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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 Caffeine in the control treatment was 21.9 -1. Absence of P induced the lowest content, 17.5
Bragantia, Campinas, 58(2):387-391, 1999
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 Caffeine in the control treatment was 21.9 -1. Absence of P induced the lowest
content, 17.5
Index terms: caffeine, Coffea arabica L., mineral nutrition.
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 O conteúdo do alcalóide
no tratamento-controle foi de 21,9 A ausência de P induziu maior redução, sendo o
conteúdo de 17,5
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:
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
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).
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
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 dry weight %
(1) Different letters indicate statistical significance by Duncan test (p
< 0.05).
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
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
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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Objective: This research aims to analyze the ability of robusta coffee leaves fraction extract to inhibit the growth of Staphylococcus aureus and Escherichia coli and also determine the minimum inhibitory concentration (MIC). Methods: Antibacterial activity evaluated by the disc diffusion method observed in four types of fraction of extract robusta coffee leaves (n-hexane, ethyl acetate, ethanol, and water). Each extract divided into three various concentrations, 5%, 10%, and 15%. Determination of antimicrobial activity in vitro by the disk diffusion method. Results: Ethyl acetate fraction of coffee leaves extract produced the largest diameter zone of inhibition of bacterial growth compared to other extraction fractions of 17.28 mm in E. coli and 18.58 mm in S. aureus. The MIC of coffee leaves extract fraction water, ethyl acetate, and n-hexane on E. coli and S. aureus is 5%, while the fraction ethanol MIC is 10%. Conclusion: The antibacterial effect of ethyl acetate fraction of coffee leaves extract showed an antibacterial effect that was better than the fraction of n-hexane, ethanol, and water.
... Koricheva et al. 1998). Low P supply was associated with a 20% reduction in N-based foliar caffeine concentrations in coffee seedlings (Mazzafera 1999), but had no effect on the concentration of alkaloids in tobacco leaves (Andrade et al. 2013), or on pyrrolizidine alkaloids in ragwort (Vrieling and van Wijk 1994). Most work on P nutrition effects on N-based defences has been on the agriculturally important cyanogenic crop sorghum, where consistent with the negative correlation between foliar P and CNglyc concentrations here, higher P supply is associated with decreases in shoot CNglyc concentrations (e.g. ...
Plant nutrition can affect the allocation of resources to plant chemical defences, yet little is known about how phosphorus (P) supply, and relative nitrogen (N) and P supply, affect chemical defences, especially in species with intrinsically conservative nutrient use adapted to P-impoverished soils. Waratah (Telopea speciosissima (Sm.) R.Br.), like other Proteaceae, is adapted nutrient-poor soils. It was identified as having cyanogenic glycosides (CNglycs) throughout the plant. T. speciosissima seedlings were grown for 15 weeks under two N and P concentrations. CNglycs (N-based defence) and nutrients were quantified in above- and below-ground organs; foliar carbon (C)-based phenolics and tannins were also quantified. CNglyc concentrations in roots were on average 51-fold higher than in above-ground tissues and were affected by both N and P supply, whereas foliar CNglyc concentrations only responded to N supply. Leaves had high concentrations of C-based defences, which increased under low N, and were not correlated with N-based defences. Greater root chemical defence against herbivores and pathogens may be important in a non-mycorrhizal species that relies on basal resprouting following disturbance. The differing responses of secondary chemistry in above- and below-ground organs to P and N demonstrate the importance of broadening the predominantly foliar focus of plant defence studies.
... Minerals such as phosphorus and magnesium are very important in various enzymatic processes throughout the plant, and they have large roles in promoting plant development and growth. Studies examining the effect of increases in phosphorous and magnesium have had contradictory results in regards to the effect on alkaloids, ranging from no effect to both positive and negative effects (Waller, 1978;Bramble and Graves, 1991;Mazzafera, 1999). A study that examined the effect of Phosphorus omission on P. microphyllus found no effect on the pilocarpine content; however, the authors did not look at the effect on the overall alkaloid profile (Avancini et al., 2003;Abreu et al., 2005). ...
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Studies examining the diversity of plant specialized metabolites suggest that biotic and abiotic pressures greatly influence the qualitative and quantitative diversity found in a species. Large geographic distributions expose a species to a great variety of environmental pressures, thus providing an enormous opportunity for expression of environmental plasticity. Pilocarpus, a neotropical genus of Rutaceae, is rich in alkaloids, terpenoids, and coumarins, and is the only commercial source of the alkaloid pilocarpine for the treatment of glaucoma. Overharvesting of species in this genus for pilocarpine, has threatened natural populations of the species. The aim of this research was to understand how adaptation to environmental variation shapes the metabolome in multiple populations of the widespread species Pilocarpus pennatifolius. LCMS data from alkaloid and phenolic extracts of leaf tissue were analyzed with environmental predictors using unimodal unconstrained and constrained ordination methods for an untargeted metabolomics analysis. PLS-DA was used to further confirm the chemoecotypes of each site. The most important variables contributing to the alkaloid variation between the sites: mean temperature of wettest quarter, as well as the soil content of phosphorus, magnesium, and base saturation (V%). The most important contributing to the phenolic variation between the sites: mean temperature of the wettest quarter, temperature seasonality, calcium and soil electrical conductivity. This research will have broad implications in a variety of areas including biocontrol for pests, environmental and ecological plant physiology, and strategies for species conservation maximizing phytochemical diversity.
... Rev Bras Cienc Solo 2018;42:e0170057 Zeiger, 2010). Accordingly, Mazzafera (1999) observed that, under P and K absence, caffeine content in coffee (Coffea arabica L.) plant leaves was lower compared to the contents in plants grown in a complete nutrient solution. ...
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Catharanthus roseus (L) G. Don (Madagascar periwinkle) belongs to the Apocynaceace family and is widely spread throughout tropical and subtropical regions of the world. The plant produces several important alkaloids, such as ajmalicine and serpentine, which are used in the treatment of circulatory diseases. The potential of inoculation with arbuscular mycorrhizal fungi (AMF) and nitrogen fertilization to enhance the production of alkaloids was investigated in periwinkle. A greenhouse experiment was carried out to evaluate the effects of arbuscular mycorrhizal fungi and N fertilizer dosages on plant growth, production of ajmalicine, and nutrient content in roots. The concentration of ajmalicine was determined by reverse-phase high-performance liquid chromatography with UV detection. The experiment was designed in randomized blocks in a 4 × 4 factorial scheme with four microbiological treatments (control - without mycorrhiza; Claroideoglomus etunicatum; Rhizophagus intraradices; mixed inoculum - Rhizophagus clarus + Gigaspora margarita), and four N fertilizer dosages (15, 30, 60, and 120 mg kg-1) with four replications. Catharanthus roseus growth was higher when plants were inoculated with mixed inoculum (R. intraradices + G. margarita) and C. etunicatum. The mixed inoculum (R. intraradices + G. margarita) and C. etunicatum, combined with N fertilization, enhanced ajmalicine yield. Catharanthus roseus inoculated with mycorrhiza showed increased P absorption and reduced N content.
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São apresentados dados da variação na concentração de nitrogênio, fósforo, potássio, cálcio e magnésio no fruto do cafeeiro durante a sua formação, bem como das quantidades dos elementos absorvidos pelo fruto e da acumulação de matéria sêca em diversos estágios de seu desenvolvimento. A concentração dos elementos dosados foi sempre maior na flor que em qualquer outro estágio da maturação. O inverso ocorreu com a concentração de matéria sêca, em conseqüência da redução progressiva do teor de água no fruto. A acumulação de matéria sêca no fruto intensificou-se a partir do início do 4.° mês após o florescimento. O mesmo ocorreu com a absorção de nitrogênio, fósforo e potássio. Nôvo incremento na absorção de nitrogênio e potássio e no aumento do pêso sêco foi observado no 6º e no 7º mês após o florescimento. Nos dois meses que antecedem o estado final de maturação, o fruto do cafeeiro acumula ou elabora 43% do seu pêso sêco, absorvendo 49% do nitrogênio, 36% do fósforo e 39% do potássio, relativamente às quantidades que contém quando da maturação completa.
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High performance liquid chromatography has been used to measure the quantities of caffeine, theobromine, and theophylline in aqueous extracts of endosperm from immature and mature fruits of Coffea arabica and six other species of Coffea. Caffeine was the alkaloid present in largest amounts and, with one exception, in concentrations that were broadly similar in immature and mature fruit. The highest concentrations of caffeine were found in C. canephora at 35.1 and 24.5 mg g−1, respectively, in immature and mature endosperm. The lowest concentrations were in C. bengalensis, where caffeine was not detected in extracts from mature fruit. [8-3H]Caffeine was metabolised relatively slowly by immature endosperm of C. arabica and C. canephora. In contrast, C. dewevrei, C. eugenioides, C. stenophylla, C. salvatrix and C. bengalensis all appeared to metabolise [8-3H]caffeine much more rapidly, as the percentage recovery of the applied label was much lower and there was more extensive incorporation of radioactivity into theobromine, theophylline, 3-methylxanthine and two unidentified polar metabolites. The endogenous caffeine concentrations and the metabolism data indicate that there may be marked differences in the rate of turnover of caffeine in the various species of Coffea. Potential sources of material for the production of naturally decaffeinated coffee are discussed.
This chapter summarises the vast literature on the composition* of green coffee beans paying particular attention to those components which are peculiar to coffee. The corresponding data are given for roasted beans and where possible for soluble powders. Attention is focused on compositional factors that might be determinants of acceptability, and situations where the data are incomplete or contradictory with the intention of provoking thought, comment and further investigation.
Callus tissue culture of Coffea arabica L. cv Hybrido de Timor prepared from apical portions of orthotropic branches produced 49 to 92 times as much caffeine per unit weight of tissue as did the original explant. Cell-free extracts made from 42 to 54-day-old callus cultures in which active biosynthesis was occurring exhibited N-methyl-N 9-nucleoside hydrolase and N-methyltransferase enzyme activities. Similar cell-free extracts exhibited selective biodegradative activity in forming urea from xanthine. Biosynthetic substrate specificities are similar to those of the enzyme obtained from green coffee fruit and tea leaves, suggesting that callus cultures of C. arabica form caffeine in the same way as the coffee fruit and tea leaves.
The addition of Aspergillus niger homogenate to Catharanthus roseus cell suspension cultures produced an increment of more than 60% in the alkaloid content of two different cell lines. The use of an inhibitor of phenylalanine ammonia lyase, i. e. cinnamic acid, along with the homogenate, resulted in an appearance of 90% of the alkaloids in the medium. Furthermore, even in the absence of fungal homogenate, there was a marked increase in the alkaloid content. The exposure of the cells to an osmotic stress produced a marked increment (320%) in the total alkaloid content. When both stress treatments were applied sequentially, an additive effect on alkaloid accumulation was observed. It was 300% higher than in cells cultured without treatment, the majority of the alkaloids found in the medium.
Two dark-grown cell suspension cultures of Ruta graveolens L. were challenged with fungal elicitors. The treatment of cell line R-4 resulted in increased accumulation of hydroxy rutacridone epoxide, rutacridone epoxide and gravacridontriol. The latter alkaloid was not detectable in non-elicited cells. Another response was observed in cell line R-15. Besides rutacridone epoxide, for the first time rutacridone accumulation was stimulated either by a yeast, Phytophthora or Colletotrichum elicitor. Using cell line R-15, an elicitor-mediated induction of 5-adenosyl-L-methionine: anthranilic acid N-methyltransferase, N-methylanthranilic acid «activating» enzyme and rutacridone synthase was recorded. Enzyme activities were influenced by incubation time and amount of elicitor.
Reports that environmental stress may enhance the accumulation of secondary substances in plants led to the idea of introducing stress into tissue cultures with the aim of improving the in vitro production of pharmaceutically active compounds. The test was made with low- and high-producing cell suspension cultures of Coffea arabica. The production of the purine alkaloid caffeine was shown to be stimulated by stressors such as high light intensity and—depending on the culture type—high NACl concentration.
In buds and emergent leaflets of Coffea arabica formation of the purine alkaloids caffeine and theobromine was studied with the aim of characterizing the chemical defence strategy of a tissue with a high risk of predation. As long as the leaflets are fully covered by a resin layer and by two stipules, their alkaloid content varies between 1 and 3% dry wt. With leaflet emergence, the alkaloid formation increases and the variation decreases. Maximum content of about 4% is reached when the leaflets are fully open. In the subsequent developmental period alkaloid content decreases. A comparison between the investment in alkaloid formation with that in primary metabolic processes demonstrates that chemical defence is costly: influx of carbon atoms into caffeine is 15% of that into respiration. A defence strategy which is based on an antagonism between mechanical and chemical protection is discussed.
Datura plants were grown on a clayed support and subjected to salinity stress (153.8 mol m−3 NaCl) at the 6-leaf stage. Salt treatment increased total alkaloid content in young leaves. The results indicated that at the organ level tropane alkaloid accumulation was related to plant growth.