Introduction
Evidence for increased frequency of stress events such as heat waves has been observed over recent decades. Based on the last IPCC report (Hoegh-Guldberg et al. 2018), heat waves are expected to become more frequent, to last longer and to increase in intensity during the reproductive phase of economically important crops (Trnka et al.2014; Christidis, Jones & Stott 2015). These new climatic patterns have led to attempts to decipher crop behavior and final performance in the light of recurring stresses. While the effects of extreme/mild environmental stresses have been widely investigated from molecular to whole plant levels (Kotak et al. 2007; Wahid, Gelani, Ashraf & Foolad 2007; Ohama et al. 2016), far fewer studies have tackled the issue of understanding the effects of their recurrence throughout the crop season. Indeed, the overall magnitude of the plant response to successive stresses might not match the effects induced by individual stressing events because (i) the first stress triggers physiological and metabolic adjustments that bring the plant to a modified status (including phenology) when the stress recurs, which leads to different responses to the second stress and (ii) a mild stress prior to further similar stresses can induce stress memory that lasts the duration of the crop season and is sometimes transmitted to offspring (Bruce, Matthes, Napier & Pickett 2007; Kinoshita & Seki 2014; Crisp, Ganguly, Eichten, Borevitz & Pogson 2016; Kumar 2018). Stress memory is defined as the process of storage and retrieval of information acquired during an initial exposure to stress (Crisp et al. 2016; Hilker & Schmülling 2019). This information acts as a priming process with beneficial effects when the stress recurs and it can lead to earlier, more rapid, intense, and sensitive responses that help plants to acclimate in changing environments (Kinoshita & Seki 2014). Underlying mechanisms include epigenetic regulation, transcriptional priming, primed conformation of proteins, and/or specific hormonal or metabolic signatures. Several examples illustrate the benefits of stress memory, not only for the current generation of plants as they are challenged repeatedly during their own life cycle (i.e. intra-generational memory, Ding, Fromm & Avramova 2012) but also for their offspring when faced with stresses similar to those experienced by the parent plants (i.e. transgenerational memory, Molinier, Ries, Zipfel & Hohn 2006; Grootet al. 2016; Wang et al. 2016; Hatzig, Nuppenau, Snowdon & Schießl 2018). This means that as the climate changes the effects of repeated stresses should be harnessed as a crop improvement strategy because this approach has promise for inducing acclimation to heat stress (Wang & Liiang 2017).
In winter oleaginous crops, the reproductive phase occurs during spring, which might expose flowering, grain filling and grain maturation to high temperature events. Due to its indeterminate growth, the OSR plant displays flowers and growing pods in different proportions throughout the reproductive phase. Consequently, heat stress can impact reproductive organs by limiting their number and their size, which in turn leads to carbon (C) partitioning modification in favor of already developed organs that have passed the sensitive stage, and this makes analysis of the direct effects of heat stress on the organs more complex (Guilioni, Wery & Tardieu 1997). While heat stress at flowering limits pollination (Sage et al. 2015) and/or induces early pod abortion resulting in yield losses in oilseed rape (Morrison & Stewart 2002; Young, Wilen & Bonham-Smith 2004), heat stress that occurs during seed filling and maturation affects seed storage compounds quantitatively and qualitatively, leading to seed quality degradation. Few studies have reported the effects on seed quality in winter oilseed rape (OSR) of repeated heat stress events that might be erratic and fluctuate as predicted in climate change models (in field conditions, Deng & Scarth 1998; Baux et al. 2013; in controlled conditions Aksouh, Jacobs, Stoddard & Mailer 2001; Aksouh-Harradj, Campbell & Mailer 2006). Seed quality encompasses a range of criteria related to nutritional and physiological characteristics (i.e. germination behaviors and storage capacity). In winter OSR, oil content, fatty acid (FA) profiles, and protein content are major nutritional criteria for edible oil and cakes used in human and animal consumption, respectively. OSR oil contains high polyunsaturated FA (PUFA) content compared to other oil crops, which makes it a healthy edible oil for human consumption (Aguirrezábal, Martre, Pereyra-Irujo, Echarte & Izquierdo 2015). While contrasting variations have been observed in oil content according to the temperature intensity, the timing of stress exposure and the pools of seeds analyzed (main stem vs. bulk) (e.g. increased in Brunel-Muguetet al. (2015); decreased in Canvin (1965); Aksouh et al.(2001); Aksouh-Harradj et al. (2006)), high temperatures are known to induce decreases in PUFAs in favor of saturated FAs (mainly C16:0 and C18:0) and monounsaturated FAs, and increases in the C18:2/C18:3 ratio, as a result of temperature-triggered impairment of desaturase enzyme activity (i.e. oleic and linoleic desaturases) (Aksouh-Harradj et al. 2006; Baux, Hebeisen & Pellet 2008; Bauxet al. 2013; Schulte et al. 2013; Brunel-Muguet et al. 2015; Gauthier et al. 2017). By contrast, seed nitrogen (N) and protein concentrations in the oil-free meal is usually negatively correlated with total oil content (Aksouh et al. 2001; Aksouh-Harradj et al. 2006) as observed in other oil crops (in soybean, N concentration Chebrolu et al. (2016); protein concentration, Dornbos & Mullen (1992)). Additionally, other seed characteristics related to physiological quality, i.e. seed storage capacity and germination behavior, have been investigated, but not to any great extent (Brunel-Muguet et al. 2015). A drastic degradation of seed storage capacity has been observed using seed conductivity and the ratio of soluble sugars (sucrose and raffinose family oligosaccharides (RFOs)), abscisic acid (ABA), and gibberellic acid (GA3) as proxies (Bailly et al. 2001; Brunel-Muguet et al. 2015) in seeds from long-term heat-stressed mother plants. Other phytohormones were shown to be involved in the control of secondary dormancy, defined as failure in the germination process of mature and non-dormant seeds under adverse conditions (Pekrun, Lutman & Baeumer 1997). Recent studies have highlighted the role of indole-3-acetic acid (IAA), whose concentrations increase in dormancy-induced seeds (Shu, Liu, Xie & He 2016; Liu et al. 2019; Tuan, Yamasaki, Kanno, Seo & Ayele 2019). Its high levels have also been correlated with high jasmonic acid (JA) in seeds as a consequence of both phytohormones interacting during the expression of indole glucosinolate biosynthesis genes (Liu et al. 2019). Although several studies have reported that salicylic acid (SA) enhanced germination in Arabidopsisseeds by reducing oxidative damage (Lee & Park 2010; Chitnis et al. 2014), SA has also been shown to inhibit germination because of higher oxidative stress (Xie, Zhang, Hanzlik, Cook & Shen 2007). In Brassica species, sulfur (S) nutrition determines yield components and seed quality because of their high S requirements throughout the crop cycle (D’Hooghe et al. 2014; Brunel-Muguet et al. 2015). In addition to its well-known implication in the synthesis and signaling of stress tolerance-controlling phytohormones (Hasanuzzaman et al. 2018), S might be involved in the acquisition of thermotolerance mediated by epigenetic regulation (Bokszczanin et al. 2013). This is based on evidence for S involvement in DNA methylation (through the role of S-adenosylmethionine (SAM) as a donor of methyl groups, Menget al. 2018), one of the key epigenetic markers that supports stress memory, thus making the analysis of S supply relevant in the context of epigenetic memory.
In our study we focused on the effects of high spring temperatures on seeds at the onset of maturation to deepen our knowledge of this seed quality-determining stage in relation to S nutrition. Our assumptions were that the effects of high temperature at the onset of seed maturation can greatly vary depending on whether plants are exposed to a mild heat stress event that primes them to withstand later heat peaks and that S nutrition might impact the ability of the plants to endure heat stress.
Overall, our experimental design addresses the following questions: (i) what are the quantitative effects of different high temperature sequences applied at the onset of seed maturation on seed yield, quality criteria and stress response indicators? (ii) to what extent do the effects of successive high temperature events applied to maturing pods differ from the effect of individual events? (iii) what are the underlying defense pathways triggered by temperature stress? (iv) is sulfur nutrition controlling heat stress responses through acquisition of thermotolerance?