Discussion

Cold is a major abiotic stressor to consider when adapting agriculture to the challenges of climate change, such as avoiding drought or growing short-cycle crops in northern regions. Seed germination and emergence are crucial for establishing crops, followed by the vigor and growth of above- and below-ground parts. Optimal vigor improves nutrient competition with weeds or after early herbicide treatment, enhances resistance to pathogens and increases resilience to abiotic stress. Thus, breeding for vigor is essential to benefit from ES (Houmanatet al. , 2016; Walne and Reddy, 2022).
Assessing vigor usually involves observing above-ground parts, but assessing the root system is equally important. For instance, a robust root system that can grow into deeper soil layers allows plants to withstand more severe drought stress later in the season (Cutforthet al. , 1986; Lamichhane et al. , 2018). Understanding the influence of chilling on early developmental stages of sunflower is crucial to identify the components of early cold tolerance, which increases oil yield in the context of ES.
These field experiments highlight the impacts of ES on early morphological traits. We observed an 80% decrease in the rate of vigor growth and a 42% decrease in the rate of height growth. However, in these ES experiments, low temperatures were correlated with the daylength and solar radiation intensity. To assess impacts of low temperatures alone, we developed an indoor experiment that reproduced the night chilling stress observed and modeled in a French field environmental network (Mangin et al. , 2017).
Sunflower responded similarly to night chilling (i.e., decrease in leaf area, hypocotyl diameter, root length and number of secondary roots) as maize did in other studies (Hussain et al., 2020; Walne and Reddy, 2022). Low temperatures decreased sunflower development, resulting in smaller leaves and roots. These phenotypes likely result from direct effects of low temperatures on plant physiology: cold prolongs the cell cycle and slows the growth rate, which results in smaller organs (Rymen et al. , 2007). Low temperatures also influence the metabolism of auxin, which strongly regulates plant growth. Rahman (2013) observed that cold stress immobilized the cellular auxin transport protein and altered the auxin gradient usually associated with organ formation. This influence could explain the decrease in root length and increase in root diameter that we observed for sunflower. A decrease in the root system influences water and nutrient uptake, which is high near root tips due to increased expression of nutrient transporters and water channels. Branching is an effective way to increase the absorptive surface area of the root system (Dinneny, 2019), which ultimately decreases the yield (Hammer et al. , 2009). The decrease in leaf area under low temperatures is caused by lower cell division and cell elongation (Ben-Haj-Salah and Tardieu, 1995), which decreases light interception and thus the yield (Merrien, 1992).
Since early chilling stress decreases oil yield, we explored the morpho-physiological mechanisms underlying this long-term effect. We observed no increase in plant height at flowering despite a notable difference in the number of days required for flowering (+33.2% for ES). Oil yield increased (80%), as did seed weight and oil content (52.5% and 16.8%, respectively). Previous studies, such as those of Tahir et al. (2009), Demir (2019) and Abdelsatar (2020), also observed an increase in oil content (+7.7%), thousand kernel weight (+27.2%) and oil yield (+43.2%) with earlier planting dates for sunflower and wheat. The longer life cycle allowed plants to accumulate more photo-assimilates and remobilize them to seeds, as suggested by Evans and Wardlaw (1976) and Giannini et al. (2022) for wheat and rice. However, the relation between the duration of the vegetative phase and yield is complex. Some studies observed a positive correlation between the duration of vegetative growth and yield for sunflower (Gontcharov and Zaharova, 2008; Abdelsatar, 2020), while other studies observed a higher yield with a shorter vegetative phase and longer grain-filling phase for wheat (Sharma, 1992). This complexity is likely due to interactions among multiple abiotic stressors (e.g., early cold, late drought) and low temperatures combined with low solar radiation after ES.
In the present study, UAV and platform phenomics helped distinguish the impact of night chilling alone on late developmental stage and showed that it had no influence on leaf area or later growth rates, but did influence flowering time and thus growth duration, resulting in taller plants and more biomass. These results agree with those of previous studies of wheat (Tahir et al., 2009) and sunflower (Alkioet al. , 2003; Demir, 2019). Ferreira and Abreu (2001) observed similar results for sunflower, with a 29% increase in the number of days between emergence and flowering with ES compared to normal sowing.
These morphological changes reflect underlying physiological processes related to cell division, extension, energy pathways and abiotic signaling. One well-documented physiological process related to cold resistance is an increase the unsaturation of fatty acids in cell membranes. We observed similar results, but specifically in hypocotyls rather than roots or leaves. This hypocotyl response has not been reported for other plants, but it has been observed for rapeseed (Brassica napus) , maize and wheat leaves and roots at varying levels of polyunsaturation (Tasseva et al. , 2004; Makarenkoet al. , 2011; Nejadsadeghi et al. , 2015).
Low temperatures decrease cell membrane fluidity and permeability, which results in electrolyte leakage and alters internal cell homeostasis (Barrero-Sicilia et al. , 2017). Destabilization of the chloroplast membrane has a cascade of negative effects on chlorophyll content, photosynthetic enzyme activity and the electron-transport chain (Banerjee and Roychoudhury, 2019), resulting in the production of ROS until chlorosis symptoms appear (Wise, 1995; Suzuki and Mittler, 2006). Our results agreed with those of Fabio et al. (2022), given the 10.3% decrease in chlorophyll content and the 9.0% and 7.2% increase in H2O2 and O2·-, respectively. Hussain et al. (2020) observed a 100% and 120% increase in H2O2 and O2·-, respectively, in maize leaves. Zhu et al. (2013) observed similar results for sugarcane (Saccharum officinarum ) leaves, as did Nejadsadeghi et al.(2015) for wheat leaves. As ROS concentrations increase in organs, the risk of damaging proteins, DNA and lipids increases (Apel and Hirt, 2004). This negative impact on plant physiology is managed by the ROS scavenging mechanism, which involves superoxide dismutase, ascorbate peroxidase, catalase and glutathione peroxidase (Cassia et al. , 2018). These results demonstrate the complex and interconnected physiological responses of sunflower to cold stress. Our results indicate that night chilling decreases the stability of cell membranes, which leak electrolytes. Disruption of chloroplast membranes is followed by a decrease in chlorophyll content and production of ROS due to the decrease in the efficiency of the electron-transport chain, which results in cell damage (Fig. 10).
To reveal the molecular mechanisms underlying the physiological responses observed, we explored the transcriptomic responses of leaves, hypocotyls and roots under chilling stress. The effects of cold stress were especially pronounced in leaves. Histone genes were the most abundant group of DEGs in leaves, which is consistent with results of Kumar and Wigge (2010) for A. thaliana that showed the critical role of histone H2A.Z in sensing temperature. This histone binds to the promoters of temperature-responsive genes, which can inhibit the binding of repressors. It is therefore plausible that an epigenetic mechanism of stress memory had long-term effects throughout the sunflower life cycle in our experiments.
Many other DEGs belonged to groups which are potentially linked to chilling stress, i.e. lipases, germin-like proteins, glycine-rich proteins, chaperones, heat shock proteins (HSP), and late embryogenesis abundant proteins (Winfield et al. , 2010; Barrero-Siciliaet al. , 2017). ). Interestingly, we observed no DEGs related to the ICE-CBF-COR cascade, perhaps due to different sampling strategies. We sampled tissues after three weeks of exposure to chilling stress, while these genes are usually found to be differentially expressed after 3-48 h of treatment (Lee et al. , 2005; Song et al. , 2013; Londo et al. , 2018), which highlights the time-dependent nature of gene expression in response to chilling.
Analysis of DEGs confirmed the importance of histone and nucleosome activity, and highlighted other functions potentially related to the chilling response. This was the case for the ontologies related to carbohydrate metabolism, whose induction has been identified in peanuts (Arachis hypogaea ) (Zhang et al. , 2020) and grapevine (Vitis vinifera ) (Londo et al. , 2018). This was also observed for ontologies related to ROS metabolism, which have been described as a chilling stress response in species such as cotton (Gossypium herbaceum ) (Tang et al. , 2021) and Chinese cottonwood (Populus simonii ) (Song et al. , 2013). The increase in ROS production, particularly under high light levels, could be associated with photoinhibition (Banerjee and Roychoudhury, 2019), resulting in the degradation of chlorophyll, which may explain the decrease in chlorophyll content in leaves during the cold stress.
The transcriptomic profile of hypocotyls was characterized by two main processes. The first was lipid metabolism, including lipid unsaturation, which was represented by two genes and is consistent with the fatty acid profiles of hypocotyls. Lipid unsaturation in response to chilling stress is usually observed in leaves (Barrero-Sicilia et al. , 2017; Zhang et al. , 2020). Our study is the first to identify this process in hypocotyls. Three other lipid metabolism terms in response to chilling were alpha-linolenic acid, glycerolipid and glycerophospholipid metabolisms, all of which are related to membrane remodeling. To date, these pathways have been described only in leaves, such as those of peanuts (Zhang et al. , 2020).
The second main process that characterized hypocotyl transcriptomes was the biosynthesis of cutin, suberin and wax, which was represented by 29 DEGs and confirmed by enrichment and deregulation analyses. The leaf cuticle is the first line of physical defense against abiotic stresses (Barrero-Sicilia et al. , 2017), and activation of cutin-related pathways under chilling stress has been described, for instance in rubber trees (Hevea brasiliensis ) (Gong et al. , 2018). However, the role of cutin in plants is usually described in leaves.
In addition to these two main processes, other relevant pathways related to chilling stress were identified in hypocotyls. First, phenylpropanoid metabolism, represented by several key DEGs, is homologous to theA. thaliana EARLI1 and phenylalanine ammonia lyase genes, respectively. The latter is induced by low temperatures in the hypocotyls of A. thaliana and leaves of rapeseed (Cabane et al. , 2012) and is related to enhanced lignin synthesis, which increases the mechanical resistance of cell walls and reduces dehydration. Overexpression of EARLI1, a lipid transfer protein, reduces electrolyte leakage during freezing stress, which suggests that it helps maintain membrane stability (Bubier and Schläppi, 2004).
To better understand the response of sunflower to abiotic stresses, we adapted the ROS wheel developed for A. thaliana (Willems et al. , 2016) to the sunflower genome (Badouin et al. , 2017). Doing so highlighted the relevance of ROS metabolism in hypocotyls, with two enriched ontologies and six ROS-wheel-related deregulated pathways, and indicated that hypocotyls and leaves had similar responses to chilling. Our transcriptomic experiment also revealed a role of the anti-oxidant vitamin B1 (thiamine) (Subki et al. , 2018). Chilling stress did not influence the root transcriptome greatly under hydroponic conditions, but it did deregulate the metabolism of the anti-oxidant vitamin B2 (riboflavin). Riboflavin has anti-oxidant properties in several plants, including apples (Malus spp.) (Zha et al. , 2022). Identification of pathways related to ROS metabolism and scavenging in the three organs highlighted the major role of these processes in the cell response to chilling.
We reproduced field conditions in spring in France to characterize sunflower after three weeks of exposure to moderate cold stress (night chilling). In this context, we observed no expression of well-known genes such as those involved in the ICE-CBF-COR cascade, which are usually induced shortly after exposure to cold stress. Overall, this shows that the transcriptome is highly plastic and suggests a sequential action of genes. The literature indicates that during the first few hours of exposure, the main genes are related to signal transduction and protein synthesis, indicating a potentially active response strategy. In contrast, after three weeks, we observed genes associated with long-term changes, such as those involved in lignin and lipid metabolism, which alters membrane lipid composition, which may be a passive strategy that requires less energy to resist stress by structurally modifying plants and their reactivity through epigenetic changes.
To study long-term impacts of early night chilling, we characterized sunflower using high-throughput phenotyping platforms combined with agronomic and biochemical measurements. Our field observations (e.g., 19% increase in C16:0, 44% decrease in oleic acid, 45% increase in linoleic acid) agreed with observations of Flagella et al. (2002) for sunflower, suggesting that the activation of desaturases required in early stages to maintain membrane fluidity remained in the seeds three months later. Similarly, the chlorophyll content increased consistently throughout the life cycle in leaves that had not developed at the time of stress. This result suggests that molecular memory may be transmitted through cell division, likely epigenetic changes, such as DNA methylation, phosphorylation or histone acetylation (Trewavas, 2016), whose stability is related to memory duration (Villagómez-Arandaet al. , 2022). Although our data did not identify these epigenetic markers, such as DNA methylation, in later developmental stages, future studies could explore long-term effects of morphological modifications induced in early stages or due to epigenetic modifications.