Zeaxanthin pretreatment alleviates low-temperature stress-induced injury in flue-cured tobacco seedlings by modulating the xanthophyll cycle and photoprotection

Low temperature is a major abiotic stress that affects flue-cured tobacco ( Nicotiana tabacum L.) seedlings during the early transplanting stage. Zeaxanthin (Zea), a xanthophyll pigment closely associated with the photosynthetic apparatus has been reported to alleviate chilling injury in crops. However, its physiological roles in flue-cured tobacco under alternating chilling and rewarming conditions remain unclear. In this study, seedlings of the flue-cured tobacco cultivar Yunyan 87 were pretreated with Zea at 0, 25, 50, 75, or 100 mg L -1 and subsequently exposed to low-temperature stress at 10 °C during both the day and night, followed by rewarming. Among the concentrations tested, 75 mg L -1 was the most effective in alleviating low-temperature injury. Compared with the low-temperature control (T1), Zea pretreatment increased plant height and maximum leaf area by 14.60%–34.03% and 21.90%–39.12%, respectively, during the stress period, and these beneficial effects persisted during rewarming. Zea pretreatment also alleviated membrane damage, as indicated by reduced malondialdehyde content and relative electrical conductivity, and improved photosynthetic performance. Across the sampling periods and Zea treatments, the maximum increases in chlorophyll content, net photosynthetic rate, and maximum photochemical efficiency of photosystem II ( Fv/Fm ) were 20.35%, 216.26%, and 70.78%, respectively, relative to T1. Targeted carotenoid analysis showed that exogenous Zea increased leaf zeaxanthin content by up to 624.61% and promoted xanthophyll-cycle de-epoxidation. It also increased non-photochemical quenching by 41.40% relative to T1 during the stress period, indicating an enhanced capacity for photoprotective thermal dissipation. Furthermore, Zea pretreatment strengthened antioxidant and osmotic adjustment, as evidenced by increased catalase, peroxidase, and superoxide dismutase activities and higher soluble protein and proline contents. Hormone analysis further revealed increases in indole-3-acetic acid, jasmonic acid, and brassinosteroid contents, accompanied by a reduction in abscisic acid content. Collectively, these findings indicate that Zea pretreatment enhances the low-temperature tolerance of flue-cured tobacco seedlings and that this enhancement is associated with xanthophyll cycle modulation, improved photosynthetic performance, and strengthened antioxidant, osmotic, and hormonal adjustments.