Heat and cold stresses phenotypes of Arabidopsis thaliana calmodulin mutants : regulation of gamma-aminobutyric acid shunt pathway under temperature stress

Plants have evolved mechanisms to cope with changes in surrounding temperatures. T-DNA insertions in seven calmodulin genes of Arabidopsis thaliana were used to investigate the role of specific calmodulin isoforms in tolerance of plants to low and high temperature for seed germination, susceptibility to low and high temperature induced oxidative damage, and changes in the levels of gammaaminobutyric acid (GABA) shunt metabolites in response to temperature stress. Exposure of wild type (WT) and cam mutant seeds at 4°C showed reduction in germination of cam5-4 and cam6-1 seeds. Exposure of cam seedlings to 42°C for 2 hr showed reduction in seed germination and survival of seedlings in cam5-4 and cam6-1 mutants compared to WT and other cam mutants. Oxidative damage by heat and cold stress measured as the level of malonaldehyde (MDA) was detected increased in root and shoot tissues of cam54 and cam6-1. Oxidative damage by heat measured as the level of MDA was detected in root and shoot of most cam mutants with highest levels in cam5-4 and cam6-1. Level of GABA shunt metabolites in seedlings were gradually increased after 1 hr and 3 hr with maximum level after 6 hr and 12 hr treatments at 4oC. GABA shunt metabolites in both root and shoot were generally elevated after 30 min and 1 hr treatment at 42°C, and increased substantially after 2 hr at 42°C comparing to the control (no treatment). GABA and glutamate levels were increased significantly more than alanine in root and shoot tissues of all cam mutants and wild type compared to the control. Alanine levels showed significant decreases in all cam mutants and in WT for 30 and 60 min of heat stress. Sensitivity of cam5-4 and cam6-1 to low temperatures suggests a role of the CAM5 and CAM6 genes in seed germination and protection against cold induced oxidative damage. Increases in the level of GABA shunt metabolites in response to cold treatment after initial reduction in some cam mutants suggests a role for calmodulin protein (cam) in the activation of glutamate decarboxylase (GAD) after exposure to cold, while increased metabolite levels may indicate involvement of other factors like reduction in cytoplasmic pH in cold regulation. Initial general elevation in GABA shunt metabolites after 30 min heat treatment in cam mutants suggests regulation of GABA level by cam. These data suggest that regulation by factors other than cam is likely, and that this factor may relate to the regulation of GAD by intracellular pH and/or metabolite partitioning under heat stress.


Introduction
Plants are exposed to either rapid or gradual changes in surrounding temperature.2][3] Changes in membrane lipid composition and accumulation of sugars had been reported under low temperature acclimation. 4,5Plants have evolved strategies to prevent and repair such damages that caused by rapid changes in the surrounding temperature.Acquired thermotolerance is one such strategy and involves production of heat shock proteins. 6,7Heat shock proteins act as molecular chaperons to protect cellular proteins against heat-induced denaturation and to facilitate refolding of heat-damaged proteins. 8The Hsp100 family proteins are essentials for the acquisition of thermotolerance in Arabidopsis [9][10][11] and Zea mays. 124][15][16] A rapid increase in GABA level in response to draught in cotton, 17 heat in cultured cowpea cells; 13 heat and induced-oxidative stress in Arabidopsis, 16,18 cold stress and mechanical damage in soybean 19 and in wheat and barely under temperature stress 20 have been reported.GABA is synthesized by a cytosollocalized Ca 2+ -calmodulin-dependent glutamate decarboxylase (GAD) protein. 21,22ollowing irreversible decarboxylation from glutamate, GABA is metabolized to succinate semialdehyde by the GABA shunt pathway which bypasses two steps in the tricarboxylic acid cycle.The last two enzymes of the GABA shunt, a GABA-transaminase using either αketoglutarate or pyruvate as amino group acceptor and succinate semialdehyde dehydrogenase, 15,23 are located in the mitochondria.Calcium/calmodulin complex (Ca 2+ / cam) provide a possible level of control for the activation of the GAD enzyme activity 15,23 since this protein is demonstrates a calmodulin binding domain. 24Such activation could account for the control of the GABA shunt pathway and the concomitant accumulation of GABA during stress.Herein we have used calmodulin T-DNA insertion mutants of A. thaliana to examine the role of specific calmodulin protein in tolerance to temperature stress with respect to seed germination, seedlings growth, oxidative damage and GABA shunt metabolite levels in various cam mutants under heat and cold treatments.

Plant material and growth conditions
The wild type and mutant lines of Arabidopsis (Arabidopsis thaliana Ecotype Columbia) bearing T-DNA insertions in various calmodulin genes (cam) used in this study are listed in Table 1.Wild type, cam1 and cam4 seeds were obtained from Arabidopsis Biological Research Stock Center, Ohio State University, Columbus, OH, USA, while seeds of all other mutants were obtained from Dr. Janet Braam, Rice University, Texas, USA.Homozygous seed stocks were propagated for this study.Seeds were surface sterilized with bleach (v/v, 6% sodium hypochlorite) for 10 min followed by five washes with sterile distilled water.Seeds were plated in Petri dishes on sterile 1X Murashige and Skoog 25

Seed germination and thermotolerance assay
Seed sensitivity to heat and cold treatments was performed according to Hong and Vierling 9 with the following modifications: for cold treatment surface sterilized seeds of wild type (WT) and cam mutants were suspended in 500 µL of 4ºC sterile distilled water and incubated at 4ºC for 24 hours.For heat treatment surface sterilized seeds of WT and each of the cam mutants were suspended in 42ºC 500 µL sterile distilled water and incubated at 42ºC in a closed water bath block for 2 hours.Each tube sample contained 50 seeds.Immediately after treatments seeds were platted on square Petri dishes containing media as described above and allowed to grow vertically under continuous light (40 µmol m -2 s -1 ) at 25ºC for 7 days.Emergence of radicle from germinating seeds was recorded and compared to control without heat treatment.Percent germination of each cam mutant was compared to WT.The average of three replicate plates was used for each treatment.

Oxidative damage and thiobarbiturate reactive substances assay
Two set of two week old seedlings of WT and cam mutants were exposed to 4ºC for 12 hours in growth chamber and another Two sets of two week old seedlings of WT and cam mutants were heat treated at 42ºC for 2 hours.For each treatment, one set was used immediately, while the second set of seedlings was placed under continuous light at 25°C for a 2 day recovery period.The level of malonaldehyce (MDA) in root and shoot tissues of seedlings exposed to heat and after recovery was determined using the thiobarbiturate reactive substances (TBARS) assay. 26hree plates with 50 seeds each were used in each replicate of each treatment.Root and shoot tissues after cold and heat treatments were separated and frozen in liquid nitrogen.Tissue (0.50 g) was ground in a 1.5 mL microfuge tube, and 0.5 mL of 0.5% (w/v) thiobarbituric acid in 20% (w/v) trichloroacetic acid and 0.5 mL 175 mm NaCl in 50 mm Tris-HCl, pH 8 was added to the ground tissues.Tubes were heated to 90ºC for 25 min.The supernatant was collected after the samples were centrifuged for 20 min at full speed.The absorbance of the supernatant was measured at 532 nm.The level of MDA was determined as nmol/mgFW from a standard curve of MDA.

Gamma-aminobutyric acid-shunt metabolites extraction and determination
For cold treatments two weeks old seedlings of WT and cam mutants grown under continuous light on 1X MS agar plates at 25°C were transferred to growth chamber at 4°C for 1 hour, 3 hours, 6 hours and 12 hours.For heat treatments WT and cam seedlings of Arabidopsis grown at 25°C for two week in continuous light were exposed to 42ºC in a growth chamber for 30 min, 1 hour, and 2 hours.Root and shoot tissues were separated and used for metabolite analysis.Three plates with 50 seeds each were used for each treatment.Meta bolites from frozen tissues were extracted according to Zhang and Bown 27 with the following modifications; separately harvested root and shoot tissues were ground in 1.5 mL microfuge tubes under LN2 until a fine pow-

Mutant
Insertion location SALK line # der was obtained.To each tube 400 µL methanol was added, and the samples were mixed for 10 min.Liquid from samples were removed by vacuum drying.Five hundred µL of 70 mm lanthanum chloride was added to each tube: the tubes were mixed for 15 min, and subsequently centrifuged at full speed for 5 min.Supernatants was removed to new tubes and mixed with 160 µL of 1 m KOH.After 10 min mixing, tubes were centrifuged at full speed for 5 min.The supernatant containing metabolites was transferred to a new tube and used to determine the quantity of specific metabolites.GABA was measured according to Zhang and Bown 27 with the following modifications.The reaction mixture contained 50 µL of sample extract, 14 µL of 4 mm nicotinamide adenine dinucleotide phosphate, 19 µL of 0.5 m potassium pyrophosphate, pH (8.6), 10 µL of (2 u/µL) GABASE enzyme (GABASE enzyme was suspended in 0.1 m potassium pyrophosphate, pH 7.2 containing 12.5% Glycerol and 5 mm β-marcaptoethanol), and 10 µL of α-ketoglutarate.The change in absorbance at 340 nm after addition of αketoglutarate was recorded after 90 min incubation at 25oC using the microplate reader (BioTek power wave, Max200R, USA).The level GABA nmol/mgFW was determined using an nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) standard curve.
Alanine was measured according to Bergmeyer 28 with the following modifications: the reaction contained 180 µL of 0.05 m Na-carbonate buffer pH (10), 7 µL of 30 mm β-NAD+, 10 µL of sample extract, and 0.3 units of alanine dehydrogenase (Sigma-Aldrich) enzyme suspension.Changes in absorbance at 340 nm after addition of alanine dehydrogenase was recorded after 60 min incubation at 25°C using a microplate reader (make and model PowerWave HT, Biotek, USA).The level of alanine as nmol/mgFW was determined using an NADH standard curve.Glutamate was measured according to Bergmeyer 28 with the following modifications: the deamination reaction contained 180 µL of 0.1 m Tris-HCl, pH 8.3, 8 µL of 7.5 mm β-NAD + , 10 µL of sample extract, and 0.8 units of glutamate dehydrogenase enzyme suspension (Sigma-Aldrich).The level of glutamate was determined at 340 nm after 60 min incubation using a microplate reader.The level of glutamate was determined as nmol/mgFW using an NADH standard curve.The level of all metabolites was represented as nmol/mg FW fold increase by calculation the metabolite for each point value of each treatment in corresponding with its control according to the following equation: Fold change = value of the metabolites / Control).Table 3. Oxidative damage for all cam mutants under cold (4ºC, 12 hours) treatments in A.thaliana.The malonaldehyde (MDA) level was determined in root and shoot tissues by the thiobarbiturate reactive substances assay describe in materials and methods.Two set of seedlings were treated at 4ºC for 12 hours.One set was used immediately for measurement of MDA level, while the other set was allowed to recover for 2 days under continuous light at 25°C before determination of MDA level (nmol/mg FW).Mutants with (*) represent the significant sensitivity (P<0.05).

Data analysis
Each data was expressed as the mean ± standard deviation (SD) of three independent experiments.The values were compared and analyzed by two-way analysis of variance (ANOVA) using least significant difference (LSD) multiple comparison tests on the means.Where differences are reported, they are at the 95% confidence level (P<0.05).

Sensitivity of cam mutants to cold and heat treatments
Seeds of the cam6-1 and cam5-4 alleles exposed to low temperature at 4°C for 24 hr showed 65% and 50% reduction in germination respectively, while seeds of all other cam mutants and WT showed germination rates between 85-93% under all treatments (Figure 1).Except for cam5-4 and cam6-1, seeds of the other Arabidopsis cam mutants exposed to 42ºC for 2 hours germinated normally (Table 2).Germination of cam5-4 and cam6-1 seeds was significantly (P<0.05)inhibited by 45% and 50% after exposure to 42ºC for 2 hours, respectively (Figure 2).The sensitivity of cam6-1 and cam5-4 alleles to low and high temperature germination may be explained by the fact that the protein products of the CAM5 and CAM6 genes may be involved in protective pathways that contribute to germination and survival of plants at low and high temperatures.
The nature of the cam5-4 mutant allele was investigated by examining the mRNA level of the entire CAM genes produced in this mutant, and it was observed that the cam5-4 exonic insertion mutant fails to produce detectable levels of CAM5 mRNA and produces either the same or reduced levels of the other CAM gene mRNAs. 29Although the level of CAM6 mRNAs in the cam6-1 mutant is reduced but not eliminated and the levels of other CAM gene mRNAs are either equal to wild type levels or reduced comparable to those in cam5-4.Since CAM5 is not expressed in the cam5-4 the results presented here are consistent with CAM5 having a direct role in cold tolerance, while CAM6 acts pleiotrophically rather than directly in thermotolerance. 29The sensitivity of the cam5-4 mutant to heat treatment may due to the absence of CaM5 protein in this mutant, but since the mutation in the cam6-1 allele did not eliminate CAM6 gene expression the heat stress sensitivity of the cam6-1 mutant (and possibly the cam5-4 allele) may be due to pleiotrophic effects of one or both of these

Oxidative by low and high temperature in cam mutants
All cam mutants and the wild type were assayed for the accumulation of thiobarbituric acid reactive substances (TBARS) by measuring the accumulation of MDA in root and shoot under cold treatment at 4ºC for 12 hours, heat treatment at 42ºC for 2 hours, and after recovery for 2 days under continuous light.In response to cold treatment MDA level accumulated in many cam mutants both immediately and after recovery in root and shoot tissues (Table 3).The cam5-4 allele showed a high level of MDA (4 to 5 fold increase, P<0.5) after recovery in both root and shoot when compared to wild type while cam6-1 accumulated 1.5-fold increase, (P<0.05) in MDA in both root and shoot tissues but not when assayed after recovery (Figure 3).In Response to heat treatment seedlings of cam5-4 and cam6-1 demonstrated dramatically higher levels of MDA in both root and shoot, and the level of MDA remained higher after 2 days of recovery (P<0.05)(Table 4, Figure 4).The production of ROS by the cam5-4 allele during cold treatment was consistent with the germination phenotype of this mutant.However, the production of TBARS reactive substances does not produce a reasonable explanation of the increased cold sensitivity of cam5-4 allele since the accumulation of MDA was not greater in tissues of cam5-4 than in other cam mutants which were not more thermosensitive.The MDA accumulation that we observed in cam mutants and wild type especially in root tissues might be modulated by disruption of a mitochondrial function and changes in membrane fluidity as a result of cold treatment.The production of ROS and the oxidative damage phenotype of cam5-4 and cam6-1 as a result of heat treatment are consistent with the germination and survival sensitivity phenotype of the alleles examined.We observed the same sensitivity phenotype of both cam5-4 and cam6-1 mutants in response to paraquat and H 2 O 2 treatments during seed germination and seedling growth, and oxidative damage. 18These results possibly delineate ROS production as a part of the pleiotrophic mechanism involved in reduced thermotolerance of cam5-4 and cam6-1.1][32] Under heat stress oxidative damage has been observed in creeping bentgrass (Agrostis stolonifera), 33 and Arabidopsis seedli -ngs. 7,34,35Heat induced oxidative damage increased in Arabidopsis seedlings pretreated with Ca +2 /CaM inhibitors, 34 and higher CaM proteins levels have been linked to lower levels of heat-induced membrane damage in maize. 36The oxidative damage (high MDA level) in the cam5-4 is directly consistent with these observations provided that the CAM5 gene product is the only CAM required for thermal protection.However, the results obtained here with the cam6-1 mutant suggest a more pleotrophic explanation of the results presented here in our study.

Level of gamma-aminobutyric acid shunt metabolites in cam mutants after cold treatment
Since GABA shunt pathway has been reported to be activated following cold stress in plant systems, 19,20 the levels of glutamate, alanine, and GABA in roots and shoots during cold treatment at 4ºC was determined (Figure 5).The levels of the three amino acids gradually increased after 1 hr of cold treatment with a maximum level reached after 6 hours and 12 hours of treatment (Table 5).After 1 hour, GABA shunt metabolite levels increased up to 0.5-1-fold compared to the control (no treatment) (Figure 5A) in root and shoot tissues of all cam mutants and the wild type (Table 5).The maximum increase occurred in root of cam5-4, cam6-1 and the WT (Figure 5B).In shoot tissue, GABA levels increased (0.7-fold increase) to a greater extent than alanine and glutamate (0.2-0.5-fold increase) with higher levels in shoot of cam5-4 mutant (Figure 5C).After a 3 hours cold treatment at 4ºC, GABA levels increased 0.65-2.5-foldwhile alanine and glutamate increased less (0.5-1.5-fold) in all mutants and the wild type and in both and shoot tissue compared with the control.GABA increased in root tissue of cam5-4 mutants.Alanine and glutamate increased in all cam mutants and the wild type greatest increases in roots of cam5-4 and cam6-1.After 6 hours incubation at 4ºC, GABA and glutamate were increased 1.5-5-fold while the alanine level only increased 1-fold in root tissues of all cam mutants and the wild type compared to the cold untreated control.Glutamate levels increased 3-5-fold in roots of cam6-1 mutant (Figure 5B).In shoot tissues, alanine and glutamate increased 0.5-fold while GABA increased up to 2-fold in all mutants with maximum increases in shoots of cam5-4 mutants as shown in Figure 5.After 12 hours of cold treatment at 4ºC, GABA and glutamate accumulated 2-7-fold while alanine accumulated to a lower level of 0.2-0.5-foldincrease in root tissues of all cam mutants and the wild type compared to the cold untreated control.GABA levels increased 2-7-fold in roots of cam5-4 mutant.Glutamate levels increased 3-7-fold in roots of cam6-1 (Figure 5B).Similarly in shoot tissues, GABA and glutamate increased 0.5-3.5-foldwhile alanine increased 0.2-0.5fold in shoots of all the mutants and the wild type compared to the control.GABA levels increased 0.5-1.5-fold in shoots of cam5-4 mutants.Glutamate levels increased 1-3.5fold in shoots of cam5-4 and cam6-1 mutants and the wild type as shown in Figure 5C.
During cold treatment almost all of the mutants showed a major changes in the levels of glutamate, alanine, and GABA in both root and shoot tissues (Figure 5).The accumulation of GABA in response to low temperature may result from an altered intracellular compartmentation of glutamate, alteration glutamate decarboxylase activity, or from the intracellular damage and lowering of protoplasmic pH that might lead to the activation of glutamate decarboxylase.CaM functions as a ubiquitous Ca +2 -binding protein that may be involved in GABA shunt pathway regulation by various possible mechanisms such as: regulation of GAD activity in response to cytosol calcium accumulation, regulation of the translocation of GABA across the mitochondrial membrane, regulation of the level and the pattern of GABA, alanine and glutamate pools, and regulating translocation of GABA, alanine and/or glutamate between roots and shoots.Mazzucotelli et al. 20 in their study for characterization of GABA shunt metabolites and GABA shunt genes during cold acclimation and freezing in wheat and barely found that GABA shunt metabolite accumulation and shunt gene expression were activated in response to low temperature, while GABA accumulation, glutamate availability, and GAD activity were correlated to the severity of the stress.Since GABA shunt may play a protective role in a pH-stat mechanism counteracting cytosol acidification due to membrane leakage as a result of cold stress, CaM signaling through activation of functional GAD (H + -consuming properties) may be involved in regulating the accumulation rate of GABA and subsequently alanine and glutamate during cold stress.Increased levels of GABA shunt metabolites in response to cold treatment after an initial reduction in some cam mutants suggests that activation of GAD does occur after exposure  to cold stress in response to the intracellular damage and low cytoplasmic pH.Since GAD is a Ca 2+ /CaM-regulated enzyme; the Ca 2+ influx that occurs following temperature drop could be responsible for the activation of the CaM/GAD complex and induction of GABA shunt pathway in response to cold stress.

Levels of gamma-aminobutyric acid shunt metabolites in response to high temperature in cam mutants
The levels of GABA shunt metabolites (Lglutamate, GABA, and L-alanine) in wild type and cam mutants of Arabidopsis CAM genes were determined after 0 min, 30 min, 1 hour, and 2 hour at 42ºC.GABA shunt metabolites in both root and shoot were generally elevated after 30 min and 1 hour of heat treatment at 42°C, and increased substantially after 2 hour at 42°C.Furthermore, GABA and glutamate levels were increased more than alanine in root and shoot tissues of wild type and all cam mutants (Table 6).After 1 hour, level of GABA (0.7-3 fold increase) and glutamate (1-4.3 fold increase) were increased in all cam mutants and wild type root and shoot tissues compared to the control except in root of cam5-4 (Figure 6).After 2 hr at 42°C, the GABA level increased in all cam mutants, and WT increased up to 5-fold in root cam5-4 compared to the control.Alanine level did not increase in WT or any of the cam mutants during heat treatment.Glutamate levels increased in all cam mutants and WT (3-11 fold increase) with highest level in root of cam5-4 and cam6-1 (6-11-fold) by comparison to the glutamate level in the untreated sample (Figure 6B).Our results showed that CaM may involve the regulation and partitioning of GABA shunt metabolites in root and shoot tissue under heat treatments.
GABA accumulation under various abiotic stresses is well documented and studied in various plants system. 16,17,19,20,37,38Previously we reported that GABA, alanine and glutamate levels were significantly increased in root and shoot of the cam5-4, and cam6-1 mutants in response to paraquat (0.5, 1 and 3 μM), while they were increased only in the root tissue of the cam5-4, and cam6-1 mutants in response to H 2 O 2 (200 and 500 l m, 1 mm). 18The data presented here are consistent with the regulation of GABA shunt pathway by calcium and calmodulin by several possible regulatory mechanisms such as: regulation of GAD activity to produce GABA in response to heat stress in the cytosol, regulation of the translocation of GABA across the mitochondrial membrane for conversion into alanine or glutamate, regulation of the level and the pattern of GABA, alanine and glutamate accumulation in root and shoot tissues, and involvement of the translocation of metabolites between the root and shoot under heat stress.The GABA shunt and Ca +2 /CaM signaling through CaM isoforms may function in multiple pathways acting together to allow plant survival at high temperature.The prevention and repair or membrane and protein damage by ROS extends the ability to tolerate oxidative damage caused by heat stress.The exact balance between these components needed for survival depends both on the plant growth stage and on the duration and severity of heat stress in Arabidopsis seedlings.
In Conclusion the data presented here suggest an important role of the GABA shunt and CaM signaling in Arabidopsis seedlings under cold stress and highlights the role of CaM and CaM-mediated signaling in adaptation and tolerance to temperature stress.
medium (pH 5.7) N o n -c o m m e r c i a l u s e o n l y supplemented with 2% (w/v) sucrose, solidified with 1.2% (w/v) agar.Seedlings were grown under continuous illumination (40 µmol m -2 s -1 ) provided by cool white fluorescent lamps at 25ºC.

Figure 1 .
Figure 1.Germination of seeds of wild type, cam5-4, and cam6-1 mutants of A. thaliana after exposure to 4°C for 24 hours.Fifty seeds each of the wild type and cam mutants were surface sterilized and exposed to 4ºC for 24 hours.Treated seeds were plated on MS salt-agar medium and allowed to germinate at 25°C under continuous light for seven days.The percent of germinating seeds were scored.Error bars represent standard deviation over three replicate plates.Except for cam5-4 and cam6-1, all cam mutants showed germination of seeds comparable to wild type.

Figure 2 .
Figure 2. Germination of A. thaliana seeds of wild type and T-DNA insertion mutant alleles cam5-4 and cam6-1 after exposure to 42°C temperature.Fifty seeds each of genotype were surface sterilized and exposed to 42ºC for 2 hours.Heat treated and control seeds were allowed to germinate described in Materials and Methods.The number of germinating seeds was scored.Error bars represent the standard deviation over three replicate plates.

Article Table 6 . 1 FW
The level of of gamma-aminobutyric acid, alanine and glutamate in root and shoot tissues of two week old seedlings of wild type and cam mutants of A. thaliana was determined after exposure to various heat treatments at 42ºC as indicated in material and method.Metabolite levels in mutants were calculated as nmol mg -fold increase in root and shoot tissues separately.Mutants with (*) represent the significant level of changes in metabolite levels (P<0.05).
i a l u s e o n l y mutations rather than a direct effect on the expression of these genes.

Figure 3 .
Figure 3. Oxidative damage in cam mutants of A. thaliana after exposure to low temperature at 4°C.Malonaldehyde (MDA) level was determined in root and shoot tissues by the thiobarbiturate reactive substances (TBARS) assay describe in materials and methods.Two sets of seedlings were treated at 4ºC for 12 hr.One set was used immediately for measurement of MDA level while the other set was allowed to recover for 2 days under continuous light at 25oC before determination of MDA level.Error bars represent the standard deviation over three replicate plates with 50 seedlings on each plate.

Figure 4 .
Figure 4. Oxidative damage by high temperature exposure in cam mutants of A. thaliana.The malonaldehyde (MDA) level was determined in root and shoot tissues by the thiobarbiturate reactive substances (TBARS) assay describe in materials and methods.Two set of seedlings were treated at 42ºC for 2 hours.One set was used immediately for measurement of MDA level, while the other set was allowed to recover for 2 days under continuous light at 25°C before determination of MDA level (nmol/mg FW).Error bars represent the standard deviation over three replicate plates with 50 seedlings on each plate.

Figure 5 .
Figure 5.The level of gamma-aminobutyric acid, alanine and glutamate in root and shoot tissues of two week old seedlings of wild type and cam mutants of A. thaliana was determined after exposure to cold as indicated.Seedlings were treated for 0 hour, 1 hours, 3 hours, 6 hours and 12 hours at 4ºC.Root and shoot tissues were harvested separately and frozen in liquid nitrogen for determination of metabolite levels as described in Materials and Methods.Metabolite levels were determined and expressed as nmol/mgFW fold increase.A) No treatment; B) Root; C) Shoot.Error bars represent standard deviation over three replicate plates, each containing 50 seedlings each of wild type and cam mutants.

Figure 6 .
Figure 6.The levels of gamma-aminobutyric acid, alanine and glutamate in twoweek-old seedlings of cam mutants of A. thaliana after exposure to heat at 42°C for 30 min, 1 hour and 2 hours.Root and shoot tissues were harvested separately and frozen in liquid nitrogen for determination of the level of metabolites as described in materials and methods.Metabolite levels in wild type and cam mutants were calculated and expressed as nmol/mgFW fold increase in root and shoot tissues.A) No treatment; B) Root; C) Shoot.Error bars represent standard deviation over three replicate plates.Each sample contained 50 seedlings.