A Comprehensive Toolkit for Inducible, Cell Type-Specific Gene Expression in Arabidopsis.

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis ( <i>Arabidopsis thaliana</i> ) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic <i>pOp</i> promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the <i>pOp</i> promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types

IIndanyloxyacetic acid-sensitive chloride channels from outer membranes of skeletal muscle

Weber-Schürholz S, Wischmeyer E, Laurien M, et al. IIndanyloxyacetic acid-sensitive chloride channels from outer membranes of skeletal muscle. Journal of Biological Chemistry. 1993;268(1):547-551.In mature mammalian muscle, the chloride conductance of the membrane is an important factor in the regulation of excitability. Up to now, no ligand was available for the biochemical characterization of muscle chloride channels. In order to localize and characterize these channels, we have used indanyloxyacetic acid (IAA)-94, a ligand previously used for epithelial Cl- channels (Landry, D. W., Reitman, M., Cragoe, E. J., Jr., and Al-Awqati, Q. (1987) J. Gen. Physiol. 90, 779-798; Landry, D. W., Akabas, M. H., Redhead, C., Edelman, A., Cragoe, E. J., Jr., and Al-Awqati, Q. (1989) Science 244, 1469-1472). IAA induced myotonic responses when microinjected into mature mouse muscle fibers, indicating a blockade of Cl- channels from the cytoplasmic side. Membrane vesicles were prepared from rabbit skeletal muscle and separated by sucrose gradient centrifugation. Fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmatic reticulum (SR), and triads and mitochondria (TR/M). The fraction enriched for SL was characterized by high specific binding capacity for [H-3]saxitoxin (Na+ channel), whereas TT-rich fractions bound [H-3]PN 200-110 (dihydropyridine receptor) with high specific activity. Upon patch-clamping of lipid supplemented vesicles, IAA-sensitive Cl- channels were found in the SL fraction but not in the SR. Highest specific activities in electrical diffusion potential sensitive Cl-36 transport and [H-3]IAA-94 binding were found in the SL. SL vesicles were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and subjected to IAA-Sepharose affinity chromatography. Specifically bound protein was eluted with 100 muM IAA-94 and either analyzed by SDS-gel electrophoresis or reconstituted into phospholipid vesicles. The eluate contained four polypeptides (specifically bound, m(app) 110-120 and 60 kDa; unspecifically bound m(app) 67 and 50 kDa) and was highly enriched for IAA-sensitive chloride channels as shown by patch-clamping after reconstitution. The IAA-sensitive 100/280-picosiemens chloride channels of the sarcolemma are likely to be responsible for its major chloride conductance and thereby for the stabilization of resting potential