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Sulfur is present in plants in a large range of essential primary metabolites, as well as in numerous natural products. Many of these secondary metabolites contain sulfur in the oxidized form of organic sulfate. However, except of glucosinolates, very little is known about other classes of such sulfated metabolites, mainly because of lack of specific and quantitative analytical methods. We developed an LC-MS method to analyze sulfated flavonoids, a group of sulfated secondary metabolites prominent, e.g., in plants of the genus . The method uses a linear gradient of methanol/formic acid in water on a Restek Raptor C Core-Shell column for separation of the compounds. The sulfated flavonoids are detected by mass spectrometry (MS) in a negative mode, using a neutral loss of 80 Da after a collision induced dissociation. With this method we were also able to quantify the sulfated flavonoids. We could detect all (mono)sulfated flavonoids described before in plus a number of new ones, such as isorhamnetin-sulfate-glycoside. In addition, we showed that sulfated flavonoids represent a substantial sulfur pool in , larger than the thiols glutathione and cysteine. The individual species possess different sulfated flavonoids, but there is no correlation between the qualitative pattern and type of photosynthesis. Similar to other sulfur-containing secondary compounds, the concentration of sulfated flavonoids in leaves is reduced by sulfur starvation. The new LC-MS method will enable qualitative and quantitative detection of these secondary metabolites in plants as a pre-requisite to addressing their functions.

Sulfur plays a vital role in the primary and secondary metabolism of plants, and carries an important function in a large number of different compounds. Despite this importance, compared to other mineral nutrients, relatively little is known about sulfur sensing and signalling, as well as about the mechanisms controlling sulfur metabolism and homeostasis. Sulfur contents in plants vary largely not only among different species, but also among accessions of the same species. We previously used associative transcriptomics to identify several genes potentially controlling variation in sulfate content in the leaves of Brassica napus, including an OASC gene for mitochondrial O-acetylserine thiollyase (OAS-TL), an enzyme involved in cysteine synthesis. Here, we show that loss of OASC in Arabidopsis thaliana lowers not only sulfate, but also glutathione levels in the leaves. The reduced accumulation is caused by lower sulfate uptake and translocation to the shoots; however, the flux through the pathway is not affected. In addition, we identified a single nucleotide polymorphism in the OASC gene among A. thaliana accessions that is linked to variation in sulfate content. Both genetic and transgenic complementation confirmed that the exchange of arginine at position 81 for lysine in numerous accessions resulted in a less active OASC and a lower sulfate content in the leaves. The mitochondrial isoform of OAS-TL is, thus, after the ATPS1 isoform of sulfurylase and the APR2 form of APS reductase 2, the next metabolic enzyme with a role in regulation of sulfate content in Arabidopsis.

The evolution of C₄ photosynthesis led to an increase in carbon assimilation rates and plant growth compared to C₃ photosynthetic plants. This enhanced plant growth, in turn, affects the requirement for soil-derived mineral nutrients. However, mineral plant nutrition has scarcely been considered in connection with C₄ photosynthesis. Sulfur is crucial for plant growth and development, and preliminary studies in the genus Flaveria suggested metabolic differences in sulfate assimilation along the C₄ evolutionary trajectory. Here, we show that in controlled conditions, foliar accumulation of the reduced sulfur compounds Cys and glutathione (GSH) increased with progressing establishment of the C₄ photosynthetic cycle in different Flaveria species. An enhanced demand for reduced sulfur in C₄ Flaveria species is reflected in high rates of [³⁵S]sulfate incorporation into GSH upon sulfate deprivation and increased GSH turnover as a reaction to the inhibition of GSH synthesis. Expression analyses indicate that the 𝛾-glutamyl cycle is crucial for the recycling of GSH in C₄ species. Sulfate reduction and GSH synthesis seems to be preferentially localized in the roots of C4 species, which might be linked to its colocalization with the phosphorylated pathway of Ser biosynthesis. Interspecies grafting experiments of F. robusta (C₃) and F. bidentis (C₄) revealed that the root system primarily controls sulfate acquisition, GSH synthesis, and sulfate and metabolite allocation in C₃ and C₄ plants. This study thus shows that evolution of C₄ photosynthesis resulted in a wide range of adaptations of sulfur metabolism and points out the need for broader studies on importance of mineral nutrition for C₄ plants.

The evolution of C photosynthesis led to an increase in carbon assimilation rates and plant growth compared to C photosynthetic plants. This enhanced plant growth, in turn, affects the requirement for soil-derived mineral nutrients. However, mineral plant nutrition has scarcely been considered in connection with C photosynthesis. Sulfur is crucial for plant growth and development, and preliminary studies in the genus suggested metabolic differences in sulfate assimilation along the C evolutionary trajectory. Here, we show that in controlled conditions, foliar accumulation of the reduced sulfur compounds Cys and glutathione (GSH) increased with progressing establishment of the C photosynthetic cycle in different species. An enhanced demand for reduced sulfur in C species is reflected in high rates of [ S]sulfate incorporation into GSH upon sulfate deprivation and increased GSH turnover as a reaction to the inhibition of GSH synthesis. Expression analyses indicate that the γ-glutamyl cycle is crucial for the recycling of GSH in C species. Sulfate reduction and GSH synthesis seems to be preferentially localized in the roots of C species, which might be linked to its colocalization with the phosphorylated pathway of Ser biosynthesis. Interspecies grafting experiments of (C ) and (C ) revealed that the root system primarily controls sulfate acquisition, GSH synthesis, and sulfate and metabolite allocation in C and C plants. This study thus shows that evolution of C photosynthesis resulted in a wide range of adaptations of sulfur metabolism and points out the need for broader studies on importance of mineral nutrition for C plants.

Sulfur plays a vital role in the primary and secondary metabolism of plants, and carries an important function in a large number of different compounds. Despite this importance, compared to other mineral nutrients, relatively little is known about sulfur sensing and signalling, as well as about the mechanisms controlling sulfur metabolism and homeostasis. Sulfur contents in plants vary largely not only among different species, but also among accessions of the same species. We previously used associative transcriptomics to identify several genes potentially controlling variation in sulfate content in the leaves of Brassica napus, including an OASC gene for mitochondrial O-acetylserine thiollyase (OAS-TL), an enzyme involved in cysteine synthesis. Here, we show that loss of OASC in Arabidopsis thaliana lowers not only sulfate, but also glutathione levels in the leaves. The reduced accumulation is caused by lower sulfate uptake and translocation to the shoots; however, the flux through the pathway is not affected. In addition, we identified a single nucleotide polymorphism in the OASC gene among A. thaliana accessions that is linked to variation in sulfate content. Both genetic and transgenic complementation confirmed that the exchange of arginine at position 81 for lysine in numerous accessions resulted in a less active OASC and a lower sulfate content in the leaves. The mitochondrial isoform of OAS-TL is, thus, after the ATPS1 isoform of sulfurylase and the APR2 form of APS reductase 2, the next metabolic enzyme with a role in regulation of sulfate content in Arabidopsis.