Sulfur in Plant Physiology
  Uptake, Transport, and Assimilation of Sulfate
    - Foliar Uptake and Metabolism of Sulfurous Gases
  Major Organic Sulfur Compounds
  Secondary Sulfur Compounds
  Interactions between Sulfur and Other Minerals
    - Nitrogen–Sulfur Interactions
    - Interactions between Sulfur and Micronutrients
Sulfur in Plant Nutrition
  Diagnosis of Sulfur Nutritional Status
    - Symptomatology of Single Plants
    - Symptomatology of Monocots
    - Sulfur Deficiency Symptoms on a Field Scale
Soil Analysis
Plant Analysis
  Analytical Methods
  Assessment of Critical Nutrient Values
  Sulfur Status and Plant Health
Sulfur Fertilization

The sulfur-containing amino acids cysteine and methionine play a significant role in the structure, conformation, and function of proteins and enzymes in vegetative plant tissue, but high levels of these amino acids may also be present in seed storage proteins (37). Cysteine is the sole amino acid whose side-chain can form covalent bonds, and when incorporated into proteins, the thiol group of a cysteine residue can be oxidized, resulting in disulfide bridges with other cysteine side-chains (forming cystine) or linkage of polypeptides. Disulfide bridges make an important contribution to the structure of proteins. An impressive example for the relevance of disulfide bridges is the influence of the sulfur supply on the baking quality of bread-making wheat. Here, the elasticity and resistance to extensibility are related to the concentration of sulfur-containing amino acids and glutathione. First, it was shown in greenhouse studies that sulfur deficiency impairs the baking quality of wheat (38-41). Then, the analysis of wheat samples from variety trials in England and Germany revealed that decrease in the supply of sulfur affected the baking quality, before crop productivity was reduced (42,43). The sulfur content of the flour was directly related to the baking quality with each 0.1% of sulfur equalling 40 to 50 mL loaf volume. The data further revealed that a lack of either protein or sulfur could be partly compensated for by increased concentration of the other.

The crude protein of wheat can be separated into albumins and globulins, and gluten, which consist of gliadins and glutenins. The first, albumins and globulins, are concentrated under the bran and are thus present in higher concentrations in whole-grain flours. Their concentration is directly linked to the thousand grain weight. In the flour, gluten proteins are predominant and the gliadin/glutenin ratio influences the structure of the gluten, rheological features of the dough, and thus the baking volume (44). Gliadins are associated with the viscosity and extensibility, and glutenins with the elasticity and firmness of the dough (45). Here, the high-molecular-weight (HMW) glutenins give a higher proportion of the resistance of the gluten than low-molecularweight (LMW) glutenins (46). Sulfur deficiency gives rise to distinctly firmer and less extensible doughs (Figure 7.2). Doughs from plants adequately supplied with sulfur show a significantly higher extensibility and lower resistance than do doughs made of flour with an insufficient sulfur supply (Figure 7.2). Sulfur-deficient wheat has a lower albumin content, but higher HMW-glutenin concentration and a higher HMW/LMW glutenin ratio (47).

Consequently the baking volume of sulfur-deficient wheat is reduced significantly. A comparison of British and German wheat varieties with similar characteristics for loaf volume and falling number is given in Table 7.1. In the German classification system, varieties C1 and C2 are used as feed or as a source for starch. Varieties B3, B4, and B5 are suitable for baking but are usually mixed with higher quality wheat. The highest bread-making qualities are in the A6-A9 varieties.

The results presented in Table 7.1 reveal that the quality of British and German varieties is similar. It is relevant in this context that the British varieties gave the same results in the baking experiment at lower protein concentrations than the German ones. The reason is that there was a higher sulfur concentration and thus a smaller N/S ratio in the British varieties. This means that higher sulfur concentrations can partially compensate for a lack of wheat protein and vice versa.

Extensographs for flour with average (continuous line) and low (broken line) sulfur content
FIGURE 7.2 Extensographs for flour with average (continuous line) and low (broken line) sulfur content. +S flour: 0.146% S, 1.82% N, N:S = 12.5:1; =S flour: 0.089% -S, 1.72% N, N:S19.3:1. (From Wrigley, C.W. et al., J. Cereal Sci., 2, 15-24, 1984.)

Comparison of Quality Parameters of German and British Wheat Varieties

Sulfur supply has been recognized as a major factor influencing protein quality for a long time (48,49). Eppendorfer and Eggum (50,51), for instance, noted that the biological value of proteins in potatoes (Solanum tuberosum L.) was reduced from 94 to 55 by sulfur deficiency at high N supply, and from 65 to 40 and 70 to 61 in kale (Brassica oleracea var. acephala DC) and field beans (Vicia faba L.), respectively. Whereas the essential amino acid concentrations declined due to sulfur deficiency, the content of amino acids of low nutritional value such as arginine, asparagine, and glutamic acid increased (50, 51). Figure 7.3 shows the relationship between sulfur supply to curly cabbage (Brassica oleracea var. sabellica L.), indicated by the total sulfur concentration in fully expanded younger leaves, and the cysteine and methionine concentration in leaf protein.

Relationship between the sulfur nutritional status of curly cabbage and the concentration of cysteine and methionine in the leaf protein
FIGURE 7.3 Relationship between the sulfur nutritional status of curly cabbage and the concentration of cysteine and methionine in the leaf protein. (From Schnug, E., in Sulphur Metabolism in Higher Plants: Molecular, Ecophysiological and Nutritional Aspects, Backhuys Publishers, Leiden, 1997, pp. 109-130.)

Influence of Sulfur Fertilization on Sulfur-Containing Amino Acids, Total Protein, and Oil Content in Soybean Grains

This example shows that a significant relationship between sulfur supply and sulfur-containing amino acids exists only under conditions of severe sulfur deficiency, where macroscopic symptoms are visible. The corresponding threshold is below leaf sulfur levels of 0.4% total sulfur in the dry matter of brassica species (52,53).

In comparison, sulfur fertilization of soybean significantly increased the cystine, cysteine, methionine, protein, and oil content of soybean grain (Table 7.2) (54).

The reason for these different responses of vegetative and generative plant tissue to an increased sulfur supply is that excess sulfur is accumulated in vegetative tissue as glutathione (see below) or as sulfate in vacuoles; the cysteine pool is maintained homeostatically because of its cytotoxicity (55). In comparison, the influence of sulfur supply on the seed protein content is related to the plant species. In oilseed rape, for instance, which produces small seeds, the total protein content is more or less not influenced by the sulfur supply (56). Species with larger seeds, which contain sulfur-rich proteins, such as soybean, respond accordingly to changes in the sulfur supply (5).

The most abundant plant sulfolipid, sulfoquinovosyl diacylglycerol, is predominantly present in leaves, where it comprises up to 3 to 6% of the total sulfur (10,57,58). This sulfolipid can occur in plastid membranes and is probably involved in chloroplast functioning. The route of biosynthesis of sulfoquinovosyl diacylglycerol is still under investigation; in particular, the sulfur precursor for the formation of the sulfoquinovose is not known, though from recent observations it is evident that sulfite is the likely candidate (58).

Cysteine is the precursor for the tripeptide glutathione (γGluCysGly; GSH), a thiol compound that is of great importance in plant functioning (32,59,60,61). Glutathione synthesis proceeds in a two-step reaction. First, γ-glutamylcysteine is synthesized from cysteine and glutamate in an ATPdependent reaction catalyzed by γ-glutamylcysteine synthetase (Equation 7.1). Second, glutathione is formed in an ATP-dependent reaction from ?-glutamylcysteine and glycine (in glutathione homologs, �-alanine or serine) catalyzed by glutathione synthetase (Equation 7.2):

Influence of Sulfur Fertilization on the Glutathione Content of the Vegetative Tissue of Different Crops

Glutathione and its homologs, for example, homoglutathione (γGluCys�Ala) in Fabaceae and hydroxymethylglutathione (γGluCys�Ser) in Poaceae, are widely distributed in plant tissues in concentrations ranging from 0.1 to 3 mM. The glutathione content is closely related to the sulfur nutritional status. In Table 7.3, the influence of the sulfur supply and sulfur status and the glutathione content is summarized for different crops. The possible significance of the glutathione content for plant health is discussed in Section.

Glutathione is maintained in the reduced form by an NADPH-dependent glutathione reductase, and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) generally exceeds a value of 7 (60-67). Glutathione fulfills various roles in plant functioning. In sulfur metabolism, glutathione functions as the reductant in the reduction of APS to sulfite (Figure 7.1). In crop plants, glutathione is the major transport form of reduced sulfur between shoot and roots, and in the remobilization of protein sulfur (e.g., during germination). Sulfate reduction occurs in the chloroplasts, and roots of crop plants mostly depend for their reduced sulfur supply on shoot-root transfer of glutathione via the phloem (59-61).

Selenium is present in most soils in various amounts, and its uptake, reduction, and assimilation strongly interact with that of sulfur in plants. Glutathione appears to be directly involved in the reduction and assimilation of selenite into selenocysteine (68). More detailed information about interactions between sulfur and other minerals is given in Section.

Glutathione provides plant protection against stress and a changing environment, viz air pollution, drought, heavy metals, herbicides, low temperature, and UV-B radiation, by depressing or scavenging the formation of toxic reactive oxygen species such as superoxide, hydrogen peroxide, and lipid hydroperoxides (61,69). The formation of free radicals is undoubtedly involved in the induction and consequences of the effects of oxidative and environmental stress on plants. The potential of glutathione to provide protection is related to the size of the glutathione pool, its oxidation-reduction state (GSH/GSSG ratio) and the activity of glutathione reductase.

Plants may suffer from an array of natural or synthetic substances (xenobiotics). In general, these have no direct nutritional value or significance in metabolism, but may, at too high levels, negatively affect plant functioning (70-72). These compounds may originate from either natural (fires, volcanic eruptions, soil or rock erosion, biodegradation) or anthropogenic (air and soil pollution, herbicides) sources. Depending on the source of pollution, namely air, water, or soil, plants have only limited possibilities to avoid their accumulation to diminish potential toxic effects. Xenobiotics (R-X) may be detoxified in conjugation reactions with glutathione (GSH) catalyzed by the enzyme glutathione S-transferase (70-72).

R-X + GSH ? R-SG + X-H

The activity of glutathione S-transferase may be enhanced in the presence of various xenobiotics via induction of distinct isoforms of the enzyme. Glutathione S-transferases have great significance in herbicide detoxification and tolerance in agriculture. The induction of the enzyme by herbicide antidotes, the so-called safeners, is the decisive step for the induction of herbicide tolerance in many crop plants. Under normal natural conditions, glutathione S-transferases are assumed to be involved in the detoxification of lipid hydroperoxides, in the conjugation of endogenous metabolites, hormones, and DNA degradation products, and in the transport of flavonoids. However, oxidative stress, plant-pathogen infections, and other reactions, which may induce the formation of hydroperoxides, also may induce glutathione S-transferases. For instance, lipid hydroperoxides (R-OOH) may be degraded by glutathione S-transferases:

R-OOH + 2GSH ? R-OH + GSSG + H2O

Plants need minor quantities of essential heavy metals (zinc, copper, and nickel) for growth. However, plants may suffer from exposure to high toxic levels of these metals or other heavy metals, for example, cadmium, copper, lead, and mercury. Heavy metals elicit the formation of heavymetal- binding ligands. Among the various classes of metal-binding ligands, the cysteine-rich metallothioneins and phytochelatins are best characterized; the latter are the most abundant ligands in plants (73-78). The metallothioneins are short gene-encoded polypeptides and may function in copper homeostasis and plant tolerance. Phytochelatins are synthesized enzymatically by a constitutive phytochelatin synthase enzyme and they may play a role in heavy metal homeostasis and detoxification by buffering the cytoplasmatic concentration of essential heavy metals, but direct evidence is lacking so far. Upon formation, the phytochelatins only sequester a few heavy metals, for instance cadmium. It is assumed that the cadmium-phytochelatin complex is transported into the vacuole to immobilize the potentially toxic cadmium (79). The enzymatic synthesis of phytochelatins involves a sequence of transpeptidation reactions with glutathione as the donor of ?-glutamyl-cysteine (γGluCys) residues according to the following equation:

The number of γ-glutamyl-cysteine residues (γGluCys)n in phytochelatins ranges from 2 to 5, though it may be as high as 11. In species containing glutathione homologs (see above), the C-terminal amino acid glycine is replaced by �-alanine or serine (73-78). During phytochelatin synthesis, the sulfur demand is enhanced (80) so that it may be speculated that the sulfur supply is linked to heavy metal uptake, translocation of phytochelatins into root cell vacuoles, and finally transport to the shoot and expression of toxicity symptoms. The sulfur/metal ratio is obviously related to the length of the phytochelatin (81), which might offer a possibility to adapt to varying sulfur nutritional conditions. Hence, increasing cadmium stress (10 �mol Cd in the nutrient solution) yielded an enhanced sulfate uptake by maize roots of 100%, whereby this effect was associated with decreased sulfate and glutathione contents and increased phytochelatin concentrations (81). The studies of Raab et al. (82) revealed that 13% of arsenic was bound in phytochelatin complexes, where as the rest occurred as nonbound inorganic compounds.