@article{BlachutzikDemirKreuzeretal.2012, author = {Blachutzik, J{\"o}rg O. and Demir, Faith and Kreuzer, Ines and Hedrich, Rainer and Harms, Gregory S.}, title = {Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75433}, year = {2012}, abstract = {Background: Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results: Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion: Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.}, subject = {Arabidopsis thaliana}, language = {de} } @article{NuhkatBroscheStoezleFeixetal.2021, author = {Nuhkat, Maris and Brosch{\´e}, Mikael and Stoezle-Feix, Sonja and Dietrich, Petra and Hedrich, Rainer and Roelfsema, M. Rob G. and Kollist, Hannes}, title = {Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells' ozone response}, series = {New Phytologist}, volume = {232}, journal = {New Phytologist}, number = {4}, doi = {10.1111/nph.17711}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259646}, pages = {1692-1702}, year = {2021}, abstract = {Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O\(_{3}\) triggered a drop in whole-plant CO\(_{2}\) uptake. Within this early phase, O\(_{3}\) pulses (200-1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O\(_{3}\) induced cell death, systemic Ca\(^{2+}\) signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca\(^{2+}\)) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.}, language = {en} } @article{DindasDreyerHuangetal.2021, author = {Dindas, Julian and Dreyer, Ingo and Huang, Shouguang and Hedrich, Rainer and Roelfsema, M. Rob G.}, title = {A voltage-dependent Ca\(^{2+}\) homeostat operates in the plant vacuolar membrane}, series = {New Phytologist}, volume = {230}, journal = {New Phytologist}, number = {4}, doi = {10.1111/nph.17272}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259627}, pages = {1449-1460}, year = {2021}, abstract = {Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca\(^{2+}\) signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca\(^{2+}\) and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca\(^{2+}\) concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H\(^{+}\)/Ca\(^{2+}\) exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca\(^{2+}\) homeostat could contribute to calcium signalling when coupled to a recently discovered K\(^{+}\) channel-dependent module for electrical excitability of the vacuolar membrane.}, language = {en} } @article{HuangDingRoelfsemaetal.2021, author = {Huang, Shouguang and Ding, Meiqi and Roelfsema, M. Rob G. and Dreyer, Ingo and Scherzer, S{\"o}nke and Al-Rasheid, Khaled A. S and Gao, Shiqiang and Nagel, Georg and Hedrich, Rainer and Konrad, Kai R.}, title = {Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel GtACR1}, series = {Science Advances}, volume = {7}, journal = {Science Advances}, number = {28}, doi = {10.1126/sciadv.abg4619}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-260925}, year = {2021}, abstract = {Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO\(_2\) and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl\(^-\) and NO\(_3\)\(^-\) currents of -1 to -2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K+ channels and the extrusion of K+. As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.}, language = {en} }