Introduction CHARACTERIZATION OF TFTRES EXPRESSING PLANTS
Enzymatic trehalose determination About 200 mg of leaves was powdered within liquid nitrogen and
diluted with 900 µ
L 0.1M of phosphate buffer, pH 6.5 then was centrifuged at 10.000 x 1g for 10 minutes. Trehalose content within the
extract was digested using trehalase. A 50 µ
l of plant extract was added to 100
µ l of 0.025 M acetate buffer. A 0.1 Unit of trehalase or water as
control was added and the final volume was adjusted to 200 µ
L using aquabidest. The glucose content was detected using glucose
measurement kit. The mixture then was subjected to 525 nm wavelength on spectrophometer. Trehalose content was calculated as glucose
equivalent after subtraction of the control reaction mixture without trehalase.
Determination of trehalose synthase activity Protein was extracted as described by Avonce et al. 2004. A 200
mg of leaves was powdered within liquid nitrogen and 900 µ
l of phosphate buffer pH 6.5 then stored on freezer before used. A 300 µL protein extract
was mixed with 300 µL of 30 maltose solution in 0.1 M buffer phosphate pH 6.5 and incubated at 25
o
C for 23 h. Trehalose produced was determined Avonce et al. 2004. Trehalose within the mixture was
digested using trehalase with reaction mixture: 50 µ
L sample, 50 µ
L aquabides, 100
µ L of 25 mM acetate buffer and 0.1 Unit of trehalase.
Glucose background within plant extract was determined by the same reaction without addition of trehalase. The mixture was then incubated at
30
o
C for 5 h followed by boiling for 5 minutes to stop the reaction. The mixture was then mixed well and centrifuged for a couple of minutes
Blazqueez et al. 1994. The glucose resulted from the digestion mixture was determined using glucose determination kit. A 25 ?
µ L of digestion
mixture or 0.1 Acetate buffer: aqua bides = 3:1 as a blank reaction was added to 75
µ L deproteinase mix well followed by addition of 900
µ L of
enzyme glycosidase. The pink color shows the glucose presence. The mixture then was subjected to spectrophotometer at 525 nm wavelength.
The glucose content resulted from digestion of trehalose was the glucose content resulted from digestion mixture subtracted by glucose background
within the plant extract.
TfTreS expressing lines in response to drought
Leaf water retention and leaf recovery test In this experiment, leaves were detached from the plants and measured
their weight at zero time followed by certain interval of times after detachment, in this experiment the time course used were: 30’, 60’, 90’, 2h and 21h. Leaf Water
Loss was calculated by subtraction of the fresh weight by dry weight that was recorded after 96h incubation at 55
o
C. Seven plants each of wild type and 7 TfTreS
expressing lines were tested. Leaf recovery was tested by rehydrating excised leaves after 16h
exposure to room temperature RT. The number of leaves with fully rehydrated after 1, 2 and 24 h was recorded.
In planta TfTreS expressing plants in response to drought First experiment. TfTreS
expressing lines were tested to see their capability to face drought. A week old seedlings resistant to 50 mgl kanamycin
were transferred to ½ MS medium and kept for a week to eliminate the effect of kanamycin. The seedlings then were transferred to potting mix until they have
established growth. Drought treatment was applied by withholding water. Observation was made at day 11
th
after treatment and day 2
nd
after rehydration. Second experiment.
Fresh and dry weight of recover plants need to be measured to clarify the presence of TfTreS on plant in response to drought. For
this reason, second experiment was set up using T3 plants. Withholding water for a week was applied to 24 TfTreS expressing plants and 9 plants of Wild type. As
a control, 17 TfTreS expressing plants and 5 Wt plants were watered normally non-stressesd as the control. The number of recovered plants at the 4
th
h and 24
th
h after rehydration were recorded. Fresh weight were measured 2 weeks after rewatering to give a chance of those experienced to drought to establish
their growth.