Publikationen - Molekulare Signalverarbeitung

Dinucleoside 5′,5‴-P1,P4-tetraphosphate hydrolase (EC 3.6.1.17) has been purified to homogeneity from tomato (Lycopersicon esculentum) cells grown in suspension. The purification procedure comprised ammonium sulphate fractionation following five standard chroma­ tography steps and a final chromatography on Ap4A-Sepharose.

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Genes induced by the plant hormone auxin are probably involved in the execution of vital cellular functions and developmental processes. Experimental approaches designed to elucidate the molecular mechanisms of auxin action have focused on auxin perception, genetic dissection of the signaling apparatus and specific gene activation. Auxin‐responsive promoter elements of early genes provide molecular tools for probing auxin signaling in reverse. Functional analysis of several auxin‐specific promoters of unrelated early genes suggests combinatorial utilization of both conserved and variable elements. These elements are arranged into autonomous domains and the combination of such modules generates uniquely composed promoters. Modular promoters allow for auxin‐mediated transcriptional responses to be revealed in a tissue‐ and development‐specific manner.

The aims of this study were to demonstrate the endogenous presence of jasmonic acid (JA) in roots, stolons and periderm of new formed tubers, by means of bioassays, ELISA and GC-MS, and to test a microdrop bioassay using the leaflets of potato cuttings cultured in vitro. Our results confirm the existence of JA by bioassays and GC-MS in foliage, stolons, roots and tuber periderm.

The plant growth substance jasmonic acid and its methyl ester (JA‐Me) induce a set of proteins (jasmonate‐induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one‐dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno‐gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP‐23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP‐37 was detected in vacuoles and in the nucleoplasm; JIP‐66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP‐100 was found within the stromal fraction of chloroplasts; JIP‐70 is present in the peroxisome and the nucleus; JIP‐50 and JIP‐6 accumulate in vacuoles. The location of JIP‐66 and JIP‐6 confirms their possible physiological role deduced from molecular analysis of their cDNA.

An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for β‐glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)‐mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin‐mediated induction of chloramphenicol acetyl‐transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin‐inducible PS‐IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.

The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the t1/2 of the PS-IAA4 and PS-IAA6 proteins to be 8 and 6 min, respectively. Their most prominent feature is the presence of a beta alpha alpha motif similar to the beta-sheet DNA-binding domain found in prokaryotic repressors of the Arc family. Based on these data, we suggest that plant tissues express short-lived nuclear proteins as a primary response to IAA. We propose that these proteins act as activators or repressors of genes responsible for mediating the various auxin responses.