The effect of light intensity on the amount of chlorophyll in “Cicer arietinum”

Extended Essay

Biology (SL)

“The effect of light intensity on the amount of
chlorophyll in “Cicer arietinum”

Word
count: 4 413 words

Content

Abstract
……………………………………………………………………………… 2

Introduction
………………………………………………………………………….. 3

Hypothesis
…………………………………………………………………………… 3

Method:

Description
..………………………………………………………………………….. 8

Results
……………………………………………………………………………….. 10

Discussion
……………………………………………………………………..…….. 14

Conclusion
………………………………………………………………………..….. 14

Evaluation of the
method ………………………………………………………..…… 15

Bibliography
…………………………………………………………………………. 16

Abstract.

            Plants, growing on the shaded area has less concentrated
green color and are much longer and thinner than plants growing on the sun
areas as they are dark green, short and thick. Research question was: “How does
the amount of chlorophyll-a and chlorophyll-b, gram per gram of
plant, depends on the light intensity in which plants are placed?”

            Hypothesis suggests
that there are several inner and outer factors that affect the amount of
chlorophylls a and b in plants and that with the increase of
light intensity the amount of chlorophyll will also increase until light
intensity exceeds the value when the amount of destructed chlorophylls is
greater than formatted thus decreasing the total amount of chlorophylls in a
plant.

            The seeds of Cicer
arietinum were divided into seven groups and placed into various places
with different values of light intensities. Light intensities were measured
with digital colorimeter. After three weeks length was measured. Then plants
were cut and quickly dried. Their biomass was also measured. Three plants from
each group were grinded and the ethanol extract of pigments was prepared. The
amount of chlorophylls was measured using method of titration and different
formulas.

            The investigation
showed that plants growing on the lowest light intensity equal 0 lux contained
no chlorophyll and had the longest length. The amount of chlorophyll quickly
increased and length decreased with the increase of light intensity from 0 lux
to 1200 lux. The amount of chlorophyll in plants unpredictably decreased during
light intensity equal to 142 lux and than continued increasing and didn’t start
decreasing reaching very high value (1200 lux).

            The sudden decrease
happened due to mighty existence of some inner genetical damages of seeds which
prevented them from normal chlorophyll synthesis and predicted decrease didn’t
decrease because extremely high light intensity was not exceeded.

Word count: 300 words

I.
Introduction.

            This
theme seemed to be attractive for me because I could see that results of my
investigation could find application in real life.

            While
walking in the forest in summer I saw lots of plants of different shades of green
color: some of them were dark green, some were light green and some even
very-very light green with yellow shades, hence I became very interested in
this situation and wanted to know why it happens to be so. I also saw that
those plants that were growing on sunny parts of forest, where trees were not
very high, had dark green color and those, that were growing in shady parts of
the same forest had very light green color. They also had difference in their
length and thickness – those, that were growing on light were very short, but
thick and strong, and those, growing in shady regions were very thin and
fragile.

            Hence
I became very interested in finding scientifical description of  my
observations.

            The
aim of my project is to find out how does the changes in light intensity affect
balance of chlorophyll in Cicer arietinum.

II.
Hypothesis.

There are several factors that affect the development of chlorophyll
in plants.[1]

Inner factors. The most important one is
– genetical potential of a plant, because sometimes happen mutations that
follow in inability of chlorophyll formation. But most of the times it happens
that the process of chlorophyll synthesis is broken only partly, revealing in
absence of chlorophyll only in several parts of the plant or in general low
rate of chlorophyll. Therefore plants obtain yellowish  color. Lots of genes
participate in the process of chlorophyll synthesis, therefore different
anomalies are widely represented. Development of chloroplasts depends on
nuclear and plastid DNA and also on cytoplasmatic and chloroplastic ribosomes.

Full provision of carbohydrates seem to be essential for chlorophyll
formation, and those plants that suffer from deficit of soluble carbohydrates
may not become green even if all other conditions are perfect. Such leaves,
placed into sugar solution normally start to form chlorophyll. Very often it
happens that different viruses prevent chlorophyll formation, causing yellow
color of leaves.

Outside
factors.
The most important outside factors, affecting the formation of chlorophyll are:
light intensity, temperature, pH of soil, provision of minerals, water and
oxygen. Synthesis of chlorophyll is very sensitive to all the factors,
disturbing metabolic processes in plants.

Light. Light is very
important for the chlorophyll formation, though some plants are able to produce
chlorophyll in absolute darkness. Relatively low light intensity is rather
effective for initialization and speeding of chlorophyll development. Green
plants grown in darkness have yellow color and contain protochlorophyll –
predecessor of chlorophyll а, which needs lite to
restore until chlorophyll а. Very high light
intensity causes the destruction of chlorophyll. Hence chlorophyll is
synthesized and destructed both at the same time. In the condition of very high
light intensity balance is set during lower chlorophyll concentration, than in
condition of low light intensity.

Temperature. Chlorophyll synthesis
seems to happen during rather broad temperature intervals. Lots of plants of  умеренной зоны
synthesize chlorophyll from very low temperatures till very high temperatures
in the mid of the summer. Many pine trees loose some chlorophyll during winters
and therefore loose some of their green color. It may happen because the
destruction of chlorophyll exceeds its formation during very low temperatures.

Provision
with minerals.
One of the most common reason for shortage of chlorophyll is absence of some
important chemical elements. Shortage of nitrogen is the most common reason for
lack of chlorophyll in old leaves. Another one is shortage of ferrum, mostly in
young leaves and plants. And ferrum is important element for chlorophyll
synthesis. And magnesium is a component of chlorophyll therefore its shortage
causes lack of production of chlorophyll.

Water. Relatively low water stress
lowers speed of chlorophyll synthesis and high dehydration of plants tissues
not only disturbs synthesis of chlorophyll, but even causes destruction of
already existing molecules.

       Oxygen.  With the absence of oxygen plants do not produce chlorophyll even on
high light intensity.  This shows that aerobic respiration is essential for
chlorophyll synthesis.

            Chlorophyll.[2]
The synthesis of
chlorophyll is induced by light. With light, a gene can be transcripted and
translated in a protein.

The plants are
naturally blocked in the conversion of protochlorophyllide to chlorophyllide.
In normal plants these results in accumulation of a small amount of
protochlorophyllide which is attached to holochrome protein. In vivo at least
two types of protochlorophyllide holochrome are present. One, absorbing
maximally at approximately 650 nm, is immediately convertible to chlorophyllide
on exposure to light. If ALA is given to plant tissue in the dark, it feeds
through all the way to protochlorophyllide, but no
further. This is because POR, the enzyme that converts protochlorophyllide
to chlorophyllide, needs light to carry out
its reaction. POR is a very actively researched enzyme worldwide and a lot is
known about the chemistry and molecular biology of its operation and
regulation. Much less is known about how POR works in natural leaf development.

            
ALA                                         Portoporphyrine

                                                          
Protochlorophyllide

                                                               
 Chlophyllide      

               
Chlorophyll b                            Chlorophyll
a

Chlorophyll[3] is a green compound
found in leaves and green stems of plants. Initially, it was assumed that
chlorophyll was a single compound but in 1864 Stokes showed by spectroscopy
that chlorophyll was a mixture. If dried leaves are powdered and digested with
ethanol, after concentration of the solvent, 'crystalline' chlorophyll is
obtained, but if ether or aqueous acetone is used instead of ethanol, the
product is 'amorphous' chlorophyll.

In 1912,
Willstatter et al. (1) showed that chlorophyll was a mixture of two
compounds, chlorophyll-a and chlorophyll-b:

Chlorophyll-a (C₅₅H₇₂MgN₄O₅,
mol. wt.: 893.49). The methyl group marked with an asterisk is replaced by an
aldehyde in chlorophyll-b (C₅₅H₇₀MgN₄O₆,
mol. wt.: 906.51).

The two
components were separated by shaking a light petroleum solution of chlorophyll
with aqueous methanol: chlorophyll-a remains in the light petroleum but
chlorophyll-b is transferred into the aqueous methanol. Cholorophyll-a
is a bluish-black solid and cholorophyll-b is a dark green solid, both
giving a green solution in organic solutions. In natural chlorophyll there is a
ratio of 3 to 1 (of a to b) of the two components.

The intense
green colour of chlorophyll is due to its strong absorbencies in the red and
blue regions of the spectrum, shown in fig. 1. (2) Because of these absorbencies
the light it reflects and transmits appears green.

Fig. 1 - The uv/visible adsorption spectrum for chlorophyll.

Due to the green
colour of chlorophyll, it has many uses as dyes and pigments. It is used in
colouring soaps, oils, waxes and confectionary.

Chlorophyll's
most important use, however, is in nature, in photosynthesis. It is capable of
channelling the energy of sunlight into chemical energy through the process of
photosynthesis. In this process the energy absorbed by chlorophyll transforms
carbon dioxide and water into carbohydrates and oxygen:

CO₂ + H₂O (CH₂O) + O₂

Note: CH₂O is the
empirical formula of carbohydrates.

The chemical
energy stored by photosynthesis in carbohydrates drives biochemical reactions
in nearly all living organisms.

In the
photosynthetic reaction electrons are transferred from water to carbon dioxide,
that is carbon dioxide is reduced by water. Chlorophyll assists this transfer
as when chlorophyll absorbs light energy, an electron in chlorophyll is excited
from a lower energy state to a higher energy state. In this higher energy
state, this electron is more readily transferred to another molecule. This
starts a chain of electron-transfer steps, which ends with an electron being
transferred to carbon dioxide. Meanwhile, the chlorophyll which gave up an
electron can accept an electron from another molecule. This is the end of a
process which starts with the removal of an electron from water. Thus,
chlorophyll is at the centre of the photosynthetic oxidation-reduction reaction
between carbon dioxide and water.

Treatment of
cholorophyll-a with acid removes the magnesium ion replacing it with two
hydrogen atoms giving an olive-brown solid, phaeophytin-a. Hydrolysis of
this (reverse of esterification) splits off phytol and gives phaeophorbide-a.
Similar compounds are obtained if chlorophyll-b is used.

Chlorophyll can
also be reacted with a base which yields a series of phyllins, magnesium
porphyrin compounds. Treatment of phyllins with acid gives porphyrins.

Now knowing all these factors affecting the synthesis and
destruction of chlorophyll I propose that the amount of chlorophyll in plant
depends on light intensity in the following way: with the increase of light
intensity the amount of chlorophyll increases, but then it starts decreasing
because light intensity exceed the point when there is more chlorophyll
destructed than formed.

Variables.

Independent:

• Light
  intensity, lux

Constant:

• pH of soil


• water supply,
  ml


• temperature, t^o
  C

Dependent:

• length, cm


• amount of
  chlorophyll in gram of a plant, gram per gram

III.
Method.

Apparatus:

·    
seeds of Cicer arietinum

·    
28 plastic pots

·    
water

·    
scissors

·    
ruler (20 cm ± 0.05 cm)

·    
CaCO₃

·    
soil (adopted for home plants)

·    
digital luxmeter (± 0.05 lux)

·    
test tubes

·    
H₂SO₄ (0.01 M solution)

·    
Pipette (5 cm³ ± 0.05 cm³)

·    
mortar and pestle

·    
burette

·    
ethanol (C₂H₅OH),
98%

·    
beakers

Firstly I went to the shop and bought
germinated seeds of Cicer arietinum. Then sorted seeds and chose the
strongest ones. After that I prepared soil for them and put them in it.

As the aim of this project is to
investigate the dependence of mass of chlorophyll in plants during different
light intensities it was needed to create those various conditions. Pots with
seeds were placed into the following places: in the wardrobe with doors (light
intensity is o lux), under the sink (light intensity is 20,5 lux), in the shell
of bookcase (light intensity is 27,5 lux), above the bookcase (light intensity
is 89,5 lux), above the extractor (light intensity is 142 lux), beyond the
curtains (light intensity is 680 lux) and on the open sun (light intensity is
1220 lux). Light intensity was measured with the help of digital luxmeter. It was measured four times each day: morning, midday, afternoon, evening. During those four periods four measurements were done and the
maximum value was taken into consideration and written down. Those measurements
lasted for three weeks of the experiment as the whole time of the experiment
was three weeks. The luxmeter’s sensitive part was placed on the plants (so it
was just lying on them) in order to measure light intensity flowing directly on
plant bodies, then two minutes were left in order to get stabilized value of
light intensity and the same procedure was repeated. All those actions were
done in order to get more accurate results of light intensity. 

Growing plants were provided with the same
amount of water (15 ml, once a day in the morning) and they were situated in
the same room temperature (20^o C), pH of soil was definitely the
same because all the plants were put in the same soil (special soil for room
flowers).  

After three weeks past the length of
plants was measured with the help of ruler. Firstly the plants were not cut, so
their length had to be measured while they were in the pots. The ruler was
placed into the pot and plants were carefully stretched on it. The action was repeated
three times and only maximum value was taken into consideration. After that
plants were cut. Then those already cut plants were put into the dark place and
quickly dried.

Titration.

I have chosen three plants from each light
intensity group and measured their weight. . In order to obtain the pigments,
three plants were cut into small pieces and placed in a mortar. Calcium
carbonate was then added, together with a little ethanol (2 cm³).
The leaf was grinded using a pestle until no large pieces of leaf tissue were
left, and the remaining ethanol was poured into the mortar (3 cm³).
Then 1 ml of obtained solution was placed into the test tube and this 1 ml of
solution was then titrated against 0.01 M solution of sulfuric acid, through
the use of a pipette. The titration was complete when the green solution turned
dark olive-green[4].
This solution obtained from the first action was stored as the etalon for the
following ones. The settled olive-green coloring meant that all chlorophyll had
reacted with H₂SO₄. So the process of titration was
repeated 7 times for all light intensity groups.

The solution is titrated until the dark
olive-green color because it is known that when the reaction between
chlorophyll and sulfuric acid happens, chlorophyll turns into phaeophetin which
has grey color (see table 1), therefore when the solution is olive-green, than
the reaction has succeeded. But while searching in the internet and books I
found out that there are several opinions about the color of phaeophytin – in
the book written by Viktorov it is ssaid to have grey color, but in the
internet link http://www.ch.ic.ac.uk/local/projects/steer/chloro.htm
it is said to have brown olive-green color. Also I made chromatography in order
to investigate the color of phaeophytin and the result was that it has grey
color. It can be proposed that olive-green color is obtained because grey
phaeophetyn is mixed with other plant pigments.

 So titration is one of the visual methods that can be used in order to
find the mass of chlorophyll in plants.

All the measurements and even
chromatography were done three times and the mean value was taken, for
chromatography grey color was confirmed.

Table 1. Plant pigments.

IV.
Results.

Table 2. Raw data.

Table 3. Frequency of lengths of
3-weeks-old plants depending on light intensity.

Table 3 (alternative) Frequency of length
of 3-weeks-old plants depending on light intensity.

Calculation
of the mean length of plants.

For light
intensity equal to  20,50 lux:

The sum of
lengths of all plants in this group is 45cm + 37cm + 39cm + 49cm + 46cm + 44cm
+ 45cm + 44cm + 42cm + 37cm + 40cm + 40cm + 39cm + 43cm + 42cm + 42cm + 36cm +
45cm + 38cm + 45cm + 46cm + 40cm + 35cm + 42cm + 43cm = 1044cm

Hence mean
length is 1044cm : 25 plants = 41,76cm

Table 4.

Calculation of amount of chlorophyll in plants basing on
the results of titration

H₂ SO₄ 
+ C₅₆ O₅ N₄ Mg => C₅₆ O₅
N₄ H + MgSO₄

Concentration of
H₂SO₄ is 0,01 M

C –
concentration

V – volume

n – quantity of
substancy

m – mass

Mr – molar mass

For light
intensity equal to 20,5 lux.

n = V (in dm³)
∙ C

2 ∙ 10^-3
∙ 0,01 = 2 ∙ 10^-5

n = m / Mr =>
m = n ∙ Mr

m = 2 ∙ 10^-5
∙ 832 = 1,664 ∙ 10^-2 grams

mass of
plant                           mass of chlorophyll

1,68
grams                   -                     0,08335 grams of chlorophyll

1 gram          
               -                      x grams of chlorophyll

Hence there are
0,0496 grams of chlorophyll.

Table 5. The correlation between mean
length of plants and mean dry biomass.

Table 6. The correlation between mean
length and mass of chlorophyll per 1 g of plant.

Table 7. The correlation between mean dry
biomass and mass of chlorophyll per 1 g of plant.

V.
Discussion.

Several tendencies can be clearly seen.

For the first, with the increase of light intensity mean length of
plants is decreasing, but there are exceptions. For light intensity 142 lux the
value of mean length is approximately equal to the values of length for light
intensities 0 lux and 20,5 lux. If exclude this data it is also seen that for
light intensity equal to 680 lux mean length is also slightly falling out from
the main tendency – decreasing from 19.81 cm.

The second tendency is increase of mass of chlorophyll per 1 gram of
plant biomass with the increase of light intensity. But the values of mass of
chlorophyll of those plants under light intensities 142 lux and 680 lux are
falling out from the main tendency. The first and the second ones are too small
– approximately equal to the value corresponding to 20.5 lux light intensity
and to 89.5 lux respectively. This may happen because not all the seeds of Cicer
arietnum were of the same quality, because it is impossible to guarantee
that more than 250 seeds in one box have the same high quality. At the mean
time it was expected that starting from the light intensity more than 680 lux
the amount of chlorophyll in plants will decrease, because the value of
destructed chlorophyll with be bigger than the value of newly formatted. But
the experiments showed that the amount of chlorophyll was constantly increasing
even when the light intensity level exceeded the point 1220 lux. This could
happen because light intensity equal to 1220 lux is not so extremely high that
the amount of total chlorophyll in plants will start decreasing.

Also it is clearly seen that there are no correlations between light
intensity and values of wet and dry biomass.

            Basing
on these arguments the sudden decrease of the amount of chlorophyll in plants
placed on light intensity equal to 142 lux was likely to be insignificant and
could not be considered as a trend.

But it is
impossible to forget such important factor as plant hormones that affect the
growth and development of plants. There are five generally accepted types of
hormones that influence plant growth and development. They are: auxin,
cytokinin, gibberellins, abscic acid, and ethylene. It is not one hormone that
directly influences by sheer quantity. The balance and ratios of hormones
present is what helps to influence plant reactions. The hormonal balance
possibly regulates enzymatic reactions in the plant by amplifying them.

VI.
Conclusion.

            Due
to results of my investigation it is seen that my hypothesis didn’t confirm
fully (for example, comparing the diagram 1 and diagram 7), because I proposed
that when light intensities will be very high, mass of chlorophyll in plant
will start decreasing and due to my observations it didn’t happen. I should say
that the only reason I can suggest is that I haven’t investigated such
extremely high light intensities, so that chlorophyll start destructing. But if
we will not pay attention to that fact the other part of my hypothesis was
confirmed and mass of chlorophyll in plants increased with the increase of
light intensity. Furthermore I didn’t estimate amount of plant hormones and so didn’t
estimate their influence on results.

Questions for further investigation:

1.   
Investigating very high light intensities.

2.   
Implementation of colorimetric analysis.

3.   
Paying attention to estimation of plant hormones
level.

Those questions should be further investigated in order to get
clearer picture and more accurate results of the dependence of the amount of
chlorophyll in plants on the light intensity, knowing the fact that the amount
of chlorophyll has a tendency to decrease at extremely high light intensities.
So this statement needs an experimental confirmation and as in this
investigation conditions with extremely light intensity were not created in
further investigations they have to be created.

Implementation of colorimetric analysis is
also very important thing, because it gives much more accurate results
comparing with the titration method. The colorimetric
method suggests that as different pigments absorb different parts of light
spectrum differently, the absorbance of a pigments mixture is a sum of
individual absorption spectra. Therefore the quantity of each individual
pigment in a mixture can be calculated using absorbance of the certain colors and molecular
coefficients of each pigment. This was proposed by D.
A. Sims, and J. A. Gamon (California State University, USA)[5] with the reference on Lichtenthaler (1987).

VII.
Evaluation.

There are several results in my work, that are falling out from the
main tendencies. It may seem that such results may occur due to different
percentage of water in plants, but when I was calculating mass of chlorophyll
in 1 gram of plant I was using only values of mean dry biomass so it couldn’t
affect my results. (see table 3)

At the same time such differences in the percentage of water are
easily explained. The rate of evaporation of water from plants, which were put
under 1220 lux light intensity was much higher than of those put under 20.5 lux
light intensity, therefore percentage of water in the soil may vary, though I
provided all the plants with the same volume of water at the same periods of
time.

One more reason that could be proposed is the reason connected with
the pH of water with which flowers were provided. It was not measured but the
thing that could have happened is that it had somehow changed the pH of soil in
which seeds were placed and therefore changed the amount of synthesized
chlorophyll.

Titration is not a perfect way of obtaining results. This happens
because the method is based on visual abilities of a person – he has to decide
whether the color he obtained is dark olive-green or not so dark olive-green.
Such a situation concerns lots of mistakes due to different optical abilities
of each person, even some humans are not able to distinguish those colors,
because of the disease called Daltonism.

            Even
those who do not suffer from this disease can also make mistakes in such
experiment. It is known that people who suffer from Myopia can hardly see
objects that are far from them, but don’t have problems with objects that are
near, but it is also important to take into consideration the fact that their
ability to distinguish colors is also lower comparing with humans with normal
eyesight.

There also exist the so called human factor, which also affects the
investigation. Man can’t be absolutely objective, because sometimes it is too
hard for a person to falsify his own theory or hypothesis, so one can ignore
results that are not suitable for his statements and select only those that are
suitable, which will also affect the investigation not in good way.

 So as human’s eye is not a perfect instrument and humans are not
perfectly objective there should be other methods of investigating the amount
of chlorophyll in plant.

Moreover
titration method doesn’t distinguish between chlorophylls-a and
chlorophyll-b, phaeophytin-a and phaeophytin-b, as their
colors differ, this giving not very accurate results. Also due to this limiting
factor it is impossible to know whether the whole amount of chlorophyll reacted
with the sulfuric acid and again it adds an uncertainty to the results.
Furthermore the saturation of color depends on the extent of dilution and it is
nearly impossible to say if the solution was diluted till the same color or
not, because it is very difficult to distinguish between different shades of olive
green color.

BIBLIOGRAPHY

1)   
Allott, Biology for IB diploma (standard and higher level), Oxford
University Press, ISBN 0-19914818

2)   
M. Roberts, M. Reisse, G. Monger, Biology: principles and
approaches, Nelson, ISBN 0-17-44-8176-4

3)   
T. King, M. Reiss, M. Roberts,
Practical advanced biology, Nelson Thorns, ISBN 0-170448308-

4)   
Викторов Д. П., Практикум по физиологии растений. – 2-е изд. – Воронеж:
ВГУ, 1991

5)    http://www.ac-creteil.fr/svt/Tp/Tp2/Tp2UK2/fiches_them_choix-P2/genechloro.doc, 15/03/2004

6)    http://vcsars.calstatela.edu/esa_posters/ds/dan_esa99.html 05/05/2004

7)    http://www.agsci.ubc.ca/courses/fnh/410/colour/3_21.htm, 16/03/2004

8)    http://vcsars.calstatela.edu/esa_posters/ds/dan_esa99.html, 22/02/2004

9)   
http://www.charlies-web.com/specialtopics/anthocyanin.html. 17/04/2004

10)  http://www.ch.ic.ac.uk/local/projects/steer/chloro.htm,
11/04/2004

11) 
http://www.bonsai.ru/dendro/physiology5.html 02/04/2004

12)  http://www.iger.bbsrc.ac.uk/Publications/Innovations/in97/Ch2.pdf,
06/05/2004