Mineral nutrition and caffeine content in coffee leaves

Abstract

The effect of nutrient supply on the caffeine content of coffee (Coffea arabica L.) leaves was investigated. Seeds were germinated in nutrient-agar media lacking N, P, K, Ca, Mg, S, Zn, B or Mo. The control treatment contained all essential nutrients. The caffeine concentration was determined seven months after seed sowing when the seedlings have 3 to 4 pair of leaves. The omission of K induced the highest caffeine content in the leaves (24.5 g.kg-1). Caffeine in the control treatment was 21.9 g.kg -1. Absence of P induced the lowest content, 17.5 g.kg-1.

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MINERAL NUTRITION AND CAFFEINE CONTENT IN COFFEE LEAVES 387
Bragantia, Campinas, 58(2):387-391, 1999
NOTA
MINERAL NUTRITION AND CAFFEINE CONTENT
IN COFFEE LEAVES
(1)
PAULO MAZZAFERA(2)
ABSTRACT
The effect of nutrient supply on the caffeine content of coffee (Coffea arabica L.)
leaves was investigated. Seeds were germinated in nutrient-agar media lacking N, P, K, Ca,
Mg, S, Zn, B or Mo. The control treatment contained all essential nutrients. The caffeine
concentration was determined seven months after seed sowing when the seedlings have 3 to
4 pair of leaves. The omission of K induced the highest caffeine content in the leaves (24.5
g.kg-1). Caffeine in the control treatment was 21.9 g.kg -1. Absence of P induced the lowest
content, 17.5 g.kg-1.
Index terms: caffeine, Coffea arabica L., mineral nutrition.
RESUMO
NUTRIÇÃO MINERAL E CONTEÚDO DE CAFEÍNA EM FOLHAS DE CAFÉ
O efeito do suprimento de nutrientes sobre o conteúdo de cafeína em folhas de café
(Coffea arabica L.) foi estudado. Sementes foram germinadas em meios nutrientes de ágar
deficientes em N, P, K, Ca, Mg, S, Zn, B ou Mo. O meio-controle continha todos os nutrien-
tes essenciais. A concentração de cafeína foi determinada sete meses após a colocação das
sementes nos meios, quando três a quatro pares de folhas haviam sido emitidos. A omissão
de K induziu o maior conteúdo de cafeína nas folhas (24,5 g.kg-1). O conteúdo do alcalóide
no tratamento-controle foi de 21,9 g.kg-1. A ausência de P induziu maior redução, sendo o
conteúdo de 17,5 g.kg-1.
Termos de indexação: cafeína, Coffea arabica L., nutrição mineral.
(1) Received for publication in January 27th 1999 and approved in July 7th, 1999.
(2) Departamento de Fisiologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Caixa Postal 6.109, 13083-970 Campinas
(SP). E-mail: pmazza@obelix.unicamp.br

P. MAZZAFERA388
Bragantia, Campinas, 58(2):387-391, 1999
Caffeine is the most abundant and important
alkaloid in coffee. Despite its taste, this alkaloid does
not contribute for more than 10% of the coffee bitter-
ness (Clifford, 1985). The importance of caffeine in
the coffee beverage seems to be solely as a stimulant,
which is the main reason for its popularity over the
centuries.
Although there is thousands of alkaloids in
nature some have economic importance because of
their physiological effects on humans. Consequently,
in view of commercial interests, many studies have
been carried out to investigate the factors that would
increase their contents in plant tissues and, especially,
in cell suspensions. For this purpose different types
of stress have been used such as osmotic stress
(Godoy-Hernández & Loyola-Vargas, 1991; Saenz et
al., 1993), salt stress (Brachet & Cosson, 1983) and
fungal elicitors (Baumert et al., 1991; Godoy-
Hernández & Loyola-Vargas, 1991).
Few studies have been carried out with caffeine
from coffee plants. Light, in amounts of 400 µmol.m-2.s-1,
induced caffeine accumulation in coffee cell suspen-
sions (Frischknecht & Baumann, 1985). Depending
on the size of the cell aggregate, NaCl can also cause
an increase on caffeine contents (Frischknecht &
Baumann, 1985). However, as often to most alkaloids,
coffee tissue culture or cell suspension does not ac-
cumulate caffeine in amounts comparable to those
found in plant tissues (Waller et al., 1983;
Frischknecht & Baumann, 1985). Therefore, it would
be interesting to learn more about the environmental
and agricultural influences on the caffeine contents
of coffee beans and leaves.
A coffee shrub usually takes 2 to 3 years to
produce the first fruits, fact that is quite inconvenient
for studying the effect of mineral deficiencies on the
caffeine contents of beans. Carelli et al. (1989) have
demonstrated the difficulties of growing coffee un-
der hydroponics untill a stage were the plant is pro-
ductive. On the other hand field experiments would
not permit full control of nutrient deficiencies. There-
fore, it would be interesting to evaluate caffeine varia-
tion in leaves as a response to mineral supply in seed-
lings growing in nutrient-agar medium.
Material and Methods
Ripe fruits of C. arabica cv. Catuaí Vermelho
were collected from a shrub growing outdoors in the
experimental plots of Universidade Estadual de
Campinas, State of São Paulo, Brazil. Under aseptic
conditions, they were surface-sterilized by immersion
in 80% ethanol for 5 min followed by washing with
sterile H2O and immersion in commercial NaOCl so-
lution (2% Cl) for 30 min. After extensive washing
with sterile H2O the fruits were manually depulped
and seeds were treated with 0.5 N NaOH for 30 min,
to remove the adhered mucilage. The fruits were
washed 10 times with sterile H2O and the excess H2O
was removed by laying the seeds on sterile paper filter.
Variations of the complete Hoagland solution
(Hoagland & Arnon, 1950) were used to induce defi-
ciencies. Solutions lacking N, K, P, Ca, Mg, S, B, Zn
or Mo were prepared with deionized H2O. As con-
trol, a complete Hoagland nutrient-agar was prepared.
The nutrient-agar media were prepared by boiling the
nutrient solutions containing 1% agar in a microwave
oven. Before the addition of agar and boiling, all nu-
trient solutions were passed through 0.2 µm filters.
The nutrient-agar media were distributed in 500 mL
flasks (100 mL per flask) in an aseptic chamber. When
NH4 was included, it was added as a filtered solution
concentrated 10 times after the agar temperature had
dropped to approximately 50oC.
Three selected seeds were placed in each flask
with the flat face down. The flasks were sealed with
parafilm and left in a growth chamber (temperature
20-25oC and photon flux density of ca 250 µmol.m-2.s-1)
until most of the seedlings produced 3 to 4 pair of
leaves. Five replicates were prepared for each treat-
ment and control.
For caffeine analyses, the leaves of the three
seedlings of each flask were collected and dried at
80oC. This occurred 210 days after transfer of the
seeds to the flasks. The leaves finely ground in
a mortar with pestle, extracted and analyzed by
reversed-phase high-performance chromatography
according to Mazzafera et al. (1994).

MINERAL NUTRITION AND CAFFEINE CONTENT IN COFFEE LEAVES 389
Bragantia, Campinas, 58(2):387-391, 1999
Results and Discussion
Two months elapsed before the emergence of
the radicles. Probably, this long period occurred be-
cause the endocarp was not removed (Válio, 1980),
and also because of the sterile conditions, which pre-
vented microbial action on this physical barrier. Usu-
ally radicle emergence in germinating coffee seeds
takes 15 to 20 days under non-sterile conditions.
At harvesting time, the leaves were smaller than
usually found on seedlings of the same age growing
in greenhouse or nursery. This might have occurred
because of the low light intensity of the growth cham-
ber compared to natural day light. On the other hand,
the light intensity of the growth chamber (250 µmol.
m-2.s-1) might have not interfered with caffeine bio-
synthesis. Frischknecht & Baumann (1985) observed
that a similar light intensity (400 µmol.m-2.s-1) induced
caffeine accumulation in coffee cell suspensions com-
pared to cells grown in the dark.
Except for the leaves of seedlings from the
treatment -N, which were slightly chlorotic, no typi-
cal visible symptoms of deficiency were observed.
This might have happened because there was not
enough time for the development of typical deficiency
symptoms. Because N in coffee is required in higher
amounts compared to other nutrients (Moraes &
Catani, 1964), it became limiting in the -N treatment,
and leaf chlorosis could be observed in young leaves.
The leaves did not differ in dry matter.
The caffeine content found in the leaves (Table
1) was higher than values reported in the literature
for C. arabica (Mazzafera & Magalhães, 1991). How-
ever, this would be expected since it has been dem-
onstrated that younger leaves have higher alkaloid
contents than older leaves (Frischknecht et al., 1986).
Although statistically similar to the control
treatment, omission of K induced the greatest increase
(12%) of caffeine contents in leaves. Except for Mo,
all other nutrient omissions led to lower values than
the control. Treatments -N and -P showed the lowest
contents.
There is no report in the literature regarding
the influence of mineral deficiencies on the caffeine
metabolism. Regarding the methyltransferases in-
volved in the caffeine biosynthesis it is not of our
knowledge any study indicating that these enzymes
are dependent on a specific ion. However, the oppo-
site occurs with the biodegradation route. Caffeine
degradation in fruits and leaves of coffee follows the
sequence: caffeine theophylline(1,3-dimethylxan-
thine) 3-methylxanthine xanthine uric acid
000allantoin -> allantoic acid -> urea + glyoxylic acid
-> NH4 + CO2. Urease, responsible for urea degrada-
tion, is dependent on nickel (Stebbins & Polacco,
1995), has very low activity in coffee fruits and leaves
(Vitória & Mazzafera, 1999), and is positioned in
the very end of the caffeine catabolism (Suzuki et al.,
1992). Vitória & Mazzafera (1999) observed that the
activity of xanthine oxidase in vitro assays with pro-
tein extracts from coffee fruits and leaves of C.
arabica was improved by addition of Mo in the reac-
tion mixture. However, here it was observed that the
accumulation of caffeine due to Mo deficiency did
not differ from the control treatment.
Table 1. Caffeine content in leaves of coffee seedlings
grown in nutrient agar media deficient in nutrients
-K................................ 24.5 + 0.1a 112
-Mo ............................. 22.5 + 0.3ab 103
Complete (control).... 21.9 + 0.3abc 100
-B ................................ 21.6 + 0.5a-d 98
-S ................................ 20.2 + 0.2bcd 92
-Zn .............................. 20.0 + 0.2bcd 91
-Ca .............................. 18.5 + 0.3bcd 84
-Mg ............................. 18.2 + 0.3cd 83
-N................................ 17.9 + 0.2cd 82
-P ................................ 17.5 + 0.3d 80
Treatments Caffeine(1) Increase/
/Decrease
g.kg-1 dry weight %
(1) Different letters indicate statistical significance by Duncan test (p
< 0.05).
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Bragantia, Campinas, 58(2):387-391, 1999
Lukaszewski et al. (1992) observed that boric
acid inhibited allantoate amidohydrolase in soybean
leaves, causing accumulation of allantoic acid. This
enzyme is involved in caffeine catabolism (Suzuki et
al., 1992). If the opposite is true, B deficiency would
cause decrease of allantoate and therefore, in caffeine.
However, the alkaloid level in the leaves of -B treat-
ment was similar to the control. At the same time,
allantoate amidohydrolase is dependent on manga-
nese, but this nutrient was not tested in the present
work.
Since alkaloids are N-containing compounds,
low caffeine content might be expected in the plants
of -N treatment. Although there is few exceptions,
several reports have shown an increase in alkaloid
content due to N fertilization (Waller & Nowacki,
1978). On the other hand, the response may vary de-
pending on the nutrient source (nitrate, ammonium
or urea) and the alkaloid type.
Regarding other nutrients, Cu amendments en-
hanced and Zn depressed nicotine contents in Nico-
tiana tabacum (Tso et al., 1973). B, Mo, Mn and Cu
amendments caused a decrease on alkaloid contents
in Lupinus species (Mironenko, 1965). In most cases,
depletion of potassium reduces alkaloid contents in
plants (Waller & Nowacki, 1978). However, in all
such cases it is not known the mechanisms leading to
alkaloid accumulation.
Recently, Yun et al. (1999) showed that K and
Mg fertilizer application increased caffeine in tea
leaves (Camellia sinensis). However, the experiment
was carried out in the field in a soil with low pH and
low organic matter, which may have also interfered
with the results.
To our knowledge only one investigation re-
ports on the effect of mineral nutrition on caffeine
contents of leaves and seeds of coffee (Rodriguez,
1961). The omission of Fe, Mn, B, Zn and Mo was
tested throughout one year in three-year old coffee
shrubs (C. arabica cv. Bourbon) by spray or soil fer-
tilization associated or not with gypsum or lime ap-
plication in the soil. Reduction in caffeine contents
(3.2%), although not statistically significant, was
observed in the seeds with gypsum omission and an
increase (17.5%) with Zn omission. The control plants
were sprayed with these micronutrients. The author
did not report any nutritional deficiency symptoms
probably because the experiment was carried out
during a short period and also because the nutritional
conditions of the soil may have interfered with the
results.
It is very likely that these interferences did not
occur in the present work. From our data it is not pos-
sible to speculate the way each nutrient affected caf-
feine metabolism. However, it is clear that caffeine
contents in coffee is leveled by a balanced mineral
nutrition. Considering that the metabolism of caffeine
is very similar in fruits and leaves of C. arabica
(Suzuki & Waller, 1984; Mazzafera et al., 1994;
Ashihara et al., 1996) it is possible that the responses
observed here might be observed also in fruits.
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