Lethicia Magno

Massuia De Almeida

Mathias Coulon

Jean-Christophe Avice

Annette Morvan-Bertrand

Jean-Jacques Bessoule

Marina Le Guedard

Tae Hwan Kim

Alseny Niare

Alain Mollier

Nadia Bertin

Lethicia Magno Massuia De Almeida

Annette Morvan- 5 Bertrand

Marina Le Guédard

Mollier

Sophie Brunel-Muguet
email: sophie.brunel-muguet@unicaen.fr

Effects of two-generational heat stress exposure at the onset of seed maturation on seed yield and quality in Brassica

Keywords: intergenerational effect, oilseed rape, seed quality, high temperature, repeated stresses, 20 priming, stress memory, thermotolerance, sulphur. 21 22 sulphur, C: carbon, N: nitrogen, FA: fatty acid, UFA: unsaturated FA, SFA

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction 1

Crop adaptation to ever-changing environment has become one of the major issues for agriculture to 2 ensure food safety and maintain or improve the quality of harvested products. The Intergovernmental 3 Panel on Climate Change report [START_REF] Hoegh-Guldberg | Impacts of 1.5 o C Global Warming on Natural and Human 12 Systems[END_REF] indicates that extreme events, such as 4 heat waves, are expected to become more frequent, to last longer and to increase in intensity (Christidis 5 et al., 2015;[START_REF] Trnka | Adverse weather conditions for European wheat production will become more frequent 10 with climate change[END_REF]. Therefore the ability to acclimate to repeated and fluctuating 6 environmental stressful conditions has become a challenging objective in crop breeding, not only at the 7 crop cycle but also across generations (Delgado et from molecular to the whole plant levels. 12 Within reasonable limits, the ability to resist to a lethal temperature stress can be conferred by exposure 13 to a previous mild nonlethal temperature stress, which induce the ability to survive. This response is 14 known as acquired thermotolerance in the case of temperature stress [START_REF] Saidi | Heat perception and signalling in plants: A tortuous path to 29 thermotolerance[END_REF]Sung et al., 15 2003). It underlies that plants are able to store and retrieve information that they have acquired upon an 16 initial exposure to the stress, which defines stress memory [START_REF] Crisp | Reconsidering plant 39 memory: Intersections between stress recovery, RNA turnover, and epigenetics[END_REF]Hilker and Schmülling, 17 2019). This information acts as a priming process with beneficial effects when the stress recurs and it 18 can lead to earlier, more rapid, intense and sensitive responses that help plants to acclimate in changing 19 environments [START_REF] Kinoshita | Epigenetic memory for stress response and adaptation in plants[END_REF]. This concept was originally developed to describe plant 20 defense mechanisms against biotic stresses [START_REF] Dowen | Widespread dynamic DNA methylation in response to biotic stress[END_REF][START_REF] Frost | Plant Defense Priming against 23 Herbivores: Getting Ready for a Different Battle[END_REF][START_REF] Pastor | Primed plants do not forget[END_REF], 21 such that plants undergoing a second attack by pathogens or herbivores have faster and more robust 22 responses than upon the first exposure. Then, this concept has been extended to abiotic stresses in view 23 of other studies which pointed out that repeated mild stressing events, such as drought, cold, heat or salt 24 stresses, could help stimulating stress memory and alleviating negative effects of a stressing event alone. 25 A large body of literature described the mechanisms that contribute to stress memory, which act at 26 different levels of regulation e.g. epigenetic marks, transcriptional priming, primed conformation of 27 proteins, and/or specific hormonal or metabolic signatures that are maintained after the stress exposure 1 [START_REF] Bokszczanin | Perspectives on deciphering mechanisms underlying plant 23 heat stress response and thermotolerance[END_REF][START_REF] Crisp | Rapid recovery 43 gene downregulation during excess-light stress and recovery in arabidopsis[END_REF][START_REF] Groot | Effects of Multi-Generational Stress Exposure and Offspring Environment on the 30 Expression and Persistence of Transgenerational Effects in Arabidopsis thaliana[END_REF][START_REF] Hatzig | Drought stress has 3 transgenerational effects on seeds and seedlings in winter oilseed rape (Brassica napus L.)[END_REF][START_REF] Wang | Heat Priming Induces Trans-20 generational Tolerance to High Temperature Stress in Wheat[END_REF]. 2

In the case of heat, it has been found that plants exposed to short periods of mild heat early in their 3 development can later tolerate normally-lethal high temperatures, which is likely to be explained by 4 epigenetic based regulatory mechanism [START_REF] Sung | Acquired tolerance to temperature extremes[END_REF]. 5

At the plant cycle level, heat stress memory (i.e. intra-generational or somatic memory) has been 6

observed in several crops. In bread wheat, pre-anthesis high temperatures improved carbohydrates 7 remobilization and grain starch accumulation when they faced post-anthesis intense heat stress (Wang 8 et al., 2014) or heat shock pre-treatment during germination reduced seed yield losses in post-anthesis 9 heat stressed plants [START_REF] Zhang | Physiological and 32 transcriptional analyses of induced post-anthesis thermo-tolerance by heat-shock pretreatment on 33 germinating seeds of winter wheat[END_REF]. In maize, exposure to high temperature at the seedling stage 10 was demonstrated to prevent from permanent damage on plants later subjected to a short heat shock 11 [START_REF] Sinsawat | Effect of heat stress on the photosynthetic 43 apparatus in maize (Zea mays L.) grown at control or high temperature[END_REF]. In winter oilseed rape, effects of heat priming applied at the onset of seed 12 maturation led to higher seed nitrogen content and seed desiccation tolerance for plants later exposed to 13 heat peaks [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF]. 14

Beneficial effects of priming have also been investigated across generations to assess whether positive 15 effects of a pre-acclimation treatment applied on plants could pass on to the offspring (i.e. inter-16 generational memory when the memory effect is observed only in the first stress-free generation, or 17 transgenerational memory when observed in at least two stress-free generations, Lämke and Bäurle, 18 2017). To date, few studies have demonstrated the effects of abiotic stresses across several generations 19 and how stress memory can be transmitted to offspring [START_REF] Crisp | Reconsidering plant 39 memory: Intersections between stress recovery, RNA turnover, and epigenetics[END_REF][START_REF] Hatzig | Drought stress has 3 transgenerational effects on seeds and seedlings in winter oilseed rape (Brassica napus L.)[END_REF]; Kinoshita 20 and Seki, 2014; [START_REF] Kumar | Epigenetic memory of stress responses in plants[END_REF][START_REF] Molinier | Transgeneration memory of stress in plants[END_REF][START_REF] Wang | Heat Priming Induces Trans-20 generational Tolerance to High Temperature Stress in Wheat[END_REF]. Most of them characterized 21 responses of Arabidopsis thaliana as in [START_REF] Whittle | Adaptive epigenetic memory of 23 ancestral temperature regime in Arabidopsis thaliana[END_REF], where high temperature on parental and 22 F1 generations resulted in accelerated flowering and in increased seed yield for plants exposed to heat 23 in the F3 generation. Likewise, accelerated flowering was also observed in F5 Arabidopsis plants after 24 two stress-free generations [START_REF] Suter | Environmental Heat and Salt Stress Induce Transgenerational Phenotypic 7 Changes in Arabidopsis thaliana[END_REF] and transgenerational effects of mild heat stress 25 were observed over two generations of heat-stressed Arabidopsis plants on seed yield (Groot et al., 26 2017). However, studies of multi-generational heat stress exposure were much less investigated in 27 valuable crops. In bread wheat, heat priming applied at the pre and post-anthesis stages to the first 1 generation of plants resulted in greater grain yield and photosynthesis related parameters in the next 2 generation of plants challenged with post-anthesis stress [START_REF] Wang | Heat Priming Induces Trans-20 generational Tolerance to High Temperature Stress in Wheat[END_REF]. More transgenerational 3 studies on crops were designed to assess the effects of other abiotic stresses, such as heavy metals in 4 rice (enhanced tolerance to mercury of Progenies from stressed Mother plants, [START_REF] Ou | 14 Transgenerational Inheritance of Modified DNA Methylation Patterns and Enhanced Tolerance 15 Induced by Heavy Metal Stress in Rice (Oryza sativa L.)[END_REF] and 5 drought in oilseed rape (positive effects on seedling vigor of seeds from stressed Mother plants, Hatzig 6 et al., 2018), or biotic stresses such as herbivory attacks in tomato plants (decreased caterpillars growth 7 on the subsequent generations from parents subjected to caterpillar feeding, [START_REF] Rasmann | Herbivory in the previous generation primes plants for enhanced insect 24 resistance[END_REF]. 8

Because oilseed rape plants display indeterminate growth, different processes will be impacted by heat 9 stress according to its earliness of occurrence during the reproductive stage. While heat stress at 10 flowering limits pollination [START_REF] Sage | The 26 effect of high temperature stress on male and female reproduction in plants[END_REF] and/or induces early pod abortion resulting in yield losses 11 [START_REF] Morrison | Heat stress during flowering in summer Brassica[END_REF][START_REF] Young | High temperature stress of Brassica napus 29 during flowering reduces micro-and megagametophyte fertility, induces fruit abortion, and 30 disrupts seed production[END_REF], heat stress that occurs during seed filling and 12 maturation affects seed storage compounds qualitatively and quantitatively , leading to seed quality 13 alteration [START_REF] Baux | Insights into temperature 14 effects on the fatty acid composition of oilseed rape varieties[END_REF][START_REF] Brunel-Muguet | Heat stress during seed filling interferes with sulfur restriction 31 on grain composition and seed germination in oilseed rape (Brassica napus L.)[END_REF][START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF]. In addition, sulphur (S) 14 nutrition impacts yield components and seed quality in Brassica species because of their high S 15 requirements [START_REF] Scherer | Sulphur in crop production -Invited paper[END_REF]Postma et al., 1999). Sulphur not only contributes to the synthesis and the 16 signaling of stress tolerance-controlling phytohormones [START_REF] Hasanuzzaman | Interaction of sulfur with phytohormones and signaling molecules in conferring abiotic 41 stress tolerance to plants[END_REF], but it might also 17 be associated to epigenetic regulatory mechanisms of thermotolerance [START_REF] Bokszczanin | Perspectives on deciphering mechanisms underlying plant 23 heat stress response and thermotolerance[END_REF], such 18 as DNA methylation which implies S-adenosylmethionine as a donor of methyl groups (Meng et al., 19 2018). 20

In the present study, we analyzed the effects of heat stress on the quality of seeds collected from the 21 second generation of stressed plants (F2, Progeny), also exposed to the same stressing events as the ones 22 applied to the first generation (F1, Mother). The underlying hypothesis is that the history of stressful 23 events experienced by the Mother plants would determine the performances of future generations. 24

Consequently, the monitoring of temperature sequences in terms of stress intensity, duration and 25 frequency, could be a promising strategy to design transmissible thermotolerance protocols, along with 26 adequate S supply in Brassica species.

Materials and Methods 2

Experimental treatments and growth conditions 3

Mother plants (cv. Aviso) were grown under greenhouse conditions in 2016-2017 as described in Magno 4 et al. (2021), . They were subjected to five different temperature modalities (Mother-modalities) for 17 5 days from stage GS72. F1 seeds were provided by the Center of Biological Resources BrACySol 6 (Brassica-Allium-Cynara-Solanum), supervised by the INRAE Center of Rennes (Brittany, France). For 7 the purpose of our intergenerational study, Progeny plants (from F1 seeds) were grown under similar 8 conditions as the ones applied to the Mother plants from October 2018 until July 2019, which included 9

two N applications at the end of vernalization and flowering (100 kg N/ha and 50 kg N/ha respectively) 10 and a single S application at the end of vernalization (75 kg SO3/ha for the High Sulphur treatment and 11 25 kg SO3/ha for the Low Sulphur treatment). In the same way as for the Mother plants (F1), Progeny 12 plants (F2) were subjected to five different temperature modalities (Progeny-modalities) as illustrated 13 in Figure 1a. The complete design resulted in 16 Mother x Progeny stress combinations (Figure 1b), that 14 is, 64 plants for each Sulphur treatment. Because the F1 seeds from the 4LHP Mother-modality were 15 the most negatively impacted, they were not used as a Mother-modality in the Mother x Progeny 16 combinations. 17

Briefly the different temperature modalities aimed at highlighting any beneficial priming effect of an 18 early mild stress prior to later heat peaks over the crop cycle [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF]. Therefore, we applied 19 similar modalities on the Progeny plants as follows: (i) the Control modality with natural thermoperiod 20 conditions, (ii) the Early Mild Stress (EMS) modality that included 5 mild stress days [i.e. 25.8°C ±1.4 21 (day, 16h)/22.2°C ± 1.2 (night, 8h)] followed by 12 days under natural thermoperiod (mean, maximum 22 and minimum temperatures being 16.6°C ±2.4, 20.8°C and 12.4°C, respectively), (iii) the 3 Late Heat 23 Peaks (3LHP) modality that included 14 days under natural thermoperiod followed by 3 mild stress days 24 with one daily heat peak applied for 5 hours (i.e. 31.1°C ±2.6 between 10 am until 3 pm), (iv) the 4 Late 25

Heat Peaks (4LHP) modality that included 10 days under natural thermoperiod followed by 7 mild stress 26 days with one daily heat peak on the last 4 days, and (v) the Priming modality which is the combination 1 of the EMS and the 4LHP modalities. developmental stage at the beginning of the temperature modalities exposure. Indeed, due to their 14 indeterminate growth, oilseed rape plants displayed mixed-aged pods over the 17 day-temperature 15 treatments. Therefore, to avoid any age effect over temperature effect, we collected two categories of 16 pods separately: pods whose length was greater than 5 cm (podsL≥5cm), and pods whose length was 17 shorter than 5 cm (podsL<5cm) following the protocol set up in [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF]. When the pods started 18 desiccating, each branch was wrapped with plastic pouches to avoid the mixing of seeds between the 1 different pod categories. 2 3

Seed yield and components 4

After freeze-drying, the seeds from the two categories of pods were weighed for dry weight (DW) 5 measurements. To determine the Thousand Seed Weight (TSW), we photographed the seeds so as to 6 score their number using image analysis algorithms (ImageJ Software, [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. 7 8

Biochemical characteristics of seeds from podsL≥5cm 9

In the following sections, biochemical characteristics of each individual plant were measured solely on 10 seeds from podsL≥5cm. Three homogeneous plants out of the four replicates were harvested and used to 11 perform the analyses (n=3). After harvest, seeds from podsL≥5cm of each individual plant were pooled 12 and lyophilized for 48h. Then, seeds were manually ground and the resulting powder was used to 13 perform the biochemical analyses, in different amounts as indicated in the following detailed protocols. 14 15

Seed C/N/S concentrations and protein content 16

Seed powder (around 3 mg per plant) was placed into tin capsules for elemental analysis. The percentage 17 of carbon, nitrogen and sulphur was determined with a C/N/S analyzer (EA3000, Euro Vector, Milan, 18

Italy) linked to a continuous flow isotope mass spectrometer (IRMS, Isoprime, GV Instrument, 19

Manchester, UK). Protein content was calculated using the nitrogen-to-protein conversion factor of 5.5 20 [START_REF] Ezeagu | Seed protein 18 contents and nitrogen-to-protein conversion factors for some uncultivated tropical plant seeds[END_REF]. 21 22

Seed fatty acid concentrations and profiles 23

Oil content and fatty acids profiles were determined as described in [START_REF] Marchand | Trace element bioavailability, yield and seed 41 quality of rapeseed (Brassica napus L.) modulated by biochar incorporation into a contaminated 42 technosol[END_REF]. 24

Approximately 30 mg of seeds powder from each plant were suspended in 1 mL of 25 methanol/toluene/H2SO4 solution (100:20:2.5; v/v) containing C17:0 as internal standard (25 µg mL -1 ), 26 remaining 1h at 80°C for transmethylation. After cooling, 750 µL of hexane and 1.5 mL of water were 27 added, and the hexane phase containing the resulting Fatty Acid Methyl Esters (FAMES) was recovered 1 for gas chromatography analysis combined with flame ionization detection (GC-FID). The FAMES (1 2 µL) were injected into an Agilent 7890A gas chromatograph equipped with a DB-WAX column (15m 3

x 1µm, 0.53 mm, Agilent) and FID system. FAMES were identified by comparing their retention times 4 with those of commercial standards (Sigma, St. Louis, MO) and quantified using ChemStation (Agilent) 5 to calculate the peak areas.

Germination time courses at sub (5°C) and optimal (20°C) temperatures 20

Because the requirements for the germination tests (2 temperatures x 50 seeds per replicate) were 21 substantial, we used the seeds from podsL<5cm which could provide enough material. Their 22 developmental stage during the 17 day-stress period (younger stage compared to seeds from podsL≥5cm) 23 has been considered for the results interpretation. For each tested temperature, 3 replicates of 50 seeds 24 of each Progeny plants were disposed homogeneously in a Petri dish, on a double layer of blotting paper 25 and with 10 mL of osmotic water [START_REF] Ling | Cold plasma treatment enhances 32 oilseed rape seed germination under drought stress[END_REF]. The Petri dishes were sealed with parafilm to 26 maintain constant moisture. They were put in a growth chamber (Binder KBWF 720, BINDER GmbH, 27 Tuttlingen, Allemagne), monitored at 5 or 25°C and 80% of humidity. Both temperatures were chosen 1 to track the germination time courses under a sub-optimal temperature (i.e. 5°C) and an optimal 2 temperature (i.e. 25°C). Observations and scoring of germinated seeds were regularly carried out in the 3 light, as prior tests were performed to ensure that the seeds were not photosensitive (data not shown). 4

The Gompertz function was fitted to the germination rates for each replicate batch of 50 seeds: 5

G(t) = a exp(- b c . exp -ct) 6
Were G(t) is the germination percentage at t time (in hour) after sowing. a, b and c are three parameters 7 of the model: a refers to the maximum germination rate, b and c are shape parameters of the simulated 8 curve [START_REF] Brunel | Using a 34 model-based framework for analysing genetic diversity during germination and heterotrophic 35 growth of Medicago truncatula[END_REF]. This function is an adjustment model usually used to simulate the growth of 9 living organisms [START_REF] Brunel | Using a 34 model-based framework for analysing genetic diversity during germination and heterotrophic 35 growth of Medicago truncatula[END_REF][START_REF] Dantigny | Mould germination: Data treatment and 4 modelling[END_REF]. Then germination times were deduced and 10 used to compare Progeny-plants germination behavior. Statistical analyses were performed with Rstudio 11 (R version 4.0.3 Rstudio, PBC) for the final germination rates and for 3 germination times (i.e. T25, T50 12 and T75, which correspond to the time to observe 25, 50 and 75% of germinated seeds respectively). 13 14

Statistical analysis 15

Both sulphur conditions (i.e. High Sulphur and Low Sulphur) results were analyzed separately. To 16 highlight the intergenerational effects, two-way ANOVAs to test the Mother and Progeny temperature 17 stress (i.e. the Mother-modalities and the Progeny-modalities) effects were performed on the measured 18 variables, both considered as independent factors. Residues independence and normality, and 19 homogeneity of variances were tested before performing the ANOVAs (using Dubin-Watson, Shapiro-20

Wilk and Bartlett tests respectively). Mother-modalities, Progeny-modalities and Mother x Progeny 21 interactions effects were analyzed using R software (version 4.0.2, R Core Team, 2020), following a 22

Type III two-way ANOVAs. To have a complete and balanced model, the [Mother-control x Progeny-23 control] combination has been left out, as the Mother-control was only combined to Progeny-control. 24

As interaction effects were not observed in this experiment, Tukey tests were performed for the 25 significant factor (i.e. Mother and/or Progeny temperature stress effect) among the 5 Progeny-modalities 26 or among the 4 Mother-modalities (Tables 1, 2, 3 and 4), also taking in account the [Mother-control x 27 Progeny-control] treatment. In addition, we performed mean comparison tests among Progeny-1 modalities for a given Mother-modality (Supplemental Data, Tables 1 and2). Different Progeny-2 modalities rankings mean that the Mother-modality modulates the effect of Progeny-modality. 3 4

Results 5

Because S supply is a secondary factor in this study, the results under the Low Sulphur condition are 6 commented after the results under High Sulphur condition, and the tables and graphs are given in 7 Supplemental Data. In doing so, we meant to simply observe to what extent S limitation modifies the 8 plant performances under heat stress. 9 10

Yield components 11

Table 1 displays total yield and yield components (i.e. seed number and TSW) for the whole plant seeds 12 and for both categories of seeds collected separately (from podsL≥5cm and podsL<5cm) under the High 13 Sulphur condition. No significant Progeny x Mother interaction effect was observed for any of the yield-14 related variables. Total seed yield among the Progeny-modalities ranged from 5.53 g plant -1 (Progeny-15 priming) to 7.92 g plant -1 (Progeny-control) with a significant Progeny effect (p<0.001). The decrease 16 in the total seed yield was only significant for the Progeny-priming (-30% relative to Progeny-control) 17 as a consequence of the seed yield penalties for podsL<5cm which was important in this treatment (-43% 18 relative to Progeny-control) and globally affected by all the Progeny-modalities (p<0.001). A significant 19

Progeny effect was also observed on TSW for total pods and both pod categories. Decreases in the TSW 20 were observed for all the Progeny-modalities compared to the Progeny-control, with the Progeny-21 priming modality being the most penalizing modality (-29%, -32% and -27% for total pods, podsL<5cm 22 and podsL≥5cm respectively from the Progeny-control value). No Mother effect was observed except for 23 TSW from podsL≥5cm (p<0.05), with the Mother-EMS being 22% lower than the Mother-control. 24

Regarding the Progeny-modalities ranking for a given Mother-modality under High Sulphur condition 25 (Supplemental Data, Table 1), no difference in rankings were observed for total seed yield nor total 26 TSW, as for every Mother-modality the Progeny-priming and the Progeny-control were respectively the 27 most and the less penalizing modalities. For seeds from podsL≥5cm differences can be observed for 1

Mother-3LHP, with Progeny-control having the lowest values (along with Progeny-priming and 2

Progeny-EMS for seed yield from podsL≥5cm). For seeds from podsL<5cm, different rankings among 3 Progeny-modalities were observed for seed yield and TSW i.e. lowest seed yield for Progeny-priming 4 under Mother-EMS and Mother-priming only, and lowest TSW for Progeny-priming under Mother-5 3LHP and Mother-priming only. 6

As observed under the High Sulphur condition, no significant Progeny x Mother interaction effect was 7 observed for any of the yield-related variables under Low Sulphur condition (Supplemental Data, Table 8 3). Total seed yield among the Progeny-modalities ranged from 5.60 g plant -1 (Progeny-priming) to 7.25 9 g plant -1 (Progeny-control) with a significant Progeny effect (p<0.001). The decrease of seed yield under 10 the temperature modalities resulted from decreased yield of seeds from podsL<5cm (p<0.01). Seed number 11 (i.e. total, podsL<5cm and podsL≥5cm) were not impacted by the Progeny nor by the Mother modalities, 12 although the total seed number displayed a significant Mother effect (p<0.01) with the Mother-priming 13 having the highest value (+30% from the Progeny-control value). A significant Progeny effect was 14 observed on TSW for total pods and both pod categories (p<0.001) and a Mother effect was also 15 observed for total pods and podsL<5cm (p<0.001, Supplemental Data, Figure 1). Both Progeny and Mother 16 effects led to decreased TSW with the Mother-priming and Progeny-priming being the most penalizing 17 sequences (Supplemental Data, Table 3). Regarding the Progeny-modalities ranking for a given Mother-18 modality under Low Sulphur condition (Supplemental Data, Table 2), differences in rankings were 19 observed for total seed yield under Mother-EMS and Mother-3LHP, with the Progeny-priming being 20 the most penalizing modality. The Progeny-priming modality was also the most impacting on TSW 21 whatever the Mother-modalities. As for the total seed number, Progeny rankings were different only 22 under the Mother-priming modality, with Progeny-EMS having the lowest value. For both pods 23 categories, the Mother-priming and Mother-3LHP modalities impacted the Progenies ranking for TWS 24 while the Mother-EMS modality only affected TSW of seeds from podsL≥5cm. 25 26

Nutritional seed quality criteria 27

Seed C and S concentrations 1

Under High Sulphur condition, the seed C and S concentrations (Table 2) were slightly impacted by 2

Progeny-modalities (Table 2, p<0.05 and p<0.01, respectively), but neither Mother nor Progeny x 3 Mother interaction effects were not observed. The Progeny-priming modality displayed the lowest C 4 concentration (5% lower than the Progeny-control), whereas the Progeny-EMS modality displayed the 5 highest value. Adversely, the seeds from the Progeny-priming modality plants displayed the highest 6 seed S concentration (32% higher than the Progeny-control). Regarding the Progeny-modalities ranking 7

for a given Mother-modality under High Sulphur (Supplemental Data, Table 1), rankings among 8

Progeny-modalities differed under Mother-3LHP and Mother-EMS modalities. For C concentration, 9

while Progeny-control displayed the highest value among the Progeny-modalities for the Mother-3LHP, 10 its value was the lowest among the Progeny-modalities for the Mother-EMS. 11

As observed under the High Sulphur condition, only a Progeny effect was observed on C and S 12 concentrations under the Low Sulphur condition (Supplemental Data, Table 4). Similarly, the Progeny-13 priming displayed the lowest C concentration (-7% from the Progeny-control). By contrast, while the 14 Progeny-priming significantly displayed the highest value in S under High Sulphur condition, the Low 15 Sulphur condition alleviated the increase under the Progeny-priming and negatively impacted the value 16 under the Progeny-EMS (-19% from the Progeny-control). Regarding the Progeny-modalities ranking 17 for a given Mother-modality under Low Sulphur (Supplemental Data, Table 2), the Progeny-priming 18 was the most negatively impacted modality regarding the C concentration under the Mother-EMS and 19

Mother-3LHP modalities, while it was the Progeny-EMS under the Mother-priming modality. For S 20 concentration, the Progeny-modalities were significantly ranked only under Mother-EMS, with the 21 Progeny-EMS displaying the lowest value (-36% from the Progeny-control). 22 23

Seed N and total protein concentrations 24

Table 2 displays seed N and protein concentrations. Similar to C and S, the seed N and protein 25 concentrations were only affected by Progeny-modalities (p<0.001). The highest N and protein 26 concentration values were observed for the Progeny-priming modality (+24% compared to Progeny-27 control). These observations lead to conclude that the early mild stress had a positive effect over the late 1 heat peaks, which suggested an alleviating effect. Rankings among the Progeny-modalities under High 2 Sulphur condition (Supplemental Data, Table 1) for N concentrations were not changed whether the 3 Mother-modality, with Progeny-priming and Progeny-control seeds having the highest and the lowest 4 values respectively. 5

As observed under the High Sulphur condition, only a Progeny effect was observed to N and protein 6 concentrations under the Low Sulphur condition (Supplemental Data, Table 4), with the Progeny-7

priming modality displaying the highest values (+12% compared to Progeny-control). Rankings among 8

the Progeny-modalities under Low Sulphur condition (Supplemental Data, Table 2) for N concentrations 9

were not changed for the Mother-EMS, while Progeny-priming displayed the highest values under 10

Mother-priming and Mother-3LHP. 11 12

Fatty acids concentration and profiles 13

Total FAs, saturated FAs (SFAs) and unsaturated FAs (UFAs) concentrations under High Sulphur 14 condition are displayed in Table 2. The 3 variables were strongly impacted by the Progeny-modalities 15 (p<0.001), but no Mother nor Progeny x Mother interaction effects were observed. Total FAs ranged 16 from 11.00 %DW (Progeny-priming) to 28.67 %DW (Progeny-control) among the Progeny-modalities 17 and decreases in total FAs concentration were observed on seeds from all the heat stressed plants 18 compared to Progeny-control, although only the Progeny-priming mean was significantly lower (-61% 19 compared to Progeny-control). The SFAs (including C16:0, C18:0, C20:0, C22:0) concentrations ranged 20 from 1.90 %DW (Progeny-priming) to 2.51 %DW (Progeny-3LHP) and the UFAs (including C16:1, 21 C18:1, C18:2, C18:3, C20:1) concentrations ranged from 15.11 %DW (Progeny-priming) to 26.26 22 %DW (Progeny-control). Overall, the Progeny-priming modality had the greatest impact on decreasing 23

FAs concentration, as a result of both SFAs and UFAs decreased concentrations, meaning that the early 24 mild stress event did not alleviate the negative effects of the later heat peaks. These decreases are in line 25 with increased proteins concentration as usually observed in oilseed crops for which oil and protein 26 contents are inversely proportional. The ω6:ω3 ratio (i.e. C18:2/C18:3 ratio) is commonly used as an 27 indicator of the health quality of vegetable oils. This ratio was significantly impacted by the Progeny 1 and Mother-modalities (p<0.001 and p<0.05, respectively), but no significant Progeny x Mother 2 interaction effects were observed (Table 2 and Figure 2). The lowest ratios were observed on both 3

Mother-control and Progeny-control modalities. The highest ω6:ω3 ratios were observed for the 4 Progeny-priming (+43% compared to Progeny-control) and for the Mother-EMS (+18% compared to 5

Mother-control). These results indicated that the number of desaturations decreased with greater 6 duration of stress exposure. Whatever the Mother-modality, ranking among Progeny-modalities under 7

High Sulphur (Supplemental Data, Table 1) remained unchanged for the total FAs, SFAs and UFAs 8 concentrations, with the Progeny-priming being the most impacting modality (lowest values). Similarly, 9 extreme rankings among Progeny-modalities for the ω6:ω3 ratio remained unchanged with Progeny-10 priming and Progeny-control having the highest and the lowest values whatever the Mother-modality. the treatments (including the control). P-values and levels of significance are given on Table 2. EMS: 4

Early Mil Stress modality, 3LHP: 3 late heat peaks modality, 4LHP: 4 late heat peaks modality. 5 6

As observed under the High Sulphur condition, only a Progeny effect was observed on FA, SFA and 7

UFA concentrations under the Low Sulphur condition (Supplemental Data, Table 4), with the Progeny-8 priming displaying the lowest values (-44%, -24% and -46% compared to Progeny-control, 9 respectively). In addition, the Progeny-EMS also led to significant decrease (-18%, -12% and -19% 10 compared to the Progeny-control, respectively). Under Low Sulphur, the ω6:ω3 ratio was only affected 11 by Progeny-modalities (unlikely to the High Sulphur condition, for which a significant Mother effect 12 was observed). As under High Sulphur condition, the Progeny-control displayed the lowest values and 13 the Progeny-priming displayed the highest values (+49% compared to Progeny-control). Rankings 14 among the Progeny-modalities under Low Sulphur condition (Supplemental Data, Table 2) for FA, SFA 15 and UFA were not changed whatever the Mother-modality, with Progeny-priming having the lowest 16 values, and the Progeny-control and the Progeny-3LHP modalities having the highest values. 17

Physiological seed quality-related criteria 19

Soluble sugar ratio as indicator of desiccation tolerance 20

Table 3 gives the concentrations of three soluble sugars found in seeds of oilseed rape (i.e. sucrose, 21 raffinose and stachyose) under High Sulphur condition. For all of them, only Progeny effects were 22 observed (p<0.01 for sucrose and raffinose, and p<0.001 for stachyose). Sucrose concentrations ranged 23 from 2.83 %DW (Progeny-priming) to 3.98 %DW (Progeny-4LHP). The late heat peaks tended to 24 increase the sucrose concentration, as the Progeny-3LHP and Progeny-4LHP modalities displayed the 25 highest values. The lowest sucrose concentrations were observed in the Progeny-priming modality (-26 12% than in the Progeny-control). Raffinose and stachyose concentrations were the highest in the 27 Progeny-control and Progeny-EMS modalities. Under the Progeny-priming modality, raffinose and 1 stachyose concentrations were respectively 25% and 53% lower than under the Progeny-control. The 2

[raffinose+stachyose]:sucrose ratio was used as an indicator of drying tolerance i.e. the higher the value, 3 the more tolerant to desiccation [START_REF] Bailly | 7 Changes in oligosaccharide content and antioxidant enzyme activities in developing bean seeds 8 as related to acquisition of drying tolerance and seed quality[END_REF]. Results ranged from 0.21 (Progeny-priming) to 4 0.39 (Progeny-EMS) (Table 3), thus indicating indicate that whatever the Mother-modality, the 5 Progeny-priming modality was the most negatively impacted for the acquisition of seed desiccation 6 tolerance. When comparing the Progeny-modalities for a given Mother-modality under High Sulphur 7

(Supplemental Data, Table 1), the lowest [raffinose+stachyose]:sucrose ratio was obtained under the 8

Progeny-priming for all the Mother-modalities, but the highest ratio was not obtained for the same 9

Progeny-modality i.e. the 4 other Progeny-modalities (including Progeny-control) for the Mother-10 priming but only the Progeny-EMS for the Mother-EMS and the Mother-3LHP. 11

As observed under the High Sulphur condition, only a Progeny effect (p<0.001) was observed to the 12 three soluble sugars and the [raffinose+stachyose]:sucrose ratio under Low Sulphur condition 13 (Supplemental Data, Table 5), with the Progeny-priming modality displaying the lowest values. 14

Similarly, the late heat peaks tended to increase the sucrose concentration, as the Progeny-3LHP and 15

Progeny-4LHP modalities displayed the highest values. The rankings among the Progeny-modalities 16 under the Low Sulphur condition were almost similar to those observed under the High Sulphur 17 condition, thus leading to conclude that the thermal stress is far more impacting on soluble sugars than 18 sulphur limitation as applied in our experimental conditions (Supplemental Data, Table 5). When 19 comparing the Progeny-modalities for a given Mother-modality under Low Sulphur (Supplemental 20 Data, Table 2), the lowest [raffinose+stachyose]:sucrose ratio was obtained under the Progeny-priming 21 for all the Mother-modalities. 22 23

ABA:GA3 ratio as indicator of temperature-induced seed dormancy 24

Because the dynamics of ABA and GA3 controls the balance between dormancy and germination, the 25 ABA:GA3 ratio was used as a proxy for seed dormancy under stress condition i.e. a high ABA:GA3 26 ratio indicates increased secondary seed dormancy, which is induced by thermo-inhibition (Debeaujon 27 and Koornneef, 2000;[START_REF] Finkelstein | Abscisic Acid Synthesis and Response[END_REF]. Table 3 displays the values of the ABA:GA3 ratio under High 1 Sulphur condition. As expected, under high temperature this ratio increased, but only Progeny effects 2 were observed (p<0.01). The ABA:GA3 ratio ranged from 0.44 (Progeny-3LHP) to 1.66 (Progeny-3 priming), thus indicating that the most negatively impacting modality (highest ratio) corresponded to 4 the longest cumulated heat days during the Progenies generation. Rankings were different according to 5 the Mother-modality under High Sulphur (Supplemental Data, Table 1). Significant lower values of the 6 ratio were observed for Progeny-4LHP and Progeny-3LHP under Mother-priming, and for Progeny-7

EMS and Progeny-control under Mother-EMS. 8

Similar to the High Sulphur condition, only a Progeny effect (p<0.01) was observed to the ABA:GA3 9 ratio under Low Sulphur condition (Supplemental Data, Table 5). When comparing the Progeny-10 modalities for a given Mother-modality under Low Sulphur (Supplemental Data, Table 2), different 11 rankings can be observed. While under Mother-priming the highest and lowest values were observed on 12

Progeny-3LHP and Progeny-priming respectively, under Mother-EMS the highest and lowest values 13 were observed to Progeny-control and Progeny-EMS respectively, and no differences among the 14 Progenies were observed under the Mother-3LHP. 15 16

Germination capacity as indicator of seed vigor 17

The germination time courses (T25, T50 and T75) and the final rates obtained at optimum (25 °C) and 18 suboptimum (5 °C) temperatures under the High Sulphur condition are presented in Table 4. The final 19 germination percentages were high (over 90%) under both temperatures and only the Progeny effect 20 was significant (p<0.01). The short duration temperature stress (early mild stress or late heat peaks) 21

have not affected the final rate, as the Progeny-EMS, Progeny-3LHP and Progeny-4LHP modalities 22 displayed the highest values. However, under both temperatures (i.e. 25 °C and 5 °C) the Progeny-23 priming displayed the lowest final rates, thus suggesting that the longer the stress exposure, the more 24 negative the impacts on final germination rates. Regarding the time courses, only T25 was significantly 25 affected by the Progeny-modalities at both germination temperatures (p<0.05), with the Progeny-26 priming displaying the lowest values (faster germination). When comparing the Progeny-modalities for 27 a given Mother-modality under High Sulphur (Supplemental Data, Table 6), different Mother effect on 1 Progeny rankings were observed according to the germination temperature. At 25 °C, the Progeny-2 priming modality displayed the lowest final rates whatever the Mother-modality, followed by the 3 Progeny-control. Differences among the Progeny T25 values were only observed under Mother-EMS, 4

with the Progeny-control and the Progeny-priming displaying the highest and lowest values respectively. 5

For T50, differences among the Progeny-modalities were observed under Mother-priming and Mother-6 EMS, with the Progeny-control displaying the highest values. The differences in T75 among the 7 Progeny-modalities were only observed under Mother-priming, with the Progeny-control and the 8

Progeny-3LHP displaying the highest and lowest values respectively. In contrast to the results under 9 optimal temperature, the time courses under 5 °C were only affected under Mother-3LHP, with Progeny-10 EMS displaying the lowest T25, T50 and T75 values. Globally, heat stress under High Sulphur condition 11 accelerated germination time (Figure 3). Although the thermal modalities applied on the Progeny plants 12 were more impacting than those applied on the Mother plants, the history of the Mother plants modified 13 the Progeny-modality effects on the germination time courses and final rates of the seeds from the 14

Progeny plants. 15

Under Low Sulphur condition, no significant Progeny, Mother nor interaction effects were observed for 16

any germination variables at 25 °C, except for T75 which was impacted by the Mother modalities 17 (p<0.05) (Supplemental Data, Table 7). By contrast, under 5 °C T25, T50 and T75 were affected by the 18 Progeny-modalities (p<0.01) and a slight Mother effect (p<0.05) can be observed on T75. The Progeny-19 EMS modality displayed the lowest values of T25, T50 and T75, thus suggesting a faster germination. 20

The final rates were only impacted by a Progeny x Mother interaction effect, thus suggesting that the 21 final germination rate was the result of the stress exposure of both generations. When comparing the 22 Progeny-modalities for a given Mother-modality under Low Sulphur (Supplemental Data, Table 8), 23 different Mother effects were observed. At 25 °C, differences in the final rate among the Progenies were 24 observed under Mother-priming and Mother-3LHP, with Progeny-EMS displaying the highest values in 25 both Mother-modalities. Differences in germination times were observed only under Mother-priming, 26 with the Progeny-priming displaying the lowest values to T25, T50 and T75 (faster germination). At 5 27 °C, the final rates were different under Mother-priming and Mother-3LHP, with lower values for the 1 Progeny-3LHP and the Progeny-control respectively. Differences in T25 and T50 were observed only 2 under the Mother-priming, with Progeny-EMS displayed the lowest values (faster germination). The 3 slowest germination rates (i.e. higher T75) were observed for Progeny late heat peaks modalities 4 irrespective of the Mother-modalities. Globally, heat stress under the Low Sulphur condition accelerated 5 germination to some extent according to the Mother-modalities (Supplemental Data, Figure 8), but these 6 observations were less obvious than under the High Sulphur condition. Regarding yield components, the Progeny effects were observed on yield and TSW from total seeds and 14 seeds from podsL<5cm, and TSW of seeds from podsL≥5cm, with the Progeny-priming being the most 15 penalizing modality under both S conditions. Heat stress during flowering was shown to reduce seed 16 yield as a consequence of alterations of gametogenesis, fertilization and damage on post-fertilization 17 structures in canola [START_REF] Elferjani | Canola responses to drought, heat, and combined stress: 15 shared and specific effects on carbon assimilation, seed yield, and oil composition[END_REF]. Other studies described the trade-off 18 between grains number and grain weight and highlighted a hierarchy in the plasticities of these 19 components such that the grain number was the only component able to adjust to massive stressful 20 changes in contrast with grain weight (wheat, [START_REF] Slafer | Coarse and fine regulation of wheat yield components in 2 response togenotype and environment[END_REF]. By contrast, our results indicated that 21 the number of seeds could not be adjusted anymore at the time of stress exposure whatever the pod 22 categories and that the weight of seeds from podsL≤5cm were the most impacted by stresses, in line with 23 our previous conclusions [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF]. Under Low Sulphur, the other Progeny-modalities also 24 impacted these seed yield components, but to a lesser extent (Table 1 and Supplemental Data, Table 3). 25

The one-off Mother effects were only observed on TSW of seeds from podsL≥5cm under High Sulphur, 26 and total TSW and TSW from podsL<5cm under Low Sulphur. These observations suggest that the Low 27 Sulphur condition applied on the Progeny plants might have delayed flowering and further seed 1 development and maturation, thus leading to Mother effects observed on younger seeds (from podsL<5cm) 2 at the timing of heat stress. They also indicate that two consecutive generations of plants growing in a 3 S limiting environment are more susceptible to high temperature stress, thus leading to lower 4 thermotolerance compared to well-supplied plants. In addition, although Mother effects were weak, 5

Mother-modalities impacted the Progenies performances in response to the Priming treatment in 6 different directions (Supplemental Data, Tables 1 and2). For instance, under the High Sulphur 7 condition, while the Mother-3LHP induced contrasting progeny rankings for seed yield and seed number 8 from podsL≥5cm, the Mother-EMS alleviated the differences among the Progeny-modalities for TSW of 9 seeds from podsL≥5cm (as the Progeny-priming did not differ anymore from the other Progeny-10 modalities). Under the Low Sulphur condition, the effects of the Mother-modalities on the Progeny 11 rankings were also observed although distinct Mother-modalities alleviated or exacerbated the Progeny 12 effects among the Progeny-modalities. The lowest total seed yield was also observed for seeds from two 13 generations of plants challenged with the Priming treatment i.e. which underwent the longest cumulated 14 duration of stressing days, as previously shown at the plant cycle level in [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF]. 15 Similar to the yield-related variables, Progeny effects also prevailed over Mother effects on the 16 concentrations of FAs-related variables and N under both sulphur conditions, as a one-off Mother effect 17 was observed on the ω6:ω3 ratio under the High Sulphur condition (Table 2 and Supplemental Data, 18 In consistence with the observations on yield components and nutritional quality criteria, seed 1 desiccation tolerance, seed dormancy and germination behavior were only impacted by Progeny-2 modalities. For both sulphur conditions, Progeny values pointed out that the longest the cumulated stress 3 duration (over the two generations), the more negative the effects i.e. low [raffinose+stachyose]:sucrose 4 ratio suggesting low desiccation tolerance, and high ABA:GA3 ratio indicating high seed dormancy. 5

Table 4). As observed in

Nevertheless, under Low Sulphur Mother effects were observed on T75 at both germination 6 temperatures along with interaction effects on the final rate at 5°C (Supplemental Data, Table 7). A 7 slight positive effect of short heat stress applied on the Progeny plants (EMS, 3LHP and 4LHP) was 8 observed on the final rate at both temperatures under High Sulphur in contrast to the sulphur limiting 9 conditions, for which the germination times courses were slowed down (Supplemental Data, Table 8, 10 with most prior results, the lowest final germination rates at the optimal temperature were observed for 12 seeds from the modalities combination that matched the longest stress duration over the two plant 13 generations (Supplemental Data, Table 6). 14

In terms of features of the stress events, the shortest duration combination over the two generations, 15 which also matched with the latest events [Mother-3LHP x Progeny-3LHP] tended to be the least 16 penalizing scenario for the yield and FAs-related variables (except for the ω6:ω3 ratio), which was less 17 remarkable for N concentration, soluble sugars and the ABA:GA3 ratio under High Sulphur. As 18 expected, the longest stress duration over the two generations which also matched with the earliness of 19 stress application [Mother-priming x Progeny-priming] tended to be the most negative, except on N 20 concentration. Under the sulphur limiting conditions, these observations slightly differed as a 21 consequence of a delay in the phenology which impacted younger pods than under the non-limiting 22 sulphur condition (e.g. total seed yield). 23 24

Intergenerational thermopriming protocols as climate acclimation strategy 25

One outcome of our study deals with the identification of Mother x Progeny stress combinations that 26 penalize or improve yield and quality of seeds from two stressed generations, hence guiding towards 27 thermotolerance acquisition breeding schemes. Figure 4 displays the values of yield components and 1 nutritional and physiological quality criteria for the 15 Mother x Progeny modalities combinations, 2 expressed as the relative difference to the [Mother-control x Progeny-control] combination (i.e. both 3

Mother and Progeny plants were grown under natural conditions). Similar patterns of responses to these 4 combinations were observed for both sulphur conditions, such that the results will be discussed 5 regardless of the S conditions and specifications will be provided when differences are observed. 6

Regarding yield components, slight increases in total yield were only observed under Progeny-control 7 that derived from Mother-priming or Mother-EMS, which also demonstrate the overruling effect of the 8 Progeny-modalities. The most negative combinations implied either the longest stress duration over the 9 two generations or late stress events which impaired the filling phase of seeds from lately-fertilized 10 flowers. In contrast, the number of seeds increased at the expense of averaged seed weight as usually 11 observed for R-selected species such as the wild relative Arabidopsis that favors the dispersal of a large 12 number of seeds which is not predetermined before the reproductive stage as a consequence of 13 adjustments to later stresses [START_REF] Bennett | The role of the pod in seed development: Strategies 17 for manipulating yield[END_REF]. This trade-off between seed size and number was 14 particularly exacerbated under our stressing conditions as it was also observed under drought stress 15 (sesame, Najafabadi and Ehsanzadeh, 2017; wheat, [START_REF] Slafer | Coarse and fine regulation of wheat yield components in 2 response togenotype and environment[END_REF]). As previously mentioned, grain 16 number appears to be a coarse-tuning stress-adjustment variable where grain weight might to act as a 17 fine-tuning mechanism. 18

Under Low Sulphur, the features of the combinations that led to decreased yield and decreased seed 19 weight gathered late events over the two generations which also matched the most intense events. This 20 was likely explained by the delay and slowdown in the phenology induced by sulphur limitation 21 (although moderate) which extended the seed development period until the late heat peaks event, thus 22 resulting in longer heat stress sensitive window and penalties on later-formed pods and seeds. 23

The total FA concentration was negatively impacted under almost all combinations, with the most 24 pronounced effects being induced by a high cumulated duration (i.e. for Progeny-priming modalities 25 regardless of the history of the Mother plants). Indeed, two combinations under High Sulphur, led to 26 unchanged concentrations i.e. [Mother-EMS x Progeny-EMS] and [Mother-3LHP x Progeny-3LHP] 27 which indicates that the shortest the duration over the two generations, the less impacting. The little 1 impact of an early stress is consistent with the mismatch with timing of seed storage compounds 2 biosynthesis which starts by lipids along with starch [START_REF] Baud | An integrated overview of seed 11 development in Arabidopsis thaliana ecotype WS[END_REF][START_REF] Borisjuk | Seed 27 architecture shapes embryo metabolism in oilseed rape[END_REF]. As 3 expected, the concentrations in SFA benefited from high temperature as a consequence of the 4 impairment in the desaturase enzyme activity [START_REF] Menard | Genome wide analysis of fatty acid desaturation and its 45 response to temperature[END_REF] with logically more pronounced 5 effects upon late events during the Progeny cycle, regardless of the history of the Mother plants. 6 Therefore, increased values of the ω6:ω3 ratio, which is a negative diet characteristic (Simopoulos, 7 2006), were more prone to be observed under the late heat peaks modalities. By contrast, an early mild 8 stress on the Progenies, regardless of the history of the Mother plants, slightly decreased the ω6:ω3 ratio, 9 thus improving this targeted characteristic. As expected, due to the extension of the heat stress sensitive 10 window resulting from sulphur limitation, these negative effects on FAs concentrations were 11 exacerbated especially when late heat peaks were applied over the two generations. By contrast with FA 12 concentrations, the Progeny-priming displayed expectedly the highest N concentrations regardless of 13 the history of the Mother plants, under both sulphur conditions. This effect was more pronounced under 14 High Sulphur. The effective priming effect observed on N concentration on the first stressed plants 15 generation [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF] was not reset under the temperature modalities applied to the offspring, 16

as the values of the different Progeny-modalities from the Mother-priming plants were higher than the 17 [Mother-control x Progeny-control] combination except for seeds from Progeny-control. There might 18 be a slight dilution of the beneficial priming effect, as the value of the seeds from the Priming treatment 19 at the first generation [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF] was higher than the values of the seeds from the [Mother-20 priming x Progeny-control] combination. This dilution effect over successive generations was also 21 observed in [START_REF] Groot | 33 Transgenerational effects of mild heat in Arabidopsis thaliana show strong genotype specificity 34 that is explained by climate at origin[END_REF], where transgenerational effects were usually weaker and less commonly 22 observed than effects observed of the stress applied on the last generation of plants (offspring). 23

The priming effect observed on the first stressed plants generation for the [raffinose+stachyose]:sucrose 24 ratio used as a proxy of seed desiccation tolerance [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF] was not observed on the offspring 25 as lower values were observed for all the combinations compared to the [Mother-control x Progeny-26 control] combination, except for seeds from two generations of plants challenged with early mild stress 27 only under High Sulphur. These results also suggested that the late heat peaks were the most detrimental 1 events to the acquisition of desiccation tolerance under both sulphur supplies, whether they occurred at 2 the first or the second plant generation, as the soluble sugars biosynthesis was likely to overlap the 3 timing of the soluble sugars biosynthesis and/or maturation. 4

Regarding seed S concentrations under High Sulphur, higher values were mostly observed, meaning that 5 heat stress did not impair sulphur allocation to the seeds while the seed weight decreased. Whether the 6 amount of S in the seeds was assimilated or stored under mineral forms remains to be investigated so as 7

to claim the benefits of heat stress on S-containing proteins (including napins sand cruciferins) which 8 are of interest in high protein-oil cakes used for cattle feeding. Seed dormancy was the most impacted 9

by heat stress at both sulphur conditions although in contrasting ways, since relative increased 10 ABA:GA3 ratios were mainly observed under High Sulphur unlike under Low Sulphur. However, 11 several combinations led to reduce the relative ABA:GA3 ratio, thus breaking seed dormancy, but no 12 straightforward features (in terms of timing, duration nor intensity) could be identified. As observed in 13Magno et al. ( 2021), the Progeny-priming led to the highest increases regardless of the Mother-14 modalities under High Sulphur. Low Sulphur had negative impact on the ratio, which was also observed 15 when comparing the [Mother-control x Progeny-control] modalities under both sulphur supplies. 16

However, heat sequences tended to alleviate the negative impact of S limitation as for most combination, 17 the relative ABA:GA3 ratios increased. This might be another consequence of the delay in the phenology 18 thus making the developing seeds more prone to germinate as already observed under heat stress and 19 eventually to undergo pre-harvest sprouting [START_REF] Brunel-Muguet | Heat stress during seed filling interferes with sulfur restriction 31 on grain composition and seed germination in oilseed rape (Brassica napus L.)[END_REF]. 20

Finally, the final germination rate remained almost unchanged under both sulphur conditions. The early 21 mild stress applied on the Mother plants led to a slight decrease in final rates at optimal temperature 22 under High Sulphur. Although little effects on relative final rates were observed, germination times 23 courses were much more affected (Supplemental Data, Tables 6 and7) which is important for base 24 temperature calculations, thus meaning that repeated stresses over several plant generations can lead to 25 changes in the base temperature value usually defined at the species, and sometimes, genotype levels. 26

Overall, increases times courses were observed under heat stress meaning that the germination behavior 27 is tightly related to the history with the seed's ancestors and that their environmental conditions could 1 help interpreting the germination behavior of seedlots produced in the fields. 2

These results clearly pointed out the complexity of priming effects which are criteria-dependent in 3 addition to stress features-determined. Indeed, as observed in other inter/transgenerational studies, the 4 beneficial effects of prior stress exposures are observed on specific behavior and stage on the progeny. 5

Yadav et al. (2020) demonstrated positive impacts of multigenerational exposure to heat stress at the 6 mature plant-stage (i.e. survival rate under heat stress at bolting) but not at the germination stage in 7

Arabidopsis. In addition, although we observed that the offspring performances were mainly driven by 8 the stress sequence applied on the Progeny, the history of the Mother plants leads to different extent of 9 the Progeny responses to heat stress. For some variables, the accumulation of stress days over the two 10 generations leads to amplified negative impact (e.g. seed weight, ω6: ω3 ratio), or to a negative impact 11 while the stress sequence at the Mother plant level had a priming effect (e.g. [raffinose+stachyose]: 12 sucrose ratio). For other variables, the negative effect of the stress sequence applied on the Mother plants 13 disappeared when the Progenies were not stressed (e.g. total fatty acids), which might indicate that the 14 effects were not memorized and rather reset when passed on to the next generation. However, although 15 positive effects of heat treatment across multiple generations could be highlighted on reproductive 16 outputs (e.g. seed production) in Arabidopsis [START_REF] Whittle | Adaptive epigenetic memory of 23 ancestral temperature regime in Arabidopsis thaliana[END_REF], in our conditions, no priming effect 17 of a stress sequence applied on the Mother plants, could be observed on the offspring performances 18 when challenged with heat stress. 19 20

Conclusion 21

The underlying objective of this work was to pinpoint, in a crop species, whether the temperature stress 22 history experienced by the Mother plants impacted the Progeny responses to heat stress. Our results 23 highlighted several significant results: (i) the Progeny effects overruled the Mother effects on most 24 criteria, but the history of the Mother plants had an impact on the extent of the offspring responses to 25 heat stress; (ii) the effects of heat stress sequences were strongly correlated with the duration of high 26 temperature events over the two generations and they should be interpreted regarding the synchrony 27 between the timing of the stress event and the dynamics of the seed storage compound synthesis; (iii) 1 priming effects observed on the first stressed-plant generation for specific quality criteria (seed N 2 concentrations and soluble sugar ratio used as a proxy of desiccation tolerance) were not systematically 3 maintained (for the proxy of desiccation tolerance only) as a result of negative accumulated effects of 4 late heat peaks or a reset of the stress induced modifications; and (iv) sulphur limitation had little effect 5 on the Progeny responses to heat stress, and when the effects were amplified, it was due to the delay and 6 slowdown in the phenology which resulted in a longer heat stress sensitive window. Interestingly, S 7 restriction led to lower heat stress-induced seed dormancy. 8

Eventually, these highlights should be bear in mind in the perspective to define multiple generations 9

priming protocols in oilseed rape, and more widely demonstrated the challenge of defining priming-10 based acclimation strategies due to trade-offs between criteria and the fine tuning (i.e. in terms of heat 11 sequence features and adequate S-fertilization supply to target the desired seed characteristics). In 12 conclusion, our results suggest different processes that support intergenerational memory, as their 13 extent, persistence and impact (positive or negative) vary according to seed characteristics.

Total pods

Pods 7 and8). EMS: Early Mil Stress modality, 3LHP: 3 late heat peaks modality, 4LHP: 4 late heat peaks modality.

1

 1

2 3 4 Figure 1 :

 41 Figure 1: Experimental design which illustrates the 16 Mother x Progeny stress combinations from 5

Figure 1b 6 11 12

 611 shows the recordings of hourly temperatures in the greenhouse for each temperature sequence applied 7 in 2018-2019. As applied on the Mother plants, two contrasting S conditions (High Sulphur and Low 8 Sulphur) were applied on the Progeny plants (128 plants). Graphs are displayed by level of sequence 9 complexity: (i) Control modality, (ii) Early Mild Stress modality (EMS), (iii) 3 Late Heat Peaks (3LHP), 10 (iv) 4 Late Heat Peaks (4LHP) and (v) Priming modality. As described in Magno et al. (2021), we distinguished two categories of pods according to their 13

6

 6

7 2. 3 . 3 . 14 2. 3 . 4 .

 7331434 Seed soluble sugar concentrations 8 Soluble sugars were extracted from 50 mg of seeds powder under water, with three stages of water bath 9 and centrifugation. A solution with 40 µL melicitose was used as the internal standard. Sucrose, 10 raffinose and stachyose contents were identified using High Performance Liquid Chromatography 11 (HPLC), and quantified using a refractive index detector (2410 Differential Refractometer, Millipore 12 Waters, Waters Corporation, MA, USA) as described in Magno et al. (2021). 13 Seed dormancy-related phytohormones 15 For each treatment, 50 mg of seeds powder were mixed with 500 µL of extraction solvent [2-16 propanol/H2O/ concentrated HCl (2:1:0.002, v/v/v)], and then analyzed by HPLC-MS as described in 17 Pan et al. (2010). 18 ( 19

11

 11

Figure 2 :

 2 Figure 2: Boxplots of the ω6:ω3 ratio measured in seeds from podsL≥ 5cm under High Sulphur condition 1

18

 18

7 8 1 Figure 3 : 8 4. Discussion 1 4. 1 .

 13811 Figure 3: Germination time courses at 25•C (3 top graphs) and 5•C (3 bottom graphs) under High 2

  [START_REF] Magno | High temperature patterns at the onset of seed maturation determine 36 seed yield and quality in oilseed rape ( Brassica napus L .) in relation to sulphur nutrition[END_REF], the Progeny-priming (i.e. the longest stress duration) was 19 also the most negative to FAs, SFAs, UFAs concentrations (low values) and to ω6:ω3 ratio (high value), 20 which resulted in opposite effects on seed N and protein concentrations which is a well-known negative 21 relationships in oil crops (Si et al., 2003 and references herein; Hammac et al., 2017). These observations 22 confirm that high temperature throughout seed filling induced negative impacts on FAs contents and 23 quality i.e. decreases in poly-UFAs in favor of SFAs and mono-UFAs, thus leading to high ω6:ω3 ratio 24 as a result of temperature-triggered impairment of desaturase enzyme activity (Aksouh-Harradj et al., 25 2006; Baux et al., 2013; Brunel-Muguet et al., 2015; Gauthier et al., 2017). 26

Figure 2 )

 2 Figure 2), in line with prior studies (Brunel-Muguet et al., 2015; D'Hooghe et al., 2019). Consistently 11

14 15 16

 1416 AcknowledgmentsThis work was supported by the metaprogramme Adaptation of Agriculture and Forest to Climate 17 Change (AAFCC) of the French National Research Institute for Agriculture, Food and Environment 18 (INRAE). The authors would like to thank Anne-Françoise Ameline, Magali Bodereau, Josiane Pichon 19 and Josette Bonnefoy for their technical assistance, and Aurelia Lornac for her support concerning the 20 HPLC. They also thank Dr. Bae for the hormone analyses, Nicolas Elie from the CMABio3 (Centre for 21 Microscopy Applied to Biology) for seed image analyses, the platform Platin' (Plateau d'Isotopie de 22 Normandie) for the elemental analyses and Candice Kerdreux for the soluble sugar analyses and 23

Figure 4 : 1 :

 41 Figure 4: Summary of the effects of the combined temperature sequences (Mother x Progeny) in yield components, nutritional and physiological quality criteria 10

SD, Figure 1 :

 1 Boxplot to the Thousand Seed Weight (TSW) criteria measured in seeds from total pods under Low Sulphur condition. The TSW is the most important yield component impacted significantly by both Mother and Progeny modalities. Letters indicate the ranking amongst all the treatments (including the control). P-values and levels of significance are given on Supplemental Data (SD,

SD, Figure 2 :

 2 Germination time courses at 25•C (left) and 5•C (right) under Low Sulphur condition. Each graph represents one Mother-modality and each curb represents one Progeny-modality. Lines are fittings to a Gompertz function. Vertical bars denote standard errors. P-values of the germination time courses (T25, T50 and T75) and final germination rates are given in Supplemental Data (SD, Table

  al., 2011; Henry, 2020; Janni et al., 2020; Wang and 8 Liiang, 2017; Kakoulidou et al. 2021). Nevertheless, in contrast to single-long lasting or single-short 9 extreme/mild environmental stresses (Hassan et al., 2021; Janni et al., 2020; Kotak et al., 2007; Ohama 10 et al., 2016; Wahid et al., 2007; Zhao et al., 2021), repeated stresses remained much less documented 11

Table 1 :

 1 Yield components distinguishing the two pools of pods (i.e. podsL< 5cm and podsL≥ 5cm at the beginning of the temperature stress application). Results 3 are presented by factor (Progeny-modality and Mother-modality) under High Sulphur condition. For a given variable, different letters (Tukey multiple 4 comparisons test) indicate the ranking among Progeny or Mother modalities (including the control). P-values and levels of significance are given for Progeny, 5 Mother and Progeny x Mother interaction effects. Levels of significance: ns non-significant, p<0.05*, p<0.01**, p<0.001***. SE: standard error, TSW: 6

	L < 5cm	Pods L ≥ 5cm

Table 2 :

 2 Nutritional seed quality criteria measured in seeds from podsL≥ 5cm. Results are presented by factor (Progeny-modality and Mother-modality) under 3 High Sulphur condition. For a given measured variable, different letters (Tukey multiple comparisons test) indicate the ranking among Progeny or Mother 4 modalities (including the control). P-values and levels of significance are given for Progeny, Mother and Progeny x Mother interaction effects. Levels of 5

	6	significance: ns non-significant, p<0.05*, p<0.01**, p<0.001***. SE: standard error, DW: dry weight, FA: fatty acid, UFA: unsaturated FA, SFA: saturated
	7	FA.

Table 3 :

 3 Seed physiological quality values measured in seeds from podsL≥ 5cm. Results are presented by factor (Progeny-modality and Mother-modality) under 4 High Sulphur condition. For a given measured variable, different letters (Tukey multiple comparisons test) indicate the ranking among Progeny or Mother 5 modalities (including the control). P-values and levels of significance are given for Progeny, Mother and Progeny x Mother interaction effects. Levels of 6

	7	significance: ns non-significant, p<0.05*, p<0.01**, p<0.001***. SE: standard error, DW: dry weight, ABA: abscisic acid, GA3: gibberellic acid.

Table 4 :

 4 Germination variables measured at optimal (25•C) and suboptimal (5•C) temperatures. Results are presented by factor (Progeny-modality and Mother-3

Table 2 :

 2 Values of each Mother-modality x Progeny-modality combination for the measured variables in the Low Sulphur condition. Letters indicate the

	Mother-modalities	Control			Priming					EMS					3LHP
	Progeny-modalities	Control Control Priming	EMS	4LHP	3LHP	Control Priming	EMS	4LHP	3LHP	Control Priming	EMS	4LHP	3LHP
	Seed yield -pods L≥5cm (g/pl)	1.77	2.05 a	2.69 a	2.02 a	2.98 a	1.35 a	3.07 a	2.21 a	2.73 a	3.72 a	3.07 a	2.11 b	1.97 b	2.27 b	3.49 ab	4.06 a
	Seed number -pods L≥5cm (per pl)	347	402 a	791 a	467 a	701 a	312 a	751 a	704 a	691 a	1017 a	770 a	464 b	568 ab	525 ab	810 ab	935 a
	TSW -pods L≥5cm (g)	5.10	5.16 a	3.54 b	4.46 ab	4.82 a	4.50 ab	4.20 a	3.28 a	4.17 a	3.72 a	4.40 a	4.58 a	3.64 b	4.31 a	4.38 a	4.37 a
	Seed yield -pods L<5cm (g/pl)	6.15	5.90 a	2.47 b	4.26 ab	4.09 ab	5.95 a	5.62 a	3.28 b	4.79 ab	2.98 b	4.37 ab	5.0 a	3.97 a	4.45 a	3.48 a	3.79 a
	Seed number -pods L<5cm (per pl)	1394	1333 a	1155 a	1086 a	1079 a	1562 a	1438 a	1088 a	1265 a	904 a	1126 a	1222 a	1224 a	1100 a	927 a	1029 a
	TSW -pods L<5cm (g)	4.42	4.57 a	2.38 b	3.90 a	3.74 ab	3.91 a	3.93 a	3.08 a	3.76 a	3.44 a	3.85 a	4.07 a	3.29 b	4.05 a	3.76 ab	3.60 ab
	Total seed yield (g/pl)	7.92	7.98 a	5.16 b	6.28 ab	7.07 ab	7.29 a	8.69 a	5.49 c	7.52 ab	6.69 bc	7.45 ab	7.11 ab	5.93 b	6.72 ab	6.97 ab	7.85 a
	Total seed number (per pl)	1741	1735 a	1946 a	1552 a	1780 a	1875 a	2190 a	1792 a	1956 a	1921 a	1896 a	1686 a	1792 a	1625 a	1738 a	1965 a
	Total TSW (g)	4.55	4.66 a	2.75 b	4.07 a	4.09 a	3.97 a	3.97 a	3.16 b	3.91 ab	3.59 ab	3.97 a	4.19 a	3.39 b	4.14 a	4.05 a	3.99 a
	Fatty acids (%DW)	29.15	28.99 a	16.73 b	25.11 a	27.25 a	26.75 a	28.71 a	14.68 b	29.14 a	24.36 a	28.11 a	27.83 a	19.61 b	25.77 a	26.55 a	29.12 a
	UFA (%DW)	26.74	26.55 a	14.87 b	22.97 a	24.66 a	24.29 a	26.23 a	12.92 b	26.64 a	21.97 a	25.56 a	25.52 a	17.54 b	23.55 a	24.05 a	26.60 a
	SFA (%DW)	2.41	2.43 ab	1.86 c	2.14 bc	2.59 a	2.46 ab	2.48 a	1.76 b	2.50 a	2.39 a	2.55 a	2.31 ab	2.07 b	2.21 ab	2.50 a	2.52 a
	ω6:ω3 ratio	1.72	1.72 b	2.51 a	1.70 b	2.13 b	1.91 b	1.76 cd	2.57 a	1.68 d	2.29 b	1.88 c	1.68 c	2.32 a	1.68 c	2.06 b	1.84 bc
	C concentration (%DW)	48.5	47.2 a	45.2 a	47.6 a	47.3 a	47.9 a	44.2 c	44.8 bc	49.0 a	46.2 abc	47.8 ab	50.2 a	44.9 b	47.8 ab	47.2 ab	47.5 ab
	N concentration (%DW)	3.21	3.11 b	4.00 a	3.71 ab	3.51 ab	3.51 ab	3.26 b	4.37 a	3.18 b	3.59 b	3.45 b	3.62 ab	3.91 a	3.69 ab	3.59 ab	3.21 b
	S concentration (%DW)	0.35	0.33 a	0.49 a	0.38 a	0.39 a	0.37 a	0.33 b	0.43 a	0.34 b	0.36 b	0.36 b	0.35 ab	0.43 a	0.38 ab	0.36 ab	0.32 b
	Protein (mg/g DW)	176.7	170.8 b	220.3 a	204.1 ab	192.9 ab	193.3 ab	179.3 b	240.5 a	174.9 b	197.3 ab 189.6 ab	199.0 ab	215.2 a	203.0 ab	197.4 ab	176.6 b
	[Raff+stach] : suc ratio	0.41	0.39 a	0.2 b	0.35 a	0.32 a	0.33 a	0.37 b	0.2 d	0.46 a	0.26 cd	0.31 bc	0.31 ab	0.25 b	0.37 a	0.28 ab	0.33 ab
	ABA:GA3 ratio	0.67	1.21 ab	2.09 a	0.87 ab	0.63 b	0.24 b	0.31 b	1.68 a	0.66 b	0.79 ab	0.79 ab	0.25 a	0.99 a	0.75 a	0.53 a	0.28 a

Table 3 :

 3 Yield components distinguishing the two pools of pods (i.e. podsL< 5cm and podsL≥ 5cm at the beginning of the temperature stress application). Results are presented by factor (Progeny-modality and Mother-modality) under Low Sulphur condition. For a given variable, different letters (Tukey multiple comparisons test) indicate the ranking among Progeny or Mother modalities (including the control). P-values and levels of significance are given for Progeny, Mother and Progeny x Mother interaction effects. Levels of significance: ns non-significant, p<0.05*, p<0.01**, p<0.001***. SE: standard error, TSW:

	Thousand Seed Weight.

Table 3

 3

). EMS: Early Mil Stress modality, 3LHP: 3 late heat peaks modality, 4LHP: 4 late heat peaks modality.

Seed C, S, N

and protein concentrations (%DW) Oil content and fatty acids (%DW)

	Factor\Variables	Carbon	Sulphur	Nitrogen	Protein (mg.g -1 )	FA	SFA	UFA	ω6:ω3 ratio
	Progeny-modalities
	Control	45.61 a	0.31 a	3.37 b	185.29 b	30.22 a	2.52 a	27.69 a	1.76 d
	Early mild stress	44.10 ab	0.25 b	3.62 ab	199.04 ab	24.76 b	2.21 b	22.55 b	1.79 cd
	3 late heat peaks	45.61 a	0.28 ab	3.35 b	184.06 b	28.94 a	2.62 a	26.33 a	1.96 c
	4 late heat peaks	44.65 a	0.31 a	3.65 ab	200.57 ab	24.70 b	2.49 a	22.21 b	2.41 b
	Priming	42.43 b	0.29 ab	3.76 a	206.78 a	16.88 c	1.91 c	14.96 c	2.62 a
	se	0.40	0.01	0.08	4.57	0.85	0.06	0.78	0.04
	Mother-modalities
	Control	45.43 ns	0.34 ns	3.48 ns	191.12 ns	29.83 ns	2.44 ns	27.39 ns	1.73 ns
	Early mild stress	44.71 ns	0.30 ns	3.58 ns	196.94 ns	24.40 ns	2.28 ns	22.12 ns	2.12 ns
	3 late heat peaks	44.22 ns	0.28 ns	3.62 ns	199.74 ns	25.08 ns	2.38 ns	22.71 ns	2.14 ns
	Priming	44.56 ns	0.27 ns	3.41 ns	187.60 ns	25.89 ns	2.41 ns	23.48 ns	2.09 ns
	se	0.57	0.01	0.11	6.04	1.33	0.08	1.25	0.08
	Progeny effect	0.000 ***	0.017 *	0.004 **	0.004 ***	0.000 ***	0.000 ***	0.000 ***	0.000 ***
	Mother effect	0.584	0.170	0.069	0.067	0.394	0.243	0.407	0.703
	Progeny x Mother	0.262	0.231	0.950	0.950	0.915	0.848	0.915	0.983
	SD,

Table 4 :

 4 Nutritional seed quality criteria measured in seeds from podsL≥ 5cm. Results are presented by factor (Progeny-modality and Mother-modality) under Low Sulphur condition. For a given measured variable, different letters (Tukey multiple comparisons test) indicate the ranking among Progeny or Mother modalities (including the control). P-values and levels of significance are given for Progeny, Mother and Progeny x Mother interaction effects. Levels of significance: ns non-significant, p<0.05*, p<0.01**, p<0.001***. SE: standard error, DW: dry weight, FA: fatty acid, UFA: unsaturated FA, SFA: saturated FA.

	Soluble sugars	Hormones

Table 5 :

 5 Seed physiological quality values measured in seeds from podsL≥ 5cm. Results are presented by factor (Progeny-modality and Mother-modality) under Low Sulphur condition. For a given measured variable, different letters (Tukey multiple comparisons test) indicate the ranking Progeny or Mother modalities (including the control). P-values and levels of significance are given for Progeny, Mother and Progeny x Mother interaction effects. Levels of significance: ns non-significant, p<0.05*, p<0.01**, p<0.001***. SE: standard error, DW: dry weight, ABA: abscisic acid, GA3: gibberellic acid.

	SD,

Table 6 :

 6 Values of each Mother-modality x Progeny-modality combination for the germination variables in the High Sulphur condition. Letters indicate the ranking among Progeny-modalities for a given Mother-modality (mean pairwise comparison test, LSD test). T25, T50 and T75 represent respectively the time (in hours) for 25%, 50% and 75% of germinated seeds, as adjusted from the observations with the Gompertz functions. Final rate is the observed final rate (in percentage). EMS: Early Mil Stress modality, 3LHP: 3 late heat peaks modality, 4LHP: 4 late heat peaks modality.

	Mother-modalities	Control			Priming					EMS					3LHP
	Progeny-modalities	Control Control Priming EMS	4LHP	3LHP Control Priming EMS	4LHP 3LHP Control Priming	EMS	4LHP 3LHP
	25°C	T25 (h)	14.1	15.3 a	14.3 a	13.8 a	13.4 a	13.4 a	15.8 a	12.0 c	14.5 ab	13.0 bc	14.3 ab	13.6 a	12.1 a	11.8 a	13.1 a	14.8 a
		T50 (h)	16.8	17.3 a	16.5 ab	16.5 ab	15.4 ab	14.9 b	17.3 a	14.5 c	16.7 ab	15.0 bc	15.9 abc	15.2 a	15.6 a	14.2 a	15.1 a	16.1 a
		T75 (h)	20.6	21.2 a	19.8 ab	20.0 ab	17.9 bc	16.9 c	19.2 a	17.9 a	19.6 a	17.5 a	17.9 a	15.2 a	15.6 a	14.2 a	15.1 a	16.1 a
		Final rate (%)	97	91 b	91 b	100 a	98 a	98 a	87 b	95 ab	100 a	99 a	98 a	99 a	95 b	99 a	99 a	100 a
	5°C	T25 (h)	95.6	95.5 a	84.6 a	84.3 a	98.6 a	93.1 a	107.0 a	82.9 a	85.4 a	94.9 a	99.5 a	91.0 ab	70.3 b	76.0 b	107.7 a	106.5 a
		T50 (h)	111.4	111.8 a	111.7 a	102.6 a	113.6 a	105.7 a	118.9 a	103.4 a	105.4 a	105.9 a	111.9 a	103.7 ab	99.0 ab	88.7 b	117.5 a	116.6 a
		T75 (h)	133.4	134.6 a	147.3 a	126.6 a	132.7 a	121.8 a	134.9 a	131.8 a	130.8 a	119.8 a	128.0 a	121.6 ab	138.9 a	105.0 b	130.7 a	129.4 a
		Final rate (%)	96	98 a	96 a	98 a	99 a	99 a	99 a	93 b	99 a	100 a	100 a	98 a	94 a	99 a	97 a	99 a

Table 7 :

 7 Germination variables measured at optimal (25•C) and suboptimal (5•C) temperatures. Results are presented by factor (Progeny-modality and Mother-modality) under Low Sulphur condition. T25, T50 and T75 represent respectively the time (in hours) for 25%, 50% and 75% of germinated seeds, as adjusted from the observations with the Gompertz functions. Final rate is the observed final rate (in percentage). For a given measured variable, different letters (Tukey multiple comparisons test) indicate the ranking Progeny or Mother modalities (including the control). P-values and levels of significance are given for Progeny, Mother and Progeny x Mother interaction effects. Levels of significance: ns non-significant, p<0.05*, p<0.01**, p<0.001***. SE: standard error.

	SD,

Table 8 :

 8 Values of each Mother-modality x Progeny-modality combination for the germination variables in the Low Sulphur condition. Letters indicate the ranking among Progeny-modalities for a given Mother-modality (mean pairwise comparison test, LSD test). T25, T50 and T75 represent respectively the time (in hours) for 25%, 50% and 75% of germinated seeds, as adjusted from the observations with the Gompertz functions. Final rate is the observed final rate (in percentage). EMS: Early Mil Stress modality, 3LHP: 3 late heat peaks modality, 4LHP: 4 late heat peaks modality.

	Mother-modalities	Control			Priming					EMS					3LHP
	Progeny-modalities	Control Control Priming EMS	4LHP	3LHP Control Priming EMS	4LHP 3LHP Control Priming	EMS	4LHP 3LHP
	25°C	T25 (h)	16.7	18.2 a	14.9 b	16.9 ab	17.7 a	17.3 a	16.1 a	15.2 a	16.4 a	16.7 a	16.3 a	17.7 a	17.2 a	17.1 a	16.3 a	16.5 a
		T50 (h)	18.5	19.5 a	16.7 b	18.7 a	19.8 a	19.8 a	17.3 a	18.1 a	18.9 a	18.7 a	18.1 a	19.8 a	19.1 a	19.7 a	18.8 a	19.2 a
		T75 (h)	20.8	21.3 ab	19.3 b	21.0 ab	22.4 a	22.3 a	18.9 a	21.5 a	22.2 a	21.4 a	20.5 a	23.3 a	21.7 a	23.0 a	22.1 a	22.7 a
		Final rate (%)	96	95 b	95 b	100 a	99 ab	99 ab	100 a	97 a	97 a	97 a	99 a	90 b	96 ab	99 a	98 ab	97 ab
	5°C	T25 (h)	89.9	104.9 a	90.5 ab	83.9 b	99.1 ab	104.5 a	92.8 a	82.2 a	80.8 a	101.5 a	96.1 a	101.2 a	91.9 a	86.5 a	95.9 a	100.2 a
		T50 (h)	104.3	115.3 a	103.8 ab	95.8 b	114.3 ab	120.3 a	101.7 a	96.6 a	97.2 a	119.2 a	108.9 a	121.2 a	104.9 a	101.9 a	114.5 a	119.7 a
		T75 (h)	123.8	128.6 ab	120.9 ab	110.8 b	133.8 a	141.3 a	113.1 b	115.7 b	118.8 ab	142.7 a	125.2 ab	149.4 a	121.4 b	121.8 b	138.1 ab	144.4 a
		Final rate (%)	98	100 a	99 a	100 a	99 a	98 b	100 a	99 a	98 a	97 a	100 a	97 b	100 a	100 a	100 a	100 a


DÉTAILS EXPÉRIMENTAUX

Deux nanofibres optiques ont été étudiées dans ce travail, la première correspond à une nanofibre fabriquée à partir d'une fibre standard monomode télécom (G652D) nommée par la suite G652D-NF, la seconde à partir d'une fibre standard monomode dans la gamme de longueurs d'onde visible (SM450) et nommée par la suite SM450-NF. Elles sont caractérisées par un diamètre de 690 nm uniforme sur une longueur de 20 mm et reliée par deux sections coniques de longueur d'environ 48 mm aux deux extrémités non-étirées de la fibre standard (comme montré sur la figure 2(b)). En régime non-linéaire de propagation, la difficulté des mesures correspond à la détection d'un signal de diffusion très faible. Cependant, la détection a été rendue possible par le faible niveau de détection du détecteur CCD refroidi à -85°C du spectromètre, et par un temps long d'exposition (entre 3 et 10 min). En outre, pour l'étude de la cascade Raman, les mesures distribuées ont étés réalisées uniquement pour la partie homogène de la nanofibre, là où le processus de diffusion est maximale et constante [ 1,2 ]. Il est important de noter ici qu'aucun traitement du signal a été réalisé et que les mesures brutes donnent directement une comparaison quantitative entre les spectres.

MESURES DISTRIBUÉES D'UNE CASCADE RAMAN DANS UNE NANO FIBRE

Nous avons étudié en premier lieu la nanofibre G652D-NF en régime de cascade Raman. La source correspond à un laser Nd-YAG doublé en fréquence, de durée d'impulsion 500 ps, et de taux de répétition 21 kHz. La figure 1

  (a) montre l'évolution du spectre le long de la partie homogène de la nanofibre pour une puissance crête de 120 W. Le processus de cascade Raman est clairement visible avec la génération de plusieurs ordres Raman Stokes (ORS) et anti-Stokes (ORAS). Même si la longueur de la nanofibre est courte (2 cm), l'amplification Raman est efficace en raison du fort confinement des modes guidés induit par la réduction de leurs aires effectives. Par ailleurs, la figure 1(b) compare le premier et le dernier spectre, et montre la dynamique de la mesure (environ 36 dB) permettant l'observation claire de l'évolution spectrale. La figure 1(c) représente l'évolution du logarithme naturel des intensités des trois 1 ers ORS en fonction de la distance de propagation.exponentielle des ordres Raman est clairement visible, depuis leur génération jusqu'à la déplétion de leurs pompes. La pente linéaire représente le gain Raman par millimètre (pointillé sur Fig.1(c)). Le 1 er ORS présente une pente d'environ 91 𝑚 -1 , 199 𝑚 -1 pour le 2 nd ORS, et 280 𝑚 -1 pour le 3 ième ORS. La théorie prédit que la pente est multipliée par un facteur deux entre le 1 er et le 2 nd ORS, et par un facteur trois entre le 1 er et le 3 ième ORS. Par ailleurs, le calcul théorique du gain du 1 er ORS [ 3 ] donne un encadrement entre 30 et 90 𝑚 -1 selon l'estimation de la puissance pompe au niveau de l'entrée de la NF. Les mesures expérimentales montrent donc que l'évolution des spectres est en bon accord quantitatif avec la théorie[ 3,4 ].

Figure 1 .

 1 Figure 1. (a) Évolution de la cascade Raman le long de la région de la NF. (b) Le spectre d'entrée (courbe bleue) et de sortie (courbe rouge) pour la NF. (c) Évolution du logarithme naturel des intensités des différents ORS avec la distance de propagation X. Les lignes pointillées sont utilisées pour estimer les pentes. 4. MESURES DISTRIBUÉES DE LA GÉNÉRATIO N DE SUPERCO NTINUUM La génération de supercontinuum a été réalisé dans la nanofibre SM450-NF avec un pompage par un laser Nd:YAG doublé en fréquence, de durée d'impulsion 36 ps, et de taux de répétition de 100 kHz. Le laser est d'abord injecté dans une fibre à petit coeur (SM600) permettant de contrôler l'injection dans la fibre SM450. La Fig.2(a) montre les positions des différents spectres mesurés le long de la nanofibre avec soit l'analyseur de spectre optique OSA (spectres 1 et 5) soit avec notre technique (spectres 2 à 4). La Fig. 2(b) montre les spectres correspondants pour une puissance crête de 340 W. Le temps d'exposition du détecteur du spectromètre est typiquement de 3 min pour les deux spectres 3 et 4, et de 10 min pour le spectre 2 afin d'augmenté le niveau du signal détecté. Cette différence de temps d'exposition s'explique par le fait que le coeur de la fibre standard présente un niveau de diffusion très faible par rapport à la diffusion au niveau de la nanofibre [ 1 ]. Tout d'abord, avant la fibre étirée (entre spectres 1 et 2), le spectre se développe côté Stokes et anti-Stokes par cascade Raman classique dans la fibre standard. Cette évolution spectrale résulte de la propagation du mode fondamental guidé le long de la fibre SM450 de longueur d'environ 1 m, et caractérisé par une aire effective faible augmentant par conséquent l'intensité du champ. Puis, dans la

Figure 2 .

 2 Figure 2. (a) géométrie de la nanofibre et positions respectives des spectres réalisés, (b) spectres correspondants le long de la nanofibre. 5. CO NCLUSIO N Dans ce travail nous avons observé pour la première fois l'évolution longitudinale du champ guidé le long d'une nanofibre optique en régime non linéaire. Nous avons ainsi pu faire une mesure directe du gain Raman pour 3 ordres Raman Stokes. De plus, dans le cas de la génération d'un supercontinuum, notre étude montre le rôle important de la transition d'entrée et de la région de la nanofibre sur la génération du SC. RÉFÉRENC ES 1 Y. Haddad et al., "Microscopic imaging along tapered optical fibers by right-angle Rayleigh light scattering in linear and nonlinear regime," Optics Express, 29(24), 39159-39172 (2021) 2 Y .-H. Lai et al., "Fiber taper characterization by optical backscattering reflectometry", Opt. Exp. 25, 22312 (2017) 3 M.D. Turner et al., "A full vectorial model for pulse propagation in emerging waveguides with subwavelength structures part II: stimulated Raman scattering ", Opt. Express 17(14), 11565-11581 (2009) 4 R.W, Boyd, "Nonlinear Optics," Academic press (2020)

Mesures spectrales distribuées le long d'une nanofibre optique en régime non linéaire Yosri Haddad, Jean-Charles Beugnot, Samuel Margueron & Gil Fanjoux

  Ce travail expérimental et numérique présente l'évolution spectrale en régime non linéaire (cascade Raman, et supercontinuum) du champ guidé le long d'une nanofibre optique de longueur centimétrique et de taille submicrométrique par analyse de la diffusion Rayleigh.Les évolutions spectrales sont principalement réalisées à l'heure actuelle en sortie de guide d'onde en fonction de la puissance injectée, et comparées aux simulations numériques pour prédire la propagation longitudinale du champ le long du guide. Cependant, les mesures simultanées spatiales et spectrales de la lumière guidée en régime linéaire et non linéaire représentent un défi expérimental crucial pour la rétro-ingénierie, afin d'améliorer la fabrication des guides d'ondes, ou pour la comparaison avec les prédictions théoriques de la propagation du champ. Dans ce but, nous avons récemment développé une méthode expérimentale non-destructive et non-invasive afin de caractériser spatialement et spectralement la lumière guidée le long d'une nanofibre optique [ 1 ]. Cette technique non-destructive et non-invasive repose sur l'analyse de la lumière rayonnée par un guide en fonctionnement par diffusion Rayleigh (DR) à l'aide d'un micro-spectromètre confocal.

	FEMTO-ST Institute, UMR 6174 CNRS / Université Bourgogne Franche-Comté, 15B avenue des
	Montboucons, 25030 Besançon -France
	gil.fanjoux@univ-fcomte.fr
	RÉSUMÉ
	Ce travail présente l'évolution longitudinale d'une cascade Raman ainsi que la génération d'un
	supercontinuum (SC) le long d'une nanofibre optique de longueur centimétrique et de largeur
	submicrométrique. Ces analyses nous ont permis de réaliser une mesure directe du gan Raman pour
	3 ordres Stokes, ainsi que d'observer pour la 1 ière fois le développement d'un supercontinuum le long
	d'une nanofibre.

MOTS-CLEFS : nanofibre optique; Diffusion Rayleigh; Cascade Raman; Supercontinuum

1. INTRO DUCTIO N


Yosri Haddad

Jean-Charles Beugnot

Samuel Margueron

Gil Fanjoux
email: gil.fanjoux@univ-fcomte.fr

Distributed spectral measurement of supercontinuum generation along an optical nanofiber

this work presents preliminary experimental results concerning distributed measurements along an optical nanofiber of the generation of a supercontinuum in the visible range, for a spatial and spectral dynamics analysis.

Introduction

The generation of spectrally-broad single-mode supercontinuum (SC) light in optical fiber is well studied in the literature [START_REF]Supercontinuum generation in optical fibers[END_REF]. Indeed, SC has been the subject of various studies in a wide variety of nonlinear media, including solids, liquids and gases, and various types of waveguides, such as optical fibers in particular photonic crystal fibers (PCF) or sub-micrometric tapered fibers called nanofiber [START_REF]Supercontinuum generation in optical fibers[END_REF][START_REF] Birks | Supercontinuum generation in tapered fibers[END_REF]. However, the spectral evolutions of the SC studies are mainly limited to the measurements at the waveguide output as a function of the injected power, and numerical simulations to predict the longitudinal propagation of the field along the fiber. Therefore, there is no experimental investigation of the SC generation along an optical nanofiber. For that purpose, we have recently developed a non-destructive and non-invasive experimental method in order to characterize spatially and spectrally the guided light along an optical waveguide [START_REF] Haddad | Microscopic imaging along tapered optical fibers by right-angle Rayleigh light scattering in linear and nonlinear regime[END_REF]. By using this method, we present in this paper preliminary results of a distributed longitudinal measurement of SC generation along a nanofiber of millimeter length and sub-micrometric size.

Experimental setup and results

The experimental setup is based on the detection of far-field right-angle Rayleigh light scattering radiated out the waveguide using a micro-spectrometer [START_REF] Haddad | Microscopic imaging along tapered optical fibers by right-angle Rayleigh light scattering in linear and nonlinear regime[END_REF] allowing a high spatial resolution given by the confocal microscope, and a high spectral resolution given by the spectrometer. Notch filters are used to remove the pump spectral components and to allow simultaneous Stokes and anti-Stokes measurements. The waveguide studied in this work corresponds to a nanofiber manufactured by heating and pulling a standard single mode fiber in visible wavelength range (SM450) until its diameter has reached a target value. After the tapering process, the nanofiber have a uniform waist of about 690 nm over 20 mm long accurately measured by Brillouin scattering method and SEM [START_REF] Haddad | Microscopic imaging along tapered optical fibers by right-angle Rayleigh light scattering in linear and nonlinear regime[END_REF][START_REF] Godet | Brillouin spectroscopy of optical microfibers and nanofibers[END_REF]. The nanofiber region is connected to the untapered fiber by taper transitions of 37 mm long each (Fig. 1(a)). After manufacturing process, the nanofiber is placed inside a closed transparent box in order to protect it from external air vibrations and to reduce any kind of contamination. The optical losses just after manufacturing were measured at 532 nm of wavelength and equal to 0.32 dB. This low level of losses ensures that the transitions are adiabatic at that wavelength. The waveguide is pumped at 532 nm frequency-doubled Nd:YAG laser source, with a repetition rate of 200 kHz, a 36 ps of pulse duration and a peak power of around 340 W. The laser beam is first injected in a small core fiber (SM600), connected to the SM450 fiber in order to control the injection preferentially in the fundamental mode of the non-stretched part of the SM450 fiber.

Fig. 1(a) shows the locations of the different spectra performed along the waveguide either with the OSA (spectra 1 and 5) or with our technique (spectra 2 to 4), and Fig. 1(b) shows the corresponding spectra. First, due to the weak scattered intensity with a picosecond laser source, the exposure time for the spectrometer detector is typically 3 min for the two spectra 3 and 4, and 10 min for the spectrum 2. This difference of duration is explained by the fact that the standard fiber core presents a very low scattering level compared to the scattering at the nanofiber level [START_REF] Lai | Fiber taper characterization by optical backscattering reflectometry[END_REF]. It is important to note that the experimental data were recorded in the same experimental conditions with any normalization or correction of the intensity of the different spectrums. Therefore, the evolution of the scattered intensity represent the real evolution of the SC along the waveguide.

Figure 1(b) shows that the spectrum is developed in both Stokes and anti-Stokes regions between the end of the SM600 fiber and the input side of the non-stretched part of the taper (cyan and green spectra). This feature is due to the propagation of the high optical power of the guided mode along of about 1 m fiber with a small effective area (SM450), which increases the field intensity and therefore the nonlinearity. After propagation along the first transition, the recorded spectrum at the end of the transition 1 (spectrum 3) is well developed in the anti-Stokes region without any significant change for the spectral components in the Stokes region. This is due to the dispersion variation along the nanofiber with a zero dispersion wavelength decreasing from 1300 nm to 490 nm. This process seems to appear at the end of the transition 1 as a measurement in the middle of the transition 1 (not shown here) presents a spectrum quite similar to spectrum 3. After the propagation along the nanofiber, the spectrum 4 shows the presence of a dispersive wave located at a wavelength of about 415 nm. This dispersive wave can be predicted by analytical phase matching equation with the dispersion coefficients of the nanofiber. Moreover, the comparison between the spectrum 4 and the spectrum 5 shows that the whole features of the SC spectrum are already present at the end of the nanofiber region, a small difference is nevertheless observed in the anti-Stokes region certainly due to the difference of the dynamics of the measurements for our system (43 dB) in comparison to the dynamics obtained with the OSA (53 dB). This observation indicates that the influence of the second transition is weak or even negligible in the generation of the SC, and that, even with a small length of 20 millimeters, the large nonlinearity and the specific dispersion at the level of the nanofiber play a key role in the development of the whole SC features. In order to confirm these observations, we will perform numerical simulations based on the nonlinear Schrödinger equation, by including the two transitions and the nanofiber in order to take into account the continuous evolution of the linear and non linear parameters of the whole waveguide.

Conclusion

Primarily results provide for the first time to our knowledge distributed measurements for the evolution of a SC along a tapered fiber with a sub-micrometric diameter and millimetric length. This work shows the important role of the input transition and the nanofiber region on the SC generation due to the management of the dispersion and the large increase of the field intensity due to the strong confinement. It is noteworthy that the non-destructive and non-invasive experimental method can be used for other nonlinear optical waveguides working in nominal operation.

Fig 1 .

 1 Fig 1. (a) geometrical data of the nanofiber, and (b) longitudinal evolution of the SC along the nanofiber.

The authors would like to acknowledge the financial support of Conseil régional de Bourgogne-Franche-Comté and Agence Nationale de la Recherche (ANR-15-IDEX-0003, ANR-16-CE24-0010, ANR-17-EURE-0002).