Sunflower (Helianthus annuus L.) is an oilseed crop widely spread throughout the world. It is mainly used for nutritional and cosmetic oils production. In Argentina, the area cultivable with sunflower covers around two million hectares. Planted area and average yields per hectare of the latest campaigns denotes a displacement of the sunflower growing areas towards the west of the Pampas region and the Chaco area, in addition to their consolidation in the southeast region of Buenos Aires (Rossi et.al., 2021). There is great genotypic variability and commercial hybrids adapted to different environmental conditions. Particularly in Argentina, the use of high-oleic (HO) sunflowers has expanded. The sunflower oil obtained from HO cultivars is one of the most appreciated oils on the market. An oil with a high concentration of omega 9 fatty acids is produced from seeds HO sunflower, which does not contain trans fats and has a low proportion of saturated fatty acids compared to others (Alberio et al., 2016). High-oleic cultivars are generally less sensitive to cold temperatures, and therefore have more stable oleic acid concentrations than traditional ones (Dill et al., 2019). It has been identified genotypes with different germinative responses to cold temperatures. These genotypes were commercial cultivars: hybrid Sierra HO and Pampero, which have shown to be tolerant and sensitive to cold, respectively (Fabio and Díaz, 2012). The average sunflower cycle comprises 125 days depending on thermal sums, genotypes, sowing dates, latitude and availability of water and nutrients (ASAGIR, 2022). Sunflower base-growth temperature is around 8°C. Sunflower is a summer crop, planting should occur as early as possible in the spring season to avoid crop critical period exposure to water deficit and heat stress in the hotter summer months (Alline et al., 2009; García-López et al., 2014; Hussain et al., 2018). But this early sowing management practice to maximize seed and oil yield often expose seedlings to cold temperatures. It is important to increase sunflower cold-tolerance in the early stages of growth and development to allow early successful sowing (Škorić, 2016).
Low temperature represents one of the most harmful abiotic stressors affecting temperate plants (Janská et al., 2013). Cold stress or low temperature exposure, can decrease plant metabolism activity and even impose severe yield penalty on crop plants (Stitt and Hurry 2002; Nord and Lynch 2009). The damage caused by low temperatures during sunflower seedling stage slows down crop growth. Some traits, such as plant chlorophyll content and dry weight, have been positively associated with cold-stress tolerance in sunflower crop (Allinne et al., 2009). Hewezi et al. (2006) demonstrated that low temperatures modify plant morphology through changes in some physiological mechanisms, such as biosynthesis of proteins and sugars; they also reported a decrease in the expression of genes encoding glutamate and synthesis of sugars (sucrose, fructose and mannose), which are compounds involved in carbon fixation. One way to evaluate cooling tolerance is through measuring physiological, biochemical and growth parameters that allow to identify contrasting responses and integrate relationships with a better development and growth of the seedling (Pittaro, 2015). The most common physiological parameters used are the loss of membrane electrolytes and the content of chlorophyll, since they are highly affected by stress and are highly correlated with growth. Loss of stability of cell membranes due to low temperatures in turn produces a reduction in their capacity to maintain intracellular content. The transfer of intracellular content to the extracellular medium has been frequently reported. A high percentage value means a substantial ion loss through membranes, which become unstable when affected by cold. The relative loss of electrolytes is usually measured to evaluate cell damage caused by chilling, both via low positive (Campos et al., 2003; Janowiak et al., 2003) and low negative temperatures (Pan et al., 2021).
On the other hand, cold temperatures generate oxidative stress in plants by accumulating reactive oxygen species (ROS) and the regulation of oxidative metabolism is analyzed by the oxidative damage and the antioxidant defense. Oxidative stress is a common phenomenon involved in abiotic and biotic stress in plants. Under stress conditions, such as extreme high or low temperatures, ROS are generated through metabolic processes, acting as change signals regulating gene expression (Pastori and Foyer, 2002; Desikan et al., 2004; Mittler et al., 2004) and ion channel activity (Foreman et al., 2003). ROS including superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (•OH), are one of the earliest known biochemical responses of eukaryotic cells to abiotic stresses. ROS are produced mostly in chloroplasts, mitochondria, and peroxisomes (Apel and Hirt, 2004). In higher plants, increased ROS production is a characteristic inherent to a stressed metabolism under diverse stress types (Wei et al., 2019). Stress response mechanisms are accompanied by the production of antioxidant enzymes involved in ROS degradation. The main anti-stress enzymes are Superoxide dismutase (SOD), Catalase (CAT), Ascorbate peroxidase (APX), and Glutathione reductase (GR) (Cassia et al., 2018). Inadequate ROS degradation causes oxidative stress, characterized by damaging ROS reactions with biologically important macromolecules such as proteins, lipids and DNA, likely producing cell damage (Inze and Van Montagu, 1995). Oxidative stress causes damage at the cellular membrane level via lipid peroxidation, which is manifested in malondialdehyde (MDA) content. Plant growth morphological traits, such as height, dry weight and fresh weight, are also reduced under stress conditions (Eremina et al., 2016). These parameters have been used as selection criteria in genotypes of Chloris gayana K. and Cenchrus ciliaris L. under high temperatures, salinity and drought conditions (Luna et al., 2005; Lanza Castelli et al., 2010; Tommasino et al., 2018). However, there is no information about the use of these indicators to characterize cold-tolerant sunflower genotypes.
Studying physiological and biochemical responses to low temperature might allow to identify tolerant materials (Gombos et al., 1994; Wada et al., 1994; Guinchard et al., 1997; Maury et al., 2000; Campos et al., 2003; Allinne et al., 2009; Sallam et al., 2019). The objective of this work was to evaluate the physiological and biochemical responses of two contrasting sunflower genotypes in their cold-tolerance after being exposed to a period of cold stress, in order to find relationships with growth performance in the seedling stage.