The identification and quantification of cyclohexenone glycoside derivatives from the model legume Lotus japonicus revealed far higher levels than expected according to the stoichiometric relation to another, already determined carotenoid cleavage product, i.e., mycorradicin. Mycorradicin is responsible for the yellow coloration of many arbuscular mycorrhizal (AM) roots and is usually esterified in a complex way to other compounds. After liberation from such complexes it has been detected in AM roots of many, but not of all plants examined. The non-stoichiometric occurrence of this compound compared with other carotenoid cleavage products suggested that carotenoid biosynthesis might be activated upon mycorrhization even in plant species without detectable levels of mycorradicin. This assumption has been supported by inhibition of a key enzyme of carotenoid biosynthesis (phytoene desaturase) and quantification of the accumulating enzymic substrate (phytoene). Our observations suggest that the activation of carotenoid biosynthesis in AM roots is a general phenomenon and that quantification of mycorradicin is not always a good indicator for this activation.

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H2O2 accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl3, intracellular accumulation of H2O2 was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H2O2 was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

We used two approaches for studying the relationships between wine consumption, wine composition and cancer In the first approach, a transgenic mouse model of human neurofibromatosis, combined with the use of well-defined, chemically purified diets, showed that red wine contains nonalcoholic components that can delay tumor onset. In additional studies, catechin, the main monomeric polyphenol of red wine, delayed tumor onset in this mouse model in a positive, linear relationship when incorporated into the diet at levels of 0.5-4 mmol/kg diet. In the second approach, low doses of the chemical carcinogen 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine (PhlP) were administered to rats, and formation of DNA adducts was evaluated by accelerator mass spectrometry. Consumption of red wine solids (the residue from red wine remaining after removal of alcohol and water) and the wine polyphenol quercetin did not influence PhlP-DNA adduct levels or induce liver enzymes (glutathione-S-transferase and quinone reductase). However, quercetin did alter distribution of PhlP in the rat tissues compared to control animals and animals fed other potential dietary chemopreventive agents, including phenylethyl isothiocyanate and sulforaphane. These studies demonstrate the feasibility of these approaches for studying the chemopreventive potential of dietary components at physiologic levels in

A rapid multiresidue method to quantify three different classes of plant hormones has been developed. The reduced concentrations of these metabolites in real samples with complex matrixes require sensitive techniques for their quantification in small amounts of plant tissue. The method described combines high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Deuterium-labeled standards were added prior to sample extraction to achieve an accurate quantification of abscisic acid, indole-3-acetic acid, and jasmonic acid in a single run. A simple method of extraction and purification involving only centrifugation, a partition against diethyl ether, and filtration was developed and the analytical method validated in four different plant tissues, citrus leaves, papaya roots, barley seedlings, and barley immature embryos. This method represents a clear advantage because it extensively reduces sample preparation and total time for routine analysis of phytohormones in real plant samples.

A facile and flexible approach for the preparation of macrocyclic molecules containing different carbohydrate moieties is presented, employing the reaction cascade: click-dimerization and ring-closing metathesis.

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2), a non‐radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of 1O2. Vitamin B6 that quenches 1O2 in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of 1O2, indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild‐type level. The flu mutant was also crossed with the jasmonic acid (JA)‐depleted mutant opr3 , and with the JA, OPDA and dinor OPDA (dnOPDA)‐depleted dde2‐2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA‐induced suppression of H2O2/superoxide‐dependent cell death reported earlier, JA promotes singlet oxygen‐mediated cell death in flu , whereas other oxylipins such as OPDA and dnOPDA antagonize this death‐inducing activity of JA.

Various octadecanoids and derived compounds have been identified in potato leaves. However, information regarding jasmonate hydroxylated forms in stolons or tubers is scarce. We investigated endogenous jasmonates in stolon material ofSolarium tuberosum cv. Spunta. Stolons and incipient tubers were collected from 8 weeks old plants. The material was cut into apical regions and stolons. We identified jasmonic acid (JA), methyl jasmonate, 11-OH-JA, 12-OH-JA, 12-oxo-phytodienoic acid (OPDA) and a conjugate. The content of JA and 12OH-JA decreased in the apical region but remained high in stolons during tuberization. Thus the apical region might be a site of JAs-utilization or metabolization and stolons might supply JAs to that region. The content of 12-OH-JA was higher than that of 11-OH-JA in all stages analyzed, both in apical regions and stolons. However, these compounds showed a different time-course in the apical region: while 11-OH-JA increased, 12-OH-JA decreased. Thus, JA from leaves or roots could be transported as 12-OH-JA to the apical region, stimulating tuber formation.

In this study, we characterize the evolutionarily conserved TOUGH (TGH) protein as a novel regulator required for Arabidopsis thaliana development. We initially identified TGH as a yeast two-hybrid system interactor of the transcription initiation factor TATA-box binding protein 2. TGH has apparent orthologs in all eukaryotic model organisms with the exception of the budding yeast Saccharomyces cerevisiae. TGH contains domains with strong similarity to G-patch and SWAP domains, protein domains that are characteristic of RNA binding and processing proteins. Furthermore, TGH colocalizes with the splicing regulator SRp34 to subnuclear particles. We therefore propose that TGH plays a role in RNA binding or processing. Arabidopsis tgh mutants display developmental defects, including reduced plant height, polycotyly, and reduced vascularization. We found TGH expression to be increased in the amp1-1 mutant, which is similar to tgh mutants with respect to polycotyly and defects in vascular development. Interestingly, we observed a strong genetic interaction between TGH and AMP1 in that tgh-1 amp1-1 double mutants are extremely dwarfed and severely affected in plant development in general and vascular development in particular when compared with the single mutants.

Thioredoxin reductases catalyse the reduction of thioredoxin disulfide and some other oxidised cell constituents. They are homodimeric proteins containing one FAD and accepting one NADPH per subunit as essential cofactors. Some of these reductases contain a selenocysteine at the C terminus. Based on the X‐ray structure of rat thioredoxin reductase, homology models of human thioredoxin reductase were created and subsequently docked to thioredoxin to model the active complex. The formation of a new type of a catalytic triad between selenocysteine, histidine and a glutamate could be detected in the protein structure. By means of DFT (B3LYP, lacv3p**) calculations, we could show that the formation of such a triad is essential to support the proton transfer from selenol to a histidine to stabilise a selenolate anion, which is able to interact with the disulfide of thioredoxin and catalyses the reductive disulfide opening. Whereas a simple proton transfer from selenocysteine to histidine is thermodynamically disfavoured by some 18 kcal mol −1 , it becomes favoured when the carboxylic acid group of a glutamate stabilises the formed imidazole cation. An identical process with a cysteine instead of selenocysteine will require 4 kcal mol −1 more energy, which corresponds to a calculated equilibrium shift of ∼1000:1 or a 10 3 rate acceleration: a value close to the experimental one of about 10 2 times. These results give new insights into the catalytic mechanism of thioredoxin reductase and, for the first time, explain the advantage of the incorporation of a selenocysteine instead of a cysteine residue in a protein.