Glycine max (L.) Merr. cv 'Maple Arrow' and cv 'Evans' were grown from seed in pots .... Arrow,' which is considered to be more sensitive (3), and in some other ...
Received for publication June 8, 1988 and in revised form November 4, 1988
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
While searching for additional warning signs of chillinginduced 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.
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 lowwave, indicates specific thresholds of low temperature stress. Its occurrence under controlled chilling can be regarded as a quantitative marker for screening chilling susceptibility in angiosperms.
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 University 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 measurements, leaves were taken from greenhouse plants of Saintpaulia ionantha hybrids and from young developing shoots of Saccharum officinarum.
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 temperature 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
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) manufactured 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 thermoelement connected to a digital voltmeter (accuracy: ± 0.1 K).
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 fluorescence 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.
Determination of Chi a Fluorescence
Photosynthetic induction transients were recorded before and during cooling of the leaves. The leaves were predarkened 740
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 Schreiber 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 fluorescence curve, saturation pulses were triggered every 10 s. Transients were plotted by a potentiometric chart-recorder (SE 130, Goerz, Wien). The photochemical quenching coefficient 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 enhanced 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
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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 temperature, 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 Lwaves, 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 angiosperm 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
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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, lowwave; Fo, basic fluorescence; (Fv)m, maximal fluorescence of a darkadapted 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.
ii2 h
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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.
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LARCHER AND NEUNER
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Plant Physiol. Vol. 89, 1989
may be connected with chilling-induced changes in the location of components of the thylakoid membrane (6). Furthermore, it was observed (Fig. iB; Fig. 2) that the initial (Fv)m, before application of actinic light, was lower at low temperatures than at 20C. The reduction in fluorescence yield in the first light pulse may be explained as a type of nonphotochemical quenching recently described by Schreiber and Neubauer (8, 10); they attribute this saturating light effect to donor sidedependent quenching. For diagnostic purposes the L-wave phenomenon can, under controlled treatment with cold, and given a knowledge of 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 abnormal alterations of vital functions at low temperatures in chilling sensitive species. the occurrence oflow-waves appears to be particularly promising on account of the ease of recognition and sensitivity ofthe response. The nonintrusive nature of the measurements and the speed with which they can be carried out make the method particularly suitable for screening tests.
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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 (shadeadapted, 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 abovementioned 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
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. LITERATURE CITED 1. Havaux M (1987) Effects of chilling on the redox state of the primary electron acceptor QA of photosystem II in chilling sensitive and resistant plant species. Plant Physiol Biochem 25:
735-743 2. Hethereugton SE, Oquist G (1988) Monitoring chilling injury: A comparison of chlorophyll fluorescence measurements, postchilling growth and visible symptoms of injury in Zea mays. Physiol Plant 72: 241-247 3. Hume DJ, Jackson KH (1981) Frost tolerance in soybeans. Crop Sc 210: 689-692 4. Larcher W, Bodner M (1987) Criteria for chilling stress in Saintpaulia ionantha. Angew Botanik 61: 309-323 5. Mac Rae EA, Hardacre AK, Ferguson IB (1986) Comparison of chlorophyll fluorescence with several other techniques used to assess chilling sensitivity in plants. Physiol Plant 67: 659-665 6. Mienpaa P, Aro E, Somersalo S, Tyystjrvi E (1988) Rearrangement of the chloroplast thylakoid at chilling temperature in the light. Plant Physiol 87: 762-766 7. Malkin S, Fork DC (1980) A comparative study of the photosynthetic unit sizes in sun and shade plants. Carnegie Inst Wash Year Book 79: 187-189 8. Neubauer C, Schreiber U (1987) The polyphasic rise of chlorophyll fluorescence upon onset of strong continuous illumination: I. Saturation characteristics and partial control by the photosystem II acceptor side. Z Naturforsch 42c: 1246-1254 9. Schreiber U, Schliwa U, Bilger W (1986) Continuous recording of photochemical and non-photochemical quenching with a new type of modulation fluorometer. Photosynth Res 10: 5162 10. Schreiber U, Neubauer C (1987) The polyphasic rise of chlorophyll fluorescence upon onset of strong continuous illumination: II. Partial control by the photosystem II donor side and possible ways of interpretation. Z Naturforsch 42c: 1255-1264 11. Smillie RM (1979) The useful chloroplast: A new approach for investigating chilling stress in plants. In JM Lyons, D Graham, JK Raison, eds, Low Temperature Stress in Crop Plants. Academic Press, New York, pp 178-202 12. Smillie RM, Hetherington SE (1983) Stress tolerance and stress induced injury in crop plants measured by chlorophyll fluorescence in vivo. Chilling, freezing, ice cover, heat and high light. Plant Physiol 72: 1043-1050
