Fluorescence Imaging of Water and Temperature Stress in Plant Leaves*

Fluorescence Imaging of Water and Temperature Stress in Plant Leaves*

MICHAEL LANG!, HARTMUT

K.

LICHTENTHALER!, MALGORZATA SOWINSKA2 , FRANCINE HEISEL2

, andJOSEPH

A.

MIEHE2

1 Botanisches Institut, Universitat Karlsruhe, Kaiserstr. 12, 0-76128 Karlsruhe, Germany

2 Groupe d'Optique Appliquee, Centre de Recherches Nucleaires, 23 rue de Loess, F-67037 Strasbourg Cedex 2, France Received July 12, 1995 . Accepted September 5, 1995

Summary

Fluorescence images of leaves from tobacco plants (green wild type and aurea mutant) were determined in the blue (F440), green (F520), red (F690) and far-red region (F740), and also expressedasfluorescence ratio images. Under long-term water stress tobacco plants initially showed constant ratios of the blue to red fluorescence (F440/F690) and the blue to far-red fluorescence (F440/F740). Below a distinct thres- hold in water content (84%in green and 88 % in aurea tobacco), however, a linear increase of the fluores- cence ratios blue/red and blue/far-red was observed. This was due to a distinct increase in the bluegreen fluorescence emission, whereas the red and far-red chlorophyll fluorescence increased to a lower propor- tion. These changes in fluorescence ratios could easily be monitored by high resolution fluorescence imag- ing of whole leaves. For each point of the leaf, the fluorescence ratio can be read from the fluorescence ratio images of the leaves. In contrast, a short-term heat plus water stress in green tobacco plants was very fast detected via fluorescence imaging as a significant increase of red and far-red chlorophyll fluorescence emission (F690 and F740) on the leaf rim, whereas the central part of the leaf still exhibited the regular fluorescence signatures of photosynthetically active leaves. A combined outdoor stress (light, heat and water stress) at a dry sunny summer period was detected in Rhododendron by fluorescence imaging dueto a much reduced red and far-red chlorophyll fluorescence. The latter was caused by UV-absorbing sub- stances (e.g. flavonols) which accumulated primarily in the epidermis of these stressed leaves. These com- pounds seemed to act as UV-radiation filter, thus reducing the amount of the UV-excitation radiation, which could penetrate the mesophyll and which resulted in a reduced chlorophyll fluorescence excitation and emission. These results demonstrate that fluorescence imaging of leaves in the blue, green, red and far- red emission bands is an excellent tool for an early stress detection in plants, which is much superior to the hitherto applied spectral point data measurements.

Key words: Blue-green fluorescence, chlorophyll fluorescence, fluorescence emission spectra, stress detection, fluorescence ratios blue/red and bluelfar-red

Abbreviations: F450

=

blue fluorescence; F530

=

green fluorescence; F690

=

red chlorophyll fluores- cence; F740= far-red chlorophyll fluorescence; F440/F520= ratio of blue to green fluorescence; F440/

F690

=

fluorescence ratio blue/red; F440/F740

=

fluorescence ratio blue/far-red; F690/F740

=

chloro- phyll fluorescence ratio red/far-red.

* This paper is dedicated ^to Prof. Dr. Eberhard Schnepf, Hei- delberg, on the occasion of his 65th birthday.

© 1996 by Gustav Fischer Verlag. Stuttgart

614 MICHAEL LANG, HARTMUTK.LICHTENTHALER, MALGORZATA SOWINSKA, FRANCINE HEISEL, and JOSEPHA.MIEHE Introduction

In their natural environment plants are often exposed to stress conditions (cf. to Larcher, 1984 and 1987; Lichtentha- ler, 1988 and 1996; Strasser, 1988). Examination of plant stress by applying techniques of chlorophyll fluorescence spectroscopy is well established (Lichtenthaler, 1987 a and 1988; Lichtenthaler and Rinderle, 1988; Strasser et al., 1988;

Schreiber et al., 1988). When excited by UV-A or UV-B ra- diation, green leaves exhibit a fluorescence emission with maxima in the blue (F440), green (F520), red (F690) and far-red (F740) range (Chappelle et al., 1984; Goulas et al., 1990; Lang and Lichtenthaler, 1991; Lichtenthaler et al., 1992; Stober and Lichtenthaler, 1993).It has been estimated that more than 90% of the ultraviolet radiation falling on a leaf is absorbed by the chlorophyll-free epidermis (Caldwell et al., 1983). Bluegreen fluorescing plant phenolics are cov- alently bound to the cell walls (Harris and Hartley, 1976), also in the epidermis walls, which are the main source of bluegreen fluorescence emission of leaves (Stober and Lich- tenthaler, 1993 b). Removal of the epidermis results in an in- crease of the UV-induced green, red and far-red fluorescence emission (Lang et al., 1991; Lang 1995) indicating the UV filter effect of the epidermis. Without the epidermis the excit- ing UV-radiation can directly pass into the leaf mesophyll cells and excite the red and far-red chlorophyll fluorescence in the mesophyll. These results also demonstrated that the mesophyll essentially contributes to the green fluorescence of leaves as is also assumed by Cerovic et al. (1994). The con- tribution of the mesophyll cells to the blue fluorescence emis- sion of the intact leaf, in turn, is negliable (Lang et al., 1992;

Cerovic et al., 1993).

The conventional laser-induced fluorescence (LIF) emis- sion spectra (LIF spectra) only allowed point measurements of randomly selected distinct leaf parts. The advantage of point measurements is that they provide information on the whole fluorescence spectra including position and intensity of fluorescence maxima. The great disadvatage of such punc- tuated measurements is the fact that neither local fluores- cence differences nor fluorescence gradients over the whole leaf surface can easily be detected, since one leaf part only yields one spectral information. In contrast, the high resolu- tion fluorescence imaging system, developed for near and far remote sensing of vegetation within the EUREKA project no.

380 LASFLEUR (Lang et al., 1994; Lichtenthaler and Lang, 1995 b), provides fluorescence information on the spatial re- solution of the distinct fluorescence bands in the blue (F440), green (F520), red (F690) and far-red region (F740) over the whole leaf surface (Lang et al., 1994; Lichtenthaler et al., 1996). The information obtained by high resolution fluo- rescence imaging is of much higher quality and superior to the hitherto applied sensing of fluorescence emission spectra of particular leaf points.

There exists an inverse relationship between total chloro- phyll content and the chlorophyll fluorescence ratio red/far- red (F690/F740) due to the partial overlapping of the red chlorophyll absorption band between 660 and 685 nm with the red chlorophyll fluorescence emission band in the 690 nm region. Thus, changes of the in vivochlorophyll content can be monitored via the chlorophyll fluorescence ratio red/far-

red F690/F740 (Lichtenthaler, 1987a; Lichtenthaler and Rin- derle, 1988; Hal< et al., 1990; D'Ambrosio et al., 1992). The chlorophyll fluorescence ratio F690/F740 can also be sensed by high resolution fluorescence imaging over the whole leaf surface (Lang, 1995; Lichtenthaler et al., 1996).

Similar to the chlorophyll fluorescence, the bluegreen fluo- rescence emission of leaves is reduced by a partial reabsorp- tion of the emitted fluorescence by the chlorophylls and ca- rotenoids, which possess bluegreen absorption bands. Conse- quently, green tobacco leaves show lower values for the fluo- rescence ratios bluelred (F440/F690) and blue/far-red (F440/F740) than the chlorophyll-poor aurea-mutant of to- bacco, which contains less photosynthetic pigments (Santru- cek et al., 1992; Lang et al., 1994). The values of the fluores- cence ratios bluelred and blue/far-red, which are increased under drought stress in maize or wheat (Dahn et al., 1992), are higher in field plants than greenhouse plants (Stober and Lichtenthaler, 1993). In contrast, treatment with the photo- synthesis herbicide diuron (DCMU) decreased the fluores- cence ratios blue/red and blue/far-red due to an increased chlorophyll fluorescence emission at the herbicide-inhibited photosynthetic quantum conversion (Edner et al., 1995).

Testing the significance of the fluorescence ratios bluelred and blue/far-red under defined plant stress situations via the newly developed fluorescence imaging is the objective of the present work. Changes in the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) of tobacco leaves in the course of long-term water stress and a short-term heat stress were studied in tobacco as well as a combined light, heat and water stress inRhododendron.

Materials and Methods Plants

Plants of a green tobacco and its aurea mutant(Nicotiana tobacum L.; Schmidt, 1971; Santrucek et al., 1992) were cultivated in the greenhouse of the Botanical Garden (University of Karlsruhe) at a quanta fluence rate of 600 Ilmol photons m-2s-l, a temperature of approximately 25'C and 60% reI. humidity.

Rhododendronspec. was grown in a private garden at the Turm- berg-site of Karlsruhe.

Determination ofpigments and water content

Photosynthetic pigments were determined spectrophotometri- cally using the redetermined pigment coefficients of Lichtenthaler (l987b). The level of total flavonol ofRhododendronleaves was de- termined spectrophotometrically in methanol after a AlClrinduced bathochromic shift of the absorption maximum to 405 nm using a rutin (quercetin-3-rhamnoglucoside) standard.

Water content of leaves was determined by drying the leaves in an oven at 95 'C for 3 h. Itwas calculated as [(fresh weight - dry weight)/(fresh weight)]*100%.

Fluorescence spectroscopy

A Perkin-Elmer LS-50 Luminescence Spectrometer (Perkin-El- mer, Dberlingen, Germany) was applied for measuring fluorescence emission spectra. Leaf samples (ca. 1^X2 cm) were pre-illuminated for 5 min in the spectrofluorometer with red light (660 nm, ca.

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Fig. 1: UV-A (355 nm) excited fluorescence emission spectra of young tobacco leaves from the fully green wild type plant (su/su) and the yellowish-green aurea mutant (Su/su).

500 limol m-2s-I) in order to achieve the steady state of the chloro- phyll fluorescence induction kinetic (Kautsky effect, as reviewed in Lichtenthaler, 1992). Fluorescence emission spectra of a leaf area of ca. 0.35 cm²were excited with UV-A radiation of 355 nm, which corresponded to the 3rd harmonic of the Nd:YAG-laser emission which has been applied as excitation source in the fluorescence imaging system (see below). The slits of excitation and emission of the monochromator of the Perkin-Elmer were adjusted to a spectral bandwidth of 15 nm. Excitation radiation was filtered using a UV- bandpass filter (center wavelength 340 nm, full-width at half max- imum (FWHM) 70 nm; Schott, Mainz, Germany) in order to re- duce stray light. The UV-A induced fluorescence emission spectra of leaves were recorded separately for the bluegreen fluorescence from 400 to 600 nm applying a 390 nm cut-off filter (Perkin-Elmer, Oberlingen, Germany) and for the chlorophyll fluorescence from 600 to 800 nm using a 530 nm cut-off filter. Both spectra were com- bined yielding the complete fluorescence emission spectrum from 400 to 800 nm.

The KarlsruhelStrasbourgfluorescence imaging system

Excitation source was a tripled Nd:YAG laser emitting 355 nm pulses at a 1kHz repetition rate. Each pulse had the energy of 10Ii]

and a FWHM of 150 ps. By expanding the laser beam the complete leaf surface even of large leaves was irradiated. Day light conditions (steady state of chlorophyll fluorescence induction kinetics) were simulated by illumination with white light of 1000 limoIm-²s-l.

The fluorescence emission bands were sensed from a distance of ap- proximately 0.2 to 0.6 m viaa gated image intensifier (gate width 20 ns, Philips XX1414M/E image intensifier tube) and a digital CCD camera with a CCD array of 288x384 elements, operating with 50 frame transfers per second. Fluorescence images of the blue (F440), green (F520), red (F690) and far-red fluorescence bands (F740) were recorded separately using the appropriate changeable interference filters (center wavelength 440 nm, 520 nm, 690 nm or 740 nm; full width at half maximum 10 nm; Oriel, France). The re- solution of the imaging system provided a high resolution of 0.2 to 0.5 mm² leaf area, depending on the distance of the CCD camera from the plant.

For image processing, the software Animater VI (A.R.P., Stras- bourg, France) was applied. The fluorescence images with 288 x 384 pixels were corrected for the inhomogenity of the excitation radia- tion and for the spectral sensitivity of the instrument. For a quanti- tative determination of the fluorescence ratios the fluorescence in- tensities were calculated from rectangular areas of 100 to 400 pixels (ca. 1-2 cm²) leaf area. For quantitative data and fluorescence ratio determinations of each leaf, the fluorescence of at least six of such rectangles were averaged.

Results

Fluorescence emission spectra

TheUV-A radiation (355 nm) induced fluorescence emis- sion spectra of leaves of a normal green tobacco plant and a yellowish-green aurea mutant are shown in Fig. 1. The aurea mutant exhibits a considerably lower chlorophyll content, and also a slightly lower carotenoid content (Lang et al., 1994; Santrucek et al., 1992). The fluorescence emission spectra showed maxima/shoulders in the blue region near 440 nm (F440), the green region near 520 nm to 530 nm (F520) and the red and far-red chlorophyll fluorescence bands near 690 nm (F690) and between 730 to 740 nm (F740). The aurea mutant exhibited a higher yield in blue-

green fluorescence and red+far-red chlorophyll fluorescence than the fully green leaf of the wild-type tobacco. This is caused by the fact that in green tobacco the emitted fluores- cence is reabsorbed by chlorophylls and carotenoids to a larger extent than in the aurea mutant which contains lower amounts of photosynthetic pigments. The lower content of chlorophyll in the aurea mutant was also documented by a higher value of the chlorophyll fluorescence ratio F690/F740 (value of 1.3) as compared to the green tobacco (value of 0.8).

Long-term water stress

Tobacco plants, green form and the aurea mutant, were kept unwatered for 3 weeks in the greenhouse, whereas con- trol plants were regularly watered. The major effect of water stress and increasing water loss of the leaf tissue was a shrink- ing of the leaves. The decreasing percentage of water content of leaves was determined together with fluorescence emission spectra and fluorescence images. TheUV-Ainduced (355 nm) fluorescence emission spectra of leaves of green tobacco did not yet change at a decreasing water content from 92% (con- trol) down to 84% (Fig. 2). In contrast, at 82% and lower percentages of water content the bluegreen fluorescence and the red +far-red chlorophyll fluorescence of green tobacco leaves increased. The relative fluorescence increase was larger for the bluegreen than for the chlorophyll fluorescence (Fig. 2).

The fluorescence ratios were also determined from fluores- cence images. The ratios blue/red (F440/F690) and blue/far- red (F440/F740) exhibited constant values in the beginning of water loss (Fig. 3). Below a certain threshold in water con- tent, both fluorescence ratios F440/F690 and F440/F740, however, linearly increased. This threshold value was 84% water content for the green tobacco and 88%water content for the aurea form. The chlorophyll fluorescence ratio F690/

F740, in turn, remained more or less constant at values from about 0.4 to 0.6 (green tobacco), indicating that a distinct breakdown of chlorophyll had apparently not occurred. The fluorescence ratio F440/F520, in turn, proved to be very var-

616 MICHAEL LANG, HARTMUTK. LICHTENTHALER, MALGORZATA SOWINSKA, FRANCINE HEISEL,andJOSEPHA.MIEHE

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a+b content per leaf area was reduced by 25% in the sun-ex- posed leaf (Table 1). The level of total flavonols was 2.4 times higher than in the shade leaf, and the largest proportion of these additional flavonols was found in the upper epidermis layer of the sun-exposed leaf

Blue and green fluorescence were higher in sun-exposed stressed leaves as compared to shade leaves (control) (Fig. 7).

The relative increase in green fluorescence was more pro- nounced than that of the blue fluorescence (Table1).In con- trast, the intensity of the emitted red and far-red chlorophyll fluorescence considerably decreased as shown in the fluores- cence images (Fig. 7) and in Table 1. Apparently, the fluores- cence exciting UV-radiation was absorbed to a large extent by the chemically modified epidermis (e.g. higher flavonol con- tent of the sun-exposed stressed leaf) and this matter drasti- Fig. 3: Changes of the fluorescence ratios bluelred (F440/F690), blue/far-red (F440/F740), red/far-red (F690/F740) and blue/green (F440/F520) ofNicotiana tabacumL.during a 3 week water stress.

A green tobacco, B aurea tobacco. A linear increase of the fluores- cence ratios F440/F690 and F440/F740 started as soon as the water content dropped below 84%and 88%for green and aurea tobacco, respectively. The ratios were read from fluorescence ratio images of the leaves.

....•. medium water stress ... low water stress --control 5

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Combined light, heat and water stress in Rhododendron Leaves ofRhododendronwere measured after a 4 week dry and sunny period in summer 1994 with midday temperatures of 34 to 36 ·C and a high photon flux density of 2200 Ilmol m-²s-l • Sun-exposed leaves exhibited a yellowish-green col- our on the upper leaf side, but a full green colour on the lower leaf side. Shaded leaves of the same plant showed a green colour on upper and lower leaf side. The yellowish- green colour of the upper leaf side of the sun-exposed leaf originated mainly from the epidermis, but the chlorophyll

Nicotiana tabacumL.

Short term temperature stress

In one week unwatered tobacco plants, additionally treated for 6 h with 40·C at an irradiance of 600 Ilmol quantam-²s-\ no visual differences were detectable to leaves of control plants. Fluorescence images, however, showed a gradient in both chlorophyll fluorescence bands (F690 and F740) from the leaf rim (highest chlorophyll fluorescence) to the central part (lowest fluorescence yield) in the leaf of the heat treated plant (Fig. 4). The chlorophyll fluorescence ratio F690/F740 did, however, not increase at the leaf rim areas (Fig. 5 b). In the lateral and leaf rim parts the red (F690) and far-red (F740) chlorophyll fluorescence were 2.5 times higher than in the central leaf parts. The latter still showed the same low chlorophyll fluorescence emission as the control leaf of the watered and non-heat treated plant (Fig. 4 c and d). The green fluorescence emission at F520 slightly increased to a 1.3 times higher intensity by heat treatment, whereas the blue fluorescence (F440) remained constant. Due to the increase of chlorophyll fluorescence at the leaf rims, the fluorescence ratios bluelred (F440/F690) and blue/far-red (F440/740) sig- nificantly decreased at the leaf rim with a gradient from the center leaf part (highest values) to the leaf rim (lowest values) (Figs. 5 and 6).

iable in green and aurea tobacco, not showing any clear tend- ency (Fig. 3).

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Fig.2: UV-A (355 nm) induced fluorescence emission spectra of green tobacco leaves with different water content. Control: 88 % water content, low water stress (84 % water content) and medium water stress (82 % watet content).

Fig. 4: False-colour images of rhe laser-in- duced blue (F440), green (F520, red (F690) and far-red (F740) fluorescence gra- dient in a green tobacco leaf after a 6 h heat plus water stress treatment ar 40·C. Fluo- rescence intensities increase from) blue via green and yellowto red (see colour scale in the figure).

Fig.5: False-colour images of the fluores- cence ratios blue/red (F440/F690), blue/far- red (F440/F740), red/far-red (F690/F740) and blue/green (F440/F520) in a green to-

bacco leaf after a 6 h heat stress at 40°C.

The fluorescence ratios increase from blue via green and yellow to red (see colour scale).

cally reduced the yield of chlorophyll fluorescence in the sub- epidermal mesophyll cells (Figs. 7 and 8 A). Consequently, the fluorescence ratios blue/red and blue/far-red increased several fold due to the decreased chlorophyll fluorescence (Figs. 8 band 9), whereas the fluorescence ratio blue/green decreased. The red/far-red chlorophyll fluorescence ratio F690/F740 also increased in the stressed sun-exposed leaf, thus reflecting the lower chlorophyll content (TableI).

Discussion

Long-term water stress, as simulated in the greenhouse by slowly drying out tobacco plants, was monitored by fluores- cence imaging and detected by an increase in the fluorescence emission in the blue (F440), green (F520), red (F690) and far-red (F740) spectral region. Since the bluegreen fluores- cence increased to a higher extent than the red + far-red

618 MICHAEL LANG, HARTMUTK. LICHTENTHALER, MALGORZATA SOWINSKA, FRANCINE HEISEL, and JOSEPHA.MIEHE

Fig. 7: False-colour images of the blue (F440), green (F520), red (F690) and far-red (F740) fluorescence distribution of a shaded leaf (left, control) and a sunexposed leaf (right, stressed) ofRhododen- dron. Fluorescence intensities increase from blue via green and yel- low to red (see colour scale in the figure).

fluorescence ratios remained constant for a longer period in the course of water loss, but below a distinct threshold in wa- ter content both fluorescence ratios increased linearily. Al- though the increase of the fluorescence ratios blue/red and blue/far-red in plants under drought stress was demonstrated before by spectral point data measurements (Oahn et al., 1992), the linear increase in the fluorescence ratios blue/red and blue/far-red below a threshold in water content, during drying of attached leaves, was observed and described here for the first time as well as its monitoring and detection by the newly developed fluorescence imaging technique. These re- sults emphasize the superiority of fluorescence images of complete leaf areas over the hitherto applied fluorescence emission spectra of randomly selected small leaf parts. The advantage of the Karlsruhe/Strasbourg fluorescence imaging system is that it permits to directly image various fluorescence ratios and to recognize gradients and local disturbances in these ratios over the leaf area.

The increase in blue fluorescence during the induced water loss might have been caused, at least to some extent, by a transitory accumulation of such blue fluorescence emitting substances, which are known to be formed as intermediates in chlorophyll breakdown (Krautler et al., 1992). Shrinking of cells as a consequence of turgor loss, however, also results in the change of optical leaf properties with an increased re- flectance in the visible spectral range (Thomas et al., 1971).

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Fluorescence intensities:

F440 275±18 419±13 ***

i

F520 223±11 645±38 ***

i

F690 1708±47 263±11 ***

-1

F740 3268±76 370±12 ***

-1

Fluorescence ratios:

F440/F690 0.16±0.02 1.60±0.10 ***

i

F440/F740 0.09±0.01 1.16±0.19 ***

i

F690/F740 0.54±0.09 0.75±0.11 *

i

F440/F520 l.27±0.17 0.66±0.11 ***

-1

Flavonol content [Ilg cm-2] 54±5 124±9 ***

i

Photosynthetic pigments

-1

Chlorophyllsa+b[Ilg cm-2] 33.5±3.5 25.0±2.2 ***

Carotenoids x+c [Ilg cm-2] 11.5±1.4 11.0±1.1 H

Ratio Chialb 2.7±0.2 2.2±0.2 *

-1

Ratio (a+b)/(x+c) 2.9±0.2 2.3±0.2 *

-1

* and ***: significantly different to the control: *** p<O.OOl;

* p<0.05.

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Table 1: Fluorescence characteristics and pigment data (with stand- ard deviation) ofa shaded (control) and a sun-exposed leaf (stressed) of a Rhododendron hybrid. Intensities of the blue (F440), green (F520), red (F690) and far-red fluorescence (F740) were determined from fluorescence images together with the fluorescence ratios bluet red (F440/F690), blue/far-red (F440/F740), red/far-red (F6901 F740) and blue/green (F440/F520). Mean values of 6 determina- tions. The values of chlorophylls, carotenoids and flavonols (given in Ilg cm-²leaf area) are based on 4 determinations. The arrows indi- cate increase or decrease in the sun-exposed leaf as compared to the shadeleaE

chlorophyll fluorescence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) changed, which could easily be monitored via images of the fluorescence ra- tios. Long-term water stress resulted in a particular behaviour of the fluorescence ratio bluelred and blue/far-red. These rwo

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F440/F690 F440/F740 F690/F740 F440/F520 BLUEIRED BLUEJFAR-REO REOIFAR-REO BLUEIGREEN Fig.6: Differences in the fluorescence ratios bluelred (F440/F690) and blue/far-red (F440/F740) [left scale], and in the fluorescence ra- tios red/far-red (F690/F740) and blue/green (F440/F520) [right scale] between stressed/damaged leaf parts of the leaf rim and central leaf parts of a tobacco leaf after a 6 h heat stress treatment at 40°C.

Mean values of 10 determinations (based on 180 pixels each) with standard deviation. ***These differences are highly significant (p<O.OOl).

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tios blue/red (F440/F690) and blue/far-red (F440/F740), which decreased. Gradients in fluorescence emission and fluorescence ratios from the leaf rim to the central part as well as a certain patchiness of the leaf area concerning the emission of fluorescence signatures are easily to be screened by fluorescence imaging. This method is the only one to de- tect such gradients or local disturbances and changes in fluo- rescence emission and fluorescence ratios. The non-invasive fluorescence imaging procedure also allows to early detect whether a regeneration of a stress effect is still possible and will proceed when the stressor, e.g. heat stress or water stress, will be removed.

The strong decrease of the chlorophyll fluorescence emis- sion together with the increase in the bluegreen fluorescence in light, heat and water stressed, sun-exposed Rhododendron leaves resulted again in increased values of the fluorescence ratios bluelred (F440/F690) and blue/far-red (F440/F740).

The much stronger increase of the green fluorescence in the sun-exposed leaves with respect to the blue fluorescence indi- cated that UV-absorbing substances emitting green fluores- cence were accumulated in the epidermis of the sun-exposed leaves, as compared to the control shade leaves. The much lower chlorophyll fluorescence emission in these sun-exposed leaves indicated that their photosynthetic apparatus in the subepidermal mesophyll cells was better protected against ul- traviolet exposure than the non-stressed shade leaves ofRhodo- F740

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Blue/Red BluelFar-Red RedIFar-Red Blue/Green Fig.8: Blue, green, red and far-red fluorescence yield (A) as well as fluorescence rarios blue/red (F440/F690), blue/far-red (F440/F740, red/far-red (F690/F740) and blue/green (F440/F520) (B) based on fluorescence images of sun-exposed and shaded leaves ofRho,wden- dron.Mean values of 10 determinations (based on 200 pixels each).

The differences are significant: ***p<O.OOl and *p<0.05.

An enhanced reflection of the emitted blue fluorescence in the mesophyll and epidermis cells may have also contributed to the increase in blue fluorescence emission. Besides the changes in absolute fluorescence yield, the linear increase in the fluorescence ratios F440/F690 and F440/F740 proved to be good indicators of the progressing water stress in green and aurea tobacco leaves.

Short-term heat stress in tobacco plants was also detected by determination of fluorescence images in the four fluores- cence bands blue, green, red and far-red. The fast detection of the increase of the chlorophyll fluorescence bands at the leaf rim indicated an impairment of photosynthetic quantum conversion at the lateral and leaf rim parts, whereas the pho- tosynthetic processes in the leaf center part were still in full function. This demonstrates the excellent suitability of the fluorescence imaging system in early stress detection in plants. Since after 6 h of heat exposure (40 ·C) virtually no changes were found in the blue fluorescence emission of the leaf, the latter can be taken as internal fluorescence standard.

Thus the heat stress induced impairment of the photosyn- thetic quantum conversion yielding an increased chlorophyll fluorescence can be quantified in form of the fluorescence ra-

Fig. 9: False-colour fluorescence ratio-images blue/red (F440/F690), blue/far-red (F440/F740), red/far-red (F690/F740) and blue/green (F440/F520) of a shaded leaf (control) and a sun-exposed stressed leaf ofRho,wdendron.The values of rhe fluorescence ratios increase from blue^tored as indicated in the scale.

620 MICHAEL LANG, HARTMUTK. LICHTENTHALER, MALGORZATA SOWINSKA, FRANCINE HEISEL, and JOSEPHA.MIEHE

dendron.The flavonol quercetin exhibits a green fluorescence, and other plant phenolics like catechin, coumarins and hy- droxycinnamic acids possess broad bluegreen emission bands (Lang et al., 1991). Though an increase of the total flavonol content in sun-exposed as compared to shaded leaves was demonstrated in Rhododendron (Table 1), further investiga- tions are required to prove whether these additionally ac- cumulated flavonols caused the increase in bluegreen fluores- cence. However, it is evident that the additional flavonols, ac- cumulated in sun-exposed leaves (and predominantly in the leaves' upper epidermis), functioned as UV-absorbing filter and thus prevented the major part of the ultraviolet radiation (here: 355nm) to penetrate through the epidermis into the mesophyll cells and there to excite chlorophyll fluorescence.

This is the cause for the much lower chlorophyll fluorescence emission observed in sun-exposedRhododendronleaves.

Conclusion

High resolution fluorescence images of complete leaf sur- faces provide faster and more precise information on stress- induced changes in the fluorescence yields and the fluores- cence ratios blue/red, blue/far-red, red/far-red and blue/green than conventional point measurements of leaves. This has been demonstrated here for long-term water stressed tobacco plants, in a short-term heat stress experiment with tobacco and also in a combined light, heat and water stress in Rhodo- dendronplants. The high resolution fluorescence imaging, ap- plied in this investigation, was shown to be an excellent tool for early stress detection, whereby gradients and a certain leaf patchiness in fluorescence emission over the leaf surface can easily be detected. Besides differences in the absolute fluores- cence yields in the four fluorescence bands, changes in the fluorescence ratios bluelred and blue/far-red proved to be the best indicators for the detection of stress conditions in plants.

Once a stress or damage has been recognized, secondary and complementary methods, such as fluorescence kinetics or chemical analysis including HPLC, can be applied to further define the stress effects in plants.

Acknowledgements

We thank Gabrielle Johnson for correcting the English text.

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