Application of the Karlsruhe CCD-OMA LIDAR- Fluorosensor in Stress Detection of Plants

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j.Plant Physiol. \tOl.148.pp.548-554 (1996)

Application of the Karlsruhe CCD-OMA LIDAR- Fluorosensor in Stress Detection of Plants

CLAUS BUSCHMANNl

, JOACHIM SCHWEIGER!, HARTMUT

K.

LICHTENTHALERl ,

and

PETER RrCHTER2

1 Botanisches Institut II, University of Karlsruhe, Kaiserstr. 12, D-76128 Karlsruhe, Germany

2 Institute of Physics, Department of Atomic Physics, Technical University of Budapest, Budafoki ut 8, H-llll Budapest, Hungary

Received August12,1995 . Accepted October 20, 1995

Summary

The Karlsruhe CCD-OMA LIDAR-Fluorosensor (excitation: cw HeNe laser, 632.8 nm; detection:

complete chlorophyll fluorescence spectra between 650-800 nm within 10 ms) was developed in a joint project by the Technical University of Budapest and the University of Karlsruhe for a non-destructive stress detection of plants. The computer-aided fluorosensor permits to measure the full fluorescence spectra at 8 different time intervals during the chlorophyll fluorescence induction kinetics, from which the the variable chlorophyll fluorescence ratio Rfd as plant vitality index can be calculated. The position of the cWorophyll fluorescence emission maxima of green leaves are found in the red region near 690 nm and the far-red region near 735 nm. The wavelength position of the two maxima remained unchanged during the light-induced fluorescence induction kinetics (Kautsky effect). The absolute fluorescence intensity and the ratio of the two chlorophyll fluorescence bands (F690/F735) depended on the chlorophyll content and the photosynthetic activity of a leaf as has been demonstrated here with the CCD-OMA LIDAR fluorosensor.

With increasing chlorophyll content of the leaf the fluorescence intensity of the red band near 690 nm decreased, whereas that of the far-red band near 735 nm remained constant or slightly increased. Conse- quently, the fluorescence ratio F690/F735 increased with decreasing chlorophyll concentration of leaves (curvi-linear relationship: y= a.x-^b). Changes in the fluorescence ratio F690/F735 can be taken as an indicator of long-term stress affecting the chlorophyll content of leaves. The inverse relationship between the intensity of the chlorophyll fluorescence and the rate of photosynthesis (Kautsky effect) can be used for detecting, via Rfd-values, a short-term damage to plants, which affects photosynthetic activity, but does not yet decrease the chlorophyll content of the leaf. Damage or stress is indicated by low Rfd-values (ratio fluorescence decrease) or low Ap-values (stress adaptation index). Examples are shown for different types of damage and/or stresses (water stress, forest decline phenomena, application of a herbicide inhibit- ing photosynthesis, and biological stress, e.g. damage by mites). The Karlsruhe CCD-OMA LIDAR-Fluo- rosensor proved to be a valuable tool for fast detection of stress to plants in the laboratory, but can also be applied for ground-truth control measurements during remote sensing of the state of health of terrestrial vegetation.

Key

words: Laser-induced chlorophyllfluorescence, stress detection ofplants, Kautsky effect, ratio F6901F735, Rfd-values.

Abbreviations:a

+

b=total chlorophylla+ bcontent; Ap=stress-adaptation index; CCD=charged coupled device; DCMU = 3-(3A-dichlorophenyl)-I, I-dimethyl urea (diuron); F690/F735 ratio of chlorophyll fluorescence at 690 and 735 nm; fmax=chlorophyll fluorescence maximum of the induction kinet-

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Plant Stress Detection with a CCD-OMA F1uorosensot

549

ics (Kautsky effect); fs = chlorophyll fluorescence in the steady state of the induction kinetics (Kautsky effect); LIDAR= light-induced detection and ranging; OMA=optical multichannel analyzer; Rfd= ratio of the variable chlorophyll fluorescence (ratio fluorescence decrease); x+c

=

total carotenoid content (xan- thopyhylls + carotenes).

Introduction forest decline test site near Freudenstadt (Black Forest) at 800 m above sea level.

All vegetation is subjected to different kinds of natural and anthropogenic stresses. The plant may adapt to low-level and short-term stress, but high-level stress and long-term stress lead to damages (see Lichtenthaler, 1996). Damage effects on plants may also be dangerous to human health. By means of optical methods stress and damage effects to terrestrial vegetation can be monitored remotely. In contrast to reflection measurements, which are widely used today for remote sensing of vegetation, the red and far-red chlorophyll fluorescence emitted by leaves after absorption of light is a specific charac- teristic of plants. The shape of the chlorophyll fluorescence emission spectra and the variation of fluorescence intensity in the second and minute range during the fluorescence induction kinetics change under stress and can be applied to char- acterize the state of health of plants (e.g. Lichtenthaler et al., 1986; Rinderle and Lichtenthaler, 1988; Hal<: et aI., 1990;

Lichtenthaler et al., 1990; D'Ambrosio et al., 1992). Com- plete fluorescence emission spectra of leaves have been measured before by means of diode array detectors (Buschmann and Schrey, 1981; Lichtenthaler and Buschmann, 1987;

Buschmann and Lichtenthaler, 1988). Diode array detectors have a serial read-out "Of the data, thus, the signals are not col- lected at the same time and small spectral changes may occur during the read-out procedure, in particular when the measuring times are short and approach the read-out times. In contrast, CCD detectors (Charged Coupled Device) have the advantage that the read-out of the data proceeds in parallel, and thus occurs simultaneously for all wavelengths. In a joint project between physicists and plant physiologists we developed a CCD-OMA LIDAR-Fluorosensor (OMA = Optical Multichannel Analyzer, LIDAR

=

light-induced detection and ranging), which is able to record the complete spectrum of the chlorophyll fluorescence between 650 and 800 nm within 10 milliseconds or also longer time periods if desired (Szab6 et al., 1992). Here we give several examples for the detection of various stress constraints to plants by means of this laser-equipped CCD-OMA fluorosensor.

Materials and Methods Plants

Leaves of 4 to 6-week-old plants of tobacco (Nicotiana tabacum L.)and bean(Phaseolus vulgarisL.),grown in the gteenhouse of the Botanical Garden of the University of Karlsruhe were examined.

Leaves of healthy bean plants were compared to those of plants affected by mites(Tetranychus urticae). Leaves of ivy(Hedera helixL.) were taken from a plant growing on the campus of the University of Karlsruhe. On May 30, 1995 we examined young, only several week old needles of this year and one-year-old needles of spruce (Picea abiesKarst.) from a healthy and a stressed tree at the «Schollkopf"

DCMU Treatment

The herbicide diuron, known as DCMU (3-(3.4-dichlorophenyl)-I, I-dimethyl urea), was applied by wetring the lower leaf side of a tobacco leaf with a 0.1 mM solution of DCMU dissolved in a buf- fer by means of a hair brush.

Pigments

The content of chlorophylls and carotenoids was determined from 100%acetone extracts of leaves and needles using the redeter- mined extinction coefficients and equations of Lichtenthaler (1987) which permit simultaneous determinations of both pigment groups in one extract solution. The pigment values given (e.g. Table I) are based on 2 determinations with a standard deviation of<5%.In the case of spruce 20 needles were extracted from the one-year-old needles, and 80 needles from the fresh this year needles. The pigments in the needles were referred to the projected needle area determined by computer-aided image analysis (Buschmann et al., 1990).

Fluorescence Measurements

The Karlsruhe CCD-OMA fluorescence system (Fig. l) consists of a10mW HeINe laser (632.8 nm) as light source for excitation of the fluorescence, a spectrograph (grating: 600 lines per mm; en- trance slit: 2 mm providing an optical resolution of10nm; 6 nm per mm dispersion at the image plane) and a CCD-line as detector (2048 pixel elements). For a more detailed description see Szab6 et al. (1992). Before the fluorescence measurements the leaves were adapted to darkness for abour 15 min. The 10mW HeINe laser pro- vides a high photon flux density of ca. 40,000 /lmol m-2s-Iat which the chlorophyll fluorescence emission is saturated. Despite this high irradiance, we did not find any damage in leaves and the measurements could be repeated several times. The leaves were fixed with the upper leaf side facing the excitation light and the detection system. The onset of illumination of the leaf sample was starred by opening a fast electromechanical shutter (opening time of 1 ms) about 20 ms after starting the measurement. A personal compurer (AT) controled the measurement and the data acquisition from the detector via an interface card developed and constructed in the Bu- dapest laboratoty. Each spectrum consisted of 150 measuring points which were detected via a parallel read-out at a minimum integration time of10ms. Eight spectra can be consecutively measured at any time during illumination which can freely be chosen by the ex- perimentor. For example 3 spectra before, directly at and shortly after reaching the maximum chlorophyll fluorescence fmax and then 5 spectra during the slow decrease of the chlorophyll fluorescence induction kinetics to the steady state fluorescence fs, which is reached after ca. 5 min of illumination. Each data point represents the sum of four neighbouring detector pixels. The variable fluorescence ratios known as Rfd-values, ratio (fmax - fs)/fs, and the chlorophyll fluorescence ratioF690/F735were calculated using10data points (i.e. a 3 nm range) in the fluorescence emission maxima near 690 and also near 735 nm, thus reducing the arbitrary influence of signal variation due to the measuring noise. Data processing and the display of

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550

CLAUS BUSCHMANN, JOACHIM SCHWEIGER, HARTMUTK. L!CHTENTHALER, and PETER RiCHTER

leaf

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Fig. 1: Scheme of the Karlsruhe laser- eqipped CCD-OMA LIDAR-fluorosensor which enables to measure complete emission spectra of the red and far-red chlorophyll fluorescence (650-800 nm) in the millisecond range. I) excitation light 632.8 nm of a 10 mWHeINelaser; 2) fluorescence emitted from the lea£ For further details see Materials and Methods.

Phaseolus vUlga.ris

Ratio F690/F735asindicator ofchlorophyll content ofa leaf With increasing chlorophyll content of the leaf the band at 690 nm decreases, whereas the band at 735 nm increases slightly or does not change. This can be seen by comparing the spectra (Fig. 3) of a bean leaf before and after damage by mites(Tetranychus urticae). Mites populate the lower leaf side and suck our the chloroplasts from cells and produce yellow- confirmed here with the CCD-OMA fluorosensor characterized by a parallel read-our of data. Changes in the relative intensity of the fluorescence and in the ratio of the two fluorescence bands F690/F735, however, occur during the induction period. In the early chlorophyll fluorescence studies, when the signals were detected through a filter in a more or less wide spectral range, it could not be ruled out with cer- tainty whether changes in the intensity of the fluorescence signal might have been due to a variation of the position of the emission maxima. But this possibility can be excluded completely with the present fast recording, high resolution fluorometers, such as the CCD-OMA fluorosensor.

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Fig. 2: Example for the fast measurement of complete chlorophyll fluorescence emission spectra of an intact predarkened leaf during the light-induced induction of photosynthesis. The spectra of a healthy dark green tobacco leaf(Nicotiana tabacum^L.)were taken with an integration time of 10 ms. The measuring time of the fluorescence spectrum after onset of the illumination (from 40 ms to 5 min) is given for each spectrum. Maximum chlorophyll fluorescence fmax was reached 200 ms after onset of illumination, the steady state fluorescence fs was reached 5 min after onset of illumination (chlorophyll content: 381lg per cm²leaf area).

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Fig. 3: Chlorophyll fluorescence emission spectra of a healthy bean leaf(Phaseolus vulgarisL.) and a bean leaf damaged by mites as measured in the kinetic fluorescence maximum 200 ms after onset of illumination (integration time: 20 ms). Control leaf: 35 Ilg chlorophyll per cm² leaf area; leaf with heavy mite damage: 151lg chlorophyll per cm²leaf area.

Emission spectrum ofthe chlorophyllfluorescence ofa leaf The in vivoemission spectrum of the chlorophyll fluorescence of a leaf taken at ambient temperature is characterized by two bands with maxima in the regions at 690 and 735 nm (Fig. 2). Ithas been shown before that the positions of these maxima remain unchanged during the chlorophyll fluorescence induction kinetics regardless of the chlorophyll content and the photosynthetic activity of the leaf (Buschmann and Schrey, 1985; Lichtenthaler and Buschmann, 1987), which is the resulting spectra is carried out by a computer programme writ- ten by K. Szabo. The data are transferred into a worksheet programme (LOTUS-123) and then plotted by means of graphic soft- wate (SIGMAPLOT).

Results and Discussion

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Plant Stress Detection with a CCD-OMA F1uorosensor

551

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Fig. 4: Chlorophyll fluorescence emission spectra of spruce needles (Picea abiesKarsr.) from a healthy tree (control) and a tree affected by forest decline phenomena at the Schollkopf test-site closeto Freudenstadt (Black Forest). From each tree the youngest light green needles (needle year 1995) were compared with the dark green one-year-old needles (needle year 1994). see TableI.The spectra wete taken with an integration time of 100 ms at different times of the fluorescence induction kinetics: 100 ms, 200 ms (maximum fluorescence fmax), 20 s, 60 s, and 5 min (steady state fluorescence fs) as shown in the spectra curves I to 5, respectively.

ish-white spots distributed over the leaves. The chlorophyll fluorescence emission spectra of such mite-affected leaves with a lower chlorophyll content show a higher 690 nm maximum than that of the undamaged controls which possess a higher chlorophyll content. Thus mite attack can be monitored via increases in the fluorescence ratioF690/F735.

Another example is shown in Figure 4 for the comparison of needles from a healthy spruce tree and a spruce affected by forest decline damage symptomes. The young and small needles with a (still) low and almost similar chlorophyll content for both trees (Table I) show a higherF690/F735 ratio than the larger, dark-green one-year-old needles. For the lat- ter the fluorescence ratio F690/F735, however, is signifi- cantly higher in the needles of the stressed tree as compared

to those of the healthy tree. This can be explained by the lower chlorophyll content of the needles of the stressed tree (Table I), which is one stress symptom.

The decrease of the 690 nm maximum in a healthy leaf or needle with increasing chlorophyll content is caused by the overlap of the fluorescence emission spectrum in the 690 nm region with the absorption spectrum of the leaf chlorophyll which leads to a partial reabsorption of the 690 nm region fluorescence inside the leaf tissue (Rinderle and Lichtenthaler, 1988). It has been demonstrated before by several authors (Rinderle and Lichtenthaler, 1988; Hak et al., 1990; D'Am- brosio et aI., 1992) that the ratio of the two fluorescence maxima (F690/F735) is decreasing with increasing chloro-

phyll content in a curvi-linear relationship which can be described by the equation:

where chI is the chlorophyll content per leaf area determined from the leaf extract, and k_l as well as k2are constants which vary within narrow limits depending on the plant species or leaf structure. A theoretical treatment of this relationship of the two fluorescence maxima was presented by Strasser et al.

(1988). Since long-term stress often leads to a reduced chlorophyll content, the chlorophyll fluorescence ratio F6901 F735 can be used for stress detection and chlorophyll decline in terrestrial vegetation. In addition, the detection of the ratio F690/F735 can be applied for a fast, non-destructive deter- mination of the chlorophyll content (C_chl) of a leaf. For this purpose the equation given above must be transformed:

C _ (_{chi -} _F690/F735k

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Fluorescence induction kinetics as indicators ofphotosynthetic activity

When plants kept in the dark are illuminated the photosynthetic processes are first impaired, and several minutes are required for the induction of full photosynthetic function.

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552

CLAUS BUSCHMANN, JOACHIM SCHWEIGER, HARTMUTK.LICHTENTHALER, and PETER RICHTER

needle year needle year needle year needle year

1995 1994 1995 1994

Table 1: Pigment content and various chlorophyll fluorescence char- acteristics of spruce needles(Picea abiesKarst.) from a healthy control tree and a stressed tree at the Schollkopf forest decline test-site closeto Freudenstadt (Black Forest). From each tree the small light green needles of the youngest year (1995) are compared with the dark green one-year-old needles (needle year 1994). The emission spectra of the chlorophyll fluorescence of the needles are shown in Figure 4.

needle area [mmz] 3.2 23.8 4.2 14.4

chlorophyll (a+b) 140 660 110 420

[mgm-z]

carotenoid (x+c) 35 139 33 94

[mg m-z]

chlorophyllalb 2.61 3.07 3.72 3.53

(a +b)/(x+c) 4.03 4.72 3.35 4.51

fluorescence signals [reI. untis]

F690 at fmax 595 510 345 610

F690 at fs 138 75 80 100

F735 at fmax 505 880 310 840

F735 at fs 130 165 80 160

fluorescence ratios

F690/F735at fmax 1.18 0.58 1.11 0.73 F690/F735at fs 1.06 0.45 1.00 0.63

Rfd at690 nm 3.31 5.80 3.30 5.10

Rfd at735 nm 2.88 4.33 2.87 4.25

Ap 0.10 0.22 0.10 0.14

control tree stressed tree

tween leaves. When the photosynthetic electron transport around photosystem II is specifically blocked by the herbicide diuron (DCMU), which binds to the O13-binding protein in- stead of

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the chlorophyll fluorescence does not decline to the steady state of controls. Then fs equals fmax and the Rfd- values become zero (Fig. 5). Short term stress blocking photosynthetic electron transport can also be detected via the fluorescence ratio F690/F735, which increased by about 20 to 30% at diuron treatment. The smaller decrease of the fluorescence signal indicating a reduced photosynthetic activity is also shown in the chlorophyll fluorescence spectra of the older spruce needles taken from the stressed tree as compared to those of the unstressed tree (Fig. 4 and Table 1).Another example is given in Figure 6 for an ivy leaf subjected to water stress. The chlorophyll fluorescence intensity decreased as well as the Rfd-values which can be calculated from the spectra at fmax and fs.

There are many further examples given in the literature for the detection of photosynthetic activity and the in vivo chlorophyll content via Rfd-values, and the fluorescence ratio F690/F735 (e.g. for the development of photosynthetic activity in greening seedlings: Buschmann, 1981; Babani et ai., 1996) showing that the changes in the induction kinetics expressed as Rfd-values can be taken as an indicator of short- term stress, which does not necessarily lead to reduced chlorophyll content of the lea£ Since the red 690 nm fluorescence shows a larger amplitude during the induction kinetics than the far-red 735 nm fluorescence the Rfd-values deter-

wavelength [nm]

Fig. 5: Chlorophyll fluorescence emission spectra of a tobacco leaf (Nicotiana tabacum L.) taken 200 ms (at maximum fluorescence fmax) and 5 min (at steady-state fluorescence fs) after onset of illumination before (control) and after treatment with0.1 mM DCMU (integration time:20 ms).

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The measurement of induction kinetics (<<Kautsky effect», Kautsky and Hirsch, 1931) shows a fast chlorophyll fluorescence rise within the first second of illumination to a maximum (fmax) and then - parallel to the onset of photosynthesis - a slow decrease within about 5 min to a steady state (fs). By means of the CCD-OMA fluorosensor (Fig. 1) the fluorescence emission spectra can be measured during fluorescence rise and decline.

The decline of the fluorescence during the induction kinetic, sometimes also called variable fluorescence, is related to the photosynthetic electron transport around photosystem II and to state I/state 2 transitions (for a review see Krause and Weis, 1991). The decrease of fluorescence is ascribed to two components: the photochemical and the non-photochemical quenching (Schreiber et al., 1986). This decline of the chlorophyll fluorescence from the fluorescence maximum fmax to the steady state fs is often expressed as Rfd value (ratio of fluorescence decrease fd, Lichtenthaler et al., 1982;

Lichtenthaler and Rinderle, 1988): (fmax-fs)/fs or as fd/fs.

The height of the Rfd-values, which can be measured in the 690 and 735 nm region (Rfd690 and Rfd735), is propor- tional to the leaves' net CO₂assimilation rate P_N(Tuba et al., 1994; Babani et al., 1996).

The Rfd-values at 690 nm amount to 3.0 to 5 for leaves with a medium to high photosynthetic capacity, and are zero in leaves with fully inhibited photosynthesis. The Rfd735- values are always lower than the Rfd690-values, but completely reflect the differentially photosynthetic activity be-

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Plant Stress Detection with a CCD-OMA Fluorosensor

553

and lower values for one-year-old needles of the stressed tree as compared to one-year-old needles of the unstressed tree (Table 1). The stress adaptation index may vary between values of 0 and 0.4 indicating a stressed or damaged plant (low stress adaptation) and a healthy plant, respectively. Assuming that the chlorophyll fluorescence at 735 nm is partially emitted by photosystem I (Lombard and Strasser, 1984) a high Ap-value may be an expression for a healthy plant with a functioning state l!state 2-transition of the photosynthetic apparatus. The leaf, which is in state 1 after a dark period of about 30 min, reaches the functional state 2 of photosynthesis after a light period of several minutes when the chlorophyll fluorescence is in its steady state. State 1 is characterized by an energy transfer predominantly towards photosystem II, whereas state 2 is characterized by a balanced energy transfer towards photosystem I and II (Bonaventura and Myers, 1969;

Murata, 1969). During a light-induced induction kinetic, when state l!state 2-transitions occur, more and more energy will be transferred towards photosystem I and thus, the fluorescence band at 735 nm containing a photosystem I component will decrease to a lower extent than the pure photosystem II fluorescence component at 690 nm, which, in addition, is reabsorbed by the leaf chlorophyll.

wavelength [nm]

Fig. 6: Chlorophyll fluorescence emission spectrum of an ivy leaf (Hedera helixL.)taken 200 ms (at maximum fluorescence fmax) and 5 min after onset of illumination (at steady-state fluorescence fs) before (control) and after 8 hours of water stress induced by detaching the leaf (integration time: 20 ms).

mined at 690 nm are always higher than those at 735 nm (Ta-.

ble 1).

During the induction kinetics of the chlorophyll fluorescence the position of the two fluorescence maxima remained constant, but the fluorescence at 690 nm decreased more strongly than the fluorescence at 735 nm, and thus the ratio of the two maximaF690/F735 decreased (Fig. 2 and Fig. 4).

The stronger decrease of the 690 nm fluorescence band during the fluorescence induction kinetics was earlier explained a) by the assumption that the 735 nm fluorescence band may have a higher contribution from deeper cell layers, where the intensity of the excitation light is lower, and thus the photosynthetic induction and the decrease of the fluorescence may be less pronounced than in the upper layers and b) by a possible energy transfer towards chlorophylls emitting at longer wavelengths (Buschmann and Schrey, 1981). Another ex- planation is the reabsorption of the red 690 nm fluorescence by the leaf chlorophyll which does not apply to the 735 nm fluorescence.

Strasser et al. (1987) found that damage effects of long- term, low-level ozone to beech and poplar showed up earlier when comparing the Rfd-values at 690 and 735 nm than by calculating only one Rfd-value. They therefore proposed to use a stress adaptation index Ap = 1-(1+Rfd735)/(1+

Rfd690). This Ap-value has also been determined for other plants and stress constraints (Lichtenthaler and Rinderle, 1988). The Ap-value for the spruce needles shows higher values for the older needles compared to the younger needles,

Conclusions

The shape of the emiSSIOn spectrum of the chlorophyll fluorescence as well as the intensity changes (fluorescence induction kinetics after onset of illumination) can be taken as an indicator of chlorophyll content (ratio F690/F735) and physiological activity of a leaf and a plant (Rfd690 and Rfd735 values). These parameters clearly change during stress or damage to plants and can therefore be used for early stress detection in plants. The capability of the Karlsruhe CCD- OMAfluorosensor system to measure complete fluorescence emission spectra within milliseconds permits to monitor both long-term and short-term stress damage to the photosynthetic apparatus and plants. This instrument promises to be a valuable tool for laboratory and field measurements as well as for outdoor ground-truth control measurements during remote sensing of vegetation.

Acknowledgements

This joint study was carried out within the cooperation agree- ment between the Technical University of Budapest and the Univer- sity of Karlsruhe. We wish to thank the Hungarian and German grant agencies for fincancial support. The supply of spruce needles from the Schollkopf site by Dorothea Siefermann-Harms is great- fully acknowledged. We would like ^to thank Gabriele ]ahnson for checking the English text.

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