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Plant Physiol. (1998) 118: 1369-1378
Inhibition of Auxin Movement from the Shoot into the Root
Inhibits Lateral Root Development in Arabidopsis1
Robyn C. Reed2,
Shari R. Brady, and
Gloria K. Muday*
Department of Biology, Wake Forest University, Box 7325, Winston-Salem, North Carolina 27109-7325
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ABSTRACT |
In
roots two distinct polar movements of auxin have been reported that may
control different developmental and growth events. To test the
hypothesis that auxin derived from the shoot and transported toward the
root controls lateral root development, the two polarities of auxin
transport were uncoupled in Arabidopsis. Local application of the
auxin-transport inhibitor naphthylphthalamic acid (NPA) at the
root-shoot junction decreased the number and density of lateral roots
and reduced the free indoleacetic acid (IAA) levels in the root and
[3H]IAA transport into the root. Application of NPA to
the basal half of or at several positions along the root only reduced
lateral root density in regions that were in contact with NPA or in
regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root
development in Arabidopsis roots was also inhibited by excision of the
shoot or dark growth and this inhibition was reversible by IAA.
Together, these results are consistent with auxin transport from the
shoot into the root controlling lateral root development.
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INTRODUCTION |
Polar auxin transport in higher plants is a directional and
regulated process. In stems auxin transport is from cell to cell and
moves from the shoot apex toward the base (Lomax et al., 1995 ). Appropriate distribution of auxin has been shown to be necessary for a
number of developmental processes. Normal embryogenesis, for example,
requires auxin transport (Cooke et al., 1993), and the Arabidopsis
mutants lop1, pin1-1, and monopterous
(Okada et al., 1991; Carland and McHale, 1996; Przemeck et al., 1996),
which may be altered in auxin transport, have altered vascular, floral, and pattern development, respectively.
In roots the movement of auxin is more complex. Analysis of the
distribution of radiolabeled auxin applied to plants indicates that
auxin is transported acropetally (from the base of the root toward the
root tip) in the central cylinder of the root (Mitchell and Davies,
1975; Tsurumi and Ohwaki, 1978). However, there is also evidence for
basipetal auxin transport near the root apex. In bean plants this
transport appears to occur only between the root tip and the
elongation zone (Mitchell and Davies, 1975), and microautoradiography
suggests that auxin transport occurs in the root epidermis (Tsurumi and
Ohwaki, 1978). Both polarities of auxin transport have been shown to be
sensitive to inhibition by the auxin-transport inhibitor
2,3,5-triiodo-benzoic acid (Tsurumi and Ohwaki, 1978), suggesting that
a similar mechanism may control both movements. The root apex may be
capable of IAA biosynthesis (Feldman, 1980), but it is unknown whether
auxin transported basipetally in the root originates in the shoot, the
root apex, or both. Auxin transport is required for root elongation,
gravity response, and lateral root development (Katekar and
Geissler, 1980; Muday and Haworth, 1994). An important question is
whether the two polarities of auxin movement in roots separately
control these growth and developmental processes.
Lateral root development is highly dependent on auxin and auxin
transport. Lateral roots originate in the root pericycle, in which
individual quiescent cells are stimulated to dedifferentiate and
proliferate to form the lateral root primordium (Blakely and Evans,
1979). Cells in the lateral root primordium differentiate and elongate,
causing the lateral root to emerge through the primary root epidermis.
Mature lateral roots structurally resemble the primary root and are
themselves capable of producing new lateral roots, allowing for
recursive branching and eventual development of a complex root system.
Several lines of evidence indicate that auxin is necessary for the
development of lateral roots. Application of IAA to growing plants
stimulates lateral root development and lateral root elongation
(Torrey, 1950; Blakely et al., 1982; Muday and Haworth, 1994).
Conversely, growth of tomato roots on agar containing auxin-transport
inhibitors, including NPA, decreases the number of lateral roots (Muday
and Haworth, 1994). Natural variation in auxin transport may lead to
differences in the development of lateral roots. Donaldson (1993) found
a negative correlation between the degree of branching in root systems
and the amount of NPA-binding activity present in roots in different
species of plants (Lomax et al., 1995).
Genetic approaches have also established the connection between auxin
and lateral root development. Mutants with reduced sensitivity to auxin
exhibit reduction or loss of lateral roots. The tomato mutant
diageotropica (dgt), isolated for its horizontal
growth pattern, does not produce lateral roots (Zobel, 1974; Muday et al., 1995). This mutant appears to be reduced in auxin sensitivity in
both the shoot (Kelly and Bradford, 1986) and the root (Muday et al.,
1995). Like dgt, the dominant auxin-insensitive Arabidopsis mutant Dwf produces no lateral roots and displays no
gravitropic response (Mirza et al., 1984). The Arabidopsis mutant
aberrant lateral root formation-4 (alf-4) does
not respond to exogenous auxin; this mutant also produces no lateral
roots (Celenza et al., 1995). axr-1 and axr-2,
which have reduced auxin sensitivities, produce fewer lateral roots
than the wild type (Estelle and Somerville, 1987).
Conversely, increased lateral and adventitious rooting has been
reported in plants with elevated auxin content. A transgenic tobacco
plant transformed with a construct expressing bacterial auxin
biosynthetic genes has a higher number of lateral roots (Sitbon et al.,
1992). Extensive proliferation of adventitious and lateral roots in the
mutant alf1-1, which is allelic to superroot and
rooty, has been linked to elevated free IAA levels (Boerjan et al., 1995; Celenza et al., 1995; King et al., 1995). Genetic approaches have also linked auxin transport to lateral root
development. The tir3-1 mutant was isolated based on its
reduced sensitivity to growth inhibition by auxin-transport inhibitors
(Ruegger et al., 1997). This mutant has reduced polar auxin transport
in floral inflorescences and fewer NPA-binding sites. This mutation
also leads to a loss of lateral roots.
To fully understand how auxin controls lateral root development,
knowledge of how auxin reaches its target tissues is necessary. An
experiment in which radiolabeled IAA was applied to the shoot apical
bud of pea seedlings found that radioactivity accumulates in lateral
root primordia (Rowntree and Morris, 1979; Kerk and Feldman, 1995). In
addition, removing the cotyledons and epicotyls from pea seedlings
causes a decrease in the number of lateral root primordia, as well as
in the percentage of primordia that develop into emergent lateral roots
(Wightman et al., 1980). This excision-induced decrease in lateral root
development can be partially rescued by applying IAA to the cut sites
(McDavid et al., 1972; Hinchee and Rost, 1986). Localized application
of auxin-transport inhibitors has been used to block one of the two
polarities of auxin movement, but mixed results have been reported with
this technique (McDavid et al., 1972; Hinchee and Rost, 1992). In
experiments designed to prevent shoot-derived auxin from reaching the
roots, auxin-transport inhibitors applied to pea plants at the base of the stem reduced lateral root development. However, these applications led to scorching and withering of the plant (McDavid et al., 1972), and
with some auxin-transport inhibitors, the effect of inhibitor application was not position-specific (Hinchee and Rost, 1986).
To determine whether auxin from the shoot drives lateral root
development in Arabidopsis wild-type plants, we prevented shoot-derived auxin from reaching the root through a variety of treatments in living
Arabidopsis. These treatments were designed to block auxin transport
with minimal damage to the plants. Lateral root inhibition by all of
the treatments was reversible by localized application of IAA to the
root-shoot junction.
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MATERIALS AND METHODS |
Chemicals and Radiochemicals
[2,3,4,5(n)-3H]NPA (58 Ci/mmol) was
purchased from American Radiolabeled Chemicals (St. Louis, MO). NPA was
from Chemical Services (West Chester, PA). ScintiVerse scintillation
fluid, Triton X-100, and Suc were purchased from Fisher Scientific.
Absolute ethanol was purchased from McCormick Distilling Co. (Weston,
MO). All other chemicals were purchased from Sigma.
Seed Germination and Plant Growth
Wild-type Arabidopsis (ecotype Landsberg erecta) seeds
were purchased from Lehle Seeds (Round Rock, TX). Seeds were soaked in
distilled water for 30 min and surface sterilized with 95% ethanol for
5 min and 10% bleach with 0.01% Triton X-100 detergent for 5 min.
After five washes in distilled water, seeds were germinated and grown
on 9-cm Petri plates containing sterile control medium (0.8% agar
[Sigma type M, plant tissue culture], 1× Murashige and Skoog salts,
pH 6.0, 1.5% Suc, 1 µg mL 1 thiamine, 0.5 µg mL1 pyridoxine HCl, 0.5 µg
mL1 nicotinic acid, and 50 µg
mL1 sterile filtered ampicillin). Seeds were
grown in vertically oriented Petri dishes in continuous fluorescent
light (94 µmol m2 s1)
at room temperature (22°C) for 4 to 5 d, until cotyledons
had emerged and roots were 1.0 to 1.5 cm long. For experiments in which
plants were grown in the dark, Petri dishes were placed in light-tight
boxes in the same room as light-grown plants to ensure consistent
temperature.
Seeds were germinated on control agar and after growth for the
indicated number of days, 10 seedlings were transferred to fresh plates
containing control agar or agar-containing compounds at the indicated
final concentrations. In experiments involving localized application of
compounds, plants were grown on vertical control plates for an
additional 3 to 6 d after application of compounds.
Preparation of Treatments in Agar
Control agar (0.8%), as described above, was supplemented with
NPA, IAA, Lucifer Yellow, or Suc, and the agar was poured into Petri
dishes or applied locally to plants under varying conditions. Compounds
were added to cooled (50°C) molten control agar and poured into
plates. NPA dissolved in DMSO was added to agar with a final DMSO
concentration of 0.1%, and IAA dissolved in absolute ethanol was added
to agar with a final ethanol concentration of 0.1%. Lucifer Yellow at
0.1% was dissolved in water and added to agar. Additional Suc was
added to agar as a concentrated, sterile, filtered solution. In
experiments using plates containing one-half control and one-half
supplemented agar, sterile Petri plates divided into quadrants were
used. Supplemented agar was poured into two adjacent quadrants, and
control agar into the other two.
Treatments to Block Auxin Movements
In experiments involving localized application of compounds in
agar, agar was allowed to harden in a sterile Pasteur pipette. The agar
was dispensed directly from the pipette in 1- to 2-mm lines across the
root-shoot junction. Lines of either control agar or agar containing
NPA were applied to the root-shoot junction and 10 and 20 mm below the
root-shoot junction. At the time of application, the roots were just
over 20 mm long, so the lowest application of agar was at the root tip.
The roots were then allowed to grow for the indicated period, and the
density of lateral roots in each of the three zones was determined.
These regions were designated zones 1, 2, and 3 for the areas between 0 and 10, 10 and 20, and 20 and 30 mm from the root-shoot junction,
respectively.
In excision experiments the plants were cut with sterilized scissors
and either the entire shoot at the root-shoot junction or the terminal
1 to 2 mm of the root tip were removed. After excision the plants were
then grown on vertical plates, as indicated. In addition, the length of
primary roots was measured using a ruler and dissecting microscope. The
average number of lateral roots and the average length were calculated.
The density of lateral roots along the primary root was calculated by
dividing the number of lateral roots by the primary root length for
each root. These paired values were then averaged and the
SE was calculated.
Diffusion Controls during Local Applications of Compounds
Diffusion of NPA from localized applications was measured by
applying agar supplemented with 10 µM cold NPA and 5 nM [3H]NPA to the root-shoot
junction of the seedlings. For each plate of 20 seedlings, the total
volume of applied agar was approximately 2 mL. The plates were placed
in a vertical position and the plants were allowed to grow for 3 d. The seedlings were removed from the plates and uniform samples of
agar were taken, using a transfer pipette and gentle suction to cut and
draw out cylindrical pieces of agar. Five samples were taken at the
site of [3H]NPA agar application and every 1 cm
down the plate. The radioactivity in each agar sample was determined by
scintillation counting.
Uptake and diffusion of NPA into plants was measured by applying agar
supplemented with 10 µM cold NPA and 8 nM
[3H]NPA to the root-shoot junction of
seedlings. The assay was performed using 20 seedlings per plate. The
total volume of applied agar was approximately 2 mL. The plates were
placed in a vertical position and the plants were allowed to grow for 2 or 8 d. Lateral root inhibition by localized NPA application was
visible within 2 d. At 2 and 8 d, the seedlings were removed
from the plates and any agar clinging to them was removed. The
seedlings were cut into 1-cm sections, beginning at the root-shoot
junction, with a razor blade. All of the sections that were at a
constant distance from the root-shoot junction were combined and
radioactivity was determined by scintillation counting.
Measurement of [3H]IAA Transport in Roots
To assess auxin-transport inhibition by NPA when applied locally
to the root-shoot junction, 100 nM
[3H]IAA was mixed with unlabeled IAA to reach a
final IAA concentration of 10 µM. Unlabeled IAA was
added, because IAA concentrations greater than 1 µM lead
to significantly higher amounts of auxin transport (data not shown).
Four-day-old seedlings were transferred to control plates, with 10 seedlings per plate. One percent agar blocks, which contained the
[3H]IAA, were placed along the root-shoot
junction of the intact plant, and agar blocks with or without 100 µM NPA were placed directly below them. The plates were
oriented vertically, with the plants inverted, so the roots were above
the shoots; thus, transport could be differentiated from diffusion or
wicking along the surface in the direction of gravity. IAA transport
was measured after 24 h by cutting the roots 2 mm below the NPA
block and placing the roots into 2.5 mL of scintillation fluid (five
roots per vial). Radioactivity in the samples was determined by
scintillation counting with a LKB-Wallac counter (Wallac, Inc.,
Gaithersburg, MD) for 2 min.
Quantification of Endogenous Free IAA Levels
Endogenous free IAA levels in roots of Arabidopsis seedlings were
determined after application of agar with or without 10 µM NPA to the root-shoot junction. All of the plants
treated with NPA had qualitative reductions in numbers of lateral
roots, compared with the controls. After treatment, roots were
harvested from 60 to 120 plants, the fresh weight was determined, and
the samples were frozen in liquid nitrogen and stored at  70°C.
Free IAA was purified and quantified using the procedure of Chen et al.
(1988) in the laboratory of Dr. Jerry Cohen (USDA, Beltsville, MD).
Between 50 and 100 mg of Arabidopsis root tissue was used for isolation
of free IAA. Tissue was frozen in liquid nitrogen and ground with a
mortar and pestle using IAA extraction buffer (65% isopropanol and
35% 0.2 M imidazole buffer, pH 7.0). [13C]IAA was used as an internal standard with
either 25 ng/g fresh weight of tissue or with 20 ng total.
[3H]IAA was used as a radiotracer using 50,000 dpm for each sample. Samples were extracted for 1 h at 4°C and
the extract was centrifuged at 10,000g for 5 min. The
supernatant fluid was then analyzed for free IAA. IAA was purified by
an amino column (Prep Sep, Fisher Scientific) with several organic
washes and eluted in methanol with 5% acetic acid. After
concentration, the sample was purified by HPLC, methylated using
ethereal diazomethane, and then analyzed by GC-SIM-MS. The GC-SIM-MS
was used for selected ion measurements to quantify the free IAA
concentrations in the root extracts relative to the
[13C]IAA internal standard.
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RESULTS AND DISCUSSION |
Localized Application of NPA at the Root-Shoot Junction
The effect of local application of NPA concentrations ranging from
5 to 100 µM on lateral root development is visible in
Figure 1 and quantified in Table
I. There was a dose-dependent decrease in
the number of lateral roots with increasing concentration of NPA, with
an almost complete inhibition at the highest NPA concentration. In
contrast, growth was reduced less than 2-fold at the highest NPA
concentration. The density of lateral roots was also calculated by
dividing the number of lateral roots by the length to normalize for the
effects of the treatment on length. Alone, the average number of
lateral roots per plant may be misleading in some cases because the
average primary root length varied according to treatments, allowing
various lengths of root along which lateral roots can form. In this
case, lateral root density decreased parallel to the decrease in the
number of lateral roots.
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| Figure 1.
Comparison of lateral root development with
localized application of control agar (left) or agar containing 100 µM NPA (right) to the root-shoot junction. Roots were
grown for 4 d after application of agar.
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Table I.
Effects of localized NPA application on lateral root
development and root length
Four-day-old seedlings were treated by application of agar containing
NPA at the indicated concentrations to the root-shoot junction. After 6 additional d of growth, lateral roots were counted and the length of
the primary roots was measured. The reported values are averages ± SE of 10 plants.
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To verify that NPA application at the root-shoot junction acted to
reduce auxin movement into the roots, two approaches were used. First,
endogenous levels of free IAA were measured after treatment with 10 µM NPA or control agar and compared with changes in
lateral root number under similar conditions. The number of lateral
roots and the levels of free IAA, as measured by GC-SIM-MS, were
reduced in roots treated with NPA relative to those in control roots,
as shown in Table II. Free IAA levels
were 28% lower in samples treated with NPA. This reduction is
statistically significant, as determined by a one-tailed Mann-Whitney U
test (P = 0.04). This measurement reflects the differences in free
IAA in the entire root, and there may be greater differences in cells
and tissues of the root that form lateral roots not detectable by this
analysis.
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Table II.
Effects of localized NPA application at the
root-shoot junction on free IAA concentration
Seven- or 8-d-old seedlings were treated by application of agar with or
without NPA to the root-shoot junction. After 3 additional d of growth,
roots were harvested and the free IAA concentration was determined by
GC-SIM-MS or the number of lateral roots was determined.
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The second approach to demonstrate that application of NPA at the
root-shoot junction reduced IAA movement was to measure [3H]IAA transport into the root from an
application site at the root-shoot junction. The results from samples
treated with or without NPA below the site of
[3H]IAA application are shown in Table
III. After 24 h there was 25% less
[3H]IAA transport into roots treated with NPA,
compared with controls. IAA-transport measurements with and without NPA
show a statistically significant reduction in IAA movement, as
determined by a Mann-Whitney U test (P = 0.04).
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Table III.
Effects of localized NPA application on
[3H]IAA movement
Agar blocks containing [3H]IAA were applied to 4-d-old
seedlings with and without NPA-containing blocks, and radioactivity in
the roots was determined after 24 h. The reported values are
averages ± SE of four replicates, each containing
five plants.
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To verify that NPA remained localized during these treatments, 10 µM NPA containing [3H]NPA was
applied to seedlings in agar at the root-shoot junction. Samples of
agar at the site of application and along the length of the plate were
analyzed by liquid scintillation counting. Less than 10% of the
tritiated NPA diffused into the agar at any point on the plate during a
3-d period (data not shown). Seedlings were also sectioned and analyzed
by liquid scintillation counting after application of tritiated NPA to
the root-shoot junction. Of the tritiated NPA that was taken up by the
plants, 69% of the radioactivity was recovered in agar within 1 cm of
the site of application 2 d after application and 47% remained
within 1 cm of the site of application after 8 d. This indicates
limited diffusion of NPA from the root-shoot junction to other areas of
the plant. As another test of diffusion, the fluorescent dye Lucifer
Yellow, used at 0.1%, was applied in agar at the root-shoot junction.
Under fluorescence microscopy the dye could be visualized in plants no
farther than 1 cm from the point of application. Substances applied in
agar in this way appear to have limited rates of bulk transport and diffusion, and do not appear to wick along the surface of the roots.
The effects of NPA applied at the root-shoot junction are therefore
consistent with localized inhibition of auxin transport.
Effect of Position of NPA Application
Because elongating root tips tend to grow out from under a line of
applied agar, two other approaches were used to examine the effect of
local application of NPA to other positions on the root. In the first
approach NPA was applied to either the apical or the basal halves of
the root by growth in Petri dishes containing 10 µM NPA
in one half and containing control agar in the other half. Although
this approach led to a broader application of NPA, it did allow NPA to
remain in continuous contact with the root tip and to be in contact
with only part of the root. After transfer of plants to half-plates so
that root tips were just below the middle line, the plates were placed
in a vertical position in the light. When applied to the upper half of
the root, NPA inhibited lateral root development in all parts of the
root, relative to controls, as shown in Table
IV. Almost no lateral roots formed on the
lower half of these roots, although they were not in contact with the
NPA. Conversely, application of NPA to the lower root half had no
statistically significant effect on lateral root number or density in
the upper half of the root, although it did significantly reduce
lateral root number in the lower half. Thus, whereas NPA always has a
local effect on the root tissue it contacts, NPA can only influence
lateral root development at distant sites when it is in contact with
the basal part of the root. This result is consistent with the
direction of auxin movement controlling lateral root development moving
from the shoot toward the root tip.
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Table IV.
Effects of application of NPA to the upper and
lower halves of seedling roots
Six-day-old seedlings were transferred to plates containing 10 µM NPA in the upper or lower half, as indicated. After 4 additional d of growth in the light, lateral roots in the upper and
lower halves were counted and new primary root growth was measured. The
reported values are averages ± SE of 10 plants.
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New root growth, measured as the change in primary root length after
transfer to new plates, was also determined in these experiments.
Plants treated with NPA in the upper half of the plate showed a modest
22% inhibition of elongation, whereas those treated with NPA in the
lower half were inhibited by 60% (Table IV). Although both NPA
treatments inhibited elongation, application of NPA at the root tip had
a more profound inhibitory effect, suggesting a role for auxin
originating near the root tip in root elongation. NPA application to
the lower half of the root also reduced the gravitropic growth of the
seedlings (data not shown). In contrast, application of NPA to the
upper half of the root or to the root-shoot junction did not affect the
gravitropic response.
In the second approach, used to examine the effects of NPA applied to
the root tip, lines of either control agar or agar containing 10 µM NPA were applied to the root-shoot junction and to
positions 10 and 20 mm below the root-shoot junction. At the time of
application, the roots were just over 20 mm long, so the most apical
application of agar was at the root tip. The roots were then allowed to
grow for 3 d and the density of lateral roots in each of three
zones was determined. These regions were designated zones 1, 2, and 3 for the area between 0 and 10, 10 and 20, and 20 and 30 mm from the
root-shoot junction, respectively. The results of this experiment are
shown in Figure 2, and the site of
application of NPA relative to the zone of measurement is indicated. In
all cases there is a statistically significant reduction in lateral
roots in regions apical to the site of application of NPA, with a range
of 60% to 83% for lateral root inhibition. When the effect on lateral roots in regions basal to the site of application is examined, there
are slight decreases in lateral root number, ranging from 5% to
22% inhibition. These data are consistent with auxin moving toward the
tip controlling lateral root development.
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| Figure 2.
The effect of NPA application at several positions
on the Arabidopsis primary root. Five-day-old Arabidopsis seedlings
were treated with a line of agar containing 100 µM NPA at
several positions along the root. The root was divided into 10-mm
zones, with zone 1 beginning at the root-shoot junction. The lines of
agar were placed above zone 1, 2, or 3, as indicated. The number and
density of lateral roots were determined after 3 d of growth. The
data were analyzed by Student's t test, and P values
were determined for each sample compared with the controls and are
indicated with asterisks (*P  0.015 and  0.038;
**P 0.005).
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Rescue of NPA Inhibition of Lateral Roots with Exogenous Auxin
Double lines of control agar and agar containing 100 µM NPA or 10 µM IAA were applied in
different combinations to the root-shoot junction of seedlings. The
number and density of lateral roots were determined, as shown in Table
V. IAA application led to a greater than
2-fold, statistically significant (P < 0.0001) increase in
lateral root density, whereas NPA application resulted in a
statistically significant 10-fold decrease (P < 0.0001) in the
lateral root density relative to control agar alone.
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Table V.
Reversal of NPA-induced lateral root inhibition by
localized IAA application
After the indicated treatments, lateral root number and root length
were determined after 4 d of additional growth in the light. The
reported values are averages ± SE of 10 plants.
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When IAA and NPA were applied together in double lines to the
root-shoot junction, the result depended on the placement of the two
compounds. When IAA was applied apical to NPA, IAA reversed the
inhibitory effect of NPA on lateral root development, as shown in Table
V. These plants produced 14-fold more lateral roots than plants treated
with NPA alone, and the increase in lateral root density was
statistically significant (P < 0.0001). Therefore, plants
apparently take up and transport the exogenously applied auxin below
the site of local NPA inhibition, leading to a partial rescue of
lateral root development. Conversely, when NPA was applied below IAA,
the number and density of lateral roots were inhibited 6- and 5-fold,
respectively, relative to plants treated with IAA alone (Table V). This
was a statistically significant decrease in lateral root density
(P = 0.0006). This NPA-mediated inhibition of lateral roots is not
as complete when exogenous IAA is applied as when NPA is applied alone,
presumably because NPA cannot completely block the movement of high
levels of exogenous auxin.
The amount of IAA taken up by roots when the compound is applied at the
root-shoot junction can be estimated by examination of the measurement
of [3H]IAA transport in Table III. The average
weight of an Arabidopsis root of the age in this analysis is
approximately 0.6 mg (determined from the harvest of many plants
for GC-SIM-MS analysis). Conversion of the femtomole values in Table
III to picomoles per gram fresh weight suggests that radiolabeled IAA
reaches a concentration of 3.5 ng/g fresh weight in the root after
24 h of application. Roots of this age had 26 ng/g fresh weight
free IAA distributed throughout the entire root, so it is conceivable
that several days of treatment with exogenous IAA led to
physiologically significant changes in IAA levels in the pericycle
cells in which lateral root initiation occurs.
Localized IAA Application
Table VI shows that when the basal
half of the root was in contact with agar containing 0.1 µM IAA, there was a greater than 1.5-fold increase in
number and density of lateral roots in both the upper and lower root
halves, compared with plants on control agar. The increase in lateral
root density was significant only in the lower half of these plants
(P = 0.004). In contrast, exposure of the apical end of the root
to IAA significantly increased the number and density of lateral roots
2-fold in the region of the root in contact with IAA (P 0.002 for root densities). The effect of IAA applied at the apical end of the
root appeared to be local, in that lateral root number and density on
the base of the root, which was in contact with control agar, did not
differ significantly from the controls. Application of IAA using
half-plates was less precise and presumably allowed loading of more IAA
than the local application of IAA with lines of agar. The results are
consistent with auxin transport from the shoot toward the root tip
playing a controlling role in lateral root development and the opposite polarity of auxin movement not playing a role in regulation of this
process.
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Table VI.
Effects of application of IAA to the upper and
lower halves of roots
Six-day-old seedlings were transferred to plates containing 0.1 µM IAA in the upper or lower half, as indicated. After 4 additional d of growth in the light, lateral roots in the upper and
lower halves were counted and new primary root growth was measured. The
reported values are averages ± SE of 10 plants.
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Shoot and Root-Tip Excision
Table VII shows the effects of shoot
and root-tip excision of seedlings on the number and density of lateral
roots, measured 3 d after excision and growth in continuous light.
Shoot excision caused a statistically significant (P < 0.0001)
greater than 4-fold decrease in both the total number of lateral roots
and the lateral root density, compared with intact plants (Table VII).
Root-tip excision did not appear to disrupt the capacity of seedlings
to form lateral roots. In fact, removing the root tip caused a
statistically significant (P = 0.001) increase in the lateral root
density. This is consistent with previous reports of lateral root
stimulation by root-tip excision in pea seedlings (Wightman and
Thimann, 1980). The effect of root-tip excision suggests that wounding
responses from excision do not inhibit lateral root development, and
that the root tip is not necessary for lateral root development.
However, a substance coming from the shoot, presumably auxin, does
appear to be necessary for lateral root development.
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Table VII.
Effects of excision and localized IAA application
on lateral root development
Plants were treated by shoot or root-tip excision and application of
agar containing IAA at the indicated concentrations to the root-shoot
junction. After 3 additional d of growth, lateral roots were counted
and the length of the primary roots was measured. The reported values
are averages ± SE of 10 plants.
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The apical 0.5 to 1.0 mm of the primary root, which included the root
cap, root tip, and part or all of the elongation zone, was removed,
leading to complete cessation of elongation of the remaining root. In
contrast, both the control roots and the shoot-excised roots elongated
throughout the growth period. The continued growth of roots from
shoot-excised plants demonstrates that excision and any subsequent
wounding response did not kill roots or prevent root growth. Removal of
the shoot did slightly reduce elongation of the primary root,
suggesting that either auxin or nutrients required for growth were
depleted. Because Suc did not reverse this elongation inhibition by
removal of the shoot (data not shown), the effect is not solely at the
level of nutrient limitation.
To determine whether auxin may be the substance transported from the
shoot that induces lateral roots, lateral root development in plants
was inhibited by shoot excision. Then, agar containing IAA at
concentrations from 0.1 to 10.0 µM was applied to the cut surfaces of shoot-excised plants. This experiment was performed in the
dark to reduce light-induced IAA breakdown. IAA at the concentrations
tested had a dose-dependent, stimulatory effect on the number of
lateral roots formed by plants with the shoot excised (Table VII),
although even at the highest concentration complete rescue was not
possible. The increases in lateral roots were statistically significant
at concentrations of 1 and 10 µM (P = 0.035 and
P < 0.00001, respectively).
Dark Growth
Dark-grown seedlings formed fewer lateral roots than light-grown
seedlings, as shown in Table VIII and as
reported previously by Jensen et al. (1998). Auxin was applied to the
root-shoot junction of whole seedlings at concentrations from 0.1 to 10 µM. IAA stimulated the development of lateral roots in
dark-grown plants relative to plants treated with control agar. Table
VIII shows a dose-dependent relationship between the concentration of
IAA applied and the number of lateral roots formed. IAA at 1 µM, applied at the root-shoot junction, was sufficient to
stimulate significant increases in lateral root development (P 0.0008), although greater number and density of lateral roots were
obtained by application of 10 µM IAA. IAA application
also increased the number and density of lateral roots in light-grown
seedlings, but the magnitude of the effect was much less (Table V).
This result is consistent with dark growth reducing the concentration
of IAA that reaches the root, so that roots require exogenous IAA to
form lateral roots.
View this table:
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Table VIII.
Effects of localized IAA application on lateral
root development in dark-grown plants
Seven-day-old seedlings were treated by application of agar containing
IAA at the indicated concentrations to the root-shoot junction. After 3 additional d of growth, lateral roots were counted and the length of
the primary roots was measured. The reported values are averages ± SE of 10 plants.
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Several lines of evidence suggest that the inhibition of lateral root
development by dark growth is not solely at the level of carbon
availability attributable to photosynthesis. Although Suc partially
reverses the dark inhibition of lateral root development, auxin is more
effective at reversing the effect. Plants on high-Suc agar plates
(7.5% Suc, compared with control plates, which contained 1.5% Suc)
produced an average of 4.5 ± 0.6 lateral roots after 5 d in
the dark, compared with plants on control plates that formed no lateral
roots (data not shown). When plants are grown in the dark on agar
containing IAA and extra Suc, the effect of IAA seems to mask that of
Suc. In the dark, plants grown on agar containing a high-Suc
concentration (7.5% Suc) and IAA applied at the root-shoot junction
have an average number of lateral roots that was not significantly
different from that of plants treated with IAA alone (data not shown).
It should be noted that this high level of Suc may cause additional
alterations in plant growth through the action of sugars as signaling
molecules (Jang and Sheen, 1997). High levels of Glc (up to 6%) alter
the root growth and development of light-grown Arabidopsis plants (Jang
et al., 1997), although Suc is much less potent in its effect than Glc
(Jang and Sheen, 1997). Also, growth of Arabidopsis plants in the light
on agar containing norflurazon, which inhibits photosynthesis, reduces but does not abolish lateral root development (M.-R. Cha and R. Hangarter, personal communication). This result suggests that dark
growth prevents the movement of a substance from the shoot into the
root, other than photosynthate, which mediates lateral root
development. These results are consistent with the auxin signal being
an important determinant of lateral root development, although carbon
levels also play a role in this process.
| |
CONCLUSIONS |
This study examined whether lateral root development depends on
specific, directional auxin transport in vivo. Disruption of polar
auxin transport from the shoot through a variety of treatments resulted
in inhibition of lateral roots. The effectiveness of the most potent
and specific of these treatments, application of NPA to the root-shoot
junction, was verified in two ways. Both free IAA levels and
[3H]IAA transport into the root were reduced by
local NPA application to the root-shoot junction. All of the treatments
that reduced lateral root development were reversible by application of
IAA to the root-shoot junction. In contrast, auxin movement from the root tip does not appear to be necessary for lateral root development. Two Arabidopsis mutants, agr1 and eir1, which are
altered in root gravity response and may have reduced auxin transport
specifically in the root, have normal lateral root development
(Luschnig et al., 1998; R. Chen and P. Masson, personal
communication). In contrast, the tir3 mutant has a defect in
auxin transport in the inflorescence stem and does not form any lateral
roots (Ruegger et al., 1997). These results are consistent with auxin
transport from the shoot into the root being the sole source of the
auxin required for lateral root development in Arabidopsis.
Why should the direction of auxin transport matter to the responding
tissues? Perhaps the simplest explanation for this phenomenon is that
acropetal and basipetal polar auxin transport occur in different root
tissues. Whereas auxin moves basipetally in the epidermis, acropetal
transport occurs in the central cylinder (Mitchell and Davies, 1975;
Tsurumi and Ohwaki, 1978). Lateral roots originate in the pericycle,
the ring of cells closest to the central cylinder (Schiefelbein and
Benfey, 1994), and auxin moving acropetally is found in tissues with
close proximity to the pericycle (Mitchell and Davies, 1975). Auxin
moving through the central cylinder may have significantly better
access to the pericycle than auxin moving through the epidermis, which
is at least three cell layers removed from the pericycle in Arabidopsis (Schiefelbein and Benfey, 1994).
Polar auxin transport from the shoot into the root may be a means by
which root and shoot developmental programs can be coordinated in
response to environmental stimuli. The shoot is much more exposed than
the root to variables such as light levels. Experimental evidence
indicates that environmental variables, including light (Behringer and
Davies, 1992) and production of ethylene (Suttle, 1988), can influence
auxin movement in shoots. Therefore, regulation of transport of
shoot-derived auxin into the root may be a mechanism by which
environmental changes sensed by the shoot can be communicated to
control root growth and development. These experiments clearly demonstrate that dark growth reduces lateral root development. Jensen
et al. (1998) reported that NPA inhibits elongation of light-grown but
not dark-grown hypocotyls, indicating changes in auxin transport or its
regulation by NPA under different light conditions.
If auxin moving from the shoot into the root controls lateral root
development, then what is the function of auxin moving from the tip
toward the root base? Root elongation and gravity response have also
been shown to be blocked by auxin-transport inhibitors (Katekar and
Geissler, 1980; Muday and Haworth, 1994) and reduced in mutants
proposed to be altered in auxin transport (Lushnig et al., 1998).
Treatments parallel to those in this work indicate that inhibition of
auxin moving from the tip toward the base will specifically block
elongation and gravity response (S.R. Brady, R.C. Reed, and G. Muday,
unpublished results). Therefore, these two distinct polarities of auxin
movement may control different root growth and developmental processes.
| |
FOOTNOTES |
1
This work was supported by the National
Aeronautics and Space Administration (grant no. NAGW 4052 to G.K.M.)
and a Sigma Xi Grant-in-Aid of Research to R.C.R.
2
Present address: Duke University Medical Center,
P.O. Box 2776, Durham, NC 27708.
*
Corresponding author; e-mail muday{at}wfu.edu; fax
1-336-758-6008.
Received May 29, 1998;
accepted September 9, 1998.
| |
ABBREVIATIONS |
Abbreviations:
GC-SIM-MS, gas chromatography-single ion
monitoring-mass spectroscopy.
NPA, naphthylphthalamic acid.
| |
ACKNOWLEDGMENTS |
We appreciate Jerry Cohen's generosity in allowing us to
conduct the GC-SIM-MS measurements of free IAA in his laboratory, through support from the U.S. Department of Energy (grant no. DE-AI02-94-ER20153). We thank Brian Tague for helpful advice and Roger
Hangarter for discussion in preparation of the manuscript. We also
appreciate Dave Anderson's assistance with the statistical analyses.
| |
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Abstract
Introduction