Extended Data Fig. 8: Pollen Ca²⁺-oscillation phenotype prior to germination in the osca2.1/2.2 double mutant is rescued under the hypo-osmotic media (350 mOsm) and exacerbated under hyper-osmotic media (680 mOsm).

a,b, Cumulative GCaMP6-fluorescence images were pooled together at the intervals of 8–33 min (left) and 138–163 min (right) for a total of 25 min, respectively, after pollen grains were spread on the hypo-osmotic (350 mOsm) germination media from WT and osca2.1/2.2 plants (a), and quantified (b). Scale bars are 50 μm. GCaMP6-fluorescence images were collected every 30 s for 300 min, and are scaled by a pseudo-color bar. Data are mean ± s.d. (n_WT = 69 grains, n_osca2.1/2.2 = 72 grains). Under the hypo-osmotic media WT pollen grains displayed an accelerated hydration, and at the time of 8–33 min the [Ca²⁺]_i were already increased to the level of these at the time of 138–163 min (as a saturated status), as shown in a and b. However, osca2.1/2.2 pollen grains displayed a “normal” hydration as seen for the WT at the standard 535 mOsm media (Fig. 4a), and at the time of 8–33 min the [Ca²⁺]_i were lower, and then increased to the level at the time of 138–163 min. c,d, Cumulative pollen Ca²⁺ images for 300 min from the same experiments as in a and b were shown in 3D surface pseudo-color images (c) or bar graphs (d), respectively. Representative 3D surface plots of cumulative Ca²⁺ signals for 300 min after pollen grains were spread on the 350 mOsm germination media from WT and osca2.1/2.2 plants. Data are mean ± s.d. (n_WT = 69 grains, n_osca2.1/2.2 = 72 grains). Similar results were seen in > 10 times. e,f, Representative Ca²⁺ oscillations in a WT (e) or an osca2.1/2.2 (b) pollen grain expressing GCaMP6 on the hypo-osmotic germination media (350 mOsm). GCaMP6-fluorescence images were taken every 30 s for 300 min. The time-lapse acquisitions show the peaks, troughs and resting phases of Ca²⁺ oscillations. Similar to the patterns Ca²⁺ oscillations in WT pollen grains in Fig. 4a, the resting phases (RePh₁, RePh₂ and RePh₃) were separated with Ca²⁺ oscillations with small amplitude (CaOsc^S) and large amplitude (CaOsc^L) in both WT and osca2.1/2.2 pollen grains. P_L/P_S, peak amplitude of CaOsc^L/CaOsc^S; T_L/T_S, trough amplitude of CaOsc^L/CaOsc^S. Similar results were seen in > 10 times. g,h, Cumulative GCaMP6-fluorescence images were pooled together at the intervals of 8–33 min (left) and 165–190 min (right) for a total of 25 min, respectively, after pollen grains were spread on the hyper-osmotic germination media (680 mOsm) from WT and osca2.1/2.2 plants (g), and quantified (h). Scale bars are 50 μm. GCaMP6-fluorescence images were collected every 30 s for 300 min, and analyzed (mean ± s.d.; n_WT = 91 grains, n_osca2.1/2.2 = 100 grains). i,j, Cumulative pollen Ca²⁺ images for 300 min from the same experiments as in g and h were shown in 3D surface pseudo-color images (i) or bar graphs (j), respectively. Data are mean ± s.d. (n_WT = 91 grains, n_osca2.1/2.2 = 100 grains). k,l, Representative Ca²⁺ oscillations in a WT (k) or an osca2.1/2.2 (l) pollen grain expressing GCaMP6 on the hyper-osmotic germination media (680 mOsm). GCaMP6-fluorescence images were taken every 30 s for 300 min. Largely altered patterns Ca²⁺ oscillations especially for osca2.1/2.2 pollen grains feature extended the resting phase (RePh₁) for osca2.1/2.2 pollen grains and Ca²⁺ oscillations with small amplitude (CaOsc^S) for WT pollen grains. P_L/P_S, peak amplitude of CaOsc^L/CaOsc^S; T_L/T_S, trough amplitude of CaOsc^L/CaOsc^S. Similar results were seen in > 10 times.

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