Transgenic ipt tobacco overproducing cytokinins overaccumulates

Plant Physiology and Biochemistry 44 (2006) 526–534
www.elsevier.com/locate/plaphy
Research article
Transgenic ipt tobacco overproducing cytokinins
overaccumulates phenolic compounds during in vitro growth
Renáta Schnablováa,b, Helena Synkováb,*, Anna Vičánkovác, Lenka Burketováb,
Josef Ederc, Milena Cvikrovác
a
Department of Plant Anatomy and Physiology, Faculty of Sciences, Charles University, Viničná 5, 128 44 Praha 2, Czech Republic
b
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 2006
Available 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 related
proteins (PR proteins), and increase in peroxidase (POD) activities. Immunolocalization of zeatin and also PR-1b protein on leaf cryo-sections
proved their accumulation in all mesophyll cells of transgenic tobacco contrary to control non-transgenic plants. Intensive blue autofluorescence
of phenolic compounds induced by UV in cross-sections of leaf midrib showed enhanced contents of phenolics in transgenic tobacco compared
with controls, nevertheless, no significant difference between both plant types was found in leaf total lignin content. Transgenic plantlets exhibited higher peroxidase activities of both soluble and ionically bound fractions compared with controls. HPLC analysis of phenolic acids confirmed the increase in all phenolic acids in transgenic tobacco except for salicylic acid (SA). The effect of high phenolic content on rooting of
transgenic 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 factors such as irradiance, temperature, ventilation, and components of a cultivation medium such as sucrose and/or growth
regulators result in the formation of abnormal morphology,
anatomy, and physiology of in vitro grown plantlets [1]. However, internal factors such as cell type, size, age, and the state
Abbreviations: C, control type rooted tobacco; CK, cytokinins; DAB, 3,3′diaminobenzidine; FM, fresh leaf mass; GPOD, guaiacol peroxidase; IAA,
indole-1,3-acetic acid; ipt, the gene for isopentenyl transferase; POD,
peroxidase; PR proteins, pathogenesis related proteins; Pssu, promoter
sequence of the gene coding for small subunit of ribulose-1,5-bisphosphate
carboxylase/oxygenase; SA, salicylic acid; SPOD, syringaldazine peroxidase;
T, transgenic non-rooted plants.
* Corresponding author. Tel.: +420 2 333 20338; fax: +420 2 243 10113.
E-mail address: synkova@ueb.cas.cz (H. Synková).
0981-9428/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2006.09.004
of differentiation of the explants play an important role in their
organogenic capacity. The basic regulatory mechanism underlying plant organ formation involves a balance between auxin
and cytokinin (CK) contents. A relatively low content of auxin
and high content of CKs result in a shoot differentiation, while
a reverse situation results in a root initiation. This could be
demonstrated in a few Arabidopsis mutants with altered CK
metabolism [2] or in transgenic plants with the gene for isopentenyltransferase (ipt) introduced under various promoters
[3,4]. Those plants are characterized by high endogenous CK
contents resulting in the high shoot forming capacity and the
low rooting capacity that cannot be improved by exogenous
auxin treatment [5]. Thus, transgenic Pssu-ipt tobacco with ca.
10-fold enhanced content of endogenous CKs is unable to form
roots during in vitro cultivation [3]. Pssu-ipt tobacco plants
exhibited increased activities of antioxidant enzymes, peroxidases, several enzymes of intermediary metabolism, and a pre-
R. Schnablová et al. / Plant Physiology and Biochemistry 44 (2006) 526–534
sence of pathogenesis related (PR) proteins such as PR-1b protein and proteins with chitinase activity in extracellular fluid
[6]. These findings clearly indicate that elevated CK content
rather than conditions of cultivation caused the stress and stimulated defense mechanisms in transgenic tobacco. The interaction between CKs and pathogenesis related proteins (PR protein) production was shown by Sano et al. [7]. CKs interfered
with the signal transduction mechanisms participating in PR
proteins synthesis by controlling endogenous level of salicylic
acid (SA) and jasmonic acid. SA belongs to a diverse group of
secondary metabolites, generally called phenolic compounds,
(e.g. flavonoids, tannins, hydrocinnamate esters, and lignin)
that are synthetized normally during plant growth and development.
Phenolic compounds have been shown to serve as signaling molecules (e.g. SA, [8]), to modulate the action of auxins
[9], and to play an important role in the resistance of plants to
biotic and abiotic stresses [10]. There are still some unanswered questions about the precise role of phenolic substances in the processes of differentiation and morphogenesis.
Through the modulation of endogenous indole-1,3-acetic acid
(IAA) content phenolics might influence the hormonal balance required, e.g. for root induction. Correlations have been
observed between phenolic content and root formation in in
vitro culture [11] or in cuttings of many species [12]. Antioxidative properties of polyphenols arise from their high reactivity as hydrogen or electron donors and from the ability of the
polyphenol-derived radical to stabilize and delocalize the unpaired electron and from their ability to chelate transition metal ions, i.e. termination of Fenton reaction [13]. Takahama
and Oniki [14] have proposed that the peroxidase/phenolics/
ascorbic acid system can function as a hydrogen peroxide
scavenging system in vacuoles and apoplast, because phenolics, ascorbic acid and peroxidase are normal components of
those compartments.
Other phenolic biopolymers, lignins, are located in the primary and secondary walls of specific plant cells as well as in
the middle lamella [15]. They are synthetized for mechanical
support and water transport of terrestrial vascular plants and in
response to pathogen attack. The monomers of lignin derived
from three hydroxycinnamyl alcohols or monolignols: pcoumaryl, coniferyl, and sinapyl are synthetized in the cytoplasm (Golgi or endoplasmic reticulum) and released into the
cell wall from vesicles. Enzymes located within the cell wall
during lignification, in either free or bound state, include various types of peroxidase (POD) and oxidase (including laccase). Oxidase activity may be associated with the earliest
stages of lignification and POD with the later stages [15].
To our knowledge, there is no information available on contents of phenolics in transgenic plants overproducing endogenous CKs and/or on a relationship between both groups of compounds. In our paper we aimed to investigate a role of phenolics
in in vitro grown non-rooting Pssu-ipt transgenic tobacco overproducing cytokinins (CK). We localized CKs, PR proteins, and
cell wall bound phenolics on transverse leaf or midrib sections
in transgenic tobacco by immunohistological methods. Furthermore, we carried out a complete HPLC analysis of phenolic
527
acids with the aim to elucidate their role in rooting of transgenic
tobacco.
2. Results
2.1. Detection and localization of zeatin
Immunolocalization with specific antibodies against zeatin
was performed on cryo-sections to test whether a specific site
of CK localization in Pssu-ipt plants exist. On cryo-sections
from C plants, the level of zeatin was very low and under the
detection limit (Fig. 1A). In sections from T, zeatin was localized in all mesophyll cells (Fig. 1B). Anti-CK label was also
detected in chloroplasts of transgenic tobacco cells (marked by
arrows).
2.2. Detection and localization of PR-1b protein
Immunocytochemical examination carried on cryo-sections
of transverse leaf samples proved that in Pssu-ipt plants the
synthesis of PR proteins was induced (Fig. 1C, D). PR-1b protein was synthesized in all cells of in vitro grown T (Fig. 1D)
contrary to C plants, where no or trace amounts of PR-1b protein around the vascular bundles was found (Fig. 1C).
2.3. Tissue localization of phenolic compounds
Autofluorescence of the cross-sections of the leaf midrib
revealed differences in the localization of cell wall phenolic
compounds (Fig. 1E, F). In C, the blue autofluorescence
(induced by UV light) was detected only in the xylem vessel
walls and it attributed particularly to lignin (Fig. 1E). In T plants
(Fig. 1F), the blue autofluorescence was significantly stronger
than in C and it was also detected in cells surrounding the
xylem vessels. It probably originated also from ferulic acid
bound 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 were
predominantly localized in cell walls, particularly of epidermal cells in C plants, while in T plants the stain intensity was
comparable in all cell types (Fig. 1G, H). Moderately stronger
stain intensity was observed around the veins in T compared
to C.
2.5. Activity of soluble and ionically bound peroxidases
The significantly higher peroxidase activities were found in T
compared with C irrespective of substrate used for activity assay
(Fig. 2). Generally, soluble peroxidase fraction exhibited significantly higher activities than ionically bound peroxidases. When
calculated per fresh leaf matter (FM), ionically bound peroxidase activities took 19% (guaiacol peroxidase, GPOD) and
15% (syringaldazine peroxidase, SPOD) of total peroxidase
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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 for
peroxidase 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.
R. Schnablová et al. / Plant Physiology and Biochemistry 44 (2006) 526–534
529
lic acids (F4) was done. The differences found in the HPLC
spectrum of phenolic acids were only quantitative between C
and T (Fig. 4). The total content of phenolic acids increased
ca. five times in T compared with C (Table 1). The most pronounced enhancement was observed particularly in the contents
of free phenolic acids (F1) and in the glycoside-bound phenolics
(F4) in T plants compared with C (Table 1 and Fig. 5). As
regards the individual phenolic acids, the most abundant phenolics in both types of plants were caffeic and chlorogenic acids
(Fig. 5). T plants contained significantly higher amounts of caffeic (F4) and chlorogenic acids (F1) and increased contents of pcoumaric, 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 was
found in T compared to C.
The total content of lignin was assayed in leaf samples by
derivatization with thioglycolic acid (Table 1). Lignin content
was moderately higher in T plants, although the difference was
not statistically significant.
3. Discussion
Fig. 2. Activities of soluble (F) and ionically bound (B) peroxidases measured
with guaiacol (GPOD) or syringaldazine (SPOD) as substrates in control (C)
and transgenic (T) tobacco. Activities were calculated per gram of fresh leaf
matter (FM). The values are the mean ± S.E. Statistical significant differences
at P = 0.05 are marked by different letters.
activity in C (Fig. 2A, B). In T, ionically bound peroxidase
activity was 11% (GPOD) and 0.5% (SPOD) of total peroxidase
activity (Fig. 2A, B). Thus, peroxidase activity measured with
syringaldazine (SPOD) was lower in ionically bound fraction
relatively to soluble peroxidases in T compared with C.
In T, more isozymes of peroxidases were present in soluble
fraction analyzed by non-denaturating polyacrylamide gel electrophoresis stained for enzyme activity compared with C
(Fig. 3). The most significant difference was found in less
mobile isozymes in the upper part of the gels.
2.6. Contents of phenolic acids and lignin
In order to characterize changes in phenolic acid composition, detailed analysis of free phenolic acids (F1), ester-bound
methanol-soluble phenolic acids (F2), ester-bound cell wall phenolic acids (F3), and glycoside-bound methanol-soluble pheno-
3.1. Phytohormones
There is substantial evidence that the process of rooting is
influenced by exogenous and endogenous contents of growth
hormones, by content of phenolics, and by activities of enzymes
involved in their metabolism. In our previous experiments we
proved that transgenic Pssu-ipt tobacco produced ca. 10 times
more endogenous CKs than control type both under in vitro
and ex vitro conditions [16,6]. Our immunohistological localization of the most abundant CK in Pssu-ipt tobacco, zeatin, carried
on leaf cryo-sections proved the presence of this CK type in all
mesophyll cells and in chloroplasts contrary to control tobacco,
where its content was under a detection limit (Fig. 1A, B). It is
in agreement with our previous findings, when higher CK contents were found in isolated chloroplasts from Pssu-ipt tobacco
[17]. The significant increase in CKs usually affects the balance
with other plant hormones, particularly auxins. The disturbances
caused by high endogenous CKs and/or auxins were observed in
various transgenic plants overproducing one of those hormones
[17]. CK overproduction decreases the content of auxin apparently by decreasing its rate of synthesis and/or transport, rather
than by increasing rates of turnover or conjugation [18].
Although our present experiments did not involve auxin determination, our previous results confirmed at least four times
higher CK/auxin ratio in Pssu-ipt tobacco than in control plants
[19].
3.2. Peroxidases
Fig. 3. Peroxidase isozyme patterns obtained after non-denaturating PAGE in
control (C) and transgenic tobacco (T).
The soluble forms of POD are cytoplasmic, whereas bound
forms are generally thought to be associated with cell walls [20].
However, under stress conditions, the enhanced POD activity in
the intercellular spaces, stimulating cell wall stiffening, probably
reduces cell growth, which might represent a mechanical adapta-
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Fig. 4. HPLC chromatogram of methanol-soluble glycoside-bound phenolic acids extracted from control (C) and transgenic (T) tobacco. Dotted line represents
acetonitril and acetic acid gradient used for the elution of phenolic acids. Each profile represents an equivalent amount of extract, normalized on a volume of extract
per 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 1
Total content of phenolic acids and lignin in control and Pssu-ipt transgenic
tobacco grown in vitro. F1 = free phenolic acids, F2 = ester-bound phenolic
acids, F3 = ester-bound cell-wall phenolic acids, F4 = glycoside-bound phenolic acids. FM = fresh leaf matter, DM = dry leaf matter. The values of F1–F4
represent the means of three replicates. The S.E. values averaged 8% and did
not 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 by
different letters
Fractions of phenolic acids
(μg g–1 FM)
F1
F2
F3
F4
Total sum (μg g–1 FM)
Lignin content (mg g–1 DM)
Control
Transgenic
1.195a
1.508a
0.295a
16.506a
22.207a
21.386 ± 1.94a
8.67b
6.212b
0.479b
95.465b
110.826b
26.028 ± 1.3a
tion [21]. This kind of action has been attributed mainly to POD
whose activity can be detected by using syringaldazine as a specific substrate. There is histochemical and biochemical evidence
that only cell walls that are undergoing lignification are able to
oxidize syringaldazine [22]. In Pssu-ipt tobacco, activity of soluble POD measured with syringaldazine (SPOD) increased six
times compared with controls, whereas it was only three times
higher when measured with guaiacol as a substrate. This would
support the hypotheses that cell wall stiffening is undergoing in
transformants. Although we expected more significant difference between T and C, we found only moderately enhanced
lignin content in Pssu-ipt tobacco compared with control plant
type. Nevertheless, the samples for our lignin assay included
Fig. 5. Contents of individual phenolic acids calculated per gram of FM in
control (C) and transgenic (T) tobacco. F1 = free phenolic acids;
F2 = methanol-soluble ester-bound phenolic acids; F3 = methanol insoluble
ester-bound cell wall phenolic acids; F4 = methanol-soluble glycoside-bound
phenolic acids. Other details see Fig. 4 for abbreviations. The values are the
means of each fraction. The S.D. did not exceed 15% of the mean.
particularly leaves and upper parts of the plantlets and not the
lower base of the stem, where rooting takes place and where the
difference could be more pronounced.
3.3. Phenolic acids
We have found ca. fivefold higher content of phenolic acids
in transgenic tobacco compared with controls (Table 1). The
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considerable enhancement was observed in contents of all
indentified phenolic acids except for SA. Caffeic and chlorogenic acids and their glycosides represented the most abundant
phenolics that we have detected in both types of tobacco and
that increased considerably in T. According to [23] chlorogenic
acid and its isomers are present in the apoplast of tobacco leaves
and the levels increase sigmoidally as a function of leaf age,
whereas levels of the caffeic acid esters of the symplast do not
significantly change during aging. Compounds such as chlorogenic, caffeic, and ferulic acids have been shown to interact with
IAA oxidase to reduce the rate of auxin oxidation [24,9]. Nevertheless, Faivre-Rampant et al. [25] suggested that the high content of chlorogenic acid exceeding a certain threshold concentration could lead to opposite effect and cause the inhibition of root
development. Furthermore, monophenolic acids such as pcoumaric acid were also shown to stimulate IAA oxidase activity [26]. Higher levels of free caffeic, chlorogenic, and pcoumaric acids found in T plants might influence through lowering the endogenous IAA content the appropriate hormonal balance required for the root induction.
Contradictory, lower content of SA was found in in vitro
grown Pssu-ipt tobacco when compared with control plants.
SA is discussed as an important signaling molecule associated
with the establishment of SA-mediated defense and a systemic acquired resistance, and the activation of genes encoding PR proteins [27]. However, there is a very little information available on interactions among SA, CKs, and PR
proteins except for those from Sano et al. [7]. We have previously found de novo synthesis of several PR proteins in
extracellular fluid in Pssu-ipt tobacco [6]. In this paper, we
proved by immunolocalization particularly the accumulation
of PR-1b proteins in all mesophyll cells of Pssu-ipt tobacco
contrary to controls. Although the content of SA was lower in
in vitro grown transgenic tobacco than in controls, the synthesis of PR proteins was higher (see also [6]). Therefore we
may hypothesize that CKs directly might be involved in the
activation of PR-1 protein synthesis.
531
promoter is light activated and therefore CK overproduction
is constitutive and permanent [31,16]. This probably suppresses or overrides all transient changes in auxin concentrations needed for the normal root growth initiation.
Furthermore, ca. six times higher POD activities were found
in Pssu-ipt tobacco in our experiment and previously also both
under in vitro or ex vitro cultivation of the primary transformant
(see also [32,6]). In spite of high POD activities, we also found
ca. five times higher content of phenolic acids in Pssu-ipt
tobacco (Table 1). The cause for this enhancement is not clear,
but it seems that high phenolics and POD activity is associated
with a certain threshold level of CKs. While it stays very high in
primary transformants, F1 generation of Pssu-ipt plants contains
lower amount of CKs in early stages of plant development. The
activity of POD and the content of phenolics are lower and
plants are able to form small root system [32]. As PODs play
the important role in auxin catabolism [33], their activity considerably affects also auxin contents, which was higher in transgenic rooted plants [19].
3.5. Conclusions
Transgenic Pssu-ipt plants showed various signs of stress
both in altered metabolism and on the ultrastructural level [6,
17]. Now we have found that in vitro grown non-rooting Pssuipt tobacco is characterized not only by the high CK content, the
high peroxidase activity, the presence of PR proteins, moderately higher lignin content, but also five times higher content
of 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 without
possible transient changes needed for the proper stimulation of
rooting process. We suppose that the simultaneous increase in
peroxidase activity and in the content of phenolics might represent the stress response to the overproduction of CKs in transgenic Pssu-ipt tobacco.
3.4. Rooting process
The involvement of auxin in nodule organogenesis is likely
in the stimulation of cell divisions and regulation of root differentiation [28]. The rooting process might be subdivided
into several interdependent phases, where also other factors
such as peroxidase activities and content of phenolics affect
the process [29,30]. There is always a transient increase in the
endogenous auxin content during the inductive phase (corresponding to a minimum level of peroxidase activity), followed by a decrease in auxin levels to a minimum at the initiation phase [31]. The period of higher POD activity
corresponded to the early events of the initiation phase. Phenolic content changes inversely to the POD activity. This has
been reported by several authors who suggested that phenolics may act by modulating enzyme activity and preventing
POD oxidation of auxin during root induction [12,30]. The
process of rooting in Pssu-ipt tobacco is strongly influenced
by the permanent disproportion of CKs and auxins as Pssu
4. Materials and methods
4.1. Plant material
Control tobacco (Nicotiana tabacum L. cv. Petit Havana
SR1) was referred as C. Transgenic tobacco (T) containing a
supplementary ipt-gene under a control of the promoter for the
small subunit of RuBPCO (Pssu-ipt) was generated by means of
the Agrobacterium tumefaciens transformation system and
grown in vitro as shoots unable to form roots as described by
Beinsberger et al. [3]. During in vitro precultivation all plants
were grown in agar with Murashige and Skoog basal salt mixture (Sigma-Aldrich, Prague, Czech Republic) in ventilated
Magenta GA-7 vessels as described in Semorádová et al. [34]
and Synková et al. [6]. Leaf samples were taken from the plants
after 3–4 weeks of in vitro precultivation.
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4.2. Fluorescence microscopy
Blue autofluorescence (induced by UV light) was used for a
localization of cell wall phenolic compounds. Fluorescence of
unfixed, hand-cut leaf sections mounted in water was analyzed
by epifluorescence light microscope (Nikon Eclipse E600,
Japan) with the filters UV-2A (EX 330–380, DM 400, BA
420). Photographs were taken by CCD camera using identical
exposure times.
4.3. Immunohistology for light microscopy
The random leaf blade samples were fixed in 3% paraformaldehyde and 0.5% glutaraldehyde in PBS (135 mM NaCl,
2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4, pH 7.2) for
2.5 hours at 4 °C, washed in PBS and dehydrated in graded
sucrose series (from 0.1 to 1.76 M in PBS at 4 °C and frozen
at – 75 °C). Dehydrated leaf pieces were cut to 8 μm thick tissue
sections on a Cryotome Cryostat (Shadon, Pittsburg, USA).
Collected sections attached to microscopic slides were rehydrated in solutions of decreasing sucrose concentration (from
1.76 to 0.11 M in PBS) followed by washing in PBS. The sections were transferred to TBS (Tris 50 mM, NaCl 150 mM, pH
7.6) containing 1% (v/v) Triton X-100 for 30 min. After blocking in blocking solution 3 × 20 min (blocking solution: TBS
containing 20 mM glycine, 0.2% gelatine (v/v), 0.1% Tween
20 (v/v), 10% goat preimmune serum (v/v)), the sections were
incubated in the primary rabbit polyclonal antibodies (antizeatin—antibodies purified by protein A, Professor Strnad, Olomouc; anti PR-1b—Dr. J. Antoniw, IACR). Sections were
washed in TBS and incubated with the secondary goat antirabbit antibody coupled to alkaline phosphatase. Following
washing with TBS and buffer (2 mM MgCl2 in Tris–HCl, pH
9.5), CKs and PR-1b protein were visualized with nitroblue tetrazolium (NBT)/5-brom-4-chlor-3-indolylphophate (BCIP) substrate (37.0 mM NBT, 35.0 mM BCIP). The reaction was
stopped by incubation of sections in EDTA (2.0 mM in TBS)
followed by fixation in 25% glutaraldehyde in TBS. Immunohistological controls were run parallel and treated with blocking
solution instead of primary antibodies. As a control for a specific CK labeling, the sections were incubated in the saturating
mixture of the antibody with free t-zeatinriboside. The stained
cryo-sections were viewed in the light microscope Nikon
Eclipse E600 equipped with CCD camera.
4.4. In situ localization of peroxidases
Histological staining for peroxidase activity was carried out
using 3,3′-diaminobenzidine (DAB) on cryo-sections made from
leaf 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) for
15 min in darkness. After thorough washing by deionized
water and dehydration through a series of solutions with increasing ethanol concentration permanent preparations were made.
The samples were examined by light microscopy (Nikon Eclipse
E600) equipped with a CCD camera.
4.5. Peroxidase extraction and activity assay
Samples of tobacco leaves (0.5 g) were frozen in liquid nitrogen, 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 supernatant, activity of soluble peroxidase (POD; E.C. 1.11.1.7) was
determined. POD ionically bound to cell walls was extracted
with 1 M NaCl from a purified pellet, which was washed once
with phosphate buffer and several times with distilled water until
no peroxidase activity was detected.
POD activities were measured with guaiacol (GPOD) or syringaldazine (SPOD) as substrates. Oxidation of guaiacol was
determined spectrofotometrically by an increase in absorbance
at 436 nm [35]. SPOD was determined as an increase in absorbance at 535 nm [36]. All activities were calculated per g of
fresh leaf matter (FM), where the rates were given in 1 μmol
of 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. Aliquots
of supernatants corresponding to 25 μg of protein per lane were
used. POD isozymes were detected in situ by staining gels in
1 M acetate buffer, pH 4.6 with 0.04% benzidine and 10 mM
H2O2 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 and
ground to a fine powder (with liquid N2). To obtain cell walls,
the powder was suspended in 1M NaCl with 0.5% Triton X-100
and stirred for 30 min. Then it was washed twice with distilled
water, twice with 100% methanol, twice with 100% acetone
(each step 30 min). Total lignin content was assayed by derivatization with thioglycolic acid (modified method of [38,39]).
Aliquots of 10 mg of the cell wall preparations were placed in
Eppendorf tube and treated with 1.5 ml of 2 N HCl and 0.3 ml
of thioglycolic acid for 4 h at 95 °C. Samples were cooled and
centrifuged for 10 min at 15,000 × g. The supernatant was
removed and pellet was washed three times with distilled
water. Thereafter, the pellet was suspended in 1 ml of 0.5 N
NaOH for 18 h on a shaker at room temperature. The suspension
was centrifuged for 10 min at 15,000 × g. The supernatant
obtained after centrifugation and the second supernatant
obtained after re-extraction the pellet with 0.4 ml NaOH were
combined and acidified with 0.3 ml concentrated HCl and lignothioglycolic acid was allowed to precipitate at 4 °C. The mixture was centrifuged as above, the supernatant removed and the
pellet solubilized in 1 ml of 0.5 N NaOH and diluted before
measuring absorbance at 280 nm. The amount of lignin was
calculated according to conversion made by [40]: 100 μg of lignin in 1 ml produce an A280 (commercial alkali lignin) of 0.60 in
a 1-cm cell.
R. Schnablová et al. / Plant Physiology and Biochemistry 44 (2006) 526–534
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) phenolic acids were obtained from a methanol extract of tissue
ground in liquid nitrogen. The fraction of cell wall-bound phenolic acids (F3) was obtained after alkaline hydrolysis of the
residual material following methanol extraction. The 2,6ditercbutyl β-cresol was used as antioxidant to minimize the
oxidation of phenolic acids during alkaline hydrolysis (4 h at
room temperature in darkness) and nitrogen was immediately
bubbled through the sample after addition of 2 N NaOH. In
spite of adding the antioxidant, the contents of caffeic and
chlorogenic (3-O-(caffeoyl) quinate) acids in the fractions of
ester-bound phenolics (F2, F3) were lowered as indicated by
the degradation of internal standards. For this reason the values
of ester-bound fractions of these two acids are not shown in
Table 1 and Fig. 5. Phenolic acids were analyzed by means
of HPLC using a Dionex Liquid Chromatograph (P660HPLC Pump, ASI-100 Automated Sample Injector, TCC-100
Termostated Column Compartment, PDA-100 Photodiode
Array Detector, Chromeleon Software 6.5) with C 18 Spherisorb 5 ODS column (25.0 × 4.6 mm). For elution was used
acetonitril and acetic acid gradient. The phenolic acids were
detected in their absorption maximum. λmax was detected
from authentic compounds (Sigma-Aldrich) that were used as
references for quantitative analyses.
4.8. Statistical evaluation
Leaf samples for the activity and lignin determination were
taken from five plants of both plant types cultivated in four
independent series. Immunohistology was carried out on the
leaf samples from three independent series. HPLC analysis of
phenolic acids was done in the leaf samples from two independent series. Statistically significant differences in the mean
values were tested by Student’s t-test at P = 0.05.
Acknowledgements
This work was supported by the grants of Grant Agency of
the Czech Republic No. 206/03/0310 and AV0Z50380511.
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