Induction of hypoxic root metabolism results from physical limitations in O2 bioavailability in microgravity

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the root zone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates: (1) the inhibition of gravity-driven convective transport can reduce the O2 bioavailability to the root tip, and (2) the perturbation of gravisensing by clinostat rotation does not induce a nonspecific stress response involving ADH. Together these experiments support the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight.