Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth

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Plant Physiology and Biochemistry 44 (2006) 526–534

Research article

Abbrevidiaminobenindole-1,3-aperoxidase;sequence ocarboxylaseT, transgeni

* CorrespE-mail a

0981-9428/doi:10.1016

Transgenic ipt tobacco overproducing cytokinins

Renáta Schnablováa,b, Helena Synkováb,*, Anna Vičánkovác, Lenka Burketováb,Josef Ederc, Milena Cvikrovác

aDepartment of Plant Anatomy and Physiology, Faculty of Sciences, Charles University, Viničná 5, 128 44 Praha 2, Czech Republicb Institute of Experimental Botany, Academy of Sciences of the CR, Na Karlovce 1a, 160 00 Praha 6, Czech Republic

c Institute of Experimental Botany, Academy of Sciences of the CR, Rozvojová 135, 165 02 Praha 6, Lysolaje, Czech Republic

Received 10 January 2006; accepted 12 September 2006Available online 29 September 2006

Abstract

We present evidence that overproduction of endogenous cytokinins (CK) caused stress response in non-rooting Pssu-ipt transgenic tobacco(Nicotiana tabacum L.) grown in vitro. It was demonstrated by overaccumulation of phenolic compounds, synthesis of pathogenesis relatedproteins (PR proteins), and increase in peroxidase (POD) activities. Immunolocalization of zeatin and also PR-1b protein on leaf cryo-sectionsproved their accumulation in all mesophyll cells of transgenic tobacco contrary to control non-transgenic plants. Intensive blue autofluorescenceof phenolic compounds induced by UV in cross-sections of leaf midrib showed enhanced contents of phenolics in transgenic tobacco comparedwith controls, nevertheless, no significant difference between both plant types was found in leaf total lignin content. Transgenic plantlets exhib-ited higher peroxidase activities of both soluble and ionically bound fractions compared with controls. HPLC analysis of phenolic acids con-firmed the increase in all phenolic acids in transgenic tobacco except for salicylic acid (SA). The effect of high phenolic content on rooting oftransgenic tobacco is discussed.© 2006 Elsevier Masson SAS. All rights reserved.

Keywords: Pssu-ipt tobacco; Phenolic acids; Cytokinins; In vitro cultivation; Peroxidases

1. Introduction

Various factors influence in vitro propagation. External fac-tors such as irradiance, temperature, ventilation, and compo-nents of a cultivation medium such as sucrose and/or growthregulators result in the formation of abnormal morphology,anatomy, and physiology of in vitro grown plantlets [1]. How-ever, internal factors such as cell type, size, age, and the state

ations: C, control type rooted tobacco; CK, cytokinins; DAB, 3,3′-zidine; FM, fresh leaf mass; GPOD, guaiacol peroxidase; IAA,cetic acid; ipt, the gene for isopentenyl transferase; POD,PR proteins, pathogenesis related proteins; Pssu, promoter

f the gene coding for small subunit of ribulose-1,5-bisphosphate/oxygenase; SA, salicylic acid; SPOD, syringaldazine peroxidase;c non-rooted plants.onding author. Tel.: +420 2 333 20338; fax: +420 2 243 10113.ddress: [email protected] (H. Synková).

$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved./j.plaphy.2006.09.004

of differentiation of the explants play an important role in theirorganogenic capacity. The basic regulatory mechanism under-lying plant organ formation involves a balance between auxinand cytokinin (CK) contents. A relatively low content of auxinand high content of CKs result in a shoot differentiation, whilea reverse situation results in a root initiation. This could bedemonstrated in a few Arabidopsis mutants with altered CKmetabolism [2] or in transgenic plants with the gene for iso-pentenyltransferase (ipt) introduced under various promoters[3,4]. Those plants are characterized by high endogenous CKcontents resulting in the high shoot forming capacity and thelow rooting capacity that cannot be improved by exogenousauxin treatment [5]. Thus, transgenic Pssu-ipt tobacco with ca.10-fold enhanced content of endogenous CKs is unable to formroots during in vitro cultivation [3]. Pssu-ipt tobacco plantsexhibited increased activities of antioxidant enzymes, peroxi-dases, several enzymes of intermediary metabolism, and a pre-

R. Schnablová et al. / Plant Physiology and Biochemistry 44 (2006) 526–534 527

sence of pathogenesis related (PR) proteins such as PR-1b pro-tein and proteins with chitinase activity in extracellular fluid[6]. These findings clearly indicate that elevated CK contentrather than conditions of cultivation caused the stress and sti-mulated defense mechanisms in transgenic tobacco. The inter-action between CKs and pathogenesis related proteins (PR pro-tein) production was shown by Sano et al. [7]. CKs interferedwith the signal transduction mechanisms participating in PRproteins synthesis by controlling endogenous level of salicylicacid (SA) and jasmonic acid. SA belongs to a diverse group ofsecondary metabolites, generally called phenolic compounds,(e.g. flavonoids, tannins, hydrocinnamate esters, and lignin)that are synthetized normally during plant growth and develop-ment.

Phenolic compounds have been shown to serve as signal-ing molecules (e.g. SA, [8]), to modulate the action of auxins[9], and to play an important role in the resistance of plants tobiotic and abiotic stresses [10]. There are still some unan-swered questions about the precise role of phenolic sub-stances in the processes of differentiation and morphogenesis.Through the modulation of endogenous indole-1,3-acetic acid(IAA) content phenolics might influence the hormonal bal-ance required, e.g. for root induction. Correlations have beenobserved between phenolic content and root formation in invitro culture [11] or in cuttings of many species [12]. Antiox-idative properties of polyphenols arise from their high reactiv-ity as hydrogen or electron donors and from the ability of thepolyphenol-derived radical to stabilize and delocalize the un-paired electron and from their ability to chelate transition me-tal ions, i.e. termination of Fenton reaction [13]. Takahamaand Oniki [14] have proposed that the peroxidase/phenolics/ascorbic acid system can function as a hydrogen peroxidescavenging system in vacuoles and apoplast, because pheno-lics, ascorbic acid and peroxidase are normal components ofthose compartments.

Other phenolic biopolymers, lignins, are located in the pri-mary and secondary walls of specific plant cells as well as inthe middle lamella [15]. They are synthetized for mechanicalsupport and water transport of terrestrial vascular plants and inresponse to pathogen attack. The monomers of lignin derivedfrom three hydroxycinnamyl alcohols or monolignols: p-coumaryl, coniferyl, and sinapyl are synthetized in the cyto-plasm (Golgi or endoplasmic reticulum) and released into thecell wall from vesicles. Enzymes located within the cell wallduring lignification, in either free or bound state, include var-ious types of peroxidase (POD) and oxidase (including lac-case). Oxidase activity may be associated with the earlieststages of lignification and POD with the later stages [15].

To our knowledge, there is no information available on con-tents of phenolics in transgenic plants overproducing endogen-ous CKs and/or on a relationship between both groups of com-pounds. In our paper we aimed to investigate a role of phenolicsin in vitro grown non-rooting Pssu-ipt transgenic tobacco over-producing cytokinins (CK). We localized CKs, PR proteins, andcell wall bound phenolics on transverse leaf or midrib sectionsin transgenic tobacco by immunohistological methods. Further-more, we carried out a complete HPLC analysis of phenolic

acids with the aim to elucidate their role in rooting of transgenictobacco.

2. Results

2.1. Detection and localization of zeatin

Immunolocalization with specific antibodies against zeatinwas performed on cryo-sections to test whether a specific siteof CK localization in Pssu-ipt plants exist. On cryo-sectionsfrom C plants, the level of zeatin was very low and under thedetection limit (Fig. 1A). In sections from T, zeatin was loca-lized in all mesophyll cells (Fig. 1B). Anti-CK label was alsodetected in chloroplasts of transgenic tobacco cells (marked byarrows).

2.2. Detection and localization of PR-1b protein

Immunocytochemical examination carried on cryo-sectionsof transverse leaf samples proved that in Pssu-ipt plants thesynthesis of PR proteins was induced (Fig. 1C, D). PR-1b pro-tein was synthesized in all cells of in vitro grown T (Fig. 1D)contrary to C plants, where no or trace amounts of PR-1b pro-tein around the vascular bundles was found (Fig. 1C).

2.3. Tissue localization of phenolic compounds

Autofluorescence of the cross-sections of the leaf midribrevealed differences in the localization of cell wall phenoliccompounds (Fig. 1E, F). In C, the blue autofluorescence(induced by UV light) was detected only in the xylem vesselwalls and it attributed particularly to lignin (Fig. 1E). In T plants(Fig. 1F), the blue autofluorescence was significantly strongerthan in C and it was also detected in cells surrounding thexylem vessels. It probably originated also from ferulic acidbound to the cell walls.

2.4. In situ localization of peroxidases

Leaf cryo-sections stained by 3,3′-diaminobenzidine(DAB) for peroxidase activity showed that peroxidases werepredominantly localized in cell walls, particularly of epider-mal cells in C plants, while in T plants the stain intensity wascomparable in all cell types (Fig. 1G, H). Moderately strongerstain intensity was observed around the veins in T comparedto C.

2.5. Activity of soluble and ionically bound peroxidases

The significantly higher peroxidase activities were found in Tcompared with C irrespective of substrate used for activity assay(Fig. 2). Generally, soluble peroxidase fraction exhibited signif-icantly higher activities than ionically bound peroxidases. Whencalculated per fresh leaf matter (FM), ionically bound peroxi-dase activities took 19% (guaiacol peroxidase, GPOD) and15% (syringaldazine peroxidase, SPOD) of total peroxidase

Fig. 1. Immunohistochemical localization of zeatin (A, B), PR-1b protein (C, D) in transverse leaf cryo-sections of control (A, C) and Pssu-ipt tobacco (B, D).Autofluorescence induced by UV in hand-cut fresh sections of control (E) and transgenic (F) tobacco. Histochemical staining with DAB in leaf cryo-sections forperoxidase activity in control (G) and transgenic (H) tobacco. Scale bars: A–D, G, H = 50 μm; E, F = 100 μm. Small arrows indicate chloroplasts in B.

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Fig. 2. Activities of soluble (F) and ionically bound (B) peroxidases measuredwith guaiacol (GPOD) or syringaldazine (SPOD) as substrates in control (C)and transgenic (T) tobacco. Activities were calculated per gram of fresh leafmatter (FM). The values are the mean ± S.E. Statistical significant differencesat P = 0.05 are marked by different letters.

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activity in C (Fig. 2A, B). In T, ionically bound peroxidaseactivity was 11% (GPOD) and 0.5% (SPOD) of total peroxidaseactivity (Fig. 2A, B). Thus, peroxidase activity measured withsyringaldazine (SPOD) was lower in ionically bound fractionrelatively to soluble peroxidases in T compared with C.

In T, more isozymes of peroxidases were present in solublefraction analyzed by non-denaturating polyacrylamide gel elec-trophoresis stained for enzyme activity compared with C(Fig. 3). The most significant difference was found in lessmobile isozymes in the upper part of the gels.

2.6. Contents of phenolic acids and lignin

In order to characterize changes in phenolic acid composi-tion, detailed analysis of free phenolic acids (F1), ester-boundmethanol-soluble phenolic acids (F2), ester-bound cell wall phe-nolic acids (F3), and glycoside-bound methanol-soluble pheno-

Fig. 3. Peroxidase isozyme patterns obtained after non-denaturating PAGE incontrol (C) and transgenic tobacco (T).

lic acids (F4) was done. The differences found in the HPLCspectrum of phenolic acids were only quantitative between Cand T (Fig. 4). The total content of phenolic acids increasedca. five times in T compared with C (Table 1). The most pro-nounced enhancement was observed particularly in the contentsof free phenolic acids (F1) and in the glycoside-bound phenolics(F4) in T plants compared with C (Table 1 and Fig. 5). Asregards the individual phenolic acids, the most abundant pheno-lics in both types of plants were caffeic and chlorogenic acids(Fig. 5). T plants contained significantly higher amounts of caf-feic (F4) and chlorogenic acids (F1) and increased contents of p-coumaric, ferulic and sinapic acid soluble esters and glycosides,precursors of lignin biosynthesis compared with C plants(Fig. 5). However, a significant decrease in content of SA wasfound in T compared to C.

The total content of lignin was assayed in leaf samples byderivatization with thioglycolic acid (Table 1). Lignin contentwas moderately higher in T plants, although the difference wasnot statistically significant.

3. Discussion

3.1. Phytohormones

There is substantial evidence that the process of rooting isinfluenced by exogenous and endogenous contents of growthhormones, by content of phenolics, and by activities of enzymesinvolved in their metabolism. In our previous experiments weproved that transgenic Pssu-ipt tobacco produced ca. 10 timesmore endogenous CKs than control type both under in vitroand ex vitro conditions [16,6]. Our immunohistological localiza-tion of the most abundant CK in Pssu-ipt tobacco, zeatin, carriedon leaf cryo-sections proved the presence of this CK type in allmesophyll cells and in chloroplasts contrary to control tobacco,where its content was under a detection limit (Fig. 1A, B). It isin agreement with our previous findings, when higher CK con-tents were found in isolated chloroplasts from Pssu-ipt tobacco[17]. The significant increase in CKs usually affects the balancewith other plant hormones, particularly auxins. The disturbancescaused by high endogenous CKs and/or auxins were observed invarious transgenic plants overproducing one of those hormones[17]. CK overproduction decreases the content of auxin appar-ently by decreasing its rate of synthesis and/or transport, ratherthan by increasing rates of turnover or conjugation [18].Although our present experiments did not involve auxin deter-mination, our previous results confirmed at least four timeshigher CK/auxin ratio in Pssu-ipt tobacco than in control plants[19].

3.2. Peroxidases

The soluble forms of POD are cytoplasmic, whereas boundforms are generally thought to be associated with cell walls [20].However, under stress conditions, the enhanced POD activity inthe intercellular spaces, stimulating cell wall stiffening, probablyreduces cell growth, which might represent a mechanical adapta-

Fig. 4. HPLC chromatogram of methanol-soluble glycoside-bound phenolic acids extracted from control (C) and transgenic (T) tobacco. Dotted line representsacetonitril and acetic acid gradient used for the elution of phenolic acids. Each profile represents an equivalent amount of extract, normalized on a volume of extractper mg of tissue basis. Only traces of SA were found in F4 extracted from T plants. CaA = caffeic acid; pCA = p-coumaric acid; ChA = chlorogenic acid;pHBA = p-hydroxybenzoic acid; FA = ferulic acid; SA = salicylic acid; SiA = sinapic acid; VA = vanillic acid.

Table 1Total content of phenolic acids and lignin in control and Pssu-ipt transgenictobacco grown in vitro. F1 = free phenolic acids, F2 = ester-bound phenolicacids, F3 = ester-bound cell-wall phenolic acids, F4 = glycoside-bound phe-nolic acids. FM = fresh leaf matter, DM = dry leaf matter. The values of F1–F4represent the means of three replicates. The S.E. values averaged 8% and didnot exceed 17% of the mean. The values of lignin content are the mean ± S.E.Statistically significant differences found by t-test at P = 0.05 are marked bydifferent letters

Fractions of phenolic acids(μg g–1 FM)

Control Transgenic

F1 1.195a 8.67b

F2 1.508a 6.212b

F3 0.295a 0.479b

F4 16.506a 95.465b

Total sum (μg g–1 FM) 22.207a 110.826b

Lignin content (mg g–1 DM) 21.386 ± 1.94a 26.028 ± 1.3a

Fig. 5. Contents of individual phenolic acids calculated per gram of FM incontrol (C) and transgenic (T) tobacco. F1 = free phenolic acids;F2 = methanol-soluble ester-bound phenolic acids; F3 = methanol insolubleester-bound cell wall phenolic acids; F4 = methanol-soluble glycoside-boundphenolic acids. Other details see Fig. 4 for abbreviations. The values are themeans of each fraction. The S.D. did not exceed 15% of the mean.

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tion [21]. This kind of action has been attributed mainly to PODwhose activity can be detected by using syringaldazine as a spe-cific substrate. There is histochemical and biochemical evidencethat only cell walls that are undergoing lignification are able tooxidize syringaldazine [22]. In Pssu-ipt tobacco, activity of solu-ble POD measured with syringaldazine (SPOD) increased sixtimes compared with controls, whereas it was only three timeshigher when measured with guaiacol as a substrate. This wouldsupport the hypotheses that cell wall stiffening is undergoing intransformants. Although we expected more significant differ-ence between T and C, we found only moderately enhancedlignin content in Pssu-ipt tobacco compared with control planttype. Nevertheless, the samples for our lignin assay included

particularly leaves and upper parts of the plantlets and not thelower base of the stem, where rooting takes place and where thedifference could be more pronounced.

3.3. Phenolic acids

We have found ca. fivefold higher content of phenolic acidsin transgenic tobacco compared with controls (Table 1). The

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considerable enhancement was observed in contents of allindentified phenolic acids except for SA. Caffeic and chloro-genic acids and their glycosides represented the most abundantphenolics that we have detected in both types of tobacco andthat increased considerably in T. According to [23] chlorogenicacid and its isomers are present in the apoplast of tobacco leavesand the levels increase sigmoidally as a function of leaf age,whereas levels of the caffeic acid esters of the symplast do notsignificantly change during aging. Compounds such as chloro-genic, caffeic, and ferulic acids have been shown to interact withIAA oxidase to reduce the rate of auxin oxidation [24,9]. Never-theless, Faivre-Rampant et al. [25] suggested that the high con-tent of chlorogenic acid exceeding a certain threshold concentra-tion could lead to opposite effect and cause the inhibition of rootdevelopment. Furthermore, monophenolic acids such as p-coumaric acid were also shown to stimulate IAA oxidase activ-ity [26]. Higher levels of free caffeic, chlorogenic, and p-coumaric acids found in T plants might influence through low-ering the endogenous IAA content the appropriate hormonal bal-ance required for the root induction.

Contradictory, lower content of SA was found in in vitrogrown Pssu-ipt tobacco when compared with control plants.SA is discussed as an important signaling molecule associatedwith the establishment of SA-mediated defense and a sys-temic acquired resistance, and the activation of genes encod-ing PR proteins [27]. However, there is a very little informa-tion available on interactions among SA, CKs, and PRproteins except for those from Sano et al. [7]. We have pre-viously found de novo synthesis of several PR proteins inextracellular fluid in Pssu-ipt tobacco [6]. In this paper, weproved by immunolocalization particularly the accumulationof PR-1b proteins in all mesophyll cells of Pssu-ipt tobaccocontrary to controls. Although the content of SA was lower inin vitro grown transgenic tobacco than in controls, the synth-esis of PR proteins was higher (see also [6]). Therefore wemay hypothesize that CKs directly might be involved in theactivation of PR-1 protein synthesis.

3.4. Rooting process

The involvement of auxin in nodule organogenesis is likelyin the stimulation of cell divisions and regulation of root dif-ferentiation [28]. The rooting process might be subdividedinto several interdependent phases, where also other factorssuch as peroxidase activities and content of phenolics affectthe process [29,30]. There is always a transient increase in theendogenous auxin content during the inductive phase (corre-sponding to a minimum level of peroxidase activity), fol-lowed by a decrease in auxin levels to a minimum at the in-itiation phase [31]. The period of higher POD activitycorresponded to the early events of the initiation phase. Phe-nolic content changes inversely to the POD activity. This hasbeen reported by several authors who suggested that pheno-lics may act by modulating enzyme activity and preventingPOD oxidation of auxin during root induction [12,30]. Theprocess of rooting in Pssu-ipt tobacco is strongly influencedby the permanent disproportion of CKs and auxins as Pssu

promoter is light activated and therefore CK overproductionis constitutive and permanent [31,16]. This probably sup-presses or overrides all transient changes in auxin concentra-tions needed for the normal root growth initiation.

Furthermore, ca. six times higher POD activities were foundin Pssu-ipt tobacco in our experiment and previously also bothunder in vitro or ex vitro cultivation of the primary transformant(see also [32,6]). In spite of high POD activities, we also foundca. five times higher content of phenolic acids in Pssu-ipttobacco (Table 1). The cause for this enhancement is not clear,but it seems that high phenolics and POD activity is associatedwith a certain threshold level of CKs. While it stays very high inprimary transformants, F1 generation of Pssu-ipt plants containslower amount of CKs in early stages of plant development. Theactivity of POD and the content of phenolics are lower andplants are able to form small root system [32]. As PODs playthe important role in auxin catabolism [33], their activity con-siderably affects also auxin contents, which was higher in trans-genic rooted plants [19].

3.5. Conclusions

Transgenic Pssu-ipt plants showed various signs of stressboth in altered metabolism and on the ultrastructural level [6,17]. Now we have found that in vitro grown non-rooting Pssu-ipt tobacco is characterized not only by the high CK content, thehigh peroxidase activity, the presence of PR proteins, moder-ately higher lignin content, but also five times higher contentof phenolic acids. The disbalance among phytohormones,which was shifted considerably in favor of CKs in T plants,caused probably the permanent decline in auxin content withoutpossible transient changes needed for the proper stimulation ofrooting process. We suppose that the simultaneous increase inperoxidase activity and in the content of phenolics might repre-sent the stress response to the overproduction of CKs in trans-genic Pssu-ipt tobacco.

4. Materials and methods

4.1. Plant material

Control tobacco (Nicotiana tabacum L. cv. Petit HavanaSR1) was referred as C. Transgenic tobacco (T) containing asupplementary ipt-gene under a control of the promoter for thesmall subunit of RuBPCO (Pssu-ipt) was generated by means ofthe Agrobacterium tumefaciens transformation system andgrown in vitro as shoots unable to form roots as described byBeinsberger et al. [3]. During in vitro precultivation all plantswere grown in agar with Murashige and Skoog basal salt mix-ture (Sigma-Aldrich, Prague, Czech Republic) in ventilatedMagenta GA-7 vessels as described in Semorádová et al. [34]and Synková et al. [6]. Leaf samples were taken from the plantsafter 3–4 weeks of in vitro precultivation.

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4.2. Fluorescence microscopy

Blue autofluorescence (induced by UV light) was used for alocalization of cell wall phenolic compounds. Fluorescence ofunfixed, hand-cut leaf sections mounted in water was analyzedby epifluorescence light microscope (Nikon Eclipse E600,Japan) with the filters UV-2A (EX 330–380, DM 400, BA420). Photographs were taken by CCD camera using identicalexposure times.

4.3. Immunohistology for light microscopy

The random leaf blade samples were fixed in 3% paraformal-dehyde and 0.5% glutaraldehyde in PBS (135 mM NaCl,2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4, pH 7.2) for2.5 hours at 4 °C, washed in PBS and dehydrated in gradedsucrose series (from 0.1 to 1.76 M in PBS at 4 °C and frozenat – 75 °C). Dehydrated leaf pieces were cut to 8 μm thick tissuesections on a Cryotome Cryostat (Shadon, Pittsburg, USA).Collected sections attached to microscopic slides were rehy-drated in solutions of decreasing sucrose concentration (from1.76 to 0.11 M in PBS) followed by washing in PBS. The sec-tions were transferred to TBS (Tris 50 mM, NaCl 150 mM, pH7.6) containing 1% (v/v) Triton X-100 for 30 min. After block-ing in blocking solution 3 × 20 min (blocking solution: TBScontaining 20 mM glycine, 0.2% gelatine (v/v), 0.1% Tween20 (v/v), 10% goat preimmune serum (v/v)), the sections wereincubated in the primary rabbit polyclonal antibodies (anti-zeatin—antibodies purified by protein A, Professor Strnad, Olo-mouc; anti PR-1b—Dr. J. Antoniw, IACR). Sections werewashed in TBS and incubated with the secondary goat anti-rabbit antibody coupled to alkaline phosphatase. Followingwashing with TBS and buffer (2 mM MgCl2 in Tris–HCl, pH9.5), CKs and PR-1b protein were visualized with nitroblue tet-razolium (NBT)/5-brom-4-chlor-3-indolylphophate (BCIP) sub-strate (37.0 mM NBT, 35.0 mM BCIP). The reaction wasstopped by incubation of sections in EDTA (2.0 mM in TBS)followed by fixation in 25% glutaraldehyde in TBS. Immuno-histological controls were run parallel and treated with blockingsolution instead of primary antibodies. As a control for a speci-fic CK labeling, the sections were incubated in the saturatingmixture of the antibody with free t-zeatinriboside. The stainedcryo-sections were viewed in the light microscope NikonEclipse E600 equipped with CCD camera.

4.4. In situ localization of peroxidases

Histological staining for peroxidase activity was carried outusing 3,3′-diaminobenzidine (DAB) on cryo-sections made fromleaf tissue similarly as for immunohistological examination.Staining was done by incubation of the sections with DAB(50 mg per 100 ml) in the presence of H2O2 (5 mM) for15 min in darkness. After thorough washing by deionizedwater and dehydration through a series of solutions with increas-ing ethanol concentration permanent preparations were made.

The samples were examined by light microscopy (Nikon EclipseE600) equipped with a CCD camera.

4.5. Peroxidase extraction and activity assay

Samples of tobacco leaves (0.5 g) were frozen in liquid nitro-gen, homogenized in 2.5 ml of phosphate buffer (0.1 M, pH 7.0)and centrifuged at 4 °C for 10 min at 20,000 × g. In the super-natant, activity of soluble peroxidase (POD; E.C. 1.11.1.7) wasdetermined. POD ionically bound to cell walls was extractedwith 1 M NaCl from a purified pellet, which was washed oncewith phosphate buffer and several times with distilled water untilno peroxidase activity was detected.

POD activities were measured with guaiacol (GPOD) or syr-ingaldazine (SPOD) as substrates. Oxidation of guaiacol wasdetermined spectrofotometrically by an increase in absorbanceat 436 nm [35]. SPOD was determined as an increase in absor-bance at 535 nm [36]. All activities were calculated per g offresh leaf matter (FM), where the rates were given in 1 μmolof respective product formed per min [U g–1 (FM)].

Soluble protein content was determined according to [37].Soluble POD isozyme patterns were obtained after separation

by 10% non-denaturating acryl amide electrophoresis. Aliquotsof supernatants corresponding to 25 μg of protein per lane wereused. POD isozymes were detected in situ by staining gels in1 M acetate buffer, pH 4.6 with 0.04% benzidine and 10 mMH2O2 for 90 min at 30 °C.

4.6. Extraction of cell walls and determination of lignin

Samples of tobacco leaves were cut into small pieces andground to a fine powder (with liquid N2). To obtain cell walls,the powder was suspended in 1M NaCl with 0.5% Triton X-100and stirred for 30 min. Then it was washed twice with distilledwater, twice with 100% methanol, twice with 100% acetone(each step 30 min). Total lignin content was assayed by deriva-tization with thioglycolic acid (modified method of [38,39]).Aliquots of 10 mg of the cell wall preparations were placed inEppendorf tube and treated with 1.5 ml of 2 N HCl and 0.3 mlof thioglycolic acid for 4 h at 95 °C. Samples were cooled andcentrifuged for 10 min at 15,000 × g. The supernatant wasremoved and pellet was washed three times with distilledwater. Thereafter, the pellet was suspended in 1 ml of 0.5 NNaOH for 18 h on a shaker at room temperature. The suspensionwas centrifuged for 10 min at 15,000 × g. The supernatantobtained after centrifugation and the second supernatantobtained after re-extraction the pellet with 0.4 ml NaOH werecombined and acidified with 0.3 ml concentrated HCl and lig-nothioglycolic acid was allowed to precipitate at 4 °C. The mix-ture was centrifuged as above, the supernatant removed and thepellet solubilized in 1 ml of 0.5 N NaOH and diluted beforemeasuring absorbance at 280 nm. The amount of lignin wascalculated according to conversion made by [40]: 100 μg of lig-nin in 1 ml produce an A280 (commercial alkali lignin) of 0.60 ina 1-cm cell.

[4]

[5]

[6]

[7]

[8]

[9]

[10

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[12

[13

[14

[15

[16

[17

[18

[19

[20

[21

[22

[23

[24

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4.7. Phenolic acid analysis

Phenolic acids were extracted as described in [41]. Briefly,free (F1), ester-bound (F2, released after alkaline hydrolysis)and glycoside-bound (F4, released after acid hydrolysis) phe-nolic acids were obtained from a methanol extract of tissueground in liquid nitrogen. The fraction of cell wall-bound phe-nolic acids (F3) was obtained after alkaline hydrolysis of theresidual material following methanol extraction. The 2,6-ditercbutyl β-cresol was used as antioxidant to minimize theoxidation of phenolic acids during alkaline hydrolysis (4 h atroom temperature in darkness) and nitrogen was immediatelybubbled through the sample after addition of 2 N NaOH. Inspite of adding the antioxidant, the contents of caffeic andchlorogenic (3-O-(caffeoyl) quinate) acids in the fractions ofester-bound phenolics (F2, F3) were lowered as indicated bythe degradation of internal standards. For this reason the valuesof ester-bound fractions of these two acids are not shown inTable 1 and Fig. 5. Phenolic acids were analyzed by meansof HPLC using a Dionex Liquid Chromatograph (P660-HPLC Pump, ASI-100 Automated Sample Injector, TCC-100Termostated Column Compartment, PDA-100 PhotodiodeArray Detector, Chromeleon Software 6.5) with C 18 Spheri-sorb 5 ODS column (25.0 × 4.6 mm). For elution was usedacetonitril and acetic acid gradient. The phenolic acids weredetected in their absorption maximum. λmax was detectedfrom authentic compounds (Sigma-Aldrich) that were used asreferences for quantitative analyses.

4.8. Statistical evaluation

Leaf samples for the activity and lignin determination weretaken from five plants of both plant types cultivated in fourindependent series. Immunohistology was carried out on theleaf samples from three independent series. HPLC analysis ofphenolic acids was done in the leaf samples from two indepen-dent series. Statistically significant differences in the meanvalues were tested by Student’s t-test at P = 0.05.

Acknowledgements

This work was supported by the grants of Grant Agency ofthe Czech Republic No. 206/03/0310 and AV0Z50380511.

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