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WELCOME

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Content

Introduction

Sulphur uptake & Transport

Sulphur Activation

Sulfate Assimilation Pathway

Sulphate Reduction

Regulation of sulphur metabolism

Biosynthesis of Glutathione

Sulphur Di-oxide toxicity in air

Recent Work

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Introduction Sulfur is an essential macronutrient required for plant growth. It is

primarily used to

synthesize cysteine, methionine and numerous essential and

secondary

metabolites derived from these amino acids.

Sulfate enters a plant primarily through the roots by way of an

active uptake

mechanism. Gaseous sulfur dioxide readily enters the leaves,

where it is

assimilated.

To reach the chloroplasts, where most of the reduction to sulfide

takes place, a

sulfate molecule must traverse at least three membrane systems:

the plasma

membrane of root cell at the soil-plant interface, the plasma

membranes of

internal cells involved in transport, and the chloroplast membranes.

Sulfate is assimilated into organic molecules in one of two oxidation

states. Most

sulfur is reduced to sulfide by the multistep process. Sulfide is then

incorporated

into cysteine, becoming the thiol group.

The bacteria from Thiobacillus species-Rhodanese & sulfogens are sulfate or

thiosulfite solubilising bacteria.

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Sulfate uptake and transport

The phenotype of sulfate starvation typically consists of pale-green young leaves,

while mature leaves remain dark-green.

This phenomenon is different from the symptoms of nitrogen and phosphate

deficiency, where young leaves remain green due to support by degrading

mature leaves, and has been interpreted as an inefficient activation of cellular

sulfate pools.

Plants can also metabolize sulfur dioxide taken up in the gaseous form through

their stomata.

Nonetheless, prolonged exposure (more than 8 hours) to high atmospheric

concentrations (greater than 0.3 ppm) of SO2 causes extensive tissue damage

because of the formation of sulfuric acid.

Sulfur assimilated in leaves is exported via the phloem to sites of protein

synthesis (shoot and root apices, and fruits) mainly as glutathione

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The expression patterns of the three genes nicely fit assumptions summarized

from earlier observations: the high affinity transporters are root-specific and

inducible by exogenous sulfate defficiency, while the low affinity transporter

mRNA is less abundant, but present in roots and leaves.

The latter form is only slightly inducible in roots and is repressed in leaves upon

sulfate deprivation, suggesting a function in cell-to-cell or intracellular

transport.

It will be interesting to see whether other types of sulfate transporters exist

and to investigate the molecular basis of such essential processes as root

uptake, vascular loading and unloading, and intracellular transport of sulfate.

The identification of the metabolites and the signal transduction pathway that

mediate the transcriptional and post-transcriptional induction and repression of

sulfate uptake in the observed tissue-specific and developmental pattern will be

crucial for understanding the regulation of sulfate assimilation.

Continue…

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Fig:

Subcellular compartmentation of major reactions and compounds of sulfur metabolism

in a typical plant cell from an autotrophic source tissue. Membrane transport processes

(open circles) are indicated in their main direction attributed to this cell type.

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Plasma Membrane Transport

Anionic sulfate (So42-) is relatively abundant in the environment and

serves as the primary sulfur source for plants.

It is actively transported into roots where it can remain or be distributed

to other sites.

Transport into cells is mediated by plasma membrane–localized H+/So42-

co-transporters that are driven by the electrochemical gradient

established by the plasma membrane proton ATPase .

Seven genes encoding sulfate transporters have been identified from

Arabidopsis Thaliana.

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Transport Into Plastids

Sulfate must be transported into plastids where reduction and most of

assimilation takes place.

Sulfide or thiosulfide are probably exported from plastids since

isoenzymes for cysteine synthesis, but not for sulfate reduction, are

localized outside of plastids.

The nature of the plastid sulfate transporter has been the subject of

much speculation but it has not been conclusively identified.

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Sulfate activation Inorganic sulfate is chemically very stable and therefore has to be activated prior

to reduction to sulfate or esterification with stable organic compounds.

The enzyme ATP-sulfurylase catalyzes the formation of adenosine 5’-

phosphosulfate (APS), an energy-rich mixed anhydride of phosphate and sulfate.

Adenosine5’-phosphosulfate is an efficient inhibitor of ATP-sulfurylase , but the

physiological significance of this possible feed-back mechanism is unclear, since

APS is labile at cellular pH and its production is kinetically unfavourable, as

indicated by its equilibrium constant (Keq≈ 10-7).

To achieve substantial product formation the equilibrium has to be pulled

forward, but inorganic pyrophosphatase hydrolysis of the product

Pyrophosphate (PPi) alone is not sufficient.

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Sulfur is among the most versatile elements in living organisms (Hell 1997).

Disulfide bridges in proteins play structural and regulatory roles.

Sulfur participates in electron transport through iron–sulfur clusters.

The catalytic sites for several enzymes and coenzymes, such as urease and

coenzyme A, contain sulfur.

Secondary metabolites (compounds that are not involved in primary pathways of

growth and development) that contain sulfur range from the rhizobial Nod factors

antiseptic alliin in garlic and anticarcinogen sulforaphane in broccoli.

The versatility of sulfur derives in part from the property that it shares with

nitrogen multiple stable oxidation states.

SULFUR ASSIMILATION

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Consumption of APS by high affinity enzymes is required to pull substrate flow,

either by channelling into sulfate reduction, or by a second ATP-dependent

activation which results in 3’-phosphoadenosine 5’-phosphosulfate (PAPS) and

is

catalyzed by APS-kinase .

3’-Phosphoadenosine 5’-phosphosulfate serves as the preferred donor in sulfate-

transfer reactions for esterification of hydroxyl residues, hence animals are able

to synthesize PAPS as well, e.g. as donor in tyrosine- sulfation reactions.

These organisms circumvent the thermodynamic implications of sulfate

activation using a bifunctional PAPS-synthetase to channel the intermediate APS.

Recent cloning of cDNAs from a marine worm, Urechis caupo, and rat revealed

the existence of polypeptides with structural homology to APS-kinase at their

N-terminus and to ATP-sulfurylase at their C-terminus. A similar association of

independent proteins may exist in plants.

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Sulfate reduction in plants:-

The long-held dogma concerning sulfate reduction stated that, in plants, APS is

the substrate for this reaction and the enzyme APS sulfotransferase (APSSTase).

For a number of reasons, however, the APS pathway in plants was not

universally accepted despite years of research, definitive evidence for the

existence of APSSTase was not forthcoming.

One report of its purification from Euglena gracilis was confounded by the

unreasonably low specific activity of the pure enzyme.

Subsequently, another group reported that, in vitro, plant APS kinase displays

APS sulfotransferase activity as a side reaction.

This result, combined with several physical similarities, prompted the idea that

perhaps APS sulfotransferase is a kinetic object.

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In contrast to this uncertainty, it is widely believed that prokaryotes (including

cyanobacteria) and fungi use PAPS(3’-phosphoadenosine 5’-phosphosulfate) for

sulfate reduction via the enzyme PAPS reductase. Recently, however, direct

evidence was published by three independent groups. confirming the existence

of an APS-dependent pathway in plants.

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Sulphate Reduction

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As reflected by its name, APSSTase was proposed to catalyze the transfer of

sulfate from APS to a thiol acceptor molecule forming a thiosulfonate .

The physiological acceptor was envisaged to be glutathione, primarily because it

is an efficient substrate and is the most abundant thiol in the chloroplast stroma

in which APSSTase is localized.

The algal enzyme shows a kinetic constant for glutathione of 0.6 mM, a value that

is well below the physiological concentration in the stroma, reported to be

between 3 and 10 mM.

This is a key finding that supports much of the earlier work on this enzyme. For

example, it has long been known that sulfite production from sulfate.

Kanno isolating the enzyme from a marine macroalga by maintaining high levels

of ammonium sulfate throughout the purification procedure.

The lability of APSSTase and that it could be stabilized with high concentrations

of sulfate salts , but this finding was never incorporated into a purification scheme.

APS sulfotransferase

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Regulation of Sulphur metabolism

A. Rate-Limiting Steps in S Pathways

Sulfate assimilation is regulated by S status.

When the amount of S in the plant is low, many enzymes involved in S acquisition

and reduction are up-regulated, including sulfate permease, ATP sulfurylase and

APS reductase.

Expression of the gene encoding APS reductase is most closely correlated with S

status and this enzyme is suspected to be a rate-controlling enzyme for the

pathway.

There is also indication that ATP sulfurylase may be limiting for sulfate uptake and

assimilation, because over-expression of the gene resulted in higher plant levels of

both reduced and total S.

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Regulatory pathway of sulphur metabolism

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Another potentially limiting enzyme for Cys formation may be serine

acetyltransferase, because over-expresion in cytosol and plastids resulted in 3-fold

and 6-fold higher Cys levels, respectively.

The regulatory enzyme for GSH synthesis under unstressed conditions is thought to

be -Glutamylsynthetaseγ .

Under metal stress, -glutamylsynthetaseγ activity is up-regulated both at the

transcription level and the enzyme activity level, and GSH synthetase may become

co-limiting.

B. Regulation of S Metabolism in Response to the Environment

As mentioned above, S limitation induces sulfate uptake and assimilation at the

transcriptional level, with GSH as an important signal molecule.

While uptake and reduction of S are enhanced under S limitation, the synthesis of

secondary S compounds (e.g. sulfation) is down-regulated, and secondary S

compounds such as glucosinolates are even broken down to provide S for essential

compounds.

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Sulfur limitation also affects the expression of seed storage proteins the rate

of photosynthesis and protein turnover.

Conversely, when photosynthesis is reduced, sulfate assimilation is reduced

as well.

Accumulation of AMP and ADP were reported to inhibit ATP sulfurylase,

offering a partial explanation of the mechanism involved.

The S assimilation pathway is also regulated in coordination with nitrogen (N)

assimilation and the ratio of reduced S to reduced N is typically maintained at

1:20.

Reduced S compounds activate the key enzyme of N reduction, nitrate

reductase. Similarly, reduced N compounds stimulate the key enzymes of S

reduction, ATP sulfurylase.

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Methionine is generally regarded as a member of the aspartate family of

amino acids however, most of the metabolic functions of methionine are

connected with its sulfur moiety.

Prime examples are the role of S-adenosylmethionine (SAM) in numerous

methyl-transfer reactions and the observation that the cycle underlying

ethylene biosynthesis essentially recovers the methylthio group, but not

the ammonia and carbon backbone of methionine.

Cystathionine- -synthaseɣ is exclusively plastid localized and catalyzes the

first committed step of methionine synthesis, the formation of

cystathionine from O-phosphohomoserine and cysteine.

Regulation of methionine synthesis is connected to the other routes of the

aspartate family at the metabolic level.

At the branch point of the pathways, threonine synthase requires SAM

as an allosteric activator also acts as an inhibitor of aspartate kinase at

the entry of the pathway.

Methionine biosynthesis

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Homocysteine is converted to methionine, catalyzed by the enzyme methionine synthase

THF- Tri hydro folate

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O-Succinylhomoserine

Fig:- Methionine synthesis

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Enzymes involved in methionine biosynthesis:

1. aspartokinase

2. ß-aspartate semialdehyde dehydrogenase

3. homoserine dehydrogenase

4. homoserine O-transsuccinylase

5. cystathionine-γ-synthase

6. cystathionine-ß-lyase

7. methionine synthase (in mammals, this step is

performed by homocysteine methyltransferase)8. Methyl transferase

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Methionine Degradation

Methyl transferase

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Methionine Biosynthesis Inhibitior

L-Propargylglycine produced growth Inhibition of exogenous methionine and

cystathionine γ-synthase activity.

L-Aminoethoxyvinylglycine also produced growth inhibtion and morphological

change partially preventable by exogenous methionine.

L-Aminoethoxyvinylglycine impairs the cleavage of cystathionine to homocysteine.

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Recycling of methionine

SAM is used in a wide variety of biological reactions and represents a major

pathway of methionine metabolism.

The flux through methionine was analysed in Lemna and it was determined that

over 80% of methionine is metabolised into SAM, of which approximately 90% is

used for transmethylation reactions.

The product of these methylation reactions in higher plants is S-

adenosylhomocysteine. is recycled to homocysteine by adenosylhomocysteinase.

prior to the re incorporation of a methyl group by methionine synthase and

regeneration of the methionine molecule.

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Cysteine biosynthesis

In animals cysteine is synthesized from homocysteine, a produce of the essential

amino acid methionine.

+ + H2O 

+ H2O  + + NH3

cystathionine

cystathionine cysteine

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In the absence of dietary methionine, animals cannot make cysteine. Bacteria and

plants however produce cysteine by a different biosynthetic route.

The sulfur used to produce cysteine originates from inorganic sulfur taken up

from the environment as sulfate.

The inorganic sulfur in sulfate is activated by forming a sulfated ADP analog, PAPS.

The sulfur is reduced and released to form sulfite and further reduced to form

sulfide (S2-).

The carbon backbone in cysteine is derived from serine. Serine is

activated through acetylation by serine acetyltransferase.

The acetyl group is then exchanged with a sulfur from sulfide to create cysteine.

From cysteine other sulfur containing molecules are synthesized, including

methionine.

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Since cysteine is an essential amino acid, manipulation of cysteine content in

transgenic plants may be a valuable means of increasing the nutritional content

of agricultural plants.

Manipulation of enzymes such as serine acetyltransferase may provide one

strategy to do this. In a related experiment, bacterial genes for cysteine

biosynthesis were engineered into sheep in an attempt to improve wool

production.

Inhibitors

Serine acetyltransferase catalyzes the formation of O-acetylserine from L-Ser and

acetyl-CoA in plants and bacteria. In plants, two types of SATase have been

described. It act as cysteine feedback inhibitor.

One is allosterically inhibited by L-Cys, and the second is not sensitive to L-Cys

inhibition.

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Fig: cysteine Biosynthesis pathway.

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Cysteine Degradation

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Biosynthesis of glutathione

GSH (y-glutamyl-cysteinyl-glycine) and GSSH, reduced and oxidized forms of

glutathione, respectively, are readily interchangeable.

This tripeptide (y -Glu-Cys-Gly) is the dominant non-protein thiol in

plants and can play a role in regulating the uptake of So42- by plant roots.

It is also a substrate for GSH-S-transferases, which are important for

detoxification of xenobiotics , and is the precursor of phytochelatins,

peptides that enable plant cells to cope with heavy metals in the

environment.

GSH is an abundant antioxidant in cells and supports redox buffering .

The synthesis of GSH occurs in plastids by a two-step reaction catalyzed

by y-glutamylcysteine synthetase and GSH synthetase, genes encoding

both have been isolated from Arabidopsis.

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Reduced Glutathione

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Exposure of plants to cadmium induces phytochelatin synthesis. This

heavymetal chelator is synthesized from GSH by phytochelatin synthase

and consists of repetitions of the y-glutamylcysteine dipeptide that

terminates with a glycine.

Mutants defective in phytochelatin synthesis are sensitive to heavy metals

whereas overexpression of y-glutamylcysteine synthetase or GSH

synthetase in Brassica juncea allowed increased cadmium tolerance.

Glutathione accumulates after excess feeding of sulfur compounds if the

normal regulatory control mechanisms are circumvented , suggesting that

glutathione functions as a storage pool for excess cysteine.

GSH is synthesized by a -glutamyl-cysteine synthase γ and has been

characterized from Nicotiana tabacum .

This compound is condensed with glycine by the glutathione synthase,

forming GSH.

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Ecological significance of H2Semissions by plants

The emission of several volatile reduced sulfur gases (H2S, COS, DMS, CS2 and

methylmercaptan ) from various plant species was determined in various

experiments.

From these volatile substances H2S is one of the most important sulfur gases

emitted by higher plants in response to an excess of sulfur.

Soil applied sulfur fertilization and H2S emission of agricultural crops was not

proven, but it was shown in field experiments that sulfur fertilization and the

sulfur nutritional status, respectively had a significant effect on fungal infections in

oilseed rape.

These findings underline the concept of sulfur-induced resistance (SIR) of plants.

H2S is highly fungi toxic and therefore a relationship between increasing hydrogen

sulfide emissions of plants and a higher resistance of crops against pests and

diseases can be assumed.

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SO2 Toxicity in plants

Major sources of sulfur dioxide are coal-burning operations, especially those

providing electric power and space heating.

Sulfur dioxide emissions can also result from the burning of petroleum and the

smelting of sulfur containing ores.

Sulfur dioxide enters the leaves mainly through the stomata and the resultant

injury is classified as either acute or chronic.

Acute injury is caused by absorption of high concentrations of sulfur dioxide in a

relatively short time.

The symptoms appear as 2-sided lesions that usually occur between the veins and

occasionally along the margins of the leaves.

The colour of the necrotic area can vary from a light tan or near white to an

orange-red or brown depending on the time of year, the plant species affected

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Chronic injury is caused by long-term absorption of sulfur dioxide at sub-lethal

concentrations.

The symptoms appear as a yellowing or chlorosis of the leaf, and occasionally as a

bronzing on the under surface of the leaves.

Some crop plants are generally considered susceptible to sulfur dioxide: alfalfa,

barley, buckwheat, clover, oats, pumpkin, radish, rhubarb, spinach, squash, Swiss

chard and tobacco.

Fig:- Acute sulfur dioxide injury to raspberry. the injury occurs between the veins and that the tissue nearest the vein remains healthy.

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Recent Work On Sulphur Metabolism

Metabolic control of sulphate uptake & assimilation . A series of feedback loops are proposed in which cellular concentration of pathway to repress or activate expression of genes encoding the protein controlling some of the individual steps in pathway.

ATP sulfurylase

ATP reductaseSulfite reductase

OAS Thiol lyase Serine acetyltransferase

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In addition there is also allostearic regulation of serineacetyltransferase(SATase ) by

o-acetylserine (OAS) & cysteine solid line represent metabolic fluxes,grey lines are

feedback control loop.

The state of knowledge has been significantly influenced by the isolation of genes for

each of the metabolic steps, but not all areas have benefited equally from molecular

methods.

There is still a clear opportunity for applying gene-cloning methods to learn more

about how glutathione and glutathione S-conjugates are transported and degraded.

Although significant progress has been made toward elucidating the structure,

organization, and regulation of GSTs, the in vivo catalytic function of most GSTs is

unknown.

It has become increasingly apparent that sulfation reactions play a critical role in

controlling developmental signals, but this process is still poorly understood; only a

few systems have been described.

There has been significant progress in defining through genetics the signaling

pathway that regulates sulfur response in Chlamydomonas.

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Thank You