Genetic engineering of metabolic pathways in

abiotic stress resistance

Abiotic stresses:
Drought
Salinity
Chilling
Temperature Freezing
High temperature
Oxidative stress
Drought Stress
Drought will be most important abiotic stress in the next 20 years
Water will become scarce and probably wars will start
on this between countries.
 Findings of NCAR (National Centre for Atmospheric Research)
(Published in J. of Hydrometeorology, Dec. 2004)
Area hit by severe drought increased from nearly 12%
in 1970 to 30% in early 2000.

Down to Earth, Feb 15, 2005

50% of this change is due to global warming rather than decrease

in rainfall or snowfall.

Drought increased in last 30 years : much of Europe, Asia, Western

And South Africa, eastern Australia and Canada.
 Water stress

Signal perception

Signal transduction

Gene expression

Gene Products

Functional Proteins Regulatory Proteins

• Water channel proteins * Transcription factors
•Osmolyte biosynthetic enzymes * Protein Kinases
•Chaperons * Phospholipase C
•LEA * 14-3-3protein
•Proteinases
•Detoxifying enzymes

Stress Tolerance Stress response

Root to shoot signaling of drought in drying soils

Role of the two stress hormones

 Abscisic acid (ABA) Cytokinins 

 Root to shoot signaling:
a) ‘Message’ synthesized by roots
b) Role of this ‘message’ in the devt. of the root itself under stress.

With the help of the message (chemical or electrical) shoots ‘sense’

soil drying through roots.

This message ‘instructs’ shoots to conserve water for use

during later stages of growth.
by

Closing stomata Decreases transpirational area

Decreasing water loss as a by inhibiting leaf expansion
‘First line of defence’
What happens to the ABA when it reaches the stomata
after traveling through the xylems?
During the last decade, intensive studies at the molecular level have unravelled
the signal intermediates in ABA guard cells drought signaling pathway.
From the Agriculture perspective we need to develop
desiccation tolerance plants.

WHAT SHOULD BE THE

APPROACH?
TRANSGENIC APPROACHES TO ENHANCE DROUGHT
TOLERANCE BY INCORPORATING MULTIPLE GENES.
 What is the best source of
these genes?

Answer is

RESURRECTION PLANTS
 What is the Unique feature
of these plants?
• Can withstand complete dryness
(RWC 1%)

• Still viable after rehydration

• Full physiological activity resume

within several hours
 Unique features of these plants…

Can loose over 95% of their

water content survive in their
dried state for prolonged
periods and revive rapidly
when water is available.

Myrothamnus flabellifolia

Many survive even at RWC of

4% while the lethal RWC of
most crop plants and mesophytes
is 30-50%. [32% Pigeon pea,
50% Soyabean; Ref.Sinclair (1980)]
 Craterostigma plantagineum
Hydrated Dried Rehydrated

ABA induces desiccation tolerance in Craterostigma calli.

This and many other studies shows that ABA responsive genes
expressed in dehydrating plants of these species may be responsible
for their amazing desiccation tolerance.
Epicuticular wax

SUN
Pest tolerance

reflectance

cooling

transpiration
PHWL

Drought tolerance
 Epicuticular wax

• Protection from mechanical damage (Walker 1998,Eigenborde 1996)

• Primary defence against pathogens (Carver et al 1996)

• Site of interaction with insect and microbes

• Scatter and reflect light (less heat load,better mesophyll function)

• Restricts non-stomatal water loss

• Can contribute for drought tolerance and WUE (Samdur et al.,2003)

 VLCFA

Aldehydes Aldehydes

Primary alcohol Alkanes

Esters Secondary alcohol

Ketone

Acyl reduction Decarbonylation

 C 18

Fatty acid
Elongation
C 26
Cer 6
C 28

C 30

C 32

OH OH
O O
Aldehydes Cer 1
Aldehydes O

CO Alkanes
2

OH

1 o Alcohols
OH 2 o
Alcohols

Esters O
Ketones

Acyl Reduction Decarbonylation

Figure2: The flow chart representing the Epicuticular wax

Biosynthetic pathway (Kunst and Samuels,2003)
WXP1- Wax production 1

March 2005
Medicago truncatula a novel model plant for legume biological studies.

 Small diploid genome

 Self fertile

 Short life cycle

 A large number of Ests have been sequenced

 Whole genome sequencing is in progress

 Closely related to the world’s most important forage legume alfalfa

 It is the fourth most widely grown crop in U.S.

Growth and development

Transgenics were obtained under 35S promoter by Agrobacterium

Mediated Transformation.

Relatively slow growth rate delayed the flowering time

Trifoliates had more glaucous appearance

Difference in glaucous was more prominent in adaxial side

Impact of WXP1 overexpression on cuticular wax production

wing coiled crystalline structure decreases

Complemented by increase in density of tubular and plate like

wax crystals
Total wax load is significantly higher in transgenic leaves compared
to control.
PHWL

Top second and third leaf showed decreased water loss

Drought tolerance

WT T47
T47 is performing better 3days of drought stress
 WT T47

Recovery after 10days of drought stress and rewatering

 WT T47

Recovery after 3 cycles of drought- rewatering treatments in green house

 Wax Transcription factor

The SHINE Clade of AP2 Domain Transcription Factors

Activates Wax Biosynthesis, Alters Cuticle Properties, and
Confers Drought Tolerance when Overexpressed in
Arabidopsis

Asaph Aharoni,a Shital Dixit,a Reinhard Jetter,b Eveline Thoenes,a Gert van
Arkel,a and Andy Pereiraa
The plant cell, 2004
 Wax composition

Shn 6 fold increase in wax coverage

WT-equal amounts of compounds from both pathway
Shn-partial towards decarbonylation pathway
Checking the drought tolerance

15d old seedlings of Arabidopsis

Exposed to 9 to 11d dehydration

Seedlings were watered

Recovery for a week

 Salinity Stress
Salt Stress – Caused by concentrations greater than that required for
optimum growth of a typical crop plant (1500 ppm or 25 mM Na+)

Oceans are the principal sources of salt –

99.991% of water is in the oceans where typically Na+ is 460 mM
and Cl- is 540 mM.
Biogeochemical cycling – water droplets containing salt are carried by wind over land
where these evaporate and deposit salt onto the soil and increase soil salt content.
Salinity Impact on Crop Production Worldwide
World Land Surface Area 150 x 106 km2
Salt affected 9 x 106 km2 (6%)

Cultivated Land 15 x 106 km2

*Salt affected 2 x 106 km2 (13%)

Irrigated Land 2.4 x 106 km2

*Salt affected 1.2 x 106 km2 (50%)
*Problem is increasing

Negative Impacts of Salinity on Agriculture

Reduced yields on land that is presently cultivated
Limited expansion into new areas
Na+ external

Na+
I A: Suaeda maritima, Atriplex nummularia
I B: Atriplex hastata, Spartina townsendii and sugar beet
II : Cotton, Barley, Tomato, Common bean and soyabean
III : Fruit trees, Avacado, stone fruits etc.,
 How does salinity effect plant growth?
Salinity decreases plant growth, yield and market quality.
1. Short-term effect (minutes to days)
 External osmotic effects
 Reduced leaf expansion
 Lowered stomatal conductance
2. Long-term effects (weeks to months)
 Internal ionic effect-specific ion toxicity
Injurious conc.of ions:Na+, Cl-, SO4

Non-saline Saline
Cytosolic Concentration: Na (1-10 mM) Ratio shifts.
K (100-200 mM) Degree of shift determines the
degree of reduction in growth.
 Chlorosis and Necrosis.
 Eventual death.
Transgenics Expressing Different Salt Stress Genes

Osmoprotectants Transporters
 Proline Biosynthesis

Glutamic Acid.

P5C-Synthase
Glutamic gama
semi aldehyde
P5C -Synthase Stress Constitutive Clone
inducible Promoter
promoter Camv-35 into a
Pyroline- 5 -Carboxylase ABRE (35S-P5CS) vector
(AB – P5CS)

Proline
Introduce to
the plant
Free proline accumulation in finger millet plants over expressing P5CS

70
WT
Proline (m mol/g FW) 60 AB-P5CS

50 35S-P5CS

40

30

20

10

0
100 % FC 50 % FC 30 % FC Recovery

Three weeks old finger millet seedlings were subjected to water deficit
stress for 5 days. The extent of accumulation of free proline were
determined at the end of stress and also one day after re-watering.
 Effect of Salt stress on Seedling growth in finger millet
WT AB-P5CS 35S-P5CS

0 mM NaCl

200 mM NaCl

400 mM NaCl

Seeds of wild type and transgenic finger millet over expressing P5CS were
subjected to salt (NaCl) stress for 5 days. The growth of the P5CS transformed
finger millet seedlings was far better than the untransformed control.
Phenotype of 6-week old wild type and P5CS seedlings as affected by salinity (200mM NaCl)
stress. Seeds were germinated and maintained on MS medium containing 200mM NaCl. The
plates were kept in a controlled environment at 240C under constant light.
 1. Plants
CH2OH O2 2H2O COO-
CHO

CH2 CH2 CH2

H3C-N+-CH3 H3C-N+-CH3 H3C-N+-CH3

2Fd (red) 2Fd (ox) NAD+ NADH
CH3 CH3 CH3
CMO BADH
Choline Betaine Betaine
aldehyde
2. Escherichia coli
CH2OH CHO COO-

CH2 CH2 CH2

NAD+ NADH NAD+ NADH

H3 C-N+-CH 3 H3C-N+-CH3 H3C-N+-CH3
CDH BADH
CH3 CH3 CH3

Choline Betaine Betaine

aldehyde
3. Arhrobacter globiformis
CH2OH COO-

CH2 CH2

Biosynthetic pathway of Glycine betaine H3C-N+-CH3 H3C-N+-CH3

2O2 2H2O2
synthesis in some natural accumulators.
CH3 CH3
COD
Choline Betaine
 BETAINES Pathway in Microorganism-
Arthrobacter globiformis
Pathway in plants
Chloine
Chloine CODA
CMO Glycine betaine

Betaine aldehyde
Cloned into
a vector
BADH

Introduce into
Glycine betaine
plants

Transgenic plants
 Glycine betaine production in transgenic plants:
Transgene Host plant Accumulation of glycine Stress tolerance tested
betaine
Barley badh Tobacco Not tested Not tested
peroxisome
Spinach badh Tobacco 20mmol g-1 FW Not tested
chloroplast
Spinach cmo Tobacco < 0.05 mmol g-1 FW Not tested
chloroplast
E.coli betB Tobacco Not tested Not tested
Chloroplast
E.coli betA Tobcco Not tested Salt
Cytosol
betA/betB Tobacco 0.035 mmol g-1 FW Chilling, Salt
betA Rice 5.0 mmol g-1 FW Drought, Salt
A.globiformis Arabidopsis 1.2 mmol g-1 FW Salt, chilling, Freezing,
codA Chloroplast Heat
CodA Rice 5.3 mmol g-1 FW Salt, chilling
A.pascens cox Arabidopsis 19 mmol g-1 DW Freezing, Salt
cox Brassica napus 13 mmol g-1 DW Drought, Salt
cox Tobacco 13 mmol g-1 DW Salt
 In Plants

Non-specific phosphatase
Pi
mtlD
(Mannitol dehydrogenase)
NADH
Fructose-6-phosphate
Mannitol-1-phosphate Mannitol

NAD

HPr P-HPr

(pHPr-phosphorylated heat stable protein)

In E.coli

Mannitol biosynthetic pathway in transgenic tobacco

Phenotype of mtlD transgenic finger millet under water deficit stress

100%FC 50%FC 30%FC 100%FC 50%FC 30%FC

MTLD-1 MTLD-3
100%FC 50%FC 30%FC 100%FC 50%FC 30%FC

MTLD-5 WT
 Cyclicitols: (Sugar Alcohols)
IMT1
Glucose 6 Phosphate Myoinositol D-Ononitol

Trehalose: D-Pinnitol

Non-reducing disaccharides
TPS1 from yeast---transformed to tobacco.
(Trehalose 6 PO4 synthase)

Fructan:
Soluble storage polysaccharides
Levan sucrase
Sucrose Fructan
Transgenic plants engineered to synthesize osmoprotectants other than glycine betaine:
Osmoprotectant Transgenes Crop plants Accumulation Stress tolerance

Mothbean P5CS Tobacco - Salt,

Rice - Drought, Salt
Proline soyabean 4 mg g-1 FW Osmotic, Heat
Anti-proDH Arabidopsis 0.6 mg g-1 FW Salt

Mannitol E.coli mtlD Arabidopsis 10 mg g-1 FW Salt

Tobacco m mol g-1 FW Salt
Sorbitol Apple s6pdh Tobacco Oxidative stress
Persimmon 61.5 m mol g-1 FW Salt
Trehalose Yeast tps1 Tobacco 3.2 m g g-1 FW Drought
Potato Drought
D-Ononitol Ice plant imt1 Tobacco 35 m mol g-1 FW Drought, Salt

Fructans B.subtilis sacB Tobacco 0.35 mg g-1 FW Drought

Sugarbeet 5 mg g-1 FW Drought
Glutamine GS2 Rice - Salt, Chilling

Osmotin Osm1-Osm4 Tobacco - Drought, Salt

Salt stress tolerance of
transgenic plants
Transgenics Expressing Different Salt Stress Genes

Osmoprotectants Transporters
 SALT STRESS

Ca

Displacement of Ca2+ by Na+ from membranes

Ca Ca Ca
Membrane permeability 
K efflux 
+
Ca Ca2+ uptake 
Na+ influx Ca

Ca Intracellular Ca pools

disturbed
Membrane potential depolarized

H+-ATPase activity  Cell elongation 

Cell production 
H+ efflux 

Membrane potential restored

Initial and short-term effects of salt stress on the

plasmalemma of root cells
(Cramer et al., 1985)
 Ca imparts saline tolerance by increasing K/Na ratio.
-Both by restricting Na entry as well as by decreasing
K+ efflux.
Control NaCl NaCl + CaCl2
Na (mol.m-3) 3.0 74 44 (40%)
Maize
K (mol.m-3) 180 132 151 (20%)
K/Na ratio 60 1.8 3.4 (50% )
(Ref: Munns, R., 1999)

Treatment K+ efflux
% of Control
Cotton 225mM NaCl 686±20
225mM NaCl + 10mM 300±133
CaCl2

(Ref:Cramer, G.R., 1985)

 1 2 3 4 5 6

1: Control 2: 10mM CaCl2 3: 100mM NaCl

4: 100mM NaCl +10mM CaCl2 5: 200mM NaCl 6: 200mM NaCl +10mM CaCl2
Cellular Homeostasis is the most important aspect and involves osmoregulation,
compartmentation of Na, Calcium singatures and Calcium homeostasis.
Two good approaches has paid rich dividends for salt tolerance…

a) Compartmentalization of the excess Na into the vacuole

b) Restrict the entry of Na by inactivating the

gene HKT1, regulating Na entry.
 High Na+ H+

? SOS1
SOS3 Na+
H2O
Ca2+ SOS2

V-ATPase
ACA4 PPase H+
Transcriptional &
Post transcriptional
Vacuole H+ gene regulation

LCA1 Na+
Ca2+
SOS2
SOS3
Na+
? HKT1
K+
Regulation of ion (e.g., Na+ and K+) Homeostasis by the SOS pathway
(Zhu, 2000)
 Transport Mode of Membrane Tools and systems K:Na
protein transport location used for Selectivity
characterization
KAT/AKT Passive Plasma Molecular and Highly selective
Inward K+ diffusion membrane electrophysilology in for K+
channels planta and in
heterologous systems
HKT1 Na+ Not known Molecular and Transports both
High affinity K+ energized electrophysilogy in Na+ and K+
transporters heterologous systems
KUP or HAK Not known Not known Molecular and Some Na+
High affinity K+ radioisotopes in permeability
transporter heterologous systems
NSC Passive Plasma Electro physiology in High Na+
Non-Selective diffusion membrane planta permeability
cation channels
AtNHX1 H+ energized Vacuole and Molecular and Not known
Na+-H+ exchanger plasma radioisotopes in planta
membrane and in heterologous
systems
LCT1 Not known Not known Molecular and Transports both
Low-affinity radioisotopes in Na+ and K+
cation transporter heterologous systems
 Salient features of Calcium transporters…
ACA4 LCA1 CAX1 CAX2
Type of Transporter Ca2+-ATPases Ca2+-ATPases Ca2+/H+ Ca2+/H+
exchangers exchangers
Salt induced Yes Yes Not known Not known

mRNA transcripts Increases Increases Increases Transcripts

under stress under cold stress
Nil Nil
Phenotype changes Normal Normal Not known Not known
when over expressed transgenic transgenic plants
plants
Over expression Done Done Not done Not done
studies
Effects on over Salt tolerance Salt stress Not known Not known
expression increased increased
Other functions Only calcium Only calcium Also involved in Also involved in
transport transport low temperature metal transport
acclimation

Efficiency of Highly Highly efficient, Highly efficient, Low efficient,

sequestration efficient, high high affinity to High affinity to Low affinity to Ca
affinity to Ca Ca Ca
Two lines of evidence suggest that plant Ca2+-ATPases
are involved in salt stress adaptation.
Expression of LCA1 in Tomato Expression of ACA4 in Arabidopsis
 AtNHX1 over-expression
in Brassica
(Salinity tolerance)
Expression of Na/H+ antiporter
in tomato for salinity tolerance

WT Transgenic
X10E1 X10E2 X10E3 WT

SCIENCE VOL. 280 19 JUNE 1998

 Ionic Ion transporters
SOS3SOS2 Ion homeostasis
stress e.g. SOS1

Salt Homeostasis
stress
Osmotic MAPK Osmotic
Osmolytes
stress cascade? homeostasis
Cold
Drought
ABA Detoxification

Secondary
Stress proteins
stresses CBF/DREB
e.g. RD29A
e.g. oxidation
Cell division
and expansion

The three aspects of salt tolerance in plants- Homeostasis,

detoxification and growth control (Zhu, 2001).
 Species and genes
Used in transformation
 Temperature stress:
Chilling stress:
physiological and/or structural damage (including plant death) induced
by non-freezing temperatures (0 - 120 C)

Freezing stress:
physiological and/or structural damage induced by ice formation within
tissues or organs

Heat stress:

metabolic and physiological damage induced by short or long term exposure

to elevated temperature (>400 C)
 Effect of temperature on plant growth:

Optimal growth temperature is a species characteristic.

• Cool season crops complete most of their life cycle under ‘cool’
conditions, 4 - 120C (broccoli, wheat, bluegrass)

•Warm season crops require higher temperatures for optimal

growth , 22 - 300C (tomato, corn, melons)

•Perennial crops have adapted mechanisms for coping with

temperature stress.
 During slow freezing, ice forms outside the plasmalemma. This
lowers the vapor pressure, and pulls water from the cell until equilibrium
-20C is met. This phenomenon is known as freeze dehydration.
Because water leaves the cell until the osmotic pressure inside
equilibrates with outside, the organelles and membranes are exposed
to high concentrations of salts and other cellular metabolites.
Cells with higher concentrations of protective solutes such as sugars
shrink less to reach equilibrium, and lose less water. The protective
solutes probably stabilize the membrane and proteins by H-bonding and
by lowering the relative concentrations of salts and other damaging
metabolites to which cell organelles are exposed.

-100C

During rapid freezing, ice may

form intracellularly because water
cannot exit the cell fast enough to
equilibrate. Intracellular freezing (in
the absence of cryoprotectants like
DMSO and glycerol) is usually lethal
to plant cells, resulting in total

Freezing Stress
membrane disruption and collapse of
the cellular organization.
 Approach: Mechanisms of freezing resistance.

During cold acclimation- plant produce a number of cold induced proteins that are
assumed to play a role in the subsequent cold resistance.

About 50 cold induced proteins have been identified in diff. Plant species.

According to their patterns of expression…

LEA proteins COR genes

(cold responsive)

There are some examples of the Expression of Cold induced proteins in transgenic plants.

Constitutive expression of Chloroplast targeted COR protein COR15a in Arabidopsis

improved freezing tolerance.
 Tolerance to heat stress:

Heat shock proteins (HSPs)

HSP100 HSP90 HSP70 HSP60 SmHSP

Members appear to function as molecular chaperones.

Individual heat shock proteins have been transformed into plants in order to
enhance heat tolerance.

The rapid heat shock response is co-ordinated by a heat-shock transcription factor (HSF)
 Transgenes used to manipulate heat tolerance:

Gene Protein Transgenic plant

AtHSF1 Heat shock transcription factor Arabidopsis

Hsp101 HSP100 class heat-shock protein Arabidopsis

Hsp70 HSP70 class heat-shock protein Arabidopsis

Hsp17.7 SmHSP (small heat-shock protein family) Carrot

TLHS1 Class I smHSP Tobacco

 Secondary effect of abiotic stress:

Production of reactive oxygen species…

 Drought High Light Heat & Cold

Heavy metals Wounding

Ozone
Pathogens

Senescence

Reactive Oxygen
Species

Oxidative stress
Antioxidants
Antioxidants Scavenging mechanism
Enzymes
O2.-
Ascorbate-Glutathione cycle (Hlti-Well Asada cycle)
SOD

Oxidized

Reduced

Hydrogen peroxide is removed by ascorbate peroxidase and ascorbate is

regenerated by this cycle
APX: Ascorbate Peroxidase
MDHA: Mono DeHydro Ascorbate
DHAR: DeHydro Ascorbate reductase
GR: Glutathione reductase
DHA: DeHydro Ascorbate
 SOD enzyme family:
SOD catalyzes the dismutation of superoxide to H2O2 and O2.

SOD is present in most subcelluar compartments of the plant cell and is assumed
to play a central role in the defence against oxidative stress.

3 distinct types of SOD: based on metal cofactor.

Cu/Zn SOD Cytosol/chloroplast

Mn-SOD Mitochondria

Fe-SOD Chloroplast

However, they are not regulated coordinately, but independently according to the
degree of oxidative stress experienced in the respective subcelluar compartments.
 Transgenes used to engineer tolerance to oxidative stress:
Gene Host Stress tolerance
Mitochondrial Mn-SOD Alfalfa chloroplast 2 X increase in SOD
Tobacco Increased field drought tolerance
Increased freezing tolerance
Chloroplast Cu/Zn- SOD Tobacco chloroplast 3-15 X increase in SOD
Increased tolerance to high light
and chilling
Cytosolic Cu/Zn-SOD Tobacco cytosol 1.5-6 X increase in SOD
Reduced damage from acute
ozone exposure
Fe-SOD Arabidopsis Tobacco Protected plants from ozone
damage
Apx3 Tobacco Increased protection against
oxidative stress
Apx1 Arabidopsis Heat tolerance
GST/GPX Tobacco Increase stress tolerance
Nt107 (GST) Tobacco Sustained growth under cold and
salinity stress
NtPox (GPX) Arabidopsis Protects against oxidative stress
NATURE BIOTECHNOLOGY VOL 23, MARCH 2004
(Plants exposed to a temp of –60C for 2 days and returned to 220C for 5 days)

(water withheld for 2 weeks)

(Plants soaked in 600mM NaCl solution for 2 hrs and transferred to pots)
 These observations suggest that both the DREB1 cDNA and the rd29A
promoter used to improve the dehydration, salt and freezing tolerance of
agriculturally important crops by gene transfer.
 The future transgenic approaches is uncertain:

Reluctance of most consumers to accept transgenically modified food.

Single gene change may not make any difference.

In the future, pyramiding regulatory genes controlling various aspects of

tolerance (i.e., ionic, osmotic homeostasis and damage control) in a single
transgenic plant is expected to yield very high levels of tolerance to salt and
other related stresses.
THANK YOU