Anoxia-induced elevation of cytosolic Ca2+ concentration depends on different Ca2+ sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca2+ concentration, [Ca2+](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca2+](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca2+](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca2+ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca2+](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La3+ and EGTA) and intracellular membrane Ca2+-channel antagonists (Li+, ruthenium red and cyclosporine A) reduced the anoxic Ca2+-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca2+](cyt). In wheat leaf protoplasts, the amplitude of the Ca2+-shift little depended on the external level of Ca2+. Wheat root protoplasts were characterized by a small shift of [Ca2+](cyt) under anoxia. Plasmalemma Ca2+-channel blockers had little effect on the elevation of cytosolic Ca2+ in wheat protoplasts. Intact rice seedlings absorbed Ca2+ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca2+. Verapamil abolished the Ca2+ influx in rice roots and Ca2+ efflux from wheat roots. Anoxia-induced [Ca2+](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca2+ stores are important for anoxic [Ca2+](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca2+](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.