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Transgenic Poplar Plants for the Investigation of ABA-Dependent Salt and Drought Stress Adaptation in Trees

American Journal of Plant Sciences 2016年第9期7卷 1337-1356页

作者： Domenica Hamisch david kaufholdt Jennifer-Christin Kuchernig Florian Bittner Ralf R. Mendel Robert Hänsch Jennifer Popko Institut für Pflanzenbiologie Technische Universitat Braunschweig Braunschweig Germany Albrecht-von-Haller-Institutfür Pflanzenwissenschaften Abteilung Biochemie der Pflanzen Georg-August-Universitat Gottingen Germany

Important functions of the plant hormone abscisic acid (ABA) in stress reactions, growth and photosynthetic processes are extensively studied in the model plant Arabidopsis thaliana. This paper investigates the importance of Moco-sulphurase ABA3 and aldehyde oxidase (AO) on ABA-biosynthesis in Populus × canescens. ABA3 is essential for activation of the molybdenum enzymes AO and xanthine dehydrogenase (XDH). AO itself catalyzes the last step in ABA-biosynthesis. Generation of transgenic poplar plants altered in ABA3 and AO-activity using RNAi knock down and overexpression was performed. Whereas RNAi-AO plants show a specific loss of AO activity, the RNAi-ABA3 plants has a strongly reduced activity of both molybdenum enzymes: AO and XDH. Constructs of AO and ABA3-promoters fused to β-glucuronidase provide the basis to investigate transcriptional regulation of ABA-biosynthetic processes under stress conditions. Application of high salt concentrations and different drought stress intensities does change the endogenous AO or XDH neither on the side of transcription nor on protein activity. On phytohormone level however, water loss leads to increased ABA-amounts regardless of whether transgenic or wildtype plants are studied. Salt application resulted in higher ABA-levels in all analyzed plant lines. The down regulation of AO in the two different RNAi-plant lines strongly prevented a wildtype-like increase of ABA-levels. Whereas the WT plants accumulated up to 6000 ng ABA g-1 FW-1 after 16 h of salt stress exposure, plants of the RNAi lines revealed a markedly lower increase of only up to 2000 ng ABA g-1 FW-1. Opposing to these observations, ABA-levels increased during drought without any influence by the RNAi-effect. These results revealed that although stresses did not result in a visible increased AO-activity, ABA-production was influenced by AO and ABA3 at least under salinity.

关键词： ABA Biosynthesis Drought Stress Populus × canescens Transgenic Poplar Salt Stress

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Split-HaloTag imaging assay for sophisticated microscopy of protein–protein interactions in planta

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Plant Communications 2021年第5期2卷 148-159页

作者： Rieke Minner-Meinen Jan-Niklas Weber Andreas Albrecht Rainer Matis Maria Behnecke Cindy Tietge Stefan Frank Jutta Schulze Henrik Buschmann Peter Jomo Walla Ralf-R.Mendel Robert Hänsch david kaufholdt Institut für Pflanzenbiologie Technische Universität BraunschweigHumboldtstrasse 138106 BraunschweigGermany Institut für Physikalische und Theoretische Chemie Technische Universität BraunschweigHagenring 30.023c38106 BraunschweigGermany Botany Department Universität OsnabrückBarbara Strasse 1149076 OsnabrückGermany Center of Molecular Ecophysiology(CMEP) College of Resources and EnvironmentSouthwest UniversityTiansheng Road No.2Beibei District400715 ChongqingP.R.China

An ever-increasing number of intracellular multi-protein networks have been identified in plant ***-GFP-based protein–protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex *** of their simple protocols,they have become some of the most frequently used ***,standard fluorescent proteins present several drawbacks for sophisticated *** the HaloTag system,these drawbacks can be overcome,as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent *** can be used in adjustable concentrations and are suitable for advanced microscopy ***,we have established the Split-HaloTag imaging assay in plants,which is based on the reconstitution of a functional HaloTag protein upon protein–protein interaction and the subsequent covalent binding of an added fluorescent *** suitability and robustness were demonstrated using a well-characterized interaction as an example of protein–protein interaction at cellular structures:the anchoring of the molybdenumcofactor biosynthesis complex to filamentous *** addition,a specific interactionwas visualized in a more distinctivemannerwith subdiffractional polarizationmicroscopy,Airyscan,and structured illumination microscopy to provide examples of sophisticated ***-GFPand Split-HaloTag can complement one another,as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo ***,this promising new Split-HaloTag imaging assay provides a unique and sensitive approach formore detailed characterization of protein–protein interactions using specific microscopy techniques,such as 3D imaging,single-molecule tracking,and super-resolution microscopy.

关键词： advanced microscopy cytoskeleton photostable fluorescent dyes gateway cloning molybdenum cofactor biosynthesis complex protein-protein interaction Split-HaloTag imaging assay

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