Planta
Planta 141, 135-139 (1978)
9 by Springer-Verlag 1978
Light-induced Fluorescence Decay during the Greening of Normal and Lincomycin-treated Maize Leaves 1~. Sfirvfiri, G. Hal/tsz*, Sz. T6r6k**, and F. L/mg Department of Plant Physiology,E6tv6s University, P.O. Box 324, H-1445 Budapest 8, Hungary
Abstract. Light-induced fluorescence decay was exam-
ined during the greening of control and lincomycintreated maize (Zea mays L.) leaves. Assuming that this decay to a first approximation is the result of two parallel first-order reactions, the fluorescence induction curves were linearized on the logarithm plot and the parameters were determined. The variable fluorescence increased, and the parameters of the two linear sections of the fluorescence d e c a y - t h a t is, the kinetics of the induction c u r v e s - c h a n g e d during the greening of the control leaves. Lincomycin treatment caused some chlorophyll deficiency and the lowering of the chlorophyll a/b ratio, changed the fluorescence emission spectra and the effect of Mg 2 + on the regulation of the excitation energy distribution. The structure of the thylakoids and the kinetics of the fluorescence decay were also changed in the treated leaves. The possible relationship between the change of the kinetics of the fluorescence decay and the change of spillover during greening and after lincomycin treatment is discussed. Key words: C h l o r o p h y l l (fluorescence) - Fluorescence
(chlorophyll) - Lincomycin - Zea.
Introduction
Some induction phenomena involving fluorescence induction (Kautsky effect) are triggered when exposed Present address:
* Research Institute for Technical Chemistry ** Hungarian Academy of Sciences Central Research Institute for Physics Abbreviations: LHC = light-harvesting complex; Chl = chlorophyll; LM =lincomycin; PS =photosystem; DCMU=3-(3,4-dichlorophenyl)-1,1-dimethylurea
to light. The fast fluorescence transients (ms range) seem closely connected with the changes of electron flow through PS II, while the slower changes (s range) may reflect processes in which the ultrastructure of the thylakoid membrane plays a fufidamental role (Papageorgiou, 1975). The slow fluorescence decay of isolated chloroplasts is, under certain circumstances, resistant to D C M U and sensitive to uncouplers obliterating the proton gradient (Papageorgiou et al., 1972; Telfer et al., 1975). It is affected by the concentration of the different divalent cations in the medium that have been proposed to control the spillover of quanta (Murata et al., 1970; Krause, 1974; Mills and Barber, 1975) affecting the configuration of thylakoid membranes (Murata, 1971; Mohanty and Govindjee, 1973; Arntzen, 1977). We analyzed the slow fluorescence intensity changes during the greening of maize seedlings. The greening process involves a series of compositional and configurational changes of the photosynthetic membranes (Boardman etal., 1972), which, as expected, strongly affect the induction of slow fluorescence. Changes of fast fluorescence induction kinetics during the greening of a dark-grown Chlorella mutant (Dubertret and Joliot, 1974) and during the greening of normal and mutant maize leaves (Klimov et al., 1972) have been published, and there are some indications that the development of the variable fluorescence is connected with the activation of the latent water-splitting system (Strasser and Sironval, 1974; Ichikawa et al., 1975). Treatment with LM (a plastid protein synthesis inhibitor), which has a strong effect on the structural organization of thylakoids (Thomson and Ellis, 1972 ; Sfirvfiri et al., 1976), was also employed to study the relations between the structural deficiencies and fluorescence properties.
0032-0935/78/0141/0135/$01.00
136
l~. Sfirvhri et al. : Light-induced Fluorescence Decay
Materials and Methods Maize (Zea mays L.) plants were grown on filter paper moistened with Hoagland solution of 1/4 strength in the dark at 2 4 + 2 ~ for 7 8 days. The seedlings were placed on Hoagland solution with or without LM. After about 16 h treatment in the dark with 100 pg/mi LM (Ellis, 1970), the seedling were transferred to continuous white light (650 Ix) for different lengths of time at 24+_2~ C. Fluorescence emission spectra, fluorescence induction curves, and Chl content were measured as described earlier (Shrvfiri et al., 1976). Mg 2 +-effect on fluorescence spectra (measured at 77 ~ K) was determined according to Davis et al. (1976). The Chl content of samples was 10 lag/ml, Mg 2+ content was 10 15 mM during the treatment. The measured samples contained 5 ~tg/ml Chl, and 50% glycerol was added before freezing. Theoretical treatment of the fluorescence induction curves: The fluorescence induction curves were measured on 25 identical samples. The 25 values obtained for every given time were digitalized, averaged, and their standard deviation computed. Assuming that the fluorescence induction curves are the results of two, parallel first-order reactions, giving the same end product (fluorescence intensities),
~r
A
0
25
50 time,seconds
75
100
75
100
2 xx
1 o_J
i
T
o
....................
25
50 time,seconds
z~F
C
the fluorescence intensity normalized to the constant fluorescence (F0) may be written as:
AF=e-Att+B~+e A2t+B2 where A1, A=, B1, and B2 are parameters. The A 1 and A 2 parameters are the slopes of the linear sections, and they are related to the rates of the decay processes. The B 1 and B2 parameters are quantities proportional to the logarithms of the fluorescence intensities extrapolized to the zero time of the first and the second processes (exp B 1 is proportional to P). If A1 >>A2, then the fluorescence induction curves can be linearized on the logarithm plot. A computer program employing the weighted least-squares method fitted two lines on this log plot, the breaking point of which was also determined by the program. Samples for electron microscopy were cut from the middle of the primary leaves, fixed with 5% glutaraldehide in 0.07 M K - N a phosphate buffer (pH 7.2) for 3 h at 20~ C, and postfixed with 1% buffered OsO4 for 2 h. After fixation the samples were dehydrated in an alcohol series, stained with 1% uranyl acetate (w/v in absolute alcohol), and embedded in durcupan (Fluka). The sections were cut with a Porter-Blum nltramicrotome, stained with lead citrate, and examined with a KEM-1 electron microscope.
Results The areas under the fluorescence induction curves and the maximal light-induced relative fluorescence intensity (P) increased d u r i n g the greening o f the cont r o l l e a v e s (Fig. 1 A , B, C). T h e k i n e t i c s o f the i n d u c t i o n c u r v e s ( F i g . 2), c h a r a c t e r i z e d b y t h e A1 a n d A2 p a r a m e t e r s o f t h e t w o l i n e a r s e c t i o n s ( T a b l e 1),
2
o--
65o 0
25
50
time, seconds
75
100
Fig. I.A-C. The light-induced relative fluorescence intensity changes measured at room temperature. The fluorescence induction curves of the control ( - - ) and LM-treated ( - - - ) leaves were measured after 12 (A), 24 (B), and 48 (C) h of illumination. The measuring light was about 650 nm and the fluorescence was measured at 725 nm. The actinic light (/) came from a well-focused 1000 W tungsten projection lamp through a 5 cm water bath. The curves are averages of about 25 induction curves normalized to the fluorescence induced by the measuring light (F0). The standard deviations of the points are about t0% of the values. Arrows indicate the switching on of the measuring and actinic lights, respectively
changed simultaneously with the increasing fluorescence intensity: The light-induced fluorescence was quenched more and more rapidly during the greening process. The time o f the b r e a k i n g p o i n t o f the lineari z e d c u r v e s ( T a b l e 1), i.e., t h e p e r i o d a f t e r w h i c h t h e MT process becomes dominant, gradually increased during greening.
1~. S~trvf.ri
137
et aI. : Light-induced Fluorescence Decay
In(aF) 150'
Table 2. Chlorophyll content and chlorophyll a/b ratio of control (Co) and LM-treated leaves after different times of illumination
.~
o ~
100"
~ o
50
Illu- Chl a/b mi- !-tg/gfresh weight
9
Chl
na-
tion Co (h)
LM
Inhibition
~
12 24 48
-5C
~ ~ n D a~rl~m D m : ~,-m9
0
25
Co
LM
3.72_+0.09 3.52_+0.06 3.28_+0.09
3.18_+0.23 2.69_+0.13 2.31_+0.09
(%)
nnn
~
a/b
50
19.5 22.6 31.0
Q
F 1.0
75
time, seconds
305.8_+ 36.0 246.3_+16.9 698.4_+ 69.5 540.3_+40.2 1004.5_+109.3 692.8_+96.9
Fig. 2. Logarithm plots of the light-induced relative fluorescence intensities [In(zxF)] of control ( - - ) and LM-treated ( - - - ) leaves after 12 (o), 24 (Ez), and 48 (o) h of illumination. The standard deviations of the points are 10%
The constant part of the fluorescence increased rapidly during the greening of the LM-treated leaves. After 48 h illumination, it reached 3-4 times that of the corresponding control level. The variable part of fluorescence (P) increased in the first stage of greening (between 12 and 24 h), then decreased (Fig. 1A, B, C). The decay of the first process in the LM-treated leaves was slower than in the control ones (Table 1:A1 parameters); however, the light-induced fluorescence of the treated leaves dropped below the F0 level 5 15 s after switching on the actinic light. This change was reversible in the dark. In the first stage of greening (up to 24 h) the time of the breaking point increased, after which it practically did not change. The Chl content of the control and LM-treated leaves increased significantly, and the Chl a/b ratio decreased during the greening (Table 2). LM treat-
05
/ /
i
g60
t II
I
S
i
I
700 %0 wavelength, nm
780
Fig. 3. Fluorescence emission spectra of control ( - - ) and LMtreated ( - - - ) leaves measured at 77 ~ K after 48 h of illumination. The curves are normalized to the long wavelength form and corrected for the photomultiplYer sensitivity. Fluorescence in arbitrary units. Insert: Difference spectra between control and LMtreated leaves
Table 1. Parameters of the two linear sections Illumination
B1
B2
A2
As
Time of the breaking point (s)
(h) 12 24 48
Co
LM
Co
LM
Co
LM
Co
LM
Co
LM
0.97 1.17 1.46
0.41 0.58 0.36
0.54 0.62 0.75
--0.36 - 0.43 -0.58
-0.058 -0.043 -0.032
-0.110 -0.085 -0.086
-0.013 - 0.008 -0.008
+0.004 -0.002 -0.005
15 25 47
12 20 20
Co = control, LM = lincomycin treated
138
1~. Sfirv/triet al. : Light-induced Fluorescence Decay
Table 3. Effect of Mgz+ on the fluorescence emission spectra (at 77~K) of control (Co) and LM treated chloroplasts isolated from leaves illuminated for 48 h
Fluorescence ratios
- M g 2+
+Mg 2+
+Mg2+ 100 _ Mg2 +
0.40_+0.04 0.19_+0.04
0.57+_0.07 0.30-+0.05
140.2_+ 8.5 156.2+20.2
0.23_+0.03 0.20-+0.02
0.27_+0.02 0.23+_0.02
116.5_+ 7.6 116.8_+ 4.0
% of the Co
Co
686/735 695/735 LM 683/730 697/730
41.9 29.9
detected at a 3-5 nm shorter wavelength (Fig. 3), and the shorter wavelength forms of Chl-a were decreased in the emission spectra of the treated leaves. F r o m the difference spectrum (Fig. 3, insert) an extra fluorescence at a b o u t 720 nm could be seen. Mg 2+-treatment caused a b o u t 40-50% increase of the fluorescence ratios 686/735 or 695/735. In chloroplasts isolated from LM-treated leaves, the Mg 2§ induced change of the fluorescence ratios reached only 30-40% that of the controls (Table 3). T h e electron micrographs of the control leaves illuminated for 48 h (Fig. 4, above) were typical for green maize leaves. In the LM-treated leaves, stroma thylakoids of the mesophyll plastids were smashed to fragments, and instead of thylakoids, vesicles occupied a large part of the volume of the bundle sheath plastids (Fig. 4, below). A more detailed demonstration and discussion of the p h e n o m e n o n will be presented in another report. Discussion
Fig. 4. Electron micrograph of mesophyll (M) and bundle sheath plastids (BS) from control (above) and LM-treated (below) leaves after 48 h of illumination, x 8800. Solid bar = 1 ~tm ment caused some Chl deficiency and the lowering of the Chl a/b ratio relative to the control. Fluorescence emission spectra of control maize leaves illuminated for 48 h differ significantly from the treated ones. The long wavelength m a x i m u m was
Examining the fluorescence induction curves o f intact maize leaves, we found that the slower fluorescence decay may be dissolved into two parallel first-order processes within a 10% error. This is smaller than the mean standard deviations of the measured points, that is, in such a first approximation the two processes can be considered to be exponential. Sokolove and Marsho (1977) also found that the slow fluorescence quenching of type A spinach chloroplasts is composed of two processes. Under our conditions the major part of the variable fluorescence is quenched by the first process. The kinetics of both processes changed during greening (Table 1), though the development of the second one came to an end earlier (see the A2 parameters in Table 1). During greening many structural and functional changes occur in the chloroplasts. In maize seedl i n g s - a t illumination with a light intensity of 2000-2500 l x - the stacking, the Hill activity (Horvfith et al., 1975) and the acid-base phosphorylation activity (Forger and Bogorad, 1973) increase markedly, and the Chl a/b, PTo0/Chl ratios, and the total fluorescence intensity (Horvfith et al., 1975) sharply decrease up to 24 h. Somewhat slower changes of all of these properties occur during the following 36 h. Similar stepwise changes were observed in grana stacking, in divalent cation effect on fluorescence emission spectra, and in the forrhation of L H C during continuous illumination of the previously intermittently illuminated pea leaves (Armond et al., 1976; Davis et al., 1976). F r o m data in the' literature we suppose that the development of the photophosphorylation and spillover regulating processes (Mohanty and Govindj ee, 1973 ; Vernotte et al., 1974 ; Papageorgiou,
1~. Sflrvf.ri et al. : Light-induced Fluorescence Decay
1975) seem to be the main components influencing the changes of the fluorescence decay during the greening process. The results of Schreiber et al. (1977) show that the size of the photosynthetic unit may also have an effect on the kinetics of the slow fluorescence transients. LM treatment, while causing marked structural changes (changes of the Chl a/b ratio, fluorescence spectra, and electron-microscopic pictures), had a very significant effect on slow fluorescence decay and modified the regulating effect of Mg 2+ on the spillover processes as well. The first process of the fluorescence decay is about two times slower in LMtreated leaves than in the controls. The fluorescence decreases very rapidly below the F 0 level, which may be attributed to a light-induced, dark-reversible building of an extra quencher. In chloroplasts isolated from the LM-treated leaves, the spillover reguIating effect of Mg 2+ decreased 2-3 times relative to the controls. It is interesting, however, that the LHC, which is the most likely candidate for the Mg 2 +-binding factor (Davis et al., 1976), is present in the LM-treated leaves with the same electrophoretic properties as in the controls (Ellis, 1975; Hiller et al., 1977). In this case the microstructural changes of the thylakoid membranes caused by LM treatment evoke a smaller Mg2+-regulability of energy distribution processes. This may also cause the changes of fluorescence decay kizetScs m the treated ~eaves. We are indebted to Dr. A. Keresztes for assistance with the electron microscopy.
References Armond, P.A., Arntzen, C.J., Briantais, J.-M., Vernotte, C.: Differentiation of chloroplast lamellae. Light harvesting efficiency and grana development. Arch. Biochem. Biophys. 175, 54-63 (I976) Arntzen, C.L : Dynamic structural features of chloroplast lamellae. In: Current topics in bioenergetics, Vol. VII. p p 1-35, Sanadi, D.R., Vernon, L.P. eds, New York: Academic Press I977 Boardman, N.K., Anderson, J.M., Hiller, R.G., Kahn, A., Roughan, P.G, Treffry, T.E., Thorne, S.W.: Biosynthesis of the photosynthetic apparatus during chloroplast development in higher plants. In: Proc. 2nd Int. Congress on Photosynthesis Research, Stresa, Vol. 3. pp. 2265 2287, Forti, G , Avron, M., Melandri, A., eds. The Hague: Dr. W. Junk N.V. 1972 Davis, D.J., Armond, P.A., Gross, E.L., Arntzen, C.J. : Differentiation of chloroplast lamellae. Onset of cation regulation of excitation energy distribution. Arch. Biochem. Biophys. 175, 64 70 (1976) Dubertret, G., Joliot, P.: Structure and organization of system II photosynthetic units during the greening of a dark-grown Chlorella mutant. Biochem. Biophys. Acta 357, 399-41l (1974) Ellis, R.J.: F~rther similarities between chloroplast a n d bacterial ribosomes. Planta 91, 329-335 (1970) Ellis, R.J. : Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamid. Phytochemistry 14, 89-93 (I975) Forger, J.M., Bogorad, L.: Steps in aquisition of photosynthetic
139 competence by plastids of maize. Plant Physiol. 52, 491-497 (1973) Hiller, R.G., Pilger, T.B.G., Genge, S.: Effect of lincomycin on the chlorophyll-protein complex I content and photosystem I activity of greening leaves. Biochim. Biophys. Acta 460, 431-444 (1977) Horvath, G., Garab, Gy.I., Mustfirdy, L.A., Halitsz, N., FaludiDfiniel, A. : The development of thylakoids and photochemical properties of mesophyll and bundle sheath chloroplasts of greening maize leaves. Plant Sci. Lett. 5, 239-244 (1975) Ichikawa, T., Inoue, Y., Shibata, K. : Delayed light emission and variable fluorescence from intermittently illuminated wheat leaves under continuous illumination related to activation of the latent water-splitting system. Plant Sci. Lett. 4, 369-376 (1975) Klimov, V.V., Lhng, F., Karapetyan, N.V., Krasnovsky, A.A. : Fluorescence induction during the greening of etiolated leaves : normal and mutant maize seedlings. Fiziol. Rast. 19, I51-159 (1972) Krause, G.H.: Changes in chlorophyll fluorescence in relation to light-dependent cation transfer across thylakoid membranes. Biochim. Biophys. Acta 333, 301 313 (1974) Mills, J., Barber, J.: Energy-dependent cation-induced control of chlorophyll a fluorescence in isolated intact chloroplasts. Arch. Biochem. Biophys. 170, 306-314 (1975) Mohanty, P., Govindjee : Light induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans. I. Relationship of slow fluorescence changes with structural changes. Biochim. Biophys. Acta 305, 95-104 (1973) Murata, N.: Control of excitation energy transfer in photosynthesis. V. Correlation membrane structure to regulation of excitation transfer between two pigment systems in isolated chloroplasts. Biochim Biophys. Acta 245, 365 372 (1971) Murata, N., Tashiro, H., Takamiya, A. : Effects of divalent metal ions on chlorophyll a fluorescence in isolated spinach chloroplasts. Biochim. Biophys. Acta 197, 250-256 (1970) Papageorgiou, G.: Chlorophyll fluorescence: An intrinsic probe of photosynthesis. In: Bioenergetics of photosynthesis, pp. 319-371. Govindjee, ed. New York-San Francisco-London : Academic Press 1975 Papageorgiou, G., Isaakidou, J., Argoudelis, C. : Structure dependent control of chlorophyll a excitation density: the role of oxygen. FEBS Lett. 25, 139-142 (1972) S~trvfiri, l~., HalS_sz, G., Nyitrai, P., Lfing, F.: Effect of lincocin treatment on the greening process in bean (Phaseolus vulgaris) leaves. Physiol. Plant. 36, 187-192 (I976) Schreiber, U., Fink, R., Vidaver, W.: Fluorescence induction in whoole leaves: Differentiation between the two sides and adaptatic>~ to differen~ light regimes. Planta 133, 121-129 (1977) Sokolove, P.M., Marsho, T.V.: Slow fluorescence quenching of type A chloroplasts resolution into two components. Biochim. Biophys. Acta 459, 27-35 (1977) Strasser, R.J., Sironval, C.: Correlation between the induction of oxygen evolution and of variable fluorescence in flashed bean leaves. Plant Sci. Lett. 3, 135 141 (1974) Teller, A., Barber, d., Nicolson, J.: Energy-dependent quenching of chlorophyll a fluorescence. Evidence for coupled cyclic electron flow in isolated intact chloroplasts. Plant Sci. Lett. 5, 171 176 (1975) Thomson, W.W., Ellis, R.J.: Inhibition of grana formation by lincomycin. Planta 108, 89-92 (1972) Vernotte, C., Briantais, J.-M., Arntzen, C.J.: Comparison of excitons transfers changes induced by cations and by adaptation in states I and II. In: Proc. 3rd, Int. Congress on Photosynthesis, Rehovot, pp, 183-193, Avron, M. ed. Amsterdam: Elsevier 1974 Received 11 January ; accepted 18 March 1978
Planta 141, 135-139 (1978)
9 by Springer-Verlag 1978
Light-induced Fluorescence Decay during the Greening of Normal and Lincomycin-treated Maize Leaves 1~. Sfirvfiri, G. Hal/tsz*, Sz. T6r6k**, and F. L/mg Department of Plant Physiology,E6tv6s University, P.O. Box 324, H-1445 Budapest 8, Hungary
Abstract. Light-induced fluorescence decay was exam-
ined during the greening of control and lincomycintreated maize (Zea mays L.) leaves. Assuming that this decay to a first approximation is the result of two parallel first-order reactions, the fluorescence induction curves were linearized on the logarithm plot and the parameters were determined. The variable fluorescence increased, and the parameters of the two linear sections of the fluorescence d e c a y - t h a t is, the kinetics of the induction c u r v e s - c h a n g e d during the greening of the control leaves. Lincomycin treatment caused some chlorophyll deficiency and the lowering of the chlorophyll a/b ratio, changed the fluorescence emission spectra and the effect of Mg 2 + on the regulation of the excitation energy distribution. The structure of the thylakoids and the kinetics of the fluorescence decay were also changed in the treated leaves. The possible relationship between the change of the kinetics of the fluorescence decay and the change of spillover during greening and after lincomycin treatment is discussed. Key words: C h l o r o p h y l l (fluorescence) - Fluorescence
(chlorophyll) - Lincomycin - Zea.
Introduction
Some induction phenomena involving fluorescence induction (Kautsky effect) are triggered when exposed Present address:
* Research Institute for Technical Chemistry ** Hungarian Academy of Sciences Central Research Institute for Physics Abbreviations: LHC = light-harvesting complex; Chl = chlorophyll; LM =lincomycin; PS =photosystem; DCMU=3-(3,4-dichlorophenyl)-1,1-dimethylurea
to light. The fast fluorescence transients (ms range) seem closely connected with the changes of electron flow through PS II, while the slower changes (s range) may reflect processes in which the ultrastructure of the thylakoid membrane plays a fufidamental role (Papageorgiou, 1975). The slow fluorescence decay of isolated chloroplasts is, under certain circumstances, resistant to D C M U and sensitive to uncouplers obliterating the proton gradient (Papageorgiou et al., 1972; Telfer et al., 1975). It is affected by the concentration of the different divalent cations in the medium that have been proposed to control the spillover of quanta (Murata et al., 1970; Krause, 1974; Mills and Barber, 1975) affecting the configuration of thylakoid membranes (Murata, 1971; Mohanty and Govindjee, 1973; Arntzen, 1977). We analyzed the slow fluorescence intensity changes during the greening of maize seedlings. The greening process involves a series of compositional and configurational changes of the photosynthetic membranes (Boardman etal., 1972), which, as expected, strongly affect the induction of slow fluorescence. Changes of fast fluorescence induction kinetics during the greening of a dark-grown Chlorella mutant (Dubertret and Joliot, 1974) and during the greening of normal and mutant maize leaves (Klimov et al., 1972) have been published, and there are some indications that the development of the variable fluorescence is connected with the activation of the latent water-splitting system (Strasser and Sironval, 1974; Ichikawa et al., 1975). Treatment with LM (a plastid protein synthesis inhibitor), which has a strong effect on the structural organization of thylakoids (Thomson and Ellis, 1972 ; Sfirvfiri et al., 1976), was also employed to study the relations between the structural deficiencies and fluorescence properties.
0032-0935/78/0141/0135/$01.00
136
l~. Sfirvhri et al. : Light-induced Fluorescence Decay
Materials and Methods Maize (Zea mays L.) plants were grown on filter paper moistened with Hoagland solution of 1/4 strength in the dark at 2 4 + 2 ~ for 7 8 days. The seedlings were placed on Hoagland solution with or without LM. After about 16 h treatment in the dark with 100 pg/mi LM (Ellis, 1970), the seedling were transferred to continuous white light (650 Ix) for different lengths of time at 24+_2~ C. Fluorescence emission spectra, fluorescence induction curves, and Chl content were measured as described earlier (Shrvfiri et al., 1976). Mg 2 +-effect on fluorescence spectra (measured at 77 ~ K) was determined according to Davis et al. (1976). The Chl content of samples was 10 lag/ml, Mg 2+ content was 10 15 mM during the treatment. The measured samples contained 5 ~tg/ml Chl, and 50% glycerol was added before freezing. Theoretical treatment of the fluorescence induction curves: The fluorescence induction curves were measured on 25 identical samples. The 25 values obtained for every given time were digitalized, averaged, and their standard deviation computed. Assuming that the fluorescence induction curves are the results of two, parallel first-order reactions, giving the same end product (fluorescence intensities),
~r
A
0
25
50 time,seconds
75
100
75
100
2 xx
1 o_J
i
T
o
....................
25
50 time,seconds
z~F
C
the fluorescence intensity normalized to the constant fluorescence (F0) may be written as:
AF=e-Att+B~+e A2t+B2 where A1, A=, B1, and B2 are parameters. The A 1 and A 2 parameters are the slopes of the linear sections, and they are related to the rates of the decay processes. The B 1 and B2 parameters are quantities proportional to the logarithms of the fluorescence intensities extrapolized to the zero time of the first and the second processes (exp B 1 is proportional to P). If A1 >>A2, then the fluorescence induction curves can be linearized on the logarithm plot. A computer program employing the weighted least-squares method fitted two lines on this log plot, the breaking point of which was also determined by the program. Samples for electron microscopy were cut from the middle of the primary leaves, fixed with 5% glutaraldehide in 0.07 M K - N a phosphate buffer (pH 7.2) for 3 h at 20~ C, and postfixed with 1% buffered OsO4 for 2 h. After fixation the samples were dehydrated in an alcohol series, stained with 1% uranyl acetate (w/v in absolute alcohol), and embedded in durcupan (Fluka). The sections were cut with a Porter-Blum nltramicrotome, stained with lead citrate, and examined with a KEM-1 electron microscope.
Results The areas under the fluorescence induction curves and the maximal light-induced relative fluorescence intensity (P) increased d u r i n g the greening o f the cont r o l l e a v e s (Fig. 1 A , B, C). T h e k i n e t i c s o f the i n d u c t i o n c u r v e s ( F i g . 2), c h a r a c t e r i z e d b y t h e A1 a n d A2 p a r a m e t e r s o f t h e t w o l i n e a r s e c t i o n s ( T a b l e 1),
2
o--
65o 0
25
50
time, seconds
75
100
Fig. I.A-C. The light-induced relative fluorescence intensity changes measured at room temperature. The fluorescence induction curves of the control ( - - ) and LM-treated ( - - - ) leaves were measured after 12 (A), 24 (B), and 48 (C) h of illumination. The measuring light was about 650 nm and the fluorescence was measured at 725 nm. The actinic light (/) came from a well-focused 1000 W tungsten projection lamp through a 5 cm water bath. The curves are averages of about 25 induction curves normalized to the fluorescence induced by the measuring light (F0). The standard deviations of the points are about t0% of the values. Arrows indicate the switching on of the measuring and actinic lights, respectively
changed simultaneously with the increasing fluorescence intensity: The light-induced fluorescence was quenched more and more rapidly during the greening process. The time o f the b r e a k i n g p o i n t o f the lineari z e d c u r v e s ( T a b l e 1), i.e., t h e p e r i o d a f t e r w h i c h t h e MT process becomes dominant, gradually increased during greening.
1~. S~trvf.ri
137
et aI. : Light-induced Fluorescence Decay
In(aF) 150'
Table 2. Chlorophyll content and chlorophyll a/b ratio of control (Co) and LM-treated leaves after different times of illumination
.~
o ~
100"
~ o
50
Illu- Chl a/b mi- !-tg/gfresh weight
9
Chl
na-
tion Co (h)
LM
Inhibition
~
12 24 48
-5C
~ ~ n D a~rl~m D m : ~,-m9
0
25
Co
LM
3.72_+0.09 3.52_+0.06 3.28_+0.09
3.18_+0.23 2.69_+0.13 2.31_+0.09
(%)
nnn
~
a/b
50
19.5 22.6 31.0
Q
F 1.0
75
time, seconds
305.8_+ 36.0 246.3_+16.9 698.4_+ 69.5 540.3_+40.2 1004.5_+109.3 692.8_+96.9
Fig. 2. Logarithm plots of the light-induced relative fluorescence intensities [In(zxF)] of control ( - - ) and LM-treated ( - - - ) leaves after 12 (o), 24 (Ez), and 48 (o) h of illumination. The standard deviations of the points are 10%
The constant part of the fluorescence increased rapidly during the greening of the LM-treated leaves. After 48 h illumination, it reached 3-4 times that of the corresponding control level. The variable part of fluorescence (P) increased in the first stage of greening (between 12 and 24 h), then decreased (Fig. 1A, B, C). The decay of the first process in the LM-treated leaves was slower than in the control ones (Table 1:A1 parameters); however, the light-induced fluorescence of the treated leaves dropped below the F0 level 5 15 s after switching on the actinic light. This change was reversible in the dark. In the first stage of greening (up to 24 h) the time of the breaking point increased, after which it practically did not change. The Chl content of the control and LM-treated leaves increased significantly, and the Chl a/b ratio decreased during the greening (Table 2). LM treat-
05
/ /
i
g60
t II
I
S
i
I
700 %0 wavelength, nm
780
Fig. 3. Fluorescence emission spectra of control ( - - ) and LMtreated ( - - - ) leaves measured at 77 ~ K after 48 h of illumination. The curves are normalized to the long wavelength form and corrected for the photomultiplYer sensitivity. Fluorescence in arbitrary units. Insert: Difference spectra between control and LMtreated leaves
Table 1. Parameters of the two linear sections Illumination
B1
B2
A2
As
Time of the breaking point (s)
(h) 12 24 48
Co
LM
Co
LM
Co
LM
Co
LM
Co
LM
0.97 1.17 1.46
0.41 0.58 0.36
0.54 0.62 0.75
--0.36 - 0.43 -0.58
-0.058 -0.043 -0.032
-0.110 -0.085 -0.086
-0.013 - 0.008 -0.008
+0.004 -0.002 -0.005
15 25 47
12 20 20
Co = control, LM = lincomycin treated
138
1~. Sfirv/triet al. : Light-induced Fluorescence Decay
Table 3. Effect of Mgz+ on the fluorescence emission spectra (at 77~K) of control (Co) and LM treated chloroplasts isolated from leaves illuminated for 48 h
Fluorescence ratios
- M g 2+
+Mg 2+
+Mg2+ 100 _ Mg2 +
0.40_+0.04 0.19_+0.04
0.57+_0.07 0.30-+0.05
140.2_+ 8.5 156.2+20.2
0.23_+0.03 0.20-+0.02
0.27_+0.02 0.23+_0.02
116.5_+ 7.6 116.8_+ 4.0
% of the Co
Co
686/735 695/735 LM 683/730 697/730
41.9 29.9
detected at a 3-5 nm shorter wavelength (Fig. 3), and the shorter wavelength forms of Chl-a were decreased in the emission spectra of the treated leaves. F r o m the difference spectrum (Fig. 3, insert) an extra fluorescence at a b o u t 720 nm could be seen. Mg 2+-treatment caused a b o u t 40-50% increase of the fluorescence ratios 686/735 or 695/735. In chloroplasts isolated from LM-treated leaves, the Mg 2§ induced change of the fluorescence ratios reached only 30-40% that of the controls (Table 3). T h e electron micrographs of the control leaves illuminated for 48 h (Fig. 4, above) were typical for green maize leaves. In the LM-treated leaves, stroma thylakoids of the mesophyll plastids were smashed to fragments, and instead of thylakoids, vesicles occupied a large part of the volume of the bundle sheath plastids (Fig. 4, below). A more detailed demonstration and discussion of the p h e n o m e n o n will be presented in another report. Discussion
Fig. 4. Electron micrograph of mesophyll (M) and bundle sheath plastids (BS) from control (above) and LM-treated (below) leaves after 48 h of illumination, x 8800. Solid bar = 1 ~tm ment caused some Chl deficiency and the lowering of the Chl a/b ratio relative to the control. Fluorescence emission spectra of control maize leaves illuminated for 48 h differ significantly from the treated ones. The long wavelength m a x i m u m was
Examining the fluorescence induction curves o f intact maize leaves, we found that the slower fluorescence decay may be dissolved into two parallel first-order processes within a 10% error. This is smaller than the mean standard deviations of the measured points, that is, in such a first approximation the two processes can be considered to be exponential. Sokolove and Marsho (1977) also found that the slow fluorescence quenching of type A spinach chloroplasts is composed of two processes. Under our conditions the major part of the variable fluorescence is quenched by the first process. The kinetics of both processes changed during greening (Table 1), though the development of the second one came to an end earlier (see the A2 parameters in Table 1). During greening many structural and functional changes occur in the chloroplasts. In maize seedl i n g s - a t illumination with a light intensity of 2000-2500 l x - the stacking, the Hill activity (Horvfith et al., 1975) and the acid-base phosphorylation activity (Forger and Bogorad, 1973) increase markedly, and the Chl a/b, PTo0/Chl ratios, and the total fluorescence intensity (Horvfith et al., 1975) sharply decrease up to 24 h. Somewhat slower changes of all of these properties occur during the following 36 h. Similar stepwise changes were observed in grana stacking, in divalent cation effect on fluorescence emission spectra, and in the forrhation of L H C during continuous illumination of the previously intermittently illuminated pea leaves (Armond et al., 1976; Davis et al., 1976). F r o m data in the' literature we suppose that the development of the photophosphorylation and spillover regulating processes (Mohanty and Govindj ee, 1973 ; Vernotte et al., 1974 ; Papageorgiou,
1~. Sflrvf.ri et al. : Light-induced Fluorescence Decay
1975) seem to be the main components influencing the changes of the fluorescence decay during the greening process. The results of Schreiber et al. (1977) show that the size of the photosynthetic unit may also have an effect on the kinetics of the slow fluorescence transients. LM treatment, while causing marked structural changes (changes of the Chl a/b ratio, fluorescence spectra, and electron-microscopic pictures), had a very significant effect on slow fluorescence decay and modified the regulating effect of Mg 2+ on the spillover processes as well. The first process of the fluorescence decay is about two times slower in LMtreated leaves than in the controls. The fluorescence decreases very rapidly below the F 0 level, which may be attributed to a light-induced, dark-reversible building of an extra quencher. In chloroplasts isolated from the LM-treated leaves, the spillover reguIating effect of Mg 2+ decreased 2-3 times relative to the controls. It is interesting, however, that the LHC, which is the most likely candidate for the Mg 2 +-binding factor (Davis et al., 1976), is present in the LM-treated leaves with the same electrophoretic properties as in the controls (Ellis, 1975; Hiller et al., 1977). In this case the microstructural changes of the thylakoid membranes caused by LM treatment evoke a smaller Mg2+-regulability of energy distribution processes. This may also cause the changes of fluorescence decay kizetScs m the treated ~eaves. We are indebted to Dr. A. Keresztes for assistance with the electron microscopy.
References Armond, P.A., Arntzen, C.J., Briantais, J.-M., Vernotte, C.: Differentiation of chloroplast lamellae. Light harvesting efficiency and grana development. Arch. Biochem. Biophys. 175, 54-63 (I976) Arntzen, C.L : Dynamic structural features of chloroplast lamellae. In: Current topics in bioenergetics, Vol. VII. p p 1-35, Sanadi, D.R., Vernon, L.P. eds, New York: Academic Press I977 Boardman, N.K., Anderson, J.M., Hiller, R.G., Kahn, A., Roughan, P.G, Treffry, T.E., Thorne, S.W.: Biosynthesis of the photosynthetic apparatus during chloroplast development in higher plants. In: Proc. 2nd Int. Congress on Photosynthesis Research, Stresa, Vol. 3. pp. 2265 2287, Forti, G , Avron, M., Melandri, A., eds. The Hague: Dr. W. Junk N.V. 1972 Davis, D.J., Armond, P.A., Gross, E.L., Arntzen, C.J. : Differentiation of chloroplast lamellae. Onset of cation regulation of excitation energy distribution. Arch. Biochem. Biophys. 175, 64 70 (1976) Dubertret, G., Joliot, P.: Structure and organization of system II photosynthetic units during the greening of a dark-grown Chlorella mutant. Biochem. Biophys. Acta 357, 399-41l (1974) Ellis, R.J.: F~rther similarities between chloroplast a n d bacterial ribosomes. Planta 91, 329-335 (1970) Ellis, R.J. : Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamid. Phytochemistry 14, 89-93 (I975) Forger, J.M., Bogorad, L.: Steps in aquisition of photosynthetic
139 competence by plastids of maize. Plant Physiol. 52, 491-497 (1973) Hiller, R.G., Pilger, T.B.G., Genge, S.: Effect of lincomycin on the chlorophyll-protein complex I content and photosystem I activity of greening leaves. Biochim. Biophys. Acta 460, 431-444 (1977) Horvath, G., Garab, Gy.I., Mustfirdy, L.A., Halitsz, N., FaludiDfiniel, A. : The development of thylakoids and photochemical properties of mesophyll and bundle sheath chloroplasts of greening maize leaves. Plant Sci. Lett. 5, 239-244 (1975) Ichikawa, T., Inoue, Y., Shibata, K. : Delayed light emission and variable fluorescence from intermittently illuminated wheat leaves under continuous illumination related to activation of the latent water-splitting system. Plant Sci. Lett. 4, 369-376 (1975) Klimov, V.V., Lhng, F., Karapetyan, N.V., Krasnovsky, A.A. : Fluorescence induction during the greening of etiolated leaves : normal and mutant maize seedlings. Fiziol. Rast. 19, I51-159 (1972) Krause, G.H.: Changes in chlorophyll fluorescence in relation to light-dependent cation transfer across thylakoid membranes. Biochim. Biophys. Acta 333, 301 313 (1974) Mills, J., Barber, J.: Energy-dependent cation-induced control of chlorophyll a fluorescence in isolated intact chloroplasts. Arch. Biochem. Biophys. 170, 306-314 (1975) Mohanty, P., Govindjee : Light induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans. I. Relationship of slow fluorescence changes with structural changes. Biochim. Biophys. Acta 305, 95-104 (1973) Murata, N.: Control of excitation energy transfer in photosynthesis. V. Correlation membrane structure to regulation of excitation transfer between two pigment systems in isolated chloroplasts. Biochim Biophys. Acta 245, 365 372 (1971) Murata, N., Tashiro, H., Takamiya, A. : Effects of divalent metal ions on chlorophyll a fluorescence in isolated spinach chloroplasts. Biochim. Biophys. Acta 197, 250-256 (1970) Papageorgiou, G.: Chlorophyll fluorescence: An intrinsic probe of photosynthesis. In: Bioenergetics of photosynthesis, pp. 319-371. Govindjee, ed. New York-San Francisco-London : Academic Press 1975 Papageorgiou, G., Isaakidou, J., Argoudelis, C. : Structure dependent control of chlorophyll a excitation density: the role of oxygen. FEBS Lett. 25, 139-142 (1972) S~trvfiri, l~., HalS_sz, G., Nyitrai, P., Lfing, F.: Effect of lincocin treatment on the greening process in bean (Phaseolus vulgaris) leaves. Physiol. Plant. 36, 187-192 (I976) Schreiber, U., Fink, R., Vidaver, W.: Fluorescence induction in whoole leaves: Differentiation between the two sides and adaptatic>~ to differen~ light regimes. Planta 133, 121-129 (1977) Sokolove, P.M., Marsho, T.V.: Slow fluorescence quenching of type A chloroplasts resolution into two components. Biochim. Biophys. Acta 459, 27-35 (1977) Strasser, R.J., Sironval, C.: Correlation between the induction of oxygen evolution and of variable fluorescence in flashed bean leaves. Plant Sci. Lett. 3, 135 141 (1974) Teller, A., Barber, d., Nicolson, J.: Energy-dependent quenching of chlorophyll a fluorescence. Evidence for coupled cyclic electron flow in isolated intact chloroplasts. Plant Sci. Lett. 5, 171 176 (1975) Thomson, W.W., Ellis, R.J.: Inhibition of grana formation by lincomycin. Planta 108, 89-92 (1972) Vernotte, C., Briantais, J.-M., Arntzen, C.J.: Comparison of excitons transfers changes induced by cations and by adaptation in states I and II. In: Proc. 3rd, Int. Congress on Photosynthesis, Rehovot, pp, 183-193, Avron, M. ed. Amsterdam: Elsevier 1974 Received 11 January ; accepted 18 March 1978
