Planta

Planta (1989) 177:409~416

9 Springer-Verlag 1989

Photoinhibition at chilling temperature Fluorescence characteristics of unhardened and cold-acclimated spinach leaves S. Somersalo* and G.H. Krause** Institute for Biochemistry of Plants, University of Dfisseldorf, Universit/itsstrasse 1, D-4000 Dfisseldorf 1, Federal Republic of Germany

Abstract. The effects of moderate light at chilling temperature on the photosynthesis of unhardened (acclimated to + 18 ~ C) and hardened (cold-acclimated) spinach (Spinacea oleracea L.) leaves were studied by means of fluorescence-induction measurements at 20 ~ C and 77K and by determination of quantum yield of 02 evolution. Exposure to 550 gmol p h o t o n s . m - Z . s -1 at + 4 ~ induced a strong photoinhibition in the unhardened leaves within a few hours. Photoinhibition manifested by a decline in quantum yield was characterized by an increase in initial fluorescence (Fo) and a decrease in variable fluorescence (Fv) and in the ratio of variable to maximum fluorescence (Fv/FM), both at 77K and 20 ~ C. The decline in quantum yield was more closely related to the decrease in the Fv/ FM ratio measured at 20 ~ C, as compared with Fv/ FM at 77K. Quenching of the variable fluorescence of photosystem II was accompanied by a decline in photosystem-I fluorescence at 77K, indicating increased thermal de-excitation of pigments as the main consequence of the light treatment. All these changes detected in fluorescence parameters as well as in the quantum yield of O2 evolution were fully reversible within 1-3 h at a higher temperature in low light. The fast recovery led us to the view that this photoinhibition represents a regulatory mechanism protecting the photosynthetic apparatus from the adverse effects of excess light by increasing thermal energy dissipation. Long-term cold acclimation probably enforces other protective mechanisms, as the hardened leaves were insensitive to * P e r m a n e n t address: Department of Biology, University of Turku, SF-20500 Turku, Finland ** To whom correspondence should be addressed Abbreviations: Fo = initial fluorescence; F~ = maximum fluores-

cence; Fv=variable fluorescence (FM-Fo); PFD=photon flux density; PS =photosystem

the same light treatment that induced strong inhibition of photosynthesis in unhardened leaves. Key words: Chlorophyll fluorescence Cold acclimation - Photoinhibition - Photosynthesis (inhibition) Quantum yield - Spinacia (cold acclimation)

Introduction When the light flux absorbed by plant leaves exceeds the photosynthetic utilization of quanta, photoinhibition may occur. There is wide agreement that photoinhibition is caused primarily by alterations of photosystem (PS) II (see Powles 1984; Kyle et al. 1987). An extensively studied molecular change is the inactivation and degradation of the D-1 (32-kDa) protein in the PSII reaction center. Typically, photoinhibition is manifested as a decrease of quantum yield of photosynthetic 02 evolution and changes in chlorophyll a fluorescence characteristics (see Baker and H o r m n 1987; Bj6rkman 1987). Not all processes related to photoinhibition may be regarded as adverse effects of high light. Rather, conditions of excess excitation supposedly induce mechanisms to protect the chloroplast against destruction of the photosynthetic system. One such mechanism, apparently adjusting the thermal de-excitation of photosynthetic pigments to light conditions, has been proposed on the basis of ' energy-dependent' fluorescence qtlenching (Krause and Behrend 1986; Krause and Laasch 1987; Weis and Berry 1987; Horton and Hague 1988). This mechanism seems to involve ia reversible increase in the rate-constant of thermM deactivation (Krause et al. 1983). The effect occ~urs within seconds to minutes upon exposure to ,excess

410

S. Somersalo and G.H. Krause: Photoinhibition at chilling temperature

light. A slower, at least partly reversible response to high light, occurring within minutes to hours, is a photoinhibitory fluorescence quenching, represented by a decline in the ratio of variable to maxim u m fluorescence (Fv/FM). This decline correlates with the decrease of quantum yield of O2 evolution (Demmig and Bj6rkman 1987). The fluorescence characteristics of this effect indicate that it is also based on increased thermal deactivation (Powles and Bj6rkman 1982; Ogren and Oquist 1984; Bar6nyi and Krause 1985; Demmig and Bj6rkman 1987) and thus may be regarded as a protective mechanism, even though it is related to a transient inhibition of photosynthesis. The photoinhibition symptoms are pronounced when in addition to high light the plant is exposed to chilling temperatures (Powles et al. 1983 ; Greer et al. 1986; Oquist et al. 1987) that slow down energy-consuming carbon metabolism and possibly also 'repair' processes in the chloroplasts. As low temperature thus imposes conditions of excess light, we expected cold acclimation to enforce mechanisms that prevent damaging reactions. We studied the susceptibility of a chilling-resistant plant (Spinacia oleracea L.) to photoinhibition at low temperature. Unhardened plants, acclimated to + 18 ~ C, and hardened plants, cold-acclimated to + I ~ under the same light conditions, were used. We found that cold acclimation which reSulted in increased frost hardiness (see Klosson and Krause 1981), strongly diminished the susceptibility to photoinhibition at chilling temperature. In contrast, unhardened plants developed pronounced photoinhibition which, however, was fully reversible. A detailed investigation of this chilling-induced reversible photoinhibition is presented. Material and methods

Lynn, Norfolk, UK.) at 20~ and a CO2 concentration of 5% (v/v) in air. Light was provided by a set of photodiodes (Hansatech; 2m~x= 660 nm, P F D 220 g m o l - m - 2 , s-1).

Photoinhibition and recovery' treatments. Detached spinach leaves were exposed to white light, P F D 550 Ixmol-m-Z.s -1, in moistened normal air cooled to + 4 ~ C. The temperature of the leaves (measured at their lower side with a copper-constantan thermocouple) was between + 5.5 and + 6.0 ~ C during the treatment. Recovery of unhardened leaves from photoinhibition was followed either at + 18 ~ C or at + 4 ~ C, P F D being 2.5 to 5.0 Ixmol.m-Z.s -1. The recovery was initiated after exposing the leaves for 3 h to the photoinhibitory treatment described above.

Quantum yield of 02 evolution. Rates of photosynthetic 0 2 evolution were measured at + 20~ with the leaf-disc-electrode unit (see above) at a CO2 concentration of 5% in air. The P F D was changed with a set of neutral-density filters, N G (Schott, Mainz, F R G ) . Measurement of 02 evolution at each P F D lasted 7 to 8 rain. Optimal quantum yield was calculated from the slope of the plot of rate versus absorbed P F D (Bj6rkman and Demmig 1987). The absorptance of the leaves (85%) was determined with an Ulbricht sphere. Room-temperature fluorescence. Fluorescence induction at 20 ~ C in leaf discs of 10 c m 2 w a s measured with a Hansatech fluorometer attached to the leaf-disc electrode (see above). Exciting light was 220 ~tmol. m - 2. s 1 (2max= 660 rim). In order to reach the maximum level of fluorescence, the leaf discs were kept for 10 rain in darkness in moistened CO2-free air. Fluorescence at 77K. Fluorescence induction at liquid-nitrogen temperature was measured from leaf discs (1.5 cm 2) at 694 n m and 735 nm (half-band width 11.5 and 7.0 nm, respectively), using blue exciting light (filters 5030 and 5433, Coming, New York, USA; 7.6 [xmol photons- m - 2. s - 1). The leaf discs were kept for 5 min in complete darkness at room temperature before freezing with liquid nitrogen. The method was similar to that of Ogren and Oquist (1984).

Chlorophyll determination. The leaf discs were extracted with 10 ml 80% (v/v) acetone and the chlorophyll content was determined according to A r n o n (1949).

Results

Plant material. Plants of Spinacia oleracea L. cv. Monatol were

Frost hardiness. Acclimation of spinach plants to

grown in a greenhouse for about five weeks and thereafter for 10 d in a climate chamber at + 18 ~ C, photoperiod 8 h, p h o t o n flux density (PFD) 260 300 ~tmol. m - z. s - 1 and relative humidity 75% (unhardened plants). To obtain ' h a r d e n e d ' , cold-acclimated plants, batches were taken to a climate chamber with the same light conditions and the temperature was kept for 2-d periods at 18 ~ C/15 ~ C, 15~ C/IO ~ C, 10~ C/3 ~ C, 3 ~ C/1 ~ C and 1~ C / I ~ (day/night).

low temperature ( + 1~ C) increased the frost tolerance of the leaves significantly. The minimum temperature that leads to 50% inactivation of photosynthetic Oz evolution (Tso) was - 6 . 5 ~ C for the leaves acclimated to + 18~ C, and - 1 1 ~ C for the cold-acclimated leaves. Estimating the frost damage visually from water infiltration of the tissue gave about the same Tso values. In the following, the leaves are termed 'unhardened' and 'hardened ', respectively.

Test of frost hardiness. Detached spinach leaves were cooled in a cryostat from + 4 ~ C at a rate of 6 ~ C . h - 1 to different minimum temperatures in darkness. After keeping the leaves for 2 h at the minimum temperature, they were warmed to +4~ at the same rate. Control leaves were kept at + 4 ~ in darkness for the duration of frost treatments. The frost damage to the leaves was determined by measuring the photosynthetic 0 2 evolution with a leaf-disc electrode (Hansatech, Kings

Room-temperature fluorescence. Figures 1 and 2 show the effects of photoinhibitory treatment on room-temperature fluorescence of unhardened and

S. Somersalo and G.H. Krause : Photoinhibition at chilling temperature i

I

I

i

i

I

o

I

411

f

i

i

Fig. 1 a, b. Effects of photoinhibition and recovery treatments of unhardened (a) and hardened (b) spinach leaves o n the Fv/FM ratio recorded at r o o m temperature. Photoinhibition treatment consisted of exposure o f detached leaves to a P F D o f 5 5 0 g m o l . m Z . s - 1 at + 4 ~ for the times given (9 Recovery o f the unhardened leaves after 3 h exposure was followed at

b

L_

o,8

O

to

0.8

i /

{3.

It

E

f

o

O.6

\,.

o

i

0.6

t

FI

0--

0 0

f

E o

0

o

o

o~

o

0 0

0 0~0%'~'--

0

-53%

0.4

+ 18 ~ C, P F D 2.5 to 5.0 gmo][. m - 2. s - 1 (m). Standard deviations

0.4i J I

I

I

400

Time of pretreotment ,

i

T,

I

200

i

I

I

0

(min)

I

400

Time of p r e t r e o t m e n t

i

I

,

1

i

for the control leaves are given;

I

200

I

n=79

(rain)

I

b

O in ,I

c

CD

t

o~

i'

o

%o o ;o

2

CD

0

0

0

x~o 0

0 0

0

o

~

0

Fig. 2 a, b. Time course of the changes :in Eo o

0.8

o .~o/0

c =

*60%

0.~

~\

0

0.6

CO

0

\x

O.E

9

O.z I

0

I

I

200

I

I

400

Time of pretreotment (rain

0

O0 O0

{~o o i

I

0

o

o I

I

I

R

and Fv recorded at room temperature, induced by exposing unhardened (a) and hardened (b) spinach leaves to photoinhibition (o) and recovery (m) treatments. Experimental conditions of pretreatment as for Fig. 1. Standard deviations of the control leaw~s are given; n = 5

0 200 400 Time of pretreotment (minl

hardened leaves and the course of 'recovery' in dim light at + 18 ~ C of unhardened leaves. The ratio of variable to maximum fluorescence (Fv/FM) was above 0.8 in unhardened control leaves, as expected for uninhibited leaves (Bj6rkman and Demmig 1987). The ratio decreased drastically during exposure to moderate light (550 gmol. m - 2. s - t) at chilling temperature (Fig. i a). After 5 h photoinhibitory treatment the ratio was lowered by about 50%. The decrease in Fv/FM results from a decrease of maximum variable (Fv) and an increase of initial fluorescence (Fo; Fig. 2 a). The changes are not caused by chilling per se, as incubating the leaves in total darkness at + 4 ~ C for several hours did not have any effect on the parameters measured (results not shown). Neither did a PFD of 550 g m o l . m - 2 . s - t induce any changes at + 18 ~ C during 3 h exposure (data not shown). The recovery at + 1 8 ~ in weak light was remarkably fast, most of it occurring within 50 min; complete recovery was reached after about 3 h (Figs. i a, 2a). Notably, at + 4 ~ there was

Table 1. Recovery of unhardened spinach leaves at + 4 ~ C ( P E D 2 . 5 - 5 . 0 g m o l . m - 2 . s -1) for 2.5 5 h , after 3 h e x p o s u r e to 550 gmol. m - 2. s - 1 at + 4 ~ C. Standard deviations are indicated; n = 3 to 9. For corresponding data o f 3-h photoinhibited leaves and recovery at + 18 ~ C see Figs. 1-3, 5

Control

Recovered

0.50 -t-0.08 2.79 _+0.32 0.84 • 0.01

0.58 + 0 . 0 5 1.52 _+0.31 0.72 _ 0.04

Fluorescence at 20 ~ C Fo Fv

Fv/FM

Fluorescence at 77 K (694 ran) Fo Fv

Fv/FM Quantum yield

1.74 • 6.90 + 0 . 2 5 0.82 _+0.02

1.75 _+0.21 6.17 + 0 . 3 0 0.78 + 0 . 0 2

0.094-+ 0.007

0.065 • 0.003

a partial recovery of Fv and Fv/FM, which was correlated with a full recovery of Fo (Table 1). These reversions occurred within about I h, whereas prolonged recovery treatment (2.5-5h) at +4~ did not induce further changes (recovery kinetics not shown).

412

S. Somersalo and G.H. Krause: Photoinhibition at chilling temperature

Table 2. Characteristics of room-temperature fluorescence induction of hardened spinach leaves excited and measured from the upper and lower sides of the leaves, respectively, before and after 3 h treatment at + 1 8 ~ (PFD 35 g m o l . m 2.s-~). Standard deviations are given, n = 3-6

Fo

Fv

Fv/F~

0.8

(~:SO ~

g

9 9

\o

0.7 > 0.6

0~0

IJ_

I

Upper side Before treatment After 3 h at + 18 ~ C

O

L

I

7

0.46_+ 0.05 0.44_+0.00

1.08 _+0.01 1.55_+0.03

0.70 + 0.04 0.78_+0.003

d, t.o

5

0.35-+0.05 0.37+0.03

1.11 -+0.11 1.32_+0.05

0.76-+0.02 0.78_+0.01

0 0'0'0~0 0 ~

E

,'0

0

~ - - 0 - 0 O0 O

(

A

I

. m~--m-i i .~-

o

,,>

Lower side Before treatment After 3 h a t + 1 8 ~

~"

I

I

O0

2.4

- ~5 ~

I

0 O -m--

J

T

+61%

2.0

u_o

The Fv/FM ratio of hardened control leaves was m o r e varied and on average lower (around 0.7) than that of the unhardened leaves (Fig. 1 b). This lowered ratio indicates that photoinhibition to some degree had taken place during the cold-acclimation process. This is confirmed by the finding that raising the temperature to + 18 ~ C in dim light (35 gmol photons . m - 2 . s-~) caused an increase in Fv and Fv/FM in the fluorescence signal from the upper side of the leaf within 3 h, but not much change was seen in signals from the lower side of the leaf (Table 2). The larger variation in the data from hardened leaves indicates that they are photoinhibited to different degrees during the cold acclimation. This might be the consequence of variations in the P F D intercepted by different leaves in the growth chamber. The scattering of data was seen in all the measurements made with hardened leaves. Figure 2b illustrates Fv and Fo of hardened spinach leaves. The Fv was very low compared with that of unhardened control leaves. Supposedly, this is partly caused by the weak photoinhibition mentioned above and partly by increased light scattering in the hardened leaves. The latter should alter Fo proportionally to Fv. A decreased Fo as a result of cold acclimation is indeed seen in Fig. 2b. The apparent slight increase in Fo during light pretreatment (Fig. 2b) is not significant and was absent in other experiments (data not shown).

Fluorescence at 77K. The effects of photoinhibition treatment and recovery of unhardened leaves on the fluorescence induction at 77K are shown in Figs. 3 and 4. The photoinhibitory treatment of hardened leaves did not induce any changes in fluorescence parameters (data not shown). In PSII fluorescence at 694 nm of unhardened leaves (Fig. 3), qualitatively the same changes are

O

m m

- --

1.6 I

I

I

I

200

0

400

Time of p r e t r e a t m e n t ( m i n )

Fig. 3. Time course of the changes in 77K fluorescence emission of PSII at 694 nm, induced by exposing unhardened spinach leaves to photoinhibition (o) and recovery treatments (l). For conditions see legend to Fig. 1. Standard deviations of the control leaves are indicated; n = 8

A

E

c Lo co

0.3

IE LL

0.2

"~~ %5~176L-o-~0O/o o

0.1 :

N L'-,-

I

3

,~.

I

I

I

o

9 ..... /miom

2

I

-'i o

'

I

l

I

I

I

I

I

]

11 o

o

u_o I

1

0 200 400 Time of pretreotment {rain)

Fig. 4. Time course of the changes in 77K fluorescence emission of PSI at 735 nm, induced by exposing unhardened spinach leaves to photoinhibition (o) and recovery (m) treatments. Conditions of pretreatment as for Fig. 1. Standard deviations of the control leaves are indicated; n = 8

seen as at 20 ~ C. The decrease in Fv/FM of PSII results from a decrease in Fv and an increase in Fo. These changes were fully reversible at + 18 ~ C in weak light (Fig. 3). The Fo also fully recovered at + 4 ~ (Table 1). However, the alterations in fluorescence measured at 77K were considerably

S. Somersalo and G.H. Krause: Photoinhibition at chilling temperature Table 3. Ratios of Fv/FM measured at 77K and at 20 ~ C from the upper and lower sides of unhardened spinach leaves before and after exposure of the upper leaf side to 550 gmol photonsm - z . s - t at + 4 ~ C for 3 h. Standard deviations are given, n = 35

Fv/FM Upper side

Mean Mean %

413 i

I

99

,~

o

L~ 0.6

o

9

oo, XS~oo

._o

Lower side

"6 rr"

1

0.82___0.01 0.79___0.01 0.81 0.69_+0.02 0.75_+0.02 0.72

1

~ " o

oo

I

I

100 89

0.7 0.82_+0.01 0.80_+0.02

0.83 0.64

100 76

l

i

r,-

0.6

o

O~

Oo

+39%

-

0.5 O.Z,

i

b' O

[.L >

smaller than those observed at 20 ~ C (see Figs. 1, 2). Partly, this difference can be explained by a higher contribution of the upper, more strongly inhibited layers of the leaves to room-temperature fluorescence. The difference between Fv/FM ratios of upper and lower sides of photoinhibited leaves (Table 3) was more pronounced at 2 0 ~ than at 77K. It seems that 77K fluorescence measured from the upper side of the leaf is closer to an average emission of all chloroplasts in the leaf section. However, a small difference between Fv/FM ratios measured at 77K and 2 0 ~ is seen also in the mean values of upper- and lower-side recordings (Table 3). The photoinhibitory treatments also caused changes to PSI fluorescence emission at 735 nm (Fig. 4). The decrease in the Fv/FM ratio of PSI was caused by a decrease in Fv; Fo did not change significantly. The quenching of variable fluorescence in both photosystems is in agreement with an increase in the rate constant of thermal de-excitation of PSII (see Butler 1978). Figure 5a illustrates the change in the ratio of the maximum fluorescence of PSII to the maximum fluorescence of PSI during the photoinhibition and recovery treatments. The decrease in this ratio gives an indication of relatively increased excitation of PSI, which would be expected from increased thermal energy dissipation in PSII. However, as demonstrated by Fig. 5 b, the variable fluorescence of PSI decreased less than that of PSII. This might, in addition, indicate a relative increase in excitation-energy transfer from PSII to PSI (see Butler 1978). When PSI fluorescence is plotted versus PSII fluorescence using the induction signals at 77K, a straight line is obtained (Kitajima and Butler 1975). The slope of this line is proportional to the rate-constant for energy transfer from PSII to PSI and to the probability of PSI fluorescence (see Ogren and (~quist

_33.

0 200 L00 Time of pretreotment (rnin) l

20 ~ C H Before exposure 0.84_+0.01 After 3 h exposure 0.48_+0.02

u

_ --m

/

uY 0.7

77K (694 nm) Before exposure After 3 h exposure

I

0.8 a

i__ u 2_

9

O9 I

0

i

I

200

I

I

400

Time of pretreatment (rain)

Fig. 5a, b. Effects of photoinhibition (o) and recovery (=) treatments on PSII (694 nm) and PSI (735 nm) fluorescence emission (77K) of unhardened spinach leaves, a Ratio of maximum fluorescence emission at 694 nm to maximum fluorescence at 735 nm. b Slope obtained from induction signals when 735-nm fluorescence is plotted versus 694-nm fluorescence. Conditions of pretreatment as for Fig. 1. Standard deviations of the control leaves are indicated; n = 3

1984). Photoinhibition treatment caused an increase of this slope (Fig. 5b). The recovery at + 18~ of the FM694/FM735ratio and of the slope F73s versus F694 shown in Fig. 5 had kinetics similar to that of the other fluorescence parameters.

Quantum yield o f O 2 evolution. In Fig. 6a the lightresponse curves of 0 2 evolution of unhardened and hardened spinach leaves are shown. It can be deduced from the figure that the O2 evolution at saturating light, as well as the optimal quantum yield was lower in the hardened leaves. T h i s may be an indication of a slight photoinhibition taking place during the acclimation process (see ',above). The light absorptance of both types of leaves was the same (85%). The photoinhibitory treatment strongly affected the quantum yield of the unhardened leaves (Fig. 6b), but not of the hardened ones (Fig. 6c). Like the fluorescence changes, the decrease in quantum yield of unhardened leaves was t~Lfllyreversible at + 18 ~ C in weak light. At + 4~ C recovery was only partial (Table 1). Relationship between quantum yield and Fv/FMratio of PSII. The relationship between quantum yield

414

S. Somersalo and G.H. Krause: Photoinhibition at chilling temperature i

i

i

i

i

i

l

i

C

0.10 O

T/

"Tul

9

_~

9 ---

9

0.1(

/r

~

O

o

_~_ooO o

0.08

o o

o

0 0

E

0

0

0.05

E

.o

s

8

O

(20L

D

c~ c o

T

I

I

0 o

Time of

I

I

200

pre~reatment

I

400

(min)

I

T

I

I

0

Time

I

I

200

of

preireatment

I

400

{rain)

0

;

20 do ' ' 120 160 PFD (jum01 m -2 S -I )

' 200

Fig. 6a-c. Effects of cold acclimation and photoinhibition treatments on photosynthetic 02 evolution of spinach leaves, a Lightresponse curve of unhardened (o) and hardened (e) leaves, b Time course of the changes in quantum yield induced by exposing unhardened leaves to photoinhibition (o) and recovery (m) pretreatments as for Fig. 1. c Quantum yield of hardened leaves after different times of photoinhibition treatments; conditions as for Fig. 1. Standard deviations are given; (in b and e, of the control leaves); n = 5

i

0.10

i

I

I

-Ill

I

a

s

,

,

R O

o

a08F

-o &08 E, &06 E

g ao~

gO6}-

9~oo~8 o o

/

}/~ o

02o

004P

O

~[i

,

0.10}- b

0./,

i

01.6

I

T

01.8

F v / FM ( room temperature )

LII,

9

,

.6 0.8 F v / F M ( 694 nm,77K }

Fig. 7 a, b. Relationship between quantum yield of Oz evolution of unhardened spinach leaves and Fv/FM ratios recorded at room temperature (a) and at 77K (b); n, untreated control leaves; o, leaves exposed to photoinhibition pretreatment for different times as for Fig. 1. The regression equations are y = 0.106x-0.0001; r=0.951 (a) and y = 0 . 2 7 7 x - 0 . 1 3 2 ; r=0.851

(b) and Fv/FM ratio measured at 20~ or at 77K (694 nm) was linear, with a significantly steeper slope in the case of 77K fluorescence (Fig. 7). It should be noted that the extrapolation of the regression lines goes through the origin only for the room-temperature fluorescence. Discussion

As shown here, spinach plants grown in low light and acclimated to + 18~ exhibited a strong reversible photoinhibition when exposed to moderate light at chilling temperature. In contrast, after acclimation to + 1~ the plants were insensitive to the same light treatment.

Reversible photoinhibition of unhardened spinach leaves. The photoinhibition of unhardened leaves

was characterized by a decrease in Fv and increase in Fo, resulting in a decrease of the Fv/FM ratio measured at 20 ~ C (Figs. 1, 2) and at 77K (Fig. 3). Similar responses to light at chilling temperatures were found by 0gren and Oquist (1984) in the chilling-insensitive plant Lemna gibba and by Greer et al. (1986) in the chilling-sensitive Phaseolus vulgaris. The different sizes of changes seen in 77K and 20~ fluorescence parameters partly seem to result from the relatively higher contribution of upper, strongly inhibited leaf layers to fluorescence signals at 20 ~ C (Table 3). This is consistent with the data on the linear relationship between decrease in Fv/FM and quantum yield of photosynthesis (Fig. 7). Since quantum yield is measured in low, strictly limiting light, the data obtained will predominantly be determined by the upper leaf layers. This may explain why the plot of Fv/FM versus quantum yield extrapolates to the origin for 20~ fluorescence only (Fig. 7), and thus the Fv/FM ratio at 20 ~ C indicates more sensitively the decline in quantum yield in photoinhibition. A linear relationship between room-temperature fluorescence and quantum yield that extrapolates to the origin has also been reported for winter-stressed pine needles (Leverenz and 0quist 1987). In contrast, the 77K-fluorescence studies of Demmig and Bj6rkman (1987) showed a deviation from the origin in such plots (however, smaller than in Fig. 7 b). The decline in Fv/FM and Fv of PSII fluorescence was accompanied by a corresponding decrease in PSI fluorescence (Fig. 4), which according to previous studies (see Introduction) indicates

S. Somersalo and G.H. Krause: Photoinhibition at chilling temperature

increased thermal energy dissipation. The data obtained here are in agreement with the view that high-light treatment transforms part of the PSII reaction centers to fluorescence quenchers (Cleland et al. 1986). These centers would still be able to trap excitation energy but convert it to heat. However, the pronounced increase of Fo (Figs. 2, 3) also indicates decreased trapping efficiency of PSII centers. The response of Fo to photoinhibition seems to be complicated and differs depending on material and experimental conditions (Powles and Bj6rkman 1982; Bar6nyi and Krause 1985 ; Krause et al. 1985; Bradbury and Baker 1986; Demmig and Bj6rkman 1987). The latter authors reported that photoinhibition related to decreased Fo recovered fully, whereas photoinhibition that leads to an increase of Fo was largely irreversible. Our results show, however, that this is not necessarily the case and that also photoinhibition characterized by increased Fo may recover totally in a short time (Fig. 3). Notably, in weak light, Fo recovered completely both at + 4 ~ C (Table 1) and at + 18 ~ C (Figs. 2, 3), whereas other fluorescence parameters and quantum yield recovered fully only at + 18 ~ C. This phenomenon seems to indicate that two mechanisms are involved in the photoinhibition: one is characterized by increased initial fluorescence of PSII and recovers at low temperature; the other is characterized by a decrease of variable fluorescence and needs higher temperatures, and thus probably certain metabolic activities to recover. In addition to decreased fluorescence emission of both photosystems, the exposure to moderate light at low temperature also led to an increased ratio of PSI to PSII fluorescence (Fig. 5a) and to an increase in the slope of the PSI versus PSII fluorescence plot (Fig. 5 b). This can be interpreted as a relative increase of energy transfer from PSII to PSI, if one assumes that the probability of PSI fluorescence emission is not changed as a consequence of the photoinhibition (compare Ogren and {)quist 1984). The increase in the slope may be based on a conversion of PSII~ to PSIIe (Sundby et al. 1986; M/ienp/i/i et al. 1988) or simply on predominant photoinhibition of PSII~ centers (reported by Cleland et al. 1986 and M/ienp/ifi et al. 1987). Both effects would increase the relative contribution of PSIIp units to variable fluorescence. Most of the PSIIp units supposedly reside in the appressed thylakoid regions (compare Sundby et al. 1986). Thus PSII~ units are in closer contact with PSI and should be mainly responsible for the energy transfer.

415

From the fast and complete recovery of the quantum yield of 02 evolution and of all the fluorescence parameters studied here, we conclude that the alterations obtained during the light treatment should be seen as a regulatory protective mechanism against excess light energy, rather than a damage. This regulatory system supposedly serves for increased thermal dissipation of excitation energy. It allows a response of the photosynthetic apparatus to changing light conditions within minutes to hours. An even faster response, also resulting in increased thermal energy dissipation is indicated by the energy-dependent fluorescence quenching (see Introduction). When this becomes light-saturated, the reversible photoinhibition might be induced to give further protection. As a result of the transient decline in photosynthesis, this mechanism may lower the productivity of the plant, but provide protection until long-term acclimation to excess light takes effect. Whether the inactivation and degradation of D-I protein is involved in the reversible photoinhibition of spinach leaves is presently under study. According to Chow et al. (1988), PSII activity can be affected during photoinhibitory treatments before a decrease in the number of atrazine-binding sites (indicating functional D-1 protein) is seen. Thus it appears that photoinhibition includes more than one phase, and inactivation of the D-1 protein is not necessarily involved in the first one. It has been shown that recovery from photoinhibition required protein synthesis in a higher plant (Greer et al. 1986), green algae (see Kyle 1987) and cyanobacteria (Samuelsson et al. 1987). As the turnover of the D-1 protein is fast and, moreover, a pool of free D-I seems to be available in the thylakoids (see Kyle 1987), the postulated regulatory mechanism could well include a role of this protein in the transformation of PSII centers to 'quenchers' and their restructuring during recovery.

Low susceptibility of hardened plants to photoinhibition. Hardened spinach leaves showed a slightly lowered quantum yield (Fig. 6 a) and Fv/FM ratio (Fig. 1 b), compared with the unhardened state. However, the light treatment at chilling temperature that induced a strong photoinhibit~on in unhardened leaves did not have any effect after the hardening procedure (Figs. 1, 2, 6). Only considerably higher light induced photoinhibition in the hardened leaves (not shown). Thus, cold acclimation resulted in a significantly increased resistance to photoinhibition. The data of Greer etal. (1986) and Samuelsson et al. (1987) indicate that the susceptibility to photoinhibition depends on the re-

416

S. Somersalo and G.H. Krause: Photoinhibition at chilling temperature

covery rates. One may speculate that the rates of protein degradation, resynthesis and replacement at chilling temperatures are increased in hardened leaves. Furthermore, cold-acclimation may lead to increased activities of other protective systems, e:g. of scavengers for active oxygen species. Preliminary experiments (data not shown) indicate that the decreased susceptibility to photoinhibition of hardened leaves is, indeed, related both to increased repair rates and to enforced scavenger systems. The authors thank the Deutsche Forschungsgemeinschaft and the Academy of Finland for support of the study.

References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1 15 Baker, N.R., Horton, P. (1987) Chlorophyll fluorescence quenching during photoinhibition. In: Photoinhibition, topics of photosynthesis, vol. 9, pp. 145 168, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Barrnyi, B., Krause, G.H. (1985) Inhibition of photosynthetic reactions by light. A study with isolated spinach chloroplasts. Planta 163, 218-226 Bjrrkman, O. (1987) Low-temperature chlorophyll fluorescence in leaves and its relationship to photon yield in photoinhibition. In: Photoinhibition, topics of photosynthesis, vol. 9, pp. 123-144, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Bj6rkman, O., Demmig, B. (1987) Photon yield of 02 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170, 489 504 Bradbury, M., Baker, N.R. (1986) The kinetics of photoinhibition of the photosynthetic apparatus in pea chloroplasts. Plant Cell Environ. 9, 289-297 Butler, W.L. (1978) Energy distribution in the photochemical apparatus of photosynthesis. Annu. Rev. Plant Physiol. 29, 345-378 Chow, W.S., Osmond, C.B., Huang, L.K. (1988) Photosystem II function and herbicide binding sites during photoinhibition of spinach chloroplasts in-vivo and in-vitro. Photosynth. Res., in press Cleland, R.E., Melis, A., Neale, P.J. (1986) Mechanism of photoinhibition: photochemical reaction center inactivation in system II of chloroplasts. Photosynth. Res. 9, 79-88 Demmig, B., Bjrrkman, O. (1987) Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution of leaves of higher plants. Planta 171, 171-184 Greer, D.H., Berry, J.A., Bj6rkman, O. (1986) Photoinhibition of photosynthesis in intact bean leaves: a role of light and temperature, and requirement for chloroplast-protein synthesis during recovery. Planta 168, 253-260 Horton, P., Hague, A. (1988) Studies on the induction of chlorophyll fluorescence in isolated barley protoplasts. IV. Resolution of non-photochemical quenching. Bioehim. Biophys. Acta 932, 107-115 Kitajima, M., Butler, W.L. (1975) Excitation spectra for photosystem II in chloroplasts and the spectral characteristics of the distribution of quanta between the two photosystems. Biochim. Biophys. Acta 408, 297-305 Klosson, R.J., Krause, G.H. (1981) Freezing injury in cold accli-

mated and unhardened spinach leaves. [. Photosynthetic reactions of thylakoids isolated from frost-damaged leaves. Planta 151, 339-346 Krause, G.H., Behrend, U. (1986) ApH-dependent chlorophyll fluorescence quenching indicating a mechanism of protection against photoinhibition of chloroplasts. FEBS Lett. 200, 298-302 Krause, G.H., Briantais, J.-M., Vernotte, C. (1983) Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77K. ApH-dependent quenching. Biochim. Biophys. Acta 723, 169-175 Krause, G.H., K6ster, S., Wong, S.C. (1985) Photoinhibition of photosynthesis under anaerobic conditions studied with leaves and chloroplasts of Spinacia oleracea L. Planta 165, 430-438 Krause, G.H., Laasch, H. (1987) Energy-dependent chlorophyll fluorescence quenching in chloroplasts correlated with quantum yield of photosynthesis. Z. Naturforsch. 42e, 581584 Kyle, D.J. (1987) The biochemical basis for photoinhibition of photosystem II. In: Photoinhibition, topics Of photosynthesis, vol. 9, pp. 197-226, Kyle, D.L, Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. (1987) Photoinhibition, topics in photosynthesis, vol. 9. Elsevier, Amsterdam Leverenz, J.W., Oquist, G. (1987) Quantum yields of photosynthesis at temperatures between - 2 ~ and 35~ in the course of one year. Plant Cell Environ. 10, 287-295 M/ienp/i/i, P., Andersson, B., Sundby, C. (1987) Difference in sensitivity to photoinhibition between photosystem II in appressed and non-appressed thylakoid regions. FEBS Lett. 215, 31-36 MS.enp/i~, P., Art, E.-M., Somersalo, S., Tyystj/irvi, E. (1988) Rearrangement of the chloroplast thylakoid at chilling temperature in the light. Plant Physiol. 87, 762--766 Ogren, E., Oquist, G. (1984) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III. Chlorophyll fluorescence at 77K. Physiol. Plant. 62, 193-200 Oquist, G., Greer, D.H., Ogren, E. (1987) Light stress at low temperatures. In: Photoinhibition, topics of photosynthesis, voi. 9, pp. 67 87, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Powles, S.B. (1984) Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35, 15-44 Powles, S.B., Berry, J.A., Bj6rkman, O. (1983) Interaction between light and chilling temperature on the inhibition of photosynthesis in chilling-sensitive plants. Plant Cell Eviron. 6, 117-123 Powles, S.B., Bj6rkman, O. (1982) Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of Nerium oleander. Planta 156, 97-107 Samuelsson, G., L6nneborg, A., Gustavsson, P., Oquist, G. (1987) The susceptibility of photosynthesis to photoinhibition and the capacity of recovery in high and low light grown cyanobacteria, Anacystis nidulans. Plant Physiol. 83, 438-441 Sundby, C., Melis, A., M/ienpfi/i, P., Andersson, B. (1986) Temperature-dependent changes in the antenna size of photosystern II. Reversible conversion of photosystem [[~ to photosystem II~. Biochim. Biophys. Acta 851, 475-483 Weis, E., Berry, J.A. (1987) Quantum efficiency of photosystem II in relation to 'energy'-dependent quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 894, 198-208 Received 22 August; accepted 7 November 1988