Iron deficiency interrupts energy transfer from a disconnected part of theantenna to the rest of Photosystem II
Fermın Morales1,∗, Nicolae Moise2,3, Rebeca Quılez1, Anunciacion Abadıa1, Javier Abadıa1 &Ismael Moya2
1Department of Plant Nutrition, Aula Dei Experimental Station, Consejo Superior de Investigaciones Cientıficas,Apartado 202, E-50080 Zaragoza, Spain; 2Groupe Photosynthese et Teledetection, LURE/CNRS, Bat 203, CentreUniversitaire Paris-Sud, B.P. 34, 91898, Orsay cedex, France; 3Permanent address: The National Institute forLasers, Plasma and Radiation Physics, Lasers Department, 76900 Bucharest-Magurele, Romania; ∗Author forcorrespondence (e-mail: [email protected]; fax: +34-976-716145)
Received 13 February 2001; accepted in revised form 5 July 2001
Key words: Chl fluorescence induction, energy transfer, iron deficiency, phase fluorometry, Photosystem IIefficiency, sugar beet, time-resolved chlorophyll fluorescence
Abstract
Iron deficiency changed markedly the shape of the leaf chlorophyll fluorescence induction kinetics during a dark–light transition, the so-called Kautsky effect. Changes in chlorophyll fluorescence lifetime and yield were observed,increasing largely the minimal and the intermediate chlorophyll fluorescence levels, with a marked dip betweenthe intermediate and the maximum levels and loss of the secondary peak after the maximum. During the slowchanges, the lifetime–yield relationship was found to be linear and curvilinear (towards positive lifetime values)in control and Fe-deficient leaves, respectively. These results suggested that part of the Photosystem II antennain Fe-deficient leaves emits fluorescence with a long lifetime. In dark-adapted Fe-deficient leaves, measurementsin the picosecond–nanosecond time domain confirmed the presence of a 3.3-ns component, contributing to 15%of the total fluorescence. Computer simulations revealed that upon illumination such contribution is also presentand remains constant, indicating that energy transfer is partially interrupted in Fe-deficient leaves. PhotosystemII-enriched membrane fractions containing different pigment–protein complexes were isolated from control andFe-deficient leaves and characterized spectrophotometrically. The photosynthetic pigment composition of thefractions was also determined. Data revealed the presence of a novel pigment–protein complex induced by Fedeficiency and an enrichment of internal relative to peripheral antenna complexes. The data suggest a partialdisconnection between internal Photosystem II antenna complexes and the reaction center, which could lead toan underestimation of the Photosystem II efficiency in dark-adapted, low chlorophyll Fe-deficient leaves, usingchlorophyll fluorescence.
Chl fluorescence yield (or lifetime) during energization; Fo and O – minimal Chl fluorescence yield (or lifetime) inthe dark; Fp and P – maximal Chl fluorescence yield (or lifetime) during the Kautsky effect; Fpl and I – intermediateChl fluorescence yield (or lifetime) plateau during the Kautsky effect; Fs and T – steady-state Chl fluorescence yield(or lifetime) during the Kautsky effect; Fv/Fm – optimal quantum yield of PS II photochemistry; ϕ – phase shift;FWHM – full width at half maximum; OIDPSMT – levels of Chl fluorescence during the Kautsky effect; � – Chlfluorescence yield; �PSII=�F/F′
m – effective PS II quantum yield; τ – Chl fluorescence lifetime; LHC – light-harvesting complexes; m – demodulation factor; PAR – photosynthetic active radiation; PPFD – photosyntheticphoton flux density; PS I and PS II – Photosystem I and II, respectively; PS IIm – Photosystem II-enrichedmembranes; SPAD – portable Chl meter; V + A + Z – violaxanthin (V) + antheraxanthin (A) + zeaxanthin (Z)
208
Introduction
Iron deficiency decreases markedly photosyntheticrates. In sugar beet, Fe-deficient leaves have a reducednumber of granal and stromal lamellae per chloro-plast (Spiller and Terry 1980). This is accompaniedby decreases in all membrane components, includingelectron carriers of the photosynthetic electron trans-port chain (Spiller and Terry 1980; Terry 1980) andlight-harvesting pigments chlorophylls and caroten-oids (Abadía and Abadía 1993; Morales et al. 1990,1994). Because of these changes, thylakoids fromFe-deficient plants show characteristics of a ‘diluted’photosynthetic membrane (Terry and Abadía 1986).Also, Fe deficiency in sugar beet decreases RuBPcarboxilation capacity, through reduced Rubisco en-zyme activation (Taylor and Terry 1986; Winder andNishio 1995) and down-regulation of gene expression(Winder and Nishio 1995). Winder and Nishio (1995)proposed that the Fe deficiency-mediated decreases inlight harvesting, electron transport and carbon fixationare well co-ordinated.
In sugar beet, Spiller and Terry (1980) reportedthat Fe deficiency does not decrease photosyntheticenergy conversion efficiency; the decreases in pho-tosynthesis were due to reductions in the number ofphotosynthetic units per unit leaf area. Since then,different research groups have reported decreases ofphotosynthetic energy conversion efficiency in re-sponse to Fe deficiency. Evidence comes from higherplants (Morales et al. 1990, 1991), cyanobacteria(Guikema 1985) and eukaryotic marine algae (Greeneet al. 1992; Falkowski et al. 1995) using chlorophyll(Chl) fluorescence. This apparent discrepancy couldbe explained because Fe deficiency affects photosyn-thetic energy conversion efficiency only below a Chlconcentration threshold level (Morales et al. 1991,2000).
One of the most obvious characteristic of Fe-deficient leaves is chlorosis, due to low concentrationsper area of chlorophylls and carotenoids (Morales etal. 1990, 1994; Abadía and Abadía 1993). However,Fe deficiency does not decrease to the same extent allphotosynthetic pigments, Chl b being more affectedthan Chl a, and lutein and the V + A + Z xanthophyllsbeing less affected than the other carotenoids (Moraleset al. 1991, 1994, 2000). These data indicate a prefer-ential loss of Chl b and therefore of light-harvestingantenna complexes (LHC). Taking into account thecharacteristic photosynthetic pigment composition ofthe different LHCs (Ruban et al. 1994; Verhoeven
et al. 1999), these data may suggest different Fedeficiency-mediated rates of degradation of the differ-ent LHCs in leaves from higher plants. On the otherhand, it has been reported a novel pigment–proteincomplex associated with PS II induced by Fe defi-ciency in cyanobacteria (Riethman and Sherman 1988;named CP43′ in Burnap et al. 1993) and marked re-ductions in reaction center and core proteins relative tothe peripheral LHC II in Fe-limited algae (Vassiliev etal. 1995). Quite a few papers have investigated similaraspects in higher plants, some of them related to Fe-resupply to Fe-deficient plants (Nishio et al. 1985a, b;Abadía et al. 1989; Fodor et al. 1995).
The aim of this work was to investigate causes ofthe Fe deficiency-mediated decreases in dark-adaptedPS II efficiency that are found only in leaves below aChl concentration threshold level. Membrane prepara-tions enriched in PS II were obtained and fractionatedand their spectroscopic characteristics and photosyn-thetic pigment composition have been investigated. Acombined approach of phase fluorometry and the time-correlated single-photon counting techniques in thepicosecond–nanosecond time domain have been usedto investigate the relationship between mean chloro-phyll fluorescence lifetime (τ ) and yield (�) duringthe Kautsky effect and the heterogeneity of fluores-cence. Evidence is presented for a partially interruptedenergy transfer from the antenna to the PS II reactioncenter, which leads to an underestimation of PS II ef-ficiency in dark-adapted Fe-deficient sugar beet leaveswith low Chl concentrations.
Materials and methods
Plant material
Sugar beet (Beta vulgaris L. Monohil hybrid fromHilleshög, Landskrona, Sweden) was grown in growthchamber with a PPFD of 350 µmol m−2s−1 PAR ata temperature of 25 ◦C, 80% relative humidity and aphotoperiod of 16 h light/8 h dark. Seeds were germin-ated and grown in vermiculite for 2 weeks. Seedlingswere grown for two more weeks in half-strength Hoag-land nutrient solution with 45 µM Fe(III)-EDTA andthen transplanted to 20-l plastic buckets (four plantsper bucket) containing half-strength Hoagland nutrientsolution (Terry 1980) with either 0 or 45 µM Fe(III)-EDTA. The pH of the Fe-free nutrient solutions wasbuffered at approximately 7.7 by adding 1 mM NaOHand 1 g l−1 of CaCO3. This treatment simulates condi-tions usually found in the field leading to Fe deficiency
209
(Susín et al. 1994). Leaves sampled were young andexpanded recently.
Purified PS II membrane fractions (PS IIm) wereobtained as described previously (Berthold et al.1981; Dunahay et al. 1984) with some modifica-tions. Leaf samples were homogenized in an Osterizerkitchen blender in 400 mM NaCl, 20 mM Tricine,2 mM MgCl2, 0.2% BSA, pH 8.0. The brei wasfiltered through 2 Miracloth layers, and the filtratecentrifuged at 300 g for 1 min. The supernatant wascentrifuged at 10 000 g for 10 min. The pellet wasresuspended in 150 mM NaCl, 20 mM Tricine, 5 mMMgCl2, 0.2% BSA, pH 8.0, and centrifuged at 9000g for 10 min. Membranes were treated in 10 mMNaCl, 50 mM MES, 5 mM MgCl2, pH 6.0, with TritonX-100 (final Chl concentration 2 mg ml−1; Triton X-100:Chl, 25:1), incubated with stirring for 25 min at4 ◦C and centrifuged at 3500 g for 5 min. The super-natant was spun in an ultracentrifuge (23 000 rpm in a70Ti fixed angle rotor – 40 000 g –, 30 min), and thepellet (thereafter referred to as PS II-enriched mem-branes; PS IIm) was resuspended in 400 mM sucrose,10 mM NaCl, 50 mM MES, 5 mM MgCl2, pH 6.0,and frozen in liquid N2 until use.
Sucrose gradient separation of pigment-proteincomplexes was carried out as described previously(Bassi and Dainese 1989) with some modifications.Aliquots of 90 µl of PS IIm (approximately 4.5 µg Chlml−1) were resuspended in 100 µl of 2 mM EDTA–NaOH, pH 7.5, and centrifuged at 10 000 g in anEppendorf centrifuge for 5 min. A total of 250 µl of1% n-dodecyl-β-D-maltoside (DDM) in 5 mM Hepes-NaOH, pH 7.6, was added to the supernatant. TheDDM:Chl ratio was 5.6:1, 8:1 and 10:1 for control,severely and extremely Fe-deficient leaves, respect-ively. Final DDM concentration was approximately0.5%. After 40 min on ice and darkness, samples werecentrifuged in an Eppendorf centrifuge for 10 min; thesupernatant was then layered on the top of ultracentri-fuge tubes containing sucrose gradients (5 mM Hepes,0.06% DDM, pH 7.6) from 0.1 to 1 M. Tubes werecentrifuged (39 000 rpm in a SW41Ti rotor, 40 000 g)for 22 h and 30 min. Different colored bands were ob-tained. Bands were concentrated (Centricon-10, 4300g in a SM-24 rotor for 4–5 h). Once the volume wasreduced 10-fold, bands were recovered by centrifuga-tion at 2500 g for 5 min. Aliquots were frozen in liquidN2 until use.
Leaf chlorophyll determination and photosyntheticpigment composition of Photosystem II-enrichedmembrane preparations
Leaf chlorophyll concentration was estimated non-destructively with a SPAD-502 device (Minolta,Osaka, Japan). The SPAD-502 device uses two light-emitting diodes (650 and 940 nm) and a photodiodedetector to measure sequentially transmission throughleaves of red and infrared light, and once calibratedcan be used as an in situ Chl meter. For calibration,leaf disks with different degrees of Fe deficiency werefirst measured with the SPAD, then extracted with100% acetone in presence of Na ascorbate and finallychlorophyll measured spectrophotometrically (Abadíaand Abadía 1993). Control (300 µmol Chl m−2),severely (60 µmol Chl m−2) and extremely (20 µmolChl m−2) Fe-deficient leaves were used.
A volume of up to 100 µl of each band ob-tained by the sucrose gradient method described abovewas mixed up with de-ionized water (up to 200 µl).800 µl of methanol were then added (in presenceof Na ascorbate) and the mixture was introduced ina methanol-washed C18 Sep-Pak cartridge (WatersCorp., Milford, Massachusetts). Band 1 from controlleaves required 70% methanol instead of 80%. Pig-ments were retained on the top of the column andwere eluted with acetone. Pigments were analyzedby HPLC as described previously (de las Rivas et al.1989).
Spectroscopic characterization of the fractions
Spectra were run from 400 to 750 nm at room temper-ature in a Shimadzu 2101PC spectrophotometer witha 1-nm slit width.
Kinetic chlorophyll fluorescence lifetimemeasurements by phase fluorometry on leaves
Mean fluorescence lifetime and yield measurementsduring the Kautsky effect (rate constants in milli-seconds to seconds range) were made by using a newphase and modulation fluorometer designed and de-veloped at LURE (Orsay, France). The system is basedon a sinusoidally modulated (54.472 MHz) laser di-ode emitting at 635 nm (Philips CQL 845/D, 5 mW;Eindhoven, The Netherlands). The on-set of the excit-ation (PPFD 50 µmol m−2 s−1) was driven by a rapidshutter (opening time < 400 µs; UniBlitz ElectronicVS14, Raleigh, North Carolina). The illuminated leafarea was approximately 20 mm2. Fluorescence was
210
detected by an avalanche photodiode (HamamatsuS2384, Japan) after passing through a red cut-off fil-ter (Schott RG 665, 4 mm). After amplification, thehigh frequency signal supplied by the photodetectorwas analyzed by a home-built analog phase-sensitivedetector (3 kHz bandwidth, 0.01◦ phase sensitivity).Briefly, the system operates as follows. A sinusoid-ally intensity-modulated laser excitation at a circularfrequency, ω, results in a sinusoidally modulated fluor-escence, that is phase-shifted and demodulated relativeto the excitation. Therefore, three signals were sim-ultaneously acquired: phase shift (ϕ), demodulationfactor (m) and total fluorescence intensity (�). Bymeasuring ϕ and m we further compute two apparentmean lifetimes (Spencer and Weber 1969):
τP = 1
ωtg(ϕ)
τm = 1
ω
√1
m2− 1
where τm > τ p. It should be noted here that a multi-exponential fluorescence emission decay gives alwaysvalues of τm > τ p, and phase and modulation lifetimeswhich decrease with increasing frequency (Spencerand Weber 1969).
Instead of using a scattering sample, the phase ori-gin was determined for each measurement using Walzstandard (Effeltrich, Germany) and oxazine (Sigma,St Quentin Fallavier, France) as standards of fluor-escence, having calibrated lifetimes of 0.5 and 2.25ns, respectively (measured with the time-correlatedsingle-photon counting system described below). Sig-nals were monitored approximately during 3.6 (OIDPchanges; 512 points, 7 ms per point) and 86 s (PSMTchanges; 4300 points, 20 ms per point). Time resolu-tion was estimated to be between 0.3 and 3 ps, fromthe reproducibility of ϕ and m measurements. A moredetailed description of this set-up will be presented ina forthcoming paper (N. Moise, M. Bergher, S. Tostiand I. Moya, in preparation).
Picosecond chlorophyll fluorescence measurements atFo level on leaves
Chlorophyll fluorescence measurements at Fo levelwere made by using a time-correlated single-photoncounting system (see Briantais et al. 1996) developedat LURE (Orsay, France). The system is based on alaser diode emitting at 635 nm (Philips CQL 840/D,
Eindhoven, The Netherlands); the beam was de-focused to illuminate a leaf area of approximately4 × 8 mm (32 mm2). The excitation PPFD was0.01 µmol m−2s−1, 5-fold lower than that inducinga detectable actinic effect (Briantais et al. 1996). Ahome-made frequency generator/amplifier drives thediode in the pulsed mode; pulses of < 70 ps (FWHM)were obtained. The fluorescence collection efficiencywas improved by using large aperture optics (two anti-reflection coated plane-convex lenses, Melles Griot, φ
= 50 mm, f = 70 mm) and a high pass fluorescencefilter (Schott RG665, 4 mm). The lense system im-ages the illuminated area into the S20 photocathodeof a microchannel plate photomultiplier (HamamatsuR 3809U, Japan). The instrumental response function(< 80 ps FWHM) was recorded by exchanging thelong pass filter RG 665 by a 635-nm interference fil-ter. Deconvolution was made by using the FluomarqtII software developed at LURE (Orsay, France) anddescribed by Camenen et al. (1996); time resolutionbetter than 10 ps was obtained. The quality of thefits was judged by the reduced χ2 criterion and bythe random distribution of the weighted residuals (notshown). The mean Chl fluorescence lifetime (τmean) ofa multiexponential fluorescence emission was definedaccording to Lakowicz and Szmacinski (1996):
τmean = �ni=1αiτ
2i
�ni=1αiτi
= �ni=1fiτi
where τ i represent the individual lifetimes, αi, thepre-exponential factors and fi the fractional intensity.The fractional contribution of each component to thesteady-state total emission intensity, fi, is given by
fi = αiτi
�jαjτj= �i
�total
where �i is the fluorescence yield of each componentand �total is the total fluorescence yield. τmean givesalways values between τ p and τm (τ p < τmean < τm).
Results
Effects of Fe deficiency on the chlorophyllfluorescence lifetime–yield relationship
Phase fluorometry has been used to kinetically ana-lyze Chl fluorescence lifetimes (τ ) during a dark–light transition (Kautsky effect – OIDPSMT changes;Kautsky and Hirsch 1931), in which concomitantchanges in Chl fluorescence quantum yield (�) andlifetimes (τ ) occur (see below).
211
Figure 1. Changes in �, τp and τm during the OIDP (A and C) and PSMT (B and D) changes in control (A and B) and Fe-deficient (C andD) sugar beet leaves. OIDPSMT are the Chl fluorescence yields or lifetimes after approximately 7, 35, 168 ms and 2, 10, 17 and 80 s ofillumination, respectively. Insets of A and C are the first 500 ms of each Chl fluorescence curve upon illumination (from the uppermost to thelowest curve τm, � and τp, respectively).
Dark-adapted control leaves (approximately 300µmol Chl m−2) showed typical Chl fluorescence in-duction curves, both � and τ increasing (Figure 1A)from Fo to Fp (due to progressive QA reduction) anddecreasing (Figure 1B) from Fp to Fs (due to QAre-oxidation and non-photochemical quenching devel-opment). During the whole induction, τ p and τmchanged concomitantly but τm was always 100–200ps higher than τ p (Figures 1A, B). This indicates thatthe Chl fluorescence of dark-adapted control leavesis heterogeneous, containing components fluorescingwith different lifetimes (Spencer and Weber 1969).
Low Chl, Fe-deficient leaves (approximately40 µmol Chl m−2) showed, after dark-adaptation, re-latively high Fo and Fpl levels in the Kautsky Chlfluorescence induction curve when compared to thecontrols. These changes were observed both in � andτ (Figure 1C). In these leaves, there were marked
differences between τm and τ p. Differences betweenτm and τ p (�τ = 400–1000 ps) during the inductioncurve were maximal at Fo and Fs (Figures 1C, D).This suggests the presence in the Fe-deficient leavesof an important contribution of a component witha long Chl fluorescence lifetime (in the nanosecondrange, see below). In these leaves, � and τ p changedconcomitantly with time of illumination, whereas τmremained fairly constant after 30 s of illumination andincreased slightly at the end of the illumination period(Figure 1D). During the PSMT change, the secondarymaximum at M was not marked in Fe-deficient leaves(Figure 1D), contrary to the marked peak found in thecontrols (Figure 1B).
During the PSMT change, the τ p = f(�) and τm =f(�) relationships were linear in control leaves (Fig-ure 2A). Linear τ–� relationships have been previ-ously reported in algae or chloroplasts (Briantais et al.
212
Figure 2. τp = f(�) and τm = f(�) relationships during the PSMTchanges in control (A) and Fe-deficient (B) sugar beet leaves.
1972; Malkin et al. 1980; Moya et al. 1986) and havebeen assumed as a proof of a homogeneous natureof the Chl fluorescence emission (see ‘Discussion’for interpretations of the heterogeneous–homogeneousduality of the Chl fluorescence emission). In Fe-deficient leaves, however, there was a slight curvatureof the τ p = f(�) relationship towards positive τ values(Figure 2B). The τm = f(�) relationships were typ-ically curvilinear, curving towards positive τ values(Figure 2B). Data from the literature and our com-puter simulations (see ‘Discussion’) revealed that itwas due to the presence of a long lifetime, constantPS II fluorescence during the whole induction curve.Furthermore, the extrapolation at � = 0 was positivein the Fe-deficient leaves (Figure 2B), whereas in thecontrols it was negative for τp (Figure 2A). This hasbeen recently ascribed to a constant PS I emission,enhanced by Chl reabsorption (see ‘Discussion’). All
these data indicated that the heterogeneity of the Chlfluorescence in the case of low Chl, Fe-deficient leaveswas larger than in the controls.
Effects of Fe deficiency on the heterogeneity of thechlorophyll fluorescence at Fo
Time-correlated single-photon counting technique wasused to investigate such Chl fluorescence heterogen-eity. At Fo, four kinetic components were resolvedin control leaves (Table 1) with Chl fluorescence life-times of 60, 272, 550 ps and 1.30 ns, contributingto approximately 35, 44, 20 and 1% of the totalChl fluorescence, respectively. This gave a mean Chlfluorescence lifetime, τmean, of 265 ps (Table 1).Taking into account such Chl fluorescence heterogen-eity, calculated values at Fo (Lakowicz et al. 1984)for phase fluorometry and modulation at 54.472 MHzwere 260 (τ 54
p) and 387 ps (τ 54m). These values
agree with those measured with phase fluorometry anddemonstrate that the first point measured with phasefluorometry was not far from the real Fo point.
Using the time-correlated single-photon countingtechnique, four kinetic components were also resolvedin low Chl, Fe-deficient leaves (Table 1). Chlorophyllfluorescence lifetimes were 49, 302 ps, 1.11 and 3.3ns and contributing approximately to 20, 29, 36 and15% of the total Chl fluorescence at Fo, respectively.This gave a mean Chl fluorescence lifetime, τmean, of980 ps (Table 1). Taking into account such Chl fluores-cence heterogeneity, calculated values at Fo (Lakowiczet al. 1984) for phase fluorometry and modulation at54.472 MHz were 758 ps (τ 54
p) and 1.43 ns (τ 54m).
These values agree with those measured with phasefluorometry obtained from the first point of the Chlfluorescence inductions in Fe-deficient leaves.
Effects of Fe deficiency on the fractionation of thePhotosystem II-enriched membranes
Eight, five and three colored bands were resolvedwhen membranes enriched in PS II (PS IIm) isol-ated from control, severely and extremely Fe-deficientleaves respectively were centrifuged in a sucrosegradient. Bands from control leaves were numberedfrom 1 to 8 (Figure 3A). Percentages of Chl contentwere 2.1, 29.4, 39.8, 7.0, 3.8, 3.3, 8.7 and 5.8, re-spectively. Severely and extremely Fe-deficient leavesshowed bands 1, 2∗, 2, 3 and 4 (7.2, 18.7, 20.8, 23.2and 30% of the total Chl), and bands 1 (main band), 3and 4, respectively (Figures 3B, C).
213
Table 1. Chlorophyll fluorescence lifetimes (τ1 to τ4, τmean, τ54p and τ54
m in ps or ns) and fractional intensities (f, in % of total fluorescence)of control and Fe-deficient sugar beet leaves at Fo level. τ1 to τ4, f1 to f4 and τmean were measured with a time-correlated single-photoncounting device, and τ54
p and τ54m were calculated for phase fluorometry and modulation at 54.472 MHz taking into account the Chl
fluorescence heterogeneity measured with the time-correlated single-photon counting device (see text for further details). Measurements arethe mean ± SD of 5–7 replications
Table 2. Absorption maxima (in nm) of the fractions isolated from Photosys-tem II-enriched membranes from control, severely and extremely Fe-deficientsugar beet leaves. See text for assignment of pigment–protein complexesto each band. The absorption maxima of the non-fractionated PhotosystemII-enriched membranes from control and Fe-deficient sugar beet leaves havebeen reported elsewhere (Quılez et al. 1992)
Band no. Treatment Absorption maxima
1 Control 445.1 479.3 671.3
Severe −Fe 439.8 478.8 671.8
Extreme −Fe 444.8 480.6 674.0
2∗ Control
Severe −Fe 439.8 480.4 673.4
Extreme −Fe
2 Control 436.7 468.5 676.6
Severe −Fe 436.0 471.0 676.4
Extreme −Fe
3 Control 437.0 472.8 651.3 674.5
Severe −Fe 437.0 472.0 651.4 674.4
Extreme −Fe 436.0 473.4 651.8 674.8
4 Control 436.4 674.5
Severe −Fe 418.0 436.6 674.2
Extreme −Fe 418.0 436.0 674.0
5 Control 436.5 676.8
Severe −Fe
Extreme −Fe
6 Control 436.2 674.6
Severe −Fe
Extreme −Fe
7 Control 435.9 467.8 675.8
Severe −Fe
Extreme −Fe
8 Control 436.0 469.1 676.2
Severe −Fe
Extreme −Fe
214
Figure 3. Absorption spectra of the fractions isolated from Pho-tosystem II-enriched membranes from control (A), severely (B) andextremely (C) Fe-deficient sugar beet leaves. See text for assign-ment of pigment–protein complexes to each band. The absorptionspectra of the non-fractionated Photosystem II-enriched membranesfrom control and Fe-deficient sugar beet leaves have been reportedelsewhere (Quılez et al. 1992).
Spectroscopic characterization of the PhotosystemII-enriched fractions
All bands were characterized by their absorptionspectra (Figure 3). In PS IIm isolated from controlleaves, band 1 had a Chl maximum shifted to shorterwavelengths when compared to the other fractions(Table 2), suggesting the presence of free pigments.The high relative absorbance of this band in the 400–500-nm region (Figure 3A) reflects its high carotenoidcontent. Bands 2 and 3 showed an important relat-ive absorbance at 650 and 470 nm (corresponding toChl b and xanthophyll absorption), which could reflectthe presence of antenna pigment–protein complexes(Dainese et al. 1990). Band 2 had a shoulder at ap-proximately 650 nm (Figure 3A). Band 3 spectrum,with maxima at 651.3 and 674.5 nm and shoulderat approximately 480 nm (Figure 3A, Table 2), isanalogous to that of LHC II (Dainese et al. 1990).Bands 7 and 8 also had shoulders at 650 and 470nm (Figure 3A), which may indicate a relatively im-portant antenna content. Bands 4, 5 and 6 had thelowest relative absorbance at around 650 and 470 nm,and had a marked shoulder at approximately 420 nm,which is one of the characteristic absorption band ofβ-carotene. This suggests that these bands contain thePS II core complex (Green 1988).
In PS IIm isolated from severely Fe-deficientleaves, there was a band, 2∗, with no spectral ana-logy to any of the bands present in control PS IIm.Band 2∗ had a high carotenoid content (Figure 3B)and a red maxima at 673.4 nm (Table 2). Bands 2and 3 were spectrally analogous to those found in con-trol PS IIm (Figure 3B). Band 4 had spectrum with amarked shoulder at approximately 420 nm, a secondshoulder around 620 nm and a very low relative ab-sorbance in the 650 and 470 nm regions; all thesespectral properties are characteristic of the PS II corecomplex.
In the case of PS IIm isolated from extremelyFe-deficient leaves, there was a marked reduction infractions related to PS II antenna and only bands 1, 3and 4 were detected (Figure 3C). Band 1 showed theshift towards shorter wavelengths in the red maximaand had a high carotenoid content, similar to whathappens in control PS IIm (Figure 3C). Bands 3 and4 had spectral properties characteristic of antenna andcore complex, respectively (Figure 3C, Table 2).
215
Table 3. Photosynthetic pigment composition (in mmol pigment (mol Chl a)−1), and Chl a/Chl b and Z + A/V + A + Z ratiosof the fractions isolated from Photosystem II-enriched membranes from control and severely Fe-deficient sugar beet leaves. Seetext for assignment of pigment–protein complexes to each band. The photosynthetic pigment composition of the non-fractionatedPhotosystem II-enriched membranes from control and Fe-deficient sugar beet leaves have been reported elsewhere (Quılez et al.1992)
Band no. Treatment Pigment V A Lutein Z Chl b β-Carotene V + A + Z Chl a/Chl b Z + A/V + A + Z
neoxanthin
1 Control 37 489 33 985 0 145 110 522 7.0 0.06
Severe −Fe 70 80 167 600 261 152 283 508 6.6 0.84
2∗ Control
Severe −Fe 79 69 151 511 188 326 115 408 3.1 0.83
2 Control 53 133 12 310 0 561 17 145 1.8 0.08
Severe −Fe 69 43 75 283 92 462 40 210 2.2 0.80
3 Control 101 26 1 288 0 811 1 27 1.2 0.04
Severe −Fe 84 9 19 232 23 706 28 51 1.4 0.82
4 Control 19 16 1 77 0 183 100 17 5.6 0.06
Severe −Fe 1 2 5 12 2 33 143 9 30.3 0.78
5 Control 22 23 2 99 0 222 101 25 4.6 0.08
Severe −Fe
6 Control 17 13 2 72 0 152 148 15 6.7 0.13
Severe −Fe
7 Control 34 19 4 115 0 273 110 23 3.7 0.17
Severe −Fe
8 Control 44 24 5 141 0 336 90 29 3.0 0.17
Severe −Fe
Photosynthetic pigment composition of thePhotosystem II-enriched fractions
In control PS IIm, band 1 had the accessory pigmentslutein and violaxanthin as free pigments, and otherpigments such as Chl a and β-carotene (Table 3).The photosynthetic pigment composition of bands 2and 3, with a high content of xanthophylls and Chl band low β-carotene content, suggests that they con-tain antenna complexes. It should be noted that band2 had a larger proportion of violaxanthin and anther-axanthin and a higher Chl a/Chl b ratio than band 3(Table 3). Bands 4, 5 and 6 had relatively low con-centrations of pigments characteristic of PS II antenna(Chl b and xanthophylls) whereas they had importantamounts of β-carotene and rather high Chl a/Chl bratios (Table 3). These data would indicate that thesebands contain reaction center components. Bands 7and 8 had a relatively high concentration of Chl b andxanthophylls, but they had also a high concentrationof β-carotene (Table 3), indicating that they containa mixture of antenna and core PS II pigment–proteincomplexes. Band 8 had proportions of antenna pig-
ments (xanthophylls and Chl b) approximately 20%higher and proportions of β-carotene 20% lower thanband 7, which indicates that band 8 contains relativelymore antenna complexes than band 7.
In the case of PS IIm isolated from severely Fe-deficient leaves, it should be noted that bands 1 to 3had similar Z + A/(V + A + Z) ratios (0.80–0.84) anda gradual reduction in the V + A + Z/Chl a ratios frombands 1–3 (Table 3). Band 1 from Fe-deficient leaveshad more neoxanthin, antheraxanthin, zeaxanthin andβ-carotene, and less lutein and violaxanthin than thecontrol, whereas the Chl a/Chl b ratio was quite sim-ilar. The photosynthetic pigment composition of band2∗ was different from any of the PS IIm fractionsisolated from control leaves. Band 2∗ had signific-ant amounts of zeaxanthin, xanthophyll that was notpresent in control PS IIm (Table 3); it had approxim-ately double amount of V + A + Z than band 2, thefollowing band in the sucrose gradient; and it had morethan double and less than half the amount of Chl b andβ-carotene than band 1, the preceeding band in thesucrose gradient. Band 3 from Fe-deficient leaves wasanalogous in its photosynthetic pigment composition
216
to band 3 in the controls, which suggests that it alsocontains PS II external antenna polypeptides, LHC II;it had, however, slightly lower amounts of neoxanthin,lutein and Chl b than band 3 in the controls, and itshould be noted the presence of some β-carotene, al-most absent in the band 3 of the controls (Table 3).Band 4 from Fe-deficient leaves had high contents ofChl a and β-carotene, low concentrations of the an-tenna accessory pigments (neoxanthin, violaxanthinand lutein) and a Chl a/Chl b ratio of approximately30 (Table 3). This photosynthetic pigment composi-tion is characteristic of the PS II core complex and hadcertain similarity to bands 4 and 6 of control PS IIm.
Discussion
Most of the time-resolved Chl fluorescence measure-ments reported to date have been carried out in plantmaterials in which fluorescence has reached a steady-state level, including dark-adapted (Fo) and illumin-ated (Fm and Fs, in presence and absence of DCMU,respectively) samples (Briantais et al. 1996; Gilmoreet al. 1995; Holzwarth et al. 1985; Moya et al. 1986b;Schmuck et al. 1992). Such reports have shown the ex-istence of different (in most cases 4) Chl fluorescencecomponents with lifetimes ranging from picosecondsto nanoseconds. In sugar beet leaves (this work), fourkinetic components have been resolved. The fastestlifetime component (τ 1, 60 and 49 ps in control andFe-deficient leaves, respectively) is associated withPS I antenna fluorescence (Agati et al. 2000; Hodgesand Moya 1986; Holzwarth et al. 1985; Schmuck andMoya 1994). The higher contribution of τ 1 to the Fofluorescence in control leaves (34.5%) relative to lowChl, Fe-deficient leaves (20%) can be ascribed to twodifferent causes: Chl reabsorption and Fe deficiency-mediated changes in PS II/PS I stoichiometry. On onehand, Chl reabsorption enhances PS I contribution forwavelengths > 700 nm (Agati et al. 2000; Genty et al.1990; Pfundel 1998; Schmuck and Moya 1994; Trisslet al. 1993). On the other hand, Fe deficiency affectsmore PS I than PS II (Abadía et al. 1989; Fodor etal. 1995; Nishio et al. 1985b). τ 2 (272 and 302 psin control and Fe-deficient leaves, respectively) andτ 3 (550 ps in control leaves) are components modi-fied by PS II photochemistry and usually associatedwith PS II (Hodges and Moya 1986). In Fe-deficientleaves, τ 3 was 1.11 ns and affected by far-red illumin-ation (data not shown), which confirms the presenceof some closed PS II reaction centers in dark-adapted
Fe-deficient leaves (Belkhodja et al. 1998). Also, along lifetime component (3.3 ns) was observed in Fe-deficient leaves, which was not present in the controls.All these data indicate that the Chl fluorescence emis-sion is heterogeneous. However, the linear τ = f(�)relationship obtained using phase fluorometry may beassumed as a proof of a homogeneous nature of theChl fluorescence emission (Tumerman and Sorokin1967). Our data also showed that in control leaves dur-ing the PSMT changes the τ = f(�) relationship waslinear, in line with previous reports on algae or chloro-plasts (Briantais et al. 1972; Malkin et al. 1980; Moyaet al. 1986). This apparent ‘paradox’ can be explainedwhen taking into account that the PSMT changes aredominated by the development of non-photochemicalquenching, which shows roughly a dynamic quench-ing for the different components of the PS II Chlfluorescence emission (Genty et al. 1992), and there-fore affects similarly the quantum yield (�) and thelifetime (τ ).
In control, sugar beet leaves, the τ = f(�) rela-tionships gave negative values for τ at � = 0. Thiseffect has been recently ascribed to a constant PS Iemission, since when only PS II fluorescence was de-tected, using an interference filter centered at 682 nm(FWHM = 20 nm) instead of the long pass RG 665filter, the τ = f(�) relationship did extrapolate to zero(Apostol et al. 2001). Also, it should be mentionedthat in control leaves the extrapolation to � = 0 isshifted towards more negative values by Chl reabsorp-tion, that enhances PS I contribution for wavelengths> 700 nm (see above). A simulation was carried outbased on a simple mathematical model including oneshort lifetime, constant component superimposed toone variable component having a linear τ = f(�) re-lationship. Simulations (see ‘Appendix’) gave a linearτ = f(�) relationship with a negative value for τ at� = 0, as it was observed experimentally in phasefluorometry measurements in control leaves.
A marked curvature towards positive τ valuesof the τ = f(�) relationship was observed in Fe-deficient leaves during the PSMT changes. Moyaet al. (1977) reported a similar observation in isol-ated lettuce chloroplasts, which was interpreted as aconstant fluorescence emission superimposed to thevariable one, due to Chl disconnected during thechloroplast extraction. A similar curvature has beenobserved recently in dark-adapted leaves illuminatedwith a series of over-saturating pulses (Apostol et al.2001). Simulations were carried out based on simplemathematical models (see ‘Appendix’) including one
217
long lifetime, constant component superimposed toone variable component having a linear τ = f(�) re-lationship. The results of these simulations confirmedthat the curvature of the τ = f(�) relationship meas-ured with phase fluorometry in Fe-deficient leaves wasdue to the presence of a long lifetime and constantPS II fluorescence during the whole induction curve.All these data are in line with the presence in Fe-deficient leaves of a long lifetime component (3.3 ns)contributing to approximately 15% of the total fluor-escence. Therefore, the complementary use of phasefluorometry and time-correlated single-photon count-ing measurements indicates that approximately 15%of the PS II fluorescence in Fe-deficient leaves re-mained constant (unaffected by illumination) duringthe whole induction. We hypothesize that Fe defi-ciency interrupts energy transfer from a disconnectedpart of the antenna to the PS II reaction centers.
Possible candidates to be disconnected are the an-tenna pigment–protein complexes contained in band2∗, since this band was not present in control leaves.Alternatively, some internal antenna complexes maybe disconnected, because Fe deficiency decreasedmore peripheral than internal antenna complexes insugar beet. On one hand, band 2∗ has approximately19% of the total Chl in severely Fe-deficient leaves andthe long lifetime component contributes to approxim-ately 15% of the total Chl fluorescence at Fo level.On the other hand, band 2∗ was highly enriched inV + A + Z pigments (41 V + A + Z molecules per100 Chl a molecules; 34 out of 41 as A + Z), whichshould promote the formation of protein aggregates inthe antenna complexes (Ruban et al. 1997) that wouldemit Chl fluorescence with a short lifetime (Gilmore etal. 1995). We therefore hypothesize that some internalantenna complexes contained in band 4 of Fe-deficientleaves may be disconnected. These disconnected in-ternal antenna complexes could be responsible for theChl fluorescence with a lifetime of 3.3 ns. This Chlfluorescence lifetime is close to the 3.5 ns lifetimeobtained when these antenna complexes are discon-nected from the PS II reaction center (Bassi et al.1991).
Spiller and Terry (1980) reported that Fe deficiencydoes not decrease the photosynthetic energy conver-sion efficiency, estimated from gas exchange meas-urements. Since then, different research groups havereported decreases of photosynthetic energy conver-sion efficiency in response to Fe deficiency, includinghigher plants (Morales et al. 1990, 1991), cyanobac-teria (Guikema 1985) and eukaryotic marine algae
(Falkowski et al. 1995; Greene et al. 1992), using Chlfluorescence. Low, apparent PS II efficiency (Fv/Fm)in dark-adapted, low Chl Fe-deficient leaves is easilyexplained by the presence of a constant PS II emis-sion contributing to approximately 15% of the totalfluorescence (see above). When this constant fluor-escence was removed, values of 0.66 in Fe-deficientleaves become 0.73–0.83 (note that some of these val-ues are still lower than the controls, possibly due to thepresence of some closed PS II reaction centers in dark-adapted Fe-deficient leaves; Belkhodja et al. 1998). Asalready mentioned, the presence of some closed PSII reaction centers in dark-adapted Fe-deficient leavesincreased the τ 3 at Fo up to 1.11 ns. τ 3 was affectedby far-red illumination but far-red illumination hadno effect on the constant PS II fluorescence emission(data not shown). Therefore, there are two independ-ent reasons that lead to underestimate the Fv/Fm ratiosin Fe-deficient leaves: the presence of (i) some closedPS II reaction centers (Belkhodja et al. 1998) and (ii)constant PS II fluorescence emission (this work).
There are other cases in the literature where Chlfluorescence does not estimate accurately PS II elec-tron flow. For instance, in intermittent light greenedpea leaves (Fv/Fm = 0.62) there have been reportedtwo long lifetimes (1.7 and 3.8 ns) components (Bri-antais et al. 1996); once subtracted, assuming thatthey are ‘dead fluorescence’ (Briantais et al. 1996),Fv/Fm became 0.80. Barley etiochloroplasts have beenreported to have low Fv/Fm ratios, possibly result-ing from the contribution of a very long (3.5–6 ns)component ascribed to free Chl (Franz et al. 1995;Mysliwa-Kurdziel et al. 1997; Raskin and Murder1995). Also, Apostol et al. (2001) have reported appar-ent PS II electron transport rates estimated from Chlfluorescence measurements markedly reduced relativeto those estimated from gas exchange, in dark-adaptedleaves treated with a series of over-saturating pulses.In their work, it was reported one long lifetime com-ponent (1.2 ns) contributing with 38% of the totalfluorescence at Fs, that also led to underestimation ofthe PS II electron flow (Apostol et al. 2001). There-fore, in some plant materials or under certain stressconditions the Fv/Fm or �PSII (�F/F′
m; Genty et al.1989) parameters may not be accurate indicators of thePS II electron flow, because of the existence of a PS IIconstant fluorescence emission.
218
Figure 4. The effect of the addition of a constant fluorescence com-ponent (�2) to a variable component with a linear τ1 = f(�1)relationship. The constant component corresponds to: A) a shortlifetime of 60 ps (PS I contribution) or B) a long lifetime of 3.3ns (measured in Fe-deficient leaves). The inset shows the kineticsduring a simulating fluorescence induction of the variable compon-ent (�1), the constant component (�2) and the total fluorescenceemission (� = �1 + �2).
Acknowledgements
This work was supported by grants AGL-2000-1721from the Spanish Comisión Interministerial de Cienciay Tecnología to A.A., PB97-1176 from the Span-ish Dirección General de Investigación Científica yTécnica and INCO-Copernicus PL97-1176 from theCommission of European Communities to J.A. andAccess to Research Infrastructure action of the Im-proving Human Potential Program of the EuropeanCommunity to F.M. Support to R.Q. was provided bya fellowship from the Spanish Ministry of Educationand Science.
Appendix
Computer simulations using Mathematica 4.0 software (WolframResearch Inc., Champaign, Illinois) were made to investigate the ef-fect of superposing a constant fluorescence component (in yield andlifetime) to a variable component (with a linear τ = f(�) relation-ship). Results are shown in Figure 4. Two cases were considered.First, the constant component corresponds to the PS I fluorescencecontribution, with a lifetime of 60 ps and a fractional contributionof 35% at Fo level (accordingly to the time-correlated single-photon
counting results shown in Table 1). The PS I contribution didnot curve significantly the τ–� relationship on a visual inspec-tion, but did lead to a negative zero interception (Figure 4A). Thesecond case considered was a constant component correspondingto the long lifetime contribution obtained by time-correlated single-photon counting in the case of Fe-deficient leaves (3.3 ns lifetimeand 15% fractional contribution, accordingly to results shown inTable 1). Simulation results gave a noticeable curvature of the τm= f(�) relationship (Figure 4B). This curvature is similar to thatobserved in Fe-deficient leaves. The τp = f(�) relationship was lesscurved, in agreement also with phase and modulation fluorometrymeasurements.
It is worth noting that this simple model was used to investigatethe effect of the addition of a constant fluorescence component onthe τ = f(�) relationship, rather than as a model of the heterogeneityof the leaf Chl fluorescence emission. Anyway, the PS II fluor-escence emission is more complex showing at least two variablecomponents.
References
Abadía J and Abadía A (1993) Iron and plant pigments. In: Bar-ton LL and Hemming BC (eds) Iron Chelation in Plants andSoil Microorganisms, pp 327-344. Academic Press, San Diego,California
Abadía A, Lemoine Y, Trémolières A, Ambard-Bretteville F andRémy R (1989) Iron deficiency in pea: Effects on pigment, lipidand pigment-protein complex composition of thylakoids. PlantPhysiol Biochem 27: 679–687
Agati G, Cerovic ZG and Moya I (2000) The effect of decreas-ing temperature up to chilling values on the in vivo F685/F735chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisumsativum: The role of the Photosystem I contribution to the 735nm fluorescence band. Photochem Photobiol 72: 75–84
Apostol S, Briantais J-M, Moise N, Cerovic ZG and Moya I (2001)Photoinactivation of the photosynthetic electron transport chainby accumulation of over-saturating light pulses given to darkadapted pea leaves. Photosynth Res 67: 215–227
Bassi R and Dainese P (1989) The role of light harvesting complexII and of the minor chlorophyll a/b proteins in the organizationof the Photosystem II antenna system. In: Baltscheffsky M (ed)Current Research in Photosynthesis, Vol II, pp 209–216. KluwerAcademic Publishers, Dordrecht, The Netherlands
Bassi R, Silvestri M, Dainese P, Moya I and Giacometti GM (1991)Effects of a non-ionic detergent on the spectral properties andaggregation state of the light-harvesting chlorophyll a/b proteincomplex (LHC II). J Photochem Photobiol B Biol 9: 335–354
Belkhodja R, Morales F, Quílez R, López-Millán A-F, Abadía A andAbadía J (1998) Iron deficiency causes changes in chlorophyllfluorescence due to the reduction in the dark of the PhotosystemII acceptor side. Photosynth Res 56: 265–276
Berthold DA, Babcock GT and Yocum CA (1981) Highly re-solved oxygen-evolving Photosystem II preparation from spin-ach thylakoid membranes. FEBS Lett 134: 231–234
Briantais J-M, Merkelo H and Govindjee (1972) Lifetime of theexcited state (τ ) in vivo. III. Chlorophyll during fluorescenceinduction in Chlorella pyrenoidosa. Photosynthetica 6: 133–141
Briantais J-M, Dacosta J, Goulas Y, Ducruet J-M and Moya I(1996) Heat stress induces in leaves an increase of the minimumlevel of chlorophyll fluorescence, Fo: A time-resolved analysis.Photosynth Res 48: 189–196
Burnap RL, Troyan T and Sherman LA (1993) The highly abundantchlorophyll-protein complex of iron-deficient Synechococcus sp.
219
PCC7942 (CP43′) is encoded by the isiA gene. Plant Physiol 103:893–902
Camenen L, Goulas Y, Guyot G, Cerovic ZG, Schmuck G and MoyaI (1996) Estimation of the chlorophyll fluorescence lifetime ofplant canopies: Validation of a deconvolution method based onthe use of a 3-D canopy mockup. Remote Sens Environ 57: 79–87
Dainese P, Hoyer-Hansen G and Bassi R (1990) The resolution ofchlorophyll a/b binding proteins by a preparative method basedon flat bed isoelectric focusing. Photochem Photobiol 51: 693–703
de las Rivas J, Abadía A and Abadía J (1989) A new reversed phaseHPLC method resolving all major higher plant photosyntheticpigments. Plant Physiol 91: 190–192
Dunahay TG, Staehelin LA, Seibert M, Ogilvie PD and Berg SP(1984) Structural, biochemical and biophysical characteriza-tion of four oxygen-evolving Photosystem II preparations fromspinach. Biochim Biophys Acta 764: 179–193
Falkowski P, Behrenfeld M and Kolber Z (1995) Variations in photo-chemical energy conversion efficiency in oceanic phytoplankton:scaling from reaction center to the global ocean. In: Mathis P (ed)Photosynthesis: From Light to Biosphere, Vol V, pp 755–759.Kluwer Academic Publishers, Dordrecht, The Netherlands
Fodor F, Böddi B, Sárvári E, Záray G, Cseh E and Láng F(1995) Correlation of iron content, spectral forms of chlorophylland chlorophyll-proteins in iron deficient cucumber (Cucumissativus). Physiol Plant 93: 750–756
Franz F, Schoefs B, Batthélemy X, Mysliwa-Kurdziel B, StrzalkaK and Popovic R (1995) Protection of native chlorophyll(ide)forms and of Photosystem II against photodamage during theearly stages of chloroplast differentiation. Acta Physiol Plant 17:123–132
Genty B, Briantais J-M and Baker N (1989) The relationshipbetween the quantum yield of photosynthetic electron transportand quenching of chlorophyll fluorescence. Biochim BiophysActa 990: 87–92
Genty B, Wonders J and Baker N (1990) Non-photochemicalquenching of Fo in leaves is emission wavelength depend-ent: Consequences for quenching analysis and its interpretation.Photosynth Res 26: 133–139
Genty B, Goulas Y, Dimon B, Peltier G, Briantais J-M and MoyaI (1992) Modulation of efficiency of primary conversion inleaves, mechanisms involved at PS2. In: Murata N (ed) Researchin Photosynthesis, pp 603-610. Kluwer Academic Publishers,Dordrecht, The Netherlands
Gilmore AM, Hazlett TL and Govindjee (1995) Xanthophyll cycledependent quenching of Photosystem II chlorophyll a fluores-cence: formation of a quenching complex with a short lifetime.Proc Natl Acad Sci USA 92: 2273–2277
Green BR (1988) The chlorophyll-protein complexes of higherplant photosynthetic membranes or just what green band is that?Photosynth Res 15: 3–32
Greene RM, Geider RJ, Kolber Z and Falkowski PG (1992) Iron-induced changes in light harvesting and photochemical energyconversion processes in eukaryotic marine algae. Plant Physiol100: 565–575
Guikema JA (1985) Fluorescence induction characteristics ofAnacystis nidulans during recovery from iron deficiency. J PlantNutr 8: 891–908
Hodges M and Moya I (1986) Time-resolved chlorophyll fluor-escence studies of photosynthetic membranes: Resolution andcharacterisation of four kinetics components. Biochim BiophysActa 849: 193–202
Holzwarth AR, Wendler J and Haehnel W (1985) Time-resolvedpicosecond fluorescence spectra of the antenna chlorophylls inChlorella vulgaris. Resolution of Photosystem I fluorescence.Biochim Biophys Acta 807: 155–167
Kautsky H and Hirsch A (1931) Neue versuche zur kohlenstof-fassimilation. Naturwissenschaften 19: 964
Lakowicz JR and Szmacinski H (1996) Imaging applications oftime-resolved fluorescence spectroscopy. In: Wang XF and Her-man B (eds) Fluorescence Imaging Spectroscopy and Micro-scopy, pp 273–311. John Wiley & Sons, New York
Lakowicz JR, Laczko G, Cherek H, Gratton E and Limkeman M(1984) Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data. Biophys J 46:463–477
Malkin S, Wong D, Govindjee and Merkelo H (1980) Parallelmeasurements on fluorescence life-time and intensity changesfrom leaves during the fluorescence induction. PhotobiochemPhotobiophys 1: 83–89
Morales F, Abadía A and Abadía J (1990) Characterization of thexanthophyll cycle and other photosynthetic pigment changes in-duced by iron deficiency in sugar beet (Beta vulgaris L.). PlantPhysiol 94: 607–613
Morales F, Abadía A and Abadía J (1991) Chlorophyll fluorescenceand photon yield of oxygen evolution in iron-deficient sugar beet(Beta vulgaris L.) leaves. Plant Physiol 97: 886–893
Morales F, Abadía A, Belkhodja R and Abadía J (1994) Irondeficiency-induced changes in the photosynthetic pigment com-position of field-grown pear (Pyrus communis L.) leaves. PlantCell Environ 17: 1153–1160
Morales F, Belkhodja R, Abadía A and Abadía J (2000) Pho-tosystem II efficiency and mechanisms of energy dissipationin iron-deficient, field-grown pear trees (Pyrus communis L.).Photosynth Res 63: 9–21
Moya I, Govindjee, Vernotte C and Briantais J-M (1977) Antag-onistic effect of mono- and divalent-cations on lifetime (τ ) andquantum yield of fluorescence (�) in isolated chloroplasts. FEBSLett 75: 13–18
Moya I, Hodges M and Barbet J-C (1986a) Modification of room-temperature picosecond chlorophyll fluorescence kinetics ingreen algae by Photosystem II trap closure. FEBS Lett 198:256–262
Moya I, Sebban P and Hahenel W (1986b) Lifetime of excited statesand quantum yield of chlorophyll a fluorescence in vivo. In:Govindjee et al. (eds) Light Emission by Plants and Bacteria,pp 161–190. Academic Press, San Diego, California
Mysliwa-Kurdziel B, Barthélemy X, Strzalka K and Franck F(1997) The early stages of Photosystem II assembly monitoredby measurements of fluorescence lifetime, fluorescence induc-tion and isoelectric focusing of chlorophyll-proteins in barleyetiochloroplasts. Plant Cell Physiol 38: 1187–1196
Nishio JN, Abadía J and Terry N (1985a) Chlorophyll-proteinsand electron transport during iron nutrition-mediated chloroplastdevelopment. Plant Physiol 78: 296–299
Nishio JN, Taylor SE and Terry N (1985b) Changes in thylakoidgalactolipids and proteins during iron nutrition-mediated chloro-plast development. Plant Physiol 77: 705–711
Pfundel E (1998) Estimating the contribution of Photosystem I tototal leaf chlorophyll fluorescence. Photosynth Res 56: 185–195
Quílez R, Abadía A and Abadía J (1992) Characteristics ofthylakoids and photosystem II membrane preparations from irondeficient and iron sufficient sugar beet (Beta vulgaris L.). J PlantNutr 15: 1809–1819
220
Raskin VI and Murder JB (1995) Chlorophyll organization in dark-grown and light-grown pine (Pinus brutia) and barley (Hordeumvulgare). Physiol Plant 101: 620–626
Riethman HC and Sherman LA (1988) Purification and character-ization of an iron stress-induced chlorophyll–protein from thecyanobacterium Anacystis nidulans R2. Biochim Biophys Acta935: 141–151
Ruban AV, Young AJ, Pascal AA and Horton P (1994) The effects ofillumination on the xanthophyll composition of the PhotosystemII light-harvesting complexes of spinach thylakoid membranes.Plant Physiol 104: 227–234
Ruban AV, Philip D, Young AJ and Horton P (1997) Carotenoid-dependent oligomerization of the major light harvesting complexof Photosystem II in plants. Biochemistry 36: 7855–7859
Schmuck G and Moya I (1994) Time resolved chlorophyll fluores-cence spectra of intact leaves. Remote Sens Environ 47: 72–76
Schmuck G, Moya I, Pedrini A, Van der Linde D, LichtenthalerHK, Strober F, Schindler C and Goulas Y (1992) Chlorophyllfluorescence lifetime determination of water-stressed C3 and C4plants. Radiat Environ Biophys 31: 141–151
Spencer RD and Weber G (1969) Measurements of subnanosecondfluorescence lifetimes with a cross-correlation phase fluorometer.Ann N Y Acad Sci 158: 361–376
Spiller S and Terry N (1980) Limiting factors in photosynthesis. II.Iron stress diminishes photochemical capacity by reducing thenumber of photosynthetic units. Plant Physiol 65: 121–125
Susín S, Abián J, Peleato ML, Sánchez-Baeza J, Abadía A, Gelpí Eand Abadía J (1994) Flavin excretion from iron deficient sugar
beet (Beta vulgaris L.). Planta 193: 514–519Taylor SE and Terry N (1986) Variation in photosynthetic elec-
tron transport capacity and its effect on the light modulation ofribulose bisphosphate carboxilase. Photosynth Res 8: 249–256
Terry N (1980) Limiting factors in photosynthesis. I. Use of ironstress to diminish photochemical capacity in vivo. Plant Physiol65: 114–120
Terry N and Abadía J (1986) Function of iron in chloroplasts. J PlantNutr 9: 609–646
Trissl H-W, Gao Y and Wulf K (1993) Theoretical fluorescenceinduction curves derived from coupled differential equations de-scribing the primary photochemistry of Photosystem II by anexciton/radical pair equilibrium. Biophys J 64: 984–998
Tumerman LA and Sorokin EM (1967) Fotosinteticheskaya edin-itsa: ‘fizicheskaya’ ili ‘statisticheskaya’ model? [The photosyn-thetic unit: A ‘physical’ or ‘statistical’ model?]. Mol Biol 1:628–638
Vassiliev IR, Kolber Z, Wyman KD, Mauzerall D, Shukla VK andFalkowski PG (1995) Effects of iron limitation on Photosystem IIcomposition and light utilization in Dunaliella terciolecta. PlantPhysiol 109: 963–972
Verhoeven AS, Adams III WW, Demmig-Adams B, Croce R andBassi R (1999) Xanthophyll cycle pigment localization and dy-namics during exposure to low temperature and light stress inVinca major. Plant Physiol 120: 727–737
Winder TL and Nishio J (1995) Early iron deficiency stress responsein leaves of sugar beet. Plant Physiol 108: 1487–1494
