Far-Red Light-Induced Changes in Intracellular Potentials of Spinach

Plant Physiol. (1983) 73, 671-676 0032-0889/83/73/0671 /06/$00.50/0

Far-Red Light-Induced Changes in Intracellular Potentials of Spinach Mesophyll Cells INTERACTION WITH RED LIGHT Received for publication December 30, 1982 and in revised form July 8, 1983

MICHEL MONTAVON, BENJAMIN A. HORWITZ, AND HUBERT GREPPIN

Plant Physiology Laboratory, University ofGeneva, 1211 Geneva 4, Switzerland (M. M, H. G.); and Department of Plant Genetics, The Weizmann Institute of Science, 76100 Rehovot, Israel (B. A. H.) membrane rearrangements depending on the spillover state of the photosystems (3). In green plants, the large bioelectric changes that photosynthetically In pulvinar motor cells of (light-grown) Samanea (21) and in active light stimulates make it difficult to observe electrical potential subepidermal cells of green Lemna (14), R and FR reversible changes related to phytochrome photoconversion. As a first step towards potential changes have been recorded with intracellular elecdistinguishing between photosynthetic and phytochrome effects, we trodes. In Samanea, R shifts the potential to a more negative showed that red light enhances far-red stimulated intracellular potential value, and FR restores the previous level. In light-grown Lemna changes in spinach (Spinacia oleracca) leaf mesophyll cells. treated with DCMU to block noncyclic electron flow, R alone For a dark-adapted leaf, the response to far-red light increased during has no effect, FR shifts the potential to a more negative value, the first 10 to 30 exposures of 2.5 minutes, after which it was constant. and R after FR restores the resting potential (14). R and FR The intracellular potential depolarized by an average of 0.3 millivolts interact in the light-induced membrane depolarization in Nitella during each 2.5-minute far-red light period, and returned to the resting (26). Photoreversibility was observed in leaves of spinach grown value during each subsequent dark period. Continuous supplementary red in SD photoperiods. A dark to FR transition increased the surface light (at 1-5% of the fluence rate of the far-red light that stimulated the potential of a leaf electrode relative to the electrically grounded depolarizations) increased the response to far-red 2- to 3-fold. Supple- root system (leaf more positive in the light than in the dark). A mentary red light did not amplify the response to alternating 702 nano- pretreatment with R increased this response to FR, while a meters light and dark periods. The Emerson enhancement effect thus pretreatment with R + FR had no effect, consistent with phytodoes not seem to explain amplification of the response to 730 nanometers chrome control (1 1). light by supplementary red light. This does not prove that photosynthetic The experiments reported here were designed to determine pigments are not involved in some other way. whether the intracellular potential of leaf cells exhibits any photoreversible control by R and FR. Although the potential changes caused by FR are small compared to the resting potential, the stability of the measuring apparatus used permitted better characterization of the FR effect than was possible with contact electrodes. The measurements were done in leaf mesophyll cells of intact plants. The physiological significance of small potential changes could Dark to light and light to dark transitions induce potential changes in cells of green leaves (5, 15, 16, 24). These have been be questioned; our data do not support the idea that phytochrome correlated (24) with transient changes in ion flux across chloro- mediates a large change of the intracellular potential. These plast membranes (during photosynthesis), and can be accounted measurements, nevertheless, form the basis for further, detailed for by the activity of photosynthetic pigments. FR' causes much action spectroscopy to look for phytochrome-specific effects. A smaller changes; for example, Luttge and Pallaghy (15) found no significant local charge separation might be expressed as only a response to wavelengths greater than 705 nm. Still, the effect of small change in the vacuole to medium potential difference. FR is of interest because of the possible correlation of phytochrome photoconversion with membrane potential changes. In MATERIALS AND METHODS etiolated seedlings, R and FR reversible electrical changes attributed to phytochrome have been shown using several intracellular Plants and Cultivation. Spinach plants (Spinacia oleracea L. or extracellular techniques (13, 17, 19, 20, 23). cv Nobel) were grown under 8-h light, 16-h dark photoperiods In green material, the situation is greatly complicated by the in a growth room. Under these standard conditions (SD), the presence of large concentrations of Chl. It would be useful to plants were vegetative. Four-week-old plants were transferred to have an electrophysiological assay for phytochrome in green the measuring apparatus described below, 1 d before the start of tissue. If electrical changes prove to be part of the transduction the experiment. The temperature was 20 ± 1.4°C in the growth pathway for sensing light, such studies would be even more room. The RH was 70 ± 3% during the light periods and 50 + important. If purely photosynthetic in origin, R- and FR-stimu- 3% during the dark periods. Illumination for the photoperiods, lated potential changes may be a consequence of chloroplast 20.4 ± 4 w m-2 from 400 to 700 nm at the location of the plants, was from Sylvania TL-33 40-w daylight fluorescent tubes. 'Abbreviations: FR, far-red light (730 nm); R, red light (660 nm); Measuring Apparatus. External electrical interference was APW, electrophysiological solution, 'artificial pond water';' Hz, Hertz, minimized by isolating the plant and measuring electrodes in a frequency in cycles per second. light-proof Faraday cage (see Fig. 1). The air in the laboratory ABSTRACT

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Plant Physiol. Vol. 73, 1983 MONTAVON ET AL. was kept at 20 ± 0.6°C and 80 ± 2% RH). These fluctuations plexiglass cuvette (Fig. 1). The cuvette was filled with electrowere further damped by linking the interior of the Faraday cage physiological solution, 1 mM KCI, 1 mM Ca(NO3)2, 0.25 mM (thermally insulated) with the ambient air through a covered 10- MgSO4, 0.23 mm NaH2PO4, 0.39 mm Na2HPO4, pH 7 (APW, cf cm tube with a water reservoir (ENTR in Fig. 1). The tempera- 7); this solution was constantly renewed at a rate of 100 ml/24 ture in the measuring chamber was 20 ± 0.2C and the RH was h. The reference electrode and the contact with the microelec80 ± 1%. The temperature, RH, and light fluence rate near the trode were both Ag/AgCl, in direct contact with the APW; the cuvette as well as signals from the control unit, were simultane- reference Ag/AgCl electrode could also be connected to the APW ously recorded along with the intracellular potentials. Light through a salt bridge (1-mm-diameter capillary filled with 0.5 M fluence rate was measured using a laboratory-constructed pho- KCI in 2% agar). Ag/AgCI electrodes were protected from light. tocell circuit, temperature with a Yellow Springs 42SC electronic The tip potential of the microelectrode, defined as the differthermometer, and humidity with a laboratory-constructed elec- ence between the potential measured with the tip immersed in tronic hygrometer. Vibrations were suppressed by placing the 0.5 M KCI and that obtained in APW, averaged 4 to 5 mv and plant and recording inputs on a table suspended on plastic foam never exceeded 10 mv. This DC potential was offset to zero in inside the Faraday cage. Narrow band irradiations were obtained using Balzers B-40 APW before insertion (DC offset of the electrometer). Potential model M-707 interference filters (40% transmission at the peak; bandwidth changes were measured with a WP Instruments 15-20 nm) with a projector (Leitz, 250 w) as light source. Light electrometer, input impedance: 10" Q with 1 kHz bandwidth from the projector was first filtered through a circulating water filter and a notch filter (50 or 60 Hz). This electrometer also has layer and a Balzers Calflex X interference filter (95% transmis- a current injection system for microiontophoresis of marker dyes, sion from 350-770 nm) to reduce IR, then a wide band red etc. The electrode resistance when the tip is in APW can be read plexiglass blocking filter. Supplementary (background) illumi- directly, and was 10 to 30 MQ before penetration into the cell, nation of the leaf was from an incandescent source variable up and values measured after the electrode was withdrawn from the to 20 w, filtered in the same way as the projector. Spectral fluence tissue showed changes of 10 to 40% (unless the electrode was rates of the sources with the wide band filters at the location of broken or obstructed). the leaf were measured with an ISCO spectroradiometer, and The output of the electrometer was recorded with a Tektronix fluence rates calculated using the transmission spectra of the 511 3N dual beam bistable storage oscilloscope and a multichanfilters. nel Watanabe MC641 pen recorder (pen response: maximum A programmable control unit was constructed to facilitate speed, 80 cm s'; acceleration, 200 cm s-2 with preamplifiers of giving a precisely timed series of treatments free from mechanical 1 MQ input impedance). The recorder inputs have optimal filters perturbations. Changing of interference filters, switching on and eliminating AC frequencies above 0.5 Hz. The oscilloscope has off light sources, and cooling the heat filter were automatic. a 5B1ON time base unit and two 5A22N differential amplifiers. Recording of Intracellular Potentials. Intracellular potentials The microelectrodes were inserted with a Hugo Sachs Elecwere measured using glass microelectrodes filled with 0.5 M KCI tronics micromanipulator with a variable stepping motor (7-100 from 1.5-mm-diameter capillaries with a Getra (Munich) elecs-', 1 Mm for a single step) at 10 Mm s-'. The microelectrode trode puller. As seen under the microscope, the microelectrodes Mm always implanted during observation with a binocular was had a short point (80 Mm from tip to the start of the second at 160 x, in a region of parenchyma with very few (Olympus) to resistant breakage. them making of the capillary) narrowing but 1 to 4 mm from the central vein. The elements, vascular This 'candle' shape gave the best results among various shapes not visible once inside the leaf, so its position was electrode tip and than before less 1 was The diameter always tested. tip Am was followed during insertion by observing the (unfiltered) signal after use (average, 0.5 ,um). The leaf, still attached to the intact plant, was held in a on the oscilloscope screen. The number of stable hyperpolarizations during stepwise insertion of the microelectrode (Fig. 3) indicated the number of cells crossed. The experimental protocol is described in Figure 2. Specific light treatments during the experiments are listed in each figure legend. I

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FIG. 1. Plan of measuring apparatus. PROJ, projector, 1, lamp; 2, lenses; 3, heat filter; 4, plexiglass filter; 5, interference filter. MOT, motor to rotate filter support disc. D, filter support disc. ENTR, air entrance with heat and humidity exchanger. FT, fluorescent tubes. IL, incandescent lamp. MI, mirror reflecting projector beam. FC, Faraday cage. ME, microelectrode. R, reference electrode-(shielded from light). CU, cuvette with leaf in position. PL, plant. EL, electrophysiological solution (see text). M, marble slab. AV, anti-vibration pads. E, multifunctional electrometer (WPI M-707). PROC, 'microprocessor' based on rotating contacts. OSC, oscilloscope (Tektronix 5113N). REC, multichannel chart recorder (Watanabe MC641). P, Photocell. T, electronic thermometer. H, electronic hygrometer.

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FIG. 2. Experimental protocol: light treatments and impaling relative photoperiod. Light and dark are indicated on the time scale. 1, Test of resistance, tip potential, and capacitance of microelectrode in APW; 2, electrode implanted in leaf; 3, after stabilization of potential, response to white light (20.4 w m-2) recorded; 4 and 5, dark adaptation period before start of experiment; 6 and 7, light on and off response checked; 8, microelectrode properties measured; 9, leaf removed from APW appears normal.

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FAR-RED LIGHT AND INTRACELLULAR POTENTIALS

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RESULTS

Electrophysiology of Leaf Cells. As the electrode is advanced into the leaf, the first hyperpolarization of -52.6 ± 4.5 mv (average and SD for 27 cells) should correspond to the intracellular (tonoplast + plasmalemma) potential of an epidermal cell. After a few min, this potential decreased to a relatively stable value between -15 and -30 mv; the electrode was then lowered to the mesophyll below. The second hyperpolarization of -86.3 ± 5.9 mv (average and SD for 57 cells in all regions of mesophylls of leaves of different plants) is the initial intracellular potential of the parenchyma cell. Current microinjection of 3.5% Evans Blue (10) from the microelectrode after successful insertions (as inferred from the potential) confirmed that the electrode was in the vacuole of the first or second mesophyll cell below the epidermis, and that the tonoplast was still intact after removal of the microelectrode. In APW, the vacuole normally occupies 95% of the volume of these cells; in partially plasmolyzed cells, the cytoplasm could be occasionally labeled with the dye. The total input resistance was measured using the 'single intracellular microelectrode technique' ( 1, 8) with a 1 kHz square wave. Using an estimate of 5.2 10-5 cm2 for the cell surface area, and the measured resistance, 29.13 ± 4.83 MQ for 23 cells, one obtains a total (plasmalemma + cytoplasm + tonoplast) resistance per unit area of 1.57 kQ cm2. The initial intracellular potential of -100 to -75 mv depolarizes to a level between -40 and -20 mv (Fig. 3). During the next 1 h, it may become more negative again by -5 to -10 mv; most measurements were made with a (dark) resting potential of about -35 to -40 mv. This value remains stable; after as long as 2 d (16), the 'light-on' reaction to high-fluence white light was still normal. The pattern shown in Figure 3a was always obtained when impaling mesophyll cells, with electrodes of various shapes, filled with either 0.5 M or 3 M KCI. The micromanipulator could be either hand-driven or motor-driven with the same result. Heat-killed cells (Fig. 3b), in contrast to viable cells, depolarize

FIG. 3. Intracellular potentials of leaf cells. The (unfiltered) potentials were traced from the

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microelectrode in APW and during successive insertion into an epidermal cell ( 1), then the adjacent mesophyll cell (12) of a spinach leaf under white light (see Fig. 2). At (t3), the electrode was removed from the leaf. a, Three insertions into a normal leaf; b, heat-killed (5 min at 70C) leaf. Microelectrode tip potentials compensated to zero (left side of figure).

FIG. 4. Photosynthetically active light causes large potential changes. Representative recordings of changes in intracellular potential due to light and dark periods of 5 mm. White light, 20.4 w m2, 400 to 700 nm. In the last part of the series, the light was left on (-) or offi(-a- -n. The left side of the figure is preceded by 1 h dark adaptation (see Fig. 2).

irreversibly to a value close to zero. Plants grown for 7 weeks under a 9-h photoperiod at 28 w m2 have larger and greener leaves than the plants used here. The 7week-old plants had initial mesophyll intracellular potentials of -95 to -1 15 mv, decreasing to -55 to -70 mv after 20 min in the light. One h after switching off the light, a new stable value of -45 to -65 mv was reached. In the dark, addition of 50 uM CN- to the APW caused a depolarization to a level of -20 to -30 mv for 7-week-old plants, while for the standard 4-week-old plants by only 10 mv. This indicates (2, 9) that, in the dark, the intracellular potentials of 7-week-old plants have a large active component which is smaller for 4-week-old plants. While part of the behavior of an impaled cell during the first 0.5 h can undoubtedly be explained by wounding, the above results suggest that the stable value is a meaningful intracellular potential. In cells with large vacuoles, relatively low values similar to those reported here have been observed, and were recently given an explanation (4) according to the electrical properties of the tonoplast and plasmalemma. Effect of White Light: 'Light-On' and 'Light-Off' Reactions. After a 1 h dark adaptation (Fig. 2), the first dark-light transition causes a small depolarization followed by a repolarization, then a depolarization. In continuous light, the potential follows a damped oscillation (16, 18), stabilizing at -40 to -60 mv after 3 h illumination. If the light is turned off after 5 min, the lightoff response has opposite phases to the light-on response, of different amplitude and duration, stabilizing after 15 min to about -35 mv. The amplitude of the various phases depends on the preceding light treatment. Under alternating light-dark periods of 5 min (Fig. 4), the amplitude becomes constant. We found no difference in light-on/off responses between cells of 4week-old plants (stable potentials about -30 mv) and those of 7week-old plants (stable potentials about -50 mv). Effects of Red and Far-Red Light. Light restricted to narrow wavelength bands also causes complex oscillations. We therefore chose a standard period of 150 s, and exposed the leaf to alternating light-dark periods. Such light sequences gave consistent, repeating patterns. Under alternating 150-s exposures of R (2.9 w m-2) and dark, the intracellular potential oscillates in time with four distinct phases (Fig. Sa). Replacing dark by FR (2.9 w m-2) has no significant effect on this pattern. A 30% reduction in the fluence rate of the R leads to a biphasic oscillation (Fig. Sb) of double amplitude. Here too, replacing dark by FR did not change the resulting pattern. In some cells, a biphasic oscillation was found also at 2.9 w m-2; this diversity might be explained by cell location in the tissue or electrode tip location within the cell. Intracellular potentials recorded from a leaf sprayed with 10 AM DCMU 12 h before microelectrode insertion, or killed by heating (Fig. 5, c and d) showed no response to the light treatments, indicating that the illuminations did not generate artifacts picked up by the electrodes and measuring apparatus. Turning on and off the temperature and humidity control installations of

674

MONTAVON ET AL.

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Plant Physiol. Vol. 73, 1983

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FIG. 5. Alternating R/dark or R/FR periods have the same effect. Representative recordings of changes in intracellular potential during light and dark sequences: D, dark; FR, 730 nm; R, 662 nm. At the end of each periodic sequence, the potential changes in continuous R ) are shown. The start of each period is ( ), D ( - ), or FR ( marked with a vertical line. Absolute values of the intracellular potential at the end of the first 150-s dark period indicated at the left of each tracing (a) and (b), same mesopyll cell. Treatments: a, R and FR at 2.9 w m-2; b, R at 0.87 w m-2, FR at 2.9 w m-2; c, leaf in APW + 10 ,M DCMU for 12 h, R and FR as in (b); d, heat-shocked leaf (5 min at 70C) in APW, R and FR as in (b). .

the laboratory also has no effect on the potential. If the periodic light treatments were stopped (Fig. 5, a and b), leaving the leaf in continuous R, the potential followed a lighton transient typical ( 15, 16) for the R fluence rate used; continuous dark (or continuous FR) after the periodic treatments gave a typical light-off transient. Thus, no effect of FR on the potential could be observed in this way. In contrast, an effect of FR alone was found by alternating FR and dark periods. After 10 to 30 light-dark cycles, a stable oscillation was obtained with a peak-to-peak amplitude (0.2-0.3 mv for 2.9 w m-2) constant for at least several hours (Fig. 6a). This pattern stopped if the light was left on or off. The oscillation is clearly significant and its amplitude is easy to measure, though the exact kinetics are not clear. Alternating FR treatments have no effect on cells of a DCMU- or heat-treated leaf (Fig. 6, c and d). Up to 2.9 w m2, the highest fluence rate used, the response was not saturated (Fig. 7). We then looked for an effect of continuous R. The depolarization during the 150 s FR period was taken as a quantitative measure of the effect of FR. Switching on a continuous R light in addition to the alternating FR periods causes a light-on oscillation. The size of this perturbation depends on the fluence rate of R. Therefore, the effect of supplementary R was measured beginning 10 min after R was added. At the highest R fluence rate (1.21 w m-2, Fig. 8), the light-on oscillation did not damp out completely, leading to a drift in the baseline. Continuous R supplementary light amplifies the response to repetitive exposures to FR described above. The effect is maximal for supplementary light of 0.054 w m-2 (Fig. 8). Note that at 1.21 w m-2, the small responses to the FR periods are opposite in sign to

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FIG. 6. Induction of responses to alternating FR and dark periods. Portions of original recordings of intracellular potential in a cell showing response to FR (see text), in a dark-adapted leaf( I h; see Fig. 2). Potential at the end of the first 150-s dark period indicated at the left. FR, 2.9 w m-2, 730 nm. a, Leaf in APW. Upper trace, three sequences: at the start, after several periods, and after complete stabilization. Lower trace, stable repetitive pattern. At the end of both series, the complete transient in FR (-) or D (-- -) is presented. b, Same as lower trace in (a), recording without filtering the input of the recorder, showing noise of frequencies above 0.5 Hz. c, Leaf in APW + 10 AM DCMU (12 h). d, Heat-killed leaf (5 min at 70°C) in APW.

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FIG. 7. The response to FR was not saturated at the light levels used. Average potential change during 150 s FR at fluences up to 2.9 w m-2 as recorded in Figure 6a. Averages with SD for six cycles of FR/dark on five cells, six cycles on each (error bars marked with an x). The smaller error bars (-) indicate, for comparison, SD for one of the individual cells.

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by supplementary R; for these cells, the response to FR was reversibly (at least 50 repetitions) enhanced by the supplementary light. We tested background light at 660, 725, and 702 nm for an effect on the response to alternating FR treatments. In addition, we measured the response to alternating 702 nm light in the presence of 660 nm background light. Figure 9 gives average responses, for seven cells showing the amplification effect, to alternating light treatments as a function of the fluence rate of the background light. The data for FR exposures with R supplementary light are averages of potential changes such as those shown in Figure 8. Supplementary light (702 nm) can somewhat enhance the response to FR, but only at higher fluence rate (0.23 w m-2) than that needed at 660 nm. Supplementary light at 725 nm, on the other hand, decreased the response to FR. Finally, the response to 702 nm light periods could not be enhanced by R supplementary light at any of the fluence rates tested.

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FIG. 9. The enhancement effect is specific to FR light supplemented by R. Average amplitude of intracellular potential variations in response to on-off cycles at various wavelengths with continuous supplementary light at different fluence rates: each symbol represents the average of 25 measurements, five cycles each on five cells in different plants, taken after damping out of the 'light-on' response to the supplementary light, with SD. In each experiment, the fluence rate was adjusted to give about 0.2 mv response to on/off cycles with no supplementary light (control). Each set was then normalized to exactly 0.2 mv for the control. Solid lines, FR (730 nm) light on-off cycles and supplementary light at the following wavelengths: (0), 662 nm; (A), 702 nm; (U), 725 nm. Dotted line: (V), 702 nm light on-off cycles and supplementary light at 662 nm. Average (dark) resting potential, -38.5 mv.

those obtained at lower supplementary light fluences. Although all cells apparently successfully impaled respond to R or white light, only about 70% responded to FR light; those that did not were not included in calculations of standard deviation. Out of the responding cells, about 60% showed the amplification effect

DISCUSSION Several lines of evidence suggest that the intracellular potential measured is a meaningful one. The values for 4-week-old plants are lower than many reported for higher plant cells, but are stable in the dark and show large changes in response to photosynthetically active light. Studies were carried out on 4-week-old plants under SD to allow comparison with biochemical and ultrastructural studies on similar plants. The decay of the initial value obtained when a cell is impaled (Fig. 3) could be explained by resealing of the tonoplast; particularly with high input resistances, the vacuole potential may be more positive compared to the cytoplasm than was previously assumed (4). Similar behavior of intracellular potential was observed in pollen tubes with vacuoles (27). The effect of alternating R and FR treatments at equal energy resembles a photoreversible response. Nevertheless, it can be explained by the effect of R alone. FR given immediately after R does not cause any potential change different from that observed during a dark period following R (Fig. 5). The light-on, light-off responses to photosynthetically active light have been well characterized (15). Hartmann (12) also observed no specific effect of FR given immediately after a R treatment in green bean

seedlings. We could, however, detect a light-on, light-off response to 730 nm light alone in spinach leaf mesophyll cells. The combination of interference and wide-band filters used did not pass detectable light of wavelength less than 720 nm. This, and the enhancement by R supplementary light, strongly indicate that contamination by shorter wavelengths is not responsible for the response to FR. Furthermore, as shown in Figure 9, the enhancement effect is specific to FR-stimulated transients in continuous R. Not all cells respond to FR, and, of these, not all show the amplification effect. Limitations of the measurement technique might, of course, be the source of this variation. Another explanation would be heterogeneity between cells that appear similar in structure and function. After 1 h in the dark, the FR-stimulated transients are smaller than the constant value reached after a large number of light-dark cycles (Fig. 6a). Only the steady state was studied here. This 'induction' phenomenon might be linked to photosynthetic induction (25) or to some synchronization of the potential changes. Low fluence rate R significantly enhanced the response to FR. The enhancement was maximal for continuous R of less than 2% of the energy in the FR. One explanation for this enhancement is that increased Pfr increases the effect of FR on the membrane potential. For example, absorption of FR by Chl might cause the response to FR by a reaction changing ion transport; Pfr might increase the yield of this reaction. Another explanation is that only Chl are involved, and the

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MONTAVON ET AL.

enhancement is due to the Emerson effect. In photosynthesis (for Chlorella, 6), the enhancement of quantum efficiency in the FR region by shorter wavelength light is observed above 680 nm, in particular, the effect is evident at 700 nm. The membrane depolarizations due to periodic exposures to 702 nm light are not enhanced by continuous 660 nm light (Fig. 9). This makes the enhancement of the response to 730 nm by R appear incompatible with the Emerson effect. The precise action spectrum of photosynthetic enhancement in intact spinach leaves is not, however, available yet for direct comparison. The responses to R and FR are blocked by DCMU treatment (Figs. 5c and 6c), supporting a hypothesis that the energy for these changes comes from the action of photosynthetic pigments. There is much evidence (cf 5, 15, 24) for a direct link between photosynthesis and the effect of photosynthetically active light on intracellular potentials. FR supplementary light (725 nm, Fig. 9) slightly inhibits the response to alternating FR and dark periods. This means that the enhancement by continuous supplementary light is wavelength specific. Also, continuous 702 nm light causes enhancement only at higher fluence rate than that sufficient using R light. Detailed action spectroscopy for both the enhancing light and the light used to stimulate potential changes may help determine the contributions of phytochrome and photosynthetic pigments. The technique and results described here allow quantitative measurements of the effect of FR and can form the basis for action spectroscopy. A relatively high fluence rate is used to stimulate responses to FR, while continuous R, 50 times weaker, causes enhancement. A preliminary equal-energy action spectrum for the supplementary light (Montavon and Greppin, 1983, in preparation) shows a peak at 660 nm for enhancement with inhibition in the FR peaking near 725 nm, and little effect in the 400 to 640 nm region. Much further work, perhaps with mutants lacking phytochrome, will be needed. Uncertainty about the intracellular localization of phytochrome and its relation (optically and physiologically) to the large amounts of Chl in green leaves limit the conclusions from action spectroscopy. Correlation (or lack of agreement) with known photosynthetic processes is not enough to understand transient membrane effects. The use (14) of DCMU to inhibit overall photosynthesis does not rule out participation of photosynthetic pigments (herbicide bleached Lemna might be better). Red and FR light cause changes in redox state of a Cyt in a Lemna mutant in which noncyclic electron transport is blocked just after PSII (22). These changes have half-times compatible with those of membrane potential transients. The possibility of such reactions must be considered in explaining our results, as well as those of Loppert et al. (14) for Lemna. Regardless of the pigment system involved, the FR-stimulated potential change could be significant in transducing the R/FR ratio of ambient light into a change in membrane properties. The FR-induced depolarization represents only a small fraction of the total light-on reaction in white light. The relatively small response to FR is actually not surprising in view of the low absorption in this region. FR might also cause only a small potential change because the principal action of the photoreceptor is at a site other than the membrane. Even such a secondary effect will be of interest in elucidating the transduction pathway of R/FR photoreception in green plants. It will be interesting to follow the FR response and

Plant Physiol. Vol. 73, 1983

its enhancement by red during the course of floral induction, and during the daily light-dark cycle. Acknowledgments-We are grateful to Drs. J. Gressel (Rehovot), E. Hartmann (Mainz), D.-P. Hider (Marburg), and M. H. Weisenseel (Karlsruhe) for their helpful criticisms and suggestions.

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20.

21. 22. 23. 24. 25. 26. 27.

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