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

麝香百合（Lilium longiflorum）花粉在合成培养基上萌发的培养条件为：琼脂培养基需要1mMCaCl_2，1mMH_3BO_3和0.3M蔗糖;液体培养基需要0.1mMCa（NO_3）_2，0.5mMH_3BO_3和0.3M蔗糖;用X射线能谱仪测定花粉中的钙和硼，由于样品制备困难，结果不理想，螯合剂EDTA和EGTA及蛋白激酶C抑制剂H-7在花粉萌发早期能促进花粉萌发，而蛋白合成抑制剂放线菌酮（CHI）则抑制花粉萌发。花粉萌发过程中磷脂酰肌醇（PI）的脂肪酸组成发生了变化，18：2和18：3等不饱和脂肪酸的分布减少，16：0等不饱和脂肪酸的比例增加。PI的这种变化与磷脂酰胆碱不同。本实验还没有成功地检测到三磷酸肌醇（inositol triphspate，IP_3）在花粉萌发过程中的存在，但上述结果表明，PI介导的钙调节系统在授粉过程中可能起着重要作用。多聚半乳糖醛酸酶和β-1，3-葡聚糖酶的活性显著。然而，在培养基中添加钙既不刺激也不抑制这些酶的活性。本实验成功地从L.麝香草叶片给药家兔制备抗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.