Introduction
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).

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 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 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.

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).

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 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).

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 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 1500- 2000 mm in the year, the average of maximum temperature is about 22ْ c, the average of minimum temperature is about 10ْ c, the average of humidity is about 74.68%.

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.

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 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).

Investigated Traits
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.
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
A _663.6= Absorption at 663.6nm.
A _646.6= Absorption at 646.6nm.
A ₄₇₀= Absorption at 470nm.

Anthocyanin content: The anthocyanin content was measured as following (Pharr et al., 2006):
Sepals from hydrangea cultivars were harvested at full bloom, weigh about 1g of sepals and they were torn at least twice.
Add 7ml of 1% Hcl in methanol(it prepared by mixing 99ml of methanol and 1 ml of Hcl).

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.

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.

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
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).

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 550ْc.
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 .

Results and Discussion
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.

Table 1 Effect of Aluminum on the content of chlorophyll a in the studied cultivars of Hydrangea at complete flowering stage (µg/ml) 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) 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) 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 4 Effect of Aluminum on the amount of anthocyanin in sepals in the studied cultivars of Hydrangea at complete flowering stage (mg/g) 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

Figure 1 Effect of Aluminum on the content of chlorophyll a in the studied cultivars of Hydrangea at complete flowering stage (µg/ml)

Figure 2 Effect of Aluminum on the content of chlorophyll b in the studied cultivars of Hydrangea at complete flowering stage (µg/ml)

Figure 3 Effect of Aluminum on content of Carotenoids in the studied cultivars of Hydrangea at complete flowering stage (µg/ml)

Figure 4 Effect of Aluminum on the amount of anthocyanin in sepals in the studied cultivars of Hydrangea at complete flowering stage (mg/g)

The results also showed that the amount of total aluminum increased in leaves and flowers when the 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)

Table 5 Concentration of Aluminum in the leaves of the studied cultivars of Hydrangea at complete flowering stage (mg/kg) 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 6 Concentration of Aluminum in the flowers of the studied cultivars of Hydrangea at complete flowering stage (mg/kg) 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

Figure 5 Concentration of Aluminum in the leaves of the studied cultivars of Hydrangea at complete flowering stage (mg/kg)

Discussion
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 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).

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).

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 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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