brassinosteroids were determined by means of detailed NMR investigations and molecular-modeling studies. Vacuum conformations obtained by simulated annealing calculations and Boltzmann statistical analysis were compared with solution conformations derived from NOE experiments and molecular dynamic simulations, and with X-ray structures. In general, results from simulated annealing calculations and NMR-supported molecular dynamics simulations are in good agreement. For some of the compounds investigated the conformation was less well-defined at the end of the side-chain. It could be shown that the energetically most favorable and most probable conformations also include the conformations obtained by NMR supported molecular-dynamics calculations and by X-ray analysis. For the most bioactive compound brassinolide (1) the majority of conformations show a side-chain bent towards the β-face of the steroid skeleton, whereas for the less bioactive brassinosteroids, conformations with straight side-chains or side-chains bent towards the α-face are more frequent.

Cathepsin H is involved in intracellular protein degradation and is implicated in a variety of physiological processes such as proenzyme activation, enzyme inactivation, hormone maturation, tissue remodeling, and bone matrix resorption. A model of the tertiary structure of the human lysosomal cysteine protease cathepsin H was constructed. The protein structure was built from its amino acid sequence and its homology to papain, actinidin, and cathepsin L for which crystallographic co-ordinates are available. The model was generated using the COMPOSER module of SYBYL.The position and interaction behavior of the so called mini-chain, the octapeptide EPQNCSAT, to the active-site cleft of cathepsin H could be determined by docking studies. Refinement was achieved through interactive visual and algorithmic analysis and minimization with the TRIPOS force field. The model was found to correlate with observed empirical data regarding ligand specificity. The model defines possible steric, hydrophobic, and electrostatic interactions. We anticipate that the model will serve as a tool to understand substrate specificity and may be used for the development of new specific ligands.

The human immunodeficiency virus 1 Tat protein suppresses antigen-, anti-CD3-and mitogen-induced activation of human T cells when added to T cell cultures. This activity is important for the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV). To find out the amino acid sequence important for the inhibition of the DP IV enzymatic activity we investigated N-terminal Tat(1–9) peptide analogues with amino acid substitutions in different positions. Interestingly, the exchange of Pro6 with Leu and Asp5 with Ile strongly diminished the DP IV inhibition by Tat(1–9). Based on data derived from one-and two-dimensional 1H NMR investigations the solution conformations of the three nonapeptides in water were determined by means of molecular dynamics simulations. These conformations were used for studies of the docking behavior of the peptides into a model of the active site of DP IV. The results suggest that several attractive interactions between the native Tat(1–9) and DP IV lead to a stable complex and that the reduced affinity of both L6-Tat(1–9) and I5-Tat(1–9) derivatives might be caused by conformational alterations in comparison to the parent peptide.