0.22 μm nylon filter

TN were collected in June 2019 at the orchards of “Giaccio Frutta” society (Vitulazio, Caserta, Italy, 41°10′ N–14°13′ E), at 20–25 days after full bloom, coinciding with the fruit thinning stage. The whole fruits were frozen at −80 °C, freeze-dried and ground to obtain a uniform powder that represented the production batch used for the analysis. For TN polyphenols extraction, 1 g of homogenized sample was suspended in 5 mL of 80% aqueous methanol containing 1% formic acid for 10 min, mixed on a vortex mixer for 1 min, sonicated (Branson Fisher Scientific 150E Sonic Dismembrator) for 10 min, and centrifuged for 10 min at 9000× g. The supernatant was decanted, and the pellet was re-extracted with 5 mL of the previous solution. Finally, the combined supernatants were filtered with a 0.22 μm nylon filter (CellTreat, Shirley, MA, USA) and stored at −20 °C until analysis [14 (link)].

TNs were harvested in June 2022 at the orchards of “Giaccio Frutta” society (Vitulazio, Caserta, Italy, 41°10′ N–14°13′ E) about one month after full bloom in concomitance with the fruit thinning stage. The whole fruits (pulp, peel, and seed) were frozen at −20 °C, lyophilized, and ground to obtain a homogenous powder. With regards to the extraction procedure, a previous method reported by Schiano et al. was used with slight modifications [23 (link)]. Briefly, 2.5 g of homogenized sample was suspended in 25 mL of a 100% ethanolic solution and thoroughly vortexed for 1 min. The suspension was then sonicated for 10 min using a VWR Ultrasonic cleaner bath USC300T (VWR International Limited, Lutterworth, UK) and stirred lightly for 40 min at 25°, 600 RPM speed. After centrifugation (10 min, 9000× g, 25°), the supernatant was decanted, filtered with a 0.22 μm nylon filter (CellTreat, Shirley, MA, USA), and evaporated using a rotary evaporator (Hei-VAP Advantage Rotary Evaporator, Heidolph, Germany). The ABA content of the dried extract was evaluated as reported in the following section. For all experiments described below, the dried extract was dissolved with a defined volume of media (MEM) in order to obtain the equivalent ABA concentrations contained in free ABA and NF-ABA samples.