Thermoluminescence studies on the facultative ... - Springer Link

5 downloads 0 Views 258KB Size Report
the 46 °C-band is observed in the morning after onset of the light and in the evening. At around 12 a.m. it is suppressed. The intensity of the 46 °C-band relates ...
Planta (1998) 205: 587±594

Thermoluminescence studies on the facultative crassulacean-acidmetabolism plant Mesembryanthemum crystallinum L. Anja Krieger1,2,*, Susanne Bolte1, Karl-Josef Dietz1, Jean-Marc Ducruet2 1

Julius-von-Sachs Institut fuÈr Biowissenschaften, UniversitaÈt WuÈrzburg, Mittlerer Dallenbergweg 64, D-97082 WuÈrzburg, Germany Section de BioeÂnergeÂtique, DBCM, CNRS URA 2096, BaÃt. 532, CEA Saclay, F-91191 Gif-sur-Yvette, France

2

Received: 7 August 1997 / Accepted: 22 December 1997

Abstract. Thermoluminescence (TL) signals were measured from leaves of the facultative CAM (crassulacean acid metabolism) plant Mesembryanthemum crystallinum L.. Following the induction of CAM by salt treatment, a TL band at 46 °C was induced, which was charged by a single-turnover ¯ash. The intensity of the 46 °C-band depends on the number of excitation ¯ashes and oscillates with a period of four. A similar band was induced in C3 plants by far-red illumination. Under CAM conditions, the intensity of the 46 °C-band underlies a diurnal rhythm. The maximal intensity of the 46 °C-band is observed in the morning after onset of the light and in the evening. At around 12 a.m. it is suppressed. The intensity of the 46 °C-band relates to diurnal changes in the ratio of dihydroxy acetone phosphate/3-phosphoglycerate (DHAP/PGA) which is an indicator of the energy status of the chloroplast. During high-intensity illumination, the 46 °C-band disappears, but it is restored in the dark. We propose that the 46 °C-band is an indicator of the metabolic state of the leaf, originating from photosystem II centres initially in the S2(S3)QB oxidation state, in which the electron acceptor QB becomes reduced either by reverse electron ¯ow or reduction of the plastoquinone pool via an NAD(P)H plastoquinone oxidoreductase. We present evidence that the redox state of the electron-transport chain is di€erent under conditions of CAM compared to C3 metabolism and that changes induced by CAM can be monitored by measuring the amplitude of the 46 °Cband after ¯ash excitation.

*

Present address: Institut fuÈr Biologie II/Biochemie der P¯anzen, UniversitaÈt Freiburg, SchaÈnzlestr. 1, D-79104 Freiburg, Germany Abbreviations: CAM ˆ crassulacean acid metabolism ; DHAP ˆ dihydroxyacetone phosphate; FR ˆ far red light, k 730 nm; PGA ˆ 3-phosphoglycerate; PFD ˆ photon ¯ux density; PQ ˆ plastoquinone; QB ˆ the second quinone acceptor in PSII; S-states ˆ oxidation states of the manganese cluster of PSII; TL ˆ thermoluminescence; Tm ˆ maximum emission temperature of a TL band Correspondence to: A. Krieger; E-mail: [email protected]; Fax: 49 (761) 2032601

Key words: Crassulacean acid metabolism ± Mesembryanthemum ± Photosystem II ± Thermoluminescence

Introduction Crassulacean acid metabolism (CAM) is an adaptation mechanism facilitating plant growth in arid environments. Uptake of CO2 from the atmosphere and photosynthesis are separated from each other in time and show a diurnal rhythm (e.g. Osmond 1978; Osmond et al. 1996; Winter and Smith 1996). During the night, CO2 uptake and malate accumulation in the vacuole take place; during the day, malate is decarboxylated and CO2 is ®xed in the Calvin cycle. The metabolic intensity changes during the day. In greenhouse-grown KalanchoeÈ pinnata, light-saturated photosynthetic electron ¯ow is highest between 9 a.m. and 2 p.m., declines in the afternoon and is lost at 10 p.m. (Heber et al. 1996). Photosynthetic carbon assimilation via the detour of CO2 ®xation by phosphoenolpyruvate (PEP)-carboxylase in the dark has a higher requirement for ATP and NADPH than C3 photosynthesis. In the light during the day, the net energy requirement varies in di€erent types of CAM plants between values of 4.8 ATP and 3.2 NADPH to 3.8 ATP and 2.6 NADPH per CO2 assimilated, depending on whether the storage carbohydrate is starch or soluble hexose (Winter and Smith 1996). During the night, 0.5±1 ATP is additionally needed per malate accumulated in the vacuole (Winter and Smith 1996; Black et al. 1996). Active mechanisms of CO2 uptake, which consume ATP, are also found in algae exposed to low CO2 concentrations in the growth medium. Under this condition, a so-called long-lived luminescence component or `afterglow' has been observed after white-light illumination and was related to reverse electron ¯ow (Mellvig and Tillberg 1986; Sundblad et al. 1986; Sundblad 1988). Reverse electron ¯ow was ®rst described by Schreiber

588

and Avron (1977; see also Schreiber 1980). Addition of ATP in the dark to preilluminated thylakoid membranes from spinach leads to a hydrolysis of ATP and thereby to a reduction of the plastoquinone (PQ) pool. The PQ pool is in equilibrium with the quinone acceptors QA and QB of PSII, thus QB becomes partially reduced in the dark. The long-lived luminescence component originates from a recombination reaction of the electrons back-transferred (Qÿ B ) with the S2 or S3 oxidation state of the Mn cluster at the donor side of PSII. In C3 plants and green algae grown at normal CO2 concentrations, the same long-lived luminescence component is generally not observed after white-light illumination or xenon ¯ash sequences but can be stimulated by far-red (FR) illumination (Bertsch and Azzi 1965; BjoÈrn 1971; Palmqvist et al. 1986; Schmidt and Senger 1987; Nakatomo et al. 1988; Sundblad 1988; Sundblad et al. 1988; Hideg et al. 1991; Miranda and Ducruet 1995). An alternative method of studying this reaction mechanism is to measure the thermoluminescence (TL) produced when luminescence emission is stimulated by progressively heating the sample, in order to facilitate recombination reactions in PSII. Thermoluminescence can be used as a probe of the behaviour of PSII reaction centres, both in isolated systems and in whole leaves (for reviews, see Sane and Rutherford 1986; Vass and Inoue 1992). Using this method, samples are illuminated to generate charge pairs within the PSII reaction centre and then rapidly cooled down to trap those charge-separated states. Alternatively, samples can be cooled down ®rst and then illuminated at the lower temperature. Subsequent warming reveals several peaks of luminescence emission. These emission bands originate from recombination of di€erent types of trapped charge pairs, which can be identi®ed by their maximum emission temperature, Tm. For example, recombination of the semiquinone, QB), with the S2 state of the water-splitting complex yields a TL band centered around 30 °C, the socalled B-band (Rutherford et al. 1982). In whole leaves, at higher temperatures (40±46 °C) an additional TL band, the afterglow band, has been reported (Desai et al. 1983) and ascribed to the `afterglow' luminescence emission induced by reverse electron ¯ow (Miranda and Ducruet 1995). This afterglow band is suppressed by either the addition of 3-(3¢,4¢-dichlorophenyl)-1,1-dimethylurea (DCMU) or an uncoupler or by freezing the sample. The main advantage of measuring TL rather than luminescence is that the components are more easily resolved than luminescence decay phases. Furthermore, warming of a leaf at an appropriate rate fully reveals the `afterglow' emission, while in luminescence measurements only a fraction of the long-lived component is seen. In the present study TL emission was measured from leaves of the facultative CAM plant Mesembryanthemum crystallinum. This is a model plant for studying changes correlated with CAM because of its metabolic ¯exibility which enables it to shift from C3-carbon assimilation to the CAM mode under high-salinity conditions. Within a few days of irrigation with a 500 mM NaCl solution, six-

A. Krieger et al.: Thermoluminescence studies on the facultative plant

week-old M. crystallinum plants show the ®rst features of CAM (Winter and Von Willert 1972; Winter 1973). In this communication, changes in TL bands were investigated during induction of CAM. To study the origin of the afterglow band, ¯ash excitation and FR light were used to induce TL in salt-treated and control plants. The number of excitation ¯ashes was varied to follow the oscillation pattern of TL bands. In addition, the diurnal variations in ¯ash-induced TL emission were measured in M. crystallinum and the obligate CAM plant KalanchoeÈ daigremontiana. The TL bands were related to the actual metabolic state of the leaf by determining the concentrations of dihydroxyacetone phosphate (DHAP) and 3-phosphogylcerate (PGA). Furthermore, the e€ect of high-light illumination on M. crystallinum was investigated by measuring chlorophyll ¯uorescence and TL immediately after high-light stress and during dark recovery. Materials and methods Plant material. Mesembryanthemum crystallinum L. and KalanchoeÈ daigremontiana Hamet et Perr. were grown from seed in potting soil in a greenhouse at a photon ¯ux density (PFD) of 400 lmol quanta á m)2 á s)1 (15-h light period). Seedlings were selected for uniform size and transferred to single pots with standard potting soil after three weeks. Six-week-old M. crystallinum plants were treated with either water or NaCl solution (500 mM) for several days. Measurements of TL and malate content were carried out using the second foliar leaf pair. Seeds were kindly provided by K. Winter (Smithsonian Tropical Research Institute, Balboa, Panama). Thermoluminescence measurements. Thermoluminescence was measured with a custom-made apparatus in WuÈrzburg. The sample holder consisted of a horizontal copper chamber sealed by a glass window. For cooling and heating, a three-stage Peltier element (Marlow Instruments, Dallas, Texas, USA) was mounted below the chamber. The Peltier element itself was embedded into a copper block and cooled by water which ¯owed through a spiral tube system inside the copper block. The sample was illuminated via a ®bre optic by either a halogen lamp or a single-turnover ¯ash lamp (Walz, E€eltrich, Germany) or FR light (LED conus, k 730 nm, 20 nm half-bandwidth). After illumination, the ®bre optic was removed and replaced by a red-sensitive photomultiplier (H570150; Hamamatsu, Shizuoka-ken, Japan) as detector. The measuring window of the detector was the same size as the cuvette. The sample was cooled down and heated via the Peltier element, controlled by a temperature-control box (Marlow Instruments). Temperature was measured with a thermistor at the highest step of the Peltier element (temperature controlling) and with a thermocouple on top of the sample. The samples (excised pieces of a leaf, 1 cm ´ 0.5 cm) were incubated for 2 min in the dark at 20 °C, then cooled within 1 min to 1 °C and illuminated with a single-turnover ¯ash. After a short dark incubation time (20 s at 1 °C), the sample was warmed up to 70 °C at a heating rate of 0.5 °C á s)1 and light emission was measured during the heating. Sample incubation and temperature regulation, data acquisition, handling and graphical simulation were as described in Ducruet and Miranda (1992). In order to allow comparison of di€erent leaves, the integrated area of the 46 °C-band was normalised to the integrated area of the B-band. The relative intensities of the TL bands can change from plant to plant and depend especially on the time of day when the measurements were performed. Unless otherwise speci®ed, measurements were

A. Krieger et al.: Thermoluminescence studies on the facultative plant

589

performed after 4 p.m. In the following, integrated peak areas of TL bands are called intensities and peak heights are called amplitudes. All TL measurements were repeated three to ®ve times with leaf pieces from di€erent plants. Fluorescence measurements. Fluorescence was measured using a PAM 101 ¯uorimeter (Walz) and the same sample holder as for TL measurements. Saturating ¯ashes were provided by a Schott (Walz) lamp, controlled by a PAM 103 ¯uorimeter; for continuous illumination a halogen lamp was used. During illumination (and, where appropriate, during dark relaxation) leaves were maintained at 20 °C. Malate-measurements. Malate was measured in aqueous leaf extracts. Fresh leaf (0.1 g) was homogenised with distilled water and boiled for 10 min in a heating block. After centrifugation (6000 g, 10 min), the clear extract was diluted 100-fold. Malate was determined by isocratic suppressed anion chromatography (Biotronik, Maintal, Germany). Determination of DHAP and PGA levels in non-aqueously isolated chloroplasts. Metabolism of leaves was stopped by rapid freezing in liquid nitrogen at the time points indicated. The leaf tissue was freeze-dried at )40 °C and used for fractionation in non-aqueous media consisting of carbon tetrachloride and petroleum ether. Chloroplasts were separated from residual leaf material and puri®ed by density-gradient centrifugation and sedimentation as described for spinach (Dietz and Heber 1984). Levels of DHAP and PGA were determined spectrophotometrically by enzymatic coupling and related to chlorophyll contents of the non-aqueous chloroplast fraction (Dietz and Heber 1984). The measurements were repeated three times; the absolute error was between 10 and 20%.

Results Figure 1 shows TL curves from leaves of Mesembryanthemum crystallinum. Induction of CAM by treating the plants with 500 mM NaCl solution resulted in a change to the shape of the TL curves. Leaves of control plants (0 d salt, Fig. 1) showed mainly a TL band with an emission peak at around 27 °C, characteristic of a B-band which originates from QB) S2 or QB) S3 recombination (Rutherford et al. 1982). In addition, there was a small shoulder at higher temperatures (approx. 46 °C). After 2 d of salt treatment, a broader emission arose, consisting of two maxima (B-band and 46 °C-band), which could be ®tted as two components by signal analysis using the ®tting procedure described by Ducruet and Miranda (1992). The increase in the intensity of the 46 °C-band in relation to the B-band depended on the duration of the salt treatment as shown in Fig. 2. During prolonged salt treatment, the intensity and sharpness of the 46 °C-band increased up to 7 d of salt treatment. The intensity of the 46 °C-band was highest when TL was measured at 4 p.m. or later (data not shown). After 4 d of salt treatment, these plants accumulated 15 mmol malate á (kg Fw))1 and after 7 d up to 35 mmol malate á (kg Fw))1. Longer salt treatment (16 and 22 d) led to a decrease in the high-temperature TL band, concomitant with obvious damage to the leaf (data not shown). Excitation with a single-turnover ¯ash led mainly to the formation of a B-band in control plants. However, it

Fig. 1. Thermoluminescence signals from leaves of Mesembryanthemum crystallinum. The plants were treated (watered) with 0.5 M NaCl solution for the time indicated. Leaves were dark-adapted for 2 min at 20 °C, then cooled to 1 °C. Thermoluminescence was excited by one single-turnover ¯ash at 1 °C. Measurements were performed after 4 p.m. The scale of the TL amplitude is the same for all curves; for clarity, the curves have been displaced vertically, a.u., arbitrary units

has been reported by Miranda and Ducruet (1995) that an afterglow band with a maximal emission around 43 °C is stimulated by FR illumination in leaves of C3 plants. Far-red light mainly excites PSI and leads to an oxidation of the PQ pool. To a small extent, it also excites PSII and causes a complete randomisation of the S-states of the Mn cluster. To see if the 46 °C-band

Fig. 2. Dependence of the 46 °C-band on the length of salt treatment. The TL signals were measured as in Fig. 1, the TL curve was ®tted by two components with a Tm at 25 °C (B-band) and a Tm at 46 °C (46 °C-band), and the integrated area of the 46 °C-band was divided by the integrated area of the B-band for normalisation

590

observed in salt-treated M. crystallinum is related to this afterglow band in C3 plants, we performed TL measurements after excitation with FR light on leaves of a 6 dsalt-treated plant and an untreated control plant (Fig. 3). Far-Red illumination for 30 s at 10 °C gave rise to the 46 °C-band in the salt-treated plant while the B-band was almost completely quenched (Fig. 3A). The amplitude of the 46 °C-band after FR illumination was as high as after excitation with one single-turnover ¯ash at 1 °C. In a control (not salt-treated) plant, charging TL with a single-turnover ¯ash resulted only in a B-band near 30 °C and no afterglow band (Fig. 3B). However, FR illumination of the control plant at 10 °C produced an afterglow band of amplitude comparable to that observed for the 46 °C-band in the salt-treated plant. The emission peak was at a lower temperature (Tm at 40 °C) compared to the salt-treated plant. The Tm might be di€erent because of changes in the lipid composition or an accumulation of osmolytes, e.g. proline, in the lumen induced by salt treatment and concomitant water stress. The temperature maximum of a TL band can be in¯uenced by the presence of osmolytes and cryopro-

Fig. 3A,B. Thermolumincescence signals of Mesembryanthemum crystallinum. Thermoluminescence was excited either by a single-turnover ¯ash at 1 °C or by 30 s FR light at 10 °C, and leaves were subsequently cooled to 1 °C. A The TL signals of a plant that was treated with salt solution for 6 d. B The TL signals of a control (not salt-treated) plant. Di€erent plants were used from those in Fig. 1 and measurements were performed in the morning from 8.30 to 10 a.m., hence signals obtained are slightly di€erent from those in Fig. 1

A. Krieger et al.: Thermoluminescence studies on the facultative plant

Fig. 4. Flash dependency of the integrated area of the 46 °C-band ( ®lled circles) and the ratio of 46 °C-band/B-band (open circles) of Mesembryanthemum crystallinum plants that were treated for 6 d with salt solution. Zero to ®ve ¯ashes were given at 1 °C after 2 min dark adaptation of the leaf. Measurements were performed between 4.30 p.m. and 7 p.m.

tectants as has been shown recently for the B-band (Krieger et al. 1998). To further characterise the 46 °C-band, we measured the oscillation pattern of TL induced by varying the number of ¯ashes in leaves of M. crystallinum plants which had been treated with salt for 6 d. Figure 4 shows an oscillation of the 46 °C-band area with a period of four and a maximum after the second ¯ash. Interestingly, heating a dark-adapted leaf without previous excitation also gave rise to a low intensity 46 °C-band (Fig. 4, ®lled circles, 0 ¯ash). The intensity of TL emission in this case depended on the time of dark adaptation. Dark adaptation for 30 min led to a total loss of the 46 °Cband. For the measurements shown in Fig. 4 leaves were dark-adapted for 2 min, the time needed for the B-band to disappear. The ratio of the area of 46 °C-band/Bband has its maximum at the third ¯ash, as reported by Miranda and Ducruet (1995) for young pea leaves exhibiting a ¯ash-induced afterglow band. The results of Figs. 3 and 4 led to the conclusion that the 46 °C-band in CAM plants re¯ects the same recombination reaction as the afterglow band described by Miranda and Ducruet (1995). In the following, we investigated the intensity of the 46 °C-band as a function of daytime. As shown in Fig. 5 the intensity of the 46 °C-band oscillated during the day. Upon darkening the plant, the 46 °C-band ®rst stayed high but was then suppressed in the morning; after onset of the light it reached its maximum, decreased to its minimum at 12 a.m., increased for the next 4 h and then stayed more or less constant. In the evening the intensity of the 46 °C-band was relatively high. This diurnal change was also seen in leaves of KalanchoeÈ diagremontiana, an obligatory CAM plant which was not subjected to any kind of salt treatment (Fig. 5). The concentrations of DHAP and PGA in rapidly frozen leaves of K. diagremontiana were determined by non-aqueous methods. The concentrations of both substances also varied during the day; the diurnal

A. Krieger et al.: Thermoluminescence studies on the facultative plant

Fig. 5. Diurnal variation of TL signals. The TL signals were measured as in Fig. 1, the integrated area of the 46 °C-band was divided by the integrated area of the B-band for normalisation. In addition the quotient DHAP/PGA is shown. Dark bars indicate the dark period, white bars the light period. Filled circles, Mesembryanthemum crystallinum, salt-treated for 3 d; ®lled triangles, M. crystallinum, salt-treated for 4 d; open circles, KalanchoeÈ daigremontiana; Metabolites were determined on K. daigremontiana leaves

changes in the ratio DHAP/PGA are shown in Fig. 5 (upper part). The close relation between the ratio of DHAP/PGA and the inclination to form a 46 °C-band suggests a close interaction of the metabolic state of the chloroplast with the redox state of the electron-transport chain. The diurnal changes in the intensity of the 46 °Cband were completely suppressed in salt-treated M. crystallinum grown for 2 d in a CO2-free atmosphere. Under these conditions, the intensity of the 46 °C-band was high during the day and increased even in the middle of the day (data not shown). In order to perturb the steady state of the electrontransport chain, we exposed the leaves to high light intensities which induce non-photochemical quenching of chlorophyll ¯uorescence (for review, see Krause and Weis 1991; Demmig-Adams 1992; Horton et al. 1996). Fluorescence and TL measurements were conducted successively on the same leaf section (Fig. 6). In a darkadapted leaf of a salt-treated plant, one ¯ash induced a B-band and a 46 °C-band (Fig. 6, right side) as reported above (Figs. 1, 3A). During illumination with white light of PFD 2100 lmol quanta á m)2 á s)1, chlorophyll ¯uorescence was quenched by non-photochemical quenching mechanisms (Fig. 6, left side). Directly after the illumination, the amplitude of the B-band was quenched and the 46 °C-band was absent. A 5-min dark adaptation led to a restoration of the variable ¯uorescence to one-third of the original value (Fig. 6, left side), to a restoration of the amplitude of the B-band and to a large increase in the 46 °C-band (Fig. 6, right side). We investigated this e€ect at PFDs ranging from 500 to 7500 lmol quanta á m)2 á s)1 (data not shown). The increase in the 46 °C-band was maximal after 10 min illumination at 2100 lmol quanta á m)2 á s)1 followed by a dark recovery period of 10 min. To study the readjustment of the system towards the situation prior to the high light stress, we performed TL measurements

591

Fig. 6. Fluorescence (left panel ) and TL measurements (right panel ) of a 6-d salt-treated Mesembryanthemum crystallinum plant. Nonphotochemical ¯uorescence quenching was induced by illumination with white light (2100 lmol quanta á m)2 s)1); the leaves were kept at 20 °C. The TL signals were measured (i) on a dark-adapted leaf, (ii) after 10 min illumination with white light and (iii) after 10 min illumination and 5 min dark recovery. The TL was excited by one single-turnover ¯ash at 1 °C. Measurements were performed between 4 p.m. and 5 p.m.

after di€erent times of recovery in the dark (Fig. 7). The amplitude of the B-band was quenched immediately after illumination but fully recovered to its maximal value within 2 min. The recovery of the 46 °C-band took longer and was related to the relaxation of the nonphotochemical quenching. The amplitude of the 46 °Cband increased during 10 min dark adaptation to a higher amplitude than was observed before the highintensity illumination, and decreased to its previous amplitude during a longer time of dark adaptation. This phenomenon can be considered as an overshoot before

Fig. 7. Dependence of the amplitude of the 46 °C-band ( ®lled circles) and the B-band (open circles) after 10 min illumination with white light (2100 lmol quanta á m)2 á s)1) on the time of dark recovery. Prior to the continuous illumination, the amplitude of the 46 °C-band was 0.56 on the given scale. The TL was excited by one single-turnover ¯ash at 1 °C. Measurements were performed between 4 p.m. and 7 p.m.

592

the steady-state level is reached. High-intensity illumination induced the same phenomenon in K. diagremontiana and K. pinnata (data not shown). Discussion The data presented in this paper show that induction of CAM leads to the appearance of an additional band with a maximum in TL emission at 46 °C after excitation by a single-turnover ¯ash. A TL band with the same temperature maximum as the 46 °C-band reported here for CAM plants has been observed previously in C3 plants after FR illumination (Fig. 3, see also Desai et al. 1983; Miranda and Ducruet 1995). We suggest that the 46 °C-band originates from the same charge recombination reaction in both CAM and C3 plants, although it was not exclusively charged after FR but was also charged after white-light illumination in CAM plants. The formation of a 46 °C-band can be explained by assuming that the PQ pool, and thereby QB, becomes rereduced after illumination and that, subsequently, TL emission due to charge recombination between a positive charge on the donor-side of PSII and QB) can take place. A reduction of PQ in the dark could occur either via reverse electron ¯ow (Schreiber and Avron 1977; Schreiber 1980) or via an NAD(P)H dehydrogenase reaction (see Bendall and Manasse 1995). In isolated thylakoids, reduction of the PQ pool in the dark could be shown by addition of either ATP (Schreiber and Avron 1977; Schreiber 1980) or DHAP (Schreiber 1980) or NADPH (Mills et al. 1979). We performed measurements varying the number of excitation ¯ashes to determine whether the 46 °C-band originates from a S2/S3QB) recombination. In darkadapted leaves, the distribution of S-states of the Mn cluster is approx. 75% S1, 25% S0 (Fowler 1977) and a certain amount (25% in chloroplasts) of QB is singly reduced at the acceptor side (Rutherford et al. 1982, see Appendix). Excitation of PSII by one single-turnover ¯ash leads to the formation of approx. 75% S2, which is distributed among (i) S2QB), the charges responsible for B-band emission, and (ii) S2QB, which results from the double reduction of singly reduced QB). The doubly reduced QB is replaced by an oxidised PQ. However, via reverse electron ¯ow, a reduction of the PQ pool and thereby formation of QB) takes place and charge recombination occurs in these otherwise `silent' centres. The highest yield of the 46 °C-band is formed after two ¯ashes. Higher S-states, S2 and S3, are present in the majority of reaction centres and recombine with darkreduced QB and form the 46 °C-band (for more detail, see Appendix). The question arises why a 46°C-band can be formed by a single-turnover ¯ash in salt-treated M. crystallinum and why it is absent in control plants (Figs. 1, 2). The 46 °C-band is likely to re¯ect some speci®c metabolic features of CAM plants, since a 46 °C-band was also found in K. daigremontiana (Fig. 5) and K. pinnata (data not shown). Crassulacean acid metabolism requires more ATP and NADPH per CO2 ®xed than C3

A. Krieger et al.: Thermoluminescence studies on the facultative plant

photosynthesis. Therefore higher concentrations of ATP and NADPH are available. This might allow ATP hydrolysis and/or PQ reduction via an NADPH dehydrogenase. The 46 °C-band might be regarded as an indicator of this extra ATP and/or NADPH which cause PQ reduction in the dark. The intensity of the 46°C-band changes during the day; additionally the DHAP/PGA ratio varies (Fig. 5). This ratio was high in the morning, low at noon, when the rate of photosynthesis is maximal and it increased again in the afternoon. Dihydroxyacetone phosphate may serve as source for ATP and NADPH via its conversion to PGA: PGA+NADPH+H‡ +ATP()DHAP+NADP‡ ADP+Pi.

This reaction is close to its chemical equilibrium in chloroplasts (Dietz and Heber 1986, 1989). The ratio of DHAP/PGA, therefore, is an indicator of the phosphorylation potential and the reducing potential realised in the chloroplast during photosynthesis. Based on this indicator, the probability for dark reduction of the PQ pool is high in the morning, low at noon and again higher in the afternoon and evening. It is noteworthy that the DHAP content was higher and the PGA content lower in chloroplasts of the CAM plant than usually observed in plants performing C3 photosynthesis. After an overnight dark adaptation, the intensity of the 46 °Cband was low and increased to its maximal level when the plant was illuminated for approx. 10 min. This e€ect might be due to an inactivation of the ATPase and other enzymes during the night. KoÈster and Winter (1985) measured the ATP/ADP ratio during the diurnal rhythm of CAM in KalanchoeÈ pinnata. They observed high ATP/ADP ratios in the early and late light period and the lowest ATP/ADP ratio in the middle of the light period. These measurements strongly support the interpretation that the 46 °Cband re¯ects a high ATP/ADP (NADPH/NADP) ratio in chloroplasts. High-intensity illumination evoking acidi®cation and related non-photochemical quenching of ¯uorescence leads to a suppression of the 46 °C-band (Fig. 6). As can be seen in Fig. 7, the amplitude of the B-band is also lowered immediately after the light treatment but quickly recovers to its maximal level within less than 2 min. The quenching of the B-band is probably due to a transient light-induced lumen acidi®cation. The restoration of the 46 °C-band takes a longer time (10 min) and seems to be closely related to the recovery of ¯uorescence. This may indicate that the metabolic status, including ATP and NADPH concentrations, is readjusted after a transient imbalance in the rate of ATP and NADPH turnover. The presented data reveal in CAM plants a tight control of the reduction state of the photosynthetic electron-transport chain and the formation of the 46 °Cband after single-turnover ¯ash excitation. The 46 °C-band seems to re¯ect a reduction state which can be perturbed, e.g. by high light stress. It might be interesting to extend this study and to investigate the

A. Krieger et al.: Thermoluminescence studies on the facultative plant

46 °C-band in other physiologically interesting systems. In C4 plants and algae grown under CO2-limiting conditions, CO2-concentrating mechanisms are active and the electron-transfer chain should be in a reduction state comparable to that in CAM plants. Additionally, in water-stressed C3 plants with limited photosynthesis due to lack of electron acceptors, a 46 °C-band should be present, and it might be interesting to relate the appearance of this band to the metabolic state of the plants. Appendix Flash dependency of B-band and 46 °C-band, starting from dark-adapted material. 1F

2F

3F

4F

5F

ÿ S1 QB ! S 2 QB ÿ ! S3 QB ! S0 Qÿ B ! S1 QB ! S2 QB ÿ ÿ S0 QB ! S1 Qÿ B ! S2 QB ! S3 QB ! S0 QB ! S1 QB ÿ ÿ S1 Qÿ B ! S2 Q B ! S 3 Q B ! S 0 Q B ! S 1 Q B ! S2 Q B ÿ ÿ S0 Qÿ B ! S1 QB ! S 2 QB ! S3 QB ! S0 QB ! S1 QB

The S-state distribution in dark-adapted leaves is assumed to be 75% S1 and 25% S0; 25% QB) is assumed to be present in the dark. Bold, states which lead to emission of a 46 °C-band; italics and underlined states which lead to emission of a B-band; the other states are not detected by TL. We thank U. Heber (Julius-von-Sachs Institut, WuÈrzburg, Germany) for giving support for building the TL machine in WuÈrzburg and for the critical discussion of this paper. Furthermore, we thank U. Schreiber and U. Schliwa (both Julius-von-Sachs Institut, WuÈrzburg) for help in building the TL machine. We would also like to thank A.W. Rutherford (CEA Saclay, Gif-sur-Yvette, France) for stimulating discussions and C. JegerschoÈld (CEA Saclay, Gifsur-Yvette) for the critical reading of the manuscript. Support by the Deutsche Forschungsgemeinschaft (SFB 251) is gratefully acknowledged. A.K. was the recipient of a DFG fellowship.

References Bendall DS, Manasse R (1995) Cyclic photophosphorylation and electron transport. Biochim Biophys Acta 1229: 23±38 Bertsch WF, Azzi JR (1965) A relative maximum in the decay of long-term delayed light emission from the photosynthetic apparatus. Biochim Biophys Acta 94: 15±26 BjoÈrn LO (1971) Far-red induced long-lived afterglow from photosynthetic cells. Size of afterglow unit and paths of energy accumulation and dissipation. Photochem Photobiol 13: 5±20 Black CC, Chen J-Q, Doong RL, Angelov MN, Sung SJS (1996) Alternative carbohydrate reserves used in the daily cycle of crassulacean acid metabolism. In: Winter K, Smith JAC (eds) Crassulacean acid metabolism (Ecological studies, vol 114) Springer, Heidelberg pp 31±45 Demmig-Adams B (1990) Carotenoids and photoprotection: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 1± 24 Desai TS, Rane SS, Tatake VG, Sane PV (1983) Identi®cation of far-red-induced relative increase in the decay of delayed light emission from photosynthetic membranes with thermolumines-

593 cence peak V appearing at 321 K. Biochim Biophys Acta 724: 485±489 Dietz K-J, Heber U (1984) Rate limiting factors in leaf photosynthesis. 1. Carbon ¯uxes in the Calvin cycle. Biochim Biophys Acta 767: 432±443 Dietz K-J, Heber U (1986) Light and CO2 limitation of photosynthesis and states of the reactions regenerating ribulose-1,5bisphosphate or reducing 3-phosphoglycerate. Biochim Biophys Acta 848: 392±401 Dietz K-J, Heber U (1989) Assimilatory force and regulation of photosynthetic carbon reduction in leaves. In: Barber J (ed) Techniques and new developments in photosynthesis (NATO ASI Series). Plenum Press, New York London Washington Boston, pp 341±363 Ducruet JM, Miranda T (1992) Graphical and numerical analysis of thermoluminescence and ¯uorescence emission in photosynthetic material. Photosynth Res 33: 15±27 Fowler CF (1977) Proton evolution from photosystem II. Stoichiometry and mechanistic considerations. Biochim Biophys Acta 462: 414±421 Heber U, Neimanis S, Kaiser W (1996) Regulation of Crassulacean acid metabolism in KalanchoeÈ pinnata as studied by gas exchange and measurements of chlorophyll ¯uorescence. In: Winter K, Smith JAC (eds) Crassulacean acid metabolism (Ecological studies, vol 114). Springer, Heidelberg, pp 78±96 Hideg E, Kobayashi M, Inaba H (1991) The far-red induced slow component of delayed ¯uorescence from chloroplasts is emitted from photosystem II. Evidence from emission spectroscopy. Photosynth Res 29: 107±112 Horton P, Ruban AV, Walters RG (1996) Regulation of light harvesting in green plants. Annu Rev Plant Physiol Plant Mol Biol 47: 655±658 KoÈster S, Winter K (1985) Light scattering as an indicator of the energy state in leaves of the crassulacean acid metabolism plant Kalanchoe pinnata. Plant Physiol 79: 520±524 Krause GH, Weis E (1991) Chlorophyll ¯uorescence and photosynthesis: the basics. Annu Rev Plant Physiol Plant Mol Biol 42: 313±349 Krieger A, Rutherford AW, JegerschoÈld C (1998) Themoluminescence measurements on chloride - depleted and calcium depleted photosystem II. Biochim Biophys Atca (in press) Mellvig S, Tillberg JE (1986) Transient peaks in the delayed luminescence from Scenedesmus obtusiusculus induced by phosphorus starvation and carbon dioxide de®ciency. Physiol Plant 68: 180±188 Mills JD, Crowther D, Slovacek RE, Hind G, McCarty RE (1979) Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark. Biochim Biophys Acta 547: 127±137 Miranda T, Ducruet JM (1995) Characterization of the chlorophyll thermoluminescence afterglow in dark-adapted or far-redilluminated plant leaves. Plant Physiol Biochem 33: 689±699 Nakamoto H, Sundblad L-S, GardestroÈm P, Sundbom E (1988) Far-red stimulated luminescence from barley protoplasts. Plant Sci 55: 1±7 Osmond CB (1978) Crassulacean acid metabolism: a curiosity in context. Annu Rev Plant Physiol 29: 379±414 Osmond CB, Popp M, Robinson SA (1996) Stoichiometric nightmares: studies of photosynthetic O2 and CO2 exchanges in CAM plants. In: Winter K, Smith JAC (eds) Crassulacean acid metabolism (Ecological studies, vol 114). Springer, Heidelberg, pp 19±30 Palmqvist K, Sundblad L-G, Samuelsson G, Sundbom E (1986) A correlation between changes in luminescence decay kinetics and the appearance of a CO2-accumulating mechanism in Scenedesmus obliquus. Photosynth Res 10: 113±123 Rutherford AW, Croft AR, Inoue Y (1982) Thermoluminescence as a probe of photosystem II photochemistry. The origin of the ¯ash-induced glow peaks. Biochim Biophys Acta 682: 457± 465

594 Sane PV, Rutherford AW (1986) Thermoluminescence in photosynthetic membranes. In: Govindjee, Amesz J, Fork DC (eds) Light emission in plants and bacteria. Academic Press, New York, pp 329±360 Schmidt W, Senger H (1987) Long-term delayed luminescence in Scenedesmus obliquus. Biochim Biophys Acta 890: 15±27 Schreiber U (1980) Light-induced ATPase and ATP-driven reverse electron ¯ow in intact chloroplasts. FEBS Lett 122: 121±124 Schreiber U, Avron M (1977) ATP-induced chlorophyll luminescence in isolated spinach chloroplasts. FEBS Lett 82: 159±162 Sundblad LG (1988) Secondary chlorophyll a luminescence decay kinetics from green algae and higher plants; mechanisms and applications. Thesis, University of Umeaà Sundblad LG, Palmqvist K, Samuelsson G (1986) Luminescence decay kinetics in relation to the relaxation of the transthylakoid DpH from high and low CO2 adapted cells of Scenedesmus obliquus. FEBS Lett 209: 28±32

A. Krieger et al.: Thermoluminescence studies on the facultative plant Sundblad LG, SchroÈder WP, Akerlund HS (1988) S-state distribution and redox state of QA in barley in relation to luminescence decay kinetics. Biochim Biophys Acta 973: 47±52 Vass I, Inoue Y (1992) Thermoluminescence in the study of photosysteme II. In: Barber J (ed) The photosystems: structure, function and molecular biology. Elsevier, Amsterdam, pp 259±294 Winter K (1973) Zum Problem der Ausbildung des CrassulaceensaÈuresto€wechsels bei Mesembryanthemum crystallinum unter NaCl-Ein¯ub. Planta 109: 134±145 Winter K, Smith JAC (1996) Crassulacean acid metabolism: current status and perspectives. In: Winter K, Smith JAC (eds) Crassulacean acid metabolism (Ecological studies, vol 114). Springer, Heidelberg, pp 389±426 Winter K, von. Willert DJ (1972) NaCl-induzierter CrassulaceensaÈuresto€wechsel bei Mesembryanthemum crystallinum. Z P¯anzenphysiol 67: 166±170