Plant & CellPhysiol.

17: 627-631 (1976)

Short communication

Localization of aluminium in tea leaves

Hideaki Matsumoto, Eiji Hirasawa, Seiichiro Morimura and Eiichi Takahashi

Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

(Received February 2, 1976)

More than 30,000 ppm of aluminium (Al) was found accumulated in old tea leaves.
The localization of Al in epidermal cells was detected by light microscopic observation
and electron microprobe X-ray analysis. A distinct thickening of epidermal cell wall
was seen in old leaves. The addition of AICI3 to the cultural solution promoted the
growth of tea seedlings.

Aluminium (Al) is one of the major soil constituents. Solubilized Al3+ in

acid soil can be absorbed via roots and thereby affect plant growth (5). The Al
content in plants growing in neutral soil are usually not high. Exceptional plants
containing large amounts of Al are known as Al-accumulating plants (10,12). They
often grow in acid soil. Tea (Camellia sinensis) is a well-known Al-accumulating
plant, whose old leaves often contain more than 10,000 ppm of Al. However, the
localization and physiological role of Al in plants has not been studied except for
investigations of the toxicity of Al given to plants or plant tissues artificially (1, 2).
It has been reported that Al was localized in nuclei and cell walls of various plant
roots cultured in Al3+ solution (1, 3, 7). Several hypotheses were proposed re-
garding Al toxicity (1-3, 7, 11, 13). However, no work has been done on
localization of Al absorbed from natural soil. Therefore, this work was conducted
to find the localization of Al in tea leaves growing naturally.
10-year-old tea (Camellia sinensis, cv. Yabukita) growing on Kyoto University
farm land was used in this study. Old leaves developed in 1974 and young ones
developed in 1975 were harvested in August, 1975. Aflamelessatomic absorption
spectrophotometer (Hitachi 518) was used for the measurement of Al as reported
previously (7). The Al content was 30,690 ppm in the old leaf and 600 ppm in the
young one. Al in the leaves was observed with a light microscope after making
a pink chelate complex of Al with aluminon (/). For the electron microprobe
X-ray (EMX) analysis and the staining of the sample with iodine, leaf sections
were embedded in epoxy resin by the method of Fujita et al. (4). Leaf samples
embedded in epoxy resin were stained with iodic reagents following the method of
Fujita et al. (4). This staining method is based on the fact that iodine preferentially
stains the embedded epoxy resin without staining the object to be observed, which is
similar to the negative background staining method for electron microscopy. The
EMX (Shimadzu-ARL) technique was the same as reported earlier (7).
627
628 H. Matsumoto, E. Hirasawa, S. Morimura and E. Takahashi

Fig. 1. Localization of Al in the old tea leaf showing the specific accumulation of Al in epidermal cells. X 400.
The transverse sections were stained by aluminon, and Al is indicated by the pink color. Upper
picture (old leaf), UE: upper epidermis, PC: palisade cells, SC: spongy cells, LE: lower epidermis.
Lower picture (young leaf).

As shown in Fig. 1, the pink color formed by the chelation of Al with aluminon
was found specifically in the epidermis of the old leaf. It was also seen that Al was
accumulated preferentially in the upper epidermis. Unlike the old leaf, no Al was
detected in the young leaf with this staining method. These results indicate that
Al absorbed from the soil was transported to the leaves and accumulated in the
epidermis gradually. This characteristic localization of Al in the epidermis of the
old leaf was further investigated by EMX analysis. The line scan of Fig. 2 indicates
the distribution of Al in the old leaf clearly. Again Al was found to be localized in
the upper and lower epidermis as observed by light microscopy (Fig. 1). No Al was
detected in the young leaf by EMX analysis either. The characteristic preferential
accumulation of Al in the upper epidermis of the old leaf led us to investigate
structural differences in the upper epidermis between young and old leaves.
Samples were embedded in epoxy resin, stained with iodic reagents, and observed
with a light microscope. The photographs in Fig. 3 were taken at the same
magnification. As can be seen, palisade cells in the old leaf are much bigger than
those in the young one. The shape of upper epidermal cells is also quite different.
In the old leaf, the cells expanded horizontally, and the lateral length is greater than
the vertical. The reverse is seen in the young leaf. It is characteristic of old
 Localization of Al in tea leaf 629

Fig. 2. Electron microprobe X-ray line scan of Al in the transverse section of the old tea leaf. X400. Electron
beams were irradiated on the line indicated as E. UE: upper epidermis, LE: lower epidermis.

epidermal cells that they have well thickened cell walls. The thickening of cell walls
might be related to the lateral expanding of the epidermal cells in the old leaf. Al
as well as other secondary materials may have entered the cell wall during thicken-
ing. Al is absorbed from the soil via roots and then passes into the vessels of the
xylem and moves upward to the leaves. At the end of the vein, Al diffuses into
neighboring mesophyll cells, passing from one cell to the other, and reaches the

Fig. 3. Upper epidermal cells and palisade cells of old (left) and young (right) tea leaves. X2800. Trans-
verse sections were embedded in epoxy resin and stained with iodine. UE: upper epidermal cells,
PC: palisade cells.
630 H. Matsumoto, E. Hirasawa. S. Morimura and E. Takahashi

Fig. 4. Tea seedlings grown in cultural solution for 100 days with {right) or without (lift) J0~3 M AICI3.

epidermal cells. It might be accumulated in these cell walls. Until now no one
has worked on the distribution of Al in leaves. In several crop plants such as
Alaska pea (7) and Zea mays (9), Al was found to be precipitated on the
surface of the epidermal cells of roots when they were immersed in a solution con-
taining Al 3+ . It is interesting that old tea leaves accumulate as much as 30,000
ppm of Al which is localized specifically in the epidermis, though the accumulation
seems to take a long time. The high concentration in old leaves is quite enough
for to inhibit root elongation in some other plants (1,2,7). What is the physiological
meaning of such slow but vast accumulation of Al in old tea leaves? To investigate
this point, tea seedlings were cultured in a solution (8) with or without 10~ 3 M AICI3.
The solutions were changed every 10 days, and pH was kept at approximately 6.0
during culture. The chemical form of Al present at this pH is A1(OH)3. Dissolved
Al 3+ could be far less than 10~3 M, although it is affected by coexisting ions in the
culture solution. This indicates that the physiological function of Al 3+ might be
quite large. It was found that Al clearly promoted the growth of seedlings. First
the formation of new roots was greatly accerelated after about 1 month of Al treat-
ment and thereafter the growth of tops were affected positively. The effect of Al
on growth appeared most conspicuously about 50 days after Al treatment began.
The positive effect of Al on tea plant growth was also reported by Matsuda et al.
 Localization of Al in tea leaf 631

(6). The content of Al in the leaves was measured every 20 days after the Al treat-
ment began. Accumulation of Al as high as 1,000 ppm was not detected before
100 days, by which time the positive effects of Al on growth have been shown clearly
(Fig. 4). At 100 days of Al-treatment, leaves of 3 different ages (upper, middle
and lower) of a 12-leaved plant were used to measure Al content, which was 262,
428 and 1249 ppm, respectively. These results tell us that the accumulation of AJ
in the old tea leaves is a slow process and could not have a positive physiological
role in the growth of tops. Al probably has a physiological role in the accerelation
of new root formation, and might be transfered to the leaves and accumulated in
the epidermal cells as waste material. The increased growth of the tops (Fig. 4)
might depend primarily on the positive function of Al 3+ in the roots.

The authors would like to thank to Dr. M. Fujita, Department of Wood Science and Technology,
Kyoto University, for embedding the samples in epoxy resin and for taking microscopic pictures of
iodime-stained samples, and to Dr. N. Wakiuchi, Department of Agricultural Chemistry, Kobe Univ-
ersity, for the EMX analysis procedures.
We also express our thanks to Dr. S. Nakamura, Department of Agricultural Biology, Kyoto
University, for the reproduction of the color print used in Fig. 1.

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