Planta (1993)189:433-439 Planta �9 Springer-Verlag 1993

Evidence for loss of D1 protein during photoinhibition of Chenopodium rubrum L. culture cells Christian Schiifer*, Gerd Vogg, and Volkmar Schmid

Lehrstuhl f/Jr Pflanzenphysiologie, Universit/it Bayreuth, Universit/itsstrasse 30, W-8580 Bayreuth, FRG

Received 8 July; accepted 14 September 1992

Abstract. The effects of high-light stress on chlorophyll- fluorescence parameters, D 1-protein turnover and the actual level of this protein were analysed in nitrogen-defi- cient and nitrogen-replete cells of Chenopodium rubrum L. Changes in the number of atrazine-binding sites and in the D 1-protein immunoblot signal indicated that a net loss of D1 protein occurred in high light and was partly reversible in low light. Nitrogen deficiency did not exac- erbate these changes. The involvement of Dl-prote in turnover was shown in pulse-chase experiments with [3sS]-methionine and by the application of a chloroplas- tic protein-synthesis inhibitor (chloramphenicol). The slowly reversible non-photochemical fluorescence quench- ing increased pronouncedly when D1 protein was lost at high irradiances, but its increase was only small when a net loss of D1 protein was produced at moderate irradiances by addition of chloramphenicol. The ratio of variable to maximum fluorescence, Fv/Fm, and the num- ber of atrazine-binding sites were correlated but a proportionali ty between these parameters could not be observed. We conclude from these results that (i) degra- dation of D1 protein was not always coupled to its resynthesis, (ii) the actual level of D 1 protein reflected the balance between degradation and resynthesis of D1 pro- tein and (iii) changes in the level of D1 protein did not depend on a pronounced increase of the slowly reversible non-photochemical quenching.

Key words: Chenopodium (cell culture) - Chlorophyll fluorescence (nonphotochemical quenching) - D1 pro- tein (turnover) - Nitrogen deficiency (D1 protein) Pho- toinhibition (photosynthesis)

* To whom correspondence should be addressed; FAX: 49 (921) 552642

Abbreviations: Fo, Fro, F~ = fluorescence yield when all PSII centers are open, when all PSII centers are closed and difference between these values; PFD = photon flux density

Introduction

The D1 protein holds a central position in the PSII reaction center (Nanba and Satoh 1987). It is synthesized in the chloroplast and shows a continuous, light-depen- dent turnover which includes inactivation, degradation and resynthesis steps (Mattoo et al. 1984; Ohad et al. 1990a). In high light this turnover is accelerated, and exceeds that of all other cellular proteins (Prasil et al. 1992). Although degradation of D1 protein is probably a secondary event during photoinhibit ion (Cleland et al. 1990), an insufficient capacity for repair of this protein should result in photoinhibitory damage. Limitations in the repair cycle could occur in the degradation step and in the replacement of degraded D 1 protein. In the latter case a net loss of D1 protein should be detectable. Therefore, the present study analyses the effects of light stress on PSII functionality, and on the turnover and content of D1 protein. To obtain further information on the role of D 1-protein replacement in PSII functionality, chloroplastic protein synthesis was blocked using chloramphenicol. The experiments were done with sus- pension-cultured cells of a higher plant (Chenopodium rubrum), allowing a homogeneous light treatment and a reproducible application of inhibitors and effectors. Ni- trogen-deficient cells were included in the investigation since they show an increased susceptibility to long-lasting light stress (Sch/ifer and Heim 1993).

Material and methods

Plant material and growth conditions. A photoautotrophic cell line of Chenopodium rubrum L. which had been established by Hiise- mann and Barz (1977) was used for the experiments. The cells were cultivated in two-tiered fasks on a gyratory shaker. The exact growth conditions were as follows (Sch/ifer and Schmidt 1991): 16 h light and 8 h darkness; 25-27 ~ C; 60-100 ~tmol �9 m -2 �9 s -I photon flux density (PFD) from fluorescent tubes (Cool White; Osram, M/inchen, FRG); 2% atmospheric CO2 (carbonate-bicarbonate buffer in lower flask); and two-weekly subculture in MS-medium (Murashige and Skoog 1962). Nitrogen-deficient cells were

434 C. Schiller et al.: Dl-protein loss during photoinhibition

produced by subculture in nitrogen-free medium (NH4NO3 omit- ted, NH4C1 replaced by KCI). These cells have lower chlorophyll contents and photosynthetic capacities than control cells (Sch~ifer and Heim 1993).

Experimental manipulations. Cell cultures were used for experiments when two to four weeks old. Photoinhibitory treatment was at a PFD of 700-900 lamol �9 m - 2. s- 1 using low-voltage halogen lamps (Q 50 MR16/WFL; General Electric, Cleveland, Ohio, USA). The flat-topped cylindrical culture vessels created a homogeneous light field and heating was counteracted by additional heat filters and air ventilation (maximum temperature about 30 ~ C). Chloramphenicol was added to the cell suspension at a final concentration of 0.1 kg �9 m -3 (ethanolic stock solution with 34 kg �9 m-3).

Chlorophyll-fluorescence parameters and oxygen evolution. Chlo- rophyll-fluorescence parameters were determined with a pulse-am- plitude-modulation fluorometer (PAM 101; H. Walz, Effeltrich, FRG) as described in Schfifer and Schmidt (1991). Minimum fluo- rescence yield (F o; all PSI I centers open) and maximum fluorescence yield (Fro; all PSII centers closed) in the dark were determined after a dark period of at least 5 min at 1.6 kHz and 100 kHz, respectively. Additional irradiation with far-red light did not change the F o level, and Fo fluorescence quenching in high light was not observed. The ratio of variable to maximum fluorescence yield (Fv/Fm) was cal- culated as (FmFo)/F m. This parameter can be used as an estimate of the maximum photochemical efficiency of PSII (Bj6rkman 1987). Changes in the Fm value of dark-adapted cells were taken as an estimate of the occurence of slowly reversible non-photochemical quenching (Krause 1988).

The rate of oxygen exchange was measured with a liquid-phase Clark-type oxygen electrode (DW2; Hansatech, Kings Lynn, Nor- folk, UK) and photosynthetic oxygen evolution was calculated as the difference between oxygen exchange in the light and in the subsequent dark period. These measurements were done at limiting PFD and used as an estimate for maximum efficiency of photosyn- thetic oxygen evolution.

Isolation (~f thylakoid membranes. Thylakoid membranes were iso- lated as described by Schmid and Schfifer (1993). The cells were broken with a French pressure cell (Aminco, Silver Spring, Md., USA) at 4.8 MPA, using the homogenisation medium of Camm and Green (1982). The homogenate was filtered through two layers of Miracloth (Calbiochem, La Jolla, Calif., USA). After centrifuga- tion (1 min, 3000 9) the pellet was washed with the same medium. The chloroplasts were broken in 2 mM TRIS, pH 7.5 (maleate), and the thylakoid fraction was washed once in the same medium. It was used immediately (determination of atrazine-binding sites) or snap- frozen and stored at - 4 0 ~ C or - 8 0 ~ C in 12.5 mM TRIs-maleate (pH 7.5), 30% (v/v) glycerine (electrophoresis).

Atrazine-binding sites. The number of QB-binding sites was quan- tified using the kinetic method of Tischer and Strotmann (1977) as described in Paterson and Arntzen (1982).

Thylakoids were incubated at a chlorophyll concentration of 50 or 80 g �9 m -3 in a buffer containing 0.1 M sorbitol, 10 mM NaC1, and 10 mM N-Tris[hydroxymethyl]methylglycine (Tricine), pH 7.8 (NaOH). Ring-labelled UL-[14C]atrazine (specific activity 166kBq.lamol 1) was added to different final concentrations (0.08 0.58 laM). After at least 5 min incubation on ice the mixture was centrifugated (5 min, 16000 9) and an aliquot of the supernatant was scintillation-counted (2500 TR; Packard, Downers Grove, Ill., USA). The number of atrazine-binding sites was calculated from double-reciprocal plots of bound versus fiee atrazine for the dif- ferent atrazine concentrations.

Radioactive labelling. [35S]-Methionine (specific activity 45.6 TBq. mmol 1) was added to the dark-adapted cell suspension (I 00 GBq " m 3 cell suspension) and the culture vessel then exposed to photoinhibitory light (pulse experiment). After a 40-min incor- poration period, label uptake was stopped by addition of excess

unlabelled methionine (l mM). Subsequently, the cells were washed, resuspended in fresh medium containing 1 mM unlabelled methionine and returned to the high light (chase experiment).

Electrophoresis. Thylakoids were solubilised in the presence of 2.5 % (v/v) sodium dodecyl sulfate (SDS) and 100 mM DL-dithiothreitol at 80 ~ C for 10 min. The gels [15% (w/v) separation gel, 5% (w/v) stacking gel, acrylamide to N,N'-methylene-bis-acrylamide ratio of 37.5] were prepared essentially according to Fling and Gregerson (1986). The separation gel contained 750 mM TRIs-HC1 (pH 8.4), 0.1% (w/v) SDS, 0.02% (w/v) ammonium persulfate and 0.5% (v/v) N,N,N',N'-tetramethyl-ethylendiamine (Temed); the stacking gel contained 125 mM TRIS-HC1 (pH 6.7), 0.1% (w/v) SDS, 0.05 % (w/v) ammonium persulfate and 0.1% (v/v) Temed. In gels which were used for autoradiography, 6 M urea was added to both the stacking and the separation gel. In some cases, samples for Western blots were prepared with the gel system described in Schmid and Schfifer (1992). Electrophoresis was performed with the Laemmli (1970) electrode buffer at 4 ~ C.

Autoradiography. Gels were dried on 3 MM Chr paper (Whatman, Maidstone, Kent, UK) and exposed at room temperature, using x-ray films (NIF RX; Fuji, Tokyo, Japan). The autoradiographs were analysed with an UltroScan XL laser densitometer and Gel- Scan XL evaluation software (Pharmacia LKB, Uppsala, Sweden). Turnover of D1 protein was estimated from changes in the area of the Dl-protein peak.

Western blotting. Following electrophoresis the proteins were trans- ferred to a polyvinylidenedifluoride membrane (Immobilon P; Mil- lipore, Eschborn, FRG) by semi-dry blotting (8 A �9 m 2, 3-4 h); the transfer buffer contained 48 mM TRIS and 39 mM glycine (pH 8.4). Blocking, antibody incubation (dilution 1:1000 for both DI anti- body and anti-rabbit IgG-peroxidase conjugate) and washing was in a buffer containing 20 mM TRIs-HC1 (pH 7.4), 500 mM NaC1 and 0.05% (v/v) polyoxyethylene-sorbitan monolaurate (Tween 20). Tween 20 was omitted at the final washing and the blots were developed with 4-chloro-1-naphthol and H202. They were evaluat- ed in the same way as the autoradiographs.

Other procedures. Chlorophyll was determined after extraction with dimethylformamide according to Porra et al. (1989). Photon flux density was measured with a quantum sensor (LI 190 SB; Li-Cor, Lincoln, Neb., USA). If results of several experiments were av- eraged, they are given as means :t: SD (n).

Results

High- l i gh t t r e a t m e n t o f C. rubrum cells resu l ted in a decrease o f the m a x i m u m efficiency o f p h o t o s y n t h e t i c o x y g e n e v o l u t i o n a n d o f Fv/Fm (dark) (Fig. 1A, C). Bo th changes are typica l fea tures o f p h o t o i n h i b i t i o n ( K r a u s e 1988). T h e l i gh t - s a tu r a t ed p h o t o s y n t h e t i c ra te was ba re ly affected (Sch~fer a n d S c h m i d t 1991) a n d the r e d u c t i o n s in p h o t o s y n t h e t i c eff iciency were m o s t l y revers ib le in low l ight (Fig. 1B, D). In these s h o r t - t e r m e x p e r i m e n t s the effects o f h igh l ight were o n l y s l ight ly m o r e p r o n o u n c e d in n i t rogen -de f i c i en t t h a n in n i t r o g e n - r e p l e t e cells (Fig. 1).

The ex ten t o f D l - p r o t e i n t u r n o v e r d u r i n g p h o t o i n - h ib i t o ry t r e a t m e n t was m e a s u r e d in n i t r o g e n - r e p l e t e cells by pu lse -chase e x p e r i m e n t s wi th [35S]-methionine (Fig. 2A, B). I n c o r p o r a t i o n o f label d u r i n g the pu lse was m a i n l y i n to the b a n d c o n t a i n i n g D1 p ro t e in , i n d i c a t i n g t ha t this p ro t e in was p r e d o m i n a n t l y synthes ized . Degra - d a t i o n o f D1 p r o t e i n was de tec tab le by the loss o f label

C. SchS.fer et al. : D l-protein loss during photoinhibition 435

lOO

~= 8o z o '~ 60

d~ 40

20

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A 0.6

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0.2

Inhibition Recovery

(800 prnol.m'2-s -t ) (60 pmol.m'2-s "1 )

\\N\ 0 \ \ \ \ \ \ �9

A "~-~>---o I I I I

\ o\x-', ~

c I I I

0 100 200 300

y B

I I I I

I I I

400 0 200 400

Time (min)

D I I

600 800 1000

Fig. 1A-D. Changes in the efficiency of photosynthetic oxygen evolution (A, B) and in Fv/Fm (dark) (C, D) during high-light exposure (800 gmol. m -2- s-a; A, C) and during a subsequent recovery period in low light ( 6 0 p m o l . m - Z . s - ~ ; B, D) of C. rubrum cell cultures (circles). Controls (squares) were kept in low light during the whole experiment. The experiment was per- formed with nitrogen-replete (closed symbols) and with nitrogen- deficient (open symbols) cells

Table 1. The effects of high-light treatment on chlorophyll-fluore- scence parameters after dark adaptation, the number of atrazine- binding sites, and the Dl-protein immunoblot signal of C. rubrum cells. Measurements were done prior to and following a 5-h high- light treatment, and for each experiment data were calculated as percentages of the initial values. Nitrogen-replete (+N) and nit- rogen-deficient ( - N ) cells were analysed. Data are means • SD, number of experiments in parentheses

Percentage changes during high-light treatment (5 h, 800 gmol. m - 2 " s -1)

+ N cells - N cells

Fig. 2A, B. Determination of the half-life (t1/2) for degradation of D1 protein from pulse-chase experiments with C. rubrum cells. Cultures were transferred to high light (850 ~mol - m-2 . s-1) and radioactively labelled with [35S]methionine for 40 min. Thylakoids were isolated at the end of this pulse period (lane 1), and after a 90-min (lane 2) and a 360-min (lane 3) chase period. After electro- phoresis, autoradiographs were prepared (position of the radioac- tive labelled molecular-weight markers on the left margin) and label intensities quantified by densitometry (A). These data were used to determine tl/2 from semilogarithmic plots (B)

Fv/Fm - 24.7 + 11.7 (7) - 36.6 • 2.6 (3) Fo + 15.8+ 10.3 (7) + 13.8:~ 10.0 (3) F m - 33.9 • 9.3 (7) - 42 .9 • 2.6 (3)

Atrazine -35.9~:20.1 (4) - 31 .1 • 7.1 (4) binding

Western -24 .6 • 8.2 (6) - 33 .5 • 12.9 (2) blot

du r ing the chase pe r iod and semi loga r i t hmic p lo ts o f label in tens i ty versus t ime gave half-l ife t imes o f a b o u t 325 rain (mean o f two exper iments) .

In o r d e r to check whe the r D l - p r o t e i n synthesis cou ld keep up wi th this d e g r a d a t i o n rate, D 1 p ro t e in was quan - tified by d e t e r m i n a t i o n o f a t r a z i n e - b i n d i n g sites a n d by i m m u n o b l o t t i n g (Fig. 3). Bo th p a r a m e t e r s ind ica t ed a loss in D1 p ro t e in du r ing p h o t o i n h i b i t i o n (Fig. 4). The

Fig. 3A, B. Immunoblot indicating changes in Dl-protein content of C. rubrum cells during high-light exposure and recovery (A) and during chloramphenicol application at moderate light levels (B). In A, thylakoid membranes were isolated before light exposure (lane 1), after 5 h of high-light exposure (800 gmol -m -2" S -1, lane 2) and after an additional 5-h recovery period in low light (60 lamol" m -2" s -x, lane 3). In B, thylakoid membranes were isolated after 24h exposure of cells to moderate light (80 gmol" m - 2 ' s -1) in the absence (lane 1) and in the presence (lane 2) of chloramphenicol. 0.72 gg of chlorophyll were applied in each well

kinet ics however var ied cons ide r ab ly in the different ex- per iments , and a p p a r e n t l y this was due to sca t te r ing o f the ini t ial value which was used for no rma l i s a t i on . A f t e r 5 h o f h igh- l ight t r e a t m e n t a r educ t i on was obse rved for bo th the n u m b e r o f a t r a z i n e - b i n d i n g sites and the Wes t -

436 C. SchS.fer et al.: Dl-protein loss during photoinhibition

~

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Atraz ine binding lOO 80

60

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Time (min) Fig. 4. Reduction in the number of atrazine-binding sites and the D l-protein immunoblot signal during exposure of C. rubrum cul- tures to high light levels (about 800 gmol �9 m -2 �9 s-X). The experi- ments were performed with nitrogen-deficient (open symbols) and with nitrogen-replete (closed symbols) cells and the data were cal- culated as percentages of the initial value. The curves are from different experiments (different symbols)

T a b l e 2. The effects of recovery in low light after 5 h high-light exposure (800 gmol.m 2.s 1) on chlorophyll-fluorescence pa- rameters after dark adaptation, the number of atrazine-binding sites, and the Dl-protein immunoblot signal of C. rubrum cells. Measurements were done prior to and following a 5-h recovery period, and for each experiment data were calculated as percentages of the initial values. Nitrogen-replete (+ N) and nitrogen-deficient ( - N ) cells were analysed. Data are means + SD, number of experi- ments in parentheses

Percentage changes during recovery (5h, 60gmol 'm -2 . s 1)

+ N cells - N cells

Fv/F m + 2 8 . 9 + 2 2 . 6 (7) +28.04- 6.3 (3) F o - 3.94- 9.8 (7) + 7.3 + 14.2 (3) Fm +46.34-20.5 (7) +38.94- 9.8 (3) Atrazine + 40.8 4- 12.1 (4) + 37.0 4- 27.0 (4) binding Western + 28.7 4- 31.7 (6) not determined blot

ern-blot signal (Table 1). This effect was not significantly different in nitrogen-replete and in nitrogen-deficient cells (95% confidence limits). The reductions in Fm and Fv/F m (dark) were again somewhat higher in the latter cell type.

These results indicate that during light stress in vivo and without application of a protein-synthesis inhibitor, degradation of D 1 protein may still surpass its synthesis, thus resulting in a net loss of this protein. Hence the degradat ion and the resynthesis processes do not seem to be tightly coupled.

During recovery in low light, non-photochemical quenching decreased again, resulting in an increase of F~ and Fv/F~, (dark) (Table 2). The number of atrazine-

Table 3. The effects o f chloramphenicol application (0.1 kg " m - 3) in the light on chlorophyll-fluorescence parameters after dark ad- aptat ion, the number of atrazine-binding sites, and the D1- protein immunoblo t signal o f C. rubrum cells. Chloramphenicol was added and the cells exposed either to 5 h o f high light ( 8 0 0 g m o l ' m - Z ' s -1) or to 2 4 h of modera te light (80 lamol �9 m 2. s - 1). Data were calculated as percentages of the initial values (800 gmol �9 m -z �9 s -1) or o f those obtained for cul- tures without chloramphenicol addit ion (80 lamol - m -2 - s-1) . The experiments were performed with nitrogen-replete cells. Da ta are means 4- SD, number o f experiments in parentheses

Percentage changes during chloramphenicol treatment

5h, 800gmol 'm-2"s 1 24h, 80gmol .m-Z-s - I

Fv/Fm -44.4• 11.8 (3) -18.54- 6.5(4) Fo +23.64-23.3 (3) +43.04- 14.0 (4) F= -48.4+ 13.3 (3) -9.64- 9.8 (4) Atrazine not determined -43.74- 5.0 (4) binding Western - 41.0 4- 3.0 (3) - 45.14- 10.4 (3) blot

binding sites increased, and in nitrogen-replete cells an increase in the D 1-protein immunoblot signal could also be observed. Obviously the high-light effects were at least partially reversible during low-light recovery. Significant differences between nitrogen-replete and nitrogen-defi- cient cells could not be detected (95 % confidence limits).

Both the changes in fluorescence parameters and the reductions in Dl-pro te in immunoblot signal were more pronounced when chloramphenicol was added during the high-light treatment to inhibit chloroplastic protein syn- thesis (compare Tables 1 and 3). The half-life of 325 min for the D1 protein, as estimated from the chase experi- ments, indicates a loss of 47% during the 5-h high-light period. This value corresponds quite well to the observa- tions in nitrogen-replete cells (Table 3).

Thus far, the results indicate a correlation between flu- orescence quenching and the reduction in D 1-protein con- tent. However, when chloramphenicol treatment was ap- plied in moderate light levels (about 80 ~tmol �9 m -2 �9 s-1), Dl -pro te in content decreased considerably with only small reductions in the Fm value of dark-adapted cells (Table 3). Hence slowly reversible, non-photochemical quenching was only small in these conditions.

Figure 5 shows the correlation between Fv/F m (dark) and the number of atrazine-binding sites. There was no proportionali ty between the two parameters, and the regression line extrapolated to a value of 0.523 for total loss of atrazine-binding sites.

The use of chloramphenicol as an inhibitor of chloro- plastic protein synthesis has been critizised recently (Okada et al. 1991) because it affected photosynthetic efficiency and chlorophyll fluorescence also when applied in the dark, and served as an electron acceptor for PSI in the light. Aro and co-workers (personal communica- tion; E.-M. Aro, Depar tment of Biology, University of Turku, Turku, Finland) also observed that chloram- phenicol can act as an electron acceptor for PSI. How- ever, it had the same effects on turnover of D1 protein

c. Schfifer et al.: Dl-protein loss during photoinhibition 437

1.0

0 , 8 --

0.6

"- 0 . 4 -

0 , 2 - -

I I I I I I

0 I I I I I I

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Number of atrazine binding sites

( mmol. tool'1 Chl) Fig. 5. Correlation between the number of atrazine-binding sites and Fv/Fm (dark) in C. rubrum cells. The data are from several experiments and Dl-protein loss was caused either by high-light treatment (800 gmol �9 m-2. s-1 ; open symbols; recovery data also included) or by the presence of chloramphenicol in moderate light (about 80 gmol �9 m- 2. s- t ; closed symbols). The calculated regres- sion line is also shown. All experiments were performed with nitro- gen-replete cells

as lincomycin, a chloroplastic protein-synthesis inhibitor which cannot function as electron acceptor. We could not observe effects of chloramphenicol on photosynthetic oxygen evolution or fluorescence parameters after a 5-h dark period. Following 24 h dark incubation, reductions in photosynthetic oxygen evolution and Fm were detect- able in the presence of chloramphenicol but this treat- ment had only a small effect on the number of atrazine- binding sites (data not shown).

Discussion

Our results indicate that a partly reversible net loss of D1 protein can occur during light stress (Tables 1, 2; Figs. 3, 4). Evidence for a net loss of D 1 protein during photoinhibition of intact cells was also obtained in inves- tigations with algae (Schuster et al. 1988) and higher plants (Chow et al. 1989; Tyystj/irvi and Aro 1990). However, other studies, some using the same species, did not support the occurrence of such a net loss of D1 protein (Neale and Melis 1990; Kettunen et al. 1991).

A net loss of D1 protein and its enhancement by chloroplastic protein-synthesis inhibitors (Tables 1, 3; Ohad et al. 1984) corroborate the view that degradation and resynthesis of D 1 protein are independent processes which might have different locations. Hence it is possible that D1 protein is degraded while the inactivated PSII centers are still in the grana region. Then, the residual proteins could move to the stroma region where reinser- tion of newly synthesized D 1 protein occurs (Mattoo and Edelman 1987). This interpretation corresponds to the model by Hundal et al. (1990) which was developed on

the basis of in-vitro studies. In contrast to this, Kettunen et al. (1991) conclude from the lack of net D 1-protein loss that PSII centres with inactivated D 1 protein move as a complex to the stroma thylakoids where Dl-protein deg- radation and the reinsertion of newly synthesized D1 protein occur (Mattoo and Edelman 1987).

It has been proposed that prior to degradation the D1 protein is modified, slightly changing its electrophoretic mobility (DI*; Callahan et al. 1990; Kettunen et al. 1991; Elich et al. 1992). The upper Dl-protein band in the autoradiogramms from chase experiments (Fig. 2) probably represents this modified form.

Chase experiments showed that the half-life of D1- protein degradation was rather long in our experimental system. The mean value in the present study was about 325 min for cells which were grown at moderate light levels (Fig. 2). It corresponds quite well to the value of about 340 rain which can be calculated from an earlier study (Schmid and Schfifer 1993). Other investigators, using various species, observed considerably shorter half- life times (27 120 rain; Ohad et al. 1990b; Shochat et al. 1990; Godde et al. 1991). Notwithstanding the slow rate of degradation of D 1 protein, its synthesis might actually be the rate-limiting step in the repair cycle of C. rubrum PSII since a net loss of this protein occurred. A low capacity for synthesis of D1 protein could also explain why the blockage of chloroplastic protein synthesis did not increase net Dl-protein loss by more than about 20% (compare Tables 1 and 3). Unfortunately, pulse experi- ments cannot be used as an estimate for the rate of Dl-protein synthesis, because the specific activity of the intracellular methionine pool and the maximum extent of labelling are not known.

We observed decreases in F~, and Fv/Fm (dark), and increases in Fo when cells were exposed to high light (Fig. 1, Table 1). These are typical effects of high-light treatment (Krause 1988). When Dl-protein loss was caused by addition of chloramphenicol in moderate light the reduction in Fm (dark) was only small. Hence the oc- currence of slowly reversible non-photochemical quench- ing is not an absolute characteristic of Dl-protein loss. This was also suggested by Briantais et al. (1988). Slowly reversible non-photochemical quenching has been re- lated to additional energy-dissipating mechanisms (Krause 1988). The results obtained with chloram- phenicol treated cells indicate that loss of D1 protein also occurs when these mechanisms are not operative.

A decrease in Fv/Fm (dark) was correlated to the de- crease in the number of atrazine-binding sites, but we did not observe a proportionality between these parameters (Fig. 5). Oquist et al. (1992) described such a propor- tionality between Fv/F m (dark) and the number of func- tional PSII centers. According to our results this relation- ship could be fortuitous, resulting from the parallel oc- currence of strong non-photochemical quenching (result- ing in a reduction of Fm and Fv/Fm (dark)) and inactiva- tion or degradation of D1 protein.

The observation that net Dl-protein loss does occur in vivo during light stress indicates that limitations in the repair-cycle capacity could also determine photosynthet- ic performance in high-light environments (Kyle et al.

438 c. SchS.fer et al.: Dl-protein loss during photoinhibition

1984). The direct effect o f a partial loss o f D 1 protein on the l ight-saturated photosynthe t ic rate is p robab ly low, because this rate did no t decrease noticeably during pho- toinhibi t ion (Sch~ifer and Schmidt 1991). Limitat ions in D l - p r o t e i n tu rnover were discussed as possible causes for the synergistic effect o f ni t rogen deficiency and high- light stress (Henley et al. 1991 ; Sch~ifer and Heim 1993). The present data indicate that in short - term experiments (up to 5 h durat ion) ni t rogen deficiency has no significant effect on the loss o f a t razine-binding sites in high light and on subsequent recovery in low light (Tables 1 and 2). Hence, even this stress factor, which should affect protein synthesis directly, did no t disturb the repair cycle for D1 protein. Possibly, the amino acids which are released dur ing D 1-protein degrada t ion are largely recycled. This would minimize the addit ional demand for ni trogen dur ing photo inh ib i t ion and could explain the lack o f a p r o n o u n c e d effect o f ni t rogen deficiency. The long- term effects o f light stress in nitrogen-deficient cells might represent the summing-up o f small changes which are not necessarily related to D l - p r o t e i n content . It is possible that these effects are indicated by the slightly more pro- nounced reduct ion in Fv/Fm (dark) in nitrogen-deficient cells which was observed in shor t - term experiments.

Finally, it mus t be kept in mind that at present it canno t be decided whether net loss o f D1 protein really represents damage to the PSII react ion center. I t could well be that the shift in D l - p r o t e i n level indicates some adapt ive response or tha t it is accompanied by some favorable changes in the propert ies o f D1 protein. Such changes have been discussed in relation to the occurrence o f PSII heterogenei ty (Aro et al. 1990; Neale and Melis 1991) and they p robab ly merit fur ther consideration.

We thank Helga Simper for excellent technical assistance and Stefan Peter for his enthusiastic help in some of the experiments. Further- more, we thank Professor E. Beck (Lehrstuhl fiir Pflanzenphysiolo- gie, Bayreuth, FRG) for critical reading of the manuscript, Profes- sor M. Sprinzl (Lehrstuhl fiJr Biochemie, Bayreuth, FRG) for the opportunity to use the laser densitometer and Professor A. Trebst (Lehrstuhl ffir Biochemie der Pflanzen, Bochum, FRG) for the generous gift of D1 antibody. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137).

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