Publikationen - Molekulare Signalverarbeitung

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).

Pflanzen müssen gegen vielfältige biotische und abiotische Umwelteinflusse eine Abwehr aufbauen. Aber gleichzeitig müssen sie wachsen und sich vermehren. Jasmonate sind neben anderen Hormonen ein zentrales Signal bei der Etablierung von Abwehrmechanismen, aber auch Signal von Entwicklungsprozessen wie Blüten‐ und Trichombildung, sowie der Hemmung von Wachstum. Biosynthese und essentielle Komponenten der Signaltransduktion von JA und seinem biologisch aktiven Konjugat JA‐Ile sind gut untersucht. Der Rezeptor ist ein Proteinkomplex, der “JA‐Ile‐Wahrnehmung” mit proteasomalem Abbau von Repressorproteinen verbindet. Dadurch können positiv agierende Transkriptionsfaktoren wirksam werden und vielfältige Genexpressionsänderungen auslösen. Dies betrifft die Bildung von Abwehrproteinen, Enzymen der JA‐Biosynthese und Sekundärstoffbildung, und Proteinen von Signalketten und Entwicklungsprozessen. Die Kenntnisse zur JA‐Ile‐Wirkung werden in Landwirtschaft und Biotechnologie genutzt.

Jasmonates are lipid-derived compounds which are signals in plant stress responses and development. They are synthesized in chloroplasts and peroxisomes. An endogenous rise occurs upon environmental stimuli or in distinct stages of development such as that of anthers and trichomes or in root growth. Hydroxylation, carboxylation, glucosylation, sulfation, methylation, or conjugation of jasmonic acid (JA) leads to numerous metabolites. Many of them are at least partially biologically inactive. The most bioactive JA is the (+)-7-iso-JA–isoleucine conjugate. Its perception takes place by the SCFCOI1-JAZ-co-receptor complex. At elevated levels of JAs, negative regulators such as JAZ, or JAV are subjected to proteasomal degradation, thereby allowing positively acting transcription factors of the MYC or MYB family to switch on JA-induced gene expression. In case of JAM negative regulation takes place by anatagonism to MYC2. JA and COI1 are dominant signals in gene expression after wounding or in response to necrotrophic pathogens. Cross-talk to salicylic acid, ethylene, auxin, and other hormones occurs. Growth is inhibited by JA, thereby counteracting the growth stimulation by gibberellic acid. Senescence, trichome formation, arbuscular mycorrhiza, and formation of many secondary metabolites are induced by jasmonates. Effects in cold acclimation; in intercropping; during response to herbivores, nematodes, or necrotrophic pathogens; in pre- and post-harvest; in crop quality control; and in biosynthesis of secondary compounds led to biotechnological and agricultural applications.

We studied the genetic variability of three genomic regions (p23, p25 and p27 genes) from 11 field Citrus tristeza virus isolates from the two main citrus growing areas of Argentina, a country where the most efficient vector of the virus, Toxoptera citricida, is present for decades. The pathogenicity of the isolates was determinated by biological indexing, single-strand conformation polymorphism analysis showed that most isolates contained high intra-isolate variability. Divergent sequence variants were detected in some isolates, suggesting re-infections of the field plants. Phylogenetic analysis of the predominant sequence variants of each isolate revealed similar grouping of isolates for genes p25 and p27. The analysis of p23 showed two groups contained the severe isolates. Our results showed a high intra-isolate sequence variability suggesting that re-infections could contribute to the observed variability and that the host can play an important role in the selection of the sequence variants present in these isolates.