Plant Science 223 (2014) 49–58

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Filter strip as a method of choice for apoplastic fluid extraction from

maize roots
Jelena J. Dragišić Maksimović a,∗ , Branka D. Živanović a , Vuk M. Maksimović a ,
Miloš D. Mojović b , Miroslav T. Nikolic a , Željko B. Vučinić a
a
Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11030 Belgrade, Serbia
b
Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: Apoplastic fluid was extracted from maize (Zea mays L.) roots using two procedures: collection from
Received 10 December 2013 the surface of intact plant roots by filter paper strips (AF) or vacuum infiltration and/or centrifugation
Received in revised form 24 February 2014 from excised root segments (AWF). The content of cytoplasmic marker (glucose-6-phosphate, G-6-P)
Accepted 5 March 2014
and antioxidative components (enzymes, organic acids, phenolics, sugars, ROS) were compared in the
Available online 13 March 2014
extracts. The results obtained demonstrate that AF was completely free of G-6-P, as opposed to AWF
where the cytoplasmic constituent was detected even at mildest centrifugation (200 × g). Isoelectric
Keywords:
focusing of POD and SOD shows the presence of cytoplasmic isoforms in AWF, and HPLC of sugars and
Apoplastic fluid
Filter paper strips
phenolics a much more complex composition of AWF, due to cytoplasmic contamination. Organic acid
Infiltration/centrifugation composition differed in the two extracts, much higher concentrations of malic acid being registered in
Isoelectric focusing AF, while oxalic acid due to intracellular contamination being present only in AWF. EPR spectroscopy
HPLC of DEPMPO spin trap in the extracts showed persistent generation of hydroxyl radical adduct in AF. The
Spin trapping electron paramagnetic results obtained argue in favor of the filter strip method for the root apoplastic fluid extraction, avoiding
resonance (EPR) the problems of cytoplasmic contamination and dilution and enabling concentration measurements in
minute regions of the root.
© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction enzymes and proteins attached, dissolved, or embedded in or to

The apoplast is a complex plant compartment, delimited from
the symplast by the plasma membrane. It consists of the rigid
cell wall fibrillar polymer network, the external surface of plasma
membrane, and the liquid- and gas-filled spaces within this net-
work, which provide interactions between the environment and
the plasma membrane enclosed cytoplasm. All of these three com-
ponents of the apoplast are rich in various organic molecules,
Abbreviations: AF, apoplastic fluid; AWF, apoplastic washing fluid; DEPMPO,
5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; DEPMPO/OH, DEPMPO
adduct with hydroxyl radical; EPR, spin trapping electron paramagnetic resonance;
G-6-P, glucose-6-phosphate; IEF, isoelectric focusing; MDH, malate dehydroge-
nase; • OH, hydroxyl radical; PPB, potassium phosphate buffer; POD, peroxidase (EC
1.11.1.7); SOD, superoxide dismutase (EC 1.15.1.1).
∗ Corresponding author. Tel.: +381 112078460; fax: +381 113055289.
E-mail addresses: draxy@imsi.rs, draxy@imsi.bg.ac.rs (J.J. Dragišić Maksimović),
vunduk@imsi.bg.ac.rs (B.D. Živanović), maxivuk@imsi.bg.ac.rs (V.M. Maksimović),
milos@ffh.bg.ac.rs (M.D. Mojović), mnikolic@imsi.bg.ac.rs (M.T. Nikolic),
vucinic@imsi.bg.ac.rs (Ž.B. Vučinić).
them.
The apoplast plays a major role in a wide range of physiological
processes, including transport of water, nutrients and metabolites
[1], gas exchange, growth regulation, plant-pathogen interactions,
and perception and transduction of environmental signals [2]. As a
thermodynamically open system, in direct contact with the envi-
ronment, the root apoplast is the first to encounter varying and
sometime adverse environmental conditions that are determining
the response of the whole plant [3,4].
For each of the three apoplastic constituents specific isolation
procedures have been developed, allowing a detailed in depth anal-
ysis of their composition, molecular organization, biochemistry and
physiology. The application of perfusion and infiltration and/or
centrifugation method initially developed by Söding and Klement
[5,6], and further refined by numerous authors, is the most fre-
quently used technique for the isolation of the apoplastic fluid.
It is a simple, quick and inexpensive method that can yield suffi-
cient quantities of the so called “Apoplastic Washing Fluid (AWF)”.
Besides the unknown dilution capacity, the infiltration of buffers
or distilled water into the plant tissue alters the ionic composition,

http://dx.doi.org/10.1016/j.plantsci.2014.03.009
0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.
50 J.J. Dragišić Maksimović et al. / Plant Science 223 (2014) 49–58
pH and metabolite concentration in the apoplastic fluid [7,8], and
inevitably alters the chemical equilibrium existing in the apoplast
[9]. Another drawback of the method is the inherent inability to
accurately determine the physiological concentration of different
metabolites and molecules present in the apoplastic fluid ex situ.
The major disadvantage of this technique is a risk of contam-
ination with cytosolic or vacuolar components. The centrifugal
force that is applied in the procedure raises the question of
membrane rupture and cellular injury. Also, the excision of plant
tissue results in inevitable contamination from injured cells at the
cut surface. Furthermore, cell injury caused by mechanical stress
leads to H2 O2 production triggering biochemical and physiolog-
ical ‘cascades’ which can disrupt physiological homeostasis [10].
This ‘cascade’ reaction shows that initially, a puzzling variety of
responses follow from a single event. Thus, a simple signal, such
as cell trauma, can generate a diversity of biochemical and phys-
iological consequences, altering the status of the metabolites and
biochemical reactions under study. The assessment of the cellu-
lar damage is usually performed by the use of specific markers
characteristic for the symplast. Contamination of the apoplastic
fluid has been quantified by comparing the activities of marker
enzymes (e.g. malate dehydrogenase, glyceraldehyde-3-phosphate
dehydrogenase, glucose-6-phosphate dehydrogenase) or concen-
tration of cytosolic metabolites (e.g. glucose-6-phosphate, G-6-P)
in AWF with that of crude extracts [7,8,11], or as a function of the
applied centrifugal force [12].
Composition of the apoplastic fluid is highly variable and
depends on plant species, age, time of a day and nutritional sta-
tus [11]. It should be emphasized that most of the studies on AWF
have been performed on the leaf tissue. Content of the leaf apoplast
has been explored in detail in terms of low molecular weight com-
pounds such as sugars [11,13–15], amino acids [11,16] and organic
acids [17–19], as well as phenolic compounds [20–22]. As opposed
to the leaf apoplastic fluid, the content of the root apoplastic fluid
has not been studied in detail, only a couple of reports being found
in the literature [22–25].
The total volume of AWF (Vinf ) was usually determined as the
difference in fresh weight before and after infiltration. Due to the
fact that infiltration of the apoplastic air space leads to a dilution
of the apoplastic fluid, the solute concentrations in the apoplastic
washing fluid were corrected by the ratio of the volume of infiltra-
tion solution (which corresponds to the volume of the apoplastic air
space Vair ) to the volume of the apoplastic water space (Vwater ). The
ion concentration in the apoplast was calculated by multiplying the
measured ion concentration in the apoplastic washing fluid by the
dilution factor (Fdil = (Vair + Vwater )/Vwater ). Vair being determined by
the silicone oil method [26] and Vwater by the [14 C] sorbitol-labeled
solution method [27] or using a plasma membrane impermeable
dye (e.g. Indigo carmine) [28]. Blue dextran 2000 has also been
recently used [29], based on the assumption that it does not enter
the symplast. Thus, determination of the dilution requires addi-
tional demanding procedures for its evaluation [11] making the
whole procedure complex and expensive.
In order to overcome the disadvantages of this most commonly
applied infiltration and/or centrifugation technique, we used fil-
ter paper strips (hereinafter referred to filter strips) to collect root
apoplastic fluid [22]. This sampling technique for organic com-
pounds based on sorption media placed onto the root surface of
plants was previously applied for determination of organic acids
collected from rhizosphere soil solution [30,31]. The filter strip
method, as a non-invasive technique for root apoplastic fluid col-
lection allows experiments with intact plants. It is suitable for
monitoring changes of metabolic compounds of the root apoplast,
especially in hydroponically grown plants, avoiding any significant
mechanical stress, implying that it is more reliable than the infil-
tration/centrifugation method for studying processes occurring in
apoplastic compartment of the root, providing adequate quantities
of the apoplastic fluid for analysis.
In the present work apoplastic fluid was collected from roots
of hydroponically grown maize plants employing the two isola-
tion techniques viz., filter strips from intact roots and infiltration
and/or centrifugation from excised roots. Different components of
the antioxidative system (enzymes, phenolics, ROS, sugars, organic
acids) present in the apoplastic fluid were analyzed using HPLC for
quantitative and qualitative determination of non-enzymatic com-
ponents, electrophoretic visualization and specific activity of POD
and SOD, and EPR spectroscopy of DEPMPO spin-trap for detection
of oxygen-centered radicals. The results obtained by the two col-
lection methods of the root apoplastic fluid were compared and
critically evaluated.
2. Material and methods
2.1. Plant material and growth conditions
Maize (Zea mays L., inbred line Va35, Maize Research Institute
“Zemun Polje”, Serbia) seeds were surface sterilized in 10% hydro-
gen peroxide, washed in distilled water and germinated on double
layer filter paper moistened with 0.5 mM CaSO4 in darkness at
27 ◦ C. Three days after sowing the seedlings were transferred to
plastic pots containing 1/4-strength modified unbuffered nutri-
ent solution [32] pH 5.9 and grown hydroponically with continual
aeration of bathing solution around the roots under controlled con-
ditions (light/dark 12 h/12 h; RH 70%; 25 ◦ C) until the second leaf
was fully developed. The nutrient solution was completely changed
every second day in order to avoid root growth reduction and lat-
eral root formation due to decreasing pH, as previously reported
for maize root [33], and nutrient deficiencies.
2.2. Isolation of apoplastic washing fluid (AWF) by infiltration
and/or centrifugation technique
Apoplastic washing fluid (AWF) was recovered from excised
root segments according to a modified procedure previously
described [25]. Briefly, apoplastic soluble components were
obtained from maize root segments whose fresh weight was mea-
sured immediately before the extraction procedure of AWF. The
following procedures were carried out at 4 ◦ C to reduce evaporation
and, thus, minimize changes in the composition of the AWF. Excised
and blotted root segments were positioned with the cut ends fac-
ing down into a 10-ml plastic vessel located over a centrifuge
tube and immediately after centrifugation at indicated speeds (200,
500, 1000 and 2000 × g denoted as CF200 , CF500 , CF1000 and CF2000 ,
respectively) for 15 min, while those denoted as IC2000 were first
vacuum infiltrated in 50 mM potassium phosphate buffer (PPB) pH
5.5 and then centrifuged at 2000 × g for 15 min too. Excised root
segments denoted as RIC2000 were kept in continuously aerated
1/4-strength nutrient solution to recover from wounding for 90 min
in a growth chamber. Then, the segments were washed in afore-
mentioned PPB, wiped with filter paper and vacuum infiltrated
with the same buffer in a vacuum desiccator, reducing the pressure
to -45 kPa, followed by slow relaxation to atmospheric pressure.
Infiltrated segments were wiped and put into plastic vessel, and
instantly centrifuged at 2000 × g for 15 min.
2.3. Collection of root apoplastic fluid (AF) by filter paper strips
For collection of fluid present in the roots apoplastic space,
small paper strips were placed on the top surface of the root pre-
viously blotted with filter paper to remove any remaining liquid
on the root surface after removing the plants from the hydro-
ponic medium. The fluid was extracted from the roots using
 J.J. Dragišić Maksimović et al. / Plant Science 223 (2014) 49–58
capillary forces, and named the “apoplastic fluid (AF)”. The filter
paper strips used for sample application for isoelectric focus-
ing (Sample application pieces, 10 mm × 5 mm, Serva, Heidelberg,
Germany), which are characterized by a high absorptive capacity
(more than 120 ␮l cm−2 ), were employed as sorption media. These
strips of inert composition (checked using HPLC chromatography,
EPR spectroscopy and UV–vis spectrophotometry) were weighted
immediately prior to their positioning on the root surface and
kept there for 30 min. The remaining root system was covered
with filter paper moistened with nutrient solution to prevent root
drying. Filter strips with absorbed AF were removed from root seg-
ments, re-measured and used for further analysis after extraction
in 100 ␮l of 50 mM PPB pH 5.5. The volume of AF collected by filter
paper strips was calculated as the difference in their weight before
application and after their removal from the root surface. Further
calculations were performed in order to calculate molarities (for
G-6-P and HPLC data) or protein content (for specific enzyme activ-
ities). Immediately after the collection of AF by filter strips, root
segments previously covered with the filter strips were excised
with a razor blade and weighed.
2.4. Contamination assays
Contamination of AWF or AF by symplast constituents was
assessed using the assay for malate dehydrogenase (MDH) activ-
ity as the cytosolic marker enzyme [28], and the presence of the
metabolite glucose-6-phosphate (G-6-P) [34].
MDH catalyzes the interconversion of l-malate and oxaloacetate
using ␤-NAD as coenzyme. MDH activity is assayed spectrophoto-
metrically (Shimadzu UV-2501 PC Kyoto, Japan) by measuring the
decrease in absorbance at 340 nm resulting from the oxidation of
␤-NADH. Taking into account that the optimum pH for the reac-
tion is 7.4–7.5, the assay mixture consists of 0.17 mM oxaloacetate
and 0.094 mM NADH dissolved in 0.1 M PPB pH 7.4. Reaction was
initiated by addition of 10 ␮l of sample to 1 ml of assay medium.
Cytoplasmic contamination of all samples was quantified by com-
paring the MDH activity in the apoplastic fluid with total root
homogenate.
G-6-P was detected using commercial glucose-6-phosphate
dehydrogenase (G-6-PDH; EC 1.1.1.49) from yeast. The reduction
of NADP was measured at 334 nm (ε334 nm = 6.18 mM−1 cm−1 ) in
a kinetic assay (Shimadzu UV-2501 PC Kyoto, Japan), where the
apoplastic fluid (AWF and AF) was the source of G-6-P substrate.
The assay medium (1 ml) contained 50 mM HEPES-KOH pH 6.9,
1.5 mM MgCl2 , 0.2 mM NADP, with a 15 ␮l of apoplastic fluid. The
reaction was initiated by the direct addition of 1 ␮l G-6-PDH dis-
solved in 50 mM HEPES-KOH pH 6.9 and 50% (w:v) glycerol. The
rise to a final maximum absorbance at 334 nm was complete within
2 min when G-6-P was present.
2.5. Enzymes assays
To determine peroxidative activity of peroxidase (POD; EC
1.11.1.7) coniferyl alcohol (A262 ; ε = 13.4 mM−1 cm−1 ) was used as
hydrogen donor. The reaction mixture (1 ml) consisted of 0.1 mM
coniferyl alcohol, 2.5 mM H2 O2 in 50 mM PPB, pH 6.5 and 10–50 ␮l
of sample, except where otherwise indicated. Initial rate of the
absorbance changes at 262 nm was measured, and corrected for the
amount of phenolic oxidation without H2 O2 . The oxidative activity
of POD was determined in 50 mM PPB at pH 5.5 by monitoring the
decrease of NADH absorbance at 340 nm in the assay mixture con-
taining 0.2 mM NADH, 0.2 mM p-coumaric acid, 0.25 mM MnCl2 ,
and 10–50 ␮l of sample in a volume of 1 ml. Spectrophotometric
measurements were performed with diode array spectrophotome-
ter (Hewlett Packard 8451A, Palo Alto, CA, USA) at 30 ◦ C. One unit
51
(U) of PODs was defined as the amount of the enzyme that oxidizes
1 ␮M of substrate per min per ml at above described conditions.
Total SOD (EC 1.15.1.1) specific activity was determined by inhi-
bition of oxygen-dependent reduction of cytochrome c (Cyt c) at
550 nm, according to the method of McCord and Fridovich [35].
One unit (U) of SOD was defined as the concentration of enzyme
needed for 50% inhibition of Cyt c reduction in 3 ml of reaction
volume. SOD specific activities were determined using a Shimadzu
spectrophotometer (UV-2501 PC Kyoto, Japan). Activities of differ-
ent SOD forms were identified and measured using KCN, by the
method of Bridges and Salin [36].
Protein concentration in maize root extracts was determined by
Bradford total protein assay [37] using BSA (bovine serum albumin)
as a standard, at 595 nm (LKB 5060-007, Micro plate Reader, GDV,
Rome, Italy). Total protein concentration of AWF was determined
immediately after isolation, while in AF it was conducted after ini-
tial extraction of filter strips in 100 ␮l of 50 mM PPB (see Section
2.3).
2.6. Protein separation and activity staining
POD and SOD isoenzymes were separated by isoelectric focusing
(IEF; LKB 2117 Multiphor II, LKB Instruments Ltd., South Croydon,
Surrey, UK) on a 7.5% polyacrylamide gel containing 3% ampho-
lite solution on a pH gradient from 3.5 to 10. Filter paper strips
containing the absorbed AF were placed directly on a gel, or AWF
containing 10 ␮g protein was applied to each well.
After completion of electrophoresis, POD isoenzymes were
stained with 10% 4-chloro-1-naphthol and 0.03% H2 O2 in 50 mM
PPB pH 6.5 for 10 min at 25 ◦ C.
Following focusing, SOD isoforms were stained by incubation in
2.45 mM NBT, 28 ␮M riboflavin, 250 mM EDTA and 2 mM TEMED
in 100 mM PPB (pH 7.8) for 30 min in darkness. Visualization of
SOD isoforms was achieved 20 min after the exposure to UV light
at room temperature. Identification of MnSOD isoforms on gel was
achieved by its incubation in 100 mM PPB (pH 7.8) containing 3 mM
KCN (inhibitor of Cu/ZnSOD), 30 min before staining by foregoing
procedure.
2.7. HPLC analysis
Phenolic compounds, organic acids and sugars were extracted
from single filter paper strips with 100 ␮l methanol acidified
with phosphoric acid and centrifuged for 10 min at 10 000 × g.
Aliquots of AF supernatant or isolated AWF were injected into a
Waters HPLC-ECD system (Waters binary pump system, thermostat
and autosampler connected to the Waters 2465 electrochemical
detector-ECD; Waters, Milford, MA, USA). The analysis of each type
of compound was done in triplicate.
Signals of phenolic compounds were detected in direct mode
at a constant potential of +0.75 V; the signal filter time was 0.2 s
and the sensitivity 10–100 nA for the full mV scale. Separation of
phenolic compounds was performed on a Symmetry C-18 RP col-
umn (125 mm × 4 mm with 5 ␮m particle size; Waters) with an
appropriate guard column. HPLC method was performed according
to the procedure previously described [38]. The data acquisition
and quantitative determination of the confirmed sample peaks
were carried out using Waters Empower Software. Results were
expressed as ␮M of phenolic compound of an isolated apoplastic
fluid.
Organic acids were separated using Aminex HPX-87H (Bio-Rad
Lab., Hercules, CA, USA) column 250 mm × 4 mm, 5 mM H2 SO4
being used as the mobile phase. Organic acids were isocratically
eluted with a flow of 0.6 ml min−1 , at constant temperature (30 ◦ C).
The signal of the organic acids was detected in the pulsed mode with
signal form: E1 = +0.3 V during 280 ms; E2 = +1.4 V during 150 ms;
52 J.J. Dragišić Maksimović et al. / Plant Science 223 (2014) 49–58
E3 = −0.4 V during 280 ms and 40 ms integration time. The results
are given in ␮M of organic acid of an isolated apoplastic fluid.
Concentration of sugars (glucose, sucrose and fructose) in the
samples was calculated from the peak size, using pure substances
(Sigma Co. St. Louis, MO, USA) as standards. Sugar separation
was performed using CarboPac PA1 (Dionex, Sunnyvale, CA) col-
umn (250 mm × 4 mm) and appropriate CarboPac PA1 precolumn.
The mobile phase was 0.2 M NaOH, the sugars being isocractically
eluted for 20 min, with a flow rate of 1 ml min−1 at constant temper-
ature (30 ◦ C). The sugar signals were detected in the pulsed mode
with signal form: E1 = +0.05 V during 400 ms; E2 = +0.75 V during
200 ms; E3 = −0.15 V during 300 ms and 180 ms integration time.
The mobile phase was prepared using NaOH solution (50%, w/w,
with low carbonate content, J.T. Baker, Deventer, The Netherlands)
and deionised water which had been vacuum degassed previously.
All the analyses were performed in triplicate. The results are pre-
sented as ␮M of sugar of an isolated apoplastic fluid.
2.8. EPR detection of • OH formation
Detection of • OH radicals was performed by EPR spin-trapping
method using DEPMPO (5-(diethoxyphosphoryl)-5-methyl-1-
pyrroline-N-oxide; ENZO Life Sciences AG, Lausen, Switzerland)
purified according to the method of Jackson et al. [39]. This spin-
trap is capable of forming different spin-adducts with • OH and
• O − radicals, thus differentiating between oxygen-centered free
2 The protein concentration in relation to the volume of iso-
radical species present in solution [40,41]. Either concentrated or
diluted DEPMPO were added to the AWF samples (60 ␮l), to a
final concentration of 240 mM or 12 mM, respectively. The mixture
was injected into a 10 cm long gas-permeable Teflon tube (wall
thickness 0.025 mm and i.d. 0.6 mm; Zeus Industries, Raritan, NJ,
USA) and folded into 2.5 cm long segments to improve the signal-
to-noise ratio [42]. Filter strips moistened with diluted (12 mM)
DEPMPO were placed on the root surface, and after 30 min AF
absorption were inserted into a quartz cuvette and placed inside
the resonator cavity. The EPR spectra were recorded at room tem-
perature by a Varian E104-A spectrometer operating at X-band
(9.3 GHz) with the following settings: modulation amplitude, 2G;
modulation frequency, 100 kHz; microwave power, 10 mW; scan
range, 200 G; scan time, 4 min. Spectra were recorded and ana-
lyzed using the EW software (Scientific Software International, Inc.,
Lincolnwood, IL, USA).
2.9. Experimental design, data collection and statistical analysis
The variability of each analytical method was measured repeat-
ing the analyses three times to confirm reproducibility. A quality
control sample (blank-containing all reagents except the test sam-
ple) was also analyzed together with the batch of samples and
recorded on a control chart. The results of all control samples were
within control limits thus confirming the reliability of analysis.
Data were subjected to analysis of variance using the statistical
software Statistica 6 (StatSoft, Inc., Tulsa, OK, USA) and means were
compared for significance by Mann–Whitney non-parametric test
at P < 5%.
3. Results
In order to determine whether different experimental proce-
dures used for the collection of AWF affect the content of the liquid
thus obtained a number of protocols were tested.
The fresh weight of maize root segments (Table 1) that were
only centrifuged at different speeds was not statisticaly different,
and slightly lower than that of the vacuum infiltrated root segments
(both, IC2000 and RIC2000 ). A much lower quantity of roots were used
for AF.
Fig. 1. Concentration of glucose-6-phosphate (G-6-P) as cytosol marker in apoplas-
tic washing fluid obtained from maize roots using different methods of extraction:
centrifugation at 200, 500, 1000 and 2000 × g, CF200 , CF500 , CF1000 and CF2000 respec-
tively; IC2000 , buffer infiltration and centrifugation at 2000 × g; RIC2000 , 90 min
recovery in nutrient solution, buffer infiltration and centrifugation at 2000 × g, and
apoplastic fluid (AF) obtained using filter strips. Bars are means of three replications
(n = 3) ± SE. Different letters denote a significant difference at P < 5%.
Maximal volume of AWF was extracted from root segments
previously vacuum infiltrated with buffer in surplus, followed by
blotting and centrifugation at 2000 × g (Table 1; IC2000 ). Surpris-
ingly, minimal volume was obtained by centrifugation at 2000 × g
(Table 1; CF2000 ). The protein concentration increased at the highest
centrifugation speed, and in the vacuum infiltrated samples.
lated apoplastic fluid, presented as total protein amount (TPA),
demonstrates the difference between the compared methods. The
traditional, centrifugation-based provides 8.4–38 ␮g protein in the
extract, while the filter strip method retrieves 9 ng protein (Table 1).
Initially, we measured malate dehydrogenase (MDH) activity,
in order to assess the cytoplasmic contamination of the apoplas-
tic fluid, as previously reported by Husted and Schjoerring [28].
AWF isolated by infiltration and centrifugation contained low lev-
els of MDH activity (approximately 1% of the activity measured in
the crude root extract, data not shown), whereas the AF obtained
using filter strips did not show any noticeable activity. According
to Dannel et al. [12], values below 1.6% are considered to be free of
cytoplasmic contaminations. However, results on isolated cell walls
[43,44] and plasma membranes [45], demonstrated the presence of
MDH in these isolates. Also, Li et al. [46] reported the occurrence of
soluble apoplastic MDH in leaves of barley and oats. All this raises
doubts on the use of MDH as a cytoplasmic contamination marker.
In subsequent experiments we focused on the presence of
glucose-6-phosphate (G-6-P), an intracellular metabolic pathway
intermediate, as a more appropriate marker. The highest concen-
tration was detected in AWF obtained only by centrifugation at
2000 × g (approx. 12 ␮M), the concentration in other centrifuged
and infiltrated samples being in the range from 2 to 4 ␮M (Fig. 1). As
opposed to AWF, in which all the samples demonstrated the pres-
ence of significant quantities of G-6-P, the AF samples did not show
the presence of this compound, indicating that these isolates are not
contaminated with cytoplasmic constituents at all. Due to the low
amount of starting material, the sensitivity of the assays was veri-
fied for other enzymes (SOD, POD), as well as metabolites (organic
acids, sugars and phenolic compounds), which were shown to be
of more than adequate and measureable quantities particularly in
the AF samples. Thus, one can state that the truancy of the activity
of marker enzymes in the case of AF samples is due to their absence
in the extracted fluid, and not due to the low detection limit of the
method.
An analysis of the peroxidase activity in the AWF extracts shows
that highest specific activities were obtained in least damaged sam-
ples, i.e. those centrifuged at lowest speed (CF200 ) and recovered
(RIC2000 ) samples, although the differences were not so pronounced
(Fig. 2a). The apoplastic fluid collected by filter strips showed a
 J.J. Dragišić Maksimović et al. / Plant Science 223 (2014) 49–58 53

Table 1
Root fresh weight (FW), volume (V), total protein concentration (TPC) and total protein amount (TPA) of apoplastic washing fluid obtained from maize roots using different
methods of extraction: (i) centrifugation at 200, 500, 1000 and 2000 × g, CF200 , CF500 , CF1000 and CF2000 respectively; (ii) IC2000 , buffer infiltration and centrifugation at 2000 × g;
(iii) RIC2000 , 90 min recovery in nutrient solution, buffer infiltration and centrifugation at 2000 × g, and (iv) apoplastic fluid (AF) obtained using filter strips.

FW (mg) V (␮l) TPC (␮g ml−1 ) TPA (␮g)

CF200 543.8 ± 43.3a 78.7 ± 0.3a 106.6 ± 30.5a 8.38

CF500 588.1 ± 53.3a 127.8 ± 19.3b 127.7 ± 1.9b 16.19
CF1000 539.8 ± 18.7a 102.9 ± 3.6b 116.0 ± 40.9a,b,c 11.83
CF2000 657.2 ± 77.9a 49.3 ± 8.8d 266.8 ± 32.5d 13.15
IC2000 811.7 ± 61.2b 180.9 ± 12.6c 211.8 ± 2.5c 38.31
RIC2000 888.0 ± 76.5b 119.1 ± 9.4b 195.2 ± 22.5c 23.24
AF 11.1 ± 0.6c 1.1 ± 0.1e 8.2 ± 0.7e 9.02 × 10−3

Data are means (n = 3) ± SE. Significant differences at P < 5% are indicated by different letters.

significantly higher specific activity of the peroxidase (3–15 times
greater than AWF samples). Isoelectric focusing of AF revealed that
they had a completely different isoenzyme pattern in the acidic
region, compared to AWF (Fig. 3a), no noticeable differences being
observed between different AWF samples. These results argue in
favor of cytoplasmic cationic peroxidase isoenzyme release into the
apoplast during AFW extraction, even at the lowest centrifugation
speeds.
As opposed to the peroxidases, SOD specific activity was
affected much more by different procedures of AWF isolation
(Fig. 2b). Similar to the peroxidases, this class of enzymes showed
the highest specific activity in the AF. The intensity of the acidic
SOD isoforms varied depending on whether infiltration was
performed, as well as the centrifugation force used (Fig. 3b). Using
KCN and H2 O2 as inhibitors, we detected MnSOD isoform at pI 8.2,
which could be observed only in AWF isolated at speeds lower than
1000 × g. This isoform, characteristic for the cytoplasm [47] could
not be detected in AF, in spite of the much greater specific enzyme
activity of this isolate. Overall, the specific enzyme activity and
isoenzyme patterns again demontrate the absence of cytoplasmic
contamination in the case of AF extraction, but also indicate that
the isoenzyme pattern can be altered due to either cytoplasmic
contamination of AWF samples, or interaction of the cytoplasmic
intermediates with the apoplastic enzymes.
A quantitative and qualitative analysis of non-enzymatic com-
ponents such as organic acids, sugars and phenolics in root
apoplastic fluid isolates was performed using HPLC chromatog-
raphy. In Fig. 4 we show representative chromatograms obtained
from AWF and AF extracts. It is obvious from these chromatograms
that AWF contains a higher number of constituents in all three ana-
lyzed classes of compounds and that their content is more complex
than the apoplastic fluid obtained using filter strips.
In Table 2 we presented the concentrations of the three classes
of analyzed compounds that have been identified and quantified
(organic acids, sugars and phenolics, respectively). In AWF obtained
from excised root by centrifugation at 2000 × g oxalic, malic and
succinic acids were detected. Oxalic acid could not be detected
in the case of the AF collected with filter strips, but the samples

Fig. 2. Peroxidase (a) and total superoxide dismutase (b) activity in apoplastic washing fluid obtained from maize roots using different methods of extraction: centrifugation
at 200, 500, 1000 and 2000 × g, CF200 , CF500 , CF1000 and CF2000 respectively; IC2000 , buffer infiltration and centrifugation at 2000 × g; RIC2000 , 90 min recovery in nutrient
solution, buffer infiltration and centrifugation at 2000 × g, and apoplastic fluid (AF) obtained using filter strips. POD, peroxidase; SOD, superoxide dismutase; U, unit. Bars
are means of three replications (n = 3) ± SE. Different letters denote a significant difference at P < 5%.
54 J.J. Dragišić Maksimović et al. / Plant Science 223 (2014) 49–58

Fig. 3. Isoelectrofocusing (IEF) pattern of soluble proteins stained for peroxidase (a) and superoxide dismutase (b) activity of maize root apoplastic washing fluid obtained
using different methods of extraction: centrifugation at 200, 500, 1000 and 2000 × g, CF200 , CF500 , CF1000 and CF2000 respectively; IC2000 , buffer infiltration and centrifugation
at 2000 × g; RIC2000 , 90 min recovery in nutrient solution, buffer infiltration and centrifugation at 2000 × g, and apoplastic fluid (AF) obtained using filter strips. IEF was done
in a pH gradient of 3–10. 10 ␮g protein was applied to each well. pI 8.2 (marked bold) represents MnSOD identified using KCN as inhibitor.

contained besides malic and succinic acids, also citric acid. The a loss of signal (Fig. 5b and c), indicating that the assumed pres-
concentration of malic acid was higher in the case of AF. Glucose ence of intracellular constituents that have been released into the
and fructose concentrations were higher in the AWF, while sucrose apoplast during the extraction procedure, quenched the signal and
concentration was slightly higher in the AF. The concentration of free radical generation. Also, as previously shown, much greater
phenolic compounds were of one to two orders of magnitude higher concentration of the oxidative metabolism enzymes, phenolics and
in AF obtained using filter strips, compared to the AWF of excised other metabolic intermediates in the apoplastic fluid, enable sus-
roots centrifuged at 2000 × g (Table 2). tained generation • OH for prolonged periods of time.
Spin trapping electron paramagnetic resonance (EPR) spec-
troscopy showed that AWF, regardless of the centrifugation speed
and spin-trap concentration, had no capacity to generate hydroxyl 4. Discussion
radicals (• OH, as judged by their capacity to form DEPMPO/OH
adduct) (Fig. 5a–c). Also, no discernible generation of • O2 − radi- The technique commonly used for the analysis of apoplastic
cals (i.e. formation of DEPMPO/OOH adducts) could be observed. fluid is the centrifugation and/or vacuum infiltration of the excised
Surprisingly, the generation of • OH in AF could be registered plant tissue. Different authors developed different variants of the
30 min following extraction, even the diluted spin-trap showing extraction procedure, involving only centrifugation [22,48,49], vac-
DEPMPO/OH spectra (Fig. 5f). Higher centrifugation speeds led to uum infiltration [50], or a combination of both [51,52], most of the
experiments being performed on excised leaf tissue.
The centrifugation/infiltration method has been the subject of
Table 2 critical evaluation by a number of authors [23,53]. The issues raised
Concentration of representative organic acids, sugars and phenolic compounds (in
␮M) in maize root apoplastic fluid detected by HPLC. Apoplastic washing fluid (AWF)
are the possibility of intracellular content contamination of the
was obtained from excised roots by centrifugation at 2000 × g. Apoplastic fluid (AF) apoplast (both outflow at the injured site as well as cellular mem-
was collected with filter strips from intact roots. brane rupture and leakage due to centrifugal force), modification
of its constituents, and the possibility of wrong estimate of the
Compound AWF (␮M) AF (␮M)
dilution, making exact measurements of the concentration of the
Oxalic acid 5.75 ± 0.73 nd
extracellular compounds questionable.
Citric acid nd 11.96 ± 4.81
Organic acids
Malic acid 1.13 ± 0.34 26.67 ± 8.96 The difference in total protein amount (TPA) is 3 orders of
Succinic acid 287.51 ± 8.55 276.07 ± 6.17 magnitude higher in AWF compared to AF (Table 1). Its sudden
increase in AWF, from 8 to 38 ␮g, is a direct consequence of
Glucose 365.65 ± 24.60 144.67± 21.95
Sugars Fructose 263.31 ± 66.05 40.63 ± 1.17 structural damage made by both the high centrifugation speed and
Sucrose 2.76 ± 0.99 3.93 ± 0.83 buffer infiltration. Also, TPC dramatically increased at the maximal
Chlorogenic acid 0.008 ± 0.003 0.45 ± 0.03
centrifugation speed, regardless to the method used (CF2000 , IC2000 ,
Caffeic acid 0.006 ± 0.002 0.52 ± 0.03 RIC2000 ). Thus, obviously harmful application of centrifugal force
Phenolics Coniferyl alcohol 1.471 ± 0.447 140.71 ± 19.85 and vacuum infiltration dramatically increases the protein content
p-Coumaric acid 0.013 ± 0.003 0.25 ± 0.03 in AWF samples, masking the real in situ situation by the release a
Isoferulic acid 0.299 ± 0.091 4.85 ± 1.19
number of different intracellular proteins (as it can be observed in
Data are means (n = 3) ± SE. the case of POD and SOD in Fig. 3 by the appearance of a numerous
 J.J. Dragišić Maksimović et al. / Plant Science 223 (2014) 49–58
Fig. 4. Representative HPLC chromatograms of apoplastic washing (AWF at
2000 × g) and apoplastic (AF) fluids showing differences amongst three major classes
of small weight metabolites. Inset with S marked compounds is PAD chromatograms
of sugars: S1 polyols, S2 rhamnose S3 trechalose, S4 arabinose, S5 mannose, S6 galac-
tose, S7 glucose, S8 fructose, S9 ribose, S10 sucrose, S11 raffinose; Inset with O marked
compounds represents UV chromatograms of organic acids: O1 oxalic, O2 citric,
O3 malic and O4 succinic. Inset with P marked compounds represents UV chro-
matograms of phenolic compounds: P1 chlorogenic acid, P2 caffeic acid, P3 coniferyl
alcohol, P4 p–coumaric acid and P5 isoferulic acid. Note difference scaling for AWF
(right ordinates) and AF (left ordinates).
Fig. 5. EPR spectra of DEPMPO/OH adducts from apoplastic fluid collected with different methods: infiltration/centrifugation (AWF) or filter strips (AF). (a) AWF collected
by centrifugation at 200 × g with 240 mM DEPMPO; (b) AWF collected by centrifugation at 2000 × g with 12 mM DEPMPO; (c) AWF collected by centrifugation at 2000 × g
with 240 mM DEPMPO; (d) filter strip (control spectra); (e) AF collected using filter strip without DEPMPO; (f) AF collected using filter strip with 12 mM DEPMPO.
55
additional isoforms). Much lower protein concentration in AF did
not prevent differentiation of the analyzed POD and SOD isoforms,
for which we can, with much greater assurance, claim to be
devoid of intracellular contamination. Also, measurements of POD
and SOD activities show that AF samples exhibited much higher
specific activity. Thus, lower protein content in AF did not affect
the analysis completeness for the enzymes analyzed in this study.
Contamination of the AWF with cytoplasmic or vacuolar com-
ponents due to tissue injury induced by excision with a razor blade
and mechanical stress response has been addressed by a number of
authors [11,12,54]. Such contamination can be reduced by rinsing
in buffer or nutrient solution prior to measurements [55]. This was
also demonstrated in the present study (Table 1, Fig. 1). Although a
decrease of protein and G-6-P marker concentrations was evident
in the fluid collected from the root segments incubated prior to
centrifugation, this did not completely remove the cytosolic con-
taminants.
A comparison of the amounts of hexose, pentose, proteins, and
malate dehydrogenase in extracellular solution from pea epicotyl
sections obtained by applying different centrifugal forces showed
that most of these substances were released from intracellular
sources at 500 × g [54], and contamination of AWF is enhanced
by an increase of centrifugal force [11]. Dannel et al. [12] demon-
strated damage to cell walls and membranes of sunflower leaves at
centrifugal forces between 2000 and 2500 × g. In our experiments,
even a centrifugal force of 200 × g resulted in the appearance of the
marker substrate (G-6-P) in AWF, significantly increasing at higher
speeds (Fig. 1).
Dilution of the apoplastic fluid induced by the infiltration pro-
cedure results in an inevitable change in the chemical equilibrium
existing in the apoplast, which can lead to inaccuracies in measure-
ments of apoplastic ion and metabolite concentrations. Infiltration
of water or buffer into the apoplast modifies the ionic content, so
that the concentration of ions is not proportional to their activity
[3]. As a result of internal ionic interactions the effective concentra-
tion of an ion is often very different from the actual concentration.
In this way, ions behave chemically like they are less concentrated
than they really are (or measured). In fact, with this technique in
56 J.J. Dragišić Maksimović et al. / Plant Science 223 (2014) 49–58
most cases ion- or element concentration, and not ion activity, is
determined. Lohaus et al. [11] tested the infiltration-centrifugation
method for parameters affecting the ions and metabolites for dif-
ferent plant species, showing that the procedure must be adjusted
for each species. Also different pH of the infiltration medium can
alter the concentrations of carbohydrates, primarily sucrose [11].
In order to bypass these issues, we employed the filter strip
technique as a method that uses intact plants avoiding mechani-
cal stress (tissue cutting, vacuum infiltration and centrifugation),
and the dilution effects. Originally, the filter strip technique was
used only for localized root exudate sampling for the analysis of
organic acids [19,30,31]. In our previous article we have reported
that such a technique is also suitable for monitoring changes of the
metabolic components in apoplastic fluid of maize root, and that it
is sensitive enough to discern metabolic changes which occur along
the maize root [22]. In the current study we extended its applica-
tion to the analysis of antioxidative systems present in the apoplast
(enzymes, phenolics, ROS, sugars and organic acids), and the results
have been compared to those of AWF samples.
Our results demonstrate that the enzyme activity and the isoen-
zyme composition of apoplastic fluid depends on the techniques
used for its isolation. Centrifugation and infiltration affected both
the POD and SOD specific activities of AWF. In the case of POD
the specific activity decreased with increasing centrifugal force
all the way to 2000 × g, while SOD specific activity gradually
decreased with increasing centrifugal force up to 1000 × g, and
greatly increased at 2000 × g (Fig. 2). Vacuum infiltration further
increased specific enzyme activity, and “recovered” samples had a
slightly aleviating effect. Thus, the difference observed in specific
enzyme activity can be explained by dilution due to leakage from
intracellular compartment during centrifugation. The increase in
SOD specific activity at 2000 × g can be explained by the release of
intracellular SOD into the apoplast. A much higher specific activity
of both enzymes detected in AF clearly demonstrates the advantage
of using the filter strips for apoplast fluid extraction and enzyme
analysis, bypassing all the problems associated with drastic isola-
tion procedures used to obtain AWF (Fig. 2).
Ionically bound cationic peroxidases have been shown to be
present in isolated cell walls [56,57] which could be readily secreted
to the apoplastic solution and serve as an emergency enzyme
involved in plant wound response [58]. In our results, the appear-
ance of root cationic isoforms only in AWF, but not in AF, could
be due to possible extraction of some weakly ionically bound per-
oxidases by infiltration buffer, since cationic group of peroxidases
was shown to be present in cell wall ionically bound fraction [54].
The other possibility is that some cationic isoforms are expressed
as the response to stress induced by wounding during AWF extrac-
tion, which was avoided by the filter strip method of extracting AF.
Different pattern of the root cationic isoforms observed in AWF and
AF in our experiments argue strongly in favor of partial intracellular
contamination in the case of AWF, irrespective of the centrifugation
and infiltration procedure. Previously published data suggested
that soluble strongly cationic apoplastic peroxidases of Norway
spruce, with isoelectric point pI 9 or higher, participate in lignin for-
mation [59]. Also, Martinez et al. [60] demonstrated in cotton the
presence of a number of cationic extracellular peroxidase isoen-
zymes at pI 9–9.3. As mentioned by these authors, the transient
alkalinization that takes place in the apoplasm of cotyledons could
favor the activity of a wall-bound cationic peroxidase, leading to
H2 O2 synthesis required for lignification. In our filter strips only
one cationic isoform of peroxidase at pI 9.3 was detected (Fig. 3a)
in maize root apoplast.
Isoelectric focusing of the isolates differentiated Cu/ZnSOD
anionic isoforms with pI from 3.6 to 5.1 and cationic isoforms with
pI from 8.6 to 9.3. Using specific inhibitors (KCN and H2 O2 ), we
have shown that MnSOD isoform with pI 8.2, characteristic for the
mitochondrial matrix of eukaryotic cells [47], was unequivocally
present in CF200 and absent in AF (Fig. 3b). However, in CF500 , CF1000
and CF2000 under supposedly more damaging conditions, there is
poorly or no visible 8.2 isoform, probably due to interference with
inter- or intracellular content. Considering that TPC was similar in
CF200 , CF500 and CF1000 but significantly higher in CF2000 (Table 1),
it is clear that high centrifugal force, causing damage of cellular
structures, released a great quantity of different proteins. Such an
altered protein profile decreased the portion of specific MnSOD
in electrophoretic sample rendering its band less visible. Since a
same amount of total protein was applied to the gel for all sam-
ples (10 ␮g, see Section 2.6), the absence of MnSOD isoform with
pI 8.2 more likely is a consequence of lower concentration of the
specific enzyme applied to the gel. This is yet another argument
for intracellular contamination of the AWF, irrespective of the iso-
lation procedure. Further research could determine whether the
increase in protein concentration applied to the gel (15 or 20 ␮g)
would result in more distinct appearance of this isoform.
The study of organic acid metabolism in the plant root is limited
due to their constant exchange with soil solution, cellular com-
partmentation and unreliable methods for determining their true
concentration in the apoplastic space. The release of organic acids
into the rhizosphere has been documented [61,62], and it has been
shown that their cytosol concentration (0.5–10 mM) is about 1000-
fold greater than that present in the soil solution (0.5–10 ␮M) [62].
Typically, the total concentration of organic acids in roots is in the
range of 10–20 mM (1–4% of total dry weight), since the concentra-
tion of metabolic intermediates such as malate and citrate in the
cytosol rarely exceeds 5 mM, whereas concentrations in the vacuole
can be 1–10 fold higher [63]. Data for organic acids concentration
in root apoplast are scarce.
In the present study, dominant organic acids identified in AWF
were oxalate, malate, and succinate in micromolar range (Table 2,
Fig. 4). In AF we did not detect oxalic acid, but citric acid could be
observed. Also, the concentration of malic acid was 25-fold greater
in AF, when compared to AWF. Oxalic acid can combine with var-
ious elements and compounds to form acid and neutral salts, a
monoamide (oxamic acid), and a diamide (oxamide) [64]. Oxalic
acid can also react with alkali and other metal ions such as Li+ ,
Na+ , K+ , and Fe2+ to form soluble salts or with Ca2+ to form Ca-
oxalate deposits in the vacuoles. Ca-oxalate formation has been
shown to be a rapid and reversible process in Lemna minor, indi-
cating that oxalate crystal formation is a highly controlled process
and providing a mechanism for regulating intracellular calcium, as
well as oxalate levels in plant organs [65]. Considering the vacuo-
lar localization of oxalate under normal physiological conditions, its
presence in AWF in our experiments is further proof of membrane
damage and leakage from the vacuoles to the apoplast. Again, oxalic
acid absence in AF samples argues in favor of the non-destructive
filter strip technique approach. Lower concentrations of malate
observed in AWF can be explained by leakage of intracellular reduc-
tants and enzymes, consuming the malic acid that was initially
present in the apoplast.
The presence of 11 identified sugar compounds in AWF, of which
only 4 (galactose, glucose, fructose and sucrose) were detected in
AF, once again argues in favor of apparent contamination of AWF
originating from intracellular content leakage due to the forceful
isolation procedure (Fig. 4). Higher concentration of glucose and
fructose observed in the present study in AWF compared to AF is
in line with this argument (Table 2).
In our previous article using the filter strips [22] we have shown
that phenolics varied both spatially and temporally in the apoplast
along the root; higher concentrations being observed in the mature
zone associated with intense lignification. Quantitative and quali-
tative analysis of monomeric phenolic compounds in maize root
apoplastic fluid demonstrated the presence of coniferyl alcohol,
 J.J. Dragišić Maksimović et al. / Plant Science 223 (2014) 49–58
chlorogenic, caffeic, p-coumaric, and isoferulic acid (Table 2, Fig. 4).
Coniferyl alcohol was shown to be the major component of both
AWF and AF. The concentrations of all phenolic compounds in AWF
were exceptionally low when compared to those in AF, where two
orders of magnitude higher concentrations could be observed. This
again is due to dilution with intracellular content because of the
applied extraction procedures used for obtaining AWF, and possi-
ble interaction with them. The presence of additional unidentified
phenolics not present in AF further corroborates this conclusion.
Liszkay et al. [66] have shown, on relatively large samples of cells
or cut tissue incubated with the POBN spin trap (not able to differ-
entiate different oxygen centered radicals) up to 2 h on a shaker,
that free radicals are present in the incubation medium. Such an
experimental approach gives an indication for the generation of
free radicals in the apoplast, but it does not exclude the possibility
of the participation of numerous free radical reactions originating
from the wounded tissue and released intracellular intermediates,
nor the interaction of different intermediates with specific cell wall
bound redox systems. In our measurements, we demonstrate sus-
tained generation of • OH in AF samples (Fig. 5). EPR spectroscopy
performed with AWF regardless of the centrifugation speed and
spin-trap concentration gave no signal (Fig. 5a–c), as well as control
spectra (Fig. 5d) and AF collected using filter strip without spin-trap
(Fig. 5e). It seems that DEPMPO/OH complex formed on filter strips
was more stable, which is prerequisite for this type of analysis.
Thus, the filter strip technique in combination with EPR spectrom-
eter provides us with a tool to study in vivo production of • OH and
other free radical species in the apoplastic fluid, without interaction
with cellular, or fibrillar cell wall components.
All the presented observations argue in favor of filter strip
method, as being more reliable than infiltration/centrifugation
method, for studying processes occurring in apoplastic compart-
ment of the root. Thus, filter strips in combination with the high
sensitivity HPLC-ECD, EPR and electrophoretic techniques provide
us with a tool to study components of antioxidative system in
the apoplast of developing plant organs and their spatial-temporal
changes. It enables distinct concentration measurements of root
metabolites in the apoplast, and does not require pre-concentrating
and purifying samples. Such an experimental technique provides a
powerful non-invasive analytical tool for studying metabolical pro-
cesses occurring in the apoplast and local changes in small regions
of the intact root tissue, especially in hydroponically grown plants.
To conclude, our results unequivocally demonstrate the advan-
tage of using filter strips for collection of the apoplastic fluid
from the intact plant roots, giving small amounts of uncontam-
inated liquid containing much higher specific enzyme activities
and metabolite concentrations, such as organic acids and phenolics.
Compared with the centrifugation/vacuum infiltration technique,
our data argue against the use of this method, using even the small-
est centrifugation force, because such procedures coupled with
excision and mechanical wounding unavoidably release a whole
range of different intracellular compounds and proteins into the
apoplast during the extraction procedure.
Acknowledgments
The authors are very grateful to Dr Günter Neumann from
Hohenheim University (Stuttgart, Germany) for method demon-
stration and training. This work was supported by the Serbian
Ministry of Education, Science and Technological Development
(grants 173040 and 173028).
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