5500 vapor pressure osmometer

Constructs were introduced into onion epidermal cells, spinach leaves, and Bienertia chlorenchyma cells by particle bombardment according to [72 (link)] and the manufacture’s protocol (Bio-Rad). For Bienertia bombardment, the epidermis of the leaf was first gently removed using forceps to grab the tip of the leaf and a small plastic pestle to gently press out chlorenchyma and internal leaf cells, resulting in an epidermis-less leaf. Cells were bombarded with 1 μm plasmid coated gold micro-carriers, using 1100 p.s.i. rupture discs at a distance of 6 cm from the stop screen. After bombardment epidermal free leaves were incubated overnight in a buffer (MES-NaOH, pH 5.8) that was osmotically adjusted with glycine betaine to match the osmolality of the leaf (determined with a 5500 Vapor Pressure Osmometer, Wescor). Bombarded onion epidermal cells, and spinach leaves were incubated overnight at room temperature on water moistened filter paper.

PLs and LUVs are subjected to a hyperosmotic solution in a stopped-flow apparatus (SFM-300 or μ-SFM, Bio-Logic, Claix, France) at 4 °C and the intensity of scattered light is monitored at 90° at a wavelength of 546 nm if not otherwise stated.^{1,18–20 (link)} Water permeability values P_f are, except explicitly mentioned, calculated using our recently calculated analytical solution.^{19 (link)} For self-quenching experiments, monochromatic light of 480 nm wavelength is used to illuminate the sample. The emitted fluorescent light passes a 515 nm longpass filter and is detected at an angle of 90°. Averaged self-quenching curves are then fitted, according to eqn (5), to the following function: where F(t) is the fluorescence intensity and B and D are fitting parameters.
The normalized intensity I_norm is determined either by I_norm(t) = I(t)/I_max or, for normalization between [0,1], by I_norm(t) = (I(t) − I_min)/(I_max − I_min), where I(t) is the measured intensity at time t and I_max and I_min are the averaged maximum and minimum intensities. In all experiments, buffer osmolarities were determined with a Wescor 5500 Vapor Pressure Osmometer.

Physiological parameters were measured in the leaves, 2-month after treatment establishment.
Gas exchange parameters were measured from 10:00 to 12:30 h on one ML per plant leaves using an open infrared gas exchange analyzer system (Li- 6400; Li-Cor Inc., Lincoln, NE, United States). Leaf chamber fluorometer (Li-6400-40, Li-Cor Inc.) conditions were PPFD of 1.500 μmol m^–2 s^–1, with 10% of blue light, and a vapor pressure deficit of 2.0–3.0 kPa at a CO₂ concentration of 400 μmol mol^–1(air). The leaf temperature was set at 25°C, and the relative humidity of the incoming air was approximately 50% throughout all measurements.
To quantify chlorophyll and leaf protein concentrations, samples of the leaves used for gas exchange measurement (ML) were frozen in liquid nitrogen and stored at −80°C. Photosynthetic pigments were extracted using 96% ethanol. Chlorophyll a, chlorophyll b and total chlorophyll content were calculated according to Lichtenthaler and Wellburn (1983) (link). Leaf protein content was determined following the method described by Bradford (1976) (link).
Leaf osmotic potential was measured in two mature leaves per plant. Frozen samples of ML were thawed and grinded for 30 s. A sap volume of 10 μL was used (Gucci et al., 1991 (link)) to determine the leaf osmotic potential using a Wescor 5500 vapor pressure osmometer (Wescor Inc., Logan, UT, United States).