Dynamics of endogenous levels and subcellular localization of ABA and cytokinins during pollen germination in spruce and tobacco

We used the enzyme-linked immunosorbent assay (ELISA)to assess the level of endogenous hormones in spruce pollen, and immunolocalization and confocal microscopy to study hormone localization in spruce and tobacco pollen. During pollen activation, the levels of ABA, zeatin, and its riboside significantly decreased. After the initiation of polar growth, the levels of all cytokinins increased sharply; ABA level also increased. In dormant spruce pollen grains, zeatin and ABA were localized uniformly throughout the cytoplasm. Zeatin was not detected in the nuclei, and the antheridial cell showed higher levels than the vegetative cell; ABA signal was detected in the cytoplasm and the nuclei. In germinating pollen, both hormones were detected mainly in plastids. The similar pattern was found in growing pollen tubes; signal from ABA also had a noticeable level in the cytosol of the tube cell, and was weaker in the antheridial cell. Zeatin fluorescence, on the other hand, was more pronounced in the antheridial cell. In non-germinated grains of tobacco, zeatin was localized mainly in organelles. ABA in dormant pollen grains demonstrated uniform localization, including the nuclei and cytoplasm of both cells. After germination, zeatin was accumulated in the plasmalemma or cell wall. ABA signal in the cytoplasm decreased; in the nuclei, it remained high. In growing tubes, the strongest zeatin and ABA signals were observed at the plasma membrane. The differences in ABA and cytokinin localization between species and dynamic changes in their level in spruce pollen highlight the key spatial and temporal parameters of hormonal regulation of gymnosperm pollen germination.