Publikationen - Molekulare Signalverarbeitung

A strategy to detect and quantify the polar ethylene precursor 1-aminocyclopropan-1-carboxylic acid (ACC) along with the more apolar phytohormones abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), jasmonic acid-isoleucine conjugate (JA-Ile), 12-oxo-phytodienoic acid (OPDA), trans-zeatin, and trans-zeatin 9-riboside using a single extraction is presented. Solid phase resins commonly employed for extraction of phytohormones do not allow the recovery of ACC. We circumvent this problem by attaching an apolar group to ACC via derivatization with the amino group specific reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl). Derivatization in the methanolic crude extract does not modify other phytohormones. The derivatized ACC could be purified and detected together with the more apolar phytohormones using common solid phase extraction resins and reverse phase HPLC/electrospray negative ion tandem mass spectrometry. The limit of detection was in the low nanomolar range for all phytohormones, a sensitivity sufficient to accurately determine the phytohormone levels from less than 50 mg (fresh weight) of Arabidopsis thaliana and Nicotiana benthamiana tissues. Comparison with previously published phytohormone levels and the reported changes in phytohormone levels after stress treatments confirmed the accuracy of the method.

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX-95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX-97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.

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1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The enzyme is encoded by a divergent multigene family in Arabidopsis thaliana, comprising at least five genes, ACS1-5 (Liang, X., Abel, S., Keller, J. A., Shen, N. F., and Theologis, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11046-11050). In etiolated seedlings, ACS4 is specifically induced by indoleacetic acid (IAA). The response to IAA is rapid (within 25 min) and insensitive to protein synthesis inhibition, suggesting that the ACS4 gene expression is a primary response to IAA. The ACS4 mRNA accumulation displays a biphasic dose-response curve which is optimal at 10 μM of IAA. However, IAA concentrations as low as 100 nM are sufficient to enhance the basal level of ACS4 mRNA. The expression of ACS4 is defective in the Arabidopsis auxin-resistant mutant lines axr1-12, axr2-1, and aux1-7. ACS4 mRNA levels are severely reduced in axr1-12 and axr2-1 but are only 1.5-fold lower in aux1-7. IAA inducibility is abolished in axr2-1. The ACS4 gene was isolated and structurally characterized. The promoter contains four sequence motifs reminiscent of functionally defined auxin-responsive cis-elements in the early auxin-inducible genes PS-IAA4/5 from pea and GH3 from soybean. Conceptual translation of the coding region predicts a protein with a molecular mass of 53,795 Da and a theoretical isoelectric point of 8.2. The ACS4 polypeptide contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. An ACS4 cDNA was generated by reverse transcriptase-polymerase chain reaction, and the authenticity was confirmed by expression of ACC synthase activity in Escherichia coli.