Tris(methylthio)methane produced by Mortierella hyalina affects sulfur homeostasis in Arabidopsis

Abstract

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Incorporation of the sulfur from the fungal volatile into plant metabolism was shown by ³⁴S labeling experiments. Under sulfur deficiency, TMTM down-regulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

Figure 1

GC–MS chromatogram of the headspace of M. hyalina (black) and the growth medium alone (blue). Identified signals are not present in the headspace of the growth medium. The three strong signals in the chromatogram of the headspace of the growth medium could be identified by MS and RI as benzaldehyde (7.13 min) nonanal (9.44 min), and decanal (11.00 min).

Figure 2

Sulfur atoms from fungal volatiles are incorporated into plant tissues. (A,B) ³⁴S/³²S ratios of glucosinolates and glutathione in shoot (A) and root (B) tissues. (C) ³⁴S/³²S ratio of cysteine (Cys) and methionine (Met) from Arabidopsis shoot. M. hyalina was grown on modified KM plates with the addition of ³²S-ammonium sulfate (unlabeled) or ³⁴S-ammonium sulfate (labeled), and co-cultivated with Arabidopsis seedlings in a desiccator. Error bars represent SEs from 3 biological replicates, each contains at least 3 technical replicates from 16 seedlings. Asterisks indicate significance level from Student’s t-test between unlabeled and labeled samples (*p < 0.05; **p < 0.01; ***p < 0.001). n.d. not detected.

Figure 3

Fungal volatile influences A. thaliana growth under sulfur deficiency. (A,B) Differences in fresh weight (A) and in dry weight (B) of seedlings grown on high sulfate (HS) or on low sulfate (LS) MGRL medium with the addition of TMTM (10, 100 and 1000 µg) to seedlings grown on LS without TMTM. Error bars represent SEs from at least 5 independent biological replicates, each with 8 seedlings. Statistical significance was determined by Duncan’s multiple range test with p-value < 0.05, and indicated with lower-case alphabets.

Figure 4

TMTM contributes positively to root growth. Wild-type (A) or slim1 (B) seedlings’ root growth on high sulfate medium (HS) or on low sulfate medium (LS) with addition of 0, 100 or 1000 µg TMTM was measured 7 days after application. Error bars represent SEs from at least 6 biological replicates for wild-type and 8 biological replicates for slim1. Statistical significance was determined by Duncan’s multiple range test with p-value < 0.05, and indicated with lower-case letters.

Figure 5

TMTM reduces plant response towards sulfur deficiency. Gene expression was analyzed 2 days (A) and 7 days (B) after TMTM application. Values were normalized to seedlings grown on low sulfate (LS) MGRL medium without TMTM (0 µg), and expressed as fold change. RNA from each treatment was extracted from total seedlings (combining root and shoot). Error bars represent SEs from 3 biological replicates, each with 8 seedlings. Statistical significance was conducted on dCq values, determined by Duncan’s multiple range test with p-value < 0.05, and indicated with lower-case alphabets.

Figure 5

TMTM reduces plant response towards sulfur deficiency. Gene expression was analyzed 2 days (A) and 7 days (B) after TMTM application. Values were normalized to seedlings grown on low sulfate (LS) MGRL medium without TMTM (0 µg), and expressed as fold change. RNA from each treatment was extracted from total seedlings (combining root and shoot). Error bars represent SEs from 3 biological replicates, each with 8 seedlings. Statistical significance was conducted on dCq values, determined by Duncan’s multiple range test with p-value < 0.05, and indicated with lower-case alphabets.

Figure 6

TMTM maintains sulfur-containing metabolites under sulfur deficiency. (A) Relative glutathione (GSH) level and (B) total glucosinolate (GSL) level in seedlings grown on low sulfate (LS) MGRL medium with addition of TMTM (0, 100 and 1000 µg) and seedlings grown on high sulfate (HS) MGRL medium 2, 4 and 7 days after treatment. Error bars represent SEs from at least 5 biological replicates, each with 8 seedlings. Statistical significance was determined by Duncan’s multiple range test with p-value < 0.05, and indicated with lower-case alphabets.

Figure 7

Incorporation of TMTM requires more than OASTLs. Cysteine biosynthesis was monitored in 4 parallel samples. In each sample, either Na₂S, water, TMTM or both Na₂S and TMTM was added as substrate for OASTLs. Error bars represent SEs from 3 independent measurement using total protein extract from 3 different biological replicates.