RESEARCH PAPER

Changes in intracellular and apoplastic peroxidase activity,

ascorbate redox status, and root elongation induced by
enhanced ascorbate content in Allium cepa L.
Mar a del Carmen Co rdoba-Pedregosa
1
, Jose Manuel Villalba
1
, Francisco Co rdoba
2
and Jose Antonio Gonza lez-Reyes
1,
*
1
Departamento de Biolog a Celular, Fisiologa e Inmunolog a, Universidad de Cordoba, Campus de Rabanales,
Edicio Severo Ochoa, Planta 3
a
, 14014 Cordoba, Spain
2
Departamento de Biolog a Ambiental y Salud Publica, Universidad de Huelva, Spain
Received 7 July 2004; Accepted 8 October 2004
Abstract
Onions (Allium cepa L.) treated with external ascorbic
acid or with the immediate precursor of its synthesis
L-galactono-c-lactone show a stimulated elongation
rate of the roots and an increase in the number of
new radicles appearing at the bulb base. Treatment
with both molecules resulted in an enhanced accumu-
lation of ascorbate and dehydroascorbate along the
root axis, but the distribution of these redox forms was
not uniform along the root, as detected in intracellular
(symplastic) and extracellular (apoplastic) compart-
ments. Thus, those radicular zones metabolically
more active, such as the meristem and the elongation
zone, accumulated the highest amount of both redox
forms of ascorbate. On the other hand, ascorbate and
L-galactono-c-lactonealsostimulatedcytosolicglucose-
6-phosphate dehydrogenase activity and inhibited per-
oxidase activity as deduced from in vivo and in vitro
experiments. Differences were also found when com-
paring apoplastic and symplastic activities. These
results are compatible with the idea of an ascorbate-
mediated stimulation of root growth by inhibiting cell
wall stiffening and increasing root metabolism.
Key words: Ascorbate, dehydroascorbate, glucose-6-phosphate
dehydrogenase, L-galactono-g-lactone, onion roots, peroxidase,
root elongation.
Introduction
Ascorbic acid (ascorbate; ASC) is an essential molecule for
higher plant cells. It is known that ASCis involved in several
defence mechanisms against oxidative stress (Noctor and
Foyer, 1998), as well as in the regulation of cell proliferation
(Arrigoni and De Tullio, 2002; De Tullio et al., 1999) and
elongation (Hidalgo et al., 1989; Gonzalez-Reyes et al.,
1994; Cordoba-Pedregosa et al., 1996). In addition, ASC
has an important role in excess energy dissipation in
thylakoids (Muller-Moule et al., 2003).
As ASC seems to play different roles depending on the
organ, it can be assumed that each plant tissue has de-
veloped different mechanisms to maintain the appropriate
concentration of this molecule to achieve optimal metab-
olismin each part of the plant. This can include a differential
ASC synthesis rate in every tissue, different efciency of
its transport to other parts, as well as differential ASC
recycling systems (Horemans et al., 2000; Smirnoff, 2000;
Smirnoff et al., 2001).
It is well known that extracellular ascorbate (i.e. apo-
plastic ascorbate) may function as a redox buffer, and that
many processes occurring in that compartment depend to
a great extent on ASC oxidation (Pignocchi and Foyer,
2003). Thus, the maintenance of an appropriate balance
of the reduced ASC and oxidized dehydroascorbate (DHA)
forms (i.e. redox status) in the apoplast seems to be
essential in extracellular matrix dynamics. The participation
of apoplastic ASC in the regulation of cross-linking be-
tween structural cell wall components has been proposed
* To whom correspondence should be addressed. Fax: + 34 957 218634. E-mail: bc1gorej@uco.es
Abbreviations: AF, apoplastic fraction; ASC, ascorbate; DHA, dehydroascorbate; DTT, dithiothreitol; GalL, L-galactono-c-lactone; G6PDH, glucose-6-
phosphate dehydrogenase; GPX, guaiacol peroxidase; MDHA, monodehydroascorbate; SSF, soluble symplastic fraction.
Journal of Experimental Botany, Vol. 56, No. 412, Society for Experimental Biology 2004; all rights reserved
This paper is available online free of all access charges (see http://jxb.oupjournals.org/open_access.html for further details)
Journal of Experimental Botany, Vol. 56, No. 412, pp. 685694, February 2005
doi:10.1093/jxb/eri051 Advance Access publication 6 December, 2004

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(Cordoba and Gonzalez-Reyes, 1994; Cordoba-Pedregosa
et al., 1996; Pignocchi and Foyer, 2003). In this respect,
ASC might facilitate elongation by inhibiting apoplastic
peroxidases involved in cell wall stiffening (Takahama,
1993; Takahama and Oniki, 1992). Furthermore, hydroxyl
radicals produced by ASC-mediated reactions could facili-
tate elongation by the non-enzymatic scission of cell wall
polysaccharides (Fry, 1998).
Intracellular ascorbate (i.e. symplastic ascorbate) can act
by regulating cell proliferation (De Tullio et al., 1999;
Arrigoni and De Tullio, 2002), and constitutes the main
soluble cytosolic antioxidant (Noctor and Foyer, 1998;
Smirnoff and Wheeler, 2000). Therefore, even in the same
organ, ASC may have different roles depending on whether
it is apoplastic or symplastic.
Recently, it has been shown that there is a differential
distribution of ascorbate along the root axis in onion, and
that its presence in the apoplast may also differ depending
on the radicular zone. Furthermore, both ASC and DHA
can be found at different concentrations depending on the
root zone and compartment (Cordoba-Pedregosa et al.,
2003a). It was concluded that ASC concentration and redox
status (i.e. the ratio between the reduced and oxidized
forms) may vary depending on the metabolic activity in
each part of the root.
In this paper, it is shown that increasing the ascorbate
content in onion roots by adding exogenous ascorbate or
L-galactono-c-lactone (GalL), the immediate precursor in
the ascorbate synthesis pathways (Wheeler et al., 1998), to
the culture media leads to the stimulation of root primordia
sprouting in the onion bulb bases, and to an enhanced
elongation rate in the growing roots. These effects were
accompanied by increased glucose-6-phosphate dehydro-
genase (G6PDH) activity and protein content, inhibition of
peroxidase activity, and accumulation of ASC and DHA in
specic regions of the roots, basically in the tips. Moreover,
it is shown that these results were different when apoplastic
and symplastic compartments were compared and that they
also depended on the root zone evaluated. The results are
discussed on the basis of the different ascorbate require-
ments for relevant functions developed in the different
zones of the root, such as proliferation and elongation in the
root apex and gradual differentiation to the root base.
Materials and methods
Growth conditions and treatments
Onion (Allium cepa L.) roots were grown hydroponically in the dark
at 25 8C in a culture medium consisting of 1 mM phosphate buffer at
pH6.5. Once roots had reached about 3 cmin length, some bulbs were
transferred to culture media containing 1 mM ASC or 2 mM GalL
(both from Sigma, Spain) in the same buffer, at pH 6.5, for 48 h.
Solutions were renewed every 24 h. Then, roots were detached from
the bulbs and cut into three zones each 2 cm long. The zone size was
the minimumthat enabled the tissue to be handled without appreciable
damage to the root and yielding apoplastic uids with low amounts of
cytosolic contamination. Zone I comprised the apical region of the
root, while zones II and III were sequentially closer to the onion base.
Root length measurements
Onions were cultured as explained above. The number of roots
visible at the onion crown, and the exact length of each one, were
measured using a plastic ruler prior to the treatments. The same
measurements were carried out after 24 and 48 h. Data were divided
into several classes depending on their respective lengths as follows:
1, up to 1.5 cm length; 2, from 1.6 to 2.7 cm; 3, from 2.8 to 3.9 cm; 4,
from 4 to 5.1 cm; 5, from 5.2 to 6.3 cm; 6, longer that 6.3 cm. Once
the roots had been classied by length, statistical analysis was carried
out as described below.
Isolation of apoplastic uids and intracellular soluble fractions
About 2 g of tissue from each root zone was quickly washed in
distilled water, placed in Petri dishes in 10 mM sodium phosphate
buffer, pH 6, containing 1.5% polyvinylpolypyrrolidone, 1 mM
EDTA and 0.5 mM phenylmethylsulphonyl uoride, and then sub-
mitted to vacuum (60 kPa) for 5 min at 4 8C. Afterwards, tissues were
carefully dried with lter paper and placed in syringes, which were
then placed in centrifugation tubes. Roots were centrifuged at 150 g
for 5 min and the apoplastic uids (AF) recovered at the bottom of the
tubes. Using this procedure about 70 ll of AF were obtained for 1 g of
each zone. The remaining root tissue was used to obtain the soluble
symplastic fraction (SSF) after homogenization in the same buffer
with an Ultraturrax T-25 (Ika, Germany) and centrifugation at 15 000 g
for 30 min. Cytosolic contamination of AF was monitored by
assaying glucose-6-phosphate dehydrogenase (G6PDH) activity as
marker.
Ascorbate and dehydroascorbate determination
To determine apoplastic and symplastic ASC and DHA, root samples
were not vacuum-inltrated, since previous assays had shown
a signicant loss of ASC and DHA during the inltration process
(Cordoba-Pedregosa et al., 1996). Instead, segments were obtained
and quickly blotted dry on lter paper and introduced into the
syringes. In this case, to avoid oxidation of ASC contained in
samples, AF were collected in centrifuge tubes containing a concen-
trated solution of metaphosphoric acid, so that its nal concentration
was 5% once the apoplast had been obtained. In another set of
experiments, intact root segments were homogenized in 5% meta-
phosphoric acid to determine the total ASC and DHA contents in
each root zone. The ASC content was estimated using the dipyridyl
method described by Knorzer et al. (1996). Previously, an ASC
standard calibration curve was run and an extinction coefcient of
16.5 mM
1
was obtained. Samples of AF and total homogenate were
preincubated in 0.5 mM dithiothreitol (DTT) to reduce DHA and then
total ascorbate concentration was measured. DHA contents were
estimated as the difference of ASC content measured in this assay
(with DTT preincubation) minus ASC determined without DTT
preincubation. Finally, ASC and DHA contents were also determined
in homogenates obtained from the root cap and meristems as well as
in the elongation zone. For this purpose, roots were detached from the
bulbs and two small portions about 2 mm long were obtained for each
root. The rst piece contained the root cap as well as the meristem,
while the second one included the elongation zone. By this procedure
about 0.1 g of each part was collected. Pieces shorter than this size
were impractical for quantitative determinations.
Enzymatic activities
Enzymatic activities were determined by spectrophotometric assays.
Reactions were developed at 25 8C, with constant stirring, in a nal
volume of 1 ml containing 2535 lg of protein. The following
686 Cordoba-Pedregosa et al.

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enzymatic activities were assayed for AF and SSF obtained from each
root zone.
Guaiacol peroxidase (GPX) was determined according to Zheng
and Van Huystee (1992). The reaction mixture contained 10 mM
sodium phosphate (pH 6), 0.1 ml of 0.3% (v/v) H
2
O
2
, and 0.1 ml of
1% (v/v) guaiacol. Reaction was followed at 470 nm (extinction
coefcient = 26.6 mM
1
cm
1
) for 5 min at 30 8C.
Glucose-6-phosphate dehydrogenase (G6PDH) assay was devel-
oped in 100 mM TRIS-HCl, pH 8, containing 1 mM MgCl
2
, 0.2 mM
NADP
+
, and 1 mM glucose-6-phosphate. Measurements were at
340 nm (Weimar and Rothe, 1986). Enzyme activities were expressed
as nmol of substrate metabolized min
1
g
1
FW. The presence of this
activity in AF was used as an estimation of cytosolic contamination.
It should be noted that all values given in this work concerning
apoplastic constituents have been corrected for cytosolic contamina-
tion according to the values of G6PDH activity in AF and the
corresponding SSF.
In vivo detection of total peroxidase activity
The method of De Pinto and Ros-Barcelo (1997) was used. Some
onions growing under normal conditions and after treatment for 48 h
with 1 mM ASC or 2 mM GalL were transferred to media consisting
of 0.1 M TRIS-acetate, 0.1 mM 4-chloro-naphthol, and 0.9 mM
H
2
O
2
, at pH 5. After several minutes of incubation a dark reaction
began to be appreciable in the roots, and pictures were immediately
obtained.
Protein determination
Protein was determined in both AF and SSF by the dye-binding
method of Bradford (1976), using c-globulin as a standard.
Statistical analysis
The effects of ASC and GalL on root sprouting and elongation were
tested by both analysis of variance (ANOVA) and the KruskalWallis
test. In all the other experiments mean values were compared using
Students t-test. Signicance levels of 95% (P <0.05) or 99%
(P <0.01) are indicated in the table and gure legends.
Results
The effect of ASC and GalL on root sprouting and
elongation
Treatments with 1 mM ASC and 2 mM GalL resulted in the
stimulation of both the mean number of roots per bulb and
root elongation rates (Fig. 1). At the beginning of the
experiment all the bulbs showed approximately the same
number of roots at the base (about 3862 roots per bulb;
mean value 6standard error; n=9), and they were also
similar in length (Fig. 1A). However, after 24 h those bulbs
treated with ASC showed a signicantly higher number of
longer roots (classes 4 and 5), while treatment with 2 mM
GalL resulted in a discrete increase in this parameter (Fig.
1B). Nevertheless, the mean number of roots per bulb
increased to practically the same extent in all the three
experimental conditions to reach values of about 4863.
After 48 h treatment with GalL and especially with ASC, an
increased number of roots per bulb was found (7264 for
control; 9568 for ASC; and 8365 for GalL; P <0.01 and P
<0.05 compared with the control, respectively). Also, the
number of longer roots (classes 5 and 6) increased signif-
icantly compared with the control, which denotes stimula-
tion of the elongation of growing roots (Fig. 1C). In
conclusion, 48 h treatments with GalL or ASC resulted in
stimulation of the rooting at the onion crown and in the
Fig. 1. Evolution of the radicular system from control bulbs and during treatment with 1 mM ascorbate (ASC) or 2 mM L-galactono-c-lactone (GalL) for
48 h. Root lengths of three bulbs per treatment were measured and classied into several classes at the beginning of the experiment (A) and after 24 h (B)
and 48 h (C) treatment. The classes are represented by different bar textures as indicated in the key (see also Materials and methods). The number of roots
included in each class is represented on the ordinate axis. Signicant differences were found using ANOVA and KruskalWallis test analysis as follows:
a, P <0.05 versus classes 4 and 5 from control; b, P <0.01 versus classes 5 and 6 from control and GalL; c, P <0.05 versus classes 5 and 6 from control.
Enhanced ascorbate and onion root growth 687

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stimulation of the root elongation rate, resulting in a higher
number of longer roots per bulb.
The effect of ASC and GalL on protein content and
G6PDH activity in the different root zones
Roots were treated with ASC and GalL as described in the
Materials and methods and then separated into three 2-cm-
long segments. From these zones apoplastic (AF)
and soluble symplastic fractions (SSF) were obtained. In
both fractions total protein content and G6PDH activity
were measured. The results are displayed in Tables 1
and 2.
For protein content, differences were found when com-
paring symplastic with apoplastic fractions (Table 1). In
control conditions, SSF protein content gradually decreased
from the root apex towards the base, as it did in all three
experimental conditions. However, AF proteins showed
the opposite tendency, increasing their concentration in the
same direction. These ndings were similar to those
obtained after ASC and GalL treatments. When the differ-
ent culture conditions were compared between them, small
differences were found for AF since ASC and GalL induced
a slight increase in protein content from zone I. However,
SSF obtained from zones II and III after incubations with
ASC and GalL, showed a signicant increase in protein
content compared with control roots. Also SSF from zone I,
after GalL treatment, showed a discrete increase of protein
compared with the control (Table 1).
G6PDH activity followed a distribution pattern strongly
similar to that shown for total proteins. Thus, in SSF the
highest activity corresponded to zone I and then progres-
sively decreased in zones II and III (Table 2). However, the
activity released to the AF during preparation of the
samples (i.e. cytosolic contamination), revealed a small
amount in zone I and gradually higher contents in zones II
and III (Table 2). In controls, the percentage activity with
respect to the corresponding SSF ranged between 0.15 and
1.93, being similar to or less than those reported in other
species (see Discussion). This indicates a low contamina-
tion with intracellular components and therefore the purity
of AF obtained with the procedure used. Following ASC
and GalL treatments, G6PDH activity followed the same
distribution pattern as in the control, but for each zone the
SSF value was signicantly higher than in untreated roots
(Table 2) and, consequently, values of apoplastic G6PDH
were higher in some cases. However, the percentage of AF
activity with respect to the corresponding SSF was signi-
cantly less in ASC pretreatments. In this respect, only zone
I from GalL-treated roots showed a signicantly reduced
percentage of G6PDH activity compared with controls
(Table 2).
Table 1. Protein content at the different zones of the root in control conditions and after 48 h treatments with 1 mM ASC or 2 mM
GalL
In SSF data are expressed in mg protein g
1
FW; in AF data are expressed in lg protein g
1
FW. In both cases, values are means 6SE from at least seven
different experiments.
Control ASC GalL
SSF AF SSF AF SSF AF
Zone I 10.060.50
a
33.063.0 10.660.77
a
45.764.3 12.261.1
a,
* 41.863.7
Zone II 4.360.33 89.567.0
b
6.760.56* 110.0610.2
b
6.760.66* 121.1611.3
b
Zone III 4.660.39 114.567.4
b
6.360.48* 114.8612.3
b
6.4260.62* 123.5610.7
b
a
P <0.01 versus zones II and III.
b
P <0.01 versus zone I.
* P <0.05 versus control.
Table 2. G6PDH activity measured in SSF and AF from the different root zones in control and after 48 h treatments with ASC and
GalL
Data are expressed in nmol min
1
g
1
FW and represent mean values 6SE (n=6). The percentage of apoplastic activity with respect to the corresponding
SSF is presented in parenthesis.
Control ASC GalL
SSF AF SSF AF SSF AF
Zone I 425645
a
0.6260.15
a
(0.1760.058) 6836114
a,
* 0.3260.08
a
(0.0560.017)* 6686107
a,
* 0.5760.14
a
(0.0860.01)*
Zone II 252632 3.0060.39 (1.2860.17) 376656* 3.2160.77 (1.0060.3)* 360652* 4.5060.47 (1.1360.1)
Zone III 197626 3.8260.37 (1.7560.09) 334658* 4.9061.06 (1.3260.28)* 325644* 6.3760.98 (1.5160.2)
a
P < 0.01 versus zones II and III.
* P < 0.05 versus control.
688 Cordoba-Pedregosa et al.

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The ascorbate system along the root axis
In control conditions the distribution pattern of ASC varied
along the root axis, both in total homogenates and in
apoplasts, as shown in Figs 2 and 3. Thus, in total homoge-
nates the highest amounts of both forms (ASC plus DHA)
were found in zone I and decreased towards the onion crown.
In all the evaluated zones ASC concentration was higher
than DHA, so that the ascorbate redox status remained
constant at a reduced state along the root axis (Fig. 2A).
Pretreatments with ASC and GalL induced a signicant
increase of both forms in all the three root zones, being higher
after GalL treatment (Fig. 2B, C). However, the relative
concentrations of ASCandDHAand, consequentlythe redox
status, remained unaltered along the root axis (Fig. 2B, C).
In the apoplast of control roots ASC content decreased
from zone I to zones II and III, while DHA increased in the
Fig. 2. ASC (white bars) and DHA (grey bars) content in total
homogenates from the different zones of control roots (A) and roots
treated with 1 mM ASC (B) and 2 mM GalL (C) for 48 h. Total ascorbate
content (ASC+DHA) is also included (solid bars). The numbers in
parenthesis represent the ascorbate redox ratio (ASC/ASC+DHA) at
every root zone. The data are expressed in lmol g
1
FW and are mean
values 6SE from at least four different experiments: a, P <0.01 versus
zone III; b, P <0.05 versus zones II and III; *, P <0.01 versus control; **,
P <0.01 versus control and ASC pretreatments.
Fig. 3. ASC (white bars) and DHA (grey bars) content in the apoplastic
fraction from the different zones of control roots (A) and roots treated
with 1 mM ASC (B) and 2 mM GalL (C) for 48 h. The total ascorbate
content (ASC+DHA) is also included (solid bars). The numbers in
parenthesis represent the ascorbate redox ratio (ASC/ASC+DHA) in
every root zone. The data are expressed in nmol g
1
FW and are mean
values 6SE from at least four different experiments: a, P <0.05 versus
zone I; b, P <0.01 versus zones II and III; *, P < 0.01 versus control; **,
P <0.05 versus control and ASC-pretreated roots.
Enhanced ascorbate and onion root growth 689

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same direction (Fig. 3A). In this compartment DHA
concentration was generally higher, and the redox status
was shifted to the oxidized form compared with total homo-
genates (Fig. 3A). Apoplastic fractions from treated roots
showed increased amounts of ASC and DHA as occurred
in total homogenates. Thus, in all the three zones ASC and
DHA contents were higher than in the control, but compar-
atively higher after GalL treatment (Fig. 3B, C), as occurred
in total homogenates.
Nevertheless, the concentration of both forms along the
root axis was different, depending on the treatment. Thus,
AF obtained after GalL showed a distribution pattern very
similar to that described for the control. However, treatment
with 1 mM ASC resulted in an increased concentration of
both ASC and DHA towards the root base (Fig. 3). On the
other hand, the redox status was different when compared
with controls. For example, in ASC treatments, redox status
in zone I was about 0.13, indicating a more oxidized status
than the same zone in controls. However, after treatments
with GalL, this parameter was signicantly higher than in
the other conditions, especially in zone I in which the redox
status was even more reduced (Fig. 3).
Peroxidase activity along the root axis
Peroxidase activity from SSF and AF was measured along
the root axis using guaiacol as substrate (GPX activity). In
control conditions, GPX was higher in SSF from zone I and
drastically decreased in zones II and III (Fig. 4A). In AF
this activity was found to represent about 3% of SSF
activity and followed the same tendency higher values in
zone I and a decrease towards the root base (see Fig. 4B).
Treatments with ASC or GalL, induced several changes
with respect to controls. First, both treatments resulted in
increased GPX activity in SSF, especially in zones II and III
(Fig. 4A). For ASC treatment, the increase in these zones
resulted in a homogeneous distribution pattern of the activ-
ity along the root axis, while GalL-treated roots showed
a nearly identical pattern to that found in controls (Fig. 4A).
For AF, GalL-treated roots had strongly similar GPX
activities along the root axis compared with controls, but
ASC signicantly increased the activity in all the radicular
zones (Fig. 4B).
An in vivo staining of peroxidase activity was performed
using 4-chloro-naphthol as susbtrate in the control and
treated roots. In these cases, the activity was revealed as
a dark stain in the root. The results of this assay are depicted
in Fig. 5. As observed, in control conditions the stain was
Fig. 4. Guaiacol peroxidase activity (GPX) in SSF (A) and AF (B) from
control roots (white bars) and roots treated with ASC (grey bars) or GalL
(solid bars) for 48 h. The data represent mean values 6SE from at least
four different experiments: a, P <0.01 versus zones II and III; *, P <0.01
versus control; **, P <0.01 versus control and GalL-pretreated roots.
Fig. 5. In vivo detection of peroxidase activity (using 4-chloro-naphthol
as substrate) in onion roots from control (C) and after 48 h treatments
with 1 mM ascorbate (ASC) or 2 mM L-galactono-c-lactone (GalL). The
whole set of roots from a typical bulb in every experimental condition is
shown in the upper part. Note that in ASC- and GalL-treated roots the
staining intensity is weaker than in the control, indicating a decreased
peroxidase activity. At the bottom is shown a magnied single root from
every treatment. In the apical zone, the root caps (RC) from ASC and
GalL treatments were not stained. The meristem (M) and the beginning of
the elongation zone (EZ) were not revealed at all with this technique,
indicating low peroxidase activity in these regions.
690 Cordoba-Pedregosa et al.

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not uniform along the root it was more intense in zone
I and gradually clearer in zones II and III. This effect was
also observed for roots treated with ASC and GalL with an
important difference roots were less stained than controls.
This effect was specially detected in GalL treatments (Fig.
5). Moreover, when control root tips were examined at
higher magnication, the staining in zone I was not uniform,
since the root cap was dark-coloured and the meristem and
elongation zone were almost free of the stain. After that
zone, at about 3 mm from the apex, the stain intensity was
similar to that in the root cap and then progressively cleared.
However, in ASC and GalL pretreatments all the root tip,
including the root cap, was clearer than in the control,
revealing less peroxidase activity (Fig. 5).
Ascorbic acid has been proposed as an effective inhibitor
of peroxidase activity (Takahama and Oniki, 1992; Takahama,
1993; Cordoba-Pedregosa et al., 1996). Therefore, a possi-
ble relationship was investigated between the scarcity of
stain obtained at the root tip and a possible accumulation of
ASC at that location. To address this point root caps plus
meristems and elongation zones were collected from
control and treated roots, and the ASC/DHA content
measured. The results are shown in Table 3. In all three
cases the root apex, which contained the rootcap and the
meristem, showed the highest values for both forms, and
the elongation zone also contained higher amount of ASC
and DHA than the whole zone I (Table 3). As occurred in
the whole root, the redox status was highly reduced in both
regions. A similar pattern was found for apical root pieces
from treated specimens (Table 3). However, in these cases
both ASC and DHA concentrations were higher than in the
control, as described for the whole root (see above). The
redox status was also similar (highly reduced) to that found
in controls (Table 3).
Discussion
In this paper a correlation is shown between the distribution
of ascorbate along the radicular axis and the stimulation
of elongation in onion roots growing hydroponically.
Although similar ideas have been proposed previously,
new data concerning the axial distribution of ascorbate, as
well as the effect of enhanced ASC content in onion root
physiology, are shown here.
The effect of ascorbate on onion root development
The increase in the mean number of roots per bulb and the
enhanced root elongation rate shown here, support the role
of ascorbate on cell proliferation and expansion as pre-
viously reported. Thus, the appearance of new roots at the
bulb base as a consequence of root primordia sprouting has
been observed in onions (De Cabo et al., 1993, 1996;
Citterio et al., 1994) and an active role of ascorbate on cell
extension through different molecular mechanisms has
been proposed as well (Cordoba and Gonzalez-Reyes,
1994; Cordoba-Pedregosa et al., 1996). However, the
direct observation of both phenomena in the same plants
has not been reported before. Furthermore, signicant
differences were found depending on the procedure to
increase the ascorbate content. As discussed below, the
capacity of ASC to be oxidized in the apoplast may have an
important role in the observed effects.
Enhanced ascorbate, G6PDH activity and protein
content in onion roots
In this paper, it is also shown that treatment with ASC or
GalL resulted in a signicant increase in G6PDH activity in
soluble symplastic fractions (SSF). This activity is widely
used to assess the purity of apoplastic fractions, since this
protein has been shown to be present in several isoforms in
the cytosol and plastids, but not in the extracellular matrix
(Vanacker et al., 1998a, b; Turcsa`nyi et al., 2000; Ranieri
et al., 2003). This is an important point since this enzyme
plays an essential role in the oxidative pentose phosphate
pathway and, therefore, in plant metabolism (Emes and
Fowler, 1979; Esposito et al., 2001; Hauschild and von
Schaewen, 2003). In the present experiments, the stimula-
tion of this enzyme activity ranged from 40% to 70%
depending on the root zone and treatment (Table 2),
Table 3. ASC and DHA contents in onion root tips from control and roots treated with ASC or GalL for 48 h
C+M represents the rootcap plus the meristematic zone; EZ is the elongation zone. Data are expressed in lmol g
1
FW and represent mean values 6SE
from at least four different experiments.
Total homogenate
ASC DHA Total Redox status
Control C+M 4.2860.90 1.0360.80 5.3160.28 0.8060.05
EZ 3.4060.15 0.9560.16 4.3560.48 0.7860.08
ASC C+M 6.0860.47
a
1.3560.07
a
7.4460.95
a
0.8260.16
EZ 6.7560.12
a
1.5160.08
a
8.2760.63
a
0.8260.07
GalL C+M 12.6560.60
b
1.4360.10
a
14.0861.64
b
0.9060.1
EZ 8.2861.26
b
1.2660.077
a
9.5461.09
b
0.8660.1
a
P < 0.01 versus control.
b
P < 0.01 versus control and ASC-pretreated roots.
Enhanced ascorbate and onion root growth 691

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indicating a clear stimulation of tissue metabolism. Fur-
thermore, Hauschild and von Schaewen (2003) reported
that 24 and 48 h treatments with metabolizable sugars
resulted in the stimulation of activity and mRNA expres-
sion of G6PDH in Solanum tuberosum leaves. Although the
present results showing increased protein content and
G6PDH activity t well with their results, the concentration
used for the various sugars tested in their paper (2550
mM) make quantitative comparisons difcult. However,
a possible partial effect of ASC and GalL as nutrients
cannot be discarded.
On the other hand, the G6PDH activity found in the
apoplastic fraction of control roots was very similar to that
reported by other authors (Vanacker et al, 1998a, b;
Veljovic-Jovanovic et al., 2001), suggesting that the AF
isolation procedure used by the authors was highly satis-
factory. However, it is interesting to note that after ASC or
GalL treatments the activity detected in AF from the
different root zones was only slightly higher than in the
control and, in some cases, less in terms of percentage from
the corresponding SSF. This effect was clearly detected in
AF from zone I and also from zones II and III in ASC-
treated roots, and suggests not only changes in the cytosolic
oxidative pentose phosphate pathway ratio, but also a pro-
tective effect of ascorbate on membrane integrity. So, root
centrifugation to obtain AF resulted in less leakage of the
enzyme from the cytosolic compartment. In this regard,
Buettner (1993) and Beyer (1994) have shown that ASC
can exert a protective effect on membrane components such
as a-tocoferol, thus preserving membrane integrity and
permeability properties. Therefore, the 48 h treatment with
ASC and, to some extent with GalL (zone I), could have
had a protective effect on membrane integrity inducing
a decrease in cytosolic leakage.
The ascorbate system along the radicular axis
Both ASC and GalL treatments resulted in an increase in
the content of ASC and DHA in roots. However, it should
be noted that GalL treatment resulted in signicantly higher
amounts of ASC/DHA compared with ASC-treated roots. It
has been reported that ASCis oxidized prior to its uptake by
the cells (Horemans et al., 2000). However, GalL is quickly
transported through the plasma membrane and used as a
substrate for ascorbate synthesis in mitochondria (Arrigoni,
1994; Siendones et al., 1999). Thus, apparently, GalL
treatments are more effective than ASC in enhancing
ascorbate accumulation in onion roots, but treatment with
ASC resulted in a higher stimulation of root elongation. A
similar result was reported by Arrigoni et al. (1997) in
Lupinus albus seedlings. Therefore, it is possible to assume
that the role of ascorbate in the stimulation of cell expansion
does not depend entirely on its accumulation in the sym-
plastic compartment. Instead, the apoplastic ASC seems to
play a more critical role in this respect. In fact, it was reported
previously that the semi-oxidized form monodehydroascor-
bate (MDHA), also called ascorbate free radical, induced an
accelerated cell expansion and root elongation in onion
(Hidalgo et al., 1989, 1991; Gonzalez-Reyes et al., 1994). It
was also shown that ASC oxidation resulted in the stimu-
lation of root growth (Gonzalez-Reyes et al., 1995). Thus,
treatments with ASC can lead immediately to MDHA
formation after its addition, while GalL has to be transported
and metabolized before ASCis delivered to the extracellular
matix. Thus, direct treatment with ASC should result in
a local increase in MDHAconcentration in the apoplast with
a consequent decrease in ASCcontent and a shift-down of its
redox ratio as is shown here in zone I.
Enhanced ascorbate and apoplastic peroxidase
action in roots
Peroxidases are a large group of proteins in plants with
a large number of isoenzymes, some of them located in the
apoplastic compartment (Andrews et al., 2000; Cordoba-
Pedregosa et al., 2003b). Although they have been shown to
be involved in growth processes, the precise role of apo-
plastic peroxidases in cell expansion has not been unequivo-
cally established. On the one hand, some peroxidases have
been shown to increase their expression levels in hypocotyls
under active elongation processes (Dunand et al., 2003).
However, more evidence has been accumulated showing
that peroxidase activity increases cross-linking between
structural components of the cell wall, thus promoting cell
wall stiffening and the cessation of elongation (Andrews
et al., 2002; Cordoba-Pedregosa et al., 2003a). Also,
Lagrimini et al. (1997) have shown that overexpression of
ananionic peroxidase inroots results ina signicant decrease
in radicular system development and in enhanced plant
wilting.
The present results show that zone I contains the highest
GPX activity. This is apparently in disagreement with the
inhibitory action of peroxidases in cell elongation, since
zone I contains the meristem and the elongation zones,
which are characterized by rapid enlargement of the cells.
However, activity staining invivo revealed that these regions
had very little peroxidase activity. It has been reported that
ASC is a strong inhibitor of peroxidases both in vivo and
in vitro (Takahama and Oniki, 1992; Takahama, 1993;
Cordoba-Pedregosa et al., 1996). Since these apical regions
of the roots contain the highest ASC concentrations, it is
very likely that peroxidase activity in the meristems and
elongation zones may be locally inhibited by the high ASC
concentration contained in these regions. An accumulation
of ascorbate in the apical region of the roots has been
reported to occur in other species (Franceschi and Tarlyn,
2002). Furthermore, when AFextracted fromonion roots are
assayed for GPX activity, increase of absorbance is not
linear with time. Instead, an initial phase with very low or
even no activity is observed, and the duration of this initial
phase is proportional to the ASC content (Cordoba-
Pedregosa et al., 2000). The same result was obtained by
692 Cordoba-Pedregosa et al.

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adding a known concentration of ascorbate to the GPXassay
cuvette (Cordoba-Pedregosa et al., 1996).
The local effect of ASC on peroxidase activity can
explain the results of in vivo staining in roots treated with
ASC or GalL, and supports the idea of a direct effect of
ASCtreatment on cell wall metabolism. Moreover, continu-
ous inhibition of apoplastic peroxidase by treatments with
ASC but not with GalL could result in an increase in
peroxidase protein synthesis and secretion in a typical
feedback mechanism. Thus, when the corresponding AF
are obtained and GPX assayed, a higher activity can be
found, as indicated in Fig. 4C, but only after the lag-phase
has nished. This fact can explain the increase in GPX
activity detected in apoplasts after ASC treatment and the
lack of staining in intact roots using 4-chloro-naphthol.
Recently, it has been proposed that ASC is also involved
in cell wall loosening via its participation in non-enzymatic
scission of cell wall polysaccharides (Fry, 1998; Miller
and Fry, 2001). This mechanism includes the formation of
hydroxyl radicals, which can be produced in the cell wall
from hydrogen peroxide formed by a reaction involving
ASC, Cu
2+
, and oxygen. In this regard, it has been proposed
that there is participation of a peroxidase in the formation
of the peroxide (Liszkay et al., 2003). This hypothesis, which
has been partially tested in vitro and in vivo (Schopfer,
2001; Schopfer et al., 2002), provides an additional explan-
ation of the role of ASC as a regulator of cell expansion.
In the present experiments, the highest ASC accumulations
have been found in those zones with the higher elonga-
tion rates. As stated above, treatment with ASCdid not have
the same effect as GalL, and most probably the immersion
of the roots directly in ASC could accelerate the reaction
and the trigger of mechanisms involved in the stimulation
of root growth.
Conclusions
ASC and GalL treatments induce stimulation of root
sprouting and elongation probably by increasing metab-
olism in the symplastic compartment and through a more
direct effect on the apoplast. In this compartment, ASC oxi-
dation seems to be important and, therefore, direct incu-
bation with ASC was more effective than GalL. Consistent
with its effect on root growth, increased ASC content modi-
es peroxidase activity in the different zones of the root.
Investigations are currently being developed to ascertain
the role of the enzymes involved in ascorbate metabolism in
plants and in its maintenance in an appropriate redox status
in each compartment in roots with increased elonga-
tion rates.
Acknowledgements
This work was supported by the Spanish Ministerio de Educacion y
Cultura (grant BMC2002-01078) and by the Junta de Andaluc a
(grant CVI-276). MdCC-P was supported by grant CVI-276).
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