Blue, green, and red fluorescence emission signatures of green, etiolated, and white leaves

REMOTE SENS. ENVIRON. 47:65-71 (1994)

Blue, Green, and Red Fluorescence Emission Signatures of Green, Etiolated, and White Leaves Fred Stober,* Michael Lang,* and Hartmut K. Lichtenthaler* Laser-induced fluorescence emission of green terrestrial vegetation has become of high interest in remote LIDAR techniques in recent years. The remote sensing of blue and red fluorescence signatures appears to be a suitable future tool to determine the state of health of plants. In this contribution we report about major factors which determine the intensities of the UV-laser (337 nm) excited blue fluorescence near 450 nm and the red chlorophyll fluorescence between 650 nm and 800 nm as well as the ratio of the blue to the red fluorescence F450/F690. In contrast to green plants grown in a phytochamber or at low light intensities, plants grown in the field at a high photon flux density (PFD) showed high values for the ratio of the blue to the red fluorescence F450/F690, which was due to a relatively low intensity of the red chlorophyll fluorescence. The strongly increased ratio F450/F690 was primarily caused by a much reduced penetration depth of the exciting UV light into the leaf, which seemed to be due to substances absorbing in the epidermal layers. Etiolated wheat leaves exhibited a stronger blue fluorescence intensity than green leaves and showed also a small maximum around 530 nm (green fluorescence). White leaves of wheat treated with the bleaching herbicide norfluorazone (10 -4 M) and leaves from which the photosynthetic pigments had been extracted by acetone showed a fourfold and tenfold increase of the blue fluorescence, respectively. Isolated chloroplasts and thylakoids did not exhibit a blue-green fluorescence. It is concluded that the blue fluorescence is mainly caused by phenolic plant substances located in the cell wall and / or vacuoles of leaves. Partial reabsorption of the emitted

*Botanisches Institut II, Universit/it Karlsruhe, Germany Address correspondence to Prof. Dr. Hartmut K. Lichtenthaler, Botanisches Institut II, Universit/it Karlsruhe, Kaiserstr. 12, D-76128 Karlsruhe, Germany. Received 9 September 1992; revised 1 May 1993.

0034-4257 / 94 / $6.00 ©Elsevier Science Inc., 1994 655 Avenue of the Americas, New York, NY 10010

blue and red fluorescence by the photosynthetic pigments modifies the shape of the fluorescence spectra. INTRODUCTION Fluorescence LIDAR techniques have attained large interest in recent years with respect to the clarification of the state of health of terrestrial vegetation. In contrast to passive reflectance measurements, the fluorescence LIDARs as active remote sensing techniques use laser as excitation source and are independent of sunlight. They can complement the information on plants obtained by reflectance measurements. Their developement is the topic of the EUREKA Research Program LASFLEUR for remote-sensing the state of health of plants in agriculture and forestry (G/inther et al., 1991; Lichtenthaler et al., 1992). UV-laser-illuminated leaves not only emit a red chlorophyll fluorescence, they also show blue and green fluorescence emission (Chappelle et al., 1984; Lichtenthaler and Stober, 1990). The red chlorophyll fluorescence spectra of green leaves, which has been used in photosynthesis research for 60 years (Kautsky and Hirsch, 1934; Lichtenthaler and Rinderle, 1988; Krause and Weis, 1984), possess two maxima near 690 nm (F690) and 735 nm (F735) (Lichtenthaler and Buschmann, 1987; Lichtenthaler and Rinderle, 1988). The ratio of the chlorophyll fluorescence maxima F690 / F735 can be used as a stress indicator of the photosynthetic apparatus and can be applied as a nondestructive indicator of the in vivo chlorophyll content (Hak et al., 1990; D'Ambrosio et al., 1992). The blue fluorescence of green vegetation, which was detected as a genuine plant signature almost 60 years ago (Kautsky and Hirsch, 1934), possesses a maximum near 450 nm (F450) and often a shoulder in the green region near 530 nm (F530). It was redetected by Chappelle et al. (1984). The nature of the blue fluorescing substance(s) is not fully clear,

65

66 Stober et al.

but there is agreement that various plant phenolics are the major responsible compounds (Goulas et al., 1990; Lang et al., 1991; Lichtenthaler et al., 1991a)./~-Carotene, NADPH, and riboflavine are discussed as possible candidates by Chappelle et al. (1991); purified ]~-carotene, however, does not show any blue or green fluorescence (Lang et al., 1991). The ratio of the blue to red fluorescence F450/ F690 can differ from plant type to plant type due to growth conditions; thus it appears to be a suitable stress indicator of plants and is accessible for remote sensing (Chappelle et al., 1985; Giinther et al., 1991; Lichtenthaler et al., 1991b; 1992). First reports indicate that, under increasing stress conditions, the ratio F450 / F690 can increase (Lichteflthaler et al., 1991a). With respect to the active remote sensing of the blue and red fluorescence bands, it is necessary to know the various factors which influence the blue and red fluorescence signatures of plants. Here we describe the influence of different growth irradiances of plants of the ratio F450 / F690 measured in green leaves of wheat. In addition, we investigated for the first time whether etiolated and white leaves, which are free of photosynthetic pigments, exhibit a blue and green fluorescence emission. We also studied whether isolated chloroplasts and thylakoids contribute to the blue-green fluorescence of green leaves.

METHODS Plants Seedlings of wheat (Triticum aestivum L. var. Rector) and soy bean (Glycine max L. var. Maple Arrow) were grown for 14 days in a phytochamber at 20°C and 55% relative humidity on peat soil with full complements of nutrition for growth. Field plants of wheat and soy bean were grown under natural conditions in pots (5 1) containing also peat soil. The mean outdoor temperature in June 1991, when the measurements were performed, was 16.6°C and the light intensity at noon on sunny days was at maximum 1600/zmol m -2 s -1. Wheat seedlings were grown in a phytochamber in the presence of 10 -4 M of the herbicide SAN 9789 [norfluorazon, 4-chloro-5(methylamino)-2-(a,a,a-trifluorom-tolyl-3(2H)pyridazone] to the nutrition solution. The herbicide blocked the synthesis of carotenoids, and the chlorophylls bleached out, thus causing the development of white leaves without carotenoids and chlorophylls. Absorbance and fluorescence spectra of etiolated leaves of wheat were taken from plants which were grown for 8 days in the dark and which had seen between 2 min and 5 min of continuous white light before the performance of the measurements. Tobacco (Nicotiana tabacum L. cv. "White burley") was grown under greenhouse conditions on peat soil for 5 months at 18-25°C.

Isolation of Chloroplasts and Thylakoids Tobacco leaves (5 g) were ground with 50 mL medium I (0.33M sorbitol, 1 mM MgCI2, 1 mM MnC12, 50 mM Hepes-KOH, pH 7.5, 2 mM isoascorbate) in a Turrax homogenizer (Janke & Kundel, Germany). After filtration through gauze, the filtrate was centrifuged at 5000 x g for 5 min. The pellet was resuspended in medium II (0.33 mM sorbitol, 2 mM EDTA, 1 mM MgClz, 1 mM MnC12, 50 mM Hepes-KOH, pH 7.5) and recentrifuged at 5000 x g for 10 min. The chloroplasts were resuspended in medium II. Thylakoids were prepared by an osmotic shock of the isolated chloroplasts in medium II without sorbitol and two consecutive centrifugations at 5000 × g for 5 min.

Fluorescence Emission Spectra Fluorescence emission spectra at room temperature were taken from the upper side of intact leaves by a self-constructed laser/OMA III fluorosensor (Fig. 1). A pulsed nitrogen laser (377 nm, UV-12, Laser Photonics) was used to excite the blue, green, and red fluorescences, which were sensed by an optical multichannel analyzer (OMA-III, EG&G) containing a diode array with 512 intensified detection elements. In some case a cw-emitting H e / N e laser (632.8 nm, 5 mW, Spectra Physics, Darmstadt) was applied to excite the chlorophyll fluorescence. The Karlsruhe laser/OMA III fluorosensor enables the simultaneous measurement of the fluorescences between 400 nm and 800 nm via a polychromator containing a grating with 150 lines per mm and an entrance slit width of 25/~m, which permits a resolution of ca. 1 nm. The UV laser was operated at 10 Hz with a pulse width of 10 ns and a pulse energy of 2.5 mJ. The integration time of the gateable detector was 100 ms per single scan with a gating time of ca. 160 ns using the pulsed UV laser. The fluorescence measurements were performed in the presence of strong white light (1800/zmol m -2 s -1) in order to measure

Figure 1. Instrument setup of the laser/OMA III fluorosensor for measuring the UV-laser-induced fluorescence emission spectra of leaves.

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Fluorescence Signatures of Leaves 67

the chlorophyll fluorescence spectra in the steady state of the fluorescence induction kinetics (Kautsky effect). The optical multichannel analyzer (OMA III) triggered the UV laser as well as the pulse amplifier and was applied to determine the fluorescence intensities at 450 nm, 530 nm, 690 nm, and 735 nm with bandwidths of 10 nm as shown in Figure 2. These fluorescence intensities were used to determine the fluorescence ratios F450/ F690, F690 / F735, and F450 / F530. Leaf fluorescence was excited and sensed at an angle of 45 ° to the upper side of the leaf plain. For one fluorescence emission spectrum of a leaf, a total of 50 scans (the result of the fluorescence of 50 UV-laser shots) was accumulated. Each fluorescence ratio represents the mean of at least six measurements with different leaves. The fluorescence intensity of the spectra is expressed as counts per one lasershot (cts lasershot-1). For the c w - H e / N e laser the detector was not gateable. Therefore, the integration time for one scan was 100 ms, and the presented spectra were the mean of measurements from six different leaves. Low-temperature fluorescence emission spectra were measured on the Fluorolog Spectrofluorometer (Spex, USA) using an excitation wavelength of 337 nm and a 350 nm cutoff filter. The spectral bandwidths for excitation and emission were 10.4 nm and 3.6 nm, respectively. The samples were placed on nonfluorescent sample holders and measured in liquid nitrogen (77 K).

Absorbance Spectra Absorbance spectra were taken from the upper side of the leaves with the single-beam VIRAF-spectrometer (visible and infrared absorbance, reflectance and fluorescence) described by Buschmann et al. (1991). It enables the measurements of absorbance and reflectance spectra between 400 nm and 800 nm as well as blue-lightexcited chlorophyll fluorescence spectra between 650

Figure 2. UV-laser-induced (337 nm) fluorescence emission spectrum of a green leaf of wheat with the fluorescence bands in the blue (F450), green (F530), and red regions (F690 and F735) used to determine the fluorescence ratios blue / red F450 / F690 and blue / green F450 / F530, and the red chlorophyll fluorescence ratio F690/F735. T

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Pigment Determination The pigments of the photosynthetic apparatus (chlorophyll a + b and the total carotenoids x + c) were extracted in 100% acetone and determined by a spectrophotometer (Shimadzu UV 200) using the redetermined extinction coefficients and equations of Lichtenthaler (1987), which allow the simultaneous determination of chlorophylls a and b as well as the total carotenoids in the same pigment extract solution. RESULTS The fluorescence emission spectrum excited by a UV laser (337 nm) of a green leaf of wheat, which was grown under low light conditions in the greenhouse, was characterized by a maximum in the blue spectral region near 450 nm and by two maxima in the red region near 685 nm and 735 nm, which is representative of the chlorophyll fluorescence (Fig. 2). In contrast to some other plants, no peak or shoulder was recognizable in the green region around 530 nm. The blue-green fluorescence is not bound to green plants only, but also a genuine signature of yellowish etiolated leaves and white chlorophyll and carotenoid-free wheat leaves. The etiolated wheat leaf (illuminated for 2 min with white light) exhibited blue fluorescence 1.5 times stronger than the green leaf and also a distinct shoulder around 530 nm (green fluorescence) (Fig. 3). The chlorophyll fluorescence of the etiolated leaf was characterized only by one peak near 685 nm due to the very low chlorophyll content of 0.5/tg cm - 2. Correspondingly the chlorophyll fluorescence ratio F690/F735 amounted to ca. 8 in the etiolated leaf as compared to 0.93 in the green leaf (30/tg chlorophyll a + b per cm 2 leaf area). When the synthesis and accumulation of the carotenoids in wheat

Figure 3. UV-laser-induced (337 nm) fluorescence emission spectra of a green leaf of wheat (grown under low light conditions), an etiolated leaf (which had been illuminated for 2 min), and a white primary leaf of wheat treated with the bleaching herbicide norfluorazone (SAN 9789). T'~ 6 0 0 0

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68 Stober et al.

leaves was blocked by the herbicide norfluorazone (SAN 9789), whereby the chlorophylls became bleached, one obtained white leaves in which the blue fluorescence (F450) was increased fourfold as compared to green leaves. No red chlorophyll fluorescence was detectable in these white leaves. A similar high increase of the blue fluorescence (F450) was obtained when all the photosynthetic pigments (chlorophylls and carotenoids) were removed by treating and submersing the green wheat leaves with acetone for 3 days (Fig. 4). The blue fluorescence of the acetone-washed white leaves increased ll-fold from 1600 (green leaD to 18,000 cts lasershot-~ (white leaD. The chlorophyll fluorescence in the red region was missing, indicating the total extraction of the chlorophylls. In order to determine whether the in vivo photosynthetic pigments, as bound to the particular chlorophyllcarotenoid-proteins (Lichtenthaler, 1987), contribute to the blue-green fluorescence emission of intact leaves, we isolated whole chloroplasts from tobacco leaves as well as thylakoid membranes. In contrast to intact leaves, neither isolated chloroplasts nor isolated thylakoids exhibited a blue-green fluorescence emission (Fig. 5). Since the fluorescence measurements were performed at liquid nitrogen temperature (77 K), the leaves showed the additional chlorophyll fluorescence band near 695 nm, which has been described before (Siffel and Sestak, 1988). This experiment also demonstrated that the blue fluorescence is not only emitted at room but also at liquid nitrogen temperatures. The excitation spectra of the blue fluorescence F450 of the green, etiolated, and white wheat leaves exhibited a broad maximum near 330 nm and seemed to come from the fluorescing substances. In order to obtain information on the influence of the photosynthetic pigments on the emitted blue-green fluorescence by possibly reabsorbing part of the emitted blue-green fluorescence, we compared the fluorescence and absorbance

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[nm] Figure 5. UV-light-induced fluorescence emission spectra taken with the spectrofluorometer of a green intact tobacco leaf, isolated chloroplasts and thylakoids taken at 77 K. The spectra were normalized at 685 nm. spectra of etiolated and green leaves of wheat. The fluorescence emission spectrum of the etiolated leaf (illuminated for 2 min with white light) exhibited a broad maximum in the blue spectral region with two depressions (arrows in Fig. 6), which coincide with the absorbance peaks of carotenoids as seen in the absorbance spectrum. The fluorescence emission spectrum of the green leaf was characterized by a single Figure 6. A) Absorbance and UV-laser-induced fluorescence emission spectra of etiolated primary wheat leaves illuminated for 2 min with white light (chlorophyll content: 0.5 pg (a + b) cm-2). B) Absorbance and UV-laser-induced fluorescence emission spectra of fully green primary leaves of wheat (chlorophyll content: 30 I~g (a + b) cm-2). 1500

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Fluorescence Signatures of Leaves

maximum in the blue region near 450 nm and a slight depression in the green region near 520-530 nm (Fig. 6b). The decrease of the total blue fluorescence intensity and the slight depression in the green region as compared to etiolated leaves may be caused by a partial reabsorption of the emitted fluorescence. The absorbance spectrum of the green leaf showed a high chlorophyll absorbance band in the red region with a maximum near 675 nm. The shorter wavelength chlorophyll fluorescence emission near 690 nm showed an overlapping region with the absorbance spectrum of the green leaf, whereas the longer wavelength chlorophyll fluorescence near 735 nm, however, is little influenced by the absorbance of the wheat leaf. The fluorescence emission spectra of green leaves of wheat plants grown under controlled conditions in a phytochamber and under natural conditions in the field are shown in Figure 7. The fluorescence spectra of wheat grown in the phytochamber exhibited a strong blue fluorescence and a well-developed red chlorophyll fluorescence emission with two distinct maxima near 690 nm and 735 nm. The blue fluorescence emission of wheat grown in the field was, however, reduced by 25%, whereas the red chlorophyll fluorescence was completely suppressed. No spectral characteristics of the red chlorophyll fluorescence were obtained; apparently the exciting 337 nm laser light did not penetrate through the epidermis into the subepidermal chlorophyll layer of the leaf of the field wheat. The same result was obtained with green leaves of soy bean grown outdoors (Fig. 8). In contrast to UV-light excitation, soy bean leaves, which were excited by a H e / N e laser (632.8 nm), exhibited a strong chlorophyll fluorescence with two typical maxima (Fig. 8). Due to the fact that in field plants the exciting UV light (337 nm) does not penetrate the epidermis, which results in an extremely low chlorophyll fluorescence emission, the various fluorescence ratios blue/red and

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blue/green and the chlorophyll fluorescence ratio F690/ F735 exhibited quite different values, as shown in Table 1. The different growth conditions mainly affected the ratio F450//7690, whereas the other ratios were less or only little changed. DISCUSSION

A typical UV-light-induced fluorescence emission spectrum of green leaves at room temperature can show four distinct peaks: the blue fluorescence near 450 nm (F450), the green fluorescence near 530 nm (F530), as well as the chlorophyll fluorescence emission near 695 and 735 nm, F695 and F735, respectively (Chappelle et al., 1984; Lichtenthaler and Stober, 1990; Lichtenthaler et al., 1992). The green leaves of wheat and soy bean (phytochamber and field plants) as well as the green leaves of tobacco investigated here did not show a clear

Table 1. Fluorescence Ratios and Total Chlorophyll Figure 7. UV-laser-induced fluorescence emission spectra of

the primary leaf of wheat grown in the field and in a phytochamber. The UV-laser light (337 nm) cannot penetrate into the mesophyll of the leaf of the field plant, and almost no chlorophyll fluorescence is excited.

Content of Leaves of Wheat Grown in a Phytochamber under Controlled Conditions and in the Field under Natural Conditions Including UV Light Phytochamber

Fluorescence Ratio F450/F690

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70

Stober et al.

shoulder in their fluorescence spectrum in the green region near 530 nm. In contrast, etiolated leaves (illuminated for 2 min), which possess only a low chlorophyll content, exhibited a green fluorescence emission band, which seems to be caused by a partial reabsorption of the emitted blue fluorescence above the 525-530 nm region by the leaf carotenoids. Green leaves with a fully intact photosynthetic apparatus, possess a high chlorophyll content and more carotenoids than etiolated leaves. This results in a partial decrease of the fluorescence in the green region around 530 nm, which appears to be due to partial reabsorption of the emitted fluorescence. Experiments during greening of etiolated wheat leaves demonstrate a linear increase of the ratio of the blue to the green fluorescence F450/F530 (Stober and Lichtenthaler, 1992). In fact, with increasing pigment content there exist correlations of F450/F530 to the chlorophyll content as well as to the ratio of the chlorophylls to the carotenoids [(a + b) / (x + c)]. White leaves treated with the herbicide norfluorazone which did not possess photosynthetic pigments, exhibited a much stronger blue fluorescence (fourfold increase) as compared to green leaves. This strong increase of F450 is due to the lack of reabsorption processes as caused by the absence of blue-fluorescence light-absorbing pigments (chlorophylls and carotenoids). Also in white acetone-treated wheat leaves, where the photosynthetic pigments as well as some of the UV-lightabsorbing acetone-soluble substances of the cell wall and the cuticle such as cutin, lipids, and waxes (Fry, 1982) were washed out, exhibited a tenfold increase of the blue fluorescence intensity. The absence of such lipid-soluble UV-light-absorbing, but not blue-fluorescing substances, as well as altered scattering features of the pigment-free leaves seem to cause this strong enhancement of blue fluorescence (F450), which we assume to derive from phenolic oligo- and polymers of the cell wall and the vacuoles. Goulas et al. (1990) assume free and esterified ferulic acid as source for the blue fluorescence. Coumarins such as the widely distributed aesculetin as well as scopoletin, chlorogenic acid, and other plant phenolics possess, however, a much stronger blue fluorescence and are the more likely candidates for the blue-green plant fluorescence emission (Lang et a]., 1991). Plants grown in the field, for example, the crop plants wheat and soybean, showed a dramatic decrease of the chlorophyll fluorescence yield between 650 nm and 800 nm when excited with UV-laser light of 337 nm, which resulted in a strong increase of the fluorescence ratio blue/red F450 [ F690, as was shown here. This also applies to other crop plants such as deciduous trees like maple and beech as well as coniferous trees like spruce and fir [data not shown, or partly contained in Lichtenthaler et al. (1991a; 1992)].

In contrast, the same leaves from outdoors plants exhibited a high chlorophyll fluorescence yield after excitation with red He/Ne-laser light (632.8 nm). It is well known that field plants t h a t - i n contrast to greenhouse or phytochamber plants-were exposed to an enhanced UV-B radiation exhibited an increased synthesis of UV-light absorbing pigments, such as phenylpropanoids or furocoumarins (Tevini and Teramura, 1989). A higher content of plant phenolics appears to be the cause of the decrease of the penetration depth of the 337-nm UV-laser light applied in this investigation, whereas the penetration of the red 632.8-nm light of the H e / N e laser is not affected. Bornman and Vogelmann (1988) also demonstrated for needles of fir and spruce that UV light (360 nm) attenuates faster than light in the visible range. When illuminated with UV light at liquid nitrogen temperatures, green leaves also exhibited a blue fluorescence and a red chlorophyll fluorescence. Isolated chloroplasts and thylakoids measured at 77 K showed the typical red chlorophyll fluorescence, but exhibited no blue fluorescence. These results are indicators that neither plastid proteins nor the plastid-bound NADPH or/~-carotene contribute to the blue fluorescence emission of green leaves. In our opinion, phenolic plant compounds, which absorb UV light and emit blue fluorescence and which cannot be extracted by acetone, are the most likely pigments for blue light emission of leaves. These pigments may be mainly located in the cell wall and in the vacuoles not only of the epidermal layer but also in the mesophyll cells of the leaves. Our results indicate that the intensity of the emitted blue and red fluorescence is controlled, on the one hand, by the blue fluorescence and the blue-light-absorbing photosynthetic pigments and, on the other hand, by the amounts of UV-light-absorbing substances located in the epidermal layer and represents a penetration barrier for the fluorescence exciting UV light. CONCLUSION Blue fluorescence excited by UV light is not only emitted by green but also by etiolated yellow leaves and pigment-free white leaves of wheat. Isolated chloroplasts or thylakoids do not exhibit any blue fluorescence after UV-light excitation, which excludes the plastid-bound proteins, NADPH, and ]~-carotene as sources of the blue fluorescence. Field plants (wheat and soybean) exhibit a strong blue fluorescence but only a very low chlorophyll fluorescence when excited with a UV laser (337 nm). Since this also applied to other crop plants and to forest trees, one should apply in the future LIDAR remote sensing of terrestrial plants a laser that is able to simultaneously excite the blue-green fluorescences as well as the red chlorophyll fluorescence. This means that the

Fluorescence Signatures of Leaves

LIDAR lasers to be applied should emit in the longwavelength UV-A region or in the short-wavelength region of the VIS between 380 nm and 400 nm, which is a suitable excitation region for the blue and red fluorescence. The blue to red fluorescence ratio F450 / F690 changes considerably due to the light intensity during growth and the pigment co~atent of leaves as shown here as well as under stress conditions (Lichtenthaler et al., 1991a, b). Simultaneous excitation of the blue fluorescence as well as the red chlorophyll fluorescence emission of vegetation and its remote sensing therefore appears to be a very suitable technique to determine the state of health or damage of terrestrial vegetation via the fluorescence ratio F450/F690 and the chlorophyll fluorescence ratio F690 //7735. The work described here was sponsored by a grant from the BMFT Bonn within the EUREKA Research Program No. 380 (LASFLEUR), which is gratefully acknowledged. The lowtemperature spectra were measured in cooperation with Dr. Pavel Siffel of the Institute Plant Molecular Biology of the CS Academy of Sciences in Ceske Budejovice, CSFR, during a research stay of M. Lang, which is gratefully acknowledged. REFERENCES Buschmann, C., Rinderle, U., and Lichtenthaler, H. K. (1991), Detection of stress of coniferous forest trees with the VIRAF spectrometer, IEEE Trans. Geosci. Remote Sens. 29: 96-100. Bornman, J. F., and Vogelmann, T. C. (1988), Penetration of blue and UV radiation measured by fiber optics in spruce and fir needles, Physiol. Plant. 72:699-705. Chappelle, E. W., Wood, F. M., McMurtey, Y. E., and Newcomb, W. W. (1984), Laser induced fluorescence of green plants. 1: A technique for remote detection of plant stress and species differentiation, Appl. Opt. 23:134-138. Chappelle, E. W., Wood, F. M., Newcomb, W. W., and McMurtey, Y. E. (1985), Laser induced fluorescence of green plants. 3: LIF spectral signatures of five major plants types, Appl. Opt. 24:74-80. Chappelle, E. W., McMurtey, Y. E., and Kim, M. S. (1991), Identification of the pigment responsible for the blue fluorescence band in the laser induced fluorescence (LIF) spectra of green plants, and the potential use of this band in remotely estimating rates of photosynthesis, Remote Sens. Environ. 36:213-218. D'Ambrosio, N., Szabo, K., and Lichtenthaler, H. K. (1992), Increase of the chlorophyll fluorescence ratio F690 / F735 during the autumnal chlorophyll breakdown, Radiat. Environ. Biophys. 31:51-62. Fry, S. C. (1982), Phenolic compounds of the primary cell wall, Biochem. J. 203:493-504. Goulas, Y., Moya, I., and Schmuck, G. (1990), Time-resolved spectroscopy of the blue fluorescence of spinach leaves, Photosynth. Res. 25:299-307.

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Giinther, K. P., Liidecker, W., and Dahn, H.-G. (1991), Design and testing of a spectral resolving fluorescence LIDAR system for remote sensing of vegetation, in Proc. 5. Int. Colloq. Phys. Measurements and Signatures in Remote Sensing, Courchevel, ESA Publications Division, Noordwijk, The Netherlands, pp. 723-726. Hak, R., Lichtenthaler, H. K., and Rinderle, U. (1990), Decrease of the chlorophyll fluorescence ratio F690/F730 during greening and development of leaves, Radiat. Environ. Biophys. 29:329-336. Kautsky, H., and Hirsch, A. (1934), Chlorophyllfluoreszenz und Kohles~iureassimilation. I. Mitteilung: das Fluoreszenzverhalten griiner Pflanzen, Biochem. Z. 247:423-434. Krause, G. H., and Weis, E. (1984), Chlorophyll fluorescence as a tool in plant physiology. II. Interpretation of fluorescence signals, Photosynth. Res. 5:139-157. Lang, M., Stober, F., and Lichtenthaler, H. K. (1991), Fluorescence emission spectra of plant leaves and plant constituents, Radiat. Environ. Biophys. 30:333-347. Lichtenthaler, H. K. (1987), Chlorophylls and carotenoids, the pigments of the photosynthetic biomembranes, Methods Enzymol. 148:350-382. Lichtenthaler, H. K., and Buschmann, C. (1987), Chlorophyll fluorescence spectra of green bean leaves, J. Plant Physiol. 129:137-147. Lichtenthaler, H. K., and Rinderle, U. (1988), Role of chlorophyll fluorescence in the detection of stress conditions in plants, CRC Crit. Rev. Anal. Chem. 19 [Suppl. /]:29-85. Lichtenthaler, H. K., and Stober, F. (1990), Laser-induced chlorophyll fluorescence and blue fluorescence of green vegetation, in Proceedings l Oth EARSeL Symposium, Toulouse, EARSeL, Boulogne-Billancourt, pp. 234-241. Lichtenthaler, H. K., Lang, M., and Stober, F. (1991a), Nature and variation of blue fluorescence spectra of terrestrial plants, in Proceedings Int. Geoscience and Remote Sensing Symposium IGARSS "91, Helsinki, Espoo, pp. 2283-2286. Lichtenthaler, H. K., Lang, M., and Stober, F. (1991b), Laserinduced blue fluorescence and red chlorophyll fluorescence signatures of differently pigmented leaves, in Proc. 5. Int. Colloq. Phys. Measurements and Signatures in Remote Sensing, Courchevel, ESA Publications Division, Noordwijk, pp. 727-730. Lichtenthaler, H. K., Stober, F., and Lang, M. (1992), The nature of the different laser-induced fluorescence signature of plants, EARSeL Adv. Remote Sens., 1(2-11):20-32. Siffel, P., and Sestak, Z. (1988), Low temperature fluorescence spectra of chloroplasts: methodical aspects and possible applications, in Applications of Chlorophyll Fluorescence (H. K. Lichtenthaler, Ed.), Kluwer Academic, Dordrecht, 1988, pp. 55-61. Stober, F., and Lichtenthaler, H. K. (1992), Changes of the laser-induced blue, green and red fluorescence signatures during greening of etiolated leaves of wheat, J. Plant Physiol. 140:673-680. Tevini, M., and Teramura, A. H. (1989), UV-B effects on terrestrial plants, Photochem. Photobiol. 50:479-487.