Are catechins localized in vacuoles in tea leaves?
Yuji Moriyasu, Takao Suzuki and Yasuhiro Miyoshi
School of Food and Nutritional Sciences, University ofShizuoka, 52-1 Yada Shizuokashi, Shizuoka 422-8526, Japan
One ofthe most striking biochemical features oftea (Camellia sinensis) leaves is
a high content of polyphenols such as catechins and the polymers of polyphenols
(called tannin). Although catechins and tannins have been thought to finally accumulate
in a large central vacuole after their biosynthesis, their intracellular localizations have
not been fully understood. Furthermore, some plant cells have recently been shown to
have different types ofvacuoles simultaneously during their developmental processes.
There are also reports showing the existence of special vacuoles containing an osmiumstainable material, presumably tannins, in some plants. But there has been no report
showing that tea leaves have such special vacuoles containing catechins and/or tannins.
We have started to investigate cellular morphology in tea leaves by
conventional electron microscopy with our attention to the existence of special
vacuoles containing a high amount ofcatechins and/or tannins.
[Materials and Methods]
The leaves oftea (Camellia sinensis cv. Yabukita) grown in a field were used. In
a preliminary experiment, we found Spurr's resin used for embedding did not seem to
enter the tissue ofmature leaves presumably because mature leaves are rich in cuticle.
Thus we selected young, unmatured leaves as a material. The leaves were cut into small
pieces with a razor blade and immediately immersed in 2% glutaraldehyde and 1%
formaldehyde in 100 roM phosphoric-Na (pH 7.0). After they were fixed at 4 C for a
few days, they were post-fixed in 1% osmium tetroxide. After fixation, the specimens
were dehydrated by ethanol series and embedded in Spurr's resin. Sections (about 1 um
thick for light microscopy and about 70 to 90 om thick for electron microscopy) were
prepared using an ultramicrotome. The sections for light microscopy were observed
without any staining or after staining with toluidine blue; those for electron microscopy
were stained with uranyl acetate and lead citrate.
[Results and Discussion]
By light microscopic observation ofthe prepared sections, we found that a
highly osmiophilic material, which was dark brown, existed in the mesophyll tissue
(Fig. lA). The presence ofthis material was easily seen without staining by toluidine
blue (Fig. 2B). We consider this osmiophilic material as tannins although there is a
possibility that catechins also contribute to make up this osmiophilic material. All
mesophyll cells did not contain tannins: some cells contained, but others did not.
Epidermal cells did not seem to have tannins.
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Fig. I Light micrographs of the sections of tea leaves. Observed after staining with toluidine
blue (A) or withiout any staining (B). Tannins look black.
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Session IT
To further clarify the intracellular localization oftannins, we examined the
sections by electron microscopy. But, the region ofthe tissue containing a large amount
oftannins was liable to be damaged when sectioning was done. Thus, we had to try to
make thicker sections, although the resulting electron micrographs became to lose high
resolution. By electron microscopy, tannins became electron-dense and they looked
black on positive prints (Fig. 2). Tannins seemed to be localized in large vacuoles which
are usually called the central vacuoles. In the lumen ofthe central vacuole, tannins were
not evenly distributed. They were existed only in restricted regions ofthe vacuolar
lumen, taking various shapes. In some cells, tannins seemed'to be present only in the
peripheral region ofthe vacuolar lumen and looked like a black ring (Fig. 2C). In other
cells, tannins occupied almost all lumenal space ofthe central vacuole, but in this case
many particles (0.1 to 10tJ.Dl in diameter) existed inside the tannin precipitate (Fig. 2D,
E). These particles were more electron-translucent than tannins and as a result, the
accumulation oftannins looked like a black disk with many white holes. We further
found that two types ofparticles which are differently electron-dense existed: one was as
electron-translucent as the hinten ofthe central vacuole of epidermal cells, which did
not have tannins; the other was a little electron-denser than the former type. We also
found relatively small vacuoles (0.1 to 0.5tJ.Dl in diameter) existing in mesophyll cells.
Some ofthese vacuoles had tannins in their whole lumens, thus can be classified into
what is usually called tannin vacuoles. The contents ofthe other vacuoles seemed to
correspond to those ofthe two types of particles observed in the tannin precipitate of
the central vacuole. We tentatively classify these small vacuoles into two types
accordingly. In addition, we found fusion profiles between a large central vacuole and
these small vacuoles (Fig. 2D, E).
From these results, we think that the mesophyll cells oftea leaves have at least
three kinds of small vacuoles, and that the large central vacuoles are formed through the
processes of fusion among these three small vacuoles.
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Electron micrographs ofthe sections oftea leafcells. Tannins look black.
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