Oxidative Signals in Tobacco Increase Cytosolic Calcium

Tobacco (Nicotiana plumbaginifolia) seedlings genetically transformed to express apoaequorin were incubated in h-coelenterazine to reconstitute the calcium-sensitive luminescent protein aequorin. Treatment of these seedlings with hydrogen peroxide resulted in a transient burst of calcium-dependent luminescence lasting several minutes. Even though the hydrogen peroxide stimulus was persistent, the change in cytosolic free calcium concentration ([Ca2+](cyt)) was transient, suggesting the presence of a refractory period. When seedlings were pretreated with hydrogen peroxide, there was no increase in [Ca2+](cyt) upon a second application, which confirmed the refractory character of the response. Only when the two treatments were separated by 4 to 8 hr was full responsiveness recovered. However, treatment with hydrogen peroxide did not inhibit mobilization of [Ca2+](cyt) induced by either cold shock or touching, suggesting that these three signals mobilize different pools of intracellular calcium. To examine whether [Ca2+](cyt) is regulated by the redox state of the cytoplasm, we pretreated seedlings with buthionine sulfoximine (to modify cellular glutathione levels) and inhibitors of ascorbate peroxidase. These inhibitors modify the hydrogen peroxide-induced transients in [Ca2+](cyt), which is consistent with their effects on the cellular prooxidant/antioxidant ratio. Treatment with hydrogen peroxide that elicited [Ca2+](cyt) increases also brought about a reduction in superoxide dismutase enzyme activity. This reduction could be reversed by treatment with the calcium channel blocker lanthanum. This indicates that there is a role for calcium in plant responses to oxidative stress.