Molecular Role of Calcium and Boron for Pollen Germination of Higher Plants

建立了长花百合花粉在合成培养基上萌发的培养条件：琼脂培养基为1mM CaCl_2、1mM H_3BO_3和0.3 M蔗糖，液体培养基为0.1 mM Ca(NO_3)_2、0.5mM H_3BO_3和0.3 M蔗糖，花粉密度为0 ~ 3 mg/ml。由于花粉管样品制备困难，用x射线能谱仪对钙和硼进行微量分析并不成功。螯合剂EDTA和EGTA以及蛋白激酶C抑制剂H-7在萌发初期促进花粉萌发，而蛋白合成抑制剂环己亚胺（CHI）抑制花粉萌发。然而，先用CHI处理花粉，再用EDTA处理花粉，不仅没有抑制萌发，反而促进了萌发。这表明发芽所需的蛋白质作用可以被EDTA取代，钙可能与细胞壁和/或质膜相互作用。花粉萌发过程中磷脂酰肌醇（PI）脂肪酸组成发生变化，18:2和18:3等不饱和脂肪酸的分布减少，16:0的比例增加。这种变化与磷脂酰胆碱不同。虽然本实验未在花粉萌发系统中成功检测到三磷酸肌醇（IP_3），但上述结果提示pi介导的钙调节系统可能在授粉过程中发挥重要作用。为了研究钙对细胞壁松动的影响，测定了细胞壁降解酶的活性。聚半乳糖醛酸酶和- 1,3 -葡聚糖酶活性显著。然而，在培养基中添加钙既不刺激也不抑制这些酶的活性。目前，我们成功地从长花L. longiflorum叶片中纯化出β - 1,3 -葡聚糖酶，用于制备抗β - 1,3 -葡聚糖酶血清，并对花粉终止与钙、硼作用的关系进行免疫组织学研究。

Culture conditions for pollen germination of Lilium longiflorum on a synthetic medium were established: 1 mM CaCl_2, 1mM H_3BO_3 and 0.3 M sucrose were required for an agar medium, 0.1 mM Ca(NO_3)_2, 0.5mM H_3BO_3 and 0.3 M sucrose for a liquid medium, with a pollen density of 0-3 mg/ml. Microanalyses of calcium and boron using X-ray energy spectrometer were not successful because of difficulty in pollen tube sample preparation.EDTA and EGTA, a chelator, and H-7 as protein kinase C inhibitor, stimulated pollen germination at the early stage of germination, whereas protein synthesis inhibitor cycloheximide (CHI) inhibited the germination. However, treatment of pollen with CHI followed by EDTA treatment stimulated the germination rather than inhibited it. This suggests that protein actions required for the germination can be replaced with EDTA and that calcium may interact with cell wall and/or plasma membrane.Fatty acid composition of phosphatidylinositol (PI) changed during the pollen germination, distribution of unsaturated fatty acids such as 18:2 and 18:3 reduced while percentage of 16:0 increased. This change with PI was different from that of phosphatidylcholine. Although detection of inositol triphsphate (IP_3) has not been successful in the pollen germination system in our experiment, the above result suggests that PI-mediated calcium regulation system may play an important role during pollination.To examine calcium effect on cell wall loosening, cell wall degrading enzgme activities were assayed. Significant activities of polygalacturonase and beta-1, 3-glucanase were detected. However, addition of calcium to the medium neither stimulated nor inhibited these enzyme activitie. Now we successfully purified beta-1, 3-glucanase from L. longiflorum leaves to administrate to rabbit for preparation of anti-beta-1, 3-glucanase serum and immuno-histological investigation of pollen termination in connection with calcium and boron action.