© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Interrelationship between phosphorus toxicity and sugar metabolism in
Verticordia plumosa L
, Jaacov Ben-Jaacov
, Alexander Ackerman
, Asher Bar-Tal
, Irit Levkovitch
, Dvora Swartzberg
, Josef Riov
& David Granot
Institute of Soils, Water and Environmental Sciences,
Institute of Field
Phosphorus, an essential plant nutrient, may become toxic when accumulated by plants to high concentrations.
Certain plant species such as Verticordia plumosa L. suffer from P toxicity at solution concentrations far lower than
most other plant species. In this study, exposure of V. plumosa plants to a solution containing as low as 3 mg l
P resulted in signiﬁcant growth inhibition and typical symptoms of P toxicity. In a wide range of P levels studied,
Notably, tomato plants with high hexokinase activity due to overexpression of Arabidopsis hexokinase (AtHXK1)
exhibited senescence symptoms similar to those of P toxic V. plumosa. The resemblance in senescence symptoms
between P-toxic tomato plants and those with high hexokinase activity suggested that increased sugar metabolism
could play a role in P toxicity in plants. To test this hypothesis, we determined the amount of hexose phosphate, the
product of hexokinase, in V. plumosa leaves grown at various P levels in the nutrient solution. Positive correlations
were found between concentration in the medium, P concentration in the plant, hexose phosphate concentration in
leaves and P toxicity symptoms. Foliar Zn application suppressed P toxicity symptoms and reduced the level of
hexose phosphate in leaves. Furthermore, Zn also inhibited hexokinase activity in vitro. Based on these results we
suggest that P toxicity involves sugar metabolism via increased activity of hexokinase that accelerates senescence
The genus Verticordia (Myrtaceae) consists of about
150 species of the most spectacular plants of the
Western Australian ﬂora (Cochrane and McChesney,
1995). These are bushy shrubs under 2 m tall with
small leaves and 5-petal ﬂowers in white, cream, yel-
low,orange, pink and red. Verticordias are sold as cut
ﬂowers in the local and international markets, but des-
pite their popularity and attractiveness as cut ﬂowers,
most of the production comes from natural bush pick-
ing and not from culture. Little is known about the
response of Verticordia spp. to fertilizers, or about
their other nutritional demands (Burton et al., 1996).
In the last two decades efforts have been made in Is-
rael to cultivate new woody ﬂowers originated from
FAX No.: +972-3-9604017;
the southern hemisphere, mainly for the European cut
When grown in preliminary experiments in the
ﬁeld or on an artiﬁcial growth substrate Verticor-
dias were chlorotic and died shortly after planting.
Growth impairment and leaf necrosis or chlorosis of
Australian plants was attributed to phosphorus tox-
icity (Goodwin, 1983; Handreck, 1997; Nichols and
Beardsell, 1981; Parks et al., 2000).
Phosphorus is an essential nutrient for all living
organisms. High internal phosphorus concentrations
cause typical symptoms of P toxicity in many plant
species such as alfalfa (Parker, 1997), Arabidopsis
(Delhaize and Randall, 1995; Dong et al., 1998),
corn (Parker, 1997; Safaya, 1976), cotton (Cakmak
and Marschner, 1986, 1987; Marschner and Cakmak,
1986), okra (Loneragan et al., 1982), soybean (Parker,
1997), subterranean clover (Loneragan et al., 1979),
tomato (Jones, 1998; Parker et al., 1992; Parker,
1997) and wheat (Parker, 1997; Webb and Loneragan,
1988). P toxicity symptoms include growth inhibition,
interveinal chlorosis and necrosis of leaves, and ac-
celerated senescence. Although the mechanism of P
toxicity in plants is poorly understood, it is accepted
that P toxicity is associated with P–Zn interactions,
either in the soil or in the plant (Loneragan and Webb,
1993). Phosphorus–Zn interactions in the soil are far
better understood. High P concentrations may reduce
Zn availability because of: (i) precipitation of Zn–
P compounds (Barrow, 1987a, b; Lindsay, 1979);
(ii) enhancement of speciﬁc adsorption of Zn on the
charged surfaces of oxides and hydroxides follow-
ing increase in negative charges (Barrow, 1993); (iii)
decrease in Zn solubilization following decrease in ex-
cretion of organic acids (Bar-Yosef, 1996; Marschner
and Romheld, 1996); (iv) decrease in root hair dens-
ity (Bates and Lynch, 1996; Ma et al., 2001) and/or
changes in root system architecture (Liao et al., 2001;
Lorenzo and Forde, 2001; Williamson et al., 2001);
and (v) reduction in vesicular-arbuscular mycorrhizae
(Loneragan and Webb, 1993). Yet, P–Zn interactions
within the plant remain a complex, confusing and an
ambiguous topic (Loneragan and Webb, 1993).
Observations that increasing the Zn supply can
ameliorate P toxicity symptoms have led to the hy-
pothesis that high P levels raise the Zn requirement
of plants, a phenomenon described as ‘P-enhanced Zn
requirement’ (Loneragan and Webb, 1993; Marschner,
1995). Cakmak and Marschner, (1986, 1987) sug-
gested a mechanism of ‘P-induced Zn deﬁciency’,
whereby a high uptake of P induces Zn deﬁciency in
the shoots due to precipitation of Zn–P compounds
(Cakmak and Marschner, 1986, 1987; Marschner and
Cakmak, 1986). Formation of Zn–phytate and immob-
ilization of Zn in the roots, reported by Van Steveninck
et al. (1993), are consistent with the above mechanism.
Cakmak and Marschner (1987) suggested that Zn is a
crucial element in the feedback mechanism that con-
trols the uptake of P by the roots, and/or transport of P
from the roots to the shoots. They suggested that Zn
deﬁciency disrupts this control mechanism and pre-
vents P retranslocation in the phloem, causing toxic
accumulation of P in the leaves. Improving the Zn
nutritional status either by increasing the soil solu-
tion Zn concentration or through foliar application can
reduce the toxicity. Alternatively, overcoming P tox-
icity symptoms through Zn addition could result from
increased CuZnSOD (superoxide dismutase) activity
(Cakmak and Marschner, 1987, 1993; Marschner
and Cakmak, 1989). Furthermore, Zn has a distinct
role in maintaining membrane integrity as Zn deﬁ-
ciency increases the peroxidation of the plasma cell
membrane in the root, leading to leakage of nutri-
tional elements and organic metabolites (Cakmak and
Marschner, 1988a, b Parker et al., 1992; Pinton et
al., 1994; Welch et al., 1982). Membrane integrity
probably depends on the overall Zn status of the plant
and not on Zn levels in roots, since foliar application
has been found to correct membrane leak-
the physiological mechanism and the biochemical pro-
cesses by which high P concentrations impair plant
development, as well as the suppressive role of Zn in
P-induced toxicity remain obscure.
Tomato plants that overexpress Arabidopsis hexok-
(1999) to exhibit toxicity symptoms similar to those
of P-toxic V. plumosa plants. Hexokinase (HXK) cata-
lyzes the ﬁrst enzymatic step of sugar metabolism, i.e.,
the formation of hexose phosphates such as glucose 6-
phosphate (Glc6P) and fructose 6-phosphate (Fru6P).
In addition to its enzymatic activity, HXK is involved
in the regulation of photosynthesis, growth and sen-
escence in higher plants (Dai et al., 1999; Jang and
Sheen, 1997; Xiao et al., 2000). Overexpression of
AtHXK1 in tomato plants reduced photosynthesis, in-
hibited growth and accelerated senescence of mature
leaves (Dai et al., 1999). The fact that these transgenic
tomato plants exhibited similar senescence symptoms
to those of P-toxic plants led to the hypothesis that
increased HXK activity may play a role in P tox-
icity in plants. HXK activity is affected by P status:
P-deﬁcient bean roots and suspension-cultured Cath-
decreased hexose phosphate levels (Li and Ashihara,
1990; Rychter and Randall, 1994). To assess the hypo-
thesis that HXK is involved in P toxicity in V. plumose
L. plants, we examined the effects of solution-P con-
centration on P toxicity symptoms and on the level
of hexose phosphate. Because P toxicity symptoms,
particularly leaf-yellowing and shedding, are sim-
ilar to those induced by high levels of ethylene, we
also measured ethylene production in P-treated plants.
In addition, since Zn suppresses P toxicity, we ex-
amined the effect of foliar application of Zn on hexose
phosphate and on P toxicity in V. plumosa plants.
house (10% shade) in Bet Dagan, Israel (35
50 m altitude) during two consecutive years. One-
obtained from the ‘Nir Nursery’ in Israel were planted
on 6 June 1998 in 8-l plastic pots ﬁlled with perlite.
The experimental design comprised of ﬁve treatments,
allocated to ﬁve randomized blocks, each with six
plants. The treatments started on 1 August 1998, and
included ﬁve levels of P: 0, 1, 3, 10 and 30 mg l
to the nutrient solution. The nutri-
ent solution contained (mg l
) 100 N (NH
= 2:1) and 80 K in tap water containing 60 Ca, 25
Mg, 80 Na, 20 S, 150 CO
, 150 C1 and 0.1 P. The
ratio was slightly decreased or increased as
the pH in the leachates decreased below 5.5 or rose
above 6.5, respectively. Micronutrient concentrations
) applied were Fe 1, Mn 0.6, Zn 0.5, Cu 0.35,
(EC) of the nutrient solutions was 1.6
±0.2 dS m
and their pH was 7.5
used were NH
the evapotranspiration rate, with a 25% excess to leach
excessive salts from the root zone. Leachates from the
pots (with and without plants) were collected and the
volume monitored daily. The EC, pH values, and P and
other nutrient element concentrations in the leachates
were analyzed once a week. Transpiration was calcu-
lated from the difference between leachate volumes
of the containers with and without plants. Flowering
intensity was estimated by four persons, according to
a scale ranging from 1 (no open ﬂowers) to 5 (full
On 14 April 1999, all plants were pruned to a
height of 20 cm, measured from the rim of the bucket.
The shoots (including ﬂowers) were collected, washed
with distilled water, dried and stored for chemical ana-
lysis. After pruning, plants were irrigated for 2 weeks
with tap water and thereafter with the experimental
solutions described above. In November 1999 and in
April 2000 two plants per replicate were collected and
separated into ﬂowers (only on April 2000), leaves,
stems and roots. The plants were washed with dis-
tilled water, dried in a ventilated oven at 60
C for 1
material (DM) was ground to pass a 20-mesh sieve.
Samples (100 mg) were wet ashed with H
and analyzed for Na, K, organic-N and P. HC1O
ashing was used for Ca, Mg, and micronutrients
analysis. Organic-N and P were determined with an
injector Lachat Autoanalyzer; and K, Ca, Mg, Fe,
Zn, and Mn by ICP. Water-soluble Zn in the leaves
was determined according to Cakmak and Marschner
Effects of P concentration in the nutrient solutions on
hexose phosphate and ethylene production in the
Rooted cuttings of Verticordia plumosa L. were
planted on 21 March 2000 and irrigated with the same
nutrient solution as in experiment I. The treatments
started on 27 April 2000 and included ﬁve levels of
P: 0, ,1, 3, 10, and 30 mg l
added as H
the nutrient solution. Samples of young leaves were
analyzed for hexose phosphate after 10 days exposure
to the experimental solutions. No visual indications
of any stress or P toxicity symptoms could be detec-
ted at that time. Ethylene concentrations in mature
leaves were measured 8 days later, when the ﬁrst vis-
ible symptoms of leaf yellowing appeared in plants
exposed to P at 30 mg l
, and after 35 days exposure
of P toxicity appeared in plants exposed to P at 10 mg
The treatments began on 6 June 2000 and included
four levels of P: 0, 1, 3, and 10 mg l
foliar applications (3 ml l
every week) of commer-
5 M NH
, or a solution con-
taining 6 M NH
+ 1 M CO(NH
Ethylene production and the concentration of hexose
phosphate in mature leaves were measured on the 35th
day, when clear symptoms of P toxicity appeared in
plants exposed to P at 10 mg l
Protein extraction from leaves of HK4 transgenic to-
mato plants that overexpress Arabidopsis HXK (Dai
et al., 1999) was carried out as described in Schaffer
and Petreikov (1997). Approximately 1 g of a mature
solution (mg l
fresh leaf material was extracted twice with 400 ml of
10 mM KCl, 3 mM MgCl
, 1 mM phenylmethylsulf-
0.2% polyvinyl polypyrrolidone). The mixture was
centrifuged for 30 min at 12 000 g, 4
C, and, fol-
was combined with three volumes of 80% ammonium
sulfate and incubated at 4
C for 15 min. After a second
C, the pellet was resus-
7.5, 1 mM EDTA and 1 mM DTT) and desalted on a
G-25 Sephadex column (55
× 11 mm).
Hexose kinase activity was measured by the
enzyme-linked assay according to Schaffer and Pet-
reikov (1997). The assays contained 100 µl of protein
extract or 0.375 U of yeast HXK (Roche), in a total
volume of 0.5 ml 30 mM Hepes–NaOH (pH 7.5),
3 mM MgCl
, 0.6 mM EDTA, 9 mM KCl, 1 mM
dehydrogenase (G6PDH, from Leuconostoc). For the
assay of glucose phosphorylation, the reaction was ini-
tiated with 10 mM glucose. Reactions were carried
out at 37
C and absorption at 340 nm was monitored
in similar manner using 0.02, 0.1, 0.2, 0.4 or 1 mM
. As a control, HXK activity was measured
in the medium and only minor
Glucose, fructose, Glc6P and Fru6P in Verticordia
leaves were extracted and assayed spectrophotomet-
rically as described by Tobias et al. (1992) with minor
modiﬁcations. Brieﬂy, 1 g fresh weight of leaves was
homogenized with 3 ml of 5% (v/v) HClO
11 000 g for 5 min at 4
C, and 1 ml of the supernatant
of 3 M KOH was added. The salt was precipitated by
centrifugation at 12 000 g for 5 min at 4
C and the
supernatant was transferred to a new tube. Active char-
coal was added until a clear supernatant was obtained
following centrifugation as above. Glc6P, Fru6P, gluc-
ose (Glc) and fructose (Fru) were measured spectro-
photometrically by the enzyme-linked assay (Tobias
et al., 1992). The level of Glc was measured fol-
lowing phosphorylation with commercial yeast HXK
(Roche). Subsequent addition of phosphoglucose iso-
merase (PGI) that converts Fru6P into Glc6P allowed
measurement of Fru.
Ethylene production in excised mature leaves of V.
ated for several hours in sealed test tubes at 25
in the dark. At the end of the incubation, 1-ml gas
and ethylene in the samples was measured with a gas
chromatograph equipped with an alumina column and
a ﬂame ionizing detector.
Data were subjected to analysis of variance (ANOVA)
by the GLM procedure (SAS-User’s Guide, 1985).
Model parameters were ﬁtted by the NLIN procedure
of SAS, using the DUD routine of SAS (SAS-User’s
Growth and dry weight of V. plumosa during the ﬁrst
year was increased with increasing phosphate concen-
tration up to 1–3 mg l
. However, irrigation with
resulted in a sig-
and low yield (Figure 1). The plants exhibited typical
symptoms of P toxicity such as chlorosis of new leaves
and necrosis of older ones. With time, the older leaves
abscised, and leaves remained only on the upper parts
of the stems. Finally, the whole plant was defoliated
and after 3 months treatment with 30 mg P l
The root/shoot ratio (g g
) decreased dramatic-
(Figure 2). The
is consistent with other published results (Lynch and
of P concentration in the nutrient solutions. Vertical bars represent
the standard errors (not shown where their size is smaller than the
of Verticordia plumosa and on ﬂowering intensity
Signiﬁcant at P
≤ 0.001, respectively.
Data from the second harvest, April 00.
Qualitative evaluation of ﬂowering stage: (1) no open ﬂowers to
(5) full opening stage.
Brown, 1997; Marschner, 1995; Plaxton and Carswell,
1999), yet the decrease observed for Verticordia was
remarkably steeper than those for other plant species.
Whereas the dry matter weight (DM) of shoots, roots
and ﬂowers increased during the second year with in-
creasing P from 0 to 1 mg l
, and decreased at higher
gressively delayed as P increased (only data for the
last sampling date are presented in Table 1).
Effect of P application on leaf P and other nutrient
Leaf-P concentration in young plants increased signi-
ﬁcantly with increasing levels of solution P (Figure 3).
Leaf concentration in plants exposed to 10 and 30 mg
in the nutrient solutions. Vertical bars represent the standard errors
(not shown where their size is smaller than the symbols).
10 g kg
DM (Jones, 1998; Marschner, 1995; Parker
et al., 1992). A signiﬁcant quadratic regression was
obtained between shoot weight and shoot-P concentra-
tions in the ﬁrst year (Figure 4). Based on the quadratic
equation, maximum shoot weight was achieved when
shoot-P concentration approached 3.7 g kg
al., 1991). However, V. plumosa plants reached that
value at a calculated solution concentration of 4.6 mg
. Plant growth was impaired above this shoot-P
value, and yield fell to zero as shoot-P concentration
approached 9 g kg
Although P affected the concentration of some
concentrations of all nutritional elements were within
the appropriate range for normal plant growth (Jones
et al., 1991; Marschner, 1995). Hence, contrary to the
traditional ‘P-induced Zn deﬁciency’ theory (Cakmak
and Marschner, 1986, 1987; Marschner and Cak-
mak, 1986), no inverse relationship could be detected
between leaf Zn concentration and leaf P concentra-
tion or P application. Even at the uppermost, youngest
part of shoots, the most sensitive part to Zn deﬁ-
ciency, Zn concentration was always above 50 mg
concentrations in leaves of Verticordia plumosa at harvest
≤0.001; ns – not signiﬁcant.
tration at the end of the ﬁrst year (April 1999). Symbols represent
experimental results. The plotted line was calculated according to
the equation: DM= 77 + 893X –3793X
. The standard errors of the
P toxicity symptoms in V. plumosa were similar to
those of tomato plants with high HXK activity re-
ported by Dai et al. (1999). To test whether V.
centrations as a function of leaf-P concentration. Vertical bars
represent the standard error of each treatment (not shown where
their size is smaller than the symbols). The standard errors of the
regression parameters were: (a) 8.1 and 0.69, respectively; (b) 12.6
and 0.02, respectively.
ity, we measured the levels of phosphorylated and
nonphosphorylated glucose (product and substrate of
HXK, respectively) in V. plumosa plants exposed to
various P levels in the solution. The level of phos-
phorylated glucose (Glc6P) increased and that of non-
phosphorylated glucose (Glc) decreased as a function
of leaf P (Figures 5a and b, respectively). We did
not measure HXK activity in protein extracts because
we could not obtain satisfactory protein extracts from
Verticordia leaves. Ethylene production in V. plumose
leaves also increased as a function of P concentration
(Figure 6). in accord with earlier ﬁndings in which
overexpression of Arabidopsis HXK in tomato plants
caused increased production of ethylene (Granot et al.,
tration. Vertical bars represent the standard error of each treatment
(not shown where their size is smaller than the symbols). The
standard errors of the regression parameters were 0.36 and 0.03 2,
Effects of Zn on P toxicity and ethylene production
Foliar Zn treatments were shown to prevent P toxicity
symptoms (Loneragan and Webb, 1993; Marschner,
1995). To assess the effect of Zn on P toxicity, V.
with Zn. As expected, foliar application of Zn did not
affect plants exposed to low P levels (below 3 mg l
plants exposed to P at 10 mg l
. The latter became
than control plants (Figure 7). The concentrations of
none of the nutrients were affected by the foliar Zn
application, all of which remained similar to those
presented in Table 1, except for of the Zn concen-
tration in the youngest leaves, which increased from
±4 to 90±24 mg kg
DM. This increase, however,
might have resulted from external Zn contamination.
Since P toxicity is associated with increased produc-
tion of ethylene, we examined the effect of Zn on
ethylene level. As expected, Zn treatment reduced
ethylene production in plants exposed to 10 mg P l
Zn was reported to inhibit the activity of mammalian
HXK (Canesi et al., 1998). Since we could not obtain
satisfactory extracts of protein from V. plumosa, we
extracts prepared from tomato plants overexpressing
of Zn on yeast HXK. In both plant and yeast HXKs,
low concentrations of Zn (<1 mM) signiﬁcantly in-
hibited HXK activity (Figure 9). To ﬁnd whether Zn
also reduces the level of hexose phosphate in vivo, we
measured the level of Glc and Glc6P in V. plumosa
following foliar application of Zn. Plants sprayed with
Zn had lower levels of Glc6P and increased levels of
Gic, especially at high leaf-P concentration (Figure
10). These results are in accord with the visual symp-
toms of P toxicity and ethylene production (Figures 7
Vertical bars represent the standard error of each treatment (not
shown where their size is smaller than the symbols).
Figure 9. HXK activity in yeast and transgenic tomato plant overex-
pressing Arabidopsis HXK (AtHXK1) as a function of Zn concen-
tration. Vertical bars represent the standard error of each treatment
(not shown where their size is smaller than the symbols).
glucose (Glc6P) and glucose (Glc) concentrations. Vertical bars rep-
resent the standard error of each treatment (not shown where their
size is smaller than the symbols).
P toxicity in V. plumosa plants was clearly demon-
strated here by the signiﬁcant relationships found
between growth parameters, such as dry weight pro-
duction and ﬂowering intensity, and P levels in the
irrigating solution and in leaves (Table 1, Figures 2–
4). These relationships highlight the crucial role of
P in Verticordia development. The concentrations of
all other elements were within the appropriate ranges
for normal plant development, and no signiﬁcant cor-
relations were found between growth parameters and
concentrations of elements other than P, neither in the
irrigation solution nor in the plant tissues. Total Zn and
water-soluble Zn concentrations in Verticordia leaves
at all studied P levels were within the range considered
adequate for optimal growth, and signiﬁcantly higher
than the critical deﬁciency levels of 15–25 (Marschner,
1995; Welch, 1995) or 5–7 mg kg
ﬁndings in tomato and A. thaliana showing typical
P toxicity symptoms even in plants containing nor-
mal concentrations of total Zn or water-soluble Zn in
their tissues (Delhaize and Randall, 1995; Parker et
al., 1992). Therefore, in these cases the mechanism of
‘P-induced Zn (or any other micronutrient) deﬁciency’
could not account for P toxicity.
P toxicity-like symptoms, such as growth inhibi-
tion and accelerated senescence, were previously ob-
served in tomato plants that overexpress AtHXK1 (Dai
et al., 1999). These symptoms were correlated with
HXK activity and with the level of hexose phosphate
in the plant. The resemblance between senescence
symptoms observed in transgenic tomato plants over-
expressing AtHXK1 and P toxicity symptoms in V.
with Zn deﬁciency, as observed in this and other stud-
ies (Delhaize and Randall, 1995; Parker et al., 1992),
gave rise to the hypothesis that P toxicity is related
to sugar metabolism rather than to a nutritional dis-
order. Indeed, similar to tomato plants that overexpress
AtHXK1, V. plumosa plants grown at high solution
P levels had higher levels of hexose phosphate and
reduced levels of nonphosphorylated hexoses.
It has been shown that HXK activity and hexose
levels are affected by P level in the plant. P-deﬁcient
bean roots and suspension cultured Catharanthus ros-
trations of hexose phosphate and increased concentra-
tions of hexose (Li and Ashihara, 1990; Rychter and
Randall, 1994). These changes were reversed by the
addition of P. Phosphorus taken up by plants is used to
form ATP, either by photo-phosphorylation or by other
biochemical reactions. Indeed, increased levels of Pi
and ATP have been observed in suspension-culture of
and Ashihara, 1990). ATP is a substrate of HXK and is
required for HXK activity. Possibly, P raised the level
of ATP in Verticordia plants and consequently accel-
erated the activity of HXK. However, it is plausibly to
assume that in addition to sugar metabolism described
above, other possible modes or sites of action were
affected by P nutrition as well.
Zn treatments that suppressed P toxicity reduced
the level of hexose phosphate in V. plumose. Zn in-
hibits the activity of mammalian HXK (Canesi et
al., 1998) and causes ATP hydrolysis by modulat-
ing the activity of yeast pyrophosphatase (Schlesinger
and Coon, 1960). It is possible that Zn acceler-
ates hydrolysis of ATP by modulating the activity of
pyrophosphatase in Verticordia plants and as a con-
sequence decreases HXK activity. Alternatively, Zn
may interfere directly with HXK activity, as demon-
strated for tomato and yeast HXKs (Figure 9). HXK
requires Mg as a co-factor, and Zn, being a divalent
cation, may compete with Mg and thereby inhibit
HXK activity. Phosphorus and Zn appear to have op-
posite effects on HXK: whereas P increased HXK
activity in suspension culture of Catharanthus roseus
(Li and Ashihara, 1990) and raised the level of hex-
ose phosphate in V. plumosa plants (Figure 5), Zn,
at in planta physiological concentration (1–2 mM),
decreased HXK activity (Figure 9) and reduced the
level of hexose phosphate. Hence we suggest that Zn
suppressed P-toxicity symptoms by inhibition of HXK
High P levels signiﬁcantly increased ethylene pro-
duction in Verticordia leaves (Figure 6), whereas Zn,
which suppressed P toxicity, reduced ethylene produc-
tion (Figure 8). There are conﬂicting reports on the
effect of high P levels on ethylene production. High
P levels in the incubation medium inhibited ethyl-
ene production in tomato fruit slices, an effect that
was particularly pronounced during ripening, when
ethylene production was high (Chaluz et al., 1980; So-
bolewska and Plich, 1986). In one study, application of
P at 0.1 M signiﬁcantly increased ethylene production
in apple disks (Sobolewska and Plich, 1986), though
in a similar experiment no increase was observed for
the same concentration in apple and avocado disks
(Chaluz et al., 1980). In olive branches, application
of P at 75 mM through the base of cut stems enhanced
ethylene production, but apparently independently of
P-induced leaf abscission (Yamada and Martin, 1994).
Phosphorus-toxicity symptoms observed in V.
also characteristic of plant responses to high ethylene
levels (Abeles et al., 1992). Reduced ﬂowering intens-
ity at high P levels might also be a response to elevated
levels of ethylene, which have been shown to inhibit
ﬂowering in short-day plants (Abeles, 1967; Reid and
Wu, 1991). A number of studies have suggested that
ethylene mediates plant responses to stress conditions,
including nutrient stress (Lynch and Brown, 1997;
Morgan and Drew, 1997). However, at present, it is not
clear whether the elevated levels of ethylene at high
solution P levels play a role in the development of P
toxicity symptoms. Yet, the fact that increased ethyl-
ene production in V. plumosa was observed at early
stages of P toxicity symptoms suggest that ethylene
may play a role in this process.
The positive correlation between the levels of hex-
ose phosphate, ethylene production and the severity of
P toxicity symptoms (Figures. 1, 7a, and 8, respect-
ively) suggests association between sugar metabol-
ism and ethylene production. Transgeni tomato plants
that overexpress AtHXK1 also produce more ethyl-
ene (Granot et al., unpublished result). However, in
all these cases, a cause and effect relations between
sugar metabolism, ethylene production and the pheno-
type have not been demonstrated. Interaction between
glucose and HXK activity on one hand and ethylene
signal transduction on the other hand was found for
Arabidopsis plants (Zhou et al., 1998). It is possible
that tissue P levels affect these interactions as well.
Many crops, such as alfalfa, corn, cotton, to-
mato, wheat, soybean and cotton exhibit P toxicity
symptoms (Cakmak and Marshner, 1986; Marschner
and Cakmak, 1986, 1987; Parker, 1997; Webb and
Loneragan, 1988; Safaya, 1976). However, unlike
30 mg l
. Yet, whereas the other species are fostered
in culture at high (30 mg l
) P-levels, V. plumosa is a
P level was probably a limiting factor that shaped its
evolution. It is possible, therefore, that V. plumosa ac-
quired a very efﬁcient mechanism to absorb P, which,
in addition to the known mechanisms described above,
may involve an efﬁcient HXK activity and sugar meta-
bolism. Supplementing V. plumosa with ‘normal’ (30
) P levels resulted in high HXK activity, which
led to inhibited growth and accelerated senescence.
The authors are grateful to Dr. B. Bar-Yosef, Prof.
U. Kafkaﬁ, Dr. E. Delhaize and anonymous reviewers
for critical reading of the article and their construct-
ive comments. This paper is contribution No. 605/01
(2001 series) of the Agricultural Research Organiza-
tion of the Volcani Center. This work was supported
by Binational Agricultural Research and Development
(BARD) grant IS-2894-97 and by Binational Science
Foundation (BSF) grant 97-00250.
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