Plant & Cell Physiol.

13: 365-375 (1972)

Metabolic conversion of JV-methyl carbon of

T'-glutamylmethylamide to caffeine
in tea plants

Shigeki Konishi1, Michiko Ozasa and Eiichi Takahashi

Department of Agricultural Chemistry, Kyoto University, Sakyo-ku,

Kyoto 606, Japan

(Received September 25, 1971)

Tea seedlings were treated with 14C-methylamine to cause synthesis of 14C-j"-glu-

tamylmethylamide (Ar-methyl-'*C). The metabolic conversion of f-glutamylmethyl-
amide was studied by tracing 14C.
"C-f-Glutamylmethylamide (Ar-methyl-14C) translocated from roots and cotyledons
to shoots of tea seedlings, was converted almost entirely into caffeine. Conversion
was greater in light-exposed samples. For those grown in the dark, the converted
amount did not correspond to the total caffeine produced. More "C-f-glutamyl-
methylamide was present in stems than in leaves, but with l4C-caffeine, the opposite
was found.
When "C-f-glutamylmethylamide or 14C-methylamine was applied to leaf disks,
14
C-caffeine was biosynthesized from both substances.

The existence and synthesis of r-glutamylmethylamide (GMA) in tea plants

were radiochemically ascertained in our previous paper (/), which was the first to
report the existence of GMA in plants. Kung and Wagner (2) reported later that
this substance was synthesized as the early product in the metabolism of methyl-
amine by Pseudomonas MS.
r-Glutamylethylamide (theanine), which contributes to the taste of green tea,
has been shown to be specifically synthesized by tea roots (3) and translocated to the
shoot where iV-ethyl carbons are converted almost entirely to the polyphenol in
light. This conversion is repressed in the dark (4-7).
GMA, like theanine, is considered to contribute to the taste of green tea.
Therefore, it is very important to clarify its metabolism and physiological role in
the tea plant.
We investigated the metabolism of GMA in tea plants, with special reference
to the light to probe into its physiological significance.
Abbreviations: GMA, ^-glutamylmethylamide; 14C-GMA, uC-7--glutamylmethylamide (JV-
methyl-l4C).
1
Present address: Department of Agricultural Chemistry, Shizuoka University, Iwata, Shizuoka
438, Japan.
365

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 366 S. Konishi, M. Ozasa and E. Takahashi

Materials and methods

Plant material
Tea (Thea sinensis L.) seeds were sterilized in Uspulun solution for 4 hr, soaked
in running tap water overnight, seeded in washed sand and stored at 20°C in the
dark for 20 days. The seedling roots were approximately 10 to 20 mm long at
this stage.

Reagents
14
C-Methylamine hydrochloride (specific activity: 2.61 mCi/mmole) was
purchased from New England Nuclear Corp. 14 C-GMA (iV-methyl-14C) was
obtained from seedlings treated with 14C-methylamine, as reported in our previous
paper (1). 14C-GMA was separated by a one-dimensional paper chromatography
line system with n-butanol-acetic acid-water ( 4 : 1 : 1, v/v) as solvent. The radio-
active area was detected with a G. M. counter and eluted with distilled water.
The concentrate was further separated by the line system with phenol-water
( 4 : 1 , w/w, N£b). To obtain higher purity, the further separation was repeated
as above first procedure.

Application of radiochemicals and incubation

a) Tea seedlings T e n seedlings, 20 day old, were incubated in the dark at 20°C
for 3 days in the following medium: 14C-methylamine hydrochloride, 10 yttCi; sodium
glutamate, 10,umoles; 0.1 M phosphate buffer, pH 5.8, 1.4 ml; brought to a final
volume of 7.0 ml. These seedlings were called 0-day-old seedlings. They were
grown for 60 days on washed sand at 20°C under natural light or in the dark.
We have previously (1) confirmed that in 0-day-old seedlings, 14C-methylamine
is incorporated almost entirely into GMA. The seedlings were sampled on the
10, 20, 30, 40, 50 and 60th days. Three plants were harvested for each sampling
and divided into three sections; root ( + hypocotyl), cotyledon, and shoot.
Dark and shade treatment was simulated by covering the chamber with black
paper or by shading 75% of the chamber with a dark polyetylene-cloth.
b) Tea leaf disks Leaves of 82 day old seedling were cut into 1 X 0.5 cm disks
from the interveinal areas. Ten gauze bags were each filled with one g (fresh
weight) of leaf disks. Five bags each were treated with 14 C-GMA, 10,uCi or
14
C-methylamine, lO^Ci; 0.2 M potassium phosphate buffer, pH 5.8; in a total
volume of 30 ml.
The labelled compounds were made to infiltrate the leaf disks by putting the
samples in a vacuum desiccator for 3 min under reduced pressure. The disks were
then spread on wet filter paper in a petri dish and stored at 25°C in light (8000 lux).
Samples were taken after 0.5, 1.5, 3, 6, and 12 hr.
Analytical methods
a) Extraction and fractionation of ethanol-soluble materials T o study the translocation
and the metabolism of 14C-GMA during growth of tea seedlings, 14C-GMA and
other 14C-products were separated as follows: Each sample was sectioned into
small pieces and macerated in a mortar with 40 ml of 80% ethanol (v/v). The
ethanol-soluble fraction was separated with a centrifuge and the insoluble material

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 Conversion from GMA to caffeine in tea plants 367

washed twice with 30 ml 80% ethanol. The ethanol-soluble fraction was separated
into cationic and other fractions on a cationic exchange resin column (Amberlite
IR-120, H + form). The cationic fraction adsorbed on the column was eluted with
2 M NH^OH. The effluent was dried at 40°C under reduced pressure and dissolved
in 6 ml of water. The caffeine present was removed with 20 ml of chloroform.
Paper chromatography revealed that 14C in the chloroform-insoluble cationic
fraction was present almost entirely in GMA. 14C in the chloroform-soluble fraction
was combined with the "other fractions".
The ethanol extract containing 14C was also separated by two-dimensional
paper chromatography to investigate the metabolites of 14C-GMA. The filter
paper used was Toyo No. 51A (36x36 cm). The solvent system was similar to
that described by Kalberer (8).
b) Extraction and identification of caffeine Caffeine in tea was extracted and identified
microanalytically by paper electrophoresis and ultraviolet absorption spectrophoto-
metry as described by Torii and Ota (9, 10). Each sample of fresh tissue was cut
into small pieces, dried at 100°C for 20 min then at 60°C for 12 hr and placed in a
vacuum desiccator. The samples were macerated in a glass mortar. The powder
(0.2-0.5 g) was moistened with an equal weight of 5% sodium carbonate solution
and stored for 1 hr. Samples having dry weight below 0.2 g were fortified with a
commercial tea powder with known caffeine content. The samples were extracted
further with 20 ml of hot chloroform for 2-3 hr using a Soxhlet-extracter-like ap-
paratus.
Caffeine in the hot chloroform extract was separated by paper electrophoresis
(filter paper, Toyo No. 51A; 0.01 M sodium borate, 500-600 V, 0.3 mA/cm) for
3-4 hr. The position of caffeine was differentiated as a dark spot under ultraviolet
light after drying. Paper strips ( 2 x 3 cm) containing caffeine were cut out and
caffeine extracted with hot water (80°C) for 5 hr. Absorbancy of the solution was
measured with a spectrophotometer (Shimadzu QV-50) at 272.5 nm and 305 nm.
Absorbancy at 305 nm was taken to correct the value at 272.5 nm for impurities of
the filter paper. Estimated impurity (y) calculated from the equation below, was
subtracted from the absorbancy at 272.5 nm (10).

y = 1.63x+0.00361
y = absorbancy at 272.5 nm of impurity
x = absorbancy at 305 nm of impurity
c) Determination of radioactivity To determine 14C-radioactivity, samples of extracts
or eluates were plated onto metal planchets, dried and counted with a gas flow
counter (Ten SA-250).
Radioautograms of paper chromatograms and of electrophoresis were made
with Fuji X-ray film (Type KX). The paper strip containing 14C-caffeine was
immersed in liquid scintillation solution and counted with a liquid scintillation
counter (Nuclear-Chicago, Model 6804). The fluid contained 4 g of 2,5-diphenyl-
oxazole (PPO) and 0.1 g of l,4-bis-2-(4-methyl-5-phenyloxazolyl) benzene in one
liter of toluene. The value of radioactivity recorded was corrected to that measured
on the gas flow counter.

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 S. Konishi, M. Ozasa and E. Takahashi

Results
Metabolisvi of l4C-GMA in different parts of tea seedlings during growth
Fig. 1 shows the radioautogram of 14C in the ethanol-soluble fraction of shoots
grown in light for 60 days. High amounts of radioactivity were observed in GMA
and caffeine which was detected by ultraviolet absorption. This suggests that
the JV-methyl-14C of "C-GMA was metabolically converted to caffeine. Table 1
presents changes in amounts of 14C-GMA during various growing periods of tea
seedlings. Decrease of the cationic fraction and increase of "other fractions"
were observed with age; the increase being especially apparent in shoots grown in
light. l4C-Activity of the cationic fraction existed mainly in GMA. Little activities
were observed in two unknown substances. "Other fractions" were comprised
mostly of caffeine.

Conversion oj' HC-GMA to caffeine in growing lea shoots

Fig. 2 indicates translocation of l4 C-GMA synthesized in the root and the
cotyledon to the shoot and its subsequent transformation. The amount of 14C
translocated in light and the dark was nearly the same. Howerver, 80% 14C was
found in the hot chloroform-soluble fraction of 60-day plants grown in light and
only 20% in those grown in the dark. The hot chloroform-soluble fraction was

i
Fig. 1. Radioautogram distribution of "C-radioactivity on paper chromatogram
of 80% ethanol extract of tea shoots grown in light for 60 days< 1: GMA,
2: Caffeine. "C-GMA and 14C-caffeine were confirmed by co-chromato-
graphy of authentic substances. Solvents used: I, n-butanol-acetic
acid-water ( 4 : 1 : 1, v/v); II, ethanol-acetic acid-water (81 : 5 : 14,
v/v). For further details, see text.

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 Conversion IVom GMA to caffeine in tea plank 369

Table 1
Metabolism of xtC-CMA in different parts of growing tea seedlings"
GMA 4 Other fractions' Total 14C
Days Treatment Part (cpm/Shoot) (cpm/Shoot) (cpm/Shoot)
X102 XlO2 XlO2

0 Root 419 3 422

Cotyledon 432 29 461

Root 585 17 602

Light Cotyledon 195 25 221
Shoot 123 12 135
20
Root 465 15 479
Dark Cotyledon 251 27 278
Shoot 139 19 158

Root 364 7 371

Light Cotyledon 278 15 293
Shoot 163 119 282
40
Root 479 9 489
Dark Cotyledon 174 7 181
Shoot 198 28 229

Root 241 8 249

Light Cotyledon 30 12 42
Shoot 58 221 279
60
Root 299 9 308
Dark Cotyledon 95 17 113
Shoot 188 112 300
0
Seedlings were sampled at four periods.
* Cationic fraction. All 14C-activity of the chloroform-insoluble portion in the cationic frac-
tion existed mainly in GMA.
c
Ethanol-insoluble portion was not included in the "other fractions". For further explana-
tion, see text.

separated by paper electrophoresis. The distribution of 14C was determined by

scanning and by radioautogram as shown in Fig. 3. The position where ultraviolet
absorption was detected, coincided with the peak of radioactivity. Our findings
corresponded well with data for authentic caffeine. From these observations we
concluded that the JV-methyl carbon of GMA was used in the biosynthesis of caffeine.
The change in caffeine content in shoots of young growing tea plants is shown
in Fig. 4. With growth caffeine content increased gradually. Almost the same
amount was formed in light as in the dark, but after 40 days greater amounts were
formed in the dark. Twice as much caffeine per dry weight was formed in the
dark after 50 and 60 days, which agrees with previous studies (11, 12). Fig. 5 shows,
however, that the amount of 14C-caffeine converted from 14C-GMA, nearly equaled
the total amount of caffeine produced in the light, while in the dark, conversion was
very slight and never corresponded to the total content. Fig. 6, showing changes

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 370 S. Konishi, M. Ozasa and E. Takahashi

• CHCIs Extract
(x|04)
(Light] D E t 0 H Extract

8 3

t o
>
n
[Dark]
nn ill
Q o
2
2
r, n
10 20 30 40 50 60
DAYS
Fig. 2. Translocation of liC from roots and cotyledons to shoots and its
subsequent transformation. | : Hot chloroform extract. £]: 80%
Ethanol extract after hot chloroform extraction.

— Pole Pole

o
M
OS

•>••• '•>' I,
-15 -10 -5 +5 DISTANCE cm)

Sample

Mud i omit

Authentic

I U.V. Absorption

Fig. 3. Distribution of l*C-cajjeine separated by paper electrophoresis. Filter

paper: Toyo No. 51A. Solution: 0.01 M sodium borate. Voltage:
500-600 V. Current: 0.3 mA/cm. Time: 3-4 hr.

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 Conversion from GMA to caffeine in tea plants 371

I-

8
LLJ

0 10 20 30 40 50 60
' DAYS
Fig. 4. Changes in caffeine content of tea shoots during growth.

0 10 20 30 40 50 60
DAYS
Fig. 5. Conversion of UC-CMA to caffeine in tea shoots during growth.

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 372 S. Konishi, M. Ozasa and E. Takahashi

0 10 20 30 40 50 60
DAYS
Fig. 6. Changes in specific activity (cpmlmg) of l*C-cqffeine
in growing tea shoots.

in the specific activity (cpm/mg) of 14C-caffeine, clearly reveals that after 30 days,
when a difference in growth began to appear, light-exposed tea seedlings synthesized
more 14C-caffeine from 14C-GMA than those grown in the dark.
Very little caffeine was found in the roots and conversion of 14C-GMA to caffeine
was very slight. The cotyledon exhibited neither presence of caffeine nor synthesis
of 14C-caffeine.
Effects of shading on the conversion of UC-GMA to caffeine in tea stems and leaves
The effect of shading (75% and dark) on conversion of GMA to caffeine in
stems and leaves was studied. Table 2 presents the results. When samples were
grown in 75% shade or in the dark, the amount of caffeine per dry weight was higher
in stems and leaves than for those grown in the light. The brighter the light, the
higher the value of specific activity in both stems and leaves. The ratio of 14C-GMA

Table 2
14
Effects of shading on the conversion of C-GMA to caffeine
in tea stems and leaves (60 days)

Ur
Amount Caffeine Amount
. .>'.•. of /Dry of U C "C- 14
C- Sperific Caffeine
Treat- Part activity / c p m / g . \
ment weignt caff< . ine w e i g n t / Cpm \ GMA' Caffeine iD.Wt. j
(\ Shoot
mg \ I mg \ \Shoot/ (%) (%) (cpm/mg) (X10*)
(
ot / \ Shoot / '"'
Control Stem 124 0.18 0.15 30 71.0 29.0 4906 70
(Light) Leaf 290 3.10 1.07 404 14.2 85.8 11184 1196

75% Stem 101 0.22 0.22 35 82.0 18.0 4553 97

Shaded Leaf 183 3.38 1.85 361 17.3 82.7 8831 1634
Stem 83 0.87 0.71 147 80.0 20.0 3388 239
uarK
Leaf 122 3.42 4.10 386 40.1 59.9 6759 2773

80% Ethanol extract after hot chloroform extraction.

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 Conversion from GMA to caffeine in tea plants 373

cc

0 0.51.5 12

Fig. 7. Incorporation of l*C into caffeine from "C-GMA and

u
C-methylamine in tea leaf disks.

to 14C-caffeine was higher in stems than in leaves, while the percentage ot'14G-caffeine
was extremely high in the leaves compared to 14 C-GMA. Increased shading caused
greater amounts of 14C-GMA to remain and lesser amounts of 14C-caffeine to be
produced in both stems and leaves.
Our results may indicate that GMA is not very actively converted to caffeine
in the stem but is rapidly metabolized when translocated to the leaf. Therefore,
conversion of iV-methyl carbon of GMA to caffeine is strongly reduced by shading.
Role of GMA as a precursor for caffeine biosynthesis
Fig. 7 presents the time course of 14C-caffeine synthesis from "C-GMA or
14
C-methylamine. Synthesis increased with time and although nearly the same
amount was produced in 12 hr, synthesis from 14C-GMA was rapid while that from
14
C-methylamine, gradual. This indicates that GMA is a more favorable precursor
of caffeine than methylamine.

Discussion
Caffeine is a methylated xanthine with a purine ring. Previously it was
presumed that the biosynthesis of caffeine was analogous to that of uric acid. That
is, a purine ring is first synthesized, then in its catabolic process methyl groups are
attached to xanthine at the 7, 3 and 1 positions, respectively; thus proceeding via
7-methyl xanthine and theobromine to caffeine. Recently Ogutuga and Northcote
(13) reported that there are two pathways in the biosynthesis of caffeine: "Pathway
I " is a process from purine pool via xanthine, 3-methylxanthine and theophylline
to caffeine; and "Pathway I I " is from purine pool via nucleic acids, 7-methylguanylic
acid, 7-methylguanosine, 7-methylxanthosine, 7-methylxanthine, and theobromine
to caffeine. The latter is predominant in tea callus tissues. In these various
methylated compounds, the methyl donor is a methyl moiety of methionine as
suggested by Anderson and Gibbs (14) and Inoue and Adachi (15). Methylamine
(16-19), have been identified as other possible precursors but their mode of incor-
poration has not yet been clarified.

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 374 S. Konishi, M. Ozasa and E. Takahashi

We have demonstrated that the TV-methyl carbon of GMA contributes to

caffeine biosynthesis as a new precursor. Methylamine was also confirmed to be
a precursor, but GMA is presented as a more favorable one. Generally, a basic
substance such as methylamine would hardly be assumed to be present in vivo in
the free state. The methylamine that is produced, forms an amide like GMA, and
the amide gives its iV-methyl carbon to caffeine.
Others have stated that light does not affect the synthesis of caffeine because
seedlings or shoots of tea plants grown in the dark or shade exhibited greater ac-
cumulation and increase of caffeine when compared to those grown in light (77, 12).
This study supports this thesis because shoots of tea seedlings produced more caffeine
in the dark than in the light. Even on a dry weight basis more accumulation of
caffeine was observed in the dark-treated samples (Fig. 4, Table 2). However, in
light-exposed samples, the amount of 14C-caffeine synthesized from Ar-methyl-14C
of 14C-GMA increased correspondingly with the increase in total caffeine. In
dark-treated samples, little 14G-caffeine was synthesized (Fig. 5, Table 2), the
difference being apparent when specific activities (cpm/mg) of 14C-caffeine are
compared (Fig. 6, Table 2). This can be attributable to the following factors:
1) dilution effect caused by increased production of endogenous GMA in the dark,
or
2) supply of precursors for caffeine synthesis, produced in the dark from other
substances.
Since the former has been rejected experimentally (20), the latter could be
the reason for no corresponding production of 14C-caffeine and caffeine. That is,
other precursors and biosynthetic systems which promote caffeine synthesis in the
dark, are probably present.
When discussing the effects of light on caffeine synthesis, not only the total
amount of caffeine, but also compounds in the light-sensitive caffeine biosynthesis
pathway, should be considered. We found that light strongly influences the con-
version of iV-methyl carbon of GMA to caffeine.
We thank Mr. M. Hirano of the Research Institute of Tea Industry, Kyoto, for the gift of tea
seeds.

References
( 1) Konishi, S. and E. Takahashi: Existence and synthesis of L-glutamic acid j--methylamide
in tea plants. Plant & Cell Physiol. 7: 171-175 (1966).
(2) Kung, H. and C. Wagner: p-Glutamylmethylamide, a new intermediate in the metabolism
of methylamine. J. Biol. Chem. 244: 4136-4140 (1969).
(3) Konishi, S. and Z. Kasai: Synthesis of theanine from 14CCh in tea plants and sites of the
synthesis. J. Sci. Soil & Manure, Japan 39: 439-443 (1968).
(4) Konishi, S. and E. Takahashi: Degradation of theanine labeled with ethylamine-l-14C in
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( 5 ) Konishi, S. and E. Takahashi: Metabolism of 14C labeled theanine (iV-ethyl-14C) and its
metabolic redistribution in the tea plant, ibid. 40: 479^t84 (1969).
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 Conversion from GMA to caffeine in tea plants 375

( 8) Kalberer, P.: Untersuchungen zum Abbau des Kaffeins in den Blattern von Coffea arabica.
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861 (1963).
(19) Preusser, E.: Zur Biosynthese des Coffeins. Biologischts Zentralblatt 86: 485-494 (1967).
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