Multi‑omics dissection reveals that fumarate serves as a metabolic hub linking Pro-Ca application to salt tolerance in rice tillers

AbstractBackgroundSoil salinization is a major abiotic constraint limiting rice growth and productivity worldwide. Rice (Oryza sativa L.) is moderately salt-sensitive, and salt stress significantly inhibits tillering, a key determinant of panicle number and final yield. Prohexadione calcium (Pro-Ca) is a novel plant growth regulator that inhibits gibberellin biosynthesis and has been shown to alleviate salt stress in rice seedlings. However, the molecular mechanisms by which Pro-Ca regulates salt tolerance in rice tillers remain poorly understood. In this study, we investigated the regulatory effects of exogenous Pro-Ca on salt-stressed tillers of the indica rice variety 9311 using integrated physiological, transcriptomic, and metabolomic approaches.ResultsExogenous Pro-Ca treatment significantly alleviated salt-induced inhibition of tillering, increasing tiller number by 64.7%–137.5% compared with salt stress alone, while its effect on stem basal width was relatively modest. Pro-Ca enhanced antioxidant enzyme activities (SOD, APX, POD, CAT) by 4.9%–30.6%, reduced MDA accumulation by 15.2%–26.0%, and partially restored soluble sugar and protein levels. Ion analysis revealed that Pro-Ca decreased Na⁺ content by 50.7%, increased K⁺ content by 13.7%, and elevated the K⁺/Na⁺ ratio by approximately 1.3-fold, while IAA and SA contents increased by 40.0% and 22.5%, respectively. Transcriptomic analysis showed that Pro-Ca shifted the transcriptional response from passive damage repair (photosynthesis inhibition, cell wall degradation) toward active defense signaling, with significant enrichment of salicylic acid response, ethylene-activated signaling, and defense-related pathways. Metabolomic profiling revealed that Pro-Ca redirected metabolic flux from amino acid and energy metabolism toward alpha-linolenic acid metabolism, while Z-score analysis identified N-Carbamoylputrescine as a consistently upregulated key metabolite and 4-hydroxy-2-nonenal as a downregulated oxidative stress marker. Integrated multi-omics analysis pinpointed N-Carbamoylputrescine and Vanillin as central hubs linking gene expression changes with metabolic adjustments, and pathway integration revealed that the TCA cycle functions as a carbon–nitrogen metabolic hub, with fumarate significantly elevated across three interconnected pathways. Furthermore, Pro-Ca activated cutin, suberine, and wax biosynthesis pathways, suggesting physical barrier reinforcement.ConclusionOur findings demonstrate that exogenous Pro-Ca enhances salt tolerance in rice tillers through a multi-layered synergistic strategy: reconstructing ion homeostasis by reducing Na⁺ accumulation while preserving K⁺ levels and coordinately elevating IAA (growth promotion) and SA (defense activation) contents; reprogramming the transcriptome from passive repair to active defense signaling, coupled with metabolic reprogramming of polyamine biosynthesis, lipid peroxidation alleviation, and TCA cycle maintenance; and reinforcing physical barriers through cutin, suberine, and wax biosynthesis. This study provides novel insights into the molecular mechanisms by which Pro-Ca regulates salt tolerance in rice tillers and offers a theoretical basis for the application of plant growth regulators in saline-alkaline rice production. Keywords: metabolome; rice; salt stress; tillering; transcriptome