Vol. XVI, Issue 1 Annals of RSCB
CHLOROPHYLL CONTENT QUANTIFICATION IN
ACCLIMATED “IN VITRO” PLUM PLANTS
(PRUNUS DOMESTICA, L.)
Zs. Jakab-Ilyefalvi, D. Pamfil
UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE,
DEPARTMENT OF BIOTECHNOLOGY, CLUJ-NAPOCA
Summary
The apparition of the symptoms caused by the plum pox virus through formation of
mosaic surfaces and necrotic zones at the stone fruits are known as reduction factors of the
chlorophyll content and influences negatively the physiological factors inclusively the
affection of the metabolic processes in the accumulating of sugar content. Spectroscopy
results revealed the fact that the chlorophyll “a” content is higher in the case of healthy
plants, this varying from 7.02 mg/l to the quantity 12.93 mg/l in comparison with the infected
plants at which the chlorophyll “a” content was at a lower level. The content in chlorophyll
“a” of the infected leaves varied between 4.46 mg/l respectively 6.38 mg/l, concluding the
fact that the chlorophyll content was significantly reduced regarding the analyzed in vitro
plant leaves. The analyzed leaves presented chlorothic bands, circles, and the reduction of the
chlorophyll content cannot due because of the physiological stadium of leaves , the in vitro
plantlets have presented a high juvenile stadium being regenerated by meristematic tissue
culture technique. The low chlorophyll content was probable due to alterations in the
biosynthesis of chlorophylls. Similar phenomena were observed in the case of the chlorophyll
“b” content analysis at the healthy and infected plum leaves. According the analysis the
chlorophyll “b” content varied 2.81 – 5.07 mg/l at the healthy in vitro plants. In the case of
the infected in vitro plants the chlorophyll “b” content varied between 1,5 – 3.11 mg/l.
Results show that the total chlorophyll content in the plum leaves had lower values in the
case of infected samples , comparative with the healthy ones.
Key words: chlorophyll, plum, leaves, plum pox virus [email protected]
infection with PPV, influencing thus the
main vital processes of the plants ( Zhang,
et al, 2000, Wang, et al., 2007). The
complex photosynthetic process also in
greenhouse conditions are open field
conditions are influenced by the
degradation of chlorophylls , thus the
quantification of the exact chlorophyll
content of the two types of chlorophyll a
and b can be used in the characterization of
the photosynthetic capacity of the plants.
The photo-inhibition in additional way is
associated but not necessary accompanied
by the degradation of the chlorophylls
Introduction
The apparition of the symptoms
caused by the plum pox virus through
formation of mosaic surfaces and necrotic
zones at the stone fruits are known as
reduction factors of the chlorophyll content
and influences negatively the physiological
factors inclusively the affection of the
metabolic processes in the accumulating of
sugar content (Bulgaru and Isac, 1988).
Many studies showed the fact that the
whole photosynthetic and metabolic system
can be affected in the case of severe
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Vol. XVI, Issue 1 Annals of RSCB
the acclimation process. There have been
effectuated serological and molecular tests
in order to confirm the presence of the PPV
virus. Healthy and PPV infected leaves
were sampled according to the effectuated
tests. Leaves were put in zip bags and
introduced in a refrigerating box and
transported to the Biochemistry Department
of USAMV Cluj-Napoca where took place
the extraction and determination of
chlorophyll
content.
Samples
were
weighted, 0.1 g for every individual sample.
Refrigerated 80 % cc. acetone was used in
order to rive the protein-chlorophyll
complexes and to extract the chlorophylls.
The vegetal material was introduced in a
grinding mortar and 80 % cc. acetone was
used for extraction. The chlorophyll extract
were put inb Falcon tubes and introduced in
an ultrasonic cleaner (Tellsonic, TPC-25)
for 15 minutes.
(Critchley, 1998). The study effectuated by
Baumgartnerova and Slovakova, 1998,
regarding the chlorophyll containment and
the viral concentration of PPV particles
have concluded the fact that in the
symptomatic leaves of apricot the
assimilatory pigment containment was
affected by the plum pox viral infection.
The affected leaves presented a lower level
of chlorophylls and the carotene content
was higher than those used for control.
Material and methods
In order to elucidate the a and b
chlorophyll amount from the in vitro plum
plants at the cultivars Ivan, Iulia, Geta,
Jubileu 50, it has been proceeded to the
extraction
and
determination
of
chlorophylls after the method described by
Arnon , (1948), and Baumgartnerova,
(1998). Micropropagated in vitro plantlets
were labeled and numbered starting with
Fig. 1 - Preparing of vegetal samples
Fig. 2. Weighing of the leaf samples
Fig. 3 Extraction of chlorophylls a+b
in 80 % acetone
Fig. 4 Centrifugation of the plant extract at
7000 rpm
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Vol. XVI, Issue 1 Annals of RSCB
Fig.5 Chlorophyll extracts ready for UV-VIS spectroscopy
The homogenate was centrifuged at
7000 rpm, decanted, and procedure was
repeated once again. The final volume was
measured for every sample in order to
effectuate
the
final
chlorophyll
concentration. The quantification of the
chlorophyll content was effectuated based
on the absorbance registered by UV VIS
spectroscopy tehnique. Based by the
formula published by Arnon, (1948)
subsequently there were effectuated the
exact chlorophyll content calculations.
Total chlorophyll (µg/ml) = 20.2 (A645) + 8.02 (A663)
Chlorophyll a (µg/ml) = 12.7 (A663) - 2.69 (A645)
Chlorophyll b (µg/ml) = 22.9 (A645) - 4.68 (A663)
TABLE I. Determination of the optical density by spectroscopy of the chlorophyll extracts
Sample
number
acclimated
plant
84
90
89
77
117
71
112
97
114
103
Sample
No.
Phytoviral Optical
Status
Density
A663
Optical
Density
A645
Optical
Density
A433
Optical
Density
A450
1
3
8
9
7
10
2
4
5
6
Healthy
Healthy
Healthy
Healthy
Healthy
Infected
Infected
Infected
Infected
Infected
0,81109
0,604948
0,96816
1,11287
0,6628
0,44535
0,38517
0,4364
0,33505
0,55504
0,32612
0,24627
0,39147
0,44874
0,29147
0,19118
0,15982
0,19212
0,13418
0,24943
0,9398
0,75622
1,13951
1,27022
0,91384
0,59371
0,4927
0,62485
0,46241
0,79003
1,44574
1,13182
1,73854
1,95072
1,31525
0,87006
0,73297
0,89268
0,67734
1,12508
103
77
117
89
84
90
6
9
7
8
1
3
Infected
Healthy
Infected
Healthy
Healthy
Healthy
0,3376
0,29747
0,62416
0,47154
0,36333
0,29622
0,14681
0,12807
0,25022
0,19322
0,14905
0,12174
0,4681
0,40834
0,71718
0,55258
0,3987
0,36265
0,6757
0,5955
1,0624
0,8450
0,63028
0,54228
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Vol. XVI, Issue 1 Annals of RSCB
Results and discussion
There have been analyzed the
individual content of “a” chlorophylls
respectively “ b” type chlorophylls at both
of the plant categories also at the healthy
ones and those infected with plum pox
virus.
The major importance in the light
assimilation process is due to chlorophyll
“a“ due to the fact that these are the main
responsible for the satisfactory functioning
of the PS II photo-system in natural light
conditions. The “b” type chlorophylls the
main components of the proteins which
collect LHCP light and the content of this
pigment in the leaves is important for the
capacity of the leave to accommodate in
shadow conditions (conditions typical in
greenhouse).
Comparative analyses of chlorophyll "a" in plum leaves
(Prunus domestica,L.)
14
9
8
12
mg/ L
10
1
7
3
8
6
10
6
4
2
5
4
2
0
Healthy leaves
Infected leaves
Fig.6 Comparative analysis of the chlorophyll “a” content in the analyzed samples
Comparative analyses of chlorophyll "b" in plum leaves
(Prunus domestica,L.)
6
9
5
mg/ L
4
3
8
1
7
6
3
4
10
2
2
5
1
0
Healthy leaves
Infected leaves
Fig. 7. Comparative analysis of the chlorophyll “b” content in the analyzed samples
Analyzing the fig. 6 it is revealed
the fact that the chlorophyll “a” content is
higher in the case of healthy plants , this
varying from 7.02 mg/l to the quantity
12.93 mg/l in comparison with the infected
plants at which the chlorophyll “a” content
was at a lower level. The content in
chlorophyll “a” of the infected leaves varied
between 4.46 mg/l respectively 6.38 mg/l,
concluding the fact that the chlorophyll
content was significantly reduced regarding
the analyzed in vitro plant leaves. The
analyzed leaves presented chlorothic bands,
mosaicated circles, and the reduction of the
chlorophyll content cannot due because of
the leaves sencescence, the in vitro plantlets
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Vol. XVI, Issue 1 Annals of RSCB
have presented a high juvenile stadium
being regenerated by meristematic tissue
culture technique. The low chlorophyll
content was probable due to alterations in
the biosynthesis of chlorophylls. Similar
phenomena was observed in the case of the
chlorophyll “b” content analysis at the
healthy and infected plum leaves ( Fig. 7).
According the analysis the chlorophyll “b”
content varied 2.81 – 5.07 mg/l at the
healthy in vitro plants. In the case of the
infected in vitro plants the chlorophyll “b”
content varied between 1,5 – 3.11 mg/l . A
lower value of chlorophylls
can be
influenced by an excess of irradiation of
these pigments at the plants grown in open
field condition (Jason et al., 2004).
The analysis of the absorbance
spectra. The chlorophyll molecules are
components in different photo-systems
integrated in the interior of the tylacoid
membranes of the chloroplasts. The
chlorophylls are absorbing the most intense
light from the blue
region of the
electromagnetic spectra of light, followed
by the red region, but they absorb very
weak in the green region , from this derives
the fact that the vegetal tissues containing
the chlorophylls are becoming the green
color after this pigment. The absorption of
the chlorophylls are between the 400-700
nm , and this region is named the
photosynthetically active region (PAR).
sursa: http://image.tutorvista.com/content/photosynthesis/electromagnetic-spectrum-of-chlorophyll.jpeg
Fig. 8 Spectral absorbance of chlorophylls in the visible light
Chlorophyll “a” molecules extracted in
acetone in our experiences have absorbed at
optical densities of A=663nm respectively
A=443 nm , and the chlorophyll “b” at
optical densities of A=645 nm and A=450
nm for the two extremities of the spectra.
Proba nr.1 - Nediluata
Absorbance
1
0
400
500
600
700
Wavelenght (nm)
Fig. 9 Absorbance of the chlorophyll extract , plum cultivar Ivan, healthy plant
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Vol. XVI, Issue 1 Annals of RSCB
In fig.9 it is shown the absorption
spectra of the sample no.1 , the specific
absorption at the level of chlorophylls
having a value of 0.811090 OD at the
maximum value of A=663 nm respectively
1.44574 OD at the maximum value of
A=433. This sample has been diluted to an
appropriate reading at the maximum value
of A=433 nm obtaining optimal values
which could be interpreted properly
obtaining the value 0,36333 at the
maximum value of A=633 nm respectively
0,63028 at the maximum value of A=433
nm. The specific absorbance at the
chlorophyll “b” at the absorbance
maximum of A=645 nm has presented the
values 0,32612 at the absorbance of A=450
nm the optical density was 0,9398.
at the maxima of A=633 nm presented the
value of 0,15982 DO and the A=433 nm the
optical density was 0,4927.
As shown above in our experiences,
the total chlorophyll content in the plum
leaves had lower values in the case of
infected samples, comparative with the
healthy ones.
Proba nr.2 - Nediluata
Absorbance
1
Comparison between the absorbance of
chlorophyll extracts of healthy / infected
plum leaves
0
400
500
600
700
Wavelenght (nm)
The comparative analysis of the
spectra of the two plant categories reveals
the fact that there are notable differences
between the absorbance of the chlorophyll
content of healthy and infected plants , in
the frame of the same cultivar ‘Geta’ there
being differences between the infected
plants and healthy plants.
Fig. 10 Absorbance of the chlorophyll extract ,
plum cultivar ‘Iulia’, infected plant
In the case of sample no.2 the
registred specific absorbance had the value
0,38517 OD at the A=633 nm point
respectively 0,73297 OD at the maxima of
A=433 nm at the chlorophyll “a” level. The
specific absorbance at the chlorophyll “b”
2
Proba nr. 5 - Nediluata
Proba nr. 9 - Nediluata
2
Absorbance
Absorbance
Proba nr. 4 - Nediluat
Proba nr. 8 - Nediluat
1
0
1
0
400
500
600
700
400
Wavelenght (nm)
500
600
700
Wavelenght (nm)
Fig.11 Comparations between the absorbance of healthy and infected samples at cultivar ‘Geta’
The spectral curve of the healthy
plants where noted with red color and the
spectra of the infected leaves were noted
with black color, being revealed the
existing differences. The symptoms of
healthy leaves without the mosaic shaped
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Vol. XVI, Issue 1 Annals of RSCB
Critchley,
C.,
Photoinhibition
in
photosynthesis,
a
comprehensive
treatise,
Cambridge University Press, Cambridge, 264271998.
Jason, J.G., Thomas and D.M. Pharr,
Photosynthesis, chlorophyll florescence, and
carbohydrate content of illicium taxa grown under
varied irradiance, J. Am. Soc. Hort. Sci., 129:46-53,
2004.
Hong, W., Falin Wang, Gang Wang, Khalid
Majourhat, The responses of photosynthetic
capacity, chlorophyll fluorescence and chlorophyll
content of nectarine ( Prunus persica var Nectarina
Maxim) to greenhouse and field grown conditions,
Scientia Horticulturae, 112:66-72, 2007.
Bulgaru, L., Isac, M. Some biochemical
changes induced by plumn pox in plum , Acta
Horticulturae,Vol.235,125-129, 1988.
Zhang, Y., Simeone , A. M., Cappelini, P.,
Physiological modifications caused by plum pox
virus in the leaves of peach and apricot tree, Agris,
ISMEA
(Italy), 39(3), p. 447-450, 2000.
surfaces on the leaves have revealed higher
levels of chlorophyll content.
According to the effectuated studies
, we consider that among other factors
which contributes to the decrease of the
chlorophyll content , the incidence of the
plum pox virus influences the biosynthesis
and metabolic system of chlorophylls,
experiences confirmed by other researches
(Zhang, at al., 2000).
References
Arnon, D.I., Copper enzymes in isolated
chloroplast, polyphenoloxidase in beta vulgaris,
Plant physiology, Vol. 24, Number 1, 1-6, 1949.
Baumgartnerova, H., Slovakova, L.,
Petrusova, N., Relationship between concentration
of plum pox virus and content of pigments in virusinfected symptomatic apricot leaves, Acta Virol., 42
(4):216-218, 1998.
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