As key components of bioregenerative life-support systems, plants will be exposed to multiple stresses associated with spaceflight. Experiments carried out in space have dramatically improved our…

This is a $273,707.50 grant award from the National Aeronautics and Space Administration (NASA) Shared Services Center to the University of Wisconsin System for a research project investigating the effects of low-speed clinorotation on the root growth behavior of the plant Brachypodium distachyon. The project aims to develop high-throughput imaging techniques to study auxin distribution and signaling in the plant roots under simulated microgravity conditions, as well as use genome-wide association studies and RNA sequencing to identify genes involved in the plant's response to clinorotation. The research is intended to improve understanding of how plants perceive and respond to gravity, which is a key objective of NASA's Space Biology program. The grant does not have a set-aside designation and is intended to be completed by July 21, 2023.

As key components of bioregenerative life-support systems, plants will be exposed to multiple stresses associated with spaceflight. Experiments carried out in space have dramatically improved our understanding of plant responses to microgravity. In turn, this novel knowledge has allowed the design of better culturing methods that enable plant development and reproduction in space. Yet, to design even better growth strategies and develop optimized genotypes favorable to plant growth and productivity during spaceflight, we need to better understand the molecular machinery that contribute to the perception of, and response to gravity and/ or microgravity. Unfortunately, the large cost associated with space experimentation, and limited accessibility to the resources needed for such experimentation, limit our ability to acquire this new knowledge. Consequently, utilization of devices that mimic microgravity is viewed as a viable alternative. The 2D-clinostat has often been utilized for this purpose. By constantly changing the orientation of biological specimens relative to gravity, these devices prevent gravity-vector detection, thereby triggering behavioral responses that are similar to those expressed under true microgravity. The 2D-clinostat has often been used to estimate the sensitivity of plant organs to different doses of gravi-stimulation. In this case, low doses of gravi-stimulation are provided at the beginning of the experiment. Stimulated seedlings are subsequently clinorotated for 4-5 hours, allowing for a curvature to develop in response to the initial stimulus. Yet, the data derived from these analyses are likely to be significant only if the clinorotation does not introduce artifacts of its own. In experiments involving arabidopsis thaliana seedlings, we previously observed a strong clinostat effect on root skewing on hard agar surfaces. Follow-up experiments involving brachypodium distachyon confirmed a clear impact of low-speed clinorotation on directional root-tip bending. These experiments also suggested an important role for the first cycle of clinorotation in setting up a specific direction of gravistimulation as long as IT is provided for a sufficiently long time period. The general objective of this research project will be to investigate the effect of low speed clinorotation on brachypodium root growth. Specific objectives include: aim 1: develop high throughput time-lapse imaging to describe the development of b. Distachyon root curvature responses to clinorotation at high spatio-temporal resolution; aim 2: use transgenic b. Distachyon seedlings that express auxin-level, auxin-function, and auxin-efflux carrier reporters to investigate the effect of clinorotation on auxin distribution and signaling along the root tip, and on the distribution of auxin transporters between flanks of clinorotated root tips; AIM3: use a combination of genome wide association study (gwas) and RNA SEQ strategies to identify candidate genes that may contribute to root-tip response to clinorotation, and use reverse genetics to investigate the role(s) of such candidates and of gravitropism-related genes in the process. Overall, this project will improve our understanding of how low-speed clinorotation affects root growth behavior. While our observations suggest a need for greater caution in using a low-speed 2D clinostat as a microgravity-mimic system, they also suggest a novel method to investigate the molecular mechanisms that contribute to gravity sensing in plants, one of the main objectives of the space biology program. This project will introduce undergraduate students to the idiosyncrasies of scientific research in space biology, while also putting a strong emphasis on undergraduate and k-12 education and public outreach.