J. Abadı´a,* and I. Moya

Fluorescence during Photosynthetic

Induction in Iron-Deficient Sugar Beet Leaves

F. Morales,* R. Belkhodja,* Y. Goulas,

J. Abadı´a,* and I. Moya

A

Iron plays important roles in the structure and function techniques has been carried out during photosynthetic

of the photosynthetic apparatus of plants (Terry and Ab- induction in iron-deficient sugar beet leaves. Iron defi-

adı´a, 1986). The most obvious characteristic of Fe-defi- ciency caused increases in the mean chlorophyll fluores-

cient leaves is their greenish-yellow color, which is due cence lifetime. These increased chlorophyll fluorescence

to low concentrations per area of the photosynthetic pig- lifetimes occurred in dark-adapted leaves, during a sud-

ments chlorophylls (Chls) and carotenoids (Morales et den increase in light intensity and also at steady-state

al., 1990; 1994; Abadı´a and Abadı´a, 1993). However, not photosynthesis. Chlorophyll fluorescence lifetimes were

all photosynthetic pigments are decreased to the same correlated with the extent of nonphotochemical and/or

extent by Fe deficiency, xanthophylls (lutein and xantho- photochemical quenching. During most of the photosyn-

phyll cycle pigments) being less affected than Chls and thetic induction period, Fe-deficient leaves showed lower

b-carotene (Morales et al., 1990; 1994). Iron deficiency actual PSII efficiencies than control leaves, due to de-

decreases the leaf photosynthetic rate (Terry, 1980) by creases in photochemical quenching and intrinsic PSII ef-

reducing the number of photosynthetic units per area ficiency. During photosynthetic induction Fe deficiency

(Spiller and Terry, 1980) and by lowering the actual PSII decreased the proportion of light absorbed by the PSII

efficiency of the remaining units (Morales et al., 1998).

antenna that is used in photochemistry and increased the

Upon illumination of a dark-adapted leaf photosyn- proportion dissipated thermally within the PSII antenna,

thesis increases progressively for several minutes. On the the later being well correlated with nonphotochemical

way to the steady-state level, the photosynthetic appara- quenching. Laser instrumentation offers new perspectives

tus passes through different transitory stages, constituting for monitoring effects of stress conditions in plants at

the so-called photosynthetic induction. This is accompa- large spatial scales. Elsevier Science Inc., 1999

nied by changes in Chl fluorescence yield (Kautsky, 1931). The Kautsky effect has been traditionally sepa- rated in two phases, a fast one (with a time scale of sec- onds) and a slow one (with a time scale of minutes). The rapid kinetics of dark-adapted leaves has been shown to

* Departamento de Nutricio´n Vegetal, Estacio´n Experimental de

be affected by Fe deficiency (Morales et al., 1991), and

Aula Dei, Consejo Superior de Investigaciones Cientı´ficas, Zaragoza,

this was found later to be due in part to a dark reduction

Spain

† Laboratoire pour l’Utilisation du Rayonnement Electromag- of the plastoquinone pool (Belkhodja et al., 1998). The

ne´tique, Universite´ de Paris XI, Orsay, France

slow kinetics, which contains complex information on

Address correspondence to Fermı´n Morales, Departamento de

regulatory mechanisms that cannot be obtained by steady-

Nutricio´n Vegetal, Estacio´n Experimental de Aula Dei, Consejo Superior

state studies, has not been investigated so far in Fe-defi-

de Investigaciones Cientı´ficas, Apdo. 202, E-50080 Zaragoza, Spain.

E-mail: jmorales@eead.csic.edu cient leaves.

Preliminary results of this work were presented at the Interna- Chlorophyll fluorescence has become a useful, non-

tional Colloquium on Photosynthesis and Remote Sensing, 28–30 Au-

destructive and nonintrusive tool in photosynthesis re-

gust 1995, Montpellier, France.

Received 1 July 1998; revised 18 January 1999. search and the early detection of stress conditions in

REMOTE SENS. ENVIRON. 69:170–178 (1999)

Elsevier Science Inc., 1999 0034-4257/99/$–see front matter

655 Avenue of the Americas, New York, NY 10010 PII S0034-4257(99)00015-2

plants (Krause and Weis, 1984; 1991). The use of Chl an initial pH of approximately 7.7 by adding 1 mM NaOH and 1 g L²¹ of CaCO3. This treatment simulates condi- fluorescence has been extended to remote sensing of ter-

restrial vegetation (Cerovic et al., 1996, and references tions usually found in the field which lead to Fe defi- ciency. Plants were used for measurements 1 week after therein). Specific LIDAR (light detection and ranging)

apparatus have been developed for the remote sensing transferring the plants to an iron-free nutrient solution.

Plants were grown under 350lmol photons m²²s²¹PAR, of Chl fluorescence of terrestrial vegetation (Rosema et

al., 1988; Cecchi et al., 1994; Goulas et al., 1994; Moya at a temperature of 258C, 80% relative humidity and a photoperiod of 16 h light/8 h dark. Young, rapidly ex- et al., 1995). These devices use laser sources and gated

light detection to discriminate changes in Chl fluores- panding leaves were used for all measurements. All chlo- rotic leaves sampled had no green veins, and showed a cence yield from scattered and reflected solar radiation,

and also from Chl fluorescence excited by sunlight. This homogeneous color throughout the leaf. Leaves receiving a similar PPFD on their surface were chosen for analysis.

approach is similar to that used in the pulse amplitude

modulation (PAM) technique applied in near-contact flu- Measurements were performed in the greenhouse with intact plants. One leaf was attached to a stand and orimeters (Schreiber et al., 1986; Schreiber and Bilger,

exposed to radiation from the s-LIDAR, the modified 1993). The main limitation of both LIDARs and PAM-

PAM-fluorimeter and a light-supplementing projector fluorimeters in remote sensing applications is that they

(350 lmol photons m²² s²¹ PAR).

measure signal amplitude, which depends on the dis- tance from the sample to the measuring device and also

on the degree of atmospheric light transmission. This Pigment Analysis

problem can be solved by performing simultaneous fluo- Chlorophyll concentration per area was estimated nonde- rescence measurements at two wavelengths and then us- structively by using an SPAD-502 Minolta device (a por- ing the fluorescence ratio as signature (Lichtenthaler and table Chl meter). For calibration of the apparatus, 40 Rinderle, 1988; Cecchi et al., 1994). An alternative ap- leaf disks across all the Chl range used were first mea- proach is to measure fluorescence lifetime, which does sured with the SPAD, then frozen in liquid N2, extracted not depend on the factors mentioned above (Moya et al., in 100% acetone as described previously (Abadı´a and Ab- 1992; Cerovic et al., 1996). adı´a, 1993; Morales et al., 1994) and the extracts ana- The main aim of this work was to characterize the lyzed spectrophotometrically according to Lichtenthaler photosynthetic induction of sugar beet leaves affected by (1987). The relationship between SPAD and Chl concen- Fe deficiency by using the s-LIDAR, an apparatus that tration per area was logarithmic (r²50.95; Abadı´a and measures fluorescence lifetimes by using picosecond la- Abadı´a, 1993). Curve fitting was made with Kaleidagraph ser pulses (Goulas et al., 1994; Moya et al., 1995). We v.3.08 for Macintosh (Synergy Software, Reading, PA).

compared the results obtained with the s-LIDAR and a

modified PAM-fluorimeter (Cerovic et al., 1996) to in- Remote Sensing of Chlorophyll Fluorescence vestigate the photosynthetic induction of sugar beet Measurements of Chl fluorescence yields were performed leaves affected by Fe deficiency. This allowed us to mea- using a modified PAM-fluorimeter (Cerovic et al., 1996).

sure simultaneously and compare Chl fluorescence life- The adaptation was based on an emission-detection unit, times (measured with thes-LIDAR) and Chl fluorescence consisting of a laser diode (Philips CQL840/D, Eindhoven, yields (measured with the modified PAM-fluorimeter). The Netherlands) as excitation source (k5635 nm), a Quenching analyses were also performed in separate ex- Fresnel lens (0.15 m of diameter), and a photodiode periments during the photosynthetic induction of sugar (S3590-01, Hamamatsu, Japan) protected by a red high- beet leaves affected by Fe deficiency. pass filter (RG-665, Schott, France). This unit permitted the measurement of Chl fluorescence yields from a dis- tance of 0.5 m to 1 m using the PAM principle and elec- MATERIAL AND METHODS

tronics (PAM-101, Walz, Effeltrich, Germany). Data were Plant Material automatically collected by a computer every 15 s by

means of a home-made acquisition program.

Sugar beet (Beta vulgaris cv. Monohil, Hillesho¨g, Land-

skro¨na, Sweden) was grown in a growth chamber. Seeds The operation principle of the s-LIDAR (light de- tection and ranging) has been described previously (Gou- were germinated and grown in vermiculite for 2 weeks.

Seedlings were grown for 2 more weeks in 3/8-strength las et al., 1994; Moya et al., 1995). Excitation light was provided by a tripled mode-locked Nd-YAG laser (35 ps Hoagland’s nutrient solution with 22.4lM Fe and then

transplanted to 20 L plastic buckets (4 plants per bucket) pulse duration of 1 mJ at 335 nm) (Quanta System, Mi- lano, Italy) from a distance of 15 m, which produced a lined with polyethylene bags and containing half-strength

Hoagland’s solution (Young and Terry, 1982) with either spot 6 cm in diameter. Thes-LIDAR is capable to carry out measurements up to 50 m of distance from the leaf 0 or 44.8 lM Fe(III)-EDTA. The nutrient solutions of

some of the buckets containing no Fe were buffered at (Goulas et al., 1994). Light coming from the leaf surface

(emitted Chl fluorescence plus reflected light) was col- (F9m2F)/F9v according to van Kooten and Snel (1990).

lected through a Fresnel lens (diameter 0.38 m) placed Therefore, FPSII is equivalent to the product qP3Fexc.. in the laser beam axis, and measured with a high speed Nonphotochemical quenching (NPQ) was calculated as crossed field photomultiplier (Sylvania, Model 502). Chlo- (Fm/F9m)21, according to Bilger and Bjo¨rkman (1990).

rophyll fluorescence and the backscattered signal were se- The fractions of light absorbed that are dissipated in the lected with a red highpass filter (RG-665, Schott, France) PSII antenna (D) and utilized in PSII photochemistry (P) and an interference 335 nm filter (Oriel, Stratford, Connec- were estimated from 12(F9v/F9m) and (F9v/F9m)·qP(Demmig- ticut), respectively. The current pulse from the photomul- Adams et al., 1996). It should be noted that, although tiplier output was digitized with a high bandwidth tran- the parameter P is equivalent to FPSII, both are com- sient analyzer (Tektronix SCD 1000, 1-GHz bandwidth). monly used in the literature. The fraction of light ab- Two ways of estimating the mean Chl fluorescence sorbed by PSII that is not used in photochemistry nor lifetime were used: i) the barycenter lifetime (sbar, de- dissipated in the PSII antenna (X) may reflect deexcita- fined as the difference between the barycenters of the tion of singlet excited Chl via the triplet pathway and was Chl fluorescence and the backscattered signals), which estimated from (F9v/F9m)·(12qP), according to Demmig- can be estimated easily during the experiment, and ii) Adams et al. (1996).

the deconvoluted lifetime (sm, mean Chl fluorescence lifetime calculated by deconvolution), which requires fur-

ther data analysis. Backscattered signals from 8 pulses RESULTS AND DISCUSSION were averaged and compared to the average of 8 Chl flu-

Effects of Iron Deficiency On The Mean orescence signals. This sequence was repeated every

Chlorophyll Fluorescence Lifetime at the 2 min, and sbar was calculated as described previously

Fo (Dark-Adapted) and Fs (Steady-State) (Cerovic et al., 1996). Deconvolution and calculation of

Fluorescence Levels s_m were also performed every 30 min as described in

The results obtained show that Fe deficiency increased Goulas et al. (1994) using the Fluomarqt II program

the mean Chl fluorescence lifetime both in dark-adapted (LURE, Orsay, France). In some experimentssm was the

leaves and at steady-state photosynthesis. These increases only parameter recorded to estimate the mean Chl fluo-

in Chl fluorescence lifetimes can be detected from a rescence lifetime. The correctness of the deconvolution

long distance with thes-LIDAR apparatus. After 30 min was judged from the examination of the distribution of

of dark-adaptation, control leaves (400–500 lmol Chl the weighed residuals between the calculated and the ex-

m²²) had mean Chl fluorescence lifetimes (sm) of approx- perimental function, and the autocorrelation function of

imately 0.40 ns (Fig. 1A). Dark-adapted, Fe-deficient the residue (Grinwald and Steinberg, 1974).

leaves showed marked increases ins_m when compared to the controls; moderately (150 lmol Chl m²²) and se- Near-Contact Modulated Chlorophyll

verely (50lmol Chl m²²) Fe-deficient leaves had s_m val- Fluorescence Measurements

ues of approximately 0.60 ns and 1.10 ns, respectively.

Modulated Chl fluorescence was measured at room tem-

At steady-state photosynthesis (350 lmol m²² s²¹ PAR), perature with a standard PAM-fluorimeter (Walz, Effel-

there was a linear increase insm with Fe deficiency from trich, Germany). Currently available fiber optic-based fluo-

the control values of 0.45 ns to 0.95 ns in severely Fe- rimeters can monitor Chl fluorescence and perform

deficient leaves (Fig. 1B). Higher sm values may be re- quenching analysis only at a distance of a few cm from the

lated to closure of PSII reaction centers and/or decreases leaf (Bolha`r-Nordenkampf et al., 1989). The experimental

of intrinsic PSII efficiency; both have been shown to oc- protocol for the analysis of the Chl fluorescence quench-

cur in illuminated, Fe-deficient sugar beet leaves (Mo- ing was essentially as described by Genty et al. (1989).

rales et al., 1998).

White light was obtained from Scho¨tt halogen lamps.Fm,

Little information is currently available in the litera- the maximal fluorescence yield in the dark, and F9m, the

ture on changes induced by other stresses on Chl fluo- maximal fluorescence yield during energization, were mea-

rescence lifetimes in intact leaves. Schneckenburger and sured at 100 kHz with a 1 s pulse of 5300 lmol photons

Frenz (1986) reported increases in Chl fluorescence life- m²² s²¹ of white light. F9m and F(Fs at steady-state photo-

times in spruce and pine needles after exposition to high synthesis) were measured during the photosynthetic induc-

O3 doses. Heat stress induced increases in Chl fluores- tion. The actual PSII efficiency (PSII efficiency at steady-

cence lifetime in barley leaves (Briantais et al., 1996).

state photosynthesis,FPSII) and the intrinsic PSII efficiency

Dark-adapted, water-stressed leaves had similar Chl fluo- (PSII efficiency with all PSII centers open,Fexc.) were cal-

rescence lifetimes than controls; however, once illumi- culated as (F9m2F)/F9m andF9v/F9m, respectively (Genty et al.,

nated water-stressed leaves showed decreases in the mean 1989; Harbinson et al., 1989), whereF9v is the variable part

Chl fluorescence lifetimes when compared to the con- of Chl fluorescence at any time of the photosynthetic in-

duction. Photochemical quenching (qP) was calculated as trols (Schmuck et al., 1992; Cerovic et al., 1996).

Figure 1. Mean chlorophyll fluorescence lifetime (sm, in ns) versus leaf total Chl (lmol m²²) in sugar beet leaves affected by Fe deficiency after dark adaptation for 30 min (A, solid circles) or at steady-state photosynthesis under 350lmol pho- tons m²²s²¹of white light (B, open circles). Data are mean6 SD of at least four replications. Curve-fitting methods were polynomial order 2 in A (r²50.82) and linear in B (r²50.94).

Figure 2. Changes in mean chlorophyll fluorescence lifetime (sbarin ns) and in chlorophyll fluorescence yield, during adap- Effects of Iron Deficiency on Chlorophyll

tation to a sudden increase in light intensity from 40–50 (natu- Fluorescence Lifetimes and Yields during a ral sunlight in the greenhouse at leaf level) to 350lmol pho- Sudden Increase in Light Intensity _{tons m}2²s²¹PAR. Control (400lmol Chl m²²; A), moderately (150lmol Chl m²²; B) and severely (50lmol Chl m²²; C) Fe- Iron deficiency also increased the Chl fluorescence life-

deficient leaves were used. The light level was increased or times during a sudden increase in light intensity from

decreased (indicated by arrows) by using a slide projector.

40–50 (natural sunlight in the greenhouse at leaf level) to 350lmol photons m²²s²¹PAR (carried out by switch-

ing on a slide projector). Under natural greenhouse light sbarvalues of approximately 0.45 ns, 0.70 ns, and 0.80 ns, control and Fe-deficient leaves had similar Chl fluores- respectively. After switching off the projector, Chl fluo- cence yields (approximately 1.0 relative units; Fig. 2). rescence yields and s_bar values became similar to those However, s_bar was higher in Fe-deficient leaves than in found at the beginning of the experiment.

controls; control, moderately and severely Fe-deficient The relationships between Chl fluorescence yields leaves had s_bar values of approximately 0.35 ns, 0.45 ns, and lifetimes could be described with polynomial curves and 0.50 ns (Figs. 2A, B, and C), respectively. Switching (Fig. 3). Close to linear, positive relationships between on the projector caused a transient increase in Chl fluo- Chl fluorescence yields and Chl fluorescence lifetimes rescence yield that was also accompanied by a transient have been reported when these parameters were changed increase insbarvalues. It should be mentioned that maxi- by photochemical quenching in algae (Moya, 1974) and mum fluorescence values are not depicted in Figure 2 by nonphotochemical quenching in nonstressed leaves because the sampling rate (1 data every 15 s) was too (Genty et al., 1992). Close to linear relationships were small to detect the peak maximum. At steady-state pho- recently found for several plant species in both control tosynthesis control and Fe-deficient leaves had Chl fluo- and water-stressed leaves (Cerovic et al., 1996). All these rescence yields of approximately 1.0 and 1.2 relative reports were carried out with green samples having nor- units. Thesbar values increased with Fe deficiency; con- mal photosynthetic pigment composition. A curvilinear relationship between fluorescence yields and lifetimes trol, moderately and severely Fe-deficient leaves showed

Figure 3. Relationships between chlorophyll fluorescence yield and mean chlorophyll fluorescence lifetime (sbar, in ns) during photosynthetic induction. Symbols were solid squares for controls (400lmol Chl m²²), open squares for moder- ately Fe-deficient leaves (150lmol Chl m²²) and open circles for severely Fe-deficient leaves (50lmol Chl m²²). Curve- fitting methods were polynomial order 3 (r²50.68, 0.95, and 0.95 in control, moderately and severely Fe-deficient leaves, respectively).

was found with changes in both photochemical (qP) and nonphotochemical quenching (NPQ) during fluorescence induction of non-stressed leaves (Malkin et al., 1980).

Iron deficiency induced larger changes in Chl fluo- rescence lifetimes than in fluorescence yields. Iron-defi- cient leaves had, for a given fluorescence yield, highersbar

values than control leaves, and this effect was more marked with severe Fe deficiency (Fig. 3). Larger changes in Chl fluorescence lifetimes than in fluorescence yields were previously found in detached branches of Quercus pu- bescens(Gu¨nther et al., 1994) and water-stressed leaves

of several plant species (Cerovic et al., 1996). Figure 4. Changes in actual PSII efficiency (FPSII; A), photo- chemical quenching (qP; B) and intrinsic PSII efficiency (Fexc.; C) during photosynthetic induction in control, moderately and Effects of Iron Deficiency on Chlorophyll

severely Fe-deficient leaves. Symbols as in Figure 3. White Fluorescence Quenchings during _{light (350}lmol photons m²²s²¹PAR) was switched on and Photosynthetic Induction off at 0 and 30 min, respectively.

In separate experiments we investigated the changes in Chl fluorescence quenchings, measured with near-contact

modulated fluorescence, during photosynthetic induction creases in both qPandFexc.. Severely Fe-deficient leaves showed, during the full time of induction, lower qPand in control and Fe-deficient leaves (Fig. 4). During most

of the photosynthetic induction, when the different types Fexc.values than the controls, and therefore had the low- est FPSII. After switching off the actinic lightFPSIIvalues of Chl fluorescence quenching develop, Fe-deficient

leaves had lower actual PSII efficiencies (FPSII) than con- increased in both control and Fe-deficient leaves, due to increases in bothqPandFexc.. In accordance with earlier trol leaves. Only during the first 4 min of induction did

moderately Fe-deficient leaves have similar FPSII values results (Belkhodja et al., 1998) qP decreased in severely Fe-deficient leaves with time during dark adaptation, to those found in control leaves (Fig. 4A). This was be-

cause moderately Fe-deficient leaves developed higher due to the reduction of the PQ pool.

Up to 12 min of photosynthetic induction nonphoto- photochemical quenching (qP; Fig. 4B) but had lower in-

trinsic PSII efficiency (Fexc.; Fig. 4C) than control leaves. chemical quenching (NPQ) development was quite simi- lar in control and Fe-deficient leaves (Fig. 5). In all After 4 min of induction, moderately Fe-deficient leaves

had lower FPSII values than control leaves, due to de- leaves there was a transient increase in NPQ, which

Figure 5. Changes in nonphotochemical quenching (NPQ) during photosynthetic induction in control, moderately and severely Fe-deficient sugar beet leaves. Symbols as in Figure 3. White light (350 lmol photons m²²s²¹PAR) was switched on and off at 0 and 30 min, respectively.

reached values of 1.50–2.00 after 2–4 min of induction.

At steady-state photosynthesis NPQ values were approxi- mately 0.60, 0.75, and 1.00 in control, moderately and severely Fe-deficient leaves, respectively. Ten minutes after switching off the light, NPQ values had decreased to approximately 0.13 in control and moderately Fe-defi- cient leaves, and to 0.25 in severely Fe-deficient leaves.

Effects of Iron Deficiency on the Distribution of the Energy Absorbed by Photosystem II during Photosynthetic Induction

Our data indicate that during photosynthetic induction Fe-deficient leaves modified the allocation of the light

Figure 6. Fractions of the light absorbed by the PSII absorbed by the PSII antenna, decreasing the proportion

antenna used in photochemistry (P), thermally dissi- used in photochemistry and increasing the proportion of pated (D) and not used in photochemistry nor dissi- light dissipated thermally. At the beginning of photosyn- pated in the antenna (X) during photosynthetic in- thetic induction all leaves had a transient increase in the duction in control (A), moderately (B) and severely

(C) Fe-deficient sugar beet leaves. Symbols as in Fig- amount of light dissipated thermally (D; Fig. 6). In con-

ure 3. In each plot the lower series of points repre- trol and Fe-deficient leavesD was maximum at 2–4 min

sents Dand the middle series of points represents of illumination (0.50–0.70), to reach steady-state values _D1X.White light (350lmol photons m²²s²¹ PAR) of 0.30, 0.40, and 0.50 in control, moderately and se- was switched on and off at 0 and 30 min, respectively.

verely Fe-deficient leaves, respectively. The amount of

light absorbed by PSII that was used in photochemistry control and Fe-deficient leaves. These data are in line (P, equivalent toFPSII) increased with the time of illumi- with results reported previously for Fe-deficient leaves at nation, reaching at steady-state photosynthesis values of steady-state photosynthesis (Morales et al., 1998).

approximately 0.58, 0.40, and 0.25 in control, moderately The amount of light absorbed by PSII that was ther- and severely Fe-deficient leaves, respectively. The amount mally dissipated (D) was linearly correlated to the extent of light absorbed by PSII that was not used in photo- of nonphotochemical quenching (NPQ) (Fig. 7). A signif- chemistry nor thermally dissipated (X) was maximum just icant amount of light (0.20–0.30) was dissipated ther- after switching on the light (0.70–0.75), and decreased to mally in control and Fe-deficient leaves at very low NPQ approximately 0.10–0.27 at steady-state photosynthesis. values. A value of 0.20 is the usual constitutive dissipa- This may indicate deexcitation of singlet excited Chl via tion level in the PSII antenna (Demmig-Adams et al., the triplet pathway during the first moments of illumina- 1996). It should be noted that dissipation of a given tion (Demmig-Adams et al., 1996). After switching off amount of light was done at lower NPQ values in Fe-

deficient leaves than in control leaves.

the light, P increased and D and X decreased in both

Figure 7. Relationships between the fraction of light ab- sorbed by the PSII antenna thermally dissipated (D) and non-photochemical quenching (NPQ) during photosyn- thetic induction in control, moderately and severely Fe- deficient sugar beet leaves. Symbols as in Figure 3. The relationships were linear (r²51.00 in all cases).

Relationship between Chlorophyll Fluorescence Lifetimes and Chlorophyll Fluorescence

Quenching Parameters

Our data show that for each type of leaf the correlation between NPQ or qP (measured by near-contact fluores- cence) and Chl fluorescence lifetime (sbar values, mea- sured by remote sensing) was linear and significant (Fig.

8). For a given NPQ value the Fe-deficient leaves always

Figure 8. Relationships between mean chlorophyll fluores- had higher sbar values than the controls, and this effect

cence lifetime (sbar, in ns) and nonphotochemical quench- was more marked in severely Fe-deficient leaves (Fig.

ing (NPQ; A) and photochemical quenching (qP; B) in con- 8A). The correlations betweens_barandqP(Fig. 8B) were trol, moderately and severely Fe-deficient sugar beet leaves.

negative in the case of severely deficient and control Symbols as in Figure 3. Chlorophyll fluorescence lifetimes leaves and positive in the case of moderately deficient were measured during adaptation to a sudden increase in light intensity from 40–50 (natural sunlight in the green- leaves. The data seem to indicate that during the photo-

house at leaf level) to 350lmol photons m²²s²¹PAR.

synthetic induction of control and Fe-deficient leaves the

Nonphotochemical quenching (NPQ) was measured during changes in mean Chl fluorescence lifetime were related photosynthetic induction at 350lmol photons m²²s²¹PAR.

to NPQ-mediated thermal dissipation and/or closure of Both parameters were plotted for a given time after illumi-

the PSII reaction centers. nation.

CONCLUSION

eccio´n General de Ensen˜anza Superior e Investigacio´n Cientı´fica and AIR3-CT94-1973 from the Commission of European Com- In summary, our data show that Fe deficiency increases

munities to J. A. The authors are indebted to Alexandre Zawad- the mean Chl fluorescence lifetime. This effect can be

zki for translating the computer program in Pascal, and to the measured from a long distance with the s-LIDAR de-

Laboratory of Plant Ecology (University Paris XI) for the use vice. In illuminated leaves, changes in the mean Chl flu- of their controlled growth chambers. F. M. and R. B. were sup- orescence lifetime seem to be associated to NPQ-medi- ported by a contract from the Spanish Ministry of Science and ated energy dissipation and/or closure of PSII reaction Education (MEC) and from the Spanish Institute of Coopera-

tion with the Arab World (ICMA), respectively.

centers. These data provide further support for the view that laser instrumentation, in combination with remote sensors, offers new perspectives for monitoring the ef-

REFERENCES fects of stress on photosynthesis at large spatial scales.

Abadı´a, J., and Abadı´a, A. (1993), Iron and plant pigments. In This work was supported by the EUREKA Project No. 380

(LASFLEUR) to I.M. and by Grants PB97-1176 from the Dir- Iron Chelation in Plants and Soil Microorganisms (L. L.

Barton, and B. C. Hemming, Eds.), Academic, New York, Kautsky, H., and Hirsch, A. (1931), Neue versuche zur kohlen- stoffassimilation.Naturwissenschaften 19:964.

pp. 327–343.

Belkhodja, R., Morales, F., Quı´lez, R., Lo´pez-Milla´n, A.-F., Ab- Krause, G. H., and Weis, E. (1984), Chlorophyll fluorescence as a tool in plant physiology. II. Interpretation of fluores- adı´a, A., and Abadı´a, J. (1998), Iron deficiency causes

changes in chlorophyll fluorescence due to the reduction in cence signals.Photosynth. Res.5:139–157.

Krause, G. H., and Weis, E. (1991), Chlorophyll fluorescence the dark of the photosystem II acceptor side.Photosynth.

Res.56:265–276. and photosynthesis: the basics. Annu. Rev. Plant Physiol.

Plant Mol. Biol. 42:313–349.

Bilger, W., and Bjo¨rkman, O. (1990), Role of the xanthophyll

cycle in photoprotection elucidated by measurements of Lichtenthaler, H. K. (1987), Chlorophyll and carotenoids: pig- ments of photosynthetic biomembranes. Meth. Enzymol.

light-induced absorbance changes, fluorescence and photo-

synthesis in leaves of Hedera canariensis. Photosynth. Res. 148:350–382.

Lichtenthaler, H. K., and Rinderle, U. (1988), The role of chlo- 25:173–185.

Bolha`r-Nordenkampf, H. R., Long, P. S., Baker, N. R., O¨quist, rophyll fluorescence in the detection of stress conditions in plants. CRC Crit. Rev. Anal. Chem.19:29–85.

G., Schreiber, U., and Lechner, E. G. (1989), Chlorophyll

fluorescence as a probe of the photosynthetic competence Malkin, S., Wong, D., Govindjee, and Merkelo, H. (1980), Par- allel measurements on fluorescence life-time and intensity of leaves in the field: a review of current instrumentation.

Funct. Ecol.3:497–514. changes from leaves during the fluorescence induction.Pho- tobiochem. Photobiophys. 1:83–89.

Briantais, J.-M., Dacosta, J., Goulas, Y., Ducruet, J.-M., and

Moya, I. (1996), Heat stress induces in leaves an increase of Morales, F., Abadı´a, A., and Abadı´a, J. (1990), Characterization of the xanthophyll cycle and other photosynthetic pigment the minimum level of chlorophyll fluorescence,Fo: a time-

resolved analysis.Photosynth. Res.48:189–196. changes induced by iron deficiency in sugar beet (Beta vul- garisL.).Plant Physiol.94:607–613.

Cecchi, G., Mazzinghi, P., Pantani, L., Valentini, R., Tirelli, D.,

and De Angelis, P. (1994), Remote sensing of chlorophyll a Morales, F., Abadı´a, A., and Abadı´a, J. (1991), Chlorophyll flu- orescence and photon yield of oxygen evolution in iron-defi- fluorescence of vegetation canopies: 1. Near and far field

measurement techniques.Remote Sens. Environ.47:18–28. cient sugar beet (Beta vulgaris L.) leaves. Plant Physiol.

97:886–893.

Cerovic, Z. G., Goulas, Y., Gorbunov, M., Briantais, J.-M., Ca-

menen, L., and Moya, I. (1996), Fluorosensing of water Morales, F., Abadı´a, A., Belkhodja, R., and Abadı´a, J. (1994), Iron deficiency-induced changes in the photosynthetic pig- stress in plants: diurnal changes of the mean lifetime and

yield of chlorophyll fluorescence, measured simultaneously ment composition of field-grown pear (Pyrus communisL.) leaves. Plant Cell Environ.17:1153–1160.

and at distance with as-LIDAR and a modified PAM-fluo-

rimeter, in maize, sugar beet, and kalanchoe¨.Remote Sens. Morales, F., Abadı´a, A., and Abadı´a, J. (1998), Photosynthesis, quenching of chlorophyll fluorescence and thermal energy Environ.58:311–321.

Demmig-Adams, B., Adams, W. W., III, Barker, D. H., Logan, dissipation in iron-deficient sugar beet leaves.Aust. J. Plant Physiol. 25:403–412.

B. A., Bowling, D. R., and Verhoeven, A. S. (1996), Using

chlorophyll fluorescence to assess the fraction of absorbed Moya, I. (1974), Dure´e de vie et rendement de fluorescence de la chlorophylle in vivo. Leur relation dans differents mo- light allocated to thermal dissipation of excess excitation.

Physiol. Plant.98:253–264. deles d’unite´s photosynthe`tique. Biochim. Biophys. Acta 368:214–227.

Genty, B., Briantais, J.-M., and Baker, N. R. (1989), The rela-

tionship between the quantum yield of photosynthetic elec- Moya, I., Guyot, G., and Goulas, Y. (1992), Remotely sensed blue and red fluorescence emission for monitoring vegeta- tron transport and quenching of chlorophyll fluorescence.

Biochim. Biophys. Acta990:87–92. tion. ISPRS J. Photogramm. Remote Sens.47:205–231.

Moya, I., Goulas, Y., Morales, F., Camenen, L., Guyot, G., and Genty, B., Goulas, Y., Dimon, B., Peltier, G., Briantais, J.-M.,

and Moya, I. (1992), Modulation of efficiency of primary Schmuck, G. (1995), Remote sensing of time-resolved chlo- rophyll fluorescence and back-scattering of the laser excita- conversion in leaves, mechanisms involved at PS2. In Re-

search in Photosynthesis (N. Murata, Ed.), Kluwer Aca- tion by the vegetation. EARSeL Adv. Remote Sens.

3:188–197.

demic, Dordrecht, pp. 603–610.

Goulas, Y., Camenen, L., Schmuck, G., Guyot, G., Morales, F., Rosema, A., Cecchi, G., and Pantani, L. (1988), Results of the LIFT project: air pollution effects on the fluorescence of and Moya, I. (1994), Picosecond fluorescence decay and

backscattering measurements of vegetation over distances. douglas fir and poplar. InApplications of Chlorophyll Fluo- rescence(H. K. Lichtenthaler, Ed.), Kluwer Academic, Dor- In Laser in Remote Sensing (C. Werner, and W. Waide-

licheds, Eds.), Springer-Verlag, New York, pp. 89–94. drecht, pp. 306–317.

Schmuck, G., Moya, I., Pedrini, A., Van Der Linde, D., Stober, Grinwald, A., and Steinberg, I. (1974), On the analysis of fluo-

rescence decay kinetics by the method of least-squares. F., and Schindler, C. (1992), Chlorophyll fluorescence life- time determination of water-stressed C3and C4plants.Rad- Anal. Biochem.59:583–598.

Gu¨nther, K. P., Dahn, H.-G., and Ludeker, W. (1994), Remote iat. Environ. Biophys.31:141–151.

Schneckenburger, H., and Frenz, M. (1986), Time-resolved flu- sensing vegetation status by laser-induced fluorescence.Re-

mote Sens. Environ.47:10–17. orescence of conifers exposed to environmental pollutants.

Radiat. Environ. Biophys.25:289–295.

Harbinson, J., Genty, B., and Baker, N. R. (1989), Relationship

between the quantum efficiencies of photosystem I and II Schreiber, U., and Bilger, W. (1993), Progress in chlorophyll fluorescence research: major developments during the past in pea leaves.Plant Physiol.90:1029–1034.

years in retrospect. In Progress in Botany (H. D. Behnke, Terry, N. (1980), Limiting factors in photosynthesis. I. Use of iron stress to control photochemical capacity in vivo. Plant U. Lu¨ttge, K. Esser, J. W. Kadereit, and M. Runge, Eds.),

Springer-Verlag, New York, pp. 151–173. Physiol. 65:114–120.

Terry, N., and Abadı´a, J. (1986), Function of iron in chloro- Schreiber, U., Schliwa, U., and Bilger, W. (1986), Continuous

recording of photochemical and non-photochemical chloro- plasts. J. Plant Nutr.9:609–646.

van Kooten, O., and Snel, J. F. H. (1990), The use of chloro- phyll fluorescence quenching with a new type of modulation

fluorometer.Photosynth. Res.10:51–62. phyll fluorescence nomenclature in plant stress physiology.

Photosynth. Res. 25:147–150.

Spiller, S., and Terry, N. (1980), Limiting factors in photosyn-

thesis. II. Iron stress diminishes photochemical capacity by Young, T. F., and Terry, N. (1982), Transport of iron into leaves following iron resupply to iron-stressed sugar beet reducing the number of photosynthetic units. Plant Phys-

iol.65:121–125. plants. J. Plant Nutr. 5:1273–1283.