Plant Physiol. (1999) 120: 705-716
Linking Development and Determinacy with Organic Acid Efflux from
Proteoid Roots of White Lupin Grown with
Low Phosphorus and Ambient
or Elevated Atmospheric
CO2 Concentration1
Michelle Watt and
John R. Evans*
Environmental Biology Group, Research School of Biological
Sciences, Australian National University, P.O. Box 475, Canberra,
ACT, Australia 2601
 |
ABSTRACT |
White lupin (Lupinus
albus L.) was grown in hydroponic culture with 1 µM phosphorus to enable the development of proteoid roots
to be observed in conjunction with organic acid exudation. Discrete
regions of closely spaced, determinate secondary laterals (proteoid
rootlets) emerged in near synchrony on the same plant. One day after
reaching their final length (4 mm), citrate exudation occurred over a
3-d pulse. The rate of exudation varied diurnally, with maximal rates
during the photoperiod. At the onset of citrate efflux, rootlets had
exhausted their apical meristems and had differentiated root hairs and
vascular tissues along their lengths. Neither in vitro
phosphoenolpyruvate carboxylase nor citrate synthase activity was correlated with the rate of citrate exudation. We suggest
that an unidentified transport process, presumably at the plasma
membrane, regulates citrate efflux. Growth with elevated (700 µL
L
1) atmospheric [CO2] promoted earlier
onset of rootlet determinacy by 1 d, resulting in shorter rootlets
and citrate export beginning 1 d earlier as a 2-d diurnal pulse.
Citrate was the dominant organic acid exported, and neither the rate of
exudation per unit length of root nor the composition of exudate was
altered by atmospheric [CO2].
| |
INTRODUCTION |
Proteoid roots develop on a range of plant species, including
white lupin (Lupinus albus L.), adapted to environments with poorly available phosphorus (Gardner et al., 1981
; Dinkelaker et al.,
1995). First described in 1960 in a study of the Proteaceae (Purnell,
1960), proteoid roots have discrete clusters of closely spaced laterals
(rootlets) along their lengths that greatly increase the surface area
for nutrient uptake compared with the proteoid root axis (Lamont et
al., 1984). These clusters of rootlets release exudates, notably
organic acids that can solubilize phosphorus by chelating the metal
ions that immobilize it (Gardner et al., 1983; Gerke et al., 1994). The
organic acids exported from white lupin proteoid roots can include
citrate, malate, succinate, and fumarate (Gardner et al., 1983;
Dinkelaker et al., 1989; Johnson et al., 1994; Keerthisinghe et al.,
1998) and can account for a substantial portion of the total plant
carbon. For example, Dinkelaker et al. (1989) showed that citrate
exported from white lupin growing in a calcareous soil was 23% of the
plant dry weight at 13 weeks, and Johnson et al. (1996b) measured
malate, succinate, and citrate totaling 12% of the plant dry weight at
3 weeks.
The metabolism linked to the synthesis and efflux of organic acids from
proteoid roots has been studied recently in white lupin (Johnson et
al., 1994, 1996a, 1996b). Johnson et al. (1996a) showed that
approximately 30% of the carbons released from the roots as malate or
citrate were fixed within the proteoid roots by the enzyme PEPC (EC
4.1.1.31). The increased activity and expression of PEPC and malate
dehydrogenase (EC 1.1.1.37) coincided with the start of organic acid
efflux and was considered to be part of an altered metabolism within
proteoid roots that was required for the synthesis of the exported
organic acids (Johnson et al., 1996b).
The morphology and anatomy of proteoid rootlets was reported previously
for some members of the Proteaceae (for summary, see Dinkelaker et al.,
1995; Skene et al., 1996, 1998a, 1998b). These studies showed that a
cluster of rootlets starts as many meristematic primordia that
subsequently mature into determinate rootlets with no apical meristem
and root hairs around their tips. In Hakea obliqua,
approximately 5 d is required for the rootlets to reach their
final determinate length (Dell et al., 1980). To our knowledge, there
are no published studies of the anatomy of white lupin proteoid rootlets, although macrographs of white lupin rootlets growing in
calcareous soil show root hairs extending around their tips (Dinkelaker
et al., 1989), suggesting that the rootlets also reach a determinate
stage.
There is evidence that the efflux of exudates, including organic acids,
occurs when rootlets are young and that this efflux is transient
(Dinkelaker et al., 1995; Neumann et al., 1995; Keerthisinghe et al.,
1998). Keerthisinghe and co-workers (1998) collected exudates along a
proteoid root axis of white lupin and found that most of the efflux
occurred in the youngest portion of the root axis, where the rootlets
were young, and that the in vitro activities of PEPC and malate
dehydrogenase were not strictly correlated with citrate fluxes from the
same portion of root. The transition from primordial tissue to fully
differentiated, determinate root tissue during rootlet growth suggests
that enzymatic changes associated with development could be confused
with those associated with exudation and, as suggested by Dinkelaker et
al. (1995), elucidation of the mechanisms related to exudation requires
a detailed time-course study of rootlets of different ages. To our
knowledge, there have been no such studies linking anatomical changes
with biochemical changes and the efflux of organic acids on the
proteoid rootlets of any species.
In the present study, we applied the root-incubation chamber used by
Keerthisinghe et al. (1998) to study proteoid rootlets of white lupin
from the time of emergence through d 8. Our first objective was to
resolve the developmental and metabolic stages associated with efflux
of organic acids by performing a detailed time-course study of rootlet
anatomy and biochemistry for plants grown with low phosphorus and
ambient atmospheric [CO2].
Increases in atmospheric [CO2] can alter root
growth and turnover (for review, see Rogers et al., 1994; for example,
see Fitter et al., 1996) and can increase the amount of
carbon-containing compounds exported to the rhizosphere (Paterson et
al., 1997). However, there have been few studies measuring the quantity
and quality of exudates on a per-root basis under ambient and elevated [CO2] (Sadowsky and Schortemeyer, 1997),
particularly those exudates that function in nutrient acquisition, such
as organic acids. If such exudates are increased under elevated
atmospheric CO2, they may confer an advantage to
those species with these processes (Gifford et al., 1996; DeLucia et
al., 1997). Because white lupin exports a large amount of carbon as
citrate from proteoid lateral roots with defined, determinate
development, they are an ideal system with which to study the effects
of atmospheric [CO2] on root and exudate
processes. Our second objective was to investigate the effect of
atmospheric [CO2] on proteoid root growth,
development, and efflux.
| |
MATERIALS AND METHODS |
Plant Growth
White lupin (Lupinus albus L. cv Kiev mutant) was grown
in ambient (350 µL L1) or elevated (700 µL
L1) atmospheric CO2 in
climate-controlled growth cabinets with a 12-h photoperiod, 600 µmol
m2 s1 light at leaf
level, 70% RH, and 15°C/22°C night/day temperatures. Seeds were
germinated in damp sand and at d 6 (2 d after cotyledon emergence) were
transferred to black, 22-L hydroponics tanks. Four seedlings per tank
were supported by removable foam discs that fit into the lid. Each tank
contained a solution of 1 µM KH2PO4, 0.25 mM CaCl2, 0.7 mM KNO3, 0.25 mM MgSO4, 11 µM
H3BO3, 2 µM MnSO4, 0.35 µM ZnSO4, 0.2 µM CuSO4, and 6 µM ferric EDTA and was adjusted daily to pH 6.0 (Keerthisinghe et al., 1998). Each day, phosphate levels were assayed
using Malachite green dye and replenished to 1 µM (Irving and McLaughlin, 1990; Keerthisinghe et al., 1998). The complete solution in the tanks was changed weekly or
biweekly. Preliminary experiments indicated that nitrate had been
depleted by less than 20% between solution changes. The nutrient
solution was aerated continuously from rings with small perforations
supported at the bottom of the tanks.
Scoring Proteoid Root Development
The working definition of a proteoid root was a primary lateral
root with defined clusters of more than 10 secondary lateral roots
(proteoid rootlets) per centimeter (Figs.
1 and 3; Johnson et al., 1996b). The
emergence of these clusters of proteoid rootlets was scored daily for 3 to 4 weeks by removing a plant from the hydroponics tank, spreading the
root system in a shallow dish of water, and counting clusters that had
recently emerged from the root cortex (rootlets 0.5-1.0 mm long).
View larger version (120K):
[in this window]
[in a new window]
| Figure 1.
Root system of an 18-d-old white lupin plant
germinated on sand for 6 d and then transferred to nutrient
culture with 1 µM phosphorus and 350 µL
L1 atmospheric [CO2]. All of the primary
basal laterals have become proteoid roots and the first cluster of
secondary laterals (proteoid rootlets) has fully emerged. The basal
laterals are longer and thicker than the thinner, shorter, acropetal
primary laterals. Scale bar = 1 cm.
|
|
View larger version (100K):
[in this window]
[in a new window]
| Figure 3.
Clusters of rootlets along proteoid roots of white
lupin grown in nutrient solution with 1 µM phosphorus and
350 µL L1 atmospheric [CO2]. A cluster is
defined as a length of root with more than 10 rootlets per centimeter
that have emerged in near synchrony. A, Proteoid root of a 32-d-old
plant. Clusters of emerged rootlets are numbered. Studies relating
development with citrate efflux were done on the fourth cluster of
rootlets. Arrow indicates the sixth cluster. B, Developmental series of
a proteoid root cluster. Rootlets emerged in near synchrony on d 1 and
developed to similar final lengths of approximately 4 mm on d
4.
|
|
In Situ Collection of Root Exudates
Exudates were collected from incubated clusters of proteoid
rootlets attached to the plant in their growing environments (Ryan et
al., 1993; Keerthisinghe et al., 1998, see figure 1 therein). Plants
were transferred to tanks partially filled with nutrient solution and
the proteoid roots were supported on trays in the tanks. A Perspex
resin incubation ring, 2 cm in diameter and 1.2 cm in height, with two
small notches to fit over the axis of the lateral root, was sealed
around a cluster of developing rootlets with silicon grease. Nutrient
solution (2 mL) was placed around the isolated cluster and the tank was
filled to cover the rest of the root system. In all experiments the
solution in the ring was replaced every 6, 12, or 18 h with fresh
nutrient solution. Keerthisinghe et al. (1998) reported that
degradation of citrate did not occur in the incubation rings, so no
precautions were taken to prevent the breakdown of organic acids during
exudate collection in the experiments reported here. Any breakdown
would have resulted in an underestimation of exported organic acids. Once collected from the rings, the solutions with the exudates were
immediately frozen and stored at 
20°C until organic acid analysis.
Exudates were collected from the fourth cluster of proteoid rootlets
developing on the basal laterals on both ambient- and elevated-[CO2]-grown plants when the plants
were 26 d old (Figs. 2 and
3). In one experiment plants were grown
with either ambient or elevated [CO2], and
exudates were collected continuously for 6 to 8 d, from the time
that the rootlets emerged from the cortex and were 0.5 to 1 mm long
(Fig. 3B). The cluster was photographed daily in the incubation ring
for correlation of rootlet growth with exudate efflux. Rootlet length
was measured directly from photographs using a digitizing tablet. Eight
rootlets per cluster and 12 clusters from four plants per
CO2 treatment were measured.
View larger version (23K):
[in this window]
[in a new window]
| Figure 2.
Emergence of clusters of proteoid rootlets scored
daily on a white lupin plant grown in nutrient solution with 1 µM phosphorus and 350 µL L1 atmospheric
[CO2]. Newly formed clusters emerged simultaneously in
discrete pulses on all of the proteoid roots of the root system. The
plot is of one representative plant; eight other plants exhibited
similar patterns of cluster emergence. The primary laterals emerged
when seedlings were 8 d old.
|
|
In the second experiment plants were grown with ambient
[CO2] only. After the developmental time course
shown in Figure 3B, exudates were collected for 24 h, and then the
cluster was harvested. A sample of two to three rootlets plus
approximately 1 mm of the adjoining main axis was excised from each
harvested cluster and immediately fixed in glutaraldehyde on ice for
anatomical studies. The remaining tissue was immediately frozen in
liquid nitrogen for enzymatic studies.
Analysis of Exudates
Enzyme Assay for Citrate
Glycylglycine buffer (100 mM with 0.2 mM
ZnCl2, pH 7.9), 0.3 mM NADH, 600 kilounits L1 lactate dehydrogenase (EC
1.1.2.3), and 600 kilounits L1 malate
dehydrogenase were combined with 1 mL of thawed exudate, mixed
thoroughly, and read at 340 nm in a spectrophotometer. Citrate lyase
(EC 4.1.3.6) was added (final activity in cuvette 40 kilounits L1), and the absorbance was monitored until it
stabilized. The decrease in absorbance was proportional and
stoichiometric to the amount of citrate present in the reaction
(Möllering, 1985; Keerthisinghe et al., 1998).
HPLC for Organic Acids
Exudates were thawed and 1 mL was passed through a 0.45-µm
pore-size filter with a syringe. The filtrate was evaporated to dryness
in a freeze drier (Speed Vac, Savant Instruments, Holbrook, NY),
redissolved with 40 to 100 µL of 13 mM
H2SO4, and spun at 13,000 rpm for 5 min. Twenty-five microliters of the supernatant was injected
into an HPLC (model 1090M, Hewlett-Packard) that was fitted with an
ion-exclusion column (300 × 7.8 mm; HPX-87H Aminex, Bio-Rad) and
an organic acid guard column (Bio-Rad). The mobile phase was 13 mM H2SO4 run at
0.5 mL min1 at 60°C. The acids were detected
at 210 nm with a photodiode-array UV detector. Standards for oxalic
acid, citric acid, 
-ketoglutaric acid, malic acid, succinic acid,
pyruvic acid, and fumaric acid were made in distilled water at
concentrations within the ranges found in the exudates. As with the
samples, the standards were filtered, evaporated, suspended in 13 mM H2SO4,
centrifuged, and then run through the HPLC system individually or as a
mixture. Linear standard curves were generated for each acid from the
areas under the peaks corresponding to different concentrations. The standard curves were used to quantify acids in the exudates. To verify
running conditions, a mixture of the standard acids was run before each
batch of samples was analyzed.
Specific Enzyme Activities and Protein Content
Each root sample was frozen, transferred to a chilled, 2-mL glass
homogenizer, and ground on ice for 1 to 2 min in 250 µL of freshly
prepared grinding solution (50 mM Hepes, 5 mM
MgCl2, 1 mM EDTA, 1 mM
EGTA, 0.1% Triton X-100, 10% glycerol, 0.5 mM PMSF
dissolved in isopropanol, 5 mM DTT, 2 mM
benzamidine, and 2 mM 
-amino-n-caproic acid).
The ground sample was divided into three aliquots, snap-frozen in
liquid nitrogen, and stored at 80°C until analysis. Just before
analysis, samples were thawed on ice and centrifuged at 13,000 rpm for
10 min at 4°C; the supernatant was maintained on ice.
PEPC activity in the tissue supernatant was measured by monitoring the
oxidation of NADH at 340 nm in a spectrophotometer (Vance et al.,
1983). The tissue supernatant (40-80 µL) was initially incubated for
10 min at 25°C in 100 mM Bicine, pH 8.0, with 5 mM MgCl2, 10 mM
NaHCO3, 0.16 mM NADH, and 60 units of
malate dehydrogenase. Next, 15 µL of 100 mM PEP was added
and the decrease in absorbance per unit of time was measured.
Citrate synthase (EC 4.1.3.7) activity was measured by following the
rate of 3-acetylpyridine adenine dinucleotide reduction at 365 nm in a
spectrophotometer (Stitt, 1983). Tissue supernatant (60-100 µL) was
incubated with 100 mM triethanolamine, pH 8.5, 3.5 mM malate, 0.3 mM 3-acetylpyridine adenine
dinucleotide, and 30 units of malate dehydrogenase at 25°C until the
absorbance had stabilized (approximately 15 min). Next, 15 µL of 10 mM acetyl-CoA was added and the increase in absorbance per
unit of time was measured. Protein in the tissue supernatant was
estimated using the Coomassie Plus protein assay reagent kit (Pierce).
Tissue Preservation and Staining for Anatomy
Rootlets were fixed in 3% glutaraldehyde in 25 mM potassium-phosphate buffer, pH 6.8, on ice overnight,
rinsed four times for 15 min each in buffer, postfixed in 1% osmium
tetroxide for 2 h, and rinsed three times for 15 min each in
buffer. The tissue was taken through a gradual dehydration series from
4% to 100% ethanol over 2 d on ice and then at room temperature
and slowly infiltrated with Spurr's resin starting with 2.5% in
ethanol and reaching 100% in 2 d. The Spurr's resin was replaced
daily for 5 d and then the resin-embedded tissue was polymerized
at 70°C overnight.
Transverse and longitudinal sections (2-3 µm thick) of
resin-embedded material were cut with a glass knife, transferred to drops of water on gelatin-coated glass slides, and dried on a hot plate
for 1 h. Sections were first stained with 1% toluidine blue in
borate, pH 11.0, and viewed with bright-field optics to show general
anatomy and development. To visualize phloem differentiation, the resin
was etched from the sections with sodium ethoxide (1-2 min), rinsed
with 70% ethanol, and then rinsed for 1 min in running tap water. The
sections were then stained with Schiff's reagent for 4 min to reduce
background wall autofluorescence, rinsed for 5 min in running tap
water, and stained with 0.05% aniline blue in 67 mM
potassium-phosphate buffer, pH 8.6, for 2 h. Sections were
mounted in fresh aniline blue and viewed with UV fluorescence optics (Axioplan microscope, Zeiss). The callose deposited in the walls
and plates of the sieve tubes fluoresces bright blue/green and could
therefore be distinguished from lignin and suberin autofluorescence (O'Brien and McCully, 1981).
| |
RESULTS |
Root System Architecture and Proteoid Root Morphology
We found in white lupin a taproot system with approximately 20 thick, indeterminate, basal primary laterals in addition to the
thinner, shorter, acropetal primary laterals (Fig. 1). All of the basal
primary laterals became proteoid roots, developing distinct clusters of
closely spaced, determinate, secondary laterals or proteoid rootlets.
Few of the thinner, acropetal laterals became proteoid roots as the
plant aged. The clusters of proteoid rootlets emerged in near synchrony
on all of the proteoid roots of the root system, regardless of root
length (Fig. 2). The emergence of the first cluster of rootlets was
predictable, occurring 7 d after the emergence of the basal
laterals when the plant was approximately 2 weeks old. This first
cluster often appeared on 7 to 15 basal primary laterals; the second
and third clusters recruited more basal laterals. By the third cluster,
a plateau was reached in the number of primary laterals becoming
proteoid roots (Fig. 2).
An example of a basal primary lateral from a 32-d-old plant is shown in
Figure 3A. Five root clusters are shown, with a sixth cluster just
emerging from the cortex. The length and spacing of the root axis of
each cluster varied; for the earlier clusters the morphology was more
variable. For this study, all data were collected from the fourth
cluster. A time course of rootlet development of this cluster is shown
in Figure 3B. Within a cluster, almost all rootlets emerged from the
cortex in near synchrony; they grew and developed at similar rates,
with an occasional rootlet reaching 2 to 3 times the length of other
rootlets.
Effect of Atmospheric [CO2] on Proteoid Rootlet
Elongation and Efflux of Organic Acids
Rootlet length was measured on clusters restrained in the
incubation rings. Rootlets grew rapidly for the first 2 d,
reaching their final 4-mm length 4 d after emergence from the
cortex in plants grown with ambient [CO2] (Fig.
4). Elevated atmospheric [CO2] resulted in the rootlets stopping growth
after only 3 d, reaching only a 2.5-mm final length.
View larger version (19K):
[in this window]
[in a new window]
| Figure 4.
Lengths of proteoid rootlets of white lupin grown
in nutrient culture with 1 µM phosphorus. Rootlets grown
with 700 µL L1 atmospheric [CO2] ( )
reached a shorter final length 1 d earlier than rootlets grown
with 350 µL L1 atmospheric CO2 ( ). Each
point represents the mean ± SE of 12 roots from four
plants per CO2 treatment.
|
|
At both CO2 concentrations, the onset of citrate
efflux occurred after the rootlets stopped elongating; onset was 1 d earlier in plants grown with elevated atmospheric
[CO2] (Fig. 5).
Citrate efflux continued for 2 d in the
elevated-[CO2]-grown plants and for 3 d in
the ambient-[CO2]-grown plants. Peaks in efflux
occurred during the day under both CO2
treatments, and the peak efflux rate per unit of length of proteoid
root was not altered by atmospheric [CO2].
Citrate was the dominant organic acid exuded from the roots, being
60-fold higher than malate at peak efflux of citrate (Table I). Other organic acids were detected at
very low concentrations and were unaffected by the atmospheric
[CO2].
View larger version (42K):
[in this window]
[in a new window]
| Figure 5.
Citrate efflux from developing clusters of
proteoid roots of white lupin grown in nutrient culture with 1 µM phosphorus. Shading indicates dark periods. Each point
represents the mean ± SE of 12 roots from four plants
per CO2 treatment. A, Plants grown with 350 µL
L1 atmospheric [CO2]. Citrate efflux began
4 d after rootlet emergence, when rootlets had stopped elongating,
and lasted 3 d, with strong peaks during light periods. B, Plants
grown with 700 µL L1 atmospheric [CO2].
Citrate efflux began 3 d after rootlet emergence when rootlets had
stopped elongating and lasted 2 d with strong peaks during light
periods.
|
|
View this table:
[in this window]
[in a new window]
|
Table I.
Rates of efflux of organic acids detected around
proteoid roots before, during, and after peaks in efflux activity
Plants were grown with ambient (350 µL L1) or elevated
(700 µL L1) atmospheric [CO2], and
exudates were collected in situ with incubation rings on roots for
1 week. Each value represents the mean ± SE of
exudates from one root from each of three to six plants.
|
|
Rootlet Development and Anatomy
The development and anatomy of the proteoid rootlets are
illustrated in Figures 6 and
7 and summarized in Table
II. One day after emergence from the
proteoid root, the rootlets were almost entirely meristematic (Figs. 6A
and 7F); only the cells of the rootlet cortex within the proteoid root
had started to vacuolate and elongate. The phloem sieve tubes were
visible at approximately six cell lengths from the phloem of the stele
of the proteoid root axis, and extended over only 25% of the rootlet
length. The root cap adhered tightly to the rootlet tip; at this stage
no root hairs had developed. On d 2, many rootlet cells were developing vacuoles and elongating, whereas cells toward the tip of the stele and
cortex still had high amounts of cytoplasm (Fig. 7G). Root hairs were
beginning to develop; root-cap cells were sloughing from the epidermis
and could be seen along the length of the rootlet; and callose in the
phloem sieve tubes had differentiated along 60% of the rootlet length
(Fig. 7, G and L).
View larger version (84K):
[in this window]
[in a new window]
| Figure 6.
Cross-sections (3 µm thick) through the tips of
resin-embedded proteoid rootlets of white lupin grown in nutrient
culture with 1 µM phosphorus and 350 µL
L1 atmospheric [CO2]. A, Rootlet 1 d
after emergence from the cortex of the proteoid root. All of the cells
shown are meristematic except for the root-cap cells, which are tightly
anchored to the epidermis. Magnification = ×280. B, Rootlet
4 d after emergence from the cortex. The apical meristem is no
longer present and all cells in the rootlet have vacuolated and
differentiated. The epidermis has differentiated root hairs to the tip
of the rootlet, and the root-cap cells are loosely anchored.
Magnification = ×300. C, Rootlet 8 d after emergence from
the cortex. The stele has two xylem and two phloem poles and is
surrounded by an endodermis with a Casparian band. Sections from d 1 and 4 were stained with toluidine blue and viewed with bright-field
optics. The section from d 8 was etched after sectioning to remove the
resin, stained with Schiff's reagent, mounted in aniline blue, and
viewed with fluorescence optics. Black arrowheads, Root hairs; white
arrowheads, Casparian bands; white arrow, phloem sieve tube; c, root
cap; x, xylem vessel. Magnification = ×1000.
|
|
View larger version (162K):
[in this window]
[in a new window]
| Figure 7.
Development from 1 d after emergence
from the cortex of proteoid rootlets from white lupin grown in nutrient
culture with 1 µM phosphorus and 350 µL
L1 atmospheric [CO2]. Left, Whole mounts of
glutaraldehyde- and osmium-fixed rootlets embedded in resin. Center,
Longitudinal sections (3 µm thick) through rootlets shown at left
stained with toluidine blue (F, G, and I) or left unstained and viewed
with differential image contrast bright-field optics (H and J). Right,
Longitudinal sections (3 µm thick) etched to remove resin, stained
with Schiff's reagent followed by aniline blue, and viewed with UV
fluorescence optics. Arrows, Points of visible callose deposition in
the phloem; x, xylem secondary wall; arrowheads, root hairs; c, root
cap. At d 1, rootlets are almost entirely meristematic. At d 2, most of
the cells are developing vacuoles, root hairs have started to develop
at the base, and the phloem callose extends to 75% of the rootlet
length. At d 3, the apical meristem is completely exhausted, and root
hairs and vascular tissues have differentiated along the length of the
rootlet. Days 4 and 6 are similar to d 3. Refer to text for more
details. Rootlet diameter was approximately 225 µm. Magnification: A
to E, ×60; F and G, ×100; H and M, ×180; I, J, L, and O, ×170; K,
×150; and N, ×425. The root tip in L, J, and O is pointing up.
|
|
View this table:
[in this window]
[in a new window]
|
Table II.
Development and citrate efflux of proteoid
rootlets, ranging from none detected () to maximum (+++), in
plants grown with ambient [CO2]
|
|
By d 3, rootlets were approaching their final length (Fig. 4), all
cells had vacuolated, and the apical meristem was no longer present.
Root hairs were long and dense toward the base of the rootlet, and
epidermal cells around the tip were just developing hairs (Fig. 7, C
and H). The distribution of root-hair growth along the rootlet varied,
however, with some rootlets showing very dense proliferation toward the
tip and others toward the base. The phloem sieve tubes and protoxylem
secondary walls were visible to within five cell lengths of the rootlet
tip, indicating that the stele had differentiated along more than 95%
of the rootlet length (Fig. 7H).
By d 4, citrate efflux had started (Fig. 5A) and root hairs continued
to expand (Fig. 7D). The root cap was loosely anchored and sloughed
clumps of root-cap cells were often associated with root hairs (Figs.
6B and 7I). Rootlet development from d 5 to 8 was similar to that seen
at d 4, although root hairs continued to develop until d 6. A
completely differentiated rootlet had sloughed root-cap cells and an
epidermal layer with root hairs had differentiated around the tip of
the rootlet. Four layers of cortical cells (the innermost being
an endodermal layer with a suberized Casparian band) are shown in
Figure 6C, and a stele occupying approximately 10% of the rootlet (in
cross-section) is shown in Figure 6B. The stele had two phloem poles
and two protoxylem poles (with spiral secondary wall thickenings; Figs. 6C and 7H).
The dry weight of clusters of rootlets increased between d 1 and 5, plateaued, and then dropped at d 8 (Fig.
8A). Fresh weight increased abruptly on d
3 and 4 because of root-hair development and vacuolation of cells (Fig.
8B); as a consequence, the soluble protein content per unit of fresh
weight fell sharply (Fig. 8C). The soluble protein content per unit of
root length increased steadily to a peak on d 3 before declining again
(Fig. 9C).
View larger version (14K):
[in this window]
[in a new window]
| Figure 8.
Dry weight (A), fresh weight (B), and soluble
protein content (C) of proteoid root clusters of white lupin grown in
nutrient culture with 1 µM phosphorus and 350 µL
L1 atmospheric [CO2]. Each point represents
the mean ± SE of one root from each of four to six
plants.
|
|
View larger version (15K):
[in this window]
[in a new window]
| Figure 9.
In vitro PEPC (A) and citrate synthase activities
(B) per unit of soluble protein of developing proteoid root clusters
from white lupin grown in nutrient culture with 1 µM
phosphorus and 350 µL L1 atmospheric
[CO2]. C, Soluble protein peaks 3 d after rootlet
emergence. Each point represents the mean ± SE of one
root from each of four to six plants.
|
|
Enzyme Activities and Citrate Efflux
In vitro PEPC activity per unit of protein peaked 3 d after
rootlet emergence, doubling in activity between d 1 and 3 before decreasing to 25% of peak activity by d 8 (Fig. 9A). Citrate synthase activity per unit of protein declined by 50% on d 1, after which it
plateaued for 5 d before declining again (Fig. 9B).
Both PEPC and citrate synthase had maximal activity per unit length of
proteoid root 3 d after rootlet emergence, preceding the onset of
citrate efflux by 1 d and the peak of efflux by 2 d (Fig.
10). PEPC and citrate synthase had
approximately 4- and 3-fold increases in activity, respectively,
between d 1 and 3. Citrate efflux from the developmental time-course
samples that were harvested daily lasted 3 d, with efflux peaks
during photoperiods (Fig. 10C). This length of time was similar to that
of the efflux from developing roots maintained in the incubation rings
for 8 d (Fig. 5A); however, efflux from roots collected from rings
in place for just 24 h was twice that of the roots maintained in rings for 8 d. The total amount of citrate exudation was
equivalent to 10% of the proteoid-root dry weight. In vitro activities
of PEPC and citrate synthase were well in excess of citrate-exudation rates at all times.
View larger version (16K):
[in this window]
[in a new window]
| Figure 10.
In vitro PEPC activity (A), citrate synthase
activity (B), and citrate efflux (C) per unit length of white lupin
proteoid root grown in nutrient culture with 1 µM
phosphorus and 350 µL L1 atmospheric
[CO2]. PEPC and citrate synthase activities per unit
length of main root peaked 3 d after rootlet emergence, 1 d
before onset of citrate efflux and 2 d before the peak in citrate
efflux on d 5. Each point represents the mean ± SE of
one root from each of four to six plants.
|
|
| |
DISCUSSION |
Synchronous Development of Proteoid Roots
A striking finding of this study was the predictable and
synchronous development of clusters of rootlets on the proteoid roots in white lupin grown in solution culture (Fig. 2). This implicates a
central signaling cascade for development of clusters of rootlets. Studies showing that the internal phosphorus status of the plant can
determine proteoid root development in white lupin (Marschner et al.,
1987; Keerthisinghe et al., 1998) and wax myrtle (Louis et al., 1990)
support a central signaling mechanism. The signal may start in the
shoot and radiate down to the roots, reaching the basal laterals first.
It is possible that the signaling process is delivered to the roots in
pulses related to the plant phosphorus status, if so, the frequency and
duration would determine the spacing and length of each cluster of
rootlets along the proteoid root axis. As the severity of phosphorus
stress grew, signaling would intensify, thus increasing the proportion
of the root system covered with proteoid rootlets (Keerthisinghe et
al., 1998).
The signals involved in proteoid root development probably include
auxin. Gilbert et al. (1997) were able to use auxin to induce the
production of proteoid rootlets in white lupin when phosphorus was
supplied at a level that normally suppresses their development, and
they could suppress proteoid rootlet formation in minus-phosphorus
treatments by supplying auxin-transport inhibitors to the nutrient
solution. Auxin plays a role in lateral root initiation in other
species (Thimann, 1936; Wightman et al., 1980); in the Arabidopsis
mutant, superroot is responsible for a phenotype that is
very similar to a proteoid root (Boerjan et al., 1995).
The rootlets that developed within clusters along a proteoid root
reached a similar, determinate length, although an occasional rootlet
extended two to three times the length of the other rootlets (Fig. 3A).
Determinacy has been reported in all species that form proteoid roots
(Dinkelaker et al., 1995) but has also been observed in roots from
other types of species (Varney and McCully, 1991; Dubrovsky, 1997). The
average final length of a rootlet varies within and among proteoid
species; recently Skene et al. (1998a) showed that rootlets of
Grevillea robusta were shorter when they developed in
hydroponics compared with development in vermiculite. To our knowledge,
we are the first to report that determinacy varies with environmental
treatment ([CO2]) in a common rooting medium
(Fig. 4), indicating that root determinacy is under internal control.
Controls for root determinacy are unknown, but the switch from
indeterminacy to determinacy in stem nodules has been linked to the
environment and the presence of the hormone ethylene
(Fernández-López et al., 1998). Although auxin is
responsible for initiating lateral root growth, continued exposure to
auxins can inhibit elongation of laterals (Thimann, 1936). In addition,
cytokinins have been shown to suppress lateral root formation (Wightman
et al., 1980). The effects of cytokinins or ethylene on proteoid root
development are not yet known.
Rootlet Development and Citrate Efflux
The anatomy of white lupin proteoid rootlets is similar in most
respects to that of members of the Proteaceae, in which the mature
rootlets have a differentiated apex, root hairs to the tip, a loosely
anchored root cap, an endodermis with a Casparian band, and a diarch
stele (for review, see Dinkelaker et al., 1995; Skene et al., 1998b).
Root-hair development in white lupin differs from that of G. robusta (Skene et al., 1996, 1998a) and Hakea obliqua
(Dell et al., 1980). In G. robusta, root hairs were produced only after the rootlet had reached its final length; they then developed back from the tip. In H. obliqua, few hairs were
produced in water culture, whereas they were produced extensively in
soil.
Citrate efflux began shortly after the rootlets reached their final
length. By that stage, phloem and xylem tissues had differentiated to
the tips of the rootlets, enabling the import of photosynthates and the
export of nutrients mobilized by the citrate exudation. Root hairs
started to develop 2 d after emergence from the cortex, which was
also 2 d before the onset of citrate efflux. This suggests that
the presence of root hairs does not alone determine organic acid
export.
Citrate export lasted only 2 to 3 d and then stopped. Neumann et
al. (1995) also reported transient release of organic acids from a
proteoid root cluster of Hakea undulata, although they did
not document root development. Keerthisinghe et al. (1998) found that
citrate efflux was maximal 1 to 3 cm from the tip and was only
one-tenth that rate from either the 0- to 1-cm or the 5- to 9-cm
region. The protein content of the rootlets peaked 3 d after
emergence and then declined during citrate efflux (Fig. 9C). The
links among determinacy, longevity, metabolism related to senescence,
and efflux of organic acids remain to be investigated.
Some plants export organic anions from their roots upon exposure to Al;
the organic acids chelate the Al, conferring tolerance. The fact that
white lupin can export large amounts of citrate, an excellent chelator
of Al, suggests that it may be tolerant to soils with high levels of
available Al. However, citrate is not exported to the rhizosphere until
the rootlets are fully mature, 2 to 4 d past the time that the
rootlets are dividing and meristematic and are thought to be most
susceptible to Al damage (Ryan et al., 1993). If the emerging rootlets
are not protected from damage by exposure to Al, they will be hindered
in their ability to access phosphorus when they are mature, imposing a
double stress in very acidic soils. To confer Al tolerance, organic
acid efflux from roots should coincide with the time that they are
meristematic.
Enzyme Activities and Citrate Efflux
We did not find a correlation between PEPC activity and the
magnitude of citrate efflux, because the peak in in vitro PEPC activity
preceded the onset of citrate efflux by 1 d and preceded peak
efflux by 2 d (Fig. 10). Furthermore, in vitro citrate synthase activity per unit of protein did not increase during citrate efflux, although citrate was the dominant organic acid exported. Our results contrast with those of Johnson et al. (1994, 1996b), who suggested that
increases in PEPC activity per unit of protein, as well as mRNA
expression and abundance, coincide with organic acid efflux from the
roots, and thus provide necessary carbons for the anapleurotic functioning of the tricarboxylic acid cycle during exudation. In the
studies by Johnson et al. (1994, 1996b), enzyme activities were not
measured on the same tissues from which exudates were collected;
exudates were collected from the entire root system by flushing the pot
with nutrient solution every 2 d, whereas enzyme activities were
measured on root portions pooled from many plants. Although PEPC
activity is clearly involved in citrate synthesis, it does not appear
to determine the rate, onset, or duration of citrate exudation.
Keerthisinghe et al. (1998) made concomitant measurements of citrate
efflux and PEPC activity from 2-cm regions along a proteoid root axis
and found that rates of citrate efflux did not vary proportionally with
PEPC activity, which is in agreement with our findings.
In vitro activities of both PEPC and citrate synthase exceeded the
rates of citrate exudation at every stage; however, these activities
may not reflect the actual rates realized in vivo. The white lupin PEPC
may be regulated by phosphorylation, similar to the regulation of PEPC
in other species (for review, see Vidal and Chollet, 1997). Neumann et
al. (1999) measured similar citrate concentrations (20 µmol
g1 fresh weight) in juvenile, mature, and
senescent proteoid root segments of white lupin, but significant
citrate exudation was observed only in mature proteoid roots.
Therefore, PEPC activity and tissue citrate concentration must not
control the rate of citrate exudation.
We have made preliminary measurements of respiration rates of whole
pieces of proteoid roots that greatly exceed the peak rate of exudation
(4 µmol O2 g1 dry
weight min1 for 0.025 µmol citrate
g1 dry weight min1),
suggesting that the flux through the TCA cycle is unlikely to limit the
rate of citrate exudation. Neumann et al. (1999) measured similar
respiration rates by mature and senescent proteoid root segments of
white lupin, despite significant citrate exudation by mature but not
senescent proteoid roots. Most of the respiration in hydroponically
grown, mature proteoid roots is therefore involved in other metabolic
processes such as ion uptake and amino acid synthesis (Jeschke and
Pate, 1995; Johnson et al., 1996b) and maintenance rather than in
citrate synthesis for exudation.
In the present study, there was a strong effect of day and night on
citrate efflux, with peaks in efflux occurring during the light
periods. To our knowledge, this is the first time a diurnal rhythm has
been reported for organic acids exported from roots. A diurnal rhythm
has been reported for the export of phytosiderophores from graminaceous
roots in response to Fe deficiency (Ma and Nomoto, 1996). The highest
tissue concentrations and effluxes of phytosiderophores also occur in
the light, but it is unclear whether the mechanisms are directly linked
to light or temperature (Ma and Nomoto, 1996).
Our data strongly suggest that citrate export is not simply a result of
PEPC activity supplying the tricarboxylic acid cycle. Another key step
must be regulating the export of citrate from the roots. In a study by
Ryan et al. (1995), neither malate dehydrogenase nor PEPC showed
enhanced specific activities associated with malate efflux from wheat
root tips. The same investigators showed that efflux of malate is
likely to be linked to the activity of an anion transporter that was
detected on the plasma membrane of the root tip cells (Ryan et al.,
1997). The transport mechanism for organic acid efflux from proteoid
roots is not known, although Dinkelaker et al. (1989) concluded that
citrate is excreted concomitantly with protons. It seems likely that an
anion transporter in the plasma membrane is synthesized and becomes
active as rootlet elongation ceases. The transporter is then
inactivated after 2 to 3 d, stopping further exudation. This
transporter could be located in specific cells, but whether those are
in the cortex, the epidermis, or elsewhere is unknown. Incubation of
proteoid roots in solutions of anion-channel blockers reduced citrate
exudation by 50% (Neumann et al., 1999), supporting the existence of
an anion channel.
Effect of Elevated Atmospheric [CO2]
Growth under elevated atmospheric [CO2]
altered the development of root clusters. Rootlets were shorter,
reaching their final length and beginning citrate exudation 1 d
earlier than ambient-[CO2]-grown plants (Figs.
4 and 5). It is unclear how atmospheric [CO2]
alters rootlet determinacy. Atmospheric CO2 can
enhance lateral root turnover in some species and in certain soil
environments (Pregitzer et al., 1995; Berntson and Bazzaz, 1996; Fitter
et al., 1996) and may do so in proteoid rootlets by promoting
exhaustion of the apical meristem and shortening growing time. By
advancing the onset of determinacy and the onset of citrate efflux,
residency time of a proteoid root cluster in a patch of soil may be
decreased. In the present study, elevated atmospheric
[CO2] did not change the rate of citrate efflux
or the composition of background organic acids per unit of length of
proteoid root, although it did shorten the period over which this
efflux occurred (Fig. 5). Unfortunately, the present experimental
technique was not able to provide an integrated picture of efflux for a
whole plant such as can be gained by the method of Johnson et al.
(1996b).
There is a paucity of data regarding measurements of root exudation in
the literature, but there is a great deal of speculation. The data
shown in Figure 5 suggest that an increased efflux from roots under
elevated atmospheric [CO2] would occur if
growth of the basal lateral roots were enhanced. However, given that
the exudation pulse was shorter, this is by no means certain. The white
lupin system illustrates the transient nature of exudation, its
dependence on a specific stage of root development and time of day,
and, even at peaks in efflux, the variability among samples from
similar atmospheric CO2 environments.
Whipps (1985) showed that carbon export per unit of length of maize
root was unaltered by ambient CO2 treatment.
Similarly, Norby et al. (1987) concluded that there was no consistent
effect of elevated [CO2] on root exudation from
pine seedlings, either per unit mass of fine root or as a percentage of
photosynthate. Gifford et al. (1996), measuring citrate efflux per unit
of dry weight of Danthonia root tips, were unable to detect
a significant difference among atmospheric CO2
treatments, although exudation was consistently greater at elevated
[CO2]. DeLucia et al. (1997) detected an
increase in oxalic acid in the rhizosphere of pine seedlings in
response to increased atmospheric [CO2];
however, these investigators did not measure efflux per unit of root
length and did not differentiate between root-derived and
hyphae-derived exudates. Using stable-isotope techniques, Hungate et
al. (1997) showed that the total labile carbon pool, including root
exudation and respiration, was larger in grassland growing with
elevated atmospheric [CO2].
In conclusion, with the limited amount of data available, it is not
possible to conclude that elevated atmospheric
[CO2] alters the rate of carbon exudation from
a given length of root. However, if root production were to be enhanced
by elevated atmospheric [CO2], then overall
exudation could also be enhanced.
| |
FOOTNOTES |
1
This study was funded in part by an Overseas
Postgraduate Award to M.W. from the Australian Government.
*
Corresponding author; e-mail evans{at}rsbs.anu.edu.au; fax
61-2-6249-4919.
Received November 24, 1998;
accepted March 9, 1999.
| |
ABBREVIATIONS |
Abbreviation:
PEPC, PEP carboxylase.
| |
ACKNOWLEDGMENTS |
We are grateful to Gamini Keerthisinghe (Commonwealth Scientific
and Industrial Research Organization Plant Industry, Canberra, Australia) for help with establishing the hydroponics and exudate collection system and to Charlie Hocart (Research School of Biological Sciences, Australian National University) for help with the HPLC analyses.
| |
LITERATURE CITED |
Berntson GM,
Bazzaz FA
(1996)
The allometry of root production and loss in seedlings of Acer rubrum (Aceraceae) and Betula papyrifera (Betulaceae): implications for root dynamics in elevated CO2.
Am J Bot
83:
608-616
Boerjan W,
Cervera MT,
Delarue M,
Beeckman T,
Dewitte W,
Bellini C,
Caboche M,
Van Onckelen H,
Van Montagu M,
Inzé D
(1995)
superroot, a recessive mutation in Arabidopsis, confers auxin overproduction.
Plant Cell
7:
1405-1419
[Abstract]
Dell B,
Kuo J,
Thomson GJ
(1980)
Development of proteoid roots in Hakea obliqua R. Br. (Proteaceae) grown in water culture.
Aust J Bot
28:
27-37
[CrossRef]
DeLucia EH,
Callaway RM,
Thomas EM,
Schlesinger WH
(1997)
Mechanisms of phosphorus acquisition for Ponderosa pine seedlings under high CO2 and temperature.
Ann Bot
79:
111-120
[Abstract/Free Full Text]
Dinkelaker B,
Hengeler C,
Marschner H
(1995)
Distribution and function of proteoid roots and other root clusters.
Bot Acta
108:
183-200
[Web of Science]
Dinkelaker B,
Römheld V,
Marschner H
(1989)
Citric acid excretion and precipitation of calcium citrate in the rhizosphere of white lupin (Lupinus albus L.).
Plant Cell Environ
12:
285-292
[CrossRef]
Dubrovsky JG
(1997)
Determinate primary-root growth in seedlings of Sonoran desert Cactacaea: its organization, cellular basis, and ecological significance.
Planta
203:
85-92
[CrossRef][Web of Science]
Fernández-López M,
Goormachtig S,
Gao M,
D'Haeze W,
Van Montagu M,
Holsters M
(1998)
Ethylene-mediated phenotypic plasticity in root nodule development on Sesbania rostrata.
Proc Natl Acad Sci USA
95:
12724-12728
[Abstract/Free Full Text]
Fitter AH,
Self GK,
Wolfenden J,
van Vuuren MMI,
Brown TK,
Williamson L,
Graves JD,
Robinson D
(1996)
Root production and mortality under elevated atmospheric carbon dioxide.
Plant Soil
187:
299-306
Gardner WK,
Parbery DG,
Barber DA
(1981)
Proteoid root morphology and function in Lupinus albus.
Plant Soil
60:
143-147
[CrossRef]
Gardner WK,
Parbery DG,
Barber DA
(1983)
The acquisition of phosphorus by Lupinus albus L. III. The probable mechanism by which phosphorus movement in the soil/root interface is enhanced.
Plant Soil
70:
107-124
[CrossRef]
Gerke J,
Römer W,
Jungk A
(1994)
The excretion of citric and malic acid by proteoid roots of Lupinus albus L.: effects on soil solution concentrations of phosphate, iron, and aluminum in the proteoid rhizosphere in samples of an oxisol and luvisol.
Z Pflanzenernähr Dueng Bodenkd
157:
289-294
Gifford RM,
Lutze JL,
Barrett D
(1996)
Global atmospheric change effects on terrestrial carbon sequestration: exploration with a global C- and N-cycle model (CQUESTN).
Plant Soil
187:
369-387
[CrossRef]
Gilbert GA,
Knight DJ,
Vance CP,
Allan DL
(1997)
Does auxin play a role in the adaptations of white lupin roots to phosphate deficiency (abstract no. 67)?
Plant Physiol
114:
S-31
Hungate BA,
Holland EA,
Jackson RB,
Chapin FS III,
Mooney HA,
Field CB
(1997)
The fate of carbon in grasslands under carbon dioxide enrichment.
Nature
388:
576-579
[CrossRef]
Irving GCJ,
McLaughlin MJ
(1990)
A rapid and simple field test for phosphorus in Olsen and Bray no. 1 extracts.
Commun Soil Sci Plant Anal
21:
2245-2255
Jeschke WD,
Pate JS
(1995)
Mineral nutrition and transport in xylem and phloem of Banksia prionotes (Proteaceae), a tree with dimorphic root morphology.
J Exp Bot
46:
895-905
[Abstract/Free Full Text]
Johnson JF,
Allan DL,
Vance CP
(1994)
Phosphorus stress-induced proteoid roots show altered metabolism in Lupinus albus.
Plant Physiol
104:
657-665
[Abstract]
Johnson JF,
Allan DL,
Vance CP
(1996a)
Root carbon dioxide fixation by phosphorus-deficient Lupinus albus. Contribution to organic acid exudation by proteoid roots.
Plant Physiol
112:
19-30
[Abstract]
Johnson JF,
Allan DL,
Vance CP
(1996b)
Phosphorus deficiency in Lupinus albus. Altered lateral root development and enhanced expression of phosphoenolpyruvate carboxylase.
Plant Physiol
112:
31-41
[Abstract]
Keerthisinghe G,
Hocking PJ,
Ryan PR,
Delaize E
(1998)
Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.).
Plant Cell Environ
21:
467-478
[CrossRef]
Lamont BB,
Brown G,
Mitchell DT
(1984)
Structure, environmental effects on their formation, and function of proteoid roots in Leucadendron laureolum (Proteaceae).
New Phytol
97:
381-390
Louis I,
Racette S,
Torrey JG
(1990)
Occurrence of cluster roots on Myrica cerifera L. (Myricaceae) in water culture in relation to phosphorus nutrition.
New Phytol
115:
311-317
Ma JF,
Nomoto K
(1996)
Effective regulation of iron acquisition in graminaceous plants: the role of mugineic acids as phytosiderophores.
Physiol Plant
97:
609-617
[CrossRef]
Marschner H,
Römheld V,
Cakmak I
(1987)
Root-induced changes of nutrient availability in the rhizosphere.
J Plant Nutr
10:
1175-1184
[Web of Science]
Möllering H
(1985)
Citrate.
In
HU Bergmeyer,
eds, Methods of Enzymatic Analyses, Ed 3, Vol VII.
VCH, Weinheim, Germany, pp 2-12
Neumann G,
Dinkelaker B,
Marschner H
(1995)
Kurzzeitige Abgabe organischer Säuren aus Proteoidwurzeln von Hakea undulata (Proteaceae).
Ökophysiologie des Wurzelraumes
6:
128-136
Neumann G, Marsonneau A, Martinoia ER, Römheld V (1999)
Physiological adaptations to phosphorus deficiency during proteoid root
development in white lupin. Planta (in press)
Norby RJ,
O'Neill EG,
Hood WG,
Luxmoore RJ
(1987)
Carbon allocation, root exudation and mycorrhizal colonization of Pinus echinata seedlings grown under CO2 enrichment.
Tree Physiol
3:
203-210
[Abstract]
O'Brien TP, McCully ME (1981) The Study of Plant Structure.
Principles and Selected Methods. Termarcarphi, Melbourne, Australia
Paterson E,
Hall JM,
Rattray EAS,
Griffiths BS,
Ritz K,
Killham K
(1997)
Effect of elevated CO2 on rhizosphere carbon flow and soil microbial processes.
Global Change Biol
3:
363-377
Pregitzer KS,
Zak DR,
Curtis PS,
Kubiske ME,
Teeri JA,
Vogel CS
(1995)
Atmospheric CO2, soil nitrogen and turnover of fine roots.
New Phytol
129:
579-585
[CrossRef]
Purnell HM
(1960)
Studies of the family Proteaceae. I. Anatomy and morphology of the roots of some Victorian species.
Aust J Bot
8:
38-50
[CrossRef]
Rogers HH,
Runion GB,
Krupa SV
(1994)
Plant responses to atmospheric CO2 enrichment with emphasis on roots and the rhizosphere.
Environ Pollut
83:
155-189
[CrossRef][Medline]
Ryan PR,
Delhaize E,
Randall PJ
(1995)
Characterization of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots.
Planta
196:
103-110
[Web of Science]
Ryan PR,
DiTomaso JM,
Kochian LV
(1993)
Aluminum toxicity in roots: an investigation of spacial sensitivity and the role of the root cap.
J Exp Bot
44:
437-446
[Abstract/Free Full Text]
Ryan PR,
Skerrett M,
Findlay GP,
Delhaize E,
Tyerman SD
(1997)
Aluminum activates an anion channel in the apical cells of wheat roots.
Proc Natl Acad Sci USA
94:
6547-6552
[Abstract/Free Full Text]
Sadowsky MJ,
Schortemeyer M
(1997)
Soil microbial responses to increased concentrations of atmospheric CO2.
Global Change Biol
3:
217-224
Skene KR,
Kierans M,
Sprent JI,
Raven JA
(1996)
Structural aspects of cluster root development and their possible significance for nutrient acquisition in Grevillea robusta (Proteaceae).
Ann Bot
77:
443-451
[Abstract/Free Full Text]
Skene KR,
Raven JA,
Sprent JI
(1998a)
Cluster root development in Grevillea robusta (Proteaceae). I. Xylem, pericycle, cortex, and epidermis development in a determinate root.
New Phytol
138:
725-732
[CrossRef]
Skene KR,
Sutherland JM,
Raven JA,
Sprent JI
(1998b)
Cluster root development in Grevillea robusta (Proteaceae). II. The development of the endodermis in a determinate root and in an indeterminate, lateral root.
New Phytol
138:
733-742
[CrossRef]
Stitt M (1983) Citrate synthase. In HU Bergmeyer, ed,
Methods of Enzymatic Analysis, Ed 3, Vol IV. Verlag Chemie, Weinheim,
Germany, pp 353-358
Thimann KV
(1936)
Auxins and the growth of roots.
Am J Bot
23:
561-569
[CrossRef]
Vance CP,
Stade S,
Maxwell CA
(1983)
Alfalfa root nodule carbon dioxide fixation. I. Association with nitrogen fixation and incorporation of amino acids.
Plant Physiol
72:
469-473
[Abstract/Free Full Text]
Varney GT,
McCully ME
(1991)
The branch roots of Zea. II. Developmental loss of the apical meristem in field-grown roots.
New Phytol
118:
535-546
[CrossRef]
Vidal J,
Chollet R
(1997)
Regulatory phosphorylation of C4 PEP carboxylase.
Trends Plant Sci
2:
230-237
[CrossRef][Web of Science]
Whipps J
(1985)
Effect of CO2 concentrations on growth, carbon distribution and loss of carbon from the roots of maize.
J Exp Bot
36:
644-651
[Abstract/Free Full Text]
Wightman F,
Schneider EA,
Thimann KV
(1980)
Hormonal factors controlling the initiation and development of lateral roots. II. Effects of exogenous growth factors on lateral root formation in pea roots.
Physiol Plant
49:
304-314
[CrossRef]
Top
Abstract
Introduction