Highly efficient uptake of phosphorus in epiphytic bromeliads
Uwe Winkler
1,
* and Gerhard Zotz
1,2
1
Functional Ecology Group, Universita¨t Oldenburg, D-26111 Oldenburg, Germany and
2
Smithsonian Tropical Research
Institute, Balboa, Panama
Received: 29 August 2008 Returned for revision: 22 September 2008 Accepted: 17 October 2008 Published electronically: 25 November 2008
† Background and Aims Vascular epiphytes which can be abundant in tree crowns of tropical forests have to cope
with low and highly intermittent water and nutrient supply from rainwater, throughfall and stem flow. Phosphorus
rather than nitrogen has been suggested as the most limiting nutrient element, but, unlike nitrogen, this element
has received little attention in physiological studies. This motivated the present report, in which phosphate uptake
kinetics by leaves and roots, the subsequent distribution within plants and the metabolic fate of phosphate were
studied as a step towards an improved understanding of physiological adaptations to the conditions of tree
canopies.
† Methods Radioactively labelled [
32
P]phosphate was used to study uptake kinetics and plant distribution of phos-
phorus absorbed from bromeliad tanks. The metabolism of low molecular phosphorus metabolites was analysed
by thin-layer chromatography followed by autoradiography.
† Key Results Uptake of phosphate from tanks is an ATP-dependent process. The kinetics of phosphorus uptake
suggest that epiphytes possess effective phosphate transporters. The K
m
value of 1
.
05 mM determined for leaves
of the bromeliad Aechmea fasciata is comparable with values obtained for the high affinity phosphate transporters
in roots of terrestrial plants. In this species, young leaves are the main sink for phosphate absorbed from tank
water. Within these leaves, phosphate is then allocated from the basal uptake zone into distal sections of the
leaves. More than 80 % of the phosphate incorporated into leaves is not used in metabolism but stored as phytin.
† Conclusions Tank epiphytes are adapted to low and intermittent nutrient supply by different mechanisms. They
possess an effective mechanism to take up phosphate, minimizing dilution and loss of phosphorus captured in the
tank. Available phosphorus is taken up from the tank solution almost quantitatively, and the surplus not needed
for current metabolism is accumulated in reserves, i.e. plants show luxury consumption. Young, developing
leaves are preferentially supplied with this nutrient element. Taken together, these features allow epiphytes the
efficient use of scarce and variable nutrient supplies.
Key words: Epiphytic bromeliads, phosphorus uptake, forest canopies, luxury consumption, phytotelms, plant
nutrition, Aechmea fasciata.
INTRODUCTION
Vascular epiphytes form a highly diverse group of plants
which are especially common in humid tropical forests
(Benzing, 1990). Even in these habitats, which are character-
ized by abundant absolute precipitation, growth in tree
crowns lacking soil is equivalent to highly intermittent water
and nutrient supply. A suite of anatomical, morphological
and physiological adaptations allows epiphytes to cope with
this irregular resource supply, a particularly remarkable adap-
tation being the ‘tank’ found in many bromeliads, an impound-
ing structure formed by overlapping leaf bases. On the one
hand, such an impoundment improves the supply of nutrients
by acting as a catchment area for water and organic debris;
on the other hand, catchment fluids prolong the time available
to take up the very same water and nutrient resources. In the
extreme case, this uptake is achieved exclusively by special-
ized leaf trichomes, whereas the function of roots is reduced
to that of non-absorbing holdfasts (Benzing, 2000).
Although alleviating the resource limitations in the epiphy-
tic habitat, available evidence indicates that in situ many if not
most individual tanks may fall dry for extended periods of time
(Zotz and Thomas, 1999). Moreover, tank bromeliads gener-
ally show low contents of nutrient elements (Stuntz and
Zotz, 2001) and grow very slowly even under near-optimal
conditions (Hietz et al., 2002; Schmidt and Zotz, 2002).
These are all typical features of stress-tolerant plants associ-
ated with nutrient-poor habitats (Grime, 2001). In this group
of plants, nutrient uptake capacities are normally tuned
towards the capture of short pulses, and such a combination
of high uptake of nutrient elements and slow potential
growth frequently leads to an accumulation of reserves
(termed ‘luxury consumption’, Chapin, 1980).
Most of the information on the nutrient ecology of epiphytic
plants is related to nitrogen. A number of studies suggested
that organic nitrogen forms such as amino acids or urea are
more important for the nutrition of tank bromeliads than for
terrestrial plants (Benzing, 1970; Nyman et al., 1987; Endres
and Mercier, 2001; Endres and Mercier, 2003), and a recent
study by Inselsbacher et al. (2007) reported detailed uptake
kinetics of various nitrogen compounds. For example, tanks
of Vriesea gigantea showed a marked preference for the
uptake of NH
4
þ
compared with NO
3
–
, the uptake rates of
glycine being intermediate. Uptake of most nitrogen com-
pounds followed Michaelis–Menten kinetics, and the* For correspondence. E-mail u.winkler@uni-oldenburg.de
# The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Annals of Botany 103: 477–484, 2009
doi:10.1093/aob/mcn231, available online at www.aob.oxfordjournals.org

estimates of the Michaelis–Menten constants (K
m
) were in the
range of those of roots of terrestrial plants, indicating an effec-
tive uptake system for NH
4
þ
.
This relative wealth of information on nitrogen uptake in
vascular epiphytes contrasts with the few data on the uptake
of other important nutrient elements such as phosphorus (see
Benzing, 1970; Benzing and Renfrow, 1980). However, in
many tropical forests, phosphorus rather than nitrogen limits
productivity, and this seems also to be true for many vascular
epiphytes. For example, there is evidence that phosphorus is
most limiting for reproduction in some bromeliads (Benzing,
1990; Zotz and Richter, 2006). Similarly, a major role for
phosphorus in vegetative function is also indicated by higher
resorption efficiencies and proficiencies (sensu Killingbeck,
1996) for phosphorus than for nitrogen during leaf senescence
in a large suite of epiphytes (Zotz, 2004), or by a sharp,
10-fold, decrease in the N : P ratio of field-grown bromeliads
when fertilized in the laboratory (Benzing and Renfrow,
1974). Hence, the present study had a 2-fold goal. First, the
hypothesis that epiphytic bromeliads show highly efficient
uptake of phosphorus was tested with a detailed analysis of
the uptake kinetics of phosphorus in leaves and roots.
Secondly, the metabolic fate of phosphorus within the plant
was investigated as an additional step towards a more mechan-
istic understanding of phosphorus metabolism.
MATERIALS AND METHODS
Plant material
The main part of this study was carried out with Aechmea fas-
ciata ‘Primera’ plants (Bromeliaceae) that were kindly sup-
plied by a commercial nursery (Corn. Bak B.V., Asseldelft,
The Netherlands). An additional experiment was conducted
with other bromeliads, Vriesea splenriet and Vriesea duvali-
ana (also from Bak B.V.), and two species collected in tropical
lowland forests in Panama: Tillandsia elongata and Werauhia
sanguinolenta. All plants had 6–8 leaves, were 8–10 cm high
and had a tank volume of approx. 1 mL. In Aechmea, only the
youngest fully developed leaf has immediate contact with the
tank water by its completely rounded and overlapping leaf
bases. Older leaves are less connected to the tank solution.
Plants were kept in the greenhouse at 25 8C, at a relative
humidity of approx. 40 % and a light dark regime of
12 : 12 h, being illuminated by natural sunlight, supplemented
with artificial light (400 W metal halide lamps, master HPI-T
plus; Philips, The Netherlands) when necessary to achieve a
photosynthetic photon flux density of at least 150–180 mmol
m
–2
s
–1
at the level of the plants.
Plants received a nutrient solution containing 28 mg L
–1
NO
3
–
,65mgL
–1
NH
4
þ
,63mgL
–1
PO
4
3–
,63mgL
–1
K
þ
and 13 mg L
–1
Mg
2þ
once a week, and were otherwise irri-
gated daily with water. Before the onset of experiments,
plants were not fertilized for 14 d.
Phosphorus uptake
Experiments were performed in the radionuclide laboratories
of the University of Oldenburg. During experiments, plants
were kept in Plexiglas boxes under conditions comparable with
those in the greenhouse. In experiments lasting up to 24 h,
plants were illuminated during the complete experiment. Before
starting the uptake studies, tanks were thoroughly rinsed with
the phosphate buffer used later in the experiments. For the exper-
iments, carrier-free [
32
P]phosphoric acid (Hartmann Analytic,
Germany), containing 37 MBq in 100 mL of water (initial
specific activity: 5
.
5 MBq nmol
–1
), was used. Phosphate (P
i
)
uptake was measured as
32
P depletion from tank solutions.
The activity of the
32
P in the tank solution was adjusted to
25  10
6
dpm by addition of 0
.
1–1
.
0 mL from the radioactive
preparation to 0
.
5 mL of unlabelled phosphate buffer, pH 6
.
1.
In all mixtures, buffer capacity was sufficient to maintain the
pH value. Final concentrations of 0
.
1–50mM P
i
were used in
experiments to measure the concentration dependence of P
i
uptake in Aechmea, and 100 mM P
i
in experiments to analyse
the distribution of P
i
in the plant tissue. We compared the
uptake rates of five different bromeliads at a concentration of
10 mM P
i
. This concentration is regarded as an average value
of naturally occurring P
i
concentrations available to bromeliads
from rainwater; throughfall and stem flow waters, and in tanks
of epiphytes, ranging from 2
.
6to24mM P
i
(Benzing, 2000;
Richardson et al., 2000). For kinetic measurements, samples
were taken every 30 min and uptake rates were calculated
from the initial linear phase of
32
P depletion. Experiments
were performed at a constant tank volume of 0
.
5 mL. Before
sampling, plants were weighed, and weight loss was compen-
sated by adding water. After correcting the tank volume, tank
fluids were mixed with Pasteur pipettes and aliquots of 5 mL
taken at regular intervals, put into liquid scintillation vials and
mixed with 8 mL of scintillation cocktail (Lume gel save,
Lumac, Germany). Measurements were done in a Wallac
1415 liquid scintillation counter, supplied with an external stan-
dard to calculate dpm rates. Furthermore, in one set of experi-
ments, carbonyl cyanide m-chlorophenylhydrazone (CCCP) at
a concentration of 20 mM was used as an ATPase inhibitor to
separate active and passive uptake (Dahlman et al., 2004). In
this experiment, tanks were pre-incubated with inhibitor and
phosphate buffer for 1 h before starting the measurement of
phosphate uptake.
In experiments lasting for .2 d, counting rates were cor-
rected for the half-life of
32
P. In one set of experiments,
uptake rates of the roots were measured in a similar way,
except that incubation solutions were placed into an
Erlenmeyer flasks. Plants were tied above the flask in such a
way that only the roots came into contact with the radioactive
incubation solution. At a wide range of P
i
concentrations
applied to tanks, the uptake rates were not dependent on the
ratio of labelled/unlabelled phosphate.
Cumulative uptake of phosphate from the tanks was calcu-
lated according to eqn (1):
uptake (nmol) ¼ PSðC Vol
Tank
 PS=V
C
=A
total
Þð1Þ
where PS is nmol phosphate in the tank, C is the counting rate
in dpm, Vol
Tank
is the tank volume in mL, V
C
is the volume
used for measuring the counting rate in mL and A
total
is the
sum of activity in the incubation solution in dpm.
Rates were calculated on a whole-plant basis for time
kinetics of P
i
uptake or on a dry weight (d. wt) basis for
Winkler & Zotz — Phosphorus uptake by bromeliad tanks478