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Plant Physiol. (1999) 119: 1349-1360
Proline Accumulation in Maize (Zea mays L.)
Primary Roots at Low Water Potentials. II. Metabolic Source of
Increased Proline Deposition in the Elongation Zone1
Paul E. Verslues2 and
Robert E. Sharp*
Department of Agronomy, Plant Science Unit, 1-87 Agriculture
Building, University of Missouri, Columbia, Missouri 65211
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ABSTRACT |
The
proline (Pro) concentration increases greatly in the growing region of
maize (Zea mays L.) primary roots at low water potentials ( w), largely as a result of an increased net
rate of Pro deposition. Labeled glutamate (Glu), ornithine
(Orn), or Pro was supplied specifically to the root tip of intact
seedlings in solution culture at high and low w to
assess the relative importance of Pro synthesis, catabolism,
utilization, and transport in root-tip Pro deposition. Labeling with
[3H]Glu indicated that Pro synthesis from Glu did not
increase substantially at low w and accounted for only a
small fraction of the Pro deposition. Labeling with
[14C]Orn showed that Pro synthesis from Orn also could
not be a substantial contributor to Pro deposition. Labeling with
[3H]Pro indicated that neither Pro catabolism nor
utilization in the root tip was decreased at low w. Pro
catabolism occurred at least as rapidly as Pro synthesis from Glu.
There was, however, an increase in Pro uptake at low w,
which suggests increased Pro transport. Taken together, the data
indicate that increased transport of Pro to the root tip serves as the
source of low-w-induced Pro accumulation. The possible
significance of Pro catabolism in sustaining root growth at low
w is also discussed.
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INTRODUCTION |
Accumulation of Pro is a widespread plant response to
environmental stresses, including low w
(Yancey et al., 1982 ). Because of the high concentrations often
observed, Pro has a clear role as an osmoticum. In particular, because
of its zwitterionic, highly hydrophilic characteristics, Pro acts as a
"compatible solute," i.e. one that can accumulate to high
concentrations in the cell cytoplasm without interfering with cellular
structure or metabolism (Yancey et al., 1982; Samaras et al., 1995).
Other functions of Pro accumulation have also been proposed, including
radical detoxification (Smirnoff and Cumbes, 1989) and regulation of
cellular redox status by Pro metabolism (Hare and Cress, 1997).
The metabolic source of the Pro accumulated at low
w is unclear. One potential mechanism is an
increase in Pro synthesis. Two possible pathways of Pro synthesis, one
using Glu and the other using Orn as a precursor, have been shown to
exist in plants (Delauney and Verma, 1993). Studies of Pro metabolism
have suggested that Pro synthesis from Glu can increase in response to
low w (Boggess et al., 1976; Hanson and Tully,
1979b; Rhodes et al., 1986). More recent studies have focused on the
expression of genes encoding Pro-synthesizing enzymes. Transcription of
mRNA encoding P5CR and P5CS, which catalyze Pro synthesis from Glu, has
been found to be induced by water deficits and salt stress (Delauney and Verma, 1990; Hu et al., 1992; Williamson and
Slocum, 1992; Verbruggen et al., 1993; Yoshiba et al., 1995; Strizhov
et al., 1997). Increased transcription was not found for Orn
 -aminotransferase, which catalyzes Pro synthesis from Orn (Delauney
et al., 1993). The investigation of Pro synthesis from Glu has also
been extended to include transgenic plants that overexpress P5CS
(Kishor et al., 1995). Although these experiments did produce plants
that had greater Pro accumulation, the effect of the enhanced Pro
production on resistance to drought or salt stress is controversial
(Blum et al., 1996; Sharp et al., 1996; Verma and Hong, 1996).
In addition to increased Pro synthesis, decreased Pro catabolism could
also contribute to Pro accumulation at low w.
Labeling studies such as those by Stewart et al. (1977) and Stewart and Boggess (1978) found a suppression of Pro oxidation, and other studies
(Kiyosue et al., 1996; Verbruggen et al., 1996) found a decrease in Pro
dehydrogenase mRNA accumulation at low w.
Transport of Pro within the plant may also be important, as indicated
by high Pro concentrations in the phloem sap of drought-stressed alfalfa (Girousse et al., 1996) and increased transcription of a
Pro-specific amino acid transporter in response to water deficit or
salt stress (Rentsch et al., 1996). To our knowledge, there have been
no studies in which the effects of low w on
the various possible contributors to Pro accumulation have been
examined in the same organ under comparable
conditions.
Previous work in our laboratory has focused on mechanisms of growth
maintenance in the maize primary root at low
w. Although root growth is inhibited at low
w, it is much less inhibited than shoot growth
(Sharp et al., 1988). Maintenance of root elongation occurs
preferentially toward the root apex (Sharp et al., 1988), in
association with dramatic increases in Pro concentration to as much as
120 mmolal at a w of  1.6 MPa (Voetberg and
Sharp, 1991). The accumulation of Pro in the apical region was shown to
be largely attributable to an increased net rate of Pro deposition (Voetberg and Sharp, 1991). In contrast, increased deposition was not
observed for K+ and hexoses (Sharp et al., 1990);
increases in the concentrations of these solutes occurred primarily in
the more basal regions of the elongation zone and could be accounted
for by decreased dilution resulting from growth inhibition.
The net Pro deposition rates reported by Voetberg and Sharp (1991) were
calculated by combining spatial distributions of elongation rate and
Pro content (see ``Materials and Methods''). Although this analysis
demonstrated unambiguously that more Pro was added to the solute pool
in the root elongation zone at low w, it could
not provide information concerning the metabolic processes responsible
for the increased Pro deposition. This increase could be caused by
increased synthesis of Pro in the elongation zone, increased Pro import
from other parts of the seedling, decreased catabolism or utilization
of Pro in the elongation zone, or a combination of these factors. In
this study, we assessed the relative importance of these factors by
applying labeled Pro or the Pro precursors Glu and Orn specifically to
the apical region of intact roots growing at high or low
w in solution culture. Rates of label
incorporation into Pro and other amino acids were monitored and used to
assess rates of Pro synthesis, catabolism, and utilization. The results
show that none of these factors was responsible for Pro accumulation;
however, Pro uptake did increase at low w.
Taken together, our results indicate that increased transport of Pro to
the root tip is the major source of Pro accumulated in the root
elongation zone at low w.
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MATERIALS AND METHODS |
Seedling Culture and Labeling
Conditions for maize (Zea mays L. cv FR27 × FRMo17) seed germination, transplanting to solution culture, low
w imposition, and seedling growth were as
described by Verslues et al. (1998). Two w
treatments were used: high w (0.02 MPa, no
PEG added) and low w (1.6 MPa PEG). In both
treatments, supplemental oxygenation was provided to increase the
solution oxygen partial pressure from 20.4 (ambient) to 43 kPa, at
which level tissue oxygen partial pressure within the root elongation
zone was shown to be similar to that in roots growing in well-aerated
vermiculite (Verslues et al., 1998). Clear plastic root guides were
used to prevent damage from the vigorous aeration and to hold the roots
in position for spatial growth analysis and labeling. In the
low-w treatment, the solution
w was reduced to 1.6 MPa over approximately
10 h by pumping PEG solution into the solution-culture box. The
boxes, each containing 23 seedlings, were essentially the same as those described by Verslues et al. (1998).
After transfer to solution culture, the seedlings were allowed to grow
for approximately 32 h in the high-w
treatment and for 52 h in the low-w treatment. At
these times, root elongation rates (measured by monitoring the position
of the root apex on the side of the box) and root-tip amino acid
concentrations were steady (see ``Results''). Average primary root
lengths were approximately 150 mm at high w
and 115 mm at low w; seedlings with primary roots that were substantially shorter or longer than this were removed
from the box. Sixteen seedlings were left in the box and were height
adjusted so that their primary root apices were all the same distance
(±1 mm) from the bottom of the box. Height adjustments were made by
gently lifting the caryopsis of each seedling and supporting it using a
toothpick inserted into a Plexiglas pegboard. The solution in the box
was then drained until only the apical 12 to 15 mm of each primary root
remained submerged. The aeration rate was reduced to 550 mL
min 1 to avoid excessive splashing of the
solution onto more basal parts of the roots, and the seedlings were
allowed to acclimate to these conditions for approximately 15 min.
To label the root apical region, 20 µCi of
3H-labeled Glu or Pro or 9 µCi of
14C-labeled Orn was added to a 25-mL aliquot of
solution removed from the root box
(L-[2,3,4-3H]Glu, 60 Ci
mmol1;
L-[3,4-3H]Pro, 40 Ci
mmol1; and
L-[1-14C]Orn, 50 mCi
mmol1; American Radiolabeled Chemicals, St.
Louis, MO; a specific activity of 40 Ci mmol1
is equivalent to 8.8 × 107 dpm
nmol1). This resulted in the following amino
acid concentrations in the labeling solution: Glu, 1.3 × 105 mM; Pro, 2.0 × 105 mM; and Orn, 7.2 × 103 mM. The solution containing
labeled amino acid was put into a second root box (the "labeling
box"), and the Plexiglas holder containing the height-adjusted
seedlings was then transferred to the labeling box, which was
configured so that the 25 mL of labeling solution covered the apical 12 to 15 mm of each primary root. Total aeration rate in the labeling box
was kept at 550 mL min1.
Sections of four primary roots were collected 10, 30, 60, and 120 min
after transfer of seedlings to the labeling box. At each sampling time,
four seedlings were removed from the box and their root tips submerged
for 3 min in an ice-cold aliquot of growth medium of the same
w as that in the box. This allowed efflux of
labeled amino acids that had entered the root apoplast but had not yet
been taken up by root cells. Three minutes was chosen because
preliminary experiments showed that there was a rapid efflux of
radioactivity from the root for 3 min, which was followed by a slower,
steady efflux, presumably from the symplast (data not shown). The
seedlings were then removed and blotted dry. After excision of the
apical 0.5 mm to remove the majority of the root cap, root sections
were collected using a razor-blade holder with the razor blades
adjusted to correspond to the positions shown in Figure
1. The sections were placed in preweighed
microcentrifuge vials and immediately frozen in liquid nitrogen.
Samples were then weighed, freeze dried, and reweighed to obtain the
weight of water by difference.
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| Figure 1.
Time courses of location and dimensions of
sections harvested in root-tip labeling experiments. Displacement
velocities and strain rates (see Fig. 4A) for roots at high (0.02
MPa) (A) or low (1.6 MPa) (B) w were used to calculate
the section of the root tip that encompassed the same tissue at each
sampling time. At all time points, the apical section was entirely
within the region of the root that had high Pro deposition rates at low
w (Fig. 4B) and the basal section was in the region that
had negative or zero Pro deposition rates at low w (see
Fig. 4B). Harvesting times were 10, 30, 60, and 120 min after the start
of labeling. Displacement immediately behind the root cap (a distance
of 0 mm from the root apex in this figure) was negligible during the
time frame of these experiments.
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Determining a time course of labeling of root sections for these
experiments required that expansion and displacement of tissue within
the root tip be taken into account. The effect of displacement on
labeling experiments was discussed by Silk et al. (1984), who showed
that local variation in labeling rates is lost when tissue displacement
is not considered. The approach that we used to determine a true time
course of tissue labeling was to track the movement of tissue through
the root tip. Therefore, we performed labeling only over relatively
short periods (2 h) so that tissue displacement would not be so great
that the labeling could not be assigned to a specific region. Sections
of the apical 4 mm of the primary root, where Pro deposition was high
at low w, were compared with tissue basal to 5 mm, where Pro deposition was negative or zero. The expansion and
displacement of these sections was taken into account by sampling
different sections of the root tip over time (Fig. 1); the boundaries
of the sections were calculated from displacement velocity and
longitudinal strain rate profiles (see below). The initial sizes of the
apical and basal sections were chosen to provide adequate tissue for
analysis while ensuring that the apical section was not displaced into
the region of negative Pro deposition during the experiment. This
approach allowed Pro metabolism to be compared in regions of high and
low Pro deposition. Pro uptake from the labeling solution was measured
by removing small aliquots at the same times that root tip samples were
removed. H activity in these aliquots was
determined by scintillation counting and used to calculate the rate of
3H uptake per root tip.
The results of the Glu-, Orn-, and Pro-labeling experiments are
presented as means of two replicates except as noted otherwise. Calculations in ``Results'' are based on these means, but in all
cases the same calculations for the two experiments individually led to the same conclusions, demonstrating the reproducibility of the results.
Before actual labeling experiments were conducted, it was necessary to
ensure that the necessary manipulations did not inhibit the root
elongation rate. Seedlings were transferred to the labeling box as
described above, and root elongation was measured at 30-min intervals
using a razor blade to mark the position of the root apex (viewed with
a magnifying lens) on a clear plastic sheet mounted on the side of the
box. The distance between the marks was then measured using the
eyepiece reticle of a stereomicroscope. This procedure allowed more
accurate quantification of small root length increases than marking the
side of the box as was routinely used.
Spatial Growth Analysis and Calculation of Pro Deposition Rate
Spatial growth analysis was performed as described by Silk et al.
(1984) and Sharp et al. (1988), with some modifications. Seedlings at
high or low w were grown for 32 or 52 h,
respectively, as described above. At these times, most of the primary
roots had reached the ends of the root guides through which they were growing; seedlings with shorter roots were removed from the box. The
Plexiglas holder and attached root guides containing the seedlings were
then removed from the root box so that the tips of the primary roots
could be reached through slots cut in the ends of the guides. The
apical 15 mm of each root was gently blotted dry and marked at
approximately 1-mm intervals with waterproof ink (no. 17 black, Pelikan, Hannover, Germany) using a small paintbrush. The seedlings were then returned to the solution and allowed to recover for 15 min. A
series of five photographs of the whole box was then taken at 15- or
30-min intervals. The photographs were scanned, and for each root the
mark displacement was measured using image-analysis software
(SigmaScan, Jandel Scientific, San Rafael, CA).
Roots that elongated at a rate near (within 0.4 mm
h1) the premarking mean during photography and
that had retained a sufficient quantity of ink were selected for growth
analysis. Mark position over time was used to calculate displacement
velocities; these values were then interpolated to 0.5-mm intervals
using cubic splines and differentiated to yield the spatial
distribution of the longitudinal strain rate (Sharp et al., 1988). In
similar experiments, the apical 12 mm of 15 to 20 roots was harvested into 1-mm sections (collected by position) and used to determine the
profile of Pro content via a ninhydrin-based assay (Bates et al.,
1973). The spatial growth and Pro-content data were combined to
calculate profiles of net Pro deposition rate using the continuity equation, as described previously (Silk et al., 1984; Sharp et al.,
1990).
The boundaries of the root sections harvested in labeling experiments
were determined by fitting a polynomial equation to the high- and
low-w displacement velocity profiles. These
equations were then put into a computer program that calculated the
displacement of a specific point on the root surface over time. From
these data, the positions of the apical and basal boundaries of the section at each sampling time were determined.
HPLC Analysis and Quantification of Amino Acid Labeling
HPLC separation of amino acids was performed as described
previously (Verslues et al., 1998). Derivatized amino acids were separated by reversed-phase HPLC (modified from Yang and Sepulveda, 1985; Ebert, 1986). HPLC results were corrected for recovery (always within the range of 60%-80%) of the internal standard
 -aminoadipic acid. The amino acids Pro, Glu, Asp, Asn, Ser, Gln,
Arg, GABA, Thr, and Ala were routinely quantified using this method.
However, the method was unable to quantify Orn because of its low
concentration in the root apical region (Voetberg and Sharp, 1991) and
coelution with other, unidentified peaks. In labeling experiments,
individual amino acid peaks were collected in 7-mL scintillation vials
by a computer-controlled fraction collector (Cygnet, Isco, Lincoln, NE). The 3H or 14C activity
of each collected peak was determined by scintillation counting for 10 min (LS-6000-IC, Beckman) using Opti-Fluor scintillation fluid (Packard
Instruments, Meriden, CT); all samples were well above the limit of
detection. Counting results were corrected for background
3H or 14C activity.
For samples in which labeled Pro was applied to the root apical region,
incorporation of Pro into the aqueous insoluble fraction was measured
by drying the residual organic phase and hydrolyzing the residue in 6 M HCl at 110°C for 12 to 15 h. HPLC fractions were
collected and 3H activity was quantified as
described above.
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RESULTS |
Root Growth and Amino Acid Concentrations
After the seedlings were transferred to solution culture at high
w, the root elongation rate accelerated and reached a
steady value at approximately 30 h (Fig.
2). In the low-w
(1.6 MPa PEG) treatment, the root elongation rate first decreased and
then recovered, reaching a near-steady value at approximately 35 h. These results are similar to those obtained in similar experiments by Verslues et al. (1998). Thirty-two hours (high
w) and 52 h (low
w) were chosen as the appropriate times for
analysis of Pro deposition and for labeling experiments because of the
steady root elongation rates and root-tip amino acid concentrations
(see below). Because root-tip labeling involved transfer of the
seedlings to another box and was done over a short time interval, it
was also necessary to examine the root elongation rate immediately after transfer to the labeling box to ensure that substantial fluctuations did not occur. Figure 2, inset, shows that root elongation was stable in the 2 h after transfer of seedlings at 1.6 MPa. In
the high-w treatment, there was a small
decrease in the root elongation rate during the initial 90 min after
transfer, followed by recovery to the normal rate. These root
elongation rates were judged to be sufficiently stable for the labeling
experiments.
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| Figure 2.
Time course of root elongation rate at high
(0.02 MPa) or low (1.6 MPa) w. Arrows show the time
at which the addition of PEG was started and the time at which solution
w reached 1.6 MPa in the low-w
treatment; solution w was constant at later times.
Boldface arrows indicate the times after transfer when 3H
or 14C labeling and quantification of the spatial patterns
of Pro concentration and growth within the root tip were performed.
Root elongation rates are means ± SE
(n = 50-100) from three to five experiments.
Inset, Root elongation rate measured in 30-min intervals after
seedlings were transferred to the labeling box. Data are means ± SE (n = 25-55) from four
experiments.
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It was also necessary to measure time courses of root-tip amino acid
concentrations in the high- and low-w
treatments before and after transfer to the labeling box to ensure that
interpretation of labeling was not complicated by increases or
decreases in concentration over time. Time courses of root-tip (apical
10 mm) concentrations of Pro, Glu, Gln, Arg, GABA, Asp, Asn, Ala, Ser,
Thr, and Gly were measured. The results for Glu, Arg, and Pro are shown
in Figure 3 because these amino acids
were the focus of much of the labeling studies and because the response
of Glu and Arg concentrations to low w was
similar to that of the other amino acids measured.
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| Figure 3.
Time courses of Glu (A), Arg (B), and Pro (C)
concentrations in the root tip. Data are means ± SE
of three to eight samples from different experiments. Each sample
contained the apical 10 mm of two roots. The inset in each panel shows
the time course of amino acid concentration after transfer to the
labeling box. In the insets, open symbols represent the apical section
and closed symbols represent the basal section (harvested as described
for Fig. 1). Data in the insets are means ± SE of six
samples, each from a separate experiment. Time courses of the amino
acids Asp, Asn, Ser, Gln, Arg, GABA, Thr, and Ala were also analyzed
and exhibited responses to low w similar to those of Glu
and Arg.
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With the exception of Pro, all of the amino acids examined exhibited a
rapid increase in concentration after the addition of PEG, which was
followed by a decline either to the prestress value (Arg [Fig. 3B])
or to a concentration greater than the prestress level (Glu [Fig.
3A]). It is likely that the initial increase in concentration was
attributable at least in part to continued amino acid deposition while
growth (and hence water deposition) was reduced (Fig. 2), causing the
solutes to "pile up" in the elongation zone. The insets in Figure
3, A and B, show that in both the apical and basal sections Arg and Glu
concentrations (as well as those of the other amino acids measured)
were stable after height adjustment, solution draining, and transfer to
the labeling box. Transfer was performed at the times indicated by the
arrows in the main figure.
The behavior of Pro in response to decreasing solution
w differed strikingly from that of the other
amino acids. Pro concentration increased immediately after
low-w imposition (note the different scales in
Fig. 3, A-C), but unlike the other amino acids, the most rapid rate of
Pro accumulation did not occur until 15 to 35 h after the start of
PEG addition (Fig. 3C). During this time, solution
w was steady at 1.6 MPa (Fig. 2) and
concentrations of the other amino acids had either stabilized or begun
to decline. The time course of the Pro response was very similar to
that reported for seedlings transplanted to vermiculite at 1.6 MPa
(Ober and Sharp, 1994). The late increase in Pro concentration showed
that Pro did not accumulate merely because root growth slowed; indeed, the root elongation rate was increasing during the period when Pro
concentration increased most rapidly (compare Figs. 2 and 3C). The
lag before the most rapid phase of Pro accumulation suggests either
that induction of metabolic or transport components is necessary or
that Pro is catabolized rapidly during the period of initial adaptation
to low w. Figure 3C, inset, shows that the Pro
concentration was stable after transfer to the labeling box.
Pro Deposition
Vermiculite was the medium used in previous studies in which Pro
deposition in the root elongation zone increased at low
w (Voetberg and Sharp, 1991; Ober and Sharp,
1994). Therefore, for the present study it was first necessary to show
that similar results could be obtained in solution culture. To
calculate Pro deposition rates, the spatial distributions of the
longitudinal strain rate (relative elongation rate) and Pro content
were measured at the same times after transfer to solution culture as
the labeling experiments. Figure 4A shows
that the length of the elongation zone was reduced at low
w to approximately 6 mm compared with 11 mm at
high w. The maximum strain rate was also
reduced at low compared with high w; however,
the strain rate in the apical 3 mm was not affected. These results are
very similar to those obtained with vermiculite-grown roots at the same
w (Sharp et al., 1988). Profiles of Pro
content (not shown) and concentration (Fig. 4B, inset) were also
similar to those obtained in vermiculite-grown roots (Voetberg and
Sharp, 1991), except that in the 1.6 MPa treatment Pro concentrations
were even higher in the apical few millimeters but lower in the 8- to
12-mm region.
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| Figure 4.
A, Longitudinal strain rate profiles in the apical
12 mm of roots at high (0.02 MPa) or low (1.6 MPa)
w. Data are means ± SE
(n = 6 or 7) from two experiments. B, Spatial
distribution of net Pro deposition rate in the apical 12 mm of roots at
high or low w. Inset, Spatial distribution of Pro
concentration used in calculating Pro deposition rates. Data are
means ± SE (n = 3-4) from three
or four experiments.
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The strain rate and Pro profiles were used to compute the pattern of
the net Pro deposition rate (Fig. 4B). At high
w, a low positive rate of Pro deposition
occurred over the entire elongation zone. At low
w, Pro deposition was greatly increased in the
apical 3 mm and then declined steeply, such that deposition rates were negative from 4 to 9 mm. A negative deposition rate indicates a net
loss of Pro from that section of the root by catabolism, by utilization
in the synthesis of protein or other compounds, or by export. These
results are similar to those obtained in vermiculite-grown roots
(Voetberg and Sharp, 1991), except that in the latter, deposition rates
in the basal region were close to zero rather than negative.
By integrating the Pro deposition rate over distance from the apex, it
could be calculated that the total Pro deposition rate for the apical 9 mm of the root was 19 nmol h1 at low
w, compared with 5.1 nmol
h1 at high w. In the
apical 4 mm, the Pro deposition rate was 36.4 nmol
h1 at low w, compared
with 1.9 nmol h1 at high
w. For the 5- to 9-mm region, the Pro
deposition rate was 14.3 nmol h1 at low
w, compared with 2.8 nmol
h1 at high w. These
deposition rates represent the minimum rates (because Pro-consuming
processes in the root tip, such as Pro catabolism and utilization in
protein synthesis, are not accounted for in the calculation) of Pro
synthesis or import needed to maintain the root-tip Pro concentration
over time. Thus, these deposition rates are useful in interpreting the
results of the root-tip-labeling experiments (see below).
Pro Synthesis from Glu
Pro synthesis from Glu has been proposed to be the major source of
Pro accumulated under drought or salinity stress (Delauney and Verma,
1993). We examined this possibility in our system by applying
[3H]Glu to the apical region of roots growing
at high or low w. Figure
5 shows Glu and Pro content per root
section (A and D), labeling of Glu and Pro (B and E), and specific
activity of Glu and Pro (C and F). Amino acid content per root section
changed over time because of the expansion of the root section and
displacement of the tissue through the gradient of amino acid
concentration in the elongation zone. These changes in amino acid
content make it essential to analyze specific activity when
interpreting the labeling data.
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| Figure 5.
Labeling of Glu (upper panels) and Pro (lower
panels) by 3H supplied as Glu in apical and basal sections
of the root tip at high (0.02 MPa) or low (1.6 MPa)
w. Peaks corresponding to individual amino acids were
collected after HPLC separation, and 3H activity was
quantified. A and D show Glu and Pro contents; B and E show total
3H activity; and C and F show specific activity. Each data
point is the mean of two samples from two separate experiments, and
each sample contained four sections. Apical and basal sections were
harvested as shown in Figure 1.  , High-w apical
section;  , high-w basal section;  ,
low-w apical section; and  , low-w
basal section.
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Glu specific activity was greater at low w
than at high w in both the apical and basal
regions of the elongation zone (Fig. 5C). Despite this, the specific
activity of Pro at low w was less than that at
high w (Fig. 5F). Based on the simplifying assumptions that in the apical root section at low
w the only fate of Pro is accumulation as free
Pro (i.e. Pro does not turn over and is not catabolized) and synthesis
from Glu is the source of all of the accumulated Pro (i.e. the rate of
Pro synthesis from Glu equals the Pro deposition rate), the predicted
specific activity of Pro can be conservatively estimated. This was done by taking the Pro deposition rate (36.4 nmol
h1, the total deposition rate for the apical 4 mm, as calculated above) and multiplying it by the Glu specific
activity (130 dpm nmol1, the specific activity
observed in the apical section at the 30-min time point; Fig. 5C) to
calculate the amount of label predicted to be converted to Pro. This
result was then divided by the Pro content of the root section (111 nmol [Fig. 5D]) to obtain the predicted Pro specific activity. The
predicted Pro specific activity at the 2-h time point for the apical
section at low w would be approximately 67 dpm
nmol1 using this method. This is already much higher than
the observed Pro specific activity of 3.9 dpm
nmol1 after 2 h of labeling (Fig. 5F). Pro
turnover and catabolism (which, based on labeling with
[3H]Pro, is known to occur at both high and low
w; see below) could further increase the
predicted specific activity by requiring the use of a Pro synthesis
rate higher than the net Pro deposition rate. Thus, synthesis from Glu
could not have been the major source of Pro accumulated in the apical
section at low w.
In addition, if Pro synthesis from Glu in the apical section was
responsible for the high Pro deposition at low
w, it would be expected that Pro specific
activity would be much less in the basal section, where the Pro
deposition rate was negative or zero (thus having an expected labeling
of zero using our simplifying assumptions). This was not the case, and
in fact, Pro specific activity was slightly higher in the basal
section, reaching 7.7 dpm nmol1 after 2 h
of labeling (Fig. 5F). Labeling of Pro in the basal section, despite
negative Pro deposition, could be explained by Pro turnover or by the
synthesis and export of Pro. However, the negative rate of Pro
deposition in the basal section (14.3 nmol h1) was insufficient to account for the
increased Pro deposition in the apical section (36.4 nmol
h1) without additional Pro synthesis. If
significant quantities of Pro had been synthesized in the basal section
and then transported to the apical section, this would have resulted in
high Pro specific activities in both the apical and basal sections. The
similarity of labeling in the two regions suggests that there is a
basal rate of Pro synthesis that is unrelated to treatment differences in Pro deposition.
At high w, the same calculation of predicted
Pro labeling in the apical section yields a somewhat different result.
Using the Pro deposition rate in the apical 4 mm of 1.9 nmol
h1, as calculated above, and the Glu specific
activity at 30 min of 40 dpm nmol1 (Fig. 5C),
the predicted Pro specific activity at the 2-h time point would be
approximately 115 dpm nmol1. Compared with the
actual Pro specific activity after 2 h of labeling (approximately
60 dpm nmol1), this calculation indicates that
Pro synthesis from Glu can account for a substantial fraction (as much
as half) of the Pro deposited. For the basal (5- to 9-mm) section, Pro
deposition was 2.8 nmol h1 and the predicted
Pro specific activity would be 47 dpm nmol1 at
the 2-h time point. However, the actual Pro specific activity remained
low (5.7 dpm nmol1 at the end of the
experiment), indicating that synthesis from Glu accounts for a smaller
fraction of Pro deposition in the basal section than in the apical
section. As was the case at low w, the
predicted Pro specific activities are probably underestimates because
Pro turnover and catabolism are not accounted for.
It should be noted that the much higher 3H
labeling in Pro in the low-w treatment (Fig.
5E) was not indicative of increased Pro synthesis, because the specific
activity of Pro was much lower than at high w
(Fig. 5F). The higher 3H activity can be
accounted for by labeled Pro being "trapped": The presence of a
large pool of Pro makes it unlikely that labeled Pro that enters this
pool will be catabolized. Thus, total 3H activity
can accumulate faster in the low-w treatment
with little or no increase in the rate of Pro synthesis.
It is also informative to look at the labeling of other amino acids
metabolically related to Glu (Table I).
Both Gln and GABA are synthesized from Glu by a single enzymatic
reaction that does not remove any of the 3H
label. Arg is synthesized from Glu by a series of steps that also leave
the 3H-labeled carbon backbone of Glu intact. All
three of these amino acids were labeled to a much higher specific
activity than Pro at low w. This indicates
that the relatively low Pro labeling at low w
was not caused by the labeled Glu being unable to participate in
biosynthetic reactions. Also, if the total 3H
activity in Gln, Arg, and GABA is summed, it is greater than the total
3H activity in Pro at either high or low
w and in either the apical or basal section.
Although the labeling of Gln, Arg, and GABA is also influenced by
catabolism and compartmentation that we cannot account for, the
relatively heavy labeling of these amino acids suggests that, in all of
our samples, synthesis of Pro is not the major metabolic fate of Glu.
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Table I.
Labeling of amino acids by 3H
supplied as Glu in apical and basal sections of the root tip at high
(0.02 MPa) or low (1.6 MPa) w.
Apical and basal sections were harvested after 120 min of labeling, as
shown in Figure 1. Data are means of two samples from two experiments.
RS, Root section.
|
|
Pro Synthesis from Orn
Orn can also serve as a precursor of Pro via the action of Orn
-aminotransferase (Delauney and Verma, 1993). Some studies have
suggested that the Orn pathway of Pro synthesis is of minor importance
in water-stressed plants (Delauney et al., 1993), although studies of
cotyledons have indicated that Orn can be a major precursor of Pro in
certain tissues (Hervieu et al., 1995). When we applied [14C]Orn to the root apical region, the pattern
of Pro labeling (Fig. 6, D-F) was
qualitatively similar to that obtained by labeling with Glu (Fig. 5,
D-F) in both the apical and basal sections. Total
14C labeling in Pro was higher at low than at
high w (Fig. 6E), and yet the specific
activity remained low in the low-w treatment (Fig. 6F). Because the HPLC analysis used here was unable to quantify Orn, it was not possible to measure the specific activity of Orn directly. Instead, the labeling of Arg is presented (Fig. 6, A-C). Arg
is synthesized from Orn through the urea cycle.
14C incorporation into Arg was greater than that
into Pro (Fig. 6, B and E), despite the much lower Arg content of the
root sections (Fig. 6, A and D). This observation provides an initial
indication that conversion to Pro may not be a major metabolic fate of
Orn in the maize primary root tip at either high or low
w.
View larger version (31K):
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| Figure 6.
Labeling of Arg (upper panels) and Pro (lower
panels) by 14C supplied as Orn in apical and basal sections
of the root tip at high (0.02 MPa) or low (1.6 MPa)
w. Sampling and data presentation are as described for
Figure 5. In F, the plot of the low-w apical section is
obscured by the low-w basal section data. ,
High-w apical section; , high-w basal
section; , low-w apical section; ,
low-w basal section.
|
|
If we make the assumption that the specific activity of Arg is
equivalent to that of Orn, the predicted specific activity of Pro can
be calculated in the same manner as described above for Glu labeling.
In the apical section at low w, the predicted specific activity of Pro after 2 h of labeling would be
approximately 318 dpm nmol1. The actual
specific activity was 12.4 dpm nmol1. In the
apical section at high w, the predicted
specific activity of Pro would be 1544 dpm
nmol1, whereas the actual specific activity was
347 dpm nmol1. Thus, synthesis from Orn cannot
account for more than a small fraction of Pro deposition in the apical
section. Similar results were obtained for the basal sections. It
should be emphasized that the assumption that the specific activity of
Orn is similar to that of Arg is highly conservative. In reality, the
specific activity of Orn should be considerably higher than that of Arg because of the small pool size of Orn (Voetberg and Sharp, 1991) and
the time needed to synthesize Arg from Orn. Thus, the predicted specific activities of Pro were almost certainly lower than they would
have been if they had been calculated directly from the specific
activity of Orn. As was the case for Glu labeling, if Pro turnover were
taken into account, the predicted specific activity of Pro would be
even higher. It should be noted that at low w Pro
labeling was similar in the apical and basal sections. As detailed
above for Glu labeling, this is not what would be expected if synthesis
from Orn were a major source of the increase in Pro deposition in the
apical section.
Because of the different specific activities of the
[3H]Glu and [14C]Orn
and the different pool sizes of Glu and Orn in the root tip, it is not
valid to directly compare the labeling or specific activity in Figure
5, E and F, and Figure 6, E and F, and draw any conclusions about
whether Pro was synthesized more rapidly from Glu or Orn. Nonetheless,
the relatively low rate of Pro synthesis from either Glu or Orn shows
that Pro synthesis could not have accounted for more than a small
fraction of the Pro deposition in the root elongation zone at low
w. This leaves decreased Pro catabolism and/or
utilization and increased Pro transport to the root tip as the possible
sources of Pro accumulation.
Pro Catabolism, Utilization, and Uptake
To assess Pro catabolism, [3H]Pro was
applied to root tips and the appearance of 3H in
Glu and other amino acids was monitored. After 30 min of labeling, the
specific activity of Pro in the apical section was 11-fold higher at
high w than at low w
(Fig. 7F). However, the specific activity
of Glu was only 3.4-fold higher at high w
(Fig. 7C). If Pro catabolism to Glu had been inhibited at low
w, it would be expected that the difference in
Glu specific activity between high and low
w would be greater than the difference in Pro specific activity. This would occur because the lower flux from Pro
to Glu at low w would limit the amount of
3H being converted from Pro to Glu. Thus, the
results indicate that Pro catabolism was not decreased in the apical
section at low w and may even have been
increased. Conversely, in the basal section, Pro specific activity at
30 min was 2-fold higher at high than at low w
but Glu specific activity was 14.2-fold higher at high
w, indicating that a suppression of Pro
catabolism occurred at low w. The labeling of
Pro at high w (Fig. 7E) decreased slightly at
later times during the experiments, complicating interpretation. Because of the low Pro content (Fig. 7D) in this treatment, the decrease in labeling had a relatively large effect on Pro specific activity (Fig. 7F). Caution must be used in interpreting these results
quantitatively because of the high turnover rate of Glu, indicated by
extensive labeling of other amino acids (see below). This high turnover
rate of Glu and the fact that we do not know the Glu deposition rate
made it impractical to perform the calculations of predicted specific
activity that were described for Glu and Orn labeling.
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| Figure 7.
Labeling of Glu (upper panels) and Pro (lower
panels) by 3H supplied as Pro in apical and basal sections
of the root tip at high (0.02 MPa) or low (1.6 MPa)
w. Sampling and data presentation are as described for
Figure 5. , High-w apical section; ,
high-w basal section; , low-w apical
section; , low-w basal section.
|
|
The labeling of amino acids other than Glu and Pro lends additional
support to a relatively high rate of Pro catabolism in the root apex.
In both the high- and low-w treatments, all of the other amino acids analyzed, with the exception of Gln, were labeled
more heavily in the experiments in which 3H was
supplied as Pro (Table II) than when
3H was supplied directly as Glu (Table I),
despite a lower Glu specific activity in the Pro-labeling experiments
(compare Figs. 5C and 7C). These other amino acids also had greater
labeling than was found in Pro after labeling with Glu (compare Table
II and Fig. 5E). This shows that the rate of flux in the direction of
Pro to Glu exceeded the rate from Glu to Pro in both the apical and
basal sections, suggesting that, instead of being a relatively inert
solute or storage form of carbon and reductant, Pro is actively catabolized at both high and low w.
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|
Table II.
Labeling of amino acids by 3H
supplied as Pro in apical and basal sections of the root tip at high
(0.02 MPa) or low (1.6 MPa) w.
Apical and basal sections were harvested after 120 min of labeling, as
shown in Figure 1. Data are means of two samples from two experiments.
RS, Root section.
|
|
The differential labeling between 3H applied as
Glu and Pro may also show the effect of metabolic compartmentation.
Several studies have shown that Pro catabolism occurs in the
mitochondria (Stewart and Lai, 1974; Elthon et al., 1984), whereas Pro
synthesis is cytoplasmic (Szoke et al., 1992). Therefore, the Glu
produced from Pro catabolism is produced inside the mitochondria, where it can be rapidly deaminated for entry into the TCA cycle. This would
explain the heavier labeling of amino acids such as Ala and Asp when
3H is supplied as Pro instead of Glu, and it may
also explain the high specific activity of some amino acids such as Arg
(Tables I and II). It is also consistent with Pro catabolism serving as
a source of energy and reductant in the root tip (see
``Discussion'').
Hydrolysis of the water-insoluble portion of the root-section extracts
showed that decreased Pro utilization in protein synthesis also did not
contribute to increased deposition of free Pro at low
w. Pro released by hydrolysis contained more
3H activity at low w
than at high w in both the apical and basal sections after 2 h of labeling (823 and 1636 dpm per root section in the apical section at high and low w,
respectively, and 303 and 656 dpm per root section in the basal
section at high and low w, respectively).
Increased Pro transport to the root tip is left as the most likely
source of the Pro accumulated at low w. In
support of this conclusion, we observed that the rate of loss of
3H activity from the Pro-labeling solution was
greater at low than at high w, indicating that
Pro uptake by the root tips increased at low
w. Additional uptake experiments were
performed to verify this observation, and the results are shown in
Figure 8. The root tips at low
w took up Pro at nearly twice the rate as
those at high w. The high rate of
3H uptake in the first 10 min after
[3H]Pro was applied is consistent with an
influx of Pro into the root apoplast. The later, sustained higher rate
of 3H uptake at low w
suggests an increased rate of Pro import into the root cells. It should
be noted that the amount of root tissue exposed to the labeling
solution at high and low w was roughly the
same at the beginning of the experiments, but the roots at high
w elongated at a higher rate than the roots at
low w (Fig. 2). Thus, by the end of the
experiments, it is likely that more root tissue was taking up Pro at
high than at low w. Accordingly, the
difference between high and low w in the rate
of Pro uptake per unit of root tissue was probably greater than that
indicated in Figure 8. Typically, less than 20% of the
3H in solution was taken up by the end of the
experiment. Measurements of label disappearance from the solutions in
the Glu- and Orn-labeling experiments did not indicate increased uptake
of Glu or Orn at low w (data not shown),
suggesting that the increase in Pro uptake was not caused by a general
increase in amino acid uptake. However, the increased Pro uptake must
be interpreted with caution because it is not known how accurately the
uptake of exogenous Pro from the apoplast reflects delivery of
endogenous Pro to the root tip.
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| Figure 8.
Uptake of [3H]Pro by the root tips
at high or low w measured as the rate of loss of
3H activity from the labeling solution in which the roots
were growing. Data are means ± SE of four
experiments.
|
|
| |
DISCUSSION |
The combined results of this study argue strongly that Pro
transported to the root tip is the main source of the dramatic increases in Pro deposition rate and Pro concentration that occur in
the maize primary root elongation zone at low
w. The results showed no evidence for a
substantial increase in Pro synthesis at low
w. In the apical section, where the highest
Pro deposition rates were found, there was also no evidence for a
decrease in Pro catabolism or utilization. Preliminary evidence in
support of increased Pro transport to the root tip at low
w was seen in the form of increased Pro uptake
from solution.
Metabolism and Function of Root-Tip Pro
Several studies of Pro metabolism have shown that Pro synthesis
from Glu is increased at low w in leaves
(Boggess et al., 1976; Hanson and Tully, 1979b) and in cell cultures
(Rhodes et al., 1986). In addition, expression of both P5CS and P5CR
mRNA has been shown to increase in response to salinity or dehydration (Delauney and Verma, 1990; Hu et al., 1992; Williamson and Slocum, 1992; Verbruggen et al., 1993; Yoshiba et al., 1995). A possible explanation for the difference between the conclusion reached in those
studies and that reached here is our examination of a very specific
region, the primary root elongation zone. Oaks (1966) examined Pro
synthesis in excised maize primary root tips at high w. Consistent with the results presented here,
it was found that C applied as acetate was
incorporated into Pro slowly. It was suggested that Pro synthesis in
the root tip was insufficient to meet the requirements for Pro and,
therefore, that Pro must be transported to the root tip to make up for
this deficit. The increase in Pro deposition at low
w would compound this deficit in Pro synthesis
unless a large shift occurred in the metabolic fate of Glu or Orn. Our
experiments showed no evidence of such a shift. In support of our
conclusion, preliminary experiments in which seedlings at high and low
w were grown in the presence of
2H2O and
2H incorporation into root-tip amino acids was
analyzed showed that Pro labeling in the root apex was approximately
one-half the amount expected if all of the Pro had been synthesized
since germination. The unlabeled Pro must have been released from seed storage and transported to the root tip (D. Rhodes, P.E. Verslues, and
R.E. Sharp, unpublished data).
Pro catabolism has often been proposed to be suppressed under water or
salt stress. Labeling studies such as those by Stewart et al. (1977)
and Stewart and Boggess (1978) found a suppression of Pro oxidation,
and in other studies (Kiyosue et al., 1996; Verbruggen et al., 1996) a
decrease in Pro dehydrogenase mRNA accumulation was reported. Again, it
is important to consider the tissues involved. Stewart et al. (1977)
and Stewart and Boggess (1978) used mature leaves, and Kiyosue et al.
(1996) and Verbruggen et al. (1996) analyzed either whole Arabidopsis
plants or whole root systems. The results presented here show that in
the apical root section Pro catabolism was not decreased at low
w, and at both high and low
w, the flux from Pro to Glu exceeded the flux from Glu to Pro. These observations agree with those of Barnard and
Oaks (1970), who showed that applied Pro is rapidly catabolized in
excised 5-mm-long maize root tips at high w.
They suggested that Pro is required by the root tip as an energy and
nitrogen source. It should be noted that a lack of suppression of Pro
catabolism relative to high w does not imply a
lack of regulation during water stress. It seems unlikely that Pro
could accumulate to such high levels if Pro catabolism were allowed to
increase in proportion to Pro concentration. Also, Pro catabolism may
be suppressed at low relative to high w in
other tissues that serve as a source of the Pro transported to the root
tip.
Our observations also fit well with some of the ideas of Hare and Cress
(1997) and Kohl et al. (1988). Hare and Cress (1997) suggested that
under water stress Pro from "effector cells" is transported to
"target cells," which have a high energy requirement. The effector
cells, which export Pro, are proposed to be cells that use the
synthesis of Pro from Glu to regenerate NADP+ for
processes such as the synthesis of purine nucleotides. The apical
growing region of the primary root is likely to be a tissue with a high
energy requirement and so would be a good candidate for such a
"target tissue." A similar system has been described in soybean
nodules by Kohl et al. (1988). Soybean nodules export nitrogen in the
form of purine derivatives. The authors proposed that Pro synthesis in
the cytoplasm of bacteroid-infected plant cells generates
NADP+ used in the synthesis of purines. The Pro
in the plant cytoplasm is then transported into the bacteroid, where it
is catabolized as an energy source to fuel nitrogen fixation. It is
interesting to note that Pro dehydrogenase activity in bacteroids is
increased at low w (Kohl et al., 1991),
whereas in other tissues Pro dehydrogenase activity or mRNA expression
is suppressed (Sudhakar et al., 1993; Kiyosue et al., 1996; Verbruggen
et al., 1996). It has also been demonstrated that bacteroid Pro
catabolism is significant in terms of plant performance: Soybean plants
inoculated with a Bradyrhizobium japonicum strain unable to
catabolize Pro had a greater reduction in yield after moderate water
stress than plants inoculated with wild-type B. japonicum
(Straub et al., 1997). This suggests that it would be of interest to
specifically modify Pro metabolism in the root tip and measure the
resulting effects on root growth and plant performance at low
w.
Pro Transport
Pro transport at low w has been much less
studied than Pro metabolism. Despite the smaller number of studies,
there are still seemingly contradictory reports. A series of studies
using barley leaves (Hanson and Tully, 1979a; Tully and Hanson, 1979;
Tully et al., 1979) found that the Pro content of phloem sap increased only slightly in response to water stress and that Pro could account for only a small fraction of the nitrogen exported from the stressed leaves. In contrast, Girousse et al. (1996) found an increase of up to
60-fold in Pro concentration of phloem sap collected from stems of
water-stressed alfalfa plants. Rentsch et al. (1996), working with
Arabidopsis, found that Pro transporter mRNA expression could be
induced by dehydration, whereas expression of a general amino acid
permease was suppressed. In addition, expression of P5CR associated
with the vascular tissue in Arabidopsis was shown to increase under
water stress (Hare and Cress, 1996; Hua et al., 1997). To our
knowledge, no studies of Pro transport have been performed using a
seedling system comparable to the system used here. Our
characterization of the metabolic mechanisms responsible for Pro
deposition in the maize primary root tip makes this an attractive
system for further studies of the regulation of Pro transport and
deposition. Previous work demonstrated that ABA accumulation is
required for the increased Pro deposition at low w (Ober and Sharp, 1994), suggesting that ABA
may play a role in regulating Pro transport to the root tip.
| |
FOOTNOTES |
1
This work was supported by National Science
Foundation grant no. IBN-9306935 to R.E.S. and Eric S. Ober. P.E.V. was
supported by a fellowship from the University of Missouri Maize Biology Training Program, a unit of the Department of Energy/National Science
Foundation/U.S. Department of Agriculture Collaborative Research in
Plant Biology Program (grant no. BIR-9420688). This is contribution no.
12,859 from the Missouri Agricultural Experiment Station journal
series.
2
Present address: Department of Botany and Plant
Sciences, University of California, Riverside, CA 92521.
*
Corresponding author; e-mail sharpr{at}missouri.edu; fax
1-573-882-1469.
Received August 10, 1998;
accepted December 20, 1998.
| |
ABBREVIATIONS |
Abbreviations:
GABA,  -aminobutyrate.
P5CR,  1-pyrroline-5-carboxylate reductase.
P5CS, 1-pyrroline-5-carboxylate synthetase.
w, water potential(s).
| |
ACKNOWLEDGMENTS |
We thank Dr. Eric Ober for advice and discussion during
the course of these experiments and Dr. David Rhodes (Purdue
University) for advice and critical comments concerning the manuscript.
| |
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Top
Abstract
Introduction