ISSN 1021-4437, Russian Journal of Plant Physiology, 2007, Vol. 54, No. 5, pp. 698–701. © Pleiades Publishing, Ltd.

, 2007.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 5, pp. 786–789.

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Expression and Location of Caffeine Synthase in Tea Plants1

Yuan-Hua Lia, b, Wei Gub, and Sheng Yec
a College
of Life Sciences, Wuhan University, Wuhan 430072, China
b College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070 China;
fax: +86-27-87-28-2010; e-mail: Liyuanhua@mail.hzau.edu.cn
c Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China

Received August 10, 2006

Abstract—Tea caffeine synthase (CS) is a key enzyme in tea plant for synthesis of caffeine. We firstly system-
atically investigated the gene expression of CS. Northern blot analysis showed that CS gene was not expressed
in stems and roots but efficiently expressed in leaves of tea plants. The expression level of CS gene in summer-
grown leaves was much higher than in spring leaves. Its expression level in leaves of the shaded or fertilized tea
plants was significantly higher. Along the same shoot, CS gene was expressed at much higher level in the young
leaves (bud and the first leaf) than in the more mature second and third leaves. RNA in situ hybridization indi-
cated that tea CS gene was mainly expressed in the palisade parenchyma and the epicuticle of leaves but less
expressed in the spongy parenchyma and the hypoderm.
DOI: 10.1134/S1021443707050196

Key words: Camellia sinensis - tea caffeine synthase - gene expression - northern blot - RNA in situ hybridiza-
tion - tea leaves

INTRODUCTION CS in caffeine synthesis, in this study we systematically

Caffeine is a major and important purine alkaloid in
tea plant. In tea plants, 99% of caffeine is present in
leaves, its content being 2–5% [1, 2]. Caffeine is not
synthesized in stem, root, and cotyledon of tea plant [3],
but can be produced in tea flower buds [4–6]. Caffeine
synthase, comprising 3-N-methyltransferase (3-NMT)
and 1-N-methyltransferase (1-NMT) activities, cataly-
ses the final two steps of caffeine biosynthesis, when
two methyl groups are added successively to 7-meth-
ylxanthine to produce theobromine and then caffeine
[7, 8]. In 1976, Suzuki et al. firstly extracted the crude
tea caffeine synthase from fresh tea leaves [9]. Since
then, the research of tea CS has been one of the issues
in tea science due to the great health benefits of tea caf-
feine. Only in 1999, Kato et al. purified the enzyme and
characterized it [8]. The tea CS gene, a 1438 bp
sequence of cDNA, was also successfully cloned from
tea leaves [10]. In order to breed new tea species, which
produce low levels of caffeine to meet the increased
demand for low-caffeine tea, the studies on the biolog-
ical characteristics of tea CS were performed with the
use of new biotechnologies, such as antisense RNA
technology.
To characterize the relationship between caffeine
distribution and CS and further determine the role of
1 The text was submitted by the authors in English.
Abbreviations: CS—caffeine synthase; DIG—digoxigenin;
NMT—N-methyl transferase.
investigated the expression of tea CS gene.
MATERIALS AND METHODS
Plant materials. Fresh young leaves (the bud and the
first three leaves), stems (lignified twigs), and feeder
roots were collected from the plants of four tea cultivars
(Fuding Dabai Cha, Fuding Dahao Cha, Fuan Dabai
Cha, and Meizhan) in the experimental farm of Hua-
zhong Agricultural University. Fuding Dabai Cha is the
standard cultivar, whereas Fuding Dahao Cha, Fuan
Dabai Cha, and Meizhan are typical tea cultivars pro-
ducing high grade green tea, black tea, and oolong tea,
respectively. Fuding Dahao Cha has the great amount
of floss and high level of amino acids, especially thea-
nine, and it is suitable for producing of high-grade
green tea. Fuan Dabai Cha has the large leaves and high
level of tea polyphenols, and is selected to make black
tea. Meizhan is for making oolong tea, because it has
the hard and crisp leaves, thick epidermis, as well as
much water-extracted amino acids and aromatic con-
stituents in young stems, other than in leaves.
To compare the effect of cultivation conditions,
some tea plants were fertilized with the nitrogen fertil-
izer (46% N, Hubei Yihua Group, China) and shaded
with the sunshade net (Zhejiang Taizhou Sunshade Net
Factory, China). Fertilization and shading were carried
out prior to the leaf collection in summer. Nitrogen fer-
tilizer was once applied, and its rate was 5.8 kg/acre.
The extent of shading was 70%.

698
 EXPRESSION AND LOCATION OF CAFFEINE SYNTHASE IN TEA PLANTS
RNA isolation and northern blotting. Total RNA
from leaves, stems, and roots of tea plants was extracted
using the TRIzol® reagent according to the procedure
recommended by the manufacturer (Gibco-BRL, Ger-
many). Northern blotting was carried out as described
in [11]. Briefly, a 20 µg sample of total RNA was
loaded and separated on a 1.5% formaldehyde agarose
gel and blotted onto a nylon membrane (NYTRAN®,
Schleicher and Schuell, United States). The treated
membrane was further incubated with the α-32P-labeled
cDNA probe at 65°ë overnight and then washed under
high stringency condition. Tea CS gene expression in
different vegetative organs was firstly investigated.
Then, tea CS expression in leaves from tea cultivars
under various cultivation conditions was estimated.
RNA in situ hybridization and detection. The first
leaves of tea plants were fixed in 4% paraformalde-
hyde, dehydrated in ethanol series, cleared in xylene,
and embedded in paraffin as described by Drews [12].
Eight µm-thick sections were attached to slides coated
with polylysine hydrobromide. Digoxigenin (DIG)-
labeled antisense and sense RNA probes were tran-
scribed from either BamHI or XbaI-digested TCS
cDNA using either T7 (antisense) or T3 (sense) RNA
polymerase. The antisense RNA of 3'-untranslated
region of tea CS gene was used as the probe, and in situ
hybridization of tea CS mRNA was performed in leaf
sections on slides with the labeled RNA probe. Hybrid-
ization was carried out at 5°C overnight. Slides were
washed twice in 2 × SSC (standard saline citrate) at
room temperature, then in 1 × SSC and 0.1 × SSC at
57°C, each for 15 min. DIG-labeled RNA was detected
using an anti-DIG alkaline phosphatase conjugate
according to the manufacturer’s instructions (Boe-
hringer Mannheim, Germany). Nitroblue tetrazolium
(NBT) mixture with 5-bromo-4-chloro-3-indoyl phos-
phate (BCIP) were used with following addition of
polyvinyl alcohol for alkaline phosphatase indoxyl–
NBT reaction according to Block and Debrouwer [13].
After enzyme-catalyzed color reaction, an insoluble
blue precipitate was observed. Slides were visualized
and photographed under an Olympus BX60 micro-
scope (OLYMPUS, Japan) using a 100ASA color neg-
ative film.
RESULTS
Northern Blotting for Tea CS Gene
To investigate the expression of CS gene in vegeta-
tive organs of tea plant, the mRNA levels in leaves,
stems, and roots of cv. Fuding Dabai Cha were ana-
lyzed by northern-blot technique using a probe target-
ing the 3'-untranslated region of tea CS cDNA
(GeneBank accession no. AB031280). Figure 1a
showed no expression of tea CS gene in twigs and roots
but an effective expression in tea leaves. Tea CS gene
expression was manifested in the bud and first three
leaves of four tea cultivars (Fig. 1b). The expression of
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 54
699
tea CS gene in the bud and first three leaves in summer
was much higher than in spring (Fig. 1c). The expres-
sion level in the bud and first three leaves of shaded and
fertilized tea plants was much higher than in the
untreated plants (Fig. 1d).
The CS gene expression was compared also in the
young leaves of various ages in Fuding Dabai Cha tea
plants. Tea CS gene was expressed at a higher level in
the buds and first leaves than in the older second and
third leaves of the same shoot (Fig. 1e).
RNA In Situ Hybridization and Location of CS
in Tea Tissues
The results obtained by RNA in situ hybridization
technique are shown in Fig. 2. In the first leaves from
the plants of three cultivars tested, tea CS gene was
mainly expressed in the palisade tissue and the epicuti-
cle of leaves but less in the spongy tissue and the hypo-
dermis.
DISCUSSION
Besides tea, at least 60 plant species around the
world contain caffeine, and some of them contain a
high level of caffeine, for example, 1–2% of caffeine
was found in Coffea arabica, 0.03% in Theabroma
cacao, 1.5% in Cola acuminata, > 4% in Paullinia
capana, and 0.7% in Ilex paraguariensis [14]. On the
other hand, in tea plants most caffeine exists in tea
leaves, while in other plants most caffeine accumulates
in their fruits [14].
As almost all caffeine is found in tea fresh leaves, it
is believed that caffeine is synthesized only in tea
leaves but not in stems and roots [1–3]. It was found
that theobromine (a precursor of caffeine) exists only in
freshly collected leaves and a lot of 8-14C-adenine can
be transferred to theobromine and caffeine [15, 16]. It
was also stated that caffeine is present in the all tea
plants, its content being higher in tea buds and first
leaves than in second and third leaves; besides, it was
higher in summer than in spring and in fresh leaves
after plant shading and fertilization than without shad-
ing and fertilization [1–4]. According to the previous
data, we studied the expression of tea CS gene to under-
stand the role of tea CS gene. As the fertilization or
shading can increase caffeine level by changing the rate
of caffeine metabolism, the nitrogen fertilizer and sun-
shade net were used. Our results showed that tea CS
mRNA was not expressed in twigs and roots, but only
in leaves, its expression level being higher in younger
leaves, in summer, and in leaves of the shaded and fer-
tilized plants. It may imply that expression of tea CS
gene may greatly influence the flavor formation of tea
plants.
Our results confirmed that tea CS gene is mainly
expressed in palisade tissue of leaves. Tea CS existing
in chloroplasts might take part in the synthesis of caf-
feine. Kato et al. [17] isolated chloroplasts from tea
No. 5 2007
700 LI et al.

1 2 3 4 5 6 7

CS CS

rRNA rRNA
(a) (b)

8 9 10 11 12 13

CS CS
CS

rRNA rRNA rRNA

(c) (d) (e)
Fig. 1. Northern blotting.
The blots were probed with cDNA probe corresponding to tea CS gene (the upper panel). The lower panel is the rRNA for loading
control.
(a) Tea CS gene expression in cv. Fuding Dabai Cha plants. (1) Buds and first three leaves; (2) lignified twigs; (3) feeder roots.
(b) Tea CS gene expression in young leaves of four tea cultivars: (4) Fuding Dabai Cha; (5) Fuding Dahao Cha; (6) Fuan Dabai Cha;
(7) Meizhan.
(c) Tea CS gene expression in young leaves of cv. Fuding Dabai Cha. (8) Summer leaves; (9) spring leaves.
(d) Tea CS gene expression in young leaves of cv. Fuding Dabai Cha. (10) Leaves of shaded and fertilized plants; (11) leaves of
untreated plants.
(e) Tea CS gene expression in the leaves of different age on the same stem. (12) The bud and the first leaf; (13) the second and third
leaves.

pt pt
pt

(a) (b) (c)

Fig. 2. Analysis of tea CS mRNA expression by RNA in situ hybridization.

The samples of first leaves were collected from the plants of cvs. (a) Fuding Dabai Cha, (b) Fuding Dahao Cha, and (c) Fuan Dabai
Cha. The antisense RNA of 3'-untranslated region of tea CS gene was used as the probe. The dark blue coloration indicates tea CS
mRNA expression in the leaf sections. pt—palisade tissue. Magnification 156×.

leaves and determined the activity of 3-NMT, which
indicated that 3-NMT might be in chloroplasts.
Mosli et al. [18] believe that tea caffeine accumulates in
cell vacuoles, being mostly bound to chlorogenic acid.
Furthermore, some researchers believe that caf-
feine also is produced in tea flowers because 25% of
adenine in isolated stamens and petals was found to
be conversed into theobromine and caffeine [4–6].
On the other hand, five enzymes catalyzing the syn-
thesis of 5-phosphoribosyl-1-pyrophosphate, the
most important precursor for purine nucleosides, are
found in stamens and petals of tea flowers, and their

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 54 No. 5 2007

 EXPRESSION AND LOCATION OF CAFFEINE SYNTHASE IN TEA PLANTS
amounts are much higher than in the organs where
caffeine cannot be synthesized [6]. However, the
study on the activity of CS in tea flowers needs fur-
ther investigation.
ACKNOWLEDGMENTS
This project was supported by National Natural Sci-
ence Foundation of China, China Postdoctoral Science
Foundation (grant no. 20060390833) and Department
of Science and Technology of Hubei.
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