The document outlines the complex process of sulfur metabolism in plants, focusing on sulfate assimilation primarily occurring in the chloroplasts of leaves. It details the multi-step biochemical pathways that convert sulfates absorbed by roots into cysteine and other sulfur-containing metabolites, highlighting the roles of various enzymes and regulatory mechanisms involved. Additionally, it discusses the influence of environmental factors, such as heavy metal stress and circadian rhythms, on sulfur assimilation and its integration with nitrogen metabolism.

1. DR. VIBHA KHANNA
Asso. Prof. (Botany)
SPC GOVERNMENT COLLEGE
AJMER (Rajasthan)

2. PLANT BIOCHEMISTRY
• BLOCK 2. : SULPHUR METABOLISM
– PRESENTATION 3:
•Sulphate Assimilation

3. Site Of Sulphate Assimilation
• Sulphate assimilation takes place chiefly in leaves in
chloroplasts.
• The sulphate absorbed by roots from soil solution is
translocated through xylem to shoots for assimilation.
• To reach the chloroplast, where most of the reduction to
sulphide takes place , a sulphate molecule must traverse
atleast three membrane system;
– the plasma membrane of root cell at the soil-plant interface ,
– the plasma membrane of internal cells involved in transport
and
– the chloroplast membranes .

4. Transport into plastids
 Sulphate must be transported into plastids where
reduction and most of assimilation takes place .
 Sulphide or thiosulphide are probably exported
from plastids since iso-enzymes for cysteine
synthesis , but not for sulfate reduction, are
localized outside of plastids .
 The nature of the plastids sulphate transporter
has been dealt with in
https://www.slideshare.net/vibhakhanna1/sulph
ur-uptake-and-transport-in-plants

5. Sulphur is assimilated in one of two
oxidation states.
 SO4
2− can be added to a hydroxyl group of an organic
molecule. The reaction is referred to as sulphation and it is
catalyzed by sulphotransferases.
 By contrast, Cys contains reduced sulphur, which is
produced from SO4
2− in a multistep pathway in which eight
electrons are added to form sulphide (S2−).
 Cys, the end product of the reductive pathway, is the
starting material for production of Met, glutathione, and
other metabolites containing reduced sulphur.
 In higher plants sulfation is a relatively minor fate for sulfur
when compared with the reductive pathway. However, in
marine algae, which produce large amounts of sulfated
extracellular polysaccharides such as agar, sulfation
accounts for a much greater proportion of the total
assimilated sulfur.

6. Scheme of Sulphate Assimilation

7. Activation of Sulphate For Assimilation
 For assimilation, sulphate is initially activated to APS, in
which sulphate is linked by an anhydride bond to a
phosphate residue by consumption of ATP and release
of pyrophosphate. This reaction is catalyzed by ATP
sulphurylase.
 Subsequently, APS is further reduced by APS reductase
or phosphorylated by APS kinase.
 APS kinase catalyzes the formation of PAPS.
 PAPS serves as a source of activated sulphate for
sulphotransferases that catalyzes sulphation of a
variety of compounds such as flavonoids,
glucosinolates, and jasmonates.

8. Reduction Of Activated Sulphate
• The sulphate residue of APS is reduced to sulphite by APS reductase
[APS sulfotransferase]
• APS reductase catalyzes a thiol-dependent two-electron reduction
of APS to sulfite. The enzyme bound S-sulfo intermediate is
presumed to be involved in the reduction of APS.
• A reduced Cys residue in APS reductase reacts with APS to form an
enzyme-(Cys)-S-SO3
− intermediate with the concomitant release of
AMP, and then this intermediate is reduced with GSH to liberate
SO3
2− and the enzyme-(Cys)-S-SG.
• The active enzyme is regenerated by reduction of the enzyme-(Cys)-
S-SG with a second molecule of GSH. In some lower plants such as
the moss Physcomitrella patens, in addition to an APS reductase-
dependent pathway, PAPS can be reduced to sulphite by PAPS
reductase as in bacteria

9. Sulphite Is Reduced To Sulphide By
Ferredoxin
• Sulphite reductase catalyzes the transfer of six electrons from ferredoxin
to sulphite to produce sulphide, S2−. Sulphite reductase is localized in
plastids of both photosynthetic and nonphotosynthetic tissues. Electrons
are supplied to ferredoxin from PSI in photosynthetic cells and from
NADPH in nonphotosynthetic cells.
• Sulphite is directly utilized as the sulphur donor for the formation of UDP-
sulphoquinovose (6-deoxy-6-sulfo-Glc) from UDP-Glc.
• UDP-sulphoquinovose is the precursor of the sulpholipid,
sulphoquinovosyl diacylglycerol, present in photosynthetic membranes
representing one of the few naturally occurring sulphonic acid (R-CH2-
SO3
−) derivatives.
• A molybdenum enzyme, sulphite oxidase, catalyzes oxidation of sulphite
to sulphate in peroxisomes. This enzyme is widely distributed in higher
plants and is likely responsible for detoxification of sulphite rather than for
chloroplast-based sulphur assimilation.

10. Sulphide Incorporation Into Cys
Requires Ser
• Incorporation of sulphide into the β-position of amino acids
is the terminal step of sulphur assimilation, leading to the
formation of Cys. Two enzymes, Ser acetyltransferase and
OAS(thiol)-lyase (Cys synthase), are committed for this step.
• These two enzymes are found in three major
compartments of plant cells, e.g. the cytosol, chloroplast,
and mitochondrion, in contrast to specific localization of
the enzymes of sulphate reduction in plastids.
• Ser acetyltransferase catalyzes the formation of OAS from
Ser and acetyl-CoA. Since this reaction is located at the
connection between nitrogen and sulfur metabolism, Ser
acetyltransferase plays a regulatory function in sulfur
assimilation.

11. Cys Is The Pivotal Compound In
Sulphur Assimilation
• Cys is the central compound for production of a
variety of metabolites containing reduced sulfur,
such as Met, GSH, phytochelatins, and
glucosinolates.
• OAS(thiol)-lyase, catalyzing the formation of Cys
from OAS and hydrogen sulphide, is in the same
three subcellular compartments as Ser
acetyltransferase.
• The catalytic activity of OAS(thiol)-lyase requires
pyridoxal phosphate as a cofactor.

12. Cys formation is controlled through a multiple
regulatory circuit
• Concentrations of OAS(thiol)-lyase are in excess of Ser acetyltransferase.
• The bound OAS(thiol)-lyase positively modulates the activity of Ser
acetyltransferase in the protein complex.
• The free form of OAS(thiol)-lyase actually catalyzes the formation of Cys
that negatively acts on Ser acetyltransferase.
• Sulphur deficiency causes the increase of OAS eventually resulting in
dissociation of the complex. The free form of Ser acetyltransferase has
only limited activity. In turn, the increased sulphur supply accumulates
sulphide to promote the formation of the complex.

13. Sulphur Is Assimilated Through
Multiple Steps: An Overview

14. Reduced sulfur, is produced from SO4
2− in a multistep pathway in which
eight electrons are added to form sulfide
The low reactivity of SO4
2− is a barrier to assimilation that is overcome by
formation of a phosphate-SO4
2−-anhydride bond in the compound APS. The
reaction is catalyzed by ATP sulfurylase
PAPS is formed through ATP-dependent phosphorylation of the 3′-hydroxyl
group of APS, catalyzed by APS kinase (Reaction 3).
The APS sulfotransferase transfers SO4
2− from APS to a thiol compound,
generating a thiosulfonate. If GSH were used, the product would be S-
sulfoglutathione (Reaction 4).
By contrast, a reductase would be expected to transfer electrons from two GSH
to APS, generating free sulfite (SO3
2−) and GSSG (Reaction 5).

15. Plant SO3
2− reductase catalyzes the reduction of SO3
2− using electrons
donated from reduced Fd (Reaction 7).
The incorporation of S2− into Cys is the last step in reductive
SO4
2−assimilation. The reaction is catalyzed by O-acetylserine(thiol)lyase
from S2− and OAS (Reaction 8). The synthesis of OAS is catalyzed by Ser
acetyltransferase (Reaction 9).
Glutaredoxin catalyzes reduction of disulfide substrates through a
thioltransferase mechanism, i.e. the thiol is transferred with the formation
of a glutathione-mixed disulfide intermediate. The analogous reaction for
APS sulfotransferase would be the one depicted in Reaction 4. SO3
2− can be
produced under the reducing conditions in the chloroplast becauseS-
sulfoglutathione is readily reduced nonenzymatically in the presence of
excess GSH (Reaction 6)

16. Sulphur assimilatory metabolism in the subcellular
compartments of plant cells.
• Black indicates name of metabolites: Ac-CoA, acetyl-CoA; APS, adenosine 5′-phosphosulfate; CN-Ala, β-cyano-
Ala; GSH, reduced glutathione; GS-X, glutathione conjugate; OAS, O-acetyl-Ser; PAPS, 3′-phosphoadenosine-5′-
phosphosulfate (3′-phosphoadenylylsulfate); SMM, S-methyl-Met. Blue indicates names of cofactors: GSH,
reduced glutathione; Fdred, reduced ferredoxin. Red indicates the name of proteins: APK, APS kinase; APR,
adenosine 5′-phosphosulfate reductase; APS, ATP sulfurylase; BCS, β-cyano-Ala synthase; OASTL, OAS(thiol)-
lyase; SAT, Ser acetyltransferase; SIO, sulfite oxidase; SIR, sulfite reductase; SULTR, sulfate transporter.

17. Positive and negative regulation of
sulphur assimilatory metabolism
• Sulphur starvation and
increased demand of sulphur
metabolites induce
assimilatory metabolism.
• OAS also acts as a positive
factor for induction.
• Plant development, circadian
rhythms, and hormones
influence sulphur metabolism
either positively or negatively.
• As negative factors, Cys and
GSH regulate specific steps of
sulphur metabolism.
• Arrow indicates the positive
effect, and bar indicates the
negative effect.

18. Hormonal Control Is Involved In
Regulation Of Assimilation:
•Methyl jasmonate induces a cluster of sulphur
assimilation genes but not sulphate transporters,
•Auxin signaling is presumably involved, at least as
an example, through activation of nitrilase leading
to changes in root morphology .
• Cytokinin signaling also appears to be involved in
gene expression related to sulphur metabolism.
Cytokinins have been shown to down-regulate the
expression of high-affinity sulphate transporter
genes in Arabidopsis roots

19. Other Factors Influencing Sulphur
Assimilation
• Sulphur assimilation is highly active in growing
tissues where high levels of Cys and Met are
required for protein synthesis.
• Specialized cells such as trichomes exhibit high
activities of enzymes for synthesis of Cys and GSH
presumably due to the demand for high
production of GSH and phytochelatins for
detoxification of heavy metals.
• Developing seeds seem to assimilate sulphur
directly from sulphate inside the seed, rather
than translocating Cys or GSH from other tissues.

20. Other Factors Influencing Sulphur
Assimilation
• Some sulphur assimilation genes, but not all, are regulated by
circadian rhythm .
• The expression of sulphate transporters, APS reductase, Ser
acetyltransferase, and 3-phosphoglycerate dehydrogenase, the first
enzyme of the plastidic Ser biosynthetic pathway, are at a peak just
before the onset of the light period.
• In leaves, ATP, electrons, and 3-phosphoglycerate are generated as
soon as the onset of light by photosynthesis, and these are ready
for use in APS formation, sulphite reduction, and Ser formation,
respectively.
• The coordinate circadian control of a cluster of genes involved in
sulphur assimilation and related pathways seems to ensure the
efficient production of Cys by the preparation of sulphate and key
enzymes just before the generation of reductants and substrates
from photosynthesis.

21. Other Factors Influencing Sulphur
Assimilation
• Abiotic stresses such as heavy metals and oxidative stress affect
sulphur assimilation. Once plants are exposed to heavy metals such
as cadmium, phytochelatins (γ-Glu-Cys)n-Gly are synthesized from
GSH, which consequently consumes Cys. In fact, heavy metal stress
promotes the expression of sulphur assimilation and transporter
genes .
• To mitigate oxidative stress, GSH functions as a direct antioxidant and
also as a reducing agent for other antioxidants such as ascorbic acid.
Since Cys availability is a limiting factor for GSH synthesis, sulfur
assimilation is also controlled by the cellular oxidative state. Most
notably, an isoform of APS reductase is activated by oxidation of two
hydrogen sulphides of Cys in the enzyme into a disulphide bond by
oxidized glutathione. This is a mechanism that enzymes of sulfur
assimilation and subsequent GSH synthesis are post-translationally
modified and thus promptly activated by after consumption of
reduced GSH by oxidative stress mitigation.

22. Regulation Of Sulphur Metabolism:
Rate-Limiting Steps in S Pathways
 Sulphate assimilation is regulated by S status .
 When the amount of S in the plant is low , many
enzymes involved in S acquistion and reduction are up-
regulated , including sulphate permerase 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 sulphurylase may be
limiting for sulphate uptake and assimilation , because
over-expression of the gene resulted in higher plant
levels of both reduced and total S .

23. Regulation Of Sulphur Metabolism:
Rate-Limiting Steps in S Pathways
 Sulphur limiting also affects the expression of seed storage
proteins, the rate of photosynthesis and protein turnover .
 Conversely , when photosynthesis is reduced , sulphate
assimilation is reduced as well .
 Accumulation of AMP and ADP were reported to inhabit
ATP sulphurylase , offering a partial explanation of the
mechanism involved .
 The S assimilation pathway is also regulated in co-
ordination 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 of N reduction ,
nitrate reductase . Similarly , reduced N compound
stimulate the key of S reduction, ATP sulphurylase .

24. GSH AND S-METHYL-Met REPRESENT MAJOR
TRANSPORTED ORGANIC SULFUR-CONTAINING
METABOLITES
• GSH represents a major thiol-containing metabolite often present in
millimolar concentrations, thus far exceeding Cys levels.
• Two enzymes, γ-glutamyl-Cys synthethase and GSH synthethase,
are responsible for synthesis of GSH from Cys, Glu, and Gly with the
consumption of two molecules of ATP. The activities of both
enzymes have been found in the cytosol and in chloroplasts.
• Feedback inhibition of γ-glutamyl-Cys synthethase activity by GSH
and the availability of Cys are the regulatory factors for GSH
synthesis.
• Transport of GSH and its derivatives is mediated by a recently
characterized proton cotransporter. However, uptake of GSH
conjugates with xenobiotics into vacuole is mediated by ATP-
energized transport via ABC proteins.
• S-Methyl-Met is also found to be a dominant transported form in
the phloem. However, S-methyl-Met is not indispensable in sulfur
transport, but the cyclic reaction of Met ↔ S-methyl-Met
presumably contributes to short-term control ofS-adenosyl-Met
levels

26. References
• REVIEW ARTICLE Molecular mechanisms of
regulation of sulfate assimilation: first steps on
a long road: Koprivova and Kopriva; Front.
Plant Sci., 29 October 2014 |
• Sulfur Assimilatory Metabolism. The Long and
Smelling Road: Kazuki Saito; Plant
Physiology Sep 2004, 136 (1) 2443-2450.
• Biochemistry And Molecular Biology Of Plants:
Buchanan, Gruissem, Jones