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Luminescence techniques for monitoring of forest decline
190
NEWS AND VIEWS
1 Pursuant to Article 6 of the Montreal Protocol on Substances that Deplete the Ozone Layer under the Auspices of the United Nations Environment Programme in J. C. van der Leun and M. Tevini (eds.), Environmental Effects Panel Report, United Nations Environment Programme, Nairobi, 1989.
Luminescence Herbert
techniques
for monitoring
of forest
decline
Schneckenburger
Fachhochschule Aafen, Fachbereich Optoe~ek~on~ 3eethove~trusse, I, W-7080 Aalen, and I~t~tut j2r Lase~e~hnolo~en in der redden an der Unive~it~t Ulm, Postfach 4066, W-79~ (~e~any)
Werner
Utm
Schmidt
Fachhochschule Aalen, Fachbereich Optoelektronik, Beethovenstrasse I, W-7080 Aalen, and Universitir’t Konstanz, Fakultiit ji2r Biologic, W-7750 Konstanz (Germany)
Trees are submitted to various stress factors such as environmental pollutants, mineral deficiencies, climate or parasite infection, giving rise to the current phenomenon of “forest decline”. To assess the diverse impacts of these factors, luminescence techniques offer many advantages. They are fast, non-invasive and highly selective for specific plant pigments, and they require only small samples. Apart from some blue and green fluorescent pigments which are mainly localized within the stomata [I], the luminescence of the chlorophyll antenna molecules of the two photosystems may be used as a measure of vitality. Previous measurements include the following. (1) The detection of erosion spectra and kinetics under continuo~ illu~ina~on [Z]. The so-called “Kautsky measurements” are based on the fact that the intensi~ of chlorophyll fluorescence of dark adapted organisms increases to a maximum fmax at the beginning of light exposure and then - during the onset of photosynthesis decreases within about 5 min to a steady state value fs.The ratio (f,~-fJ)lfs=f& may therefore reflect the photosynthetic activity. This ratio, however, is only a “global measure” including all steps of photosynthesis. (2) Picosecond spectroscopy. The primary steps of energy transfer within the photosystems are selected [3-51. A longer fluorescence decay time is expected if the energy transfer from the antenna molecules to the reaction centres is obstructed. (3) The detection of delayed luminescence. This may be due to a “reverse electron flow” in the thylakoid membrane and seems to reflect the membrane potential [6]. To measure photosynthetic defects in spruce needles we developed appropriate apparatuses including a fibre-optic fluorescence sensor [7], picosecond devices with ultrafast laser diodes and single-photon detection [l, 81 and two set-ups for measuring the induction and decay of long-term delayed luminescence (LDL). The induction curve was obtained by quasi-continuous excitation of dark adapted needles with about 1000 pulses of a sequent-doubIed ne~~ium-doped yttrium aluminium garnet laser and subsequent luminescence detection within a time window of 10-90 ms after each laser pulse; the LDL decay was measured in the second range after a single light shot.
NEWS AND VIEWS
191
Samples were taken from individual spruces belonging to different damage classes and accessible from high wooden structures (Freudenstadt, Black Forest), as well as from healthy-looking spruces in so-called open-top chambers, where air pollutants such as SO*, NO, and O3 could be excluded (Edelmannshof, Welzheimer Wald; part of the spruces were exposed to filtered, and another part to environmental air). All measurements were carried out between September 1989 and October 1991 with green needles without any visible defects. Picosecond fluorescence decay curves detected at 695-800 nm showed a superposition of three exponentially decaying ~mponents with time constants I; = MO-200 ps, I’*= 300-500 ps and T3 =2.0-3.5 ns. In agreement with literature [3, 51 the latter was attributed to chlorophyll molecules associated with “closed” reaction centres mainly of photosystem II. The relative intensity 1, of this long-lived component showed a pronounced seasonal course with values ranging between 2-S% in winter and 3-15% in summer. This implies that the spruces are submitted to more stress - possibly owing to high light doses, drought or increased ozone concentrations - during the summer period. As demonstrated in Fig. 1, higher intensities Is were obtained from spruces attributed to damage class 1 and 3 than from those of damage class 0. Even two of the healthy-looking spruces at Edelmannshof showed increased & values in summer 1990. This was an early indication of drought of one spruce and parasite infection of the other (observed in September of the same year) which seemed to be independent of environmental pollutants. Stationary measurements of the induction and decrease of chlorophyll fluorescence (“Kautsky curves”) showed pronounced variations in the ratio fd/fswith the season, as well as the location and vitality of each tree IS]. These ratios usually flu~~ated between 2.5-3.5 in winter and spring and 3.5-5.0 in summer and autumn. The lowest ratios were obtained for the most damaged spruces at Freudenstadt, as well as for
10
T---------
~-
I damage
1
class
2
~-----
3
6
damage
+
---_
-~~-
7
class
12
1
31
_
_._
damage
*
13
14
15
_---..-
class
16
-__
0
I7
24
Spruce No. Fig. 1. Relative intensity of the long-lived component of chlorophyll fluorescence obtained from about ten green needles of the second age class of 12 spruces with different degrees of damage (Freudenstadt; March 1991). The reproducibility of individual values was within flO%.
NEWS AND VIJZWS
192
the most stressed trees at Edelmannshof in summer 1990. Similar information as from picosecond kinetics may be deduced from this kind of measurement; however, the differences between the individual spruces appear less pronounced than those obtained from the picosecond decay curves. LDL showed a three-exponential decay pattern (similar to that observed in the picosecond fluorescence decay) after excitation by a single red light pulse (high-pass filter; A> 64.5 nm) according to the equation An&
=Af
exp(
-
W
+A,
eq(
-
kJ)
+A
exp(
-W
with the decay rates kf, k, and k, and the corresponding amplitudes Af, A,,, and A, respectively [9]. Size and seasonal for the “fast”, “middle” and “slow” components variations were found to be most pronounced for the amplitudes Af, and less for A, and A,. The Af variations were typically up to a factor of 4 for the healthy spruces and only about 2 for the most declined spruces (ratio of summer to winter values). The reaction constants k,,, and k, barely depend on the season and damage class. Another clear difference between spruces of the damage classes 0,l and 3 was observed for the induction kinetics (Fig. 2). After dark adaptation the delayed Iumines~nce increased to a maximum after about 10 s before declining asymptotically to a steady state value. Both the maximum intensity and the ratio of the maximum to the steady state value were lowest for the most declined spruces 1 and 2 and highest for the “most healthy” spruce 13. The time shift of the maximum of spruce 24 remains to be investigated further. In spite of some unresolved questions it may be concluded that the various luminescence techniques presented, i.e. Kautsky kinetics, picosecond spectroscopy and
19.2
I
I
I
I
time
[s]
Induction kinetics CDL}
38.4
44.8
51.2
57.6
Fig. 2. Induction kinetics of delayed luminescence of various trees belonging to damage class 0 (spruces 13, 17 and 24), 1 (spruce 31) and 3 (spruces 1 and 2) at the location Freudenstadt.
NEWS AND VIEWS
193
delayed luminescence (in the long-term seconds range and as induction kinetics) offer a valuable tool for diagnosis of photosynthetic organisms such as conifers, particularly in an early and invisible state of damage. 1 H. Schneckenburger and W. Schmidt, Fluorescence
2 3
4 5
6
in forest decline studies, in 0. S. Wolfbeis (ed.), Methods and Applications of Fluorescence Spectroscopy, Springer, Berlin, 1992, in the press. H. K. Lichtenthaler, C. Buschmann, U. Rinderle and G. Schmuck, Applications of chlorophyll fluorescence in ecophysiology, Radiat. Environ. Biophys., 25 (1986) 297-308. A. R. Holzwarth, Time-resolved chlorophyll fluorescence - what kind of information does it provide? in H. K. Lichtenthaler (ed.), Applications of Chlorophyll Fluorescence, Kluwer, Dordrecht, 1988, pp. 21-31. M. Hodges and I. Moya, Time resolved chlorophyll fluorescence studies on pigment-protein complexes from photosynthetic membranes, Biochim. Biophys. Acta, 935 (1988) 41-52. R. Sparrow, E. H. Evans, R. G. Brown and D. Shaw, Time-resolved spectroscopy of photosynthetic systems using synchrotron radiation: photosystem II preparations from lettuce, J. Photochem. Photobiol. B: Biol., 3 (1989) 65-69. J. Barber, Stimulation of millisecond delayed light emission by KC1 and NaCl gradients as a means of investigating the ionic permeability properties of the thylakoid membranes, Biochim. Biophys. Acta, 275 (1972) 105-116.
7 H. Schneckenburger, W. Schmidt, P. Hammer, K. Hudelmaier and R. Pfeifer, Fiber-optic fluorescence sensor for in-vivo measurements of photosynthetic defects, in W. Waidelich (ed.), Optoelectronics in Engineering, Springer, Berlin, 1990, pp. 403407. 8 H. Schneckenburger and W. Schmidt, Time-resolving luminescence techniques for possible detection of forest decline (II) Picosecond chlorophyll fluorescence, Radiut. Environ. Biophys., 31 (1992) 73-81. 9 W. Schmidt and H. Schneckenburger, Time-resolving luminescence techniques for possible detection of forest decline (I) Long term delayed luminescence, Radiut. Environ. Biophys., 31 (1992) 63-72.
NEWS AND VIEWS
1 Pursuant to Article 6 of the Montreal Protocol on Substances that Deplete the Ozone Layer under the Auspices of the United Nations Environment Programme in J. C. van der Leun and M. Tevini (eds.), Environmental Effects Panel Report, United Nations Environment Programme, Nairobi, 1989.
Luminescence Herbert
techniques
for monitoring
of forest
decline
Schneckenburger
Fachhochschule Aafen, Fachbereich Optoe~ek~on~ 3eethove~trusse, I, W-7080 Aalen, and I~t~tut j2r Lase~e~hnolo~en in der redden an der Unive~it~t Ulm, Postfach 4066, W-79~ (~e~any)
Werner
Utm
Schmidt
Fachhochschule Aalen, Fachbereich Optoelektronik, Beethovenstrasse I, W-7080 Aalen, and Universitir’t Konstanz, Fakultiit ji2r Biologic, W-7750 Konstanz (Germany)
Trees are submitted to various stress factors such as environmental pollutants, mineral deficiencies, climate or parasite infection, giving rise to the current phenomenon of “forest decline”. To assess the diverse impacts of these factors, luminescence techniques offer many advantages. They are fast, non-invasive and highly selective for specific plant pigments, and they require only small samples. Apart from some blue and green fluorescent pigments which are mainly localized within the stomata [I], the luminescence of the chlorophyll antenna molecules of the two photosystems may be used as a measure of vitality. Previous measurements include the following. (1) The detection of erosion spectra and kinetics under continuo~ illu~ina~on [Z]. The so-called “Kautsky measurements” are based on the fact that the intensi~ of chlorophyll fluorescence of dark adapted organisms increases to a maximum fmax at the beginning of light exposure and then - during the onset of photosynthesis decreases within about 5 min to a steady state value fs.The ratio (f,~-fJ)lfs=f& may therefore reflect the photosynthetic activity. This ratio, however, is only a “global measure” including all steps of photosynthesis. (2) Picosecond spectroscopy. The primary steps of energy transfer within the photosystems are selected [3-51. A longer fluorescence decay time is expected if the energy transfer from the antenna molecules to the reaction centres is obstructed. (3) The detection of delayed luminescence. This may be due to a “reverse electron flow” in the thylakoid membrane and seems to reflect the membrane potential [6]. To measure photosynthetic defects in spruce needles we developed appropriate apparatuses including a fibre-optic fluorescence sensor [7], picosecond devices with ultrafast laser diodes and single-photon detection [l, 81 and two set-ups for measuring the induction and decay of long-term delayed luminescence (LDL). The induction curve was obtained by quasi-continuous excitation of dark adapted needles with about 1000 pulses of a sequent-doubIed ne~~ium-doped yttrium aluminium garnet laser and subsequent luminescence detection within a time window of 10-90 ms after each laser pulse; the LDL decay was measured in the second range after a single light shot.
NEWS AND VIEWS
191
Samples were taken from individual spruces belonging to different damage classes and accessible from high wooden structures (Freudenstadt, Black Forest), as well as from healthy-looking spruces in so-called open-top chambers, where air pollutants such as SO*, NO, and O3 could be excluded (Edelmannshof, Welzheimer Wald; part of the spruces were exposed to filtered, and another part to environmental air). All measurements were carried out between September 1989 and October 1991 with green needles without any visible defects. Picosecond fluorescence decay curves detected at 695-800 nm showed a superposition of three exponentially decaying ~mponents with time constants I; = MO-200 ps, I’*= 300-500 ps and T3 =2.0-3.5 ns. In agreement with literature [3, 51 the latter was attributed to chlorophyll molecules associated with “closed” reaction centres mainly of photosystem II. The relative intensity 1, of this long-lived component showed a pronounced seasonal course with values ranging between 2-S% in winter and 3-15% in summer. This implies that the spruces are submitted to more stress - possibly owing to high light doses, drought or increased ozone concentrations - during the summer period. As demonstrated in Fig. 1, higher intensities Is were obtained from spruces attributed to damage class 1 and 3 than from those of damage class 0. Even two of the healthy-looking spruces at Edelmannshof showed increased & values in summer 1990. This was an early indication of drought of one spruce and parasite infection of the other (observed in September of the same year) which seemed to be independent of environmental pollutants. Stationary measurements of the induction and decrease of chlorophyll fluorescence (“Kautsky curves”) showed pronounced variations in the ratio fd/fswith the season, as well as the location and vitality of each tree IS]. These ratios usually flu~~ated between 2.5-3.5 in winter and spring and 3.5-5.0 in summer and autumn. The lowest ratios were obtained for the most damaged spruces at Freudenstadt, as well as for
10
T---------
~-
I damage
1
class
2
~-----
3
6
damage
+
---_
-~~-
7
class
12
1
31
_
_._
damage
*
13
14
15
_---..-
class
16
-__
0
I7
24
Spruce No. Fig. 1. Relative intensity of the long-lived component of chlorophyll fluorescence obtained from about ten green needles of the second age class of 12 spruces with different degrees of damage (Freudenstadt; March 1991). The reproducibility of individual values was within flO%.
NEWS AND VIJZWS
192
the most stressed trees at Edelmannshof in summer 1990. Similar information as from picosecond kinetics may be deduced from this kind of measurement; however, the differences between the individual spruces appear less pronounced than those obtained from the picosecond decay curves. LDL showed a three-exponential decay pattern (similar to that observed in the picosecond fluorescence decay) after excitation by a single red light pulse (high-pass filter; A> 64.5 nm) according to the equation An&
=Af
exp(
-
W
+A,
eq(
-
kJ)
+A
exp(
-W
with the decay rates kf, k, and k, and the corresponding amplitudes Af, A,,, and A, respectively [9]. Size and seasonal for the “fast”, “middle” and “slow” components variations were found to be most pronounced for the amplitudes Af, and less for A, and A,. The Af variations were typically up to a factor of 4 for the healthy spruces and only about 2 for the most declined spruces (ratio of summer to winter values). The reaction constants k,,, and k, barely depend on the season and damage class. Another clear difference between spruces of the damage classes 0,l and 3 was observed for the induction kinetics (Fig. 2). After dark adaptation the delayed Iumines~nce increased to a maximum after about 10 s before declining asymptotically to a steady state value. Both the maximum intensity and the ratio of the maximum to the steady state value were lowest for the most declined spruces 1 and 2 and highest for the “most healthy” spruce 13. The time shift of the maximum of spruce 24 remains to be investigated further. In spite of some unresolved questions it may be concluded that the various luminescence techniques presented, i.e. Kautsky kinetics, picosecond spectroscopy and
19.2
I
I
I
I
time
[s]
Induction kinetics CDL}
38.4
44.8
51.2
57.6
Fig. 2. Induction kinetics of delayed luminescence of various trees belonging to damage class 0 (spruces 13, 17 and 24), 1 (spruce 31) and 3 (spruces 1 and 2) at the location Freudenstadt.
NEWS AND VIEWS
193
delayed luminescence (in the long-term seconds range and as induction kinetics) offer a valuable tool for diagnosis of photosynthetic organisms such as conifers, particularly in an early and invisible state of damage. 1 H. Schneckenburger and W. Schmidt, Fluorescence
2 3
4 5
6
in forest decline studies, in 0. S. Wolfbeis (ed.), Methods and Applications of Fluorescence Spectroscopy, Springer, Berlin, 1992, in the press. H. K. Lichtenthaler, C. Buschmann, U. Rinderle and G. Schmuck, Applications of chlorophyll fluorescence in ecophysiology, Radiat. Environ. Biophys., 25 (1986) 297-308. A. R. Holzwarth, Time-resolved chlorophyll fluorescence - what kind of information does it provide? in H. K. Lichtenthaler (ed.), Applications of Chlorophyll Fluorescence, Kluwer, Dordrecht, 1988, pp. 21-31. M. Hodges and I. Moya, Time resolved chlorophyll fluorescence studies on pigment-protein complexes from photosynthetic membranes, Biochim. Biophys. Acta, 935 (1988) 41-52. R. Sparrow, E. H. Evans, R. G. Brown and D. Shaw, Time-resolved spectroscopy of photosynthetic systems using synchrotron radiation: photosystem II preparations from lettuce, J. Photochem. Photobiol. B: Biol., 3 (1989) 65-69. J. Barber, Stimulation of millisecond delayed light emission by KC1 and NaCl gradients as a means of investigating the ionic permeability properties of the thylakoid membranes, Biochim. Biophys. Acta, 275 (1972) 105-116.
7 H. Schneckenburger, W. Schmidt, P. Hammer, K. Hudelmaier and R. Pfeifer, Fiber-optic fluorescence sensor for in-vivo measurements of photosynthetic defects, in W. Waidelich (ed.), Optoelectronics in Engineering, Springer, Berlin, 1990, pp. 403407. 8 H. Schneckenburger and W. Schmidt, Time-resolving luminescence techniques for possible detection of forest decline (II) Picosecond chlorophyll fluorescence, Radiut. Environ. Biophys., 31 (1992) 73-81. 9 W. Schmidt and H. Schneckenburger, Time-resolving luminescence techniques for possible detection of forest decline (I) Long term delayed luminescence, Radiut. Environ. Biophys., 31 (1992) 63-72.