Photodestruction of Chloroplast Ultrastructure by Red Light: Location of Chlorophyll* Patricia L

Proceedings of the National Academy of Sciences Vol. 67, No. 3, pp. 1501-1504, November 1970

Photodestruction of Chloroplast Ultrastructure by Red Light: Location of Chlorophyll* Patricia L. Walne, Alan H. Haber, and Larry L. Triplett

BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY, OAK RIDGE, TENN. 37830, AND DEPARTMENT OF BOTANY, UNIVERSITY OF TENNESSEE, KNOXVILLE, TENN. 37916 Communicated by W. A. Arnold, August 11, 1970 Abstract. A lethal dose (1 Mrad) of gamma rays was given to wheat leaf tissue to restrict migration of the energy of red light photoexcitation to the photosynthetic pigment system. This dose of gamma radiation had no apparent effect on chloroplast ultrastructure in tissue that received no illumination. Subsequent illumination with red light, used to induce chlorophyll-sensitized photodestruction of ultrastructure, permitted inferences about the location of chlorophyll within the chloroplast. The red light produced extensive disruption in chloroplasts but not in any other cellular organelles. The sequence of events in this photodestruction was different from that which occurs when chloroplasts are destroyed by methods that do not involve illumination. The red light caused peculiar abnormalities only in the internal lamellar system of the chloroplast and not in the envelope or any other regions. These findings support other studies which suggest that chlorophyll is located exclusively in grana and stroma lamellae. An acute lethal dose of gamma rays (1 i\Jrad) does not affect chloroplast ultrastructure in green wheat leaf tissue; such a dose actually suppresses the slow, normal disintegration of chloroplasts that occurs in darkness.1 2 Studies of glow curves and delayed light emission indicated that after a dose of 1 Mrad there was a normal capacity to put the energy from the photoreaction into electron traps in the photosynthetic pigment system, but the normal energy storage was abolished in the photosynthetic system that is coupled to the enzymatic re- actions of electron transport.2 Since the light-induced bleaching of chlorophyll in the massively gamma-irradiated leaf tissue is oxygen-dependent,1 and since the energy of photoexcitation is restricted within or very close to the photosynthetic pigment system,2 we suggest in this paper that red-light photodestruction after gamma irradiation may be used to locate the sites of chlorophyll within the chloroplast. Materials and Methods. 3-cm apical leaf tips were excised from 7-day-old seed- lings of wheat, Triticum vulgare, var. Monon, that was grown under conditions de- scribed elsewhere.3 Unless otherwise indicated, the freshly excised leaf tips were irradiated with gamma rays. Some were fixed immediately for electron microscopy, and others were illuminated with red light before fixation. Gamma irradiation: The leaf tips were irradiated in darkness in ice water at 00C in a "Gammacell 200" unit (Atomic Energy of Canada, Ltd., Ottawa) at 5 krad/min. Red-light illumination: Some leaf tips were exposed to 1, 2, or 3 hr of red light in an apparatus in which radiation from a 750-W projection bulb was collimated, re- 7 1501 Downloaded by guest on October 1, 2021 1502 BOTANY: WALNE ET AL. PROC. N. A. S.

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flected, condensed, and passed through a Corning glass filter (no. 2403) that cuts off radiation with wavelengths less than 600 nm. The temperature of the illuminated material was 29-30'C. The peak intensity was at 687 nm and ranged from about 200-300 ,uW/cm2 per nm, depending upon position within the illuminated area. The intensities at 650 and 675 nm were 67 and 99%, respectively, of the intensity at 687 nm. Electron microscopy: 2-mm-square pieces of leaf blade were fixed in 2.5% glutaraldehyde buffered to pH 7.2 with sodium cacodylate buffer for 24 hr at 100C and were postfixed in 2% buffered OS04 for 2 hr at the same temperature. After dehydration in a series of graded ethanols and acetone, specimens were embedded in a plastic mixture of ERI4206, according to the methods of Spurr.4 Sections were cut on a Sorvall MT-2 ultramicrotome, were post-stained with uranyl acetate and lead citrate, and were examined in JEM 6C and Siemens Elmiskop I electron microscopes. Results and Discussion. If disruption of chloroplasts is truly caused by photodestruction mediated by absorption of light by chlorophyll, gamma-irradiated material not exposed to light should experience no ultrastructural disruption. This expectation is verified in Fig. 1, which confirms our earlier study in showing that, despite its lethality, 1 Mrad of gamma rays has no apparent effect on chloroplast ultrastructure. Also, as expected, subsequent extensive photodestruction by red light (Figs. 2-4) was found only in the chloroplast but not in other cellular organelles. Again, consistent with the interpretation that we are examining a chlorophyll-sensitized photodestruction, the sequence of events as shown in Figs. 2-4 is in sharp contrast with the sequence of events either (a) during the slow and normal disintegration of chloroplasts during physiological senescence in darkness or (b) immediately after receiving doses of 2-7 Mrad of gamma rays. In these two instances (a and b), the internal lamellae reorient toward the periphery of the chloroplasts, and large numbers of osmophilic gran- ules accumulate in the severely disintegrated chloroplasts and in grana-stroma lamellar complexes, which remain after rupture of the chloroplast envelope.2'3 By-contrast, the first lesion from red-light illumination is a separation and swelling of stroma lamellae, rather than a reorientation of internal lamellae (Fig. 2). Subsequently, similar swelling and separation occur in the grana lamellae (Fig. 3). Eventually, definite grana and stroma membrane configurations cannot be discerned, but continuous swollen and distorted membrane profiles traverse the entire chloroplast stroma, in line with the long axis of the plastid (Figs. 3 and 4).

FIG. 1. Chloroplast from leaf immediately after 1 Mrad and no illumination. Note that, despite its lethality,2 1 Mrad of gamma rays has no apparent effect on chloroplast ultrastructure. This chloroplast is indistinguishable from controls that received no gamma irradiation (X 20,000). FIGS. 2-4. Chloroplasts from leaves given 1 Mrad of gamma rays, followed by illumination with red light. The sequence of events in chlorophyll-sensitized photodestruction is shown. FIG. 2. Separation and swelling of stroma lamellae (arrows) indicate the first lesions from red-light illumination (X 22,000). FIG. 3. Somewhat later, similar swelling and separation occur in the grana lamellae (arrows). In the upper chloroplast, swelling is so severe that most definite grana configurations are difficult to discern. Note also the presence of small striations (arrowheads) extending from the membranes (X 14,000). Insert at upper right shows higher magnification of area enclosed by box (X31,000). FIG. 4. Shortly before complete disintegration of chloroplasts after red-light illumination, well-defined grana and stroma configurations are scarcely evident. Swollen, distorted membrane profiles traverse the matrix, in line with the long axis of the plastid. Small striations (arrow) extend from the membranes (X 16,000). Insert at lower right shows details at higher magnification of area enclosed by box (X 40,000). Downloaded by guest on October 1, 2021 1504 BOTANY: WALNE ET AL. PROC. N. A. S.

Examination of all swollen and separated lamellar profiles, grana and stroma, reveals the presence of small fibrils extending from the membranes (Figs. 3 and 4). All effects of illumination on chloroplasts appear to be centered in membranes, since the stroma or matrix regions of illuminated and dark-control chloroplasts appear identical. As an added control, non-gamma-irradiated tissue exposed to intense red light showed chloroplast photodestruction similar to that shown in Figs. 2-4. The internal lamellar system but no other cellular membranes, including even the chloroplast envelope, show the photodestructive effects reported here. These findings provide an independent line of evidence supporting other studies which suggest that chlorophyll is in stroma lamellae as well as in grana lamellae.5 6 Extension of this approach to freeze-etched material should provide more pre- cise localization of the locus of red light absorption and therefore of chlorophyll location. We thank Dr. 0. L. Miller, Jr. for making his electron microscope facilities available to us. * Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. 1 Haber, A. H., and P. L. Walne, Radiat. Bot., 8, 389 (1968). 2 Walne, P. L., and A. H. Haber, Radiat. Bot., 8, 399 (1968). 3 Haber, A. H., P. J. Thompson, P. L. Walne, and L. L. Triplett, Plant Physiol., 44, 1619 (1969). 4Spurr, A. R., J. Ultrastruct. Res., 26, 31 (1969). 5 Weier, T. E., and A. A. Benson, in Biochemistry of Chloroplasts, ed. T. W. Goodwin (New York: Academic, Press, 1966), vol. 1, p. 91. 6 Menke, W., in Biochemistry of Chloroplasts, ed. T. W. Goodwin (New York: Academic Press, 1966), vol. 1, p. 3. Downloaded by guest on October 1, 2021