Abstract
Early studies revealed a highly predictable pattern of gravity-directed growth and development in Ceratopteris richardii spores. This makes the spore a valuable model system for the study of how a single-cell senses and responds to the force of gravity. Gravity regulates both the direction and magnitude of a trans-cell calcium current in germinating spores, and the orientation of this current predicts the polarization of spore development. In order to make Ceratopteris richardii cells easier to transform and image during this developmental process, a procedure for isolating protoplasts from Ceratopteris richardii gametophytes has been developed and optimized. These protoplasts follow the same developmental pattern as Ceratopteris richardii spores and can be used to monitor the molecular and developmental processes during single-cell polarization. Here, we describe this optimized procedure, along with protocols for sterilizing the spores, sowing them in solid or liquid growth media, and evaluating germination and polarization.
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References
Edwards ES, Roux SJ (1994) Limited period of graviresponsiveness in germinating spores of Ceratopteris richardii. Planta 195:150–152
Chatterjee A, Roux SJ (2000) Ceratopteris richardii: a productive model for revealing secrets of signaling and development. J Plant Growth Regul 19(3):284–289
Roux SJ, Chatterjee A, Hillier S, Cannon T (2003) Early development of fern gametophytes in microgravity. Adv Space Res 31(1):215–220. https://doi.org/10.1016/s0273-1177(02)00749-4
Edwards ES, Roux SJ (1997) The influence of gravity and light on developmental polarity of single cells of Ceratopteris richardii gametophytes. Biol Bull 192(1):139–140. https://doi.org/10.2307/1542588
Edwards ES, Roux SJ (1998) Gravity and light control of the developmental polarity of regenerating protoplasts isolated from prothallial cells of the fern Ceratopteris richardii. Plant Cell Rep 17(9):711–716. https://doi.org/10.1007/s002990050470
Chatterjee A, Porterfield DM, Smith PS, Roux SJ (2000) Gravity-directed calcium current in germinating spores of Ceratopteris richardii. Planta 210(4):607–610. https://doi.org/10.1007/s004250050050
Salmi ML, ul Haque A, Bushart TJ, Stout SC, Roux SJ, Porterfield DM (2011) Changes in gravity rapidly alter the magnitude and direction of a cellular calcium current. Planta 233(5):911–920. https://doi.org/10.1007/s00425-010-1343-2
ul Haque A, Rokkam M, De Carlo AR, Wereley S, Roux SJ, Irazoqui PP et al (2007) A MEMS fabricated cell electrophysiology biochip for in silico calcium measurements. Sens Actuators B 123:391–399
Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7(5):417–427. https://doi.org/10.1111/j.1364-3703.2006.00346.x
Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C et al (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40(3):428–438. https://doi.org/10.1111/j.1365-313X.2004.02219.x
Faraco M, Di Sansebastiano GP, Spelt K, Koes RE, Quattrocchio FM (2011) One protoplast is not the other! Plant Physiol:474–478
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675. https://doi.org/10.1038/nmeth.2089
Acknowledgments
Work on Ceratopteris is supported by NASA grant NNX13AM54G.
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Cannon, A.E., Sabharwal, T., Roux, S.J. (2022). Spore Preparation and Protoplast Isolation to Study Gravity Perception and Response in Ceratopteris richardii . In: Blancaflor, E.B. (eds) Plant Gravitropism. Methods in Molecular Biology, vol 2368. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1677-2_3
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DOI: https://doi.org/10.1007/978-1-0716-1677-2_3
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