Download Effect of Aluminum Sulfate on the Chlorophyll a, Chlorophyll b

International Journal of Horticulture 2015, Vol.5, No.9, 1-8
http://ijh.biopublisher.ca
Research Article
Open Access
Effect of Aluminum Sulfate on the Chlorophyll a, Chlorophyll b, Carotenoids
and Anthocyanin Content in Some Cultivars of Hydrangea (Hydrangea
macrophylla)
Eid G.M.1, , Albatal N.2, Haddad S.2
1. PhD student, Horticulture department, Agriculture college, Damascus university, Syria.
2. Professors, Horticulture department, Agriculture college, Damascus university , Syria.
Corresponding author email: [email protected]
International Journal of Horticulture, 2015, Vol.5, No.9
doi: 10.5376/ijh.2015.05.0009
Received: 24 May, 2015
Accepted: 10 Jun., 2015
Published: 12 Aug., 2015
Copyright © 2015 Eid et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Eid G.M., Albatal N and Haddad S., 2015, Effect of Aluminum Sulfate on the Chlorophyll a, Chlorophyll b, Carotenoids and Anthocyanin Content in Some
Cultivars of Hydrangea (Hydrangea macrophylla), International Journal of Horticulture, 2015, Vol.5, No.9 1-8 (doi: 10.5376/ijh.2015.05.0009)
Abstract The experiment was conducted in Hama governorate (Bammraa village) during the spring and summer seasons 2011- 2012
to study the effect of Aluminum on the content of chlorophyll a, b and Carotenoids in leaves and also the content of anthocyanin in
sepals. Aluminum in leaves and flowers was measured to estimate its amount in all treatments.
Aluminum sulfate was supplied in different concentrations (control, 50 mg/l, 100 mg/l, 150 mg/l) to two cultivars of Hydrangea
macrophylla. Nikko Blue (a cultivar with blue color flower), Pia (a cultivar with deep pink color flower).These plants were two years
old and planted in pots. It was used four treatments, there was three replications in every treatment and in every replication there was
six plants. The media was peat moss and perlite (2:1).
Results indicated that increasing Aluminum sulfate more than 50 mg/l caused a decrease in the content of chlorophyll a, b and the
content of Carotenoids in leaves, Also anthocyanin was best in the concentration 50 mg/l in the two cultivars with significant
differences.
In addition, the results showed that the cultivar (Nikko blue) was more responsive in some studied indicators. Also it was proved that
the amount of Aluminum in leaves was more than in the flowers of the two cultivar .
Keywords Hydrangea;Cultivars; Aluminum; Chlorophyll a; Chlorophyll b; Carotenoids; Anthocyanin.
Introduction
Hydrangea macrophylla is a species of Hydrangea and
it is the only species that is spread in Syria in the
coastal and mountaineer areas. Common names include
big leaf Hydrangea, French Hydrangea, Lacecap
Hydrangea, Mophead Hydrangea, Penny Mac and
Hortensia (Adkins and Dirr, 2003). It is widely cultivated
in many parts of the world in many climates (Jessica,
2008). In most species the flowers are white in color,
but in others the flowers can be changed to blue, red,
pink, or purple (Smith et al., 2008). Hydrangeas are
very popular plants for ornamental purposes, people
seem to love them for their large flower heads
(Albatal, 2003).
The Hydrangea genus contains about 70-75 species of
various flowering plants (Jones and Reed, 2006). This
scientific name comes from two Greek words “Hydro”
and “aggeino” which means “water vessel”(Lassiter,
2000).
Most of these species are shrubs, some are small trees,
while others are of the climbing variety (Bailey, 1998).
It has a beautiful, perennial bushes with huge flower
heads. These deciduous bushes profusely produce
huge, round flower heads which have two kinds of
florets: Sterile, or ray florets, are male and form the
large, colorful sepals on the outside of the flower head.
The fertile florets bear the male and female parts and
are usually found in the center of the cluster (Mojahed
et al., 1969). Flowering of these plants are produced
by the plant from early spring to late autumn. Hydrangea
are native to North and South America, Himalayas,
central and eastern Asia (Dirr, 2004).
The color of hydrangeas will vary considerably due to
the pH of the soil they are growing in (Tilt, 2008).
Kikelly (2006) reported that the blue hues are best in
acid soil and the degree of blueness is controlled by
the amount of available aluminum and the capacity of
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International Journal of Horticulture 2015, Vol.5, No.9, 1-8
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a particular variety to draw it up. The reds and pinks
enjoy an alkaline or neutral soil where aluminum is
not actively absorbed. The whites stay white but
usually enjoy the same conditions as the reds and pinks.
in leaves of Hydrangea and the amount of anthocyanin
in sepals.
Materials and Methods
Used Plants: In this work, two cultivars were used of
the species Hydrangea macrophylla. The first cultivar
is Nikko Blue (a cultivar with blue color flower), the
second cultivar is Pia (a cultivar with deep pink color
flower).
Bailly found in (1992) that to encourage "bluing" of
the flowers, we need to raise the acidity of the soil.
Acidity levels need to be around 5.5 - 6.0 on the pH
scale. To lower the pH and increase the amount of
aluminum in the soil, one should apply around the
hydrangea aluminum sulfate several times at intervals
in the spring and again in the fall if the desired color is
not achieved. The amount of aluminum sulfate really
depends on the concentration because aluminum is
toxic in large doses (Ghanati et al., 2005).
These plants were tow years old and planted in pots
(one plant in every pot), the media was peat moss and
perlite (2:1).
Study Area: The study was in Bammraa village in
Hama in Syria. Bammraa is considered as a mountaineer
According to Kochain (1995), Al is the third most
abundant element in the earth´s crust, comprising
about 7% of the total mass of the earth (Delhaize and
Ryan, 1995; Zhang et al., 2007). Lidon and Barreiro
(2002) reported that rocks contain from 0.45 to 10%
Al. (Yakimova et al., 2007) considered that Al is one
of the most abundant toxic elements with the ability to
contaminate soil, water and trophic chains. Nonetheless,
the specific biological functions of Al for plants are
unknown, and so it is not regarded as an essential
nutrient (Poschenrieder et al., 2008).
Hydrangea (Hydrangea macrophylla) is also a well
known Al-accumulating plant, and the relationship
between Al and the blue coloration of Hydrangea
sepals has been thoroughly investigated (Takeda et al.,
1985a, 1985b). Hydrangea plants can accumulate as
much as 5 mg Al/ g dry weight in the leaves within
several months (Martin, 1988). It is well known that
the color of Hydrangea sepals changes from red to
blue when soil pH is shifted from weak alkaline or
neutral to acidic. However, it was found that it is the
Al dissolved in acid soils that responsible for the blue
color of the sepals, not soil pH itself (Allen, 1943).
However, the blue and pink color of hydrangea flower
sepals due to only one anthocyanin, delphinidin
3-glucoside. In the presence of aluminum, a blue color
will form due to the aluminum binding with the
anthocyanin. The reason for the blue color under acid
conditions is due in part to an increase in availability and
uptake of aluminum from the soil (yoshida et al., 1995).
The aim of this research is to study the effect of
Aluminum on chlorophyll a, b and Carotenoids content
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village which its altitude is more than 900 m above the
sea, this height cause a very cold winter and a cool
summer. The climate is wet and rainy for about eight
months in a year, the rainfall average is about 15002000 mm in the year, the average of maximum
temperature is about 22◌ْ c, the average of minimum
temperature is about 10◌ْ c, the average of humidity is
about 74.68%.
Place in dark(fluorescent lights will degrade chl-a, b)
and cold (4°C; a fridge).
After an extraction period of 8-24 h, measure chl-a
content in the extract using spectrophotometer instrument.
Calculate the chl-a, b and Carotenoids concentrations
in the water sample as following Equations (Porra, 2002):
Chlorophyll a (µg/ml) = 12.25 (A663.6)- 2.55 (A646.6)
Chlorophyll b (µg/ml) Chlorophyll - = 20.31 (A646.6)4.91 (A663.6)
Carotenoids (µg/ml) (1000A470- 3.27 (chl a) – 104
(chl b))/227
A663.6= Absorption at 663.6nm.
A646.6= Absorption at 646.6nm.
A470= Absorption at 470nm.
the chemical analysis was done in the laboratories of
Agriculture college in Damascus university, Syria.
The experiment was laid out according to Randomized
Complete Block Design (RCBD) with three replications
(three blocks), the data were analyzed using SPSS
program to find the differences between the means of
all the studied treatments, two way ANOVA was used
and least significant differences (LSD) at 0.05 level of
significances.
Anthocyanin content: The anthocyanin content was
measured as following (Pharr et al., 2006):
Sepals from hydrangea cultivars were harvested at full
bloom, weigh about 1 g of sepals and they were torn at
least twice.
Add 7ml of 1% Hcl in methanol(it prepared by mixing
99 ml of methanol and 1 ml of Hcl).
Treatments: There were four treatments:
1. The control, without adding aluminum to the irrigation
water.
2. Adding aluminum to the irrigation water to the
concentration (50 mg/l).
3. Adding aluminum to the irrigation water to the
concentration (100 mg/l).
4. Adding aluminum to the irrigation water to the
concentration (150 mg/l).
The sepal-extract slurry was vigorously mixed on a
magnetic stirrer for about thirty minutes, after which
the sepals had lost essentially all their color and the
extract turned red-pink(independent of the initial color
of the sepals) then the extract was decanted.
The aluminum was supplied to the water irrigation in
two times, the first time was in the early spring (march),
and the second time was after four weeks from the
first time (Akira et al., 2004). It was used three
replications in every treatment and in every replication
there were six plants so in every treatment there were
eighteen pots of the plants one plant in every pot.
Every pot was irrigated with water until 90 % of the
field capacity, the media was peat moss and perlite
(2:1).
Buffers at pH1 (0.025 M Kcl) and pH4.5(0.400 M
NaC2H3o2) were prepared and adjusted to be within ±
0.01 of the desired value (NH4OH was added to
increase the basicity of the buffer, or Hcl to increase the
acidity).
Exactly 2 ml of each buffer were pipette into separate
small beakers, followed by adding 4 ml of anthocyanin
extract to each beaker and the resulting extract-buffer
solutions were equilibrated 15 minutes, then filtered to
remove any particulates.
Investigated Traits
Spectra for both solution were obtained from 700 to
533 nm on a scanning spectrophotometer.
Calculate the absorption A as in the following equation:
A=(A533-A700)pH1-(A533-A700)pH4.5
Calculate the concentration of delphinidin-3-glucoside
in mg anthocyanin per g fresh sepals as following
equation:
(delphinidin-3-glucoside),mg/g
sepal=(A.M.DF.10000.v)/(ε. m)
A: Absorption
1 Chlorophyll a, b and Carotenoids content: The
content of chlorophyll a, b and Carotenoids were measured
as following (Lichtenthaler, 1983):
Weigh 5 g of leaves and grind them for about three
replications for each treatment.
Add about 45 ml of acetone [Prepare 80% acetone by
adding 200 ml of distilled water to 800 ml of acetone.]
and put them in a tube after filtering them since spillage
will occur more frequently otherwise.
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M: Molar mass of 465.2 g/mole
ε:molar extinction coefficient of 29 000
DF: dilution factor(the total volume of extract- buffer
solution divided by the volume of extract).
V: The volume of acidic methanol solution in ml used
in the extraction.
m: the mass of sepals (mg).
Table 1 Effect of Aluminum on the content of chlorophyll a in
the studied cultivars of Hydrangea at complete flowering stage
(µg/ml)
Treatment
control
Al (50 mg/L)
Al (100 mg/L)
Al (150 mg/L)
Mean
SD
SE
Variables
LSD 0.05
The amount of total Aluminum: It was measured in
the leaves and the flowers as the following (Jones,
2001) .Weigh 2 g of planting sample in a porcelain pot
which it was weighed when it was empty then take the
weight of all ( planting sample + porcelain pot).
Put the full porcelain pot in the ashing instrument for
two hours at 550◌c.
ْ
Chill the samples, weigh them and calculate the percent
of ashing.
Move the ashing to a flask (100 ml) then add 3 ml of
Hcl 25% and 50 ml of distilled water and heat them for
one hour.
The samples were measured by the atomic absorption
instrument .
Nikko blue
1.83
1.83
1.19
0.90
1.33a
0.34
0.04
cultivars
0.08
Pia
1.33
1.55
0.99
0.78
1.16b
0.30
0.04
treatment
0.11
Mean
1.37b
1.69a
1.09c
0.84d
1.25
0.33
0.03
interaction
1.15
Different letters indicate that mean difference between
treatments are significant at 0.05 level in the same column and
between cultivars are significant at 0.05 level in the same row
Table 2 Effect of Aluminum on the content of chlorophyll b in
the studied cultivars of Hydrangea at complete flowering stage
(µg/ml)
Treatment
Nikko blue
Pia
Mean
control
2.58
2.32
2.45b
Al (50 mg/l)
3.05
2.55
2.80a
Results and Discussion
Al (100 mg/l)
2.26
2.00
2.13c
Results in tables 1, 2, 3, 4 and also in figures 1, 2, 3, 4,
indicate that the content of chlorophyll a, b and
Carotenoids increased by adding some concentrations
of aluminum to the irrigation water. This was true in
the two studied cultivars, however the cultivar Nikko
blue recorded the highest values of them (1.33, 2.48,
1.14). The results showed that the second treatment
was the best treatment in the two cultivars with
significant effects in the above mentioned parameters
(1.69, 2.80, 2.48). It was noticed that when the
concentration of aluminum increased more than 50
mg/l, the chlorophyll a, chlorophyll b, Carotenoids
and anthocyanin content were decreased in the two
cultivars. The results also showed that the cultivar
Nikko Blue have chlorophyll a, b and Carotenoids
content more than the cultivar Pia in all treatments,
and recorded the highest values of the previous
parameters under aluminum concentration of 50 mg/L
(1.83, 3.05, 2.63). But the anthocyanin content in
sepals was more in the cultivar Pia, it was recorded in
the second treatment 50 mg/L (0.63 my/l) while it was
recorded in Nikko Blue (0.20 mg/l) and it was
decreased when the concentration of Aluminum was
increased to more than 50 mg/l in the two cultivars.
Al (150 mg/l)
2.03
1.93
1.98d
Mean
2.48a
2.20b
2.34
SD
0.39
0.25
0.35
SE
0.05
0.03
0.03
Variables
cultivars
treatment
interaction
LSD 0.05
0.08
0.11
0.16
Different letters indicate that mean difference between
treatments are significant at 0.05 level in the same column and
between cultivars are significant at 0.05 level in the same row
Table 3 Effect of Aluminum on content of Carotenoids in the
studied cultivars of Hydrangea at complete flowering stage
(µg/ml)
Treatment
Nikko blue
Pia
Mean
control
0.77
0.56
0.67b
Al (50 mg/l)
2.63
2.33
2.48a
Al (100 mg/l)
0.59
0.44
0.52b
Al (150 mg/l)
0.55
0.22
0.39b
Mean
1.14a
0.89b
1.02
SD
0.87
0.85
0.87
SE
0.10
0.10
0.07
Variables
cultivars
treatment
interaction
LSD 0.05
0.20
0.29
0.41
Different letters indicate that mean difference between
treatments are significant at 0.05 level in the same column and
The results also showed that the amount of total
aluminum increased in leaves and flowers when the
between cultivars are significant at 0.05 level in the same row
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Table 4 Effect of Aluminum on the amount of anthocyanin in
sepals in the studied cultivars of Hydrangea at complete
flowering stage (mg/g)
Table 5 Concentration of Aluminum in the leaves of the studied
Treatment
Nikko blue
Pia
Mean
Treatment
control
440.68
390.00
415.00d
Al (50 mg/l)
479.00
470.00
474.50c
Al (100 mg/l)
590.00
500.00
545.00b
Al (150 mg/l)
610.93
532.06
571.49a
Mean
530.15a
473.01b
501.58
SD
72.57
53.13
69.56
SE
8.55
6.26
5.80
Variables
cultivars
treatment
interaction
LSD 0.05
0.67
0.95
1.35
Nikko blue
Pia
cultivars of Hydrangea at complete flowering stage (mg/kg)
Mean
b
control
0.05
0.05
0.05
Al (50 mg/l)
0.20
0.63
0.42a
c
Al (100 mg/l)
0.06
0.45
0.26
Al (150 mg/l)
0.06
0.14
0.1d
Mean
0.09b
0.32a
0.21
SD
0.07
0.24
0.20
SE
0.01
0.03
0.02
Variables
cultivars
treatment
interaction
LSD 0.05
0.006
0.009
0.01
Different letters indicate that mean difference between
Different letters indicate that mean difference between
treatments are significant at 0.05 level in the same column and
between cultivars are significant at 0.05 level in the same row
treatments are significant at 0.05 level in the same column and
between cultivars are significant at 0.05 level in the same row
Table 6 Concentration of Aluminum in the flowers of the
studied cultivars of Hydrangea at complete flowering stage
(mg/kg)
concentration of aluminum increased in all treatments
in the two studied cultivars (Tables 5, 6). and (Figure
5, 6). However, it was noticed that leaves are different
to flowers as far as the accumulation of aluminum.
The amount of total aluminum in leaves increased in
the two cultivars with the gradual increscent of its
concentration in the irrigation water (Table 5) and
(Figure 5). The situation in the flowers was rather
different. The amount of aluminum increased in the
treatment of 50 mg/l but the higher concentration in the
water reflected a decrease in its concentration in the
flowers in the two cultivars (Table 6) and (Figure 6).
Discussion
Treatment
Nikko blue
Pia
Mean
control
192.00
180.00
186.00d
Al (50 mg/l)
432.00
378.00
405.00a
Al (100 mg/l)
400.00
370.00
385.00b
Al (150 mg/l)
356.00
346.00
351.00c
Mean
345.00a
318.50b
331.75
SD
93.06
81.44
88.14
SE
10.97
9.60
7.35
Variables
cultivars
treatment
interaction
LSD 0.05
0.67
0.95
1.34
Different letters indicate that mean difference between
treatments are significant at 0.05 level in the same column and
between cultivars are significant at 0.05 level in the same row
Although aluminum is not an essential element for
plant growth, the study showed that there were visible
differences in growth between the plants treated with
and without Al as it was reported by (Kumar et al.,
1988). But (Jian et al., 1997) reported that the only
effect of aluminum in Hydrangea plant is that the
sepals of plants grown in the solution containing Al
were blue, whereas those of plants grown at the same
solution but without Al were pink.
The study here showed that when the concentration of
Al was high (more than 50 mg/l), the chlorophyll a,
chlorophyll b, Carotenoids and anthocyanin content
were decreased. This agree with many previous
studies indicating to the toxicity of the element of
aluminum especially in higher concentration. However,
plants differ from each other in the ability of tolerance
(Foy, 1992, Jian et al., 1997, Pietraszewska, 2001).
And also agree with (Tohidi et al., 2014) who assured
Figure 1 Effect of Aluminum on the content of chlorophyll a in
the studied cultivars of Hydrangea at complete flowering stage
(µg/ml)
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Figure 6 Concentration of Aluminum in the flowers of the
studied cultivars of Hydrangea at complete flowering stage
(mg/kg)
that when aluminum increases more than the available
limit, it will cause a decreasing in chlorophyll, flavones
and Carotenoids. That is because the aluminum will
control on the activity of photosynthesis enzymes and
cause a decreasing in chlorophyll compound and
destroy the Carotenoids as it was mentioned by
(Haag-Kerwer et al., 1999, Lagriffoul et al., 1998,
Okhi, 1986, Loboda et al., 2006).
Figure 2 Effect of Aluminum on the content of chlorophyll b in
the studied cultivars of Hydrangea at complete flowering stage
(µg/ml)
This may explain the effect of aluminum on the pH of
soil, soluble Al present in the soil when the pH begins
to drop below 6.0. However, it is inconsequential in
the vast majority of soils until the pH drops below 5.5
Even then, it is rarely a problem until the soil pH
drops below 5.0. However, the concentration of
soluble Al increases dramatically in nearly all soils as
the soil pH drops below pH 5.0. In these extremely
acid soils, only those species adapted to acid soils
(such as blueberries, cranberries, and acid-loving
ornamentals) or the few crop species bred to tolerate
high soil Al levels can be expected to do well (Abreu
et al., 2003).
Figure 3 Effect of Aluminum on content of Carotenoids in the
studied cultivars of Hydrangea at complete flowering stage
(µg/ml)
Our research mentioned that anthocyanin in deep pink
sepals was more than in blue sepals and that’s agree
with a previous study(Asen et al., 1977) and (Robinson,
1939) that explained that red and pink sepals had six
times as much anthocyanin as blue sepals. But, (Pharr
et al., 2006) reported that there was no difference
among the anthocyanin contents of red, purple, and
blue sepals for the same cultivar. However, if only
those samples are included in which the red, purple
and blue sepals were measured from blooms in the
same plant, there was a tendency for the blue sepals to
have slightly (but not significantly) higher anthocyanin
contents. Moreover, the results in this study indicated
that the Al in leaves was more than in the flowers
(sepals) and that’s similar to the finding of (Jian et al.,
1997) who reported that leaves of Hydrangea contain
Al more than cell-sap and sepals (flowers) and they
Figure 4 Effect of Aluminum on the amount of anthocyanin in
sepals in the studied cultivars of Hydrangea at complete
flowering stage (mg/g)
Figure 5 Concentration of Aluminum in the leaves of the studied
cultivars of Hydrangea at complete flowering stage (mg/kg)
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http://dx.doi.org/10.1021/ja01875a510
Smith K., chenault J.A., and Tilt K., 2008, Pruning Hydrangeas. Horticulture
associate, Aubum University, Alabama MG Program Coordinator.
www.ag.aubum.edu/landscape/kerry smith.html.
Takeda K., Kariuda M., and Itoi H., 1985a, Blueing of sepal color of
Hydrangea macrophylla, Phytochemistry, 24: 2251-2254
http://dx.doi.org/10.1016/S0031-9422(00)83019-8
Takeda K., Kubota R., and Yagioka C., 1985b, Copigments in the blueing of
sepal color of Hydrangea macrophylla. Phytochemistry, 24: 1207-1209
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Tilt K., 2008, Extension Specialist and professor, Horticulture, Department,
Auburn-University.
www.whatgrowsthere.com/grow/
indicated in the same publication that in Hydrangea
plant with blue sepals there was Al more than in the
pink ones. This agrees too with the results of this work.
Conclusion
Although micro-nutrient (Functional nutrient) aluminum
available for uptake by the plant has an effect on the
color of Hydrangea plant, the results of this work
proved that Al has an influence on some other
parameters.
Increasing the concentration of Al more than 50 mg/l
will affect the content of chlorophyll a, b, Carotenoids
and anthocyanin amount.
Many questions concerning plant response to Al can be
posed but very few answers can be given, so many
researches must be done to know more about Al and its
influences on Hydrangea.
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