Sulfur nutrition: impacts on plant development, metabolism, and stress responses

Sulfur is an essential element for all organisms and has a wide variety of functions. Plant sulfur nutrition is particularly important as plants are our primary source of the essential amino acid methionine. Sulfur deficiency affects the growth, development, disease resistance, and performance of plants and has a great impact on the nutritional quality of crops. In this special issue, a collection of papers based mainly on contributions from the 11th International Plant Sulfur Workshop gives an overview of different facets of sulfur functions in plants and highlights the importance of sulfur nutrition for different parts of the plant life cycle. From a biochemical point of view, sulfur is a remarkably interesting mineral element because it transitions into a wide range of oxidation states, allowing sulfur-containing compounds to be involved in diverse chemical reactions. In addition, sulfur utilization is dependent on assimilation pathways that incorporate sulfur into organic molecules in either reduced form of organic thiols or as organic sulfated compounds (Box 1) (Takahashi et al., 2011). The amino acid cysteine is a product of the reductive sulfate assimilation pathway. Cysteine is a source of reduced sulfur for many other essential metabolites, including methionine. Furthermore, the reactivity of the thiol group (-SH) makes cysteine residues in proteins important for their structural maintenance as they form disulfide bridges. They are also important for enzyme reaction mechanisms and for regulation directed through either changes in redox states or covalent modifications. Thiol residues of glutathione (GSH), phytochelatins, and some other functional polypeptides such as thioredoxins and metallothioneins are essential for detoxification of heavy metals, xenobiotics, pathogen/pest-borne molecules, and reactive oxygen species. 3´-Phosphoadenosine 5´-phosphosulfate (PAPS) produced in the sulfate assimilation pathway provides activated sulfate for sulfation of metabolites that play characteristic roles in the mitigation of both abiotic and biotic stress (Box 1). In addition to the essential amino acid methionine, a number of plant sulfur metabolites are important ingredients in human diets, such as glucosinolates and their derivatives in Brassicaceae species, and are responsible for distinctive tastes or flavors as well as also being recognized as compounds that are beneficial to health (Traka and Mithen, 2009). Plants take up sulfate (SO42-) from soil and synthesize S-amino acids cysteine (Cys) and methionine (Met). In the reductive sulfate assimilation pathway, SO42- is first activated to adenosine 5’-phosphosulfate (APS) using ATP, which is followed by reduction to sulfite (SO32-) and then to sulfide (S2-). Cys is formed by condensation of S2- and an amino acid Ser derived carbon skeleton O-acetylserine (OAS). SO32- is required for the sulfolipid biosynthesis. S2- is involved in protein modification and redox control. Met and S-adenosylmethionine (SAM) are synthesized downstream of the Cys biosynthetic pathway. SAM serves as a methyl donor for various methylation reactions and a substrate for ethylene biosynthesis. Glutathione (GSH; γ-glutamyl-cysteinylglycine) synthesized from Cys plays pivotal roles in redox control and detoxification of cytotoxic molecules, such as reactive oxygen species (ROS) and heavy metals. In plants, following SO42- activation to APS, the sulfate assimilation pathway bifurcates into two pathways – one for the SO42- reduction as mentioned above, and the other for the 3’-phosphoadenosine 5’-phosphosulfate (PAPS) biosynthesis. PAPS serves as the sulfate donor for sulfation of peptides, hormones, and specialized metabolites. Sulfation thus modulates functions of peptides and metabolites involved in biologically important processes in the plant life cycle, such as abiotic/biotic stress mitigation and growth regulation. 3’-Phosphoadenosine 5’-phosphate (PAP), the byproduct of sulfation, acts as a retrograde signal for stress responses. Specialized metabolites derived from Cys and sulfation contribute to tastes and flavors of foods, and are also known as nutraceuticals. The diagram illustrates an overview of the S metabolic pathways highlighting their connections to essential biological processes. Only the representative pathways are shown. The S compounds displayed in clockwise order: adenosine 5’-phosphosulfate (1), sulfoquinovosyl dipalmitoylglycerol (2), cysteine (3), methionine (4), glutathione (5), S-adenosylmethionine (6), allicin (7), glucosinolate (8), phytosulfokine (9). Thus, there is clear value in fundamental research aimed at understanding how plants utilize sulfate (the primary mineralized inorganic form in which sulfur occurs in soils) in order to produce an array of sulfur-containing metabolites, and in understanding how this utilization is controlled. Increasing our knowledge will lead to potential agronomical, ecological, and nutritional benefits. Accordingly, plant sulfur research has been positioned at the forefront of plant science, adopting new experimental approaches to investigate underlying molecular mechanisms, and in addition exploring potential applications in several important areas associated with global sustainability and human well-being. Some specific questions addressed in plant sulfur research have dealt with crop quality beyond productivity issues, such as studies with wheat and potato that have examined the effect of sulfur deficiency on the accumulation of free asparagine, which correlates with acrylamide formation in foods that occurs through the Maillard reaction during cooking (Halford et al., 2012), and studies that have evaluated the genetic potential for increasing contents of health-promoting sulfur metabolites in broccoli (Traka et al., 2013). This special issue illustrates recent progress that has been made in diverse fields of plant sulfur research and provides perspectives on major future directions and routes toward applications based on concepts developed in the post-genomic era. Sulfur research entered the post-genomic era after decades of primarily using biochemical approaches, then molecular genetics and systems biology. Rapid progress has been made during the past two decades, particularly with the emergence of systems biology, where we have seen early pioneering studies focused on responses to sulfate deficiency that have pushed the boundaries and shown the power of ‘omics’ approaches in identifying new genes, gene regulatory networks, and gene functions (Hirai et al., 2003, 2005, 2007; Maruyama-Nakashita et al., 2003; Nikiforova et al., 2003, 2004, 2005). Precise information on genome structures and sequences, which became available in the post-genomic era alongside the development of high-throughput sequencing technologies, has greatly accelerated the procedures used to identify causal genes for variation in many plant traits and candidate genes of interest for trait development. The increasing availability of reference genome sequences and transcriptome datasets across the plant kingdom has allowed us to search for homologs and to explore the evolutionary history of genes and complex traits. This technological development and the rapid increase in genomic information that occurred alongside it broke the existing boundaries of our research areas and extended the depth of our knowledge, allowing us to tackle more complex research questions and to investigate and engineer specific traits directly in non-model plant species, including crops. Among the new approaches that were enabled by the increase in data, genome-wide association studies (GWAS) have had a particularly strong impact on the field of sulfur research (Chan et al., 2011; Chao et al., 2014). GWAS demonstrate their power not only in identifying genes responsible for variation in, for example, sulfate content or glucosinolate composition, but also in providing ideas and further questions about ecological drivers of adaptations in sulfur metabolism. Several GWAS have taken advantage of the development of the ionomics approach, a high-throughput method for determining the elemental composition of plants (Chao et al., 2014). The impact of ionomics on sulfur research has been demonstrated by the discovery of a unique epigenetic regulatory mechanism (Huang et al., 2016), and the approach also helps us understand the interconnection of multiple nutrients, as described by Courbet et al. (2019) in this special issue. Much sulfur research in non-model plant species has been aimed at addressing questions relevant to the agricultural impact of sulfur and the traits connected with the nutritional quality of crops. There are multiple examples presented in this special issue, such as metabolism of non-proteinogenic sulfur-containing amino acids in legumes (Joshi et al. 2019), analysis of the effects of sulfur deficiency and water stress on protein composition in peas (Henriet et al., 2019), and a detailed examination of the synthesis of organosulfur compounds in onion and garlic (Yoshimoto and Saito, 2019). Interest in non-model species is not limited to crops, and Jobe et al. (2019) present a review with a focus on sulfur metabolism in C4 plants. However, despite this diversification in the range of species that are being studied in the post-genomic era, traditional topics and approaches in model plant species still remain highly relevant in sulfur research. Elucidating the functions and regulation of transporters and enzymes involved in sulfur metabolism has been the main research topic for decades. A collection of reviews in this special issue illustrates recent progress and perspectives. Takahashi (2019) presents the concept of regulatory coordination of the signals that modulate the expression and activity of transporters for sulfate uptake and internal distribution, while Jez (2019) reviews the unique fine-tuned switching mechanisms that control the activity of sulfur assimilatory enzymes that have been found in studies based on protein structure analysis. Selles et al. (2019) address a less well-known component of sulfur metabolism by discussing the diverse functionalities of sulfurtransferases in sulfur metabolism and, more broadly, in processes involved in mineral utilization. As noted above, genome sequencing has contributed greatly to our knowledge and has increased the number of genes and proteins to be functionally characterized. Transcriptomics, proteomics, and metabolomics have then produced large and complex datasets. Watanabe and Hoefgen (2019) provide an overview of such data collections and indicate potential approaches to identify novel gene or protein functions based on ‘omics’ data analyses. The majority of information on regulatory mechanisms published in relation to sulfur research is derived from studies that have explored the phenotypic consequences of genetic alterations. Recent studies, however, indicate that epigenetic regulatory mechanisms also have substantial impacts on the pathways that control sulfate transport and utilization, as reviewed by Huang et al. (2019). Growing attention is being paid to specialized sulfur-containing metabolites that impart distinctive flavors and potential health benefits to food (Traka and Mithen, 2009; Nakabayashi and Saito, 2017). However, many of these metabolites are often specific to non-model plant species and while their uniqueness in chemistry is of inherent interest, the lack of adequate genomic sequence data has hitherto limited the power of gene discovery. As noted above, progress in genome sequencing and the increasing availability of transcriptome datasets now are now helping us to study the molecular basis of these unique metabolic traits in non-model species. Yoshimoto and Saito (2019) highlight the biosynthesis of S-alk(en)ylcysteine sulfoxide in the genus Allium and present predictive approaches that can be implemented to identify reaction mechanisms and the relevant pathway enzymes. Joshi et al. (2019) review pathways for biosynthesis of cysteine-derived non-proteinogenic sulfur amino acids and their γ-glutamyl dipeptides and homoglutathione derivatives that are found in legume species. The biosynthetic pathways for specialized sulfur-containing metabolites begin with S-amino acids or via a connection with the primary sulfate assimilation pathway at several steps downstream of derivatization. Because of these metabolic connections, the pathways for primary and specialized metabolism show regulatory coordination that depends on sulfur availability and the specific demands for the bioactive compounds. Amir et al. (2019) discuss the potential for enhancing the levels of the essential amino acid methionine in seeds, either by increasing the sink capacity of seed storage proteins or by manipulating the flux of methionine synthesis and catabolism. Chan et al. (2019) review sulfation of metabolites (e.g. glucosinolates), hormones, and peptides as part of the specialized metabolism, and discuss their regulatory coordination and possible cooperative roles in stress mitigation, as they all require PAPS as the sulfate donor. Remarkable progress has been made in determining the pathways that control plant nutrient uptake, metabolism, and homeostasis. However, the mechanisms that coordinate the acquisition of individual nutrients and their integration in cellular metabolic networks are less well understood. In this special issue, three reviews summarize progress in understanding the integration of sulfur nutrition with other nutrient uptake and metabolic pathways. Courbet et al. (2019) address the coordination of multiple macro- and micronutrients from the perspective of a balanced ionome. Mendoza-Cózatl et al. (2019) discuss the interplay of sulfur more specifically with a single nutrient, iron. A more general integration of sulfur nutrition with primary plant productivity is given by Jobe et al. (2019). From all three papers it may be concluded that sulfur metabolism is necessarily modulated for the improvement of various plant traits that are seemingly not directly associated with sulfur. In line with this concept, the activity of target-of-rapamycin (TOR) kinase has been shown to control plant growth under sulfur-limited conditions (Dong et al., 2017). The requirement for cysteine for the biosynthesis of abscisic acid and subsequent stomatal closure implicates sulfur nutrition and metabolism as being important for in plant water relations (Batool et al., 2018). Thiol groups of GSH and protein cysteine (Cys) residues are essential for redox homeostasis of cells. Enzyme activity and protein function can be modulated by modification of Cys residues. Several review articles in this special issue describe how changes in redox status control sulfur assimilation together with targets in other cellular processes to affect plant responses under stress conditions. Telman and Dietz (2019) review redox regulation of key enzymes in sulfur assimilation and discuss a link between the sulfur and oxilipin signaling pathways. The redox switches of these key enzymes are also discussed from the structural perspective by Jez (2019). Gotor et al. (2019) highlight chemical features of sulfide and cyanide produced in the sulfur assimilation and downstream metabolic pathways that promote post-translational protein persulfidation and S-cyanylation. Umbreen et al. (2019) discuss another example of redox-based post-translational modification with a focus on nitric oxide acting on S-nitrosation of protein Cys residues. Chan et al. (2019) examine the roles of 3′-phosphoadenosine 5′-phosphate (PAP), a by-product of sulfation, in acting as a retrograde signaling molecule to control abiotic stress responses. The redox control of nucleotide phosphatase that is responsible for PAP catabolism further indicates multifaceted connections with sulfate assimilation (Chan et al., 2016). Kaufmann and Sauter (2019) provide an overview of peptide sulfation, with a discussion of the key roles of sulfated small signaling peptides in the regulation of plant immunity and developmental processes, such as the formation of the Casparian strip in roots (Doblas et al., 2017; Nakayama et al., 2017). Two research articles in this special issue report new findings in relation to the mitigation of abiotic and biotic stress. Henriet et al. (2019) describe the combinatorial effects of water and sulfur limitation on reproductive growth, seed production, and the seed transcriptome and proteome in Pisum sativum, while Hunziker et al. (2019) report the discovery of numerous plasmodesmata that form connections between the glucosinolate storage cells (S-cells) and neighboring cells that synthesize glucosinolate in the vasculature of Arabidopsis. Our knowledge of sulfur metabolism has increased exponentially in the past two decades. Similar to nitrogen and phosphorus, sulfur is an element that is present in the macromolecules that are essential for plant growth and development, such as amino acids, proteins, and membrane lipids. In face of global sustainability issues, improvements in nutrient-use efficiency and yield performance are considered important targets of crop breeding in the 21st century. Sulfur is clearly one of the essential macronutrients that needs to be studied. Sulfur has unique features, for example it is indispensable for the activities of redox modulators, and for some defense chemicals and attractants that play important roles in stress mitigation, and thus it affects crop productivity. Together with the impact of sulfur metabolites on nutritional quality, this makes sulfur research important for achieving global food security. However, in order to be able to contribute to solving the great challenges that we face in crop production, precise and detailed information on biological and molecular mechanisms needs to be collected and thoroughly analysed. Plant biology and biochemistry are the core subject areas for the study of these key biological questions – more specifically, we need to understand the molecular functions of the genes, proteins, and metabolites that are involved in sulfur sensing, signaling, metabolism, and regulation. Combinations of biochemistry, molecular genetics, genomics, and systems biology will continue to drive fundamental research, allowing us to develop ideas and strategies to explore new features of gene–protein–metabolite regulatory networks. Integrative approaches using ‘omics’ tools and information resources will further help us to build hypotheses and models to be tested against empirical knowledge. Recent developments in genome-editing technologies have great potential to rationalize ideas for trait improvement in crop breeding. However, we may encounter major obstacles due to the complexities of genome architecture, life-cycle, fertility, and feasibility for genetic transformation of crop species. Besides these technological issues, significant gaps in our knowledge still remain in a number of areas. Given the importance of the geochemical cycle of sulfur in nature, the roles of sulfur nutrition and metabolites in the environmental adaptation of plants need to be studied with more attention given to ecophysiological aspects. The effects of availabilities of other mineral nutrients on sulfur utilization also need to be studied in more detail by focusing on their interactions. The functions of sulfur metabolites in assisting mutualistic interactions between living organisms (i.e. microbes, animals, and plants) are not fully understood. With the exception of a number of known examples characterized so far for essential biological or nutritional functions, the diversity of sulfur metabolites appears to be an unexplored area worthy of further study in order to build up a comprehensive ‘S-chemicals’ catalogue. Analyses of proteomes, post-translational modifications, and protein structures are areas to be exploited to gain more information. Sulfur has unique characteristics in various aspects, some of which provide potential areas for practical applications, as summarized in this special issue. We endeavor to explore the biology of sulfur to gain further mechanistic understanding of plant systems and networks in nature. This special issue is thus not a final report of sulfur research but rather a summary of current progress with future outlooks that illustrates the dynamic nature of this topic. Most of the contributions in this special issue derive from presentations given at the 11th International Plant Sulfur Workshop held on 16–20th September 2018 in Conegliano, Italy. We are grateful to the plant sulfur research community, which has been actively supporting the workshop series since the first meeting in 1989 in Groningen, in the Netherlands. We also thank the Journal of Experimental Botany for supporting our research community and for providing us with an opportunity to present updates in this second special issue 15 years after the publication of the first (Hawkesford, 2004).