Extended Data Fig. 1: Schematic illustration of the perception of extracellular stimuli by cell-surface receptors, and the characteristics of analyzing hypo-osmosensors in plants.

a, The extracellular stimuli (first messengers) are perceived by cell-surface receptors/sensors, and converted to cytosolic signals (second messengers). It is essential for living organisms to sense environmental signals to program accordingly growth, development and survival^{1,4,5,21,23,34,46,89,90,91,92,93,94}. The environmental signals include physical, chemical, and biological stimuli, which are called the first messengers. The cytosolic signals include diffusible small molecules and ions, which are called the second messengers⁸⁹. These receptors are usually ion channels, receptor kinases (RTKs)/receptor-like kinases (RLKs), and G-protein-coupled receptors (GPCRs) in animals and plants, and their activation often leads to increases in [Ca²⁺]_i through either Ca²⁺ influx across the plasma membrane or Ca²⁺ release from internal stores^4,5,23,90. It is well known that Ca²⁺ ion serves as a versatile second messenger for various external stimuli in living organisms^{1,19,21,89,90}. b,c, Comparison between the assays for the activities of hyper-osmosensors and hypo-osmosensors in plants. With respect to the analysis of cytosolic signals, for example, [Ca²⁺]_i, the stable baseline at the resting state should be established first. Subsequently, the extracellular stimulus, such as hyper-osmotic stress (b) or hypo-osmotic shock (c), could be applied as indicated by the arrow, and the dynamic of cytosolic signals could be recorded continuously, which reflects the activity of the cell-surface receptor corresponding to the stimulus applied. These terms of “osmosensing”, “osmosensitive” and “osmosensor” have been widely used in microorganisms, plants, and animals^2,4,11,12,87. In general, as long as cells face the amount of available water less than “normal” in the extracellular spaces, which causes cells to lose water from the cytosol, this is called as hyper-osmotic stress/stimulation. On the other hand, if cells face the amount of water more than “normal”, which leads to water influx into the cell, this is called as hypo-osmotic shock/stimulation. Note that, the definition of hyper- or hypo-osmolarity is not about the absolute values to some extent, rather the relationship to the osmolarity in the cytosol, similar to the de-polarization and hyper-polarization in relative to the resting membrane potential. Note that, under the normal growth conditions without any osmotic stress, vegetative tissues (seedlings) are well hydrated, and thus by default, the vegetative tissues are at the stable baseline of the hydrated state (b). Hyper-osmotic stress could be applied directly to these vegetative tissues, and cytosolic signaling processes could be recorded accordingly^6,85,87. In contrast, also under the normal growth conditions, pollen grains and seeds are well dehydrated. By default, they are at the stable baseline of the dehydrated state, and hypo-osmotic shock could be applied directly to analyze hypo-osmosensitive activities (c)^{41,43,48,72,95}. Apparently, without the need of any pretreatment, vegetative tissues are good systems for the analysis of hyper-osmotic stress, while pollen grains and seeds are good systems for the analysis of hypo-osmotic stress. d, The assay for hypo-osmotic shock-induced signaling or physiological changes in vegetative tissues is much more challenging technically. The analyses of hypo-osmotic shock-induced responses in vegetative tissues have been well described in a number of studies^16,17,18,84. Essentially, these vegetative tissues have to be pre-treated with hyper-osmotic conditions for a relatively long period of time (hours or days) first to establish the stable baseline of the dehydrated state, then hypo-osmotic shock could be applied for the analysis of hypo-osmotic signaling processes (in minutes) or physiological changes (in hours and days). Therefore, it has been hard to establish Ca²⁺-imaging-based genetic screens for mutants defective in HOSCA, in contrast to mutants defective in hyper-osmotic stress-induced Ca²⁺ increases⁶. Evidently, if vegetative tissues are treated with drought stress by withdraw water supply for days to establish the dehydrated state, and then water is resupplied to allow rehydration¹⁴, the phenotypes of growth and survival rate could reflect the complex effects on both the dehydration and/or rehydration processes.