photoinhibition and d1 protein degradation in peas acclimated … · photoinhibition and d1 protein...

Plant Physiol. (1993) 103: 835-843

Photoinhibition and D 1 Protein Degradation in Peas Acclimated to Different Growth Irradiances'

Eva-Mari Aro*, Stephanie McCaffery, and Jan M. Anderson

Commonwealth Scientific and Industrial Research Organization, Divison of Plant lndustry and Cooperative Research Centre for Plant Science, G.P.O. Box 1600, Canberra, Australian Capital Territory 2601, Australia

lhe relationship between the susceptibility of photosystem II (PSII) to photoinhibition in vivo and the rate of degradation of the D1 protein of the PSll reaction center heterodimer was investigated in leaves from pea plants (Pisum safivum 1. cv Creenfeast) grown under widely contrasting irradiances. lhere was an inverse linear relationship between the extent of photoinhibition and chlorophyll (Chl) a /b ratios, with low-light leaves being more susceptible to high light. In the presence of the chloroplast-encoded protein synthesis inhibitor lincomycin, the differential sensitivity of the various light-acclimated pea leaves to photoinhibition was largely removed, demonstrating the importance of D1 protein turnover as the most crucial mechanism to protect against photoinhibition. In the differently light-acclimated pea leaves, the rate of D1 protein degradation (measured from [35S]methionine pulse-chase experiments) increased with increasing incident light intensities only if the light was not high enough to cause photoinhibition in vivo. Under moderate illumination, the rate constant for D1 protein degradation corresponded to the rate constant for photoinhibition in the presence of lincomycin, demonstrating a balance between photodamage to D1 protein and subsequent recovery, via D1 protein degradation, de novo synthesis of precursor D1 protein, and reassembly of functional PSII. In marked contrast, in light sufficiently high to cause photoinhibition in vivo, the rate of D1 protein degradation no longer increased concomitantly with increasing photoinhibition, suggesting that the rate of D1 protein degradation is playing a regulatory role. lhe extent of thylakoid stacking, indicated by the Chl a/b ratios of the differently light- acclimated pea leaves, was linearly related to the half-life of the D1 protein in strong light. We conclude that photoinhibition in vivo occurs under conditions in which the rate of D1 protein degradation can no longer be enhanced to rapidly remove irreversibly damaged D l protein. We suggest that low-light pea leaves, with more stacked membranes and less stroma-exposed thylakoids, ate more susceptible to photoinhibition in vivo mainly due to their slower rate of D1 protein degradation under sustained high light and their slower repair cycle of the photodamaged PSll centers.

Photoinhibition of PSII is a phenomenon that occurs in a11 oxygenic photosynthetic organisms under high irradiance (Powles, 1984; Prasil et al., 1992). Although light is the driving force of PSII, it also inactivates electron transport and destroys structural components of the PSII reaction center. Susceptibility of plants to photoinhibition under any given

' This research was supported by the Academy of Finland and the Cooperative Research Centre for Plant Science, Canberra, Australia.

* Corresponding author; Department of Biology, University of Turku, SF-20500 Turku, Finland; fax 358-21-6335549.

835

PPFD greatly depends also on environmental conditions other than light (Powles, 1984; Krause, 1988). If plants suffer from stress such as nutrient deficiency, drought, or low temperature, photoinhibition of PSII may occur even at a moderate light intensity. Furthermore, the light acclimation of plants or individual leaves greatly affects the susceptibility of PSII to photoinhibition.

The underlying molecular mechanism for photoinhibition of PSII is at present under intense study (for review, see Aro et al., 1993). According to current knowledge, primarily based on in vitro studies, photoinhibition of PSII can be separated into severa1 partia1 and consecutive reactions. The first event, photoinactivation of PSII electron transport, is thought to be followed by oxidative damage to the D1 protein, one of the heterodimeric polypeptides of the PSII reaction center complex. This damage, either directly or indirectly, exposes the D1 protein to an intrinsic protease (Virgin et al., 1991; De Las Rivas et al., 1992) by a mechanism not yet known.

Rapid light-dependent tumover of the D1 protein in vivo was reported as early as two decades ago (Eaglesham and Ellis, 1974; Mattoo et al., 1984), long before its central role as a reaction center polypeptide of PSII was known (Nanba and Satoh, 1987). Now, this fast D1 protein turnover is also often connected to in vivo photoinhibition of PSII and subsequent recovery of electron transport activity via de novo protein synthesis (Prasil et al., 1992). Indeed, in Chlamydo- monas cells, accelerated rates of D1 protein degradation have been measured under photoinhibitory light conditions (Kyle et al., 1984; Ohad et al., 1988; Schuster et al., 1988). How- ever, involvement of D1 protein damage and tumover in photoinhibition of higher plants is not universally accepted; contrasting views exist that attribute only a minor role for D1 protein turnover in the susceptibility of leaves to photoinhibition and during subsequent recovery (e.g. Demmig-Adams and Adams, 1992; Syme et al., 1992).

In the present paper we have compared the susceptibility of PSII to photoinhibition and the rate of D1 protein degradation in pea (Pisum sativum L.) plants acclimated under different-growth light irradiances. Plants differ in their susceptibility to photoinhibition in vivo, with shade and low- light plants being more susceptible to photoinhibition than

Abbreviations: D1, D1 reaction center protein of PSII; D1*, phos- phorylated form of the D1 protein; F, and F,, variable and maximal fluorescence, respectively; kp, and kD1 dep., first-order rate constants for photoinhibition and D1 protein degradation, respectively; LHCII, light-harvesting Chl a / b proteins.

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836 Aro et al. Plant Physiol. Vol. 103, 1993

sun and high-light plants (Demmig and Bjorkman, 1987; Greer and Laiing, 1988; Tyystjarvi et al., 1991, 1992; Oquist et al., 1992a). Moreover, with varying acclimation, even in the level of D1 protein tumover, plants appear to invoke rather different strategies for protection against photoinhibition (Oquist et al., 1992a; Tyystjarvi et al., 1992). Since these studies have either compared different species or used only two high and low light-acclimated plants, we have compared five light treatments of peas to cover the range of growth irradiances. Our results demonstrate that the rate of D1 protein degradation clearly increases with increasing light intensity in a11 the differently light-acclimated pea leaves. However, in light strong enough to induce photoinhibition in vivo, the rate of D1 protein degradation cannot be further increased. Moreover, the capacity of low-light leaves to increase the rate of D1 protein degradation with increasing PPFD is more limited than in high-light leaves. We suggest that this difference in the capacity for D1 protein degradation is an underlying reason for higher sensitivity of low-light leaves to photoinhibition in vivo.

MATERIALS AND METHODS

Plant Material

Peas (Pisum sativum L. cv Greenfeast) were grown in a compost:perlite mixture in growth chambers under photon flux densities of 65, 250, and 700 pmol m-' s-', photoperiod of 12 h, and temperature at 23OC in a glasshouse under full light or under shade cloth that allowed only 7% transmission of the natural light. It must be stressed, however, that from September to December, 1992, when peas were grown in the glasshouse, only a few sunny days were included in each growth cycle of peas. Two to 5-week-old seedlings, depend- ing on the PPFD during growth, were used for experiments. To ensure the uniformity of leaves, the fourth leaf pair representing the youngest fully developed pair of leaves was always used.

Light Treatment of Leaf Discs

Leaf discs, floating adaxial side up on water (or lincomycin or DTT solutions in respective experiments) were illuminated with an HMI studio lamp. Light was passed through a heat filter and temperature was controlled at 23OC. Different photon flux densities (2800, 1600, 400, and 50 pmol photons m-2 -1 s ) were obtained by using neutra1 density filters.

To study the role of chloroplast-encoded protein synthesis in the tolerance of pea leaves to photoinhibition, the petioles were immersed in 0.6 m~ lincomycin solution in the hood at a PPFD of 20 pmol m-2 s-' before leaf discs were punched and illuminated at different photon flux densities. The volume of lincomycin solution taken up by the leaves during a 3- to 4-h period corresponded to 100 to 150% of the leaf water content.

The role of the xanthophyll cycle in protection against photoinhibition was tested with leaves taken from growth chambers at the end of the dark period. Before photoinhibitory illumination, the petioles of these leaves were immersed in 1.5 m~ DTT solution as described for lincomycin uptake.

Pulse and Chase Experiments with [~-~%]Met: D 1 Protein Degradation

Leaf discs (diameter of 1.9 cm) were pressed extremely gently against coarse sandpaper to facilitate incorpora tion of [35S]Met. For the radioactive labeling of thylakoid proteins, leaf discs floating on 20 mL of solution containing 1 mCi of [35S]Met in 0.4% Tween 20 were illuminated for 1.5 In at 50 to 200 pmol photons m-' s-'. The light intensity used (did not induce photoinhibition in any of the light-acclimated leaves. Immediately after the pulse period, leaf discs were washed twice in 10 m~ unlabeled Met, 0.4% Tween 20 solution and further illuminated in the presence of cold Met for 1.5 h before taking the first chase sample (chase O min). The decrease in radioactivity of the D1 protein was followed at 50, 400, 1600, and 2800 pmol photons m-'s-', generally for 45, 90, 135, 180, and 225 min. Two leaf discs at each time point were combined and thylakoid membranes were rapidly isolated at 2OC. Leaves were ground in ice-cold 50 m~ Hepes, pH 7.6, 0.3 M sorbitol, 10 mM NaCI, 5 mM MgCI2. The homogenates were filtered through four layers of Miracloth and centrifuged at 4500g for 5 min, and pellets were washed once with washing buffer (similar to isolation buffer, except for 0.1 M sorbitol). Thylakoid pellets were resuspended in a small volume of washing buffer, and after taking the s,3mples for Chl determinations (Porra et al., 1989), the thylakoids were immediately frozen in liquid nitrogen.

Thylakoid polypeptides were solubilized (65OC, 5 mjn) and electrophoretically separated, essentially according to Ljung- berg et al. (1986), including 4 M urea in both the solubilizing and gel buffers. A gradient of 12 to 22.5% acrylamide was used in the separation gel and 4% acrylamide was uised in the stacking gel. D1 protein was identified in polyacrylamide gels by immunoblotting; after electrophoresis, thylakoid polypeptides were blotted to nitrocellulose membranes and im- munodetected with D1 protein specific antibody (kindly provided by Prof. I. Ohad) and alkaline phosphatase conjugate.

To analyze the radioactivity in thylakoid membrane polypeptides, the gels were first stained with Coomassie brilliant blue, destained, treated with amplifier, and dried. Dricrd gels were exposed to Molecular Dynamics (Sunnyvale, CA.) stor- age phospho screens for approximately 82 h. The screens were scanned on a Molecular Dynamics 400s Phosphor- Imager and quantification of the radioactivity in D1 protein and LHCII polypeptides was made using the ImageQuant volume integration software. When the rate of D1 protein degradation was calculated, the radioactivity in LHCII polypeptides was generally used as an interna1 standard. This method was chosen because light treatments did not change the level of radioactivity in LHCII polypeptides during the chase period.

Fluorescence Measurements

Fluorescence measurements of the leaf discs were made with a Hansatech Plant Efficiency Analyzer. Before meiasure- ments, the leaf discs were dark adapted for 30 min to al- low relaxation of easily reversible fluorescence-queriching components.



Photoinhibition and D1 Protein Degradation in Light-Acclimated Peas

Chl Determinations

Chl was extracted in 80% buffered acetone (25 m~ Hepes, pH 7.5) and quantified by using the extinction coefficients and wavelengths determined by Porra et al. (1989).

Curve Fitting

Both the decrease in Fv/Fm during illumination in the presence of lincomycin and the decrease in the radioactivity of the D1 protein during the chase period were fitted to a first-order equation, weighing the data points individually according to their SD values. The Fig. P software (Biosoft, Cambridge, UK) was used for curve fitting.

RESULTS

Photoinhibition in Vivo of Light-Acclimated Pea Leaves

During photoinhibition, Chl fluorescence Fv/Fm ratios are linearly correlated with both the quantum yields of light- limited O2 evolution (Demmig and Bjorkman, 1987) and the number of functional PSII reaction centers (Oquist et al., 199213). When differently light-acclimated pea leaves were exposed to strong illumination at a PPFD of 1600 Imo1 m-’ s-l, the inhibition of PSII, measured as the Fv/Fm ratio, varied with respect to growth irradiance. As expected, the low light- acclimated plants (65 pmol photons m-* s-’) became more severely photoinhibited than the leaves of high light-grown peas (700 wmol photons m-’ s-’). Peas grown either in growth chambers or under natural sunlight behaved similarly (Fig. 1, A and B, open symbols). In a11 cases, the exponential decline in Fv/Fm ratios with time during photoinhibition (Fig. 1) equilibrated to a steady level of photosynthetic efficiency of PSII within 3 to 4 h of illumination; almost no further decline in Fv/F,,, in Figure 1 (open symbols) was observed if illumination was continued up to 5 h (data not shown). Such an equilibration to a steady-state level of active PSII centers is expected if a first-order recovery reaction is counteracting a first-order inhibition reaction during the high-light treatment (Tyystjarvi et al., 1992).

Growth of plants in varying light intensities of constant quality induces marked modulation in photosynthetic composition and function, as well as in chloroplast structure (Anderson, 1986). With pea plants, there is a striking correlation of Chl a / b ratios of differently light-acclimated leaves, not only with the distribution of Chl among the various Chl proteins, but also with photosynthetic function (Anderson, 1986). Susceptibility of pea leaves to photoinhibition was also linearly correlated with Chl a/b ratios; the lower the Chl a / b ratio, the more susceptible pea leaves were to photoinhibition in vivo (Fig. 2).

To assess the importance of xanthophyll-associated energy dissipation (Demmig-Adams, 1990) to explain the differences in the susceptibility to photoinhibition between the differently light-acclimated pea leaves, we also inhibited the de- epoxidation of violaxanthin to zeaxanthin with DTT prior to photoinhibitory illumination. Bilger et al. (1989) demonstrated that 1 m~ DTT administered through the cut petioles of leaves completely inhibits zeaxanthin formation. Although we observed that the xanthophyll cycle indeed protects pea

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Figure 1. Photoinhibition of PSll (decrease in FJF, ratios) during illumination of differently light-acclimated peas at 1600 pmol photons m-’ s-’. A, Peas grown in growth chambers at PPFDs of 65 (O, O), 250 (O, W), and 700 (A, A) pmol m-’ s-’, and B, under 7% (O, O) and 100% (A, A) natural light. Open symbols indicate the absence and closed symbols indicate the presence of lincomycin. Control values of F,/F, varied from 0.82 to 0.83 f 0.01 (maximum SD) in differently light-acclimated peas. In the presence of lincomycin, control values of F,/F, varied from 0.80 to 0.82 zk 0.01 (maximum SD). Data obtained in t h e presence of lincomycin are fitted to a first-order equation. Mean f SD; n = 6.

leaves against photoinhibition, the extent of protection between the differently light-acclimated peas was nearly the same (Table I). Therefore, even in the absence of the xanthophyll cycle (DTT-treated leaves), significant differences per- sisted in the susceptibility of pea leaves to photoinhibition.

Role of Chloroplast-Encoded Protein Synthesis in Proteding against Photoinhibition

To analyze the role of chloroplast-encoded protein synthesis (tumover of the D1 protein) in the susceptibility to photoinhibition in differently light-acclimated pea leaves, high illumination of leaves was repeated in the presence of the chloroplast-encoded protein synthesis inhibitor lincomycin. We have previously shown that lincomycin has no side effects on photosynthesis in pumpkin (Tyystjiirvi et al., 1992) or pea leaves (data not shown). The presence of lincomycin clearly




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Figure 2. Relatioiiship between susceptibility of PSll to photoinhibition (F,/F,, % of control after illumination of 5 h at 1400 pmol photons m-’ s-’) and the Chl a/b ratio in differently light-acclimated pea leaves. Peas in order of increasing Chl a/b ratios were grown at 7% sunlight, 65 pmol photons m-’ s-’, 250 pmol photons m-’ s-’, 100% sunlight, arid 700 pmol photons m-’ s-’. Mean f SD; n = 4 to 5.

enhanced the rate of photoinhibition in a11 pea plants (Fig. 1). Moreover, the significant difference in the susceptibility of PSII to photoinhibition at 1600 pmol photons m-’ s-’ among peas grown under various light regimes was greatly diminished when photoinhibition was induced in the pres- ente of lincomycin (Fig. 1, closed symbols). When chloroplast-encoded protein synthesis was inhibited, declines in Fv/F, ratios during high illumination fit a first-order equation, as previously demonstrated with pumpkin leaves (Tyystjarvi et al., 1992).

We next focused on PSII function in pea leaves illuminated with moderate light of 400 pmol photons m-’s-’. Moderate light did not induce photoinhibition with four of the light- acclimated peas; weak photoinhibition was induced only in peas grown under 7% sunlight, which had the lowest maximum photosynthetic capacity and Chl a / b ratio (Fig. 3, A and B, open symbols). However, if chloroplast-encoded protein synthesis was inhibited with lincomycin, significant photo-

Table 1. The role of xanthophyll-associated energy dissipation a5 a means to protect against sustained photoinhibition in pea leaves acclimated to varying PPFDs

Photoinhibition of PSll was measured as a decrease in F,/F, in the presence and absence of DTT (1.5 m M ) during illumination of pea leaves grown under different irriadiances. lllumination at 1400 pmol photons m-’s-’ for 5 h. Mean f SD; n = 6.

FvIFm PPFD during Crowth of llluminated for 5 h

-DTT +DTT Peas Dark control

pmol m-’s-’

65 0.81 f 0.002 0.43 f 0.048 0.30 f 0.051 250 0.83 f 0.003 0.54 f 0.026 0.45 f 0.030 700 0.82 f 0.003 0.70 f 0.028 0.60 f 0.020

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Figure 3. Effect of illumination at 400 pmol photons m-’ s-’ on t h e photochemical efficiency of PSll (F,/F,) in peas grown under’different light regimes. A, Peas grown in growth chambers at PPI-Ds of 65 (O, O), 250 (O, W), and 700 (A, A) pmol m-’ s-’, and B, at 7% (O, O) and 100% (A, A) natural light. Open symbols represent experiments without lincomycin and closed symbols indicate the presence of lincomycin. Data obtained in the presence ofllinco- mycin are fitted to a first-order equation. Mean f so; n = 5 to 8.

inhibition of PSII was evident, even at 400 pmol photons m-2 -1 (Fig. 3, A and 8, closed symbols) in a11 our light- acclimated peas. These photoinhibition results, obtained in the presence and absence of lincomycin, demonstrate ii crucial role for chloroplast protein tumover in the protection of leaves against photoinhibition of PSII (Greer et al., 1986; Schuster et al., 1988), even at moderate light levels.

Since a11 leaf discs were dark adapted for 30 min after light treatments and before the F.,/F,,, ratios were recorded, nearly a11 dark-reversible fluorescence quenching, which probably represents the reversible down-regulation of PSII, was relaxed (data not shown). Thus, the decline in F J F , diuring illumination of the differently light-acclimated peas in the presence of lincomycin was due mainly to photodamage to PSII complexes. However, a minor component, which com- prised only about 10% of the decline in F,/F,,, after various time periods of illumination, relaxed in low light conditions, even in the presence of lincomycin (data not shown).

s



Photoinhibition and D1 Protein Degradation in Light-Acclimated Peas 839

Table II. Comparison of rate constants for photoinhibition of PSII ofpea leaves acclimated to varying growth irradiances

Rate constants for photoinhibition of PSII (/CP,) were calculatedfrom a decrease in Fv/fm at 400 and 1600 photons m~the presence of lincomycin. Values were calculated from lincomy-cin data (Figs. 1 and 3) fitted to first-order reaction kinetics. Half-times for photoinhibition (t,/2, min) are given in parentheses.

(cm X 103, min"',

PPFD duringGrowth

tiMo! m s65

2507007% sunlight100% sunlight

and (d/2, min)

PPFD of treatment

400 nphotons

4.53.53.64.94.3

molm-2 s-

(154)(196)(193)(141)(160)

1 600 nmolphotons m"2 s"1

16.7 (42)14.5 (48)11.9 (58)16.8 (41)13.8 (50)

We next determined rate constants for photoinhibition (kP{)from first-order fits of the decline in Fv/Fm during illuminationof light-acclimated peas in the presence of lincomycin, atboth high and moderate light (Table II). In all cases thecloseness of the fit was within 95% of the expected limits fora correct model of the data. Rate constants for photoinhibi-tion, determined in the presence of lincomycin, are nearly 4-fold higher at 1600 than at 400 /imol photons m~2 s"1 (TableII). However, there are only relatively small differences inthe rate constants for photoinhibition at either 400 or 1600/imol photons m~2 s"1 when differently light-acclimated pealeaves are compared; low light-acclimated peas are slightlymore susceptible to photoinhibition than high light-accli-mated peas in the presence of lincomycin (Table II).

There has been criticism of the use of prokaryotic proteinsynthesis inhibitors to evaluate the role of chloroplast-en-coded protein synthesis in the susceptibility to photoinhibi-tion of thylakoid membranes or intact leaves (Okada et al.,1991). Although our earlier results (Oquist et al., 1992a;Tyystjarvi et al., 1992) show that in intact leaves theseprecautions are not always necessary, we wanted to verifythe crucial role of Dl protein turnover in photoinhibitionwith totally independent experiments by measuring the ratesof Dl protein degradation.

Rates of D1 Protein Degradation in Light-Acclimated Peas

Leaf discs were pulse-labeled with [35S]Met under condi-tions that did not induce photoinhibition of PSII. This pre-caution was taken to ensure that minimal amounts of pho-todamaged PSII centers were present at the beginning of thechase period. Figure 4 shows a typical labeling pattern ofthylakoid membrane polypeptides after the pulse period (lane1), demonstrating that Dl protein and LHCII polypeptidesare the most heavily labeled polypeptides. Both nonphos-phorylated and Dl* forms of the Dl protein were resolvedin our SDS-PAGE, but in chase experiments they were treatedas one entity. Decreases in the radioactivity of pulse-labeledDl protein during the chase period (Fig. 4) at 50, 400, 1600,or 2800 nmol photons m"2 s~' were fitted to first-order

reaction kinetics (Fig. 5). Then the rate constants for degra-dation of the Dl protein and the half -life values of the Dlprotein were determined in differently light-acclimated pealeaves (Table III). Several important features were observed.The rate of degradation of the Dl protein increased with anincrease in incident light intensity (Fig. 5, Table HI). At 50^mol photons m~2 s"1, the calculated half-life for the Dlprotein was over 6 h, whereas an increase in light intensityto 400 /trnol photons m~2 s"1 enhanced the rate of Dl proteindegradation, giving the half-life of about 2.5 h in low (65

m~2 s"1), medium (250 i^mol m~2 s"1), and high (700m~2 s~!) light-acclimated peas. Indeed, in low and

moderate light levels, the rate of Dl protein degradation isdetermined mainly by the prevailing incident PPFD, not bythe light acclimation of leaves.

However, under photoinhibitory light of 1600 jumol pho-tons m~2 s"1, the rates of Dl protein degradation were strictlydependent on the light acclimation of pea leaves (Table III).The lower the growth irradiance, the slower the rate of Dlprotein degradation under the photoinhibitory light of 1600/imol photons m~2 s"1. High light-acclimated peas were ableto enhance the rate of Dl protein degradation at 1600photons m"2 s"1 2.3-fold from that observed at 400 jimolphotons m~2 s"1, whereas only a 1.4-fold increase was meas-ured in low-light peas. The rate of Dl protein degradationincreased 1.7-fold in moderate-light peas at 1600 /urno! m~2

s"1, as compared with that at 400 j*mol irT2 s"1. The slowestrate of Dl protein degradation during illumination at 1600/imol photons m~2 s"1 was measured in 7% sunlight peas(Table III). These plants also possessed the lowest Chl a/bratio and were most susceptible to photoinhibition (Fig. 2).

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Figure 4. Phosphorlmager printout of thylakoid membrane poly-peptides resolved by SDS-PACE after pulse labeling with [35S]Met(lane 1) and subsequent chase of radioactivity for 45 min (lane 2),90 min (lane 3), 135 min (lane 4), and 180 min (lane 5). Leaf discsof peas grown at 65 /imol photons m"2 s"' were pulse labeled at200 ftmol photons m"2 s"' for 1.5 h, washed with unlabeled Met,and chased at a PPFD of 1600 Mmol m"2 s"1.




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Figure 5. Rate of degradation of t h e D1 protein at different incident PPFDs in peas grown at 700 pmol photons m-' s-I. Leaf discs were pulse labeled with ["SIMet and the radioactivity in the D1 protein was chased at 50, 400, 1600, and 2800 pmol photons m-' s-'. Data obtained from two to five independent experiments were fitted to first-order equations. Data points were weighed according their SD

values.

When the incident light was increased to very high irradiance (2800 pmol photons m-' s-'), a limited additional increase in the rate of D1 protein degradation occurred in high- light peas (grown at 700 pmol m-2 s-'), giving the half-life of 54 min for the D1 protein (Fig. 5, Table 111). Illumination of high-light leaves for 4 h at a PPFD of 2800 pmol m-' s-' induced a steady-state level of 45% photoinhibition in the photochemical efficiency of PSII. In low-light peas, a PPFD of 2800 pmol m-2 s-' was too high to give reliable results, since photooxidative Chl bleaching was observed during the course of illumination.

Interestingly, in high-light peas the rate constant for photoinhibition (determined in the presence of lincomycin) and the rate constant for D1 protein degradation at 1600 pmol photons m-' s-' are very similar (Tables I1 and 111). These data suggest that photodamaged D1 protein in the PSII

Table ll l . Comparison of the rate constants for degradation of the D7 protein (kol deg,.) of pea plants acclimated to varying photon flux densities

k,, degr. was calculated from t h e decrease in radioactivity in D1 protein during the chase at different PPFDs. Values were calculated from data fitted to íirst-order reaction kinetics as in Figure 5. Half- life values of the D1 protein ( t l12, min) are given in parentheses.

k,, degr. x 103, min-', and (tl12, min)

PPFD during Crowth lncident irradiance during chase (pmol photons m-2 s-')

50 400 1600 2800

pmol photons m-2 s-' 65 1.9 (372) 4.2 (165) 5.8 (120)

250 1.8 (383) 4.2 (165) 7.1 (98) 700 1.8 (397) 4.5 (154) 10.1 (68) 12.9 (54) 7% sunlight 5.5 (126) 100% sunlight 8.3 (84)

centers of high-light leaves is rapidly degraded. However, in peas acclimated to low irradiance (65 pmol photons TIL-' s-' or 7% sunlight), the half-life values of the D1 protein are nearly three times more than the half-life values for photoinhibition; thus, the degradation of the D1 protein is nearly three times slower than photoinhibition of PSII in the pres- ente of lincomycin (Tables I1 and 111). With differently light- acclimated pea leaves, there was a general trend suggesting that the lower the Chl a/b ratio of the leaves, the greater the difference between the half-time for photoinhibition arid the half-life of the D1 protein (Fig. 6).

The importance of the capacity for D1 protein degradation in determining the apparent steady-state level of photoinhibition in differently light-acclimated pea leaves is also demonstrated in Figure 7. Note that a11 three parameters of Figure 7 were determined independently of each other. Photoinhi- bition of PSII was measured from the decrease in F,/F,, after illumination at 400 or 1600 pmol photons m-* s-' of intact leaves for 3 h. kPl, which in our experimental conditions mainly represent the damaging effect of light without concomitant repair, were determined from the first-order l'its of the decline in FJF, in leaves in the presence of linconiycin. Finally, kD1 d e g were determined from [35S]Met experinients, as described in 'Materials and Methods." Figure 7 demon- strates that the susceptibility of differently light-acclirnated leaves to photoinhibition is linearly related to the ratio kpI/kD1 de-, that is to the capacity of the leaves for degradation of photodamaged D1 protein induced by incident irradiance. The ratio kPI/kD1 degr is almost 1 in a11 the light-acclimated pea leaves when illuminated in moderate light, suggesting that photodamaged PSII centers are rapidly repaired and no photoinhibition is induced in intact leaves (Fig. 7, open symbols). At high irradiance, however, the kP,/kD1 degr ratio varies widely among differently light-acclimated pea leaves and is directly related to the susceptibility of pea leaves to photoinhibition (Fig. 7, closed symbols).

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Figure 6. Relationship of the Chl a/b ratio of peas grown under contrasting light regimes with the half-time for photoinhibition (determined in the presence of hncomycin) and with the half-life of the D1 protein. Chl a/b ratios of differently light-acclimated ,peas are as in Figure 2. Half-times for photoinhibition and half-life values of t h e D1 protein were taken from Tables II and 111, respectively.



Photoinhibition and D1 Protein Degradation in Light-Acclimated Peas 84 1

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o 60 c O .- c.

40 .- e c c .- E 20 O c a

O 0.5 1.0 1.5 2.0 2.5 3.0 3.5

kPlIkD1 degr.

Figure 7. Relationship between photoinhibition of PSll (inhibition of F,/F,,, after illumination of 3 h) and the ratio of the rate constant for photoinhibition (determined in the presence of lincomycin) to that for D1 protein degradation (kpl/kOl degr.). Pea leaves were illuminated at either 400 pmol photons m-2 s-' (O) or at 1600 pmol photons m-'s-l (O).

DISCUSSION

Photoinhibition of photosynthesis is an extremely complex phenomenon, comprising both damaging and protective events, including down-regulation of PSII and reversible and irreversible photoinactivation of PSII electron transport, as well as the concomitant repair of photodamaged PSII centers via de novo synthesis of the D1 protein (Krause, 1988; Prasil et al., 1992; Aro et al., 1993). In the present paper we have primarily focused on the rate of D1 protein degradation in five differently light-acclimated pea leaves from plants grown under a wide range of irradiances. We have compared the rate of D1 protein degradation under varying PPFDs, both at moderate and high incident irradiances. It is known that sun and high-light plants have different strategies to help combat photoinhibition, including a higher capacity for photosynthesis (Demmig and Bjorkman, 1987), a more active PSII repair cycle (Greer et al., 1986; Oquist et al., 1992a), and greater xanthophyll-associated energy dissipation (Demmig- Adams, 1990; Demmig-Adams and Adams, 1992; Horton and Ruban, 1992). Shade and low-light plants have very limited capacity for the PSII repair cycle (Oquist et al., 1992a; Tyystjarvi et al., 1992). Oquist et al. (1992b) suggest that photoinhibited PSII centers (with photodamaged D1 protein) as they accumulate confer increased protection of the re- maining connected, functional PSII centers by controlled, nonphotochemical dissipation of excess excitation energy. This strategy, of stable down-regulation of PSII under sustained high irradiance to regulate resistance to irreversible photodamage, is particularly evident in shade and low-light plants.

Our results corroborate many studies made with sun or high-light and shade or low-light leaves (Demmig and Bjork- man, 1987; Greer and Laing, 1988; Tyystjarvi et al., 1991, 1992; Oquist et al., 1992a, 1992b); they clearly indicate that low light-grown peas are more susceptible to photoinhibition than high light-grown peas. Indeed, we demonstrate for the

first time a significant inverse correlation (Fig. 2) between sensitivity to photoinhibition and the Chl a / b ratios of pea leaves, a sensitive index of acclimation to growth inadiance. Low-light leaves have fewer PSII units with larger light- harvesting antenna compared with PSI, lower maximal photosynthetic capacity on a Chl basis, and relatively more stacked thylakoid membranes compared with high-light leaves (Anderson, 1986).

Photoinhibition of PSII was greatly enhanced when chloroplast-encoded protein synthesis was inhibited with lincomycin. Significantly, the distinct difference in the susceptibility to photoinhibition between high and low light-grown peas was greatly diminished in both glasshouse peas and in peas grown in growth chambers (Fig. 1). For example, a 3- fold difference in the sensitivity to photoinhibition between high-light and low-light peas illuminated at a PPFD of 1600 pmol m-2 s-' (estimated from the steady-state level of photoinhibition; Fig. 1) declined to only a 1.4-fold difference in the presence of lincomycin (calculated from the rate constants for photoinhibition) (Table 11). Corresponding values were obtained when comparing 7% and 100% sunlight peas grown in the glasshouse. Our results clearly demonstrate that the smaller size of the LHCII antenna and the more efficient photosynthesis of high-light peas are only minor factors contributing to the better tolerance of PSII against photoinhibition in high-light peas compared with low-light peas. Further, at least under conditions of sustained high light, our results (Table I) do not lend support to the hypothesis that high-light pea leaves are more tolerant toward photoinhibition only because of higher capacity for formation of zeaxanthin via the xanthophyll cycle (Demmig-Adams and Ad- ams, 1992).

When pea leaves were illuminated at a moderate PPFD (400 pmol m-* s-') at which no photoinhibition occurs, the rate of D1 protein degradation (measured by chasing the radioactivity in the Dl protein) has severa1 significant features. First, the rate of D1 protein degradation is not influ- enced by the previous light acclimation of the leaves. Second, it corresponds to the rate of photoinhibition obtained from totally independent experiments that measured the decline in F,IF, in the presence of lincomycin (Tables I1 and 111).

These results demonstrate that under moderate illumination there is a balance between photodamage to PSII and subsequent recovery of photodamaged PSII centers via D1 protein degradation, de novo synthesis of a new D1 protein, and reassembly of functional PSII. This has also been pro- posed earlier in Chlamydomonas cells (Schuster et al., 1988). However, an increase in PPFD sufficient to induce marked photoinhibition (e.g. 1600 Kmol photons m-2 s-' for low-light peas and 2800 pmol photons m-2 s-' for high-light peas) was not directly accompanied by enhanced degradation of the D1 protein (Table 111). Therefore, it seems likely that the rate of D1 protein synthesis per se is not the only factor limiting the repair of photodamaged PSII centers in higher plants, but also the rate of D1 protein degradation plays a regulatory role. Indeed, at these much higher light intensities the rate of D1 protein degradation remained rather constant and at the same level that had been reached at the highest light intensity that did not induce photoinhibition of PSII. Previously, with Brassica napus acclimated to three growth irradiances, we




found that the maximum rate of D1 protein degradation was achieved already at growth irradiance and was not enhanced at higher irradiances (Sundby et al., 1992). In contrast, this is not the case with peas, since the rate of degradation of the D1 protein is clearly enhanced far above the growth light irradiance (Table 111). However, the light intensities at which the rate of D1 protein degradation becomes saturated and photoinhibitiori is evoked are greatly dependent on the light acclimation of pea leaves.

The kPI/kD1 deip ratio is indeed an important parameter that reflects the susceptibility of differently light-acclimated pea leaves to photoinhibition (Fig. 7). Remarkably, the higher the kPl/kD1 degr ratio, the more susceptible the pea leaves are to photoinhibition. Under PPFDs that do not induce photoinhibition (400 pmol photons m-’s-’ for a11 peas grown in growth chambers) this ratio is dose to 1. A kpl/kD1 degr ratio of 1 means that the rate of D1 protein degradation matches the rate of photoinhibition in the presence of lincomycin and only marginal photoinhibition is evoked. When the PPFD is increased to 1600 pmol m-’ s-’ and photoinhibition becomes manifest, the kPl/kD1 degr ratio also increases, indicating that photodamage to PSII centers exceeds the rate of D1 protein degradation; the higher the ratio, the more severely evoked is photoinhibition (Fig. 7). Peas grown at 7% sunlight have the highest kPl/kD1 degr rabo and are the most susceptible to photoinhibition, whereas high-light peas grown at 700 pmol photons m-’ s-’ have the lowest value of this ratio; conse- quently they are the most tolerant against photoinhibition induced by illumination at a PPFD of 1600 pmol m-’ s-I

(Fig. 7). It has been suggested earlier that photoinhibition will occur

when the rate of light-induced damage to PSII centers is greater than the repair capacity (Greer et al., 1986). Our data indeed corroborate this definition. However, the repair of photodamaged PSII centers constitutes a complex cycle, including degradation of photodamaged D1 protein, de novo synthesis, and insertion of newly synthesized D1 protein into a PSII complex, possible migration of PSII complexes between appressed and nonappressed thylakoid regions, and finally, the activation of the PSII complex (for review, see Aro et al., 1993). Because in the present paper we have focused on the first step of the repair cycle of the PSII reaction centers, i.e. the degradation of the photodamaged D1 protein, we can specify the definition of Greer et al. (1986) and suggest that photoinhibition becomes manifest under conditions where the rate of D1 protein degradation cannot be further enhanced so as to rapidly remove a11 of the irreversibly damaged D1 protein.

What then sets the limit for D1 protein degradation? This is a particularly interesting question because in vitro studies with isolated PSII complexes suggest that a D1 protein- specific protease is an integral component of the PSII reaction center complex (Virgin et al., 1991; De Las Rivas et al., 1992). However, there is strong evidence that even rather severe photoinhibition in vivo does not lead to a net loss of the D1 protein from thylakoid membranes (Cleland et al., 1990; Kettunen et al., 1991; Adamska et al., 1992; Schnettger et al., 1993), provided that the strong illumination is not prolonged enough to induce bleaching of the bulk Chls. Therefore, it seems likely that D1 protein degradation and insertion of

new copies of the D1 protein to the D1 protein-depleted PSII reaction centers are somehow coordinated in vivo.

It is well known that D1 protein synthesis occurs on ribosomes attached to nonappressed regions of the thylakoid membranes (Hemn and Michaels, 1985; Mattoo and Edel- man, 1987), but the site of D1 protein degradation in vivo still remains to be identified. Adir et al. (1990) have suggested that the rate of the repair cycle of PSII is controlled by the migration of photodamaged PSII reaction centers from the appressed to nonappressed membrane regions for repa Ir. Our experiments with differently light-acclimated peas suggest that the degree of thylakoid stacking may not only influence the susceptibility of pea leaves to photoinhibition (Fig. 2), but may also control the rate of D1 protein degradation (Fig. 6). In peas, the most severe photoinhibition occurs in leaves acclimated to the lowest growth irradiance, since the ]*ate of degradation of the D1 protein, and hence the total repair cycle of photodamaged PSII reaction centers, is very slow. Degradation fragments of the D1 protein in intact leaves have been found only in nonappressed thylakoid rltgions (Ghirardi et al., 1990; Kettunen et al., 1992), giving iridirect evidence that the final degradation of the photodamaged D1 protein possibly occurs in nonappressed thylakoid membranes in vivo. Therefore, we suggest that the slow rate of D1 protein degradation in low-light pea leaves is due to their high degree of thylakoid stacking and scarcity of nonappressed stroma thylakoids. Further, high light-induced phosphorylation of the D1 protein in the appressed thyllakoid regions possibly prevents premature degradation of the D1 protein (Tyystjarvi et al., 1992), thereby allowing no net loss of the D1 protein from thylakoid membranes in spite jof the accumulation of photodamaged PSII centers.

We conclude that the main factor that makes low light- acclimated pea leaves more susceptible to photoinhibition compared with high light-grown peas is a slower rate tof D1 protein degradation at high light, and therefore, their slower repair cycle of the photodamaged PSII centers. This slower rate of D1 protein degradation is consistent with the stable long-term accumulation of photoinhibited PSII reactiori centers in low-light and shade plants (Oquist et al., 1992b). Controlled degradation of the D1 protein might be a sui-vival strategy of low-light leaves, since the uncontrolled degradation of the D1 protein in appressed membranes could result in total disassembly and degradation of a11 the polypeptides of photoinhibited PSII complexes. It is possible that D1 protein is degraded by an intrinsic protease only when a photodamaged PSII complex enters the nonappressed thylakoid regions, and the degree of thylakoid stacking probably regulates this migration (Tyystjarvi et al., 1992). The mechanism of the migration and the possible role of D1 protein phosphorylation in the appressed membranes of higher plants in regulation of D1 protein degradation remain to be elucidated.

ACKNOWLEDCMENTS

We thank Prof. I. Ohad for his generous gift of D1 protein antibody and Dr. E. Tyystjarvi for stimulating discussions.

Received April 19, 1993; accepted July 7, 1993. Copyright Clearance Center: 0032-0889/93/103/0835/09.



Photoinhibition and D1 Protein Degradation in Light-Acclimated Peas 843

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