Diurnal Changes of the Mean Lifetime and Yield of Chlorophyll Fluorescence, Measured Simultaneously and at Distance With a

ELSEVIER

Fluorosensing of Water Stress in Plants:

Diurnal Changes of the Mean Lifetime and Yield of Chlorophyll Fluorescence, Measured Simultaneously and at Distance With a

r-LIDAR and a Modified PAM-Fluorimeter, in Maize, Sugar Beet, and KalanchoO

Z. G. Cerovic,* Y. Goulas,* M. Gorbunov,*'* J.-M. Briantais, t L. Camenen,* and I. Moya*

W e modified a PAM fluorimeter for remote detection from 0.5 m to 1 m distance by using a laser diode for excitation. It permitted us to compare directly and simultaneously measurements of relative fluorescence yield to the measurements of lifetinae with the r-LIDAR performed under natural conditions. The existence of a linear relationship, and therefore the equivalence between the lifetime and yield for chlorophyll fluorescence estima- tion in vivo was confirmed here for several plant types and under both optimal conditions and conditions of water stress. We have also shown that fluorescence life- time measurements, with the r-LIDAR, can be used to perform complex fluorescence quenching analysis in flu- orosensing, like the one developed in the laboratory for near-contact measurements with PAM-fluorimetry. Water stress effects on fluorescence changes are especially pro- nounced in maize plants due to the C4 type of metabolism and the absence of photorespiration. This would permit the use

of

the steady state level of fluorescence (Fs, rs) for

"Laboratoire pour l'Utilisation du Rayonnement Electromag- ndtique

* Laboratoire d'Ecologie V6g6tale, Universit6 de Paris XI, 91405 Orsay, France

*Present address: Moscow State University, Department of Chemistry, Moscow 119899, Russia.

Address correspondence to Zoran G. Cerovic, Laboratoire pour l'Utilisation du Rayonnement Electromagndtique, BSt. 209 D, Centre Universitaire Paris-Sud, 91405 Orsay, France.

Received 25 September 1995; revised 6 April 1996.

REMOTE SENS. ENVIRON. 58:311-321 (1996)

©Elsevier Science Inc., 1996

655 Avenue of the Americas, New York, NY 10010

stress detection, provided that the irradiance is known or is estimated. © Elsevier Science Inc., 1996

I N T R O D U C T I O N

Chlorophyll fluorescence has been used successfully as a nondestructive and nonintrusive signal in plant biochemistry, physiology, and ecology (Lichtenthaler and Rinderle, 1988; Krause and Weis, 1991; Schreiber and Bilger, 1993; Govindjee, 1995; Joshi and Mohanty, 1995). The use of this signal has been extended lately to r e m o t e sensing of terrestrial vegetation (see the spe- cial issue of Remote Sensing of Environment, Vol. 47 (1), 1994, dedicated to this topic). Specific LIDARs (light detection and ranging) were developed for the r e m o t e sensing of chlorophyll fluorescence of terrestrial vegeta- tion (Rosema et al., 1988; GUnther et al., 1991; Anderson et al., 1994; Cecchi et al., 1994; Gorbunov and Cheka- lyuk, 1994; Goulas et al., 1994; Moya et al., 1995). They use laser sources and gated detection to discriminate changes in fluorescence yield against solar radiation (scattering and reflectance) and fluorescence excited by the sun. This approach is similar to the pulse amplitude modulation (PAM) technique applied in near-contact fluorimeters (Schreiber et al., 1986; Schreiber and Bilger, 1993), and therefore the results are directly comparable.

T h e main limitation of both PAM-fluorimeters and LIDARs in r e m o t e sensing applications is that they measure signal amplitude (an extensive parameter), 0034-4257/96 / $15.00 PII S0034-4257(96)00076-4

312 Cerovic et al.

which depends on distance and atmospheric transmis- sion. This problem can be solved by performing simulta- neous measurements at two wavelengths and using fluo- rescence ratios as signatures (Lichtenthaler and Rinderle, 1988; Gfinther et al., 1991; Cecchi et al., 1994). The alternative approach in fluorosensing is to measure the fluorescence lifetime, which is an intensive parameter (Moya et al., 1988; Moya et al., 1992). This approach was effectively introduced in fluorosensing of terrestrial vegetation with the development of a z-LIDAR, which uses picosecond laser pulses (Goulas et al., 1994; Moya et al., 1995).

In the present study we adapted a PAM-fluorimeter, by using a laser diode as excitation source, which does not induce photosynthesis, to be able to measure fluo- rescence yield from a distance of 0.5-1 m. This enabled us to perform simultaneous measurements with the z-LIDAR and the PAM-fluorimeter and to compare in- formation (signatures) obtained from fluorescence life- time and fluorescence yield measurements under natu- ral conditions. Automated procedure enabled us to follow the fluorescence parameters during the whole diurnal cycle and for several days. The close-to-linear relationship between chlorophyll fluorescence lifetime and yield (Briantais et al., 1972; Moya, 1974; Malkin et al., 1980; Haehnel et al., 1982; Moya et al., 1986) could be verified under natural conditions. The whole formal- ism of fluorescence quenching analysis developed in near contact measurements (Schreiber et al., 1986;

Sehreiber and Bilger, 1993) could be implemented to z-LIDAR measurements. As an example, we analyzed in this study the relationship between chlorophyll fluo- rescence and water availability, one of the major limita- tions for plant development and crop yield.

As water stress induces stomata closure, hence CO2 supply, changes in chlorophyll fluorescence yield and lifetime are expected to occur. From coupled measure- ments of CO2 assimilation and chlorophyll fluorescence (Cornic and Briantais, 1991) it was concluded that, for a given irradiance, desiccation must induce an increase in nonphotochemical quenching of fluorescence espe- cially under nonphotorespiratory conditions. It was therefore attractive to follow changes in fluorescence during a diurnal cycle in well-watered and water- stressed plants having different types of carbon meta- bolism.

Preliminary results of this work were presented at the International Colloquium on Photosynthesis and Remote Sensing, 28-30 August 1995, Montpellier, France.

MATERIAL A N D M E T H O D S Plant Material

Sugar beet (Beta vulgaris L. cv Monohill) was grown in a growth chamber in half-Hoagland nutrient solution.

Plants were grown with a photosynthetic photon flux density (PPFD) of 350 gmol m -2 s -l at a temperature of 20°C (day) and 15°C (night), 80% relative humidity.

Measurements were performed on the fourth pair of leaves. Maize (Zea mays L.) was grown in pots on soil (3 L, one plant per pot) in the greenhouse with supplemented light from sodium lamps (350/~mol m-'2 s-1 PPFD). Plants were 4 weeks old at the beginning of the experiment. The succulent CAM plant (Kalancho~

sp.) was grown in pot on soil in the greenhouse without additional light and was 2 years old.

F l u o r e s c e n c e M e a s u r e m e n t s

Fluorescence measurements were all performed in the greenhouse on intact plants with one leaf attached to a stand and exposed to radiation from the T-LIDAR, the PAM-flnorimeter, and projectors supplementing day light (natural irradiance on a vertically positioned leaf in December being too low) or providing saturating flashes (1 s at 3000 gmol m -2 s-l).

The measurements of relative fluorescence yield were performed using an adaptation of the PAM- fluorimeter. We have made a new emission-detection unit consisting of a laser diode (2--635 nm) (Philips CQL840/D, Eindhoven, The Netherlands), a Fresnel lens (0.15 m diameter), and a photodiode ($3590-01 Hamamatsu, France) protected by a red highpass filter (RG 665, Schott, France). This unit permitted us to collect information on fluorescence yield from a distance of 0.5-1 m using the PAM principle and electronics (PAM-101, H. Walz, Effeltrich, Germany).

The operation principle of the r-LIDAR was de- scribed elsewhere (Goulas et al., 1994; Moya et al., 1995). The excitation of the leaf was provided by a tripled mode-locked Nd-YAG laser (35 ps pulse duration of i mJ at 355 nm) (Quanta System, Milano, Italy) from a distance of 15 m, which made the spot 6 cm in diameter. Light emitted or reflected from the leaf was collected through a 0.38 m diameter Fresnel lens in the axis of the laser beam, and detected with a high speed crossed field photomultiplier (Sylvania, Model 502). The fluorescence signal was selected with a red highpass filter (RG 665, Schott, France) and the backscattered signal with an interference filter at 355 nm (Oriel, Stratford, Connecticut). The current pulse at the output of the photomultiplier was digitized with a high band- width transient analyzer (Tektronix SCD 1000, 1-GHz bandwidth). Backscattered signal from eight pulses were averaged and compared to the average of eight fluores- cence signals. This sequence was repeated every 2 rain, the barycenter lifetime (l'bar) w a s calculated on line as described below, and stored. Deconvolution and calcu- lation of the mean lifetime were also performed every 30 rain as described in Goulas et al. (1994) using the FLUOMARQT II program (LURE, Orsay, France). Fig-

Fluorosensing of Water Stress Plants

313

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, , , , , i , , , , , r , , , ,

1:02:08:20 1:09:00:00 1:15:S1:40 day & time of day (d:h:min:s)

Figure 1.

Comparison of two types of chlorophyll fluorescence lifetime determination using the decon- volution or the barycenter approach. A night-day cy- cle is presented with measurement performed every 30 min on a well-watered maize plant.

ure 1 illustrates the quality of the two types of lifetime determination. It can be seen that, although the disper- sion of points is greater in the case of 2"~r, the same type of information is obtained. This is true for all plants and conditions tested as illustrated for well-watered maize and sugar b e e t in Fig. 2.

Indeed, when the excitation flash is a Dirac func- tion, the mean lifetime (2") is defined by

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(1)

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®f(t), (2) w h e r e ® stands for the convolution product. On the other hand, it can be easily calculated that

Figure 2.

Relationship between barycenter and deconvoluted lifetime obtained during diurnal variations in leaves of sugar beet and maize.

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have a limit equal to zero in the time window. The 2"bar can be very easily calculated on line during the experiment, greatly diminishing the amount of data to be stored.

This permitted us to collect data on fluorescence life- time (2"bar) automatically every 2 min and to save com- plete decay curve only every 0.5 h for control or analysis of the decay components.

L e a f t e m p e r a t u r e was measured using a YSI plati- num t e m p e r a t u r e probe (Yellow Springs, Ohio) pressed against the leaf on the side opposite to illumination.

Irradiance at the level of the leaf was measured using a quantum sensor (Laboratory for Plant Ecology, Orsay, France) calibrated to yield P P F D in gmol m -2 s -~. The data on fluorescence yield, temperature, and P P F D were automatically recorded every 15 s by means of an acquisition program developed in our laboratory.

RESULTS

Diurnal Changes in Sugar Beet Plants

W e first analyzed the behavior of sugar beet grown hydroponically in order to minimize the environmental variables. This is our test plant, representative of the C3 family, whose physiology is readily controlled and whose behavior and optical properties are well known (Cerovic et al., 1994). Measurements are performed on leaves attached to the m o t h e r plant in the greenhouse.

Figure 3 illustrates the changes of fluorescence yield and lifetime during a daily cycle before and after a decrease of the water potential of the hydroponic solu- tion. By adding sorbitol in the solution in the dark (final concentration 0.3 M) the water potential was decreased to - 0 . 7 3 MPa. This ehange did not bring any change in fluorescence in the dark, but influenced markedly the next illumination cycle. Both the lifetime and yield increased in the morning like in the control, but at the irradiance above 150/zmol m-2 s-1 fluorescence started to decrease and remained constant for several hours.

The proportionality between fluorescence yield and life-

3 1 4 Cerovic et al.

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Figure 3. Diurnal changes in control and water-stressed sugar beet. For details see text.

Figure 5. Diurnal changes in chlorophyll fluorescence in a water-stressed and rehydrated maize plant.

time was maintained both under control conditions and low water potential conditions (Fig. 4).

Diurnal Changes in Maize

Maize was chosen as a representative of the C4 plant family. Measurements we performed on plants grown in pots that experienced gradual induction of water stress by withholding watering of pots. In Figure 1 we have seen the behavior of chlorophyll fluorescence for plants in moist soil, where the lifetime increased with irradiance during the day. The situation is quite different in water stressed plants, 10 days after withholding of watering (Fig. 5). The midday depression specific to

water stressed plants (Schulze, 1986) was reflected in fluorescence changes (Fig. 5, first day). The closing of stomata was accompanied by a substantial decrease in fluorescence level (both lifetime and yield) after a transient increase of fluorescence early in the morning.

These changes were reversible, as seen in Figure 5 during the second

day

(first day after rewatering). Again, the linear relationship between yield and lifetime seen in sugar beet is observed in maize (Fig. 6). The main difference is that the midday quenching of fluorescence is far more pronounced in maize than in sugar beet, and the fluorescence can even be lower than the night level (the F0 level, see below). In Figures 3 and 5 we

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water stressed /

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Figure 4. Proportionality between the chlor- ophyll fluorescence yield and lifetime in control and water-stressed sugar beet.

Fluorosensing of Water Stress Plants 315

deliberately presented the lifetime curves without noise reduction, to be able to appreciate the discriminative power of single measurements. The use of r~a~ (see Material and Methods) permitted a sampling of fluores- cence so frequent that, for diurnal changes, averaging (curve smoothing) can be applied without loss of infor- mation on changes. This yields smooth curves very close to that for yield measurements (not shown).

Analysis of Fluorescence Quenching in Maize Leaves In a nonstressed maize leaf we analyzed the individual components of fluorescence quenching by applying the saturation pulse method as described earlier (Schreiber et al., 1986; Schreiber and Bilger, 1993). The character- istic fluorescence levels, quenching coefficients and ex- pressions for PSII quantum yield, derived from measure- ments with the PAM-fluorimeter (Fig. 7) can be applied for r-LIDAR measurements (Fig. 8) due to the linear relation between lifetime and yield (Fig. 6). The corre- sponding nomenclature was obtained by exchanging the F with "z", therefore, r0 and rs are the lifetimes during the night and day and rm and rm' the lifetime during the saturating pulse applied at night and day, respectively (Fig. 8). Results presented in Figures 7 and 8 are repre- sentatives of measurements made on several leaves un- der well watered and very mild water stress. It can be seen that the photochemical quenching (qe) decreases very early in the morning (at very low irradiance) and does not change much during the day. It is the nonpho- tochemical quenching (NPQ) that is following the in- crease in irradiance during the day. These changes are more pronounced in the PAM-fluorimeter measure- ments (yield) than r-LIDAR measurements (lifetime).

The discrepancy might originate either from a static type of quenching of absorbed light not reflected in lifetime [carotenoid related energy deactivation (Dem- mig-Adams and Adams, 1992)], or from a variation of optical effects (chloroplast movements (Brugnoli and Bj6rkman, 1993), light focusing (Myers et al., 1994), and state 1-state 2 transition (Allen, 1992)) that will also

affect the yield measurements but will not affect the lifetime measurements.

The level of fluorescence Fs increases less than r8 because of this "static" quenching. This leads to a higher AF / Fro' than Ar / ^{~'m !} during the day, although it is identi- cal during the night (Fig. 9), and at maximum NPQ in the presence of water stress (not shown). Even under control conditions, Figure 9, the minimal values of AF/

Fm' and Ar / rm' approach the one-to-one line.

Fluorescence quenching analysis, and especially the use of the AF/Fm' parameter, is amiable in maize be- cause of the absence of photorespiration. Thanks to the absence of this alternative sink for absorbed photons, a linear relationship can be seen between the effective quantum yield of PSII measured by AF/Fro' and by gas exchange methods (oxygen evolution or CO2 fixation) (Genty et al., 1989; Edwards and Baker, 1993). There- fore, in maize it is possible to calculate the relative rate of electron transport and rate of CO2 fixation (assuming 12 quanta are used to fix one molecule of COQ from the fluorescence data (AF/Fm') and accompanying PPFD (Edwards and Baker, 1993). From the diurnal changes in fluorescence lifetime (Fig. 8) light response curves of photosynthesis could be obtained (Fig. 10). Here they end at 300 pmol m -2 s -1, but during a summer day complete saturation curves could be obtained from re- mote fluorescence measurements only, yielding informa- tion on the adaptation of the plant to the environmental conditions (Schreiber and Bilger, 1993).

Diurnal Changes in Kalanchoi~

In Figure 11 the diurnal changes of fluorescence are presented for a CAM (crassulacean acid metabolism) plant Kalancho6 compared to the two other types of plants under exactly the same variation of irradiance.

Kalancho6 is the representative of the third major group of terrestrial plants, which are well adapted to dry climates. They use a metabolic adaptation that permits carboxylation during the night with the stomata open, and closed stomata during the day (e.g., Winter and

Figure 6. Proportionality between the chlor- ophyll fluorescence yield and lifetime in water-stressed and rehydrated maize plants.

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316 Cerovic et al.

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day & time of day (d:h:min:s) Figure 7. Analysis of chlorophyll fluorescence quench- ing using the saturation pulse method (yield). The characteristic fluorescence yield levels were continuously recorded during a night and day by applying a

Lesch, 1992). In this case induction of water stress was not even attempted. The results of changes in control plants are shown for comparison of the fluorescence changes among the three groups of plants. The lifetime of fluorescence during the night was the same as in the representatives of C3 and C4 plants (0.38-0.40 ns in all three cases). The changes of fluorescence during the day were much smaller than in sugar beet and maize, but, although in a well-watered plant, the diurnal changes resembled the one seen in sugar beet and maize, which experienced water stress and closure of stomata during the day (midday dip) (Figs. 3 and 5). This is in agreement with the constitutive closure of stomata in these plants during the day (Winter and Lesch, 1992).

Relationship between Chlorophyll Fluorescence and Irradiance

Continuous measurements of fluorescence, during the whole day, allowed us to study the relationship between chlorophyll fluorescence and irradiance under natural daily cycles. In Figure 12, for sugar beet, and Figure 13, for maize, we plotted the Fs level of chlorophyll fluorescence as a function of PPFD. Basically, the same results were obtained when r, was plotted, but with a larger noise (not shown, but cf. Figs. 3 and 5). These plots are a good signature of the development of water stress in maize (Fig. 13). Water stress is associated with lower fluorescence at higher irradiance due to pronounced nonphotochemical quenching (see above).

Also, the increase of fluorescence with irradiance (in the morning) becomes steeper under water stress. The same is true for sugar beet (Fig. 12).

Two features can be noticed on the light-fluores- cence curves that develop with the water stress. First, the fluorescence level (F,) starts to decrease above a certain threshold (around 200 pmol m -2 s -~) and de- clines more with increasing water stress (development of nonphotoehemical quenching, see above). Second, during the afternoon decrease in PPFD the change in fluorescence does not follow the same path any more;

lower levels are attained, which indicates the presence of photoinhibition (Long et al., 1994) in addition to nonphotochemical quenching. The plants recovered from photoinhibition during the night, as can be seen from the light-fluorescence curve on the next morning (Fig. 13).

DISCUSSION

Currently available fluorimeters based on fiber optics cannot be used to record chlorophyll fluorescence and

saturating flash every 12 min. Quenching coefficients were calculated as described in the text.

Fluorosensing of Water Stress Plants 317

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day & time of day (d:h:min:s) Figure 8. Analysis of chlorophyll fluorescence quench- ing using the saturation pulse method (lifetime). The characteristic fluorescence lifetime levels were contin- uously recorded during a night and day by applying a saturating flash every 12 min in parallel to yield

p e r f o r m q u e n c h i n g analysis f r o m a d i s t a n c e of m o r e t h a n a f e w c e n t i m e t e r s ( B o l h ~ r - N o r d e n k a m p f et al., 1989). W e t h e r e f o r e d e v e l o p e d a laser d i o d e - b a s e d flu- orimeter operating from 0.5 m to 1 m distance. It permitted us to compare directly and simultaneously measurements of relative fluorescence yield to the mea- surements of lifetime performed with the r-LIDAR un- der natural conditions. A near-linear relation exists be- tween the fluorescence yield and average lifetime when they are changed either by photochemical quenching, as seen in algae (Moya, 1974), nonphotochemical quenching, in leaves (Genty et al., 1992; Goulas, 1992), or both, during fluorescence induction in leaves (Malkin et al., 1980). This linear relationship, and therefore the equivalence between the lifetime and yield for chloro- phyll fluorescence estimation in vivo, were confirmed here for several plant types under both optimal condi- tions and conditions of stress.

Simultaneous measurements permitted us to imple- ment the saturation pulse method (Schreiber et al., 1986; Schreiber and Bilger, 1993) to the r-LIDAR, and to test it. Remotely sensed changes in fluorescence lifetime could be analyzed by the type of quenching, and photochemical efficiency of PSII could be deduced.

This extended the use of the lifetime approach to the detailed analysis of fluorescence quenching at the plant level, for ecophysiology. In addition, this confirmed the advantage of the lifetime approach in remote sensing of fluorescence compared to amplitude measurements.

These encouraging results allowed us to envisage to extend the r-LIDAR to a lifetime pulse-and-probe type of LIDAR (Gorbunov and Chekalyuk, 1994), by adding a second laser for saturation pulses. In general, LIDARs based on the lifetime approach would give preferentially an information on photosynthesis functioning, and the one based on the amplitude approach, would give infor- mation on pigment concentration. Still, without any changes in chlorophyll concentration, the red/far-red fluorescence ratio showed pronounced diurnal variation (Cecchi et al., 1994), indicating that LIDAR measure- ments of fluorescence amplitude ratio can contain infor- mation on functioning of photosynthesis. In this particu- lar case, the decrease of the red/far-red ratio could be explained by the presence of nonphotochemical quench- ing and photoinhibition during the midday dip as seen here for water-stressed maize, both in fluorescence life- time and yield.

The importance of measurements of diurnal changes cannot be over stressed. The fluorescence lifetime dur- ing the night (F0) was remarkably stable among individu- als and plant species, either in the presence or absence

measurements. Quenching coefficients were calculated as described in the text.

3 1 8 Cerovic e t al.

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Fv/Fm (night) or AF/Fm' (day) Figure 9. Comparison of the effec- tive quantum yield of PSII photo- chemistry calculated using measure- ments with the r-LIDAR or the PAM-fluorimeter.

of stress. This "nighC fluorescence would not be a useful signature, except perhaps during the first hour in the evening after a severe water stress, when photoinhibi- tion was accompanied by a quenching of F0.

The effect of water stress on fluorescence depends on irradiance and plant type. The higher the irradiance

Figure 10. Calculated light response curves of photosynthesis from fluorescence measure- ments. Photosynthesis (CO2 fixation) was cal- culated by multiplying the effective quantum yield of PS II (AF/Pro), the photosynthetic photon flux density (PPFD), the fraction of PPFD absorbed by the leaf (0.8) and the known ratio (Edwards and Baker, 1993) of quantum yield of PSII and quantum yield of CO2 fixation (12).

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o xm'-'~s 0.8 PPFD

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1 O0 1 SO 2 0 0 2 5 0 3 0 0

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Figure 11. Diurnal changes of chlorophyll fluorescence in Kalancho6 compared to sugar beet and maize.

during the day, the larger is the effect of water stress on fluorescence. A much larger amplitude of F~ (r,) decrease is induced by water stress in maize (a C4 plant) than in sugar beet (a C3 plant), at similar light

Figure 12. Light-fluorescence curves for well watered and water-stressed sugar beet.

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0.5

0.4

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Fluorosensing of Water Stress Plants 31 9

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PPFD (~a'nol m -2 s-l) PPFD (lamol m-2 s'l)

Figure 13. Light-fluorescence curves during slowly de- veloping water stress in maize. Numbering of days was started after the cessation of watering of well-saturated compost of the pot. On the 8th day the soil was still hu- mid at the surface.

intensities. This can be easily explained by the absence of photorespiration in maize. Indeed in sugar beet, as observed in other C3 plants (Cornic and Briantais, 1991), a water-deficit (stomata closure) will increase drastically the electron drainage by oxygen reduction;

therefore, at a given light intensity, discrepancy between photon input and the utilization of the electrons pro- duced by Photosystem II photochemistry will be much lower than in dehydrated C4 plants. The deactivation of excitons as heat, that is, nonphotochemical quench- ing, will be comparatively smaller in C3 plants. This corroborates the finding of Schmuck et al. (1992) on the strong decrease of fluorescence lifetime by water- stress in maize, even below the dark level, but not in wheat (another C3 plant).

Although the quenching analysis was useful to un- derstand the phenomenon, for remote sensing purposes it is very important to notice that the steady state fluores- cence lifetime (rs) is already a good indicator of the closure of stomata, and consequently, the presence of stress. It could be used for fluorosensing on board aircraft.

The opening of stomata is dictated by the availability of water and the irradiance of the leaf. The midday depression in photosynthesis (CO2 fixation) is the conse- quence of this combined influence (Schulze, 1986). The simple r~ level of fluorescence reflected the closure of stomata both in water stressed representatives of C3 and C4 plants and the well watered CAM plant. The generally accepted assumption that high fluorescence indicates impeded photosynthesis, and consequently stress (Rosema et al., 1991), should be revised on the basis of measured diurnal changes. For a whole day

fluorescence (Fs, r~) the opposite can be said, that is, a higher level of fluorescence is an indication of good health. Giinther et al. (1994) came to a similar conclu- sion for detached branches of Quercus pubescens.

Water shortage is the primary constraint to plant growth and productivity over much of the land surface.

The two main causes of loss of production under water stress are the reduction in the crop leaf area due to fewer, smaller leaves, and the lower rates of photosynthesis per unit leaf area (Hall, 1990). Remote sensing can help to optimize irrigation and to increase the productivity. Sev- eral characteristics of the diurnal changes in zs can be used to fine-tune the scheduling of irrigation (before visible symptoms appear): the presence of the noon dip in rs; lower level of r, in the light than in the dark (for maize); the extent of rs recovery at the beginning of the night and the shape of the light-fluorescence curve.

Indeed it was shown in agronomic studies that, for soybean cultivars, the midday depression is related to final yield and that this is related to precipitations during the year (Kokubun and Shimada, 1994). Two passive remote sensing methods involving narrow-waveband mea- surements of reflectance (Gamon et al., 1992), or fluores- cence (McFarlane et al., 1980), have also been proposed to follow diurnal changes in crops under stress, and could be used to detect the midday dip. Presently, their discrim- inative potential is smaller than that of LIDARs.

Part of the work described here was supported by the EUREKA project No. 380 (LASFLEUR). The authors are indebted to Alexandre Zawadzki, who translated the computer program in Pascal, the Laboratory of Plant Ecology (University Paris XI) for the use of their controlled growth chambers, and Professors E. Govindjee and G. Guyot for a critical reading of the manu- script.

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