TMTM reduces sulfur deficiency responses
To test whether TMTM serves as sulfur source and affects the sulfur homeostasis of Arabidopsis seedlings, we tested the effect of the volatile on the expression of sulfur-responsive genes and the sulfur metabolite dynamics. Under sulfur limitation conditions, expression of sulfur transporters SULTR1;1 , SULTR1;2 and SULTR2;1was upregulated. Two days after exposure to the volatile, we observed a gradual decrease of their transcript levels and the effect increased with increasing TMTM amounts. Furthermore, the expression of the GSL repressor genes SDI1 and SDI2 was significantly down-regulated by TMTM, again in a dosage-dependent manner (Figure 5A). We further examined the expression of genes involved in the GSL and GSH metabolisms (i.e., BRANCHED-CHAIN AMINOTRANSFERASE4, BCAT4; SULFOTRANSFERASE, SOTs; GLUTAMATE-CYSTEINE LIGASE, GSH1; GLUTATHIONE SYNTHETASE, GSH2; two CYTOCHROME P450, CYP79B2 and CYP79F2 ) . With 10 and 100 µg TMTM, their expression levels were similar to those in seedlings grown on HS medium, and with 1000 µg TMTM, their expression levels increased slightly.
Seven days after volatile application, SDI1 was significantly up-regulated with 10 µg TMTM, while with 100 or 1000 µg TMTM, bothSDI1 and SDI2 remained down-regulated compared to LS without TMTM (Figure 5B). The expression levels of the GSL metabolism genes CYP79B2 and SOTs increased to the levels in seedlings grown on LS without TMTM, and with 1000 µg TMTM, they showed the highest expression.
In conclusion, after the application of 100 µg TMTM to LS-exposed seedlings, expression of the examined genes is similar to that of the seedlings grown on HS medium, and this is observed from the second to the 7th day. We propose that low doses of TMTM (10 and 100 µg) diminish sulfur stress by adjusting the expression of the analyzed genes to the expression levels found under HS conditions. Upregulation of SDI1 and SDI2 in LS-grown seedlings exposed to 10 µg TMTM for 7 days indicates that this doses is too low to repress the sulfur-deficiency response after longer time periods.
TMTM maintained high GSH and GSL levels under sulfur deficiency
Cysteine is the first metabolite synthesized during sulfur assimilation, while GSH and GSLs contain large portions of the total sulfur pool. Under sulfur deficiency, these metabolites are broken down, and the sulfur is recycled for primary growth (Falket al. , 2007; Sugiyama et al. , 2021). We measured the GSH and GSLs level to investigate whether TMTM influences the plant sulfur homeostasis at this level.
Two, 4 and 7 days after application of 1000 µg TMTM, the GSH level was significantly increased compared to the untreated control. Even 100 µg TMTM stimulated the GSH level, which was similar to that found in seedlings grown on HS (Figure 6A).
A similar pattern was observed for the total GSL levels. After 2 days, the GSLs slightly increased with increasing TMTM concentrations (Figure 6B). The effect broadened after 4 days. On LS without TMTM, total GSL level was significantly lowered compared to the rest of the treatments (Figure 6B). Similar to the results obtained for GSH, application of 100 µg TMTM maintained the total GSL level at the same level found in seedlings grown on HS medium without the volatile after 7 days (Figure 6B). We conclude that 100 µg TMTM established sulfur homeostasis in LS-grown seedlings which is comparable to the conditions in seedlings grown on HS. Furthermore, incorporation of TMTM can be observed in seedlings treated with 1000 µg TMTM, since they showed significantly higher amounts of GSH and GSLs than the unexposed controls.