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Plant Science 223 (2014) 49–58

Contents lists available at ScienceDirect

Plant Science

j ourna l ho me pa ge: www.elsev ier .com/ locate /p lantsc i

ilter strip as a method of choice for apoplastic fluid extraction fromaize roots

elena J. Dragisic Maksimovic a,∗, Branka D. Zivanovic a, Vuk M. Maksimovic a,ilos D. Mojovic b, Miroslav T. Nikolica, Zeljko B. Vucinic a

Institute for Multidisciplinary Research, University of Belgrade, Kneza Viseslava 1, 11030 Belgrade, SerbiaFaculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia

r t i c l e i n f o

rticle history:eceived 10 December 2013eceived in revised form 24 February 2014ccepted 5 March 2014vailable online 13 March 2014

eywords:poplastic fluidilter paper stripsnfiltration/centrifugation

a b s t r a c t

Apoplastic fluid was extracted from maize (Zea mays L.) roots using two procedures: collection fromthe surface of intact plant roots by filter paper strips (AF) or vacuum infiltration and/or centrifugationfrom excised root segments (AWF). The content of cytoplasmic marker (glucose-6-phosphate, G-6-P)and antioxidative components (enzymes, organic acids, phenolics, sugars, ROS) were compared in theextracts. The results obtained demonstrate that AF was completely free of G-6-P, as opposed to AWFwhere the cytoplasmic constituent was detected even at mildest centrifugation (200 × g). Isoelectricfocusing of POD and SOD shows the presence of cytoplasmic isoforms in AWF, and HPLC of sugars andphenolics a much more complex composition of AWF, due to cytoplasmic contamination. Organic acidcomposition differed in the two extracts, much higher concentrations of malic acid being registered in

soelectric focusingPLCpin trapping electron paramagneticesonance (EPR)

AF, while oxalic acid due to intracellular contamination being present only in AWF. EPR spectroscopyof DEPMPO spin trap in the extracts showed persistent generation of hydroxyl radical adduct in AF. Theresults obtained argue in favor of the filter strip method for the root apoplastic fluid extraction, avoidingthe problems of cytoplasmic contamination and dilution and enabling concentration measurements inminute regions of the root.

© 2014 Elsevier Ireland Ltd. All rights reserved.

. Introduction

The apoplast is a complex plant compartment, delimited fromhe symplast by the plasma membrane. It consists of the rigidell wall fibrillar polymer network, the external surface of plasmaembrane, and the liquid- and gas-filled spaces within this net-

ork, which provide interactions between the environment and

he plasma membrane enclosed cytoplasm. All of these three com-onents of the apoplast are rich in various organic molecules,

Abbreviations: AF, apoplastic fluid; AWF, apoplastic washing fluid; DEPMPO,-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; DEPMPO/OH, DEPMPOdduct with hydroxyl radical; EPR, spin trapping electron paramagnetic resonance;-6-P, glucose-6-phosphate; IEF, isoelectric focusing; MDH, malate dehydroge-ase; •OH, hydroxyl radical; PPB, potassium phosphate buffer; POD, peroxidase (EC.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. Dragisic Maksimovic),unduk@imsi.bg.ac.rs (B.D. Zivanovic), maxivuk@imsi.bg.ac.rs (V.M. Maksimovic),ilos@ffh.bg.ac.rs (M.D. Mojovic), mnikolic@imsi.bg.ac.rs (M.T. Nikolic),

ucinic@imsi.bg.ac.rs (Z.B. Vucinic).

ttp://dx.doi.org/10.1016/j.plantsci.2014.03.009168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

enzymes and proteins attached, dissolved, or embedded in or tothem.

The apoplast plays a major role in a wide range of physiologicalprocesses, including transport of water, nutrients and metabolites[1], gas exchange, growth regulation, plant-pathogen interactions,and perception and transduction of environmental signals [2]. As athermodynamically open system, in direct contact with the envi-ronment, the root apoplast is the first to encounter varying andsometime adverse environmental conditions that are determiningthe response of the whole plant [3,4].

For each of the three apoplastic constituents specific isolationprocedures have been developed, allowing a detailed in depth anal-ysis of their composition, molecular organization, biochemistry andphysiology. The application of perfusion and infiltration and/orcentrifugation 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 buffersor distilled water into the plant tissue alters the ionic composition,

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H and metabolite concentration in the apoplastic fluid [7,8], andnevitably alters the chemical equilibrium existing in the apoplast9]. Another drawback of the method is the inherent inability toccurately determine the physiological concentration of differentetabolites and molecules present in the apoplastic fluid ex situ.The major disadvantage of this technique is a risk of contam-

nation with cytosolic or vacuolar components. The centrifugalorce that is applied in the procedure raises the question of

embrane rupture and cellular injury. Also, the excision of plantissue results in inevitable contamination from injured cells at theut surface. Furthermore, cell injury caused by mechanical stresseads to H2O2 production triggering biochemical and physiolog-cal ‘cascades’ which can disrupt physiological homeostasis [10].his ‘cascade’ reaction shows that initially, a puzzling variety ofesponses follow from a single event. Thus, a simple signal, suchs cell trauma, can generate a diversity of biochemical and phys-ological consequences, altering the status of the metabolites andiochemical reactions under study. The assessment of the cellu-

ar damage is usually performed by the use of specific markersharacteristic for the symplast. Contamination of the apoplasticuid has been quantified by comparing the activities of markernzymes (e.g. malate dehydrogenase, glyceraldehyde-3-phosphateehydrogenase, glucose-6-phosphate dehydrogenase) or concen-ration of cytosolic metabolites (e.g. glucose-6-phosphate, G-6-P)n AWF with that of crude extracts [7,8,11], or as a function of thepplied centrifugal force [12].

Composition of the apoplastic fluid is highly variable andepends on plant species, age, time of a day and nutritional sta-us [11]. It should be emphasized that most of the studies on AWFave been performed on the leaf tissue. Content of the leaf apoplastas been explored in detail in terms of low molecular weight com-ounds such as sugars [11,13–15], amino acids [11,16] and organiccids [17–19], as well as phenolic compounds [20–22]. As opposedo the leaf apoplastic fluid, the content of the root apoplastic fluidas not been studied in detail, only a couple of reports being found

n the literature [22–25].The total volume of AWF (Vinf) was usually determined as the

ifference in fresh weight before and after infiltration. Due to theact that infiltration of the apoplastic air space leads to a dilutionf the apoplastic fluid, the solute concentrations in the apoplasticashing fluid were corrected by the ratio of the volume of infiltra-

ion solution (which corresponds to the volume of the apoplastic airpace Vair) to the volume of the apoplastic water space (Vwater). Theon concentration in the apoplast was calculated by multiplying the

easured ion concentration in the apoplastic washing fluid by theilution factor (Fdil = (Vair + Vwater)/Vwater). Vair being determined byhe silicone oil method [26] and Vwater by the [14C] sorbitol-labeledolution method [27] or using a plasma membrane impermeableye (e.g. Indigo carmine) [28]. Blue dextran 2000 has also beenecently used [29], based on the assumption that it does not enterhe symplast. Thus, determination of the dilution requires addi-ional demanding procedures for its evaluation [11] making thehole procedure complex and expensive.

In order to overcome the disadvantages of this most commonlypplied infiltration and/or centrifugation technique, we used fil-er paper strips (hereinafter referred to filter strips) to collect rootpoplastic fluid [22]. This sampling technique for organic com-ounds based on sorption media placed onto the root surface oflants was previously applied for determination of organic acidsollected from rhizosphere soil solution [30,31]. The filter stripethod, as a non-invasive technique for root apoplastic fluid col-

ection allows experiments with intact plants. It is suitable for

onitoring changes of metabolic compounds of the root apoplast,

specially in hydroponically grown plants, avoiding any significantechanical stress, implying that it is more reliable than the infil-

ration/centrifugation method for studying processes occurring in

t Science 223 (2014) 49–58

apoplastic compartment of the root, providing adequate quantitiesof the apoplastic fluid for analysis.

In the present work apoplastic fluid was collected from rootsof hydroponically grown maize plants employing the two isola-tion techniques viz., filter strips from intact roots and infiltrationand/or centrifugation from excised roots. Different components ofthe antioxidative system (enzymes, phenolics, ROS, sugars, organicacids) present in the apoplastic fluid were analyzed using HPLC forquantitative and qualitative determination of non-enzymatic com-ponents, electrophoretic visualization and specific activity of PODand SOD, and EPR spectroscopy of DEPMPO spin-trap for detectionof oxygen-centered radicals. The results obtained by the two col-lection methods of the root apoplastic fluid were compared andcritically 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 doublelayer filter paper moistened with 0.5 mM CaSO4 in darkness at27 ◦C. Three days after sowing the seedlings were transferred toplastic pots containing 1/4-strength modified unbuffered nutri-ent solution [32] pH 5.9 and grown hydroponically with continualaeration of bathing solution around the roots under controlled con-ditions (light/dark 12 h/12 h; RH 70%; 25 ◦C) until the second leafwas fully developed. The nutrient solution was completely changedevery second day in order to avoid root growth reduction and lat-eral root formation due to decreasing pH, as previously reportedfor maize root [33], and nutrient deficiencies.

2.2. Isolation of apoplastic washing fluid (AWF) by infiltrationand/or centrifugation technique

Apoplastic washing fluid (AWF) was recovered from excisedroot segments according to a modified procedure previouslydescribed [25]. Briefly, apoplastic soluble components wereobtained from maize root segments whose fresh weight was mea-sured immediately before the extraction procedure of AWF. Thefollowing procedures were carried out at 4 ◦C to reduce evaporationand, thus, minimize changes in the composition of the AWF. Excisedand blotted root segments were positioned with the cut ends fac-ing down into a 10-ml plastic vessel located over a centrifugetube 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 firstvacuum infiltrated in 50 mM potassium phosphate buffer (PPB) pH5.5 and then centrifuged at 2000 × g for 15 min too. Excised rootsegments denoted as RIC2000 were kept in continuously aerated1/4-strength nutrient solution to recover from wounding for 90 minin a growth chamber. Then, the segments were washed in afore-mentioned PPB, wiped with filter paper and vacuum infiltratedwith the same buffer in a vacuum desiccator, reducing the pressureto -45 kPa, followed by slow relaxation to atmospheric pressure.Infiltrated segments were wiped and put into plastic vessel, andinstantly 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 liquidon the root surface after removing the plants from the hydro-ponic medium. The fluid was extracted from the roots using

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apillary forces, and named the “apoplastic fluid (AF)”. The filteraper strips used for sample application for isoelectric focus-

ng (Sample application pieces, 10 mm × 5 mm, Serva, Heidelberg,ermany), which are characterized by a high absorptive capacity

more than 120 �l cm−2), were employed as sorption media. Thesetrips of inert composition (checked using HPLC chromatography,PR spectroscopy and UV–vis spectrophotometry) were weightedmmediately prior to their positioning on the root surface andept there for 30 min. The remaining root system was coveredith filter paper moistened with nutrient solution to prevent rootrying. Filter strips with absorbed AF were removed from root seg-ents, re-measured and used for further analysis after extraction

n 100 �l of 50 mM PPB pH 5.5. The volume of AF collected by filteraper strips was calculated as the difference in their weight beforepplication and after their removal from the root surface. Furtheralculations were performed in order to calculate molarities (for-6-P and HPLC data) or protein content (for specific enzyme activ-

ties). Immediately after the collection of AF by filter strips, rootegments previously covered with the filter strips were excisedith a razor blade and weighed.

.4. Contamination assays

Contamination of AWF or AF by symplast constituents wasssessed using the assay for malate dehydrogenase (MDH) activ-ty as the cytosolic marker enzyme [28], and the presence of the

etabolite glucose-6-phosphate (G-6-P) [34].MDH catalyzes the interconversion of l-malate and oxaloacetate

sing �-NAD as coenzyme. MDH activity is assayed spectrophoto-etrically (Shimadzu UV-2501 PC Kyoto, Japan) by measuring the

ecrease in absorbance at 340 nm resulting from the oxidation of-NADH. Taking into account that the optimum pH for the reac-

ion is 7.4–7.5, the assay mixture consists of 0.17 mM oxaloacetatend 0.094 mM NADH dissolved in 0.1 M PPB pH 7.4. Reaction wasnitiated by addition of 10 �l of sample to 1 ml of assay medium.ytoplasmic contamination of all samples was quantified by com-aring the MDH activity in the apoplastic fluid with total rootomogenate.

G-6-P was detected using commercial glucose-6-phosphateehydrogenase (G-6-PDH; EC 1.1.1.49) from yeast. The reductionf NADP was measured at 334 nm (ε334 nm = 6.18 mM−1 cm−1) in

kinetic assay (Shimadzu UV-2501 PC Kyoto, Japan), where thepoplastic fluid (AWF and AF) was the source of G-6-P substrate.he assay medium (1 ml) contained 50 mM HEPES-KOH pH 6.9,.5 mM MgCl2, 0.2 mM NADP, with a 15 �l of apoplastic fluid. Theeaction was initiated by the direct addition of 1 �l G-6-PDH dis-olved in 50 mM HEPES-KOH pH 6.9 and 50% (w:v) glycerol. Theise to a final maximum absorbance at 334 nm was complete within

min when G-6-P was present.

.5. Enzymes assays

To determine peroxidative activity of peroxidase (POD; EC.11.1.7) coniferyl alcohol (A262; ε = 13.4 mM−1 cm−1) was used asydrogen donor. The reaction mixture (1 ml) consisted of 0.1 mMoniferyl alcohol, 2.5 mM H2O2 in 50 mM PPB, pH 6.5 and 10–50 �lf sample, except where otherwise indicated. Initial rate of thebsorbance changes at 262 nm was measured, and corrected for themount of phenolic oxidation without H2O2. The oxidative activityf POD was determined in 50 mM PPB at pH 5.5 by monitoring theecrease of NADH absorbance at 340 nm in the assay mixture con-

aining 0.2 mM NADH, 0.2 mM p-coumaric acid, 0.25 mM MnCl2,nd 10–50 �l of sample in a volume of 1 ml. Spectrophotometriceasurements were performed with diode array spectrophotome-

er (Hewlett Packard 8451A, Palo Alto, CA, USA) at 30 ◦C. One unit

t Science 223 (2014) 49–58 51

(U) of PODs was defined as the amount of the enzyme that oxidizes1 �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) at550 nm, according to the method of McCord and Fridovich [35].One unit (U) of SOD was defined as the concentration of enzymeneeded for 50% inhibition of Cyt c reduction in 3 ml of reactionvolume. SOD specific activities were determined using a Shimadzuspectrophotometer (UV-2501 PC Kyoto, Japan). Activities of differ-ent SOD forms were identified and measured using KCN, by themethod of Bridges and Salin [36].

Protein concentration in maize root extracts was determined byBradford 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 determinedimmediately after isolation, while in AF it was conducted after ini-tial extraction of filter strips in 100 �l of 50 mM PPB (see Section2.3).

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 stripscontaining the absorbed AF were placed directly on a gel, or AWFcontaining 10 �g protein was applied to each well.

After completion of electrophoresis, POD isoenzymes werestained with 10% 4-chloro-1-naphthol and 0.03% H2O2 in 50 mMPPB pH 6.5 for 10 min at 25 ◦C.

Following focusing, SOD isoforms were stained by incubation in2.45 mM NBT, 28 �M riboflavin, 250 mM EDTA and 2 mM TEMEDin 100 mM PPB (pH 7.8) for 30 min in darkness. Visualization ofSOD isoforms was achieved 20 min after the exposure to UV lightat room temperature. Identification of MnSOD isoforms on gel wasachieved by its incubation in 100 mM PPB (pH 7.8) containing 3 mMKCN (inhibitor of Cu/ZnSOD), 30 min before staining by foregoingprocedure.

2.7. HPLC analysis

Phenolic compounds, organic acids and sugars were extractedfrom single filter paper strips with 100 �l methanol acidifiedwith phosphoric acid and centrifuged for 10 min at 10 000 × g.Aliquots of AF supernatant or isolated AWF were injected into aWaters HPLC-ECD system (Waters binary pump system, thermostatand autosampler connected to the Waters 2465 electrochemicaldetector-ECD; Waters, Milford, MA, USA). The analysis of each typeof compound was done in triplicate.

Signals of phenolic compounds were detected in direct modeat a constant potential of +0.75 V; the signal filter time was 0.2 sand the sensitivity 10–100 nA for the full mV scale. Separation ofphenolic compounds was performed on a Symmetry C-18 RP col-umn (125 mm × 4 mm with 5 �m particle size; Waters) with anappropriate guard column. HPLC method was performed accordingto the procedure previously described [38]. The data acquisitionand quantitative determination of the confirmed sample peakswere carried out using Waters Empower Software. Results wereexpressed as �M of phenolic compound of an isolated apoplasticfluid.

Organic acids were separated using Aminex HPX-87H (Bio-RadLab., Hercules, CA, USA) column 250 mm × 4 mm, 5 mM H2SO4

being used as the mobile phase. Organic acids were isocraticallyeluted 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 withsignal form: E1 = +0.3 V during 280 ms; E2 = +1.4 V during 150 ms;

5 . / Plant Science 223 (2014) 49–58

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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 minrecovery in nutrient solution, buffer infiltration and centrifugation at 2000 × g, and

2 J.J. Dragisic Maksimovic et al

3 = −0.4 V during 280 ms and 40 ms integration time. The resultsre given in �M of organic acid of an isolated apoplastic fluid.

Concentration of sugars (glucose, sucrose and fructose) in theamples was calculated from the peak size, using pure substancesSigma Co. St. Louis, MO, USA) as standards. Sugar separationas performed using CarboPac PA1 (Dionex, Sunnyvale, CA) col-mn (250 mm × 4 mm) and appropriate CarboPac PA1 precolumn.he mobile phase was 0.2 M NaOH, the sugars being isocracticallyluted for 20 min, with a flow rate of 1 ml min−1 at constant temper-ture (30 ◦C). The sugar signals were detected in the pulsed modeith signal form: E1 = +0.05 V during 400 ms; E2 = +0.75 V during

00 ms; E3 = −0.15 V during 300 ms and 180 ms integration time.he mobile phase was prepared using NaOH solution (50%, w/w,ith low carbonate content, J.T. Baker, Deventer, The Netherlands)

nd deionised water which had been vacuum degassed previously.ll the analyses were performed in triplicate. The results are pre-ented as �M of sugar of an isolated apoplastic fluid.

.8. EPR detection of •OH formation

Detection of •OH radicals was performed by EPR spin-trappingethod using DEPMPO (5-(diethoxyphosphoryl)-5-methyl-1-

yrroline-N-oxide; ENZO Life Sciences AG, Lausen, Switzerland)urified according to the method of Jackson et al. [39]. This spin-rap is capable of forming different spin-adducts with •OH andO2

− radicals, thus differentiating between oxygen-centered freeadical species present in solution [40,41]. Either concentrated oriluted DEPMPO were added to the AWF samples (60 �l), to anal concentration of 240 mM or 12 mM, respectively. The mixtureas injected into a 10 cm long gas-permeable Teflon tube (wall

hickness 0.025 mm and i.d. 0.6 mm; Zeus Industries, Raritan, NJ,SA) and folded into 2.5 cm long segments to improve the signal-

o-noise ratio [42]. Filter strips moistened with diluted (12 mM)EPMPO were placed on the root surface, and after 30 min AFbsorption were inserted into a quartz cuvette and placed insidehe resonator cavity. The EPR spectra were recorded at room tem-erature by a Varian E104-A spectrometer operating at X-band9.3 GHz) with the following settings: modulation amplitude, 2G;

odulation frequency, 100 kHz; microwave power, 10 mW; scanange, 200 G; scan time, 4 min. Spectra were recorded and ana-yzed using the EW software (Scientific Software International, Inc.,incolnwood, IL, USA).

.9. Experimental design, data collection and statistical analysis

The variability of each analytical method was measured repeat-ng the analyses three times to confirm reproducibility. A qualityontrol sample (blank-containing all reagents except the test sam-le) was also analyzed together with the batch of samples andecorded on a control chart. The results of all control samples wereithin control limits thus confirming the reliability of analysis.

Data were subjected to analysis of variance using the statisticaloftware Statistica 6 (StatSoft, Inc., Tulsa, OK, USA) and means wereompared for significance by Mann–Whitney non-parametric testt P < 5%.

. Results

In order to determine whether different experimental proce-ures used for the collection of AWF affect the content of the liquidhus obtained a number of protocols were tested.

The fresh weight of maize root segments (Table 1) that were

nly centrifuged at different speeds was not statisticaly different,nd slightly lower than that of the vacuum infiltrated root segmentsboth, IC2000 and RIC2000). A much lower quantity of roots were usedor AF.

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 segmentspreviously vacuum infiltrated with buffer in surplus, followed byblotting 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 highestcentrifugation speed, and in the vacuum infiltrated samples.

The protein concentration in relation to the volume of iso-lated apoplastic fluid, presented as total protein amount (TPA),demonstrates the difference between the compared methods. Thetraditional, centrifugation-based provides 8.4–38 �g protein in theextract, 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 inthe crude root extract, data not shown), whereas the AF obtainedusing filter strips did not show any noticeable activity. Accordingto Dannel et al. [12], values below 1.6% are considered to be free ofcytoplasmic contaminations. However, results on isolated cell walls[43,44] and plasma membranes [45], demonstrated the presence ofMDH in these isolates. Also, Li et al. [46] reported the occurrence ofsoluble apoplastic MDH in leaves of barley and oats. All this raisesdoubts on the use of MDH as a cytoplasmic contamination marker.

In subsequent experiments we focused on the presence ofglucose-6-phosphate (G-6-P), an intracellular metabolic pathwayintermediate, as a more appropriate marker. The highest concen-tration was detected in AWF obtained only by centrifugation at2000 × g (approx. 12 �M), the concentration in other centrifugedand infiltrated samples being in the range from 2 to 4 �M (Fig. 1). Asopposed to AWF, in which all the samples demonstrated the pres-ence of significant quantities of G-6-P, the AF samples did not showthe presence of this compound, indicating that these isolates are notcontaminated with cytoplasmic constituents at all. Due to the lowamount of starting material, the sensitivity of the assays was veri-fied for other enzymes (SOD, POD), as well as metabolites (organicacids, sugars and phenolic compounds), which were shown to beof more than adequate and measureable quantities particularly inthe AF samples. Thus, one can state that the truancy of the activityof marker enzymes in the case of AF samples is due to their absencein the extracted fluid, and not due to the low detection limit of themethod.

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. Dragisic Maksimovic et al. / Plant Science 223 (2014) 49–58 53

Table 1Root fresh weight (FW), volume (V), total protein concentration (TPC) and total protein amount (TPA) of apoplastic washing fluid obtained from maize roots using differentmethods 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 8.38CF500 588.1 ± 53.3a 127.8 ± 19.3b 127.7 ± 1.9b 16.19CF1000 539.8 ± 18.7a 102.9 ± 3.6b 116.0 ± 40.9a,b,c 11.83CF2000 657.2 ± 77.9a 49.3 ± 8.8d 266.8 ± 32.5d 13.15IC2000 811.7 ± 61.2b 180.9 ± 12.6c 211.8 ± 2.5c 38.31RIC 888.0 ± 76.5b 119.1 ± 9.4b 195.2 ± 22.5c 23.24

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ata are means (n = 3) ± SE. Significant differences at P < 5% are indicated by differen

ignificantly higher specific activity of the peroxidase (3–15 timesreater than AWF samples). Isoelectric focusing of AF revealed thathey had a completely different isoenzyme pattern in the acidicegion, compared to AWF (Fig. 3a), no noticeable differences beingbserved between different AWF samples. These results argue inavor of cytoplasmic cationic peroxidase isoenzyme release into thepoplast during AFW extraction, even at the lowest centrifugationpeeds.

As opposed to the peroxidases, SOD specific activity wasffected much more by different procedures of AWF isolationFig. 2b). Similar to the peroxidases, this class of enzymes showedhe highest specific activity in the AF. The intensity of the acidicOD isoforms varied depending on whether infiltration waserformed, as well as the centrifugation force used (Fig. 3b). UsingCN and H2O2 as inhibitors, we detected MnSOD isoform at pI 8.2,

hich could be observed only in AWF isolated at speeds lower than

000 × g. This isoform, characteristic for the cytoplasm [47] couldot be detected in AF, in spite of the much greater specific enzymectivity of this isolate. Overall, the specific enzyme activity and

ig. 2. Peroxidase (a) and total superoxide dismutase (b) activity in apoplastic washing flut 200, 500, 1000 and 2000 × g, CF200, CF500, CF1000 and CF2000 respectively; IC2000, buffeolution, buffer infiltration and centrifugation at 2000 × g, and apoplastic fluid (AF) obtare means of three replications (n = 3) ± SE. Different letters denote a significant differenc

e 8.2 ± 0.7e 9.02 × 10−3

ers.

isoenzyme patterns again demontrate the absence of cytoplasmiccontamination in the case of AF extraction, but also indicate thatthe isoenzyme pattern can be altered due to either cytoplasmiccontamination of AWF samples, or interaction of the cytoplasmicintermediates with the apoplastic enzymes.

A quantitative and qualitative analysis of non-enzymatic com-ponents such as organic acids, sugars and phenolics in rootapoplastic fluid isolates was performed using HPLC chromatog-raphy. In Fig. 4 we show representative chromatograms obtainedfrom AWF and AF extracts. It is obvious from these chromatogramsthat AWF contains a higher number of constituents in all three ana-lyzed classes of compounds and that their content is more complexthan the apoplastic fluid obtained using filter strips.

In Table 2 we presented the concentrations of the three classesof analyzed compounds that have been identified and quantified

(organic acids, sugars and phenolics, respectively). In AWF obtainedfrom excised root by centrifugation at 2000 × g oxalic, malic andsuccinic acids were detected. Oxalic acid could not be detectedin the case of the AF collected with filter strips, but the samples

id obtained from maize roots using different methods of extraction: centrifugationr infiltration and centrifugation at 2000 × g; RIC2000, 90 min recovery in nutrient

ined using filter strips. POD, peroxidase; SOD, superoxide dismutase; U, unit. Barse at P < 5%.

54 J.J. Dragisic Maksimovic 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 obtainedu , CF20

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sing different methods of extraction: centrifugation at 200, 500, 1000 and 2000 × gt 2000 × g; RIC2000, 90 min recovery in nutrient solution, buffer infiltration and cenn a pH gradient of 3–10. 10 �g protein was applied to each well. pI 8.2 (marked bo

ontained besides malic and succinic acids, also citric acid. Theoncentration of malic acid was higher in the case of AF. Glucosend fructose concentrations were higher in the AWF, while sucroseoncentration was slightly higher in the AF. The concentration ofhenolic compounds were of one to two orders of magnitude higher

n AF obtained using filter strips, compared to the AWF of excisedoots centrifuged at 2000 × g (Table 2).

Spin trapping electron paramagnetic resonance (EPR) spec-roscopy showed that AWF, regardless of the centrifugation speednd spin-trap concentration, had no capacity to generate hydroxyladicals (•OH, as judged by their capacity to form DEPMPO/OHdduct) (Fig. 5a–c). Also, no discernible generation of •O2

− radi-als (i.e. formation of DEPMPO/OOH adducts) could be observed.

urprisingly, the generation of •OH in AF could be registered0 min following extraction, even the diluted spin-trap showingEPMPO/OH spectra (Fig. 5f). Higher centrifugation speeds led to

able 2oncentration of representative organic acids, sugars and phenolic compounds (inM) in maize root apoplastic fluid detected by HPLC. Apoplastic washing fluid (AWF)as obtained from excised roots by centrifugation at 2000 × g. Apoplastic fluid (AF)as collected with filter strips from intact roots.

Compound AWF (�M) AF (�M)

Organic acids

Oxalic acid 5.75 ± 0.73 ndCitric acid nd 11.96 ± 4.81Malic acid 1.13 ± 0.34 26.67 ± 8.96Succinic acid 287.51 ± 8.55 276.07 ± 6.17

SugarsGlucose 365.65 ± 24.60 144.67± 21.95Fructose 263.31 ± 66.05 40.63 ± 1.17Sucrose 2.76 ± 0.99 3.93 ± 0.83

Phenolics

Chlorogenic acid 0.008 ± 0.003 0.45 ± 0.03Caffeic acid 0.006 ± 0.002 0.52 ± 0.03Coniferyl alcohol 1.471 ± 0.447 140.71 ± 19.85p-Coumaric acid 0.013 ± 0.003 0.25 ± 0.03Isoferulic acid 0.299 ± 0.091 4.85 ± 1.19

ata are means (n = 3) ± SE.

0, CF500, CF1000 and CF2000 respectively; IC2000, buffer infiltration and centrifugationation at 2000 × g, and apoplastic fluid (AF) obtained using filter strips. IEF was doneresents MnSOD identified using KCN as inhibitor.

a loss of signal (Fig. 5b and c), indicating that the assumed pres-ence of intracellular constituents that have been released into theapoplast during the extraction procedure, quenched the signal andfree radical generation. Also, as previously shown, much greaterconcentration of the oxidative metabolism enzymes, phenolics andother metabolic intermediates in the apoplastic fluid, enable sus-tained generation •OH for prolonged periods of time.

4. Discussion

The technique commonly used for the analysis of apoplasticfluid is the centrifugation and/or vacuum infiltration of the excisedplant tissue. Different authors developed different variants of theextraction procedure, involving only centrifugation [22,48,49], vac-uum infiltration [50], or a combination of both [51,52], most of theexperiments being performed on excised leaf tissue.

The centrifugation/infiltration method has been the subject ofcritical evaluation by a number of authors [23,53]. The issues raisedare the possibility of intracellular content contamination of theapoplast (both outflow at the injured site as well as cellular mem-brane rupture and leakage due to centrifugal force), modificationof its constituents, and the possibility of wrong estimate of thedilution, making exact measurements of the concentration of theextracellular compounds questionable.

The difference in total protein amount (TPA) is 3 orders ofmagnitude higher in AWF compared to AF (Table 1). Its suddenincrease in AWF, from 8 to 38 �g, is a direct consequence ofstructural damage made by both the high centrifugation speed andbuffer infiltration. Also, TPC dramatically increased at the maximalcentrifugation speed, regardless to the method used (CF2000, IC2000,RIC2000). Thus, obviously harmful application of centrifugal force

and vacuum infiltration dramatically increases the protein contentin AWF samples, masking the real in situ situation by the release anumber of different intracellular proteins (as it can be observed inthe case of POD and SOD in Fig. 3 by the appearance of a numerous

J.J. Dragisic Maksimovic et al. / Plan

Fig. 4. Representative HPLC chromatograms of apoplastic washing (AWF at2000 × g) and apoplastic (AF) fluids showing differences amongst three major classesof small weight metabolites. Inset with S marked compounds is PAD chromatogramsof 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 markedcompounds 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 coniferylalcohol, 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 differentby centrifugation at 200 × g with 240 mM DEPMPO; (b) AWF collected by centrifugationwith 240 mM DEPMPO; (d) filter strip (control spectra); (e) AF collected using filter strip

t Science 223 (2014) 49–58 55

additional isoforms). Much lower protein concentration in AF didnot prevent differentiation of the analyzed POD and SOD isoforms,for which we can, with much greater assurance, claim to bedevoid of intracellular contamination. Also, measurements of PODand SOD activities show that AF samples exhibited much higherspecific activity. Thus, lower protein content in AF did not affectthe 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 bladeand mechanical stress response has been addressed by a number ofauthors [11,12,54]. Such contamination can be reduced by rinsingin buffer or nutrient solution prior to measurements [55]. This wasalso demonstrated in the present study (Table 1, Fig. 1). Although adecrease of protein and G-6-P marker concentrations was evidentin the fluid collected from the root segments incubated prior tocentrifugation, this did not completely remove the cytosolic con-taminants.

A comparison of the amounts of hexose, pentose, proteins, andmalate dehydrogenase in extracellular solution from pea epicotylsections obtained by applying different centrifugal forces showedthat most of these substances were released from intracellularsources at 500 × g [54], and contamination of AWF is enhancedby an increase of centrifugal force [11]. Dannel et al. [12] demon-strated damage to cell walls and membranes of sunflower leaves atcentrifugal forces between 2000 and 2500 × g. In our experiments,even a centrifugal force of 200 × g resulted in the appearance of themarker substrate (G-6-P) in AWF, significantly increasing at higherspeeds (Fig. 1).

Dilution of the apoplastic fluid induced by the infiltration pro-cedure results in an inevitable change in the chemical equilibriumexisting in the apoplast, which can lead to inaccuracies in measure-ments of apoplastic ion and metabolite concentrations. Infiltrationof water or buffer into the apoplast modifies the ionic content, sothat 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 concentratedthan they really are (or measured). In fact, with this technique in

methods: infiltration/centrifugation (AWF) or filter strips (AF). (a) AWF collected at 2000 × g with 12 mM DEPMPO; (c) AWF collected by centrifugation at 2000 × gwithout DEPMPO; (f) AF collected using filter strip with 12 mM DEPMPO.

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ost cases ion- or element concentration, and not ion activity, isetermined. Lohaus et al. [11] tested the infiltration-centrifugationethod for parameters affecting the ions and metabolites for dif-

erent plant species, showing that the procedure must be adjustedor each species. Also different pH of the infiltration medium canlter the concentrations of carbohydrates, primarily sucrose [11].

In order to bypass these issues, we employed the filter stripechnique as a method that uses intact plants avoiding mechani-al stress (tissue cutting, vacuum infiltration and centrifugation),nd the dilution effects. Originally, the filter strip technique wassed only for localized root exudate sampling for the analysis ofrganic acids [19,30,31]. In our previous article we have reportedhat such a technique is also suitable for monitoring changes of the

etabolic components in apoplastic fluid of maize root, and that its sensitive enough to discern metabolic changes which occur alonghe maize root [22]. In the current study we extended its applica-ion to the analysis of antioxidative systems present in the apoplastenzymes, phenolics, ROS, sugars and organic acids), and the resultsave been compared to those of AWF samples.

Our results demonstrate that the enzyme activity and the isoen-yme composition of apoplastic fluid depends on the techniquessed for its isolation. Centrifugation and infiltration affected bothhe POD and SOD specific activities of AWF. In the case of PODhe specific activity decreased with increasing centrifugal forcell the way to 2000 × g, while SOD specific activity graduallyecreased with increasing centrifugal force up to 1000 × g, andreatly increased at 2000 × g (Fig. 2). Vacuum infiltration furtherncreased specific enzyme activity, and “recovered” samples had alightly aleviating effect. Thus, the difference observed in specificnzyme activity can be explained by dilution due to leakage fromntracellular compartment during centrifugation. The increase inOD specific activity at 2000 × g can be explained by the release ofntracellular SOD into the apoplast. A much higher specific activityf both enzymes detected in AF clearly demonstrates the advantagef using the filter strips for apoplast fluid extraction and enzymenalysis, bypassing all the problems associated with drastic isola-ion procedures used to obtain AWF (Fig. 2).

Ionically bound cationic peroxidases have been shown to beresent in isolated cell walls [56,57] which could be readily secretedo the apoplastic solution and serve as an emergency enzymenvolved in plant wound response [58]. In our results, the appear-nce of root cationic isoforms only in AWF, but not in AF, coulde due to possible extraction of some weakly ionically bound per-xidases by infiltration buffer, since cationic group of peroxidasesas shown to be present in cell wall ionically bound fraction [54].

he other possibility is that some cationic isoforms are expresseds the response to stress induced by wounding during AWF extrac-ion, which was avoided by the filter strip method of extracting AF.ifferent pattern of the root cationic isoforms observed in AWF andF in our experiments argue strongly in favor of partial intracellularontamination in the case of AWF, irrespective of the centrifugationnd infiltration procedure. Previously published data suggestedhat soluble strongly cationic apoplastic peroxidases of Norwaypruce, with isoelectric point pI 9 or higher, participate in lignin for-ation [59]. Also, Martinez et al. [60] demonstrated in cotton the

resence of a number of cationic extracellular peroxidase isoen-ymes at pI 9–9.3. As mentioned by these authors, the transientlkalinization that takes place in the apoplasm of cotyledons couldavor the activity of a wall-bound cationic peroxidase, leading to

2O2 synthesis required for lignification. In our filter strips onlyne cationic isoform of peroxidase at pI 9.3 was detected (Fig. 3a)n maize root apoplast.

Isoelectric focusing of the isolates differentiated Cu/ZnSODnionic isoforms with pI from 3.6 to 5.1 and cationic isoforms withI from 8.6 to 9.3. Using specific inhibitors (KCN and H2O2), weave shown that MnSOD isoform with pI 8.2, characteristic for the

t Science 223 (2014) 49–58

mitochondrial matrix of eukaryotic cells [47], was unequivocallypresent in CF200 and absent in AF (Fig. 3b). However, in CF500, CF1000and CF2000 under supposedly more damaging conditions, there ispoorly or no visible 8.2 isoform, probably due to interference withinter- or intracellular content. Considering that TPC was similar inCF200, CF500 and CF1000 but significantly higher in CF2000 (Table 1),it is clear that high centrifugal force, causing damage of cellularstructures, released a great quantity of different proteins. Such analtered protein profile decreased the portion of specific MnSODin electrophoretic sample rendering its band less visible. Since asame amount of total protein was applied to the gel for all sam-ples (10 �g, see Section 2.6), the absence of MnSOD isoform withpI 8.2 more likely is a consequence of lower concentration of thespecific enzyme applied to the gel. This is yet another argumentfor intracellular contamination of the AWF, irrespective of the iso-lation procedure. Further research could determine whether theincrease 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 limiteddue to their constant exchange with soil solution, cellular com-partmentation and unreliable methods for determining their trueconcentration in the apoplastic space. The release of organic acidsinto the rhizosphere has been documented [61,62], and it has beenshown 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 therange of 10–20 mM (1–4% of total dry weight), since the concentra-tion of metabolic intermediates such as malate and citrate in thecytosol rarely exceeds 5 mM, whereas concentrations in the vacuolecan be 1–10 fold higher [63]. Data for organic acids concentrationin root apoplast are scarce.

In the present study, dominant organic acids identified in AWFwere oxalate, malate, and succinate in micromolar range (Table 2,Fig. 4). In AF we did not detect oxalic acid, but citric acid could beobserved. Also, the concentration of malic acid was 25-fold greaterin AF, when compared to AWF. Oxalic acid can combine with var-ious elements and compounds to form acid and neutral salts, amonoamide (oxamic acid), and a diamide (oxamide) [64]. Oxalicacid 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 beenshown to be a rapid and reversible process in Lemna minor, indi-cating that oxalate crystal formation is a highly controlled processand providing a mechanism for regulating intracellular calcium, aswell as oxalate levels in plant organs [65]. Considering the vacuo-lar localization of oxalate under normal physiological conditions, itspresence in AWF in our experiments is further proof of membranedamage and leakage from the vacuoles to the apoplast. Again, oxalicacid absence in AF samples argues in favor of the non-destructivefilter strip technique approach. Lower concentrations of malateobserved in AWF can be explained by leakage of intracellular reduc-tants and enzymes, consuming the malic acid that was initiallypresent in the apoplast.

The presence of 11 identified sugar compounds in AWF, of whichonly 4 (galactose, glucose, fructose and sucrose) were detected inAF, once again argues in favor of apparent contamination of AWForiginating from intracellular content leakage due to the forcefulisolation procedure (Fig. 4). Higher concentration of glucose andfructose observed in the present study in AWF compared to AF isin line with this argument (Table 2).

In our previous article using the filter strips [22] we have shownthat phenolics varied both spatially and temporally in the apoplast

along the root; higher concentrations being observed in the maturezone associated with intense lignification. Quantitative and quali-tative analysis of monomeric phenolic compounds in maize rootapoplastic fluid demonstrated the presence of coniferyl alcohol,

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hlorogenic, caffeic, p-coumaric, and isoferulic acid (Table 2, Fig. 4).oniferyl alcohol was shown to be the major component of bothWF and AF. The concentrations of all phenolic compounds in AWFere exceptionally low when compared to those in AF, where two

rders of magnitude higher concentrations could be observed. Thisgain is due to dilution with intracellular content because of thepplied extraction procedures used for obtaining AWF, and possi-le interaction with them. The presence of additional unidentifiedhenolics not present in AF further corroborates this conclusion.

Liszkay et al. [66] have shown, on relatively large samples of cellsr cut tissue incubated with the POBN spin trap (not able to differ-ntiate different oxygen centered radicals) up to 2 h on a shaker,hat free radicals are present in the incubation medium. Such anxperimental approach gives an indication for the generation ofree radicals in the apoplast, but it does not exclude the possibilityf the participation of numerous free radical reactions originatingrom the wounded tissue and released intracellular intermediates,or the interaction of different intermediates with specific cell wallound redox systems. In our measurements, we demonstrate sus-ained generation of •OH in AF samples (Fig. 5). EPR spectroscopyerformed with AWF regardless of the centrifugation speed andpin-trap concentration gave no signal (Fig. 5a–c), as well as controlpectra (Fig. 5d) and AF collected using filter strip without spin-trapFig. 5e). It seems that DEPMPO/OH complex formed on filter stripsas more stable, which is prerequisite for this type of analysis.

hus, the filter strip technique in combination with EPR spectrom-ter provides us with a tool to study in vivo production of •OH andther free radical species in the apoplastic fluid, without interactionith cellular, or fibrillar cell wall components.

All the presented observations argue in favor of filter stripethod, as being more reliable than infiltration/centrifugationethod, for studying processes occurring in apoplastic compart-ent of the root. Thus, filter strips in combination with the high

ensitivity HPLC-ECD, EPR and electrophoretic techniques provides with a tool to study components of antioxidative system inhe apoplast of developing plant organs and their spatial-temporalhanges. It enables distinct concentration measurements of rootetabolites in the apoplast, and does not require pre-concentrating

nd purifying samples. Such an experimental technique provides aowerful non-invasive analytical tool for studying metabolical pro-esses occurring in the apoplast and local changes in small regionsf the intact root tissue, especially in hydroponically grown plants.

To conclude, our results unequivocally demonstrate the advan-age of using filter strips for collection of the apoplastic fluidrom the intact plant roots, giving small amounts of uncontam-nated liquid containing much higher specific enzyme activitiesnd metabolite concentrations, such as organic acids and phenolics.ompared with the centrifugation/vacuum infiltration technique,ur data argue against the use of this method, using even the small-st centrifugation force, because such procedures coupled withxcision and mechanical wounding unavoidably release a wholeange of different intracellular compounds and proteins into thepoplast during the extraction procedure.

cknowledgments

The authors are very grateful to Dr Günter Neumann fromohenheim University (Stuttgart, Germany) for method demon-

tration and training. This work was supported by the Serbianinistry of Education, Science and Technological Development

grants 173040 and 173028).

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