General Introduction

Sulfur is an essential element for growth and physiological functioning of plants, however, its content strongly varies between species and it ranges from 0.1 to 6 % of the dry weight (0.03 to 2 mmol g^-1 dry weight; De Kok et al., 2002a). Sulfate taken up by the roots is the major sulfur source for growth, though it has to be reduced to sulfide before it is further metabolized. Root plastids contain all sulfate reduction enzymes, however, the reduction of sulfate to sulfide and its subsequent incorporation into cysteine takes predominantly place in the shoot in the chloroplast (Figure 1). Cysteine is the precursor or reduced sulfur donor of most other organic sulfur compounds in plants. The predominant proportion of the organic sulfur is present in the protein fraction (up to 70 % of total S), as cysteine and methionine residues. In proteins cysteine and methionine are highly significant in the structure, conformation and function of proteins. Plants contain a large variety of other organic sulfur compounds, as thiols (glutathione), sulfolipids and secondary sulfur compounds (alliins, glucosinolates, phytochelatins), which play an important role in physiology and protection against environmental stress and pests (De Kok et al., 2002a). Sulfur compounds are also of great importance for food quality and for the production of phyto-pharmaceutics. Sulfur defi­ciency will result in the loss of plant production, fitness and resistance to environmental stress and pests. Plants may have to deal with temporary or prolonged periods of excessive sulfur or sulfur deficiency. Excessive sulfur from both pedospheric and atmospheric origin may be utilized as sulfur source for plants (De Kok et al., 2002a, b). On the other hand, it may cause physiological imbalances and negatively affect plant growth.

 

Plants' sulfur requirement for growth

The uptake of sulfate by the roots and its reduction and further assimilation in the shoots, is under normal conditions highly regulated on ''a whole plant level" and it will be in tune with the actual sulfur requirement of a plant species for biomass production (De Kok et al. 2002a). The sulfur requirement strongly varies between species and it may strongly vary at different developmental stages of the plant (vegetative growth, seed production). The overall plant sulfur requirement(S _requirement ) can be estimated as follows (De Kok et al., 2002a; Durenkamp and De Kok, 2004):

 

S _requirement ( µ mol g^-1 plant day^-1 ) =RGR (% day^-1 )xS _content ( µ mol g^-1 plant )

 

where RGR represent the relative growth rate and S _content the total plant tissue sulfur content. The RGR can be estimated as follows:

 

RGR = (lnW₂ - lnW₁)/(t₂ - t₁)

 

where W₁ and W₂ represent the total weight (g) at timet₁ and t₂, respectively, and t₂ -t ₁the time interval (days) between harvests. The rate of sulfate uptake by the roots necessary to meet the plants' sulfur requirement for growth can be estimated as follows:

 

Sulfate _uptake ( µ mol g^-1 rootday^-1) = S _requirement ( µ mol g^-1 plant day^-1 )x (S/R _ratio + 1)

 

where S/R_ratio represents the shoot (S) to root (R) biomass partitioning of the plant.

At optimal growth conditions the sulfur requirement (equivalent to sulfur flux,) of different crop species ranges from 2 to 10 µ mol g^-1 plant fresh weight day^-1 (0.08 to 0.4 µ mol g^-1 plant fresh weight h^-1, Figure 1). Generally the major proportion of the sulfate taken up is reduced and metabolized into organic compounds essential for structural growth. However, seedlings of some plant species, e.g. Brassica oleracea, may contain relatively high sulfate contents and here the organic sulfur content might be used for the estimation of the sulfur requirement needed for structural growth (Castro et al., 2003).

 

Uptake and assimilation of sulfate

Sulfate is taken up by the roots with high affinity and the maximal sulfate uptake rate is generally already reached at pedospheric sulfate levels of 0.1 mM and lower (Hawkesford, 2000; Hawkesford and Wray, 2000; Hawkesford et al., 2003a, b). The uptake of sulfate by the roots and its transport to the shoot is strictly controlled and it appears to be one of the primary regulatory sites of sulfur assimilation (Figure 1).

Sulfate is actively taken up across the plasma membrane of the root cells, subsequently loaded into the xylem vessels and transported to the shoot by the transpiration stream. The uptake and transport of sulfate is energy dependent (driven by a proton gradient generated by ATPases) through a proton/sulfate (presumably 3H⁺/SO₄ ^2-) co-transport (Clarkson et al., 1993). In the shoot the sulfate is unloaded and transported to the chloroplasts where it is reduced. The remaining sulfate in plant tissue is predominantly present in the vacuole, since the cytoplasmatic concentrations of sulfate are kept rather constant.

 

Distinct sulfate transporter proteins mediate the uptake, transport and subcellular distribution of sulfate. According to their cellular and subcellular expression, and possible functioning the sulfate transporters gene family has been classified in up to 5 different groups (Davidian et al., 2000; Hawkesford 2000; Hawkesford et al. 2003a, b; Buchner et al., 2004). Some groups are expressed exclusively in the roots or shoots or expressed both in the roots and shoots. Group 1 are 'high affinity sulfate transporters', which are involved in the uptake of sulfate by the roots (Figure 2). Group 2 are vascular transporters and are 'low affinity sulfate transporters'. Group 3 is the so-called 'leaf group', however, still little is known about the characteristics of this group. Group 4 transporters may be involved in the transport of sulfate into the plastids prior to its reduction, whereas the function of Group 5 sulfate transporters is not known yet (Buchner et al., 2004).

 

Figure 1: An overview of sulfate reduction and assimilation in plants (APS, adenosine 5'-phosphosulfate; Fd_red, Fd_ox, reduced and oxidized ferredoxin; RSH, RSSR, reduced and oxidized glutathione) and the rates of sulfate uptake by the roots and its reduction and assimilation in the shoots of a variety of plant species grown under optimal sulfur supply (adapted from De Kok et al., 2002a).

Regulation and expression of the majority of sulfate transporters are controlled by the sulfur nutritional status of the plants. Upon sulfate deprivation, the rapid decrease in root sulfate is regularly accompanied by a strongly enhanced expression of most sulfate transporter genes (up to 100-fold), accompanied by a substantially enhanced sulfate uptake capacity (Hawkesford, 2000; Hawkesford and Wray, 2000; Hawkesford et al., 2003a, b; Buchner et al., 2004).It is still unresolved, whether sulfate itself or metabolic products of the sulfur assimilation (viz. O-acetyl-serine, cysteine, glutathione) act as signals in the regulation of sulfate uptake by the root and its transport to the shoot, and in the expression of the sulfate tranporters involved (Davidian et al., 2000; Hawkesford, 2000; Hawkesford et al., 2003a, b; Buchner et al., 2004).

Even though root plastids contain all sulfate reduction enzymes, sulfate reduction takes predominantly place in the leaf chloroplasts. The reduction of sulfate to sulfide occurs in three steps (Figure 1). Sulfate needs to be activated to adenosine 5'-phosphosulfate (APS) prior to its reduction to sulfite. The activation of sulfate is catalyzed by ATP sulfurylase, which affinity for sulfate is rather low (K_m approximately 1 mM) and the in situ sulfate concentration in the chloroplast is most likely one of the limiting/regulatory steps in sulfur reduction (Stulen and De Kok, 1993). Subsequently APS is reduced to sulfite, catalyzed by APS reductase with likely glutathione as reductant (Leustek and Saito, 1999; Kopriva and Koprivova, 2003). The latter reaction is assumed to be one of the primary regulation points in the sulfate reduction, since the activity of APS reductase is the lowest of the enzymes of the sulfate reduction pathway and it has a fast turnover rate (Brunold, 1990, 1993; Leustek and Saito, 1999; Kopriva and Koprivova, 2003; Saito, 2003). Sulfite is with high affinity reduced by sulfite reductase with ferredoxin as a reductant and the formed sulfide is incorporated into cysteine, catalyzed by O-acetylserine(thiol)lyase, with O-acetylserine as substrate (Figure 1). The synthesis of O-acetylserine is catalyzed by serine acetyltransferase and together with O-acetylserine(thiol)lyase it is associated as enzyme complex named cysteine synthase (Droux et al., 1998; Hell, 2003). The formation of cysteine is the pre-dominant direct coupling step between sulfur and nitrogen assimilation in plants (Brunold, 1990, 1993; Brunold et al., 2003)

The remaining sulfate in plant tissue is transferred into the vacuole. The remobilization and redistribution of the vacuolar sulfate reserves appears to be rather slow and sulfur-deficient plants may still contain detectable levels of sulfate (Cram 1990; Davidian et al . , 2000;Hawkesford, 2000; Buchner et al., 2004).

Metabolism of atmospheric sulfur gases

The r apid economic growth, industrialization and urbanization are associated with a strong increase in energy demand and emissions of gaseous pollutants including SO₂ (Shen et al., 1995; Feng et al., 2000; Emberson et al., 2001; Yang et al., 2002) . As a consequence agricultural crop yields are at most risk from current levels of sulfurous air pollutants, viz. SO₂, since they are grown close to sources of emissions, where the annual average SO₂ concentrations may exceed 0.1 µ l l^-1. However, the impact of sulfurous air pollutants on plant functioning is paradoxical, since theymay both act as toxin and nutrient (De Kok 1990; De Kok et al., 1998, 2000, 2002a, b ; De Kok and Tausz, 2001). Plants even may benefit from elevated levels of atmospheric sulfur gases , since they contribute to plants sulfur nutrition and exposure may result in enhanced yields , especially when sulfate is deprived in the root environment (Ernst, 1993; Van Der Kooij et al., 1997; De Kok et al., 1997, 2000).

Plant shoots form a sink for atmospheric sulfur gases , which can directly be taken up by the foliage. The foliar uptake of SO₂ is generally directly dependent on the degree of opening of the stomates, since the internal resistance to gas is low. SO₂ is highly soluble in the apoplastic water of the mesophyll, where it dissociates under formation of bisulfite (HSO₃ ^-) and sulfite (SO₃ ^2-). Sulfite may directly enter the sulfur reduction pathway and be reduced to sulfide, incorporated into cysteine, and subsequently into other sulfur compounds (Figure 2). Sulfite may also be oxidized to sulfate, extra- and intracellularly by peroxidases or non-enzymatically catalyzed by metal ions or superoxide radicals and subsequently reduced and assimilated again. Excessive sulfate is transferred into the vacuole; enhanced foliar sulfate levels are characteristic for SO₂-exposed plants. The foliar uptake of H₂S appears to be directly dependent on the rate of H₂S metabolism into cysteine and subsequently into other sulfur compounds (De Kok et al., 1998, 2000, 2002a.b; Figure 2). There is strong evidence that O-acetyl-serine (thiol)lyase is directly responsible in the active fixation of atmospheric H₂S by plants. Plants are able to transfer from sulfate to foliar absorbed SO₂ or H₂S as sulfur source (De Kok, 1990, De Kok et al., 1998, 2000, 2002a.b, Yang et al., 2003) and levels of 0.06 µ l l^-1 appear to be sufficient to cover the sulfur requirement of plants (Yang et al., 2003; Buchner et al., 2004). There is an interaction between atmospheric and pedospheric sulfur utilization. For instance, H₂S exposure resulted in a decreased activity of APS reductase and a depressed sulfate uptake in Brassica oleracea (Westerman et al., 2000, 2001; De Kok et al., 2002b). However, H₂S solely affected the expression of the different sulfate transporters in the shoot, but not in the roots (Buchner et al., 2004).

 

Figure 2: Metabolism of SO₂ and H₂S in the plant shoots and possible sites of feedback inhibition of sulfate uptake (adapted from De Kok et al. 2002a).

  Synthesis and physiological functions of sulfur metabolites

Cysteine is sulfur donor for the synthesis of methionine, the major other sulfur-containing amino acid present in plants (Giovanelli, 1990; Noji and Saito, 2003). Both sulfur-containing amino acids are of great significance in the structure, conformation and function of proteins and enzymes, but high levels of these amino acids may also be present in seed storage proteins (Tabatabai, 1986). The thiol groups of the cysteine residues in proteins can be oxidized resulting in disulfide bridges with other cysteine side chains (and form cystine) and/or linkage of polypeptides. Disulfide bridges make an important contribution to the structure of proteins. The thiol groups are also of great importance in substrate binding of enzymes, in metal-sulfur clusters in proteins (e.g. ferredoxins) and in regulatory proteins (e.g. thioredoxins).

Sulfoquinovosyl diacylglycerol, is the predominant sulfur-containing lipid present in plants. In leaves its content comprises up to 3 - 6 % of the total sulfur present (Heinz, 1993; Benning, 1998; Harwood and Okanenko, 2003). This sulfolipid is present in plastid membranes and likely is involved in chloroplast functioning. The route of biosynthesis and physiological function of sulfoquinovosyl diacylglycerol is still under investigation. From recent studies it is evident that sulfite it the likely sulfur precursor for the formation of the sulfoquinovose group of this lipid (Harwood and Okanenko, 2003).

Glutathione ( g Glu-Cys-Gly; GSH) or its homologues, e.g. homoglutathione ( g Glu-Cys- b Ala) in Fabaceae; hydroxymethylglutathione ( g Glu-Cys- b Ser) in Poaceae) are the major water-soluble non-protein thiol compounds present in plant tissue and account for 1-2 % of the total sulfur (De Kok and Stulen 1993; Rennenberg, 1997; Grill et al., 2001). The content of glutathione in plant tissue ranges from 0.1 - 3 mM. Cysteine is the direct precursor for the synthesis of glutathione (and its homologues). First, g -glutamylcysteine is synthesized from cysteine and glutamate catalyzed by g -glutamylcysteine synthetase. Second, glutathione is synthesized from g -glutamylcysteine and glycine (in glutathione homologues, b -alanine or serine) catalyzed by glutathione synthetase (2). Both steps of the synthesis of glutathione are ATP dependent reactions:

 

cysteine +glutamate +ATP - > g -glutamylcysteine +ADP + Pi(1)

g -glutamylcysteine synthetase

 

g -glutamylcysteine + glycine + ATP -> GSH + ADP + P(2)

glutathione synthetase

 

NADPH + H⁺ + GSSG - > 2GSH + NADP⁺(3)

glutathione reductase

 

Glutathione is maintained in the reduced form by an NADPH-dependent glutathione reductase (3) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) generally exceeds a value of 7 (Rennenberg, 1997; Foyer and Noctor, 2001; Tausz 2001).

Glutathione fulfils various roles in plant functioning.In sulfur metabolism it functions as reductant in the reduction of APS to sulfite (Figure 1). It is also the major transport form of reduced sulfur in plants. Roots likely largely depend for their reduced sulfur supply on shoot/root transfer of glutathione via the phloem, since the reduction of sulfur occurs predominantly in the chloroplast (De Kok et al., 1993; Rennenberg, 1997; Grill et al., 2001). Glutathione is directly involved in the reduction and assimilation of selenite into selenocysteine (Andersen and McMahon, 2001). Furthermore glutathione is of great significance in the protection of plants against oxidative and environmental stress and it depresses/scavenges the formation of toxic reactive oxygen species, e.g. superoxide, H₂O₂ and lipid hydroperoxides (Grill et al., 2001; Tausz et al., 2003). Glutathione functions as reductant in the enzymatic detoxification of reactive oxygen species in the glutathione-ascorbate cycle and as thiol buffer in the protection of proteins via direct reaction with reactive oxygen species or by the formation of mixed disulfides. The potential of glutathione as protectant is related to the pool size of glutathione, its redox state (GSH/GSSG ratio) and the activity of glutathione reductase. Glutathione is the precursor for the synthesis of phytochelatins (( g Glu-Cys)_nGly), which are synthesized enzymatically by a constitutive phytochelatin synthase. The number of g -glutamyl-cysteine residues ( g Glu-Cys)_n in the phytochelatins may range from 2 - 5, sometimes up to 11. Despite the fact that the phytochelatins form complexes which a few heavy metals, viz. cadmium, it is assumed that these compounds play a role in heavy metal homeostasis and detoxification by buffering of the cytoplasmatic concentration of essential heavy metals (Rauser, 1993, 2000, 2001; Verkleij et al., 2003). Glutathione is also involved in the detoxification of xenobiotics, compounds without direct nutritional value or significance in metabolism, which at too high levels may negatively affect plant functioning. Xenobiotics may be detoxified in conjugation reactions with glutathione catalyzed by glutathione S-transferase, which activity is constitutive; different xenobiotics may induce distinct isoforms of the enzyme (Schröder, 1998, 2001; Gullner and Kömives, 2001). Glutathione S-transferases have great significance in herbicide detoxification and tolerance in agriculture and their induction by herbicide antidotes (safeners) is the decisive step for the induction of herbicide tolerance in many crop plants. Under natural conditions glutathione S-transferases are assumed to have significance in the detoxification of lipid hydroperoxides, in the conjugation of endogenous metabolites, hormones and DNA degradation products, and in the transport of flavonoids.

Some plant species contain so-called secondary sulfur compounds, viz. glucosinolates in Brassica (Schnug, 1990, 1993; Rosa, 1997; Graser et al., 2001, Glawisching et al., 2003) and γ-glutamyl peptides and alliins (S-alk(en)yl cysteine sulfoxides) in Allium (Randle et al., 1993, 1995; Randle, 2000; Randle and Lancaster, 2002; Coolong and Randle, 2003a, b).In shoot and roots of Brassica the glucosinolate content accounted for 1 - 2 % of the total sulfur, however, there is a great diversity in glucosinolates between cultivars based on differences in amino acid derived side chains and their elongated derivatives (Castro et al., 2004). Glucosinolates, are composed of a ß-thioglucose moiety, a sulfonated oxime and a side chain. The synthesis of glucosinolates starts with the oxidation of the parent amino acid to an aldoxime, followed by the addition of a thiol group (through conjugation with cysteine) to produce thiohydroximate. The transfer of a glucose and a sulfate moiety completes the formation of the glucosinolates (Schnug, 1990; Rosa, 1997, 1999; Graser et al., 2001).

The physiological significance of glucosinolates is still ambiguous, though they are considered to function as sink compounds in situations of sulfur excess (Schnug, 1990, 1993; Ernst, 1993). However, when Brassica was exposed to H₂S (Westerman et al., 2001) and Arabidopsis to SO₂ (Van der Kooij et al., 1997), the sink capacity of the glucosinolate fraction seemed to be rather limited. Upon tissue disruption, glucosinolates are enzymatically degraded by myrosinase and may yield a variety of biologically active products such as isothiocyanates, thiocyanates, nitriles and oxazolidine-2-thiones (Rosa, 1997, 1999; Kushad et al., 1999; Graser et al., 2001; Petersen et al., 2002; Reichelt et al., 2002; Wittstock and Halkier 2002). The glucosinolate-myrosinase system is assumed to play a role in plant-herbivore and plant-pathogen interactions. Furthermore, glucosinolates are responsible for the flavor properties of Brassicaceae and recently have received attention in view of their potential anticarcinogenic properties (Kushad et al., 1999; Graser et al., 2001; Petersen et al., 2002; Reichelt et al., 2002).

The content of γ-glutamyl peptides and alliins in Allium species strongly depends on stage of development of the plant, temperature, water availability and the level of nitrogen and sulfur nutrition (Randle et al., 1993, 1995; Randle, 2000; Randle and Lancaster, 2002; Coolong and Randle 2003a, b; Durenkamp and De Kok, 2002, 2003, 2004). In onion bulbs their content may account for up to 80 % of the organic sulfur fraction (Schnug, 1993). Less is known about the content of secondary sulfur compounds in the seedling stage of the plant. It is assumed that alliins are predominantly synthesized in the leaves, from where they are subsequently transferred to the attached bulb scale (Lancaster et al., 1986). The biosynthetic pathways of synthesis of γ-glutamylpeptides and alliins are still ambiguous. γ-Glutamylpeptides can be formed from cysteine (via γ-glutamylcysteine or glutathione) and can be metabolized into the corresponding alliins via oxidation and subsequent hydrolization by γ-glutamyl transpeptidases (Lancaster and Boland, 1990; Randle and Lancaster 2002). However, other possible routes of the synthesis of γ-glutamylpeptides and alliins may not be excluded (Granroth, 1970; Lancaster and Boland, 1990; Edwards et al., 1994; Randle and Lancaster, 2002). Alliins and γ-glutamylpeptides are known to have therapeutic utility and might have potential value as phytopharmaceutics (Haq and Ali, 2003). The alliins and their breakdown products (e.g. allicin) are the flavor precursors for the odor and taste of species. Flavor is only released when plant cells are disrupted and the enzyme alliinase from the vacuole is able to degrade the alliins, yielding a wide variety of volatile and non-volatile sulfur-containing compounds (Lancaster and Collin, 1981; Block, 1992). The physiological function of γ-glutamylpeptides and alliins is rather unclear (Schnug, 1993).

Various other sulfur metabolites, e.g. alliins, glucosinolates, phytoalexins, the release of volatile sulfur compounds as H₂S, the production of sulfur-rich proteins (thionins) and localized deposition of elemental sulfur are assumed to have significance in the resistance of plants against stress and pests (Schnug, 1997; Glawishnig et al., 2003; Haneklaus et al., 2003; Haq and Ali, 2003). Several aspects of sulfur metabolism and its possible significance in "sulfur-induced-resistance" need further evaluation (Schnug, 1997; Haneklaus et al., 2003).

 

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