Benzothiadiazole (BTH) activates sterol pathway and affects vitamin D3 metabolism in Solanum malacoxylon cell cultures.

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D3 metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50 μM BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3′-diaminobenzidine (DAB) for hydrogen peroxide (H2O2) detection and phloroglucinol for lignin staining showed that BTH causes H2O2 accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (β-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D3, and the hydroxylated metabolites 25-hydroxyvitamin D3 [25(OH)D3] and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D3, though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D3 and 1α,25(OH)2D3 metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol.