Extended Data Fig. 10: Impaired osmolarity regulation of [Ca²⁺]_i oscillations during pollen germination in osca2.1/2.2.

a,b, Data on Ca²⁺ spiking patterns in pollen grains placed on hypo-osmotic (350 mOsm) or hyper-osmotic media (680 mOsm) were from the same experiments in Fig. 5a,b,e,f, and quantified for the periods of CaOsc^S (a) and CaOsc^L (b) similarly as in Fig. 5c,d,g,h (mean ± s.d.; n₃₅₀ = 114 grains for WT; n₃₅₀ = 235 grains for osca2.1/2.2; n₆₈₀ = 122 grains for WT; n₆₈₀ = 198 grains for osca2.1/2.2). The time between the peaks of two Ca²⁺ spicks was calculated as a period. The oscillation periods of CaOsc^S and CaOsc^L were increased under high osmolarity media in WT, suggesting that low water availability suppressed Ca²⁺ oscillations. Overall, the oscillation periods of CaOsc^S and CaOsc^L were longer in osca2.2/2.2 than those in WT, suggesting that osca2.2/2.2 had weaker Ca²⁺ oscillations. c,d, Data on Ca²⁺ spiking patterns in pollen grains placed on hypo-osmotic media (350 mOsm) or hyper-osmotic media (680 mOsm) were from the same experiments in Fig. 5a,b,e,f, and quantified for the oscillation duration times of CaOsc^S (c) and CaOsc^L (d) similarly as in Fig. 5c,d,g,h (mean ± s.d.; n₃₅₀ = 114 grains for WT; n₃₅₀ = 235 grains for osca2.1/2.2; n₆₈₀ = 122 grains for WT; n₆₈₀ = 198 grains for osca2.1/2.2). The duration time of an individual Ca²⁺ spick was calculated as the time between the troughs immediately before and after the peak. The duration times of CaOsc^S and CaOsc^L were increased under high osmolarity media in WT, also indicating that the water availability was positively associated with Ca²⁺ oscillations. Overall, the duration times of CaOsc^S and CaOsc^L were longer in osca2.2/2.2 than those in WT, except for CaOsc^L at high osmolarity media. Note that, the shorter duration times of CaOsc^L at 680 mOsm in osca2.2/2.2 were largely due to very few or no CaOsc^L occurring as seen in Fig. 5f, preventing accurate analysis. These results suggest that OSCA2.1/2.2 are responsible for sensing water, and that Ca²⁺ oscillations are the second messengers for water. e,f, Effects of media osmolarity on the period (e) and amplitude (f) of CaOsc^S from the same experiments as in Fig. 5i,j (mean ± s.d.; n = 5 independent experiments; two-way ANOVA, P < 0.001). With the decreases in media osmolarity, i.e. increases in water availability, the oscillation amplitudes were elevated and the oscillation periods were reduced, suggesting that the water availability was positively associated with Ca²⁺ oscillations. Overall, the osca2.2/2.2 was less sensitive to the increases in water availability, consistent with OSCA2.1/2.2 being hypo-osmosensors. g–j, Complementation of defective Ca²⁺ oscillations in osca2.1/2.2 under the standard germination media (535 mOsm) by expression of genomic DNA of OSCA2.1 or OSCA2.2. Data on Ca²⁺ spiking patterns in pollen grains placed on the 535 mOsm media were from the experiments similar to these in Fig. 4, and the periods and amplitudes of CaOsc^S (g, h) and CaOsc^L (i, j) were quantified similarly as in a–d (mean ± s.d.; n = 3 independent experiments). The periods and amplitudes of CaOsc^S and CaOsc^L in the osca2.1 and osca2.1 single mutants were similar to these in WT, consistent with their phenotypes in pollen germination (Fig. 2c) and HOSCA (Extended Data Fig. 4c–e). In addition, these periods and amplitudes of CaOsc^S and CaOsc^L in the complementation lines (OSCA2.1 osca2.1/2.2 and OSCA2.2 osca2.1/2.2) were comparable to these in WT, also suggesting that OSCA2.1 and OSCA2.2 had functional redundancy in regulating Ca²⁺ oscillations. Notably and consistently, the periods of CaOsc^S and CaOsc^L in osca2.1/2.2 were significantly longer than these of the rest of genotypes, and the amplitudes of CaOsc^S and CaOsc^L in osca2.1/2.2 were also significantly smaller than these of the rest of genotypes. Taken together, these WT-like phenotypes on Ca²⁺ oscillations, germination, and HOSCA in the osca2.1 and osca2.1 single mutants and complementation lines suggest that OSCA2.1 and OSCA2.2 were redundant and function together to control pollen grain perception of hypo-osmolarity conditions. k, Model of osmosensors and water potential relationships in plant cells and their environment. Soil water potential (Ψw_soil) drops from saturated state (0 MPa) to field capacity (−0.03 MPa), at which both air and water are in the macropores, to permanent wilting point (−1.5 MPa), at which soil water is held by solid particles too tightly to be taken up by plants^7,85,87. The air water potential is about −100 MPa, which provides the driving force for water transport through the soil−plant-atmosphere continuum. Notably, the extracellular spaces (called the apoplast) are connected largely with their environments (illustrated by a dashed line surrounding cell walls and arrows). The extracellular spaces in roots are directly connected to the soil, and stomatal pores allow the extracellular spaces in the leaf to connect to the air, forming the soil-plant-atmosphere continuum. Therefore, the water potential in the extracellular spaces (outside of the cell, Ψw_o) is not set to a point, in contrast to that in mammalian cells, and varies enormously from −0.03 to −4.5 MPa (equivalent to the change in solution osmolarity from 12 to 1820 mOsm). As the osmosensing OSCA1 family has expended greatly compared to three members in animals, and co-evolved with the plant transition from water to land³⁵, these OSCAs may be major osmosensors in plants. l, Model of osmosensors and water potential relationships in mammalian cells and their environment. The extracellular spaces are separated with their environment (illustrated by a solid line and stop arrows). The water potential of the extracellular fluid (ECF, Ψw_o) is set to a point, and the ECF osmolarity is tightly regulated around this set-point of ∼ 300 ± 30 mOsm (equivalent to the change in water potential from −0.66 to −0.82 MPa)¹².

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