Changes in calcium and calmodulin levels during microsporogenesis,pollen development and germination in Gasteria verrucosa (Mill.) H. Duval

Summary The distribution of membrane calcium and calmodulin (CaM) has been fluorimetrically determined in the anther of Gasteria verrucosa with particular attention to sporogenous cells, meiocytes, microspores, pollen and stages of pollen germination and tube growth using chlortetracycline (CTC) and fluphenazine (FPZ). CTC and FPZ fluorescence in sporogenous cells is relatively higher than in the adjacent tapetal cells, indicating higher membrane calcium and CaM levels in the former cell type. However, during meiosis there is a significant increase in membrane calcium and CaM levels in the meiocytes compared to that found in the young microspores. CTC and FPZ fluorescence in the sporogenous cells, meiocytes and young microspores is punctate and slightly diffused throughout the cytoplasm. In the microspores of the tetrad and the young released microspores CTC fluorescence (CTCf) is polarized and mainly associated with the area opposite the future colporal region. FPZ fluorescence (FPZf) becomes polarized in the young microspore. Subsequently, there is a shift in the polarity, and most of the CTCf and FPZf in the old microspores and pollen is regionalized towards the colporal region, and the fluorescence is more diffused, indicating a change in the organellar-bound calcium and CaM. This final graded distribution of CTCf is maintained during pollen germination in that the growing pollen tubes invariably show a tip to base membrane-calcium gradient. In the tapetal cells a high level of Ca²⁺ is present during the microspore stage. During the preparation for anthesis the endothecium differentiation is marked by the presence of Ca²⁺. Post-treatment of labelled cells with a Ca²⁺ chelator such as EGTA resulted in a substantial decrease in diffuse and punctate CTCf. Alternatively, treatment of cells with non-ionic detergent Nonidet P-40 resulted in the total elimination of CTCf, suggesting that the observed CTC fluorescence was due to membrane-associated calcium. The cytological specification of CTC as a probe for calcium is discussed. From cytofluorometric measurements and atomic absorption, it became clear that the level of Ca²⁺ in the anther is high during the sporogenous and meiotic phases. An increase in CTCf and FPZf occurred after microspore mitosis. An interaction of Ca²⁺ transport from tapetum to the young pollen is postulated. These findings suggest that the level of Ca²⁺ in the anther during meiosis is generally relatively higher than at the sporogenous or young microspore stage. These findings are discussed in the light of available information on the role of Ca²⁺ and CaM-mediated processes such as cell division, callose synthesis and pollen-tube tip growth.