Experimental treatments and growth conditions
Seeds of Brassica napus L. (cv. Aviso) were germinated in vermiculite in October 2016 under greenhouse conditions. After 5 weeks the seedlings were transplanted into pots containing perlite and vermiculite (2:1, v/v) for 7 weeks and seedlings were grown as described in Poisson et al. (2019). Afterwards, seedlings were subjected to a 12-week period of vernalization in a climatic chamber maintained at 4°C (night) and 8°C (day) with artificial light during the day (10h day/14h night) and supplied with a 25% Hoagland solution without N or S. Then the plants were transferred into the greenhouse (Caen, France, 49°11′09 N, 0°21′32 W) and subjected to a thermoperiod of 20°C (day) and 15°C (night) without additional light. The plants were manually provided with two N applications with an NH4NO3solution: 100 units (kg N/ha) at the end of vernalization (GS30, stem elongation, Lancashire et al. 1991) and 50 units of N at (GS60, bud formation) assuming a plant density of 40 plants.m-2. The two contrasting S supplies i.e. high S (HS) and low S (LS) were manually applied at the end of vernalization (GS30) as usually provided in the field. Plants were supplied with a solution of MgSO4 containing 75 (HS) and 25 (LS) units (kg SO3/ha), which represent, respectively, 100% and 33% of the conventional supply. Four temperature modalities (T-modalities) were applied to plants at stage GS72 (i.e. 20% of the siliques having reached their maximum size) for 17 days. The T-modalities were designed to assess the effects of a mild temperature event prior to a more intense temperature event including daily heat peaks (Figure 1a, b). Our complete design allowed testing of the effects of three single-event sequences (SESs, mod2, mod3 and mod5) and of one combined-events sequence (CES, mod1): (i) an early period of mild warming (SES mod2, 25.3°C ±1.8 /21.7°C ± 0.7 day (16h) /night (8h) applied for 5 full days) (ii) late heat peaks (SES, daily 31.4°C ±1.7 applied during the period of mild warming for 5 hours over 3 days -mod5- or 4 days-mod3) and (iii) the CES combining a mild warming period for 5 full days followed by a 5-day natural thermoperiod (mod1, mean, maximum and minimum temperatures being 15.7°C ±2.7, 28.4°C and 11.5°C, respectively) and eventually a 7-day mild thermoperiod including 4 heat interrupted by 3 day without a peak. The temperature intensities were chosen according to previous studies that used similar ranges to mimic mild and intense temperature treatments under controlled conditions (Aksouh-Harradj et al. 2006). Temperatures were recorded hourly with temperature probes (105T Campbell, Campbell Scientific Ltd., Leicestershire, UK).
Because mixed-age pods were present throughout the temperature sequences, on the day before the beginning of the temperature modalities exposure we labeled the plants’ branches to identify the two categories of pods: (i) pods whose length was above 5 cm (podsL≥5cm) and (ii) pods whose length was shorter than 5 cm (podsL<5cm). Indeed, prior experiments (data not shown) indicated that 5 cm-long pods (or longer) contain seeds that have reached their final fresh weight, which coincides with late seed-filling development and the start of seed maturation (Borisjuk et al. 2013). When the pods started desiccating, they were carefully and individually wrapped with plastic pouches to avoid seed dispersal and the mixing of seeds between the pod categories.