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JBC MANUSCRIPT M2-00444 –REVISED VERSION

A TRANSIENT EXCHANGE OF THE PHOTOSYSTEM II REACTION CENTER

PROTEIN D1:1 WITH D1:2 DURING LOW TEMPERATURE STRESS OF

SYNECHOCOCCUS SP. PCC 7942 IN THE LIGHT LOWERS THE REDOX

POTENTIAL OF QB

P. V. Sanea, Alexander G. Ivanovb, Dmitry Sveshnikova, Norman P.A. Hunerb and

Gunnar Öquista*

aUmeå Plant Science Centre, Department of Plant Physiology, Umeå University, S-90187

Umeå, Sweden, bDepartment of Plant Sciences, University of Western Ontario, London,

Ontario, N6H 5B7 Canada.

Corresponding author: Dr. Gunnar Öquist, Umeå Plant Science Centre, Department of

Plant Physiology, University of Umeå, S 901 87 Umeå, Sweden. Tel: + 46-90-786 54 16,

Fax: + 46-90-786 66 76, E-mail: gunnar.oquist@plantphys.umu.se

Running title: Low temperature stress lowers the redox potential of QB in cyanobacteria

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 24, 2002 as Manuscript M200444200 by guest on February 10, 2018

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SUMMARY

Upon exposure to low temperature under constant light conditions, the

cyanobacterium Synechococcus sp. PCC 7942 exchanges the photosystem II reaction

center D1 protein form 1 (D1:1) with D1 protein form 2 (D1:2). This exchange is only

transient and after acclimation to low temperature the cells revert back to D1:1, which is

the preferred form in acclimated cells (Campbell et al. 1995). In the present work we use

thermoluminescence (TL) to study charge recombination events between the acceptor and

donor sides of photosystem II in relation to D1 replacement. The data indicate that in

cold-stressed cells exhibiting D1:2, the redox potential of QB becomes lower approaching

that of QA. This was confirmed by examining the Synechococcus sp. PCC 7942

inactivation mutants R2S2C3 and R2K1 which possess only D1:1 or D1:2 respectively.

In contrast, the recombination of QA- with the S2 and S3 states did not show any change in

their redox characteristics upon the shift from D1:1 to D1:2. We suggest that the change

in redox properties of QB results in altered charge equilibrium in favour of QA. This

would significantly increase the probability of QA- and P680+ recombination. The

resulting non-radiative energy dissipation within the reaction center of PSII may serve as

a highly effective protective mechanism against photodamage upon excessive excitation.

The proposed reaction center quenching is an important protective mechanism since

antenna and zeaxanthin cycle-dependent quenching are not present in cyanobacteria. We

suggest that lowering the redox potential of QB by exchanging D1:1 for D1:2 imparts the

increased resistance to high excitation pressure induced by exposure to either low

temperature or high light.

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The responses of Synechococcus and other unicellular cyanobacteria to various

abiotic stresses have been extensively investigated and has provided useful information in

understanding the mechanisms employed by cyanobacteria to overcome unfavourable

environmental conditions such as chilling temperatures (1, 2), excess light (3-8), and UV-

B exposure (9).

It has been shown that during environmental stress and acclimation

Synechococcus has the ability to shift between two different forms of the D1 polypeptide

of the PS II reaction centre complex (4). D1 form 1 (D1:1) is the preferred form in cells

acclimated to its normal growth environment but when stressed under either high light or

low temperatures D1:1 is exchanged for another form of the D1 protein called D1 form 2

(D1:2) (2, 6). However, this exchange is only transient, and when cells have acclimated

to the new growth conditions they revert to D1:1 (10). These shifts are governed by

changes in the relative expression of the psbAI gene encoding for D1:1 and the psbAII/III

genes encoding for D1:2 (2, 4).

The D1:1 product of the psbAI gene is different from the D1:2 product of the

psbAII/III genes (11). Of the 25 different amino acids, 13 reside in the N-terminal part,

four in helix B, three in helix C, two in helix E and the remaining three in the C-terminal

region. Both D1:1 and D1:2 have a total of 360 amino acids. Gene-inactivation mutant

strains expressing either only D1:1 (R2S2C3) or only D1:2 (R2K1) (11) have proven to

be very useful in studies of differential psbA expression in response to environmental

changes (10, 12).

Cells with D1:2 appear to be more stress resistant than those possessing D1:1

under conditions when the excitation pressure on PSII increases due to either increased

irradiance or decreased temperature (2, 3, 6). This is partly due to a high rate of D1

synthesis and expression of the psbAII/III genes forming D1:2 under high excitation

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pressure (3, 4), and partly due to a higher intrinsic resistance of PSII reaction centers with

D1:2 to photoinhibition (5, 13, 14).

Since the replacement of D1:1 by D1:2 is expected to modify the functional

characteristics of PSII at the molecular level, we hypothesized that the D1:1 to D1:2

exchange affects PSII charge stabilisation and charge recombination events as a

consequence of alteration in the redox behaviour of QA and QB as well as the water

oxidation complex (the S states). The back reactions of QA and QB with the different S

states, which reflect the charge stabilisation on both acceptors and donors of PS II was

assessed by using the technique of thermoluminescence (15, 16).

We exploited the fact that a reversible D1:1 to D1:2 exchange can be induced in

wild-type Synechococcus sp. PCC 7942 through a temperature shift from 36 to 25°C (2).

In addition, the use of two mutants, one of which expresses only D1:2 while the other

expresses only D1:1 (11), allowed us to confirm whether the changed photochemical

behaviour observed in wild type cells under conditions that permit expression of either

D1:1 or D1:2 are indeed due to the type of D1 polypeptide present in the PS II complex.

MATERIALS AND METHODS

Strains and Growth Conditions - Wild type Synechococcus sp. PCC 7942 cells

and the psbA gene-inactivation mutant strains R2S2C3 and R2K1 (11) were grown in

BG11 inorganic medium (17) at 36°C in 80 ml of fresh growth medium in rod-shaped

glass tubes under continuous illumination of 50 µmol photons m-2 s-1 of white light (400-

700 nm) (Philips TLD 18W/950 fluorescent tubes) as measured by a Li-Cor quantum meter

(Lambda Instruments, Lincoln, NB, USA) at the culture tube as described earlier (4). The

medium was buffered with 10 mM 3-N-morpholino-propane sulphonic acid (pH 7.5).

The cultures were bubbled with 5% CO2 in air (about 1 ml s-1). Log phase cultures were

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used for all experiments. For chilling stress, the culture tubes were shifted to a water bath

maintained at 25°C but otherwise with identical growth conditions. The cells were

transferred back to 36°C after 3 h. Samples were removed with a syringe under aseptic

conditions for TL measurements.

Sample Preparation and Light Treatments - For TL measurements, 10 ml cultures

were removed from the culture tubes, centrifuged and resuspended in fresh growth

medium. An aliquot equivalent to 0.2-0.3 mg Chl was spotted on to a membrane filter

(0.45 µm) using the vacuum filtration assembly from Millipore (18). The sample was

illuminated with continuous white light from a projector lamp (200W) for 30 sec and

cooled gradually in liquid nitrogen in the presence of light (19). Alternatively, for

identifying the S2QB- and S2QA

- peaks dark adapted samples were subjected to 2

consecutive saturating microsecond flashes of white light applied by a flash tube (Type

FX200, EG&G Electro Optics, Salem, MA, USA) in either the absence or presence of

DCMU. For this purpose, the sample was first relaxed for 10 min at 30°C and then

cooled to 0°C prior to exposing to the flashes. After the flash exposure, the samples were

quickly frozen in liquid nitrogen. When present, DCMU was at a final concentration of

10 µM in a cell suspension containing 0.2-0.3 mg Chl.

Thermoluminescence (TL) Measurements - TL measurements of wild type

Synechococcus and mutant cells were performed on a personal-computer-based TL data

acquisition and analysis system as described earlier (20). A photomultiplier tube

(Hamamatsu R943-02, Hamamatsu Photonics K.K., Shizuoka-ken, Japan) equipped with

a photomultiplier power supply (Model PS-302, EG&G Electro Optics, Salem MA, USA)

and a preamplifier (Model C1556-03) was used as a radiation measuring set. The

temperature was monitored by a microthermoresistor (FMF 2101, Umwelt Sensor

Teknik, Germany) placed on the surface of the sample holder and a bridge amplifier. The

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sample holder (cold finger) was placed in the TL set up and heated at a rate of 0.6°C s-1.

The output signal from the photomultiplier, which is proportional to the TL photon

emission, and the output signal from the bridge amplifier which is proportional to the

temperature of the sample holder, were connected to two different channels of an A/D

converter data acquisition card and an IBM compatible personal computer.

Decomposition analysis of the TL glow curves was carried out by a non-linear, least

squares algorithm that minimises the chi-square function using a Microcal™ Origin™

Version 6.0 software package (Microcal Software Inc., Northampton, MA, USA). The

nomenclature of Vass and Govindjee (21) was used for characterisation of the TL glow

peaks.

Immunoblot analysis - Isolation of total cellular protein, SDS-PAGE protein

separation, electrophoretic protein transfer and immunoblotting were performed

essentially as described earlier (2). Samples for electrophoretic separation were loaded on

an equal Chl basis (0.3 – 0.6 µg). Specific polyclonal antibodies were applied for

detection of D1:1 and D1:2 forms by using the ECL chemiluminescent kit (Amersham,

UK). Densitometric scanning and analysis of X-ray films from each replicate immunoblot

was performed with a Epson Perfection 1640SU Desktop scanner and Scion Image

densitometry software (Scion Corporation 1998, Fredrick, MD).

RESULTS

Representative TL curves of control and DCMU treated wild type Synechococcus

cells are presented in Figure 1. The samples were illuminated for 30 s with white light

during cooling to liquid nitrogen temperature (19). Deconvolution of the TL glow signal

of control cells grown at 36°C resolved three peaks exhibiting characteristic TM at 0°C,

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24°C and 37°C (Fig. 1A, Table 1). The major peak around 24°C accounted for about 85%

of the total luminescence, while the peak around 37°C accounted for most of the

remaining luminescence with the peak appearing near 0°C contributing to less than 0.5%

(Table 1).

To identify the recombining redox species responsible for each of the peaks as

documented in the literature (22, 16), the glow curve patterns were determined in the

presence of DCMU. In agreement with previous reports (21, 16), DCMU caused a

complete loss of the 37°C peak and appearance of a new peak at 28°C (Fig. 1B; Table 1).

In an earlier study of Synechococcus sp. PCC 7942, the peak appearing around 40°C was

assigned to the recombination of S2 and QB- (22) while the appearance of DCMU or

atrazine resistant peak around 30°C was ascribed to the recombination of S2 and QA- (21,

16). More precise identification of the characteristic temperatures of S2QA- and S2QB

-

peaks was obtained from flash induced TL measurements. It has been demonstrated

earlier that the TL band produced after two saturating flashes is related to both S2QB- and

S3QB- recombinations (36, 37). In fact, exposure of control WT sample to two

consecutive flashes of white saturating light also yielded a glow curve pattern which

could be deconvoluted in two peaks the major one appearing at 38°C and the smaller one

at 25°C, respectively (Fig. 2A). As expected, addition of DCMU drastically reduced the

peak at 38°C with the appearance of a new peak with a TM of 32°C (Fig. 2B). Hence,

since DCMU treatment causes the inhibition of the electron flow between QA and QB and

concomitant conversion of S2QB- to S2QA

- (16), the data presented above clearly indicates

that the peaks appearing around 38°C and 28°C in our study originate from S2QB- and

S2QA- recombinations respectively. The smaller peak appearing at 23.7°C is due to S3QB

-

recombination and the peak at 17.5°C from an S3QA- recombination (Table 1). We

emphasise that flash excitation at 0°C of a fully relaxed sample in the presence of DCMU

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could not generate S3 unless S2 is quite stable in the dark. Since, in these cells, S2QB-

appears at higher temperatures, we believe that S2 is also relatively more stable and can

generate S3 in the presence of DCMU. The situation is different in experiments using

continuous illumination during freezing (as was done in the present study). It is possible

that, in the presence of DCMU, S2 to S3 conversion can occur with concomitant reduction

of an acceptor like C550 at very low temperatures. Although the characteristic TM

assigned for S3QA- and S2QA

- recombinations seem higher than those reported for higher

plants (16), they are well within the range observed in cyanobacteria (23, 36, 34). In

addition, different growth temperatures and the use of different organisms could result in

shifting the peak positions to higher or lower temperatures (22, 23). It is interesting to

note that the observed temperature difference between S2QB- and S2QA

- in Synechococcus

cells is rather small compared to that in higher plant chloroplasts and suggests that the

redox potential difference between QA and QB is much lower than in higher plants.

Shifting Synechococcus cells grown at 36°C to a temperature of 25°C induced a

gradual low temperature shift of the two major glow peaks and after exposure to 25°C for

180 min the peaks exhibited TM of about 21 and 30°C, respectively. The resultant peak

temperatures were very close to those observed in 36°C acclimated cells treated with

DCMU (Fig. 1B, Table 1). Most surprising, the addition of DCMU caused only minor

effects on the TL glow peak positions in cells shifted to 25°C (Table 1). It appears that

exposing Synechococcus cells to 25°C shifted the recombination temperatures of S2QB-

and S3QB- pairs closer to those of S2QA

- and S3QA- pairs. The time course of the reversible

changes of S2QB-, S2QA

-, S3QB- and S3QA

- recombinations during the temperature shift is

presented in Fig. 3. The characteristic TM of S2QB- decreased gradually to 30°C after 180

min of incubation at low temperature (Table 1, Fig. 3A). Interestingly, transferring the

cells back to the normal growth temperature of 36°C caused very rapid shift of the S2QB-

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peak to higher temperatures. A similar trend was observed for S3QB- recombinations (Fig.

3B). In contrast, the temperatures of S2QA- and S3QA

- recombinations remained fairly

constant during the temperature shifts (Table 1, Fig. 3A, B). In addition, the relative TL

yield measured as the integrated area under the glow curves also decreased by 30% after

180 min exposure of the cells to low temperature (Fig. 3C). This effect was reversible

and the TL yield fully recovered after shifting the cultures back to the normal growth

temperature of 36°C.

Consistent with previous reports (2, 24), immunoblot analysis of the relative

polypeptide abundance of the PSII reaction center D1:1 and D1:2 forms clearly indicated

that the transfer of Synechococcus sp. PCC 7942 cells from 36°C to 25°C caused a

gradual appearance of D1:2 polypeptide and a concomitant decrease in the D1:1

polypeptide within 180 min (Fig. 4). Interestingly, shifting the culture back to the normal

growth temperature of 36°C induced the opposite effect. The amount of D1:1 increased in

parallel with the reduction in D1:2 abundance within the same time frame (Fig. 4). The

finding that the changes in glow peak positions during the temperature shift from 36°C to

25°C occurred in parallel with the transient appearance D1:2 suggests that the

recombination patterns between the S states and QB- are controlled by the different D1

forms. Regression analysis of the experimental data yielded a strong linear correlation

between the relative abundance of D1:2 polypeptide and the characteristic TM of S2QB-

TL-band (R2 = 0.851) (Fig. 5). Furthermore, the observation that cells acclimated to 36°C

in the presence of DCMU, and cells acclimated to 25°C ± DCMU show approximately

similar glow peak signatures suggest that the S2QB- and S3QB

- recombinations in cells

shifted to 25°C have almost similar activation energies as the S2QA- and S3QA

-

recombinations in 36°C grown cells.

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The observed relationship between the transient low temperature induced

appearance of D1:2 form and the corresponding changes in the redox potential of QB, was

further tested by examining the TL pattern of Synechococcus sp. PCC 7942 mutants

possessing only D1:1 (mutant R2S2C3) or only D1:2 (mutant R2K1) (11). As expected,

the mutant expressing only D1:2 showed similar glow peak characteristics as wild type

cells shifted to 25°C. The characteristic TM of the major glow peak around 22°C and the

overall TL yield were not significantly affected by a low temperature shift to 25°C (Fig.

6). It should be pointed out however, that the TL yield of control R2K1 cells was two

fold lower (TLAREA = 274.5 ± 41.0) than in wild type (TLAREA = 666.9 ± 67.2) and

R2S2C3 mutant (TLAREA = 586.6 ± 37.6) cells. Furthermore, the mutant expressing only

D1:1 showed at the growth temperature a major peak at 27°C accounting for a little over

53% of the total luminescence and another appearing at 36°C accounting for around 46%

of the total luminescence (Fig. 6). This is qualitatively similar to what we found in the

wild type cells grown at 36°C (Fig. 1, Table 1) This pattern is expected since at normal

growth temperature the R2S2C3 mutant is similar to the wild type which under the same

conditions also contains only D1: 1.

DISCUSSION

Earlier studies have shown that when cells of Synechococcus are exposed to an

increased excitation pressure by either increasing the light (6) or by lowering the

temperature under constant irradiance (2), there is a rapid and transient exchange of D1:1

with D1:2 polypeptide in the reaction centers of PSII. In full agreement with these

reports, a very rapid differential response of D1:1 and D1:2 forms to low temperature

shift was also observed in our study (Fig. 4). Furthermore, it should be pointed out that

shifting the growth temperature back to 36°C completely reversed the polypeptide

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abundance in only 3 h (Fig. 4). It has also been shown earlier that cells with D1:2 are

much more resistant to high light stress than cells possessing D1:1 because of both a

sustained high rate of D1:2 turnover and associated PSII repair (4) and intrinsically

higher resistance of cells with D1:2 (3, 13). The TL analyses of the present work

indicates that the reversible shift between the D1:1 and D1:2 forms in Synechococcus has

a major influence on the redox potential of QB (Fig. 1, 3, and Table 1). The finding that

the S2QB- peak in 25°C shifted cells (containing D1:2) appears at temperature that is

similar to S2QA- peak suggests that the redox potential of QB in the presence of D1:2 is

similar to the redox potential of QA. It has been demonstrated that the temperature at

which the TL peak appears is proportional to the changes in free energy and therefore to

the changes in the midpoint potential of the reactive species (25). Since there is little, if

any, change in the temperature at which the recombination reaction S2QA- and S3QA

-

occurs (Fig. 3), it is obvious that neither the redox potential of S2/S3, the oxidising species

participating in TL nor the redox potential of QA are affected by a shift from D1:1 to

D1:2 form. In contrast, it is evident that the redox potential of QB has changed

significantly upon transition of the cells to 25°C towards a more negative midpoint

potential approaching that of QA. These differential effects of the low temperature stress

on the redox properties of QA and QB are consistent with the fact that the QB site resides

on the D1 polypeptide, whereas the QA site resides on the D2 polypeptide (26) of PSII

reaction centers which does not undergo a change under stress conditions (2).

Changes in the TL band assigned to S2QB- recombination to lower temperature

have been well documented to occur in herbicide resistant mutants of many species (27-

29, 32, 33) or upon even minor amino acid substitutions in the D1 or D2 proteins. For

example, replacement of Serine 264 by Gly or Ala in D1 of Synechococcus PCC 7942

was accompanied by a down shift of the TM of the glow peak related to S2/S3QB-

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recombination by 10 – 15°C (30, 22). Substitution of Y112 in D1 of Synechocystis PCC

6803 with a hydrophobic amino acid L but not by F also caused charge recombination of

S2/S3QB- to occur at 15-20°C lower temperature (31). The modification in the stability of

S2QA- and S2QB

- states was also observed when a mutation in the D-de loop of D1 was

introduced in Synechocystis PCC 6803 (37). Moreover, modification of phylogenetically

conserved amino acids with no known specific function in the D loop of the D2 protein in

Synechocystis sp. PCC 6803 resulted in a temperature shift of the QB peak and alteration

of the stability of the semi-reduced QB (35). More recently, Minagawa et al. (34) have

demonstrated that mutations on the stromal side (N234D and F260S) of the D1 protein in

Synechocystis resulted in a downshift of the S2/S3QB- TL glow peak, while that of the

S2QA- peak was up-shifted in the lumenal (I6) mutant. They interpreted these results as

indicative of a decrease in the equilibrium constant for sharing an electron between QA

and QB in both types of mutants. The deletion of the PEST like sequence from D1 in

Synechocystis sp. PCC 6803 also resulted in a change in the redox equilibrium between

QA and QB (32) and the redox potential of QB/QB- (33).

The replacement of the D130 residue of D1 protein from glutamine to glutamate

has been shown to change the kinetics of primary charge separation (38). Apparently,

such a change may influence the kinetics of fluorescence emission and could also affect

the delayed light emission characteristics. Interestingly, the D130 residue of D1:1 is

glutamine while it is substituted to glutamate in D1:2 (11). This naturally occurring

substitution of D130 might well be responsible for the altered redox potential of QB

observed in our study. However, since the major changes in the amino acid residues of

D1:1 and D1:2 are in the N-terminus (11, 26), certain unspecific long range effects of the

modified N-terminus in D1:2 on the QB-binding pocket and the redox properties of QB

could not be ruled out. It is also quite possible that in a PSII assembled with D1:2 the

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binding of other low molecular mass proteins (encoded by psbE, F, I, J, H) might be also

affected and the observed temperature shift of S2QB- could not be ascribed solely to the

D1:2 effect on QB conformation. Indeed, an impairment in the electron flow between QA

and QB and a shift of the redox potential of the QB/QB- redox pair to more negative values

have been reported in a psbH null mutant of Synechocystis sp. PCC 6803 (36).

Assuming all the above, it is clear that redox states and stabilisation of

charges on both QA and QB could be altered by changes in the amino acid sequences of

either the D1 or the D2 protein. However, since D2 does not exhibit any significant

changes during the low temperature treatment (2) we believe that only the exchange of

D1:1 by D1:2 (Fig. 5), could account for the redox changes of QB. This suggestion is in

agreement with the observation that the redox potential of QB comes closer to that of QA

even at normal growth temperature when only D1:2 is present in the R2K1 mutant (Fig.

6). However, we cannot ignore the possibility that the DCMU insensitive peaks, in fact,

may result from luminescence arising from the QA related recombination only. This can

happen if the redox potential of QB comes so close to the redox potential of QA that the

charge may be stabilised preferentially on QA. Such samples, when heated for TL

measurements, will result in the oxidation of QA through S3/S2QA- recombinations.

However, since the QB in terms of its redox potential is suggested to be much closer to

QA in PSII reaction centers with D1:2, QB will reduce QA due to the changed equilibrium

in favour of QA. This would enhance the emission associated with QA recombinations.

It has been demonstrated earlier that during the transient exchange of D:1 with

D1:2 in wild type Synechococcus following a transfer from 36 to 25°C there is an

accompanying partial reduction in photosynthetic oxygen evolution (2). A preferential

localisation of the charge on QA associated with the proposed change of the redox

potential of QB approaching that of QA, would result in an increased probability of back

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reaction with the oxidised donors of the water oxidation complex. The back reaction of

the reduced QA with P680+ has been previously suggested (39, 40) and may be enhanced

when QA remains reduced (41). The accumulation of reduced QA (QA-) has been shown to

inhibit the formation of radical pair P680+Pheo- thus preventing the triplet formation of

P680 (42). Hence, it seems very likely that the increased population of QA- due to the

lowered redox potential of QB during the shift to low temperature may enhance P680+QA-

recombination and thus protect the QB site from photoinhibitory excitation pressure. This

could explain both the transient drop in photosynthesis during the transient replacement

of D1:1 with D1:2 after a low temperature shift (2), and also the observed increased

resistance to photoinhibition of photosynthesis in the mutant expressing only D1:2 (13).

In fact, Vavilin and Vermaas (41) have proposed a model suggesting the existence of two

competing pathways of QA- decay through its back reaction with P680 by radiative and

non-radiative charge recombinations.

In summary, we show for the first time that the transient exchange of D1:1 by

D1:2 leads to specific functional changes in the electron transport and charge stabilisation

within PSII reaction center altering the redox characteristics of QB such that it is similar

to that of QA. We suggest that this would increase the probability for non-radiative charge

recombination of QA- and the possibility of non-radiative reaction center quenching

within PSII. If indeed, the non-radiative pathway is enhanced a decline in the TL yield

should be expected. In support of this suggestion, the quantitative analysis of the TL glow

curves demonstrated a 30% decrease in the TL yield during a temperature shift from

36°C to 25°C (Fig. 3C). It has been previously hypothesized that the potential for non-

photochemical quenching of PSII reaction centers might protect against excess light (10).

More recently the reduction of QA has been suggested as a major requirement for

efficient reaction center quenching (43). It has been also implied that the increased

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population of QA- may enhance the dissipation of excess light energy within the PSII

reaction center during cold acclimation via its back non-radiative reaction with P680+,

thus complementing the capacity for zeaxanthin cycle - dependent energy quenching

(44). Since cyanobacteria lack zeaxanthin cycle-dependent antenna quenching (45), the

proposed mechanism of energy dissipation would be of primary importance in protecting

cyanobacterial cells from photoinhibitory damage when PSII reaction centers are exposed

to high excitation pressure induced by exposure to chilling temperatures. The transient

exchange of the D1:1 protein which appears to be the initial response may thus provide

the time required for full cellular acclimation to an increased excitation pressure through

modifications in protein (2, 6, 10) and lipid (24, 46) composition.

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Footnotes:

Abbreviations: Chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; QA,

primary electron-accepting quinone in PSII; QB, secondary electron-accepting quinone in

PSII; PSII, photosystem II; P680, primary electron donor in PSII; TL,

thermoluminescence; TM, temperature of maximum thermoluminescence emission.

Acknowledgements: This work was supported by the Swedish Research Council,

Natural Sciences and Engineering Research Council of Canada and the Swedish

Foundation for International Cooperation in Research and Higher Education (STINT).

Prof. Yuzeir Zeinalov from the Institute of Plant Physiology, Bulgarian Academy of

Sciences is acknowledged for the assistance regarding the TL apparatus.

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Figures legends:

Figure 1. A – Thermoluminescence glow curve and mathematical decomposition in

Gaussian sub-bands of Synechococcus sp. PCC 7942 cells under control (36°C) growth

conditions after illumination with continuous white light. B – Glow curves of control

(36°C) cells in the presence of DCMU (10 µM) and temperature stressed (25°C) cells.

Cultures grown at 36°C were shifted to 25°C for 3 h to subject them to a temperature

stress. Glow curve pattern was obtained after illumination with continuous white light

prior to the temperature shift and 180 min after the temperature shift. The presented glow

curves are averages from 5-7 measurements in 3 independent experiments.

Figure 2. Thermoluminescence glow curves and mathematical decompositiuon in

Gaussian sub-bands of control (36°C) (A) and DCMU-treated (10 µM) (B)

Synechococcus sp. PCC 7942 wild type cells after illumination with two single turnover

flashes of white saturating light. The presented glow curves are averages from 5-7

measurements in 3 independent experiments.

Figure 3. Time course of S2QB- and S2QA

- (A) and S3QB- and S3QA

- (B) characteristic

peaks in wild type Synechococcus sp. PCC 7942 cells during the temperature shift from

the growth temperature of 36°C to 25°C for the first 180 min and back to 36°C for the

second part of the curve. C – Relative TL yield measured as the total area under the

experimental glow curves. The peak positions were estimated by decomposition analysis

of the experimental TL curves after illumination with continuous white light. The

presented mean values ± SE are calculated from 6-8 measurements in 3-5 independent

experiments. ↑ - shift from 25°C back to 36°C.

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Figure 4. Representative immunoblots (A) and densitometric analysis (B) of D1:1 and

D1:2 polypeptides of PSII during the temperature shift of Synechococcus sp. PCC 7942

cells from 36°C to 25°C for 180 min and back to 36°C. Polypeptide abundance was

detected by immunoblotting after SDS-PAGE with D1:1 and D1:2 specific antibodies.

Mean values ± SE were calculated from 5-7 independent experiments. The data for

relative abundance of D1:1 was normalized to its maximal values in control non-treated

cells and for D1:2 to its maximal values after 180 min at 25°C.

Figure 5. Correlation between the characteristic peak temperature of S2QB- TL-band and

the relative abundance of D1:2 polypeptide in Synechococcus sp. PCC 7942 cells during

the temperature shift from 36°C to 25°C for 180 min and back to 36°C. All values

represent means ± SE from 5-8 independent measurements.

Figure 6. Thermoluminescence glow curves of R2K1 mutant cells grown at 36°C in the

absence and in the presence of DCMU and the R2K1 mutant cells subjected to 25°C for

180 min. TL curve of R2S2C3 mutant at 37°C is also presented. All TL curves were

obtained after illumination with continuous white light and the presented traces are

averages from 5-7 measurements in 3 independent experiments.

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Table 1. Peak emission temperatures (T°C) and contribution of different glow peaks to

the total area under the TL curve of wild type Synechococcus sp. PCC 7942 cells

The contribution of glow peaks represented by characteristic sub-bands was estimated by

a nonlinear least-squares fitting of the experimental glow curves obtained after

illumination with continuous white light and the data are presented as a percentage of the

total area (A, %). Mean values ± SE were calculated from 6-8 measurements in 3-5

independent experiments. The measurements were performed in the presence and

absence of DCMU following the temperature shift from 36°C to 25°C (180 min) and

back to 36°C.

Sample Control + DCMU

(Time, min) Peak T (°C) A, % Peak T (°C) A, %

Control at 36°C 0.4 ± 0.1 0.4 ± 0 -2.67 ± 0.11 2.5 ± 0.1

(0 min) 23.7 ± 0.01 84.7 ± 0.1 17.49 ± 0.06 66.2 ± 1.2

37.1 ± 0.01 14.8 ± 0.1 28.61 ± 0.28 31.3 ± 1.2

180 min -1.01 ± 0.01 1.7 ± 0.1 -2.24 ± 0.08 3.6 ± 0.1

(36°C → 25°C) 20.85 ± 0.12 79.2 ± 5.5 17.70 ± 0.12 67.3 ± 2.9

30.44 ± 2.24 19.1 ± 5.5 27.73 ± 0.72 27.0 ± 2.9

180 min 3.00 ± 0.06 5.9 ± 0.1 2.31 ± 0.14 1.9 ± 0.2

(25°C → 36°C) 24.86 ± 0.01 93.1 ± 0.1 19.84 ± 0.08 60.2 ± 11.6

42.37 ± 0.07 1.0 ± 0.1 26.90 ± 2.14 37.9 ± 11.8

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Figure 1 Sane et al., 2001

-20 0 20 40

B

Temperature (oC)

Ther

mol

umin

esce

nce

inte

nsity

(rel

ativ

e un

its)

- WT (36oC) + DCMU - WT + 180 min at 25oC

A

- WT at 36oC - Sum of gaussian

subbands - Gaussian subband

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Figure 2 Sane et al., 2001

-20 0 20 40

B - WT (36oC) + DCMU

Ther

mol

umin

esce

nce

inte

nsity

(rel

ativ

e un

its)

Temperature ( oC)

A - WT (36oC) control

- Experimental curve - Sum of gaussian subbands - Gaussian subband

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Figure 3 Sane et al., 2001

0 100 200 300 400

60

80

100

120 C

B

TL y

ield

(% o

f con

trol)

Time after transfer (min)

15

20

25 - S3QB-

- S3QA-

A

Tem

pera

ture

(o C)

25

30

35

40 - S2QB-

- S2QA-

Tem

pera

ture

(o C)

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Figure 4 Sane et al., 2001

0 60 120 180 240 300 3600.0

0.5

1.0

1.50 60 120 180 240 300 360

B

A

D1:2

D1:1

Prot

ein

abun

danc

e (re

lativ

e un

its)

Time after transfer (min)

- D1:1 - D1:2

36°C 36°C25°C

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Figure 5. Sane et al., 2001

0 50 100 150 20028

30

32

34

36

38

40

R2 = 0.851F< 0.0086

S 2QB_ p

eak

tem

pera

ture

( oC)

D1:2 abundance (relative units)

- Experimental data - Linear data fit - Upper and lower 95%

confidence limit

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Figure 6. Sane et al., 2001

-20 0 20 40

- R2K1 mutant at 36oC - R2K1 + 180 min at 25oC - R2K1 (36oC) + DCMU - R2S2C3 mutant at 36oC

The

rmol

umin

esce

nce

inte

nsity

(rel

ativ

e un

its)

Temperature (oC)

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OquistP. V. Sane, Alexander G. Ivanov, Dmitry Sveshnikov, Norman P. A. Huner and Gunnar

redox potential of QBduring low temperature stress of Synechococcus sp. PCC 7942 in the light lowers the A transient exchange of the photosystem II reaction center protein D1:1 with D1:2

published online June 24, 2002J. Biol. Chem. 

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