Heat stress induces in leaves an increase of the minimum level of chlorophyll fluorescence Fo A time resolved analysis Briantais et al

Prévia do material em texto

Photosynthesis Research 48: 189-196, 1996. 
(~) 1996 Kluwer Academic Publishers. Printed in the Netherlands. 
Regular paper 
Heat stress induces in leaves an increase of the minimum level of chlorophyll 
fluorescence, Fo: A time-resolved analysis 
J ean-Mar ie Br ianta is 2, Jose Dacosta I , Y. Gou las I , J ean-Marc Ducruet 3 & I smae l Moya 1 
i Laboratoire pour l'Utilisation du Rayonnement Electromagn#tique, Universit# Paris XI, F91405, Orsay Cedex, 
France; 2Laboratoire d'Ecologie V#g~tale, Universit~ Paris XI, F91405 Orsay Cedex, France; 3CEN Saclay, 
D#partement de Biologie, Service de Biodnerg#tique, F91190, Gif-sur-Yvette Cedex, France 
15 January 1996; accepted in revised form 26 March 1996 
Key words: heat stress, barley leaves, mutant, intermittent light, chlorophyll fluorescence, fluorescence lifetime 
Abstract 
A time-resolved study of the effects of heat stress (23 to 50 °C) on Fo level of chlorophyll fluorescence of leaves 
having different antenna content has been performed in order to elucidate the causes of heat induced increase of 
Fo in vivo. The multi-exponential deconvolution of the decays after a picosecond flash at Fo have shown that the 
best fit in both wild-type and the mutant chlorina F2 of barley leaves is obtained with three components in the 
temperature range utilized (100, 400 and 1200 ps at 23 °C). In intermittent light greened pea leaves, a fourth long 
lifetime component (4 ns at 23 °C) is needed. The comparison of the three types of leaves at 23 °C shows that the 
content of the LHCII b complex does not affect the lifetimes of the two main components (100 and 400 ps) and 
affects their preexponential factors. This result suggests that in the PS II unit the exciton transfer from LHC IIb 
to the rest of the antenna is irreversible. The effects of heat stress on individual lifetime components, Ti, included 
several changes. Utilizing for PS II unit an extended 'Reversible Radical Pair' model, having three compartments, 
to interpret the variations of Ti and Ai induced by temperature increases, it can be inferred that heat determines: (i) 
an irreversible disconnection of a minor antenna complex which is not the LHC IIb complex, this effect is induced 
by temperatures higher than 40 °C; (ii) a decrease of the quantum efficiency of Photosystem II photochemistry 
which is due to several effects: a decrease of the rate of charge separation, an increase of P+I - recombination rate 
constant and a decrease of the stabilization of charges. These effects on Photosystem II photochemistry start to 
occur above 30 °C and are partially reversible. 
Abbreviations: F- chlorophyll fluorescence (subscripts o and m define minimum and maximum yields correspond- 
ing to open and closed PS II photochemical traps); ILG-Intermittent-Light-Greened; LHC II-chlorophyll a-b 
harvesting complex of Photosystem II; LHC I Ib-major LHC II complex according to Peter and Thornber nomen- 
clature (1991); P680-primary electron donor of PS II; PS I: Photosystem I; PS I I -Photosystem II; QA-first 
quinonic acceptor of PS II 
Introduction 
In natural environmental conditions the leaf temper- 
ature can reach 40°C, and even more, especially 
if plants are water-stressed (stomata closure) (for a 
review see Berry and Bjorkman 1980). At tempera- 
tures higher than 30 °C, both the capacity and the quan- 
tum yield of CO2 assimilation start to decline (Berry 
and Bjorkman 1980). These decreases could in part 
result from the inhibition of PS II activity. Indeed, this 
photosystem, in contrast to PS I, is very sensitive to 
heat (Yamashita and Butler 1968; Berry and Bjorkman 
1980; Havaux et al. 1991). This inhibition of PS II 
leads to a decrease of the variable chlorophyll fluores- 
cence. This decline is produced by both a quenching 
of the maximum level, Fm, which starts at 30 °C, and 
190 
an increase of the minimum level Ft. This increase in 
Fo occurs in two steps, first a small increase from 30 to 
40 °C and then above this temperature, up to 50 °C, a 
large augmentation of Fo occurs (Schreiber and Berry 
1977; Bukhov and Mohanty 1993). The temperature 
at which this second phase starts is routinely used to 
screen plants sensitive and resistant to heat (Schreiber 
and Berry 1977; Pearcy et al. 1977; Raison et al 1982). 
Chlorophyll fluorescence yield at Fo is especially 
interesting as it depends upon the competition between 
fluorescence and all the other paths of PS II exciton 
deactivation: photochemistry and internal conversions, 
whereas Fm is in competition only with internal con- 
version. 
The heat-induced enhancement of Fo has been 
extensively studied and two interpretations have been 
proposed. First, it could be the result of changes 
at the antenna level. A disconnection of the large 
LHC II antenna complex was proposed (Schreiber and 
Armond 1978; Armond et al. 1980) as a cause of the 
Fo increase. A State 2 to State 1 transition could also 
explain an increase in Fo; but several authors (Sund- 
by and Andersson 1985; Havaux 1988; Ruban and 
Trach 1991) observed the opposite change i.e. a heat 
induced State 1 to State 2 transition. Second it can 
be due to changes at the level of PS II reaction cen- 
ter. An accumulation in the dark (or very dim light) 
of reduced QA has been also proposed to explain the 
heat induced increase of Fo (Bukhov et al 1990; Cat 
and Govindjee 1990; Goltsev et al. 1994). None of 
these interpretations have received definitive support. 
Therefore, there is still controversy on the mechanisms 
involved in heat-induced modifications of PS II proper- 
ties in vivo. Generally speaking this increase of Fo can 
be due to a decrease of the other paths of PS II deac- 
tivation: photochemistry and internal conversions. In 
principle, variations of internal conversion can be fol- 
lowed by changes of Fm level. As previously stated, 
heat quenches irreversibly Fm. According to Yamashita 
and Butler (1968) this quenching is explained by the 
accumulation of P680 + in PS II centers impaired on 
their electron donor side. Nevertheless, in the very dim 
light used to determine Fo, the presence of P680 + must 
be restricted to centers which contain QB- in the dark. 
One way to get more details on Fo fluorescence is 
to perform time-resolved measurements at this level; 
indeed the chlorophyll-fluorescence decay, at Fo, after 
a picosecond pulse is multi-exponential (2-3 compo- 
nents) and it has been proposed that each component 
depends on the rate constant of a limiting step of PS 
II photochemistry (Schatz et al. 1988). Therefore we 
examined the effects of heat on chlorophyll fluores- 
cence lifetime at Fo in two kinds of barley leaves, a 
wild type and a mutant deficient in chlorophyll b and 
the LHC IIb antenna complex. Most of the data on 
barley reported here have been previously presented at 
a conference (Dacosta et al. 1995). Lifetime measure- 
ments have been also performed on pea leaves greened 
under an intermittent light regime which is supposed to 
limit the biosynthesis of the PS II light-harvesting sys- 
tem to the core antenna (Glick and Melis 1988; Hartel 
and Lokstein 1995; see also Falk et al. 1994). 
Materials and methods 
Plants 
The wild-type (Triumph variety) and the chlorina F2 
mutant of barley, deficient in LHC IIb (White and 
Green 1987; Morrisey et al 1989; Peter and Thorn- 
ber 1991; Harrison and Melis 1992; Harrison et al. 
1993; Jansson 1994), were grown for two weeks in a 
growth-chamber with a 16-h photoperiod of white light 
at an intensity of 400 #Em -2 s- I. The temperature was 
20 °C during the day and 15 °C during the night. Mea- 
surements were performed on the first and the second 
leaves, attached to the plants. Peas (cv. Petit Provenqal) 
were grown at 20 °C during one week in total darkness 
and then submitted to a cycle of intermittent-light: 2min white light (150 #E m -2 s - l ) and 118 min dark 
during 5 days, according to Armond et al. (1976). Flu- 
orescence measurements were also performed on the 
two terminal leaves attached to the plant. 
Picosecond measurements on leaves 
In a previous work we have shown that fluorescence 
lifetime measurements on green leaves are meaning- 
ful, in spite of the strong chlorophyll fluorescence 
reabsorption (Schmuck and Moya 1994). A simple 
time correlated single photon counting system has been 
developed at LURE, based on a laser diode emitting at 
635 nm (Philips CQL 840/D, Eindhoven, The Nether- 
lands). A home-made frequency generator/amplifier 
drives the diode in the pulsed mode. The duration of 
the resulting light pulse is < 70 ps (FWHM). The aim 
of this system is to measure fluorescence decays under 
Fo fluorescence conditions. To do that, the excitation 
photon flux density is kept as low as possible and the 
collection efficiency of the fluorescence is improved 
by using large aperture optics and a high pass fluores- 
cence filter. The laser diode beam has been defocused 
in order to illuminate a leaf area of about 4 x 8 mm. 
The diode intensity was adjusted to a level 5 fold lower 
than the intensity which induces a detectable increase 
in the average fluorescence lifetime due to QA photore- 
duction (this was verified at both 23 and 50 °C). Thus 
at the level of the leaf the photon flux density was 0.01 
#E m -2 s -1. The fluorescence collecting optics con- 
sists of a pair of anti-reflection coated piano-convex 
lenses (Melles Griot, ~b = 50 mm, f = 70 mm) which 
images the illuminated area into the cathode of a red 
sensitive microchannel plate photomultiplier (Hama- 
matsu R 3809 U, Japan). A red filter (Schott RG 665, 
4 ram) is placed between the two lenses in order to 
select the whole fluorescence emission. The instru- 
mental response function (< 80 ps FWHM) is recorded 
by exchanging the red filter by an interference filter 
centered at 635 nm. Time resolution better than 10 ps 
is achieved through deconvolution by the Fluomarqt II 
software developed at LURE. 
Heating system and temperature control 
A leaf is pressed against a piece of brass, heated by 
a resistor (Thermocoax). Heating of the leaf was pro- 
gressive from 23 to 50 °C in steps of 3 °C. The tem- 
perature was monitored by a 16 channel thermocouple 
monitor (SR630 Stanford Research Systems). The leaf 
was maintained for 3 min at each temperature. Then the 
temperature was decreased back to 23 °C by flowing 
cold water around the piece of brass. A microcomputer 
was used to monitor changes in temperature. 
Resu l ts and d i scuss ion 
Effects of temperature on Fo and Fm levels 
As described previously by others, in many higher 
plants (for a review see Berry and Bjorkman 1980), we 
observed that the increase of the leaf temperature from 
23 to 50 °C induces a quenching of Fm and an enhance- 
ment of Fo in the three types of plants (not shown). 
The effect on Fm starts at 30°C and on Fo it becomes 
drastic above 40°C. The effect on Fm is irreversible, 
whereas the increase of Fo is partially reversible. The 
Fv/Fm ratio of dark-adapted leaves at 23 °C was 0.80 
and 0.83 in the wild-type and the chlorina F2 barley 
leaves, respectively. It was 0.62 in the ILG-pea leaves. 
As it will be shown later this low Fv/F m vahle in ILG- 
pea leaves is certainly due to a high Fo level because 
191 
2.5 
2 
1.5 
1 
o.5 
o 
i l 
. . . . , - - L ............. , -~- - . - -q* ........... ~ ................ I ................... 
! i i I 
I l i I 
J J i i 
• i [ [ . . . . . . . . 
.................. i ..................... r ................ r ................. - .................... ~ ......... 
......... ~ - . . . . . . . . . . . - t . . . . . . . . . . i . . . . . . . . :~-.-.~ . . . . . . -~ .................... 
04 o io 
. . . . i . . . . I . . . . A . . . . i . . . . . . . . 
20 25 30 35 40 45 50 
Temperature (°C) 
Figure 1. Changes of mean-lifetimes Tm, induced by increase in 
temperature in leaves of wild-type barley (O), chlorina F2 mutant 
of barley (A) and ILG pea leaves (0) . R stands for the values obtained 
after returning to 23 °C. 
2.5 
2 
~ 1.5 
~ 1 
o.5 
o 
' ' . . . . . . . . . . ! ' ' ' ' 
• iO 
• i 
• i 
io 
°c A 
i i i i ~ t i i I i t i i i i i L 
0.5 1 1.5 2 
Fo (re lat ive un i ts ) 
Figure 2. Relationship between temperature-induced changes in 
mean-lifetime Tm and yield of fluorescence at Fo, in barley leaves, 
wild-type (O), chlorina F2 mutant (A) and in ILG pea leaves (0). 
of the presence of a long lifetime component even at 
23 °C. 
Effects of temperature on the fluorescence lifetime at 
the level Fo 
The mean lifetime 
Figure 1 shows that in wild-type of barley, in the chlo- 
rina F2 mutant and in ILG-pea leaves the increase 
in Fo, induced by heat stress, is accompanied by an 
increase in the mean lifetime of chlorophyll a fluores- 
cence. Thus, the augmentation in Fo corresponds to an 
increase in quantum yield of fluorescence, not to an 
increase in the size of PS II antenna through a State 2 
to State 1 transition. Indeed, a state transition which 
192 
Table 1. Individual lifetime (ps) and pre- 
exponential factor (%) of fluorescence decays 
and values of Fv/Fm in the 3 types of leaves, 
at 23°C. The uncertaities in the lifetimes is q- 
10% for lifetimes< 500 ps and 15% for lifetimes 
>500 ps. The estimated uncertainty in preexpo- 
nential factors is 4- 15% 
Barley ILG 
Wild type Chlorina F2 
Fv/Fm 0.80 0.83 0.62 
Tm 260 280 1480 
T1 100 90 61 
T2 400 400 500 
7"3 1000 1200 1700 
T4 - - 3800 
A l 78 83 74 
A2 21 16 16 
A3 <1 <1 9 
A4 - - 1 
affects the antenna size does not necessarily affect sen- 
sitively the mean lifetime (Haworth et al. 1983; Hodges 
et al. 1987). Figure 2 shows that a linear relationship 
exists between fluorescence amplitudes and mean life- 
times, as the temperature was varied, but, in barley 
leaves, and in contrast to variations caused by QA pho- 
toreduction, the linear relationships do not extrapolate 
to zero (Moya et al. 1986a,b). Therefore, as opposed to 
earlier suggestions (Bukhov et al. 1990, Goltsev et al. 
1994), the heat-induced increase in Fo cannot be due 
solely to an accumulation of QA. 
Analysis of fluorescence decays 
The best fit of chlorophyll fluorescence decays, in both 
wild-type and the mutant of barley leaves, is obtained 
by a deconvolution with 3 components at all leaf tem- 
peratures. At 23 °C, the major component (A1 = 0.8) 
has a lifetime T1 of 100 ps. A second component (A2 
= 0.2) has a longer lifetime (T2 = 400 ps). The third 
component has a lifetime, ~, of 1200 ps, with a pre- 
exponential factor smaller than 0.01. These results are 
similar to those previously obtained with other green 
plants (for a review, see Moya et al. 1986b). In the 
ILG-pea leaves a fourth lifetime component (T4 = 4 
ns, A4 = <0.01) is necessary to fit the decays at all the 
temperatures (see Table 1). 
Figure 3 shows that heating of both wild-type 
(upper row) and mutant leaves (middle row) of barley 
significantly decreases A1 but does not modify T1. This 
decrease starts at 30 °C and is reversible. An augmen- 
tation in A2 was induced also at temperatures higher 
than 30-35 °C. An increase in T2 is observed at tem- 
peratures higher than 40 °C. These changes are par- 
tially reversible. At temperatures higher than 40 °C an 
almost irreversible increase of T3 occurs and is accom- 
panied by an augmentation of A3 which is partially 
reversible. In the ILG-leaves (Figure 3 lower row), 
heat induces very similar effects as in barley leaves on 
Tl and T2 (plus/'3) components. The increase in T4, 
observed in ILG leaves, resembles the effecton ~ in 
barley leaves. This last observation suggests that T3-A3 
component of barley could come from the same type 
of complex as T4-A4 of ILG pea leaves, the latter being 
more loosely connected to the center. It is well docu- 
mented (Avarmaa et al. 1977; Pfarrherr et al. 1991) that 
the fluorescence lifetime of chlorophyll a in solution in 
an organic solvent is about 6.5 ns; therefore, a lifetime 
longer than 6.5 ns can hardly be interpreted as prompt 
fluorescence. We suppose that our T4 = 8 ns compo- 
nent is a delayed fluorescence emission superimposed 
on the prompt fluorescence emission. However, the 
discussion of this minor emission is beyond the scope 
of this work. 
Note also that if we assume that the long lifetime 
components 7"3 and T4 of ILG leaves are dead fluo- 
rescence and if their yields are subtracted from both 
Fo and Fro, then the value of Fv/Fm in these leaves 
becomes 0.80. 
Several effects overlap to cause the increase of Fo 
induced by heat. The similarity of heat-stress effects on 
the individual fluorescent components in the wild type 
and the mutant lacking LHC IIb provides evidence that, 
in contrast to what has been often proposed, a discon- 
nection of this large antenna complex is not the cause 
of heat-induced increase in Fo. Our data on ILG-leaves 
and the data of Glick and Melis (1988) and of Har- 
tel and Lokstein (1995), who observed an absence of 
minor LHC II complexes in ILG-leaves, infer that dis- 
connection of these pigments would not be the reason 
for heat-induced enhancement of Fo. 
The multi-exponential kinetics of chlorophyll flu- 
orescence decay after a picosecond pulse have been 
extensively discussed in the past. It was mostly 
attempted to establish correlations between the dif- 
ferent lifetime components and various types of PS 
II which emerged from the multiphasic increase of 
chlorophyll fluorescence associated with the photore- 
duction of QA (for a review see Dau 1994 and for a 
review on PS II heterogeneity, see Lavergne and Bri- 
antais 1996). Another approach, proposed by Schatz 
et al. (1987, 1988), is that of the 'Reversible Radical 
2.5 
2 
1.5 
t~ 
v- 1 
0.5 
2.5 
~ '1 .5 
c 
0.5 
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........... . ....................................... i ............. i o "~ 
oR i 6 : 
.......... i .............. i ............. ~ ............ -..o....-,.i ........... 
ol o , o ~o ol ! - 
20 25 30 35 40 45 50 
Temperature (°C) 
6 
4 
2 
0 
20 
' ' ' ! ' ' ' ' I . . . . I . . . . . . . . . . . . - 
Ri i °! 
2 o .! ............ L i 
i , i , = = 
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R! i i o~ 
..... o°i ~o ,o i °° . 
o ~[, , , ~, ~, ~ ~",~,,~, [] ~ 
20 25 30 35 40 45 50 
Temperature (°C) 
~o i .... ' .... i i i 
8 . . i . . . . . . . . . . . . . . . . . . . . . . ~ ............. ! ........... ~........ 
~R i i i 
--~--i-----"=-! .............. i ........... ~: ............. i ........... 
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i e i e l o! i - 
! ~ io 6 o i :.N..:-.....@.!.....@-..@..-~!..~.-...~..---.~ 
' , , , , I . . . . I . . . . I . . . . I . . . . I . . . . " 
25 30 35 40 45 50 
Temperature ( °C) 
1 ' " ! . . . . . . . . . . . . I , ' " ! ' " , 
0.8 ...... ~ i - ~ i - ~ - - ~ ; i ............ i ........... 
R i imam 
0.6 
0.4 ........... -i ............. ~ ........................... i ........... i ........... 
ni i ~ oiO ~ o~ 
0.2 ....... oi........~.L..,?..~ ............ i .............. i ......... - 
i i i - 
0 ,,-i , , . h,_, k.,..i .., ,6, • 4 
20 25 30 35 40 45 50 
Temperature (°C) 
1 ' " l . . . . i . . . . . . . . ! . . . . . . . 
Ri [] 
0.6 ~ i ~ m, 
o.4 ............ i ............................ ! .............. i .............. i .......... 
R i i i 6 oC 
0.2 ..... 8 i .......... 6 o P a i .............. i ........... 
! ~ i i i - 
0 , . i , , , ~ , ~ , ~ , ~ i ~ , ~ , ~1 
20 25 30 35 40 45 50 
Temperature (°C) 
ml [][ ; i i 
o .6~i i i !~ 
0.4 ........... i ............. } ............. f ............. i ............ ~ .......... 
0.2 "c3 " ; 6 i o ~ ~ i~e ......... 
0 ~' '== i , , ~. i, ,A , i . , ,~ i ,A , ,,~ . . . . • 
20 25 30 35 40 45 50 
Temperature ( °C) 
< 
193 
Figure 3. Effect of temperature on individual lifetime components Ti and their corresponding pre-exponential factors Ai, from the deconvolution 
of fluorescence decays at Fo in leaves; upper row: wild-type barley, middle row: chlorina F2 mutant of barley and lower row: ILG-pea. EB: Tl, 
A t - O : 7"2, A2; • : T3, A3; • : T4, A4 R stands for the values obtained after returning to 23 o C. 
Pair' (RRP) model. Using PS II particles with ~80 
Chl/P680 isolated from Synechococcus, these authors 
found a tri-exponential fluorescence decay under Fo 
conditions. However, they discarded a ~ 1.8 ns compo- 
nent that they assign to disconnected antenna pigments 
(Schatz et al. 1987). The RRP model is based on two 
assumptions: (i) there exists a rapid excitation equi- 
librium between all PS II pigments including P680, 
and (ii) The primary charge separation is reversible in 
the nanosecond time domain. By limiting the discus- 
sion to solely two decay components, the mathematical 
representation is achieved by a model using two differ- 
ential equations. To 'invert' the model (i.e. to retrieve 
rate constants) an additional assumption is required for 
example, the rate constant of antenna deactivation is 
fixed. 
The results presented in this work concern in-vivo 
intact systems. To take into account the third slow 
fluorescence decay component (T3), the assumption 
of completely disconnected antenna pigments seems 
unlikely, at least for the wild type sample. So we added 
a third component by means of a modified RRP mod- 
el, first proposed by Goulas (1992). This corresponds 
to the scheme presented in Figure 4. We made the 
hypothesis that the new compartment (3) corresponds 
to a minor amount of pigments in incomplete equilib- 
194 
k3Tlk3 
k 'a 
• fluorescence and 
internal conversion 
antenna-centre 
complex 
kt 
Ch l*P ~ CHIP* 
k-t 
Q 
radical-pair 
electron transfer to Q A 
~ ka de-excitation of the antenna by 
- fluorescence 
- internal conversion 
Figure 4. Scheme o f the modi f ied 'Revers ib le Radica l Pa i r ' mode l 
o f PS II energy migrat ion , modi f ied f rom Schatz et al. (1988). (1) is 
the antenna-center compar tment , (2) is the radical pa i r compar tment . 
A third compar tment (3) has been added. It is a complex conta in ing 
less than 1% o f total PS II ch lorophyl l . It is loosely bound. The entire 
complex represents on ly the inner antenna o f PS II. 
rium with the main antenna (1). We fixed its amount 
to 1% of total PS II chlorophyll; however, changes of 
this amount has little effect, provided that it remains 
minor compared to (1). A second hypothesis is made 
on the deactivation rate of antenna pigments: ka and 
k'a which we fixed to ~0.5 ns -] . This value was cho- 
sen because we found it experimentally in a mutant 
without PS II reaction centers (Moya et al. 1986c); 
changing it to 0.25 ns -] , as it has been found in isolat- 
ed LHC II (Bassi et al. 1991), changes the results very 
little. The rate constants ka and/da are also supposed 
to be independent of temperature between 20 and 50 ° 
C. Following the assumptions of the RRP model, kt 
and k- t a re the rate-constants of the reversible exci- 
ton transfer within the antenna, including P. They are 
assumed to be too fast to beconsidered in the model. 
The remaining unknown rate constant: kl, k_l, k2, k3 
and k-3 are derived from the T1, Al, T2, A2, T3, A3 
experimental parameters of Figure 3, by a non linear 
least square fitting program developed at LURE for this 
purpose. The program converges to a single solution 
in all cases• 
The augmentation in temperature diminishes the 
quantum yield of PS II photochemistry (Figure 5a) by 
decreasing irreversibly the rate constant of charge sep- 
aration (kl) and of the stabilization on QA (k2) and by 
increasing the rate constant (k-l) of the P+I- recom- 
'7 
% 
.ag 
9 
8 
7 
6 
5 
4 
3 
2 
1 
20 
I . . . . . . . . ! . . . . ! . . . . . . . . t . . . . 
..................................... i _ _ . __ i . . . . . . . . . i __a_ _ 
• i " I i - • f " 
: i ~ • I i " 
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I • 
I 
r"~ ............................................. ." . . . . . . . . . . . . . . . ~ - , , . . . . . . I . . . . . . 9__ 
i • I [ • 
25 30 35 40 45 50 
Temperature (°C) 
0 .7 ' ' ' ' r . . . . . . . . ~ . . . . I . . . . . . . . 
i / A - - [ I b 
0.6 ..................... i- .............. ..'.. ..................... L . , . _ ___ ' . _ J . . . . . . . . . . . . 
• l i I 
0.5 . . . . . . . . . . . . . . . . . . F ~ ................... " . . . . . . . . . . . . . . . . . . l i . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . r- 
'7 / i 
....................... I ................. it ............ 
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,~ o.3 ................... ~ ......................................................................... 1 ........... * ............... 
I 
~0.2 ......... 5~ ! ................................................ .[ ....................... ! . ....................... ~- ............ 
] i I 
o.~ .................... t ....................... T ........................ t ......................... t .............................. . .... 
• D i i i a 
0 .................. r.~ ......................... ~ ....................... ~ ................ i_+......JL ........ ~ ............ 
e i o i • i ° i 
. . . . i . . . . i . . . . i . . . . i . . . . -0 ,1 . . . . 
20 25 30 35 40 45 50 
Temperature (°C) 
Figure 5 Variat ions wi th temperature o f rate constants o f the mode l 
depicted in F igure 4, insert ing values o f T]-AI, T2-A2 and T3-A3 
in the mode l (a) k] ( I ) , k - i (O) and K2 (&) ; (b) k3 (&) and k -3 
(O) . 
bination. This last effect is reversible. These effects 
on PS II photochemistry start to occur around 30 °C. 
At temperatures higher than 40 °C the increase of Fo 
is mostly due to the increase of the fluorescence yield 
of the third component• Figure 5b shows the varia- 
tions with temperature of the rate-constants k3 and k-3 
of excitation exchange between compartments (1) and 
(3). At temperatures higher than 40 °C a large irre- 
versible decrease of k3 occurs. This result suggests 
that compartment (3) becomes more loosely bound to 
the rest of PS II unit, at temperatures higher than 40 °C. 
We are tempted to identify compartment (3) as 
the so called LHC IIe complex isolated by Peter and 
Thornber (1991)• Indeed this complex has the small- 
est content in chlorophyll, around 2% of total PS II- 
chlorophyll. But inconsistent with this conclusion is 
the absence of this complex in ILG-leaves (Glick and 
Melis 1988; Hartel and Lokstein 1995). 
From these time-resolved measurements of chloro- 
phyll fluorescence at Fo, it emerges that two phe- 
nomena probably overlap to build up the heat-induced 
increase of Fo in vivo. First, there is a moderate increase 
of Fo from 30 °C, probably due to changes of rate- 
constants which determine a decrease of the yield of 
photochemistry. This effect is partially reversible. Sec- 
ond, there is a major increase of Fo, starting at 40 °C, 
certainly determined by a decrease of the connection 
to the rest of PS II of a chlorophyll-protein complex, 
containing a small % of total PS II-chlorophyll. This 
complex is obviously not LHC IIb; it may be not neces- 
sarily one of the minor LHC II component. This effect 
is irreversible. 
Another important conclusion arises from this 
study. Data reported in Figure 3 and Table 1 shows 
that the presence or the absence of a large antenna size 
- namely LHC IIb - does not affect the averaged flu- 
orescence lifetime nor the lifetime components (~ 100 
and 400 ps of the Fo fluorescence state at 23 °C). In this 
work we used an extended version of the RRP model 
(Schatz et al. 1988) to interpret our data. The RRP mod- 
el assumes a rapid exciton equilibrium among all anten- 
na pigments which should include peripheral antenna, 
inner antenna and P. It is also assumed that the reaction 
center of PS II constitutes a shallow trap and that the 
exciton decay is 'trap limited'. Under these conditions 
the apparent rate constant for energy transfer should 
depend on two terms: one term describing the ener- 
gy difference between the antenna chlorophylls and 
the special reaction center chlorophyll P (Bolzmann 
energy distribution) and a second term proportional to 
(Npig)- 1 which expresses the probability for the exci- 
tation to be located in the antenna. This is equivalent 
to the one formulated by other authors (Knox 1975; 
Pearlstein 1982). The chlorina F2 mutant has an anten- 
na size reduced by a 2.5 factor when compared with 
the wild type (not shown); nevertheless, the lifetime 
parameters are similar. As the difference comes from 
the reduced amount of the LHC IIb in the mutant case, 
this result suggest that excitation absorbed by LHC IIb 
is irreversibly transferred to the core antenna in a time 
domain << 100 ps. The scheme of the kinetic model 
presented in Figure 4 must be understood as concern- 
ing only energy transfer mechanisms within the inner 
antenna. A similar conclusion has been reached by 
Gilmore et al. (1996). 
195 
Acknowledgements 
This work was supported in part by C.N.R.S. and in part 
by the EUREKA project No. 380 (LASFLEUR).The 
authors would like to thank A. Zawadzki for his help 
in computer programs and Z. Cerovic for critically 
reading the manuscript 
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