TY  - JOUR
A1  - Li, Kunkun
A1  - Prada, Juan
A1  - Damineli, Daniel S. C.
A1  - Liese, Anja
A1  - Romeis, Tina
A1  - Dandekar, Thomas
A1  - Feijó, José A.
A1  - Hedrich, Rainer
A1  - Konrad, Kai Robert
T1  - An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca\(^{2+}\) and H\(^{+}\) reveals new insights into ion signaling in plants
JF  - New Phytologist
N2  - Whereas the role of calcium ions (Ca\(^{2+}\)) in plant signaling is well studied, the physiological significance of pH‐changes remains largely undefined.

Here we developed CapHensor, an optimized dual‐reporter for simultaneous Ca\(^{2+}\) and pH ratio‐imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio‐temporal relationships between membrane voltage, Ca\(^{2+}\)‐ and pH‐dynamics revealed interconnections previously not described.

In tobacco PTs, we demonstrated Ca\(^{2+}\)‐dynamics lag behind pH‐dynamics during oscillatory growth, and pH correlates more with growth than Ca\(^{2+}\). In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA‐responses and even opened stomata in the presence of ABA, disclosing an important pH‐dependent GC signaling node. In MCs, a flg22‐induced membrane depolarization preceded Ca2+‐increases and cytosolic acidification by c. 2 min, suggesting a Ca\(^{2+}\)/pH‐independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage‐, Ca\(^{2+}\)‐ and pH‐responses.

We propose close interrelation in Ca\(^{2+}\)‐ and pH‐signaling that is cell type‐ and stimulus‐specific and the pH having crucial roles in regulating PT growth and stomata movement.
KW  - abscisic acid (ABA)
KW  - calcium
KW  - flg22
KW  - guard cells
KW  - imaging
KW  - ion signaling
KW  - pH
KW  - pollen tube
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239847
VL  - 230
IS  - 6
SP  - 2292
EP  - 2310
ER  - 
TY  - JOUR
A1  - Peters, Simon
A1  - Kaiser, Lena
A1  - Fink, Julian
A1  - Schumacher, Fabian
A1  - Perschin, Veronika
A1  - Schlegel, Jan
A1  - Sauer, Markus
A1  - Stigloher, Christian
A1  - Kleuser, Burkhard
A1  - Seibel, Juergen
A1  - Schubert-Unkmeir, Alexandra
T1  - Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria
JF  - Scientific Reports
N2  - Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce 'click-AT-CLEM', a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.
KW  - antimicrobials
KW  - biological techniques
KW  - imaging
KW  - microbiology
KW  - microbiology techniques
KW  - microscopy
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259147
VL  - 11
IS  - 1
ER  -