Ratio-imaging of Ca2+i in the self-incompatibility response in pollen tubes of Papaver rhoeas

The data presented here describe ratio-imaging of in intracellular free calcium (Ca-i(2+)) during the self-incompatibility (SI) response in pollen. Use of the ratiometric indicator, fura-2 dextran, in pollen tubes of Papaver rhoeas has provided new, detailed information about the spatial-temporal alterations in Ca-i(2+), and has permitted calibration of alterations in the concentration of intracellular free calcium ([Ca2+](i)) in the SI response. Ratio images demonstrate that, like other pollen tubes, normally growing P. rhoeas pollen tubes exhibit a tip-focused gradient of Ca-i(2+bf), with levels reaching 1-2 mu M at the extreme apex of the pollen tube. Non-growing pollen tubes did not exhibit this tip-focused gradient. Basal levels of Ca-i(2+) in the shank of the pollen tube were fairly consistent and had a mean value of 210 nM, with low-level fluctuations +/- 50 nM observed. Challenge with incompatible S proteins resulted in S-specific, rapid and dramatic alterations in [Ca2+](i) within a few seconds of challenge. Increases in [Ca2+](i) were visualized in the subapical/shank regions of the pollen tube and alterations in [Ca2+](i) in this region subsequently increased for several minutes, reaching >1.5 mu M. At the pollen tube tip, a diminution of the tip-focused gradient was observed, which following some fluctuation, was reduced to basal levels within similar to 1 min. Our data suggest that some of these alterations in [Ca2+](i) might be interpreted as a calcium wave, as the changes are not global. Although the increases in [Ca2+](i) in the subapical/shank region are very rapid, because tip [Ca2+](i) oscillates during normal growth, it is difficult to ascertain whether the increases in the shank of the pollen tube precede the decreases in [Ca2+](i) at the pollen tube tip.