Role of GABA Shunt Pathway in Plant Abiotic Stress Tolerance

Dr. Nisreen A. AL-QuraanJordan University of Science and Technology

JORDAN

What is GABA?

• GABA : Gamma-aminobutyric Acid • Four carbon non-protein amino

acid• It exists in non-bound form• Highly soluble in water• Has amino group on the γ-carbon

rather than on the α-carbon• GABA is a zwitterion (carry both +

and – charges)• Express in plant tissue under stress

conditions.

COO-

|

CH2

|

CH2

|

CH2-NH+3

γγ--Aminobutyric Aminobutyric acidacid

GABA Role in plants:

• GABA level in plant are low (0.03 to 2.0 µmol/g Fresh weight

• Increases in response to:- Heat shock- Cold stress- Mechanical stimulation - Drought stress- Salinity - Biotic stress

GABA shunt Enzymes in Plants

• Glutamate Decarboxylase (GAD)

• GABA Transaminase (GABA-TK) or

(GABA-TP)

• Succinic Semialdehyde Dehydrogenase (SSADH)

GABA Shunt in Plants

MITOCHONDRIAMITOCHONDRIA CYTOSOLCYTOSOL

PyruvatePyruvate

CoACoA

AcetylAcetyl--CoACoA

CitrateCitrate

OxaloacetateOxaloacetate

IsocitrateIsocitrate

ADP+PADP+P ii

CoACoA22--ketoglutarateketoglutarate 22--ketoglutarateketoglutarate

COCO22

NADNAD++

NADH+HNADH+H++

SuccinylSuccinyl--CoACoA

NADNAD++

NADH+HNADH+H++COCO22

NADNAD++

NADH+HNADH+H++

SuccinateSuccinate

FADHFADH22 FADFAD

FumarateFumarate

MalateMalate

HH22OO

PyruvatePyruvate

Succinyl-semialdehydeSuccinylSuccinyl--semialdehydesemialdehyde

GABAGABAGABAHH++COCO22

GlutamateGlutamateGlutamate

ATPATP

CoACoACOCO22

NADH+HNADH+H++

NADNAD++

IsocitrateIsocitrate

COCO22

NADPNADP++

NADPH + HNADPH + H++

GABAGABAGABA

NADH+HNADH+H++

NADNAD++

AlanineAlanineAlanine AlanineAlanineAlanine

GADGAD

GABAGABA--TPTP (1)(1)(2)(2)

(3)(3)SSADHSSADH

AATAATcytcyt

TCA TCA CycleCycle

Mito

chon

dria

l M

itoch

ondr

ial

inne

r mem

bran

ein

ner m

embr

ane

GHBGHBGHB

Succinyl-semialdehydeSuccinylSuccinyl--semialdehydesemialdehyde

NADPNADP++

NADPHNADPH+ H+ H++

Mito

chon

dria

l M

itoch

ondr

ial

inne

r mem

bran

ein

ner m

embr

ane

GHB DH

GHB DH

Locy et al. 2000Locy et al. 2000

Glutamate Decarboxylase (GAD):

• Cytosolic enzyme optimal PH ~5.5

• Calcium/Calmodulin binding protein

• In Arabidopsis two expressed isoforms:

- GAD I : 58 KD ( exclusively in roots)

- GAD II: 56 KD ( in all organs)

• Both under Calcium/ Calmodulin regulation

• 87% identical amino acid sequences

Ca+2/CaM-GADAbiotic stress:- Ca+2 Elevation-Ca+2/CaM complex-CaM/GAD Bound state- GABA shunt- CaM regulation of other proteins (CaM-binding proteins)

CaM-Ca /GAD

Ca+2/CaM- Acidic protein (4 EF hand , 12 amino

acids Asp and Glut)- 4 Ca+2 binding sites- CaM/Ca+2 Complex - Modulation of various enzyme activities.

Molecular Plant • Volume 2 • Number 1 • Pages 13–21 • January 2009Molecular Plant • Volume 2 • Number 1 • Pages 13–21 • January 2009

Insertion sites in CAM genes

AL-Quraan et al. 2008AL-Quraan et al. 2008

• Normalized expression of nine CAM genes in root and shoot tissues of wild type A. thaliana grown at 25ºC.

0

0.5

1

1.5

2

2.5

CAM1CAM2

CAM3CAM4

CAM5CAM6

CAM7CAM8

CAM9

Nor

mal

ized

exp

ress

ion

Root Shoot

AL-Quraan et al. 2010AL-Quraan et al. 2010

• Normalized expression of CAM gene in root and shoot tissues of the corresponding cam mutants of A. thaliana at 25ºC.

00.5

11.5

22.5

33.5

44.5

55.5

cam1

cam2-1

cam2-2

cam3-2

cam3-3

cam4

cam5-1

cam5-2

cam5-3

cam5-4

cam6-1

cam7-1

Nor

mal

ized

exp

ress

ion

Root Shoot

AL-Quraan et al. 2010AL-Quraan et al. 2010

AL-Quraan et al. 2010AL-Quraan et al. 2010

Seed germination: 42 ºC

0

20

40

60

80

100

120

WT cam5-4 cam6-1

Ger

min

atio

n as

%W

T

No treatment42ºC, 2 hr

* *

AL-Quraan et al. 2010AL-Quraan et al. 2010

Seedling survival: 42ºC

0

20

40

60

80

100

120

WT cam5-4 cam6-1

Surv

ival

as

%W

T

No treatment 42ºC, 30min42ºC, 1 hr42ºC, 2hr

* **

* * *

AL-Quraan et al. 2008AL-Quraan et al. 2008

MDA level: 42 ºCHeat MAD: Root

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

No treatment 42ºC, 2hr 42ºC, 2hr- recovery

MD

A le

vel n

mol

/mgF

W

cam5-4cam6-1WT

Heat MDA: Shoot

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

No treatment 42ºC, 2hr 42ºC, 2hr- recovery

MD

A le

vel n

mol

/mgF

W

cam5-4cam6-1WT

AL-Quraan et al. 2008AL-Quraan et al. 2008

GABA shunt metabolites: 42 ºC (Root)

Heat treatments: Root

0123456789

10111213

cam5-4

cam6-1 WT

cam5-4

cam6-1 W

T

cam5-4

cam6-1 W

T

cam5-4

cam6-1 W

T

Met

abol

ites

leve

l nm

ol/m

gFW

fo

ld in

crea

seGABA Alanine Glutamate

No treatment 30 min 1 hr 2 hr

AL-Quraan et al. 2008AL-Quraan et al. 2008

GABA shunt metabolites: 42 ºC (Shoot)

No treatment 30 min 1 hr 2 hr

Heat teatments: Shoot

0

1

2

3

4

5

6

cam5-4

cam6-1 WT

cam5-4

cam6-1 W

T

cam5-4

cam6-1 WT

cam5-4

cam6-1 W

T

Met

abol

ites

leve

l nm

ol/m

gFW

fo

ld in

crea

se

GABA Alanine Glutamate

AL-Quraan et al. 2008AL-Quraan et al. 2008

AL-Quraan et al. 2010AL-Quraan et al. 2010

Expression of CAM genes in cam5-4 and cam6-1

cam5-4 : Shoot

0123456789

10111213

CAM1CAM2

CAM3CAM4

CAM5CAM6

CAM7CAM8

CAM9

Nor

mal

ized

Exp

ress

ion

Control (25ºC)Heat Treated (42ºC)

cam5-4 : Root

0123456789

101112

CAM1CAM2

CAM3CAM4

CAM5CAM6

CAM7CAM8

CAM9

Nor

mal

ized

Exp

ress

ion

Control (25ºC)Heat Treated (42ºC)

cam6-1 :Root

0123456789

1011

CAM1CAM2

CAM3CAM4

CAM5CAM6

CAM7CAM8

CAM9

Nor

mal

ized

Exp

ress

ion

Control (25ºC)Heat Treated (42ºC)

cam6-1 : Shoot

0123456789

101112

CAM1CAM2

CAM3CAM4

CAM5CAM6

CAM7CAM8

CAM9

Nor

mal

ized

Exp

ress

ion

Control (25ºC)Heat Treated (42ºC)

AL-Quraan et al. 2010AL-Quraan et al. 2010

•CAM genes showed differential expression and metabolites partition in root and shoot tissues after exposure to heat.

•Variations the expression of CAM genes in different cam mutants in control and in response to heat suggest interactions between calmodulin isoforms during expression of CAM genes in Arabidopsis.

•cam5-4 and cam6-1 mutants are sensitive to high temperature stress for seed germination, survival/ growth and oxidative damage.

•Sensitivity of cam5-4 presumably resulted from absence of CaM5 protein in cam5-4.

Summary …….Conclusion

• CAM6 was expressed in cam6-1 mutant. Sensitivity of cam6-1 to stress may not be a direct consequence of this mutation.

• Differential tissue specific expression of CAM genes in root and shoot tissues

• Regulatory and compensatory interactions between calmodulin isoforms in expression of CAM genes in Arabidopsis.

• Important role for CaM5 protein in tolerance to abiotic stress in cam5-4.

• Sensitivity of cam6-1 to all environmental stress tested may be mediated indirectly through other alternative mechanism.

• Elevation in GABA shunt metabolites in response to heat stress in cam mutants suggests regulation of GABA level by CaM through GAD activation after the stress exposure.

• Later increase in metabolite level after long exposure may indicate involvement of reduction in cytoplasmic pH/or metabolite partitioning under stress.

Major References

• Locy, R.D., Wu, S-J, Bisnette, J., Barger, T.W., MacNabb, D., Zik, M., Fromm, H., Singh, N.K., Cherry, J.H. (2000). The Regulation of GABA Accumulation by Heat Stress in Arabidopsis. Edited in J.H. Cherry et al: Plant Tolerance to Abiotic Stresses in Agriculture, Role of Genetic Engeneering, 39-52, Kluwer Academics. Netherland.

• AL-Quraan, N.I. Role of Arabidopsis thaliana Calmodulin Isoforms in Tolerance to Abiotic Stress. PhD Dissertation. Auburn University. Alabama.USA. August 2008.

• AL-Quraan. N.I., Robert D. Locy, and Narendra K. Singh. Expression of calmodulin genes in wild type and calmodulin mutants of Arabidopsis thaliana under heat stress. Plant Physiology and Biochemistry. 48: 697 –702. 2010

• Dr. Narendra K.Singh• Dr. Robert D. LocyAuburn University , Alabama, USA

• Dr. Hanan I. MalkawiYarmouk University, JORDAN

• Dr. Zuhair Amr• Dr. Nizar Abu-HarfielJordan University of Science and Technology,

JORADN

Acknowledgement