Vapro pressure osmometer

Drosophila S2 cells were plated and transfected with pMT-Myc-CarT or the pMT-Myc-CarT^del as described^{25 (link)}. Post 24-hour induction with CuSO₄, cells were washed twice in either modified Schneider’s, Na⁺ free, or Cl⁻ free media (Supplemental Table 1) as indicated and incubated with the respective media containing 0 µM or 200 µM carcinine for 1 hour. Cells were then transferred to ice and fixed with 4% ethyl-3-(-3-dimethylaminopropyl) carbodiimide (wt/vol, Sigma) in ice-cold 0.1 M phosphate buffer solution. Fixed cells were stained with Myc and carcinine antibodies as described^{25 (link)}. Quantification was performed in ImageJ by normalizing the integrated density of the carcinine signal by that of the Myc signal. Media isotonicity was measured via a Vapro pressure osmometer (model 5520, Wescor).

Bd21 kindly provided by Dr. John Vogel et al.24 (link) at USDA-ARS, was used as plant material in this study. Bd21 seeds were surface sterilized by 15% sodium hypochlorite for 3 min, and rinsed 4 times in sterile distilled water. Seeds were submerged in water for 12 h at room temperature, and then transferred to wet filter paper to germinate at room temperature (22–25 °C) for 24 h. The uniformly germinated seeds were selected to grow in plastic pots containing Hoagland solution, in which was changed every two days. In a greenhouse, the experimental conditions included daily photo cycle of 16 h light/8 h dark (26 °C/18 °C) and 65–75% air humidity. Three biological replicates were performed under the same conditions. At the three leaf stage, the seedlings for different drought treatment times (0, 6, 12, 24 and 48 h) were cultivated in Hoagland solution containing 20% PEG600070 (link) (ψs = −0.75) measured by VAPRO pressure osmometer (Wescor 5520, USA), whereas the seedlings for the control were only cultivated in Hoagland solution (ψs = −0.045). After treatment for different times, some sampled leaves for each biological replicates were used for physiological indicator measurement and ultrastructure observation, and the remaining leaves were kept frozen in −80 °C for later protein extraction.

The fresh leaf tissue from control and treated plants were frozen, thawed and centrifuged at 10,000 g to extract the sap. The solute concentration was determined using Vapro Pressure Osmometer (model-5600; Wescor, Logan UT, USA). The solute potential (Ψ_s) of the sap was calculated as —nRT/V; where n represent a number of solute molecules; R is universal gas constant; T is the temperature in ° K, and V is volume in liter.
The treated and untreated WT and transgenic seedlings were oven dried, acid [perchloric acid and nitric acid solution (3:1 v/v)] digested and heated to dryness. The digest was dissolved in deionised water and ion contents (Na⁺ and K⁺) were estimated by inductively coupled plasma optical emission spectrometer (Optima2000DV, PerkinElmer, Germany).