OXIDATIVE SIGNALS IN TOBACCO INCREASE CYTOSOLIC CALCIUM

Tobacco (Nicotiana plumbaginifolia) seedlings genetically transformed to express apoaequorin were incubated in h-coelenterazine to reconstit ute the calcium-sensitive luminescent protein aequorin. Treatment of t hese seedlings with hydrogen peroxide resulted in a transient burst of calcium-dependent luminescence lasting several minutes. Even though t he 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 res ponsiveness recovered. However, treatment with hydrogen peroxide did n ot inhibit mobilization of [Ca2+](cyt) induced by either cold shock or touching, suggesting that these three signals mobilize different pool s of intracellular calcium. To examine whether [Ca2+](cyt) is regulate d by the redox state of the cytoplasm, we pretreated seedlings with bu thionine sulfoximine (to modify cellular glutathione levels) and inhib itors of ascorbate peroxidase. These inhibitors modify the hydrogen pe roxide-induced transients in [Ca2+](cyt), which is consistent with the ir effects on the cellular prooxidant/antioxidant ratio. Treatment wit h hydrogen peroxide that elicited [Ca2+](cyt) increases also brought a bout a reduction in superoxide dismutase enzyme activity. This reducti on could be reversed by treatment with the calcium channel blocker lan thanum. This indicates that there is a role for calcium in plant respo nses to oxidative stress.