| growth protocol | For the Heliaphen phenotyping platform experiment conducted from May 31st to July 16th. Bleach-sterilized seeds were germinated on Petri dishes with Apron XL and Celeste solutions (Syngenta, Basel, Switzerland) for 3 days at 28C. Germinated plantlets were transplanted in individual pots, and each pot contained two or three plants. Pots were filled with 15 L of P.A.M.2 potting soil (Proveen distributed by Soprimex, Chateaurenard, Bouches-du-Rhone, France) and covered with a 3-mm-thick polystyrene sheet to prevent evaporation. On June 17th, plants were thinned to keep one plant per pot representative of the genotype. Plants were fertilized with 500ml of Peter's Professionnal 17-07-27 (0,6g/l) and Hortilon (0,46g/l) solution. On June 21st, Polyaxe at 5mg/l was applied on foliage against thrips. The XRQ plants were part of a larger experimental design that will not be completely studied in this article. In total, 144 plants, corresponding to 24 genotypes, two drought stress conditions and three replicates were arranged in a split-split-plot design with three blocks. The stress intensity (i.e., FTSW) was the main factor within the block, the genotype was the second factor. Each pot was fertilized and irrigated as in Rengel et al., (2012) before the beginning of the water stress application. For this study, we retained only the six XRQ plants (three irrigated and three non-irrigated plants). Pots were saturated with water on July 5th 2013, 35 days after germination. The following morning, excessive water was drained for two hours and pots were weighed to obtain the saturation mass. On July 8th, irrigation was stopped for stressed plants. Both control and stressed plants were weighed three or four times per day by the Heliaphen robot to estimate evapotranspiration. The water lost was added back automatically by the robot to control plants. Pairs of stressed and control plants were harvested when the FTSW of the stressed plant reached 0.1 which happened between July 12th to 16th. Leaves were harvested between 11:00 and 13:00. To select the leaf to harvest, the total leaf number was estimated, and leaves at the rank position corresponding to two thirds of the total leaf number from the bottom were tagged and termed the nth leaf. For transcriptomic studies, we selected the n+1 leaves. |
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