Acta Physiol Plant (2008) 30:401–405
DOI 10.1007/s11738-007-0132-4
SHORT COMMUNICATION
Caffeine affects adventitious rooting and causes biochemical
changes in the hypocotyl cuttings of mung bean (Phaseolus aureus
Roxb.)
Daizy R. Batish Æ Harminder Pal Singh Æ
Mansimran Kaur Æ Ravinder Kumar Kohli Æ
Surender Singh Yadav
Received: 23 October 2006 / Revised: 24 June 2007 / Accepted: 10 December 2007 / Published online: 9 January 2008
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2008
Abstract Caffeine (1,3,7-trimethylxanthine), a purine
alkaloid found naturally in over 100 plant species, has
recently been viewed as a safe chemical for management of
pests including molluscs, slugs, snails, bacteria, and as a
bird deterrent. It possesses phytotoxicity against plant
species, yet the mechanism of action is lacking. A study
was conducted to determine the effect of caffeine on the
rooting of hypocotyl cuttings of mung bean (Phaseolus
aureus) and the associated biochemical changes. At lower
concentrations (\1,000 lM) of caffeine, though rooting
potential was not affected, yet there was a significant
decrease in the number of roots and root length. At
1,000 lM caffeine, there was a 68% decrease in the
number of roots/primordia per cutting, whereas root length
decreased by over 80%. However, no root formation
occurred at 2,000 lM caffeine. Further investigations into
the biochemical processes linked to root formation
revealed that caffeine significantly affects protein content,
activities of proteases, polyphenol oxidases (PPO) and total
endogenous phenolic (EP) content, in the mung bean hy-
pocotyls. A decrease in rooting potential was associated
with a drastic reduction in protein content in the lower
rooted portion, whereas the specific activity of proteases
Communicated by J. Kepczynski.
D. R. Batish
M. Kaur
R. K. Kohli
S. S. Yadav
Botany Department, Panjab University,
Chandigarh 160 014, India
H. P. Singh (&)
R. K. Kohli
Centre for Environment and Vocational Studies,
Panjab University, Chandigarh 160 014, India
e-mail: hpsingh_01@yahoo.com
increased indicating that caffeine affects the protein
metabolism. Activity of PPO decreased in response to
caffeine, whereas EP content increased significantly indi-
cating its non-utilization and thus less or no root formation.
Respiratory ability of rooted tissue, as determined through
TTC (2,3,5-triphenyl tetrazolium chloride) reduction, was
impaired in response to caffeine indicating an adverse
effect on the energy metabolism. The study concludes that
caffeine interferes with the root development by impairing
protein metabolism, affecting activity of PPO (and thus
lignification), and EP content, which are the crucial steps
for root formation.
Keywords Cellular respiration
Protease activity

Protein content
Polyphenol oxidase activity

Total endogenous phenolic content
Introduction
Plants synthesize an array of bio-molecules as secondary
metabolites that exhibit a great chemical and structural
diversity. These are involved in a multitude of ecological
roles including plant defense mechanisms, vegetation pat-
terning, community structure and composition, and
allelopathy in the ecosystems and also act as crop protec-
tants (Singh et al. 2001). Among the different classes of
secondary metabolites, alkaloids constitute a large group of
nitrogen-containing compounds present in plants (Wink
2004). These play a variety of functions including defense
against insects, bacteria and fungi, and allelopathic activity
(Wink and Twardowski 1992). A number of alkaloids such
as aconitine, berberine, caffeine, scopolamine, gramine,
lupanine, hordenine, hyoscamine, theophylline, and
papaverine are allelopathic in nature and inhibit the seed
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germination, root and seedling growth of plants (Wink and
Twardowski 1992; Wink 2004). Levitt et al. (1984)
reported that tropane alkaloids like scopolamine present in
Datura stramonium inhibit the seed germination and radi-
cle growth of Helianthus annuus and alter root
ultrastructure. Liu and Lovett (1993) reported that alkaloids
gramine and hordenine present in barley (Hordeum vulgare
L.) reduce radicle growth of Sinapis alba, Stellaria media
and Capsella bursa-pastoris. Chen et al. (1995) reported
that alkaloid ellipticine inhibits the rooting in the mung
bean hypocotyls but the mechanism remains unknown.
Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid
found in about 100 plant species including coffee (Coffea
arabica L.) and tea (Camellia sinensis [L.] Kuntze)
(Ashihara 2006). Besides an important dietary component
for humans, it is biologically very active and possesses
fungicidal (Prabhuji et al. 1983), antiherbivoral (Hewa-
vitharanage et al. 1999), antimicrobial (Ibrahim et al.
2006), bird repellent (Avery et al. 2005) and mollusci-
cidal (Hollingsworth et al. 2003) activity. Further, it
imparts allelopathic behaviour to the donor plants,
thereby providing them selective advantage by suppress-
ing the surrounding competing vegetation (Friedman and
Waller 1983a, b). Wilkins et al. (1995) proposed caffeine
as a pesticide for insect control. Rizvi et al. (1981, 1987)
pointed that caffeine has herbicidal activity with species
selectivity. In fact, US Food and Drug administration
(USFDA) classified caffeine as GRAS (generally regar-
ded as safe) compound due to its water solubility and
species specificity (Hollingsworth et al. 2003). It can
prove to be an ideal agrochemical provided its mode of
action is understood. Ashihara and Crozier (2001) even
described caffeine as a less explored natural plant prod-
uct. Rizvi et al. (1987) reported that caffeine reduces the
activity of amylases in the germinating seedlings of
Amaranthus spinosus and gibberellic acid could not
counteract the inhibitory effect. Caffeine inhibits the
growth of rice and maize seedlings and the effect is more
severe on roots than on shoots (Smyth 1992; Anaya et al.
2002). It also inhibits mitosis and cell plate formation in
the root tips of Coffea arabica (Friedman and Waller
1983a). However, details about the inhibitory effect of
caffeine on the rhizogenesis process and the related
biochemical changes remain largely unknown. With this
objective in mind, a study was undertaken to find out the
effect of caffeine on the rooting potential of hypocotyl
cuttings of Phaseolus aureus (mung bean) and some
related biochemical changes (in terms of water-soluble
proteins, activities of proteases and polyphenol oxidases
and endogenous phenolic content). We selected P. aureus
as a target species, since its hypocotyls are widely used
as a bioassay material to study rooting response (Batish
et al. 2007).
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Acta Physiol Plant (2008) 30:401–405
Materials and methods
Caffeine (1,3,7-trimethylxanthine; purity 99%) was pur-
chased from Loba-Chemie, Mumbai, India. A stock
solution of 2,000 lM of caffeine was prepared in distilled
water and diluted to get working solutions of 50, 100, 200,
500, and 1,000 lM. All other chemicals were of reagent
grade and purchased from the best available sources. To
explore the effect of caffeine on the rooting of hypocotyls,
certified seeds of mung bean (Phaseolus aureus Roxb. var.
SML-32) were used. Mung bean seedlings were raised in
enamel trays under continuous cool day light fluorescent
tubes (Photosynthetic Photon Flux Density, PPFD =
approx. 180 lmol m-2 s-1) in an environmentally con-
trolled growth chamber (Caltan, Narang Scientific Works,
India). After 7 days, uniform-sized seedlings were selected
and their cotyledons removed. Cuttings were made under
water at nearly 3.5 cm below the cotyledonary node and
keeping the epicotylar portion intact. Ten mung bean
cuttings were dipped in 25 ml of respective caffeine
solution or distilled water (to serve as control). Five rep-
licates were maintained per treatment. Each replication
comprised 10 hypocotyls per vial and arranged in a ran-
domised block design (RBD) manner in the growth
chamber maintained at 25 ± 2°C, 72 ± 2% relative
humidity and a 16 h photoperiod (6.00 AM–22.00 PM) of
*180 lmol m-2 s-1 PPFD provided by cool white fluo-
rescent tubes and incandescent lamps. After a week, the
number of hypocotyls rooted and number of roots or root
primordia per hypocotyl was noted, and average root
length measured.
The lower hypocotylar region (approx. 1.5–2.0 cm;
LHR) of mung bean cuttings where roots formed was cut,
and used for estimation of protein content, activities of
proteases (EC 3.4.4.1) and polyphenol oxidases (EC
1.11.1.7; PPO), total endogenous phenolic (EP) content and
respiratory activity. For assaying enzyme activities, about
100 mg of the LHR was homogenized in 5 ml of 0.1 M
phosphate buffer (pH 7). The extracts were centrifuged at
15,000g for 25 min at 4°C rotor temperature in a Sigma
cold centrifuge and the supernatants were used for deter-
mining enzyme activities. Proteases were assayed using
casein (1%, w/v, in 0.1 M phosphate buffer, pH 6) as a
substrate following Basha and Beevers (1975). PPO
activity was assessed using catechol (0.01 M in 0.1 M
phosphate buffer, pH 6) as a substrate (Van Lelyveld and
Pretorius 1973). For each assay, there were five indepen-
dent replicated tissue samples. For determining protein
content, nearly 150 mg of LHR tissue was homogenized in
8 ml of distilled water followed by centrifugation at
15,000g for 20 min. Protein content was determined in the
supernatant using Folin–Ciocalteu reagent against a stan-
dard of bovine serum albumin (Lowry et al. 1951).
Acta Physiol Plant (2008) 30:401–405 403

Endogenous phenolic content was determined in the
LHR using Folin–Ciocalteu (Swain and Hillis 1959). LHR
(100 mg) was homogenized in 5 ml of water and filtered.
To 1 ml of extract 1 ml of Folin–Ciocalteu reagent (50%,
v/v) and 1 ml of 20% Na2CO3 were added. The mixture
was shaken and kept in the dark for 30 min and absorbance
of blue colour developed was read at 700 nm against a
standard of ferulic acid (25 mgl-1).
Respiratory activity was determined in the LHR by
using 2,3,5-triphenyl tetrazolium chloride (TTC) as per
Steponkus and Lanphear (1967). It is an indirect method of
measuring cellular respiration wherein TTC is reduced to
red formazan in the viable respiring cells and the absor-
bance is read at 530 nm. The values of treated samples
were expressed as % respiration with respect to control.
All the experiments and analyses were repeated. Since
the results obtained were reproducible (with \5% differ-
ence), the data presented is mean of both. The data were
analysed by one-way analysis of variance followed by
separation of treatment and control means using post hoc
Dunnett’s test at P \ 0.05.
Results and discussion
Results show that caffeine adversely affects the rooting
potential of hypocotyl cuttings of P. aureus. At lower
concentrations (50–500 lM) of caffeine, all hypocotyl
cuttings rooted like control, whereas the rooting potential
declined with increasing concentration (Table 1). At
2,000 lM caffeine, none of the P. aureus cutting rooted
thereby showing a complete suppression of root formation
the number of roots per cutting, and at 1,000 lM caffeine,
root number was lesser by *68%. There was a drastic
reduction in the average root length of hypocotyl cuttings
in response to caffeine (Table 1). At 50 lM caffeine
reduces average root length by nearly 42%, whereas it is
lesser by 87% at 1,000 lM (Table 1). These results indi-
cate that caffeine interferes with the rooting potential of
P. aureus.
The rooting of hypocotyl cuttings occurs after the
excision of primary root and involves enhanced meriste-
matic activity and cell division during the process of
rhizogenesis. A reduction or suppression of rooting in
mung bean hypocotyls in response to caffeine indicates an
interference with cell division. Though no specific studies
in this regard has been conducted, yet available reports
indicate that caffeine inhibits cell division and cell plate
formation in the root tips of Coffea arabica (Friedman and
Waller 1983a), Tradescantia spp. (Valster and Hepler
1997), Triticum aestivum (Thomas et al. 1997) and Allium
cepa (Gimenez-Abian et al. 1998).
To explore the mechanism of inhibition of rooting
process in the hypocotyl cuttings of P. aureus in response
to caffeine, some biochemical studies were undertaken.
Amount of water-soluble proteins decreased in the hypo-
cotyl cuttings in response to caffeine and the effect was
concentration dependent (Fig. 1). At 2,000 lM caffeine
exposure, there was a nearly 50% loss in protein content.
25 8
Total protein content
a
Protease activity
b
Total Protein Content (mg g−1 fresh tissue)

Proteases activity (µg h−1 mg−1 protein)

(Table 1). Although at lower concentrations of caffeine, 20 b b
there was no effect on the number of cuttings that rooted, 6
b
yet a drastic effect on the number of roots per cutting and
b
average root length was observed (Table 1). At 50 lM of b
15
caffeine treatment, there was a reduction of nearly 27% in b
b 4
Table 1 The effects of caffeine on the rooting potential of P. aureus
b b
hypocotyls 10 b
Concentration Cutting No. of roots Mean length
nbsp;(lM) rooted (%) per cutting of roots (cm) b
2
a a a a
0 (Control) 100 8.2 1.82 5
50 100a 6.0b (26.8) 1.06b (41.7)
a b
100 100 5.4 (34.2) 0.88b (51.6)
a b
200 100 4.6 (43.9) 0.78b (57.1)
500 100a 4.2b (48.8) 0.52b (71.4) 0 0
0 500 1000 1500 2000
a b b
1,000 96 (4.0) 2.6 (68.3) 0.24 (86.8) Concentration (µM)
2,000 0b (100) – –
Fig. 1 The effects of caffeine on total protein content and specific
Figures in parenthesis represent percent reduction over control activity of enzyme proteases in P. aureus hypocotyl cuttings.
Different letters represent significance from control at P \ 0.05 Different letters represent significance from control at P \ 0.05
applying Dunnett’s test applying Dunnett’s test

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The specific activity of enzyme proteases, on the other indicating its non-utilization for root cell wall formation. It
hand, increased with increasing caffeine concentration. At was evident from the results wherein EP content increased,
2,000 lM caffeine treatment, there was a nearly 3.6 times activity of PPO decreased and there was less or no rooting
increase in activity of proteases (Fig. 1) indicating an (Fig. 2). The decreased activity of PPO thus indicates an
enhanced protein hydrolysis. Caffeine also interacts with interference of caffeine in the root differentiation process
nucleic acids—RNA and DNA (Poltev et al. 2003) and probably by reducing lignin biosynthesis, though it was not
impairs the process of replication and translation (Wink determined in the present study.
and Twardowski 1992), and thus interferes with protein Further, the cellular respiration (measured through TTC
biosynthesis. The decreased amount of proteins could thus reduction) decreased in the hypocotyls in response to caf-
be due to both enhanced hydrolysis and decreased bio- feine treatment (data not presented). It decreased by *20%
synthesis. In other words, caffeine adversely affects protein over control at 50 lM caffeine, whereas at 1,000 lM, it
metabolism—an important step in the developmental was lesser by *88%. It is a known fact that TTC, a water-
biology. Not only the protein metabolism, but the activity soluble salt, converts into water insoluble red-coloured
of PPO, another important enzyme playing a key role in formazan in the metabolically active tissues with well
adventitious root formation, also decreased significantly in functioning electron transport chain. TTC reduction is thus
the hypocotyl cuttings in response to caffeine (Fig. 2). PPO reflective of cellular respiration besides tissue viability.
is a copper-containing enzyme localized in thylakoids of The decrease in respiratory ability indicates that tissue
plastids and catalyses the oxidation of phenolics into toxic could not cope with the caffeine-induced stress that is
substances quinines (Vaughn et al. 1988). It is also manifested in reduction/suppression of rooting in P. aureus
involved in defence mechanism of plants against environ- hypocotyl cuttings.
mental stresses (Thipyapong et al. 1995). Furthermore, The study concludes that caffeine affects the rooting
PPO plays a key role in the rhizogenesis and is involved in process in mung bean hypocotyls by impairing protein
the lignin biosynthesis during root differentiation by reg- metabolism, altering PPO activity and EP content, which
ulating the synthesis of phenolic compounds required for are the crucial steps for root formation.
lignin synthesis (Haissig 1986). In contrast to PPO activity,
EP content increased significantly in response to caffeine
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