- Email: [email protected]
Oriented chlorophyll in vivo
651
SHORT COMMUNICATIONS
sc 43 029
Oriented chlorophyll M v/vo Observations of polarized fluorescencez and dichroism ~ on chloroplasts of Euglena showed that a part of the chlorophyll was highly oriented relative to the planes of the chloroplast lamellae. The wavelength dependency of the polarized fluorescence (~max = 716 m/z) and dichroism (~max = 705 mP) 3 indicated that the oriented chlorophyll absorbed at somewhat longer wavelengths than the main bulk of the chlorophyll which was not oriented. SAUER AND CALVIN¢ found that chloroplast fragments showed electric dichroism with a wavelength maximum near 700 m/z. It was suggested z-3 that C-7o5 (ref. 5) might be the oriented chlorophyll. Recently, we obtained a mutant of Scenedesmus from Dr. N. I. BISHOP which lacked C-7o 5 (ref. 6). We report in the present note that the mutant cells show dichroism and polarized emission in the absence of C-7o5.
400
,
,
,
,
I
500
. . . .
I
. . . .
T
,
600 700 WAVELENGTH ( m~)
6~o'
,
' 7~o'
,
Fig. I. Absorption and derivative absorption spectra at --1960 of wild-type and CO 2 m u t a n t No. 8 cells of Scenedesmus.
The absorption and derivative absorption spectra of wild-type and mutant cells at --1960 are shown in Fig. I. C-7o5, which appears in the spectra of the wild-type cells as a shoulder in the absorption spectrum on the long-wavelength side of the main chlorophyll band and as a maximum near 70o mp in the derivative spectrum~ is absent from the spectra of the mutant cells. Both the wild-type and mutant cells show polarized fluorescence in the fluorescence microscope; particularly, when viewed with an infrared image converter or when photographed with infrared-sensitive film. In fact, the effect can be photographed more readily with the mutant cells because the fluorescence yield of the mutant cells is greater than that of the wild-type cells6. The cells shown in Fig. 2 (wild type) and Fig. 3 (mutant) were irradiated with unpolarized blue light (mainly at 436 rap) and photographed through a red cut-off filter and polarizer, oriented as indicated by the double-headed arrows. The polarized emission is demonstrated predominately by those chloroplasts or parts of chloroplasts which are against the cell wall and are viewed on edge. Under these conditions the planes of the chloroplast lamellae tend to be parallel to the cell wall and to the line of sight. The fluorescing chloroplasts appear brightest when the chloroplast profile is parallel to the plane of Biochim. Biophys. Acts, 88 (1964) 651-653.
652
SHORT COMMUNICATIONS
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. e. I~hotomicrographs of fluorescing w i l d - t y p e S c e n e d e s m u s cells t a k e n t h r o u g h C o r n i n g filters ~o3 o a n d 983o a n d a Nicol prism. P l a n e of polarization i n d i c a t e d b y d o u b l e - h e a d e d arrows. Fig. 3. S a m e as Fig. 2 b u t w i t h CO 2 m u t a n t No. 8. Fig. 4. P h o t o m i c r o g r a p h s of w i l d - t y p e Scened e s m u s cells t a k e n in polarized, m o n o c h r o m a t i c l i g h t a t 69o m/~. P l a n e of polarization i n d i c a t e d b y d o u b l e - h e a d e d arrows. Fig. 5- S a m e as Fig. 4 b u t with COe m u t a n t No. 8.
polarization, indicating that the oriented oscillators are in the planes of the lamellae. The same conditions of orientation were found with Euglena cellsk The wild-type and mutant cells also exhibit a polarized absorption (dichroism) which is consistent with the polarized emission. Photomicrographs of the wild-type (Fig. 4) and mutant (Fig. 5) cells taken in polarized, monochromatic light at 69o m/x show that a number of the chloroplasts, which are viewed on edge, absorb most strongly when the chloroplast profile is parallel to the plane of polarization. We must conclude that C-7o5, which is absent from the mutant cells, is not the oriented chlorophyll. Even in the wild-type ceils, the amount of C-7o5 present is probably too small to account for the polarized absorption and emission. Direct spectrophotometric measurement of the polarized emission and absorption, as was done with Euglena and Mougeotia a, is much more difficult with Scenedesmus because the chloroplasts are smaller. However, visual observation of fluorescence from Scenedesmus chloroplasts through different cut-off filters indicated that the fluorescence was more polarized at the longer wavelengths of emission and observation of polarized absorption in different wavelengths of light showed that dichroism was more pronounced at wavelengths beyond the chlorophyll absorption maximum. We conclude that the chlorophyll a in both the wild-type and mutant cells of Scenedesmus consists of an unoriented shorter-wavelength-absorbing form and an oriented longerwavelength-absorbing form. The absorption and derivative spectra of Fig. I show that the chlorophyll a in the wild-type and mutant strains is comprised of two types of chlorophyll a with absorption maxima near 670 and 68o m/x at --196°. We shall refer to chlorophyll a-67 o and chlorophyll a-68o, although, at room temperature, the absorption maxima are nearer 673 and 683 m/x, respectively7. We suggest that the oriented chlorophyll is chlorophyll a-68o. Chlorophyll a-67o and chlorophyll Biochim. Biophys. Acta, 88 (I964) 651 653
SHORT COMMUNICATIONS
653
a-68o have been postulated to be monomeric and aggregated forms of chlorophyll a, respectivelys. It is reasonable that the oriented chlorophyll should be the aggregated chlorophyll. Measurement of the wavelength dependency of the dichroic ratio 3 does not define the absorption maximum of the oriented pigment. The dichroic ratio will be greatest at those wavelengths where the absorption by the oriented oscillator is greatest relative to the absorption by unoriented oscillators. The presence of unoriented chlorophyll a-67o will shift the dichroism due to oriented chlorophyll a-68o molecules to longer wavelengths. The observation that the dichroic ratio in chloroplasts of Euglena and Mougeotia goes through a maximum and declines at longer wavelength indicates that an unoriented oscillator absorbs in the long-wavelength region, perhaps C-7o5 . A calculation of the wavelength dependency of the dichroic ratio in a model system consisting of an unoriented oscillator absorbing at 673 m/~, an oriented oscillator at 685 m/~ with a dichroic ratio of IO, and an unoriented oscillator at 703 mp. in absorbancy ratios of IO:IO:I, assuming Gaussian distributions for the absorption bands, gave a curve similar to that obtained experimentally with Euglena and Mougeotia ~. All of our measurements and observations are consistent with chlorophyll a-68o being the oriented pigment. Action spectra for the two pigment systems of photosynthesis indicate that chlorophyll a-67o is predominately associated with system 2 (ref. 9) and that chlorophyll a-68o is associated with system I. We suggest, therefore, that the chlorophyll a associated with system I is oriented in the planes of the lamellae.
Instrumentation Research Laboratory, Market Quality Research Division, Agricultural Marketing Service, U.S. Department of Agriculture, Beltsville, Md. and Laboratory of Physical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.)
W. L. BUTLER R. A. OLSON W. H. J E N N I N G S
1 R. A. OLSON, W. L. BUTLER AND W. H. JENNINGS, Biochim. Biophys. Acta, 54 (1961) 615. z R. A. OLSON, W. L. BUTLER AND W. H. JENNINGS, Biochim. Biophys. Acta, 58 (1962) 144. a R. A. OLSON, in Photosynthetic Mechanisms in Green Plants, N A S - N R C Publ. 1145, W a s h i n g t o n , 1963, P. 545. 4 K. SAUER AND M. CALVIN, J. Mol. Biol., 4 (1962) 451. 5 W. L. BUTLER, Arch. Biochem. Biophys., 93 (1961) 413 . e W. L. BUTLER AND 1~. I. BISHOP, in Photosynthetic Mechanisms in Green Plants, N A S - N R C Publ. 1145, W a s h i n g t o n , 1963, p. 91. J. S. BROWN AND C. S. FRENCH, Plant Physiol., 34 (1959) 3o5. 8 A. H. KRASNOVSKYAND L. M. KOSOBUTSKAYA, Doklady Akad. Nauk S S S R, 85 (1962) 177. 9 GOVINDJEE AND E. RABINOWlTCH, Science, 132 (196o) 355.
Received June 26th, 1964 Biochim. Biophys. Acta, 88 (1964) 651-655
SHORT COMMUNICATIONS
sc 43 029
Oriented chlorophyll M v/vo Observations of polarized fluorescencez and dichroism ~ on chloroplasts of Euglena showed that a part of the chlorophyll was highly oriented relative to the planes of the chloroplast lamellae. The wavelength dependency of the polarized fluorescence (~max = 716 m/z) and dichroism (~max = 705 mP) 3 indicated that the oriented chlorophyll absorbed at somewhat longer wavelengths than the main bulk of the chlorophyll which was not oriented. SAUER AND CALVIN¢ found that chloroplast fragments showed electric dichroism with a wavelength maximum near 700 m/z. It was suggested z-3 that C-7o5 (ref. 5) might be the oriented chlorophyll. Recently, we obtained a mutant of Scenedesmus from Dr. N. I. BISHOP which lacked C-7o 5 (ref. 6). We report in the present note that the mutant cells show dichroism and polarized emission in the absence of C-7o5.
400
,
,
,
,
I
500
. . . .
I
. . . .
T
,
600 700 WAVELENGTH ( m~)
6~o'
,
' 7~o'
,
Fig. I. Absorption and derivative absorption spectra at --1960 of wild-type and CO 2 m u t a n t No. 8 cells of Scenedesmus.
The absorption and derivative absorption spectra of wild-type and mutant cells at --1960 are shown in Fig. I. C-7o5, which appears in the spectra of the wild-type cells as a shoulder in the absorption spectrum on the long-wavelength side of the main chlorophyll band and as a maximum near 70o mp in the derivative spectrum~ is absent from the spectra of the mutant cells. Both the wild-type and mutant cells show polarized fluorescence in the fluorescence microscope; particularly, when viewed with an infrared image converter or when photographed with infrared-sensitive film. In fact, the effect can be photographed more readily with the mutant cells because the fluorescence yield of the mutant cells is greater than that of the wild-type cells6. The cells shown in Fig. 2 (wild type) and Fig. 3 (mutant) were irradiated with unpolarized blue light (mainly at 436 rap) and photographed through a red cut-off filter and polarizer, oriented as indicated by the double-headed arrows. The polarized emission is demonstrated predominately by those chloroplasts or parts of chloroplasts which are against the cell wall and are viewed on edge. Under these conditions the planes of the chloroplast lamellae tend to be parallel to the cell wall and to the line of sight. The fluorescing chloroplasts appear brightest when the chloroplast profile is parallel to the plane of Biochim. Biophys. Acts, 88 (1964) 651-653.
652
SHORT COMMUNICATIONS
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. e. I~hotomicrographs of fluorescing w i l d - t y p e S c e n e d e s m u s cells t a k e n t h r o u g h C o r n i n g filters ~o3 o a n d 983o a n d a Nicol prism. P l a n e of polarization i n d i c a t e d b y d o u b l e - h e a d e d arrows. Fig. 3. S a m e as Fig. 2 b u t w i t h CO 2 m u t a n t No. 8. Fig. 4. P h o t o m i c r o g r a p h s of w i l d - t y p e Scened e s m u s cells t a k e n in polarized, m o n o c h r o m a t i c l i g h t a t 69o m/~. P l a n e of polarization i n d i c a t e d b y d o u b l e - h e a d e d arrows. Fig. 5- S a m e as Fig. 4 b u t with COe m u t a n t No. 8.
polarization, indicating that the oriented oscillators are in the planes of the lamellae. The same conditions of orientation were found with Euglena cellsk The wild-type and mutant cells also exhibit a polarized absorption (dichroism) which is consistent with the polarized emission. Photomicrographs of the wild-type (Fig. 4) and mutant (Fig. 5) cells taken in polarized, monochromatic light at 69o m/x show that a number of the chloroplasts, which are viewed on edge, absorb most strongly when the chloroplast profile is parallel to the plane of polarization. We must conclude that C-7o5, which is absent from the mutant cells, is not the oriented chlorophyll. Even in the wild-type ceils, the amount of C-7o5 present is probably too small to account for the polarized absorption and emission. Direct spectrophotometric measurement of the polarized emission and absorption, as was done with Euglena and Mougeotia a, is much more difficult with Scenedesmus because the chloroplasts are smaller. However, visual observation of fluorescence from Scenedesmus chloroplasts through different cut-off filters indicated that the fluorescence was more polarized at the longer wavelengths of emission and observation of polarized absorption in different wavelengths of light showed that dichroism was more pronounced at wavelengths beyond the chlorophyll absorption maximum. We conclude that the chlorophyll a in both the wild-type and mutant cells of Scenedesmus consists of an unoriented shorter-wavelength-absorbing form and an oriented longerwavelength-absorbing form. The absorption and derivative spectra of Fig. I show that the chlorophyll a in the wild-type and mutant strains is comprised of two types of chlorophyll a with absorption maxima near 670 and 68o m/x at --196°. We shall refer to chlorophyll a-67 o and chlorophyll a-68o, although, at room temperature, the absorption maxima are nearer 673 and 683 m/x, respectively7. We suggest that the oriented chlorophyll is chlorophyll a-68o. Chlorophyll a-67o and chlorophyll Biochim. Biophys. Acta, 88 (I964) 651 653
SHORT COMMUNICATIONS
653
a-68o have been postulated to be monomeric and aggregated forms of chlorophyll a, respectivelys. It is reasonable that the oriented chlorophyll should be the aggregated chlorophyll. Measurement of the wavelength dependency of the dichroic ratio 3 does not define the absorption maximum of the oriented pigment. The dichroic ratio will be greatest at those wavelengths where the absorption by the oriented oscillator is greatest relative to the absorption by unoriented oscillators. The presence of unoriented chlorophyll a-67o will shift the dichroism due to oriented chlorophyll a-68o molecules to longer wavelengths. The observation that the dichroic ratio in chloroplasts of Euglena and Mougeotia goes through a maximum and declines at longer wavelength indicates that an unoriented oscillator absorbs in the long-wavelength region, perhaps C-7o5 . A calculation of the wavelength dependency of the dichroic ratio in a model system consisting of an unoriented oscillator absorbing at 673 m/~, an oriented oscillator at 685 m/~ with a dichroic ratio of IO, and an unoriented oscillator at 703 mp. in absorbancy ratios of IO:IO:I, assuming Gaussian distributions for the absorption bands, gave a curve similar to that obtained experimentally with Euglena and Mougeotia ~. All of our measurements and observations are consistent with chlorophyll a-68o being the oriented pigment. Action spectra for the two pigment systems of photosynthesis indicate that chlorophyll a-67o is predominately associated with system 2 (ref. 9) and that chlorophyll a-68o is associated with system I. We suggest, therefore, that the chlorophyll a associated with system I is oriented in the planes of the lamellae.
Instrumentation Research Laboratory, Market Quality Research Division, Agricultural Marketing Service, U.S. Department of Agriculture, Beltsville, Md. and Laboratory of Physical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.)
W. L. BUTLER R. A. OLSON W. H. J E N N I N G S
1 R. A. OLSON, W. L. BUTLER AND W. H. JENNINGS, Biochim. Biophys. Acta, 54 (1961) 615. z R. A. OLSON, W. L. BUTLER AND W. H. JENNINGS, Biochim. Biophys. Acta, 58 (1962) 144. a R. A. OLSON, in Photosynthetic Mechanisms in Green Plants, N A S - N R C Publ. 1145, W a s h i n g t o n , 1963, P. 545. 4 K. SAUER AND M. CALVIN, J. Mol. Biol., 4 (1962) 451. 5 W. L. BUTLER, Arch. Biochem. Biophys., 93 (1961) 413 . e W. L. BUTLER AND 1~. I. BISHOP, in Photosynthetic Mechanisms in Green Plants, N A S - N R C Publ. 1145, W a s h i n g t o n , 1963, p. 91. J. S. BROWN AND C. S. FRENCH, Plant Physiol., 34 (1959) 3o5. 8 A. H. KRASNOVSKYAND L. M. KOSOBUTSKAYA, Doklady Akad. Nauk S S S R, 85 (1962) 177. 9 GOVINDJEE AND E. RABINOWlTCH, Science, 132 (196o) 355.
Received June 26th, 1964 Biochim. Biophys. Acta, 88 (1964) 651-655