Pb221s

Relative water content in AZ and leaves harvested after 48 and 51 days of plant growth in drought (25% WHC) and soil with optimal moisture (70% WHC, control plants) was measured using the drying method. Plant material (2 g) was weighed (analytical weight PB221S, Sartorius, an accuracy of ± 0.0001 g), placed in zinc vessels, and dried (SUP200W, WAMED) (60 °C) for 24 h. Then, plant material was cooled down in desiccator and weighed again. The relative water content percent (RWC) was determined through the following equation: RWC [%] = (FW-DW)/SW × 100, where FW: Fresh gross weight [g]; DW: Dry gross weight [g]; and SW: Sample weight [g]. The dried material was also used for element content determination.
The influence of drought stress on flower abortion rate was presented as a percentage of separated flowers in relation to the flowers formed by the plant. Additionally, we determined the effect of drought on the number of leaves per plant and leaf area per plant using AM350 Portable Leaf Area Meter (ADC BioScientific Ltd., Hoddesdon, UK). The maximum quantum efficiency of PS II (Fv/Fm) as a reliable marker of photo-inhibition [92 (link)] was measured using an OS-30P (Opti-Sciences, Inc., Hudson, NH, USA) according to the methods described by Weng (2006) [93 (link)]. The results were presented as the mean of the sum of the areas of all leaves per plant.

To purify
PfEVs, the frozen supernatant generated above was thawed on ice and loaded on quick-seal ultracentrifugation tubes (Bechman Coulter cat# 343322), then centrifuged at 150, 000g for 2hrs using an Optima XE90 ultracentrifuge and 70.1Ti rotor (Beckman Coulter). The pellet was washed twice by re-suspending in cold PBS and centrifuging at 150,000g for 2 hours after each wash. The final pellet was loaded onto OptiPrep™ density gradient medium prepared as described
^{47 (link)–
49 (link)} and centrifuged at 250,000g for 18 hours. 1ml fractions were collected from the top of the gradient into 1.5ml Eppendorf tubes. To estimate the density of the purified vesicles, the weights of the tubes containing the fractions were measured using weighing machine (Sartorius, PB221S). Each fraction was diluted in PBS to 13.5ml and centrifuged at 150, 000g for 2 hours and the pellet re-suspended in 400µl of 8M urea, 2.5% SDS in 50 mM phosphate buffer, pH 8.0 to extract proteins, before concentrating using a 3KDa MWCO concentrator (Pierce™). A fifth of each fraction was analysed for the presence of proteins by running on SDS-PAGE and staining using silver stain (Bio-Rad).