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3 The energy costs for utilizing N₂ as a nitrogen source are much higher than for the utilization of NO⁻[sub(3)]

3 The energy costs for utilizing N₂ as a nitrogen source are much higher than for the utilization of NO⁻[sub(3)]

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11.4 Plants improve their nutrition by symbiosis with fungi



319



this reason, plant roots possess very high affinity transporters, with a half

saturation of 1 to 5 μM phosphate, where the phosphate transport is driven

by proton symport, similar to the transport of nitrate (section 10.1). In order

to increase the uptake of phosphate, but also of other mineral nutrients (e.g.,

nitrate and potassium), most plants enter a symbiosis with fungi. Fungi are

able to form a mycelium with hyphae that have a much lower diameter than

root hairs and are therefore well suited to penetrate soil particles, thereby

mobilizing the nutrients. The symbiotic fungi (microsymbionts) deliver these

nutrients to the plant root (macrosymbiont) and are in turn supplied by the

plant with carbon metabolites for maintaining their own metabolism.



The arbuscular mycorrhiza is widespread

The arbuscular mycorrhiza has been detected in more than 80% of all terrestrial

plant species. In this symbiosis the fungus penetrates the cortex of plant roots

by a plant controlled process and forms a network of hyphae, which protrude

into cortical cells and form treelike invaginations, termed arbuscules (Fig. 11.9),

or form hyphal coils. The boundary membranes of fungus and host remain

intact. The arbuscules form a large surface, enabling an efficient exchange of

compounds between the fungus and the host. The fungus delivers phosphate,

nitrate, Kϩ ions, and water, and the host delivers carbohydrates. The arbuscules have a lifetime of less than two weeks, but the subsequent degeneration



Root cortex cell



Plant

plasma membrane



Fungus hypha in

intercellular space



Plasma membrane

of fungus



Figure 11.9 Schematic

representation of an

arbuscel. The hypha

of a symbiotic fungus

traverses the rhizodermis

cells and spreads into the

intercellular space of the

root cortex. From there

tree-like invaginations

into the inner layer of the

cortex are formed. The cell

walls of the plant and of

the fungus (not shown in

the figure) and the plasma

membranes remain intact.

The large contact area

between the host and the

microsymbiont enables

an efficient exchange of

compounds.



320



11



Nitrogen fixation enables plants to use the nitrogen of the air for growth



does not damage the corresponding host cell. Therefore, the maintenance of

symbiosis requires a constant formation of new arbuscules. The arbuscular

mycorrhiza evolved at a very early stage of plant evolution about 450 million

years ago. Whereas the number of plant species capable of forming an arbuscular mycorrhiza is very large (about 80% of terrestrial plants), there are only six

genera of fungi capable of forming microsymbionts, resulting in rather unspecific plant-fungus combinations. Since the supply of the symbiotic fungi by the

roots demands a high amount of assimilates, in many plants the establishment

of mycorrhiza depends on the phosphate availability in the soil.



Ectomycorrhiza supply trees with nutrients

Many trees in temperate and cool climates form a symbiosis with fungi

termed ectomycorrhiza. In this the hyphae of the fungi do not penetrate

the cortex cells, but colonize only the surface and the intercellular space

of the cortex with a network of hyphae, termed Hartig net, which is connected to a very extensive mycel in the soil. Microsymbionts are Asco- and

Basiodiomycetae from more than 60 genera, including several mushrooms.

The plant roots colonized by the fungi become thicker and do not form root

hairs. The uptake of nutrients and water is delegated to the microsymbiont, which in turn is served by the plant with carbon metabolites to maintain its metabolism. The exchange of compounds occurs, as in arbuscular

mycorrhiza, via closely neighbored fungal and plant plasma membranes.

The ectomycorrhiza also enables a transfer of assimilates between adjacent

plants. Ectomycorrhiza are of great importance for the growth of trees, such

as beech, oak, and pine, as it increases the uptake of phosphate by a factor

of three to five. It has been observed that the formation of ectomycorrhiza

is negatively affected when the nitrate content of the soil is high. This may

explain the damaging effect of nitrogen input to forests by air pollution.

Other forms of mycorrhiza (e.g., the endomycorrhiza with orchids and

Ericaceae) will not be discussed here.



11.5 Root nodule symbioses may have

evolved from a pre-existing pathway for

the formation of arbuscular mycorrhiza

There are parallels between the establishing of arbuscular mycorrhiza

and of root nodule symbiosis. In both cases, receptor-like kinases (RLK,

section 19.1) appear to be involved, linked to signal cascades, which induce



Further reading



the synthesis of the proteins required for the controlled infection. These signal cascades probably involve G-proteins, MAP-kinases, and Caϩϩ ions as

messengers (section 19.1). For several legume species, mutants are known

that have lost the ability to establish both root nodule symbiosis and arbuscular mycorrhiza. One of the genes that causes such a defect in different

legume species has been identified as encoding an RLK, indicating that this

RLK has an essential function in the formation of both arbuscular mycorrhiza and root nodule symbiosis. Fungi and bacteria, despite their different

natures, apparently induce similar genetic programs upon infection.

Molecular phylogenetic studies have shown that all plants with the ability to enter root nodule symbiosis, rhizobial or actinorhizal, belong to a

single clade (ϭbranch of phylogenetic tree, named Eurosid I). This implies

that these species go back to a common ancestor, although not all descendants of this ancestor are symbiotic. Obviously, this ancestor has acquired a

property on the basis of which a bacterial symbiosis could develop. Based

on this property, root nodule symbiosis evolved about 50 million years

ago, not as a single evolutionary event, but reoccurred about eight times.

In order to transfer the ability to enter a root nodule symbiosis to agriculturally important monocots, such as rice, maize, and wheat by genetic engineering, it will be necessary to find out which properties of the Eurosid I

clade plants allowed the evolution of such symbiosis.



Further reading

Atkins, C. A., Smith, P. M. C. Translocation in legumes: Assimilates, nutritients, and

signaling molecules. Plant Physiology 144, 550–561 (2007).

Chalot, M., Blaudez, D., Brun, A. Ammonia: A candidate for nitrogen transfer at the

mycorrhizal interface. Trends in Plant Science 11, 263–266 (2006).

Christiansen, J., Dean, D. R. Mechanistic feature of the Mo-containing nitrogenase.

Annual Review Plant Physiology Molecular Biology 52, 269–295 (2002).

Giraud, E., Fleischmann, D. Nitrogen-fixing symbiosis between photosynthetic bacteria

and legumes. Photosynthesis Research 82, 115–130 (2004).

Govindarajulu, M., et al. Nitrogen transfer in the arbuscular mycorrhizial symbiosis.

Nature 435, 819–823 (2005).

Igarashi, R. Y., Seefeld, L. C. Nitrogen fixation: The mechanism of the Mo-dependent

nitrogenase. Critical Reviews Biochemistry Molecular Biology 38, 351–384 (2003).

Karandashov, V., Bucher, M. Symbiotic phosphate transport in arbuscular mycorrhizas. Trends in Plant Science 10, 22–29 (2005).

Karlin, K. D. Metalloenzymes, structural motifs and inorganic models. Science 701–708

(1993).

Limpens, E., Franken, C., Smit, P., Willemse, J., Bisseling, T., Geurts, R. LysM domain

receptor kinases regulating rhizobial Nod factor-induced infection. Science 302,

630–633 (2003).

MacLean, A. M., Finan, T. M., Sadowsky, M. J. Genomes of the symbiotic nitrogenfixing bacteria of legumes. Plant Physiology 144, 615–622 (2007).



321



322



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Nitrogen fixation enables plants to use the nitrogen of the air for growth



Martin, F., Kohler, A., Duplessis, S. Living in harmony in the wood underground:

Ectomycorrhizal genomics. Current Opinion Plant Biology 10, 204–210 (2007).

Mylona, P., Pawlowski, K., Bisseling, T. Symbiotic nitrogen fixation. Plant Cell 7,

869–885 (1995).

Ott, T., et al. Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root

nodules but not for general plant growth and development. Current Biology 15,

531–535 (2005).

Pauly, N., Pucciariello, C., Mandon, K., Innocenti, G., Jamet, A., Baudouin, E.,

Hérouart, D., Frendo, P., Puppo, A. Reactive oxygen and nitrogen species and glutathione: Key players in the legume-Rhizobium symbiosis. Journal Experimental

Botany 57, 1769–1776 (2006).

Prell, J., Poole, P. Metabolic changes of rhizobia in legume nodules. Trends in

Microbiology 14, 161–168 (2006).

Samac, D. A., Graham, M. A. Recent advances in legume-microbe interactions:

Recognition, defense response, and symbiosis from a genomic perspective. Plant

Physiology 144, 582–587 (2007).

Sawers, R. J., Gutjahr, C., Paszkowski, U. Cereal mycorrhiza: An ancient symbiosis in

modern agriculture. Trends in Plant Science 13, 93–97 (2008).

Smith, P. M. C., Atkins, C. A. Purine biosynthesis. Big in cell division, even bigger in

nitrogen assimilation. Plant Physiology 128, 793–802 (2002).

Smith, F. A., Smith, S. E. Structural differences in arbuscular mycorrhizal symbioses:

More than 100 years after Gallaud, where next? Mycorrhiza 17, 375–938 (2007).

Sprent, J. I., James, E. K. Legume evolution: Where do nodules and mycorrhizas fit in?

Plant Physiology 144, 575–581 (2007).

Stacey, G., Libault, M., Brechenmacher, L., Wan, J., May, G. D. Genetics and functional genomics of legume nodulation. Current Opinion Plant Biology 9, 110–121

(2006).

van der Heijden, M. G., Bardgett, R. D., van Straalen, N. M. The unseen majority:

Soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems.

Ecology Letters 11, 296–310 (2008).

White, J., Prell, J., James, E. K., Poole, P. Nutrient sharing between symbionts. Plant

Physiology 144, 604–614 (2007).

Zhu, H., Choi, H.-K., Cook, D. R., Shoemaker, R. C. Bridging model and crop legumes through comparative genomics. Plant Physiology 137, 1189–1196 (2005).



12

Sulfate assimilation enables the

synthesis of sulfur containing

compounds

Sulfate is an essential constituent of living matter. In the oxidation state -II,

it is present 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 compounds by assimilating sulfate taken up from the environment.

The animal metabolism is dependent on sulfur containing nutrients such as

methionine and cysteine. 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 present 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.



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.

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

323



324



Figure 12.1 Schematic

presentation of the sulfate

metabolism in a leaf.

Sulfate is carried by the

transpiration stream into

the leaves and is transported

into the mesophyll cells,

where it is transported

to the chloroplast via the

phosphate translocator.

Sulfate is reduced there

to H2S and subsequently

converted to cysteine.

Sulfate can also be deposited

in the vacuole. Serine is

activated as acetylserine

prior to the reaction with

H2S (Fig. 12.4).



12



Sulfate assimilation enables the synthesis of sulfur containing compounds



CHLOROPLAST

6 Ferredoxinox



6 Ferredoxin red



SO32



H2S



Serine



VACUOLE







ATP



AMP

+2P



GSSG



AMP

+2P



ATP



2 GSH



Cysteine



SO42







SO42







SO42







P



SO42







3 H+



XYLEM VESSELS



Transpiration

stream



assimilation. The activities of the enzymes involved in sulfate assimilation are

minute, a reason why it is 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 (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.



12.1 Sulfate assimilation proceeds primarily by photosynthesis



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:

SO4 2Ϫ ϩ 2 eϪ ϩ 2 Hϩ → SO32Ϫ ϩ H2 O

SO32Ϫ ϩ 6 eϪ ϩ 8 Hϩ → H2S ϩ 3 H2 O

Whereas the NH3 synthesized during nitrite reduction is fixed in the

amino acid glutamine (Fig. 10.6), the hydrogen sulfide formed during

sulfite reduction is integrated into 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.



Sulfate is activated prior to reduction

Sulfate is probably taken up into the chloroplasts via the triose phosphatephosphate translocator (section 1.9) in counter-exchange for phosphate.

Sulfate cannot be directly reduced in the chloroplasts because the redox

potential of the substrate pair SO32Ϫ/SO42Ϫ (ΔE0Ј ϭ 517 mV) is too high.

No reductant is available in the chloroplasts that could reduce SO42Ϫ to

SO32Ϫ 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 12.2a, 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 to form AMP-sulfate (APS). Since the free energy of the hydrolysis of the sulfate-phosphate anhydride bond (ΔGoЈ ϭ Ϫ71 kJ/mol) is very

much higher than that of the phosphate-phosphate anhydride bond in ATP

(ΔGoЈ ϭ Ϫ31 kJ/mol), the equilibrium of the reaction lies far towards 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.



325



326



12



Sulfate assimilation enables the synthesis of sulfur containing compounds



O

O

O



Adenine

O



ATP



CH2



O



P



O

O



O



P

O



P



O



O



O



OH



OH



O



ATP

sulfurylase



O



P



O



O

CH2



O



P



O



P



O



2P



O



O

O



O

OH



Pyrophosphatase



O



O



Adenine



Sulfate



O



O

O



S



S



APS

(AMP-sulfate)



O



O



OH



2 GSH

APS

reductase



O



GSSG



S



O



Sulfite



O

O



Adenine

O



CH2



O



P



O



AMP



O

OH



Figure 12.2a



OH



Reduction of sulfate to sulfite.



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.

Alternatively APS is phosphorylated via APS kinase to 3-phospho AMP

sulfate (PAPS) (Fig. 12.2b), resulting in an activation of the sulfate residue.

Because of its high sulfate transfer potential PAPS is an important precursor for the introduction of sulfate residues into biological molecules such as

glucosinolates (section 16.4).



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



12.1 Sulfate assimilation proceeds primarily by photosynthesis



O



Adenine

O



CH2



O



O



P



O



S



O

OH



O



APS

(AMP-sulfate)



O



327



Figure 12.2b Synthesis

of PAPS (3-phosphoAMP-sulfate), the “active

sulfate”.



OH



ATP

APS-Kinase

ADP

O



Adenine

O



CH2



O



P

O



OH



O



O

O



S



O



O



PAPS

(3-Phospho-AMP-sulfate)



OH

O

P



O



O



Light



6 Ferredoxin

reduced

Photosystem I



Sulfite reductase

4 Fe–4 S



6e –



Siroheme



6 Ferredoxin

oxidized



Figure 12.3 Reduction of sulfite to hydrogen sulfide by sulfite reductase in the

chloroplasts. Reduced ferredoxin from photosystem delivers electrons for the reaction.



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 feature allows the sulfite reduction

to occur also in heterotrophic tissues.



H2S is fixed in the amino acid cysteine

The fixation of the newly formed H2S requires the activation of serine

and for this the hydroxyl group of serine is acetylated by acetyl-CoA via a

serine transacetylase (Fig. 12.4). The latter is formed from acetate and CoA



SO32







+ 8 H+



H2S + 3 H2O



328



Figure 12.4 Activation of

serine precedes the reaction

of cysteine synthesis. The

hydrogen sulfide formed

by sulfite reduction is

incorporated into cysteine.



12



Sulfate assimilation enables the synthesis of sulfur containing compounds



COO



Serine



H C NH3



AMP

+ PP



H2C OH



ATP

Acetate + CoASH



Acetyl CoA

Acetyl CoA

synthetase



Serine

transacetylase

CoASH

COO



O-Acetylserine



HSH



Acetate



H2C SH



H2C O C CH3

O



COO

H C NH3



H C NH3



O-Acetylserine

(thiol)-lyase



Cysteine



with the consumption of ATP (which is converted to AMP and pyrophosphate) by the enzyme acetyl-CoA synthetase. As the pyrophosphate released

in this reaction is hydrolyzed by the pyrophosphatase present in the chloroplasts, the activation of the serine costs the chloroplasts in total two energyrich 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 many enzymes and a replacement by any other amino acids

would alter the catalytic properties. Moreover, cysteine residues form ironsulfur 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 γ-carboxyl group of glutamate with the amino group of the cysteine

is formed by γ-glutamyl-cysteine-synthetase accompanied by the hydrolysis of ATP; and second, a peptide bond between the carboxyl group of



12.2 Glutathione serves the cell as an antioxidant



γ -GlutamylCysteine

synthetase



Figure 12.5 Biosynthesis

of glutathione.



Glutathione

synthetase

COO



Glycine



Cysteine



H C NH3



SH



CH2



Glutamate



CH2

H

H

H2C C N C C N CH2 COO



γ -Glu-Cys



O



ATP



ADP + P



ATP



329



ADP + P



H O



γ -Glu-Cys-Gly



Glutathione



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 a 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 compounds produced by the plant or which are taken up (xenobiotics, including herbicides) are detoxified by reaction with glutathione.

Catalyzed by glutathione-S transferases, the reactive SH group of glutathione can form a thioether by reacting with electrophilic carbon double

bonds, carbonyl groups, and other reactive groups. Glutathione conjugates

(Fig. 12.6) synthesized in this way in the cytosol are transported into the

vacuole by a specific glutathione translocator against a concentration gradient. In contrast to the transport processes, 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 is also present in bacteria. Various ABC transporters with

different specificities are localized in the vacuolar membrane. The imported

conjugates are often modified (e.g., by degradation to a cysteine conjugate)



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