Tree Physiology 31, 102–113
doi:10.1093/treephys/tpq105
Research paper
Oxidative stress responses and root lignification induced by Fe
deficiency conditions in pear and quince genotypes
Silvia Donnini1, Marta Dell’Orto and Graziano Zocchi
Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy; 1Corresponding author ([email protected])
Received June 4, 2010; accepted October 31, 2010; handling Editor Roberto Tognetti
We analysed Pyrus communis cv. Conference and Cydonia oblonga BA29, differently tolerant to lime-induced chlorosis, to
identify the key mechanisms involved in their different performance under Fe deficiency induced by the absence of Fe (−Fe)
or by the presence of bicarbonate (+FeBic). Under our experimental conditions, a decrease in root elongation was observed
in BA29 under bicarbonate supply. Superoxide dismutase (SOD) and peroxidase (POD) activities were analysed and the relative isoforms were detected by native electrophoresis. The data obtained for both genotypes under −Fe and for BA29 +FeBic
suggest the occurrence of overproduction of reactive oxygen species (ROS) and, at the same time, of a scarce capacity to
detoxify them. The detection of ROS (O2− and H2O2) through histochemical localization supports these results and suggests
that they could account for the modifications of mechanical properties of the cell wall during stress adaptation. On the other
hand, in the cv. Conference +FeBic, an increase in non-specific POD activity was detected, confirming its higher level of protection in particular against H2O2 accumulation. Peroxidases involved in lignification were assayed and histochemical analysis
was performed. The results suggest that only in BA29 under bicarbonate supply can the presence of ROS in root apoplast be
correlated with lignin deposits in external layers and in endodermis as a consequence of the shift of PODs towards a lignification role. We suggest that in BA29 the decrease in root growth could impair mineral nutrition, generating susceptibility to
calcareous soils. In the cv. Conference, the allocation of new biomass to the root system could improve soil exploration and
consequently Fe uptake.
Keywords: bicarbonate, chlorosis, Cydonia oblonga, Pyrus communis.
Introduction
Soils with a relatively high lime concentration are present in
about one-third of the earth’s surface (Chen and Barak 1982).
This can be a matter of concern for the development of several
orchards and vineyards, in which plants must face Fe-deficiencyinduced chlorosis, resulting in a decrease in productivity
(Tagliavini and Rombolà 2001). However, different varieties of
a given plant species may differ appreciably in their sensitivity
to calcareous soils (Korcak 1987).
Adverse environmental conditions are reported to induce
oxidative stress in plants as a consequence of reactive oxygen
species (ROS) production (Foyer et al. 1997). However, relatively little information is available on the relationships between
Fe deficiency and secondary oxidative stress, and some data
remain controversial (Iturbe-Ormaexte et al. 1995, Ranieri et al.
2001, Lombardi et al. 2003, Chouliaras et al. 2004, Molassiotis
et al. 2005). Iron is either a constituent or a cofactor of many
antioxidant enzymes and, at the same time, can act as a prooxidant through the Fenton reaction (Halliwell and Gutteridge
1984). In its role as an enzyme ­constituent, Fe is part of
­catalase (CAT, EC 1.11.1.6), non-specific peroxidases (POD,
EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) and Fe
superoxide dismutase (Fe-SOD, EC 1.15.1.1).
Superoxide radical (O2−) is produced at any location where
an electron transfer is present and thus in every compartment
of the cell. Superoxide dismutases, converting O2− to H2O2,
constitute the first line of defence against ROS in different plant
species under several stress conditions (Alscher et al. 2002,
© The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Oxidative stress responses in pear and quince 103
Blokhina et al. 2003). In particular, studies on plants grown in
Fe-deprived conditions showed high SOD activity, mainly due
to increased Cu/Zn or Mn-SOD isoforms (Iturbe-Ormaexte
et al. 1995, Molassiotis et al. 2006).
In addition to SOD activity, the intracellular level of H2O2 is
controlled by other enzymes, the most important being CAT
and PODs. Peroxidases, by means of their hydroxylic or peroxidative activity, can regulate both ROS production and scavenging in numerous cell compartments and thus they can be
involved in many plant processes, such as growth and biotic/
abiotic stress responses (Passardi et al. 2005). In the cell wall,
PODs are present as soluble, ionically and covalently bound
forms and they can catalyse cross-linking reactions, building a
rigid cell wall, or produce ROS-generating wall-loosening reactions, to make it more flexible (Lewis and Yamamoto 1990,
Polle et al. 1994, Schweikert et al. 2000). Additionally, PODs
are enzymes directly involved in lignin biosynthesis by assembling lignin units through oxidative polymerization (Lewis and
Yamamoto 1990). Several works have well documented that
some growth conditions are responsible for the increase in
cell-wall lignification which, by reducing cell growth, may represent a plant’s adaptation to adverse conditions (Jbir et al.
2001, Lee et al. 2007). With regard to plants grown under Fe
deficiency, POD isoforms are differently affected. Ranieri et al.
(2001) showed, in Fe-deficient sunflower, a preferential reduction in the activity of those isoforms involved in H2O2 detoxification, rather than in the maintenance of cell-wall structure.
According to Molassiotis et al. (2006), increased POD activity
may be an important attribute linked to chlorosis tolerance in
peach rootstocks.
Approximately 80% of pear cultivars are budded on pear
seedlings; the rest are grafted on quince (Cydonia oblonga)
rootstock, which induces low vigour of the scion (Kobelus et al.
2007). However, pear cultivars often show symptoms of Fe
chlorosis when grafted on quince, above all when grown on
calcareous soils (Korcak 1987).
In the present work, we focused our attention on pear
(cv. Conference) and quince (clone BA29) genotypes characterized by different levels of tolerance to lime-induced chlorosis. In a previous paper (Donnini et al. 2008), we saw evidence
only in cv. Conference (tolerant to the presence of bicarbonate), a strategy of adaptation characterized by a decrease in
shoot development that could be finalized to maintain an adequate level of Fe in leaf tissues as a concentration effect.
Furthermore, the same cultivar grown in the presence of bicarbonate showed photosynthetic activity similar to that of control
plants (Donnini et al. 2009). While in control growth conditions, the photosynthetic products are basically destined for
the formation of new tissues, in the bicarbonate supply, these
compounds could essentially be targeted to the roots to ­sustain
enzymatic activities involved in Fe uptake, which are energy
consuming (Vigani and Zocchi 2009).
In this study, we hypothesize that antioxidant enzymes,
which are involved in stress adaptation/tolerance, are activated
to different degrees in relation to different chlorosis tolerance.
To test this hypothesis, the possibility that Fe deficiency could
generate oxidative stress at root level was investigated by
checking changes in the activity of some enzymes involved in
ROS production/detoxification, as well as the presence of O2−
and H2O2 at root level. Moreover, by using a specific substrate,
syringaldazine, and performing histochemical analysis with
safranin/fast green, root lignification was detected in both
­genotypes to verify whether the difference in the degree of
lignification could be related to the different adaptations to
such adverse conditions.
Materials and methods
Plant material and culture
One-year-old plants with three primary shoots propagated by
microshoots (Pyrus communis L. cv. Conference) or cuttings
(Cydonia oblonga Mill. clone BA29) were grown in a ­hydroponic
medium containing (mM) 4 Ca(NO3)2, 4 KNO3, 1.6 MgSO4,
0.8 KH2PO4 and (µM) 37 H3BO3, 7.3 MnCl2 ⋅ 4H2O, 0.23
CuCl2 ⋅ 2H2O, 0.64 ZnSO4, 0.08 (NH4)6Mo7O24 with either
80 µM Fe(III)Na-ethylenediaminetetracetic acid (EDTA) (+Fe,
control) or without Fe (−Fe). Iron shortage was also indirectly
achieved by adding 10 mM KHCO3 to +Fe medium (+FeBic).
Except for the last treatment (pH 8.30), the initial pH was
adjusted to 6.00–6.20 with NaOH. Plants were grown for 21
days under different treatments and the medium was changed
weekly. Plants were maintained in a growth chamber with a
day/night regime of 16/8 h and with a photosynthetic photon
density flux of 200 µmol m−2 s−1. The temperature was 22 °C
in the dark with relative humidity (RH) 60% and 26 °C in the
light with an RH of 80%.
Total chlorophyll determination
Leaf chlorophyll concentration was determined according to
Lichtenthaler (1987). Leaf samples were homogenized with
100% acetone, in the presence of Na-ascorbate to prevent
pigment oxidation. Extracts were filtered through Minisart
SRP15 0.2 µm filters (Sartourius, Goettingen, Germany) and
absorbance was determined at 644.8 and 661.6 nm with a
Cary 50 Bio UV–visible spectrophotometer (Varian, Walnut
Creek, CA, USA).
Root growth determination
Root elongation (in cm) was calculated as the difference
between root length at the end of treatment (Day 21) and root
length at the starting point (Day 0). Reference roots for growth
measurement were marked with tape. Six biological replicates
for each genotype and treatment were performed.
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104 Donnini et al.
SOD extraction and activity assay
Root samples were ground in liquid N2 with 10% (w/w) polyvinylpolypyrrolidone (PVPP) and homogenized in a medium containing: 50 mM K-phosphate buffer (pH 7.00), 1 mM EDTA,
0.05% (v/v) Triton X-100, 1 mM dithiothreitol (DTT), 1 mM
Na-ascorbate and 0.50 mM phenylmethylsulphonyl fluoride
(PMSF). After centrifugation at 12,000g for 30 min at 4 °C, the
supernatant was collected and dialysed overnight at 4 °C against
1:50 (v/v) diluted buffer.
Superoxide dismutase activity was assayed according to the
method of Scebba et al. (2003) at 560 nm, based on the
enzyme’s ability to inhibit the photoreduction of nitrobluetetrazolium (NBT). One enzyme unit was defined as the amount of
enzyme inhibiting 50% of NBT photoreduction.
Native PAGE and SOD isoform visualization
Superoxide dismutase isoforms were separated by 12.5%
native polyacrylamide gel electrophoresis (PAGE) at 100 V
using the method described by Beuchamp and Fridovich
(1971). Pre-treatment of the gels with 5 mM H2O2 and 3 mM
KCN before SOD staining allowed us to characterize SOD isoforms as Cu/Zn-SOD, Fe-SOD or Mn-SOD. Mn-SOD is resistant
to both inhibitors, Fe-SOD is resistant to KCN and inhibited by
H2O2, and Cu/Zn-SOD is inhibited by both inhibitors.
POD and APX extraction and activity assay
To test POD activity, roots were homogenized in 100 mM
Tricine–KOH buffer (pH 8.00), 20 mM MgCl2, 50 mM KCl,
10 mM EDTA, 1‰ (v/v) Triton X-100, 1 mM DTT 0.50 mM
PMSF and 10% (w/w) PVPP. For APX extraction, 50 mM
ascorbic acid (AA) was added to the buffer to maintain the
enzyme active during the extraction procedure. After centrifugation at 23,700g for 30 min at 4 °C, the supernatant was
collected and dialysed overnight against 2.5 mM Tricine–KOH
(pH 8.00). Ascorbic acid was added to the buffer during the
dialysis of APX extracts according to the method of Ranieri
et al. (1996).
Non-specific POD activity was assayed by using o-dianisidine as a substrate (Ranieri et al. 1997). To inactivate APX
activity, 50 mM p-chloromercuribenzoate (pCMB), a specific
APX inhibitor, was added to the reaction mixture (Miyake and
Asada 1992).
Ascorbate peroxidase activity was determined by following
the decrease in absorbance at 290 nm due to the oxidation of
AA, using an extinction coefficient of 2.8 mM−1 cm−1 for ascorbate (Ranieri et al. 1996).
Native PAGE and non-specific POD isoenzyme
determination
Native PAGE was performed through a 7.5% polyacrylamide
gel according to Laemmli (1970). Peroxidase active bands
were revealed by immersing the gels in a solution consisting of
Tree Physiology Volume 31, 2011
100 mM Na-phosphate buffer (pH 6.50), 5.55 mM H2O2 and
0.1 % o-dianisidine until all the POD bands were visible.
Separation of soluble, ionically and covalently bound
POD and syringaldazine–POD determination
The separation of soluble, ionically and covalently bound fractions extracted from root tissues was performed as reported
by Ranieri et al. (1995). Freshly harvested roots were homogenized (4 °C) with 66 mM Na-phosphate buffer (pH 6.10) and
then centrifuged at 800g for 5 min. The supernatant was again
centrifuged at 10,000g for 5 min, and the second supernatant
was considered the soluble fraction. The first pellet was washed
twice with phosphate buffer, twice with distilled water and,
after shaking for 1 h at 4 °C in 2% (v/v) Triton-X100 in water,
it was rinsed five times with distilled water. After centrifugation
(2,000g for 5 min), the pellet was treated with 1 M CaCl2 for
1 h and centrifuged at 800 g for 10 min at 4 °C. The supernatant was the ionically bound fraction, while the pellet was
washed five times with distilled water and incubated overnight
at room temperature with 0.3% cellulase, 0.3% macerase and
0.3% cellulolysin in 50 mM Na-acetate buffer (pH 5.50) to
obtain, after centrifugation (800g for 10 min), the covalently
bound fraction.
Peroxidase activity was assayed in the three fractions by
using syringaldazine, a specific substrate analogue of lignin
monomer used to test the role of POD in the lignification process (Ranieri et al. 2001, Lee et al. 2007).
The activity of syr-POD was determined by measuring the
increase in absorbance at 530 nm of the reaction mixture containing 200 mM Na-K phosphate buffer (pH 6.00), 2 mM
syringaldazine, 2.5 mM H2O2 and the protein extract (Pandolfini
et al. 1992).
Histochemical detection of H2O2 and O2−
The histochemical detection of H2O2 and O2− in root tissues
was performed as described by Hernandez et al. (2001) with
some modifications. For H2O2 determination, roots were vacuum infiltrated with 0.1 mg ml−1 3,3′-diaminobenzidine (DAB)
in 50 mM Tris-acetate buffer (pH 5.00) and then incubated in
the dark at room temperature for 24 h. For O2− determination,
roots were vacuum infiltrated with 0.1 mg ml−1 NBT in 25 mM
HEPES buffer (pH 7.60) and incubated in the dark at room
temperature for 2 h. Controls of H2O2 and O2− (data not shown)
were performed by adding to the infiltration buffer 10 mM AA
and 10 mM MnCl2, respectively, highly effective inhibitors of
the two reactive species (Hernandez et al. 2001). Freshly cut
root sections were observed by optical microscope (Leica
DMR) and acquired with a Leica DC300F Digital Camera.
Lignin visualization in root tissues
Root segments were treated according to Dell’Orto et al.
(2002) with some modifications. After fixing at 4 °C overnight
Oxidative stress responses in pear and quince 105
in 100 mM Na-phosphate buffer (pH 7.00) containing 4% para­
formaldehyde (w/v), root segments were dehydrated through
an ethanol–tertiary butanol series and embedded in paraffin
(Paraplast Plus, Sigma). Serial sections of 5 µm were cut with
a microtome, mounted on silanized slides, deparaffinized in
xylene and rehydrated thorough an ethanol series. Sections
were then stained with the safranin/fast green method according to Johansen (1940), mounted with cover slides, observed
by Optical Microscope (Leica DMR) and acquired by Leica
DC300F Digital Camera.
Protein determination
Protein content was determined by the Bradford (1976) procedure using the Bio-Rad reagent and bovine serum albumin as a
protein standard.
Table 1. Total chlorophyll content of 21-day-old leaves of cv. Conference
and BA29 grown in different conditions.
Total chlorophyll
content (μmol g−1 FW)
Cv. Conference
+Fe
−Fe
+FeBic
Treatment
Genotype
Treat × Gen
1.71 ± 0.23a
0.25 ± 0.04d
1.13 ± 0.17b
***
***
*
BA29
1.88 ± 0.36a
0.22 ± 0.03d
0.63 ± 0.06c
Data are means ± SD (n = 5). For each parameter the significance of
the F-ratio following two-way ANOVA is shown. In the case of significant interaction between treatment and genotype, values followed by
different letters are statistically different (*P < 0.05; **P < 0.01;
***P < 0.001; n.s. = not significant).
Statistical analysis
Root growth determination
All data were subjected to two-way ANOVA (treatment × genotype), using Tukey’s test at P < 0.05. The statistical analysis
was conducted with Sigma-Stat® 3.1.
With the aim of verifying the effect of the different treatments on
the growth of the two species, the length of young not lignified
roots was determined. The results put in evidence a different
behaviour between the two species (Figure 2). In fact, while no
significant change was observed in cv. Conference roots, a dramatic decrease in root elongation was recorded in BA29 under
bicarbonate treatment (−88%, with respect to the control).
Results
Leaf chlorosis symptoms and total chlorophyll content
After growing plants under different treatments (+Fe, −Fe
and +FeBic), we observed a high correlation between tolerance to Fe chlorosis reported under field conditions and
symptoms of Fe deficiency. In fact, while the absence of Fe
caused chlorosis in both genotypes (Figure 1b and e), the
presence of bicarbonate caused leaf symptoms only in BA29
(Figure 1f).
Table 1 shows the two-way ANOVA of leaf chlorophyll content expressed on a fresh weight basis. The lack of Fe and the
bicarbonate supply conditions resulted in a significant decrease
in chlorophyll content in both genotypes. However, the cv.
Conference seemed less affected than BA29 when bicarbonate was added to the medium.
Superoxide dismutase activity assay and isoform
visualization
Superoxide dismutase activity under +Fe treatment was ~4-fold
higher in BA29 roots than in the cv. Conference (Figure 3a).
In both genotypes, total SOD activity was strongly increased
in the absence of Fe, whereas no appreciable variation was
detected in the presence of bicarbonate. In more detail, in
comparison with the relative controls (+Fe), cv. Conference and
BA29 plants grown in the absence of Fe exhibited a 196 and
284% increase, respectively.
To identify a possible change in the SOD isoform pattern
induced by stress conditions, non-denaturing PAGE was
Figure 1. Representative pictures of 21-day-old leaves of P. communis L. cv. Conference (a–c) and C. oblonga Mill. (d–f) grown in different conditions: control (a, d), absence of Fe (b, e) and +FeBic treatment (c, f).
Tree Physiology Online at http://www.treephys.oxfordjournals.org
106 Donnini et al.
Figure 2. Change in root elongation (in cm) of the cv. Conference (left)
and BA29 (right) grown for 21 days in control conditions (+Fe), in the
absence of iron (−Fe) and in the presence of bicarbonate (+FeBic).
Data are given as means ± SD (the experiment was performed measuring three roots per six different plants for each treatment, n = 18).
For each parameter, the significance of the F-ratio following two-way
ANOVA is shown. In the case of significant interaction between treatment and genotype, values followed by different letters are statistically
different (*P < 0.05; **P < 0.01; ***P < 0.001; n.s. = not significant).
­ erformed. Loading of samples on the gel was performed by
p
keeping the amount of protein constant between treatments
for each genotype, but different between genotypes because
of their different activity. As shown in Figure 3b, a single SOD
isoform was detected in the control roots of the cv. Conference
(lane 1). Upon addition of KCN and H2O2 as inhibitors, this
isoform was identified as a Cu/Zn-SOD (data not shown).
A similar isoform was also detected in −Fe-treated roots; in this
condition, an additional isoform at lower molecular weight was
detected and identified as Mn-SOD (lane 2). In contrast, in
plants grown in bicarbonate, these isoforms were not active
and a new faintly stained Mn-SOD isoform was expressed
(lane 3). Concerning BA29 (Figure 3c), three different bands
were detected only in roots grown in the absence of Fe, one
corresponding to Cu/Zn-SOD and two corresponding to
Mn-SOD. No isoforms were revealed in the other treatments.
Anyway, loading the gel with a higher amount of proteins
revealed the same three isoforms for all conditions, but led to
an overcharge of the −Fe sample (data not shown).
Peroxidase activity assay and isoform visualization
Non-specific POD activity was measured using o-dianisidine as
a substrate (Ranieri et al. 1997). The activity under +Fe treatment was similar in both genotypes; however, a different trend
was observed under both stress conditions (Figure 4a). The
absence of Fe induced in the cv. Conference a decrease in
POD activity (−50%, compared with the control), whereas in
BA29 it increased by ~31%. Conversely, the presence of
­bicarbonate in the growth medium induced a strong increase
Tree Physiology Volume 31, 2011
Figure 3. (a) Total SOD activity (U mg prot−1) in cv. Conference (left)
and BA29 (right) roots grown for 21 days in control conditions (+Fe),
in the absence of iron (−Fe) and in the presence of bicarbonate
(+FeBic). One enzyme unit was defined as the amount of enzyme
inhibiting 50% of NBT photoreduction. Data are given as means ± SD
(n = 5). For each parameter, the significance of the F-ratio following
two-way ANOVA is shown. In the case of significant interaction
between treatment and genotype, values followed by different letters
are statistically different (*P < 0.05; **P < 0.01; ***P < 0.001; n.s. = not
significant). Native PAGE of SOD isoforms for cv. Conference (b) and
BA29 (c) roots. +Fe (lane 1), −Fe (lane 2) and +FeBic (lane 3). An
amount of total proteins of 15 and 10 µg for cv. Conference and BA29,
respectively, was applied.
(+74%) in the cv. Conference, while in BA29 a decrease of
~40% was observed in response to this treatment.
The separation of POD isoforms by non-denaturing PAGE
revealed a species-specific difference: in the cv. Conference
grown in the presence of Fe, three bands were detected
(Figure 4b, lane 1), while in BA29 five bands appeared, two
with high and three with low molecular weight (Figure 4c,
lane 1). In the cv. Conference, only the bicarbonate treatment
induced a generalized increase in the band staining intensity
(Figure 4b, lane 3). Conversely, in the BA29 roots grown under
–Fe treatment, enhanced intensity of POD isoforms occurred
(Figure 4c, lane 2), while under bicarbonate treatment a faint
staining of the bands at low molecular weight and the loss
of those at high molecular weight was observed (Figure 4c,
lane 3).
Concerning APX activity (Figure 5), the absence of Fe
induced similar changes in the two species, even if in BA29
the decrease was stronger than in the cv. Conference (−64
and −55%, respectively). On the contrary, the presence of
bicarbonate in the growth medium induced a significant
decrease (−56%) only in BA29. Concerning APX activity under
+Fe condition, in the cv. Conference roots it was approximately
fivefold higher than in BA29.
Oxidative stress responses in pear and quince 107
Figure 4. (a) Peroxidase activity (μmol mg prot−1 min−1) assayed by
using o-dianisine as substrate in cv. Conference (left) and BA29 (right)
roots grown for 21 days in control conditions (+Fe), in the absence of
iron (−Fe) and in the presence of bicarbonate (+FeBic). Data are
given as means ± SD (n = 5). For each parameter, the significance of
the F-ratio following two-way ANOVA is shown. In the case of significant interaction between treatment and genotype, values followed by
­different letters are statistically different (*P < 0.05; **P < 0.01;
***P < 0.001; n.s. = not significant). Native PAGE for POD isoforms of
cv. Conference (b) and BA29 (c) roots. +Fe (lane 1), −Fe (lane 2) and
+FeBic (lane 3). An amount of 10 µg of total proteins for each sample
was applied.
Figure 5. Ascorbic POD activity (μmol mg prot−1 min−1) in cv.
Conference (left) and BA29 (right) roots grown for 21 days in control
conditions (+Fe), in the absence of iron (−Fe) and in the presence of
bicarbonate (+FeBic). For each parameter, the significance of the
F-ratio following two-way ANOVA is shown. Data are given as
means ± SD (n = 5). In the case of significant interaction between
treatment and genotype, values followed by different letters are
­statistically different (*P < 0.05; **P < 0.01; ***P < 0.001; n.s. = not
significant).
Table 2. Peroxidase activity of the soluble, ionically and covalently
bound cell-wall fractions of cv. Conference and BA29 roots, measured
using syringaldazine as electron donor.
Treatment
Soluble
fraction
(Δabs min−1 mg−1)
Syr-POD determination
When the soluble, ionically and covalently bound cell-wall
­fractions were tested for syr-POD activity, a different trend
was determined in the tolerant and susceptible genotypes
(Table 2).
The activity of syr-POD determined in the soluble fraction
increased in both genotypes grown in the presence of bicarbonate although to different extents: while in the cv. Conference
an increase of 84% was observed in comparison with the control, in BA29 the increase was dramatically higher (+970%).
In the ionically bound fraction, syr-POD activity was increased
only in BA29 roots grown in the presence of bicarbonate,
whereas a decrease was observed in response to the absence
of Fe. On the other hand, no significant differences were
observed in the same fraction of cv. Conference roots. In contrast to what occurred in the soluble and ionically bound fractions, in the covalently bound ones, no significant effects of the
different treatments were detected in either genotype.
Histochemical detection of O2− and H2O2
O2−
Since
is often the first reduced form of oxygen to be generated in plant tissues, we analysed its distribution by treating
roots with NBT, which forms a dark blue formazan precipitate
Activity syr-POD
cv. Conference
​ ​ + Fe
​ ​ − Fe
​ ​ + FeBic
BA29
​ ​ + Fe
​ ​ − Fe
+FeBic
Treatment
Genotype
Treat × Gen
Ionically
bound fraction
(Δabs min−1 mg−1)
Covalently
bound fraction
(Δabs min−1 g −1 dW)
0.84 ± 0.21c
0.69 ± 0.12c
5.27 ± 0.42b
8.33 ± 2.31c
7.10 ± 2.02c
8.01 ± 2.84c
29.07 ± 3.11
30.51 ± 2.80
28.90 ± 3.18
1.50 ± 0.24c
1.95 ± 0.17c
16.12 ± 1.04a
**
*
*
25.66 ± 3.12b
8.25 ± 2.17c
69.23 ± 4.63a
*
*
*
31.71 ± 2.50
31.25 ± 1.91
38.15 ± 6.83
n.s.
n.s.
n.s.
Syringaldazine POD activity is expressed as ΔAbs530 min−1 mg−1 protein, except for the covalently bound fraction, whose activity is reported
as ΔAbs530 min−1 g−1 DW of residual cell-wall material. Data are
means ± SD (n = 5). For each parameter, the significance of the F-ratio
following two-way ANOVA is shown. In the case of significant interaction between treatment and genotype, values followed by different letters are statistically different (*P < 0.05; **P < 0.01; ***P < 0.001;
n.s. = not significant).
in contact with O2− (Bielski et al. 1980). In whole roots of both
genotypes grown under Fe deficiency, this colour appeared
intense in the meristematic and elongation zones, whereas it
was weaker in the maturation zone (data not shown). The
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108 Donnini et al.
observation of root cross-sections revealed the presence of
dark blue precipitate in the stele and in the cortex apoplast of
both species under all growth conditions (Figure 6). On the
other hand, no staining was observed in the rhizodermis of
­differently treated roots. However, in BA29 –Fe and +FeBic
(Figure 6E and F) and in Conference –Fe (Figure 6B) the blue
stain in the cortex apoplast was more evident with respect to
the controls; in BA29 –Fe (Figure 6E) it was also present in
the cortex cytosol.
The production of H2O2 was revealed after infiltration of the
roots with DAB, which reacts with H2O2 in the presence of
endogenous PODs to produce a brown polymerization product. Incubation of intact Fe-deficient roots showed a distinct
pattern of brown colour. The cells belonging to the region
encompassing the end of the elongation zone and the maturation zone appeared brown, but this colour was stronger in the
elongation and meristematic zones (data not shown). Crosssections of primary lateral roots of both genotypes grown without Fe and of BA29 exposed to bicarbonate showed a brown
colour in the cortex and, to a greater extent, in the rhizodermis,
endodermis and stele (Figure 7b, e and f). Conversely, in the
control roots weaker staining was ­evident in the endodermis, in
Figure 6. Histochemical localization of O2− in root sections of cv. Conference (a–c) and BA29 (d–f) plants grown in the presence of Fe (a, d), in
the absence of Fe (b, e) or in the presence of bicarbonate (c, f). O2− distribution is visualized in the presence of NBT, which forms a dark blue
precipitate (formazan) in contact with superoxide anion.
Tree Physiology Volume 31, 2011
Oxidative stress responses in pear and quince 109
Figure 7. Histochemical localization of H2O2 in root sections of cv. Conference (a–c) and BA29 (d–f) plants grown in the presence of Fe (a, d), in
the absence of Fe (b, e) or in the presence of bicarbonate (c, f). H2O2 distribution is visualized in the presence of DAB, which forms a dark precipitate when reacting with H2O2 in the presence of endogenous PODs.
Figure 8. Root sections of cv. Conference (a–c) and BA29 (d–f) plants grown in the presence of Fe (a, d), in the absence of Fe (b, e) or in the
presence of bicarbonate (c, f). Lignified cell walls are stained red.
the rhizodermis and in the whole stele, while cortex cells were
stained only in the apoplast (Figure 7A and D). The distribution
of H2O2 followed a similar pattern in cv. Conference roots
grown under bicarbonate except for the stele, which appeared
completely brown (Figure 7c).
The production of O2− and H2O2 was totally suppressed by
using MnCl2 and AA, respectively, indicating the specificity of
the DAB/NBT staining applied (data not shown).
Histochemical visualization of lignin
Microscopic analysis of root sections after safranin/fast green
staining allowed lignified cell walls to be visualized as red spots.
A significant presence of red stain, consistent with intense lignification, was visible in root sections of BA29 grown in the presence of bicarbonate (Figure 8f); the red colour is clearly localized
in the stele and in the endodermis, which appears totally ­lignified.
Red-coloured cell walls were also detected in the sub-­epidermis
Tree Physiology Online at http://www.treephys.oxfordjournals.org
110 Donnini et al.
zone and in the cortex of the same sample. Red spots, especially localized in the ­endodermis zone, were also present in
BA29 roots grown in the absence of Fe (Figure 8e). In the other
treatments (Figure 8a–d), sporadic spots, particularly localized
in the sub-epidermis zone, were present.
Discussion
Under our experimental conditions, the behaviour of the two
genotypes was primarily evident by the different induction of
Fe chlorosis symptoms (Figure 1). The tolerant cv. Conference
showed visual symptoms only in the absence of Fe; on the contrary, in the susceptible BA29, chlorosis was evident in
the young leaves growing under both stress conditions. In
agreement with the symptoms shown by the plants, the chlorophyll content was more affected in quince than in the cv.
Conference by the presence of bicarbonate (Table 1). In addition, Figure 2 shows that this growth condition caused a significant difference also in root elongation, which was negatively
affected only in BA29. We suggest that a better growth performance of cv. Conference under both Fe-stress conditions could
reside in specific responses to improve Fe acquisition. In the
same pear cultivar, our previous results (Donnini et al. 2008)
suggested the presence, at the shoot level, of an adaptive strategy to calcareous soils consisting in a lower branching capacity
and in a minor elongation of the main stem that could help in
maintaining an adequate level of Fe in the same tissues, avoiding its allocation to the new outgrowths of lateral shoots. Taken
together, these results suggest the presence, in the cv.
Conference, of a combined effect of limited canopy growth and
compensation in new root tissue formation, which could reduce
sink competition for photosynthates and then optimize plant
survival under bicarbonate-induced stress (Marschner 1995).
In fact, since Fe is scarcely mobile in calcareous soils, the ability to maintain ample soil volume explored by roots would confer essential benefits. On the other hand, BA29 is affected in a
different way by the presence of bicarbonate since it shows a
strong depression at root growth level (Donnini et al. 2008).
The mechanisms involved in the growth of plant organs are
very complex. Among these, factors inducing changes in cellwall plasticity have been described (Hoson 1993, Cosgrove
1999). Recently, the possibility based on the idea that cell-wall
modifications underlying organ growth are affected by ROS
production has received support by several reports (Schopfer
2001, Liszkay et al. 2004, Passardi et al. 2005). It has been
suggested that the genesis of ROS in the apoplast can drive the
oxidative cross-linking of cell-wall components such as lignin
precursors (Boudet 2000), consequently stopping cell elongation. On the other hand, wall-loosening reactions involved in
root elongation are also directly linked to the action of ROS
(Schopfer 2001, Liszkay et al. 2003). The hydroxyl radical
(OH·) produced in the ­apoplastic space of plant tissues, for
Tree Physiology Volume 31, 2011
instance, is capable of ­splitting covalent bonds of wall polymers (Fry 1998). Since the chemical determination of OH· has
rarely been attempted because of its extremely short lifetime
(Kuchitsu et al. 1995, Liszkay et al. 2004), we tested, in the
apoplast of root tissues, the presence of H2O2 and O2−, directly
involved in both OH· generation (Schopfer 2001) and cell-wall
plasticity (Halliwell 1978).
The histochemical method we have adopted, though not
quantitative, has been chosen since it is generally considered
to be one of the most conservative and sensitive methods for
plant analysis, being based on a rapid reaction directly at the
site of ROS generation (Fryer et al. 2002, Romero-Puertas
et al. 2004). Staining of freshly cut root cross-sections in the
meristematic, elongation and maturation zones extending for
the first 4–5 mm from the tip showed that O2− accumulation
preferentially occurs in roots of both genotypes grown in the
absence of Fe and in BA29 also in the presence of bicarbonate. In particular, O2− increase is mainly located in the apoplast
of stele and cortex (Figure 6). Similar results were obtained
using DAB as a probe for H2O2 in the presence of endogenous
PODs, although in this case the staining was present in the
rhizodermis, in the stele and, to a lesser extent, in the cortex
walls (Figure 7). Thus, the information collected by histochemical analysis might suggest that O2− and H2O2 accumulation
occurs in root tissues of all chlorotic plants, accounting for the
modifications of mechanical properties of the cell wall during
stress adaptation.
Reactive oxygen species accumulation detected in BA29
(both treatments) and cv. Conference grown in the absence of
Fe could be linked to an imbalance in the antioxidant machinery. Moreover, in cv. Conference +FeBic, the lack of ROS could
be due either to their limited synthesis or to their rapid degradation. To test this hypothesis, we have analysed the main
enzymes involved in ROS metabolism. Among these activities,
the SODs are involved in the responses to Fe deficiency stress,
catalysing the dismutation into H2O2 of O2−, formed to a high
extent under this condition (Ranieri et al. 1999, Molassiotis
et al. 2005). To determine whether differences between the
individual SOD isoforms were present, we have also determined SOD activity by using non-denaturating gel staining
(Figure 3a–c). Total SOD activity was induced in −Fe roots of
both genotypes, associated with Cu/Zn-SOD and Mn-SOD isoforms. However, SOD induction appears not to be sufficient to
detoxify O2−, which seems to be accumulated in the root tissues (Figure 6). In BA29 under +FeBic treatment, no increase
in total SOD activity was detected, suggesting that (i) the presence of bicarbonate could induce, in the susceptible genotype,
a loss of O2− detoxification activity, (ii) the increase in H2O2
(Figure 7) cannot be attributed to increased SOD activity. The
H2O2 overproduction could be directly triggered by the rise in
other activities, such as NAD(P)H oxidases (Romero-Puertas
et al. 2004), pH-dependent PODs (Bestwick et al. 1998,
Oxidative stress responses in pear and quince 111
Bolwell 1999) or other extracellular enzymes (Bolwell and
Wojtaszek, 1997). Alternatively, the increased level of H2O2
may be the result of insufficient activation of haem-containing
POD and CAT, due to low Fe availability. In support, specific
APX activity was reduced in both genotypes in the absence of
Fe and in BA29 also in the presence of bicarbonate (Figure 5),
probably as a consequence of insufficient Fe availability for the
enzyme, as it contains, in addition to the haem group, another
Fe atom. Different behaviour, as regards non-specific PODs,
was shown in the two genotypes under −Fe treatment, the
activity of these enzymes being diminished only in cv.
Conference (Figure 4a). These results seem to suggest that, in
the absence of Fe, BA29 tries to keep Fe-dependent PODs
functioning, probably to counter H2O2 accumulation linked to
the high SOD activity (Figures 3 and 7). In general, though
other activities could not be ruled out, the data obtained in the
absence of Fe suggest that the presence of ROS in roots of
both genotypes could be the consequence of overproduction
of these toxic species and, at the same time, of a reduced
capacity to detoxify them. An important aspect of this work
comes from the behaviour of bicarbonate-treated plants concerning non-specific POD activity. In fact, while an increase in
this activity was detected in the tolerant cv. Conference, in
BA29 it underwent a reduction (Figure 4), making a contribution to H2O2 accumulation. In general, it can be inferred that in
BA29 the presence of bicarbonate negatively affected the
enzymes considered in this work. On the other hand, in the cv.
Conference +FeBic, only an increase in non-specific POD activity was detected, confirming its higher level of protection in
particular against H2O2 accumulation. The specific role of PODs
within the lignification process was investigated by using a
specific substrate, syringaldazine, on different cellular fractions. There is both histochemical and biochemical evidence
that only cell walls that are undergoing lignification are able to
oxidize syringaldazine (Imberty et al. 1985, Christensen et al.
1998). While the H2O2 scavenging activity of soluble PODs
plays only a detoxification role, both covalently and ionically
bound PODs also catalyse the polymerization of lignin precursors and cross-links between extensins and feruloylated polysaccharides (Abeles and Biles 1991, Sato et al. 1993, Polle
et al. 1994). In our results, only the bicarbonate treatment
induces an increase in syr-POD activity (Table 2). In particular,
while in soluble PODs an increase was observed in both genotypes, PODs that are ionically bound to the cell wall underwent
an increase only in BA29. Furthermore, histochemical analysis
of roots was performed and a staining pattern consistent with
an increased process of lignification was observed in particular
in BA29 grown with bicarbonate (Figure 8). The increase in
syr-POD activity observed in this treatment could contribute to
lignin synthesis which, reducing root elongation and consequently nutrient uptake, could be correlated with susceptibility
to bicarbonate.
In the present work, POD activity was assayed using dianisidine, which is an aspecific substrate, and syringaldazine, a
specific substrate for PODs involved in lignification. This led to
the identification of a different effect of bicarbonate on the two
genotypes: while in the cv. Conference dian-POD is enhanced,
suggesting that this treatment induces an increase in H2O2
scavenging, in BA29 the syr-POD undergoes an increase, playing a role in morphological modification of roots by influencing
cell-wall plasticity.
Taken together, these results suggest that, only in BA29
grown in the presence of bicarbonate, can ROS accumulation
in root apoplast (Figures 6 and 7) be associated with lignin
deposits in external root layers and in endodermis (Figure 8),
as the consequence of the shift of PODs towards a lignification
role (Table 2). Cell-wall lignification, which provides structural
rigidity and durability to plant tissues, can occur as a stress
response (Srivastava et al. 2007, Kim et al. 2008) and has
been reported to be involved in the mechanism responsible for
plant tolerance to salt and drought stress (Jbir et al. 2001, Lee
et al. 2007). Under salt stress, the thicker the wall, the more
efficiently the cell interior is protected against ion absorption.
On the other hand, under drought stress, lignification reduces
plant growth, leading to a lower water request. Our results
could suggest another picture: we found that under bicarbonate stress the lignification process is much higher in the susceptible than in the tolerant genotype. In any case, while under
water and drought stress root lignification could be implicated
in the mechanism responsible for plant tolerance, under Fe
deficiency, it could further impair mineral nutrition generating
higher susceptibility to this constraint. Meanwhile, in the cv.
Conference, the allocation of new biomass to the organs
involved in nutrient acquisition could assure the exploration of
soil and consequently improve Fe uptake.
The results suggest that the tolerance of cv. Conference to
the presence of bicarbonate could be ascribed to the ability
to feed more appropriately for nutrients by means of: (i) unaffected root growth and (ii) increased Fe uptake activity
(Donnini et al. 2009). The absence of a similar adaptive strategy in quince could explain its susceptibility to this growth
condition.
Although future work should endeavour to link these aspects
with genetic events, the results obtained in this work can contribute to explaining the contrasting phenotypes of these species in response to the presence of bicarbonate in the growth
medium.
Acknowledgments
We thank Dr Patrizia De Nisi, Dr Gianpiero Vigani and Stefano
Mattaini for advice and critical reading of the manuscript. Silvia
Donnini was the recipient of a post-doctoral fellowship from
MIUR.
Tree Physiology Online at http://www.treephys.oxfordjournals.org
112 Donnini et al.
Funding
The work was supported by grants from MIUR and the
Università degli Studi di Milano (PUR).
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