REMOTE SENS. ENVIRON. 47:2-9 (1994)
Chlorophyll Fluorescence as a Tool for Management of Plant Resources
M. Mdthy,* A. Olioso, t and L. Trabaud*
Light-induced chlorophyll fluorescence has become a tool which has ever-increasing potential application to experimental plant physiology. The effects of frost, heat, and drought have been analyzed using the kinetics of individual leaves of two representative types of life form:
an evergreen tree (holm oak) dominant in the Mediterra- nean Basin and an annual cultivated legume (soybean).
Various indices were used to quantify their response to environmental stress. Canopy fluorescence for the two types of plants was simulated. For two levels of measure- ment, leaf or canopy, light-induced fluorescence appears to be helpful for forest or crop management in the Medi- terranean area.
INTRODUCTION
Remote or field solar reflectance measurements have application to assessment of green biomass and canopy cover. They have been successfully applied in studies of plant production for pasture management (Grouzis and M6thy, 1983). However, a technique which could detect changes in the physiological status of plants is also needed in this type of study. The fluorescence kinetics of leaves, first described by Kautsky and Hirsh (1931), were widely analyzed during the last 15 years.
The level of energy dissipated during fluorescence de- pends on the rates of the photochemical reactions of the photosynthetic process; following the Lavorel and Etienne's (1977) nomenclature, these kinetic curves show, in photosynthetically active tissues (pretreated in
*CNRS--Centre d'Ecologie Fonctionnelle et Evolutive, Mont- pellier, France
tLaboratoire I.N.R.A. associ6 ~ la chaire de Bioclimatologie de rI.N.A-P.G., 78850 Thivervat Grignon, France.
Address correspondence to M. M6thy, C N R S - C e n t r e d'Ecolo- gie Fonctionnelle et Evolutive, B.P. 5051, 34033 Montpellier Cedex 01, France.
Received 24 October 1993; revised 1 May 1993.
2
darkness), a fluorescence rise from the level O (initial fluorescence) via I (initial rise), and D (dip) to P (peak) and the decline via S (quasi-steady-state) and M (second- ary maximum) to the terminal state T. The meaning of the transients of the fluorescence induction kinetics can be described by the following way: at O-level, all pri- mary acceptors QA of photosystem II are oxidized. The
OIDP
phase reflects the redox changes of Q^, the second- ary electron acceptor Q~ and the plastoquinone pool (PQ). The /-level corresponds to the equilibriumQ~,Q~
Q^Q(.
At P-level, Q^, Q~, and the PQ pool are fully reduced by PSII. The following decline of fluorescence reflects the onset of PSI activity with the development of a number of fluorescence quenching processes.The rate of photosynthesis may be reduced by stress conditions, which disturb or block the light-driven pho- tosynthetic electron transfer system and the photosyn- thetic pigment apparatus. Whenever the process of light absorption is not affected, an increased deexcitation of the absorbed energy via chlorophyll fluorescence and heat emission can be observed. Stress conditions which can be detected and their subsequent applications have been reviewed by Lichtenthaler and Rinderle (1988).
Among them are the effects of: senescence, water deficit, mechanical injury of leaves, chilling, heat, nutrient defi- ciency, chemical stress, photoinhibition, exposure to UV-B radiation, and herbicides inhibitor of photosyn- thetic electron flow.
Depending on the nature of the stress conditions and the stage of the process which is involved, many indices can be analyzed from the shape of the fluores- cence kinetics curves. As examples, levels of O or P directly legible on curves, are useful for heat damage (Schreiber and Berry, 1977) or freezing injury (Klosson and Krauze, 1981). More elaborated indices can be used.
The maximal rate of the induced rise in chlorophyll fluorescence is an indicator of photosynthetic function and is markedly affected by chilling injury (Smillie and Hetherington, 1983). The fluorescence decrease ratio
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Chlorophyll Fluorescence as a Plant Management Tool 3
Rfd=(P- T)/T
(1)is an approximate measure of the potential photosyn- thetic activity of a leaf and is useful for screening for the presence of photosynthetic function (Lichtenthaler and Rinderle, 1988). The stress adaptation index related to the two
Rfd
values simultaneously measured in the two wavelength bands is represented by the following equation:Ap
= 1 - (1 +Rfd730)/(1
+Rfd690)
(2) It is useful to know how the leaves reorganize the structure of the photosynthetic apparatus for best adap- tation (Strasser et al., 1987).The shape of the chlorophyll-fluorescence spectra is also to be considered. The ratio of the fluorescence intensity in the two maxima of these spectra, near 690 nm and 730 nm,
K = F690 / F730, (3)
can be used in the detection of stress conditions. When photosynthetic quantum conversion is blocked, the flu- orescence emission shows a preferential increase in the 690-nm region, and there occurs an increase in K. Due to the reabsorption process of the emitted fluorescence at 690 nm, this ratio is also sensitive to the amount in chlorophyll pigments (Lichtenthaler and Rinderle, 1988).
All these chlorophyll fluorescence signatures are mainly defined for working at the leaf level, in laboratory or in the field. The use of classical indices calculated from Kautsky induction kinetics of fluorescence at the canopy level would imply some difficulties:
• Necessity of predarkening of the vegetation: for daily solar-course independent stresses, the problem could be solved by measurements in the night.
• The measurement of the terms of the slow com- ponent of the curves (from the maximum P to the steady-state 7) is not compatible with the moving speed of usual airborne systems, and fluorescence intensity, which quickly increases from O to P (fast component of the induction), is difficult to analyze.
Then approaches of fluorescence which appear to be more promising are:
• The total amount of fluorescence: The extrac- tion of fluorescence from reflection data was shown possible when applied to algae (Kim et al., 1985); sunlight-induced fluorescence in 656.3-nm Fraunhofer line can be a suitable technique for remote detection of plant stress in daylight (McFarlane et al., 1980).
• The above-mentioned ratio of fluorescence at 690 and 730 nm which is rather unaffected by instant of measurement.
However, canopy structure affects canopy fluorescence and may interact with other factors influencing fluores- cence (Hoge et al., 1983; Rosema et al., 1988; Olioso et al., 1992). Therefore, a good knowledge of fluorescence transfers through the canopy is needed: Rosema et al.
(1991) and Olioso et al. (1992) published models to describe the fluorescence of a canopy and for a laser remote fluorosensing application.
Most of the previous publications are intended for physiological or ecophysiological purposes. They tend to focus on a single application and deal with the effect of a given stress on fluorescence indices of leaves of a given species (often cultivated). This work aims to test light-induced fluorescence as a tool for management of plant resources in the Mediterranean Basin: This area is exposed to damages from fire, frost, or drought. The responses of representative types of life forms were investigated: Is light-induced fluorescence able to give an early diagnosis for major environmental stresses such as heat, cold, or water stress? Is it possible to get information about their tolerance to stress irrespective of the types of vegetation and of the level under consid- eration, leaf or canopy? Taking into account individual leaf fluorescence properties, canopy fluorescence will be simulated to test the feasibility of remote detection of plant stress.
MATERIALS AND METHODS Plant Materials
The experiments were carried out in the area of Mont- pellier. Two different types of plants were selected, a woody species and a herbaceous species:
• Holm oak,
Ouercus ilex
(L.) is a spontaneous sclerophyllous tree very frequent in the Medi- terranean Basin. Temperature affects its area of distribution.• Soybean,
Glycine max.
(L.) Merri., a wide- spread cultivated species, is known to be very sensitive to water availability.Stress Treatments and Fluorescence Measurements
Thermal Stress
Thermal resistance was analyzed on leaves of
Q. ilex
taken from small branches cut in a tree located at 100 m from the laboratory from a 50-year-old spontaneous population each time on the same aspect and at the same time (near solar noon). Branches were immediately put into water and dark-adapted (30 min minimum).Low Temperature Stress.
For cold effect, on 18-20 January 1988 (1-year-old) and on 16-18 May 1988 (1-month-old), leaves were placed in a cryogenic box with increasing exposures to several intensities of cold:- 5 ° C to - 2 0 ° C for 15 min up to 7 h. Then, leaves were put onto wet papers; fluorescene was induced from the upper side by an halogenure lamp via a pass band
4
MOthy et al.filter (Corning 4-96 10-mm blue) and detected by means
of a photomultiplier (Hamamatsu R446) equipped with 2.0- a sharp cutoff filter (Corning 2-64 3-mm red) and a
microcomputer-based fast data acquisition system de-
scribed by M6thy and Salager (1989). Excitation and o~ 1.g- emitted light were brought by means of a bifurcated ~_a:
fiber optic cable. Emphasis was placed on the maximal
rate of the rise in chlorophyll fluorescence. ~ 1.o- High Temperature Stress. For heat effect, from Oc-
tober 1990 to September 1991, three temperature levels o.5- were applied: 50°C, 55°C, and 60°C. Leaves were
separated into two categories according to the dates of sampling: New leaves younger than 3 months were collected from May to July; they will later be referred to as "young leaves," leaves aged more than 3 months being referred to as "old leaves." For each temperature, leaves were set inside aluminum cans in a thermostatted box for 5 min, 10 min, 15 min, and 30 min, respectively.
They were also put onto watered papers. The exciting irradiance was provided on their adaxial surface at 632.8 nm, by a 5 mW / He-Ne laser (Polytec PL 750) and was ranging about 400 /~mol m -2 s-L Two interference filters (Andover Corporation) centred around 693 nm (10.6 nm half-width) and 732 nm (9.2 nm half-width) were applied on top of silicon photocells with cutoff filters (Sehott RG665, 2 mm thick) excluding excitation and stray light. Excitation and emitted light were brought by means of a trifurcated fiber optic cable to sense the laser-induced chlorophyll fluorescence in the 690 nm and 730 nm bands, the fluorescence kinetics being simultaneously measured with a two-channel po- tentiometrie recorder. Fluorescence decrease ratios, the adaptation index, and the/7690/F730 ratio were calcu- lated according to Eqs. (1), (2), and (3).
Water Stress
Resistance to drought stress was analyzed on leaves of an unirrigated crop of soybean (25 plants m-2). Watering lasted for 2 months and then was stopped for 50 days.
The experiment was performed twice, once beginning 4 July 1989 and once beginning 4 July 1990. Unless otherwise indicated, measurements of fluorescence were made on the adaxial surface of lateral folioles of
younger mature leaves. The water deficit, measured on 5- the central blade by determining the water potential
with a Scholander pressure bomb, was between - 0 . 8
MPa and - 1 . 8 3 MPa. The above-mentioned laser- 4- induced fluorescence experimental design and indices
were used. 3-
RESULTS O F M E A S U R E M E N T S
~ • - 5 o c
. . . . x - 1 0 ° C
f. • -.-.-; I [
I I lilt
Time (hours)
Figure 1. Example of the effects of cold temperatures and time of exposure on the maximal rate of the rise of chloro- phyll fluorescence of 1-year-old leaves of Q. ilex. Vertical lines represent the confidence limits for 5% (from M6thy and Trabaud, 1990).
quick advent of severe data inside the photosynthetic apparatus for - 15°C and - 20°C. Injuries seem to be irreversible for leaves exposed 3 h at - 15°C or 2 h at - 2 0 ° C (MOthy and Trabaud, 1990): After treatment the leaves were left at room temperature for 2 h or 5 h;
further illumination did not restore their rate of rise in fluorescence. These effects are more pronounced on young (1-month-old) leaves, in which no photosynthetic activity was detected after 1 h at - 5 ° C .
A typical heat effect as displayed by the chlorophyll fluorescence method is shown in Figure 2 (example of oak leaf responses in April). Rfd values decrease ac- cording to a rise in temperature; the duration of expo- sure also has an effect: The longer the duration time, the greater the decrease in Rfd values. The effect is more pronounced from an exposure of 10 min at 55°C
Figure 2. Example of the effects of heat and its duration of exposure on fluorescence decrease ratio of Q. ilex leaves:
( - 0 - ) 50°C; (--- rq---) 55oc, (... /x ...) 60oc.
0
",..0 73 ',l- n , 2 -
Frost resistance of 1-year-old oak leaves, as analyzed by the maximal rate of the rise in chlorophyll fluorescence, is shown in Figure 1: This rate is mainly unaffected by
several hours of exposure at - 5 ° C or - 1 0 ° C . But o light-induced chlorophyll fluorescence can detect a
d l b 2b 3b
T i m e ( m i n u t e s )
Chlorophyll Fluorescence as a Plant Management Tool 5
on. The fluorescence decrease ratio is then a good indi- cator of the heat-induced limitation of photosynthesis.
It is well known that the photosynthetic perfor- mance of evergreens shows seasonal variation; thermal treatment also has a seasonal effect on the fluorescence decrease ratio. Figure 3 presents the example of the annual course of
Rfd730
values as measured after leaf exposure at 60°C for 10 min. The absolute values were much lower compared to those of untreated leaves, not presented here (minimum 0.65 and 1.79, maximum 1.3 and 2.86, respectively, for heated and control old leaves). The general behavior of heated leaves was ex- actly the opposite of the control leaves. The values ofRfd
for treated leaves were lower in autumn (0.69 in October) to reach a maximum in winter (1.28 in Janu- ary), then progressively decreased to the end of spring (May 0.65), at which point they are at their lowest, and then again progressively increased up to July (1.30).With this treatment the
Rfd
value of young leaves was lowest in May (0.26) when they had just appeared (younger than 1 month), and progressively increased up to July. For young leaves temperature had an extremely drastic effect: The range of the index values varied from 0.26 in May to 1.85 in July (7 times more) when in untreated young leaves values varied only slightly (mini- mum: 2.79, maximum: 3.14).The thermal treatment had a severe effect (not graphically presented here) on
Ap
(M6thy and Trabaud, 1993), particularly during winter months and until May where it was near 0. In summer for old leavesAp
increased slightly to 0.08. For young leavesAp
increased from May (0) to July (0.06).The fluorescence decrease ratios are also able to detect stress in leaves of the unwatered soybean canopy:
From both indices the first damage to the photosyn- thetic apparatus is visible on about the 15th day (Fig.
4). From the start of unwatering up to the 10th day the development of the photosynthetic apparatus appears also on this curve.
MODELING LASER-INDUCED FLUORESCENCE All the above-mentioned results concern individual leaves. Remote detection of fluorescence at canopy level should take into account the spatial arrangement of the leaves with different physiological status. At this point it becomes obvious that a model is needed for simulating the fluorescence both inside and outside the canopy.
The only main points about the simulation are given here. Details are given in Olioso et al. (1992). For a complete description of radiative transfer, emphasis is placed on the following factors:
• attenuation of downward laser beam within the canopy,
• individual leaf fluorescence response,
• attenuation of emitted upward beam,
• background reflection of exciting and down- ward emitted radiations.
dF
being the emitted fluorescence by a layerdL
at depthL,
the emitted fluorescence by the whole canopy isFc
=(dF,(L)
+dFz(L)
+dFa(L )),
(4) wheredFI(L)
= contribution of the layerdL
to the signal received by the sensor,dF2(L)
= background reflection in sensor direction of the downward emitted fluorescence,Figure 3.
Example of the seasonal course of fluorescence decrease ratio after 10 min at 60°C for young (shaded) and old (open) holm oak leaves. Bars give mean values (n = 5-6).Vertical lines indicate one s.c.
2,0
0 fO /~,
"0 '.t- n."
1.5
1.0
0 , 5
0,0
L
.' Dec ~ J a n ~ Feb _T_
Mar. Apr. M a y dun. ~ Jul
D A T E
T
i i
Aug Sept
Figure 4.
Example of water stress effect on fluorescence de- crease ratios of soybean leaves (replicate 2): ( - ) 690 nm;(- - -) 730 nm. Vertical lines indicate one s.c.
i"¢
4 . 0 -
3 . 5
3 . 0
2 . 5
2 , 0
1 . 5
r a i n f a l l
6 lb 2b 3b 4b sb
N u m b e m o f d a y s
6 M#thy et al.
dF3(L)
= emitted fluorescence from the background reflection of laser exciting radiation.For calculating these different terms, several hypoth- eses are used:
• Attenuation of exciting and emitted radiation to- ward the sensor is assumed to follow the Beer- Lambert law.
• The individual leaf fluorescence in response to exciting radiation at level L can be written as the product of the exciting irradiance by a co- efficient called fluorescence efficiency assumed to be independent of irradiance. Leaves are as- sumed to be lambertian emitters, and the can- opy is assumed to be homogeneous from the point of view of leaf optical properties and leaf angle distribution.
Simulations of canopy fluorescence are presented in Figure 5. Model parameters were selected to be representative of soybean canopies from Olioso et al.
(1992). Canopy fluorescence is strongly affected by leaf area index (LAI) and canopy structure. There are differ- ences between wavelength bands. Such effects are ex- plained by leaf absorption being more important at 690 nm, and by interception, which is more and more efficient with erectophile, spherical, uniform, and pla- nophile canopy structure.
The model was used to simulate the ratio,
K~ -- Fc690 / Fc730, (5) of a holm oak canopy with the previous data on leaf fluorescence. According to Eckardt et al.'s measure- ments (1975) near Montpellier, a LAI value of 5 was used. The K~ ratio shows a seasonal course (Fig. 6).
From higher values in autumn (0.32 in September and
Figure 5. Simulations of soybean canopy fluorescence Fc [(--) 690 nm; (- - -) 730 nm]: 1) planophile canopy; 2) uni- form; 3) spherical; 4) erectophile.
1.0
¢ 0.8
0 C
©
m 4 ~
• -~ 0,6
~ C
L ~ 0 . 4
>- co G c£ '-~
o C
0 . 2
(.3
0 , 0
0
.-" . . . . ..--r'"-7:::'-::::::::: . . . / / / / " / ' / ~ - 4 " "
1' 2 3 4 6 6
L e a f A r e a I n d e x
0 .M
C 0 CO r-- 0 la..
\ 0 13", ',13 h 0
,3
.2
.1
0
Oct. Nov. Dec. don. Feb. Mar. Apr May. Jun. Jul. Aug. Sept.
Figure 6. Simulation of the seasonal course of the K~, ratio from a holm oak canopy with (shaded) and without (open) young leaves.
October) K~ progressively decreases to reach its lowest value from January to April (0.13). Then it increases to reach values similar to the autumn ones in the end of spring and beginning of summer (0.29 in May), when the new leaves appear. In summer the value decreases again (0.26 in July). These results are to be compared with seasonal variations of chlorophyll content and sto- matal conductance in Mediterranean evergreen sclero- phyll species (Rhizopoulou et al., 1991), where the general behavior is the reverse.
The K~ ratio of soybean canopies was simulated with leaf fluorescence profiles. Figure 7 shows this simulation as a function of LAI for two dates: before and during drought stress. Due to a higher chlorophyll content, nonstressed canopy has the lowest K~ ratio. A stressed
O -H 4 J
0 CO p~
L U
0 L
Figure 7. Simulation of t h e / ~ ratio from a stressed (20 Au- gust 1990) and unstressed (4 July 1990) soybean crop can- opy as a function of leaf area index: ( - ) planophile canopy;
(- - -) erectophile canopy,
, 9
, 7
, 5 ¸
.sel
. . .. . . Z . . .
u n s t p e s s e d
.3
L e a f a m e a ± m d e x
Chlorophyll Fluorescence as a Plant Management Tool
7
canopy has generally a more erectophile structure than a nonstressed one: The differences between the two canopies are more important taking into account the stress-induced variation of canopy structure.
DISCUSSION
The above-mentioned results deal only with the specific thermal or drought resistance; the possible effect of photoinhibition was not considered here.
The survival capacities of species are a function of properties of each organ; however, such results give useful information about the whole plant physiology. It is well known that cold temperatures, often 13°C below those presented here (Larcher, 1981) for vegetative organs, limit the extension of Mediterranean species by destroying the photosynthetic apparatus. Then the isothermic (mean of minima) curve - 2 ° C (60 days of frost in winter) would limit (Le Houerou, 1974) the area of distribution of Quercus ilex. On the other hand, heat stress (high temperatures near 60°C) can limit the expansion of the species in areas with hot climate.
For example, Q. ilex grows in sites in which absolute maximum summer temperatures reach 49°C (Sevilla, Spain) but does not in hotter areas. Due to some lack of climatological data, an accurate local diagnosis of the species extension may be impossible: Fluorescence techniques can detect damages on Q. ilex leaves at the boundary of its area of distribution, by intense frost or by strong heat. Then, the technique can help to improve our knowledge about species distribution and to predict the best area of growth.
The pattern of heat tolerance during the course of the year recalls previous results about the desiccation tolerance of Q. ilex (Kyriakopoulos and Richter, 1991) and other evergreen species in Central Europe (Pisek and Larcher, 1954). As Larcher (1963) wrote about dehydration tolerance, and taking into account the high frost resistance of Q. ilex, one can emphasize a connec- tion between increased heat tolerance in winter and the ability of organs to survive extracellular freezing.
The behavior of an evergreen forest of Q. ilex with respect to CO2-exchange during the year was described by Eckardt et al. (1975) using direct measurements of gaseous exchanges by climatic cuvettes: Photosynthetic activity runs all through the year, but photosynthesis is lower according to winter and summer conditions, with vespertine decreases in this last case. The less heavy method of fluorescence signatures seems to be an im- portant contribution to this type of work.
Climatic changes have been given increased impor- tance by international research programmes and increased temperatures can induce damage on plant leaves. The Intergovernmental Panel on Climate Changes (GIEC/
IPCC, 1990) scientific assessment has identified the need for improved knowledge on the temperature in-
crease potential. The usefulness of light-induced fluo- rescence signatures of plants rises in the perspective of the greenhouse effect on climatic changes with the increase of CO2 concentration: For the example of Q.
ilex, if an increase of temperature occurred, a tempera- ture of 50°C would not be detrimental for only a short time. However, a rise to 60°C would cause rapid dam- age, preventing the recovery of the photosynthetic appa- ratus and stopping the reserve accumulation process.
With the development of the lidar system, monitor- ing of stress-induced injuries by fluorescence techniques is possible. Modeling the fluorescence transfers inside the canopy enables us to select the useful indices. Satel- lite remote sensing can make an important contribution to the study of wildland fires (Malingreau, 1990). How- ever, there have been relatively few studies which dem- onstrate the contribution of remote sensing to detect fire damage on plants. In such areas as the Mediterra- nean Basin, where fire damage to vegetation occurs fre- quently, laser-induced fluorescence techniques could detect thermal injuries due to the heat on the photosyn- thetic apparatus of the crown foliage of trees at different levels from crown scorch to total destruction. When the leaves are not totally burned-destroyed, the technique will detect the degree of damage to leaves and whether they are able to recover and function.
Remote or near detection of photosynthetic perfor- mance is able to show the time of vegetative rest needed to enable the best date for underburning to be chosen for prescribed fires. For example, in Q. ilex the photosyn- thetic system of young leaves, particularly in May when they have just appeared, is affected very quickly by temperatures near 60°C. The fluorescence technique will help to choose the time of prescribed burnings from September to late winter. On the ground, fire- killed crowns could be easily detected with their re- sponse to laser-induced fluorescence.
The use of chlorophyll fluorescence signatures pres- ently appears to be a very promising tool to enable:
• detection of stress-induced injuries,
• determination of stress-tolerance.
According to the responses of a species to extreme cold and heat, and drought, through the results from fluorescence technique, it would be possible to deter- mine the climatic areas in which it can grow and then to estimate the probabilities to be successful in an exotic afforestation or crop planting. Fluorescence response allows one to detect first symptoms of crop dryness and to predict the best time for starting of watering. The full Kautsky induction kinetics allow for an early detec- tion and a rapid assessment of environmental stress effects on leaves. They are relevant to ground truth measurements during remote sensing data acquisition.
It can be observed that related techniques, such as pulse-amplitude modulation chlorophyll fluorometry,
8 M~thy et al.
can also provide useful quantitative information on the relative quantum yield and capacity of photosynthetic electron transport (Genty et al., 1989).
For laser or sunlight-induced canopy fluorescence measurements, simulations show that it is possible to limit the effects of canopy parameters by using the K~
ratio. This ratio provides information about the behavior of the two types of plants. However, it is sensitive to the a m o u n t of chlorophyll pigments. Rosema and Verhoef (1991) have shown its limits. Some additional informa- tion could be obtained from reflectance measurement.
F o r this purpose, the laser-induced blue fluorescence or the time-resolved fluorescence may provide stress- d e p e n d e n t signatures (Theisen, 1988; Holzwarth, 1988).
However, further investigation is n e e d e d before they can be applied as a diagnosing technique in stress physi- ology and r e m o t e sensing of vegetation.
The authors wish to thank M. Obaton (INRA, Montpellier) for providing the soybean seeds, J. Fabreguettes, Ch. Fournier, F.
Jardon, J. F. Pinet, and P. Riga for their excellent technical or research assistance, and the field staff of CNRS-CEFE for maintaining facilities. Many thanks are due to A. M. M~thy and S. Oakley for their linguistic corrections.
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