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1. REGULAR PAPER
Age-dependent changes in the functions and compositions
of photosynthetic complexes in the thylakoid membranes
of Arabidopsis thaliana
Krishna Nath • Bong-Kwan Phee • Suyeong Jeong •
Sun Yi Lee • Yoshio Tateno • Suleyman I. Allakhverdiev •
Choon-Hwan Lee • Hong Gil Nam
Received: 28 March 2013 / Accepted: 30 July 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Photosynthetic complexes in the thylakoid
membrane of plant leaves primarily function as energy-
harvesting machinery during the growth period. However,
leaves undergo developmental and functional transitions
along aging and, at the senescence stage, these complexes
become major sources for nutrients to be remobilized to
other organs such as developing seeds. Here, we investi-
gated age-dependent changes in the functions and compo-
sitions of photosynthetic complexes during natural leaf
senescence in Arabidopsis thaliana. We found that Chl a/b
ratios decreased during the natural leaf senescence along
with decrease of the total chlorophyll content. The photo-
synthetic parameters measured by the chlorophyll fluores-
cence, photochemical efficiency (Fv/Fm) of photosystem II,
non-photochemical quenching, and the electron transfer
rate, showed a differential decline in the senescing part of
the leaves. The CO2 assimilation rate and the activity of
PSI activity measured from whole senescing leaves
remained relatively intact until 28 days of leaf age but
declined sharply thereafter. Examination of the behaviors
of the individual components in the photosynthetic com-
plex showed that the components on the whole are
decreased, but again showed differential decline during leaf
senescence. Notably, D1, a PSII reaction center protein,
was almost not present but PsaA/B, a PSI reaction center
protein is still remained at the senescence stage. Taken
together, our results indicate that the compositions and
structures of the photosynthetic complexes are differen-
tially utilized at different stages of leaf, but the most dra-
matic change was observed at the senescence stage,
possibly to comply with the physiological states of the
senescence process.
Keywords Arabidopsis thaliana Chl contents and
Chl a/b ratio Developmental stage and senescence
Nutrient mobilization Photosynthetic performance
Photosynthetic complexes and their components
Electronic supplementary material The online version of this
article (doi:10.1007/s11120-013-9906-2) contains supplementary
material, which is available to authorized users.
K. Nath B.-K. Phee Y. Tateno H. G. Nam ()
Department of New Biology, DGIST, Daegu 711-873,
Republic of Korea
e-mail: nam@dgist.ac.kr
S. Jeong
Department of Molecular and Life Science, POSTECH,
Pohang 790-784, Republic of Korea
S. Jeong H. G. Nam
Academy of New Biology for Plant Senescence and Life
History, Institute for Basic Science, Daejeon, Republic of Korea
S. Y. Lee
Protected Horticulture Research Station, National Institute of
Horticultural Herbal Science, RDA, Busan 618-800,
Republic of Korea
S. I. Allakhverdiev
Institute of Plant Physiology, Russian Academy of Sciences,
Botanicheskaya Street 35, Moscow 127276, Russia
S. I. Allakhverdiev
Institute of Basic Biological Problems, Russian Academy of
Sciences, Pushchino, Moscow Region 142290, Russia
C.-H. Lee ()
Department of Molecular Biology, Pusan National University,
Busan 609-735, Republic of Korea
e-mail: chlee@pusan.ac.kr
123
Photosynth Res
DOI 10.1007/s11120-013-9906-2

2. Abbreviation
BN-PAGE Blue-native polyacrylamide gel
electrophoresis
Chl Chlorophyll
ETR Electron transport rate
Fv/Fm Maximum photochemical efficiency of PSII
for dark-adapted sample
LHC Light-harvesting complex
NPQ Non-photochemical quenching
PAR Photosynthetically active radiation
PS Photosystem
RC Reaction center
Introduction
The plant leaf is a major organ for photosynthesis in higher
plants. Photoassimilates are essential for growing leaves,
and they provide nutrients to other parts of the plant. On
the other hand, leaves are under the control of develop-
mental signals, including aging. Therefore, photosynthetic
functions and development are expected to be highly
coordinated. One of the most dramatic examples of the
coordination between photosynthesis and development
occurs during leaf senescence. During leaf senescence, an
initial visible sign of senescence in the leaf is color change,
which is due to the preferential degradation of chlorophyll
(Chl) (Matile et al. 1992). The gradual loss of Chl is nor-
mally accompanied by a decrease in photosynthetic activity
as well as by differential changes in the structural com-
ponents of photosynthetic complexes in the thylakoid
membranes during senescence (Park et al. 2007; Sakuraba
et al. 2012; Zhang et al. 2012). In leaves, photosynthetic
activity is the highest at the mature stage and then declines
gradually during senescence (Stoddart and Thomas 1982;
Gepstein 1988; Feller and Fischer 1994; Smart 1994; Tang
et al. 2005). The decline in photosynthetic activity during
senescence is accompanied by the breakdown of thylakoid
membranes and stromal proteins (Gepstein 1988; Sakuraba
et al. 2012). Chloroplasts are also dismantled in an orderly
sequence, and the components in the individual photo-
synthetic complexes are likely to be adjusted in a coordi-
nated manner (Noodén et al. 1997; Park et al. 2007).
Thylakoid membranes in the chloroplast harbor most of
the photosynthetic system. The photosynthetic system in the
thylakoid membranes contains several multi-subunit pig-
ment-binding protein complexes, including photosystem I
(PSI), photosystem II (PSII), cytochrome b6/f, and ATP
synthase (Wollman et al. 1999; Nelson and Yocum 2006;
Allakhverdiev 2011, 2012). Among these, PSI and PSII are
the most abundant Chl-protein (CP) complexes and are
equipped with specific light-harvesting complexes (LHCs)
and reaction centers (RCs) (Green and Durnford 1996). PSI-
associated LHCI and PSII-associated LHCII proteins are
encoded by the Lhca and Lhcb gene families, respectively.
PSI is located in the grana margins and stroma lamellae of
thylakoid membranes, where the RC is surrounded by six
LHCI proteins (Lhca1-6) (Jansson 1999; Neilson and
Durnford 2010). Among these, Lhca1, Lhca2, Lhca3, and
Lhca4 are the main LHCI antenna proteins, assembled in
dimeric forms, and are encoded by Lhca1, Lhca2, Lhca3, and
Lhca4, respectively. In addition, Lhca5 and Lhca6, which are
encoded by Lhca5 and Lhca6, respectively, have also been
reported in LHCI (Neilson and Durnford 2010). In higher
plants, LHCI contains eight molecules of Chl a and two
molecules of Chl b (Croce and Bassi 1998). PSII is located
predominantly in the stacked region of the thylakoid mem-
brane. The most important CP complex involved in stacking
of grana is the major trimeric LHCII complex of PSII (Allen
and Forsberg 2001). Lhcb1, Lhcb2, and Lhcb3 are the major
LHCII antenna proteins and comprise either homo- or het-
ero-trimers and are encoded by Lhcb1, Lhcb2, and Lhcb3,
respectively, while monomers CP29, CP26, and CP24 are
minor LHCII antenna proteins and are encoded by Lhcb4,
Lhcb5, and Lhcb6, respectively. LHCII in higher plants is
associated with 14 Chl a molecules and eight Chl b mole-
cules (Liu et al. 2004).
Leaf senescence is a developmental process that is regu-
lated by several developmental and environmental signals
(Lim et al. 2007). The gradual loss of Chl during leaf
senescence is normally accompanied by a decrease in pho-
tosynthetic activity along with the degradation of structural
components of photosynthetic complexes in the thylakoid
membrane. Chl plays a key role in forming light-harvesting
protein complexes and the RC protein complexes of PSI and
PSII. The photosynthetic complexes in higher plants contain
Chl a and Chl b. Chl a is a major component of LHC and RC,
whereas Chl b is restricted to LHC (Green and Durnford
1996; Oster et al. 2000; Liu et al. 2004; Morita et al. 2009;
Allakhverdiev 2011, 2012). Levels of both Chl a and Chl
b were decreased during leaf senescence (Thomas et al.
2003; Huang et al. 2013). The decrease in Chl a and b is
accompanied by degradation of many photosynthetic pro-
teins. Lepeduš et al. (2010) found that the differential deg-
radation of major photosynthetic proteins causes functional
disturbance of PSII photochemistry. In addition, Cyt b6f and
Rubisco, particularly the large subunit of Rubisco (RbcL),
are also degraded during natural senescence (Holloway et al.
1983; Crafts-Brandner et al. 1990; Grassl et al. 2012).
Leaf senescence is not just a passive process. Rather, it is
an active process that serves to remobilize nutrients to the
required sinks such as young leaves and developing seeds. It
is thus critical for this process to be regulated in a highly
coordinated manner. Photosynthetic components are highly
abundant and thus serve as major sources of nitrogen to be
remobilized. The delicate balance between active
Photosynth Res
123

3. photosynthesis and degradation of the photosynthetic com-
plex is critically required during leaf senescence. Owing to
its importance in plant productivity, leaf senescence has been
extensively studied at the physiological, biochemical, and
(recently) genetic levels. Although several types of defects in
photosynthesis accompany leaf senescence, some of the
most important aspects of this process, such as the differ-
ential utilization of photosynthetic complexes and the
coordinated regulation of their components along successive
stages of leaf development, are not well understood and have
not yet been thoroughly elucidated.
In this study, we explored how the functions and com-
positions of photosynthetic complexes change during the
leaf lifespan, including senescence. Here, we report our
results on age-dependent changes in Chl content, photo-
synthetic performance, and dynamic behavior of photo-
synthetic complexes in the leaves of Arabidopsis thaliana
(ecotype Col-0).
Materials and methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Col-0 was used throughout
the experiment. Plants were grown at 22 °C at a relative
humidity of 60–70 % under a 16/8-h light/dark cycle. To
induce uniform germination, seeds were incubated at 4 °C
for 3 days before sowing on soil. Lighting was provided by
an Osram FHF32SSEX-W 32 W fluorescent lamp to
ensure a minimum of 100 lmol photons m-2
s-1
on the
upper leaf surfaces. The first set of rosette (third and
fourth) leaves of individual plants was used in each
experiment.
Measurement of Chl contents
The fresh weights of third and fourth leaves were recorded
before freezing the samples in liquid nitrogen. The frozen
leaf samples were then ground in 1 ml 80 % acetone and
incubated at 4 °C overnight to extract the Chl completely.
Leaf debris was removed by centrifugation at 14,000 rpm
(Sorvall Legend Micro 17 Microcentrifuge, Thermo Sci-
entific, USA) for 10 min and Chl a, Chl b, and total Chl
contents were calculated on a fresh tissue weight basis
according to Porra et al. (1989).
Analysis of Chl fluorescence and photosynthetic
performance
Age-dependent photosynthetic parameters were measured
using the MAXI version of IMAGING-PAM M-Series
(Heinz Walz GmbH, Effeltrich, Germany) at room
temperature after dark-adaptation for 10 min. To measure
Chl fluorescence, rosette leaves were placed onto a block
wrapped in water-moistened blotting paper. An area of
interest was selected from digital images of leaves to
record Chl fluorescence parameters including F0 and Fm.
Maximum photochemical efficiency of PSII or Fv/Fm was
calculated using the equation (Fm–F0)/Fm. The light
induction curves of electron transport rate (ETR) and non-
photochemical quenching (NPQ) were measured as
described by Li et al. (2010). Age-dependent changes in
photosynthetic CO2 assimilation or net photosynthesis
were measured using an Li-6400 XT infrared gas analyzer
(Li-Cor) at 25 °C under a CO2 concentration of 400 ppm.
Measurement of PSI activity
The redox state of P700 was measured using a pulse-
amplitude-modulated fluorometer (PAM101/102/103,
Walz, Effeldrich, Germany) in the reflectance mode as
described in Kim et al. (2005). The device was equipped
with a dual-wavelength (810/870 nm) emitter–detector unit
(ED-P700DW) consisting of an LED-driver unit and an
emitter–detector unit (Walz).
Separation of photosynthetic complexes by blue-native
polyacrylamide gel electrophoresis (BN-PAGE)
BN-PAGE was performed as previously described
(Schägger and von Jagow 1991) with some modifications.
Leaf samples were immediately frozen in liquid nitrogen
after harvest. Thylakoid membranes were isolated at 4 °C
according to Li et al. (2010) and washed with a buffer
containing 0.33 M sorbitol and 50 mM Bis–Tris (pH 7.0).
The thylakoids were then resuspended in suspension buffer
containing 20 % glycerol and 25 mM Bis–Tris (pH 7.0)
and adjusted to a final concentration (2 mg/ml) of Chls in
each sample. The thylakoids were then solubilized in
resuspension buffer with 2 % n-(dodecyl) b-D-maltoside,
20 % glycerol, 25 mM Bis–Tris (pH 7.0), and 10 mM
MgCl2. After gentle shaking for 30 min at 4 °C, insoluble
debris was removed by centrifugation at 18,0009g for
40 min at 4 °C. The supernatant was combined with 1/10
volume of 5 % Serva blue G (100 mM Bis–Tris [pH 7.0],
0.5 M 6-amino-n-caproic acid and 30 % glycerol). Finally,
an equal amount of supernatant equivalent to 10 lg of Chls
was subjected to BN-PAGE with a 4–20 % gradient gel.
Electrophoresis was carried out for 3 h at 30 mA at 4 °C.
SDS-PAGE and immunoblotting
Thylakoid membranes were isolated at 4 °C in grinding
buffer (50 mM HEPES [pH 7.6], 0.3 M sorbitol, 10 mM
NaCl, and 5 mM MgCl2). The resulting homogenate was
Photosynth Res
123

4. filtered through four layers of Miracloth (Calbiochem) and
centrifuged at 12,000 rpm (Sorvall Legend Micro 17
Microcentrifuge, Thermo Scientific, USA) for 10 min. The
pellet was suspended in washing buffer (50 mM HEPES
[pH 7.6], 0.1 M sorbitol, 10 mM NaCl, and 5 mM MgCl2)
and centrifuged at 12,000 rpm. This step was repeated
twice. The pellet was resuspended in washing buffer to
measure the Chl concentrations. Chl levels in isolated
thylakoids were measured following the method of Porra
et al. (1989). Finally, each sample, corresponding to 1 lg
of Chl, was loaded and electrophoresed at 120 V at room
temperature for immunoblotting analysis using specific
antibodies against specific proteins of the photosynthetic
protein complexes.
Results and discussion
Changes in Chl contents and photochemical efficiency
of PSII during successive stages of leaf development
In this study, we attempted to assay the changes in pho-
tosynthetic components during leaf aging. To obtain sam-
ples of leaves at a defined age, we used only the third and
fourth rosette leaves of Arabidopsis. The formation of these
two leaves primarily occurs at less than 1 day apart. We
counted the ages of the leaves as days after emergence of
leaf primordia. In Arabidopsis leaves, changes in Chl
content can be conveniently monitored to use as markers
for the progress of leaf senescence. Therefore, we first
measured total Chl contents at various stages of leaf
senescence to form an experimental basis for evaluating
leaf senescence. As shown in Fig. 1a, the levels of total Chl
in the 28-day-old leaves were 40 % of the maximal, control
(14-day-old) levels. In the 35-day-old leaves, only 10 % of
total Chl remained, indicating a dramatic decrease in total
Chl content during leaf senescence.
The initial target of leaf senescence for mobilization of
nutrients is the chloroplast (Smart 1994, Noodén et al.
1997), and therefore leaf senescence is associated with a
decrease in photosynthetic performance. Chl fluorescence
is a convenient measure of photosynthetic performance of
PSII, and changes in Chl fluorescence have been used as
quantitative senescence markers. We thus measured Chl
fluorescence at successive stages from the young stage at a
leaf age of 7 days to the late senescence stage at 35 days.
Fig. 2a shows the visible phenotypes of leaves at different
ages, as well as their digital false color images of Chl
fluorescence parameters. The leaves grew until they were
21 days old (late mature stage), and at 28 days, approxi-
mately 1/3 of the leaves turned yellow starting from the tip
of each leaf. At 35 days, the leaves turned completely
yellow; this was consistent with the changes observed in
Chl contents (Fig. 1a). Arabidopsis leaves contain both Chl
a and Chl b, and the differential degradation of Chl a and
b leads to changes in the Chl a/b ratio. The Chl a/b ratio
decreased linearly during aging, although the Chl content
decreased rapidly in leaves over 21 days old. However, we
previously reported an increase in the Chl a/b ratio during
dark induced senescence (DIS) in Arabidopsis leaves
(Oh and Lee 1996; Oh et al. 2000, Huang et al. 2013).
Therefore, we assume that the decrease in the Chl a/b ratio
observed during natural senescence is due to the mobili-
zation of nutrients, which does not occur during DIS in
detached leaves. Similarly, there are several reports of
decreases in the Chl a/b ratio during natural senescence in
Arabidopsis leaves (Weaver and Amasino 2001), wheat
flag leaves (Camp et al. 1982), and rice flag leaves (Tang
et al. 2005).
The spatial distribution of each Chl fluorescence
parameter can be monitored from digital false color ima-
ges. In leaves beginning at 28 days old, mesophyll cells in
the same leaf were not homogeneous in terms of Chl
content or Chl fluorescence characteristics. Therefore, we
selected a region at the tip of each leaf to quantitatively
monitor changes in Chl fluorescence characteristics in a
confined area. As shown in Fig. 2b, the minimum
Fig. 1 Changes in Chl content and Chl a/b ratio during successive
leaf ages in Arabidopsis thaliana. Changes in total Chl content a and
Chl a/b ratio b were measured in the third and fourth rosette leaves of
Arabidopsis thaliana (ecotype Col-0) in the 7-, 14-, 21-, 28-, and
35-day-old leaves. Chl was extracted according to Porra et al. (1989).
Each measurement was performed with the upper leaf sections of five
leaves. The measured Chl contents were normalized to the fresh
weights of the leaf disks. The means and standard errors (±SE) from
three to five replicates are shown
Photosynth Res
123

5. fluorescence (F0) of PSII did not change until the leaves
were 28 days old, when the F0 showed a sharp decrease,
reaching background levels in the 35-day-old leaves. By
contrast, the maximum Chl fluorescence (Fm) decreased
substantially in the 28-day-old leaves. The maximum
photochemical efficiency of PSII or Fv/Fm also started to
decrease in the 28-day-old leaves and then declined to
background levels in the 35-day-old leaves. The reduced
Fv/Fm value in the 28-day-old leaves was primarily due to
the decrease in Fm.
The Chl fluorescence data suggest that during develop-
ment of Arabidopsis leaves, the photosynthetic apparatus in
most of the mesophyll cells in leaves up to 21 days old is
quite functional. In the 35-day-old leaves, however, most
of the PSII in the leaf mesophyll cells is functionally dead.
At 28 days, the top parts of the leaves turned quite yellow,
and the Chl contents decreased in the leaves to 40 % of
control levels. In the area of interest, i.e., the tip, the actual
Chl content is expected to be much lower than 40 % of
control levels. Considering the Chl content that was
observed, the Fv/Fm at 28 days was much higher than
expected, and the intensity of F0 was also quite high,
considering the fact that this value is roughly proportional
to Chl content. Therefore, we assume that at 28 days, most
of the remaining PSII proteins are functionally active,
although many PSII proteins have already degraded. Some
CP complexes remained after the degradation of PSII,
which caused the unusual increase in F0.
Differential changes in photosynthetic complexes
during successive stages of development
In higher plants, Chl a is associated with both RC and
LHCs, whereas Chl b is specific to LHCs (Green and
Durnford 1996; Oster et al. 2000; Liu et al. 2004; Morita
et al. 2009; Allakhverdiev 2011). The rate of degradation
of LHCs proteins is roughly proportional to that of Chl
b (Schelbert et al. 2009). As the Chl a/b ratio dropped
during aging, we expected there to be alterations in the
structure of the photosynthetic apparatus, and thus in the
composition of the CP complexes. We therefore examined
the changes in major photosynthetic protein complexes by
BN-PAGE, including the CP complexes, ATPase, and Cyt
b6f complexes during the five successive stages of leaf
development (Fig. 3). Although no new bands appeared
during aging (including late senescence), we observed
Fig. 2 Changes in photochemical efficiency estimated from Chl
fluorescence along successive leaf ages. a. Visible symptoms (top)
and false color images of minimum fluorescence (F0), maximum
fluorescence (Fm) and maximum photochemical efficiency of PSII
(Fv/Fm) are shown for leaves at the indicated ages. The intensity scale
for Fv/Fm is shown at the bottom. b. The quantified values of F0, Fm
and Fv/Fm. The values were taken from the circled areas of the Chl
fluorescence images of the leaves, as indicated at the top panel in (a).
Each measurement was performed using the images of five leaves.
The means and standard errors (±SE) from three to five replicates are
shown. The asterisks indicate the level of statistical significance
determined by the t test (at http://vassarstats.net/) for the differences
in the F0, Fm and Fv/Fm values between leaves at 14 days and at the
indicated ages; triple (P 0.0005), double (P 0.005) and single
(P 0.05)
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123

6. some differences in the rates of disappearance of bands.
We grouped the proteins into three classes based on the
differential degree of decrease in the amount of photo-
synthetic complexes during aging: (1) PSI ? PSII dimer
and Cyt b6f complexes, which showed the highest rate of
decrease; (2) PSI core, PSII monomer, and ATPase com-
plexes, which showed the lowest rate of decrease; and (3)
LHCII trimer, which showed an intermediate rate.
To confirm the changes in major photosynthetic protein
complexes in detail, we checked the quantitative changes
in specific protein components of each photosynthetic
complex by performing an immunoblotting assay using
specific antibodies (Fig. 4). We observed a rapid decrease
in D1 and Cyt b6f in leaves over 28 days old along with a
rapid decrease in RbcL, a positive control for the senes-
cence-associated decrease in photosynthetic function. Note
that the amount of proteins loaded in each lane was relative
to the total Chl content and, thus, a moderate decrease
observed in the assay actually reflects a drastic decrease.
Indeed, we observed that there was no or only residual
amounts of D1 and Cyt b6f, respectively, in the 35-day-old
leaves.
Differential changes in LHC complex proteins
during aging
The amounts of LHCII (Lhcb1–b3) were rather stable
during aging (Figs. 3, 4), which is consistent with the
observed decrease in the Chl a/b ratio shown in Fig. 1b.
We then questioned whether there is differential usage of
proteins in LHCs for the mobilization of nutrients. To test
this notion, we analyzed the changes in the levels of each
protein component in LHCII and LHCI during leaf aging
by performing an immunoblotting assay using a specific
antibody against each of the LHC proteins (Fig. 4). We did
not observe any big changes in the electrophoretic mobil-
ities of any of the LHC proteins analyzed, suggesting that
there may be no gross changes in their characteristics
during leaf aging. This result is consistent with the result of
BN-PAGE analysis. For LHCII, we analyzed the changes
in four protein components, namely Lhcb1, 2, 3, and 4.
Among these, three (Lhcb1, 2, and 3) exhibited similar
rates of decrease in band intensities, but the other protein
(Lhcb4), a minor antenna protein in LHCII, showed the
highest rate of degradation. Lhcb1, 2, and 3 comprise
the major antenna proteins and exist in the trimeric form in
the LHCII complex of PSII. By contrast, Lhcb4 belongs to
the minor antenna proteins of LHCII and exists in the
monomeric form. Thus, our observation indicates that,
Fig. 3 Differential stability of photosynthetic protein complexes of
thylakoid membranes during successive leaf ages. Thylakoid mem-
branes were isolated from the third and fourth rosette leaves at the
indicated ages. Total Chl contents in the samples were adjusted to a
final concentration of 2 mg/ml of Chl in solubilization buffer for each
sample. The solubilized photosynthetic complexes were then resolved
by BN-PAGE. Each measurement was performed with the upper leaf
sections of five leaves. Note that total Chl amount decreases in the
later stages (Fig. 1) and thus the overall quantity of the photosynthetic
complexes is decreasing in those later stages, as we used the total Chl
amount as a loading control
Fig. 4 Differential changes in major photosynthetic proteins complex
units during successive leaf ages. D1, Cyt b6f, PsaA/B, ATPase,
Lhcb1-Lhcb4, and Lhca1-Lhca4 belong to the PSII reaction center
complex, Cyt b6f complex, PSI reaction center complex, ATP
synthase complex, LHCII complex, and LHCI complex, respectively.
Total proteins in the thylakoid fractions were isolated and subjected to
SDS-PAGE before immunoblotting with the respective antibodies.
Each measurement was performed with the upper leaf sections of five
leaves. RbcL was used as a control, representing a photosynthetic
protein that is highly sensitive to aging. The bottom panel shows
coomassie blue-stained LHCII proteins, which were used as a loading
control
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123

7. during leaf senescence, there is differential usage of major
and minor antenna proteins, and major antenna proteins are
preferentially retained in the thylakoid membranes. For
LHCI, we analyzed the patterns of age-dependent changes
in four protein components, i.e., Lhca1, 2, 3, and 4. Unlike
LHCII, there was more variability in the LHCI members in
terms of their age-dependent patterns. Lhca1 and Lhca2
exhibited the least amount of change, followed by Lhca4,
then Lhca3. Together, these results show that there is dif-
ferential usage of each protein component in the LHC
complexes of PSI and PSII during leaf aging. However, the
differential degradation of LHCI polypeptides should be
confirmed carefully, as both Lhca1/4 and Lhca 2/3 function
as heterodimers for PSI light harvesting (Wientjes et al.
2009; Wientjes and Croce 2011; Croce and van Ameron-
gen 2013). During DIS, the stability of major LHCII
complexes has been reported, but LHCI degrades more
quickly than LHCII (Oh and Lee 1996). In rice flag leaves,
LHCI is one of the most unstable LHC complexes during
natural senescence (Tang et al. 2005). However, we did not
notice any preferential degradation of LHCI in the current
study. Therefore, the reason for this discrepancy remains
unclear.
Change in photosynthetic performance during leaf
senescence
One essential activity of the photosystems is the transfer of
electrons (ETR) from PSII to the successive components
for use in electrochemical reactions. The light induction
curves of ETR at different photosynthetically active radi-
ations (PARs) are shown in Suppl. Fig. 1a, and their age-
dependent kinetics curves are shown in Fig. 5a. Compared
with the 7-day-old leaves, a significant decrease in ETR
was observed at 21 days, and the ETR after 28 days was
only 50 % of the level of the 14-day-old control leaves.
However, in the 35-day-old leaves, no ETR was detected.
These data are in agreement with previous reports showing
a drastic decline in ETR during senescence (Biswal and
Mohanty 1976; Sabat et al. 1989; Bhanumathi and Murthy
2011). The decline kinetics of ETR during aging (Fig. 5a)
were different from those of Fv/Fm (Fig. 2b), especially at
21 days. While Fv/Fm was measured using dark-adapted
leaves, ETR was measured using leaves in a light-adapted
state. An earlier decrease in ETR versus Fv/Fm can indicate
certain early changes that occur at 21 days. As shown in
Fig. 3 and 4, this early reduction in ETR during senescence
is due to decreased concentrations of Cyt b6f, as ETR
primarily reflects the functionality of PSII (Genty et al.
1989) and the Cyt b6f complex (Holloway et al. 1983). Our
observation is consistent with the rapid decrease in the
amount of D1 and Cyt b6f after 28 days shown in Fig. 4.
However, another candidate for this early change is the
photosynthetic carbon assimilation rate, indicated by the
rapid decrease in RbcL (Fig. 4), which cannot be over-
looked. As mentioned, Fv/Fm can remain high even at
28 days, if some remaining photosynthetic apparatus is still
active.
Another photosynthetic parameter that is involved in the
thermal dissipation of excitation energy of Chl fluores-
cence is NPQ. Therefore, we measured the NPQ at dif-
ferent PARs (Suppl. Fig. 1b); the age-dependent kinetics
curve of this activity is shown in Fig. 5b. Leaves at 21 days
did not show a significant decrease in NPQ (Fig. 5b).
Leaves at 28 days showed only an approximately 32 %
level of NPQ compared with the 14-day-old control sam-
ples. Unlike ETR, at the late senescence stage (35 days),
the leaves still retained approximately 30 % NPQ com-
pared with the control, which reflects the capacity of leaves
to dissipate excess amounts of excitation energy to protect
Fig. 5 Changes in ETR, NPQ, and CO2 assimilation rates during
successive leaf ages. ETR, electron transport rate. NPQ, non-
photochemical quenching. The values at each age indicate the
maximum values of ETR and NPQ obtained from light induction
curves. The values were taken from the circled areas of the Chl
fluorescence images of the leaves, as indicated at the top panel in
Fig. 2a. CO2 assimilation rates were measured from a whole leaf.
Each value is based on three replicates. Shown are the means and
standard errors (±SE). The asterisks indicate the level of statistical
significance determined by the t test (at http://vassarstats.net/) for the
differences in values between leaves at 14 days and at the indicated
ages; double (P 0.005) and single (P 0.05)
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123

8. the leaves from oxidative damage during senescence
(Fig. 5b). Overall, the NPQ declined during senescence but
remained higher than that of ETR, suggesting that NPQ
plays a role in the down-regulation of PSII during aging.
These data are in agreement with the results of Chen and
Gallie (2008).
Inhibition of CO2 assimilation during successive stages
of leaf aging
The changes in net CO2 assimilation reflect the capacity of
mesophyll cells for photosynthesis. Thus, we determined
the age-dependent changes in photosynthetic CO2 assimi-
lation. These values remained constant until day 21 and
gradually declined thereafter until the late senescence stage
at 35 days (Fig. 5c). The decrease in net photosynthetic
CO2 assimilation was consistent with the reduced amount
of RbcL, a large subunit of Rubisco, which was detected
(Fig. 4b). Since Rubisco activity decreases much more
quickly than the chloroplast population (Ono et al. 2013),
some chloroplasts in the same cell may undergo a loss of
Rubisco protein. Therefore, we assume that the first step of
nutrient mobilization from chloroplasts may involve the
degradation of Rubisco, along with D1 and Cyt b6f. We
believe that this step occurs earlier than structural degra-
dation; the former processes are reversible, while the latter
processes are irreversible. The next irreversible process
may involve the degradation of thylakoid membranes (Diaz
et al. 2005).
Reduced PSI activity during successive stages of leaf
development
As shown in Fig. 4, PSI core proteins were more stable
than PSII core proteins at 28 days, and their degradation
rates were much slower than that of D1 at 35 days.
Therefore, we measured PSI activity by examining the
relative amount of far red light-induced P700?
(Fig. 6).
The activity of PSI remained relatively stable until 28 days
and declined thereafter. These data suggest that PSII is
mobilized earlier than PSI during senescence. These results
are in agreement with those of Ghosh et al. (2001). The
relatively high stability of PSI activity is consistent with
the slower rate of reduction of PsaA/B, which are PSI core
proteins (Fig. 4).
Conclusions
As the initial target of leaf senescence in the mobilization
of nutrients is the chloroplast, leaf senescence is first
associated with a decrease in photosynthetic performance.
Here, we have provided evidence for age-dependent
differential loss of Chl a and b and age-dependent differ-
ential utilization of photosynthetic protein complexes and
their components during successive stages of leaf devel-
opment, including senescence. Although the Chl a/b ratio
increased during DIS when leaves were detached from
plants, the Chl a/b ratio decreased during natural leaf
senescence. The decrease in the Chl a/b ratio is due to a
slower degree of utilization of Chl b (along with LHCII)
compared to that of Chl a (along with RC), since the major
antenna proteins are relatively stable in thylakoid mem-
branes compared with other CP complexes. During aging,
we observed drastic decreases in the levels of major pho-
tosynthetic components including D1, Cyt b6f, and RbcL,
which in turn caused rapid decreases in ETR and CO2
assimilation. These results suggest that reversible degra-
dation processes first begin to mobilize components related
to photosynthetic function, followed by irreversible pro-
cesses that degrade structural components, including
LHCII and thylakoid membrane lipids. The nutrient
mobilization begins at the tips of leaves, and Chl fluores-
cence imaging data suggest that the degradation processes
in different chloroplasts and in different cells do not occur
homogeneously. Therefore, Chl contents are relatively
proportional to the number of degraded PSIIs, while the
non-degraded remaining PSIIs actively keep Fv/Fm and F0
relatively high compared with the Chl content. These
results suggest that the mobilization process begins in cells
that are located relatively far from vascular bundles, and
that even within a single cell, all chloroplasts do not begin
senescence simultaneously. We suggest that the differential
utilization of photosynthetic complexes by plants at
Fig. 6 Change in PSI activity during successive leaf ages. The PSI
activity is expressed as the relative amount of far red light-induced
P700?
in leaves (DA810/A810) after 5 min of pre-illumination with
120 lmol m-2
s-1
actinic white light. A810 is the absorbance signal
after the application of saturating far red light, and DA810 is the
saturating light-induced change in the absorbance signal during
illumination with actinic light. All measurements were carried out at
room temperature. Each measurement was performed with whole
leaves. Data shown are the means and standard errors (±SE) from
three replicates. Each replicate included three leaves
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123

9. different stages of leaf development and senescence likely
corresponds to the physiological state at a given stage,
since at all stages of natural senescence, photosynthetic
complexes represent sources for nutrient remobilization to
other developing organs.
Acknowledgments The authors thank Dr. Sunghyun Hong for pro-
viding critical feedback on this manuscript. This study was supported by
the Research Center Program of IBS (Institute for Basic Science,
No.CA1208) and the National Research Foundation of Korea (The
National Honor Scientist Support Program, No.20100020417) funded
by the Korea government (MEST) in Korea. SIA was supported by
grants from the Russian Foundation for Basic Research and by the
Molecular and Cell Biology Programs of the Russian Academy of
Sciences. CHL was supported by a grant from the National Research
Foundation of Korea (NRF), funded by MEST (No. 2012-0004968).
References
Allakhverdiev SI (2011) Recent progress in the studies of structure
and function of photosystem II. J Photochem Photobiol B: Biol
104:1–385
Allakhverdiev SI (2012) Photosynthesis research for sustainability:
from natural to artificial. Biochim Biophys Acta 1817:1107–1524
Allen JF, Forsberg J (2001) Molecular recognition in thylakoid
structure and function. Trends Plant Sci 6:317–326
Bhanumathi G, Murthy SDS (2011) Senescence induced alterations in
the photosynthetic electron transport activities in maize primary
leaves. Bot Res Intl 4:65–68
Biswal UC, Mohanty P (1976) Aging induced changes in photosyn-
thetic electron transport of detached barley leaves. Plant Cell
Physiol 17:323–332
Camp PJ, Huber SC, Burke JJ, Moreland DE (1982) Biochemical
changes that occur during senescence of wheat leaves. I. Basis
for the reduction of photosynthesis. Plant Physiol 70:1641–1646
Chen Z, Gallie DR (2008) Dehydroascorbate reductase affects non-
photochemical quenching and photosynthetic performance. JBC
283:21347–21361
Crafts-Brandner SJ, Salvucci ME, Egli DB (1990) Changes in
ribulose bisphosphate carboxylase/oxygenase and ribulose
5-phosphate kinase abundances and photosynthetic capacity
during leaf senescence. Photosynth Res 23:223–230
Croce R, Bassi R (1998) The light harvesting complex of photosystem
I: pigment composition and stoichiometry. In: Garab G (ed)
Photosynthesis: mechanisms and effects 1:421–424. Kluwer
Academic, Dordrecht
Croce R, van Amerongen H (2013) Light-harvesting in photosystem I.
Photosynth Res. doi:10.1007/s11120-013-9838-x
Diaz C, Purdy S, Christ A, Gaudry MJF, Wingler A, Daubresse MC
(2005) Characterization of markers to determine the extent and
variability of leaf senescence in arabidopsis. A metabolic
profiling approach. Plant Physiol 138:898–908
Feller U, Fischer A (1994) Nitrogen metabolism in senescing leaves.
Crit Rev Plant Sci 13:241–273
Genty B, Briantais JM, Baker NR (1989) The relationship between
the quantum yield of photosynthetic electron transport and
quenching of chlorophyll fluorescence. Biochim Biophys Acta
990:87–92
Gepstein S (1988) Photosynthesis in senescence and aging in plants.
In: Leopold AC, Nooden L (eds) Senescence and aging in plants.
Academic Press, San Diego, pp 85–109
Ghosh S, Mahoney SR, Penterman JN, Peirson D, Dumbroff EB (2001)
Ultrastructural and biochemical changes in chloroplasts during
Brassica napus senescence. Plant Physiol Biochem 39:777–784
Grassl J, Pruzinska A, Hortensteiner S, Taylor NL, Millar AH (2012)
Early events in plastid protein degradation in stay-green
arabidopsis reveal differential regulation beyond the retention
of LHCII and chlorophyll. J Proteome Res 11:5443–5452
Green BR, Durnford DG (1996) The chlorophyll-carotenoid proteins
of oxygenic photosynthesis. Annu Rev Plant Physiol Plant Mol
Biol 47:685–714
Holloway PJ, Maclean DJ, Scott KJ (1983) Rate-limiting steps of
electron transport in chloroplasts during ontogeny and senes-
cence of barley. Plant Physiol 72:795–801
Huang W, Chen Q, Zhu Y, Hu F, Zhang L, Ma Z, He Z, Jirong Huang
(2013) Arabidopsis thylakoid formation 1 is a critical regulator
for dynamics of PSII-LHCII complexes in leaf senescence and
excess light. Mol Plant. doi:10.1093/mp/sst069
Jansson S (1999) A guide to the Lhc genes and their relatives in
Arabidopsis. Trends Plant Sci 4:236–240
Kim JH, Kim SJ, Cho SH, Chow WS, Lee CH (2005) Photosystem I
acceptor side limitation is a prerequisite for the reversible
decrease in the maximum extent of P700 oxidation after short-
term chilling in the light in four plant species with different
chilling sensitivities. Physiol Plant 123:100–107
Lepeduš H, Jurković V, Štolfa I, Ćurković-Perica M, Fulgosi H, Cesar
V (2010) Changes in photosystem II photochemistry in senesc-
ing maple leaves. Croat Chem Acta 83:379–386
Li J, Pandeya D, Nath K, Zulfugarov IS, Yoo SC, Zhang H, Yoo JH,
Cho SH, Koh HJ, Kim DS, Seo HS, Kang BC, Lee CH, Paek NC
(2010) ZEBRA-NECROSIS, a thylakoid-bound protein, is
critical for the photoprotection of developing chloroplasts during
early leaf development. Plant J 62:713–725
Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant
Biol 58:115–136
Liu Z, Yan H, Wang K, Kuang T, Zhang J, Gui L, An X, Chang W
(2004) Crystal structure of spinach major light-harvesting
complex at 2.72 Å resolution. Nature 428:287–292
Matile P, Schellenberg M, Peisker C (1992) Production and release of
a chlorophyll catabolite in isolated senescent chloroplasts. Planta
187:230–235
Morita R, Sato Y, Masuda Y, Nishimura M, Kusaba M (2009) Defect
in non-yellow coloring 3, an a/b hydrolase-fold family protein,
causes a stay-green phenotype during leaf senescence in rice.
Plant J 59:940–952
Neilson JAD, Durnford DG (2010) Structural and functional diver-
sification of the light-harvesting complexes in photosynthetic
eukaryotes. Photosynth Res 106:57–71
Nelson N, Yocum CF (2006) Structure and function of photosystem I
and II. Annu Rev Plant Biol 57:521–565
Noodén LD, Guiamét JJ, John I (1997) Senescence mechanisms.
Physiol Plant 101:746–753
Oh MH, Lee CH (1996) Dissambly of chloropyll-protein complexes
in Arabidopsis thaliana during dark induced foliar senescence.
J Plant Biol 39:301–307
Oh MH, Kim YJ, Lee CH (2000) Leaf senescence in stay-green
mutant of Arabidopsis thaliana: disassembly process of photo-
system I and II during dark- incubation. J Biochem Mol Biol
33:256–262
Ono Y, Wada S, Izumi M, Makino A, Ishida H (2013) Evidence for
contribution of autophagy to Rubisco degradation during leaf
senescence in Arabidopsis thaliana. Plant Cell Environ
36(6):1147–1159
Oster U, Tanaka R, Tanaka A, Rudiger W (2000) Cloning and
functional expression of the gene encoding the key enzyme for
chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana.
Plant J 21:305–310
Photosynth Res
123

10. Park SY, Yu JW, Park JS, Li J, Yoo SC, Lee NY, Lee SK, Jeong SW,
Seo HS, Koh HJ, Jeon JS, Park YI, Paek NC (2007) The
senescence-induced stay-green protein regulates chlorophyll
degradation. Plant Cell 19:1649–1664
Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of
accurate extinction coefficients and simultaneous equations for
assaying chlorophyll a and chlorophyll b extracted with 4
different solvents: verification of the concentration of chloro-
phyll standards by atomic absorption spectroscopy. Biochim
Biophys Acta 975:384–394
Sabat SC, Grover A, Mohanty P (1989) Senescence induced
alterations in the electron transport in wheat leaf chloroplasts.
J Photochem Photobiol B3:175–183
Sakuraba Y, Schelbert S, Park SY, Han SH, Lee BD, Andrès CB,
Kessler F, Hörtensteiner S, Paek NC (2012) STAY-GREEN and
chlorophyll catabolic enzymes interact at light-harvesting com-
plex II for chlorophyll detoxification during leaf senescence in
Arabidopsis. Plant Cell 24:507–518
Schägger H, van Jagow G (1991) Blue native electrophoresis for
isolation of membrane protein complexes in enzymatically
active form. Anal Biochem 99:223–231
Schelbert S, Aubry S, Burla B, Agne B, Kessler F, Krupinska K,
Hörtensteinera S (2009) Pheophytin pheophorbide hydrolase
(Pheophytinase) is involved in chlorophyll breakdown during
leaf senescence in Arabidopsis. Plant Cell 21:767–785
Smart CM (1994) Gene expression during leaf senescence. New
Phytol 126:419–448
Stoddart JL, Thomas H (1982) Leaf senescence. In: Boulter D,
Parthier B (eds) Encyclopedia of plant physiology, Vol 14A.
Springer, Berlin, pp 592–636
Tang Y, Wen X, Lu C (2005) Differential changes in degradation of
chlorophyll–protein complexes of photosystem I and photosys-
tem II during flag leaf senescence of rice. Plant Physiol Biochem
43:193–201
Thomas H, Ougham HJ, Wagstaff C, Stead AD (2003) Defining
senescence and death. J Exp Bot 54:1127–1132
Weaver LM, Amasino RM (2001) Senescence is induced in
individually darkened Arabidopsis leaves, but inhibited in whole
darkened plants. Plant Physiol 127:876–886
Wientjes E, Croce R (2011) The light-harvesting complexes of
higher-plant photosystem I: lhca1/4 and Lhca2/3 form two
redemitting heterodimers. Biochem J 433:477–485
Wientjes E, Oostergetel GT, Jansson S, Boekema EJ, Croce R (2009)
The role of Lhca complexes in the supramolecular organization
of higher plant photosystem I. J Biol Chem 284:7803–7810
Wollman FA, Minai L, Nechushtai R (1999) The biogenesis and
assembly of photosynthetic proteins in thylakoid membranes.
Biochim Biophys Acta 1141:21–85
Zhang Z, Li G, Gao H, Zhang L, Yang C, Liu P, Meng Q (2012)
Characterization of photosynthetic performance during, senes-
cence in stay-green and quick-leaf-senescence Zea mays L.
Inbred Lines. PLoS One 7:e42936
Photosynth Res
123