Journal of Experimental Botany, Vol.
Page63,
1 of
1012,
2, pp.
2012
No.
pp.695–709,
4349–4358,
2012
doi:10.1093/jxb/err313
doi:10.1093/jxb/ers082
2011
doi:10.1093/jxb/ers082 Advance
AdvanceAccess
Accesspublication
publication 420November,
March, 2012
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
The
In
Posidonia
enhancement
oceanica
of cyclic
cadmium
electron
induces
flow changes
around in DNA
photosystem
methylation and
I improves
chromatin
thepatterning
recovery of severely desiccated
Porphyra yezoensis (Bangiales, Rhodophyta)
Maria Greco, Adriana Chiappetta, Leonardo Bruno and Maria Beatrice Bitonti*
1,
Department
of Ecology,
University
of Calabria,
Laboratory of Plant Cyto-physiology, Ponte Pietro Bucci, I-87036 Arcavacata di Rende,
Shan
Gao1,2
and Guangce
Wang
*
Cosenza, Italy
1
Institute of Oceanology, Chinese Academy of Sciences (IOCAS), Qingdao 266071, China
*2 To whom correspondence should be addressed. E-mail: [email protected]
Graduate School, Chinese Academy of Sciences, Beijing 100049, China
*Received
To whom
should
addressed.
E-mail:
[email protected]
29correspondence
May 2011; Revised
8 Julybe2011;
Accepted
18 August
2011
Received 1 December 2011; Revised 14 February 2012; Accepted 20 February 2012
Abstract
In
mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent
Abstract
epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with
Porphyra
yezoensis,
a representative
species
of intertidal
macro-algae,
is able tolevel
withstand
periodic
desiccation
its effect on
chromatin
reconfiguration
in Posidonia
oceanica.
DNA methylation
and pattern
were
analysed at
in
low
tide
but
is
submerged
in
seawater
at
high
tide.
In
this
study,
changes
in
photosynthetic
electron
in
actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) dosesflow
of Cd,
P.
yezoensis
during
desiccation
and
re-hydration
were
investigated.
The
results
suggested
that
the
cyclic
electron
through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach,
flow
around photosystem
I (PSI)
increased
significantly
during desiccation, (CMT)
continued
to operate
at times of severe
respectively.
The expression
of one
member
of the CHROMOMETHYLASE
family,
a DNA methyltransferase,
desiccation,
and
showed
greater
tolerance
to
desiccation
than
the
electron
flow
around
PSII.
In
addition,
PSIelectron
activity
was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission
in
desiccated
blades
recovered
faster
than
PSII
activity
during
re-hydration.
Even
though
linear
electron
flow
microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating thatwas
de
suppressed
by DCMU
[3-(3#,4#-dichlorophenyl)-1,1-dimethylurea],
flow could
still be restored. This
novo methylation
did indeed
occur. Moreover, a high dose of Cdcyclic
led toelectron
a progressive
heterochromatinization
of
process
was
insensitive
to antimycin
A were
and could
be suppressed
by dibromothymoquinone
(DBMIB).
The prolonged
interphase
nuclei
and apoptotic
figures
also observed
after long-term
treatment. The data
demonstrate
that Cd
dark
treatment
of blades
reduced
the speed
in which
the cyclic electron
flow around
PSI recovered,
suggesting
perturbs
the DNA
methylation
status
through
the involvement
of a specific
methyltransferase.
Such
changes that
are
stromal
includingreconfiguration
NAD(P)H, played
an important
role
thebalance
donation
electrons to PSI andchromatin.
were the
linked toreductants,
nuclear chromatin
likely
to establish
a in
new
of of
expressed/repressed
main
cause
of the
rapid
ofbasis
cyclictoelectron
flow in underlying
desiccatedCd
blades
during
re-hydration. These results
Overall,
the data
show
an recovery
epigenetic
the mechanism
toxicity
in plants.
suggested that cyclic electron flow in P. yezoensis played a significant physiological role during desiccation and rehydration
may be one of the most
importantcondition,
factors chromatin
allowing reconfiguration,
P. yezoensis CHROMOMETHYLASE,
blades to adapt to intertidal
Key words:and
5-Methylcytosine-antibody,
cadmium-stress
environments. Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile.
DNA-methylation,
Key words: Cyclic electron flow, desiccation, photosystem I, Porphyra yezoensis.
Introduction
In the Mediterranean coastal ecosystem, the endemic
Although not essential for plant growth, in terrestrial
Introduction
seagrass Posidonia oceanica (L.) Delile plays a relevant role plants, Cd is readily absorbed by roots and translocated into
Cyclic
electron
flow around
photosystem
I (PSI) in and
the several
reports
provided
strong
evidence
that the
cyclic
by ensuring
primary
production,
water oxygenation
aerial organs
while,
in acquatic
plants,
it is directly
taken
up
thylakoid
membranes
was discovered
years
ago (Arnon electron
chain
PSI induces
could be
organized
into
provides niches
for some
animals, >50
besides
counteracting
by leaves.transfer
In plants,
Cd around
absorption
complex
changes
et
al., 1954),
and
has been
to occur
in higher
that associate
cytochrome
and
coastal
erosion
through
its shown
widespread
meadows
(Ott,plants,
1980; supercomplexes
at the genetic, biochemical
and PSI,
physiological
levelsbf,which
reductase
(Carrillo
eukaryotic
algae,
cyanobacteria
et al.,is1993;
Piazzi et al.,
1999;and
Alcoverro
et al., (Asada
2001). There
also ferredoxin-NADP
ultimately account+ for
its toxicity
(Valleand
andValleios,
Ulmer, 1983;
1972;
Joliot and
Joliotand
2002;
Iwai et1999;
al., 2010).
Recently,
has
Ravenel
et al.,
1994; Mi
1995). plants
In particular,
considerable
evidence
thatetP.al.,
oceanica
are ablethis
to Sanitz
di Toppi
Gabrielli,
Benavides
et al., it2005;
that cyclic
flowThe
around
was
pathway
hasaccumulate
been investigated
in Arabidopsis
absorb and
metals from
sediments thaliana,
(Sanchiz been
Weberreported
et al., 2006;
Liu etelectron
al., 2008).
most PSI
obvious
photosynthesis
in plants
(Munekage
Chlamydomonas
reinhardtii, 1998;
and tobacco
et al., 1990; Pergent-Martini,
Maserti (Nicotiana
et al., 2005)tabathus essential
symptom for
of Cd
toxicity is a reduction
in plant
growth et
dueal.,
to
During ofsteady-state
photosynthesis,
electron
cum
var. Petit
Havana)
(Horváthinetthe
al.,marine
2000; Joet
et al., 2004).
influencing
metal
bioavailability
ecosystem.
an inhibition
photosynthesis,
respiration,cyclic
and nitrogen
contributes
to ATP
adjusts
ATP:
2001,
2002;
Munekage
et al., 2002,
2004; considered
DalCorso et
For this
reason,
this seagrass
is widely
to al.,
be flow
metabolism,
as well
as a synthesis
reduction and
in water
andthemineral
ratio (Arnon
et al.,
1967;
Backhausen et
2008;
Takahashi
et al.,species
2009; Iwai
et al.,
In addition,
a metal
bioindicator
(Maserti
et 2010).
al., 1988;
Pergent NADPH
uptake (Ouzonidou
et al.,
1997;
Perfus-Barbeoch
et al.,
al., 2000;
et al., 1995; Lafabrie et al., 2007). Cd is one of most Shukla et al., 2003; Sobkowiak and Deckert, 2003).
Abbreviations: AA, antimycin A; AWC, absolute water content; DBMIB, dibromothymoquinone; DCMU, 3-(3#, 4#-dichlorophenyl)-1, 1-dimethylurea; ETR, electron
widespread
heavy metals in both terrestrial and marine
At the genetic level, in both animals and plants, Cd
transport rate; F0, minimum fluorescence; Fv/Fm, maximum quantum yield of PSII; PQ, plastoquinone; Y(I), effective PSI quantum yield; Y(II), effective PSII quantum
environments.
can induce
chromosomalquantum
aberrations,
in
yield;
Y(NA), non-photochemical quantum yields of PSI caused by acceptor-side limitation;
Y(ND), non-photochemical
yields of PSIabnormalities
caused by donor-side
limitation.
2011Author
The Author(s).
ª The
[2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For
please article
e-mail:distributed
[email protected]
ThisPermissions,
is an Open Access
under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
4350 and
Wang
2 of 10| Gao
| Gao
and
Wang
Kramer et al., 2004a), which is essential for preventing
stroma over-reduction (Munekage et al., 2004). More
importantly, a number of papers have demonstrated that
cyclic electron flow around PSI plays a significant physiological role in plant responses to stresses, such as drought or
desiccation (Horváth et al., 2000; Golding and Johnson,
2003; Bukhov and Carpentier, 2004; Gao et al., 2011).
However, the above-mentioned model plants have limited
desiccation tolerance, although some bryophytes and ferns
show some tolerance (Heber et al., 2007). Therefore, it is
more appropriate to choose desiccation-tolerant species
when undertaking studies on the effects of desiccation on
the cyclic electron flow around PSI.
Porphyra spp. inhabit the high intertidal zones of rocky
seashores, are periodically submerged in seawater at high
tide, and are exposed to air at low tide (Tseng et al., 1983).
During exposure to air, especially during the daytime, they
are subjected to severe desiccation (Davison and Pearson,
1996). High intertidal Porphyra reportedly demonstrate
extreme tolerance to desiccation and can survive a loss of
up to 85–95% of their water during desiccation at daytime
low tides (Blouin et al., 2011). Hence, Porphyra spp. is an
excellent model for investigating the response and tolerance
mechanisms of intertidal algae to desiccation. Furthermore,
the PSI-driven cyclic electron flow is particularly important
in the plant response to drought or desiccation; therefore, as
a highly desiccation-tolerant species, Porphyra is an appropriate model for studying the effect of desiccation on cyclic
electron flow around PSI in plants.
Most Porphyra blades consist of a single layer of cells
during the macroscopic gametophyte blade phase of their
life cycle. This feature is quite distinct from higher plants.
Porphyra has become an important marine crop worth
;US$1.3 billion per year (Blouin et al., 2011) and has been
intensively cultivated in eastern Asian countries, including
China, Japan, and South Korea. Porphyra tolerance to
desiccation has been widely applied in Porphyra aquaculture. Nets seeded with P. yezoensis blades are raised out of
the sea by farmers to expose the blades to air for several
hours a day, several times a week. This aerial drying is used
to kill harmful seaweeds and strengthen the disease
resistance of Porphyra (Lin et al., 2009; Blouin et al., 2011).
To date, there have been several reports on the effect of
desiccation on Porphyra photosynthetic performance
(Johnson et al., 1974; Smith et al., 1986; Lipkin et al., 1993;
Zou and Gao, 2002; Lin et al., 2009; Contreras-Porcia et al.,
2011). In addition, reports show that Porphyra blades have
developed a high tolerance to desiccation (Sibbald and
Vidaver, 1987; Lipkin et al., 1993; Lin et al., 2009; Blouin
et al., 2011; Contreras-Porcia et al., 2011). The photosynthetic function of Porphyra blades under severe desiccation
can be restored rapidly when re-hydrated, but there is little
information available on the response and tolerance
mechanisms of Porphyra to desiccation. Little is known
regarding the responses of photosynthetic electron flow in
Porphyra to desiccation, although this has been documented
in lichens and higher plants (Bukhov et al., 2004; Heber and
Shuvalov, 2005; Liu et al., 2006).
Recently, it has been reported that an increase in cyclic
electron flow around PSI occurred in desiccated green macroalgae, Ulva sp, which are higher green algae with a photosynthetic apparatus similar to that of higher plants (Gao et al.,
2011). However, Porphyra is a phylogenetically basal red
algae which has special light-harvesting complexes (phycobilisomes) that are similar to those of cyanobacteria. In order
to investigate the relationship between the cyclic electron flow
around PSI and the desiccation and re-hydration of these
lower red algae, P. yezoensis blades were chosen for the study
and their physiological responses to desiccation and
re-hydration were determined. Furthermore, particular attention was paid to photosynthetic electron flows around PSI
and PSII during desiccation and re-hydration.
Materials and methods
Sample collection
Porphyra yezoensis blades were collected in the morning from the
intertidal zones of Qingdao (360#3##N, 1212#0##E), China,
between April and May 2011 when irradiance ranged between 300
lmol photons m�2 s�1 and 400 lmol photons m�2 s�1. The blades
were rinsed lightly in sterile seawater to remove any sediment or
epiphytes and then were cultured in the laboratory at 15 C and
under an irradiance regime of 150–200 lmol photons m�2 s�1.
Healthy blades with similar physiological states were chosen.
Water content determination
The absolute water content (AWC) of the P. yezoensis blades was
determined by:
AWC ¼ ðWt � Wd Þ=ðWo � Wd Þ3100%
where Wd is the weight of blades dried for 24 h at 80 C; W0 is the
wet weight of the fresh blades after the gentle removal of water
from the surface; and Wt is the weight of blades at time t after
dehydration. Tissue paper was used to remove water from the
surface of the blades in order to avoid the formation of salt
crystals. The blades were then naturally dehydrated in the
laboratory at 150–200 lmol photons m�2 s�1 at room temperature. When they reached ;13% AWC, the desiccated blades were
re-hydrated in seawater at room temperature.
Chlorophyll fluorescence and P700 measurement
The photosynthetic properties of the blades were measured during
desiccation and re-hydration. The chlorophyll fluorescence of PSII
and the redox state of P700 were determined concomitantly at
room temperature using a Dual-PAM-100 fluorometer (Walz,
Effeltrich, Germany) connected to a PC with WinControl software. In order to assess the fluorescence and P700 parameters, the
repetitive application of saturation pulses and the automated
induction and recovery curve routine in the Dual-PAM software
were used. Light from a 620 nm light-emitting diode (LED) and
blue actinic light at 100 lmol photons m�2 s�1 from 460 nm LED
arrays were delivered to P. yezoensis blades for 5 min periods by
a DUAL-DR measuring head. Additionally, saturating light pulses
of 300 ms duration and 10 000 lmol photons m�2 s�1 were
delivered. The P. yezoensis blades were dark adapted for 3–5 min
before measurement. F0 was determined and then a saturating
flash was applied to the blades in order to detect the maximum
fluorescence (Fm). The difference between Fm and F0 was referred
to as the variable fluorescence (Fv), and the maximum quantum
yield was obtained as Fv/Fm (Schreiber, 2004). Fm# was measured
when the blades were illuminated. Y(II) is the effective
Cyclic
electron
in desiccated
P. yezoensis 4351
Cyclic
electron
flowflow
in desiccated
P. yezoensis
| 3| of
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photochemical quantum yield of PSII (Kramer et al., 2004b). The
electron transport rate of PSII (ETRII) was calculated by
Dual-PAM software. The Fv/Fm and F0 images of P. yezoensis
blades during desiccation and re-hydration were examined with the
Maxi version of an IMAGING-PAM M-series (Walz, Effeltrich,
Germany).
Together with fluorescence measurement, the saturation pulse
method was used to determine P700 parameters (Klughammer and
Schreiber, 1994). P700 was measured in the dual-wavelength mode
(photodetector set to measure 875 nm and 830 nm pulsemodulated light) (Schreiber and Klughammer, 2008). The
maximum P700 signal, Pm, was determined by application of the
saturation pulse in the presence of far-red light, which was
considered as analogous to Fm. The zero P700 signal, P0, was
determined when complete reduction of P700 was induced after the
saturation pulse and cessation of far-red illumination. Pm# is the
maximum P700 signal induced by combined actinic illumination
plus saturation flash. Based on the values of Pm, P0 and Pm#, the
Y(I), as well as the quantum yield of non-photochemical energy
dissipation in PSI due to donor-side limitation and acceptor-side
limitation [Y(ND) and Y(NA), respectively] were calculated by
Dual-PAM software and saved in a report file (Pfündel et al.,
2008). The photosynthetic electron transport rates of PSI (ETRI)
were also calculated by Dual-PAM software.
Inhibitor treatment
To monitor electron flow around PSI and PSII, the inhibitors
3-(3#,4#-dichlorophenyl)-1,1-dimethylurea (DCMU) and dibromothymoquinone (DBMIB) were used to treat P. yezoensis blades at
different levels of dehydration before measurement with a
Dual-PAM-100. DCMU is known to block electron transport
after the primary acceptor in PSII (QA). Therefore, linear electron
flow was diminished or abolished in the presence of DCMU (Joet
et al., 2002). With the DCMU treatment, fully hydrated (100%
AWC) blades were treated at room temperature with 5 lM
DCMU for 1 min and the ETR was subsequently measured. After
measurement, blades were treated at room temperature for 1 min
in the presence of 80 lM DBMIB, which is known to block the
electron transport from plastoquinone (PQ) to the cytochrome b6/f
complex (Frank and Trebst, 1995; Ivanov et al., 2005; Iwai et al.,
2010) and effectively suppresses cyclic electron flow around PSI
(Herbert et al., 1990). Subsequently, the blades were measured
again. As described above, the photosynthetic performance of
blades at 69% and 20% AWC were examined after treatment with
the inhibitors.
The blades at 13% AWC were re-hydrated for 5 min in seawater
containing 5 lM DCMU before measurement, following which the
blades were incubated in 10 lM antimycin A (AA) for 5 min, which
specifically inhibits PGR5-dependent PSI cyclic electron transport
(Munekage et al., 2002; Shikanai, 2007). Then the ETR was
measured. Subsequently, they were incubated for 5 min in 80 lM
DBMIB before measurement. The ratios of cyclic electron flow to
total electron flow at varying levels of desiccation were derived
from:
physiological states of the blades after varying dark treatment
times were measured.
Statistical analysis
The results were expressed as mean values of five independent
experiments 6standard deviation (SD). Data were analysed by
analysis of variance (ANOVA) using the SPSS 18.0 statistical
software. Tukey’s multiple comparison test was used at an a¼0.05
significance level to determine any significant differences between
different levels of desiccation.
Results
Maximum quantum yield (Fv /Fm) and minimum
fluorescence (F0) of blades during desiccation and rehydration
There were large changes in Fv/Fm and F0 during desiccation
and re-hydration (Figs 1, 2). Fv/Fm decreased significantly
with increasing loss of water from the blades (Fig. 1).
Meanwhile, F0 increased steadily as the blade water content
dropped between onset of desiccation and 14% AWC where
F0 reached its maximum (Fig. 2). When the AWC of the
blades decreased to ;14%, the blades were re-hydrated.
Clearly, the Fv/Fm increased significantly during re-hydration
and was fully restored after 30 min of re-hydration (Fig. 1).
In contrast, there was a decreasing trend in F0 during rehydration (Fig. 2). In addition, the Fv/Fm and F0 blade
images were examined during desiccation and re-hydration
with a Maxi-Imaging-PAM (Figs 1A, 2A). The images for
ETRI ðtreated with DCMUÞ=ETRI ðno inhibitorÞ
Dark treatment
To investigate the influence of starch degradation on photosynthetic electron flow, the P. yezoensis blades were incubated at
15 C in darkness for 12, 24, 48, and 72 h, respectively. After dark
treatment, the blades were immediately treated with 5 lM DCMU
in darkness. Then the blades were dehydrated naturally in the
presence of DCMU to avoid the influence of linear electron flow
on cyclic electron flow around PSI. They were re-hydrated for
5 min in seawater containing 5 lM DCMU when AWC reached
;13%, after which the ETR was measured. In addition, the
Fig. 1. (A) Images of Fv/Fm for P. yezoensis blades at various
desiccation levels and different durations of re-hydration. The bar
along the top of the image indicates the relative value of each
parameter as a percentage. The scale bar represents 1 cm.
(B) Variations in Fv/Fm for P. yezoensis blades at various desiccation
levels and different durations of re-hydration (corresponding to the
images of Fv/Fm in A). The data are the mean of five independent
experiments (6SD). (This figure is available in colour at JXB online.)
4352 and
Wang
4 of 10| Gao
| Gao
and
Wang
Fig. 3. Variations in the effective quantum yields of PSI and PSII in
P. yezoensis blades during desiccation and re-hydration. The data
are the mean of five independent experiments (6SD).
Fig. 2. (A) Images of F0 for P. yezoensis blades at various
desiccation levels and different durations of re-hydration. The bar
along the top of the image indicates the relative value of each
parameter as a percentage. The scale bar represents 1 cm.
(B) Variations in F0 for P. yezoensis blades at various desiccation
levels and different durations of re-hydration (corresponding to the
images of F0 in A). The data are the mean of five independent
experiments (6SD). (This figure is available in colour at JXB online.)
Fv/Fm of fully hydrated blades (100% AWC) clearly differed
from those at 76–14% AWC (Fig. 1A). During re-hydration,
the images of Fv/Fm gradually reverted to their original
appearance, which were in accordance with the Fv/Fm values
(Fig. 1B). The images of F0 at different levels of desiccation
and different durations of re-hydration (Fig. 2A) showed that
blades with ;14% AWC had higher values for F0, which were
also similar to the F0 values shown in Fig. 2B.
Quantum yields of PSI and PSII during desiccation and
re-hydration
There were large changes in the effective quantum yields of
PSI and PSII in the blades during desiccation and
re-hydration (Fig. 3). Y(II) increased slightly (P < 0.05)
from the onset of desiccation down to 70% AWC. During
further desiccation, Y(II) declined steadily and decreased to
zero when AWC neared 23%. In comparison, Y(I) clearly
rose from the onset of desiccation down to 70% AWC,
where Y(I) reached a maximum (0.8). There were significant
differences (P < 0.05) between Y(I) at 100% AWC and 70%
AWC. Subsequently, Y(I) decreased steadily to zero as
AWC approached ;13%. Upon re-hydration, both Y(I)
and Y(II) increased sharply. However, the recovery of Y(I)
was more rapid than that of Y(II). Moreover, there was
a decreasing trend in Y(I) during further re-hydration.
There were variable tendencies in the quantum yields of
non-photochemical energy dissipation in PSI under desiccation and re-hydration (Fig. 4). During desiccation, the
Fig. 4. Variation in quantum yields of non-photochemical energy
dissipation in PSI [Y(ND) and Y(NA)] in P. yezoensis blades during
dehydration and re-hydration. The data are the mean of five
independent experiments (6SD).
Y(ND), rose gradually to a maximum (0.8) when AWC
reached ;13%. In contrast, the Y(NA) did not increase
significantly between the onset of dehydration and an AWC
of ;39%. Interestingly, it declined significantly at moderate
levels of desiccation. During further desiccation (39–13%
AWC), Y(NA) increased significantly to a maximum of
;0.55. After 5 min of re-hydration, both Y(ND) and
Y(NA) were restored to normal levels. However, both
showed decreasing trends under further re-hydration.
Electron transport rates of PSI and PSII during desiccation
and re-hydration and their response to inhibitors
The electron transport rates of PSI and PSII changed
considerably during desiccation and re-hydration (Fig. 5).
The ETRII increased slightly from the onset of desiccation
Cyclic
electron
in desiccated
P. yezoensis 4353
Cyclic
electron
flowflow
in desiccated
P. yezoensis
| 5| of
10
Fig. 5. Changes to the ETRI and the ETRII in P. yezoensis blades
during desiccation and re-hydration. The data are the mean of five
independent experiments (6SD).
to 70% AWC, where the ETRII values reached a maximum
of ;17%. With further dehydration, the ETRII dropped
steadily and decreased to zero when AWC approached 23%.
In contrast, the ETRI rose significantly and increased to
a maximum of ;34 at 70% AWC. There were significant
differences between the ETRI at 100% AWC and at 70%
AWC (P < 0.05). Subsequently, the ETRI declined
gradually and decreased to zero when AWC reached 13%.
After a 5 min re-hydration period, the ETRI recovered
rapidly to a level even higher than that of the fully hydrated
blades (100% AWC). In contrast, the ETRI decreased
slightly after a further period of re-hydration. After 30 min
of re-hydration, the ETRII had slowly recovered but had
still not reached the pre-desiccation rate.
During an investigation of the photosynthetic electron
transport in PSI and PSII in P. yezoensis blades under
desiccation, the ETRI and the ETRII of the blades were
determined in the presence of inhibitors at varying levels of
desiccation: 100% AWC, 69% AWC, and 20% AWC (Fig. 6
and Table 1). When the fully hydrated blades (100% AWC)
were treated with DCMU, the ETRII dropped sharply to
zero, suggesting that linear electron flow was effectively
inhibited (Fig. 6A). Meanwhile, the ETRI decreased significantly to a relatively low value (2.8), which then
decreased immediately to zero upon treatment with DBMIB
(Fig. 6A). When the blades at 69% AWC were treated with
DCMU, both the ETRI and the ETRII decreased strongly
to 5.3 and 0, respectively. Furthermore, the ETRI was
effectively suppressed by DBMIB (Fig. 6B and Table 1).
When AWC reached 20%, both the ETRII and the ETRI
decreased to 0 and 12.8, respectively. Clearly, the ETRI was
not affected by DCMU but was greatly reduced by
DBMIB. The ratio of cyclic electron flow to total electron
flow in desiccated blades at 69% AWC (14.9%) was much
higher than that of fully hydrated blades (9.4%; Table 1). At
20% AWC, this increased sharply to 97.7%, suggesting that
cyclic electron flow around PSI was dominant at this level
Fig. 6. Changes to the ERTI and the ERTII in P. yezoensis blades
at various desiccation levels in response to DCMU and DBMIB.
(A) The responses of ETRI and ETRII in fully hydrated blades (100%
AWC) to DCMU (5 lM) and DBMIB (80 lM); (B) the responses of
ETRI and ETRII in blades with 69% AWC to DCMU (5 lM) and
DBMIB (80 lM); and (C) the responses of ETRI and ETRII in blades
with low water content (20% AWC) to DCMU (5 lM) and DBMIB
(80 lM). The DBMIB treatment was performed after the blades
had been treated with DCMU. The data are the mean of five
independent experiments (6SD).
of desiccation. All these results suggested that cyclic
electron flow around PSI in P. yezoensis blades was less
sensitive to desiccation than PSII and continued to operate
under severe desiccation conditions.
Changes in electron transport rates in PSI in response
to varying durations of dark treatment and to inhibitors
during re-hydration
When AWC reached 13%, the ETRI decreased to its lowest
level (0), which rapidly recovered during re-hydration
(Fig. 5). More interestingly, the ETRI could still recover to
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Table 1. The ETRI of blades at varying durations of desiccation (100, 69, and 20%) and the ETRI of blades treated with the inhibitors
DCMU and DBMIB at varying levels of desiccation
The ratio of cyclic electron flow to total electron flow was calculated as ETRI (treated by DCMU)/ETRI (no inhibitor). The data are mean
values from five independent replicates.
Desiccation levels
ETRI (no inhibitor)
ETRI (DCMU)
ETRI (DBMIB)
Cyclic electron flow/total electron flow
Fully hydrated state (100% AWC)
69% AWC
20% AWC
29.861.5 (n¼5)
35.660.8 (n¼5)
12.860.7 (n¼5)
2.860.2 (n¼5)
5.360.5 (n¼5)
12.560.6 (n¼5)
0 (n¼5)
0 (n¼5)
0 (n¼5)
9.4%
14.9%
97.7%
;10 when desiccated blades, with an AWC of 13%, were
treated with DCMU to suppress PSII. The ETRI was not
influenced by AA, whereas it was effectively stopped by
DBMIB (Fig. 7A). These results indicated that as PSII was
suppressed, PSI-driven cyclic electron flow in P. yezoensis
blades could still be restored during re-hydration and that it
was insensitive to AA.
It has been proposed that starch degradation can provide
exogenous NAD(P)H, which could donate electrons to
intersystem electron carriers (Bukhov et al., 2002; Bukhov
and Carpentier, 2004). The P. yezoensis blades were treated
in darkness to determine the effects of starch degradation on
the recovery of cyclic electron flow around PSI during
re-hydration. The results showed no obvious changes in the
physiological states of blades undergoing varying durations
of dark treatment, as reflected in the similar ETRI and Fv/Fm
values at 100% AWC. There was a gradual decline in the
ETRI when desiccated blades at 13% AWC were re-hydrated
in the presence of DCMU while undergoing prolonged dark
treatment (Fig. 7B). The ETRI was much lower in the
re-hydrated blades that had undergone dark treatment for
72 h compared with those that had not undergone dark
treatment (i.e. dark 0 h). There were significant differences in
the ETRI between dark treatment times.
Discussion
The responses of Fv /Fm and F0 to desiccation and
re-hydration
There were large changes in Fv/Fm and F0 in P. yezoensis
blades during desiccation and re-hydration (Figs 1, 2).
Fv/Fm is a sensitive indicator of the photosynthetic performance of plants, and decreases significantly when plants are
subjected to stresses (Maxwell and Johnson, 2000). The
results of this study suggested that in P. yezoensis, a lower
eukaryotic photosynthetic organism, the response of blade
Fv/Fm to desiccation was similar to that of higher plants.
However, even following the loss of >85% of their water
content, the blades recovered rapidly when re-hydrated,
which is quite different from the response in higher plants
(with the exception of some bryophytes and ferns). In
addition, F0 gradually increased with the loss of water
(Fig. 2), whereas, during re-hydration, F0 decreased. It has
been proposed that the increase in F0 is a result of the
separation of the light-harvesting pigment protein complex
Fig. 7. (A) Changes to the ERTI and ETRII in desiccated
P. yezoensis blades (13% AWC) in response to re-hydration in the
presence of DCMU. The antimycin A (AA) treatment was
performed after the blades had been treated with DCMU and
subsequently the blades were treated with DBMIB before further
measurement. (B) The influence of dark treatment on the recovery
of cyclic electron flow around PSI in desiccated blades (13%
AWC). Insets show the Fv/Fm of blades having undergone varying
durations of dark treatment. The desiccated blades were rehydrated in the presence of DCMU.
from PSII core complexes in isolated chloroplasts under
heat-pre-treated conditions (Schreiber and Armond, 1978).
Cyclic
electron
in desiccated
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Alternatively, Yamane et al. (1997) reported that a partly
reversible inactivation of the PSII reaction centre at high
temperature could lead to an increase in F0. It is known that
the main light-harvesting complexes in Porphyra are phycobilisomes that are composed of predominantly hydrophilic
polypeptides and are highly mobile (Mullineaux et al., 1997;
MacColl, 1998). Nevertheless, F0 still increased significantly
as the AWC reached 23% and PSII was inactivated. It
should be noted that phycobilisomes are essentially
immobile under such desiccated conditions. These results
suggested that the reversible inactivation of the PSII
reaction centre might be the main cause of the increase in
F0 in P. yezoensis blades during desiccation.
Cyclic electron flow activity around PSI increased
significantly during desiccation
Clearly, the PSI activity of P. yezoensis blades increased
significantly (P < 0.05; Tukey’s test) during desiccation and
was still activated under severe desiccation (19–15% AWC)
when linear electron flow had stopped (Fig. 5). Cyclic
electron flow around PSI at 69% AWC was much higher
than at 100% AWC (Fig. 6A, B). Furthermore, the PSI
activity of desiccated blades was not affected by DCMU but
was specifically suppressed by DBMIB (Fig. 6C). All these
results implied that PSI-driven cyclic electron flow in
P. yezoensis blades was elevated during desiccation and still
continued to operate under severe desiccation. These results
were consistent with earlier reports that cyclic electron flow
around PSI was stimulated by many stresses such as drought
and salt stress (Jeanjean et al., 1993; Joet et al., 2002; Golding
and Johnson, 2003; Bukhov and Carpentier, 2004; Gao et al.,
2011). Indeed, cyclic electron flow around PSI plays a critical
physiological role in plant tolerance to desiccation (Canaani
et al., 1989; Herbert et al., 1990; Heber and Walker, 1992;
Horváth et al., 2000; Munekage et al., 2002; Golding and
Johnson, 2003). During photosynthesis, cyclic electron flow
around PSI could generate a pH gradient across the
thylakoid membrane that not only could be used to
synthesize ATP, but could also induce thermal dissipation
and provide protection for the photosynthetic apparatus
(Heber and Walker, 1992; Munekage et al., 2002; Golding
and Johnson, 2003; Kramer et al., 2004a). Hence, it is
possible that in P. yezoensis blades, cyclic electron flow
around PSI may contribute substantially to photosynthetic
electron flow during desiccation in order to provide ATP and
protect the photosynthetic apparatus. This could potentially
be one of the most important factors allowing P. yezoensis
blades to tolerate desiccation stress during low tide.
The up-regulation of cyclic electron flow around PSI
triggered a transient increase in photosynthetic activity
at moderate levels of desiccation
The effective quantum yields of PSI and PSII were
significantly affected by desiccation and re-hydration
(Fig. 3). PSI activity increased significantly (P < 0.05;
Tukey’s test) at a moderate level of desiccation (70%
AWC), as reflected in increased Y(I). In contrast, PSII
activity increased slightly from the onset of desiccation to
70% AWC. A transient increase in photosynthetic capability
has been reported in some Porphyra spp. (Johnson et al.,
1974; Gao and Aruga, 1987; Lipkin et al., 1993). It is
unclear what caused the increased photosynthetic capability
in Porphyra at moderate levels of desiccation.
The quantum yields of non-photochemical energy dissipation in PSI increased significantly during desiccation as
reflected by Y(ND) and Y(NA). Y(ND) and Y(NA) show
the quantum yield of non-photochemical energy dissipation in
PSI reaction centres due to a shortage of electrons and
electron acceptors, respectively (Pfündel et al., 2008). The
increase in Y(ND) correlated with the functional breakdown
of PSII (Fig. 3). The activity of PSII, which provides electrons
by water splitting, was rapidly suppressed at severe levels of
desiccation. Consequently, the rates of linear electron flow
were low and even stopped, which ultimately resulted in
elevated Y(ND). These results suggested that both donor-side
and acceptor-side electron flow were affected during desiccation. There was a slight decrease in Y(ND) at moderate levels
of desiccation (Fig. 4). More interestingly, as AWC
approached 61%, Y(NA) declined significantly but increased
steadily under further desiccation—a phenomenon also found
in desiccated Ulva spp. (Gao et al., 2011). Cyclic electron flow
around PSI increased significantly at 70% AWC (Fig. 5 and
Table 1), which could lead to the effective downstream
transport of electrons. The transient increase in Y(I) and the
decline of Y(NA) at moderate levels of desiccation demonstrated that the redox rate of P700 was elevated. These results
suggested that up-regulation of cyclic electron flow around
PSI at moderate levels of desiccation led to increased PSI
activity in P. yezoensis blades and ultimately resulted in
a transient increase in photosynthetic activity.
The recovery of cyclic electron flow around PSI as linear
electron flow was suppressed
After only 5 min of re-hydration, the ETRI of the desiccated
blades at 13% AWC rapidly recovered to former levels
(Fig. 5), suggesting that PSI recovery was much faster than
that of PSII. Furthermore, this study found that when the
blades at 13% AWC (where ETRI had decreased to zero)
were treated with DCMU, the ETRI could still be restored to
;10 (Fig. 7A). These results suggested that as PSII was
suppressed, the PSI-driven cyclic electron flow in desiccated
P. yezoensis blades could still be restored. Thus, the recovery
of cyclic electron flow around PSI in the desiccated blades
was independent of linear electron flow. It remains unclear
how cyclic electron flow around PSI was restored during rehydration if there was no electron donation from PSII.
These results indicated that prolonged dark treatment of
blades was accompanied by a gradual decline in the ETRI
when blades at 13% AWC were re-hydrated in DCMU.
Therefore, this study suggests that the dark treatment
(floridean starch degradation) of P. yezoensis blades, which
caused a decrease in the pool of stromal reductants
including NAD(P)H, profoundly influenced the recovery of
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PSI-driven cyclic flow in the desiccated blades. There is
strong evidence that exogenous NADPH and NADH can
donate electrons to intersystem electron carriers (Asada
et al., 1992, 1993; Endo et al., 1997; Bukhov et al., 2001).
For example, in the mesophyll chloroplasts of maize, the
pool of stromal reductants, such as NADPH, is capable of
rapid electron donation to P700+ (Asada et al., 1993) and
the NADPH and NADH produced are mainly generated
from starch degradation in the chloroplasts (Bukhov et al.,
2002; Bukhov and Carpentier, 2004).
The cyclic electron flow around PSI was insensitive to AA
but was effectively suppressed by DBMIB (Fig. 7A). Two
pathways of cyclic electron flow around PSI have been
proposed, including the PGR5-dependent cyclic flow, which
could be specifically inhibited by AA, and the chloroplastic
NAD(P)H dehydrogenase (NDH)-dependent cyclic flow
(Shikanai, 2007; Endo et al., 2008). The results of this study
suggested that the NDH-dependent cyclic flow played
a significant physiological role in the recovery of cyclic
electron flow around PSI in P. yezoensis blades. Furthermore, the results showed that the ETRI increased significantly when the blades were exposed to AA for >15 min.
Indeed, AA is also a known inhibitor of the respiratory
cytochrome b/c1 complex and may cause accumulation of
NADH in mitochondria, which could then be transported
into chloroplasts (Rochaix, 2011). This would cause increased
cyclic electron flow around PSI. These results further support
the notion that stromal reductants, such as NAD(P)H,
donate electrons to intersystem electron carriers. Published
papers, together with these results, suggest that stromal
reductants, such as NAD(P)H, which could accumulate by
floridean starch degradation, may contribute substantially to
the rapid recovery of PSI-driven cyclic electron flow in
re-hydrated blades. This could generate a pH gradient and
provide ATP for a variety of processes in the chloroplast and
also protect the photosynthetic apparatus.
The photosynthetic performance of P. yezoensis blades
changed significantly during desiccation and re-hydration.
The activity of PSI-driven cyclic electron flow was elevated
significantly during desiccation and continued to operate
under desiccated conditions, demonstrating much greater
tolerance to desiccation stress than PSII. The PSI in
desiccated blades was restored faster than PSII during rehydration. Most importantly, as PSII was suppressed, the
PSI-driven cyclic electron flow of the desiccated P. yezoensis
blades at 13% AWC could still be rapidly restored when rehydrated and was also insensitive to AA. Moreover, the
recovery of cyclic electron flow around PSI was influenced
by the dark treatment duration, suggesting that stromal
reductants, including NAD(P)H, played an important role
in the donation of electrons to PSI and was mainly
responsible for the rapid recovery of cyclic electron flow
around PSI in the desiccated blades during re-hydration.
Overall, this study suggests that cyclic electron flow around
PSI in P. yezoensis blades plays a significant physiological
role during desiccation and re-hydration. This may be one
of the most important factors allowing P. yezoensis blades
to adapt to intertidal environments.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China [30830015, 30970302, 40806063,
B49082401].
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