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

Abstract

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.

Expression of Arabidopsis MCA1 enhanced mechanosensitive channel activity in theXenopus laevisoocyte plasma membrane

“…Most recently, homologous genes in tobacco and rice, NtMCA1, NtMCA2 and OsMCA1, respectively, were identified; they are localized in the plasma membrane, channel Ca 2+ , and may have potential roles in cell proliferation. 16,17 However, direct evidence for mechanical stress-dependent activation of MCA proteins is still lacking.…”

Intracellular localization and physiological function of a rice Ca2+-permeable channel OsTPC1

“…[22][23][24] FM4-64 was used as a VM marker. 23,25 When GFP alone was expressed, it is localized to the nucleus and the cytoplasm (Fig.…”

The Maize MID-COMPLEMENTING ACTIVITY Homolog CELL NUMBER REGULATOR13/NARROW ODD DWARF Coordinates Organ Growth and Tissue Patterning

“…The Arabidopsis MCA1 was identified by its ability to complement the low Ca 2+ and related lethality phenotypes of mid1 (mating induced death1) yeast mutants upon mating induction (Iida et al, 1994;Fischer et al, 1997;Nakagawa et al, 2007;Yamanaka et al, 2010;Kurusu et al, 2012aKurusu et al, , 2012c. Mid is a putative regulatory subunit of a high-affinity, low-capacity Ca 2+ influx system that has Cch1 as the putative pore-forming subunit (Iida et al, 1994;Fischer et al, 1997;Muller et al, 2001;Rigamonti et al, 2015).…”

“…Mid is a putative regulatory subunit of a high-affinity, low-capacity Ca 2+ influx system that has Cch1 as the putative pore-forming subunit (Iida et al, 1994;Fischer et al, 1997;Muller et al, 2001;Rigamonti et al, 2015). Interestingly, cch1 lethality was also partially complemented by MCA expression (Yamanaka et al, 2010;Kurusu et al, 2012aKurusu et al, , 2012c. Experiments in various cell types showed that overexpression of MCAs leads to increased Ca 2+ uptake under normal and membrane-distorting conditions (Nakano et al, 2011;Kurusu et al, 2012aKurusu et al, , 2012bKurusu et al, , 2012cFuruichi et al, 2012).…”

“…Interestingly, cch1 lethality was also partially complemented by MCA expression (Yamanaka et al, 2010;Kurusu et al, 2012aKurusu et al, , 2012c. Experiments in various cell types showed that overexpression of MCAs leads to increased Ca 2+ uptake under normal and membrane-distorting conditions (Nakano et al, 2011;Kurusu et al, 2012aKurusu et al, , 2012bKurusu et al, , 2012cFuruichi et al, 2012). Furthermore, as Mid is thought to have stretch-activated Ca 2+ uptake functions and MCA1 is required for mechanoperception in roots, it was suggested that MCAs are stretch-activated Ca 2+ channels (Nakagawa et al, 2007).…”