BAPTA-CALCIUM BUFFERS MODULATE CELL PLATE FORMATION IN STAMEN HAIRS OF TRADESCANTIA - EVIDENCE FOR CALCIUM GRADIENTS

Five BAPTA buffers with differential affinities for Ca2+ have been examined for their effects on cell plate formation in stamen hair cells of Tradescantia. The five include 5,5'-dimethyl BAPTA (Kd = 0.15 mu M), BAPTA (Kd = 0.22 mu M), 5,5'-dibromo BAPTA (Kd = 1.5 mu M), 5-methyl,5'-nitro BAPTA (Kd = 22 mu M), and 5-nitro BAPTA (Kd = 40 mu M). At a concentration of 5 mM and 25 mM in the pipette, the buffers were iontophoretically microinjected into dividing stamen hair cells (2 nA for 1 min) prior to or at the onset of cell plate formation. At the lowest concentration (5 mM), only one buffer, 5,5'-dibromo BAPTA, inhibits cell plate formation, and is most effective if delivered at the moment of cell plate vesicle aggregation. The inhibitory effects appear as a slowing of cell plate expansion, the formation of distorted plates, or the complete dissolution of plates that might have initiated normally. When the pipette tip concentration is elevated to 25 mM, the effects of 5,5'dibromo BAPTA become more profound. At these levels 5,5'-dimethyl BAPTA, BAPTA, and 5-nitro BAPTA also modulate cell plate formation, producing effects similar to that of 5,5'-dibromo BAPTA at the lower concentration. Independent studies using fura-2 as a fluorescent analogue of the BAPTA buffers, indicate that the apparent effective concentration for 5,5'-dibromo BAPTA is between 1.0-1.4 mM; its threshold concentration is not known but expected to be somewhat lower. For the other buffers the threshold concentration is between 1.5-2.2 mM. The concentration dependence supports the idea that the buffers facilitate diffusion of Ca2+ away from regions of elevated concentration. The results thus provide evidence that local Ca2+ gradients may be present in the vicinity of the cell plate and that they participate in the cytokinetic process.