Plant Physiol.
(1989) 89, 740-742
0032-0889/89/89/0740/03/$01 .00/0
Communication
Cold-Induced Sudden Reversible Lowering of in Vivo
Chlorophyll Fluorescence after Saturating Light Pulses1
A Sensitive Marker for Chilling Susceptibility
Walter Larcher* and Gilbert Neuner
Institute of Botany, University of Innsbruck, SternwartestraBe 15, A-6020 Innsbruck, Austria
ABSTRACT
In chilling-sensitive plants (Glycine max, Saintpaulia ionantha,
Saccharum offkinarum) a sudden reversible drop in chlorophyll
fluorescence occurs during photosynthetic induction immediately
following saturating light pulses at low temperatures in the range
4 to 80C. A comparison of two soybean cultivars of different
chilling sensitivities revealed that this phenomenon, termed low-
wave, indicates specific thresholds of low temperature stress. Its
occurrence under controlled chilling can be regarded as a quan-
titative marker for screening chilling susceptibility in angio-
sperms.
In vivo Chl fluorescence has in many respects proved to be
a sensitive and reliable method for the detection and quanti-
fication of chilling-induced disturbances (1, 2, 4, 5, 11, 12).
A number of parameters of the photosynthetic induction
transient may be employed for rating chilling susceptibility.
Smillie and Hetherington (12) determine FR2 of induced Chl
fluorescence of leaves exposed to a constant temperature of
0.5°C and take the time required for a 50% reduction of FR
as a measure of chilling susceptibility. Havaux (1) estimates
relative chilling sensitivity by measuring the QA redox state
under low temperature conditions. Larcher and Bodner (4)
base their assessment of chilling susceptibility on the temper-
ature at which the decrease in fluorescence, vFd = Fp-FT, is
50% of its highest value measured on the same leaf before
cooling, and on the readiness and extent of recovery of
photosynthetic function after rewarming.
'Supported by Osterreichische Nationalbank, Project No. 2986.
2Abbreviations: FR, maximal rate of fluorescence rise; Fo, basic
fluorescence; Fp, peak of the induction curve upon excitation with
actinic light; FT, stationary level of variable fluorescence; F, variable
fluorescence at any given time during induction; (F,)1, minimal
variable fluorescence at appearance of L-waves; (F,)m, maximal flu-
orescence of a dark-adapted leaf upon excitation with a saturating
light pulse; (F,)S, maximal variable fluorescence at any given time
during induction observed upon application of a saturation pulse; qP,
photochemical quenching coefficient; QA, primary electron acceptor
of PS II; vFd, variable fluorescence decrease (FP-FT); L-wave, sudden
reversible lowering of in vivo Chl fluorescence after saturating light
pulses.
740
Received for publication June 8, 1988
and in revised form November 4, 1988
While searching for additional warning signs of chilling-
induced deviations from normal photosynthetic activity it
was observed that at low temperatures the leaves of various
chilling-sensitive plants exhibited a sudden undershoot in Chl
fluorescence immediately after saturating light pulses. This
phenomenon has proved to be a good diagnostic criterion for
the onset of chilling stress.
MATERIALS AND METHODS
Plant Materials
Glycine max (L.) Merr. cv 'Maple Arrow' and cv 'Evans'
were grown from seed in pots in a soil mixture of compost,
litter, and clay (2: 1: 1) in the Botanical Garden of the Univer-
sity of Innsbruck. Second trifoliate leaves of well watered
plants in the vegetative state, 38 d after seedling emergence,
were used for the experiments. For additional check measure-
ments, leaves were taken from greenhouse plants of Saintpau-
lia ionantha hybrids and from young developing shoots of
Saccharum officinarum.
Chilling Treatments
For progressive cooling, attached leaves were placed with
the abaxial surface down on a 165x95 mm aluminium plate
mounted on the thermal sink side of a thermoelectric module
(type 803-1008-01) controlled by a bipolar controller (type
809-3030) and a range extender (type 809-1019) manufac-
tured by Midland-Ross Corp. (Cambridge, MA). After each
fluorescence measurement the temperature was lowered at a
rate of 4 K. min-' in 8 steps from 20°C down to 0.9C. For
continuous long-term cooling at a preset constant temperature
(3.5°C), attached leaves were fixed with a magnet to the surface
of an insulated metal chamber, through which cold ethylene
glycol was pumped. The temperature of the cooling fluid was
regulated by means of a through-flow cooler (K 11, Haake,
Karlsruhe, FRG). The sample temperature was measured on
the upper leaf surface using a copper-constantan thermoele-
ment connected to a digital voltmeter (accuracy: ± 0.1 K).
Determination of Chi a Fluorescence
Photosynthetic induction transients were recorded before
and during cooling of the leaves. The leaves were predarkened
 COLD-INDUCED LOW-WAVES IN CHL FLUORESCENCE TRANSIENTS
in the 1 h elapsing between measurements. Fluorescence was
measured using a pulse-modulation Chl fluorometer system
(PAM 103; H.Walz, Effeltrich, FRG) as described by Schrei-
ber et al. (9). Basic fluorescence Fo was obtained by weak,
modulated light of 1.64 jimol photons. m-2 s ', maximal
fluorescence of a dark-adapted leaf (Fv)m by a saturating flash
of white light of 2000 W * m-2 of 600 ms duration. After about
10 to 20 s, continuous actinic light of 111 ,utmol photons.
m-2 s' at 650 nm was applied. After the peak of the fluores-
cence curve, saturation pulses were triggered every 10 s.
Transients were plotted by a potentiometric chart-recorder
(SE 130, Goerz, Wien). The photochemical quenching coef-
ficient qP = ([Fv]s-Fv)/(Fv)s was calculated according to
Schreiber et al. (9).
RESULTS AND DISCUSSION
If short light pulses of saturating intensity are superimposed
on a light-driven photosynthetic induction curve the en-
hanced fluorescence usually relaxes within seconds to the
level reached shortly before application of the pulse (Fig. IA).
In each of the chilling-sensitive species tested, at a certain
temperature level during cooling, the fluorescence intensity
dropped within milliseconds after saturating light pulses below
the previous level of the induction curve. Within a few seconds
after the undershoot, the fluorescence gradually rose again
(Fig. 1 B). This reversible short-term decrease in fluorescence
will be termed low-wave. If the temperature is progressively
lowered, the L-waves first appear after several saturating light
lOs
(Fv)m
jj{}4J4
( Fv)s
o FO
._ 0 12 B (FV)e | t1
>
(D
(Fv)m(n)
~~~~~~~~~~~~(Fv)s
FO
0 1 2 co
Time of induction ( min )
Figure 1. Typical fluorescence induction kinetics of second trifoliate
leaves of Glycine max cv 'Maple Arrow' measured during progressive
cooling (A) at 200C and (B) at 0.90C. Insets: Enlarged detail. L, low-
wave; Fo, basic fluorescence; (Fv)m, maximal fluorescence of a dark-
adapted leaf upon excitation with a saturating light pulse; (F,)8,
maximal fluorescence during induction observed upon application of
a saturating light pulse; F,, variable fluorescence during induction;
(Fr)j, minimal variable fluorescence immediately after saturating light
pulses during L-waves.
741
pulses. As cooling progresses, L-waves are seen after each
saturating pulse and, in addition, the extent of the undershoot
gradually increases.
If soybean leaves are cooled for a longer period (several
hours) at a constant temperature at which L-waves begin to
appear, the sudden reversible lowering of fluorescence steadily
increases the longer the cooling continues (Fig. 2). If the
relative amplitude of the L-wave in the region of the largest
undershoots, (Fv-[Fv]l)/(F,)s, is plotted against the leaf tem-
perature, the critical temperature at which the L-waves appear
can be read from the curve (Fig. 3).
A functional phenomenon can be considered as a useful
criterion of chilling stress if it is (a) specific, i.e. if it occurs at
different critical temperatures in different chilling-susceptible
species and varieties, and (b) if it is reliable, i.e. if it is
invariably observed when the temperature drops below a
certain low level but in no case at temperatures above a
critical level.
(a) In order to test the specificity of the appearance of L-
waves, fluorescence transients were measured in two soybean
varieties, 'Evans,' known to be less sensitive, and 'Maple
Arrow,' which is considered to be more sensitive (3), and in
some other chilling-sensitive and chilling tolerance angios-
perm species during cooling down to 0°C. As Figure 3 shows,
L-waves do indeed appear at lower temperatures in the variety
'Evans' than in the variety 'Maple Arrow.' In Saintpaulia
ionantha L-waves were observed from a temperature of 9 to
10C, and in Saccharum oFficinarum from 7 to 8C. The
sensitivities of these species and varieties were in the same
order if Fv/Fm was calculated. The value of Fv/Fm at 4°C, as
compared with that at 20°C, was lowered by 0.027 (2.9%) in
'Evans,' by 0.046 (5.3%) in 'Maple Arrow,' by 0.047 (5.6%)
in S. oFlicinarum, and by 0.142 (17.9%) in S. ionantha. The
photochemical quenching coefficient qP under steady state
conditions is lowered by 50% in 'Evans' at approximately
4°C, in 'Maple Arrow' at 5.5°C, in S. offcinarum at about
4°C, and in S. ionantha at 9 to 10°C (Fig. 3). Thus our results
are in good agreement with the responses of chilling-sensitive
crop plants as reported by Havaux (1). A 50% depression of
ii2 h
O 2 ~~~60s
Time of induction
Figure 2. Effect of long-term cooling of leaves of Glycine max cv
'Maple Arrow' at 3.5°C for 1, 6, and 22 h. The magnitude of L-waves
increases with the duration of cooling.
742 LARCHER AND NEUNER Plant Physiol. Vol. 89, 1989

100
% (L
0% 80
60
L-
100
%- %
a)
80
80 C
0.
60
100
% nition and sensitivity ofthe response. The nonintrusive nature
0 co
80
60 0
60
0 4 8 12 16 20
Leaf
temperature ( 0C )
Flgure 3. Relative L-wave amplitudes (0,0) expressed as (Fv-(Fv))/
(FV1, and photochemical quenching coefficients under steady-state
conditions (*, *; qP expressed in percent of the value at 200C)
plotted against leaf temperatures of Glycine max cv 'Evans' (R =
-0.86) and cv 'Maple Arrow' (R = -0.96), S. officinarum (shade-
adapted, developing, greenhouse-grown leaves; R = -0.94) and S.
ionantha (R = -0.92) during progressive cooling.
vFd was observed at temperatures 3 to 6° K below those at
which the L-waves occur. This emphasizes the early-warning
character of the L-wave. In S. ionantha the vEd was reduced
to 50% at temperatures of 5 to 6°C; below 4C, necrotic
damage was observed (4).
(b) In leaves of angiosperm species, including the above-
mentioned chilling-sensitive species, typical L-waves did not
appear at room temperature provided predarkening did not
exceed 1 h; if the leaves were kept more than a few hours in
the dark, undershoots could also be seen at temperatures
above the critical level.
Cryptogams (Conocephalum conicum, Dryopterisfilix-mas,
Pteridium aquilinum) and gymnosperms (Chamaecyparis ob-
tusa, Sciadopitys verticillata, Metasequoia glyptostroboides,
Larix decidua, Picea abies, Pinus species, Ginkgo biloba)
exhibited L-waves at room temperature. In Ginkgo biloba this
phenomenon was more pronounced in the shade leaves than
in sunlit leaves.
The investigations carried out so far do not permit us to
offer a causal explanation for the brief drops in fluorescence.
Since taxa in which the size of the photosynthetic units is
comparable to that of shade-type plants, i.e. cryptogams and
gymnosperms (7) also show L-waves at room temperature,
this phenomenon appears to be of potentially far-reaching
significance. The occurrence of L-waves at low temperatures
may be connected with chilling-induced changes in the loca-
tion of components of the thylakoid membrane (6). Further-
0r more, it was observed (Fig. iB; Fig. 2) that the initial (Fv)m,
-c before application of actinic light, was lower at low tempera-
C)
tures than at 20C. The reduction in fluorescence yield in the
._ first light pulse may be explained as a type of nonphotochem-
*- ical quenching recently described by Schreiber and Neubauer
0
(8, 10); they attribute this saturating light effect to donor side-
dependent quenching.
0)
C
For diagnostic purposes the L-wave phenomenon can, un-
der controlled treatment with cold, and given a knowledge of
C. the fluorescence response to saturating light pulses at room
temperature, be regarded as a marker for thresholds of specific
chilling susceptibility. Of the various criteria indicating ab-
normal alterations of vital functions at low temperatures in
chilling sensitive species. the occurrence oflow-waves appears
E to be particularly promising on account of the ease of recog-
of the measurements and the speed with which they can be
0 carried out make the method particularly suitable for screen-
ing tests.
(
ACKNOWLEDGMENTS
We are grateful to Dr. U. Schreiber, University of Wuirzburg, for
critically reading the manuscript and for valuable suggestions; further
we thank Dr. Maria Bodner for her helpful cooperation.
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