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Publikation

Ziegler, J., Brandt, W. & Geißler, R. Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis J Biol Chem 284, 26758-26767, (2009) DOI: 10.1074/jbc.M109.030957

Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel -helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also

revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalRwas created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.

Publikation

Lee, C-W., Efetova, M., Engelmann, J.C., Kramell, R., Wasternack, C., Ludwig- Müller, J., Hedrich, R. & Deeken, R. Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsis thaliana. The Plant Cell 21, 2948 - 2962, (2009) DOI: 10.1105/tpc.108.064576

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis–Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not.

This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumorgrowth initiated by agrobacteria.

Bücher und Buchkapitel

Wasternack, C. Jasmonates in Stress, Growth, and Development. In: Plant Stress Biology (H. Hirt). WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 91 - 118, (2009) ISBN: 978-3-527-32290-9

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Bücher und Buchkapitel

Dorka, R., Miersch, O., Hause, B., Weik, P. & Wasternack, C. Chronobiologische Phänomene und Jasmonatgehalt bei Viscum album L.. In: Die Mistel in der Tumortherapie 2 (Scheer, R.; Bauer, R.; Bekker, A.; Berg, P. A.; Fintelmann, V.). KVC-Verlag Essen 49-56, (2009) ISBN: 978-3-933351-82

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Publikation

Flores, R., Gas, M.E., Molina-Serrano, D., Nohales, M.A., Carbonell, A., Gago, S., de la Peña, M. & Daròs, J.A. Viroid replication: rolling-circles, enzymes and ribozymes Viruses 1, 317-334, (2009) DOI: 10.3390/v1020317

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Publikation

Wasternack, C. & Hause, B. Emerging complexity: jasmonate-induced volatiles affect parasitoid choice J Exp Bot 60, 2451-2453, (2009)

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Publikation

Mugford, S.G., Yoshimoto, N., Reichelt, M., Wirtz, M., Hill, L., Mugford, S.T., Nakazato, Y., Noji, M., Takahashi, H., Kramell, R., Gigolashvili, T., Flügge, U.-I., Wasternack, C., Gershenzon, J., Hell, R., Saito, K. & Kopriva, S. Disruption of Adenosine-5'-Phosphosulfate Kinase in ArabidopsisReduces Levels of Sulfated Secondary Metabolites Plant Cell 21, 910-927, (2009)

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 59-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 39-phosphoadenosine 59-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normalgrowth rates.

Publikation

Parry, G., Calderón Villalobos, L.I., Prigge, M., Peret, B., Dharmasiri, S., Itoh, H., Lechner, E., Gray, W.M., Bennett, M. & Estelle, M. Complex regulation of the TIR/AFB family of auxin receptors PNAS USA 106(52), 22540-22545, (2009)

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.

Publikation

Gago, S., Elena, S.F., Flores, R. & Sanjuán, R. Extremely high mutation rate of a hammerhead viroid Science 322, 1308, (2009)

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Publikation

Ticconi, C.A., Lucero, R.D., Sakhonwasee, S., Adamson, A.W., Creff, A., Nussaume, L., Desnos, T. & Abel, S. ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability Proc Natl Acad Sci USA (PNAS) 106, 14174-14179, (2009)

Inadequate availability of inorganic phosphate (Pi) in the rhizosphere is a common challenge to plants, which activate metabolic and developmental responses to maximize Pi acquisition. The sensory mechanisms that monitor environmental Pi status and regulate root growth via altered meristem activity are unknown. Here, we show that phosphate deficiency response 2 (PDR2) encodes the single P5-type ATPase of Arabidopsis thaliana. PDR2 functions in the endoplasmic reticulum (ER) and is required for proper expression of scarecrow (SCR), a key regulator of root patterning, and for stem-cell maintenance in Pi-deprived roots. We further show that the multicopper oxidase encoded by low phosphate root 1 (LPR1) is targeted to the ER and that LPR1 and PDR2 interact genetically. Because the expression domains of both genes overlap in the stem-cell niche and distal root meristem, we propose that PDR2 and LPR1 function together in an ER-resident pathway that adjusts root meristem activity to external Pi. Our data indicate that the Pi-conditional root phenotype of pdr2 is not caused by increased Fe availability in low Pi; however, Fe homeostasis modifies the developmental response of root meristems to Pi availability.

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