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

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