H2S-dependent trehalose-induced salt tolerance in tomato involves phenylpropanoid and cyanoamino acid pathways.

Soil salinity limits tomato growth. Trehalose (Tre) and hydrogen sulfide (H₂S) individually enhance salt tolerance, but their combined effect is unknown. Here, we investigated seedling physiological and transcriptomic responses to exogenous Tre and the H2S donor sodium hydrosulfide (NaHS), alone or in combination, under salt stress. Our results demonstrated that both Tre and NaHS significantly alleviated salt-induced growth inhibition, with the combined treatment showing the most pronounced effects. Pharmacological experiments using the Tre inhibitor validamycin A (VA) and the H2S scavenger hypotaurine (HT) revealed that the protective effect of Tre was largely dependent on H2S, whereas NaHS efficacy was independent of Tre. RNA-seq analysis indicated that Tre modulated the salt stress response of tomato at the transcriptional level via H₂S, with the phenylalanine biosynthesis as a core metabolic target and cyanogenic amino acid metabolism pathway also annotated as a potential output. Compared with the NaCl+Tre treatment, the addition of HT significantly reversed the induced accumulation of protective metabolites, including p-coumaryl alcohol, coniferyl alcohol in the phenylpropanoid pathway and amygdalin and prunasin in the cyanogenic amino acid metabolism pathway. Consistent with the metabolite profiles, the NaCl+Tre treatment significantly elevated the activities of enzymes in the phenylpropanoid biosynthesis pathway (4CL, CCoAOMT, POD) and the cyanoamino acid metabolism pathway (MDL, bglX), as well as the transcript levels of their corresponding encoding genes (4CL1, CCoAOMT, POD9, POD43, MDL3 and bglX1). By contrast, the NaCl+Tre+HT treatment significantly reversed the Tre-induced enhancement of both enzyme activities and gene expression, thereby confirming at the molecular level that H₂S serves as a central mediator in Tre during salt stress alleviation. This study shows that exogenous Tre improves salt tolerance in tomato seedlings, and that inhibition of H2S attenuates this effect. The transcriptomic, metabolomic, and enzyme activity data indicate that H2S is involved in Tre-induced regulation of phenylpropanoid biosynthesis and cyanoamino acid metabolism under salt stress. However, based on the current dataset, our results mainly support a requirement for H2S in Tre action, rather than demonstrating that H2S alone is sufficient to reproduce the full Tre response. Further transcriptomic and functional analyses will be necessary to clarify the precise hierarchical relationship between Tre and H2S.