Figure 6

Involvement of OsMCA1 in the regulation of hypo-osmotic shock-induced [Ca²⁺] _cyt changes. (A) RT-PCR analysis of OsMCA1 in three independent RNAi lines expressing apoaequorin mRNA. (B) Hypo-osmotic shock-induced [Ca²⁺]_cyt changes in cultured rice cells. Cells were diluted four-fold with water at 0 min. A representative result of five experiments is shown. (C) Pharmacological analyses of hypo-osmotic shock-induced [Ca²⁺]_cyt changes. Cells were diluted four-fold with water at 0 min. BAPTA (5 mM), GdCl₃ (5 mM), LaCl₃ (5 mM) and verapamil (1 mM) were added to the cell suspension 15 min before hypo-osmotic shock treatment. No treatment prior to hypo-osmotic shock in the "control." Peak intensities of aequorin chemiluminescence were compared; relative luminescence level in the control was standardized as 100% (C and D). Data are mean ± SE for five independent experiments. ***P < 0.005; significantly different compared with control. (D) Effects of OsMCA1 suppression on N-acetylchito-oligosaccharides-induced [Ca²⁺]_cyt changes. Cells were treated with N-acetylchitoheptaose (1 μM). Error bars indicate SE of the mean of five experiments. (E-a) 1 mL of water, medium, or diluted medium was added to OsMCA1-suppressed cells harboring apoaequorin at 0 min to generate wide-ranging changes in extracellular osmotic pressure. The relative amount of luminescence accumulated for 10 min after hypo-osmotic shock was plotted versus relative extracellular osmotic pressure. Data are means ± SE, n = 3-7. ⁺*P < 0.05; ⁺**P < 0.01; significantly different compared with two control lines (No. 20-5 and 5-3). *P < 0.05; significantly different compared with the control line (No. 20-5). (E-b) Hypo-osmotic shock-induced Ca²⁺ signature in OsMCA1-suppressed lines. Cells were diluted four-fold with water at 0 min. A representative result of several experiments is shown.