Dark-stimulated calcium ion fluxes in the chloroplast stroma and cytosol

Using transgenic Nicotiana plumbaginifolia seedlings in which the calcium reporter aequorin is targeted to the chloroplast stroma, we found that darkness stimulates a considerable flux of Ca2+ into the stroma. This Ca2+ flux did not occur immediately after the light-to-dark transition but began similar to5 min after lights off and increased to a peak at similar to20 to 30 min after the onset of darkness. Imaging of aequorin emission confirmed that the dark-stimulated luminescence emanated from chloroplast-containing tissues of the seedling. The magnitude of the Ca2+ flux was proportional to the duration of light exposure (24 to 120 h) before lights off; the longer the duration of light exposure, the larger the dark-stimulated Ca2+ flux. On the other hand, the magnitude of the dark-stimulated Ca2+ flux did not appear to vary as a function of circadian time. When seedlings were maintained on a 24-h light/dark cycle, there was a stromal Ca2+ burst after lights off every day. Moreover, the waveform of the Ca2+ spike was different during long-day versus short-day light/dark cycles. The dark-stimulated Ca2+ flux into the chloroplastidic stroma appeared to affect transient changes in cytosolic Ca2+ levels. DCMU, an inhibitor of photosynthetic electron transport, caused a significant increase in stromal Ca2+ levels in the light but did not affect the magnitude of the dark-stimulated Ca2+ flux. This robust Ca2+ flux likely plays regulatory roles in the sensing of both light/dark transitions and photoperiod.