Extended Data Fig. 4: Medium Ca2+ effects on pollen germination, and the subcellular localization of OSCA2.1/2.2 and their requirement for hypo-osmolarity-induced [Ca2+]i increases (HOSCA) in pollen grains.
From: Osmosensor-mediated control of Ca2+ spiking in pollen germination
a,b, Representative micrographs of pollen grains from wild-type and osca2.1/2.2 plants (a). Pollen grains were placed on the hyper-osmotic solid germination media (680 mOsm) containing variable Ca2+ concentrations of 2 mM, 5 mM, or 10 mM CaCl2 (black bars at the bottom) for 6 h, and micrographs were taken. Scale bars are 50 μm. Germination rates of wild-type and osca2.1/2.2 pollen grains were quantified (b). Data are presented as mean ± s.d. (n = 3 trails). Similar results were seen in > 10 times. c–e, hypo-osmolarity-induced [Ca2+]i increases (HOSCA) in pollen grains were not affected in the osca2.1 and osca2.1 single mutants and the osca2.1/2.2 complementation lines (OSCA2.1 osca2.1/2.2 and OSCA2.2 osca2.1/2.2). Pollen grains expressing the Ca2+ indicator GCaMP6 were hydrated in the germination media containing 300 mM sorbitol for 1 h, and GCaMP6 fluorescence images were taken every 1 s after the treatment with the germination solution containing 0 mM sorbitol, similar to these experiments in Fig. 3a,b. Representative GCaMP6 images scaled by a pseudo-color bar before and after hypo-osmotic treatment were shown (c). Scale bars are 20 μm. HOSCA in wild-type, osca2.1, osca2.2, OSCA2.1 osca2.1/2.2 and OSCA2.2 osca2.1/2.2 pollen grains from experiments as in c were analyzed for kinetics, respectively (d), and for peak changes in HOSCA (e). Data were mean ± s.d. (n = 3 independent experiments). f, OSCA2.1-GUS and OSCA2.2-GUS expression levels in pollen grains from plants expression pOSCA2.1::OSCA2.1-GUS and pOSCA2.2::OSCA2.2-GUS, respectively, were placed on the common germination media at 0 min and 120 min, and the GUS assay was performed. Scale bars are 50 μm. g, Pollen grains over-expressing GFP alone (pLAT52::GFP) were placed on the standard germination media at 0 min and 120 min, respectively, and GFP fluorescence was analyzed using confocal microcopy. pLAT52::GFP was used as a control for pOSCA2.1::OSCA2.1-YFP and pOSCA2.2::OSCA2.2-YFP localization in pollen grains in Fig. 3f. Scale bars are 5 μm. h, The plasma-membrane localization of OSCA2.1/2.2 in pollen tubes. The fluorescence of pOSCA2.1::OSCA2.1-YFP, pOSCA2.2::OSCA2.2-YFP, or FM4-64 fluorescence was analyze using confocal microscopy in newly developed pollen tubes from experiments similar to these in Fig. 3f. Merged images showed FM4-64 red fluorescence was well associated with OSCA2.1-GFP or OSCA2.2-GFP fluorescence in both the cell surface and the cytosol, but not GFP-alone fluorescence, suggesting that OSCA2.1-GFP and OSCA2.2-GFP were localized to the plasma membrane, and underwent typical endocytosis, which was seen in pollen tube tips43,71,72,73. Scale bars are 10 μm.
