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Studies on the primary process in photosynthesis. I. Photosynthetic luminescence: Multiple reactants
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
70,
507-526 (1957)
Studies on the Primary Process in Photosynthesis. Photosynthetic Luminescence : Multiple Reactants W. E. Arthur From the Department
and B. L. Strehler
of Biochemistry Chicago, Received
I.
(Fels Fund), Illinois
July
University
of Chicago,
10, 1956
INTRODUCTION
Photosynthesis encompasses a variety of discrete physical and chemical processes. The physical act of light absorption and the subsequent transformation of radiant energy into chemical energy comprises the first portion of the process; the second group of reactions is concerned with the storage of the intermediate chemical energy as carbohydrate or in some other stable chemical form (through the fixation and reduction of CO,).
Due to the development of radioactive tracer techniques and an accessible supply of useful carbon isotopes, a great amount of information on the path of carbon in photosynthesis has been accumulated in the last decade, particularly in the laboratories of Calvin and colleagues (1, 2), Horecker and co-workers (3, 4), and in this laboratory (5, 6). At present the general pattern of the intermediate transformations of fixed CO2 in green plants may be considered established, at least in outline. By contrast, there is much less definitive information and a considerable lack of unanimity concerning the nature of the physical and chemical events during the early portion of the process. Chemical studies on this latter area involve various difficulties, e.g.: (a) Since the photochemical processes are probably cyclic in nature and relatively fast, the instantaneous concentrations of intermediates are low; and (b) the specific environment in which a photochemical reaction occurs inevitably influences the outcome. Stated otherwise, while thermally activated, catalytic biological re507
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actions may be and generally are highly specific, photochemistry is in general unspecific-i.e., all reactions possible will take place provided that the light energy is sufficient to overcome the existing activation barrier. This principle has been discussed in detail by Franck (7). Consequently, while there have been many elegant studies [eg. (S-lo)] on the photochemistry of chlorophyll solutions, one cannot a priori decide on the pertinence of these findings to the process of photosynthesis. The specific chemical milieu in which the photochemical process or processes in photosynthesis takes place is not known. What takes place in solution may or may not have a relation to what occurs in the organized structure, the chloroplast, during photosynthesis. Nevertheless, experimental progress has been made in this difficult area of the problem. A. For example, Hill’s finding (11) that illuminated chloroplasts can produce oxygen when the proper photochemical oxidant is present, is one such tool and advance. B. Similarly, the examination of rapid spectral changes which occur when photosynthetic organisms are illuminated, such as the studies of Duysens (12), Lundegardh (13), Strehler and Lynch (14), and Chance and Smith (15) have demonstrated, may eventually prove extremely useful in understanding the early processes. C. A third tool is the finding that all photosynthesizing green plants and chloroplasts obtained from them emit light immediately following illumination and for some time thereafter (16). This bioluminescence is believed to represent a small but significant reversal of the early steps in photosynthesis. The following equations represent an interpretation of the findings in these three areas: Chl
+ hv XH
x+yc XH
+
4YOH
+
ZHill
+ YOH’
---) ZH + X
02 + 2H,O
+ 4Y
(1)
(2) (3)
A. The Hill reaction embodies reactions (1)) (2)) and (3). B. Spectral changes result if the absorption spectra of either chloro1 Although the notation indicates that water is a reactant in process (l), this need not be so literally, inasmuch as reaction (1) might, for example, simply consist of an electron transfer from Y to X with a subsequent attack on the water molecule or ions derived from it.
PHOTOSYNTHESIS.
I
509
phyll or any of the intermediate oxidants or reductants (e.g., XH, YOH, ZH) are modified by the occurrence of the process. C. Luminescence is thought to occur through the reversal of reaction (1) and to be modified by any reactions which can affect the concentrations of XH and YOH [e.g., reactions (2) and (3)]. The present paper describes a number of additional properties of the luminescent reaction in plants. The results are consistent with and extend earlier interpretations of the phenomenon of light emission by green plants. MATERIALS
AND
METHODS
A. Light Detection In these studies a photomultiplier (lP22) operated at liquid nitrogen temperature was used as a light detector, essentially as described in an earlier publication (7). Individual photoelectron pulses were amplified through an AlA preamplifier and an AlC linear amplifier manufactured by Radiation Counter Laboratories. The amplified pulses were passed through the discrimination circuit and then used to drive a decade scaler and a counting rate meter. The output of the latter was used to drive a Brown recorder. By this arrangement it was possible to obtain the number of counts per second and to record the time course of the reaction simultaneously. The photomultiplier was housed in a light-tight box equipped with a rotary shutter, as illustrated in Fig. 1. The load resistor on the photomultiplier was 2 megohms and the voltage per stage was 90 v.
B. Phosphoroscope In order to measure the time course of luminescence decay and induction curves, a specially designed phosphoroscope was built. Its unique feature is the absence of any glass parts. In earlier unpublished experiments glass had been found to phosphoresce to an extent which interfered with the accurate measurement of plant luminescence. The general design of this apparatus is shown in Fig. 2. Various shutter speeds were achieved by using a series-wound, fs horsepower Robbins and Myers motor and a variable transformer (Variac). The speed of rotation of the shutter was continuously monitored with a centrifuge-type tachometer attached to the motor shaft. Shutter speeds from 50 to 6000 r.p.m. (corresponding to a dark time between illumination and measurement of 360 to 3.3 msec., respectively) were obtained through a special speed reduction gear system which was used to achieve slow shutter speeds.
C. Preparation of Plant Material 1. Chlorella was grown at 25°C. in modified Knopp’s solution according to published procedures (IS), except as noted herein. 2. Chloroplasts were prepared from spinach in 0.35 M NaCl solution by grind-
510
ARTHUR
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STREHLER
LIQUID N2TRAP
FOR FILLING
HIGH VOLTAGE
CONNECTOR
SIGNAL OUTPUT
-HOUSING
IP22 PHOTOMULTIPLIER
DEWAR-
FIG. 1. Arrangement
of “quantum
counter.”
ing with sand in a mortar at 0°C. for 1 min. (19). They were separated from most other constituents by filtration through cheescloth, and centrifugation at 1000 r.p.m. for 1 min. in an International centrifuge, followed by a centrifugation of the supernatant solution at 1000 X g. for 7 min. The chloroplasts were then taken up in NaCl solution and kept at 0°C. until they were used. 3. Leaf Materials. Spinach leaves (flat and crinkly varieties) were obtained commercially. Marine algae of various types were collected by the Supply Department of the Woods Hole Marine Biological Laboratory. Various land plants including conifers were collected in Woods Hole. 4. Leaf Powders. The preparation of stable dried powders of flat-leaved spinach was as follows: The crude chloroplasts were prepared by homogenizing the leaves rapidly in a Waring blendor at room temperature in distilled water, filtering the homogenate through cheesecloth, and lyophilizing this filtrate immediately. It was found that such preparations lost most of their activity in 15-20 min. if they were not freeeedried. But by contrast, when such extracts were immediately lyophilized, preparations were obtained which retained (stored at 4°C.) for months (up to 1 year) the
511
PHOTOSYNTHESIS. I
.-\ ‘ \ I , ‘- d ,-* ;
.-’
\ ’
G
a ,-1 ‘, .-.’
*i-i-; --*
--
I-___._
/--:
9’234fiq7tj SCALE
FIG. 2. Phosphoroscope
(INCHES)
used to measure
decay of photosynthetic
luminescence.
ability to luminesce when mixed with water or buffer solutions. It was found flat-leaved spinach yielded brightly luminescent and stable preparations, crinkly-leaved spinach was generally unsatisfactory. Surprisingly, powder arations from flat-leaved spinach were considerably more stable when they suspended in water than was the original unlyophilized filtered homogenates which the powders were obtained.
EXPERIMENTAL
that while prepwere from
RESULTS
The general questions which we have attempted to answer in these experiments are the following: A. Is this chemiluminescence a universal accompaniment of photosynthesis? B. Is more than one compound involved, and can the different reactants be distinguished on the basis of kinetic behavior, response to inhibitors, etc.? C. What is the nature of the products produced in illuminated photosynthesizing plants which are responsible for this chemiluminescence? The experimental results will be presented in the light of these questions.
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A. Generality of the Phenomenon In earlier experiments (16) about 20 different species of photosynthesizing plants had been examined for ability to luminesce. All had been found to luminesce after exposure to visible light. Most of these species were agricultural varieties of green plants. In the present work we have examined about 40 additional photosynthesizing species of widely different habitat and origin and have found, in all cases, that luminescence accompanies photosynthesis. All monocotyledenous, dicotyledenous, and coniferous plants and blue-green (20) and brown algae tested gave off light after illumination. Marine algae, fresh water algae, and most land plants emitted approximately the same amount of light under equivalent conditions. Juniper and pine (conifers) were only about 10% as luminous as the other species tested. The marine alga, MUCUS,was especially bright. B. Evidence of Multiple
Components
Photosynthesis obviously consists of a number of consecutive and/or parallel reactions. A number of earlier studies have been concerned with the measurement of the relative rates of different reactions in the process. Of particular experimental significance in this connection are the studies of the effects of flashing light by Warburg (21), Emerson and Arnold (22)) and a great number of later investigators (23-25). Emerson and Arnold demonstrated that, under their conditions of flash illumination the predominant time constant for the “Blackman reaction” is around xoo sec. Inasmuch as the luminescence affords a direct window into the early photochemistry, in these experiments we have measured the time course of luminescence after illumination. For these studies we employed the phosphoroscope described in Materials and Methods. 1. E$ect of Light Intensity. The findings on the decay of photosynthetic luminescence are illustrated in Fig. 3, in which the dark interval between illumination and measurement of luminescence is plotted against the intensity of light emission. The several curves represent different illuminating intensities. From this figure it is seen that there is a shortlived component which has a half-life of about x00 sec. which appears at higher illumination intensities but is very low when the exciting light
PHOTOSYNTHESIS.
Time after Illumination
I
513
WInseconds)
FIG. 3. The decay of photosynthetic luminescence in chloroplasts (left) and Chlorella (right) at different light intensities. Chloroplasts suspendedin 0.35 M NaCl, 0.01 M KCl. Light intensity: Chlorella Chloroplasts n = 606 ft.-candles 0 = 600 ft.-candles x = 120ft.-candles n = 300 ft.-candles A = 6 ft.-candles A = 120ft.-candles X = 30 ft.-candles Luminescence intensity: 9000 counts/set. at reading of 100 for chloroplasts; 18,000 counts/set.
at reading
of 100 for ChloTells.
Temperature = 25°C. intensity is low. The long-lived component persists even at lower intensities.2 As is also indicated in Fig. 3, this division into slow and rapid decay reactions is also apparent in chloroplasts. Therefore, the phenomenon must be due to reactions taking place in the chloroplasts and not to reactions mediated or primarily influenced by cytoplasmic pools of intermediates or catalysts. The response of the luminescence of ChZoreZZaand of the chloroplasts to variations in illuminating intensity is shown in Fig. 4.a The relationship of these presumably different reactions to each other, * The decay curves as plotted do not represent the actual time course of luminescent decay, since the measurements at high shutter speeds include some longerlived components carried over from prior flashes of illumination. 3 Some differences between the decay curves of chloroplasts and ChZoreZZawere expected because the former are, of course, incapable of all the reactions in photosynthesis. Whether the differences observed are principally due to their different chemical capacities or whether they reflect changes produced in the photochemical apparatus during extraction, etc., we cannot now say.
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ARTHUR
300
450
AND STREHLER
a0
Incident
FIQ. 4. Luminescence vs. illuminating
0
150
300
450
600
Light Intensity (Ft.Candles)
intensity for chloroplasts ChZoreZZa(right). Conditions same as for Fig. 3. Time elapsed between flash and measurement: Chloroplasts Chlorella * = 3.3 msec. 0 = 3.3 msec. A = 180 msec. l = 6.6 msec. n = 360 msec. n = 30 msec. A = 60 msec. X = 360 msec.
(left) and
to photosynthesis, and to the effect of environmental factors was approached experimentally by affecting them separately through the addition of inhibitors, heat treatment, and through a study of the effect of other environmental factors such as pH on the processes. If, during exposure to higher temperatures, one reaction is more rapidly inhibited than another, it might be possible to study separately the behavior of individual reactions. Similarly, the use of inhibitors which affect one process but not the other may give evidence bearing on the nature of each. 2. E$ect of Temperature. Figure 5 shows the effect of temperature on the shape of luminescence decay curves in Chlorella at two different light intensities. Note particularly that low temperature selectively depresses the long-lived luminescence. The effect of partial thermal inactivation on luminescence decay is shown in the succeeding figure (Fig. 6). From these data it is clear that the most sensitive reaction to thermal inactivation is that responsible for the long-lived decay. Also note that the short exposure to higher temperature increases the relative contribution of the long-lived luminescence.
PHOTOSYNTHESIS.
60
3600
160
60
Time after llluminotion
515
I
180
360
(Milliseconds)
FIG. 5. Effect of temperature on decay curves for Chlorella at two different light intensities. Figure at left: 600 ft.-candles; 100% emission = ca. 18,000 counts/set. . = 40°C. * = 27°C. = 14W. A ;
I
Figure at right: 60 ft.-candles. Emission = 0 + = = A
g: at 14 = ca. 1050 counts/set. 40°C. 27°C. 14°C.
3. E$ect of Inhibitors. The effect of several inhibitors (dinitrophenol, hydroxylamine, and cyanide) on luminescence decay is shown in Fig. 7. Note first that dinitrophenol selectively inhibits the long-lived luminescence in intact Chlorella, but that it stimulates the long-lived light emission in chloroplasts. Secondly, hydroxylamine selectively inhibits the tong-lived luminescence at all concentrations in both Chlorebla and chloroplasts. Third, cyanide selectively decreases the contribution of the short-lived luminescence to the over-all emission in both Chlorella and chloroplasts. 4. Effect of PH. Another agent which selectively affects the two processes is the hydrogen-ion concentration. In an earlier paper (17) it was
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ARTHUR
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180 60 360 Time after Illumination (Milliseconds) FIG. 6. Effect of heat treatment on decay curves of ChZoreZZu.Algae exposed to 50°C. for the following times: Time of exposure Maximum count rate counts/see + = 0 min. 3,450 =lmin. 5,550 0 = 2 min. 3,050 =4min. 2,150 ii 930 A = 8 min. and then measured at 25°C. The counting rate per second at the 100% line for each experiment is shown at the right. suggested that the double pH optimum observed for the chemiluminescence of chloroplasts is due to a differential effect of pH on reactions forming and consuming the luminescence substrates. The present results are consistent with the earlier proposal if we assume that the rapid decay is a reffection of the rate of formation of an early product and that the slow decay is an index of the pool sizes of later intermediates. In these studies we have observed only a single pH optimum for the short-lived luminescence. The effect of pH on huninescence decay is shown in Fig. 8. When Iyophilized chloroplasts are dissolved in buffer and then exposed to light at different pH’s there are typical changes in the lumines-
PHOTOSYNTHESIS.
517
I
DNP Chloroplosts
DNP Chlorello
NH20H
NHZOH Chlorella
Chloroplasts
NoCN Chlwello
NaCN Chloroplosts
Milliseconds
FIG. 7. Effect of inhibitors on the decay of chloroplast and Chlorella luminescence. Inhibitors added (0.1 ml.) to 0.9 ml. of ChZoreZZain dark cu. 10 min. before measurement. ChZoreZZa were obtained by the normal culture procedure. Chloroplasts were prepared by a special procedure as follows: 200 g. spinach ground in a Waring blendor with ca. 100 ml. 0.5 M sucrose, 0.01 M KCl; filtered through cheesecloth, centrifuged 10 min. at 2000 r.p.m. to bring down debris. Then supernatant was centrifuged at 3000 r.p.m. for 20 min. Chloroplasts were taken up in 100 ml. sucrose-KCI, divided into l-ml. aliquots in test tubes, and immediately frozen and stored on Dry Ice. For use in these experiments, 1 ml. of frozen chloroplasts was mixed with 1
518
ARTHUR
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FIG. 7.-continued ml. of the following: 0.5 M sucrose, 0.01 M KCl, 0.05 M phosphate buffer, pH 7.1 plus inhibitor in twice desired concentration. These mixtures were used immediately after thawing and remeasured after 1 hr. in dark at room temperature, Symbols Upper left Upper right = Control = Control ; =3XlVMDNP ; -3X10-6MDNP =3X10-4MDNP A =3X10-“MDNP A . = 3 X IO-’ M DNP Middle left Middle right * = Control t - = Control = lo+ M NHzOH n = 10-s M NHzOH A = 10-3 M NH,OH = 1O-4M NHSOH X X l = 10-a M NHzOH Lower left Lower right *-c Control * - = Control = IO+ M NaCN l = 10-E Af N&N = 1O-2M NaCN = lo-* M N&N X :: = lWa M NaCN A 0 = 10-r M NaCN 80 25
ru oz
i
I 60
I 180
b I 360
Time after Illumination (Milliseconds) FIG. 8. Effect of pH on the decay of chloroplast luminescence. Chloroplasts prepared by standard procedure, then suspended in 0.5 M sucrose and 0.01 M KC1 plus phosphate buffer (0.05 M) at the desired pH. . = pH 4.4 A = pH 6.6 x = pH 7.1 n = pH 10.6 5000 counts/set. at scale reading of 60.
PHOTOSYNTHESIS.
I
12 13 14 15 16 I7 I8 19 20 0 Time o,lll”miMllion v.%““tes~
519
, , , ( 5 IO 15 20 Tomeme, lll”M”DflMWlllbeCWS,
course of luminescence when lyophiliced chloroplasts are added to solutions of varying pH. At left: All experiments run at 25”C., 300 ft.-candles. A small aliquot (cu. 25 mg.) of lyophilized powder added to 1 ml. of solution containing 1 M glucose, 0.1 M phosphate at desired pH. pH measured at end of experiment. Note that activation occurs during illumination at pH 4.3-5.6. pH’s as indicated in figure. Inset at right: Decay curves during course of photoactivation. l = 0-4min. n = 4-8 min. X = 12-16 min. at pH 5.45. FIG.
9. Time
cence with time as illustrated in Fig. 9. At certain pH values there is a marked increase in luminescence with continuing illumination. This increase is a photoactivation. If the decay curves for luminescence are measured during different portions of the photoactivation period, it is observed that the short-lived component is selectively stimulated during photoactivation (see Fig. 9). 5. E$ect of Induction Period on Decay. The probability that the slow and fast decays are due to two different components is further supported by studies on the induction curves for chemiluminescence of intact Chlorella and a photoactivation of luminescence in chloroplasts (see Fig. 9). Figure 10 illustrates the induction curves of ChZoreZZaluminescence in the presence and absence of inhibitors. In order to determine whether the induction effects reported earlier are primarily due to a slow-
520
ARTHUR
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60
40
=
.
10-3h4 NH20H
6 X lU4M DNP
FIQ. 10. The effect of inhibitors on the induction curves for Chorella. Chtoretla kept in dark with or without inhibitors for 10 min.; 0.1 ml. inhibitor solution added to 0.9 ml. algal suspension to give appropriate inhibitor concentration, and then exposed to 600 ft.-candles red light during measurement. Maximum light emission at induction maximum = ca. 14,000 counts/see. Measurements at 25°C.
or fast-decaying component, the shapes of induction curves were measured at high and low speeds of the phosphoroscope. It was found that the increased luminescence during the induction maximum is largely due to changes in a component intermediate in lifetime between the short- and long-lived substances. Typical results are shown in Fig. 11. From the foregoing it is evident that a number of different environmental factors can affect the rates of the two or more reactions respon-
PHOTOSYNTHESIS.
I
Time after tllumination
(Milliseconds]
521
The decay curves of Chlorella at different portions of the induction period. Left figure: high light intensity (600 ft.-candles). Right figure : low light intensity (60 ft.-candles). Upper 0 = decay at induction peak maximum. Lower 0 = decay at steady state. l = difference between peak and steady-state decay. Temperature = 25°C. FIG.
11.
sible for the decay curve of luminescence. This evidence, along with the decay of light emission exhibited by normal algae, suggests that the early steps in photosynthesis consist of a number of consecutive reactions, even in the chloroplasts and in the absence of COz fixation and reduction. The first of these reactions possesses a time constant for its decay reminiscent of that observed by Emerson and Arnold. Following this step is another, also capable of feeding back to form excited chlorophyll. This latter step has, by contrast, a half life of seconds, and it is this reaction which has been discussed in the earlier papers on the subject. The induction curves suggest the existence of a third component of intermediate lifetime. C. Nature of the Reactants The ideal approach to the question of chemical intermediates in luminescence of plants would have been the classical one in luminescence
522
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work, i.e., a separation of a “luciferin” and a “luciferase.” Therefore, a number of attempts were made to separate from the illuminated chloroplasts compounds which could cause chloroplasts kept in the dark to luminesce. These attempts were uniformly unsuccessful. Under no circumstances could we obtain extracts of illuminated materials which would exhibit a “luciferin-luciferase” reaction with chloroplasts kept in the dark. Illuminated leaves and chloroplasts were extracted with boiling water, cold acetone, methanol, alcohol, and ether, both separately and serially. When the single or pooled extracts were dissolved in water and added to a chloroplast preparation capable of emitting light after illumination, no luminescence resulted. Several possible interpretations of these negative results are possible. It may be that the primary oxidant and reductant produced are an integral part of the chloroplast and cannot be reinserted into other chloroplasts. Or the products may be so unstable as to defy extraction of the sort here attempted. The next approach met with more success. It was found that leaves lyophilized during illumination retained an ability to emit a small amount of light when they were wetted, even after a month’s storage in the dry state. These findings clearly demonstrate that the compounds formed during illumination are stable for long periods in the dried state and presumably that water is necessary for their recombination, probably because it is required as a diffusion medium. The present results are consistent with earlier studies (unpublished) by Arnold, who has demonstrated that leaves cooled to liquid nitrogen temperature after illumination would retain for hours the ability to luminesce when thawed. The leaves or chlorplasts prepared by freezedrying retain for many months their ability to luminesce when they are suspended in water and illuminated. In this respect the lyophilized powders are more satisfactory than frozen chloroplasts. The experiments here discussed have clearly shown that it is possible to trap early photoproducts by these freeze-drying procedures. Attempts to obtain more information on the chemical nature of these compounds through this type of study have been unsuccessful until now. Therefore, other approaches to the chemical nature of these compounds were attempted. Since, possibly, one or more of the primary photoproducts may be iron-containing compounds such as cytochromes, Chlwella cells were grown in iron-deficient media and the intensity of
PHOTOSYNTHESIS.
I
523
their luminescence as well as time curves for luminescence were compared with Chlorellu grown under standard conditions. First, it was found that the luminescence of iron-deficient Chlorella was considerably lower than that exhibited by normal cells. Secondly, the striking induction curves exhibited by normal cells were reduced in the deficient cells in a number of experiments. Iron compounds may therefore be associated with the early steps in photosynthesis, perhaps even in the photochemical step. At the suggestion of Dr. H. Gaffron, evidence was obtained that luminescence is not inhibited by agents, such as phthiacol, which poison the oxygen-liberating enzyme. This finding suggests that phthiacol acts on the reactions yielding O2 from the primary photo-produced oxidant but does not inhibit the photochemical formation of these precursors. The present results, although clearly showing that a number of reactions can and do affect the luminescence, have not led to any clear idea of what that chemical reaction is. DISCUSSION
The phenomenon of light emission by green plants and functional chloroplasts obtained from them is clearly a universal property of photosynthetic organisms. The general significance of this reaction also seems established: Luminescence results from the chemical reaction of early photoproducts, and it is highly likely that these photoproducts are intermediates in the photochemically driven sequence of reactions in photosynthesis. In the course of the present studies a number of new phenomena of general significance have been uncovered. The first is that the luminescence decay curve is made up of several components: (a) a fast-disappearing luminescence of about 0.01 sec. duration, and (b) the luminescence which was observed in the earlier experiments of Strehler and Arnold. This short luminescence (which was independently discovered by Arnold and suggested by the findings of Franck and Brugger) is reminiscent of the Emerson-Arnold (22) time constant in its decay characteristics and differs strikingly from the earlier described long-lived decay. It has not been found to saturate as a function of incident intensity under our experimental conditions. It is highly probable that this luminescence reflects the concentration of the first oxidized and reduced products in photosynthesis, perhaps radicals (e.g., XH, YOH). The luminescence of longer duration, by contrast, probably is due
524
ARTHUR
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to a later-reducing intermediate, such as ZH. It is also possible, of course, that these two reactions represent two parallel processes, but we believe this to be improbable because we have never succeeded in obtaining the long-lived component in the complete absence of the shorter-lived species. The present studies have shown that these reactions can be affected independently. Generally, it is easier to affect the long-lived decay adversely (i.e., inhibit it). For example, most inhibitors, pH variations, heat treatment, and other experimental variables all reduce the proportionate contribution of the slow decay process. Conversely, only a few experimental conditions selectively inhibit the short-lived luminescence. These facts, in our opinion, speak against a parallel reaction mechanism for the two processes, but rather suggest that they are interdependent and consecutive. The second newly described phenomenon concerns the photoactivation of chloroplast luminescence. This occurs only under restricted physiological pH range and certain osmotic conditions. It is mainly due to an increase in the short-lived luminescence and may represent (a) an increased efficiency of the enzymes concerned in this process; (b) augmentation of luminescence by newly accumulated side products which also are capable of luminescence; (c) the coupling into the reaction sequence of a new pool of hydrogen or OH acceptors; (d) the destruction of an inhibitor, or a combination of these and other factors. Of these, it seems most likely to us that it represents an increase in the YOH pool through reduction of some acceptor such as ZH. The third finding is the fact that the time courses of the fast and slow decay luminescences during the induction period are quite different. The major portion of the induction effects are a result of changes in the slowly decaying luminescence. The nature of XH and YOH is the subject of considerable speculation. It seems unlikely on energetic and kinetic grounds that XH is anything as reduced as the nicotinic coenzyme systems (26, 27). Moreover, the concentrations of these acceptors seem to be too low to permit efficient photochemical transfer (28). Rather, some other constituent, e.g., riboflavine derivatives, carotenoids, or a cytochrome, perhaps in the cytochrome b region, would seem possible oxidants. YOH may be an iron porphyrin similar to cytochrome oxidase or cytochrome f which may have oxidase properties in tivo (29). Of particular pertinence in this connection is the observation (30) that chloroplasts can carry on a
PHOTOSYNTHESIS. I
525
rapid respiration in the presence of certain chelating agents. An alternative possibility, suggested by J. Franck (unpublished) is that YOH might be a modified chlorophyll molecule, perhaps the oxidized hydrate radical. (This possibility is consistent with the recent finding that chlorophyll itself is capable of a comparatively bright and sustained chemiluminescence in the presence of aldehydes, base, and oxygen.) The somewhat contrasting interpretations of flashing-light effects (23-25) are also understandable in terms of this hypothesis. If, as it now appears, there are two different consecutive reactions with different relaxation times, it seems possible that Emerson and Arnold observed the cu. +~OO sec. constant (corresponding to the short luminescence decay) because their flashes were so short that only the short decay intermediates (XH, YOH) had an opportunity to accumulate. In Tamiya’s experiments, on the other hand, the flashes were of sufficient duration to permit the accumulation of later intermediates, thus increasing both the flash saturation limits and the yield per flash and the apparent dark-time constant. ACKNOWLEDGMENTS The authors wish to thank Drs. James Franck, Hans Gaffron, and William Arnold for many helpful discussions and suggestions; Mr. Charles Soderquist, Mr. John Hanacek and Mr. George Gibson for furnishing expert advice and generous help in the construction of the apparatus; Mr. William Keeton, Mr. A. Gene Ferrari, and Mr. Robert Cahn for assistance in certain phases of the work; and Mrs. Clara Gaffron for supplying us with algae cultures for use throughout these experiments. This work was assisted by a grant from the U. S. Atomic Energy Commission, and part of the investigations was pursued while the junior author was a Lalor Fellow at the Woods Hole Marine Biological Laboratory during the summer of 1954. REFERENCES 1. CALVIN, M., BASSHAM, J. A., BENSON, A. A., KAWAQUCHI, S., LYNCH, V. H., STEPKA, W., AND TOLBERT, N. E., U. S. Atomic Energy Comm., Unclassified Report UCRL No. 1386, 1951. 2. CALVIN, M., Federation Proc. 13, 697 (1954). 3. HORECKER, B., AND SMYRNIOTIS, I., J. Am. Chem. Sot. ‘76, 1009 (1953). 4. HORECKER, B., SMYRNIOTIS, P., AND KLENOW, H., J. Biol. Chem. 206, 661 (1953). 5. FAQER, E. W., ROSENBERG, J. L., AND GAFFRON, H., Federation PTOC.9, 535 (1950). 6. FAOER, E. W., Biochem. J. 67,264 (1954). 7. FRANCK, J., Arch. Biochem. Biophys. 46,190 (1963).
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8. LIVINGSTON, R., AND RYAN, V. A., J. Am. Chem. Sot. 76,2176 (1953). LINSCHITZ, A,, AND RENNERT, J., Nature 169, 193 (1952). KRASNOVSKY, A. A., Compt. rend. acad. sci. U.R.S.S. 60, 421 (1948). HILL, R., Proc. Roy. Sot. (London) Bl27, 192 (1939). DUYSENS, L. N. M., Science 120, 353 (1954). LUNDEGARDH, H., Physiol. Plantarum 7. 375 (1954). STREHLER, B. L., and LYNCH, V. H., Science 133, 462 (1956). CHANCE, B., and SMITH, L., Nature 1’76, 803 (1955). STREHLER, B. L., and ARNOLD, W. A., J. Gen. Physiol. 34, 809 (1951). STREHLER, B. L., Arch. Biochem. Biophys. 34, 239 (1951). PRINGSHEIM, F. G., “Pure Cultures of Algae.” Cambridge Univ. Press, Eng-
9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
land, 1946. 19. SPIKES, J. D., Arch Biochem. Biophys. 36, 101 (1952). 20. ARNOLD, W., and THOMPSON, J., J. Gen. Physiol. 37, 677 (1954). 21. WARBURO, O., Biochem. 2. 166, 386 (1925). 22. EMERSON, R., and ARNOLD, W. A., J. Gen. Physiol. 16, 191 (1933). Photosynthesis Symposium.” (1955), Interscience, 23. KOK, B., “Gatlinburg New York, to be published in 1957. 24. GAFFRON, H., and RIEKE, F. F., J. Phys. Chem. 47, No. 4, 299 (1943). 25. TAMIYA, H., Studies Talcagawa Inst. 6, No. 2 (1949). 26. VISNIAC, W., and OCHOA, S. J. Biol. Chem. 196, 75 (1952). 27. TOLMACH, L. J., Arch. Biochem. Biophys. 33, 120 (1951). Photosynthesis Symposium.” (1955), Interscience, 28. FRANCE, J., “Gatlinburg New York, to be published in 1957. 29. KAMEN, M., “Gatlinburg Photosynthesis Symposium.” (1955), Interscience, New York, to be published in 1957. 30. OHMURA, T., Arch. Biochem. Biophys. 67, 187 (1955).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
70,
507-526 (1957)
Studies on the Primary Process in Photosynthesis. Photosynthetic Luminescence : Multiple Reactants W. E. Arthur From the Department
and B. L. Strehler
of Biochemistry Chicago, Received
I.
(Fels Fund), Illinois
July
University
of Chicago,
10, 1956
INTRODUCTION
Photosynthesis encompasses a variety of discrete physical and chemical processes. The physical act of light absorption and the subsequent transformation of radiant energy into chemical energy comprises the first portion of the process; the second group of reactions is concerned with the storage of the intermediate chemical energy as carbohydrate or in some other stable chemical form (through the fixation and reduction of CO,).
Due to the development of radioactive tracer techniques and an accessible supply of useful carbon isotopes, a great amount of information on the path of carbon in photosynthesis has been accumulated in the last decade, particularly in the laboratories of Calvin and colleagues (1, 2), Horecker and co-workers (3, 4), and in this laboratory (5, 6). At present the general pattern of the intermediate transformations of fixed CO2 in green plants may be considered established, at least in outline. By contrast, there is much less definitive information and a considerable lack of unanimity concerning the nature of the physical and chemical events during the early portion of the process. Chemical studies on this latter area involve various difficulties, e.g.: (a) Since the photochemical processes are probably cyclic in nature and relatively fast, the instantaneous concentrations of intermediates are low; and (b) the specific environment in which a photochemical reaction occurs inevitably influences the outcome. Stated otherwise, while thermally activated, catalytic biological re507
508
ARTHUR
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actions may be and generally are highly specific, photochemistry is in general unspecific-i.e., all reactions possible will take place provided that the light energy is sufficient to overcome the existing activation barrier. This principle has been discussed in detail by Franck (7). Consequently, while there have been many elegant studies [eg. (S-lo)] on the photochemistry of chlorophyll solutions, one cannot a priori decide on the pertinence of these findings to the process of photosynthesis. The specific chemical milieu in which the photochemical process or processes in photosynthesis takes place is not known. What takes place in solution may or may not have a relation to what occurs in the organized structure, the chloroplast, during photosynthesis. Nevertheless, experimental progress has been made in this difficult area of the problem. A. For example, Hill’s finding (11) that illuminated chloroplasts can produce oxygen when the proper photochemical oxidant is present, is one such tool and advance. B. Similarly, the examination of rapid spectral changes which occur when photosynthetic organisms are illuminated, such as the studies of Duysens (12), Lundegardh (13), Strehler and Lynch (14), and Chance and Smith (15) have demonstrated, may eventually prove extremely useful in understanding the early processes. C. A third tool is the finding that all photosynthesizing green plants and chloroplasts obtained from them emit light immediately following illumination and for some time thereafter (16). This bioluminescence is believed to represent a small but significant reversal of the early steps in photosynthesis. The following equations represent an interpretation of the findings in these three areas: Chl
+ hv XH
x+yc XH
+
4YOH
+
ZHill
+ YOH’
---) ZH + X
02 + 2H,O
+ 4Y
(1)
(2) (3)
A. The Hill reaction embodies reactions (1)) (2)) and (3). B. Spectral changes result if the absorption spectra of either chloro1 Although the notation indicates that water is a reactant in process (l), this need not be so literally, inasmuch as reaction (1) might, for example, simply consist of an electron transfer from Y to X with a subsequent attack on the water molecule or ions derived from it.
PHOTOSYNTHESIS.
I
509
phyll or any of the intermediate oxidants or reductants (e.g., XH, YOH, ZH) are modified by the occurrence of the process. C. Luminescence is thought to occur through the reversal of reaction (1) and to be modified by any reactions which can affect the concentrations of XH and YOH [e.g., reactions (2) and (3)]. The present paper describes a number of additional properties of the luminescent reaction in plants. The results are consistent with and extend earlier interpretations of the phenomenon of light emission by green plants. MATERIALS
AND
METHODS
A. Light Detection In these studies a photomultiplier (lP22) operated at liquid nitrogen temperature was used as a light detector, essentially as described in an earlier publication (7). Individual photoelectron pulses were amplified through an AlA preamplifier and an AlC linear amplifier manufactured by Radiation Counter Laboratories. The amplified pulses were passed through the discrimination circuit and then used to drive a decade scaler and a counting rate meter. The output of the latter was used to drive a Brown recorder. By this arrangement it was possible to obtain the number of counts per second and to record the time course of the reaction simultaneously. The photomultiplier was housed in a light-tight box equipped with a rotary shutter, as illustrated in Fig. 1. The load resistor on the photomultiplier was 2 megohms and the voltage per stage was 90 v.
B. Phosphoroscope In order to measure the time course of luminescence decay and induction curves, a specially designed phosphoroscope was built. Its unique feature is the absence of any glass parts. In earlier unpublished experiments glass had been found to phosphoresce to an extent which interfered with the accurate measurement of plant luminescence. The general design of this apparatus is shown in Fig. 2. Various shutter speeds were achieved by using a series-wound, fs horsepower Robbins and Myers motor and a variable transformer (Variac). The speed of rotation of the shutter was continuously monitored with a centrifuge-type tachometer attached to the motor shaft. Shutter speeds from 50 to 6000 r.p.m. (corresponding to a dark time between illumination and measurement of 360 to 3.3 msec., respectively) were obtained through a special speed reduction gear system which was used to achieve slow shutter speeds.
C. Preparation of Plant Material 1. Chlorella was grown at 25°C. in modified Knopp’s solution according to published procedures (IS), except as noted herein. 2. Chloroplasts were prepared from spinach in 0.35 M NaCl solution by grind-
510
ARTHUR
AND
STREHLER
LIQUID N2TRAP
FOR FILLING
HIGH VOLTAGE
CONNECTOR
SIGNAL OUTPUT
-HOUSING
IP22 PHOTOMULTIPLIER
DEWAR-
FIG. 1. Arrangement
of “quantum
counter.”
ing with sand in a mortar at 0°C. for 1 min. (19). They were separated from most other constituents by filtration through cheescloth, and centrifugation at 1000 r.p.m. for 1 min. in an International centrifuge, followed by a centrifugation of the supernatant solution at 1000 X g. for 7 min. The chloroplasts were then taken up in NaCl solution and kept at 0°C. until they were used. 3. Leaf Materials. Spinach leaves (flat and crinkly varieties) were obtained commercially. Marine algae of various types were collected by the Supply Department of the Woods Hole Marine Biological Laboratory. Various land plants including conifers were collected in Woods Hole. 4. Leaf Powders. The preparation of stable dried powders of flat-leaved spinach was as follows: The crude chloroplasts were prepared by homogenizing the leaves rapidly in a Waring blendor at room temperature in distilled water, filtering the homogenate through cheesecloth, and lyophilizing this filtrate immediately. It was found that such preparations lost most of their activity in 15-20 min. if they were not freeeedried. But by contrast, when such extracts were immediately lyophilized, preparations were obtained which retained (stored at 4°C.) for months (up to 1 year) the
511
PHOTOSYNTHESIS. I
.-\ ‘ \ I , ‘- d ,-* ;
.-’
\ ’
G
a ,-1 ‘, .-.’
*i-i-; --*
--
I-___._
/--:
9’234fiq7tj SCALE
FIG. 2. Phosphoroscope
(INCHES)
used to measure
decay of photosynthetic
luminescence.
ability to luminesce when mixed with water or buffer solutions. It was found flat-leaved spinach yielded brightly luminescent and stable preparations, crinkly-leaved spinach was generally unsatisfactory. Surprisingly, powder arations from flat-leaved spinach were considerably more stable when they suspended in water than was the original unlyophilized filtered homogenates which the powders were obtained.
EXPERIMENTAL
that while prepwere from
RESULTS
The general questions which we have attempted to answer in these experiments are the following: A. Is this chemiluminescence a universal accompaniment of photosynthesis? B. Is more than one compound involved, and can the different reactants be distinguished on the basis of kinetic behavior, response to inhibitors, etc.? C. What is the nature of the products produced in illuminated photosynthesizing plants which are responsible for this chemiluminescence? The experimental results will be presented in the light of these questions.
512
ARTHUR
AND
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A. Generality of the Phenomenon In earlier experiments (16) about 20 different species of photosynthesizing plants had been examined for ability to luminesce. All had been found to luminesce after exposure to visible light. Most of these species were agricultural varieties of green plants. In the present work we have examined about 40 additional photosynthesizing species of widely different habitat and origin and have found, in all cases, that luminescence accompanies photosynthesis. All monocotyledenous, dicotyledenous, and coniferous plants and blue-green (20) and brown algae tested gave off light after illumination. Marine algae, fresh water algae, and most land plants emitted approximately the same amount of light under equivalent conditions. Juniper and pine (conifers) were only about 10% as luminous as the other species tested. The marine alga, MUCUS,was especially bright. B. Evidence of Multiple
Components
Photosynthesis obviously consists of a number of consecutive and/or parallel reactions. A number of earlier studies have been concerned with the measurement of the relative rates of different reactions in the process. Of particular experimental significance in this connection are the studies of the effects of flashing light by Warburg (21), Emerson and Arnold (22)) and a great number of later investigators (23-25). Emerson and Arnold demonstrated that, under their conditions of flash illumination the predominant time constant for the “Blackman reaction” is around xoo sec. Inasmuch as the luminescence affords a direct window into the early photochemistry, in these experiments we have measured the time course of luminescence after illumination. For these studies we employed the phosphoroscope described in Materials and Methods. 1. E$ect of Light Intensity. The findings on the decay of photosynthetic luminescence are illustrated in Fig. 3, in which the dark interval between illumination and measurement of luminescence is plotted against the intensity of light emission. The several curves represent different illuminating intensities. From this figure it is seen that there is a shortlived component which has a half-life of about x00 sec. which appears at higher illumination intensities but is very low when the exciting light
PHOTOSYNTHESIS.
Time after Illumination
I
513
WInseconds)
FIG. 3. The decay of photosynthetic luminescence in chloroplasts (left) and Chlorella (right) at different light intensities. Chloroplasts suspendedin 0.35 M NaCl, 0.01 M KCl. Light intensity: Chlorella Chloroplasts n = 606 ft.-candles 0 = 600 ft.-candles x = 120ft.-candles n = 300 ft.-candles A = 6 ft.-candles A = 120ft.-candles X = 30 ft.-candles Luminescence intensity: 9000 counts/set. at reading of 100 for chloroplasts; 18,000 counts/set.
at reading
of 100 for ChloTells.
Temperature = 25°C. intensity is low. The long-lived component persists even at lower intensities.2 As is also indicated in Fig. 3, this division into slow and rapid decay reactions is also apparent in chloroplasts. Therefore, the phenomenon must be due to reactions taking place in the chloroplasts and not to reactions mediated or primarily influenced by cytoplasmic pools of intermediates or catalysts. The response of the luminescence of ChZoreZZaand of the chloroplasts to variations in illuminating intensity is shown in Fig. 4.a The relationship of these presumably different reactions to each other, * The decay curves as plotted do not represent the actual time course of luminescent decay, since the measurements at high shutter speeds include some longerlived components carried over from prior flashes of illumination. 3 Some differences between the decay curves of chloroplasts and ChZoreZZawere expected because the former are, of course, incapable of all the reactions in photosynthesis. Whether the differences observed are principally due to their different chemical capacities or whether they reflect changes produced in the photochemical apparatus during extraction, etc., we cannot now say.
514
ARTHUR
300
450
AND STREHLER
a0
Incident
FIQ. 4. Luminescence vs. illuminating
0
150
300
450
600
Light Intensity (Ft.Candles)
intensity for chloroplasts ChZoreZZa(right). Conditions same as for Fig. 3. Time elapsed between flash and measurement: Chloroplasts Chlorella * = 3.3 msec. 0 = 3.3 msec. A = 180 msec. l = 6.6 msec. n = 360 msec. n = 30 msec. A = 60 msec. X = 360 msec.
(left) and
to photosynthesis, and to the effect of environmental factors was approached experimentally by affecting them separately through the addition of inhibitors, heat treatment, and through a study of the effect of other environmental factors such as pH on the processes. If, during exposure to higher temperatures, one reaction is more rapidly inhibited than another, it might be possible to study separately the behavior of individual reactions. Similarly, the use of inhibitors which affect one process but not the other may give evidence bearing on the nature of each. 2. E$ect of Temperature. Figure 5 shows the effect of temperature on the shape of luminescence decay curves in Chlorella at two different light intensities. Note particularly that low temperature selectively depresses the long-lived luminescence. The effect of partial thermal inactivation on luminescence decay is shown in the succeeding figure (Fig. 6). From these data it is clear that the most sensitive reaction to thermal inactivation is that responsible for the long-lived decay. Also note that the short exposure to higher temperature increases the relative contribution of the long-lived luminescence.
PHOTOSYNTHESIS.
60
3600
160
60
Time after llluminotion
515
I
180
360
(Milliseconds)
FIG. 5. Effect of temperature on decay curves for Chlorella at two different light intensities. Figure at left: 600 ft.-candles; 100% emission = ca. 18,000 counts/set. . = 40°C. * = 27°C. = 14W. A ;
I
Figure at right: 60 ft.-candles. Emission = 0 + = = A
g: at 14 = ca. 1050 counts/set. 40°C. 27°C. 14°C.
3. E$ect of Inhibitors. The effect of several inhibitors (dinitrophenol, hydroxylamine, and cyanide) on luminescence decay is shown in Fig. 7. Note first that dinitrophenol selectively inhibits the long-lived luminescence in intact Chlorella, but that it stimulates the long-lived light emission in chloroplasts. Secondly, hydroxylamine selectively inhibits the tong-lived luminescence at all concentrations in both Chlorebla and chloroplasts. Third, cyanide selectively decreases the contribution of the short-lived luminescence to the over-all emission in both Chlorella and chloroplasts. 4. Effect of PH. Another agent which selectively affects the two processes is the hydrogen-ion concentration. In an earlier paper (17) it was
516
ARTHUR
AND
STREHLER
180 60 360 Time after Illumination (Milliseconds) FIG. 6. Effect of heat treatment on decay curves of ChZoreZZu.Algae exposed to 50°C. for the following times: Time of exposure Maximum count rate counts/see + = 0 min. 3,450 =lmin. 5,550 0 = 2 min. 3,050 =4min. 2,150 ii 930 A = 8 min. and then measured at 25°C. The counting rate per second at the 100% line for each experiment is shown at the right. suggested that the double pH optimum observed for the chemiluminescence of chloroplasts is due to a differential effect of pH on reactions forming and consuming the luminescence substrates. The present results are consistent with the earlier proposal if we assume that the rapid decay is a reffection of the rate of formation of an early product and that the slow decay is an index of the pool sizes of later intermediates. In these studies we have observed only a single pH optimum for the short-lived luminescence. The effect of pH on huninescence decay is shown in Fig. 8. When Iyophilized chloroplasts are dissolved in buffer and then exposed to light at different pH’s there are typical changes in the lumines-
PHOTOSYNTHESIS.
517
I
DNP Chloroplosts
DNP Chlorello
NH20H
NHZOH Chlorella
Chloroplasts
NoCN Chlwello
NaCN Chloroplosts
Milliseconds
FIG. 7. Effect of inhibitors on the decay of chloroplast and Chlorella luminescence. Inhibitors added (0.1 ml.) to 0.9 ml. of ChZoreZZain dark cu. 10 min. before measurement. ChZoreZZa were obtained by the normal culture procedure. Chloroplasts were prepared by a special procedure as follows: 200 g. spinach ground in a Waring blendor with ca. 100 ml. 0.5 M sucrose, 0.01 M KCl; filtered through cheesecloth, centrifuged 10 min. at 2000 r.p.m. to bring down debris. Then supernatant was centrifuged at 3000 r.p.m. for 20 min. Chloroplasts were taken up in 100 ml. sucrose-KCI, divided into l-ml. aliquots in test tubes, and immediately frozen and stored on Dry Ice. For use in these experiments, 1 ml. of frozen chloroplasts was mixed with 1
518
ARTHUR
AND
STREHLER
FIG. 7.-continued ml. of the following: 0.5 M sucrose, 0.01 M KCl, 0.05 M phosphate buffer, pH 7.1 plus inhibitor in twice desired concentration. These mixtures were used immediately after thawing and remeasured after 1 hr. in dark at room temperature, Symbols Upper left Upper right = Control = Control ; =3XlVMDNP ; -3X10-6MDNP =3X10-4MDNP A =3X10-“MDNP A . = 3 X IO-’ M DNP Middle left Middle right * = Control t - = Control = lo+ M NHzOH n = 10-s M NHzOH A = 10-3 M NH,OH = 1O-4M NHSOH X X l = 10-a M NHzOH Lower left Lower right *-c Control * - = Control = IO+ M NaCN l = 10-E Af N&N = 1O-2M NaCN = lo-* M N&N X :: = lWa M NaCN A 0 = 10-r M NaCN 80 25
ru oz
i
I 60
I 180
b I 360
Time after Illumination (Milliseconds) FIG. 8. Effect of pH on the decay of chloroplast luminescence. Chloroplasts prepared by standard procedure, then suspended in 0.5 M sucrose and 0.01 M KC1 plus phosphate buffer (0.05 M) at the desired pH. . = pH 4.4 A = pH 6.6 x = pH 7.1 n = pH 10.6 5000 counts/set. at scale reading of 60.
PHOTOSYNTHESIS.
I
12 13 14 15 16 I7 I8 19 20 0 Time o,lll”miMllion v.%““tes~
519
, , , ( 5 IO 15 20 Tomeme, lll”M”DflMWlllbeCWS,
course of luminescence when lyophiliced chloroplasts are added to solutions of varying pH. At left: All experiments run at 25”C., 300 ft.-candles. A small aliquot (cu. 25 mg.) of lyophilized powder added to 1 ml. of solution containing 1 M glucose, 0.1 M phosphate at desired pH. pH measured at end of experiment. Note that activation occurs during illumination at pH 4.3-5.6. pH’s as indicated in figure. Inset at right: Decay curves during course of photoactivation. l = 0-4min. n = 4-8 min. X = 12-16 min. at pH 5.45. FIG.
9. Time
cence with time as illustrated in Fig. 9. At certain pH values there is a marked increase in luminescence with continuing illumination. This increase is a photoactivation. If the decay curves for luminescence are measured during different portions of the photoactivation period, it is observed that the short-lived component is selectively stimulated during photoactivation (see Fig. 9). 5. E$ect of Induction Period on Decay. The probability that the slow and fast decays are due to two different components is further supported by studies on the induction curves for chemiluminescence of intact Chlorella and a photoactivation of luminescence in chloroplasts (see Fig. 9). Figure 10 illustrates the induction curves of ChZoreZZaluminescence in the presence and absence of inhibitors. In order to determine whether the induction effects reported earlier are primarily due to a slow-
520
ARTHUR
AND
STREHLER
60
40
=
.
10-3h4 NH20H
6 X lU4M DNP
FIQ. 10. The effect of inhibitors on the induction curves for Chorella. Chtoretla kept in dark with or without inhibitors for 10 min.; 0.1 ml. inhibitor solution added to 0.9 ml. algal suspension to give appropriate inhibitor concentration, and then exposed to 600 ft.-candles red light during measurement. Maximum light emission at induction maximum = ca. 14,000 counts/see. Measurements at 25°C.
or fast-decaying component, the shapes of induction curves were measured at high and low speeds of the phosphoroscope. It was found that the increased luminescence during the induction maximum is largely due to changes in a component intermediate in lifetime between the short- and long-lived substances. Typical results are shown in Fig. 11. From the foregoing it is evident that a number of different environmental factors can affect the rates of the two or more reactions respon-
PHOTOSYNTHESIS.
I
Time after tllumination
(Milliseconds]
521
The decay curves of Chlorella at different portions of the induction period. Left figure: high light intensity (600 ft.-candles). Right figure : low light intensity (60 ft.-candles). Upper 0 = decay at induction peak maximum. Lower 0 = decay at steady state. l = difference between peak and steady-state decay. Temperature = 25°C. FIG.
11.
sible for the decay curve of luminescence. This evidence, along with the decay of light emission exhibited by normal algae, suggests that the early steps in photosynthesis consist of a number of consecutive reactions, even in the chloroplasts and in the absence of COz fixation and reduction. The first of these reactions possesses a time constant for its decay reminiscent of that observed by Emerson and Arnold. Following this step is another, also capable of feeding back to form excited chlorophyll. This latter step has, by contrast, a half life of seconds, and it is this reaction which has been discussed in the earlier papers on the subject. The induction curves suggest the existence of a third component of intermediate lifetime. C. Nature of the Reactants The ideal approach to the question of chemical intermediates in luminescence of plants would have been the classical one in luminescence
522
ARTHUR
AND
STREHLER
work, i.e., a separation of a “luciferin” and a “luciferase.” Therefore, a number of attempts were made to separate from the illuminated chloroplasts compounds which could cause chloroplasts kept in the dark to luminesce. These attempts were uniformly unsuccessful. Under no circumstances could we obtain extracts of illuminated materials which would exhibit a “luciferin-luciferase” reaction with chloroplasts kept in the dark. Illuminated leaves and chloroplasts were extracted with boiling water, cold acetone, methanol, alcohol, and ether, both separately and serially. When the single or pooled extracts were dissolved in water and added to a chloroplast preparation capable of emitting light after illumination, no luminescence resulted. Several possible interpretations of these negative results are possible. It may be that the primary oxidant and reductant produced are an integral part of the chloroplast and cannot be reinserted into other chloroplasts. Or the products may be so unstable as to defy extraction of the sort here attempted. The next approach met with more success. It was found that leaves lyophilized during illumination retained an ability to emit a small amount of light when they were wetted, even after a month’s storage in the dry state. These findings clearly demonstrate that the compounds formed during illumination are stable for long periods in the dried state and presumably that water is necessary for their recombination, probably because it is required as a diffusion medium. The present results are consistent with earlier studies (unpublished) by Arnold, who has demonstrated that leaves cooled to liquid nitrogen temperature after illumination would retain for hours the ability to luminesce when thawed. The leaves or chlorplasts prepared by freezedrying retain for many months their ability to luminesce when they are suspended in water and illuminated. In this respect the lyophilized powders are more satisfactory than frozen chloroplasts. The experiments here discussed have clearly shown that it is possible to trap early photoproducts by these freeze-drying procedures. Attempts to obtain more information on the chemical nature of these compounds through this type of study have been unsuccessful until now. Therefore, other approaches to the chemical nature of these compounds were attempted. Since, possibly, one or more of the primary photoproducts may be iron-containing compounds such as cytochromes, Chlwella cells were grown in iron-deficient media and the intensity of
PHOTOSYNTHESIS.
I
523
their luminescence as well as time curves for luminescence were compared with Chlorellu grown under standard conditions. First, it was found that the luminescence of iron-deficient Chlorella was considerably lower than that exhibited by normal cells. Secondly, the striking induction curves exhibited by normal cells were reduced in the deficient cells in a number of experiments. Iron compounds may therefore be associated with the early steps in photosynthesis, perhaps even in the photochemical step. At the suggestion of Dr. H. Gaffron, evidence was obtained that luminescence is not inhibited by agents, such as phthiacol, which poison the oxygen-liberating enzyme. This finding suggests that phthiacol acts on the reactions yielding O2 from the primary photo-produced oxidant but does not inhibit the photochemical formation of these precursors. The present results, although clearly showing that a number of reactions can and do affect the luminescence, have not led to any clear idea of what that chemical reaction is. DISCUSSION
The phenomenon of light emission by green plants and functional chloroplasts obtained from them is clearly a universal property of photosynthetic organisms. The general significance of this reaction also seems established: Luminescence results from the chemical reaction of early photoproducts, and it is highly likely that these photoproducts are intermediates in the photochemically driven sequence of reactions in photosynthesis. In the course of the present studies a number of new phenomena of general significance have been uncovered. The first is that the luminescence decay curve is made up of several components: (a) a fast-disappearing luminescence of about 0.01 sec. duration, and (b) the luminescence which was observed in the earlier experiments of Strehler and Arnold. This short luminescence (which was independently discovered by Arnold and suggested by the findings of Franck and Brugger) is reminiscent of the Emerson-Arnold (22) time constant in its decay characteristics and differs strikingly from the earlier described long-lived decay. It has not been found to saturate as a function of incident intensity under our experimental conditions. It is highly probable that this luminescence reflects the concentration of the first oxidized and reduced products in photosynthesis, perhaps radicals (e.g., XH, YOH). The luminescence of longer duration, by contrast, probably is due
524
ARTHUR
AND
STREHLER
to a later-reducing intermediate, such as ZH. It is also possible, of course, that these two reactions represent two parallel processes, but we believe this to be improbable because we have never succeeded in obtaining the long-lived component in the complete absence of the shorter-lived species. The present studies have shown that these reactions can be affected independently. Generally, it is easier to affect the long-lived decay adversely (i.e., inhibit it). For example, most inhibitors, pH variations, heat treatment, and other experimental variables all reduce the proportionate contribution of the slow decay process. Conversely, only a few experimental conditions selectively inhibit the short-lived luminescence. These facts, in our opinion, speak against a parallel reaction mechanism for the two processes, but rather suggest that they are interdependent and consecutive. The second newly described phenomenon concerns the photoactivation of chloroplast luminescence. This occurs only under restricted physiological pH range and certain osmotic conditions. It is mainly due to an increase in the short-lived luminescence and may represent (a) an increased efficiency of the enzymes concerned in this process; (b) augmentation of luminescence by newly accumulated side products which also are capable of luminescence; (c) the coupling into the reaction sequence of a new pool of hydrogen or OH acceptors; (d) the destruction of an inhibitor, or a combination of these and other factors. Of these, it seems most likely to us that it represents an increase in the YOH pool through reduction of some acceptor such as ZH. The third finding is the fact that the time courses of the fast and slow decay luminescences during the induction period are quite different. The major portion of the induction effects are a result of changes in the slowly decaying luminescence. The nature of XH and YOH is the subject of considerable speculation. It seems unlikely on energetic and kinetic grounds that XH is anything as reduced as the nicotinic coenzyme systems (26, 27). Moreover, the concentrations of these acceptors seem to be too low to permit efficient photochemical transfer (28). Rather, some other constituent, e.g., riboflavine derivatives, carotenoids, or a cytochrome, perhaps in the cytochrome b region, would seem possible oxidants. YOH may be an iron porphyrin similar to cytochrome oxidase or cytochrome f which may have oxidase properties in tivo (29). Of particular pertinence in this connection is the observation (30) that chloroplasts can carry on a
PHOTOSYNTHESIS. I
525
rapid respiration in the presence of certain chelating agents. An alternative possibility, suggested by J. Franck (unpublished) is that YOH might be a modified chlorophyll molecule, perhaps the oxidized hydrate radical. (This possibility is consistent with the recent finding that chlorophyll itself is capable of a comparatively bright and sustained chemiluminescence in the presence of aldehydes, base, and oxygen.) The somewhat contrasting interpretations of flashing-light effects (23-25) are also understandable in terms of this hypothesis. If, as it now appears, there are two different consecutive reactions with different relaxation times, it seems possible that Emerson and Arnold observed the cu. +~OO sec. constant (corresponding to the short luminescence decay) because their flashes were so short that only the short decay intermediates (XH, YOH) had an opportunity to accumulate. In Tamiya’s experiments, on the other hand, the flashes were of sufficient duration to permit the accumulation of later intermediates, thus increasing both the flash saturation limits and the yield per flash and the apparent dark-time constant. ACKNOWLEDGMENTS The authors wish to thank Drs. James Franck, Hans Gaffron, and William Arnold for many helpful discussions and suggestions; Mr. Charles Soderquist, Mr. John Hanacek and Mr. George Gibson for furnishing expert advice and generous help in the construction of the apparatus; Mr. William Keeton, Mr. A. Gene Ferrari, and Mr. Robert Cahn for assistance in certain phases of the work; and Mrs. Clara Gaffron for supplying us with algae cultures for use throughout these experiments. This work was assisted by a grant from the U. S. Atomic Energy Commission, and part of the investigations was pursued while the junior author was a Lalor Fellow at the Woods Hole Marine Biological Laboratory during the summer of 1954. REFERENCES 1. CALVIN, M., BASSHAM, J. A., BENSON, A. A., KAWAQUCHI, S., LYNCH, V. H., STEPKA, W., AND TOLBERT, N. E., U. S. Atomic Energy Comm., Unclassified Report UCRL No. 1386, 1951. 2. CALVIN, M., Federation Proc. 13, 697 (1954). 3. HORECKER, B., AND SMYRNIOTIS, I., J. Am. Chem. Sot. ‘76, 1009 (1953). 4. HORECKER, B., SMYRNIOTIS, P., AND KLENOW, H., J. Biol. Chem. 206, 661 (1953). 5. FAQER, E. W., ROSENBERG, J. L., AND GAFFRON, H., Federation PTOC.9, 535 (1950). 6. FAOER, E. W., Biochem. J. 67,264 (1954). 7. FRANCK, J., Arch. Biochem. Biophys. 46,190 (1963).
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