LAB 5 : HORMONAL CONTROL OF LEAF SENESCENCE

INTRODUCTION :

Senescence of leaves is widespread, and can be the easiest aspect of senescence to

see because of the sheer number of leaves all around us, especially in fall. The
different colors are caused by the chlorophyll in the leaves decaying and becoming
inactive.Chlorophyll is the chemical that makes leaves green and enables the plant to
conduct photosynthesis.

Leaves can die and fall off from damage, such as when they are eaten by pests or
get a disease. However, natural senescence is programmed into the plant even without
these, and it is regulated through interactions of various hormones.There are several
factors that can trigger the hormones that cause senescence in plants. Some are
environmental factors, such as drought, or the changing of seasons. Senescence can
also be triggered in only a small area of the plant. This might happen if the plant
became infected with a disease. It could kill off only that small area and prevent the
disease from spreading, thereby saving the plant as a whole.The senescence delaying
effect of cytokinin is well known, however, the details behind how this process occurs
remain unclear. Efforts to improve understanding of this phenomenon have led to the
identification in Arabidopsis of specific cytokinin signaling components through
which senescence signal responses are regulated.

At the mechanistic end of this process, it was found that increased cell-wall
invertase activity which occurs in response to cytokinin is both necessary and
sufficient for the inhibition of senescence. Yet, a direct link between the signaling and
mechanistic steps of a cytokinin regulated senescence process has yet to be
demonstrated. This may be in part because the relationship between senescence and
primary metabolism implied by the key role of cell-wall invertase is the subject of two
apparently opposing bodies of evidence. Here we briefly summarize and propose a
model in which cytokinin mediated changes in sink or source relationships leads to
delayed senescence which is consistent with existing evidence both for and against
sugars as a trigger for developmental senescence.

OBJECTIVE :

To study the role of cytokinins in leaf senescence

PROCEDURE :

First Week :

1) Cork borer was used to cut 25 discs from two primary eaves. The leaves was laid
topside down on paper towel then cork borer was pressed down firmly.

2) The cut discs was placed into 200ml of distilled water.

3) 5 petri dish was obtained and numbered 1-5. 15ml of distilled water was added to
each dishes 1 and 2. 15ml of N6-benzyladenine (BAP) solution added at 1.3x10-
4M,1.3x10-5M AND 1.3X10-6M TO dishes 3,4,5 respectively.

4) 5 discs was dried on tissues then the discs was weighted and recorded. The 5 discs
was floated adaxial side up on the solution number 1.

5) Step 4 was repeated to dishes number 2-5.

6) Another 5 discs was dropped into an empty screw top test tube labeled “6 initial”.
The tube was wrapped with aluminium foil and stored in freezer.

7) Dish 1 was incubated in blower room for light source while dishes 2-5 was
incubated in the cabinet for dark.
Second week :

1) The discs was harvested after 7 days incubation. 6 test tubes was labeled with
number 1-6 and the 5 discs from each dish was transferred into the corresponding
tube.

2) 10ml 0f 80% ethanol was added to the discs in each of the tubes. Each tubes was
capped with marble.

3) The tubes was placed in 75-78℃ water bath for 35 minutes to extract the
chlorophyll from leaf discs.

4) The tubes was removed from the bath after 35 minutes and allowed to cool.

5) The discs was discarded from the tubes. The volume was checked and 80% ethanol
was added to restore the volume to 10ml.

6) The absorbance at 645nm and 663nm was measured and recorded for each pigment
extract. The spectrometer was calibrated to A645=0.000 and A663nm=0.000 on blank
composed of 80% ethanol.

7) Concentration of chlorophyll a and b, final mass of chlorophyll and the final

amount of chlorophyll was calculated. The result was tabulated.
CALCULATIONS :

Plate 1 :

645 : 0.518

663 : 0.407

W = 44.7 mg

Ch1 (a+b) = 20 (0.518) + 8(0.407)

Final Ch1 (a+b) = 13.616 x 10/44.7

= 3.046 µg/mg

Final

Amount (% ) = 3.046 x100/0.664

= 4,587x100

= 458.73%
 RESULTS :

Dish/Tube Treatments Mass of 5 A645 A663 Ch1 (a+b) Chl (a+b) Ch1 (a+b)
number discs fresh mass retained
combined (µg/ml)
(mg)
(µg/mg) Final/Initial

x100%

1 Water and 44.7 0.518 0.407 13.616 3.046 458.73

light

2 Water and 52.7 0.032 0.115 1.56 0.296 44.6

dark

3 1.3X10^-4 47.9 0.091 0.271 3.988 0.833 125

BAP and
dark

4 1.3x10^-5 42.6 0.114 0.301 4,.688 1.1004 165.7

BAP and
dark

5 51.3x10^-6 44.5 0.073 0.260 3.54 0.796 119.81

BAP and
dark

6 Initial 45 0.073 0.191 2.988 0.664

DISCUSSION

Based on the experiment, it was shown that cytokinin plays an important role
especially in regulation of plant growth. Cytokinin function is delaying the process of
senescence of the leaves part and other organs.Also, this aid in increasing the cell
expansion in dicot cotyledons.Cytokinin one of the regulators of senescence that
cytokinin content decrease rapidly during the progression of the cytokinin processes.
This show the meaning that with the presence of high concentration of cytokinin will
cause the process of senescence slowing down. In addition, role of cytokinin found to
regulate biological processes towards the environmental stress such as rising in
temperature, drought and condition of darkness

As for the result, for dish 1 condition for water +light treatment has 458.73% that
showed the highest of the percentage of chlorophyll a+b. This is because in
chlorophyll, the process of photosynthesis is still being carried as long as there are
presence of light in the surrounding. For dish 2 that have condition water +dark
treatment, the result obtained was the lowest percentage of chlorophyll a+b which is
44.6%. The reason because there are absence of light sources thus no light was
received by the chlorophyll. Hence, no photosynthesis process is done.

The result for 3,4 and 5 BAP + DARK treatment was recorded. For this time, there
are addition of BAP and the function of N6-benzyladenine (BAP) is the first
generation of the synthetic cytokinin that aid to elicit the plant growth and responses
of development, stimulating fruit richness also the flower blossom by stimulating the
cell division. Also it act as the inhibitor of the respiratory kinase in plants that
increase the harvest life of green vegetables. The presence of cytokinin is high as they
helping in promoting cell divison by stimulating process of mitosis. Next , in dish 4
the concentration solution of BAP is 1.3x10^-5 which is the highest amount of
cytokinin so it will slow down the process of senescence.But in dish 5 with the
concentration of 1.3x10^-6 is more than dish 3 with treatments 1.3x10^-4 BAP.
Supposedly based on theories, the highest sholud be 1.3x10^-4 BAP + dark and this
indicated high amount of number cytokinin in plants with higher concentration in the
solution. But the result was not obtained that way. So there are the presence of errors
while conducting this experiment. There errors might be from the spectrometer itself
as the machine do not absorb fully the light during the experiment.

CONCLUSION :

As the conclusion, dishes that contained the synthetic cytokinin or known as BAP
have more percentage of chlorophyll compared with the dishes contained distilled
water. This is proven from the results obtained which is in 1.3x 10^-4 and dark
condition with the final amount of chlorophyll is 125%. This can be concluded there
are high amount of cytokinin hormone in leaf that help to slow down the rate of
senescence process in the leaf discs.

REFERENCES

Solomyanny, R., Mrug, G., Frasinyuk, M., Shablykin, O., & Brovarets, V. Study of
Auxin, cytokinin and gibberellin-like activity of heterocyclic compounds
derivatives of pyrimidine, pyridine, pyrazole and isoflavones.

De Leo, P., & Sacher, J. A. (1970). Senescense: association of synthesis of Acid

phosphatase with banana ripening. Plant physiology, 46(2), 208-211.

Gan, S., & Amasino, R. M. (1997). Making sense of senescence (molecular genetic
regulation and manipulation of leaf senescence). Plant physiology, 113(2), 313.