Fine-Tuning of the Cytoplasmic Ca2+ Concentration Is Essential for Pollen Tube Growth

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca²⁺ concentration (Ca²⁺]) gradient and regular oscillations of the cytosolic Ca²⁺] (Ca²⁺]_cyt) in the tip region. Whether this Ca²⁺] gradient and/or Ca²⁺]_cyt oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored Ca²⁺]_cyt dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused Ca²⁺]_cyt gradient was always observed in growing pollen tubes. Regular oscillations of the Ca²⁺]_cyt, however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar Ca²⁺]_cyt in the tip region is essential for pollen tube growth, whereas a regular Ca²⁺] oscillation is not. Next, we monitored Ca²⁺] dynamics in the endoplasmic reticulum (Ca²⁺]_ER) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca²⁺ compared with yellow cameleon 3.60. The Ca²⁺]_ER in pollen tubes grown under the semi-in vivo condition was between 100 and 500 μm. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca²⁺-ATPases, inhibited growth and decreased the Ca²⁺]_ER. Our observations suggest that the ER serves as one of the Ca²⁺ stores in the pollen tube and cyclopiazonic acid-sensitive Ca²⁺-ATPases in the ER are required for pollen tube growth.In many flowering plants, a pollen grain that lands on the top surface of a stigma will hydrate and germinate a pollen tube. Following germination, the pollen tube enters the style and grows through the wall of transmitting tract cells on the way to the ovary, where the tube emerges to release the sperm for double fertilization. Therefore, pollen tube growth is essential for reproduction in flowering plants.Since Brewbaker and Kwack (1963) revealed that Ca²⁺ is essential for in vitro pollen tube cultures, the relationship between the Ca²⁺ concentration (Ca²⁺]) and pollen tube growth has been further examined under in vitro germination culture conditions. Ratiometric ion imaging using fluorescent dye has revealed that the apical domain of a pollen tube grown in vitro contains a tip-focused Ca²⁺] gradient (Pierson et al., 1994, 1996; Cheung and Wu, 2008) and that the cytoplasmic Ca²⁺] (Ca²⁺]_cyt) in the tip region and the growth rate oscillate with the same periodicity (Pierson et al., 1996; Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997). Therefore, oscillation of the Ca²⁺]_cyt has been thought to correlate with pollen tube growth. It is not clear, however, whether regular Ca²⁺]_cyt oscillations in the tip region occur in pollen tubes growing through stigmas and styles.The Ca²⁺]_cyt is controlled temporally and spatially by transporters in the membranes of intracellular compartments and in the plasma membrane (Sze et al., 2000). Studies using a Ca²⁺-sensitive vibrating electrode revealed Ca²⁺ influx in the tip region of the pollen tube (Pierson et al., 1994; Holdaway-Clarke et al., 1997; Franklin-Tong et al., 2002). Stretch-activated Ca²⁺ channels have been found in the plasma membrane using patch-clamp electrophysiology (Kuhtreiber and Jaffe, 1990; Dutta and Robinson, 2004). Recently, CNGC18 was identified as a Ca²⁺-permeable channel in the plasma membrane that is essential for pollen tube growth (Frietsch et al., 2007). The intracellular compartments that store Ca²⁺ in the pollen tube and the relevant Ca²⁺ transporters, however, have yet to be identified.Yellow cameleons are genetically encoded Ca²⁺ indicators that were developed to monitor the Ca²⁺] in living cells (Miyawaki et al., 1997). These indicators are chimeric proteins consisting of enhanced cyan fluorescent protein (ECFP), calmodulin (CaM), a glycylglycine linker, the CaM-binding domain of myosin light chain kinase (M13), and enhanced yellow fluorescent protein (EYFP). When the CaM domain binds Ca²⁺, the domain associates with the M13 peptide and induces fluorescence resonance energy transfer (FRET) between ECFP and EYFP. Several types of cameleons have been developed by tuning the CaM domain binding affinity for Ca²⁺. Yellow cameleon 2.1 (YC2.1) is a high-affinity indicator that has been used to monitor the Ca²⁺]_cyt in Arabidopsis (Arabidopsis thaliana) guard cells (Allen et al., 1999, 2000, 2001), Lilium longiflorum and Nicotiana tabacum pollen tubes (Watahiki et al., 2004), and the root hair of Medicago truncatula (Miwa et al., 2006). YC3.1 is a low-affinity indicator that has been used to monitor the Ca²⁺]_cyt during pollen germination and in papilla cells of Arabidopsis (Iwano et al., 2004).Recently, YC3.60 was developed as a new YC variant (Nagai et al., 2004), in which the acceptor fluorophore is a circularly permuted version of Venus rather than EYFP (Nagai et al., 2002). YC3.60 has a monophasic Ca²⁺ dependency with a dissociation constant (K_d) of 0.25 μm. Compared with YC3.1, YC3.60 is equally bright with a 5- to 6-fold larger dynamic range. Thus, YC3.60 results in a markedly enhanced signal-to-noise ratio, thereby enabling Ca²⁺ imaging experiments that were not possible with conventional YCs. On the other hand, YC4.60 was developed by mutating the Ca²⁺-binding loop of CaM in YC3.60. Because YC4.60 has a significantly lower Ca²⁺ affinity with a biphasic Ca²⁺ dependency (K_d: 58 nm and 14.4 μm), it allows changes in Ca²⁺] dynamics to be detected against a high background Ca²⁺] (Nagai et al., 2004).To examine whether the Ca²⁺]_cyt oscillates in pollen tubes growing through a stigma after pollination (in vivo condition), in those placed in germination medium immediately after passing through a style (semi-in vivo condition), or in those grown in germination medium (in vitro condition), we generated transgenic Arabidopsis and N. tabacum lines expressing the YC3.60 gene in their pollen grains and monitored Ca²⁺ dynamics in the pollen tube tip. We also examined how inhibitors of pollen tube growth affect Ca²⁺ dynamics in pollen tubes growing under the semi-in vivo condition. To examine Ca²⁺ dynamics in the endoplasmic reticulum (ER), we generated transgenic Arabidopsis plants expressing YC4.60 in the pollen tube ER. The results are discussed in relation to the physiological relevance of Ca²⁺] oscillations for pollen tube growth.