Planta (2005) 221: 567–579
DOI 10.1007/s00425-004-1464-6
O R I GI N A L A R T IC L E
Jean-Marc Ducruet Æ Miruna Roman Æ Michel Havaux
Tibor Janda Æ André Gallais
Cyclic electron flow around PSI monitored by afterglow luminescence
in leaves of maize inbred lines (Zea mays L.): correlation with chilling
tolerance
Received: 1 August 2004 / Accepted: 2 November 2004 / Published online: 2 February 2005
Springer-Verlag 2005
Abstract Maize (Zea mays L.) inbred lines of contrasting
chilling sensitivity (three tolerant, three sensitive lines)
were acclimated to 280 lmol photons m2 s1 white
light at a 17C sub-optimal temperature. They showed
no symptoms of photoinhibition, despite slight changes
in photosystem II (PSII) fluorescence and thermoluminescence properties in two tolerant lines. A luminescence
‘‘afterglow’’ emission [Bertsch and Azzi (1965) Biochim
Biophys Acta 94:15–26], inducible by a far-red (FR)
illumination of unfrozen leaf discs, was detected either
as a bounce in decay kinetics at constant temperatures
or as a sharp thermoluminescence afterglow band at
about 45C, in dark-adapted leaves. This band reflects
the induction by warming of an electron pathway from
stromal reductants to plastoquinones and to the QB
secondary acceptor of PSII, resulting in a luminescenceemitting charge recombination in the fraction of centres
that were initially in the S2/3QB non-luminescent state. A
5-h exposure of plants to growth chamber light shifted
this luminescence emission towards shorter times and
lower temperatures for several hours in the three chill-
J.-M. Ducruet (&) Æ M. Roman
Service de Bioénergétique, INRA/CEA-Saclay,
91191 Gif-sur-Yvette cedex, France
E-mail: ducruet@dsvidf.cea.fr
M. Roman
Lasers Department, INFLPR,
POB MG-36, Bucuresti-Magurele, Romania
M. Havaux
Laboratoire d’Ecophysiologie de la Photosynthèse,
CEA-Cadarache, DSV, DEVM,
UMR 6191 CNRS-CEA-Aix-Marseille II,
13108 St Paul-lez-Durance, France
T. Janda
Agricultural Research Institute of the Hungarian
Academy of Sciences, 2462 Martonvasar, POB 19, Hungary
A. Gallais
INRA/INAPG/U-Paris-Sud: Génétique Végétale,
ferme du Moulon, 91190 Gif-sur-Yvette, France
ing-tolerant lines. This downshift was not observed, or
only transiently, in the three sensitive lines. In darkness,
the downshifted afterglow band relaxed within hours to
resume its dark-adapted location, similar for all maize
lines. A faster dark re-reduction of P700+ oxidized by
FR light (monitored by 820-nm absorbance) and an
increase of photochemical energy storage under FR
excitation (determined by photoacoustic spectroscopy)
confirmed that a cyclic pathway induced by white actinic
light remained activated for several hours in the tolerant
maize lines.
Keywords Chlororespiration Æ Ferredoxineplastoquinone-reductase Æ Photoinhibition Æ
Thermoluminescence Æ Photosystem II Æ
Photosystem I-cyclic electron transfer
Abbreviations DCMU: 3-(3¢,4¢-Dichlorophenyl)-1,
1-dimethylurea (diuron), an inhibitor of photosystem II
that binds to the QB pocket Æ Ea: Apparent activation
energy (enthalpy) of charge recombination Æ 1F, 2F: 1 or
2 flashes Æ FQR: Ferredoxine-plastoquinonereductase Æ F0, FV, FM: Basal, variable and maximal
chlorophyll fluorescence levels Æ PSI: Photosystem I Æ
PSII: Photosystem II Æ QB: Secondary quinonic acceptor
of photosystem II Æ NDH: NAD(P)H dehydrogenase
(plastoquinone reductase) Æ R, S: Chilling-tolerant and
sensitive maize lines Æ TL: Thermoluminescence Æ Tm:
Temperature maximum of a thermoluminescence band
Introduction
Cyclic electron flow driven by photosystem I (PSI)
produces ATP and no NADPH (Arnon 1959; for a review see Bukhov and Carpentier 2004). A related phenomenon is chlororespiration (Bennoun 1982; Garab
et al. 1989; for a review see Peltier and Cournac 2002),
which consists of an electron transfer in darkness from
568
the reducing stroma-exposed side of the PSI to the
plastoquinone pool, then to oxygen via one or several
oxidases. In the plastoquinone reducing part of the
chlororespiratory chain, electrons come from NAD(P)H
via an NDH enzymatic complex, but there is another
pathway, ferredoxine-plastoquinone-reductase or FQR,
that also causes a dark reduction of the plastoquinones
(PQ) (Ravenel et al. 1994; Bendall and Manasse 1995;
Scheller 1996; Shikanai et al. 1998). The NDH is a large
protein complex of which inactive NDH mutants are
available (Shikanai et al. 1998), but the FQR pathway
had no known molecular support until recently, when a
PGR5 protein was found necessary for this activity
(Munekage et al. 2002).
Photoacoustic spectroscopy provides the most direct
measurement of cyclic flow as an increase in energy
storage (ES) under excitation by far-red (FR) light almost exclusively absorbed by PSI (Malkin and Canaani
1994). A more indirect method relies on the re-reduction
kinetics of P700+, the primary donor to PSI monitored
by its absorbance change at 820 nm, after an oxidation
of P700 by FR light (Schreiber et al. 1988). An activation of a cyclic pathway that provides electrons to
P700+ accelerates this reduction rate. Increase in light
scattering at 530 nm reflects the pumping of protons
into the lumen by the cyclic flow (Cornic et al. 2000).
Chlororespiration can be detected in leaves essentially
through the dark reduction of the PQ, either from a
decrease of the complementary area above the chlorophyll fluorescence-induction curve (Bennoun 1982) or
from an increase of F0 fluorescence monitored by a weak
analytical light after an FR excitation (Feild et al. 1998;
Shikanai et al. 1998). A very early discovered chlorophyll luminescence emission appearing as a delayed
bounce in darkness following an FR illumination
(Bertsch and Azzi 1965) has also been shown to be related to the dark reduction of plastoquinone (Sundblad
et al. 1988), on which it can provide complementary
information.
A charge pair separated photochemically is stabilized
on electron carriers by activation of energy barriers that
limits the back-reaction, i.e. charge recombination,
within the temperature adaptation range of every living
organism. In oxygen-evolving organisms, the recombination of charge pairs stabilized on photosystem II
(PSII) centres generates, with a low yield, a delayed
fluorescence emission called luminescence. When recorded at a constant temperature after an illumination,
it consists of multiexponential luminescence decays difficult to analyse. Thermoluminescence (TL) is a higher
resolving technique that consists in cooling the sample
before or just after an illumination at a sufficiently low
temperature—not necessarily freezing. The low temperature makes the recombination of the charge pairs
investigated negligibly slow, so they appear as successive
emission bands during a progressive warming (for a review, see Ducruet 2003).
In an unstressed photosynthetic material, the luminescence-emitting pairs produced photochemically con-
sist mainly of an electron stabilized on the quinonic
secondary acceptor QB and of positive charges stored on
the manganese oxygen-evolving complex (S states) on
the lumen side of PSII. S4+ that allows oxygen evolution
from water is short-lived. Two of the four S0–S3 states
form the S2Q
B and S3QB luminescence-emitting pairs
producing the B-band emission. However, the initial
charge pattern created by light may evolve during the
subsequent dark period, generating complex non-exponential decay phases. In intact photosynthetic systems
such as whole chloroplasts, algal cells or leaf tissues, a
luminescence bounce—or afterglow—superimposed to
the tail of the initial decay phases, can most often be
induced by an FR pre-illumination (Bertsch and Azzi
1965). This emission may also occur after white light or
xenon-flash excitation, depending on species and growth
conditions of plants or algae (Sundblad et al. 1988;
Miranda and Ducruet 1995; Krieger et al. 1998).The
emission results from the progressive back-transfer of
electrons, originating from a pool of reducing compounds present in the stroma towards the plastoquinone
pool and QB in PSII centres, with the need for a thylakoid proton gradient (Sundblad et al. 1988). Whereas
PSII centres initially in the luminescence-emitting state
S2/3Q
B produce an exponential decay or a B band
(Rutherford and Inoue 1984), no luminescence can be
emitted by centres initially in the S2/3 QB state, unless
they become enabled for that when QB is reduced by
electron back-transfer: this corresponds to the afterglow
emission.
In all higher plant species assayed so far, darkadapted leaves submitted to FR light exhibit both (1) a
TL B band (S2/3Q
B ) downshifted by lumen acidification
and (2) a band at about 45C that behaves similarly to
the afterglow superimposed to luminescence decay at
constant temperatures (Miranda and Ducruet 1995).
The afterglow emission is enhanced by increasing temperature (Bertsch and Azzi 1965), so that the TL
warming transforms the broad afterglow bounce seen at
constant temperature into a sharper and more intense
TL band usually at 45C, as will be further demonstrated in this paper. The fluorescence complementary
area, related to FV, decreases at temperatures that induce the afterglow emission, confirming a heat-induced
reduction of PQ (Constantin-Roman 2000). Indeed, it
has been shown by different techniques that temperatures above 30C activate the electron transfer from
stroma reductants to quinones (Havaux 1996; Lajko
et al. 1997). The 30–40C temperatures, although below
the 40C threshold of heat damage to PSII detectable by
the rise of F0 fluorescence (Schreiber and Berry 1977),
induce a re-organization of thylakoid membranes with
an unstacking of grana (Weis 1984), an increase of b
non-connected centres relatively to a PSII centres
(Sundby et al. 1986) and a modification of PSII properties (Havaux 1993). These reports suggest that grana
unstacking favours the cyclic electron pathway around
the PSI centres located in stroma lamellae (for a review,
see Albertsonn 2001). The afterglow emission is sup-
569
pressed by uncouplers, by freezing and by DCMU-like
inhibitors of PSII. Its oscillation with period 4 after flash
sequences demonstrates the role of S states in the
emission mechanism (Sundblad et al. 1988; Miranda and
Ducruet 1995). An essential property of this afterglow
emission is to be suppressed by low concentrations of
antimycin A (Nakamoto et al. 1988), an inhibition that
we could reproduce in peeled tobacco leaves and in
Chlamydomonas reinhardtii. Antimycin A also inhibits
the FQR activity and the cyclic electron flow at about
1 lM (Bendall and Manasse 1995). This suggests that
the FQR and cyclic pathways share a common electron
carrier blocked by 1 lM antimycin A and hence are
mechanistically closely related.
The physiological roles of PSI-dependent cyclic and
of chlororespiratory electron flows remain unclear.
There is still questioning about the role of cyclic photophosphorylation in addition to linear electron flow to
maintain the ATP/NADPH ratio needed for CO2
assimilation: cyclic electron transport would come into
play only at higher photon flux densities and its role
seems to control the activity of PSII by increasing the
proton gradient, hence the non-photochemical quenching (Heber et al. 1995; Cornic et al. 2000).
In this paper, we show in maize leaf discs that white
light modifies this afterglow emission, which reflects the
dark reduction of QB, in the same way as it triggers the
cyclic pathway, as monitored by both the dark rereduction kinetics of P700+ after an FR illumination
and by photoacoustic spectroscopy. Furthermore, the
relaxation of the afterglow band towards its darkadapted location occurs much faster in the chillingsensitive than in the chilling-tolerant maize inbred lines
assayed here, indicating that the cyclic and/or chlororespiratory pathways might play a role in chilling
tolerance.
Materials and methods
Plant material
Maize (Zea mays L.) inbred lines F2, F618, Co329 and
Io were obtained from the Station d’Amélioration des
Plantes INRA (Ferme du Moulon, F-91190 Gif-surYvette). Line F2 is one of the early European flint inbreds obtained from altitude population Lacaune in
1951 and has been used in combination with more
productive American dent inbreds to extend maize cultivation towards Northern Europe (Messmer et al.
1993). Blondon et al. (1981) have shown that a 10-day
hardening at 10C shifts the lowest edge of the thermal
photosynthetic optimum of the F2·F7 hybrid from
19.1C to 16.9C, instead of 19.3C to 18.6C for the
American WH·WJ. The Canadian line Co329 has been
selected as a moderately chilling-tolerant line following
field assays in Northern France (C. Giauffret, personal
communication; FRASEMA company, unpublished results). Io and INRA F618 lines are chilling-sensitive.
Inbred lines 1872 (INRA F217) and 1194, provided by
Euralis Company, are chilling-tolerant and chillingsensitive, respectively. Havaux (1987) has demonstrated
a greater chilling-sensitivity of photosynthetic electron
transfer in 1194 than in 1872. Differences in phosphorylation of the CP29 sub-unit of PSII antenna have been
found between these two lines by Mauro et al. (1997),
after exposure at +5C under light.
Maize seeds were placed on a water-soaked filter
paper in Petri dishes and left to germinate at 25C for
3 days, then planted 1 cm deep in Perlite in pots with
a water reservoir containing Hydrokani H2 fertilizing
solution diluted 5/1,000 and mixed just before watering with 100 mg l1 Sequestrene (complexed iron) to
prevent chlorosis. They were grown in a Conviron E7
climatic chamber under a 280–300 lmol m2 s1 light
intensity from Sylvania ‘‘Cool White 4450’’ fluorescent
tubes, with a daily cycle of 16-h day, 8-h night.
Tungsten lamps remained turned off. Up to the 2- to
3-leaves stage, the temperature was 22C day/18C
night, then it was decreased to 17C day/15C night.
Measurements were done using plants from a 5- to
7-leaves stage, on leaves 3 or 4, in a room air-conditioned at approximately 21C (±2C), maintained
in the dark or under a weak light when handling
samples.
Luminescence and TL measurements
Thermoluminescence measurements were made with a
TL apparatus built in our laboratory, as previously
described (Miranda and Ducruet 1995; Ducruet 2003),
in which the temperature of the sample is regulated by
a thermoelectic Peltier plate (Marlow DT1089-12).
Luminescence is now collected by the common arm of
a PAM-Walz fibre-light guide placed in front of the
sample, then conveyed through the main arm of the
guide to a red-sensitive photomultiplier. The four
other arms remain available for various illuminations
of the sample, and that with the thickest fibre core was
selected for FR or flash illumination to achieve saturation. A 24-mm disc was punched in one-half of a
leaf if large enough; otherwise the central vein was
removed to achieve a good contact between the TL
plate and two half discs. Then, the disc was placed in
the 25-mm-diameter well of the TL sample holder on a
drop of water for thermal contact. The sample with
the adaxial face up was covered by a Hellma 202-OS
disc window and an O-ring narrowing the emitting
area to 15 mm diameter, in front of the 14-mmdiameter light guide. Discs from dark-adapted leaves
were first maintained for 2 min at 20C in complete
darkness for relaxation of residual charge pairs, then
the temperature was decreased to 10C, 5C or 0C for
1 min, avoiding any overshoot to negative temperatures. Thereafter, the sample was submitted either to a
30-s FR illumination or to a flash sequence (1 s1) just
before recording TL.
570
Kinetic spectrophotometry of P700+ reduction
A modulated PAM-100 fluorimeter (Walz-Effeltrich)
was used to monitor kinetics of both chlorophyll fluorescence induction and P700+ reduction. The redox
state of P700 was measured in leaves by its absorption at
820 nm, using a dual-wavelength detector Walz EDP700DW-E. In both types of measurements, the attached leaf was softly pressed in front of the light guide
at an ambient temperature maintained at about 20C.
Decompositions of re-reduction kinetics into exponentials were done with the Sigmaplot 8.0 software.
the FV/FM ratio, then the fluorescence induction
was triggered by a continuous irradiation with
250 lmol photons m2 s1 white light, with saturating
pulses every 30 s. The only difference between R and S
lines was a slightly lower FV/FM in dark-adapted leaves
of F2 (0.763±0.008 compared to F618 (0.796±0.007).
FV/FM ratio in control maize plants is usually close to
0.8, so no significant decrease of FV/FM could be detected here after illumination at 17C, except for the
tolerant F2 line. This decrease of FV/FM is small,
therefore unlikely to be the result of photoinhibition but
more likely of some adaptation in PSII centres, as further indicated by slight changes in the B TL band.
Photoacoustic spectroscopy
Energy storage by cyclic electron flow around PSI was
measured in vivo using the photoacoustic technique, as
described elsewhere (Joët et al. 2002). Leaf discs, placed
in the photoacoustic chamber, were illuminated with
modulated FR light (>715 nm). The FR-light fluence
rate (15 W m2) was measured with a LiCor radiometer
(Li-185B/Li-200SB, LiCor, Lincoln, USA). PSI photochemistry was saturated with a strong background FR
light (>715 nm, 320 W m2). ES was calculated from
the amplitude of the maximal photothermal photoacoustic signal (Apt+), measured when the strong FR
light was added to the modulated measuring light and
the actual photothermal signal amplitude (Apt):
ES ¼ ðAptþ AptÞ=Aptþ
Actinic light sources
Single turn-over flashes were produced by a Walz XST
103 flash unit and fired 1 s apart to allow full reload of
the capacitor. FR light was provided by an FR-101 Walz
LED connected to a PAM-102 unit, generally set at
intensity 10. Both flashes and FR light were conveyed to
the sample through the Walz light guide, the detector
being closed, and triggered by the computer. White light
illumination for fluorescence induction kinetics and
P700 experiments (Fig. 5) were carried out with a
tungsten lamp with anti-heat filters, through an arm of
the Walz light guide (250 lmol photons m2 s1).
Results
Plants were illuminated for at least 5 h in the growth
chamber, then they were transferred to the dark room
(at about 20C) and measurements were done at different time intervals.
Fluorescence induction kinetics
On attached leaves at ambient temperature (20C), a
saturating light pulse was given in darkness to determine
Properties of the flash-induced B band in unfrozen
leaves of the maize lines
The B band of TL is the main band observed in untreated photosynthetic material after flash excitation. It
reflects the charge pair stabilization on PSII with positive charges stored on the manganese oxygen-evolving
complex and an electron on the secondary quinonic
acceptor. In the first step, we characterized the B-band
emission of maize leaf discs after 1 and 3 single turn-over
flashes, producing predominantly the S2Q
B and S3QB
charge pairs, respectively, owing to a 25% S0/75% S1
distribution in dark-adapted material. Table 1 shows
that the temperature maximum Tm of B band after 1F is
located at 30C for all lines except F2 peaking at 26.5C.
The activation energy of recombination Ea is close to
1.0 eV for the S lines and the 1872 R line, but decreases
to 0.8 eV in the R lines F2 and Co329. After 3F, a significantly lower Tm was confirmed for F2 only when
compared to other lines. It should be noted that a TL
band is determined by both the activation energy Ea and
a pre-exponential factor, so that Tm is not strictly related
to Ea. Essentially the very tolerant F2 line exhibited
modified characteristics of the B band, and the mediumtolerant Co329 to a lesser extent, but not the third tolerant line 1872, so that these slight modifications of PSII
cannot be considered as essential to the hardening
mechanism at 17C (notwithstanding their possible role
at lower chilling temperatures). In addition to the B
band, the TL signal contained a shoulder near 45C
ascribable to an afterglow band, which was stronger
after 3F than after 1F as expected (Miranda and
Ducruet 1995). However, this flash-induced afterglow
was always much weaker in maize compared to some
other species such as pea, cucumber or tobacco.
Optimizing experimental conditions to characterize
the afterglow emission
In a second step, flash excitation was replaced by FR
excitation in order to induce a strong afterglow emission
(Bertsch and Azzi 1965). Although FR light excites
mainly PSI, its relatively weak absorption by the PSII
571
Table 1 Characteristics of B and afterglow TL bands in freshly excised disks of maize plantlets illuminated 5 h (280 lmol photons m2 s1, 17C), then placed in the dark
Inbred
lines
F2
Co329
1872
Io
F618
1194
Origin
France
Canada
France
USA
France
France
Chilling
R
MR
R
S
S
S
Grain
texture
Flint
Flint-Dent
Dent
Dent
Flint-Dent
Flint
Precocity
(1–4)
1
2
2
3
2
1
B band (1F) or S3Q
B (3F)
Tm of afterglow band after 30-s FR
Ea 1F
(eV)
Tm 1F
(C)
Tm 3F
(C)
Dark: 20 min
to 1 h 30 min
1 h 30 min to
3h
20 h to
24 h
0.79±0.04
0.82±0.10
0.99±0.07
0.99±0.05
0.98±0.06
0.99±0.08
26.5±1.0
29.8±2.2
30.5±3.2
30.2±1.2
29.0±2.1
31.6±3.2
25.1±1.1
29.4±1.1
28.0±2.4
29.1±1.3
29.6±3.0
28.4±2.3
33.0±1.4
37.1±1.1
36.1±1.5
42.5±1.2
43.0±1.1
41.3±1.1
33.8±1.3
42.1±1.5
37.1±3.2
44.1±4.6
42.5±2.6
41.5±2.9
41.4±2.9
43.6±1.3
43.7±2.0
43.7±4.0
46.7±2.4
42.5±0.9
The B band was induced by one or three single turn-over xenon
flashes, after 20 min to 1 h 30 min in darkness. The afterglow band
was induced by 30-s FR light at 10C, on leaf discs excised from
plants maintained from 20 min to 1 h 30 min, 1 h 30 min to 3 h
and 20 h to 24 h in the dark. ±SE at 5%
antenna is sufficient to induce several charge separation
events. This creates a uniform distribution of the S0–S3
oxidation steps of the oxygen-evolving manganese
complex, half of them in the S2 and S3 states able to
generate luminescence by recombining with Q
B . Immediately after FR illumination at +5C, the detector was
opened and two temperature profiles were assayed
comparatively while recording luminescence emission
(Fig. 1). (a) T-jump (Fig. 1a, see also Fig. 6a): the
sample was suddenly heated up within a few seconds to a
constant measurement temperature in the 20–40C
range, (b) TL (Fig. 1b): the sample was progressively
warmed.
The fast temperature jump favouring charge recombination within PSII centres induced an initial rise of
luminescence, which started to decrease as soon as a
constant measuring temperature was reached (Fig. 1a).
This is due to the recombination of S2/3Q
B charge pairs
stabilized on PSII, i.e. homologous to a B band of TL
(Rutherford and Inoue 1984). Then, a transient burst of
luminescence superimposed to the exponential decay
became noticeable at 25C and grew faster and sharper
as the measuring temperature was increased. Increasing
the measurement temperature above 30C proved necessary to fully reveal this afterglow emission but also
increased the overlap of the afterglow transient with the
initial decay, making a separation difficult. Replacement
of the temperature jump by a progressive TL warming
from the illumination temperature at 5C up to 70C
revealed two well-resolved emission bands, a downshifted B band near 20C and an intense and sharp afterglow band (Fig. 1b) peaking at about 45C when
recorded at 0.5C s1. Decreasing the heating rate to
0.35C s1 or 0.2C s1 caused a downshift of this
afterglow band, due to a longer recombination time per
C (Miranda and Ducruet 1995). A heating rate of
0.5C s1 was further used because it provided a better
resolution of the B band downshifted to 20C (Fig. 1b)
by lumen acidification, which decreases the stability of
S2 and S3 (Bennoun 1982; Miranda and Ducruet 1995).
As a control, a B band, generated by one single turnover flash that converts most of the 75% S1 states stable
R Chilling-tolerant, S chilling-sensitive, Precocity 1 very short cycle
to 4 very long cycles
Fig. 1 Effect of temperature on the afterglow luminescence
emission in leaves of maize inbred line Io, after overnight dark
adaptation. a Excitation by 30 s FR light at +5C, followed by a
few seconds of jump up to constant temperatures (20C to 35C)
while recording luminescence emission. b Same 30 s FR excitation
(lines) at +5C, followed by a progressive warming at different
heating rates to reveal TL bands. Lines Recording at 0.5C s1
(30C min1), 0.33C s1 (20C min1) and 0.2C s1. Open circles
Excitation by 1 Xenon flash just before TL recording at 0.5C s1.
Ordinates correspond to TL analog amplitudes (photon counting
signals would be multiplied by 1, 1.5 and 2.5, respectively)
572
in the dark into the luminescence-emitting S2, is shown
to peak at 30C (Fig. 1b, Table 1). The effect of FR
compared to flash excitation is both to stimulate the
afterglow emission and to shift the B band towards
lower temperatures by acidifying the lumen. However, in
dark-adapted plants, this proton influx is not due to
cyclic electron flow, since the S2Q
A recorded at +1C in
atrazine-infiltrated leaves retained the same half-time
after FR or 2F illumination (data not shown): this
demonstrates that FR light does not drive any proton
influx when the PSII is inhibited. So, lumen acidification
results from the weak linear electron flow supported by
an FR without atrazine.
Effect of white light pre-illumination on the afterglow
emission and relaxation kinetics in the dark
Figure 2 shows the evolution of the FR-induced afterglow emission in darkness following illumination in the
growth chamber. Luminescence decay kinetics were recorded periodically at 30C every 45 min during the first
2 h of dark adaptation (Fig. 2a). The S line Io exhibited
first a luminescence decay which started bouncing up at
about 50 s to reach a maximum at 80 s, without any
significant change in the successive kinetics recorded
during the 2-h dark adaptation. In contrast, the R line
F2 produced this luminescence bounce much earlier,
appearing first merged with the initial decay when recorded after a 15-min dark adaptation, then progressively evolving as a shoulder peaking 35 s after the end
of FR illumination, as the plant stayed for 2 h in the
dark. In order to examine the influence of temperature
on the afterglow emission in both maize lines, the
emission induced by 30 s FR was also recorded during a
linear warming from 0C to 70C at 0.5C s1. This
Fig. 2 Evolution of FRinduced (thermo)luminescence
emission during dark
adaptation of maize plants
following at least a 5-h
illumination under 280
lmol m2 s1 in the growth
chamber. a Luminescence
decay, at 25C after 30 s of FR
at 5C, of leaf discs from plants
maintained for different times
in the dark. The arrows show
the time-dependent evolution
for the inbred lines F2 and Io.
b, c Thermoluminescence after
2 h 30 min in the dark of F2
and Io, respectively, the same
30-s FR excitation at 5C.
Thick line Signal. Thin line
Simulation (mostly hidden).
Dotted lines Simulated
components B (S2/3Q
B ) and AG
(afterglow)
revealed a sharp and intense afterglow band centred at
42C in the Io line, well separated from the B band at
26C (Fig. 2c), whereas the TL emission from the F2 line
appeared as a broad band with a maximum at about
30C (Fig. 2b). The Io TL signal could be decomposed
into two bands, B and AG. However, the single broad
band in F2 could not be simulated by one B component,
and a second strongly overlapping component had to be
added. This suggests that there is also an afterglow
bounce in F2 too (Tm=37C), close to the B band
(Tm=27C). This is consistent with the afterglow band
being almost fused with initial luminescence decays in
Fig. 1a. It should be noted that the afterglow emission,
although not of a Randall-Wilkins type (Ducruet 2003),
can be fitted by one component, which suggests that the
temperature contribution predominates over the time
contribution in inducing this emission, at least at a
0.5C s1 warming rate.
Hence, white light illumination before dark adaptation accelerates the afterglow kinetics in luminescence
decays at 30C, in the R F2 line, which corresponds to a
downshift of the afterglow band when recorded in TL
mode. An almost complete fusion of the B and afterglow
emissions was observed at the beginning of the dark
period (Fig. 2a). The Tm of the TL signal provides an
index of this downshift and Table 1 confirms that it was
large in the three tolerant lines in the 20 min to 1 h
30 min period after transfer from the illuminated growth
chamber. Between 1 h 30 min and 3 h, only the very
tolerant line F2 remained strongly downshifted, the 1872
had partly relaxed and the moderately tolerant Co329
line was undistinguishable from the S lines. After 20 h in
the dark, almost no downshift could be detected, except
sometimes in F2.
Figure 3 shows the TL emission of the tolerant F2
and Co329 lines and the sensitive line Io after 3 h in
573
Fig. 3 Thermoluminescence after 30 s of FR excitation of the
inbred lines F2 (tolerant), Co329 (moderately tolerant) and Io
(sensitive), after 5 h of illumination under 280 lE m2 s1 and 3 h
in darkness
darkness. Consistent with data in Table 1, the moderately tolerant Co329 had almost recovered its darkadapted afterglow maximum above 40C, similar to that
of F618, but the band was broader in Co329 than in
F618. In F2, B and afterglow bands began to separate,
but the latter remained broad and still strongly downshifted below 40C. After more than 20 h of dark
adaptation (sometimes 30 h for F2), all lines (data not
shown) exhibited the same sharp afterglow band near
45C as Io in Fig. 3.
As illustrated in Fig. 3, the Tm of the TL band, i.e.
initially that of the unique broad band evolving into that
of a distinct afterglow band, can be taken as an index to
follow the kinetics of dark relaxation of the downshifted
afterglow band after transferring the plant from the
illuminated chamber to darkness. The tolerant lines F2,
Co329 (Fig. 4a) and 1872 (Fig. 4b) relaxed much slowly
than the three sensitive lines. The fastest relaxing was the
moderately tolerant Co329 then, 1872 and finally F2, the
slowest to resume its dark-adapted afterglow location.
The sensitive lines Io and F618 also exhibited a downshift of afterglow band at the beginning of the dark
adaptation, but it faded away in less than 1 h.
After a long dark adaptation, no plastoquinonereducing pathway is active at ambient temperatures in
all lines, and the sharp afterglow band peaking around
45C reflects an induction of back-electron flow by
warming.
Monitoring the cyclic electron flow by P700+ (DA820)
re-reduction kinetics after FR illumination
In attached leaves of maize plants, P700 was oxidized
by 100 s of FR light (Fig. 5). The FR-light intensity
provided by the 102-FR LED with a setting of 10 on
PAM-102 was found here to saturate P700 oxidation. A
sufficiently long FR duration was chosen to stabilize
the reduction rates of rapidly and slowly donating
Fig. 4 Evolution of the apparent Tm of the FR-induced TL signal
according to time in darkness following 5 h of light in the growth
chamber. One rising exponential was fitted on Tm data from 0 h to
20 h and is shown for a F2, F618 and Co329. b 1872 (1194 could
not be fitted). Relaxation kinetics of Io and F618 were similar. For
clarity, the timescale of the figure is cut at 5 h and the fitted Tm
curves are indicated at 20 h by the horizontal line at right (dashed
lines link the fittings from 5 h to 20 h)
PSI units (Bukhov et al. 2002). Then, 5 min of
250 lmol photons m2 s1 white light was given
through the light guide, followed again by 100 s of FR
(Fig. 5a). The kinetics of post-FR re-reduction of
P700+ (a, b, v, d in Fig. 5a) are enlarged and related to
the decay amplitude of DA820 in Fig. 5b. The first
reduction kinetics was much faster in the F2 than in the
F618 line, indicating that a cyclic pathway provides
electrons faster to P700+ in F2. White light had a
stronger enhancing effect on the second post-FR P700+
reduction in F618 than in F2, which was already very
fast before white light illumination. It should be noted
that the P700 oxidation during 5 min of white light
574
Fig. 5 P700 oxidation by FR or white light (WL) and its rereduction in the dark followed by absorption at 820 nm in F2 (R)
and F618 (S) maize lines. Plants transferred from an illuminated
growth chamber were maintained in darkness. Measurements on
attached leaves at approximately 21C room temperature. a P700
redox kinetics during a light sequence 100 s FR fi dark fi
5 min WL fi 100 s FR fi dark. Kinetics ad are enlarged in
(b). b Reduction kinetics following 100 s FR light, before and after
5 min of white light
increased less for F2 than for F618, which also suggests
a faster supply of electrons to P700+ in F2 under these
light conditions.
Similar differences were also observed between the R
1872 and S 1194 lines. After more than 20 h of dark
adaptation, the P700+ reduction rate decreased and
became similar for F2 and F618 (not shown) as well as
Table 2 Reciprocal of time
constant of the two fast and
slow exponential phases of
P700+ re-reduction after
oxidation by FR light of plants
illuminated in growth chamber
and placed for 45 min in the
dark (t1/2)=0.69s
Fig. 6 Comparison of the luminescence afterglow emission and
P700+ re-reduction following an FR illumination, in 1872 (R) and
1194 (S) maize leaves, at 25C. a After 30 s of FR illumination of
leaf discs at 10C, the temperature was immediately raised to 25C
while the luminescence decay was recorded. Arrows pointing to
maximum of afterglow emission; Upper traces actual temperature
rise. b Kinetics of P700+ reduction after oxidation by FR light in
attached leaves at ambient temperature (22C), 45 min (two lower
traces, 1872 thick line, 1194 dots) and 22 h/23 h (two overlapping
upper traces) in darkness after transfer from an illuminated growth
chamber. Arrows Evolution in the dark for each maize line
for 1872 and 1194 (Fig. 6b). In luminescence decays at
25C, the afterglow also rose faster for the tolerant 1872
than for the sensitive 1194 (Fig. 6a), and they became
similar after more than 20 h of dark adaptation (not
shown for clarity). The reduction kinetics could be
decomposed into two exponentials, the parameters of
which are given in Table 2. Assays with one exponential
Inbred lines
n
(s)
sf=k1
f
F2 R
F618 S
1872 R
1104 S
9
10
8
9
0.80±0.27
2.10±0.31
0.98±0.32
2.80±0.63
(0.55)
(1.45)
(0.68)
(1.93)
ss=k1
(s)
s
Amplitude fast
phase (%)
6.3±3.1 (4.35)
13.0±2.0 (8.97)
4.6±0.4 (3.17)
12.7±2.2 (8.76)
59±1
57±5
71±7
67±10
575
generally could not fit the decay. The amplitudes of the
fast and slow phases were approximately in a 2/1 ratio.
Growth chamber pre-illumination accelerated both the
fast and the slow phases, as evidenced by the sf and ss
reciprocals of time constants. Hence, it is difficult to
ascribe a particular P700+ reduction pathway to these
two phases. These bi-exponential decay parameters are
in agreement with those determined in the C3 plant.
(Cornic et al. 2000; Bukhov et al. 2002).
The data on P700+ re-reduction support the conclusion deduced from the afterglow downshift. A cyclic
pathway contributing both to the afterglow emission
and to re-reduction of P700+ is induced at ambient
temperature by white light. This induction lasts much
longer in the R than in the S maize inbred lines.
to the measurements substantially increased ES in the F2
line, while ES in F618 leaves was slightly reduced compared to 25C-grown leaves, resulting in a 75% augmentation of ES in F2 relatively to F618. This effect was
due to both an increase in the maximal ES efficiency and
a less rapid saturation of ES by FR light in F2 (not
shown). Increasing the temperature during the photoacoustic measurements to 35C stimulated the cyclic PSI
activity in both maize lines. However, ES in F2 leaves
remained higher than the ES value measured in F618.
Thus, the photoacoustic data show that white light preillumination, when done at 17C and not at 25C, enhanced cyclic electron transport around PSI in the tolerant maize line.
Discussion
Photoacoustics
PSI-mediated cyclic electron flow was monitored in vivo
using photoacoustic spectroscopy—a technique which
measures the conversion of light energy to heat in an
absorbing sample and hence the storage of light energy
as chemical energy (ES, photochemical energy storage,
see Materials and methods) (Malkin and Canaani 1994).
When the photoacoustic measurements are performed in
FR light, the ES measured is specifically related to the
PSI function, reflecting ES in photochemical products
associated with the cycling of electrons around PSI. A
change in cyclic electron transport can manifest as a
change in the maximal efficiency of photochemical ES
and/or a modification of the saturation of ES by
increasing the FR-light intensity (Ravenel et al. 1994).
Therefore, in this study, a moderately elevated fluence
rate was used (15 W m2) to take into account both
phenomena. When maize plants were grown at 25C,
cyclic PSI activity (measured at 25C) was similar in the
F2 and F618 lines (Table 3). Increasing leaf temperature
during the photoacoustic measurements to 35C stimulated cyclic electron flow in both lines. Exposing plants
to 280 lmol photons m2 s1 at 17C for 3 days prior
Table 3 Photoacoustic study of ES by cyclic electron transfer
around PSI in F2 and F618 maize lines
Treatment
Chamber at 25C/20C
Measured at 25C
Measured at 35C
Chamber 3 days at 17C/15C
Measured at 25C
Measured at 35C
ES (%)
F2
F618
5.9±0.5
6.9±2.0
6.4±0.7
7.9±1.0
8.7±0.2
11.7±0.3
5.0±0.1
8.0±0.1
Photochemical ES was measured in FR light (715–750 nm,
15 W m2). Plants were grown at 25C/20C, then submitted to
17C/15C for 3 days. They were illuminated for 5 h in the growth
chamber under 300 lmol photons m2 s1, then maintained for
1 h in darkness before measurements that were carried out at 25C
and 35C
Maize chilling tolerance and activation of cyclic electron
flow
Low temperatures may have a major effect on the photosynthetic apparatus under daylight because photochemical conversion of absorbed light energy is slowed
down, leading to detrimental effects of singlet oxygen
and free radicals. For plants from warm climates such as
maize, the chilling stress causing permanent photoinhibitory damage in the day occurs between 0C and
12C, whereas sub-optimal temperatures from 12C to
18C for long periods impair plant growth and crop
yield without loss of PSII centres (Allen and Ort 2001).
However, it must be emphasized that this absence of
apparent PSII damage at sub-optimal temperatures does
not preclude a faster repair cycle of proteins, especially
the fast turn-over D1 sub-unit of PSII, with an increased
demand in ATP. Thus, 17C is a threshold stress temperature for maize, at which differential effects on the
linear electron transfer activity and on the Calvin cycle
become detectable between the R and S inbred lines,
1872 and 1194, studied here (Havaux 1987), Z7 and
Penjalinan lines (Haldimann et al. 1996; Janda et al.
1998).
The different tolerance of maize genotypes to chilling
is a complex phenomenon involving various mechanisms
such as photosynthetic activity, antioxidant mechanisms
and much more (Allen and Ort 2001, Foyer et al. 2002).
In a preliminary work (Ducruet et al. 1997), we detected
a downshift of the afterglow band in two resistant lines,
compared to two susceptible lines of those obtained by
Stamp in Zürich (Haldimann et al. 1996). However, this
band shift was not as large and long lasting as in the
three tolerant lines considered here (Constantin-Roman
2000).
Effect of light at sub-optimal temperatures on PSII
Illumination at 17C under 280 lmol photons m2 s1
did not cause a significant decrease of the FV/FM ratio,
576
except for a slight decrease in the F2 line. As shown in
Table 1, the properties of the flash-induced B band were
also different in Co329 (lower Tm) and F2 (lower Tm and
Ea). Decrease in Ea and Tm have already been observed
in cold-tolerant plants such as spinach (Briantais et al.
1992) and Arabidopsis thaliana (Sane et al. 2003). These
changes can be tentatively ascribed to a phosphorylation
of PSII centres. Modifications of the antenna have also
been reported in maize submitted to chilling, with the
appearance of a 31-kDa protein and a decrease of the
29-kDa chlorophyll protein, leading to a modified 77-K
fluorescence emission spectrum of LHCII particles
(Hayden et al. 1988). A phosphorylation of the CP29
sub-unit was found in a maize chilling-tolerant hybrid
but not in a sensitive one (Bergantino et al. 1995); in the
1872 line, but not in the 1194 line (Mauro et al. 1997).
However, this phosphorylation of CP29 resulted from
incubation at temperatures (5C) much lower than 17C,
which may activate mechanisms of tolerance of PSII to
photoinhibition different from those activated at suboptimal but non-photoinhibitory temperatures. As the B
band of the 1872 tolerant line was similar to that of the
sensitive lines, the decreases in Ea, Tm and FV/FM in F2,
or only Tm in Co329 do not appear directly related to
the downshift of the afterglow band.
Light activation of a cyclic/chlororespiratory pathway
Luminescence, P700 and photoacoustic measurements
consistently demonstrate that both cyclic and plastoquinone-reducing electron pathways are induced when
plants are submitted to 280 lmol photons m2 s1
white light at 17C and that this induction lasts longer in
R compared to S, all the more as the tolerance is strong
(e.g. F2 > 1872 > Co329). The cyclic electron flow,
measured by both photoacoustics and P700+ re-reduction, and the back-flow of electrons towards the PSII
acceptor side in darkness evidenced by luminescence, are
enhanced by light. This activation of cyclic electron flow
by light is consistent with the necessity to complement
the linear electron transfer by some cyclic transfer to
reach the ratio ATP/NADPH=3 needed for carbon
assimilation (Heber et al. 1995; Cornic et al. 2000).
The afterglow emission originates from PSII centres
that are located essentially in mesophyll cells in C4
plants, so the luminescence provides a tool to specifically
observe the mesophyllic cyclic pathway without interference of the strong cyclic flow in bundle sheaths.
However, the overall PSI cyclic activity in both the
mesophyll and bundle sheath is increased in a similar
way by white light, as monitored by P700 and photoacoustic measurements, which would suggest that cyclic
electron flow in maize bundle sheaths becomes inactive
in the dark and has to be re-activated by white light.
Furthermore, the two decay phases of P700+ re-reduction are both accelerated by pre-illumination (Table 2),
so no relation can be established between one of these
phases and the afterglow emission. They can be ascribed
to two types of rapidly and slowly reducing PSI units
(Bukhov et al. 2002).
The afterglow downshift, although it could be detected when plants were illuminated in a growth chamber at 25C, was stronger and long lasting when the
chamber was set at 17C. This was also evidenced by the
photoacoustic data (Table 3) showing a stimulation of
the cyclic PSI activity in the F2 maize line after 3 days at
17C. This can be explained by a less-efficient drainage
of absorbed light energy at 17C than at 25C, due to a
slower rate of the Calvin cycle, which would trigger
tolerance mechanisms to different extents depending on
maize lines.
Role of cyclic/chlororespiratory electron flow in stress
and chilling tolerance
Cyclic electron pathway driven by the PSI in light is
activated in many stress situations (Bukhov and Carpentier 2004), including light at cool temperatures (Kim
et al. 2001; Barth and Krause 2002; Munekage et al.
2002; Li et al. 2004; Quiles and Lopez 2004; Bukhov
et al. 2004). This may correspond to an increased demand in ATP, for protein synthesis or for other tolerance mechanisms, which is fulfilled by cyclic electron
flow: this has been shown to occur in a mitochondrial
mutant (Cardol et al. 2003. Cyclic flow also contributes
to pumping protons into the lumen, thus producing a
stronger non-photochemical quenching to dissipate the
excess light energy. Such a role has also been considered
for chlororespiration, which would maintain an acidic
lumen pH to protect the oxygen-evolving complex in the
dark (Peltier and Cournac 2002). The induction of cyclic
electron flow is related to a state 1 to state 2 transition
(Finazzi et al. 2002), state 2 being also less sensitive to
photoinhibition, measured by D1 degradation (Finazzi
et al. 2001). This light-induced state transition is concomitant to a fast grana unstacking (Rozak et al. 2002).
Temperature elevation above 30C produces similar
structural changes (Weis 1984; Sundby et al. 1986;
Havaux 1993) as well as an induction of the cyclic
electron flow reflected by the 45C band in dark-adapted
material. This afterglow emission would tend to merge
with the B band at a lower temperature when state 2 is
induced either by an illumination with white or blue
light exciting preferentially PSII or by other treatments,
such as anerobiosis (Joët et al. 2002). Phosphorylation
of PSII antenna and centre proteins is a complex phenomenon following four different regulatory patterns
induced by environmental cues (Pursiheimo et al. 2003)
and some of them play a role in the state transition,
hence in the afterglow emission. Phosphorylation of
LHCII appears very flexible, whilst that of CP29 is induced only by harsher cold treatments (Bergantino et al.
1995; Mauro et al. 1997).
The afterglow emission is suppressed by low concentrations of antimycin A (Nakamoto et al. 1988) that
also specifically inhibit the FQR pathway and the cyclic
577
electron flow (Bendall and Manasse 1995). In contrast,
the afterglow band is unchanged in NDH mutants of
tobacco (L. Cournac and J. M. Ducruet, unpublished
results; A. Krieger-Liszkay, personal communication).
This contrasts with the fluorescence F0 bounce at the
onset of darkness, which is partly suppressed, although
not completely, in tobacco NDH mutants (Shikanai
et al. 1998). Hence, the FQR pathway, if not a third
unknown pathway also sensitive to antimycin A, would
support the back-transfer of electrons leading to the
afterglow emission. However, work in progress suggests
that, in contrast to tobacco, the NDH pathway might
also contribute the afterglow emission in A. thaliana (M.
Havaux, unpublished data). Munakage et al. (2002)
have obtained a PGR5 mutant of A. thaliana deprived of
FQR activity, which appears more cold-sensitive. They
suggest that FQR electron transfer plays a protective
role under high light by pumping protons in the thylakoid lumen and increasing non-photochemical quenching. Consistently, the FQR inhibitor antimycin A
induces a photoinhibition of PSI (Quiles and Lopez
2004).
The results reported here are not specific to C4 plants.
We found similar downshifts of the afterglow band in
tolerant varieties from two other species adapted to
warm climates, French bean and grapevine, but not in
rice. In the chilling-tolerant Purley King and chillingsusceptible Kilt bean varieties, a downshift of the
afterglow band was observed in the tolerant variety, not
in the sensitive one, and lasted approximately 2 h. We
also compared grapevine cultivars susceptible (Meunier,
Carignan, Pinot) or tolerant (Chardonnay, Chasselas,
Merlot) to flower abortion after a spring chilling period.
A downshift of the afterglow band could be observed
only in the tolerant genotypes during cool sunny days,
not during warm days (A. Toulouse, unpublished results).
Thermoluminescence detection of the afterglow emission
The afterglow emission has been until recently detected
as a ‘‘bounce’’ superimposed to luminescence decays
recorded at constant temperatures. The advantage of
recording TL while warming the sample is, in addition to
a better resolution, to detect the activation of the cyclic
pathway from the downshift and ultimately from the
apparent suppression of the afterglow emission when it
becomes undistinguishable from the B band. Hence, the
sharp 45C band usually found in dark-adapted healthy
leaves after FR illumination provides an essential control. Its modification by various treatments reveals the
induction of a cyclic pathway at lower temperatures that
depletes the pool of stroma reductants and/or the S2/3
states. Various stresses cause complex changes of leaf TL
signal (Janda et al. 1999) and photoinhibitory conditions
result in a loss of PSII centres and related TL bands in
maize (Janda et al. 2000). Here, our conditions (17C
and 280 lmol photons m2 s1) correspond to an ear-
lier, hardening stage, without any significant damage to
PSII detectable from fluorescence induction kinetics and
B-band characteristics. Thus, afterglow luminescence
would allow us to detect subtle changes in photosynthetic metabolism before the emergence of apparent
symptoms.
Conclusion
In maize plants submitted to white actinic light at a 17C
sub-optimal temperature, the upper threshold of chilling
stress, (thermo)luminescence, post-FR P700+ re-reduction and photoacoustics consistently showed a stimulation of cyclic/chlororespiratory pathway(s) lasting
longer in the dark in the chilling-tolerant than in the
chilling-sensitive lines. A chlororespiratory flow during
the night may bring some yet hypothetical advantages,
such as maintaining the lumen acidic. However, maize
leaves are damaged by chilling temperature during the
day, much more than during the night (Allen and Ort
2001). Although our chilling-sensitive genotypes had
also their cyclic electron flow transiently induced by
light, this long-lasting induction in the tolerant ones
might enable them to undergo sudden sunlight rises
(sunflecks) at chilling temperatures.
Acknowledgements This work was supported by a ACC-SV grant
from the Ministry of Research and Technology and by the Comité
Technique Permanent de la Sélection (CTPS). The Mumm-PerrierJouët company provided initial support to investigate spring
chilling in grapevine. Seeds of the 1872 and 1192 inbred lines were
kindly given by Euralis. We thank Dr. Sridharan Govindachary for
the critical review of the manuscript.
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