Analysis on root physiology and cold-tolerant gene expression differences in watermelon seedlings with distinct cold resistance

【Objective】In recent years, the climate in northern China has been featured by high variability, with extreme low- temperature events occurring frequently. Watermelon is a typical thermophilic crop, and its seedling stage is highly sensitive to low temperatures, making chilling injury a common issue during early spring cultivation. As a key organ involved in plant growth and metabolism, the root system shows a significant reduction in cellular metabolic activity when the soil temperature drops below 5 ℃. This impairment hinders the effective absorption of nutrients (e.g., nitrogen, phosphorus, and potassium), minerals, and water, thereby inhibiting the normal growth and development of watermelon plants. The impacts range from moderate decreases in yield and quality to severe crop failure in extreme cases. Low-temperature stress has thus become a major constraint on the sustainable development of the watermelon industry in northern China. Clarifying the response mechanism of watermelon seedlings to low-temperature stress is of great significance for breeding cold-tolerant varieties and improving the cold tolerance of this crop. Currently, systematic studies focusing on watermelon seedling roots as the core organ for perceiving low-temperature stress remain insufficient. The physiological metabolic patterns, molecular regulatory mechanisms, and differential responses among varieties with varying cold tolerance under low-temperature conditions have not been fully elucidated. This study investigated the changes in root growth, antioxidant enzyme system activity, endogenous hormone contents, and expression levels of cold-tolerance-related genes in two watermelon varieties with contrasting cold tolerance under low-temperature stress. The objective was to systematically clarify the intrinsic mechanisms underlying the response of watermelon seedling roots to low-temperature stress from morphological, physiological, and molecular perspectives, thereby providing a theoretical basis for the improvement of coldtolerant watermelon varieties and the rational regulation of chilling injury during the seedling stage. 【Methods】Two watermelon cultivars, the cold-tolerant Siweite No. 2 and the cold- sensitive Jinguan, previously screened for cold tolerance, were used as experimental materials. Plump seeds with uniform quality germinated and were raised until the three-true-leaf and one-terminal-bud stage. Seedlings with uniform growth vigor were transferred to artificial incubators and subjected to low-temperature at 5± 1 ℃, with a light intensity of 8000 lx and a 12 h/12 h light-dark cycle; seedlings maintained at 25±1 ℃ served as the control. After 8 days of treatment, root morphological indices (root tip number, total root length, average root diameter, total root surface area, and total root volume) were determined using a root scanner, and root activity was measured via the triphenyltetrazolium chloride (TTC) reduction method. Total RNA was extracted from the roots, reverse-transcribed into cDNA, and quantitative realtime PCR (qRT-PCR) was performed to detect the expression levels of cold-tolerance-related genes, including ClCOR47, ClCNGC20, ClCBF2, ClCBF1, and ClCNGC2. On days 0, 2, 4, 6, and 8 of the treatment, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined by the nitroblue tetrazolium photoreduction method, guaiacol colorimetric method, and ammonium molybdate colorimetric method, respectively. The contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) were measured using the thiobarbituric acid method and spectrophotometry, respectively. High-performance liquid chromatography (HPLC) was employed to determine the changes in endogenous hormones, including indole-3-acetic acid (IAA), gibberellin (GA), and abscisic acid (ABA). 【Results】Low-temperature stress at 5 ℃ significantly inhibited root growth in watermelon seedlings, and all root morphological indices and root activity of both cultivars decreased significantly compared with the control. In Siweite No. 2, the root tip number, total root length, total root surface area, and root activity decreased by 30.59%, 36.24%, 37.12%, and 28.37%, respectively, all of which were significantly lower than the corresponding reductions in Jinguan (35.08%, 43.42%, 47.33%, and 32.57%, respectively). Under low-temperature stress, the activities of antioxidant enzymes in the roots of both cultivars increased to varying degrees. On the 8th day of stress, the SOD, POD, and CAT activities in Siweite No. 2 increased by 6.28%, 10.47%, and 24.74% compared with the control, respectively, which were significantly higher than those in Jinguan (1.32%, 9.56%, and 20.79%, respectively). Regarding endogenous hormone changes, Siweite No. 2 established a balanced regulatory system characterized by“high IAA, low GA3, and high ABA”. On the 8th day of stress, the IAA content increased by 26.74% compared with the initial level, and the ABA content increased to 1.40 times its initial level. In contrast, the IAA content in Jinguan decreased significantly in the late stage of stress, accompanied by insufficient ABA accumulation. Significant differences were also observed in the expression of cold-tolerance-related genes between the two cultivars. In Siweite No. 2, the expression levels of ClCOR47, ClCBF1, and ClCBF2 were significantly or extremely significantly up- regulated; among them, ClCNGC20 and ClCNGC2 were up-regulated to 2.45 and 2.56 times the control level, respectively. In contrast, the expression of ClCBF2 in Jinguan was extremely significantly down- regulated, and the up- regulation of ClCNGC20 was only 2.22 times the control level.【Conclusion】Cold-tolerant watermelon cultivars can enhance their adaptability to low temperatures by maintaining a stable root structure, efficient reactive oxygen species scavenging capacity, coordinated endogenous hormone regulation, and rapid activation of cold-tolerance-related gene expression. In contrast, cold-sensitive cultivars exhibit abnormal regulation at both the physiological and molecular levels, resulting in weak cold resistance. These findings can provide a theoretical basis for the breeding of cold-tolerant watermelon varieties and the regulation of field cultivation.