Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed arabidopsis

Abstract

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.

Figure 1

Osmotic stress-induced PtdIns(4,5)P₂ production in Arabidopsis plants. Anion-exchange HPLC analysis of deacylated myo-[2-³H]inositol-labeled lipids from plants that were untreated (A, B, and C, ×) or treated with 0.25 m NaCl (A, ●), 0.25 m KCl (B, ▴), and 0.5 m sorbitol (C, ♦) for 30 min. The total counts in each sample were 7 × 10⁵.

Figure 2

Concomitant increase in osmotic stress-induced PtdIns(4,5)P₂ and IP₃ levels. Sorbitol (at a final concentration of 1 m) was added to myo-[2-³H]inositol-labeled plants and an equivalent number of plants were withdrawn at 0, 15, and 30 min. A, The elution profile of gPI(4,5)P₂ (elution volume of 41–46 mL) is shown. B, The elution profile of inositol 1,4,5-trisphosphate (elution volume of 55–60 mL) is shown. For each HPLC run, the aqueous or organic sample each contained 1 × 10⁶ cpm.

Figure 3

Time course of salt stress-induced PtdIns(4,5)P₂ and IP₃ production. NaCl (at a final concentration of 0.25 m) was added to myo-[2-³H]inositol-labeled plants and an equivalent number of plants was withdrawn from solution at 0, 1, 5, 15, 30, and 60 min. A, The extracted and deacylated phosphoinositide head groups (organic) were separated by anion-exchange HPLC and the total number of counts in each gPI(4,5)P₂ peak was calculated. B, The aqueous IP₃-containing samples were analyzed by anion-exchange HPLC and the total number of counts in each IP₃ peak was calculated. For each HPLC run, the aqueous or organic sample each contained 1 × 10⁶ cpm.

Figure 4

U-73122 blocks IP₃ accumulation in salt-stressed plants. Myo-[2-³H]inositol-labeled plants were incubated in the absence (control) or presence of the PLC inhibitor (+ U-73122) and were then exposed to 0.25 m NaCl for 0 (□) or 15 (▪) min, respectively. A, The extracted and deacylated phosphoinositide head groups (organic) were separated by anion-exchange HPLC and the total number of counts in each PtdIns(4,5)P₂ peak was calculated. B, The aqueous IP₃-containing samples were analyzed by anion-exchange HPLC and the total number of counts in each IP₃ peak was calculated. The aqueous and organic samples contained 1 × 10⁶ and 3 × 10⁶ cpm, respectively.

Figure 5

Calcium mobilization in root-tip cells. A, Root tips were loaded with Fluo-3 and were untreated (▴), treated with 0.25 m NaCl (▪), or treated with 0.25 m NaCl in the presence of the PLC inhibitor U-73122 (♦). NaCl was added at t = 0 and data for these experiments was collected simultaneously from plants laying adjacent to each other. For the untreated experiment (▴), 100 μL of buffer was added at t = 0. B, Mean and sd calculations of root tip cell fluorescence (n = 9 for each treatment). Roots were treated as described above, images collected, and relative fluorescence presented; control (black), salt stressed (light gray), and salt stressed plus U-73122 (white). Salt stressed indicates exposure to 0.25 m NaCl. C, Mean and sd calculations of fluorescence ratios using Fura-2-loaded 8-d-old root tips (n = 7 for each treatment). Root tips were untreated (black) or were treated with 0.25 m NaCl (light gray) or 10 μm 4-Bromo A-23187 (dark gray; as a positive control).