TMTM maintains the sulfur homeostasis under sulfur limitation
Plants respond to sulfur limitation in various ways. The first response is up-regulation of sulfate transporters (SULTRs ) to increase sulfate uptake from root (Takahashiet al. , 2011; Shibagaki et al. , 2002; Yoshimoto et al. , 2002; Kataoka et al. , 2004; Takahashi et al. , 1997). On the other hand, genes for GSL biosynthesis (e.g., BCAT4 ,CYP79B2 and CYP79F2 ) are down-regulated, while those repressing GSL biosynthesis (SDI1 and SDI2 ) are up-regulated. These responses help plants to remobilize sulfur to sustain growth (Lewandowska and Sirko, 2008; Frerigmann and Gigolashvili, 2014; Borpatragohainet al. , 2016). Among the inspected genes, the sulfur starvation genes SULTR1;1 , SULTR1;2 , SULTR2;1 and bothSDI1 and SDI2 were down-regulated in a TMTM dose-dependent manner in plants which suffer from sulfur limitation (Figure 5A and 5B). Since the sulfur in TMTM can be incorporated into the plant material, the expression of the above-mentioned genes and those involved in GSL and GSH metabolism is similar to seedlings grown under HS condition without TMTM. Moreover, excessive TMTM results in the upregulation of these genes, indicating that these plants are actively moving excess sulfur to secondary metabolites. This is in accordance with a recent study by Sugiyamaet al. (2021), showing a retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis. Interestingly, the mRNA levels for GSL and GSH metabolism genes was higher in seedlings after 2 days on LS medium without TMTM compared to seedlings grown on HS medium. This might be caused by higher sulfate influx from the medium due to up-regulation of SULTRs . The plants actively metabolize the assimilated sulfate into various metabolites and utilize this as a store to sustain growth under sulfur limitations.
The effect is also observed at the metabolic level. Under sulfur limitation, the GSH and GSL levels decreased. However, 100 µg TMTM maintained the levels high under sulfur starvation conditions (Figure 6). Again, besides maintaining sulfur homeostasis, excess sulfur from the high TMTM dose is largely metabolized into secondary metabolites.