Addition of histidine or n-propyl gallate lead to significant protection of reaction-center proteins as well as Lhca against strong illumination.. The specific action of histidine and n-pr
Trang 1degradation and photochemical function in photosystem I submembrane fractions during light stress
Subramanyam Rajagopal*, David Joly, Alain Gauthier, Marc Beauregard and Robert Carpentier Groupe de Recherche en Biologie Ve´ge´tale, Universite´ du Que´bec a` Trois-Rivie`res, Que´bec, Canada
Excessive light causes the inactivation of
photosyn-thesis The detailed mechanism of PSII inactivation
has been extensively characterized both in vivo and
in vitro [1–5] and it has been suggested that the
inac-tivation develops at either acceptor or donor side of
the photosystem [2,6] PSI was first believed to be
tolerant to strong light However, several in vitro
studies suggested that PSI photochemical activity
could be inhibited under strong illumination [7,8] In
these early experiments, photoinactivation of PSI was
not observed in the absence of oxygen Later, Satoh
& Fork [9] demonstrated the inactivation of PSI in intact Bryopsis chloroplasts under strongly reducing conditions The authors suggested the photoinactiva-tion site to be either P700 itself [9] or close to the PSI reaction center [10] under both aerobic and anaerobic conditions The main site of PSI photo-inhibition under aerobic conditions was then shown
to be the inactivation of iron–sulfur centers [11], whereas anaerobic photoinhibition resulted in the blocking of electron transfer between A0 and FX [12]
Keywords
photosystem I; submembrane fraction;
active oxygen; P700; light stress
Correspondence
R Carpentier, Groupe de Recherche en
Biologie Ve´ge´tale, Universite´ du Que´bec a`
Trois-Rivie`res, C.P 500, Trois-Rivie`res,
Que´bec, Canada, G9A 5H7
Fax: 1 819 376 5057
E-mail: Robert_Carpentier@uqtr.ca
*Present address
School of Life Sciences and Center for the
Study of Early Events in Photosynthesis,
Arizona State University, Tempe, Arizona
85287, USA
(Received 17 August 2004, revised 23
November 2004, accepted 2 December
2004)
doi:10.1111/j.1742-4658.2004.04512.x
The protective role of reactive oxygen scavengers against photodamage was studied in isolated photosystem (PS) I submembrane fractions illumin-ated (2000 lEÆm)2Æs)1) for various periods at 4C The photochemical activity of the submembrane fractions measured as P700 photooxidation was significantly protected in the presence of histidine or n-propyl gallate Chlorophyll photobleaching resulting in a decrease of absorbance and fluorescence, and a blue-shift of both absorbance and fluorescence maxi-mum in the red region, was also greatly delayed in the presence of these scavengers Western blot analysis revealed the light harvesting antenna complexes of PSI, Lhca2 and Lhca1, were more susceptible to strong light when compared to Lhca3 and Lhca4 The reaction-center proteins PsaB, PsaC, and PsaE were most sensitive to strong illumination while other polypeptides were less affected Addition of histidine or n-propyl gallate lead to significant protection of reaction-center proteins as well as Lhca against strong illumination Circular dichroism (CD) spectra revealed that the a-helix content decreased with increasing period of light exposure, whereas b-strands, turns, and unordered structure increased This unfolding was prevented with the addition of histidine or n-propyl gallate even after
10 h of strong illumination Catalase or superoxide dismutase could not minimize the alteration of PSI photochemical activity and structure due to photodamage The specific action of histidine and n-propyl gallate indicates that 1O2 was the main form of reactive oxygen species responsible for strong light-induced damage in PSI submembrane fractions
Abbreviations
Chl, chlorophyll; LHC I, light-harvesting complex I; P700, primary electron donor; PSI and II, photosystem I and II; SOD, superoxide
dismutase.
Trang 2Recently, Velitchkova et al [13] reported that
PSI-mediated electron transport, P700 content, and
reac-tion-center proteins, PsaA⁄ B, were altered by strong
light illumination at room or low temperatures in
isolated spinach thylakoid membranes Also, these
authors observed that under these conditions toxic
hydroxyl radicals were generated Inactivation of
PSI-mediated electron flow was also reported in isolated
PSI core particles such as spinach 180 and
PSI-100, as well as cyanobacterial PSI membranes exposed
to strong light [14] In PSI core particles illuminated
with strong light, damage to the light-harvesting
com-plex (LHC) and degradation of reaction-center
pro-teins as well as acceptor side propro-teins were observed
[15] We have recently shown that exposure of PSI
submembrane fractions to strong light under low
tem-perature altered the structure of chlorophyll
(Chl)–pro-tein (CP) complexes and decreased the photochemical
activity and the efficiency of excitation energy
migra-tion [16–18] The damages were associated with the
formation of reactive oxygen species It was also found
that the above photoinactivation of PSI was retarded
by glycinebetaine and sucrose [18]
In intact leaves, PSI was also found to be
inacti-vated by light In Cucumis sativus leaves, weak light
induced the photoinactivation of PSI at chilling
tem-peratures, while practically no damage to PSII was
reported [19,20] Sonoike et al [20] demonstrated that
electron carriers located on the acceptor side of PSI
(A1, FX, FA, and FB) were damaged during the
photo-inhibition of PSI The loss of PSI activity in thylakoids
isolated from spinach leaves exposed to weak light
illu-mination at room temperature is believed to be
associ-ated with the degradation of PsaB protein, one of the
PSI reaction-center subunits [21] In barley and
cucum-ber leaves exposed to weak light at chilling
temper-ature, photoinhibition of PSI coincided with the
damage of both reaction-center proteins, PsaA and
PsaB, and the proteins PsaD and PsaE on the acceptor
side of the photosystem [22]
In plants, reactive oxygen species (ROS) are
associ-ated with normal physiological processes as well as with
responses to adverse conditions ROS are implicated in
many ways with stressful conditions: as primary
elici-tors, as products and propagators of oxidative damage,
or as signal molecules initiating defense or adaptation
[23–25] Several authors proposed that oxidative
mecha-nisms are at the basis of PSI photodamage in intact
leaves [19,22], isolated chloroplasts [7,8,11], and isolated
PSI fragments [15–18] Two types of active oxygen
spe-cies with their derivative products are proposed to be
involved in photooxidative damage, superoxide anion
radicals, ÆO2 , and singlet oxygen, 1O2 [26–29] Hence,
addition of glucose oxidase, glucose, and catalase to scavenge dissolved oxygen suppressed the photodamage
of PSI submembrane fractions [17]
However, the protective role of specific active oxy-gen scavengers against photo-induced changes in the photosystems was discussed only in a limited number
of reports LHCII protein degradation was analyzed during strong light illumination of isolated LHCII or BBY PSII subcomplexes [28] Random cleavage, start-ing in the NH2 terminal region resulted in the complete degradation of the antenna proteins The addition of scavengers such as histidine, DABCO, and n-propyl gallate, retarded the above damages to the antenna proteins indicating mainly 1O2 was involved [28] In spinach thylakoids, illumination at low light intensity resulted in the degradation of PsaB gene product into two fragments of 51 and 45 kDa [30] These fragments were absent with added n-propyl gal-late, which removes hydroxyl radicals [30] There are
no reports regarding the protective role of oxygen scavengers against reactive oxygen species under strong light in PSI submembrane fractions
We used PSI submembrane particles as model sys-tem to obtain a better insight into the photo-induced changes in PSI The submembrane particles used con-tain all the components of PSI, including the cyto-chrome b6⁄ f complex and plastocyanin [31,32] In the present study, the degradation of each polypeptide and the protein conformation changes are assessed in con-nection with alterations in photochemical activity dur-ing strong light illumination of the PSI submembrane fractions It is concluded from the specificity of the various active oxygen scavengers used that only the generation of1O2is responsible for the photooxidation effects The role of active oxygen scavengers in the protection of the structure and function of the photo-systems against excess light indicates they should also
be beneficial under natural conditions
Results
PSI submembrane fractions were exposed to strong light (2000 lEÆm)2Æs)1) for various time durations at
4C The action of several active oxygen scavengers was analyzed for all the parameters studied in order to emphasize the role of reactive oxygen species in the photooxydative damage
Changes inDA830
Exposure of isolated PSI submembrane fractions to strong white light at 4C resulted in the loss of ability
of P700 to undergo reversible redox changes Similar
Trang 3results were obtained at room temperature (data not
shown) Figure 1 shows the magnitude ofDA830signal
remained stable during the first 4 h of strong light
treatment of PSI submembrane fractions in the
absence of active oxygen scavengers It indicated an
unchanged amount of photooxidizable P700 during
this period However, further irradiation caused a
rapid decline in DA830 The addition of histidine and
n-propyl gallate to the submembrane fractions
signifi-cantly prevented the decline of absorbance changes
(DA830) The magnitude ofDA830 was almost constant
until 10 h of light exposure with the above reactive
oxygen scavengers In contrast, in presence of
cata-lase or superoxide dismutase, or both, the amplitude
of DA830 was similar to control values (data not
shown)
Absorption changes
Figure 2A illustrates the changes in the room
tempera-ture absorption spectra of PSI submembrane fractions
exposed to various periods of strong white light
illumin-ation at 4C The spectra measured in untreated
sub-membrane fractions showed typical absorption peaks at
680 and 440 nm corresponding to Chl a and shoulders
at 650 and 470 nm originating from Chl b [16] As the
period of illumination increased, the magnitude of the
absorption peak at 680 nm declined (Fig 2) In addi-tion, the position of that peak was finally shifted by
6 nm towards shorter wavelengths after a 6-h
illumin-Fig 1 The changes in the magnitude of light-induced absorbance
at 830 nm during strong light illumination in PSI submembrane
frac-tions Results are means ± SE n ¼ 3 All experimental conditions
are given in Experimental procedures.
Fig 2 Room temperature absorption spectra of the isolated PSI submembrane fractions illuminated for various periods of time with strong WL (2000 lmolÆm)2Æs)1) at 4 C (A) Control (no addition of any additives), (B) in the presence of histidine, and (C) in the pres-ence of n-propyl gallate These experiments were repeated three times and yielded identical spectra; a typical spectrum is presented Insets: the first derivative of the absorption maxima in the red is presented to show peak position.
Trang 4ation This becomes particularly clear from the changes
in first derivative spectra that further demonstrate the
gradual time-dependent alteration in the position of the
major peak in the red region (Fig 2, insert) On the
other hand, in the presence of the scavengers histidine
or n-propyl gallate, the absorption maximum at 680 nm
was less affected by the strong illumination and even
after a 10-h exposure this absorption was higher than in
control (6 h) Consequently, the blue shift of the
maxi-mum absorption peak at 680 nm was also less
pro-nounced (Fig 2B,C) In the presence of histidine the
peak shift was about 5 nm after 6 h, while in n-propyl
gallate this peak shift was only 3 nm after 10 h
Fluorescence emission changes
The 77 K fluorescence emission spectra and their first
derivatives measured in PSI submembrane fractions
illuminated for various times at 4C are shown in
Fig 3 The intensity of the peak at 736 nm associated
to the PSI complex decreased by about 24% after
1 h of strong light illumination It declined with
further light exposure, with only 30% of its initial
magnitude remaining after a 3-h illumination Similar
to the absorbance spectra, a blue shift of the position
of the major fluorescence peak was observed The
extent of that shift was larger in the fluorescence
emission spectra and reached 15–18 nm after 6 h
(Fig 3A,C) The shift in fluorescence maximum was
evident from both absolute (Fig 3A) and first
deriv-ative (Fig 3C) spectra The latter also demonstrates
that the half-width of the emission band peaking at
736 nm in untreated preparations was not affected by
light exposure despite the marked changes in the
peak position
In the presence of histidine or n-propyl gallate the
changes in the fluorescence maximum at 736 nm were
delayed In the presence of histidine, the fluorescence
maximum declined by 80% after 10 h of illumination
In the case of propyl gallate this fluorescence was
reduced by 70% (Fig 3B) Also, the peak shift was
delayed by these reactive oxygen scavengers The
fluor-escence maximum peak shifts were 18 and 11 nm in
the presence of histidine and n-propyl gallate,
respect-ively, after 10 h of illumination (Fig 3C) In the
pres-ence of catalase and SOD the changes in fluorescpres-ence
and peak shift resembled the control experiment (data
not shown)
Changes of polypeptide composition
Specific antibodies of PSI polypeptides were used to
study the degradation of proteins in PSI submembrane
Fig 3 (A) Low temperature fluorescence emission spectra of the isolated PSI submembrane fractions illuminated for various periods
of time with strong WL (2000 lmolÆm)2Æs)1) at 4 C Inset: the first derivative of the absorption maxima in the red is presented to show peak position (B) Changes in fluorescence intensity meas-ured at the maximum in the presence or absence of scavengers Results are means ± SD n ¼ 3 (C) Changes in the position of the fluorescence maximum obtained from the first derivative of the fluorescence spectra of PSI complexes Control (s), histidine (h), and n-propyl gallate (,) For further details see Experimental proce-dures These experiments were repeated three times and yielded identical spectra; a typical spectrum is presented.
Trang 5particles during strong illumination The
reaction-center protein PsaA was stable until 4 h and then
decreased very slightly with further illumination
How-ever, PsaB protein degradation started after a 1-h
exposure and a degradation product appeared, whereas
after a 5-h exposure this protein mostly disappeared
(Fig 4) These reaction-center proteins were protected
in the presence of histidine and n-propyl gallate (data
not shown) The stromal ridge constituted of PsaC,
PsaD, and PsaE polypetides provides the docking site
for the soluble electron acceptors ferredoxin and
flavo-doxin [33,34] Among these polypeptides, PsaC was
relatively sensitive to strong light and this protein
star-ted to degrade even after 1 h of illumination and
fur-ther illumination accelerated the degradation (Fig 4,
Table 1) PsaD was less sensitive (Fig 4) and after 6 h
of exposure this polypeptide degraded only by 30%
(Table 1) PsaE was the most sensitive, after a 3-h
exposure more than 60% of this protein content was
decreased (Table 1) In comparison, the integral mem-brane protein PsaF was more stable (Fig 4) Addition
of reactive oxygen scavengers significantly protected the above proteins against photodegradation (Fig 5, Table 1)
The outer antenna system of higher plant photo-system I, LHCI, is composed of four proteins with molecular masses of 20–24 kDa, the products of the genes Lhca1–4, which are associated with the PSI core [33–35] We have analyzed the photodegradation pro-file of each polypeptide in Fig 4 Lhca1 is degraded linearly with 30% loss of this protein after 3 h, and it completely disappeared after a 5-h exposure (see also Table 1) However, with the addition of histidine, this degradation was negligible and after 6 h only 20% of this subunit was lost (Fig 5, Table 1) Surprisingly, this protein was not degraded in the presence of n-pro-pyl gallate (Fig 5, Table 1) Lhca2 was more sensitive
to strong light and started to degrade after 1 h and completely vanished after 3 h With histidine, this pro-tein declined by 45 and 60% after 3 and 6 h of illu-mination, respectively, while with n-propyl gallate, the degradation of this submit was only about 40% after 3 and 6 h illumination The Lhca3 subunit linearly degraded with the illumination period After 3 h of illumination, this protein was lost by 40% and almost completely disappeared after 6 h In the presence of histidine this protein declined by only 15 and 60% after 3 and 6 h of illumination, respectively, whereas,
in presence of n-propyl gallate the protein was stable after 3 h and 20% loss was noticed after 6 h of illu-mination Lhca4 was the most stable subunit, after 3 h this subunit was altered by 25 and by 50% after 6 h of
Table 1 Quantification of photosystem I polypeptides from
immuno-blots obtained from Fig 5.
Control (%)
Histidine (%)
n-Propyl gallate (%)
Fig 4 Quantitative immunoblot analysis of PSI submembrane
frac-tions illuminated with strong light at 4 C for different time
dur-ation All experimental conditions are given in Experimental
procedures.
Fig 5 Quantitative immunoblot analysis of PSI submembrane frac-tions illuminated with strong light at 4 C for different time duration
in the presence of histidine or n-propyl gallate All experimental conditions are given in Experimental procedures.
Trang 6illumination Histidine and n-propyl gallate retarded
the photodegradation The sensitivity of LCHI
pro-teins to strong illumination was Lhca2 > Lhca1 >
Lhca3 > Lhca4 (Fig 4)
Analysis of CD changes
As shown in Fig 6A, strong light induced significant
alterations in the secondary structure of proteins from
PSI submembrane fractions After a 6-h exposure, a
clear decrease in elipticity measured at 222 and 190 nm
in the CD spectra was observed, which is typical of a decrease in helical content In the presence of histidine and n-propyl gallate the above elipticities were not affected after a 6-h illumination, and after a 10-h expo-sure these changes were still minor (not shown) The CD curve-analyzing algorithm (see Experimen-tal procedures) revealed that untreated (0 h) PSI sub-membrane fractions were composed of a-helix (24%), b-sheet (25%), turns (22%), and unordered (29%) structures (calculated from Fig 6A) Performing the same spectral analysis after various treatments allowed quantifying the change in secondary structures in PSI fractions The proportion of a-helix decreased by 33% after 6 h of exposure to strong light (Fig 6B) However, there was no change in a-helix even after a 6-h illumination in the presence of histidine or n-pro-pyl gallate After 10 h in the presence of both agents, the a-helix helix percentage dropped slightly, although the amplitude of this decrease was comparable to the error margin of the method used for its computation (Fig 6B) These results clearly reveal that protein con-formation is protected against photodamage in the presence of histidine and n-propyl gallate
Discussion
The primary objective of this work was to evaluate the protective role of reactive oxygen scavengers against strong light in PSI submembrane fractions Our data clearly demonstrated that some specific scavengers sig-nificantly protected both the structure and function
of PSI
Several authors postulated that oxidative mecha-nisms are the basis of PSI photodamage in intact leaves [19,20,22], isolated chloroplasts [7,8,11], or iso-lated PSI fragments [15–18] The selective action of reactive oxygen scavengers used here indicated that the species involved in the photodamage of proteins and photochemical functions in the PSI submembrane frac-tions were 1O2, OH, and alkoxyl radicals However,
H2O2 and •
O2 were apparently not implicated in the damaging processes Addition of n-propyl gallate pro-vided more protection than histidine (Figs 1–3) It is known that n-propyl gallate protects against OH and alkoxyl radicals, and histidine scavenges 1O2 [28,29] Generally, •
OH radicals are formed by the reaction between hydrogen peroxide and reduced metal ions during the so-called Fenton reaction In the present case, addition of SOD and⁄ or catalase did not show any protection against strong light Nonetheless, this was not due to a temperature-dependent inhibition of SOD or catalase as similar changes of absorption, fluorescence, and P700 oxidation under strong
illumin-Fig 6 (A) Room temperature CD spectra of PSI submembrane
fractions illuminated for various time duration in control PSI
sub-membrane particles illuminated without active oxygen scavengers.
(B) Content in a-helical structures calculated from CD spectra
obtained from experiments such as shown in Fig 6 A with no
additive (s) or with histidine (,), and n-propyl gallate (h) Results
are means ± SD n ¼ 3 All experimental conditions are given in
Experimental procedures.
Trang 7ation were observed at both room and low
tempera-ture in the PSI submembrane fractions (results not
shown) Thus, OH and alkoxyl radicals originated
from1O2 It is well known that •
OH and alkoxyl radi-cals can be produced from reactions of 1O2 or •
OH with organic molecules [36,37]
Generation of singlet oxygen by Chl is expected
dur-ing strong illumination as it depends on the population
of excited Chls molecules and is formed by energy
transfer from Chl molecules in triplet state to oxygen
[27 and references therein, 38] The involvement of
P700 triplet states appearing as a consequence of
charge recombination between P700+and reduced A1
or A0 [39] could not represent a significant source for
1O2 production in isolated submembrane fractions
because of the low rates of electron flow through PSI
in the absence of an electron donor system (Fig 1) As
P700+is known to be a very efficient quencher of
exci-ted singlet states of Chls close to the PSI reaction
cen-ter [40], excited states available for oxygen must have
appear mostly in the light-harvesting complexes, not in
the PSI core In such case,1O2 derivatives had first to
attack antenna Chls [41] This might explain the faster
Chl breakdown compared to the loss of PSI
photo-chemical activity (Figs 1 and 2)
The native PSI complex from higher plants contains
about 200 Chls per P700 Among them, LHCI binds
about 70–110 Chl a + b molecules and serves as an
accessory antenna to harvest light and funnel its energy
to the reaction center, P700 The latter is located in the
PsaA⁄ B proteins of the core component (CCI), where
96 Chls are bound [33,34] The Chl a⁄ b binding
peri-pheral antenna of plants PSI (LHCI) is composed of
four nuclear gene products, Lhca1–4, with molecular
masses of 20–24 kDa [33,34] Each Lhca protein binds
approximately 10 Chls [42–44] A total of eight Lhca
proteins are thought to be present per PSI: Lhca1 and
Lhca4 are present as heterodimers, whereas Lhca2 and
Lhca3 are likely present as homodimers [33,34]
Immu-noblot analysis showed that among the Lhca proteins,
Lhca2 is more sensitive and Lhca4 is stable to strong
light The sequence of the changes observed in Lhca
proteins was: Lhc2 > Lhca1 > Lhca3 > Lhca4
(Fig 4)
As in the PSI core antenna, excitonically coupled
dimers or trimers of Chl a or b in the Lhca were also
suggested to form a pool of red pigments of
low-energy [45–47], more specifically in the Lhca4 subunit
of the LHCI-730 complex [45,48,49] Recent findings
confirmed that Lhca2 and Lhca3 are also having
low-energy red pigments [44] but this is more pronounced
in Lhca3 [35] If the absorbed energy migrates towards
PSI holochromes with higher-absorption wavelengths
[27], then, the Chl molecules with an absorption max-ima in the red located at a relatively long wavelength
in Lhca3 and Lhca4 should be bleached first A blue shift in absorption and fluorescence maximum in the red was clearly observed in the PSI submembrane frac-tions (Figs 2 and 3, see also [16–18]) Thus, the pig-ment aggregates absorbing at these long-wavelengths could be involved in photoprotection [16,17,27] This phenomenon is in agreement with our results showing that Lhca4 and Lhca3, which have red pigments with higher absorption wavelength, are more stable More-over, Lhca1 and Lhca2 have a higher content in bulk Chl Thus, the fast degradation of Lhca1 and Lhca2 is due to generation of reactive oxygen species during strong light illumination which leads to some alter-ation in the Chl–protein interaction Hui et al [15] reported in PSI core complexes that LHCI-680 is more sensitive due to decreased interaction between Chl–Chl
or Chl–protein The discrepancy in sensitivity to strong light between LHCI-680 and LHCI-730 could be explained by the organization of Chl–Chl or Chl–pro-tein in the Lhca subunits The degradation of Lhca protein subunits was reduced with the addition of his-tidine or n-propyl gallate in PSI submembrane parti-cles exposed to strong light even after 10 h, with a more pronounced action of n-propyl gallate (Fig 5, Table 1) The above results are consistent with previ-ous reports of strong light-induced degradation of LHCII proteins where histidine and n-propyl gallate also retarded the photooxidative effects with a more efficient action of n-propyl gallate [28,29]
The PSI core is a large pigment–protein complex composed of 11–13 protein subunits, the largest two
of which, PsaA and PsaB, comprises the molecular masses of 83.2 and 83.4 kDa [33,34] This is a het-erodimer to which the majority of the core antenna pigments, as well as most of the reaction center cofactors, are bound In PSI core preparations, strong light exposure induced changes to both reaction-cen-ter proteins [14] In the present study using submem-brane fractions, PsaB was more sensitive to strong light and also produced degradation products (Fig 4) Similar degradation products of PsaB were observed with no apparent modification of PsaA in spinach thylakoids or cucumber leaves illuminated with weak light at low or room temperature [21,29] These authors proposed that the degradation of PsaB was mainly due to formation of superoxide and hydroxyl radicals and this was protected by n-propyl gallate In another report using barley leaves illumin-ated with weak light at low temperatures, damage to both reaction-center proteins of PsaA and PsaB was reported [23] The involvement of reactive oxygen
Trang 8species such as superoxide, hydrogen peroxide, and
hydroxyl radicals was suggested They also showed
that 1O2 was involved in the damage of
reaction-center proteins In vivo, the photooxidative damages
in PSI may occur with a similar pattern as in PSII
where reactive oxygen species are thought to initiate
protein degradation that is completed by chloroplast
proteases [21,30] It was recently shown that the
deg-radation of PsaA⁄ B proteins and alteration of
photo-chemical activity in spinach thylakoids are more
intensive at room temperature than at low
tempera-ture under strong light [13], which suggested that the
photoinactivation may involve an enzymatic
contribu-tion to the phenomenon
As mentioned above, in PSI submembrane fractions,
PsaA was more resistant to strong light compare to
PsaB PsaA was stable until 4 h of illumination and
then started degradation (Fig 4) The PSI core is
mainly composed of bulk antenna Chl that absorbs in a
broad band with a maximum at 680 nm, but also
con-tains 3–10% of low-energy Chl (red) The quenching of
excitations located on the red Chls by P700+could
pro-vide a pathway to prevent the generation of Chl triplet
states, which can lead to the formation of harmful1O2
under strong light illumination [46] The lower
sensitiv-ity of PsaA to photooxidative damage compare to PsaB
may indicate that it contains less low energy Chl
aggre-gates Alternatively, PsaA may be less closely associated
with the antenna Chl of the light harvesting complexes
and thus receive less excess energy that leads to the
generation of 1O2 Addition of histidine and n-propyl
gallate protected these two reaction-center proteins
even after 10 h of illumination (data not shown), which
supports the involvement of1O2
Apart from PsaA and PsaB, other smaller
polypep-tides of PSI were degraded during strong light
illumin-ation PsaC together with PsaD and E comprises the
stromal side of PSI PsaC anchors the two Fe4S4
clus-ters FAand FB, which are needed to carry out the
elec-tron transfer from Fx The order of sensitivity to
strong light illumination was PsaE > PsaC > PsaD
(Fig 5, Table 1) Similar results were observed in PSI
core particles [15] This order closely corresponds with
the degradation profile observed during disassembly
studies using urea treatment [50] The crystal structure
of plant and cyanobacterium PSI revealed that loops
at the stromal surface of PsaA⁄ B partly contribute the
binding interface to the PsaC, D and E subunits
[51,52] These observations are in agreement with our
data showing that the degradation of PsaC subunit
ensue almost simultaneously the degradation of Lhca2
and PsaB subunits (Figs 4 and 5) PsaD is stable even
after 5 h of illumination, whereas, PsaE is more
susceptible to strong light The structural model of Thermosynechococcus PSI showed that part of PsaE is sandwiched between the PsaA⁄ B heterodimer and PsaC [33,53] The higher plant reaction-center structure retains the same organization as the cyanobacterial complex [52] Thus, it seems possible that PsaE affects the intracomplex electron transport under some condi-tions From our data it is clear that the degradation of the PSI stromal ridge starts at PsaE Most likely,
in plants PsaE would determine the susceptibility to photodamage of the PSI complex Interestingly, the present data also indicate that, even though PsaC seems to be required during the assembly of PsaD and PsaE with the PSI complex [50], PsaC can be lost inde-pendently from the two other extrinsic proteins (Figs 4 and 5)
Excess light clearly altered the secondary protein structure of the PSI submembrane fractions With increased time of exposure a significant portion of the a-helices was lost (Fig 6) The protein unfolding was significantly prevented by histidine and n-propyl gallate (Fig 6) The alteration of protein secondary structure components as revealed from the CD spectral studies is likely to play a central role in the active oxygen-depend-ent photodegradation processes of the PSI complex It is possible that the observed structural perturbations involved altered pigment–protein and protein–protein interaction, contributing to the observed decreased efficiency of energy migration and capture, and leading
to Chl degradation
Experimental procedures
Isolation of photosystem I submembrane fractions
PSI submembrane particles were isolated from fresh spin-ach leaves obtained from the local market, according to the procedure of Peters et al [31] with some modifications [32] The isolated preparations with Chl content of 1–2 mg
PSI submembrane fractions Chl was determined in 80% acetone according to Porra et al [54]
Light treatment
projector lamp WL was passed through a 5-cm layer of
Trang 9water containing CuSO4 to cut the infrared radiation The
samples were illuminated in the presence of various active
oxygen scavengers to selectively remove the reactive oxygen
species The scavengers used were histidine at a
for•O2 and H2O2, respectively [28,29]
Measurement of P700 redox state
The changes in the redox state of P700, the primary donor
of PSI, were measured by the light-induced absorbance
ED-P700DW connected to a PAM fluorometer (Walz,
Effeltrich, Germany) as described by Schreiber et al [55]
All measurements were carried out at an identical
sensitiv-ity of the PAM fluorometer The absorbance changes at
830 nm represented only the oxidation and reduction of
P700 as no contribution to the absorbance changes due to
plastocyanin redox transformations can be detected with
the ED-P700DW unit White actinic light was obtained
from the KL 1500 projector (Walz, Effeltrich, Germany)
The aliquots of 60 lL taken from the suspension of PSI
submembrane particles during strong light treatment were
added to 140 lL of suspension buffer containing 200 lm
ascorbate as an artificial donor Chl concentration during
experiments (prior to the illumination)
Measurement of absorption spectra
The room temperature absorption spectra and their first
derivatives were recorded using a PerkinElmer Lambda 40
spectrophotometer (Wellesley, MA, USA) Ten microlitres
of the suspension of submembrane particles were repeatedly
taken during the illumination Such aliquots taken from the
suspension of untreated particles contained 5 lg of Chl,
whereas the Chl content decreased gradually in the samples
taken during strong illumination
Fluorescence spectroscopy
Low temperature (77 K) spectra of fluorescence emission
excited at 436 nm were measured as reported previously
[16] using a PerkinElmer LS55 spectrofluorometer The Chl
excitation and emission slit width were set at 5 and 2.5 nm,
respectively
Circular dichroism spectroscopic analysis
CD spectra were measured in a Jasco-720
spectropolari-meter (Easton, MD, USA) in a cell with a 0.1 mm optical
path length over a wavelength range of 190–260 at a
and a 1 nm spectral band width The base line was correc-ted with blank buffer for each spectrum The secondary structure of PSI proteins was determined from the CD spectra using the cd pro program as described previously
con-trol measurements (prior to the illumination)
Immunoblot analysis
PSI submembrane polypeptides were separated by poly-acrylamide gel electrophoresis (PAGE) according to Raja-gopal et al [32] Electrophoresis was performed on a 13% separating and 4% stacking gel of polyacrylamide The sus-pension (10 lL aliquots) of submembrane fractions were repeatedly taken during the course of photoinhibition Such aliquots contained 5 lg Chl obtained from the suspension
of untreated preparations An equal volume of 2· buffer was added to the aliquots
To identify and quantify the PSI polypeptides, immuno-blotting was carried out essentially as described by Towbin
et al [57] Western blotting was performed by electropho-retic transfer of proteins to nitrocellulose membranes (0.45 lm, Millipore, Billerica, MA, USA) The membrane was incubated with polyclonal antibodies raised in rabbits against PSI complex Subsequently, secondary antibodies ligated to alkaline phosphate were applied Bromo-chloro-indolyl-phosphate and tetrazolium blue were used for the coloring reaction The developed membranes were analyzed
by using Bio-Rad Gel-Doc 2000 system (Hercules, CA, USA)
Acknowledgements
The authors wish to thank Drs J.H Golbeck, K Sonoike and P Chitnis for antibodies and J Harnois for helpful professional assistance This research was supported by the Natural Sciences and Engineering Research Council of Canada and by Fonds Que´be´cois
de la Recherche sur la Nature et les Technologies
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