Sampling

In order to analyse short- and long-term responses for each treatment, 3 control and 3 heat-stressed berries per cluster (25 clusters per condition) were sampled in the evening (7:00 p.m.) at 3 different time points after treatment (1, 7, and 14 d), immediately frozen in liquid nitrogen and stored at −80°C. To obtain 3 experimental replicates, each of the 3 berries collected per cluster was used to constitute 3 independent groups of 25 berries for each condition. The pool of deseeded berries from each group was used as a biological replication and underwent independent RNA extractions.

For the biochemical analysis conducted around harvest, 3 biological replicates were prepared as described above, with berries collected from the same 25 fruiting cutting used for each treatment. Then, frozen berries (5 berries per replicate) were slightly thawed and separated quickly into skin, pulp, and seed. The skin and pulp were immediately ground into fine powder in liquid nitrogen using mortar and pestle.