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Sulfur is essential for plant growth and development, and the molecular systems for maintaining sulfur and thiol metabolism are tightly controlled. From a biochemical perspective, the regulation of plant thiol metabolism highlights nature's ability to engineer pathways that respond to multiple inputs and cellular demands under a range of conditions. In this review, we focus on the regulatory mechanisms that form the molecular basis of biochemical sulfur sensing in plants by translating the intracellular concentration of sulfur-containing compounds into control of key metabolic steps. These mechanisms range from the simple (substrate availability, thermodynamic properties of reactions, feedback inhibition, and organelle localization) to the elaborate (formation of multienzyme complexes and thiol-based redox switches). Ultimately, the dynamic interplay of these regulatory systems is critical for sensing and maintaining sulfur assimilation and thiol metabolism in plants.

The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 Å resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Sitedirected mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2-methylthio)-ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2-methylthio)ethylmalate ϳ100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.

Sulfur is an essential plant nutrient and is metabolized into the sulfur-containing amino acids (cysteine and methionine) and into molecules that protect plants against oxidative and environmental stresses. Although studies of thiol metabolism in the model plant Arabidopsis thaliana (thale cress) have expanded our understanding of these dynamic processes, our knowledge of how sulfur is assimilated and metabolized in crop plants, such as soybean (Glycine max), remains limited in comparison. Soybean is a major crop used worldwide for food and animal feed. Although soybeans are protein-rich, they do not contain high levels of the sulfur-containing amino acids, cysteine and methionine. Ultimately, unraveling the fundamental steps and regulation of thiol metabolism in soybean is important for optimizing crop yield and quality. Here we review the pathways from sulfur uptake to glutathione and homoglutathione synthesis in soybean, the potential biotechnology benefits of understanding and modifying these pathways, and how information from the soybean genome may guide the next steps in exploring this biochemical system.

In plants, exposure to temperature extremes, heavy metal-contaminated soils, drought, air pollutants, and pathogens results in the generation of reactive oxygen species that alter the intracellular redox environment, which in turn influences signaling pathways and cell fate. As part of their response to these stresses, plants produce glutathione. Glutathione acts as an anti-oxidant by quenching reactive oxygen species, and is involved in the ascorbate–glutathione cycle that eliminates damaging peroxides. Plants also use glutathione for the detoxification of xenobiotics, herbicides, air pollutants (sulfur dioxide and ozone), and toxic heavy metals. Two enzymes catalyze glutathione synthesis: glutamate–cysteine ligase, and glutathione synthetase. Glutathione is a ubiquitous protective compound in plants, but the structural and functional details of the proteins that synthesize it, as well as the potential biochemical mechanisms of their regulation, have only begun to be explored. As discussed here, the core reactions of glutathione synthesis are conserved across various organisms, but plants have diversified both the regulatory mechanisms that control its synthesis and the range of products derived from this pathway. Understanding the molecular basis of glutathione biosynthesis and its regulation will expand our knowledge of this component in the plant stress response network.

The redox active peptide glutathione is ubiquitous in nature, but some plants also synthesize glutathione analogs in response to environmental stresses. To understand the evolution of chemical diversity in the closely related enzymes homoglutathione synthetase (hGS) and glutathione synthetase (GS), we determined the structures of soybean (Glycine max) hGS in three states: apoenzyme, bound to g-glutamylcysteine (gEC), and with hGSH, ADP, and a sulfate ion bound in the active site. Domain movements and rearrangement of active site loops change the structure from an open active site form (apoenzyme and gEC complex) to a closed active site form (hGSH d ADP d SO 4 22 complex). The structure of hGS shows that two amino acid differences in an active site loop provide extra space to accommodate the longer b-Ala moiety of hGSH in comparison to the glycinyl group of glutathione. Mutation of either Leu-487 or Pro-488 to an Ala improves catalytic efficiency using Gly, but a double mutation (L487A/P488A) is required to convert the substrate preference of hGS from b-Ala to Gly. These structures, combined with site-directed mutagenesis, reveal the molecular changes that define the substrate preference of hGS, explain the product diversity within evolutionarily related GS-like enzymes, and reinforce the critical role of active site loops in the adaptation and diversification of enzyme function.

Summary• Ozone (O 3 ) causes significant agricultural losses, with soybean (Glycine max) being highly sensitive to this oxidant. Here we assess the effect of elevated seasonal O 3 exposure on the total and redox proteomes of soybean.• To understand the molecular responses to O 3 exposure, soybean grown at the Soybean Free Air Concentration Enrichment facility under ambient (37 ppb), moderate (58 ppb), and high (116 ppb) O 3 concentrations was examined by redox-sensitive thiol labeling, mass spectrometry, and targeted enzyme assays.• Proteomic analysis of soybean leaf tissue exposed to high O 3 concentrations reveals widespread changes. In the high-O 3 treatment leaf, 35 proteins increased up to fivefold in abundance, 22 proteins showed up to fivefold higher oxidation, and 22 proteins increased in both abundance and oxidation. These changes occurred in carbon metabolism, photosynthesis, amino acid synthesis, flavonoid and isoprenoid biosynthesis, signaling and homeostasis, and antioxidant pathways.• This study shows that seasonal O 3 exposure in soybean alters the abundance and oxidation state of redox-sensitive multiple proteins and that these changes reflect a combination of damage effects and adaptive responses that influence a wide range of metabolic processes, which in some cases may help mitigate oxidative stress.

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