Effects of red and far-red light on the fluorescence yield of chlorophyll in vivo

309

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 3 7 3 2

E F F E C T S OF R E D A N D F A R - R E D THE FLUORESCENCE

YIELD

L I G H T ON

OF CHLOROPHYLL

I N VIVO

W A R R E N L. B U T L E R

Instrumentation Research Laboratory, Market Quality Research Division Agricultural Marketing Service, U.S. Department of Agriculture, BeltsviUe, Md. U.S.A.) (Received April 9th, 1962)

SUMMARY

The fluorescence of chlorophyll a and C-7o 5 in green leaves has been investigated during the induction period of photosynthesis in air and in nitrogen. A brief illumination of a leaf with red light causes an increased yield of chlorophyll a fluorescence which persists for a longer period in nitrogen than air. The increased fluorescence yield, produced b y red light, can be inhibited by a subsequent irradiation with far-red light. The action spectrum for the effect of far-red light shows a m a x i m u m near 7o5 mix. The effects of red and far-red light on the fluorescence yield of chlorophyll a are thought to be another manifestation of the second EMERSON effect.

INTRODUCTION

Fluorescence-excitation spectra of green leaves 1 revealed a small amount of a 7o5-mixabsorbing form of chlorophyll (C-7o5) which had a fluorescence m a x i m u m in the region of 73o mix. The 7o5-mix band in the fluorescence-excitation spectrum was somewhat unexpected because previous work on the fluorescence of green plants had assumed that the emission of wavelengths longer than 68o mix emanated solely from chlorophyll a. The presence of these two fluorescing components suggested that the fluorescence of a green leaf should be reinvestigated during the induction period of photosynthesis when the overall fluorescence yield is changing in order to determine the relative fluorescence-yield changes of chlorophyll a and C-7o 5. The fluorescence of C-7o 5 was previously found to be excited b y the transfer of energy from chlorophyll a. The proposal was advanced 1 that the C-7o 5 molecules were the reaction centers for one of the two photochemical reactions demonstrated by the "second EMERSON effect ''~. The present paper provides more evidence for the participation of C-7o 5 in the photochemistry of photosynthesis. METHODS

Methods for measuring fluorescence-excitation spectra have been described previously 1. Low-intensity monochromatic light, incident on one side of a leaf, excites fluorescence that is viewed from the other side of the leaf through a cut-off filter which transmits wavelengths longer than 73o mix. Limiting the fluorescence measureBiochim. Biophys. Acta, 64 (1962) 309 317

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w . L. BUTLER

ment to wavelengths longer than 730 m/~ reduces self-absorption to negligible levels and permits the excitation spectra to be measured to about 720 m~. The photometer records the logarithm of the intensity of light reaching the phototube (EMI 9558) versus the wavelength of the exciting light on an X-Y recorder. A multitapped potentiometer corrects the excitation spectra for the quantum flux variation of the exciting light with wavelength. The previously described spectrophotometer 3, 4, used for these measurements, has been modified by using a half-silvered mirror to reflect the exciting light onto the sample and by the addition of a hole in the top of the sample compartment to permit irradiation of the sample with an actinic beam through the half-silvered mirror. Actinic radiation was obtained with a tungsten lamp and the appropriate filter. RIBBON FILAMENT LAMP

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Biochim. Biophys..4cta, 64 (1962) 3o9-317

FLUORESCENCE-YIELD CHANGES OF CHLOROPHYLL

311

A 65o-m/~ interference filter (Schott, DAL) gave a red source with a pass band of 2om/~ and an intensity of lO 4 ergs/sec/cm 2 at the sample. Four layers of blue and red cellophane were used for a far-red source of wavelengths longer than 720 m ~ with an intensity of lO 5 ergs/sec/cm 2. To determine action spectra, an actinic monochromator was constructed (Fig. i) from two 25 × 200 m m wedge interference filters, a first-order band filter with a dispersion of 2.5 m/~/mm and a second-order line filter with a 3.3 m ~ / m m dispersion (Schott, Veril B2oo and Veril $2oo, respectively). These filters were coupled together so as to stay in wavelength synchronization as they moved over a 3 ~: 25 m m slit. The ribbon filament of a 6 V, 18 A tungsten lamp was focused on the slit with an F/4 lens system. With a 3-mm wide slit the monochromatic beam had an area of 1.2 × 2.2 cm at the sample position, a pass band of 15 mtz (i.e. the width between the half-maximum intensity values) and stray light of less than one part in lO4 at wavelengths corresponding to the higher transmission orders of the wedge interference filters. The energy of the beam was measured as a function of wavelength, after transmission through the half-silvered mirror, with a Kipp and Zonen thermopile (320 ergs/sec/cm2/~V)and a chopper-stabilized direct current amplifier for various operating voltages on the lamp. With 115 V on the lamp transformer, the energy was constant at 17oo ± 5 ° ergs/sec/cm 2 from 600 to 800 m/~. The beam from the fluorescence-excitation monochromator was approx, i cm ~ at the sample and lay within the larger actinic beam. The pass band and energy of the exciting beam varied monotonously from 7.5 m/~ and 7 ergs/sec/cm2, respectively, at 600 m/, to 12 m/z and 15 ergs/sec/cm ~ at 700 m/,. Measurements were made on leaves from 7- to Io-day-old, greenhouse-grown bean plants. The experiments under anaerobic conditions were carried out in a gastight chamber which was flushed with nitrogen. RESULTS

The fluorescence measurements presented here differ from most previous reports in the use of rather low intensities of exciting light. In previous fluorescence investigations of green plants the intensities of lO4-1o 8 ergs/sec/cm 2, which were generally used to excite fluorescence, caused appreciable photosynthesis. The low intensity of excitation used here (IO ergs/sec/cm 2 at 65om~) serves only to determine the fluorescing condition of the leaf. The effects due to high light intensities are produced with the actinic beam and are determined immediately after the actinic beam is turned off.

The effects of red actinic radiation KAUTSKY5 first noted that the fluorescence yield of chlorophyll in vivo rises rapidly during the first few seconds of illumination and decays to a steady-state value during the next 1- 3 min depending on the length of the induction period of photosynthesis. The transient rise and decay of fluorescence in continuous illumination is known as the KAUTSKY effect. The time course of the fluorescence yield (measured at low light intensity) following high intensity irradiations (lO 4 ergs/sec/cm 2) is shown in Fig. 2. Immediately after a 5-sec irradiation, when the transient fluorescence burst is near its peak, the fluorescence yield is 1.8 times as great as it was before the irradiation (Z]EF- 0.25) Biochim. Bioph),s..4cta, 64 (I962) 3o9-317

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W . L . BUTLER

and decays back to the dark level in 2-3 min. After 5 min of high-intensity illumination, the fluorescence yield of the leaf in light has decayed to its steady-state value which is still somewhat higher than the fluorescence yield of the dark leaf. LATIMER et al. 6 and BRUGGER7 have previously shown that at low excitation intensities the steady-state fluorescence yield of chlorella increases with increasing light intensity.

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Fig. 2. Time course of the fluorescence (2F > 73 ° m/z) of a leaf in air excited at 650 m/~ (lO ergs/sec/cm 2) following a 5-sec and a 5-rain irradiation with a red actinic source (lO4 ergs/ sec/cm 2 at 65 ° m/~).

Fig. 3. lquorescence excitation spectra of a leaf in air. I n t e n s i t y of fluorescence (2F > 73 ° m#) excited b y equal n u m b e r s of q u a n t a of incident light as a function of the wavelength of the exciting light before irradiation and i m m e d i a t e l y after irradiation for 5 sec and for 5 min w i t h the red actinic source. Scan time from 03o to 700 m/~: 6 sec.

The fluorescence excitation spectra of a leaf before, during, and after the KAUTSKY effect are shown in Fig. 3. The steep rise in the curves at 720 m~ marks the beginning of the cut-off filter transmission. The excitation spectrum for the dark-conditioned leaf shows nearly equal bands for chlorophyll b (650 mF), chlorophyll a (680 mF), and C-7o5". The leaf was irradiated with the red actinic source for 5 sec to produce the fluorescence burst of the KAUTSKY effect and the spectrum was recorded immediately after the actinic beam was turned off. The spectra were scanned from 630 to 700 m F in 6 sec so that the fluorescence decay during the scan time was small compared to the total change. The increased intensity of fluorescence is demonstrated but the increase is due solely to an increase in the fluorescence yield of chlorophyll a. At all wavelengths shorter than 685 m F there is a uniform increase in the efficiency of excitation which results from the increased efficiency of emission from chlorophyll a, regardless of whether the excitation was direct or via resonance transfer of excitation energy from chlorophyll b. T h e long wavelength tail of the chlorophyll a excitation band extends to 720 m F. Any change in the fluorescence-excitation band of C-7o 5 * Limiting the fluorescence m e a s u r e m e n t to wavelengths longer t h a n 730 m # enhances the excitation b a n d of C-7o 5 relative to the excitation b a n d s for chlorophyll a because a greater p o r t i o n of the C-7o5 emission extends beyond 730 m/z. The greater efficiency in the m e a s u r e m e n t of the fluorescence emission from C-7o 5 over t h a t from chlorophyll a c o m p e n s a t e s for the lesser a b s o r p t i o n of light b y C-7o5. These m e a s u r e m e n t s do not p e r m i t a direct c o m p a r i s o n of the fluorescence yields of chlorophyll a and C-7o5.

Biochim. Biophys. Acta, 64 (1962) 3o9-317

313

FLUORESCENCE-YIELD CHANGES o F CHLOROPHYLL

is too small to be noted with certainty. After an illumination period of 5 min with the red actinic source the fluorescence intensity has decayed to its steady-state vahie in light. The excitation spectrum measured immediately after the light was turned off shows that the fluorescence yield of chlorophyll a is less than after 5 sec of light but is still somewhat greater than that of the dark-conditioned leaf. After much

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Fig. 4. T i m e course of t h e fluorescence (;tF ~ 730 m/*) of a leaf in n i t r o g e n exc i t e d a t 650 m/* (i o e r g s / s e c / c m 2) following 5-sec i r r a d i a t i o n s w i t h t h e red a c t i n i c source (64o-66o m/z, to 4 e r g s / s e c / c m 2) a n d t h e far-red source ( ~ 720 mH, ~o~ ergs/ sec/cm2).

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Fig. 5. F l u o r e s c e n c e e x c i t a t i o n s p e c t r a of a leaf in n i t r o g e n before i r r a d i a t i o n a n d a f t e r a 5-sec i r r a d i a t i o n w i t h t h e red a n d far-red a c t i n i c source.

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stronger actinic radiation than has been used here (a focused, unfiltered, tungsten lamp) the excitation spectrum shows that C-7o 5 is irreversibly bleached and the fluorescence yield of chlorophyll a is increased and remains constant thereafter (see Fig. 9 of ref. I). Since the emission m a x i m u m of chlorophyll a is at 685 m/,, while that of C-7o 5 is near 73 ° m~, the emission spectrum must change in response to the change of the fluorescence yield of chlorophyll a which occurs during the course of the KAUTSKY effect. VIRGIN8 has measured the emission spectrum during the initial period of high fluorescence yield and during the steady-state light condition and has shown that the intensity of emission at 685 m/~, relative to that at 730 m~, is greater initially. During the decay to the steady-state, tile decrease is proportionally greater at 685 m~ than at 730 mt~. Similar measurements have been made in the laboratory of Dr. J. ROSENBERG9. VIRGIN attributed the changing emission spectrum to an unspecified Biochirn. [3iopt~y,~. ,4cta, 6 4 (19~-') 3o0 3 17

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W. L. BUTLER

scatter change on the basis of the emission changes which occurred when the leaf was infiltrated. However, the fluorescence-excitation spectra, which are not confounded by such scatter artifacts, show that there is an actual change in the spectral quality of the fluorescence emission during the KAUTSKY effect.

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Fig. 7. A, T i m e course of fluorescence (~F > Fig. 6. Effect of far-red light in r e v e r s i n g t h e fluorescence e n h a n c e d b y red light. I n t e n s i t y of 73 ° m#) of a leaf in n i t r o g e n excited b y io ergs/ fluorescence (~F > 730 m # ) of a leaf in n i t r o g e n s e c / c m ~ at 650 m/z following a 5-sec, red actinic irradiation. T h e e x c i t a t i o n w a s s u s p e n d e d excited at 65o rot* following 5-sec i r r a d i a t i o n s d u r i n g t h e I 2 - m i n period indicated. B, T i m e w i t h t h e actinic m o n o c h r o m a t o r . I r r a d i a t i o n s course of fluorescence excited b y 5 ° e r g s / s e c / c m 2 a t t h e w a v e l e n g t h s i n d i c a t e d were a l t e r n a t e d w i t h irradiation a t 65o m/~. A, actinic i n t e n s i at 650 m/t following 5-sec i r r a d i a t i o n s w i t h t h e red a n d far-red sources. ties of 400 e r g s / s e c / c m ~ ; B, actinic intensities of 17oo ergs/sec/cm 2. L o w e r p a r t : t h e differences b e t w e e n t h e i n t e n s i t y levels following t h e a l t e r n a t e i r r a d i a t i o n s (65o a n d 2) as a f u n c t i o n of f r o m A a n d B.

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Fig. 8. T i m e course of fluorescence (~F > 730 m # ) of a leaf excited b y 65 ° m/~ (IO e r g s / s e c / c m s) following t h e i n t r o d u c t i o n of n i t r o g e n a n d t h e reversal of t h e e n h a n c e d fluorescence b y air.

Biochim. Biophys, Acta, 64 (t962) 3o9-317

FLUORESCENCE-YIELD CHANGES OF CHLOROPHYLL

315

The effects of far-red actinic radiation The decay period of the KAUTSKY effect is much prolonged if the leaf is anaerobic 1°. The decay of the enhanced fluorescence yield in the dark following illumination is also much slower in nitrogen. Fig. 4 shows the enhanced fluorescence yield following a 5-sec irradiation of a leaf in nitrogen with red light. The noteworthy feature of the experiment shown in Fig. 4 is that a subsequent irradiation with a far-red source decreases the fluorescence yield. The enhanced and depressed fluorescence yields can be repeatedly demonstrated by a series of alternating exposures to red and far-red light. The fluorescence-excitation spectra for the anaerobic leaf in the red- and far-red-irradiated conditions as well as the dark condition are shown in Fig. 5- Again the changes in the fluorescence yield of the leaf are due to changes in the fluorescence yield of chlorophyll a while that of C-7o 5 appears relatively unchanged.

Action spectra for the far-red effect The action spectrum for the effectiveness of far-red radiation in reversing the enhanced fluorescence yield caused by a previous red irradiation was determined with the actinic monochromator. The leaf in nitrogen was irradiated 5 sec alternately with the actinic monochromator set at 650 m/~ and at a longer wavelength. The intensity of fluorescence (~F > 730 m/~) was recorded against time for about io sec after each irradiation to establish the intensity of fluorescence. The results of two experiments, one with actinic intensities of 400 ergs/sec/cm 2, the other with 17oo ergs/sec/cm 2, are shown in Fig. 6A and B. The action spectra for these two experiments plotted as the differences in the intensity levels between the red- and far-red-irradiated states as a function of the actinic wavelength are shown in the lower part of the figure. The same results are obtained with a 69o-m/~ cut-off filter instead of the 73o-m~ cut-off filter. The shorter wavelength cut-off filter transmits a larger light signal to the phototube but the fluorescence-intensity changes are proportionately the same. The results are also independent of the fluorescence-excitation wavelength provided it is less than 680 m/~. For longer wavelengths of excitation the fluorescence-yield changes are smaller as Fig. 5 shows.

The effects of the measuring light Even the very low intensity of excitation used in these experiments (IO ergs/ sec/cm 2) has a discernible influence on the photochemical state of the leaf. The rate of the decay of the enhanced fluorescence yield in nitrogen is a function of the intensity of the exciting light. In Fig. 7 A the measuring beam was blocked and the leaf was in complete darkness for 12 min. The fluorescence decay was suspended during the dark period and was resumed when the measurement resumed. In Fig. 7 B, increasing the intensity of excitation to 50 ergs/sec/cm 2 b y focusing the exciting light on the leaf to an area approx. I x IO m m increased the rate of fluorescence decay in the measuring light.

The effect of oxygen When air is flushed out of the leaf chamber with nitrogen, the fluorescence yield of chlorophyll a increases and slowly decays to a level somewhat higher than the Biochim. Biophys. Ac/a, 64 (I96~) 309 317

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steady-state level in air (Fig. 8). If air is admitted to the chamber while the leaf is in nitrogen, the enhanced fluorescence yield caused by red light is lost rapidly (Fig. 8). DISCUSSION

The photochemical basis for the fluorescence-yield changes is unknown. A previous publication 1 reported that as an etiolated leaf greens, the ratio of C-7o 5 to chlorophyll a increases and the fluorescence yield of chlorophyll a decreases; also that excitation energy is transferred from chlorophyll a to C-7o 5. This supported the hypothesis that C-7o 5, acting as an energy sink, quenches the fluorescence of chlorophyll a. On this basis the results of the present paper suggest that the action of red light inhibits the transfer of energy from chlorophyll a to C-7o 5 while far-red light enhances the transfer. The coincidence between the absorption m a x i m u m of C-7o 5 and the action m a x i m u m for the fluorescence inhibition indicates that C-7o 5 is responsible for the far-red action. The red-light effect of enhancing the fluorescence yield is presumably mediated by chlorophyll a. The report by KOK AND HOCH11 that light absorbed by chlorophyll a results in the photochemical bleaching of a pigment absorbing near 700 m/~ (presumably the same pigment we designate C-7o5) would account for the action of red light in enhancing the fluorescence yield of chlorophyll a. The opposition of red and far-red photoeffects contributes to the "red drop" in the fluorescence yield li beyond 680 m/~ i n vivo. As the wavelength of excitation is increased beyond 680 m/~, a smaller portion of the light is absorbed by chlorophyll a which enhances the fluorescence yield and a greater fraction is absorbed by C-7o 5 which opposes the enhanced fluorescence. The results reported here relate to some recent fluorescence studies b y GOVINDJEE et al. 13. They found that the intensity of fluorescence (plus scatter) emitted by chlorella, when illuminated with 670- and 7oo-mt~ light simultaneously, was less than the sum of the intensities obtained with the 670- and 7oo-mt~ beams separately. Their interpretation that the 7oo-mt, beam decreased the yield of fluorescence excited by the 67o-m/~ beam is confirmed in the results reported here. Previous measurements of the reversible bleaching of chlorophyll a i n vivo n , 14 by high-intensity actinic light m a y have been confounded by changes of the fluorescence yield. Fluorescence excited by the measuring beam in these bleaching experiments was undoubtedly a detectible part of the light signal measured by the phototube (see DISCUSSION following ref. II). The red or white actinic beam would increase the fluorescence yield of chlorophyll a and thus increase the light signal during the measuring cycle. Assuming that the transmitted light and the fluorescence have the same geometry (this condition would be approached if the sample diffused the transmitted light or if the phototube was close to the sample), calculation shows that an increase of the fluorescence yield of chlorophyll a from o.oi to o.o125 would appear as an absorbancy decrease of o.ooi for a sample that absorbed 50 % of the light at 68o mt~. If the sample absorbed 9 ° % of the light, the absorbaney decrease at 680 m/~ would be o.oi. The magnitude of these fluorescence changes is more than enough to account for the observed chlorophyll a bleaching. Chlorophyll b would also appear to be bleached because it sensitizes chlorophyll a fluorescence. The changes in the yield of chlorophyll a fluorescence cannot account for the observed bleaching at 705 m/~, however n, 24 Biochim. Biophys. ,4cta. ~4 (1962) ,309 317

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A l t h o u g h t h e r e l a t i o n s h i p of t h e f l u o r e s c e n c e y i e l d of c h l o r o p h y l l a t o t h e p h o t o c h e m i s t r y of p h o t o s y n t h e s i s is n o t e s t a b l i s h e d , i t s e e m s l i k e l y t h a t t h e r e d a n d farr e d effects of l i g h t o n t h e f l u o r e s c e n c e y i e l d h a v e t h e s a m e p h o t o c h e m i c a l b a s i s as t h e t w o w a v e l e n g t h effects of t h e s e c o n d EMERSON effect. T h e c o i n c i d e n c e b e t w e e n t h e a c t i o n m a x i m u m for t h e f a r - r e d effect o n t h e f l u o r e s c e n c e y i e l d a n d t h e a b s o r p t i o n m a x i m u m of C-7o 5 f u r t h e r i m p l i c a t e s C-7o 5 as o n e of t h e p i g m e n t s w h i c h c o n t r i b u t e s t o a s e c o n d EMERSON effect.

REFERENCES 1 W. 2 R. a K. 4 W.

L. BUTLER, Arch. Biochem. Biophys., 93 (19611 413 . EMERSON, R. CHALMERS AND C. CEDERSTRAND, Proc. Natl. Acad. Sci. U.S., 43 (1957) II3. H. NORRIS AND W. L. BUTLER, I R E Trans. Bio-Med. Electronics, B M E - 8 (196o) 153. L. BUTLER AND K. H. NORRIS, Arch. Biochim. Biophys., 87 (196o) 31. 5 H. tX~AUTSKY AND E. EBERLEIN, Biochem. Z., 302 (I939) 137. 6 p. LATIMER, T. T. BANISTER AND E. RABINOWITCH, in H. GAFFRON, Research in Photosynthesis, Interscience Publ., New York, 1955, p. lO77 j. E. BRUGGER, in H. GAFFRON, Research in Photosynthesis, Interscience Publ., New York, 1955, p. II3. 8 H. I. VIRGIN, Physiol. Plantarum, 7 (1954) 56o. 9 j. ROSENBERG, personal communication. 1o E. D. McALISTER AND J. MYERS, Smithsonian Misc. Collections, 99 (194 o) I. 11 B. KOK AND G. HOCH, in W. D. MCELROY AND n. GLASS, Light and Life, Johns Hopkins Press, 1961, P. 397. 12 L. M. N. DUYSENS, Thesis, Univ. of Utrecht, Holland, 1952 p. 45. 13 GOVINDJEE, S. SCHIMURI, C. CEDERSTRAND AND E. RABINOWITCH, Arch. Biochem. Biophys., 89 (196o) 322. 1~ j . W. COLEMAN, A. S. HOLT AND E. RABINOWlTCH, Science, 123 (1956) 795. Biochim. Biophys. Acta, 64 (1962) 3o9-317