Introduction
Sulfur is an indispensable macronutrient required for proper plant
growth, development and physiology. It is first incorporated into
cysteine, and further into methionine, or glutathione (GSH), vitamins
and cofactors, such as thiamine and biotin, to carry out important
biochemical processes. Notable examples are the iron-sulfur (Fe-S)
clusters which are required for electron transport in photosynthesis,
reduction and assimilation of sulfur and nitrogen
(Ravenet al. , 1999; Lancaster et al. , 1979; Krueger and Siegel,
1982). In Brassicales , assimilation of sulfur contributes to the
biosynthesis of glucosinolates (GSL), which are essential defense
molecules against herbivores and pathogens
(Bakhtiari
and Rasmann, 2020; Halkier and Gershenzon, 2006; Ting et al. ,
2020; Wittstock et al. , 2016). Although being classified as
secondary metabolites, GSLs can hold up to 30% of total sulfur content
in the plant body and serves as sulfur reservoir
(Falket al. , 2007; Aghajanzadeh et al. , 2014).
In natural environments, microorganisms play an important role in
providing sulfate (SO42-), the primary
sulfur source accessible, to roots for the biosynthesis of
sulfur-containing compounds in plants. As early as in 1877, scientists
already knew that elemental sulfur (S0) can be
oxidized to sulfate, and microbes were thought to be an essential part
of it
(Lipmanet al. , 1916). It was few decades later that scientists isolated
the S-oxidizing bacteria Thiobacillus denitrificans and T.
thioparus , and showed that they produce sulfate from
S0(Beijerinck,
1904; Lipman et al. , 1916; Waksman and Joffe, 1922). It is now
known that microorganisms possess sulfatases to mineralize organic
sulfur, thereby releasing sulfate into the rhizosphere
(Deng
and Tabatabai, 1997; Kertesz, 2000). Furthermore, fungi were shown to
mobilize sulfate-esters and activate arylsulfatase activity under
sulfur-limiting conditions
(Fitzgerald,
1976; Marzluf, 1997; Omar and Abd-Alla, 2000; Baum and Hrynkiewicz,
2006). Fungal symbionts are also crucial in supporting plants with
sulfur. Mycorrhizal fungi are notable example for the promotion of
sulfur uptake, as shown in maize, clover and tomato
(Gray
and Gerdemann, 1973; Cavagnaro et al. , 2006). The expression of
sulfate transporters in plants can also be influenced by mycorrhizal
fungi, resulting in improved sulfur status in host plants under sulfur
deficient condition
(Giovannettiet al. , 2014).
Volatile organic compounds (VOCs) from microorganisms present another
possible route to provide sulfur to plants. Dimethyl disulfide (DMDS) is
produced by the bacteria Serratia odorifera and Bacillus
spp. B55. Under sulfur deficiency, DMDS can sustain plant growth and
increase root branching
(Meldauet al. , 2013). Labeling experiment demonstrated that the
S-containing volatile is taken up by the plants
(Kaiet al. , 2010; Meldau et al. , 2013). Compared to bacteria,
much less is known about sulfur-containing volatiles produced by fungi
(Dickschat,
2017). Besides DMDS, mercaptoacetone, 3-methylsulfanylpropan-1-ol,
benzothiazole, 2-acetylthiazole, 3,5-dimethyl-1,2,4-trithiolane,
5-(1-propynyl)-thiophen-2-carbaldehyde and sulfur dioxide
(SO2) were identified from various fungi
(Splivalloet al. , 2007; Seifert and King, 1982; Nemcovic et al. ,
2008; Schalchli et al. , 2011; Larsen, 1998; Dickschat, 2017;
Citron et al. , 2012; Birkinshaw and Chaplen, 1955; Brock et
al. , 2011). Not
much is known about the mechanisms of their incorporation into the plant
metabolism, but SO2 can cross cell membranes directly
from the surrounding air and influence sulfur distribution within leaf
tissue
(Randewiget al. , 2012; Pfanz et al. , 1987; Rennenberg and Polle,
1994).
Incorporation of sulfur is a multi-step process. In soil, it starts
primarily with the assimilation of sulfate by sulfate transporters
(SULTRs) in the root cells. SULTR1;1 and SULTR1;2 act as the primary
sulfate transporters. SULTR2;1 is located in the xylem and pericycle and
responsible for root-shoot sulfur transport
(Takahashiet al. , 2011; Shibagaki et al. , 2002; Yoshimoto et
al. , 2002; Kataoka et al. , 2004; Takahashi et al. , 1997).
Once the sulfate is in root tissue, it is incorporated alongside with
ATP into adenosine-5′-phosphosulfate (APS) via the enzyme ATP
sulfurylase (ATPS). APS serves as the branching point between primary
and secondary metabolism. Through APS reductase, APS is transformed into
sulfite (SO32-), and subsequently
reduced to sulfide (S2-) by sulfite reductase. With
O-acetyl-serine(thiol)lyase (OASTL), sulfide is further incorporated
into O-acetylserine (OAS) to form the amino acid cysteine for primary
metabolism
(Mugfordet al. , 2011). On the other hand, APS goes into secondary
metabolism through APS kinase, which catalyzes the formation of
3’-phosphoadenosine-5’-phosphosulfate (PAPS). PAPS serves as the
molecule required for the last step of glucosinolate biosynthesis
(Mugfordet al. , 2009).
Sulfur assimilation and dynamics are highly regulated under sulfur
deficiency. In Arabidopsis , SULFUR LIMITATION1(SLIM1 ) is a central regulator of sulfur deficiency. The
transcription factor of the EIL family induces the expression of genes
for sulfur uptake transporters. Furthermore, genes for GSL catabolism
are stimulated while those for GSL biosynthesis are repressed, thereby
releasing sulfur from the GSL storage for proper plant growth
(Maruyama-Nakashitaet al. , 2006). Correspondingly, mutants defect in SLIM1cannot respond to sulfur deficiency, and show reduced root growth
(Maruyama-Nakashitaet al. , 2006). Finally, SULFUR DEFICIENT INDUCED(SDI )1 and SDI2 are often used as marker genes to
monitor sulfur deficiency
(Aarabiet al. , 2016). SDI1 is localized in the nucleus, and can repress
GSL biosynthesis by interacting with MYB28, a major transcription factor
for aliphatic GSL biosynthesis
(Aarabiet al. , 2016; Hirai et al. , 2007; Gigolashvili et
al. , 2007). All these components fine tune the sulfur status in the
plant body to optimize plant competence in response to sulfur
limitation.
Mortierella hyalina belongs to the phylum Mucoromycota . It
possesses a distinctive garlic-like smell in synthetic culture. In the
co-cultivation experiments with Arabidopsis thaliana seedlings,M. hyalina promoted plant growth
(Johnsonet al. , 2019). Similar results were obtained for three otherMortierella strains with garlic-like smells, while the growth
responses were less for two strains which did not smell (Figure S1). In
this study, we address the question whether the volatile from M.
hyalina interferes with the plant metabolism and might be involved in
the regulation of plant growth. The headspace of M. hyalina was
analyzed by GC-MS to identify those VOCs which are potentially involved
in plant nutrition. By NMR, a sulfur-containing volatile,
tris(methylthio)methane (TMTM; CAS Number 5418-86-0), was identified as
the major chemical in the fungal headspace. Incorporation of the sulfur
from the fungal volatile into plant metabolism was shown with stable
sulfur isotope labeling experiments. Under sulfur deficiency, TMTM
promoted plant growth, reduced the consumption of sulfur-containing
metabolites, and reduced the response of seedlings to sulfur deficiency.
We propose that TMTM maintains sulfur homeostasis in the plant under
sulfur limitation condition. Finally, biochemical analyses examining
cysteine biosynthesis did not show direct incorporation of TMTM into
O-acetylserine (OAS), suggesting that additional biochemical steps are
involved before the sulfur from TMTM is incorporated into cysteine, or
non-canonical incorporation mechanisms different from sulfate
assimilation are involved.