PhotosynthesisResearch 44: 271-275, 1995. © 1995KluwerAcademicPublishers. PrintedintheNetherlands. Regular paper
Chlorophyll fluorescence changes at high temperatures induced by linear heating of greening barley leaves Petr Ilfk 1, Jan Naug 1, D a n i e l Cik~inek 1 & R a d k o Novotn3) 2
1Department of Experimental Physics, Faculty of Science, Palaclo] University, 771 46 Olomouc, Czech Republic; 2Center for Microscopic Methods, Faculty of Medicine, Palacl~ University, L P. Pavlova 35, 775 20 Olomouc, Czech Republic Received 13 June 1994;acceptedin revisedform 13 April 1995
Key words: fluorescence temperature curve, LHC II, pigment-protein complex, Photosystem, thylakoid membrane
Abstract
A relative decrease of the high temperature part (above 60 ° C) of the chlorophyll fluorescence temperature curve during 3 h to 10 h greening period of barley (Hordeum vulgare L.) leaves was found to be concomitant to a decrease of Chl a/b ratio and to a gradual increase of LHCP/core ratio found by electrophoresis and the ratio of granal to total length of thylakoid membranes. It is suggested that the high temperature part of the fluorescence temperature curve depends inversely on the relative amount of LHC II in thylakoid membranes.
Abbreviations: Chl a(b) - chlorophyll a(b); CPa- chlorophyll a protein complex of PS II; CP 1 - P700 chlorophyll a protein complex of PS I; FP-free pigments; FTC-fluorescence temperature curve; F(T30)-fluorescence intensity at 30 °C; LHC II-light harvesting complex II; L H C P - light harvesting chlorophyll protein; LHCP3 (LHCPm)monomeric form of LHC II; LHCPo-oligomeric form of LHC II complex; M1-first maximum of FTC; M 2 second maximum (region) of FTC; PAA-polyacrylamide; PAR-photosynthetically active radiation; PS I(II)Photosystem I(II); SDS-PAGE-sodium dodecyl sulfate polyacrylamide gel electrophoresis Introduction
Upon linear heating from the room to higher temperatures and under weak or analytic light excitation of intact chloroplasts or whole leaves, a characteristic chlorophyll fluorescence response - fluorescence temperature curve (FTC) - is registered (Schreiber and Armond 1978; Downton and Berry 1982; Havaux 1992; Kuropatwa et al. 1992; Naug et al. 1992a, b etc.). Two characteristic peaks or regions of higher fluorescence intensity are discernible within FTC. The first peak (M1) located at lower temperatures (40--55 °C) has been thoroughly studied and used as an thermostability indicator of photosynthetic apparatus (Armond et al. 1978; Berry and BjOrkman 1980; Bilger et al. 1984). The notion that this maximum is caused by blocking of PS II electron transport (Schreiber and Armond
1978) was recently strongly supported by Bukhov et al. (1990). The second FTC region of increased fluorescence intensity (usually situated above 60 ° C) is still a matter of controversy. A relatively high second maximum (M2) of the FTC appeared in P S I enriched preparations (Fork 1976), in bundle sheath chloroplasts of C4 plants having non-appressed thylakoids and in Atriplex plants grown in higher light intensities (Downton and Berry 1982). All these samples were also characterized by a higher Chl a/b ratio. This led Downton and Berry (1982) to a suggestion that this FTC region can be related to PSI content in the studied material or directly to the emission of PS I. A high PS UPS II ratio was inferred for samples with high M2 region. At that time, this idea was supported by the paper of Melis and Brown (1980) where a correlation between Chl
272 a/b ratio and PS I/PS II stoichiometry was found for their spinach plants. Recent findings, however, indicate that neither the general correlation between M2 region and PS I/PS II stoichiometry nor between M2 region and Chl a/b region can be expected. The proportionality between Chl a/b ratio and PS I/PS II stoichiometry was not found for several plants species grown in different lights of different qualities and quantities (for review see Anderson et al. 1988 and Chow et al. 1990). Instead, a lower PS I/PS II ratio and higher Chl a/b have been found in leaves exposed to high light conditions as compared to the plants of the same species cultivated at low light (e.g. spinach, pea - see Anderson et al. 1988). These facts lead to a quite opposite expectation regarding the relations between the M2 relative intensity and the PS I/PS II stoichiometry than mentioned above, i.e. the M2 region should be higher for samples of lower PS UPS II ratio. Thus the discrepancies described above indicate that no unequivocal general relation between M2 FTC region and the photosystem stoichiometry is valid. Mannan et al. (1986) found also no general relation between the Chl a/b ratio and the high temperature part of FTC upon screening a series of species. The relative height of the M2 region was found to be species specific. However, a detailed inspection of literature data shows that within a particular species a relation is conserved. The plants grown in higher light intensities have a higher Chl a/b ratio and higher M2 FTC region in comparison with the same species grown in lower light intensities (Downton and Berry 1982 forAtriplex, Mannan et al. 1986 for Thunbergia grandiflora leaves and Spunda et al. 1993 for spruce needles). In this paper a connection between the relative intensity of the high temperature fluorescence (M2 region of the FTC) and the relative amount of LHC II complexes on chlorophyll basis in thylakoid membranes is demonstrated during greening of barley leaves.
Material and methods
Plant material Barley seedlings (Hordeum vulgare L. cv. Akcent) were cultivated 5.5 days in peat substrate at 25 °C in dark. The etiolated seedlings were transferred to continuous white light and greened at 24 4- 1 °C. The light intensity was about 90 #mol m -2 s -1 of PAR at the
top of the seedlings. After several periods of illumination, the 1.5 cm long middle parts of the primary leaf (1.5 cm below the top) were used for various measurements.
Fluorescence temperature curve (FTC) A laboratory made spectrofluorimeter, with computer driven linear heating of samples, was used for FTC measurements (Nau~ et al. 1992a). The leaf segment was immersed in distilled water and heated at a rate of 4 °C/min up to 70 °C. Weak actinic light (about 6 #mol m -2 s -1) of 436 nm with 15 nm spectral half-width was used for the chlorophyll fluorescence excitation. Fluorescence intensity was detected at 685 nm with spectral half-width of 4 nm.
Electrophoretic separation (SDS-PA GE) of PPC Thylakoids from greening leaves were isolated according to Argyroudi-Akoyunoglou and Akoyunoglou (1979) with small modifications. Middle parts of leaves were homogenised in liquid nitrogen and subsequently in cold homogenisation buffer. The slurry was filtered through nylon mesh (diameter 50 #m). The suspension was centrifuged at 3000 x g for 8 min. The thylakoids were resuspended and washed once in 5 ml of cold homogenisation buffer, collected at 1500 x g for 10 min and than resuspended in 0.5 ml of 50 mM HEPES buffer (pH 8.0). Plastid membranes for mild SDS-PAGE were prepared according to Burkey (1986) with the exception of using HEPES buffer instead of the phosphate buffer. The thylakoid pellet was resuspended just before electrophoretic run with ice cold solubilization buffer (0.3 M Tris-HC1, pH 8.8, 10% (v/v) glycerol, 1% (w/v) SDS). Constant SDS/Chl ratio of 40 was used. The contents of Chl a and Chl b were determined spectrophotometrically in 80% acetone according to Lichtenthaler (1987). Insoluble white matter was removed by short centrifugation (1 rain at 15 000 x g). The electrophoresis was run according to Anderson (1980). Gel scans were recorded at 675 nm and 750 nm (background) on a modified Schnellphotometer G U (Carl Zeiss, Germany). Individual bands were identified according to their absorption spectra and molecular weight of apoproteins.
273
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Fig. 1. Fluorescence temperature curves (FTC) detected in different phases of barley greening (the period of greening is shown at each curve, 'green' represents the FTC of fully green barley leaf) with detection at 685 nm under excitation at 436 nm. All PTCs are normalised for fluorescenceintensity at 30 oC - F(T30). Linear heating at rate of 4 oC/min. Very similar FTC changes were observed during five independent greening processes.
°°O, 2
i 0
Transmission electron microscopy The middle part o f the primary leaf was placed in 3% (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH = 7.2. After rinsing in phosphate buffer and postfixing in 1% (w/v) OsO4 in the same buffer, samples were dehydrated in ethanol series and propylene oxide and embedded in Durcupan ACM. Thin sections were stained with uranyl acetate and Reynold's lead citrate. Samples were examined under the transmission electron microscope Tesla BS 613 (Brno, Czech Republic) at an accelerating voltage o f 80 kV, and direct magnification of 16 000 and 22 000. The ultrastructural chloroplast parameter - granal/total thylakoid length - was measured by means of the computer program Videoplan ( K O N T R O N Elektronik GmbH, Germany).
Results and discussion
Fluorescence temperature curves of initially etiolated barley leaves were measured at various stages of greening process (from 3rd to 10th hour). The increasing M 1 peak and its shift to higher temperatures occurring during the greening (Fig. 1) can be explained by improving
8 'v -I
0 2
4
6
8
10
time of greening [h]
F/g. 2. FTC parameter M2/F(T30) (A), Chl a/b ratio (B) during greening of barley leaves. Ratio of granal to total thylakoid length (C) at various phases of barley greening was determined from the electron micrographs.The mean values and SD were calculated from 10 - 20 chloroplast thin sections. Ratio of LHCP/core (D) during greening was estimated from densitometric scans of gels (Fig. 3), as a ratio of Chl a amount in LHCP (LHCPm + LHCPo) to a sum of Chl a in CP1 and CPa complexes.
photochemical function of PS II (results from fluorescence induction measurements not shown) and their thermal stabilisation (Armond et al. 1978, etc.). During the greening a gradual decrease of the relative height of the M2 region appeared (Fig. 1). The onset of fluorescence increase to M2 region concomitantly shifted to lower temperatures and fluorescence decrease from
274 E c
CPI
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LHCPm
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FP
LHCPo
< 0.2' m
Chlorophyll fluorescence changes at high temperatures induced by linear heating of greening barley leaves Petr Ilfk 1, Jan Naug 1, D a n i e l Cik~inek 1 & R a d k o Novotn3) 2
1Department of Experimental Physics, Faculty of Science, Palaclo] University, 771 46 Olomouc, Czech Republic; 2Center for Microscopic Methods, Faculty of Medicine, Palacl~ University, L P. Pavlova 35, 775 20 Olomouc, Czech Republic Received 13 June 1994;acceptedin revisedform 13 April 1995
Key words: fluorescence temperature curve, LHC II, pigment-protein complex, Photosystem, thylakoid membrane
Abstract
A relative decrease of the high temperature part (above 60 ° C) of the chlorophyll fluorescence temperature curve during 3 h to 10 h greening period of barley (Hordeum vulgare L.) leaves was found to be concomitant to a decrease of Chl a/b ratio and to a gradual increase of LHCP/core ratio found by electrophoresis and the ratio of granal to total length of thylakoid membranes. It is suggested that the high temperature part of the fluorescence temperature curve depends inversely on the relative amount of LHC II in thylakoid membranes.
Abbreviations: Chl a(b) - chlorophyll a(b); CPa- chlorophyll a protein complex of PS II; CP 1 - P700 chlorophyll a protein complex of PS I; FP-free pigments; FTC-fluorescence temperature curve; F(T30)-fluorescence intensity at 30 °C; LHC II-light harvesting complex II; L H C P - light harvesting chlorophyll protein; LHCP3 (LHCPm)monomeric form of LHC II; LHCPo-oligomeric form of LHC II complex; M1-first maximum of FTC; M 2 second maximum (region) of FTC; PAA-polyacrylamide; PAR-photosynthetically active radiation; PS I(II)Photosystem I(II); SDS-PAGE-sodium dodecyl sulfate polyacrylamide gel electrophoresis Introduction
Upon linear heating from the room to higher temperatures and under weak or analytic light excitation of intact chloroplasts or whole leaves, a characteristic chlorophyll fluorescence response - fluorescence temperature curve (FTC) - is registered (Schreiber and Armond 1978; Downton and Berry 1982; Havaux 1992; Kuropatwa et al. 1992; Naug et al. 1992a, b etc.). Two characteristic peaks or regions of higher fluorescence intensity are discernible within FTC. The first peak (M1) located at lower temperatures (40--55 °C) has been thoroughly studied and used as an thermostability indicator of photosynthetic apparatus (Armond et al. 1978; Berry and BjOrkman 1980; Bilger et al. 1984). The notion that this maximum is caused by blocking of PS II electron transport (Schreiber and Armond
1978) was recently strongly supported by Bukhov et al. (1990). The second FTC region of increased fluorescence intensity (usually situated above 60 ° C) is still a matter of controversy. A relatively high second maximum (M2) of the FTC appeared in P S I enriched preparations (Fork 1976), in bundle sheath chloroplasts of C4 plants having non-appressed thylakoids and in Atriplex plants grown in higher light intensities (Downton and Berry 1982). All these samples were also characterized by a higher Chl a/b ratio. This led Downton and Berry (1982) to a suggestion that this FTC region can be related to PSI content in the studied material or directly to the emission of PS I. A high PS UPS II ratio was inferred for samples with high M2 region. At that time, this idea was supported by the paper of Melis and Brown (1980) where a correlation between Chl
272 a/b ratio and PS I/PS II stoichiometry was found for their spinach plants. Recent findings, however, indicate that neither the general correlation between M2 region and PS I/PS II stoichiometry nor between M2 region and Chl a/b region can be expected. The proportionality between Chl a/b ratio and PS I/PS II stoichiometry was not found for several plants species grown in different lights of different qualities and quantities (for review see Anderson et al. 1988 and Chow et al. 1990). Instead, a lower PS I/PS II ratio and higher Chl a/b have been found in leaves exposed to high light conditions as compared to the plants of the same species cultivated at low light (e.g. spinach, pea - see Anderson et al. 1988). These facts lead to a quite opposite expectation regarding the relations between the M2 relative intensity and the PS I/PS II stoichiometry than mentioned above, i.e. the M2 region should be higher for samples of lower PS UPS II ratio. Thus the discrepancies described above indicate that no unequivocal general relation between M2 FTC region and the photosystem stoichiometry is valid. Mannan et al. (1986) found also no general relation between the Chl a/b ratio and the high temperature part of FTC upon screening a series of species. The relative height of the M2 region was found to be species specific. However, a detailed inspection of literature data shows that within a particular species a relation is conserved. The plants grown in higher light intensities have a higher Chl a/b ratio and higher M2 FTC region in comparison with the same species grown in lower light intensities (Downton and Berry 1982 forAtriplex, Mannan et al. 1986 for Thunbergia grandiflora leaves and Spunda et al. 1993 for spruce needles). In this paper a connection between the relative intensity of the high temperature fluorescence (M2 region of the FTC) and the relative amount of LHC II complexes on chlorophyll basis in thylakoid membranes is demonstrated during greening of barley leaves.
Material and methods
Plant material Barley seedlings (Hordeum vulgare L. cv. Akcent) were cultivated 5.5 days in peat substrate at 25 °C in dark. The etiolated seedlings were transferred to continuous white light and greened at 24 4- 1 °C. The light intensity was about 90 #mol m -2 s -1 of PAR at the
top of the seedlings. After several periods of illumination, the 1.5 cm long middle parts of the primary leaf (1.5 cm below the top) were used for various measurements.
Fluorescence temperature curve (FTC) A laboratory made spectrofluorimeter, with computer driven linear heating of samples, was used for FTC measurements (Nau~ et al. 1992a). The leaf segment was immersed in distilled water and heated at a rate of 4 °C/min up to 70 °C. Weak actinic light (about 6 #mol m -2 s -1) of 436 nm with 15 nm spectral half-width was used for the chlorophyll fluorescence excitation. Fluorescence intensity was detected at 685 nm with spectral half-width of 4 nm.
Electrophoretic separation (SDS-PA GE) of PPC Thylakoids from greening leaves were isolated according to Argyroudi-Akoyunoglou and Akoyunoglou (1979) with small modifications. Middle parts of leaves were homogenised in liquid nitrogen and subsequently in cold homogenisation buffer. The slurry was filtered through nylon mesh (diameter 50 #m). The suspension was centrifuged at 3000 x g for 8 min. The thylakoids were resuspended and washed once in 5 ml of cold homogenisation buffer, collected at 1500 x g for 10 min and than resuspended in 0.5 ml of 50 mM HEPES buffer (pH 8.0). Plastid membranes for mild SDS-PAGE were prepared according to Burkey (1986) with the exception of using HEPES buffer instead of the phosphate buffer. The thylakoid pellet was resuspended just before electrophoretic run with ice cold solubilization buffer (0.3 M Tris-HC1, pH 8.8, 10% (v/v) glycerol, 1% (w/v) SDS). Constant SDS/Chl ratio of 40 was used. The contents of Chl a and Chl b were determined spectrophotometrically in 80% acetone according to Lichtenthaler (1987). Insoluble white matter was removed by short centrifugation (1 rain at 15 000 x g). The electrophoresis was run according to Anderson (1980). Gel scans were recorded at 675 nm and 750 nm (background) on a modified Schnellphotometer G U (Carl Zeiss, Germany). Individual bands were identified according to their absorption spectra and molecular weight of apoproteins.
273
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...........
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5h
B
3h e-
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2
4
.........i- - 7 - \ 25
35
45
55
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65
75
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temperature [*C] 0,4 ¸
Fig. 1. Fluorescence temperature curves (FTC) detected in different phases of barley greening (the period of greening is shown at each curve, 'green' represents the FTC of fully green barley leaf) with detection at 685 nm under excitation at 436 nm. All PTCs are normalised for fluorescenceintensity at 30 oC - F(T30). Linear heating at rate of 4 oC/min. Very similar FTC changes were observed during five independent greening processes.
°°O, 2
i 0
Transmission electron microscopy The middle part o f the primary leaf was placed in 3% (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH = 7.2. After rinsing in phosphate buffer and postfixing in 1% (w/v) OsO4 in the same buffer, samples were dehydrated in ethanol series and propylene oxide and embedded in Durcupan ACM. Thin sections were stained with uranyl acetate and Reynold's lead citrate. Samples were examined under the transmission electron microscope Tesla BS 613 (Brno, Czech Republic) at an accelerating voltage o f 80 kV, and direct magnification of 16 000 and 22 000. The ultrastructural chloroplast parameter - granal/total thylakoid length - was measured by means of the computer program Videoplan ( K O N T R O N Elektronik GmbH, Germany).
Results and discussion
Fluorescence temperature curves of initially etiolated barley leaves were measured at various stages of greening process (from 3rd to 10th hour). The increasing M 1 peak and its shift to higher temperatures occurring during the greening (Fig. 1) can be explained by improving
8 'v -I
0 2
4
6
8
10
time of greening [h]
F/g. 2. FTC parameter M2/F(T30) (A), Chl a/b ratio (B) during greening of barley leaves. Ratio of granal to total thylakoid length (C) at various phases of barley greening was determined from the electron micrographs.The mean values and SD were calculated from 10 - 20 chloroplast thin sections. Ratio of LHCP/core (D) during greening was estimated from densitometric scans of gels (Fig. 3), as a ratio of Chl a amount in LHCP (LHCPm + LHCPo) to a sum of Chl a in CP1 and CPa complexes.
photochemical function of PS II (results from fluorescence induction measurements not shown) and their thermal stabilisation (Armond et al. 1978, etc.). During the greening a gradual decrease of the relative height of the M2 region appeared (Fig. 1). The onset of fluorescence increase to M2 region concomitantly shifted to lower temperatures and fluorescence decrease from
274 E c
CPI
greer
r-.
LHCPm
to 0,4."
FP
LHCPo
< 0.2' m
