Plant growth and productivity are adversely affected by nature in the form of various abiotic stress condition such as drought, flooding, salt, low and high temperature, oxidative stress factors, their cells protect themselves from high concentrations of intracellular salts by accumulating a variety of small organic metabolites that are collectively referred to as compatible solutes/osmoprotectants Compatible solutes are soluble in water and non-toxic even at higher concentration. These metabolites allow cells to retain water and help in avoiding disturbances in their normal function when exposed to abiotic stresses. Compatible solutes are classified into three major groups; (1)Polyol(e.g.mannitol, etc), (2)Amino acids (e.g. proline) (3)Quaternary amines (e.g. glycine betaine)

1. B Y
T A N V I C H A U H A N
WELCOME

2. “ENGINEERING OF GLYCINE BETAINE
PATHWAY ITS IMPORTANT EFFORTS AND
ASSOCIATED PROBLEMS”

3. HIGHLIGHTS
 Introduction
 Osmoprotectants
 Definition of GB
 Structure & Function of GB
 Availability of the precursors of glycine betaine pathways
 Pathway for Biosynthesis of Glycine Betaine
 Mechanism and protective role of glycine betaine pathway
 GB protection of photosynthesis machinery and ROS detoxification during abiotic stress
 Stratgeies for genetic engineering in case of drought
 Genetic Engineering of Glycine Betaine Pathway In Plants
 Transgenic plants with GB
 Application of glycine betaine pathway
 Future prospects
 Case study
 Conclusion
 References

4. Introduction
Plant growth and productivity are adversely affected by nature in the form of
various abiotic stress condition such as drought ,flooding ,salt ,low and high
temperature ,oxidative stress factors ,their cells protect themselves from high
concentrations of intracellular salts by accumulating a variety of small organic
metabolites that are collectively referred to as compatible
solutes/osmoprotectants
Compatible solutes are soluble in water and non toxic even at higher
concentration.
 These metabolites allow cells to retain water and help in avoiding
disturbances in their normal function when exposed to abiotic stresses.

5. Osmoprotectants:
Osmoprotectants are small, nontoxic molecules that raise the osmotic
potential of the cytoplasm without disrupting metabolism and also stabilize
protein and membrane structures.
Osmoprotectants are small molecules that can benefit osmotically stressed
cells in two ways:
(1) By acting as nontoxic cytoplasmic osmolytes to raise osmotic pressure
(2) By protecting enzymes and membranes against damage by salt levels..
osmotic
osmoprotectant

6.  Compatible solutes are classified into three major groups;
(1)Polyol(e.g.mannitol, etc),
(2)Amino acids (e.g. proline)
(3)Quaternary amines (e.g. glycine betaine)
Many higher plants do not accumulate glycine betaine or any
their Osmoprotectants, and this has led to interest in the
metabolic engineering of the glycine betaine biosynthesis
pathway as an approach for enhancing stress resistance.
Higher plants synthesize glycine betaine in chloroplasts via the
pathway.

7. (1)Polyols
Polyols is a chemical class of osmoprotectent.
It occurs in small families of higher plants like Rice,Spinach etc.
 Accumulation of polyols in various plant species is related to
high tolerance to salt and drought stress (Bohnert and Jensen,
1996).
Polyols protect membranes and enzyme complexes from
reactive oxygen (ROS) species mainly by interacting with
enzymes.
For e.g.Mannitol, an important member of the polyols, studied in
model plants such as Arabidopsis and tobacco.

8. (2)Amino acids (e.g. proline)
Proline accumulation has been reported in various plant species during a
wide range of abiotic stresses (Hayat et al, 2012).
In plants, proline accumulation has been reported during osmotic stress
induced by salt and drought stresses (Delaney and Verma, 1993).
The primary function of proline in plants is to counteract the osmotic
effects by stabilizing protein structures and scavenging free radicals. Apart
from the above, proline also serves to store carbon and nitrogen.
Application of proline play an important role in enhancing plant stress
tolerance. This role can be in the form of either osmoprotection or
cryoprotection.
For example, in various plant species growing under saline conditions,
exogenously supplied proline provided osmoprotection and facilitated
growth.

9. (3) Quaternary amines (e.g. glycine betaine)
Betaines are quaternary ammonium compounds in which the
nitrogen atom is fully methylated.
Trimethylglycine was the first betaine discovered; Originally it was
simply called betaine because, in the 19th century, it was discovered in
sugar beets.
The most common betaines in plants include glycine betaine (GB;
the most widely studied betaine).
e.g of betaines: proline betaine, β-alanine betaine, choline-O-sulfate
and 3-dimethylsulfoniopropionate these are also termed as by specific
name glycine betaine.
 A sweet tasting crystalline alkaloid, C5H11NO2, found in sugar beets
and other plants, used to treat certain metabolic disorders, especially
an enzyme defect that causes excessive levels of homocysteine in the
blood and urine.

10. DEFINITION OF GB Its Structure
 Glycine betaine (GB) is
a small, water-soluble
organic molecule that is
essential to protect
plants, animals, and
bacteria against abiotic
stress.
 Glycine betaine is a quaternary
ammonium compound that occurs
naturally in a wide variety of plants,
animals and microorganisms.
 It is a dipolar but electrically neutral
molecule at physiological pH
Glycine betaine

11. Function of GB:
The major role of Glybet in plants exposed to saline soil is probably protecting
plant cells from salt stress by osmotic adjustment & protein stabilization.
A physiological role of betaine in alleviating osmotic stress was proposed i.e.
based on enhanced accumulation of betaine in some plants subjected to osmotic
stress.
Betaine may also stabilize the photosystem II protein-pigment complex in the
presence of high NaCl concentrations.
Glycine betaine (GB) is zwitter ionic fully N-methyl substituted derivatives of
glycine.
It Plays important role in higher plants such as maize, barley, sugarbeet and
spinach.
It protects various component of photosynthetic machinery and oxygen evolving
photosystem II and maintains highly order state of membranes at non
physiological temperature and high salt concentration.
The major role of GB in plants exposed to saline soil and reduction of oxygen
radical scavengers.

12. Availability of the precursors of glycine betaine pathways
Choline
Choline [(CH3)3N+CH2CH2OH] is a methylated nitrogen compound that is a
common constituent of eukaryotic membranes in the form of
phosphatidylcholine and therefore should be widespread in the marine
environment.
 It is a precursor of glycine betaine [(CH3)3N+CH2COOH] , one of the most
potent Osmoprotectants known.
Choline biosynthesis occurs in the cytosol and is then transported to chloroplast for
GB production in higher plants.
In transgenic plants, choline availability is one of the main factors that limit GB
accumulation.
Based on these findings, choline availability in the chloroplast is crucial to GB
biosynthesis and subsequent stress tolerance.

13. Pathway for Biosynthesis of Glycine Betaine:
site of glycine betaine accumulation:
The tolerance of plant to abiotic stress is influenced by two factors that are
concentration and localization of GB in cells.
There are many reports engineered Accumulation of GB in plants in which GB
biosynthetic enzyme have been targeted to chloroplast.
However, there are few studies shows that the enzymes have been
targeted to either cytosol or mitochondria, or to both cytosol and chloroplast
simultaneously.
GlyBet occurs in some but not all higher plants, as well as in bacteria and
other organisms
Rice plant do not accumulate GB naturally but the highest level of
accumulated GB have found in leaves of codA transgenic rice plants 53umol-
1FW while in a natural GB Accumulator maize the highest reported level of GB
in leaves of bet A transgenic maize plant is 5.753umol-1FW

14. Biosynthesis of glycine betaine pathway
It is synthesized by a two-step oxidation of choline via betaine aldehyde, but different
types of enzymes are involved.
 In E.coli, a membrane-bound, electron transfer-linked choline dehydrogenase (CDH)
oxidizes choline to betaine aldehyde. The aldehyde is then oxidized to GlyBet by a soluble,
NAD-linked betaine aldehyde dehydrogenase (BADH). Bacteria have a soluble choline
oxidase (COX) that carries out both oxidation steps and generates H2O2.
In plants, the first oxidation is mediated by a ferredoxin-dependent choline
monooxygenase (CMO) and the second by BADH. the enzyme choline monooxygenase
(CMO) first converts choline into betaine aldehyde and then a NAD dependent enzyme,
betaine aldehyde dehydrogenase (BADH) produces glycinebetaine. These enzymes are
mainly found in chloroplast stroma and their activity is increased in response to salt stress.
All these enzymes have been used to engineer tobacco and other plants that lack GlyBet,
generally by placing the transgenes under the control of the CaMV 35S promoter.

15. Recently, a novel pathway for GB synthesis from glycine was found in two
extremely halophilic microorganisms, Actinopolyspora halophilia and
Ectothiorhodospira halochloris.
In these microorganisms a three-step successive methylation of the amino residue is
catalyzed by two enzymes, glycine sarcosine methyltransferase (GSMT) and
sarcosine dimethylglycine methyltransferase (SDMT), with S-adenosylmethionine as
the methyl group donor. Both GSMT and SDMT are capable of catalyzing the three
steps of methylation because of their partially overlapping specificity for the
substrates.
Genes that encode the enzymes involved in the biosynthesis of GB have been
cloned. They include genes for choline monooxygenase (CMO) and BADH from
higher plants;
CDH and BADH from Escherichia coli;
choline oxidase (i.e. codA from Arthrobacter globiformis and
cox from Arthrobacter pascens; and
 glycine sarcosine methyltransferase (GSMT) and sarcosine dimethylglycine
methyltransferase (SDMT) from both Actinopolyspora halophilia and
Ectothiorhodospira halochloris.

17. Mechanism and protective role of glycine betaine pathway

18. Main mechanisms have been proposed for GB's responsibility for enhanced stress
tolerance:
Osmotic adjustment controlling the absorption of water from the surroundings,
Reactive oxygen species (ROS) scavenging
Stabilization by GB of the highly ordered structures of certain complex proteins to
prevent denaturation when plants or plant cells are exposed to stress conditions.
Induction by GB of the expression of specific genes that encode reactive oxygen
species (ROS) scavenging enzymes and subsequent depression of levels of ROS in
plant cells and Prevention by GB of the accumulation of excess ROS, resulting in
protection of the photosynthetic machinery NADPH from the combined effects of
light stress and other kinds of stress as well as of ionchannel proteins and the
integrity of cell membranes.
The increased production of glycine betaine (GB) improves plant tolerance to
various abiotic stresses without strong phenotypic changes, providing a feasible
approach to improve stable yield production under unfavourable conditions.

19. GB protection of photosynthesis machinery and ROS
detoxification during abiotic stress
GB has also been implicated in protection of quaternary structure
of proteins from damaging effects of environmental stresses.
Many proteins are prone to aggregation under heat and salt stress
there by, losing their native structure and activity.
 GB has been shown to protect the photosynthesis machinery by
stabilizing the activity of repair proteins under high concentrations
of NaCl.
 The role of GB in ROS detoxification is also evident by reduced
accumulation of ROS in transgenic plants under water deficit stress

20. Strategies For Genetic Engineering
e.g. In case of Drought Tolerance:

21. Genetic Engineering Of Glycine betaine pathway in
plants:
GB confers osmoproctection in bacteria, plants and animals and
protects cell components against harsh condition in vitro.
 Major cereals like wheat, maize and barley do not accumulate
significant amount of GB naturally. This could be due to the production of
transmuted transcripts for GB biosynthesizing enzyme (BADH) among
these, rice is the only cereals that does not accumulate GB naturally as
well as under stress condition.
Like rice, many crop plants such as Arabidopsis, Mustard, Tobacco and
Tomato do not accumulate GB and are therefore potential target for
engineering the GB biosynthesis.

22. Use of GB biosynthetic genes in Rice
transgenic plants
In Rice has two BADH and one CMO encoding genes,
however, no GB accumulation occurs in rice under stress.
The BADH transcripts are processed in an unusual manner in
rice resulting in removal of translational initiation codon, loss of
functional domains and premature stop codons.
 However, some correctly spliced BADH transcripts have
also been reported from rice. Exactly similar observations were
made for CMO transcripts in rice by same group.

23. The BADH transcripts are processed in an unusual manner in rice resulting in removal
of translational initiation codon, loss of functional domains and premature stop codons.
However, some correctly spliced BADH transcripts have also been reported from rice.
Exactly similar observations were made for CMO transcripts in rice by same group.
However, transgenic rice plants expressing functional BADH gene from barley could
convert exogenously applied betaine aldehyde to GB at a level better than WT plants.
Like rice many crop plants lack the ability to accumulate GB naturally during abiotic
stress
. Identification of genes of GB biosynthetic pathways has made it easy to engineer GB
biosynthesis into non accumulators by transgenic approach for improved stress tolerance.
This approach has been successfuly used in diverse plant species, e.g., Arabidopsis,
tobacco, Brassica, Persimmon, tomato, maize, rice, potato and wheat to improve their
abiotic stress tolerance.
Availability of endogenous choline, therfore, could limit the GB biosynthesis in
transgenic plants. However, levels of endogenous choline were not changed significantly
in transgenic Arabidopsis and rice plants expressing codA gene.
Therefore, availability of choline does not affect the GB synthesis in these
transgenic plants probably due to synergism in demand and supply of choline
metabolism.

24. Heat tolerance
Figure
 High temperatures also limit the growth and
productivity of plants.
 in vitro experiment indicated that GB protects
some enzymes and protein complexes from
heat induced destabilization. Therefore, it has
been postulated that GB increases resistance to
high temperature stress.
 More recent experiments showed that
transformed Arabidopsis that accumulated GB
exhibited enhanced tolerance to high
temperatures during the imbibition and
germination of seeds, as well as during the
growth of young seedlings.
 It also seems likely that GB might alleviate
the effects of heat shock because the extent of
the induction of Heat shock proteins was
significantly reduced in these transgenic
plants.

25. Salt tolerance: Figure
 It has been demonstrated, through studies
of both plant physiology and genetics, that
the level of accumulated GB is correlated
with the degree of salt tolerance.
 Transgenic Arabidopsis plants that
produced COD in their chloroplasts not
only acquired resistance to high
concentrations of NaCl during
germination but also were able to tolerate
high levels of salt during the subsequent
growth of seedlings and mature plants.
 Transformation of tobacco with a gene for
CDH also enhanced plant growth under
salt stress, although the level of GB was
much lower than that in ‘COD engineered’
Arabidopsis.

26. Transgenic plants with GB
 The GB biosynthetic genes in transgenic plants proved very effective in conferring stress tolerance compared to
that of other osmoprotectant genes. Several studies have reviewed the important roles of GB in transgenic plants
under various abiotic stresses.
 Transgenic plants such as Arabidopsis, eucalyptus, tobacco, rice, tomato, potato and wheat with GB biosynthetic
genes have showed increased GB accumulation and Metabolic engineering of plants for GB biosynthesis .
 A number of transgenic plants with GB biosynthetic genes have been tested for GB accumulation and the
resultant salt, drought and temperature tolerance .
 The GB accumulation was targeted in the chloroplast, in most of the transgenic plants, where its increased
concentration conferred protection against various abiotic stresses, particularly salt, and drought stresses.
 Overall, in transgenic plants the accumulated GB content and the resultant stress tolerance is believed to be
influenced by three factors:
 choline (precursor for GB) availability,
 type of transgene of the GB biosynthetic pathway and
 the type of promoter (constitutive and stress-inducible).
 In some GB-transgenic plants, it was reported that the accumulated GB not only conferred stress tolerance but
also improved reproductive and yield components such as flowers and fruits.

28. Transformation of tobacco with a gene for
CDH also enhanced plant growth under salt
stress, although the level of GB was much
lower than that in ‘COD-engineered’
Arabidopsis
Arabidopsis plants that
produced COD in their
chloroplasts not only
acquired resistance to
high concentrations of
NaCl during
germination but also
were able to tolerate
high levels of salt
during the subsequent
growth of seedlings
and mature plants.
In addition, Brassica juncea and Japanese
persimmon ( Diospyros kaki ) have been successfully
transformed to tolerate salt stress through the
introduction and overexpression of a gene for COD

29. APPLICATION OF GLYCINE BETAINE PATHWAY
Drought in case of glycine betaine pathway-
Drought trigger a wide variety of plant responses, Ranging from cellular
metabolism to changes in growth rate and crop yields.
The naturally occurring quartenary ammonium compound GB has received
attention as a compatible solute that may aid in drought tolerance by allowing
mentainance of turgor pressure.
Salinity and glycine betaine-
 GB give adverse effect of salt stress by changing photosynthetic activity in
many crop like Tomato, Maize Wheat and Sunflower which mainly occur
due to stomatal limitation.
Net photosynthetic rate and stomatal conductance showed a significant
positive relationship and positively correlated with substomatal carbon
dioxide.

30. Low temperature and glycine betain-
 Chilling injury that cause physical and physiological changes induced by
exposure to low temperature is another primary factor which limit crop
production worldwide.
Exogenous GB is effective in inducing cold tolerance in unhardened and
cold hardening plant of Strawberry.
Oxidative stress and Glycine betaine
Reactive oxygen species (ROS) are chemically reactive molecules
containing oxygen.
R O S inactivate enzymes and damage important cellular components
This may result in significant damage to cell structures. Cumulatively, this
is known as oxidative stress. ROS are also generated by exogenous sources
such as ionizing radiation.

31. Future prospects
Glycine betaine appears to play an important role in the responses of plant
cells to a variety of stresses, and transgenic approaches have shed some light
on the ways in which GB protects plants from stress.
In present scenario, Current research efforts are focused on the elucidation
of the mechanisms by which GB protects the cellular machinery in vivo and
how, as a result, it enhances the tolerance of whole plants to environmental
stress.
Transgenic plants accumulate GB at levels of 50–100 m M at most, with
substantial effects on stress tolerance. Further studies of transgenic plants are
necessary, particularly at the cellular level, to address this issue and resolve
the discrepancy.
Finally, the application of new technologies such as DNA arrays and
proteome analysis should help to reveal additional possible functions of GB in
vivo, for instance the maintenance of transcription and translation under stress
conditions

32. CASE
STUDIES

33. Effect of water stress on proline metabolism and leaf relative water content in
two high yeilding genotypes of groudnut( Arachis hypogaea L. ) with contrasting
drought tolrence.(Ranganayakulu, G.S., et al., 2015. Jour. Of Exp. Bio. And Agri.
Sci. Vol.3 no.1 pp)
Objective:
Comparative study was done out for two
groundnut cultivars (cv K-134 and cv JL-
24, drought tolerant and drought
sensitive, respectively) during water
stress at different soil moisture levels
[100 (control), 75, 50 and 25%]
The, activities of pyrroline-5-carboxylate
reductase (P-5-CR), proline oxidase,
proline dehydrogenase (PDH) along with
levels of quaternary ammonium
compounds, leaf relative water content
and chlorophyll stability were
investigated
Case study 1

34. free proline content
quantification
Water stress resulted in a significant accumulation of free
proline content in leaves of both groundnut cultivars
cv K-134 accumulated relatively higher amounts of proline than cv JL-24
Water stress resulted 2.5 fold higher accumulation of proline in cv K-134 and
2.0 fold in cv JL-24 on day-5 at 25% SMLs.
Procedure:

35. Furthermore, the tolerant cv K-134 shows a greater activity of pyrroline-5-carboxylate
reductase and lesser inhibition of proline oxidase and proline dehydrogenase than
susceptible cv JL-24.
The greater proline levels were due to both the higher rates of proline synthesis and
lower rate of proline oxidation in cv K-134 compared to cv JL-24
This study showed that water stress altered the proline metabolism and this alternation was
significantly varied between the cultivars.
Further, drought tolerance of cv K-134 can be justified by the higher accumulation of
quaternary ammonium compounds (glycine betaine) which helps in the maintenance of
leaf relative water content and higher chlorophyll stability during water stress as compared
to cv JL-24.
Findings

36. Case study 2
Accumulation of Glycinebetaine in Rice Plants that Overexpress Choline
Monooxygenase from Spinach and Evaluation of their Tolerance to Abiotic
Stress(Shirasawa .K., et al., 2006. Annals of Botany 98: 565–571)
Objective:
Glycinebetaine (GB),is synthesized from
choline (Cho) via betaine aldehyde (BA). The
first and second steps in the biosynthesis of
GB are catalysed by choline monooxygenase
(CMO) and by betaine aldehyde
dehydrogenase (BADH), respectively. Rice
(Oryza sativa), which has two genes for
BADH, does not accumulate GB because it
lacks a functional gene for CMO. Rice plants
accumulate GB in the presence of
exogenously applied BA, which leads to the
development of a significant tolerance to salt,
cold and heat stress. The goal in this study
was to evaluate and to discuss the effects of
endogenously accumulated GB in rice.

37. Transgenic rice plants that over expressed a gene for CMO from spinach
(Spinacia oleracea) were produced by Agrobacterium-mediated
transformation
After Southern blotting
western blotting analysis
Quantitation of glycine betaine and choline
in rice leaves was quantified by NMR
spectroscopy
The tolerance of GB-accumulating plants
to abiotic stress was investigated.

38. Findings
Transgenic plants that had a single copy of the transgene and expressed spinach CMO
accumulated GB at the level of 029–043 umol /g d. wt and had enhanced tolerance to
salt stress and temperature stress in the seedling stage.
In the CMO-expressing rice plants, the localization of spinach CMO and of
endogenous BADH might be different and/or the catalytic activity of spinach
CMO in rice plants might be lower than it is in spinach.
These possibilities might explain the low levels of GB in the transgenic rice
plants. It was concluded that CMO expressing rice plants were not effective for
accumulation of GB and improvement of productivity.
The transformants also exhibited tolerance to salt stress and temperature stress
in the seedling stage, not enough GB is accumulated in the plants to improve
their productivity. The CMO from a higher plant, spinach, has proved to be less
effective for the accumulation of GB in non-GB-accumulating rice plants than
bacterial COD and CDH.

39. Transgenic potato plants (Solanum tuberosum L. cv. Superior) with the ability to synthesize
glycinebetaine (GB) in chloroplasts (referred to as SC plants) developed via the
introduction of the bacterial choline oxidase (codA) gene under the control of an oxidative
stress-inducible SWPA2 promoter.
CASE STUDY 3
Stress-induced expression of choline oxidase in potato plant
chloroplasts confers enhanced tolerance to oxidative, salt, and
drought stresses(R. Ahmad. etal., Plant Cell Rep (2008)
27:687–698)
Increasing salinity can also significantly reduce
the total and average yields of different potato
cultivars, and the presence of 50 mM NaCl can
result in a 50% reduction in the growth of
potato plants
Potato (Solanum tuberosum L.) is one of
the major food crops in many world
regions, and ranks fourth in production
after wheat, maize, and rice.
Objective

40. Plant transformation and regeneration
Plantlets, which were capable of developing good root systems on selection
medium, were screened further for the presence of the codA gene via PCR. All
subsequent experiments were conducted on the T0-generation of transgenic
plants.
Total RNA was extracted RT-PCR
analysis
Glycinebetaine analysis*
Salt-stress treatment
Drought-stress treatment
PROCEDURE

41. Glycinebetaine analysis
These extracts were treated with the strong anion exchange resin,
AG 1-X8 (Bio-Rad, Hercules, CA, USA),
liquid nitrogen frozen leaves were powdered with a ceramic mortar and
pestle
This powder (2 g) was then suspended in 2 mL of ice-cold
methanol: chloroform: water (60:25:15) and thoroughly vortexed.
An equal volume of distilled water was added to the tubes.
The resultant homogenate was shaken gently for 10 min, and then
centrifuged for 10 minutes at 5000rpm at room temperature.
The upper methanol-water phase was transferred to clean tubes.
The extracts were freeze-dried and dissolved in distilled water (2 mL).

42. Micro Bio-spin chromatography columns (Bio-Rad, USA) were packed with AG 1-X8
resin via the addition of 1 mL of resin slurry and centrifuged at 5000rpm at room
temperature
Afterward, 1 ml of crude GB extract was loaded into
the resin bed and centrifuged for 3 minutes at 5,000rpm.
The resin was washed with 0.5 mL of distilled water and
mixed with the previous flow-through fraction.
GB was measured via high performance
liquid chromatography,
Purified GB was detected .

43. The GB peak and quantification was monitored using a UV detector .
FINDING
The transgenic lines harboring the codA gene evidenced enhanced tolerance to
a variety of stresses. As the codA gene was driven by a stress-inducible
promoter, the induction pattern of the codA gene under different stress
conditions, including Methyl viogent salt, and drought verifies the stable
integration and transcription of foreign genes in SC Plants tolerance of
transgenic plants expressing stress induced and constitutive GB-synthesizing
genes, indicating that the accumulation of biomass tends to be greater in the
case of stress-induced GB producers than in constitutively GB-producing plants
during salt stress.

44. CASE STUDY 4
Exogenous Proline and Glycine Betaine Mediated Up regulation of Antioxidant Defense
and Glyoxalase Systems Provides Better Protection against Salt-Induced Oxidative
Stress in Two Rice (Oryza sativa L.) Varieties( H.Mirza., etal., BioMed Research
Internlt. Vol. 2014, 17 pg)
Objective:
The roles of exogenous proline (Pro, 5mM) and glycine betaine (GB, 5mM) in improving
salt stress tolerance in salt sensitive (BRRI dhan49) and salt tolerant (BRRI dhan54)
rice (Oryza sativa L.) varieties.
Salt stresses (150 and 300mM NaCl for 48 h)
•endogenous Pro and increased
lipid
•peroxidation and H2O2 levels.
reduced
• significantly leaf relative water
(RWC)
• chlorophyll (chl) content
increased

45. Plant Materials and Stress Treatments.
Measurement of Relative Water
Content.
Determination of Proline Content.
Measurement of Lipid Peroxidation.
Measurement of H2O2.
Extraction and Measurement of
Ascorbate and Glutathione.
Determination of Protein.
Enzyme Extraction and Assays.
Statistical Analysis.

46. They suggests that exogenous application of Pro and GB increased rice
seedlings’ tolerance to salt-induced oxidative damage by upregulating their
antioxidant defense system where these protectants rendered better
performance to taken rice variety and Pro can be considered as better
protectant than GB.
FINDINGS

47. Conclusion
 According Engineering transgenic plants with abiotic stress
tolerance that utilized genes encoding osmoprotectants, and other
stress-related functional proteins. In comparison to other genes,
biosynthetic accumulation of glycine betaine, proline and other
osmoprotectant genes in several transgenic crop plants have
shown some improvement in abiotic stress tolerance. However,
the success of these genes has been limited in the sense that most
of the developed transgenic plants have been tested under
controlled laboratory conditions response pathway

48. References
 Bohnert HJ, Sheveleva E. Plant stress adaptations—Making metabolism move.
Curr
 Opin Plant Biol 1998;1:267–74.http://dx.doi.org/10.1016/S1369-5266(98)80115-5.
 Bray EA, Bailey-Serres J, Weretilnyk E. Responses to abiotic stresses. In:
Gruissem
 W, Buchannan B, Jones R, editors. Biochemistry and molecular biology of plants.
 American Society of Plant PhysiologistsRockville, MD: Wiley & Sons; 2000.
 p. 1158–249 [ISBN 0-943088-39-9].
 Ingram J, Bartels D. The molecular basis of dehydration tolerance in plants.
Annu Rev
 Plant Biol 1996;47:377–403.http://dx.doi.org/10.1146/annurev.arplant.47.1.377.
 Vierling E. The roles of heat-shock proteins in plants. Annu Rev Plant Biol
1991;42: 579–620.http://dx.doi.org/10.1146/annurev.pp. 42.060191.003051.

49.  Shinozaki K, Yamaguchi-Shinozaki K. Gene expression and signal
transduction in water-stress response. Plant Physiol 1997;115:327–34.
http://dx.doi.org/10.1104/pp. 115.2.327.
 Shinozaki K, Yamaguchi-Shinozaki K, Seki M. Regulatory network of gene
expression in the drought and cold stress responses. Curr Opin Plant Biol
2003;6:410–7. http://dx.doi.org/10.1016/S1369-5266(03)00092-X.
 Munns R. Genes and salt tolerance: Bringing them together. New Phytol
2005;167:
 645–63.http://dx.doi.org/10.1111/j.1469-8137.2005.01487.x.
 Flowers TJ, Flowers SA. Why does salinity pose such a difficult problem for
plant breeders?AgricWaterManag2005;78:15–
24.http://dx.doi.org/10.1016/j.agwat.2005.04.015.

50.  Bustan A, Sagi M, Malach YD, Pasternak D. Effects of saline irrigation water
and heat waves on potato production in an arid environment. Field Crop Res
2004;90: 275–85.http://dx.doi.org/10.1016/j.fcr.2004.03.007.
 Levy D, Veilleux RE. Adaptation of potato to high temperatures and salinity—
Areview.Am J Potato Res 2007;84:437–
56.http://dx.doi.org/10.1007/BF02987885.
 Chinnusamy V, Zhu J, Zhu JK. Salt stress signaling and mechanisms of plant
salt tolerance. Genet Eng 2006;27:141–77.http://dx.doi.org/10.1007/0-387-
25856-6_9.
 Boyer JS. Plant productivity and environment. Science 1982;218:443–8.
 http://dx.doi.org/10.1126/science.218.4571.443.
 Chaves MM, Oliveira MM. Mechanisms underlying plant resilience to water
deficits: Prospects for water-saving agriculture. J Exp Bot 2004;55:2365–84.

51. THANK YOU