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Nutrient availability is of paramount importance for the growth, performance, yield and reproductive success of plants, including crop plants, which are particularly dependent on a balanced nutrient supply (Marschner, 1995). At the molecular physiological level, several single nutrient ion studies have been performed (Amtmann & Armengaud, 2009), which have shown that nutrient starvation responses are widely interconnected (Astolfi et al., 2010; Watanabe et al., 2010). Sulfur (S) is usually taken up as sulfate by the plant roots, with any excess sulfate being stored in vacuoles. Minor amounts of volatile S compounds (e.g. H2S, SO2 and dimethyl sulfate (DMS)) can also be taken up and metabolized by the leaves (Durenkamp & De Kok, 2004). In addition to the water mobile pedospheric sulfate, organic matter contains substantial amounts of sulfuric compounds, for example, organic esters, which are released through the activity of soil bacteria, potentially providing substantial amounts of S for the soil (Schmalenberger et al., 2009).
Sulfur starvation leads to strong starvation phenotypes in plants, including reduced growth, yellowing of the leaves, and decreased seed number and viability. Recently, the impact of S starvation on the yield of crop plants has been receiving more attention as the reduced atmospheric input of S-containing compounds from industrial and other types of pollution is reducing the amount of S available to crop plants (Lehmann et al., 2008). This has led to a change from an excess supply of S to moderate or even starvation conditions in some areas.
The formation of an arbuscular mycorrhizal (AM) symbiosis leads to an improved nutritional status of the plant partner under low nutrient availability (Marschner & Dell, 1994; Clark & Zeto, 2000). Several studies showed the transfer of mineral nutrients such as phosphate and nitrate from the surrounding area through the fungal hyphae to the plant partner (Ames et al., 1983; Smith et al., 1994, 2003; Javot et al., 2007a; Leigh et al., 2009). The impact of AM symbiosis on phosphate uptake is quite well understood, and it has been demonstrated that plants possess a special, symbiotic phosphate uptake pathway (Javot et al., 2007b; Nagy et al., 2009). Pioneering work in the 1970s and 1980s also showed the uptake of sulfate by the mycorrhizal fungus and its translocation to the plant root and shoot (Rhodes & Gerdemann, 1978a,b; Hepper, 1984). This often resulted in increased growth, especially under S starvation conditions. A recent study using carrot (Daucus carota) root organ cultures also demonstrated the symbiotic transfer of organic S-containing compounds such as cysteine, methionine and glutathione (Allen & Shachar-Hill, 2009). The fact that S, as well as organic S-containing compounds, can be transported via the mycorrhizal pathway suggests that the AM symbiosis might also be beneficial for the plant's S nutrition. However, because S availability has not been a limiting factor in agriculture, the impact of mycorrhizal S transfer on plant metabolism is poorly understood. As leaves are the major sinks for plant S compounds and the main site of sulfate assimilation, the analysis of leaf metabolites was included in our study.
In previous decades, plant model systems such as Medicago truncatula were established for the analysis of the molecular background of the AM symbiosis. However, little information is available about the S assimilation pathway in M. truncatula. The influence of S nutrition on root nodule symbiosis has been studied in white cloverTrfolium repens (L.), and it was shown that the amount of S supplied to clover plants influenced the number of nodules and nitrogen (N)-fixing activity, similar to the starvation effects of phosphorus (P) and potassium (K) (Varin et al., 2010). Additionally, a symbiotic sulfate transporter, Sst1, of Lotus japonicus is necessary for nodule formation and essential for the symbiotic supply of S to the bacteria (Krusell et al., 2005). Little is known about the physiological impact of S transfer during the AM symbiosis. We assume that, under S starvation, the mycorrhizal S supply plays a significant role in plant S nutrition and thus also influences other cellular functions. Recent elemental analysis of amounts of S, N and P confirmed that mycorrhizal symbiosis is able to increase sulfate uptake and assimilation, and an analysis of the transcription level of predicted sulfate transporters showed a very complex picture of sulfate uptake pathways in M. truncatula (Casieri et al., 2012). However, S and P metabolisms are strongly connected and, in addition to N, the availability of P and S considerably influences plant growth (Watanabe et al., 2012). To investigate the correlation between phosphate nutrition, mycorrhizal colonization and S nutrition, we investigated the influence of mycorrhizal S uptake on plant S nutrition and growth, taking into account its link to phosphate availability. We determined the response of M. truncatula to S deprivation in the background of phosphate nutrition at the physiological and molecular levels. Another aim of the study was to investigate known sulfate starvation response genes, whose homologs were analyzed in M. truncatula, as well as the metabolite responses of S-containing metabolites and amino acids.
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Most vascular plants are able to participate in a symbiotic interaction with AM fungi. The main benefit for both organisms in this relationship is an exchange of nutrients and carbon. In the past, the primary focus has been on mycorrhizal phosphate transfer, mainly because low phosphate concentrations in soils are often the main growth-limiting factor. Evidence for symbiotic uptake pathways for additional nutrients emerged from the recent identification of a number of other nutrient transporter genes, which are also specifically expressed or induced in mycorrhizal roots (Guether et al., 2009; Benedito et al., 2010; Gaude et al., 2012). Although a specific transporter involved in the symbiotic uptake of S compounds has not been identified to date, in vitro and in vivo experiments clearly demonstrated a symbiotic uptake pathway for sulfate (Rhodes & Gerdemann, 1978a,b; Hepper, 1984).
Indicative of sulfate starvation in plants are reduced thiol, protein and chlorophyll contents and, therefore, reduced biomass. There are also diverse pleiotropic effects on metabolism and gene expression (Nikiforova et al., 2006; Watanabe et al., 2010). This typical S starvation phenotype was also observed in Medicago truncatula. However, the response to S starvation was directly dependent on the plant's phosphate status. Under strong phosphate starvation, S fertilization treatments did not influence shoot fresh weights. This was probably related to the greater impact of phosphate nutrition on plant metabolism and the reduced demand for S in phosphate-starved plants (Hawkesford & De Kok, 2006). By contrast, when grown with 1 mM phosphate fertilization, plants developed severe symptoms of S starvation when no S was added. Mycorrhizal colonization had a significant impact on the S starvation phenotype when plants were grown under conditions of 1 mM phosphate fertilization.
The frequency of mycorrhizal colonization of plants grown under conditions of 1 mM phosphate fertilization was clearly reduced compared with severely phosphate-starved plants, which is consistent with the fact that the phosphate status of a plant systemically influences the frequency of mycorrhizal colonization (Breuillin et al., 2010; Balzergue et al., 2011). However, the reduced degree of mycorrhizal colonization was apparently sufficient to provide enough S via a symbiotic uptake pathway to reduce visible and molecular phosphate starvation responses. This may be related to the c. 2-fold lower concentrations of S compared with P in plant shoot dry matter (Marschner, 2005).
Although increased mycorrhizal colonization levels were found in phosphate-starved plants, a similar effect was not observed in response to changing S nutrition, with −S conditions not significantly enhancing the frequency of mycorrhizal root colonization.
The reduced starvation symptoms of mycorrhizal plants grown under S starvation were accompanied by a significant reduction in the transcription level of two APS reductase genes. APS reductase is involved in the reduction of sulfate, the step preceding S assimilation in plants. APS reductase transcription is strongly regulated by sulfate availability, with increased transcript levels during S starvation (Gutierrez-Marcos et al., 1996; Takahashi et al., 1997). The decrease in transcript levels of two APS reductase genes in leaves of mycorrhizal plants compared with nonmycorrhizal plants is presumably a consequence of increased S nutrition mediated by the AM symbiosis. As in A. thaliana and other plant species, sulfate deprivation leads to the induction of sulfate transporters in M. truncatula (Hawkesford & De Kok, 2006).
We identified nine putative sulfate transporter genes in M. truncatula, which correspond well to the known classifications in other species. In A. thaliana, transporters of the first group are transcriptionally regulated by the sulfate availability in the soil and induced by sulfate deficiency in plants (Rouached et al., 2009). The pattern of decreased transcript levels of four putative sulfate transporter genes in roots of mycorrhizal plants compared with roots of nonmycorrhizal plants probably reflects the reduced S starvation in mycorrhizal plants as a result of symbiotic uptake of S. We also found decreased transcript levels of four putative sulfate transporters in leaves of mycorrhizal plants, which presumably also reflects decreased S starvation of mycorrhizal plants. However, it is worth mentioning that we did not find a putative sulfate transporter with expression in mycorrhizal roots under conditions of 1 mM phosphate fertilization. This is consistent with the findings of a recent work analyzing M. truncatula S transporter gene regulation in mycorrhizal roots under strong phosphate starvation (Casieri et al., 2012). The mycorrhizal phosphate uptake pathway is mediated by mycorrhizal-inducible or mycorrhizal-specific phosphate transporters (Bucher, 2007; Javot et al., 2007a) and it is very likely that plants have an analogous, mycorrhizal-specific transporter for symbiotic S uptake. The currently available M. truncatula genome sequence covers c. 94% of expressed genes (Young et al., 2011), and it is possible that a mycorrhizal-specific sulfate transporter might be located in the part of the genome not yet available. Another possible explanation for the missing mycorrhizal sulfate transporter is that this putative protein is not similar to annotated plant sulfate transporters. Moreover, we cannot exclude the possibility that organic S compounds are also transferred via a mycorrhizal uptake pathway to the host plant, which would require different transporter systems.
It is obvious that reduced sulfate availability will eventually result in reduced levels of S-containing compounds. This is also true for M. truncatula. The concentrations of thiols such as glutathione and cysteine, as well as derived metabolites such as methionine and hence proteins in general, are reduced. It has been shown that sulfate starvation affects other pathways in a pleiotropic manner (Nikiforova et al., 2005) and is also linked to N metabolism (Kopriva et al., 2002), affecting in particular amino acids (Nikiforova et al., 2006), which is, as we have shown, also true for M. truncatula.
The dominance of phosphate starvation compared with S starvation was demonstrated by the more marked changes observed in the leaf metabolite concentrations of phosphate-starved plants. Additional S starvation caused only moderate changes in leaf metabolites, which presumably reflects reduced demand for S in phosphate-starved plants. Remarkably, in phosphate-starved plants, mycorrhizal colonization led to greater changes in leaf metabolite concentrations compared with additional S starvation. This underlines the increased phosphate nutrition of mycorrhizal plants and hence, as mentioned previously, the primary importance of phosphate as compared with S for plant metabolism.
A different effect of mycorrhizal colonization was observed in the leaf metabolite pattern of plants grown under conditions of 1 mM phosphate fertilization. Here, mycorrhizal colonization of S-starved plants led to a shift in the metabolite pattern toward S-replete plants, demonstrating the improved S nutrition of mycorrhizal plants and the importance of mycorrhizal S uptake for plant metabolism, when the plant's phosphate status is high enough for the plant to benefit from increased S nutrition.