Journal of Experimental Botany, Vol. 55, No. 406, pp. 2269–2280, October 2004
DOI: 10.1093/jxb/erh246 Advance Access publication 27 August, 2004
RESEARCH PAPER
Spatial mapping of phosphorus influx in bean root systems
using digital autoradiography
Gerardo Rubio1,2, Agostino Sorgonà1,3 and Jonathan P. Lynch1,*
1
Department of Horticulture, 102 Tyson Building, The Pennsylvania State University, University Park, PA 16802,
USA
2
Facultad de Agronomia, Universidad de Buenos Aires, 1417 Buenos Aires, Argentina
Dipartimento di Biotecnologie per il Monitoraggio Agroalimentare ed Ambientale, Università degli Studi
‘Mediterranea’ di Reggio Calabria, Feo di Vito 89060 Reggio Calabria, Italy
3
Received 10 January 2004; Accepted 12 July 2004
Abstract
A novel technique was developed to spatially map
the phosphorus net influx capacity in intact root
systems. The method is based on digital autoradiography and permits the quantification of phosphorus
influx at high spatial resolution (2 mm). Roots of 18d-old common bean plants were exposed to 32P-labelled
orthophosphate, quickly frozen, excised, lyophilized,
scanned, and exposed to a storage phosphor screen.
Plots of 32P content versus root length (distance from
the root tip or from the base of the root) were obtained
for three different root classes: basal, basal laterals,
and taproot laterals. Radioactivity detected by filmless
autoradiography correlated well (r250.99) with measurements made by scintillation counting. Basal roots
absorbed 2.5 times and 1.9 times more phosphorus
than the taproot lateral and basal lateral root classes,
respectively, in the first 20 mm from the root apex.
External phosphorus markedly affected influx: roots
averaged 5, 16, and 34 pmol P min21 in the apical 20
mm when exposed to 1, 5, and 10 lM P solutions,
respectively. The spatial pattern of phosphorus influx
along the root axes of the different root classes was
rather homogeneous when measured on a root surface
area basis. Phosphorus influx in the older segments of
basal roots (those next to the hypocotyl) did not differ
from the newer segments close to the root apex.
However, a heterogeneous pattern was detected for
basal roots when measured on a length basis, indicating that both root class and diameter constitute main
factors controlling the spatial pattern of net influx.
Key words: Digital autoradiography, mineral nutrition, phosphorus uptake.
Introduction
Root systems are composed of a complex array of distinct
root classes (Fitter, 1991; Lynch, 1995). Different root
classes exploit different soil domains, are subject to
different external signals, and may have different intrinsic
capacity to absorb nutrients. Russel and Sanderson (1967)
found large variation in the phosphorus influx among
seminal, nodal, and lateral roots of barley. Root class
‘specialization’ for nutrient uptake could be of practical
relevance. For example, a greater intrinsic phosphorus
influx capacity of adventitious roots would be beneficial
for the plant since this root class generally explores the
topsoil where the majority of available soil phosphorus is
located (Pothuluri et al., 1986; Lynch and Brown, 2001;
Rubio et al., 2001).
Spatial heterogeneity in phosphorus influx capacity may
also be expected along the root axis. Such information could
be important in the construction of models of root growth
(Lynch and Nielsen, 1996), nutrient uptake (Clarkson,
1996; Silberbush, 1996), and overall root function
(Aguirrezabal et al., 1993; Eshel and Waisel, 1996).
However, current models usually assume a constant pattern
of influx along the root axis (Smethurst and Comerford,
1993; Rubio et al., 2001). Most root classes have marked
morphological and anatomical differentiation from tip to
base. These sections are typically the root tip, the elongation zone, the maturation zone, and the mature zone. The
* To whom correspondence should be addressed. Fax: +1 814 863 6139. E-mail: [email protected]
Journal of Experimental Botany, Vol. 55, No. 406, ª Society for Experimental Biology 2004; all rights reserved
2270 Rubio et al.
root tip, which includes the root cap and the meristematic
region, is thought to have a large nutrient demand
(Clarkson, 1996). In the maturation zone, the development
of root hairs from the epidermis and a functional Casparian
Band in the endodermis facilitate nutrient uptake. In the mature region of the root, the cortex may senesce, lateral roots
may emerge, and in dicots, secondary growth becomes important. The degree of cell vacuolization increases from the
root tip (where no cell vacuoles are present) to the base of
the root. The relationship of the anatomical heterogeneity
along the root axis with nutrient acquisition has concerned
researchers for some time (Prevot and Steward, 1936), and
appears to be nutrient-specific. For example, the absorption
of iron appears to be concentrated at the root tip (Clarkson,
1996) and that of sulphur in the elongation zone immediately behind the meristematic region (Holobrada, 1977).
Colmer and Bloom (1998) found consistent differences in
the pattern of nitrogen uptake between maize and rice roots,
probably brought about by the presence of a layer of
sclerenchymatous fibres in rice roots. In the case of
phosphorus, contrasting results have been found. Some
researchers observed that all regions of the root are effective
in absorbing phosphorus (Burley et al., 1970), whereas
others reported that phosphorus is absorbed more actively
at the apical regions of the root axis (Bar-Yosef et al., 1972).
To quantify nutrient uptake capacity at high spatial
resolution, either stationary or vibrating ion-selective microelectrodes and radionuclide tracers have been the most
common approaches. Ion-selective stationary microelectrodes have been used to detect ammonium and nitrate intake
in barley (Colmer and Bloom, 1998; Henriksen et al., 1990)
and potassium in maize (Newman et al., 1987). Vibrating
microelectrodes were originally used to measure extracellular calcium gradients and then used to measure potassium
and calcium fluxes in maize roots (Kochian et al., 1992).
Microelectrode approaches have not been useful for studies of
phosphorus uptake because of the lack of good phosphorussensing electrodes. Root uptake capacity for phosphorus
has been measured mainly with labelled ions (Epstein et al.,
1963; McNaughton and Chapin, 1985; Rubio et al., 1997).
Standard autoradiographic techniques consist of the exposure of the studied object to a radioactive compound. The
radiolabelled object is then exposed to a photographic emulsion to obtain a map of radionuclide distribution (Yeung,
1984). Standard autoradiography has been extensively used
to study the dynamics and extension of nutrient depletion
zones (Jungk, 1987; Krauss et al., 1987; Owusu-Bennoah
and Wild, 1979), the radial distribution of phosphorus in
different regions of the roots (Ferguson and Clarkson, 1976),
and the rate of phosphorus uptake by various root zones
(Clarkson et al., 1978). More recently, digital autoradiography has been used to describe hyphal 32P transport in
mycorrhizal root cultures (Nielsen et al., 2002) and root
morphological characteristics such as root diameter, length,
and branching patterns quantitatively (Moran et al., 2000).
The objective of this work was to evaluate the variation
of phosphorus influx along the axis of the different root
classes of common bean. The experimental approach used
here included two phases. First, the experimental conditions
in which phosphorus translocation along the root axis can
be neglected were analysed so that net influx in each root
section can be estimated. With these results, in the second
phase, a new method using quantitative autoradiography
was developed to estimate phosphorus influx at high spatial
resolution.
Materials and Methods
Plant cultivation
The root system of the common bean (Phaseolus vulgaris L.) is
composed of a taproot, adventitious roots, and an umbrella of basal
roots upon which the rest of the root system develops through lateral
branching (Fig. 1). For common bean roots, Wanke et al. (1998)
reported that the location of the different zones along the axis is as
follows (distances from the root apex): 0–7 mm root tip, 7–20 mm
elongation zone, 20–25 mm maturation zone. Seeds of common bean
genotype BAT 477 were surface-sterilized for 1 min in 10% NaOCl
and germinated at 25 8C for 2 d. BAT 477 is a medium-seeded
genotype (22 g per 100 seeds) and belongs to race M of the
Mesoamerican gene pool (Singh et al., 1991). Uniform seedlings
were transplanted into 20 l containers filled with solid-phase-buffered
pure silica sand (Lynch et al., 1990) maintaining either low (1 lM) or
high (30 lM) phosphorus availability. Plants were irrigated every 2 d
with a nutrient solution lacking phosphorus and composed of (in
lM): 1500 KNO3, 1200 Ca(NO3)2, 400 NH4NO3, 25 MgCl2, 5 FeNa-EDTA, 500 MgSO4, 300 K2SO4, 300 (NH4)2SO4, 1.5 MnSO4,
1.5 ZnSO4, 0.5 CuSO4, 0.5 Na2B4O7, and 0.143 (NH4)6Mo7O24. To
minimize the accumulation of nutrients in the growth media, a 100%
leaching fraction was maintained during irrigation. Plants were
grown in a temperature controlled (22–30 8C) greenhouse in
University Park, Pennsylvania (408859 N; 778839 W), with average
midday photosynthetically active radiation of 1000–1600 lmol
photons mÿ2 sÿ1. A general description of the growth and architecture of seedling root systems of common bean is provided in Lynch
and van Beem (1993).
Axial phosphorus transport
Because the proposed method implies a mass balance at the root
segment level, the exposure time for which translocation can be
neglected was defined first. To this end, an experiment was conducted
in which discrete sections of roots were exposed to labelled
Fig. 1. Scheme of the root system of common bean showing the different
root classes.
Phosphorus influx in roots
phosphorus in order to estimate phosphorus translocation along the
root axes and the experimental conditions where it can be neglected.
After 18 d of growth, basal roots of bean plants were placed on
331833 cm Plexiglass blocks with 12 cells, each 1.5 cm deep and
0.5 cm wide (Fig. 2). Plants were grown under high phosphorus
conditions, in which maximum levels of phosphorus mobilization
from the roots occurs in common bean (Snapp and Lynch, 1996).
Each cell was connected to the next cell by a shallow bridge so that
a root could be laid out across the block traversing all the cells. All the
cells contained 5 lM potassium phosphate and 0.5 mM CaSO4 to
maintain membrane integrity (Chapin and Van Cleve, 1989). In the
second cell, 3 lCi 32P were added. To detect possible differences of
translocation between mature and immature zones, two different
sections of the basal root were exposed to the labelled phosphorus of
the cell containing the radioactive source. In one group of plants, the
exposed section was located 80 mm from the root tip (Fig. 2I). In
a second group, the first mm from the root tip of basal roots was in
contact with the labelled cell (Fig. 2II). At 4, 30, and 60 min after the
addition of 32P, the roots were excised in several sections. Basal roots
were in contact with the solution in each cell and silicon grease (Dow
Corning, Midland, MI, USA) was used to isolate each cell. During the
exposure to the labelled source, the complete root system remained
attached to the shoot and lateral roots were not excised. The
remaining root system was maintained in a solution equivalent to
the one used to fill the plexiglass cells. The ‘exposure zone’ (Fig. 2)
included the portion (‘cell with 32 P’ in Fig. 2I and II, 0.5 mm length)
of the root actually immersed in the cell with 32P and the adjacent
20 mm segments. The ‘measurement zone’ included the 60 mm root
portion next to the ‘exposure zone’. After dividing the root into
20 mm long sections, they were rinsed for 4 min in 1 mM KH2PO4
and 0.5 mM CaSO4 at 4 8C to exchange 32P from the root free space,
followed by a second 4 min rinse in 0.5 mM CaSO4 at 4 8C (Chapin
Fig. 2. Scheme of the sampling method used for estimating the
phosphorus movement along the root axis. Two different sections of
the basal root were exposed to 32P located 80 mm (I) or 10 mm (II) from
the root tip. At 4, 30, and 60 min after exposure to 32P, roots were cut off
in several sections. The ‘exposure zone’ included the portion of the root
immersed in the cell with the radioactive source (0.5 mm length) and the
adjacent 20 mm segments). The ‘measurements zone’ included the
60 mm root portion next to the ‘exposure zone, and was cut at 2 mm
intervals to perform the measurements.
2271
and Van Cleve, 1989). Radioactive phosphorus in the root sections
was analysed using a scintillation counter (Packard 1500, Packard
Instruments Co, Downer’s Grove, IL, USA).
Phosphorus influx
To evaluate the variation of phosphorus influx along the axis of the
different root classes of common bean, a new experimental set-up
was devised with quantitative autoradiography (Fig. 3). After 18 d of
growth, root systems were carefully washed out from the soil but kept
attached to the shoot and transferred to an aerated solution containing
0.5 mM CaSO4. Roots remained in this solution for 30–45 min and
were then placed for 4 min in plastic containers filled with uptake
solutions (100 ml) of 0.5 mM CaSO4 and 1, 5, or 10 lM KH2PO4
labelled with 32P (1 lCi per container). For each uptake solution, 1 ml
sample was collected and analysed for 32P to calculate the specific
activity. Average specific activity was 1.4 mCi mmolÿ1 P. The length
of the period of root exposure to the radioactive nutrient was
determined after performing the transport experiment described
above. After being exposed to the labelled solution, root systems
were rinsed for 4 min in 1 mM KH2PO4 and 0.5 mM CaSO4 at 4 8C to
exchange 32P from the root free space, followed by a second 4 min
rinse in 0.5 mM CaSO4 at 4 8C. Roots were then separated from the
shoot and placed in dry ice for 2 h. Frozen root systems were then
placed overnight in a lyophilizer.
Estimating 32P uptake along the root axes
For each lyophilized root system, basal roots, basal laterals, and
taproot laterals were carefully excised at their base and then placed on
blue germination paper, maintaining a minimum distance of 2 cm
among them to avoid the overlapping of emitted radiation on the
imaging plate (Fig. 3). These roots were scanned at 1200 dpi with
WinRhizo v. 4.0 software (Regent Systems, Quebec, Canada) to
measure surface area and length and then exposed overnight on
a photostimulatable storage phosphor screen (Amersham Biosciences
Corporation, Piscataway, NJ, USA). The screen captures latent image
formation produced by the ionizing radiation released from the
labelled roots. Phosphorescence (blue light) is then generated by the
screen in proportion to the amount of radioactivity in the sample. At
the end of the exposure period, the screens were laser scanned at
a spatial resolution of 100 lm per pixel and 16 bit grey level range
with the PhosphorImager 445 SI system (Amersham Biosciences
Corporation, Piscataway, NJ, USA). During scanning, phosphorescence emitted by the screen is collected by a fibre optic bundle and
conducted to a photomultiplier tube and, finally, a graphical representation of the localization of absorbed labelled ions is obtained.
Exposure for 10 h was sufficient to obtain accurate images of the 32P
accumulated by the labelled roots (Fig. 4). To quantify 32P accumulation, the root segment of interest was selected by drawing a band
over its image and analysed with ImageQuant 5.0 (Amersham
Biosciences Corporation, Piscataway, NJ, USA). The software
calculates the area under the curve created by a plot of pixel
intensities versus pixel locations along the band and expresses it as
counts. The width of the band was selected by the operator and
background radioactivity is discounted automatically by the software.
In each exposure, a calibration curve was used to convert data
collected from ImageQuant (expressed as counts) to lCi. The
calibration curve was made with different volumes of a solution with
known 32P radioactivity. Pots of counts versus root length (distance)
were obtained for each individual group of roots scanned. To test the
accuracy of filmless autoradiography to detect radioactivity, results
from the calibration curve were compared with results from a similar
standard curve, but measured with the standard method to quantify
emissions (scintillation counting).
In the examples shown in this paper, roots were divided into 2 mm
segments, starting from the root tip, to obtain the phosphorus influx
2272 Rubio et al.
Fig. 3. Scheme of the phosphorus influx experiment.
Fig. 4. Labelled phosphorus absorbed by individual segments of bean
roots after 4 min of exposure to 1 lM phosphate labelled with 1 lCi 32P
and exposed to a phosphor screen for 10 h. The grey intensity is
proportional to the phosphorus accumulation. The image was laser
scanned with the filmless autoradiography system. A standard curve
made to calculate the specific activity is shown (upper left).
rate. An example of the calculations for an individual segment of basal
roots is shown in the Appendix. Results can also be expressed on an
area basis. This last case requires data on surface area from each
segment of root. From ImageQuant the number of counts every 2 mm
were obtained and, from WinRhizo output, the root surface area in each
2 mm segment was measured. To test if the 2 mm segments were
equivalent in the two procedures, the length of the root segments
measured from the tip obtained by Imagequant and WinRhizo were
compared.
To obtain precise quantification and localization of radionuclides in
the root, several preliminary experiments were conducted to determine
the appropriate combination of radioactivity, length of the period of
contact between the roots and the labelled solution, and the length of
the period of exposure of the root to the phosphor screen. If 32P
accumulation in the root is not in the optimum range of detection, either
under-exposure or over-saturation of the image would be problematic.
Based on these preliminary experiments, the conditions used in the
main experiment were 1 lCi in 100 ml of 1–10 lM of KH2PO4, 4 min
contact with the labelled source, and 10 h of root exposure to the
phosphor screen. If the whole root system is exposed to the phosphor
screen, the uptake rate of individual segments of roots cannot be
calculated due to the overlap of the radiation emitted by adjacent root
segments. That is the reason why individual root segments were
separated from each other after being in contact with the labelled
phosphorus and before being exposed to the phosphor screen. The
range of grey intensity in Fig. 4 represents different influx values.
Statistical analysis
In the influx experiment, 30 plants (two phosphorus treatments during
the growth period by three phosphorus concentrations in the nutrient
Phosphorus influx in roots
solution by five blocks) were arranged following a completely
randomized block design. For each plant, results from several (2–8)
units of each root class were averaged. Repeated-measures ANOVA
and regression analysis were performed using SYSTAT v. 8.0 (SPSS
Inc., Evanston, IL, USA).
Results
Accuracy of filmless autoradiography
The close relationship (r2=0.99) (Fig. 5) between the
number of counts of a standard curve (as shown in Fig.
4) measured by ImageQuant software and the amount of
radioactivity measured by scintillation counting shows that
digital autoradiography can give an accurate value of the
32
P radioactivity present in the sample. The ‘spatial’
accuracy of filmless autoradiography to quantify phosphorus influx at a high spatial resolution (every 2 mm of dried
root length) is indicated by the almost exact relationship
(r2=0.99) (Fig. 6) between the length of the root segments
obtained from the radioactivity images (as in Fig. 4) and the
length of the same root measured with WinRhizo.
Axial phosphorus transport: After 4 min of root exposure to
a labelled source of phosphorus, the proportion of phosphorus that moved to the ‘measurement zone’ (Fig. 2)
represented 0.05–0.12% of the phosphorus found in the
exposed segment (Table 1). When the exposure period was
extended to 60 min, the proportion of transported phosphorus increased to almost 25% (Table 1). In every case,
the basipetal movement was greater than the acropetal
movement. It was concluded that labelled phosphorus
detected in a given root section after 4 min of exposure
accurately reflects the amount of phosphorus influx in that
root section. Thus, roots were exposed to the labelled
phosphorus source for 4 min in the phosphorus net influx
experiment.
2273
Fig. 7 shows the total phosphorus influx in the apical 20 mm
of each root class. Among root classes, the basals showed the
highest influx: they absorbed 2.5 times and 1.9 times more
phosphorus than the taproot lateral and basal lateral root
classes, respectively (Fig. 7; Table 2). The higher apparent
effectiveness of the basal roots was observed in both plants
that had been grown under high and low phosphorus
conditions and at the three phosphorus concentrations
offered in the labelled nutrient solutions (Fig. 7). Basal
Fig. 5. Relationship between the number of counts of the standard curve
as shown in Fig. 3 measured by digital autoradiography and by the
standard method (liquid scintillation counting).
Phosphorus influx
Basal laterals and taproot laterals were around 20 mm long
and basal roots were approximately ten times longer. To
make direct comparisons among the three root classes,
Fig. 6. Relationship between the root length measured by analysing the
digital autoradiography images of the ionizing radiation emitted by the
roots (as in Fig. 3) and the root length analysed by a root analysis software
(WinRhizo).
Table 1. Effects of the length of exposure time and labelling position on the basipetal (i.e. towards the base of the root) and acropetal
(i.e. towards the root tip) movement of 32P
Movement is expressed as a proportion of the amount of labelled phosphorus found in the labelled area. Standard error between brackets (n=5). For
details see Fig. 2.
Exposure time
to 32P (min)
Labelling position
(mm from root tip)
P uptake in the labelled
area (pmol P gÿ1 fresh
weight minÿ1)
Basipetal P movement
(% of P detected in
labelled area)
Acropetal P movement
(% of P detected
in labelled area)
4
30
4
30
60
80
80
10
10
80
100 (23)
27 (4.3)
99 (25)
43 (19)
55 (21)
0.05
0.37
0.12
1.91
24.17
NDa
0.05 (0.01)
NAb
NA
0.10 (0.02)
a
b
ND, non-detectable.
NA, non-applicable.
(0.02)
(0.22)
(0.05)
(0.55)
(7.21)
2274 Rubio et al.
laterals and taproot laterals had comparable phosphorus
influx (Fig. 7). The only differences between these two
classes were found in plants grown under low phosphorus
conditions and exposed to the two lowest concentrations in
the labelled solutions (1 lM and 5 lM). In these treatments,
basal laterals showed higher total phosphorus influx than
taproot laterals (Fig. 7). Phosphorus concentration in the
labelled nutrient solution was the factor that exerted the
largest effect over the influx of phosphorus into plants (Fig.
7; Table 2). On average, phosphorus influx was 6.9 times and
2.1 times greater at the highest concentration (10 lM), than
at 1 lM and 5 lM, respectively.
Analysis of variance revealed significant effects of root
class and plant phosphorus status on phosphorus influx
expressed on a root length basis (i.e. pmol P mmÿ1 minÿ1)
or on a root area basis (pmol P cmÿ2 minÿ1) (Table 2).
Interactive effects were observed between phosphorus
status and phosphorus concentration in the labelled nutrient
solution: Figures 8 and 9 show that phosphorus stress only
affected influx when plants were exposed to low phosphorus concentrations (see also Table 2). The effect of the
position of the root segment along the root axis was
statistically significant when phosphorus influx was expressed on a root length basis, but not when expressed on
a root area basis (Table 2). When phosphorus influx was
expressed on a length basis, basal roots had reduced influx
in those root sections close to the root tip (Figs 8, 9). This
effect was statistically significant for plants grown at both
low and high phosphorus conditions exposed to 1 lM
labelled phosphorus (Figs 8, 9). Maximum influx per unit
length in basal roots was observed at the sections located
20 mm behind the root tip. No consistent spatial pattern was
observed when phosphorus influx along the root axis of
basal roots was calculated on an area basis (Figs 8, 9).
Phosphorus influx was rather constant in the basal root
segments next to the hypocotyl (which represents the oldest
segments of this root class) (Fig. 10) and it did not differ
from the segments located 20–80 mm from the root tip as
shown in Figs 8 and 9. No clear effects of the position of the
root section along the root axis were observed for lateral
taproot and lateral basal roots when expressed as either an
area or a length basis (Figs 8, 9).
Discussion
Fig. 7. Phosphorus influx rate in the first 20 mm (from the root tip) of
basal (closed bars), lateral basal (open bars), and lateral tap (stacked bars)
roots of common bean. Plants were grown for 18 d under low (1 lM) or
high (30 lM) phosphorus supply and then exposed for 4 min to 1, 5, and
10 lM phosphate labelled with 1 lCi 32P. Values represent the mean of
five plants and are expressed on a dry root basis. For each plant, results
from several (2–8) units of each root class were averaged. Bars represents
SE of the mean values. Numbers above bars indicate the ratio of influx
between high and low phosphorus status.
Digital autoradiography
Digital autoradiography permitted the spatial mapping of
phosphorus influx capacity along root axes of common
bean plants. Roots do not need to be separated from the
shoot, a common requirement of many current analytical
methods (Chapin and Van Cleve, 1989). Root excision
could affect phosphorus influx because (i) shoot demand is
the main driving force for nutrient uptake and this signal
cannot be detected by excised roots; (ii) root excision leads
to rapid loss of carbohydrates, which reduces nutrient
uptake (Bloom and Caldwell, 1988). For calcium, White
et al. (1992) found that the net influx of intact roots was
almost 8-fold greater than excised roots.
Because the phosphor screen efficiently detects radioactivity in a limited range, some precautions should be taken
into account when using digital autoradiography. If experiments are made using a specific activity that does not result
in 32P accumulation in the optimum range of detection,
either the under-exposure or the over-saturation of the image
would not allow correct quantification and localization of
the labelled ions present in the roots. To avoid this problem,
the amount of radioactivity in the nutrient solution and the
length of the exposure to the screen must be adjusted
carefully. Low radioactivity (or very short exposure times)
leads to an under-exposure of the screen, making it difficult
to distinguish the actual nutrient influx from natural background radiation. Excessive radioactivity or very long
periods of exposure lead to greater experimental error due
to over-saturation of the image. Over-saturation results in
dark spots on the phosphor screen, from which it is not
possible to estimate the real dimensions of the analysed
Phosphorus influx in roots
2275
Table 2. ANOVA table of a phosphorus net influx experiment with common bean plants
Parameters analysed: (i) total phosphorus influx (measured in pmol P minÿ1) in the first 20 mm from the root tip, (ii) rate of phosphorus influx per unit of
root area (measured in pmol P cmÿ2 minÿ1), and (iii) rate of phosphorus influx per unit of root length (measured in pmol P mmÿ1 minÿ1). Factors
analysed: (A) root type (three levels: basal root, lateral basal, lateral tap); (B) phosphorus supply during the growth period (two levels: high and low); (C)
position along the root axes (10 levels: 2 mm sections from the root type up to 20 mm); (D) phosphorus concentration in the labelled nutrient solution
(three levels: 1, 5, and 10 lM phosphate).
Degrees of freedom
Root type (A)
Phosphorus (B)
Position (C)
Pconc (D)
A3B
A3C
A3D
B3C
B3D
C3D
a
2
1
9
2
2
18
4
9
2
18
F value and significancea
Phosphorus influx
Phosphorus influx
(area basis)
Phosphorus influx
(length basis)
9.05***
0.7 NS
NA
20.83***
0.11 NS
NA
1.43 NS
NA
0.85 NS
NA
12.88***
10.83**
0.33 NS
256.83***
0.80 NS
0.59 NS
4.21**
0.06 NS
3.20*
0.58 NS
87.06***
7.22**
2.85**
197.32***
1.25 NS
0.53 NS
13.80***
0.07 NS
7.80***
1.03 NS
NA, non-applicable; NS, non-significant; *,**,*** significant at the 0.05, 0.01, and 0.001 probability level, respectively.
Fig. 8. Phosphorus influx rate along the axis of basal, lateral basal, and lateral tap roots of common bean. Left column: influx per unit root surface area;
right column: uptake per unit root length. Plants were grown for 18 d under low phosphorus supply (1 lM phosphate) and then exposed for 4 min to 1, 5,
and 10 lM phosphate labelled with 1 lCi 32P. Values represent the mean of five plants and are expressed on a dry root basis. For each plant, results from
several (2–8) units of each root class were averaged. Bars represents the critical value (P<0.05) for comparison among means of each root class according
to the LSD test.
segment of root. Preliminary experiments should be conducted before using this method in order to determine the
most convenient combination of (i) the amount of radioactivity of the labelled solution, (ii) the length of the period of
contact of the root with the labelled solution, and (iii) the
length of the period of exposure of the labelled root to the
phosphor screen. In this case, 1 lCi in 100 ml of 1–10 lM of
KH2PO4, for 4 min, and 10 h, respectively, was used. As
a general rule, the time of exposure to the labelled solution
should be long enough to allow nutrients to pass the free
space of the apoplasm and to reach the symplasm, and it
should be short enough to avoid the translocation of the
nutrient to the adjacent root segment. To determine the
length of the absorption period, the length of the smallest
segment of root to be analysed must be taken into account.
For example, if 2 mm of root is taken as the unit of analysis,
the length of the absorption period should be short enough to
avoid translocation of 32P through a distance of 2 mm. For
determining the velocity of translocation, simple experiments, like the contiguous chamber method described in this
paper, can be made. The radioactivity of the nutrient solution
and the time of exposure to the phosphor screen should be
2276 Rubio et al.
Fig. 9. Phosphorus influx rate along the axis of basal, lateral basal, and lateral tap roots of common bean. Left column: influx per unit root surface area;
right column: uptake per unit root length. Plants were grown for 18 d under high phosphorus supply (30 lM phosphate) and then exposed for 4 min to 1,
5, and 10 lM phosphate labelled with 1 lCi 32P. Values represent the mean of five plants and are expressed on a dry root basis. For each plant, results
from several (2–8) units of each root class were averaged. Bars represents the critical value (P<0.05) for comparison among means of each root class
according to the LSD test.
Fig. 10. Phosphorus influx rate along the axis of basal roots of common bean, starting from the base of the root (where the basal root is joined to the main
root axis). Plants were grown for 18 d under low (1 lM, left column) or high (30 lM, right column) phosphorus supply and then exposed for 4 min to 1,
5, and 10 lM phosphate labelled with 1 lCi 32P. Values represent the mean of five plants and are expressed on a dry root basis. For each plant, results
from several (2–8) basal roots were averaged. Bars represents SE of the mean values.
adjusted simultaneously. The method described here could
be useful for other radionuclides important in plant biology,
including 33P, 45Ca, 35S, 14C, 3H, and 36Cl. The parameters
of the method (mainly radioactivity and time of exposure)
would have to be adjusted to the energetic characteristics of
each radionuclide.
Phosphorus influx along the axis of different root
classes of common bean
In the process of nutrient uptake, four different processes
must be distinguished: influx, storage, translocation, and
efflux. The last process is considered relevant only under
conditions of low nutrient availability (Elliot et al., 1984;
Jackson et al., 1976). Influx is associated with the activity
of carriers, which has been characterized with classic
enzyme kinetics (Epstein, 1972; see also Nissen, 1996).
Storage is associated with the presence of cell vacuoles, and
thus with root sections with some degree of maturation (Lee
and Ratcliffe, 1993). Translocation also requires some level
of maturation, since it depends upon mature xylem and
phloem elements. In this paper, to quantify influx independently of translocation, a 4-min exposure period to the
Phosphorus influx in roots
labelled solution was used, in which time negligible translocation occurred. When the exposure time was extended to
60 min, a large amount of phosphorus was transported
basipetally to the shoot from root sections located 80 mm
from the root tip, where the xylem is completely mature.
These results, as well as previous reports, focus on the
movement of recently absorbed ions. It would be interesting to investigate if there is any difference in the translocation of recently and previously absorbed ions.
It was observed that phosphorus deficiency increased
phosphorus uptake capacity, which is consistent with the
well-known fact that nutritional status has an important
influence on phosphorus uptake (Aerts and Chapin, 2000;
Cartwright, 1972; Clarkson and Scattergood, 1982; Clarkson et al., 1978; Drew et al., 1984). This modulation
constitutes an effective feedback regulation and varies
greatly among species (Cogliatti and Clarkson, 1983; Drew
et al., 1984; Jungk et al., 1990). In this study, it was observed
that this regulation was not consistent either among root
classes or among phosphorus concentrations in the nutrient
solution. Phosphorus deficiency increased phosphorus influx only in basal roots and their laterals. There are few
antecedents of this kind about the dissimilar effect of
nutritional status on the various root classes of plants grown
in homogeneous media (Russel and Sanderson, 1967;
Waisel and Eshel, 1992). By contrast, in heterogeneous
media with phosphorus ions concentrated in patches, an
increase in the phosphorus uptake capacity of the roots
located in the patch has been well documented (Jackson
et al., 1990; Snapp et al., 1995). Regarding the interactive
effects of the nutritional status of the plant and ion concentrations in the nutrient solution, two different patterns of
response have been reported (Cartwright, 1972; Drew et al.,
1984; Dunlop et al., 1997). The different responses were
related to changes in the kinetics of the ion-uptake mechanism: increases in Vmax (i.e. maximal absorption rate)
without appreciable changes in Km (i.e. substrate ion
concentration giving half Vmax) were found responsible for
increased phosphorus influx promoted by phosphorus stress
in barley (Drew et al., 1984) and Arabidopsis (Dunlop
et al., 1997). By contrast, Cartwright (1972) found that
Vmax remained relatively constant but Km decreased in
phosphorus-starved barley. In this last report, differences
in phosphorus influx between phosphorus-stressed and
phosphorus-sufficient plants were smaller at higher phosphorus concentrations in the nutrient solution due to the lack
of adjustments in Vmax. These results suggest that the
influence of the nutritional status of the plant on phosphorus
influx declines at higher nutrient concentrations in the soil
solution. This study’s results are clearly more compatible
with this last model rather than with the first one, since
differences between control and stressed plants were detectable at 1lM and 5 lM but not at 10 lM phosphorus (Fig. 7).
Basal roots had greater phosphorus influx rates, estimated
on a length basis, than lateral roots. This was observed in all
2277
the experimental regimes used. When influx was measured
on an area basis, the differences among root classes were
smaller, although basal roots remained as the group with the
highest phosphorus influx at low phosphorus concentrations
in the uptake solution. The lowest phosphorus concentration
used in this study (1 lM) resembles the typical concentration of phosphorus in the soil solution in most field
conditions (Barber, 1995). Greater phosphorus uptake
capacity in basal roots could be a beneficial trait in
genotypes in which basal roots and their laterals are
concentrated in the topsoil, like BAT 477, the genotype
used in this study’s experiment (Bonser et al., 1996; Liao
et al., 2001). It was observed that this genotype had almost
40% of the basal roots (and their laterals) concentrated in the
upper layer of the soil (Liao et al., 2001). Since phosphorus
availability is normally highest in the topsoil and decreases
with depth (Chu and Chang, 1966; Pothuluri et al., 1986),
genotypes with shallow basal roots have superior phosphorus acquisition capacity, as observed in the greenhouse and
in the field (Lynch and Brown, 2001; Rubio et al., 2003).
Although the main focus of the present work is on individual roots, a rough estimation of phosphorus influx
capacity on a whole plant basis can be made using the
phosphorus influx data obtained here and length data of
different root classes in 14-d-old bean plants (cv. Carioca,
total root length 35.14 m) reported previously (Lynch and
van Beem, 1993). An influx capacity of 3.8 nmol P minÿ1
plantÿ1 is estimated using both sources of information if
roots in contact with a 5 lM P solution are considered as an
example. Uptake rates at a given external nutrient concentration can be derived from published values of uptake
kinetics (Km, Vmax, Cmin). For example, from data reviewed
by Tinker and Nye (2000; see their Table 5.4), uptake rates
of 5.5, 2.8, and 6.1 nmol P minÿ1 plantÿ1 can be estimated
for onion, rape, and barley, respectively. For Paspalum
dilatatum, a wild plant, a rate of 1.7 nmol P minÿ1 plantÿ1
can be estimated from data published by Rubio et al. (1997).
To calculate these rates, total root length and external
phosphorus concentration were unified to values used for
the common bean example (35.14 m and 5 lM P).
It has been proposed that phosphorus is either rather
evenly absorbed over the entire root surface of annual plants
(Bowen, 1969; Rovira and Bowen, 1970; Russel and
Clarkson, 1975; Russel and Sanderson, 1967) or is absorbed
most actively in the apical regions of the root axis (Clarkson
et al., 1978). In agreement with the first group of reports,
relatively homogenous influx rates were observed along root
axes. However, basal roots showed a heterogeneous pattern
of phosphorus influx along the root axes, but only when
influx was calculated on a root length basis. The fact that this
spatial heterogeneity disappeared when influx was calculated on an area basis indicates that root diameter is a primary
factor regulating the pattern of influx along the root axis.
These results cannot be directly compared with most
published reports because a great majority of them consider
2278 Rubio et al.
longer periods of exposure to the labelled source, commonly
more than 20 min and up to 24 h in some cases (Clarkson
et al., 1978). As noted above, in such a period of time, not
only influx but also translocation processes are responsible
for the pattern of phosphorus localization in different root
sections. Experiments with long periods of exposure are
reliable indicators of all the processes involved in nutrient
acquisition acting in conjunction and of the capacity of the
different root sections to export phosphorus from the root to
the shoot. However, they are not appropriate for distinguishing the spatial pattern of phosphorus influx. In this study’s
results, root diameter variations along the root axis accounted for the differences in influx per unit length, since no large
differences were found when the results were expressed as
per unit surface area. A similar tendency was observed by
Russel and Sanderson (1967), who analysed phosphorus
localization in the root axis after 24 h exposure to a labelled
source. The substantial homogeneity in the pattern of
phosphorus influx detected along the root axis expressed
in this way could be related to the fact that the plants in this
study were all young (18 d) and all their roots were actively
growing. It is interesting to note, however, that no substantial
differences were found in the influx between the vicinity of
the apex and the vicinity of the base of the basal roots.
Considering that the basal roots emerge from the hypocotyl
3–4 d after germination, the age difference between the tip
and the base of the basal roots of the plants was approximately 2 weeks. By contrast, in maize, Gao et al. (1998)
observed a sharp decrease in the phosphorus uptake capacity
between the 5th and the 10th day of root growth, while older
roots showed a more gradual decline. The results of this
study are consistent with recent findings showing that
phosphorus carriers are present in all parts of the root
system, in spite of the anatomical dissimilarities and levels of
maturation of the different sections (Muchhal and Raghothama, 1999). In the field, the actual pattern of phosphorus
influx in the root system will result from the interplay of
phosphorus influx capacity with the spatial pattern of soil
phosphorus availability. In this context, Figs 7, 8, and 9 are
illustrative of the large dependence of phosphorus influx on
the phosphorus concentration in the soil solution. It can be
expected that the newest segments of the root system (those
close to the apex) in the field will face soil domains richer in
phosphorus than the oldest ones, which experience a rhizosphere already depleted of phosphorus.
Conclusions
The method described in this paper offers a convenient
protocol to study phosphorus influx into roots. The method
uses digital autoradiography to visualize and quantify
phosphorus influx into intact root systems. It permits
accurate quantification of the spatial pattern of phosphorus
influx. Following the protocol described here, maps of
nutrient net influx capacity and nutrient kinetic parameters
can be obtained and applied to the study of a broad range of
issues. This information may assist in the construction of
models of nutrient uptake by indicating that, for young
plants, it is reasonable to assume a constant phosphorus
influx along the root system but differences among root
classes must be taken into account.
Acknowledgements
We acknowledge support from USDA/NRI grant 9900632 and NSF
grant IBN-0135872 to JPL and KM Brown. We thank Robert H
Snyder for his technical assistance and Robert T Simpson for
permitting access to his laboratory facilities.
Appendix
Example of calculations of phosphorus influx for an individual
segment of root
Individual: plant no. 56
Root type: basal root
Root segment analysed: 0–2 mm (from root tip)
Phosphorus treatment: High
Concentration of uptake solution: 1 lM P
Radioactivity of the uptake solution: 7573 cpm mlÿ1 (measured by
scintillation counting).
Volume of the uptake solution: 180 ml
Total radioactivity: 75733180=1 363 140 cpm in 180 ml
Plant no. 56 was assigned to 1 lM P, hence in a volume of 180 ml
there is 0.18 lmol P
Specific activity is the cpm of 32P lmolÿ1 P: 1 363 140/0.18=7 573
000 cpm lmolÿ1 P
Pimager measurement of basal root no. 56 at 0–2 mm: 441.2 counts
Transformation of Pimager counts to cpm by standard curve
calibration (this calibration is made for each group of roots as shown
in Fig. 4):
For plant no. 56: cpm=0.0233Pimager counts–0.29;
since counts=441.2, then cpm=9.857
Transformation from radioactivity (cpm) to lmol phosphorus by
specific activity:
9.857 cpm/7 573 000 cpm lmolÿ1 P=1.29310ÿ6 lmol P
lmol P in the first 2 mm: 1.29310ÿ6 lmol P
Time of exposure to nutrient solution: 4 min
P influx per min=1.29310ÿ6/4
P influx rate: 3.23310ÿ7 lmol P mmÿ1 min ÿ1
P influx rate: 0.323 pmol P mmÿ1 min ÿ1
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