Low temperature, as a major abiotic stress, impacts the formation of high-quality tobacco seedlings. It is urgent to take appropriate measures to improve the low-temperature tolerance of tobacco seedlings. A hydroponics experiment was conducted with a tobacco cv. Y2001 under 25 °C (control temperature) and 10 °C (low-temperature stress). Three phosphorus (P) levels including the traditional P concentration (2 mM PO43−) and higher P levels (3 mM PO43− and 4 mM PO43−) were applied to investigate their effects on antioxidant metabolism and carbohydrate metabolism in low-temperature-stressed tobacco seedlings. The results showed that the low temperature decreased plant height, stem diameter, and biomass of shoots and roots, while the higher P levels promoted plant height and shoot biomass of low-temperature-stressed tobacco seedlings compared to the traditional P level. The leaf net photosynthetic rate (AN) was decreased by the low temperature, while the AN of low-temperature-stressed tobacco leaves was increased by 38.6–61.3% for the higher P levels than the traditional P level. Higher O2− and H2O2 were observed in tobacco leaves exposed to low-temperature stress, damaging the AN, although the low temperature upregulated the expression of encoding superoxide dismutase (NtSOD), peroxidase (NtPOD), and catalase (NtCAT). However, compared with the traditional P level, the higher P levels further upregulated the expression of NtSOD and NtCAT in low-temperature-stressed tobacco leaves to accelerate O2− and H2O2 removal. Higher leaf sucrose content was detected since the low temperature significantly downregulated the expression of NtSuSy, NtCWINV, and NtNINV encoding sucrose synthase, the cell wall, and alkaline invertases, respectively, inhibiting sucrose hydrolysis. Compared with the traditional P level, higher P levels downregulated the expression of NtCWINV in low-temperature-stressed tobacco leaves, further promoting leaf sucrose content. The low temperature downregulated the expression of NtAGP encoding ADP-glucose pyrophosphorylase, NtSSS encoding soluble starch synthase, and NtGBSS encoding granule-bound starch synthase, thereby restricting starch biosynthesis. Additionally, the low temperature upregulated the expression of α-amylase and β-amylase, accelerating starch hydrolysis. These led to a lower starch content in low-temperature-stressed tobacco leaves. The higher P levels further upregulated the expression of α-amylase in low-temperature-stressed tobacco leaves than the traditional P level, further lowering the starch content. Moreover, the leaf soluble sugar content was higher under the low temperature than the control temperature, which helped the tobacco plants resist low-temperature stress. And higher P levels further promoted the soluble sugar content in low-temperature-stressed tobacco leaves compared with the traditional P level, further improving tobacco seedlings’ low-temperature tolerance. Therefore, these results indicated that increasing the P application level can alleviate the adverse impacts of cold stress on antioxidant metabolism and carbohydrate metabolism in tobacco seedlings.
