| dc.description.abstract | Cold stress causes substantial losses in global agricultural productivity by impairing plant growth, physiology, and survival. Safflower (Carthamus tinctorius L.), an ancient, marginalized, neglected yet multipurpose domesticated oilseed crop, is known for its cold and drought tolerance. However, the crop has shown phenological stage-dependent susceptibility to cold stress, especially under Mediterranean type climates, posing a major threat to safflower growers. Moreover, the underlying physiological, biochemical, and molecular mechanisms of cold signal transduction in different developmental stages of safflower are poorly understood due to a lack of systematic research on cold signalling pathways and gene regulatory networks. To address this knowledge gap and issues of chilling injury (CI), this study investigatedchilling incidence, physiological, biochemical, and molecular responses of safflower to chilling stress at two phenological stages (rosette and bolling) under both controlled (artificial) and natural (field) conditions. The experimental designs were 2 x 4 x 4 factorial design in artificial chilling condition and 2 x 4 factorial laid down in a completely randomized block design (RCBD) in natural conditions. The results demonstrated that chilling injury incidence (CII) occurred when safflower plants at the bolling stage were exposed to 0 and 4°C for durations of 8 and 12 hours, respectively under artificial chilling stress (refrigerator). While safflower plants at the rosette stage under artificial chilling stress induction did not show any symptoms of CI. On the other hand, plants naturally chilled did not exhibit any signs of CII at either the rosette or bolling stage. Moreover, safflower plants chilled at 4°C for 8 to 12 hours showed moderate increases in relative electrolyte leakage (REL), up to 17.1% and reduced proline levels from 4.79 to 5.40 µmol/g FW under artificial cold stress. While at 0°C for 12 hours, plants at the bolling stage experienced severe membrane damage as indicated by a sharp increase in REL to 65.7%, compared to12.0% rise in rosette plants. This was accompanied by a drastic decline in total chlorophyll content (Chl a+b), from 2.64 to 1.19 mg/g FW and stomatal conductance (gs) reducing accumulation peaked at 8.84 µmol/g FW in rosette plants but remained low (~4.9 µmol/g FW) in bolling plants. Carotenoids increased in rosette plants from 1.04 to 1.77 mg/g FW but decreased in bolling plants from 0.91 to 0.37 mg/g FW. For the field experiment, exposure of plants to 2°C at night in the bolling stage increased REL (20.18%) compared to rosette plants (6.81%), with gs declining significantly but partially recovering by mid-day from 311.35 to Safflower plants at the rosette stage exposed xx to chilling stress at 0°C for 12 hours significantly (P < 0.05) produced higher proline content of 8.84 µmol/g FW than plants in both development stages exposed to low temperatures of 0 and 4°C for 8 and 12 hours and control. Proline levels increased to 11.65 µmol/g FW in rosette and 10.70 µmol/g FW in bolling plants under field conditions. Safflower genotypes in both phenological stages exposed to chilling stress of 2°C at night under had low leaf chlorophyll content in the morning than control plants. The reduction in leaf chlorophyll content in all genotypes was much higher in plants at the bolling stage compared to the rosette stage. The genotype Sina had higher leaf chlorophyll contents of 73.87 and 61.97 (SPAD) at rosette and bolling stages, respectively than other genotypes under field conditions. In artificial cold induced stress experiments, safflower plants at the rosette stage exhibited enhanced ) increasing from 0.35 (control) to 0.71 after exposure to 4°C for 12 hours, while plants at the bolling stage showed a decrease from 0.40 to 0.19 under the same conditions. Similarly, non-photochemical s at the rosette stage but dropped sharply from 0.56 to 0.23 in plants at the bolling stage exposed to 4°C for 8 hours. At 0°C for 8 hours, the bolling was later reversed with a 45.42% decrease after 12 hours. Nondeclined in safflower plants at the rosette stage from 0.21 (control) to 0.15 and 0.18 at 4°C and 0°C, respectively, but increased in plants at the bolling stage from 0.21 to 0.31 and 0.26. Cold stress drastically reduced ETR II from 220.17 (control) to 0.22 at both 0°C and 4°C across all stages. Under field conditions, morning cold exp to 0.57 for plants at the rosette stage, while plants at the bolling stage experienced a reduction from 0.57 to 0.51. Concurrently, plants at the rosette 0.16 to 0.17) than plants at the bolling stage (from 0.16 to 0.28). While mid-day recovery showed higher ETR II in plants at the rosette stage (312.23) than plants at the bolling stage (237.92). Among genotypes, Sina exhibited the highest ETR II (300.97), followed by Gila (272.02), PI 537636 (265.77), and Turkey (261.54). Antioxidant enzyme activities (CAT, SOD, POD) also varied significantly. Under artificial cold stress, plants at the rosette stage showed peak activities at 0°C for 12 hours: CAT (1.80 U/mL FW), POD (2.29 U/mL FW), and SOD (4.45 U/mL FW). In contrast, plants at the bolling stage under prolonged cold exposure of 0°C for 12 hours led to a sharp decrease in CAT (0.31 U FW) and SOD (0.41 U FW) activities, and a significant reduction in POD activity (0.27 U FW), indicating differential stress responses across growth stages. Meanwhile, under natural cold stress, CAT, SOD, and POD activities were notably influenced by genotype and time of day. xxi Gila demonstrated the highest CAT activity of 1.58 U/mL FW in plants at the bolling stage, while Sina exhibited the highest mid-day SOD (3.21 U/mL FW) and POD (0.92 U/mL FW) activities. At the molecular level, expression analysis identified three cold-responsive genes: two E3 ligase genes (CtAH10T0001700.1, CtAH10T0001500.1) with RING finger domains, and a GRAS family gene (CtAH10T0000500). These genes successfully exhibited similar cold responses as per their respective family. Overexpression of some E3 genes like CIP8 and HOS 1 could be associated with the cold susceptibility of plants at the bolling stage. Additionally, CtAH10T0001700.1 expression was found to be positively correlated with electrolyte leakage and proline content in safflower plants at the rosette stage. Meanwhile CtAH10T0001500.1, a negative cold regulator significantly correlated with electrolyte leakage and proline content. While CtAH10T0000500, a positive cold regulator significantly correlated with electrolyte leakage and proline at the rosette stage but negatively associated with the two parameters in plants at the bolling stage. Overall, the genes were found to play a significant role in cold responsiveness of safflower plants at the rosette and bolling phenological stages. In conclusion, safflower exhibited developmental stage-specific responses to cold stress, with plants at the rosette stage demonstrating better physiological resilience, antioxidant defence, and gene regulation than plants at the bolling stage. These findings provide novel insight into the cold signal transduction pathways and identified molecular markers for breeding cold-tolerant safflower genotypes. Therefore, the study recommended that future research and breeding efforts should focus on enhancing safflower cold tolerance, particularly at the bolling stage, by targeting physiological, biochemical, and molecular traits associated with resilience at this and yield stability under chilling conditions. | en_US |
