Journal of Experimental Botany, Vol. 47, No. 299, pp. 793-803, June 1996
Journal of
Experimental
Botany
Phosphate fluxes, compartmentation and vacuolar
speciation in root cortex cells of intact Agrostis capillaris
seedlings: effect of non-toxic levels of aluminium
A.E.S. Macklon1, D.G. Lumsdon, A. Sim and W.J. McHardy
Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB9 2QJ, UK
Received 13 July 1995; Accepted 8 March 1996
Abstract
Introduction
32
From compartmental analysis of P elution measurements, concentrations and fluxes of orthophosphate
were estimated for root cortical cells of intact seedlings of the indigenous grass Agrostis capillaris L. cv.
Highland when in complete nutrient solution containing 10 mmol m 3 or 100 mmol m 3 phosphate with
or without 3.7 and 37 mmol m 3 Al, respectively,
during loading and elution. When plotted as counts
min~ 1 remaining in the tissue as a function of time,
the data failed to meet the criteria for first order kinetics. Transformation of the data to meet the kinetic
criteria gave corrected values for compartmental concentrations and fluxes of phosphate, and estimates of
the otherwise unresolved slowly exchanging compartment within the vacuole considered to be the cause
of the discrepancy in flux analysis. In the control, the
discrepancy was considered due to sequestration of
phosphate with Ca in the vacuole and a small, but not
significant, increase in sequestered P occurred in the
presence of Al, the presence of which was confirmed
by X-ray microanalysis. A chemical speciation model
was used to demonstrate, for various values of pH and
carboxylic acid concentration, the possibility that
phosphate was precipitated in root cell vacuoles as
Ca and Al phosphates (hydroxy-apatite and variscite).
The ecological significance of the ability of A. capillaris
plants to make efficient use of scarce P resources by
minimizing the sequestration of P by Al in vacuoles,
compared with Lolium perenne, was recognized.
Key words: Phosphate sequestration, phosphate use
efficiency, X-ray microanalysis, super-saturation.
1
To whom correspondence should be addressed. Fax: +44 1224 311566.
© Oxford University Press 1996
The great majority of work with aluminium in plant
growth and nutrition studies is concerned with aluminium
toxicity (Luttge and Clarkson, 1992), but effects of Al
have been found, particularly on P uptake, exchange and
transport using levels similar to those observed in nutrient-poor mineral soils in the uplands of N.E. Scotland,
and at which no toxic symptoms were found (Macklon
et al., 1994a). In our work with Lolium perenne L. cv.
Premo (Macklon and Sim, 1992a, b) evidence was found
for the sequestering of P in root cell vacuoles in the
presence of Al so that a significant part of the resource
in the sward was effectively unavailable for nutrition.
This effect of Al could have considerable consequences
for the productivity of nutrient-poor natural grasslands
receiving no managed inputs.
In this paper, an extension of conventional compartmental analysis is employed (Cram and Laties, 1974;
Macklon et al., 1990; Macklon and Sim, 19926) to study
an indigenous grass species (Agrostis capillaris L. cv.
Highland) with the objective of understanding how low
levels of Al affect P uptake and efficiency of use where P
supply may be critical. In such soils, much of the soluble
P is in organic form (Shand et al., 1994a, b; Macklon
et al., 19946), but if this is hydrolysed by root surface
phosphatases before uptake, Al could have a similar effect
on the sequestration of P as found in L. perenne seedlings.
In earlier papers on Al effects on P sequestration in
vacuoles (Macklon and Sim, 1992a, b; Macklon et al.,
1994a), there has been speculation about the mechanisms
involved. In this paper, estimates have been made of
vacuolar pH, and concentrations of inorganic nutrient
and organic acid ions to reveal the possible speciation
effects leading to the solubility products of Al and Ca
794 Macklon et al.
phosphates being exceeded, resulting in precipitation. A
comparison between A. capillciris and L. perenne
(Macklon and Sim, 1992a, b) is made.
Materials and methods
Seeds of Agrostis capillaris L. cv. Highland were surfacesterilized in 0.5% peracetic acid for 5 min, after soaking the
seeds in sterile water for 1 h. The seeds were thoroughly rinsed
and set to germinate in the dark (day 0) in a controlled
environment at 20±l°C on Tygan mesh (Simkin Machin,
Sheffield) fixed to PTFE supports. The seeds (c. 100) were
grown aseptically in 2.0 dm3 extraction flasks closed with
crystallizing dishes, over 1.0 dm3 nutrient solution comprising
(molm' 3 ) KC1, 1.0; Ca(NO3)2, 1.0; MgSO4, 0.25.
Orthophosphate was added as NaH2PO4 to give 10 or 100 mmol
m~3 P. Trace elements were included as follows (mmol m" 3 )
Cu, 0.1; Zn, 0.77; Mn, 18.2; B, 1.85; Mo, 0.21; V, 0.20; Fe,
18.0. From day 4, the plants were illuminated (440 ^mol
m" 2 s"1 at plant level) by Philips slim 'white' fluorescent tubes
(TLD 36 W), using a 16 h light, 8 h dark regime. On day 14,
continuous light was given and continued throughout the
experimental period starting on day 15. The nutrient solution
was renewed on day 11 and on day 15 when 32P was introduced
at 925 MBqm" 3 in flasks containing plants for 32P uptake
experiments. Except in the controls, Al was added with the 32P.
The radioisotope was obtained as orthophosphate in dilute HC1
solution from Amersham International. Controls and solutions
containing Al were adjusted to and maintained at pH 4.3 to 4.6
by addition of HC1 as necessary.
Efflux experiments
Plants were loaded overnight (days 15-16) with 32P, and then
transferred unrinsed on their grids, each to a purpose-made
shallow-based funnel. This arrangement ensured that the roots
were totally immersed in 30 cm3 nutrient solution, containing
unlabelled phosphate, which could be easily drained off and
replaced. The funnels were mounted on a reciprocating shaker
(80 oscillations min"1) to ensure adequate washing of the roots.
Radioisotope was eluted from the roots over a period of 9 h
with numerous changes of washing solution, which became
progressively less frequent. The intervals are illustrated in Fig. 1.
This part of the experiment was carried out in a controlled
environment cabinet at 95% relative humidity and 20 °C, to
reproduce the conditions in which the seedlings were grown (in
closed vessels) and also to minimize the effects of transpiration
on transport. Changes of solution in the funnels required access
to drainage taps via a small port in the cabinet door, but
replacement solutions were dispensed from outside the cabinet
viaflexibletubing to minimize any disturbance of the conditions.
Further details can be found in Macklon (1975) and Macklon
and Sim (19926).
Four treatments, replicated three times, were used.
(1) Control plants loaded and eluted in nutrient solution
containing 10 mmol m" 3 P.
(2) Plants loaded and eluted in nutrient solution containing
10 mmol m~3 P and 3.7 mmol m" 3 Al (as A1C13).
(3) Control plants loaded and eluted in nutrient solution
containing 100 mmol m~3 P.
(4) Plants loaded and eluted in nutrient solution containing
100 mmol m" 3 P and 37 mmol m~3 Al.
Parallel sets of seedlings were used to determine continued 32P
uptake in the same conditions as the eluted plants, except that
Hours
Fig. 1. An example of treatments of elution data for roots of plants
loaded with 32 P in nutrient solution, containing 100 mmol m~ 3 P
(control) and eluted with the same solution unlabelled. (A) Original
data: log10 32 P tracer content as a function of time: a, intercept of
linear 'vacuolar' phase extrapolated to zero time. Inset: after subtraction
of the slowest exchange phase; a1, intercept of linear cytoplasm phase
at zero time. (B) log lo tracer efflux as a function of time; b, intercept
of vacuolar phase at zero time. Inset: after subtraction of the slowest
exchanging phase; b 1 , intercept of cytoplasm phase at zero time. (C)
Modified data: log, 0 32 P tracer content as a function of time, after the
deduction of the same value ('complexed 32 P') from each point to give
a rate constant similar to that for (B); c, intercept of vacuolar phase at
zero time. Inset: after subtraction of the slowest exchanging phase; c1,
intercept of cytoplasm phase at zero time. The rate constant (k) for
each linear phase is indicated. Legends on .y-axis also apply to respective
inset graphs.
the roots were bathed in 150 cm3 32P-labelled nutrient solution
amended in the same way as the elution solutions and changed
every 4 h.
Each set of seedlings was separated into roots and shoots,
ashed, and taken up in acid (Ron et al., 1988). The resultant
extracts and elution solutions were assayed for 32P content by
liquid scintillation spectrometry. After radioisotope decay, P
Phosphate fluxes and aluminium
795
and Al contents were analysed by ICP spectrometry. Values for
total concentrations of cations and anions given in Table 6 were
also determined by ICP spectrometry except chlorine which
was determined by a direct potentiometric method using hot
water extracts (Macklon, 1975).
complexes were included in the MINTEQA2 calculation
(Macklon et al.. 1994a). The results indicated whether the
vacuolar solution was super-saturated with respect to various
precipitated phases.
Statistics
Results and discussion
A one-way analysis of variance was carried out on the tabulated
data after transformation to natural logarithms, to stabilize the
variances. A r-test was applied by comparing the results of the
calculation n1— n2/sed to the tabulated values of t (sed =
standard error of the difference, «' and n2 = means of
treatments to be compared). The least significant difference
related to logged values, so the significant differences are
expressed by way of superscript letters, different letters indicating
significant differences between treatments. No superscript letters
are shown for ^(s" 1 ) values since they are related to t± values
by the factor 0.693/ft, and hence significant differences are the
same as shown for ti values.
Cell dimensions
Mean dimensions of 190 cortical parenchyma cells were
measured randomly in longitudinal sections by light microscope
in root systems of six A. capillaris seedlings. To compute the
volume occupied by each cell component (%) and the membrane
surface areas {g~l) it was assumed that cell wall and cytoplasm
each had a thickness of 0.5 ^m and that the cells approximated
right cylinders.
X-ray microanalysis
X-ray microanalysis was carried out on roots from two or three
plants of each treatment. Spectra were obtained for five cortical
cells in each of three specimens for seedlings treated with
37 mmol m " 3 Al in nutrient solution for 3 d. Spectra were also
obtained from root cortical cells in the control (no Al)
treatment. Micro-analysis was carried out on some roots treated
with 100 mmol m " 3 Al, in nutrient solution, for 4 d. All these
spectra were for vacuolar contents. Where cell walls, or
junctions thereof, were thick enough, spectra for walls were
obtained which excluded vacuolar and cytoplasmic contents.
Roots were thoroughly rinsed in deionized water before
0.5 cm segments were cut about 1.5 cm from the tip. The
specimens were mounted in a drop of water and frozen in liquid
nitrogen (—196.8 °C) and fractured for imaging in a Philips
scanning electron microscope. To obtain acceptable images and
spectra the surface of the fracture (uncoated) was etched by
raising the temperature of the cryostage temporarily to — 80 °C
from the working temperature of — 150 °C. The operating
voltage was 10 keV, the working distance 36 mm and the X-ray
resolution for Al was better than 3 fim for the conditions used
here. Spectra were obtained over a period of 100 s, after
allowing for a dead time which varied between 4% and 9%.
Speciation modelling
To model the speciation occurring in root cortex cell vacuoles,
in terms of equilibrium thermodynamics, information was
assembled on the total concentrations of all vacuolar inorganic
and carboxylic acid components, pH of the vacuolar sap and
complexation formation constants. The basis for estimates of
these values and the references for formation constants are
considered in the Results and discussion section. To facilitate
computations the computer program MINTEQA2 was used
(Allison et al., 1991). In addition to the aluminium organic
acid complexes the formation of soluble aluminium phosphate
Fluxes and compartmentation
Figure 1 illustrates the graphical analysis of data from a
typical elution experiment. In the conventional compartmental analysis (Fig. 1A), the final radioisotope content
of the tissue (counts min"1 g"1) fixed the final (9 h) point
on the curve, which was constructed on a semi-log plot
by addition of counts in progressively earlier elution
samples, so that at zero time the initial 32P content was
found. The curve, therefore, represents counts min ~'
remaining g" 1 tissue as a function of time. In the later
stages of elution the graph becomes linear, the slope
giving the rate constant (kv) for the slowest exchanging
compartment, normally equated with the vacuole.
Extrapolation of the slope to zero time gave an apparent
content of this compartment at the start of the wash-out.
Subtraction of values on the linear extrapolation from
the total counts remaining at each interval gave a second
linear phase on a semi-log plot (Fig. 1A, inset) equated
with the cytoplasm (Macklon, 1975; Ron et al., 1988;
Macklon et al., 1990; Macklon and Sim, 19926). Further
treatment of the data in this manner revealed three further
compartments identified with the Donnan free space
(DFS), water free space and superficial solution.
Mean values for rate constants (k), half-times of
exchange (^) and apparent contents, for vacuole, cytoplasm and DFS are given in Table 1, for each treatment.
Rate constants are more easily understood in terms of
half-times of exchange (to which they are related by a
factor of 0.693/k). All treatments had half-times for
exchange in the DFS, clearly separable from slower
exchanging phases. Half-times of exchange were faster in
the 100 mmol m~3 P treatments than in the 10 mmol m~3
treatments (P<0.001), but the order of magnitude was
that usually found for DFS exchange rate values (Ron
et al., 1988; Macklon and Sim, 19926). The content of
the DFS compartment in the presence of Al was larger
than the control for the higher P/Al treatment (P<0.05),
but no significant effect was observed for the lower P/Al
treatment. There were no significant differences between
cytoplasmic half-times of exchange and apparent contents
of the cytoplasm reflected closely the 10-fold difference
in P concentration between the high and low P treatments.
At both levels of P supply, Al made no difference to the
apparent amount of exchangeable P in the vacuole,
though treatment with 100 mmol m~3 P resulted in a
higher P content (P<0.00\). Higher P also led to halftimes of exchange across the tonoplast an order of
magnitude faster than with the low P treatment.
796
Macklon et al.
Table 1. Original values for rate constants (k, s~[), half-times of exchange (tt, h or ruin) and apparent contents of P (ixmol P g~l
fw) in each compartment of root cortical cells, loaded and washed in complete nutrient solution, with and without Al; standard error
of the mean for each value is given (n = 3)
Parameter
Treatments (used for both loading and elution of 32P)
lOmmol m" 3 P,
control
lOmmol m" 3 P.
3.7 mmol m" 3 Al
100 mmol m
control
3
P.
100 mmol m" 3 P,
37 mmol m~ 3 Al
Efflux constants (k)
Vacuole
Cytoplasm
Donnan free space
9.78 + 0.65 xlO" 7
1.24 + 0 33 x 10" 4
6.53±0.88x 10^4
5.11 +0.55 x 10"7
1.64 +0.30 x 10~4
5.22±0.19xl0" 4
4.09 + 0 55 xlO~ 6
1.68+0.04xl0" 4
9.77±0.45xl0~ 4
4.98 + 0.45 xlO" 6
1.65 + 0.04 xlO" 4
1.02±0.06xl0" 3
Half-times of exchange (t±)
Vacuole (h)
Cytoplasm (min)
Donnan free space (min)
199 ±14"
115 + 42"
18.3 + 2.2*
385 + 40"
74.7+11.7"
22.2 ±0.8"
49.0 + 7.1°
68.9 + 1.5"
11.9±0.5"
39.3 + 3.5°
70.1 + 1.8"
11.4 ±0.8"
1.96 + 0.05"
0.098 ±0.050"
0.175 ±0.022"
2.19 + 0.36"
0.091 ±0.019"
0.161 ±0.019"
4.65±0.18"
0.963 + 0.107"
0.527 + 0.098"
6.00 + 0.48"
1.37 + 0.03"
0 761 ±0.006°
Apparent contents (/)
Vacuole
Cytoplasm
Donnan free space
Significant differences (P<0.05) between treatments are denoted by superscript letters. Values with the same letter were not significantly different.
In Fig. IB, efflux is expressed as counts min 1
m i n ^ g " 1 on a semi-log plot. The slope of the linear
phase (kv) in each treatment (Table 2) differed from kv in
the respective original analyses by at least an order of
magnitude, representing half-times of exchange of several
hundreds of minutes rather than several thousands of
minutes. To meet the criteria forfirstorder kinetics (Cram
and Laties, 1974) rate constants for kv in the
'counts min ~1 remaining' plot should be similar to that
in the 'counts min" 1 min" 1 ' plot. Further, the ratio of
the intercepts for vacuole and cytoplasm (a/a1 Fig. 1A),
bore a different proportionality to the ratio of the associated rate constants than was the case for the efflux-time
graphs (b/b1, Fig. IB). Thus, the original data failed to
obey the criteria for first order kinetics on two counts.
This is considered due to the vacuolar slope being representative of two compartments, rather than one. The data
plotted in Fig. 1C are calculated to be those which yielded
a final rate constant similar to that in Fig. IB, by subtraction of an appropriate constant number of counts min "'
from the value at each time interval in Fig. 1A. This
amount was considered to represent slowly exchanging
assimilated or complexed P, which masked the true
exchange rate for P in the vacuole. Mean values for
transformed kv and complexed P are given in Table 3.
Values of k and t± for the pool of very slowly exchanging
P, probably complexed in the vacuole, were estimated
from the initial compartmental analysis (Table 1). The
impact of the transformations on the kinetic data for the
cytoplasm was relatively small, as can be seen by comparing the cytoplasmic t^ values for each treatment between
Tables 1 and 3. The significant effects of Al on vacuolar
half-times of exchange (/><0.01) in the low P treatments
are also apparent in Tables 1 and 3.
It is onerous to compute a deduction from the original
elution points which results in a kv matching exactly the
Table 2. Rate constants (k, s 1), half times of exchange (tit min) and zero time intercept values (I, nmol g 'J from P tracer efflux
analysis, plotting log counts min~' appearing in eluent min~l g" 1 , as a function of time (as in Fig. IB); standard error of the mean
for each value is given (n = 3)
Parameter
Treatment
lOmmol m" 3 P,
control
Efflux constants (k)
Cytoplasm
Vacuole
Half-times of exchange (t^)
Cytoplasm
Vacuole
Apparent P content (/)
Cytoplasm
Vacuole
lOmmol m~3 P,
3.7 mmol m" 3 Al
100 mmol m" 3 P,
control
100 mmol m~3 P,
37 mmol m~ 3 Al
1 05 + 0 lOx 10" 4
2.11±0.20xl0" 5
1.15 + 0.11 x 10"4
2.68±0.33xl0" 5
7.44 + 0.80x 10" 5
1.46±0.10xl0" 5
1.10 + 0.07x 10~4
2.26±0.68x 10" 5
158+16"
804 + 60"
106 + 7"
672 + 276"
112+12"
558 + 54"
103 + 10"
444 + 48"
0.314 + 0.122"
0.149±0.018"
0.489 + 0.151"
0.090 ±0.023"
3.55 + 0.34"
1.81 ±0.27"
5.88 + 0.85"
3.15 + O.31b
Values with the same superscript letters (among treatments) are not significantly different (/ ) <0.05).
Phosphate fluxes and aluminium
797
Table 3. Rate constants (k, s~'), half-times of exchange (t^) and apparent contents (fimol g~* fwj for P in cytoplasm and vacuole.
after taking into account the theoretical considerations of Cram and Laties (1974)
Also given is uptake rate to the vacuole ((/»//up, /j.mol g" 1 s" 1 ). Standard error of the mean is given for each value (n = 3)
Parameter
Treatment
lOmmol m " 3 P,
control
Efflux constants (k)
Cytoplasm
Vacuole
Complexed
Half-times of exchange (tt)
Cytoplasm (min)
Vacuole (h)
Complexed (h)
Apparent contents (/)
Cytoplasm
Vacuole
Complexed
AA.P
lOmmol m " 3 P.
3.7 mmol m " 3 Al
1.13 + 0.01 x l O ~ 4
1.46 + 0.17x 10" 5
9.78 + 0.65 x l O 7
1.45 + 0.08 x 10" 4
3.60 + 0.08 x l O " 5
5.11+0.55x 10" 7
101 + ]•
13.5±1.7 a
198±13 a
80 + 5"
5.4 + 0.12b
385 ±40"
0.067 + 0.027°
0.167±0.009 a
1.79 + 0.05a
3.23 + 0 . 0 9 x l 0 " 5 a
0.075+0.018 a
0.085+0.023 b
2.11+0.33"
3.59 + 0 5 8 x 10~ 5 a
100 mmol m " 3 P,
control
1.78 + 0.28 x l O " 4
2 64 + 0.27 x 10" 5
4.09 + 0 55 x 10""6
69+13"
7 5 + 0.8"
50 + 7c
slowest rate constants in Table 2. The values shown in
Table 3 are, however, reasonably close approximations
and lead to a closer proportionality between intercepts
for vacuole and cytoplasm (c/c1, Fig. 1C) and the ratio
of associated rate constants, to that established for the
efflux-time graphs (Fig. IB). It has to be recognized,
however, that the estimate of the content of the very
slowly exchanging component is, in each treatment,
approximate.
The transformed values for rate constants and apparent
contents have been used as the basis for all further
calculation. Unidirectional fluxes and 'real' contents,
expressed as concentrations in cytoplasm and vacuole,
were calculated from rate constants and apparent content
data in Table 3, using the relationships given by Macklon
(1975), and the cell dimensions shown in Table 4. The
results are shown in Table 5. As with our other work on
roots (Macklon et al., 1990; Macklon and Sim, 19926)
the results are considered to represent values for the
average cortical cell. Such cells constitute about 75% of
the root volume in A. capillaris (Table 4).
Complexed P constituted the major vacuolar P component in the control and Al treatments, at both levels
of P supply (Table 5). In the presence of Al, at a
concentration in the medium of 100 mmol m " 3 P, complexed P was higher than in the control but at
10 mmol m " 3 P, the effect of Al was rather small.
However, in neither treatment were the control and 'plus
Al' values significantly different, unlike the effects of Al
on the amount of P complexed in L. perenne root cell
vacuoles (Macklon and Sim, 19926). Nevertheless, the
concentration of free P in the vacuole, in the 10 mmol m~ 3
P treatments was significantly (P<0.01) lower in the
presence of 3.7 mmol m~ 3 Al. In the 100 mmol m~ 3 P
2.16 + 0.29 xlO~ 4
3.49±0.53xl0"5
4.98±0.45xl0"6
55 + 7"
5.8+0.8"
39±3 C
0.66 + 0.14"
1.38+0.21 c
3.47 + 0.17"
7.87 + 0.30 x l O " 5 "
Treatment values with the same superscript letter are not significantly different. Significant differences for
rate constants, between treatments.
100 mmol m " 3 P.
37 mmol m~ 3 Al
1.00 + 0.19"
1.97 + 0.02°
4.47 + 0.60"
10.51+0.95 x l 0 " i b
values (/><0.05) are the same for
Table 4. Mean dimensions of cortical parenchyma cells measured
randomly in root systems of six Agrostis capillaris seedlings
It was assumed that cell wall and cytoplasm each has a thickness of 0.5
;um. There were no significant differences between cells in plants grown
in 100 mmol m " 3 P and cells in those grown in 10 mmol m~ 3 P. SEMs
are shown for cell length and width («= 190) and for stele as percentage
root volume (n= 12).
Component
Cell volume
(mm3xl05)
Volume of
cortex (%)
Cell
Protoplast
Vacuole
Cytoplasm
Wall
Air spaces
2.37
2.08
1.82
0.26
0.28
—
97.0
85.4
74.6
10.8
11.6
3.0
Cell number g " ' cortex (density 1 gcm~ 3 )
Mean cell length (jim)
Mean cell diameter (/xm)
Plasmalemma surface area
Tonoplast surface area
Volume of stele as a percentage whole root
4.09 x 107
98.5 + 2.5
17.5 + 0.3
2133cm 2 g - '
1983 cm2 g" 1
25.0± 1.8
treatments, free P tended to be higher in the presence of
Al. This relates to the stimulating effect of Al on the rate
of P absorption (Macklon et al., 1994a). The data in
Table 5 suggest that the effect of the Al treatment was to
increase the unidirectional and net fluxes into the cytoplasm itself, but to reduce a little the amount of P
transported to the shoot, compared with control levels,
although transport fluxes remained several times higher
than found in L. perenne (Macklon and Sim, 19926).
However, none of these treatment effects, found in
A. capillaris, were significant.
The interpretation of the compartmental analyses has
to take account of three factors. A significant component
798 Macklon et al.
Table5. Compartmentctl concentrations (mol m~3) and unidirectional fluxes (xlO10 mol m~2s~{) of P calculated from the data in
Table 3 using the relationships given bv Macklon (1975) and the volumetric and membrane surface area data given in Table 4
SEMs are based on 3 values. Total influx across the plasmalemma includes P transported to shoots.
Parameter
Cytoplasmic concentration
Vacuolar concentration
Complexed P
Plasmalemma total influx
Influx to root only
Efflux
Net flux
Tonoplast influx
Efflux
Net flux
Transport to shoot
Treatment
lOmmol m~ 3 P,
control
lOmmol m~ 3 P,
3.7 mmol m~ 3 Al
100 mmol m~ 3 P,
control
100 mmol m " 3 P,
37 mmol m~ 3 Al
6.53 + 1.22"
0.40 + 0.02"
2.40 + 0.08"
7.53 + 0.70"
1.83 + 0.21"
0.52 + 0.18"
+ 1.31
3.33+0.74"
1.92 + 0.77"
+ 1.41
5.70 + 0.60"
3.68 + 0.26"
0.10 + 0.01"
2.83 + 0.44"
7.20 + 0.563
2.20 ±0.42"
0.66 ±0.17"
+ 1.54
2.16 + 0.19"
0.54 + 0.08"
+ 1.64
5.00 + 0.50"
12.48+1.48°
2.37 + 0.46'
4 65±0.23 b
17.44 ±1.02"
9.44+1.96"
6.71+2.02"
+ 2 73
4.73 + 0.25'
1.79 + 0.18"
+ 2.94
8.00+1.00"
16.06+1.34'
3.11+0.18'
5.99 + 0.80"
20.83 ±0.56"
15 44 + 2.40"
12.18 + 3.40'
+ 3.26
6.04 + 0.40'
2.54 + 0.81"
+ 3.50
5.39±0.51"
Treatment values bearing the same superscript letter are not significantly different (/ ) <0.05).
of influx was the quantity of phosphate destined for
transport to the shoot. Secondly, there was, in the control,
a pool of very slowly exchanging 32P and, thirdly, in
treatments particularly of L. perenne exposed to Al
(Macklon and Sim, 19926) an additional quantity was
added to this very slowly exchanging pool. It is likely
that transport, assimilation and complexation all played
a part in causing the failure of the original data to meet
the criteria for first order kinetics.
In absolute terms, phosphorus transport to the shoots
in Agrostis (Table 5) was considerably larger than that
reported for Lolium (Macklon and Sim, 19926), and
represented about 69% and 76% of total plasmalemma
influx in plants given 10 mmol m~3 P and 26% and 46%
in plants given 100 mmol m" 3 P, with or without Al,
respectively. A full discussion of the effect of transport in
modifying other measured fluxes is given by Macklon
and Sim (19926). Suffice it to say that Pitman (1971)
found (for C\~ in wheat) that even in a system where
transport to the shoots was 76-80% of total flux, values
for plasmalemma influx and fluxes across the tonoplast,
after taking account of transport, were only 10-20%
different from fluxes calculated using the conventional
model. The main difference was in the value for efflux at
the plasmalemma, which was much smaller when the
transport flux was included in thefluxanalysis. Since the
turnover time for most P assimilates is less than the
slowest P exchange rate (Table 3) (Bieleski and Ferguson,
1983) they would be kinetically invisible, so it seems that
this factor can not account for the discrepancy between
kv for counts remaining in tissue and ky for
counts min ~' min" l (efflux) in the way that it did for
NH^ in onion roots (Macklon et al., 1990). The evidence,
therefore, points to the very slowly exchanging compartment being due to complexation of P within the vacuole,
possibly with Al, the presence of which was demonstrated
by X-ray microanalysis.
The microanalysis was undertaken to demonstrate specifically whether or not Al was present in root cortical
cell vacuoles (Fig. 2). The presence of Ca in vacuoles is
well established (Macklon, 1984), but could not be confirmed in these spectra since the Ca peak was obscured
by the K/3 peak in vacuoles. In cell wall spectra (Fig. 3)
where the K peak was negligible, no Ca peak was evident
in the presence of Al.
The results for X-ray microanalysis for cortical vacuoles
of control roots, and those grown in 37 or 100 mmol m~3
are shown in Fig. 2. In the control (Fig. 2) there was no
discernible peak for Al, whereas the vacuoles from roots
given Al at 37 mmol m~3 revealed variable, but definite
peaks for Al (Fig. 2B-D), although occasionally in
smaller cells, no Al peak was seen (Fig. 2E). Figure 2F
Table 6. Total concentrations of species in vacuoles, estimated
from analysis of A. capillaris roots after determination of
fresh weights
Contributions from the cytoplasm were considered not to significantly
bias the values. Organic acid concentrations are assumed values. For
the purpose of speciation calculations, these values were also adopted
for L. perenne root cell vacuoles.
Total concentration
(mol m~ 3 )
Ca
K
Na
Mg
Cl
SO 4
Malic acid
Citric acid
PO 4
Al
4
40
8
II
20
5
20
1. 5, 10
variable
variable
Phosphate fluxes and aluminium
799
B
^^*T^^*»
0
1
2
3
4
»lw
5
Fig. 2. X-ray microanalysis spectra for mature root cortical cell vacuoles, using fresh frozen root specimens. Vertical scales all 0-4000 counts. (A)
Control (B-D) Examples of spectra for three roots from plants given 37 mmol m~ 3 Al in nutrient medium for 2 d prior to sampling. (E) Some
smaller cells in Al-treated roots occasionally showed no evidence of vacuolar Al content. (F) Example of spectra from roots given 100 mmol irT 3
Al for 4 d prior to sampling. For details of operating conditions see text.
shows an example of vacuolar spectra for cells treated in
100 mmol rrT 3 Al. The Al peak was a little larger than
found generally in vacuoles of roots treated in
37 mmol m " 3 Al. Although quantification of these results
is difficult to achieve, peaks for Al in the vacuole were
clearly demonstrated.
Speciation modelling
Aluminium and phosphate can exist in several forms in
biological systems. These may include inorganic soluble
complexes such as Al(OH)^, A1H2PC>4+, soluble complexes with organic ligands (Ohman and Sjoberg, 1988)
or complexes bound to cell walls (McCormick and
Borden, 1974). The complexes of Al and Ca likely to
occur in vacuoles were examined to see whether the
vacuolar solution could be super-saturated with respect
to various precipitated phases, which could account for
the formation of extremely slowly exchanging forms of
phosphate.
Vacuolar inorganic solute values (Table 6) were estimated from analysis of whole fresh roots, on the assumption
that in mature cells, the cytoplasmic volume and content
were too small significantly to affect values attributed to
the vacuole, even if cytoplasmic concentrations were very
different from vacuolar values. The influence of root tips
on content values was considered small since they were
only 1-2% of the whole root. The contribution from cell
walls was also small (Fig. 3A). Organic acid ions will
also have been present and, in the range pH 5-6 commonly encountered in the vacuole (Raven and Smith,
1974; Mathieu et al., 1989), complexation of aluminium
with organic acids is likely to have been strong (Ohman
and Sjoberg, 1988). The concentrations of organic acids
assumed are critical for the calculation of aluminium
800 Macklon et al.
I
B
4.6
0
1 2
3
keV
4
5
Fig. 3. X-ray microanalysis spectra for cell walls of root cortical cells
treated in 37mmolm~ 3 Al (A) and lOOmmolm" 3 Al (B). Scale as
in Fig. 2.
activity. However, the carboxylic acids present and their
concentrations are poorly denned for vacuoles, and it is
necessary to make some simplifying assumptions. Values
for vacuolar malate have been found to vary from
<5molm" 3 to >140molm~ 3 in epidermis of barley
(Fricke etai, 1995) and up to 70 mol m" 3 in Catharanthus
roseus cells (Guern et al., 1991). Osmond (1976), quoting
data from MacLennon, gives values for vacuolar malate
storage pools between 0.34 and 15.1 mol m~3. Citrate
storage pools have been found in vacuoles at between 0.5
and 10 mol m" 3 (Osmond, 1976; Guern et al., 1991).
Storage pools of other carboxylic acids are generally
found at considerably lower concentrations than for
citrate (Osmond, 1976). In the calculations of speciation
in the vacuole a value of 20 mol m " 3 for malate has been
assumed, and the outcome has been examined with citrate
values set at 1, 5 or lOmolm" 3 with a pH range
of 4.8-6.0.
The total amounts of phosphate in the vacuole were
based on experimental data (Table 5), and vacuolar concentrations of Al were estimated from slopes of the
accumulation phase of Al uptake, which avoids misalloca-
6.2
Fig. 4. Per cent phosphate precipitation, at three levels of citric acid,
as a function of pH, calculated for the vacuolar milieu given in Table 6.
(A) A. capillaris; (B) L. perenne
tion of Al adsorbed on cell walls (Macklon et al., 1994a).
No Al was transported to shoots. The Ca value in Table 6
is similar to that established by compartmental analysis
in roots of other species (Macklon, 1984). The other ions
in Table 6 do not much affect the outcome.
A particular interest was to determine the most likely
mechanism for vacuolar accumulation of P in both
A. capillaris (this paper) and L. perenne roots (Macklon
and Sim, 19926) in a non-exchangeable form when
exposed to non-toxic levels of Al, and the role of pH and
organic acids in this mechanism. To this end malic acid
protonation and aluminium complexation constants were
taken from Perrin (1979) and those for the citric acid
system from Ohman (1988). Formation constants for
hydroxy-Al species were those given by Nordstrom and
May (1989).
Possible precipitated aluminium phosphate phases that
were thought likely to form included A1PO4.2H2O (variscite) according to the reaction:
»A1PO4.2H2O log A:so =
(1)
The value of log Kso is based on the value reported by
Stumm and Morgan (1981). Several other log Kso values
for the solubility of variscite are reported in the literature.
Based on the reaction stoichiometry in equation (1) these
Phosphate fluxes and aluminium
include 22.05 (Lindsay, 1979), 21.5-22.5 (Taylor and
Gurney, 1964) and 22.0 (Sillen and Martell, 1964). Since
the Stumm and Morgan (1981) value of log Kso = 2l
represents the most soluble and, therefore, the least likely
to precipitate, the calculations to test whether the vacuole
solutions were super-saturated with respect to a solid
aluminium phosphate phase used this value.
The calculations indicate that, in the absence of organic
acids, all the phosphate in the vacuole would be precipitated as either calcium or aluminium phosphate at pH 6.
Examples of the effects of organic acids in controlling the
activity of Al and Ca in the model system are illustrated
for A. capillaris (Fig. 4A), where vacuolar concentrations
of Al and PO4 were estimated to be 8.0 mol m~3 and
9.1 mol m~3, respectively (Table 7, system 4). Data for
L. perenne (Macklon and Sim, 1992ft) were also reviewed
(Fig. 4B), by way of comparison, using vacuolar concentrations of Al and PO4 estimated to be 10.9 mol m~3 and
7.3 mol m~3, respectively (Table 7, system 6). The percentage of total vacuolar phosphate which was precipitated increased with increasing pH and decreased with
increasing citric acid concentration. Since vacuoles of
Lolium contained more phosphate than those of Agrostis,
grown in similar conditions (Macklon and Sim, 19926;
Macklon et al., 1994a), the consequences of precipitation
will be greater in Lolium. At pH in excess of 5.3, some
Ca and Al phosphates were super-saturated even at citric
acid concentrations of 10 mol m~3. Figures 5 and 6 show
separately the predicted amounts of PO4 that would be
precipitated as either Al or Ca phosphates for different
concentrations of citric acid. At each of the concentrations
of citric acid used, the solubility of Ca phosphate was
not exceeded until the pH was raised above about pH 5.2.
For A. capillaris (Fig. 5) the amount of P precipitated as
Ca phosphate at pH 6 ranged from 20-25% of the total
P. For L. perenne (Fig. 6) the range was 6-30%. The
highest values were found when citric acid was set at
o
4.6
SO
54
801
o
53
4.6
Fig. S. Contributions of variscite (A1PO4.2H2O) and hydroxy-apatite
(Ca s (PO 4 ) 3 (OH)) to percentage phosphate precipitation in A. capillaris
vacuoles as functions of pH and citric acid concentration.
5 mol m 3 (Agrostis) or 10 mol m 3 (Lolium). At 10 mol
m~3 citric acid Ca phosphate (hydroxy-apatite) exceeded
Al phosphate (variscite) precipitated in both species. At
other concentrations of citric acid tested in the model,
Table 7. Summary of P and Al concentrations in loading solutions and estimated P and Al concentrations in root cortex cell vacuoles
for each treatment
Estimated contents of non-exchangeable P are given in absolute values and as a percentage of total vacuolar P. Predicted values, at two levels of
citric acid, are those calculated from assumptions affecting speciation, at pH 6.0. L. perenne values are taken from Macklon and Sim (19926).
Loading solution
(mmol m " 3 )
Total in vacuole
(mol m"
P
Al
P
A. capillaris
1
2
3
4
10.0
10.0
100
100
0
3.7
0
37
2.8
2.9
7.0
9.1
L. perenne
5
6
100
100
0
37
3.1
7.3
Species and
system number
•
3
Non-exchangeable
P in vacuole
(mol m~ 3 )
Non-exchangeable
P as a percentage of
total P
Predicted precipitation
as a percentage of total P in vacuole
1 mol m~ 3
citrate
5 mol m~ 3
citrate
2.8
5.9
1.7
8.0
2.4
2.8
4.6
6.0
85.7
96.5
65.7
65 9
83.9
95.9
97.3
90.6
46.6
72.5
21.7
55.7
1.7
10.9
2.5
6.5
80.6
89.0
67.4
98.7 •
35.7
91.6
)
Al
In practice, Al was not entirely excluded from the roots (systems 1, 3 and 5) while in the growing solution.
Fig. 6. Contributions of vanscite (A1PO4.2H2O) and hydroxy-apatite
(Ca5(PO4)3(OH)) to percentage phosphate precipitated in L perenne
vacuoles as functions of pH and citric acid concentration.
the proportion of Al phosphate precipitated was much
greater than for Ca phosphate, rising at pH 6 to 70% in
Agrostis and to 93% in Lolium, when citrate was set at
1 mol m~3. These effects occurred because, at the higher
citrate concentrations, the aluminium was complexed with
citrate, thus lowering the activity of Al3 + . As the concentration of citrate was lowered the concentration of Al3 +
increased and the solubility of Al phosphate was exceeded.
The size of these trends would account for the smaller
increases in non-exchangeable vacuolar phosphate found
in Agrostis (this paper) in the presence of Al than were
evident in Lolium (Macklon and Sim, 19926).
Table 7 gives a comparison of the percentage of PO4
in each experimental system that is non-exchangeable
compared to the predicted amount that would be precipitated. The calculated quantities are based on a pH of 6
and total citric acid content of 1 mol m~3 or 5 mol m" 3 .
For experimental systems 4 and 6, when citric acid is set
at 5 mol m~3, there is quite a good match between
observed and predicted precipitation. However, for the
other systems, with 5 mol m~3 citric acid, the predicted
amount precipitated is less than the observed non-
exchangeable fraction. One possible explanation could be
that the experimental systems contained lower amounts
of organic acids. With citric acid set at 1 mol m" 3 ,
systems 1 and 2 show close matches between experimental
and predicted values, but when no Al was present in the
lOOmmol m" 3 P loading solutions predictions are too
high or too low.
Flux analysis has been used as an indirect way of
getting an insight into ion contents and fluxes in plant
cell compartments, remote from the tissue surface, for
nearly 40 years (MacRobbie and Dainty, 1958; Bell et al,
1994). It has always been recognized that a number of
assumptions have to be made in making such an analysis.
When using transformations to elicit more information
(Cram and Laties, 1974), further assumptions are made.
This approach has been used to extend the understanding
of vacuolar nutrient dynamics from a point reached in
earlier work (Macklon and Sim, 1992a, b; Macklon et al,
1994a), a location notoriously difficult to access in intact
cells of higher plants. The speciation analysis is based on
the best data available from our own experiments and
from the literature and gives the first estimates of some
effects of Al and Ca on the phosphorus economy of an
upland grass compared with a species used in reseeded
improved pastures.
The results suggest that for solutions containing the
total amounts of Al and PO4 used in our experimental
systems, vacuolar Al phosphate and Ca phosphate phases
are super-saturated for a wide range of organic acid
concentrations and pH, and can explain the mechanism
which results in a proportion of P in root vacuoles being
non-exchangeable (as hydroxy-apatite) and this proportion increasing in the presence of Al (as variscite). It is
clear, however, that the degree of precipitation is sensitive
to citric acid concentration.
Conclusions
In the control plants, a slowly exchanging pool of P
already existed (Tables 5, 7), due, it is proposed, to
complexation with Ca. Increases in the complexed pool
due to Al treatment were in addition to the Ca effect or
in replacement of it.
The effect of non-toxic levels of Al on P sequestration
in root cells of Agrostis (Table 5) was small (and not
statistically significant) compared with Lolium (Macklon
and Sim, 19926). Notwithstanding the amount of complexed P present in the absence of Al, this difference
between species is ecologically significant. L. perenne
plants bred for high productivity in pastures, receiving
some degree of fertilizer addition, are still able to yield
well with a significant proportion of P sequestered in root
vacuoles, caused by low levels of Al in soil solution.
A. capillaris which is an indigenous species, often growing
on unimproved upland sites with poor nutrient status,
Phosphate fluxes and aluminium
has a carboxylic biochemistry which minimizes absorbed
Al further sequestering scarce phosphorus. A considerably
higher level of free orthophosphate was present in
A. capillaris root cortical cell vacuoles than was present
in the root cells of L. perenne (Table 5 and Macklon and
Sim, 19926). This factor, together with a greater turnover
of P and a proportionally greater transport of P to the
shoot all contribute to the greater phosphorus efficiency
of A. capillaris, compared with L. perenne, identified in
uptake experiments (Macklon et al., 1994a).
Acknowledgements
Thanks are due to Karen Clements for able assistance with
experiments and to Elizabeth Duff(BioSS) for statistical advice.
This work was funded by the Scottish Office Agriculture,
Environment and Fisheries Department.
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