One of the most long-lived models in plant science is the belief that the long-distance transport and ratio of two plant hormones, auxin and cytokinin, at the site of action control major developmental events such as apical dominance. We have used in vivo deuterium labeling and mass spectrometry to investigate the dynamics of homeostatic cross talk between the two plant hormones. Interestingly, auxin mediates a very rapid negative control of the cytokinin pool by mainly suppressing the biosynthesis via the isopentenyladenosine-5 -monophosphate-independent pathway. In contrast, the effect of cytokinin overproduction on the entire auxin pool in the plant was slower, indicating that this most likely is mediated through altered development. In addition, we were able to confirm that the lateral root meristems are likely to be the main sites of isopentenyladenosine-5 -monophosphate-dependent cytokinin synthesis, and that the aerial tissue of the plant surprisingly also was a significant source of cytokinin biosynthesis. Our demonstration of shoot-localized synthesis, together with data demonstrating that auxin imposes a very rapid regulation of cytokinin biosynthesis, illustrates that the two hormones can interact also on the metabolic level in controlling plant development, and that the aerial part of the plant has the capacity to synthesize its own cytokinin independent of long-range transport from the root system.

Organophosphorus compound-based nerve agents inhibit the essential enzyme acetylcholinesterase (AChE) causing acute toxicity and death. Clinical treatment of nerve-agent poisoning is to use oxime-based antidotes to reactivate the inhibited AChE. However, the nerve agent tabun is resistant to oximes. To design improved oximes, crystal structures of a tabun-conjugated AChE in complex with different oximes are needed to guide the structural modifications of known antidotes. However, this type of structure is extremely challenging to obtain because both deamidation of the tabun conjugate and reactivation of AChE occur during crystallographic experiments. Here we report, for the first time, the crystal structures of Ortho-7 and HLö-7 in complex with AChE that is conjugated to an intact tabun. These structures were determined by our new strategy of combining crystallographic and mass spectrometric analyses of AChE crystals. The results explain the relative reactivation potencies of the two oximes and offer insights into improving known medical antidotes.

Studies of de novo cytokinin biosynthesis in isopentenyltransferase (ipt)-transformed Arabidopsis thaliana, involving in vivo deuterium labeling and mass spectrometry, showed that the biosynthetic rate of zeatinriboside-5 -monophosphate was around 66-fold higher than that of isopentenyladenosine-5 -monophosphate (iPMP), the proposed primary product of the Agrobacterium ipt. Double tracer analysis, using [ 2 H6] isopentenyladenosine and deuterium oxide, provided evidence for an alternative, iPMPindependent, biosynthetic pathway for zeatin-type cytokinins, present in both ipt-expressing and wild-type Arabidopsis thaliana. Reduction of the biosynthetic flux in the alternative pathway by use of mevastatin, an inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase, indicated a terpenoid origin for the side-chain precursor of the iPMP independent pathway. C ytokinins are an important class of plant growth regulators, defined by their ability to promote cell division in tissue culture in the presence of auxins (1, 2). Virtually all naturally occurring cytokinins identified to date are adenine species substituted at N 6 with an isoprenoid or aromatic side chain. In this text, cytokinins will refer solely to the isoprenoid cytokinin bases and their sugar conjugates. Cytokinins affect many plant developmental processes including cell division, cell differentiation, chlorophyll senescence, and apical dominance (3).Efforts to elucidate the biosynthetic origin of cytokinins in plants have been inconclusive. Early suggestions that tRNA degradation could be the major source of free, active cytokinins (4) were disproved when calculations of tRNA turnover rates showed that a tRNA-independent de novo biosynthetic pathway also must be present in plants (5). A major breakthrough was the discovery of a cytokinin biosynthetic enzyme in the slime mold, Dictyostelium discoideum (6). Cell-free extracts from this organism can convert AMP and dimethylallyl-pyrophosphate (DMAPP) to the free cytokinins isopentenyladenosine-5Ј-monophosphate (iPMP) and the corresponding nucleoside (isopentenyladenosine, iPA). This finding, and studies on the metabolism of isopentenyl-type cytokinins (7,8), led to the proposal that iPMP is also the primary cytokinin intermediate in plants, and zeatin cytokinins are formed by hydroxylation of iPMP and its derivatives (9, 10) ( Fig. 1).Later, the product of the T-DNA gene 4 (ipt) of the crown gall-forming bacterium, Agrobacterium tumefaciens, also was described as a DMAPP:AMP isopentenyltransferase (ipt) (11,12). AMP was found to be the preferred adenylic substrate for the Agrobacterium ipt enzyme but searches for alternative side-chain donors have been limited. However, when de novo biosynthesis in crown gall tissue of Vinca rosea was traced with 14 C-adenine, in vivo production of iPMP was undetectable, whereas zeatinriboside-5Ј-monophospate (ZMP) production was strong (13). After studies of 14 C-isopententyladenine metabolism in this system, it was proposed that the very low levels of iPMP were caused by rapi...

We have developed a method for analyzing polar compounds by reversed-phase LC-ESI-MS following esterification of the analytes' free hydroxyl groups with propionyl or benzoyl acid anhydride. The method was applied to members of the plant hormone group cytokinins, which includes adenine bases, ribosides/glycosides, and nucleotides substituted at N-6 with an isoprenoid side chain, spanning a wide range of polarity. It was also used to analyze other compounds of biological importance, e.g., the nucleotides AMP, ADP, and ATP. The formation of more hydrophobic derivatives had a significant impact on two aspects of the analysis. The retention on a reversed-phase material was greatly increased without the use of any acetate/formate buffer or ion pairing reagent, and the ESI response was enhanced, due to the higher surface activities of the derivatives. Detection limits of propionylated cytokinins were in the high-attomole to low-femtomole range, an improvement by factors of 10-100 compared to previously reported figures. Using an automated SPE-based purification method, 12 endogenous cytokinins were quantified in extracts from 20- to 100-mg samples of leaves (from the plant Arabidopsis thaliana) with high accuracy and precision. Furthermore, the chromatographic properties of the benzoylated AMP, ADP, and ATP in the reversed-phase LC-MS system were much better in terms of retention, separation, and sensitivity than those of their underivatized counterparts, even without the use of any ion pairing reagent. Our data show that derivatization followed by LC-ESI-MS is an effective strategy for analyzing low molecular weight compounds, enabling compounds with a wide range of polarity to be determined in a single-injection LC-MS analysis.

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The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A1, B1 and E1, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A1, B1, E1 and F1 were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.

). ² These authors contributed equally to this work. SummaryTransgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins. All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross. Indole-3-acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside-5¢-monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type. Unexpectedly, hormone levels in the cross were very similar to wild-type levels. Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced. The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly Kassociated with IAA and cytokinin overproduction, and observed in the iaa ¥ ipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs. As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.T ype 2 ribosome-inactivating proteins (RIP-II toxins) are a class of plant toxins that includes a number of potent chem-bio threat agents, such as ricin (Ricinus communis), abrin (Abrus precatorius), viscumin (Viscum album), modeccin (Adenia digitata), and volkensin (Adenia volkensii). RIP-II toxins are heterodimeric proteins that consist of an enzymatically active N-glycosidase A chain connected to a B chain via a disulfide bond. The B chain is a lectin with carbohydrate binding domains that have an affinity for galactose-terminated surface receptors on eukaryotic cells. Binding promotes the uptake of the toxin into the cell by endocytosis, where a part intriguingly finds its way via the Golgi complex to the endoplasmatic reticulum. From there the A subunit is translocated into the cytosol, where it hydrolyses the Nglycosidic bond between an adenine residue and ribose at a specific position in 28S rRNA and thereby inhibits protein synthesis, eventually causing cell death.