Sulfate assimilation enables sulfurcontaining substances

the

synthesis

of

Sulfate is an essential constituent of living matter. In the oxidation state -II, it is contained in the two amino acids cysteine and methionine, in the detoxifying agent glutathione, in various iron sulfur redox clusters, in peroxiredoxins, and in thioredoxins. Plants, bacteria, and fungi are able to synthesize these substances by assimilating sulfate taken up from the environment. Animal metabolism is dependent on nutrients to supply amino acids containing sulfur. Therefore sulfate assimilation of plants is a prerequisite for animal life, just like the carbon and nitrate assimilation discussed previously. Whereas the plant uses nitrate only in its reduced form for syntheses, sulfur, also in the form of sulfate, is an essential plant constituent. Sulfate is contained in sulfolipids, which comprise about 5% of the lipids of the thylakoid membrane (Chapter 15). In sulfolipids sulfur is attached as sulfonic acid via a C-S bond to a carbohydrate residue of the lipid. The biosynthesis of this sulfonic acid group is, to a great extent, not known.

12.1 Sulfate assimilation proceeds primarily by photosynthesis

Sulfate assimilation in plants occurs primarily in the chloroplasts and is then a part of photosynthesis, but it also takes place in the plastids of the roots. However, the rate of sulfate assimilation is relatively low, amounting to only about 5% of the rate of nitrate assimilation and only 0.1% to 0.2% of the rate of CO2 assimilation. The activities of the enzymes involved in sulfate assimilation are minute, making it very difficult to elucidate the reactions involved. Therefore our knowledge about sulfate assimilation is still fragmentary.

Sulfate assimilation has some parallels to nitrogen assimilation Plants take up sulfate via a specific translocator of the roots, in a manner similar to that described for nitrate in Chapter 10. The transpiration stream in the xylem vessels carries the sulfate to the leaves, where it is taken up by a specific translocator, probably a symport with three protons, into the mesophyll cells (Fig. 12.1). Surplus sulfate is transported to the vacuole and is deposited there. The basic scheme for sulfate assimilation in the mesophyll cells corresponds to that of nitrate assimilation. Sulfate is reduced to sulfite by the uptake of two electrons and then by the uptake of another six electrons, to hydrogen sulfide:

Whereas the NH3 formed during nitrite reduction is fixed by the formation of the amino acid glutamine (Fig. 10.6), the hydrogen sulfide formed during sulfite reduction is

fixed to form the amino acid cysteine. A distinguishing difference between nitrate assimilation and sulfate assimilation is that the latter requires a much higher input of energy. This is shown in an overview in Figure 12.1. The reduction of sulfate to sulfite, which in contrast to nitrate reduction occurs in the chloroplasts, requires in total the cleavage of two energy-rich phosphate anhydride bonds, and the fixation of the hydrogen sulfide into cysteine requires another two. Thus the ATP consumption of sulfate assimilation is four times higher than that of nitrate assimilation. Let us now look at the individual reactions. Sulfate is activated prior to reduction. Sulfate is probably taken up into the chloroplasts in counter-exchange for phosphate. Sulfate cannot be directly reduced in the chloroplasts, because the redox potential of the substrate pair SO32-/SO42- (DE0/= -517 mV) is too high. No reductant is available in the chloroplasts that could reduce SO42- to SO32- in one reaction step. To make the reduction of sulfate possible, the redox potential difference to sulfite is lowered by activation of the sulfate prior to reduction. As shown in Figure 12.2, activation of sulfate proceeds via the formation of an anhydride bond with the phosphate residue of AMP. Sulfate is exchanged by the enzyme ATP-sulfurylase for a pyrophosphate residue of ATP, and AMP-sulfate (APS) is thus formed. Since the free energy of the hydrolysis of the sulfate-phosphate anhydride bond (DGo = -71 kJ/mol) is very much higher than that of the phosphate-phosphate anhydride bond in ATP (DGo = -31 kJ/mol), the equilibrium of the reaction lies far toward ATP. This reaction can proceed only because pyrophosphate is withdrawn from the equilibrium by a high pyrophosphatase activity in the chloroplasts. Sulfate present in the form of APS is reduced by glutathione (Figs. 12.5, 3.38) to sulfite. The APS reductase involved in this reaction catalyzes not only the reduction, but also the subsequent liberation of sulfite from AMP. The redox potential difference from sulfate to sulfite is lowered, since the reduction of sulfate is driven by hydrolysis of the very energy-rich sulfite anhydride bond. The mechanism of the APS reductase reaction remains to be elucidated.

Sulfite reductase is similar to nitrite reductase As in nitrite reduction, six molecules of reduced ferredoxin are required as reductant for the reduction of sulfite in the chloroplasts (Fig. 12.3). The sulfite reductase is homologous to the nitrite reductase, it also contains a siroheme (Fig. 10.5) and a 4Fe-4S cluster. The enzyme is half saturated at a sulfite concentration in the range of 10-6 mol/L and thus is suitable to reduce efficiently the newly formed sulfite to hydrogen sulfide. The ferredoxin required by sulfite reductase, as in the case of nitrite reductase (Fig. 10.1), can be reduced by NADPH. This makes it possible for sulfite reduction to occur in heterotrophic tissues also.

H2S is fixed in the form of cysteine The fixation of the newly formed H2S requires the activation of serine, and for this its hydroxyl group is acetylated by acetyl-CoA via a serine transacetylase (Fig. 12.4). The latter is formed from acetate and CoA with the consumption of ATP (which is converted to AMP) by the enzyme acetyl-CoA synthetase. As pyrophosphate formed in this reaction is hydrolyzed by the pyrophosphatase present in the chloroplasts; the activation of the serine costs the chloroplasts in total two energy-rich phosphates.

Fixation of H2S is catalyzed by the enzyme O-acetyl serine (thiol) lyase. The enzyme contains pyridoxal phosphate as a prosthetic group and has a high affinity for H2S and acetyl serine. The incorporation of the SH group can be described as a cleavage of the ester linkage by H-S-H. In this way cysteine is formed as the end product of sulfate assimilation. Cysteine has an essential function in the structure and activity of the catalytic

site of enzymes and cannot be replaced there by any other amino acids. Moreover, cysteine residues form iron-sulfur clusters (Fig. 3.26,) and are constituents of thioredoxin (Fig. 6.25).

12.2 Glutathione serves the cell as an antioxidant and is an agent for the detoxification of pollutants
A relatively large proportion of the cysteine produced by the plant is used for synthesis of the tripeptide glutathione, (Fig. 12.5). The synthesis of glutathione proceeds via two enzymatic steps: first, an amide linkage between the g-carboxyl group of glutamate with the amino group of the cysteine is formed by g-glutamyl-cysteine-synthetase accompanied by the hydrolysis of ATP; and second, a peptide bond between the carboxyl group of the cysteine and the amino group of the glycine is produced by glutathione synthetase, again with the consumption of ATP. Glutathione, abbreviated GSH, is present at relatively high concentrations in all plant cells, where it has various functions. The function of GSH as a reducing agent was discussed in the previous section. As an antioxidant, it protects cell constituents against oxidation. Together with ascorbate, it eliminates the oxygen radicals formed as by-products of photosynthesis (section 3.9). In addition, glutathione has a protective function for the plant in forming conjugates with xenobiotics and also as a precursor for the synthesis of phytochelatins, which are involved in the detoxification of heavy metals. Moreover, glutathione acts as a reserve for organic sulfur. If required, cysteine is released from glutathione by enzymatic degradation.

Xenobiotics are detoxified by conjugation Toxic substances formed by the plant or which it has taken up (xenobiotics) are detoxified by reaction with glutathione. Catalyzed by glutathione-S transferases, the reactive SH group of glutathione can form a thioether by reacting with carbon double bonds, carbonyl groups, and other reactive groups. Glutathione conjugates (Fig. 12.6) formed in this way are transported into the vacuole by a specific glutathione translocator against a

concentration gradient. In contrast to the transport processes so far, where metabolite transport against a gradient proceeds by secondary active transport, the uptake of glutathione conjugates into the vacuole proceeds by an ATP-driven primary active transport (Fig. 1.20). This translocator belongs to the superfamily of the ABC-transporter (ATP binding cassette), which is ubiquitous in plants and animals and also occurs in bacteria. In the vacuolar membrane, various ABC transporters with different specificities are present. The conjugates taken up are often modified (e.g., by degradation to a cysteine conjugate) and are finally deposited in this form. In this way plants can also detoxify herbicides. Herbicide resistance (e.g., resistance of maize to atrazine) can be due to the activity of a specific glutathione-S transferase. In an attempt to develop herbicides that selectively attack weeds and not crop plants, the plant protection industry has produced a variety of different substances that increase the tolerance of crop plants to certain herbicides. These protective substances are called safeners. Such safeners, like other xenobiotics, stimulate the increased expression of glutathione-S transferase and of the vacuolar glutathione translocator, and in this way the herbicides taken up into the plants are detoxified more rapidly. Formation of glutathione conjugates and their transport into the vacuole is also involved in the deposition of flower pigments (section 18.6).

Phytochelatins protect the plant against heavy metals

Glutathione is also a precursor for the formation of phytochelatins (Fig. 12.7). Phytochelatin synthase, a transpeptidase, transfers the amino group of glutamate to the carboxyl group of the cysteine of a second glutathione molecule, accompanied by liberation of one glycine molecule. The repetition of this process results in the formation of chains of up to 11 Glu-Cys residues. Phytochelatins have been found in all plants investigated so far, although sometimes in a modified form as iso-phytochelatins, in which glycine is replaced by serine, glutamate, or b-alanine.

Phytochelatins protect plants against toxicity from heavy metals compounds for Cu
++

and

are

storage

and Zn

++

. Through the thiol groups of the cysteine residues, they form

tight complexes with metal ions such as Cd++, Ag+, Pb++, Cu++, Hg++, and Zn++ as well as the nonmetal As3+ (Fig. 12.8). The phytochelatin synthase present in the cytosol is activated by the ions of at least one of the heavy metals listed previously. Thus, upon the exposure of plants to heavy metals, within a very short time the phytochelatins required for detoxification are synthesized de novo from glutathione. Exposure to heavy metals can therefore lead to a dramatic fall in the glutathione reserves in the cell. The phytochelatins loaded with heavy metals are pumped, in a similar manner to the glutathione conjugates, at the expense of ATP into the vacuoles. Because of the acidic environment in the vacuole, the heavy metals are liberated from the phytochelatins and finally deposited there as sulfides. Phytochelatins are essential to protect plants against heavy metal poisoning. Mutants of Arabidopsis have been found with a defect in the phytochelatin synthase, which showed an extreme sensitivity to Cd++. The capacity of plants to sequester heavy metal ions by binding them to phytochelatins has been utilized in recent times to detoxify soils polluted with heavy metals. On such soils plants are grown, which by breeding or genetic engineering have a particularly high capacity for heavy metal uptake by the roots and of phytochelatin biosynthesis, and in this way are able to extract heavy metals from the polluted ground. This procedure, termed phytoremediation, may have a great future, since it is much less costly than other methods to remediate heavy metal polluted soils.

12.3 Methionine is synthesized from cysteine

Cysteine is a precursor for methionine, another sulfur-containing amino acid. OPhosphohomoserine, which has already been mentioned as an intermediate of threonine synthesis (Fig. 10.14), reacts with cysteine, while a phosphate group is liberated to form cystathionine (Fig. 12.9). The thioether is cleaved by cystathionine-b-lyase to form homocysteine and an unstable enamine, which spontaneously degrades into pyruvate and NH4+. The sulfhydryl group of homocysteine is methylated by methyltetrahydrofolate (methyl-THF) (see Fig. 7.6), and thus the end product methionine is formed.

S-Adenosylmethionine is a universal methylation reagent

The origin of the methyl group provided by tetrahydrofolic acid (THF) is not clear. It is possible that it is derived from formate molecules, reacting in an ATP-dependent reaction with THF to form formyl-THF, which is reduced by two molecules of NADPH to methylTHF. Methyl-THF has only a low methyl transfer potential. S-Adenosylmethionine, however, has a more general role as a methyl donor. It is involved in the methylation of nucleic acids, proteins, carbohydrates, membrane lipids, and many other substances and can therefore be regarded as a universal methylating agent of the cell.

Figure 12.10 Sadenosylmethionine formed from methionine and ATP is a methylating agent.

S-Adenosylmethionine is formed by the transfer of an adenosyl residue from ATP to the sulfur atom of methionine, with the release of phosphate and pyrophosphate (Fig. 12.10). The methyl group to which the positively charged S-atom is linked is activated and can thus be transferred by corresponding methyl transferases to other acceptors. The remaining Sadenosylhomocysteine is hydrolyzed to adenosine and homocysteine and from the latter methionine is recovered by reduction with methyltetrahydrofolate (Fig. 12.9). Sadenosylmethionine is a precursor for the synthesis of the phytohormone ethylene (section 19.7).

12.4 Excessive concentrations of sulfur dioxide in air are toxic for plants
Sulfur dioxide in the air, which is formed in particularly high amounts during the smelting of ores containing sulfur, but also during the combustion of fossil fuel, can cover the total nutritional sulfur requirement of a plant. In higher concentrations, however, it leads to dramatic damage in plants. Gaseous SO2 is taken up via the stomata into the leaves, where it is converted to sulfite:

Plants possess protective mechanisms for removing the sulfite, which has been formed in the leaves. In one of these, sulfite is converted by the sulfite reductase, discussed in section 12.1, to hydrogen sulfide and then further into cysteine. Cysteine formed in increasing

amounts can be converted to glutathione. Thus one often finds an accumulation of glutathione in the leaves of SO2- polluted plants. Excessive hydrogen sulfide can leak out of the leaves through the stomata, although only in small amounts. Alternatively, sulfite can be oxidized, possibly by peroxidases in the leaf, to sulfate. Since this sulfate cannot be removed by transport from the leaves, it is finally deposited in the vacuoles of the leaf cells as K+ or Mg++-sulfate. When the deposit site is full, the leaves are abscised. This explains in part the toxic effect of SO2 on pine trees: The early loss of the pine needles of SO2- polluted trees is to a large extent due to the fact that the capacity of the vacuoles for the final deposition of sulfate is exhausted. In cation-deficient soils, the high cation demand for the final deposition of sulfate can lead to a serious K+ or Mg++ deficiency in leaves or pine needles. The bleaching of pine needles, often observed during SO2 pollution, is partly attributed to a decreased availability of Mg++ ions.

Further reading
Clemens, S., Palmgren, M. G., Kraemer, U. A long way ahead: Understanding and engineering plant metal accumulation. Trends Plant Sci 7, 309315 (2002). Cobbett, C., Goldsbrough, P. Phytochelatins and metallothioneins: Roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53, 159182 (2002). Cole, J. O. D., Blake-Kalff, M. M. A., Davies, T. G. E. Detoxification of xenobiotics by plants: Chemical modification and vacuolar compartmentation. Trends Plant Sci 2, 144151 (1997). Dixon, D. P., Cummins, I., Cole, D. J., Edwards, R. Glutathione-mediated detoxification systems in plants. Plant Biol 1, 258266 (1998). Foyer, C. H., Theodoulou, F. L., Delrot, S. The functions of inter- and intracellular glutathione transport systems in plants. Trends Plant Sci 6, 486492 (2001). Heber, U., Kaiser, W., Luwe, M., Kindermann, G., Veljovic-Iovanovic, S., Yin, Z-H., Pfanz, H., Slovik, S. Air pollution, photosynthesis and forest decline. Ecol Stud 100, 279296 (1994). Hesse, H., Hoefgen, R. Molecular aspects of methionine biosynthesis. Trends Plant Sci 8, 259262 (2003). Higgins, C. F., Linton, K. J. The xyz of ABC transporters. Science 293, 17821784 (2001). Howden, R., Goldsbrough, C. R., Anderson, C. R., Cobbett, C. S. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 107, 10591066 (1995). Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F.-L., Kim, S.-H. Crystal structure of the ATP-binding subunit of an ABC transporter. Nature 396, 703707 (1998). Kreuz, K., Tommasini, R., Martinoia, E. Old enzymes for a new job: Herbicide detoxification in plants. Plant Physiol 111, 349353 (1996). Leustek, T., Martin, M. N., Bick, J.-A., Davies, J. P. Pathways and regulation of sulfur metabolism revealed through molecular and genetic studies.

Annu Rev Plant Physiol Plant Mol Biol 51, 141165 (2000). Ma, L. Q., Komart, K. M., Tu, C., Zhang, W., Cai, Y., Kennelley, E. D. A fern that hyperacculmulates arsenic. Nature 409, 579 (2001). Saito, K. Regulation of sulfate transport and synthesis of sulfur-containing amino acids. Curr Opin Plant Biol 3, 188195 (2000). Zenk, M. H. Heavy metal detoxification in higher plants. Gene 179, 2130 (1996). ASIMILASI SULFAT