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.