Utilization of the micro gravity condition to examine the cellular process of formation of the gravity sensor and the molecular mechanism of gravity sensing (Plant Gravity Sensing 1) - 09.17.14
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Plant Gravisensing continues research, sponsored by the Japan Aerospace Exploration Agency since STS-95, using microgravity to examine how plants determine up and down as they grow, something that is confounded in microgravity. Thale cress, used as a genetic model in many plant studies, is cultivated both in microgravity and artificial gravity as a control in the Kibo experiment module. Researchers analyze starch-filled bodies from cells in the root tips to understand the role of the MCA1 protein in activating a calcium channel essential for plant growth.
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OpNom Plant Gravity Sensing 1
Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)
ISS Expedition Duration
September 2014 - March 2015
Previous ISS Missions
Plants exhibit cytoplasmic calcium concentration ([Ca2+]c) increases in response to a variety of endo- and exogenous stimuli such as phytohormones, temperature and touch, the mechanisms of which have been extensively investigated. However, few studies have assessed the relationship between [Ca2+]c-increase and gravistimulation, because gravity is a ubiquitous force and difficult to control on Earth. The details of underlying cellular/molecular mechanisms leading the gravitropic response , especially the sensing machinery for gravity in plants remain totally unknown. Our recent preliminary experiments suggest that a mechanosensitive channel, e.g., MCA1, and actin cytoskeleton are involved in a gravistimulation-induced [Ca2+]c increase in Arabidopsis seedlings. The hypothesis proposed by researchers including us is that the amyloplasts (or heavy mass particles) sediment in the direction of the gravity vector, causes tension increases in the actin stress fibers, and it activates the mechanosensitive Ca2+ channels in the cell membrane. These subcellular components (channels and actin fibers) must be assembled and form the gravity-sensing machinery in the subcellular space under the 1-g condition, but it is unknown whether the machinery is assembled under the micro-g condition or not.
This research is needed because it tests the above hypothesis and the possible assembly of a gravisensing machinery in a micro-g space envelopment by performing four experiments as follows. (1) a gravistimulation induced [Ca2+]c-increase in Arabidopsis seedlings grown in the micro-g condition to clarify whether the gravity sensing machinery can be formed under the micro-gravity or not, (2) image analyses of the subcellular distribution of the mechanosensitive Ca2+ permeable channel candidate protein, MCA1, in root cells of the seedlings grown under the micro-g and the 1-g condition to examine weather the localization of the mechanosensitive channel is gravity dependent, (3) mRNA contents (including MCA1) in seedlings in the 1-g and micro-g conditions is examined, which will show the expression of the MCA1 and other gravisensor related proteins requires gravity, and (4) parabolic flight experiments with the MCA1 knockout and overexpressing seedlings to explore the role of MCA1 in gravity sensing.
The space experiments will clarify whether seedlings grown under the micro-gravity condition response to the changes in gravity and elevate [Ca2+]c or not. If the [Ca2+]c does not increase, or the amplitude or the duration of the [Ca2+]c response differs from that of plants grown under 1-g conditions, we can suggest that the proper assembly of the gravity sensing machinery requires gravity. The analysis of the subcellular distribution of the GFP-labeled mechanosensitive channel candidate protein Mca1 in root cells of the seedlings grown under the micro-g condition will provide the reason of the deficient (or modulated) [Ca2+]c response in plants grown under micro-g condition, e.g., if the mechanosensitive channel is not localized to the cell membrane, the tension changes in the cytoskeleton and/or cell membrane may not be transduced to activate MCA1, which will induce Ca2+ influx via the channel, and lead to the [Ca2+]c-increase. If the mRNA content in seedlings is down regulated in plants grown under micro-g condition, we can suggest the component of the gravity sensing machinery (e.g., MCA1) is not properly synthesized in plants under the micro-g condition. If the [Ca2+]c-increases in the mutant seedlings of MCA1 (knock out and overexpressor) in ground parabolic flight experiments show the important roles of MCA in gravisensing, the results of space experiments with those of the ground ones will strongly support our hypothesis that Mca1 is involved in the gravity sensing machinery in plants.
Gravitropism, plants sense the gravity vector and regulate their growth direction accordingly, was first identified nearly 200 years ago by Knight and was later characterized by Darwin. However, the molecular mechanism of gravity sensing in higher plants is a long-standing question. This research will allow us to investigate the process of the assembly of the gravity sensing machinery under micro-g, and also to examine the hypothesis of a gravisensing machinery. We may obtain some evidence in the space research to support the model that the change in the gravity vector is sensed by the mechanism consisting of amyloplast-actin filaments-mechanosensitive channels (e.g., MCA1) super complex subcellular structures.
Long-term space missions ultimately must be able to grow their own food, which starts with plants. Since artificial gravity may not be available on these missions, it is important to know what controls their growth. Results from the gravisensing experiment may allow the growth of plants under low-g or intermittent gravity during long space missions.
Understanding how plants determine which way to grow may allow bioengineering of new breeds that can right themselves more readily after being flattened by high winds or floods. In turn, this could improve crop yields and reduce food shortages. The findings may carry over to human health and the treatment of osteoporosis and muscle loss.
For Plant Growth:
Carefully put the sheets of oblaat with Arabidopsis seeds on “the center” of the wet surface of the medium plate. Avoid the bank of oblaat.
Carefully transfer the meshes with Arabidopsis seedlings, do not smash the seedlings.
Arabidopsis seedlings in RNAlater® should be kept in -95 Degree Celcius.
4 percent PFA should be prepared and warmed to 22 Degree Celcius, immediately before its use.
Arabidopsis seedlings in PBS (fixed with 4 percent PBS) should be kept in 4 Degree Celcius; the seedling should not be frozen.
Samples should be returned less than 6 months to the Earth.
For Run2-1 and 2-2:
Stock solution of Coelenterazine-CP (in ethanol) should be kept in the dark, in freezer.
1/2 MS medium should be warmed to 22 °C before adding stock solution of Coelenterazine-CP.
The Coelenterazine-CP solution (10 M in 1/2 MS medium) should be prepared, immediately before its use, handle under the dark or a dim light.
Do not push or smash the seedlings by the liquid absorbing devices.
Plant Seeds and growing medium:
1. Transgenic Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) expressing apoaequorin, a Ca2plus-reporting photoprotein, in cytoplasm under the control of the 35S promoter of Cauliflower mosaic virus will be used to detect the gravity-induced [Ca2plus]c increase.
2. Transgenic Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) expressing MCA1-GFP under the control of the 35S promoter of Cauliflower mosaic virus will be used to analyze the subcellular localization of MCA1 in microgravity condition.
3. Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) will be used to analyze the transcript level of MCA1 and the other potential components of gravity-sensing machineries.
4. Sheets of oblaat, thin edible layer of starch sheet, with the Arabidopsis seeds (ca. 50 seeds).
5. Plant Experiment Culture Dish (60 mm diameter, 20 mm height, filled with filled with 1/2 Murashige-Skoog (MS) medium containing 0.3percent [w/v] Gelrite ca. 25 cc in a gel form; termed medium plate) will be used to grow Arabidopsis seedlings in in the Japanese Kibo experiment module.
1. Put the sheets of oblaat with Arabidopsis seeds on the wet surface of the medium plate, incubated at 4 Degree Celcius in the dark for 2 days for the vernalization.
2. Cultivate at 22°C in PEUs under continuous light at approximately 50 mol m−2 s−1 with LED (Red : Blue = 4 : 1) for 10 days. For Run2-1 and 2-2, cultivate under µg or 1 g by CBEF, respectively. Sprouting of seedlings is seen 2-3 days of cultivation at 22°C in a growth chamber under continuous light. After 10 days, 5-10 mm seedlings (height) will be seen.
1. Cultivate wild type and Mca1-GFP expressing seedlings with the different color nylon meshes in the containers under the micro-g condition for 10 days.
2. Gently remove the meshed with seedlings (5-10 mm high with green leaves) from the medium plate. Seedlings is attached onto the mesh. When the mesh is gently pulled off, the seedlings will come off with the mesh.
3. Insert the mesh with seedlings into the plastic bag for the fixation (RNAlater® or PFA). The same experiment with the seedlings cultivated under the 1-g condition generated by the centrifuge should be done as a control.
4. Incubate the seedlings in RNAlater® at -30 °C overnight, and then store them at -95 °C.
5. Incubate the seedlings in PFA at room temperature (22-28 °C) for 1 h, remove PFA with absorbing polymer in the bag, wash the seedlings with PBS, and then store them at 4 °C. The seedlings should not be frozen.
Run2-1 and 2-2:
1. Containers with Arabidopsis seedlings are gently filled with 10 mL of the Coelenterazine-CP solution, avoid shaking and vibrations during the 4-12 h of incubation at 22 °C, in the dark.
2. Remove the Coelenterazine-CP solution gently from the dish 1-2 h before experiments with a liquid absorbing device containing water absorption polymer donut. Do not push or smash the seedlings by the donut.
3. Set Culture Dish into the Plant Experiment Photomultiplier in CBEF to give 1-g gravitational acceleration in the direction from leaves to roots. Next, detach Plant Exp Photomultiplier from CBEF, take out Culturer Dish from Plant Exp Photomultiplier and upset. Set Culture Dish into the Plant Experiment Photomultiplier in CBEF, 1-2h later, to give 1-g gravitational acceleration in the direction from roots to leaves.
4. Do not give the specimen any mechanical shock such as shaking or hitting the Plant Exp Photomultiplier 10-30 sec after the start of the centrifugation (micro-g to 1 g), the number of the emitted photons from seedlings will increase transiently from 100 counts/500ms to 1000 counts/500ms, and the number will decline within 3-5 min of recordings, or different amplitude (or different time course) of photon emission might be recorded.