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Theantagonisticeffectofhydroxylradicalon the
development ofahypersensitiveresponsein tobacco
Sheng Deng
1,2,
*, Mina Yu
1,2,
*, Ying Wang
1
, Qin Jia
1
, Ling Lin
2
and Hansong Dong
1
1 Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agricuture of R. P. China, Department of
Plant Pathology, College of Plant Protection, Nanjing Agricultural University, China
2 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
Introduction
In the course of their development, plants are often
confronted with various potential pathogens. To pre-
vent infection by these pathogens, plants have devel-
oped a set of inducible defence response systems. The
activation of plant defence responses is initiated
through the recognition of pathogen-associated
molecular patterns by plant receptors or the recogni-
tion of pathogen effectors (avirulence proteins) by
plant resistance gene products (R proteins) [1,2]. The
defence responses include the production of reactive
oxygen species (ROS) and phytoalexins; the reinforce-
ment of cell walls; the deposition of callose; the
Keywords
elicitin; hydroxyl radicals; hypersensitive
response; reactive oxygen species;
riboflavin
Correspondence
H. Dong, Key Laboratory of Monitoring and
Management of Crop Diseases and Pest
Insects, Ministry of Agricuture of R. P.
China, Department of Plant Pathology,
College of Plant Protection, Nanjing
Agricultural University, Nanjing 210095,
China
Fax: +86 25 8439 5325
Tel: +86 25 8439 9006
E-mail: hsdong@njau.edu.cn
*These authors contributed equally to this
work and are regarded as joint first authors
(Received 19 June 2010, revised 24 August
2010, accepted 12 October 2010)
doi:10.1111/j.1742-4658.2010.07914.x
Reactive oxygen species (ROS) are important signalling molecules in living
cells. It is believed that ROS molecules are the main triggers ofthe hyper-
sensitive response (HR) in plants. Inthe present study oftheeffectof ribo-
flavin, which is excited to generate ROS in light, onthedevelopmentof the
HR induced by the elicitin protein ParA1 intobacco (Nicotiana tabacum),
we found that the extent ofthe ParA1-induced HR was diminished by
hydroxyl radical (OH
•
), a type of ROS. As compared with the zones trea-
ted with ParA1 only, the HR symptom inthe zones that were infiltrated
with ParA1 plus riboflavin was significantly diminished when the treated
plants were placed inthe light. However, this did not occur when the
plants were maintained inthe dark. Trypan blue staining and the ion leak-
age measurements confirmed HR suppression inthe light. Further experi-
ments proved that HR suppression is attributed to the involvement of the
photoexcited riboflavin, and that the suppression can be eliminated with
the addition of hydrogen peroxide scavengers or OH
•
scavengers. Fenton
reagent treatment and EPR measurements demonstrated that it is OH
•
rather than hydrogen peroxide that contributes to HR suppression. Accom-
panying the endogenous OH
•
formation, suppression ofthe ParA1-induced
HR occurred inthetobacco leaves that had been treated with high-level
abscisic acid, and that suppression was also removed by OH
•
scavengers.
These results offer evidence that OH
•
, an understudied and little appreci-
ated ROS, participates in and modulates biologically relevant signalling in
plant cells.
Abbreviations
ABA, abscisic acid; DAB, 3,3¢-diaminobenzidine; G + GOX, glucose plus glucose oxidase; H
2
O
2
, hydrogen peroxide; HR, hypersensitive
response; O
2
•)
, superoxide radical; OH
•
, hydroxyl radical; PA, ParA1 plus adenine; PC, ParA1 plus catalase; PR, ParA1 plus riboflavin;
PT, ParA1 plus thiourea; PV, ParA1 plus ascorbic acid; ROS, reactive oxygen species; SOD, superoxide dismutase; WIPK, wounding-induced
protein kinase.
FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS 5097
expression of pathogenesis-related proteins; and, most
drastically, thehypersensitiveresponse (HR), which is
a form of programmed cell death. The HR leads to cell
death at the infection sites, thus limiting pathogen
growth and establishing systemic acquired resistance in
the whole plant [3,4].
Although numerous studies have been carried out
regarding thedevelopmentofthe HR induced by plant
pathogens or pathogen-derived elicitors, the underlying
molecular networks have not been well elucidated.
Undoubtedly, the early stage of HR development
involves some or all ofthe key events, including Ca
2+
influx, ROS burst, mitogen-activated protein kinase
cascades, nitric oxide production, cytochrome c release,
lipid peroxidation and phytohormone imbalance [2,5–
7]. In these events, ROS burst is most often considered
to be the protagonist to the occurrence ofthe HR
[8,9].
ROS, especially superoxide radical (O
2
•)
), hydrogen
peroxide (H
2
O
2
) and hydroxyl radicals (OH
•
), are very
important signalling molecules [6]. They play pivotal
roles in plant processes as diverse as development,
growth, theresponse to biotic and abiotic stimuli, and
programmed cell death [10–12]. In plant cells, O
2
•)
can
be converted into H
2
O
2
by superoxide dismutase
(SOD), and H
2
O
2
can be converted into highly toxic
OH
•
by the Fenton reaction or by peroxidase under
certain conditions [11,13]. Plant cells can sense the var-
iation of ROS production in location, amount, type,
rate and duration; these variations direct the subse-
quent responses of cells [12,14–17].
To date, the signalling roles of H
2
O
2
and O
2
•)
have
been intensively analysed, whereas other types of ROS,
such as OH
•
and singlet oxygen (
1
O
2
), have been lar-
gely ignored [11]. O
2
•)
,H
2
O
2
and
1
O
2
can be detoxi-
fied by the internal antioxidants or by some specific
enzymes. However, no specific scavenger for OH
•
has
been identified in plants. As a result of its high reac-
tiveness, OH
•
is under strict control in plants [14,18].
However, this does not mean that OH
•
has no other
roles than that ofa destroyer in cells. Foreman et al.
[19] reported that ROS are involved in plant cell
growth and root hair elongation. Specifically, OH
•
can
loosen cell walls, thus helping plant organs to elongate
and seeds to germinate [20–22]. The natural killer cell,
which is an essential constituent of host defence sys-
tems in humans, is activated by OH
•
exclusively, and
OH
•
scavengers can inhibit the activity of this type of
cell [23]. The present study showed that OH
•
can nega-
tively regulate the HR development induced by elicitin
and bacterium (Xanthomonas spp.) intobacco ( Nicotiana
tabacum L. cv NC89), thereby suggesting that under
certain circumstances, OH
•
may differ significantly
from other species of ROS with regard to biological
activity.
Purified elicitors are usually applied to given plants
for the elucidation of plant defence mechanisms.
ParA1, an elicitor derived from Phytophthora parasiti-
ca, belongs to the elicitin family, which comprises a
group of 10 kDa small proteins secreted by oomycetes
of the genera Phytophthora and some Pythium [24–26].
In most Nicotiana species, elicitins such as cryptogein,
ParA1 and INF can trigger various defence responses,
including the HR [25,27–29].
Riboflavin, which is a water-soluble vitamin essen-
tial to living cells, can reversibly accept or lose a pair
of hydrogen atoms. Therefore, its two derivatives,
flavin adenine dinucleotide and flavin mononucleotide,
can perform key metabolic functions as coenzymes in
electron transfer processes. It is generally believed that
photoexcited riboflavin can generate various ROS
under visible light [30–33]. Specifically, O
2
•)
,H
2
O
2
and OH
•
are generated through a type I reaction,
whereas
1
O
2
is produced through a type II reac-
tion [34]. Inthe present study, by exploiting the
character of riboflavin, we found that OH
•
can inhibit
the developmentofthe HR triggered by ParA1 in
tobacco leaves.
Results
The antagonisticeffectof photoexcited
riboflavin onthedevelopmentofthe HR induced
by ParA1
It is known that ParA1 can induce the HR in tobacco
leaves [25]. However, inthe current experiment, HR
development was suppressed by photoilluminated ribo-
flavin. The purified ParA1 protein (obtained from the
Pichia pastoris expression system; see Materials and
methods section) and ParA1 plus riboflavin (PR) were
infiltrated into different parts ofa leaf (simplified as
the same leaf or leaves inthe following sections). After
48 h exposure to light, the HR spread over the entire
ParA1-infiltrated zones, whereas just some small HR
lesion spots were scattered inthe PR-treated zones. By
contrast, inthe dark treatment, the infiltration of the
two materials yielded similar effects on HR develop-
ment (Fig. 1A). Additionally, inthe zones infiltrated
with riboflavin and the control (ParA1 purification
buffer), no HR-like lesion spots could be found, either
in the light or inthe dark (Fig. 1A).
To identify the most effective concentration of ribo-
flavin for the suppression ofthe HR, ParA1 protein
was mixed with riboflavin at concentrations ranging
from 5 to 100 mgÆL
)1
(data not shown). The most
Suppression effectofhydroxylradicalon HR S. Deng et al.
5098 FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS
effective riboflavin concentration was found to be
50 mgÆL
)1
, which was used to produce the results
shown in Fig. 1A.
The trypan blue staining also supported the pheno-
types. The HR extent inthe PR zone was diminished
as compared with that inthe ParA1 zone, and the
riboflavin zone was not stained as expected
(Fig. 1B,C). Furthermore, ion leakage measurement
exhibited the cell death progression in different infil-
trated zones at the indicated time points (Fig. 1D).
The extent ofthe HR inthe PR zone was significantly
suppressed as compared with that inthe ParA1 zone.
In the ParA1 zone, the ion leakage increased dramati-
cally within 12–24 h, whereas inthe PR zone the ion
leakage rose to a peak of 25% at 12 h and then
started to decline. The further developmentof HR
symptom in these zones was monitored from 48 to
96 h, but no changes were noted, except that the dead
zones inthe leaves became dry and crisp. The results
indicate that the photoexcited riboflavin can suppress
the HR rather than simply delay its development.
Photoexcited riboflavin suppresses the HR
development by disturbing the HR signalling
Riboflavin did not affect the HR development of
plants that were kept inthe dark (Fig. 1A). These find-
ings suggest that riboflavin did not disturb the HR sig-
nalling and the initial contact recognition between
ParA1 and its receptor under dark condition.
PR and ParA1 were infiltrated into the same leaf
and the treated plants were placed inthe dark. After
2, 4, 8 and 12 h, at least six plants at each time were
transferred from the dark to the continuous light
(termed predark treatment). Forty-eight hours after
infiltration, ion leakage in each treated zone was mea-
sured (Fig. 2B). The ParA1 zones were set as controls,
with similar ion leakage levels found for all groups. In
the PR zones, the ion leakage ratios ofthe first three
predark groups climbed from 12% inthe 2 h group to
25% inthe 8 h group, and then the ratio rose dras-
tically inthe 12 h group, approximating to the level of
ParA1 zones. It is known that the fluorescence inten-
sity of riboflavin provides a means of assessing its level
of concentration [35]. Inthe PR zones, the increase in
ion leakage from 4 to 12 h could not be attributed to
the decrease in riboflavin content, because no signifi-
cant changes in fluorescence intensity had been found
in these zones during predark treatment (Fig. 2B). In
addition, four other groups were treated ina reverse
manner (Fig. 2A; prelight treatment). Inthe PR-trea-
ted zones of these plants, only the 2 h prelight group
had a high ion leakage ratio ( 50%); in other prelight
groups the ratio reduced to 10% or even lower.
The mRNA levels of several elicitin response genes
were investigated by semiquantitative RT-PCR
A
B
D
C
Fig. 1. Theantagonisticeffectof riboflavin on HR development
occurred under light. (A) ParA1, Rf (50 mgÆL
)1
riboflavin), PR
(ParA1 + 50 mgÆL
)1
riboflavin) and Control (ParA1 purification buffer)
were infiltrated into different interveinal segments inthe same
tobacco leaves. The treated leaves were photographed 48 h after
they were placed under continuous light (lower row) or inthe dark
(upper row). The experiment was repeated five times (four plants per
repeat, with two kept inthe dark and two kept inthe light) with simi-
lar results (scale bars = 1 cm). (B) Dead cells were stained in situ
with trypan blue. The leaf was stained 12 h after treatment, and infil-
trated areas were encircled by a black line. (C) The details of each
zone were observed and recorded under a universal microscope
(scale bars = 500 lm). (D) The time course of cell death was moni-
tored by ion leakage measurements inthe different treatment zones.
The ion leakages from leaf discs obtained from the corresponding
zones were measured at the indicated time points after infiltration.
Con represents control treatment. Mean values ± standard error of
at least three replicates of ion leakage measurements are presented.
S. Deng et al. Suppression effectofhydroxylradicalon HR
FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS 5099
(Fig. 2D). With regard to the HR hallmark genes,
including hypersensitive-related (hsr) Hsr515, Hsr203J
and sensitivity-related (str) Str319 [36], the accumula-
tion of their transcripts was inhibited and delayed sig-
nificantly during the first 9 h inthe PR-treated zones
as compared with the ParA1-treated zones. Similar
results were found for alternative oxidase, which is a
marker gene for mitochondrial dysfunction [37]. Lipox-
ygenase-1, a key HR-dependent gene [38], was substan-
tially suppressed within 6–12 h. The transcript levels of
the wounding- induced protein kinase (WIPK) gene and
the 3-hydroxy-3-methylglutaryl CoA reductase gene,
which represent the activation of WIPK and salicylic
acid-induced protein kinase signalling pathways,
respectively [5,39], were lower inthe PR zones than in
the ParA1 zones from 6 to 12 h. However, the tran-
script levels of pathogenesis-related proteins 1a were
not significantly different in ParA1 and PR treatment.
Cytosolic ascorbate peroxidase was upregulated in all
treatments, whereas catalase was only induced by ribo-
flavin treatment.
These results demonstrate that the photoexcited
riboflavin can suppress HR development by disturbing
the HR signalling rather than by blocking the contact
recognition between ParA1 and its receptor.
The ROS generated from photoexcited riboflavin
are involved in HR suppression
The photolytic products of riboflavin obtained by plac-
ing riboflavin under light for 24 h failed to inhibit the
development ofthe HR (data not shown). This result
indicated that some active species for HR suppression
had disappeared after riboflavin photolysis. Attention
was then focused onthe ROS that were generated dur-
ing the riboflavin photolysis process, such as O
2
•)
,
H
2
O
2
,OH
•
and
1
O
2
.
In Fig. 3A,B, the riboflavin-infiltrated zone, which
was covered by aluminium foil, could not be stained
by the H
2
O
2
probe 3,3¢-diaminobenzidine (DAB).
However, the uncovered zone could be stained by the
probe, as was the zone that was infiltrated with glucose
plus glucose oxidase (G + GOX; taken as the positive
control) [15]. Further proof was obtained by H
2
O
2
measurement. Compared with the zone infiltrated by
H
2
O, the relative content of H
2
O
2
in the riboflavin-
infiltrated zone was increased by 40% (Fig. 3C).
The findings indicate that riboflavin can generate ROS
in tobacco leaves inthe presence of light irradiation.
To investigate the role of different types of ROS in
HR suppression, we introduced various free radical
scavengers, including catalase (scavenger of H
2
O
2
) [40],
SOD (scavenger of O
2
•)
) [11], ascorbic acid (redox reg-
A
B
C
Fig. 2. Photoexcited riboflavin suppresses HR development by dis-
turbing HR signalling. (A) Inthe predark and prelight groups, the ion
leakage inthe ParA1- and PR-treated zones was measured 48 h
after infiltration. After infiltration, at least six plants at each time
were first placed inthe dark for the indicated times (2, 4, 8 and
12 h) and then transferred to the continuous light (labelled as pre-
dark treatment) and others were treated reversely (labelled as pre-
light treatment). The treated plants kept inthe dark for 48 h after
infiltration were used as the control and labelled as ‘dark’. Mean
values ± standard error of three replicates are presented. (B) In
PR-infiltrated zones, the fluorescence emitted from riboflavin was
observed at the indicated times (0, 4, 8 and 12 h) after predark
treatment. (C) The levels of different transcripts inthe zones, which
were treated with ParA1 (P), riboflavin (R) or PR, were assayed by
semiquantitative RT-PCR. After infiltration, the plants were placed
under continuous light and the corresponding zones were collected
at the indicated times (3, 6, 9 and 12 h) for RNA isolation. Con rep-
resents the leaves that did not receive any treatments, and were
taken as the control. The constitutively expressed gene elongation
factor 1-alpha was used as an internal reference transcript. Three
independent experiments were completed with similar results.
Suppression effectofhydroxylradicalon HR S. Deng et al.
5100 FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS
ulator of cells) [11], l -histidine (scavenger of
1
O
2
and
OH
•
) [41], adenine and thiourea (scavengers of OH
•
)
[20,23]. Leaves treated with these scavengers or with
the mixture of each scavenger plus riboflavin mani-
fested no symptoms 72 h after infiltration (data not
shown). However, inthe zones infiltrated with ParA1
plus each scavenger, HR cell death occurred as it did
in the ParA1-treated zones (Fig. 3D). These results
reflect that HR development induced by elicitins is
independent of ROS, which is consistent with previ-
ously reported results [42–45].
The ROS scavengers, when coinfiltrated with PR,
were able to restore the HR symptom, with the excep-
tion of SOD (Fig. 3D). l-histidine, a scavenger of
1
O
2
and OH
•
, had little effecton HR restoration even at
100 mm. For comparison, the specific scavengers of
OH
•
, adenine and thiourea, can restore the HR sub-
stantially at 1 and 50 mm, respectively (Fig. 3D).
These findings suggest that H
2
O
2
or OH
•
(but not
O
2
•)
or
1
O
2
) is involved inthe suppression ofthe HR.
It is noteworthy that the HR symptom was further
suppressed inthe PRS (PR + SOD) zone, which is
probably an outcome of much more H
2
O
2
and OH
•
derived from SOD catalysing O
2
•)
(Fig. S2). Because
H
2
O
2
is the precursor of OH
•
[11,46], and because the
OH
•
scavengers as well as catalase and ascorbic acid
could restore the HR symptom (Fig. 3D), it was
hypothesized that OH
•
was the HR suppressor.
The suppressor role of OH
•
in HR development
is verified by the Fenton reaction and EPR
measurements
OH
•
is introduced and generated by a Fenton-type
reaction that takes place inthe presence of Fe
2+
and
H
2
O
2
[13,47]. To obtain stable and moderate H
2
O
2
generation, we used the G + GOX system again.
In the presence or absence of Fe
2+
, ParA1 and
G + GOX at increasing concentrations were coinfil-
trated into the same leaves. Because Fe
2+
⁄ Fe
3+
may
interfere with the results of ion leakage measurement,
five grades were adopted to assess the severity of the
HR induced by ParA1 (Fig. 4A). Inthe Fe
2+
-present
groups, the HR symptoms were significantly sup-
pressed, with grade 0 (i.e. no visible HR lesion)
accounting for 60% ofthe treated leaves or for even
higher proportions in some treatments (Fig. 4A). Con-
versely, inthe Fe
2+
-absent groups, grade IV accounted
for more than 60%. Moreover, the compromised HR
was observed when ParA1 was coinfiltrated with Fe
2+
(Fig. 4A). All the suppression effecton HR develop-
ment could be eliminated with the addition of the
scavengers of H
2
O
2
or OH
•
(Fig. S3A).
A
C
B
D
Fig. 3. The ROS generated from photoilluminated riboflavin influ-
ence HR development. (A) In situ detection of H
2
O
2
was per-
formed by DAB staining 1.5 h after treatment. Riboflavin (Rf;
50 mgÆL
)1
) and the positive control (G + GOX; 14 mM glucose and
2.5 unitsÆmL
)1
glucose oxidase) were infiltrated into tobacco
leaves. The upper part ofthe leaf was covered with aluminium
foil (shaded area inthe left-hand picture), and the lower part of
the leaf was exposed to light. The experiment was repeated three
times (two leaves per repeat) with similar results. (B) The details
of DAB staining were observed under a universal microscope. The
control was taken from the untreated zone (scale bars = 100 lm).
(C) As compared with the H
2
O treatment, the relative content of
H
2
O
2
in the riboflavin-infiltrated zone was measured 1.5 h after
treatment. Mean values ± standard error of at least three repli-
cates are presented. (D) Different ROS scavengers were used to
investigate the role of corresponding type of ROS in HR suppres-
sion. After infiltration, the treated plants were placed inthe light,
and the HR extent was evaluated by the ion leakage ratio at 12
and 48 h after infiltration. Mean values ± standard error of three
replicates are presented. The treatment by protein purification buf-
fer was regarded as the control. [P, ParA1; R, riboflavin
(50 mgÆL
)1
); C, catalase (2000 unitsÆmL
)1
); S, SOD (100 uni-
tsÆmL
)1
); V, ascorbic acid (50 mM); T, thiourea (50 mM); A, adenine
(1 m
M); H, L-histidine (100 mM); RC, riboflavin + catalase; PRC,
ParA1 + riboflavin + catalase; other abbreviations follow the same
pattern.]
S. Deng et al. Suppression effectofhydroxylradicalon HR
FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS 5101
The EPR method, a reliable way to analyse the for-
mation of OH
•
[48,49], was applied to the evaluation
of the OH
•
level during riboflavin photolysis in vitro
and in vivo. In Fig. 4B, a strong signal was detected,
which means that OH
•
is generated by riboflavin
in vitro under light irradiation. The addition of the
OH
•
scavenger adenine, and the dark maintenance of
the sample could substantially reduce the signal. As
expected, riboflavin also had similar effects in vivo
(Fig. 4C). Against the H
2
O infiltration, the relative
content of OH
•
in the riboflavin-treated zone was
increased by 50% (Fig. 4D). These results supported
A
B
C
D
a
b
c
d
e
a
b
c
d
e
Suppression effectofhydroxylradicalon HR S. Deng et al.
5102 FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS
the previous findings that OH
•
is involved in HR sup-
pression. It should also be noted that light irradiation
can affect the background signal of spin trapping
reagent [Fig. 4B (c) and (e)]. However, the signal is not
produced by OH
•
generation, because the OH
•
scaven-
ger cannot reduce the signal [Fig. 4B (d) and (e)].
As a result of endogenous OH
•
generation, HR
development is suppressed by exogenous
application of high-level abscisic acid
Previous studies have proved that the exogenous appli-
cation of high-level abscisic acid (ABA) can induce the
accumulation of catalytic Fe, which is critical for the
generation of OH
•
through the Fenton reaction [50].
ParA1, PR, PA (ParA1 plus adenine), PT (ParA1 plus
thiourea), PV (ParA1 plus ascorbic acid) and PC
(ParA1 plus catalase) were infiltrated into the same
leaves oftobacco plants, which had been sprayed with
100 lm ABA, 400 lm ABA or H
2
O and kept inthe light
for 24 h. Twenty-four hours after ParA1 infiltration, the
development ofthe HR inthe leaves that had been pre-
treated with ABA was significantly suppressed as com-
pared with the leaves that had been pretreated with H
2
O
(Fig. 5A,D). After 48 h, the HR further developed in
the leaves pretreated with 100 lm ABA, but less in the
leaves pretreated with H
2
O and 400 lm ABA. The EPR
assays provided the evidence that the 400 lm ABA-pre-
treated leaves had a higher OH
•
level after ParA1 treat-
ment, and the level increased by 20% against H
2
O
treatment (Fig. 5B,C). These findings suggest that the
HR induced by ParA1 can be suppressed by the exoge-
nous application of high-level ABA, probably as a result
of the endogenous OH
•
formation. Additionally, in the
PR-infiltrated zone, because ofthe ABA pretreatment,
the HR suppression effectof photoexcited riboflavin
was synergistic (Fig. 5A).
In the zones infiltrated with OH
•
scavengers, both
adenine and thiourea could restore HR development in
ABA-pretreated leaves, although the formation of HR
lesions inthe PT zone lagged behind that seen in the
PA zone, where the HR lesion could be observed 16 h
after infiltration (Fig. 5A,D). Similarly, in PV and PC
zones, the HR in ABA-pretreated leaves could be res-
cued by the application of ascorbic acid and catalase
(Fig. 5A,D).
The phytopathogenic bacterium Xanthomonas oryzae
pv. oryzicola (stain RS105), can cause disease in their
host rice; in nonhost plants, such as tobacco, they can
trigger the HR [51]. ABA- and H
2
O-pretreated leaves
were infiltrated with stain RS105 and RS105 plus ade-
nine. As expected, against H
2
O-pretreated leaves, the
HR induced by RS105 was suppressed in ABA-pre-
treated leaves, and this suppression could be restored
by the addition of adenine (Fig. 5D). In addition, it
was affirmed that the suppressed HR cannot be
explained by the preferential decrease inthe viable
bacterial population, which was caused by the unfa-
vourable environment in plant cells (data not shown).
These findings indicate that the exogenous application
of high-level ABA suppressed the HR development
triggered by ParA1 and RS105 intobacco leaves
through the involvement of OH
•
, which was generated
by the accumulation of ROS and catalytic Fe.
Discussion
In the present study, the HR induced by ParA1 was sig-
nificantly suppressed when ParA1 and riboflavin were
coinfiltrated, and the suppression only took place in the
light and not inthe dark. Further analysis suggested
that the major suppressor was OH
•
derived from photo-
excited riboflavin. In addition, the endogenous OH
•
could also suppress thedevelopmentofthe HR induced
by ParA1 and the bacteria strain RS105 in tobacco.
Accordingly, it is proposed that OH
•
, an understudied
and little appreciated ROS, has an antagonisticeffect in
HR development by disturbing the HR signalling.
Fig. 4. OH
•
was involved inthe suppression ofthe HR. (A) The HR induced by ParA1 could also be suppressed by the Fenton reaction
reagent. Five grades of HR severity were depicted and labelled as 0, I, II, III and IV. PG
0.5
O
0.5
(ParA1 mixed with 0.5 mM glucose and
0.5 unitsÆmL
)1
glucose oxidase), PG
1
O
1
,PG
5
O
2.5
were infiltrated into the same tobacco leaves inthe presence or absence of Fe
2+
(0.5 mM,
FeSO
4
). ParA1 and ParA1 plus Fe
2+
were infiltrated as controls. The data were recorded and compiled 48 h after infiltration. Leaves that
showed the HR extent of grade IV or III in ParA1-treated zones were selected for calculation (in total 21 leaves from 10 plants). (B) The EPR
method was used to detect OH
•
generation during riboflavin photolysis in vitro 1.5 h after exposure to light. (a) Riboflavin under light; (b)
riboflavin plus adenine (1 m
M) under light; (c) riboflavin inthe dark; (d) adenine (1 mM) with spin trapping assay reagent under light; and (e)
spin trapping assay reagent under light. (C) OH
•
generated from riboflavin photolysis in vivo was measured by EPR 1.5 h after treatment. (a)
Leaf tissues treated with H
2
O plus spin trapping assay reagent as a negative control; (b) leaf tissues treated with riboflavin; (c) leaf tissues
cotreated with adenine (1 m
M) and riboflavin; (d) leaf tissues treated with the Fenton reaction reagent (5 mM glucose, 2.5 unitsÆmL
)1
glucose
oxidase and 0.5 m
M Fe
2+
) as a positive control; and (e) leaf tissues obtained from the untreated zone as a background for the spectra. All
spectra were representative of at least three measurements under indicated conditions. (D) Against the H
2
O treatment, the relative content
of OH
•
in the riboflavin zone and the riboflavin plus adenine zone were calculated from the above spectra. Mean values ± standard error of
three replicates are presented.
S. Deng et al. Suppression effectofhydroxylradicalon HR
FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS 5103
Riboflavin is one ofthe pivotal vitamins for living
organisms. It is also an excellent photosensitizer that
can generate ROS under light irradiation. Over the
years, due to its photosensitization, riboflavin has been
extensively studied in medicine, pharmaceutical chemis-
try, foodstuffs, nutrition and other fields [33,34,52–54],
but less so in plants. As plants are the major source of
riboflavin taken up by animals and also that, to date,
little evidence in plants has been reported about the
involvement of photoexcited riboflavin (including its
two derivatives) in light stress and light injury, the field
in question is worthy of research effort.
To identify the real HR suppressor, we used various
ROS scavengers, because the photoilluminated ribofla-
vin can generate varied species of ROS, including
O
2
•)
,H
2
O
2
,OH
•
and
1
O
2
. It seems a little complicated
on the surface. However, in fact, the application of
SOD failed to restore HR symptoms and l-histidine
had little effecton HR restoration (Fig. 3D), which
suggests that O
2
•)
and
1
O
2
do not participate in HR
suppression. Further experiments provided more evi-
dence: (a) only the Fenton reaction can significantly
affect HR development (Fig. 4A); (b) the signal
strength of EPR is correlated with HR suppression
(Figs 4C,D and 5B,C); (c) the presence of Fe
2+
can
also suppress the HR induced by ParA1 to a large
extent (Fig. 4A). This evidence indicates that the chem-
ical species involved in HR suppression is OH
•
.
It is believed that plants pretreated with sublethal
stress (e.g. ozone exposure, ultraviolet irradiation and
methyl viologen treatment) can build up a resistance to
the subsequent lethal stress and pathogen infections
with less cell death. These phenomena are termed
cross-tolerance or acclimation [55,56]. Initially, we
A
D
B
C
Fig. 5. The exogenous application of ABA counteracted HR development. (A) The HR extent was affected by ABA pretreatment. Twenty-
four hours before the infiltration with ParA1, PR, PA, PT, PV and PC, tobacco plants were sprayed with H
2
O and ABA (100 lM, A100;
400 l
M, A400). After infiltration, the HR extent in leaves was recorded at the indicated times in terms ofthe grade depicted in Fig. 4A
(upper panel). Each treatment was repeated in 15 leaves from eight plants, and all recorded data were compiled. Furthermore, the HR
extents were also assessed by ion leakage measurement (lower panel). Mean values ± standard error of at least three replicates are pre-
sented. (B) The EPR measurements indicated that the leaves pretreated with ABA had a higher OH
•
level after ParA1 treatment. H
2
O, H
2
O
pretreatment; H + ParA1, ParA1 infiltration after H
2
O treatment; A400, 400 lM ABA pretreatment; A + ParA1, ParA1 infiltration after ABA
treatment. All the spectra were representative of at least three measurements under the indicated conditions. (C) The relative content of
OH
•
in the H + ParA1, A400 and A + ParA1 zones against the H
2
O zone. Mean values ± standard error of three replicates are presented.
(D) With ABA pretreatments, thedevelopmentofthe HR was compromised or suppressed. 1, ParA1; 2, PV; 3, PA; 4, PC; 5, PT; 6, RS105
(colony-forming unit = 10
8
); 7, RS105 + A; 8, PR. Pictures were taken for each treated leaf at the indicated times, and they represent the
general results from 15 infiltrated leaves for each treatment.
Suppression effectofhydroxylradicalon HR S. Deng et al.
5104 FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS
assumed that the suppression ofthe HR in this study
was due to acclimation, but the evidence refuted the
assumption. First, it takes hours for the preparation of
more severe stress or pathogen infection after pretreat-
ment. However, inthe PR-infiltrated zone, the ribofla-
vin and ParA1 functioned almost simultaneously.
Second, the ABA-pretreated leaves only showed small
HR lesions after infiltration with ParA1, which could
be regarded as probably acclimation. However, the
application of ROS scavengers can restore the HR,
and the addition of riboflavin, which can generate
ROS in light, suppresses the HR completely. These
outcomes are not feasible in accordance with the
classical concept of acclimation.
Liu et al. [57] found that the autophagy process
negatively regulates the HR triggered by tobacco
mosaic virus intobacco plants with N protein. The
reason offered by the authors was that autophagy
can restrict prodeath signal(s) from diffusing. Xiong
et al. [58] confirmed that autophagy can be induced
by oxidative stress in Arabidopsis. These results are
strongly reminiscent ofthe HR suppression that was
found inthe present study. However, when ParA1
was infiltrated into the leaf zones that had been trea-
ted with 10 or 20 mm H
2
O
2
5 h previously, no HR
suppression occurred (Fig. S3B). Therefore, the HR
suppression presented here has nothing to do with
autophagy.
Nitric oxide may be involved in HR suppression in
the present work, because the production balance
between nitric oxide and H
2
O
2
is crucial to trigger the
HR [59]. However, increasing the H
2
O
2
level by adding
the G + GOX system or by eliminating H
2
O
2
with
specific scavengers had no effectonthe final HR
symptoms induced by ParA1 (Figs 3D and 4A), which
probably means that the imbalance of nitric oxide and
H
2
O
2
is not the key reason for the HR suppression.
In the present study, the ROS seemed to be a sup-
pressor ofthe HR. In Fig. 3D, 12 h after infiltration,
the ratios of iron leakage inthe PC, PV and PA zones
were increased against ParA1 treatment, which sug-
gests that the HR symptoms were promoted in these
zones. Similar results were obtained by other research
groups. Ruste
´
rucci et al. [60] reported that the devel-
opment of cell death was strongly delayed and dimin-
ished when leaves were exposed to continuous light
(7000 lux of white light) 24 h before elicitin treatment.
A similar phenomenon was also observed by Tronchet
et al. [61]. Moreover, correlating with the HR inhibi-
tion, the activity of 9-lipoxygenase and galactolipases
as well as their mRNA levels w as significantly suppressed
by high light exposure (350 lmol quantaÆm
)2
Æs
)1
) after
cryptogein treatment [62]. These findings imply that at
least one species of ROS may negatively regulate the
development ofthe HR.
The short-lived OH
•
is the most reactive species of
ROS. It is very unstable, and it can rapidly attack bio-
molecules that are located at the site of its generation
[13,63]. So far, in plant cells, research regarding the
functions of OH
•
has mainly focused on cell wall loos-
ening and its actions related to oxidative stress,
whereas other fields involving OH
•
have rarely been
addressed [18,22,64]. For example, is it involved in any
signalling pathway, and how does it perform its func-
tion as a signalling molecule? Conversely, the movable
and relatively stable H
2
O
2
has been extensively studied.
It is prevailingly believed that H
2
O
2
can diffuse in cells
and between organelles, and that it can conduct its
functions by modifying thiol groups in certain pro-
teins, such as transcription factors and protein kinases
[11,14,46].
In addition to ParA1, HrpN
Ea
protein (UniProt
accession number: Q01099), a well-known HR trigger
in tobacco [65], was investigated inthe present study.
The experimental results prove that photoexcited ribo-
flavin fails to suppress the HrpN
Ea
-induced HR, which
is ROS dependent and mitochondrial dysfunction
dependent [66,67] (Fig. S4). To some extent, the photo-
excited riboflavin promotes HR development (data not
shown). Because the HR induced by elicitin is chloro-
plast dependent and fatty acid peroxidation dependent
[38], theeffectof OH
•
produced by photoexcited ribo-
flavin on HR suppression should be signalling pathway
dependent.
In plant cells, the signature of fatty acid peroxida-
tion can be changed after OH
•
treatment [68]. Fatty
acid peroxidation can be performed either by free radi-
cals (not including H
2
O
2
and O
2
•)
) or by lipoxygenase
pathways. The former process yields 9(R,S), 12(R,S),
13(R,S) and 16(R,S) fatty acid hydroperoxides,
whereas enzymatic oxygenation yields 9(S) and 13(S)
fatty acid hydroperoxides exclusively [69]. It has been
reported that 9(S)-hydroperoxides are the crucial sig-
nalling molecules for the execution ofthe HR, and
that the enantiomer composition of different fatty acid
peroxidation products determines cell fates (i.e. death
or survival) after cryptogein treatment [38,70]. In the
present study, it was hypothesized that the enantiomer
composition and signature of fatty acid hydroperox-
ides were changed, probably as a result of modification
by OH
•
, thus leading to HR suppression.
As plants in nature are subject to biotic and abiotic
stress simultaneously, it should not be ignored that
OH
•
formed under abiotic stress will probably exert
disturbing effects on biotic stress response pathways,
especially onthe fatty acid-dependent response pathways.
S. Deng et al. Suppression effectofhydroxylradicalon HR
FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS 5105
However, whether the interference is positive or negative
has to b e d etermined on t he basis of g iven conditi ons
and mechanisms.
Materials and methods
Chemicals
Riboflavin was obtained from Calbiochem (Merck KGaA,
Darmstadt, Germany), reduced glutathione from Roche
(Basel, Switzerland). Catalase, SOD, ascorbic acid and
ABA were purchased from Sigma (St Louis, MO, USA).
All other mentioned reagents were of analytical grade.
Plant culture condition
Tobacco plants (N. tabacum L. cv NC89) were grown in
a growth chamber at 25 °C with 16 h of light (50 lmol
quantaÆm
)2
Æs
)1
). All treatments were performed on plants
14–16 weeks old.
Treatments oftobacco leaves with ParA1,
riboflavin and other reagents
Before treatment, all operations involving riboflavin were
performed in subdued light to protect riboflavin from deg-
radation. ParA1 (UniProt accession number: P41801), ribo-
flavin and different reagents were infiltrated by needleless
syringes inthe second, third and fourth bottom leaves,
which were intact but not etiolated. The infiltrated zones
were marked with a black marking pen. Unless there were
special requirements, all the experimental treatments were
as follows: the intensity of light was 50 lmol quantaÆm
)2
Æs
)1
(cool white light) and the concentrations of ParA1 and
riboflavin were 3 nm and 50 mgÆL
)1
, respectively.
H
2
O
2
detection in situ
In situ H
2
O
2
generated by photoexcited riboflavin was
detected with the use ofthe DAB staining method (Sigma)
described by Thordal-Christensen et al. [71], with the
following modifications. Treated leaves were vacuum
infiltrated in 1 mgÆmL
)1
DAB solution, pH 3.8, for 15 min.
After incubation at 25 °C for 12 h inthe dark, samples
were transferred in 95% ethanol at 95 °C for the removal
of chlorophyll. Samples were observed under a universal
microscope and stored in 70% ethanol.
The observation ofthe fluorescence emitted from
riboflavin
After infiltration, the treated plants were placed inthe dark.
Then at indicated times (0, 4, 8 and 12 h), the fluorescence
emitted from riboflavin in PR-infiltrated zones was
observed in 480 nm light under a stereomicroscope
SZX12 (Olympus, Tokyo, Japan) equipped with a mercury
lamp fluorescence system and an excitation filter (460–
490 nm).
Measurement of H
2
O
2
in treated tobacco leaves
In accordance with a ferrous ammonium sulphate ⁄ xylenol
orange method [72] with some modification, the content of
H
2
O
2
in the treated leaves was measured. The relative
increase in H
2
O
2
in riboflavin-treated zones was calculated
in comparison with the H
2
O-treated zones inthe same
leaves. An area of 3–5 cm
2
in the infiltrated zone was cut
and crushed into a coarse powder in liquid nitrogen.
Approximately 7.5 mg powder was loaded into a tube that
contained 1.5 mL precooled 5% trichloroacetic acid. After
gentle vibration to mix, the tube was placed inthe dark at
room temperature for 2 min. The homogenate was then
centrifuged at 10 000 g,4°C for 2 min. Next, 500 lL
supernatant was taken and mixed with 500 lL assay solu-
tion (containing 500 lm ferrous ammonium sulphate,
200 lm sorbitol, 200 lm xylenol orange, 50 mm H
2
SO
4
and
2% ethanol). The mixtures and the blank sample (500 lL
5% trichloroacetic acid and 500 lL assay solution) were
placed at room temperature inthe dark for 30 min. The
absorbance ofthe sample was then measured at 560 nm
using a spectrophotometer (U-2800; Hitachi, Tokyo,
Japan). Onthe basis ofthe absorbance value, the standard
curve (obtained by adding the concentration gradient of
H
2
O
2
) and the loaded weight of plant tissue, the content of
H
2
O
2
was calculated.
Protein expression, purification and
concentration evaluation
The full-length cDNA, which encodes ParA1 protein (Uni-
Prot accession number: P41801), had been cloned from
Phytophthora parasitica inthe previous study. The synthetic
ParA1 gene (together with seven histidine codons attached
at the 3 ¢ end), which was flanked by EcoRI and KpnI
restriction sites at the 5¢ and 3¢ ends, respectively, was
obtained by PCR with the following oligonucleotide prim-
ers: 5¢-TGAATTC
AATAATGTCTAACTTCCGCGCTCT-
GTTC-3¢ and 5¢-AGGTACCTCAATGATGATGATGAT
GATGATGCAGTGACGCGCACGTAGA-3¢. For the suc-
cessful protein expression, a yeast expression consensus
sequence (including ATG) must be added to the 5¢ end pri-
mer (underlined). The correct ParA1 gene sequence was
cloned into pPICZ-A, the expression plasmid, through
EcoRI and KpnI restriction sites.
In accordance with the manual inthe EasySelect
TM
Pichia Expression Kit bought from Invitrogen (Carlsbad,
CA, USA), the yeast strain KM71H was used for the
expression of ParA1 protein, and the Escherichia coli strain
Suppression effectofhydroxylradicalon HR S. Deng et al.
5106 FEBS Journal 277 (2010) 5097–5111 ª 2010 Nanjing Agricultural University. Journal compilation ª 2010 FEBS
[...]... the basis ofthe gradient concentration of bovine serum albumin, the ParA1 concentration was evaluated by quantity one, a software contained in Bio-Rad Gel Documentation System 2000 Finally, we obtained 75 ngÆlL)1 ( 6500 nm) ParA1 protein (Fig S 1A, B), which can induce the HR at 6000· dilution ontobacco leaves in 24 h Measurement of ion leakage from leaf discs Cell death can be assayed by measuring... respectively The spectra shown were obtained from the accumulation of five single scans RNA isolation and RT-PCR ParA1, riboflavin and PR were in ltrated into different interveinal segments inthe same tobacco leaves, and the samples were obtained from the corresponding in ltrated regions at the indicated times (four leaves from two plants per time point) The sample from the untreated plant leaves was taken as the. .. supplementary material is available: Table S1 Information about the genes investigated in this study for RT-PCR Fig S1 The plasmid constructed for the expression of ParA1 in Pichia pastoris and the tricine–SDS-PAGE analysis of purified ParA1 Fig S2 The synergized effectof HR suppression inthe PRS (ParA1, riboflavin and SOD) zone Suppression effectofhydroxylradicalon HR Fig S3 Thein uence ofthe Fenton... water for 3 h ina growth chamber The conductivity ofthe bathing solution was measured with a conductivity meter (SY-2, Institute of Soil Science, Chinese Academy of Sciences, China): value AThe leaf discs were then returned to the bathing solution and incubated at 95 °C for 25 min After cooling to room temperature the conductivity ofthe bathing solution was measured again: value B The result of. .. mixture was placed into a glass capillary with an inside diameter of 1.1 mm for the EPR assay The parallel control without riboflavin was carried out ina similar way For thein vivo assay of photoexcited riboflavin, the mixture was in ltrated into leaves with needleless syringes, and the treated plants were placed inthe light After 1.5 h, two pieces of leaf tissue (6 mm in width, 15 mm in length) obtained... peroxide J Invest Dermatol 105, 608–612 31 Frati E, Khatib AM, Front P, Panasyuk A, Aprile F & Mitrovic DR (1997) Degradation of hyaluronic acid by photosensitized riboflavin in vitro Modulation oftheeffect by transition metals, radical quenchers, and metal chelators Free Radic Biol Med 22, 1139–1144 32 Hasan N, Ali I & Naseem I (2006) Photodynamic inactivation of trypsin by the aminophylline-riboflavin system:... precipitated inthe presence of phosphate For the plants that were pretreated with ABA or H2O, ParA1 and the spin trapping assay reagent were coinfiltrated into the leaves, and the plants were placed under light After 6 h, the levels of OH• inthe leaf tissues obtained from in ltrated zones were measured by EPR ina similar way to that described above EPR measurements The measurements were performed with an EMX... component ofthe cryptogein signaling pathway leading to defense responses and hypersensitive cell death intobacco Plant Cell 14, 1937–1951 Takahashi Y, Berberich T, Miyazaki A, Seo S, Ohashi Y & Kusano T (2003) Spermine signalling in tobacco: activation of mitogen-activated protein kinases by spermine is mediated through mitochondrial dysfunction Plant J 36, 820–829 ´ Rusterucci C, Montillet JL, Agnel... al TOP10 F¢ was used for the construction ofthe expression plasmid pPICZA::ParA1 Ni2+ affinity columns (HisTrapÔ HP; Amersham Bioscience, Buckinghamshire, UK) were applied for ParA1 protein purification In accordance with the instructions, the optimal concentrations of imidazole for binding (100 mm) and elution (350 mm) were chosen to purify the protein, and most ofthe purified ParA1 was collected in. .. inthe second tube and stored at )80 °C The purified recombinant ParA1 protein was determined by tricine–SDS ⁄ PAGE with the use of 4% stacking gels and 10% separation gels in accordance with the method introduced by Schagger [73] The protein electrophoresis ¨ was performed by constant current (10 mA in stacking gels, 15 mA in separation gels) with PowerPAC Universal (Bio-Rad, Hercules, CA, USA) Onthe . basis of the gradi-
ent concentration of bovine serum albumin, the ParA1 con-
centration was evaluated by quantity one, a software
contained in Bio-Rad Gel. ParA1 zone, the ion leakage increased dramati-
cally within 12–24 h, whereas in the PR zone the ion
leakage rose to a peak of 25% at 12 h and then
started