TY - JOUR A1 - Hyun, Tae Kyung A1 - van der Graaff, Eric A1 - Albacete, Alfonso A1 - Eom, Seung Hee A1 - Grosskinsky, Dominik K. A1 - Böhm, Hannah A1 - Janschek, Ursula A1 - Rim, Yeonggil A1 - Ali, Walid Wahid A1 - Kim, Soo Young A1 - Roitsch, Thomas T1 - The Arabidopsis PLAT Domain Protein1 is Critically Involved in Abiotic Stress Tolerance JF - PLOS ONE N2 - Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty. KW - salicylic acid KW - gene expression KW - signal transduction KW - cold stress KW - salt stress KW - abscisic acid KW - endoplasmatic reticulum KW - transcription factors KW - pseudomonas syringae KW - plants response Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114648 VL - 9 IS - 11 ER - TY - THES A1 - Ali, Walid Wahid T1 - Screening of plant suspension cultures for antimicrobial activities and characterization of antimicrobial proteins from Arabidopsis thaliana T1 - - N2 - Die zunehmende Resistenz humanpathogener Mikroorganismen gegen bekannte Antibiotika bedingt die Notwendigkeit, nach neuen Quellen für die Produktion antimikrobieller Stoffe zu suchen. Als eine solche Quelle gelten besonders Pflanzen, da viele antimikrobielle Stoffe bei der Abwehr gegen invasierende Mikroorganismen bilden. Das Ziel der vorliegenden Arbeit besteht in der Charakterisierung von pflanzlichen Zellkulturen im Hinblick auf ihre Fähigkeit, anitimkrobielle Aktivität gegen humanpathogene Mikroorganismen zu entwickeln. Dabei sollen aktive Proteine aufgereinigt und die kodierenden Gene isoliert werden. Dazu wurden zehn verschiede pflanzliche Suspensionskulturen in Anwesenheit von neun Elicitoren auf ihre antimikrobielle Aktivität gegen fünf humanpathogene Mikroorganismen getestet. Dabei erwiesen sich die heterotrophen Kulturen im Vergleich zu den autotrophen als aktiver. Die höchste antimikrobielle Aktivität wurde bei der intrazellulären Fraktion der mixotrophen Kultur von Arabidopsis thaliana nach Elicitierung mit Salicylsäure nachgewiesen. Da in einem Präzipitat mit Ammoniumsulfat Aktivität gegen Candida maltosa nachgewiesen wurde, konnte angenommen werden, dass es sich bei der aktiven Komponente um ein Protein handelt. Durch Hochgeschwindigkeitszentrifugation wurde eine partielle Aufreinigung dieser aktiven Komponente erreicht. Die proteinoide Natur wurde durch Bioautographie bestätigt und das Molekulargewicht auf ca. 26kDa geschätzt. Mittels Gelfiltration und Massenspektrometrie wurde das Protein aufgereinigt. Die Mikrosequenzierung ergab ein Protein mit bisher unbekannter Funktion, das eine pflanzliche Stressdomäne (PLAT) enthält. Das Protein wurde daraufhin als AtPDP1 (Arabidopsis thaliana Plat-Domain Protein 1) bezeichnet. Das Gen und ein zweites mit hochgradiger Homologie (AtPDP2) wurden in E. coli kloniert. Der Digital Northern zeigt an, das beide Gene durch verschiedene Pathogene induziert werden, sowie von Chemikalien, die pflanzliche Abwehr hervorrufen und weiterhin von Phytohormonen. Der Versuch, AtPDP1 unter die Kontrolle eines Promors einer Proteinase zu stellen, der Induzierbarkeit durch Elicitoren vermittelt, blieb erfolglos. Weiterhin wurden 13 Thaumatingene aus Arabidopsis thaliana in E. coli kloniert, da ihre antimikrobielle Aktivität bekannt ist, und ihre Expression durch verschiedene Stimuli induziert wird. Von diesen Genen zeigt der Digital Northern bei allen Stimuli eine maximale Expression für At1g75800, während At1g75050 minimal induziert ist. Diese Gene stehen für zukünftige Studien zur Verfügung. N2 - The continuously increase in resistance of human pathogenic microorganisms to the known antibiotics leads to the necessity for searching new sources for production of new active antimicrobial compounds from different natural sources especially plants, since many plants have been found to be able to produce antimicrobial compounds as a defense phenomenon against invading microorganisms. The aim of this work is to screen cultures for production of antimicrobial activity against representative of human pathogenic microorganisms and selection the most active cell culture producing antimicrobial protein(s) which are active against these pathogenic microorganisms and also isolation ,purification of the active protein(s) and cloning of its/their genes. Ten different plant suspension cultures have been screened in presence of nine elicitors for their antimicrobial activity against five selected human pathogenic microorganisms, and it has been found that the heterotrophic cultures are more active against the tester isolates than the autotrophic ones. The intracellular fraction of the mixotrophic Arabidopsis thaliana culture elicited with salicylic acid showed the highest antimicrobial activity against the tester isolates. The presence of proteinous antimicrobial activity has been elucidated by testing the activity of ammonium sulphate precipitate against Candida maltosa. High speed centrifugation technique has been used for partial purification of the active protein. The proteinous nature of the isolated compound has been confirmed by using bioautography technique and its molecular weight could be estimated to be around 26KDa. The active protein has been purified using gel filtration, and using mass spectrometry technique, for microsequencing of the active protein, it has been found that the function of the protein is unknown and we have termed it as AtPDP1 according to Arabidopsis thaliana Plat-Domain Protein1, since it contains a plant stress domain termed PLAT domain. It has been found that a second protein from the same plant with high homology level to AtPDP1 with the same domain, we termed it as AtPDP2. Genes for AtPDP1 and AtPDP2 have been cloned in E. coli using PGEM-T easy vector. The expression of both genes have been tested using Digital Northern program, and it has been observed that both genes are induced by different pathogens, chemicals known to induce defense in plant cells and also different hormones. We tried to clone the gene for AtPDP1 in PBI121 binary vector under the control of an elicitor inducible promoter of a proteinase inhibitor gene, to test its function in plant by overexpression, but we did not succeeded. Also the work aims to cloning the different known thaumatin genes from Arabidopsis thaliana for future work which represented by testing their expression under different stimuli, since most thaumatins have antimicrobial activity and some of them are active against Candida spp..Thirteen genes of known thaumatins from Arabidopsis thaliana have been cloned in PGEM-Teasy vector in DH5-alpha cells. coli cells. The expression of the thirteen genes has been done using Digital Northern program and it has been found that different genes show different expressions under different stimuli and the expression of At1g75800 gene was the maximum under all stimuli. The minimum expression of genes was for At1g75050. The rest of thaumatin genes showed moderate expressions under different stimuli. KW - - KW - Plant antimicrobial proteins KW - Arabidopsis thaliana KW - PLAT-Domain KW - Thaumatins Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-24358 ER -