Salt-stress secondary metabolite signatures involved in the ability of Casuarina glauca to mitigate oxidative stress

The model actinorhizal plant Casuarina glauca is characterized by its capacity to establish nitrogen-fixing root-nodule symbiosis with Frankia bacteria and by its resilience to thrive under extreme environments, such as high salinity. Previous studies have shown that salt tolerance in C. glauca is linked to photosynthetic, primary metabolic adjustments and to an effective antioxidant machinery. This study aimed at analyzing the secondary metabolite composition of tissues from nodulated (NOD⁺) and non-nodulated (KNO₃ ⁺) C. glauca plants (nodules, roots, and branchlets) with liquid-chromatography-high resolution mass spectrometry (LC-HRMS) to evaluate the effect of three NaCl concentrations (200, 400 and 600 mM) on the secondary metabolite composition of these two plant groups. In the branchlets, significant accumulation of almost all annotated secondary metabolites, including flavonoids and their glycosides, was mainly induced by the highest salt concentration. Furthermore, the different secondary metabolite levels between roots and nodules were associated to the previously reported impairment of symbiotic activity from 200 mM NaCl onwards. Ionome analysis in C. glauca branchlets revealed salt stress-induced changes in ion homeostasis, which in turn, affected the K⁺/Na⁺ ratio. Nevertheless, despite these changes C. glauca plants can still maintain its primary metabolic activity. Moreover, our results suggest that C. glauca plants activate a flavonoid-based secondary antioxidant system to mitigate the impacts of oxidative stress, thereby enhancing their salt stress tolerance.