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

Abstract

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 μ m. 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.

The Arabidopsis Synaptotagmin1 Is Enriched in Endoplasmic Reticulum-Plasma Membrane Contact Sites and Confers Cellular Resistance to Mechanical Stresses

“…1E), and signal movement through transvacuolar strands was observed (Supplemental Movie S1). Transient colocalization in Nicotiana benthamiana of a Red Fluorescent Protein (RFP)-tagged SYT1 fused to its endogenous promoter(SYT1proSYT1-RFP) with the ER-targeted yellow cameleon 4.60 (Iwano et al, 2009) showed that the SYT1 signal was enriched at specific ER subdomains (Supplemental Fig. S3).…”

“…The genomic fragments of the SYT1 and VAP27 coding sequence were amplified from Col-0 DNA and recombined into pDONR221 (Invitrogen). The ER-targeted yellow cameleon 4.60 was subcloned from pBI121 vector (Iwano et al, 2009) into the multicloning site of pGEM3Zf(+) using restriction fragments generated by XbaI and SacI, allowing for further subcloning into pENTR1A using the resulting SalI and EcoRI restriction fragment. For the C-terminal XFP fusions (GFP, pEN-R2-F-L3,0; mRFP, pEN-R2-R-L3,0 [Karimi et al, 2007]; and TagRFP, pEN-R2-TagRFPSTOP-L3 [Mylle et al, 2013]), the respective entry vectors were recombined together with the respective promotor and coding sequence entry vectors into pKm43GW or pBm43GW (Karimi et al, 2005).…”

Cyclic nucleotide-gated channel 18 is an essential Ca 2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

“…4A) and cngc18-22 (Fig. 4A) ] cyt oscillation, are required for pollen tube growth and guidance (24,30). Therefore, the irregular drift of the basal [Ca 2+ ] cyt levels (i.e., the disturbance of the Ca 2+ gradients in pollen tube tips) might be the main cause of pollen tube guidance defects in cngc18-17 and cngc18-22.…”

“…Ca 2+ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19)(20)(21)(22)(23)(24)(25)(26)(27). Spatial modification of the Ca 2+ gradients leads to the reorientation of pollen tube growth in vitro (28,29).…”

Fluorescence Resonance Energy Transfer-Sensitized Emission of Yellow Cameleon 3.60 Reveals Root Zone-Specific Calcium Signatures in Arabidopsis in Response to Aluminum and Other Trivalent Cations

“…The higher dynamic range of YC3.60 is due to the use of a circularly permutated YFP called Venus (cpVenus) that is capable of absorbing a greater amount of energy from CFP (Nagai et al, 2004). Recently, the utility of YC3.60 for monitoring [Ca 2+ ] cyt was demonstrated in Arabidopsis roots and pollen tubes using ratiometric imaging approaches (Monshausen et al, 2007(Monshausen et al, , 2009Haruta et al, 2008;Iwano et al, 2009). Here, we further evaluated YC3.60 as a [Ca 2+ ] cyt sensor in plants using confocal microscopy and FRET-sensitized emission imaging.…”