BY ROBERT T. FURBANK AND DAVID A. WALKER Research Institute for Photosynthesis, University of Sheffield, Sheffield SIO 2TN, UK

Transcription

1 New Phytol. (1986) 104, O7 CHLOROPHYLL A FLUORESCENCE AS A QUANTITATIVE PROBE OF PHOTOSYNTHESIS: EFFECTS OF CO^ CONCENTRATION DURING GAS TRANSIENTS ON CHLOROPHYLL FLUORESCENCE IN SPINACH LEAVES BY ROBERT T. FURBANK AND DAVID A. WALKER Research Institute for Photosynthesis, University of Sheffield, Sheffield SIO 2TN, UK {Accepted 12 June 1986) SUMMARY The relationship between changes in chlorophyll afluorescenceand changes in CO^ concentration in spinach leaves is analyzed. The height of the fluorescence excursion, when plotted against the CO., concentration during the transient, results in a hyperbola. When these data are replotted on an inverse-reciprocal plot, an apparent K^ (COg) for the fluorescence transient can be obtained which closely approximates the K^ (COj) for carbon assimilation under similar conditions. Transitions in CO^ concentration at 2 % O^ result in deviation from this hyperbolic relationship, reducing the apparent K^ (CO2) for this process. The relationship between carbon assimilation and chlorophyll fluorescence is discussed with reference to the two components of fluorescence quenching. This technique raises the possibility that chlorophyll fluorescence could he used as a quantitative as well as a qualitative tool in plant screening. Key words: Chlorophyll a fluorescence, CO., concentration, spinach, gas transients, photosynthesis. INTRODUCTION When a leaf is subjected to a sudden variation in CO^, O^ or irradiance, large-scale changes in chlorophyll a fluorescence yield are seen (Heber, 1969; Krause, 1973; Sivak, Prinsley & Walker, 1983; Bradbury, Ireland & Baker, 1985). The response of fluorescence to these changes has already been reported and, qualitatively, a close relationship between fluorescence quenching and the rate of CO2 assimilation has been found (see e.g. Sivak et al., 1983; Sivak & Walker, 1984, 1985a, b, c; Walker & Sivak, 1985). The characteristics of these transients in chlorophyll fluorescence are extremely sensitive to the metabolic state of the leaf, and recently it has been demonstrated that chlorophyll fluorescence during gas transitions can be used as an indicator of the phosphate status of the cytosol in an intact leaf (Sivak & Walker, 1985c; Sivak & Walker, 1986; Walker & Sivak, 1986). Other studies have shown that the magnitude of steady-state fluorescence yield closely follows the rate of ATP utilization in intact leaves, providing a potential measure of the factors limiting photosynthesis at various CO2 concentrations and irradiances (Sharkey, 1985). In view of these close correlations between chlorophyll fluorescence and carbon assimilation, measurement of this parameter can be regarded as a valuable in vivo probe of photosynthesis. However, owing to the complexity of the fluorescence signal, little quantitative use of this technique has been made. The present study examines chlorophyll a fluorescence in intact spinach leaves under X/86/ $03.00/ The New Phytologist

2 2O8 R. T. FURBANK AND D. A. WALKER strictly defined conditions and postulates a quantitative relationship between the size of the fluorescence transient observed during a change in gas phase and the rate of COg assimilation. MATERIALS AND METHODS Fluorescence detection equipment consisted of a photodiode (Hansatech, King's Lynn, UK) at an angle of 45 to the leaf surface, protected from actinic light by a narrow band-pass filter (Balzers IF 740 filtraflex B-40) and a red glass filter (Schott RG 715). Actinic Hght was provided by an array of red (peak 635 nm) photodiodes (Hansatech, King's Lynn, UK) giving an irradiance of 70 W m~^. A thermostatically controlled leaf chamber (volume 5 cm^) regulated at 25 C was used in all experiments. Leaves of spinach {Spinacia oleracea L.) were detached from the plant, the petioles immersed in water and placed in the fluorescence chamber. A gas stream of 180//I \~^ COj, 21 % O^ (or 2% Og as indicated) was saturated with water vapour and passed over the leaf for 10 min. The COj concentration was then switched to another value for 30 s, then replaced by 180/tl r^ CO2 for 5 min and the procedure was repeated. Each 30 s pulse of a given COj concentration was repeated three times before another change was made. In this way, transitions from 180 ja \~^ to a series of COj concentrations in the range 0 to 1000/^11~^ were made. Switching of gas streams was controlled by a set of solenoid valves connected between the leaf chamber and gas supplies of various CO2 concentrations. The operation of these valves was controlled by a Commodore 3016 microcomputer. The COj concentration of the gas streams was adjusted by selective absorption from a stock gas (1000 /tl 1~^ COj in air or 2% O^ using a pair of COj Diluters (Analytical Development Corporation, Hoddesdon, UK). Infrared gas analysis experiments were carried out in a system described elsewhere (Furbank & Walker, 1985) comprising a CO2 analyzer and HjO analyzer (mk III, Analytical Development Corporation, Hoddesdon, UK) interfaced to a Commodore 3016 microcomputer. The partial pressure of water vapour entering the leaf chamber in these experiments was lombar. Intercellular COg was calculated from assimilation and transpiration rates as described by von Caemmerer & Farquhar (1981). RESULTS Figures l(a) and (b) show the general characteristics of the fluorescence transient observed after a transition in COj concentration of gas stream. A transition to a lower CO2 concentration resulted in a transient increase in fluorescence yield, while the inverse occurred when the CO2 concentration was increased. These changes in fluorescence began to reverse after 2 to 3 s (see Sivak & Walker, 1985c). The data of Figure l(a) were obtained in 20% O^ and those of Figure l(b) in 2% O2. Figure 2(a) is a plot of the height of the peak (or trough) in fluorescence [observed during the gas transients shown in Figure l(a)] against the COj concentration used for the 30 s pulse (a decrease in fluorescence is indicated by a negative value). Two separate experiments are shown using two different leaves, indicating that these results are highly reproducible. The plot shown in Figure 2(a) is generally hyperbolic in nature and its similarity to a plot of COj assimilation rate against CO2 concentration (Fig. 3) is striking. This is in accordance with the fact that the kinetics of the change in fluorescence yield during oscillations following a

3 Chlorophyll a fluorescence as a probe of photosynthesis 209 0) u c Qi 0) o 3 (a 300 I " 180 r ( 1 t J 1 V ^ *- Relative 390 F 1 J "" """"" 180 j 1 t -CO \ 80 1 mi n Fig. 1. Representative traces of transient changes in chlorophyll fluorescence during the transition from 180 /d 1"' to a series of COj concentrations. The pulses of COj were maintained for 30 s. The arrows indicate changes in gas phase. CO.^ concentrations (in /(I 1""') used for these transitions are indicated on the figure. Experiments in (la) were at 20% Oj; those in (lb) at 2% Oj. perturbation of the steady state are broadly reciprocal to the rate of assimilation (Sivak & Walker, 1985c). A double reciprocal plot of these data [Fig. 2(b)] gives an apparent K ^ (CO2) for the change in fluorescence yield. The data were normalized to the minimum fluorescence yield to avoid negative values. When the apparent K^ (CO.^) is calculated from both sets of data, a value of approximately \6Q fi\\~^ is obtained. This Lineweaver-Burk plot is essentially linear except at higher COg concentrations, a phenomenon also seen to a more extreme degree when attempts are made to calculate the apparent X^ (COj) for photosynthesis with infrared gas analysis techniques (Farquar, von Caemmerer & Berry, 1980). The apparent K^ (COj) for photosynthesis in this leaf tissue as determined by infrared gas analysis at similar light intensities was between 140 and 150/ill 1 (from Fig. 3). When these gas transitions were repeated using 340/il l~i as the steady-state gas phase, K ^ values of 160 to 180/tll~^ were found; however, a longer equilibration time between transitions was required for reproducible results. Figure 4 is a repeat of the experiment shown in Figure 2(a), except that 2 % O^ was used in place of 21 % O^. From gas exchange measurements, a reduction in O2 tension has been observed to decrease the apparent K^ (COg) of photosynthesis and hence causes the initial slope of the response of assimilation to COj concentration to increase. This behaviour is seen in Figure 4 where the initial slope of the response of fluorescence yield to COj concentration is much steeper under 2 % O2 than at 21 % O^ and a K,^ (COg) is almost halved. Similar values (around 90 /A ri) were obtained using conventional gas exchange techniques. Presumably, this is due to the reduced competition between CO^ and O^, the catalytic site of

4 2IO R. T. FuRBANK AND D. A. WALKER 1 40' \ \ 20 (a) (b) U CO, {ul r') r' xio-") Fig. 2. A plot of the size of the fluorescence peak or trough (resulting from a gas transient) against the COj concentration during the 30 s pulse is shown in (2a). The Oj tension was 21%. The separate experiments are shown, (b) is an inverse-reciprocal plot of the data of (a). 800 Fig. 3. The response of COj assimilation in the spinach leaves used here to COj concentration (measured in a gas exchange system). The COj concentration plotted is the intercellular value calculated from transpiration measurements. Conditions used were as in Figure 2 (a). 140 > nee esc Fig. 4. Response of the magnitude of change in fluorescence yield during COj transients, as in Figure 2(a), with the exception that the O2 tension was 2% to reduce photorespiration. As in Figure 2, leaves from two separate plants were used as replicates.

5 Chlorophyll a fluorescence as a probe of photosynthesis 211 RuBP carboxylase (Farquahar et al., 1980). Also, the transition to lower CO2 concentrations resulted in a greater transient increase in fluorescence at 2% O2. It has been suggested that this is due to a further relaxation in qq in the absence of photorespiratory dissipation of reducing power (Sivak et al., 1983). Another factor contributing to the larger fluorescence rise at low O2 may be the reduced rate of pseudocyclic electron flow under these conditions resulting in less photochemical quenching (Sivak & Walker, 1985c). DISCUSSION Owing to the technical difficulties involved in measuring CO2 assimilation during a transition in COj concentration, it was not possible to measure fluorescence and assimilation simultaneously in these experiments. Thus, the rate of carbon assimilation at a particular point in the transient cannot be strictly related to the fluorescence trace. Despite this limitation, a close quantitative relationship between the height of the peak or trough in fluorescence during a gas transition and the rate of carboxylation of RuBP is suggested by these data. This is supported by the close correlation between the behaviour of CO2 assimilation under similar conditions at steady state. The K^ (CO2) calculated from the fluorescence transients (Fig. 2) closely approximates that obtained by traditional methods at low irradiance, as does the response of the fluorescence peak heights to CO2 at low O2 (Fig. 3). However, the Kj^{CO,^ obtained by infrared gas analysis is, in fact, a value for the intercellular CO2 concentration. This value is typically only 60 to 80 % of the CO2 concentration outside the leaf. It seems likely that during the transitory change in CO2 carried out here, intercellular CO2 closely approximates the CO2 concentration of the gas supplied, possibly due to the slow response of stomata. The data shown here present the possibility that comprehensive data on the response of leaf carbon assimilation to CO2 concentration may be obtained very rapidly using simple fluorescence detection equipment. However, further investigation of the effect of light intensity on the relationship between fluorescence and carbon assimilation is required before this technique could be applied routinely. A further advantage of this technique is the likelihood that such a rapid pulse measurement may reduce RuBP limitation of the carboxylation reaction, i.e. the limitation of photosynthetic carbon assimilation by light driven regeneration of carbon acceptor. This phenomenon is thought to be responsible for the deviation of assimilation vs CO2 plots (such as in Fig. 4) from the Michaelis-Menten kinetics of the carboxylase in vitro and pulse type measurements have been shown to reduce such non-hyperbolic behaviour (Laisk, 1983). Thus, the application of this technique to screening of plants for differing ratios of carboxylation to oxygenation by ribulose bisphosphate carboxylase could be of considerable interest. Analysis of the fluorescence quenching mechanisms which produce the kinetics observed is quite difficult. From simultaneous measurements of light scattering and fluorescence (Sivak & Walker, 1985c), it appears that during the gas transition the following sequence of events occurs. In the case of the transition to low CO2, fluorescence initially rises due to a decrease in qq quenching as the regeneration of NADP via 3-PGA reduction decreases. This process is rapidly overtaken by qe quenching of fluorescence which increases due to a fall in ATP demand. During a transition to a CO2 concentration higher than that used in steady state the inverse will apply, i.e. qq will rise and q^ will fall. The resulting height of the peak

6 212 R. T. FuRBANK AND D. A. WALKER or trough measured here is a product of the magnitude of both these processes and the relative rates at which the two quenching mechanisms act (i.e. the product of two simple exponential curves). Studies of light-scattering during transitions to and from CO^-free air (Sivak & Walker, 1985c) and light-doubling measurements made under similar conditions (Bradbury et al., 1985) suggest that the two component quenching processes may not be as clearly resolved, as qg in some circumstances may relax simultaneously with qq (see also Krause et al., 1977). Many of these experiments should not be extrapolated to the data shown here without careful consideration. The effects of totally and simultaneously removing potential electron acceptors (i.e. COj-free air and low or no O^) may differ considerably from those obtained during a transition in CO2 concentration at a given O2 tension. Considering the complexity of this system, it is unlikely that there is a universally simple relationship between this fluorescence parameter and the rate of CO2 fixation. The relationship between carbon assimilation and steady state room temperature fluorescence has recently been investigated with respect to COj concentration (Sharkey, 1985). The relationship was far from simple and certainly not flrst orjder and reciprocal as seen here. This was probably due to the combination of high light intensity and the fact that the present study was made using non-steady state techniques. An attempt to describe mathematically the responses of fluorescence seen here with reference to the component quenching mechanisms is currently underway. Given the obvious need for appropriate circumspection and scepticism at this stage, it is nevertheless clear that there are apparent quantitative relationships between chlorophyll a fluorescence and carbon assimilation which require further elucidation. The present findings also strengthen our previously expressed conviction that fluorescence, as a cheap, easy and non-intrusive probe, is one which ought to command the attention of the plant physiologist with an interest in photosynthetic carbon assimilation every bit as much as the biophysicist preoccupied with the earliest events in photosynthesis. REFERENCES BRADBURY, M., IRELAND, C. R. & BAKER, N. R. (1985). An analysis of the chlorophyll-fluorescence transients from pea leaves generated by changes in atmospheric concentrations of COj and Oj. Biochimica et Biophysica Acta, 806, FARQUHAR, G. D., VON CAEMMERER, S. & BERRY, J. A. (1980). A biochemical model of photosynthetic COj assimilation in leaves of C, species. Planta, 149, FuRBANK, R. T. & WALKER, D. A. (1985). Photosynthetic induction in C4 leaves. An investigation using infra-red gas analysis and chlorophyll fluorescence. Planta, t63, HEBER, U. (1969). Conformational changes of chloroplasts induced by illumination of leaves in vivo. Biochimica et Biophysica Acta, 180, KRAUSE, G. H. (1973). The high energy state of the thylakoid system as indicated by chlorophyll fluorescence and by chloroplast shrinkage. Biochimica et Biophysica Acta, 292, KRAUSE, G. H., LORIMER, G. H., HEBER, U. & KIRK, M. R. (1977). Photorespiratory energy dissipation in leaves and chloroplasts. In: Proceedings of the Fourth International Congress on Photosynthesis, pp LAISK, A. Kh. (1983). Biochemical structure and kinetic function of the photosynthetic apparatus of plants. Fysiologiya Rastenii, 30, SHARKEY, T. D. (1985). Steady-state room temperature fluorescence and COj assimilation rate in intact leaves. Photosynthesis Research, 7, SIVAK, M. N. & WALKER, D. A. (1984). What can be learned about the regulation of photosynthesis from multiple measurements? State of the art and perspectives. In: Regulation of Sources and Sinks in Crop Plants (Ed. by B. Jeffcoat, A. F. Hawkins & A. D. Stead), pp Proceedings of Symposium 25/26 Sept British Plant Growth Regulator Group, Long Ashton, Bristol.

7 Chlorophyll ^fluorescence as a probe of photosynthesis 213 SiVAK, M. N. & WALKER, D. A. (1985a). Theory and practice of chlorophyll a fluorescence in its relation to photosynthesis; the state of the art and perspectives. In: Photosynthesis and Physiology of the Whole Plant (Proceedings of a workshop held at Volkenrode, Braunschweig 24/26 Sept. 1984), pp Paris. SiVAK, M. N. & WALKER, D. A. (1985b). Chlorophyll a fluorescence; can it shed light on fundamental questions in photosyntheic carbon dioxide fixation? Plant, Cell and Environment, 81, SiVAK, M. N. & WALKER, D. A. (1985c). Can in vivo photosynthesis be modified? In: Plant Products and the New Technology (Ed. by K. W. Fuller and J. R. Gallon), pp Annual Proceedings of the Phytochemical Society of Europe, vol. 26. Oxford University Press, Oxford. SiVAK, M. N. & WALKER, D. A. (1986). Photosynthesis in vivo can be limited by phosphate supply. New Phytologist, 102, SiVAK, M. N., PRINSLEY, R. T. & WALKER, D. A. (1983). Some effects of changes in gas phase of the steady state chlorophyll a fluorescence exhibited by illuminated leaves. Proceedings of the Royal Society London, B, 217, VON CAEMMERER, S. & FARQUHAR, G. D. (1981). Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta, 153, WALKER, D. A. & SIVAK, M. N. (1985). 7nt)it;o chlorophyll a fluorescence transients associated with changes in the CO2 content of the gas-phase. In: Regulation of Carbon Partitioning in Photosynthetic Tissue. Proceedings of the Eighth Annual Symposium in Plant Physiology (Ed. by R. L. Heath and J. Preiss), pp University of California, Riverside. American Society of Plant Physiologists, Rockville, Maryland. WALKER, D. A. & SIVAK, M. N. (1966). Photosynthesis and phosphate - a cellular affair. Trends in Biological Sciences, 11,

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