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.