The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] is a key signaling molecule in animal cells. It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP 3 ), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively. In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P 2 binds to proteins that modify cytoskeletal architecture and phospholipid constituents. Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P 2 synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol. These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides. We also show data demonstrating that whole-plant IP 3 levels increase significantly within 1 min of stress initiation, and that IP 3 levels continue to increase for more than 30 min during stress application. Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments. Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P 2 is hydrolyzed to IP 3 . The accumulation of IP 3 occurs during a time frame similar to that observed for stress-induced calcium mobilization. These data also suggest that the majority of the PtdIns(4,5)P 2 synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP 3 signaling pathway.Phosphoinositides are a class of membrane phospholipids that serve numerous roles in eukaryotic cellular processes. The family of phosphoinositides includes phosphatidylinositol monophosphate species phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 4-phosphate [PtdIns(4)P], phosphatidylinositol bisphosphate species phosphatidylinositol 3,4-bisphosphate, phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P 2 ], and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ], and the phosphatidylinositol trisphosphate species phosphatidylinositol 3,4,5-trisphosphate. PtdIns(3)P and PtdIns(4)P regulate vesicle-mediated protein transport to the vacuole/lysosome and protein secretion,

Stomatal responses to light and CO2 were investigated using isolated epidermes of Tradescantia pallida, Vicia faba and Pisum sativum. Stomata in leaves of T. pallida and P. sativum responded to light and CO2, but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO2. However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO2. Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO2. Epidermes from V. faba did not respond to light or CO2 when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO2 rather than being sensitized to light and CO2 by the mesophyll. The data suggest that most of the stomatal response to CO2 and light occurs in response to a signal generated by the mesophyll.

Phosphoinositide signaling regulates events in endocytosis and exocytosis, vesicular trafficking of proteins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis. Obtaining mechanistic insights in living cells is impeded by the membrane impermeability of these anionic lipids. We describe a carrier system for intracellular delivery of phosphoinositide polyphosphates (PIP ns) and fluorescently labeled PIP ns into living cells, such that intracellular localization can be directly observed. Preincubation of PIPns or inositol phosphates with carrier polyamines produced complexes that entered mammalian, plant, yeast, bacterial, and protozoal cells in seconds to minutes via a nonendocytic mechanism. Time-dependent transit of both PIPns and the carrier to specific cytosolic and nuclear compartments was readily visualized by fluorescence microscopy. Platelet-derived growth factor treatment of NIH 3T3 fibroblasts containing carrier-delivered phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2]-7-nitrobenz-2-oxa-1,3-diazole resulted in the redistribution of the fluorescent signal, suggesting that fluorescent PtdIns(4,5)P 2 was a substrate for phospholipase C. We also observed a calcium flux in NIH 3T3 cells when complexes of carrier and PtdIns(4,5)P 2 or inositol 1,4,5-trisphosphate were added extracellularly. This simple intracellular delivery system allows for the efficient translocation of biologically active PIP ns, inositol phosphates, and their fluorescent derivatives into living cells in a physiologically relevant context. P hosphatidylinositol polyphosphates (PIP n s) and inositol polyphosphates (IP n s) serve as signaling molecules in numerous eukaryotic cellular processes (1-5), including tyrosine kinase growth factor and G-protein receptor signaling pathways (6, 7). PIP n s are crucial components for endocytic, exocytic, and Golgi vesicle movement (8), and in remodeling of the actin cytoskeleton (9). Moreover, cellular phosphoinositide composition is dynamic in time and space. An ideal technique would permit visualization of both exogenously introduced and endogenously synthesized PIP n s in membrane-associated, nuclear, and cytosolic domains.Investigators have sought cell-permeant derivatives to deliver active PIP n s or IP n s intracellularly without disruption of the cell membrane. Several lipid-soluble analogs of inositol trisphosphates [Ins(1,3,4)P 3 , Ins(1,4,5)P 3 ] and phosphoinositides [PtdIns(3,4,5)P 3 ] (10 -12) and ''caged'' Ins(1,3,4,5)P 4 , Ins(1,4,5)P 3 , and InsP 6 have been synthesized and used in cellular studies (13). Extensive chemical synthesis (11) was required, and deprotection by light and͞or esterase action was necessary to release the free IP n or PIP n . Because multiple protecting groups were attached to each cell-permeant molecule (13), the rate of release of the active chemical signal could not be readily controlled, and a heterogeneous mixture of potential agonists was produced.The ability to monitor changes in ce...

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 m thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.Stomatal aperture in leaves is controlled by the turgor pressures of the guard cells (P g ) and the surrounding epidermal cells (P e ). Increases in P g open the pore and increases in P e close the pore, but the exact roles of these two parameters in determining aperture are complex (Franks et al., 1995(Franks et al., , 1998. Equal increases in guard cell and epidermal turgor pressure generally close the pore; thus, epidermal cells have a "mechanical advantage" over guard cells (Glinka, 1971; Edwards et al., 1976; Franks et al., 1998). Although some progress has been made toward understanding the relationships between aperture, P g , and P e (Meidner and Edwards, 1975; Franks et al., 1995 Franks et al., , 1998, less is known about how various perturbations affect P g and P e . P g and P e are functions of their respective water potentials (⌿ g and ⌿ e ) and osmotic pressures ( g and e ). Therefore, efforts to understand and predict the effects of environmental perturbations on stomatal aperture should focus on ⌿ g , ⌿ e