Effects of calcium concentration, calcium chelators, calcium channel-blockers on Hsp70a expression in Chlamydomonas reinhardtii

Year 2022, , 10 - 16, 15.06.2022

Tuba Sevgi Elif Demirkan

https://doi.org/10.38042/biotechstudies.1069555

Abstract

In this study, calcium concentration, calcium chelators, and calcium channel blockers that could be effective in triggering the heat shock response in Chlamydomonas reinhardtii were investigated. For this purpose, continuously expressed and heat-inducible transformant C. reinhardtii strains were used, and heterologously expressed arylsulfatase activities were detected. After a short time of heat shock at 40°C, cultures were shifted to 23°C and different concentrations of calcium (0-1 M CaCl2), EGTA (0-50 mM), BAPTA (0-2 mM), lanthanum (0-300 µM), gadolinium (0-350 µM), and verapamil (0-100 µM) applications were performed. To compare the arylsulfatase activity results at the transcript level, HSP70A expression level was analyzed. Arylsulfatase activity was increased with the increase of the calcium concentration, in the presence of calcium chelators, blockers, and parallel results were obtained in HSP70A expression level. These findings support that both extracellular and intracellular calcium influx is effective in the heat shock response of C. reinhardtii.

Keywords

Arylsulfatase, Heat shock response, Membrane fluidity

Supporting Institution

TUBITAK

Project Number

116Z892

Thanks

We thank to Michael Schroda, Prof. Dr., Technische Universität Kaiserslautern, Germany for the supply of the strains and the constructs.

References

• Fuhrmann, M., Oertel, W., & Hegemann, P. (1999). A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. The Plant Journal, 19(3), 353-361. https://doi.org/10.1046/j.1365-313X.1999.00526.x
• Fuhrmann, M., Hausherr, A., Ferbitz, L., Schödl, T., Heitzer, M., & Hegemann, P. (2004). Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant molecular biology, 55(6), 869-881.
• Gao, F., Han, X., Wu, J., Zheng, S., Shang, Z., Sun, D., & Li, B. (2012). A heat‐activated calcium‐permeable channel–Arabidopsis cyclic nucleotide‐gated ion channel 6–is involved in heat shock responses. The Plant Journal, 70(6), 1056-1069. https://doi.org/10.1111/j.1365-313X.2012.04969.x
• Goff, S.A. & Goldberg, A.L. (1985) Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell, 41, 587-595. https://doi.org/10.1016/S0092-8674(85)80031-3
• Gong, M., van der Luit, A. H., Knight, M. R., & Trewavas, A. J. (1998). Heat-shock-induced changes in intracellular Ca2+ level in tobacco seedlings in relation to thermotolerance. Plant Physiology, 116(1), 429-437. https://doi.org/10.1104/pp.116.1.429
• Harris, E. H. (2001). Chlamydomonas as a model organism. Annual review of plant biology, 52(1), 363-406. https://doi.org/10.1146/annurev.arplant.52.1.363
• Hostos, E. L. de, Schilling, J., & Grossman, A. R. (1989). Structure and expression of the gene encoding the periplasmic arylsulfatase of Chlamydomonas reinhardtii. Molecular and General Genetics MGG, 218(2), 229-239.
• Kindle, K. L. (1990). High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences 87 (3): 1228-1232. https://doi.org/10.1073/pnas.87.3.1228
• Kurepa, J., Walker, J.M., Smalle, J., Gosink, M.M., Davis, S.J., Durham, T.L.,Sung, D.Y., &Vierstra, R.D. (2003) The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and _2 conjugates is increased by stress. J. Biol. Chem. 278,6862-6872. https://doi.org/10.1074/jbc.M209694200
• Lancaster, B., & Batchelor, A. M. (2000). Novel action of BAPTA series chelators on intrinsic K+ currents in rat hippocampal neurones. The Journal of Physiology, 522(2), 231-246. https://doi.org/10.1111/j.1469-7793.2000.t01-1-00231.x
• Link, V., Sinha, A. K., Vashista, P., Hofmann, M. G., Proels, R. K., Ehness, R., & Roitsch, T. (2002). A heat‐activated MAP kinase in tomato: a possible regulator of the heat stress response. FEBS letters, 531(2), 179-183. https://doi.org/10.1016/S0014-5793(02)03498-1
• Liu, H. T., Li, B., Shang, Z. L., Li, X. Z., Mu, R. L., Sun, D. Y., & Zhou, R. G. (2003). Calmodulin is involved in heat shock signal transduction in wheat. Plant physiology, 132(3), 1186-1195. https://doi.org/10.1104/pp.102.018564
• Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25(4), 402-408. https://doi.org/10.1006/meth.2001.1262
• Meehl, G. A., & Tebaldi, C. (2004). More intense, more frequent, and longer lasting heat waves in the 21st century. Science, 305(5686), 994-997. 10.1126/science.1098704
• Morimoto, R. I. (1998). Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes & development, 12(24), 3788-3796. https://doi.org/ 10.1101/gad.12.24.3788
• Ohresser, M., Matagne, R. F., & Loppes, R. (1997). Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii. Current genetics, 31(3), 264-271. https://doi.org/10.1093/nar/20.12.2959
• Porra, R. J., Thompson, W. A., & Kriedemann, P. E. (1989). Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 975(3), 384-394. https://doi.org/10.1016/S0005-2728(89)80347-0
• Rasala, B. A., Lee, P. A., Shen, Z., Briggs, S. P., Mendez, M., & Mayfield, S. P. (2012). Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PLoS ONE, 7, e43349. https://doi.org/10.1371/journal.pone.0043349
• Rasala, B. A., Barrera, D. J., Ng, J., Plucinak, T. M., Rosenberg, J. N., Weeks, D. P., ... & Mayfield, S. P. (2013). Expanding the spectral palette of fluorescent proteins for the green microalga C hlamydomonas reinhardtii. The Plant Journal, 74(4), 545-556. https://doi.org/10.1111/tpj.12165
• Rütgers, M., Muranaka, L. S., Schulz‐Raffelt, M., Thoms, S., Schurig, J., Willmund, F., & Schroda, M. (2017). Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii. Plant, cell & environment, 40(12), 2987-3001. Doi: https://doi.org/10.1111/pce.13060
• Saidi, Y., Finka, A., Muriset, M., Bromberg, Z., Weiss, Y. G., Maathuis, F. J., & Goloubinoff, P. (2009). The heat shock response in moss plants is regulated by specific calcium-permeable channels in the plasma membrane. The Plant Cell, 21(9), 2829-2843. https://doi.org/10.1105/tpc.108.065318
• Saidi, Y., Peter, M., Finka, A., Cicekli, C., Vigh, L., & Goloubinoff, P.(2010) Membrane lipid composition affects plant heat sensing and modulates Ca2+-dependent heat shock response. Plant Signal. Behav. 5, 1530-1533. https://doi.org/10.4161/psb.5.12.13163
• Saidi, Y., Finka, A., & Goloubinoff, P. (2011). Heat perception and signalling in plants: a tortuous path to thermotolerance. New Phytologist, 190(3), 556-565. https://doi.org/10.1111/j.1469-8137.2010.03571.x
• Sangwan, V., Örvar, B. L., Beyerly, J., Hirt, H., & Dhindsa, R. S. (2002). Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways. The Plant Journal, 31(5), 629-638. https://doi.org/10.1046/j.1365-313X.2002.01384.x
• Saoudi, Y., Rousseau, B., Doussiere, J., Charrasse, S., Gauthier‐Rouvière, C., Morin, N., & Job, D. (2004). Calcium‐independent cytoskeleton disassembly induced by BAPTA. European Journal of Biochemistry, 271(15), 3255-3264. https://doi.org/10.1111/j.1432-1033.2004.04259.x
• Schmollinger, S., Schulz-Raffelt, M., Strenkert, D., Veyel, D., Vallon, O., & Schroda, M. (2013). Dissecting the heat stress response in Chlamydomonas by pharmaceutical and RNAi approaches reveals conserved and novel aspects. Molecular Plant, 6(6), 1795-1813. https://doi.org/10.1093/mp/sst086
• Schroda, M., Blöcker, D., & Beck, C. F. (2000). The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. The Plant Journal, 21(2), 121-131. Doi: https://doi.org/10.1046/j.1365-313x.2000.00652.x
• Schroda, M., & Vallon, O. (2009). Chaperones and proteases. In The Chlamydomonas Sourcebook (pp. 671-729). Academic Press. https://doi.org/10.1016/B978-0-12-370873-1.00027-7
• Schroda, M., Hemme, D., & Mühlhaus, T. (2015). The Chlamydomonas heat stress response. The Plant Journal, 82(3), 466-480. https://doi.org/10.1111/tpj.12816
• Schulz‐Raffelt, M., Lodha, M., & Schroda, M. (2007). Heat shock factor 1 is a key regulator of the stress response in Chlamydomonas. The Plant Journal, 52(2), 286-295. https://doi.org/10.1111/j.1365-313X.2007.03228.x
• Shao, N., & Bock, R. (2008). A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii. Current Genetics, 53(6), 381-388.
• Sevgi, T., Demirkan, E. (2021) Evaluation of the effects of temperature, light and UV-C radiation on HSP70A expression in Chlamydomonas reinhardtii. Turkish Journal of Botany, 45: 671-680. https://doi.org/10.3906/bot-2012-43
• Sugio, A., Dreos, R., Aparicio, F., & Maule, A.J. (2009) The cytosolic protein response as a subcomponent of the wider heat shock response in Arabidopsis. Plant Cell, 21, 642-654. https://doi.org/10.1105/tpc.108.062596
• Sung, D. Y., Kaplan, F., Lee, K. J., & Guy, C. L. (2003). Acquired tolerance to temperature extremes. Trends in Plant Science, 8(4), 179-187. https://doi.org/10.1016/S1360-1385(03)00047-5
• Suri, S. S., & Dhindsa, R. S. (2008). A heat‐activated MAP kinase (HAMK) as a mediator of heat shock response in tobacco cells. Plant, Cell & Environment, 31(2), 218-226. https://doi.org/10.1111/j.1365-3040.2007.01754.x
• Wu, H. C., Luo, D. L., Vignols, F., & JINN, T. L. (2012). Heat shock‐induced biphasic Ca2+ signature and OsCaM1‐1 nuclear localization mediate downstream signalling in acquisition of thermotolerance in rice (Oryza sativa L.). Plant, Cell & Environment, 35(9), 1543-1557. https://doi.org/10.1111/j.1365-3040.2012.02508.x
• Zheng, S. Z., Liu, Y. L., Li, B., Shang, Z. L., Zhou, R. G., & Sun, D. Y. (2012). Phosphoinositide‐specific phospholipase C9 is involved in the thermotolerance of Arabidopsis. The Plant Journal, 69(4), 689-700. https://doi.org/10.1111/j.1365-313X.2011.04823.x