Osmomat 030

The hemolymph samples were firstly thawed and homogenized using an IKA homogenizer (T10B, IKA Co., Germany). The resultant homogenates were then centrifuged at 10,000 × g for 20 min at 4°C, and the supernatant was collected for analysis. The hemolymph osmolality was analyzed using a freezing-point osmometer (OSMOMAT 030, Gonotec, Berlin, Germany). The concentrations of Na⁺, K⁺, Ca²⁺, and Cl⁻ were detected using an electrolyte analyzer (K-Lite 5, Meizhou Kangli high-tech Co., Ltd, Guangzhou, China), while the concentration of Mg²⁺ was determined with a spectrophotometer (T6 New Century, Beijing Purkinje General Instrument Co., Ltd, Beijing, China) and a commercial test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The 11-DAS seedlings were frozen in liquid nitrogen and then centrifuged for 20 min at 1,500 g in microcentrifuge tubes. Further separation of the cellular fluid from plant debris was obtained by centrifugation at 9,600 g for 10 min, and osmotic potential was measured using 30 µL samples at freezing-point depression with an Osmomat 030 (Gonotec, Berlin, Germany). Since the WT and PtoPIP1;1 OE plants all grew in plates with 100% relative humidity, the water potential of the plants was used as the value of the osmotic media. Based on the above osmotic potential and water potential, the turgor pressure was calculated and could be regarded as an estimated value of turgor pressure in plants [62 (link)]. PtoPIP1;1 OE Arabidopsis plants were grown together with their corresponding WT on the same plate. Osmotic potential was measured by using six biological replicates with multiple leaves pooled in each replicate.

The osmolality studies were performed using Osmomat 030 (Gonotec GmbH, Berlin, Germany). Each point was measured at least three times, and the mean values and SD were calculated. MilliQ water was used to determine background, and standard NaCl solutions of 300 and 400 mOsm (Knauer GmbH, Berlin, Germany) were used for initially osmometer calibration. Recalibrations were performed between measurements.

On D 0, in both periods, plasma concentrations of glycerol, glucose, insulin, NEFA, urea, plasma osmolality and, in whole blood, RBC indices were determined from samples drawn from jugular vein before morning feeding at 08:00. On D 3, plasma concentrations of glycerol, glucose, insulin, NEFA, urea, and plasma osmolality were also determined from eight consecutive samples, while RBC indices from two samples (at 08:00 and at 10:00). Repeated sampling on D 3 were collected after jugular cannulation. At each sampling, from each ewe, two blood samples were collected: one using 2-mL vacuum collection tubes with glycolytic inhibitor (5.0 mg sodium fluoride, 4.0 mg pot. ox. - Vacutainer Systems Europe; Becton Dickinson, MeylanCedex, France) for glucose assay; one using 2.0 mL vacuum collection whole blood tube with spray-coated K₂EDTA (Vacutainer Systems Europe; Becton Dickinson, MeylanCedex, France) for other metabolites, insulin and RBC indices determination. Immediately after recovery, samples were cooled to 4 °C. RBC indices were determined within 2 h of blood collection. The other blood samples were centrifuged at 1500 g for 15 min at 4 °C degrees. Individual plasma was removed and stored in vial at − 20 °C until assayed.
Plasma osmolality (Osm/kg) was measured using a freezing point osmometer (Osmomat 030, Gonotec, Berlin, Germany).