Underlying Mechanism of Prohexadione Calcium-Mediated Alleviation of Salt Stress-Induced Damage via Modulation of Leaf Lipid Metabolism, Chloroplast Structure and Photosynthetic Performance in Rice Seedlings

Abstract Rice ( Oryza sativa L.) is a staple food crop in China, where salt stress poses a severe constraint on rice growth and development. Prohexadione calcium (Pro-Ca) has been recognized as an effective regulator in mitigating salt stress damage in rice. In this pot experiment, two rice cultivars—the salt-sensitive Huanghuazhan (HHZ) and the salt-tolerant Guanghong No. 3 (GH3)—were used as plant materials. Foliar application of Pro-Ca was conducted at the three-leaf stage, followed by NaCl treatment at the four-leaf stage. Three treatments were established: (1) 0 mM NaCl + 0 mg·L -1 Pro-Ca (control, CK); (2) 70 mM NaCl + 0 mg·L -1 Pro-Ca (salt stress, S); (3) 70 mM NaCl + 100 mg·L -1 Pro-Ca (salt stress + Pro-Ca, SP). On day 11 after NaCl exposure, untargeted lipidomics profiling was performed. Chloroplast ultrastructure was observed via transmission electron microscopy on day 21, and photosynthetic gas exchange parameters 【net photosynthetic rate(Pn), intercellular CO₂ concentration ༈Ci༉, stomatal conductance༈Gs༉, transpiration rate ༈Tr) and photosynthetic pigment concentrations (chlorophyll a, chlorophyll b, carotenoids, total chlorophyll)】 were quantified on day 31 after salt treatment. The underlying mechanism by which Pro-Ca alleviates salt stress-induced damage was elucidated based on the sequential alterations in lipid metabolites, chloroplast ultrastructure, and photosynthetic function.Our results revealed that at the lipid metabolic level, GH3 exhibited a much stronger lipidomic response to salt stress than HHZ, with 283 and 35 differentially accumulated lipids identified, respectively. GH3 showed coordinated remodeling across multiple lipid classes, including dysregulated galactolipids (up-regulated DGDG and partially down-regulated MGDG), reduced phospholipids, and accumulated glycerolipids. By contrast, the lipid response in HHZ was dominated solely by the passive accumulation of glycerolipids (TG and DG). At the chloroplast ultrastructural level, HHZ chloroplasts displayed more severe disintegration of thylakoid lamellae and greater accumulation of osmiophilic plastoglobules, whereas GH3 chloroplasts retained better overall morphology and intact grana architecture. With respect to photosynthetic performance, HHZ was characterized by a marked decline in stomatal parameters accompanied by only a minor reduction in photosynthetic pigment contents, while GH3 exhibited substantial degradation of photosynthetic pigments.Our findings demonstrated that foliar application of Pro-Ca conferred stepwise alleviation of salt stress damage via the regulation of leaf lipid homeostasis. Specifically, Pro-Ca first prioritized the remodeling of leaf lipid profiles and restored membrane lipid composition by modulating glycerolipid metabolism and phospholipid biosynthesis pathways. This lipidomic remodeling subsequently facilitated the restoration of ordered thylakoid grana stacking, reduced the accumulation of osmiophilic plastoglobules, and preserved chloroplast structural integrity. Ultimately, these improvements translated into elevated photosynthetic gas exchange parameters and photosynthetic pigment contents, thereby mitigating salt-induced photosynthetic damage.Notably, the alleviatory effects of Pro-Ca exhibited pronounced genotype specificity. In HHZ, Pro-Ca predominantly targeted the clearance of excess accumulated glycerolipids, leading to a comprehensive recovery of all eight measured photosynthetic traits. In GH3, by contrast, Pro-Ca exerted synergistic protective effects by activating multiple biosynthetic pathways including galactolipid and phospholipid metabolism, resulting in a substantial increase in net photosynthetic rate (Pn).Pathway enrichment analysis indicated that glycerolipid metabolism was the only significantly enriched KEGG pathway in response to Pro-Ca treatment in HHZ (Q < 0.05). The phospholipid biosynthesis pathway showed a strong enrichment trend in the SMPDB database across both cultivars.Collectively, this study elucidates the mechanism by which Pro-Ca mitigates salt-induced photosynthetic damage in rice through modulating leaf lipid homeostasis and maintaining chloroplast structural integrity. The marked genotype-specific regulatory patterns identified herein provide a theoretical basis for the stress-resistant cultivation of rice in saline-alkali soils.