2.5.

4 Determination of Free Proline

Free proline was determined according to the method of Bates (1973). A Known
weight of dry plant material was homogenized in 1.5 ml of 3% Sulfosalicylic
acid and the residue was removed by centrifugation. 1 ml of the extract was
reacted with 2 ml glacial acetic acid and 2ml Acid Ninhydrin (1.25gm Ninhydrin
warmed in 30ml glacial acetic acid and 20ml 6M Phosphoric Acid until
dissolved) for 1h at 100C and the reaction was then terminated in an ice bath.
The reaction mixture was extracted with 10 ml toluene. The chromophorecontaining toluene was warmed to room temperature and its optical density was
measured at 520nm. The amount of proline was determined from a standard
curve in the range of 20 - 100 g/ml.

2.5.5 Determination of Glycinebeteine

The amount of GB was estimated according to the method of Grieve and Grattan
(1983). The plant tissue was finely ground, mechanically shaken with 20ml of
deionised water for 24h at 25C. The samples were then filtered and filtrates
were diluted to 1:1 with 2 N H2SO4. Aliquots were kept in centrifuge tubes and
cooled in ice water bath for 1h. Cold KII2 reagent was added, and the reactants
were gently stirred with a vortex mixture. The tubes were stored at 4C for 16 h
and then centrifuged at 10,000rpm for 15 min at 4C. The supernatant was
carefully aspirated with a fine glass tube. The periodide crystals were dissolved
in 9ml of 1, 2-dichloroethane. After 2h, the absorbance was measured at 365 nm
using GB as a standard and expressed in mg g1 DW.

2.5.6 Determination of Free Amino Acids

The total free amino acid content was determined by the ninhydrin test,
according to the method of ircelj et.al. (2005). A known weight of dry
powdered plant material of each sample was extracted in 80% ethanol and
centrifuged at 8,000g for 15min. The supernatant containing the alcohol-soluble
fraction was collected. A reaction mixture, consisting of 0.1ml extract, 0.9 ml of
double-distilled water and 1ml of ninhydrin reagent was shaken vigorously and
then heated in a boiling water bath for about 20 min and added to 5ml diluents
(equal volumes of water and n-propanol). After cooling the tubes to room
temperature, the color intensity was measured at 570nm. The blank set was
prepared by adding 0.1ml of 80% ethanol in place of sample extract. The
standard curve was prepared by using leucine as a standard amino acid. The
results were expressed in terms of mg of amino acid per g of dry tissue.

2.7 Antioxidants Activity Assay

Frozen leaf samples were ground in liquid nitrogen and homogenized in 50mM
sodium phosphate buffer (pH 7.0) containing 2mM EDTA and 5mM mercaptoethanol and 4% (w/v) polyvinylpyrrolidine-40 (PVP-40). The
homogenate was centrifuged at 30,000g for 30 min at 4 C. The supernatant
was used for antioxidant enzyme (SOD, CAT, POX and APX) analysis. Protein
content was determined as described by Bradford (1976).

2.7.1 Catalase Activity

Catalase activity was assayed by determining the rate of change in the
absorbance at 240 nm in a reaction mixture (3ml) that consisted of 50mM
potassium phosphate, pH 6.9, 11.6 mM H2O2 and 10 mM DTT at 25C, and the
decreased absorbance of H2O2 ( = 39.4mM1 cm1) at 240 nm was recorded
1min later. One unit of CAT activity was defined as the amount of enzyme

required for catalyzing the conversion of 1mmol H2O2 into water per min (U =
1mM of H2O2 reduction min1 mg1 of protein) according to the method of Aebi
(1984).

2.7.2 Guiacol Peroxidase Activity

Peroxidase (POX) activity was assayed in a reaction mixture that consisted of
50mM potassium phosphate, pH 6.4, 0.3mM guaiacol, 0.14mM H2O2 at 470nm
by its ability to convert guaiacol to tetraguaiacol ( = 26.6mM1 cm1) according
to the method of Chance and Maehly (1955).

2.7.3 Superoxide Dismutase Activity

SOD activity was measured according to Giannopolitis and Ries (1977). The
activity of SOD was assayed by monitoring its ability to inhibit the
photochemical reduction of nitrobluetetrazolium (NBT). Each 3mL reaction
mixture contained 50mM sodium phosphate (pH7.8), 13mM methionine, 2mM
riboflavin, 75mM NBT, 100nM EDTA, and 100mL of the enzyme extract.
Careful monitoring of the increase in absorbance at 560 nm was carried out
following the production of blue formazan. Identical tubes with the reaction
mixture were kept in the dark that served as blanks. Reaction was carried out in
test tubes at 25C under illumination from a fluorescent lamp (l5W). Riboflavin
was added at the last and the tubes were shaken and placed in the racks. The
reaction was started by switching on the light and was run for 10min, before
being stopped by switching off the light. Under the experimental conditions, the
initial rate of reaction, as measured by the difference in increase in absorbance at
560nm in the presence and absence of extract, was proportional to the amount of
enzyme. Enzyme activity (unit mL-1) was proportional to (V/v-1), where V
equals the change in absorbance per min in the absence of SOD, and v equals
the change in absorbance per min in the presence of SOD. The unit of SOD

activity was defined as the amount of enzyme that inhibits the

nitrobluetetrazolium photoreduction by 50%. SOD activity values are given in
units per mg of protein.

2.7.4 Ascorbate Peroxidase Activity

For ascorbate peroxidase (APX) activity, separate extraction was carried out
with the above-mentioned buffer containing additional 5mM ascorbate in order
to protect APX activity. APX activity was determined spectrophotometrically by
monitoring the decrease in ascorbate at A290 as described by Nakano and Asada
(1981). The reaction mixture consisted of 50mM potassium phosphate buffer
(pH7.0), 0.5mM ascorbate 0.1mM H2O2, 0.5mM EDTA and extract in a final
volume of 3ml ( = 2.8mM1 cm1).
'ai du utiliser une mthode rcemment pour le dosage des sucres totaux. Pour ma part j'ai
utilis la mthode phnol-sulfurique. Voici le rsum de mon mmoire :Les glucides en milieu
acide sulfurique et chaud sont dshydrats en drivs du furfural qui se combine facilement
avec le phnol et donnent une coloration rose-saumon (le glucose fournit de
l'hydroxyfurfural). L'absorbance est lue la longueur d'onde de 490 nm
par spectrophotomtrie. La coloration est permanente. Cette mthode est trs sensible
puisqu'elle permet de dtecter des quantits de glucides pouvant atteindre 1 g.Cette mthode
permet la dtermination de la teneur en glucides totaux (sucres simples, sucres complexes et
polyols). Une hydrolyse acide chaud (acide sulfurique 96% dans un bain 90C pendant
5min) est ralise sur la calibration au glucose et les chantillons doser. Les glucides totaux
librs sont quantifis par spectrophotomtrie UV (A=492nm) aprs raction colorimtrique
au phnol 5%.La quantification des sucres totaux se fait par destruction des liaisons
glycosidiques des sucres complexes (polysaccharides) qui se convertissent alors en sucres
simples (monosaccharides).L

ajout du phnol se fait directement aprs l

ajout de l

acide sulfurique (aprs destruction des sucres complexes).La concentration des sucres totaux
dans les chantillons doser a t calcule par rapport la calibration au glucose. Une
calibration l

amidon et la cellulose ont galement tralise pour vrifier la raction complte de la

mthode (destruction des sucres complexes)

Dosage des sucres totauxLe dosage des sucres totaux solubles est ralis par la mthode de
Dubois et al.(1956).II.3.6.1 PrincipeLes oses sont stables en milieu acide. Cependant s

ils sont chauffs en milieu acide concentr, ils donnent des furfuraldhydes par cyclisation et
dshydratation. Les furfurals etses drivs ont la proprit de se condenser avec le phnol
pour former des complexesmarron.II.3.6.2 Mode opratoireDans une srie de tubes essai, 25
l d

extrait sont additionns 0,5 ml de phnol (5%) et 1,5 ml de solution d

acide sulfurique concentre (H2SO4). Le mlange est chauff aubain marie 100 C pendant
5 mn. Aprs refroidissement dans la glace fondante, la densitoptique est mesure 490 nm
contre un blanc dans lequel 25 l d

alcool 80 % remplacel

extrait brut. Un talon est construit grce une gamme (0 35 mg/ml) de concentrationd

une solution mre de glucose 1 mg/ml (Fig. 8). Les quantits sont exprimes en mg/g de poids
frais et dtermines par la formule suivante