Joint European Partial-G Parabolic Flight Campaign-Calcium Analysis in Arabidopsis Thaliana Cell Cultures
Abstract
Callus cells derived from stem tissue suspension cultures of Arabidopsis thaliana (cv. Columbia) were exposed to parabolic flights on board of an Airbus A300 (Novespace). The cells were either wild type or expressed a fluorescent probe for the quantification of cytosolic calcium (green fluorescent protein (GFP)-based Cameleon). The wild type cells were used for both, fluorescence background control, and the analysis of gene expression. With respect to fluorescence measurements, changes in the amounts of Ca2+, an important component of signalling chains, could be assayed in vivo in real time. The technique used takes advantage of a shift in fluorescence from 480 to 535 nm with increasing Ca2+ content. During the experiment, fluorescence data were monitored at Mars, Moon and micro gravity producing flight profiles, each at 1g, pull up (1.8g), about 20 to 26 s of mars (0.36g), moon (0.16g) or micro gravity, and pull out (1.8g) for 12/12/6 consecutive parabolas at different days. Transition from hypergravity to microgravity resulted in a typical increase in cytosolic Ca2+. The flight profile “Moon” (0.16g) exhibited a very similar behaviour as microgravity, whereas simulation of “Mars” gravitation (0.36) resulted in a weaker signal. This can also be deduced from minimal/maximal values of the ratio between hyper-g and onset of reduced g. Obviously, the threshold gravitation for a Ca2+ response is above 0.36g. Increasing gravity by centrifugation, in contrast, induced a decrease in cytosolic calcium. Here, a threshold in response was obvious between 3 and 4g. In order to assay changes in gene expression, we additionally quenched parabolic flight samples by the injection of RNAlater. A microarray analysis of these samples showed a clear impact of the different profiles. Both Moon and Mars profiles exhibited less response than the μg profile. However, the latter responded also less compared to previous “μg only” flights. In those we had much higher numbers of altered transcripts. For example, after 20 s of μg we found about 400 transcripts increased and about 500 transcripts decreased in amount; this compares to 140 /100 only in the present example. We thus assume that the perturbations induced by Moon and Mars parabolas decrease the sensitivity towards μg. We can, however, directly compare the Moon profile with our “μg only” experiments, because these were the first ones within the whole series to experience reduced gravitation. Here, we find only about 20 transcripts up- and about 50 transcripts down-regulated, which is much less than observed for μg. In summary, we find that Ca2+ responses have a threshold above Mars gravitation (0.38 g), while prominent changes in gene expression occur below Moon gravitation (0.16 g).
- Publication:
-
Life in Space for Life on Earth
- Pub Date:
- February 2013
- Bibcode:
- 2013ESASP.706E..18N