| Apoplast CaM has been found to exist in animals and plants and has various biological functions. Based on systemic studies on extracelluar CaM, we put forward a view that the biological functions of extracellular CaM may act as a polypeptide signal. Despite intensive investigation on the detection, localization, biological roles and signal transduction pathway of extracellular CaM in plants, the mechanism by which extrallular CaM is activated by Ca2+ has been unclear, mainly because of specific of extrallular milieu. As mentioned earlier, the concentration of Ca in extracellular milieu is millimolar, while the intracellular Ca2+ is of the order of O.lWnol/L in a resting cell, but as a result of an external stimulus, the level of Ca2+ may transiently rise to l-10Mmol/L leading to the activation of CaM. Due to 100-1000 fold differtence in Ca2+ concentration between the cytosal after stimulus and exterior of cell, one can deduce that extracellular CaM will be highly occupied with Ca2+. We also found that the pH in the apoplast is much lower than that in cytosol. In order to understand fully the mechanism of extrallular CaM at high Ca2+ level and low pH, it is essential to establish quantitative relationship of Ca2+, pH and CaM concentration.Tang et al(1999) have studied the influence of pH on the binding of calcium to calmodulin and the activation effects of calmodulin to its target enzyme by using the methods of equilibrium dialyze and activation of NAD kinase. They found that H+ decrease the binding affinity of calcium to calmodulin, also inhibit calmodulin active NAD kinase. Xiao measured and calculated the successive binding constants of Ca2+ to arabidopsis CaM (isoform II) and cauliflower CaM (mixed isoforms) by Ca2+-selective electrode combined with Excel program solving method. The result shows that the binding constants of Ca2+ to CaM reduce about more than 10 times when the pH changes from 6.5 to 5.5 or 5.5 to 4.5. Although these results demonstrates the activation of extracellular CaM is regulated by pH in vitro, it is essential to be verified furtherly by in vivo experiments.In this paper, we used the pollen germination percentage of Nicotiana tabacum asexperiment model to analyze the activation relationship of H+, Ca2+ and CaM in vivo. The main results are as followings.Firstly, during the course of pollen germination, the concentration of Ca2+ in the medium increased with the medium decreased pH. This indicates that the increased H+ icon can replace the bound Ca2+ in cell-wall milieu, making free Ca2+ concentration higher than that in neutral pH.Secondly, extracellular CaM has the same functional mechanism in acidic (pH=4.8,5.5) and neutral (pH=6.5) conditions. This is because in three pH conditions exogenous CaM can promote pollen germination, and CaM's antigonist Wy-agarose and antibody can inhibited the promoted effect, and added extracellular CaM can recover the above inhibited effect.Thirdly, Ca2+- H+ antagonism in CaM activation has been demonstrated in vivo by pollen germination system. Although proton antagonism in Ca2+ binding to CaM was established from the in vitro data of xiao et al. Up to now there is no in vivo evidence. In our experiment, when adding lower concentration of Ca2+ in the medium less than 200Hmol/L Ca2+, the percentage pollen germination decreased with the decreased pH. When adding higher concentration of Ca2+ such as 500Mmol/L, lOOOWnol/L or 2000Mmol/L in the medium, the percentage pollen germinations in different medium pH value have no significant difference. In the other hand, different amounts of Ca2+ can promote pollen germination in a fixed pH (such as pH=4.8, 5.5 or 6.5). The profile of germination percentage to Ca2+ concentration in three pH values increased up to 500Mmol/L Ca2+, before the onset of a plateau and the maxium germination percentage is almost the same value. When Ca2+ is less than 200umol/L, reaching the same germination percentage needs more Ca2+ concentration in acidic condition than that in neutral condition. This is because H+ can reduce...
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