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) - 10.21.14
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Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense the gravity vector and regulate their growth direction accordingly. Utilization of the microgravity condition to examine the cellular process of formation of the gravity sensor and the molecular mechanism of gravity sensing (Plant Gravity Sensing 1) examines whether the gravity sensing machinery can be formed under the micro-gravity condition on board the International Space Station, and examines the gravity sensing mechanism in plant seedlings.
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OpNom Plant Gravity Sensing 1
JAXA TKSC Space Environment Utilization Center, Tsukuba, , Japan
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 gravity stimulation, 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. Recent preliminary experiments suggest that a mechanosensitive channel, e.g., MCA1, and actin cytoskeleton are involved in a gravity stimulation-induced [Ca2+]c increase in Arabidopsis seedlings. The hypothesis proposed by researchers is that the amyloplast (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 calcium ion (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 gravity sensing machinery in a micro-g space environment. By performing four experiments, as follows, a gravity stimulation induced [Ca2+] increase in Arabidopsis seedlings grown in the micro-g condition can clarify whether the gravity sensing machinery is 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 whether 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 can show that the expression of the MCA1 and other gravity sensor 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 experiment seeks to clarify whether seedlings grown in micro-gravity conditions respond 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, researchers can suggest that the proper assembly of the gravity sensing machinery requires gravity. The analysis of the subcellular distribution of the Green Flourescence Protein (GFP)-labeled mechanosensitive channel candidate protein MCA1 in root cells of the seedlings grown under the micro-g condition provides the reason of the deficient (or modulated) [Ca2+]c response in plants grown under micro-g conditions, 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 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, it can be suggested that the component of the gravity sensing machinery (e.g., MCA1) is not properly synthesized in plants under micro-g conditions. 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 MCA1 in gravity sensing, the results of space experiments with those of the ground ones strongly support the hypothesis that MCA1 is involved in the gravity sensing machinery in plants.
Gravitropism, the process in which 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 allows the investigation of the process of the assembly of the gravity sensing machinery under micro-g, and also to examine the hypothesis of a gravity sensing machinery. Evidence may also be obtained 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.
Future manned space flight to Mars or asteroid fields need to cultivate plants for food. The gravitational acceleration to the plants may affect their morphology and physiological functions, the gravitational acceleration to maintain the plant growth is hard to control during the space flight. Unfortunately, the molecular mechanism of gravity sensing in plants is poorly understood at present. If the hypothesis of plant gravity sensing is proved to be correct by the space research, there may be tools to modify the gravity sensitivity of plants by regulating the expression of mechanosensitive channels or other gravity sensing related proteins. In other words, healthy plants may be cultivated in a relatively low magnitude of gravitational accelerations, or even under the high magnitude of gravitational acceleration (e.g., on the surface of heavy planets) by up- or down-regulation of the plants sensitivity to gravity.
Plants sense changes in the gravity vector and regulate their growth direction accordingly. According to the hypothesis, it may be possible to modify the gravity sensitivity of plants on the earth, also. Plants may be designed that respond to gravity vector changes more efficiently than wild ones. These plants could recover from collapse by winds or by flood flows more rapidly than wild ones. Thus, the crop yield (or the agricultural output) of the designed plants can be greatly increased, which may solve in part the shortage of crops in the near future. In the field of medical sciences, the understanding of the gravity sensing machinery in cells may also bring profound progresses in exploring the mechanism of gravity stimulation related diseases, such as osteoporosis and muscle loss.
For Plant Growth: Carefully place the sheets of oblate, with Arabidopsis seeds, on the center of the wet surface of the medium plate. Avoid the bank of oblate.
For the Experiment Run: Carefully transfer the meshes with Arabidopsis seedlings, do not smash the seedlings. Arabidopsis seedlings in RNAlater® should be kept at -95°C. A mixture of paraformaldehyde and glutaraldehyde should be prepared and warmed to 22°C, immediately before its use. Arabidopsis seedlings in paraformaldehyde and glutaraldehyde (fixed with 4 % paraformaldehyde and 0.1% glutaraldehyde) should be kept in 2°C; the seedlings should not be frozen. Samples should be returned less than 6 months to the Earth.
For the Plant Seeds and Growing Medium: Transgenic Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) expressing apoaequorin, a Calcium ion reporting photoprotein, in cytoplasm under the control of the 35S promoter of Cauliflower mosaic virus is used to detect the gravity-induced Calcium ion increase. Transgenic Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0), expressing MCA1-GFP under the control of the 35S promoter of Cauliflower mosaic virus, is used to analyze the subcellular localization of MCA1 in microgravity conditions. Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) is used to analyze the transcript level of MCA1, and the other potential components of gravity-sensing machinery. Sheets of oblate, a thin edible layer of starch sheet, with the Arabidopsis seeds and a culture dish filled with 1/2 Murashige-Skoog (MS) medium containing 0.3% [w/v] Gelrite in a gel form, is used to grow Arabidopsis seedlings in the Japanese Kibo experiment module. Plant Growth: Place the sheets of oblate with Arabidopsis seeds on the wet surface of the medium plate, incubated in the dark for 2 days for the growth period. Cultivate in the PEU under continuous light for 10 days. Sprouting of seedlings is seen 2-3 days after cultivation in the growth chamber under continuous light. After 10 days, seedlings are seen. For the Experiment Run: Cultivate wild type and Mca1-GFP expressing seedlings with different color nylon meshes in the containers under microgravity conditions for 10 days. Gently remove the meshed with seedlings from the medium plate. Seedlings are attached onto the mesh. When the mesh is gently pulled off, the seedlings come off with the mesh. Insert the mesh, with seedlings, into the plastic bag for the fixation process (RNAlater or a mixture of paraformaldehyde and glutaraldehyde). The same experiment with the seedlings cultivated under 1-g conditions generated by the centrifuge should be done as a control. Incubate the seedlings in RNAlater® at 2°C overnight, and then store. Incubate the seedlings in a mixture of paraformaldehyde and glutaraldehyde at room temperature overnight, then store. The seedlings should not be frozen.