Role of GABA Shunt Pathway in Plant Abiotic Stress Tolerance

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1 Role of GABA Shunt Pathway in Plant Abiotic Stress Tolerance Dr. Nisreen A. AL-Quraan Jordan University of Science and Technology JORDAN

2 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 γ-aminobutyric acid

3 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

4 GABA shunt Enzymes in Plants Glutamate Decarboxylase(GAD) GABA Transaminase (GABA-TK) or (GABA-TP) Succinic Semialdehyde Dehydrogenase (SSADH)

5 GABA Shunt in Plants Oxaloacetate NADH+H + NAD + TCA Cycle MITOCHONDRIA NADH+H + Acetyl-CoA NAD + Citrate Isocitrate NAD + CO 2 CO 2 CoA NADH+H + 2-ketoglutarate CoA NAD + Pyruvate (2) GABA GABA-TP Alanine Mitochondrial inner membrane CYTOSOL Pyruvate GABA CO 2 Isocitrate NADP + CO 2 Alanine GAD (1) 2-ketoglutarate H + NADPH + H + Glutamate AAT cyt Malate Fumarate Succinate SSADH SSADH (3) NADH+H + Mitochondrial inner membrane CO 2 NADH+H + Succinyl-CoA Succinyl- ADP+P i semialdehyde H 2 O FADH 2 FAD CoA ATP NAD + Succinyl- semialdehyde NADP + GHB GHB DH DH NADPH + H + GHB Locy et al. 2000

6 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

7 Ca +2 /CaM-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. Abiotic 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

8 Molecular Plant Volume 2 Number 1 Pages January 2009

9 Insertion sites in CAM genes AL-Quraan et al. 2008

10 Normalized expression of nine CAM genes in root and shoot tissues of wild type A. thaliana grown at 25ºC. 2.5 Root Shoot Normalized expression CAM1 CAM2 CAM3 CAM4 CAM5 CAM6 CAM7 CAM8 CAM9 AL-Quraan et al. 2010

11 Normalized expression of CAM gene in root and shoot tissues of the corresponding cam mutants of A. thaliana at 25ºC. Normalized expression Root Shoot cam1 cam2-1 cam2-2 cam3-2 cam3-3 cam4 cam5-1 cam5-2 cam5-3 cam5-4 cam6-1 cam7-1 AL-Quraan et al. 2010

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13 Seed germination: 42 ºC 120 No treatment 42ºC, 2 hr 100 Germination as %WT * * 0 WT cam5-4 cam6-1 AL-Quraan et al. 2010

14 Seedling survival: 42ºC No treatment 42ºC, 30min 42ºC, 1 hr 42ºC, 2hr Survival as %WT * * * * * * 20 0 WT cam5-4 cam6-1 AL-Quraan et al. 2008

15 MDA level: 42 ºC Heat MAD: Root 1.4 cam5-4 cam6-1 WT MDA level nmol/mgfw No treatment 42ºC, 2hr 42ºC, 2hr- recovery Heat MDA: Shoot 1.4 cam5-4 cam6-1 WT 1.2 MDA level nmol/mgfw No treatment 42ºC, 2hr 42ºC, 2hr- recovery AL-Quraan et al. 2008

16 GABA shunt metabolites: 42 ºC (Root) Heat treatments: Root GABA Alanine Glutamate Metabolites level nmol/mgfw fold increase cam5-4 cam6-1 WT cam5-4 cam6-1 WT cam5-4 cam6-1 WT cam5-4 cam6-1 WT No treatment 30 min 1 hr 2 hr AL-Quraan et al. 2008

17 GABA shunt metabolites: 42 ºC (Shoot) Heat teatments: Shoot GABA Alanine Glutamate Metabolites level nmol/mgfw fold increase cam5-4 cam6-1 WT cam5-4 cam6-1 WT cam5-4 cam6-1 WT cam5-4 cam6-1 WT No treatment 30 min 1 hr 2 hr AL-Quraan et al. 2008

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19 Expression of CAM genes in cam5-4 and cam6-1 cam5-4: Root Normalized Expression CAM1 cam5-4 : Shoot CAM2 CAM3 CAM4 CAM5 CAM6 Control (25ºC) Heat Treated (42ºC) CAM7 CAM8 CAM9 Control (25ºC) Heat Treated (42ºC) cam6-1 :Root Normalized Expression CAM1 cam6-1 : Shoot CAM2 CAM3 CAM4 CAM5 CAM6 Control (25ºC) Heat Treated (42ºC) CAM7 CAM8 CAM9 Control (25ºC) Heat Treated (42ºC) Normalized Expression Normalized Expression CAM1 CAM2 CAM3 CAM4 CAM5 CAM6 CAM7 CAM8 CAM9 CAM1 CAM2 CAM3 CAM4 CAM5 CAM6 CAM7 CAM8 CAM9 AL-Quraan et al. 2010

20 Summary.Conclusion 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.

21 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.

22 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.

23 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 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:

24 Acknowledgement Dr. Narendra K.Singh Dr. Robert D. Locy Auburn University, Alabama, USA Dr. Hanan I. Malkawi Yarmouk University, JORDAN Dr. Zuhair Amr Dr. Nizar Abu-Harfiel Jordan University of Science and Technology, JORADN