Effects of Superimposed Temperature Stress on in vivo Chlorophyll Fluorescence of Vigna unguiculata under Saline Stress

J. PlantPhysiol. Vol. 136. pp. 92-102 (1990)

Effects of Superimposed Temperature Stress on in vivo Chlorophyll Fluorescence of Vigna unguiculata under Saline Stress W. LARCHER\ 1 2

J. WAGNER\ and A. THAMMATHAWORN2

Institute of Botany, University of Innsbruck, SternwartestraBe 15, A-6020 Innsbruck (Austria) Department of Biology, Khon Kaen University, 40002 Khon Kaen (Thailand)

Received December 4, 1989 . Accepted December 18, 1989

Summary

Cowpea [Vigna unguiculata (L.) Walp. var. IT 82 D-889] plants were exposed to a double stress whereby salinization (4-6 weeks at 100, 150 or 200 mM NaCI in the substrate) was the predisposing, and low and high temperatures were the additional constraints. Stress responses were assessed by measuring deviations from normal photosynthetic function by in vivo chlorophyll fluorescence. Salinization alone resulted in considerable changes in morphology, ion concentration and osmolality of the cellular sap of leaves, but had remarkably little effect on fluorescence characteristics at 20°C. Decreasing and increasing temperatures caused marked changes in the fluorescence quenching kinetics and revealed that temperatures of 5-10 °C are suboptimal, and those around 40°C, supraoptimal. In non-salinized plants, severe temperature stress at 3 - 6°C and 45 - 50°C was reflected by break-points in the temperature courses of the quenching coefficients at steady state, of Fv/FM' of (Fv)~ark/(Fv')~h\ and of the heat-induced rise of Fo. Mild superimposed temperature stress enhanced the unfavourable effect of saline stress on the kinetics of qN, indicating an increased impairment of the photosynthetic process in response to the combined stress. With severe superimposed temperature stress, however, the salinized plants appeared to be less susceptible than the control plants, since the threshold temperatures for abrupt deviations of Fv/FM and (Fv)~ark/(Fv')~h\ and for injury indicators (chilling symptoms, tissue freezing temperatures, Tc) shifted by 2 - 3 K in the direction of the stressing temperature. We discuss possible mechanisms for these contrasting effects, i.e. the enhancement of disturbances at sub- and superoptimal temperatures but coadaptive adjustments at extreme stress temperatures. The results demonstrate the value of employing superimposed stressors for intensifying stress expression and for providing more information on interactions between environmental factor combinations as they exist in nature.

Key words: Vigna unguiculata; chlorophyll fluorescence; saline, chilling, and heat resistance; multiple stress monitoring. Abbreviations: Fo = basic fluorescence; FM = maximal fluorescence of a dark-adapted leaf; Fp = peak of the induction transient; Fs = fluorescence intensity at steady state; Fv - variable fluorescence; Fv' = variable fluorescence upon application of saturation pulses at any given time during induction; (Fv)~ark = maximal variable fluorescence of a dark-adapted leaf; (FV')~ht = maximal variable fluorescence upon application of saturation pulses during induction; L-wave = sudden reversible undershoot of variable fluorescence after saturating light pulses; NaCl ext = NaCI concentration in the root medium; PhAR = photosynthetically active radiation; PS I, II - photosystem I, II; QA = primary electron acceptor of PS II; qN = non-photochemical quenching coefficient; (qN)max = highest value of qN during in© 1990 by Gustav Fischer Verlag, Stuttgart

Chlorophyll fluorescence under combined stress

93

duction; (qN)S = level of qN at steady state 8 min after start of induction; qP = photochemical quenching coefficient; (qp)s = steady state level of qP; Rfd = ratio between fluorescence decrease and steady state fluorescence at saturating light; tg ex = slope of initial rise to the transient peak of qN; tg (3 = decline after the peak of qN; Tc = critical temperature for heat-induced fluorescence rise; T p = temperature of heat-induced peak fluorescence; T f = tissue freezing temperature; 7r = solute potential.

1. Introduction A physiological approach to the study and analysis of response patterns to constraints on plants usually employs methods in which the plants are increasingly subjected to a defined stress factor. Such an approach provides the best means of obtaining reproduce able, quantitative values for specific stress responses, as well as a basis for the causal analysis of injury and resistance mechanisms. Most of our knowledge of the behaviour of plants under stress and their susceptibility to external constraints is based on one-factorone-response concepts. Under natural conditions, covariation of and interaction between stress factors is the norm (Osmond et al. 1987) under which the plant's response to a particular environmental factor undergoes modification in trend and quantity (Larcher 1987). It is known that a combination of different stress factors can bring about intensification, overlapping and reversal of stress effects (Burian et al. 1982). Under field conditions, stress may elicit more pronounced effects than a single stressor in a controlled experiment (Larcher and Bodner 1987). Furthermore, if a low-level stressor is combined with another stressor, the response to the first factor may become quantitatively more expressed (Wieser et al. 1974). Observations of this nature indicate that after the investigation in the form of a one-factor test, the applicability of the results should be further verified under the more complex conditions existing in the natural habitat. The method of controlled multiple-stress application provides a link between highly precise laboratory experiments and field studies. The investigation reported here was carried out in order (1) to study the effects of a double stress (i.e. climatic stress superimposed upon chemical-edaphic stress) on photosynthetic functions of Vigna unguiculata leaves, monitored by in vivo fluorescence measurements, and (2) to ascertain whether this procedure yields substantial new information and provides a more differentiated kind of stress monitoring. Vigna unguiculata is a subtropical crop plant that, due to its drought resistance (Hall and Schulze 1980) and a tendency to moderate halophily (Imamul Huq and Larher 1984), is of considerable agricultural importance in semiarid regions. Salinization was therefore selected as the predisposing stress, with high and low temperatures as additional physical stress factors, since temperature stress reaches all cell components immediately and to the same degree. Extreme temperatures therefore appear to be especially well suited for application as superimposed stress in the investigation of complex effects.

2. Material and Methods 2.1. Matenal

Cowpea [Vigna unguiculata (L.) Walp. var. IT 82 D-889] seeds were germinated in rectangular plastic pots of 3 litre capacity on artificial support media (vermiculite: perlite: 1: 1, v/v). The pots were soaked in half-strength HOAGLAND nutrient solution containing 5 mM KN0 3 , 1.5 mM Ca (N0 3)z, 1 mM NH4 H 2P0 4 , 1 mM MgS0 4 , and standard micronutrients. The nutrient solution was supplied weekly. Salinization was started after the plants had attained the developmental stage of first trifoliate leaf. For the salt treatment, NaCl was added to the nutrient solution in order to obtain final concentrations of 0 (control), 100, 150 and 200 mM; pH was 5.5. The level of solution in the containers was adjusted by adding deionizised water in order to maintain constant salinity (monitored with conductometer LF 21, WTW Weilheim, FRG). The plants were cultivated in a glass-covered screen house in the Botanical Garden of the University of Innsbruck under summer conditions at 15/9 h day/night, and a mean temperature of 18°C fluctuating between 11- 31 0c. Measurements were made on plants of the flowering stage that had been exposed to salinity stress for at least 1 month. 2.2. Methods Low and high temperature treatments: For the application of superimposed stress the intact plants were transferred to the laboratory in their pots. A leaflet of an attached trifoliate leaf was placed on a 165 x 95 mm aluminium plate connected with the thermal sink side of a thermoelectric modul (Type 803-1008-01), which was controlled by a bipolar controller (Type 809-3030) and a range extender (Type 809-1019) manufactured by Midland-Ross Corp. (Cambridge, USA). The roots and the shoot (except for the temperature-stressed leaves) were kept at a constant temperature of 20 to 22°C throughout the experiment. At the beginning of each series of measurements a fluorescence transient was measured at 20 °C as a reference. The temperature control system was then switched to the cooling mode and the leaf temperature lowered at a rate of 4 K . min -1 to the first cooling step of 10°C. After 30 min exposure to this temperature a fluorescence transient was again recorded, after which the temperature was lowered to 5 °C and then stepwise to 1 0C. At each temperature step, 30 min were allowed for the establishment of temperature equilibrium before measurements were made. Heat treatment was effected with the same Peltier system, in the heating mode, commencing with a temperature of 30°C, which was raised in steps of 5 K up to 50°C. The sample temperature was measured on the upper leaf surface using a copper-constantan thermocouple connected to a digital thermometer (accuracy: ±O.lK). Each series of measurments was repeated on 4 - 5 leaves. In vivo chlorophyll fluorescence: Variable fluorescence was measured after 30 min predarkening at the same position on the same leaflet before and during each cooling or heating experiment. Time courses of fluorescence emission were measured using a pulse-modulation

94

W. LARCHER, J. WAGNER, and A. THAMMATHAWORN

chlorophyll fluorometer system (PAM 101-103; H. Walz, Effeltrich, FRG) as described by Schreiber et al. (1986), and recorded with a potentiometric chart recorder (SE 130, Goerz, Wien). Basic fluorescence was determined by a weak, modulated light of 1.6/lmol photons' m - 2. S - 1, maximal fluorescence of a dark-adapted leaf by a saturating flash of strong white light of 104 /lmol'm- 2 's- 1 (PhAR) of 600 ms duration. After a lag phase of 20 s a fluorescence transient of 8 min duration was induced by continuous actinic light of 40/lmol photons' m - 2 • S - 1 at 650 nm. To analyse quenching mechanisms, saturation pulses were triggered every 20 s. Data were digitized (Digi-Pad 7, GTCO Corporation, Rockville USA) from the original traces, and fluorescence parameters (see Fig. 3), quenching coefficients, mean values and standard deviations were calculated with a computer programme adapted by Dr. M. Bodner (Institute of Botany, Innsbruck). The following fluorescence characteristics were evaluated: (FvJf:rk = (FM-F o), maximal variable fluorescence in the nonener~ized state of a dark-adapted leaf; (Fv'l'i}"' = maximal variable fluorescence after application of saturating flashes during photosynthetic induction by actinic light; the quotient Fv/FM = (Fv)~rk/((Fv)~rk + Fo), according to Kitajima and Butler (1975), as a measure of the potential efficiency of PSII (Bjorkman and Demmig 1987, Somersalo and Krause 1988); the ratio {FvJ}Jrk/{Fv'fJJh' which by deviation from a value of 1±0.2 indicates membrane-bound disturbances resulting from low and high temperatures (see Fig. 10); the relative low-wave amplztude, ((Fs-Fo)-(F,-Fo))/(Fv')s at steady state fluorescence, as defined by Larcher and Neuner (1989); the ratio between the fluorescence decrease and the steady state fluorescence, Rfd, according to Lichtenthaler et al. (1986), determined using fluorescence yield of saturating light pulses (FM and FM'); Fluorescence quenching coefficients were calculated according to Schreiber and Bilger (1987) as follows: Photochemical quenching coefficient qP = (FM'-F)/Fv'. Nont.hotochemical quenching coefficient qN = ((Fv) M-Fv')/(Fv) M; if (Fv)Jrk was lower than (Fv')l:fht at low temperatures, qN was calculated on the basis of (Fv')Rfht. Heat·induced chlorophyll fluorescence: For determination of the critical temperature for heat injury to the photosynthetic apparatus the method of heat induction of fluorescence was applied as described by Schreiber and Berry (1977). As in the light-induced treatments, the leaves were predarkened for 30 min and, attached to the plant, were placed on the thermoelectric module. During heating from 20 to 60 °C at a rate of 1 K . min -1 the temperature course was monitored by a digital thermocouple thermometer. Fo was excited by modulated light of extremely low intensity (1.6 /lmol photons' m - 2 • S - 1), PS I was maintained in a state of oxidation by application of additional far-red background light (Schott RG 715). The Foltemperature traces were evaluated for the critical temperature for onset of the heat-induced rise in fluorescence, Tc, and the temperature of peak fluorescence, Tp, according to Schreiber and Berry (1977).

Tissue freezing temperature: Determination of the freezing temperature, Tf , of leaves was made on strips (1 x 2 cm) excised from lateral leaflets of fully expanded trifoliate leaves. Eight such strips at a time were cooled until frozen on a thermoelectric cooling plate at a rate of 1 K . min -1 and the accompanying exotherms determined by differential thermal analysis (Burke et al. 1976). For this purpose the temperature course up to total freezing was registered via thermocouples by means of a data acquisition device (Mikromet-1; Cernusca 1987), which simultaneously evaluates and prints the values using a specially designed computer programme (see Fig. 12). This cryometric system requires 0.66 s for recording and evaluating each individual measurement and permits a reliable identification of exotherms down to a temperature difference of 0.2 K. The set-up was calibrated with ice-water and ice-salt mixtures to exactly 0.1 K.

When fully frozen, the tissue strips were thawed and the dead tissues were refrozen for determination of the freezing point depression of the cellular sap.

Osmometry: The cellular sap of shock frozen leaves was squeezed out and centrifuged at 10,000 g for 5 min (Biofuge A, Heraeus Sepatech, Osterode, FRG). The osmolality of the supernatant was determined with a cryoscopic osmometer (Osmomat 030, Gonotec Berlin, FRG). Ten replicates of control leaves and at least 5 replicates of stressed leaves of each salinity level were used each time. Ion concentration in the leaves: Dried leaf material was despatched to the Institute of Botany, University of Wiirzburg (Prof. Dr. W. M. Kaiser), where Na + content was measured by atomic absorption spectroscopy (Beckmann Instruments, Miinchen, FRG), and Clby anion chromatography (IC 1000, Biotronic, Maintal, FRG), as described by Schroppel-Meier and Kaiser (1987, 1988). Measurements are based on 4-5 replicate leaves. Morphometry: On at least 8 leaves from control plants and plants of each salt level the thickness of leaf tissues between the vascular bundles was measured by microscopy of sections with an ocular micrometer, and the saturated water content and dry weight were determined gravimetrically after drying at 80°C.

3. Results and Discussion 3.1. Single stress effects 3.1.1. Salinization Fluorescence measurements were made when the plants had been exposed to salt treatment for 4 - 6 weeks and were showing distinct signs of salt stress, as described by Imamul Huq and Larher (1984, 1985) for Vigna unguiculata and by Salim and Pitman (1988) for Vigna radiata. Due to poor growth and shorter internodes the plants of all salinization levels were at most half the height of the controls. At 100 mM NaCI the plants produced only half as many leaves as the controls, and at 200 mM NaCI only a third as many. After 6 weeks, necrotic damage became visible as small brown dots with a yellow periphery at 150 mM NaCl, and as dried leaf tips at 200 mM NaCl. The leaves of plants exposed to the highest salt levels were more succulent than those of only slightly stressed plants: at 200 mM NaCI the thickness of their mesophyll amounted to 390/tm, and at 100 mM to 365/tm, as compared to a mean of 305 /tm in the controls. The internal concentrations of N a + and CI-, and the osmolality increased in proportion to the substrate salt concentration (Fig. 1). Chloride accumulated in the leaves to a greater extent than sodium, the latter being retained mainly in the basal stems and roots (Imamul Huq and Larher 1985). Surprisingly, in view of the pronounced inhibition of growth and even the occurrence of damage, appreciable differences between controls and the various salt levels were seen in only a few of the fluorescence characteristics. At 20°C, the fluorescence indicators Fv/FM (see Fig.9), the ratio (Fv)~rk/(Fv')l:fht (see Fig. 10) and the steady state levels of qP and qN (Fig.2) remained practically unaffected. The peak of the induction curve, Fp (expressed as fraction of F M), was 20 % higher for salt-stressed plants than for controls, and the Rfd values (at 30°C) for salt-stressed plants were 2.5 as

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Fig. 3: Fluorescence transients at 20°C for leaves of control and salinized (200 mM NaCl ext) plants of Vigna unguiculata. Fo = fluorescence intensity with all PS II reaction centres open in non-energized state; F~rk = fluorescence intensity with all PS II reaction centres closed in non-energized, dark-adapted state; F~h! = maximal fluorescence intensity during induction by actinic light; FM' = fluorescence intensity with all PS II centres closed in energized state; F = fluorescence intensity at an, given time during induction; Fp = fluorescence at P level; (Fv)~r = maximal variable fluorescence in the non-energized state; Fyi = variable fluorescence upon application of saturation pulses at any given time during induction.

It is known that in intact leaves considerable impairment of the photosynthetic capacity (Schroppel-Meier and Kaiser 1988, Flanagan and Jefferies 1989) and of primary processes only occurs at excessive sodium chloride levels (Down ton and Millhouse 1985). One explanation might be that potentially toxic ions are effectively compartmentalized in the vacuoles and that few N a+ and Cl - ions penetrate the chloroplasts (Ball et al. 1985, Flowers 1985, Kaiser et al. 1983, Schroppel-Meier and Kaiser 1988). 3.1.2. Temperature

Evaluation of the fluorescence parameters suggests that the optimal temperature is between 20 and 30°C: within this range all processes involved in photosynthesis exhibit adjusted activity (d. kinetics of qP and qN; Figs. 5 A and B). At temperatures between 20 and 40°C, a value of more than 3 for the ratio between the fluorescence decrease and steady state fluorescence, Rfd, (which is an indicator for the activity of the secondary photosynthetic processes; Lichtenthaler 1986, 1988, Haitz and Lichtenthaler 1988) signalizes high rates of photosynthesis. By measuring the gaseous exchange of CO 2 under strong irradiation (300 W· m -2 PhAR) Ludlow and Wilson (1971) found the temperature optimum of photosynthesis for Vigna luteola to be 31°C.

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With decreasing or increasing temperature the shape of the fluorescence transients changes in a typical manner, examples of which are shown in Fig. 10 (insens). Marked changes in the fluorescence characteristics reveal the region around 10°C to be suboptimal; at high temperatures, photosynthesis remains efficient up to about 35 °C, but becomes increasingly impaired above 40°C. The «photosynthesisstress-thermogramme» for Vigna unguiculata (Fig. 4) shows the temperature ranges that, on the basis of fluorescence criteria, can be considered as optimal, sub- and supraoptimal.

Fig. 6: Temperature dependence of photochemical, (qp)s, and nonphotochemical, (qN)s, quenching of fluorescence in the steady state after 8 min application of actinic light (40 Jtmol . m - 2 • S - 1) on leaves of non-salinized cowpea plants. Ranges of optimal, suboptimal, supraoptimal and stress temperatures (cross hatched areas) are as defined in Fig. 4. Data points represent the mean of 3 -10 measurements. Regression lines for (qp)s between 0 and 5°C: y 0.088+0.153, r = 0.964, and between 10 and 40°C: y = 0.822+0.002, r = 0.943.

Quenching coefficients: The time courses of the quenching coefficients qP and qN at various temperatures are shown in Fig. 5. With decreasing temperatures the time courses of qP indicate an increasing delay in reoxidation of QA; at 5°C, where qP still attains the steady state level of the optimal temperature, electron transpon between PS II and NADPH 2 is greatly limited, causing reductions in quantum efficiency (Baker et al. 1983, Oquist and Manin 1986). A fun her temperature drop leads to accumulation of reduced QA, which in chilling sensitive species is only slowly reoxidized and remains elevated even under steady state conditions. Below

5°C, (qp)s drops abruptly (Fig. 6) and at 4°C it attains 78 % of the value for optimal temperatures; thus Vigna unguiculata can be considered as moderately chilling sensitive (as compared with the crop plants screened by Havaux (1987) at somewhat higher light intensity during measurements). Below 2°C the activity level of PS II remains permanently low (Fig. 5 A). The non-photochemical quenching coefficient (Fig. 5 B) rises to a high peak of thylakoid energization at and below 10°C, which reaches its maximal level at 5°C, indicating, just as the slowing down of the relaxation after (qN)max, a retardation of the energy consumption of ATP and NADPH2

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Chlorophyll fluorescence under combined stress Table 1: Threshold temperatures (0C) for limitation of photosynthetic activity of Vigna unguiculata grown on nutrient medium without NaCI at a mean temperature of 18°C. Chilling Heat Fluorescene indicator 4 45 Fv/F M
associated with the Calvin cycle (Krause et al. 1988). Below about 6°C the relaxation of qN disappears, and below 5 °C there is a rapid collapse of the build-up of high energization of the thylakoid membranes (Fig. 6). At supraoptimal temperatures above 40°C, thermal alterations of the thylakoid membranes (Berry and Raison 1981) and the related impairment of photosynthesis (Bilger et al. 1987) become apparent. The rapid rise of qP, in connection with the reduced speed of relaxation of qN, reflects the imbalanced distribution of excitation energy in favour of PS I, at the expense of the pronounced thermal sensitivity of the PSII complex (Santarius 1975, Berry and Bjorkman 1980, Krause and Weis 1984, Santarius and We is 1988) and the fact that non-radiative dissipative processes are becoming more important (Weis and Berry 1988). Maximal variable fluorescence: Strong reduction of the quantum yield of the PS II is signalized at temperatures of about 4°C and 45°C by a lowering of Fv/FM to 0.7 (see Fig. 9). The decrease of variable fluorescence at supraoptimal temperatures can be related to a limitation of electron donation to PSII (Laasch 1987, Santarius and We is 1988). The variable fluorescence of dark-adapted leaves, (Fvmrk, drops continuously at temperatures below 6°C as compared with (Fv')Rfh" observed upon application of saturating light pulses during induction. The ratio between these two fluorescence characteristics indicates this temperature as the threshold for the onset of stress effects on photosynthesis by a sharp break (Fig. 10). A possible explanation for the drop in this ratio with decreasing temperature might be a kind of non-photochemical donor-side dependent quenching described by Schreiber and Neubauer (1987) and Neubauer and Schreiber (1987). At lower temperatures there is a rearrangement of active centres of the thylakoid membrane (Maenpaa et al. 1988) so that non-radiative dissipation of energy takes place and the fluorescence yield in the first flash of light is small. Hence the ratio (Fv)~\lfk/(Fv')Rfh' appears to be an appropriate indicator for membrane-bound disturbances due to chilling. Threshold indicators for chilling, freezing, and heat stress: In Fig. 4 the ranges for stressful heat and cold are seen each to

97

extend over a temperature zone of 5 K. For the most important fluorescence indicators, threshold values for onset of cold- and heat-dependent depression of photosynthesis and for so % reduction in optimal activity are given in Table1. The critical temperature below which low-waves appear is SoC, as derived from the intersection of the extended regression line with the abscissa (d. Larcher and Neuner 1989); this threshold temperature corresponds to the break to the steep drop in (qp)s (d. Fig. 6). At 2.5-3°C, pronounced low-waves are seen, and the photosynthetic activity indicated by qP at steady state and the slope of the increase of qN is reduced to SO % of that at optimum temperature. At 3°C, Rfd also approaches 1, from which it can be deduced that there is no longer a net CO 2 uptake by the leaves (Lichtenthaler 1988). In a number of tropical Fabaceae, Ludlow and Wilson (1971) found temperature minima for CO 2 uptake to be between 6 and 7°C. In preliminary tests the chilling lethality of cowpea shoots was determined in refrigerated boxes. The plants survived overnight chilling (12 h) at 4 and 2°C without visible injuries; the first sign of damage affecting 20 - 25 % of the total leaf area occurred at 0.5 0C. Due to the time factor involved in the development of chilling injuries (Larcher and Bodner 1980) the lethality thresholds rose with increasing duration of chilling (48 h); then, the first visible damage to the leaves was recorded at 3°C (chiefly in the region of the vascular bundles), and at 1.5°C all leaves were killed. Kaku and Iwaya-Inoue (1988) found slight damage to seedlings of Vigna radiata and Vigna mungo after 2-3 days at 2.5 °C, although early biomembrane alterations as primary events of chilling disturbances were already detectable (from determinations of water proton NMR relaxation times) at temperatures of 7 -10°C. The average tissue freezing temperature, Tr, of the leaves is -1.3°C±0.16 (d. Fig. 13). On freezing, the cells are killed immediately and thus the Tr of Vigna unguiculata serves as a marker for instantaneous membrane rupture (Sakai and Larcher 1987). Within the heat range, the fluorescence characteristics become dramatically altered. When a temperature of 45°C is exceeded Rfd becomes 1. Ludlow and Wilson (1971) found the heat limit for CO 2 uptake to be 49°C for Vigna luteola, and an average of so °C for the tropical Fabaceae investigated. The critical temperature for the beginning of a heat-induced rise in fluorescence, Te, is SO °C (see Fig. 11). At this temperature, dissociation from the light-harvesting system takes place, as well as direct inactivation of the PS II reaction centres (Schreiber and Armond 1978, Havaux et al. 1988). According to Bilger et al. (1984) there is a clear correlation between Tc and so % necrotic heat injuries due to 30 min exposure to heat. Leaves of Vigna unguiculata used for measurement of light-induced transients and heated stepwise to SO °C showed some necrotic damage. With a heat limit for photosynthesis of between 45 and so °C and with damage at about so °C, Vigna unguiculata proves to be a species with fairly good heat tolerance (d. data in Berry and Bjorkman 1980, Kappen 1981, Larcher 1983), with a higher Te threshold than a number of tropical plants studied by Smillie and Nott (1979).

W. LARCHER, J. WAGNER, and A. THAMMATHAWORN

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3.2. Combined stress effects Under the influence of low and high temperatures, a number of fluorescence criteria show up the effect of saline stress on photosynthesis much more clearly than at temperatures within the optimal range. Particularly, the kinetics of qN appear to be a good indicator at sub- and supraoptimal temperatures. Fv/FM' the ratio (Fv)~rk/(Fv')Rfht and, under heat, Fa, only deviate from the norm in the stress regions below 5 °C and above 45°C. Mild imposed temperature stress enhances the increasing delay in energization of thylakoids that results from salt stress (Fig. 7). Characteristics of the course of quenching kinetics, which at 20°C differ only slightly for the different salinization levels, become much more pronounced outside the optimal temperature range. At 10 °C (not shown) the time required for the energization peak to be reached increases gradually from controls up to the highest salinization level. The slope, tg ex, to (qN)max, in the range of stress temperatures, becomes proportionally smaller with increasing saline stress (Fig. 8). All of these changes in the quenching kinetics indicate a deterioration of photosynthetic functions in response to combined stress. NaCI toxicity has to be avoided by salt sequestration in the vacuole. Since this requires a low membrane conductance and metabolic energy (Kaiser et al. 1988), sub- and supraoptimal temperatures might impair salt compartimentation. At low temperatures,

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longed (Fig. 12). Both changes are correlated with a higher osmolality of the cellular sap (Fig. 13). Schmidt et al. (1986) found that in salt-treated spinach leaves the killing temperature changed from - 6.5 °C (control) to -10 °C (at 150 mM NaCI) and -13 °C (at 200 mM NaCI), accompanied by a corresponding rise in the cellular solute concentration. On the basis of equations defining the dehydration effect of extracellular ice formation (Gusta et al. 1975, Rajashekar and Burke 1982), Schmidt et al. (1986) calculated the approximate relative contraction in cell volume at the frost killing temperature using the formula Vr/Vo = 1. 86("lT01 - Tr). 100, whereby Vr/Vo represents the residual volume of the frozen cell (as percent of the unfrozen cell volume), "lTo the initial osmolality (as osmol· kg-I) and Tr the freezing temperature (in 0q. Frost killing occurred in all samples at almost the same unfrozen cell volume. Calculating Vr/Vo for Vigna unguiculata gave a critical residual volume of 42-46%, irrespective

Fig. 10: Temperature course of the ratio (Fv)~rk/(Fv')l:fht in cowpea leaves: (e) control, (¢) 100mM, ('~) 150mM, and (0) 200mM NaClext . SE as in Fig. 9. Inserts: typical induction kinetics with saturating light pulses for control plants at 3, 20 and 45°C indicating changes of the relative heights between F dark and F"ght. L = low waves. M M

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tures than that of the controls. This is also reflected in the lower threshold temperatures for the appearance of lowwaves (2-3 °C for salinized plants), and in the displacement towards higher temperatures (by 2 K) of Tc in the salinized plants relative to the controls (Fig. 11). The extent to which stability to low and high temperatures is increased is apparently independent of the salt concentration and appears to be of an all-or-none nature. In the presence of chronic saline stress coadaptive processes might be able to effect a rise in heat resistance. Harrington and Aim (1988) provided evidence that salt shock results in synthesis of polypeptides similar to heat shock proteins, thus demonstrating cross tolerance between salt and heat stress (d. also Sachs and Ho 1986). With increasing saline stress the tissue freezing temperature becomes significantly lowered and supercooling is pro-

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100

W. LARCHER, J. WAGNER, and A. THAMMATHAWORN

of the different tissue freezing temperatures at the various levels of salinization. Schmidt et al. (1986) attribute this effect to an accumulation of thylakoid protecting compatible solutes that maintain osmotic equilibrium between the cell compartments. Indeed, Imamul Huq and Larher (1984) found a large accumulation of sugars, polyols, proline and quaternary ammonium compounds in Vigna unguiculata under saline conditions.

4. Conclusions The results presented in this paper show that an existing stress, in this case saline stress, which under optimal temperature conditions is only revealed by a few fluorescence indicators, becomes much more clearly recognizable in the presence of superimposed stress, such as heat or cold, as applied here. The value of employing combined stress and the greater information provided by multiple-stress fluorogrammes are clearly demonstrated by the foregoing results. In one and the same plant the application of combined stress factors can lead to opposing reactions, such as increased impairment of photosynthetic capacity and extension of the critical limits for function and viability: The delayed energization of thylakoids and the signs of reduced photosynthetic capacity at sub- and supraoptimal temperatures can be regarded as the reaction of a strained energy turnover system which, already predisposed by salt treatment, now responds to the additional stress of unfavourable temperatures in a detrimental manner. Disturbances in the photosynthetic system due to combined stresses can be detected readily by quenching analysis, as demonstrated by Bilger et al. (1987) for heat stress and by Krause and Somersalo (1989) for freezing stress. The broader temperature range between the inactivation thresholds resulting from pretreatment with salt reflects coadaptive resistance promoting changes in the molecular structure of biomembranes and proteins. Such changes are mainly indicated by fluorescence characteristics such as Fv/FM' (Fv)~ark/(Fv')kfht and the thermoinduced increase in Fa. Acknowledgements We are indebted to U. Schreiber and W. M. Kaiser, University of Wiirzburg, for helpful cooperation and valuable suggestions. Eva Wirth (Wiirzburg) determined the ion concentrations in the plant material. We thank B. B. Singh, Copea Breeder, IITA Ibadan, Nigeria, and Ponpimon Suriyajantratong, Khon Khaen University, Thailand, for supplying Vigna unguiculata seeds. Maria Bodner most kindly placed the evaluation programme at our disposal; G. Neuner determined chilling lethality. Our thanks are also due to J. Wiedermann for rearing the plants. Joy Wieser carefully translated the manuscript.

References BAKER, N. R., T. M. EAST, and S. P. LONG: Chilling damage to photosynthesis in young Zea mays. II. Photochemical function of thylakoids in vivo. J. Exp. Bot. 34, 189-197 (1983).

BALL, M. C. and J. M. ANDERSON: Sensitivity of photosystem II to NaCI in relation to salinity tolerance. Comparative studies with thylakoids of the salt-tolerant mangrove, Avicennia marina, and the salt-sensitive pea, Pisum sativum. Aust. ]. Plant Physio!. 13, 689-698 (1986). BALL, M. c., R. ]. MEHLHORN, N. TERRY, and L. PACKER: Electron spin resonance studies of ionic permeability properties of thylakoid membranes of Beta vulgaris and Avicennia germinans. Plant Physio!. 78, 1-3 (1985). BERRY, J. A. and O. BJORKMAN: Photosynthetic response and adaption to temperature in higher plants. Ann. Rev. Plant Physio!. 31, 491-543 (1980). BERRY, ]. A. and]. K. RAISON: Responses of macrophytes to temperature. In: LANGE, O. L., P. S. NOBEL, C. B. OSMOND, and H. ZIEGLER (eds.): Physiological Plant Ecology I, 277 -338, Encyc!. Plant Physiol. 12, Springer, Berlin (1981). BILGER, H.-W., U. SCHREIBER, and O. L. LANGE: Determination of leaf heat resistance: comparative investigation of chlorophyll fluorescence changes and tissue necrosis methods. Oecologia 63, 256-262 (1984). BILGER, W., U. SCHREIBER, and O. L. LANGE: Chlorophyll fluorescence as indicator of heat induced limitation of photosynthesis in Arbutus unedo L. In: TENHUNEN, J. D., F. M. CATARINO, O. L. LANGE, and W. C. OECHEL (eds.): Plant Responses to Stress, 391-399, Springer, Berlin (1987). BJORKMAN, O. and B. DEMMIG: Photon yield of O 2 and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170, 489-504 (1987). BURIAN, K., W. PUNZ, and R. SCHINNINGER: Efficiency of pollutant combinations on plants. In: STEUBING, L. and H. ]. JXGER (eds.): Monitoring of air pollutants by plants. Methods and Problems, 131-135, Junk, The Hague (1982). BURKE, M. J., L. V. GUSTA, H. A. QUAMME, c.]. WEISER, and P. H. LI: Freezing and injury in plants. Ann. Rev. Plant Physio!. 27, 507-528 (1976). CERNUSCA, A.: Application of computer methods in the field to assess ecosystem function and response to stress. In: TENHUNEN, J. D., F. M. CATARINO, O. L. LANGE, and W. C. OECHEL (eds.): Plant Responses to Stress, 157 -164, Springer, Berlin (1987). DOWNTON, W. J. S. and]. MILLHOUSE: Chlorophyll fluorescence and water relations of salt-stressed plants. Plant Sci. Lett. 37, 205-212 (1985). FLANAGAN, L. B. and R. L. JEFFERIES: Photosynthetic and stomatal responses of the halophyte, Plantago maritima L. to fluctuations in salinity. Plant, Cell and Environm. 12, 559-568 (1989). FLOWERS, T. J.: Physiology of halophytes. Plant and Soil 89, 41- 56 (1985). GUSTA, L. V., M. J. BURKE, and A. C. KApOOR: Determination of unfrozen water in winter cereals at subfreezing temperatures. Plant Physiol. 56, 707-709 (1975). HAITZ, M. and H. K. LICHTENTHALER: The measurement of Rfd-values as plant vitality indices with the portable field fluorometer and the PAM-fluorometer. In: LICHTENTHALER, H. K. (ed.): Applications of chlorophyll fluorescence, 249-254. Kluwer Acad. Pub!., Dordrecht (1988). HALL, A. E. and E.-D. SCHULZE: Drought effects on transpiration and leaf water status of cowpea in controlled environments. Aust.]. Plant Physiol. 7, 141-147 (1980). HARRINGTON, H. M. and D. M. ALM: Interaction of heat and salt shock in cultured tobacco cells. Plant Physiol. 88, 618-625 (1988). HAVAUX, M.: Effects of chilling on the redox state of the primary electron acceptor QA of photosystem II in chilling-sensitive and resistant plant species. Plant Physio!. Biochem. 25, 735-743 (1987).

Chlorophyll fluorescence under combined stress HAVAUX, M., M. ERNEZ, and R. LANNOYE: Correlation between heat tolerance and drought tolerance in cereals demonstrated by rapid chlorophyll fluorescence tests. J. Plant Physiol. 133, 555-560 (1988). IMAMUL HUQ, S. M. and F. LARHER: Osmoregulation in higher plants: Effect of maintaining a constant N a : Ca ratio on the growth, ion balance and organic solute status of NaCI stressed cowpea (Vtgna sinensis L.). Z. Pflanzenphysiol. 113, 163 -176 (1984). - - Dynamics of Na +, K +, and proline accumulation in salttreated Vigna sinensis (L.) and Phaseolus aureus (L.). J. Plant Physiol. 119, 133-147 (1985). KAISER, W. M., H. WEBER, and M. SAUER: Photosynthetic capacity, osmotic response and solute content of leaves and chloroplasts from SpmaCta oleracea under salt stress. Z. Pflanzenphysiol. 113, 15-27 (1983). KAiSER, W. M., G. KAISER, E. MARTINOIA, and U. HEBER: Salt toxicity and mineral deficiency in plants: Cytoplasmic ion homeostasis, a necessity for growth and survival under stress. In: KLEINKAUF, H., R. v. DOHREN, and L. JAENICKE (eds.): The roots of modern biochemistry, 721-733, de Gruyter, Berlin (1988). KAKU, S. and M. IWAYA-INOUE: Monitoring primary response to chilling stress in etiolated Vigna radtata and V. mungo seedlings using thermal hysteresis of water proton NMR relaxation times. Plant Cell Physiol. 29, 1063 -1068 (1988). KApPEN, L.: Ecological significance of resistance to high temperature. In: LANGE, O. L., P. S. NOBEL, C. B. OSMOND, and H. ZIEGLER (eds.): Encyclopedia of Plant Physiology, Vol. 12A, Physiological Plant Ecology I, 439-474, Springer, Berlin (1981). KITAjIMA, M. and W. L. BUTLER: Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochim. Biophys. Acta 376, 105 -115 (1975). KRAUSE, G. H. and S. SOMERSALO: Fluorescence as a tool in photosynthesis research: application in studies of photoinhibition, cold acclimation and freezing stress. Phil. Trans. R. Soc. Lond. B323, 281- 293 (1989). KRAUSE, G. H. and E. WEIS: Chlorophyll fluorescence as a tool in plant physiology. II. Interpretation of fluorescence signals. Photosynth. Res. 5,139-157 (1984). KRAUSE, G. H., S. GRAFFLAGE, S. RUMICH-BAYER, and S. SOMERSALO: Effects of freezing on plant mesophyll cells. In: LONG, S. F. and F. I. WOODWARD (eds.): Plants and Temperature, 311-327. Compo BioI. Ltd., Cambridge (1988). LAASCH, H.: Non-photochemical quenching of chlorophyll a fluorescence in isolated chloroplasts under conditions of stressed photosynthesis. Planta 171, 220-226 (1987). LARCHER, W.: Physiological Plant Ecology, 2nd. Ed. Springer, Berlin (1983). - StreB bei Pflanzen. Naturwissenschaften 74, 158-167 (1987). LARCHER, W. and M. BODNER: Dosisletalitat-Nomogramm zur Charakterisierung der Erkaltungsempfindlichkeit tropischer Pflanzen. Angew. Botanik 54,273-278 (1980). - - Criteria for chilling stress in Samtpaulta tonantha. Angew. Bot. 61, 309-323 (1987). LARCHER, W. and G. NEUNER: Cold-induced sudden reversible lowering of m VtVO chlorophyll fluorescence after saturating light pulses. A sensitive marker for chilling susceptibility. Plant Physiol. 89, 740-742 (1989). LICHTENTHALER, H. K.: Laser-induced chlorophyll fluorescence of living plants. Proc. IGARSS Symp. Zurich, ESA SP-254, ESA Publ. Div. (1986). - In VIVO chlorophyll fluorescence as a tool for stress detection in plants. In: LiCHTENTHALER, H. K. (ed.): Applications of chlorophyll fluorescence, 129 -142 (1988).

101

LICHTENTHALER, H. K., C. BUSCHMANN, U. RINDERLE, and G. SCHMUCK: Application of fluorescence in ecophysiology. Radiat. Environm. Biophys. 25, 297 -308 (1986). LUDLOW, M. M. and G. L. WILSON: Photosynthesis of tropical pasture plants: I. Illuminance, carbon dioxide concentration, leaf temperature, and leaf-air vapour pressure difference. Aust. J. BioI. Sci. 24, 449-470 (1971). MAENPAA, P., E. ARO, S. SOMERSALO, and E. TYYSTjARVI: Rearrangement of the chloroplast thylakoid at chilling temperature in the light. Plant Physiol. 87, 762-766 (1988). NEUBAUER, C. and U. SCHREIBER: 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 (1987). NEUNER, G. and W. LARCHER: Determination of differences in chilling susceptibility of two soybean varieties by means of in VIVO chlorophyll fluorescence measurements. J. Agron. Crop Sci., in press (1990). OQUIST, G. and B. MARTIN: Cold climates. In: BAKER, N. R. and S. P. LONG (eds.): Photosynthesis in contrasting environments, 237-293. Elsevier, Amsterdam (1986). OSMOND, C. B., M. P. AUSTIN, J. A. BERRY, W. D. BILLINGS, J. S. BOYER, J. W. H. DACEY, P. S. NOBEL, S. D. SMITH, and W. E. WINNER: Stress physiology and the distribution of plants. BioScience 37,38-48 (1987). RAjASHEKAR, C. and M. J. BURKE: Liquid water during slow freezing based on cell water relations and limited experimental testing. In: LI, P. H. and A. SAKAI (eds.): Plant Cold Hardiness and Freezing Stress, 2, 211-220. Academic Press, New York (1982). SACHS, M. M. and T.-H. D. Ho: Alteration of gene expression during environmental stress in plants. Ann. Rev. Plant Physiol. 37, 363 - 376 (1986). SAKAI, A. and W. LARCHER: Frost survival of plants. Responses and adaptation to freezing stress. Ecological Studies 62, Springer, Berlin (1987). SALIM, M. and M. G. PITMAN: Salinity tolerance of mung bean (Vtgna radzata L.): seed production. BioI. Plant. 30, 53 - 57 (1988). SANTARIUS, K. S.: Sites of heat sensitivity in chloroplasts and differential inactivation of cyclic and noncyclic photophosphorylation by heating. J. Thermal Biology 1, 101-107 (1975). SANTARIUS, K. A. and E. WEIS: Heat stress and membranes. In: HARWOOD, J. L. and T. J. WALTON (eds.): Plant Membranes - Structure, Assembly and Function, 97 -112. Biochem. Soc., London (1988). SCHMIDT, J. E., J. M. SCHMITT, W. M. KAISER, and D. K. HINCHA: Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach. Plant a 168,50-55 (1986). SCHREIBER, U. and P. A. ARMOND: Heat-induced changes of chlorophyll fluorescence in isolated chloroplasts and related heatdamage at the pigment level. Biochim. Biophys. Acta 502, 138-151 (1978). SCHREIBER, U. andJ. A. BERRY: Heat-induced changes of chlorophyll fluorescence in intact leaves correlated with damages of the photosynthetic apparatus. Plant a 136, 233-238 (1977). SCHREIBER, U. and W. BILGER: Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. In: TENHUNEN, J. D., F. M. CATARINO, O. L. LANGE, and W. C. OECHEL (eds.): Plant response to stress. Functional analysis in mediterranean ecosystems, 27 -53, Springer, Berlin (1987). SCHREIBER, U. and C. NEUBAUER: 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 (1987).

102

W. LARCHER, J. WAGNER, and A. THAMMATHAWORN

SCHREIBER, U., U. SCHLIWA, and W. BILGER: Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth. Res. 10, 51-62 (1986). SCHROPPEL-MEIER, G. and W. M. KAISER: Amperometric titration largely overestimates chloride concentrations in chloroplast extracts. Z. Naturfosch. 42c, 1109-1112 (1987). - - Ion homeostasis in chloroplasts under salinity and mineral deficiency. 1. Solute concentrations in leaves and chloroplasts from spinach plants under NaCl or NaN0 3 salinity. Plant Physio!. 87, 822-827 (1988). SMILLIE, R. M. and R. NOTT: Heat injury in leaves of alpine, temperate and tropical plants. Aust. J. Plant Physio!. 6, 135 -141 (1979).

- - Salt tolerance in crop plants monitored by chlorophyll fluorescence in vivo. Plant Physio!. 70, 1049 -1054 (1982). SOMERSALO, S. and G. H. KRAUSE: Changes in chlorophyll fluorescence related to photoinhibition of photosynthesis and cold acclimation of green plants. In: LICHTENTHALER, H. K. (ed.): Applications of chlorophyll fluorescence, 157-164. Kluwer Acad. Pub!., Dordrecht (1988). WEIS, E. and J. A. BERRY: Plants and high temperature stress. In: LONG, S. F. and F. I. WOODWARD (eds.): Plants and Temperature, 329-346. Compo Bio!. Ltd., Cambridge (1988). WIESER, W., J. OTT, F. SCHIEMER, and E. GNAIGER: An ecophysiological study of some meiofauna species inhabiting a sandy beach at Bermuda. Marine Bio!. 26, 235-248 (1974).