TITLE : HORMONAL CONTROL OF LEAF SENESCENCE

INTRODUCTION

Plant senescence is the final stage of development during which the plant recycles the valuable

cellular building blocks that have been deposited in the leaves and other parts of the plant during

growth. These reusable nutrients are then stored in the plant until they can be used in new growth

or sent to the seed to provide a nutrient store for the next generation. Maintaining an efficient

senescence process is therefore essential for the fitness of the plant or its seed. Senescence is a

complex, highly regulated process that requires new gene expression and involves the interactions

of many signalling pathways. In crops inappropriately timed senescence can reduce final crop

yield, and in many vegetable crops significant postharvest loss is due to senescence. Unravelling

the regulatory mechanisms that underlie senescence may have significant impact on increasing

future food production.

Cytokinins are plant hormones that cause increased cell division by stimulating the process of

mitosis. They are made naturally by plants but have been synthesized by humans. Increased mitosis

results in plant growth and the formation of shoots and buds, as well as the development of fruits

and seeds.

OBJECTIVE

1. To study the role of cytokinins in leaf senescence.

MATERIALS

Plant, cork borer, marble, paper towel, deionized water, 5 petri dishes, N6-benzyladenine (BAP),

screw-top test tube, aluminum foil, gooseneck lamp, freezer, ethanol, spectrophotometer.

METHODS

The first week of plant hormone lab

1. A sharp cork borer is used to cut 35-40 discs from the two primary leaves. To do this, the

primary leaves was removed from the plant and was laid topside down on several layers of

paper towel. The cork borer was pressed down firmly and evenly on the desired area of the

leaf.

2. The discs was placed in the 400ml beaker containing 200ml of deionized water.

3. 5 petri dishes was obtained and labelled 1-5. 15ml of deionized water was added to dishes

1 and 2. 15 ml of N6-benzaldenine (BAP) solution was added at 1.3x10−4 M, 1.3x10−5M,

1.3x10−6M to dishes 3,4 and 5.

4. 5 leaf discs was transferred from the beaker to tissue by using forceps. 5 discs was gently

blotted dry with the tissue. The mass of 5 discs was measured and recorded.

5. After weighing, the 5 discs adaxial side up was floated on solution on each petri dishes.

6. The discs in petri dish was incubated at about 25°C. dish 1 was incubated in continuous

light while dish 2-5 was incubated in the dark.

The second week of the plant hormone lab

7. The discs was harvested after 7 days of incubation. 5 test tube was labelled with number

1-5, and 5 discs was transferred from each dish into the corresponding tube. Tube number

6 was retrieved containing the 5 discs that were stored in the freezer.

8. 10ml of 80% ethanol was added to discs in each of the 6 tubes. Each tube was capped with

a marble.

9. The tube was placed in 75-78°C water bath for 35 minutes to extract the chlorophyll from

the leaf discs.

10. After 35 minutes, the tube was removed from the bath and allowed to cool.

11. The leaf discs was removed from the tube and discarded by using a forcep. The volume of

each extract was checked with a 10ml graduated cylinder and 80% ethanol was added to

restore the volume to 10ml.

12. For each pigment extract, the absorbance at 645 nm and 663 nm was measured and

recorded.

13. For each extract, calculate combined concentrations of chlorophyll a and n being

calculated according to the formula:

(chl a + b)(µg/ml) = 20A645 + 8A663

14. For each extract, the final mass of chlorophyll a and b per mg of initial fresh mass are

calculated by using the combined mass of the 5 discs that was measured in previous week

and the fact that the alcohol extract 10-ml volumes. Thus:

(chl a + b)µg (chl a + b)(µg/ml) × (10ml)

=
𝑓𝑟𝑒𝑠ℎ 𝑚𝑎𝑠𝑠 𝑚𝑔 𝑚𝑎𝑠𝑠 𝑜𝑓 5 𝑑𝑖𝑠𝑐 (𝑚𝑔)
 15. For extracts 1-5, final amount of chlorophyll per fresh mass as a percentage of the initial

amount is calculated, i.e., by dividing (chl a + b)/fresh mass for each extract by the

dividing (chl a + b)/fresh mass of extract

CALCULATIONS

Plate 1

645 : 0.518

663 : 0.407

W : 44.7 mg

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

= 13.616

13.616 × 10
Final Chl (a+b) = 44.7

= 3.046 µg/mg

3.046 ×100
Final amount (%) =
0.664

= 4.587 × 100

= 458.73 %
 RESULTS

Dish / Treatment Mass of A645 A663 Chl (a+b) Chl (a+b) Chl (a+b)
tube combined 5 discs (µg/ml) fresh mass retained
numbef (mg) (µg/ml) Final x
100% initial
1 Water 44.7 0.518 0.407 13.616 3.046 458.73

Light

2 Water 52.7 0.032 0.115 1.56 0.296 44.6

Dark

3 1.3x10−4 BAP 47.9 0.091 0.271 3.988 0.833 125

Dark

4 1.3x10−5 BAP 42.6 0.114 0.301 4.688 1.1004 165.7

Dark

5 1.3x10−6 BAP 44.5 0.073 0.260 3.54 0.796 119.81

Dark

6 Initial 45 0.073 0.191 2.988 0.664 100

DISCUSSION

Senescence is the final stage of development of leaf in plant. Plant undergo senescence so

that it can recycle the nutrient to other parts of the plant. That’s the reason why senescence is

frequently occur at lower parts of plant leaves. Leaf senescence is not destructive process in plant

but it is very significant in plant growth. The purpose is for recycle of nutrient. Usually it only

occur at the bottom part of leaves. Usually bottom parts of leaves in covered from getting sunlight.

Therefore it cannot undergo the photosynthesis process effectively. It will be waste of nutrient to

supply it to the leaf that cannot undergo the photosynthesis process efficiently. Therefore in will

undergo programmed cell death and the nutrient can be transported to the upper parts of plant

where this part receive huge amount of sunlight can able to carry out photosynthesis process

effectively.

Senescence process starts when the chlorophyll of leaf is degenerated. The it will be

followed with degeneration of protein, nucleus, and other organelles. Therefore, in this experiment

the indication that have been used to measure the leaf senescence is the presence of chlorophyll in

each plant. To measure the concentration of chlorophyll in solution spectrophotometer has been

used in this experiment. Spectrophotometer is an instrument that have been used to measure the

concentration of solutes (in this experiment is chlorophyll) by measuring the amount of light that

have been absorbed. If the spectrophotometer reading is high, it indicates that the amount of

chlorophyll is high and the process if leaf senescence is slow and vice versa.

Leaf senescence is actually influenced by internal and external factors. The external factors

is such as light intensity whereas internal factors is such as concentration of hormone. Therefore,

in this experiment light factors and concentration of hormone are used as independent variable to

study how these factors will influence the process of leaf senescence.
 Light played as a major factor that contributing leaf senescence. With the presence of light,

plant will be able to carry out the photosynthesis process and producing their product which is

oxygen and glucose. The absence of light will initiate the senescence process in leaves. Plant will

be unable to produce oxygen and glucose for respiration process. Respiration process cannot occur

and cell inside of the plant unable to gain energy therefore senescence process will occur faster.

Internal factor that influence the leaf senescence which is hormone. It was observed that

when we applied a N6-benzyladenine (BAP) it gives a significant reading towards the

concentration of chlorophyll. In this experiment, when the leaf discs are not exposed to light it

will gives a least reading of chlorophyll which is 44.6%. The reading for the leaf discs that are

exposed to light gives the highest which is 458.73. Under dark condition with the hormone with

different concentration which are BAP 1.3 × 10-4 M, 1.3 × 10-5 M and 1.3 × 10-6 M will gives a

reading 125%, 165.7% and 119.81%. BAP is type of cytokinin hormone. The function of

cytokinin are promote cell division in plant, promote cell differentiation, maintaining cell

meristem and the most significant in this experiment is delay the leaf senescence. Supposedly,

the higher the concentration of cytokinin the senescence process should be slower. However, in

this experiment 1.3 × 10-5 M shows the highest concentration of chlorophyll compared to 1.3 ×

10-4 M. error might be occurred in this experiment.

CONCLUSION

In a conclusion, when the leaf discs are not exposed to light it will gives a least reading of

chlorophyll which is 44.6%. The reading for the leaf discs that are exposed to light gives the

highest which is 458.73%. However, when we applied BAP even thought under dark condition it

will gives a higher number of chlorophyll. This is because the function of cytokinin are promote

cell division in plant, promote cell differentiation, maintaining cell meristem and the most

significant in this experiment is delay the leaf senescence.

REFERENCES

Aloni, R., Aloni, E., Langhans, M., & Ullrich, C. I. (2006). Role of cytokinin and auxin in shaping

root architecture: regulating vascular differentiation, lateral root initiation, root apical

dominance and root gravitropism. Annals of botany, 97(5), 883-893.

Greenboim-Wainberg, Y., Maymon, I., Borochov, R., Alvarez, J., Olszewski, N., Ori, N., ... &

Weiss, D. (2005). Cross talk between gibberellin and cytokinin: the Arabidopsis GA

response inhibitor SPINDLY plays a positive role in cytokinin signaling. The Plant

Cell, 17(1), 92-102.

Leopold, A. C. (1961). Senescence in plant development. Science, 134(3492), 1727-1732.

 BIO 611
PLANT PHYSIOLOGY

LAB 5
HORMONE CONTROL OF LEAF SENESCENCE

NAME : SARAH AFIQAH BINTI YAHAYA

MATRIC NO : 2016589421
GROUP : AS2014B1
LECTURER’S NAME : DR. NOR’AISHAH BINTI ABU SHAH