Real-time Recording of Cytosolic Calcium Levels in Arabidopsis thaliana Cell Cultures during Parabolic Flights

Abstract

In plants, like in other organisms, calcium (Ca2+) is an important second messenger which participates in the conversion of environmental signals into molecular responses. There is increasing evidence, that sensing of changes in gravitation or reorientation of tissues is an example for such signaling cascades in which Ca2+ is involved. In order to determine g-dependent changes in the cytosolic calcium (Ca $^{2+}_{\text {cyt}})$ concentration of plant cells, semisolid transgenic callus cell cultures of Arabidopsis thaliana (A.t.), expressing the calcium sensor YC3.6 (cameleon), were exposed to g-forces between 1.8g and μg during parabolic flights. Using such cells, intracellular calcium transients can be monitored by FRET in vivo and in real-time. Interestingly we observed a slight decrease of the Ca $^{2+}_{\text {cyt}}$ level during the hypergravity phases of a parabola but a significant increase of the Ca $^{2+}_{\text {cyt}}$ concentration during microgravity. Application of known Ca2+ inhibitors and antagonists yielded the following effects: nifedipine (Ca2+ channel blocker) showed no effect, whereas LaCl3, GdCl3 (both inhibitors of uptake at the plasma membrane), DPI (inhibitor of NADP oxidase), and DMSO (solvent) diminished the gravity-alteration-related Ca $^{2+}_{\text {cyt}}$ response. EGTA (binding of Ca2+) and eosin yellow (inhibitor of a plasma membrane-located Ca2+ pump) suppressed the respective Ca $^{2+}_{\text {cyt}}$ changes entirely. We thus conclude that the significant increase in Ca $^{2+}_{\text {cyt}}$ under microgravity is largely due to extracellular Ca2+ sources.