Involvement of the putative Ca2+-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca2+ uptake, Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells
- 600 Downloads
To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca2+-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca2+ uptake defects of yeast mutants lacking mechanosensitive Ca2+ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca2+ influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca2+ uptake, and significantly alleviated growth inhibition under Ca2+ limitation. NtMCA1-overexpressing BY-2 cells showed higher sensitivity to hypo-osmotic shock than control cells, and induced the expression of the touch-inducible gene, NtERF4. We found that both NtMCA1-GFP and NtMCA2-GFP were localized at the plasma membrane and its interface with the cell wall, Hechtian strands, and at the cell plate and perinuclear vesicles of dividing cells. NtMCA2 transcript levels fluctuated during the cell cycle and were highest at the G1 phase. These results suggest that NtMCA1 and NtMCA2 play roles in Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca2+ influx through the plasma membrane.
KeywordsCalcium ion Cell proliferation Hechtian strand Mechanosensitive ion channel Plasma membrane Tobacco (Nicotiana tabacum) BY-2 cells
We would like to thank Dr. Yasuhiro Kadota, Ms. Yui Miki and Mr. Yasuhiro Sakurai for technical assistance, and Dr. Dierk Wanke for discussion. This work was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas (21200067) to T. K., for Scientific Research on Priority Area (21026009 and 23120509) to H. I., for Scientific Research B (19370023) to K. K. and (21370017) to H. I., for Exploratory Research (21658118) to K. K., and by grants from Japan Science and Technology Agency, for CREST to H. I. and K. K.
- Higaki T, Kurusu T, Hasezawa S, Kuchitsu K (2011) Dynamic intracellular reorganization of cytoskeletons and the vacuole in defense responses and hypersensitive cell death in plants. J Plant Res 124:315–324Google Scholar
- Iida H, Yagawa Y, Anraku Y (1990) Essential role for induced Ca2+ influx followed by [Ca2+]i rise in maintaining viability of yeast cells late in the mating pheromone response pathway. A study of [Ca2+]i in single Saccharomyces cerevisiae cells with imaging of fura-2. J Biol Chem 265:13391–13399PubMedGoogle Scholar
- Kanzaki M, Nagasawa M, Kojima I, Sato C, Naruse K, Sokabe M, Iida H (1999) Molecular identification of a eukaryotic, stretch-activated nonselective cation channel. Science 285:882–886; report clarification (2000) Science 288:1347Google Scholar
- Kurusu T, Hamada J, Nokajima H, Kitagawa Y, Kiyoduka M, Takahashi A, Hanamata S, Ohno R, Hayashi T, Okada K, Koga J, Hirochika H, Yamane H, Kuchitsu K (2010) Regulation of microbe-associated molecular pattern-induced hypersensitive cell death, phytoalexin production and defense gene expression by calcineurin B-like protein-interacting protein kinases, OsCIPK14/15, in rice cultured cells. Plant Physiol 153:678–692PubMedCrossRefGoogle Scholar
- Lefebvre B, Furt F, Hartmann MA, Michaelson LV, Carde JP, Sargueil-Boiron F, Rossignol M, Napier JA, Cullimore J, Bessoule JJ, Mongrand S (2007) Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system. Plant Physiol 144:402–418PubMedCrossRefGoogle Scholar
- Nakagawa Y, Katagiri T, Shinozaki K, Qi Z, Tatsumi H, Furuichi T, Kishigami A, Sokabe M, Kojima I, Sato S, Kato T, Tabata S, Iida K, Terashima A, Nakano M, Ikeda M, Yamanaka T, Iida H (2007) Arabidopsis plasma membrane protein crucial for Ca2+ influx and touch sensing in roots. Proc Natl Acad Sci USA 104:3639–3644PubMedCrossRefGoogle Scholar
- Nakano M, Iida K, Nyunoya H, Iida H (2011) Determination of structural regions important for Ca2+ uptake activity in Arabidopsis MCA1 and MCA2 expressed in yeast. Plant Cell Physiol. doi: 10.1093/pcp/pcr131