Journal of Experimental Botany, Vol. 56, No. 418, pp. 2085–2093, August 2005
doi:10.1093/jxb/eri207 Advance Access publication 13 June, 2005
RESEARCH PAPER
Hydrogen peroxide and expression of hipI-superoxide
dismutase are associated with the development of
secondary cell walls in Zinnia elegans
Marlene Karlsson1, Michael Melzer2, Isabella Prokhorenko3, Thorsten Johansson4 and Gunnar Wingsle1,*
1
Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Science,
Umeå Plant Science Centre, 90183 Umeå, Sweden
2
Department of Molecular Cell Biology, Institute of Plant Genetics and Crop Plant Research,
06466 Gatersleben, Germany
3
Institute of Basic Biological Problems, Russian Academy of Sciences, Pushtchino, Moscow Region, 142292 Russia
Swedish Defense Research Agency, FOI, NBC, Defense Department of Threat Assessment,
901 82 Umeå, Sweden
4
Received 16 December 2004; Accepted 22 April 2005
Abstract
A special form of a CuZn-superoxide dismutase with
a high isoelectric point (hipI-SOD; EC 1.15.1.1) and
hydrogen peroxide (H2O2) production were studied
during the secondary cell wall formation of the inducible tracheary element cell-culture system of Zinnia
elegans L. Confocal microscopy after labelling with
29,79-dichlorofluorescin diacetate showed H2O2 to be
located largely in the secondary cell walls in developing tracheary elements. Fluorescence-activated cell
sorting analysis showed there were lower levels of
H2O2 in the population containing tracheary elements
when H2O2 scavengers such as ascorbate, catalase,
and reduced glutathione were applied to the cell
culture. Inhibitors of NADPH oxidase and SOD also
reduced the amount of H2O2 in the tracheary elements.
Furthermore, addition of these compounds to cell
cultures at the time of tracheary element initiation
reduced the amount of lignin and the development of
the secondary cell walls. Analysis of UV excitation
under a confocal laser scanning microscope confirmed
these results. The expression of hipI-SOD increased as
the number of tracheary elements in the cell culture increased and developed. Additionally, immunolocalization of a hipI-SOD isoform during the tracheary
element differentiation showed a developmental buildup of the protein in the Golgi apparatus and the
secondary cell wall. These findings suggest a novel
hipI-SOD could be involved in the regulation of H2O2
required for the development of the secondary cell
walls of tracheary elements.
Key words: CuZn-superoxide dismutase, hydrogen peroxide,
immunolocalization, tracheary element, Zinnia cell-culture system.
Introduction
Hydrogen peroxide in plants has proposed roles in many
different processes. For instance, besides its traditionally
accepted role as a reactive oxygen species (ROS), oxidizing
cellular compounds, it has suggested roles in plant defence
mechanisms against pathogens, in signalling pathways
associated with systemic-acquired acclimation, in the onset
of secondary wall (sCW) formation, and the regulation of
plant cell growth (Bestwick et al., 1997; Karpinski et al.,
1999; Potikha et al., 1999; Foreman et al., 2003).
Another process in which H2O2 has a proposed role is in
lignification of the sCW. The classical view of lignin polymerization is that cell wall oxidases convert the monolignols
* To whom correspondence should be addressed. Fax: +46 90 786 5901. E-mail: [email protected]
Abbreviations: ASA, ascorbic acid; CAT, catalase; CN, sodium cyanide; cp-SOD, chloroplastic CuZn-SOD; cyt-SOD, cytosolic CuZn-SOD; DCFH-DA, 29,79dichlorofluorescin diacetate; DDC, N,N-diethyldithiocarbamate; DPI, diphenyleneiodonium; FACS, fluorescence-activated cell sorting; GSH, reduced
glutathione; hipI-SOD, high-isoelectric-point CuZn-superoxide dismutase; IEF, isoelectrofocusing; ROS, reactive oxygen species; sCW, secondary wall;
SHA, salicylhydroxamic acid; SOD, superoxide dismutase; TE, tracheary element.
ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2086 Karlsson et al.
(coumaryl, coniferyl, and sinapyl alcohols) into free radicals, which then spontaneously polymerize to give lignin.
Both laccase (H2O2-independent) and peroxidase (H2O2dependent) activities are associated with differentiating
xylem, and these enzymes have been suggested to be
responsible for free radical formation (Liu et al., 1994;
Boudet et al., 1995; Whetten et al., 1998; Pomar et al.,
2002). The presence of H2O2 in the cell wall has been
demonstrated with histochemical staining (Olsen and
Varner, 1993; Schopfer, 1994; Bestwick et al., 1997),
but the mechanism of its generation is not yet clear.
Much accumulated evidence indicates the involvement of
a membrane-bound NADPH oxidase producing superoxide (Oÿ
2 ) that spontaneously forms H2O2 (Doke, 1985;
Auh and Murphy, 1995; Ros-Barceló et al., 2002). Other
H2O2-generating systems have also been suggested, e.g.
cell wall cationic peroxidases (Bolwell et al., 1995) and
amine-oxidases (Goldberg et al., 1985). It has also been
suggested that the H2O2 generated during auto-oxidation
of coniferyl alcohol drives the oxidase activity of a basic
peroxidase in Zinnia (Pomar et al., 2002; López-Serrano
et al., 2004). However, this conversion may also be
mediated in the cell wall by superoxide dismutase (SOD;
EC 1.15.1.1), which catalyses the dismutation of Oÿ
2 into
molecular oxygen and H2O2 (Ogawa et al., 1997).
A novel isoform of CuZn-SOD was discovered recently
in Scots pine and found to be localized in sieve elements,
sCWs of the xylem, and intercellular spaces in pine needles
by immunohistochemistry (Karpinska et al., 2001). A
similar enzyme was also found in poplar (Schinkel et al.,
2001). Due to its high isoelectric point (pI), it was denoted
high-isoelectric-point CuZn-superoxide dismutase (hipI-SOD)
and its localization in secondary walls of xylem implies
that it may be involved in processes other than ‘ordinary’
oxygen defence mechanisms.
To study the underlying mechanisms of the regulation of
H2O2, in the sCW and to acquire further information about
the enzymes involved in different cell wall processes of xylem,
an obvious tool to use is the well-characterized Zinnia
cell-culture system (Kohlenbach and Schmidt, 1975; Fukuda
and Komamine, 1980; Milioni et al., 2001; Demura et al.,
2002). In the present study, the Zinnia system was used in
combination with fluorescence-activated cell sorting (FACS),
immunolocalization, and western blots. This paper presents data suggesting that H2O2 is required for the development and lignification of the secondary walls of tracheary
elements (TEs) in the Zinnia cell-culture system. In addition, it is proposed that a novel isoform of a CuZn-SOD,
localized in the sCW, is a regulator of H2O2 in this process.
Materials and methods
Plant material
Seedlings of Zinnia elegans (L. cv. Canary bird; Park Seed,
Greenwood, USA) were grown with an 18 h photoperiod in a growth
chamber at 25 8C, at 80% relative humidity and a photon flux
density of 300 lmol mÿ2 sÿ1 (HQITS 400; Orsam). The first true
leaves were harvested 12–15 d after sowing. They were surfacesterilized with 1% w/v calcium hypochlorite and 0.01% Triton-X
100, and then repeatedly rinsed with sterile distilled water. The
leaves were ground in medium and released cells were isolated as
described by Fukuda and Komamine (1980). The isolated cells were
cultured in 50 ml Erlenmeyer flasks containing 12 ml of the medium
described by Fukuda and Komamine (1980) which contains the
plant growth regulators a-naphthalene-acetic acid (0.1 mg lÿ1) and
6-benzylaminopurine (0.2 mg lÿ1). The flasks were placed on an
orbital shaker in a dark incubator at 27 8C and shaken at 90 rpm.
Under these conditions mesophyll cells trans-differentiate after
approximately 72 h, via a form of programmed cell death (Fukuda,
1996; Groover et al., 1997), into TEs, which are dead, hollow cells
with lignified sCWs. Fluorescein diacetate (Sigma) was used to
label living cells under a fluorescence microscope (Gahan, 1984),
and cells were counted in a Bürker chamber.
Confocal laser scanning microscopy
The system used to detect lignin in the sCW of fresh and fixed
Zinnia cells was a Zeiss Axiovert 135M microscope (Carl Zeiss)
attached to a confocal laser scanning system (CLSM; model Zeiss
LSM 410). Lignin autofluorescence was induced by excitation light
of 351/364 nm produced by an Ar-UV-laser, and detected using
a 400–510 nm emission filter to eliminate most of the autofluorescence signals from other cell components. As well as detecting
fluorescence signals, images were obtained in the transmission
mode. To examine cells by CLSM after staining with 29,79dichlorofluorescin diacetate (DCFH-DA) the 488 nm and 568 nm
lines of the Ar-Kr laser in the Zeiss LSM 510 system were used,
giving, respectively, the ‘green’ signal detected at 505–530 nm and
the autoflourescence signal detected above 585 nm. These signals
were detected in separate channels.
FACS and lignin analyses
One and a half millilitre aliquots of cell culture were pretreated in
an Eppendorf tube for 2 h with different compounds, including
reduced glutathione (GSH; 5 mM; Sigma), ascorbic acid (ASA;
5 mM; Merck), diphenyleneiodonium (DPI; 10 lM; Sigma), N,Ndiethyldithiocarbamate (DDC; 0.5 mM; Sigma), salicylhydroxamic
acid (SHA; 0.5 mM; Sigma), bovine CuZn-SOD (400 units mlÿ1),
and catalase (CAT; 100 units mlÿ1; Sigma). After these incubations,
the samples were centrifuged at 46 g for 5–10 min and washed once
with HBSS-EDTA, pH 7.4 buffer (0.4 mM KH2PO4, 0.3 mM
Na2HPO4, 4.2 mM NaHCO3, 5.4 mM KCl, 137 mM NaCl, 5.6 mM
D-glucose, and 3 mM EDTA). The cell preparations were then
mixed with 1 ml of HBSS-EDTA buffer and 10 ll of a stock
solution of DCFH-DA (Sigma; 4 mM in ethanol), protected from
light, mixed, and incubated for 20 min at 22 8C. The cells were then
washed twice and kept on ice until analysis by FACS. Before
analysing the samples in the flow cytometer [FACSort (Becton
Dickinson Immuno Systems) equipped with an argon laser giving
a 488 nm primary emission line] it was calibrated by analysing the
signals from labelled and unlabelled beads (Becton Dickinson) with
FACSComp software (Becton Dickinson). Data from unlabelled
cells were adjusted for forward scatter (relative size) and side scatter
(relative granularity), while FL1 (green), FL2 (red), and FL3 (deep
red) were adjusted by compensation, as appropriate (Shapiro, 1994).
The measuring time was 30 s with a medium rate of 35 l sÿ1. For
each sample 5000–10 000 cells were typically collected. Data were
analysed using Cell Quest software (Becton Dickinson).
At the time when the sCW started to form in the TEs, after
approximately 72 h, similar compounds used for the FACS experiment and also sodium cyanide (CN; Sigma) were added to the
HipI-superoxide dismutase and secondary cell wall development 2087
Erlenmeyer flasks. The cells were stained by phloroglucinol–
hydrochloric acid after 48 h of treatment to detect lignin (Sherwood
and Vance, 1976). The data from the staining were analysed by paired
t-test using the software package from Microsoft Excel (Microsoft R
Excel 2002). All statistically significant differences were tested at
the P=0.05, P=0.01, and P=0.001 levels.
Specimens were stained with uranyl acetate prior to examination in
a transmission electron microscope (Zeiss CEM 902A) at 80 kV.
Results
TE differentiation
Protein extraction
Fully developed leaves (50 g) of Zinnia, tobacco (Nicotiana tabacum
L.), barley (Hordeum vulgare L.), pea (Pisum sativum L.), and Scots
pine (Pinus sylvestris L.) were homogenized and further processed to
give a partially purified protein fraction after separation by anionexchange chromatography according to Karpinska et al. (2001).
Proteins from isolated Zinnia cells (2 ml) were extracted during the
developmental process of TEs in a similar way, except that the anionexchange column used in this separation was a Mono Q HR 5/5
column (Amersham Pharmacia Biotech AB).
SOD activity was determined by the direct KO2 assay (Marklund,
1985). Staining for SOD activity after isoelectrofocusing (IEF) was
performed as described by Beauchamp and Fridovich (1971). Protein
content was estimated from the absorbance at 280 nm, assuming that
A=1 corresponds to 1 mg of protein per ml.
Isolated mesophyll cells showed signs of sCW formation,
visible as thickenings of the cell wall, after approximately
72 h incubation (Fig. 1c). The first significant autofluorescence signals from lignin were detected at the same time
under the CLSM using UV-excitation (Fig. 1d). Cells from
early stages showed a low signal from organelles (Fig. 1b),
whereas the UV-excitation of lignified cell-wall thickenings
Preparation of antibodies
Synthesized peptides obtained from the sequence of the N-terminal of
chloroplastic CuZn-SOD (cp-SOD) and cytosolic CuZn-SOD (cytSOD) isolated from pine (Karpinski et al., 1992) were used to prepare
antibodies by intramuscular injection into rabbit (Agrisera). The cpSOD and cyt-SOD amino acid sequences were AAKKAVAVLKGDSQVEGC and GLLKAVVVLNGAADVKGC, respectively, both
of which were conjugated to keyhole limpet haemocyanin. The
peptide (200 lg) was emulsified with Freud’s complete adjuvant
before injection. Subsequent injections (50 lg) were given in the
third and sixth weeks, and serum was collected 6 weeks after the
last booster. For hipI-SOD analyses, the polyclonal antibodies described by Karpinska et al. (2001) were used.
Electrophoresis and immunoblotting
SDS-PAGE (10–15%), IEF (pH 3–9), and western blotting were
performed at 15 8C using a Phast system (Amersham Biosciences)
and Lumi-LightPLUS western blotting kit (Boerhringer Mannheim) as
previously described by Karpinska et al. (2001).
Immunoelectron microscopy
Isolated cells of the cell cultures of Zinnia elegans were fixed for 2 h
at room temperature in 50 mM cacodylate buffer (pH 7.2) containing 0.5% (v/v) glutaraldehyde and 2.0% (v/v) formaldehyde. After
fixation the cells were washed and transferred into 1.5% agar. Pieces
of agar (1 mm3) were then dehydrated in stepwise fashion by adding
progressively increasing concentrations of ethanol and concomitantly
lowering the temperature using an automated freeze substitution
unit (Leica). The steps used were as follows: 30% (v/v), 40% (v/v),
and 50% (v/v) ethanol for 1 h each at 4 8C; 60% (v/v) and 75% (v/v)
ethanol for 1 h each at ÿ15 8C; 90% (v/v) ethanol, and two times
100% (v/v) ethanol for 1 h each at ÿ35 8C. The samples were
subsequently infiltrated with Lowycryl HM20 resin (Plano GmbH)
by incubating them in the following mixtures: 33% (v/v), 50% (v/v),
and 66% (v/v) HM20 resin in ethanol for 4 h each and then 100% (v/v)
HM20 overnight. Samples were transferred into gelatine capsules,
incubated for 3 h in fresh resin and polymerized at 35 8C for 3 d under
indirect UV light. Thin sections, 70 nm thick, were cut with a diamond
knife and used for immunogold labelling with anti-hipI-SOD, cytSOD, cp-SOD, and hipI-SOD pre-immune serum as primary antibody and 10 nm protein A-gold as described by Teige et al. (1998).
Fig. 1. Confocal laser scanning microscopy of TEs. Transmission (a, c,
e, g, i) and autofluorescence images (b, d, f, h, j) after UV-excitation of
isolated mesophyll cells of Zinnia elegans during induced transdifferentiation into TEs: 24 h (a, b), 72 h (c, d), and 120 h (e, f ) after
induction. Cells (120-h-old) after induction and inhibition of SOD (g, h),
and NADPH oxidase (i, j) by the addition of 0.5 mM DDC and 10 lM DPI,
respectively, to the growth medium at day 3. Scale bars represent 20 lm.
2088 Karlsson et al.
increased significantly until the TEs were fully developed
(Fig. 1f). The number of TEs formed was generally
equivalent to 20–60% of the number of isolated cells 120 h
after the start of the incubation. Thus, the differentiation
kinetics and efficiency parameters were similar to the figures
reported by Groover and Jones (1999). Treatment with
the NADPH oxidase inhibitor DPI and the SOD inhibitor
DDC after 72 h of induction reduced both the lignification
(Fig. 1g–j) and the development of TEs, giving their sCW
a ‘looser’ appearance, and results in less intense labelling
of hipI-SOD (Fig. 6h, i).
FACS and lignin analysis of Zinnia cells
A confocal image of a newly formed TE stained with
DCFH-DA is shown in Fig. 2. The non-fluorescent DCFH
is oxidized to a highly fluorescent compound, DCF, in
the presence of ROS, especially H2O2 (Oyama et al., 1994).
Fig. 2. Confocal laser scanning microscope images of 29,79-dichlorofluorescin (DCF)-stained Zinnia mesophyll cell after 24 h (left) and TE
after 72 h (right) of differentiation in the Zinnia in vitro system. Scale
bars represent 10 lm.
A living cell is required for the DCFH-DA probe to be
deacetylated and subsequently oxidized by an appropriate
compound, e.g. H2O2 (Oyama et al., 1994). Intense green
fluorescence from the sCW structures was observed in the
developing TEs. A more diffuse fluorescence was also
observed in the vicinity of the sCW thickenings. The
undifferentiated mesophyll cells generate green fluorescence from various organelle compartments, including the
chloroplasts and the plasmalemma area (Fig. 2).
Figure 3 shows the results of FL1 (green fluorescence)
FACS analyses of a gated cell population selected for high
relative size and high relative granularity after 72 h of
induction following DCFH-DA staining (control), and
unstained cells (auto-fluorescence). The mesophyll cell
expands to approximately double the size during TE development (Fukuda and Komamine, 1980). The cell population with high green fluorescence (DCF) was shown to
belong to the population selected on the basis of size and
granularity (data not shown). A reduction in the green
fluorescence intensity from the TEs was also found
when old TEs (144 h) were stained with DCFH-DA and examined by fluorescence microscopy (data not shown). The
results from these experiments show clearly that the selected gated cell population contains developing TEs.
After approximately 72 h, when the first signs of TEformation were observed, the cell culture was exposed to
a variety of compounds. Incubation with the ROS scavengers GSH and ASA for 2 h prior to staining with DCFHDA dramatically reduced the number of cells, and the mean
fluorescence intensity of the remaining population, in
a selected range of fluorescence intensity in FL1 (labelled
M1 in Fig. 3) (Table 1). This range (M1) was selected
according to the fluorescence range obtained by the control
(Fig. 3). Addition of inhibitors of NADPH oxidase (DPI),
Fig. 3. FACS analysis of Zinnia cells. The graph shows cell counts after treatment with reagents (for 2 h) that alter the catabolism of cellular H2O2
according to the DCF fluorescence (FL1) constructed from a gated cell population selected by size and granularity after 72 h of differentiation. Autofluorescence, not stained with DCF; Control, cells stained with DCF; GSH, cells stained with DCF after treatment with 5 mM GSH; DPI, cells stained
with DCF after treatment with 10 lM DPI; M1, the fluorescence intensity range obtained by the gated control cells and used for statistical analysis of
different treatments (see Table 1).
HipI-superoxide dismutase and secondary cell wall development 2089
peroxidases (SHA), and CAT reduced the mean fluorescence intensity in the selected M1 range (Fig. 3 and Table 1).
The SOD inhibitor DDC also showed a significant reduction
in the mean fluorescence intensity of the gated population.
On the contrary, when CuZn-SOD was added to the cell
culture the mean intensity of the fluorescence increased.
To analyse the lignin development in TEs, the cell
culture was treated with various compounds after approximately 72 h differentiation as described above. After
a further 48 h incubation the cells were stained and studied
under a light microscope. Changes in the degree of lignification could be detected by phloroglucinol staining in
the cell walls of mature TEs. Ascorbate, GSH, and CAT, as
well as DPI and SHA, inhibited the lignification process of
the sCW in the TEs significantly (Fig. 4), as did the SOD
inhibitors DDC and CN. Extra SOD added to the solution
did not have any impact on the lignin content of the TEs.
If the substances were applied when the sCW started to
appear, the number of TEs was not significantly reduced by
the different treatments in comparison with the controls.
However, when cells were treated with DPI, DDC, SHA,
GSH, or ASA before the formation of the sCW (after 40–45 h
of cell culture; concentration of compounds as in Fig. 4)
the frequency of TEs that developed was low or negligible
(equivalent to 0–7% of the cell count; data not shown).
Fig. 4. Lignification of TEs analysed by phloroglucinol staining. Effects
of adding the following reagents and enzymes after 72 h of differentiation
on lignification in a Zinnia cell culture: DPI, DDC, ASA, SHA, GSH,
CN, CAT (100 units mlÿ1), and SOD (400 units mlÿ1). At least 100 TEs
were counted from each individual vial after 48 h of treatment. The values
shown are relative to phloroglucinol-stained TEs (percentage of total
TEs 6standard error; n=3–4). Statistical significance is indicated as:
*, P <0.05; **, P <0.01; ***, P <0.001.
Western blots and immunolocalization of hipI-SOD
To assess whether hipI-SOD is present in Zinnia, a polyclonal antibody obtained from Pinus sylvestris (Karpinska
et al., 2001) was used to analyse a total protein extract from
Zinnia leaves by western blotting. A single band at pI >9
was detected by IEF gel electrophoresis (Fig. 5a). The
presence of this hipI-SOD was also found in tobacco,
barley, and pea leaves. Furthermore, IEF and SDS-PAGE
analyses were performed to study the amount of the hipI–
SOD present during the differentiation of the TEs (Fig. 5b).
Table 1. FACS analysis of Zinnia cells after 72 h of
differentiation
The cells were stained with 29,79-dichlorofluorescin (DCF) after 2 h
incubation with different agents and enzymes. M1 refers to a selected
range of green fluorescence intensity (FL1) of the control cell population,
as shown in the graph in Fig. 3. The table indicates the percentage of
detected cells and the mean fluorescence intensity of the gated population
in M1.
Compound
% Cells in M1
Mean fluorescence
intensity in M1
Auto-fluorescence (see Fig. 3)
Control (see Fig. 3)
GSH (5 mM, see Fig. 3)
DPI (10 lM, see Fig. 3)
ASA (5 mM)
DDC (0.5 mM)
SHA (0.5 mM)
CAT (100 units mlÿ1)
SOD (400 units mlÿ1)
0
95
1
90
6
90
79
98
87
0
3849
799
1260
868
2508
1535
2063
4318
Fig. 5. Western blot analysis of hipI-SOD. (a) Western blot analysis of
isoelectric point of hipI-SOD from leaf protein extracts from pea, Scots
pine, tobacco, barley, and Zinnia after IEF, pH 3–9, using polyclonal
antibody from Pinus sylvestris. Six units of SOD were loaded. (b)
Western blot analysis using protein obtained from Zinnia cells after
various periods (24, 48, 60, 72, 96, and 120 h) of culture in the Zinnia in
vitro system. A 1.4 lg aliquot of protein was loaded onto IEF (pH 3–9)
and 1.1 lg onto SDS-PAGE gels (10–15%). Isoelectric point and
molecular mass in kilodaltons (kDa) are indicated to the left.
The antibody recognized a protein at pI >9, which was
only weakly expressed during the first 48 h (before any
TEs were visible), but was present at progressively higher
levels as the number of TEs in the cell culture increased
and developed (Fig. 5b). The SDS-PAGE analysis confirmed this pattern of expression and also indicated that
2090 Karlsson et al.
the antibody recognized a 16–17 kDa protein, the expected
size for a monomeric unit of CuZn-SoD (Fig. 5b).
The subcellular distribution of hipI-SOD in Zinnia
mesophyll cells during the development of TEs was investigated by immunogold electron microscopy, using a pine
hipI-SOD antibody. Pre-immune serum, cyt- and cp-SOD
antibodies were used as controls in these experiments. After
24 h, signals from the cells were very weak or undetectable
(Fig. 6a). By contrast, in cells prior to and during the first
stages of sCW formation, after 48–72 h, strong gold
labelling of hipI-SOD was observed in the Golgi apparatus
and in the plasmalemma in close vicinity to them (Fig. 6b).
By contrast, the cytoplasm, mitochondria, endoplasmatic
reticulum, and nuclei did not show significant labelling at
any time. Peroxisomes were weakly labelled after 48 h of
differentiation (data not shown). The sCW of mature TEs
after 120 h appeared electron dense, with strong labelling
of hipI-SOD (Fig. 6c, d), whereas neither hipI-SOD preimmune serum (data not shown) nor antibodies raised
against cp-SOD caused any significant accumulation of
gold particles (Fig. 6e). The labelling of cp-SOD and cytSOD was restricted to the chloroplasts (Fig. 6f) and the
cytoplasm (Fig. 6g), respectively.
Discussion
Hydrogen peroxide has been found and given important roles
during cell wall development in plants (Thordal-Christensen
et al., 1997; Ros-Barceló, 1998; Liu et al., 1999, Potikha
Fig. 6. Immunogold labelling of isolated mesophyll cells of Zinnia elegans during induced trans-differentiation into TEs. Localization of hipI-SOD in
cells 24 h (a), 72 h (b), and 120 h (c, d) after induction. Immunogold labelling of the secondary cell wall (e) and chloroplasts (f ) with antibodies raised
against cp-SOD and localization of cyt-SOD (g) of 72-h-old cells. HipI-SOD labelling of the secondary cell wall of 120-h-old Zinnia cells after inhibition
of SOD (h) and NADPH oxidase (i) by the addition of 0.5 mM DDC and 10 lM DPI, respectively, to the growth medium at day 3. Cy, Cytoplasm; D,
dictyosome; ER, endoplasmatic reticulum; M, mitochondrion; Pl, plasmalemma; pCW, primary cell wall; sCW, secondary cell wall. Scale bars represent
0.3 lm (a, b, d, e, g, h, i), 4 lm (c), and 0.1 lm (f ).
HipI-superoxide dismutase and secondary cell wall development 2091
et al., 1999; Liszkay et al., 2004). However, the mechanism of its generation and regulation is still largely
unknown. This study initially focused on H2O2 and its
possible roles in the development of the sCW. The Zinnia
cell-culture system has proved to be very useful for
studies of physiological and biochemical events during
TE differentiation, and thus also for sCW differentiation
(Sato et al., 1995; Fukuda 1996; Groover et al., 1997;
Nakashima et al., 1997; Milioni et al., 2001).
In this study, Zinnia cell cultures were labelled with the
probe DCFH-DA to monitor H2O2 when the first signs of
sCW formation were visible under the light microscope
after approximately 72 h. This resulted in green fluorescence from the sCWs in newly developed TEs (Fig. 2). The
main agent responsible for oxidation of the probe has been
shown previously to be H2O2 (Rothe, 1990; LeBel, 1992)
but it has also been criticized as a specific probe for H2O2
(Liochev and Fridovich, 2001). FACS analysis and fluorescence microscopy with DCFH-DA were also employed
to look for Zinnia cells during their differentiation into TEs
(Fig. 3 and Table 1). At the time of the sCW development,
scavenger molecules of H2O2 such as GSH and ASA were
applied to the cell culture and a dramatic reduction in the
green fluorescence intensity of the gated cell population
was observed. Application of a NADPH oxidase inhibitor
also significantly reduced the fluorescence of the gated cell
population. A plausible explanation is that inhibition of
NADPH oxidase by DPI leads to a reduction of Oÿ
2 , and
thus decreases the level of H2O2. However, since DPI
inhibits flavin oxidases it may have affected the results.
For instance, Bolwell et al. (1998) found that DPI inhibits peroxidase-mediated generation of H2O2 as well as
NAD(P)H oxidase-mediated generation of Oÿ
2 . SHA also
decreased the fluorescence intensity and may indicate the
presence of a H2O2-generating basic peroxidase in Zinnia,
as previously discussed (Pomar et al., 2002; López–
Serrano et al., 2004). Addition of SOD to the medium
increased, but the inhibition of SOD by DDC reduced the
fluorescence intensity of the cell population. However, it
has been reported that DDC will inhibit xanthine oxidase
which may also lead to a lower level of H2O2 production
(Kober et al., 2003). The reduction in the fluorescence
intensity of DCF induced by DPI and DDC is consistent
with observations made in tobacco and mammalian brain
neurons (Oyama et al., 1994; Allan and Fluhr, 1997). The
present findings point to the presence of H2O2 in the sCW
during the development of TEs in Zinnia and suggest that
NADPH oxidase, SOD, and peroxidases might all be involved in its generation. The time-course of the expression
of cDNAs coding for both a putative NADPH-like oxidase
and a peroxidase showed a burst prior to the development
of the sCW in a micro-array analysis of the trans-differentiation of Zinnia mesophyll cells (Demura et al., 2002).
Confocal laser scanning microscopy and immunolocalization studies showed that inhibition of TE development
occurred after treatment with DPI and DDC after approximately 72 h, causing the sCW to have a more loose
appearance compared with the controls (Figs 1g–j, 6h, i). If
cells were treated with DPI, DDC, SHA, GSH, or ASA after
48 h, a very low frequency of TEs developed. This indicates
that ROS are necessary for the complete development of
TEs and supports former reports about the requirement
for H2O2 in the differentiation of sCWs in cotton fibres
(Potikha et al., 1999), and also that the actual redox state of
the cell culture might be of importance for TE differentiation. Henmi et al. (2001) showed that if GSH and GSSG
were applied at an advanced state to a Zinnia cell culture
medium (after 48 h of culture) TE frequency was reduced.
The first signs of sCW formation in differentiating TEs
were accompanied by the development of lignin, as
visualized by UV-confocal microscopy (Fig. 1d). If scavengers of H2O2 such as ASA, GSH, and CAT were applied
at the time when the sCW had started to develop, the
amount of lignin in the mature TEs was reduced. Furthermore, if ROS like Oÿ
2 and H2O2 were inhibited by DPI,
SHA, and DDC, as indicated in the FACS experiments,
reductions in the lignin content of the sCW was detected.
Cyanide, which is known to inhibit SODs as well as peroxidases (Weisiger and Fridovich, 1973; Gajhede, 2001),
also reduced the lignin content. These results suggest
that H2O2 plays an important role in the lignification
process and supports earlier reports in which peroxidases
have been suggested to oxidize monolignols into radicals
(Østergaard et al., 2000), thereby initiating the polymerization into lignin.
Since both the substrate (Oÿ
2 ) and the product (H2O2) of
the enzymatic reaction determined by SOD have been
suggested here as playing a role in the development of the
sCW it encouraged the study of a novel form of SOD, hipISOD, during TE differentiation. This form of SOD was
selected due to its localization to the xylem and intercellular
spaces in pine needles (Karpinska et al., 2001) and using
this hipI-SOD antibody the corresponding protein(s) was
monitored during the developmental process of TE
formation (Fig. 5b). Levels of a protein migrating to pI 9,
on pH 3–9 IEF gels, rose simultaneously during the transdifferentiation of the mesophyll cell into TEs. A very weak
band was also detected at this position just 1 d after the start
of the culture. When gels were stained for SOD activity,
a band was detected at the same pI position as the antibody
reaction (data not shown). These results indicate that Zinnia
contains one or more hipI-SOD form(s) as also shown here
for pea, barley, and tobacco, and previously shown for
Scots pine and poplar (Karpinska et al., 2001; Schinkel
et al., 2001). In addition, it suggests having a specific
function during TE morphogenesis in Zinnia.
To acquire information on the localization of this type
of SOD, the TE developmental process was studied by immunolocalization. Prior to differentiation of TEs, 48–72 h
after start of cell culture, Golgi bodies in mesophyll cells
2092 Karlsson et al.
contained significant amounts of the enzyme label (Fig. 6b).
The label was also found in the plasmalemma area, often
close to the Golgi apparatus. This was also the case when
the first signs of sCW formation appeared, while the
vacuole was still present, supporting the conclusion that
hipI-SOD is actively secreted to the cell wall. The accumulation of the gold label to the plasmalemma and in the
sCW continued during TE development. These findings
indicate clearly that there was a developmental build-up of
the hipI-SOD protein in the sCW in the Zinnia cell system,
as was also supported by the western blot analysis. Coregulation and co-localization of the hipI-SOD with the TE
differentiation process suggest that this isoform might
participate in regulation of ROS during the development
of the sCW even though that this form of CuZn-SOD was
not identified in over 8000 cDNA clones during the transdifferentiation of Zinnia mesophyll cells (Demura et al.,
2002). However, both a putative cytosolic and a chloroplastic isoform of CuZn-SOD were identified in micro-array
analysis.
In essence, it was found that reductions in H2O2 content
influenced the developmental process and reduced lignin
formation in the sCW during the differentiation of Zinnia
TEs. Although no causal relationship was established
between SOD activity and the regulation of H2O2 or the
lignification process, the results suggest that the enzyme
might have an essential function as a regulator of ROS,
as previously discussed for spinach (Ogawa et al., 1996,
1997). Here it is reported that a ‘unique’ hipI-SOD isoform
might be one of the ROS-regulating enzymes that are
putatively involved in sCW differentiation of TEs, even if
a more ‘traditional’ protective role as a scavenger of Oÿ
2
during TE development cannot be ruled out.
Acknowledgements
We thank Dr Madoka Gray-Mitsumune for reading the manuscript
and giving valuable comments, Veronica Bourquin for technical
assistance at the CSLM, and Josephine Wernersson for technical
assistance with the Zinnia cultures. This research was funded by
grants from the Swedish Research Council for Environment,
Agricultural Sciences and Spatial Planning (Formas), the Swedish
Foundation for Strategic Research (SSF), and by a grant from the
Kempe Foundation. This work was also supported in part by Grant
436 RUS 17/94/02 from the Deutsche Forschungsgemeinschaft
(DFG).
References
Allan CA, Fluhr R. 1997. Two distinct sources of elicited reactive
oxygen species in tobacco epidermal cells. The Plant Cell 9,
1559–1572.
Auh CK, Murphy TM. 1995. Plasma membrane redox enzyme is
involved in the synthesis of superoxide and hydrogen peroxide by
Phytophthora elicitor stimulated rose cells. Plant Physiology 107,
1241–1247.
Beauchamp CO, Fridovich I. 1971. Superoxide dismutase: improved
assay and an assay applicable to acrylamide gels. Analytical
Biochemistry 44, 276–287.
Bestwick CS, Brown IR, Bennet MHR, Mansfield JW. 1997.
Localization of hydrogen peroxide accumulation during the
hypersensitive reaction of lettuce cells to Pseudomonas syringae
pv phaseolicola. The Plant Cell 9, 209–221.
Bolwell GP, Buti VS, Davies DR, Zimmerlin A. 1995. The origin
of the oxidative burst in plants. Free Radical Research 23,
517–532.
Bolwell GP, Davies DR, Gerrish C, Auh CK, Murphy TM. 1998.
Comparative biochemistry of the oxidative burst produced by rose
and French bean cells reveals two distinct mechanisms. Plant
Physiology 116, 1379–1385.
Boudet AM, Lapierre C, Grima-Pettenati J. 1995. Biochemistry
and molecular biology of lignification. New Phytologist 129,
203–236.
Demura T, Tashiro G, Horiguchi G, et al. 2002. Visualisation by
comprehensive microarray analysis of gene expression programs
during transdifferentiation of mesophyll cells into xylem cells.
Proceedings of the National Academy of Sciences, USA 99,
15794–15799.
Doke N. 1985. NADPH-dependent Oÿ
2 generation in membrane
fractions isolated from wounded potato tubers inoculated with
Phytophthora infestans. Physiological Plant Pathology 27, 311–322.
Foreman J, Demidchik V, Bothwell JHF, et al. 2003. Reactive
oxygen species produced by NADPH oxidase regulate plant cell
growth. Nature 442, 442–446.
Fukuda H. 1996. Xylogenesis: initiation, progression, and cell death.
Annual Review of Plant Physiology and Plant Molecular Biology
47, 299–325.
Fukuda H, Komamine A. 1980. Establishment of an experimental
system for the tracheary element differentiation from single cells
isolated from the mesophyll of Zinnia elegans. Plant Physiology
65, 57–60.
Gahan PB. 1984. Plant histochemistry and cytochemistry. Experimental botany. An international series of monographs. London:
Academic Press.
Gajhede M. 2001. Plant peroxidases: substrate complexes with
mechanistic implications. Biochemical Society Transactions 29,
91–99.
Goldberg R, Hule T, Catesson AM. 1985. Localisation and
properties of cell wall enzyme activities related to the final
stages of lignin biosynthesis. Journal of Experimental Botany 36,
503–510.
Groover A, DeWitt N, Heidel A, Jones A. 1997. Programmed
cell death of plant tracheary elements: differentiating in vitro.
Protoplasma 196, 197–211.
Groover A, Jones AM. 1999. Tracheary element differentiation
uses a novel mechanism coordinating programmed cell death and
sCW synthesis. Plant Physiology 119, 375–384.
Henmi K, Tsuboi S, Demura T, Fukuda H, Iwabuchi M, Ogawa K.
2001. A possible role of glutathione and glutathione disulfide in
tracheary element differentiation in the cultured mesophyll cells of
Zinnia elegans. Plant and Cell Physiology 42, 673–676.
Karpinska B, Karlsson M, Schinkel H, Streller S, Süss K-H,
Melzer M, Wingsle G. 2001. A novel superoxide dismutase with
a high isoelectric point in higher plants: expression, regulation and
protein localisation. Plant Physiology 126, 1668–1677.
Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G,
Mullineaux P. 1999. Systemic signaling and acclimation in
response to excess excitation energy in Arabidopsis. Science
284, 654–657.
HipI-superoxide dismutase and secondary cell wall development 2093
Karpinski S, Wingsle G, Olsson O, Hällgren J-E. 1992. Characterization of cDNAs encoding CuZn-superoxide dismutases in
Scots pine. Plant Molecular Biology 18, 545–555.
Kober T, König I, Weber M, Kojda G. 2003. Diethyldithiocarbamate inhibits the catalytic activity of xanthine oxidase. FEBS
Letters 551, 99–103.
Kohlenbach HW, Schmidt B. 1975. Cytodifferenzierung in Form
einer direkte Umwandlung isolierter Mesophyllzellen zu Tracheiden. Zeitschrift für Pflanzenphysiologie 7, 369–374.
Lebel CP, Isochiropoulosi H, Bondy SC. 1992. Evaluation of the
probe 29,79-dichorofluorescin as an indicator of reactive oxygen
species formation and oxidative stress. Chemical Research in
Toxicology 5, 227–231.
Liochev SI, Fridovich I. 2001. Copper, zinc superoxide dismutase
as a univalent NO-oxidoreductase and as a dichlorofluorescin
peroxidase. Journal of Biological Chemistry 276, 35253–35257.
Liszkay A, van der Zalm E, Schopfer P. 2004. Production of
reactive oxygen intermediates (Oÿ
2 , H2O2, and OH) by maize roots
and their role in wall loosening and elongation growth. Plant
Physiology 136, 3114–3123.
Liu L, Dean JFD, Friedman WE, Eriksson KEL. 1994. A laccaselike phenoloxidase is correlated with lignin biosı́ntesis in Zinnia
elegans stem tissues. The Plant Journal 6, 213–224.
Liu L, Eriksson KEL, Dean JFD. 1999. Localization of hydrogen
peroxide production in Zinnia elegans L. stems. Phytochemistry
52, 545–554.
López-Serrano M, Fernandez MD, Pomar F, Pedreno MA,
Barcelo AR. 2004. Zinnia elegans uses the same peroxidase
isoenzyme complement for cell wall lignification in both singlecell tracheary elements and xylem vessels. Journal of Experimental Botany 55, 423–431.
Marklund SL. 1985. Direct assay with potassium superoxide. In:
Greenwald R, ed. Handbook of methods for oxygen radical
research. Boca Raton, FL: CRC Press, 249–255.
Milioni D, Sado PE, Stacey NJ, Domingo C, Roberts K,
McCann MC. 2001. Differential expression of cell-wall-related
genes during the formation of tracheary elements in the Zinnia
mesophyll cell system. Plant Molecular Biology 47, 221–238.
Nakashima J, Takabe, Fujita M, Saiki H. 1997. Inhibition of
phenylalanine ammonia-lyase activity causes the depression of
lignin accumulation of secondary wall thickening in isolated
Zinnia mesophyll cells. Protoplasma 196, 99–107.
Ogawa K, Kanematsu S, Asada K. 1996. Intra and extra-cellular
localisation of ‘cytosolic’ CuZn-superoxide dismutase in spinach
leaf and hypocotyls. Plant and Cell Physiology 37, 790–799.
Ogawa K, Kanematsu S, Asada K. 1997. Generation of superoxide
anion and localisation of CuZn-superoxide dismutase in vascular
tissue of spinach hypocotyls: their association with lignification.
Plant and Cell Physiology 38, 1118–1126.
Olsen PD, Varner JE. 1993. Hydrogen peroxide and lignification.
The Plant Journal 4, 887–892.
Oyama Y, Hayashi A, Ueha T, Maekawa K. 1994. Characterization
of 29,79-dichlorofluorescin fluorescence in dissociated mammalian
brain neurons – estimation on intracellular content of hydrogenperoxide. Brain Research 635, 113–117.
Østergaard L, Teilum Kaare, Mirza O, Mattsson O, Petersen M,
Welinder KG, Mundy J, Gajhede M, Henriksen A. 2000.
Arabidopsis ATP A2 peroxidase. Expression and high-resolution
structure of a plant peroxidase with implications for lignification.
Plant Molecular Biology 44, 231–243.
Pomar F, Caballero N, Pedreno MA, Ros-Barceló A. 2002. H2O2
generation during the auto-oxidation of coniferyl alcohol drives the
oxidase activity of a highly conserved class III peroxidase involved
in lignin biosynthesis. FEBS Letters 529, 198–202.
Potikha TS, Collins CC, Johnson DI, Delmer DP, Levine A. 1999.
The involvement of hydrogen peroxide in the differentiation of
secondary walls in cotton fibers. Plant Physiology 119, 849–858.
Ros-Barceló A. 1998. The generation of H2O2 in the xylem of Zinnia
elegans is mediated by an NADPH-oxidase-like enzyme. Planta
207, 207–216.
Ros-Barceló A, Pomar F, Lópes-Serrano M, Martinez P,
Pendreno MA. 2002. Developmental regulation of the H2O2producing system and basic peroxidase isoenzyme in the Zinnia
elegans lignifying xylem. Plant Physiology and Biochemistry
40, 325–332.
Rothe G, Valet G. 1990. Flow cytrometric analysis of respiratory
burst activity in phagocytes with hydroethidine and 2979dichlorofluorescin. Journal of Leukocyte Biology 47, 440–448.
Sato Y, Sugiyama M, Komamine A, Fukuda H. 1995. Separation
and characterization of the isoenzymes of wall-bound peroxidase
from cultured zinnia cells during tracheary element differentiation.
Planta 196, 141–147.
Schinkel H, Hertzberg M, Wingsle G. 2001. A small family of
novel CuZn-superoxide dismutases with a high isoelectric point in
hybrid aspen. Planta 213, 272–279.
Schopfer P. 1994. Histochemical demonstration and localisation of
hydrogen peroxide in organs of higher plants by tissue printing
on nitrocellulose paper. Plant Physiology 104, 1269–1275.
Shapiro HM. 1994. Practical flow cytometry, 3rd edn. New York:
Wiley Liss.
Sherwood RT, Vance CP. 1976. Histochemistry of papillae formed
in reed canarygrass leaves in response to noninfecting pathogenic
fungi. Phytopathology 66, 503–510.
Teige M, Melzer M, Süss K-H. 1998. Purification, properties and
in situ localization of the amphibolic enzymes D-ribulose-5phosphate 3-epimerase and transketolase from spinach chloroplasts. European Journal of Biochemistry 252, 237–244.
Thordal-Christensen H, Zhang ZG, Wei YD, Collinge DB. 1997.
Subcellular localization of H2O2 in plants. H2O2 accumulation in
papillae and hypersensitive response during the barley–powdery
mildew interaction. The Plant Journal 11, 1187–1194.
Whetten RW, Mackay JJ, Sederoff RR. 1998. Recent advances in
understanding lignin biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 49, 585–609.
Weisiger RA, Fridovich I. 1973. Superoxide dismutase. Organelle
specificity. Journal of Biological Chemistry 248, 3582–3592.