Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

OSCA1 is an osmotic specific sensor: a method to distinguish Ca2+ -mediated osmotic and ionic perception.

Genetic mutants defective in stimulus-induced Ca2+ increases have been gradually isolated, allowing the identification of cell-surface sensors/receptors, such as the osmosensor OSCA1. However, determining the Ca2+ -signaling specificity to various stimuli in these mutants remains a challenge. For instance, less is known about the exact selectivity between osmotic and ionic stresses in the osca1 mutant. Here, we have developed a method to distinguish the osmotic and ionic effects by analyzing Ca2+ increases, and demonstrated that osca1 is impaired primarily in Ca2+ increases induced by the osmotic but not ionic stress. We recorded Ca2+ increases induced by sorbitol (osmotic effect, OE) and NaCl/CaCl2 (OE + ionic effect, IE) in Arabidopsis wild-type and osca1 seedlings. We assumed the NaCl/CaCl2 total effect (TE) = OE + IE, then developed procedures for Ca2+ imaging, image analysis and mathematic fitting/modeling, and found osca1 defects mainly in OE. The osmotic specificity of osca1 suggests that osmotic and ionic perceptions are independent. The precise estimation of these two stress effects is applicable not only to new Ca2+ -signaling mutants with distinct stimulus specificity but also the complex Ca2+ signaling crosstalk among multiple concurrent stresses that occur naturally, and will enable us to specifically fine tune multiple signal pathways to improve crop yields.

Hydrogen peroxide sensor HPCA1 is an LRR receptor kinase in Arabidopsis.

Hydrogen peroxide (H2O2) is a major reactive oxygen species in unicellular and multicellular organisms, and is produced extracellularly in response to external stresses and internal cues1-4. H2O2 enters cells through aquaporin membrane proteins and covalently modifies cytoplasmic proteins to regulate signalling and cellular processes. However, whether sensors for H2O2 also exist on the cell surface remains unknown. In plant cells, H2O2 triggers an influx of Ca2+ ions, which is thought to be involved in H2O2 sensing and signalling. Here, by using forward genetic screens based on Ca2+ imaging, we isolated hydrogen-peroxide-induced Ca2+ increases (hpca) mutants in Arabidopsis, and identified HPCA1 as a leucine-rich-repeat receptor kinase belonging to a previously uncharacterized subfamily that features two extra pairs of cysteine residues in the extracellular domain. HPCA1 is localized to the plasma membrane and is activated by H2O2 via covalent modification of extracellular cysteine residues, which leads to autophosphorylation of HPCA1. HPCA1 mediates H2O2-induced activation of Ca2+ channels in guard cells and is required for stomatal closure. Our findings help to identify how the perception of extracellular H2O2 is integrated with responses to various external stresses and internal cues in plants, and have implications for the design of crops with enhanced fitness.

Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca2+ concentration, which activate Ca2+-binding proteins and upregulate the Na+/H+ antiporter in order to remove Na+. Salt-induced increases in Ca2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca2+-imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca2+]i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca2+ spikes and waves, Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.

OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis.

Water is crucial to plant growth and development. Environmental water deficiency triggers an osmotic stress signalling cascade, which induces short-term cellular responses to reduce water loss and long-term responses to remodel the transcriptional network and physiological and developmental processes. Several signalling components that have been identified by extensive genetic screens for altered sensitivities to osmotic stress seem to function downstream of the perception of osmotic stress. It is known that hyperosmolality and various other stimuli trigger increases in cytosolic free calcium concentration ([Ca(2+)]i). Considering that in bacteria and animals osmosensing Ca(2+) channels serve as osmosensors, hyperosmolality-induced [Ca(2+)]i increases have been widely speculated to be involved in osmosensing in plants. However, the molecular nature of corresponding Ca(2+) channels remain unclear. Here we describe a hyperosmolality-gated calcium-permeable channel and its function in osmosensing in plants. Using calcium-imaging-based unbiased forward genetic screens we isolated Arabidopsis mutants that exhibit low hyperosmolality-induced [Ca(2+)]i increases. These mutants were rescreened for their cellular, physiological and developmental responses to osmotic stress, and those with clear combined phenotypes were selected for further physical mapping. One of the mutants, reduced hyperosmolality-induced [Ca(2+)]i increase 1 (osca1), displays impaired osmotic Ca(2+) signalling in guard cells and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels, revealing that OSCA1 may be an osmosensor. OSCA1 represents a channel responsible for [Ca(2+)]i increases induced by a stimulus in plants, opening up new avenues for studying Ca(2+) machineries for other stimuli and providing potential molecular genetic targets for engineering drought-resistant crops.