Journal of Experimental Botany, Vol. 51, No. 350
WD Special Issue, pp. 1555–1562, September 2000
Growth of Arabidopsis thaliana seedlings under water
deficit studied by control of water potential in
nutrient-agar media
Corine M. van der Weele1, William G. Spollen2, Robert E. Sharp2 and Tobias I. Baskin1,3
1 Division of Biological Sciences, 109 Tucker Hall, University of Missouri-Columbia, Missouri 65211, USA
2 Department of Agronomy, Plant Science Unit, University of Missouri-Columbia, Missouri 65211, USA
Received 12 November 1999; Accepted 8 March 2000
Abstract
We have characterized the growth responses of
Arabidopsis thaliana seedlings to water deficit. To
manipulate the water potential, we developed a
method whereby the nutrient-agar medium could be
supplemented with polyethylene glycol (PEG 8000);
PEG was introduced into gelled media by diffusion,
which produced media with water potential as low as
−1.6 MPa. For dark-grown plants, hypocotyl growth
had a hyperbolic dependence on water potential, and
was virtually stopped by −1 MPa. In contrast, primary
root elongation was stimulated by moderate deficit
and even at −1.6 MPa was not significantly less than
the control. That these results did not depend on a
direct effect of PEG was attested by obtaining indistinguishable results when a dialysis membrane impermeable to PEG was placed between the medium and
the seedlings. For light-grown seedlings, moderate
deficit also stimulated primary root elongation and
severe deficit reduced elongation only partially. These
changes in elongation were paralleled by changes in
root system dry weight. At moderate deficit, lateral
root elongation and initiation were unaffected and at
higher stress levels both were inhibited. Primary root
diameter increased steadily with time in well-watered
controls and under water deficit increased transiently
before stabilizing at a diameter that was inversely proportional to the deficit. Along with stimulated primary
root elongation, moderate water deficit also stimulated
the rate of cell production. Thus, A. thaliana responds
to water deficit vigorously, which enhances its use
as a model to uncover mechanisms underlying plant
responses to water deficit.
Key words: Arabidopsis thaliana, cell division, polyethylene
glycol, root and shoot growth, water deficit.
Introduction
Plants live in a variable environment to which they
acclimate their growth and development. Plant growth is
often limited by water deficiency; typically, however, roots
are affected less than shoots. In fact, even under mild
water deficit, shoots may stop growing completely while
roots continue to grow. Continued root growth allows
the plant to plumb the soil for water and can be especially
important for seedling establishment.
Although the responses of plants to water deficit have
been studied in many species, we sought to use Arabidopsis
thaliana to take advantage of the potent molecular and
genetic tools available for this species. In addition, roots
of A. thaliana seedlings have a well-defined anatomy
(Dolan et al., 1993) and cells in the epidermis and cortex
are visible in living roots using Nomarski microscopy,
which is helpful when looking at growth responses at the
cellular level. Furthermore, Beemster and Baskin
developed a method for A. thaliana in which the expansion
and division parameters of the root meristem can be
quantified with high spatial and temporal resolution
(Beemster and Baskin, 1998).
Despite the advantages of A. thaliana, to our knowledge
only one previous study has used this species to investigate
growth at low water potential. Vartanian et al. examined
‘drought rhizogenesis’, the production of short, tuber-like
lateral roots in response to soil drying ( Vartanian et al.,
1994). The authors also presented data on total root
system biomass; shoot growth was not assayed.
3 To whom correspondence should be addressed. Fax: +1 573 882 0123. E-mail: [email protected]
© Society for Experimental Biology 2000
1556 van der Weele et al.
Unfortunately, comparative data for well-watered controls were not included, so how water deficit affected root
growth could not be assessed.
The limited use of A. thaliana for studies of growth
responses to water deficit has been, perhaps, because its
small size compounds the difficulty of controlling plant
water status reproducibly and quantitatively. In addition,
because the roots are extremely thin, it is difficult to
image them for growth measurement in soil or vermiculite.
Therefore, the objective of this study was to develop a
method to grow A. thaliana seedlings at constant and
defined water potentials on an agar-solidified nutrient
medium. Nutrient-agar media are often used in studies
of A. thaliana, and hence a large amount of physiological
data have been obtained for this condition. By placing
the agar medium vertically, the root grows on the surface;
this minimizes the possibility of oxygen limitation, which
may occur in solution culture when water potential is
manipulated with high-molecular-weight solutes ( Verslues
et al., 1998). An agar medium also facilitates the method
developed previously (Beemster and Baskin, 1998), which
requires marking and imaging the roots on the surface of
a growth substrate.
The difficulty encountered here was introducing a highmolecular-weight solute into the medium. Whereas lowmolecular-weight compounds are readily incorporated
into an agar medium at even rather high concentrations,
larger compounds interfere with the entanglement of the
polysaccharide chains and prevent the medium from
gelling. Although some authors have decreased water
potential with low-molecular-weight solutes, this
approach suffers because small molecules may be taken
up into cells, particularly in long-term experiments, and
because they penetrate the cell wall readily, thus removing
water only from the cell. In drying soil, water is lost from
the cell wall as well as from the cell, and this condition
is reproduced using high-molecular-weight solutes. We
describe here how a high-molecular-weight solute (polyetheylene glycol 8000) was successfully introduced into
agar media; and, using this system, we characterized
several growth responses of A. thaliana seedlings to
water deficit.
Materials and methods
Plant growth
Seeds of Arabidopsis thaliana L. (Heynh), ecotype Columbia,
were surface-sterilized with 15% household bleach and grown
vertically on nutrient-agar media in 10 cm Petri plates. The
medium comprised: 1.2% agar, 0.5% sucrose, 4 mM KNO ,
3
1 mM Ca(NO ) , 0.3 mM MgSO , 2 mM KH PO , 89 mM iron
3 2
4
2 4
citrate, 46.3 mM H BO , 9.1 mM MnCl , 0.77 mM ZnSO ,
3 3
2
4
0.31 mM CuSO , and 0.11 mM MoO . To minimize evaporation
4
3
but still permit gas exchange, plates were wrapped with one
layer of bandage tape (Micropore, 3M Company, St Paul, MN ).
For light-grown plants, seeds were stored at 4 °C and on day
zero, seeds on plates were placed in a growth chamber under
constant conditions (19 °C, 135 mmol m−2 s−1), as described
previously (Baskin and Wilson, 1997). Six days after plating,
similar seedlings (roots 1.5–2.0 cm long, cotyledons expanded)
were transferred to plates with lowered water potential or
control plates with the same water potential. The plates were
kept under a clear, acrylic cover to reduce evaporation. For
each experiment, three plates with six to seven seedlings each
were prepared. For dark-grown plants, seeds on plates were put
in a light-tight box, held at 4 °C for 5 d to break dormancy,
and then transferred to a 20 °C growth chamber. The time of
transfer to 20 °C defined day zero. After 6 d, seedlings with a
root length of 4–7 mm and a hypocotyl length of 3–12 mm
were transferred to plates with lowered water potentials or to
the same water potential (controls). During transplantation,
plants were exposed to the fluorescent lighting in the laboratory
(approximately 3 mmol m−2 s−1) for about 10 min. The plates
were returned to the light-tight box and put back in the
chamber. For each experiment, two plates with 7–11 plants
each were prepared per treatment.
For experiments with membranes, seeds were sown on
thoroughly cleaned and autoclave-sterilized dialysis tubing
(molecular weight cutoff 3500 Da; SpectraPor 3, Fisher
Scientific, Pittsburgh, PA) that was cut open and spread as a
single layer on top of the agar medium. Seedlings that were
outside the desired size range were removed from the membrane,
and the remaining seedlings were transplanted by transferring
the membrane.
Water deficit treatment
Water potential was lowered by the addition of various amounts
of polyethylene glycol (PEG) (molecular weight 8000; Sigma,
St Louis, MO) to the growth medium. Approximately 20 ml of
PEG solution was poured on top of an equal volume of
solidified nutrient-agar in a Petri plate, and after 24 h, the
solution on top of the plate was poured off and the plate used
for experiments. During the 24 h period, the PEG diffused into
the agar medium, thus lowering its water potential. That the
24 h period was long enough to reach an approximate
equilibrium was verified by measurements of water potential of
the top and bottom surfaces of the agar medium. The PEG
solution had the same nutrient composition as the growth
medium; nutrient solution without PEG was poured on the
plates for the ‘well-watered’ control medium, which gave a
water potential for the agar medium of approximately
−0.10 MPa. Water potentials were measured using isopiestic
thermocouple psychrometry (Boyer and Knipling, 1965) at the
end of each experiment, and the data are reported as the mean
of the measured potentials.
PEG changes when autoclaved: the water potential of a
−1.6 MPa PEG solution decreased by 20% after autoclaving,
while the water potential of a comparable sodium chloride
solution decreased by only 4%. Therefore, the PEG-nutrient
solution was sterilized by passing it through a filter (0.22 mm).
In some experiments, solid non-sterile PEG was mixed with
sterile nutrient solution without leading to detectable
contamination.
Growth measurement
Root and hypocotyl elongation were measured by scoring the
back of the plastic plate with a razor blade every 24 h, starting
immediately after transplanting. For scoring plates in the
experiments on dark-grown plants, plates were illuminated for
less than 1 min by light from a flashlight filtered through green
acrylic (0.2 mmol m−2 s−1). At the end of treatment, the plates
were photocopied at 1.4 times enlargement and elongation was
measured with a digitizing tablet or a ruler as the distance
Water-stress in Arabidopsis thaliana
1557
along the root or hypocotyl between each mark. For lightgrown plants, root and shoot dry weight, number of laterals
emerged, length of the laterals, and primary root diameter were
also measured at the end of the 5 d treatment. For measuring
dry weight, shoots and roots were pooled per plate, dried for
8 h at 60 °C and weighed on a microbalance (C-31, Cahn
Instruments, Cerritos, CA). The number of laterals that were
visible through a dissecting microscope was counted. The length
of the laterals was measured on the photocopies of the plates
with a digitizing tablet. Root diameter was measured at each
score mark by viewing the roots on the agar plates through a
compound microscope at low magnification. Diameters were
measured with a video digitizer (Image 1/AT, Universal
Imaging, West Chester, PA).
To determine cell flux on days 2, 3, and 4 after transplanting,
at the end of the experiment the lengths of 20 cortical cells
were measured in parts of the primary root that passed out of
the elongation zone on those days. Beemster and Baskin
determined that in well-watered A. thaliana roots, cells resided
in the zone of rapid elongation for 6–8 h (Beemster and Baskin,
1998). To ensure that the measured cells had matured on the
day in question, cell lengths were measured only in the region
of the root that had grown during the latter half of that day.
For each day, the elongation rate of a root was divided by the
relevant average cell length of the same root, and this ratio was
averaged over the sample. Four to six roots were used on each
of the three plates per treatment.
Results
To study the effect of low water potential on the growth
of A. thaliana seedlings, seeds were germinated and plants
grown for 6 d under well-watered conditions (i.e. nutrientagar medium, water potential approximately −0.10 MPa)
and then transplanted onto plates with lowered water
potentials. To prepare medium with lowered water potential, high-molecular-weight polyethylene glycol (PEG
8000) was used. The PEG could not be mixed with molten
agar because at higher concentrations of PEG, agar does
not solidify. Therefore, as described in Materials and
methods, the water potential was lowered by allowing the
PEG to diffuse into already solid agar media. In this way,
water potentials in the agar medium as low as −1.6 MPa
were attained reproducibly, and remained approximately
constant over at least 5 d in the growth chamber.
We studied growth responses that were elicited by water
deficit in both light-grown and dark-grown A. thaliana
seedlings because each is known to respond distinctly to
various stimuli. Results for dark-grown seedlings over a
3 d treatment period are shown in Fig. 1A. Hypocotyl
elongation had an approximately hyperbolic response to
water deficit, decreasing to 60% of the control at a water
potential of −0.2 MPa and to near zero below −0.9 MPa.
In contrast, primary root elongation was stimulated by
lowered water potential between −0.2 and −0.9 MPa,
and even at −1.6 MPa was not lower than the wellwatered control. Results for light-grown roots over a 5 d
treatment period are shown in Fig. 1B (hypocotyl elongation was already too low by the time of transplanting to
be measurable on the photocopied images of the plates).
Fig. 1. Increase in length of the hypocotyl and primary root of A.
thaliana seedlings after transplanting to nutrient-agar media as a function
of the water potential of the medium. Water potential was lowered by
increasing concentrations of PEG 8000, introduced by diffusion as
described in Materials and methods. Seedlings were 6-d-old at the start
of treatment. (A) Dark-grown seedlings treated for 3 d. Filled symbols
plot results for experiments in which a dialysis membrane was placed
between the plants and the medium. Data are means ±SE of two plates
from one experiment. (B) Light-grown seedlings treated for 5 d. Data
are means±SE for six experiments, each with three plates per treatment.
Horizontal error bars report the standard deviation of the water
potentials from six plates per treatment (one from each experiment).
Primary root elongation, similarly to dark-grown seedlings, was stimulated by moderate water deficit. Root
elongation was reduced by water potentials below
−0.5 MPa, although at the lowest water potential
(−1.2 MPa), elongation was still 50% of the control.
To determine whether there was a direct effect of PEG
on growth, plants were grown on a dialysis membrane
spread on the agar. The molecular weight of the PEG
(8000 Da) was larger than the cut-off of the membrane
(3500 Da). The membrane did not affect the total elongation over 3 d of hypocotyls or roots ( Fig. 1A) or the
kinetics of the response (data not shown), indicating that
PEG did not affect growth directly.
Because root elongation in the light was more vigorous
than in the dark, further investigation used light-grown
plants. The dry weights of the whole plant and the shoot
decreased with increasing stress ( Fig. 2). The dry weight
of the whole root system paralleled the changes in primary
root elongation, being increased at moderate deficit and
decreased at the most severe deficit. Increased root-system
1558 van der Weele et al.
Fig. 2. Shoot and root system dry weight as a function of water
potential for light-grown seedlings. Data are means ±SE of six plates
from two experiments.
dry weight was not explained by stimulated initiation or
elongation of lateral roots, because there was no significant difference in the number or length of lateral roots of
seedlings grown under moderate stress compared to wellwatered conditions ( Table 1). Severe stress reduced both
the number and the length of the laterals.
To gain further insight into the response of primary
root elongation to water deficit, the kinetics of the
response during the 5 d exposure were studied (Fig. 3).
For the control, elongation rate accelerated steadily
throughout the experiment, whereas under water deficit,
rates leveled off and approached steady state. At high
stress levels (−0.8 and −1.2 MPa), elongation rates
throughout the experiment were lower than the control;
however, under moderate stress (−0.23 and −0.51 MPa),
elongation rates during the first 3–4 d of treatment were
substantially greater than the control.
Water deficit is known to cause maize primary roots to
become thinner (Sharp et al., 1988; Liang et al., 1997).
Thinning is believed to be adaptive so that, under limited
Table 1. The number and total length of lateral roots for
light-grown seedlings as a function of water potential
Data are means ±SE of six plates from two experiments.
Water potential (MPa)
Number
Length (cm)
−0.10
−0.23
−0.49
−0.92
−1.21
4.3±0.7a
4.9±1.0a
3.6±0.7a
1.3±0.3
0.7±0.2
0.87±0.17
0.77±0.12
0.06±0.03
Nmb
Nm
aNo evidence to reject equivalence of means based on ANOVA
followed by unpaired t-tests.
bNm, Not measured. Laterals were too short to appear on
photocopied images.
Fig. 3. Primary root elongation rate as a function of time for lightgrown seedlings at various water potentials. Data are means ±SE for
six experiments, each with three plates per treatment.
water supply, roots can concentrate their use of resources
to maintain elongation (Sharp et al., 1990). Therefore,
we determined whether water deficit also caused root
thinning in A. thaliana. We expected that, at least at
moderate stress levels where elongation rate was stimulated, root diameter would decrease for the sake of
conserving water. Primary root diameter was measured
for each day at the end of the 5 d water-stress treatment.
For the control, diameter increased steadily during the
experiment (Fig. 4). Under water stress, root diameter
increased over the first day of the experiment, and for all
deficits except the most severe, the roots actually became
thicker than the control. However, thickening was not
sustained, and except for the −0.23 MPa treatment in
which diameter continued to increase, by the end of the
experiment root diameter had stabilized at a level that
was roughly inversely proportional to the stress level and
less than that of the well-watered roots.
The total increase of volume in the primary root was
calculated from volume increases per day, assuming the
shape of the root was a cylinder. Compared to controls,
roots at moderate water deficit added significantly more
volume, and only at the highest stress was water uptake
reduced ( Table 2). At moderate deficit, volume increased
because of the transient stimulation of elongation and
radial expansion; by the end of the experiment, the rate
of volume increase for all deficits was less than the control.
In well-watered primary roots, the length of mature
cortical cells increased slightly (Table 3). Moderate water
deficit increased cell length modestly whereas severe stress
had little effect. For the three treatments, however, the
changes in cell length, if any, were smaller than the
concomitant changes in elongation rate. This approximate
Water-stress in Arabidopsis thaliana
Fig. 4. Primary root diameter as a function of time for light-grown
seedlings at various water potentials. Data are means ±SE of six plates
from two experiments.
Table 2. Primary root volume increase over 5 d of growth in the
light as a function of water potential
Volume added to the root each day was calculated from the length
increase and diameter measured for that day, and the volume increments
for each day were summed. Data report means ±SE of three plates
from one experiment. Similar results were obtained in a second
experiment.
Water potential (MPa)
Volume (mm3)
−0.10
−0.23
−0.47
−0.82
−1.31
1.05±0.12
1.33±0.11
1.29±0.07
0.94±0.16
0.48±0.03
constancy suggests that mature cell length may be regulated to fall within a preferred range.
To study how water deficit affected cell production, we
measured cell flux at the end of the elongation zone.
When a root elongates at steady state, cell production
rate for a file of cells equals the rate at which cells in that
file exit the elongation zone, i.e. the cell flux, and is
1559
calculated as the ratio of elongation rate to mature cell
length. However, when root elongation rate and mature
cell size increase over time, this cell flux underestimates
cell production rate. In work on roots grown on 3%
sucrose, in which elongation rate and mature cortical cell
length increase over time similarly to the well-watered
roots studied here, cell production rate was quantified
kinematically, accounting for non-steady state behaviour
(Beemster and Baskin, 1998). Cell production rates thus
obtained exceeded the cell flux calculated as the ratio of
elongation rate to mature cell length by approximately
10% only (Beemster and Baskin, unpublished data).
Because the kinematic analysis is tedious, cell production
rate was estimated here as the ratio of root elongation
rate to mature cell length without correcting for nonsteady state behaviour.
In well-watered roots, the flux of cortical cells increased
over time in parallel with elongation rate ( Table 3),
similar to the increase previously reported for roots grown
on medium containing 3% sucrose (Beemster and Baskin,
1998). At the severest water deficit assayed (−1.28 MPa),
cell flux was approximately 50% of the control; however,
at moderate deficit (−0.22 MPa), cell flux was stimulated
significantly during the second day of the response. Note
that under moderate deficit, elongation rate and cell
length increased more than they did under well-watered
conditions; therefore, accounting for non-steady-state
behaviour would result in calculating cell production to
have been increased by moderate deficit to an even
greater extent.
Discussion
As with many plants, the shoot of A. thaliana seedlings
was acutely sensitive to water deficit, growing slowly at
moderate stress and not at all at severe stress. The
response of the primary root was opposite in that moderate deficit stimulated elongation. Even at more negative
water potentials, primary roots still grew at appreciable
rates. Increased primary root elongation rate was correlated with an increased cell production rate. Water stress
Table 3. Cortical cell length and cell flux of primary roots grown at selected water potentials on consecutive days of treatment
The lengths of newly matured cortical cells were measured as described in Materials and methods. Data report means ±SE of three plates from
one experiment. Similar results were obtained in a second experiment.
Water potential (MPa)
−0.09
−0.22
−1.28
Cell length (mm)
Cell flux (cells d−1)
Day 2
Day 3
Day 4
Day 2
Day 3
Day 4
213±6
227±3
194±14
223±2
261±5
218±5
228±2
249±12
234±7
39.4±3.1*
53.1±2.8*
17.9±1.2
45.6±2.3*
50.3±0.4
23.6±2.3
53.8±1.8*
56.7±3.9
24.1±1.3
* Cell flux differed significantly, at the 95% level or better, for the −0.09 MPa treatment on each day, and between the −0.09 and −0.22 MPa
treatments on day 2, but not on days 3 or 4, as determined by repeated measures ANOVA followed by paired t-test across days and unpaired
t-tests within days. For the −1.28 MPa treatment, t-tests were not done.
1560 van der Weele et al.
also prevented the steady increase in root diameter that
occurred in the control so that by the end of the experiment, stressed roots were thinner than controls. These
results suggest that the species A. thaliana is relevant for
studies of plant response to water deficit not only because
of its well-developed molecular genetics, but also because
the species acclimates vigorously to water deficit.
The use of PEG in agar to limit water availability
The addition of PEG to agar-solidified media provided a
growth substrate with consistent and reproducible water
potential. However, the use of PEG might be problematic.
In solutions it increases viscosity, and in solution-culture
even moderate concentrations of PEG 8000 have been
shown to hinder oxygen diffusion to the root ( Verslues
et al., 1998). A. thaliana roots grown on the surface of
an agar medium often have a film of solution around the
root, which could become an increasing barrier to
diffusion with increasing concentrations of PEG.
However, under severe stress (<−0.8 MPa) few if any
roots had a visible film of solution around them, and
under moderate stress, roots grew faster than control
roots, not slower as would be expected for oxygendeficient roots. Moreover, when plants were grown on a
membrane impermeable to PEG 8000, any water film
around the root would have been free of PEG; for all
PEG concentrations no differences in root or shoot
growth rates were found with or without the membrane.
Therefore, the effects reported here most likely result
from the changed water status of the medium rather than
from some other consequence of PEG addition.
Solid growth media supplemented with PEG are
also frequently used for tissue culture experiments
( Tschaplinski et al., 1995; Capuana and Debergh, 1997;
Linossier et al., 1997). Not only is the response of cells
and explants to water deficit interesting intrinsically,
lowering the water potential of the culture medium often
enhances regeneration and hence is important practically.
However, the deficits imposed in tissue-culture studies
have been limited by the inability of agar to gel when
containing more than about 15% PEG. Although gellan
gum (phytagel ) does gel with higher concentrations of
PEG, in our hands, gellan gum-media led to irreproducible
root elongation rates among controls and, in stress treatments, to effects on root elongation rate as a function of
the age of the medium. These effects might be related in
some way to the fact that gellan gum-media increase in
gel strength over time ( Klimaszewska and Smith, 1997).
Interestingly, elongation was affected to the same extent
when a dialysis membrane was interposed between the
gellan gum-medium and the seedlings, which appears to
rule out a direct effect of any gel property on the roots.
In the light of these effects on elongation, gellan gummedia seem to be inadvisable for physiological experiments. Therefore, the diffusion-based method used here
for adding PEG to agar media used here may be useful,
not only for studies on the minuscule seedlings of A.
thaliana but also for studies of any plant in culture.
Sucrose supplement
Often, nutrient-agar media for A. thaliana contain up to
3% sucrose because this promotes plant growth (Baskin
and Wilson, 1997). It is assumed that growth is improved
because sucrose is a source of carbohydrate; however, the
addition of 3% sucrose to a medium would lower its
water potential by about 0.2 MPa. It was found that
lowering the water potential of the growth medium by
this amount with PEG improved root growth. Therefore,
the enhancement of root growth by sucrose might occur
because of an osmotic rather than a nutritional effect.
Initially, we did experiments in a medium that lacked
sucrose completely; germination and growth were irregular to such an extent that it was difficult to select enough
uniform seedlings for transplanting. Therefore, 0.5% sucrose was added to the media, which lowered the water
potential by a small amount only. However, sucrose,
often compared to a hormone, is considered to play a
prominent role in regulating plant metabolism ( Koch,
1996), and in this context even 0.5% could have profound
physiological consequences, particularly for dark-grown
plants, which presumably have less endogenous sucrose.
By the same token, the macronutrients in the agar medium
also influence seedling growth and may have affected the
behaviour of the plant in response to water deficit. The
connections between responses to levels of various nutrients and water are knotted and untangling them is an
active area of research (McDonald and Davies, 1996).
With the method demonstrated here, such studies can be
extended to A. thaliana; the availability of mutants with
defined alterations in specific components of these
responses may support particularly incisive experiments.
Root growth of A. thaliana under water deficit
Water deficit increased the dry weight of the A. thaliana
root system, except at the highest deficit tested. Water
deficit nearly always increases the ratio of root to shoot
dry weight, and there are many examples where water
deficit increases the absolute dry weight of the root system
(Sharp and Davies, 1979; Meyer and Boyer, 1981). In
this respect, A. thaliana responds typically to water deficit,
which validates its use as a model system. Not typical is
the strikingly enhanced primary root elongation. Most
studies have found that water deficit inhibits primary root
elongation rate (Gingrich and Russell, 1956; Mirreh and
Ketcheson, 1973; Sharp and Davies, 1989; Materechera
et al., 1992; Spollen et al., 1993), although stimulation
has been shown for Pinus pinaster (Nguyen and Lamant,
1989; Triboulot et al., 1995) and Glycine max (Creelman
et al., 1990). The experiments on pine lowered water
potentials with PEG whereas those on soybean used
Water-stress in Arabidopsis thaliana
vermiculite culture and withheld water; therefore,
enhanced primary root growth at moderate stress is not
unique to agar or to the use of PEG as an osmoticum.
However, stimulated root elongation is not necessarily
unusual in the context of the whole root system. Water
deficit often stimulates root system growth, as assessed
by the increase of length density of the whole or partial
root system (Read and Bartlett, 1972; Klepper et al.,
1973; Sharma and Ghildyal, 1977; Jupp and Newman,
1987; Schmidhalter et al., 1998). Increased root system
length requires that either root elongation rate or proliferation increases. In many of the above studies, water
deficit did increase root proliferation, but also appeared
to stimulate the elongation rate of at least some roots
within the system, although elongation rate was not
measured directly. Under water stress, the stimulated
primary root elongation of A. thaliana seedlings thus
resembles the enhanced root system growth of the older
plants of other species, perhaps reflecting an accelerated
development associated with A. thaliana’s short life cycle.
In addition to stimulating elongation of the A. thaliana
primary root, moderate water deficit also stimulated the
rate of cell production. To our knowledge, this is the first
report of stimulated rates of cell production under water
deficit. In species where water deficit inhibited primary
root elongation rate, cell production rate was similarly
inhibited ( Fraser et al., 1990; Dubrovsky et al., 1998). In
A. thaliana at both moderate stress where root elongation
rate increased, as well as at severe stress where elongation
rate decreased, the change in elongation rate was paralleled closely by the change in cell production rate. This
suggests that the root’s elongation rate at given levels of
water deficit is determined principally by the supply of
cells to the zone of rapid elongation, as recently argued
to account for the steady increase in elongation rate of
well-watered A. thaliana roots (Beemster and Baskin,
1998). In contrast, in pine seedling primary roots
responding to water deficit, cell production rate stayed
constant despite increases or decreases in root elongation
rate at specific stress levels ( Triboulot et al., 1995).
Apparently, regulating the supply of cells plays a pivotal
role in executing the response to water deficit in the
primary roots of some species but not in others; it would
be interesting to determine the effect of water stress on
cell production in the root systems of older plants.
Root diameter
For many years, root systems under water deficit have
been observed to produce thin roots, although such
observations have rarely distinguished between new roots
being initiated in narrower diameter classes and individual
roots lessening their radial expansion. However, several
studies have measured the diameter of the primary root
of water-stressed plants, and consistently reported that it
was thinner than that of the well-watered control ( Taylor
1561
and Ratliff, 1969; Eavis, 1972; Read and Bartlett, 1972;
Sharp et al., 1988). Thus, the thin primary roots of A.
thaliana under water deficit resemble the response seen in
other plants.
Despite the presumed importance of limiting radial
expansion to conserve water, few reports have examined
how roots become thinner at low water potential. In the
primary root of maize, individual roots became thinner
with time under stress, and the rates of radial expansion
were not correlated with rates of elongation (Liang et al.,
1997). In A. thaliana, however, individual roots did not
become thinner; instead, water deficits below −0.23 MPa
prevented the steady increase in diameter that occurred
under well-watered conditions. In fact, the diameter of
the root appeared to be closely correlated with elongation
rate, so much so that, except under severe stress, the root
simultaneously elongated faster and grew fatter than the
control, failing to minimize water uptake. Root diameter
and elongation rate have been correlated in other species
for various circumstances ( Wilcox, 1972; Hackett and
Rose, 1972; Thaler and Pagès, 1996). Thus, in A. thaliana,
root diameter and elongation rate may be reduced under
water deficit by a common mechanism, whereas in maize,
the thinning of roots at low water potential appears to
reflect the action of a specific mechanism for limiting
radial expansion.
That A. thaliana roots did not thin over time may be
a consequence of the small absolute diameter of the root,
which is scarcely more than 100 mm, roughly a tenth of
the maize root. First, in the A. thaliana root meristem,
where final root diameter is attained in this species, cells
may already be close to the minimal cytoplasmic volume
needed to function. Second, decreased root diameter may
be significant not only because it reduces the quantity of
water that must be absorbed to support elongation but
also because it increases the surface to volume ratio, thus
facilitating water uptake. Given that the surface to volume
ratio of the thread-like roots of A. thaliana is already
high, there is relatively less to gain by reducing root
diameter further.
Acknowledgements
We thank Steve Wells for help with constructing thermocouples
used in measuring water potential, and Mike Keller for help
with statistics. This work was supported in part by grant
No. IBN 9817132 from the US National Science Foundation
(to TIB), and by award No. 95-37100-1601 from the US
Department of Agriculture (to WGS and RES ). This is
contribution No. 13 006 from the Missouri Agricultural
Experiment Station journal series.
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